Synthesis and Evaluation of Tetrahydroprotoberberines As Dopamine Receptor Ligands

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Synthesis and Evaluation of Tetrahydroprotoberberines as Dopamine Receptor Ligands

Satishkumar V. Gadhiya The Graduate Center, City University of New York

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SYNTHESIS AND EVALUATION OF TETRAHYDROPROTOBERBERINES AS DOPAMINE RECEPTOR LIGANDS

SATISHKUMAR GADHIYA

A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York. 2017

SATISHKUMAR V GADHIYA

Synthesis and Evaluation of Tetrahydroprotoberberines as Dopamine Receptor Ligands

Satishkumar Gadhiya

This manuscript has been read and accepted for the Graduate Faculty in Chemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy

Date Dr. Wayne Harding

Chair of Examining Committee

Date Dr. Brian Gibney

Executive Officer

Dr. Shengping Zheng Dr. Barbara Zajc Dr. Wayne Harding Supervisory Committee

THE CITY UNIVERSITY OF NEW YORK

iii ABSTRACT Synthesis and Evaluation of Tetrahydroprotoberberines as Dopamine Receptor Ligands By Satishkumar Gadhiya

Advisor: Dr. Wayne Harding

Dopamine (DA) receptors belong to the G-protein coupled receptors (GPCRs) family, divided in to two groups based on their high homology transmembrane domains; D1-like DA receptors (D1, D5) and D2-like DA receptors (D2-D4). DA receptor specific ligands have been exploited as a means for studying the prognosis and curing several CNS disorders. Though several efforts have been devoted to discover selective and potent DA receptor ligands, complete selectivity within the DA receptor subtypes remains a challenge.

Tetrahydroprotoberberines (THPBs) are a group of naturally occurring tetracyclic alkaloids that belong to the tetrahydroisoquinoline family. A wide range of biological activities are associated with the THPB scaffold. Noted examples of THPB alkaloids, (-)-stepholidine 2.1

(SPD), (-)-tetrahydropalmatine 2.2 (THP), (-)-isocorypalmine 2.3 (ICP), and (-)-canadine 2.4, are known to possess high affinity for DA receptors and have been studied widely for their utility in various CNS disorders. (-)-Stepholidine is endowed with DA D1 agonist and D3 antagonist dual pharmacological profile, which makes it a potential candidate for the treatment of schizophrenia and psychostimulant drug abuse. Limitations on the unavailability of efficient synthetic methods, scarcity of structure-activity relationship (SAR) data at DA receptors and metabolic instability

iv need to be addressed to make (-)-SPD 2.1 an ideal drug candidate for schizophrenia and psychostimulant drug addiction.

In our work we have addressed prominent issues and missing gaps towards the synthesis and SAR studies of THPBs at dopamine D1, D2 and D3 receptors. A novel route to synthesize

(±)-SPD (2.1) was developed over eight synthetic steps with 30% overall yield via the intermediacy of easily obtainable diester 3.7. In addition to the superior yield, this approach is advantageous in terms of conciseness and ease of synthetic manipulations. This route was deployed to produce C10 THPB analogues for SAR studies at DA receptors. Employing vital intermediate lactone 2.102, the enantioselective synthesis of (-)-SPD (2.1) was accomplished in

23% overall yield from commercially available starting materials in a fourteen step sequence. The crystal structure of (-)-SPD (2.1) was determined by single crystal X-ray diffraction. Synthesis of lactone 2.102 was straightforward, scalable and high yielding. This synthetic pathway was applied to generate C3 chiral THPB analogues of THPB for bioactivity studies.

In order to understand the structural tolerance of the THPB core required for selective

D1/D3 receptor binding, a thorough SAR study was designed. Consequently, a diverse library of

C3 and C10 THPB analogues was synthesized and evaluated for affinity at DA D1, D2 and D3 receptors. Results from the SAR studies demonstrate that the C10 THPB analogues exhibit a general preference for the D1 receptor. Binding affinity evaluation at D3 receptor indicates that larger alkoxy groups are not tolerated at C10 position. In general, small alkoxy substituents are well tolerated at C10 position of THPB scaffold for D1 and D3 receptor affinity. Novel enantiopure

C3 analogues of THPB were designed and synthesized to assess the optimum size of the alkoxy group at this position required for DA D1 and D3 receptor affinity. The results for binding affinity data suggest that C3 alkoxy substitution, decreases D1 and D3 receptor affinity while improving

v selectivity at D3 versus D2 receptor when compared with parent compound (-)-SPD (2.1). Reduced affinity at D2 receptor for these analogues signifies the importance of the C3 alkoxy group to suppress affinity at the D2 receptor. These compounds will further expand our understanding of the tolerance of the THPB core required for DA D1 and D3 receptor activity.

In order to rationalize the affinity of ligands, molecular docking studies were conducted at the D3 receptor. The ligand binding energies from the D3 receptor docking studies show some similarities and minor discrepancies compared to the experimentally derived affinities. These docking studies have revealed key receptor–ligand interactions, including the critical protonated tertiary N—

Asp110 salt bridge motif, H-bonds to Ser192 and Cys181 and hydrophobic interactions to Phe106 and Phe345.

Novel series of conformationally flexible analogues of (-)-SPD (2.1) were synthesized and evaluated in order to identify potent and selective DA D1 and D3 receptor ligands. Two groups of analogues were synthesized and assayed for binding affinity at DA receptors, one group having truncated THPB scaffold while the other featured a tetrahydroisoquinoline (THIQ)/arylamide hybridized pharmacophore core. Results of this study indicate that de-rigidification of the THPB scaffold nullifies the DA receptor activity and the intact THPB scaffold of (-)-SPD is necessary for the DA receptor activity. On the other hand, THIQ/arylamide hybrid analogues have demonstrated high potency and selectivity for D3 receptor.

Representative compounds were studied in order to test the metabolic stability of synthesized THPB analogues. The results of this metabolic study indicate that having phenolic hydroxyl group at C2 and/or C10 on THPB scaffold engenders susceptibility to hepatic metabolism.

Dedicated to

Rev. Dadaji and Rev. Taiji, the source of my inspiration and blessings

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Acknowledgement

This dissertation could not have been completed without the great support that I have received from so many people over the years.

First and foremost, I offer my sincerest gratitude to my advisor, Professor Wayne Harding, for his patience, motivation, enthusiasm and immense knowledge. He patiently provided the vision, encouragement and advice necessary for me to proceed through the doctoral program and complete my dissertation. He has been generous with his knowledge, approachable to clarify doubts and willing to assist me overcome whatever hurdles I came across during my doctoral research. He showed genuine interest in my career development. He understood my career goals and provided me with ample opportunities to develop the skills required to achieve my goals. He always guided me to work towards substantial long-term objectives, structure my research, present my ideas effectively, and promote both myself and my research results. I could not have imagined having a better and more concerned mentor for my PhD study.

I am also very grateful that Professor Barbara Zajc and Professor Shengping Zheng have been my committee members since 2012. They have provided valuable suggestions on my proposal and research, which guided me to interpret the research project from different perspectives and to improve my scientific skills.

I am very much thankful to all the members of Harding lab who have been helpful and cheering at many different levels of graduate study. A special thanks to my senior colleagues

Shashi, Sudharshan and Nirav for their continuous support and encouragement. I have learnt a lot from you guys, specially many tedious organic reactions and laboratory techniques. Anupam,

Piarpaolo and Raj, thank you all for making the last phase of my PhD a joyous and cheerful period.

viii

I am thankful to all the undergrads of the Harding lab for their support and all the help they provided in my different research projects.

I would like to thank all the members of Dr. Mootoo’s lab, Dr. Zheng’s Lab, Dr.

Kawamura’s lab and Dr. Drain’s lab for their cooperation, direct and indirect favors during my graduate study. I am sincerely grateful to the Chemistry Department at Hunter College for providing the environment and equipment I have needed to produce and complete my thesis work.

I would like to thank Prof. Thomas Kurtzman and his lab members at Lehman College for doing all the docking studies for my research project. I am very thankful to Dr. Bryan Roth and his colleagues at the University of North Carolina School of Medicine for screening compounds of my research projects. The research work I have done would have been not possible without the funding provided by National Institute of Health.

A very special gratitude and regards to my family members and friends for their continued love, advice and support. My parents Vijyaben and Vallabhbhai not only raised me with love of education and science but always encouraged me to move forward and believing in me. Mohit and

Sheetal thanks for your constant support, you guys gave me the strength when I most needed during many tough situations.

Finally, to my wife Chetna, without you I would have never accomplished this. You always stood by me with love and care during all the favorable and unfavorable situations in this journey.

You were there to cherish and share happy moments and to support me during difficult time as well. Thanks for being in my life, love you.

Table of Contents

Chapter 1: Dopamine receptor: subtypes, selective ligands and therapeutic relevance

1.1 Introduction ...... 1

1.2 Dopamine receptors ...... 2

1.2.1 Structures of dopamine receptor subtypes ...... 3

1.2.2 Distribution of brain dopamine receptors ...... 4

1.2.3 Functions of brain dopamine receptors ...... 6

1.2.4 Regulation of dopaminergic system in schizophrenia and drug abuse ...... 9

1.2.4.1 Role of dopamine receptors in schizophrenia...... 9

1.2.4.2 Abnormal dopamine transmission in drug abuse ...... 11

1.3 Dopamine receptor ligands ...... 13

1.3.1 Clinical significance of dopamine receptor ligands ...... 13

1.3.1.1 Therapeutic significance of D1 receptor selective ligands ...... 13

1.3.1.2 Therapeutic significance of D2 receptor selective ligands ...... 15

1.3.1.3 Therapeutic significance of D3 receptor selective ligands ...... 17

1.3.1.4 Therapeutic significance of D4 receptor selective ligands ...... 18

1.3.1.5 Therapeutic significance of D5 receptor selective ligands ...... 19

1.3.2 Selective D1 receptor ligands ...... 21

1.3.2.1 Arylbenzazepines...... 21

1.3.2.2 Dihydrexidine derivatives...... 27

1.3.3 Selective D2 receptor ligands ...... 29

1.3.3.1 Dihydroindenes and oxime analogues ...... 29

1.3.3.2 Aporphine analogues ...... 30

1.3.3.3 Arylpiperazine/piperidine analogues ...... 32

1.3.3.4 Dibenzazepine analogues ...... 36

1.3.4 Selective D3 receptor ligands ...... 37

1.3.4.1 2-Aminotetralin derived D3 ligands ...... 37

1.3.4.2 4-Phenylpiperazines and analogues ...... 41

1.4 Limitations with dopaminergic agents related to their clinical use ...... 46

Chapter 2: Tetrahydroprotoberberine alkaloids and derivatives

2.1 Introduction ...... 48

2.2 Occurrence ...... 49

2.3 Synthesis of THPBs ...... 52

2.3.1 Via late stage closure of ring B ...... 53

2.3.1.1 Ring B closure via bond formation between ring A and C14 carbon...... 53

2.3.1.2 Ring B closure via bond formation between ring A and C5 carbon...... 55

2.3.1.3 Ring B closure by bond formation between C6 carbon and nitrogen ...... 56

2.3.2 Via late stage closure of ring C ...... 58

2.3.2.1 Ring C closure via adding C8 carbon between ring D and nitrogen ...... 58

2.3.2.2 C-N bond formation between nitrogen and C8 carbon pre-established on ring

D ...... 61

2.3.2.3 Ring C closure via bond formation between ring D and C13 carbon...... 65

2.4 Biological activities of tetrahydroprotoberberine alkaloids ...... 67

2.4.1 CNS activities of THPB alkaloids ...... 68

2.4.2 Non-CNS activities of THPB alkaloids ...... 70

2.5 Structure-activity relationship studies at dopamine receptors ...... 71

Chapter 3: Synthesis and evaluation of stepholidine and its analogues at dopamine receptors

3.1 Background ...... 77

3.2 Synthesis of (±)-SPD ...... 79

3.2.1 Retrosynthesis ...... 79

3.2.2 Synthesis ...... 80

3.3 Enantioselective synthesis of (-)-SPD ...... 81

3.3.1 Retrosynthesis ...... 81

3.3.2 Synthesis ...... 82

3.3.2.1 Unsuccessful attempts via ester 3.10 ...... 82

3.3.2.2 Synthesis via lactone 2.62 ...... 88

3.3.2.3 Discussion ...... 90

3.4 Synthesis and evaluation of (±)-C10 analogues ...... 91

3.4.1 Rationale ...... 91

3.4.2 Results and discussion ...... 91

3.4.2.1 Synthesis ...... 91

3.4.2.2 Structure-activity correlations ...... 92

3.4.3 Molecular docking study at D3 receptor ...... 95

3.4.4 Structure-activity relationship study at σ receptors ...... 99

3.4.4.1 Background ...... 99

3.4.4.2 Structure-activity correlations ...... 101

3.5 Synthesis and evaluation of (-)-C3 analogues ...... 103

3.5.1 Rationale and analogue design...... 103

3.5.2 Results and discussion ...... 104

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3.5.2.1 Synthesis ...... 104

3.5.2.2 Structure-activity correlations ...... 105

3.6 Metabolic stability study ...... 107

3.6.1 Rationale ...... 107

3.6.2 Results ...... 108

3.7 Conclusions ...... 112

3.8 Experimental ...... 114

3.8.1 Chemistry ...... 114

3.8.2 Biological evaluation: primary and secondary radioligand binding assays ...... 133

3.8.3 Metabolic stability study ...... 134

Chapter 4: Synthesis and evaluation of de-rigidified THPB analogues at dopamine receptors

4.1 Background and rationale ...... 136

4.2 Results and discussion ...... 139

4.2.1 Synthesis ...... 139

4.2.2 Structure-activity correlations ...... 141

4.3 Molecular docking study at D3 receptor ...... 145

4.4 Metabolic stability study ...... 148

4.5 Conclusion ...... 150

4.6 Experimental ...... 152

4.6.1 Chemistry ...... 152

4.6.2 Biological evaluation ...... 162

4.6.3 Metabolic stability study ...... 162

Appendix: NMR spectra of final analogues………………………………………………..…...164

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References………………………………………………………………………………………200

xiv

List of Symbols and Abbreviations

1H NMR Proton Nuclear Magnetic Resonance

5-HT 5-hydroxytryptamine

5-HT1A Serotonin receptor - subtype 1A

5-HT2A Serotonin receptor - subtype 2A

5-OH-DPAT 5-Hydroxy-N,N-Dipropyl-2-aminotetralin

7-OH-DAPT 7-Hydroxy-N,N-Dipropyl-2-aminotetralin

13C NMR Carbon Nuclear Magnetic Resonance

α1A Alpha 1A adrenergic receptor

δ Chemical shift in ppm

µM micromolar

ơ1 Sigma receptor subtype 1

ơ2 Sigma receptor subtype 2

Å Angstrom

Ac2O Acetic anhydride

AChE AcetylCholinesterase

AcOH Acetic acid

ADHD Attention Deficit Hyperactivity Disorder

AgOTf Silver trifluoromethanesulfonate

AlCl3 Aluminium trichloride

AlMe3 Trimethylaluminium

APO Apomorphine

Asp Aspartic acid

AsPh3 Triphenylarsine

AUC Area under the curve

BBE Berberine Bridge Enzyme

BDNF Brain-derived Neurotrophic Factor

BF3.Et2O Boron trifluoride etherate

BH3-THF Borane tetrahydrofuran

BCl3 Boron trichloride

BnBr Benzyl bromide

(BoC)2O Di-tert-butyl dicarbonate bp Base pair

Br2 Bromine

Bu Butyl n-Bu3SnH Tributyltin hydride n-BuLi n-Butyllithium s-BuLi s-Butyllithium t-BuLi t-Butyllithium t-BuOH t-Butanol

C2H5OH Ethanol calcd. Calculated cAMP Cyclic Adenosine Monophosphate

CDCl3 Deuterated chloroform

CDI 1,1′-Carbonyldiimidazole

xvi

CH3CN Acetonitrile

CH3COOH Acetic acid

CH3I Methyl iodide

CH3NO2 Nitromethane

CF3COOH Trifluoro acetic acid

ClCOOEt Ethyl chloroformate

CNS Central Nervous System

Conc. Concentrated

COOH Carboxylic acid group

Cu(OAc)2 Copper (II) acetate

Cys Cysteine d Doublet

D2L Dopamine receptor-subtype D2 long isoform

D2S Dopamine receptor-subtype D2 short isoform

DA Dopamine

DA D1 Dopamine receptor - subtype D1

DA D2 Dopamine receptor - subtype D2

DA D3 Dopamine receptor - subtype D3

DA D4 Dopamine receptor - subtype D4

DA D5 Dopamine receptor - subtype D5

DCC N,N'-Dicyclohexylcarbodiimide

DCM Dichloromethane

DIBAL Diisobutylaluminium hydride

xvii

DIPA Diisopropylamine

DIPEA N,N-Diisopropylethylamine

DLPFC Dorsolateral Prefrontal Cortex

DMAP 4-Dimethylaminopyridine

DMF Dimethyl formamide

DMM Dimethyl malonate

DMSO Dimethyl sulfoxide

EC50 Half maximal effective concentration

EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EPS Extrapyramidal Motor Symptoms

Et Ethyl

EtOH Ethanol

Et3SiH Triethylsilane

Fe Iron

FLIPR Fluorescence Imaging Plate Reader

FRT Forelimb Retraction Time

Foral Oral bioavailability g Gram

GABA gamma-Aminobutyric Acid

GAFF Generalized Amber Force Field

Glu Glutamic acid

GPCR G-Protein Coupled Receptor h Hour

xviii

H2 Hydrogen gas

H2SO4 Sulfuric acid

HCHO Formaldehyde

HCl Hydrogen chloride

HCOOH Formic acid

Hex Hexyl

His Histidine

HOBt Hydroxybenzotriazole

HPLC High Performance Liquid Chromatography

HRMS (ESI) High Resolution Electrospray Ionisation Mass Spectroscopy

HRT Hindlimb Retraction Time

Hz Hertz

I2 Iodine

IC50 Half maximal inhibitory concentration

ICP Isocorypalmine

J Coupling constant

K2CO3 Potassium carbonate kcal Kilocalorie

Ke Apparent binding affinity

KI Potassium Iodide

Ki Binding affinity

KOH Potassium hydroxide

LAH Lithium Aluminum Hydride

xix

LDA Lithium DiisopropylAmide

LiBH4 Lithium borohydride m Multiplet m/z mass to charge ratio

M Molar

MDA MB 231 Human breast cancer cell line

Me Methyl

MeI Methyl Iodide

MeOH Methanol mg Milligram

MgSO4 Magnesium sulfate

MHz MegaHertz min Minute mL Milliliter mmol Millimoles mp Melting point mRNA messenger Ribonucleic Acid

NA Not Applicable

Na(AcO)3BH Sodium triacetoxyborohydride

Na2CO3 Sodium carbonate

N2H4·H2O Hydrazine monohydrate

NaBH4 Sodium borohydride

NaCN Sodium cyanide

NaCNBH3 Sodium cyanoborohydride

NaHCO3 Sodium bicarbonate

NaOAc Sodium acetate

NaOH Sodium Hydroxide

NBS N-Bromosuccinimide

NCI National Cancer Institute nd Not determined

NH2 Amino group

NH3 Ammonia gas

NH4OAc Ammonium acetate

NIH National Institutes of Health

NIMH National Institute of Mental Health nM Nanomolar

NMR Nuclear Magnetic Resonance

NPA N-n-propylnorapomorphine

NPT Constant number, pressure and temperature

NVT Constant number, volume and temperature

Pd(dba)3 Tris(dibenzylideneacetone)dipalladium(0)

Pd(OAc)2 Palladium (II) acetate

Pd(OH)2 Palladium (II) hydroxide

Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium (0)

PDSP Psychoactive Drug Screening Program

Pen Pentyl

xxi

PET Positron Emission Tomography

PGRMC1 Progesterone Receptor Membrane Component 1

Ph3P Triphenylphosphine

PhB(OH)2 Phenylboronic acid

Phe Phenylalanine

PG Protecting Group

POCl3 Phosphorus oxychloride

Pr Propyl i-PrBr Isopropyl bromide

PTSA p-Toluenesulfonic acid q Quartet quin Quintet

RAMP (R)-1-amino-2-methoxymethylpyrrolidine red Al Sodium bis(2-methoxyethoxy)aluminum hydride

RMSD Root Mean Square Deviation

(S)-Ru(OAc)2(BINAP) Diacetato[(S)-(−)-2,2′-bis(diphenylphosphino)-1,1′

binaphthyl]ruthenium(II) s singlet

SAR Struture-Activity Relationship

SCL Scoulerine

SEM Standard Error of the Mean sex Sextet

Ser Serine

xxii

SN2 Bimolecular nucleophilic substitution

SnCl4 Tin tetrachloride

SOCl2 Thionyl chloride

SPD Stepholidine t triplet

TBAF Tetra-n-butylammonium fluoride

TBDPSCl tert-Butyldiphenylsilyl chloride

TBSCl tert-Butyldimethylsilyl chloride

TEA Triethylamine tert tertiary

TFA Trifluoroacetic acid

TFAA Trifluoroacetic anhdyride

THF Tetrahydrofuran

THIQ Tetrahydroisoquinoline

THP Tetrahydropalmatine

THPB Tetrahydroprotoberberine

Ti(Oi-Pr)4 Titanium isopropoxide

TLC Thin Layer Chromatography

TM Transmembrane

TMEDA Tetramethylethylenediamine

TMSCl Trimethylsilyl chloride

Trp Tryptophan

TsCl p-Toluenesulfonyl chloride

xxiii

Tyr Tyrosine vmPFC Ventromedial Prefrontal Cortex

VTA Ventral Tegmental Area

V/V Volume /volume

ZnBr2 Zinc Bromide

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List of Figures

Chapter 1

Figure 1.1: Mesolimbic dopamine hypothesis for positive and negative symptoms of

schizophrenia…………………………………………..…………………………....11

Figure 1.2: Examples of psychostimulant drugs………………………….……………………..12

Figure 1.3: Schematic representation of mechanism of action of a) cocaine

b) methamphetamine and c) nicotine...……………………………….…………….12

Figure 1.4: Examples of a) D1 receptor agonists shown to improve cognition and working

memory in schizophrenia b) D1 partial agonist agents studied for psychostimulant

drug abuse…………………………………………………………………………...14

Figure 1.5: Examples of D2 receptor antagonists (1.11-1.117) and partial agonist (1.18) those

being used as antipsychotic…………………………...…………………………….16

Figure 1.6: Structures of D2 receptor agonists (1.19-1.21) and levodopa (1.22)…………….…16

Figure 1.7: Selective D3 receptor antagonists and partial agonist for cocaine addiction…….…17

Figure 1.8: Examples of selective D4 receptor antagonists………………………………….….18

Figure 1.9: Examples of selective D5 receptor antagonists……………………………………..19

Figure 1.10: Phenyl tetrahydrobenzazepine containing D1 receptor ligands…………………...22

Figure 1.11: Structure of conformationally constrained arylbenzazepine…………………..…..24

Figure 1.12: D1 receptor ligands having bioisostere of benzazepine……………………….…..26

Figure 1.13: Dihydrexidine and dinapsoline derivatives as D1 receptor ligands……………….28

Figure 1.14: Dihydroindenes and oxime containing D2 receptor ligands……………………....29

Figure 1.15: Aporphine analogues as D2 receptor ligands……………………………………...31

xxv

Figure 1.16: Examples of haloperidol analogues……………………………………….……….33

Figure 1.17: Arylpiperazine D2 receptor ligands……………………………………………….35

Figure 1.18: Dibenzazepine D2 receptor ligands………………………………………..………36

Figure 1.19: Advancement of dopamine to aminotetralin scaffold……………………………..38

Figure 1.20: Aminotetralin D3 receptor ligands………………………………………….……..40

Figure 1.21: Pramipexole analogues as D3 receptor ligands……………………………………40

Figure 1.22: Miscellaneous D3 receptor ligands……………………………………….……….44

Chapter 2

Figure 2.1: Structure of general scaffold of naturally occurring THPB alkaloids…………..…..48

Figure 2.2: THPB alkaloids derived from genus Stephania………………………………….....49

Figure 2.3: THPB alkaloids from Corydalis species and other species…………………………50

Figure 2.4: End ring closure position for ring B (a, b, c) and ring C (d, e, f)………………...…52

Figure 2.5: Intermediates for the Pummerer and Friedel-Crafts reaction……………………….55

Figure 2.6: Tetrasubstituted intermediates employed in the synthesis of THPB compounds…..62

Figure 2.7: 2,3,9 and 2,3,11 trisubstituted THPB analogues………………………………...…72

Figure 2.8: Diversely substituted THPB analogues…………………………………………….73

Figure 2.9: Structural requirements of THPB scaffold for DA receptor activity based on

previous SAR………………………………………………………………….....…75

Chapter 3

Figure 3.1: Synthetic strategy for (±)-SPD synthesis…………………………………….……..79

Figure 3.2: Retrosynthetic analysis of (-)-SPD……………………………………………….…82

xxvi

Figure 3.3: Structure of imide 3.21 and primary alcohol 3.22……………………………….….86

Figure 3.4: Alternate strategy for the synthesis of (-)-SPD……………………………………..88

Figure 3.5: Crystal structure of (-)-SPD monohydrate………………………………………….89

Figure 3.6: C10 analogues of (±)-SPD…………………………………………………….……91

Figure 3.7: (a) Docked poses of the lead molecule SPD (2.1) and the C10 analogues

3.25a-3.25f and 3.25h. (b) Compound 3.25a docked in binding pocke..……..……97

Figure 3.8: Docked pose of the hydroxypropyl analogue, compound 3.25g……………………98

Figure 3.9: Structural classes of ơ2 ligands and representative examples………………….….101

Figure 3.10: C3 analogues of (-)-SPD…………………………………………………………104

Figure 3.11: Representative THPB analogues for metabolic stability study……………..……109

Figure 3.12: % of total AUC at given time period of incubation for representative

analogues……………………………………………..…………………….……...110

Figure 3.13: Major metabolites analyzed in LC/MS during metabolic stability study……...…110

Chapter 4

Figure 4.1: Structural likeness between dopamine and THPB scaffold……………………….137

Figure 4.2: De-rigidified THPB analogues…………………………………………………….138

Figure 4.3: THIQ hybridized D3 receptor ligand…………………………………………...…139

Figure 4.4: THIQ-arylamide hybridized D3 receptor ligands………………………………....140

Figure 4.5: Docked pose of the flexible analogues, a) compound 4.1 and b) compound

4.2a and 4.2b…………………………………….………………………………...147

Figure 4.6: RMSD between all atom coordinates at each frame, compared to the

xxvii

coordinates of the docked pose of flexible compounds a) 4.1, b) 4.2a, and c)

4.2b………………………………………………………………………….……148

Figure 4.7: Distance between flexible compounds a) 4.1, b) 4.2a, and c) 4.2b center

of mass and the key aspartate Asp110 over the course of a 100 ns simulation….149

Figure 4.8: % of total AUC at given time period of incubation for representative

analogues………………………………………………………………………...... 150

xxviii

List of Tables

Chapter 1

Table 1.1: Potential therapeutic applications of dopamine receptor ligands……………………20

Table 1.2: Structure modification and binding affinities of catechol containing

arylbenzazepines at DA D1 and D2 receptors……………………………………….22

Table 1.3: Structure modification and binding affinities of arylbenzazepines at DA D1 and D2

receptors………………………….…………………………………………………..23

Table 1.4: Structure modification and binding affinities of conformationally

constrained arylbenzazepines at DA D1 and D2 receptors……...... ……………25

Table 1.5: Binding affinities of bioisosteres of arylbenzazepines at DA D1 and D2 receptors…26

Table 1.6: Binding affinities of aporphine analogues at DA D1 and D2 receptors……………..32

Table 1.7: Binding affinities of haloperidol analogues at DA D2, D3 and D4 receptors……….34

Table 1.8: Binding affinities of arylpiperazine analogues at DA D2 and 5-HT2A receptors……35

Table 1.9: Structure modification and binding affinities of aminotetralin

derivatives at DA D2 and D3 receptors…………………………...…………………38

Table 1.10: Binding affinities of pramipexole analogues at DA D2 and D3 receptors…………41

Table 1.11: Structure modification and binding affinities of 4-phenylpiperazine

analogues at DA D2 and D3 receptors…………...…………………………………43

Table 1.12: Summary of dopaminergic properties displayed by different chemical scaffold..…45

xxix

Chapter 2

Table 2.1: THPB alkaloids from genus Stephania …………………………………….………..50

Table 2.2: THPB alkaloids found in Corydalis species and other plants…………………...…..51

Table 2.3: Binding affinity data of 2,3,9 and 2,3,11 substituted THPBs……………………..…72

Table 2.4: Structures of and binding affinity data for THPB analogues……………………..….74

Chapter 3

Table: 3.1 Bischler-Napieralski conditions (step e, Scheme 3.1)…………………………....….81

Table 3.2: Unsuccessful esterification conditions for compound 3.16 (step b, Scheme 3.4)…...84

Table 3.3: Reduction of compound 3.10 (Scheme 3.6)………………………………………....86

Table 3.4: Hydrogenation conditions (step a, Scheme 3.7)…………………………………..…87

Table 3.5: Binding affinity data (Ki nM) for C10 analogues at dopamine receptors…………....94

Table 3.6: Predicted binding affinity from Glidescore for C10 analogues in D3

receptor docking study……………………………………………………………….96

Table 3.7: Ki data for C10 analogues at dopamine and σ receptors…………………………....102

Table 3.8: Binding affinity (Ki nM) for C3 analogues at dopamine receptors……………..….106

Table 3.9: Metabolic stability study data for representative compounds…………………...…109

Chapter 4

Table 4.1: Binding affinity data (Ki nM) for de-rigidified analogues at dopamine receptors…141

Table 4.2: Binding affinity (Ki nM) data for THIQ-arylamide hybridized D3 receptor

ligands………………………………………………………………………………145

Table 4.3: Metabolic stability study data for representative compounds……………………...150

xxx

List of Schemes

Chapter 2

Scheme 2.1: Synthesis of THPB scaffold via ring B closure in the end……………………...…53

Scheme 2.2: Synthesis of (±)-THP via ring B end closure step…………………………………54

Scheme 2.3: Synthesis of (-)-THP via Pomeranz-Fritsch reaction……………………………...55

Scheme 2.4: Synthesis of (-)-SPD via ring B closure by C-N bond formation……………….....57

Scheme 2.5: Synthesis of THPBs using Mannich condensation………………………………...58

Scheme 2.6: Synthesis of THPB scaffold via silyl directed Pictet-Spengler reaction…………..60

Scheme 2.7: Synthesis of THPB scaffold using sulfinyl directed Pictet-Spengler reaction…….60

Scheme 2.8: Bio-catalytic organic synthesis of optically pure THPB………………………..…61

Scheme 2.9: First enantioselective synthesis of (-)-SPD via tetrasubstituted lactone………..…62

Scheme 2.10: Enantioselective synthesis of (-)-SPD via chiral auxiliary protected amide……..63

Scheme 2.11: Synthesis of (-)-THP via chiral formamidine carbanion………………………....64

Scheme 2.12: Synthesis of (+)-THP using chiral b-amino sulfoxide………………………...….66

Scheme 2.13: Synthesis of (±)-THP via Pd(II)-catalyzed intramolecular tandem olefin

amidation/C-H activation……………………………………………………..….66

Chapter 3

Scheme 3.1: Synthesis of (±)-SPD………………………………………………………………81

Scheme 3.2: Enantioselective reduction of intermediate imine with Noyori's catalyst………....83

Scheme 3.3: Synthesis of oxypalmatine……………………………………………………..…..83

Scheme 3.4: Synthesis of t-butyl ester 3.17……………………………………………………..84

Scheme 3.5: Formation of enamine amide 3.18……………………………………………...….85

xxxi

Scheme 3.6: Reduction of ester 3.10 to alcohol 3.19……………………………………………86

Scheme 3.7: Reduction of imide 3.23 to primary alcohol 3.24……………………………...…..86

Scheme 3.8: Reported asymmetric hydrogenation of enamide 3.25………………………...…..87

Scheme 3.9: Asymmetric hydrogenation of enamide 3.14…………………………………..….87

Scheme 3.10: Synthesis of (-)-SPD via lactone intermediate………………………………...…89

Scheme 3.11: Synthesis of C10 analogues of (±)-SPD…………………………………...……..92

Scheme 3.12: Synthesis of C3 analogues of (-)-SPD…………………………………………..105

Chapter 4

Scheme 4.1: Synthesis of flexible analogues……………………………………………….…141

Scheme 4.2: Synthesis of flexible analogues with THIQ moiety………………………...……141

Scheme 4.3: Synthesis of THIQ-arylamide hybridized D3 receptor ligands……………….….142

xxxii

Chapter 1: Dopamine receptor: subtypes, selective ligands and therapeutic relevance

1.1 Introduction

Dopamine (DA) is a vital catecholamine neurotransmitter in the mammalian brain that controls important functions including motor co-ordination, cognition, working memory, learning, neuroendocrine regulation and positive reinforcement. Moreover, it contributes to the regulation of several autonomic and peripheral functions including cardiovascular, renal and hormone secretion. 1-4

Much research over the last 50 years has been directed towards understanding the role of the dopaminergic system in several pathological conditions such as Parkinson’s disease, schizophrenia, psychostimulant drug abuse, restless leg syndrome, attention deficit hyperactivity disorder, hyperprolactinemia, and Tourrete’s syndrome.5-8

Efforts have been made to modulate the dopamine activity in these ailments through different dopamine receptor agonist and antagonist agents, but adverse drug effects have stymied positive clinical outcome due to lack of selectivity of the drugs. The etiology of neurological disorders has not been clearly resolved, but the study of dopaminergic systems with various dopamine receptor ligands has enabled a greater understanding of the dopaminergic system.9

A new momentum to the exploration in the DA field came from gene-cloning procedures to receptor biology brought into play three decades ago, which discovered more complicated structure of DA receptors than previously thought. 2, 10

1.2 Dopamine receptors

Dopamine receptors belong to the G-protein coupled receptors (GPCRs) family, which are prime targets for >25% of marketed drugs.11 One prominent physiological response of dopamine is to increase the synthesis of cAMP by activating the adenylyl cyclase enzyme.12 Various compounds have also been demonstrated to mimic this effect of dopamine to increase adenylyl cyclase activity.13 On the other hand, there are physiological responses of dopamine not associated with stimulation of adenylyl cyclase or elevation of intracellular cAMP. An example here is dopamine induced inhibition of prolactin release from mammotrophs. The biochemical, physiological and pharmacological evidence have shown the presence of dopamine receptors on the mammotrophs and work as endogenous hypothalamic factor to inhibit prolactin release while there is no evidence found that dopamine mediated inhibition of prolactin release is associated with increased cAMP level.14 Amongst various other examples of studies where dopamine receptors have shown discrepancy in physiological response, one includes a considerable range of potency at DA receptors from micromolar to nanomolar in different tissues by dopamine itself.15,

16 In 1978, Kebanian and Calne concluded this diversity into the classification of two different DA receptor populations, one positively coupled to adenylyl cyclase is the D1 receptor and one independent of this factor is D2 receptor.17

The original classification of DA receptors served as the base for the study of DA receptors for a decade. However, molecular cloning and recombinant DNA techniques revealed the existence of five DA receptor subtypes (D1-D5).18-20 Structural, pharmacological and biochemical studies of the cloned receptors put them into two families based on their high homology in transmembrane domains; D1-like DA receptors (D1, D5) and D2-like DA receptors (D2-D4).2, 3,

21-24

1.2.1 Structures of dopamine receptor subtypes

The structural analysis of the cloned DA receptors revealed that they are members of the seven transmembrane (TM) domain G protein-coupled receptor families and share most of their structural characteristics. In their TM segment, the D1 and D5 receptors share 78% homology, while D3 receptor and D4 receptor have respectively 75% and 53% identity with the parent D2 receptor.21, 25

The NH2-terminal stretch has a similar number of amino acids in all the receptor subtypes while having a variable number of N-glycosylation sites. The D1 and D5 receptors possess two such sites, the D2 receptor has four, the D3 has three, and the D4 possesses only one N- glycosylation site.22, 23, 26 The COOH terminal is about seven times longer for the D1-like receptors than for the D2-like receptors and is rich in serine, threonine, and cysteine residues. In the D1-like receptors, this cysteine residue is located near the beginning of the COOH terminus, whereas in the D2-like receptors it is located in end of the terminus. As in all G protein-coupled receptors,

DA receptors possess two cysteine residues in extracellular loops 2 and 3, which have been suggested to form an intramolecular disulfide bridge to stabilize the receptor structure.27, 28 The

D2-like receptors have a long third intracellular loop, which is common to receptors inhibiting adenylyl cyclase, whereas the D1-like receptors have a short third loop. The D1 and D5 receptor third intracellular loop and the COOH terminus are similar in size but divergent in their sequence.

The external loop between TM4 and TM5 is considerably different in the two receptor subtypes, shorter (27 amino acids) in the D1 receptor than in the D5 receptor (41 amino acids).22, 23, 26 Highly conserved residues are present in the core of the protein and define a binding pocket that is most likely the agonist binding site.29

The D2 receptor exists as two alternatively spliced isoforms D2S and D2L.30, 31 In spite of several efforts, no obvious differences have emerged so far between the two isoforms. Both variants share the same distribution pattern, both isoforms inhibited adenylyl cyclase and revealed the same pharmacological profile.30-32

Splice variants of the D3 receptor have also been identified wherein one transcript carries a 113-bp deletion in TM3 and the second variant derives from a deletion of 54 bp between TM5 and TM6 of the D3 receptor.33, 34

1.2.2 Distribution of brain dopamine receptors

In situ hybridization techniques and immunohistochemical analysis have been employed to study the distribution of DA receptor mRNAs in the brain. The dopaminergic neurons in substantia nigra pars compacta, ventral tegmental area, and hypothalamus give rise to three major dopaminergic pathways in the brain, the nigrostriatal, the mesolimbocortical, and the tuberoinfundibular.

The DA D1 receptors are more widespread than any other receptors. Using in situ hybridization and receptor autoradiography, expression and distribution of dopamine D1 receptor in the brain was examined. The mRNA for D1 receptor is most abundant in cortical, limbic, and hypothalamic brain regions as well as caudate, nucleus accumbens and olfactory tubercule .35 36

This indicates its important role in cognitive, affective and neuroendocrine functions of the brain.37

In contrast, no mRNA has been detected in the substantia nigra pars reticulata even though D1 receptor protein is highly expressed in this region, suggesting that D1 receptors in this area are on terminals of the striatonigral pathway neurons.36, 38

In contrast to DA D1 receptors, DA D5 receptors are poorly expressed in rat brain. mRNAs of this receptor are expressed restrictively to the hippocampus, parafascicular nucleus of thalamus and the lateral mamillary nucleus.39, 40 Further studies have discovered the distribution of DA D5 receptors in various forebrain regions including striatum, cerebral cortex, lateral thalamus, and diagonal band area.41-43 Progress in finding the DA D1 and D5 receptor specific antibodies have led to better insight about cellular and subcellular localization of these receptors.44

In the neostriatum, both DA D1 and D5 receptor antibodies tagged the medium spiny neurons, on the other hand D5 receptor antibodies labeled the large spiny neurons only. In the caudate nucleus, D5 antibodies mostly labeled the shafts, and rarely, the spines of these cells, whereas D1 receptor labeling was more intense in the spines and shafts of projection neurons. 44

Within the same pyramidal neuron cells, D1 receptors are located on dendritic spines and D5 receptors are on dendritic shafts.44, 45 The D1 receptors have been distributed into the pars reticulate of the substantia nigra, but D5 receptors are not detected in this structure. 46 These dissimilarities between D1 and D5 receptors at subcellular localization suggest that even though having analogous pharmacology, functionally they are different.

In the rat brain, the highest concentration of D2 receptor mRNA was found in substantia nigra, nucleus accumbens, olfactory tubercle, neostriatum and ventral tegmental area.47 D2 receptor is also distributed in granule cells of hippocampal formation, in the septal region, in cingulate, prefrontal, enthorinal and temporal cortex.23 In ventral tegmental area, substantia nigra, and hypothalamus regions, D2 receptor is expressed by dopaminergic neurons.47, 48

Immunohistochemical analysis study has suggested that D2 receptors are more concentrated in spine heads and dendrices than in somata in the spiny neurons of striatum.46, 49 The

5 immunoreactivity of D2 receptor with specific monoclonal antibodies is also found in the central nucleus of amygdala and internal plexiform layers.49

In situ hybridization histochemistry revealed the localization of DA D3 mRNAs in the islands of Calleja and in the nucleus accumbens, as well as low level of D3 mRNAs was spotted in putamen and in anterior caudate.50 In the rat brain, D3 receptor mRNAs, mainly detected in the islands of Calleja, hippocampus, nucleus accumbens, and stria terminalis.48 The granule cells of islands of Calleja are enriched with both D3 receptor binding RNAs and mRNAs, while in nucleus accumbens, they are mainly expressed in neurons of the ventromedial shell subdivisions and rostral pole.51

The D4 receptor mRNAs shows a limited expression in the central nervous system. The hypothalamus, olfactory bulb, thalamus and frontal cortex are the main areas where mRNAs for

D4 receptors are significantly expressed.52 Subtype specific antibodies binding study for these receptors revealed the presence of GABAergic interneuron modulator D4 receptors in cerebral cortex and in hippocampus as well as in globus pallidus and reticular nucleus of thalamus. 53

The D5 receptor mRNA is most abundant in hippocampal subfields, in discrete cortical areas

(layers II, IV and VI), and in the dentate gyrus.54 in the rat brain, this receptor mRNAs are found to localized in parafascicular nucleus of the thalamus and in hippocampus.39

1.2.3 Functions of brain dopamine receptors

The importance of DA in the control of movements is well demonstrated in pathological conditions such as Parkinson’s disease. The role of DA receptors in motor functions, such as forward locomotion, grooming, catalepsy, rearing, and sniffing has been studied.55 D1, D2 and D3 receptors in ventral striatum are primarily involved in forward locomotion. Decreased locomotor

6 activity is associated with decreased DA release triggered by activation of D2 autoreceptors.23

Individual D1 receptor activation has barely or no effect on producing locomotor activity.56

However, pharmacological studies have shown that the simultaneous stimulation of D1 receptor is crucial to produce maximal locomotor effect by D2 agonists, which indicates forward locomotion involves synergistic interaction between D1 and D2 receptors.57, 58 Targeted gene inactivation of D1 receptor in the mouse has confirmed these observations as well.59

The D3 receptors present in nucleus accumbens have an inhibitory role on locomotor activity. In fact, supported by physiological actions of selective agents, DA D3 agonists inhibit while, DA D3 antagonists induce the locomotion.24, 60 The opposite roles of DA D2 and D3 receptors on neurotensin gene expression in nucleus accumbens may have some correlation with their differing effect on locomotion.61

Although some biases are present in literature, the mesolimbocortical DA is considered as an important neurological system responsible for cognition and memory. In the monkey, DA neurons in the area A10 have been reported to be involved in the cognitive behavior and learning process via transient changes in impulsive activity.62 Dopaminergic neurons in the prefrontal cortex are known to play an important role in working memory. The activation of DA D1 and D2 receptors in the hippocampus by post training administration of agonists has improved the working memory tasks in animal models, suggesting important role of hippocampus DA receptors in the retention of working memory.63, 64 The stimulation of DA D1 and D2 receptors in prefrontal cortex has been shown to enhance performance in working memory in the monkey.37, 65

Increased DA transmission in mesolimbic areas after administration of drugs of abuse and psychostimulants and decreased level of DA after withdrawal of these drugs implies that mesolimbic DA is primarily involved in reward and reinforcement mechanisms.66-69 Amongst

7 various models developed for the study of drug abuse and dopamine release, drug self administration and intracranial self stimulation are commonly used in this area. In the case of drug self administration, both DA D1 and D2 receptors are associated with reinforcing effect of drugs, with D1 receptor playing an acquiescent role while D2 receptor mediates drug reinforcement.70, 71

D1 and D2 receptors are mainly involved in this behavior as agonists and antagonists increase and decrease self administration of drug of abuse respectively.72, 73 Previous studies suggest that D1 receptors in the nucleus accumbens are important for the reinforcing properties of cocaine.71 The opposite role of DA D1 receptor and DA D2 receptor was observed while studying the effects of agonists on cocaine seeking behavior. D1 agonists inhibited the cocaine seeking behavior, while

D2 receptor agonists promoted this behavior.74 D3 receptors are also involved in modulating reward and reinforcement. Recently, D3 receptor partial agonists and antagonists have shown to inhibit cocaine self administration in animal models for cocaine addiction study.75, 76 Several additional behaviors, including yawning, hypothermia, ejaculatory behavior, alcohol consumption, and inhibition of sniffing are associated with nucleus accumbens D3 receptor.77

The role of D4 and D5 receptors in the physiology of dopaminergic system is still mostly unknown. Studies have found pharmacological evidences to establish possible links between mood disorders and DA D4 gene polymorphism, however the findings are not consistent and need to be reevaluated.78-80 Because of a lack of selective agents that can differentiate the D1 receptor from

D5 receptor, scarce data is available to decide the physiological role of D5 receptors in the CNS.

Currently, gene knockout techniques are being used to study the involvement of D5 receptors in attention deficit hyperactivity syndrome (ADHD) and in the locomotor stimulant effect of cocaine.81, 82 Very recently, it has been revealed that DA D5 receptor is linked to the activation of brain-derived neurotrophic factor (BDNF) and protein kinase B signaling which indicates a

8 possible association with mood stabilizers and antidepressant effect.83 Further research into the role of DA D5 receptor in prefrontal cortex as a therapeutic target in neurological disorders is required, with the development of selective DA D5 receptor therapeutic agents.

1.2.4 Regulation of dopaminergic system in schizophrenia and drug abuse

1.2.4.1 Role of dopamine receptors in schizophrenia

Schizophrenia is a psychological disorder characterized by disturbed thought process, poor emotional flow, auditory hallucinations, delusion and disorganized speech and thinking.84, 85 It is a heterogeneous mental disorder, prevailing in about 0.85% of the population worldwide.86

Schizophrenia syndromes can be classified into positive symptoms and negative symptoms.

Positive symptoms reflect excessive expression of normal functions, exemplified by hallucination, bizarre behavior and delusions. Loss of normal functions or decreased intensity of normal functions is associated with negative symptoms, characterized by defect in attention, social withdrawal and cognitive impairment, lack of interest, flattened affect and poverty of speech.87, 88

The initial hypothesis of schizophrenia was based on the consideration that psychological outcomes in this ailment are due to a hyperdopaminergic state of the CNS.89 During the 1970s, a direct relation between the therapeutic effectiveness of neuroleptics and their affinity for DA receptors, further solidified this hypothesis.90, 91 At that time, the spotlight was on hyperdopaminergia and halting the transmission at dopaminergic neurons to treat schizophrenia.

The study of various DA agonists and antagonists on schizophrenia provided indirect evidences to this hypothesis. However, inconsistent results obtained during the treatment as well as in different studies have turned this hypothesis inaccurate. Resistance in the treatment, no induction of schizophrenia even after augmentation of dopaminergic system in nonschizophrenic individual

9 and partial effectiveness of neuroleptics in alleviating negative symptoms are some examples to support this fact. In 1991, Davis et al revised this concept, calling it “a modified dopamine hypothesis of schizophrenia”, wherein they explained multiple facets of the hypothesis based on a subcortical hyperdopaminergia and prefrontal specific hypodopaminergia.92 Elevated levels of DA and its metabolite, homovanillic acid was observed in the striatum after lesions in prefrontal cortex, while decreased DA metabolite level in striatum by presence of DA agonists in prefrontal areas has provided more precise definition of schizophrenia i.e. prefrontal hypodopaminergia resulting in striatal hyperdopaminergia.93, 94

Further evidence in schizophrenia in this line comes from PET imaging of DA receptors using different radiotracers. Numerous studies have confirmed the elevated DA D2 and D3 receptor density in striatum because of intrinsic abnormalities in schizophrenia but not due to rebound effects of antipsychotics.95-98

Figure 1.1: Mesolimbic dopamine hypothesis for positive and negative symptoms of th schizophrenia (Source: Stahl’s Essential Psychopharmacology, 4 Edition)

DA D1 receptors are rich in prefrontal cortex and mediate major dopaminergic transmission in this region, and D1 hypofunction is found to be connected to negative symptoms

10 and cognitive impairment in schizophrenia.99, 100 That concludes, impaired activity of dopamine

D1 receptors in the medial prefrontal cortex is an important factor in producing the negative symptoms and cognitive disorders of schizophrenia, whereas D2 receptor hyperactivity might lead to the positive symptoms of the disorder (Schematic diagram shown in Figure 1.1).

1.2.4.2 Abnormal dopamine transmission in drug abuse

Psychostimulant drug abuse is understood as a special case of operant attitude bolstered by the positive reinforcing properties of the abused drugs.101-103 Among the neural pathways that contribute in incentive the ones that operate dopamine emerge as that most diffusely involved in the action of drugs of abuse. As examined by brain microdialysis in rats, most drugs of abuse of various pharmacological classes increase extracellular DA in different areas of the brain, preferentially in nucleus accumbens and ventral tegmental area.104 This is explained in cocaine

(1.1) like, amphetamine (1.2) like, nicotine (1.3), narcotic analgesics and phencyclidine (1.4) drug abuse and their reward enhancing properties.105-110

Figure 1.3: Schematic representation of mechanism of action of a) cocaine b) methamphetamine and c) nicotine (Source: NIH Curriculum Supplement Series, The Brain: Understanding Neurobiology through the Study of Addiction)

Different drugs of abuse have varied mechanism of action by which they stimulate DA transmission directly or indirectly (shown in Figure 1.3). Amphetamine operates through carrier mediated non-exocytotic release of newly synthesized DA, while phencyclidine increase synaptic

DA concentration by blocking the reuptake process.111 Nicotine activates the firing activity of DA units by directly stimulating excitatory nicotine receptors localized on the somato-dendritic region of DA neurons.112 Hyperpolarization of the interneurons by narcotic analgesic acting on opioid receptors attenuate the GABA mediated synaptic input to the DA cells, which lead to excitation of

12 the dopamine cells by disinhibition.113 Cocaine binds to the presynaptic DA transporters and inhibits its reuptake mechanism.114

1.3 Dopamine receptor ligands

The development of selective DA receptor agents has provided necessary tools to assess the role of these receptors in the nervous system. Over the several decades, DA receptor specific ligands have been exploited as the means for studying the prognosis and curing several CNS disorders. Agents possessing selectivity at one type of receptor may demonstrate distinct type of clinical actions in the neurological disorders. Though some sort of selectivity is achieved between main classes of receptors i.e. D1 versus D2 types, complete selectivity within the DA receptor subtypes remains a challenge.

1.3.1 Clinical significance of dopamine receptor ligands

1.3.1.1 Therapeutic significance of D1 receptor selective ligands

The insufficient D1 receptor signaling in prefrontal region results in cognitive deficits in psychosis. Low doses of selective D1 receptor agonists, such as dihydrexidine (1.5), A77636 (1.6) and SKF81297 (1.7), have been reported to have cognitive-enhancing actions in nonhuman primates.115-117 The finding that full D1 receptor agonists can elevate working memory suggests that such type of agents can be a newer approach to treat negative and cognitive symptoms of schizophrenia.118, 119 Animal laboratory data indicate that D1 receptor agonists may have possible applicability for the treatment of cocaine abuse. D1 agonists, such as, SKF81297 (1.7) and dihydrexidine (1.5) have been shown to decrease cocaine craving in humans and motivation to seek cocaine, and masks the reinforcing effects of cocaine during self-administration.120, 121 These

D1 agonists could serve as “methadone like” compound for their capacity to therapeutically

13 substitute psychomotor stimulant drugs.122 However, considering the addiction potential, development of tolerance and severe adverse effects limits their therapeutic liability as antipsychostimulant drug.

The alternate approach along with this line is of using partial D1 agonist agents to avert the addiction potential of psychomotor stimulants. D1 partial agonists would be expected to act primarily as antagonists in the presence of cocaine, whereas in the absence of cocaine they would be expected to function as weak agonists.123 Various pharmacological studies have exhibited the antidrug abuse potential of D1 partial agonist agents like SKF 83959 (1.8), SKF 77434 (1.9) and

SKF 38393 (1.10) (shown in Figure 1.4b). 123-125

Based on preclinical studies, D1 antagonists are not found much effective as antipsychotics, but they are developed for the treatment of self-harming behavioral disturbances arise in Lesch−Nyhan Disease, as well as for the treatment of Tourette syndrome in children and for obesity.126-128

1.3.1.2 Therapeutic significance of D2 receptor selective ligands

Since the discovery of chlorpromazine (1.11), D2 antagonism is considered a fundamental necessity to positively modulate psychosis.129, 130 Since then, without exception, currently available antipsychotics all have some degree of D2 receptor antagonist property.131, 132 As shown in Figure 1.5, other examples of antipsychotics having D2 antagonist effect include fluphenazine

(1.12), haloperidol (1.13), thiothixene (1.14), risperidone (1.15), clozapine (1.16), and quetiapine

(1.17).

Brain imaging studies including positron emission tomography (PET) indicate that the blocking of at least 65% of D2 receptors is necessary to achieve antipsychotic effects, at the same time, passing the threshold of 72%-78% blockade of D2 receptors, increases the risk of extrapyramidal motor symptoms (EPS) and hyperprolactinemia.130, 133

A second approach to treat schizophrenia is to prevent the excessive D2 receptor activity using D2 partial agonists. D2 receptor antagonists block the action of endogenous DA, while partial agonists like aripiprazole (1.18), diminish the hyperactivity of the receptor by competing with endogenous DA to produce lower degree of action at DA receptors.134 D2 agonist compounds

(shown in Figure 1.6) like bromocriptine (1.19), cabergoline (1.20), and quinagolide (1.21) are being used for the management of hyperprolactinaemic disorders and combination therapy with levodopa (1.21) for Parkinson’s disease.135, 136

The enhanced reinforcing effect and self administration of cocaine was observed after the administration of D2 agonist as well as, clinical and pharmacological studies indicate that D2 agonist agents are not effective against cocaine addiction because of the abuse potential in humans.74, 137, 138

1.3.1.3 Therapeutic significance of D3 receptor selective ligands

Cocaine and methamphetamine overdose fatalities have been associated with upregulation of D3 receptors in the ventral striatum and other D3 receptor rich regions in brain.139, 140 D3 receptor antagonists SB277011A (1.23), have shown promise in reversing acquisition and expression of drug-seeking and cue-induced relapse in animals.141, 142 Rewarding effects of cocaine and cocaine-induced reinstatement of drug-seeking behavior was shown to be inhibited by D3 receptor antagonist NGB 2904 (1.24).143 As shown in Figure 1.7, other examples of D3 receptor antagonists include SR21502 (1.25), PG01037 (1.26), and YQA14 (1.27) that have demonstrated to inhibit cocaine seeking behavior and cocaine self administration in animal models.144-146

Moreover, it has been demonstrated that the D3 selective partial agonist BP 897 (1.28) inhibits cocaine seeking behavior caused by the presentation of drug-associated cues.76

Elevated expression of D3 receptor was observed in a postmortem study of untreated schizophrenic patients, whereas D3 receptor level was normal in subjects having antipsychotic drugs treatment.147 There is increasingly strong evidence that D3 receptor antagonists will be effective antipsychotic agents. In this regard, refinement of the cognitive and negative symptoms

17 of schizophrenia holds the most promise for D3 receptor antagonists.148 Studies have shown that the D3 receptor antagonism can inhibit extrapyramidal symptoms and can positively affect the social behavior in schizophrenia.149

1.3.1.4 Therapeutic significance of D4 receptor selective ligands

Considerable work has been progressed to find selective D4 receptor binding compounds based on the hypothesis that the D4 receptors are likely involved in a psychosis, ADHD and erectile dysfunction. There are many studies suggesting that the selective D4 receptor antagonists might be a novel strategy towards schizophrenia.150, 151 Various D4 receptor selective antagonists

(Figure 1.8) have reached clinical trials, for instance, sonepiprazole (1.29), NGD-94-1 (1.30), and

L-745, 870 (1.31) but none of them have successfully demonstrated antipsychotic activity in schizophrenic patients.152-154 The effect of stimulating the D4 receptor using selective agonist agents in erectile dysfunction is under investigation.155

1.3.1.5 Therapeutic significance of D5 receptor selective ligands

A few studies have implicated the D5 receptor in the pathophysiology of hypertension and drug abuse, making it of pharmacological interest.156-158 It was shown in D5 knockout mice that the D5 receptor seems to be involved in the locomotor-stimulant effects of cocaine, whereas there is only little involvement in the discriminative stimulus effects of cocaine suggesting therapeutic relevance of D5 receptor in cocaine addiction.81 However, discrimination between D1 and D5 receptors remains largely unexplored due to lack of highly selective ligands and of specific methods of biological characterization. There are some examples of selective D5 receptor antagonists (compounds 1.32 and 1.33 shown in Figure 1.9) under investigation for their anti- addiction potential against cocaine.159

Potential clinical importance of selective dopamine receptor ligands for different CNS disorders is summarized in Table 1.1.

Table 1.1: Potential therapeutic applications of dopamine receptor ligands

DA receptor ligand Therapeutic significance D1 receptor agonist - Parkinson’s disease - Cognitive and working memory deficits in schizophrenia - Psychostimulant drug abuse partial agonist - Psychostimulant drug abuse antagonist - Tourette syndrome - self-harming behavioral disturbances in Lesch−Nyhan Disease D2 receptor agonist - Hyperprolactinaemic disorders - Parkinson’s disease - Prolactinoma - Restless legs syndrome partial agonist - Parkinson’s disease - Restless legs syndrome - Schizophrenia - Bipolar disorders - Depression antagonist - Schizophrenia D3 receptor agonist - Hyperprolactinaemic disorders - Parkinson’s disease partial agonist - Psychostimulant drug abuse antagonist - Psychostimulant drug abuse - Schizophrenia D4 receptor agonist - Cognitive disorders - Erectile dysfunction antagonist - Schizophrenia - ADHD D5 receptor agonist - Antagonist - Psychostimulant drug abuse

1.3.2 Selective D1 receptor ligands

1.3.2.1 Arylbenzazepines

1.3.2.1.1 Simple arylbenzazepines

Initial findings of D1 antagonists like SCH-23390 (1.34), followed by a number of selective

D1 receptor ligands were developed in 1980s.160 All of these D1 receptor ligands (1.7, 1.8, 1.10, and 1.34-1.36) share a common template of phenyl tetrahydrobenzazepine (shown in Figure 1.10).

Many useful D1 receptor selective compounds having agonist or antagonist activity with high D1 affinity, for example SCH and SKF series (Figure 1.10), were acquired from this class. More diverse D1 receptor ligands were developed to enhance binding affinity and selectivity at D1 receptor as well as to improve intrinsic efficacy and metabolic stability. In these compounds, β- catechol-substituted ethylamine moiety, which resembles the catecholamine neurotransmitter DA, is associated with D1 receptor affinity. The chiral center at C1 position should be in the R configuration to be active at DA receptors.161 Variant substitutions on C1 phenyl ring, C6 or N3 can modify the binding affinity and selectivity between D1 and D2 receptors. Usually, catechol- benzazepines are D1 agonists, while C7 substituent can govern the functional activity from agonism to antagonism predominantly. As demonstrated by benzazepine analogues 1.34 and 1.35, replacing the catecholic 7-OH group with halogen produces D1 antagonist activity.160, 162 SKF

83959 (1.8) is one of the primary D1 receptor agonist with high binding affinity and selectivity.

To overcome the limitations of having low intrinsic activity and poor metabolic stability, various analogues of SKF 83959 (compounds 1.37-1.44) were synthesized and studied for their affinity at

163, 164 DA receptors. These SAR studies have revealed that 3’ or 2’ CH3 substitution on 1-phenyl ring and H, CH3 or allyl N-substituents as well as C6 halogens are important for the D1 receptor activity.163 Most of these

Table 1.2: Structure modification and binding affinities of catechol containing arylbenzazepines at DA D1 and D2 receptors

Ki (nM) Comp. Isomer R1 R2 R3 D1 D2 (±)-1.8 RS-(±) Cl CH3 3’-CH3 1.18 920 (+)-1.8 R-(+) Cl CH3 3’-CH3 0.49 515 1.37 R-(+) Br CH2CH=CH2 H 2.29 2.09 1.38 RS-(±) Cl CH3 2’-CH3 0.46 226 1.39 RS-(±) Cl H 3’-CH3 0.60 ≥5000 1.40 RS-(±) Br CH3 3’-CH3 0.19 440 1.41 RS-(±) Br CH3 2’-CH3 1.10 409 1.42 RS-(±) Br H 2’-CH3 1.81 19.5 1.43 RS-(±) Cl CH2CH=CH2 3’-CH3 0.52 119 1.44 RS-(±) Br CH2CH=CH2 3’-CH3 0.11 83.8 1.45 RS-(±) 3-toluyl H H 39 >10000 1.46 RS-(±) 2-naphthyl H H 56 >10000 1.47 RS-(±) 3-toluyl H 3’-CH3 6.81 >10000 1.48 RS-(±) 2-naphthyl H 3’-CH3 4.88 >10000

agents exhibited higher D1 receptor selectivity over D2 receptor with subnanomolar binding affinity (Table 1.2). For the same molecule (1.8), another series of analogues (1.45-1.48, Table

1.2) having an aryl group at 6 position, has been synthesized. The tolerance of larger aryl group at this position indicate a large hydrophobic pocket on D1 receptor around the C6 position of phenylbenzazepine.164 In the parent molecule SCH 23390 (1.34), various substituents were introduced at para position of pendant phenyl ring to generate novel D1 antagonists (1.49a-1.49f,

Table 1.3).165 Most of these analogues have shown higher preference for D1 over D2 receptors with subnanomolar (0.5-0.9 nM) binding affinity (Table 1.3).

Table 1.3: Structure modification and binding affinities of arylbenzazepines at DA D1 and D2 receptors

Ki (nM) Comp. Isomer R1 R2 D1 D2 1.49a R-(+) H n-Bu 0.9 2000 1.49b R-(+) Ac n-Bu 0.2 159 1.49c R-(+) SO2CH3 n-Bu 0.5 2000 1.49d R-(+) SO2CH3 CH3 0.5 6000 1.49e R-(+) SO2Ph CH3 0.6 331 1.49f R-(+) Bn 2,4-F2C6H3 0.7 552

1.3.2.1.2 Conformationally constrained arylbenzazepines

Arylbenzazepines retain a noticeable mobility, and the proposed position of the aryl moiety in the binding pocket of D1 receptor is postulated to be equatorial.166, 167 One of the binding intercations of phenylbenzazepines with the D1 receptor was believed to be π-π interaction between C1 aryl group and Tyr, Phe or Trp terminals in the receptor active site.168 In the parent

23 compound SCH23390 (1.34), introducing an ethylene bridge between C2 and C2’ results in conformationally restrained benzazepine compound 1.50 (Figure 1.11).166 Binding study at D1 receptor reveals that (R)-trans isomer has the highest affinity (Ki 1.9 nM) amongst all four isomers, supporting the fact that the pharmacologically active arylbenzazepine possesses R- configuration at C1 chiral center.166 Compound 1.50a (SCH 39166), also known as ecopipam, was studied extensively for the treatment of obesity, but it faces limitations of poor oral bioavailability.127

To further explore this scaffold, novel D1 antagonist analogues (1.51a-1.51j, 1.52a-1.52c, Table

1.4) having variant ring D substitutions at 3’ and 4’ positions in SCH 39166 were synthesized and their activity at D1 receptor was studied.165, 169 These compounds have shown nanomolar to subnanomolar D1 affinity (0.2-3.1 nM, Table 1.4) with better selectivity at D1 versus D2 receptor and improved pharmacokinetic properties as compared to parent compound 1.50a. Highest binding affinity was demonstrated by 4’ phenyl analogue 1.51g (0.2 nM) while compound 1.51c has shown

>3000-fold selectivity at D1 (3.1 nM) versus D2 receptor (>10000 nM). 3’-sulfonyl derivative

1.52b, showed to improved pharmacokinetics and oral bioavailability (Foral = 29% for 1.52b versus

0.6% for 1.50a).165

Table 1.4: Structure modification and binding affinities of conformationally constrained arylbenzazepines at DA D1 and D2 receptors

Ki (nM) Comp. R D1 D2 1.50a H 1.2 980 1.51a CH2OH 2.0 1647 1.51b OH 0.6 440 1.51c 2-oxopurrolidine-1-yl 3.1 >10000 1.51d NH2 2.8 2100 1.51e NHCONHEt 2.4 900 1.51f NHCO2Et 2.3 500 1.51g Ph 0.2 79 1.51h 4-OMePh 0.7 550 1.51i Thien-2-yl 0.9 93 1.51j 1H-indol-5-yl 0.6 51 1.52a H 7.0 5400 1.52b SO2Me 0.5 3000 1.52c CONH(2,6-Cl2Ph) 0.7 1093

1.3.2.1.3 Bioisosteres of benzazepines

The metabolic susceptibility of arylbenzazepines arises because of O-glucuronidation of the catechol hydroxyl group and N-dealkylation of N-methyl functionality. The main approach to tackle this drawback is to replace catechol with its bioisosteres, which can mimic the hydrogen bond complement of phenol at the receptor binding site.170 Wu et al. developed heterocycle-fused phenol isostere compounds (Figure 1.12) as DA D1 receptor ligands having triazole (1.53, 1.54), indole (1.55), imidazole (1.56), imidazolone (1.57a, 1.57b) and thiazolone (1.58a, 1.58b).171

Considerably poor binding affinity (Table 1.5) was displayed by the benzotriazole (1.53, 1.54) and

25 benzimidazole (1.56) analogues with Ki values of 583, 146, and 248 nM respectively. Remarkable higher binding affinity (Table 1.5 Ki = 2.1 to 16.5 nM) shown by benzimidazolone and benzthiozolone compounds (1.57a-b, 1.58a-b) signifies the importance of orientation and acidity of the NH moiety in these heterocycles. Based on this observation they conclude that, suitably aligned heterocycle with an NH group would be able to serve as surrogates for the phenolic OH group. As expected, these D1 receptor antagonists exhibited better pharmacokinetic profiles as compared to parent benzazepines.

Table 1.5: Binding affinities of bioisosteres of arylbenzazepines at DA D1 and D2 receptors

Ki (nM) Comp. D1 D2 1.53 583 3000 1.54 146 1530 1.55 24.7 232 1.56 248 984 1.57a 7 1023 1.57b 16.5 3270 1.58a 2.1 257 1.58b 6.5 661

1.3.2.2 Dihydrexidine derivatives

Compound 1.59 represents a class of 4-phenyl substituted tetrahydroisoquinolines which has moderate, but selective DA D1 receptor affinity.172, 173 As shown in Figure 1.13, introduction of ethylene-bridge has rigidified this scaffold to give novel D1 receptor ligands 1.5, 1.5a, and

1.5b.174 The trans configured compound 1.5, also known as dihydrexidine, is a highly selective and potent D1 receptor ligand. More significantly, compound 1.5 is a full DA D1 agonist with

EC50 of 70 nM to activate adenylate cyclase. The cis isomer (1.60) of 1.5 is found inactive at DA receptors. The N-Me (1.5a) and N-Pr (1.1.5b) analogues demonstrated much lower binding affinity of 91 nM and 651 nM, respectively, with the reduction of intrinsic activity at the same time. Switching the substitution from 10, 11 dihydroxy in ring D of 1.60 to 9, 10 dihydroxy

(compound 1.61) results in vanished D1 receptor activity.

Applying the original idea of rigidifying compound 1.59 to develop dihydrexidine, similar

D1 ligands (1.62a-1.62d) were designed wherein, two phenyl rings were connected through methylene-bridge and ethylene-bridge was removed in dihydrexidine. Binding affinity of compound 1.62a (also known as dinapsoline) was found to be 5.9 nM, comparable to 1.5, and it displayed D1 full agonistic profile.175 The C6-methyl (1.62b) and C6-ethyl (1.62c) compounds have nearly equal affinity to the D1 receptor with Ki of 11 and 14 nM, respectively, while C6- fluoro analogue (1.62d) has biological activity consistent with dinapsoline. 176

Replacing methylene bridge with ether linkage to tether phenyl rings in dinapsoline produced compound 1.63a, also known as dinoxyline.177 This novel ligand was found to possess remarkable affinity (3.9 nm) for D1 receptor and greater selectivity (22-fold) for D1 versus D2 receptor. It also retained full agonistic profile (EC50 87 nM) at D1 receptor similar to dinapsoline.

It has shown high binding affinity at D5 receptor with Ki value of 1.0 nM as well as at D3 receptor

(Ki 6.5 nm). Reduction in affinity was observed after N-alkyl insertion (compound 1.63b and

1.63c) in the dinoxyline template. Compound 1.63a serves as the key example of ligands with improved affinity for all DA receptors and also endowed with the functional aspects akin to dopamine itself. Dinoxyline is under investigation for the treatment of Parkinson’s disease.178

D1 agonists have been investigated as potential treatments for Parkinson’s disease and hypertension, and D1 antagonists have been tested in the treatment of psychotic disorders, including schizophrenia, though both efforts have yielded only limited success. Fenoldopam

(Figure 1.13, 1.64), a benzazepine analog, remains the only D1-selective compound to reach the pharmaceutical market in the US for short-term management of severe hypertension.179

1.3.3 Selective D2 receptor ligands

1.3.3.1 Dihydroindenes and oxime analogues

To develop metabolically stable DA receptor ligands, trans-2-amino, 1-phenyl-2,3- dihydroindene derivatives were designed. Compounds 1.65-1.67 (Figure 1.14) belong to this class of DA receptor ligands, having C2-amino group and C1-phenyl moiety in trans configuration to attain the receptor activity. Amino group substitution affects the activity at D2 receptors, as demonstrated by N,N di-alkyl substituted indenes having greater affinity and selectivity at D2 receptors. Indene analogues 1.65, 1.66, and 1.67 have D2 affinity 650, 270, and 170 nM respectively and they possess D2 agonist effect.180, 181

Aminotetralin compound 1.68 and its derivatives are known mixed DA D1/D2 agonists explored for the potential utility in the treatment of Parkinson’s disease. 182-184 To combat against the metabolic instability, series of oxime analogues (1.69-1.71, Figure 1.14) were developed as prodrug of 60.185 These compounds exhibited apparent and extended dopaminergic effects in

Parkinson’s disease behavioral model. Novel compounds 1.72 and 1.73 developed as DA agonists, since the thienylethylamine moiety can act as bioisosteres of DA.186 Though these compounds have moderate DA D2 receptor affinity (Ki 27 nM for 1.72 and Ki 20 nM for 1.73), they have improved oral bioavailability. The benzoquinoline compound 1.74, is a potent DA D1 and D2 receptor agonist with EC50 value of 25 nM (D1 receptor) and 0.64 nM (D2 receptor). The enone compound 1.75 is the prodrug of 1.74, which is an interesting lead compound for potential development of anti-Parkinson agents.187, 188

1.3.3.2 Aporphine analogues

Based on the fact that 1,2,3,4-tetrahydroisoquinoline compounds maintain the integrity of the phenethylamine portion of dopamine, several naturally occurring as well as synthetic tetrahydroisoquinoline derivatives have been screened for dopamine receptor activity. 1-benzyl-

1,2,3,4-tetrahydroisoquinoline alkaloids, for example, nor-roefractine (1.76, Figure 1.15) and nor armepavine (1.77, Figure 1.15) have affinity for both DA D1 and D2 receptors.189, 190 Among the tetrahydroisoquinolines and congeners, the conformationally rigid aporphines have been extensively studied with regard to their dopamine receptor activity.191 Apomorphine and some of its derivatives were among the earliest pharmacological tools used to characterize DA receptors.192

Clinically, R-(-)-apomorphine (1.78) is being used as anti-Parkinson drug and for the treatment of male erectile dysfunction as D1 and D2 agonist.

To resolve the drawback of poor bioavailability and short duration of action, diverse synthetic analogues (1.80-1.91, Figure 1.15) were developed and studied at DA receptors.193-195

The optimum activity at D2 receptor was found with n-propyl alkyl chain on tertiary nitrogen of

30 aporphines, for example (R)-(-)-N-n-propylnorapomorphine (NPA, 1.79 Ki = 4.80 nM). For DA receptor activity, apart from the structural requirements of tertiary nitrogen and 6a-R configuration,

C-2 substitution is also considered a significant factor in aporphines. Differences in affinity (Table

1.6) was observed between alkyloxy and 4-hydroxyphenyl substitutions at C-2 position. When compared to the parent compound APO (1.78), C2 alkoxy analogues (1.80, 1.81) were 4 to 8-fold less potent, while, hydrophilic substituent 4-hydroxyophenyl analogues (1.82, 1.83) were 5-fold more potent.196, 197 Insertion of alkylthio moiety at C2 position in aporphine scaffold yielded novel compounds 1.84-1.86 (Table 1.6). Methylthio analogue (1.84) was found to have improved potency (Ki 3.73 nM), while ethyl and propyl analogues have shown reduced affinity (Ki 7.8 and

198 15.6 nM respectively) compared to NPA (1.79 Ki 4.80 nM) at DA D2 receptors.

Table 1.6: Binding affinities of aporphine analogues at DA D1 and D2 receptors

Ki (nM) Comp. D1 D2 1.78 214 13.2 1.79 733 4.8 1.80 2900 44 1.81 3500 75 1.82 124 1.5 1.83 94 2.0 1.84 - 3.73 1.85 - 7.8 1.86 - 15.6 1.87 >10000 >10000 1.88 >10000 >10000 1.89 >10000 97 1.90 130 28.0 1.91 1690 44.0

To improve the in vivo metabolic stability profile of aporphines, exclusion of one or both catecholic hydroxyl group or replacing them with bioisosteres was envisaged. Switching the catechol component with its aminothiazole bioisosteres, results in complete loss of activity at D2 receptor (1.87, 1.88), while mono hydroxyl analogues (1.89-1.91) have shown to reduce the potency 3 to 8-fold as corresponds to their lead compounds 1.78 or 1.79.199

1.3.3.3 Arylpiperazine/piperidine analogues

Arylpiperazines or arylpiperidines belong to one of the largest groups of chemical structure with dopaminergic activities. Most of the recent widely used antipsychotics which have D2 antagonistic activity (for example haloperidol, risperidone, olanzapine and clozapine) fall into this category. Current attempts to develop D2 ligands as superior antipsychotics with limited EPS, have targeted agents bearing moderate D2 antagonism or D2 partial agonistic effects, combined with interactions at other DA receptors as well as serotonin receptors.

Various analogues (1.92-1.104, Figure 1.16) were generated by modification of haloperidol

(1.13) scaffold. The nitrogen/silicon exchange at the C-4 position of the piperidinyl ring, has produced sila-haloperidol (1.92) and sila-trifluperidol (1.93). Switching the chloro group of haloperidol with trifluoro moiety produces novel the compound 1.94. Binding affinity at D2 receptor for compound 1.92 and 1.93 is comparable to haloperidol, while compound 1.94 is 15- fold less potent than haloperidol (Table 1.7).200 The role of hydroxyl group and other features in haloperidol were assessed through probing the D2 receptor activity of analogues 1.95-1.98.201 In this series, conformationally constrained tropane agents 1.95 and 1.96 have shown high D2 affinity

(Ki = 0.31nM and 2.3 nM, respectively) while having low selectivity between D2 and D3 receptors.

Removal of hydroxyl group has substantial effect on DA receptor activity as demonstrated by analogues 1.97 and 1.98 which have reduced affinities for all DA receptors as shown in Table 1.7.

Another library of analogues (1.99-1.104, Figure 1.16) was generated by replacing the butyrophenone portion of haloperidol with 3-methylindole and 3-methylbenzofuran

33 heterocycles. Compounds 1.99 and 1.100 bind at D2 receptors with high affinity (Ki = 2.3 and 5.5 nM) with more than 50-fold (1.99) and 100-fold (1.100) D2/D3 selectivity, and were found to be

D2 antagonists.202, 203 Compounds 1.101-1.104 possess binding affinities ranging from 6.9 to 23.9 nM and 10- to 28-fold selectivity for D2 versus D3 receptors.203

Table 1.7: Binding affinities of haloperidol analogues at DA D2, D3 and D4 receptors

Ki (nM) Comp. D2 D3 D4 1.13, haloperidol 1.1 5.5 12.7 1.92 3.0 - - 1.93 15.0 - - 1.94 1.5 - - 1.95 0.31 0.81 12.1 1.96 2.3 3.2 19.1 1.97 16.3 46.0 25.9 1.98 254 403 17.5 1.99 2.3 190 840 1.100 5.5 580 567 1.101 10 104 449 1.102 23.9 638 319 1.103 6.9 111 1100 1.104 21.0 28.0 167

Because the “pure D2 antagonism” engenders extrapyramidal side effects, researchers tried to bring about molecules having weaker D2 antagonist profile mixed with other receptor activity,

204 for instance 5-HT2A, to avoid the risk of EPS. Some compounds with phenylpiperazine connected to benzotriazole have exhibited antipsychotic effects, for example, analogue 1.105 have low D2 receptor affinity (Ki = 52 nM) and high 5-HT2A receptor affinity (Ki = 5.1 nM). Other examples include, compounds 1.106-1.109 (Figure 1.17) which having benzoisoxazole and

34 benzoisothiazole attached to piperazine ring have similar profile as atypical antipsychotics i.e.

205 moderate D2 and high 5-HT2A affinity (Table 1.8).

Table 1.8: Binding affinities of arylpiperazine analogues at DA D2 and 5-HT2A receptors

Ki (nM) Comp. D2 5-HT2A 1.105 51 5.1 1.106 25.2 0.73 1.107 27.7 0.12 1.108 15 0.17 1.109 4.0 1.07 1.18 8.0 40 1.110 2.6 7.4 1.111 5.0 16 1.112 0.14 0.81

Enlarging the piperazine ring in aripiprazole (1.18) provided novel analogues 1.110 and

1.111. With improved affinity for D2 receptor (Ki = 2.6 and 5.0 nM) and moderate affinity for

35 other DA as well as serotonin receptors (Table 1.8), these compounds possess potent antipsychotic activity.206 Structurally distinct from traditional antipsychotics, novel compound 1.112

(blonanserin) has a dual D2/5HT2A receptor activity (Ki = 0.14 and 0.81 nM), but has similar risk of EPS as haloperidol.207

1.3.3.4 Dibenzazepine analogues

Dibenzazepines are another class, different from arylpiperazines or piperidines, found in several antipsychotics including clozapine (1.16), olanzapine (1.113), and sertindole (1.114).

Considerable amount efforts have been made to enhance the antipsychotic activity of this class by optimizing the structural requirements of dibenzazepine core.208

Clozapine analogues (Figure 1.18) 1.115, 1.116, 1.17 (quetiapine), and 1.117 were developed as D2 antagonists for the treatment of schizophrenia. Most of these compounds except

1.117, exert EPS and prolactin elevating effects.209 A novel series of pyrrolobenzazepines and

36 pyrrolobenzothiazepine analogues (1.118a-1.118d, 1.119a-1.119c) have been developed.210 All these analogues demonstrated moderate to low affinity (Ki = 3.1 nM to 79 nM) at DA D2 receptors.

1.3.4 Selective D3 receptor ligands

1.3.4.1 2-Aminotetralin derived D3 ligands

The concept of rigidifying the free aminoethyl side chain of dopamine led to development of 2-aminotetralin class of DA ligands. As shown in Figure 1.19, α- and β-rotamers (1.120a,

1.120b) of dopamine were advanced to conformationally restrained 5-hydroxy and 7-hydroxy analogues 1.123 and 1.124. 2-aminotetralin derivative 7-OH-DPAT (1.122) was recognized as selective D3 agonist and suitable radioligand ([3H] 7-OH-DPAT) to analyze the pharmacological characteristics and brain distribution of DA D3 receptors.211 Later on, it was found out that, R-(+)-

7-OH-DPAT binds with 74-fold higher affinity to DA D3 receptors (Ki = 0.57 nM) than the corresponding S-(−)-enantiomer (42.1 nM), while 5-OH derivative resulted in the opposite trend, wherein the S-(−)-enantiomer was 140-fold more active than the R-(−)-enantiomer.212, 213 The S-(-

)-5-OH-DPAT analogue S-(−)-N-0437 (1.123, rotigotine) is known for its potent agonistic activity at DA D2 and D3 receptors and exhibited similar efficacy in Parkinson’s disease symptoms and restless leg syndrome.214, 215 In a similar way, the iodinated analogue (1.124) of (+)-7-OH-DPAT

(1.122) was developed to have improved selectivity (143-fold) at D3 over D2 receptors as compared to 1.122 (60-fold).216

Table 1.9: Structure modification and binding affinities of aminotetralin derivatives at DA D2 and D3 receptors

Ki (nM) Comp. Isomer X R R1 D2/D3 D2 D3 1.125a RS 5-OH H H ND 340 ND 1.125b RS 5-OH H n-Pr 0.50 0.75 0.67 1.125c S 5-OH n-Pr n-Pr 14 0.54 26 1.125d RS 5-OH n-Pr 2-thienylethyl 0.06 4.0 0.015 1.125e RS 5-OH n-Pr phenylethyl 0.5 3.4 0.15 1.126a RS 7-OH H H 3.3 5.1 0.64 1.126b RS 7-OH H n-Pr 3.8 0.66 5.8 1.126c R 7-OH n-Pr n-Pr 34 0.57 60 1.126d RS 7-OH n-Pr 2-thienylethyl 0.76 0.75 1.0 1.126e RS 7-OH n-Pr phenylethyl ND 2.0 ND

SAR studies of 2-aminotetralins at DA receptors suggest that meta OH (5 or 7 position) is significant for DA receptor activity.162, 182, 217 Moreover, n-propyl group is most favorable in size as N-alkyl substituents to increase the activity at D3 receptors. The affinity of the 5-OH derivative at D3 receptors has greatly increased by having mono substitution on nitrogen with n-propyl (Ki

340 nM for compound 1.125a to 0.75 nM for compound 1.125b). As shown in Table 1.9, adding a second n-propyl group to the nitrogen significantly improved selectivity for D3 over D2 receptors in both the 5-OH (1.125c, 26-fold) and 7-OH derivatives (1.126c, 60-fold).213 Replacement of one n-propyl group with a sterically more demanding 2-thienylethyl or phenylethyl substituent results in minor to moderate decreases in D3 affinity (1.125d, 1.125e, 1.126d, 1.126e), but also in a loss of the D3 selectivity relative to the D2 receptor.

Further development of 2-amonitetralins as D3 ligands turned to replacing the phenolic

OH group with metabolically stable isosteres. An exemplary prototype for this transformation is pramipexole (1.127), wherein an aminothiazole component replaces the catechol of aminotetralin.218, 219 Advancement in this direction lead to development of potent mixed D2-D3 agonists like 1.128, ropinirole (1.129), quinelorane (1.130), quinpirole (1.131), and pergolide

(1.132) (shown in Figure 1.20).220-223

Replacing the catechol OH group with enyne or endiyne functionalities produced novel nonaromatic tetralin analogues 1.133 and 1.134 (Figure 1.21).224, 225 These compounds have high affinity and selectivity at D3 receptor with Ki value of 9.1 nM and 3.2 nM for 1.133 and 1.134 respectively. Another modification, having pyrazole and pyrazolopyridine element as isostere of phenol, results in compounds 1.135-1.138.226, 227 The fact that S-enantiomer of these analogues have demonstrated higher binding affinity (6.0 nM to 0.54 nM, Table 1.10) as well as higher selectivity (15 to 157-fold) at D3 receptor over D2 receptor, suggests that the sp2 nitrogen of

39 pyrazole 1.136 and thiazole of pramipexole (1.127) have significant role as pharmacophoric element. Introducing the enyne component in to the hybrids of arylpiperazine and aminotetralin produced new D3 receptor ligands. Representative compounds 1.139 and 1.140 have very high affinity and selectivity at D3 receptor (Table 1.10).

Table 1.10: Binding affinities of pramipexole analogues at DA D2 and D3 receptors

Ki (nM) Comp. D2 D3 D2/D3 1.127, pramipexole 2.07 0.49 4 1.133 110 9.1 12 1.134 54 3.2 17 1.135 90 6.0 15 1.136 180 4.0 45 1.137 85 0.54 157 1.138 230 16 14 1.139 40 0.21 190 1.140 610 1.3 469

1.3.4.2 4-Phenylpiperazines and analogues

In last 20 years, 4-phenylpiperazines have been thoroughly studied regarding their usage as potent and selective D3 ligands. Structural congener of DA antagonist nafadotride (1.141),

1.142 has been developed, in which basic 4-phenylpiperazine is linked to naphthamide through ethyl chain.228 Even though having only two carbon alkyl linker, compound 1.142 has good binding affinity (Ki 8 nM) and 1250-fold selectivity at D3 receptor (Table 1.11). In this chemical class, numerous analogues having diverse arylamide group and alkyl linker with 2,3- dichlorophenylpiperazine or 2-methoxyphenylpiperazine moiety have been designed and studied for D3 receptor activity. Extending the alkyl chain and modifying the arylamide group to dichlorinated phenyl ring led to identification of novel tricyclic D3 receptor antagonists 1.24 (NGB

2904) and 1.143 (NGB 2849) with a remarkable binding profile (1.24 Ki = 1.4 nM, 1.143 Ki = 0.9 nM) with more than 160 to 290-fold selectivity for D3 receptor (Table 1.11).229 SAR examinations relating fluorene and oxo-fluorene derivatives 1.144 and 1.145 show modest and nonselective binding profile (Ki = 19 nM at D3 and Ki = 10 nM at D2) for 1.144, while 1.145 has nanomolar

41 affinity (1.4 nM) and 64-fold selectivity at D3 receptor.230 In this chemical class, the naphthamide derivative 1.28, (BP 897) is a high affinity D3 receptor ligand (Ki 0.92 nM) with 66-fold selectivity over D2 receptor having partial agonistic effect at D3 receptors.231 Though results are not promising, it is one of the most studied D3 partial agonist that had entered clinical trials for the treatment of drug addiction.232

Following the parent compounds 1.28 (BP 897) and 1.24 (NGB 2904), other new selective

D3 partial agonists and antagonist have been identified (Table 1.11). Replacing the naphthamide aryl ring system in BP 897 with benzofuran (1.146, 1.147) or benzothiophene (1.148, 1.149) results in a rise in D3 binding affinity (Ki 0.23–1.5 nM). More importantly, thiophene compound 1.149, is found to be one of the most selective D3 antagonists (7200-fold over D2 receptor) with greater potency (Ki 0.5 nM) while compound 1.148 is even more potent (Ki 0.23 nM) than 1.149 and having D3 partial agonist activity.233 With similar approach, two new compounds (1.150, 1.151) were discovered having indolcarboxamide as isostere of benzamide. Both of these compounds were recognized as highly potent and selective D3 receptor ligands (1.150 Ki = 0.56 nM, 5500-

234 fold vs D2, 1.151 Ki = 0.25 nM, 640-fold vs D2). Other remarkable compounds acquired by variation of arylamide portion are 1-methoxynaphthamide analogue (1.152) and 7- methoxybenzofuran analogue (1.153), which have subnanomolar D3 affinities (1.152 Ki = 0.60 nM and 1.153 Ki = 0.13 nM) and significant selectivities over D2 receptors (1400-fold for 1.152 and 2800-fold for 1.153).235 Apart from modification at phenylpiperazine moiety, replacing the flexible alkyl spacer moiety with rigid cyclic structures has been studied towards the identification of novel D3 ligands.

Table 1.11: Structure modification and binding affinities of 4-phenylpiperazine analogues at DA D2 and D3 receptors

Ki (nM) Comp. R R1 R2 X D2/D3 D2 D3 1.141 3.0 0.3 10 1.142 - - - - >10000 8.0 >1250 1.24 Cl Cl - 220 1.4 160 NGB 2904 1.143 Cl Cl - 260 0.9 290 NGB 2849

1.144 Cl Cl - 10 19 0.53

1.145 Cl Cl - 89 1.4 64

1.28 OMe H - 61 0.92 66 BP 897

1.146 OMe H O 110 1.1 100 1.147 Cl Cl O 320 1.5 210 1.148 OMe H S 87 0.23 380 1.149 Cl Cl S 3600 0.50 7200 1.150 Cl Cl H 3100 0.56 5500 1.151 OMe H CN 160 0.25 640

1.152 Cl Cl - 830 0.60 1400

1.153 Cl Cl - 370 0.13 2800

Introduction of the cyclohexylethyl linker in place of simple alkyl chain generated compounds such as 1.154 and 1.155 (Figure 1.22).236 The 2-thienylcarboxamide analogue (1.154) has shown noticeable D3 affinity (Ki 0.02 nM), however D3 receptor selectivity is only 30-fold over D2 receptor. It has antagonist functional activity at D3 receptor. Stereochemical conformation change in cyclohexyl ring has prominent impact on D3 receptor activity. Cis isomer of 1.155 has 90-fold reduced (1.8 nM) D3 affinity compared to trans isomer.

Tetrahydroisoquinoline derivative 1.156 has somewhat low D3 affinity, while, 1.23 (SB-277011) is a potent and selective D3 receptor antagonist (Ki = 4.0 nM, >60-fold selective over other DA receptors).237, 238 Novel D3 antagonists having pyrazolyl substituted thiopropyltetrahydrobenzazepine (1.157) and its congener (1.158) having fused tricyclic benzazepine have been developed, possessing binding affinities 1.0 nM (1.157) and 2.1 nM (1.158)

44 with improved bioavailability profile.239, 240 The similar series of “C-linked” analogues were found to preserve high activity and preference at D3 receptor. The representative compounds 1.159 and

1.160 have D3 Ki values of 3.2 nM and 2.5 nM and better selectivity (>100-fold) versus D2 receptors. In addition both compounds displayed high oral bioavailability (84%) and longer half life (> 6h), which make them attractive lead compounds for further assessment.241

Having discussed broadly, variant dopaminergic receptor activities present in several core molecular structures can be summarized as shown in Table 1.12.

Table 1.12: Summary of dopaminergic properties displayed by different chemical scaffold D1 receptor ligand D2 receptor ligand D3 receptor ligand Molecular Scaffold Ag. Ant. P. Ag. Ag. Ant. P. Ag. Ag. Ant. P. Ag. Arylbenzazepine √ √ √ Conformationally constrained √ arylbenzazepine Dihydrexidine, √ √ dinapsoline Aporphine √ √ √ Arylpiperazine/ √ √ √ √ piperidine Dibenzazepine √ √ √ Aminotetralin √ √ √ √ 4-phenylpiperazine √ √ √ √ √ √ Ag. = agonist, Ant. = antagonist, P. Ag. = partial agonist

1.4 Limitations with dopaminergic agents related to their clinical use

With respect to the clinical use of D1 dopamine agonists, poor pharmacokinetic profiles and adverse effects of the agent are likely to limit its clinical value. Hypotension was the major reason for the early termination of the pilot human trial on intravenous dihydrexidine (1.5). 242

Several compounds, with high binding affinity and selectivity for the D1 receptor had even entered clinical trials as anti-parkinson or anti-psychotic drugs, but were discontinued later. These ligands include, for example, SKF-81297 (1.7), SKF-83959 (1.8), SKF-77434 (1.9), and SKF-38393

(1.10).243-245 A-77636 (1.6) has rewarding and antiparkinson effects in animal studies, but its high

246 potency and long duration of action causes D1 receptor downregulation and tachyphylaxis. In addition to their low intrinsic partial agonist activity, the failure of these compounds was largely due to poor oral bioavailability and rapid tolerance. The catechol moiety, that seems a necessary component of D1 agonist activity, causes major problems related to low bioavailability. Ecopipam

(1.50a) was studied extensively for the treatment of obesity, but it faces limitations of poor oral bioavailability. Moreover, it lacks apparent antipsychotic effect and fails to attenuate the subjective effects of cocaine.247, 248 Apomorphine (1.78) is one of the clinically available dopamine agonist that has been shown to be markedly efficacious for patients with advanced PD. Its oral use is limited by both pharmacokinetics and drug-related azotemia. Parenteral use has been limited by side effects including severe nausea, vomiting, orthostatic hypotension, local skin irritation, plus the inconvenience of the pump required for drug administration.249, 250

In terms of D2 receptor binding, for pure D2 antagonists, there is a narrow window between the threshold for antipsychotic efficacy and that for side effects like EPS and hyperprolactinemia.

To avoid these adverse effects, atypical antipsychotics are endowed with mixed receptor binding

46 profile. However, some of these agents (especially clozapine 1.16, olanzapine 1.113, and quetiapine 1.17) strongly promote weight gain and result in adverse metabolic effects, including type 2 diabetes mellitus, hyperlipidemia, and hypertension.251

Apart from the mixed D2 and D3 agonists, pramipexole (1.127), ropinirole (1.129), and rotigotine (1.123), selective high-potency D3 ligands (especially D3 partial agonists and antagonists) entering clinical trials are rare. SB-277011A (1.23) is a highly selective D3 receptor antagonist with good oral availability and significant effects in many models of drug seeking and reinstatement. However, this compound was not studied clinically due to in vitro metabolism studies in liver microsomes that demonstrated rapid metabolism by aldehyde oxidase, predicting low bioavailability in humans.252 In a similar way, NGB2904 (1.24), could not enter in clinical trials because of its limited bioavailability and poor pharmacokinetics owing to its high lipophilicity.75 The most studied D3 partial agonist BP-897 (1.28) had entered clinical trials for the treatment of drug addiction, but the results are not promising. Nevertheless, its intrinsic efficacy at D3 receptors has been disputed, depending on what functional assay is used for assessment.253, 254

Chapter 2: Tetrahydroprotoberberine alkaloids and derivatives

2.1 Introduction

Tetrahydroprotoberberines (THPBs) are a class of naturally occurring tetracyclic alkaloids that contain an isoquinoline core, and are a subclass of the protoberberine alkaloids. The general structure (Figure 2.1) contains two isoquinoline rings fused in [3,2-a] fashion to generate a stereocenter at C14 position. More commonly ring A and ring D are substituted with oxygen functionalities at C2, C3, C9 and C10 positions in THP natural products, for example, (-)- stepholidine ((-)-SPD, 2.1), (-)-tetrahydropalmatine ((-)-THP, 2.2), (-)-isocorypalmine ((-)-ICP,

2.3), (-)-canadine or (-)-tetrahydroberberine ((-)-THB, 2.4), (-)-scoulerine ((-)-SCL, 2.5), (-)- stylopine (2.6), (-)-sinactine (2.7), and (-)-cheilanthifoline (2.8). While, less common is the class of “pseudo-THPBs” wherein the oxygen substitutions are on C2, C3, C10 and C11 positions, for example, (-)-corytenchine (2.9), (-)-xylopinine (2.10), and (-)-govadine (2.11).

2.2 Occurrence

The tetrahydroprotoberberines, occur in at least seven plant families including

Menispermaceae, Papaveraceae, Annonaceae, and Euphorbiaceae.255-258 THPB alkaloids are widely distributed in various species of genus Stephania (Menispermaceae) and genus Corydalis

(Papaveraceae). The genus Stephania is a large family of about 350 species, distributed in warmer parts of the world. The members of this family are mostly slender herbs or shrubs but rarely trees with peltate leaves and umbelliform flowers. In traditional medicine, these plants have been used for the treatment of tuberculosis, dysentery, asthma, inflammation fever, and intestinal complaints.259-261 Over the last 50 years, an ample amount of work has been done on several of these plants because of their medicinal importance. More than 200 alkaloids including tetrahydroprotoberberines along with other minor constituents have been isolated from these plants. Different THPB alkaloids isolated from different species of genus Stephania are listed in

Table 2.1 and their structures are shown in Figure 2.2.

Table 2.1: THPB alkaloids from genus Stephania

Stephania species THPB alkaloid/s

Stephania bancroftii262 (-)-THP (2.2), (-)-corydalmine (2.12)

Stephania elegans263 (-)-N-methylcorydalmine (2.13), (-)-cyclanoline(2.14) (-)-ICP (2.3), (-)-capaurine (2.15), (-)-corydalmine (2.12), (-)-SPD (2.1), Stephania glabra264, 265 (-)-corynoxidine (2.16), (-)-tetrahydrojatrorrhizine (2.17), (-)- tetrahydropalmatrubine (2.18) (-)-N-methyltetrahydropalmatine (2.19), (-)-xylopinine (2.10), (-)- Stephania pierrei266 capaurine (2.15), (-)-THP (2.2) (-)-SPD (2.1), (-)-THP (2.2) Stephania rotunda267 (-)-corynoxidine (2.16), (-)-xylopinine (2.10) (-)-corydalmine (2.12)

The genus Corydalis contains about 350 species such as C. melifolia, C. ambigua, C. decumbens, C. ternate, C. impatiens and C. yanhusuo in the family Papaveraceae. It is most diverse in China, which has over 290 species, mainly distributed in the southwest regions. Up to now, different classes of isoquinoline alkaloids have been found in Corydalis species including

50 aporphine, protopine, protoberberine, tetrahydroprotoberberine, benzo[c]phenanthridine, benzylisoquinoline, and morphinan. THPB alkaloids derived from Corydalis species as well as from other plants are listed in detail in Table 2.2 and the structures of these alkaloids are shown in

Figure 2.3.

Table 2.2: THPB alkaloids found in Corydalis species and other plants

Plant source THPB alkaloid/s (-)-cavidine (2.20), (-)-stylopine (2.6) (-)-tetrahydrocorysamine (2.21), (-)-apocavidine (2.22), (-)- Corydalis impatiens268 tetrahydroepiberbeine (2.23) (-)-cheilanthifoline (2.24), (-)-isoapocavidine (2.25) (-)-corydaline (2.26), (-)-THP (2.2), (-)-stylopine (2.6), (-)- canadine (2.4) Corydalis yanhusuo269 (-)-ICP (2.3), (-)-corypalmine or tetrahydrojatrorrhizine (2.17) (+)-sinactine ((+)-2.7), (-)-apocavidine (2.22) Corydzlis meifolia270 (-)-stylopine (2.6), (+)-cavidine (2.20) (-)-cheilanthifoline (2.24) (+)-stylopine (2.6), (+)-corydaline (2.26) Corydalis turtschaninovii 271 (-)-demethylcorydalmine (2.27), (-)-ICP (2.3) Corydalis clarkei272 caseanidine (2.28), clarkeanidine (2.29)

Corydalis remota273 corymotine (2.30) (-)-gusanlung E (2.31), (-)- THP (2.2) Arcangelisia gusanlung oxotetrahydroplamatine (2.32), gusanlung B (2.33), (Menispermaceae)274 corydaline (2.26), tetrahydrothalifendine (2.34) Annona spinescens (-)-pessoine (2.35), (-)-spinosine (2.36) (Annonaceae)275 Annona cherimolia Mill. (-)-corytenchine (2.9) (Annonaceae)276 Croton hemiargyreus. hemiargyrine (2.37) (Euphorbiaceae) 277

2.3 Synthesis of THPBs

Several approaches have been adopted to synthesize tetrahydroprotoberberines. Significant challenges that still persist in the synthesis of the THPB scaffold, are introducing the four substituents ortho to each other on the future ring D and introduction of the stereocenter at C14 position. While building the THPB scaffold, based on which ring closure takes place last as shown in Figure 2.4, currently available synthetic methods can be categorized as below;

2.3.1 Via late stage closure of ring B

2.3.1.1 Ring B closure via bond formation between ring A and C14 carbon

2.3.1.2 Ring B closure via bond formation between ring A and C5 carbon

2.3.1.3 Ring B closure by bond formation between C6 carbon and nitrogen

2. 3.2 Via late stage closure of ring C

2.3.2.1 Ring C closure via adding C8 carbon between ring D and nitrogen

2.3.2.2 C-N bond formation between nitrogen and C8 carbon pre-established on ring D

2.3.2.3 Ring C closure via bond formation between ring D and C13 carbon

2.3.1 Via late stage closure of ring B

2.3.1.1 Ring B closure via bond formation between ring A and C14 carbon

One strategy for late stage construction of ring B of the THPB core structure, involves synthesizing the benzylisoquinoline portion in the end with the formation of a bond between the

C14 carbon and aromatic ring A.278 According to this method (shown in Scheme 2.1), o-vanillin

(2.54) was transformed to tosyl protected aminoacid (2.55) and this acid, after chlorination underwent Friedel-Craft condensation to get tetrahydroisoquinoline (2.56). In compound 2.56, deprotection and reduction was achieved in same step using sodium bis-methoxyethoxyaluminum dihydride (red Al) to furnish 2.57. Linkage with phenylethyl iodide (2.58) followed by ring closure with concentrated HCl produced THPB scaffold in compound (2.60). The final cyclization step was quite low yielding (30 %) in this synthetic method.

Amongst several approaches, one of the most commonly used to form the ring A/ring B tetrahydroisoquinoline motif of the THPB scaffold, is via using an appropriate Bischler

Napieralski cyclization condition.279-281 Yasuda et al. designed and synthesized (±)-THP (2.2) using a similar synthetic strategy wherein, the ring B was formed towards the end of synthesis via

Bischler Napieralski condition in the cyclic amide 2.64.282 As shown in Scheme 2.2, secondary

53 amine 2.62 derived after reductive amination of primary amine 2.61 and aldehyde 2.54, was acylated with α-chloro-α-(methylthio)acetyl chloride and subjected to intramolecular Friedel-

Crafts cyclization in presence of stannic chloride to yield isoquinoline compound 2.64. A secondary amine similar to 2.62 but

without the bromine atom on ring A, proved unsuccessful due to providing a mixture of desired isoquinoline compound and the seven-membered lactam benzazepinone 2.65 having cyclization on ring A. Blocking the para position of methoxy group in ring A prevented this undesirable cyclization and produced product 2.64 solely. The Bischler-Napieralski cyclization of 2.64 followed by reduction of intermediate iminium ion produced (±)-THP (2.2).

2.3.1.2 Ring B closure via bond formation between ring A and C5 carbon

Another connectivity that has been utilized to form ring B, is through a bond formation between C-5 carbon and C-5α carbon; the Pomeranz-Fritsch reaction remained the prominent one to serve this purpose. Using this strategy (shown in Scheme 2.3), (-)-THP (2.2) was synthesized from (R)-1-amino-2-methoxymethylpyrrolidine (RAMP) hydrazone 2.66 and 2,3-dimethoxy-6- methylbenzamide 2.67.283 In this synthesis, deprotonation of 2.67 was achieved by using the mixture of tetramethylethylenediamine (TMEDA) and sec-butyllithium. Nucleophilic addition of lithiated 2-methylbenzamide to the CN double bond of hydrazone (R)-2.66 from less hindered face followed by cyclization produced (S,R)-2.68. Chiral auxiliary on nitrogen was removed by using

BH3·THF followed by HCl with concomitant reduction of carbonyl group to prepare isoquinoline compound (S)-2.69. Pomeranz-Fritsch precursor 2.70 was obtained by addition of two carbon chain of bromoacetaldehyde diethyl acetal using SN2 condition. Pomeranz-Fritsch cyclization was carried out using the mixture of conc. HCl and acetone to render intermediate 2.71 as a diastereomeric mixture which was subjected to dehydration to afford (-)-THP (2.2) with 98% ee.

Transformation from (S)-2.69 to (-)-THP (2.2) was also effected via the Pummerer reaction using

2.72 (Figure 2.5), but after the cyclization, reductive desulfurization of the intermediate was a low yielding (30%) step. On the other hand, Friedel-Crafts cyclization conditions were not effective to induce the cyclization in compound 2.73 (Figure 2.5) to provide (-)-THP (2.2).283

2.3.1.3 Ring B closure by bond formation between C6 carbon and nitrogen

An alternate plan to construct ring B of the THPB scaffold is via forming the C-N bond in the final stage of the synthesis as demonstrated by Yang et al to synthesize (-)-SPD (Scheme

2.2).284 This method to synthesize (-)-SPD (2.1) exploits a similar approach to install the chiral center as adopted by Enders to synthesize (-)-THP (2.2) (Scheme 2.3), i.e. chiral auxiliary aided nucleophilic addition of a tetrasubstituted future ring D moiety to an appropriate imine (2.66 in the case of (-)-THP and 2.79 in the case of (-)-SPD). As shown in Scheme 2.4, primary alcohol 2.75 derived from substituted phenylacetic acid, was brominated and protected to grant 2.76. Lithium- halogen exchange followed by reaction with DMF introduced the aldehyde group in compound

2.77. After condensation with chiral sulfinylamide 2.65, aldehyde 2.77 was transformed to sulfinimine 2.79. Nucleophilic addition of lithiated tetrasubstituted benzonitrile 2.80 to sulfinimine

2.79 followed by sequential steps of concomitant removal of silyl group and N-sulfinyl auxiliary, and hydrolysis of nitrile group produced 2.83. In final steps, tosylation of hydroxyl group rendered

56 cyclization of ring B which was followed by reduction and deprotection to procure (-)-SPD (2.1).

Apart from having a lengthy procedure to synthesize 2.66 (5 steps) and 2.80 (6 steps) as well as their further transformation to (-)-SPD (2.1) (6 steps), the nucleophilic addition step was low yielding because of self condensation of 2.80.

2.3.2 Via late stage closure of ring C

2.3.2.1 Ring C closure via adding C8 carbon between ring D and nitrogen

The most commonly used approach to synthesize the THPB scaffold is via an intramolecular Mannich cyclization of a relevant benzyltetrahydroisoquinoline with formaldehyde. The major complication in this method is regiochemical control in the closure of ring C and getting a mixture of para and ortho cyclized products or exclusively giving the former product. This approach is the most widely utilized route to synthesize “pseudo-THPBs” in a racemic and enantioselective manner.279, 280, 285 Using this approach, Battersby et al. synthesized (-

)-SCL (2.5) and coreximine (2.85) (Scheme 2.5) wherein, benzyltetrahydroisoquinoline (2.84) bearing 3-hydroxy substituted benzyl group underwent Mannich condensation to produce a 2:1 mixture of THPBs scoulerine (2.5) and coreximine (2.85).286 Varying the pH gave a higher yield of the desired 9,10 substituted product; however tedious chromatographic separation remained necessary.287

To tackle this problem, a strategy was used wherein compound 2.84 was brominated to block the para position (2.86) for Pictet-Spengler cyclization to afford the single product 2.87 and it was transformed to (-)-SCL (2.5) by debromination. The disadvantage in this method apart from adding two more steps in the synthesis, was that the cyclization step was low yielding, most likely due to ring deactivation by halogen atom.288

Another similar approach in the synthesis of 9,10 substituted THPBs is to use an preestablished ipso directing group in ring D. Schore et al. synthesized five different THPB alkoids using a trimethylsilyl moiety as an ipso-directing ring closure functionality.289 As shown in

Scheme 2.6, aryl silyl agent 2.90, which mimics the substitution patern of ring D of THPB, was synthesized from tertiary amine 2.89 by introducing a TMS group into the benzene ring with the aid of n-BuLi. Subsequent synthetic transformation lead to benzyltetrahydroisoquinoline 2.92 with ipso-directing silyl substituted benzyl moiety. The Pictet-Spengler cyclization was accomplished using formaldehyde in acidic condition to produce (-)-THP (2.2) in 46% overall yield and 55% ee.

In the case of (-)-sinactine (2.7) synthesis, the final cyclization no longer displayed regioselectivity and the product obtained was almost a 1:1 mixture of both (-)-sinactine (2.7) and 2.94. That indicates the ineffectiveness of this method when having strongly activating substituent like methylenedioxy group on ring D.

In a similar manner, Pictet-Spengler cyclization involving the participation of the sulfinyl group as an ipso-director in the electrophilic aromatic substitution reaction, opened new synthesis of THPBs (shown in Scheme 2.7).290 Using this method, the synthesis of optically pure (-)-THP

(2.2) and (-)-canadine (2.4) was performed with overall isolated yields of 33% and 34%, respectively. This synthesis was commenced by reaction of ortho-sulfinyl benzyl carbanion derived from 2.95 and N-sulfinylimines 2.96 and 2.97 to cater intermediates 2.98 and 2.99.

Subsequent sulfinyl- directed microwave assisted Pictet-Spengler cyclization produced (-)-THP

(2.2) and (-)-canadine (2.4).

Kroutil and group members came up with a unique approach in the synthesis of THPBs.

As shown in Scheme 2.8, the biocatalytic property of berberine bridge enzyme (BBE) for oxidative

C-C bond formation was employed to synthesize steroselectively (-)-SCL (2.5) from benzylisoquinoline (2.100).291 This transformation takes place via oxidative C-H activation of the substrate’s N-methyl group at the expense of molecular oxygen, a reaction unparalleled in organic synthesis.292

2.3.2.2 C-N bond formation between nitrogen and C8 carbon pre-established on ring D

To construct the continuously tetrasubstituted ring D is the most eminent problem towards the synthesis of a 9,10 substituted THPB scaffold. Having a tetrasubstituted pattern on ring D and closure of ring C during the late stage of synthesis is a common approach taken up in many synthesis of THPB scaffold, especially for (-)-SPD (2.1).293-295 Several examples of tetrasubstituted intermediates (shown in Figure 2.6) have been employed as a surrogate to ring D in the synthesis of the THPB core.

By executing a similar strategy, Yang and group members reported the first enantioselective synthesis of (-)-SPD (2.1) (Scheme 2.9).293 The key intermediate lactone (2.102) was synthesized from the substituted phenylacetic acid (2.101) through hydroxymethylation directed by phenylboronic acid in the presence of paraformaldehyde. Aminolysis of the lactone with substituted phenylethylamine produced amide (2.103). It was eventually transformed to (-)-

SPD (2.1) employing the key steps of Bischler-Napieralski cyclization, Noyori’s asymmetric reduction followed by ring C closure via chlorination of primary alcohol. This route seemed promising to apply further for the synthesis of diverse THPB compounds to study their biological activities. However, transformation from phenylacetic acid (2.101) to lactone (2.102) was inefficient and troublesome and it was the bottleneck for this synthetic route.

Recently, the same group synthesized (-)-SPD (2.1) using bromo compound 2.109 which was derived from chiral auxiliary protected amine 2.108 and bromo substituted phenylacetic acid

2.107 (Scheme 2.10).294 Serving as an alternate of lactone 2.102, tetrasubstituted phenylacetic

acid 2.107 was employed in this synthesis to construct the ring D of (-)-SPD (2.1). Dihydroxy benzaldehyde 2.104 was selectively protected with an isopropyl group, which was followed by ortho bromination to produce tetrasubstituted benzaldehyde 2.105. Sequential steps of reduction of aldehyde, chlorination of alcohol, cyanation, and hydrolysis of intermediate nitrile compound produced acid 2.107 from aldehyde 2.105. Acid 2.107 was coupled with chiral auxiliary protected

63 secondary amine 2.108 to afford amide 2.109. A sequence of reactions including Bischler-

Napieralski cyclization and reduction installed the chiral center in compound 2.110. The C-8 carbon of ring D was introduced in compound 2.110 via lithium-halogen exchange followed by treatment with methyl chloroformate to provide compound 2.111. Removal of the chiral auxiliary proceeded with simultaneous closure of ring C to present the tetracyclic scaffold of compound

2.112 which was further transformed to (-)-SPD (2.1) via reduction of the carbonyl group and deprotection of the phenolic hydroxyl groups.

Engaging a similar strategy to synthesize (-)-THP (2.2) (as shown in Scheme 2.11), to furnish ring D, Meyers et al. employed carboethoxy compound 2.114 derived from tertiary amine

2.113.295 Transformation from 2.113 to 2.114 was carried out via metalation followed by addition of a formyl group to afford intermediate alcohol which was protected with TBS group. Chiral formamidine 2.117 was synthesized from isoquinoline 2.115 by transamination with a valine

64 derivative 2.116. In this route, the crucial step involves addition of the carbanion of 2.114 to chiral formamidine 2.117 to furnish intermediate 2.118, which was advanced to compound 2.119 via removal of the formamidine auxiliary. Deprotection of the silyl group was accompanied by ring closure to yield (-)-THP (2.2). Carbanions having an ortho carbonyl substituent in place of a methyl silyl ether in compound 2.114 were tried first, but owing to an unexplainable problem of racemization of the chiral center, this was switched to compound 2.114.

2.3.2.3 Ring C closure via bond formation between ring D and C13 carbon

Having stereocenter established in isoquinoline moiety and addition of ring D part followed by closing the ring C at C13 carbon is an interesting way to construct THPB core. Pyane employed chiral β-amino sulfoxide (2.120), conveniently prepared by conjugate addition of amine to chiral vinyl sulfoxide296, 297, to synthesize (+)-THP (2.2).298 Chiral aminosulfoxide 2.120 was condensed with aldehyde to provide intermediate compound 2.12, which was subjected to an intramolecular

Pummerer reaction to grant a single diastereomer 2.122. Reductive desulfurization in compound

2.122 provided (+)-THP (2.2).

Very recently, Mhaske et al. synthesized various THPB and 8-oxoprotoberberine alkaloids via a unique intramolecular Pd-catalyzed tandem oxidative olefin amidation/C−H activation.299

The corresponding amide 2.123 for (±)-THP (2.2), (derived after coupling of primary

amine and dimethoxy benzoic acid) was iodinated using iodine and silver triflate to afford iodoamide 2.124. A vinyl unit was inserted into compound 2.124 via Stille coupling to yield vinylamide 2.125. After optimizing reaction conditions, C−N and C−C bonds were formed in a single step by intramolecular tandem amidation/C−H activation sequence to produce 8- oxoprtoberberine 2.32 which was reduced to (±)-THP (2.2).

Owing to their widespread occurrence in nature, diverse biological activity, and unique chemical properties, THPB alkaloids have become interesting targets for organic synthesis. In the past two decades a large number of chiral and racemic synthetic methods have been reported for the synthesis of THPB scaffold. General approaches adopted to synthesize THPB alkaloids include,

Bischler-Napieralski cyclization/simple reduction or catalytic hydrogenation (for enantioselective methodologies) followed by either Mannich type condensation or ipso directing strategies to close the second ring. Newer trends have been based on diastereoselective syntheses using chiral auxiliaries, annexed either to or around the nitrogen atom. In this context, the addition of carbon nucleophile from a tetrasubstituted benzyl moiety to the isoquinoline C=N double bond has been the most often explored strategy.

Although THPB compounds are readily synthesized in a racemic manner, still there is a demand to develop simple and efficient methods that would be applicable to the preparation of enantiopure compounds. Generally the available methodologies bear various limitations such as: e.g., multistep procedures, lack of diversity, moderate to poor yields, unsatisfactory regio- and stereo-selectivity, etc. Therefore, exploration of more efficient and simpler synthetic strategies still need to be undertaken.

2.4 Biological activities of tetrahydroprotoberberine alkaloids

Plants containing THPBs alkaloids have long been used traditionally in China, India, Japan and other Eastern Asian countries as analgesics, anti-inflammatory, anti-infectious, anti-allergic, anxiolytic, anti-venom, anti-arrhythmic, and in the treatment of rheumatism, gastric issues, and duodenal ulcers.267, 269, 271, 300 Major attention has been developed towards the pharmacological importance of THPB alkaloids (-)-SPD (2.1), (-)-THP (2.2), and (-)-govadine (2.11) have been

67 studied for schizophrenia and psychostimulant drug abuse because of their dopamine receptor pharmacological profiles.

2.4.1 CNS activities of THPB alkaloids

In functional assays, (-)-SPD (2.1) has been identified as an agonist at the dopamine D1 receptor and as an antagonist at the dopamine D2 and D3 receptor.301, 302 Also, the online database of the National Institute of Mental Health (NIMH), Psychoactive Drug Screening Program (PDSP) revealed (-)-SPD (2.1) to have high affinity mostly for dopaminergic receptors (Ki value for D1 =

5.9, D2 = 974.3, D3 = 30.1, D4 = 3748, D5 = 4.4 nM). (-)-SPD (2.1) significantly potentiated the therapeutic effects of typical antipsychotic drugs (phenothiazenes or butyrophenones) and markedly reduced the EPS induced by these drugs. Additionally, (-)-SPD (2.1) alone also exhibited a significant antipsychotic effect.303 (-)-SPD (2.1) was demonstrated to be superior to perphenazine

(2.1) in antipsychotic efficacy and treating the negative symptoms, and, unlike perphenazine, (-)-

SPD induced no extrapyramidal symptoms.304 The effects of (-)-SPD (2.1) in increasing forelimb and hindlimb retraction time (FRT and HRT) in the paw test and in reversing the apomorphine induced disruption of prepulse inhibition was investigated.305 In the paw test, all antipsychotics increase the HRT and only the typical antipsychotics affect the FRT allowing a differentiation between typical and atypical antipsychotics. The data show that (-)-SPD (2.1) behaved like an antipsychotic with more similarity to clozapine. In the paw test, (-)-SPD (2.1) dose dependently increased the HRT, while it only increased the FRT at the highest dose tested.305 Since theoretically, an agent with partial D1 receptor agonistic and D3 antagonistic properties may reduce drug abuse liability, the potential role of THPBs in the treatment of drug abuse has recently received greater attention. Clinical trial and animal studies demonstrated that THPBs including (-

)- SPD (2.1), (-)-ICP (2.3), and (-)-THP (2.2) are potential candidates for the treatment of drug abuse. A study in animals showed that (-)-SPD (2.1) inhibits acquisition, maintenance, and re- acquisition of morphine conditioned place preference (CPP).306 The effect of pretreatment with (-

)-SPD (2.1) on heroin-seeking behavior induced by heroin priming was investigated. Pretreatment with (-)-SPD (2.1) inhibited the heroin-induced reinstatement of heroin-seeking behavior.

Importantly, (-)-SPD (2.1) did not affect locomotion, indicating that the observed effects of (-)-

SPD (2.1) on reinstatement are not the result of motor impairments. 307 In a pharmacological study in mice, (-)-SPD (2.1) was shown to increase dose-dependently c-Fos gene expression in neurons of the ventrolateral preoptic area, a sleep center in the anterior hypothalamus. That indicates, (-)-

SPD initiates and maintains non rapid eye movement sleep with activation of the sleep center, suggesting its potential application for the treatment of insomnia.308 In Parkinson’s disease rat model, (-)-SPD (2.1) was demonstrated to attenuate the development and expression of L-DOPA induced dyskinesia.309

In various investigations, the effects of (-)-THP (2.2) on cocaine self-administration and cocaine-induced reinstatement have exhibited promising results towards the treatment of cocaine addiction.310, 311 (±)-THP (2.2) possesses anxiolytic effects avoiding sedation and myorelaxation when administrated orally to mice at low dose regime. Results from the elevated plus-maze show that (±)-THP (2.2) increases the percentage of entries and time spent by rats in open arms to basal levels in this model for anxiety.312 The antinociceptive effect of (±)-THP (2.2) was evaluated using different pain models including formalin-induced nociception, writhing test and bee venom- induced hyperalgesia. Results suggested that (±)-THP (2.2) more effectively inhibited visceral nociception and hyperalgesia than persistent nociception.313, 314

Effect of (-)-ICP (2.3) on cocaine addiction was evaluated using animal models.

Administration of (-)-ICP (2.3) before cocaine reduced cocaine-induced locomotor sensitization and conditioned place preference (CPP).315 Pseudo-THPB alkaloid (-)-govadine (2.11) exhibited antipsychotic efficacy and rodent models of positive symptoms through antagonism of DA-D2 receptors.316 (-)-govadine (2.11) demonstrated its ability to improve performance of the dizocilpine-induced impairment in the paired associate learning task. This behavioral effect correlates the ability of (-)-govadine to its antipsychotic potential.317

2.4.2 Non-CNS activities of THPB alkaloids

Investigations also suggest that (-)-SPD (2.1) possesses pronounced hypotensive effect

318 mediated through α1-adrenoceptor inhibition. In vitro, (-)-SPD (2.1) inhibited lipid peroxidation and scavenged hydroxyl free radicals due to its free hydroxyl group as well as other properties.319

(-)-SPD (2.1) improved cell differentiation, contact suppression, and expression of the tumour suppressor gene protein (p53), and it inhibited cell growth, colony formation, and expression of proto-oncogenes (C-myc bcl-2).320 Lin and group members studied the anti-hypertensive effects

321 of (±)-THP (2.2) and found it to act via 5-HT2A and/or D2 receptor antagonism. In a similar study, (-)-THP (2.2), (-)-THB (2.4) and (-)-SPD (2.1) showed anti-ischemia and cardio-protective property, acting directly on cardiomyocytes.322 The anti-inflammatory effect of (-)-THP (2.2) was studied in vitro using human monocytic cell line. It was found that (-)-THP (2.2) inhibited lipopolysaccharide (LPS) induced interleukin-8 (IL-8) production in a dose-dependent manner via mitogen activated protein kinase (MAPK) inhibition.323

Significant inhibition of spore germination was seen against several phytopathogenic fungi by (-)-ICP (2.3) and (-)-corydalmine (2.12).324 Recently, corydalmine (2.12), and other THPB

70 alkaloids were demonstrated to have antibacterial activity toward methicillin-resistant

Staphylococcus aureus.325 Lee and co-workers isolated various THPB alkaloids from C. turtschaninovii and studied their inhibition activity against low density lipoprotein oxidation. (-)- demethylcorydalmine (2.27) and (+)-stylopine (2.6) were the most active alkaloids with IC50 values of 2.1 and 2.0 µM.326 Antiplasmodial activity of (-)-SCL (2.5) was studied along with other alkaloids isolated from C. dubia and it has shown remarkable activity against TM4/8.2 (transgenic

-1 327 plasmodium) strain with IC50 values of 1.78 µg ml . In a similar manner coraximine (2.85) exhibited antimalarial activity against P.falciparum.328 Experimental results obtained in a study demonstrate that (±)-canadine (2.4) is effective in anti-lipid peroxide production and acts as an antioxidant agent.329 (-)-canadine (2.4) also showed more than 80% inhibition in platelet aggregation induced by different platelet aggregating agents.330 Many different THPB alkaloids including (-)-THP (2.2), (-)-ICP (2.3), (-)-canadine (2.4), and (-)-stylopine (2.6) have displayed chemopreventive and anti-tumor activity.331

2.5 Structure-activity relationship studies at dopamine receptors

Since THPBs and dopamine share some structural similarities, various natural and synthetic derivatives of THPBs have been studied for their binding affinity at dopamine receptors, more specifically at DA D1 and DA D2 receptors.

In one study, the influence of the trisubstitution with methoxy, hydroxyl or chloro at the 2,

3- and 9 or 11 positions in THPB was evaluated for dopaminergic activity. Eleven trisubstituted analogues of THPB (2.72–2.82, shown in Figure 2.7) were synthesized and tested for their binding affinity (Table 2.3) at DA D1 and D2 receptors.332 Results of this SAR study indicate that protected phenolic hydroxyls showed a lower affinity for DA D1 and D2 receptors than their corresponding

71 homologues with free hydroxyl groups (see 2.72 vs 2.73, 2.74 vs 2.75, 2.77 vs 2.79, 2.78 vs 2.80 in Table 2.3). Introduction of the chlorine atom in to ring A of THPBs increases affinity for both

D1 and D2 receptors (2.73 vs 2.79, Table 2.3). A decrease in affinity

Table 2.3: Binding affinity data of 2,3,9 and 2,3,11 substituted THPBs

Ki (µM) Compound Substitution D2/D1 D1 D2 2.72 3-Cl-2,11-diMeO 1.80 2.05 1.1 2.73 3-Cl-2,11-diOH 0.10 0.19 1.9 2.74 2,3-diBnO-11-MeO 40.60 40.87 1.0 2.75 2,3-diOH-11-MeO 1.10 0.08 0.07

2.76 2,3-OCH2O-11-MeO 2.40 1.33 0.5 2.77 2,3,11-triMeO 4.31 8.54 2.0 2.78 2,3,9-triMeO 3.923 0.39 0.1 2.79 2,3,11-triOH 2.96 0.40 0.1 2.80 2,3,9-triOH 3.78 0.09 0.02

2.81 2,3-OCH2O-11-OH 33.00 7.51 0.2

2.82 2,3-OCH2O-9-OH 0.34 0.04 1.1

at the D2 receptor was observed in 2.76c and 2.81e after the introduction of a methylenedioxy group at 2, 3 positions. Compounds 2.73b and 2.82f in this series displayed the highest affinity for

72 both DA D1 and D2 receptors. This study demonstrated that affinity for DA receptors was higher with analogues having hydroxyl group in position 9 than in position 11 (2.82 versus 2.81).

In two sets of SAR studies, Hong Liu and group members designed and synthesized a series of novel as well as known tetrahydroprotoberberine compounds (representative novel analogues

2.83-2.89 are shown in Figure 2.8) by introducing various substituents (such as methyl, methoxy, hydroxymethyl, and methylenedioxy groups) at different positions of the THPB pharmacophore.333, 334. The binding affinity data for these analogues at dopamine D1 and D2 receptors is summarized in Table 2.4.

Tetra-alkoxy analogues ((-)- THP 2.2 and (-)-canadine 2.4) exhibited reduced binding affinity for both DA D1 (Ki = 231 nM and 66 nM respectively) and D2 receptors (Ki = >5000 nM and 119 nM respectively). Monohydroxy compound 2.3 ((-)-ICP) shows higher selectivity than parent compound (-)-SPD (2.1) at D1 receptor over D2. Changing the C9, C10 di-methoxy substitution in ring D of (-)-ICP (2.3) to C9, C11 di-methoxy produced highly D1 receptor selective and potent compound 2.83. In addition, a congener of 2.83, compound 2.84 with C10-C11 methylenedioxy substitution possess similar high binding affinity at the D1 and D2 receptors (4.2 nM for D1 receptor and 32.1 nM for D2 receptor) with (-)-ICP (2.3). However, analogue 2.84 shows functional antagonism at DA D1 receptor in contrast to (-)-ICP (2.3), which is a D1 partial agonist.

Table 2.4: Structures of and binding affinity data for THPB analogues

Ki (nM) No. R1 R2 R3 R4 R5 R6 D2/D1 D1 D2 2.1 H OH CH3O CH3O OH H 3.4 11 3.2

2.2 H CH3O CH3O CH3O CH3O H 231 >5000 >21.6

2.4 H OCH2O CH3O CH3O H 66 119 1.8

2.3 H OH CH3O CH3O CH3O H 4.5 91 20.2

2.83 H OH CH3O CH3O H CH3O 2.5 - -

2.84 H OH CH3O H OCH2O 4.2 32 7.6

2.34 H OCH2O CH3O OH H 6.3 145 23.0

2.85 H OCH2CH2 CH3O OH H 7.5 - -

2.18 H CH3O CH3O OH CH3O H 443 - -

2.86 H OCH2O OH CH3O H 60 >5000 >83

2.17 H CH3O OH CH3O CH3O H 310 - -

2.87 H CH3O OH OH CH3O H 530 >5000 >9.4 82% 2.88 CH3O H CH3O CH3O H CH3O 8.0 - Inhib. 52% 2.89 CH3O CH3O CH3O CH3O H CH3O 23.8 - Inhib.

These results suggest that the hydroxyl group at C2 may be essential to maintain potency at the D1 and D2 receptors. Compounds 2.34 and 2.85 showed a similar high binding affinity (Ki

= 6.3 and 7.5 nM) and selectivity to the D1 receptor, indicating that apart from the C2 hydroxyl, the C10 hydroxyl group is also crucial for D1 receptor activity. Compounds 2.18 and 2.86 containing only a hydroxyl group at C9 showed reduced binding affinity for the D1 receptor with

Ki values of 334 and 60 nM. Compound 2.17 having a hydroxyl group at C3 retained a moderate affinity for the D1 receptor, with a Ki value of 310 nM. On the contrary, 2.87 containing two

74 hydroxyl groups at C3 and C9 with a Ki value of 530 nM were less potent than 2.17 at the D1 receptor. Compound 2.88 and 2.89 in which two phenolic hydroxy groups on the A- and D-rings were removed, exhibited high D1 receptor binding affinity (Ki = 8.0 nM and 23 nM) and selectivity. Functional assays revealed that both these analogues were full D1 receptor antagonists.

Similar to the naturally derived alkaloids, R enantiomer of all these compounds showed remarkably weakened binding affinity to the dopamine receptors, indicating that the C14-S configuration in THPBs is an important determinant of receptor binding affinity.

As shown in Figure 2.9 and explained below, the previous SAR study of THPB scaffold at

DA receptor can be summarized as below;

• Either C2 or C10 hydroxyl group is necessary for dopamine D1 receptor activity (example

2.3, 2.34, and 2.85). This indicates, the hydrogen bonding donor (OH) at C2 and C10 is a

determining factor to D1 receptor binding.

• Antagonist activity shown by compound 2.83, 2.88 and 2.89 reveals that 2, 3, 9, 10

tetrasubstituted pattern is crucial for D1 agonistic activity

• Reduced affinity of compound 2.17, 2.18, 2.86, and 2.87 signifies the importance of C3

and C9 alkoxy group for the dopamine receptor activity

• S configuration of the C14 position in the THPB skeleton is a critical factor determining

receptor binding affinity

Chapter 3: Synthesis and evaluation of stepholidine and its analogues at dopamine receptors

3.1 Background

(-)-SPD (2.1) shows a strong preference for binding to dopamine receptors as revealed by data from the Psychoactive Drug Screening Program (PDSP) and by many previous studies

(discussed in chapter 2). (-)-SPD (2.1) has high affinity for D1 and D3 receptors (Ki = 5.9 and 30

335 nM respectively) and moderate affinity for the D2 receptor (Ki = 974 nM). (-)-SPD (2.1) has been studied primarily due to its multi-receptor targeted profile.302, 336-338 In this regard, its interesting pharmacological profile has led to a number of animal studies where it has shown potential as an antipsychotic and as an anti-drug abuse agent.

The THPB scaffold of (-)-SPD (2.1), which is characterized by a 2,3,9,10 tetra-substituted pattern and the presence of two phenol groups, makes it a distinct THPB scaffold.339 Although several syntheses of THPBs have been reported (discussed in chapter 2, section 2.3), an efficient, high yielding, and concise synthesis of (-)-SPD (2.1) is yet to emerge. Moreover, the extremely low content (~0.1%) of this compound in natural sources265 and the need for large amounts of the compound for further pharmacological research necessitate its total synthesis.

Though having an interesting novel, pharmacological profile, there is a need to further optimize the potency of (-)-SPD (2.1) at dopamine receptors. In that regard compounds with <10 nM affinity at D1 and D3 receptors and with higher degrees of selectivity versus D2 receptor, will be valuable to study in vivo.

Until now, limited SAR data is available for this THPB scaffold332-334, 337 and it is restricted to structural modifications in the core pharmacore including:

I. Switching the position of phenolic hydroxyl group and methoxy moiety in aromatic

rings

II. Introducing methoxy, methylenedioxy and benzyloxy substituents

III. Insertion of a halogen atom in the ring A

IV. Effect of mono, di and tri hydroxy substitution on binding affinity

Additionally, all SAR studies have been performed (discussed in chapter 2, section 2.5) at DA

D1 and D2 receptors, no SAR data is available at the D3 receptor. Deficiency of enough structure activity relationship (SAR) data at DA D1 and D3 receptor for this THPB scaffold has to be filled.

The pharmacokinetics of (-)-SPD (2.1) and its delivery to the brain was studied in rats.340 Key findings in this study reveal that although (-)-SPD (2.1) exhibits good brain penetration, it has a very poor oral bioavailability (< 2%) because of extensive pre-systemic metabolism. Findings suggest that rapid and extensive pre-systemic metabolism of (-)-SPD (2.1), predominantly by O- glucuronidation and O-sulphation of phenolic hydroxyl groups restricts its favorable biological profile.341

Our work addresses the above mentioned shortcomings via;

I. Developing a practical and versatile synthetic methodology which can provide SPD

(2.1) and its analogues in larger amount for further pharmacological studies

II. Synthesizing and studying binding affinity at dopamine receptors of diversely

substituted C3 and C10 THPB analogues

III. Studying the effect of various substitutions on THPB scaffold on metabolic stability

against hepatic enzymes

Our efforts and the results we have obtained are discussed in this chapter.

3.2 Synthesis of (±)-SPD

3.2.1 Retrosynthesis

For the synthesis of (±)-SPD (2.1), late stage construction of the berberine bridge (ring C) via a Mannich-type reaction is challenging because there is a substituent ortho (i.e. the methoxy group) to the site of ring closure. In our retrosynthetic analysis (as shown in Figure 3.1), we visualized constructing the berberine bridge in the final stage of synthesis. We planned to synthesize (±)-SPD (2.1) from compound 3.1, which has previously established berberine bridge carbon on ring D that can be utilized to close ring C. We envisaged that the tetrahydroisoquinoline moiety in compound 3.1 could be prepared from amide intermediate 3.2 via Bischler-Napieralski cyclization/reduction conditions. Amide intermediate 3.2 is derivable from acid 3.3 (via amide coupling with the corresponding amine). Compound 3.3, we reasoned would serve as an easily reachable synthetic surrogate for the ring D of SPD (2.1). The acid 3.3 would be prepared from aryl bromide 3.4.

3.2.2 Synthesis

Commercially available bromo cresol 3.5 was protected as the ethoxymethyl ether by reaction with chloromethoxyethane affording 3.6. Thereafter reaction of 3.6 with Lithium diisoproylamide in the presence of dimethylmalonate gave diester 3.7.342, 343 This reaction presumably proceeds via nucleophilic attack of malonate anion on benzyne intermediate generated from dehydrobromination of 3.6.344 Selective hydrolysis of the aliphatic ester group was accomplished with K2CO3 in refluxing H2O/MeOH to give acid 3.8. This acid was coupled to readily available amine 3.9 with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) to provide key amide precursor 3.10. Bischler-Napieralski cyclization on amide 3.10 proved to be successful with POCl3 as dehydrating agent and acetonitrile as solvent.

This cyclization was accompanied by simultaneous removal of the ethoxymethyl protecting group. Other dehydrating conditions (shown in Table 3.1) and solvents

(dichloromethane or toluene) were tried but gave inferior yields of imine product. Bischler-

Napieralski cyclization was followed by immediate reduction (due to instability of the intermediate imines of this type) with NaBH4 to afford the 8-oxotetrahydroprotoberberine 3.11. Reduction of the 8- oxo group of 3.11 with LAH provided an intermediate tertiary amine, which was subsequently heated with concentrated HCl, thus effecting debenzylation to give (±)-SPD (2.1).

Thus we accomplished synthesis of (±)-SPD (2.1) with overall yield of 30% over 8 synthetic steps starting from bromocresol 3.5.

Table 3.1: Bischler-Napieralski conditions (step e, Scheme 3.1) Conditions Reagent Yield Solvent Time Temperature

PCl5 (1.5 eq.) DCM 10 h 0 ̊ C – rt No reaction

P2O5 (10 eq.) DCM 10 h rt No reaction 80 ̊ C POCl3 (neat) POCl3 10 min 63% (MW-150W)

POCl3 (6 eq.) Toluene 2 h 100 ̊ C 58%

POCl3 (6 eq.) CH3CN 3 h 60 ̊ C 82%

3.3 Enantioselective synthesis of (-)-SPD

3.3.1 Retrosynthesis

In this section we describe our attempt to develop a practical route to synthesize (-)-SPD

(2.1). It is not restricted to only synthesis of (-)-SPD (2.1); the main consideration behind our aim is to develop a versatile synthetic method that can be extended to synthesize optically pure C3

81 analogues of (-)-SPD (2.1). Following a similar approach employed in our racemic synthesis of

(±)-SPD (2.1) (section 3.2), we planned to synthesize (-)-SPD (2.1) from compound 3.11, which has a previously established berberine bridge carbon on ring D (Figure 3.2). Compound 3.11 can be synthesized from amide ester 3.10 via Bischler-Napieralski cyclization followed by Noyori’s asymmetric hydrogenation of the intermediate imine.345 Amide ester 3.10 can be synthesized from acid 3.8 and amine 3.9 as shown in Scheme 3.1.

3.3.2 Synthesis

3.3.2.1 Unsuccessful attempts via ester 3.10

Bischler-Napieralski cyclization of amide 3.10 was achieved using POCl3 to give intermediate imine 3.12 (shown in Scheme 3.2). The imine formed was then subjected to Noyori’s asymmetric hydrogenation conditions. To our surprise this did not yield the desired chiral tetrahydroisoquinoline 3.13. Instead the 8-oxoprotoberberine 3.14 was produced. One possible cause for failure of the reduction is due to formation (and subsequent isomerization) of an acyl iminium ion formed from reaction of the imine nitrogen (from Bischler-Napieralski reaction) with the aryl ester group. Another possibility is that when the imine derived from 3.10 (i.e., compound

3.12) is subjected to Noyori’s reduction conditions, isomerization of the imine to an enamine precedes the cyclization of ring C. We found that treatment of 3.12 with triethylamine (the base

82 used in the Noyori’s reduction) gives 3.14. Presumably, the isomerization/cyclization steps occur at a faster rate than Noyori’s reduction.

Nevertheless, through this attempt to achieve an enantioselective synthesis of (-)-SPD

(2.1), it was recognized that naturally occurring 8-oxoprotoberberines could be synthesized by variations of this method. Previous syntheses of the 8-oxoprotoberberine skeleton have required several synthetic steps and/or suffer from low yielding steps.346-349 Thus to demonstrate the pliability of the route, compound 3.14 was transformed to the natural product oxypalmatine (3.15,

Scheme 3.3) by removal of the C2 benzyl group followed by Williamson ether methylation. Insofar as synthesis of oxypalmatine is concerned, this method is comparable to published methods in both

83 yield and number of steps (seven steps and 22% overall yield for the present synthesis; nine steps

10% yield for the previous synthesis).350

It was postulated that the reason for formation of 3.14 was due to nucleophilic attack by the imine nitrogen on the methyl ester in intermediate 3.12. This would form an acyl iminium intermediate that later isomerized to form 3.14. To avert this transformation, we planned to make t-butyl ester 3.17 which can provide steric hindrance against approaching nitrogen. To synthesize t-butyl ester 3.17, we attempted transesterification using t-BuOH and ester 3.10 in presence of sulfuric acid but this was unsuccessful. In another attempt at synthesizing 3.17 (shown in Scheme

3.4), the ester 3.10 was hydrolyzed to acid 3.16 and then various conditions for esterification

(shown in Table 3.2) were applied on acid 3.16. However, none of the conditions that were tried worked and we recovered starting material. Steric hindrance might be the factor at play here.

Table 3.2: Unsuccessful esterification conditions for compound 3.16 (step b, Scheme 3.4) Entry Reagents Solvent 1 DCC, DMAP t-BuOH

2 H2SO4, MgSO4 t-BuOH

3 (t-BuO)2O DCM

After unsuccessfully attempting to synthesize 3.17, our efforts were directed towards synthesis of di-amide 3.18. It was envisaged that the aromatic amide group in 3.18 would not be

84 as susceptible to ring closure via the intermediate imine (akin to compound 3.12). Thus 3.18 was synthesized as shown in Scheme 3.5. Bischler-Napieralski cyclization of diamide 3.18 followed by Noyori’s catalytic hydrogenation produced enamine 3.19. This result tends to suggest that imine 3.12 first isomerizes to an enamine which then cyclizes to form the enamide 3.14, contrary to our initial postulate that cyclization preceded isomerization.

After unsuccessful attempts to stop the formation of enamine 3.14, we planned to reduce ester 3.10 to primary alcohol 3.20 and the resultant alcohol could then be advanced to synthesize

(-)-SPD (2.1). We tried various conditions to reduce ester 3.10 (as shown in Table 3.3); in which

DIBAL (at -78 ̊ C) and NaBH4 (in refluxing THF) produced a complex TLC profile, while treating with LAH at 0 ̊ C gave cyclic imide 3.21. Formation of imide 3.21 after the reaction with LAH and NaBH4 revealed that cyclization of ring C occurs before ester reduction.

Reduction with LiBH4 at 0 ̊ C to room temperature did not furnish the desired alcohol 3.20, but we received unexpected primary alcohol 3.22. We found similar literature precedence wherein cyclic imide 3.23 was shown to transform to an opened ring aliphatic alcohol 3.24 in the presence

351 of excess NaBH4 (Scheme 3.7). We tried to cyclize imide 3.21 using POCl3, having thought that cyclization would occur at aliphatic carbonyl and further advancing the intermediate iminium ion to (-)-SPD (2.1) synthesis. But we observed a complex TLC profile after cyclization of imide 3.23.

Table 3.3: Reduction of compound 3.10 (Scheme 3.6) Reducing agent Temperature Solvent Product Yield

NaBH4 rt to reflux THF/MeOH NA NA DIBAL -78 ̊ C THF NA NA LAH 0 ̊ C THF 3.21 46%

NaBH4 0 ̊ C to rt MeOH 3.21 54%

LiBH4 0 ̊ C to rt THF 3.22 52%

Finally, we planned to reduce enamide 3.14 (derived from ester 3.10) via enantioselective hydrogenation using ruthenium catalyst as per the reported procedure (Scheme 3.8), wherein enamide 3.25 was reduced to formamide 3.26.352 As shown in Scheme 3.9, enamide 3.14 was

Table 3.4: Hydrogenation conditions (step a, Scheme 3.9)

Hydrogen Temperature ( ̊ C) Pressure (atm) 1 rt 5 rt 10 rt 20 rt 40 rt 50 rt 20 50 40 50 50 50 a. All conditions were unsuccessful treated with (S)- Ru(OAc)2(BINAP) under hydrogen pressure in a stainless steel high pressure reactor. We applied different hydrogen pressure ranging from 1atm to 50 atm at different temperature (shown in Table 3.4); however none of the conditions furnished the expected product

(-)-3.11. As per our assumption the cyclic conjugation between two aromatic rings makes this enamide double bond quite inert against hydrogenation.

3.3.2.2 Synthesis via lactone 2.62

After all ineffective efforts to reduce ester 3.10 to primary alcohol 3.20 as well as asymmetric hydrogenation of enamide 3.14, we redesigned our route (shown in Figure 3.4) to synthesize (-)-SPD (2.1) via lactone 2.102, which can be synthesized from diester 3.7 via selective reduction of aromatic ester.

As depicted in Scheme 3.10, our synthesis of (-)-SPD (2.1) commenced with compound

3.7 which was synthesized as per Scheme 3.1 from 4-bromoguaiacol (3.5). The ethoxymethyl group of 3.7 was exchanged for a benzyl protecting group in two high yielding steps. Here, 3.7 was refluxed in acidic methanol to effect deprotection of the ethoxymethyl group and this was followed by protection of resulting phenol as the benzyl ether to give 3.27. Global hydrolysis of the ester functionalities of 3.27 and subsequent selective esterification of the aliphatic acid functionality, generated acid 3.28. The acid 3.28 was converted to an intermediate activated ester via reaction with EDCI/HOBt. This ester group was selectively reduced with NaBH4 to provide an alcohol which was then cyclized to the lactone 2.62. Thereafter the route developed by Yang was implemented to prepare (-)-SPD (2.1).293

To provide unequivocal stereostructural proof, we determined the 3-dimensional structure of 2.1 by single crystal X-ray diffraction. Figure 3.5 shows a representation of the x-ray structure, in which a water molecule co-crytallizes with 2.1. The synthesis of 2.1 was accomplished in 23% overall yield from compound 3.5 in a fourteen step sequence. This route involves an alternative preparation of the vital intermediate compound 2.102. The synthetic manipulations to afford 2.102 are straightforward, scalable ( > 5 g) and generally high yielding.

Figure 3.5: Crystal structure of (-)-SPD monohydrate

3.3.2.3 Discussion

Herein, a new synthesis of (±)-SPD (2.1) is delineated, which features the use of the easily obtainable diester intermediate 3.7. (±)-SPD (2.1) was prepared in eight steps from a readily available precursor 4-bromoguaiacol in 30% overall yield. The route is attractive due to its efficiency and practical ease; no chromatographic purification was necessary for a number of steps.

The present approach has the advantages of brevity, ease of synthetic manipulations and superior overall yield as compared to previous reported syntheses of (±)-SPD (2.1). This method has addressed a solution to the key problem in synthesis of THPBs, to set up the 2,3,9,10 tetrasubstituted pattern, by employing diester 3.7. The key amide intermediate (3.10), that is, used for (±)-SPD (2.1) synthesis can also be used to prepare a 9,10-oxygenated-8-oxoprotoberberine core, and we adapted this method to prepare oxypalmatine (3.15). Hence, the route examined here is a versatile one for the preparation of 9,10-oxygenated THPBs as well as their 8- oxoprotoberberine analogues.353

The enantioselective synthesis of (-)-SPD (2.1) was accomplished in 23% overall yield from compound 3.5 in a fourteen step sequence. The crystal structure of (-)-SPD (2.1) was determined by single crystal X-ray diffraction, and validated the success of this method. This is comparable to Yang’s first synthesis where a 24% yield of (-)-SPD (2.1) was obtained in eleven steps (from a 4-benzyloxy-3-hydroxy phenylacetic acid precursor).293 This, route involves an alternative, reliable preparation293 of the vital intermediate compound 2.102. The synthetic manipulations to afford 2.102 are straightforward, scalable and generally high yielding. This synthetic pathway was applied to generate of C3 chiral analogues of (-)-SPD (2.1) for bioactivity studies.

3.4 Synthesis and evaluation of (±)-C10 analogues

3.4.1 Rationale

As mentioned previously in section 3.1, others have investigated the replacement of the C2 and C10 hydroxyl groups of (±)-SPD (2.1) with methoxy, methylenedioxy and benzyloxy motifs.

Based on the results obtained in previous studies, either C2 or C10 hydroxyl group are pre- requisites for dopamine D1 activity. Base to this premise, in this project, we planned to probe the tolerance of larger lipophilic substituents and steric bulk as well as hydroxyl group homologs at

C10 position while keeping the C2 hydroxyl group free. The rationale behind this study was to get an insight about optimum size of the alkoxy group required at C10 position for dopamine D1 and

D3 receptor affinity and selectivity. To this end, molecules 3.29a-3.29h (Figure 3.6) were designed to test the effect of extended alkyl chain, three carbon cyclic ring and hydroxyl homologs on the affinity and activity at D1, D2 and D3 receptors.

3.4.2 Results and discussion

3.4.2.1 Synthesis

Using our newly developed synthetic method (Scheme 3.1), we synthesized amide 3.11 in large amount from diester 3.7, and it was reduced to tertiary amine 3.30 using LAH. Compound

3.30 was deployed to synthesize C10 analogues (3.29a-3.29h) of (±)-SPD (2.1) via alkylation with

91 appropriate alkyl halide followed by debenzylation using concentrated HCl (as shown in Scheme

3.11).

3.4.2.2 Structure-activity correlations

This series of analogues along with (±)-SPD (2.1) were assayed for binding affinity to human dopamine D1, D2 and D3 receptors by the Psychoactive Drug Screening Program (PDSP).

This data is presented in Table 3.5. (±)-SPD (2.1) displayed approximately 20-fold stronger affinity for the D1 receptor as compared to the D2 and D3 receptors (Ki of 5.6, 115.5 and 101 nM at D1, D2 and D3 respectively). Alkylation tends to reduce D1 affinity, with the analogues showing a 1.2 to 7-fold decrease in affinity as compared to (±)-SPD (2.1). The C10 phenol plays role but is not absolutely required for D1 affinity, given the relatively small drop in affinity seen for some analogues (for example 3.29c and 3.29g). This result is in accordance with observations from previous SAR work on the THPB framework by others.19

Methyl and ethyl substituents resulted in an approximately 2-fold decrease in affinity

(compounds 3.29a and 3.29b respectively). In the n-propyl (3.29c) to n-hexyl (3.29f) homologous series, a progressive decrease in affinity was observed. The D1 receptor affinity seen for the

92 hydroxypropyl analogue 3.29g was comparable to that observed for (±)-SPD (2.1) and the n-butyl analogue 3.29d (6.7, 5.6 and 11 nM respectively). This suggests that the hydroxyl group in 3.29g plays a relatively minor role in binding to the D1 receptor. The presence of the hydroxypropyl substituent in 3.29d restores the D1 affinity of (±)-SPD (2.1), improves the selectivity versus the

D2 receptor (20.6 versus 31.5 for D2/D1 selectivity for (±)-SPD (2.1) and 3.29d respectively) and retains the selectivity versus the D3 receptor (18 versus 16-fold for D3/D1 selectivity). Among the alkylated analogues, a 3 carbon chain length seems to be optimal for D1 affinity (consider 3.29c and 3.29g). The cyclopropylmethyl analogue (3.29h) had similar affinity to the n-butyl analogue

3.29d and ethyl analogue 3.29b. Thus some degree of branching on the alkyl chain may be tolerated for D1 affinity. On a whole, the D2 affinity of the analogues was lower than their D1 affinities. Thus the compounds retained selective affinity for the D1 receptor, as was observed in the lead molecule (±)-SPD (2.1). However, most compounds had lower than the 20-fold D1 selectivity seen for (±)-SPD (2.1) (except for compounds 3.29f and 3.29g).

Compounds in the homologous series from a methyl group to an n-butyl chain (3.29a-

3.29d), had D2 affinities that are similar to (±)-SPD (2.1), ranging from 99 to 159 nM. However, compounds with the larger n-pentyl and n-hexyl chains had reduced affinities; in fact, the n-hexyl analogue (3.29f) did not show any appreciable affinity for the D2 receptor (<50% inhibition) in the primary assay. The cyclopropylmethyl analogue (3.29h) was the only compound in this series that displayed a significantly higher affinity for the D2 receptor than (±)-SPD (2.1).

Table 3.5: Binding affinity data (Ki nM) for C10 analogues at dopamine receptors

a Ki (nM) Selectivity Compound R D1b D2c D3d D2/D1 D3/D1 D2/D3 (±)-2.1 H 5.6e 115.5e 101e 20.6 18.0 1.1 3.29a Me 15 102 37 6.8 2.5 2.8 3.29b Et 12.4 159 44 12.8 3.5 3.6 3.29c n-Pr 7.8 99 53 12.7 6.8 1.9 3.29d n-Bu 11 121 65 11 5.9 1.9 3.29e n-Pent 19 283 101 14.9 5.3 2.8 3.29f n-Hex 40 NAf 220 NDg 5.5 NDg 3.29g Hydroxypropyl 6.7 211 110 31.5 16.4 1.9 3.29h Cyclopropylmethyl 12 54 54 4.5 4.5 1 a b Experiments carried out in triplicate - SEM values are within 13% of reported Ki; [3H]SCH2390 used as radioligand/(+)-butaclamol c used as reference compound - Ki = 2.3 nM; [3H]N-methylspiperone used as radioligand/haloperidol used as reference compound - d e Ki = 9.6 nM; [3H]N-methylspiperone used as radioligand/chlorpromazine used as reference compound - Ki = 11.0 nM; experiments carried out in duplicate; f NA- not active (<50% in primary assay); g ND-not determined.

There was a clear trend in the methyl to n-hexyl series at the D3 receptor, where an increase

in the alkyl chain length was associated with a progressive decrease in affinity for the receptor.

However, methyl to n-butyl analogues demonstrated improved binding affinity at the D3 receptor

as compared to the lead compound (±)-SPD (2.1) (37 to 65 nM versus 101 nM). The hydroxypropyl

analogue had similar affinity for the D3 receptor as compared to (±)-SPD (2.1). In the case of the

94 cyclopropylmethyl analogue, D3 receptor affinity was improved two-fold as compared to (±)-SPD

(2.1) (54 nM vs 101 nM).

3.4.3 Molecular docking study at D3 receptor

The phenolic group at C10 of SPD (2.1) does not seem to play a major role in its binding to dopamine D1 and D2 receptors (given the similarity in D1 and D2 affinities of the analogues as compared to SPD (2.1)). In the case of the D3 receptor, a number of analogues had moderately improved affinity as compared to SPD (2.1). Therefore it seems that the C10 phenolic group is not an absolute necessity for affinity of SPD (2.1) to the D3 receptor. Based on the SAR trend observed

(increasing alkyl chain length decreases D3 affinity), it is apparent that the C10 alkoxy substituents of the analogues might project into a shallow hydrophobic pocket in the receptor. To get further clarity into this possibility and to aid in rationalizing the D3 affinity trend observed, docking evaluations were performed on SPD (2.1) and analogues 3.29a-3.29h at the D3 receptor.

Investigation of the docked ligand poses and recognition of key protein–ligand interactions provides insights into the SAR outcomes of the series of C10 analogues and the de-rigidified compounds with respect to D3. In this study, ligand docking was performed retrospectively in order to elucidate and rationalize the measured D3 affinity data. Overall, the calculated binding energies for SPD (2.1) and the C10 analogues are in reasonable agreement with activities derived from the experimental data as shown in Table 3.6.

Compounds SPD (2.1) and 3.29a-3.29h were docked into a pre-prepared 3-dimensional structure of the D3 receptor derived from the crystal structure with PDB code 3PBL.354 A representation of the docked poses for the ligands SPD (2.1), 3.29a-3.29f and 3.29h within the D3 receptor binding pocket is depicted in Figure 3.7. Each ligand generated multiple binding

Table 3.6: Predicted binding affinity from Glidescore for C10 analogues in D3 receptor docking study

Ki (nM) at D3 Compound R Glidescore receptor 3.29h Cyclopropylmethyl -7.3 54 3.29f n-Hex -7.3 220 3.29b Et -7.4 44 3.29d n-Bu -7.4 65 3.29a Me -7.5 37 3.29c n-Pr -7.5 53 3.29e n-Pent -7.5 101 SPD (2.1) H -7.9 101 3.29g Hydroxypropyl -8.1 110

modes and the structures shown display the top ranked poses according to Glidescore (+ligand strain). These poses maintain key receptor–ligand interactions, including the critical protonated tertiary N—Asp110 salt bridge motif, H-bonds to Ser192 and Cys181 and hydrophobic interactions to Phe106 and Phe345 (Figure 3.7). The analogues are positioned such that the aryl substituents project into hydrophobic cavities in the receptor (depicted in Figure 3.7b for compound 3.29a). The estimated binding energies according to Glidescore are very similar for this series of C10 analogue ligands, covering a narrow range from -7.9 to -7.3 kcal/mol. Overall, the relatively small decrease in the predicted binding energy as the length of the alkyl chain increases for compounds 3.29a-3.29f qualitatively matches the decrease in the measured affinity for these ligands. Quantitatively, the binding energy differences are very small compared to the wider, yet still relatively low, variation in measured affinities. The Glidescore value for SPD (-7.9 kcal/ mol) was found to be lower than that for the other C10 analogues, including the cyclopropylmethyl analogue (-7.3 kcal/mol), in contrast to the experimental measurements.

Figure 3.7: (a) Docked poses of the lead molecule SPD (2.1) and the C10 analogues 3.29a-3.29f and 3.29h. Key hydrogen bonding interactions are given by the yellow dashed lines and hydrophobic interactions by the turquoise dashed lines (b) Compound 3.29a docked in binding pocket.

However, in a similar manner to the measured affinities, the hydroxypropyl analogue, with docked pose illustrated in Figure 3.8, had a predicted binding energy that is close to that of SPD (2.1) (-

8.1 kcal/mol vs -7.9 kcal/mol, respectively). Examination of the D3 receptor binding site shows that the alkyl, cyclopropylmethyl and hydroxypropyl groups of the C10 analogues fit into the extracellular hydrophobic region of the binding pocket, consisting of the extracellular loops and the junction of several helices, without incurring any significant clashes with the protein structure.

Hence, the small difference in predicted binding energies for these systems is unsurprising. The hydroxyl group in compound 3.29g also forms an additional H-bond to Glu90 (Figure 3.8).

Figure 3.8: Docked pose of the hydroxypropyl analogue, compound 3.29g

Others have investigated docking of SPD (2.1) at the D3 receptor computationally.355 As compared to prior work, we found similarities in the key residues that are important for affinity of

SPD (2.1) (and the C10 analogues), particularly Asp110, Ser192 and Phe345. Apart from the key

98 protonated nitrogen- Asp110 salt bridge interaction, in both studies Ser192 interacts with an aromatic hydroxyl group via a hydrogen bond and Phe345 interacts with an aromatic ring via aromatic stacking (Figure 3.6a). The main difference is that the orientation of SPD (2.1) in our

Glide docking study is reversed compared to the AutoDock derived pose in the previous work.

Thus in our case Ser192 is H-bonded to the phenolic hydroxyl in ring A and Phe345 interacts with ring A, whereas in prior work Ser192 and Phe345 have interactions with the ring D hydroxyl and ring D respectively. Given the close to symmetric structure of the molecule and its high rigidity, this reversal has little impact on the interactions with the receptor pocket, except for the formation of an additional H-bonding interaction between the phenolic group in ring D of SPD (2.1) and the backbone O of Cys181 in our docked pose.

3.4.4 Structure-activity relationship study at σ receptors

3.4.4.1 Background

There are two types of σ receptors - σ1 and σ2, as borne out by differential binding of selective radioligands as well as other biochemical and pharmacological studies.356-358 σ receptors are distributed in the central nervous system (CNS) and in peripheral tissue.359, 360 Within the CNS,

σ1 receptors are concentrated in the hippocampus and other limbic regions which control cognition and emotion.361, 362 Thus these receptors have been implicated in a number of neuropsychiatric disorders and have been targets for the development of treatment interventions for such diseases.363-365 σ2 receptors are found predominantly in regions responsible for motor functions in the CNS and are also highly expressed in the lung, liver and kidneys.361, 362 The σ1 receptor has been cloned but the σ2 receptor has not, and there is some debate about the identity of the σ2 receptor.366 Recent studies suggest that the σ2 receptor is Progesterone Receptor Membrane

Component 1 (PGRMC1).367, 368 σ2 receptors are overexpressed in tumor cells and are upregulated in rapidly proliferating cells and this receptor has attracted interest as a biomarker for cancer proliferation.369, 370 Some σ2 ligands (particularly σ2 receptor agonists) display cytotoxic activity.371 Thus, σ2 ligands have been of interest for the development of "stand alone" antitumor drugs, targeted drug delivery agents and Positron Emission Tomography (PET) tools for imaging the proliferative status of various cancers.367, 372-374 σ2 receptor ligands have been predominantly from four structural classes: 6,7-dimethoxytetrahydroisoquinolines, indoles, tropanes and cyclohexylpiperazines (Figure 3.9). Many of these compounds lack sufficient selectivity or are otherwise hampered by unfavorable pharmacokinetic drawbacks that limit their usefulness as drugs, anticancer drug delivery vehicles or tools. New, potent and selective σ2 receptor ligands are sought after to fully exploit the potential of the σ2 receptor as a target for cancer diagnosis and therapy.

(-)-SPD (2.1) also shows good affinity for σ2 (Ki = 54 nM), as revealed by data from the

Psychoactive Drug Screening Program (PDSP) database.335 Given the comparatively high σ2 affinity of (-)-SPD (2.1), we considered that the C10 THPB analogues we made are also exploitable towards the development of novel potent and selective σ2 receptor ligands. Since, no SAR studies have been performed on THPBs in relation to their σ2 receptor affinity, such studies could propel our understanding of the potential utility of this scaffold towards obtaining novel, selective σ2 receptor ligands.

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3.4.4.2 Structure-activity correlations

All C10 THPB analogues assayed for binding affinity to human σ1 and σ2 receptors by the

Psychoactive Drug Screening Program (PDSP). Binding affinity data is shown in Table 3.7. At the

σ1 receptor, all analogues tested showed increased affinity as compared to (±)-SPD (2.1) (ranging from approximately 2-fold to 22-fold increase in σ1 affinity). Therefore, the C10 phenol functionality is not required for σ1 receptor affinity. No clear SAR trend could be discerned in the homologous series 3.29a-3.29f. The n-butyl analogue 3.29d had the highest affinity of the compounds in this series (Ki = 13 nM for 3.29d vs 269 nM for (±)-SPD (2.1)). The hydroxypropyl analogue 3.29g had lower affinity than 3.29d (53 vs 13 nM) which suggests that the hydroxyl group in 3.29g does not have any significant binding interactions with the σ1 receptor (akin to observations at the D1 receptor). The cyclopropylmethyl and n-butyl analogues had similar σ1 affinities- approximately 12 nM (a situation that mirrors results for 3.29h at the D1 receptor). It

101 thus appears that the cyclopropylmethyl group may function as a less hydrophobic bioisostere of the n-butyl group in this series at σ1 receptors. A clear trend was seen at the σ2 receptor for analogues 3.29a-3.29f, wherein there was a progressive decrease in σ2 receptor affinity as the alkyl chain was extended. Compounds 3.29a and 3.29b had the highest affinity of all compounds assayed (1 nM and 1.3 nM respectively), representing an approximately 7- to 9-fold higher σ2 receptor affinity than (±)-SPD (2.1). With respect to

Table 3.7: Ki data for C10 analogues at dopamine and σ receptors

Ki (nM) Compound R σ1/ σ2 σ1a σ2b (±)-2.1 H 269 9 30 3.29a Me 106 1 106 3.29b Et 124 1.3 95 3.29c n-Pr 57 6.7 8.5 3.29d n-Bu 13 49 0.27 3.29e n-Pent 119 97 1.22 3.29f n-Hex 37 107 0.35 3.29g hydroxypropyl 53 3.8 13.9 3.29h cyclopropylmethyl 12 2.4 5

a [3H](+)-pentazocine used as radioligand/haloperidol used as reference compound - Ki = 1.1 b nM; [3H]DTG used as radioligand/haloperidol used as reference compound - Ki = 10.0 nM;

102 binding at σ receptors, both 3.29a and 3.29b exhibited higher σ2 receptor selectivity than compound (±)-SPD (2.1) (approximately 100-fold for 3.29a and 3.29b versus 30-fold for compound (±)-SPD (2.1). All other analogues had decreased selective affinity for the σ2 receptor as compared to the σ1 receptor. In fact, this σ receptor selectivity was reversed for compounds

3.29d and 3.29f which both showed approximately 3-fold higher selectivity for σ1 versus σ2 receptors. Analogues 3.29a-3.29c had higher affinity than (±)-SPD (2.1), indicating that small C10 alkoxy groups are better tolerated than the C10 phenolic group at the σ2 receptor. Compounds

3.29g and 3.29h displayed very high σ2 receptor affinity, comparable to 3.29a and 3.29b; thus there is some tolerance for small polar or hydrophobic groups at C10 for σ2 affinity.

As alluded to before, some compounds have shown cytotoxic actions that are attributable to their interaction with σ receptors. We were curious to determine the extent to which our newly identified high affinity σ2 receptor ligands displayed cytotoxicity. Therefore, 3.29a, the most potent and selective σ2 ligand identified in this study, was selected for cytotoxicity evaluation in an Alamar

375 Blue assay. Compound 3.29a lacked significant cytotoxic activity in this assay, with an IC50 of

178 μM. Since σ2 receptor agonists are generally cytotoxic,376-378 this result tends to imply that compound 3.29a is a σ2 receptor antagonist (although this needs to be ratified with further assays).

3.5 Synthesis and evaluation of (-)-C3 analogues

3.5.1 Rationale and analogue design

Substituent pattern investigated at the C3 position in previous SAR studies was limited to methoxy, methylenedioxy or hydroxy functionalities (discussed in chapter 2, section 2.5). These

SAR studies indicate that having hydroxyl group at C3 position diminishes the activity at dopamine receptors and an alkoxy group is necessary for the activity. Still much chemi9cal diversity space is present to explore this site using various alkoxy chains having different carbon

103 length. Filling this deficient element, our project was aimed to design, synthesize and evaluate C3 alkoxy analogues of (-)-SPD (2.1) at dopamine receptors. Examining the binding affinity of these variant bulk analogues would provide some idea about the size of the binding pocket in the vicinity of C3 at the various receptors.. Compounds 3.35a-3.35f (Figure 3.10) have been designed to test the effect of extended alkyl chain and fluorine-containing homologs on the affinity and activity at

D1, D2 and D3 receptors.

3.5.2 Results and discussion

3.5.2.1 Synthesis

Compounds 3.35a-3.35f were synthesized using the procedure developed for (-)-SPD (2.1) as shown in Scheme 3.12. Commercially availably dihydroxy benzaldehyde (3.36) was selectively protected with benzyl group as per the reported procedure to give compound 3.37.379 The phenolic group of aldehyde 3.37 was protected with silyl group and the intermediate was subjected to

Henry’s condensation reaction to give nitrostyrene 3.38. Reduction of nitro compound 3.38 using

LiBH4 yielded primary amine 3.39. Aminolysis of lactone 2.102 with primary amine 3.39 was carried out to give amide alcohol 3.40, which was acetylated to afford 3.41. Ring B of the THPB scaffold was formed via Bischler-Napieralski cyclization followed by asymmetric hydrogenation using Noyori’s catalyst and formic acid/triethylamine mixture to generate 3.42 with good yield

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(88%).345 Hydrolysis of acetyl group and subsequent chlorination endowed the tetracyclic scaffold of THPB in compound 3.43. The enantiomeric excess of this precursor was found to be 90.2%

(chiral HPLC) and it was used for further analogue generation. Alkylation of compound 3.43 followed by debenzylation provided the C3 analogues 3.35a-3.35f.

3.5.2.2 Structure-activity correlations

All C3 analogues were assayed for binding affinity to human dopamine D1, D2 and D3 receptors by the PDSP. This data is presented in Table 3.8. The highest activity at D1 receptor was demonstrated by compounds 3.35b and 3.35a, which were 2.5 and 3-fold lower than parent compound (-)-SPD (2.1) respectively. This result suggests that an increase in the alkyl chain length at C3 position decreases affinity at D1 receptor, the same trend also exhibited by other compounds.

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In comparison to other analogues, compound 3.35b showed 2.3 to 3.5 times higher binding affinity at the D1 receptor. D1 receptor affinity was increased 1.2 to 1.7 fold while moving from four carbon chain to five and six carbon chain (3.35c vs 3.35d and 3.35e). As displayed by compound

3.35f, terminal introduction of the fluoro group to the ethyl chain decreases the affinity 2 times when compared with 3.35a. The selectivity for D1 versus D2 receptor was slightly improved for compound 3.35b when compared with (-)-SPD (2.1). Though 3.35f and 3.35a have slightly decreased selectivity at D1 receptor, they are comparable with (-)-SPD (2.1).

Table 3.8: Binding affinity (Ki nM) for C3 analogues at dopamine receptors

a Ki (nM) Selectivity Compound R D1b D2c D3d D2/D1 D2/D3 D3/D1 3.35a Et 17.0 2089.0 40.0 122.9 52.2 2.4 3.35b n-Pr 15.0 2682.0 46.0 178.8 58.3 3.1 3.35c n-Bu 53.0 2739.0 51.0 51.7 59.5 1 3.35d n-Pen 31.0 1433.0 33.0 46.2 43.4 1.1 3.35e n-Hex 42.0 1491.0 26.0 35.5 57.4 0.6 3.35f 2-fluoroethyl 34.0 5045.0 86.0 148.4 58.7 2.5 (-)-SPD 2.1 Me 5.9 974.3 30.1 165.1 32.4 5.1 a b Experiments carried out in triplicate - SEM values are within 13% of reported Ki; [3H]SCH2390 used as radioligand/(+)- c butaclamol used as reference compound - Ki = 2.3 nM; [3H]Nmethylspiperone used as radioligand/haloperidol used as reference d compound - Ki = 9.6 nM; [3H]N-methylspiperone used as radioligand/chlorpromazine used as reference compound - Ki = 11.0 nM.

D1 versus D2 receptor selectivity progressively decreases from four carbon alkoxy chain to six carbon alkoxy chain (3.35c-3.35e). All C3 analogues displayed a reduced affinity at D2

106 receptor from 1.47 to 5.2 fold as compared to (-)-SPD (2.1). This reveals the importance of these substitutions in suppressing the dopamine D2 receptor activity. The affinity observed at D3 receptor was comparable between C3 analogues and (-)-SPD (2.1). Slightly improvement in affinity was seen in 3.35e (26.0 nM vs 30.1 nM), while a 2.8 fold reduced activity was found in compound 3.35f. At the same time, the selectivity at D3 receptor versus D2 receptor is enhanced by 1.3 to 1.8 fold as measured against (-)-SPD for all C3 compounds. Compounds 3.35e (8.5 times), 3.35c (5.1 times) and 3.35d (4.6 times) have higher selectivity for D3 versus D1 receptor than (-)-SPD (2.1). That confers that larger lipophilc chains are more tolerated at this site for D3 receptor activity. Other compounds (3.35a, 3.35b, 3.35f) have comparable selectivity between 1.6-

2.1 fold for D3 receptor versus D1 receptor as (-)-SPD (2.1).

3.6 Metabolic stability study

3.6.1 Rationale

Although (-)-SPD (2.1) possesses favorable properties for delivery to the CNS, rapid and extensive pre-systemic metabolism restricts its application. Previous pharmacokinetic and metabolic stability study of (-)-SPD (2.1) suggests that the phenolic hydroxyl groups of (-)-SPD

(2.1) are molecular targets for the unwanted pre-systemic metabolism via O-glucuronidation and

O-sulfation.340

It is very crucial to evaluate the effect of substitutions at C3 and C10 position on metabolic stability of phenolic hydroxyl groups of (-)-SPD (2.1). In this project we aimed to study the metabolic stability of the representative C3 and C10 analogues that we have synthesized using rat hepatocytes. Among the routinely used in vitro systems, such as microsomes and hepatocytes,380 microsomes are usually used to determine cytochrome P450-mediated metabolism (phase I).

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Hepatocytes, having intact cell membranes and physiological concentrations of enzymes and cofactors, are believed to be a model close to whole liver for drug clearance measurements.381-384

Though human hepatocytes mimic the ideal environment of hepatic metabolism,385 we chose rat hepatocytes because of their cost effectiveness and because they are easily available as compared to human hepatocytes. Our next goal in this context is to study metabolic stability study using human hepatocytes.

This study would provide us a valuable comparison of metabolic stability pattern of (-)-

SPD (2.1) and its various analogues. It would add a significant piece of information while searching for a metabolically stable THPB scaffold having superior biological activity at dopamine receptors. As shown in Figure 11, compounds (-)-SPD (2.1), 3.29a, 3.29f, 3.35e, 3.35f, and 3.44 as representative analogues from C3 and C10 series were studied for in vitro metabolic stability using rat hepatocytes. Analogue 3.44 was selected to serve as a member of tetra-alkoxy substituted

THPB scaffold.

3.6.2 Results

For tested compounds, the % AUC was calculated from the LC/MS peak abundance in comparison with that total AUC. The % AUC data for each compound during the 36 h time course is shown in Table 3.9 and the time versus % AUC graph is depicted in Figure 3.12.

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Table 3.9: Metabolic stability study data for representative compounds % of total AUC at given time point Time (h) (-)-SPD 3.29a 3.29f 3.35e 3.35f 3.44 (2.1) 0 100 100 100 96 100 100 1 98 95 95 81 97 100 2 91 92 86 89 89 100 3 82 74 72 78 66 100 6 52 23 31 47 26 100 12 10 22 3 10 0 100 24 10 8 3 1 3 99 36 11 8 1 0 3 100

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120 SPD 3.29a 3.29f 3.35f 3.35e 3.44 100

% of total AUC at given point given timeAUC attotal of % 0 0 5 10 15 20 25 30 35 40 Time (h)

Figure 3.12: % of total AUC at given time period of incubation for representative analogues

According to % AUC derived at particular time point, it was found that within six hours, all of the compounds were metabolized to more than 50% except tetrasubstituted analogue 3.44.

Mono glucuronidated metabolite was predominant for all the tested compounds (shown in Figure

3.13; 3.45 for 2.1, 3.46 for 3.29a, 3.47 for 3.29f, 3.48 for 3.35e, and 3.49 for 3.35f). Peak abundance of sulfate metabolites for each compound was very minor (< 0.5% of total AUC), which suggests that O-glucuronidation is the major pathway for THPB metabolism in liver. These results are in accordance with the previous study in terms of the major metabolite observed i.e. O- glucuronidated.340

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No difference was demonstrated in the rate of metabolism between dihydroxy and monohydroxy analogues (3.29a, 3.29f versus 2.1, 3.35e, 3.35f). As per our expectation, no metabolism was observed during the whole course of experiment for tetrasubstituted analogue

3.44. Previous study has revealed that C10 hydroxyl group is the primary site for metabolism and it get glucuronidated first in (-)-SPD (2.1).341 This trend can be applied to other C3 analogues

(3.35e and 3.35f) having C2 and C10 free phenolic hydroxyl group similar to (-)-SPD (2.1). C10 analogues 3.29a and 3.29f do not have a free C10 hydroxyl group, but those having the C2 hydroxyl group free, get glucuronidated at similar rates as (-)-SPD. This indicates that the C2 phenol is also susceptible to glucuronidation to a similar extent as the C10 phenolic hydroxyl.

Based on results of previous study and our results here, we surmise that having C2alkoxy/C10 hydroxyl pattern in the THPB scaffold affords a similar rate of metabolism as the C2, C10- dihydroxy analogues (2.1, 3.35e, 3.35f).

Tetrasubstituted analogue (3.44) remained stable during the 36 hour time period of the assay with same abundance (% AUC); no metabolism was observed during whole experiment. We reasoned that the unavailability of free phenolic OH group is responsible for inertness of this compound towards metabolism. Steric hindrance due to the bulkier benzyl moieties at C2 and C10 position is also another possibility for metabolic stability of compound 3.44, as this could slow down oxidative dealkylation of the alkoxy groups. To examine this prospect, compounds having smaller alkoxy tetrasubstitutions, for instance (-)-THP (2.2), need to be tested for stability in the hepatic environment.

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3.7 Conclusions

In our work we have addressed prominent issues and missing gaps towards the synthesis and SAR studies of THPBs at dopamine D1, D2 and D3 receptors. We have developed a novel route to synthesize (±)-SPD (2.1) in eight synthetic steps with 30% overall yield via the intermediacy of easily obtainable diester 3.7. Compared to previous reported syntheses, this approach is advantageous in terms of conciseness, ease of synthetic manipulations and superior overall yield. Additionally, this route is applicable to prepare 8-oxoprotoberberine analogues as exemplified by oxypalmatine (3.15).

The enantioselective synthesis of (-)-SPD (2.1) was accomplished in 23% overall yield from compound 3.5 in a fourteen step sequence. The crystal structure of (-)-SPD (2.1) was determined by single crystal X-ray diffraction, which validated the success of this method. This is comparable to Yang’s first synthesis where a 24% yield of (-)-SPD (2.1) was obtained in eleven steps (from a 4-benzyloxy-3-hydroxy phenylacetic acid precursor).293 This, route involves an alternative, reliable preparation of the vital intermediate compound 2.102. The synthetic manipulations to afford 2.102 are straightforward, scalable and generally high yielding. This synthetic pathway we applied to generate of C3 chiral analogues of (-)-SPD (2.1) for bioactivity studies.

While searching for selective D1/D3 receptor ligands, we have designed and synthesized

C10 alkoxy THPB analogues 3.29a-3.29h. The C10 THPB analogues seem to exhibit a general preference for the D1 receptor. The lowest D1 affinity seen for any analogue was 40 nM (for compound 3.29f). Thus the C10 position seems to be fairly tolerant of alkoxy substituent groups with respect to D1 affinity. Among the 3 dopamine receptors, affinity was generally highest for

112 the D1 receptor and lowest for the D2 receptor. Overall, the general order of affinities for C10 analogues at the dopamine receptors evaluated is D1>D3>D2. The clear trend observed in the methyl to n-hexyl series at the D3 receptor, where an increase in the alkyl chain length was associated with a progressive decrease in affinity for the receptor, indicates tolerance of smaller alkoxy substituents at C10 position of THPB core. Methyl to n-butyl analogues have demonstrated improved binding affinity at D3 receptor as compared to lead compound (±)-SPD (2.1) (37 to 65 nM versus 101 nm).

The ligand binding energies from the D3 receptor docking studies show some similarities and minor discrepancies compared to the experimentally derived affinities. This is not surprising as empirical scoring functions, such as Glidescore used in this study, cannot rank order correctly and quantitatively discriminate all protein-ligand complexes based on the predicted scores. Further exploration of a significantly larger and structurally more diverse ligand library would be required to provide deeper understanding and quantitative evaluation of the effectiveness of our in silico modeling process.

Using our enantioselective method to synthesize (-)-SPD (2.1), we have produced optically pure C3 analogues (3.35a-3.35f) of THPB. The results for binding affinity data suggest that introducing the alkyl chain substitution at C3 position, somewhat decreases D1 affinity, maintains or slightly decreases D3 receptor affinity while improving selectivity at D3 versus D2 receptor when compared with (-)-SPD (2.1). All C3 analogues displayed a reduced affinity at the D2 receptor from 1.47 to 5.2 fold as compared to (-)-SPD (2.1). This reveals the significance of C3 alkoxy substituents to suppress the dopamine D2 receptor affinity. In contrast to the C10 analogue series, larger lipophilic substituents are well tolerated at the C3 position for D3 receptor affinity as demonstrated by n-hexyl (3.35e) and n-pentyl (3.35d) compounds.

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In our efforts to examine the effect of C10 and C3 alkoxy substituents on the metabolic stability against hepatic enzymes, we studied representative analogues having di, tri and tetra alkoxy substituents on the THPB scaffold. The results of this metabolic study indicate that having phenolic hydroxyl group at C2 and/or C10 on THPB scaffold engenders susceptibility to hepatic metabolism. Since this study was done using rat hepatocytes, further studies need to be done using either fresh or cryopreserved human hepatocytes to mimic the actual human hepatic metabolism environment.

3.8 Experimental

3.8.1 Chemistry

4-bromo-1-(ethoxymethoxy)-2-methoxybenzene (3.6)

Compound 3.5 (8.0 g, 39.4 mmol) and diisopropylethyl amine (13.7 mL, 78.8 mmol) were stirred in DCM (100 mL) at 0 ̊ C for 15 min. Ethoxycholoromethyl ether (5.48 mL, 59.1 mmol) was added dropwise to the solution at 0 ̊ C. The solution was allowed to stir at rt for 1 h. The reaction mixture was washed with 0.1N HCl (120 mL). The organic layers were combined, dried over sodium sulfate and evaporated to dryness to give 3.6 (11.1 g, quantitative) as a brownish oil.

This product was used in the subsequent reaction without any further purification: 1H NMR

(CDCl3, 500 MHz) δ 7.06-7.00 (m, 3H), 5.24 (s, 2H), 3.86 (s,3H), 3.76 (q, J = 7.1 Hz, 2H), 1.22

13 (t, J = 7.1 Hz, 3H); C NMR (CDCl3, 125 MHz) δ 150.5, 145.9, 123.6, 117.7, 115.2, 114.4, 94.2,

+ 64.5, 56.1, 15.1; HRMS (ESI) m/z calcd. for C10H13BrO3 ([M+Na] ), 282.9940, found 282.9942.

Methyl-3-(ethoxymethoxy)-2-methoxy-6-(2-methoxy-2-oxoethyl)benzoate (3.7)

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Diisopropylamine (11.49 mL, 84.3 mmol) was stirred in dry THF (100 mL) at -78 ºC in an oven-dried round bottom flask for 15 min. n-butyllithium (2.5 M in hexane, 100 mL) was added and the solution allowed to stir at -78 ºC for 30 min. Dimethylmalonate (18.85 mL, 164.7 mmol) in 60 mL dry THF was added. After stirring the solution at -78 ºC for 1 h, a solution of 3.6 (10.0 g, 38.3 mmol) in 50 mL dry THF was added. After 45 min, the reaction mixture was quenched with a saturated solution of ammonium chloride. THF was evaporated and the crude mixture was extracted with dichloromethane (4 × 100 mL). The solvent was reduced in vacuo and the crude product was purified via flash chromatography on silica gel (10% acetone/hexanes) to afford

1 compound 3.7 (6.7 g, 56%) as a yellowish oil: H NMR (CDCl3, 500 MHz) δ 7.20 (d, J = 8.5 Hz,

1H), 6.96 (d, J = 8.5 Hz, 1H), 5.26 (s, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.75 (q, J = 7.1 Hz, 2H),

13 3.68 (s, 3H), 3.62 (s, 2H), 1.23 (t, J = 7.1 Hz, 3H); C NMR (CDCl3, 125 MHz) δ 171.5, 167.6,

149.7, 147.6, 129.0, 126.4, 125.6, 118.0, 93.8, 64.6, 61.7, 52.2, 52.1, 38.4, 15.1; HRMS (ESI) m/z

+ calcd. for C15H20O7 ([M+Na] ), 335.1101, found 335.1104.

2-[4-(ethoxymethoxy)-3-methoxy-2-(methoxycarbonyl)phenyl]acetic acid (3.8)

Compound 3.7 (5.8 g, 18.4 mmol) and potassium carbonate (5.1 g, 36.8 mmol) were refluxed in a 1:1 water/methanol mixture (150 mL) for 1 h. The methanol was evaporated and the crude mixture was acidified with 0.1N HCl. The solution was extracted with ethyl acetate (3 × 100 mL) and dried over sodium sulfate. Filtration and evaporation of the ethyl acetate extract afforded

3.8 (5.4 g, 98%) as a yellowish oil. This was used in the next step without purification: 1H NMR

(CDCl3, 500 MHz) δ 7.21 (dd, J = 8.4, 2.7 Hz, 1H), 6.97 (dd, J = 8.4, 3.2 Hz , 1H), 5.26 (d, J =

1.8 Hz, 2H), 3.90 (d, J = 3.7 Hz, 3H), 3.88 (d, J = 2.0 Hz, 3H), 3.76 (q, J = 1.75 Hz, 2H), 3.64 (s,

13 2H), 1.27-1.21 (m, 3H); C NMR (CDCl3, 125 MHz) δ 171.1, 168.8, 150.0, 147.9, 128.3, 126.6,

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+ 125.4, 118.5, 93.7, 64.6, 61.7, 52.7, 38.9, 15.1; HRMS (ESI) m/z calcd. for C14H18O7 ([M+Na] ),

321.0945, found 321.0948.

Methyl-6-(2-((4-(benzyloxy)-3-methoxyphenethyl)amino)-2-oxoethyl)-3-(ethoxymethoxy)-2- methoxybenzoate (3.10)

Compound 3.8 (5.0 g, 16.8 mmol) was dissolved in 60 mL DCM and stirred at 0 ̊ C for 15 min. EDCI (3.2 g, 16.8 mmol) was added portion-wise to the solution at 0 ̊ C and the mixture allowed to stir at rt for 1 h. A solution of 3.9 (4.3 g, 16.8 mmol) in 60 mL DCM was added to the above reaction mixture at 0 ̊ C and allowed to stir overnight at rt. 50 mL DCM was added to the reaction mixture and it was washed with sodium bicarbonate and 0.1N HCl consecutively. The organic layer was dried, filtered and concentrated in vacuo to afford 3.10 (7.3 g, 81%) as a brown

1 oil : H NMR (CDCl3, 500 MHz) δ 7.43 (d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.3 Hz , 2H), 7.30 (d, J =

7.4 Hz, 1H), 7.17 (d, J = 8.5, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.68 (d, J =

1.9 Hz, 1H), 6.51 (dd, J = 8.1, 1.9 Hz, 1H), 5.25 (s, 2H), 5.11 (s, 2H), 3.88 (s, 6H), 3.84 (s, 3H),

3.75 (q, J = 7.05 Hz, 2H), 3.43-3.39 (m, 4H), 2.67 (t, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); 13C

NMR (CDCl3, 125 MHz) δ 170.3, 168.4, 149.7, 149.6, 147.3, 146.7, 137.4, 132.1, 128.8, 128.5,

128.5, 127.8, 127.3, 127.3, 126.8, 126.1, 120.6, 118.5, 114.2, 112.4, 93.8, 71.1, 64.7, 61.6, 55.9,

+ 52.6, 41.2, 40.9, 35.2, 15.1; HRMS (ESI) m/z calcd. for C30H35NO8 ([M+Na] ), 560.2255, found

560.2260.

2-(benzyloxy)-10-hydroxy-3,9-dimethoxy-5,6,13,13a-tetrahydro-8H-isoquinolino[3,2- a]isoquinolin-8-one (3.11)

Compound 3.10 (5 g, 9.3 mmol) and POCl3 (5.2 mL, 55.8 mmol) were refluxed in 80 mL acetonitrile for 2 h. The solvent was evaporated and the residue was dissolved in 120 mL DCM.

The resulting solution was washed with sodium bicarbonate and then dried over sodium sulfate.

116

The organic solvent was removed in vacuo and methanol (100 mL) was added. Sodium borohydride (0.7 g, 18.6 mmol) was added at 0 ̊ C and the mixture allowed to stir overnight. The reaction was quenched with water and methanol was evaporated. The residue was extracted with

DCM (3 × 80 mL). The organic layer was dried, filtered and evaporated to dryness to afford 3.11

(3.3 g, 82% over two steps). It was used in the next step without further purification: 1H NMR

(CDCl3, 500 MHz) δ 7.44 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz , 2H), 7.33 (d, J = 7.3 Hz, 1H),

7.06 (d, J =8.1 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.71 (s, 1H), 6.68 (s, 1H), 5.15 (s, 2H), 5.00-4.98

(m,1H), 4.68 (dd, J = 13.0, 3.4, 1H), 4.00 (s, 3H), 3.90 (s, 3H), 2.95-2.71 (m, 5H); 13C NMR

(CDCl3, 125 MHz) δ 162.5, 149.0, 148.8, 147.3, 147, 137.0, 130.7, 128.6, 128.6, 128.1, 127.9,

127.51, 127.4, 127.4, 121.5, 122.8, 118.1, 112.6, 111.9, 71.6, 62.4, 56.1, 54.9, 38.9, 38.3, 29.4;

+ HRMS (ESI) m/z calcd. for C26H25NO5 ([M+H] ), 432.1805, found 432.1809.

(±)-SPD (2.1)

Lithium aluminum hydride (40 mg, 1.1 mmol) was stirred in THF at 0 ̊ C for 10 min.

Compound 3.11 (0.3 g, 0.7 mmol) in THF was added to the reaction mixture at 0 ̊ C and it was allowed to reflux for 2 h. The reaction mixture was allowed to cool to rt and excess of lithium aluminum hydride was quenched with water. THF was evaporated and crude mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The crude product was refluxed in mixture of methanol (5 mL) and concentrated hydrochloric acid (5 mL) for 3 h. The reaction mixture was basified by ammonia solution and extracted with dichloromethane (20 mL × 2). The combined organic layer was dried over sodium sulfate and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (2%-6% methanol/dichloromethane) to give compound 2.1

1 (0.29 g, 81% over two steps): Yellowish white crystals, mp 157 ̊ C; H NMR (CDCl3, 400MHz)

117

δ 6.83-6.78 (m, 3H), 6.60 (s, 1H), 4.20 (d, J = 15.8 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.58-3.53

13 (m, 2H), 3.27-3.10 (m, 3H), 2.80 (dd, J = 15.9, 11.4 Hz, 1H), 2.70-2.62 (m, 2H); C NMR (CDCl3,

400MHz) δ 146.4, 145.1, 144.0, 143.1, 130.4, 127.8, 127.3, 125.9, 125.0, 114.1, 111.3, 110.6,

+ 60.7, 59.3, 55.9, 58.9, 51.7, 36.1, 29.1; HRMS (ESI) m/z calcd. for C19H21NO4 ([M+H] ),

328.1551, found 328.1543.

2-(benzyloxy)-10-hydroxy-3,9-dimethoxy-5,6-dihydro-8H-isoquinolino[3,2-a]isoquinolin-8- one: (3.14)

Compound 3.10 (0.2 g, 0.37 mmol) and POCl3 (0.28 mL, 2.97 mmol) were refluxed in 10 mL acetonitrile for 3 h. The solvent was evaporated and the residue was dissolved in 25 mL DCM.

The resulting solution was washed with sodium bicarbonate and then organic layer was dried over sodium sulfate. The organic solvent was removed in vacuo and the residues were dissolved in 10 mL dry DCM. Triethylamine (1.6 mL, 0.103 mmol) was added to the solution and allowed to stir at rt for 2 h. Reaction mixture was washed with 0.1N HCl and extracted with DCM (3 × 15 mL).

The combined organic layer was evaporated and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (1.5%-3% methanol/dichloromethane)

1 to give compound 3.14 (0.11 g, 70% over two steps): H NMR (CDCl3, 500MHz) δ 7.49 (d, J

=7.4 Hz, 2H), 7.40 (t, J = 7.3 Hz , 2H), 7.35-7.30 (m,2H), 7.27 (s, 1H), 7.20 (d, J = 8.6 Hz, 1H),

6.75 (s, 1H), 6.60 (s, 1H), 5.21 (s, 2H), 4.31 (t, J = 6.1 Hz, 2H), 4.04 (s, 3H), 3.93 (s, 3H), 2.92 (t,

13 J = 6.1 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 159.7, 151.0, 147.7, 147.6, 145.5, 136.9, 135.3,

132.2, 129.0, 128.5, 128.5, 127.9, 127.4, 127.4, 122.9, 122.3, 121.1, 118.1, 111.3, 111.0, 101.4,

+ 71.9, 62.6, 56.0, 39.4, 28.2; HRMS (ESI) m/z calcd. for C26H23NO5 ([M+H] ), 430.1649, found

430.1652.

Oxypalmatine (3.15)

118

Compound 3.14 (0.1 g, 0.23 mmol) was refluxed in a mixture of methanol (5 mL) and concentrated hydrochloric acid (3 mL) for 3h. Methanol was evaporated and the resultant mixture was basified with ammonia solution and extracted with dichloromethane (3 × 10 mL). The combined organic layer was dried and concentrated to give a crude product, which was used in the next step without further purification. Crude residues were dissolved in 5 mL DMF. Potassium carbonate (63 mg, 0.46 mmol) and methyl iodide (0.04 mL, 0.7 mmol) were added and reaction mixture was stirred at rt for 6 h. DMF was evaporated and 0.1 N HCl (10 mL) was added in it. The crude mixture was extracted with dichloromethane (3 × 10 mL). The combined organic layer was dried over sodium sulfate the crude product was purified by flash column chromatography on silica gel (1% - 3% methanol/dichloromethane) to yield oxypalmatine (3.15) (0.07 g, 79%) as yellowish

1 solid. mp: 178-185 ̊ C. H NMR (CDCl3, 500 MHz) δ 7.33 (d, J =8.8 Hz, 1H), 7.3 (d, J = 8.7 Hz

, 1H), 7.23 (s, 1H), 6.76 (s, 1H), 6.73 (s, 1H), 4.32 (t, J = 6.1 Hz, 2H), 4.02 (s, 3H), 3.98 (s, 3H),

13 3.96 (s, 3H), 3.94 (s, 3H), 2.93 (t, J = 6.1 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 160.3, 151.4,

150.1, 149.6, 148.4, 135.6, 132.4, 128.5, 122.3, 122.1, 119.4, 118.9, 110.5, 107.5, 100.9, 61.6,

+ 56.9, 56.24, 56.0, 39.4, 28.2; HRMS (ESI) m/z calcd. for C21H21NO5 ([M+H] ), 368.1492, found

368.1497.

2-(4-(benzyloxy)-3-methoxyphenethyl)-7-(ethoxymethoxy)-8-methoxyisoquinoline-

1,3(2H,4H)-dione (3.21)

Compound 3.10 (0.1 g, 0.18 mmol) was dissolved in MeOH (5 mL) and reaction mixture was allowed to cool at 0 ̊C. NaBH4 (14 mg, 0.36 mmol) was added slowly in to reaction mixture and it was stirred for 1 hr at rt. Excess NaBH4 was quenched with water and then solvent was evaporated. Crude mixture was extracted with DCM (2 X 10 mL), combined organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude residue, which was

119 purified by column chromatography on silica gel using 1:99 MeOH:DCM as eluent to afford

1 compound 3.21 (0.05 g, 54%); H NMR (500 MHz, CDCl3) δ 7.44 (m, 3H), 7.37 (t, J = 5.0 Hz,

2H), 7.29 (t, J = 7.3 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 1.2 Hz, 1H), 6.81-6.77 (m,

2H), 5.29 (s, 2H), 5.12 9s, 2H), 4.17-4.14 (m, 2H), 3.94-3.88 (m, 8H), 3.78 (q, J = 7.1 Hz, 2H),

13 2.86-2.83 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H); C NMR (500 MHz, CDCl3) δ 169.6, 162.6, 151.9,

150.7, 149.5, 146.7, 137.3, 131.9, 128.5 X 2, 128.3, 127.7, 127.3, 122.9, 122.4, 121.0, 119.6,

114.2, 114.1, 112.6, 94.2, 71.1, 64.7, 61.5, 56.0, 41.6, 36.4, 33.7, 15.1; HRMS (ESI) m/z calcd for

+ C29H31NO7 ([M+H] ), 506.2179, found 506.2168.

2-(4-(benzyloxy)-3-methoxyphenethyl)-7-(ethoxymethoxy)-8-methoxyisoquinoline-

1,3(2H,4H)-dione (3.22)

Solution of compound 3.10 (0.1 g, 0.18 mmol) in THF (5 mL) was added to the suspension of LiBH4 (8 mg, 0.36 mmol) in THF at 0 ̊C and reaction mixture was allowed to stir for 2 hr at rt.

Excess LiBH4 was quenched with water and then solvent was evaporated. Crude mixture was extracted with DCM (2 X 10 mL), combined organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude residue, which was purified by column chromatography on silica gel using 2:98 MeOH:DCM as eluent to afford compound 3.22 (0.05 g,

1 52%); H NMR (500 MHz, CDCl3) δ 7.42 (d, J = 7.3 Hz, 2H), 7.3 (t, J = 7.7 Hz, 2H), 7.30-7.27

(m, 1H), 7.17 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.82-6.81 (m, 2H), 6.72 (dd, J = 8.2,

2.0 Hz, 1H), 6.29 (s, 1H), 5.24 (s, 2H), 5.13 (s, 2H), 3.90-3.84 (m, 4H), 3.80-3.71 (m, 8H), 2.87

13 (t, J = 7.0, 2H), 2.73 (t, J = 6.0, 2H), 1.23 (t, J = 7.1 Hz, 3H); C NMR (500 MHz, CDCl3): δ

167.7, 149.8, 148.7, 146.9, 146.3, 137.3, 131.9, 131.8, 128.5, 127.8, 127.3, 126.0, 120.7, 118.0,

114.4, 112.5, 93.8, 71.1, 64.5, 63.8, 61.8, 56.0, 41.0, 35.5, 35.2, 15.1; HRMS (ESI) m/z calcd for

+ C29H35NO7 ([M+H] ), 510.2486, found 510.2489.

120 methyl 3-(benzyloxy)-2-methoxy-6-(2-methoxy-2-oxoethyl)benzoate (3.27): Compound 3.7 (2 g, 6.4mmol) was refluxed in methanol with conc. HCl (1 mL) for 2 hr.

Solvent was evaporated and the residues were dissolved in acetonitrile (50 mL). K2CO3 (1.7g, 12.8 mmol) and benzyl bromide (0.77 mL, 6.5 ) were added. Reaction mixture was refluxed for 3 hrs.

Solvent was evaporated and residues were extraced with water (30 mL) and ethyl acetate (20 mL x3). Ethyl acetate layer was dried over sodium sulfate and evaporated to dryness to give compound

3.27 ( 2.2 g, quant.) as brown oily liquid. This product was used in the subsequent reaction without

1 any further purification: H NMR (CDCl3, 400 MHz) δ 7.44-7.33 (m, 5H), 6.98-6.92 (m, 2H), 5.12

13 (s, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.67 (s, 3H), 3.62 (s, 3H) C NMR (CDCl3, 100 MHz) δ 171.56,

167.67, 151.11, 147.42, 136.65, 129.06, 128.64 x 2, 128.05, 127.24 x 2, 126.27, 124.79, 115.81,

+ 70.95, 61.62, 52.25, 52.06, 38.34; (ESI) m/z calcd. for C19H20O6 ([M+Na] ), 367.1152, found

367.1161.

3-(benzyloxy)-2-methoxy-6-(2-methoxy-2-oxoethyl)benzoic acid (3.28):

Compound 3.27 (2.1 g, 6.1 mmol) was relfuxed with 10% NaOH (10 mL ) in ethanol (20 mL ) for

1 hr. Solvent was evaporated and the residues were acidified with 3 N HCl and it was extraced with ethyl acetate (20 mL x 3). Organic layer was dried over sodium sulfate and evaporated to dryness. Residues were dissolved in to methanol and SOCl2 (0.11 mL, 1.5 mmol) was added and allowed it to stir overnight at rt. Mehanol was evaporated and residues were washed with water

(50 mL) and ethyl acetate (20 mL x 3). Ethyl acetate layer was dried over sodium sulfate and evaporated to dryness to give compund 3.28 (1.97 g, 98%) as yellowish viscous liquid. This

1 product was used in the subsequent reaction without any further purification: H NMR (CDCl3,

400 MHz) δ 7.45-7.34 (m, 5H), 7.08 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 5.15 (s, 2H),

13 4.04 (s, 3H), 3.88 (s, 2H), 3.71 (s, 3H); C NMR (CDCl3, 100 MHz) δ 172.20, 166.79, 150.88,

148.53, 136.16, 128.75, 128.41 x 2, 128.31, 128.15, 127.35 x 2, 124.50, 117.09, 71.16, 62.28,

121

+ 52.03, 39.78; (ESI) m/z calcd. for C18H18O6 ([M+Na] ), 335.0996, found 335.1003. 2-(benzyloxy)-

3,9,10-trimethoxy-5,8,13,13a-tetrahydro-6H- isoquinolino[3,2-a]isoquinoline (3.30)

Compound 3.11 (3.3 g, 7.6 mmol) in 50 mL THF was added to a suspension of lithium aluminum hydride (0.6 g, 15.2 mmol) at 0 ̊C and the reaction mixture allowed to come down at room temperature and then it was refluxed for 1 h. The reaction mixture was allowed to cool to rt and excess of lithium aluminum hydride was quenched with water. THF was evaporated and the crude mixture was extracted with dichloromethane (4 × 50 mL). Combined organic layers were dried over sodium sulfate and concentrated in vacuo and the crude product was purified via flash chromatography on silica gel (2% MeOH/DCM) to afford compound 3.30 (2.7 g, 84%) 1H NMR

(CDCl3, 500 MHz ) δ 7.46 (d, J = 7.4 Hz , 2H), 7.39-7.35 (m, 2H), 7.31 (t, J = 7.4 Hz, 1H), 6.78

(dd, J = 10.3, 8.3 Hz, 2H), 6.74 (s, 1H), 6.64 (s, 1H), 5.14 (s, 2H), 4.19 (d, J = 15.3 Hz, 1H), 3.88

13 (s, 3H), 3.80 (s, 3H), 3.56-3.49 (m, 2H), 3.20-3.06 (m, 3H), 2.73-2.61 (m, 3H); C NMR (CDCl3,

125 MHz) δ 148.3, 146.5, 146.5, 143.1, 137.3, 129.6, 128.5, 128.5, 128.0, 127.8, 127.5, 127.5,

127.4, 127.3, 125.0, 114.0. 112.2, 111.9, 71.6, 60.7, 59.3, 56.0, 53.9, 51.5, 36.2, 29.1 HRMS (ESI)

+ m/z calcd. for C26H27NO4 ([M+H] ), 418.2013, found 418.2007.

General procedure for synthesis of compounds 3.29a-3.29h (as described for compound

3.29a):

3,9,10-trimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol (3.29a)

Compound 3.30 (0.1 g, 0.24 mmol) was dissolved in DMF. Potassium carbonate (0.07 g,

0.48 mmol) and methyl iodide (0.02 mL, 0.36 mmol) were added and the reaction mixture was stirred overnight at rt. The crude reaction solution was filtered and the filtrate was evaporated to dryness. The residue was refluxed in a mixture of MeOH (5 mL) and concentrated HCl (3 mL) for

122

3 h. The MeOH was evaporated and the crude mixture was basified with ammonia solution and extracted with DCM (3 × 15 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (2% MeOH/DCM) to give compound 3.29a (0.052 g, 64%) as an off-white solid; mp

1 208-210 ̊ C; H NMR (CDCl3, 400 MHz) δ 6.87-6.77 (m, 3H), 6.60 (s, 1H), 5.56 (s, 1H), 4.24

(d, J = 15.7 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 6H), 3.54-3.50 (m, 2H), 3.26-3.11 (m, 3H), 2.81 (dd, J

13 = 16, 11.5 Hz, 1H), 2.68-2.61 (m, 2H); C NMR (CDCl3, 100 MHz) δ 150.2, 145.1, 143.9, 143.9,

130.6, 128.7, 127.9, 126.1, 124.0, 111.3, 111.0, 110.6, 60.2, 59.3, 59.3, 55.9, 54.0, 51.7, 36.3, 29.2;

+ HRMS (ESI) m/z calcd. for C20H23NO4 [M+H] , 342.1700, found 342.1704.

10-ethoxy-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol

(3.29b)

1 Yield 67%; off-white powder, mp 186-188 ̊ C ; H NMR (CDCl3, 400 MHz) δ 6.85-6.77

(m, 3H), 6.60 (s, 1H), 4.24 (d, J = 15.8 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.87 (s,

3H), 3.52 (d, J = 15.3 Hz, 2H), 3.27-3.10 (m, 3H), 2.84-2.61 (m, 3H), 1.44 (t, J = 7.0 Hz, 3H); 13C

NMR (CDCl3, 100 MHz) δ 147.5, 143.5 143.2, 142.0, 128.7, 126.7, 125.9, 124.2, 122.0, 110.5,

109.5, 108.7, 62.5, 58.2, 57.4, 54.0, 52.2, 49.8, 34.4, 27.3, 13.1; HRMS (ESI) m/z calcd. for

+ C21H25NO4 [M+H] , 356.1856, found 356.1861.

3,9-dimethoxy-10-propoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol

(3.29c)

1 Yield 68%; brown solid, mp 87-90 ̊ C; H NMR (CDCl3, 500 MHz) δ 6.84-6.78 (m, 3H),

6.60 (s, 1H), 4.23 (d, J = 15.8 Hz, 1H), 3.94 (dt, J = 6.7, 1.8 Hz, 2H), 3.87 (s, 6H), 3.51 (d, J =

15.3 Hz, 2H), 3.25-3.10 (m, 3H), 2.80 (dd, J = 15.6, 11.4 Hz, 1H), 2.68-2.60 (m, 2H), 1.84 (sex, J

13 = 7.1 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H); C NMR (CDCl3, 125 MHz) δ 149.6, 145.3, 145.1, 144.0,

123

130.6, 128.6, 127.8, 126.0, 123.8 112.3, 111.3, 110.6, 70.4, 60.2, 59.2, 55.9, 54.1, 51.7, 36.3, 29.2,

+ 22.8, 10.7; HRMS (ESI) m/z calcd. for C22H27NO4 [M+H] , 370.2013, found 370.2020.

10-butoxy-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol

(3.29d)

1 Yield 73%; orange oil; H NMR (CDCl3, 400 MHz) δ 6.84-6.77 (m, 3H), 6.60 (s, 1H), 4.24

(d, J = 15.7 Hz, 1H) 3.98 (dt, J =1.0, 6.45 Hz, 2H), 3.86 (s, 6H), 3.52 (d J = 15.0 Hz, 2H), 3.25-

3.10 (m, 3H), 2.80 (dd, J = 15.2, 11.3 Hz, 1H), 2.67-2.60 (m, 2H), 1.80 (quin, J = 6.9 Hz, 2H),

13 1.52 (sex, J = 7.6 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 149.6, 145.3,

145.1, 143.9, 130.5, 128.5, 127.7, 126.0, 123.8, 112.2, 111.3, 110.6, 68.5, 60.1, 59.2, 55.9, 54.0,

+ 51.6, 36.2, 31.5, 29.2, 19.4, 13.9; HRMS (ESI) m/z calcd. for C23H29NO4 [M+H] , 384.2169, found

384.2173.

3,9-dimethoxy-10-(pentyloxy)-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol

(3.29e)

1 Yield 65%; brown oil; H NMR (CDCl3, 400 MHz ) δ 6.84-6.76 (m, 3H), 6.60 (s, 1H), 4.23

(d, J = 15.8 Hz, 1H), 3.97 (dt, J =1.5, 6.55 Hz, 2H), 3.87 (s, 6H), 3.51 (d, J = 15.2 Hz, 2H), 3.26-

3.10 (m, 3H), 2.80 (dd, J = 15.7, 11.5 Hz, 1H), 2.68-2.60 (m, 2H), 1.82 (quin, J = 6.7 Hz, 2H),

13 1.49-1.36 (m, 4H), 0.93 (t, J = 7.2 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 149.6, 145.3, 145.0,

143.9, 130.6, 128.6, 127.7, 126.1, 123.8, 112.3, 111.3, 110.6, 68.9, 60.2, 59.2, 55.9, 54.6, 51.6,

+ 36.3, 29.2, 29.1, 28.3, 22.5, 14.0; HRMS (ESI) m/z calcd. for C24H31NO4 [M+H] , 398.2326, found

398.2326.

10-(hexyloxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinolin-2-ol

(3.29f)

124

1 Yield 70%; dark brown oil; H NMR (CDCl3, 500MHz ) δ 6.84-6.76 (m, 3H), 6.60 (s, 1H),

4.23 (d, J = 15.7 Hz, 1H), 3.97 (t, J = 6.6 Hz , 2H), 3.86 (s, 6H), 3.53-3.50 (m, 2H), 3.26-3.10 (m,

3H), 2.80 (dd, J = 15.8, 11.6 Hz, 1H), 2.68-2.60 (m, 2H), 1.81 (quin, J = 6.7 Hz, 2H), 1.48 (quin,

13 J= 7.2, 2H), 1.34 (sex, J = 3.8 Hz, 4H), 0.91 (t, J= 7.0 Hz, 3H); C NMR (CDCl3, 125 MHz) δ

149.6, 145.3, 145.0, 143.9, 130.6, 128.6, 127.7, 126.1, 123.8, 112.3, 111.4, 110.6, 68.9, 60.2, 59.2,

55.9, 54.1, 51.7, 36.3, 31.6, 29.4, 29.2, 25.8, 22.6, 14.0; HRMS (ESI) m/z calcd. for C25H33NO4

[M+H]+, 412.2482, found 412.2429.

10-(3-hydroxypropoxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2- a]isoquinolin-2-ol (3.29g)

1 Yield 76%; reddish-brown solid, mp 73-78 ̊ C; H NMR (CDCl3, 500MHz ) 6.87-6.80 (m,

3H), 6.60 (s, 1H), 4.22 (d, J = 15.7 Hz, 1H), 4.16 (td, J = 6.1, 1.5 Hz, 2H), 3.91-3.84 (m, 8H),

3.52 (d, J = 15.4 Hz, 2H), 3.27-3.10 (m, 3H), 2.80 (dd, J = 15.7, 11.6 Hz, 1H), 2.68-2.61 (m, 2H),

13 2.07 (quin, J = 5.8 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 149.4, 145.4, 145.1, 143.9, 130.5,

128.8, 128.4, 126.1, 124.1, 112.5, 111.3, 110.6, 67.5, 61.0, 60.3, 59.2, 55.9, 54.0, 51.6, 36.3, 32.1,

+ 29.2; HRMS (ESI) m/z calcd. for C22H27NO5 [M+H] , 386.1884, found 386.1897.

10-(cyclopropylmethoxy)-3,9-dimethoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2- a]isoquinolin-2-ol (3.29h)

1 Yield 81%; yellow oil; H NMR (CDCl3, 500 MHz ) δ 6.83-6.76 (m,3H), 6.60 (s, 1H),

4.23 (d, J = 15.7 Hz, 1H), 3.90- 3.83 (m, 8H), 3.51 (d, J = 15.1 Hz, 2H), 3.26-3.11 (m, 3H), 2.80

(dd, J = 15.0, 12.0 Hz, 1H), 2.68-2.62 (m, 2H), 1.31-1.27 (m, 1H), 0.61 (q, J = 5.7 Hz, 2H), 0.36

13 (q, J = 4.85 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 149.5, 145.7, 145.0, 143.9, 130.6, 128.6,

128.1, 126.1, 123.8, 113.1, 111.3, 110.6, 73.9, 60.2, 59.2, 55.9, 54.1, 51.6, 36.3, 29.2, 10.5, 3.2,

+ 3.2; HRMS (ESI) m/z calcd. for C23H27NO4 [M+H] , 382.2013, found 382.2019.

125

4-(benzyloxy)-3-hydroxybenzaldehyde (3.37)

To a stirring solution of 3,4-dihydroxybenzaldehyde, 3.36 (2.5 g, 18.1 mmol) in anhydrous

Acetonitrile (50 mL), was added K2CO3 (2.5 g, 18.1 mmol) followed by benzyl bromide (3.2 mL,

27.15 mmol) slowly, at room temperature, under an inert atmosphere. Resulting reaction mass was heated to reflux temperature and stirring was continued for 2 h. The reaction solvent was removed by evaporation under reduced pressure and to the resulted residue was added cold 10% NaOH solution and stirred for 10 min. and added the ethyl acetate (2 x 50 mL). Resulted biphasic mixture was separated and aqueous layer was acidified using 4N HCl and extracted with DCM (3 x 30 mL), combined organic layer was washed with brine solution, water, dried on Na2SO4, and concentrated under reduced pressure to obtain the residue, which was purified by crystallization

1 using ethylacetate to afford, 3.37 (2.7 g, 65%); H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 7.46-

13 7.38 (m, 7H), 7.03 (d, J = 8.2 Hz, 1H), 5.81 (s, 1H), 5.20 (s, 2H); C NMR (400 MHz, CDCl3) δ

190.9, 150.9, 146.3, 135.2, 130.8, 128.9 X 2, 128.8, 127.9 X 2, 124.3, 114.4, 111.5, 71.2; HRMS

+ (ESI) m/z calcd for C14H12O3 ([M+H] ), 229.0859, found 229.0863.

2-(benzyloxy)-5-(2-nitrovinyl)phenoxy)(tert-butyl)diphenylsilane (3.38)

Compound 3.37 (2.7 g, 11.8 mmol) was dissolved in anhydrous DCM (50 mL). Imidazole

(1.6 g, 23.6 mmol) and DMAP (0.1 g, 0.12 mmol) were added in to reaction mixture and allowed it to stir at 0 ̊C for 10 minute. To the reaction mixture, TBDPSCl (4.6 mL 17.7 mmol) was added and the reaction was allowed to stir overnight at rt. Reaction mixture was acidified with 2N HCl and extracted with (2 x 40 mL) DCM. Combined organic layer was washed with brine solution, water, dried on Na2SO4, and concentrated under reduced pressure to obtain the crude residue,

126 which was used in the next step without further purification. Residues were dissolved in 50 mL acetic acid, NH4OAc (1.0 , 12.98 mmol) and CH3NO2 (2.5, 47.2 mmol) were added in it and allowed it to reflux for 8 hr. Solvent was evaporated and the residues were basified using 10%

NaOH and extracted with DCM (2 x 50 mL). Combined organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain a crude residue, which was purified by column chromatography on silica gel using 10:90 EtOAC:Hexanes as eluent to afford compound 3.38 (5.1

1 g, 85% over two steps); H NMR (400 MHz, CDCl3) δ 7.73-7.63 (m, 5H), 7.46-7.27 (m, 11H),

13 7.05-7.00 (m, 2H), 6.86-6.77 (m, 2H), 4.97 (s, 2H), 1.10 (s, 9H); C NMR (400 MHz, CDCl3) δ

153.5, 145.8, 139.1, 136.1 X 2, 135.4 X 2 , 135.3 X 2, 135.0, 134.8, 132.8, 130.1, 129.7, 128.5 X

3, 128.0 X 2, 127.8 X 4, 127.4 X 2, 124.8, 122.7, 119.5, 113.5, 70.5, 26.6; HRMS (ESI) m/z calcd

+ for C31H31NO4Si ([M+H] ), 510.2101, found 510.2098.

2-(4-(benzyloxy)-3-((tert-butyldiphenylsilyl)oxy)phenyl)ethan-1-amine (3.39)

In a dry round bottom flask, LiBH4 (1.8 g, 82.4 mmol), was suspended in THF (30 mL) and TMSCl (5.3 mL, 41.2 mmol) was added slowly. Solution of compound 3.38 (5.1 g, 10.3 mmol) in THF (30 mL) was added slowly in to reaction mixture and allowed to reflux overnight. Reaction was allowed to cool at rt and excess LiBH4 was quenched with MeOH. 10% NaOH (20 mL) was added to the reaction and it was filtered through celite. Solvent was evaporated and the crude mixture was extracted with DCM (2 x 50 mL). Combined organic layer was dried over Na2SO4,

1 and concentrated under reduced pressure to obtain compound 3.39; H NMR (400 MHz, CDCl3)

δ 7.72-7.68 (m, 5H), 7.41-7.27 (m, 10H), 6.74 (d, J = 6.6 Hz, 1H) 6.62 (dd, J = 1.4, 6.5 Hz, 1H),

6.4 (d, J = 1.5 Hz, 1H), 4.88 (s, 2H), 2.72 (t, J = 5.8 Hz, 2H), 2.59 (t, J = 5.8 Hz, 2H), 1.08 (s, 9H);

13 C NMR (400 MHz, CDCl3) δ 147.5, 144.4, 136.1, 134.4 X 2 , 135.3 X 2, 133.7, 132.2, 129.1,

127

128.7 X 3, 128.6, 127.2 X 4, 126.8 X 2, 126.7, 126.4 X 2, 120.7, 119.7, 113.2 X 2, 69.6, 40.9,

+ 34.5, 25.5; HRMS (ESI) m/z calcd for C31H35NO2Si ([M+H] ), 482.2515, found 482.2509.

2-(4-(benzyloxy)-2-(hydroxymethyl)-3-methoxyphenyl)-N-(4-(benzyloxy)-3-((tert- butyldiphenylsilyl) oxy)phenethyl)acetamide (3.40)

Compound 2.102 (1.5 g, 5.3 mmol) and primary amine 3.39 (2.5g, 5.3 mmol) were refluxed in to ethanol (20 mL) for 16 h. Solvent was evaporated and the residues were purified by column

1 chromatography (2 % MeOH/DCM) to yield 3.40 (3.2g, 78%). H NMR (CDCl3, 600 MHz) δ 7.69

(dd, J = 8.1, 1.3 Hz, 4H), 7.43-7.36 (m, 6H), 7.33-7.28 (m, 10H), 6.82 (d, J = 8.4 Hz, 1H), 6.72 (d,

J = 8.4 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H), 6.42-6.38 (m, 2H), 5.56 (s, 1H), 5.10 (s, 2H), 4.93 (s,

2H), 4.64 (d, J = 6.2 Hz, 2H), 3.93 (s, 3H), 3.55 (t, J = 6.2 Hz, 1H), 3.35 (s, 2H), 3.11 (q, J = 6.4

13 Hz, 2H), 2.41 (t, J = 6.6 Hz, 2H), 1.09 (s, 9H); C NMR (CDCl3, 150 MHz) δ 171.4, 151.1, 148.7,

145.5, 137.3, 137.0, 135.5 X 3, 134.2, 133.4, 131.4, 129.8 X 3, 128.6 X 3, 128.3 X 3, 128.0 X 3,

127.7 X 2, 127.6 X 2, 127.4 X 2, 127.2 X 2, 125.6, 121.8, 120.6, 114.3, 114.1, 70.8 X 2, 61.8,

+ 56.6, 40.7, 40.6, 34.3, 26.6 X 3, 19.7; (ESI) m/z calcd. for C48H51NO6Si ([M+Na] ), 788.3378, found 788.3416.

3-(benzyloxy)-6-(2-((4-(benzyloxy)-3-((tert-butyldiphenylsilyl)oxy)phenethyl)amino)-2- oxoethyl)-2-methoxybenzyl acetate (3.41)

To the solution of compound 3.40 (3.1 g, 4.1 mmol) in DCM (20 mL) was added pyridine

(0.6 mL, 8.2 mmol) and DMAP (10 mg, 0.08 mmol). The mixture was cooled to 0 ̊ C and acetic anhydride (0.8 mL, 8.2 mmol) was added dropwise. The solution was allowed to stir for 1.5 h at room temperature, the reaction mixture was diluted with DCM (20 mL), washed with 1 N HCl and dried over sodium sulfate and concentrated. The residue was purified by flash chromatography

1 (1.5% MeOH/DCM) to afford 3.41 (3.1 g, 92%). H NMR (CDCl3, 500 MHz) δ 7.66 (dd, J = 8.1,

128

1.3 Hz, 4H), 7.41-7.36 (m, 6H), 7.33-7.27 (m, 10H), 6.88 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 8.4 Hz,

1H), 6.68 (d, J = 8.4 Hz, 1H), 6.40-6.38 (m, 2H), 5.14 (s, 2H), 5.08 (s, 2H), 4.88 (s, 2H), 3.88 (s,

3H), 3.45 (s, 2H), 3.10 (q, J = 6.7 Hz, 2H), 2.40 (t, J = 6.8 Hz, 2H), 1.97 (s, 3H), 1.07 (s, 9H););

13 C NMR (CDCl3, 125 MHz) δ 170.8, 170.5, 151.1, 149.2, 148.3, 145.4, 137.2, 136.6, 135.4 X 3,

133.3, 131.2, 129.7 X 3, 128.6 X 3, 128.3 X 3, 128.0 X 3, 127.6 X 4, 127.4 X 2, 127.2 X 2, 126.2,

121.6, 120.5, 114.8, 114.1, 70.7 X 2, 61.4, 58.4, 40.5, 40.4, 34.5, 26.6 X 3, 20.9, 19.7; (ESI) m/z

+ calcd. for C50H53NO7Si ([M+Na] ), 830.3489, found 830.3491.

(S)-3-(benzyloxy)-6-((7-(benzyloxy)-6-hydroxy-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)-

2-methoxybenzyl acetate (3.42)

To the solution of 3.41 (3.0 g, 3.7mmol) in dry acetonitrile (20 mL) was added POCl3 (2.1 mL, 22.3 mmol) and the mixture was refluxed for 1 h. The reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was dissolved in DCM (30 mL) and washed with saturated NaHCO3 (15 mL). The organic layer was dried over sodium sulfate and evaporated to dryness. The yellow solid residue was dissolved in DMF (15 mL) and RuCl[(R,R)-

TsDPEN(P-cymene)] (20 mg, 0.03 mmol) was added under nitrogen. The solution was purged

Nitrogen gas for 15 minutes. The mixture of formic acid (0.8 mL, 20.7 mmol ) and triethyl amine

(0.3 mL 2.21 mmol) was added in to the reaction mixture and allowed it to stir overnight at rt. The reaction mixture was adjusted pH to 8 with saturated NaHCO3 and extracted with ethyl acetate (3

X 35 mL). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography on silica gel using 3:97 to 5:95 MeOH:DCM as eluent to afford compound, 3.42 (1.4 g, 67% from two steps).

(S)-2,10-bis(benzyloxy)-9-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2- a]isoquinolin-3-ol (3.43)

129

Compound 3.42 (1.4 g, 2.5 mmol) was dissolved in ethanol (20 mL) and added 10% NaOH

(10 mL) to the solution then the mixture was refluxed for 1 h. The mixture was cooled and ethanol was evaporated. The residue was extracted with ehylacetate (2 X 30 mL) and the combined organic layer was washed dried over Na2SO4 and evaporated to dryness. The residues were dissolved in dry DCM (15 mL) and the solution was cooled 0 ̊ C. Thionyl chloride (0.9 mL, 12.5 mmol) was added dropwise to the reaction mixture and allowed it to stir for 1 hr. Reaction solution was cooled to 0 ̊ C and saturated NaHCO3 (30 mL) was added to it and allowed it stir for 1 h. Reaction mixture was extracted with DCM (2 X 50 mL), combined organic layer was dried over sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography on silica gel using 1:99 MeOH:DCM as eluent to afford compound, 3.43 (1.1 g, 88% over two

1 steps). H NMR (CDCl3, 500 MHz) δ 7.45-7.37 (m, 10H), 6.83 (s, 2H), 6.78 (s, 1H), 6.71 (s, 1H),

5.58 (s, 1H), 5.14-5.06 (m, 4H), 4.25 (d, J = 15.8 Hz, 1H), 3.90 (s, 3H), 3.55-3.52 (m, 2H), 3.20-

13 3.08 (m, 3H), 2.80 (dd, J = 11.5, 15.4 Hz, 1H), 2.67-2.60 (m, 2H); C NMR (CDCl3, 120 MHz) δ

149.3, 145.6, 144.3, 144.2, 137.2, 136.4, 129.2, 128.9, 128.7 X 2, 128.5 X 2, 128.4, 128.2, 128.0,

127.9, 127.8 X 2, 127.3 X 2, 123.7, 114.4, 113.1, 109.4, 71.4, 70.9, 60.3, 59.3, 54.0, 51.5, 36.5,

+ 28.9; (ESI) m/z calcd. for C32H31NO4 ([M+H] ), 494.2326, found 494.2330.

General synthetic procedure for the compounds 3.35a-3.35f as demonstrated for 3.35a

(S)-3-ethoxy-9-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,10-diol

(3.35a)

Compound, 3.43 (1.0 eq) was dissolved in anhydrous DMF and added K2CO3 (2.0 eq) followed by ethyl bromide (1.2 eq). Resulted reaction mass was allowed to stir at rt for 5 h. The reaction mass was quenched with cold water and extracted with ethyl acetate (3 x 50 mL).

Combined organic layer was washed with brine solution, dried over Na2SO4 and concentrated to

130 obtain a residue, which was used in the next step without further purification. Residues were dissolved in ethanol (10 mL) and refluxed with conc. HCl (4 mL) for 3 hr. The reaction mixture was concentrated in vacuo and residue was basified using aqueous ammonia solution and extracted with ethyl acetate (2 X 20 mL). Combined organic layer was washed with brine solution, dried over Na2SO4 and concentrated under reduced pressure to obtain a residue, which was purified by column chromatography on silica gel using 2:98 MeOH:DCM as eluent to afford compound 3.35a.

1 Yield 71 %, white solid, Mp 104 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.84-6.79 (m, 3H), 6.59 (s,

1H), 4.20 (d, J = 15.4 Hz, 1H), 4.10 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 3.58-3.53 (m, 2H), 3.27-3.09

(m, 3H), 2.80 (dd, J = 11.4, 15.9 Hz, 1H), 2.67-2.62 (m, 2H), 1.45 (t, J = 7.0, 3H); 13C NMR

(CDCl3, 125 MHz) δ 146.4, 144.3, 144.0, 143.1, 130.3, 127.9, 127.3, 125.8, 125.0, 114.0, 111.4,

+ 111.2, 64.4, 60.7, 59.3, 53.9, 51.7, 36.2, 29.1, 14.0; (ESI) m/z calcd. for C20H23NO4 ([M+H] ),

342.1700, found 342.1697.

(S)-9-methoxy-3-propoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,10- diol (3.35b)

1 Yield 75%, dark brown solid, Mp 82 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.85-6.81 (m, 3H),

6.59 (s, 1H), 4.28 (d, J = 15.4 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.82 (s, 3H), 3.65-3.58 (m, 2H),

3.28-3.24 (m, 2H), 3.15 (td, J = 5.6, 16.7 Hz, 1H), 2.84 (dd, J = 11.8, 16.0 Hz, 1H), 2.70-2.66 (m,

13 2H), 1.84 (sext, J = 7.1 Hz, 2H), 1.05 (t, J = 7.5, 3H); C NMR (CDCl3, 125 MHz) δ 146.5, 144.5,

144.1, 143.2, 130.3, 127.9, 127.3, 125.8, 125.0, 114.2, 111.5, 111.2, 70.4, 60.7, 59.4, 53.9, 51.7,

+ 36.1, 29.1, 22.6, 10.5; (ESI) m/z calcd. for C21H25NO4 ([M+H] ), 356.1856, found 356.1849.

(S)-3-butoxy-9-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,10-diol

(3.35c)

131

1 Yield 76%, yellowish powder, Mp 85 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.85-6.81 (m,

3H), 6.59 (s, 1H), 4.28 (d, J = 15.4 Hz, 1H), 3.99 (t, J = 6.6 Hz, 2H), 3.82 (s, 3H), 3.65-3.58 (m,

2H), 3.28-3.24 (m, 2H), 3.15 (td, J = 5.6, 16.7 Hz, 1H), 2.84 (dd, J = 11.8, 16.0 Hz, 1H), 2.70-2.66

13 (m, 2H), 1.84 (sext, J = 7.1 Hz, 2H), 1.05 (t, J = 7.5, 3H); C NMR (CDCl3, 125 MHz) δ 146.5,

144.5, 144.0, 143.1, 130.2, 127.8, 127.3, 125.8, 125.0, 114.2, 111.4, 111.1, 68.6, 60.7, 59.4, 53.9,

+ 51.7, 36.1, 31.3, 29.1, 19.2, 13.8; (ESI) m/z calcd. for C22H27O4 ([M+H] ), 370.2013, found

370.2006.

(S)-9-methoxy-3-(pentyloxy)-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-

2,10-diol (3.35d)

1 Yield 73%, brown solid, Mp 73 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.83-6.79 (m, 3H), 6.59

(s, 1H), 5.56 (s, 1H), 4.20(d, J = 15.3 Hz, 1H), 4.02 (t, J = 6.6 Hz, 2H), 3.81 (s, 3H), 3.58-3.55 (m,

2H), 3.27-3.10 (m, 3H), 2.80 (dd, J = 11.6, 15.8 Hz, 1H), 2.67-2.62 (m, 2H), 1.82 (pent, J = 6.6

13 Hz, 2H), 1.48-1.36 (m, 4H), 0.94 (t, J = 7.2, 3H); C NMR (CDCl3, 125 MHz) δ 146.4, 144.5,

144.1, 143.1, 130.2, 127.8, 127.3, 125.8, 125.0, 114.1, 111.4, 111.1, 68.9, 60.7, 59.4, 53.9, 51.7,

+ 36.1, 29.0, 28.9, 28.2, 22.4, 14.0 ; (ESI) m/z calcd. for C23H29NO4 ([M+H] ), 384.2169, found

384.2165.

(S)-3-(hexyloxy)-9-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2-a]isoquinoline-2,10- diol (3.35e)

1 Yield 70%, dark brown powder, Mp 61 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.83-6.79 (m,

3H), 6.59 (s, 1H), 4.22 (d, J = 15.3 Hz, 1H), 4.02 (t, J = 6.6 Hz, 2H), 3.81 (s, 3H), 3.58(d, J = 14.7

Hz, 2H), 3.28-3.12 (m, 3H), 2.84-2.79 (m, 1H), 2.69-2.64 (m, 2H), 1.81 (pent, J = 6.7 Hz, 2H),

13 1.49-1.43 (m, 2H), 1.34 (sext, J = 3.7, 4H), 0.91 (t, J = 7.0, 3H); C NMR (CDCl3, 125 MHz) δ

132

146.4, 144.5, 144.0, 143.1, 130.2, 127.9, 127.3, 125.8, 125.0, 114.1, 111.4, 111.1, 68.9, 60.7, 59.4,

+ 53.9, 51.7, 36.2, 31.5, 29.2, 29.1, 25.7, 22.6, 14.0; (ESI) m/z calcd. for C24H31NO4 ([M+Na] ),

398.2326, found 398.2319.

(S)-3-(2-fluoroethoxy)-9-methoxy-5,8,13,13a-tetrahydro-6H-isoquinolino[3,2- a]isoquinoline-2,10-diol (3.35f)

1 Yield 87%, yellow powder, Mp 95 ⁰C; H NMR (CDCl3, 500 MHz) δ 6.85-6.79 (m, 3H),

6.61 (s, 1H), 4.82 (t, J = 3.9 Hz, 1H), 4.73 (t, J = 3.9 Hz, 1H), 4.32-4.19 (m, 3H), 3.81 (s, 3H),

3.58-3.54 (m, 2H), 3.27-3.10 (m, 3H), 2.80 (dd, J = 11.5, 15.7 Hz, 1H), 2.68-2.62 (m, 2H); 13C

NMR (CDCl3, 125 MHz) δ 146.4, 144.3, 143.8, 143.1, 131.5, 127.8, 127.2, 126.0, 125.0, 114.2,

112.4, 111.9, 82.4, 81.0, 68.5, 68.3, 60.7, 59.3, 53.8, 51.6, 36.0, 29.0; (ESI) m/z calcd. for

+ C20H22FNO4 ([M+Na] ), 360.1606, found 360.1599.

3.8.2 Biological evaluation: primary and secondary radioligand binding assays

Both the assays were done at the PDSP facility. In the primary binding assays, compounds were tested at single concentrations (10 μM) in quadruplicate in 96-well plates. Compounds that showed a minimum of 50% inhibition at 10 μM were tagged for secondary radioligand binding assays to determine equilibrium binding affinity at specific targets. In the secondary binding assays, selected compounds were usually tested at 11 concentrations (0.1, 0.3, 1, 3, 10, 30, 100,

300 nM, 1, 3, 10 μM) and in triplicate (3 sets of 96-well plates). Both primary and secondary radioligand binding assays were carried out in a final of volume of 125 μl per well in appropriate binding buffer. The hot ligand concentration was usually at a concentration close to the Kd (unless otherwise indicated). Total binding and nonspecific binding were determined in the absence and presence of 10 μM appropriate reference compound, respectively. In brief, plates were usually

133 incubated at room temperature and in the dark for 90 min (unless otherwise indicated). Reactions were stopped by vacuum filtration onto 0.3% polyethyleneimine (PEI) soaked 96-well filter mats using a 96-well Filtermate harvester, followed by three washes with cold wash buffer. Scintillation cocktail was then melted onto the microwave-dried filters on a hot plate and radioactivity was counted in a Microbeta counter.For detailed experimental details please refer to the PDSP website http://pdsp.med.unc.edu/ and click on ‘Binding Assay’ or ‘Functional Assay’ on the menu bar.

3.8.3 Metabolic stability study

The procedure is described below that was used for in vitro metabolic stability study.

A 48-well plate seeded with fresh Sprague Dawley rat hepatocytes, having cell density

0.75-1.5 X 105 cells/well was obtained from TRL, LLC (Durham, NC, USA). After receiving the plated hepatocytes, the shipping media was aspirated and replaced with maintenance media and the plate was incubated in 5% CO2 incubator at 37 ⁰C for 16 h to resume the cellular activity.

After 16 h, the maintenance media was replaced with 250 µL of fresh maintenance media.

2 µL of stock solution of each compound prepared in DMSO was diluted with cell culture media

(198 µL) to achieve the final incubation concentration 2 µM of compound and 0.4 % DMSO V/V.

Each of these solutions (200 µL) were added in to plate wells in duplicate and the plate was placed into the 37 ⁰C incubator, set at 95% relative humidity and 5% CO2. The sample aliquots were taken at the time point of 0h, 1h, 2h, 3h, 6h, 12h, 24h and 36h, and transferred in to microcentrifuge tube preloaded with 100 µL of cold acetonitrile as stop solution. The microcentrifuge tubes were centrifuged at 13,000 rpm for 6 min and the supernatant layer was separated and each sample was analyzed via LC/MS in duplicate.

3.8.4 Alamar Blue Cytotoxicity Assay

134

Pre-assay optimization protocol

Cells (MDA MB 231) were plated in a range from (105-102) per well in triplicates in 96 well plate and they are incubated at 37 oC for 24 h. 25 µl of Alamar blue [10% v/v of 440µM solution of resazurin (Sigma #R7017)] was added to each well and incubated at 37 oC for 5 h.

Fluorescence intensity was measured (Alamar blue, 561 dichroic mirror from bottom). A standard graph between log (fluorescence intensity) and log (number of cells) was plotted and the optimal number of cells required for the assay was determined from the linear range of the standard graph.

Cytotoxicity assay protocol

10000 cells were plated (optimal cell number obtained from the standard graph) in 100 µL wells in 96 well plate and were incubated at 37 oC for 24 h. Serial dilutions of the compound (30 mM - 300 nM) were made in the cell growth media. The media in each well was replaced with media containing the compound, with triplicates for each dilution. The cells were then incubated at 37 oC for 24 h. Subsequently, 25 µl of Alamar blue (10%v/v of 440 µM solution of resazurin) was added to each well and incubated at 37 oC for 5 h. Fluorescence intensity was measured

(Alamar blue, 561 dichroic mirror from bottom). The number of cells after treatment in each well was determined from the standard graph and IC50 values were determined using GraphPad Prism software.

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Chapter 4: Synthesis and evaluation of de-rigidified THPB analogues at dopamine receptors

4.1 Background and rationale

THPBs share the common structure consisting of a four-ring main structural backbone

(isoquinoline ring) with substituent methoxyl or hydroxyl groups at the two benzene rings. The dopaminergic activity of THPB scaffold resides in the structural alikeness between dopamine and

THPB (as shown in Figure 4.1). Based on previous SAR studies it was found that C2 or C10 hydroxyl groups of THPB play key roles in determining the intrinsic activities and affinity at DA receptors (Chapter 2, Section 2.5).332-334 The, rigid THPB pharmacophore is devoid of flexibility of the free ethtylamine chain of dopamine. It is quite interesting to know the results of de- rigidifying this rigid scaffold on dopaminergic affinity and activity. We hypothesized that the rigid tetracyclic THPB core is not required for affinity and activity at the dopamine receptors and that de-rigidification would engender increased affinity and selectivity for dopamine D3 receptors.

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To test this hypothesis, we planned to prepare compounds 4.1, 4.2a-4.2c and 4.3a-4.3d

(shown in Figure 4.2) having a flexible core structure and which do not have an intact THPB scaffold. Compound 4.1 has broken ring C although having similar carbon skeleton as of SPD

(2.1). Analogues 4.2a-4.2c posses complete flexibility and are devoid of an intact ring B and ring

C of the THPB core. While having complete THIQ portion of THPB scaffold, analogues 4.3a-

4.3d carry rest of the segment of THPB impaired. These molecules will test the necessity of the rigid core structure of THPB for the dopaminergic receptor activity. This approach is attractive because if it is found that rigidity is not required, compound library synthesis and hence ligand optimization would be more efficient since the synthesis of these molecules would be shorter and simpler.

In the design and development of novel D3 receptor ligands, a primary challenge is achieving a high degree of selectivity for D3 receptor over the highly homologous D2 receptor for ligands with druglike characteristics. These issues, as well as the progress made in the development of D3 receptor selective ligands, have been the subject of several reviews.75, 142, 386-389 A typical pharmacophore for dopamine D3 receptor ligands, possess a general pattern, which is divided into three subunits (as shown in Figure 4.2): an aryl moiety, which is connected to an amide (first

137 subunit) and further linked via an alkyl or aryl spacer (second subunit) to a basic residue with aryl substituent (third subunit).390 For example, as shown in Figure 4.3, SB-277011A or BP-897 contain arylpiperazine or tetrahydroisoquinoline basic subunit and n-butyl or cyclohexyl ethyl spacer attached to arylamide group.233, 238, 391

In our efforts towards the discovery of novel, selective and potent D3 receptor ligands, we planned to incorporate the THIQ portion of the THPB scaffold into typical pharmacophore of D3 ligands to generate a hybridized D3 receptor ligand as shown in Figure 4.3. This hybrid moiety is different from previously made analogues in having a mono-protected catechol moiety on the

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THIQ ring and we presumed that this would add potency and preference for the D3 receptor.392

Based on this hybridization pattern, we planned to synthesize series of compounds 4.4a-4.4l

(Figure 4.4) with variant arylamide functionalities.

4.2 Results and discussion

4.2.1 Synthesis

As shown in the Scheme 4.1, we deployed secondary amine 4.4 to synthesize de-rigidified analogues 4.1 and 4.2a-4.2c. Starting with primary amine 3.9, it was subjected to reductive amination with 3-benzyloxy-2-methoxy benzaldehyde derived from 2, 3-dihydroxybenzaldehyde to give secondary amine 4.5.393 Pictet-Spengler reaction with 1,1, diethoxyethane followed by debenzylation produced compound 4.1 from secondary amine 4.5.394, 395 Reductive amination of an appropriate aldehyde and amine 4.5 and subsequent debenzylation gave the desired tertiary amine analogues 4.2a-4.2c.

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THIQ based flexible analogues 4.3a-4.3d were synthesized from primary amine 3.9 via tetrahydroisoquinoline derivative 4.6 (Scheme 4.2). Primary amine 3.9 was subjected to Pictet-

Spengler reaction with paraformaldehyde in presence of formic acid to afford THIQ derivative

4.6.396 Reductive amination with appropriate aldehyde followed by acid catalyzed debenzylation gave analogues 4.3a-4.3d from compound 4.6.

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Synthesis of tetrahydroisoquinoline-arylamide hybrid analogues was carried out as depicted in Scheme 4.3. Compound 4.7 was prepared from compound 4.6 via four carbon chain elongation with 4-bromobutyronitrile followed by reduction of the intermediate nitrile group to primary amine 4.7 using LAH.237 Appropriate acids were coupled with primary amine 4.7 and the final analogues 4.4a-4.4l were synthesized after debenzylation of intermediate amide compounds.

4.2.2 Structure-activity correlations

The data for binding affinity to human dopamine D1, D2 and D3 receptors is presented in

Table 4.1. Dismantling the THPB scaffold via truncation of ring B and ring C (in compounds 4.2a-

4.2c) results in significantly lower affinity for all dopamine receptors. Thus, intact THPB core seems to be very important for affinity to dopamine receptors. Compounds 4.2a-4.2c which lacked a tetrahydroisoquinoline motif had lower affinities for the dopamine receptor than compounds 4.1 and 4.3a-4.3d possess such motif. THIQ derivatives 4.3a, 4.3c and 4.3d have shown good affinity and selectivity at D3 receptor, specifically compound 4.3c has notable selectivity at D3 versus D1 receptor (286.9) and D2 receptors (33.3). Therefore it would appear that the presence of an intact

THIQ moiety contributes significantly to better D3 receptor affinity and selectivity.

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Compounds 4.4a-4.4l were submitted to the PDSP for evaluation of D1, D2 and D3 receptor affinity and activity. The binding affinity data is shown in Table 4.2. The primary binding assay data suggest that the % inhibition at D3 receptor is very high when compared with % inhibition at D1 and D2 receptor. Many compounds including 4.4d-4.4g, 4.4k and 4.4l have exhibited less than 50% inhibition (10 µM concentration) at D1 and D2 receptor in the primary binding assay so they could not carried further for secondary binding assay as per PDSP protocol.

In the secondary binding assay analogues 4.4a and 4.4b having bulkier aryl substituents have shown high and similar potency (2.7 nM) at D3 receptor while retaining some affinity at D1 and D2 receptors. Higher binding affinity for these compounds suggests the possibility of presence of hydrophobic pocket at this position of the D3 binding site. Very similar binding profile was demonstrated by p-chlorobenzyl analogue 4.4h, which possess the highest binding affinity in the series at D3 receptor (2.1 nM) and having low affinity at D1 (683 nM) and D2 (1060 nM) receptors. Compound 4.4c retain heteroatom in the ring showed 8 fold reduced affinity at D3 receptor as compare to similar analogue (4.4b) without heteroatom. In a similar manner, 4.4d having pyridine heteroaromatic ring, displayed the least D3 receptor affinity (24 nM) among all analogues tested. Surprisingly, 4-methoxybenzyl analogue 4.4k presented higher affinity at D3 receptor as compared to 4-hydroxybenzyl analogue 4.4e (Ki value of 5.9 nM for 4.4k versus 8.7 nM for 4.4e) and both these analogues were inactive at D1 and D2 receptors. Compound

4.4f with 3-methoxy substitution exhibited 2 fold reduced affinity as compared to 4-methoxy analogue 4.4k (12 nM for 4.4f versus 5.9 nM for 4.4k).

Table 4.1: Binding affinity data (Ki nM) for de-rigidified analogues at dopamine receptors

a Ki (nM) Selectivity Compound D1b D2c D3d D2/D3 D1/D3 4.1 833 1095 1367 1.2 0.6

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4.2a >10,000 >10,000 1811.0 5.5 5.5 4.2b 3111.0 >10,000 2680.0 3.7 1.1 4.2c >10,000 >10,000 >10,000 1 1 4.3a 6479.0 1593.0 92.0 17.3 70.4 4.3b 3318.0 3450.0 191.0 18.1 17.4 4.3c 4590.0 533.0 16.0 33.3 286.9 4.3d 3255.0 1972.0 41.0 48.1 79.4 a b Experiments carried out in triplicate - SEM values are within 13% of reported Ki; [3H]SCH2390 used as radioligand/(+)- c butaclamol used as reference compound - Ki = 2.3 nM; [3H]Nmethylspiperone used as radioligand/haloperidol used as reference d compound - Ki = 9.6 nM; [3H]N-methylspiperone used as radioligand/chlorpromazine used as reference compound - Ki = 11.0 nM. Analogues 4.4g, 4.4k, and 4.4l bearing 4-fluoro, 4-methoxy and 4-cayno benzyloxy substituent possess similar range of binding affinity at D3 receptor (Ki value 4.4 nM, 5.9 nM and

6.3 nM respectively). Additionally, none of 4.4g, 4.4k, and 4.4l is active at dopamine D1 and D2 receptors. Amongst all 4-halogenated analogues, fluoro compound 4.4g have shown the highest selectivity for D3 receptor while, its chloro congener 4.4h expressed highest potency (2.1 nM) at

D3 receptor with some retention of affinity at D1 and D2 receptors (683 nM and 1060 nM). The general order of affinities for halogenated analogues evaluated at the D3 receptors was chloro>bromo>fluoro>iodo. Compound 4.4i with bromo substitution have shown noticeable selectivity (20 fold) at D1 receptor over D2 receptor apart from D3 receptor selectivity (2941 fold).

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Table 4.2: Binding affinity (Ki nM) data for THIQ-arylamide hybridized D3 receptor ligands

a Ki (nM) Compound R D1b D2c D3d 4.4a 1-Biphenyl 788.0 787.0 2.7 4.4b 2-Naphthyl 809.0 668.0 2.7 4.4c 4-Quinolyl 1808.0 NAe 22.0 4.4d 4-Pyridyl NAe NAe 24.0 4.4e 4-Hydroxybenzyl NAe NAe 8.7 4.4f 3-methoxybenzyl NAe NAe 12.0 4.4g 4-Fluorobenzyl NAe NAe 4.4 4.4h 4-Chlorobenzyl 683.0 1060.0 2.1 4.4i 4-Bromobenzyl 495.0 >10,000 3.4 4.4j 4-Iodobenzyl 1573.0 NAe 10.0 4.4k 4-Methoxybenzyl NAe NAe 5.9 4.4l 4-Cyanobenzyl NAe NAe 6.3 a b Experiments carried out in triplicate - SEM values are within 13% of reported Ki; [3H]SCH2390 used as radioligand/(+)- c butaclamol used as reference compound - Ki = 2.3 nM; [3H]Nmethylspiperone used as radioligand/haloperidol used as reference d compound - Ki = 9.6 nM; [3H]N-methylspiperone used as radioligand/chlorpromazine used as reference compound - Ki = 11.0 nM. ND = not determined; eNA- not active (<50% in primary assay).

Overall, all of the para substituted benzyl amide analogues (4.4e-4.4l) endowed with <10 nM D3 receptor binding affinity and most of them were only active at D3 receptor except 4.4h,

4.4i, and 4.4j. In general, this series of analogues have demonstrated very high potency and selectivity at D3 receptors amongst all dopamine receptor subtypes.

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4.3 Molecular docking study at D3 receptor

Molecular docking evaluations were performed on analogues 4.1, 4.2a, and 4.2b at the D3 receptor. Investigation of the docked ligand poses and recognition of key protein-ligand interactions provides insights into the SAR outcomes of the series the de-rigidified compounds with respect to D3. In this study, ligand docking was performed retrospectively in order to elucidate and rationalize the measured D3 affinity data. The docked posses maintain key receptor– ligand interactions, including the critical protonated tertiary N—Asp110 salt bridge motif and H- bonds to Ser192 in flexible compound 4.1 (Figure 4.5a). As shown in Figure 4.5b, hydrophobic interaction was observed between compounds 4.2a and 4.2b and imidazole ring of His349 as well as phenyl ring of Tyr373.

In order to assess the binding stability of flexible compounds 4.1, 4.2a, and 4.2b the ligands were simulated in complex with D3 (crystal structure 3pbl).354 Ligand parameters were generated in Generalized Amber Force Field (GAFF),397 protein parameters were assigned from

AMBERFF14SB,398 and the system was solvated in a 10 Å buffer of Tip4pew (~12000 water molecules). Ligands were allowed to move freely, however, protein heavy atoms were restrained with 2.5 kcal/Å2 harmonic restraints to maintain model accuracy (in the absence of lipids). The starting configuration utilized for these simulations was the top ranked docked poses discussed above. These configurations were minimized and heated to 300 K, then equilibrated for 20 ns in

NPT, and finally run in a 100 ns production NVT molecular dynamics simulation sampling every

1 ps. The results of these simulations show that the docking poses are stable within this D3 configuration. Two metrics were utilized to assess ligand stability throughout the production simulations: ligand RMSD and the distance between the ligand’s center of mass and Asp110

145 a)

Figure 4.5: Docked pose of the flexible analogues, a) compound 4.1 and b) compound 4.2a and 4.2b

146

(critical for binding shown in Figure 4.5). Ligand RMSD was computed for each frame of the production trajectories compared to the ligand configuration in the original docked pose. This analysis shows that while ligand positions fluctuated over the course of the simulation, however, this did not differ greatly from their starting poses (Figure 4.6). The distance between ligand center of mass and Asp110 was utilized to determine whether the ligand maintained this key contact over the course of the simulation. Though the distance does change by a small amount in compounds

4.1 and 4.2b, all three ligands maintain a bound state during the simulation (Figure 4.7).

a) b)

Figure 4.6: RMSD between all atom coordinates at each frame, compared to the coordinates of the docked pose of flexible compounds a) 4.1, b) 4.2a, and c) 4.2b

These data suggest that compounds 4.1, 4.2a, and 4.2b maintain a configuration similar to the predicted docked pose during a molecular dynamics simulation and that these predicted poses

147 are stable with respect to the crystal D3 configuration. This supports the assertion that the difference in binding affinity is not likely due to destabilized binding, but rather the added ligand flexibility and decreased lipophilic interactivity.

a) b)

Figure 4.7: Distance between flexible compounds a) 4.1, b) 4.2a, and c) 4.2b center of mass and the key aspartate Asp110 over the course of a 100 ns simulation.

4.4 Metabolic stability study

As discussed in Chapter 3 (Section 3.6b), metabolic susceptibility of (-)-SPD (2.1) against hepatic enzyme needs to be resolved to make it orally bioavailable. It is very crucial to evaluate the metabolic stability of synthesized analogues in order to get a comparative idea between metabolism of rigid scaffold of (-)-SPD (2.1) and flexible conformation of these analogues. To test the effect of structural flexibility in the metabolism we have studied the in vitro metabolic stability

148 of representative flexible and THIQ/arylamide analogues (4.3c and 4.4l) along with C-3 and C-10 analogues of THPB (Chapter 3, Section 3.6).

Table 4.3: Metabolic stability study data for representative compounds % of total AUC at given time point Time (h) (-)-SPD (2.1) 4.3c 4.4l 0 100 100 100 1 98 99 93 2 91 98 96 3 82 94 92 6 52 73 48 12 10 25 46 24 10 2 2 36 11 4 2

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l-SPD 100 4.3c 80 4.6l

% of total AUC at given timepoint given at AUC total of % 0 0 5 10 15 20 25 30 35 40 Time (h)

Figure 4.8: % of total AUC at given time period of incubation for representative analogues For tested compounds, the % AUC was calculated from the LC/MS peak abundance in comparison with that total AUC. The % AUC data for each compound during the 36 h time course

149 is shown in Table 4.3 and the time versus % AUC graph is depicted in Figure 4.8. In this study the major metabolite observed was O-glucuronidated one as similar to (-)-SPD (2.1) and other rigid

THPB analogues. Results of the metabolic stability study show that flexible compounds have slightly slower rate of metabolism than parent compound (-)-SPD (2.1). At 6 hr time point, 52 % of active (-)-SPD (2.1) was present in the sample aliquot while 73% of compound 4.3c was present in active form at the same time point. In a same manner, 46% of active analogue 4.4l was present as compare to 10% of (-)-SPD (2.1) at 12 hr time point. These results indicate that, though it does not affect drastically, conformational flexibility has some impact on metabolic stability of THPB scaffold against hepatocyte enzymatic environment. Further study needs to be done to evaluate this fact using more diversely flexible analogues of (-)-SPD (2.1).

4.5 Conclusion

In conclusion, we have completed structure–activity relationship studies on various conformationally flexible analogues of (-)-SPD (2.1) with the goal of identifying a potential and selective DA D1 and D3 receptor ligand. Two categories of analogues were synthesized and assayed for binding affinity at DA receptors, one group (4.1, 4.2a-c and 4.3a-4.3d) having flexibility in (-)-SPD scaffold and second one (4.4a-4.4l) based on THIQ/arylamide hybridization concept.

The results of this study revealed that intact THPB scaffold of (-)-SPD is necessary for all dopamine receptor activity and de-rigidification is detrimental to DA receptor activity. On the other hand, having THIQ moiety annexed with flexible arylamide subunit, generates high potency for and preference at DA D3 receptor. Analogue 4.4h was identified as the most potent compound in this series with Ki value of 2.1 nM. All compounds in this series (4.4a-4.4l) displayed high

150 selectivity at D3 along with very little affinities at other DA receptors (D1, D2, D4, and D5), however, compound 4.4l was found to be active solely at D3 receptor. To the best our knowledge, this is the first ever selective D3 ligand which does not have affinity at other DA receptors at all.

Results from docking study (4.1, 4.2a, 4.2b) at D3 receptor revealed the key receptor–ligand interactions, including the protonated tertiary N—Asp110 salt bridge motif, H-bonds to Ser192, and hydrophobic interaction with imidazole ring of His349 as well as with phenyl ring of Tyr373.

Overall, structure-activity relationship study of de-rigidified and THIQ/arylamide analogues revealed potent and selective D3 receptor ligands. Still more diverse analogues need to be synthesized and tested for D3 receptor affinity in order to retrieve compounds having subnanomolar potency. Also, compounds having metabolic stability against hepatic metabolism need to be discover as a potential candidate for the treatment of drug addiction. There is plenty of room for exploration to modify THIQ-arylamide hybrid ligands, for instance, replacement of the n-butyl spacer link with ethyl cyclohexane and replacing the catechol moiety with its bioisosteres.

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4.6 Experimental

4.6.1 Chemistry

N-(3-(benzyloxy)-2-methoxybenzyl)-2-(4-(benzyloxy)-3-methoxyphenyl)ethan-1-amine (4.5)

Amine 3.9 (0.25 g, 0.97 mmol) and 3-(benzyloxy)-2-methoxybenzaldehyde (0.24 g ,0.97 mmol) were dissolved in 10mL DCM and allowed to stir the solution at 0 ⁰C for 10 minutes.

Sodium triacetoxy borohydride (0.31 g, 1.5 mmol) was added in to the reaction mixture at 0 ⁰C and allowed it to stir for 3 h at room temperature. Reaction mixture was washed with saturated

NaHCO3 and organic layer was dried over sodium sulfate and concentrated to give the compound

1 4.5 (0.4 g, 83%)%) as brown oil; H NMR (CDCl3, 500MHz ) δ 7.40-7.27 (m, 10H), 6.98-6.95

(m, 1H), 6.89-6.6.85 (m, 2H), 6.79-6.74 (m, 2H), 6.66 (dd, J = 1.6, 8.1 Hz, 1H), 5.12 (s, 2H), 5.10

13 (s, 2H), 3.85-3.79 (m, 8H), 2.86 (t, J = 7.3, 2H), 2.77 (t, J = 7.3 Hz, 2H); C NMR (CDCl3,

500MHz) δ 151.6, 149.5, 147.7, 146.5, 137.3, 137.0, 133.5, 133.0, 128.6, 128.5, 128.0, 127.7,

127.3, 127.2, 123.8, 122.2, 122.1, 121.3, 120.6, 114.7, 114.1, 113.3, 112.4, 111.9, 71.1, 70.1, 60.7,

+ 55.9, 50.3, 48.8, 35.7 ; HRMS (ESI) m/z calcd. for C31H33NO4 ([M+H] ), 484.2488, found

484.2475.

7-(benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline (4.6)

Mixture of primary amine 3.9 (4 g, 15.6 mmol) and formic acid (20 mL) was stirred at 0 ̊

C for 15 minutes. Paraformaldehyde (0.47 g) was added into the reaction mixture and allowed it stir at 50 ̊ C for 12 h. reaction mixture was concentrated in vacuo and residues were washed with saturated NaHCO3 and extracted with ethyl acetate (4 X 50 mL). Organic layer was combined, dried over sodium sulfate and concentrated. Crude product was purified by flash column chromatography on silica gel (3% methanol/dichloromethane) to give the compound 4.6 (3.65 g,

152

1 87%) as brown oil; H NMR (CDCl3, 500 MHz) δ 7.41 (d, J = 7.1 Hz, 2H), 7.37 (t, J = 7.6 Hz,

2H), 7.31 (t, J = 7.1 Hz, 1H), 6.62 (s, 1H, 6.57 (s, 1H), 5.09 (s, 2H), 4.15 (s, 2H), 3.85 (s, 3H),

13 3.65 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 6.0 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 149.3, 147.4,

136.7, 128.6 X 2, 128.0, 127.3 X 2, 124.2, 119.6, 111.9, 111.6, 71.1, 56.0, 44.0, 41.6, 25.2; (ESI)

+ m/z calcd. for C17H19NO2 ([M+H] ), 270.1489, found 270.1485.

2-(3-hydroxy-2-methoxybenzyl)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol

(4.1)

Compound 4.5 (0.3 g, 0.6 mmol) was dissolved in 10 mL DCM. 1,1 diethoxyethane (0.13 mL, 0.9 mmol) and TFA (0.14 mL, 1.8 mmol) were added in to the reaction mixture and allowed it to stir overnight. The reaction mixture was washed with 10 mL saturated NaHCO3 and organic layer was dried over sodium sulfate and evaporated to dryness. The residues were refluxed in to the mixture of methanol (5 mL) and concentrated HCl (3 mL) for 3 h. Methanol was evaporated and the crude mixture was basified with ammonia solution, and extracted with dichloromethane

(10 mL × 3). The combined organic layers were dried over sodium sulfate and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (3% methanol/dichloromethane) to give the compound 4.1 (0.12 g, 60%) as yellow powder, mp 56-58

1 ̊ C; H NMR (CDCl3, 500MHz) δ 7.00-6.98 (m, 2H), 6.88 (dd, J =6.85, 2.8 Hz, 1H), 6.64 (s, 1H),

6.55 (s, 1H), 3.85-3.79 (m, 8H), 3.67 (d, J = 13.6 Hz, 1H), 3.07-3.02 (m, 1H), 2.83-2.77 (m, 1H),

2.68 (dt, J = 12.5, 4.5 Hz, 1H), 2.58 (dt, J = 13, 4.5 Hz, 1H), 1.37 (d, J = 6.6 Hz, 3H); 13C NMR

(CDCl3, 500MHz) δ 149.0, 145.9, 144.9, 143.6, 133.0, 132.7, 125.6, 124.6, 122.1, 114.3, 113.0,

+ 110.6, 61.8, 56.3, 55.9, 52.2, 44.0, 27.0, 20.0; HRMS (ESI) m/z calcd. for C19H23NO4 ([M+H] ),

330.1700, found 330.1707.

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General procedure to synthesize compounds 4.2a-4.2c and 4.3a-4.3d is described for 4.2a:

3-(((4-hydroxy-3-methoxyphenethyl)(methyl)amino)methyl)-2-methoxyphenol (4.2a)

Amine 4.5 (0.2 g, 0.41 mmol) and formaldehyde (0.06 mL, 1.64 mmol) were dissolved in

10 mL DCM and allowed to stir the solution at 0 ⁰C for 10 minutes. Sodium triacetoxy borohydride

(0.13 g, 0.62 mmol) was added in to the reaction mixture at ⁰C and allowed it to stir for 3 h at room temperature. Reaction mixture was washed with saturated NaHCO3 and organic layer was dried over sodium sulfate and concentrated. The residues were refluxed in to the mixture of methanol (5 mL) and concentrated HCl (3 mL) for 3 h. Methanol was evaporated and the crude mixture was basified with ammonia solution, and extracted with dichloromethane (10 mL × 3).

The combined organic layers were dried over sodium sulfate and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (3%

1 methanol/dichloromethane) to give the compound 4.2a (0.1 g, 74%) as yellow oil; H NMR

(CDCl3, 500MHz) δ 7.0-6.89 (m, 3H), 6.82 (d, J = 8.1 Hz, 1H), 6.68-6.66 (m, 2H), 3.86 (s, 3H),

13 3.80 (s, 3H), 3.64 (s, 2H), 2.79 (s, 2H), 2.70 (s, 2H), 2.33 (s, 3H); C NMR (CDCl3, 500MHz) δ

149.0, 146.3, 145.9, 143.8, 124.7, 122.5, 121.2, 121.2, 114.2, 114.1, 114.0, 111.2, 61.7, 55.9, 55.7,

+ 52.2, 42.0, 33.3; HRMS (ESI) m/z calcd. for C18H23NO4 ([M+H] ), 318.1700, found 318.1693

3-((ethyl(4-hydroxy-3-methoxyphenethyl)amino)methyl)-2-methoxyphenol (4.2b)

1 Yield 68%, brown oil; H NMR (CDCl3, 500MHz ) δ 6.98-6.93 (m, 2H), 6.87 (dd, J = 2.0,

7.5 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.65-6.64 (m, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.67 (s, 2H),

13 2.71 (s, 4H), 2.62 (q, J = 6.8 Hz, 2H), 1.1 (t, J = 7.0 Hz, 3H); C NMR (CDCl3, 500MHz) δ 149.0,

146.3, 145.7, 143.7, 124.6, 122.3, 121.3, 121.3, 114.3, 114.1, 114.1, 111.2, 61.7, 55.8, 55.3, 51.9,

+ 47.5, 33.0, 11.8; HRMS (ESI) m/z calcd. for C19H25NO4 ([M+H] ), 332.1856, found 332.1851.

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3,3'-(((4-hydroxy-3-methoxyphenethyl)azanediyl)bis(methylene))bis(2-methoxyphenol)

(4.2c)

1 Yield 67%, dark brown solid, mp 61 ̊ C; H NMR (CDCl3, 500 MHz) δ 7.00-6.95 (m, 4H),

6.85 (dd, J = 2.1, 7.4 Hz, 2H), 6.72-6.68 (m, 2H), 6.56 (dd, J = 2.0, 8.2 Hz, 1H), 3.84 (s, 3H), 3.76

13 (s, 6H), 3.70 (s, 4H), 2.74 (q, J = 6.0 Hz, 2H), 2.67 (q, J = 4.5 Hz, 2H); C NMR (CDCl3, 125

MHz) δ 148.8 X 2, 145.7, 145.3 X2, 144.8, 133.7, 132.5, 124.7, 121.9 X 4, 120.1, 115.0, 114.2 X

+ 2, 110.5, 61.6 X 2, 56.0 X 2, 55.7, 52.2, 32.6; (ESI) m/z calcd. for C25H29NO6 ([M+H] ), 440.2068, found 440.2063.

6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol (4.3a)

1 Yield 72%, white powder, mp 164 ̊ C; H NMR (CDCl3, 500 MHz) δ 6.57 (s, 1H), 6.56

(s, 1H), 3.85 (s, 3H), 3.46 (s, 2H), 2.83 (t, J = 7.4 Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.43 (s, 3); 13C

NMR (CDCl3, 125 MHz) δ 145.1, 143.6, 127.3, 125.0, 112.0, 110.6, 57.4, 55.9, 53.0, 46.1, 28.9;

+ (ESI) m/z calcd. for C11H15NO2 ([M+H] ), 194.1176, found 194.1172.

2-ethyl-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol (4.3b)

1 Yield 65%, brown solid, mp 90 ̊ C; H NMR (CDCl3, 500 MHz) δ 6.59 (s, 1H), 6.7 (s,

1H), 3.85 (s, 3H), 3.59 (s, 2H), 2.87 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.7 Hz, 2H), 2.63 (q, J = 7.1

13 Hz, 2H), 1.22 (t J = 7.2 Hz, 3H); C NMR (CDCl3, 125 MHz) δ 147.8, 146.2, 129.8, 128.0, 114.9,

+ 113.1, 58.4, 57.7, 54.7, 53.3, 31.3,14.9; (ESI) m/z calcd. for C12H17NO2 ([M+H] ), 208.1332, found 208.1327.

2-(3-hydroxy-2-methoxybenzyl)-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol (4.3c)

155

1 Yield 68%, yellow solid, mp 150 ̊ C; H NMR (CDCl3, 500 MHz) δ 7.01-6.97 (m, 2H),

6.91-6.89 (m, 1H), 6.57 (s, 2H), 3.85 (s, 6H), 3.69 (s, 2H), 3.57 (s, 2H), 2.79 (t, J = 5.7 Hz, 2H),

13 2.73 (t J = 5.6 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 149.0, 145.9, 145.1, 143.6, 131.5, 127.5,

125.6, 124.6, 122.3, 114.5, 112.2, 110.6, 61.9, 56.4, 55.9, 55.6, 50.6, 28.9; (ESI) m/z calcd. for

+ C18H21NO4 ([M+H] ), 316.1543, found 316.1538.

2-(4-hydroxy-3-methoxybenzyl)-6-methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol (4.3d)

1 Yield 70%, light brown powder, mp 62 ̊ C; H NMR (CDCl3, 500 MHz) δ 6.97 (d, J = 1.6

Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.81 (d J = 8.2, 1H), 6.57 (s, 1H), 6.54 (s, 1H), 3.89 (s, 3H),

3.84 (s, 3H), 3.61 (s, 2H), 3.55 (s, 2H), 2.82 (t, J = 5.3 Hz, 2H), 2.74 (t J = 5.2 Hz, 2H); 13C NMR

(CDCl3, 125 MHz) δ 145.8, 145.4, 145.1, 143.6, 125.5, 120.8 X 2, 115.4, 112.2, 110.6, 110.6,

+ 110.4, 62.0, 56.0 X 2, 55.3, 50.4, 28.5; (ESI) m/z calcd. for C18H21NO4 ([M+H] ), 316.1543, found

316.1537.

4-(7-(benzyloxy)-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-amine (4.7)

Compound 4.6 (3.0 g, 11.2 mmol), K2CO3 (3 g, 22.4 mmol) and 4-bromobutyronitrile (1.1 mL, 11.2 mmol) were refluxed in acetonitrile (30 mL) for 2 h. Reaction was cooled to room temperature and it was filtered. The filtrate was concentrated in vacuo and the residue was washed with sat. NaHCO3 and extracted with ethyl acetate (3 X 40 mL). Combined organic layer was dried over sodium sulfate and evaporated to get the crude product which was used in next step without further purification. The crude mass (3.4 g) was dissolved in THF and the solution was cooled to

0 ̊ C for 10 minutes. LAH (0.6 g, 16.8 mmol) was added portion wise in to the solution and allowed it to sir for 2 h at rt. Excess LAH was quenched with water and 10 % aqueous NaOH was added.

Reaction mixture was filtered through celite, filtrate was evaporated to dryness and the residue was extracted with ethyl acetate (3 X 40 mL). The combined organic layers were dried over sodium

156 sulfate and concentrated to give the crude product, which was purified by flash column chromatography on silica gel (5% methanol/dichloromethane) to give the compound 4.7 (2.9 g,

1 76%) as yellow oil. H NMR (CDCl3, 500MHz) δ 7.43 (d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.8 Hz,

2H), 7.29 (t J = 7.1 Hz, 1H), 6.61 (s, 1H), 6.56 (s, 1H), 5.08 (s, 2H), 3.83 (s, 3H), 3.65 (s, 2H),

13 2.86-2.81 (m, 6H), 2.57 (t, J =5.8 Hz, 2H), 1.82-1.73 (m, 4H); C NMR (CDCl3, 125 MHz) δ

148.5, 146.7, 137.1, 128.6 X 2, 127.8, 127.3, 125.9, 124.4, 124.4 X 2, 111.9, 71.1, 56.5, 56.0, 54.8,

+ 49.7, 39.7, 27.2, 26.9, 24.9; (ESI) m/z calcd. for C21H28N2O2 ([M+H] ), 341.2224, found 341.2225.

General procedure to synthesize compounds 4.4a-4.4l is described for 4.4a:

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-[1,1'-biphenyl]-4- carboxamide (4.4a)

Compound 4.7 (100 mg, 0.3 mmol), EDCI (86 mg, 0.45 mmol) and biphenyl-4-carboxylic acid (89 mg, 0.45 mmol) were added in to DCM (5 mL). The reaction mixture was allowed to stir overnight at rt. Extra 10 mL of DCM was added in to the reaction and it was washed with sat.

NaHCO3 (15 mL). Organic layer was dried over sodium sulfate and evaporated to dryness. The crude mass was dissolved in EtOH (5 mL). Conc. HCl (2 mL) was added into the reaction mixture and it was refluxed for 3 h. The solvent was evaporated and residue was basified with aqueous ammonia solution and extracted with ethyl acetate (2 X 15 mL). Combined organic layer was dried over sodium sulfate and evaporated to dryness. The crude product was purified using column chromatography (1% MeOH/DCM) to give 4.4a as yellow solid (94 mg, 73 %) mp 85 ̊ C; 1H

NMR (CDCl3, 500MHz) δ 7.71 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 7.5 Hz, 2H), 7.46-7.35 (m, 5H),

6.58 (s, 1H), 6.52 (s, 1H), 3.78 (s, 3H), 3.57-3.51 (m, 4H), 2.77 (dd, J = 5.3, 22.1 Hz, 4H), 2.61 (s,

13 2H), 1.79 (d, J = 2.9 Hz, 4H); C NMR (CDCl3, 125 MHz) δ 167.2, 145.4, 143.9, 143.5, 140.0,

157

133.3, 128.8 X 2, 127.8 X 2, 127.4 X 2, 127.1 X 2, 126.8 X 2, 125.3, 112.2, 110.5, 57.2, 56.0,

+ 55.9, 50.0, 39.8, 28.3, 27.3, 24.8; (ESI) m/z calcd. for C27H30N2O3 ([M+H] ), 431.2329, found

431.2328.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-naphthamide (4.4b)

1 Yield 76%, yellow solid, mp 82 ̊ C; H NMR (CDCl3, 500MHz) δ 8.21 (s, 1H), 7.81-7.66

(m, 5H), 7.49 (td, J = 7.9, 23.7 Hz, 2H), 6.52 (s, 1H), 6.41 (s, 1H), 3.77 (s, 3H), 3.56 (s, 4H), 2.75

13 (s, 4H), 2.62 (s, 2H), 1.81 (s, 4H); C NMR (CDCl3, 125 MHz) δ 167.8, 145.3, 143.8, 134.5,

132.5, 132.0, 128.9 X 2, 128.1, 127.6 X 2, 127.3 X 2, 126.3, 123.7, 112.2, 110.4, 57.3, 55.8, 55.7,

+ 50.2, 40.0, 28.1, 27.2, 24.7; (ESI) m/z calcd. for C25H28N2O3 ([M+H] ), 405.2173, found 405.2177.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)quinoline-4- carboxamide (4.4c)

1 Yield 66%, yellow powder, mp 89 ̊ C H NMR (CDCl3, 500MHz) δ 8.80 (s, 1H), 8.62 (d,

J = 4.3 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.70(t, J = 7.2 Hz, 1H), 7.54

(t, J = 7.8 Hz, 1H), 7.16 (d, J = 4.3 Hz, 1H), 6.27 (s, 1H), 6.13 (s, 1H), 3.78 (s, 3H), 3.56 (d, J =

5.5 Hz, 2H), 3.34 (s, 2H), 2.55 (pent, J = 7.3 Hz, 4H), 2.35(t, J = 5.6 Hz, 2H), 1.84-1.80 (m, 4H);

13 C NMR (CDCl3, 125 MHz) δ 167.4, 149.6, 148.3, 145.3, 143.7, 129.6, 129.5, 127.2 X 2, 125.5

125.3, 124.5, 124.3, 118.3, 111.8, 110.1, 57.8, 55.7, 55.3, 50.5, 40.1, 27.9, 27.8, 25.1; (ESI) m/z

+ calcd. for C24H27N3O3 ([M+H] ), 406.2125, found 406.2134.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)isonicotinamide (4.4d)

1 Yield 70%, brown solid, mp 104 ̊ C H NMR (CDCl3, 600MHz) δ 8.44 (dd, J = 1.6, 3.7

Hz, 2H), 8.29 (s, 1H), 7.46 (dd, J = 1.3, 3.8 Hz, 2H), 6.54 (s, 1H), 6.50 (s, 1H), 3.84 (s, 3H), 3.59

158

(s, 2H), 3.50 (q, J = 5.4 Hz, 2H), 2.79 (s, 4H), 2.65 (t, J = 6.1 Hz, 2H), 1.83-1.76 (m, 4H); 13C

NMR (CDCl3, 150 MHz) δ 165.6, 150.1 X 2, 145.8, 144.3, 141.7, 124.6 X 2, 121.0 X 2, 112.3,

+ 110.6, 56.8, 56.0, 55.6, 50.0, 39.7, 27.8, 27.0, 24.4; (ESI) m/z calcd. for C20H25N3O3 ([M+H] ),

356.1969, found 356.1976.

4-hydroxy-N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)benzamide

(4.4e)

1 Yield 65%, yellow powder, mp 80 ̊ C H NMR (C5D5N, 500MHz) δ 9.03 (s, 1H), 8.42 (d,

J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 6.75 (s, 1H), 3.87 (s, 2H), 3.75-3.71 (m,

13 5H), 3.97 (s, 4H), 2.79 (s, 2H), 1.88-1.77 (m, 4H); C NMR (CD3OD, 125 MHz) δ 169.1, 161.1,

147.6, 145.4, 129.2 X 2, 125.3, 123.3, 123.1, 115.0 X 2, 112.8, 111.3, 57.0, 55.3, 54.2, 50.7, 39.2,

+ 27.2, 26.5, 22.9; (ESI) m/z calcd. for C21H26N2O4 ([M+H] ), 371.1965, found 371.1969.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methoxybenzamide

(4.4f)

1 Yield 80%, yellow solid, mp 68 ̊ C H NMR (CDCl3, 500MHz) δ 7.38 (s, 2H), 7.22 (d, J

=7.5 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.96 (dd, J = 2.3, 8.1 Hz, 1H), 6.53 (s, 2H), 3.83 (s, 3H),

3.81 (s, 3H), 3.67 (s, 2H), 3.49 (q, J = 6.0 Hz, 2H), 2.88 (dd, J = 4.0, 13.3 Hz, 4H), 2.73 (t, J =

13 6.6 Hz, 2H), 1.81 (pent, J = 6.9 Hz, 2H), 1.73 (pent, J = 6.3 Hz, 2H); C NMR (CDCl3, 125 MHz)

δ 167.5, 159.6, 145.9, 144.2, 136.0, 129.3 X 2, 124.2, 118.8, 117.6, 112.3, 112.1, 110.6, 56.4, 55.9,

+ 55.4, 54.6, 50.1, 39.4, 27.2, 26.9, 23.6; (ESI) m/z calcd. for C22H28N2O4 ([M+H] ), 385.2122, found 385.2126.

4-fluoro-N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)benzamide

(4.4g)

159

1 Yield 78%, yellow solid, mp 57 ̊ C H NMR (CDCl3, 500MHz) δ 7.75 (s, 1H), 7.61-7.58

(m, 2H), 6.81 (t, J = 8.7 Hz, 2H), 6.54 (s, 1H), 6.50 (s, 1H), 3.85 (s, 3H), 3.50-3.45 (m, 4H), 2.75-

13 2.68 (m, 4H), 2.56 (t, J = 6.1 Hz, 2H), 1.76 (t, J = 5.8 Hz, 4H); C NMR (CDCl3, 125 MHz) δ

166.6, 164.3 (d, J = 249 Hz), 145.4, 143.9, 130.9 X 2, 129.2, 129.1, 126.8, 125.1, 115.2 (d, J =

21.8 Hz), 112.2, 110.5, 57.5, 56.2, 55.9, 50.2, 40.0, 28.5, 27.3, 24.9; (ESI) m/z calcd. for

+ C21H25FN2O3 ([M+H] ), 373.1922, found 373.1927.

4-chloro-N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)benzamide

(4.4h)

1 Yield 76%, brown solid, mp 63 ̊ C H NMR (CDCl3, 500MHz) δ 7.93 (s, 1H), 7.53 (d, J =

8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.51 (s, 1H), 6.48 (s, 1H), 3.84 (s, 3H), 3.50-3.46 (m, 4H),

13 2.70 (t, J = 3.0 Hz, 4H), 2.56 (s, 2H), 1.76 (s, 4H); C NMR (CDCl3, 125 MHz) δ 166.7, 145.5,

144.0, 137.0, 133.1 X 2, 128.4, 126.5 X 2, 125.1 X 2, 112.2, 110.5, 57.4, 56.1, 55.9, 50.1, 40.0,

+ 28.3, 27.3, 24.; (ESI) m/z calcd. for C21H25ClN2O3 ([M+H] ), 389.1626, found 389.1624.

4-bromo-N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)benzamide

(4.4i)

1 Yield 72%, yellow powder, mp 89 ̊ C H NMR (CDCl3, 500MHz) δ 7.98 (s, 1H), 7.48 (d,

J = 8.4 Hz, 2H), 7.25 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H), 6.48 (s, 1H), 3.84 (s, 3H), 3.53 (s, 2H), 3.46

13 (d, J = 5.3 Hz, 2H), 2.73 (s, 4H), 2.59 (t, J = 5.6 Hz, 2H), 1.77 (s, 4H); C NMR (CDCl3, 125

MHz) δ 166.7, 145.6, 144.0, 133.5, 131.3 X 2, 128.6, 126.1 X 2, 125.5, 125.0, 112.2, 110.5, 57.3,

+ 55.9 X 2, 50.0, 39.9, 28.1, 27.2, 24.7; (ESI) m/z calcd. for C21H25BrN2O3 ([M+H] ), 433.1121, found 433.1108.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-4-iodobenzamide (4.4j)

160

1 Yield 69%, brown solid, mp 95 ̊ C H NMR (CDCl3, 500MHz) δ 7.86 (s, 1H), 7.65 (dd, J

= 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 6.55 (s, 1H), 6.53 (s, 1H), 3.86 (s, 3H),

3.75 (s, 2H), 3.48 (q, J = 5.5 Hz, 2H), 2.96-2.95 (m, 2H), 2.86-2.84 (m, 2H), 2.80 (t, J = 6.9 Hz,

13 2H), 1.86 (pent, J = 6.7 Hz, 2H), 1.75 (pent, J = 6.3 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 166.9,

145.9, 144.4, 137.4, 137.2, 134.0, 131.3 X 2, 128.7 X 2, 124.0, 112.2, 110.5, 97.9, 56.0, 55.8, 54.5,

+ 49.5, 39.1, 26.7, 23.4; (ESI) m/z calcd. for C21H25IN2O3 ([M+H] ), 481.0983, found 481.0986.

N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-4-methoxybenzamide

(4.4k)

1 Yield 83%, brown powder, mp 62 ̊ C H NMR (CDCl3, 500MHz) δ 7.70 (d, J = 8.3 Hz,

2H), 7.41 (s, 1H), 6.75 (d, J = 8.3 Hz, 2H), 6.57 (s, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.72 (s, 2H),

3.49 (q, J = 5.5 Hz, 2H), 2.93-2.89 (m, 4H), 2.76 (s, 2H), 1.84 (pent, J = 6.6 Hz, 2H), 1.74 (pent,

13 J = 6.2 Hz, 2H); C NMR (CDCl3, 125 MHz) δ 167.1, 161.8, 145.8, 144.2, 128.8 X 2, 126.8 X 2,

124.2 X 2, 113.5, 112.3, 110.6, 56.1, 55.9, 55.3, 54.6, 49.7, 39.1, 29.5, 26.9, 23.5; (ESI) m/z calcd.

+ for C22H28N2O4 ([M+H] ), 385.2122, found 385.2122.

4-cyano-N-(4-(7-hydroxy-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)benzamide

(4.4l)

1 Yield 68%, yellow solid, mp 115 ̊ C H NMR (CDCl3, 500MHz) δ 8.43 (s, 1H), 7.80 (d,

J = 8.0 Hz, 2H), 6.45 (d, J = 8.2 Hz, 2H), 6.53 (s, 1H), 6.49 (s, 1H), 3.86 (s, 3H), 3.71 (s, 2H), 3.50

(d, J = 5.3 Hz, 2H), 2.93-2.75 (m, 6H), 1.88 (pent, J = 6.3 Hz, 2H), 1.79 (pent, J = 6.4 Hz, 2H);

13 C NMR (CDCl3, 125 MHz) δ 166.0, 146.1, 144.5, 138.5, 132.0 X 2, 127.8 X 2, 123.9 X 2, 118.2,

114.3, 112.1, 110.4, 56.2, 56.0, 54.9, 49.6, 39.3, 26.9, 26.6, 23.5; (ESI) m/z calcd. for C22H25N3O3

([M+H]+), 380.1969, found 380.1971.

161

4.6.2 Biological evaluation

As per Section 3.8.2

4.6.3 Metabolic stability study

As per Section 3.8.3

162

Appendix: NMR spectra of final compounds

2.1

163

3.29a

164

3.29b

3.29c

165

166

3.29d

167

3.29e

168

3.29f

169

3.29g

3.29h

170

171

3.15

3.35a

172

173

3.35b

174

3.35c

175

3.35d

176

3.35e

177

3.35f

178

4.1

179

4.2a

180

4.2b

181

4.2c

182

4.3a

183

4.3b

184

4.3c

185

4.3d

186

4.4a

187

4.4b

188

4.4c

189

4.4d

190

4.4e

191

4.4f

192

4.4g

193

4.4h

194

4.4i

195

4.4j

196

4.4k

197

4.4l

198

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