Synthesis and Biological Evaluation of Potential CGRP Antagonists: Application to Chronic Pain

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Synthesis and biological evaluation of potential CGRP antagonists : application to chronic pain Nishanth Kandepedu Hemachandra

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Nishanth Kandepedu Hemachandra. Synthesis and biological evaluation of potential CGRP antagonists : application to chronic pain. Other. Université Blaise Pascal - Clermont-Ferrand II, 2015. English. NNT : 2015CLF22627. tel-03081193

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Numéro d’ordre : DU 2627

UNIVERSITE BLAISE PASCAL

(U.F.R. Recherche Scientifique et Technique) ECOLE DOCTORALE DES SCIENCES FONDAMENTALES

THESE

présentée pour obtenir le grade de

DOCTEUR D’UNIVERSITE

(Spécialité : Chimie Organique Biologique)

par

Nishanth H. KANDEPEDU

Master of Science in Pharmaceutical Chemistry, Vellore Institute of Technology (VIT), Vellore (Inde)

Synthèse et Évaluation de nouveaux squelettes de molécules potentiellement antagonistes du récepteur au CGRP : Application à la douleur chronique

Soutenue à huis clos le 27 Novembre 2015, devant la Commission d’Examen :

Président :

Pascale MOREAU Professeur/Université Blaise Pascal de Clermont-Ferrand

Rapporteurs :

Damien PRIM Professeur / Université de Versailles-St. Quentin en Yvelines

Maria SANTOS Assistant Researcher/ Université de Lisbonne, Portugal

Jean SUFFERT Directeur de Recherches CNRS/ Université de Strasbourg

Examinateurs :

Denis ARDID Professeur/Université d’Auvergne

Gérald GUILLAUMET Professeur/Université d’Orléans

Isabelle THOMAS Maître de conférences/ ENS de Chimie de Clermont-Ferrand

Yves TROIN Directeur de thèse / Professeur/ ENS de Chimie de Clermont-Ferrand

ACKNOWLEDGMENT

This work has been carried out in the laboratory of Conception Et Synthèse de Molécules Antalgiques (CESMA) affiliated to the Institut de Chimie de Clermont-Ferrand (ICCF-UMR 6296) of Université Blaise Pascal, Clermont-Ferrand under the supervision of Professor Yves Troin and Doctor Isabelle Abrunhosa Thomas.

First of all, I would like to thank Professor Yves Troin, the director of my thesis, for accepting me to enroll as a PhD student to work under his direction. He was encouraging and was always ready to help when I faced some problems in chemistry. He provided me enough freedom in exploring the areas of my research. He encouraged me not just to confine in the day-to-day laboratory work but also to be autonomous in other areas of our research.

I would like to thank Doctor Isabelle Abrunhosa Thomas, without whom I would have had tough time not just in lab but also in day-to-day life. I learnt a lot of chemistry concepts from her during the course of these three years and she was always available for me when I need some advice or help in chemistry. She created an ambient environment for me to work these three years and she had atmost faith on my work. Apart from this, she was kind and took care of me in these three years not just in the lab but also outside the laboratory .

I would also like to thank Professor Jean Suffert of Université de Strasbourg, Professor Damien Prim of Université de Versailles and Professor Maria M. M. Santos of Universidade de Lisboa for accepting as referees to judge my thesis. I would like to extend my thanks to Pr. Santos for accepting to be part of my jury even though she has to travel a long distance.

I would like to thank Professor Gérald Guillaumet of Université d'Orléans, Professor Pascale Moreau of Université Blaise Pascal, Professor Denis Ardid of Université d'Auvergne for accepting to be part of the jury for my thesis.

I would like to extend my thanks to Professor Denis Ardid, one of the principle investigator and collaborator of our project who has encouraged us to conduct the pharmacological activity of our molecules and helped in lot of ways to enhance this project according to the clinical setting. I would like to thank Miss. Julie Barbier, Laboratory Technician at Université d'Auvergne for accompanying me during the biological evaluation part of our project and the rapport & humour we shared at times. I would like to thank Professor Sylvie Ducki and Doctor Kasi Sankar who i consider as mentors for my scientific career. They encouraged me to pursue research as my career and whatever the aptitude i have developed towards science, is because of them.

I would like to extend my thanks to the present and ex-members of CESMA, Dr. Khalil Bennis, Dr. Isabelle Ripoche, Dr. Pierre Chalard, Dr. Jean-Philippe Roblin and students like Ostache Cosmin, Marie Harel, Li Jie, Dr. Oana-Patriciu, Dr. Tomas Carillo Marquez, Romain Jouve, Dr. Naoual Bouzidi, Dr. Wahid-Bux Jatoi, Quentin Lenin, Ombeline Danton, Camille Monnier etc who all helped me in the course of these three years.

I would like to specially thank Doctor Delphine Vivier and Gary Vallon with whom i shared most of the time during my stay in CESMA. Since my arrival, Delphine and Gary helped me in uncountable number of aspects and never said no when i needed them.

I also would like to thank Safia Laid, Gaelle Framery and other members of ENSCCF for their help during these three years.

I would like to thank my parents K. L. Hemachandra and K. H. Indira without whom i would have never been in the stage where i am now. They supported and helped in all stages of my life.

LIST OF ABBREVATIONS

ATP: Adenosine triphosphate DBU : 1,8-Diazabicyclo[5.4.0]undec-7-ene AT/ET: Angiotensin/Endothelin DEPBT : (3-(diethoxyphosphoryloxy)- Ar: Aromatic 1,2,3-benzotriazin-4(3H)-one) ADs: Antidepressants DEPT : Distortionless enhancement by Boc: Di-tert-Butyl carbamate polarization transfer BuLi: Butyl lithium DMF : N,N-Dimethylformamide BINAP: (2,2'-bis(diphenylphosphino)-1,1'- DPPA: Diphenylphosphoryl azide binaphthyl) ee: enantiomeric excess CNS: Central Nervous System er: enantiomeric ratio CSD: Cortical Spreading Depression E: Entgegen CGRP: Calcitonin Gene Related Peptide EDC:1-Ethyl-3-(3- CALCRL: Calcitonin Receptor Like dimethylaminopropyl)carbodiimide Receptor GBD: Global Burden Data COX: Cyclooxygenase GPCR: G-protein Coupled Receptor cAMP: Cyclic Adenosine monophosphate HPA: Hypothalamic pituitary adrenal Cbz: Benzyl carbamate IASP: International Association for the COMU: (1-Cyano-2-ethoxy-2- study of pain oxoethylidenaminooxy)dimethylam IBS: Irritable Bowel Syndrome ino-morpholino-carbenium iPr: Isopropyl hexafluorophosphate LOX: Lipoxygenase CY: Cyclic L-Asp: Levo-Aspartic acid de: diastereomeric excess MAO: Monoamine Oxidase dr: diastereomeric ratio MW: Microwave DIAD: Diisopropyl azodicarboxylate NSAIDS: Non-steroidal Antiinflammatory DIPEA: N,N-Diisopropylethylamine Drugs D-Asp : Dextro-Aspartic acid NDM: Noerpinephrine Dopamine receptor DCC : N,N'-dicyclohexylcarbodiimide NCS: N-chlorosuccinimide DMAP : Dimethylaminopyridine NMR:Nuclear Magnetic Resonance HBD : Hydrogen bond donor HWE: Horner-Wadsworth-Emmons HBA : Hydrogen bond acceptor Hz: Hertz HIV : Human immune virus HOBT: Hydroxybenzotriazole HCl : Hydrogen chloride

HBTU:3- RT: Room temperature Bis(dimethylamino)methyliumyl]-3H- SNRI:Serotonin-Norepinephrine Reuptake benzotriazol-1-oxide hexaﬂuorophosphate Inhibitor HY: Hydrogenated SSRI: Selective Serotonin Reputake PNS: Peripheral Nervous system Inhibitor PKA: Protein Kinase A TCA : Tricyclic antidepressants PGE: Proftaglandin E Tert : Tertiary PDB: Protein Data Bank THF : Tetrahydrofuran Ph: Phenyl TrCl : Trityl chloride ppm: parts per million TFA : Trifluoroacetic acid p-TsOH: para toluenesuphonic acid TMSI : Iodotrimethylsilane PG: Protecting group T3P: 1-Propanephosphonic anhydride PS: privileged structure USFDA : United States Food & Drug RAMP: Receptor Activity Modifying Administration Protein Z: zusammen

CONTENTS CHAPTER 1: INTRODUCTION ...... 21 I. PAIN ...... 22 A. Classification: ...... 23

1. Classification based on time period: ...... 23

1.1. Acute pain: ...... 23 1.2 Chronic pain: ...... 24 i. Nociceptive pain: ...... 24 ii. Neuropathic pain: ...... 25 2. Intensity ...... 26

II. PAIN ORIGIN, TRANSMISSION AND MODULATION ...... 27 A. Nervous system ...... 27

B. Pain pathway ...... 28

2. Transmission ...... 30

1.1 Peripheral transmission: ...... 31 1.2 Synaptic transmission: ...... 31 1.3 Central transmission: ...... 31 3. Perception ...... 31

4. Modulation ...... 32

4.1 Gate control theory of pain ...... 32 4.2 Descending modulation ...... 32 C. Global data on pain ...... 33

III. MIGRAINE & VISCERAL INFLAMMATORY PAIN ...... 35 A. Migraine ...... 35

1. Key facts & figures ...... 35

2. Pathophysiology of migraine ...... 36

3. Treatment of migraine ...... 37

3.1 5-HT1B/1D receptor antagonists for migraine therapy; the triptans ...... 37 3.2 Calcitonin Gene Related Peptide ...... 38 i. CGRP receptor antagonism ...... 39 ii. Discovery of peptide antagonists for CGRP receptor ...... 40

iii. Non peptide CGRP antagonists ...... 41 iv. Antibodies & biologics in contemporary migraine market ...... 43 B. Visceral pain ...... 44

1. Causes of visceral pain ...... 44

2. Clinical manifestation of visceral pain ...... 44

2.1 True visceral pain ...... 45 2.2 Visceral hyperalgesia...... 45 2.3 Viscera-viscero hyperalgesia ...... 45 2.4 Referred pain without hyperalgesia ...... 46 2.5 Referred pain with hyperalgesia ...... 46 3. Pathophysiology of visceral pain ...... 46

3.1 Central sensitization ...... 46 3.2 Peripheral sensitization ...... 47 IV. RATIONALE FOR THE PROJECT ...... 47 A. Dual activity ...... 48

1. Drugs for same diseases/disorders: ...... 49

2. Drugs for different diseases/disorders: ...... 52

B. Design of the project ...... 54

C. Depression and its management ...... 55

1. Depression ...... 55

Chronic pain and depression ...... 56

2. Classification of depression ...... 56

2.1 Major depressive disorder or Major depression ...... 56 2.2 Dysthymia or Dysthymic disorder ...... 56 2.3 Minor depression ...... 56 3. Biochemistry of depression ...... 56

3.1 Monoamine neurotransmitters ...... 57 3.2 Mechanism of action ...... 57 4. Treatment of depression ...... 58

4.1 Pharmacotherapy ...... 58 i. Mechanism of action ...... 59

D. Depression vs chronic pain ...... 62

1. Biochemistry of antidepressants in chronic pain management...... 62

2. Antidepressants and pain ...... 63

V. CONTEXT OF THE PROJECT ...... 65 CHAPTER 2: CALCITONIN GENE RELATED PEPTIDE RECEPTOR, MOLECULAR MODELING STUDIES & LIGAND DESIGN ...... 70 I. CALCITONIN GENE RELATED PEPTIDE RECEPTOR ...... 71 A. DRUGGABILITY ...... 72

1. Druggability study of CGRP receptor ...... 72

2. Druggability validation of CGRP receptor ...... 73

B. DRUG-RECEPTOR INTERACTION STUDY: ...... 76

C. SPECIES SELECTIVIY DECISIVE RESIDUES ...... 76

II. MOLECULAR MODELING STUDIES ...... 77 A. Modelling of the receptor ...... 78

B. Modelling of the ligands ...... 79

1. Structure based ligand designing ...... 79

1.1 Ligand designing ...... 80

1.2 Application of medicinal chemistry principles ...... 81

C. Docking ...... 83

D. Identification and classification of potential hits ...... 85

E. Receptor pocket volume Vs ligand volumes ...... 87

F. Drug-likeliness properties ...... 89

III. RETROSYNTHETIC DESIGN FOR SYNTHESIS OF HITS ...... 92 CHAPTER 3: RESULTS AND DISCUSSION ...... 96 I. LITERATURE INVESTIGATION ...... 97 A. Synthesis of 2, 6-disubstituted-4-hydroxy piperidines ...... 97

1. Synthesis by using metal catalysis ...... 97

1.1 Application of Iridium complex: ...... 98 1.2 Application of Gold complex: ...... 99

1.3 Miscellaneous metal complex: ...... 100

2. Synthesis by intramolecular reductive amination ...... 100

3. Synthesis by intramolecular nucleophilic displacement ...... 103

3.1 Activation in the form of mesylate or tosylate ...... 103 4. Miscellaneous methods ...... 105

4.1 Asymmetric hetero Diels-Alder reactions ...... 105 B. SYNTHESIS OF 4-HYDROXY-6-SUBSTITUTED PIPECOLIC ACID/ESTER ...... 113

1. Synthesis using chiral auxiliaries from amino acids ...... 114

2. Synthesis using chiral auxiliaries from chiral alcohols ...... 116

C. CONCLUSION ...... 120

II. STRATEGY FOR THE SYNTHESIS OF FAMILY 1 ...... 121 Synthon A ...... 121

Synthon B ...... 122

A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME ...... 123

1. Synthesis of 6-methyl-4-oxopiperidine-2-carboxylic acid from aspartic acid ...... 123

1.1 Fischer-Speier esterification of aspartic acid ...... 123 1.2 Protection of amine group in dimethyl ester of aspartic acid ...... 123 1.3 Synthesis of β-keto phosphonate ester...... 125 1.4 Synthesis of (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate ...... 126 Stereoselectivity of HWE ...... 126

Mechanism involved in HWE to give E-alkene ...... 127

1.5 Synthesis of methyl 6-methyl-4-oxopiperidine-2-carboxylate ...... 129 1.6 Protection of amine in 6-methyl-4-oxopiperidine-2-carboxylic acid ...... 134 2. Synthon B ...... 136

3. Precedent work and optimizations ...... 137

4. Application of the previously optimized conditions...... 142

5. Peptide coupling for amide bond formation ...... 143 6. Deprotection of mono-peptides ...... 151 6.1 Deprotection of tert-Butyl carbamate ...... 151 6.2 Deprotection of the benzylcarbamate on the piperidine ...... 155 7. C-N cross coupling reaction ...... 156

8. Deprotection of ketal ...... 161

III. STRATEGY FOR THE SYNTHESIS OF FAMILY 3 ...... 162 A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME ...... 165

2.6 Piperidine Synthon III ...... 165

1.1 Synthesis of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate ...... 165 1.2 Synthesis of (S)-4-methoxy-4-oxobutan-2-aminium acetate ...... 167 1.3 Protection of (S)-3-methoxy-3-oxobutan-1-aminium acetate ...... 168 1.4 Synthesis of ketophosphonates ...... 169 1.5 Synthesis of α,β-unsaturated ketones: application of Horner-Wadsworth-Emmons condition ...... 170 i. Synthesis of biaryl aldehydes ...... 170 ii. Horner-Wadsworth-Emmons ...... 171 1. Aza-Michael intramolecular cyclization ...... 173

2. Formation of tetracyclic moiety ...... 178

2.1 Subclass A ...... 178 i. Application of designed route C ...... 180 2.2 Subclass B ...... 185 2.3 Subclass C ...... 192 IV. FUNCTIONAL GROUP INTERCONVERSION...... 198 V. CONVERGENCE OF THE TWO FRAGMENTS ...... 206 CHAPTER 4: PHARMACOLOGICAL EVALUATION ...... 225 BIOASSAY ...... 226 A. Pain bioassay ...... 226

1. Hot plate method ...... 227

1.1 Rationale ...... 227 1.2 General procedure ...... 227 1.3 Assessment ...... 228 1.4 Significant points to be noted ...... 228 2. Antinociceptive property of the synthesized CGRP antagonists ...... 229

B. Locomotory activity bioassay...... 233

1. Photoactometer- an infrared photocell-based detection ...... 234

1.1 General procedure ...... 234

1.3 Assessment ...... 235 2. Locomotory activity of the synthesized CGRP antagonists ...... 235

C. Antidepressant bioassay ...... 239

1. Porsolt Forced Swimming Test (FST) ...... 239

1.1 General procedure ...... 239 1.2 Assessment ...... 240 GENERAL CONCLUSION ...... 246

CHAPTER 5: EXPERIMENTAL PART ...... 250

LIST OF FIGURES

Figure 1.Deaths due to untreated pain associated with disease/disorder ...... 22 Figure 2.Classification of pain based on time period of occurrence ...... 23

Figure 3.Drugs used for acute pain ...... 24

Figure 4.Drugs used for chronic pain ...... 24 Figure 5.showing ascending pain pathway ...... 25

Figure 6.showing pain scaling and experience of a patient ...... 26

Figure 7.showing signalling pathways of CNS & PNS ...... 27

Figure 8.showing signalling pathways from receptor to the effector organ ...... 28 Figure 9.showing signalling pathways from receptor to spinal cord by transmission ...... 30

Figure 10.showing areas of brain implicated in thermal pain signalling ...... 31

Figure 11.showing gate control theory of pain ...... 32 Figure 12.Drugs acting at different levels as analgesics ...... 33

Figure 13.screenshot showing global ranking with respect to Years Lived with Disability (YLD) ...... 34

Figure 14.screenshot showing global ranking with respect to deaths ...... 34 Figure 15.screenshot showing global deaths due to Non-infectious inflammatory bowel syndrome . 35

Figure 16.Distribution & implications of CGRP receptor ...... 39

Figure 17.Molecular level cascade events of CGRP receptor activation ...... 40

Figure 18.Peptide antagonists for CGRP receptor...... 40 Figure 19.Sequence of modulations resulting in BIBN-4096 ...... 41

Figure 20.Non peptide antagonists ...... 41

Figure 21.Non peptide antagonists by Merck ...... 42 Figure 22.Non peptide antagonists by Boehringer Ingelheim ...... 42

Figure 23.showing various forms of visceral pain ...... 45

Figure 24.Showing the account of analgesics developed recently ...... 47 Figure 25.Dual cyclooxygenase/5-lipoxygenase inhibitors ...... 49

Figure 26.Indomethacin derived dual COX/5-LOX inhibitors ...... 50

Figure 27.Tepoxaline as dual COX/5-LOX inhibitors ...... 50 Figure 28.Licofelone as dual COX/5-LOX inhibitors ...... 51

Figure 29.Dual Angiotensin II and Endothelin I receptors antagonist development ...... 51

Figure 30.Isomers of Tramadol ...... 52

Figure 31.Dual activity of Tapentalol...... 53

Figure 32.Dual activity design for the project ...... 54 Figure 33.Showing correlation of pain and major depression ...... 55

Figure 34.Serotonin and norepinephrine function ...... 56

Figure 35.Biological action of monoamines ...... 58

Figure 36.context of the project ...... 65

Figure 37.(a).GPCR Tree (b) Class-B Secretin family (c) CGRP receptor (d) CALCRL-protein interactions ...... 71

Figure 38.Depicting the Tractability, Druggability and Ensemble druggability of CALCLR and RAMP-173

Figure 39.showing the CALCRLs site 1 & the fragment of Olcegepant® embedded inside the pocket 75

Figure 40.Crystal structure of CGRP receptor stabilized with Olcegepant® studied using Molecular Operating Environment® (MOE) software [21] ...... 76

Figure 41.showing the important water molecules and its interactions ...... 78

Figure 42.Showing the hybridization of two leads ...... 79 Figure 43.showing structure based ligand designing ...... 80

Figure 44.Showing importance of stereocenters ...... 81

Figure 45.showing medicinal chemistry aspects applied for ligand design ...... 82

Figure 46.showing the Olcegepant® and Telcagepant® in the docked site ...... 83 Figure 47.showing the SARmap graph of potential hits & different zones with lead as reference ...... 84

Figure 48.showing the filers and descriptors applied to identify potential hits ...... 85

Figure 49.showing the general structure of family 1 ...... 85 Figure 50.showing the general structure of family 2 ...... 86

Figure 51.showing the general structure of family 3 ...... 86

Figure 52.showing the plot using drug-likeliness model score ...... 91 Figure 53.Showing (+)-241D alkaloid, (2S,4R)4-hydroxy pipecolic acid and Palinavir ...... 97

Figure 54.Showing the L-pipecolic acid and its analogues ...... 113

Figure 55.showing steric hindrance due to mono and di-boc (diterbutylester) PG ...... 124

Figure 56.showing protection of dimethyl ester of aspartic acid with trityl PG ...... 125 Figure 57.showing preparation of energy states of intermediates during HWE ...... 127

Figure 58.showing preparation of β -keto phosphonate ester as E isomer ...... 128

Figure 59.showing preparation of β -keto phosphonate ester as E isomer ...... 128

Figure 60.showing preparation of cis/trans piperidine with 75/25 ratio ...... 131

Figure 61.showing splitting pattern with coupling constants for H2e proton of trans piperidine ..... 133

Figure 62.showing amide rotamers for secondary amides ...... 134 Figure 63.showing amide rotamers for tertiary amides...... 135

Figure 64.showing amide rotamers for compound 158 ...... 135

Figure 65.showing rotamer of cis/trans piperidine in DEPT NMR ...... 136

Figure 66.showing the DEPT NMR showing N-acylurea ...... 145 Figure 67.showing the 1H NMR showing piperidinone ...... 152

Figure 68.showing the 1H NMR showing piperidine with ketal at 4th position ...... 153

Figure 69.showing the disappearance of rotamers on cyclizing ...... 160 Figure 70.showing the diastereomeric selectivity for the formation of compound 1 ...... 166

Figure 71.showing two routes to obtain compound V ...... 166

Figure 72.showing three subclasses of family 3 ...... 178 Figure 73.Contributors for Buchwald-Hartwig C-N coupling ...... 188

Figure 74.Choice of different catalytic systems on Buchwald-Hartwig C-N coupling ...... 188

Figure 75. Showing the conformation of sublcass C in CGRP pocket ...... 192

Figure 76.showing the formation of equatorial and axial alcohols ...... 201 Figure 77.Showing the CALCRL fragments used for potent CGRP antagonists ...... 206

Figure 78.Showing the privileged structure we used for CGRP antagonists ...... 207

Figure 79.showing the carbamate like antagonist synthesized by David K.L et al ...... 209 Figure 80.showing the carbamate like antagonist BMS846372 ...... 210

Figure 81.showing the 1H NMR of the carbonate CGRP antagonists ...... 213

Figure 82.showing different nociceptive bioassays ...... 226 Figure 83.Showing Hot plate device ...... 228

Figure 84.showing time of response before and after administration of antagonists in Ist batch ...... 229

Figure 85.showing time of response before and after administration of antagonists in IInd batch .. 231 Figure 86.showing photoactometer ...... 235

Figure 87.showing photoactometer results in Ist batch ...... 235

Figure 88.showing photoactometer results in IInd batch ...... 237

Figure 89.showing FST ...... 239 Figure 90. showing first and total immobilization times in Ist batch of FST ...... 240

Figure 91.showing first and total immobilization times in IInd batch of FST ...... 242

LIST OF TABLES

Table 1 .Showing comparison of different primary afferent nerve fibres ...... 30 Table 2.Symptoms in migraine patients ...... 36

Table 3.Structure & activity of triptans in clinical stage ...... 38

Table 4.showing different monoamine neurotransmitters ...... 57 Table 5.Antidepressants based on their generation ...... 60

Table 6.Antidepressants based on their mechanism ...... 61

Table 7.Antidepressant’s effectivity against chronic pain...... 63 Table 8.CALCRL interaction with various proteins generated using STRING-8 software [13] ...... 72

Table 9.Druggability and Tractability scoring criterion [18] ...... 73

Table 10.Visualizing the druggable sites of CALCRL & RAMP-1 generated by DrugEBility® software [18] ...... 75

Table 11.showing the total scores of family 1 potential hits ...... 85 Table 12.showing the total scores of family 2 potential hits ...... 86

Table 13.showing the total scores of family 3 potential hits ...... 86

Table 14.Showing the volume of potential hits in comparison with the druggable & tractable sites of CGRP receptor [28] ...... 88 Table 15.showing the drug-likeliness of potential hits generated using Molsoft® ...... 90

Table 16.Showing the effect of configuration modulation from 38 to 39 ...... 104

Table 17.Showing the reaction scale and substrates used by Sutherland et al...... 129 Table 18.showing optimization to form compound 7 ...... 130

Table 19.showing splitting pattern with coupling constants for cis piperidine ...... 132

Table 20.Showing the effect of solvent on rotational barrier ...... 135 Table 21.conditions to synthesize compounds 11 and 12 ...... 137

Table 22.Showing the optimization conditions used for coupling the synthon A & B ...... 140

Table 23.showing application deprotection in route B to obtain synthon D ...... 141

Table 24.showing optimization of cross coupling reaction ...... 141 Table 25.showing application of cross coupling reaction ...... 141

Table 26.showing the employed coupling agents (-not worked, *not tried) ...... 147

Table 27.Showing the employed substrates for DEPBT mediated coupling ...... 150 Table 28.showing the 4th position affecting C-N cross coupling ...... 158

Table 29.showing the deprotection of ketal to ketone ...... 162

Table 30.showing different PGs used with their yield ...... 168

Table 31.showing different ketophosphonates with their yield ...... 169

Table 32.showing different biaryl aldehydes with their yield ...... 170 Table 33.showing different alkenes with their yield ...... 173

Table 34.showing application of Michael intramolecular reaction for ethyl and benzyl carbamate PGs ...... 176

Table 35.showing application of Michael intramolecular reaction for tert-Butyl carbamate PG ...... 177

Table 36.Ratios of piperidine formed ...... 180 Table 37.showing different various Buchwald-Hartwig conditions ...... 182

Table 38.Showing the variance of substrate ...... 186

Table 39.showing contributors for Buchwald-Hartwig C-N coupling ...... 188 Table 40.Showing problems and how they can be solved according to Buchwald ...... 190

Table 41.showing different various Buchwald-Hartwig conditions we tried ...... 190

Table 42.Showing the alkylation of subclass A with different conditions ...... 195 Table 43.Cyclization optimization for subclass C ...... 197

Table 44.showing the deprotection substrates and products ...... 198

Table 45.showing the activation of alcohols using mesylate for family 1 ...... 204

Table 46.showing the conversion of mesylate to amine for family 1 ...... 205 Table 47.showing the route A to obtain the carbonate CGRP antagonists...... 212

Table 48.showing the route B we used to obtain the carbamate CGRP antagonists ...... 215

Table 49.showing time of response before and after administration of antagonists in Ist batch ...... 230 Table 50.showing time of response before and after administration of antagonists in IInd batch.... 232

Table 51.showing cut-off counts for administration of antagonists in Ist batch ...... 236

Table 52.showing cut-off counts for administration of antagonists in IInd batch ...... 238 Table 53.showing immobilization times on administration of antagonists in Ist batch ...... 241

Table 54.showing immobilization times on administration of antagonists in IInd batch ...... 243

LIST OF SCHEMES

Scheme 1.showing a generalized retrosynthetic design ...... 92 Scheme 2.Showing the formation of piperidine using chiral ligand L ...... 98

Scheme 3.Showing the formation of (+)-241D alkaloid ...... 98

Scheme 4.Showing the synthesis of (+)-241D alkaloid using gold complex ...... 99 Scheme 5.Showing the synthesis of (+)-241D alkaloid using gold complex ...... 99

Scheme 6.Showing the application of gold complex for another example ...... 100

Scheme 7.Showing the application of reductive amination by Chandrasekhar and coll...... 100

. Scheme 8.Showing the application of reductive amination by Das and coll...... 101

Scheme 9.Showing the application of reductive amination by Rao and coll...... 102

Scheme 10.Showing the application by Toyooka and coll...... 103

Scheme 11.Showing the application by A.B.Smith and coll...... 103 Scheme 12.Showing the inoculation of 3rd asymmetric centre ...... 104

Scheme 13.Showing the synthesis of 2, 4, 5 trisubstituted piperidines ...... 105

Scheme 14.Showing the synthesis of piperidine moiety using Diels Alder reaction ...... 105 Scheme 15.Showing the application of Diels Alder reaction ...... 106

Scheme 16.Showing application of dipolar cycloaddition reaction by Chattopadhyay and coll...... 106

Scheme 17.Showing the application to yield (+)- and (-)-241D alkaloid ...... 107

Scheme 18.Showing the application of Aza-Michael cyclization by Hong and coll...... 108 Scheme 19.Showing the synthesis of (-)-epimyrtine 56 and (+)-myrtine 57...... 108

Scheme 20.Showing the synthesis of (+)-myrtine by Del Pozo and coll...... 108

Scheme 21.Showing the synthesis of (−)-andrachcinidine by Krishna and coll...... 109 Scheme 22.Showing the application of Aza-Michael by Akiyama and coll...... 109

Scheme 23.Showing the application of Aza-Michael by Troin and coll...... 110

Scheme 24.Showing the application of Aza-Michael by Troin and coll...... 110 Scheme 25.Showing the application of electrochemistry by Hurvois et al ...... 111

Scheme 26.Showing the application of (+)-myrtine and (+)-241D by Hurvois and coll...... 112

Scheme 27.Showing the substituted piperidine synthesis Dieter and coll...... 112

Scheme 28.Showing the 2, 6 trans substituted piperidine ...... 113 Scheme 29.Showing the 2, 6 cis substituted piperidine...... 113

Scheme 30.Showing the synthesis of compound 88 proposed by Aitken and coll...... 114

Scheme 31.Showing the synthesis of compound ent-89 proposed by Hou and coll...... 115

Scheme 32.Showing the synthesis of compound 108 proposed by Haufe and coll...... 115

Scheme 33.Showing the synthesis of compound 89 proposed by Occhiato and coll...... 116 Scheme 34.Showing the synthesis of compound 116 proposed by Occhiato et al...... 116

Scheme 35.Showing the synthesis of compounds 117 and 118 proposed by Occhiato et al...... 117

Scheme 36.Showing the synthesis of compound 120 proposed by Occhiato and coll...... 117

Scheme 37.Showing the synthesis of compound 89 proposed by Riera and coll...... 118 Scheme 38.Showing the synthesis of compound 134 proposed by Merino and coll...... 118

Scheme 39.Showing the synthesis of compound 138 proposed by Sutherland and coll...... 119

Scheme 40.Showing the synthesis of compound 140 proposed by Sutherland et al...... 119 Scheme 41.showing retro synthetic scheme for family 1 ...... 121

Scheme 42.showing retro synthetic scheme for synthon I ...... 122

Scheme 43.showing retro synthetic scheme for synthon II ...... 122 Scheme 44.showing esterification of aspartic acid ...... 123

Scheme 45.showing protection of dimethyl ester of aspartic acid ...... 124

Scheme 46.showing preparation of β -keto phosphonate ester ...... 125

Scheme 47.showing preparation of β -keto phosphonate ester ...... 126 Scheme 48.showing preparation of β -keto phosphonate ester ...... 127

Scheme 49.showing preparation of 6-methyl-4-oxopiperidine-2-carboxylate salt ...... 129

Scheme 50.showing preparation of methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate...... 130 Scheme 51.showing protection of methyl compound 157 ...... 134

Scheme 52.showing reductive amination to synthesize compounds 160 and 161 ...... 137

Scheme 53.Showing the optimization using L-proline ...... 138 Scheme 54.showing application of optimized condition on our substrate...... 138

Scheme 55.showing newly proposed design to obtain synthon C ...... 139

Scheme 56.showing application of copper in route A to obtain synthon D ...... 139 Scheme 57.showing application of palladium in route A to obtain synthon D ...... 140

Scheme 58.showing application of coupling agents in route B to obtain synthon E ...... 140

Scheme 59.showing application deprotection in route B to obtain synthon E ...... 141

Scheme 60.showing application of route B to obtain synthon C ...... 142 Scheme 61.showing previously optimized condition to chiral substrate ...... 142

Scheme 62.showing the peptide coupling using DCC/DMAP ...... 143

Scheme 63.showing the peptide coupling mechanism using DCC ...... 144

Scheme 64.showing the intramolecular rearrangement ...... 144

Scheme 65.showing the DCC peptide coupling giving N-acylurea ...... 145 Scheme 66.showing the general peptide coupling reaction ...... 146

Scheme 67.showing the T3P® peptide coupling giving desired compound ...... 147

Scheme 68.showing the mechanism of T3P® peptide coupling ...... 148

Scheme 69.showing the DEPBT peptide coupling giving desired compound ...... 149 Scheme 70.showing the mechanism of DEPBT peptide coupling ...... 149

Scheme 71.showing the deprotection of tert-Butyl carbamate from the substrate ...... 151

Scheme 72.showing the protection of ketone to cyclic ketal ...... 154

Scheme 73.showing the protection of ketone to methoxy ketal & in situ deprotection of tert-Butyl carbamate ...... 154

Scheme 74.showing the protection of pTsOH quantity affecting the deprotection ...... 155

Scheme 75.showing the deprotection of benzyl carbamate by TMSI ...... 155 Scheme 76.showing the protection of ketone to methoxy ketal ...... 156

Scheme 77.showing the deprotection of benzyl carbamate with TMSI ...... 156

Scheme 78.showing the C-N cross coupling using Pd(OAc)2/Xantphos ...... 157

Scheme 79.showing the C-N cross coupling using Pd2(dba)3/BINAP ...... 157

Scheme 80.showing the C-N cross coupling using Pd2(dba)3/BINAP ...... 157 Scheme 81.showing the 2, 6 cis affecting C-N cross coupling ...... 158

Scheme 82.showing the 2, 6 cis affecting C-N cross coupling ...... 158 Scheme 83.showing the deprotection of ketal to ketone ...... 161

Scheme 84.showing the retrosynthetic strategy for family 3 ...... 162

Scheme 85.showing retro synthetic scheme for family 3 (Y=NH, O, propan-1-ol) ...... 163 Scheme 86.showing retro synthetic scheme for synthon III ...... 164

Scheme 87.showing Aza-Michael addition of Davies amine to α,β-unsaturated ester ...... 165

Scheme 88.showing hydrogenation of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate .... 167 Scheme 89.showing protection of (S)-4-methoxy-4-oxobutan-2-aminium acetate ...... 168

Scheme 90.showing synthesis of ketophosphonates...... 169

Scheme 91.showing synthesis of biaryl aldehydes ...... 170

Scheme 92.showing routes to obtain compound 198 ...... 171 Scheme 93.showing Horner-Wadsworth-Emmons reaction ...... 172

Scheme 94.showing general approach for Aza-Michael intramolecular cyclization ...... 173

Scheme 95.showing effect of alcohol used for protection of ketone ...... 174

Scheme 96. Showing mesomeric effect in cyclization ...... 174 Scheme 97.showing Michael intramolecular reaction in general context ...... 175

Scheme 98.showing multiple approaches to reach subclass A ...... 179

Scheme 99.Showing formation of amine ...... 180

Scheme 100.showing route C to reach subclass A ...... 181 Scheme 101. Design for the synthesis of subclass A ...... 181

Scheme 102.showing investigation on first C-N cross coupling ...... 183

Scheme 103.showing prime site for C-N cross coupling ...... 183 Scheme 104.Mechanism for Buchwald-Hartwig C-N coupling...... 183

Scheme 105.showing trans C-N cross coupling ...... 184

Scheme 106.showing design for synthesis of subclass B ...... 185 Scheme 107.Showing the catalytic cycle for subclass B ...... 186

Scheme 108.Hydrogenation of compound 248 ...... 187

Scheme 109.Optimization of catalytic systems for subclass B ...... 190

Scheme 110.protection of HY ...... 191 Scheme 111. Another way to achieve Subclass B ...... 191

Scheme 112.Showing the designed synthetic scheme for family 3 subclass c ...... 193

Scheme 113.Showing the synthesis of O-protected iodoethanol ...... 194 Scheme 114.showing the O-protected amine through Gabriel synthesis ...... 194

Scheme 115.showing the alkylation designed according to route C ...... 195

Scheme 116.Showing route A ...... 196 Scheme 117. Showing route B ...... 196

Scheme 118.showing cyclization of subclass C ...... 196

Scheme 119.showing the general deprotection method ...... 197 Scheme 120.showing general synthetic scheme to obtain amine from ketone ...... 198

Scheme 121.Showing the reduction of piperidone using sodium borohydride and L-selectride ...... 199

Scheme 122.Showing the configuration achieved after reduciton ...... 199

Scheme 123.showing the diasteroisomeric ration of alcohols formed ...... 201 Scheme 124.showing the application of Mitsunobu condition for racemic structure ...... 202

Scheme 125.showing the application of Mitsunobu condition for subclass A of family 3 ...... 202

Scheme 126.showing the activation of alcohols of family 1 ...... 203

Scheme 127.showing the activation of alcohols using mesylate for family 1 ...... 203

Scheme 128.. showing the activation of alcohols using mesylate for family 1 ...... 205 Scheme 129.Showing the privileged structure’s design for synthesis ...... 207

Scheme 130.Showing the synthesis of compound 1a ...... 208

Scheme 131.Showing the synthesis of compound 1 ...... 208

Scheme 132.Coupling of the fragments ...... 209 Scheme 133.showing the synthesis of BMS846372 ...... 210

Scheme 134.showing the route A and B to obtain the CGRP antagonists ...... 210

Scheme 135.showing the route A to obtain the carbonate CGRP antagonists...... 211 Scheme 136.showing the application of BMS846372 synthetic methodology ...... 211

Scheme 137.showing the route B to obtain the carbamate CGRP antagonists ...... 214

Scheme 138.showing deprotection of Boc in entry 1 ...... 215 Scheme 139.showing deprotection and debromination in entries 2, 3, 4 ...... 216

CHAPTER 1: INTRODUCTION

I. PAIN

Pain, in broad-spectrum is not classified as a disease but a symptom associated with a disease or a disorder. According to International Association for Study of Pain (IASP), pain can be defined as

“An unpleasant sensory and emotional experience associated with actual and potential tissue damage, or described in terms of such damage, or both [1].”

Even though pain is not the main reason for mortality, in most of the cases it is undeniably associated with a form of disease or a disorder (Cancer, HIV, Ischemic heart disease etc.) responsible for the major causes of death in the world [2]. Every year, around 7.2 million people suffering from pain originated due to HIV or cancer are prone to death worldwide (Figure.1). In 2012, not less than 2.4 million deaths are due to untreated pain [3].

Figure 1.Deaths due to untreated pain associated with disease/disorder And so, treating pain might not directly cure a disease but will surely diminish the effect of the associated disease or disorder and thus results in relatively better quality of life for the patient.

Some other forms of pain (migraine, Irritable Bowel Syndrome, rheumatoid arthritis) won’t result in the death of a patient instead will decrease the quality of life [4]. Especially chronic pain, which is majorly responsible for many of such cases and treating it might be cumbersome as some patients as well as physicians can’t different between diverse forms of pain and treat one instead of the other with an improper adjuvants and dosage regimen[5].

Thus, a proper understanding of different forms of pain is important which will help for better treatment of a disease or a disorder.

A. Classification:

Classifying pain into different forms is complicated as there are different rationales for sorting them. They are classified according to their time period (Figure.2), frequency, location, intensity and cause of pain attack [6, 7].

Figure 2.Classification of pain based on time period of occurrence 1. Classification based on time period: Based on time course of existence, pain is classified into [8]

1.1. Acute pain: Pain which lasts for a period of 3-6 months duration is called as acute pain [9]. Acute pain is physiological pain and serves as protective function. Alongside this, acute pain has clearer and visible association with injury or disease. For example, post- surgical pain is a form of acute pain and the reason behind it is clear i.e. because of surgical wound. Nociceptive pain can be a better word and/synonymic for acute pain as nociceptive pain is caused by noxious stimulus due to thermal (Heat or cold), mechanical (cuts, crushing etc.) and chemical (chilli powder on cuts or eyes etc.) in whose case, the reason for pain is clear and expected to fade with the time period as the noxious stimuli diminishes. Henceforth, it serves as a protective function by alarming the brain that something

deleterious process is happening to the body and withdrawal is obligatory to protect the body from further damage [10].

Not just treating the cause of pain and/ injury, it is also equally important to treat the pain itself. By doing this, we can lessen the effect of disease or injury and also not to transform acute pain to chronic [11]. Depending on the intensity of pain, medication used for acute pain includes Acetaminophen, Codeine, Ibuprofen, Hydromorphone etc. (Figure.3).

O HO HO O HO O H O H N O N N H H H HO O Acetaminophen Ibuprofen Codeine Hydromorphone

Figure 3.Drugs used for acute pain 1.2 Chronic pain: It can be designated as more ‘complicated’ form of pain and lasts for a period of more than 6 months [10]. Even though it is also associated with some injury like acute pain, with the time period, the reason becomes less clear and persists for longer time than the injury. Along with the common NSAIDS used against pain, chronic pain treatment includes antidepressants, anticonvulsants, corticosteroids etc. (Figure 4).

O H HO OH N O O O H OH NH2 N S H H O Amitriptyline Duloxetine Prednisone Gabapentin Antidepressant Antidepressant corticosteroid Anticonvulsants

Figure 4.Drugs used for chronic pain Chronic pain can be further classified based on the cause and/ origin of pain [12] i. Nociceptive pain: It is caused by the noxious stimuli as a result of activation of nociceptive receptors called as nociceptors. Based on its profundity, it is sorted out as

a) Superficial pain is a response due to the activation of nociceptors distributed in skin or superficial tissues as in the case of first degree burns and minor cuts and bruises.

b) Deep pain occurs in tissues deep inside and is further classified into

1) Deep somatic pain occurs in the tendons, ligaments, bones, blood vessels etc. and is fading, vaguely localized pain as in the case of pain during broken bones, sprains.

2) Deep visceral pain occurs in the visceral organs and is generally localized but hard to locate as ‘referred pain (pain response in region other than injured location)’ might also be present in other visceral space (apart from the actually injured organ) due to the inflammatory response by the damaged tissue as in the case of Irritable Bowel Syndrome. ii. Neuropathic pain: It is caused by the damage (due to lesions, injury etc.) of the nervous system, to be exact, somatosensory nervous system. It is co-associated with allodynia (pain from non-painful stimulus) or dysesthesia (abnormal sensations) and occurs as episodic or continuous.

a) Central neuropathic pain is found in multiple sclerosis and spinal cord injuries. Amongst a cluster of theories resulting in central pain, central sensitization is more acceptable one.

Figure 5.showing ascending pain pathway The dorsal horn neurons which forms the spinothalamic tract contributes the major ascending nociceptive pathway (Figure 5). Due to the ongoing spontaneous peripheral rise in activity, spinothalamic tract also develops increased background activity and

responses to afferent impulses including normal touch stimuli. All these cascade reactions synergise to form a state called central sensitization.

b) Peripheral neuropathic pain is due to a lesion in the nerve ending which modulates the nerve activity at the injured space. These modulations include, abnormal excitability, increased sensitivity to physical, chemical stimuli. These cascades contribute to form the state of peripheral sensitization.

Along with the classification of pain using time frame as the rationale, there are other basis which includes frequency, location, cause of pain etc. But all give the same explanations which have been covered in the above classification.

Intensity is another classification criteria set by some clinicians and it shows some interesting facts as below [13].

2. Intensity: Based on intensity, pain is rated on a scale of 1 to 10, 1 being normal and 10 being in severe pain (Figure.6).

Figure 6.showing pain scaling and experience of a patient There is ambiguity in this classification as most of the patients over time gets adapted to the pain experiences and cannot express the same pain anguish which they showed in the beginning. It lacks the consistency as the rating changes from one individual to the other. This kind of classification is least desirable by most of the scientific community but preferred by physicians in some countries.

Moving further, whatever the pain is, there is a means of transportation of signalling from the place of injury to the brain and vice versa for the modulation. Thus, a look into the pain origin, transformation and modulation will further help us to understand better physiology of pain and ultimately for better designing of drugs for pain.

II. PAIN ORIGIN, TRANSMISSION AND MODULATION

Pain is a role of the nervous system in alarming the body and cautioning of potential or actual injury. And hence, this complex pain signalling system has many elements involved and nociceptors are considered to be one of the important elements of all in pain pathway.

Before discussing about the nociceptors, a brief preface about the nervous system and their characteristics helps for a better understanding of the pain signalling pathways.

A. Nervous system

The nervous system can be divided into two segments i.e. central and peripheral nervous system. Central nervous system (CNS) consists of brain and spinal cord and this is the region of the nervous system where the processing of received information and subsequent influence on the resulting activity takes place. On the other hand, peripheral nervous system (PNS) consists of spinal and cranial nerves and acts as the bridge between CNS and rest of the body (Figure 7) [14].

Figure 7.showing signalling pathways of CNS & PNS These signalling pathways are mainly attributed to three kinds of neurons [15];

 Afferent neuron sends the signal from the place of stimuli to CNS. These neurons are distributed in PNS and consist of long axons and sensory receptor (for example, nociceptors) on one of their ends.

 Efferent neuron takes back the signal from CNS to effector organs. In PNS, it has long axon.

 Interneurons are confined completely to CNS and acts as a link between afferent and efferent neurons.

The sensory afferent nerve fibre helps in reception of various stimuli such as touch, pain, pressure, temperature changes etc. These stimuli conduction start from the receptors of the sensory afferent neurons and ends in the effector organ (Figure 8).

Figure 8.showing signalling pathways from receptor to the effector organ This conduction of stimulus results in transmitting the concerned signal to the CNS for processing and for a subsequently resulted action which again passes from CNS to PNS and finally to motor efferent division through which the signal passes to the effector organ such as muscles/glands [16].

Thus, the review of nervous system as above can be useful in further study aspects involved in pain signalling pathways.

B. Pain pathway

Pain pathway and processing occurs in 4 different steps as follows;

1. Transduction 2. Transmission 3. Perception 4. Modulation

Each step has its own importance and variety of drugs acts on different stages of this pain signalling pathway. These stages can be summarized as follows;

1. Transduction:

Transduction can be defined in general as a process of conversion of one form of energy into another form by a transducer (for example, conversion of electrical energy into light energy by an electric bulb). In case of pain, the noxious stimuli like physical and chemical are converted into a pain signal by a transducer called as nociceptor. Nociceptors are distributed all along the body and exposed to different noxious stimuli.

Nociceptors: They are sensory receptors which have an ability to identify noxious stimuli and convert the stimuli into pain signal (to be exact, electrical signal) [16]. The nociceptors are formed by free nerve endings of A-δ and C fibres of the nervous system. They can be activated by thermal, mechanical and chemical stimuli [17]. Even some inflammatory mediators like bradykinins, prostaglandins, cytokines, Calcitonin Gene Related Peptide, Serotonin etc. can activate nociceptors. These mediators are also responsible for primary sensitization, where the activation threshold of the nociceptors becomes less.

Nociceptors are distributed in

 Somatic structures like muscles, skin, bones, joints, connective tissue etc.  Visceral structures like liver, intestine etc.

In conjunction with A-δ and C fibres, other primary afferent fibres like A-β exists but they carry non-noxious stimuli. Along with the formation of nociceptors, there are certain characteristics of them which make these fibres active in specific cases (Table.1).

Character A-β fibres A-δ fibres C fibres

Function Motor Sensory Autonomic

Diameter Large Small 2-5µm Smallest <2µm

Myelinated Highly Thinly Unmyelinated

Conduction velocity > 40 ms-1 5-15 ms-1 < 2 ms-1

Receptor activation Low High and low High thresholds

Sensation on Light touch, non- Rapid, sharp, Slow, diffuse, dull stimulation noxious localized pain pain

Table 1 .Showing comparison of different primary afferent nerve fibres

A-δ and C fibres contribute majorly for carrying the pain nociception and they vary from each other in case of many aspects. C fibres conduct the pain signal slower than any A-δ fibre and this can be the reason when someone is injures, at first stance they feel a sharp and localized pain (due to A-δ fibre) and when the time passes, the pain becomes more dull and diffused (due to C fibres) [18]. But C fibres contribute 70% of nociceptors and hence it is the major fibre making up the volume for nociception.

2. Transmission

The transduced signal from previous step reaches the next step i.e. transmission. Transmission is the process by which impulses are sent to the dorsal horn of the spinal cord and then along the sensory tracts to the brain (Figure 9) [19].

Figure 9.showing signalling pathways from receptor to spinal cord by transmission Transmission of the impulse to the CNS is also done by two major primary afferent neurons i.e. A-δ fibres and C fibres through three different types of transmission and the

30 transmitted nerve impulse also called as ‘action potential’ is resulted from the activation of specific ionic channels like sodium channel.

Transmission occurs in three different stages as follows [20];

1.1 Peripheral transmission: The noxious stimuli travel alongside within peripheral nerve fibres and terminate at dorsal horn of the spinal cord.

1.2 Synaptic transmission: The signal reaching dorsal horn through peripheral nerve fibres synapse with the second order neurons.

1.3 Central transmission: The signal after synaptic transmission further conducted through neurons which bridges spinal cord and rise to the thalamus and in turn branches to the brainstem.

Finally, the transmitted nociceptive signal reaches different areas of brain. This terminates the transmission step and advances to the next step i.e. perception.

3. Perception

The noxious stimuli which reach the brain is processed and in turn recognized as a pain. The precise region of brain involved in perception is unclear but there are certain areas of brain which can be labelled as prime regions where defining of pain process takes place [21].

Figure 10.showing areas of brain implicated in thermal pain signalling In Figure 10, the involvement of numerous regions of brain in thermal pain clearly states a complex pain perception processes taking place in brain. The somatosensory cortex

(both primary and secondary) is important for the localization and identification of form of pain as well as degree or intensity of it. And limbic system involves in emotional response for a pain signal. Other regions include insula, frontal lobes etc. After the processing of pain signal, the next step involves the reaction of brain to the pain stimulus received in the form of modulation.

4. Modulation

The perceived noxious stimulus by brain is processed and an inhibitory pathway starts resulting in inhibition of nociceptive signal. During the modulation phase, the descending neurons from the brain release certain substances that inhibit the transmission of noxious stimuli [22]. There are many theories involved in pain modulation and among those, more accepted theories are as follows;

4.1 Gate control theory of pain: This inhibitory pathway theory for pain was proposed by Melzack and wall [23]. It states that by activation of A-α fibres by non-noxious and touch stimulus, results in activation of interneurons thus resulting in inhibition of noxious stimuli conducting through C fibres.

Figure 11.showing gate control theory of pain 4.2 Descending modulation: The received and processed noxious stimulus in brain is further answered as a reaction by the brain in the form of modulation. Periaqueductal grey in midbrain and rostral ventromedial medulla are two important regions of brain involved in descending modulation [24]. The above named regions have high concentrations of opioid receptors and endogenous opioids answering to the question, why opioids are potent pain killers. The pathway reaches dorsal horn of spinal cord and is monoaminergic, which uses serotonin and norepinephrine as prime neurotransmitters. Even though believed that the

mechanism involves activation of descending pathway and release of opioids endogenously results in inhibition, the clear and complex mechanism is not well understood till date.

As we saw that the pain pathway is complex and interconnected, there are certain analgesics which act at different levels of this signalling pathway to inhibit the nociception.

Figure 12.Drugs acting at different levels as analgesics Hence the drugs used against pain in day-to-day life can be summarized as above (Figure 12). Point to be noted, not just analgesics but other categories of drugs are also can be used to suppress the nociceptive stimuli. In the future chapters, some of the antidepressants acting as pain can be discussed in brief to validate this rationale.

A brief overview of pain origin and signalling along with drugs acting at different levels paves a path for the next important and hot topic, the pain and its sufferers worldwide.

C. Global data on pain

Pain is one of the most prevalent symptoms associated with many disease/disorders. Among pain, acute pain does have sufficient medication as well as the analgesics currently available are efficacious and potent. On the contrary, the chronic pain and its current medication is one amongst most other disorders lacking potent treatments. As we know that

33 the chronic patient has deprived quality of life, the global data poses some interesting facts (Figure 13).

Figure 13.screenshot showing global ranking with respect to Years Lived with Disability (YLD) The data retrieved from Global Burden Data (GBD) [25] clearly depicts that chronic pain conditions like low back pain ranks 1 in most of the patients. It averages about 80,666,900 (55,065,800 to 108,723,000) globally. Other chronic visceral pain condition such as neck pain ranks 4 with a mean of about 32,650,800 (22,782,900 to 44,857,400) and somatic pain condition, migraine ranks 8 with a mean of 22,362,500 (14,828,600 to 31,244,900). Major depressive disorder (ranks 2, mean-63,239,300) is a condition which coexists with most of the chronic pain condition but even though vice versa is not factual. Having a closer look on the deaths global data (Figure 14), shows the results as follows.

Figure 14.screenshot showing global ranking with respect to deaths The ischemic heart disease caused by visceral inflammation as one of the reasons ranks 1st with a mean of about 7,029,270 (6,577,160 to 7,431,130). ‘Non-infective inflammatory bowel syndrome’ is a chronic visceral inflammatory condition and a major group of diseases accounting for deaths and Disability-adjusted life years (DALYs) in recent years (Figure 15). This group of diseases includes Crohn’s disease, Ulcerative colitis etc.

Figure 15.screenshot showing global deaths due to Non-infectious inflammatory bowel syndrome Around 34,000 deaths and 2,875,360 DALYs have been reported in 2010 and making the situation worse, the rate is increasing rapidly since 1990. And hence, the major problem when it comes to chronic pain includes visceral pain and migraine which turns our spotlight on to it.

III. MIGRAINE & VISCERAL INFLAMMATORY PAIN

A. Migraine

Migraine is a complex vasodilatative and incapacitating disorder occurring in almost 15% of the adults throughout the world among whom the female population are affected majorly. It is a periodic affliction characterised by one-sided, pulsating headache associated with photo & phonophobias [26]. Typical migraine attack ranges from 1.5 times a month and reaches its toll around an age of 40 years during patient’s lifetime [27].

1. Key facts & figures

 ≈15% of world population suffers from migraine among which 70% are women.  Migraine is a syndrome with a group of symptoms among which, all of them or some of them are shown during one migraine attack. A study among 4000 migraine patients have shown symptoms as follows [28];

Symptom Percentage Pulsating ache 85 Photophobia 80 Phonophobia 76 Gastric related disturbances 73 One-sided pain 59 Visual disturbances 44 Aura 36

Table 2.Symptoms in migraine patients  50% of the migraine in patients remains un-investigated and not properly treated of which half of the subjects remains unconsulted with the doctor [29].  Migraine is ranked amongst top 20 disabilities throughout the world [30].  In United Kingdom, 0.19 million migraine flare-ups each day [31].  Depression is 3 times more often in migraine patients compared to the healthy volunteers.  Most notably, migraine research is the least funded neurobiological disorder when equated with its financial impact on public [32].

2. Pathophysiology of migraine

The initiation of migraine even after years of research is still unclear. But many hypothetical theories are proposed by different scientists making it a difficult target to work on. One of the main theories which have an adequate theoretical basis is Cortical Spreading Depression (CSD) [33]. CSD onsets as a wave of electrical signal in cerebral cortex. The key event in unveiling and dissemination of CSD can be related to drastic decrease of neuronal membrane resistance in association with a series of events like extracellular increase of neurotransmitters & potassium (K+) ions and intracellular decrease of sodium (Na+) and calcium (Ca++) ions [34]. The modulation of CSD threshold is influenced by several factors like environment, genetic makeup, age, hormones etc. However the recent preclinical & clinical investigations showed a clear coherence of migraine with Calcitonin Gene-Related Peptide (CGRP), a potent vasodilator. Hence most research groups working on migraine shifted their focus onto CGRP in the recent years.

3. Treatment of migraine

Drugs used for migraine therapy are majorly triptan class even though CGRP receptor antagonists are potent. This accounts for no CGRP blockers in market till date but still in the drug developmental stages. Apart from triptans, other NSAIDS such as acetaminophen, ibuprofen, and aspirin are used for mild migraines. Some more drugs such as opioids, antidepressants, corticosteroids, ergotamines proved to be effective but their usage is limited due to the side-effects shown by them. And hence, the drugs which are both effective and showing least side effects are as follows;

3.1 5-HT1B/1D receptor antagonists for migraine therapy; the triptans

The triptans class of the drugs are the efficacious, potent and mostly used drugs for migraine therapy even at present [35]. The name ‘Triptans’ indicates its core chemical moiety i.e. tryptamine. All the class of triptans have 5-Hydroxy tryptamine as their core moiety. Some triptans are as follows (table 3);

Name Core R R1 Dose I S C T moiety (mg)

5 = = = = N Rizatriptan N N

10 + + ++ =

N Almotriptan S 12.5 = + + ++ O O

2.5 = = = = Zolmitriptan H H N O R O 1 5 = = = = R

N H Sumatriptan 25 - =/- - +

H N S O 50 = = =/- = O

Naratriptan N 2.5 - - - ++

20 - - - =

Eletriptan H O S 40 =/+ =/+ = = N O

80 ++ + = -

(I = Initial 2 h relief, S = Sustained pain-free, C = Consistency, T = Tolerability) (= indicates equal, - indicates less & + indicates more activity when compared with Sumatriptan 100 mg, N° of subjects - 24089) [27] Table 3.Structure & activity of triptans in clinical stage i. Complications of triptans

Alongside its efficacy, triptans have side effects like myocardial infarction, hypertension, tachycardia, angina pectoris, renal & hepatic failures, serious allergic reactions, dry mouth, gastro & heart related problems etc [36]. Increased doses of sumatriptan results in sulfhaemoglobinemia, a reversible state where blood turns into greenish-black due to the co- ordination of sulphur onto the haemoglobin moiety [37]. A life menacing medical condition called ‘serotonin syndrome’ is resulted due to drug-drug interaction of triptans with either mono amino oxidase class of anti-depressant drugs or serotonergic drugs [38].

3.2 Calcitonin Gene Related Peptide

Calcitonin Gene-Related peptide, a 37 amino acid neuropeptide which is involved in several physiological processes came into the lime-light after extensive studies on migraine has shown some interesting facts [39]. Among the migraine patients, the brain plasma showed abnormal increase in CGRP concentration during the attack which infers the involvement of CGRP in migraine [40]. This observation has shifted whole migraine research onto CGRP and its receptor antagonists.

There has been some extensive studies on the distribution of CGRP receptors in recent times stating that they are widely distributed in central and peripheral nervous system [41]. In central nervous system (CNS), it acts as a neurotransmitter. In peripheral nervous system (PNS) its action has been exploited for its importance in the dilation of smooth muscles which results not only in migraine but also in some neglected visceral disorders like Irritable Bowel Syndrome (IBS) and Crohn’s disease (Figure 16).

Figure 16.Distribution & implications of CGRP receptor Therefore, current migraine therapy rationale is based on its mechanism of dilation of cranial blood vessels.

i. CGRP receptor antagonism

CGRP receptor belongs to the class B of G-protein coupled receptors. It is a heterodimeric protein with an association between Calcitonin-receptor Like Receptor (CLR) and Receptor Activity Modifying Protein-1 (RAMP-1) [42]. The orthosteric activation of the receptor by the natural ligand CGRP results in cascade of reactions like activation of Adenylate cyclase enzyme by the G-protein, conversion of Adenosine Triphosphate (ATP) to cyclic Adenosine Monophosphate (cAMP) by the activated adenylate cyclase, activation of Protein Kinase A (PKA) enzyme by cAMP, phosphorylation of ionic channels by the PKA resulting in opening of ionic channels and consequently an intracellular increase of K+ and Ca++ (Figure 17). Thus these alterations results in hyperpolarisation or vasodilatation [43, 44]. In case of migraine, due to the vasodilatation or distension of the cranial blood vessels and resistance & limitation from the skull bone results in the pulsating and severe pain in head.

Figure 17.Molecular level cascade events of CGRP receptor activation In order to counteract this vasodilatation, the binding site of natural ligand is masked using antagonists [45] and the cascade of reactions can be turned off resulting in no vasodilatation step.

ii. Discovery of peptide antagonists for CGRP receptor

CGRP receptor was first antagonised by CGRP8-37, a peptide chain of 30 amino acid which was discovered from the intertwining of the natural ligand i.e. CGRP [46] (Figure 18).

Br OH

Br O O H N N H O

NH2

(R)-Tyr(S)-Lys Dipeptide lead IC50 = 17000 nM

Figure 18.Peptide antagonists for CGRP receptor Further research at Boehringer Ingelheim has developed some small peptides derived from the C terminus of the natural ligand [47]. Later on by High Throughput Screening, a (R) Tyr (S) Lys peptide was identified as a lead. A series of modulations like rigidification of C & N terminus, bio-isosteric replacement keeping the peptide pharmacophore untouched resulted in the first potent CGRP receptor antagonist called as BIBN-4096 (Olcegepant®®) (Figure 19).

Br Br Br OH OH OH Br Br O O O Br O O H O O H H N N N N N N N N N H N N H O N H O N O N N N N N N O N O O H NH2 NH2 NH2 IC50 = 0.03 nM IC50 = 1000 nM IC50 = 44 nM BIBN4096

Figure 19.Sequence of modulations resulting in BIBN-4096 Olcegepant® discriminates the rodent and primate CGRP receptors [47]. It has more affinity towards primate receptor with Ki = 0.014 nM compared with that of rodent receptor th showing Ki = 3.4 nM. This is due to the replacement of Tryptophan with Lysine at 74 position of the receptor in rodent species implying the importance of tryptophan interaction to have potency for small molecules. Even though BIBN4096 is potent and selective antagonist, the peptidic nature of the molecule has limited its usage due to poor lipophilicity.

iii. Non peptide CGRP antagonists

MK-0974 called as Telcagepant®® (Figure 20) is orally active, potent antagonist developed by Merck & Co. Being a non-peptide, it is more lipophilic with equal potency as BIBN4096 hence advantageous over latter [48]. Like BIBN4096, it is 1500 folds more selective towards primate CGRP receptor.

F3C

F N O O H2N N O F O O N N N N NH O N NH H F N F N

MK0974 Ki = 0.8 nM BMS927711 Ki = 0.027 nM

Figure 20.Non peptide antagonists MK-0974 was terminated from its phase III clinical trials as there were cases showing increased serum transaminases, which inclines to hepatotoxicity [49]. BMS-927711 by Bristol

Myers Squibb is another orally active, potent and selective with Ki = 0.027 nM has recently finished 2nd of the 3 tests for the approval into the market [50]. Adding to this list, some more non peptide antagonists are currently in their preclinical evaluation [51].

With the inspiration from development of potent CGRP antagonist in the form of Telcagepant®, Merck continued their research on this receptor and fabricated MK-3207.

O O HN O NH N N N H

F F

MK-3207 Ki = 0.021 nM

Figure 21.Non peptide antagonists by Merck MK-3207 showed 50-100 folds increased potency than MK-0974 (Telcagepant®) in preclinical studies [51]. These positive results have been validated even on clinical studies showing an efficacy better than many other triptans as well as CGRP antagonist counterparts for a dose of 200 mg. Also to consider, the clinical studies have been conducted on a small sample size of the dose group and hence gave a significant overlap between confidence intervals between each dose. And so, a possible conclusion on its efficacy can’t be drawn as of now and has to be validated further.

The other candidate from Boehringer Ingelheim, BI-44370 was also evaluated clinically [51].

H O N

O N O O N N N O

O O OH

BI-44370 Ki = 0.3 nM

Figure 22.Non peptide antagonists by Boehringer Ingelheim In phase II study, it showed efficacy on acute migraine and compared with placebo and eletriptan. 400 mg showed statistical significance over placebo with a response rate of 27.4% whereas 200 mg showed no statistical significance. The current development of BI- 44370 is in uncertain as Boehringer Ingelheim announced no progress further from phase II.

Not just BIBN-4096, MK-0974, BMS-927711, MK-3207 and BI-44370, but other CGRP antagonists have also been under developmental stages of drug discovery process. But above stated 5 have entered clinical stages and proved effective in the preclinical as well as preliminary clinical trials. But with several reasons ranging from solubility and bioavailability

(as in the case of BIBN-4096) to hepatotoxicity (MK-0974), there was always a problem posing in CGRP antagonist development.

Thus, the antagonists further developed should keep in mind all the positives and negatives of the previous CGRP antagonists and develop a better moiety with more desired and least side effects.

iv. Antibodies & biologics in contemporary migraine market

LB101 (previously RN307), a monoclonal antibody which is used for periodic and long term migraine is now in phase IIb clinical trial. Teva pharmaceuticals acquired it for $825 million from labrys biologics [52]. Zecuity® by NuPathe Inc, an advanced transdermal patch loaded with sumatriptan delivers a dose of 6.5mg over a period of four hours and demonstrated to be effective for migraine. Allergan’s Botox® usage is continued as a preventive treatment for chronic migraine. Another monoclonal antibody LY2951742 developed by Arteaus biotech is also used as a preventive medication. It has recently finished the phase II trials sponsored by Eli lilly and showed significant efficacy and tolerability [53]. ALD403 by Alder biopharmaceuticals finished phase II clinical trials and showed radical decrease of Migraine headache days by 75% [54].

Along with medication for treating migraine (chronic), there are certain class of drugs which now-a-days are prescribed as preventive [51]. But they are not as potent as above classes.

 Cardiovascular drugs like beta blockers (Propranolol, Metaprolo, Timolol) and calcium channel blockers (Verapamil) which are used for high blood pressure are prescribed as preventive medication for migraine.

 Tricyclic antidepressants like Amitriptyline, Venlafaxine.

 Antiepileptics like Valproate, Topiramate.

 OnabotulinumtoxinA i.e. botox also proved to be an efficient preventive medication.

Thus, migraine medication which currently used has more side-effects relative to their beneficial & desired therapeutic effect. Thus validating a new target for treating migraine seems to be inevitable.

After having a gaze at migraine, let us have a brief outlook about visceral pain which is also our target to act on.

B. Visceral pain

Visceral pain is a form of pain in viscera i.e. internal hollow organs and marks a prime grave problem when it comes to viscera [55]. It is one of the chronic pain conditions and is caused mainly due to the activation of nociceptors of abdominal, pelvic or thoracic viscera [68]. Point to be remembered that unlike in other cases of pain perception by nociceptors, the visceral pain nociceptor activation is mainly caused by stretching, inflammation, ischemic conditions etc. and relatively insensible to cuts and burns [56].

Neuropathic and other chronic pains might or might not happen during lifetime of an individual but visceral pain has become inevitable now-a-days like abdominal pain due to IBS, angina pectoris during ischemia etc.

1. Causes of visceral pain

 Nociception caused by direct injury of visceral organs as in the case of ischemia.

 Inflammation, either acute or chronic dilation of viscera as in IBS, Ulcerative colitis.

Crucial observation is not all the internal organs are prone to visceral pain. Solid organs like liver, kidneys are not prone to this form of pain [57]. Considering an example as in the case of lesions in liver/pancreas, they are not transmitted as pain signal to the CNS. Instead they are discovered when there is other abnormal condition of liver as in like jaundice (which is painless) to identify a lesion or carcinomas of pancreas.

2. Clinical manifestation of visceral pain

Identifying a visceral pain is most difficult out of all in clinical setting. At the same time, it can’t be ignored as they are sometimes associated with prime life threatening disease like ischemia.

Classifying a visceral pain is also cumbersome as explained earlier due to their vague symptoms and complex nature. But in a whole, they are classified as below (Figure 23);

Figure 23.showing various forms of visceral pain Even though referred pain is not a direct manifestation of visceral pain, it indirectly shows up the condition of the viscera [58].

2.1 True visceral pain

Poorly localized, diffused randomly and unclearly defined form of pain [59]. It typical place of exhibition includes anterior abdominal region, below the sternum and around the diaphragm and is manifested as heavy and compressed. It co-exhibited with severe perfusion, gastro-intestinal and psychological disturbances. E.g. Myocardial infarction.

2.2 Visceral hyperalgesia

It is also poorly localized and slow. Hyperalgesia is a condition in which the sensitivity towards the noxious stimuli increases [60]. Visceral hyperalgesia shows similar increased sensitivity to pain subsequent to an inflammation and with/without injury internally. E.g. IBS, chronic pelvic pain, fibromyalgia etc.

2.3 Viscera-viscero hyperalgesia

It occurs due to the sensory interactions between two visceral organs which shares same portion of afferent pathway [61]. As in the case of coronary heart disease and gallbladder calculosis, the patient experiences synergistically double the pain with these two conditions existing together because of same afferent pathways.

2.4 Referred pain without hyperalgesia

Referred pain can be defined as the condition in which the symptoms of pain occur in completely different place than that of the affected visceral organ. E.g. Pain during a heart attack. In the above said case, the referred pain occurs in the upper part of left arm and hand. This happens due to the misinterpretation in processing of signals by the brain due to similar sensory signalling pathways.

2.5 Referred pain with hyperalgesia

A referred pain associated with hyperalgeisa occurs in superficial muscles. It is also termed as viscera-somatic hyperalgesia. This form of pain occurs due to central sensitization posed due to viscera-convergent neurons.

Thus, all forms of visceral pains are unclear, poorly localized, not easily detectable and complicated. All these reasons make visceral pain one of the gravest problems to treat.

3. Pathophysiology of visceral pain

Visceral pain in general is transmitted in two different mechanisms. They are classified according to their sensitization properties. Sensitization is a non-associative study process in which a repeated administration of stimuli results in increase of the shown response. This sensitization can be central or peripheral depending on which, visceral pain is classified as follows

3.1 Central sensitization

Central sensitization phenomenon happens due to the increased excitability of neurons in CNS which results in abnormal firing of responses even though the signal received is normal. This abnormal excitability is attributed to the nociceptors which alter the strength of connection of synapses between nociceptors and the neurons of spinal cord. Even though we feel that pain originates from periphery, it actually arises due to abnormality of processing in CNS. The tactile allodynia due to light touch happens due to central sensitization. For example, in migraine, slight touch of hair feels so painful so as the case after various surgeries.

3.2 Peripheral sensitization

It is a result of decreased threshold and henceforth increased responsiveness of the peripheral ends of the nociceptors [60]. For example, as in the case of sun burns, the feeling of heat increases even the burnt area is exposed to normal water. This sensitization is due to the action of released chemical messengers around the affected area. These messengers increase the transmission of noxious stimuli resulting in sensitization.

The release of prostaglandin PGE 2 is due to the activation of neutrophils which further releases the enzyme cyclooxygenase-2 (cox-2). This cox-2 is responsible for production of PGE 2. NSAIDS like aspirin acts by inhibiting cox-2 [62].

Medication for visceral pain

Almost all the drugs used against migraine are prescribed for treating visceral pain. Most common drugs include NSAIDS, opioids, antidepressants, etc.

Recent studies have shown the distribution of CGRP receptors in visceral organs [63]. Due to its potent vasodilatative action and least side effects, the research groups are hitting on finding a target for this receptor.

IV. RATIONALE FOR THE PROJECT

The main rationale for the project can be attributed in simple words as acting at two different levels for an effective treatment of chronic pain. We nominated these two effects to be concurrent, synergistic and more practical to achieve. According to the study conducted by Burgess et al, [64] the recent identification and development of analgesics can be accounted as follows

19 4

Figure 24.Showing the account of analgesics developed recently

In their study, they stated that the design and synthesis of new analgesic molecules in recent times (blue region of pie chart) accounts for 7% of the drugs which entered the market. Whereas, the reformulation (crimson red region of pie chart), where the pharmaceutical companies modulates the actual formulation of potent drugs to increase the bioavailability and in turn increase the drug efficacy accounts for 57% of the drugs in current market.

The remaining 37% (green region of pie chart) account for the drugs which are combinations of two varied mechanism of actions. And hence, the pharmaceutical companies play safe by choosing two varied and well know potent and efficacious moieties as leads and develop a new analgesic molecule from them. The pharmaceutical companies and research groups started using this particular rationale due to the reasons like

 Due to synergistic activity, this method decreases the usage of dose of two drugs.

 It decrease the side effects posed by two different drugs.

 It decrease the cost of production of two different drugs

As a result, the dual activity drugs identification has been enrolled as an active area and a hot topic in current drug discovery and development of not just analgesics but many other drugs. Hence, a superficial outlook on dual activity drugs development in current market will be helpful for our better understanding.

A. Dual activity

Dual activity molecules tend to a single moiety possessing two pharmacological effects, which can be used to treat same disease or two different diseases [65]. The approach of incorporating two desired pharmacological effects in one chemical structure is not just a fashionable thematic, but also an important pharmaceutical application now-a-days. With this approach, the advantages include the following;

1. Applicability of combinatorial methodology by using already existing and efficacious drugs to profit a variety of new generation dual activity molecules.

2. Easier management of a disease with simplified drug and dose regimen for the patient.

3. Decrease the dose of two different drugs (In case the two desired effects are synergistic).

4. Decrease the side effects of two different drugs.

5. In industrial point of view, decrease the cost of production of two drugs.

Currently, there are varieties of pharmacologically dual active chemical entities in the pharmaceutical market ranging from analgesics to anti-cancerous agents. A quick overview of those entities and developmental strategies are not just attention-grabbing, but also hints a lead for further research in pharmaceutical research and development.

1. Drugs for same diseases/disorders: There exists certain drugs which are combination of two different mechanisms/different targets but implicates for treating the same disease or disorder. For example,

Dual Cyclooxygenase/5-Lipoxygenase inhibitors: They act on both cyclooxygenase and 5- lipoxygenase enzymes which are responsible for synthesis of pro-inflammatory bodies [66]. It includes drugs like Tepoxaline, Licofelone, Flufenamic acid derivatives etc.

Arachidonic acid is chemically polyunsaturated fatty acid and is abundant in phospholipid cell membrane. They are synthesised by the phospholipase-A2 mediated conversion of membrane phospholipids. Thus formed arachidonic acid can be metabolised by the influence of two enzymes cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). The subsequent products from these two enzyme catalysed pathways are pro-inflammatory bodies prostaglandins and leukotrienes (Figure 25).

Figure 25.Dual cyclooxygenase/5-lipoxygenase inhibitors

The Cyclooxygenase (COX) enzyme inhibition by Non-Steroidal Anti Inflammatory Drugs (NSAIDs) results in declined synthesis of vasodilatory and gastro-protective prostaglandins which ultimately leads to an up regulation of arachidonic acid metabolism by the 5-LOX pathway, increasing the formation of leukotrienes and contributing to inflammation and NSAIDs-induced adverse effects such as asthma.

Thus the design of dual cyclooxygenase/5-lipoxygenase inhibitors has been proposed and lime lighted in order to bestow a superior anti-inflammatory nature as well as decrease of side effects due to 5-LOX pathway. The antagonists used to design these kind of dual moieties are mostly already existing drugs. Thus, a brief over view about the dual active COX/5-LOX is discussed further.

Modified NSAIDs

Indomethacin is well-versed NSAIDs and is modified to a dual COX/5-LOX inhibitors by converting the carboxylic group to an N-hydroxyurea [70].

O H N 2 N OH HOOC HO NH MeO MeO 2 N O N N S

O Cl O Cl Zileuton

Indomethacin Compound 1 % inhibition of 10 µM COX-1=9% COX-2=48% IC50 5-LOX=0.4 µM

Figure 26.Indomethacin derived dual COX/5-LOX inhibitors This N-hydroxyurea, obtained from Zileuton (a 5-LOX inhibitor) plays as a chelator by complexing with the non-heme iron of 5-LOX. Thus resulted compound 1 is potent dual COX/5-LOX inhibitor.Another potent dual COX/5-LOX inhibitor is Tepoxaline which again incorporates two individual activities in same moiety (Figure 27).

MeO

N Selective COX-2 N N OH 5-LOX iron inhibitors pattern chelating pattern O

Cl Tepoxaline IC50 COX=4.2 µM 5-LOX=1.7 µM

Figure 27.Tepoxaline as dual COX/5-LOX inhibitors

Tepoxaline shows 1,5-diphenyl-1H-pyrazole pattern (red circle) which tends for selective COX-2 inhibition and N-hydroxy-N-methylpropionamide (blue circle) which is for 5-LOX iron chelating pattern. This tepoxaline has undergone clinical evaluation for psoriasis and rheumatoid arthritis and proved to be effective.

There are some other miscellaneous dual COX/5-LOX inhibitors like Licofelone.

COOH

Cl Licofelone IC50 COX-1=0.16 µM COX-2=0.37 µM 5-LOX=0.21 µM

Figure 28.Licofelone as dual COX/5-LOX inhibitors Licofelone is therapeutically applied for osteoarthritis. It showed a potent dual activity without showing any side effects [68].

Along with anti-inflammatory agents, there are other class of drugs which are used to treat other diseases/disorders as follows.

Dual Angiotensin II and Endothelin receptor antagonists: They act on both Angiotensin II and Endothelin I receptors which are implicated in hypertension [69]. These two endogenous potent vasoconstrictors acts in a positive feedback loop thus potentiating each other’s release and action.

The rationale for the design of Dual Angiotensin II and Endothelin receptor antagonists was done keeping in mind the key pharmacophore scaffolds of both classes of drugs. In both class of these drugs, the biphenyl moiety is constant and positions series of modifications leads to compound 2 (Figure 29).

N N N O N N N N

O Dual O Lead O active lead modification + N O NH O N O O N O O S O S O O S N N N O N HN HN HN

BMS-193884 Irbesartan Compound 6 Compund 7 ET K = 79 nm ETA K i = 1.4 nm ETA K i > 10 µm A i ETA K i = 1.9 nm AT K = 4.7 nm AT1 Ki > 10 µm AT1 K i = 0.8 nm 1 i AT1 K i = 10 nm

Figure 29.Dual Angiotensin II and Endothelin I receptors antagonist development

BMS 193884 is a potent endothelin receptor antagonist and Irbesartan on the other hand is angiotensin receptor antagonist [69]. The combination of these two results in compound 2 which is potent dual active AT/ET antagonist.

Not just above stated dual active therapeutic agents, there are other diseases/disorders being treated. For example, dual topoisomearse I/II inhibitors uses dual active molecule strategy to treat cancer [66].

As presented at the beginning of the dual activity discussion, there is applicability of dual active therapeutics for different disease/disorders as shown below.

2. Drugs for different diseases/disorders: On contrary to the first group of dual active drugs, there exists certain drugs which are combination of two different mechanisms/different targets and also implicates for treating two different disease or disorder. For example,

Dual μ-opioid receptor and serotonin/norepinephrine reuptake inhibitors: They act on both opioid receptor and as a serotonin/norepinephrine reuptake inhibitor [68]. Thus the first action results in analgesic effect and the latter results for its antidepressant action. As a result, these dual opioid receptor and serotonin/ norepinephrine reuptake inhibitors are effectively used for chronic pain for its better management.

For example, Tramadol acts as opioid receptor and as a serotonin reuptake inhibitor and Tapentadol acts as opioid receptor and as a norepinephrine reuptake inhibitor. These two drugs not only treat pain but also can be used to combat depression.

Tramadol

Tramadol is dual active opioid receptor (central acting) and serotonin reuptake inhibitor [68]. Chemically it is 2-((dimethylamino)methyl)-1-(3-methoxyphenyl)cyclohexanol and occurs in both R- and S-stereoisomers but commercially, tramadol is marketed as a racemic mixture because of the complementary action of both isomers.

N N HO HO

O O

(1R,2R) Tramadol (1S,2S) Tramadol

K i 2.1-57.6 µM Figure 30.Isomers of Tramadol

Analgesic activity is accredited to low but efficient affinity for the µ-opioid receptor as well as reuptake inhibition of the monoamine, serotonin. It shows no selectivity between µ, k,

δ-opioid and shows highest affinity with µ-opioid (Ki 2.1 µM) but the active metabolite of tramadol, o-desmethyl tramadol shows higher affinity than tramadol itself. Along with opioid action for pain relief, it acts with a secondary pathway for relieving pain using descending inhibitory system (this part has been discussed in-depth in depression and its management chapter). Venlafaxine is a SNRI used to treat depression, whose structure is intimately related to tramadol.

As seen in the previous chapters, antidepressants act mainly by recapture of serotonin or norepinephrine. Thus, tramadol due to its descending inhibitory activity on monoaminergic system acts as a moderate antidepressant in treatment of chronic pain.

Tapentalol

It is another dual active opioid receptor and norepinephrine reuptake inhibitor which acts in a similar way as that of tramadol. Its analgesic property is more than tramadol but less than morphine. But its norepinephrine reuptake activity is poor compared to its preceded counterpart i.e. tramadol.

HO N

(2R,3R) Tapentalol

K i = 0.096 µM-mu opioid 0.97 µM- delta opioid 0.91 µM-kappa opioid Figure 31.Dual activity of Tapentalol Tapentadol’s dual activity has been validated by studying the inhibition of norepinephrine reuptake with a Ki of 0.48 µM and serotonin reuptake with a Ki = 2.37 µM, clearly showing its affinity towards norepinephrine than that of serotonin.

Along with above discussed analgesics, there are other categories of drugs which are applied with dual activity principle and thus can be used as a treatment for two different drugs/diseases.

With the above described rationale, we have designed the prototype of the project.

B. Design of the project

As presented earlier, the first target for acting on chronic conditions like migraine and visceral pain was CGRP receptor antagonism. For selecting the second target to achieve a dual active moiety, we have done some more literature investigation and presented the full prototype of the project as below

Figure 32.Dual activity design for the project We have chosen tricyclic antidepressant property to be our secondary effect to be incorporated into the dual active molecule along with CGRP antagonism. This has been proposed due to the effect of tricyclic antidepressants on chronic pain implication. As reported in the previous subsection [refer figure 12], tricyclic antidepressants along with its antidepressant property also have analgesic effect due to its action on descending pain modulation pathway.

Thus, we can sum-it-up and present that we wanted to incorporate CGRP antagonism and tricyclic antidepressant property into a single moiety to effectively treat chronic conditions like migraine and visceral pain.

As we have selected antidepressant property as our second target to achieve, we will have a brief introduction about the depression and effect of antidepressant on chronic pain.

C. Depression and its management

1. Depression: It is a state of low mood and aversion to the activity that can affect a person’s behaviour, thoughts, feelings and sense of well-being. It is potentially a life-threatening disorder that affects millions of people around the world irrespective of the age group. Symptoms includes mood swing (majorly depressed mood), low in energy, anhedonia, suicidal tendencies, psychomotor as well as gastrointestinal disturbances [71]. Depression is a complex phenomenon which shows fluctuating symptoms (depending on patient), all as well as few and mild to severe symptoms.

Thus all the above ins and outs interpret for non-suitability of ADs against acute pain. There are definite points which show us the correlation between depression and chronic pain;

 Pain and major depression mostly overlap each other. A statistical study was done using depression and painful symptoms, which includes Headaches, Neck pain, abdominal pain, chest pain etc.

Figure 33.Showing correlation of pain and major depression If we consider the case of chronic pain vs. depression, statistical data between some chronic pains with a tinge of depression like migraine, back pain and arthritis are as above[72].  Depression and chronic pain shares a common neurobiology and neurochemistry i.e. involvement of serotonin, norepinephrine along with similar Hypothalamic-Pituitary-Adrenal (HPA) axis, autonomic nervous system and inflammatory cytokine (Tissue necrosis factor-α and interleukin-6) disturbances.  Antidepressants can recover chronic pain symptoms with or without the existence of major depression in the patient. Even the vice versa condition applies i.e. antidepressants can recover major depression with or without the existence of chronic pain in the patient.

Chronic pain and depression: Recent studies showed that patients suffering from pain have a risk of inclining towards depression 2 to 5 times more than a normal individual. But the assessment or identification of these two syndromes (pain and depression) is cumbersome as both of them have shared symptoms like gastrointestinal disturbances, fatigue, sleep disturbances etc. The comorbidity between pain and depression paves a rationale for using antidepressants again chronic pain.

2. Classification of depression [73]:

2.1 Major depressive disorder or Major depression: It disables a person from functioning normally. It interferes in every aspect of patient’s life like working, sleeping, eating, studying etc. Some people may experience an episodic form of major depression only once in their lifetime but mostly it recurs as multiple episodes in most of the patients.

2.2 Dysthymia or Dysthymic disorder: It won’t disable a person from normal functioning and shows the symptoms of depression (mildly intense) for long-term i.e. 2 years or more. People with dysthymia experiences one or more episodes of major depression in their life time.

2.3 Minor depression: It lasts for two weeks or longer and can’t be categorized into major depression due to its symptoms. When left untreated, there is high risk for developing major depression.

Along with the above stated depressions, there are some other unclassified forms like Bipolar disorder, psychotic depression, postpartum depression, seasonal affective disorder etc. which are rare and affects minority of population.

3. Biochemistry of depression:

Depression in a general context is due to the decreased monoamine neurotransmitter concentration in the synaptic cleft.

Figure 34.Serotonin and norepinephrine function

3.1 Monoamine neurotransmitters: They are neurotransmitters or neuromodulators that has one amino group and has a bridge of two carbon chain connecting to the aromatic ring. All monoamine transmitters are biosynthesized from aromatic amino acids like phenylalanine, tryptophan, tyrosine and thyroid hormones by the enzymatic action of amino acid decarboxylase [74].

Monoamine transmitters

Histamine Catecholamines Tryptamines Trace amines Phenethylamines

HO H N 2 NH HO OH Phenethylamine Adrenaline HO NH2 NH N H2N H Serotonin OH N-Methylphenethylamine HO N NH NH2 Histamine Tryptamines Dopamine

H O N N O H

H2N N HO H2N H Tryptamine Melatonin HO H OH N Noradrenaline

N H N-Methyltryptamine

Table 4.showing different monoamine neurotransmitters These monoamines play an important role in cognitive processes such as mood, emotions, memory etc. Thus the drugs used to increase the monoamines in the synaptic cleft are used as first line medication for depression and other related disorders.

3.2 Mechanism of action:

Transmission of signal from one neuron to other neuron involves a cascade of steps ranging from transport of amino acids from blood to the brain where the neurotransmitters are biosynthesized from their respective amino acids with the help of enzymes [74]. Thus formed

57 amino acids are stored in the synaptic vesicles and later get released into the synaptic cleft when there is a Ca2+ dependent process (which depends on the firing rate of neurons). After the release of the neurotransmitters from the presynapse, they won’t cross the postsynapse instead activate the receptors on the surface of postsynapse to induce cascade of cellular response.

Figure 35.Biological action of monoamines Once activating the receptors, the functioning of the neurotransmitters are terminated for this cycle and the neurotransmitters are either transported back to the presynapses from the synaptic cleft using specific monoamine transporters which are selective for each monoamine or simply metabolized by the enzyme Monoamine Oxidase (MAO) in presynapses. Thus these are the two probable mechanisms through which the concentration of the monoamines in the synaptic cleft are decreased and in turn results in variety of mood related disorders especially depression. Hence these are the potential targets for therapy of depression.

4. Treatment of depression [75]:

The treatment for depression can be classified based on various methods used to treat it. Out of all, the three most important and effective treatment methods include psychotherapy, electroconvulsive therapy and pharmacotherapy.

Pharmacotherapy is proved to me most effective compared to the rest and is considered as first line treatment for depression.

4.1 Pharmacotherapy: It can be defined as the therapy to treat the depression using pharmaceutical drugs. It includes the drugs used to treat major depressive disorders like

Obsessive Compulsive Disorder (OCD), chronic pain, Neuropathic pain, Migraine, Substance abuse, Eating and sleeping disorders etc.

i. Mechanism of action: As discussed in the aetiology of depression, the main principles in anti-depressant drug design and target validation, the limelight is completely focused on two targets i.e. Monoamine reuptake and Monoamine Oxidase inhibition (MAOI) which tends to increase the intracellular concentration of monoamines. Anti-depressants acts by selectively binding to monoamine transporters like specific serotonin, norepinephrine or dopamine and thus directly increase the monoamine concentration in the synapses. On the contrary, the inhibition of Monoamine Oxidase (MAO) enzyme using anti-depressants also proved to be effective as it inhibits the monoamine metabolism by MAO in the presynapses after their reuptake. Hence it decreases the metabolism of monoamines and makes them available for the next cycle of signal transmission and thus indirectly participates in the increase of monoamines.

Based on their mechanism of action, the generation, structure, selectivity, anti-depressants are classified as follows;

Anti-depressant drugs

Tricyclic Antidepressants Mono Amino Oxidase (MAO) First Imipramine, Inhibitors Generation Clomipramine, Phenelzine, Amytriptiline. Moclobemide, Selegiline.

Selective Serotonin and Allosteric Norepinephrine and Serotonin Norepinephrine Serotonin Dopamine Modulator Reuptake Reuptake Reuptake (NDM) Inhibitors Inhibitors Inhibitors (SSRI) (SNRI) (SSRI) Bupropion

Second Citalopram, Venlafaxine, Escitalopram Generation Fluoxetine, Milnacipran,

Fluvoxamine, Duloxetine. Paroxetine, Norepinephrine Sertraline. Reuptake Inhibitor (NRI)

Reboxetine

Noradrenergic and Specific Serotonergic Antidepressant (NaSSA)

Mirtazapine

Third Melatonergic Generation Agomelatine Table 5.Antidepressants based on their generation All anti-depressants are equally effective and hence choosing one amongst solely depends on bearing in mind its side effects, drug interactions, cost etc.

Pharmacotherapeutic drugs:

TCAs MOA inhibitors SSRIs SNRIs Classification Chemical structure of the ligands Mechanism of action Selectivity towards receptor Selectivity towards receptor Mechanism Serotonin and Norepinephrine Blocking the Monoamine Serotonin reuptake inhibition by Norepinephrine reuptake of action reuptake inhibition by blocking oxidase mediated selectively blocking the reuptake inhibition by selectively the reuptake sites. enzymatic metabolism of sites. blocking the reuptake sites. serotonin and norepinephrine Place in Pillar stone for depression, First Not first-line, rarely used First-line for depression in older Used for SSRI non- therapy line for depression adults responsive patients Effective Major depression, Masked Depression non- Efficacious as TCAs, once daily Used mostly for patients towards depression, pseudo-dementia responsive to TCAs, dosing, safe in case of over- who are not responding for Depressive phase of dosing. other antidepressants bipolar disorder, Anxiety Precautions Low therapeutic index, Cardiac Drug-drug interactions Sexual dysfunction, Gastro- Sexual dysfunction, conduction disturbances, (with Opiates, SSRIs, intestinal disturbances, Insomnia, Gastrointestinal Ischemic heart disease SNRIs, Triptans etc.), Drug interactions, disturbances, dry mouth, Sedation, sexual Increase in blood pressure. dysfunction Serotonin syndrome Drug interactions

F F F N F F H N Examples HN HN Cl H F NH H2N N N O H N O Cl O N NH N 2 H O fluoxetine Sertraline N HO H O S H N Amytriptyline Nortriptyline Imipramine 2 Venlafaxine Duloxetine Phenelzine Tranylcypromine Selegiline Fluvoxamine Table 6.Antidepressants based on their mechanism D. Depression vs chronic pain

Antidepressants (ADs) are one of the important adjuvant in the dosage regimen of pain management, especially chronic pain. Reasons for ADs as not an adjuvant for acute pain include [76];

 The chemical changes (neuroplasticity) in brain due to pain are shown only in chronic pain subjects but not in their counterpart i.e. acute pain subjects.  Among the two pain components i.e. sensory and affective, the affective component is not revealed in acute pain.  Acute treatment of pain involves medication which are given in a single or multiple doses of small range (in days) but whereas ADs need to be given for long-term medication.

1. Biochemistry of antidepressants in chronic pain management:

As summarized above, antidepressants can be effectively used for treating pain symptoms even though there is no existence of major depression. This shows that the antidepressants alone possess analgesic effect. The analgesic effect of antidepressants can be shown at two levels i.e. at central and peripheral.

The central effect can be accounted because of their action on descending pain pathway through spinal cord [77]. Even though multiple mechanisms are proposed, the potentiation or synergistic action of the antidepressants in pain management is still unclear till date. Probably the most accepted theory with stronger support is their action on serotonin and norepinephrine receptors, in precise, on the descending pain pathway through spinal cord. Other theories include antidepressant’s adjunctive therapeutic actions through histamine receptors and/ sodium channel modulation. Norepinephrine and serotonin’s are the involved in the nociceptive signalling at multiple levels. In chronic patients, the activity of descending inhibitory bulbospinal pathway is compromised and ADs are considered to enhance their activity by blocking the 5HT1A receptor. Not just with this, ADs manipulate several other areas of pain circuit (like locus coeruleus nucleus, the dorsal horn of spinal cord etc.). All these manipulations finally results in increased nociceptive threshold of the subject.

The peripheral effect is quite contrary to what happens in the central pathway. ADs can’t act on serotonin or norepinephrine, as these neurotransmitters increases tends to enhance the nociceptive signalling instead of decreasing [77]. Therefore, competitive inhibition of serotonin, noradreno, histamine or muscarinic acetylcholine (ACH) receptors decreases the nociceptive signal transmission. ADs like amitriptyline, captivatingly, enhance the adenosine transmission by increasing extracellular adenosine.

2. Antidepressants and pain: Most of the antidepressants can be used in the management of pain. But not all of them show same degree of effectiveness in treating the chronic pain. Some of the examples in different classes of antidepressants and they effectiveness towards chronic pain management are as follows [76];

2.1 Tricyclic antidepressants (TCAs) are the prime choice in the management of chronic pain. The dose to relieve or to treat chronic pain is relatively less than that used for its antidepressant action. To be accurate, tertiary amine TCAs are more efficacious when compared to Secondary amine TCAs. But their use is limited by weight gain, cardiovascular effects, overdose complications etc. 2.2 Selective Serotonin Reuptake Inhibitors (SSRIs) shows moderate to no efficacy in chronic pain management. Even though they relatively show least side effects when compared with TCAs, they are not efficacious for chronic pain management. Paroxetine and citalopram showed moderate efficacy whereas fluoxetine showed no effectiveness in chronic pain relief.

Antidepressants Efficacy Evidence based Dose Side effect support loading Tertiary amine tricyclic Low to antidepressants Yes +++ standard +++ (Amitriptyline, Imipramine) Venlafaxine Yes ++ Standard + Duloxetine Yes ++ Standard + Bupropion Yes + Standard + Secondary amine tricyclic Yes ++ Low to +++ antidepressants(Desipramine, Standard Nortriptyline) Paroxetine, citalopram Modest + Standard + Fluoxetine No -- Standard +

Table 7.Antidepressant’s effectivity against chronic pain

2.3 Serotonin Norepinephrine Reuptake inhibitors (SNRIs) Showed better efficacy than SSRIs with standard dose. With an effective dose of 150mg/day, venlafaxine can be effectively used against chronic pain management and acts on both key neurotransmitters i.e. Serotonin and Norepinephrine which are involved in pain signalling. Duloxetine is another candidate from the same class which showed similar efficacy and the key point to be noted is, it is the only antidepressant for chronic pain management approved by United Stated Food and Drug Administration (US FDA). For optimal chronic pain management, the effective dose is 60-120mg/day. Like other antidepressants, their use is limited by drug interactions, anti-cholinergic effects, Gastro intestinal disturbances etc. 2.4 Norepinephrine and dopamine modulators showed better efficacy than SSRIs but not more than TCAs & SNRIs. 300mg/day of bupropion showed similar efficacy as TCAs and considered to be optimal for chronic pain management. As discussed earlier, action on serotonin and norepinephrine is important for chronic pain management. Instead, bupropion proved it wrong by showing effectiveness in chronic pain management even though it acts on norepinephrine and dopamine (instead of serotonin). But their usage can be limited because of cases of seizures in the subjects.

Hence, from the above proof based efficacy results, we can make out that the tricyclic antidepressants are deemed to be the best drug entities to treat chronic pain. They proved to be potent as well even on low doses. The other class of drugs, SNRIs also showed potential activity again chronic pain. This also proves that, to act on chronic pain, the drug molecules should act on both serotonin and norpinephrine which are the main neurotransmitters involved in both depression and pain signalling.

Henceforth, we had an outlook of both primary and secondary targets of our dual activity project. Now, let’s sum-up all together and have a preface about the project.

V. CONTEXT OF THE PROJECT

Calcitonin Gene Related Peptide receptor is one such target which is implicated in treating chronic pain conditions like visceral pain and migraine. Telcagepant®® is a prime molecule when it comes to CGRP antagonism.

Tricyclic antidepressants are drugs which are not just used for treating depression but also as an analgesic for treating chronic pain. Imipramine is one of the TCAs, which even in low doses showed potent analgesic property.

Thus the concept of the project was to create a hybrid molecule by combining Telcagepant®® and Imipramine in a single moiety for a better treatment of chronic pain.

Figure 36.context of the project We want to combine the Telcagepant®® and Imipramine in a single moiety to recreate a hybrid structure which can be potentially used for treating some of the chronic pain conditions.

As we have made our first stage of drug discovery process by outlining the concept and approach towards the project, we proceeded to the second stage where the molecular modeling and docking studies have been performed. This will be explained in detail in the next chapter starting from design till validating the potential hits.

Take-home message from this chapter

In this chapter, we have seen ‘pain’ in broader context as one of the grave forms of illness in current generation to be treated. The data of global sufferers due to pain are more dynamic like never before.

Even among pain, acute pain does have improved and efficacious medication available in market. On contrary, even though chronic pain affects almost 36 % of Europeans and 43% of Americans, there are still inefficacious pharmacotherapeutics available. And if potent and efficacious drugs are available, they are posing some potent side effects as well.

Migraine and visceral pain are two of the most dreaded conditions under the roof of chronic pain. On one hand, migraine causes diminished quality of life whereas in the other hand, visceral pain reduces quality of life and sometimes being fatal. Even in 2010, with the current technology in diagnosis and treatment methods, the mortality and years passed with disability rates are climbing rampantly.

Calcitonin Gene Related Peptide receptor is one such target which is implicated in treating chronic pain conditions like visceral pain and migraine. Telcagepant® is a prime molecule when it comes to CGRP antagonism.

Depression is associated in most of the cases with chronic pain and patient has to take multiple doses of medication to treat both chronic pain and depression individually. The doses of antidepressants used are also high ranging between 150-300 mg.

Hence dual active moieties which can use the synergistic principle of treating chronic pain are potent future generation molecules to be developed in treating them.

Thus the concept of the project was to create a hybrid molecule by combining Telcagepant® and Imipramine in a single moiety for a better treatment of chronic pain.

Winding up with the concept derived from this chapter, we will look into the next stage of drug discovery i.e. crystal structure of protein and molecular modeling studies in the next chapter.

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CHAPTER 2: CALCITONIN GENE RELATED PEPTIDE RECEPTOR, MOLECULAR MODELING STUDIES & LIGAND DESIGN

I. CALCITONIN GENE RELATED PEPTIDE RECEPTOR

Calcitonin Gene Related Peptide (CGRP) receptor is structurally a heterodimeric protein with CALCitonin Receptor Like receptor (CALCRL) and Receptor Activity Modifying Protein-1 (RAMP-1) as its subunits [1-3]. These receptors on activation by the endogenous natural ligand results in various physiological disorders like Migraine, Crohn’s disease, Irritable Bowel Syndrome, Systemic Inflammatory Response Syndrome etc [4-6]. Among the two subunits involved, CALCRL belongs to the Class B (Figure 37(b)) of G- Protein Coupled Receptors (GPCR) (Figure 37(a)) [7]. Whereas RAMP is an activity modulator associated with different GPCRs like Calcitonin receptor or Calcitonin receptor like receptor to incarnate various dimeric receptors [8, 9]. There are three different RAMPs; RAMP-1, RAMP-2 and RAMP-3 [10].

Figure 37.(a).GPCR Tree (b) Class-B Secretin family (c) CGRP receptor (d) CALCRL-protein interactions CALCRL associating with RAMP-1 gives CGRP receptor (Figure 37(c)) while RAMP-2 and 3 results in Adrenomedullin receptor or CGRP/Adrenomedullin dual activating receptor respectively [11, 12]. The notch up of protein-protein interaction of CALCRL with different partner proteins (Figure 37(d)) is as follows (Table 8);

Partner protein Score

RAMP-1 (Receptor Activity Modifying Protein-1) 0.999

CALCA (Calcitonin related peptide alpha) 0.995

RAMP-2 (Receptor Activity Modifying Protein-2) 0.994

CALCB (Calcitonin related peptide beta) 0.977

RAMP-3 (Receptor Activity Modifying Protein-3) 0.976

ADM (Adrenomedullin) 0.915

GPR-182 (G-Protein Coupled Receptor 182) 0.829

CRCP (CGRP Receptor component) 0.809

GNAS (Guanine Nucleotide Binding Protein Alpha 0.809 Stimulating)

ADM-2 (Adrenomedullin-2) 0.750

Table 8.CALCRL interaction with various proteins generated using STRING-8 software [13] Like for the other GPCRs, hitting a hotspot of CGRP receptor is a challenging task due to the least reported and exploited binding sites on the receptor. Hence we will try to validate different aspects of the CGRP receptor with the aid of in silico tools to draw some possible hotspots of the receptor and craft a pathway in CGRP research.

A. DRUGGABILITY

Druggability of a protein can be defined as a virtual recognition or derivation of the pocket of a protein, which acts as a site of interaction for probable ligand [14]. From this recognition, it paves a pathway for drug discovery process of a disease or a disorder. This virtual screening of druggable site is considered as the foundation for a thriving drug discovery process [15]. On an average, only ≈15% of the total available proteins in humans is found to be modulators of a disorder or disease out of which only around 15% are druggable [16]. Hence finding a new target for newly developed drugs is least probable. However these figures can be increased if the number of protein-protein interactions can be disturbed by the ligands so that new vicinities of the protein are exposed increasing the chances of the druggable sites [17].

1. Druggability study of CGRP receptor

The Druggability of CGRP receptor was identified using DrugEBility® online software by European Molecular Biology Laboratory-European Bioinformatics Institute [18]. To begin with, the crystal structure of the protein was retrieved from the Protein Data Bank (PDB) by Royal Collaboratory for Structural Bioinformatics with PDB ID 3N7S [19]. Initial study

includes general druggability of the receptor’s two subunits individually (Figure 38). Hence the proteins CALCRL and RAMP-1 preliminary impressions are as follows;

Figure 38.Depicting the Tractability, Druggability and Ensemble druggability of CALCLR and RAMP-1 Druggability score can be defined as likelihood of small molecules which satisfies the Lipinski’s rule of five to have a good binding in the pocket. Tractability is similar to druggability but with more flexibility in some of the Lipinski’s rules. Ensemble Druggability is mean of druggability score but calculated in various models (Table 9).

Score Molecular Hydrogen Hydrogen Calculated Number Others Weight Bond Bond LogP of Donors Acceptors rotatable bonds Tractability 200

Both the subunits have relatively same nature in their sites (Figure 38). They showed 0% of Druggability and Ensemble Druggability. However, interestingly they showed 50% of Tractability in their binding sites once again proving the hypothesis that relatively bulkier ligands might serve as a good hits for this receptor.

2. Druggability validation of CGRP receptor

Following a brief study on the general druggability and tractability of CGRP receptor family in general, the individual druggability validation of the receptor is investigated using the DrugEBility® online software. In this method, the DrugEBility® software takes up the receptor replica via uploading manually the protein model or by entering the PDB code directly. After a run-through and scrutinizing the database, the software provides the top four probable binding sites of a protein. As CGRP receptor is a heterodimeric protein, two individual runs on both CALCRL & RAMP-1 is required.

Retrieving the results & further exploration at the CALCRL & RAMP-1 proteins with their four binding sites (Table 10), it shows quite clearly that Olcegepant® is interacting in the vicinity of the site 1 of CALCRL whereas RAMP-1 has a tractable site 1 which opens the port for persuading new and important interactions.

Site Site on protein V D C T C E (Å3)

1 674.1 0.00 0.73 0.00 0.71 -0.98

2 498.2 0.00 0.96 0.00 0.86 -0.99

CALCRL 3 288.9 0.00 0.96 0.00 0.86 -0.99

4 207.5 0.00 0.96 0.00 0.86 -0.99

1 1468.5 0.00 0.73 1.00 0.83 -0.98

1 -

2 434.9 0.00 0.96 0.00 0.86 -0.99

- RAMP

3 RAMP 394.8 0.00 0.96 0.00 0.86 -0.97

4 300.3 0.00 0.96 0.00 0.86 -0.99

(V-volume, D-druggable, C-confidence, T-tractable, E-ensemble) Table 10.Visualizing the druggable sites of CALCRL & RAMP-1 generated by DrugEBility® software [18]

By having a closer look at the 4 sites of the CALCRL & RAMP-1 subunits, there is a clear visualization of the probable binding sites on CALCRL showed relatively smaller druggable volumes (674.1, 498.2, 288.9 and 207.5Å3) compared to that of RAMP-1(1468.5, 434.9, 394.8, 300.3Å3). And furthermore, the site 1 of RAMP-1 is envisaged as a tractable site with a volume of 1468.5Å3. Thus designing an antagonist with CALCRL interacting fragment relatively smaller to that of RAMP-1 is more persuasive & reasonable theory. This hypothesis can be validated using a well known CGRP antagonist Olcegepant® and its binding inside the receptor pocket.

Figure 39.showing the CALCRLs site 1 & the fragment of Olcegepant® embedded inside the pocket The fraction of the ligand which was embedded in the site 1 of CALCRL is 3- (piperidin-4-yl)-3, 4-dihydroquinazolin-2(1H)-one. The binding of this fragment of Olcegepant® into the receptor is considered as inevitable and important interaction (Figure 39). Hence this fragment has been well thought-out as universal for all the CGRP antagonists from there on [20]. Whereas the uninteracted fragment of Olcegepant® in CALCRL is thought to be interacting with the associated protein RAMP-1. The site 1 of RAMP-1 (Table 10) shows the tractability score hence proves that (S)-2-amino-N-((R)-6-amino-1-oxo-1-(4-(pyridin-4- yl)piperazin-1-yl)hexan-2-yl)-3-(3,5-dibromo-4-hydroxyphenyl) propanamide (the fragment not shown in the red dotted line of Olcegepant®) is having an interaction with it. The large volume of the site 1 (1468.5 Å3) also proves that considerable bulky interacting fragment is required for a healthy fit as well as interactions.

B. DRUG-RECEPTOR INTERACTION STUDY:

The crystal structure of CGRP receptor with Olcegepant® as the ligand showed the significance of various interactions [19]. The fragment interacting with CALCRL protein showed some key interactions like Thr122, Asp94. Thr122, a polar residue acts as a backbone donor for the carbonyl oxygen of 3-(piperidin-4-yl)-3, 4-dihydroquinazolin-2(1H)-one and Asp-94, an acidic residue shows a side chain acceptor interaction with the nitrogen on the pyridine (Figure 40).

Figure 40.Crystal structure of CGRP receptor stabilized with Olcegepant® studied using Molecular Operating Environment® (MOE) software [21] Whereas the (S)-2-amino-N-((R)-6-amino-1-oxo-1-(4-(pyridin-4-yl) piperazin-1-yl) hexan-2-yl)-3-(3, 5-dibromo-4-hydroxyphenyl) propanamide interacting with RAMP-1 illustrate key interactions with Asp71, Arg38 & Arg67. Asp71, an acidic residue acts as side chain acceptor from the amine of the pentan-1-amine and Arg38, Arg67, both being basic residues shows water dependent hydrogen bond with the oxygen of the 2, 6-dibromo-4- ethylphenol.

C. SPECIES SELECTIVIY DECISIVE RESIDUES

The importance of some amino acids apart from those listed above is studied using deletion or mutational investigation. The significance of various amino acid residues as well as the binding of CGRP antagonists specifically to CALCRL/RAMP-1 complex but not for

76 the other protein-protein complexes like RAMP-2 or RAMP-3 has been documented using the genetic engineering methods.

The radioligand-isotope binding assay illustrated that rodent CGRP receptor (both Mice & Rats) showed less affinity for the CGRP antagonists compared to that of primate receptor. This decrease of affinity in rodent CGRP receptor is attributed to the fact that at 74th position of the RAMP-1, the Tryptophan is replaced with Lysine. The crystal structure clearly showed the Trp74 has formed a hydrophobic roof in the RAMP-1 and this observation is found to be crucial to derive lot of important conclusions. Olcegepant® showed 100 times less affinity in rodent species [22] whereas Telcagepant® showed 1500 [23]. This variation of affinity for these antagonists might be endorsed due to the protein-ligand hydrophobic pocket in rodents has been decreased [24], the other functionalities of Olcegepant® like 4- (pyridin-4-yl)piperazin-1-yl, Amino hexyl moieties might act as steric hindrance and thus reducing the ligand-Trp74 interaction. Hence the replacement of Tryptophan with Lysine couldn’t result in huge impact. In the case of Telcagepant®, the antagonist interacting with RAMP-1 is small and non-sterically hindered, thus the Tryptophan replacement can easily show the impact on affinity. Not only with affinity facet, tryptophan 74 might also implicate in selectivity of CGRP antagonists towards CALCRL/RAMP-1 complex [25]. This Trp74 is expressed exclusively in RAMP-1. However, in RAMP-2 or RAMP-3 it is replaced by Glutamic acid. Hence Tryptophan 74 might play a crucial role both in selectivity and affinity of the antagonists. Alanine 70, Aspartic acid 71, Histidine 75, Phenylanlanine 83, Tryptophan 84 and Proline 85 also are other miscellaneous amino acids whose interactions can serve in answering a better CGRP antagonism. Henceforth, with the knowledge obtained through the study of CGRP receptor, we performed molecular modelling & docking studies in the next stage to identify potential hits for the CGRP receptor.

II. MOLECULAR MODELING STUDIES

With the innate knowledge from the previous chapter on the CGRP receptor, its druggability, hotspots and key residues, we further proceeded to the molecular modelling and docking studies using the crystal structure of CGRP receptor. This particular chapter can be discussed in three different subsets as;

1. Modelling of the receptor and validation of the model 2. Modelling of the ligands 3. Docking

A. Modelling of the receptor

The crystal structure of the CGRP has been retrieved from Protein Data Bank (PDB) of Research Collaboratory for Structural Bioinformatics (RCSB). CGRP complex with Telcagepant® or Olcegepant® with PDB ID 3N7R and 3N7S respectively are currently available crystal structures in the data bank. We nominated 3N7S as our model because of its highest resolution CGRP structure till date. After retrieving from the PDB, the receptor was re-refined with the help of Phenix macromolecular crystallographic software [26], using which the important aspects like solvation of the system and ligand interactions in the receptor have been visualized.

During the refinement step, we prepared our molecule ready for docking by removing the already interacting ligand (in our case, Olcegepant®), chains B and C as well as the water molecules leaving the following system i.e. HOH201A; HOH206D; HOH203D; HOH210D; HOH203A; HOH210A; HOH225D; HOH216A (Figure 41).

Figure 41.showing the important water molecules and its interactions . Finally, this model is minimized using molecular mechanics (refer appendix for details). Hence this energy minimization step results in a low energy and refined crystal structure which will be engaged in the docking simulation.

B. Modelling of the ligands

As described in general during the course of first chapter, we have recreated a hybrid structure which has been derived from Telcagepant®® and Imipramine cross-over. The hybridization can be depicted as below

Figure 42.Showing the hybridization of two leads The fragment coloured in red is carried as a privileged structure from CGRP receptor antagonists and the fragment in blue was designed according to the structure of the receptor.

During the design of the ligands, we kept in mind various important points. Hence, first we will look into the aspects and design of ligands for CGRP antagonism.

1. Structure based ligand designing

As stated, the crystal structure of CGRP receptor with highest possible resolution has been retrieved from PDB. Using this model, structure based ligand designing of the potential hits was done (Figure 43). During this process of designing the hits, we followed different set of medicinal chemistry rules for drug designing in order to provide an optimized potential hits.

Figure 43.showing structure based ligand designing The region in white represents RAMP-1 where as in yellow is CALCRL. The fragment in our hits interacting with CALCRL is highly potent and is most common fragment of currently available CGRP antagonists.

Whereas, the most important region for CGRP antagonism is RAMP-1 and further in that, Tryptophan 74 is highly important for affinity towards the receptor. As this RAMP-1 requires hydrophobic interactions, the fragment of the hits interacting here has to be hydrophobic. And last but not the least, both the fragments can’t be tightly bound as this may result in high crash and strain in the pocket and hence there has to be flexibility between both the fragments which can be obtained by using a linker to have easy rotation.

1.1 Ligand designing

The fragment designed is solely for the RAMP-1 region as we use optimized and already used privileged fragment for CALCRL part.

Points considered during designing:

 No same volume fragments: The RAMP-1 is big in volume and we can’t design a fragment of same volume as they will be facing problem for drug transportation to reach the place of interaction. Even if it reaches, they can’t fit because of strain and crash inside the pocket.

 Chiral carbons incorporation: If we can incorporate chiral carbons, depending on the interaction required, we can reach both sides of the plane of the molecule. Even if

tough to reach the required interaction, homologation can help to fill the desired interactions (Figure 44).

Figure 44.Showing importance of stereocenters  No flat molecules: For the same reason as stated in the point above, a flat molecule can’t reach the desired interaction (Figure 44). Instead, using conformational isomers might help in reaching the amino acids by modulating the substituants on them.

Thus along with the above points, we have chosen piperidine to be the core moiety as the laboratory has expertise in synthesis of chiral piperidines and indigenous developed methodologies for using different substituants.

1.2 Application of medicinal chemistry principles

Hydrophobic RAMP-1 fragment has been designed as shown below.

O O O HN N HN N O HN N O O Replacement N N N with fluorine NH Ring opening O NH O NH O N N N N H N N N N N

II I III

Benzo cracking Ring attachment

O O O O HN N O O HN N HN N Divalent isosteric Divalent isosteric N NH N NH replacement N NH replacement N N N N N N O NH Y

VI IV V

Homologation Ring equivqlent

O O O O O HN N HN N O N O N NH Peptide isostere N NH N O NH N N N N N N N N N N N

OH VII VIII I Figure 45.showing medicinal chemistry aspects applied for ligand design 1. Molecule I has been developed first from which rest of the molecules have been derived using various principles of drug design. Replacement with fluorine from I to II was done in order to increase the metabolic stability of the molecule, aqueous solubility, antidepressant property. Molecule III is obtained by ring opening principle from I with increased aqueous solubility, flexibility inside the pocket and increasing the hydrogen bond donor by exposing NH. These modifications gave us family 1.

2. Further modification of molecule I ring attachment principle gave general structure of family 2 and 3. Molecule I by peptide isosteric replacement gives VIII and thus derives family 2. Family 2 can also be obtained by ring equivalent replacement of molecule IV.

3. From molecule IV, using diequivalent isosteric replacement, V and VI have been derived contributing family 3. Molecule VI on further homologation gave VII and hence contributing for the formation of family 3.

In conclusion, all the families are derived using structure based ligand designing by using medicinal chemistry principles as described above.

And so, these designed molecules are taken to the next level i.e. docking studies.

All the ligands docked in this current study are modelled using same protocol as described in the appendix.

C. Docking The designed ligands have been forwarded to a in silico interaction studies using docking procedure. This part of the study has been done with the help of ‘Molecular modellization’ team representing Mr. Lionel Nauton and Dr. Vincent Thery. This doking has been performed using the energy minimized receptor model and designed ligands. The procedure followed has been briefed in the appendix.

At the end of the docking simulation, all obtained results were combined in a single data file, in order to make statistical studies on obtained scores. Telcagepant® and Olcegepant® were also included in the docked ligands as they serve as markers or references. These docked conformers are consistent with the crystallographic structures which we retrieved previously from PDB (Figure 46).

Figure 46.showing the Olcegepant® and Telcagepant® in the docked site Superposition of the crystal structure of Telcagepant® (the one available in PDB) with the result of docking;

Purple: Telcagepant® conformation in crystal structure retrieved through PDB.

Blue: Telcagepant® conformation obtained in the course of docking.

The docking results were regrouped into various zones and further classified into different family of ligands in the sarmap as shown below (Figure 47). Further the results were sorted with the help of the module ‘MDB’ in sybylx2.0.

Figure 47.showing the SARmap graph of potential hits & different zones with lead as reference Zone 1: This region corresponds to low electrostatic interactions between the ligand and the receptor. The hits found in this zone were expected to have least interaction with the pocket and consequently in the receptor.

Zone2: This area depicts a very strong or too many electrostatic interactions. Most potent CGRP antagonist, Olcegepant® lies here. Hence hits from this region can be interesting when it comes to considering electrostatic interaction scores alone.

Zone 3: This area can’t be ignored either as the previous two. We have found the grip (good placement in the pocket) as we wanted. We can consider the hits in this zone as well.

Zone 4: This zone corresponds to the hits with desired interactions as well as total scores and we also found Telcagepant® in this region. After this initial screening, and after checking remaining solutions and applying different filters, we shortened the hits from 2225 to 36 possibilities with complete disappearance of several compounds of different families.

D. Identification and classification of potential hits

The docking results were further scrutinized using various filters (Figure 48). The most probable bioactive forms were hypothesised by the above discussed factors like electrostatic interactions, crash and strain (grouped together as ‘Total score’).

Figure 48.showing the filers and descriptors applied to identify potential hits Alongside total score, the final prioritized list of ‘36 potential hits’ was selected by fixing a benchmark of total score to be 10.5(score of Telcagepant® is 10.8). These 36 probable hits can be classified into 3 families based on their structures.

The total scores of three families are as follows:

O O N X N NH 4 N 6 2 O R4 N N R1

R2 Figure 49.showing the general structure of family 1

Table 11.showing the total scores of family 1 potential hits

Family-1 (Figure 49) docking scores along with their substituants and configurations in different chiral positions (Table 11) suggest the equality of both benzyl and trifluoro ethyl substituants for interactions in receptor.

O O N O N NH 4 N 6 2 N N N N

Figure 50.showing the general structure of family 2

Table 12.showing the total scores of family 2 potential hits Family-2 (Figure 50) docking scores along with their configurations in different chiral positions (Table 12) shows their receptor interactions are as strong as family-1.

Family 3

O O N X N NH 4 N 6 2 R4 N

Figure 51.showing the general structure of family 3

Table 13.showing the total scores of family 3 potential hits

Family-3 (Figure 51) docking scores along with their substituants and configurations in different chiral positions (Table 13) gives an observation over the chirality as well as importance of substitutions at position ‘Y’ for new electrostatic interactions.

Thus the above docking results paved a rationale for identifying the potential hits for the CGRP antagonists. And in the next subunits, we can have a brief over view on some more aspects like pocket volume importance, drug-likeliness etc.

E. Receptor pocket volume Vs ligand volumes

During the preliminary stages of this chapter, we have studied the binding pocket along with their volumes. Thus the volume of the pocket suggests that mere ‘flat and small molecules’ are not sufficient for a proper drug-receptor interaction. Instead, the ligands have to be designed by considering the volume (to be precise, size) and amino acids in the vicinity of the pocket. Along with the volume aspect, we also have to note down the point that incorporating chiral carbons wherever required will increase the chances of effortless reaching of the substituants of the ligand towards the amino acids of the receptor. As we already saw the volume of the binding pockets, now let’s see the volume of the ligands which we have considered as probable hits (Table 14).

RAMP-1; Top 4 binding site volumes are 1468.5, 434.9, 394.8, 300.3 Å3

CALCRL; Top 4 binding site volumes are 674.1, 498.2, 288.9 and 207.5Å3

Exemplified ligand in CGRP interacting RAMP-1interacting CALCLR the binding site ligand volume v (Å3) ligand volume v (Å3) interacting ligand volume v(Å3)

O O NH N N HN N 474.13 267.47

O O NH N N HN N 477.55 270.89

O O NH N N HN N 519.55 312.89

N N

O 198.81 O NH N N HN N 530.51 323.86 O N N

O O NH N N HN N 540.98 334.32 O N H N

O O NH N N HN N 490.40 283.75 O N N F F F

O O NH N N HN N 500.86 294.21 O N H N F F F

Table 14.Showing the volume of potential hits in comparison with the druggable & tractable sites of CGRP receptor [28] The pocket volume for CALCRL interacting fragment (198.81) vs pocket of CALCRL (674.1) is constant for all the molecules and hence the interacting hit’s volumes inside the pocket pose no issues. On the other hand, the fragment of our hits interacting with RAMP-1 is relatively big when compared with their counterparts interacting with CALCRL. The RAMP- 1 volume (1468.5) can play crucial role as we saw in the previous chapters about the importance of RAMP interaction with hits for a potent activity. And thus a bigger in volume fragments is designed for a better interaction.

With this, the molecular studies on the receptor and their ligands can be concluded. There is one more aspect called drug-likeliness of molecules which play important part in pharmacokinetics which will be discussed in the next subset.

F. Drug-likeliness properties:

Drug-likeliness properties are ideal characteristics of a molecule to be qualified as a drug entity. Prof. Lipinski postulated a group of rules to quantify and validate the drug- likeliness of a drug. Drugs satisfying these rules posses optimized pharmacokinetic properties. These rules are called Lipinski’s rule of 5 [29].

Lipinski’s rule of 5

According to Lipinski’s rule of 5, an ideal drug should have following characters

 A molecular weight (MW) of not more than 500 daltons.

 Partition coefficient LogP not more than 5.

 No more than 10 hydrogen bond acceptors (HBA) like N, O.

 No more than 5 hydrogen bond donors (HBD) like NH, OH.

Note than even though there are only 4 rules, Lipinski’s rule of 5 was named so because all the descriptors are multiples of 5.

Hence the drug-likeliness of the designed molecules in general is shown in Table 15.

Ligand MW HBA HBD Log PSA vol stereocentres score 2 3 P (A ) (A )

O O O NH N N F3C N NH N F 566.21 4 2 3.69 79.31 533.94 2 1.47

Telcagepant®

O O NH N N HN N 579.30 4 2 4.48 80.40 591.89 3 1.40 O N N

O O NH N N HN N 571.25 4 2 3.78 80.67 555.25 3 1.23 O N N F F F

O O NH N N HN N 581.31 5 3 3.41 90.21 600.21 3 1.50 O N H N

O O NH N N HN N 573.27 5 3 2.70 90.48 563.57 3 1.45 O N H N F F F

O O NH N N HN N 523.27 3 3 4.94 75.20 521.62 3 1.12

O O NH N N HN N 524.25 4 2 5.80 70.93 530.09 3 0.99

N O

O O NH N N HN N 567.30 4 3 4.45 82.07 574.21 3 1.31

Table 15.showing the drug-likeliness of potential hits generated using Molsoft® The potential hits studied for drug-likeliness properties by Molsoft® showed that most of the molecules complies the Lipinski’s rule of 5. Some observations noted from the results are as follows;

 Molecular weights of the potential hits have slightly surpassed the proposed limit but comparing with the lead molecule Telcagepant®, they are in concordance.

 Hydrogen bond acceptors and donors of all the molecules are within the range of rule of 5.

 Partition coefficient LogP is a determining factor for both absorption and distribution of the drug molecule. If they are more than 5, they are hydrophobic and won’t be completely soluble in body system and on the other hand, way less than 5 (around 0-1) results in hydrophilic molecules posing the same problem of solubility and bioavailability. All the hits designed showed optimized LogP values around 3-5.

 Polar surface area shows the sum of surface volume of all the polar atoms of the molecule. PSA value less than 90 is required to cross Blood-Brain-Barrier when they want to act as CNS drugs. All the designed hits are well within the range of rule of 5.

Along with that, the Molsoft® online tool gives a score for each molecule submitted. These are called model score. They provide a graph taking into consideration 5000 of marketed drugs from World Drug Index (positives) and 10000 of carefully selected non-drug compounds (negatives).

Figure 52.showing the plot using drug-likeliness model score The Gaussian distribution curve of drug vs non-drugs was plotted using the drug- likeliness model scores. The blue plot has drug-like molecules and green plot gives non- drugs. The plot generated using our hit molecules showed that all of them are actually distributed in the mean area of the drug-like blue plot. And other point to be noted, these molecules are having a model score quite close to the lead molecule Telcagepant®.

And so on, the potential hits are having drug-likeliness as well as their scores are almost same as that of Telcagepant®. Hence all the hits are hypothesized to have good pharmacokinetic properties.

In conclusion, potential hits have been designed using structure based ligand designing and later validated for their interactions using docking simulation as well as all other possible Insilco validation methods such as pocket vs ligand volumes, drug-likeliness properties etc. finally providing a list of 36 potential Calcitonin Gene Related Peptide antagonists.

Thus obtained hits have to be synthesized to validate the hypothesized concept in living systems, which brings us to the next part of drug discovery process i.e. designing of synthetic methodology for the potential hits.

III. RETROSYNTHETIC DESIGN FOR SYNTHESIS OF HITS

The potential hits obtained through molecular modeling and docking studies have been initially scrutinized to propose a generalized retrosynthetic design. The following design seems to be legit of all to achieve the desired moiety.

Potential dual active molecules

O O O O O O N N N N X N NH X N NH X NH

N N N O N R4 N R4 N N R4 N N N R1 Y 1 2 R 2 R2 R R

Family 1 Family 2 Family 3

Convergent synthesis

O O

N 4 + HN NH R N Z CALCRL fragment RAMP-1 fragment Y N R2

O Ph Ph O O OH O N O HO 4 3 NH O R OMe R N R 2 4 R4 OMe R6 Aspartic Acid D or L Choice of the Core piperidine moiety stereochemistry R or S Scheme 1.showing a generalized retrosynthetic design

The prototypical design proposes that we can reach the potential hit starting from two different sources, either aspartic acid or methyl crotonate depending on the family to be synthesized. The core piperidine moieties can be achieved using a Aza-Michael intramolecular reaction to give a piperidine ring with desired configuration and substituants. Finally, a convergent synthesis can be followed to couple with the privileged structure in order to accomplish the potential dual active molecules.

Thus, in the next chapter, we will have an in-depth analysis and investigate about the piperidine synthesis proposed by different research groups and retrieve some of the available methods and optimize according to our desire to synthesize dual active molecules.

Take-home message from this chapter:

In the current chapter we tried to cross-examine all possible aspects of CGRP receptor ranging from basic information on the association of different proteins to form the receptor to more complex Druggability, Tractability and Drug-receptor interaction studies. With the aid of bioinformatics, we have put forth an outcome which proves to be more feasible to achieve the CGRP antagonists with increased affinity and potency. We have placed up front some key amino acids which are impeccable for an optimal drug-receptor interaction as well as crucial for species & receptor selectivity of ligands. Hence all these descriptors can be clubbed and publicized as the ‘hotspots’ and consequently showing how to proceed in targeting these ‘hotspots’ will be viable for providing an optimal drug interaction in CGRP receptor.

Along with the study of the receptor, molecular modeling studies have also been conducted to put forth a database 2225 molecules which were further engaged in docking simulation. Finally the prioritized list of molecules has been selected based on the benchmark i.e. score more than the ‘lead’ molecule Telcagepant®. These filters finally provided us with 36 ‘potential hits’ which can be classified into three structural families.

From thereon, we saw some more important aspects such as pocket vs ligand volumes, drug-likeliness properties etc. through which the concept for designing the hits have been validated.

In final units we saw the retro synthetic design of synthetic methodology for the potential hits in general. Thus forwarding our drug discovery process to the next aspect i.e. ‘chemistry as well as synthesis of the hits’.

REFERENCES 1. Ter Haar E., Koth CM., Abdul-Manan N., Swenson L., Coll J.T., Lippke J.A., Moore J.M., Structure, 2010, 18, 1083–1093. 2. McLatchie L.M., Fraser N.J., Main M.J., Wise A., Brown J., Thompson N., Solari R., Lee M.G., Foord S.M., Nature, 1998, 393, 333-339. 3. Kunso S., Kukimoto-Niino M., Akasaka R., Toyama M., Terada T., Shirouzu M., Shindo T., Yokoyama S., Prot. Sci., 2008, 17, 1907-1914. 4. Vecchia D., Pietrobon D., Trends Neurosci., 2012; 35, 507–520. 5. Delafoy L., Gelot A., Ardid D., Eschalier A., Bertrand C., Doherty AM., Diop, L., Gut 2005, 55, 940–945. 6. Snider R.H., Nylen E.S., Becker K.L., J. Investig. Med., 1997, 45, 552-560. 7. Schelstraete C., Paemeleire K., Acta Neurol. Belg., 2009, 109, 252–261. 8. Steven M.F., Roger K.C., Eur. J. Biochem., 1988, 170, 373-379. 9. Skofitsch G., Jacobowitz D.M., Peptides, 1985, 6, 975-986. 10. Armour S.L., Foord S.M., Kenakin T., Chen W.J., J. Pharmacol. Toxicol. Methods, 1999, 42, 217-224. 11. Champion H.C., Santiago J.A., Murphy W.A., Coy D.H., Kadowitz P.J., Am. J. Physiol., 1997, 272, 234-242. 12. Kamitani S., Asakawa M., Shimekake Y., Kuwasako K., Nakahara K., Sakata T. FEBS Lett., 1999, 448, 111-114. 13. Jensen L.J., Kuhn M., Stark M., Chaffron S., Creevey C., Muller J., Doerks T., Julien P., Roth A., Simonovic M., Bork P., Von Mering C., Nucleic Acids Res., 2009, 37, 412-416. 14. Hajduk P.J., Huth J.R., Tse C., Drug Discov. Today, 2005, 10, 1675-1682. 15. Joanna O., Nat. Rev. Drug. Discov., 2007, 6, 187-191. 16. Stockwell B., Outsmarting Cancer: why it’s so tough. 20th September 2011. http://www.scientificamerican.com/article/outsmarting-cancer. Last accessed on 17th January, 2015. 17. Buchwald P., IUBMB Life, 2010, 62, 724-731. 18. ChEMBL: a large-scale bioactivity database for drug discovery"" Nucleic Acids Research., 2011, 40, D1100-D1107. 19. Ter Haar E., Koth C.M., Abdul-Manan N., Swenson L., Coll J.T., Lippke J.A., Moore J.M., Structure, 2010, 18,1083–1093. PDB ID:3N7S 20. Leahy D.K., Desai L.D., Deshpande R.P., Mariadass V.A., Org. Process Res. Dev., 2012, 16, 244-249.

21. Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015. 22. Doods H., Hallermayer G., Wu D., Entzeroth M., Rudolf K., Engel W., Eberlein, W., Brit. J. Pharmacol., 2000, 129, 420-423. 23. Salvatore C.A., Mallee J.J., Bell I.M., Zartman C.B., Williams T.M., Koblan K.S., Kane S.A., Biochemistry, 2006, 45, 1881-1887. 24. Hay D.L., Christopoulos G., Christopoulos A., Sexton P.M., Mol. Pharmacol., 2006, 40, 1984-1991. 25. Hay D.L., Howitt S.G., Conner A.C., Schindler M., Smith D.M., Poyner D.R., Brit. J. Pharmacol., 2003, 140, 477-486. 26. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart., Acta Cryst., 2010, D66, 213-221. 27. SYBYL-X 1.2, Tripos International, 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA. 28. Molinspiration Cheminformatics 2015; Nova ulica, SK-900 26, Slovensky Grob, Slovak Republic. Retrived from www.molinspiration.com/cgi-bin/properties on 28th July 2015. 29. Lipinski C.A., Drug Discovery Today, 2004, 1, 337–341.

CHAPTER 3: RESULTS AND DISCUSSION

I. LITERATURE INVESTIGATION

The piperidine moiety is a structural unit which is most common in skeleton of numerous alkaloids and biologically active molecules [1]. The key objective of many groups working on this skeleton includes modulation of the stereoselectivity of the skeleton [2]. Keeping in mind the large number of applied methodologies, we have cited in this chapter some of the most recent work in the synthesis of piperidines and in particular with the substitutions at 2, 4 and 6 positions with principle lead being the alkaloid (+)-241D, a compound isolated from an American frog Dendrobates speciosus [3] which showed potent inhibition of nicotinic acetylcholine receptor [4] and 4-hydroxy pipecolic acid which has been also obtained from leaves of Calliantra pittieri and Strophantus scandeus[5]. (2S,4R)-4- hydroxy pipecolic acid and 6-substituted analogs are also the core of biologically important compouds as, in particular the Palinavir structure an HIV-protease inhibitor [6].

OH OH O H CONHt-Bu N N N H N O OH N C9H19 N CO2H H H O N (+)-241D alkaloid (2S,4R)-4-hydroxy pipecolic acid Palinavir

Figure 53.Showing (+)-241D alkaloid, (2S,4R)4-hydroxy pipecolic acid and Palinavir Due to their different structures and consequently to their synthetic approaches ( the amino acid functionality), this chapter is divided in two parts : the former is devoted to 2,6- disubstituted 4-hydroxy piperidines and the second to 4-hydroxy pipecolic acid derivatives.

A. Synthesis of 2, 6-disubstituted-4-hydroxy piperidines

1. Synthesis by using metal catalysis

Majority of the methods followed for the formation of piperidine skeleton involves an intramolecular cyclization principle to form a C-N bond. This step determines and accomplish with the maximum stereoselectivity inclusion in the resulted piperidine ring with substitution at 2 and 6 positions. In the current chapter, we will deal with the methodologies and developments concerning the synthesis of 2, 4 and 6 substituted piperidines.

1.1 Application of Iridium complex:

The method developed by Helmchen and coll. [7] allows to access (+)-241D alkaloid relatively in limited steps compared with other methodologies developed in the recent times.

OH Boc Boc NH NH OH TFA OCO Me 2 NH2 CH2Cl2 OCO2Me 1 2 E/Z=9/1 3

OH (S,S,aS)-L

96% ee Ar N O H 4 [Ir(cod)cl]2 L = P N 3 O Ar OH

92% ee Ar=2-MeOC6H4 N H (R,R,aR)-L 5

Scheme 2.Showing the formation of piperidine using chiral ligand L

Starting from the key intermediate 3 which has retained the stereoselectivity from N- protected amine 1, a metal catalysed allylic cyclization was done in presence of Iridium complex and then a chiral ligand L helps in accessing piperidines 4 and 5 with desired chiralities. The chirality of the piperidines thus formed depends on the chirality of the ligand L used during the reaction (scheme 1).

OH OAc OH

1. H2,Rh/C

N N C9H19 2. 0.5M NaOH N C9H19 H Cbz H 4 (+)-241D alkaloid Scheme 3.Showing the formation of (+)-241D alkaloid Thus obtained piperidine 4 was transformed into (+)-241D alkaloid using optimized conditions like protection of amine & hydroxyl functionalities followed by homologation using metathesis with desired carbon chain, reduction and finally a saponification to give the desired compound (scheme 2). The same strategy has been applied for piperidine 5 to give (+)-6-epi 241D.

1.2 Application of Gold complex:

Synthesis of (+)-241D alkaloid has also been described using an intramolecular cyclization reaction catalysed by gold complex [8].

OH OH OH OH O nBuLi N ClAuPPh3 1. HCl C9H19 NHBoc Undecyne NHBoc N C9H19 2. H Pd/C N C9H19 AgSbF6 Boc 2, H 6 7 83% 8 66% (+)-241D

Scheme 4.Showing the synthesis of (+)-241D alkaloid using gold complex This involves an alkylation of Weinreb’s amide 6 in presence of butyllithium and undecyne giving an adduct 7. Finally, the cyclization occurs in presence of coordinated chlorine complexed with gold/triphenylphosphine associated with silver to obtain the cyclic enamine compound 8. Lastly, washing with acidic medium followed by reduction gives alkaloid (+)-241D.

Another synthetic method using gold complex for the synthesis of (+)-241D alkaloid has been recently described in the literature [9]. The first chiral centre has been introduced by alkylation of sulfinimine [10] to give compound 9 followed by deprotection of amine and later acylating using acid chloride gives corresponding amide 10.

O SOR" NH Acid 2 R'COCl S N  N R'' MgBr  + R R R 9

R' R' R' R' Y Y [Au(PPh3)NTf2] HN O N O reduction N O N O Y-H R CH2Cl2, MsOH R R R

10 11 12

R' R' Y Y N Reduction N

R O Hydrolysis R OH 13 14

Scheme 5.Showing the synthesis of (+)-241D alkaloid using gold complex In presence of gold complex, an intermediate 11 has been derived using cyclization which was later selectively reduced to give α-amino ether 12 which spontaneously rearrange to give 4-piperidone 13 using the Petasis-Ferrier type mechanism [11]. Compound 14 was

99 obtained from 13 using simple reduction. Based on the reducing agent used, they were able to modulate the stereochemistry at position 4.

An example to investigate the efficacy of this method has been applied to synthesize a natural compound as depicted below.

iPr iPr HN 1. [Au(PPh )NTF ], CH Cl , MsOH O 3 2 2 2 HN Ph de (25/1) 2. Catecholborane Ph OH

Scheme 6.Showing the application of gold complex for another example 1.3 Miscellaneous metal complex:

The intramolecular reaction has also been studied using various alcohols and derivatives of allylic alcohols to synthesize stereoselective piperidines using other metal complexes like palladium [12], or Iron (III) chloride hexahydrate [13] etc.

2. Synthesis by intramolecular reductive amination

The formation of piperidine cycle through reductive amination remains one of the most exploited methods for the reason being impeccable control over the stereochemistry of the retrieved piperidines.

 Chandrasekhar and coll. [14] used this method starting from homoallyl amine 15 which has been obtained by using decanal and valine as the starting materials.

Cbz CO2Me NHCbz NH H OsO4 Decanal + NH 2 C9H19 C9H19 O NaIO4 15 16

Cbz Cbz D-proline NH OH O NH OH O +

Acetone C9H19 C9H19 17a 17b

OH OH

H , Pd/C H2, Pd/C 2 17b 17a 95% C H N 95% C9H19 N 9 19 H H (-)-241D (-)-epi 241D Scheme 7.Showing the application of reductive amination by Chandrasekhar and coll.

100

The compound 15 was transformed into amino aldehyde 16, which being unstable reacts spontaneously with D-proline through aldol condensation to give a ketone 17a and 17b with an overall yield of 56%.

The deprotection of the amine as well as intramolecular cyclization through reductive amination was carried as a two-step & one pot synthesis by using palladium on carbon as catalyst. This method, when applied to 17a gave (-)-241D alkaloid and 17b gave 4-epi-(-) isomer.

 Das and coll. [15] used a method which was mostly similar to that of the Chandrasekhar and coll., except varying the chirality on the final molecule.

O O S S N In, THF HN Decanal + H2N S C9H19 H C9H19 d.r : 96/4 O Br (S)-tert-butane sulfinamide 18 19

1. 4M HCl Cbz Cbz NH RuCl3 NH O

2. CbzCl, Na2CO3 NaIO4 C9H19 C9H19 H 20 21

Cbz Cbz Acetone NH OH O NH OH O + NaOH C9H19 C9H19

22a 22b

OH OH

H2, Pd/C H2, Pd/C 22b 22a C H N C9H19 N 9 19 H H (+)-241D (+)-epi-241D Scheme 8.Showing the application of reductive amination by Das and coll. In this case, decanal was treated with (S)-tert-butanesulfinamide for the synthesis of imine 18 which was later alkylated using allyl bromide in presence of Indium which gave the amine 19 with high stereoselectivity (96/4). For feasibility during the later stages of the reaction, protecting group was being replaced to give homoallyl amine 20. The oxidation of 20 to 21 was done using ruthenium chloride and aldol reaction was done in the presence of acetone and sodium hydroxide to recover a mixture of 22a and 22b in 1/1 and was finally cyclized according to the procedure cited above to give (+)-241D and 4-epi-(+)-241D alkaloids.

101

 Following the same strategy, Rao and coll. [16] described a stereoselective synthesis of alkaloid (+)-241D. This method is almost of same which was described above and the only difference here is the formation of the first asymmetric centre

Starting from the decanal, an enantiomeric allylation has been carried with complex of titanium and (S,S) BINOL and in presence of allyltributyltin [17] which gave homoallylic alcohol 23 in an enantiomeric excess of 98%.

TiCl (S,S)-BINOL 4, OH N3 Decanal 1. TsCl, pyridine C H C9H19 Ag2O, allyltributyltin 9 19 Br 23 24 d.r : 96/4 e.e : 98%

1. LiAlH4/THF Cbz Cbz Cbz NH AD-mix-beta NH OH NH OH OH + OH 2. CbzCl, Na2CO3 C9H19 tBuOH-H2O C9H19 C9H19 25 26 80% 27 20%

Cbz Cbz 1. NaH/THF, 0 °C, TsCl NH O NH OH 26 C H C H 2. M g B r ,CuI 9 19 9 19 28

Cbz OH NH OH O PdCl2, CuCl H2, Pd/C C9H19 O2 C9H19 N 22b H (+)-241D Scheme 9.Showing the application of reductive amination by Rao and coll. This was followed by activation of alcohol functionality of 23 with tosylate and later a nucleophilic substitution reaction using sodium azide to give compound 24 which was further reduced with lithium aluminium hydride. Thus formed amine was protected with benzyl carbamate to give 25. An asymmetric dihydroxylation of alkene was carried out using the protocol proposed by K.B. Sharpless [18] which gave diols 26 and 27 in 80/20 ratio. The major compound 26 was further treated with sodium hydride in presence of tosyl chloride at 0 °C to give an epoxide which was immediately engaged to react with vinyl cuprate to obtain homoallylic alcohol 28. The oxidation of 28 according to the protocol developed by Wacker gave 22b and this act as a substrate for reductive amination to give the alkaloid (+)-241D.

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3. Synthesis by intramolecular nucleophilic displacement

An alternative and frequently used way to synthesize piperidine cycle consists of an intramolecular cyclization of an amine with a potent leaving group (SN2).

3.1 Activation in the form of mesylate or tosylate

 Toyooka and coll. [19] described the synthesis of polyhydroxypiperidines as potential inhibitors of α-L- fucosidase. In this synthesis, the chirality appears from using the L-alanine and branching of the different carbon chains with asymmetric centres has been studied.

OMOM BocHN OMOM 1. MsCl, Et3N MOMO OMOM H2N CO2H Oxidation OMOM MOMO 2. NaH, DMF N anodic L-alanine Boc MeOH OH 29

OMOM OMOM OH MOMO OMOM TMS MOMO OMOM Cl Cl HO OH

MeO N BF ·OEt N N 3 2 H Boc Boc Cl 30 31 32

Scheme 10.Showing the application by Toyooka and coll. An anodic oxidation of the compound 29 gives 30 which was later reacted with allyltrimethylsilane to give 31. The double bond was later functionalized to obtain α-L- fucosidase inhibitor, 32 (IC50 = 0.0005µM, Ki = 0.0011 µM).

 A.B.Smith III and coll. [20] has described the synthesis of 2, 4, 6 trisubstituted piperidines by using an anion transferring via rearrangement reaction.

1. tBuLi, tBuOK O OTBS S S S S S S S S R THF, -78 °C R1 TBS 1 R1 H S 33 2. 35 36 S S S O  TBS 34

TsN  R OTBS S S 2 S S  37 1. TBAF R1 S   S S  2. MsCl, TEA R2 N R1 TsHN S 3. NaH/THF Ts R2 38 39 Scheme 11.Showing the application by A.B.Smith and coll.

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The anion derived from dithiane 33 reacts with the epoxide 34 to give an alcoholate 35 which further undergoes a Brook rearrangement [21] to give 36. This intermediate 36 further reacts with aziridine 37 to form the compound 38. A cascade of reaction succeeds starting from deprotection of alcohol followed by an activation of the alcohol by mesylate and later a cycliclization in basic medium to retrieve 2, 4, 6 trisubstituted piperidine 39.

According to the configuration required, change of epoxide and aziridine results in modulation of configurations at 2 and 6 positions as described in the following table.

compound R1 R2 Resultant molecule Configuration Percentage

38 (S,S) 39 (R,S) 2, 6 cis 87

38 (R,R) Methyl Benzyl 39 (S,R) 2, 6 cis 85

38 (S,R) 39 (R,R) 2, 6 trans 55

38 (R,S) 39 (S,S) 2, 6 trans 58

Table 16.Showing the effect of configuration modulation from 38 to 39 According to the cited experimental conditions, in both 2, 6 cis piperidine and trans, the selective deprotection of one or the other or two dithiane groups are possible (scheme 11).

S S O OH NCS O L-selectride O   S S Ph Ph   Ph   N AgNO3 N N Ts Ts Ts

Scheme 12.Showing the inoculation of 3rd asymmetric centre The choice of the reducing agent permits the formation of an alcohol with desired configuration and thus introducing another asymmetric carbon in the final molecule.

3.2 Activation through a Mitsunobu reaction

 Krische and coll. [22] has described recently the access of stereoselective 2, 4, 5 trisubstituted piperidines (scheme 12). The ultimate step for final cyclization uses the classic Mitsunobu reaction [23].

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OH OH Iridium complex OH OH DIAD, PPh3 OH CH2Cl2 N NHNs Ns 40 TsN 62% 41 42 d.r : 4/1 Ns = p- nitrophenylsulfonyl

Scheme 13.Showing the synthesis of 2, 4, 5 trisubstituted piperidines

An example stated above shows the diol 40 reacting with vinyl aziridine in presence of Iridium complex and a chiral ligand to give 41. The compound 41 thus obtained has good stereoselectivity and finally was cyclized in presence of DIAD and triphenylphosphine to form compound 42.

4. Miscellaneous methods

4.1 Asymmetric hetero Diels-Alder reactions

This reaction accounts as a method being exploited the most because of its control over regio and steroselectivities. This also allows for an amplification to give polysubstituted piperidine cycles. It is a well known fact that the imines are reactive compounds involved in Diels Alder reaction carried in presence of dienes like Danishefsky's diene(for example) [24].

Lewis or OTMS Bronsted O EWG acid N +  MeO R2 N R2 EWG

Scheme 14.Showing the synthesis of piperidine moiety using Diels Alder reaction On the other hand, this reaction had proved to be unpredictable and depends a great deal on the engaged substrates. The inoculation of a chiral centre in the final product is interesting if we can control the absolute configuration which can be done via the transition state of the reaction by using chiral inducer.

The selected way by He and coll. [25] consists of using a cyclic sulfonylimine in presence of a chiral inducer which was derived from natural Cinchona alkaloids.

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OMe O O F O O R2 S S H O N O O N CO H + 2 O NH2 R2 chiral catalyst N

dr >19/1 chiral catalyst ee >90%

Scheme 15.Showing the application of Diels Alder reaction The overall yield of the reaction (>65%) was good with an excellent diastereomeric ratio and enantiomeric excess. Thus final steps include deprotection of the nitrogen atom, stereoselective reduction of ketone to alcohol which allows the access of 2, 4, 6 substituted and chiral piperidine.

4.2 Application of nitrones

Nitrones are the synthetic intermediates which can access heterocycles with nitrogen. 1, 3 dipolar cycloaddition of nitrones on alkenes permits for the formation of the isoxazolidines and also leads for formation of many other heterocyclic derivatives.

However, this method is limited by a weak diastereoselectivity owing to the nature of utilized alkene during the reaction.

Chattopadhyay and coll. [26] has used the methodology first described by Merino and coll.[27 ] for synthesizing two enantiomers of the alkaloid 241D. The reaction of (R)-2,3-O- cyclohexylidene glyceraldehyde with allylamine gave a corresponding imine 43 which later was alkylated using organozinc and organomagnesium to give 44 and 45 respectively.

MgBr Allylamine H202 O O O O O O O O O O MgSO4 44: 9%; 45: 74% Na2WO4 Toluene O O N NH 86% N reflux 43 O N H 49 ZnBr 45 47 (R)-2,3-0- cyclohexylidene 44: 66%; 45: 15% glyceraldehyde

H202 O O O O O O Na WO Toluene O NH 2 4 84% N reflux N O 44 46 48

Scheme 16.Showing application of dipolar cycloaddition reaction by Chattopadhyay and coll. On subsequent oxidation of 44 and 45 with the help of hydrogen peroxide gave the nitrenes 46 and 47 with a good yield. The key step i.e. 1, 3 dipolar cycloaddition of nitrenes

106 was carried in toluene reflux to yield respective isoxazolidines (2,6-syn-disubstituted-1-aza-7- oxabicyclo[2,2,1]heptane [28]) 48 and 49.

O OH Zn 48 N C H 9 19 AcOH N C9H19 50 92% H (-) 241 D

OH O Zn 49 N C H 9 19 AcOH C9H19 N H 51 92% (+) 241 D

Scheme 17.Showing the application to yield (+)- and (-)-241D alkaloid Thus obtained 48 has been transformed into 50 which on subsequent ring opening reaction using zinc in acid medium gave (2S, 4R, 6R)-(-)-241D alkaloid.

In a pretty similar way, 49 was used to obtain (2R, 4S, 6S)-(+)-241D alkaloid through intermediate 51.

4.3 Application of Aza-Michael type intramolecular cyclization

Intramolecular cyclization reaction between an amine and an α,β unsaturated ketone (Aza-Michael) is also a method of choice for the synthesis of piperidines. This particular methodology is mostly used to synthesize recemic mixture and few also describe to obtain them with stereoselectivity. Recently enantioselective synthesis using organocatalysts has been described [29] but the reported reactions mostly used the intramolecular reaction. This intramolecular reaction has been exploited a great number [30] and in this sub-section, we try to put forth a few of them.

 Hong and coll. has described the preparation of (+)-myrtine and the (-)-epimyrtine starting from a precursor with an asymmetric centre in α-postition to the nitrogen [31].

Starting from an allylic alcohol 52, an oxidation with manganese dioxide gave the α,β unsaturated aldehyde 53. The Aza-Michael reaction was then carried in presence of a chiral inducer. The configuration of the inducer determines the absolute configuration on the final molecule giving 2, 6 cis (54) or 2, 6 trans (55).

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(R)-I* S S Benzoic acid H dr: 11/1 BnO 92% N O S S S S Ts 54 MnO H (Z) 2 BnO BnO NH OH CH2Cl2 NH O H Ts Ts S S 52 53 (S)-I* H BnO dr: 3/1 N O Benzoic acid Ts 92% 55 Ph I* = N Ph H OTMS

Scheme 18.Showing the application of Aza-Michael cyclization by Hong and coll. This reaction was applied for the synthesis of (-)-epimyrtine 56 and (+)-myrtine 57 starting from 54 and 55 respectively.

O S S H N N O Ts 54 56 (-)epimyrtine

S S H N N O Ts 55 57 (+)myrtine

Scheme 19.Showing the synthesis of (-)-epimyrtine 56 and (+)-myrtine 57. Del Pozo and coll. has described the synthesis of (+)-myrtine 57 by using same methodology as above.

Ar Ar N OTMS CHO H MgBr H CHO N N N OH Boc THF PhCO2H Boc Boc 58 59 60 Ar: 3,5-(CF3)2-C6H3

O Dess-Martin Et3N 1. TFA N O N O N CH2Cl2 2. K2CO3 Boc Boc 61 62 57 Scheme 20.Showing the synthesis of (+)-myrtine by Del Pozo and coll.

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Commencing from aldehyde 58 in presence of benzoic acid and a chiral catalyst gave the piperidine 59 (63% yield and 94% ee) which on further reaction with allyl magnesium bromide gave the alcohol 60. Compound 60 was oxidized using Dess-Martin reagent to give the ketone 61 which on reconjugation gave 62. After the déprotection of protecting group in acidic condition, a new cyclization was carried out using Michael reaction to give (+)-myrtine 57 [32].

 Krishna and coll. [33] has described synthesis of (−)-andrachcinidine through Aza- Michael cyclization. In this case, the absolute configuration on asymmetric centres were controlled during the construction of the intermediate 63 which later was cyclised without any catalyst to give exclusively 2, 6 cis piperidine 64.

Bn Cbz O HN O 1. TFA, iPrOH OH O

N 2. H2,Pd/C H 63 64

Scheme 21.Showing the synthesis of (−)-andrachcinidine by Krishna and coll. Akiyama and coll. [34] has described that the Aza-Michael reaction can also be carried out without the protection of the amine. But using the chiral catalyst however is necessary to inoculate the asymmetric centres. In this case, they used a phosphoric acid derivative as the catalyst. The solvent used in this reaction proved to be crucial. Thus, the dihydro 2, 3-quinol- 4-one 66 was prepared from the precursor 65.

C6F5 O O

Ph Catalyst-R* O O 93% ee R= P OH NH2 C6H6/C6H12 (1/1) N Ph O H 65 95% 66 C6F5

Scheme 22.Showing the application of Aza-Michael by Akiyama and coll.  Troin and coll. has also used Aza-Michael intramolecular reaction to synthesize substituted piperidines starting from compound like 67. In presence of ethylene glycol, para- toluene sulfonic acid and trimethylorthoformate, this reaction gave derivatives of 2, 6 trans in majority 68 [35].

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O O O O Cbz (CH OH) NH O 2 2 + Ph pTSOH Ph N Ph N CH(OCH3)3 Cbz Cbz

67 68 69 68 / 69 : 86 / 14

68 Ph N H

(-)-(2R,6S)-2-methyl-6-phenylpiperidine

Scheme 23.Showing the application of Aza-Michael by Troin and coll. A systemic study on the parameters affecting the formation of major configuration was also conducted. This study has notable showed the importance of alcohol used during the reaction as shown in the below examples [36]

MeO OMe MeO OMe Cbz NH O MeOH + C H 9 19 pTSOH N C9H19 N C9H19 CH(OCH3)3 Cbz Cbz

70 71 72 71 / 72 : 15 / 85 OH

72 N C9H19 H

(+)-241D

O O O O Cbz NH O (CH OH) 2 2 + C H 11 23 pTSOH N C11H23 N C11H23 CH(OCH3)3 Cbz Cbz

73 74 75 74 / 75 : 82 / 18

74 N C11H23 H

(-)-solenopsine A

Scheme 24.Showing the application of Aza-Michael by Troin and coll. In the first case, the cyclization of compound 70 in presence of methanol gave 2, 6 cis 72 isomer as major compound which was later transformed into (+)-241D alkaloid. Whereas

110 in the second case, the cyclization of compound 73 driven in presence of ethylene glycol gave 2, 6 trans 74 in majority which subsequently was transformed into (-)-solenopsine A.

 Hurvois and coll. [37] has described a method to synthesize (+)-241D and (+)-myrtine by using electrochemistry. Starting from N-protected piperidone (R configuration) 76a, a voltammetric step in presence of sodium cyanide gave stereoisomer piperidines 77a,b. The treatment of the mixture with iodomethane after the reaction with LDA gave exclusively 78.

CN CN O O O O O O O O

-e H -H H -e N N H N N Ep= + 1.0 volt Ph Ph Ph Ph 76a radical cation amino radical iminium cation

O O O O O O O O O O O O 1. LDA/THF -80 °C NaBH4 + CN + N CN N CN 2. Iodomethane N CN EtOH N 91% N N 90% Ph Ph Ph Ph Ph Ph 77a (41%) 77b (40%) 78 79a (79%) 79b(12%)

O O O O 1. H2,Pd/C 79a 76b 2. (Boc)2O N N Hunig base Boc Boc 90% 80a 80b

Scheme 25.Showing the application of electrochemistry by Hurvois et al Compound 78 on reduction with sodium borohydride (with a iminium transition state) gave sterioisomers 79a,b which are separated through column chromatography using silica gel. The major isomer 79a was deprotected by hydrogenation and later reprotected to form N-Boc 80a. A similar methodology starting now from N-protected piperidone (S configuration) 76b was followed to give 80b.

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O O O O O O O Cl 1. HCl 5M, acetone S-BuLi I N 86% N Cl 2. NaHCO3 5M N CuCN/LiCl N CuLiCN Boc Boc Boc (+)-myrtine 80a 81

S-BuLi DMF

OH O O O O 1. Wittig 1. HCl 5M, acetone

2. H2,Pd/C 2. NaBH4 N CHO N C9H19 N C9H19 H Boc Boc 82 83 (+)-241D Scheme 26.Showing the application of (+)-myrtine and (+)-241D by Hurvois and coll. Synthesis of (+)-myrtine 57 was done using 80a as a starting material. The cyano cuprate was obtained through metallation using s-BuLi as well as treatment with copper(I) cyanide and lithium chloride. The alkylation with iodo-4-chloro butane gave the desired 2, 6 disubstituted 81 which after transacetalation in acidic medium forwards the intramolecular cyclization to form (+)-myrtine 57.

Synthesis of alkaloid (+)-241D starts with the same compound 80a. The alkylation in presence of s-BuLi and DMF gave the piperidine 82, a mixture of cis/trans-80/20. The major isomer was separated through column chromatography and further engaged in Wittig reaction followed by reduction of the double bond which finally gave compound 83. An acidic treatment results in formation of ketone and freeing the amine simultaneously. This has been succeeded by a hydrogenation reaction to retrieve the alkaloid (+)-241D.

 Dieter and coll. [38] reported the alkylation of N-Boc-4-pyridone 84 through Grignard reaction in presence of trimethylsilane chloride. This procedure avoided the use of copper salts customarily used to forward this transformation [39]. The dihydropyridine thus obtained depends on the nature of organomagnesium utilized during the reaction.

O O RMgX

N TMSCl (3 eq) N R -78 °C Boc Boc

84 85 R = nBu-60% 86 R = 2-CH3-C6H4-95% Scheme 27.Showing the substituted piperidine synthesis Dieter and coll.

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The same strategy has been applied to dihydropyridines which predominantly gave access to 2, 6 disubstituted piperidines. The formation of 2, 6 trans was found to be major as showed in following cases

O O iPrMgCl

N nBu TMSCl iPr N nBu Boc Boc 85 88%

O O iPrMgBr

N TMSCl iPr N Boc Boc 86 85% Scheme 28.Showing the 2, 6 trans substituted piperidine However, the nature of the reagents employed has great influence on stereoselectivity of the recovered products. One of their application which gave 2, 6 cis majorly is as follows [40].

O O

nBu2CuLi

N nBu Me2S nBu N nBu Boc Boc 85 86%

Scheme 29.Showing the 2, 6 cis substituted piperidine

B. SYNTHESIS OF 4-HYDROXY-6-SUBSTITUTED PIPECOLIC ACID/ESTER

Natural amino acid, L-pipecolic acid 87 plays an important role as a biologically active moiety especially at CNS level of mammalians [41]. The oxygenated derivative at 4th position, 4-oxo-pipecolic acid 88, the (2S,4R) and (2S,4S) 4-hydroxypipecolic acid 89 and 90 are also important as building blacks for pharmacologically active molecules [42].

O OH OH

N CO2H N CO2H N CO2H N CO2H H H H H

87 88 89 90 Figure 54.Showing the L-pipecolic acid and its analogues

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They can also act as constrained amino acids and by this way they can replace natural peptides with the advantage of a greater metabolic stability [43]. In this context synthesis of constrained peptides is still an important challenge in medicinal chemistry [44].

For the above listed reasons, a number of research groups have committed themselves for the enantioselective synthesis for 88, 89, 90 and also their analogues either by resolution of the racemic intermediates or by using a chiral starting material. Recently, number of ways to reach the 4-substituted pipecolic acid and its analogues has been reported [45]. In this current subsection, we tried to present some of the synthetic strategies which have been developed in the past 10 years.

1. Synthesis using chiral auxiliaries from amino acids

The amino acids are the most utilized precursors in chiral synthesis. Indeed, the amino acids comprise a stereocenter at α position and there are several possible strategies to modulate and substitute the core are at hand.

 Aitken and coll. [46] used the N-(cyanomethyl)-4-phenyloxazolidine 91, prepared from phenylglycinol, as an intermediate. The condensation in basic medium resulted in the formation of anion derivative which subsequently reacts with 2-(methoxymethoxy)prop-1-ene to form oxazolidine epimers 92 and 93. The major isomer 92 in presence of Lewis acid forms the bicyclic compound 94.

Ph Ph Ph 1) LDA/THF O O O + NC NC 2) MOMO NC 91 79% 92 93

OMOM OMOM 92/93 : 70/30 70% 30%

Ph Ph Ph O NC N H2NOC N H O 1) NaOH,MeOH N H Pd/C BF3.OEt2 2 2 O 2, 92 (S) -70 °C, 84% K2CO3 2) HCl,MeOH 70% N COOH O O H MOMO 86% MOMO O 94 95 96 (S)-88 Scheme 30.Showing the synthesis of compound 88 proposed by Aitken and coll.

The nitrile functionality was later transformed into amide in presence of hydrogen peroxide to give 95. A basic workup followed by an acid hydrolysis results in the formation of a lactone 96. This compound 96, on hydrogenation gave (S)-4-oxopipecolic acid 88.

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 According to one of the reviewed literature, Hou and coll. [47] used the chiral synthon 97 which was also obtained from phenylglycinol [48]. The alkylation of 97 with 2- (iodomethyl)allyl benzoate 98 resulted in the formation of compound 99. This has been followed by the deprotection of nitrogen and subsequent alkylation with allyl bromide giving compound 100.

O O O O I OBz O O O O 98 1) TFA GRUBBS N Ph N Ph N Ph N Ph 2) 96% Boc NaHMDS, THF Boc Br 46% 97 99 100 101 OBz OBz Bz

(tBu)2P

O O O O OH

OsO4, NaIO4 N Ph N Ph 3 [(n -C3H5)PdCl2]2 , HCOOH N COOH H 102 O 103 ent-89 Scheme 31.Showing the synthesis of compound ent-89 proposed by Hou and coll.

This was followed with a metathesis reaction (GRUBBS 2nd generation) which allowed to recover the bicyclic compound 101 with an excellent yield. The intermediate 102 was obtained by a palladium catalyzed reaction in presence of formic acid and a ligand which was finally transformed into 103 by an oxidative cleavage using Osmium tetraoxide. The compound 103 was finally transformed into enantiomer of 89.

 According to the strategy proposed and developed by Seebach and coll. [49], Haufe and coll. used the imidazoline 104 [50] to synthesize the compound 108.

N O N O LDA/THF F MeOH/H2O tBu tBu N N F HCl 4N Boc OTs Boc 104 105 106 95%, 99% e.e

H F F OH H3C N O H H C F N O 3 tBu F N NH NH N tBu N tBu N H H H tBu O O

O OH - HF 1) NaBH4 / MeOH NH OH tBu N 2) 6N HCl, reflux tBu N H H O O 107 108 Scheme 32.Showing the synthesis of compound 108 proposed by Haufe and coll.

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The alkylation of the compound 104 using 2-fluoroallyltosylate 105 gave the alkylated imidazoline 106 with an excellent yield and high diastereoselectivity. Treatment of 106 in aqueous acidic medium resulted in the formation of 107. Thus formed compound 107was reduced and hydrolyzed to give 6-tert-butyl-4-hydroxy pipecolic acid 108.

2. Synthesis using chiral auxiliaries from chiral alcohols

 Occhiato and coll. [51] has described the synthesis of (2S,4R)-4-hydroxy pipecolic acid 89 starting from (3S)-4-chloro-3-hydroxy-ethylbutanoate 109. Transformation of chloro functionality to a nitrile followed by protection of alcohol gave compound 110. The hydrolysis of the nitrile functionality to form an amide resulted in intramolecular cyclization giving away 2-piperidone 111. This compound 111 was later protected to form N-carbamate and then to an enol triflate which, by methoxy carbonylation reaction catalyzed by palladium salt, furnished compound 112. Changing of protection group of alcohol (e.g 113) succeeding by a hydrogenolysis of the double bond and deprotection gave the compound 89 in salt form.

OPMB OH O OPMB OPMB NaBH4 Cl CO2Et CO Et NC CO2Et H N 2 NiCl 2 N O 2 H 109 110 111

OPMB OPMB OTBDMS OH

1) H , Pd/c 1) nBuLi, ClCO2Me Pd(OAc)2 2 2) KHMDS, PhNTf Ph P, CO, MeOH N CO Me 2) 2N HCl 2 N OTf 3 N CO2Me 2 N CO2H CO Me H CO2Me CO2Me 2 .HCl 112 113 89. HCl

Scheme 33.Showing the synthesis of compound 89 proposed by Occhiato and coll.

The same research group has proposed another method which allows accessing a new way to synthesize 4-hydroxy-pipecolic acid using biocatalysts [52]. Starting from N-protected 2-piperidone 114, and as described before, a palladium catalyzed methoxy carboxylation resulted in the formation of an enamine 115 which on subsequent allylic oxidation gave an allylic alcohol 116.

N O N CO2Me N CO2Me CO2Me CO2Me CO2Me 114 115 116 Scheme 34.Showing the synthesis of compound 116 proposed by Occhiato et al.

116

Kinetic de-racemization of 116 in presence of the enzyme lipase (Candida antarctica) preferentially gave the isomer R with good enantiomeric excess.

OH O Me OH Lipase +

N CO2Me N CO2Me N CO2Me CO2Me CO2Me CO2Me 116 117 118 Scheme 35.Showing the synthesis of compounds 117 and 118 proposed by Occhiato et al.

After the separation of mixture, the isomers 117 and 118 were transformed into corresponding pipecolate esters 120 and ent-120 in few steps.

O OMe OH OH NaOMe

MeOH N CO2Me N CO2Me N CO2Me H CO Me CO Me 2 2 (2S, 4R) 117 119 120

OH OH

N CO2Me N CO2Me H CO2Me (2R, 4S) 118 ent-120 Scheme 36.Showing the synthesis of compound 120 proposed by Occhiato and coll.  Riera and coll. has used chiral epoxyde 121 to introduce the amine counterpart for providing compound 122 This was followed with a metathesis reaction (GRUBBS 2nd generation) which allowed to recover compound 123. Oxydative cleavage with sodium periodate followed by iodolactonisation reaction gave 124 which dehalogenation reaction with tributyl tin provided bicyclic compound 125 Finally, basic hydrolysis and deprotection of the amino group gave (2S,4R)-4-hydroxy pipecolic acid 89 [53].

117

1) allylamine N-Boc GRUBBS N Boc OH O 2) (Boc) O, MeOH 2 OH OH OH 121 122 OH 123

I 1) NaIO4, THF/H2O N Boc I2, KI, NaHCO3 N Boc

2) NaClO2, t-BuOH/THF COOH 124 O 125 O

NaOH/H O Bu3SnH, AIBN N Boc 2 N Boc TFA NH

HO COOH HO COOH O 126 127 89 O Scheme 37.Showing the synthesis of compound 89 proposed by Riera and coll.  Merino and coll. has used the dimethyl acetal of glyceraldehyde 128 as source of chirality. Condensation of benzyl hydroxylamine gave nitrone 129 which was alkylated with allyl magnesium bromide in the presence of ZnBr2, furnishing compound 130 in excellent yield.

Oxydation with MnO2 gave a new nitrone 131 which cyclized in toluene to the oxazolidine 132. Functionnal group interconversion led to 133 which, by treatment with zinc in acidic medium provided the (2R,4S,6S) 6-phenyl-4-hydroxy pipecolic acid 134 [27].

MgBr O O Ph-CH2-NHOH O O O O MnO2 O O H ZnBr O MgSO4 2 quant. quant. 80% N N N H d.r. : 95/5 Ph O Bn HO Bn O 128 129 130 131 OH Toluene O O O reflux O O 1) H5IO6 Zn 75% 2) NaOCl HOC H OC AcOH N Ph N Ph 2 N Ph HOOC N Ph H 132 133 134

Scheme 38.Showing the synthesis of compound 134 proposed by Merino and coll.  Sutherland and coll. [53] has utilized the aspartic acid as the building blocks for the synthesis of 4-hydroxypipecolic acid derivatives. After the protection and activation, the ester 135 was transformed into β-ketophosphonate 136 which, during Horner-Wadsworth-Emmons with a range of alcohols gave corresponding enones 137 with good yields.

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O O O P OMe MeO C HO2C 2 (OMe)2P-CH3 OMe R-CHO O nBuLi/THF R HN CO2Me H N CO H HN CO2Me 2 2 Tr Tr HN CO2Me Aspartic acid 135 Tr 136 137

OH O O CO2Me H NaBH3CN N R N CO Me R N CO Me Ph 2 2 R Bn Ph 138 H Scheme 39.Showing the synthesis of compound 138 proposed by Sutherland and coll.

The compound 137 was treated with acidic medium (TFA), inducing the deprotection on nitrogen which reacts with benzaldehyde giving an imine intermediate which on reduction with sodium cyanoborohydride gave the compound 138 (2S,4S,6R) 6-substituted-4-hydroxy methyl pipecolate) which exclusively was trans . The compound 138 passes through a transition state during the 6-Endo-Trig cyclization.

On the other hand, if the nitrogen deprotection was followed by a treatment in basic medium results in the formation of 2,6 cis isomer 139 through an Aza-Michael intramolecular reaction [54].

O OH O CO2Me 1) TFA NaBH(OAc)3 137 R NH2 2) Hünig's base R N CO2Me R N CO2Me H H H 139 140 Scheme 40.Showing the synthesis of compound 140 proposed by Sutherland et al.

The compound 139 was then reduced stereoselectively in presence of a bulky reducing agent to retrieve (2S,4S,6S) 6-substituted-4-hydroxy methyl pipecolate 140 in good yields.

This method is found to be general and can be applied to access piperidines with variety of substitutions at 6th position. This can be achieved by engaging the required aldehyde during the HWE reaction to incorporate requisite substitution on 6th position.

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C. CONCLUSION

Far from being exhaustive, this section shows the principle methods for the preparation of 2,6 disubstituted piperidines and pipecolic derivatives, with diversified approaches (asymmetric version, application to natural products synthesis, methodology,..) which render the diversity and richness of all the research which are done in this area. Although a great number of syntheses permit the preparation of piperidines possessing one or two asymmetric centres, a few of them are able to control all the potential asymmetric centres of this class of heterocycles. So, as we have demonstrated that the synthetic route developed in our laboratory was relevant for the asymmetric synthesis of piperidines derivatives, we have choosen this route for compounds issued from family 3. On the other hand, for family 1, we have used the procedure described by Sutherland since it seems to be the most applicable process for our purpose.

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II. STRATEGY FOR THE SYNTHESIS OF FAMILY 1

The retro synthetic strategy for the access of family 1, with the core benzodiazepanone moiety can be framed as depicted in the scheme 41.

HN X NH2 O  

   O O FGI O N  N  N  N N N R R R

141 143 144

O O  O Br H N  N + OH N R N PG R O

144 B A

142= N N NH R=phenyl, CF3

Scheme 41.showing retro synthetic scheme for family 1 This proposed scheme portray that the desired urea like molecule 141 can be obtained by pairing the fragment 142 with that of 143. Whereas in fragment 143, the stereochemistry is modulates as we saw in the previous chapter, incorporation of asymmetric carbons deemed to be fruitful in a better reach and interaction with the amino acids in the binding pocket of CGRP receptor. Therefore, stereochemistry is fixed according to the molecules prioritized based on the docking results. This fragment 143 with the desired stereochemistry on the piperidinic moiety can be obtained from the desired ketone intermediate 144 by using the Functional Group Interconversion (FGI) principle. The intermediate 144 is possible from a reaction between key synthons A and B.

Synthon A

To obtain the synthon A, the retro synthetic scheme proposed is as follows;

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O O O HO     OH OH OH N HN H2N PG O PG O O Aspartic A A' acid Scheme 42.showing retro synthetic scheme for synthon I Synthon A is N-protected 6-methyl-4-oxopiperidine-2-carboxylic acid synthon. In previous chapter, various methodologies concerning how to synthesize the synthon A have been discussed in detail. According to the bibliographic conclusion from previous section, to reach the key piperidine skeleton of family 1, a pipecolic acid derivative, we have nominated the process proposed by Sutherland et al [53,54].

The proposed retro synthetic scheme shows that synthon A can be derived from a Michael acceptor A’. The key intermediate alkene A’ is the precursor for obtaining synthon A through Michael intramolecular reaction (6-Endo-Trig-cyclization) [55]. Whereas, the whole synthesis starts from commercially available aspartic acid, either D or L, depending on the required configuration on final potential hit molecule.

Synthon B

To obtain synthon B, the retro synthetic scheme designed is as follows;

Br O Br H N H H2N R R +

B 2-bromo B' benzaldehyde Scheme 43.showing retro synthetic scheme for synthon II The proposed retro synthetic scheme shows that the synthon B can be derived from a simple reductive amination of the commercially available 2-bromobenzaldehyde and amine

B’ with desired substituants [56, 57]. This substituent can either be a phenyl or CF3 based on the desired substitution on the final potential hit molecule.

Imperative points derived from this synthetic strategy  The synthesis of final potential hit is achieved by ‘convergent synthesis’  The Michael intramolecular reaction for obtaining the synthon A act as key step to introduce stereocenters on the final molecule in a single step.

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 Substituants on the final molecule 141 depends on the amine B’ which we use in the formation of the synthon B.  Another key asymmetric center on the final molecule 141 is obtained by FGI from 144 to 143.

A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME

In the previous section, the synthetic scheme to obtain the final hit molecule 141 has been successfully designed. In the current segment, the application of the designed scheme will be discussed step-by-step in detail. Synthon A The piperidine synthon A with substitution at 2 and 6, a methyl and an ester/acid functionality will be obtained in 5 steps. In the first step, the aspartic acid will be esterified followed by the N-protection. This N-protected compound will participate in nucleophilic substitution reaction with phosphonate followed by Horner-Wadsworth-Emmons reaction to give α, β-unsaturated ketone. The last step involves a 6-Endo-Trig-cyclization to form piperidine moiety. Now let’s focus on each step in facet.

1. Synthesis of 6-methyl-4-oxopiperidine-2-carboxylic acid from aspartic acid

We have used the same methodology for both D- and L-aspartic acid until we reach the synthon A. Hence throughout the course of this discussion, we will exemplify the steps using D-aspartic acid.

1.1 Fischer-Speier esterification of aspartic acid The first step involves the esterification of commercially available D-aspartic acid. We used the classical Fischer-Speier esterification method [58] because of its simple yet effective esterification to recover the desired product.

O O SOCl2 HO O OH O H N MeOH, Reflux -Cl+H N 2 5h, Quantitative 3 O O

D-Aspartic 145 acid Scheme 44.showing esterification of aspartic acid

1.2 Protection of amine group in dimethyl ester of aspartic acid In the literature, to orient the selectivity during nucleophilic attack of the dialkylmethyl phosphonate, there are two described procedures [59]. The ester 145 from

123 previous step can be subjected to an N-protection by using rather bulky protecting groups like trityl chloride or di-tert-Butyl carbamate, which helps in regioselective reaction during the nucleophilic substitution step to access phosphonate 149 and thus prevents the side product formation.

O O O O (Boc)2O, O (Boc)2O, O NaH O Et3N O TrCl,Et3N O O HN O O - + O O HN Cl H3N CH Cl , RT, O N Dry THF, RT, Dry THF, RT, 2 2 O O O 86% O 72% 82% O O O

145b 145a 145 146 Scheme 45.showing protection of dimethyl ester of aspartic acid If the PG used was trityl, it was one-step process whereas in case of tert-Butyl carbamate protection, it was a two-step synthesis to do a double protection of nitrogen to duly induce selectivity on the nucleophilic attack of the phosphonate. The first protection of amine using tert-Butyl carbamate gave 82% of compound 147 which on further deprotonation by a strong base and reaction with another equivalent of di-tert-Butyl carbamate gave compound 148 with a yield of 72%. Compound 146 was obtained by a simple nucleophilic substitution reaction in one step.

The following minimum energy conformers obtained through energy minimization molecular mechanics done using ChemDraw® ultra shows that a single tert-Butyl carbamate protection results in exposure of both esters and hence it lacks in the regioselectivity during the next step. On the other hand, a di-tert-Butyl carbamate protected amine exposes only one ester leaving the other ester under the influence of steric hindrance [60].

Figure 55.showing steric hindrance due to mono and di-boc (diterbutylester) PG

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Whereas the following structure of trityl showed the steric hindrance similar to that of di-tert-Butyl carbamate protection with good yield in single step.

Figure 56.showing protection of dimethyl ester of aspartic acid with trityl PG

Hence, due to least steps, a facile reaction condition and good yield amid an excellent regioselectivity during the future steps, we selected trityl PG for the above reaction and carried onto the next step.

1.3 Synthesis of β-keto phosphonate ester

Regioselective reaction of 146 with 4.5 equiv each of dimethyl methylphosphonate and n-BuLi (which subsequently gives 4.5 equiv of lithium anion of dimethyl methylphosphonate) gave exclusively 147 with a yield of 77%.

O O O O P O O O O P O (EtO)2POMe, O (MeO)2POMe, O n-BuLi O n-BuLi O HN O HN O HN O THF, -78 °C, THF, -78 °C, 28% 77%

148 146 147 Scheme 46.showing preparation of β -keto phosphonate ester

When we tried the same protocol with diethyl methyl phosphonate as the anion source, the reaction gave the product 148 but relatively in poorer yield (28%) and purification was much difficult as described in the literature [59].

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1.4 Synthesis of (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate

Thus formed β-keto phosphonate ester 147 has been subjected to Horner-Wadsworth- Emmons (HWE) reaction in a gentle condition, which was optimized in the laboratory itself to get E form of α, β unsaturated ketone 149 in majority [61].

O O O P O O

O Ba(OH)2.H2O O HN O HN O CH3CHO

THF/H2O:40/1, 96%

147 149 Scheme 47.showing preparation of β -keto phosphonate ester This HWE reaction is considered as the key intermediate reaction in whole synthesis as the substituent on the final molecule depends on which aldehyde we use during this step.

Stereoselectivity of HWE HWE reaction predominantly gives E-alkene. Systemic study conducted by Thompson and Heathcock [62] showed that the following are key factors determining the formation of E alkene.  Li > Na > K salts during the reaction.  Increased steric hinderance or bulkiness of the aldehyde used.  Higher reaction temperatures (23 °C over −78 °C).  Preferring 1, 2-dimethoxyethane over tetrahydofuran. Along with the above factors, another study showed that bulky phosphonate and bulky electron withdrawing groups results in E alkene over Z form. Currently optimized condition gave better yields compared to other methods like Masamune and Roush or Rathke’s methodology [63, 64]. There are other modifications for HWE reaction which may result in formation of Z alkene predominantly. Still and Gennari [65] identified a methodology which produces Z:E in a ratio of 12:1 to 50:1. Use of phosphonates with electron withdrawing groups like trifluoroethyl along with strong bases like Potassium bis(trimethylsilyl)amide gave exclusively Z alkene.

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Mechanism involved in HWE to give E-alkene HWE reaction has many transition states during its course of formation of alkene. Deprotonation of phosphonate by a base to produce carbanion of phosphonate accounts for the first step in HWE. In the next stage, nucleophilic addition of carbanion onto the carbonyl group of aldehyde results in the intermediates x and y. This step as considered as rate limiting step. Finally there is an elimination step which results in the formation of alkenes. The scheme of mechanism is as follows;

H O O O O O P P O O X O O H HN O H O C C H O X O H C H CH O O P O 3 H 3 149 E-alkene 147a 147a' + H3C H Rapid Rapid O Slow H HN O O O O O O P P O O H O O H HN O X O H C C H X H C X CH 3 H 3 147 147b 147b' 149 Z-alkene

Scheme 48.showing preparation of β -keto phosphonate ester In the formation of alkenes through HWE, the oxaphosphetane (147a,b) and betaine (147a’,b’) were considered as the slowest and oxyphosphetane being the lowest energy compared to that of their corresponding betaines [66] has been confirmed by low temperature 31P NMR studies. The energy states of the starting material and the transition intermediates are as follows;

Figure 57.showing preparation of energy states of intermediates during HWE

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Thus, the above graphical study also depicts that the most stable and low energy form is the trans or E and hence the recovered major product was E-alkene. The formation of E-alkene was clearly evident with the coupling constants observed through the proton NMR. The coupling constants for the vicinal protons of trans alkene i.e. E- alkene between H6 and H7 should typically range between 11-18 Hz.

H 7 8 O 1 3 5 O 2 4 6 H HN O

149 Figure 58.showing preparation of β -keto phosphonate ester as E isomer

Practically, the E-alkene formation is noticeable through the proton NMR showing a coupling constant of 15.8 Hz, thus confirming the HWE condition used above resulted in the formation of E-isomer.

Figure 59.showing preparation of β -keto phosphonate ester as E isomer

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This large coupling constant depicts the two protons on adjacent carbon to be in trans position. Thus obtained E-alkene has been engaged in the next step where a 6-endotrig cyclization has been carried out to form the desired 2,6 disubstituted piperidone synthon A.

1.5 Synthesis of methyl 6-methyl-4-oxopiperidine-2-carboxylate

The (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate 149 formed during HWE reaction was carried forward to the next step for the synthesis of methyl 6-methyl-4- oxopiperidine-2-carboxylate 6.

O O O O 40 eq 2M HCl 2M HCl O 70 DIPEA DIPEA + HN O O O O N N N N MeOH, RT MeOH, RT H H H O O O

152 149 150 151 Scheme 49.showing preparation of 6-methyl-4-oxopiperidine-2-carboxylate salt Michael intramolecular reaction serves as the rationale for the synthesis of 6 from 5a. As reported by Sutherland et al [53, 54], this step involves 6-endo-trig cyclization of (R,E)- methyl 4-oxo-2-(tritylamino)hept-5-enoate in accordance to Baldwin’s rules [55]. They used this methodology for similar substrate as 149 but with different aliphatic substitutions like isobutyl and propyl at 6th position. The results for them were as follows

N° amount of substrate product Yield cis/trans ratio substrate (%)

O O 0.07 g 68 83/17 1 O O HN N H Tr O O 153 O O 0.1 g 59 75/25 2 O O HN N H Tr O O 154 Table 17.Showing the reaction scale and substrates used by Sutherland et al. As the conditions they followed and their diastereomeric ratios were excellent, we went after them with similar conditions. Following the procedure stated above, there was formation of 6-methyl-4-oxopiperidine-2-carboxylate salt, compound 152, instead of desired

129 compounds 150, 151. This is a consequence of saponification of 150 & 151 in situ. Compound 152 is pipecolate salt and being a salt of amino acid, its solubility in the aqueous phase played a stringent role in this step. This saponification of 150 & 151 might be due to the large excess of water (in acid as well as during the basification intermediate step) along with base. In the above stated procedure, the investigators used 40 equivalents of 2M HCl and 70 equivalents of N,N-Diisopropylethylamine base which gave 153 & 154 (condition 1 of table 18). On contrary we got compound 152 along with diisopropylethylamine hydrochloride salt in aqueous layer and no 150 & 151 compounds in organic layer but just the excess of N,N-Diisopropylethylamine. Thus further esterification was done as below and the quantification was done as tabulated;

O O O O O

SOCl2 + O O O N N MeOH, N N H Reflux, O H H 91% O O 152 155 156 Scheme 50.showing preparation of methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate. Condition 2M HCl (Eq) DIPEA (Eq) Yield of Reproducibility 155/156 (%)

1 40 70 63 Not reproducible 2 10 17 83 reproducible 3 2 3 91 reproducible

Table 18.showing optimization to form compound 152 Hence conditions 2 & 3 are well optimized and reproducible compared to 1 even thought it gave compound 152 instead of desired products 150 & 151. Thus formed compounds 155 & 156 are cis/trans isomer and both of them are easily separable through normal phase column chromatography. In our case, we experienced relative amounts of formation of cis and trans isomers as 75/25 (calculated using 1H NMR) and this can be attributed for cis being thermodynamically stable form where as trans being kinetically stable. And hence the initial formation of trans isomer slowly results in the conversion into cis form being thermodynamically stable. But this conversion depends on various factors like time, temperature, substituants etc. and hence results in not complete transformation into cis form, instead leaving some portion of trans also in the final mixture.

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Figure 60.showing preparation of cis/trans piperidine with 75/25 ratio Thus isolated (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (cis) and (2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (trans) have different splitting patterns when it comes to proton NMR and they serve as basis for identification of the isomers.

(2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (cis) Each proton experiences a different splitting pattern due to the existence of equatorial and axial protons and hence the coupling constants are complicated. The typical coupling constants (varies with the conformation and substitution at 4th position) can be attributed as stated in the following table.

Isomer H Splitting pattern

Ha O Ha O Ha O 4 4 4 H3C He O H3C He O H3C He O 6 5 6 5 6 5 Ha Ha Ha 6Ha MeOOC Ha MeOOC Ha MeOOC Ha 2 3 He 2 3 He 2 3 He HN HN HN 1 1 1 Ha Ha Ha 2.84 ppm, multiplet

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Ha O Ha O Ha O 4 4 4 H3C He O H3C He O H3C He O 6 5 6 5 6 5 Ha Ha Ha

MeOOC Ha MeOOC Ha MeOOC Ha 2 3 He 2 3 He 2 3 He HN HN HN 1 1 1 Ha Ha Ha

5He J5e-5a = 13.3 Hz

J5e-6a= 2.7 Hz

JW = 2.7 Hz

1.97 ppm, doublet of triplet O O 3He Same splitting pattern as 5He; 2.34 ppm, doublet of triplet

O Ha O Ha O N 4 4 H3C He O H3C He O H 6 5 6 5 O Ha Ha

MeOOC Ha MeOOC Ha 5Ha 2 3 He 2 3 He HN HN 155 1 1 Ha Ha

J5a-5e = 13.2 Hz

J5a-6a= 11.7 Hz 1.08 ppm, triplet

3Ha Same splitting pattern as 5Ha; 1.37 ppm, triplet

Ha O Ha O 4 4 H3C He O H3C He O 6 5 6 5 Ha Ha

MeOOC Ha MeOOC Ha 2 3 He 2 3 He HN HN 2Ha 1 1 Ha Ha

J2a-3a = 12.2 Hz

J2a-3e = 2.8 Hz

3.55 ppm, doublet of doublet J2a-3e= 2.8 Hz Table 19.showing splitting pattern with coupling constants for cis piperidine

Cis piperidine, as portrayed above has distinguishing coupling constants and splitting patterns. Protons H6a shows H6a-H5a, H6a-H5e, H6a-HCH3 which finally was shown as a

132 multiplet. On the other hand, protons H5e and H3e shows similar splitting and coupling constants. The splitting goes like H5e-H5a which are geminal protons and hence shows J =

13.3 Hz, H5e-H3e which are characteristic W pattern splitting and shows J = 2.7 Hz and finally

H5e-H6a which are vicinal protons and thus shows J = 2.7 Hz. The same kind of pattern applies for H3e also, considering the fact that both H5e and H3e are having similar environment of protons. H3a and H5a also show similarity due to the same vicinity. The splitting pattern goes like H5a-H2a which are vicinal protons and hence shows, a transdiaxial splitting J = 13.2 Hz,

H5a-H5e which are geminal protons and hence shows J = 13.2 Hz. Whereas, H2a shows splitting pattern as H2a-H3a which are vicinal protons and hence shows, a transdiaxial splitting

J = 12.2 Hz and H2a-H3e showing J = 2.8 Hz.

(2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (trans) Trans piperidine shows mostly similar kind of splitting pattern and coupling constants as that of cis isomer. Protons like H6, H5a, H5e are absolutely in accordance with that of cis piperidine’s coupling constants. But, the characteristic coupling constant was shown by H2 proton. In case of trans, there is no transdiaxial splitting which we observed in case of cis (J

12.2 Hz, H2a-H3a). Instead, there is appearance of transequatorial splitting between H2e-H3e. The splitting pattern is as follows:

Ha O 4 H3C He O 6 5 COOMe J2e-3e = 8.8 Hz

He Ha J2e-3a= 3.7 Hz 2 3 He HN 1 Ha Figure 61.showing splitting pattern with coupling constants for H2e proton of trans piperidine

Along with the above depicted splitting, the H3a-H2a (J = 12.2 Hz) in cis became H3a-H2e (J =

3.4 Hz) as well as H3e-H2a (J = 2.7 Hz) in cis became H3e-H2e (J = 8.8 Hz).

The esterification of 6-methyl-4-oxopiperidine-2-carboxylic acid 152 to compound 155 & 156 i.e. methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate was working efficiently for undersized quantities of compound 152. On a large scale (even more than a gram of compound 152), the reaction was giving poorer yields due to the presence of the large amounts of salt from previous step. And thus, a further optimization/modification of compound 152 to give synthon A directly was proposed and optimized as stated in the next reaction.

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1.6 Protection of amine in 6-methyl-4-oxopiperidine-2-carboxylic acid

The compound 152 obtained from Aza-Michael reaction was directly engaged in protection of amine to give the synthon A. This reaction was optimized as the esterification step of compound 152 to 155 & 156 was posing problems on scale-up even to gram scale.

Hence, compound 152 was protected using different protecting groups (PGs) in order to give synthon I. We tried to give synthon A with two different protecting groups based on the sensitivity of other substituants during the deprotection step. Thus we made a list of criteria for choosing protecting group as follows;  The PGs should give good quantity of desired compound, i.e. the reactivity of the PGs with the substrates same as that of what we use (eg. Alicyclics, piperidines etc) to give good yields.  The PGs shouldn’t pose any steric hindrance in the succeeding reactions.  The protection step shouldn’t alter any other functionalities of the molecule.  The PGs should have facile cleavage step.  The deprotecting step shouldn’t affect any other functionality changes of the molecule.

These main reasons made us to select di-tert-butyl dicarbonate ((Boc)2O) or benzyl chloroformate (Cbz) [67, 68].

O Cbz O O

Na2CO3 (Boc)2O - + OH O Na MeOH, OH N DCM/H2O N N 86% H Reflux, O O O O O 72% O O

159 157 158 Scheme 51.showing protection of methyl compound 157 From here, a phenomenon called rotamers has started posing hard time in the structural analysis of the formed compounds. Rotamer: These are defined as isomers which interconvert into another form by rotation among its single bonds. They are in general called as conformational isomers or conformers and to be precise, rotamers [69]. R' O R N R' H R N O H trans amide cis amide Figure 62.showing amide rotamers for secondary amides

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If R’ was a bulky substitution (i.e., tert.-Bu, Ph, O-Tol, etc.), the cis rotamer is dominant in deuterated chloroform during the NMR analysis [70]. For tertiary amides, the case is a bit staggering as there are also two forms, the second form being formed due to the lower apparent symmetry [71]. It can be explained as below;

O O CH CH3 H N 3 H N CH CH3 3 Figure 63.showing amide rotamers for tertiary amides The two forms as shown and their ratio in NMR depend completely on the solvent system as well as the temperature. Compound Rotational Barrier

N,N-Dimethylformamide 92 kJ/mol (D2O)

N,N-Dimethylacetamide 80 kJ/mol (D2O)

N,N-Dimethylbenzamide 66 kJ/mol (CDCl3)

Table 20.Showing the effect of solvent on rotational barrier Rotations along these single bonds are constrained by a ‘rotational energy barrier’ which ought to be crossed to interconvert one conformer to the other. Conformational isomerism takes place when the rotation around the single bond is relatively unhindered i.e with no bulky groups around. That is, the energy barrier should be less enough for the interconversion [72].

O O

OH OH N N O O O O O O

Figure 64.showing amide rotamers for compound 158 We even tried to change the temperature to 20, 30, 40, 50 & 60 °C and see the effect on rotational barrier energy which might result in better resolution. But we saw neither decrease of rotamers nor a better resolution. This might be perhaps because of non-interconvertable nature of the rotamers or may be due to high barrier energy.

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Figure 65.showing rotamer of cis/trans piperidine in DEPT NMR So, from the above 13C NMR, we can make out that there are two carbonyl carbons co- existing at room temperature. Hence, with this step, we have successfully reached the synthon A, an acid synthon. Now for obtaining a mono-peptide, we carried an amide bond formation with two different amines as discussed in the next segment.

2. Synthon B

Synthon II is an aryl amine synthon. As stated in the previous chapter, the amine synthon which later gets incorporated in the final molecule plays an important role for interactions. Depending on the amine used, they provide a hydrophobic interaction with tryptophan 74 of RAMP-1 or in some cases when there are fluorine substitution results in better bioavailability thus improving pharmacokinetic properties.

A generalized reductive amination has been followed to synthesize this fragment which was retrieved from the literature [56, 57]. The reductive amination condition followed was depicted below:

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Br Br O Reductive amination H X NH N X + 2 H X=phenyl CF3 2-bromo B' B benzaldehyde Scheme 52.showing reductive amination to synthesize compounds 160 and 161 N° Aldehyde Amine Condition product Yield (%)

Br NaBH4 in N 1 methanol, RT H 95 NH2 Br O 160 H Br NaBH3CN, N CF3 2 AcOH in H 72 F3C NH2 methanol, RT 161 Table 21.conditions to synthesize compounds 11 and 12 With this, two key synthons A & B were successfully optimized and synthesized in large scale. The next step involves an amide bond formation to obtain a monopeptide.

3. Precedent work and optimizations:

Initially, an exclusive study on the design and optimization of this step using another racemic substrate with a similar core piperidine ring but without analogous substituants (no methyl group on 6th position) was done within the team of Prof. Yves Troin and Dr. Isabelle Thomas.

The synthesis of 144 like synthon was first attempted by following the conditions proposed by Ma and co-workers [73]. They achieved the condensation of L-proline (similar to synthon A) into synthon similar to 141 in one pot two-step synthesis, which gave 163, a molecule very similar to the desired benzodiazepanone moiety. The first step according to the designed synthesis was metal catalyzed ‘Ullmann coupling’ of the synthon B to L-proline inorder to achieve 162. This has been achieved at 90 °C with 10 mol% of CuI and L-proline acting as ligand. Further, by increasing the temperature to 110 °C, a peptide bond formation was mediated to yield the desired product 163.

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Br N O H 160 N OH N 63% yield N 10 mol% CuI H 163 O 1.5-2 eq Cs2CO3 Proline dry-DMF

OH N N O 90°C 24h H 110°C, 21h

162 Scheme 53.Showing the optimization using L-proline When these conditions were applied into pipecolic acid to verify whether they could be feasible to get a molecule similar to synthon 144, It was observed on GC-MS that although there was a small peak corresponding to the desired product, most of amine 160 did not react, and found being de-brominated, which suggested that the Ullmann cross-coupling might have worked well but the peptide bond formation was not achieved whatsoever, as shown in scheme 54. Neither addition of DCC or L-proline as ligand was able to push the reaction to improve the yield of the desired product.

Br N O H 160 N OH N N H 10 mol% CuI O 1.5-2 eq of base dry-solvent Pipecolic acid 164 Scheme 54.showing application of optimized condition on our substrate

Therefore, a new strategy must be found to get to synthon 144. A retrosynthetic analysis showed that such tricyclic derivative could be synthesized in two different ways. In route A (in green in scheme 55) the amine derivative is coupled (C-N cross coupling) first to synthon D, followed by peptide bond formation to get the desired product. In route B (in red) is the other way around, peptide bond formation to achieve E followed by intramolecular cyclization, which could be promoted by palladium or copper catalysis.

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X X

Peptide bond O O synthesis N N OH N (Route A) N

C D

(Route B) C-N cross coupling X=H, O O

X X

O HN N Peptide bond OH + H N N synthesis H O Br

A'' 160 Br E Scheme 55.showing newly proposed design to obtain synthon C

ROUTE A: Following this synthetic pathway, the first step is the coupling of the piperidinic nitrogen atom to synthon 160. This could be promoted by copper (Ullmann reaction) [74] or palladium (Buchwald-Hartwig reaction) [75, 76]. The first attempt was to employ 10 mol% of

CuI and Cs2CO3, (as shown in scheme 56), although no traces of the product was observed whatsoever on GC-MS. We thought that the amine synthon could interfere in the cross- coupling reaction and thus we decided to protect it by a di-tert-Butyl carbamate PG. Unfortunately, when the Ullmann reaction was carried out again, no reaction occurred either.

N O O H

O O 10 mol% CuI OEt 2 eq Cs2CO3 N O OEt DMF N N H H O 165 166

DMF 10 mol% CuI O O 2 eq Cs2CO3 OEt Br Br N (Boc)2O N N O H N Et3N, O O 4-DMAP (Cat) O O DCM 160 88% yield 168 167 Scheme 56.showing application of copper in route A to obtain synthon D

139

The reactions was attempted to be carried out using palladium mediated catalysis. 10 mol% of Pd2(dba)3 and BINAP as ligand were employed [77]. The reaction was performed in dry DMF at 110°C and again no trace of the desired product was observed.

O O N Br O O 10 mol% Pd (dba) O O 2 3 10 mol% BINAP OEt N OEt 110 °C, DMF O N N H O O O 168 165

Scheme 57.showing application of palladium in route A to obtain synthon D Route B: Following this way, synthon 169 was coupled first to synthon 160 through a peptide bond formation (see scheme 58). First, pipecolic acid (synthon A with no substitution at 4th position) was protected with a di-tert-Butyl carbamate group, which was achieved employing traditional condition [16]. Bromoamine 160 was then coupled with DCC coupling agent, which has been optimized as follows.

Br Br A, B OH + N N N H N Boc Boc O O 169 160 170

Scheme 58.showing application of coupling agents in route B to obtain synthon E A B Solvent Yield (%) HOBt DCC DCM 55 DMAP DCC DCM 65 DMAP EDC DCM 60 HOBt EDC DCM 56 HOBt DCC DMF 50 DMAP DCC DMF 50 DMAP EDC DMF 56 HOBt EDC DMF 54 Table 22.Showing the optimization conditions used for coupling the synthon 169 & 160

Thus developed condition has been applied onto the synthon 160 & 165 to synthesize 171. The coupled synthon 170 has been forwarded to the next step where the deprotection of

140 tert-butyl carbamate was carried. A variety of conditions have been used to optimize this step as follows

Br Br Br O O O O O Condition + N N N N N N H Boc H O O O 172 173 171 Scheme 59.showing application deprotection in route B to obtain synthon E Conditions Yield (%) 172 173 TFA, dry DCM 90 4 1

Bu4NF, THF - - -

BF3, dry THF 62 1 99

Table 23.showing application deprotection in route B to obtain synthon E Thus, we selected the deprotection of the piperidine condition to be TFA/DCM (90%) was carried out prior to intramolecular coupling with 10 mol% of Pd(OAc)2 and 30 mol% Xantphos. We tried a range of conditions to achieve the desired product Catalyst Ligand Base Temperature Time (h) Solvent Yield (%) Dioxane - DMSO -

Pd(OAc)2 Xantphos Cs2CO3 110 °C 48h Toluene 54-63% Distille Toluene 80% CuI Toluene - Table 24.showing optimization of cross coupling reaction

Of all the conditions, the Pd(OAc)2 with Xantphos as ligand in distilled toluene reflux gave the desired compound F in the 80% of yield. Thus this deemed to be the useful catalytic system which can be applied to substrates like ours. To be noted, the reaction gave the desired product in non-polar solvent but not in polar solvents.

Br X X

0.1eq Pd(OAc)2 0.3 eq Xantphos O N X=O, O O N N H N O Cs2CO3 dry toluene, 110 °C E 80% C Table 25.showing application of cross coupling reaction

141

Application of the optimized Buchwald condition gave the desired product in same yields irrespective of the substituants on 4th position.

Hence, the overall conditions optimized can be depicted as follows

X X X (Boc) O Coupling 2 reaction OH OH N N N H Base, MeOH Boc O N O Boc O Br A'' A E'

TFA DCM X=H, O O X Pd(OAc)2 X xanthphos

Base=Et3N for x-H O N N O O toluene N NaOH for x- N 110°C H O Br

C E Scheme 60.showing application of route B to obtain synthon C

Therefore, it has been shown that route B is the suitable one to synthesize the family of molecules in which we are interested and, therefore, peptide coupling must be done prior to intramolecular Buchwald-Hartwig cyclization.

4. Application of the previously optimized conditions

Thus, learning the dos and don’ts from the optimized conditions from the past, we initially applied the same strategy and conditions for similar but not the same substrate we used. The initial design of the future steps is as follows;

O O O

C-N cross Amide bond Br O coupling O formation OH + X N N N N H H PG N N O X X A B Br G F X=CF3,Phenyl Scheme 61.showing previously optimized condition to chiral substrate

Following the above depicted scheme, the final benzodiazepanone moiety can be achieved from the monopeptide F which has been obtained using an acid synthon A and amine synthon B.

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5. Peptide coupling for amide bond formation:

As per the previously optimized condition, we initially used the coupling agent N,N'- dicyclohexylcarbodiimide (DCC) to obtain the desired monopeptide as depicted in the following scheme. We started the optimization with DCC as coupling agent not just because of the previous optimizations on racemic mixture, but also it is one of the coupling agent in market claimed to decrease the epimerization of formed peptides, as in the current case we used asymmetric substrates [78].

Br O O Br 1.1 eq DCC, X N (0.1 eq DMAP) OH + N N H N PG DMF, RT PG O O X A B X=CF3, Phenyl F PG=Boc, Cbz Scheme 62.showing the peptide coupling using DCC/DMAP Unlike in the optimization stage, when we tried on our synthon A as the substrate, there was no desired product formation. Instead, there was only formation of side product during the DCC coupling reaction (Scheme 62). Even on repeating this reaction with addition of catalytic amounts of DMAP, to fasten-up the reaction process, the results were still not satisfactory.

Mechanism of DCC mediated amide bond formation

We investigated what precisely was the side product formed by studying the mechanistic approach of DCC mediated amide bond formation. The steps involved in the DCC coupling include an initial deprotonation of the N-protected pipecolic acid substrate A by DCC followed by a nucleophilic attack of the carboxylate onto the imide which consequently gives an O-acylurea. This O-acylurea is formed as an inevitable intermediate during the course of imide coupling agent mediated amide bond formation. Thus formed O- acylurea, can form 3 subsequent products as follows

 On addition of amine, the desired mono-peptide can be formed due to the nucleophilic attack of the amine onto the activated N-protected pipecolic acid-DCC complex.

 On addition of one more equivalent of N-protected pipecolic acid, there occurs a formation of anhydride from O-acylurea. This anhydride is highly reactive which immediately reacts when an amine is added. This particular step finally gives a mono-

143

peptide as well as one equivalent of the N-protected pipecolic acid. But mostly, no one follows this step since expensive N-protected acid substrate addition is not worthy.

All these steps are depicted in the following scheme.

O O N N + C O C OH N N N N H PG PG O O A DCC

O Br O Br O N O C H N N NH N N + PG N NH NPG O O O PG O X X N-Acyl urea F B O-Acyl urea

O- N PG O O O Br O Br H H N N X=CF3, Phenyl O + N N O N NH + PG PG N O O PG O X X B anhydride F Dicyclohexyl-urea Scheme 63.showing the peptide coupling mechanism using DCC

 When the O-acylurea can’t react with the amine substrate or when there is no amine substrate at all, this rearranges and forms a stable N-acylurea.

O O O N N C Intramolecular H PG rearrangement N N O NH N PG O O N-Acyl urea

O-Acyl urea Scheme 64.showing the intramolecular rearrangement This formation of the undesired product was constant for all the N-protected pipecolic acid substrates and amines we used and we exemplified the following NMR which was obtained due to the following reaction

144

Br O O O O 1.1 eq DCC, N CF3 + H (0.1 eq DMAP) OH N CF N 3 N DMF, RT O 161 O O O O O

174 175

O O

H N N N O O O O

N-acyl intermediate 176 Scheme 65.showing the DCC peptide coupling giving N-acylurea

We used a racemic mixture of N-protected acid synthon 174 with an amine 161 using DCC/DMAP. But the product obtained was N-acylurea 176. This has been confirmed by scrutinizing the DEPT NMR.

Figure 66.showing the DEPT NMR showing N-acylurea The above shown DEPT NMR of clearly showed the formation of N-acylurea 176. This can be confirmed by the peaks in the range of 24-32 ppm and these peaks being

145 secondary/quaternary carbons. If there is formation of desired product, this area (region of alicyclics/aliphatics) can’t produce these many peaks. For further confirmation, the tertiary carbons and the carbonyl carbons match perfectly with the undesired N-acylurea.

This may be attributed due to following reasons

 Conformation achieved by the 2, 6 disubstituted pipecolic acid.

 The bulkiness of the amine synthon due to two aromatic rings.

 The electronic properties of the amine synthon as there are two aromatic rings which alters the nucleophilicity of the amine.

If the bulkiness and nucleophilicity of amine are sole reasons, this type of reaction when performed using 2-substituted pipecolic acid (as shown in scheme 58) shouldn’t work either. As it worked with 2-substituted pipecolic acid but not for 2, 6 disubstituted pipecolic acid, it shows the conformation achieved by this substrate might have been the reason which posed issues during the coupling reaction with this kind of amine (may be the secondary reason).

After failing to synthesize the mono-peptide using DCC mediated coupling, we tried many other coupling agents as tabulated below.

Br O O Br Coupling agent N X OH + N N H N PG PG O O X X=Phenyl, CF3 A B F PG-Cbz, Boc Scheme 66.showing the general peptide coupling reaction We tried a series of coupling agents based on literature [79]. We tried to optimize the condition suitable for both small scale and gram scale synthesis. The coupling agents we employed were as follows

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% Yield

Coupling agent Additive Amine

Phenyl CF3

DCC DMAP - - EDC HOBt - - HBTU TEA - * COMU® - * T3P®/DCM TEA 40 37 T3P®/DMF TEA - - DEPBT DIPEA 72 -

Table 26.showing the employed coupling agents (-not worked, *not tried) (Blue-works for both amines, Red-works for benzyl amine alone)

Coupling agents like EDC, HBTU, and COMU® showed no desired product formation whatsoever. Thus, we tried to put forth the discussion only on the coupling agents which gave the desired mono-peptide F from now on.

Propane Phosphonic Acid Anhydride-T3P®

T3P® is chemically propane phosphonic acid anhydride developed by Euticals pharmaceuticals [80]. This coupling agent is prominent for being one of the best coupling agents available in the market due to its low epimerization (1.8%), high reaction selectivity and purity along with ease of handling [80] [87].

We applied T3P® to synthesize the desired mono-peptide with the two amines 160 and 161. The reaction condition followed is as follows

Br O O Br T3P, Et3N OH + X N N N H N Cbz DCM, RT Cbz O O X B X= CF3-37% 159 phenyl-40% G

Scheme 67.showing the T3P® peptide coupling giving desired compound

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Even though the yields were not good, this coupling agent worked for both the amine substrates we used. To make a point, the same reaction when carried in DMF didn’t forward and gave the starting materials as such. The mechanism with which it proceeds is as follows

O O O O P HN N O O P P X O O O O O O O OH O N P P P N N T3P Cbz Cbz O O O O O O O O

Br O 159

N X N Cbz O G

Scheme 68.showing the mechanism of T3P® peptide coupling The reaction proceeds by an initial deprotonation by a base to form a carboxylate which attacks the cyclic propane phosphonic acid anhydride. This attack opens up the T3P® and forms a highly activated group on the acid substrate. A final nucleophilic attack by the amine B results in yielding desired amide G with side product which can be easily washed-off through water workup. Thus this reaction gives relatively clean reaction mixture at the end of the reaction as the byproduct formed is of phosphoric acid which is thoroughly soluble in water. This reaction worked well when we used T3P®/DCM 50 wt % solution. Whereas, when we tried the same reaction in T3P®/DMF 50 wt % solution, there was no desired product but the starting materials were recovered as such.

As the yield of the reaction when used with T3P® was not good, we tried another organophosphorous coupling agent, DEPBT.

3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one-DEPBT

DEPBT is an organophosphorous coupling agent belonging to the family of benzotriazine class of coupling agents [81]. It is another coupling agent which showed the best results for resisting the racemization of substrate as well as formed amides during the coupling reactions [82]. Hence, we applied DEPBT for the synthesis of our mono-peptide.

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With the condition retrieved from the literature, we have applied the same on the synthesis of our mono-peptides. The synthetic condition followed was

Br O O Br DEPBT, DIPEA OH X N N + H N DCM, RT N PG PG O O X X=CF , Phenyl A B 3 PG-Boc, Cbz F

Scheme 69.showing the DEPBT peptide coupling giving desired compound With DEPBT as coupling agent, we were able to synthesize the desired mono-peptide when the amine substrate was benzyl with an excellent yield. Whereas, if the amine substrate was CF3, the reaction wasn’t going forward and the starting materials were recovered as such. The reaction proceeds in a similar fashion as that of other benzotriazine class of coupling agents. An initial deprotonation of acid substrate follows a nucleophilic attack of the carboxylate onto the phosphoryl group of DEPBT which finally forms an activated benzotriazine-acid substrate complex. This activated complex, on nucleophilic attack of amine B onto it results in the desired mono-peptide F.

O O O N N N N EtO O N EtO O EtO P N P N O O O O OH N EtO N N PG PG O PG O O O O A DEPBT Br

O B NH Br O O N X N N N PG N O N O PG O X

X=CF3, Phenyl F Scheme 70.showing the mechanism of DEPBT peptide coupling

We tried 2, 6 piperidone acid substrates with different protecting groups and the yields are as follows

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N° Acid substrate Amine substrate Product Yield (%)

Br O

Br N N N H O O O 86

O 160 177 OH 1 N Boc O Br O 158 Br No product N N N CF3 formation H O CF O O 3

161 178

Br O

N N O O O 72 160

O 179

OH 2 N Cbz O Br O 159 N N O CF O O 3 No product formation 161

180

Table 27.Showing the employed substrates for DEPBT mediated coupling

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The DEPBT mediated coupling worked well with benzyl amine substrate and showed no product formation for CF3 amine. Even in protecting groups, it showed partiality with di- tert-Butyl carbamate giving major yield of 86% and benzyl carbamate giving 72%.

Note: The analysis of the synthesized compounds was done with the help of Electrospray ionization Mass Spectroscopy (ESI-MS) from now on because of the problem posed during the interpretation of NMR spectra due to the occurrence of 4 rotamers.

Thus formed mono-peptides have been forwarded to the next step, deprotection of the PGs to facilitate a C-N cross coupling reaction.

6. Deprotection of mono-peptides

The mono-peptides achieved in the previous step have been subjected to deprotection of the protecting groups in order to carry out a C-N cross coupling in the future steps. Well documented and mostly used methodologies have been applied to obtain the deprotection of tert-Butyl carbamate and benzyl carbamate protecting groups [67].

For deprotection of both the protecting groups, we used conditions from literature which are used on similar amide substrates as these kinds of substrates are highly prone for hydrolysis in presence of acid/base along with small amount of water.

6.1 Deprotection of tert-Butyl carbamate

The deprotection of tert-Butyl carbamate was optimized earlier with the help of trifluoroacetic acid in dichloromethane with the help of small amount of water using racemic substrate without any substitution on 6th position and cyclic acetal protection on 4th position.

This method was followed with the similar condition as reported and optimized previously as below

Br Br O O

TFA, H O N 2 N N N DCM, RT H O O O O

178 182 Scheme 71.showing the deprotection of tert-Butyl carbamate from the substrate

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Following the same condition which has been optimized, we weren’t able to reproduce the deprotection of tert-Butyl carbamate moiety. This might be due to two reasons;

 The absence of ketal at 4th position as this may change the conformation of the piperidine chair.

 The substitution at 6th position by methyl group which may be posing either a sterical hindrance for the attack or changing the conformation from that of the already optimized substrate.

The substitution of ketal at 4th position changes the entire conformation of the piperidine has been studied by using 2, 6 cis piperidine ester.

Figure 67.showing the 1H NMR showing piperidinone When there is ketone on 4th, even though it still remains in a chair conformation, the strain of the chair varies. The splitting of both vicinal and geminal protons of both axial and equatorial protons on 3rd and 5th position changes considerably.

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On the other hand, we can compare it with that of coupling constants of the piperidine ester with ketal at 4th position which clearly shows the change of conformation from each other.

Figure 68.showing the 1H NMR showing piperidine with ketal at 4th position Thus, this perhaps may be one of the reasons that the deprotection of tert-Butyl carbamate from piperidine with cycle acetal in previously optimized condition was working and for ketone due to the conformational strain, the deprotection may not be going forward.

Hence we tried to reprotect the ketone to ketal. One of the easiest methods to obtain a methoxy ketal is to do Fischer-Speier esterification using thionyl chloride. But we can’t follow this due to the presence of hydrolysable amide bond. Hence we tried other method retrieved from literature as follows [83]

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Br Br O OH OH O O

pTSOH N N N N DCM, Reflux O O O O O O

178 183

Scheme 72.showing the protection of ketone to cyclic ketal

Thus, due to the in-applicability of other harsh methods, we applied the above said condition in para toluene sulfonic acid with 1,3 propanediol with dichloromethane as solvent which gave no protection at all.

In another study, which will be discussed in family 3 of the same chapter, we showed that the 2,6 substituted piperidines are formed from a tert-Butyl carbamate α, β-unsaturated ketones via an intermediate ketone to methoxy ketal followed by cyclization into piperidine and finally deprotection of tert-Butyl carbamate to give a free amine by using p-TsOH in trimethylorthoformate [84, 85].

Hence, this also confirms that the deprotection of tert-Butyl carbamate was done after the protection of ketone to ketal. Thus, we applied the same methodology here with the conditions as below

Br Br Br O O O O O

pTSOH N N N + N N N H (CH O) CH, H O O 3 3 O O O RT 93% 184 185 178 Scheme 73.showing the protection of ketone to methoxy ketal & in situ deprotection of tert-Butyl carbamate

When applied the above condition for an in situ protection of ketone and deprotection of tert-Butyl carbamate, the reaction gave desired product with an excellent yield and minimal time (7 h).

The above proposed reaction can be monitored in another way as below

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Br Br Br O O O O 0.2 eq TFA/H O pTSOH 2 N N N N N N DCM H (CH3O)3CH, H O O O 91% O O RT 93% 184+185 186+187 178 Scheme 74.showing the protection of pTsOH quantity affecting the deprotection

When 0.2 equivalents of pTsOH was added alone, it results in protection of ketone alone but wasn’t able to deprotect the tert-Butyl carbamate. The deprotection of tert-Butyl carbamate can be achieved by the optimized condition of trifluoroacetic acid in small amounts of water. With this step, we not just monitored the proceedings of the reaction but also, we wanted to know the effect of substitution on 4th position during the C-N cross coupling reaction.

6.2 Deprotection of the benzylcarbamate on the piperidine

Benzyl carbamate protecting group can be deprotected by many conditions like saponification, acid/base hydrolysis, catalytic hydrogenation etc [67]. But the problem face again was due to the presence of amide bond in our structure which will be hydrolysed. On a simple note, we can also do a hydrogenation, but due to the presence of bromide on the aromatic ring, which gets debrominated during the hydrogenation we can’t carry this type of deprotection as well.

Hence we chose trimethylsilyl iodide which in the literature has been applied for the similar kind of substrates [67, 86] and we handled carefully to prevent hydrolysis. The condition we followed was as below

Br Br O O

1-15 equiv. N TMSI, Et3N N N N H O X O X CH3CN, 0 °C O O H

X=phenyl, CF3

G Scheme 75.showing the deprotection of benzyl carbamate by TMSI

As depicted above, the reaction gave no deprotected product even on addition up to 15 equivalents of TMSI. Hence, we thought the problem which we encountered during the tert- Butyl carbamate deprotection might have been the reason which was posing the difficulties in

155 deprotection of benzyl carbamate as well. Thus, we applied the same strategy to protect the ketone to ketal which might facilitate in deprotection of benzyl carbamate.

Br Br O O O

pTSOH N N N N (CH O) CH, O X 3 3 O X O O RT O O 91% G F X=phenyl, CF3

Scheme 76.showing the protection of ketone to methoxy ketal

Hence forth, we were able to protect the ketone in facile condition but we can’t deprotect the benzyl carbamate. We took thus formed compound and engaged in deprotection using the TMSI with similar conditions as follows

Br Br O O O 1-15 equiv. N TMSI, Et3N N N N O X CH CN, 0 °C O X O O 3 O O

F G X=phenyl, CF 3 Scheme 77.showing the deprotection of benzyl carbamate with TMSI

However, after the reaction, we were able to deprotect the ketal to form a ketone but we weren’t able to deprotect the benzyl carbamate. Hence, we came to the same compound where we started i.e. a ketone.

Thus obtained product can’t be cyclized due to the protection of amine and hence, it was engaged to synthesize the subclass of family 1 with an amide fragment as discussed earlier.

Whereas the compounds which were deprotected from tert-Butyl carbamate has been engaged in a C-N cross coupling reaction.

7. C-N cross coupling reaction

Thus formed mono-peptides from previous step have been engaged into a cyclization reaction by forming a C-N bond. From the optimizations done within the laboratory as presented in previous section, we chose Buchwald-Hartwig cross coupling reaction to be the

156 principle in forming the C-N bond. Hence, we took the same conditions as optimized previously and applied onto our amide synthon C. The reaction was followed as below

Br O O O O 0.1 Pd(OAc)2, 0.15 Xantphos O N N N Cs2CO3 N H O Toluene, 110 °C

184 188

Scheme 78.showing the C-N cross coupling using Pd(OAc)2/Xantphos

When we used the same condition developed initially, we weren’t able to forward the reaction to get our desired product. Even on doubling the catalytic load, there was no formation of desired product. Thus, keeping the solvent constant, we optimized the reaction using another catalytic system as follows;

Br O O O O 0.05 eq Pd2(dba)3, 0.075 eq BINAP O N N N NaOtBu N H O Toluene, 110 °C 52% 184 188

Scheme 79.showing the C-N cross coupling using Pd2(dba)3/BINAP

This optimization step has been inherited from the same kind of C-N cross coupling reaction of family 3. And hence, the in-depth analysis, mechanistic review and optimization conditions have been discussed in the results and discussion section of family 3.

The effect of substitution at 4th position played crucial role during various stages of our synthesis. To see the effect of the same on the C-N cross coupling reaction, we used the optimized condition and applied on various substrates as below

Br X X

0.05 eq Pd2(dba)3, 0.075 eq BINAP O N N N NaOtBu N H O Toluene, 110 °C

I J

Scheme 80.showing the C-N cross coupling using Pd2(dba)3/BINAP

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N° X Conformation Condition Yield (%)

1 O 2, 6 cis 0.05 eq Pd2(dba)3 - 0.0075 eq BINAP

O O 1.2 eq NaotBu

2 2, 6 trans Dry toluene, 110 °C, - Sealed tube

Table 28.showing the 4th position affecting C-N cross coupling

The results as tabulated above showed that the 2, 6 cis can form a C-N bond but not the 2, 6 trans and the ketone at 4th position. The keto group at 4th position can change the entire conformation as we presented earlier. Hence, this might be the reason for non formation of C- N bond. In case of 2, 6 cis, the conformer is as depicted below and due to less distance between the aromatic halogen and amine, the C-N cross coupling might have gone forward.

H O O O O O O H H C 3 O H O O H H N N HN H N N N Easy H availablility Br Br 188 2, 6 cis

Scheme 81.showing the 2, 6 cis affecting C-N cross coupling And its counterpart, the 2, 6 trans wasn’t able to form a C-N bond due to the attained conformation as below

O O O O Br O H O O O N H N N H3C O H N N Too distant H H H HN 2, 6 tr ans H Br 189

Scheme 82.showing the 2, 6 cis affecting C-N cross coupling As we can see, the distance between the aromatic halogen and amine is too long and this might affect the formation of C-N bond. And so, we weren’t able to retrieve the cyclized form of trans.

158

Meanwhile, the rotamers which were posing the problem since the protection of amine and formation of peptide bond has totally gone astray once the cyclization has been done. As a result, a spotless and uncluttered spectrum has been recorded for this cyclized compound.

159

Figure 69.showing the disappearance of rotamers on cyclizing Thus the cyclized as well as the N-protected mono-peptides has been forwarded to the next step where the deprotection of ketal to ketone has been performed.

8. Deprotection of ketal

Thus formed amides and a benzodiazepanone from previous reactions have to be finally deprotected from a ketal to a ketone at 4th position. As presented in previous sections, this has been optimized within the laboratory and thus, we applied this condition to obtain the desired ketones. The generalized reaction is as follows

O O O TFA, H2O

DCM, RT N N

K L Scheme 83.showing the deprotection of ketal to ketone

We applied this condition on many substrates and their deprotection went smoothly with good yields.

N° Substrate Product Reference Yield (%)

O O O

1 O 189 In-progress O N N N N

O O O

O O N N Cbz Cbz N 2 N 190 98 Br Br

O O O

O O N N Cbz Cbz N 3 N 191 95 Br Br

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O O O

O O N N Cbz Cbz N 4 N 192 91 Br Br

O O O

O O N N Cbz Cbz N CF3 5 N CF3 193 89 Br Br

Table 29.showing the deprotection of ketal to ketone These deprotected ketones have been forwarded to the next step, where the Functional Group Interconversion (FGI) to an amine using a series of modulations. This FGI part has been conferred after the discussion of family 3 in general.

III. STRATEGY FOR THE SYNTHESIS OF FAMILY 3

Candidate 194 of the family 3 could be obtained from the corresponding Ketal 197 after chemicals transformation and coupling with 142.

HN X NH2 O   O O R   FGI   N  N  N  N 

Y Y Y Y

194 195 196 197

Y=NH R=H, NH2 X= N N NH O, propan-1-ol N 142

Scheme 84.showing the retrosynthetic strategy for family 3 This proposed scheme portray that the desired urea like molecule 194 can be obtained by uniting the fragment 142 with that of 195. Whereas in fragment 195, the stereochemistry is modulates as we saw in the previous chapter, incorporation of asymmetric carbons deemed to

162 be fruitful in a better reach and interaction with the amino acids in the binding pocket of CGRP receptor. Therefore, stereochemistry is fixed according to the molecules prioritized based on the docking results. This fragment 195 with the desired stereochemistry on the piperidinic moiety can be obtained from ketone intermediate 196 by using the Functional Group Interconversion (FGI) principle. The retro synthetic strategy for accessing family 3 ketal, with the core tetracyclic moiety can be framed as depicted in the scheme 85 and the substitution on the starting key piperidine is the factor affecting the nature of the second heteroatom on the seven membered rings.

O O O O O O

NH2 NO2 subclass A N N N H NH R R = H, COOR 201 IIA IA 198 IIIA

O O I I O O O O O O O O N subclass B N N N Y H R O R = H, COOR 197 202 IIB IB 199 IIIB

O O O O O O R NH Br

N N N H subclass C R N R = H, COOR 203 IIC 200 IIIC IC OH Scheme 85.showing retro synthetic scheme for family 3 (Y=NH, O, propan-1-ol) The intermediate 197 of each subclass can be obtained by using intra or intermolecular cross-coupling reaction to form C-N bond from the desired synthons II of respective subclass. The synthons III of all subclasses can be obtained without any transformation from the corresponding piperidine III of each subclass or after a reduction or N-deprotection or coupling reaction. The various piperidine synthons III as presented during the bibliographic study will be synthesized using Aza-Michael intramolecular reaction [88, 89, 90]. Similar to family 1, the key intermediate reaction ‘Horner-Wadsworth-Emmons’ preceding with phosphonate insertion into the substrate was applied to transform 204 to V and finally to IV [91, 92]. Synthon VI is mostly available commercially and sometimes synthesized by nucleophilic substitution reaction. Finally, synthon 204 is obtained by Aza-Michael insertion

163 of Davies amine onto the commercially available methyl crotonate 205 [93]. The orientation/configuration of asymmetric carbon will be fixed at the first step with the help of Davies amine.

O O O PG PG NH O NH O O  OEt H   P  + N OEt Y H R Y R Y R

III IV V VI

Ph Ph  O I or Br N O O Y=NO2,Br,F,  OMe OMe

204 205 R=H,NO2 Stereoselectivity R or S Scheme 86.showing retro synthetic scheme for synthon III The retro-synthetic design for key synthon III is crucial. The attribution of both stereocenters and the desired substituants are done during the synthesis of synthon III.

Synthon III is mostly kept constant and obtained in similar way for all the three subclasses of family 3.

Imperative points derived from this synthetic strategy  The synthesis of final potential hit is achieved by ‘convergent synthesis’ same as we saw earlier in family 1.  Like family 1, there are no steps which induce 2 stereocenters in one single step.  The Aza-Michael reaction acts as key step to introduce stereocenters on the final molecule, once during conversion of 205 to 204 and again during IV to III.  Substituants on the final molecule 194 depends on the aldehyde VI which we use in the formation of the synthon IV.  Just by using corresponding aldehydes VI, all three subclasses of family 3 can be obtained.  Another key asymmetric center on the final molecule 194 is obtained by FGI from 196 to 195. Above derived strategies for the synthesis of family 3 molecules are applied to obtain them with desired stereochemistry and substituants.

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A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME

In previous discussion, the synthetic scheme to obtain the final hit molecule 194 has been successfully designed. In the current segment, the application of the designed scheme will be discussed step-by-step in detail.

2.6 Piperidine Synthon III

The synthon III has been obtained using asymmetric synthesis from commercially available starting material, methyl crotonate. The methodology has been designed and developed within the laboratory by using Michael reaction [90]. The first step in synthesis of synthon III is formation of (S)-methyl 3-(benzyl((R)-1-phenylethyl)amino)butanoate.

For all the three subclasses of family 3, the synthetic methodology remains alike until synthon III formation.

1.1 Synthesis of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate

The synthesis of family 3 molecules begins with this step which involves formation of (S)-methyl 3-(benzyl((R)-1-phenylethyl)amino)butanoate. This step results in attribution of a stereocenter at β position. Among the available methods in the literature [95, 96], the following method is most facile, takes bare minimum steps to obtain the desired product and gives highest amount of diastereomeric ratio (94%).

Ph Ph O N nBuLi (S) N O OMe + H -78 °C, (S) OMe THF anhyd. 205 S-Davies amine dr >94% 204

Scheme 87.showing Aza-Michael addition of Davies amine to α,β-unsaturated ester This step involves Aza-Michael reaction, where a conjugate addition of enantiopure lithium (S)-N-benzyl-1-phenylethanamine to an α,β-unsaturated ester i.e. methyl crotonate was done at -78 °C using anhydrous conditions to give compound 204.

The diastereomeric ratio is normally above 94% and this can be accredited to the structural conformation of Davies amine. The two phenyl groups in Davies amine attain a minimum energy conformation as a sandwiched model or a butterfly model where both of them lie parallel to each other. This is also called as π-stacking [95]. The nucleophilic attack

165 of lone pair of electron on nitrogen onto the double bond of methyl crotonate can take place in two ways, above the plane and below the plane of the α,β-unsaturated ester (Figure 70).

Figure 70.showing the diastereomeric selectivity for the formation of compound 1 The stereochemistry of the methyl group on Davies amine plays crucial role in determining the position of attack taking place and subsequently the stereochemistry obtained in the compound 204. In our case, S-Davies amine, the methyl group lies below the plane and hence the attack of the Davies amine onto the α,β-unsaturated ester happens from below the plane. This is due to the steric hindrance posed by the methyl group on Davies amine.

Compound 204 obtained from this step have been previously studied in two different ways to obtain the compound V. They are as follows,

S-Davies O amine, Ph Ph nBuLi OMe (S) N O -78 °C, THF anhyd. (S) OMe 205 dr >94% 204

R A out oute e B R H nB /C, 2 uLi OH) 2 Pd( - O H, 78 °C Ph Ph MeO THF , PSI, anh + 60 0% 7 yd. O NH3 O 8 4% N O O (S) OEt P OMe (S) OEt 206 207 O O OEt P Protection of amine Hydrogenation OEt

- PG Cl NH O PG NH + O O O O HN O O 3 OEt OEt OEt P P OMe P OEt OEt Phosphonation OEt Protection of VII V amine 209 208

Figure 71.showing two routes to obtain compound V In route A, the compound 204 was engaged into hydrogenation to give 206 which was followed by protecting the amine functionality by suitable protecting group. In final step,

166 phosphonation reaction was followed to obtain compound V. On contrary, in route B, compound 204 was allowed to undergo phosphonation as the fundamental step to obtain 207 and later applied with hydrogenation to recover a free amine in salt form 209. But the hydrogenation reaction from 207 gave 208 instead of 209 due to β-elimination and subsequent reduction of alkene. Even though variety of hydrogenation conditions like Pearlman’s catalyst with 60 pound per square inch (PSI) in methanol or Pd/C in methanol and reflux with ammonium formate as a hydrogen donor, there occurs formation of 208 [97]. To present in general, whatsoever the condition is, the final compound ultimately deemed to be 208.

As the desired product V from route B can’t be achieved, we have nominated route A as the prime way of synthesis of compound V. Route A starts with the N-debenzylation of compound 204 as discussed in the next section.

1.2 Synthesis of (S)-4-methoxy-4-oxobutan-2-aminium acetate

Compound 204 obtained in the previous step was engaged in hydrogenation reaction using Pearlman’s catalyst, Pd(OH)2/C [98, 99]. Among the available methods, the proposed method deemed to be more facile and gives desired product 211 with least by products (mono bezylated amine) [56].

Ph Ph O Pd(OH)2/C, H2 + (S) N O O NH3 O 60PSI, MeOH OMe OMe (S) H2O:AcOH/1:1 (S) 204 211 Scheme 88.showing hydrogenation of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate Not just the catalyst, but also solvent and acid used plays a crucial role in obtaining the desired product. Methanol and ethanol are considered to be the best solvent to give compound 211 faster while other solvents increase the time of product formation [98]. Even the pressure employed plays determining factor in deriving the compound 211 without any side products [99]. We followed the heterogeneous catalytic method for the N-debenzylation of compound 204 using Parr apparatus. As obtained from the literature, we used Pd(OH)2 as catalyst with methanol being the solvent and carried the reaction at 60 PSI pressure to derive compound 211 [100]. This method in general gives free amine but due to the presence of acetic acid, the acetic acid salt of amine 211 is obtained.

167

Thus obtained compound 211 without any purification was engaged in next step where the free amines have been protected using various protecting groups.

1.3 Protection of (S)-3-methoxy-3-oxobutan-1-aminium acetate

In order to prevent the side product formation in future steps, the amine functionality of compound 211 has been protected. The rationale for using protecting groups (PGs) depends on several factors as listed below.

 The PGs should give good quantity of desired compound  The PGs shouldn’t pose any steric hindrance in the succeeding reactions.  The protection step shouldn’t alter any other functionalities of the molecule.  The PGs should have facile cleavage step.  The deprotecting step shouldn’t affect any other functionality changes of the molecule.

These main reasons made us to selected di-tert-butyl dicarbonate ((Boc)2O), benzyl chloroformate (Cbz) and ethyl carbamate.

O O (RCO)2O RCO Cl, R O- + 2 O NH O NH3 O Na2CO3 O O CH2Cl2/H2O

211 VII

R=phenyl, t-butyl, ethyl Scheme 89.showing protection of (S)-4-methoxy-4-oxobutan-2-aminium acetate Yield % N° substrate PGs Time(h) Product (3 steps) O O 1 7 O NH O 55 Cl OEt O O 212 O O O O O- + NH3 O 2 O O 6 O NH O 58 O O 211 213 O O

3 O Cl 8 O NH O 52 O 214

Table 30.showing different PGs used with their yield

We followed a generalized method for protection of compound 211 using carbamate PGs. This method has been retrieved from literature [101, 102, 103] which uses a facile

168 condition as stated in the procedure. First time since the beginning of reaction, purification has been carried out and the yield was quantified. The overall percentage yields in all the cases were ranging in and around 50%. On the other hand, we used some PG like ethyl carbamate which is less bulky to overcome steric hindrance in further steps. Overall, the desired compounds were obtained in and around 52-58% in 6-8 h. We used various PGs to study a range of factors affecting Michael cyclization reaction which we applied during later stages of the synthesis. Thus obtained N-protected amines VII were subjected to phosphonation reaction in the next stage.

1.4 Synthesis of ketophosphonates

A generalized phophonation condition has been applied using the guidelines from literature to give compound VIII [85].

(EtO)2P(O)Me PG PG NH O nBuLi NH O O OEt P O -78 °C, OEt THF anhyd VII V

Scheme 90.showing synthesis of ketophosphonates

The N-protected esters VII were transformed in ketophosphonates by treating with 2.5 equiv of diethyl lithomethylphosphonate in anhydrous condition at -78 °C using THF. Substrate Time N° ketophosphonates Yield (%) (min) O O

O NH O O NH O O 1 40 OEt 55 P O OEt 215 O O

O NH O O NH O O 2 20 OEt 63 P O OEt 216 O O

O NH O O NH O O 3 35 OEt 48 P O OEt 217 Table 31.showing different ketophosphonates with their yield

Unlike in family 1, use of diethyl methylphosphonate for synthesizing ketophosphonates V didn’t pose any obstacles during purification step. Excess of diethyl methylphosphonate used in the reaction can be easily removed by distillation at reduced pressures followed by column chromatography yields ketophosphonates in the range of 48-65%.

169

Similar to that of family 1, the synthesized ketophosphonates V has been used as substrates for the key intermediate Horner-Wadsworth-Emmons reaction.

1.5 Synthesis of α,β-unsaturated ketones: application of Horner-Wadsworth- Emmons condition

The ketophosphonates synthesized in previous step has been treated with different aldehydes using Horner-Wadsworth-Emmons (HWE) reaction to get various α,β-unsaturated ketones with desired substituants. These substituants are the result of chosen aldehyde during the reaction. Most of the aldehydes we used were commercially available apart from some of them. However, they are typically aromatic aldehydes and can be synthesized using nucleophilic substitution reaction as followed [105].

i. Synthesis of biaryl aldehydes

Along with the commercially available aldehydes used to synthesize α,β-unsaturated ketones, we used some other biaryl aldehydes. These biaryl aldehydes were synthesized using facile nucleophilic substitution reaction on various substrates but from similar reagents and methodology retrieved from the literature [106].

O H O X X Y=OH, NH H K2CO3 Y 2 + X=I, Br Y F DMF anhyd Y'=O,NH 2-fluoro IX X benzaldehyde Scheme 91.showing synthesis of biaryl aldehydes N° Nucleophile Aldehyde Time (hours) Product Yield (%) H O I I 1 O 97

OH O 7 218 H O Br Br H 2 O 98 F OH 219 H O I H 3 I 10 N 20

NH2 220 Table 32.showing different biaryl aldehydes with their yield

Using the nucleophilic substitution reaction, compounds 218, 219 were synthesized in good yields.

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Whereas in the case of 220, the yield is mere 20% in 1H NMR integration and making the situation worse, on purification, perhaps due to the instability of aldehyde 220 we weren’t able to isolate the desired product. Hence, we followed route B instead of route A.

PG HN O O OEt  P OEt O IV

H Route A Route B O NH H I Horner-Wadsworth-Emmons NO2

PG Michael O O PG HN O intramolecular HN O reaction     N H O2N HN O2N I XII XIII XI

Michael Reduction intramolecular reaction

O O O O Br O O

   Br   N  N N C-N cross H C-N cross H coupling NH coupling H N HN reaction reaction 2 221 I 198 201

Scheme 92.showing routes to obtain compound 198 For route B, commercially available 2-nitrobenzaldehyde was used during HWE which can be followed by reduction from nitro to amine. The final tetracyclic skeleton 198 was achieved by a C-N cross coupling reaction. Hence, this route was found to be more feasible and thus employed to obtain 198 instead of route A. Apart from the above discussed aldehydes, we used other aldehydes based on the subclass which we have to synthesize. They include 2-Bromobenzaldehyde, 2-Fluoro-5- nitrobenzaldehyde. And thus, we retrieved all the required starting material for key reaction HWE.

ii. Horner-Wadsworth-Emmons

This key intermediate step will be determining factor as the aldehyde used during the course of this reaction results as substituants in the final molecule. We followed similar methodology as we discussed previously for family 1. We used facile and workup-friendly

171 chemicals like barium hydroxide monohydride as the base instead of some harsh chemicals like Et3N/LiCl, DBU etc used in literature [refer family-1]. The general synthetic methodology is as follows

O I or Br O PG PG NH O NH O O Ba(OH)2.H2O  OEt H Y=NO2,Br,F, P +  OEt Y THF/H O:40/1, RT R 2 Y R R=H,NO2 V VI IV Scheme 93.showing Horner-Wadsworth-Emmons reaction

Compound V was subjected to HWE using series of aldehydes VI to give compound IV. As presented earlier, HWE is highly stereoselective reaction and gives E-alkene as a major compound. Mechanism and transition states involved in HWE to give E-alkene have been discussed in detail in family 1. The yield of compound IV was variable depending on the PGs used as depicted in the following table.

N° Phosphonate IV Aldehyde Time Compound V Yield (h) (%)

O Br O NH O Br

H 1.5 88 1 O O 222 O NH O O O OEt P OEt O NH O H O 2 I O 4 91 O I 223 O 3 2 O NH O Br 90 224 O O

4 H 3 O NH O NO2 75 O2N 225 O

O NH O 5 O 2 70 O NH O O OEt O P I OEt 226

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O H O Br O NH O 6 O 1.5 86

O Br 227 O O O NH O NO2 94 7 H 1.5

O2N O 228 O NH O O O OEt P O O NH O F OEt NO 8 H 2 2 79 F

NO2 229 Table 33.showing different alkenes with their yield

The general applicability of the condition we used for HWE has been studied with various aldehydes. As we can see, the reaction times and the yields are good for all the series of phosphonates we used. Hence, the retrieved compound IV was engaged in the next step i.e. Aza-Michael intramolecular cyclization.

1. Aza-Michael intramolecular cyclization

The series synthesized during HWE was later subjected to Aza-Michael intramolecular cyclization. As shown during the bibliographical survey on 2,6 disubstituted piperidones, Aza-Michael reaction is prime choice for synthesis of 2,6 disubstituted piperidones with intramolecular type cyclization in chiral form.

We developed a simple but efficient reaction condition for Aza-Michael intramolecular reaction to obtain piperidines [85]. The reaction was carried in presence of p- toluenesulfonic acid, trimethylorthoformate and alcohol as shown below.

OH RO OR RO OR O HO n or R3O NH O MeOH R1 N R2 R1 N R2 R1 R2 (CH3O)3CH, p-TsOH R3O O R3O O R =CH -(CH ) -,-(CH ) - XI 3, 2 2 2 3 2,6-Trans 2,6-Cis n =1, 2 XII XIII Scheme 94.showing general approach for Aza-Michael intramolecular cyclization

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Diastereoselectivity of the synthesized molecules depends on various factors. Some of the most important descriptors are as follows  The nature of the alcohol used for the generation of acetal plays crucial role in stereoselectivity. It also showed that the stereochemistry of the intermediate acetal (scheme 95) was independent of the configuration (either E or Z alkene) of the α,β unsaturated ketone compound IV.

O O HB HO OH O O EtO NH O OEt OEt EtO N (CH3O)3CH H HA O O Z/E : 40/60 p-TsOH J-HAHB = 15.5 Hz 230 231

Transcetalation

O OEt (CH3O)3CH O O O p-TsOH EtO N HB H HA

J-HAHB= 8.4 Hz 232 Scheme 95.showing effect of alcohol used for protection of ketone Steric hindrance posed by the substituants played crucial role during the Aza-Michael intramolecular cyclization. A bulky and steric hindrance posing substituants results in no cyclization of the α,β unsaturated ketone compound V.  Electronic effects generated by the aromatic group positioned near β of the α,β unsaturated ketal. When the electronic effects are highly donating, it results in mesomeric effect and finally preventing cyclization.

O O O (CH O) CH O NH O 3 3 N pTsOH:H2O O O OMe OMe 233 234 Scheme 96. Showing mesomeric effect in cyclization This can be accounted as the reason to select the skeleton 199 of the subclass B to use the biaryl 202. Undeniably, doublet doublets of oxygen are shared between the two aromatic rings and thereby reduces the donor effect and could allow the cyclization on the 4-position of the Michael acceptor.

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 The quantity of the acid source used has also deemed to be important factor. The amount of the acid influence the kinetics of the reaction as well the cis to trans ratio of the formed piperidine. Thus, keeping in mind the above factors we designed our synthesis accordingly. For the synthesis of our piperidines, we used a generalized condition which we optimized to reach the 2,6-cis piperidine conformation as major compound. An initial addition of 0.2 equivalents of pTsOH with 5 to 10 equivalents of trimethylorthoformate at room temperature was followed. The methoxy substituants of the ketal were generated in situ by decomposition of the trimethylorthoformate in acidic conditions. This ketal was chosen in order to avoid a high steric hindrance due to the substituants in the ortho position of the aromatic ring.

O O 53% PG O O O O X 0.2 eq NH O X 1.2 eq X TsOH:H O X p 2 pTsOH:H2O N + (CH O) CH N N PG (CH3O)3CH 3 3 H H IV PG-Boc XIV III cis III tr ans 60-85% cis/trans Not separable 0.2 eq TsOH:H O p 2 O O PG-Cbz, (CH O) CH ethyl carbamate 3 3 X 1 eq pTsOH:H2O N Boc (CH3O)3CH cis/trans Not separable Scheme 97.showing Michael intramolecular reaction in general context We applied this on variety of E-alkenes for studying the applicability of the developed reaction. In general, this reaction gave satisfactory yields of desired product with cis/trans ratio fluctuating.

175

N° Alkene T compound cis/trans Yield (h) (%) O O O O O O NH O Br 1 0.5 Br Br 60/40 85 + N N

O O O O

235 O O O O O O NH O NO2 NO2 2 1.5 NO2 71/29 81 + N N

O O O O

236

O O O O

NO2 O NO2 + N N 3 O NH O NO2 7 72/28 76 O O O O

237

O O O O O F F O NH O F + 4 7 N N 70/30 71 O O O O NO NO2 2 NO2

238 Table 34.showing application of Michael intramolecular reaction for ethyl and benzyl carbamate PGs

As presented earlier, we tried on variety of substrates and studied the effect of substituants on the yield and ratios of isomers formed. When we tried the PG to be ethyl and benzyl carbamate, the reaction forwards by the protection of ketone to ketal and cyclization mediated by 0.2 equivalents of p-TsOH to form piperidines.

176

N° Alkene T compound cis/trans Yield (h) (%) O O O O O O NH O NO2 NO 1 4 NO2 2 67/33 65 + N N H H

239 O O O O O 2 O NH O Br 12 65/35 88 + N N H H Br Br 240 O O O O O O NH O

N N 3 10 H H 60/40 76 O + O O I I I

202 O O O O O O NH O

N 6 10 N H 65/35 72 O H + O O Br Br Br

241 Table 35.showing application of Michael intramolecular reaction for tert-Butyl carbamate PG

Whereas, when the PG is tert-Butyl carbamate, there occurs protection of ketone to ketal and cyclization mediated by 0.2 equivalents of p-TsOH to form piperidine and further addition of 1 equivalent of p-TsOH deprotects the tert-Butyl carbamate in situ. This is attributed to the acid mediated deprotection of the tert-Butyl carbamate and for the other PGs we used, the deprotection condition is not the same. Another point to be noted that, when the piperidine formed is N-carbamate, the cis and trans can’t be isolated using column chromatography. Hence, we used 1H NMR to integrate and quantify the ratios of cis/trans.

Henceforth, the piperidines formed using Michael intramolecular reaction were sorted and carried to the next step where the tetracyclic moieties were formed.

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2. Formation of tetracyclic moiety Thus formed piperidines in Aza-Michael intramolecular cyclization were categorized according to their applicability in each subclass of family 3. To brush up, we already showed that the family 3 was further classified into three subclasses. Their structures are as follows

O O O O O O R    N  N  N  R=H, NH2

NH O N

OH 198 199 200 Subclass A Subclass B Subclass C Figure 72.showing three subclasses of family 3 This family of molecules was classified into subclasses based on heteroatomic substitution in their core moiety. They were designed to study the hydrogen bond donor, acceptor and a homologated hydrogen bond donor effect towards the receptor interaction and consequently its biological activity. Thus, to avoid the perplexity over the synthesis of each of them, from now on we will deal with individual subclass of family 3 until we reach core tetracyclic moiety.

2.1 Subclass A

Subclass A represents tetracyclic core moiety with a hydrogen bond donor (NH, in our case) as the heteroatomic substitution in it. Its synthetic design was as follows

178

Not separable by column

O O O O O NO NH2 2 * * O NH O NO2 * * Route A * (CH O) CH 3 3 N N pTSOH 1.Reduction O O O O Yield= 81 % 2.Coupling 242 3.Deprotection 226 4.Coupling 243 Trans /cis : 29/71 Br O O

1.Deprotection Br * 2.Reduction Route B N * 3.Coupling O O NH NH2 244 * * O O O N H 198 O NH O NO NO2 2 * * * (CH3O)3CH Route C Subclass A N a pTSOH H 1.Reduction Yield= 79 % 2.Coupling Route Cb Trans /cis : 33/67 235 Separable by column 239

O O O NO2 * * O NH O NO2 N * (CH3O)3CH 237 pTSOH O O

228 Yield= 65 %

Trans /cis : 28/72

Not separable by column

Scheme 98.showing multiple approaches to reach subclass A Route A: It starts with the reduction of compound 226 which was followed by a C-N cross coupling reaction. This cross coupling was done by using 1,2 dihalogenated benzene (in our case; 1,2 dibromobenzene) and this mono cross coupled compound was later deprotected from ethyl carbamate which exposes free amine group. The last step is to follow-up another C-N cross coupling to give desired compound. Overall, it takes 4 steps to retrieve the desired product. Route B: It starts with the deprotection of ethyl carbamate from compound 226 which was followed by reduction of nitro functionality to amine. To finish, the concluding step is a double C-N cross coupling reaction to form a cycle. Overall, it takes 3 steps to retrieve the desired product. Route C:

Route Ca: It starts with a piperidine moiety 239 with free amine functionality and a nitro group. Thus, we start from reduction of nitro group to amine

179

Route Cb: It starts with a piperidine moiety 237 with Cbz protection and a nitro group. Hydrogenation was followed to do an in situ deprotection of Cbz as well as reduction of nitro to amine.

Both route Ca & Cb were later followed by a similar double C-N cross coupling reaction as discussed in route B. Overall, it takes just 2 steps to obtain the desired product. Hence, amongst all the routes, route C was found to be more ergonomic and feasible which persuaded us to follow this route to obtain subclass A.

i. Application of designed route C

The designed route Ca was applied for synthesizing the subclass of family 3. It can be prepared in two-step process. The first step involves reduction and second step is a C-N Palladium cross-coupling reaction using 1,2-dibromobenzene [107]. The Buchwald-Hartwig reaction have been choosen, as the prime C-N bond forming reaction for all the molecules we have synthesized because of its applicability on our substrate as portrayed during the optimization of family 1. When the protecting group is tert-Butyl carbamate, the starting material can be obtained in two-step process, a cyclisation and in situ deprotection followed by hydrogenation from nitro to amine.Whereas, if the PG is benzyl carbamate, after cyclization, hydrogenation can be performed to deprrotect and reduce nitro to amine in situ.

O O O O O O O O NO NO Pd/C, H 2 2 2 NH NH2 1 atm, RT 2 225, 228 + N + N N N H R R MeOH H Quantitativ R=H, Cbz R=H, Cbz e IIIA 244

Scheme 99.Showing formation of amine

N° Ketone Cyclisation Reduction Total Yield(%) Yield (%) Yield(%) (cis/trans) (cis/trans) 1 225 65 (67/33) Quantitative (67/33) 65 2 228 76 (72/28) Quantitative (72/28) 76

Table 36.Ratios of piperidine formed

180

Thus obtained 2, 6 cis piperidine has been forwarded to the succeeding step for the C-N cross coupling reaction.

O O O O Br

NH2 Br N N H "Pd" NH

244 198

Scheme 100.showing route C to reach subclass A The compound 244 thus obtained acts as substrate for Buchwald-Hartwig cross coupling reaction. We considered the following points before commencing the cross coupling reaction [108].  Buchwald stated that monodentate phosphine ligands are ineffective with aryl iodides as they allow them to form more stable palladium-iodide dimers.  Pd-C and Pd-P rotation barriers established to be superior for bigger halides.

is more labile than

 1,2 diiodobenzene is relatively expensive than 1,2 dibromobenzene. And thus, above stated points made us to use a bidentate ligand and also gave us a hint that bromo substrates are more favoured to give desired compound. We tried different catalysts and confined with the usage of bidentate ligands. Different product can be form during the cyclization process, the tetracycle A, the desired moiety and both mono cross coupled moieties B, C corresponding to the first insertion of the di-bromide by coupling reaction (scheme 101).

Br O O O O O O O O Br HN NH2 NH2 Br N N N N H H Br Reflux NH

244 198 245 246

Scheme 101. Design for the synthesis of subclass A The results for this optimization were as tabulated below

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N Catalyst Ligand Base(n.eq.) Time Isolated Condition ° (mol %) (mol %) (h) Yield(%)

1 Pd(OAc)2 (5) Xantphos Cs2CO3 (1,2) 48 SM (100) Toluene (110°C) (7.5)

2 Pd2(dba)3(5) BINAP (7.5) NaOtBu (1.2) 12 A(55), B+C (40) Toluene (110°C)

3 Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) 24 A(60), B+C(35) Mesitylene (110°C)

4 Pd(dba)2(10) BINAP (15) NaOtBu (1.2) 72 A(65), B+C(30) Toluene (110°C)

5 Pd(dba)2(10) BINAP (15) NaOtBu (1.2) 24 A (quantitative) Toluene sealed tube (130°C)

5 Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) 24 A (quantitative) Toluene sealed tube (130°C)

6 Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) 24 A (quantitative) Dioxane sealed tube (120°C) Table 37.showing different various Buchwald-Hartwig conditions As the results in table 37 clearly shows that using Bis(dibenzylideneacetone)palladium

(Pd(dba)2) catalyst with (2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) (BINAP) gave the desired compound in quantitative yield. The reaction can be done in a polar or non polar solvent, and the best conditions were obtained when a sealed tube was used. Then to decrease the time of the reaction we wanted to optimize further in the future to try microwave condition and to use dioxane as a solvent for better activation.

The same condition was then applied on the piperidine 2,6-trans and it give exactly the same result in dioxane as the piperidine 2,6-cis. The yield of 80 % in toluene was attributed to the lower solubility of this diastereoisomer in this solvent.

On entries 2, 3 and 4 we came across side products B & C. To understand the whole mechanism of the cyclization, we wanted to know which of the two C-N cross couplings takes place first. Either the first C-N bond insertion was on the piperidine nitrogen or on the aniline. Hence, we investigated on the piperidine 239 by trying the following method using the same catalytic system which gave us the desired product.

O O O O Pd(dba) 2 NO NO BINAP 2 2 Br N N + NaOtBu, H Br Br anhyd. Toluene sealed tube <10% yield 239 247

182

Scheme 102.showing investigation on first C-N cross coupling After 24 hours, only 10 % (calculated through 1H NMR integration) of desired compound X was observed. This concludes that the preferred formation of desired compound forwards in the following fashion.

Br O O O O O O NH 2 Br Intermolecular HN Intramolecular N + N H N Br H NH

244 245 198 Scheme 103.showing prime site for C-N cross coupling The first C-N bond formation takes place on the aniline moiety followed by the intramolecular C-N bond connection on the piperidine ring. This mechanism is similar to the formation of the benzodiazepanone family 1 ring. Merely no product was obtained if the first C-N bond was tried on the piperidine ring by intermolecular reaction. This concludes that the formation of the 7 membered ring can be reached only by intramolecular C-N formation. Then according to the above disclosed results, we can propose the following mechanism, in two cross coupling cycles to get the desired tetracyclic subclass A.

O O O O

0 O O Pd (L)2 Br 0 Pd (L)2 N HN H N Br Reductive Oxidative Br Oxidative N NH elimination Reductive H addition NH elimination addition O O Br 245 O O 245 198

N H N O O HN L Br Pd Pd L L NH HN Pd(II)L2Br Second cross coupling cycle Pd(II)L Br L First cross coupling cycle N 2 Br H

NaOtBu NaOtBu HOtBu HOtBu O O Br NaBr NaBr HN Pd(II)L2OtBu O O Pd(II)L OtBu N 2 H NH2 N 244 H Scheme 104.Mechanism for Buchwald-Hartwig C-N coupling Thus the Scheme 104 showing prime site for C-N cross coupling predicts that the Buchwald-Hartwig reaction for subclass A compounds happens in above depicted mechanism. In the first cycle, first step includes oxidative addition of 1,2 dibromobenzene to a Pd(0) species which will be followed by deprotonation, addition of amine to the oxidative

183 addition complex and finally a reductive elimination to windup the first cycle to give a mono cross coupled product. The second cycle follows the same pattern but starts with the mono coupled product formed in the first cycle which finally ends up to give the tetracyclic subclass A. We tried to study the effect of 2, 6 cis and 2, 6 trans as described in the family 1. For this study, we used the following method

O O O O Pd(dba)2 NH BINAP 2 Br N N + NaOtBu, H Br anhyd. Dioxane NH sealed tube 81% yield 244 198 Scheme 105.showing trans C-N cross coupling But, in case of family 3, the trans position is obtained due to the changing of methyl from equatorial to axial but not the substitution at 2nd position. As a result, we were easily able to cyclize the 2, 6 trans with same yields as that of 2,6 cis.

Figure 73. NMR showing the cyclized 198

184

Thus formed compound 198 as shown in above 1H NMR clearly shows the coupling of two C-N connections. This can be attributed to multiple facts that we can see an aniline like broad singlet around 9.35 ppm and most importantly, disappearance of the proton beside a C- Br bond. Henceforth, the synthesis of tetracyclic subclass A proceeded and recovered as above. We applied the same principle which we used in subclass A to the next subclass i.e. B.

2.2 Subclass B Subclass B represents tetracyclic core moiety with a hydrogen bond acceptor (O, in our case) as the heteroatomic substitution in it. According to the conclusion drawn from the possible mechanism of the formation of the 7-membered ring of subclass B, piperidine 241 (Br) and 202 (I) were synthesized which differs only on the nature of the halogen on the aromatic ring to reach the desired tetracore subclass B 199 by C-N coupling reaction. Its synthetic design was as follows and the cyclization steps was study on the piperidine 2,6-cis.

I O O O O Br O O O O O 'Pd' 'Pd' O N N H N H N O O H

199 202 248 241 Subclass B

Scheme 106.showing design for synthesis of subclass B In this cyclization, we were able to form either the desired product 199 or a secondary product 248 resulted due to beta-hydride elimination or catalyst decomposition as we showed in the catalytic cycle below as well as according to the results described in the literature.

185

O O

N O O O O Pd0(L) Reductive 2 I elimination N 199 H Oxidative addition 202

O O O O

N O O N H L Pd O L O O beta-hydride Pd(II)L I elimination N 2 H 248 NaOtBu

HOtBu O O O NaI Pd(II)L OtBu N 2 H

Scheme 107.Showing the catalytic cycle for subclass B The cycle goes in a similar fashion as shown in the subclass A for the second cross coupling cycle. It starts by an oxidative addition resulting of the insertion of the palladium (0) on the R-X bond. The intermediate is obtained after deprotonation of the amine on the piperidine ring and coupling intramolecular connection with the palladium. At this stage, two reactions are possible, either the reductive elimination to reach the desired 7 member ring or the β-hydride elimination to give the de-halogenated product 248. To be pointed out here, this side product wasn’t observed in the previous subclass A.

Firstly, we applied the optimized condition found for subclass A are show on the table 38 :

N Catalyst Ligand Base(n.eq.) Time NMR Condition ° (mol %) (mol %) (h) calculation (%)

A Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) 24 A (30) + C (70) Toluene sealed tube (130°C)

B Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) 24 B(30), C (40) + D Toluene sealed tube (30) (130°C) Table 38.Showing the variance of substrate

186

According this table, comparing the result between the bromide and iodide substrates, the cylization was observed only on the iodide substrate. The results also share the information that a lot of hydrogenated side product was generated. We weren’t able to separate the 3 molecules (Starting material, cyclic, hydrogenated) by purification on a column chromatography as all the 3 products have the same Rf on TLC. Then to precisely identify and confirm the hypothesis and to quantify using 1H NMR & 13C NMR, we had engaged the piperidine 202 in a hydrogenation reaction in presence of Pd(OH)2/C at 1 atmosphere of H2 (scheme 108).

I O O O O

O Pd(OH)2/C N N H H H 2 quantitative O 202 248 Scheme 108.Hydrogenation of compound 248 As we saw in the earlier discussions, the aryl iodides form more stable complexes with the catalytic-ligand system and even if a reductive amination reaction takes place, it will be very slow. By this time, the side reaction called β-hydride elimination happens and results in de-halogenation of the substrate as seen in our case.

Contemplations on cross coupling reaction by Buchwald:

With the persistence of the problem created due to the β-hydride elimination, we did the review of literature to identify and sort-out the problem. Some of the key facts identified are as follows  There are no catalytic system which is universally applicable for all kinds of substrates during a cross coupling reaction [109, 110].

 For instance, the amines and amides diverge in their nucleophilicity and pKa which means that the rate determining step during the catalytic cycle can vary with the substrate [111].

 Not just palladium source, ligand or the base etc determines the cross coupling reaction but taken as a whole, all of them are important contributors for the C-N bond formation [112]. Their individual contributions are as follows

187

R R X H N N R R

1.Pd source Y Y 2.Ligand 3.Temperature 4.Solvent 5.Base Figure 74.Contributors for Buchwald-Hartwig C-N coupling N° Factor Contribution 1 Palladium source Impacts efficient formation of active catalyst species 2 Ligand Choice depends on substrate combination 3 Temperature Influences rate of cross-coupling and formation of by-products 4 Solvent Determines solubility of substrates and base 5 Base Choice of base influences rate, functional group tolerance and byproduct formation 6 H Electronic & steric properties influences rate of amine N R R binding/deprotonation and reductive elimination 7 X Influences rate of oxidative addition, amine binding/deprotonation 8 Y Electronic properties influences rate of oxidative addition, amine binding/deprotonation, reductive elimination Table 39.showing contributors for Buchwald-Hartwig C-N coupling

 Although Pd(OAc)2 is attractive even on large scale amination, its reactivity poses

some drawback for using on lot of substrates [113]. On contrary, preheating Pd2(dba)3, ligand and base in solvent prior to the addition of substrates is highly beneficial and

thus highlights the formation of the L1Pd complex [114].

No additives Active required, No additives species reduction of Pd required not required Pd (dba) L1Pd0) 2 3 NH Pd 2 Not applicable dba can bind for all ligands Pd and poison L Cl reaction Anilines and L=Monodentate amides needs Economic ligands exogenous reductants

Pd(OAc)2 Figure 75.Choice of different catalytic systems on Buchwald-Hartwig C-N coupling

188

 Toluene has been proved as the prime choice of solvents even though 1,4 dioxane shows similar profile but un-favored due to its toxicity. Toluene is particularly advantageous in the coupling of aryl iodides due to its weak ability to solubilize the inorganic iodide salts formed during the course of reaction [115]. Polar solvents like DMF or DMA also proved to forward the reaction however, with low reaction yields [116].  The choice of base is always the tricky part as the conventional and traditionally used base like NaOt-Bu, even though one of the versatile of all and mostly used for Pd- catalysed amination, it can form side reactions due to its strong basic nature (pKa

17.0) [117]. Even Cs2CO3 proved to be efficient but shows similar basicity like NaOt- Bu [118].  The presence of electron donating or withdrawing substituants on ortho position affects the catalytic cycle and will be decisive in rate of the reaction [112].  Interestingly, the aryl iodides, being the easiest class of electrophile for C-C cross coupling poses difficulties during the Pd-catalyzed amination [119, 120]. Mechanistic study shows that this intricacy has been caused by the unreactive Pd dimers bridged by iodide anions. Some monodentate ligands like Brettphos and RuPhos have shown efficient amination in primary and secondary amines respectively [115].  To sum it up, a simple guide for some commonly encountered problems with its solution was proposed as follows [112] R R X H N N R R

Pd source, Y Ligand, Y Base/solvent

Problem Probable reason Solution Insufficient formation of Employ readily activable active catalytic species precatalysts Low conversion Increase catalyst loading, Low rate of reaction Perform the reaction at high temperatures Incompatibility of base with Employ a weaker base with Poor mass functional groups in substrate better functional group balance/low yield tolerance Catalyst decomposition Perform reaction at a lower Formation of temperature

189

H Inefficient reductive Use a ligand which gives elimination faster reductive elimination

Y Table 40.Showing problems and how they can be solved according to Buchwald

Overall, Buchwald proposed that the ligand like Pd2(dba)3 with bidentate ligand like BINAP, dppf, Xantphos with sodium tert-butoxide as the base and running the reaction in non-polar solvent, toluene might give desired product in good yields. Whereas, he also proposed that the reaction might not give the desired compound if the substrate is an aryl iodide and suggest that the reaction takes place if the monodentate ligands like Brettphos, XPhos and RuPhos are used for aryl iodides.

On this conclusion we decided to try the XPhos and Pd(OAc)2 as the source of Palladium. We have also decided to investigate the influence of the concentration on the intramolecular reaction and the influence of the load of catalyst used. The results are reported below on the table 109. Reactions were performed on toluene, in sealed tube at 130 °C over a period of 72h and the base used was the NaOtBu or Cs2CO3.

I O O O O O O O 'Pd' N N H N H O O

248 202 199 Scheme 109.Optimization of catalytic systems for subclass B N° Catalyst Ligand Base NMR calculation Conc. (mol %) (mol %) (1.2) (%) Mol/l

1 Pd(OAc)2 (5) Xantphos (7.5) CO3Cs2 (1,2) SM (100)/CY(0)/HY(0) 0.05

1 Pd(OAc)2 (10) XPhos (15) NaOtBu (1,2) SM (50)/CY(0)/HY(50) 0.05

2 Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) SM (30)/CY(40)/HY(30) 0.05

3 Pd(dba)2(5) BINAP (7.5) NaOtBu (1.2) SM (25)/CY(45)/HY(30) 0.05

4 Pd2(dba)3(5) BINAP (15) NaOtBu (1.2) SM (20)/CY(50)/HY(30) 0.1

5 Pd2(dba)3(10) BINAP (30) NaOtBu (1.2) SM (0)/CY(70)/HY(30) 0.1

Table 41.showing different various Buchwald-Hartwig conditions we tried

190

The first conclusion was to perform the reaction, as we did for the first subclass, in presence of bidentate ligand. The use of XPhos has not shown the formation of desired 7 member ring. The influence of the concentration was put in front in comparison with entries 2 and 4. More concentrated, the intermolecular palladium reaction following by the reductive animation was more efficient. The ratio of catalytic system also affects the formation of the desired product and unfavored the hydrogenated product. At present, we weren’t able to optimize a perfect condition to obtain the desired product. The next trials will be changing the bidentate ligands like 1,1'-Bis(diphenylphosphino)ferrocene or 1,3- Bis(diphenylphosphino)propane and trying in more polar solvents like Dioxane etc. As detailed earlier, 202, 199 and 248 have same retardation factor and thus can’t be purified. So, to recover the pure CY tetracycle the crude mixture of the entry 5 was engaged in N- protection to form the trifluoroacetamide derivative of the 248. Then 199 and HY-CF3 have been isolated separately and the yield after column of 202 was 52 % (scheme 110).

O O O O O O O O O (CF CO) O 3 2 N N N H N Et3N O O O O CF3

199 248 199 248-A

Scheme 110.protection of HY As a one last attempt, we tried another strategy based on the subclass A methodology. In here, the corresponding piperidine 240 which possess a bromide group on the aromatic ring was used as a substrate to couple with the 1,2-bromophenol. The condition applied was toluene as solvent in presence of Pd2(dBba)3/BINAP as this deemed to be the best condition for the subclass A. But this also proved to be unfruitful. And hence, when, we tried to see if this type of cyclization works using 1,2 dibromoaniline which obviously gives the Subclass A, there was no product formation whatsoever.

Pd dba O O O O 2 3 Br Binap Br HX Toluene 110°C N N + H NaOtBu X X-N,O 240 199-A Scheme 111. Another way to achieve Subclass B Hence, this shows that this type of cyclization can’t be reached with our substrate.

191

2.3 Subclass C Subclass C is an analogue of the Subclass A and it represents a tetracyclic core moiety with a hydrogen bond donor (OH) on the chain substituted to the nitrogen on the 7 membered ring. This chain will pave’s a path to reach a hydrogen interaction with a Glycine on the RAMP-1 of the CGRP receptor. Another possible interaction with a meta substitution on one of the phenyl group to reach Asp 78 in the RAMP-1 part of the CGRP receptor was also identified during the molecular studies.

Family 3 Subclass C (Total Score : 11.6)

O O N X N NH

N R4 N ASP Y Possible HH effet

O O N O N NH

N N

Polarity : 2 Hydrogens bond Polarité : 1 suplementary hydrogen bond interactions interaction • Tyrosine : H-H : 2.0 Å • Glycine : H-H • H2O : H-H : 1.9 Å Figure 76. Showing the conformation of sublcass C in CGRP pocket According to the docking scores and the results obtained for the subclass A, we have proposed 3 different ways to reach the desired molecules (scheme 112). Route A, B and C depending of the substitution (NO2 or H) on the meta position of the aromatic ring. NO2 will be the precursor of the NH2 function that we targeted to do an interaction with the aspartic acid on the pocket.

192

H2N O O O O OR1 Br OR1 HN

N N 298 Cbz Cbz Buchwald Hartwig 297 cross coupling ROUTE A Br Buchwald Hartwig cross coupling OR1= OMOM or OBn Br

R1= H or NO2

O O ROUTE C O O R 2 Alkylation R O O N N I OBn X 200 + N N R2 =H, NO2 NH 303 X=Cl, F 299 Boc OR1 198 NO2 Nucleophilic Br Buchwald Hartwig substitution cross coupling Br ROUTE B H2N OBn 300

O O O O HN OBn HN OBn N N Boc Deprotection H of Boc NO 2 302 NO2 301 Scheme 112.Showing the designed synthetic scheme for family 3 subclass c

When R2 = H

Two routes can be investigated, route A and C. From the corresponding N-benzyl carbamate piperidine 297, the tetracycle 200 can be obtained after two metalcatalyzed reaction and one N-piperidine deprotection. N insertion of the well-chosen amine followed by the Buchwald cross coupling reaction optimized for the subclass A can be used here. In the route C, a simple alkylation of the 7 member ring subclass A could be utilized.

When R2 = NO2

Similar to the route A, route B N-tert-Butyl carbamate piperidine can be used as the starting material which possess an aromatic ring substituted by chloro or fluoro in 2nd position and nitro in 5th position. In 3 steps, after nucleophilic substitution of the selected amine, N deprotection and cyclization, the desired tetracycle 200 can be obtained.

Initially, we have prepared 300 and 303 molecule needed for our different routes.

Preparation of 303

193

Two different ways [35] were described in the literature to get the precursor of mono protected iodide 303. First method involves benzaldehyde, where the transformation of the carbonyl functions into the dioxolane Y1, which was subjected to react with the DIBAL-H to form the benzyloxyethanol Y2 with an overall yield of 19 %. The second way investigated was the direct mono alkylation of the corresponding ethylene glycol in presence of benzyl bromide. According the yield, the second way was preferred. The mono alcohol was then transformed into the iodide derivative by reaction in presence of iodine and triphenyl phosphine under a “Wittig” type procedure with a yield of 53 % [122].

O O O H OH Dibal-H H HO PPh3, Imidazole HO I OBn OBn pTsOH Toluene, MeOH, I2, CH2 Cl2, 0 °C 53% Toluene, Refiux Y NaOH, 0 °C Y Y 21% 1 89% 2 3 NaH, Br DMF, 0 °C, 28%

OH HO

Scheme 113.Showing the synthesis of O-protected iodoethanol

Preparation of W

In route A, the piperidine formed during Michael intramolecular cyclization will be engaged in a C-N cross coupling reaction. The amine required for this cross coupling reaction has been prepared used well know and documented Gabriel type synthesis (scheme 114) [124]. The same Gabriel type synthesis was use to prepare W3a and W3b amines. We chose the PGs depending on the condition of reaction and deprotection during the next step.

H N O 2 O O R-X NH2NH2 OH H2N O N N EtOH Toluene OH DMF OR reflux OR O reflux O Base O 307 R-Bn-60% 306 R-Bn-55% 304 305 OMOM-50% OMOM-83%

Scheme 114.showing the O-protected amine through Gabriel synthesis After condensation of the ethanolamine on phthalic anhydride, the desired phthalimide

W1 was obtained with a quantitative yield and O alkylated with the opted protecting group.

194

The free amine was then obtained after hydrazinolysis with an overall yield ranging around 30 % respectively for the O-MOM and O-Bn derivative.

Formation of the tetracore Route C: R2 = H

From the tetracore 198 and previous optimizations and through procedures already described in the literature on the 7 member ring skeleton [140]. We have tried to N-alkylate the aniline group, and the results are showed in the table 42.

O O O O

Alkylating agent N N Base N NH OBn

Scheme 115.showing the alkylation designed according to route C N° Alkylating agent Reactifs conditions Yield (%)

1 NaNH2, Toluene Reflux 12h --

2 I OBn n-BuLi, THF -78°C to rt -- 24h

3 NaH, THF 0°C then -- reflux 12h

4 Br OMe n-BuLi, THF -78°C to rt -- O 24h

5 Ethyl glyoxilate NaBH4 overnight --

Table 42.Showing the alkylation of subclass A with different conditions As showed in the differents entries, various conditions were employed: N-alkylation by using a halogenated substrat or aldehyde subtract. None of the case, we could manage to transform the tetracore 200, certainly due to the low reactivity of the aniline fonction.

Formation of the tetracore Route A and B : R2 = H, and NO2

We shifted to route A and B as it involves the conditions of cross coupling reaction to form the 7-member ring 200 and as we optimized the conditions as presented earlier with a similar substrate, we thought of applying the same condition over here. Initially, precursor 297 and 298 were prepared to be engaged in the Buchwald cross coupling reaction.

195

The coupling reaction by using the optimized condition as described in the previous section to form a C-N bond with a yield of 88% has been followed by hydrogenation to free the amine using Pearlman’s catalyst under 1 atm of H2 304 was obtaining with an overall yield of 72%.

H2N O O O O OMOM OMOM O O Br HN OMOM HN N N N Cbz 0.01 eq Pd(dba)2 H 0.015 eq BINAP Cbz Pd(OH)2/C NaOtBu H2, RT 297 Toluene, 100 °C, 12 h 298 Quantitative 304 88% Scheme 116.Showing route A In route B, the first step to prepare the precursor XX before cyclization involves a nucleophilic substitution (scheme X) reaction. The transformation was observed only when the halogen is a fluoride. Moreover, under those conditions, the nitrogen was deprotected in situ which had allowed us to reach the desired XX in one step with 65 % of yield.

H2N O O O O OBn X OBn HN

N N H Boc Cs2CO3 reflux, Toluene NO 2 10 h NO2

299 X-F,65% 302 -Cl-No reaction Scheme 117. Showing route B

Thus formed compound was engaged into the C-N cross coupling reaction with the optimized condition used for the subclass A and the result are given below (scheme 118 and table 43):

Br O O O O OR HN 1 NO2 Br N N H 0.01 eq Pd(dba)2 0.015 eq BINAP N NaOtBu R2 Toluene, 100 °C, 48 h OR1 302 200

Scheme 118.showing cyclization of subclass C

196

N° R1 R2 Yield (%) 1 MOM H Sealed tube 0

2 Bn OBn Sealed tube 0

3 Bn OBn MW 6 (calculated by NMR)

Table 43.Cyclization optimization for subclass C Three trails were conducted on this substrate, but this reaction wasn’t going forward even on doubling the load and running the reaction for longer time. Only under microwave we were able to observe a mere transformation of 6% which was calculated through 1H NMR. We started optimizing these reactions at the end of my PhD and so, no further optimization methodologies were performed. In future, we would like to use another solvent like dioxane in microwave condition which could increase the energy to the catalytic system which might give us the desired product.

Deprotection of ketal

The ketals formed during the previous steps have been deprotected so that they can be transferred to the next step where the FGI takes place. The general deprotection methodology we followed was optimized within the team [refer 1st chapter]. Hence, the method followed was

O O O TFA/H2O

N DCM, RT N

Scheme 119.showing the general deprotection method

We tried this method on two of the moieties from family 3, one from subclass A and the other from B. Their yields are as follows

N° Substrate Product Yield (%)

O O O 1 95 N N

NH NH

198 250

197

O O O 2 91 N N

O O

199 251 Table 44.showing the deprotection substrates and products With this, we have successfully reached the ketone of both the subclass moieties, now let’s proceed to the next step where the FGI occurs and finally these fragments will be converged to obtain the potential CGRP receptor antagonists.

IV. FUNCTIONAL GROUP INTERCONVERSION

The ketones obtained from ketal are then converted into amines using a series of Functional Group Interconversion (FGI) principle. We will present here a general structure representing all our synthesized molecules. The initial design to obtain the amines from ketones is as follows

NH2 O OH N3 Reduction Reduction SN2 N N N N XVIII XV XVI XVII Scheme 120.showing general synthetic scheme to obtain amine from ketone The ketones obtained from deprotection of ketals as we saw in previous chapter, can be reduced to obtain the alcohol. These alcohols will be chemically transformed into corresponding azide according to SN2 process in one or two steps. At this stage, the stereocenter of the position 4 on the piperidine ring will be fixed in accordance with the docking calculations. Then, the azides will be reduced to form the desired amine fragment which would be advanced to the final compound on convergence with the privileged structure. This was our initial prototype to synthesize the amines from ketones. Now let us look into each individual steps in detail.

Reduction of ketone to alcohol

According the literature, differents reductives agent could be used to form alcohol from the corresponding ketone. This reduction can form a mixture of enantiomers as shown in scheme 121, and this selectivity is entirely dependent of the reducing agent used [123, 124].

198

Most preferred O H H H H H OH OH O H H H H H H H H O Na H OH H H H H H + B H H H Na H H H H H H N N H HN HN HN H H H H H Sodium Major Minor borohydride Equatorial alcohol

Li O H H H O H H OH OH OH H O H H H H H H H H Li B H H H + H H H H H H H H H N N HN HN HN H H H H H L-selectride Major Minor Axial alcohol Most preferred Scheme 121.Showing the reduction of piperidone using sodium borohydride and L-selectride If the reducing agent is a bulky one like L-selectride, due to the 1,3-diaxial interaction, the hydride attack takes place from below the plane due to steric hindrance posed by the piperidone conformation. As a result, there is formation of axial alcohol in majority. On contrary, if the hydride source is small, the attack can occur from any side of the plane but it prefers the attack from above the plane to give the more stable equatorial alcohol. Thus, a general depiction of reduction from ketone to alcohol can be shown as follows

OH O OH

NaBH4

MeOH, RT N N N + Quantitative Major XV XVI Minor Scheme 122.Showing the configuration achieved after reduciton Hence, a similar strategy is followed for reduction of all the ketones and the yields in all the cases are quantitative. But the formed diastereoisomers can’t be purified using column chromatography due to no difference in retardation factors.

199

N° Substrate Major Product Yield (%) O OH

N N N N 1 Cbz O Br Cbz O Br

281

O OH

N N N 2 N Cbz O Br Cbz O Br

285 O F F OH F CF3 N N N 3 Cbz O Br N Cbz O Br

284 Quantitative O OH 4 N Boc N Boc 283 O OH 5 N N

NH NH

252 O OH 6 N N

O O

282 Table 45. Showing reduction of ketone to alcohol For family 3 we can show the case of reduction of subclass A of family 3. The synthetic scheme followed was as given below

200

OH O OH

(S) NaBH4 (S) N N (S) + N MeOH, RT NH NH Quantitative NH

250 Major-79% Minor-21% 252 Scheme 123.showing the diasteroisomeric ration of alcohols formed The diastereoisomer formation has been identified and confirmed by using 1H NMR coupling constant (figure 76), and the ratios have been calculated using integration.

Figure 77.showing the formation of equatorial and axial alcohols The formation of equatorial alcohol as major product can be accredited because of the characteristic axial coupling constant mesure on the tdd signal at 3.38 ppm of proton at 4th position with large coupling constants of 11.2. At this stage H2, H4 and H6 are three axial proton according theirs coupling constant on the piperiding ring. A de of 60 % in favor of the equatorial alcohol (2S, 4S, 6S) have been calculated. Both diastereoisomeres wasn’t separable and used as a mixture in the following step.

Application of Mitsunobu reaction (direct SN2) formation The formed alcohols from previous steps were converted into azide directly with the help of Mitsunobu reaction [125, 126]. Till now this condition has been tested for two of our

201 substrates, one belonging to family 1 and the other for subclass A of family 3. In previous study on racemic structure belonging to family 1, the conditions we applied were as follows.

O O O O P O N3 N OH 3 EtO N N OEt N3 (dppa) O O (DEAD) O N N N N N N DBU PPh3, dppa toluene dry THF 0°C to RT RT 253 254 255 Scheme 124.showing the application of Mitsunobu condition for racemic structure This condition has been tried previously within the laboratory to convert the alcohols belonging to family 1 directly to an azide. But we weren’t able to achieve this conversion even on using two variants of azodicarboxylate sources. But when the same condition was applied for the synthesis of subclass A of family 3, the desired product was formed. The desired azide product thus formed without any purification was engaged into reduction from azide to amine to give the final fragment of subclass A belonging to family 3.

O O

OH O N N O N3 NH2 (DIAD) PPh3, DPPA N N N PPh3,dry THF THF/H2O-10/1 NH RT NH RT NH

Overall yield 252 256 257 62% Scheme 125.showing the application of Mitsunobu condition for subclass A of family 3 The first step involves the Mitsunobu reaction mediated by DIAD to convert the alcohol to azide. This conversion is SN2 type reaction and thus there is inversion of stereochemistry at 4th position from equatorial to axial [127]. Thus formed desired product without any purification was engaged into the succeeding step to reduce the azide into amine. The overall yield of this reaction was 62% after purification and occurs in a two-step process from the alcohol into the amine.

As the application of Mitsunobu failed for family 1, we have followed the previously designed step through the activation of alcohol for the formation of azides.

202

Activation of alcohol

The alcohols of family 1 obtained from ketones through reduction were then activated in order to obtain an intermediate with good leaving groups i.e. sulfonates [128]. In a previous study conducted within the laboratory, we tried two different sulfonates for the activation of alcohols as depicted below.

OMs OTs OH

TsCl, Et N O MsCl, Et N O O 3 3 N N N N N N DCM, RT DCM, RT

259 254 258 Scheme 126.showing the activation of alcohols of family 1 Hence, the activation using mesylate gave the desired activated sulfonate whereas the tosylate activation gave no desired product [129]. Thus, we used this condition to apply for our synthesis.

OH OMs

MsCl, Et3N

N DCM, RT N

XVI XIX Scheme 127.showing the activation of alcohols using mesylate for family 1 N° Substrate Product Yield (%) OH OMs

1 N N 94 N N Cbz O Br Cbz O Br

260 OH OMs

2 N N 92 N N Cbz O Br Cbz O Br

261

OH OMs CF3 CF3

3 N N 96 N N Cbz O Br Cbz O Br

203

262

OH OMs

4 97 N N Boc Boc 263

Table 46.showing the activation of alcohols using mesylate for family 1

Thus activated alcohols using mesylate were converted into azides using a nucleophilic substitution reaction.

Nucleophilic substitution reaction to form azides and sunsequent amines

The active sulfonates formed from previous step were converted into corresponding azides using an SN2 reaction, in presence of sodium azide in reflux of the DMF [130]. Due to this, an inversion of configuration happens at 4th position. Thus, all the mesylates which were in equatorial position interchanges into axial azides. Thus formed azides, mostly gave clean reaction mixture. Hence, we didn’t perform the purification step and converted the azides to amine in succeeding step by using Staudinger reaction. They were transformed into amines using triphenylphosphine mediated reduction reaction [131]. We chose this clause over the others due to the mild conditions followed during this step which won’t remove the bromide on the aromatic ring. Along with this, triphenylphosphine mediated reduction reaction on these type of chiral substrates prevents racemization of starting materials as stated in the literature [132].

After optimized all the steps retrieved as well known in the literature [124, 131], we applied the best condition to an overall 3 steps process and purified after reduction into amine:

204

NH OMs N3 2

NaN3 PPh3

DMF, 85 °C N H2O/THF-1/10 N N RT 24 h 10 h XIX XVII XVIII Scheme 128.. showing the activation of alcohols using mesylate for family 1 N° Substrate Product Yield (%)

OMs NH2

1 N N N N 72 Cbz O Br Cbz O Br

264

OMs NH2

2 N N N N 68 Cbz O Br Cbz O Br

265

OMs NH2 CF3 CF3 3 N N 76 N N Cbz O Br Cbz O Br

266

OMs NH2

4 62 N N Boc Boc 267

Table 47.showing the conversion of mesylate to amine for family 1

An overall yield ranging from 62-76% was observed over a three-step process. Thus formed amines are considered as the RAMP-1 fragments and carried onto the next step where it was coupled with a privileged structure using convergent synthetic methodology.

205

V. CONVERGENCE OF THE TWO FRAGMENTS

The amine fragments of the family 1& 3 as briefed in previous chapters have interactions with the RAMP-1 part of the CGRP receptor protein. On contrary, the ‘privileged structure’ interacting with CALCRL (refer chapter 2) which was kept constant for almost all the CGRP receptor antagonists is a 1-(Piperidin-4-yl)-1H-imidazo[4,5-b]-pyridin-2(3H)-one (as used in case of Telcagepant®). This substructure was seen in more than 1000 of the CGRP antagonists which are tested either in preclinical or clinical stages of their discovery or developmental process [133, 134].

Br OH

Br F3C O O H F N O O N F O N N N H N O N N N NH N H N N N O H NH2 K = 0.77 nM Ki = 0.0144 nM i Olcegepant Telcagepant Figure 78.Showing the CALCRL fragments used for potent CGRP antagonists Due to their discrepancy from one CALCRL interacting fragment in more potent Olcegepant® to that of Telcagepant® (refer the red-highlighted fragment), we tested both the fragments individually as well as a substructure for our antagonists (see chapter 4 : biological evaluation of molecules). Previous study within the group assayed using Writhing test in mice showed that 289 possessed potent activity than the other side chains when coupled with our RAMP-1 fragments.

O NH O NH O NH O HN N N HN N X NH X = HN

N O N N 268 269 270

14% 28% 10%

Pain inhibition

The results has been followed by another test using hot-plate method inorder to validate the results and even it showed that the fragment used in Telcagepant® is more potent

206 than that of the one used in Olcegepant® with over 5 folds of difference. This part of the results and discussion has been put forwarded in the pharmacological tests of our antagonists in the next chapter.

® These results made us to choose the fragment of Telcagepant and we carried further with our convergent synthesis to retrieve our antagonists. This 1-(Piperidin-4-yl)-1H- imidazo[4,5-b]-pyridin-2(3H)-one fragment will be further called as ‘privileged structure-PS’.

O NH N NH

1-(piperidin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one Privileged structure Figure 79.Showing the privileged structure we used for CGRP antagonists DESIGN AND SYNTHESIS OF PRIVILEGED STRUCTURE

Due to its multiple biological activities [133], the synthesis of PS has been well documented and widely used [3a, b, c, d]. There are multiple methods used for synthesizing PS which was even applied for large scale synthesis [134].

H H N N

N O NH NH2

NH N Cl 2 N Cl N Cl 271 272 273

Route A

H H N N

Route B NH2 N NH O N NH2 N N N NH H 2 274 2, 3 diamino 142 pyridine Route C

H N

NH NH2

N NO2 N NO2 275 2-nitro-3 amino pyridine

Scheme 129.Showing the privileged structure’s design for synthesis

207

Of all available routes designed and presented in figure 3, route B was reported to be the most feasible and yields the PS in minimal steps. Hence, we followed route B to synthesize compound 142.

Synthesis of 1-(Piperidin-4-yl)-1H-imidazo[4,5-b]-pyridin-2(3H)-one

Synthesis of compound 1 was started using commercially available 2,3- diaminopyridine and N-Boc piperid-4-one.

R R N N

O NaBH(OAc) , Triphosgene, NH 3 NH N 2 TFA Et3N + O THF, 0 °C N NH2 N EtOAc, RT N NH2 N N R H R=Boc-91% R=Boc-85% XIX Cbz-89% XXI Cbz-52% XXII R=Boc, Cbz

Scheme 130.Showing the synthesis of compound 1a An optimization for reagents & reaction conditions used has been done by David K. L et al [134] showing the importance of using sodium triacetoxyborane as reducing agent and trifluoroacetic acid for reductive amination in ethyl acetate. All these factors plays important role in regioselectivity of reductive amination. When used the above said conditions, the ratio of desired product to that of side products was 56/1 with a reaction time decreasing from 24 hours to 1hour.

Thus obtained 279 has been engaged in deprotection step to yield the compound 142.

H Cbz Boc N N N

N N N TFA, H O H2,Pd/C 2 O O O 1 atm, DCM N N N N N N H MeOH H H 94% 91% 276 142 277

Scheme 131.Showing the synthesis of compound 1

Hence, the compound 142 has been retrieved with good yields in 3-step process. Thus synthesized fragment was engaged for a convergent and coupling step with that of the fragments synthesized in family 1 and 3.

208

Coupling of the fragments

For obtaining the CGRP antagonists, a final coupling reaction has to be done between the RAMP-1 antagonist fragment and CALCRL interacting fragment. This particular subset, for easy comprehension and depiction has been presented in the form of a generalized approach as follows.

H N O O

NH2 HN N N NH N N + O N N N N H

CALCRL CGRP antagonist RAMP-1 interacting interacting fragment XXIII fragment XVIII 142 Scheme 132.Coupling of the fragments Urea like final molecules were obtained by a simple coupling reaction between the two fragments as stated above.

Meanwhile, David K. L et al has reported another synthetic method to obtain CGRP antagonists in minimal steps [136]. They used RAMP-1 fragment as an alcohol instead of an amine during the coupling reaction. Hence, the synthesized CGRP antagonist they retrieved was a carbonate instead of the conventional carbamate.

F3C

F N O O H2N N O F O O N N N N NH O N NH H F N F N

Telcegepant Ki = 0.77 nM BMS927711 Ki = 0.027 nM Figure 80.showing the carbamate like antagonist synthesized by David K.L et al Even though they replaced the carbamate with a carbonate which subsequently changes the hydrogen bond donor (-NH-) of Telcagepant® to a hydrogen bond acceptor (-O-) in BMS927711, they were able to increase the affinity towards the CGRP receptor.

Another carbonate linked CGRP antagonist called BMS-846372 has also shown some promising receptor affinity and currently is under clinical investigation [137].

209

O O N O F N N F NH

BMS-846372 Ki=0.070 nM Figure 81.showing the carbamate like antagonist BMS846372 This encouraged us to follow the BMS846372 and synthesize a carbonate like moiety which also decreases the number of synthetic steps during the course and thus increasing overall yield.

N O N H O O N F O N O N N N F F N CDI,DIPEA tBuOK N NH + F HN THF, RT N N THF, 10 °C N 97% .2HCl 86% O HO NH N BMS-846372 142 277 278 279 Scheme 133.showing the synthesis of BMS846372 The strategy to synthesize BMS846372 has been proposed by Luo G et al. [138]. The first reaction involves the coupling of carbonyldiimidazole with the PS to form x. The compound y was obtained starting from 2-bromonicotinaldehyde over 10 steps. Thus synthesized y has been deprotonated by using potassium tert-butoxide to form an alcoholate anion which subsequently follows a nucleophilic attack over the carbonyl group of x. This leads to the formation of BMS846372 with a yield of 97%. As the above proposed strategy looks facile with excellent yields, we took it as basis for us and designed the following routes for coupling the fragments

OH OMs N NH2 O Route B Nucleophilic 3 Reduction Activation substitution Reduction N N N N N XVIII XV XVI XIX XVII

Route A N N Coupling reaction Coupling reaction HN N NH HN N NH O O

O O O O HN O N N NH N N NH

N N N N

XX XXIII Scheme 134.showing the route A and B to obtain the CGRP antagonists 210

The planned route A gives carbonate CGRP antagonist 11 obtained starting from 10 i.e. piperidin-4-ol starting material which was obtained by a reduction from piperid-4-one 9. Hence, this route gave the final molecule in minimal 2 steps from the ketone 9. On contrary, route B takes 5 steps from the ketone 9. First step is reduction of piperid-4-one 9 to piperidin- 4-ol 10 which was followed by an activation of alcohol to a mesylate 12. This compound 12 will be further transformed into an azide 13 using nucleophilic substitution reaction and later to an amine 14 by reduction. This compound 14 was used as a fragment for coupling to synthesize carbamate CGRP antagonist 15.

Therefore, due to its minimal steps to retrieve the compound 11, we initially chose route A.

ROUTE A: This route selection has been followed by the study of coupling agents used to couple the two fragments. We carried this study using a piperidin-4-ol analog, cyclohexane-4- ol due to their similar conformations.

O O O OH Base, O N N NH HN N NH Coupling agent + N N THF, RT

XVI 142 XXIV

Scheme 135.showing the route A to obtain the carbonate CGRP antagonists As presented earlier, the synthesis of BMS846372 has been taken as methodological prototype. Thus, we applied the same to our compound of subclass A to couple it with the PS.

N O O N NH H O OH O N N O N N N N N CDI,DIPEA tBuOK + N N HN THF, RT N THF, 10 °C NH NH .2HCl 85% O NH N 280 142 277 252

Scheme 136.showing the application of BMS846372 synthetic methodology

On following the same condition, the reaction didn’t proceed forward and gave the starting materials as such. We thought that the deprotonation of the alcohol by potassium tert- butoxide was not forwarding due to the electronic property of our molecule which hamper the

211 deprotonation step. And hence, we tried a series of coupling agents and bases with different substrates. The conditions used are as follows;

N° RAMP-1 fragment CALCRL Coupling agent Base Yield fragment 1 Triphosgene 2 OH Carbonyldiimidazole 3 4-nitrochloroformate OH 4-nitrochloroformate 4 N Carbonyldiimidazole Boc H N With and No desired 283 Triphosgene without carbonate OH N O NaH formation N N N N H Cbz as major 5 Br O 142 4-nitrochloroformate compound

281 OH 6 4-nitrochloroformate LiHMDS N

NH NaH

252

Table 48.showing the route A to obtain the carbonate CGRP antagonists As tabulated above, all conditions to obtain the carbonate CGRP antagonists had failed. In some cases there is formation of product but as less as 1% (calculated through 1H NMR). This may be accounted due to the electronic properties of the alcohol substrates we used. After deprotonation by using a base, the alcoholate anion has to attack the carbonyl group quickly which might not have been the case in our compounds. This might be a slow and rate limiting step in our substrates and hence there was no reaction or very less formation of desired compound.

The following 1H NMR shows the doubled peak of the pyridine moiety and identified through their coupling constants. This signal corresponds to the proton right beside the nitrogen on pyridine. On integrating and quantifying, the desired carbonate was present in less than 1% overall yield.

212

Even the cyclohexanol substrate wasn’t able to couple with the PS made us to believe that the reaction of alcoholate anion with this type of substrates are rate limiting and if proceeds, the reaction rate might have been really slow. And being a reactive anion, the alcoholate wasn’t able to react with the substrate and instead gets protonated back and gives us the starting material as such.

Figure 82.showing the 1H NMR of the carbonate CGRP antagonists Thus, we changed our route and continued to use the route B by means of the conditions described in the literature [138].

ROUTE B: We took the condition optimized in the literature and modulated with small changes in accordance with our substrate. This optimization was done within the team by using a similar substrate. The conditions of optimization were as follows

213

HN N O NH2 O Et N, N HN N NH 3 NH CDI O O N N + N THF, Reflux N 48h 54% 142 286 268 Scheme 137.showing the route B to obtain the carbamate CGRP antagonists This optimized condition has been employed to our substrates to synthesize the carbamate CGRP antagonists.

N° RAMP-1 interacting CALCRL interacting Yielded molecule Yield (%) fragment fragment O NH 2 HN N 1 68

N Boc N Boc 287 NH2 O HN N O 2 N N O 54 N N

268 O NH2 N HN 3 N N Cbz 62 Br O N O N Cbz O HN N NH Br

N 289

NH2 O

HN N 4 N 59 N Cbz Br O N N Cbz Br O

280

214

F NH O F 2 F F HN N F 5 N 71 N F Cbz Br O N N Cbz Br O

291 NH2 O

NH N In-progress 6 N

NH N

292 Table 49.showing the route B we used to obtain the carbamate CGRP antagonists In all the cases, the yields are satisfactory and give the desired product in over a period of 24h. From the compounds obtained above, depending on the protecting group, a general methodology for corresponding PG has been applied to obtain the final compound.

Concluding scratch to give potential CGRP antagonists

The two fragments combined in the previous step have been subjected for final touch- ups to recover our potential CGRP antagonists. Mostly this last step includes deprotection and sometimes a dehalogenation step as well to retrieve the desired compound. Now let’s take each case. The entry 1 is having a tert-Butyl carbamate protecting group which was deprotected using trifluoroaceticacid as presented in the previous chapters.

O O

HN N O HN N O TFA, H2O N N NH NH .CF3COOH N DCM, RT N H N Boc N Quantitative 287 293

Scheme 138.showing deprotection of Boc in entry 1 The deprotected compound x was left in salt form as this will increase the solubility in aqueous phase of vehicle (5% Tween 80 in saline 0.9%) during the pharmacological evaluation. This easy solubility also increases the bioavailability of the tested compounds more readily in biological system.

215

Entries 2, 3, 4 and 5 were having benzyl carbamate as protecting groups and also we want to debrominate the compounds as bromine in biological system has proved to be mutagenic [139]. And thus, we did a hydrogenation reaction which deprotects the benzyl carbamate and dehaolgenates in situ.

O O

N HN N HN X X Pd(OH)2/C, H2 N N N N Cbz H Br O MeOH, RT, O 1 atm

X-phenyl, CF3

289, 290, 291 294, 295, 296

Scheme 139.showing deprotection and debromination in entries 2, 3, 4 The hydrogenation as presented before is quantitative and gives the desired product in 24 hours.

And the last entry, compound 292 needs no further steps and hence qualifies as such to be tested in biological evaluation.

216

Take home message from this chapter

In this chapter, we illustrated the conception of organic skeletons based on multistep synthesis for two hybrid families (family 1 and 3, scheme 140) that I had to construct for my PhD project. Those structures have been handpicked based on the molecular modeling and docking studies made on the CGRP receptor. The best substituents have been selected to innoculate in each family and we had proposed a convergent synthesis limelighted on the preparation of a common structure: the 2,6-piperidin-4-one.

O O O O N N N X NH X N Family 3 NH Family 1 N O N 4 R N R4 N N R1 Y

2 R R2

Potential dual active molecules

Convergent synthesis

O O N 4 + HN NH R N Z CALCRL fragment RAMP-1 fragment Y N R2

Ph Ph O O O 6 steps 5 steps OH O N O HO 4 3 NH O R4 OMe R N R 2 R4 OMe R6 Aspartic Acid D or L Choice of the Core piperidine moiety stereochemistry R or S

Scheme 140. General synthetic scheme Piperidine moieties were prepared in 6-steps form the corresponding methyl crotonate for family 3 and in 5-steps from the chiral aspartic acid for the family 1. The chirality for the family 3 has been introduced during the first step through an Aza-Michael insertion of the desired chiral Davies Amine. The piperidine skeleton was obtained for both families from an intramolecular reaction based on an Aza-Michael principle, methodologies that we had to optimize or which have been published by the laboratory.

According to the family, various procedures were made to reach RAMP-1 fragment and none of them were known in the literature. Later, several ways to construct those fragments were investigated. For the family 1, the optimal procedure started after

217 optimization of the synthesis for desired N-protected pipecolic acid. The benzodiazepanone 7 membered ring have been obtained in 4-step synthesis through an amide bond formation following by an in-situ ketone protection and N-deprotection followed by an intramolecular palladium catalysed Buchwald cross coupling reaction. Only the benzodiazepanone with R = benzyl could be reached and some peptides before cyclisation were also used as RAMP-1 fragment.

Scheme 141. showing design of family 1 For family 3, desired substituents on the piperidine ring for reaching the 7-membered skeleton were directed on the HWE reaction. Piperidine 2,6-cis and 2,6-trans were obtained in favor of the cis isomer majority. Only the cis configuration was used for the following steps. The 3 subsclass A, B and C 7 membered rings were investigated to be prepared by using an intramolecular palladium catalyzed reaction. Only candidate from the subclass A and B could be reached with an average to good yields, and concerned methods are still in optmisation progress.

218

Scheme 142. showing design of family 3

All the ketone obtained have been next transform into amine through a SN2 procedure after activation of the corresponding alcohol and branched to the CALCRL optimize fragment. Overall, 6 final molecules containing RAMP-1 fragment as well as CALCLR fragment were tested and a lot of other fragments were also tested for their in vivo pharmacological activity.

219

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CHAPTER 4: PHARMACOLOGICAL EVALUATION

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BIOASSAY

The course of earlier chapters has introduced us the diverse structure of pain along with the aetiology implicated in it. Along with this, in the subsequent chapters, we have seen the design and synthesis of the potential CGRP antagonists. In the current section, we would have a detailed outlook about the application of the synthesized potential CGRP antagonists in pain models, to be precise on nociceptive pain replica.

As we have discussed in the preceding chapters, different type of pain stimulation exists. It includes nociceptive and neuropathic in general. Hence for the study of analgesic activity of the synthesised molecules, knowledge of which form of pain we are acting on is obligatory. For this reason, the choice of biological models for the analgesic tests depends on the pain initiation mechanism, feasibility, and animal ethics along with the pharmacologist who conducts the test.

We studied the pharamacological activity of our molecules using hot-plate method for analgesic property, actophotometry for sedative property and porsolt forced swimming test for antidepressant property.

A. Pain bioassay

Analgesic activity in biological systems can be assessed using various models. Depending on the administered stimuli, the nociceptive pain models can be classified as below [1]

Figure 83.showing different nociceptive bioassays

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The choice of the model depends on various factors like sensitivity, reproducibility and region of brain involved in response for stimuli [2, 3, 4].

Advantages of thermal stimuli administered nociceptive assay

Among the three stimulations which can be administered for the mice, electrical and chemical stimuli are more aggressive and may cause internal abrasion (in case of chemical stimuli) and thus obtained results are more prone to unreliability and not bring robust due to changes during the mice behavioural studies [5, 6].

On the other hand, the thermal stimuli give more reliable and reproducible data [7]. Even amongst the thermal stimulus administered nociceptive assay, the hot plate method is found to be more reliable as the assay involves higher brain functions and is considered a supraspinal organized response [2]. Whereas, the tail flick and paw withdrawal methods involves spinal reflux to a noxious stimuli [3]. Hence, the nociceptive pain inhibitory data obtained through Hot plate method deemed to provide a deeper pain processing responsive study than that of a simple reflux as in case of other thermal stimulation tests.

As an outcome, we used the Hot plate method to be our essential principle test to envisage the nociceptive pain inhibitory properties of our designed and synthesized potential CGRP antagonists.

1. Hot plate method:

1.1 Rationale:

Rodent species are characterised by thermo-sensitivity in paws for the non damaging heat changes. Especially, rodents are able to express the heat response by wiggling, jumping and licking of the paws particularly of hind leg. Hence crafting this as a basis, the hot plate method has been developed in mid 19th century [8]. The time taken for showing the above said responses is increased (if the molecules tested are having analgesic property) in general.

1.2 General procedure:

The hot plate method was initially designed by Woolfee and McDonald in 1944. But the currently followed method is a result of series of modifications [9¸ 10]. And mostly followed method is as follows;

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Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. The commercially available hot plate will be having a temperature controlled surface, a plastic cage for enclosing the mice along with the time and temperature display.

Figure 84.Showing Hot plate device The surface in general was maintained at 52°C and is made of copper. An initial habituation to the cage for individual mice was performed with the surface being at room temperature. Then the mice are placed on the heated surface (at 52°C) and using the stop watch, the time at which the mice expresses the pain response like wiggling, jumping or licking of hind paws are noted with a cut-off time of 30 sec above which was considered as error and restarted again. When it shows the above signs, the mouse is instantaneously taken out of the hot plate. The molecule to be tested for analgesic property was administered subcutaneously. After the administration, a latency period of 15 minutes was given and the mice was kept on the hot plate again at same temperature to see if there is any increase of time of perception of pain/expressing the pain responses due to the analgesic property induced by the molecule administered.

1.3 Assessment In this particular pain model, the time of showing the response before and after the administration of the molecule can be regarded as the criteria for evaluation of analgesic property. Even the values of the control group and the experimental group can be used for statistical comparison using t-test.

1.4 Significant points to be noted  Centrally acting analgesics will increase the time of showing the responses where as peripherally acting analgesic derivatives showed no response according to the literature [11].

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 A hot plate method shows pseudo positive results when the analgesic moiety tested possesses some secondary activities like muscle relaxation, sedation or psycho- mimetic property [12].  Even among rodents, the sensitivity for temperature difference from the hot plate to that of environment was detected higher in mice whereas the rats showed no difference [13].

2. Antinociceptive property of the synthesized CGRP antagonists The designed and synthesized potential hits which were discussed in the previous sections have been tested for their antinociceptive activity using hot plate method. We carried the antinociceptive activity studies in two batches till now. The strategy for studying the analgesic property of molecules was as follows

 We tested the fragments of the synthesized hits to facilitate the easy structure activity relationship (SAR) studies.  Variety of designed RAMP-1 interacting fragments of family 1, 3 and privileged CALCRL interacting fragment has been tested individually as well as in coalition.  We predominantly tried to validate the reliability of in silico scores obtained by using the results acquired through in vivo tests in biological system.  We investigated various factors like the effect of the substituants, the configuration, the enantiomers etc. affecting the antinociceptive property. Thus, the first consignment of in vivo results is as follows

Figure 85.showing time of response before and after administration of antagonists in Ist batch (Vehicle- solution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

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The results for the first batch, on comparison with response times before and after administration of potential CGRP antagonists showed a clear inhibition of nociceptive pain. Thus obtained results on normalization to percentage pain inhibition are as follows; N° Molecule Pain inhibition (%) Conditions O NH 1A HN N .2HCl 72 N

O O N N NH 2A HN .CF3COOH 55 N

3A Control group -

NH2 .HCl Dose-5 mg/Kg O N 4A N 48 Route-Subcutaneous

(S.C) OH Solubility-0.5% DMSO

N in vehicle 5A 37 O Latency period-15 mins OH

N 6A 21 NH

O NH N O

7A N NH 41 O N N N

Table 50.showing time of response before and after administration of antagonists in Ist batch The normalization of the results to a percentage scale was done by using the following formula.

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×100

During the continuation of study in identifying the lead, we have conducted nociceptive pain inhibitory property tests for another batch of potential CGRP antagonists. Thus, the second consignment of in vivo results is as follows

Figure 86.showing time of response before and after administration of antagonists in IInd batch (Vehicle- solution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

Similar to the first batch, the second set of compounds also showed considerable amount of difference in nociceptive response before and after administration of the designed and synthesized CGRP antagonists. On normalization to a percentage pain inhibition as described in above case, the results are as follows,

N° Molecule Pain inhibition (%) Conditions

1B O 13 NH .HCl HN N

O Dose-5 mg/Kg O N NH

N 2B HN 31 N Route-Subcutaneous N N H O (S.C)

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O Solubility-0.5% DMSO O N NH in vehicle 3B HN N 47 N N N H O Latency period-15 mins

HN N O 4B N O NH N 12

O O N NH CF N 3 5B HN 20 N N H N O

6B Control group -

N 7B N 40 H O

Table 51.showing time of response before and after administration of antagonists in IInd batch Thus obtained percentage pain inhibition values from two batches have been studied to understand the structure activity relationship. Some of the key identifications are as follows

 A dose of 5 mg/kg showed significant amount of pain inhibition.  The latency period of 15 minutes proves that the tested molecules are potent and shows the desired analgesic activity in less time.  All the hits tested showed a potent nociceptive pain inhibitory activity  None of the tested molecules showed amplification of pain/induction of pain.  We maintained privileged structure as 1A instead of 1B as 1A stands out to be the best with percentage pain inhibition of 72.

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 Apart from privileged structure, 2A showed the best pain inhibition with a value of 55% and tops in the contender to be a potential lead.  The racemic structures 4A & 7A, even though a mixture of two enantiomers showed good pain inhibitory property.  The fragments belonging to family 3 also possessed antinociceptive properties and among which, the flexible fragment 5A showed better results than that of the rigid counterpart 6A. Hence, the proposed hypothesis (refer chapter 3, Family-3) on a flexible structure for this family has been justified. A final molecule coupled with privileged structure will be studied in the future tests.  The fragment 7B showed 40% of pain inhibition and hence a final molecule coupled with privileged structure will be studied in the future tests.  Ligand 2B showed 31% of pain inhibition whereas 3B showed 47% even though they both are similar structures with former being an S-enantiomer and latter being an R- enantiomer.  The ligand 5B showed a mere 20% of pain inhibition attributing the loss of analgesic

property either due to the configuration or change of substitution (phenyl with CF3) when compared with 3B. Hence, during the course of above study, we envisaged several factors involved in gain or loss of nociceptive activity. We investigated the effect of substituants, the conformation, configuration etc. Most of the time, the obtained results are in correlation with the results retrieved from the molecular modelling studies.

With thus obtained pleasing results, a IIIrd batch of potential CGRP antagonists are currently under investigation and will be presented during the thesis defence.

To authenticate that the obtained analgesic activity is not due to the sedative property of the molecules, we performed the test for sedation using actimetry assay for locomotory activity.

B. Locomotory activity bioassay Locomotion tends to the movement from one place to the other. In rodent species like mice and rats, the most important amongst the catalogue of impulsive activity is locomotion. The exploration, a most common activity of rodent species along with locomotion are implicated in many behavioural utilities and are prejudiced by environmental conditions such as light, temperature, sound etc., circadian rhythm, food/drink deprivation, age, gender and

233 many more factors [14, 15]. These locomotory and exploration behaviour is mediated by neurotransmitters which can be modulated by many drugs such as epileptics, psychomimetics, analgesics, antidepressants etc [16, 17].

Hence, we made this as a rationale to identify whether the pain inhibitory property of our molecules are solely due to its antinociceptive property or due to the sedative property. Alongside this, we also want to envisage the sedative side effect our molecules which might be possessed in their tricyclic and tetracyclic moieties as we have structural resemblance with benzodiazepines.

There are various locomotor activity assessment tests available like actimeter, circadian locomotor activity, open field test for basal global activity, emergence test, novel object test, hole board test, treadmill test etc. Among all the available bioassays for locomotor activity, actimeter with photocell-based detection is one of the quickest, robust and gives reliable data [18].

1. Photoactometer- an infrared photocell-based detection

The actimeter used for assessing the locomotor activity of the rodents are mostly automated now-a-days. The structure of actimeter includes a rectangular box which has enough space for free movement of the rodents inside it. Two photo emitters are placed on one end of the box which on other side trespasses with a pair of photo detectors. These photobeams, when broken registers a count on a digital counter. In some variants, photocells are placed on top of the cage as well in order to identify the rearing along with locomotor activity.

1.1 General procedure Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. For the preliminary testing of the equipment’s condition, the instrument was switched on and by manual cut-off, all the photocells were tested. This was followed by a subcutaneous administration of the molecules to be tested. After a latency period of 15 mins, the mice were placed in the actimeter box and left for the locomotor activity assay for a period of 10 mins. During this time, the digital counter outside the box shows the count of cut-offs. Once the test was done, the cages were cleaned and disinfected to carry the next batch of testing.

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Figure 87.showing photoactometer

1.3 Assessment In this actimeter, the difference in count of cut-offs between the mice administered with molecules and the control can be regarded as the criteria for evaluation of locomotor activity. In simple words, if the molecules don’t possess sedative property, it shows more or equivalent count as that of the control group.

2. Locomotory activity of the synthesized CGRP antagonists

The designed and synthesized potential CGRP antagonists were tested for their locomotor activity using photoactometer. Similar to the antinociceptive assay, the tests were conducted in two different batches.

The locomotor activity of first batch of compounds tested is as follows

Figure 88.showing photoactometer results in 1st batch (Vehicle- solution of 5% Tween 80 in saline) The first batch of potential CGRP antagonists showed no big difference in the cut-off count from the control group. The cut-off counts were as follows

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N° Molecule Cut-off count Conditions O NH 1A HN N .2HCl 198 N

O O N N NH 2A HN .CF3COOH 223 N

3A Control group 214

NH2 .HCl Dose-5 mg/Kg O N 4A N 217 Route-Subcutaneous

(S.C) OH Solubility-0.5% DMSO

N in vehicle 5A 200 O Latency period-15 mins OH

N 6A 196 NH

O NH N O

7A N NH 198 O N N N

Table 52.showing cut-off counts for administration of antagonists in 1st batch Unlike in hot plate method, here the results of administered molecule will be compared with that of the control group. These results showed that there was no significant change in locomotory activity because of the administered molecules.

Further, the tests on second batch of potential CGRP antagonists were conducted and the results were as follows

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Figure 89.showing photoactometer results in 2nd batch (Vehicle- solution of 5% Tween 80 in saline) Akin to the first set of compounds tested for locomotory activity changes, even the second batch of our molecules showed no significant alterations of cut-off count from that of the control group.

N° Molecule Cut-off count Conditions

1B O 248 NH .HCl HN N

O O N NH

N 2B HN 229 N N N H O

Dose-5 mg/Kg O O N NH Route-Subcutaneous 3B HN N 253 N N N H (S.C) O

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O Solubility-0.5% DMSO

HN N O in vehicle 4B N 240 O NH N Latency period-15 mins

O O N NH CF N 3 5B HN 245 N N H N O

6B Control group 267

N 7B N 262 H O

Table 53.showing cut-off counts for administration of antagonists in 2nd batch

Summing it up, the results retrieved from the locomotory activity studies showed no significant sedation which made us to draw two important and significant conclusions as following

 The antinociceptive property shown during the hot plate method was not because of sedative property of the administered molecules.  Concurrently, we also saw that our molecules, even though has a tricyclic and tetracyclic core moiety like benzodiazepines, don’t possess sedation as a side effect. With these two studies, the main goal of our project has been investigated and validated with significant and pleasing results. As discussed in the preceding section, the bioassay for depression will be studied to authenticate the concept of dual activity.

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C. Antidepressant bioassay

Depression can be defined clinically as a complex pathophysiological state, which also involves neuroendocrine symptoms which is highly difficult to replicate in animals models [19]. The foremost hurdle in identifying a novel antidepressant drug is lack of animal models which look a lot like depressive disorder as well as selectively responsive to clinically effectual antidepressant therapy [20]. During the course of period, various models like reserpine effects reversal test designed in 1960 is dated to be the first of its kind which led to the isolation of desipramine and express its antidepressant activity [21]. One of the models developed is Porsolt Forced Swimming Test (FST) by Porsalt.R.D in 1977 which is considered as most reliable, sensitive and mimics clinical treatment like response [22, 23].

1. Porsolt Forced Swimming Test (FST) Porsolt et al observed during a behavioural study of rodents in a maze that among the rats which are involved in the test, most of them found the exit within a period of 10 mins and some of them stayed floating passively. This led to the design of FST which is also called as behavioural despair in mice.

1.1 General procedure Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. The testing apparatus is cheap and even a laboratory beaker can be used. The beaker was initially filled with water at a temperature of 25 °C and maintained the same for entire period of assay. The beaker used should be in such a depth that the mice can’t touch the bottom of the beaker with its tail or feet. An ideal depth of the beaker is 30 cms.

Figure 90.showing FST

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The molecules to be tested were administered subcutaneously and the mice was given a latency period of 15 mins. After which, each mice was placed in the beaker of water and calculated for the immobility of the mice due to loss of hope to escape from the beaker. The test runs for a time of 5 mins and immediately thereafter, the mice was taken off from the water and dried completely before placing back in the cage. The beaker in general was cleaned and disinfected during constant intervals of time.

1.2 Assessment

During the course of the FST test, two parameters were noted down. One being the first immobilization time and second was overall immobile time. The obtained results from the tested molecules were compared with that of the control group. In simple terms, the immobilization time for tested compounds should be less than that of the control group to have antidepressant property.

Similar to the other tests which we performed, we conducted FST in two batches. The results of 1st batch are as follows

Figure 91. showing first and total immobilization times in Ist batch of FST (Vehicle- solution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

In first set of compounds, 2A and 5A showed some amount of antidepressant property as shown above. The immobilization of the molecules is as follow

N° Molecule Immobilization time Conditions First Total O NH 1A HN N .2HCl 0.865 2.682 N

240

O O N N NH 2A HN .CF3COOH 1.059 2.599 N

3A Control group 0.807 3.022

NH2 .HCl

O N Dose-5 mg/Kg 4A N 0.861 2.608 Route-Subcutaneous

OH (S.C)

Solubility-0.5% DMSO N 5A 1.042 2.581 O in vehicle

OH Latency period-15 mins 0.86 2.695 N 6A NH

O NH N O

7A N NH 0.95 2.84 O N N N

Table 54.showing immobilization times on administration of antagonists in 1st batch

We can consider either of the two immobilization times to predict the antidepressant property of the administered molecule. As shown in the above table, the immobilization times of all the molecules administered are less than that of the control group and hence, they showed some amount of antidepressant nature but only 2A when applied with statistics showed significance. We continued to search for a molecule with more significant antidepressant property than that of 2A. Hence we followed this study with another batch.

The results for the second batch are as follows

241

Figure 92.showing first and total immobilization times in 2nd batch of FST (Vehicle- solution of 5% Tween 80 in saline)

In 2nd batch of compounds, the compounds like 2B, 4B, 5B and 7B showed better total immobilization time reduction from that of control group. But due to lack of significance, they aren’t considered as good antidepressant molecules.

The immobilization of the molecules is as follows N° Molecule Immobilization times Conditions First Total

1B O 1.073 2.914 NH .HCl HN N

O Dose-5 mg/Kg O N NH

N 2B HN 1.227 2.289 N Route-Subcutaneous N N H O (S.C)

O Solubility-0.5% DMSO N NH O in vehicle 3B HN N 1.233 2.758 N N N H O Latency period-15 mins

242

HN N O 4B N 1.237 2.574 O NH N

O O N NH CF N 3 5B HN 1.189 2.53 N N H N O

6B Control group 1.156 2.97

N 7B N 1.319 2.64 H O

Table 55.showing immobilization times on administration of antagonists in 2nd batch

Hence, even though the molecules tested in IInd batch possessed considerable amount of desired antidepressant property, they lack of significance.

Overall, the test results for antidepressant property identified through FST showed not excellent but definitely significant amount of desired property. Molecules like 2A showed good as well as significant results where as compounds like 5A, 2B, 4B, 5B and 7B showed good but lacked the significance in them.

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Take home message from this chapter

The designed and synthesized molecules have been tested for their pharmacological properties. The prime focus was kept on antinociceptive activity and thus we did hot plate method to show that the molecules possess analgesic property. We even validated that the analgesic property showed by our molecules are not because of the sedation but due to their direct analgesic effect by conducting photoactometer test for locomotor activity. Molecules like 1A, 2A, 4A, 5A, 7A, 3B and 7B showed an excellent and significant amount of pain inhibition (>37%) for the mere amount of dose we used i.e. 5mg/kg. The other point to be kept in mind is to get the desired activity, it just took 15 minutes of time which gave us the above results. This demonstrates that our molecules possess potent analgesic property.

Photoactometer test was performed to show that our molecules don’t possess sedation as a side effect and hence the analgesic property showed by our molecules is not because of sedation.

To validate our concept of dual activity to treat chronic pain, we also tested our molecules for antidepressant effect. This test showed that 2A is both significant and persuasive where as 5A, 2B, 4B, 5B and 7B have antidepressant property but aren’t significant with their results.

Another batch of molecules is currently ready to be tested for all the above said tests and the results will be presented during the thesis defence.

“In conclusion, molecules 2A, 5A and 7B showed our desired dual activity whereas 1A, 2A, 4A, 5A, 7A, 3B and 7B showed potent antinociceptive effect. Hence, further structure activity relation (SAR) study is in process to take these as leads and make lead modifications for the amplification of the desired dual active molecules or to enhance the antinociceptive effect alone. This will be followed by in-depth mechanistic study of the identified lead to recognize how their pharmacodynamic and pharmacokinetic modulations are taking place within a biological system”

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REFERENCES 1. Le Bars D., Gozariu M., Cadden S.W., Pharmacol Rev., 2001, 53, 597. 2. Chapman C. R., Casey K. L., Dubner R. K., Foley M., Gracely R. H., Reading A. E., Pain, 1985, 22, 1-31. 3. Hoffmeister F., Kroneberg G., Methods in drug evaluation. Eds.: Mantegazza P & E Piccinini. North Holland, Amsterdam. 1966, 270-277. 4. Dennis S. G., Melzack R., Adv. Pain Res. Ther., 1979, 3, 747-760. 5. Paalzow L., Acta Pharm. Suec., 1969, 6, 193-206. 6. Paalzow G., Paalzow L., Acta Pharmacol. Toxicol., 1973, 32, 22-32. 7. Abbott E., Palmour M., Life Sci., 1988, 43, 1685- 1695. 8. Woolfe G., MacDonald A. D., J. Pharmacol. Exper. Ther., 1944, 80, 300–307. 9. O’Neill K.A., Courtney C., Rankin R., Weissman A., J. Pharmacol. Meth., 1983, 10, 13–18. 10. Tjølsen A., Rosland J.H., Berge O.G., Hole K., J. Pharmacol. Meth., 1991, 25, 241– 250. 11. Menendez L,. Lastra A., Hidalgo A., J. Neurosci. Methods, 2002, 113, 91–97. 12. Knoll J., Screening and grouping of psychopharmacological agents. In: Siegler PE, Moyer HJ (eds) Animal and Clinical Pharmacologic Techniques in Drug Evaluation. Yearbook Med Publ. Inc., Chicago, 1967, 305–321. 13. Gunn A., Bobeck E.N., Weber C., Morgan M.M., J. Pain, 2011, 12, 222-227. 14. Görtz N., Lewejohann L., Tomm M., Ambrée O., Keyvani K., Paulus W., Sachser N., Behav. Brain. Res. 2008, 19, 43-48. 15. Rubio M., Neuropharmacology, 2008, 54, 976-988. 16. Tsuchida R., Kubo M., Kuroda M., Shibasaki Y., Shintani N., Abe M., Köves K., Hashimoto H., Baba A., J. Pharmacol. Sci., 2009, 109, 396–402. 17. Lynch J.J., Vincent C., Paul C.M., Scott W.M., J. Pharmacol. Toxicol. Methods, 2011, 64, 74–80. 18. Holmes A., Mouse behavioral models of anxiety and depression. In: Crawley JN (ed) Mouse behavioral phenotyping. Society for Neuroscience, Washington D.C., 2003, 43–47. 19. Lucki I., Behav. Pharmacol., 1997, 8, 523–532. 20. Costa E., Garattini S., Valzelli L., Experientia, 1960, 16, 461–463. 21. Porsolt R.D., Bertin A., Jalfre M., Arch. Int. Pharmacodyn. Ther., 1977, 229, 327– 336. 22. Porsolt R.D., Bertin A., Jalfre M., Eur. J. Pharmacol., 1978, 51, 291–294.

245

GENERAL CONCLUSION

During the course of my PhD, we tried to find a true answer towards the treatment of chronic pain (migraine and viscerals) and on the postulate based on the use of an hybrid molecule which could possess a dual activity : antagonist for the CGRP receptor and inhibitor for the recapture of monoamines.

Before starting the synthesis of antagonists for the CGRP receptor, we initially conducted a deep survey on the receptor and its properties for designing better and efficacious antagonists. During this course, we identified that unlike other receptors, to distort the action of this receptor, we need to design ligands which are ‘out of box’ or we should be linient about the conventional Lipinski’s rule of 5. This may be attributed to the huge receptor pockets and not just one interaction but a group of interactions are important for antagonizing the CGRP receptor. And hence, we designed ligands directly inspired form two molecules known for there activities on the CGPR receptor (antagonist: Telcagepant) and as inhibitor for the recapture of monoamines (Imipramine) and conducted an in silico docking simulation and from its results, we identified potential hits from 2225 proposed molecules 39 structures which were distributed among three families (10.1 ≤ Score Total ≤ 12.9) which correspond with a total score up to the Telcagepant (Score Total = 10.08).

From then on, we were working on synthesizing the potential hits obtained from docking ranks and testing them in biological systems as well to validate our prototypes.

246

The preparation of those structures is based on a convergent synthesis from the corresponding piperidine well substituted RAMP-1 fragment to attach to the more potent CALCRL fragment.

O O O O N N N X NH X N Family 3 NH Family 1 N O N 4 R N R4 N N R1 Y

2 R R2

Potential dual active molecules

Convergent synthesis

O O N 4 + HN NH R N Z CALCRL fragment RAMP-1 fragment Y N R2

Ph Ph O O O 6 steps 5 steps OH O N O HO 4 3 NH O R4 OMe R N R 2 R4 OMe R6 Aspartic Acid D or L Choice of the Core piperidine moiety stereochemistry R or S

After a chapter of bibliography to put in front the best procedure to reach the desired disubtituted 2,6-piperidone, we have selected published and optimized, for the two families, an intramolecular Michael synthesis started from methyl crotonate or aspartic acid.

According to the family, and to access to the skeleton RAMP-1 fragment, we have to work out and optimized new process to reach the different 7-member ring heterocycle form family 1 and 3. Those procedures are focus on an intramolecular Buchwald “type” reaction in presence of palladium. Three new skeletons belonged to family 1 and family 3 have been successfully prepared. An optimization to improve yield is in progress in the laboratory.

The various piperidinone were then transformed into the corresponding amine to be further connected to the CALCLR fragment. Five finals molecules with fragment RAMP-1 and CALCLR were next tested for there potencies to act on pain. Seven other molecules, corresponding at some part of the final molecules were also tested.

247

Then till now, we have tested around twelve molecules in various tests to assay their pharmacological properties. All have shown antalgic effect, and for some (2A, 5A, 7B) a dual effect was observed.

O OH O OH N N NH HN .CF3COOH N N N N H O NH O

2A 5A 7B

Surprisingly, 5A and 7B don’t possess the desired CALCLR fragment to be well connected to the CGRP receptor, but show a good activity. From the 6 moleclues which possess the CALCLR fragment, only 2A, active molecule that could belong respectively to family 1 or to family 3, has been identify as the more potent skeleton, and give a good dual activity. 2A doesn’t posses the benzodiazepanone or tricyle core that we selected to reach the recapture inhibitor properties. 2A is more flexible and on the future work we want to identify a new generation of compounds, starting from 2A as lead, which take in part the modulations according to the amino acid interactions inside the receptor in order to enhance the potency as well as the specificity.

O N *

HN O

Scheme 2 : result of the hybrid molecule 2 on hot plate and porsolt method by using a dose of 5mg/kg (SC) R2 N R1 R3

2A : in vivo results and General Lead Structure

We proposed our work to “Société d'Accélération de Transfert de Technologie” (SATT) and they are currently considering our project to take over. During this period, we want to take 2A as lead and modulate on R1, R2 and R3 as following:

248

Figure 93. Showing the perspective of our project

As depicted above, we wanted to design in the future, a new series of CGRP antagonists using the model proposed above. We wanted to study taking 2A as the lead and conduct a rigorous Structure Activity Relationship study. The results, with the help of SATT will be patented.

249

CHAPTER 5: EXPERIMENTAL PART

250

GENERAL REMARKS

Thin layer chromatography were performed on TLC pre-coated aluminium backed silica plates Kieselgel 60 F254 (Merck) or glass backed silica Duracil 25 UV254 (Macherey nagel). Spots were visualized using UV light (254 nm) before using an ethanolic solution of para- anisaldehyde or phosphomolybdic acid (heating).

Purifications by column chromatography were carried out on silica gel Kiesgel 60 (0.073-0.230).

Melting points were measured by a Kofler Hot bench or Reichert plate-heating microscope.

Optical rotations were measured on a ADP 440 Bellingham polarimeter at the wavelength of sodium D ray (Ä = 589 nm).

1H NMR and 13C NMR spectra were recorded on a Bruker Avance spectrometer at 400.13 and 100.61 MHz respectively. Chemical shifts δ were reported in ppm relative to solvent residual signals (1H and 13C). The coupling constants J were given in Hertz (Hz). The abbreviations used for signal descriptions are as:

- ax : axial - dt: doublet of triplets

- eq : equatorial - t : triplet

- s : singlet - dq : doublet of quartets

- m : multiplet - q : quartet

- br s : broad singlet - tt : triplet of triplets

- dd: doublet of doublets - Q : quintet

- d : doublet - ddd : doublet of doublets of doublets

251

High Resolution Electro-Spray Ionisation Mass Spectra (HR-ESI-MS) were obtained from the “Service de Spectrométrie de masse du service UBP-START (Université Blaise Pascal Clermont II).

High Performance Liquid Chromatography (HPLC) analysis conditions used for progress of the reaction; column: Poroschell 120 EC-C18 2.7 µm (3.0 x 50 mm) , temperature column: 40 °C; injection: 10 µL, 1 mL/min; solvents used A-water with 0.1% formic acid and B-methanol (95:5 to 5:95, KBNUNO) method using UV using 254 nm and 280.5 nm.

Numbers placed in figures refer to NMR attributions, and were given independently from IUPAC nomenclature.

MOLECULAR MODELLING STUDIES

Docking studies have been done by Mr. Lionel Nauton and Dr. Vincent thery. Whereas the other modeling studies presented previously has been generated by myself using various online and offline tools as reported. Most of the structures were drawn using ChemDraw® and sometimes using Marvinsketch. Various experimental methods followed during the study are as follows.

Modeling of the receptor: Previously refined crystal structure was opened in ‘Biopolymer’ of sybylx2.0 to adjust the backbones in a correct way in order to add the hydrogen atoms on the receptor considering the hydrogen interactions. Finally, this model was minimized using molecular mechanics

Energy minimization of refined protein model Method: conjugate gradient Force fields: Tripos Charges: MMF94 (suitable for H interactions of protein) Gradient: 0.05 Dielectric constant: 4.0 (protein environment) Aggregates: The backbone and ‘O’ of the water molecule. Hence this energy minimization step resulted in a low energy and refined crystal structure which will be engaged in the docking simulation.

Modeling of the ligands:

All the ligands docked in this current study were modeled using same protocol as described below. Initially molecules have been drawn in 2D version using Marvinsketch with a stress on the stereochemistry on the chiral carbons. Further, their geometry in 3D were obtained again using the same program i.e. Marvinsketch. To end with, the stereochemistry was ensured before recording the structures in MOL2 format. The ligands were then reopened in sybylx2.0 and any missing hydrogen atoms were added.

Conformational studies: Each molecule thus modeled is subjected to random search geometry. The geometry of lowest energy conformation is then recovered and inserted in SDF format which later can be directly used for docking.

All these stages were passed on to reach the docking simulation where an in silico interactions was visualized.

BIOLOGICAL ASSAY

Animal models handling: Male swiss albino mice (18-22 g) were purchased from Elevage Janvier. Animals were housed under controlled environmental conditions (22 °C, 55% humidity) and kept under a 12/12 hours light/dark cycle with food and water ad libitum for a week before the commencement of the tests. The tests were performed as a double blind study in a quiet cabin by the same manipulator to minimize the errors as well as to avoid discomfort to the animals. Animals were randomly divided into groups of 10 mice each and each mouse was not treated with the same compound twice. This study was followed by euthanization of mice by concerned person. Animal care and experiments were performed in accordance with the committee for research and ethical issues of the International Association for the Study of Pain (IASP; Zimmermann, 1983). All animal procedures were approved by the local animal ethics committee of Auvergne (Comité Régional d’éthique en Matière d’expérimentation animale Auvergne). Behavioral testing in mice

These experiments were carried out at Neuro-Dol by me and Julie Barbier following the ethical principles thoroughly.

Injection: The tested compounds were dissolved in vehicle (5% Tween 80 in saline (0.9%), 0.5% DMSO), 0.5% DMSO was used to enhance the solubility of compounds. In all the

253 cases, the injection was done 15 min before the behavioral assessment. The injections were done into the peritoneal cavity of the mice through subcutaneous route of administration.

Hot plate method: After acclimatizing for 5 min on hot plate at room temperature, mice were placed on the plate set at 52 °C until they started licking their forepaws (Latencies) (cut-off time: 30 s). After having obtained two consecutive stable latencies, treatment effects were assessed after 15 min. The results were expressed as:

×100

Photoactometer: Unlike Hot plate method, no acclimatization was done. The mice were treated with the testing compound and after 15 min, they were placed in the rectangular box and closed. After the test run of 10 min, the cut-offs of the photocell beam due to the movement of mice was noted which was recorded on the counter outside the box. The results were directly compared with that of control group to assess sedative activity.

Porsolt Forced Swimming Test: The mice were treated with the testing compound and after 15 min, they were placed in a beaker of water which was maintained at 25 °C. After the test run of 5 min, the 1st time of immobilization and overall immobility times were recorded using a stop watch. The results were directly compared with that of control group to assess antidepressant activity.

254

(R)-dimethyl 2-aminosuccinate hydrochloride 145

O 4 3 2 O O 1 .HCl NH2 O

Chemical Formula: C6H12ClNO4 Molecular Weight: 197.61

Thionyl chloride (15 mL, 206.5 mmol, 0.9 equiv) was added dropwise over 10 min to a stirred mixture of D-aspartic acid (30 g, 225.4 mmol, 1.0 equiv) and Methanol (300 mL) at 0 °C. The mixture was set to reflux and stirred for 16 h. The reaction mixture was cooled down to room temperature and later the methanol was evaporated in vacuo to give the ester (110.0 g, 99% yield) as a white waxy solid.

Melting point: 107-110 °C.

1 H NMR (400 MHz, D2O) δ (ppm) 3.15 (dd, J = 18.2, 4.6 Hz, 1H, H3), 3.22 (dd, J = 18.2, 6.1

Hz, 1H, H3), 3.77 (s, 3H, OCH3), 3.86 (s, 3H, H4), 4.50-4.56 (m, 1H, H2).

CAS Registry Number: 14358-33-9, 32213-95-9, 69630-50-8.

D [α] 20 = - 13.3 (c 1.00, CHCl3).

The analysis is in accordance with the literature.

(R)-dimethyl 2-(tert-butoxycarbonylamino)succinate 145a

O 4 O 3 5 2 6 O 1 O NH O 7 O

Chemical Formula: C11H19NO6 Molecular Weight: 261.27

To a solution of (R)-dimethyl 2-aminosuccinate hydrochloride (2.0 g, 10.12 mmol, 1.0 equiv) in methanol (50 mL) at room temperature under argon was added dropwise triethylamine (4.23 mL, 30.36 mmol, 3.0 equiv) followed by di-tert-butyl-dicarbonate (2.64 g, 12.14 mmol, 1.2 equiv). The reaction mixture was continued to stir for 6 h. The mixture

255 was washed with water (3x100 mL) & dried over MgSO4 and then concentrated in vacuo to give yellow viscous oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-dimethyl 2-(tert-butoxycarbonylamino)succinate as a white crystalline sticky solid (2.16 g, 82%).

Rf 0.12 (SiO2, cyclohexane/ethyl acetate: 7/3).

Mp: 61-62 °C.

1 H NMR (400 MHz, CDCl3) δ (ppm) 1 .40 (9H, s, H 5, 6, 7), 2.70-3.10 (2H, m, H3), 3.69 (3H, s, OCH3), 3.76 (3H, s, OCH3), 4.59 (1H, m, H2).

D [α] 25 = + 27. 9 (c 1.10, CHCl3).

CAS Registry Number: 130622-08-1

The analysis is in accordance with the literature.

(R)-dimethyl 2-(bis(tert-butoxycarbonyl)amino)succinate 145b

O 4 O 5 3 O 6 2 O 1 7 N O O O O 10 8 9 Chemical Formula: C16H27NO8 Molecular Weight: 361.38 To a solution of (R)-dimethyl 2-(tert-butoxycarbonylamino)succinate (2.0 g, 5.53 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) at 0 °C was added slowly sodium hydride ( 0.15 g, 6.08 mmol,1.1 equiv) and continued to stir at 0 °C for 30 min. This was followed by bringing the reaction to the room temperature and addition of di-tert-butyl-dicarbonate (1.44 g, 6.63 mmol, 1.2 equiv). The reaction mixture was continued to stir for 10 h. The mixture was washed with water (3x100 mL) & dried over MgSO4 and then concentrated in vacuo to give white viscous oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give (R)-dimethyl 2-(tert-butoxycarbonylamino)succinate as a white crystalline solid (2.16 g, 72 %).

256

Rf 0.31 (SiO2, cyclohexane/ethyl acetate: 8/2).

Mp: 54 - 56 °C.

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.56 (18H, s, H5-10), 2.72 (1H, dd, J = 16.6, 6.4 Hz,

H3), 3.31 (1H, dd, J = 16.2, 7.2 Hz, H3), 3.76 (3H, s, OCH3), 3.81 (3H, s, OCH3), 5.48 (1H, dd, J = 7.2, 6.4 Hz, H2).

D [α] 25 = - 31.2 (c 1.00, CHCl3).

CAS Registry Number: 219617-08-0.

The analysis is in accordance with the literature.

(S)-dimethyl 2-aminosuccinate hydrochloride 145’

O 4 3 2 O O 1 .HCl NH2 O

Chemical Formula: C6H12ClNO4 Molecular Weight: 197.61

Thionyl chloride (15 mL, 206.5 mmol, 0.9 equiv) was added dropwise over 10 min to a stirred mixture of L-aspartic acid (30 g, 225.4 mmol, 1.0 equiv) and Methanol (300 mL) at 0 °C. The mixture was set to reflux and stirred for 16 h. The reaction mixture was cooled down to room temperature and later concentrated in vacuo to give the methyl ester salt (44.0 g, 98% yield) as a white waxy solid.

Mp: 114-116 °C.

1 H NMR (400 MHz, D2O) δ (ppm) 3.13 (1H, dd, J = 18.2, 4.6 Hz, H3), 3.20 (1H, dd, J =

18.2, 6.1 Hz, 1H, H3), 3.74 (3H, s, OCH3), 3.84 (3H, s, OCH3), 4.49 (1H, dt, J = 6.0, 4.7 Hz,

H2).

D [α] 20 = + 20.8 (c 1.01, CHCl3).

CAS Registry Number: 32213-95-9.

The analysis is in accordance with the literature.

257

(R)-dimethyl 2-(tritylamino)succinate 146

O 4 O 3 2 O 1 HN O

Chemical Formula: C25H25NO4 Molecular Weight: 403.47

To a solution of (R)-dimethyl 2-aminosuccinate hydrochloride (29.53 g, 150 mmol, 1.0 equiv) in dichloromethane (450 mL) at 0 °C under argon was added dropwise triethylamine (62.72 mL, 450 mmol, 3.0 equiv) followed by triphenylmethyl chloride (50 g, 180 mmol, 1.2 equiv). The reaction mixture was allowed to come down to room temperature and stirred for 6 h. The mixture was washed with water (3x300 mL), dried over MgSO4 and then concentrated in vacuo to give yellow oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9/1) to give (R)-dimethyl 2-(tritylamino)succinate as a white crystalline solid (52.3 g, 86%).

Rf 0.2 (SiO2, cyclohexane/ethyl acetate: 9/1)

Mp: 69 - 71 °C.

1 H NMR (400 MHz, CDCl3) δ (ppm) 2.53 (1H, dd, J = 14.7, 7.0 Hz, H3), 2.65 (1H, dd, J =

14.7, 5.4 Hz, H3),1.59 (1H, br s, NH), 3.28 (3H, s, OCH3), 3.70 (3H, s, OCH3), 3.72–3.76

(1H, m, H2), 7.18–7.26 (5H, m, ArH), 7.29–7.36 (4H, m, ArH), 7.49–7.52 (6H, m, ArH).

D [α] 20 = - 1.02 (c 1.05, CHCl3).

CAS Registry Number: 151521-94-7.

The analysis is in accordance with the literature.

258

(S)-dimethyl 2-(tritylamino)succinate 146’

O 4 O 3 2 O 1 HN O

Chemical Formula: C25H25NO4 Molecular Weight: 403.47

To a solution of (S)-dimethyl 2-aminosuccinate hydrochloride (29.64 g, 150 mmol, 1.0 equiv) in dichloromethane (450 mL) at 0 °C under argon was added dropwise triethylamine (62.76 mL, 450 mmol, 3.0 equiv) followed by triphenylmethyl chloride (50 g, 180 mmol, 1.2 equiv). The reaction mixture was allowed to come down to room temperature and stirred for 6 h. The mixture was washed with water (3x300 mL) & dried (MgSO4) and then concentrated in vacuo to give yellow oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9/1) to give (S)-dimethyl 2-(tritylamino)succinate as a white crystalline solid (50.8 g, 84%).

Rf 0.2 (SiO2, cyclohexane/ethyl acetate: 9/1)

Mp: 71-74 °C.

1 H NMR (400 MHz, CDCl3) δ (ppm) 2.51 (1H, dd, J = 14.7, 7.0 Hz, H3), 2.65 (1H, dd, J =

14.7, 5.4 Hz, H3), 2.94 (1H, br s, NH), 3.26 (3H, s, OCH3), 3.68 (3H, s, OCH3), 3.68–3.73

(1H, m, H2), 7.17–7.20 (3H, m, ArH), 7.24–7.31 (6H, m, ArH), 7.48–7.50 (6H, m, ArH).

D [α] 25 = + 33.2 (c 1.00, CHCl3).

CAS Registry Number: 116393-72-7.

The analysis is in accordance with the literature.

259

(R)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate 147

5 6 O O P 4 O 3 O 2 O 1 HN O

Chemical Formula: C27H30NO6P Molecular Weight: 495.50

A solution of dimethyl methylphosphonate (21.6 mL, 201.87 mmol, 4.5 equiv) in tetrahydrofuran (200 mL) was cooled to −78 °C under argon. Later, n-Butyl lithium (2.5 M in hexane, 80.75 mL, 201.87 mmol, 4.5 equiv) was added dropwise during which, the reaction mixture turns purple in color and was stirred for 20 min. In another round bottom flask, a solution of (R)-dimethyl 2-(tritylamino)succinate (18.1 g, 44.89 mmol, 1.0 equiv) in tetrahydrofuran (90 mL) was cooled to −78 °C under argon and the dimethyl methylphosphonate/n-butyl lithium solution was transferred via cannula into the flask and the reaction mixture was left for stirring at −78 °C for 60 min. During this time, a color change from purple to yellow takes place. The reaction was quenched with a saturated solution of ammonium chloride (100 mL) and allowed to warm to room temperature. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with brine (2x250 mL), water (2x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 2/8) to give (R)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2- (tritylamino)pentanoate as a yellow oil (17.1 g, 77%).

Rf 0.12 (SiO2, cyclohexane/ethyl acetate: 2/8)

1 H NMR (400 MHz, CDCl3) δ (ppm) 2.80 (1H, dd, J = 16.7, 7.0 Hz, H3), 2.90 (1H, dd, J =

16.7, 4.6 Hz, H3), 3.08 (2H, dd, J(H-C-P) = 22.7, 2.7 Hz, H4),3.30 (3H, s, OCH3), 3.67–3.73

(1H, m, H2), 3.75 (3H, s, H5), 3.77 (3H, s, H6), 7.18–7.20 (6H, m, ArH), 7.29-7.35 (3H, m, ArH), 7.46-7.57 (6H, m, Ar-H).

D [α] 20 = - 30.5 (c 1.05, CHCl3).

260

CAS Registry Number: 151491-88-2.

The analysis is in accordance with the literature.

(S)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate 147’

5 6 O O P 4 O 3 O 2 O 1 HN O

Chemical Formula: C27H30NO6P Molecular Weight: 495.50

A solution of dimethyl methylphosphonate (16 mL, 149.4 mmol, 4.5 equiv) in tetrahydrofuran (180 mL) was cooled to −78 °C under argon. Later, n-Butyl lithium (2.5 M in hexane, 60 mL, 149.4 mmol, 4.5 equiv) was added dropwise during which, the reaction mixture turns dark purple in color and was stirred for 20 minutes. In another round bottom flask, a solution of (S)-dimethyl 2-(tritylamino)succinate (13.4 g, 33.2 mmol, 1.0 equiv) in THF (70 mL) was cooled to −78 °C under argon and the dimethyl methylphosphonate/n-butyl lithium solution was transferred via cannula into the flask and the reaction mixture was left for stirring at −78 °C for 60 minutes. During this time, a color change from purple to yellow takes place. The reaction was quenched with a saturated solution of ammonium chloride (100 mL) and allowed to warm to room temperature. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with brine (2x250 mL), water (2x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 2/8) to give (S)-methyl 5- (dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate as a yellow oil (12.3 g, 75%).

Rf 0.12 (SiO2, cyclohexane/ethyl acetate: 2/8)

1 H NMR (400 MHz, CDCl3) δ (ppm) 2.81 (1H, dd, J = 16.7, 6.9 Hz, H3), 2.92 (1H, dd, J =

16.8, 4.6 Hz, H3), 3.08 (2H, dd, J(H-C-P) = 22.6, 2.0 Hz, H4),3.31 (3H, s, OCH3), 3.69–3.72

(1H, m, H2), 3.73 (3H, s, H5), 3.76 (3H, s, H6), 7.18–7.26 (6H, m, ArH), 7.28-7.32 (3H, m, ArH), 7.48-7.52 (6H, m, Ar-H).

261

D [α] 25 = + 32.3 (c 1.00, CHCl3).

CAS Registry Number: 1197335-24-2.

The analysis is in accordance with the literature.

(R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate 149

6 5 4 O O 1 8 3 9 2 O NH 7

Chemical Formula: C27H27NO3 Molecular Weight: 413.50

To a solution of (R)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate (25.77 g, 44.86 mmol, 1.0 equiv) in tetrahydrofuran (130 mL) at room temperature was added Ba(OH)2.H2O (11 g, 58.32 mmol, 1.3 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of acetaldehyde (3.3 mL, 58.32 mmol, 1.3 equiv) diluted in tetrahydrofuran/water: 40/1 (130 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate as a colorless oil (19.9 g, 96%).

Rf 0.37 (SiO2, cyclohexane/ethyl acetate: 8/2).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.92 (3H, dd, J = 6.8, 1.6 Hz, H6), 2.69 (1H, dd, J =

15.4, 7.0 Hz, H3), 2.82 (1H, dd, J = 15.3, 5.2 Hz, H3), 2.88 (1H, J = 9.5 Hz, NH), 3.30 (3H, s,

OCH3), 3.72-3.77 (1H, m, H2), 6.10 (1H, dd, J = 15.8, 1.6 Hz, H4), 6.80 (1H, dq, J = 15.7, 6.8

Hz, H5), 7.15-7.32 (9H, m, Ar-H), 7.50-7.52 (6H, m, Ar-H).

262

13 C NMR (100 MHz, CDCl3) δ (ppm) 18.4 (C6), 44.6 (C3), 51.60 (C1), 53.36 (C2), 71.01

(C7), 126.33, 127.68, 128.59, 131.90, 143.28 (CHar), 145.63 (Car), 174.20 (C9), 197.04 (C8).

25 [α] D = - 8.7 (c 0.975, CHCl3).

+ HR-ESI-MS: calculated for C27H28NO3 (M+H ): 414.2069, found: 414.2088.

(S,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate 149’

6 5 4 O O 1 3 2 O NH

Chemical Formula: C27H27NO3 Molecular Weight: 413.50

To a solution of (S)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate (20.4 g, 33.2 mmol, 1.0 equiv) in tetrahydrofuran (91 mL) at room temperature was added Ba(OH)2.H2O (8.14 g, 43.17 mmol, 1.3 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of acetaldehyde (2.4 mL, 43.17 mmol, 1.3 equiv) diluted in tetrahydrofuran/water: 40/1 (91 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over

MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give (S,E)-methyl 4-oxo-2- (tritylamino)hept-5-enoate as a colorless oil (13.0 g, 95%).

Rf 0.37 (SiO2, cyclohexane/ethyl acetate: 8/2).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.92 (3H, dd, J = 6.8, 1.7 Hz, H6), 2.69 (1H, dd, J =

15.4, 7.0 Hz, H3), 2.82 (1H, dd, J = 15.3, 5.2 Hz, H3), 2.89 (1H, J = 10 Hz, NH), 3.29 (3H, s,

OCH3), 3.72-3.80 (1H, m, H2), 6.10 (1H, dq, J = 15.8, 1.6 Hz, H4), 6.80 (1H, dq, J = 15.8, 6.8

Hz, H5), 7.17-7.27 (5H, m, Ar-H), 7.29-7.36 (4H, m, Ar-H), 7.48-7.54 (6H, m, Ar-H).

CAS Registry Number: 1313240-35-5.The analysis is in accordance with the literature.

263

Methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate

O O

O N H O

Chemical Formula: C10H19NO4 Molecular Weight: 217.26 Thionyl chloride (0.47 mL, 6.35 mmol, 0.9 equiv) was added dropwise over 10 min to a stirred mixture of sodium (6R)-6-methyl-4-oxopiperidine-2-carboxylate (1.1 g, 7.06 mmol, 1.0 equiv) and Methanol (10 mL) at 0 °C. The mixture was set to reflux and stirred for 16 h. The reaction mixture was cooled down to room temperature and later concentrated in vacuo. The resulting residue was diluted with dichloromethane (100 mL), washed with 1N NaOH

(3x100 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 3/7) to give methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate as a brown oil (1.21 g, 91%).

Note: Methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate gives 75/25 ration of cis/trans

1. (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate 155

8 9 O O 5 4 3 6 2 10 O 11 7 N 1 H O

Rf 0.3 (SiO2, cyclohexane/ethyl acetate: 3/7).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.08 (dd, J = 13.1, 2.8 Hz, 1H, H5a), 1.13 (d, J = 6.4

Hz, 3H, H7 (CH3)), 1.37 (dd, J = 16.1, 9.2 Hz, 1H, H3a), 1.97 (dt, J = 13.3, 2.7 Hz, 1H, H5e),

2.34 (dt, J = 13.1, 2.8 Hz, 1H, H3e), 2.78-2.88 (m, 1H, H6a) 3.17 (3H, s, OCH3), 3.21 (3H, s,

O-CH3), 3.55 (dd, J = 12.2, 2.8 Hz, H2a), 3.72(s, 3H, H11(CH3)).

13 C NMR (100 MHz, CDCl3) δ (ppm) 22.15 (C7), 35.78 (C3), 40.61 (C5), 47.52 (C8), 48.03

(C9), 52.13 (C6), 55.95 (C11), 57.96 (C2), 99.27 (C4), 173.30 (C10).

20 [α] D = - 2.6 (c 1.0, CHCl3).

264

+ HR-ESI-MS: calculated for C10H19NO4 (M+H ): 218.1392, found: 218.1402.

2. (2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate 156

8 9 O O 5 4 3 6 2 10 O 11 7 N 1 H O

Rf 0.16 (SiO2, cyclohexane/ethyl acetate: 3/7).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.02 (dd, J = 13.0, 2.7 Hz, 1H, H5a), 1.11 (d, J = 6.5

Hz, 3H, H7 (CH3)), 1.25 (dd, J = 16.0, 3.7 Hz, 1H, H3a), 1.85 (dt, J = 13.1, 2.8 Hz, 1H, H5e),

2.19 (dt, J = 13.2, 8.8 Hz, 1H, H3e), 2.98 (3H, s, OCH3), 3.14 (3H, s, O-CH3), 3.17-3.26 (m,

1H, H6a), H), 3.71 (s, 3H, H11(CH3)). 3.80 (dd, J = 6.2, 1.5 Hz, H2e)

13 C NMR (100 MHz, CDCl3) δ (ppm) 21.03 (C7), 36.51 (C3), 39.33 (C5), 47.10 (C8), 47.79

(C9), 51.28 (C6), 56.89 (C11), 57.31 (C2), 98.74 (C4), 172.11 (C10).

20 [α] D = + 3.1 (c 1.05, CHCl3).

+ HR-ESI-MS: calculated for C10H19NO4 (M+H ): 218.1392, found: 218.1398.

(2R,6R)-methyl 6-methyl-4-oxopiperidine-2-carboxylate 155’

5 4 3 6 2 8 O 9 7 N 1 H O

Chemical Formula: C8H13NO3 Molecular Weight: 171.19

To a solution of (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (0.2 g, 1.17 mmol, 1.0 equiv) in DCM (5 mL) and distilled H2O (0.5 mL) at 0 °C was added with trifluoroacetic acid TFA ( 0.27 mL, 2.34 mmol, 2.0 equiv) dropwise over a time of 2 min. The reaction mixture was continued to stir at room temperature for 2 h. The reaction mixture was basified using 1N NaOH till we reach a pH of 9-10 and later diluted with dichloromethane (10 mL) and washed using brine (3x10 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl

265 acetate: 3/7) to give (2R,6R)-methyl 6-methyl-4-oxopiperidine-2-carboxylate as brown oil (0.15 g, 93%).

Rf 0.15 (SiO2, cyclohexane/ethyl acetate: 3/7).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.28 (d, J = 6.2 Hz, 3H, H7(CH3)), 2.04 (brs, 1H, NH),

2.11 (ddd, J = 14.3, 11.7, 0.9 Hz, 1H, H5a ), 2.42 (ddd, J = 14.4, 7.3, 1.6 Hz, 2H, H3e,5e), 2.69

(ddd, J = 14.4, 3.4, 2.0 Hz, 1H, H3a), 2.94-3.09 (m, 1H, H6a), 3.68 (dd, J = 12.2, 3.4 Hz, 1H,

H2a), 3.79 (s, 3H, H9(CH3)).

13 C NMR (100 MHz, CDCl3) δ (ppm) 22.68 (C7), 44.15 (C3), 50.10 (C5), 51.57 (C6), 52.63

(C9), 58.05 (C2), 173.30 (C8), 207.10 (C4).

20 [α] D = - 3.4 (c 1.015, CHCl3).

+ HR-ESI-MS: calculated for C8H13NO3 (M+H ): 172.1815, found: 172.1859.

Sodium (R)-6-methyl-4-oxopiperidine-2-carboxylate 157 O

O-Na+ N H O

Chemical Formula: C7H10NNaO3 Molecular Weight: 179.14

To a solution of (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate (13 g, 31.46 mmol, 1.0 equiv) in methanol (260 mL) at room temperature was added dropwise 2M hydrochloric acid (158 mL, 314.6 mmol, 10.0 equiv) and left to stir for 60 minutes. The reaction mixture was diluted with distilled water (320 mL) followed by N, N-diisopropylethylamine (66 mL, 380 mmol, 12.0 equiv) until pH was adjusted to 10. The reaction mixture was continued to stir at room temperature for 12 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 7), washed with ethyl acetate (3x200 mL). pH of the aqueous phase was further adjusted to 14 by using 1M NaOH (200 mL), washed with ethyl acetate (3x200 mL) and aqueous phase was concentrated in vacuo to obtain sodium (2R)-6-methyl-4- oxopiperidine-2-carboxylate. The sodium salt thus obtained is further engaged in the succeeding steps without purification.Note: The attribution of chemical shift for each proton was difficult due to the presence of large amount of diisopropylethylamine hydrochloride salt with relatively less amount of desired product.

266

Sodium (S)-6-methyl-4-oxopiperidine-2-carboxylate 157’ O

O-Na+ N H O

Chemical Formula: C7H10NNaO3 Molecular Weight: 179.14

To a solution of (S,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate (11 g, 26.55 mmol, 1.0 equiv) in methanol (220 mL) at room temperature was added dropwise 2M hydrochloric acid (133 mL, 265.7 mmol, 10.0 equiv) and left to stir for 60 minutes. The reaction mixture was diluted with distilled water (270 mL) followed by N, N-diisopropylethylamine (55 mL, 315.7 mmol, 12 equiv) until pH was adjusted to 10. The reaction mixture was continued to stir at room temperature for 12 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 7), washed with ethyl acetate (3x200 mL). pH of the aqueous phase was further adjusted to 14 by using 1M NaOH (180 mL), washed with ethyl acetate (3x200 mL) and aqueous phase was concentrated in vacuo to obtain sodium (2S)-6-methyl-4- oxopiperidine-2-carboxylate. The sodium salt thus obtained is further engaged in the succeeding steps without purification.

Note: The attribution of chemical shift for each proton was difficult due to the presence of large amount of diisopropylethylamine hydrochloride salt with relatively less amount of desired product.

(R)-1-(tert-butoxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid 158

5 4 3 6 2 1 OH 7 N 8 O O O 9 12 10 11

Chemical Formula: C12H19NO5 Molecular Weight: 257.28 To a solution of sodium (2R)-6-methyl-4-oxopiperidine-2-carboxylate (1.25 g, 7.01 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added with di-tert-butyl dicarbonate (2.29 g, 10.52 mmol, 1.5 equiv). The reaction mixture was continued to stir in refluxed

267 condition for 16 h and concentrated in vacuo. The resulting residue was diluted with water

(until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 1/9) to give (R)-1-(tert-butoxycarbonyl)-6-methyl-4- oxopiperidine-2-carboxylic acid (1.29 g, 72%) as yellow oil.

Rf 0.12 (SiO2, cyclohexane/ethyl acetate: 1/9).

Note: The attribution of chemical shift for each proton was difficult due to the presence of 2 rotamers. Percentages of two rotamers were (55/45).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.22 (3H, d, J = 6.6 Hz, H7), 1.28 (3H, d, J = 6.8 Hz,

H7), 1.41 (9H, s, H10, 11, 12), 1.45 (9H, s, H10, 11, 12), 2.29-2.43 (2H, m), 2.56-2.75 (2H, m), 2.79-3.06 (4H, m), 4.30-4.67 (2H, m), 4.73-5.19 (2H, m).

Rotamer A

13 C NMR (100 MHz, CDCl3) δ (ppm) 20.82 (C7), 22.63 (C10, 11, 12), 36.49 (C3), 44.75 (C5),

49.64 (C6), 53.24 (C2), 81.63 (C9), 175.05 (C8), 176.42 (C1), 206.27 (C4).

Rotamer B

13 C NMR (100 MHz, CDCl3) δ (ppm) 20.28 (C7), 28.30 (C10, 11, 12), 39.68 (C3), 45.46 (C5),

49.64 (C6), 53.39 (C2), 81.83 (C9), 175.51 (C8), 177.17 (C1), 206.53 (C4).

25 [α] D = + 15.9 (c 1.02, CHCl3).

- HR-ESI-MS: calculated for C12H19NO5 (M+H ): 270.1341, found: 270.1369.

(2R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid 159

5 4 3 6 2 1 OH 7 N 8 O O O 9

Chemical Formula: C15H17NO5 Molecular Weight: 291.29

268

To a solution of sodium (2R)-6-methyl-4-oxopiperidine-2-carboxylate (5.63 g, 31.46 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added dropwise benzyl chloroformate (6.7 mL, 47.2 mmol, 1.5 equiv. If the reaction mixture’s pH is acidic, adjust the pH to basic using 1M NaOH followed by further addition of one more equivalent of benzyl chloroformate. The reaction mixture was continued to stir at room temperature for 16 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 1/9) to give (2R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid as an orange-yellow oil (8.14 g, 86%) as yellow oil.

Rf 0.12 (SiO2, cyclohexane/ethyl acetate: 1/9).

Note: The attribution of chemical shift for each proton was difficult due to the presence of 2 rotamers. Percentages of two rotamers were (66/33).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.18 (3H, d, J = 6.6 Hz, H7), 1.25 (3H, d, J = 6.8 Hz,

H7), 2.50-3.02 (5H, m, H6a, 3, 5), 4.33-4.69 (2H, m, H2a), 5.11 (4H, m, H9), 7.25 (10H, m, HAr).

Rotamer A

13 C NMR (100 MHz, CDCl3) δ (ppm) 20.28 (C7), 36.27 (C3), 44.53 (C5), 47.37 (C6), 53.34

(C2), 67.30 (C9), 127.85, 128.09, 128.30 (CHAr), 135.77 (CAr), 174.48 (C8), 175.49 (C1)

205.92 (C4).

Rotamer B

13 C NMR (100 MHz, CDCl3) δ (ppm) 20.72 (C7), 39.63 (C3), 45.40 (C5), 48.52 (C6), 53.41

(C2), 68.23 (C9), 127.99, 128.22, 128.57 (CHAr), 135.90 (CAr), 175.12 (C8), 177.09 (C1)

205.92 (C4).

25 [α] D = + 10.9 (c 1.00, CHCl3).

- HR-ESI-MS: calculated for C15H17NO5 (M+H ): 290.1028, found: 290.1030.

269

(2S)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid 159’

5 4 3 6 2 1 OH 7 N 8 O O O 9

Chemical Formula: C15H17NO5 Molecular Weight: 291.29

To a solution of sodium (2S)-6-methyl-4-oxopiperidine-2-carboxylate (4.75 g, 26.5 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added dropwise benzyl chloroformate (5.65 mL, 39.7 mmol, 1.5 equiv. If the reaction mixture’s pH is acidic, adjust the pH to basic using 1M NaOH followed by further addition of one more equivalent of benzyl chloroformate. The reaction mixture was continued to stir at room temperature for 16 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 1/9) to give (2S)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid as an orange-yellow oil (6.48 g, 84%) as yellow oil.

25 [α] D = - 5.1 (c 1.05, CHCl3).

Apart from the optical activity, rest of the data remains same as that of (2R)-1- (benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid.

N-benzyl-1-(2-bromophenyl)methanamine 160

1 5 6 7 2 N 8 H 3 Br 11 9 4 10

Chemical Formula: C14H14BrN Molecular Weight: 276.17

270

To a solution of 2-bromobenzaldehyde (3 g, 16.22 mmol, 1.0 equiv) and benzylamine (2.66 mL, 24.32 mmol, 1.5 equiv) in methanol (100 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.92 g, 24.32 mmol, 1.5 equiv). The reaction mixture was continued to stir at room temperature for 12 h. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9.5/0.5) to give N-benzyl-1-(2- bromophenyl)methanamine as a colorless oil (4.25 g, 95%).

Rf 0.15 (SiO2, 100% ethyl acetate).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.73 (brs, 1H, NH), 3.83 (s, 2H, H6), 3.92 (s, 2H, H5),

7.15 (td, J = 7.7, 1.7 Hz, 1H, H2), 7.28-7.45 (m, 7H, Ar-H), 7.57 (dd, J = 7.9, 1.1 Hz, 1H, H4).

CAS Registry Number: 65185-56-0.

The analysis is in accordance with the literature.

N-(2-bromobenzyl)-2,2,2-trifluoroethanamine 161

1 5 6 F F 2 N H 3 F Br 4

Chemical Formula: C9H9BrF3N Molecular Weight: 268.07

To a solution of 2-bromobenzaldehyde (1.58 g, 8.57 mmol, 1.0 equiv) and 2,2,2 trifluoroehtylamine (0.68 mL, 8.57 mmol, 1.0 equiv) in methanol (100 mL) at 0 °C was added slowly in portion wise acetic acid (1.47 mL, 25.7 mmol, 3.0 equiv) followed by sodium cyanoborohydride (2.153 g, 34.26 mmol, 4.0 equiv). The reaction mixture was continued to stir at room temperature for 12 h. The mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous solution of NaHCO3 (3x50 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give N-benzyl-1-(2-bromophenyl)methanamine as a colorless oil (4.25 g, 95%).

Rf 0.1 (SiO2, 100% ethyl acetate).

271

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.87 (brs, 1H, NH), 3.20 (q, J = 9.4 Hz, 2H, H6), 4.01

(s, 2H, H5), 7.18 (td, J = 7.8, 1.8 Hz, 1H, H2), 7.33 (td, J = 7.5, 1.2 Hz, H3), 7.41 (dd, J = 7.6,

1.5 Hz, H1)7.59 (dd, J = 8.0, 1.1 Hz, 1H, H4).

CAS Registry Number: 558483-94-6.

The analysis is in accordance with the literature.

tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate 177

N N O Br O O

Chemical Formula: C26H31BrN2O4 Molecular Weight: 515.43

To a solution of 1-(tert-butoxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.8 g, 3.11 mmol, 1.0 equiv) in dichloromethane (10 mL) at 0 °C was added N,N- Diisopropylethylamine (1.08 mL , 6.218 mmol, 2.0 equiv) followed by (3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (1.86 g, 6.218 mmol, 2.0 equiv). The reaction mixture was continued to stir at 0 °C for 60 min. The mixture was later added with a solution of N-benzyl-1-(2-bromophenyl)methanamine (0.86 g, 3.11 mmol, 1.0 equiv) in dichloromethane (5 mL) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with dichloromethane (150 mL), washed with saturated aqueous solution of NaHCO3 (3x150 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (1.28 g, 72%).

Note:Tert-butyl-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate was obtained in 70/30 ration of cis/trans.

272

(2R,6R)-tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 177 cis

12 11 O 13 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N 23 O 22 17 Br O O 18 24 27 21 19 25 26 20

Rf 0.27 (SiO2, cyclohexane/ethyl acetate: 8/2)

1 + HR-ESI-MS: calculated for C26H31BrN2O4 (M+H ): 515.1545, found: 515.1534.

(2S,6R)-tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 177 trans

12 11 O 13 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N 23 O 22 17 Br O O 18 24 27 21 19 25 26 20

Rf 0.19 (SiO2, cyclohexane/ethyl acetate: 3/7)

1 + HR-ESI-MS: calculated for C26H31BrN2O4 (M+H ): 515.1545, found: 515.1569.

(R)-Benzyl-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 179

N N O Br O O

Chemical Formula: C29H29BrN2O4 Molecular Weight: 549.45

273

Method A To a solution of (R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.2 g, 0.68 mmol, 1.0 equiv) in dichloromethane (5 mL) at 0 °C was added N,N- Diisopropylethylamine (0.24 mL , 1.37 mmol, 2.0 equiv) followed by (3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (0.41 g, 1.37 mmol, 2.0 equiv). The reaction mixture was continued to stir at 0 °C for 60 min. The mixture was later added with a solution of N-benzyl-1-(2-bromophenyl)methanamine in dichloromethane (3 mL) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous solution of NaHCO3 (3x50 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-Benzyl 2- (benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (0.27 g, 72%).

Method B

To a solution of (R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.2 g, 0.68 mmol, 1.0 equiv) and N-benzyl-1-(2-bromophenyl)methanamine (0.21 g, 0.75 mmol, 1.1 equiv) in dichloromethane (8 mL) at 0 °C was added N,N-Diethylethanamine (0.29 mL , 2.06 mmol, 3.0 equiv) followed by 1-Propylphosphonic acid cyclic anhydride, 50% (w/w) solution in dichloromethane (3.27 mL, 6.86 mmol, 10.0 equiv). The reaction mixture was continued to stir at room temperature for 14 h. The resulting mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous solution of NaHCO3 (3x50 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-Benzyl 2- (benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (0.15 g, 40%).

Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans.

Note: Due to the presence of 4 rotamers, the structural elucidation of the synthesized compound was not possible. Even on changing the solvents, the spectra still remains unpredictable. Hence, we analyzed the compound using HR-ESI-MS alone.

274

Even the performance of optical activity deemed to be difficult as there are 4 rotamers and the tested compounds optical activity were not reproducible and kept on fluctuating.

(2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 179 cis

12 11 O 13 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N 26 25 O 22 17 Br 27 24 O O 23 18 28 30 21 19 29 20

Rf 0.35 (SiO2, cyclohexane/ethyl acetate: 3/7).

+ HR-ESI-MS: calculated for C29H29BrN2O4 (M+H ): 549.1389, found: 549.1401.

(2S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 179 trans

12 11 O 13 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N 26 25 O 22 17 Br 27 24 O O 23 18 28 30 21 19 29 20

Rf 0.31 (SiO2, cyclohexane/ethyl acetate: 3/7).

+ HR-ESI-MS: calculated for C29H29BrN2O4 (M+H ): 549.1389, found: 549.1365.

(S)-Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 179’

N N O Br O O

275

Chemical Formula: C29H29BrN2O4 Molecular Weight: 549.45

We performed the reaction with the same quantity and procedure as briefed for Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate. All the obtained data are similar to that of (R)-Benzyl-2-(benzyl(2- bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate.

(S)-Benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate 180

O F F F N N O Br O O

Chemical Formula: C24H24BrF3N2O4 Molecular Weight: 541.35 To a solution of 1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (1.5 g, 5.15 mmol, 1.0 equiv) and N-(2-bromobenzyl)-2,2,2-trifluoroethanamine (1.52 mL, 5.66 mmol, 1.1 equiv) in dichloromethane (60 mL) at 0 °C was added N,N-Diethylethanamine (4.3 mL , 30.8 mmol, 6.0 equiv) followed by 1-Propylphosphonic acid cyclic anhydride, 50% (w/w) solution in dichloromethane (24.6 mL, 51.4 mmol, 10.0 equiv). The reaction mixture was continued to stir at room temperature for 14 h. The resulting mixture was diluted with dichloromethane (250 mL), washed with saturated aqueous solution of NaHCO3 (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give benzyl 2-((2- bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (1.37 g, 37%).

Note: Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans.

276

(2R,6R)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate 180 cis

O F 10 F 5 4 3 9 F 6 2 8 N 11 7 1 N 21 12 22 20 O Br O 18 O 17 19 13 23 25 16 14 24 15

Rf 0.36 (SiO2, cyclohexane/ethyl acetate: 3/7).

+ HR-ESI-MS: calculated for C24H24BrF3N2O4 (M+H ): 541.0950, found: 541.0998.

(2S,6R)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate 180 trans

O F 10 F 5 4 3 9 F 6 2 8 N 11 7 1 N 21 12 22 20 O Br O 18 O 17 19 13 23 25 16 14 24 15

Rf 0.45 (SiO2, cyclohexane/ethyl acetate: 3/7)

+ HR-ESI-MS: calculated for C10H19NO4 (M+H ): 541.0950, found: 541.0987.

tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6-methylpiperidine-1- carboxylate

O O

N N H O Br

Chemical Formula: C23H29BrN2O3 Molecular Weight: 461.39

To (2R,6R)-tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate (0.34 g, 0.66 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl orthoformate (0.36 mL, 3.30 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.14 g, 0.73

277 mmol, 1.1 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH

(3x30 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 85/15) to give (2R,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6-methylpiperidine-2-carboxamide as a colorless viscous oil (0.29 g, 95%).

Note: Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans.

(2R,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6-methylpiperidine-2-carboxamide 184 cis

23 24 12 11 13 O O 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N H O 22 17 Br 18 21 19 20

Rf 0.17 (SiO2, cyclohexane/ethyl acetate: 85/15).

The attribution of chemical shift for each proton is difficult due to the presence of rotamers.

1 H NMR (400 MHz, C6D6) δ (ppm) 0.86-0.96(4H, m, H5a), 0.98 (6H, d, J = 6.5 Hz, H7), 1.02

(6H, d, J = 6.5 Hz, H7), 1.30-1.48 (8H, m, H5e/3e+3a/6a), 1.85-2.02 (8H, m, H5e/3e+3a/6a), 2.28

(4H, ddt, J = 12.7, 9.9, 2.5 Hz, H5e/3e) 2.88 (6H, s, H23/24), 2.89 (6H, s, H23/24), 3.10 (6H, s,

H23/24), 3.13 (6H, s, H23/24), 3.92 (2H, dd, J = 11.8, 2.7 Hz, H2a), 4.11 (2H, dd, J = 11.8, 2.7

Hz, H2a), 4.33-5.15 (16H, m, H10/16), 6.71-7.36 (32H, m, Ar-H), 7.40 (2H, d, J = 7.9 Hz, H19),

7.51 (2H, d, J = 7.6 Hz, H19).

Rotamer A

13 C NMR (101 MHz, C6D6) δ (ppm) 19.09 (C7), 36.82 (C3), 41.70 (C5), 46.87 (C24, 25), 48.39

(C6), 49.04 (C9), 50.52 (C16), 54.17 (C2), 99.98 (C4), 123.06 (C-Br), 126.81, 127.82, 128.06,

128.30, 128.73, 129.05, 133.19, 136.19, 137.33(CAr).

278

Rotamer B

13 C NMR (101 MHz, C6D6) δ (ppm) 22.13 (C7), 36.87 (C3), 41.81 (C5), 47.23 (C24, 25), 48.71

(C6), 49.15 (C9), 50.65 (C16), 54.35 (C2), 100.10 (C4), 124.10 (C-Br), 126.98, 127.89, 128.51,

128.59, 128.92, 129.59, 133.02, 137.12, 137.94 (CAr).

+ HR-ESI-MS: calculated for C23H29BrN2O3 (M+H ): 461.1440, found: 461.1426.

20 [α] D = - 15.5 (c 1.05, CHCl3).

(2S,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6-methylpiperidine-2-carboxamide 185 trans

23 24 12 11 13 O O 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N H O 22 17 Br 18 21 19 20 The NMR spectra of trans isomer was not only rotamer prone but also badly resoluted. Hence it is neither predictable nor identifiable. Hence, we analyzed only using the following data.

Rf 0.10 (SiO2, cyclohexane/ethyl acetate: 85/15)

+ HR-ESI-MS: calculated for C23H29BrN2O3 (M+H ): 461.1440, found: 461.1454.

20 [α] D = - 8.8 (c 1.0, CHCl3).

(1R,4aR)-6-benzyl-3,3-dimethoxy-1-methyl-2,3,4,4a,6,7-hexahydrobenzo[f]pyrido[1,2- a][1,4]diazepin-5(1H)-one 188

23 24 O O 14 13 5 4 3 6 2 O 15 12 7 N 22 1 8 9 10 11 21 N 16 20 17 19 18

Chemical Formula: C23H28N2O3 Molecular Weight: 380.48

279

A 25 mL sealed tube was charged with tris(dibenzylideneacetone)dipalladium-Pd2(dba)3 (0.042 g, 0.068 mmol, 0.15 equiv) and (2,2'-bis(diphenylphosphino)-1,1'-binaphthyl)-BINAP (0.074 g, 0.114 mmol, 0.25 equiv) which was later added with fresh anhydrous toluene (3 mL) and left for stirring at room temperature for 10 min. This has been followed by the addition of a solution of (2R,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6- methylpiperidine-2-carboxamide (0.210 g, 0.455 mmol, 1 equiv) in anhydrous toluene (1 mL) through cannula during which, the stirring was continued. Finally, sodium tert-butoxide NaotBu (0.053 g, 0.55 mmol, 1.2 equiv) was added and the sealed tube was stoppered tightly and continued to stir at 100 °C for 10 h. The resulting mixture has been filtered through Celite® bed and thus collected filtrate was diluted with DCM (20 mL) and washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over MgSO4 and evaporated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (1R,4aR)-6-benzyl-3,3-dimethoxy-1-methyl- 2,3,4,4a,6,7-hexahydrobenzo[f]pyrido[1,2-a][1,4]diazepin-5(1H)-one as a colorless oil (0.09 g, 52%).

Rf 0.52 (SiO2, cyclohexane/ethyl acetate: 5/5).

1 H NMR (400 MHz, C6D6) δ (ppm) 0.85 (3H, d, J = 5.9 Hz, H7), 1.45 (1H, dd, J = 13.1, 11.6

Hz, H5a), 2.04-2.14 (2H, m, H3e, 3a), 3.03-3.06 (1H, m, H5e), 3.07 (3H, s, H23), 3.18 (3H, s,

H24), 3.37 (1H, d, J = 13.4, H16), 3.37-3.45 (1H, m, H6a), 4.02 (1H, dd, J = 12.4, 2.6 Hz, H2a),

4.58 (2H, d, J = 1.9 Hz, H9), 5.06 (1H, d, J = 13.1 Hz, H16), 6.54 (1H, dd, J = 7.4, 1.5 Hz,

H21), 6.75 (1H, td, J = 7.4, 1.1 Hz, H20), 6.84 (1H, d, J = 7.8 Hz, H18), 7.01 (1H, td, J = 7.5,

1.1 Hz, H19), 7.05-7.14 (5H, m, H11-15).

13 C NMR (101 MHz, C6D6) δ (ppm) 21.34 (C7), 39.32 (C3), 41.49 (C5), 47.43 (C23), 47.57

(C24), 48.40 (C9), 49.43 (C2), 52.14 (C16), 68.27 (C6), 98.06 (C4), 122.32, 123.20, 127.20,

127.51, 128.55, 128.70, 128.79 (CAr), 137.42 (C17), 138.22 (C10), 148.39 (C22), 172.27 (C8).

+ HR-ESI-MS: calculated for C23H28N2O3 (M+H ): 381.2178, found: 381.2188.

20 [α] D = + 10.6 (c 1.05, CHCl3).

280

(2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6- methylpiperidine-1-carboxylate 190

O O

N N O Br O O

Chemical Formula: C31H35BrN2O5 Molecular Weight: 595.52

To (2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate (0.350 g, 0.64 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl orthoformate (0.35 mL, 3.19 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.03 g, 0.13 mmol, 0.2 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH

(3x30 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 70/30) to give (2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6-methylpiperidine-1- carboxylate as a colorless oil (0.35 g, 91%).

Rf 0.45 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C31H35BrN2O5 (M+H ): 596.1729, found: 596.1788.

(4S,14bS)-2,2-dimethoxy-4-methyl-1,2,3,4,10,14b-hexahydrodibenzo[b,f]pyrido[1,2- d][1,4]diazepine 198

21 22

O O 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 NH 15 10 19 12 11

Chemical Formula: C20H24N2O2

281

Molecular Weight: 324.41

A 25 mL sealed tube was charged with Pd(dba)2 (0.040 g, 0.068 mmol, 0.1 equiv) and BINAP (0.064 g, 0.102 mmol, 0.15 equiv) which was later added with fresh anhydrous toluene (3 mL) and left for stirring at room temperature for 10 min. This has been followed by the addition of a solution of 2-((2S,6S)-4,4-dimethoxy-6-methylpiperidin-2-yl)aniline (0.17 g, 0.68 mmol, 1 equiv) in anhydrous toluene (1 mL) through cannula followed by 1, 2 dibromobenzene (0.17 mL, 0.75 mmol, 1.1 equiv) during which, the stirring was continued. Finally, sodium tert-butoxide NaotBu (0.079 g, 0.82 mmol, 1.2 equiv) was added and the sealed tube was stoppered tightly and continued to stir at 100 °C for 10 h. The resulting mixture has been filtered through Celite® bed and thus collected filtrate was diluted with DCM (20 mL) and washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over MgSO4 and evaporated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (4S,14bS)-2,2-dimethoxy- 4-methyl-1,2,3,4,10,14b-hexahydrodibenzo[b,f]pyrido[1,2-d][1,4]diazepine as a yellow oil (0.22 g, quantitative).

Rf 0.45 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.28 (2H, d, J = 6.0 Hz, H7), 1.31-1.38 (1H, t, J = 12.6

Hz, H5a), 1.95 (1H, t, J = 12.7 Hz, H3a), 2.11 (1H, dt, J = 13.1, 2.7 Hz, H3e), 3.07-3.18 (1H, m, H6a), 3.28 (3H, s, 0-CH3), 3.29 (3H, s, 0-CH3), 4.06 (1H, dd, J = 12.1, 2.6 Hz, H2a), 6.79

(1H, td, J = 8.0 Hz, 1.4 Hz, Har), 6.99 (1h, J = 7.4 Hz, Har), 7.26 (3H, m, Har), 7.43 (1H, d, J =

7.9 Hz, Har), 7.48 (1H, d, J = 7.9 Hz, Har), 7.63 (1H, dd, J = 7.9, 1.3 Hz, Har), 9.35 (1H, brs, NH).

13 C NMR (100 MHz, CDCl3) δ (ppm) 22.58 (C7), 35.97 (C3), 40.39 (C5), 47.47 (C21), 47.57

(C22), 53.52 (C6), 57.76 (C6), 99.78 (C4), 112.96, 116.07, 118.54, 120.40, 121.04, 127.86, 127.98, 128.57, 128.77, 131.30, 133.31, 141.74, 142.05 (Car).

25 [α]D = - 11.1(c=1.05 CHCl3)

HR-ESI-MS: calculated for C20H24N2O2 325.1916, found: 325.1907.

(4S,14bS)-2,2-dimethoxy-4-methyl-2,3,4,14b-tetrahydro-1H-dibenzo[b,f]pyrido[1,2- d][1,4]oxazepine 199

282

21 22 O O 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 O 15 10 19 12

Chemical Formula: C20H23NO3 Molecular Weight: 325.40 Following the general method as stated for 198, we synthesized 199

Rf 0.30 (SiO2, cyclohexane/ethyl acetate: 7/3).

Yield: 99%

1 H NMR (400 MHz, C6D6) δ (ppm) 1.10 (1H, d, J = 6.2, H7), 1.71 (1H, t, J = 12.6 Hz, H5a), 1.94-2.07 (2H, m, H3a, 3e), 2.37 (1H, td, J = 13.3, 3.2 Hz, H5e), 2.54 (1H, dqd, J = 12.1, 6.1 Hz, 2.8 Hz, H6e), 3.02 (3H, s, O-Me), 3.18 (3H, s, O-Me), 7.11 (2H, tdd, J = 8.5, 7.4, 1.4 Hz, Har), 7.21 (1H, t, J = 7.6 Hz, Har), 7.36 (1H, dd, J = 7.2, 1.3 Hz, Har), 7.61 (1H, dd, J = 7.6, 1.1 Hz, Har). 7.68 (1H, dd, J = 6.3 Hz, 1.3 Hz, Har), 7.75 (1H, d, J = 7.4 Hz, Har). 13 C NMR (100 MHz, C6D6) δ (ppm) 21.58 (C7), 40.11 (C3), 41.52 (C5), 47.34 (C21,22), 56.46 (C6), 60.17 (C2), 98.45 (C4), 112.11, 119.03, 120.87, 122.93, 123.89, 124.52, 125.04, 126.06, 127.34, 129.57, 156.68 (Car).

25 [α]D = - 10.8(c=1.05 CHCl3)

HR-ESI-MS: calculated for C20H23NO3 326.2413, found: 326.2407. (2S,6S)-ethyl2-(2-(2-iodophenoxy)phenyl)-4,4-dimethoxy-6-methylpiperidine-1- carboxylate 202

17 8 7 I O O 16 5 4 3 O 15 6 2 14 N 13 9 H 10 12 11 cis

Chemical Formula: C20H24INO3 Exact Mass: 453.0801

283

Physical properties : Brown oil

Yield : 76%.

Rf = 0.50 Cyclohexane/ethyl acetate 30/70

1 NMR H (400 MHz, C6D6) δ: 7.86(dd, J=7.9,1.6, 1H, H17), 7.58 (dd, J=7.6,1.8, 1H, H13), 7.11-7.20(3H, m, H10,16,15), 6.74 (td, J=7.7,1.4, 1H, H12), 6.63 (dd, J=8.0,1.3, 1H, H11), 3.11 (s, 3H, H7,8), 3.02 (s, 3H, H7,8), 3.01-3.06 (m, 1H, H6), 2.17 (dt, 1H, J=13.7et2.6, H4e), 1.91 (dt, J=12.7,2.6, 1H, H5e), 1.46 (dd, J=13.4et12.7, 1H, H4a), 1.11 (dd, J=12.2et11.7, 1H, H5a), 1.03 (d, J=6.2, 3H, H9).

13 NMR C (CDCl3): δ (ppm). 22.53 (C-9), 39.78, (C-4), 41.44(C-5), 47.3/47.4(C-7,8), 49.4(C- 3), 51.5 (C-6), 88.1(C-16), 117/118/120/124/125 (C-ar)

25C Cis: [α]D = - 2.0(c=0.917 CHCl3).

(S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate 204

11 12 10

13 7 9 1 8 2 6 N O 17 5 3 15 O 4 14 16

Chemical Formula: C20H25NO2 Molecular Weight: 311.41

To a solution of (S)-N-benzyl-N-α-methyl benzylamine (13.4.0 mL, 63.6 mmol, 1.0 equiv) in dry THF (200 mL) at 0 °C was slowly added with n-butyl lithium (28 mL, 1.6 M in hexane, 70 mmol, 1.1 equiv). The resultant pink solution of lithium amide was stirred for 30 min at 0 °C and then cooled to −78 °C before dropwise addition of a solution of methyl crotonate (7.4 mL, 70 mmol, 1.1 equiv) in dry THF (60 mL). The mixture was stirred at −78 °C for 2 h. Then, a saturated aqueous solution of ammonium chloride (80 mL) was added slowly, and the resulting solution was allowed to warm to room temperature. The solution was extracted with ethyl acetate (3x100 mL). Combined organic extracts were dried over MgSO4, filtered, and evaporated in vacuo to give (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate as yellow oil. The crude product thus obtained was engaged into the next step without any purification.

284

Rf 0.8 (SiO2, cyclohexane/ethyl acetate: 9/1)

1 H NMR (400 MHz, CDCl3)  (ppm) 1.01 (3H, d, J = 6.6 Hz, H14), 1.25 (3H, d, J = 7 Hz,

H7), 2.00 (1H, dd, J = 14.2, 7.8 Hz, H16’), 2.25 (1H, dd, J = 14.2, 6.2 Hz, 1H, H16), 3.30 (1H, m, H15), 3.35 (3H, s, H17), 3.68 (1H, d, J = 14.6 Hz, H8), 3.76 (1H, q, J = 7 Hz, H6), 7.05-7.28

(m, 10H, Har).

D [α] 20 = - 1.02 (c 1.0, CHCl3).

The analysis is in accordance with the literature.

(S)-3-methoxy-3-oxobutan-1-aminium acetate 211

O 3 - + O NH3 O 5 1 2 O 4

Chemical Formula: C7H15NO4 Molecular Weight: 177.19

To a suspension of 20% Pd(OH)2/C (3.18 g, 30 mmol, 0.5 equiv) in methanol (100 mL), a solution of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate (19.8 g, 63.6 mmol, 1.0 equiv) in methanol (100 mL), H2O (50 mL), acetic acid (20 mL) was added and the mixture was placed on a Parr apparatus and stirred under a hydrogen atmosphere (60 psi) for 24 h. The catalyst was then removed by filtration on Celite. The residue was concentrated in vacuo to give (S)-4-methoxy-4-oxobutan-2-aminium acetate as a pale yellow oil. The crude product thus obtained was engaged into the next step without any purification.

1 H NMR (400 MHz, CDCl3)  (ppm) 1.24 (3H, d, J = 6.8 Hz, H1), 1.84 (4H, s, H3), 2.64

(1H, dd, J = 17.2, 7.3 Hz, H4’), 2.70 (1H, dd, J = 17.2, 5.7 Hz, H4), 3.63 (3H, s, H5), 3.67 (1H, m, H2).

The analysis is in accordance with the literature.

(S)-methyl 3-(ethoxycarbonylamino)butanoate 212

O 6 3 O NH O 7 5 1 2 O 4

285

Chemical Formula: C8H15NO4 Molecular Weight: 189.20

To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (11.4 g, 63.6 mmol, 1.0 equiv) in DCM (320 mL) and H2O (320 mL) at room temperature was added with sodium carbonate (13.5 g, 127 mmol, 2.0 equiv) and ethyl chloroformate (12.2 mL, 127 mmol, 2.0 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 3 h. The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. The crude product was purified using column chromatography on silica (cyclohexane/ethylacetate: 7/3) to give (S)-methyl 3- (ethoxycarbonylamino)butanoate as colorless oil. (55%)

Rf 0.35 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3)  (ppm) 1.15 (3H, d, J = 6.6 Hz, H7), 1.16 (3H, d, J = 6.9 Hz,

H1), 2.54 (1H, dd, J = 17.2, 7.3 Hz, H4’), 2.60 (1H, dd, J = 17.2, 5.7 Hz, H4), 3.62 (3H, s, H5),

4.02 (3H, m, H2, 6), 5.05 (1H, brs, H3).

D [α] 25 = + 35.1 (c 1.0, CHCl3).

The analysis is in accordance with the literature.

(S)-methyl 3-(tert-butoxycarbonylamino)butanoate 213

7 6 O 3 8 O NH O 5 1 2 O 4

Chemical Formula: C10H19NO4 Molecular Weight: 217.26

To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (7.4 g, 42.0 mmol, 1.0 equiv) in DCM (160 mL) and H2O (160 mL) at room temperature was added with sodium carbonate (11.2 g, 105 mmol, 2.5 equiv) and Di-tert-butyl dicarbonate (14.0 g, 63 mmol, 1.5 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 10 h. The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. The crude product was purified using column

286 chromatography on silica (cyclohexane/ethylacetate: 7/3) to give (S)-methyl 3-(tert- butoxycarbonylamino)butanoate as yellow oil. (57%)

Rf 0.40 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3)  (ppm) 1.20 (3H, d, J = 6.8 Hz, H1), 1.43 (9H, s, H6, 7, 8), 2.49-

2.50 (2H, m, H4), 3.68 (3H, s, H5), 4.12 (1H, m, H2).

D [α] 25 = - 28.7 (c 1.1, CHCl3).

The analysis is in accordance with the literature.

(S)-methyl 3-(benzyloxycarbonylamino)butanoate 214

O 7 6 8 3 O NH O 5 9 12 1 2 O 11 4

Chemical Formula: C13H17NO4 Molecular Weight: 251.27

To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (7.4 g, 42.0 mmol, 1.0 equiv) in DCM (160 mL) and H2O (160 mL) at room temperature was added with sodium carbonate (11.2 g, 105 mmol, 2.5 equiv) and benzyl chloroformate (9.0 mL, 63 mmol, 1.5 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 10 h. The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. The crude product was purified using column chromatography on silica (cyclohexane/ethylacetate: 7/3) to give (S)-methyl 3- (benzyloxycarbonylamino)butanoate as yellow oil. (52%)

Rf 0.45 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3)  (ppm) 1.15 (3H, d, J = 7 Hz, H1), 1.16 (2H, t, J = 6.9 Hz, H6),

2.52 (2H, d, J = 5.5 Hz, H4), 3.65 (3H, s, H5), 4.11 (1H, m, H2), 5.03 (1H, brs, H3), 7.26-7.31

(5H, s, H7, 8, 9, 10, 11).

D [α] 25 = - 16.9 (c 1.15, CHCl3).

The analysis is in accordance with the literature.

287

(S)-ethyl 5-(diethoxyphosphoryl)-4-oxopentan-2-ylcarbamate 215

O 6 3 7 O NH O O 9 P 10 2 O 1 O H H H 11 4 H 4' 8 8' 12

Chemical Formula: C12H24NO6P Molecular Weight: 309.29

To a solution of diethyl methylphosphonate (4.25 mL, 29.1 mmol, 2.5 equiv) in anhydrous THF (60 mL) at −78 °C was added dropwise n-butyl lithium (11.6 mL, 1.6 M in hexane, 29.1 mmol, 2,5 equiv). After 20 min at −78 °C, a solution of (S)-methyl 3- (ethoxycarbonylamino)butanoate (2.2 g, 11.6 mmol, 1.0 equiv) in anhydrous THF (12 mL) was added dropwise. After addition, the temperature of the reaction was kept at −78 °C for 30 min, then allowed to reach 0 °C in 1 h, quenched with saturated solution of ammonium chloride, and extracted with ethyl acetate (3x100 mL). After drying over MgSO4 and concentration in vacuo, the crude oil was first distillated at low pressure to remove excess diethyl methylphosphonate, and the residue purified using column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (S)-ethyl 5-(diethoxyphosphoryl)-4-oxopentan-2- ylcarbamate as a yellow oil (3.3 g, 55% yield).

Rf 0.3 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3)  (ppm) 1.15−1.20 (6H, m), 1.33−1.21 (6H, m), 2.71 (1H, dd, J

= 17.1, 5.7 Hz, H4/4’), 2.84 (1H, dd, J = 17.1, 6.0 Hz, H4/4’), 2.99 (1H, dd, J = 22.6, 14.0 Hz,

H8/8’), 3.08 (1H, dd, J = 23.0, 14.0 Hz, H8/8’), 4.16−3.94 (7H, m), 5.03 (1H, brs, H3).

D [α] 25 = - 34.6 (c 1.17, CHCl3).

The analysis is in accordance with the literature.

288

(S)-tert-butyl (5-(diethoxyphosphoryl)-4-oxopentan-2-yl)carbamate 216

6 7 O

3 5 O NH O O 9 P 10 2 O 1 O H H H H 11 4 4' 8 8' 12

Chemical Formula: C14H28NO6P Molecular Weight: 337.34 We followed the similar methodology as optimized for (S)-ethyl 5-(diethoxyphosphoryl)-4- oxopentan-2-ylcarbamate to give (S)-tert-butyl (5-(diethoxyphosphoryl)-4-oxopentan-2- yl)carbamate as a yellow oil with an yield of 63%.

Rf 0.4 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.20 (d, J=6.8, 3H, H1), 1.33 (t, J=7.5, 6H, H10, 12), 1.41

(s, 9H, H5,6,7 ), 2.80 (dd, J= 17.3 and 5.6, 2H, H4,4’), 3.11 (dd, J= 23.0 and 13.1, 2H, H8,8’),

4.06-4.18 (m, 5H, H2, 9, 11), 4.91 (m, 1H, H3).

25 [α]D = - 47.3 (c 1.00 CHCl3).

The analysis is in accordance with the literature

(S)-benzyl (5-(diethoxyphosphoryl)-4-oxopentan-2-yl)carbamate 217

O 5 14 6 3 O NH O O 9 P O 10 7 15 2 13 1 O H H H H 11 4 4' 8 8' 12

Chemical Formula: C17H26NO6P Molecular Weight: 371.36

We followed the similar methodology as optimized for (S)-ethyl 5-(diethoxyphosphoryl)-4- oxopentan-2-ylcarbamate to give (S)-benzyl (5-(diethoxyphosphoryl)-4-oxopentan-2- yl)carbamate as a yellow oil with an yield of 50%

Rf 0.4 (SiO2, cyclohexane/ethyl acetate: 7/3).

289

1 H NMR (400 MHz, CDCl3) : δ (ppm) 1.16 (d, J= 6.7, 3H, H1), 1.26 (t, J=6.2, 6H, H10, 12),

2.70 (dd, J= 17.1, 6.0, 1H, H4,4’), 3.05 (dd, J= 22.1 and 13.3, 1H, H8,8’), 4.03 (m, 5H, H2, 9, 11) ,

7.36-7.22 (m, 5H, H5,6,7,13,15),

25 [α]D = - 41.3(c=1.05 CHCl3).

The analysis is in accordance with the literature.

GENERAL METHOD FOR HORNER-WADSWORTH-EMMONS REACTION

To a solution of dimethoxyphosphoryl (1.0 equiv) in tetrahydrofuran (80 mL) at room temperature was added Ba(OH)2.H2O (1.3 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of aldehyde (1.3 equiv) diluted in tetrahydrofuran/water: 40/1 (80 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give E-alkene.

Now let’s see each formation of alkene individually

(S,E)-ethyl 6-(2-bromophenyl)-4-oxohex-5-en-2-ylcarbamate 222

O 6 3 7 O NH O Br 14 8 2 12 1 4 13 5 9 11 10

Chemical Formula: C15H18BrNO3 Molecular Weight: 340.21 Physical property: Yellow solid M.P: 89-90 °C Yield : 88%

Rf 0.35 (SiO2, cyclohexane/ethyl acetate: 7/3).

1 NMR H (400 MHz, CDCl3): δ (ppm) 7.86 (d, J = 16.2, 1H, H8), 7.56 (d, J = 7.8, 1H, H12), 7.56 (d, J = 7.8,1H, H9,), 7.27 (t, J = 7.8 Hz, 1H, H10), 7.17 (td, J = 7.8, 1.5 Hz, 1H, H11),

290

6.57 (d, J= 16.2, 1H, H5), 5.08 (se, 1H, H3), 4.15−3.88 (m, 3H, H6), 2.96 (dd, J = 16.4,4.8, 1H, H4), 2.79 (dd, J = 16.3, 6.2, 1H, H4), 1.22 (d, J = 6.8, 3H, H1),1.16 (t, J = 7.1, 3H, H7)

13 NMR C (100 MHz CDCl3): δ (ppm) 14.5 (C7), 20.6 (C1), 43,2 (C4), 60.7 (C6), 127,135, 132, 127, 141 (C-8), 157.1 (C-14), 198.2 (C-13).

25 [α]D = - 18.5(c 1.12 CHCl3)

HRMS-ESI (M + Na), m/z: calcd. for C15H18BrNO3Na 362.0368 found 362.0371.

(S,E)-ethyl 6-(2-(2-iodophenoxy)phenyl)-4-oxohex-5-en-2-ylcarbamate 223 O 6 3 7 O NH O 8 9 2 10 1 4 5 11 O 12 I 13

16 14 15 Chemical Formula: C21H22INO4 Exact Mass: 479.0594

Yield : 91% Rf = 0.27 Cyclohexane/Ethyl acetate 70/30.

1 NMR H (400 MHz, CDCl3)::  7.89 (dd, J = 16 , 1H, H8), 7.81-7.90 (m, 1H, H16),7.66 (dd, J=8.0,1.1, 2H, H19), 7.29-7.34 (m, 2H, H14,15), 7.15(t, J=7.6, 1H, H11), 6.9 (td, J=7.4,1.5, 1H, H10), 6.78-6.80 (dd,J=7.8, 1H, H12), 6.82 (d, J = 16 , 1H, H5), 6.77 (dd, J=8.24,1.1, 1H, H13), 5.18 (m, 1H, H3), 4.05-4.17 (m, 3H, H6,2), 2.82-2.97(m, 2H, H4), 1.16-1.12 (m, 6H, H1,7).

13 NMR C (100 MHz, CDCl3):  (ppm). 14.6 (C-7), 20.8 (C-1), 43 (C-2), 46 (C-4), 60.4 (C - 6), 125.7(C-5), 88.7, 127.1, 128/165/123/132/137/122,140(C-8), 155.1(C-14), 193(C-13).

25 []D = - 22.5(c=1.02 CHCl3)

+ HRMS ESI (M + H) ion: calc. C21H23NO4I 480.0672, found. 480.0672

291

(S,E)-tert-butyl (6-(2-bromophenyl)-4-oxohex-5-en-2yl)carbamate 224

6 13O 3 7 O NH O Br 8 2 12 1 4 5 9 11 10 Chemical Formula: C17H22BrNO3 Exact Mass: 367.0783

Physical property: Yellow solid

Yield : 90%

Rf = 0.45 Cyclohexane/ethyl acetate 70/30.

1 NMR H (400 MHz, CDCl3):δ (ppm) 7.92 (d, J = 16 Hz, 2H, H8), 7.61 (td,J=8.2,1.4, 2H, H12,9), 7.33 (td,J=7.2,1.1, 1H, H11),7.23 (td, J=7.8,1.6, 1H, H10), 6.63 (d, J = 16 Hz, 1H, H5), 4.98 (m, 1H, H3), 4.09-4.12 (m, 1H, H2), 2.92 (dd, J=16, 2H, H4), 1.42 (s, 9H, H6,7,13), 1.26 (d, J=7, 3H, H7).

13 NMR C 100 MHz CDCl3: δ (ppm). 20.7(C-1), 28.8(C-6,7,13), 46.3(C-2), 49(C-4), 67.7(C 14), 127.5(C-5),131/133/127/120 (C-ar), 141(C-8).

25 [α]D = - 25.3(c=1.0 CHCl3)

+ HRMS ESI (M + H) ion: calc. C17H23NO3Br 368.0861, found. 368.0893

(S,E)-tert-butyl (6-(2-nitrophenyl)-4-oxohex-5-en-2-yl)carbamate 225

6 13O 3 7 O NH O NO 8 2 2 12 1 4 5 9 11 10

Chemical Formula: C17H22N2O5 Exact Mass: 334.1529

Physical property: Yellow solid

Yield: 75%

Rf = 0.35 Cyclohexane/Ethyl acetate 70/30.

292

1 NMR H (400 MHz, CDCl3):: δ (ppm) 8.05-8.07 (m, 2H, H12,9), 8.01 (d, J = 16, 2H, H8), , 7.63-7.66 (m, 1H, H11),7.53-7.57 (m, 1H, H10), 6.59 (d, J = 16, 1H, H5), 4.92 (m, 1H, H3), 4.08-4.10 (m, 1H, H2), 2.86 (2 dd, J=16,6, 2H, H4), 1.42 (s, 9H, H6,7,13), 1.26 (d, J=7, 3H, H7).

13 NMR C (CDCl3): δ (ppm). 20.5(C-1), 27.4(C-6,7,13), 46.6(C-2), 46.4(C-4), 75.7(C-14), 125.5(C-5),128/129/131/120 (C-ar), 138(C-8), 152.1(C-14),188.9(C-16).

25 [α]D = - 30(c=1.12 CHCl3)

+ HRMS ESI (M + H) ion: calc. C17H22N2O5 357.1426 found. 357.1405 2-(2-iodophenoxy)benzaldehyde

H O 13 I 11 10 9 O 8 1 12 2 4 5 7 3 6

Chemical Formula: C13H9IO2 Molecular Weight: 324.11

To a solution of 2-iodophenol (2 g, 9.1 mmol, 1.0 equiv) in DMF anhydride (16 mL) at room temperature was added with 2-fluorobenzaldehyde (1.128 g, 9.1 mmol, 1.0 equiv) and K2CO3 (2.01 g, 14.54 mmol, 1.6 equiv). The reaction mixture was allowed to reflux and continued to stir for 7 h. The reaction mixture was cooled to room temperature and concentrated in vacuo.

The resulting residue was diluted with ethyl acetate (200 mL), washed with NH4Cl (2x200 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give 2-(2- iodophenoxy)benzaldehyde as a brown oil (2.85 g, 97%).

Rf 0.74 (SiO2, cyclohexane/ethyl acetate: 7/3)

1 H NMR (400 MHz, CDCl3) δ (ppm) 10.06 (1H, s, H13), 7.99 (1H, dd, J = 7.8, 1.8 Hz, H1),

7.93 (1H, dd, J = 7.9, 1.5 Hz, H8), 7.52 (1H, ddd, J = 8.9, 7.4, 1.8 Hz, H2), 7.39 (1H, td, J =

8.1, 1.5 Hz, H3), 7.22 (1H, t, J = 7.5 Hz, H7), 6.96-7.04 (2H, m, H4,6), 6.77 (1H, dd, J = 8.3

Hz, H5). + HR-ESI-MS: calculated for C23H26INO4 (M+H ): 323,9647, found:

293

2-(2-bromophenoxy)benzaldehyde

H O 13 Br 11 10 9 O 8 1 12 2 4 5 7 3 6

Chemical Formula: C13H9BrO2 Molecular Weight: 277.11

To a solution of 2-bromophenol (2 g, 11.56 mmol, 1.0 equiv) in DMF anhydride (16 mL) at room temperature was added with 2-fluorobenzaldehyde (1.43 g, 11.56 mmol, 1.0 equiv) and

K2CO3 (2.29 g, 17.34 mmol, 1.5 equiv). The reaction mixture was allowed to reflux and continued to stir for 7 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was diluted with ethyl acetate (200 mL), washed with NH4Cl (2x200 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give 2-(2-bromophenoxy)benzaldehyde as a brown oil (3.13 g, 98%).

Rf 0.74 (SiO2, cyclohexane/ethyl acetate: 7/3)

1 H NMR (400 MHz, CDCl3) δ (ppm) 10.06 (1H, s, H13), 7.96 (1H, dd, J = 7.8, 1.7 Hz, H1),

7.69 (1H, dd, J = 8.0, 1.5 Hz, H8), 7.50 (1H, ddd, J = 8.1, 7.3, 1.8 Hz, H2), 7.35 (1H, ddd, J =

8.1, 7.5, 1.6 Hz, H3), 7.17-7.23 (1H, m, H7), 7.10-7.16 (1H, m, H6), 7.09 (1H, dd, J = 8.1, 1.5

Hz, H4), 6.75 (1H, dd, J = 8.4, 0.6 Hz, H5). 13 C NMR (100 MHz, CDCl3) δ (ppm) 189.93 (C=O, C13), 135.86, 134.31, 129.11, 128.67,

126.37, 123.47, 121.82, 117.04 (Ar-CH), 159.60, 152.57 (Ar-C), 126.20 (C12), 115.57 (Ar-Br,

C9).

(S,E)-tert-butyl 6-(2-(2-iodophenoxy)phenyl)-4-oxohex-5-en-2-ylcarbamate 226

O 21 20 O NH O 19 6 5 7 8 3 9 1 2 4 10 O 12 I 13 11 18 14 17 15 16

294

Chemical Formula: C23H26INO4 Molecular Weight: 507.36 To a solution of (S)-tert-butyl 5-(diethoxyphosphoryl)-4-oxopentan-2-ylcarbamate (1.77 g,

5.25 mmol, 1.0 equiv) in THF (50 mL) at room temperature was added Ba(OH)2.H2O (1.25 g, 6.56 mmol, 1.25 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of 2-(2-iodophenoxy)benzaldehyde (1.7 g, 5.25 mmol, 1.0 equiv) diluted in THF/H2O: 40/1 (30 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (150 mL), washed with water (3x150 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (S,E)-tert-butyl 6-(2-(2-iodophenoxy)phenyl)-4- oxohex-5-en-2-ylcarbamate (2.08 g, 83%).

Rf 0.43 (cyclohexane/ethyl acetate: 7/3)

Yield: 83%

1 H NMR (400 MHz, CDCl3) δ (ppm) 7.93 (1H, J = 16.4 Hz, H6), 7.91 (1H, dd, J = 7.9, 1.6

Hz, H8), 7.69 (1H, dd, J = 7.9, 1.5 Hz, H17), 7.30-7.38 (2H, m, H10, 16), 7.17 (1H, t, J = 7.2 Hz,

H15), 6.93 (1H, td, J = 7.6, 1.4 Hz, H9), 6.87 (1H, dd, J = 8.1, 1.4 Hz, H11), 6.85 (1H, d, J =

16.4 Hz, H5), 6.79 (1H, dd, J = 8.3 Hz, 0.8Hz, H14), 5.03 (1H, brs, NH), 4.02-4.17 (1H, m,

H2), 2.98 (1H, dd, J = 16.0, 4.8 Hz, H3), 2.80 (1H, dd, J = 16.2, 6.4 Hz, H3), 1.44 (9H, s, H21),

1.24 (3H, d, J = 6.7 Hz, H1). 13 C NMR (100 MHz, CDCl3) δ (ppm) 10.72 (C1), 14.75 (C21), 44.20 (C2), 45.76 (C3), 53.61

(C20), 118.21 (C5), 125.57 (C6), 119.71, 124.07, 126.02, 128.35, 128.90, 129.99, 131.91,

135.28, 137.77, 140.25 (Ar-C11-18), 155.73 (C12), 156.02 (C13), 170.61 (C19), 194.70 (C4). + HR-ESI-MS: calculated for C23H26INO4 (M+H ): 508.0985, found: 508.0993. 25 [α] D = - 20.8(c=1.01 CHCl3).

295

(S,E)-tert-butyl 6-(2-(2-bromophenoxy)phenyl)-4-oxohex-5-en-2-ylcarbamate 227

O 21 20 O NH O 19 6 5 7 8 3 9 1 2 4 10 O 12 Br 13 11 18 14 17 15 16

Chemical Formula: C23H26BrNO4 Molecular Weight: 460.36 To a solution of (S)-tert-butyl 5-(diethoxyphosphoryl)-4-oxopentan-2-ylcarbamate (4.44 g,

13.17 mmol, 1.0 equiv) in THF (80 mL) at room temperature was added Ba(OH)2.H2O (3.0 g, 16.47 mmol, 1.25 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of 2-(2-bromophenoxy)benzaldehyde (3.65 g, 13.171 mmol, 1.0 equiv) diluted in THF/H2O: 40/1 (60 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (S,E)-tert-butyl 6-(2-(2-bromophenoxy)phenyl)-4- oxohex-5-en-2-ylcarbamate (5.15 g, 85%).

Rf 0.41 (cyclohexane/ethyl acetate: 7/3) Yield: 85%

1 H NMR (400 MHz, C6D6) δ (ppm) 8.06 (1H, J = 16.4 Hz, H6), 7.34 (1H, dd, J = 8.0, 1.5 Hz,

H8), 7.26 (1H, dd, J = 7.9, 1.5 Hz, H17), 6.83 (1H, ddd, J = 8.2, 7.4, 1.7 Hz, H10), 6.70-6.77

(3H, m, H9,15,16), 6.49-6.59 (2H, m, H5,11), 6.46 (1H, dd, J = 8.2 Hz, 1.0 Hz, H14), 4.85 (1H, brs, NH), 4.10-4.24 (1H, m, H2), 2.57 (1H, dd, J = 16.1, 4.7 Hz, H3), 2.35 (1H, dd, J = 16.1,

6.3 Hz, H3), 1.44 (9H, s, H21), 0.99 (3H, d, J = 6.7 Hz, H1). 13 C NMR (100 MHz, C6D6) δ (ppm) 20.50 (C1), 28.55 (C21), 44.03 (C2), 45.06 (C3), 76.57

(C20), 115.43 (C5), 125.99 (C6), 117.84, 121.13, 123.85, 125.63, 128.84, 128.97, 131.51,

134.23, 136.99, 142.17 (Ar-C11-18), 153.63 (C12), 155.94 (C13), 173.19 (C19), 198.22 (C4). + HR-ESI-MS: calculated for C23H26INO4 (M+H ): 460.1123, found: 460.1143. 20 [α] D = - 10.55 (c=1.00 CHCl3).

296

GENERAL METHOD FOR PIPERIDINE CORE FORMATION

To alkene (0.34 g, 0.66 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl orthoformate (0.36 mL, 3.30 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.14 g, 0.73 mmol, 1.1 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH

(3x30 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 85/15) to give desired piperidine

(2R,6S)-ethyl2-(2-bromophenyl)-4,4dimethoxy6methylpiperidine-1-carboxylate and (2R,6R)-ethyl 2-(2-bromophenyl)-4,4-dimethoxy-6-methylpiperidine-1-carboxylate 235

8 7

O O O O 5 4 3 Br Br 6 2 9 N 13 N 10 12 O O 11 O O 2 cis 1 trans

Chemical Formula: C17H24BrNO4 Exact Mass: 385.0889

Physical property : Brown oil

Yield : 85%

Rf = 0.55 Cyclohexane/Ethyl acetate 70/30 trans.

1 NMR H (400 MHz, C6D6): δ (ppm) 7.51 (dd, J = 7.7, 1.2 , 1H, Har),7.47 (dd, J = 7.7, 0.7 , 1H, Har), 7.09 (t, J = 7.7 , 1H, Har), 6.79 (t, J = 7.7, 1H, Har), 5.58 (dd, J = 12.4, 5.1, 1H, H3), 4.85 (qtd, J = 7.0, 3.7 ,1H, H6), 3.91−4.08 (m, 2H, H2), 3.15 (s, 3H, H7), 2.97 (s, 3H, H8), 2.57 (dd, J = 14.3,5.1, 1H, H4), 2.11 (dd, J = 14.1, 7.5 Hz, 1H, H5), 1.93 (dd, J = 14.3, 12.4, 1H, H4), 1.72 (dd, J = 14.1, 3.7 Hz, 1H, H5), 1.40 (d, J = 7,0 Hz, 3H, H9), 0.83(t, J = 7.1, 3H, H1). cis. 1 NMR H (400 MHz, C6D6): 7.77 (d, J = 8.2, 1H, Har), 7.46 (dd, J = 7.7, 0.7 , 1H, Har),

297

7.46 (t, J = 7.7, 1H, Har), 6.76 (m, 1H, Har), 5.56(dd, J = 5.8, 5.4 , 1H, H3), 4.69 (m, 1H, H6), 3.90 (m, 2H, H2), 3.03 (s, 3H, H7),2.80 (s, 3H, H8), 2.62 (dd, J = 14.5, 5.4 , 1H, H4), 2.33 (dd, J = 14.5, 5.8, 1H, H4), 2.05 (dd, J = 14.6, 6.2 , 1H, H5), 1.89 (m, 1H, H5), 1.57 (d, J = 6.9, 3H, H9), 0.91 (t, J = 6.9 , 3H, H1) 25 [α]D = - 11.3(c=1.0 CHCl3)

+ HRMS ESI (M + H) ion: calc. C17H24NO4Br 386.0861, found. 386.0893

(2R,6S)-2-(2-bromophenyl)-4,4-dimethoxy-6-methylpiperidine and (2R,6R)-2-(2- bromophenyl)-4,4-dimethoxy-6-methylpiperidine 240

8 7

O O O O 5 4 3 Br Br 6 2 N 13 N 9 H H 10 12 11 trans cis

Chemical Formula: C14H20BrNO2 Exact Mass: 313.0677

Physical properties : Brown oil

Yield : 80%. Cis/Trans : 68/32.

NMR 1H (400 MHz, CDCl3) trans.

NMR 1H (400 MHz, CDCl3) δ :7.58 (dd, J=8.1,1.1, 1H, H13), 7.51 (dd, J=8.0,1.4, 1H, H10), 7.31 (td, J=6.8,0.8, 1H, H12), 7.11(td, J=6.9,1.0, 1H, H11), 4.54 (dd, J=11.4et2.5, 1H, H3), 3.52-3.56 (m, 1H, H6), 3.26 (s, 3H, H7,8), 3.18 (s, 3H, H7,8), 2.12 (dt ,J=13.1, 2.5, 1H, H4e), 1.93 (dt, J=13.1et2.5, 1H, H5e), 1.83 (dd, J=13.4et5.2, 1H, H4a), 1.57 (dd, J=12.1et13.3, 1H, H5a), 1.39 (d, J=6.7, 3H, H9). cis.

RMN 1H (400 MHz, CDCl3) δ: 7.61 (dd, J=8.2,1.0, 1H, H13), 7.52 (dd, J=8.0,1.4, 1H, H10), 7.29 (td, J=7.0,0.8, 1H, H12), 7.10 (td, J=7.6,1.6, 1H, H11), 4.53 (dd, J=11.2et2.3, 1H, H3), 3.52-3.56 (m, 1H, H6), 3.26 (s, 3H, H7,8), 3.18 (s, 3H, H7,8), 2.22 (dt , J=13.1et2.3, 1H,

298

H4e), 2.03 (dt, J=12.9et2.3, 1H, H5e), 1.38 (dd, J=13.0,12.3, 1H, H4a), 1.24 (dd, J=12.4et13.1, 1H, H5a), 1.12-1.14 (d, J=6.1, 3H, H9).

13 C NMR (CDCl3): δ(ppm). 21.3(C-9), 40/41(C-7,8), 48.7(C-4), 49.4(C-5), 50.0(C-3), 59.8(C-6), 90.8(C-14), 142/127/126/124(C-ar).

25 Cis: [α]D = - 14.5(c=1.07 CHCl3)

25 Trans: [α]D = + 19.8(c=1 CHCl3)

+ HRMS ESI (M + H) ion: calc. C14H20NO2Br 314.0756, found. 314.0771

(2S,6R)-4,4-dimethoxy-2-methyl-6-(2-nitrophenyl)piperidine and (2R,6R)-4,4- dimethoxy-2-methyl-6-(2-nitrophenyl)piperidine 239

8 7 O O O O 5 3 4 NO2 NO2 6 2 N 13 N 9 H H 10 12 11 trans cis

Chemical Formula: C14H20N2O4 Exact Mass: 280.1423

Physical property : Brown oil

Yield : 65%. Cis/Trans : 62/38.

Rf = 0.45 Cyclohexane/Ethyl acetate 70/30 trans.

NMR 1H (400 MHz, CDCl3) δ: 7.74(dd, J=8.04,1.1, 1H, H13), 7.38(dd, J=8.15,1.2, 1H, H10), 7.02 (m, 1H, H12), 6.75 (td, J=7.9,1.3, 1H, H11),4.73 (dd, J=11et2.5,1H, H3), 3.21 (s, 3H, H7,8), 3.07 (s, 3H, H7,8), 2.86-2.91 (m, 1H, H6), 2.42 (dt, J=13.4,3.4et1.6, 1H, H5e),1.78 (dt, J=13.0et2.6.2, 1H, H4e), 1.66 (dd, J=13.2et12.1, 1H, H4a), 1.49 (1H, m), 1.24 (d, J=7.0, 3H, H9).

299 cis.

NMR 1H (400 MHz, CDCl3) δ: 7.82 (dd, J=8.0,1.4, 1H, H13), 7.39 (dd, J=8.3,1.0, 1H, H10), 7.01 (td, J=7.6,1.1, 1H, H12), 6.75 (td, J=7.8,1.3, 1H, H11),4.32 (dd, J=11et2.6,1H, H3), 3.26 (s, 3H, H7,8), 3.14 (s, 3H, H7,8), 2.86-2.91 (m, 1H, H6), 2.58 (dt, J=13.7et2.7, 1H,H4e), 2.04 (dt, J=12.6,2.6, 1H, H5e), 1.53 (dd, J=12.2et12.3, 1H, ), 1.31 (dd, J=12.4et11.91,1H,), 0.96 (d, J=7.0, 3H, H9,).

13 NMR C (CDCl3): δ (ppm). 20.2(C-9), 36.2/37.8(C-7,8), 45.5(C-4), 47.4(C-5), 51.9(C-3), 59.8(C-6), 98.8(C-14), 118/127/126/147(C-aromatic).

25oC Cis: [α]D = + 27.9(c=1.02 CHCl3)

25C Trans: [α]D = + 10.5(c=1.01 CHCl3)

+ HRMS ESI (M + H) ion: calc. C14H21N2O4 280.1501, found. 280.1511. 2-((2S,6S)-4,4-dimethoxy-6-methylpiperidin-2-yl)aniline 244

8 7 O O O O 5 3 4 NH2 NH2 6 2 N 13 N 9 H H 10 12 11 trans cis

Chemical Formula: C14H22N2O2 Exact Mass: 250.1681

A generalized hydrogenation method as proposed for (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6- methylpiperidine-2-carboxamide has been followed.

Physical properties : Brown oil

Yield : quatitative

NMR 1H (400 MHz, CDCl3) δ: 7.00-7.03(m, H10，1H, H13), 6.73-6.77(m, 2H, H11，H12), 4.20 (dd, J=12.5et2.6, 1H, H3), 3.49 (s, 3H, H7,8), 3.20 (s, 3H, H7,8), 2.86-2.91 (, m, 1H H6), 2.57 (dt, J=13.7et2.7, 1H, H4e), 2.01 (dt, J=12.2,2.7, 1H, H5e), 1.73 (dd, 1H, J=13.5et13.3), 1.37 (d, 3H, J=7.0, H9).

300

13 NMR C 100 MHz CDCl3: δ (ppm). 19.5(C-9), 36.2/36.9(C-7,8), 46.6(C-4), 47.2(C-5), 51.5(C-3), 99.8(C-14), 116/127/147(C-aromatic).

Rf = 0.35, 100 % ethyl acetate

25 Cis: [α]D = + 20.9(c=1.16 CHCl3)

(2S,6S)ethyl2(2(2iodophenoxy)phenyl)4,4dimethoxy6methylpiperidine1carboxylate

8 7 I I O O O O 5 4 3 O O 6 2 9 N 13 N 10 12 O O 11 O O 2 trans cis 1

Chemical Formula: C23H28INO5 Exact Mass: 525.1012

Physical properties : Brown oil

Yield : 86%. Proportion Cis/Trans : 62/38.

Rf = 0.30 Cyclohexane/Ethyl acetate 70/30 cis :

1 NMR H (400 MHz, CDCl3): δ7.78 (d, J=7.8, 1H, Har), 7.29 (d, J=7.6, 1H, Har), 7.22-7.01

(m, 4H, Har), 6.77 (m, 1H, Har), 5.17 (dd, J = 7.1, 6.4, 1H, H3), 4.37 (m, 1H, H6), 4.06–3.94

(m, 2H, H2), 3.16 (s, 3H, H8), 2.89 (s, 3H, H7), 2.55 (dd, J = 14.5, 5.9, 1H, H4eq), 2.21 (dd, J

= 14.5, 5.6,1H, H4ax), 2.00 (dd, J = 14.9, 3.5, 1H, H5eq), 1.95 (dd, J = 14.9, 6.0, 1H, H5ax),1.29 (d, J = 6.9, 3H, H9), 1.04 (t, J = 7.1, 3H, H1).

13 NMR C (101 MHz, CDCl3) δ 165.1, 155.9, 149.7, 147.9, 138.1, 133.8, 128.2, 125.4, 123.1, 121.6, 120.8, 114.3, 98.4, 88.9, 62.0, 52.3, 47.9, 47.6, 47.3, 37.4, 36.7, 22.8, 14.4. trans :

1 NMR H (400 MHz, CDCl3): δ 7.73 (d, J=8.4, 1H, Har), 7.22-7.01 (m,, 4H, Har), 6.80-6.71

(m, 2H, Har), 5.14 (t, J = 5.3, 1H, H3), 4.53 (m, 1H, H6), 4.06–3.94 (m, 2H, H2), 3.11 (s, 3H,

H8), 3.07 (s, 3H, H7), 2.85 (dd, J = 14.7, 4.6, 1H, H4eq), 2.15 (ddd, J = 14.7, 8.2, 1.01, 1H,

301

H4ax), 2.01 (dd, J = 14.3, 11.8, 1H, H5ax), 1.95 (dd, J = 14.3, 3.3, 1H, H5ax),1.42 (d, J = 6.9, 3H, H9), 0.95 (t, J = 7.1, 3H, H1).

13 RMN C (CDCl3): δ 165.1, 156.1, 149.5, 147.7, 137.9, 133.5, 128.2, 125.3, 122.9, 121.5, 120.8, 115.1, 98.2, 91.2, 61.3, 51.7, 47.8, 47.6, 46.8, 37.3, 36.6, 20.6, 14.4.

+ HRMS ESI (M + H) ion: calc. C23H29NO5I 526.1118, found. 526.1091

(2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- (methylsulfonyloxy)piperidine-1-carboxylate 260

OMs

N N O Br O O

Chemical Formula: C30H33BrN2O6S Molecular Weight: 629.56

To a solution of (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6- methylpiperidine-1-carboxylate (0.38 g, 0.68 mmol, 1.0 equiv) in DCM (6 mL) at room temperature was added with triéthylamine (0.198 mL, 1.37 mmol, 2.0 equiv) followed by a dropwise addition of methanesulfonyl chloride (0.08 mL, 1.03 mmol, 1.5 equiv) and continued to stir at room temperature for 5 h. The reaction mixture was diluted with DCM (20 mL) washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over

MgSO4 and concentrated in vacuo. This gives (2S,4S,6R)-benzyl 2-(benzyl(2- bromobenzyl)carbamoyl)-6-methyl-4-(methylsulfonyloxy)piperidine-1-carboxylate as yellow oil. Due to stability issues, without any purification, the crude was engaged into the next step.

Rf 0.20 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C30H33BrN2O6S (M+H ): 629.1243, found: 629.1259.

(2R,4S,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- (methylsulfonyloxy)piperidine-1-carboxylate 261

302

OMs

N N O Br O O

Chemical Formula: C30H33BrN2O6S Molecular Weight: 629.56 We performed the reaction with the same quantity and procedure as briefed for (2R,4S,6S)- benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. All the obtained data are similar to that of (2R,4S,6S)-benzyl 2-(benzyl(2- bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate.

(2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4- (methylsulfonyloxy)piperidine-1-carboxylate 262

OMs F F F N N O Br O O

Chemical Formula: C25H28BrF3N2O6S Molecular Weight: 621.46

To a solution of (2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-4- hydroxy-6-methylpiperidine-1-carboxylate (0.34 g, 0.629 mmol, 1.0 equiv) in DCM (5 mL) at room temperature was added with triéthylamine (0.175 mL, 1.26 mmol, 2.0 equiv) followed by a dropwise addition of methanesulfonyl chloride (0.07 mL, 0.944 mmol, 1.5 equiv) and continued to stir at room temperature for 5 h. The reaction mixture was diluted with DCM (20 mL) washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over

MgSO4 and concentrated in vacuo. This gives ((2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2- trifluoroethyl)carbamoyl)-6-methyl-4-(methylsulfonyloxy)piperidine-1-carboxylate as colourless oil. Due to stability issues, without any purification, the crude was engaged into the next step.

303

Rf 0.20 (SiO2, cyclohexane/ethyl acetate: 5/5).

+ HR-ESI-MS: calculated for C25H28BrF3N2O6S (M+H ): 621.0882, found: 621.0931.

tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate 263

6 O S O O 3 4 2

5 N 1

O O 7 9 8

Chemical Formula: C11H21NO5S Molecular Weight: 279.35

To a solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (4.04 g, 20 mmol, 1 equiv) in anhydrous dichloromethane (100 mL) at 0°C was added methylsulfonyl chloride (2.32 mL, 30 mmol, 1.5 equiv), triethylamine (5.57 mL, 40 mmol, 2 equiv). The resulting mixture was stirred at room temperature for 3 h. Saturated NH4Cl and 1N HCl were added until pH 2-3 was reached and the aqueous layer is extracted with dichloromethane (3x20 mL). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo to give 5.6 g (quantitative yield) of tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate as yellow solid.

Rf = 0.22 (cyclohexane/ethyl acetate : 7/3)

1 H NMR (400 MHz, CDC13) δ(ppm) 4.75-4.82 (m, 1H, H3), 3.60-3.71 (m, 2H, H2), 3.21-

3.32 (m, 2H, H4), 2.95(s, 3H, H6), 1.78-1.85 (m, 2H, H1), 1.67-1.78 (m, 2H, H5), 1.35 (s, 9H,

H7-9).

CAS Registry Number: 141699-59-4.

The analysis is in accordance with the literature.

(2S,4R,6R)-benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1- carboxylate

304

N N O Br O O

Chemical Formula: C29H30BrN5O3 Molecular Weight: 576.48

To a solution of (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- (methylsulfonyloxy)piperidine-1-carboxylate (0.43 g, 0.68 mmol, 1.0 equiv) in anhydrous DMF (3.2 mL) at room temperature was added with sodium azide (0.1 g, 1.37 mmol, 2.0 equiv) and continued to stir at 85 °C for 24 h. The reaction mixture was evaporated in vacuo and the resulting residue was dissolved in DCM (20 mL) and washed with water (2x20 mL), dried over MgSO4 and finally evaporated in vacuo. This gives (2S,4R,6R)-benzyl 4-azido-2- (benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate as pale yellow non viscous liquid. Due to stability issues, without any purification, the crude was engaged into the next step.

Rf 0.76 (SiO2, cyclohexane/ethyl acetate: 6/4).

+ HR-ESI-MS: calculated for C29H30BrN5O3 (M+H ): 576,1610, found: 576.1671.

(2S,4R,6R)-benzyl 4-amino-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1- carboxylate 264

NH2

N N O Br O O

Chemical Formula: C29H32BrN3O3 Molecular Weight: 550.48

To a solution of (2S,4R,6R)-benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6- methylpiperidine-1-carboxylate (0.39 g, 0.68 mmol, 1.0 equiv) in THF (7.5 mL) and H2O

305

(0.75 mL) at room temperature was added with triphenylphosphine (0.27 g, 1.02 mmol, 1.5 equiv) and continued to stir at room temperature for . The reaction mixture was evaporated in vacuo and the resulting residue was diluted with ethyl acetate and H2O (20 mL). The pH was adjusted to be acidic using 1N HCL (until TLC showed no traces of product) and discarded. The combined aqueous layer was taken to 0°C and the pH carefully was adjusted to 14 with NaOH. Then it was extracted with ethyl acetate (3x30 mL). The combined organic layer was dried over MgSO4 and concentrated in vacuo. The crude thus obtained was purified using column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to yield (2S,4R,6R)-benzyl 4-amino-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate as yellow amorphous powder.

M.P: 79-81 °C

+ HR-ESI-MS: calculated for C29H32BrN3O3 (M+H ): 551.1271, found: 551.1352.

(2R,4S,6S)-benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1- carboxylate

N N O Br O O

Chemical Formula: C29H30BrN5O3 Molecular Weight: 576.48 We performed the reaction with the same quantity and procedure as briefed for (2S,4R,6R)- benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate All the obtained data are similar to that of (2S,4R,6R)-benzyl 4-azido-2-(benzyl(2- bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate.

(2R,4R,6S)-benzyl 4-amino-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1- carboxylate 265

306

NH2

N N O Br O O

Chemical Formula: C29H32BrN3O3 Molecular Weight: 550.48

We performed the reaction with the same quantity and procedure as briefed for (2S,4R,6R)- benzyl 4-amino-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate All the obtained data are similar to that of (2S,4R,6R)-benzyl 4-amino-2-(benzyl(2- bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate.

M.P: 91-92 °C

+ HR-ESI-MS: calculated for C29H32BrN3O3 (M+H ): 551.1271, found: 551.1283.

(2S,4R,6S)-benzyl 4-azido-2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6- methylpiperidine-1-carboxylate

N F 3 F F N N O Br O O

Chemical Formula: C24H25BrF3N5O3 Molecular Weight: 568.36

To a solution of (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- (methylsulfonyloxy)piperidine-1-carboxylate (0.37 g, 0.69 mmol, 1.0 equiv) in anhydrous DMF (5 mL) at room temperature was added with sodium azide (0.09 g, 1.37 mmol, 2.0 equiv) and continued to stir at 85 °C for 24 h. The reaction mixture was evaporated in vacuo and the resulting residue was dissolved in DCM (20 mL) and washed with water (2x20 mL), dried over MgSO4 and finally evaporated in vacuo. This gives (2S,4R,6S)-benzyl 4-azido-2- ((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methylpiperidine-1-carboxylate as pale

307 yellow liquid. Due to stability issues, without any purification, the crude was engaged into the next step.

Rf 0.65 (SiO2, cyclohexane/ethyl acetate: 6/4).

(2S,4R,6S)-benzyl 4-amino-2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6- methylpiperidine-1-carboxylate 266

NH F 2 F F N N O Br O O

Chemical Formula: C24H27BrF3N3O3 Exact Mass: 541.11

To a solution of (2S,4R,6S)-benzyl 4-azido-2-((2-bromobenzyl)(2,2,2- trifluoroethyl)carbamoyl)-6-methylpiperidine-1-carboxylate (0.24 g, 0.80 mmol, 1.0 equiv) in

THF (8 mL) and H2O (0.8 mL) at room temperature was added with triphenylphosphine (0.17 g, 0.83 mmol, 1.2 equiv) and continued to stir at room temperature for . The reaction mixture was evaporated in vacuo and the resulting residue was diluted with ethyl acetate and H2O (20 mL). The pH was adjusted to be acidic using 1N HCL (until TLC showed no traces of product) and discarded. The combined aqueous layer was taken to 0°C and the pH carefully was adjusted to 14 with NaOH. Then it was extracted with ethyl acetate (3x30 mL). The combined organic layer was dried over MgSO4 and concentrated in vacuo. The crude thus obtained was purified using column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to yield (2S,4R,6S)-benzyl 4-amino-2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6- methylpiperidine-1-carboxylate.

M.P: 62-64 °C

+ HR-ESI-MS: calculated for C24H27BrF3N3O3 (M+H ): 542.1266, found: 542.1284.

308

tert-butyl 4-aminopiperidine-1-carboxylate 267

6 NH2 3 4 2

5 N 1

O O 6

8 7

Chemical Formula: C10H20N2O2 Molecular Weight: 200.27 To a solution of tert-butyl 4-azidopiperidine-1-carboxylate (4.53, 20 mmol, 1 equiv) in Tetrahydrofuran and water (10+1 mL) at room temperature was added triphenylphosphine (7.86 g, 30 mmol, 1.5 equiv). The resulting mixture was stirred at room temperature for 48 h and concentrated in vacuo.

Rf = 0.11 (cyclohexane/ethyl acetate : 7/3)

1 H NMR (400 MHz, CDC13) δ(ppm) 4.02(br s, 2H, H6), 2.72-2.82 (m, 3H, H3, H2 ), 1.74-

1.78 (m, 2H, H4), 1.43 (s, 9H, H7-9), 1.16-1.27 (m, 2H, H5).

CAS Registry Number: 87120-72-7.

The analysis is in accordance with the literature.

N-((3S,4aS)-6-benzyl-5-oxo-1,2,3,4,4a,5,6,7-octahydrobenzo[f]pyrido[1,2-a][1,4]diazepin- 3-yl)-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide 268

HN N

N N O NH N O N

Chemical Formula: C32H35N7O3 Molecular Weight: 565.66

309

A 5mL one-neck round-bottomed flask was charged with N,N-carbonyldiimidazole (31mg, 0.19mmol) and flushed with argon, carefully pumped out at vacuum and refilled with argon. Such procedure was done three times. Then freshly distilled THF (2mL) was added and stirred. In the meantime, a 10mL one-neck round-bottomed flask was attached to a refrigerant and charged with 286 (50mg, 0.15mmol). Then, it was flushed with argon, the inner atmosphere carefully pumped out at vacuum and refilled with argon. Such procedure was done three times as well. Then freshly distilled THF (3.6mL) and TEA (0.022mL, 0.19mmol) were added via syringe and then stirred at r.t. a while under argon. Then, this solution was added drop-wise very slowly (within 30 min) via syringe to the previous one. The resulting solution was stirred for 1 hour under argon. Last, compound 142 (34mg, 0.19mmol) and TEA (0.051mL, 0.39mmol) were added and the solution stirred in one portion and the resulting solution stirred under argon at 65°C for overnight. The following day it was left to cool down to r.t. and AcOEt was added. The organic solution was washed with NaOH 1M and brine, dried over anhydrous magnesium sulphate and solvent removed under reduced pressure. The resulting crude was purified by flash chromatography (AcOEt/methanol) to give 47mg of 19 (57% yield, rf= 0.54 in AcOEt/methanol 8/2). 1 ( H400 MHz, CDCl3) H 7.87 (1H, d, J 4.5, Ar), 7.24-7.02 (9H, br, Ar), 6.86 (1H, d, J 7.5,

Ar), 6.81 (1H, m, ar), 6.67 (2H, m, Ar), 5.01 (1H, d, J 3.5), 4.69 (2H, t, J 15, CONCH2), 4.36 (2H, m), 4.19 (1H, br), 4.10 (2H, m), 2.23 (1H, t, J 10), 2.17-2.03 (3H, m), 1.97-1.65 (2H, br), 13 ( C100 MHz, CDCl3) 172.07 (CO), 157.1 (NCON), 153.57 (CO), 150.3 (Ar), 143.2 (Ar), 140.4, (Ar), 136.8 (Ar), 132.2 (Ar), 129.1 (Ar), 128.7 (Ar), 128.2 (Ar), 127.6 (Ar), 127.3 (Ar),123.1 (Ar), 122.0 (Ar), 119.01 (Ar), 116.09 (Ar), 115.6 (Ar), 64.3, 51.4, 50.4, 48.8, 44.6, 43.9, 43.6, 36.6, 30.9, 29.3. + HR-ESI-MS calcd for C32H35N7O3Na (M+Na )=588.2699, found=588.2699

310

(2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine- 1-carboxylate 281 A

N N O Br O O

Chemical Formula: C29H31BrN2O4 Molecular Weight: 551.47

To a solution of (2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate (0.400 g, 0.72 mmol, 1.0 equiv) in methanol (10 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.33 g, 0.86 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 min. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (50 mL), washed with water (3x50 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine- 1-carboxylate as a yellow oil (0.39 g, 98%).

Rf 0.13 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C29H31BrN2O4 (M+H ): 551.1545, found: 551.1579.

25 [α] D = + 2.6 (c 1.0, CHCl3).

(2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine- 1-carboxylate 281 B

N N O Br O O

311

Chemical Formula: C29H31BrN2O4 Molecular Weight: 551.47

To a solution of (2S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate (0.376 g, 0.68 mmol, 1.0 equiv) in methanol (9.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.30 g, 0.82 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (50 mL), washed with water (3x50 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6- methylpiperidine-1-carboxylate as a yellow oil (0.37 g, 97%).

Rf 0.11 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C29H31BrN2O4 (M+H ): 551.1545, found: 551.1526.

25 [α] D = + 3.9 (c 1.0, CHCl3).

(2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-4-hydroxy-6- methylpiperidine-1-carboxylate 284

OH F F F N N O Br O O

Chemical Formula: C24H26BrF3N2O4 Molecular Weight: 543.37

To a solution of (2S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6- methyl-4-oxopiperidine-1-carboxylate (0.35 g, 0.647 mmol, 1.0 equiv) in methanol (8.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.29 g, 0.775 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (30 mL), washed with water (3x30 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate:

312

7/3) to give (2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-4- hydroxy-6-methylpiperidine-1-carboxylate as a yellow oil (0.35 g, quantitative).

Rf 0.13 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C24H26BrF3N2O4 (M+H ): 543.1106, found: 543.1159.

25 [α] D = - 1.7 (c 1.05, CHCl3).

(2R,4S,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine- 1-carboxylate 285

N N O Br O O

Chemical Formula: C29H31BrN2O4 Molecular Weight: 551.47

To a solution of (2R,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4- oxopiperidine-1-carboxylate (0.265 g, 0.482 mmol, 1.0 equiv) in methanol (7.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.21 g, 0.578 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (20 mL), washed with water (3x20 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2R,4S,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6- methylpiperidine-1-carboxylate as a yellow oil (0.37 g, 97%).

Rf 0.17 (SiO2, cyclohexane/ethyl acetate: 7/3).

+ HR-ESI-MS: calculated for C29H31BrN2O4 (M+H ): 551.1545, found: 551.1526.

25 [α] D = - 4.7 (c 1.01, CHCl3).

(2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6-methylpiperidine-2-carboxamide

313

12 OH 11 13 10 14 5 4 3 9 15 6 2 N 8 16 7 1 N H O 22 17 18 21 19 20

Chemical Formula: C21H26N2O2 Molecular Weight: 338.44 To a solution of (2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6- methylpiperidine-1-carboxylate ( 0.3 g, 0.54 mmol, 1.0 equiv) in methanol (5 mL) at room temperature was added with Pd(OH)2 ( 0.015 g, 0.11 mmol, 0.20 equiv). The mixture was kept under hydrogen atmosphere and continued to stir for 12 h. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (30 mL), washed with water

(3x30 mL), dried over MgSO4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to yield (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6-methylpiperidine-2-carboxamide as yellow solid.

Rf 0.1 (SiO2, cyclohexane/ethyl acetate: 5/5).

Proton NMR is difficult to predict even now. And thus, we completely depend on carbon NMR.

Rotamer A

13 C NMR (100 MHz, CDCl3) δ (ppm) 19.02 (C7), 37.20 (C3), 43.58 (C5), 47.22 (C6), 52.89

(C2), 53.49 (C9), 127.64, 128.33, 129.08, 129.96, 136.73, 137.44 (CHar), 170.33 (C8).

Rotamer B

13 C NMR (100 MHz, CDCl3) δ (ppm) 20.72 (C7), 38.13 (C3), 43.98 (C5), 48.02 (C6), 53.41

(C2), 54.10 (C9), 127.94, 128.74, 129.75, 133.01, 136.85, 137.60 (CHAr), 170.33 (C8).

25 [α] D = + 17.9 (c 1.10, CHCl3).

+ HR-ESI-MS: calculated for C21H26N2O2 (M+H ): 339.2073, found: 339.2048.

314

tert-butyl 4-((2-aminopyridin-3-yl)amino)piperidine-1-carboxylate

9 10 8

O O

N 11 7 12 5 6 4 3 NH 12 2 11 N NH2 1

Chemical Formula: C15H24N4O2 Exact Mass: 292.37

2,3-Diamino pyridine (10.0 g, 91.6 mmol, 1.0 equiv), tert-butyl 4-oxopiperidine-1- carboxylate (21.9 g, 110 mmol, 1.2 equiv) and ethyl acetate (100 mL) were cooled to 0–5 °C. Trifluoroacetic acid (23.4 g, 205 mmol, 2.2 equiv) followed by sodium triacetoxyborohydride (29.1 g, 137 mmol, 1.5 equiv) were added at ≤10 °C. The reaction mixture was warmed to ambient temperature, stirred for 2 h, and then cooled to ≤10 °C. The reaction mixture was quenched with 10% sodium hydroxide solution (∼180 mL), and the pH was adjusted to 8.5– 9.0 while maintaining the reaction temperature at ≤10 °C. The reaction mixture was diluted with ethyl acetate (200 mL), and the organic layer was separated and washed with water (100 mL), dried over MgSO4 and concentrated in vacuo. Thus obtained brow coloured compound without any purification results in tert-butyl 4-((2-aminopyridin-3-yl)amino)piperidine-1- carboxylate (24g, 86%).

1 H NMR (400 MHz, DMSO-d6): δ 1.19 (2H, m, H6,12), 1.38 (9H, s, H8, 9, 10), 1.87 (2H, d, 2H,

H6,12), 2.87 (1H, brs,H7/11), 3.38 (1H, broad s, H12), 3.88 (2H, d, ), 4.48 (1H, d, H5), 5.43 (2H, broad s, H11), 6.17 (1H, dd, J = 76, 1.2 Hz, H3), 6.64 (1H, dd, J = 6.8, 1.2, H4), 7.25 (1H, dd,

J = 4.8, 1.6 Hz, H2).

CAS Registry Number: 781649-86-3.

The analysis is in accordance with the literature.

tert-butyl 4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxylate

315

O O N

N O N N H

Chemical Formula: C16H22N4O3 Exact Mass: 318.16 Tert-Butyl-4-(2-aminopyridin-3-ylamino)piperidine-1-carboxylate (5.0 g, 17.1 mmol, 1.0 equiv), and acetonitrile (50 mL) were treated with N,N-diisopropylethylamine (4.89 g, 37.8 mmol) followed by 1,1′-carbonyldiimidazole (4.16 g, 2.56 mol) at ambient temperature. The mixture was stirred for 2 h. The reaction mixture was cooled to 0–5 °C and stirred for 30 min. The resulting slurry was filtered, and the cake was washed with acetonitrile (200 mL) and dried at 50 °C in vacuo to afford Tert-butyl 4-(2-oxo-2,3-dihydro-1H-imidazo[4,5- b]pyridin-1-yl)piperidine-1-carboxylate (4.85 g, 82%) as a white solid.

1 H NMR (400 MHz, DMSO-d6) δ (ppm) 1.44 (9H, s, 9H), 1.72 (2H, d, J = 10.4 Hz), 2.13 (2H, dq, J = 12.4 Hz, 4.4 Hz), 2.78–2.84 (2H, m), 4.10 (2H, J = 12.0 Hz, d), 4.32–4.38 (1H, m), 6.99 (1H, dd, J = 7.6 Hz, 5.2 Hz), 7.53 (1H, dd, J = 8.0 Hz, 0.8 Hz), 7.90 (1H, dd, J = 5.2 Hz, 1.2 Hz), 11.54 (1H, s).

CAS Registry Number: 781649-87-4.

The analysis is in accordance with the literature.

1-(Piperidin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one dihydrochloride

H N .HCl

N O N N H .HCl

Tert-Butyl-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxylate (5.0 g, 15.7 mmol) and HCl (4.0 M in EtOH; 2.50 mL) were heated to 50–55 °C for 10 h. The reaction mixture was cooled to ambient temperature and the resulting slurry filtered. The cake

316 was washed with ethanol (100 mL) and dried at 50 °C in vacuo to afford 4.28 g, 94% of 1- (Piperidin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one dihydrochloride as an off-white solid.

1 H NMR (400 MHz, DMSO-d6) δ 1.87 (2H, d, J = 12.4 Hz), 2.60–2.69 (2H, m), 3.09 (2H, q, J = 12.0 Hz,), 3.40 (2H, d, J = 12.4 Hz), 4.60 (1H, dt, J = 12.4 Hz, 3.6 Hz), 7.05 (1H, dd, J = 7.6 Hz, 5.2 Hz), 7.89–7.94 ( 3H, m), 9.14 (1H, brs), 9.48 (1H, brs),11.67 (1H, brs).

CAS Registry Number: 1209456-28-9.

The analysis is in accordance with the literature.

tert-butyl 4-hydroxypiperidine-1-carboxylate 283

6 OH 3 4 2

5 N 1

O O 7

9 8

Chemical Formula: C10H19NO3 Molecular Weight: 201.26

To a solution of tert-butyl 4-oxopiperidine-1-carboxylate (4 g, 20 mmol, 1 equiv) in methanol (100 ml) at 0°C was added powdered sodium borohydride (0.76 g, 20 mmol, 1 equiv) slowly. The resulting solution was stirred at room temperature for 15 min and concentrated in vacuo. The white solid residue was dissolved in ethyl acetate (100 mL) and extracted with brine solution (3x30 mL). The organic extracts were dried over MgSO4, filtered and concentrated in vacuo to give 4.213 g (quantitative yield) of tert-butyl 4-hydroxypiperidine-1-carboxylate as yellow-orange oil.

Rf 0.17 (cyclohexane/ethyl acetate : 7/3).

1 H NMR (CDCl3, 400 MHz) δ (ppm) 3.87-3.76 (m, 3H, H3, H2), 3.02 - 2.96 (m, 2H, H4),

2.13 (br, 1H, H6), 1.85 - 1.79 (m, 2H, H1), 1.47 - 1.38 (m, 2H, H5), 1.43 (s, 9H, H7-9).

CAS Registry Number: 109384-19-2.

The analysis is in accordance with the literature.

317

tert-butyl 4-azidopiperidine-1-carboxylate

N N N 3 4 2

5 N 1

O O 6

8 7

Chemical Formula: C10H18N4O2 Molecular Weight: 226.27

To a solution of tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate (5.6 g, 20 mmol, 1 equiv) in anhydrous N,N-Dimethylformamide (100 mL) at room temperature was added sodium azide (2.6 g, 40 mmol, 2 equiv). The resulting mixture was stirred at 85°C overnight and concentrated in vacuo. The solid residue was dissolved in dichloromethane (100 mL) and extracted with water (3x30 mL). The organic extracts were dried over MgSO4, filtered and concentrated in vacuo to give 4.53 g (quantitative yield) of tert-butyl 4-azidopiperidine-1- carboxylate as pale yellow oil.

Rf = 0.77 (cyclohexane/ethyl acetate : 6/4)

1 H NMR (400 MHz, CDC13) δ(ppm) 3.69-3.72 (m, 2H, H2), 3.44-3.50 (m, 1H, H3), 2.95-

3.01 (m, 2H, H4), 1.73-1.77 (m, 2H, H1), 1.39-1.48 (m, 2H, H5), 1.34 (s, 9H, H7-9).

CAS Registry Number: 180695-80-1.

The analysis is in accordance with the literature.

318

(4S,14bS)-4-methyl-1,3,4,14b-tetrahydrodibenzo[b,f]pyrido[1,2-d][1,4]diazepin-2(10H)- one 250 O 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 NH 15 10 19 12 11

Chemical Formula: C18H18N2O Molecular Weight: 278.34

We used generalized method as presented for 155’.

Rf 0.45 (SiO2, cyclohexane/ethyl acetate: 7/3).

Yield: 97%

1 H NMR (400 MHz, CDCl3) δ (ppm) 0.93 (1H, d, J = 6.2 Hz, H7), 1.03 (1H, t, J = 12.3 Hz,

H5a), 1.48 (1H, m), 1.92 (1H, t, J = 12.2 Hz, H3a), 2.17 (1H, t, J = 16.3, 4.6 Hz ), 2.36 (1H, m), 2.57 (1H, m, H6), 2.64 (1H, m), 3.63 (1H, dd, J = 15.3 Hz, H2), 6.55-6.67 (1H, m, Har), 6.88-6.96 (1H, m, Har), 6.94-7.03 (1H, m, Har), 7.04-7.18 (1H, m, Har), 7.30 (1H, m, Har), 7.440 (1H, d, J = 8.0 Hz; Har), 7.50-7.69 (1H, m, Har).

13 C NMR (100 MHz, C6D6) δ (ppm) 22.52 (C7), 30.17 (C3), 44.98 (C5), 49.61 (C6), 60.58 (C2), 113.0, 116.31, 119.13, 120.97, 121.42, 127.41, 127.97, 128.62, 128.96, 130.37, 133.61, 141.90 (Car), 206.18 (C=O).

25 [α]D = - 29.1(c=1.00 CHCl3)

HR-ESI-MS: calculated for C18H18N2O 279.1916, found: 279.1968.

319

(4S,14bS)-4-methyl-1,2,3,4,10,14b-hexahydrodibenzo[b,f]pyrido[1,2-d][1,4]diazepin-2-ol 252

OH 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 NH 15 10 19 12 11

Chemical Formula: C18H20N2O Molecular Weight: 280.36 We followed a similar method as proposed for (2R,4R,6R)-benzyl 2-(benzyl(2- bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. Yield is quantitative

Rf 0.25 (SiO2, cyclohexane/ethyl acetate: 5/5).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.04 (1H, m), 1.31 (1H, m), 1.73 (1H, m), 1.92 (2H, m), 3.38 (1H, J = 11.2 Hz, H4), 6.55-6.67 (1H, m, Har), 6.88-6.96 (1H, m, Har), 6.56 (1H, m, Har), 6.96 (1H, m, Har), 7.14 (1H, m, Har), 7.44 (1H, d, J = 8.0 Hz; Har), 7.56 (1H, m, Har).

13 C NMR (100 MHz, C6D6) δ (ppm) 22.60 (C7), , 39.17 (C3), 43.63 (C5), 50.35 (C6), 59.30 (C2), 69.35 (C4), 112.93, 116.01, 119.39, 120.62, 121.41, 126.88, 127.94, 128.87, 132.22, 133.70, 142.31, 142.52(Car).

25 [α]D = - 15.1(c=1.10 CHCl3)

HR-ESI-MS: calculated for C18H20N2O 281.2314, found: 281.2328.

(4S,14bS)-4-methyl-3,4-dihydro-1H-dibenzo[b,f]pyrido[1,2-d][1,4]oxazepin-2(14bH)-one

O 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 O 15 10 19 12

Chemical Formula: C18H17NO2

320

Molecular Weight: 279.33 We used generalized method as presented for 155’

Rf 0.25 (SiO2, cyclohexane/ethyl acetate: 7/3).

Yield: 98%

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.00 (1H, d, J = 6.2, H7), 1.12 (1H, t, J = 12.6 Hz, H5a), 1.72-1.99 (2H, m, H3a, 3e), 2.10 (1H, td, J = 13.3, 3.2 Hz, H5e), 2.15 (1H, dqd, J = 12.1, 6.1 Hz, 2.8 Hz, H6e), 7.18 (2H, tdd, J = 8.5, 7.4, 1.4 Hz, Har), 7.20 (1H, t, J = 7.6 Hz, Har), 7.29 (1H, dd, J = 7.2, 1.3 Hz, Har), 7.52 (1H, dd, J = 7.6, 1.1 Hz, Har). 7.60(1H, dd, J = 6.3 Hz, 1.3 Hz, Har), 7.69 (1H, d, J = 7.4 Hz, Har).

13 C NMR (100 MHz, C6D6) δ (ppm) 21.71 (C7), 30.19 (C3), 39.39 (C5), 59.48 (C6), 63.37 (C2), 112.08, 117.77, 119.61, 120.99, 123.06, 123.99, 124.71, 125.71, 126.89, 127.57, 154.13, 156.51 (Car), 205.04 (C=O).

25 [α]D = - 9.6(c=1.00 CHCl3)

HR-ESI-MS: calculated for C18H17NO2 280.5471, found: 280.5401.

(2S,4S,14bS)-4-methyl-2,3,4,14b-tetrahydro-1H-dibenzo[b,f]pyrido[1,2-d][1,4]oxazepin- 2-ol 282

OH 5 3 4 18 6 2 17 N 20 7 8 1 16 14 9 13 O 15 10 19 12

Chemical Formula: C18H19NO2 Molecular Weight: 281.34 We followed a similar method as proposed for (2R,4R,6R)-benzyl 2-(benzyl(2- bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. Yield is quantitative

Rf 0.17 (SiO2, cyclohexane/ethyl acetate: 5/5).

1 H NMR (400 MHz, CDCl3) δ (ppm) 1.16 (1H, m), 1.56 (1H, m), 1.88 (1H, m), 2.21 (2H, m), 3.66 (1H, J = 11.2 Hz, H4), 4.03 (1H, d, J = 11.5, 4.3 Hz, H2) 7.21 (2H, m, Har), 7.32

321

(2H, m, Har), 7.54 (1H, m, Har), 7.71 (1H, m, Har), 7.79 (1H, m, Har), 7.85 (1H, d, J = 8.0 Hz; Har).

25 [α]D = - 25.1(c=1.15 CHCl3)

HR-ESI-MS: calculated for C18H19NO2 282.0017, found: 282.0178.

tert-butyl 4-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1- carboxamido)piperidine-1-carboxylate 287

O 7 8 HN N 11 O 9 2 3 6 N 10 12 NH 1 5 N 13 N O O 18 14 15 16 17

Chemical Formula: C22H32N6O4 Molecular Weight: 444.52

We followed the general method as 268.

1 H NMR (400 MHz, CD3OD) δ (ppm) 1.47 (9H, s, H16, 17, 18), 1.87 (4H, ddd, J = 15.9, 12.3, 2.3 Hz, H 1, 5), 2.31 (2H, qd, J = 12.6, 4.3 Hz), 2.95 (2H, m), 4.09 (2H, d, J = 13.4 Hz), 4.25 (2H, d, J = 13.3 Hz), 4.94 (6H, s), 7.56 (1H, J = 7.9, 1.3 Hz), 7.72 (2H, s), 7.96 (1H, dd, J = 5.3, 1.2 Hz).

13 C NMR (100 MHz, CD3OD) δ (ppm) 28.67 (C16, 17, 18), 30.09, 44.64, 52.38 (C alicyclic), 81.06, 116.71, 118.00, 124.96, 141.11 (Car), 156.55, 159.10 (C=O).

+ HR-ESI-MS calcd for C22H34N6O4 (M+H )=445.2563, found=445.2576.

322

(2S,4R,6R)-phenyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-(4-(2-oxo-2,3- dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamido)piperidine-1- carboxylate 289

O Br O N NH

HN N N N N O O O

Chemical Formula: C40H42BrN7O5 Molecular Weight: 780.70

Similar tert-Butyl carbamate deprotection method as that of 155’

Due to rotamers, we can’t asses any peak in the spectra. So, we have to depend on HR-MS again.

+ HR-ESI-MS calcd for C40H42BrN7O5 (M+H )=794.2666, found=764.2733.

(2R,4R,6S)-phenyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-(4-(2-oxo-2,3- dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamido)piperidine-1- carboxylate 290 O Br O N NH

HN N N N N O O O

Chemical Formula: C40H42BrN7O5 Molecular Weight: 780.70 Both preparation and data are identical to the (2S,4R,6R)-phenyl 2-(benzyl(2- bromobenzyl)carbamoyl)-6-methyl-4-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-

323 yl)piperidine-1-carboxamido)piperidine-1-carboxylate.

(2R,4R,6S)-phenyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4-(4-(2- oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamido)piperidine-1- carboxylate 291

O Br O N NH

HN N N N N F O O O F F

Chemical Formula: C35H37BrF3N7O5 Molecular Weight: 772.61

+ HR-ESI-MS calcd for C35H37BrF3N7O5 (M+H )=773.0123, found=773.0238.

4-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1- carboxamido)piperidinium 293

O 7 8 HN N 11 O 9 2 3 6 N 10 12 NH 1 5 N 13 H H N 14 15

Chemical Formula: C17H25N6O2 Molecular Weight: 345.41

Similar tert-Butyl carbamate deprotection method as that of 155’

1 H NMR (400 MHz, CD3OD) δ (ppm) 1.77 (4H, m), 2.10 (2H, t, J = 15.5), 2.31 (2H, q, J = 12.2 Hz), 3.01 (5H, dt, J = 25.3, 12.3 Hz, 1H), 3.43 (2H, d, J = 12.7 Hz), 4.23 (2H, d, J = 13.2 Hz), 7.08 (1H, S, Har), 7.96 (1H, s), 8.90 (2H, s).

+ HR-ESI-MS calcd for C19H25F3N6O4 (M+H )=345.2039, found=345.2039.

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N-((2S,4S,6R)-2-(dibenzylcarbamoyl)-6-methylpiperidin-4-yl)-4-(2-oxo-2,3-dihydro-1H- imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide 294

O N NH

HN N N N N H O

Chemical Formula: C33H39N7O3 Molecular Weight: 581.70

Hydrogenation procedure as followed in (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6- methylpiperidine-2-carboxamide.

Physical property: white solid

M.P: 163-165 °C.

13 C NMR (400 MHz, CD3OD) δ (ppm) 21.03, 21.11, 30.12, 30.21, 30.80, 33.87, 37.41, 44.90, 46.48, 46.54, 47.82, 51.02, 51.02, 51.19, 52.23, 52.34, 53.20, 53.28, 116.75, 116.93, 118.14, 129.10, 129.77, 130.15, 137,18, 137.89, 141.11, 144.77, 155.27, 159.76, 173.81, 173.86 (C=O)

HR-ESI-MS calcd for C33H39N7O3 for 582.3193, found 582.3204.

20 [α]D = - 10.7 (c=1.00 CHCl3)

((2R,4S,6S)-2-(dibenzylcarbamoyl)-6-methylpiperidin-4-yl)-4-(2-oxo-2,3-dihydro-1H- imidazo[4,5-b]325yridine-1-yl)piperidine-1-carboxamide 295

O N NH

HN N N N N H O

Chemical Formula: C33H39N7O3

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Molecular Weight: 581.70

All the data were similar to N-((2S,4S,6R)-2-(dibenzylcarbamoyl)-6-methylpiperidin-4-yl)-4- (2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide except the following. M.P: 149-150 °C

20 [α]D = - 7.2 (c=1.00 CHCl3) N-((2S,4S,6S)-2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methylpiperidin-4- yl)-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide 296 O Br O N NH

HN N N N N H F O F F

Chemical Formula: C28H33BrF3N7O3 Molecular Weight: 652.50

Hydrogenation procedure as followed in (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6- methylpiperidine-2-carboxamide.

1H NMR was still unpredictable. The other data are as follows

Physical property: Yellow solid

M.P: 191-193 °C.

13 C NMR (400 MHz, CD3OD) δ (ppm) 30.15, 32.93, 44.72, 45.29, 49.95, 52.60, 53.01,

53.84, 116.71, 118.19, 125.92, 128.84, 130.23, 136.72, 141.16, 144.69, 155.43, 158.92 (Car), 173.43, 173.80 (C=O)

HR-ESI-MS calcd for C28H33BrF3N7O3 for 574.2753, found 574.2810.

20 [α]D = + 1.3 (c=1.00 CHCl3)

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