The Development and SAR of Selective Sigma-2 Receptor Ligands for the Diagnosis of Cancer Mark Ashford University of Wollongong

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2010 The development and SAR of selective sigma-2 receptor ligands for the diagnosis of cancer Mark Ashford University of Wollongong

Recommended Citation Ashford, Mark, The development and SAR of selective sigma-2 receptor ligands for the diagnosis of cancer, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3634

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The Development and SAR of Selective Sigma-2 Receptor Ligands for the Diagnosis of Cancer

Mark E. Ashford B. Med. Chem. Hons (Adv)

A thesis submitted in fulfilment of the requirements for the award of the degree

Doctor of Philosophy

From

University of Wollongong

School of Chemistry

August 2010

Declaration

I, Mark Edward Ashford, declare that this thesis, submitted in fulfilment of the requirements for the award of Doctor of Philosophy, in the School of Chemistry, Faculty of Science, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution.

Mark E. Ashford 2010

Mark Ashford, PhD Thesis 2010 ii Table of Contents Declaration...... ii

List of Figures...... vi

List of Schemes ...... viii

List of Tables ...... x

Abbreviations ...... xi

Publications and Presentations ...... xiv

Acknowledgements...... xv

Abstract...... xvi

1. Introduction...... 1 1.1. Sigma Receptors...... 1 1.2. Sigma Receptor Subtypes...... 2 1.2.1. Sigma-1 Receptor...... 2 1.2.2. Sigma-2 Receptor...... 3 1.3. Synthetic Sigma-2 Ligands ...... 3 1.4. Sigma-2 Receptor Function...... 4 1.4.1. Apoptosis ...... 4 1.5. The role of Sigma-2 Receptors in Disease...... 5 1.5.1. Cancer ...... 5 1.6. Classes of Sigma-2 Ligands...... 6 1.6.1. Spiro and Phenylpiperidines...... 7 1.6.2. Azabicyclic Ligands...... 8 1.6.3. Benzamide Analogues...... 9 1.7. Radiopharmaceutical Chemistry...... 11 1.7.1. Radionuclides...... 11 1.7.2. Radiopharmaceuticals ...... 12 1.7.3. Imaging Modalities ...... 14 1.8. Radiolabelled Sigma-2 Ligands...... 15 1.8.1. 123 I Labelled Ligands...... 16 1.9. Project Aims...... 17 2. Synthesis of Region 3 Aromatic Derivatives...... 19 2.1. Rationale for targeted compounds...... 19 2.2. SAR of Region 3...... 19 2.3. Synthesis of the key intermediate 4-((3,4-dihydro-6,7-dimethoxyisoquinolin- 2(1 H)-yl)methyl)piperidine-1-yl)amine [34] ...... 21 2.4. Amine coupling reactions and preparation of [36] ...... 23 2.5. Synthesis of the 4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidine-1-yl) amides with region 3 modification ...... 24 2.5.1. Non-halogenated amides...... 24

Mark Ashford, PhD Thesis 2010 iii 2.6. Halogenated amides...... 26 2.6.1. Synthesis of 5-iodobenzofuran-2-carboxylic acid [44] ...... 29 2.7. Synthesis of target halogenated amides ...... 33 2.8. In vitro binding of amide derivatives...... 35 2.9. Lipophilicity Estimates ...... 39 2.10. Attempted further modification to Region 3...... 39 2.11. Conclusion ...... 40 3. Synthesis of Region 2 Carbon Spacer Derivatives...... 42 3.1. Modification of Region 2...... 42 3.2. General Synthetic Strategy...... 43 3.3. Synthesis of flexible benzamide intermediates...... 44 3.3.1. Synthesis of target amines [70] -[72] ...... 44 3.4. Synthesis of flexible spacer target amides [73] -[78] ...... 46 3.5. In vitro studies and log P...... 50 3.6. Amides with semi-rigid spacers...... 52 3.6.1. Synthesis of key amines [90] and [91] ...... 53 3.6.2. Synthesis of target compounds [92] -[95] with a ethyl- or propylpiperidine spacer...... …….55 3.7. Synthesis of propylpiperazine target compound [102] ...... 56 3.7.1. Method 1: Synthesis of amine intermediate [101] via the Boc protected piperazine [99] ...... 57 3.7.2. Method 2: Synthesis of piperazine [101] via the benzyl protected piperazine [100] ...... ………….60 3.8. Synthesis of target amide [104] ...... 63 3.9. In vitro studies and log P...... 64 3.10. Conclusions and future directions...... 65 4. Synthesis of Region 3 Isoquinoline Derivatives...... 67 4.1. Rationale ...... 67 4.2. Isoquinoline Synthesis ...... 67 4.3. Formation of isoquinoline intermediates ...... 73 4.3.1. Synthesis of conformationally flexible intermediates using isoquinolines [105] , [108] and [115a] ...... 73 4.3.2. Synthesis of conformationally semi-restricted intermediates using isoquinolines [108] and [115a] ...... 74 4.4. Synthesis and in vitro evaluation of amides with region 3 modification...... 75 4.4.1. Synthesis of target flexible spacer amides [134] -[136] and semi-rigid amides [137] -[138] ...... 75 4.5. In vitro studies and log P...... 77 4.6. Conclusions...... 79 5. Summary of in vitro results and Consequence to the SAR...... 80 5.1. General considerations regarding the Structure and Activity Relationship ....80 5.2. Target compounds [36] -[42] and [60] -[65] with modification to Region 3....81 5.3. Target compounds [73] -[78] , [92] -[95] , and [104] with modification to Region 2...... 83 5.4. Target compounds [134] -[140] with modification to region 1 ...... 84 6. Radiochemistry, Cell Studies and in vivo evaluation of tracers...... 87

Mark Ashford, PhD Thesis 2010 iv 6.1. Radiolabelling Methods ...... 87 6.2. Synthesis of 123 I Compounds ...... 88 6.2.1. Synthesis of radiolabelled compound [ 123 I] [65] ...... 88 6.2.2. Synthesis of [ 123 I] [78] ...... 92 6.3. In vitro Cell studies...... 95 6.4. In vivo biodistribution studies...... 97 6.4.1. In vivo biodistribution of [ 123 I] [65] ...... 97 6.4.2. In vivo biodistribution of [ 123 I] [78] ...... 101 6.5. In vivo competition studies...... 103 6.6. In vivo stability study of [ 123 I] [78] ...... 105 6.6.1. In vivo stability study of [ 123 I] [78] ...... 106 6.7. Conclusions and Future Directions...... 106 7. Conclusions and Future Directions ...... 108

8. Experimental ...... 113 8.1. General Comments...... 113 8.2. Experimental procedures for region 3 modification ...... 115 8.2.1. Intermediate Synthesis ...... 115 8.2.2. Synthesis of target compounds with region 1 modification...... 124 8.3. Experimental procedures for region 2 modification ...... 138 8.3.1. Intermediate Synthesis ...... 138 8.3.2. Synthesis of Final Compounds with Region 2 modification...... 151 8.4. Experimental procedures for region 3 modification ...... 163 8.4.1. Region 3 intermediate synthesis...... 163 8.4.2. Target compounds with Region 1 modification...... 178 8.5. Experimental procedures for the stannylation reactions and radioiodination reactions...... 184 8.6. Pharmacological Methods...... 187 8.6.1. Membrane Preparation...... 187 8.6.2. In Vitro binding Assay for Sigma-1 Receptor...... 187 8.6.3. In Vitro binding Assay for Sigma-2 Receptor...... 188 8.6.4. In Vivo Biodistribution Studies for [ 123 I] [65] and [ 123 I] [78] ...... 188 8.6.5. In Vivo Competition Studies for [ 123 I] [78] ...... 189 8.6.6. In Vivo Stability Studies for [ 123 I] [78] ...... 190 8.6.7. Lipophilicity Estimations...... 191 9. References ...... 192

Mark Ashford, PhD Thesis 2010 v List of Figures

Figure 1.1 The proposed endogenous sigma-1 ligand, dimethyltrypamine (DMT)...... 2 Figure 1.2 Structures of representative sigma receptor ligands ...... 4 Figure 1.3 Inhibition constants of Sigma-1 and Sigma-2 receptors by phenylpiperidines [11] and [12] ...... 8 Figure 1.4 11 C-labelled derivatives of PB28 [21] , [22] and [23] ...... 15 Figure 1.5 18 F labelled benzamide derivatives of selective sigma-2 ligands ...... 16 Figure 1.6 123 I labelled Sigma-2 selective benzamide derivative [26] ...... 16 Figure 1.7 Lead compound [27] with restricted carbon spacer core and modified amido group...... 17 Figure 2.1 Structure of lead compound [27] showing possible modification regions. Region 1 is the tetrahydroisoquinoline, region 2 is the spacer, and region 3 is the amide incorporating the benzofuran moiety ...... 19 Figure 2.2 Activation of alcohol via triphenylphosphine mediated iodination...... 22 Figure 2.3 Array of halogenated carboxylic acids [43] -[48] to be used in coupling to the amine [34] ...... 27 Figure 2.4 Synthesis of 5-iodobenzofuran-2-carboxylic acid [44] via diazonium intermediate [56] ...... 29 Figure 3.1 Current examples of high activity and selective sigma-2 ligands with a flexible, linear spacer of 2 [15] or 4 [17] carbons...... 42 Figure 3.2 Target compound of type [66] showing where region 2 modification will take place either by: a) lengthening the spacer or, b) making the spacer flexible. .... 43 Figure 3.3 Pathway to target compound [102] via the Boc [99] or Benzyl [100] protected analogues...... 57 Figure 5.1 General considerations regarding the Structure and Activity Relationship...... 80 Figure 5.2 Substitution changes made to region 3 of the lead compound [27] ...... 81 Figure 5.3 Variations made to region 2 of the lead compound [27] ...... 83 Figure 5.4 Substitutional changes made to region 1 of lead compound [27] ...... 85 Figure 6.1 Three common oxidants used in electrophilic radiolabelling……………………88 Figure 6.2 Highest affinity sigma-2 ligand from chapter 2, with an IC 50 value of 12.5 nM...... 88 Figure 6.3 Purification of [ 123 I] [65] using semipreparative HPLC column eluted with 40% ACN:60% H 2O:0.1% TFA at 3mL/min showing UV activity (top) and radioactivity (bottom)...... 91

Figure 6.4 The highest affinity sigma-2 ligand from chapter 3, displaying a sigma-2 IC 50 of 1.0 nM...... 92 Figure 6.5 Purification of [ 123 I] [78] (at 9.1 min) using a semipreparative RP HPLC column eluted with 50% ACN:50% H 2O:0.1% TFA at 3 mL/min...... 94

Mark Ashford, PhD Thesis 2010 vi Figure 6.6 The percentage uptake of [ 123 I] [78] in different cancer cell lines: MCF-7 (human breast adenocarcinoma cancer cell line), PC-3 (human prostate cancer cell line), A375 (human melanoma cancer cell line) and MDA (human breast adenocarcinoma cancer cell line)...... 96 Figure 6.7 Biodistribution of [ 123 I] [65] in male Sprague-Dawley rats at various time points...... 98 Figure 6.8 Biodistribution of [ 123 I] [65] in Sprague-Dawley rats in all brain regions...... 100 Figure 6.9 Biodistribution of [ 123 I] [78] in male Sprague-Dawley rats at various time points...... 102 Figure 6.10 Effect of various drugs on [ 123 I] [78] uptake in rat organs...... 104

Mark Ashford, PhD Thesis 2010 vii List of Schemes

Scheme 2.1 Divergent synthesis of amine [34] to yield the amides of type [35] ...... 21 Scheme 2.2 Varying amide bond formation conditions for the synthesis of amide [36] ...... 23 Scheme 2.3 Coupling reactions using HOBt/EDC conditions to produce [36] -[42] ...... 25 Scheme 2.4 Potassium permanganate oxidation of aldehyde [49] to the carboxylic acid [47] ...... 27 Scheme 2.5 Synthesis of carboxylic acid [48] from the indole [50] ...... 28 Scheme 2.6 The synthesis of 5-benzofuran-2-carboxylic acid [43] ...... 28 Scheme 2.7 Reduction conditions for the nitro functionality of [54] to give the corresponding aniline [55] ...... 30 Scheme 2.8 Iodination of aniline [55] via the diazonium salt [56] and subsequent hydrolysis to yield the target compound 5-iodobenzofuran-2-carboxylic acid [44] ...... 31 Scheme 2.9 Synthesis of target carboxylic acid [44] from the corresponding iodosalicylaldehyde [58] ...... 32 Scheme 2.10 Synthesis and yield of halogenated amides [60] -[65] ...... 34 Scheme 2.11 Attempted synthesis of 1 H-indene-2-carboxylic acid...... 40 Scheme 3.1 Formation of target compounds [73] -[78] with a 4,5 or 6 carbon flexible spacer...... 44 Scheme 3.2 Attempted amide formation for target amides [76] -[78] using DMAP, NsCl, Et 3N...... 46 Scheme 3.3 Synthesis and yield of Sulfonamides [79] -[81] ...... 47 Scheme 3.4 General pathways for amide formation employing NsCl coupling conditions...... 48 Scheme 3.5 Pathway of the side reaction that lead to the formation of Sulfonamides [79] -[81]...... 48 Scheme 3.6 Coupling reactions attempted using HOBt/EDC conditions...... 49 Scheme 3.7 Synthetic pathway to target ligands [92] -[95] with semi-rigid region 2 modification...... 53 Scheme 3.8 Conditions for the formation of propylpiperidine [83] from pyridine [96] ...... 54 Scheme 3.9 Synthesis of target amides [92] -[95] ...... 55 Scheme 3.10 Conditions for alkylation of Boc protected piperazine [97] to give piperazine [98] ...... 58 Scheme 3.11 Synthesis of the Boc-protected piperazine intermediate [99] by condensation of isoquinoline hydrochloride [32] and piperaize [98] ...... 59 Scheme 3.12 Varying attempts at Boc deprotection of piperazine [99] ...... 59

Mark Ashford, PhD Thesis 2010 viii Scheme 3.13 The synthesis of isoquinoline piperazine [100] via alkylpiperazine [103] ...... 61 Scheme 3.14 Deprotection Methods in an attempted to increase the yield of [101] ...... 62 Scheme 3.15 Coupling of amine [102] with carboxylic acid [44] to yield the target amide [104] ...... 63 Scheme 4.1 The synthesis of racemic 1-phenylisoquinoline [105] promoted by TFA...... 67 Scheme 4.2 Synthesis of 6-methoxytetrahydroisoquinoline [108] ...... 68 Scheme 4.3 The attempted synthesis of isoquinolines [113] and [114] ...... 69 Scheme 4.4 Synthesis of 7-methoxytetrahydroisoquinoline [115] using pathway 2...... 71 Scheme 4.5 Reaction conditions to yield 7-methoxy-1,2,3,4-tetrahydroisoquinoline [115] through selective reduction of the heterocyclic ring...... 72 Scheme 4.6 Unrestricted intermediate synthesis using isoquinolines [105] , [108] and [115a] ...... 74 Scheme 4.7 Restricted intermediate synthesis using isoquinolines [108] and [115a] ...... 75 Scheme 6.1 Reaction conditions for synthesis of stannane [144] ...... 89 Scheme 6.2 Radiolabelling conditions to synthesise [ 123 I] [65] ...... 90 Scheme 6.3 Radiosynthesis of [ 123 I] [78] ...... 93

Mark Ashford, PhD Thesis 2010 ix List of Tables

Table 1.1 Inhibition of sigma-1 and sigma-2 receptors by the spiropiperidine derivatives 40 ...... 7 Table 1.2 Inhibition constants of azabicyclic compounds for the sigma-1 and sigma-2 receptors...... 9 Table 1.3 Inhibition constants of tetrahydroisoquinoline benzamide derivatives for the sigma-1 and sigma-2 receptor...... 10 Table 1.4 Selected radionuclides of interest in nuclear medicine...... 13

Table 2.1 Sigma-1 and sigma-2 binding affinities (IC 50 ) of benzamides bearing different R groups...... 36 Table 3.1 Sigma-1 and sigma-2 binding affinities of target compounds [73] -[81] ...... 50 Table 3.2 Sigma-1 and sigma-2 binding affinities of target amides [92] -[95] and [104] ...... 64 Table 4.1 Flexible amides [134] -[138] using EDC and HOBt coupling conditions...... 76 Table 4.2 Semi-rigid target amides [139] and [140] using NsCl and DMAP or EDC and HOBt coupling conditions...... 77

Table 4.3 Sigma-2 and Sigma-1 IC 50 values for flexible ligands with modification at region 3...... 78

Table 4.4 Sigma-2 and Sigma-1 IC 50 values for semi-rigid ligands with modification at region 3...... 78

Mark Ashford, PhD Thesis 2010 x Abbreviations

% Percentage

µL Microlitre

10% Pd/C 10% Palladium on Charcoal

ACN Acetonitrile

AcOH Acetic Acid

Ar Aryl bs Broad singlet

CAT Chloramine-T

CI Chemical Ionisation

CNS Central Nervous System

DCC N,N'-Dicyclohexylcarbodiimide dd Doublet of doublets

DEPT Distorntionless Enhancement by Polarisation Transfer

DIPEA Diisopropylethylamine

DMF Dimethylformamide

DMM Dimethylmorpholine

DMSO Dimethylsulfoxide

DMT Dimethyltryptamine

DNA Deoxyribonucleic acid dt Doublet of triplets

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

EI Electron Impact

ES Electrospray

Mark Ashford, PhD Thesis 2010 xi FDG Fluorodeoxyglucose

GIT Gastrointestinal Tract

G-protein Guanine Nucleotide-binding protein h Hour

[3H]DTG [3H] 1,3-di-o-tolylguanidine

HOBt Hydroxybenzotriazole

HPLC High Performance Liquid Chromatography

HRMS High Resolution Mass Spectroscopy

Hz Hertz

IC 50 Inhibition Constant at 50%

ID/g Injected dose per gram

IP3 Inositol trisphosphate kDA Kilodalton keV Kiloelectron Volts

Ki Inhibition Constant

M Molar m Multiplet m/z Mass-to-charge ratio

MBq Megabequerel min Minute mL Millilitre mmol Milli mol mp Melting Point

MS Mass Spectrometry nM Nanomolar

Mark Ashford, PhD Thesis 2010 xii NsCl 4-Nitrobenzenesulfonamide oC Degrees Celcius

PE Petroleum Ether

PET Positron Emission Tomography psi Pounds per square inch

PTC Phase Transfer Catalyst q Quartet

RCY Radiochemical Yield rt Room temperature s Singlet

SAR structure activity relationship

SN2 Bimolecular Nucleophilic Substitution

SPECT Single Photon Emission Computed Tomography t Triplet

TBAI Tetrabutylammonium Iodide

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography

UV Ultraviolet

Mark Ashford, PhD Thesis 2010 xiii Publications and Presentations

Presentations

1. AIMECS 09 - 7th AFMC International Medicinal Chemistry Congress, 23 rd -27 th August, 2009, Cairns Convention Centre, Cairns, Australia.

2. Annual Chemistry Department Conference, Kioloa, 2006 and 2007.

Mark Ashford, PhD Thesis 2010 xiv Acknowledgements

There are many people I would like to thank in making this project would possible:

To my supervisors Associate Professor Paul Keller and Dr. Andrew Katsifis for their guidance and support throughout the duration of the project.

To everyone within the new ANSTO lifesciences (the old RRI). Specifically, I would like to thank Vu and Rachael for their help with everything biology. Also, I am also indebted to my lab buddy, Branko - it has definitely been ‘educating’!!!!!!!!

To the other PhD students who started this journey with me: Karina, Linda, Jane and

Mick F. They shared my pain and joy and were always there to have a laugh and joke with. Thankyou for the numerous coffees and chats!

To my family for all their love, belief in me and your continued, ongoing support throughout the duration of this project.

Finally to my fiancé Ammie, only you truly know what an effort this has been.

Thankyou for the constant words of encouragement, your patience, understanding and love. Thankyou for putting up with my whinging, late nights and stressful moments.

You never gave up on me when I thought things were hopeless. You truly are an amazing, wonderful person and I am very lucky to have you in my life. Love you my angel. xoxo

Mark Ashford, PhD Thesis 2010 xv Abstract

Three series of compounds formed the basis for sigma-1 and sigma-2 receptor structure activity studies. In total, thirty four compounds based on the lead benzamide compound

[27] were synthesised, incorporating a variety of isoquinoline and carboxylic acid moieties, linked together with either a linear or cyclic amine spacer. Furthermore, the incorporation of a halogen on selected carboxylic acid moieties provided a convenient strategy for the introduction of a radiohalogen for applications in pharmacological and imaging studies.

Thirteen compounds with modifications to the carboxylic acid region (region 3), bearing substituted benzoic and benzofuran carboxylic acids were synthesised in five steps.

Each of these carboxylic acid fragments were linked to a 6,7-dimethoxytetrahydroisoquinoline ring through a rigid piperidine spacer. In vitro evaluation of these compounds to determine their sigma-1 and sigma-2 receptor affinities gave IC 50 values for the sigma-2 receptor ranging from 9.8 – 955 nM. In vitro structure activity relationship (SAR) studies indicated that halogen substitution yielded compounds with greater affinity than their unsubstituted analogues and that most brominated compounds displayed higher binding affinities than their iodinated analogues. The optimum benzamide compounds contained a 5-bromo or 5- iodobenzofuran-2-carboxylic acid fragment. The highest affinity iodinated ligand, [65] with a sigma-2 affinity of 12.2 nM, was radiolabelled with 123 I in 51-82% radiochemical yield and was consequently evaluated in vivo in Sprague-Dawley rats. Biodistribution studies indicated uptake in organs known to contain sigma-2 receptors including the pancreas and lungs.

Mark Ashford, PhD Thesis 2010 xvi Fourteen compounds with spacer modifications (region 2) were synthesised. Nine of these compounds contained a flexible carbon spacer, and five contained a semi-rigid

(cyclic) spacer. The highest affinity ligand [78] bearing an iodobenzofuran fragment and displaying an IC 50 of 1.0 nM linked to the isoquinoline via a six carbon flexible spacer was radiolabelled with 123 I in 78% radiochemical yield and also evaluated in cell studies and in vivo in rats. This compound showed high uptake in breast tumour cells known to express sigma-2 receptors. Although this compound displayed high uptake in organs known to contain sigma-2 receptors including the pancreas and lungs, initial drug competition studies with the non-specific sigma-2 ligand “haloperidol” failed to displace the radioactivity corresponding to this compound. In the absence of a complete receptor screen, it suggests that this compound may have a higher affinity for other receptor types in vivo and that the uptake of radioactivity may not be due to its affinity to the sigma-2 receptor in vivo . However, metabolite studies showed that the radioactivity extracted from the cortex, lungs and spleen after 60 min was > 95% intact, whilst indicating only between 55-65% unchanged tracer in the plasma at this time point. Three novel nitrosulfonamides were also synthesised in this series, however all exhibited only moderate to weak sigma-2 receptor IC50 values of 31-800 nM.

Seven compounds containing modified isoquinolines (region 1) were synthesised and evaluated. Compound [135] bearing an iodobenzofuran fragment linked to the 6- methoxytetrahydroisoquinoline via a six carbon flexible spacer displayed an IC 50 value of 5.0 nM for the sigma-2 receptor; however, it also displayed comparable high affinity for the sigma-1 receptor (IC 50 14.0 nM). All other compounds displayed moderate sigma-2 IC 50 values ranging from 35-150 nM

Mark Ashford, PhD Thesis 2010 xvii

Chapter 1 - Introduction

1. Introduction

1.1. Sigma Receptors

Sigma receptors were discovered in 1976 and represent a class of proteins that were initially classified as a subtype of the opiate receptor. 1 Further studies revealed that the sigma receptors are unique proteins with an amino acid sequence, drug selectivity pattern, and anatomical distribution that is distinct from the opioid peptides. 2 Abundant sigma sites are found in the brain and spinal cord, as well as heart, spleen, adrenal glands, kidneys, GI tract and liver. 3

Much of the early interest in sigma receptors was driven by their involvement in CNS disorders such as schizophrenia, depression and dementia and their potential role as antipsychotic agents. This was mainly due to their high affinity for most of the typical neuroleptics, such as haloperidol, and their shared affinity for compounds known to cause psychotomimetic activity such as N-allylnormetazocine. 4 Certain sigma receptor antagonists have been shown to ameliorate the effects of cocaine and other psychostimulant drugs of abuse. 5

More recently, sigma receptors have been found in high density in many tumour cells 4 including breast, prostate, lung and melanoma compared to normal cells. Over expression in various tumour cell types, and particularly in their proliferating phase, suggested their development as potential tumour-imaging agents. 3

To date, no endogenous functional ligand for sigma receptors has been conclusively identified. Therefore, the development of specific sigma ligands would improve our understanding of the role of sigma receptors in physiological and pathophysiological

Mark Ashford, PhD Thesis 2010 1 Chapter 1 - Introduction functions. 6 In addition, as sigma receptors are associated with diverse biochemical and physiological processes, the synthesis and discovery of selective ligands may provide potential drugs for a variety of disorders such as psychosis, neuroprotection, motor control and cancer treatment. The development of radiolabelled analogues could also provide tumour and neurodegenerative imaging agents for non-invasive diagnosis and clinical research in oncology and neuroscience.

1.2. Sigma Receptor Subtypes

Pharmacological, functional and molecular studies have identified two subclasses of sigma receptors namely sigma-1 and sigma-2. 5 These two subtypes are distinct and do not seem to be co-located within the same protein macromolecule, nor do they represent different affinity states of the same receptor. 7

1.2.1. Sigma-1 Receptor

Cloning studies have confirmed that sigma-1 receptors constitute a single polypeptide containing 223 amino acids with a molecular weight of 25.3 kDa. 8 The amino acid sequence is not consistent with that of a classical G protein-coupled receptor, which makes it especially difficult to predict the effects of agonists or antagonists of the receptor. 5 It is a chaperone protein at the endoplasmic reticulum that modulates calcium signalling through the inositol trisphosphate (IP3) receptor 9 and is expressed in many different tissue types as well as the CNS. 10 The endogenous agonist is thought to be dimethyltrypamine [1] (Figure

1.1) 11 and was found through the use of a simplified conical sigma-1 receptor pharmacophore, 12 competition assays and sigma-1 receptor knockout mice. 11

Mark Ashford, PhD Thesis 2010 2 Chapter 1 - Introduction

H N

N CH3 H3C [1]

Figure 1.1 The proposed endogenous sigma-1 ligand, dimethyltrypamine (DMT) 11

1.2.2. Sigma-2 Receptor

The sigma-2 receptor is an 18-21 kDa protein whose characteristics are not yet known.

Subcellular localisation studies in EMT-6 mouse and human MDA-MB-435 human breast cancer cells involving two photon and confocal microscopy with sigma-2 fluorescent probes indicated that sigma-2 receptors are located on the plasma membrane, endoplasmic reticulum, lysosomes and the mitochondria. 13

1.3. Synthetic Sigma-2 Ligands

Although a number of sigma-2 receptor ligands have been reported, most of them have either a high selectivity for the sigma-1 site (e.g. (+)-pentazocine [2]) or bind with similar affinities to both the sigma-1 and sigma-2 receptors (eg haloperidol [3] and DTG [4] ). 14

Only a few examples, such as siramesine [5], CB-184 [6], azabicyclic and the benzamide analogues represented by WC-59 [7] and RHM-1 [8] have a moderate to high selectivity for the sigma-2 receptor (Figure 1.2). 15-23

Mark Ashford, PhD Thesis 2010 3 Chapter 1 - Introduction

H3C

CH3 N

Pentazocine [2] Cl NH OH O N N H H N CH3 CH3 Ditolyguanidine (DTG) [4] F Halperidol [3]

H C 3 N OH O Cl N N Cl

[6] Siramesine [5] CB-184

CH3 OCH3 O N F CH3O O N N O N OCH3 H H OCH3

CH3 WC-59 [7] RHM-1 [8]

Figure 1.2 Structures of representative sigma receptor ligands

1.4. Sigma-2 Receptor Function

1.4.1. Apoptosis

Studies using DNA fragmentation (TUNEL staining) and inversion of phosphatidyl serine

(Annexin V binding), 24 have shown that sigma-2 receptors are involved in the apoptotic processes of numerous tumour cells lines. These include the human breast tumour, MCF-

7, 24 human neuroblastoma, SK-N-SH, 25 and murine fibrosarcoma, WEHI-S26 cell lines.

Although the mechanism of cell death is largely unknown, studies have suggested that the

Mark Ashford, PhD Thesis 2010 4 Chapter 1 - Introduction sigma-2 receptor induces apoptosis by caspase-dependant 25 and/or caspase independent pathways. 24,26 In SK-N-SH neuroblastoma cells, sigma-2 receptor ligands reduced mitochondrial membrane potential and induced caspase-dependant apoptosis, suggesting that sigma-2 receptors play a role in the intrinsic apoptotic pathway. 13 Caspase-independent cell death may involve lysosome leakage, cathepsin activation, and oxidative stress. 26

However, it is not known whether the mode of action is a direct result of interactions with sigma-2 receptors residing in the mitochondria and lysosomes or via a downstream signalling mechanism. 13

Apoptosis is also regulated by release of intracellular calcium, and sigma-2 receptor agonists promote calcium release from the endoplasmic reticulum and mitochondrial stores 27 with subsequent cell death by caspase-independent apoptosis. 24 Apoptosis may also be induced by tumor cells by regulation of the sphingolipid pathway. 28

1.5. The role of Sigma-2 Receptors in Disease

1.5.1. Cancer

A number of studies have reported that the sigma-2 receptor may be a potential biomarker for the proliferative status of solid tumours. For example, studies using a tissue culture model of mouse mammary adenocarcinoma have shown that the density of sigma-2 receptors is approximately 10 times higher in cycling proliferating (P) cells than in the corresponding noncycling quiescent (Q) tumour cells. 29 A subsequent study using solid tumour xenografts derived from the same tumour cell line demonstrated a positive correlation between the sigma-2 receptor density and the P:Q ratio measured by flow cytometry. 30,31 The agreement between the solid tumour and tissue culture data suggested that the expression of sigma-2 receptors may serve as a potential biomarker of the

Mark Ashford, PhD Thesis 2010 5 Chapter 1 - Introduction proliferative index of solid tumours. Although these studies were conducted using a mouse mammary adenocarcinoma, the observation that there is a high density of sigma-2 receptors in a wide panel of human tumour cell lines 32 including brain 33 , bladder 34 , colon 35 and melanoma 35 suggests that this could provide an imaging strategy in many types of human tumours.

1.5.1.1. Improving the effectiveness of Cytotoxic Drugs

Most antitumour drugs have severe adverse side effects at high doses or following chronic use thus limiting their clinical utility. One of the strategies to overcome this obstacle is to use a drug with no overlapping toxicity to enhance the ability of another antitumour drug to kill tumour cells. This is known as chemosensitisation and can result in either an increase in tumour cell kill at the same level of toxicity or a decrease in toxicity at the same level of tumour cell kill. 23 Additional studies with some sigma-2 selective ligands have demonstrated their ability to enhance the cytotoxicity of anticancer drugs 24,26,36,37 by inhibiting the activity of P-glycoprotein, responsible for the active extrusion of anticancer drugs. 37 For example, cytotoxicity is increased when MCF-7 cells are treated with combinations of either doxorubicin or actinomycin and the sigma-2 selective ligand, CB-

184. 24 Consequently, there is also great interest in developing high affinity sigma-2 ligands as anticancer drugs or chemosensitising agents.

1.6. Classes of Sigma-2 Ligands

Although many different classes of compounds have demonstrated high sigma-2 affinity almost all of these exhibited comparable affinity for the sigma-1 receptor. Only three general classes of compounds have displayed useful sigma-2 affinity and selectivity over sigma-1; the phenyl-38,39 and spiropiperidines, 40 some azobicyclic analogues, 15 and the dimethoxyisoquinoline benzamides. 41

Mark Ashford, PhD Thesis 2010 6 Chapter 1 - Introduction

1.6.1. Spiro and Phenylpiperidines

1.6.1.1. Spiropiperidines

The spiropiperidines were the first class of compounds to show high affinity and selective binding to the sigma-2 receptor over the sigma-1 receptor. A large series of spiropiperidines bearing an N-fluorophenylindole and a flexible, linear carbon chain linking it to the spiropiperidine moiety were evaluated for their sigma-2 affinity. The key structural elements contributing to both high affinity and selectivity was the fluorine atom on the 3 or 6 position of the spiro ring (Table 1.1). 40

Table 1.1 Inhibition of sigma-1 and sigma-2 receptors by the spiropiperidine derivatives 40

R1 N N

R4 F

Ki (nM)

Compound X R1 R4 σ1 σ2

[5] O H H 17 0.12

[9] O F H 290 1.8

[10] O H F 150 0.58

Mark Ashford, PhD Thesis 2010 7 Chapter 1 - Introduction

1.6.1.2. Phenylpiperidines

Selective phenylpiperidine compounds linked by a four carbon flexible spacer to a N-4- fluorophenylindole were also reported to exhibit high affinity and selective binding to the sigma-2 receptor over the sigma-1 receptor.38 Restricting the conformation of the piperidine ring as in Lu 29-253 [11], it was found to enhance the overall sigma-2 selectivity, whilst retaining its high affinity (Figure 1.3).

Figure 1.3 Inhibition constants of Sigma-1 and Sigma-2 receptors by phenylpiperidines [11] and [12]

1.6.2. Azabicyclic Ligands

Compounds bearing two aromatic moieties separated by an azabicylic core have been the most extensively studied 42-49 for sigma-2 activity. The most selective azabicyclics 40 are shown in Table 1.2. The addition of two chlorine atoms at the 3 and 4 positions of the phenyl ring almost doubles the selectivity for the sigma-2 receptor, with little affect on affinity.

Mark Ashford, PhD Thesis 2010 8 Chapter 1 - Introduction

Table 1.2 Inhibition constants of azabicyclic compounds for the sigma-1 and sigma-2 receptors. H3C N OH R

R O

Ki (nM)

Compound Enantiomer R σ1 σ2

[13] (+)-1R,5R H 3 063 16.5

[6] (+)-1R,5R Cl 7 436 13.4

1.6.3. Benzamide Analogues

Compounds incorporating a functionalised aromatic benzamide structure linked to a 6,7- dimethoxytetrahydroisoquinoline ring via a flexible carbon spacer of varying length are the most selective and potent sigma-2 ligands known, with activities ranging from 1 – 102 nM and selectivity ratios over the sigma-1 receptor greater than 100. They also possess vastly superior in vivo kinetics than other reported ligands (Table 1.3). 3,41,50,51

Mark Ashford, PhD Thesis 2010 9 Chapter 1 - Introduction

Table 1.3 Inhibition constants of tetrahydroisoquinoline benzamide derivatives for the sigma-1 and sigma-2 receptor.

H3CO OR1 N R2 H3CO N n H H R4

Ki (nM)

Compound n R1 R2 R4 σ1 σ2

[14] 1 OCH 3 H Br 5 484 12.4

[15] 1 OCH 3 H CH 3 10 412 13.3

[16] 1 OCH 2CH 2F H CH 3 22 750 102

[17] 3 OCH 3 OCH 3 Br 12 900 8.2

[18] 3 OCH 3 OCH 3 I 554 1

[19] 3 OCH 2CH 2F OCH 3 Br 15 300 386

[8] 3 OCH 3 H CH 3 3 078 10.3

[20] 3 OCH 2CH 2F H Br 1 076 0.65

Table 1.3 shows the binding affinity of selected isoquinoline derivatives linked to various benzamides. The structure activity relationship (SAR) of this class suggests activity is optimal with a carbon spacer length of 4 units, and an isoquinoline 6,7-dimethoxy substitution pattern, typified by compound [17] (σ-2 K i = 8.2 nM, σ-1 Ki = 12 900 nM).

The optimum substitution pattern on the benzamide ring incorporated a halogen with either a dimethoxy or fluoroethoxy substitution.

Mark Ashford, PhD Thesis 2010 10 Chapter 1 - Introduction

1.7. Radiopharmaceutical Chemistry

The overexpression of the sigma-2 receptor in a variety of tumours and the association of this receptor with proliferation and apoptosis suggests that radiolabelled sigma-2 specific receptor ligands could have enormous potential in the imaging and diagnosis of tumours as well as providing valuable clinical information on the proliferative and growth index of cancers to complement [ 18 F]FDG studies. In addition, radiolabelled sigma-2 ligands could offer significant value in further elucidating the role and function of the sigma-2 receptor as well as providing information on the pharmacological mechanisms of selective sigma-2 ligands and the receptor. The general aspects of radiopharmaceutical chemistry will be briefly reviewed, followed by examples of applications of radiolabelled sigma-2 ligands.

1.7.1. Radionuclides

Radionuclides are unstable and undergo radioactive decay to achieve stability. During decay they either emit electromagnetic radiation or charged particles which are classified into three distinct classes:

• Alpha radiation: the emission of a helium nucleus,

• Beta radiation: the emission of a positron or electron, and

• Gamma radiation: the emission of gamma rays (photons).

Gamma radiation has a greater penetrating ability than that of alpha or beta radiation, making it suitable for imaging of the body by external detectors. Typical radionuclides used in imaging include: 11 C, 13 N, 15 O, 18 F, 99m Tc, 123 I and 76 Br.

Only a few radionuclides combine the favourable characteristics of physical decay with desirable biological characteristics to become a useful medical radioisotope. Factors such as half life, dose minimisation, detection characteristics and energy emission must be

Mark Ashford, PhD Thesis 2010 11 Chapter 1 - Introduction considered. 52 This leads to the use of radioisotopes such as iodine and fluorine, which have appropriate half-lives and radiation emission that is not detrimental to the patient. Since these halogens are not usually present in biological systems or pharmaceuticals, they must be incorporated into the target compound without negatively changing the interactions between it and the target. For example, 18 F is a bioisostere for H or OH, while 123 I can

53 often substitute for OH or CH 3 substituents. Today, many radiopharmaceuticals have incorporated 123 I or 18 F substituents. 54

1.7.2. Radiopharmaceuticals

Radiopharmaceuticals are medicinal products which are composed of a radionuclide attached to a chemical entity used primarily for diagnostic or therapeutic purposes.

Different radionuclides may be used for diagnosis or therapy, for example, 123 I is used in

SPECT imaging while 131 I is used in therapy. Table 1.4 shows the half life, decay mode, and use of selected radionuclides. 123 I decays by electron capture, a process whereby an orbital electron collapses into the nucleus and combines with a proton to produce a neutron and a neutrino. 55 The nucleus that has captured the orbital electron can emit a gamma ray that can be subsequently detected outside the body and utilised in imaging.

Mark Ashford, PhD Thesis 2010 12 Chapter 1 - Introduction

Table 1.4 Selected radionuclides of interest in nuclear medicine

Entry Nuclide Decay mode Half life Energy Use 1 11 C β+ 20.4 min 0.33 MeV PET # 2 13 N β+ 9.98 min 0.43 MeV PET 3 15 O β+ 2.03 min 0.65 MeV PET 4 18 F β+ 109.8 min 0.20 MeV PET 5 123 I Electron capture ς 13.2 hours 159 keV SPECT * 6 131 I β- 8.1 days 364 keV Therapy 7 99m Tc Electron capture 6 hours 140 keV SPECT

# PET: Positron Emission Tomography, * SPECT: Single Photon Emission Computed Tomography, ςElectron capture: A type of nuclear reaction where an orbital electron collapses into the nucleus and a proton is converted into a neutron. The nucleus that has captured the orbital electron can emit a gamma ray.

Administration of a particular radiopharmaceutical to a patient results in its selective localisation in the organ of interest, with the distribution showing if it is diseased or abnormal (such as in tumour development). This is represented by parts of the image being

‘hotter’ or ‘colder’ than the surrounding normal tissue 56 with the radiation emitted from the radiopharmaceutical the radiation emitted from the radiopharmaceutical detected outside the body in a non-invasive manner using specific detector systems. Radionuclides incorporated onto radiopharmaceuticals emitting a single photon are detected on imaging platforms called SPECT (single photon emission computer tomography) whilst those emitting dual photons are detected by PET (positron emission tomography). An efficient diagnostic radiopharmaceutical should have high uptake in the target tissue soon after injection, with rapid clearance from surrounding tissue to provide optimal imaging of the disease as well as reducing the radiation burden to the patient and carers.

Mark Ashford, PhD Thesis 2010 13 Chapter 1 - Introduction

1.7.3. Imaging Modalities

In PET imaging, positrons ( β+, positively charged electrons) are emitted from a positron- emitting radionuclide such as 11 C, 13 N, 15 O or 18 F (Table 1.4). The emitted positron travels a short distance and hits an electron. This collision causes annihilation of both particles, with the creation of two 511 keV γ-rays travelling in opposite directions 180 o to each other.

Detectors on opposite sides of the positron source detect the pair of γ-rays simultaneously and are usually arranged in a circle around the body so the full 360 o view is covered. 53

SPECT uses a scintillation camera to detect the γ-rays emitted from radionuclides such as

99m Tc and 123 I. The emitted photons are detected and the data collated to produce either a transverse (cross section), sagittal (longitudinal) or coronal (frontal) image. 57 PET is much more sensitive than SPECT, however, the radioisotopes used in PET imaging have much shorter half-lives. 58

Both PET and SPECT are powerful tools for the non-invasive study of physiological, biochemical and pharmacological functions and pathways at the molecular level. Of huge significance is the ability of these techniques to detect and measure functional receptors and binding sites at sub-nanomolar concentrations. As a consequence, monitoring the changes in receptor concentration or binding sites may provide significant insights into the progress of diseases at a molecular level. In addition to high specificity and selectivity, a radiolabelled ligand should display other essential properties including high specific activity, low non-specific binding, slow metabolism, receptor saturability, blood brain barrier permeability and safety for human use.

Mark Ashford, PhD Thesis 2010 14 Chapter 1 - Introduction

1.8. Radiolabelled Sigma-2 Ligands

In recent years, a number of sigma-2 receptor ligands have been radiolabelled with 11 C, 18 F,

76 Br or 123 I and their pharmacological and biological characterisation reported. 3,16,17,59

PB28 [21] has been radiolabelled with both 11 C59 for PET imaging as with 3H60 for pharmacological studies. The radiolabelling of PB167 17 [22] also with 11 C permitted the rapid comparative analysis of the pharmacokinetics of the structural analogues. Similarly, the aromatic analogue PB183 [23] has the potential to be radiolabelled with 11 C (Figure

1.4) and compared to PB28 and PB167, also shows superior pharmacokinetic properties to that of [21] or [22] using the 3H labelled analogue. 61

N N N N n

11 O CH3 OCH3

[11C]PB28, n = 1, [21] PB183 [23]

11 [ C]PB167, n = 2, [22]

Figure 1.4 11 C-labelled derivatives of PB28 [21] , [22] and [23]

Ligands incorporating 18 F have also been synthesised for use in PET studies16 (Figure 1.5).

Conformationally flexible benzamide analogues [24] and [25] containing an isoquinoline linked to a fluoro-ethoxy aromatic derivative have been synthesised and pharmacokinetic properties studied in rats and EMT-6 tumoured BALB/c mice. They were shown to bind specifically to sigma-2 receptors in vivo and were acceptable agents for detecting solid tumours and imaging their sigma-2 receptor status with PET.

Mark Ashford, PhD Thesis 2010 15 Chapter 1 - Introduction

18F

H CO 3 OO

N R1 H3CO N H

[24] R1 = H, R2 = CH3 [25] R1 = OCH3, R2 = I

Figure 1.5 18 F labelled benzamide derivatives of selective sigma-2 ligands

1.8.1. 123 I Labelled Ligands

The short half life of a positron emitting radioisotope limits the use of these tracers to centres with PET imaging systems and in proximity to cyclotron centres. A specific sigma-

2 ligand incorporating a longer-lived radionuclide such as 123 I may have greater clinical potential for imaging cancer using widely available SPECT cameras. To date, only one compound 3 [26] has been labelled with 123 I (Figure 1.6). This tracer [26] was evaluated in female BALB/c mice bearing EMT6 tumours. It was also labelled with 125 I for cell uptake and tumour studies for comparison against [ 18 F]FDG in EMT6 cells. These studies showed that the level of uptake of the benzamide derivative [26] was comparable to that of

[18 F]FDG, a commonly used nonselective metabolic marker 3 and that radiopharmaceuticals bearing a radiohalogen such as I-123 for SPECT imaging are feasible.

H3CO O OCH3 N OCH3 H3CO N H

123I [26]

Figure 1.6 123 I labelled Sigma-2 selective benzamide derivative [26]

Mark Ashford, PhD Thesis 2010 16 Chapter 1 - Introduction

The lack of availability of SPECT labelled radiopharmaceuticals for imaging sigma-2 receptors in disease and the limited structure-activity relationship (SAR) studies (Table

1.3) prompted the development of benzamide derivatives in our laboratories. In our previous work 62 we established the lead compound [27] which displayed excellent affinity and selectivity for the sigma-2 receptor (IC 50 = 3 nM); c.f sigma-1 (IC 50 1250 nM).

However, in this initial work structural variations were restricted to the amido group and the carbon spacer between the isoquinoline and the amide. In addition, this structure was limited in its suitability for incorporation of a suitable radionuclide (Figure 1.7).

carbon linker amido group

O H CO O 3 N N H3CO [27]

Sigma-1 IC50 = 1230 nM Sigma-2 IC = 3 nM 50

Figure 1.7 Lead compound [27] with restricted carbon spacer core and modified amido group.

1.9. Project Aims

Therefore the general aims of this project were to:

a) Modify the structure of [27] to develop highly active (sigma-2 < 10 nM) and

selective (sigma-1 > 1000 nM) ligands for the sigma-2 receptor that had the

potential to be radiolabelled with 123 I for SPECT imaging,

b) Generate knowledge from SAR studies to allow further development of PET

analogues.

Mark Ashford, PhD Thesis 2010 17 Chapter 1 - Introduction

c) Characterise lead radiolabelled sigma-2 ligands via tumour cell uptake studies,

biodistribution studies, drug competition studies and metabolite analysis in normal

and tumour bearing animals. In doing so, it could provide an insight into the SAR

pharmacophore of the sigma-2 binding site.

Specifically, the project will involve:

1. The chemical synthesis and characterisation of ligands based upon the lead

compound [27],

2. Perform in vitro binding studies to determine their affinity and selectivity for the

sigma-1 and sigma-2 binding domains,

3. Prepare appropriate precursor compounds suitable for radiolabelling with 123/125 I,

4. Undertake 123 I radiolabeling, purification and formulation studies on compounds

with high affinity and selectivity,

5. Perform in vivo biodistribution, drug competition and stability studies in rats,

6. Perform in vitro tumour cell uptake studies,

7. Perform in vivo tumour uptake studies.

Mark Ashford, PhD Thesis 2010 18 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

2. Synthesis of Region 3 Aromatic Derivatives

2.1. Rationale for targeted compounds

The favourable in vitro properties displayed by the lead compound [27] (Figure 2.1), and the limited SAR study, 62 provided an excellent structural platform for the development of sigma-2 ligands suitable for radiolabelling. This basic structure had additional benefits in that it allowed significant structural modifications covering the isoquinoline (Region 1), the spacer (Region 2) and the benzamide (Region 3) offering both structural flexibility, restricted conformations and a variety of substitution patterns to the resultant analogues.

Region 1 Region 2 Region 3 (isoquinoline) (spacer) (amide)

O H CO O 3 N N H3CO

[27]

Figure 2.1 Structure of lead compound [27] showing possible modification regions. Region 1 is the tetrahydroisoquinoline, region 2 is the spacer, and region 3 is the amide incorporating the benzofuran moiety .

2.2. SAR of Region 3

The first region to be investigated was the aromatic region 3. In addition to synthesising high affinity iodinated sigma-2 ligands, the corresponding brominated analogues (where appropriate) were also synthesised as they provided the basis for preparing the organotin derivatives for radiolabelling via electrophilic iododestannylation reactions with 123 I.

Additional compounds were prepared without a bromo or iodo substituent to gain a better

Mark Ashford, PhD Thesis 2010 19 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives understanding of the SAR for this structural class. Any active derivatives that emerged could then be further modified with a bromo or iodo substituent, as appropriate, as well as extending to the development of PET radionuclides in the future.

As part of the overall strategy towards the synthesis of the 4-((3,4-dihydro-6,7- dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidine-1-yl) amide derivatives (as outlined in

Scheme 2.1), a flexible and divergent synthesis was adopted that would yield a range of suitable candidates. Protection 63 of the commercially available 4-piperidine methanol [28] followed by conversion of the alcohol [29] into the mesylate 63 [30] or iodide 64 [31], would allow a S N2 reaction with the commercially available isoquinoline [32] to yield compound

[33]. Deprotection 63 of [33] would yield the key intermediate [34] as a free secondary amine which could then be coupled to a range of carboxylic acids to yield the targeted compounds of the type [35].

Mark Ashford, PhD Thesis 2010 20 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Scheme 2.1 Divergent synthesis of amine [34] to yield the amides of type [35] .

R HO HO 2 ditertbutyl Method 1 dicarbonate, CH2Cl2 84%

N 96% N Method 2 N H Boc 58% Boc [28] [29] [30] R2 = OMs [31] R2 = I

H CO 3 Method 3: 58% NH.HCl Method 4: 78% H CO 3 [32]

H CO 3 NH H CO Boc TFA/CH2Cl2 3 N N H CO N 3 86% H CO [34] 3 [33]

HO R1

O H CO 3 R1 = various aromatic substituents N R1 N H3CO [35]

2.3. Synthesis of the key intermediate 4-((3,4-dihydro-6,7-dimethoxyisoquinolin- 2(1 H)-yl)methyl)piperidine-1-yl)amine [34]

The Boc protection of amine [28] proceeded smoothly using di-tert -butyl dicarbonate in

63 CH 2Cl 2 and after workup afforded [29] in 96% yield. Treatment of the product with methanesulfonyl chloride and anhydrous triethylamine 63 gave the mesylate [30] in 84% yield (Scheme 2.1, Method 1). Alternatively, treatment of [29] with triphenylphosphine, imidazole and iodine 64 (Scheme 2.1, Method 2) gave the iodo derivative [31] in 58% yield.

Mark Ashford, PhD Thesis 2010 21 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

The order of addition of the reagents for iodination is important as the triphenylphosphine activates the iodine, enabling a S N2 addition to the methylene adjacent to the hydroxyl group to take place (Figure 2.2).

R I HO R Ph3P I I Ph3PI Ph3PO Ph3PO RI

Figure 2.2 Activation of alcohol via triphenylphosphine mediated iodination

The alkylated isoquinoline [33] was initially prepared by reaction of the mesylate ester [30] and isoquinoline [32] in a suspension of K 2CO 3 with a catalytic quantity of lithium iodide in 2-butanone at reflux (Scheme 2.1, Method 3). After workup, the residue was purified by silica gel column chromatography (EtOAc:MeOH, 9:1) to afford [33] in 58% yield.

Improved yields of [33] were obtained when the iodinated piperidine [31], the isoquinoline

[32], TBAI and K 2CO 3 in DMF were stirred for 16 h at rt (Scheme 2.1, Method 4). Work- up and purification gave [33] in a 78% yield.

Analysis of the 1H NMR spectrum of [33] showed the addition of singlet peaks at δ 6.52 and 6.59 ppm assigned to the two aromatic protons of the tetrahydroisoquinoline ring. The resonance attributed to the methylene CH2OMs of [30] had shifted upfield from δ 4.07 ppm to 2.33 ppm, which was consistent with substitution of the mesylate with an isoquinoline.

Analysis of the 13 C NMR spectrum revealed new peaks at δ 56.06 ppm and 56.08 ppm, assigned to the methoxy substituents. In addition, a base peak at m/ z 391 in the ES + mass

+ spectrum was assigned to the MH ion, with the molecular formula C 22 H34 N2O4 confirmed by HRMS with a peak at m/z 391.2597.

Mark Ashford, PhD Thesis 2010 22 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

63 Deprotection of the secondary amine [33] under standard conditions in CH 2Cl 2:TFA (2:1) stirred at rt for 1 h gave the key amine intermediate [34] in an overall yield of 86%

(Scheme 2.1).

Analysis of the 1H NMR of [34] indicated the disappearance of the peak at δ 1.45 ppm previously assigned to the tert -butylcarbonyl sp 3 protons. A peak at m/z 291 in the ES mass spectrum was assigned to the MH + ion.

2.4. Amine coupling reactions and preparation of [36]

The synthesis of [36] involved the coupling of amine [34] to indole-2-carboxylic acid.

Although amide bond formation conditions are well established,65 three different approaches were initially trialled for the synthesis of [36] to establish the best conditions for these substrates (Scheme 2.2).

Scheme 2.2 Varying amide bond formation conditions for the synthesis of amide [36]. O H N HO O H H CO H3CO N 3 NH N N N H3CO H3CO 30-52 % [36] [34]

Method Conditions Reaction Reaction Yield (%)

temperature time

1 DCC/HOBt/DMM/CH 2Cl 2 rt 3 days 30

2 EDC/HOBt/DIPEA/DMF rt 2 days 48

66 3 NsCl/DMAP/ACN/Et 3N reflux 3 h 52

Mark Ashford, PhD Thesis 2010 23 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Method 1 employed the use of DCC/HOBt as the coupling agents to assist in the amide formation and after 3 days gave a yield of 30%. Method 2 utilised the coupling reagents

EDC/HOBt which resulted in a faster reaction time and higher yield (48%) of [36]. The advantage of this method is the convenient removal of the urea by-products during aqueous work up due to the presence of a tertiary amine in the structure of EDC.67 Method 3 66 utilised 4-nitrobenzenesulfonamide (NsCl) and DMAP reagents to give the amide [36] in a

52% yield. Therefore, a combination of Methods 2 and 3 were used based upon superior yielding reactions and shorter reaction times.

The syntheses of tertiary amides from carboxylic acids are relatively difficult to achieve and are only obtained under forcing conditions. For example, a sterically hindered tertiary amide similar to that of [36] was prepared in 52% yield using NsCl/DMAP/ACN/Et 3N as outlined above in Method 3.66 Recently, methods involving the use of triacyloxyborane derivatives 68 or thermal amidation using molecular sieves 69 have increased yields of selected carboxylic acids and amines, however, the narrow scope of some of the reported methods limits their extensive application.

2.5. Synthesis of the 4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidine-1-yl) amides with region 3 modification

2.5.1. Non-halogenated amides

A series of non-halogenated 4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidine-1-yl) amides of type [35] with region 3 aromatic substitution were synthesised. In a typical amine condensation, amine [34], indole-2-carboxylic acid, EDC,

HOBt and DIPEA in anhydrous DMF were stirred at rt for 2 days. After column chromatography, the amide [36] was formed in 48% yield. Analysis of the 1 H NMR

Mark Ashford, PhD Thesis 2010 24 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives spectrum of [36] showed new peaks in the aromatic section at δ 6.77, 7.07, 7.20, 7.45 and

7.62 ppm assigned to the protons of the indole. Analysis of the 13 C NMR spectrum showed a new peak at δ 162.7 ppm assigned to the carbonyl group. In addition, the base peak at m/ z

434 in the ES + mass spectrum was assigned to the MH + ion of amide [36], with molecular formula of C 26 H31 N3O3 confirmed by HRMS with a peak at m/z 433.2356. The results are summarised in Scheme 2.3.

Scheme 2.3 Coupling reactions using HOBt/EDC conditions to produce [36]-[42] O

HO R O H CO HOBt, EDC, H CO 3 3 N R NH DIPEA N N H CO H3CO DMF 3 [36]-[42] [34]

Compound R Reaction time (h) Yield (%) H N [36] 42 48

S [37] 49 41

[38] 52 46

[39] 43 48 N H N [40] 45 42

[41] 46 35

[42] 40 39 NH

Mark Ashford, PhD Thesis 2010 25 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

The overall yields for these reactions were moderate to low and could be explained by the low solubility of the some of the carboxylic acids in DMF. However, sufficient material was generated to allow testing and optimisation was not pursued further.

2.6. Halogenated amides

Halogenated derivatives of amides [36] -[42] were prepared to assess the effect of halogen substitution on the sigma-2 affinity and selectivity as well as a convenient route towards the introduction and preparation of radiohalogenated sigma-2 ligands. As a variety of radioiodine and radiobromine isotopes (eg I-123, I-124, I-125, Br-76) are available for both

PET and SPECT imaging, both bromine and iodine atoms were incorporated into the architecture of compounds [36]-[42]. Consequently, a number of halogenated carboxylic acids were synthesised [43]-[48] and coupled with the amine [34] to produce compounds

[60]-[66].

As in vitro studies would indicate (discussed in Section 2.8) the incorporation of a benzofuran moiety afforded compounds with the best sigma-2 affinity and selectivity (for example compound [27]). Therefore, the 5-bromo- and 5-iodobenzofuran-2-carboxylic acids [43] and [44] were synthesised. The corresponding indole derivatives were also synthesised from commercially available bromoindole [45] and the reported 70 indole [48].

The phenyl derivative [46] was commercially available and the carboxylic acid [47], yielding benzamide [17] 41 , were also incorporated into amides after reaction with amine

[34] (Figure 2.3).

Mark Ashford, PhD Thesis 2010 26 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

O O O O HO H O N HO HO

I [43] Br [44] [45] Br

O O OCH3 O CH OCH3 HO 3 HO HO

Br NH [46] Br [48] [47]

Figure 2.3 Array of halogenated carboxylic acids [43]-[48] to be used in coupling to the amine [34].

The carboxylic acid [47] was synthesised in 98% yield from the corresponding aldehyde

[49] facilitated by a potassium permanganate promoted oxidation in acetone as reported 41

(Scheme 2.4).

Scheme 2.4 Potassium permanganate oxidation of aldehyde [49] to the carboxylic acid [47]

O OCH3 10% KMnO4 O OCH3 OCH acetone, 2 h OCH3 H 3 HO 98% Br Br

[49] [47]

The carboxylic acid [48]* was synthesised as reported 70 from 5-bromoindole [50] in an overall yield of 61% over two steps. A Vilsmeier-Haack reaction 70 employing DMF and

POCl 3 gave the aldehyde [51] in 63% yield, followed by a potassium permanganate oxidation to give carboxylic acid [48] in 96% yield (Scheme 2.5).

* Carboxylic acid [48] is commercially available, however, was not available at the time of synthesis.

Mark Ashford, PhD Thesis 2010 27 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Scheme 2.5 Synthesis of carboxylic acid [48] from the indole [50]

Br Br Br 10% KMnO4 O acetone, 2 h O POCl3, DMF H HO 63% NH NH 96% NH [50] [48] [51]

The 5-bromobenzofuran-2-carboxylic acid [43] was synthesised as reported 71 from 5- bromosalicylaldehyde [52] via the ethyl ester intermediate [53] (Scheme 2.6).

Scheme 2.6 The synthesis of 5-benzofuran-2-carboxylic acid [43]

O diethylbromomalonate O Br K CO , 2-butanone, reflux, 7 h O H 2 3 EtO

OH 48% Br [52] [53]

KOH, EtOH,

H2O, reflux 65% 5 min O O HO

Br [43]

The ethyl ester intermediate [53] was synthesised in 48% yield, comparable to that reported. 71 Hydrolysis of the ester [53] with potassium hydroxide, followed by acidification, yielded the carboxylic acid [43] in 65% yield.

Mark Ashford, PhD Thesis 2010 28 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

2.6.1. Synthesis of 5-iodobenzofuran-2-carboxylic acid [44]

The synthesis of carboxylic acid [44] had not been reported at the time. Therefore, two different routes were considered for its synthesis.

2.6.1.1. The synthesis of 5-iodobenzofuran-2-carboxylic acid [44], Method 1

The first method employed in the synthesis of the 5-iodobenzofuran-2-carboxylic acid [44] involved preparing the 5-nitrobenzofuran intermediate [54],72 followed by reduction to the corresponding aniline [55] with subsequent diazotisation to [56] in the presence of iodine to selectively give the benzofuran [57]. The selective introduction of iodine at C5 generated the target 5-iodobenzofuran-2-carboxylic acid [44] (Figure 2.4).

O O benzofuran formation O O N EtO 2 H

OH [54] NO2 reduction

O O O EtO diazotization O EtO

[56] [55] N2 NH2

halogen substitution

O O O O EtO ester hydrolysis HO

[44] [57] I I

Figure 2.4 Synthesis of 5-iodobenzofuran-2-carboxylic acid [44] via diazonium intermediate [56].

Mark Ashford, PhD Thesis 2010 29 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

The intermediate 5-nitrobenzofuran [54] was synthesised using diethylbromomalonate,

72 K2CO 3 in 2-butanone heated at reflux as reported. The nitro group of [54] was then reduced 72, 73 using either 10% Pd/C under a hydrogen atmosphere at 60 psi (Method 1) or a

Raney-nickel catalyst 74 (Method 2) to yield the aniline [55] in 50% and 85% yield respectively (Scheme 2.7).

Scheme 2.7 Reduction conditions for the nitro functionality of [54] to give the corresponding aniline [55]

O O O O EtO EtO

[54] [55] NO2 NH2

Method Conditions Yield (%)

1 MeOH, dioxane, 10 % Pd/C, H 2, 60 psi, 5 h 50

2 Raney-nickel, MeOH, H 2, rt, 24 h 85

Although TLC analysis indicated quantitative reduction of nitro [54] to the corresponding aryl amine [55] (Method 1) with no other intermediates identified, the compound was isolated in a moderate 50% yield. This was significantly improved when Raney-Nickel was employed for the reduction, and amine [55] was isolated in 85% yield.

The in-situ synthesis of the diazonium salt [56] was achieved using p-toluenesulfonic acid

o (p-TsOH) and sodium nitrite (NaNO3) in ACN at 10 C. Direct substitution with potassium iodide (KI) produced the ethyl ester [57], which, upon hydrolysis with aqueous potassium hydroxide, yielded the carboxylic acid [44] (Scheme 2.8).

Mark Ashford, PhD Thesis 2010 30 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Scheme 2.8 Iodination of aniline [55] via the diazonium salt [56] and subsequent hydrolysis to yield the target compound 5-iodobenzofuran-2-carboxylic acid [44].

O p-TsOH.H2O, O O ACN, NaNO O EtO 2 EtO 10 oC [55] [56]

NH2 N2

i) KI,

ii) H2O rt, 24 h

O KOH, EtOH, O O reflux, 5 min O HO EtO [44] 71% [57]

I I 55% overall

Formation of the ethyl ester intermediate [57] was achieved in 55% over two steps.

Analysis of the 1H NMR revealed the disappearance of the peak at δ 3.57 ppm assigned to

13 the aniline NH2 protons. The C NMR showed the upfield shift of the C5 carbon from δ

146.4 ppm to 87.4 ppm, confirming the incorporation of the iodine atom onto C5. Mass spectral analysis revealed a peak at m/z at 301, assigned to the molecular ion. Formation of the target 5-iodobenzofuran-2-carboxylic acid [44] was achieved in a 71% yield through a potassium hydroxide deprotection. Analysis of the 1H NMR revealed the disappearance of the ethyl signals assigned to the ester and a mass spectral peak at m/z 288, assigned to the molecular ion.

Mark Ashford, PhD Thesis 2010 31 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

2.6.1.2. The synthesis of 5-iodobenzofuran-2-carboxylic acid [44], Method 2

The second method employed to synthesise [44] involved iodosalicylaldehyde [58]†, which was prepared by the iodination of salicylaldehyde [59] using iodine monochloride in

75 CH 2Cl 2. Cyclisation and hydrolysis of [58] as previously shown in Scheme 2.7 for the corresponding brominated benzofuran gave the 5-iodobenzofuran-2-carboxylic acid [44]

(Scheme 2.9).

Scheme 2.9 Synthesis of target carboxylic acid [44] from the corresponding iodosalicylaldehyde [58].

O ICl, CH2Cl2, O 17 h, rt I H H

OH 42% OH [58] [59]

diethylbromomalonate K2CO3, 2-butanone, 80% reflux, 7 h

KOH, EtOH,

O H2O, reflux, O O O HO 5 min EtO

71% [44] [57] I I

Analysis of the 1H NMR and mass spectral data of the reaction mixture of [58] revealed a mixture of di- (22%) and mono-iodinated products. Recrystallisation from cyclohexane and then from isopropanol gave the 5-iodosalicylaldehyde [58] in 42% yield.

†This commercially available product was not available at time of synthesis.

Mark Ashford, PhD Thesis 2010 32 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

The observed mixture of mono- and di-iodinated products is common and attributed to the permanent dipole and hence high reactivity of the iodine monochloride species, greatly facilitating electrophilic attack. Attempts to overcome the selectivity issue through the generation of iodine monochloride in situ have been reported.76 Furthermore, iodination of phenols with moderate selectivity for the para position using combinations of ammonium iodide and oxone have also been reported.76

2.7. Synthesis of target halogenated amides

The target amides [60]-[65] were synthesised from carboxylic acids [43]-[48] as previously described in Scheme 2.3. The results are summarised in Scheme 2.10.

Mark Ashford, PhD Thesis 2010 33 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Scheme 2.10 Synthesis and yield of halogenated amides [60]-[65].

HO R O H CO HOBt, EDC, H CO 3 3 N R NH DIPEA N N H CO H3CO DMF 3 [60]-[65] [34]

Compound R Reaction time (h) Yield (%) O

[60] 48 40

Br H N [61] 52 27

Br CH3 [62] 42 55 Br OCH3 OCH3 [63] 49 41

Br Br

[64] 56 38

NH O

[65] 43 46

The common NMR spectroscopic feature of these compounds is the signal assigned to the carbon adjacent to a bromine or iodine atom due to the different electronic affects these halogens exhibit on the surrounding environment. For example, analysis of the 13 C NMR spectrum for the target amide [60] revealed a signal at δ 111.2 ppm, assigned to C5 of the

Mark Ashford, PhD Thesis 2010 34 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives benzofuran adjacent to the bromine atom, whilst the target amide [65], C5 is observed at δ

93.0 ppm as it is adjacent to an iodine atom.

2.8. In vitro binding of amide derivatives

The in vitro binding affinities (IC 50 ) of all compounds for the sigma-2 receptor were determined by measuring the displacement of [ 3H]DTG ([ 3H] 1,3-di-o-tolylguanidine), a non-selective ligand for sigma-1 and sigma-2 receptors, bound to rat brain membranes

(350-400 µg/mL) with non-tritiated pentazocine added to mask the sigma-1 receptors.

Sigma-1 binding affinities were determined using [ 3H] pentazocine (a selective sigma-1 receptor ligand) on rat brain membranes. The average disintegrations per minute (dpm) were calculated for each concentration of test compound in fractions corresponding to non- specific binding, total binding and total activity. From this, the percentage inhibition was calculated using a non-linear least squares fitting method to fit the data to a sigmoid curve, which was used to estimate the IC 50 . The IC 50 values for the compounds are shown in

Table 2.1.

Mark Ashford, PhD Thesis 2010 35 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Table 2.1 Sigma-1 and sigma-2 binding affinities (IC 50 ) of benzamides bearing different R groups. H CO R 3 N N H3CO

Log P σ2 IC 50 σ1 IC 50 Selectivity R a b c Compound (nM) (nM) (σ1/σ2)

[27] 3.22 3 1230 410

H N [36] 3.29 100 ± 12 820 ± 35 8.20

S [37] 3.38 110 ± 9 210 ± 25 1.91

[38] 3.05 155 ± 6 1800 ± 160 11.6

[39] 2.38 320 ± 2 180 ± 15 0.56 N H N [40] 2.77 955 ± 55 360 ± 30 0.38

[41] 3.08 130 ± 8 1045 ± 90 8.03

[42] 2.76 850 ± 60 285 ± 40 0.34 NH O

[60] 4.14 9.8 ± 1.1 5600 ± 200 510

Br H N [61] 4.20 45 ± 9 8500 ± 500 111

Br CH3 [62] 3.51 50 ± 6 170 ± 32 3.40 Br

Mark Ashford, PhD Thesis 2010 36 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

OCH3 OCH3 [63] 3.16 415 ± 1.0 4800 ± 300 12.0

Br Br

[64] 3.63 180 ± 40 870 ± 60 4.83

NH O

[65] 4.25 12.5 ± 2.5 1500 ± 320 120

(a) The log P7.5 values were determined by comparing HPLC retention times of test compounds with standards. (b) The concentration of tested compounds that inhibited [ 3H]DTG binding to rat brain membranes

(IC 50 ) by 50% was determined with nine concentrations of the test compounds, each performed in triplicate.

IC 50 values are the average of at least three determinations.

The compounds examined displayed a medium to high affinity for the sigma-2 receptor, ranging from 9.8 nM to 955 nM with most compounds displaying selectivity for the sigma-

2 receptor over the sigma-1 receptor, with some sigma-1 IC 50 > 5000 nM. The most potent ligands for the sigma-2 receptor in this section were [60] and [65] bearing a 5-bromo- and

5-iodobenzofuran-2-carboxamide in region 3. Both displayed a sigma-2 IC 50 of 9.8 and

12.5 nM respectively, 3-4 fold less active than the lead compound [27] (sigma-2 IC 50

3nM). In addition, the selectivity between the sigma receptor subtypes was shown to be greatest when the 5-bromobenzofuran-2-carboxamide was employed in region 3. Changing the heteroatom from oxygen [27] to nitrogen [36] or to sulphur [37] in region 3 decreased the binding affinity and selectivity for the sigma-2 receptor, with [37] being almost equipotent at both receptor sites.

Mark Ashford, PhD Thesis 2010 37 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

A comparison of the brominated benzamides [61] (sigma-2 IC 50 45 nM) and [64] (sigma-2

IC 50 180 nM) to that of their non-halogenated counterparts [36] (sigma-2 IC 50 100 nM) and

[42] (sigma-2 IC 50 850 nM) revealed that a bromine atom on an indole moiety in region 3 increases the sigma-2 affinity.

The iodobenzofuran benzamide [65] (sigma-2 IC 50 12.5 nM) had a slightly lower affinity and selectivity for the sigma-2 receptor when compared to that of its brominated counterpart [60] (sigma-2 IC 50 9.8 nM), suggesting that the smaller the halogen, the better the binding affinity.

A direct comparison can be made between [63] (sigma-2 IC 50 415 nM) and the literature

41 compound [17] (sigma-2 IC 50 8.2 nM) as both contain the same aromatic carboxylic acid in region 3. The inclusion of a piperidine moiety and removal of the amido NH group in

[63] decreased the affinity and also the selectivity for the sigma-2 receptor ([63] σ1/σ2 12.0 vs [17] σ1/σ2 1573), however, both benzamides displayed low affinities for the sigma-1 receptor. Also, the benzamide [62] containing a 5-methyl-4-bromo substituted aromatic carboxamide in region 3 gave moderate sigma-2 activity (50 nM), however, did not display the same selectivity for the receptors ( σ1/σ2 3.4) as reported for both [63] and [17] .

The 1-napthylene [41] (130 nM) and cinnamic [38] (155 nM) carboxamides displayed almost the same affinity and selectivity for the sigma-2 receptor ([41] σ1/σ2 8.03 vs [38]

σ1/σ2 11.6); however, they were not as potent or selective as the lead compound [27]. This suggested that aromatic substituents in region 3 without a heteroatom or halogen substitution decreased the affinity for the sigma-2 receptor.

Mark Ashford, PhD Thesis 2010 38 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

The 6-indole carboxamide [40] (955 nM) gave a far more potent sigma-1 ligand when compared to that of 2-indole carboxamide [36] and 3-indole carboxamide [42] , suggesting that the orientation of the nitrogen in the aromatic heterocycle affects the affinity for the sigma-1 receptor.

2.9. Lipophilicity Estimates

The lipophilicity of each compound was examined by determination of the log P7.5 value using a HPLC method.77 The HPLC retention times of test compounds were compared with standards with known log P values. The log P values are shown in Table 2.1.

Unexpectedly, varying the substitution pattern on the carboxamide significantly influenced the lipophilicity of the molecules. For example, compounds possessing the 2-carboxamide moiety possessed a higher log P value than compounds with a 3- or 6- carboxamide function, c.f. [36] (log P 3.29) vs [40] (log P 2.77) and [42] (log P 2.76). Addition of bromine to these molecules increased the log P values from 3.22 in [27], 3.29 in [36] and

2.76 in [42] to 4.14 in [60], 4.20 in [61] and 3.63 in [64] respectively. The iodinated compound [65] had a higher log P value than its brominated analogue [60] . The presence of non-halogenated carboxylic acid derivatives at R1 can deliver compounds with log P values between 2 and 3.5 and have moderate to low sigma-2 affinity, while compounds with log P values between 3.5 and 4.5 showed high sigma-2 affinity.

2.10. Attempted further modification to Region 3

To evaluate the importance of a heteroatom in compounds [27], [36] and [37] in region 3, the synthesis of 1 H-indene-2-carboxylic acid was attempted as reported78 (Scheme 2.11).

Mark Ashford, PhD Thesis 2010 39 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives

Scheme 2.11 Attempted synthesis of 1 H-indene-2-carboxylic acid. O O R R HO O

reflux, 4 h

Method R Yield

1 Br 0

2 Cl 0

However, attempts to prepare the indene carboxylic acids by reaction of oxalyl dibromide in neat indene resulted in the formation of a black tar presumably due to polymerisation of the starting material. Alternatively, the use of oxalyl dichloride based on the premise that the chlorine atom is not as good a leaving group as a bromine atom which would theoretically decrease the amount of polymerisation taking place also resulted in the formation of a black tar. The synthesis of this compound was consequently abandoned.

2.11. Conclusion

Twelve new halogenated and non-halogenated 4-((3,4-dihydro-6,7-dimethoxyisoquinolin-

2(1 H)-yl)methyl)piperidine-1-yl) amides were synthesised and a SAR study was undertaken. The SAR was based on the target compounds [36]-[42] and [60]-[65] with key differences at region 3 correlated with the measured binding affinities. Changing the orientation of the acid from the optimal 2-position decreased the sigma-2 affinity. The corresponding brominated compounds had a higher sigma-2 affinity than their non- halogenated analogues. The iodinated analogue displayed a similar affinity for the sigma-2 receptor as the brominated analogue. The heteroatom for optimal sigma-2 binding affinity was oxygen, with binding affinity decreasing from nitrogen to sulphur. Most of the compounds showed moderate to high affinity and selectivity for the sigma-2 receptor, and

Mark Ashford, PhD Thesis 2010 40 Chapter 2 – Synthesis of Region 3 Aromatic Derivatives hence, would be an applicable class of compound to potentially radiolabel with 123 I. In

Chapter 6, the radiolabelling of the most potent iodinated compound [65], will be discussed, along with in vivo evaluation of the radioiodinated compound in rats.

Mark Ashford, PhD Thesis 2010 41 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

3. Synthesis of Region 2 Carbon Spacer Derivatives

3.1. Modification of Region 2

To date, little work has been reported on the spacer between the isoquinoline and the aromatic segments in this class of compounds. Currently, analogues bearing a linear and flexible spacer of either 2 [15] or 4 [17] carbon units are described 41 (Figure 3.1).

H3CO H3CO O OCH3 O OCH3 N N OCH3 H3CO N H3CO N H H [15] CH3 [17] Br

Sigma-2 Ki: 13.3 nM Sigma-2 Ki: 8.2 nM Sigma-1 Ki: 10 412 nM Sigma-1 Ki: 12 900 nM

Figure 3.1 Current examples of high activity and selective sigma-2 ligands with a flexible, linear spacer of 2 [15] or 4 [17] carbons. 41

Modifications to region 2 involved varying the spacer length and flexibility between regions 1 and 3. Therefore, 5-bromo- and 5-iodobenzo[ b]furan-2-carboxylic acid [43] and

[44] were used in region 3 and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline in region 1 to give target compounds of type [66] (Figure 3.2).

Mark Ashford, PhD Thesis 2010 42 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Region 2 spacer for modification Region 1 Region 3

O H CO O 3 N N H3CO R

[66] R = Br, I

Figure 3.2 Target compound of type [66] showing where region 2 modification will take place either by: a) lengthening the spacer or, b) making the spacer flexible.

3.2. General Synthetic Strategy

Initially, the tetrahydroisoquinoline hydrochloride [32] was alkylated with the required bromonitriles to give the corresponding nitrogen alkylated isoquinolines [67],41 [68] and

[69]. These were then reduced to the corresponding amines [70]-[72], and coupled to either

5-bromobenzo[ b]furan-2-carboxylic acid [43] or 5-iodobenzo[ b]furan-2-carboxylic acid

[44] to give the (3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)alkyl amides [73]-[78]

(Scheme 3.1)

Mark Ashford, PhD Thesis 2010 43 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.1 Formation of target compounds [73]-[78] with a 4,5 or 6 carbon flexible spacer.

N Br n Method 1: Et3N, H3CO H3CO CH2Cl2 N NH.HCl N H3CO Method 2: TBAI, H3CO n [32] KI, K2CO3, DMF n = 2 [67], 82-85% n = 3 [68], 83% n = 4 [69], 86%

LiAlH4, THF

H CO 3 O H3CO N O H3CO N n H N H3COn NH2 n = 2 [70], 65% R n = 2, R = Br [73] n = 3 [71], 62% n = 3, R = Br [74] n = 4 [72], 68% n = 4, R = Br [75] n = 2, R = I [76] n = 3, R = I [77] n = 4, R = I [78]

3.3. Synthesis of flexible benzamide intermediates

3.3.1. Synthesis of target amines [70]-[72]

Two methods were investigated to incorporate the flexible spacer and yield the intermediate [67] via the alkylation of the isoquinoline nitrogen of [32]. The optimised reaction conditions were then applied to yield nitrile intermediates [68] and [69].

Method 1 involved the condensation of the isoquinoline hydrochloride [32] with the

41 selected nitrile in the presence of Et 3N in CH 2Cl 2 at rt for 18 h as described. For optimum yield, an additional 1.5 equivalents of Et 3N were added to the reaction mixture

Mark Ashford, PhD Thesis 2010 44 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives and was allowed to proceed for an additional 18 h to yield [67] in 82% yield. Method 2 utilised phase transfer conditions resulting in a faster reaction time (18 h) and a slightly higher yield (85%). Therefore, using the conditions outlined in Method 2, the nitrile intermediates [68] and [69] were obtained in 83 and 86% yields respectively.

The increase in yield resulting from Method 2 was attributed to the use of a phase transfer catalyst and the use of a polar aprotic solvent, DMF. In this reaction, a proton is extracted

79 from the NH of the isoquinoline. The deprotonation occurs at the surface of the K 2CO 3.

The TBAI provides a source of lipophilic cations which can undergo ion exchange to produce the lipophilic ion pair in order to bring the anion into the organic phase of the reaction. 80

The formation of the key primary amines [70]-[72] provided the framework for the generation of the final amides [73] -[78] . Therefore, a mixture of nitrile [67], and lithium aluminium hydride in THF was stirred at reflux for 18 h. After neutralisation (water, 10%

NaOH), the solution was filtered, dried (Na 2SO 4) and volatiles were removed to afford amine [70] (65%) as a honey coloured oil without the requirement for further purification.

Applying the same method, amines [71] and [72] were isolated in 62% and 68% yields respectively (Scheme 3.1).

Numerous methods for the reduction of a nitrile group to a primary amine are known, including lithium aluminium hydride or derivatives thereof, for example, lithium trimethoxyaluminum hydride. 81 However, lithium aluminium hydride was the reagent of choice for the reduction of [70]-[72] as it is reported to not generate secondary or tertiary amine byproducts. 81-83

Mark Ashford, PhD Thesis 2010 45 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

3.4. Synthesis of flexible spacer target amides [73]-[78]

To produce the target compounds [73]-[78], the amines [70]-[72] were coupled to carboxylic acids [43] or [44]. In a typical procedure, coupling of the amines [70]-[72] to carboxylic acid [44] was facilitated by the use of DMAP, Et 3N and NsCl (Scheme 3.2). The yields for the condensations were between 48-68%.

Scheme 3.2 Attempted amide formation for target amides [76]-[78] using DMAP, NsCl, Et 3N.

H3CO N H3CO n NH2 [70]-[72] n = 1,2,3

O O NsCl, DMAP, Et3N HO ACN, reflux [44] I X

H CO H3CO 3 O O N O N S H CO N H3CO N 3 n H n H O NO2 [76]-[78] [79]-[81] I n = 1,2,3 n = 1,2,3

1H NMR spectral analysis of the attempted condensation of amine [70] and acid [44] revealed two doublets at δ 7.39 ppm and 8.20 ppm which is consistent with a para substituted aromatic ring. Analysis of the 13 C NMR lacked the characteristic C-I signal between δ 85-90 ppm. Finally, mass spectral analysis revealed a signal at m/z 449 assigned as the molecular ion. These observations suggested a para-substituted nitro-sulfonamide

Mark Ashford, PhD Thesis 2010 46 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

[79] had formed. These results ‡ were also consistent for the formation of [80] and [81] and are summarised in Scheme 3.3.

Scheme 3.3 Synthesis and yield of Sulfonamides [79]-[81].

O O HO

[44] I

NsCl, DMAP, Et3N H3CO H3CO ACN, reflux O N N S H CO NH 48-68% H3CO n N 3 n 2 H O [70]-[72] [79]-[81] NO2 n = 1,2,3 n = 1,2,3

Compound n Reaction time (h) Yield (%)

[79] 1 8 48 [80] 2 6 68 [81] 3 8 60

The formation of compounds [79]-[81] can be explained by the order of addition of reagents. If the acid was added to NsCl, it should form the activated ester species in situ .

Introduction of the amine to this solution should facilitate the formation of the corresponding amide bond (Scheme 3.4).

‡ The syntheses of [79], [80] and [81] were perfomed in parallel. It was only after workup that the structures were eludicated, hence, all three compounds were synthesised.

Mark Ashford, PhD Thesis 2010 47 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.4 General pathways for amide formation employing NsCl coupling conditions.

O O O O O OH S O Cl -HCl O I OS O NO2 I R NH2 O2N

O O

I HN R amide

However, if NsCl was added to the amine, followed by the acid, the acid would not be activated and a sulfonamide product would be produced, leading to the formation of compounds [79]-[81] (Scheme 3.5).

Scheme 3.5 Pathway of the side reaction that lead to the formation of Sulfonamides [79]- [81].

O O S -HCl S R NH2 Cl RN O H O NO2 NO2 sulphonamide

The unexpected products were not discarded as they serve as precursors for other forms of radiolabelling. Also, as sulfonamide derivatives have not been reported displaying sigma-2 activity, they would be a novel class of compounds to investigate.

Mark Ashford, PhD Thesis 2010 48 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Subsequently, an alternative coupling procedure was used to give the target amides [73]-

[78]. Therefore (following Method 2, section 2.2) to a stirred solution of carboxylic acid

[44] in DMF was added HOBt, EDC, amine [70] to give the amide [76] in 47% yield

(Scheme 3.6).

Scheme 3.6 Coupling reactions attempted using HOBt/EDC conditions.

EDC, HOBt, DIPEA H CO H3CO DMF 3 O N N O H CO NH 22-47% H3CO N 3 n 2 n H [70]-[72] [73]-[78] n = 1,2,3 R n = 1,2,3

Compound R n Reaction time (h) Yield (%) [73] Br 1 18 23 [74] Br 2 17 22 [75] Br 3 16 35 [76] I 1 16 47 [77] I 2 15 30 [78] I 3 17 41

1H NMR analysis of [76] revealed signals δ 3.79 ppm (s, 3H) and 3.82 ppm (s, 3H) assigned to the two methoxy groups of the isoquinoline and signals at δ 7.05, 7.23, 7.59 and 7.90 ppm were assigned to the protons of the benzofuran. Analysis of the 13 C NMR spectrum revealed a signal at δ 87.2 ppm assigned to the carbon attached to the iodine atom. In addition, a base peak at m/ z 535 in the ES + mass spectrum was assigned to the molecular ion, with molecular formula of C 24H27 N2O4I confirmed by HRMS with a peak at m/z 534.0984.

Mark Ashford, PhD Thesis 2010 49 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

3.5. In vitro studies and log P

The six new bromo and iodo benzamides [73]-[78] and three new sulfonamides [79]-[81] were tested for their in vitro binding affinities for the sigma-1 and sigma-2 receptors as described in Chapter 2. The binding affinity results along with the log P values are shown in Table 3.1

Table 3.1 Sigma-1 and sigma-2 binding affinities of target compounds [73]-[81].

H3CO

N H CO R 3 n

Log σ2 IC 50 σ1 IC 50 Selectivity Compound R n P (nM) (nM) (σ1/ σ2) H N O [73] 1 3.81 17 ± 2.0 5230±450 295

Br H N O [74] 2 3.63 7.0 ± 0.6 900 ± 50 130

Br H N O [75] 3 3.94 9 ± 0.5 345 ± 5 40

Br H N O [76] 1 3.82 7.0 ± 2.5 1250±360 143

I H N O [77] 2 3.91 10 ± 3.0 450 ± 28 45

Mark Ashford, PhD Thesis 2010 50 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

H N O [78] 3 4.47 1.0 ± 0.8 1730±220 1000

[79] 1 2.92 800 ± 75 115 ± 3 0.15

[80] 2 3.38 36 ± 11 205 ± 45 5.7

[81] 3 3.04 31 ± 10 120 ± 5 3.8

The benzofuran compounds [73]-[78] exhibited sigma-2 affinity IC 50 values between 1.0-

17 nM with selectivity ratios ( σ1/ σ2) greater than 345. The substitution of the bromine atom for the iodine atom seemed to have a negligible effect on affinity for the sigma-2 receptor, with [78] having a sigma-2 IC 50 of 1.0 nM, giving the most potent ligand produced in this series. It also exhibited good selectivity with a sigma-1 IC 50 of >1000 nM.

Increasing the chain length from 4 to 6 carbons in the series of bromo compounds [73]-[75] decreased the selectivity for the sigma-2 receptor, however, this trend was not observed for the iodo series [76]-[78]. This suggested that the optimum length of spacer separating the isoquinoline and amide was four or six carbon units, depending on the type of halogen present on the benzofuran.

Compounds [80] and [81] in the sulfonamide series exhibited moderate sigma-2 affinity with IC 50 values of 36 nM and 31 nM respectively, with reduced selectivity than comparable compounds [74] and [75] or [77] and [78] with four and five carbon units in the benzofuran series. Compound [79] was the only sulfonamide compound to exhibit a

Mark Ashford, PhD Thesis 2010 51 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives greater selectivity for the sigma-1 receptor over the sigma-2 receptor with poor binding affinity for the sigma-2 receptor (800 nM) and moderate affinity for the sigma-1 receptor

(115 nM). This data suggested that the sulfonamide substituent linked to an isoquinoline ring through a four, five or six carbon units spacer is not the preferred conformation for binding to the sigma-2 receptor.

3.6. Amides with semi-rigid spacers

The overall strategy towards the synthesis of target amides is modelled on the synthesis of the target amides in Chapter 2 (Scheme 3.7).

Briefly, reaction of secondary amines [82] and [83] with tert -butoxycarbonyl (Boc) anhydride would give the Boc protected amines [84] and [85] , which could then be converted to the iodo derivatives [86] and [87] using iodine and triphenylphosphine, either of which could then undergo an S N2 reaction with isoquinoline [32] to yield the protected amines [88] and [89] . Deprotection of Boc amines [88] or [89] using acidic conditions would form the key intermediates [90] or [91] as free secondary amines which then could then be coupled to the 5-bromobenzo[ b]furan-2-carboxylic acid [43] or 5- iodobenzo[ b]furan-2-carboxylic acid [44] to give the 1,2,3,4-tetrahydro-6,7-dimethoxy-2-

(3-(piperidin-1-yl)alkyl amides [92]-[95].

Mark Ashford, PhD Thesis 2010 52 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.7 Synthetic pathway to target ligands [92]-[95] with semi-rigid region 2 modification.

I n HO n

PPh3, imidazole, I2

N N Boc R n = 2, [86], 56% n = 2, R = H [82] n = 3, [87], 59% n = 3, R = H [83]

n = 2, R = Boc [84], 95% n = 3, R = Boc [85], 64% [32], TBAI, K2CO3 DMF, rt, 18-72 h

H3CO H3CO Boc NH CH2Cl2/TFA N N N H CO H CO 3 n 3 n n = 2, [90], 83% n = 2, [88], 59% n = 3, [91], 94% n = 3, [89], 62%

[43], [44] NsCl, DMAP OR EDC, HOBt

O H CO O 3 N N H CO 3 n R n = 2, R = Br [92] n = 3, R = I [93] n = 2, R = Br [94] n = 3, R =I [95]

3.6.1. Synthesis of key amines [90] and [91]

The ethylpiperidine [82] was commercially available. The propylpiperidine [83] was synthesised from the corresponding 4-pyridyl propanol [96] as reported 84 in 97% yield using a palladium catalyst under a hydrogen atmosphere at 60 psi (Scheme 3.8).

Mark Ashford, PhD Thesis 2010 53 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.8 Conditions for the formation of propylpiperidine [83] from pyridine [96]

HO HO 10% Pd/C, AcOH

H2, 60 psi, 48 h

97% N N .2OAc H [96] [83]

Boc protection of [82] and [83] was facilitated as reported using di-tert -butyl dicarbonate to give [84]85 and [85]84 in 95% and 64% yields, respectively. The products [84] and [85] were iodinated 84,86 using triphenyl phosphine, imidazole and iodine to afford [86] in 56% yield and [87] in 59% yield. The iodinated piperidines [86] and [87] were then used to alkylate the isoquinoline [32] using TBAI, K 2CO 3 in DMF at rt as described in Chapter 2 to yield the isoquinoline intermediates [88] (59%) and [89] (62%).

Analysis of the 1H NMR spectrum of [88] revealed new signals at δ 3.83 ppm (s, 3H) and δ

3.84 ppm (s, 3H) assigned to the methoxy substituents on the isoquinoline ring. The signal at δ 1.45 ppm (s, 9H) was assigned to the tert -butyl CH 3 protons of the Boc protecting group. Analysis of the 13 C NMR spectrum revealed new signals at δ 56.0 ppm and δ 56.1 ppm assigned to the methoxy substituents on the isoquinoline ring with a peak at δ 155.0 ppm assigned to the carbonyl group. Mass spectral analysis revealed a peak at m/z 404 assigned to the molecular ion, and the molecular formula C 23 H36 N2O4 confirmed by HRMS with a peak at m/z 404.2672.

Formation of the key amines [90] and [91] was facilitated as previously described (Chapter

2, Scheme 2.6) using a TFA: CH 2Cl 2 solution in yields of 83% and 94% respectively.

Mark Ashford, PhD Thesis 2010 54 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Analyisis of the 1H NMR spectrum of [90] revealed the loss the signal at δ 1.45 ppm (s,

9H) that was assigned to the tert -butyl protons of the Boc protecting group. Mass spectral analysis of [90] revealed a signal at m/z 305 assigned to the molecular ion, with molecular formula C 18 H28 N2O2 confirmed by HRMS with a peak at m/z 304.2139.

3.6.2. Synthesis of target compounds [92]-[95] with a ethyl- or propylpiperidine spacer

Using the conditions outlined in Scheme 2.3, coupling of the amines [90] and [91] to the carboxylic acids [43] or [44] was promoted using either NsCl and DMAP in ACN (Method

1) or EDC, HOBt in DMF (Method 2). The results are summarised in Scheme 3.9

Scheme 3.9 Synthesis of target amides [92]-[95]

H3CO H3CO

N N R H3CO H3CO O n NH n N

n = 1 [90] [92]-[95] O n = 2 [91]

Compound Method R n Reaction time (h) Yield (%)

[92] 1 Br 1 3 51 [93] 1 Br 2 3 36 [94] 2 I 1 23 50 [95] 2 I 2 48 43

A typical analysis of the 1H NMR spectrum of [90] revealed signals at δ 3.820 ppm and

3.824 ppm assigned to the two methoxy groups on the isoquinoline ring, with signals at δ

7.13 ppm, 7.27 ppm, 7.63 ppm, and 7.96 ppm assigned to the benzofuran protons. Analysis of the 13 C NMR spectrum revealed peaks at δ 55.9 ppm and 56.0 ppm, assigned to the methoxy groups and a peak at δ 87.1 ppm assigned to carbon adjacent to the iodine on the

Mark Ashford, PhD Thesis 2010 55 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives benzofuran. Mass spectral analysis revealed a peak at m/z 574 assigned to the molecular ion, with molecular formula C 27 H31 N2O4I confirmed by HRMS with a m/z of 574.4563.

3.7. Synthesis of propylpiperazine target compound [102]

The best sigma-2 ligand produced from this project to date contained a six carbon flexible spacer, (3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexyl carboxamide [78].

Therefore, to investigate the SAR of region-2 further, compound [102] containing a semi- rigid propylpiperazine spacer was also synthesised. This would allow a direct comparison between the target compounds [78] and [95] to observe which conformation gave the best sigma-2 ligand.

The pathway selected for the synthesis of the target compound [102] is shown in Figure

3.3. Briefly, the mono-Boc protected piperazine [97] could be alkylated to give [98]. This could then undergo an S N2 reaction with isoquinoline [32] to yield [99] and deprotection to give the amine intermediate [101]. This could then be coupled to 5-iodobenzo[ b]furan-2- carboxylic acid [44] to give the target 1,2,3,4-tetrahydro-6,7-dimethoxy-2-(3-(piperazin-1- yl)propyl)amide [102].

Mark Ashford, PhD Thesis 2010 56 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

H Boc N N alkylation N Br N N N Boc H H [98] [96] [97]

alkylation

H CO P H CO 3 N 3 NH deprotection N N N N H3CO H3CO [101] P = Boc [99] P = Bn [100]

O H CO O 3 N N N H3CO

[102] I

Figure 3.3 Pathway to target compound [102] via the Boc [99] or Benzyl [100] protected analogues.

3.7.1. Method 1: Synthesis of amine intermediate [101] via the Boc protected piperazine [99] tert -Butylpiperazine-1-carboxylate [97] was synthesised as reported 87 using piperazine hexahydrate [96] and di-tert-butyldicarbonate in isopropanol to yield the mono-Boc piperazine [97] in 89% yield. The piperazine [97] was then alkylated 88 with 1,3- dibromopropane to give piperazine [98] which was spectroscopically identical to that reported. The reaction conditions were optimised by using K 2CO 3 in acetone heated at reflux and the results are summarised in Scheme 3.10.

Mark Ashford, PhD Thesis 2010 57 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.10 Conditions for alkylation of Boc protected piperazine [97] to give piperazine [98].

Br Br NH N Br N N Boc Boc [97] [98]

Time Yield Method Conditions (h) (%) 1 Et 3N, CH 2Cl 2, rt 18 20 K CO , acetone (dry), 2 2 3 48 82 reflux

Method 1 involved the reaction of the Boc-piperazine [97], 1,3-dibromopropane and Et 3N as reported 88 and after 18 h gave a yield of 20%. Method 2 involved heating a solution of tert -butylpiperazine-1-carboxylate [97], K 2CO 3 and 1,3-dibromopropane in acetone at reflux for 48 h to give a substantially improved yield of 82%. This improved yield was attributed to the heating of the reaction pushing it further to completion, and the use of a polar aprotic solvent (acetone) instead of the non-polar solvent (CH 2Cl 2) to promote product formation via the S N2 reaction mechanism.

Alkylation of the isoquinoline nitrogen of [32] was performed as described in Scheme 3.7 to give [99] in 82% yield using TBAI, KI, K 2CO 3 in DMF (Scheme 3.11).

Mark Ashford, PhD Thesis 2010 58 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.11 Synthesis of the Boc-protected piperazine intermediate [99] by condensation of isoquinoline hydrochloride [32] and piperazine [98] .

Br N N Boc [98]

TBAI, KI, K2CO3, H CO H CO Boc 3 DMF, rt, 18 h 3 N NH.HCl N N H3CO 82% H3CO [32] [99]

Analysis of the 1H NMR spectrum of [99] revealed new peaks at δ 2.70 ppm (t, 2H), 2.81 ppm (t, 2H) and 3.55 ppm (s, 2H) assigned to the methylene protons of the isoquinoline ring. A multiplet at δ 2.39 ppm was assigned to the protons on the piperazine ring. Analysis of the 13 C NMR spectrum revealed a peak at δ 154.9 ppm, assigned to the Boc carbonyl group. Mass spectral analysis revealed a peak at m/z 419, assigned to the molecular ion, with molecular formula C 23 H38 N3O4 confirmed by HRMS with a peak at m/z 420.2845.

Attempts to deprotect the piperazine [99] used the classical methods of either TFA:CH 2Cl 2

(1:2) 89 or HCl and dioxane,89 but were unsuccessful (Scheme 3.12).

Scheme 3.12 Varying attempts at Boc deprotection of piperazine [99]

H CO Boc H3CO 3 N NH N N N N H3CO H3CO [99] [101]

Method Conditions Time (h) Yield (%)

1 TFA:CH 2Cl 2 (1:2) 0.5 0

2 Dioxane:HCl (1:1) 1 0

3 4 M HCl in dioxane 1 0

Mark Ashford, PhD Thesis 2010 59 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Method 1 employed a TFA:CH 2Cl 2 (1:2) solution as previously described for N-Boc deprotection of piperidine intermediates [88] and [89], however, no target amine was recovered from the reaction mixture. Method 2 employed a dioxane:HCl solution, without any amine product being observed. Method 3 employed a less concentrated dioxane:HCl solution, however, no product was observed.

TLC analysis revealed a mixture of products that could not be separated, with no evidence of starting material remaining. Analysis of the 1H NMR spectrum for all Methods indicated degradation of the compound. Mass spectrum analysis of the reaction mixture revealed no peak consistent with that for the expected amine product or starting material.

3.7.2. Method 2: Synthesis of piperazine [101] via the benzyl protected piperazine [100]

The second synthetic strategy involved the use of 1-benzylpiperazine. The advantage of this strategy is that it employs the same synthetic route as previously described (see Figure

3.2). Briefly, 1-benzylpiperazine could be alkylated to give the chloro adduct [103] which could then be attached to the isoquinoline [32]. The isoquinoline [100] could then be deprotected to give the desired piperazine amine [101].

Therefore, the chloro adduct [103] was synthesised as reported 90 (Scheme 3.13) using 1- benzylpiperazine and 1-bromo-3-chloropropane in 60% yield. Alkylation of isoquinoline

[32] with the chloro adduct [103] using TBAI, KI, K 2CO 3 in either ACN or DMF gave the isoquinoline [100] in 13% or 75% yields respectively (Scheme 3.13).

Mark Ashford, PhD Thesis 2010 60 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Scheme 3.13 The synthesis of isoquinoline piperazine [100] via alkylpiperazine [103].

1-bromo-3-chloropropane, 2-butanone, K2CO3, N Cl N reflux, 4 h N 60% N H [103]

H CO 3 N N N H3CO [100]

Method Conditions Time (h) Yield (%)

1 KI, TBAI, K 2CO 3, DMF, rt 72 13

KI, TBAI, K 2CO 3, ACN, 2 16 75 reflux

Analysis of the 1H NMR of [100] revealed peaks at δ 6.51 ppm (s, 1H) and 6.58 ppm (s,

1H) assigned to the aromatic protons of the isoquinoline ring, and the peak at δ 7.29 ppm

(m, 5H) was assigned to the phenyl ring of the benzyl protecting group. Analysis of the 13 C

NMR spectrum revealed signals at δ 56.02 ppm and 56.04 ppm were assigned to the methoxy groups on the isoquinoline ring and a signal at δ 63.2 ppm was assigned to the benzylic carbon. Mass spectrum analysis revealed a peak at m/z 410 assigned to the molecular ion.

The yield for the synthesis of isoquinoline [100] was optimised by heating the reaction at reflux. The removal of a benzyl group is usually and conveniently facilitated by catalytic hydrogenation in the presence of a palladium catalyst.89 Consequently, a variety of reaction

Mark Ashford, PhD Thesis 2010 61 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives conditions were tried with the objective of increasing yield of the formation of [101]. The results are summarised in Scheme 3.14.

Scheme 3.14 Deprotection methods in an attempted to increase the yield of [101]

Entry Conditions Time (h) Yield (%)

1 10% Pd/C, EtOH, H 2, 60 psi 28 0

10% Pd/C, 10% Pd(OH) 2/C THF- 2 19 0 isopropanol (3:1), H 2, STP

10% Pd/C, 10% Pd(OH) 2/C THF- 3 22 0 isopropanol (3:1), H 2, 60 psi

4 10% Pd/C, HCO 2NH 4, MeOH, reflux 0.25 91

Entry 1 employed a palladium catalyst at 60 psi using classical conditions. 89 However, only starting material was recovered after 28 h. It has been reported 91 that a more efficient way to remove a benzyl group was to add a mixture of two catalysts (10% Pd/C and 10%

Pd(OH) 2/C) to the reaction mixture at standard temperature and pressure (entry 2).

However, no conversion was observed after 19 h. The reaction mixture was then subjected to a hydrogen atmosphere at 60 psi for an additional 22 h (entry 3) however, in all attempts only starting material was recovered. Finally, removal of the benzyl group was achieved using ammonium formate and 10% Pd/C 92, 93 to give [101] in 91% yield.

Mark Ashford, PhD Thesis 2010 62 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Analysis of the 1H NMR spectrum revealed the loss of the signals at δ 7.29 ppm (m, 5H) and 3.51 ppm (s, 2H) assigned to the benzyl group. Mass spectrum analysis revealed a peak at m/z 319 assigned to the molecular ion.

3.8. Synthesis of target amide [104]

Coupling of the amine [102] with the carboxylic acid [44] was facilitated using EDC,

HOBt in DMF in 26% yield (Scheme 3.15).

Scheme 3.15 Coupling of amine [102] with carboxylic acid [44] to yield the target amide [104]

H CO 3 NH N N H3CO [102]

EDC, HOBt, DMF, rt, 72 h O O HO 26%

[44] I O H CO O 3 N N N H3CO [104] I

Analysis of the 1H NMR of [104] revealed peaks at δ 6.51 ppm (s, 1H) and 6.58 ppm (s,

1H) assigned to the aromatic protons of the isoquinoline ring and signals at δ 7.18, 7.28,

7.69 and 7.97 ppm were assigned to the protons of the benzofuran. Analysis of the 13 C

NMR spectrum revealed signals at δ 56.00 ppm and 56.02 ppm which were assigned to the

Mark Ashford, PhD Thesis 2010 63 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives methoxy groups on the isoquinoline ring and a signal at δ 87.2 ppm, assigned to the carbon adjacent to the iodine atom on the benzofuran. Mass spectrum analysis revealed a peak at m/z 590 assigned to the molecular ion, with molecular formula C 27 H33 N3O4I confirmed by

HRMS with a peak at m/z 590.1511.

3.9. In vitro studies and log P

The 5 new bromo and iodo compounds [92]-[95] and [104] were tested for their in vitro binding affinities for the sigma-1 and sigma-2 receptors. The binding affinity results along with the log P values determined by the HPLC Method,77 are shown in Figure 3.4

Table 3.2 Sigma-1 and sigma-2 binding affinities of target amides [92]-[95] and [104]

H3CO

N R H3CO X O n N

σ2 IC 50 σ1 IC 50 R n X Log P Compound (nM) (nM) Selectivity ( σ1/ σ2) [92] Br 1 C 4.02 5.5 ± 0.2 790 ± 100 144 [93] Br 2 C 3.99 6.0 ± 3.0 460 ± 90 77 [94] I 1 C 3.83 12 ± 4.0 405 ± 120 34 [95] I 2 C 3.36 12 ± 3.0 110 ± 5.0 9.1 [104] I 2 N 3.88 3.2 ± 0.07 135 ± 5.0 42

Target compounds [92]-[95] displayed high sigma-2 binding affinity ranging from 5.5-12 nM. These compounds were selective for the sigma-2 receptor over the sigma-1 receptor, however, they were not as potent as their flexible spacer analogues [73]-[78]. The binding affinity was observed to be higher for the bromo analogues [92] and [93] compared to the corresponding iodo derivatives [94] and [95]. The bromo analogues were also the most selective class of compounds, suggesting that a smaller halogen is preferred for the sigma-2

Mark Ashford, PhD Thesis 2010 64 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives binding site. The effect of chain lengthening of the spacer from 5 to 6 carbons also increased the affinity for the sigma-1 receptor while having little effect on the sigma-2 affinity. Replacing the carbon atom of the piperidine ring of [95] with a nitrogen atom for the piperazine ring of [104] increased the sigma-2 affinity approximately 4-fold, with little effect observed on the sigma-1 binding affinity. This change gave the most potent ligand for this class of compounds.

Generally, compounds [92]-[95] and [104] from the semi-rigid series gave similar activity for the sigma-2 receptor as those in the flexible series [73]-[78], however, they lacked the selectivity for the sigma-2 receptor observed in the flexible spacer series.

3.10. Conclusions and future directions

Nine new compounds incorporating flexible spacer amide chains were synthesised and their sigma-2 binding affinities were determined. The more flexible amide [77] (7 nM) gave a higher sigma-2 affinity than that of its restricted cyclic compound described in

Chapter 2 [65] (12.5 nM).

Compound [78] was found to be the most potent and selective sigma-2 ligand synthesised

(1.0 nM) from this particular series and the radiolabelling of compound [78] with 123 I is discussed in Chapter 5.

An interesting result was observed with the order of addition of reagents, leading to the formation of the sulfonamide derivatives [79]-[81]. These novel compounds exhibited moderate to low sigma-2 affinity.

Mark Ashford, PhD Thesis 2010 65 Chapter 3 – Synthesis of Region 2 Carbon Spacer Derivatives

Five new halogenated piperidine and piperazine based amides were synthesised and their sigma-2 binding affinities determined. All compounds were found to display high affinity and selectivity for the sigma-2 receptor, with the piperazine analogue [104] having a higher binding affinity (3.2 nM) than the best iodinated ligand from Chapter 2 ([65] 12.5 nM).

However, [104] (sigma-1 IC 50 135 nM) was not as selective for the sigma-2 receptor as

[65] (sigma-1 IC 50 1500 nM). It was also observed that changing the atom from bromine to iodine marginally decreased the activity for the sigma-2 receptor, supporting the observation in Chapter 2 that the sigma-2 receptor may not accommodate a larger halogen.

Future directions would involve the incorporation of a piperazine ring system into ligands

[65] and [93] to observe the affects on sigma-2 activity and affinity and the radiolabelling of [78] with 123 I.

Mark Ashford, PhD Thesis 2010 66 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

4. Synthesis of Region 3 Isoquinoline Derivatives

4.1. Rationale

To date, little modification has occurred on the isoquinoline system. The one exception has been where fused methylene-, ethylene-, and propylenedioxy rings were incorporated onto the tetrahydroisoquinoline.94 Related studies in our laboratory by other members of our group suggested that the presence other di- and tri-methoxy configurations on the ring decreased sigma-2 affinity, § however, the effects of 6- and 7-monomethoxy configurations or 1-substitutions on the ring were not known. Therefore, the synthesis of 6- and 7- methoxytetrahydroisoquinolines and a racemic 1-substituted tertahydroisoquinoline was attempted.

4.2. Isoquinoline Synthesis

Tetrahydroisoquinoline [105] was synthesised as reported 95 in a toluene:TFA solution in

74% yield from 3,4-dimethoxyphenethylamine and benzaldehyde (Scheme 4.1). This reaction used a slight variation on literature methods, with TFA used in place of HCl.

Scheme 4.1 The synthesis of racemic 1-phenylisoquinoline [105] promoted by TFA. 95

i) toluene, reflux ii) TFA, reflux H3CO NH2 H3CO NH H CO H3CO 74% 3

[105]

§ Unpublished data from our laboratory

Mark Ashford, PhD Thesis 2010 67 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

6-Methoxyisoquinoline [108] was commercially available; however, it was very expensive and only available in milligram quantities which were not appropriate for this synthesis.

The synthesis of [108] is described in the literature by the Pictet-Spengler reaction 96 or

Bobbit modification of the Pomeranz-Fritz cyclisation.97 However, a draw back of these reactions are the reactions are poor yielding; workup is difficult and time consuming or generates regioisomers which are difficult to separate by chromatography.

Recently, the synthesis of 6-methoxyisoquinoline [108] was published using a modified

Pictet-Spengler synthesis. 98 Briefly, 3-methoxyphenethylamine [106] in formaldehyde and dilute HCl (1M) at 60 oC gave the bis product [107] in 41% yield which was then hydrolysed at rt using conc. HCl to give 6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride [108] in 90% yield (Scheme 4.2). Intermediate [107] and target isoquinoline

[108] were spectroscopically identical to that reported.98

Scheme 4.2 Synthesis of 6-methoxytetrahydroisoquinoline [108].98

HCHO, HCl (1M), o H2O, 60 C, 4 h NN

41% H3CO OCH3 H3CO NH2 [107] [106] HCl (conc), IPA/MTBE 90% 22 h

NH.HCl

H3CO [108]

Mark Ashford, PhD Thesis 2010 68 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

The synthesis of 7-methoxytetrahydroisoquinoline has only being reported in small quantities or as a mixture of products. 96, 99 However, for this project, it was needed in multigram quantities in a quick, convenient synthesis. The strategy initially attempted for the synthesis of isoquinoline [115] is outlined in Scheme 4.3.

Scheme 4.3 The attempted synthesis of isoquinolines [113] and [114]

NaOAc, H2SO4, KNO3, acetic anhydride, 5-10 oC-rt, 20 h reflux, 1 h NH.HCl N CH NH O N 3 32% O2N 89% 2 [109] [110] [111] O

10%Pd/C, EtOH, 88% 50 psi, H2, rt, 5.5 h

N CH N CH3 HO 3 H2N O [112] O [113]

N CH3 NH H3CO H3CO [114] O [115]

The isoquinoline intermediate [112] was synthesised as reported. 100 Briefly, nitration of isoquinoline [109] using a H 2SO 4/KNO 3 mixture afforded [110] in 32% yield which was consistent with literature yield.100 The product was then acetylated in sodium acetate and acetic anhydride at reflux to give isoquinoline [111] in 89% yield. 100 The nitro group was then reduced with H 2 at 50 psi in the presence of a palladium catalyst to give aniline [112] in 88% yield. 100

Mark Ashford, PhD Thesis 2010 69 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Attempts to synthesise the phenol [113] by hydrolysing the corresponding diazonium salt was unsuccessful. To the isoquinoline [112] was added a solution of H 2SO 4:H 2O (1:9) and

o NaNO 2 to generate the azide salt in situ followed by heating to 45 C for 30 mins. TLC analysis revealed a complex mixture of inseparable spots with no conversion to phenol

[113] observed by analysis of the mass spectrum or 1H NMR spectra. It has been reported 101,102 that benzenediazonium salts react with methanol to give anisoles under acidic conditions. Therefore, attempts to prepare the corresponding anisole [114] using these methods were attempted. However, addition of MeOH to the azide salt of [112]

(generated as described previously) gave a complex mixture of products with no conversion to [114]. In both cases, no starting material or products were observed. These results are consistent with literature observations that similar compounds to that of [112] are converted to their corresponding phenols with difficulty.103 Although, this was overcome by the use of TFA at reflux, these conditions would not have been applicable for this series as the harsh reaction conditions may have removed the acetyl protecting group.

The reaction for the conversion of aniline [112] to anisole [114] has been observed to work under acidic and basic conditions. However, under basic conditions, the homolytic cleavage of the C-N bond is affected by the presence of oxygen. 104 Also, the diazonium salt is formed under acidic conditions using H 2SO 4, making the use of base impractical.

Consequently, a new synthetic strategy was devised for the synthesis of 7- methoxytetrahydroisoquinoline [115] via the 7-methoxyisoquinoline 105 [119] (Scheme 4.4).

Mark Ashford, PhD Thesis 2010 70 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Scheme 4.4 Synthesis of 7-methoxytetrahydroisoquinoline [115] using pathway 2

Aminoacetalaldehyde- dimethyl acetal toluene, reflux, 4 h OMe H N H CO 99 % H3CO OMe 3 [116] O

Method A Method B 86% 98% Method A Ts OCH 67% 3 OMe N H H3CO OCH3 N [118] Method B H3CO OMe 80% [117] Dioxane, HCl (6M), reflux, 16 h 76%

N NH H3CO H3CO [119] [115]

Briefly, imine [116] was synthesised from the corresponding benzaldehyde using aminoacetalaldehyde and dimethyl acetal in yield (99%) consistent with that reported.105

The reported method 105 for the reduction of imine [116] utilised hydrogen in the presence of PtO 2, which was not available at the time of synthesis. Therefore the reduction to amine

[117] proceeded via two methods. Method A employed NaBH 4 in MeOH at rt and gave the amine [117] in 86% yield. Alternatively, (Method B) hydrogenation using Pt/C and H 2 at

60 psi gave the amine [117] in 98% yield. The reported method 105 (Method A) for tosylation of amine [117] used pyridine and TsCl stirred at rt for 3 days and gave [118] in

67% yield. Alternatively, modification of the literature procedure (Method B) using aqueous sodium hydroxide, tetra-n-butylammoniumhydrogen sulphate in dichloromethane gave [118] in 80% yield after 18 h. Cyclisation to isoquinoline [119] using HCl and dioxane proceeded in 76% yield (c.f. 67% 105 ) as reported. 105

Mark Ashford, PhD Thesis 2010 71 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Conversion of an isoquinoline to 1,2,3,4-tetrahydroisoquinoline has been reported using either nickel-aluminium alloy in potassium hydroxide 106 or liquid ammonia, sodium and ethanol (Birch Reduction).107 Therefore, three different reaction conditions for the synthesis of 7-methoxy-1,2,3,4-tetrahydroisoquinoline [115] were attempted (Scheme 4.5).

Scheme 4.5 Reaction conditions to yield 7-methoxy-1,2,3,4-tetrahydroisoquinoline [115] through selective reduction of the heterocyclic ring.

HCl N NH NH.HCl H3CO H3CO H3CO [119] [115] [115a]

Entry Method Time (h) Yield (%)

1 1M KOH, Ni-Al Alloy, rt 18 0

2 1M KOH, Ni-Al Alloy, reflux 20 0

3 Na, NH 3, EtOH 1 81

Entries 1 and 2 employed nickel-aluminium alloy in potassium hydroxide at either room temperature or reflux, however, only starting material was recovered from the reaction mixture. Entry three utilised liquid ammonia, sodium and ethanol to yield target compound

[115] which, after workup and exposure to 4M HCl in dioxane, was isolated as the hydrochloride salt [115a] and was spectroscopically identical to that reported.96

Therefore, following modifications and improvements to the literature procedure for the synthesis of isoquinoline [119] , the isoquinoline [115a] was isolated in a yield of 48% over the five step synthesis.

Mark Ashford, PhD Thesis 2010 72 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

4.3. Formation of isoquinoline intermediates

The most potent ligands from chapter 3 contained a flexible chain of 4, 5 or 6 carbons or a propylpiperidine spacer. Therefore, the isoquinolines [105], [108] and [115a] generated in this chapter were alkylated with flexible 4, 5 or 6 carbon spacer or a propylpiperidine spacer.**

4.3.1. Synthesis of conformationally flexible intermediates using isoquinolines [105], [108] and [115a].

The reaction conditions used were described previously in Scheme 3.1. Briefly, the isoquinoline nitrogens of [105], [108] and [115a] were alkylated with bromopentyl- or bromohexyl nitrile, using TBAI, KI, and K 2CO 3 in DMF to give the corresponding nitrile isoquinolines [120]-[124] in yields of 59-77%. Reduction with LiAlH 4 gave amines [125]-

[129] in yields of 77-94% (Scheme 4.6).

** The propylpiperazine spacer in [104] gave the most potent sigma ligand observed in Chapter 3, however, this ligand was tested at the end of this project.

Mark Ashford, PhD Thesis 2010 73 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Scheme 4.6 Unrestricted intermediate synthesis using isoquinolines [105], [108] and [115a].

N Br n R R1 1 TBAI, KI N N NH.HCl R R2 DMF, rt, 18 h 2 R n R3 3 R1 R2 R3 n R1 R2 R3 Yield % 120 2 OCH3 H H 76 108 OCH3H H 121 3 OCH3 H H 61 115a H OCH3 H 122 2 H OCH3 H 77 105 OCH3OCH3 Ph 123 3 H OCH3 H 59 124 4 OCH3 OCH3 Ph 83

R 1 LiAlH4, THF, N reflux, 18 h R2 NH2 n R3

n R1 R2 R3 Yield % 125 2 OCH3 H H 94 126 3 OCH3 H H 94 127 2 H OCH3 H 77 128 3 H OCH3 H 89 OCH OCH Ph 83 129 4 3 3

4.3.2. Synthesis of conformationally semi-restricted intermediates using isoquinolines [108] and [115a].

The synthesis of the semi-restricted mono methoxyisoquinoline intermediates were performed as described previously in Scheme 3.7. The alkylation of isoquinolines [108] and [115a] gave [130] and [131] in 77% and 75% yields respectively. Deprotection of the nitrogen under standard conditions gave [132] and [133] in 75% and 90% yields (Scheme

4.7).

Mark Ashford, PhD Thesis 2010 74 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Scheme 4.7 Restricted intermediate synthesis using isoquinolines [108] and [115a].

I N Boc R1 Boc R1 [87] N N NH.HCl R2 R2 TBAI, DMF, rt, 20 h R R Yield % R1 R2 1 2 108 OCH H 130 OCH3 H 77 3 131 H OCH 75 115a H OCH3 3

R1 TFA:CH Cl (1:2) NH 2 2 30 mins N R2

R1 R2 Yield %

132 OCH3 H 75 133 H OCH3 90

4.4. Synthesis and in vitro evaluation of amides with region 3 modification

4.4.1. Synthesis of target flexible spacer amides [134]-[136] and semi-rigid amides [137]-[138]

The target compounds were synthesised using two different sets of conditions as outlined in Scheme 2.2. Therefore, to the amines [125]-[129] was added carboxylic acid [44], EDC and HOBt in DMF to give target amides [134]-[136] in yields of 28-51%. The results are summarised in Table 4.1.

Mark Ashford, PhD Thesis 2010 75 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Table 4.1 Flexible amides [134]-[138] using EDC and HOBt coupling conditions.

R 1 O N O R2 N n H R3

Compound R1 R2 R3 n Time (h) Yield (%)

[134] OCH 3 H H 2 48 51

[135] OCH 3 H H 3 48 34

[136] H OCH 3 H 2 48 36

[137] H OCH 3 H 3 48 30

[138] OCH 3 OCH 3 Ph 3 48 28

The second set of conditions used NsCl and DMAP as these are reported to give superior yields for the formation of tertiary amides. 66 Therefore, amines [132] and [133] were reacted with acid [44] using NsCl and DMAP or EDC and HOBt to yield the target compounds [139] and [140] in 17% and 39% respectively. The results are summarised in

Table 4.2

Mark Ashford, PhD Thesis 2010 76 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Table 4.2 Semi-rigid target amides [139] and [140] using NsCl and DMAP or EDC and HOBt coupling conditions. O R O 1 N N R2 I

Compound Conditions R1 R2 Time (h) Yield (%)

[139] NsCl/DIPEA OCH 3 H 3 17

[140] EDC/HOBt H OCH 3 48 39

Initially compound [139] was synthesised using NsCl/DIPEA coupling conditions, however, this gave a poor yield of the target compound (17%). In an effort to improve the yield for compound [140], it was synthesised using EDC/HOBt to give the target compound in 39% yield. No further optimisation studies for compound [139] were attempted.

4.5. In vitro studies and log P

The seven new iodo compounds [134]-[140] were tested for their in vitro binding affinities for the sigma-1 and sigma-2 receptors. The results are shown in Tables 4.3 and 4.4.

Mark Ashford, PhD Thesis 2010 77 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives

Table 4.3 Sigma-2 and Sigma-1 IC 50 values for flexible ligands with modification at region 3

R 1 O N O R2 N n H R3

σ2 IC 50 σ1 IC 50 R1 R2 R3 n Log P Compound (nM) (nM)

[134] OCH 3 H H 2 4.08 150 ± 4.0 >1000

[135] OCH 3 H H 3 4.31 5.0 ± 2.0 14.0 ± 4.0

[136] H OCH 3 H 2 4.43 40 ± 15 >1000

[137] H OCH 3 H 3 4.33 35 ± 10 530 ± 65

[138] OCH 3 OCH 3 Ph 3 5.14 170 ± 10 >5000

Table 4.4 Sigma-2 and Sigma-1 IC 50 values for semi-rigid ligands with modification at region 3 O R O 1 N N R2 I

σ2 IC 50 σ1 IC 50 R1 R2 Log P Compound (nM) (nM)

[139] OCH 3 H 4.61 35 ± 15 250 ± 50

[140] H OCH 3 4.69 120 ± 2 50 ± 3

Generally, the compounds in this series displayed moderate affinity for the sigma-2 receptor (5.0 nM to 170 nM). However, [140] displayed a higher Sigma-1 potency and

[135] was 3-fold more active for the Sigma-1 receptor. This series suggested that changing from a 6,7-dimethoxy substitution on the isoquinoline to a 6- or 7-monomethoxy

Mark Ashford, PhD Thesis 2010 78 Chapter 4 – Synthesis of Region 3 Isoquinoline Derivatives substitution decreased the affinity for the sigma-2 receptor. Also, having a racemic 1- phenyl substituent [138] decreased the sigma-2 affinity relative to [78] ; however, it maintained the selectivity between the two receptors. This suggested that having a substituent on the isoquinoline ring could be beneficial for sigma-2 selectivity.

4.6. Conclusions

A new method to access 7-methoxy-1,2,3,4-tetrahydroisoquinoline [115a] in gram quantities was developed. This involved an improvement on the synthesis of 7- methoxyisoquinoline, using Pt/C at 60 psi under a hydrogen atmosphere to reduce the imine bond using milder conditions than reported 105 and tosylation of the nitrogen using a phase transfer catalyst in 80% yield to reduce the reaction time from 3 days 105 to 18 h. Use of the Birch reduction to give [115a] in 81% yield was also realised.

All compounds synthesised displayed moderate activity for the sigma-2 receptor with the exception of [135], which showed high activity (sigma-2 IC 50 5.0 nM) and [140] which showed higher activity for the sigma-1 receptor (sigma-1 IC 50 50 nM, sigma-2 IC 50 120 nM).

No compounds were radiolabelled with 123 I in this series as none displayed a binding affinity of less that 10 nM for the sigma-2 receptor with the exception of [135], however, it did not display the appropriate selectivity.

Future studies would involve completing the synthesis for the different substituted dimethoxy tetrahydroisoquinolines and determine their binding affinities; and producing more 1-substituted isoquinolines to observe its effect on activity and selectivity.

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5. Summary of in vitro results and Consequence to the SAR

5.1. General considerations regarding the Structure and Activity Relationship

From the three series synthesised, a SAR on ligands for the Sigma-2 receptor was described (Figure 5.1). The optimised configuration incorporated a benzofuran carboxamide moiety with a halogen substitution at C5 and a 6,7- dimethoxytetrahydroisoquinoline separated by a six carbon flexible spacer.

Figure 5.1 General considerations regarding the Structure and Activity Relationship.

Mark Ashford, PhD Thesis 2010 80 Chapter 5 – Summary of in vitro results and Consequence to the SAR

5.2. Target compounds [36]-[42] and [60]-[65] with modification to Region 3

The target compounds [36]-[42] had similar structural properties in that they all contained a tetrahydroisoquinoline ring with a 6,7-dimethoxy substitution and a methylpiperidine chain spacer bridging the ring and the amide group. Changes were made in region 3 (Figure 5.2) by varying the carboxamide group from that of the lead compound [27]. This allowed comparative relationships to be made between the amide moiety and the activity and selectivity towards the sigma-2 receptor. All compounds were prepared with the goal of increasing the affinity and selectivity, as well as to establish preliminary SAR data.

Region 3

O H CO O 3 N N H3CO

[27] H CO R 3 N N H3CO O O O O H H R = N S N HN

[40] [36] [37] [42] Sigma-2: 100 nM Sigma-2: 110 nM Sigma-2: 955 nM Sigma-2: 850 nM Sigma-1: 820 nM Sigma-1: 210 nM Sigma-1: 360 nM Sigma-1: 285 nM

O O H O O N Br

N H Br Br [60] [61] [64] Sigma-2: 9.8 nM Sigma-2: 45 nM Sigma-2: 180 nM Sigma-1: >5000 nM Sigma-1: 870 nM Sigma-1: >5000 nM

Figure 5.2 Substitution changes made to region 3 of the lead compound [27].

Mark Ashford, PhD Thesis 2010 81 Chapter 5 – Summary of in vitro results and Consequence to the SAR

The lead compound [27] has a benzofuran moiety in region 3. Compounds [36] and [37] were synthesised in order to observe the effects of changing heteroatom from an oxygen, to a nitrogen [36] or sulphur [37]. The compounds were still selective for the sigma-2 receptor; however, they decreased substantially when compared to the lead compound. In addition, the activity towards the sigma-2 receptor had decreased between 30-35 fold when compared to the lead compound [27]. Compounds [38]-[42] were synthesised to study the effect that different carboxylic acid substitution patterns have on binding affinity/selectivity. Changing the indole acid substitution of [36] to the six position of [40] or the three position of [42] gave target compounds that were more selective for the sigma-

1 receptor by approximately 4-5 fold. This trend was also observed with a quinoline substitution in [39], although it was twice as selective for the sigma-1 receptor.

Substitution of aromatic acids in [38] and [41] gave moderately active sigma-2 ligands, with 10 fold selectivity over the sigma-1 receptor. Incorporating a bromine atom at the 5 position on the indole on [36] to give [61] increased the activity for the sigma-2 receptor 2- fold and increased the selectivity at least 5 fold. This trend was also noted with [64] as it changed the selectivity of the compound to give a more active sigma-2 ligand.

Incorporating a bromine atom in [60] at the 5-position on the benzofuran of the lead [27] increased the selectivity over the sigma-1 receptor, whilst only having a small affect on activity. Furthermore, incorporation of an iodine atom in [65] at the same position gave an almost equally selective ligand with good activity for the sigma-2 receptor. Compounds

[62] and [63] were synthesised to observe the effect of having a benzamide would have on activity and selectivity. The best compound at the time contained the amide in [63] with the only change being the inclusion of the methylpiperidine core. However, it decreased the activity and selectivity for the sigma-2 receptor. Compound [62] showed moderate to high

Mark Ashford, PhD Thesis 2010 82 Chapter 5 – Summary of in vitro results and Consequence to the SAR affinity for the sigma-2 receptor, however, was not as active or selective as the lead compound [27].

5.3. Target compounds [73]-[78], [92]-[95], and [104] with modification to Region 2

The target compounds [73]-[78], [92]-[95] and [104] had similar structural properties in that they all contained a tetrahydroisoquinoline ring with a 6,7-dimethoxy substitution and a amide or sulfonamide bridged by a carbon spacer. Changes were made in region 2 (Figure

5.3) by varying the carbon spacer group from straight chain (flexible) to semi-rigid to allow observations on sigma-2 activity and selectivity.

Region 2

O H CO O 3 N N H3CO X X = Br, I

H CO O 3 O H3CO O N N O H3CO N Y n H3CO n X X n = 4, X = Br [73] n = 4, X = I [76] n = 2, X = Br [92] Sigma-2: 17 nM Sigma-2:7.0 nM Sigma-2:5.5 nM Sigma-1: >5000 nM Sigma-1:>1000 nM Sigma-1:790 nM n = 5, X = Br [74] n = 5, X = I [77] n = 3, X = I [95] Sigma-2: 7.0 nM Sigma-2:10 nM Sigma-2:12 nM Sigma-1: 900 nM Sigma-1: 450 nM Sigma-1:110 nM n = 6, X = Br [75] n = 6, X = I [78] n = 3, Y = N, X = I [104] Sigma-2: 9 nM Sigma-2: 1.0 nM Sigma-2:3.2 nM Sigma-1:135 nM Sigma-1: 345 nM Sigma-1: >1000 nM

Figure 5.3 Variations made to region 2 of the lead compound [27] .

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Compounds [73]-[78] were synthesised to observe the effect of having a flexible carbon spacer of either 4, 5 or 6 carbon units between the tetrahydroisoquinoline ring and the 5- bromo or 5-iodobenzofuran[ b]-2-carboxamide. Increasing the carbon spacer length from 4 to 6 carbon units in the bromo compounds [73]-[75] increased the activity for the sigma-1 receptor, while maintaining similar activities for the sigma-2 receptor. The same strategy was employed for the iodo compounds [76]-[78] and a similar trend observed, culminating in the most active sigma ligand for this series, [78] . Compounds [79]-[81] were unexpectedly isolated from coupling reactions using NsCl as an activating agent. They were also evaluated as compounds of this type have not been tested for sigma activity and selectivity. Compound [79] gave an active sigma-1 ligand, while compounds [80] and [81] were moderately active for the sigma-2 receptor, with poor selectivity. Compounds [92]-

[95] and [102] were synthesised to observe the effect of having a semi-rigid ethyl-, propylpiperidine or propylpiperazine spacers on sigma-2 activity and selectivity. Bromo compounds [92] and [93] gave similar sigma-2 activity, however, the selectivity for the sigma-2 receptor decreased as the length of the spacer increased. This trend was also observed for iodo compounds [94] and [95]. In comparison to their corresponding flexible spacer compounds, they gave similar activities and selectivities. The propylpiperazine iodo compound [104] was the most active compound in this group. However, it was less selective and active for the sigma-2 receptor than [78].

5.4. Target compounds [134]-[140] with modification to region 1

The target compounds from this series had similar structural properties in that they all contained a 5-iodobenzofuran[ b]-2-carboxamide in region 3. Changes were made to the tetrahydroisoquinoline (Region 1), with incorporation of a variety of carbon spacer cores from the previous sections (Figure 5.4).

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Region 1

O H CO O 3 N N H3CO n I O X X O O N N O N Y N Y H Z n n I I

X = OCH3, Y = H, Z = H, n = 3 [135] X = OCH3, Y = H, n = 3 [139] Sigma-2:5.0 nM Sigma-2:35 nM Sigma-1:14 nM Sigma-1:250 nM

X = H, Y = OCH3, Z = H, n = 3 [137] X = H, Y = OCH3, n = 3 [140] Sigma-2:35 nM Sigma-2:120 nM Sigma-1:530 nM Sigma-1:75 nM

X = OCH3, Y = OCH3, Z = Ph, n = 4 [138] Sigma-2:170 nM Sigma-1:>5000 nM

Figure 5.4 Substitutional changes made to region 1 of lead compound [27] .

Compounds [134]-[140] incorporated a 6- or 7-methoxytetrahydroisoquinoline with 4 or 5 carbon flexible-spacer units or a 6 carbon semi-rigid unit. Compound [134] contained a 6- methoxytetrahydroisoquinoline with a 4 carbon flexible-pacer that gave a moderately active sigma-2 compound with good selectivity. Increasing the length of the spacer to a 5 carbon unit [135] , gave an almost equipotent sigma-1/sigma-2 ligand. Changing the configuration to a 7-methoxytetrahydroisoquinoline ring gave compounds with moderately high sigma-2 activity and good selectivity. Changing the spacer from five carbon units to four carbon units increased the selectivity for the sigma-2 receptor at least 2 fold.

Compounds [139] and [140] contained a six carbon semi-rigid spacer and were not as

Mark Ashford, PhD Thesis 2010 85 Chapter 5 – Summary of in vitro results and Consequence to the SAR active or selective as the flexible spacer compounds [135] and [137]. Compound [138] was synthesised to observe the effect of a 1-substituted tetrahydroisoquinoline on sigma-2 activity and selectivity. This gave a moderately active sigma-2 ligand with a high selectivity.

Overall, compounds displaying the best sigma-2 activity generally contained a 6,7- dimethoxytetrahydroisoquinoline with a flexible chain spacer of 5 or 6 carbons bridging the a halogenated benzofuran group. This type of configuration also enhanced the selectivity for the sigma-2 receptor.

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6. Radiochemistry, Cell Studies and in vivo evaluation of tracers

6.1. Radiolabelling Methods

The introduction of radioiodine onto organic molecules is readily achieved using classical nucleophilic substitution or electrophilic addition reactions.108 Nucleophilic substitution reactions include halogen exchange by either interhalogen exchange or isotope exchange.

These however require moderate to high heating, long reaction times and encounter difficulties in separation of the non-radioactive precursor from the radioactive product when compared to electrophilic substitution. Furthermore, the harsh conditions employed in nucleophilic substitution reactions may also lead to decomposition of both substrates and the end product.

Electrophilic reactions on the other hand are performed in the presence of an oxidising agent to generate the iodine electrophile. 108 The iodination then proceeds via direct iodination (across a double or triple bond) or demetallation (substitution of an organometallic group) under mild conditions at room temperature. The most suitable organometallic derivatives include trialkylstannyl, trialkylsilyl or boronic acid derivatives.

Organogermanium, lead and mercury derivatives have also been extensively explored, however, their relative expensive or comparative toxicity has deterred their use. Oxidising agents of choice include chloramine-T [141], peracetic acid [142] and iodogen [143]108

(Figure 6.1). Nevertheless, the use of organometallic intermediates not only provides a convenient means of introducing radiohalogens onto molecules under mild conditions, but also provides a convenient method for separation of precursor from product using HPLC thus giving convenient access to high specific activity product.

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Cl N Na O O S O HN NH CH3(CO)3H

HN NH

Chloramine-T [141] Peracetic Acid [142] Iodogen [143]

Figure 6.1 Three common oxidants used in electrophilic radiolabelling

6.2. Synthesis of 123 I Compounds

6.2.1. Synthesis of radiolabelled compound [ 123 I][65]

The iodinated compound displaying the highest affinity for sigma-2 (IC 50 12.5 nM) from

Chapter 2 was compound [65] (Figure 6.2).

O H CO O 3 N N H3CO [65] I

Sigma-2 IC50: 12.5 nM Sigma-1 IC50: 1500 nM

Figure 6.2 Highest affinity sigma-2 ligand from chapter 2, with an IC 50 value of 12.5 nM.

The radiolabelling of this compound with 123 I would provide the first radiolabelled compound of this class bearing an iodinated benzofuran linked to the 6,7- dimethoxytetrahydroisoquinoline via a restricted piperidine spacer. The radioiodinated analogue would provide the means to assess the pharmacokinetics if this type of molecule and potentially useful properties of sigma-2 receptor ligands in vitro and in vivo .

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The most convenient method for the synthesis of [ 123 I] [65] involved an electrophilic iododestannylation reaction with 123 I via the corresponding trimethyl tin precursor [144]

(Scheme 6.1).

Scheme 6.1 Reaction conditions for synthesis of stannane [144]

O O H CO O H3CO O 3 N N N N H3CO H3CO Br Sn [60] [144]

Entry Method Time (h) Yield (%)

Pd(Ph ) (commercial source), Sn (CH ) , A 3 4 2 3 6 18-53 trace toluene, reflux B Pd(Ph 3)4 (synthesised), Sn 2(CH 3)6, toluene, reflux 18 43

The synthesis of the organostannane [144] involved the reaction of the bromo precursor

[60] with hexamethylditin in the presence of a catalytic amount of palladium tetrakistriphenylphosphine in toluene. Entry A 109 employed commercially available palladium tetrakistriphenylphosphine, however, after extensive heating, varying the catalyst source and stiochiometry, only trace amounts of the product was recovered. The low yield for this reaction was attributed to the catalyst being poisoned through exposure to air and water. Consequently, use of freshly prepared catalyst 110 (Entry B) successfully provided the desired stannane [144] in 43% yield.

Analysis of the 1H NMR spectrum of stannane [144] revealed a singlet with surrounding satellites at δ 0.32 ppm assigned to the nine protons of the three methyl groups on the tin.

Analysis of the 13 C NMR spectrum revealed a peak at δ -9.14 ppm assigned to three methyl groups on the tin. Mass spectrum analysis revealed signals at m/z 598 assigned to the

Mark Ashford, PhD Thesis 2010 89 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers molecular ion with a isotopic ratio consistent to that of a tin derivative. High resolution mass spectrometry gave a peak at m/z 598.1639 confirming the molecular formula

120 C29 H28 N2O4 Sn.

The 123 I radiolabelled compound [123 I][65] was synthesised from the stannane precursor

[144]. Varying reaction conditions were used by changing the oxidant and the results are summarised in Scheme 6.2.

Scheme 6.2 Radiolabelling conditions to synthesise [123 I][65]

O O H CO O H CO O 3 N 3 N N N H3CO H3CO [144] Sn [123I][65] 123I

Entry Oxidant Radiochemical Yield (%)

A Chloramine-T, 0.1 M HCl 82

B Chloramine-T, 1 M HCl 51

C Peracetic Acid 89

D Iodogen 76

Briefly, for Entry A, to the stannane [144] in EtOH (150 µL) was added Chloramine-T in

HCl (0.1 M, 100 µL) and Na 123 I (0.24 GBq). The reaction was performed at rt for 5 mins before being quenched with sodium metabisulphate solution (50 mg/mL, 100 µL) and neutralised with sodium bicarbonate solution (50 mg/mL, 100 µL). After dilution with

HPLC mobile phase, the entire reaction mixture subjected to reverse phase HPLC purification. Chromatographic analysis of the reaction mixture indicated the presence of three peaks (Figure 6.3) corresponding to three radioactive products. The first peak at 2.9

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123 mins was a mixture of unreacted I species, whilst the peak at (R t 25.0 min) was an unidentified radiolabelled side-product. The largest peak corresponding to the required

123 product was collected, giving [ I] [65] (R t = 22 min) in 82% radiochemical yield and

>99% radiochemical purity.

4.00

3.00

abs 2.00

1.00

0.00

400.00

300.00

200.00 mV

100.00

0.00 0.00 5.00 10.00 15.00 20.00 25.00 Minutes

Figure 6.3 Purification of [ 123 I] [65] using semipreparative HPLC column eluted with 40%

ACN:60% H 2O:0.1% TFA at 3mL/min showing UV activity (top) and radioactivity

(bottom).

To confirm that the peak at 22 min was the desired product, a sample of the radioactive product was co-injected and compared to the non-radioactive standard [65] on an analytical

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HPLC. Both peaks were found to elute at the same retention time, 8.9 mins, confirming the identity of the product. Even though the use of peracetic acid in acetic acid as the oxidant gave the best radiochemical yield, the UV elution profile indicated that a non–radioactive

UV active by-product could not be separated from the radioactive peak.

The purity of [ 123 I] [65] in saline solution was assessed by analytical HPLC immediately after purification and formulation. The analysis was repeated after 3 hours to assess the stability of the product. By integrating the area under the peaks, it was determined that the radiochemical purity of [123 I] [65] was greater than 99% after formulation in saline solution, which was reduced to 97.3% purity after 3 h. This confirmed that the product was relatively stable in saline solution and hence could be used in animal studies without appreciable degradation over the course of the experiments.

6.2.2. Synthesis of [ 123 I][78]

The highest affinity sigma-2 ligand prepared in Chapter 3 was compound [78] (IC 50 σ-2 1.0 nM (Figure 6.4).

H CO 3 O N O H3CO N H [78] I Sigma-2 IC50: 1.0 nM Sigma-1 IC50: 1000 nM

Figure 6.4 The highest affinity sigma-2 ligand from chapter 3, displaying a sigma-2 IC 50 of

1.0 nM.

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The synthesis of the [ 123 I][78] was prepared from the stannane precursor [145] which was prepared from the corresponding bromo analogue [75] in 49% yield (Scheme 6.3).

Scheme 6.3 Radiosynthesis of [ 123 I] [78].

H CO 3 O N O H3CO N H [75] Br Sn (CH ) , Pd(0)(PPh ) 2 3 3 3 4 49% toluene, reflux

H CO 3 O N O H3CO N H [145] Sn

Na123I CAT, 0.1 M HCl 72-78%

H CO 3 O N O H3CO N H [123I][78] 123I

Radiotracer [123 I] [78] was prepared from the stannane [145] using chloramine-T and 0.1 M

HCl (as described in Scheme 6.2) in radiochemical yields ranging from 72-78%. The

HPLC trace of the reaction mixture showed three radioactive peaks at 2.93 minutes, 9.1 minutes and 13.0 minutes using 50% ACN:50% H 2O:0.1% TFA as mobile phase at a flow rate of 3 mL/min with undetectable UV activity in the radioactive peak at 9.1 minutes

(Figure 6.5). The peak at 9.1 minutes was confirmed to be [123 I] [78] as it co-eluted with the non-radioactive iodo standard [78].

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3.00

2.00 abs

1.00

0.00

500.00

400.00

300.00 mV

200.00

100.00

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Minutes

Figure 6.5 Purification of [ 123 I] [78] (at 9.1 min) using a semipreparative RP HPLC column eluted with 50% ACN:50% H 2O:0.1% TFA at 3 mL/min.

The purity of [ 123 I] [78] in saline solution was assessed by analytical HPLC immediately after purification and formulation. The analysis was repeated after 3 hours to assess the stability of the product. By integrating the area under the peaks, it was determined that the radiochemical purity of [ 123 I] [78] was greater than 98.6% after formulation in saline solution, was reduced to 97.2% purity after 3 h.

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6.3. In vitro Cell studies

The radiolabelled compound [ 123 I] [78] (0.37 MBq in 500 L of phosphate buffer solution

(PBS)) was added and the uptake in four cancer cell lines: MCF-7 (human breast adenocarcinoma cancer cell line), PC-3 (human prostate cancer cell line), A375 (human melanoma cancer cell line) and MDA (human breast adenocarcinoma cancer cell line) were evaluated. The cells were incubated at 37 oC for 2, 15, 30, 60, 120, 180 and 240 min and uptake was terminated by removing the tracer solution and washing cells with ice-cold

PBS. Subsequently, the cells were lysed with NaOH (0.2 M, 500 L) and the radioactivity in the cells was measured with a gamma counter (Wallac model 1480) and the results are summarised in Figure 6.6.

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Uptake of 123 I[78] in tumour cells

40 cells

5 MCF-7 PC-3 30 A375 MDA

%uptake/1x10 20

0 0 60 120 180 240 300 Time (mins)

Figure 6.6 The percentage uptake of [123 I][78] in different cancer cell lines: MCF-7 (human breast adenocarcinoma cancer cell line), PC-3 (human prostate cancer cell line), A375 (human melanoma cancer cell line) and MDA (human breast adenocarcinoma cancer cell line).

The radiolabelled compound [ 123 I] [78] exhibited the highest uptake in the human breast cancer cell line MCF-7 at 2 hours (46%). The highest uptake observed in other cell lines were: prostate cancer cell line PC-3 at 2.5 hours (37%), human melanoma cell line A375 at

1 hour (34%) and human breast cancer cell line MDA at 2 hours (30%)

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6.4. In vivo biodistribution studies

To determine the distribution of radiolabelled compounds in the body the radiolabelled compound (0.74 Mbq in 100 µL saline) was administered into rats via the tail vein. At predetermined time points, the animals were sacrificed and their organs removed and weighed. The amount of radioactivity in each organ is counted, and the percent of injected dose per gram (%ID/g) of the organ was calculated.

6.4.1. In vivo biodistribution of [ 123 I][65]

The radiolabelled compound [123 I] [65] 0.74 Mbq in 100 L saline was injected into 24 male Sprague-Dawley rats, and the uptake into 17 organs was determined at 15 minutes, 30 minutes, 1, 3, 6 and 24 hours post injection of the radiotracer (Figure 6.7).

Mark Ashford, PhD Thesis 2010 97 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers Liver Spleen Kidney Lungs Heart Blood Pancreas Plasma Time (hours) Time I-[65] Biodistribution in Normal SDrats Normal in Biodistribution I-[65] 123

8 6 4 2 0

12 10 -1 4 9 14 19 24 D/g ID %

Figure 6.7 Biodistribution of [ 123 I] [65] in male Sprague-Dawley rats at various time points.

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After 15 minutes, there was uptake of radioactivity into organs known to contain sigma-2 receptors such as the pancreas (4.4%), lungs (1.4%) and liver (4.0%) and brain (0.23%

ID/g). There was also uptake into the thyroid (1.6%) which increased with time (24 h,

30.2%). Interestingly, these values are considerably lower than for those for a similar sigma-2 ligand 16 (liver (10.7%), lungs (3.7%) and kidneys (7.7 % ID/g)) whilst uptake in the brain (0.27% ID/g) was similar to that described. 16 The high uptake of the radiotracer in the thyroid is suggestive of in vivo deiodination. This is a common phenomenon with radioiodinated compounds as any free iodine is naturally taken up by the thyroid. After 24 h, besides the thyroid, the highest uptake of the radiolabelled product was observed in the liver (6.2%) and is partly attributed to the metabolism of the radiolabelled compound. After

24 h, clearance of the radiolabelled compound was observed from all other organs. The uptake of the radiotracer in the brain was less than 0.35% ID/g at all time points and is shown in Figure 6.8.

Mark Ashford, PhD Thesis 2010 99 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers Olf bulb Olf pons Med Cerebellum Midbrain Hypothalamus Hippocampus Striatum cortex Frontal Posterior Cortex Time (hours) I-[65] Brain Biodistribution in Normal SDRats Normal in Biodistribution Brain I-[65] 123 0 6 12 18 24

0.4 0.3 0.2 0.1 0.0 % ID/g %

Figure 6.8 Biodistribution of [ 123 I] [65] in Sprague-Dawley rats in all brain regions.

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Even though the brain is known to contain multiple sigma-2 receptors sites, initial uptake was attributed to blood flow, with activity decreasing as time increased, an observation consistent with other ligands of this type. 41 Although not shown on the graph, the amount of activity in the gastrointestinal tract increased up to 1.3% ID/g by 6 h, and decreased by

24 h. This was probably due to the compound being excreted. The amount of radiotracer in the bladder was also determined, the highest being at 6 h (0.6% ID/g), with radiotracer being excreted in the urine.

6.4.2. In vivo biodistribution of [ 123 I][78]

Radiotracer [123 I] [78] (0.74 Mbq in 100 L saline) was injected into 24 male Sprague-

Dawley rats, and the distribution throughout the body was determined at 15 minutes, 30 minutes, 1, 3, 6 and 24 hours post injection (Figure 6.9). At 15 minutes the uptake of activity was highest in the lungs (3.2%), followed by the thyroid (2.5%), pancreas (2.5%), kidney (2.3%), liver (1.5%) and spleen (1.4%). Uptake in the kidney remained around 1.9%

ID/g for the 24 h period. The uptake in the liver peaked after 6 hours (3.5%) and began to decrease after 24 hours. The uptake in the pancreas peaked at 6 hours (5.5 %) and began to decrease after 24 hours. The initial uptake in the lungs at 15 minutes rapidly decreased to

0.36% ID/g after 3 hours. Although not shown on the graph, the thyroid showed a slight increase over 24 hours from 2.5% ID/g to 2.91% ID/g, suggesting in vivo deiodination.

Also, the amount of radiotracer in the brain peaked at 0.15% ID/g after 15 minutes, with total clearance observed after 3 hours. This uptake is significantly lower than that for compound [65] and the compounds described 16 suggesting high degree of non-specific binding in the brain.

Mark Ashford, PhD Thesis 2010 101 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers Liver Kidneys Spleen Thyroid Lungs Heart Blood Pancreas Plasma Time (hours) I-[78] Biodistribution in Normal SDrats Normal in Biodistribution I-[78] 123 0 6 12 18 24

8 6 4 2 0

10 % ID/g %

Figure 6.9 Biodistribution of [ 123 I] [78] in male Sprague-Dawley rats at various time points.

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6.5. In vivo competition studies

Due to its superior in vitro results and higher uptake in the lungs and pancreas known to express the sigma-2 receptor, [123 I][78] was assessed for its specific binding.

To assess the specificity of binding of [ 123 I] [78] to sigma-2 receptors in vivo in rats, drug competition studies were performed by pre-administration of sigma-1 and sigma-2 binding drugs prior to the distribution of the radioligand. The uptake of radioactivity in the presence of these selective drugs was then examined in all organs and tissues. The competition drugs included non-radioactive [78] , (+)-pentazocine and haloperidol. (+)-

Pentazocine is a sigma-1 receptor drugs while haloperidol has affinity for both the sigma-2 and sigma-1 receptors. For this experiment four rats for each competition drug were used; an additional four were administered the [ 123 I] [78] only and hence used as controls.

Therefore, [ 123 I] [78], (+)-pentazocine and haloperidol at a concentration of 1 mg/kg (no competition drug, [ 123 I][78] only) were used. Four rats per drug were injected with 1 mg/kg in saline were each administered into groups of four rats followed by the tracer [ 123 I] [78]

(185 MBq/100 µL ) 5 min later. One group of four rats served as controls and were administered only [ 123 I] [78] (185 MBq/100 µL). All rats were sacrificed 1 h post injection of the [ 123 I][78], and the organs were removed, weighed and counted. The organs used in this study included liver, spleen, kidney, lungs, heat, bladder, stomach, gastrointestinal tract

(GIT), thyroid, pancreas, testis, thymus and brain. The amount of radioactivity in each organ was measured using an automated gamma counter and the percent injected dose (%

ID) for each organ calculated. The effects of the competition drugs on the uptake of

[123 I] [78] into selected organs is summarised in Figure 6.10

Mark Ashford, PhD Thesis 2010 103 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers

Control (+)-Pentazocine Haloperidol 123I[78]

sma

Thym

Panc

roid

GIT

r de

I[78] in normal SDrats normal in I[78]

lad B

123

loo

un L

Competition study of of study Competition

Femur

Skin

Muscle

Kidney

Spleen

Liver

kull S

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 %ID/g

Figure 6.10 Effect of various drugs on [ 123 I] [78] uptake in rat organs.

Mark Ashford, PhD Thesis 2010 104 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers

The preadministration of the sigma-2 binding drug haloperidol induced a 20% reduction in the uptake of [ 123 I] [78] in the kidneys, however, no other significant reduction in uptake was observed in any other organs. The lack of any drug-tracer competition in organs known to express the sigma-2 receptor suggests that the tracer was not binding specifically to sigma-2 receptors in vivo . There was also no significant reduction of uptake in all organs with pre-administration with the sigma-1 binding drug (+)-pentazocine, indicating there is negligible non-specific binding to the sigma-1 receptors in vivo . This supported the in vitro data results that it was not a sigma-1 receptor binding compound.

It was speculated that possible reasons for the high non-specific binding in vivo for organs known to contain sigma-2 receptors could be the result of rapid metabolism of the radiotracer, or it binding better with another receptor type in vivo . The latter is a possibility as the compounds in this project were only examined for their activity and selectivity for the two sigma receptor subtypes. It is also known that compounds of this type possess affinity for the dopamine and histamine receptors. However, the affinity for these receptors was not studied.

6.6. In vivo stability study of [ 123 I][78]

To further characterise the activity distribution of [ 123 I] [75] and help explain the lack of drug/tracer competition in the blocking studies, the fate and nature of the radioactivity in vivo was studied through metabolite analysis. Specifically, it was not known whether the radioactivity measured is of intact (non-metabolised) radiotracer, or of some radioactive metabolite or free iodine. The following in vivo study was undertaken with the objective of determining the amount of intact radiotracer in the body (organs and plasma) at specific time points after injection of the tracer into animals.

Mark Ashford, PhD Thesis 2010 105 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers

6.6.1. In vivo stability study of [ 123 I][78]

Three male Sprague-Dawley rats were each administered [123 I] [78] (37 MBq in 100 L per rat). The animals were sacrificed at 15, 30 and 60 minutes post injection, and selected organs and blood removed. The organs chosen for this study included the cortex (brain), lung, spleen as well as the plasma. After mincing the organs, the radioactivity in the tissues was extracted into acetonitrile with sonication followed by centrifugation. The acetonitrile was evaporated to dryness and the radioactivity reconstituted in methanol. The methanol extract from each tissue as well as the standard radiotracer was analysed by TLC and

HPLC.

The results of the study indicated that in the activity extracted from the cortex, lung and spleen were unchanged (>95%) [ 123 ][78] and there was no significant metabolites found. In contrast, there was no intact [ 123 I][78] detected in urine, while in the plasma, unchanged

[123 I] [78] averaged between 35-45% of total activity at all time points. These results indicate that the activity which is bound to the tissue corresponds to intact tracer while that in plasma is significantly metabolised. The activity which was excreted represents entirely metabolite fractions.

6.7. Conclusions and Future Directions

Two new high affinity sigma-2 ligands were successfully radiolabelled with 123 I. Cell studies indicated high uptake of [ 123 I] [78] in cell lines known to overexpress the sigma-2 receptor, such as the human breast adenocarcinoma cell line, MCF-7. In vivo biodistribution studies of [ 123 I] [65] and [ 123 I] [78] in male Sprague-Dawley rats indicated uptake in organs known to contain sigma-2 receptors. Pre-treatment of the sigma-2 drug haloperidol did not significantly decrease the uptake of [ 123 I] [78] in organs known to contain the sigma-2 receptor, with a stability study suggesting that this result was not due

Mark Ashford, PhD Thesis 2010 106 Chapter 6 – Radiochemistry, Cell Studies and in vivo evalutation of tracers to metabolic breakdown of the radiotracer in vivo . The lack of tracer inhibition during drug pre-treatment studies could not be explained and further studies need to be carried out, possibly employing other drugs.

Future studies would involve further optimisation of these series of compounds to develop a radiotracer with the desired in vivo properties for imaging cancerous tumours and sigma-

2 sites within the body.

Mark Ashford, PhD Thesis 2010 107 Chapter 7 – Conclusions and Future Directions

7. Conclusions and Future Directions

The synthesis of thirty-four new compounds based on a previously established lead benzamide [27] was achieved. The aim of this was to synthesise compounds exhibiting great sigma-2 activity and selectivity with the opportunity for radiolabelling with either

SPECT or PET through the incorporation of an iodine or bromine within the structure. It involved modifications at three keys regions (the tetrahydroisoquinoline (Region 1), spacer

(Region 2) and carboxamide (Region 3)) to observe the effect these changes would have on sigma-2 activity and selectivity.

In Chapter 2, the synthetic strategy for variation in the carboxamide (Region 3) included the formation of a halogenated piperidine which was installed onto a tetrahydroisoquinoline ring via N-alkylation. Deprotection of the piperidine nitrogen to the corresponding amine, which when coupled with various carboxylic acids, yielded the thirteen target compounds. The sigma-2 and sigma-1 binding affinities of the thirteen compounds were determined by measuring the displacement of [ 3H]DTG or

[3H]pentazocine from rat cortex membranes. All compounds, except for two, were selective for the sigma-2 receptor, with one, [60] exhibiting a better selectivity than that of the lead compound [ 27].

Most compounds showed moderate sigma-2 affinity with two halogenated derivatives, [60] and [65] , exhibiting high affinity and selectivity for the sigma-2 receptor. However, it was observed that the presence of iodine on the benzofuran [65] decreased the sigma-2 affinity slightly (IC 50 12.5 nM), compared to the brominated compound [60] (IC 50 9.8 nM) or the halogen free lead [27] (IC 50 <3 nM). The high affinity iodinated compound was radiolabelled for in vivo pharmacological studies in Sprague-Dawley rats. Radiolabelling

Mark Ashford, PhD Thesis 2010 108 Chapter 7 – Conclusions and Future Directions was achieved with radiochemical yields (RCY) ranging from 51-89%. This radiolabelled product, named [ 123 I] [65] showed good uptake in organs known to express sigma-2 receptors. Future studies would involve further evaluation of [ 123 I] [65] in metabolite, competition and tumour model studies.

In Chapter 3, the synthesis of fourteen new compounds with modifications to the zone designated ‘region 2’ was achieved. This involved the synthesis of compounds containing either: 1) a flexible spacer core, or 2) a semi-rigid spacer core. The synthetic strategy for the compounds containing a flexible spacer core involved the N-alkylation of the tertahydroisoquinoline with a bromo nitrile and reduction to the corresponding amine. This amine was then coupled to either 5-bromo, or 5-iodobenzo[ b]furan-2-carboxylic acid which were both synthesised in three steps from their corresponding salicylaldehydes.

These compounds were all high affinity sigma-2 ligands with good selectivity. An interesting result was observed with the order of addition of reagents to yield novel nitrosulfonamides, which were also tested for their sigma-2 binding affinity. These compounds, however, possessed poor sigma-2 binding affinity. The synthetic strategy for compounds containing a semi-rigid spacer core involved a similar process as described for region 3 modification, with an added reduction step at the beginning for the propylpiperidine spacer compounds. The propylpiperazine core was initially synthesised via the Boc protected amine using similar methods as previously described; however, the removal of the protecting group proved troublesome in this case. A new synthetic strategy based upon the benzyl protected propylpiperazine was implemented to give the target compound. Troublesome removal of the benzyl group was also experienced; however, this was overcome using hydrogen transfer conditions to give the free amine. All compounds produced in this series showed high sigma-2 activity. They were, however, not as selective for the sigma-2 receptor as the flexible spacer core compounds synthesised previously. The

Mark Ashford, PhD Thesis 2010 109 Chapter 7 – Conclusions and Future Directions most selective and active iodinated compound [78] , with an IC 50 of 1.0 nM, had the highest affinity for the sigma-2 receptor out of all compounds synthesised in this thesis and was radiolabelled with 123 I in 78% RCY. In vitro cell studies showed good uptake in cells known to express the sigma-2 receptor, such as human breast adenocarcinoma. In vivo studies showed uptake in organs known to express sigma-2 receptors, while metabolite studies showed that the compound was metabolically stable. However, blocking studies were not able to displace this compound from organs of interest, suggesting that the uptake of the radiotracer in the organs was not specifically due to its sigma-2 affinity and possibly due to a higher affinity for some other receptor found in the organs. This suggested that the ligand would not be a good marker for studying sigma-2 receptors in vivo .

In Chapter 4, the synthesis of seven new compounds was achieved with modifications to region 1. The synthesis of 6-methoxytetrahydoisoquinoline was achieved as reported in similar yields. Previously, 7-methoxytetrahydroisoquinoline had only been reported in milligram quantities or as a reaction by-product. Its synthesis was achieved in multigram quantities in 5 synthetic steps starting from 3-methoxybenzaldehyde. This was an improvement on the procedure,105 with tosylation of the amine nitrogen achieved in 18 h instead of 3 days through the use of the phase transfer catalyst tetra-n-butylammonium hydrogensulfate. Access to the 7-methoxytetrahydroisoquinoline was facilitated by the

Birch reduction of the known compound 7-methoxyisoquinoline [119] , in 81% yield. A racemic 1-phenyl-6,7-dimethoxytetrahydroisquinoline was also synthesised as reported. 95

The synthesised tetrahydroisoquinolines were then used to synthesise five flexible-spacer target compounds and two semi-rigid propylpiperidine compounds using methods established previously. All compounds from this series displayed moderate sigma-2 affinity. One compound [135] displayed an IC 50 of 5.0 nM, however it also displayed a good sigma-1 affinity (14.0 nM). The compound containing a 1-position modification was

Mark Ashford, PhD Thesis 2010 110 Chapter 7 – Conclusions and Future Directions the most selective compound from this series, and one of the most selective compounds produced in this thesis, however, its moderate sigma-2 receptor affinity did not warrant labelling with 123 I and further in vivo evaluation .

Future directions of this work could involve further SAR exploration of structural features that could enhance the affinity and selectivity of the benzamide nucleus for the sigma-2 receptor. With substantial investigations into the carboxamide derivatives already achieved, more in depth exploration of the other sites such as the spacer and isoquinoline may yield more potent and selective ligands. Such modifications may include incorporation of varying azabicyclic spacers or alkene spacers to observe the effect of increased conformational restriction or ether-type spacers in an effort to increase lipophilicity and blood brain barrier permeability. Other 1-substituted tetrahydroisoquinolines with the optimised 6,7-dimethoxy configuration could also be explored to evaluate the physiochemical properties of these candidates. Additional studies using molecular modelling could be performed to enhance the development of specific sigma-2 ligands.

Further in vivo biological testing and screening of compound [65] will determine its applicability as a potential sigma-2 receptor marker. Compound [78] showed positive results for uptake in organs expressing sigma-2 receptor sites and metabolic stability.

However, it was unable to be displaced from organs of interest in vivo and also showed low uptake in the brain. Further testing will ascertain its legitimacy as a tracer for peripheral tumour screening.

The results of this study have made significant progress towards the development of a selective sigma-2 ligand that could be used to study the receptor in vivo . Potential candidate ligands have been identified by modifying the benzamide structure, and

Mark Ashford, PhD Thesis 2010 111 Chapter 7 – Conclusions and Future Directions advanced chemistry schemes were used to synthesise an array of compounds which were evaluated using SAR.

Mark Ashford, PhD Thesis 2010 112 Chapter 8 - Experimental

8. Experimental

8.1. General Comments

All reagents purchased were used without further purification. Anhydrous solvents were purchased and used without further drying unless otherwise stated. THF and Et 2O were distilled from sodium under nitrogen gas. Air sensitive reactions were performed under a positive pressure of nitrogen gas Petroleum ether (PE) of boiling point range 40-60 oC was used. Melting points were recorded on a Gallenkamp (Griffin) melting point apparatus with temperatures reported in degrees Celsius ( oC) and are uncorrected.

Proton ( 1H) and carbon ( 13 C) nuclear magnetic resonance (NMR) spectra were recorded at

400 and 100 MHz respectively on a Bruker 400 MHz spectrometer. NMR spectra were acquired in deuterated chloroform (CDCl 3) unless otherwise noted, using chloroform ( δ

7.26) and TMS ( δ 0.00) as internal standards. Chemical shifts ( δ) in ppm were measured relative to the internal standard. Multiplicities were reported as singlet (s), broad singlet

(bs), doublet (d), doublet of doublets (dd), triplet (t), quartet (q) and multiplet (m).

Coupling constants ( J) are reported in Hertz. Protons associated with the piperidine ring are reported as Ha (axial) or He (equatorial) in the 1H spectrum.

Electron impact (EI) and electrospray (ES) mass spectra (MS) were obtained using a

Shimadzu QP-5000 GC-MS spectrometer by direct insertion technique with a 70 eV electron beam and high resolution (HR) on a VG Autospec spectrometer. Electrospray (ES)

MS were recorded on a Micromass Platform LCZ spectrometer and HR on a micromass

QTOF2 spectrometer. Ion mass to charge ( m/z ) values are stated and their relative abundances as a percentage in parentheses.

Mark Ashford, PhD Thesis 2010 113 Chapter 8 - Experimental

Analytical thin layer chromatography (TLC) was performed on Merck Kieselgel 60 F 254 precoated polyester plates with a thickness of 250 µm. Gravity column chromatography was performed using silica gel, Merck, grade 60, 70-230 mesh, 60 Å. High performance liquid chromatography (HPLC) was performed using a Waters 600 pump, a Waters 2996

Photodiode Array detector set at 254 nm and a RP X-terraC-18 4.6 x 150 mm column at a flow rate of 1 mL/min unless otherwise stated.

[3H] Pentazocine and [ 3H] DTG were purchased from Perkin-Elmer Life Sciences (Boston,

MA, USA). Carrier free Na 123 I in 0.1 M NaOH was obtained from Australian

Radioisotopes and Industrials (ARI), Australia. Male Sprague-Dawley rats were purchased from the Animal Resources Centre (Perth, WA, Australia). The rodents were housed in cages with access to standard rodent chow and water ad libitum in an animal house equipped with a high standard ventilation system and maintained at 22 ± 2 oC, 60% ± 10% humidity. Air changes were set at 15 changes/h and a 12h/12h light/dark cycle. All procedures were carried out in compliance with Australian laws governing animal experimentation.

The experimental data is divided into:

1. Experimental procedures for region 3 modification

2. Experimental procedures for region 2 modification

3. Experimental procedures for region 1 modification

4. Experimental procedures for the stannylation reactions and radioiodination

reactions.

Mark Ashford, PhD Thesis 2010 114 Chapter 8 - Experimental

Literature and commercially available compounds have their names written in italics , while new compounds have their names written in non-italics. Spectra of literature compounds are identical to that reported.

8.2. Experimental procedures for region 3 modification

8.2.1. Intermediate Synthesis

1-(tert-Butoxycarbonyl)-4-hydroxymethylpiperidine 63 [29]

HO To a stirred solution of 4-piperidinemethanol (3.25 g, 26.2 mmol) in 4 anhydrous CH 2Cl 2 (40 mL) was added di-tert -butyl dicarbonate (7.00 g, 1 N 31.1 mmol). The solution turned yellow and was stirred at rt for 30 min. (H C) C 3 3 O O The organic solvent was then removed, resulting in a pale yellow oil, which upon standing yielded [29] (6.70 g, 96%) as a pale yellow solid, mp 70-72 oC, (lit 55 73 oC). 1H NMR

(CDCl 3) δ 1.11-1.21 (m, 2H, H3a, H5a), 1.47 (s, 9H, OC(CH3)3), 1.61-1.75 (m, 3H, H3e,

H5e, H4), 2.69-2.75 (m, 2H, H2a, H6a), 3.00 (d, 2H, J = 8.0 Hz, HOCH 2), 4.12 (bs, 2H,

H2e, H6e). MS-ES + m/z 216 (MH +, 73), 160 (100%).

1-(tert-Butoxycarbonyl)-4-(methanesulfonyloxymethyl)piperidine 63 [30]

To a stirred solution of the Boc protected amine [29] (1.80 g, 8.30 mmol) H3C SO2 O in anhydrous CH 2Cl 2 (25 mL) was added anhydrous Et 3N (4.05 mL, 29.0 4 mmol) followed by the dropwise addition of methanesulfonyl chloride 1 N o (0.75 mL, 9.30 mmol). The solution was stirred at 0 C for 1 h under N 2. (H C) C 3 3 O O The solution was basified with aqueous K 2CO 3 (0.1 M, 50 mL) and the product extracted into CH 2Cl 2 (2 x 50 mL). The organic layers were combined, dried

(Na 2SO 4) and the organic solvent removed to afford a yellow oil which upon standing yielded [30] (2.06 g, 84%) as a yellow solid, mp 74-75 oC, (lit 55 75-76 oC). 1H NMR

Mark Ashford, PhD Thesis 2010 115 Chapter 8 - Experimental

(CDCl 3) δ 1.18-1.27 (m, 2H, H3a, H5a), 1.46 (s, 9H, OC(CH3)3), 1.75 (bd, J = 12.8 Hz,

2H, H3e, H5e), 1.92 (m, 1H, H4), 2.72 (bt, 2H, J = 12.5 Hz, H2a, H6a), 3.02 (s, 3H,

+ SO 2CH3), 4.06-4.08 (d, 2H, J = 8.0 Hz, OCH2), 4.14 (bs, 2H, H2e, H6e). MS-ES m/z 294

(MH +, 11), 238 (100%).

1-(tert-Butoxycarbonyl)-4-(iodomethyl)piperidine 64 [31]

To a stirred solution of the Boc protected amine [29] (4.22 g, 19.6 mmol) I

o in toluene (150 mL) at 0 C was added PPh 3 (6.68 g, 25.5 mmol),

N imidazole (4.01 g, 58.8 mmol) and iodine (6.47 g, 25.5 mmol). After (H C) C 3 3 O O addition, the reaction was allowed to warm to rt and stirred for 1.5 h. The organic solvent was removed and the residue was subjected to column chromatography

(EtOAc:PE 1:1) and dried in vacuo to yield [31] (5.58 g, 88%) as a clear oil. 1H NMR

(CDCl 3) δ 1.05-1.16 (m, 2H, H3a, H5a), 1.42 (s, 9H, OC(CH3)3), 1.51-1.63 (m, 1H, H4),

1.78 (bd, 2H, J = 12.8 Hz, H3e, H5e), 2.65 (bt, 2H, J = 12.8 Hz, H2a, H6a), 3.08 (d, 2H, J

+ + = 7.0 Hz, CH 2I), 4.05-4.11 (m, 2H, H2e, H6e). MS-ES m/z 326 (MH , 2), 270 (100 %).

tert -Butyl-4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)methyl)piperidine-1- carboxylate [33]

Nitrogen alkylation Method A: To a stirred O H CO 4a' 1 C(CH ) 3 3' N O 3 3 solution of 1-(tert -butoxycarbonyl)-4- N 4 H3CO 8a' 1' (methanesulfonyloxymethyl)piperidine [30]

(500 mg, 1.70 mmol) in 2-butanone (10 mL) was added K 2CO 3 (470 mg, 3.40 mmol), 6,7- dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride [32] (586 mg, 2.55 mmol) and LiI

(12 mg, 0.085 mmol). After heating at reflux for 18 h, the solution was cooled to rt, diluted with H 2O (30 mL) and extracted with Et 2O (3 x 15 mL). The combined organic layers were dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:MeOH, 9:1) and dried in vacuo to yield [33] (384 mg, 58%) as a

Mark Ashford, PhD Thesis 2010 116 Chapter 8 - Experimental

o 1 clear yellow oil which solidified upon standing, mp 90-92 C. H NMR (CDCl 3) δ 1.10-

1.14 (m, 2H, H3a, H5a), 1.45 (s, 9H, O(CH 3)3), 1.76-1.80 (m, 3H, H3e, H5e, H4), 2.33 (m,

2H, NC H2CH), 2.67-2.80 (m, 6H, H3 ′, H4 ′, H2a, H6a), 3.52 (s, 2H, H1 ′), 3.83 (s, 6H, 2 x

13 OCH 3), 4.08-4.10 (bs, 2H, H2e, H6e), 6.52 (s, 1H, H5 ′), 6.59 (s, 1H, H8′). C NMR δ 28.6

(OC( CH3)3), 28.8 (C4 ′), 34.1 (C3, C5), 35.2 (C4), 37.2 (CH 2), 44.2 (C2, C6), 51.5 (C3 ′),

56.06 (OCH 3), 56.08 (OCH 3), 56.4 (C9 ′), 64.4 (C1 ′), 79.4 (O C(CH3)3), 109.7 (C5 ′), 111.6

(C8 ′), 126.4 (C4a ′), 126.8 (C8a ′), 147.4 (C6 ′), 147.7 (C7 ′), 155.1 (CO). MS-ES + m/z 391

+ + (MH , 100%); HRMS-ES calculated for C 22 H34 N2O4: 391.2586, found 391.2597.

Nitrogen alkylation Method B: To a solution of 6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline hydrochloride [32] (1.29 g, 5.63 mmol), K 2CO 3 (3.06 g, 22.12 mmol) and TBAI (85 mg, 0.23 mmol) in DMF (20 mL) was added 4-(2- iodomethyl)piperidine-1-carboxylic acid tert -butyl ester [31] (1.83 g, 5.63 mmol). The solution was allowed to stir at rt for 3 days. The solution was diluted with CH 2Cl 2 (100 mL) and extracted with H 2O (3 x 20 mL), sat. NaHCO 3 (25 mL), brine (25 mL) and H 2O

(20 mL). The organic layers were combined, dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:MeOH, 9:1) and dried in vacuo to yield the title compound [33] (1.72 g, 78%) as a clear oil which was spectroscopically identical to that reported for previous methods.

1,2,3,4-Tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34]

5 Nitrogen deprotection Method A : A solution of tert - H CO 1' 3 3 NH 4' N butyl-4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- H CO 3 8a 1 yl)methyl)piperidine-1-carboxylate [33] (380 mg, 0.970 mmol) in CH 2Cl 2:TFA (6 mL, 2:1) was stirred for 30 min at rt. The product was basified with aq. K 2CO 3 (1 M, 200 mL) followed by aq. KOH (0.1 M, 50 mL) and extracted with

Mark Ashford, PhD Thesis 2010 117 Chapter 8 - Experimental

CH 2Cl 2 (3 x 100 mL). The organic layers were combined, dried (Na 2SO 4) and the solvent

1 removed to yield [34] (224 mg, 78%) as a yellow oil. H NMR (CDCl 3) δ 1.11-1.15 (m,

2H, H3a′, H5a′), 1.72-1.80 (m, 3H, H3a′, H5a′, H4 ′), 2.32 (m, 2H, NC H2CH), 2.58-2.68 (m,

4H, H4, H2a′, H6a′), 2.75-2.81 (m, 2H, H3), 3.06-3.09 (m, 2H, H2e′, H6e′), 3.51 (s, 2H,

13 H1), 3.83 (s, 6H, 2 x OCH 3), 6.51 (s, 1H, H5), 6.58 (s, 1H, H8). C NMR δ 28.8 (C4),

32.4 (C3 ′, C5 ′), 34.3 (C4 ′), 46.7 (C2 ′, C6 ′), 51.5 (C3), 56.1 (2 x OCH 3), 56.5 (N CH2CH),

65.2 (C1), 109.7 (C5), 111.6 (C8), 126.5 (C4a), 127.1 (C8a), 147.3 (C6), 147.6 (C7). MS-

+ + ES m/z 291 (MH , 40), 208 (100%); HRMS-EI calculated for C 17 H26 N2O2: 290.1994, found 290.1996.

5-Bromo-2,3-dimethoxybenzoic acid 41 [47]

To a solution of 5-bromo-2,3-dimethoxybenzaldehyde (1.03 g, 4.08

H3CO O H CO 1 mmol) in acetone (30 mL) was added a hot solution of 10% KMnO 4 3 OH

5 (10 mL). The solution was allowed to stir for 2 h at rt. The insoluble Br material was filtered off and the filter cake was washed with hot H 2O

(20 mL). The organic layer was removed and the aqueous solution was acidified using HCl

(12 M, 10 mL). The precipitate was filtered to yield [47] (1.04 g, 98%) as a white solid, mp

o 1 223-224 C. H NMR (CDCl 3) δ 3.93 (s, 3H, C3OCH3), 4.06 (s, 3H, C2OCH3), 7.25 (d,

13 1H, J = 2.4 Hz, H4), 7.83 (d, 1H, J = 2.4 Hz, H6). C NMR (CDCl 3) δ 56.4 (C3-OCH3),

62.2 (C2-OCH3), 117.3 (C1), 120.4 (C5), 123.6 (C4), 126.1 (C6), 147.7 (C3), 153.0 (C2),

165.0 (CO). MS-ES + m/z 261 (MH +, 20), 522 (100%); HRMS-EI calculated for

79 C9H9O4 Br: 260.9762, found 260.9763.

Ethyl-5-bromobenzo[b]furan-2-carboxylate 71 [53]

O OEt A mixture of 5-bromosalicylaldehyde (3.5 g, 17.5 mmol), 2 5 O Br 3

Mark Ashford, PhD Thesis 2010 118 Chapter 8 - Experimental diethylbromomalonate (35.03 g, 14.7 mmol) and K 2CO 3 (4.5 g, 31.5 mmol) was heated at reflux in 2-butanone (55 mL) for 24 h. When cool, the solvent was removed and water (100 mL) was added and then extracted with CH 2Cl 2 (3 x 20 mL). The combined organic layers were dried (MgSO 4), concentrated, subjected to column chromatography (EtOAc:PE, 1:9) to yield [53] (1.78 g, 48%) as an off white solid, mp 82-83 oC (lit 71 90 oC). 1H NMR

(CDCl 3) δ 1.43 (t, 3H, J = 7.1 Hz, OCH 2CH3), 4.44 (q, 2H, J = 7.1 Hz, OC H2CH 3), 7.45

(d, 1H, J = 0.7 Hz, H4), 7.47 (s, 1H, H3), 7.53 (dd, 1H, J = 6.8, 1.9 Hz, H6), 7.81 (d, 1H, J

= 2.0 Hz, H7). MS-ES + m/z 270 (MH +, 31), 115 (100%).

5-Bromobenzo[b]furan-2-carboxylic acid 71 [43]

A mixture of KOH (0.67 g, 1.23 mmol) in EtOH (30 mL) and H O O OH 2 2 5 O (3 mL) was added to ethyl 5-bromobenzo[b]furan-2-carboxylate Br 3 [53] (1.52 g, 6.03 mol) and was allowed to stir at reflux for 5 mins. The solvent was removed and H 2O (75 mL) was added to the residue. The pH was adjusted to pH 3 by addition of HCl (2 M). The white precipitate was filtered and crystallised (AcOH) to afford

[43] (870 mg, 65%) as a pale purple solid, mp 258-259 oC (lit 71 256-260 oC): 1H NMR

(CDCl 3) δ 7.65-7.97 (m, 2H, H7, H3), 7.72 (d, 1H, J = 8.8 Hz, H6), 8.03 (d, 1H, J = 1.8

Hz, H4). MS-ES + m/z 242 (MH +, 7), 146 (100%).

Towards Ethyl-5-iodobenzo[ b]furan-2-carboxylate

Strategy 1 :

Ethyl-5-nitrobenzo[b]furan-2-carboxylate 72 [54]

7 A mixture of 5-nitrosalicylaldehyde (2.05 g, 12.27 mmol), O O 2 diethylbromomalonate (1.54 g, 11.04 mmol) and K 2CO 3 (3.05 g, OEt O2N 3 22.08 mmol) in 2-butanone (40 mL) was heated at reflux for 19 h. The mixture was cooled to rt, diluted with CH 2Cl 2 (50 mL), and then washed successively with H 2O (3 x 50 mL),

Mark Ashford, PhD Thesis 2010 119 Chapter 8 - Experimental brine (20 mL), H 2O (20 mL) and dried (Na 2SO 4). The solvent was evaporated and the residue subjected to column chromatography (EtOAc:PE, 1:9) to yield [54] (1.42 g, 55%)

o 72 o 1 as a yellow solid, mp 103-105 C (lit 105 C). H NMR (CDCl 3) δ 1.44 (t, 3H, J = 7.1 Hz,

OCH 2CH3), 4.47 (q, 2H, J = 7.1 Hz, OC H2CH 3), 7.63 (s, 1H, H3), 7.69 (d, 1H, J = 9.2 Hz,

H7), 8.36 (dd, 1H, J = 9.2, 2.3 Hz, H6), 8.63 (d, 1H, J = 2.3 Hz, H4). 13 C NMR δ 14.4

(CH 3), 62.3 (CH 2), 113.1 (C7), 114.0 (C3), 119.6 (C4), 123.1 (C6), 127.4 (C3a), 144.9

(C5), 148.6 (C2), 158.1 (C7a), 158.8 (CO). MS-EI m/z 235 (M +, 56), 207 (100%).

Ethyl-5-aminobenzo[ b]furan-2-carboxylate [55]

Hydrogenation Method A :73 A solution of ethyl-5- 6 O O 2 OEt nitrobenzo[ b]furan-2-carboxylate [54] (470 mg, 2.00 mmol) in H2N 3 MeOH:dioxane (1:1 v/v 10 mL) and 10% Pd/C (50 mg) was subjected to a hydrogen atmosphere at 60 psi for 5 h at rt. The solution was filtered though celite and the filter cake washed with EtOAc (50 mL). The filtrate was concentrated to give a green oil that solidified on standing. The solid was recrystallised (EtOAc:PE, 1:1) to yield [55] (205 mg,

50%) as a brown solid, mp 89-91 oC (lit 73 92 oC). 1H NMR (DMSO) δ 1.37 (t, 3H, J = 7.1

Hz, CH 2CH3), 3.57 (bs, 2H, N H2), 4.38 (q, 2H, J = 7.1 Hz, C H2CH 3), 6.78 (dd, 1H, J = 8.8,

2.4 Hz, H6), 6.83 (d, 1H, J = 2.2 Hz, H4), 7.30-7.35 (m, 2H, H7, H3). 13 C NMR (DMSO) δ

14.1 (CH 3), 61.4 (CH 2), 113.3 (C6, C7), 114.0 (C3), 122.5, 127.4 (C3a), 144.2 (C2), 146.4

(C5), 153.3 (C7a), 158.4 (CO). MS-EI m/z 205 (M +, 98), 104 (100 %).

Hydrogenation Method B: A mixture of ethyl-5-nitrobenzo[ b]furan-2-carboxylate [54]

(470 mg, 2.00 mmol) and Raney-nickel (175 mg) in MeOH (10 mL) were subjected to a hydrogen atmosphere at STP for 24 h at rt. The solution was filtered through celite and the filter cake was washed with EtOAc (50 mL). The organic solvent was removed, and the

Mark Ashford, PhD Thesis 2010 120 Chapter 8 - Experimental residue was submitted to column chromatography (EtOAc:PE, 1:9-3:7) to yield [55] as a brown solid (350 mg, 85%), which was spectroscopically identical to that reported above.

Strategy 2 :

5-Iodosalicyclaldehyde 75 [59]

O To a solution of salicylaldehyde (30.6 g, 251 mmol) in CH 2Cl 2 (100 mL) I H was added ICl (250 mL, 1 M solution in CH 2Cl 2) and the reaction was OH allowed to stir for 17 h at rt. To the solution was added sat. Na 2SO 3 (300 mL) until the colour of the solution changed from dark red to clear. The solution was diluted with CH 2Cl 2 (100 mL), and extracted with H 2O (3 x 100 mL), brine (100 mL) and

H2O (100 mL). The organic layers were combined, dried (Na 2SO 4) and the organic solvent removed. The residue was recrystallised from cyclohexane and then isopropanol to yield

o 75 o 1 [59] (26.34 g, 42%) as a white solid, mp 99-101 C (lit 105 C). H NMR (CDCl 3) δ 6.79

(d, 1H, J = 8.8 Hz, H3), 7.76 (dd, 1H, J = 8.8, 2.2 Hz, H4), 7.84 (d, 1H, J = 2.2 Hz, H6),

9.83 (s, 1H, OH), 10.93 (s, 1H, CHO). MS-EI m/z 248 (M +, 5), 151 (100 %).

Ethyl-5-iodobenzo[ b]furan-2-carboxylate [57]

Method A: To a solution of p-TsOH.H O (555 mg, 2.92 mmol) in O O 2 2 5 ACN (10 mL) was added aniline [55] (200 mg, 0.97 mmol). The I 3 OEt

o resulting suspension was cooled to 10 C and to this was added a solution of NaNO 2 (135 mg, 1.95 mmol) and KI (403 mg, 2.43 mmol) in H 2O (2 mL). The reaction mixture was stirred for 10 mins at 10 oC, allowed to warm to rt and stirred for a further 24 h. To the mixture was added 10% NaOH solution until basic and was then extracted with EtOAc (3 x

20 mL). The combined organic layers were dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (10% EtOAc:PE) and dried in vacuo to yield [57] (160 mg, 55%) as a white solid, mp 134-136 oC. 1H NMR

(CDCl 3) δ 1.42 (t, 3H, J = 6.0 Hz, CH3), 4.44 (q, 2H, J = 6.0 Hz, CH2), 7.36 (bd, 1H, J =

Mark Ashford, PhD Thesis 2010 121 Chapter 8 - Experimental

8.8 Hz, H7), 7.43 (s, 1H, H3), 7.65-7.71 (m, 1H, H6), 8.01 (d, 1H, J = 1.8 Hz, H4). 13 C

NMR (CDCl 3) δ 14.4 (CH 3), 61.9 (CH 2) 87.4 (C5), 112.6 (C3), 114.5 (C7), 129.7 (C4),

131.7 (C3a), 136.3 (C6), 146.6 (C2), 155.1 (C7a), 159.3 (CO). MS-EI m/z 301 (M +, 5), 316

(100 %); HRMS-EI calculated for C 11 H9IO 3: 301.6287, found 301.6290.

Method B: A mixture of 5-iodosalicyclaldehyde [59] (1.04 g, 4.19 mmol), diethylbromomalonate (0.9 g, 3.77 mmol) and K 2CO 3 (1.04 g, 7.54 mmol) in 2-butanone

(30 mL) was heated at reflux for 20 h. The reaction mixture was allowed to cool to rt and then diluted with EtOAc (50 mL). The organic layer was extracted with H 2O (20 mL), brine (20 mL), and then H 2O (20 mL). The organic layer was dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography

(EtOAc:PE, 1:9) and dried in vacuo to yield [57] (900 mg, 80%) as a white solid which was spectroscopically identical to that reported earlier.

5-Iodobenzo[ b]furan-2-carboxylic acid [44]

To a solution of KOH (190 mg, 3.34 mmol) in EtOH:H 2O (10:1, 20 6 O O 2 b [57] OH mL) was added to ethyl-5-iodobenzo[ ]furan-2-carboxylate I 3 (500 mg, 1.66 mmol) and the mixture was heated at reflux with stirring for 5 min. The solvent was removed, and H 2O (50 mL) was added. The pH was adjusted to pH 3 by addition of HCl (2 M). The precipitate was filtered and dried to yield [44] (340 mg, 71%) as a white solid, mp 220-222 oC. 1H NMR (DMSO) δ 7.54-7.58 (m, 2H, H6, H7), 7.75-

7.78 (m, 1H, H4), 8.17 (s, 1H, H3) 13 C NMR (DMSO) δ 87.9 (C5), 112.4 (H6), 114.5 (H3),

129.7 (C4), 131.5 (C3a), 135.7 (C6), 146.9 (C2), 154.3 (C7a), 159.7 (CO). MS-EI m/z 288

+ (M , 100%); HRMS-EI calculated for C 9H5IO 3: 288.2365, found 288.2360.

Mark Ashford, PhD Thesis 2010 122 Chapter 8 - Experimental

5-Bromoindole-3-carboxaldehyde 111 [51]

To a stirred solution of POCl (6.00 mL, 63.7 mmol) in DMF (15 mL) H 3 N1 was added 5-bromoindole [50] (10.03 g, 51.0 mmol) such that the Br 5 3 O temperature did not rise above 10 oC. The solution was stirred at 10 oC H for 1 h, warmed to 35 oC, and stirred for a further 1 h. Ice was then added to the reaction mixture until a cherry red solution resulted, followed by NaOH (22.5 g, 564 mmol) in H 2O

(80 mL). The resulting mixture was heated rapidly to boiling point and allowed to cool.

The resulting precipitate was collected and recrystallised from EtOH to yield [51] (7.11 g,

63%) as a pale brown solid, mp 189-191 oC. 1H NMR (DMSO) δ 7.39 (dd, 1H, J = 8.6, 1.6

Hz, H6), 7.49 (d, 1H, J = 8.6 Hz, H7), 8.22 (d, 1H, J = 1.4 Hz, H4), 8.33 (s, 1H, H2), 9.92

(s, 1H, CHO). MS-EI m/z 224 (M +, 5), 186 (100 %).

5-Bromoindole-3-carboxylic acid 111 [48]

To a solution of 5-bromoindole-3-carboxaldehyde [51] (1.03 g, 4.46 H N1 5 mmol) in acetone (25 mL) was added a hot solution of 10% KMnO 4 Br 3 O (10 mL). The solution was allowed to stir for 2 h at rt. The insoluble HO material was filtered and the filter cake washed with hot H 2O (20 mL). The organic layer was removed and the aqueous solution was acidified using HCl (12 M). The resulting precipitate was filtered to yield [48] (1.02 g, 96%) as an off-white solid, mp 213-215 oC.

1H NMR (DMSO) δ 7.30 (dd, 1H, J = 8.6, 1.5 Hz, H6), 7.44 (d, 1H, J = 8.6, H7), 8.04 (1H, d, J = 2.8, H4), 8.13 (s, 1H, H2). MS-ES + m/z 241 (MH +, 20), 123 (100%).

Mark Ashford, PhD Thesis 2010 123 Chapter 8 - Experimental

8.2.2. Synthesis of target compounds with region 1 modification

4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)(1 H-indol-

2-yl)methanone [36]

O Coupling Method A: To a stirred solution of H H CO 4a'' 1' 3 3'' 2 N 7 N 7a indole-2-carboxylic acid (178 mg, 1.09 mmol) N H CO 4' 3 3 8'' 3a 1'' in anhydrous DMF (10 mL) was added 1,2,3,4-

tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (316 mg, 1.09

mmol), DCC (270 mg, 1.31 mmol), HOBt (147 mg, 1.09 mmol), and DMM (0.27 mL, 2.18

mmol) and the resulting solution was allowed to stir for 3 days at rt under N 2. The reaction

mixture was diluted with EtOAc (80 mL), washed with H 2O (3 x 25 mL), sat. Na 2CO 3 (15

mL), H 2O (20 mL) and brine (15 mL). The organic layer was dried (Na 2SO 4) and solvent

removed. The residue was subjected to column chromatography (EtOAc:MeOH, 95:5) and

dried in vacuo to yield the title compound [36] (142 mg, 30%) as a white solid, mp 208-

o 210 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 8.6 min).

1H NMR (DMSO) δ 1.16-1.26 (m, 2H, H3a′, H5a ′), 1.88 (bd, 2H, J = 12.3 Hz, He3 ′, He5 ′),

1.96-2.03 (m, 1H, H4 ′), 2.38 (d, 2H, J = 7.2 Hz, NC H2CH), 2.67 (t, 2H, J = 5.9 Hz, H4 ′′),

2.76 (t, 2H, J = 5.3 Hz, H3 ′′), 3.09 (bs, 2H, H2a′, H6a′), 3.52 (s, 2H, H1 ′′), 3.74 (s, 3H,

OCH 3), 3.75 (s, 3H, OCH 3), 4.47 (bs, 2H, He2 ′, He6 ′), 6.67 (s, 1H, H5 ′′), 6.70 (s, 1H, H8 ′′),

6.77 (s, 1H, H3), 7.07 (t, 1H, J = 7.2 Hz, H5), 7.20 (t, 1H, J = 7.2 Hz, H6), 7.45 (d, 1H, J =

8.2 Hz, H4), 7.62 (d, 1H, J = 8.0 Hz, H7). 13 C NMR (DMSO) δ 29.0 (C4 ′′), 31.5 (C3 ′, C5 ′),

34.1 (C4 ′), 44.1 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.41 (C6 ′′OCH3, C7 ′′OCH3) 56.44 (N CH2CH),

64.3 (C1 ′′), 104.1 (C3), 111.2 (C5 ′′), 112.7 (C7), 113.0 (C8 ′′), 120.3 (C4), 121.9 (C5),

123.7 (C6), 126.9 (C4a ′′), 127.6 (C3a), 127.7 (C8a ′′), 131.2 (C7a), 136.6 (C2), 147.8 (C6 ′′),

148.1 (C7 ′′), 162.7 (CO). FTIR ν 3237 (w), 2930 (m), 2351 (s), 1588 (m), 751 (s) cm-1.

+ + MS-ES m/z 434 (MH , 100%); HRMS-EI calculated for C 26 H31 N3O3: 433.2365, found

433.2356.

Mark Ashford, PhD Thesis 2010 124 Chapter 8 - Experimental

Coupling Method B: To a stirred solution of indole-2-carboxylic acid (178 mg, 1.09 mmol) in anhydrous DMF (10 mL) was added 1,2,3,4-tetrahydro-6,7-dimethoxy-2-

((piperidine-4-yl)methyl)isoquinoline [34] (316 mg, 1.09 mmol), EDC (252 mg, 1.31 mmol), HOBt (147 mg, 1.09 mmol), and DIPEA (0.4 mL, 2.18 mmol) and the resulting solution was allowed to stir for 2 days at rt under N 2. The reaction mixture was diluted with

EtOAc (80 mL), washed with H 2O (3 x 25 mL), sat. Na 2CO 3 (15 mL), H 2O (20 mL) and brine (15 mL). The organic layer was dried (Na 2SO 4) and solvent removed. The residue was subjected to column chromatography (EtOAc:MeOH, 95:5) and dried in vacuo to yield the title compound [36] (207 mg, 48%) as a white solid, which was spectroscopically identical to that reported above.

Coupling Method C: To a stirred solution of indole-2-carboxylic acid (178 mg, 1.09 mmol) in ACN (20 mL) was added Et 3N (0.30 mL, 2.18 mmol), DMAP (27 mg, 0.22 mmol) and NsCl (242 mg, 1.09 mmol). After stirring for a further 10 min, 1,2,3,4- tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (350 mg, 1.20 mmol) was added and the reaction mixture heated at reflux for 3 h and the solvent removed. The residue was taken up in CH 2Cl 2 (100 mL) and extracted with H 2O (30 mL), sat. NaHCO 3 (30 mL) and brine (30 mL). The organic layer was dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography

(EtOAc:MeOH, 9:1) and dried in vacuo yield the title compound [36] (225 mg, 52%) as a white solid, which was spectroscopically identical to that reported above.

Mark Ashford, PhD Thesis 2010 125 Chapter 8 - Experimental

(E)-1-(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)-3- phenylprop-2-en-1-one [38]

O The title compound [38] was prepared as a 4'' 7 H CO 4a'' 1' 1 3 N 8 white solid (210 mg, 46%) from cinnamic N 4 H3CO 8a'' 4' 1'' acid (162 mg, 1.09 mmol), HOBt (147 mg,

1.09 mmol), EDC (252 mg, 1.31 mmol), 1,2,3,4-tetrahydro-6,7-dimethoxy-2-((piperidine-

4-yl)methyl)isoquinoline [34] (316 mg, 1.09 mmol) and DIPEA (0.4 mL, 2.18 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 149-150 oC. HPLC analysis (0.1M

1 PO 4 buffer pH 7.5: MeOH, 35:65, retention time 7.1 min). H NMR (DMSO) δ 1.00-1.11

(m, 2H, Ha3 ′, Ha5 ′), 1.83 (bd, 2H, J = 12.3 Hz, He3 ′, He5 ′), 1.90-1.95 (m, 1H, H4 ′), 2.35

(d, 2H, J = 7.1 Hz, NC H2CH), 2.65 (t, 2H, J = 6.0 Hz, H4 ′′), 2.75 (t, J = 5.4 Hz, H3 ′′), 3.11

(bs, 2H, Ha2 ′, Ha6 ′), 3.50 (s, 2H, H1 ′′), 3.74 (s, 3H, OCH 3), 3.75 (s, 3H, OCH 3), 4.38 (bs,

2H, He2 ′, He6 ′), 6.66 (s, 1H, H5 ′′), 6.69 (s, 1H, H8 ′′), 7.24 (d, 1H, J = 15.5 Hz, H7), 7.37-

7.46 (m, 3H, H3, H4, H5), 7.49 (d, 1H, J = 15.5 Hz, H8), 7.70-7.73 (m, 2H, H2, H6). 13 C

NMR (DMSO) δ 29.0 (C4 ′′), 31.4 (C3 ′, C5 ′), 34.1 (C4 ′), 44.1 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.4

(C6 ′′OCH3, C7 ′′OCH3, N CH2CH), 64.3 (C1 ′′), 111.2 (C5 ′′), 113.0 (C8 ′′), 119.6 (C4), 126.9

(C4a ′′), 127.7 (C8a ′′), 128.6 (C3, C5), 129.4 (C2, C6), 130.0 (C1), 136.1 (C7), 141.7 (C8),

147.9 (C6 ′′), 148.1 (C7 ′′), 165.1 (CO). FTIR ν 2930 (w), 2372 (m), 1608 (s), 1255 (s), 982

-1 + + + (s) cm . MS-ES m/z 421 (MH , 31), 443 (MHNa) (100%); HRMS-EI calculated for

C26 H32 N2O3: 420.2413, found 420.2401.

Mark Ashford, PhD Thesis 2010 126 Chapter 8 - Experimental

(Benzo[ b]thiophen-2-yl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [37]

The title compound [35] was prepared as an O H CO 4a'' 1' 2 S 3 3'' N 7a off-white solid (200 mg, 41%) from N 3 H3CO 8a'' 4' 3a 1'' benzo[ b]thiophene-2-carboxylic acid (194 mg,

1.09 mmol), HOBt (147 mg, 1.09 mmol), EDC (252 mg, 1.31 mmol), 1,2,3,4-tetrahydro-

6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (316 mg, 1.09 mmol) and

DIPEA (0.4 mL, 2.18 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 80-

o 1 82 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 9.3 min). H

NMR (DMSO) δ 1.17-1.27 (m, 2H, H3a′, H5a′), 1.87 (bd, 2H, J = 12.2 Hz, H3e′, H5e′),

1.97-2.02 (m, 1H, H4 ′), 2.37 (d, 2H, J = 7.2 Hz, NC H2CH), 2.66 (t, 2H, J = 6.1 Hz, H4 ′′),

2.75 (t, J = 5.5 Hz, H3 ′′), 3.09 (bs, 2H, H2a′, H6a′), 3.51 (s, 2H, H1 ′′), 3.74 (s, 3H, OCH 3),

3.75 (s, 3H, OCH 3), 4.32 (bs, 2H, H2e′, H6e′), 6.66 (s, 1H, H5 ′′), 6.69 (s, 1H, H8 ′′), 7.45-

7.46 (m, 2H, H5, H6), 7.70 (s, 1H, H3), 7.94-7.97 (m, 1H, H4), 8.02-8.04 (m, 1H, H7). 13 C

NMR (DMSO) δ 29.0 (C4 ′′), 31.4 (C3 ′, C5 ′), 34.0 (C4 ′), 45.6 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.4

(C6 ′′OCH3, C7 ′′OCH3) 56.5 (N CH2CH), 64.2 (C1 ′′), 111.2 (C5 ′′), 113.0 (C8 ′′), 123.2 (C3),

125.5 (C7), 125.52 (C4), 125.53 (C5), 126.4 (C6), 126.9 (C4a ′′), 127.7 (C8a ′′), 138.1

(C3a), 139.4 (C2), 140.0 (C7a), 147.9 (C6 ′′), 148.1 (C7 ′′), 163.1 (CO). FTIR ν 3390 (w),

2934 (m), 2356 (s), 1630 (s), 754 (s) cm -1. MS-ES + m/z 451 (MH +, 100%); HRMS-EI calculated for C 26 H30 N2O3S: 450.1977, found 450.1974.

Mark Ashford, PhD Thesis 2010 127 Chapter 8 - Experimental

(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)(quinolin-

3-yl)methanone [39]

O The title compound [39] was prepared as an 4a'' 1' H3CO 3 4a 3'' N orange solid (330 mg, 48%) from quinoline- N H3CO 8a'' 4' N 8a 1'' 1 2-carboxylic acid (268 mg, 1.55 mmol),

HOBt (209 mg, 1.55 mmol), EDC (357 mg, 1.86 mmol), 1,2,3,4-tetrahydro-6,7-dimethoxy-

2-((piperidine-4-yl)methyl)isoquinoline [34] (450 mg, 1.55 mmol) and DIPEA (0.5 mL,

3.10 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 70-72 oC. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 4.4 min). H NMR

(DMSO) δ 1.19-1.29 (m, 2H, H3a′, H5a′), 1.85 (bd, 2H, J = 12.3 Hz, H3e′, H5e′), 1.96-2.00

(m, 1H, H4 ′), 2.38 (d, 2H, J = 7.1 Hz, NC H2CH), 2.66 (t, 2H, J = 5.9 Hz, H4 ′′), 2.75 (t, 2H,

J = 5.4 Hz, H3 ′′), 3.07 (bs, 2H, H2a′, H6a′), 3.50 (s, 2H, H1 ′′), 3.739 (s, 3H, OCH 3), 3.742

(s, 3H, OCH 3), 4.51 (bs, 2H, H2e′, H6e′), 6.66 (s, 1H, H5 ′′), 6.69 (s, 1H, H8 ′′), 7.69-7.73

(m, 1H, H6), 7.85-7.89 (m, 1H, H7), 8.10 (m, 2H, H2, H4), 8.44 (d, 1H, J = 2.1 Hz, H5),

8.92 (d, 1H, J = 2.1 Hz, H8). 13 C NMR (DMSO) δ 29.0 (C4 ′′), 31.2 (C3 ′, C5 ′), 34.0 (C4 ′),

47.5 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.43 (C6 ′′OCH3, C7 ′′OCH3), 56.46 (N CH2CH), 64.2 (C1 ′′),

111.2 (C5 ′′), 113.0 (C8 ′′), 126.9 (C4a ′′), 127.4 (C3), 127.6 (C8a ′′), 128.0 (C6), 129.3 (C8),

129.5 (C5), 130.2 (C7), 131.2 (C4a), 134.8 (C4), 147.9 (C6 ′′), 148.1 (C7 ′′), 148.3 (C8a),

149.2 (C2), 167.4 (CO). FTIR ν 3231 (w), 2935 (m), 2361 (m), 1615 (s), 1254 (s), 760 (s)

-1 + + + cm . MS-ES m/z 446 (MH , 100%); HRMS-ES calculated for C 27 H31 N3O3: 446.2444, found 446.2454.

Mark Ashford, PhD Thesis 2010 128 Chapter 8 - Experimental

(5-Bromo-1H-indol-2-yl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [61]

O The title compound [61] was prepared as H 1' H3CO 4a'' 2 N 3'' N 7a white solid (140 mg, 27%) from 5- N H3CO 8a'' 4' 3a 1'' 5 bromoindole-2-carboxylic acid (240 mg, Br 1.00 mmol), HOBt (135 mg, 1.00 mmol), EDC (230 mg, 1.20 mmol), 1,2,3,4-tetrahydro-

6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (290 mg, 1.00 mmol) and

DIPEA (0.2 mL, 2.00 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp

o 212-214 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 19.6 min). 1H NMR (DMSO) δ 1.15-1.26 (m, 2H, H3a′, H5a′), 1.89 (bd, 2H, J = 12.3 Hz, H3e′,

H5e′), 1.98-2.03 (m, 1H, H4 ′), 2.38 (d, 2H, J = 7.1 Hz, NC H2CH), 2.67 (t, 2H, J = 6.3 Hz,

H4 ′′), 2.76 (t, 2H, J = 5.6 Hz, H3 ′′), 3.09 (bs, 2H, H2a′, H6a′), 3.51 (s, 2H, H1 ′′), 3.74 (s,

3H, OCH 3), 3.75 (s, 3H, OCH 3), 4.42 (bs, 2H, H2e′, H6e′), 6.66 (s, 1H, H5 ′′), 6.70 (s, 1H,

H8 ′′), 6.76 (s, 1H, H3), 7.31 (dd, 1H, J = 8.7, 1.9 Hz, H6), 7.42 (d, 1H, J = 8.6 Hz, H7),

7.82 (d, 1H, J = 1.9 Hz, H4). 13 C NMR (DMSO) δ 29.0 (C4 ′′), 31.4 (C3 ′, C5 ′), 34.1 (C4 ′),

46.0 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.45 (C6 ′′OCH3, C7 ′′OCH3), 56.46 (N CH2CH), 64.2 (C1 ′′),

103.5 (C3), 111.2 (C5 ′′), 112.8 (CBr), 113.0 (C8 ′′), 114.7 (C7), 124.1 (C6), 126.3 (C4),

126.9 (C4a ′′), 127.7 (C8a ′′), 129.5 (C3a), 132.7 (C2), 135.3 (C7a), 147.9 (C6 ′′), 148.1

(C7 ′′), 162.2 (CO). FTIR ν 3222 (w), 2930 (m), 2351 (s), 1598 (s), 1138 (s), 798 (s) cm -1.

+ + + 79 MS-ES m/z 511.8 (MH , 75), 230 (100%); HRMS-ES calculated for C26 H30 BrN3O3:

512.1549, found 512.1532.

Mark Ashford, PhD Thesis 2010 129 Chapter 8 - Experimental

(5-Bromobenzofuran-2-yl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [60]

O The title compound [60] was prepared as H CO 4a'' 1' 2 3 3'' O 7a N white solid (220 mg, 40%) from 5- N 3 H3CO 8a'' 4' 1'' 5 bromobenzo[ b]furan-2-carboxylic acid [43] Br (260 mg, 1.07 mmol), HOBt (145 mg, 1.07 mmol), EDC (245 mg, 1.28 mmol), 1,2,3,4- tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (310 mg, 1.07 mmol) and DIPEA (0.35 mL, 2.13 mmol) in anhydrous DMF (10 mL) using coupling

o Method B, mp 75-78 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 15.6 min). 1H NMR (DMSO) δ 1.15-1.23 (m, 2H, H3a′, H5a′), 1.84-1.88 (m, 2H,

H3e′, H5e′), 1.97-2.01 (m, 1H, H4 ′), 2.35 (d, 2H, J = 7.1 Hz, NC H2CH), 2.64 (t, 2H, J =

6.2 Hz, H4 ′′), 2.73 (t, J = 5.6 Hz, H3 ′′), 3.06 (m, 2H, H2a′, H6a′), 3.49 (s, 2H, H1 ′′), 3.717

(s, 3H, OCH 3), 3.723 (s, 3H, OCH 3), 4.29 (m, 2H, H2e′, H6e′), 6.63 (s, 1H, H5 ′′ ), 6.67 (s,

1H, H8 ′′), 7.31 (s, 1H, H3), 7.56-7.59 (m, 1H, H6), 7.65 (d, 1H, J = 1.9 Hz, H4), 7.95 (d,

1H, J = 7.9 Hz, H7). 13 C NMR (DMSO) δ 29.0 (C4 ′′), 31.6 (C3 ′, C5 ′), 34.0 (C4 ′), 48.2

(C2 ′, C6 ′), 51.8 (C3 ′′), 56.44 (OCH 3), 56.46 (OCH 3), 56.47 (N CH2CH), 64.1 (C1 ′′), 110.0

(C3), 111.2 (C5 ′′), 113.0 (C8 ′′), 114.5 (C7), 116.6 (CBr), 125.4 (C4), 126.9 (C4a ′′), 127.7

(C8a ′′), 129.6 (C6), 129.8 (C3a), 147.9 (C6 ′′), 148.1 (C7 ′′), 150.8 (C2), 153.5 (C7a), 159.2

(CO). FTIR ν 2919 (w), 2361 (m), 1634 (s), 1517 (s), 1437 (s), 1255 (s) cm -1. MS-EI m/z

+ 79 512 (M , 7), 206 (100%); HRMS-EI calculated for C26 H29 BrN2O4: 512.1311, found

512.1309.

Mark Ashford, PhD Thesis 2010 130 Chapter 8 - Experimental

(4-Bromo-3-methylphenyl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [62]

O The title compound [62] was prepared as 1' H3CO 4a'' 1 CH3 3'' N white solid (310 mg, 55%) from 4-bromo- N H3CO 8a'' 4' 4 Br 1'' 3-methylbenzoic acid (250 mg, 1.15 mmol),

HOBt (155 mg, 1.15 mmol), EDC (265 mg, 1.38 mmol), 1,2,3,4-tetrahydro-6,7-dimethoxy-

2-((piperidine-4-yl)methyl)isoquinoline [34] (333 mg, 1.15 mmol) and DIPEA (0.4 mL,

2.30 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 50-51 oC. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 10.4 min). H NMR

(DMSO) δ 1.09-1.19 (m, 2H, H3a′, H5a′), 1.77-1.84 (m, 2H, H3e′, H5e′), 1.92-1.98 (m, 1H,

H4 ′), 2.35 (d, 2H, J = 7.1 Hz, NC H2CH), 2.41 (s, 3H, CH 3), 2.65 (t, 2H, J = 6.0 Hz, H4 ′′),

2.74 (t, J = 5.6 Hz, H3 ′′), 2.93–3.04 (m, 2H, H2a′, H6a′), 3.50 (s, 2H, H1 ′′), 3.73 (s, 3H,

OCH 3), 3.74 (s, 3H, OCH 3), 4.05 (bs, 2H, H2e′, H6e′), 6.65 (s,1H, H5 ′′), 6.69 (s, 1H, H8 ′′),

7.15 (dd, 1H, J = 8.2, 2.1 Hz, H2), 7.38 (s, 1H, H6), 7.66 (d, 1H, J = 8.1 Hz, H3). 13 C

NMR (DMSO) δ 22.9 (CH 3), 29.0 (C4 ′), 31.2 (C3, C5), 34.0 (C4), 47.5 (C2, C6), 51.7

(C3 ′), 56.36 (2 x OCH 3), 56.38 (NCH 2), 64.2 (C1 ′), 111.1 (C5 ′), 113.0 (C8 ′), 125.6 (CBr),

126.6 (C5 ′′ ), 126.9 (C4a ′), 127.7 (C8a ′), 130.0 (C2 ′′ ), 132.8 (C4 ′′ ), 136.8 (C1 ′′ ), 138.3

(C3 ′′ ), 147.8 (C6 ′), 148.1 (C7 ′), 168.6 (CO). FTIR ν 3226 (w), 2914 (s), 2361 (m), 1516 (s),

1254 (s), 753 (s) cm -1. MS-ES + m/z 488 (MH +, 100%); HRMS-EI calculated for

79 C25 H31 BrN2O3: 487.1596, found 487.1581.

Mark Ashford, PhD Thesis 2010 131 Chapter 8 - Experimental

(5-Bromo-2,3-dimethoxyphenyl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [63]

[63] O OCH3 The title compound was prepared as 4a'' 1' H3CO 3'' 1 OCH3 N white solid (250 mg, 41%) from 5-bromo- N H CO 4' 3 8a'' 1'' 5 Br 3,4-dimethoxy-carboxylic acid [47] (300 mg, 1.15 mmol), HOBt (155 mg, 1.15 mmol), EDC (265 mg, 1.38 mmol), 1,2,3,4- tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (333 mg, 1.15 mmol) and DIPEA (0.4 mL, 2.30 mmol) in anhydrous DMF (10 mL) using coupling

o Method B, mp 51-52 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 7.7 min). 1H NMR (DMSO) δ 1.09-1.19 (m, 2H, H3a′, H5a′), 1.68-1.71 (bd, 2H, J =

12.4 Hz, H3e′, H5e′), 1.90-1.96 (m, 1H, H4 ′), 2.35 (d, 2H, J = 8.0 Hz, NC H2CH), 2.64 (t,

2H, J = 5.4 Hz, H4 ′′), 2.74 (t, J = 5.6 Hz, H3 ′′ ), 3.05 (bs, 2H, H2a′, H6a′), 3.49 (s, 2H,

H1 ′′), 3.737 (s, 3H, OCH 3), 3.742 (s, 3H, OCH 3), 3.75 (s, 3H, OCH 3), 3.89 (s, 3H, OCH 3),

4.51 (bs, 2H, H2e′, H6e′), 6.65 (s, 1H, H5 ′′), 6.68 (s, 1H, H8 ′′), 6.94-6.98 (m, 1H, H4), 7.27

(s, 1H, H6). 13 C NMR (DMSO) δ 29.0 (C4 ′′), 30.8 (C3 ′, C5 ′), 34.0 (C4 ′), 47.3 (C2 ′, C6 ′),

51.8 (C3 ′′), 56.43 (OCH 3), 56.45 (OCH 3), 56.47 (OCH 3, N CH2CH), 57.1 (OCH 3), 64.2

(C1 ′′), 111.2 (C5 ′′), 113.0 (C8 ′′), 116.5 (CBr), 117.2 (C4), 121.4 (C1), 121.7 (C6), 126.9

(C4a ′′), 127.7 (C8a ′′), 133.8 (C2), 147.9 (C6 ′′), 148.1 (C7 ′′), 154.1 (C3), 165.1 (CO). FTIR

ν 3222 (w), 2924 (m), 2356 (m), 1633 (s), 1463 (s), 1256 (s), 845 (s) cm -1. MS-ES + m/z

+ 79 534 (MH , 100%); HRMS-EI calculated for C 26 H33 BrN2O5: 533.1651, found 533.1683.

Mark Ashford, PhD Thesis 2010 132 Chapter 8 - Experimental

(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1- yl)(naphthalen-1-yl)methanone [41]

The title compound [41] was prepared as O 1' 8a H3CO 4a'' 3'' N 1 4a white solid (180 mg, 35%) from 1-naphthyl N H CO 2 3 8a'' 1'' 4' carboxylic acid (200 mg, 1.15 mmol), HOBt

(155 mg, 1.15 mmol), EDC (265 mg, 1.38 mmol), 1,2,3,4-tetrahydro-6,7-dimethoxy-2-

((piperidine-4-yl)methyl)isoquinoline [33] (333 mg, 1.15 mmol) and DIPEA (0.4 mL, 2.30 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 58-60 oC. HPLC analysis

1 (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 7.3 min). H NMR (DMSO) δ

1.10-1.30 (m, 2H, H3a′, H5a′), 1.60-1.70 (m, 1H, H4 ′), 1.90-1.97 (m, 2H, H3e′, H5e′), 2.36

(m, 2H, NC H2CH), 2.64 (t, 2H, J = 5.7 Hz, H4 ′′), 2.73 (t, J = 5.6 Hz, H3 ′′), 2.93-3.10 (m,

2H, H2a′, H6a′), 3.48 (s, 2H, H1 ′′), 3.732 (s, 3H, OCH 3), 3.736 (s, 3H, OCH 3), 4.69 (bs,

2H, H2e′, H6e′), 6.64 (s, 1H, H5 ′′), 6.68 (s, 1H, H8 ′′), 7.40-7.50 (m, 1H, H6), 7.56-7.63 (m,

3H, H3, H7, H5), 7.76-7.83 (m, 1H, H2), 7.97-8.03 (m, 2H, H4, H8). 13 C NMR (DMSO) δ

29.0 (C4 ′′), 31.1 (C3 ′, C5 ′), 34.1 (C4 ′), 47.6 (C2 ′, C6 ′), 51.8 (C3 ′′), 56.46 (OCH 3), 56.49

(OCH 3), 56.5 (N CH2CH), 64.2 (C1 ′′), 111.2 (C5 ′′), 113.0 (C8 ′′), 124.1 (C2), 125.4 (C3),

126.1 (C6), 126.9 (C4a ′′), 127.1 (C7), 127.6 (C5, C8), 127.7 (C8a ′′), 129.1 (C4), 129.3

(C8a), 133.8 (C4), 135.7 (C1), 147.9 (C6 ′′), 148.2 (C7 ′′), 168.6 (CO). FTIR ν 2914 (w),

2366 (m), 1629 (s), 1517 (s), 781 (s) cm -1. MS-ES + m/z 445 (MH +, 100%); HRMS-ES + calculated for C 28 H32 N2O3: 445.2491, found 445.2488.

Mark Ashford, PhD Thesis 2010 133 Chapter 8 - Experimental

(3a,7a-Dihydro-1H-indol-3-yl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [42]

O The title compound [42] was prepared as an H CO 4a'' 1' 3 3 3'' N 7 off-white solid (175 mg, 39%) from indole- N 2 NH H3CO 4' 8'' 1'' 3-carboxylic acid (368 mg, 2.27 mmol),

HOBt (306 mg, 2.27 mmol), EDC (523 mg, 2.73 mmol), 1,2,3,4-tetrahydro-6,7-dimethoxy-

2-((piperidine-4-yl)methyl)isoquinoline [34] (660 mg, 2.27 mmol) and DIPEA (0.8 mL,

4.55 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 206-207 oC. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 5.7 min). H NMR

(DMSO) δ 1.13–1.23 (m, 2H, H3a′, H5a′), 1.81–1.84 (m, 2H, H3e′, H5e′), 1.95-2.03 (m,

1H, H4 ′), 2.37 (d, 2H, J = 6.2 Hz, NC H2CH), 2.66 (t, 2H, J = 5.9 Hz, H4 ′′), 2.74 (t, 2H, J =

5.5 Hz, H3 ′′), 2.98-3.04 (m, 2H, H2a′, H6a′), 3.51 (s, 2H, H1 ′′), 3.74 (s, 6H, 2 x OCH 3),

4.29-4.33 (m, 2H, H2e′, H6e′), 6.66 (s, 1H, H5 ′′), 6.69 (s, 1H, H8 ′′), 7.13 (t, 1H, J = 7.1 Hz,

H5), 7.19 (t, 1H, J = 7.1 Hz, H6), 7.47 (d, 1H, J = 8.1 Hz, H4), 7.64 (d, 1H, J = 2.4 Hz,

H2), 7.69 (d, 1H, J = 7.8 Hz, H7). 13 C NMR (DMSO) δ 28.1 (C4 ′′), 30.7 (C3 ′, C5 ′), 33.3

(C4 ′), 44.4 (C2 ′, C6 ′), 50.9 (C3 ′′), 55.52 (OCH 3), 55.54 (OCH 3), 56.9 (N CH2CH), 63.5

(C1 ′′), 110.3 (C7), 110.4 (C3), 111.7 (C5 ′′), 112.1 (C8 ′′), 119.8 (C4 ′), 119.9 (C6), 121.5

(C5), 125.8 (C3a), 126.0 (C2), 126.8 (C4a ′′), 127.1 (C8a ′′), 135.5 (C7a), 147.0 (C6 ′′), 147.2

(C7 ′′), 165.3 (CO). FTIR ν 3308 (w), 2924 (m), 2356 (m), 1593 (s), 1518 (s), 1453 (s), 752

-1 + + + (s) cm . MS-ES m/z 434 (MH , 100%); HRMS-ES calculated for C 26H31 N3O3:

434.2454, found 434.2444.

Mark Ashford, PhD Thesis 2010 134 Chapter 8 - Experimental

(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)(1 H-indol-

6-yl)methanone [40]

O The title compound [40] was prepared as a H H CO 4a'' 1' 6 7a 3 3'' N N 2 light brown solid (188 mg, 42%) from N H CO 3 1'' 4' 3a indole-6-carboxylic acid (368 mg, 2.27 mmol), HOBt (306 mg, 2.27 mmol), EDC (523 mg, 2.73 mmol), 1,2,3,4-tetrahydro-6,7- dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (660 mg, 2.27 mmol) and DIPEA

(0.8 mL, 4.55 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 210-213 o 1 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 5.7 min). H

NMR (DMSO) δ 1.13-1.23 (m, 2H, H3a′, H5a′), 1.80-1.83 (m, 2H, H3e′, H5e′), 1.92-1.97

(m, 1H, H4 ′), 2.38 (d, 2H, J = 7.2 Hz, NC H2CH), 2.66 (t, 2H, J = 6.2 Hz, H4 ′′), 2.75 (t, 2H,

J = 5.6 Hz, H3 ′′), 2.94-3.00 (m, 2H, H2a′, H6a′), 3.51 (s, 2H, H1 ′′), 3.74 (s, 3H, OCH 3),

3.75 (s, 3H, OCH 3), 4.15-4.24 (m, 2H, H2e′, H6e′), 6.51 (d, 1H, J = 2.9 Hz, H3), 6.66 (s,

1H, H5 ′′), 6.69 (s, 1H, H8 ′′), 7.05 (dd, 1H, J = 8.1 Hz, 1.3 Hz, H5), 7.47 (m, 2H, H4, H7 ),

7.61 (s, 1H, H2). 13 C NMR (DMSO) δ 30.0 (C4 ′′), 32.4 (C3 ′, C5 ′), 35.0 (C4 ′), 44.6 (C2 ′,

C6 ′), 52.7 (C3 ′′), 57.1 (OCH 3), 57.3 (OCH 3), 57.6 (N CH2CH), 65.4 (C1 ′′), 102.8, (C3),

111.7 (C5 ′′), 112.2 (C8 ′′), 113.4 (C7), 119.6 (C5), 121.3 (C4), 127.6 (C4a ′′), 128.4 (C8a ′′),

128.7 (C2), 130.0 (C6), 130.6 (C3a), 136.7 (C7a), 148.5 (C6 ′′), 148.8 (C7 ′′), 171.9 (CO).

FTIR ν 3236 (w), 2924 (m), 1623 (s), 1521 (s), 1254 (s), 858 (s) cm -1. MS-ES + m/z 434

+ + (MH , 100%); HRMS-ES calculated for C 26 H31 N3O3: 434.2444, found 434.2450.

Mark Ashford, PhD Thesis 2010 135 Chapter 8 - Experimental

(5-Bromo-1H-indol-3-yl)(4-((3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)methyl)piperidin-1-yl)methanone [64]

Br The title compound [64] was prepared as an O H CO 4a'' 1' 3a 6 3 3'' N off-white solid (200 mg, 38%) from 5- N 2 N 7a H3CO 4' bromoindole-3-carboxylic acid [48] (368 1'' H mg, 2.27 mmol), HOBt (306 mg, 2.27 mmol), EDC (523 mg, 2.73 mmol), 1,2,3,4- tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (660 mg, 2.27 mmol) and DIPEA (0.8 mL, 4.55 mmol) in anhydrous DMF (10 mL) using coupling

o Method B, mp 215-217 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 35:65, retention time 11.6 min). 1H NMR (DMSO) δ 1.07-1.18 (m, 2H, H3a′, H5a′), 1.78 (bd, 2H,

J = 10.9 Hz, H3e′, H5e′), 1.89-1.94 (m, 1H, H4 ′), 2.32 (d, 2H, J = 7.1 Hz, NC H2CH), 2.61

(t, 2H, J = 6.1 Hz, H4 ′′), 2.71 (t, 2H, J = 5.3 Hz, H3 ′′), 2.98 (bt, 2H, J = 11.4 Hz, H2a′,

H6a′), 3.46 (s, 2H, H1 ′′), 3.700 (s, 3H, OCH 3), 3.703 (s, 3H, OCH 3), 4.26 (bd, 2H, J = 11.1

Hz, H2e′, H6e′), 6.61 (s, 1H, H5 ′′), 6.65 (s, 1H, H8 ′′), 7.26 (dd, 1H, J = 8.6, 1.9 Hz, H6),

7.40 (d, 1H, J = 8.6 Hz, H7), 7.68 (s, 1H, H2), 7.82 (d, 1H, J = 1.9 Hz, H4). 13 C NMR

(DMSO) δ 28.1 (C4 ′′), 30.6 (C3 ′, C5 ′), 33.3 (C4 ′), 44.4 (C2 ′, C6 ′), 50.9 (C3 ′′), 55.5

(OCH 3), 55.6 (OCH 3), 56.7 (N CH2CH) 63.4 (C1 ′′), 109.7 (C3), 110.2 (C5 ′′), 112.0 (C7),

112.6 (C8 ′′), 113.7 (CBr), 122.3 (C4), 124.2 (C6), 126.0 (C3a), 126.7 (C2), 128.0 (C4a ′′),

128.5 (C8a ′′), 134.3 (C7a), 146.9 (C6 ′′), 147.2 (C7 ′′), 164.5 (CO). FTIR ν 3303 (w), 2919

(m), 2335 (M), 1588 (s), 1516 (s), 806 (s) cm -1. MS-EI m/z 512 (M +, 100%); HRMS-EI

79 calculated for C 26 H31 N3O3 Br: 512.1549, found 512.1542.

Mark Ashford, PhD Thesis 2010 136 Chapter 8 - Experimental

(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)(5- iodobenzofuran-2-yl)methanone [65]

O The title compound [65] was prepared as a 4a'' 1' H CO 2 O 3 3'' N 7a white foam (257 mg, 46%) from 5- N 6 H CO 4' 3 1'' 3a I iodobenzo[ b]furan-2-carboxylic acid [44] (300 mg, 1.04 mmol), HOBt (170 mg, 1.25 mmol), EDC (140 mg, 1.25 mmol), 1,2,3,4- tetrahydro-6,7-dimethoxy-2-((piperidine-4-yl)methyl)isoquinoline [34] (301 mg, 1.04 mmol) and DIPEA (0.4 mL, 2.08 mmol) in anhydrous DMF (10 mL) using coupling

o Method B, mp 91-92 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 50:50, retention

1 time 30.1 min). H NMR (CDCl 3) δ 1.27-1.34 (m, 2H, H3a′, H5a′), 1.93-2.00 (m, 3H, H3e′,

H5e′, H4 ′), 2.40 (d, 2H, J = 6.4 Hz, NC H2CH), 2.51 (m, 2H, H2a′, H6a′), 2.70 (t, 2H, J =

6.0 Hz, H4 ′′), 2.82 (t, 2H, J = 6.0 Hz, H3 ′′), 3.55 (s, 2H, H1 ′′), 3.83 (s, 3H, OCH 3), 3.84 (s,

3H, OCH 3), 4.48 (m, 2H, H2e′, H6e′), 6.52 (s, 1H, H5 ′′), 6.60 (s, 1H, H8 ′′ ), 7.14 (s, 1H,

H3) 7.28 (d, 1H, J = 8.4 Hz, H7), 7.64 (dd, 1H, J = 8.8, 2.0 Hz, H6), 7.97 (d, 1H, J = 2.0

13 Hz, H4). C NMR (CDCl 3) δ 28.8 (C4 ′′), 32.1 (C3 ′, C5 ′), 34.3 (C4 ′) , 44.1 (C2 ′, C6 ′), 51.6

(C3 ′′), 56.10 (OCH 3), 56.11 (OCH 3), 56.5 (N CH2CH), 64.1 (C1 ′′ ), 87.1 (CI), 109.7 (C4),

110.2 (C5 ′′), 111.6 (C8′′ ), 114.0 (C3), 126.4 (C6), 126.8 (C4a ′′), 129.9 (C8a ′′ ), 131.1 (C7),

135.0 (C3a), 147.4 (C6 ′′), 147.7 (C7 ′′), 150.3 (C2), 154.0 (C7a), 159.5 (CO). FTIR ν 2930

(w), 2352 (m), 1643 (s), 1520 (s), 760 (s) cm -1. MS-EI m/z 512.2 (M +, 100%); HRMS-EI calculated for C 26 H29 N2O4I: 560.4236, found 560.4240.

Mark Ashford, PhD Thesis 2010 137 Chapter 8 - Experimental

8.3. Experimental procedures for region 2 modification

8.3.1. Intermediate Synthesis

4(2-Hydroxyethyl)-piperidine-1-carboxylic acid tert-butyl ester 85 [84]

OH To a solution of 4-piperidine ethanol [82] (2.51 g, 19.35 mmol) in 5%

NaHCO 3:CHCl 3 (40 mL, 1:1) was added di-tert -butyl dicarbonate (4.65 g, 3 21.29 mmol). The mixture was heated at reflux for 16 h, cooled and 6 N 1

O O(CH3)3 extracted with CH 2Cl 2 (3 x 75 mL). The combined organic layers were dried (Na 2SO 4), and the organic solvent removed. The residue was distilled under reduced

1 pressure to yield [84] (4.13 g, 93%) as a clear oil. H NMR (CDCl 3): δ 1.08-1.12 (m, 2H,

H3a, H5a), 1.43 (s, 9H, O(CH3)3), 1.47-1.67 (m, 5H, H3e, H5e, H4, CH 2), 2.67 (t, 2H, J =

12.2 Hz, H2a, H6a), 3.68 (t, 2H, J = 6.5 Hz, CH 2OH), 4.04-4.09 (m, 2H, H2e, H6e). MS-EI m/z 229 (M +, 4), 128 (100%).

4-(2-Iodoethyl)piperidine-1-carboxylic acid tert-butyl ester 86 [86]

I To a solution of Boc protected amine [84] (900 mg, 3.92 mmol) in toluene

(30 mL) was added imidazole (374 mg, 5.49 mmol), PPh 3 (1.07 g, 4.08 3

6 mmol) and iodine (995 mg, 3.92 mmol). The resulting mixture was heated N1 o O O(CH3)3 at 80 C for 2.5 h, allowed to cool to rt, filtered and concentrated. The residue was subjected to column chromatography (EtOAc:PE, 1:9) and dried in vacuo to

1 yield [86] (730 mg, 56%) as a clear oil. H NMR (CDCl 3) δ 1.07-1.15 (m, 2H, H3a, H5a),

1.44 (s, 9H, O(CH3)3), 1.57-1.66 (m, 3H, H4, H3e, H5e), 1.76 (q, 2H, J = 7.1 Hz, C H2CH),

2.69 (bt, 2H, J = 12.6 Hz, H2a, H6a), 3.20 (t, 2H, J = 7.2 Hz, CH 2I), 4.07-4.12 (m, 2H,

H2e, H6e). MS-EI m/z 339 (M +, 8), 156 (100%).

Mark Ashford, PhD Thesis 2010 138 Chapter 8 - Experimental

4-Piperidylpropanol acetate 84 [83]

HO To a solution of 4-pyridylpropanol [96] (15.06 g, 110.02 mmol) in AcOH

(110 mL) was added 10% Pd/C (2.01 g). The suspension was subjected to a 3 hydrogen atmosphere at 60 psi for 48 h. After this time, the solution was 6 N 1 H filtered through celite and the filter cake washed with AcOH (50 mL). The combined organic layers were concentrated to yield [83] (33.04 g, 97%) as a clear oil. 1H

NMR (CDCl 3) δ 1.36-1.61 (m, 6H, H3a, H5a, H3e, H5e, H4), 1.82 (bd, 2H, J = 12.2 Hz,

H2a, H6a), 2.0 (s, HOAc), 2.81 (t, 2H, J = 7.0 Hz, CHC H2), 3.35 (bd, 2H, J = 12.2 Hz,

+ H2e, H6e), 3.63 (t, 2H, J = 7.0 Hz, CH2OH), 9.41 (bs, 1H, NH). MS-EI m/z 143 (M , 100

%).

4-(3-Hydroxypropyl)piperidine-1-carboxylic acid tert-butyl ester 84 [85]

HO To a solution of 4-piperidylpropanol acetate [83] (16.04 g, 111.5

mmol) in dioxane (100 mL) was added 3M NaOH (50 mL) and di-tert - 3 butyl dicarbonate (24.36 g, 111.5 mmol). The solution was allowed to 6 N 1 stir for 18 h at rt. The organic solvent was removed and the aqueous O O(CH3)3 solution was extracted with Et 2O (3 x 50 mL). The combined organic layers were washed with sat. NaHCO 3 (20 mL), brine (20 mL) and dried (Na 2SO 4). The organic solvent was removed and the residue subjected to column chromatography (EtOAc:PE, 1:9-1:1) and

1 dried in vacuo to yield [85] (17.52 g, 64%) as a clear oil. H NMR (CDCl 3) δ 1.06-1.12 (m,

2H, H3a, H5a), 1.30-1.40 (m, 3H, CH2CH), 1.45 (s, 9H, O(CH3)3), 1.51-1.62 (m, 2H, H3e,

H5e), 1.65 (bd, 2H, CH 2CH2CH 2), 2.65 (m, 2H, H2a, H6a), 3.61 (t, 2H, J = 7.0 Hz,

+ + HOC H2), 4.06 (bd, 2H, J = 12.5 Hz, H2e, H6e). MS-ES m/z 244 (MH , 27), 229 (100 %).

Mark Ashford, PhD Thesis 2010 139 Chapter 8 - Experimental

4-(3-Iodoethyl)piperidine-1-carboxylic acid tert-butyl ester 84 [87]

I To a solution of Boc protected amine [85] (10.13 g, 41.51 mmol) in

toluene (400 mL) was added PPh 3 (14.15 g, 53.96 mmol), imidazole 3 (8.47 g, 124.53 mmol) and iodine (13.65 g, 53.96 mmol) at 0 oC. The 6 N 1 solution was allowed to warm to rt and stirred for a further 3 h. The O O(CH3)3 organic solvent was removed, the residue taken up in EtOAc (200 mL) and then washed with sat. Na 2CO 3 solution (50 mL) and brine (50 mL). The organic layer was dried

(MgSO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE, 3:7) and dried in vacuo to yield [87] (8.62 g, 59%) as a clear

1 oil. H NMR δ 1.05-1.13 (m, 2H, H3a, H5a), 1.29-1.38 (m, 3H, CH2CH) 1.43 (s, 9H,

O(CH3)3), 1.58-1.63 (m, 2H, H3e, H5e), 1.78-1.86 (m, 2H, CH 2CH2CH 2), 2.64 (bt, J =

12.2 Hz, H2a, H6a), 3.16 (t, 2H, J = 7.0 Hz, IC H2), 4.02 (bs, 2H, H2e, H6e). MS-EI m/z

353 (M +, 12), 143 (100%).

tert-Butylpiperazine-1-carboxylate 87 [97]

To a solution of hydrated piperazine [96] (10.03 g, 51.5 mmol) in 4 NH 1 isopropanol (50 mL) was added a solution of di-tert -butyldicarbonate (3.74 O N

O(CH3)3 g, 17.2 mmol) in isopropanol (20 mL). The solution was allowed to stir for

5 h at rt. After this time, a solution of succinic acid (4.23 g, 34.9 mmol) in isopropanol (25 mL) was added and the resulting precipitate was stirred for 30 min. The precipitate was filtered, and the solute concentrated. The residue was dissolved in CH 2Cl 2 (100 mL), washed with 10% Na 2CO 3 (50 mL) and dried (Na 2SO 4). The solvent was removed to yield

o 1 [97] (2.84 g, 89%) as a white solid, mp 42-44 C. H NMR (CDCl 3) δ 1.44 (s, 9H,

13 O(CH 3)3), 2.17 (s, 1H, H3a), 2.80 (s, 3H, H3e, H5), 3.37 (d, 4H, J = 5.0 Hz, H2, H6). C

NMR δ 28.5 (C CH3)3), 44.1 (C3, C5), 45.9 (C2, C6), 79.6 (( CCH3)3), 154.8 (CO). MS-EI m/z 186 (M +, 10), 86 (100%).

Mark Ashford, PhD Thesis 2010 140 Chapter 8 - Experimental

4-(3-Bromopropyl)piperazine-1-carboxylic acid tert-butyl ester [98]

Nitrogen alkylation Method C:88 To a solution of tert - N Br

O N butylpiperazine-1-carboxylate [97] (1.02 g, 5.01 mmol) and Et 3N

O(CH3)3 o (0.77 mL, 5.5 mmol) in CH 2Cl 2 (15 mL) at 0 C was added 1,3- dibromopropane (0.56 mL, 5.5 mmol) dropwise over 15 min. The reaction was allowed to warm to rt and stirred for a further 18 h. The solution was diluted with EtOAc (100 mL) and washed with brine (20 mL), sat. NaHCO 3 (20 mL) and H 2O (20 mL). The organic layer was dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE, 7:3) to yield [98] (320 mg, 20%) as a yellow oil. 1H NMR

(CDCl 3) 1.42 (s, 9H, C(CH3)3), 1.99 (dt, 2H, J = 6.8 Hz, CH 2CH2CH 2), 2.34 (m, 4H, H3,

H5), 2.44 (t, 2H, J = 6.8 Hz, NCH 2), 3.37 (m, 4H, H2, H6), 3.42 (t, 2H, J = 6.8 Hz,

+ + CH 2Br). MS-ES m/z 307 (MH , 100%).

Nitrogen alkylation Method B: To a solution of 1,3-dibromopropane (3.03 g, 15.03 mmol) in anhydrous acetone (150 mL) was added K 2CO 3 (2.07 g, 15.03 mmol) and Boc protected amine [97] (1.02 g, 5.01 mmol) and the solution was heated at reflux for 18 h.

After this time, additional 1,3-dibromopropane (2.00 mL) and K 2CO 3 (2.01 g) was added and the mixture was heated at reflux for a further 18 h. The solution was allowed to cool to rt, filtered and the organic solvent removed. The residue was taken up in H 2O (100 mL) and extracted with Et 2O (3 x 20 mL). The combined organic layers were then dried

(Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE, 3:7) to yield [98] (1.25 g, 82%) as a cream solid, mp 92-96 oC that was spectroscopically identical to previously reported.

Mark Ashford, PhD Thesis 2010 141 Chapter 8 - Experimental

1-Chloro-3-(4-benzylpiperazin-1-yl)propane 90 [103]

To a solution of 1-benzylpiperazine (5.02 g, 28.37 mmol) N Cl N and 1-bromo-3-chloropropane (5.61 mL, 56.74 mmol) in 2- butanone (75 mL) was added K 2CO 3 (5.88 g, 42.56 mmol) and the mixture was stirred at reflux for 4 h. The solution was allowed to cool, filtered and the filtrate evaporated. The residue was taken up in Et 2O (100 mL) and washed with H 2O (2 x 25 mL). The organic layer was dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE:NH 4 solution, 9:1:5 drops) and dried in vacuo to yield

1 †† [103] (4.32 g, 60%) as a clear oil. H NMR (CDCl 3) δ 1.89 (dt, 2H, J = 6.8 Hz,

CH 2CH2CH 2), 2.43 (m, 10H, J = 7.2 Hz, 4 x pip CH 2, NCH 2), 3.46 (s, 2H, CH 2Ph), 3.53 (t,

13 2H, J = 6.4 Hz, ClCH 2), 7.20-7.27 (m, 5H, Ph). C NMR δ 30.1 (CH 2), 43.4 (ClCH 2), 53.2

(Pip CH 2), 53.4 (Pip CH 2), 55.6 (NCH 2), 63.2 (CH 2Ph), 127.1 (C4), 128.3 (C3, C5), 129.3

(C2, C6), 138.3 (C1). MS-ES + m/z 253 (MH +, 12), 217 (100%).

tert -Butyl-4-(2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)ethyl)piperidine-1- carboxylate [88]

4' The title compound [88] (546 mg, 62%) was H3CO 4 N 2 prepared as a clear oil from 6,7-dimethoxy-1,2,3,4- H3CO 1' N Boc tetrahydroisoquinoline [32] (500 mg, 2.18 mmol), 4-

(2-iodoethyl)piperidine-1-carboxylic acid tert -butyl ester [86] (740 mg, 2.18 mmol),

K2CO 3 (1.20 g, 8.72 mmol) and TBAI (35 mg, 0.09 mmol) in DMF (15 mL) using the

1 nitrogen alkylation Method B. H NMR (CDCl 3) δ 1.12-1.24 (m, 2H, H3a, H5a), 1.45 (s,

9H, O(CH 3)3), 1.49-1.70 (m, 5H, H3e, H5e, H4, NCH 2CH2CH), 2.52 (t, 2H, J = 8.0 Hz,

†† Ref 90 reports only mass spectral data for compound [100]

Mark Ashford, PhD Thesis 2010 142 Chapter 8 - Experimental

NC H2CH 2CH), 2.66-2.71 (m, 2H, H2a, H6a), 2.81 (t, 2H, J = 5.8 Hz, H3 ′), 3.53 (s, 2H,

H1 ′), 3.83 (s, 3H, OCH 3), 3.84 (s, 3H, OCH 3), 4.06-4.15 (m, 2H, H2e′, H6e′), 6.51 (s, 1H,

13 H5 ′), 6.59 (s, 1H, H8 ′). C NMR δ 28.6 (C(CH3)3), 28.7 (C4 ′), 32.4 (NCH 2CH2), 34.0,

(C3, C5), 34.5 (C4), 44.2 (C2, C6), 51.2 (C3 ′), 55.8 (N CH2CH 2), 56.0 (OCH 3), 56.1

(OCH 3), 64.3 (C1 ′), 79.4 ( C(CH 3)3), 109.7 (C5 ′), 111.6 (C8 ′), 126.3 (C4a ′), 126.9 (C8a ′),

147.4 (C6 ′), 147.5 (C7 ′), 156.0 (CO). MS-EI m/z 404 (M +, 8), 206 (100%); HRMS-EI calculated for C 23 H36 N2O4: 404.2675, found 404.2672.

1,2,3,4-Tetrahydro-6,7-dimethoxy-2-(2-(piperidin-4-yl)ethyl)isoquinoline [89]

5 The title compound [89] was prepared as a cream solid H3CO 3 N 4' (820 mg, 83%) from tert -butyl-4-(2-(3,4-dihydro-6,7- H3CO 1 NH 1' dimethoxyisoquinolin-2(1 H)-yl)ethyl)piperidine-1- carboxylate [88] (1.05 g, 2.59 mmol) in CH 2Cl 2:TFA (2:1, 20 mL) using the nitrogen

o 1 deprotection Method A , mp 102-103 C. H NMR (CDCl 3) δ 1.16-1.54 (m, 5H, H3a′, H5a′,

NCH 2CH2CH), 1.69-1.78 (m, 2H, H3e′, H5e′), 2.50-2.69 (m, 6H, NC H2CH 2,, Ha2, Ha6,

H4), 2.80 (bs, 2H, H3), 3.06 (bd, 2H, J = 9.8 Hz, H2e′, H6e′), 3.53 (s, 2H, H1), 3.82 (s, 6H,

13 2 x OCH 3), 6.52 (s, 1H, H5), 6.58 (s, 1H, H8). C NMR δ 29.0 (C4 ′), 33.7

(NCH 2CH2CH), 34.8 (C4), 34.9 (C3 ′, C5 ′), 46.8 (C2 ′, C6 ′), 51.4 (C3), 56.0 (2 x OCH 3),

56.20 (C1), 56.22 (N CH2CH 2CH), 109.8 (C5), 111.7 (C8), 126.5 (C4a), 127.0 (C8a), 147.5

+ + (C6), 147.8 (C7). MS-ES m/z 305 (MH , 100%); HRMS-EI calculated for C 18 H28 N2O2:

304.2151, found 304.1391.

Mark Ashford, PhD Thesis 2010 143 Chapter 8 - Experimental tert -Butyl-4-(3-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)propyl)piperidine-1- carboxylate [89]

4' The title compound [89] (703 mg, 59%) was H CO 1 Boc 3 N N prepared as a clear oil that solidified upon H CO 4 3 8' 1' standing from 6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline hydrochloride [32] (650 mg, 2.83 mmol), 4-(3-iodoethyl)- piperidine-1-carboxylic acid tert -butyl ester [87] (1.02 g, 2.83 mmol), K 2CO 3 (1.56 g,

11.32 mmol), and TBAI (30 mg, 0.11 mmol) in DMF (20 mL) using the nitrogen

o 1 alkylation Method B , mp 86-89 C. H NMR (CDCl 3) δ 1.06-1.09 (m, 2H, H3a, H5a), 1.25-

1.29 (m, 2H, NCH 2CH2CH2CH), 1.30-1.44 (m, 1H, H4), 1.42 (s, 9H, O(CH 3)3), 1.58-1.67

(m, 4H, H3e, H5e, NCH 2CH2CH2CH), 2.46 (t, 2H, J = 7.9 Hz, NCH 2), 2.65-2.69 (m, 4H,

H2a, H6a, H4 ′), 2.80 (t, 2H, J = 8.0 Hz, H3 ′), 3.52 (s, 2H, H1 ′), 3.81 (s, 3H, OCH 3), 3.82

13 (s, 3H, OCH 3), 4.06-4.11 (m, 2H, H2e, H6e), 6.50 (s, 1H, H5 ′), 6.57 (s, 1H, H8 ′). C NMR

(CDCl 3) δ 24.5 (C4), 28.6 (NCH 2CH2CH 2CH), 28.7 (NCH 2CH 2CH2CH), 32.3 (C3, C5),

34.5 (C4 ′), 44.1 (C2, C6), 51.2 (C3 ′), 55.9 (C1 ′), 56.01 (OCH 3), 56.04 (OCH 3), 58.6

(NCH 2), 79.3 ( C(CH 3)3), 109.6 (C5 ′), 111.5 (C8 ′), 126.3 (C4a ′), 126.7 (C8a ′), 147.3 (C6 ′),

147.7 (C7 ′), 155.0 (CO). MS-ES + m/z 419 (MH +, 35), 265 (100%); HRMS-ES + calculated for C 24 H39 N2O4: 419.2910, found 419.2919

1,2,3,4-Tetrahydro-6,7-dimethoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [91]

4 1' The title compound [91] was prepared as a clear oil H CO 6 3 NH N (500 mg, 94%) from tert -butyl-4-(3-(3,4-dihydro- H3CO 4' 1 6,7-dimethoxyisoquinolin-2(1 H)- yl)propyl)piperidine-1-carboxylate [89] (700 mg, 1.67 mmol) in CH 2Cl 2:TFA (2:1, 15 mL)

1 using the nitrogen deprotection Method A . H NMR (CDCl 3) δ 1.12-1.30 (m, 5H, H3a′,

H5a′, H4 ′, NCH 2CH2CH2CH), 1.56-1.71 (m, 4H, H3e′, H5e′, NCH 2CH2CH2CH), 2.45 (t,

Mark Ashford, PhD Thesis 2010 144 Chapter 8 - Experimental

2H, J = 7.7 Hz, NC H2CH 2CH 2CH), 2.58-2.62 (m, 2H, H2a′, H6a′), 2.67 (t, 2H, J = 6.1 Hz,

H4), 2.80 (t, 2H, J = 5.8 Hz, H3), 3.08 (bd, 2H, J = 12.0 Hz, H2e′, H6e′), 3.51 (s, 2H, H1),

13 3.810 (s, 3H, OCH 3), 3.813 (s, 3H, OCH 3), 6.49 (s, 1H, H5), 6.57 (s, 1H, H8). C NMR

(CDCl 3) δ 24.3 (C4 ′), 28.7 (NCH 2CH2CH 2CH), 32.8 (NCH 2CH 2CH2CH), 34.8 (C3 ′, C5 ′),

35.9 (C4), 46.2 (C2 ′, C6 ′), 51.1 (C3), 55.88 (2 x OCH 3), 55.92 (NCH2CH 2CH 2CH), 58.6

(C1), 109.6 (C5), 111.4 (C8), 126.3 (C4a), 126.7 (C8a), 147.2 (C6), 147.5 (C7). MS-EI m/z

+ 318 (M , 26), 206 (100%); HRMS-EI calculated for C 19 H30 N2O2: 318.2307, found

318.2305.

2-(3-(4-Benzylpiperazin-1-yl)propyl)-1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline

[100]

4 H CO 4' 1'' The title compound [100] was prepared as 3 3 N N N H CO 4'' a clear oil (280 mg, 13%) from 1,2,3,4- 3 8 1 1' tetrahydroisoquinoline hydrochloride [32] (1.22 g, 5.30 mmol), K 2CO 3 (2.94 g, 21.80 mmol), chlorobenzylpiperazine [103] (2.01 g, 7.95 mmol), KI (9 mg, 0.053 mmol) and

TBAI (80 mg, 0.212 mmol) in DMF (20 mL) using the nitrogen alkylation Method B. 1H

NMR (CDCl 3) δ 1.79 (m, 2H, NCH 2CH2CH 2Pip), 2.40-2.54 (m, 10H, pip,

NCH 2CH 2CH2Pip), 2.70 (t, 2H, J = 6.0 Hz, H4), 2.81 (t, 2H, J = 5.6 Hz, H3), 3.47-3.54

(m, 6H, NCH2Ph, NC H2CH 2CH2Pip, H1), 3.826 (s, 3H, OCH 3), 3.83 (s, 3H, OCH 3), 6.51

13 (s, 1H, H5), 6.58 (s, 1H, H8), 7.26-7.32 (m, 5H, Ph). C NMR (CDCl 3) δ 24.8

(NCH 2CH2CH 2Pip), 28.7 (C4), 51.1 (C3), 53.2 (Pip), 53.4 (Pip), 56.0 (OCH 3), 56.04

(OCH 3), 56.1 (NCH2CH2CH 2Pip), 56.5 (NCH2CH2CH2Pip), 56.9 (C1), 63.2 (NCH2Ph),

109.7 (C5), 111.5 (C8), 126.3 (C4a), 126.8 (C8a), 127.1 (H4 ′′ ), 128.3 (H3 ′′ , H5 ′′ ), 129.4

(H2 ′′ , H6 ′′ ), 138.3 (H1 ′′ ), 147.3 (C6), 147.7 (C7). MS-ES + m/z 410 (MH +, 2), 242 (100%).

+ HRMS-ES calculated for C 25 H36 N3O2: 410.2808, found 410.2814.

Mark Ashford, PhD Thesis 2010 145 Chapter 8 - Experimental

Nitrogen alkylation Method 3: To a solution of 1,2,3,4-tetrahydroisoquinoline hydrochloride [32] (2.06 g, 8.30 mmol) and K 2CO 3 (5.03 g, 35.00 mmol) in ACN (80 mL) was added chlorobenzylpiperazine (2.12 g, 8.3 mmol), KI (9.1 mg, 0.053 mmol) and TBAI

(80 mg, 0.212 mmol). The solution was heated at reflux for 16 h and then the solvent was removed. The residue taken up in EtOAc (100 mL) and washed with H 2O (3 x 20 mL). The organic layer was dried (Na 2SO4) and the organic solvent removed. The residue was subjected to column chromatography (CH 2Cl 2:MeOH, 9:1) to yield [100] (2.54 g, 75%) as a clear oil, which was spectroscopically identical to that reported here.

tert -Butyl-4-(3-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)propyl)piperazine-1- carboxylate [99]

The title compound [99] (310 mg, 82%) was H CO 5' 4' 1 Boc 3 N N N prepared as a brown solid from 4-(3- H3CO 4 8' 1' bromopropyl)piperazine-1-carboxylic acid tert -butyl ester [98] (340 mg, 01.16 mmol), 6,7- dimethoxy-1,2,3,4-tetrahydroisoquinoline [32] (266 mg, 1.16 mmol), K 2CO 3 (641 mg, 4.64 mmol), TBAI (17 mg, 0.046 mmol) and KI (2 mg, 0.012 mmol) in DMF (20 mL) using the

o 1 nitrogen alkylation Method B , mp 115-117 C. H NMR (CDCl 3) δ 1.44 (s, 9H, O(CH 3)3),

2.78 (m, 2H, NCH 2CH2CH 2N), 2.37-2.41 (m, 6H, H3, H5, NCH 2CH 2CH2Pip), 2.53 (t, 2H,

J = 6.2 Hz, NC H2CH 2CH2Pip), 2.70 (t, 2H, J = 5.6 Hz, H4 ′), 2.81 (t, 2H, J = 5.6 Hz, H3 ′),

3.42 (m, 4H, H2, H6), 3.55 (s, 2H, H1 ′), 3.81 (s, 3H, OCH 3), 3.82 (s, 3H, OCH 3), 6.50 (s,

13 1H, H5 ′), 6.57 (s, 1H, H8′). C NMR (CDCl 3) δ 24.7 (NCH 2CH2CH 2Pip), 28.6 (C4 ′), 51.1

(C3 ′), 53.2 (H3, H5), 55.9 (H2, H6), 56.01 (OCH 3), 56.04 (OCH 3), 56.3 (NCH2CH2CH2N),

56.8 (C1 ′), 79.7 ( C(CH 3)3), 109.6 (C5 ′), 111.5 (C8 ′), 126.2 (C4a ′), 126.6 (C8a ′), 147.4

(C6 ′), 147.7 (C7 ′), 154.9 (CO). MS-EI m/z 419 (M +, 14), 192 (100%); HRMS-ES + calculated for C 23 H38 N3O4: 420.2862, found 420.2845.

Mark Ashford, PhD Thesis 2010 146 Chapter 8 - Experimental

1,2,3,4-Tetrahydro-6,7-dimethoxy-2-(3-(piperazin-1-yl)propyl)isoquinoline [101]

To a suspension of 2-(3-(4-benzylpiperazin-1- H CO 5 4 1' 3 3 NH N N yl)propyl)-1,2,3,4-tetrahydro-6,7- H CO 4' 3 8 1 dimethoxyisoquinoline [100] (2.00 g, 4.88 mmol) and 10%Pd/C (2.00 g) in anhydrous MeOH (20 mL) was added anhydrous ammonium formate (1.55 g, 24.44 mmol) under N 2 and the reaction was heated at reflux for 15 min.

The solution was allowed to cool to rt and the organic solvent was removed. The residue was taken up in CHCl 3 (100 mL) and washed with H 2O (2 x 20 mL). The organic layer was dried (Na 2SO 4), filtered and the organic solvent removed to yield [101] (1.41 g, 91%) as a

1 yellow oil. H NMR (CDCl 3) δ 1.75-1.82 (m, 2H, NCH 2CH2CH 2Pip), 2.38-2.55 (m, 10 H,

Pip, NCH2CH2CH2Pip), 2.70 (t, 2H, J = 5.6 Hz, H4), 2.81 (t, 2H, J = 6.0 Hz, H3) 2.90 (t,

2H, J = 4.9 Hz, NC H2CH2CH2Pip), 3.55 (s, 2H, H1), 3.825 (s, 3H, OCH 3), 3.832 (s, 3H,

13 OCH 3), 6.51 (s, 1H, H5), 6.58 (s, 1H, H8). C NMR (CDCl 3) δ 24.7 (NCH 2CH2CH 2Pip),

28.8 (C4), 46.2 (C3 ′, C5 ′), 51.1 (C3), 54.8 (C2 ′, C6 ′), 56.01 (OCH 3), 56.10 (OCH 3), 56.13

(NCH2CH2CH2Pip), 56.5 (NCH2CH2CH2Pip), 57.5 (C1), 109.7 (C5), 111.6 (C8), 126.4

(C4a), 126.8 (C8a), 147.4 (C6), 147.7 (C7). MS-ES + m/z 320 (MH +, 100%); HRMS-ES + calculated for C 18 H30 N3O2: 320.2284 , found 320.2291.

5-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentanenitrile [68]

The title compound [68] (2.06 g, 83%) was prepared 5' H3CO 4 as a clear oil from 5-bromovaleronitrile (1.01 mL, N 1 H3CO 1' N 8.71 mmol), 6,7-dimethoxy-1,2,3,4- tetrahydroisoquinoline hydrochloride [32] (2.01 g, 8.71 mmol), K 2CO 3 (4.81 g, 34.82 mmol), TBAI (125 mg, 0.348 mmol) and KI (14 mg, 0.087 mmol) in DMF (20 mL) using

1 the nitrogen alkylation Method B. H NMR (CDCl 3) δ 1.73-1.77 (m, 4H, H4, H3), 2.39 (t,

2H, J = 6.6 Hz, H5), 2.53 (t, 2H, J = 6.6 Hz, H2), 2.69 (t, 2H, J = 6.0 Hz, H4 ′), 2.81 (t, 2H,

Mark Ashford, PhD Thesis 2010 147 Chapter 8 - Experimental

J = 5.8 Hz, H3 ′), 3.54 (s, 2H, H1 ′), 3.83 (s, 3H, OCH 3), 3.84 (s, 3H, OCH 3), 6.52 (s, 1H,

13 H5 ′), 6.59 (s, 1H, H8 ′). C NMR (CDCl 3) δ 17.2 (C2), 23.5 (C3), 26.0 (C4), 28.6 (C4 ′),

51.0 (C3 ′), 55.8 (C5), 55.99 (OCH 3), 56.02 (OCH 3) 57.0 (C1 ′), 109.6 (C5 ′), 111.5 (C8 ′),

119.8 (CN), 126.2 (C8a ′), 126.4 (C4a ′), 147.4 (C7 ′), 147.6 (C6 ′). MS-EI m/z 274 (M +, 33),

206 (100%); HRMS-EI calculated for C 16 H22 N2O2: 274.3206, found 274.3210.

4-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butanenitrile 41 [67]

4' The title compound [67] was prepared as a white solid H3CO N N (1.93 g, 85%) from bromobutylnitrile (0.86 mL, 8.71 H CO 3 1' 4 1 mmol), 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride [32] (2.06 g, 8.71 mmol), K 2CO 3 (4.79 g, 34.82 mmol), TBAI (123 mg,

0.348 mmol) and KI (10 mg, 0.087 mmol) in DMF (20 mL) using the nitrogen alkylation

o 1 Method B , mp 106-108 C. H NMR (CDCl 3) δ 2.47 (t, 2H, J = 7.1 Hz, H3), 2.62 (t, 2H, J

= 6.7 Hz, H4), 2.70 (t, 2H, J = 6.1 Hz, H4 ′), 2.81 (t, 2H, J = 5.8 Hz, H3 ′), 3.54 (s, 2H, H1 ′),

3.83 (s, 3H, OCH 3), 3.84 (s, 3H, OCH 3), 6.51 (s, 1H, H5 ′), 6.59 (s, 1H, H8 ′). MS-EI m/z

260 (M +, 42), 164 (100%).

6-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexanenitrile [69]

H3CO 6' The title compound [66] was prepared as a red oil N N (2.06 g, 86%) from bromohexanenitrile (1.15 mL, H3CO 1 1' 4 8.71 mmol), 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride [32] (2.02 g, 8.71 mmol), K 2CO 3 (4.84 g, 34.82 mmol), TBAI (129 mg, 0.348 mmol) and KI (12 mg, 0.087

1 mmol) in DMF (20 mL) using the nitrogen alkylation Method B . H NMR (CDCl 3) δ 1.73-

1.77 (m, 4H, H3, H4), 2.40-2.42 (m, 2H, H2), 2.52-2.55 (m, 2H, H6), 2.69 (t, 2H, J = 6.0

Hz, H4 ′), 2.81 (t, 2H, J = 5.8 Hz, H3 ′), 3.54 (s, 2H, H1 ′), 3.83 (s, 3H, OCH 3), 3.84 (s, 3H,

13 OCH 3), 6.52 (s, 1H, H5 ′), 6.59 (s, 1H, H8 ′). C NMR (CDCl 3) δ 17.2 (C2), 23.5 (C3), 25.9

Mark Ashford, PhD Thesis 2010 148 Chapter 8 - Experimental

(C4), 26.0 (C4′), 28.6 (C5), 51.0 (C6), 52.3 (C3 ′), 55.7 (OCH 3), 56.0 (OCH 3), 57.0 (C1 ′),

109.6 (C5 ′), 111.5 (C8 ′), 119.8 (C4a ′), 126.2 (CN), 126.4 (C8a ′), 147.4 (C6 ′), 147.7 (C7 ′).

+ MS-EI m/z 288 (M , 2), 206 (100%); HRMS-EI calculated for C 17 H24 N2O2: 288.6578, found 288.6583.

General Method of reduction for alkyl nitriles

4-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butan-1-amine [70]

41 4' Reduction Method A: To a suspension of LiAlH 4 H3CO 1 N (2.13 g, 56.0 mmol) in dry THF (50 mL) was added 4- H CO NH 3 1' 4 2 (3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)butanenitrile [67] (3.07 g, 11.20 mmol) in dry THF (25 mL) dropwise under a stream of

N2. The resulting mixture was heated at reflux for 18 h under N 2. To the cooled solution at

o 0 C was added iced H 2O (5 mL) and 10% NaOH (15 mL). The solution was warmed to rt and allowed to stir for 15 min. The resulting suspension was filtered through celite and the filter cake washed with EtOAc (50 mL). The organic layer was dried (Na 2SO 4) and solvent

1 removed to yield [70] (1.92 g, 65%) as a yellow oil. H NMR (CDCl 3) δ 1.49 (m, 2H, H3),

1.62 (m, 2H, H2), 2.49 (t, 2H, J = 8.0 Hz, H4), 2.71 (m, 4H, H1, H4 ′), 2.80 (t, 2H, J = 5.6

Hz, H3 ′), 3.53 (s, 2H, H1 ′), 3.81 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.50 (s, 1H, H5 ′), 6.57

13 (s, 1H, H8 ′). C NMR (CDCl 3) δ 25.0 (C3), 27.2 (C4 ′), 30.6 (C2), 41.0 (C1), 53.1 (C3 ′),

54.9 (C4), 56.0 (OCH 3), 56.1 (OCH 3), 58.9 (C1 ′) 110.9 (C5 ′), 112.5 (C8 ′), 126.1 (C8a ′),

129.1 (C4a ′), 147.2 (C7 ′), 148.8 (C6 ′). MS-ES + m/z 265.1 (MH +, 100%); HRMS calculated for C 15 H24 N2O2: 264.1838, found 264.1832.

Mark Ashford, PhD Thesis 2010 149 Chapter 8 - Experimental

5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentan-1-amine [71]

The title compound [71] was prepared as a yellow H3CO 3' N NH H CO 2 oil (1.94 g, 62%) from LiAlH 4 (2.13 g, 56.0 mmol) 3 1' 3 1 and 5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentanenitrile [68] (3.07 g, 11.20

1 mmol) in anhydrous THF (75 mL) using the reduction Method A. H NMR (CDCl 3) δ 1.23-

1.62 (m, 6H, H2, H3, H4), 2.45-2.49 (m, 2H, H5), 2.67-2.71 (m, 4H, H4 ′, H1), 2.80 (t, 2H,

J = 5.9 Hz, H3 ′), 3.52 (s, 2H, H1 ′), 3.805 (s, 3H, OCH 3), 3.81 (s, 3H, OCH 3), 6.50 (s, 1H,

13 H5 ′), 6.56 (s, 1H, H8 ′). C NMR (CDCl 3) δ 25.0 (C3), 27.2 (C4 ′), 28.7 (C4), 33.6 (C2),

42.0 (C1), 51.2 (C3 ′), 55.9 (C5), 56.0 (OCH 3), 56.02 (OCH 3), 58.4 (C1 ′), 109.6 (C5 ′),

111.5 (C8 ′), 126.3 (C4a ′), 126.8 (C8a ′), 147.3 (C6 ′), 147.6 (C7 ′). MS-ES + m/z 279 (MH +,

41), 181 (100%); HRMS-EI calculated for C 16 H26 N2O2: 278.1994, found 278.1977.

6-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexan-1-amine [72]

5' 4' The title compound [72] was prepared as a yellow H3CO 3' 5 N 1 oil (2.19 g, 68%) from LiAlH 4 (2.10 g, 55.3 H CO NH 3 8' 1' 4 2 2 mmol) and 6-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexanenitrile [69] (3.19 g,

1 11.06 mmol) in anhydrous THF (75 mL) using the reduction Method A. H NMR (CDCl 3)

δ 1.22-1.46 (m, 8H, H2, H3, H4, H5), 1.58 (bs, 2H, NH 2), 2.48 (t, 2H, J = 7.8 Hz, H6),

2.60-2.66 (m, 4H, H4 ′, H1), 2.76 (t, 2H, J = 5.6 Hz, H3 ′), 3.48 (s, 2H, H1 ′), 3.77 (s, 3H,

13 OCH 3), 3.78 (s, 3H, OCH 3), 6.51 (s, 1H, H5 ′), 6.57 (s, 1H, H8 ′). C NMR (CDCl 3) δ 26.9

(C4), 27.2 (C4 ′), 27.5 (C3), 28.7 (C2), 33.5 (C5), 42.0 (C1), 51.1 (C3 ′), 55.8 (C6), 55.9

(OCH 3), 56.0 (OCH 3), 58.4 (C1 ′), 110.6 (C5 ′), 111.9 (C8 ′), 126.8 (C4a ′), 127.1 (C8a ′),

147.6 (C6 ′), 147.9 (C7 ′). MS-ES + m/z 293 (MH +, 50), 188 (100%); HRMS-EI calculated for C 17H28N2O2: 292.2151, found 292.2144.

Mark Ashford, PhD Thesis 2010 150 Chapter 8 - Experimental

8.3.2. Synthesis of Final Compounds with Region 2 modification

5-Bromo-N-(5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentyl)-1-benzofuran-

2-carboxamide [74]

The title compound [74] was prepared Br 4'' H3CO 3a as a white solid (210 mg, 23%) from 5- H N N 2 7 H3CO O bromobenzo[ b]furan-2-carboxylic acid 8'' 1'' 3' 1' O [43] (433 mg, 1.80 mmol), HOBt (243 mg, 1.80 mmol), EDC (412 mg, 2.15 mmol), 5-(3,4-dihydro-6,7-dimethoxyisoquinolin-

2(1 H)-yl)pentan-1-amine [71] (502 mg, 1.80 mmol) and DIPEA (0.42 mL, 3.46 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 96-98 oC. HPLC analysis (0.1M

1 PO 4 buffer pH 7.5: MeOH, 25:75, retention time 5.7 min). H NMR (CDCl 3) δ 1.45-1.49

(m, 2H, H3 ′), 1.65-1.71 (m, 4H, H2 ′, H4 ′), 2.53 (t, 2H, J = 7.5 Hz, H5 ′), 2.71 (t, 2H, J = 6.1

Hz, H4 ′′), 2.81 (t, 2H, J = 5.6 Hz, H3 ′′), 3.50 (apparent q‡‡ , 2H, J = 6.3 Hz, H1 ′), 3.55 (s,

2H, H1 ′′), 3.826 (s, 3H, OCH 3), 3.832 (s, 3H, OCH 3), 6.51 (s, 1H, H5 ′′), 6.57 (s, 1H, H8 ′′),

6.66 (bt, 1H, J = 3.9 Hz, NH), 7.36 (m, 2H, H3, H7), 7.49 (dd, 1H, J = 8.8, 2.0 Hz, H6),

13 7.80 (d, 1H, J = 2.0 Hz, H4). C NMR (CDCl 3) δ 26.7 (C3 ′), 27.3 (C4 ′′), 27.8 (C4 ′), 29.6

(C2 ′), 39.5 (C1 ′), 50.7 (C3 ′′), 55.2 (C5 ′), 56.01 (OCH 3), 56.04 (OCH 3), 57.6 (C1 ′′ ), 109.5

(C3), 109.6 (H7), 111.4 (C5 ′′), 113.4 (C8 ′′), 116.9 (CBr), 125.2 (C4), 125.4 (C4a ′′), 129.6

(H6), 129.9 (C8a ′′ ), 131.2 (C3a), 147.5 (C6 ′′ ), 147.9 (C7 ′′), 150.1 (C2), 153.5 (C7a), 158.5

+ + + 79 (CO). MS-ES m/z 502 (MH , 100%); HRMS-ES calculated for C25 H28 N2O4 Br:

502.5639 found 502.5643.

‡‡ This is likely to be a dt, however, the outer peaks could not been seen

Mark Ashford, PhD Thesis 2010 151 Chapter 8 - Experimental

5-Bromo-N-(6-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexyl)-1-benzofuran-

2-carboxamide [75]

The title compound [75] was 4'' H CO 3 O 5' 1' 1 prepared as a white solid (310 mg, N O H3CO2' N 8'' 1'' H 3 6 35%) from 5-bromobenzo[ b]furan-2- 4 Br carboxylic acid [43] (412 mg, 1.71 mmol), HOBt (231 mg, 1.71 mmol), EDC (394 mg, 2.05 mmol), 6-(3,4-dihydro-6,7- dimethoxyisoquinolin-2(1 H)-yl)hexan-1-amine [72] (502 mg, 1.71 mmol) and DIPEA

(0.46 mL, 3.41 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 110-111 o 1 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time 6.9 min). H

NMR (CDCl 3) δ 1.39-1.46 (m, 4H, H3 ′, H4 ′), 1.61-1.68 (m, 4H, H2 ′, H5 ′), 2.48-2.54 (m,

2H, H6 ′), 2.70 (t, 2H, J = 6.1 Hz, H4 ′′), 2.81 (t, 2H, J = 5.8 Hz, H3 ′′), 3.47 (apparent q§§ ,

2H, J = 7.0 Hz, H1 ′), 3.54 (s, 2H, H1 ′′), 3.82 (s, 3H, OCH 3), 3.83 (s, 3H, OCH 3), 6.50 (s,

1H, H5 ′′), 6.58 (s, 1H, H8 ′′), 6.62 (t, 1H, J = 5.3 Hz, NH), 7.38 (m, 2H, H3, H7), 7.49 (dd,

13 1H, J = 8.8, 2.0 Hz, H6), 7.80 (d, 1H, J = 1.9 Hz, H4). C NMR (CDCl 3) δ 26.6 (C3 ′),

27.1 (C4 ′′), 26.8 (C4 ′), 27.8 (C2 ′), 29.6 (C5 ′), 39.5 (C6 ′), 50.7 (C3 ′′), 55.2 (C1 ′), 56.01

(OCH 3), 56.04 (OCH 3), 57.6 (C1 ′′ ), 109.5 (C3), 109.6 (H7), 111.4 (C5 ′′), 113.4 (C8 ′′),

116.9 (CBr), 125.2 (C4), 125.4 (C4a ′′ ), 129.6 (H6), 129.9 (C8a ′′ ), 131.2 (C3a), 147.5

(C6 ′′), 147.9 (C7 ′′), 150.1 (C2), 153.5 (C7a), 158.5 (CO). MS-ES - m/z 513 (M-1, 100%);

- 79 HRMS-ES calculated for C 26 H30 N2O4 Br: 513.1389, found 513.1375.

§§ This is likely to be a dt, however, the outer peaks could not been seen

Mark Ashford, PhD Thesis 2010 152 Chapter 8 - Experimental

(4-(3-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)propyl)piperidin-1-yl)(5- iodobenzofuran-2-yl)methanone [95]

O The title compound [95] was prepared 5'' 4'' H3CO 1' 2 O N as a white solid (321 mg, 43%) from 5- N 3 6 H3CO 1'' 4' 4 iodobenzo[ b]furan-2-carboxylic acid I [44] (363 mg, 1.26 mmol), HOBt (170 mg, 1.26 mmol), EDC (290 mg, 1.51 mmol),

1,2,3,4-tetrahydro-6,7-dimethoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [91] (400 mg,

1.26 mmol) and DIPEA (0.44 mL, 2.51 mmol) in anhydrous DMF (10 mL) using coupling

o Method B, mp 162-163 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75,

1 retention time 12.3 min). H NMR (CDCl 3) δ 1.23-1.29 (m, 4H, H3a′, H5a′,

NCH 2CH2CH 2CH), 1.62-1.66 (m, 1H, H4 ′), 1.82 (m, 4H, NCH 2CH2CH2CH, H3e′, H5e′),

2.87-3.00 (m, 4H, H4 ′′, H3 ′′), 2.82 (m, 2H, NC H2CH2CH2CH), 3.20 (bs, 2H, H2a′, H6a′),

3.83 (s, 3H, OCH 3), 3.85 (s, 3H, OCH 3), 4.06 (s, 2H, H1 ′′), 4.50 (bd, 2H, H2e′, H6e′), 6.52

(s, 1H, H5 ′′), 6.61 (s, 1H, H8 ′′), 7.14 (s, 1H, H3), 7.28 (d, 1H, J = 1.7 Hz, H7), 7.63 (dd,

13 1H, J = 8.7, 1.8 Hz, H6), 7.96 (d, 1H, J = 1.7 Hz, H4). C NMR (CDCl 3) δ 25.5 (C4 ′),

29.8 (NCH 2CH2CH 2CH), 32.9 (C3 ′, C5 ′), 33.6 (C4 ′′), 35.6 (NCH 2CH2CH2CH), 43.6 (C2 ′,

C6 ′), 49.6 (C3 ′′), 53.4 (NCH2CH2CH 2CH), 56.09 (OCH 3), 56.14 (OCH 3), 60.5 (C1 ′′ ), 87.1

(CI), 109.5 (C3), 110.2 (C5 ′′), 111.4 (C8 ′′), 114.0 (C7), 124.3 (C8a ′′), 129.8 (C4a ′′), 129.9

(C4), 131.1 (C3a), 135.0 (C6), 147.9 (C6 ′′), 148.3 (C7 ′′ ), 150.2 (C2), 154.0 (C7a), 159.4

+ (CO). MS-EI m/z 588 (M , 42), 341 (100%); HRMS-EI calculated for C 28 H33 N2O4I:

588.1301, found 588.1289.

Mark Ashford, PhD Thesis 2010 153 Chapter 8 - Experimental

5-Iodo -N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)butyl)-1-benzofuran-2- carboxamide [76]

The title compound [76] was prepared as a H CO 5'' 3 3'' O 3' 1' N 2 O white wax (380 mg, 47%) from 5- H3CO N 1'' H 3 6 iodobenzo[ b]furan-2-carboxylic acid [44] 4 I (435 mg, 1.51 mmol), HOBt (204 mg,

1.51 mmol), EDC (350 mg, 1.81 mmol), 4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)- yl)butan-1-amine [70] (400 mg, 1.51 mmol) and DIPEA (0.73 mL, 3.02 mmol) in anhydrous DMF (10 mL) using coupling Method B. HPLC analysis (0.1M PO 4 buffer pH

1 7.5: MeOH, 50:50, retention time 21.1 min) H NMR (CDCl 3) δ 1.73-1.74 (m, 4H, H2 ′,

H3 ′), 2.55 (m, 2H, H1 ′), 2.71 (t, 2H, J = 6.0 Hz, H4 ′′), 2.82 (t, 2H, J = 5.6 Hz, H3 ′′), 3.50

(m, 2H, H4 ′), 3.55 (s, 2H, H1 ′′), 3.79 (s, 3H, OCH 3), 3.82 (s, 3H, OCH 3), 6.47 (s, 1H, H5 ′′),

6.56 (s, 1H, H8 ′′ ), 7.05 (d, 1H, J = 8.7 Hz, H7), 7.23 (s, 1H, H3), 7.38 (m, 1H, NH), 7.59

13 (d, 1H, 8.7 Hz, H6), 7.90 (s, 1H, H4). C NMR (CDCl 3) δ 24.9 (C2 ′), 27.4 (C3 ′), 28.6

(C4 ′′), 39.5 (C1 ′), 50.7 (C3 ′′, C4 ′), 55.99 (OCH 3), 56.02 (OCH 3), 57.5 (C1 ′′ ), 87.2 (CI),

108.9 (C3), 109.6 (C5 ′′), 111.5 (C8 ′′), 113.8 (C7), 126.2 (C8a ′′ ), 126.5 (C4), 130.2 (C4a ′′),

131.4 (C3a), 135.2 (C6), 147.4 (C6 ′′), 147.7 (C7 ′′ ), 149.8 (C2), 154.0 (C7a), 158.5 (CO).

+ + MS-ES m/z 535 (MH , 100%); HRMS-EI calculated for C 24 H27 N2O4I: 534.1016, found

534.1011.

5-Iodo -N-(5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentyl)-1-benzofuran-2- carboxamide [77]

The title compound [77] was 6 H3CO 3'' I prepared as a brown wax (230 mg, H O 2' N 4' N 4 H CO 2 3 8'' 1'' 1' 3 30%) from 5-iodobenzo[ b]furan-2- O carboxylic acid [44] (411 mg, 1.43 mmol), HOBt (193 mg, 1.43 mmol), EDC (330 mg,

Mark Ashford, PhD Thesis 2010 154 Chapter 8 - Experimental

1.77 mmol), 5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentan-1-amine [71] (400 mg, 1.43 mmol) and DIPEA (0.70 mL, 2.86 mmol) in anhydrous DMF (10 mL) using coupling Method B. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time

1 6.4 min) H NMR (CDCl 3) δ 1.46-1.50 (m, 2H, H3 ′), 1.62-1.70 (m, 4H, H2 ′, H4 ′), 2.51 (m,

2H, H1 ′), 2.69 (t, 2H, J = 6.2 Hz, H4 ′′ ), 2.80 (t, 2H, J = 5.7 Hz, H3 ′′ ), 3.48 (t, 2H, J = 7.0

Hz, H5 ′), 3.53 (s, 2H, H1 ′′ ), 3.82 (s, 3H, OCH 3), 3.82 (s, 3H, OCH 3), 6.50 (s, 1H, H5 ′′ ),

6.56 (s, 1H, H8 ′′ ), 6.71 (m, 1H, NH), 7.22 (d, 1H, J = 8.7 Hz, H7), 7.34 (s, 1H, H3), 7.64

13 (dd, 1H, J = 8.7, 1.8 Hz, H6), 7.98 (d, 1H, J = 1.7 Hz, H4). C NMR (CDCl 3) δ 23.7 (C3 ′),

24.9 (C4 ′), 26.9 (C4 ′′ ), 28.7 (C2 ′), 39.4 (C1 ′), 51.1 (C3 ′′ ), 55.9 (C5 ′), 56.97 (OCH 3), 57.00

(OCH 3), 58.1 (C1 ′′ ), 87.2 (CI), 109.1 (C3), 109.6 (C5 ′′ ), 111.5 (C8 ′′ ), 113.7 (C7), 126.3

(C8a ′′ ), 126.7 (C4), 128.0 (C4a ′′ ), 131.5 (C3a), 135.4 (C6), 147.3 (C6 ′′ ), 147.6 (C7 ′′ ), 149.7

(C2), 154.0 (C7a), 158.4 (CO). MS-ES + m/z 549 (MH +, 100%); HRMS-ES + calculated for

C25 H30 N2O4I: 549.1250, found 549.1262.

5-Iodo -N-(6-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexyl)-1-benzofuran-2- carboxamide [78]

The title compound [78] was prepared H CO 3 3'' O 1' N 2 O as a brown wax (310 mg, 41%) from H3CO8'' 1'' 4' N H 3 5-iodobenzo[ b]furan-2-carboxylic 5 I acid [44] (390 mg, 1.36 mmol), HOBt

(183 mg, 1.36 mmol), EDC (312 mg, 1.63 mmol), 6-(3,4-dihydro-6,7- dimethoxyisoquinolin-2(1 H)-yl)hexan-1-amine [72] (400 mg, 1.36 mmol) and DIPEA

(0.65 mL, 2.72 mmol) in anhydrous DMF (10 mL) using coupling Method B. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 50:50, retention time 38.5 min) H NMR

(CDCl 3) δ 1.41 (m, 4H, H3 ′, H4 ′), 1.62 (m, 4H, H2 ′, H5 ′), 2.48 (m, 2H, H1 ′), 2.68 (t, 2H, J

= 6.1 Hz, H4 ′′), 2.80 (t, 2H, J = 5.7 Hz, H3 ′′), 3.45 (t, 2H, J = 7.0 Hz, H6 ′), 3.53 (s, 2H,

Mark Ashford, PhD Thesis 2010 155 Chapter 8 - Experimental

H1 ′′), 3.81 (s, 3H, OCH 3), 3.82 (s, 3H, OCH 3), 6.50 (s, 1H, H5 ′′), 6.57 (s, 1H, H8 ′′), 6.66

(m, 1H, NH), 7.24 (d, 1H, J = 8.7 Hz, H7), 7.35 (s, 1H, H3), 7.65 (dd, 1H, J = 8.7, 1.8 Hz,

H6), 7.98 (d, 1H, J = 1.7 Hz, H4). 13 C NMR δ 27.0 (C3 ′), 27.2 (C4 ′), 27.3 (C4 ′′), 28.7

(C2 ′), 29.7 (C5 ′), 39.5 (C1 ′), 51.1 (C3 ′′), 55.9 (C6 ′), 56.00 (OCH 3), 56.02 (OCH 3), 58.4

(C1 ′′ ), 87.3 (CI), 109.2 (C3), 109.6 (C5 ′′), 111.5 (C8 ′′), 113.8 (C7), 126.3 (C8a ′′), 126.8

(C4), 130.4 (C4a ′′), 131.6 (C3a), 135.5 (C6), 147.3 (C6 ′′), 147.6 (C7 ′′), 149.7 (C2), 154.1

(C7a), 158.4 (CO). MS-ES + m/z 563 (MH +, 100%); HRMS-ES + calculated for

C26 H32 N2O4I: 563.1407, found 563.1413.

4-(3-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)propyl)piperazin-1-yl)(5- iodobenzofuran-2-yl)methanone [102]

The title compound [102] was prepared O H CO 5'' 1' 2 O 3 N as a off-white wax (240 mg, 26%) from 2'' NN 3 H CO 4' 3 5-iodobenzo[ b]furan-2-carboxylic acid I [44] (451 mg, 1.56 mmol), HOBt (211 mg, 1.56 mmol), EDC (363 mg, 1.87 mmol),

1,2,3,4-tetrahydro-6,7-dimethoxy-2-(3-(piperazin-1-yl)propyl)isoquinoline [101] (508 mg,

1.56 mmol) and DIPEA (0.75 mL, 3.13 mmol) in anhydrous DMF (10 mL) using coupling

1 Method B. H NMR (CDCl 3) δ 1.80 (m, 2H, NCH 2CH2CH 2Pip), 2.44 (t, 2H, J = 7.6 Hz,

NC H2CH2CH 2Pip), 2.52-2.57 (m, 6H, 3 x Pip CH 2, NCH 2CH2CH2Pip), 2.71 (t, 2H, J = 6.1

Hz, H4 ′′), 2.81 (t, 2H, J = 5.7 Hz, H3 ′′), 3.55 (s, 2H, H1 ′′), 3.82-3.85 (m, 8H, 2 x OCH 3,

Pip CH 2), 6.51 (s, 1H, H5 ′′), 6.58 (s, 1H, H8 ′′ ), 7.18 (s, 1H, H3), 7.28 (d, 1H, J = 8.7 Hz,

13 H7), 7.64 (dd, 1H, J = 8.7, 1.8 Hz, H6), 7.97 (d, 1H, J = 1.8 Hz, H4). C NMR (CDCl 3) δ

24.8 (NCH 2CH2CH 2Pip), 28.8 (C4 ′′), 43.1 (C3 ′, C5 ′, 2 x Pip CH 2), 51.1 (C2 ′, C6 ′, 2 x Pip

CH 2), 53.6 (C3 ′′), 56.0 (OCH 3), 56.02 (OCH 3), 56.1 (NCH2CH2CH 2Pip), 56.3

(NCH 2CH2CH2Pip), 56.6 (C1 ′′ ), 87.2 (CI), 109.6 (C3), 110.8 (C5 ′′), 111.5 (C8 ′′ ), 114.0

(C7), 126.3 (C8a ′′), 126.6 (C4), 129.7 (C4a ′′ ), 131.1 (C3a), 135.1 (C6), 147.3 (C6 ′′), 147.7

Mark Ashford, PhD Thesis 2010 156 Chapter 8 - Experimental

(C7 ′′ ), 150.0 (C2), 154.0 (C7a), 159.2 (CO). MS-ES + m/z 590 (MH +, 100%); HRMS-ES + calculated for C 27 H33 N3O4I: 590.1516, found 590.1511.

(5-Bromobenzofuran-2-yl)(4-(2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)ethyl)piperidin-1-yl)methanone [92]

The title compound [92] was prepared H CO 3 3'' N 4' 5 as a white solid (215 mg, 51%), from H CO O Br 3 8'' 1'' N 2 5-bromobenzo[ b]furan-2-carboxylic 1' 3 O acid [42] (214 mg, 0.89 mmol), Et 3N (180 mg, 1.78 mmol), DMAP (24 mg, 0.20 mmol) and NsCl (197 mg, 0.98 mmol) and 1,2,3,4-tetrahydro-6,7-dimethoxy-2-(2-(piperidin-4- yl)ethyl)isoquinoline [90] (300 mg, 0.98 mmol) in anhydrous ACN (20 mL) using coupling

Method C, mp 92-95 oC . 1 H NMR (DMSO) δ 1.16-1.24 (m, 2H, H3a′, H5a′), 1.49 (q, 2H, J

= 7.2 Hz, NCH 2CH2CH), 1.67-1.70 (m, 1H, H4 ′), 1.79 (bd, 2H, J = 12.5 Hz, H3e′, H5e′),

2.45-2.51 (m, 2H, NC H2CH2CH), 2.60 (t, 2H, J = 6.0 Hz, H4 ′′), 2.70 (t, 2H, J = 5.5 Hz,

H3 ′′), 3.00 (bs, 2H, H2a′, H6a′), 3.45 (s, 2H, H1 ′′), 3.69 (s, 3H, OCH 3), 3.70 (s, 3H, OCH 3),

4.25 (bs, 2H, H2e′, H6e′), 6.62 (s, 1H, H5 ′′), 6.64 (s, 1H, H8 ′′ ), 7.28 (s, 1H, H3), 7.48 (d,

1H, J = 8.8 Hz, H7), 7.60 (dd, 2H, J = 8.8, 2.0 Hz, H6), 7.94 (d, 1H, J = 2.0 Hz, H4). 13 C

NMR (DMSO) δ 28.2 (C4 ′′), 31.9 (C4′), 32.8 (C3 ′, C5 ′), 33.4 (NCH 2CH2CH), 50.6 (C2 ′,

C6 ′), 54.6 (N CH2CH 2CH), 54.9 (C3 ′′), 55.1 (C1 ′′), 55.51 (OCH 3), 55.54 (OCH 3), 109.1

(C3), 110.2 (C5 ′′), 112.0 (C8 ′′), 113.7 (C7), 115.7 (CBr), 124.6 (C8a ′′), 126.0 (C4), 126.8

(C4a ′′), 128.7 (C3a), 128.9 (C6), 146.9 (C6 ′′), 147.2 (C7 ′′ ), 149.9 (C2), 152.6 (C7a), 158.3

+ + + 79 (CO). MS-ES m/z 528 (MH , 100%); HRMS-ES calculated for C 27 H32 N2O4 Br:

528.6352, found 528.6360.

Mark Ashford, PhD Thesis 2010 157 Chapter 8 - Experimental

(5-Bromobenzofuran-2-yl)(4-(3-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)- yl)propyl)piperidin-1-yl)methanone [93]

The title compound [93] was prepared O 4'' H CO 1' 2 O 3 N as white foam (280 mg, 36%) from 5- N H CO 4' 3 6 3 8'' 1'' bromobenzo[ b]furan-2-carboxylic acid Br [43] (342 mg, 1.42 mmol), Et 3N (288 mg, 2.85 mmol), DMAP (35 mg, 0.29 mmol), NsCl

(315 mg, 1.42 mmol) and 1,2,3,4-tetrahydro-6,7-dimethoxy-2-(3-(piperidin-4- yl)propyl)isoquinoline [91] (500 mg, 1.57 mmol) in anhydrous ACN (20 mL) using

o coupling Method C, mp 62-64 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75,

1 retention time 7.1 min) H NMR (CDCl 3) δ 1.23-1.29 (m, 4H, H3a′, H5a′,

NCH 2CH2CH 2CH), 1.62-1.66 (m, 3H, H4 ′, NCH 2CH 2CH2CH), 1.82 (bd, 2H, J = 11.7 Hz,

H3e′, H5e′), 2.47-2.51 (m, 2H, NC H2CH 2CH2CH), 2.70 (t, 2H, J = 6.1 Hz, H4 ′′), 2.82 (t,

2H, J = 5.8 Hz, H3 ′′), 3.20 (bs, 2H, H2a′, H6a′), 3.55 (s, 2H, H1 ′′), 3.825 (s, 3H, OCH 3),

3.832 (s, 3H, OCH 3), 4.52 (bd, 2H, H2e′, H6e′), 6.51 (s, 1H, H5 ′′), 6.58 (s, 1H, H8 ′′ ), 7.15

(s, 1H, H3), 7.31 (d, 1H, J = 8.7 Hz, H7), 7.46 (dd, 2H, J = 8.8, 2.0 Hz, H6), 7.76 (d, 1H, J

13 = 1.9 Hz, H4). C NMR (CDCl 3) δ 21.6 (H4 ′), 24.0 (NCH 2CH2CH 2CH), 32.2 (C3 ′, C5 ′),

33.4 (C4′′), 35.8 (NCH 2CH 2CH2CH), 43.4 (C2 ′, C6 ′), 49.1 (C3 ′′), 52.1 (N CH2CH 2CH 2CH),

54.8 (C1 ′′), 56.1 (OCH 3), 56.2 (OCH 3), 109.4 (C3), 110.6 (C5 ′′), 111.4 (C8 ′′), 113.5 (C7),

116.7 (CBr), 118.2 (C8a ′′) 122.6 (C4), 124.8 (C4a ′′), 129.1 (C3a), 129.4 (C6), 148.8 (C6 ′′),

149.4 (C7 ′′), 150.5 (C2), 153.3 (C7a), 159.4 (CO). MS-ES + m/z 542 (MH +, 100%); HRMS-

79 EI calculated for C 26 H33 N2O4 Br: 541.1689, found 541.1693.

Mark Ashford, PhD Thesis 2010 158 Chapter 8 - Experimental

5-Bromo-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)butyl)-1-benzofuran-

2-carboxamide [73]

The title compound [73] was prepared as 4'' H CO 3 O 1' white foam (187 mg, 23%) from 5- N 2 O H3CO1'' N 8'' 2' H bromobenzo[ b]furan-2-carboxylic acid 3 4 Br [43] (415 mg, 1.72 mmol), HOBt (230 mg, 1.72 mmol), EDC (660 mg, 1.72 mmol), 4-(3,4-dihydro-6,7-dimethoxyisoquinolin-

2(1 H)-yl)butan-1-amine [70] (503 mg, 1.89 mmol) and DIPEA (0.60 mL, 3.44 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 58-59 oC. HPLC analysis (0.1M

1 PO 4 buffer pH 7.5: MeOH, 40:60, retention time 20.1 min) H NMR (CDCl 3) δ 1.74 (m,

4H, H2 ′, H3 ′), 2.57 (t, 2H, J = 6.7 Hz, H4 ′), 2.74 (t, 2H, J = 6.2 Hz, H4 ′′), 2.83 (t, 2H, J =

5.6 Hz, H3 ′′ ), 3.50 (dt, 2H, J = 5.8 Hz, H1 ′), 3.56 (s, 2H, H1 ′′), 3.79 (s, 3H, OCH 3), 3.82 (s,

3H, OCH 3), 7.16 (s, 1H, H5 ′′), 7.18 (s, 1H, H8 ′′), 7.25 (m, 2H, H3, H7), 7.38 (m, 1H, NH),

7.43 (dd, 1H, J = 8.8, 2.0 Hz, H6), 7.71 (d, 1H, J = 2.0 Hz, H4). 13 C NMR δ 24.8 (C2′),

27.3 (C3 ′), 28.5 (C4 ′′), 39.4 (C1 ′), 50.7 (C3 ′′), 55.9 (C4 ′), 55.99 (OCH 3), 56.00 (OCH 3),

57.4 (C1 ′′), 109.2 (C3), 109.6 (C5 ′′), 111.5 (C8 ′′), 113.3 (C7), 116.7 (CBr), 125.2 (C8a ′′),

126.1 (C4), 126.2 (C4a ′′), 129.5 (C3a), 129.6 (C6), 147.5 (C6 ′′), 147.8 (C7 ′′), 150.2 (C2),

153.4 (C7a), 158.6 (CO). MS-ES + m/z 488 (MH +, 100%); HRMS-ES + calculated for

79 C24 H38 N2O4 Br: 488.3659, found 488.3652.

(4-(2-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)ethyl)piperidin-1-yl)(5- iodobenzofuran-2-yl)methanone [94]

The title compound [94] was prepared 4'' H CO 3 7 as brown wax (200 mg, 50%) from 5- N 4' H CO O I 3 8'' 1'' iodobenzo[ b]furan-2-carboxylic acid N 4 1' 2 3 O

Mark Ashford, PhD Thesis 2010 159 Chapter 8 - Experimental

[44] (200 mg, 0.70 mmol), Et 3N (142 mg, 1.40 mmol), DMAP (20 mg, 0.14 mmol), NsCl

(156 mg, 0.70 mmol) and 1,2,3,4-tetrahydro-6,7-dimethoxy-2-(2-(piperidin-4- yl)ethyl)isoquinoline [90] (213 mg, 0.77 mmol) in anhydrous ACN (20 mL) using coupling

Method C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time 6.5

1 min). H NMR (CDCl 3) δ 1.22-1.29 (m, 2H, H3a′, H5a′), 1.57-1.59 (m, 2H, NCH 2CH2CH),

1.79 (m, 1H, H4 ′), 1.82 (bd, 2H, J = 11.7 Hz, H3e′, H5e′), 2.55 (t, 2H, J = 8.0 Hz,

NC H2CH2CH), 2.69 (t, 2H, J = 6.4 Hz, H4 ′′), 2.81 (t, 2H, J = 5.6 Hz, H3 ′′ ), 3.13 (bs, 2H,

H2a′, H6a′), 3.54 (s, 2H, H1 ′′), 3.820 (s, 3H, OCH 3), 3.824 (s, 3H, OCH 3), 4.58 (bd, 2H,

H2e′, H6e′), 6.51 (s, 1H, H5 ′′), 6.58 (s, 1H, H8 ′′ ), 7.13 (s, 1H, H3), 7.27 (d, 1H, J = 8.9 Hz,

13 H7), 7.63 (dd, 1H, J = 8.8, 2.0 Hz, H6), 7.96 (d, 1H, J = 1.2 Hz, H4). C NMR (CDCl 3) δ

28.9 (C4 ′′), 32.9 (C4′), 33.7 (C3 ′, C5 ′), 34.5 (NCH 2CH2CH), 44.6 (C2 ′, C6 ′), 51.2 (N CH2-

CH 2CH), 55.6 (C3 ′′), 55.9 (C1 ′′), 56.91 (OCH 3), 56.99 (OCH 3), 87.1 (CI), 109.6 (C3),

110.3 (C5 ′′), 111.5 (C8 ′′), 113.9 (C7), 126.2 (C8a ′′ ), 126.6 (C4), 129.8 (C4a ′′), 131.0

(C3a ′′ ), 134.9 (C6), 147.3 (C7 ′′), 147.7 (C6 ′′), 150.2 (C2), 153.9 (C7a), 159.3 (CO). MS-EI

+ m/z 574 (M , 39), 206 (100%); HRMS-EI calculated for C 27 H31 N2O4I: 574.4526, found

574.4530.

N-[2-(3,4-Dihydroisoquinolin-6,7-dimethoxy-2(1 H)-yl)butyl]-4-nitrosulfonamide [89]

4'' The title compound [89] was prepared as H CO 3 O N 1' S 1 brown oil (156 mg, 48%) from 5- H CO N 3 1'' 4' H O 4 NO2 iodobenzo[ b]furan-2-carboxylic acid [44]

(200 mg, 0.70 mmol), Et 3N (0.23 mL, 3.44 mmol), DMAP (22 mg, 0.14 mmol), NsCl (156 mg, 0.70 mmol) and 4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)butan-1-amine

[70] (210 mg, 0.77 mmol) in anhydrous ACN (20 mL) using coupling Method C. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time 3.9 min). H NMR

(CDCl 3) δ 1.71 (m, 4H, H2 ′, H3 ′), 2.56 (m, 2H, H4 ′), 2.82 (m, 2H, H1 ′), 2.90 (m, 2H, H4 ′′),

Mark Ashford, PhD Thesis 2010 160 Chapter 8 - Experimental

3.01 (m, 2H, H3 ′′), 3.42 (s, 2H, H1 ′′), 3.86 (s, 3H, OCH 3), 3.91 (s, 3H, OCH 3), 6.42 (s, 1H,

H5 ′′), 6.72 (s, 1H, H8 ′′), 7.39 (d, 2H, J = 6.8 Hz, H2, H6), 8.20 (d, 2H, J = 7.2 Hz, H3, H5).

13 C NMR δ 25.7 (C2 ′), 28.2 (C3 ′), 29.7 (C4 ′′), 45.5 (C1 ′), 52.2 (C3 ′′), 54.4 (C4 ′), 56.0

(OCH 3), 56.2 (OCH 3), 58.7 (C1 ′′), 109.4 (C5 ′′), 111.7 (C8 ′′), 124.0 (C3, C5), 125.0 (C2,

C6), 126.4 (C4a ′′), 128.0 (C8a ′′), 146.6 (C6 ′′), 147.7 (C7 ′′), 148.3 (C1), 149.5 (C4). MS-EI

+ + m/z 449 (M , 4), 263 (100%); HRMS-ES calculated for C 21 H28 N3O6S: 450.1669, found

450.1681.

N-[2-(3,4-Dihydroisoquinolin-6,7-dimethoxy-2(1 H)-yl)pentyl]-4-nitrosulfonamide [80]

The title compound [80] was prepared 4'' H3CO 4 NO2 H O as brown oil (220 mg, 68%) from 5- N N H CO S 1 3 8'' 1'' 3' 1' O iodobenzo[ b]furan-2-carboxylic acid

[44] (200 mg, 0.70 mmol), Et 3N (0.23 mL, 3.44 mmol), DMAP (22 mg, 0.14 mmol), NsCl

(156 mg, 0.70 mmol) and 5-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)pentan-1- amine [71] (200 mg, 0.77 mmol) in anhydrous ACN (20 mL) using coupling Method C.

1 HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 40:60, retention time 12.9 min). H NMR

(CDCl 3) δ 1.24-1.53 (m, 6H, H2 ′, H3 ′, H4 ′), 2.45 (t, 2H, J = 7.6 Hz, H5 ′), 2.66 (t, 2H, J =

5.6 Hz, H1 ′), 2.78 (m, 2H, H4 ′′), 2.97 (t, 2H, J = 7.2 Hz, H3 ′′), 3.51 (s, 2H, H1 ′′), 3.81 (s,

3H, OCH 3), 3.82 (s, 3H, OCH 3), 6.51 (s, 1H, H5 ′′), 6.56 (s, 1H, H8 ′′), 7.94 (d, 2H, J = 7.6

13 Hz, H2, H6), 8.29 (d, 2H, J = 7.2 Hz, H3, H5). C NMR (CDCl 3) δ 26.2 (C3 ′), 26.7 (C5 ′),

28.6 (C2′), 29.6 (C4 ′′), 43.2 (C1 ′), 51.0 (C3 ′′), 55.7 (C5 ′), 56.01 (OCH 3), 56.03 (OCH 3),

58.0 (C1 ′′), 109.7 (C5 ′′), 111.5 (C8 ′′ ), 124.4 (C3, C5), 126.2 (C2, C6), 126.6 (C4a ′′), 128.3

(C8a ′′), 146.4 (C6 ′′), 147.3 (C7 ′′ ), 148.6 (C1), 150.0 (C4). MS-EI m/z 463 (M +, 1), 192

+ (100%); HRMS-ES calculated for C 22 H30 N3O6S: 464.1855, found 464.1867.

Mark Ashford, PhD Thesis 2010 161 Chapter 8 - Experimental

N-[2-(3,4-Dihydroisoquinolin-6,7-dimethoxy-2(1 H)-yl)hexyl]-4-nitrosulfonamide [81]

The title compound [81] was 4'' H CO 3 O prepared as brown oil (200 mg, N 1' S 1 H CO N 3 1'' 4' 8'' 6' H O 4 60%) from 5-iodo-benzo[ b]furan-2- NO2 carboxylic acid [44] (200 mg, 0.70 mmol), Et 3N (0.23 mL, 3.44 mmol), DMAP (22 mg, 0.14 mmol), NsCl (156 mg, 0.70 mmol) and 6-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexan-1-amine [72] (210 mg, 0.77 mmol) in anhydrous ACN (20 mL) using coupling Method C. HPLC analysis

1 (0.1M PO 4 buffer pH 7.5: MeOH, 40:60, retention time 9.3 min). H NMR (CDCl 3) δ 1.25-

1.25 (m, 8H, H2 ′, H3 ′, H4 ′, H5 ′), 2.45 (t, 2H, J = 6.4 Hz, H6 ′), 2.67 (t, 2H, J = 5.2 Hz, H1 ′),

2.80 (t, 2H, J = 4.8 Hz, H4 ′′), 3.00 (t, 2H, J = 4.6 Hz, H3 ′′), 3.50 (s, 2H, H1 ′′), 3.83 (s, 6H,

2 x OCH 3), 6.51 (s, 1H, H5 ′′), 6.58 (s, 1H, H8 ′′), 7.85 (d, 2H, J = 8.4 Hz, H2, H6), 8.25 (d,

2H, J = 8.4 Hz, H3, H5). 13 C NMR δ 24.1 (C3 ′), 26.2 (C2 ′, C4 ′), 28.5 (C5 ′), 29.0 (C4 ′′),

43.4 (C1 ′), 51.1 (C3 ′′), 55.8 (C6 ′), 56.0 (OCH 3), 56.1 (OCH 3), 57.6 (C1 ′′), 109.7 (C5 ′′),

111.5 (C8 ′′), 124.3 (C3, C5), 126.2 (C2, C6), 126.4 (C4a ′′), 128.2 (C8a ′′), 146.5 (C6 ′′),

147.4 (C7 ′′), 147.8 (C1), 149.9 (C4). MS-EI m/z 477 (M +, 5), 44 (100%); HRMS-ES + calculated for C 23 H32 N3O6S: 478.2012, found 478.2007.

Mark Ashford, PhD Thesis 2010 162 Chapter 8 - Experimental

8.4. Experimental procedures for region 3 modification

8.4.1. Region 3 intermediate synthesis

1-Phenyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 95 [105]

A solution of 2-(3 ′,4 ′-dimethoxyphenyl)ethylamine (1.02 g, 2.76 4 H3CO 6 4a

1 NH mmol) and benzaldehyde (590 mg, 2.76 mmol) in toluene (80 mL) H3CO 7 8a was heated at reflux for 2 h. The mixture was then allowed to cool to

rt. TFA (40 mL) was added and the solution heated at reflux for a further 2 h. The reaction mixture was cooled, quenched with H 2O (100 mL) and the mixture made basic with NaOH (5M). The aqueous layer was then extracted with CH 2Cl 2

(3 x 50 mL). The organic layers were combined, dried (MgSO 4), and concentrated. The residue was subjected to column chromatography (EtOAc:MeOH, 95:5) and dried in vacuo to yield the title compound [105] (1.11 g, 74%) as a white solid, mp 82-83 oC. 1H NMR

(DMSO) δ 2.61-2.68 (m, 1H, H4 a), 2.76-2.79 (m, 1H, H4 a), 2.82-2.87 (m, 1H, H3 a), 3.00-

3.04 (m, 1H, H3 a), 3.47 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 4.91 (s, 1H, H1), 6.19 (s, 1H,

H5), 6.70 (s, 1H, H8), 7.21-7.31 (m, 5H, Ph). MS-ES+ m/z 270 (MH +, 100%).

Di(6-methoxy-3,4-dihydroisoquin-2(1H)-yl)-methane 98 [107]

An aqueous solution of formaldehyde (37 % 4 4' H3CO OCH3 N N w.t, 795 mg, 36.45 mmol) was added to a 8 1 1' 8' solution of 2-(3-methoxyphenyl)ethylamine

[106] (1.00 g, 6.61 mmol) in aqueous HCl solution (1M, 362 mg, 9.92 mmol). The mixture was heated and stirred at 60 oC for 4h. The reaction mixture was then cooled to rt and basified with 10% NaOH solution and extracted with EtOAc (3 x 20 mL). The organic layers were combined, dried (Na 2SO 4) and concentrated to yield [107] (920 mg, 41%) as a

o 98 o 1 white solid, mp 119-121 C (lit 121 C). H NMR (CDCl 3) δ 2.83-2.89 (m, 8H, H3, H4,

Mark Ashford, PhD Thesis 2010 163 Chapter 8 - Experimental

H3 ′, H4 ′), 3.26 (s, 2H, NCH2N), 3.68 (s, 4H, H1, H1 ′), 3.78 (s, 6H, 2 x OCH3), 6.65 (d, 2H,

J = 2.6 Hz, H5, H5 ′), 6.70 (dd, 2H, J = 8.4 Hz, 2.6 Hz, H7, H7 ′), 6.95 (d, 2H, J = 8.4 Hz,

13 H8, H8 ′). C NMR (CDCl 3) δ 29.5 (C4, C4 ′), 49.1 (C3, C3 ′), 54.0 (C1, C1 ′), 55.4 (2 x

OCH 3), 80.8 (NCH 2N), 112.2 (C7, C7 ′), 113.5 (C5, C5 ′), 127.5 (C8a, C8 ′a), 127.8 (C8,

C8 ′), 136.1 (C4a, C4 ′a), 158.0 (C6, C6 ′). MS-EI m/z 162 (100%).

6-Methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride 98 [108]

4 To a solution of the bis product [107] (920 mg, 2.72 mmol) in H3CO NH.HCl isopropanol (10 mL) was added HCl (37% w.t, 605 mg, 3.58 8 1 mmol). The resulting suspension was stirred for 18 h at rt.

Methyl-tert -butyl ether (5 mL) was added and the solution stirred for a further 4 h. The precipitate was filtered, washed with MTBE/IPA (1:1, 20 mL) and air-dried to yield [108]

o 98 o 1 (628 mg, 90%) as a white solid, mp 235-236 C (lit 239 C). H NMR (CDCl 3) δ 2.98 (t,

2H, J = 6.2 Hz, H4), 3.27-3.30 (m, 2H, H3), 3.73 (s, 3H, OCH3), 4.13 (s, 2H, H1), 6.79-

13 6.83 (m, 2H, H5, H6), 7.12 (d, 1H, J = 8.5 Hz, H8). C NMR (CDCl 3) δ 25.8 (C4), 41.2

(C3), 43.9 (C1), 56.1 (OCH 3), 114.0 (C7), 114.1 (C5), 121.7 (C8a), 128.8 (C8), 134.3

(C4a), 159.3 (C6). MS-EI m/z 162 (100%).

7-Nitro-1,2,3,4-tetrahydroisoquinoline hydrochloride 100 [110]

To cooled H SO (90 mL) was added 1,2,3,4- 4 2 4 3 NH.HCl tetrahydroisoquinoline [109] (25.00 g, 187.7 mmol) in small O2N 1 portions with stirring, keeping the temperature below 5 oC. To the solution was added KNO 3 (20.22 g, 200 mmol) in small portions, keeping the temperature between 5-10 oC. The solution was allowed to stir for 18 h at rt. The mixture was then made basic with NaOH solution and extracted with CHCl 3 (2 x 300 mL). The combined organic layers were dried (MgSO 4) and the organic solvent removed. To the residue were

Mark Ashford, PhD Thesis 2010 164 Chapter 8 - Experimental added EtOH (90 mL) and HCl (37% w.t, 9 mL). The precipitate was collected and recrystallised (MeOH) to yield [110] (10.62 g, 32%) an off-white solid, mp 265-266 oC

(lit 100 268-269 oC). 1H NMR (DMSO) δ 3.17 (t, 2H, J = 6.0 Hz, H4), 3.41, (t, 2H, J = 6.2

Hz, H3), 4.38 (s, 2H, H1), 7.53 (d, 1H, J = 8.8 Hz, H5), 8.11 (dd, 1H, J = 8.4, 2.2 Hz, H6),

8.22 (d, 1H, J = 2.4 Hz, H8). MS-EI m/z 214 (M +, 100%).

N-Acetyl-7-nitro-1,2,3,4-tetrahydroisoquinoline 100 [111]

A solution of the 7-nitroisoquinoline [110] (1.01 g, 4.70 mmol) 5 4 3 and sodium acetate (385 mg, 4.70 mmol) in acetic anhydride (10 N O N 2 1 mL) was heated at reflux for 1 h and the solution was then allowed O to cool to rt. The solution was diluted with H 2O (20 mL) and then extracted with CHCl 3 (3 x 20 mL). The organic layers were combined, dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE, 9:1) to yield

[111] (920 mg, 89%) as a pale yellow solid mp, 83-84 oC (lit 100 84-86 oC). 1H NMR

*** (CDCl 3) δ 2.18 (s, 3H, NCOCH 3), 2.93-3.01 (m, 2H, H4), 3.71-3.87 (m, 2H, H3), 4.81

(s, 2H, H1), 7.28-7.33 (m, 1H, H5), 8.00-8.05 (m, 2H, H6, H8). MS-EI m/z 220 (M +, 32),

162 (100%).

N-Acetyl-7-amino-1,2,3,4-tetrahydroisoquinoline 100 [112]

To a solution of 10% Pd/C (90 mg) in EtOH (50 mL) was added 5 3 acetyl nitroisoquinoline [111] (900 mg, 4.08 mmol). The solution N H N 2 1 O was subjected to a hydrogen atmosphere at 50 psi for 5.5 h at rt.

The solution was then filtered through celite, and the filter cake washed with EtOAc (20

*** Rotomers observed in NMR specta of [111]

Mark Ashford, PhD Thesis 2010 165 Chapter 8 - Experimental mL). The organic layer was dried (Na 2SO 4) and the organic solvent removed. The residue was subjected to column chromatography (CHCl 3:MeOH, 9:1) and dried in vacuo to yield

1 ††† [112] (680 mg, 88%) as a red oil. H NMR (CDCl 3) δ 2.16 (s, 3H, NCOCH 3), 2.71-2.80

(m, 2H, H4), 3.64 (bs, 2H, NH 2), 3.65-3.80 (m, 2H, H3), 4.51 and 4.63 (s, 2H, H1), 6.43-

6.57 (m, 2H, H5, H6), 6.91-6.96 (m, 1H, H8). MS-EI m/z 190 (M +, 29), 151 (100%).

[N-(3-Methoxybenzylidene)amino]acetalaldehyde dimethyl acetal 105 [116]

To a solution of 3-methoxybenzaldehyde (10.12 g, 74.19 OCH3 N mmol) in toluene (150 mL) was added aminoacetaldehyde H3CO 3 1 OCH3 dimethyl acetal (7.83 g, 74.18 mmol). The mixture was heated at reflux for 4 h with a dean- stark trap to remove water. The organic solvent was then removed, and the residue distilled

1 under reduced pressure to yield [116] (16.5 g, 99%) as a yellow oil. H NMR (CDCl 3) δ

3.37 (s, 6H, CH(OC H3)2), 3.72 (d, 2H, J = 0.2, 5.3 Hz, NC H2CH), 3.77 (s, 3H, ArOCH3),

4.64 (t, 1H, J = 5.3 Hz, C H(OCH 3)2), 6.90-6.93 (s, 1H, H2), 7.21-7.32 (m, 3H, H4, H5,

13 H6), 8.20 (s, 1H, HCN). C NMR (CDCl 3) δ 53.9 (2 x OCH 3), 55.1 (ArOCH 3), 63.4

(CH 2), 103.8 (CH), 111.7 (C2), 117.3 (C4), 121.4 (C6), 129.4 (C5), 137.4 (C1), 159.8

(C3), 163.2 (CN). MS-EI m/z 223 (M +, 38), 160 (100%); HRMS-EI calculated for

C12 H17 NO 3: 223.1208, found 223.1218.

[N-(3-Methoxybenzyl)amino]acetalaldehyde dimethyl acetal [117]

5 Method 1:105 To a solution of imine [116] (5.32 g, 23.73 H OCH3 N mmol) in EtOH (30 mL) was added NaBH (3.59 g, 94.95 H3CO 3 1 OCH3 4 mmol) in small instalments over 10 min at rt and the solution stirred for a further 20 h under N 2. The reaction mixture was then poured onto cold H 2O (100 mL) and extracted

††† Rotomers observed in NMR spectrum of [112]

Mark Ashford, PhD Thesis 2010 166 Chapter 8 - Experimental with Et 2O (3 x 50 mL). The organic layers were combined, dried (Na 2SO 4) and the organic

1 solvent removed to yield [117] (4.61 g, 86%) as a clear oil. H NMR (CDCl 3) δ 1.44 (m,

1H, NH), 2.72 (bd, 2H, J = 10.8, NHCH2CH), 3.34 (s, 6H, CH(OC H3)2), 3.75-3.77 (m, 5H,

ArOCH3, NHCH2Ar), 4.46 (t, 1H, J = 5.5 Hz, NCH 2CH), 6.75-6.78 (m, 1H, H2), 6.86-6.88

13 (m, 2H, H4, H6), 7.20 (dt, 1H, J = 7.7 Hz, H5). C NMR (CDCl 3) δ 50.5 (2 x OCH 3),

53.78 (OCH 3), 53.84, (NH CH2Ar) 55.1 (NH CH2CH), 103.9 (CH), 112.5 (C2), 113.5 (C4),

120.4 (C6), 129.3 (C5), 141.8 (C1), 159.7 (C3). MS-EI m/z 225 (M +, 21), 121 (100 %);

HRMS-EI calculated for C 12 H19 NO 3: 225.1365, found 225.1368.

Method 2 : To a solution of Pt/C (200 mg) in toluene (200 mL) was added imine [116]

(10.1 g, 45.06 mmol). The solution was subjected to a hydrogen atmosphere at 60 psi for

18 h at rt. The solution was then filtered through celite and the filter cake washed with

EtOAc (50 mL). The organic solvent was removed to yield [117] (10.11 g, 98%) as a clear oil. The compound was spectroscopically identical as previously reported.

N-(2,2-Diethyl)-N-[(3-methoxyphenyl)methyl]-4-methylbenzenesulfonamide [118]

105 5 Toslyation Method 1: To a solution of amine [117] 4 6 Ts OCH3 3 N (10.05 g, 44.83 mmol) in pyridine (60 mL) was added a H CO 1 OCH 3 2 3 solution of p-methylbenzenesulfonyl chloride (9.40 g,

49.31 mmol) in dry pyridine (40 mL) at 0 oC. The solution was allowed to stir at rt for 3 days. The pyridine was then removed and the oil poured onto H 2O (100 mL) and stirred at

o 5 C for 1 h. The aqueous layer was then extracted with Et 2O (3 x 50 mL). The combined organic layers were extracted with brine (3 x 20 mL), dried (Na 2SO 4) and the organic

1 solvent removed to yield [118] (11.52 g, 67%) as an orange oil. H NMR (CDCl 3) δ 2.43

(s, 3H, Ts-CH3), 3.22 (d, 2H, J = 5.5 Hz, NC H2CH), 3.24 (s, 6H, CH(OC H3)2), 3.72 (s, 3H,

ArOCH3), 4.36 (t, 1H, J = 5.6 Hz, NCH 2CH), 4.45 (s, 2H, NC H2Ar), 6.71 (s, 1H, H2), 6.77

Mark Ashford, PhD Thesis 2010 167 Chapter 8 - Experimental

(m, 2H, H4, H6), 7.18 (dt, 1H, J = 6.8, 2.4 Hz, H5), 7.30 (d, 2H, J = 8.2 Hz, Ts), 7.73 (d,

2H, 8.2 Hz, Ts). MS-EI m/z 379 (M +, 23), 180 (100%).

Toslyation Method 2: To a solution of amine [117] (1.55 g, 4.08 mmol) in CH 2Cl 2 (20 mL) was added 50% NaOH (10 mL) and the solution was allowed to stir for 5 min. To this was added p-methylbenzenesulfonyl chloride (860 mg, 4.60 mmol) and tetra-n- butylammonium hydrogensulfate (70 mg, 0.204 mmol) and the solution was stirred for a further 18 h at rt. The solution was diluted with CH 2Cl 2 (100 mL), extracted with H 2O (2 x

20 mL), brine (20 mL) and NaHCO 3 (20 mL). The combined organic layers were dried

(Na 2SO 4) and the organic solvent removed. The residue subjected to column chromatography (40% EtOAc:PE) to yield [118] (1.23 g, 80%) as a red oil. The compound was spectroscopically identical as reported previous.

7-Methoxy-1,2,3,4-isoquinoline 105 [119]

4 To a solution of amine [118] (11.02 g, 29.04 mmol) in dioxane (150 3 N mL was added HCl (6 M, 30 mL). The resulting solution was heated H CO 3 8 1 at reflux for 16 h. The solution was allowed to cool to rt and H 2O (150 mL) was added and allowed to stir for a further 10 min. The solution was then extracted Et 2O (2 x 50 mL). The aqueous layer was made basic with 10% NaOH solution and then extracted with CH 2Cl 2 (4 x 50 mL). The organic layers were combined, dried (MgSO 4), and the organic solvent removed. The residue was subjected to column chromatography (EtOAc:PE, 3:7) and dried

1 in vacuo to yield [119] (3.52 g, 76%) as a brown oil. H NMR (CDCl 3) δ 3.92 (s, 3H,

OCH 3), 7.18 (d, 1H, J = 1.8 Hz, H8), 7.32 (dd, 1H, J = 9.0, 1.8 Hz, H6), 7.55 (d, 1H, J =

5.0 Hz, H3), 7.70 (d, 1H, J = 9.0 Hz, H5), 8.40 (d, 1H, J = 5.0 Hz, H4), 9.14 (s, 1H, H1).

13 C NMR (CDCl 3) δ 55.5 (OCH3), 104.8 (C8), 120.3 (C4), 123.6 (C6), 128.1 (C5), 129.9

Mark Ashford, PhD Thesis 2010 168 Chapter 8 - Experimental

(C8a), 131.5 (C4a), 141.3 (C3), 151.1 (C1), 158.5 (C7). MS-EI m/z 159 (M +, 100); HRMS-

EI calculated for C 10 H9NO: 159.0684, found 159.0679.

7-Methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride [115a]

4 To a solution of 7-methoxyisoquinoline [119] (700 mg, 4.40

NH.HCl mmol) in freshly distilled and dried liquid ammonia (30 mL) and H CO 3 8 1 EtOH (5 mL) was added Na (700 mg, 30.4 mmol) in small pieces over 5 min. The reaction was allowed to proceed for 1 h. The ammonia solution was then allowed to evaporate and to the residue was added ice H 2O (200 mL) and extracted with EtOAc (150 mL). The organic layer was further washed with H 2O (20 mL) and brine

(20 mL). The organic layer was dried (MgSO 4) and the organic solvent removed to yield a dark yellow oil. The oil was suspended in Et 2O (50 mL) and made acidic by the addition of a solution of 4 M HCl in dioxane. The resulting suspension was filtered to yield [115a]

(585 mg, 81%) as an off-yellow solid, mp 250-253 oC. 1H NMR (DMSO) δ 2.91 (t, 2H, J =

6.1 Hz, H4), 3.27 (t, J = 6.1 Hz, H3), 3.72 (s, 3H, OCH3), 4.16 (s, 2H, H1), 6.80-8.83 (m,

2H, H6, H8), 7.10 (bd, 1H, J = 8.2 Hz, H5). 13 C NMR (DMSO) δ 23.9 (C4), 40.7 (C3),

43.4 (C1), 55.1 (OCH 3), 111.3 (C8), 113.8 (C6), 123.9 (C5), 129.7 (C4a), 130.0 (C8a),

+ 157.7 (C7). MS-EI m/z 163 (M free base, 43), 134 (100 %); HRMS-EI C 10 H13 NO:

163.0997, found 163.0993.

4-(3,4-Dihydro-6-methoxyisoquinolin-2(1 H)-yl)butanenitrile [120]

5' The title compound [120] was prepared as a white solid H3CO N N (870 mg, 76%) from 6-methoxy-1,2,3,4- 1 1' 2 tetrahydroisoquinoline hydrochloride [108] (1.01 g, 5.00 mmol), 4-bromobutylnitrile (740 mg, 5.00 mmol), K 2CO 3 (2.76 g, 20.00 mmol), KI (10 mg, 0.035 mmol) and TBAI (80 mg, 0.2 mmol) in DMF (20 mL) using nitrogen alkylation

Mark Ashford, PhD Thesis 2010 169 Chapter 8 - Experimental

1 Method B . H NMR (CDCl 3) δ 1.92 (pentet, 2H, J = 6.9 Hz, H3), 2.47 (t, 2H, J = 7.1 Hz,

H4), 2.62 (t, 2H, J = 6.7 Hz, H2), 2.71 (t, 2H, J = 6.1 Hz, H4 ′), 2.87 (t, 2H, J = 5.8 Hz,

H3 ′), 3.55 (s, 2H, H1 ′), 3.77 (s, 3H, OCH 3), 6.63 (d, 1H, J = 2.6 Hz, H5 ′), 6.70 (dd, 1H, J

13 = 8.4, 2.6 Hz, H7 ′), 6.92 (d, 1H, J = 8.4 Hz, H8 ′). C NMR (CDCl 3) δ 15.0 (C2), 23.3

(C3), 29.5 (C4 ′), 51.0 (C3 ′), 55.4 (C4), 55.6 (OCH 3), 56.2 (C1 ′), 112.3 (C7 ′), 113.4 (C5 ′),

120.0 (CN), 126.8 (C8a ′), 127.6 (C8 ′), 135.5 (C4a ′), 158.2 (C6 ′). MS-EI m/z 230 (M+, 28),

176 (100%); HRMS-EI calculated for C 14 H18 N2O: 230.1340, found 230.1334.

4-(3,4-Dihydro-7-methoxyisoquinolin-2(1 H)-yl)butanenitrile [122]

5' 3' The title compound [122] was prepared as a white solid N N H CO 1 (440 mg, 77%) from 7-methoxy-1,2,3,4- 3 1' 2 tetrahydroisoquinoline hydrochloride [115a] (500 mg, 2.50 mmol), 4-bromobutylnitrile

(370 mg, 2.50 mmol), K 2CO 3 (1.42 g, 10.01 mmol), KI (5 mg, 0.025 mmol) and TBAI (40

1 mg, 0.1 mmol) in DMF (20 mL) using nitrogen alkylation Method B . H NMR (CDCl 3) δ

1.91 (pentet, 2H, J = 6.9 Hz, H3), 2.45 (t, 2H, J = 7.1 Hz, H4), 2.63 (t, 2H, J = 6.7 Hz, H2),

2.71 (t, 2H, J = 6.1 Hz, H4 ′), 2.82 (t, 2H, J = 5.8 Hz, H3 ′), 3.59 (s, 2H, H1 ′), 3.77 (s, 3H,

OCH 3), 6.56 (d, 1H, J = 2.7 Hz, H8 ′), 6.72 (dd, 1H, J = 8.4, 2.7 Hz, H6 ′), 7.01 (d, 1H, J =

13 8.4 Hz, H5 ′). C NMR (CDCl 3) δ 15.0 (C2), 23.2 (C3), 28.3 (C4 ′), 51.1 (C3 ′), 55.4 (C4),

56.0 (OCH 3), 56.3 (C1 ′), 111.3 (C6 ′), 112.7 (C8 ′), 119.9 (CN), 126.4 (C5 ′), 129.7 (C4a ′),

135.6 (C8a ′), 157.7 (C7). MS-EI m/z 230 (M+, 74), 176 (100%); HRMS-EI calculated for

C14 H18 N2O: 230.1419, found 230.1422.

Mark Ashford, PhD Thesis 2010 170 Chapter 8 - Experimental

5-(3,4-Dihydro-6-methoxyisoquinolin-2(1 H)-yl)pentanenitrile [121]

4' The title compound [121] was prepared as a white H3CO 6' 2 N 1 solid (790 mg, 61%) from 6-methoxy-1,2,3,4- 1' 3 N tetrahydroisoquinoline hydrochloride [108] (1.01 g,

5.00 mmol), 5-bromopentanenitrile (810 mg, 5.00 mmol), K 2CO 3 (2.76 g, 20.00 mmol), KI

(10 mg, 0.035 mmol) and TBAI (80 mg, 0.2 mmol) in DMF (20 mL) using nitrogen

1 alkylation Method B . H NMR (CDCl 3) δ 1.73-1.76 (m, 4H, H3, H4), 2.39-2.42 (m, 2H,

H5), 2.51-2.55 (m, 2H, H2), 2.69 (t, 2H, J = 6.0 Hz, H4 ′), 2.87 (t, 2H, J = 5.8 Hz, H3 ′),

3.54 (s, 2H, H1 ′), 3.77 (s, 3H, OCH 3), 6.63 (d, 1H, J = 2.6 Hz, H5 ′), 6.71 (dd, 1H, J = 8.4,

13 2.7 Hz, H7 ′), 7.01 (d, 1H, J = 8.4 Hz, H8 ′). C NMR (CDCl 3) δ 17.2 (C2), 23.5 (C3), 26.1

(C4 ′), 29.5 (C4), 51.0 (C2 ′), 55.3 (C5), 55.8 (OCH 3), 57.2 (C1 ′), 112.2 (C7 ′), 113.3 (C5 ′),

119.8 (CN), 127.0 (C8a ′), 127.6 (C8 ′), 135.5 (C4a ′), 158.1 (C6 ′). MS-EI m/z 244 (M+, 23),

176 (100%); HRMS-EI calculated for C 15 H20 N2O: 244.1497, found 244.1497.

5-(3,4-Dihydro-7-methoxyisoquinolin-2(1 H)-yl)pentanenitrile [123]

The title compound [123] was prepared as a white 5' 4' solid (380 mg, 59%) from 7-methoxy-1,2,3,4- N 2 1 H3CO 1' N tetrahydroisoquinoline hydrochloride [115a] (500 mg,

2.50 mmol), 5-bromopentanenitrile (405 mg, 2.51 mmol), K 2CO 3 (1.40 g, 10.01 mmol), KI

(5 mg, 0.025 mmol) and TBAI (46 mg, 0.1 mmol) in DMF (20 mL) using nitrogen

1 alkylation Method B . H NMR (CDCl 3) δ 1.74-1.78 (m, 4H, H3, H4), 2.40-2.43 (m, 2H,

H5), 2.52-2.56 (m, 2H, H2), 2.60 (t, 2H, J = 6.0 Hz, H4 ′′ ), 2.83 (t, 2H, J = 5.8 Hz, H3 ′),

3.59 (s, 2H, H1 ′), 3.78 (s, 3H, OCH 3), 6.56 (d, 1H, J = 2.6 Hz, H8 ′), 6.71 (dd, 1H, J = 8.4,

13 2.7 Hz, H6 ′), 7.01 (d, 1H, J = 8.4 Hz, H5 ′). C NMR (CDCl 3) δ 17.2 (C2), 23.5 (C3), 26.1

(C4 ′), 28.3 (C4), 51.2 (C2 ′), 55.4 (C5), 56.4 (OCH 3), 57.1 (C1 ′), 111.4 (C8 ′), 112.7 (C6 ′),

Mark Ashford, PhD Thesis 2010 171 Chapter 8 - Experimental

119.8 (CN), 126.5 (C5 ′), 129.7 (C4a ′), 135.7 (C8a ′), 157.7 (C7 ′). MS-EI m/z 244 (M+, 23),

176 (100%); HRMS-EI calculated for C 15 H20 N2O: 244.1576, found 244.1564.

6-(3,4-Dihydro-6,7-dimethoxy-1-phenylisoquinolin-2(1 H)-yl)hexanenitrile [124]

The title compound [124] was prepared as a yellow 4' H3CO 5 1 N oil (560 mg, 83%) from 1-phenyl-6,7-dimethoxy- 1' N H3CO 1'' 1,2,3,4-tetrahydroisoquinoline [105] (500 mg, 1.85

4'' mmol), 6-bromohexanenitrile (300 mg, 1.85 mmol)

1 and Et 3N (0.5 mL, 3.71 mmol) in CH 2Cl 2 (50 mL) using nitrogen alkylation Method A. H

NMR (CDCl 3) δ 1.21-1.65 (m, 6H, H3, H4, H5), 1.75-1.90 (m, 2H, H6), 2.25-2.34 (m, 2H,

H2), 2.52-2.56 (m, 1H, H4 a′), 2.72-2.78 (m, 1H, H4 b′), 2.95-3.03 (m, 1H, H3 a′), 3.11-3.17

(m, 1H, H3 b′), 3.47 (s, 3H, OCH 3), 3.72 (s, 3H, OCH 3), 4.45 (s, 1H, H1 ′), 6.16 (s, 1H, H5 ′),

13 6.60 (s, 1H, H8 ′), 7.23-7.28 (m, 5H, Ph). C NMR (CDCl 3) δ 17.3 (C2), 25.2 (C3), 26.2

(C4), 27.4 (C4 ′), 28.5 (C5), 47.2 (C3 ′), 53.6 (C6), 55.92 (OCH 3), 55.94 (OCH 3), 68.5 (C1 ′),

111.0 (C5 ′), 112.0 (C8 ′), 120.0 (CN), 127.0 (C4 ′′ ), 127.3 (C4a ′), 128.2 (C3 ′′, C5 ′′ ), 129.7

(C2 ′′ , C6 ′′ ), 130.3 (C8a ′), 144.5 (C1 ′′ ), 147.2 (C6 ′), 147.5 (C7 ′). MS-ES + m/z 365 (MH+,

51), 363 (100%); HRMS-EI calculated for C 23 H28 N2O2: 364.1916, found 364.1925.

4-(3,4-Dihydro-6-methoxyisoquinolin-2(1 H)-yl)butan-1-amine [125]

4' The title compound [125] was prepared as a yellow oil H3CO 3 1 N (830 mg, 94%) from LiAlH 4 (430 mg, 11.34 mmol) NH 8' 1' 2 and 4-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)- yl)butanenitrile [120] (870 mg, 3.78 mmol) in anhydrous THF (75 mL) using reduction

1 Method A. H NMR (CDCl 3) δ 1.54-1.56 (m, 2H, H2), 1.67 (m, 2H, H3), 2.52 (t, 2H, J =

7.6 Hz, H4), 2.64-2.77 (m, 4H, H4 ′, H1), 2.86-2.89 (m, 2H, H3 ′), 3.61 (s, 2H, H1 ′), 3.78 (s,

3H, OCH 3), 6.61 (d, 1H, J = 2.6 Hz, H5′), 6.68 (dd, 1H, J = 8.4, 2.6 Hz, H7′), 6.90 (dd, 1H,

Mark Ashford, PhD Thesis 2010 172 Chapter 8 - Experimental

13 J = 8.3, 2.8 Hz, H8 ′). C NMR (CDCl 3) δ 24.6 (C2), 29.4 (C4 ′), 31.7 (C3), 42.0 (C1), 50.9

(C3 ′), 55.2 (OCH 3), 55.7 (C4), 58.3 (C1 ′), 112.1 (C5 ′), 113.2 (C7 ′), 127.1 (C4a ′), 127.5

(C8a ′), 135.5 (C8 ′), 157.9 (C6 ′). MS-EI m/z 234 (M +, 1), 162 (100%); HRMS-EI calculated for C14 H22 N2O: 234.1654, found 234.1653.

5-(3,4-Dihydro-6-methoxyisoquinolin-2(1 H)-yl)pentan-1-amine [126]

The title compound [126] was prepared as a yellow 4' H3CO oil (760 mg, 95%) from LiAlH 4 (350 mg, 9.15 N NH2 8' 1' 3 1 mmol) and 4-(3,4-dihydro-6-methoxyisoquinolin-

2(1H)-yl)pentanenitrile [121] (790 mg, 3.18 mmol) in anhydrous THF (75 mL) using

1 reduction Method A. H NMR (CDCl 3) δ 1.34-1.62 (m, 8H, H2, H3, H4, NH 2), 2.48 (t, 2H,

J = 7.8 Hz, H5), 2.67-2.70 (m, 4H, H4 ′, H1), 2.86 (t, 2H, J = 5.8 Hz, H3 ′), 3.54 (s, 2H,

H1 ′), 3.75 (s, 3H, OCH 3), 6.61 (d, 1H, J = 2.4 Hz, H5 ′), 6.66 (dd, 1H, J = 8.4, 2.5 Hz, H7 ′),

13 6.91 (d, 1H, J = 8.4 Hz, H8 ′). C NMR (CDCl 3) δ 24.9 (C3), 27.1 (C4 ′), 29.4 (C4), 33.6

(C2), 42.0 (C1), 51.0 (C3 ′), 55.3 (OCH 3), 55.7 (NCH 2), 58.5 (C1 ′), 112.1 (C5 ′), 113.2

(C7 ′), 127.2 (C4a ′), 127.5 (C8a ′), 135.5 (C8 ′), 158.0 (C6 ′). MS-EI m/z 248 (M +, 18), 42

(100%); HRMS-EI calculated for C 15 H24 N2O: 248.3602, found 248.3612.

5-(3,4-Dihydro-7-methoxyisoquinolin-2(1 H)-yl)pentan-1-amine [128]

4' The title compound [128] was prepared as a yellow

oil (290 mg, 77%) from LiAlH 4 (175 mg, 4.58 N NH2 H3CO 8' 1' 3 1 mmol) and 4-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)-yl)pentanenitrile [123] (380 mg,

1 1.52 mmol) in anhydrous THF (75 mL) using reduction Method A. H NMR (CDCl 3) δ

1.35-1.63 (m, 6H, H2, H3, H4), 1.79 (bs, 2H, NH 2), 2.48 (t, 2H, J = 7.6 Hz, H5), 2.67-2.70

(m, 4H, H4 ′, H1), 2.81 (m, 2H, J = 5.6 Hz, H3 ′), 3.57 (s, 2H, H1 ′), 3.75 (s, 3H, OCH 3),

6.54 (dd, 1H, J = 2.4 Hz, H8 ′), 6.68 (dd, 1H, J = 8.4 Hz, 2.4 Hz, H6 ′), 6.98 (d, 1H, J = 8.4

Mark Ashford, PhD Thesis 2010 173 Chapter 8 - Experimental

13 Hz, H5 ′). C NMR (CDCl 3) δ 24.9 (C3), 27.2 (C4 ′), 28.3 (C4), 33.7 (C2), 42.2 (C1), 51.3

(C3 ′), 55.3 (OCH 3), 56.5 (C5), 58.4 (C1 ′), 111.4 (C8 ′), 112.6 (C6 ′), 126.6 (C5 ′), 129.6

(C4a ′), 136.0 (C8a ′), 157.7 (C7 ′). MS-EI m/z 248 (M +, 15), 32 (100%); HRMS-EI calculated for C 14 H22 N2O: 248.3604, found 248.3609.

4-(3,4-Dihydro-7-methoxyisoquinolin-2(1 H)-yl)butan-1-amine [127]

The title compound [127] was prepared as a yellow oil 4' 6' 1 (447 mg, 89%) from LiAlH 4 (220 mg, 5.73 mmol) and N H CO NH 3 1' 4 2 4-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)- yl)butanenitrile [122] (440 mg, 1.91 mmol) in anhydrous THF (75 mL) using reduction

1 Method A. H NMR (CDCl 3) δ 1.48-1.84 (m, 6H, NH 2, H2, H3), 2.50 (t, 2H, J = 7.7 Hz,

H4), 2.71 (m, 4H, H4 ′, H1), 2.82 (t, 2H, J = 5.8 Hz, H3 ′), 3.59 (s, 2H, H1 ′), 3.76 (s, 3H,

OCH 3), 6.55 (d, 1H, J = 2.5 Hz, H8 ′), 6.69 (dd, 1H, J = 8.4, 2.6 Hz, H6 ′), 6.99 (d, 1H, J =

13 8.4 Hz, H5 ′) C NMR (CDCl 3) δ 24.7 (C2), 28.3 (C4 ′), 31.7 (C3), 42.1 (C1), 51.3 (C3 ′),

55.3 (OCH 3), 56.4 (C4), 62.4 (C1 ′), 111.4 (C8 ′), 112.6 (C6 ′), 126.5 (C5 ′), 129.6 (C4a ′),

135.9 (C8a ′), 157.7 (C7 ′). MS-EI m/z 234 (M +, 15), 162 (100%); HRMS-EI calculated for

C14 H22N2O: 234.1732, found 234.1737.

6-(3,4-Dihydro-6,7-dimethoxy-1-phenylisoquinolin-2(1 H)-yl)hexan-1-amine [129]

4' The title compound [129] was prepared as a H3CO 1 1' N yellow oil (420 mg, 83%) from LiAlH 4 (160 mg, H CO NH 3 4 2 1'' 4.11 mmol) and 6-(3,4-dihydro-6,7-dimethoxy-1-

4'' phenylisoquinolin-2(1 H)-yl)hexanenitrile [124]

(500 mg, 1.31 mmol) in anhydrous THF (75 mL) using reduction Method A. 1H NMR

(CDCl 3) δ 1.21-1.65 (m, 8H, H2, H3, H4, H5), 2.55-2.60 (m, 1H, H4 a′), 2.75-3.08 (m, 5H,

H4 b′, H6, H1), 3.22-3.26 (m, 1H, H3 a′), 3.40-3.46 (m, 1H, H3 b′), 3.88 (s, 3H, OCH 3), 4.13

Mark Ashford, PhD Thesis 2010 174 Chapter 8 - Experimental

(s, 3H, OCH 3), 4.76 (s, 1H, H1 ′), 6.46 (s, 1H, H5 ′), 6.89 (s, 1H, H8 ′), 7.51-7.59 (m, 5H,

13 Ph). C NMR (CDCl 3) δ 26.8 (C2), 26.9 (C3), 27.2 (C4), 28.3 (C4 ′), 30.5 (C5), 42.3 (C1),

47.2 (C3 ′), 54.2 (C6), 55.90 (OCH 3), 55.92 (OCH 3), 68.1 (C1 ′), 111.0 (C5 ′), 111.9 (C8 ′),

127.10 (C4 ′′ ), 127.12 (C4a ′), 128.2 (C3 ′′ , C5 ′′ ), 129.7 (C2 ′′ , C6 ′′ ), 130.4 (C8a), 144.5 (C1 ′′ ),

147.1 (C6 ′), 147.5 (C7 ′). MS-ES + m/z 369 (MH +, 5), 367 (100%); HRMS-ES + calculated for C 23 H33 N2O2: 369.2542, found 369.2544.

tert -Butyl-4-(3-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)-yl)propyl)piperidine-1- carboxylate [130]

4' The title compound [130] (750 mg, 77%) was H CO 6' 1 Boc 3 N N prepared as a clear oil from 6-methoxy-1,2,3,4- 4 1' tetrahydroisoquinoline hydrochloride [108] (500 mg, 2.50 mmol), 4-(3-iodoethyl)piperidine-1-carboxylic acid tert -butyl ester [87] (885 mg,

2.50 mmol), K 2CO 3 (1.40 g, 10.00 mmol), and TBAI (37 mg, 0.10 mmol) in DMF (20 mL)

1 using nitrogen alkylation Method B . H NMR (CDCl 3) δ 1.06-1.12 (m, 2H, H3a, H5a),

1.23-1.29 (m, 2H, NCH 2CH2CH2CH), 1.30-1.44 (m, 10H, O(CH 3)3, H4), 1.55-1.69 (m, 4H,

H3e, H5e, NCH 2CH2CH2CH), 2.49 (t, 2H, J = 7.8 Hz, NC H2CH2CH2CH), 2.63-2.73 (m,

4H, H2a,H6a, H4 ′), 2.87 (t, 2H, J = 5.8 Hz, H3 ′), 3.57 (s, 2H, H1 ′), 3.76 (s, 3H, OCH 3),

4.06-4.14 (m, 2H, H2e, H6e), 6.62 (d, J = 2.6 Hz, 1H, H5 ′), 6.68 (dd, J = 8.4, 2.7 Hz, 1H,

13 H7 ′), 6.92 (d, J = 8.4 Hz, H8 ′). C NMR (CDCl 3) δ 24.3 (C4), 28.4 (NCH 2CH2CH 2CH),

28.9 (C( CH3)3), 29.2 (NCH 2CH 2CH2CH), 32.3 (C3, C5), 34.4 (C4 ′), 44.6 (C2, C6), 50.9

(C3 ′), 55.4 (C1 ′), 55.6 (OCH 3), 58.6 (N CH2CH 2CH2CH), 79.3 ( C(CH 3)3), 112.2 (C5 ′),

113.2 (C7 ′), 126.7 (C8a ′), 127.7 (C8), 135.4 (C4a ′), 155.0 (C6 ′), 158.1 (CO). MS-ES + m/z

+ + 389 (MH , 37), 101 (100%); HRMS-ES calculated for C 23H36 N2O4: 389.6359, found

289.6345.

Mark Ashford, PhD Thesis 2010 175 Chapter 8 - Experimental

1,2,3,4-Tetrahydro-6-methoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [132]

The title compound [132] was prepared as a red oil H CO 4 1' 3 NH N (420 mg, 75%) from tert -butyl-4-(3-(3,4-dihydro-6- 7 4' 1 methoxyisoquinolin-2(1 H)-yl)propyl)piperidine-1-carboxylate [130] (750 mg, 1.93 mmol)

1 in CH 2Cl 2:TFA (2:1, 15 mL) using nitrogen deprotection Method A . H NMR (CDCl 3) δ

1.08-1.14 (m, 2H, H3a′, H5a′), 1.24-1.27 (m, 2H, NCH 2CH2CH2CH), 1.46 (m, 1H, H4 ′)

1.56-1.71 (m, 4H, H3e′, H5e′, NCH 2CH2CH2CH), 2.10 (bs, 1H, NH), 2.45 (t, 2H, J = 7.6

Hz, NC H2CH2CH2CH), 2.54-2.58 (m, 2H, H2a′, H6a′), 2.68 (t, 2H, J = 6.0 Hz, H4), 2.86

(t, 2H, J = 5.6 Hz, H3), 3.06 (bd, 2H, J = 12.4 Hz, H2e′, H6e′), 3.54 (s, 2H, H1), 3.75 (s,

3H, OCH 3), 6.61 (s, 1H, H5), 6.66 (dd, 1H, J = 8.4, 2.4 Hz, H7), 6.91 (d, 1H, J = 8.4 Hz,

13 H8). C NMR (CDCl 3) δ 24.6 (C4 ′), 28.3 (NCH 2CH2CH 2CH), 31.9 (NCH 2CH 2CH2CH),

34.8 (C3 ′, C5 ′), 35.7 (C4), 45.9 (C2 ′, C6 ′), 51.5 (C3), 55.4 (C1), 55.7 (OCH 3), 58.6

(N CH2CH 2CH2CH), 111.5 (C5), 112.9 (C7), 126.8 (C8a), 130.0 (C8), 136.2 (C4a), 158.1

+ (C6). MS-EI m/z 288 (M , 31), 159 (100%); HRMS-EI calculated for C 18 H28 N2O:

288.2202, found 288.2205.

tert -Butyl-4-(3-(3,4-dihydro-7-methoxyisoquinolin-2(1 H)-yl)propyl)piperidine-1- carboxylate [131]

4' The title compound [131] (730 mg, 75%) was 1 Boc 6' N N prepared as a clear oil from 7-methoxy-1,2,3,4- H CO 4 3 1' tetrahydroisoquinoline hydrochloride [115a] ( 500 mg, 2.50 mmol), 4-(3- iodoethyl)piperidine-1-carboxylic acid tert -butyl ester [87] (885 mg, 2.50 mmol), K 2CO 3

(1.40 g, 10.00 mmol), and TBAI (37 mg, 0.10 mmol) in DMF (20 mL) using nitrogen

1 alkylation Method B . H NMR (CDCl 3) δ 1.06-1.10 (m, 2H, H3a, H5a), 1.25-1.39 (m, 3H,

NCH 2CH2CH2CH, H4), 1.44 (s, 9H, O(C H3)3), 1.55-1.69 (m, 4H, H3e, H5e,

NCH 2CH2CH2CH), 2.47 (t, 2H, J = 7.7 Hz, NC H2CH2CH2CH), 2.63-2.73 (m, 4H, H2a,

Mark Ashford, PhD Thesis 2010 176 Chapter 8 - Experimental

H6a, H4 ′), 2.81 (t, 2H, J = 5.8 Hz, H3 ′), 3.58 (s, 2H, H1 ′), 3.76 (s, 3H, OCH 3), 4.06-4.12

(m, 2H, H2e, H6e), 6.54 (d, J = 2.6 Hz, 1H, H8 ′), 6.69 (dd, J = 8.4, 2.7 Hz, 1H, H6 ′), 6.99

13 (d, J = 8.4 Hz, H5 ′). C NMR (CDCl 3) δ 24.5 (C4), 28.6 (NCH 2CH2CH 2CH), 28.9

(C( CH3)3), 29.9 (NCH 2CH 2CH2CH), 32.2 (C3, C5), 34.5 (C4 ′), 44.2 (C2, C6), 51.3 (C3 ′),

55.4 (C1 ′), 56.5 (OCH 3), 58.6 (N CH2CH 2CH2CH), 79.3 ( C(CH 3)3), 111.4 (C5 ′), 112.6

(C7 ′), 126.6 (C8a ′), 129.6 (C8), 135.9 (C4a ′), 155.0 (C6 ′), 157.7 (CO). MS-EI m/z 388 (M +,

21), 176 (100%); HRMS-EI calculated for C 23 H36 N2O4: 388.2726, found 388.2730.

1,2,3,4-Tetrahydro-7-methoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [133]

4 The title compound [133] was prepared as a red oil 1' 6 NH N (450 mg, 90%) tert -butyl-4-(3-(3,4-dihydro-7- H3CO 1 4' methoxyisoquinolin-2(1 H)-yl)propyl)piperidine-1- carboxylate [131] (500 mg, 1.73 mmol) in CH 2Cl 2:TFA (2:1, 15 mL) using nitrogen

1 deprotection Method A . H NMR (CDCl 3) δ 1.24-1.33 (m, 4H, H3a′, H5a′,

NCH 2CH2CH2CH), 1.44 (m, 1H, H4 ′) 1.58-1.62 (m, 2H, NCH 2CH2CH2CH), 1.76 (bd, 2H,

J = 11.4 Hz, H3e′, H5e′), 2.47 (t, 2H, J = 7.7 Hz, NC H2CH2CH2CH), 2.64-2.72 (m, 4H,

H2a′, H6a′, H4), 2.82 (t, 2H, J = 5.6 Hz, H3), 3.18 (bd, 2H, J = 9.5 Hz, H2e′, H6e′), 3.54 (s,

2H, H1), 3.76 (s, 3H, OCH 3), 6.55 (d, 1H, J = 2.6 Hz, H8), 6.70 (dd, 1H, J = 8.4, 2.7 Hz,

13 H6), 6.99 (d, 1H, J = 8.4 Hz, H5). C NMR (CDCl 3) δ 24.2 (C4 ′), 28.2

(NCH 2CH2CH 2CH), 31.6 (NCH 2CH 2CH2CH), 34.5 (C3 ′, C5 ′), 35.4 (C4), 45.6 (C2 ′, C6 ′),

51.2 (C3), 54.6 (N CH2CH2CH 2CH), 55.2 (OCH 3), 58.4 (C1), 111.3 (C5), 112.5 (C7),

126.5 (C8a), 129.6 (C8), 135.9 (C4a), 157.6 (C6). MS-EI m/z 288 (M +, 31), 159 (100%);

HRMS-EI calculated for C 18 H28 N2O: 288.2202, found 288.2205.

Mark Ashford, PhD Thesis 2010 177 Chapter 8 - Experimental

8.4.2. Target compounds with Region 1 modification

(4-(3-(3,4-Dihydro-7-methoxyisoquinolin-2(1 H)-yl)propyl)piperidin-1-yl)(5- iodobenzofuran-2-yl)methanone [140]

O The title compound [140] was 5'' 1' 2 O 3'' N prepared as a white solid (300 mg, N 3 6 H3CO 4' 39%) from 5-iodobenzo[ b]furan-2- I carboxylic acid [44] (397 mg, 1.38 mmol), HOBt (186 mg, 1.38 mmol), EDC (320 mg,

1.66 mmol), 1,2,3,4-tetrahydro-7-methoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [133]

(400 mg, 1.38 mmol) and DIPEA (0.48 mL, 2.77 mmol) in anhydrous DMF (10 mL) using

o coupling Method B, mp 151-153 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH,

1 25:75, retention time 12.0 min). H NMR (CDCl 3) δ 1.25-1.38 (m, 4H, H3a′, H5a′,

NCH 2CH 2CH2CH), 1.60-1.65 (m, 3H, H4 ′, NCH 2CH2CH 2CH), 1.82 (bd, 2H, J = 12.6 Hz,

H3e′, H5e′), 2.47-2.51 (m, 2H, NC H2CH 2CH2CH), 2.72 (t, 2H, J = 5.7 Hz, H4 ′′), 2.83 (t,

2H, J = 5.8 Hz, H3 ′′), 3.12 (bs, 2H, H2a′, H6a′), 3.59 (s, 2H, H1 ′′), 3.76 (s, 3H, OCH 3),

4.45 (bs, 2H, H2e′, H6e′), 6.55 (d, 1H, J = 2.6 Hz, H8 ′′ ), 6.70 (dd, 1H, J = 8.4, 2.7 Hz,

H6 ′′ ), 7.00 (d, 1H, J = 8.4 Hz, H5 ′′ ), 7.14 (s, 1H, H3), 7.29 (d, 1H, J = 8.9 Hz, H7), 7.64

13 (dd, 1H, J = 8.7, 1.7 Hz, H6), 7.96 (d, 1H, J = 1.7 Hz, H4). C NMR (CDCl 3) δ 24.5 (C4 ′),

28.3 (NCH 2CH2CH 2CH), 33.8 (C3 ′, C5 ′), 34.3 (C4 ′′), 36.3 (NCH 2CH 2CH2CH), 44.1 (C2 ′,

C6 ′), 51.4 (C3 ′′), 55.4 (N CH2CH 2CH 2CH), 56.5 (OCH 3), 58.5 (C1 ′′ ), 87.1 (CI), 110.0 (C3),

111.5 (C6 ′′), 112.6 (C8 ′′), 114.0 (C7), 126.6 (C5 ′′), 129.7 (C4a ′′), 129.9 (C4), 131.0 (C3a),

134.9 (C6), 135.9 (C8a ′′), 150.3 (C2), 154.0 (C7a), 157.7 (C7 ′′), 159.4 (CO). MS-EI m/z

+ 558 (M , 2), 162 (100%); HRMS-EI calculated for C 27 H31 N3O2I: 558.1379, found

558.1361.

Mark Ashford, PhD Thesis 2010 178 Chapter 8 - Experimental

5-Iodo -N-(5-(3,4-dihydro-7-methoxyisoquinolin-2(1 H)-yl)pentyl)-1-benzofuran-2- carboxamide [137]

The title compound [137] was 7 4'' 6'' 3'' O I prepared as a brown wax (310 mg, 4' H N N 4 H3CO 1' 3 36%) from 5-iodobenzo[ b]furan-2- O carboxylic acid [44] (490 mg, 1.70 mmol), HOBt (230 mg, 1.70 mmol), EDC (391 mg,

2.04 mmol), 5-(3,4-dihydro-7-methoxyisoquinolin-2(1 H)-yl)pentan-1-amine [128] (400 mg, 1.70 mmol) and DIPEA (0.82 mL, 3.40 mmol) in anhydrous DMF (10 mL) using coupling Method B. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time

1 9.1 min). H NMR (CDCl 3) δ 1.43-1.47 (m, 2H, H3 ′), 1.62-1.69 (m, 4H, H2 ′, H4 ′), 2.50 (t,

2H, J = 7.5 Hz, H5 ′), 2.69 (t, 2H, J = 6.0 Hz, H4 ′′ ), 2.80 (t, 2H, J = 5.7 Hz, H3 ′′ ), 3.46 (m,

2H, H1 ′), 3.57 (s, 2H, H1 ′′ ), 3.74 (s, 3H, OCH 3), 6.52 (d, 1H, J = 2.5 Hz, H8 ′′ ), 6.67 (dd,

1H, J = 8.4, 2.6 Hz, H6 ′′ ), 6.75 (m, 1H, NH), 6.96 (d, 1H, J = 8.4 Hz, H5 ′′ ), 7.21 (d, 1H, J

= 8.7 Hz, H7), 7.33 (s, 1H, H3), 7.63 (dd, 1H, J = 8.7, 1.7 Hz, H6), 7.96 (d, 1H, J = 1.7 Hz,

13 H4). C NMR (CDCl 3) δ 24.9 (C4 ′′ ), 26.8 (C3 ′), 28.2 (C4 ′), 29.6 (C2 ′), 39.4 (C1 ′), 51.2

(C3 ′′ ), 55.3 (C5 ′), 56.4 (OCH 3), 58.0 (C1 ′′ ), 87.2 (CI), 109.1 (C3), 111.4 (C6 ′′ ), 112.6

(C8 ′′ ), 113.8 (C7), 126.5 (C5′′ ), 129.6 (C4a ′′ ), 130.3 (C4), 131.5 (C3a), 135.4 (C6), 135.8

(C8a ′′ ), 149.7 (C2), 154.0 (C7a), 157.7 (C7 ′′ ), 158.4 (CO). MS-ES + m/z 519 (MH +, 100%);

+ HRMS-ES calculated for C 24 H28 N2O3I: 519.1145, found 519.1133.

5-Iodo -N-(5-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)-yl)pentyl)-1-benzofuran-2- carboxamide [135]

The title compound [135] was 4'' 5 H3CO 6'' I H O prepared as a brown wax (290 mg, N N 4 1'' 3' 1' 3 O 34%) from 5-iodobenzo[ b]furan-2- carboxylic acid [44] (490 mg, 1.70 mmol), HOBt (230 mg, 1.70 mmol), EDC (391 mg,

Mark Ashford, PhD Thesis 2010 179 Chapter 8 - Experimental

2.04 mmol), 5-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)-yl)pentan-1-amine [126] (400 mg, 1.70 mmol) and DIPEA (0.82 mL, 3.40 mmol) in anhydrous DMF (10 mL) using coupling Method B. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time

1 9.0 min). H NMR (CDCl 3) δ 1.47 (m, 2H, H3 ′), 1.66 (m, 4H, H2 ′, H4 ′), 2.50 (t, 2H, J =

6.7 Hz, H5 ′), 2.69 (t, 2H, J = 5.6 Hz, H4 ′′), 2.86 (t, 2H, J = 5.3 Hz, H3 ′′), 3.47 (dt, 2H, J =

6.3 Hz, H1 ′), 3.54 (s, 2H, H1 ′′), 3.76 (s, 3H, OCH 3), 6.61 (s, 1H, H5 ′′), 6.67 (m, 2H, NH,

H7 ′′), 6.91 (d, 1H, J = 8.4 Hz, H8 ′′), 7.23 (d, 1H, J = 8.7 Hz, H7), 7.35 (s, 1H, H3), 7.65 (d,

13 1H, J = 8.7 Hz, H6), 7.99 (s, 1H, H4). C NMR (CDCl 3) δ 24.9 (C4 ′′), 26.9 (C3 ′), 29.5

(C4 ′), 29.6 (C2 ′), 39.5 (C1 ′), 51.0 (C3 ′′), 55.3 (C5 ′), 55.8 (OCH 3), 58.3 (C1 ′′), 87.2 (CI),

109.2 (C3), 112.1 (C6 ′′), 113.3 (C8 ′′ ), 113.8 (C7), 127.2 (C5 ′′), 127.6 (C4a ′′ ), 130.3 (C4),

131.6 (C3a), 135.4 (C6), 135.6 (C8a ′′ ), 149.7 (C2), 154.1 (C7a), 158.0 (C7 ′′), 158.4 (CO).

+ + + MS-ES m/z 519 (MH , 100%); HRMS-ES calculated for C 24 H28N2O3I: 519.1145, found

519.1155.

5-Iodo-N-(4-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)-yl)butyl)-1-benzofuran-2- carboxamide [134]

4'' The title compound [134] was prepared as H CO 6'' 3 O 1' N 2 O a white solid (410 mg, 51%) from 5- N 1'' 4' H 3 6 iodobenzo[ b]furan-2-carboxylic acid [44]

I (437 mg, 1.52 mmol), HOBt (205 mg,

1.52 mmol), EDC (350 mg, 1.82 mmol), 4-(3,4-dihydro-6-methoxyisoquinolin-2(1 H)- yl)butan-1-amine [125] (400 mg, 1.52 mmol) and DIPEA (0.73 mL, 3.04 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp 72-74 oC. HPLC analysis (0.1M

1 PO 4 buffer pH 7.5: MeOH, 25:75, retention time 7.6 min). H NMR (CDCl 3) δ 1.74 (m,

4H, H2 ′, H3 ′), 2.56 (t, 2H, J = 6.6 Hz, H4 ′), 2.72 (t, 2H, J = 6.0 Hz, H4 ′′), 2.88 (t, 2H, J =

5.7 Hz, H3 ′′), 3.50 (m, 2H, H1 ′), 3.58 (s, 2H, H1 ′′), 3.76 (s, 3H, OCH 3), 6.61 (d, 1H, J = 2.5

Mark Ashford, PhD Thesis 2010 180 Chapter 8 - Experimental

Hz, H5 ′′), 6.69 (dd, 1H, J = 8.4, 2.6 Hz, H7 ′′), 6.91 (d, 1H, J = 8.4 Hz, H8 ′′), 7.06 (d, 1H, J

= 8.7 Hz, H7), 7.22 (s, 1H, H3), 7.40 (m, 1H, NH), 7.60 (dd, 1H, J = 8.7, 1.7 Hz, H6), 7.90

13 (d, 1H, J = 1.6 Hz, H4). C NMR (CDCl 3) δ 24.9 (C4 ′′), 27.4 (C3 ′), 29.4 (C2 ′), 39.5 (C1 ′),

50.6 (C3 ′′), 55.4 (C4 ′), 55.9 (OCH 3), 57.6 (C1 ′′ ), 87.2 (CI), 108.9 (C3), 112.3 (C6 ′′), 113.4

(C8 ′′ ), 113.8 (C7 ′), 126.9 (C5 ′′), 127.6 (C4a ′′), 130.3 (C4), 131.5 (C3a), 135.3 (C6), 135.5

(C8a ′′), 149.8 (C2), 154.1 (C7a), 158.1 (C7 ′′), 158.5 (CO). MS-ES + m/z 505 (MH +, 100%);

+ HRMS-ES calculated for C 23 H26 N2O3I: 505.0988, found 505.0982.

5-Iodo -N-(4-(3,4-dihydro-7-methoxyisoquinolin-2(1 H)-yl)butyl)-1-benzofuran-2- carboxamide [136]

4'' The title compound [136] was prepared as O 3' 1' N 2 O H CO N a brown wax (210 mg, 30%) from 5- 3 8'' 1'' H iodobenzo[ b]furan-2-carboxylic acid [44] 4 5 I (385 mg, 1.33 mmol), HOBt (180 mg, 1.33 mmol), EDC (305 mg, 1.60 mmol), 4-(3,4-dihydro-7-methoxyisoquinolin-2(1 H)-yl)butan-

1-amine [127] (450 mg, 1.33 mmol) and DIPEA (0.64 mL, 2.66 mmol) in anhydrous DMF

(10 mL) using coupling Method B. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 50:50,

1 retention time 38.3 min). H NMR (CDCl 3) δ 1.75-1.77 (m, 4H, H2 ′, H3 ′), 2.55-2.58 (m,

2H, H4 ′), 2.74 (t, 2H, J = 6.0 Hz, H4 ′′), 2.84 (t, 2H, J = 5.7 Hz, H3 ′′), 3.51-3.53 (m, 2H,

H1 ′), 3.62 (s, 2H, H1 ′′), 3.76 (s, 3H, OCH 3), 6.53 (d, 1H, J = 2.6 Hz, H8′′ ), 6.72 (dd, 1H, J

= 8.4, 2.7 Hz, H6 ′′ ), 7.00-7.07 (m, 2H, H7, H5′′ ), 7.23 (s, 1H, H3), 7.42-7.44 (m, 1H, NH),

13 7.61 (dd, 1H, J = 8.7, 1.8 Hz, H6), 7.91 (d, 1H, J = 1.7 Hz, H4). C NMR (CDCl 3) δ 24.9

(C4 ′′), 27.4 (C2 ′), 28.2 (C3 ′), 39.4 (C1 ′), 50.8 (C3 ′′), 55.4 (C4 ′), 56.6 (OCH 3), 57.4 (C1 ′′ ),

87.2 (CI), 108.9 (C3), 111.4 (C6 ′′), 112.7 (C8 ′′), 113.8 (C7), 126.5 (C5 ′′), 129.7 (C4a ′′),

130.3 (C4), 131.5 (C3a), 135.3 (C6), 135.7 (C8a ′′), 149.8 (C2), 154.1 (C7a), 157.8 (C7 ′′),

Mark Ashford, PhD Thesis 2010 181 Chapter 8 - Experimental

+ + + 158.6 (CO). MS-ES m/z 505 (MH , 100%); HRMS-ES calculated for C 23 H26 N2O3I:

505.0988, found 505.0980.

5-Iodo -N-(6-(3,4-Dihydro-6,7-dimethoxy-1-phenylisoquinolin-2(1 H)-yl)hexyl)-1- benzofuran-2-carboxamide [138]

The title compound [138] was H CO 5'' 3 3'' O 1' prepared as brown solid (205 mg, N O H CO N 3 4' 1''' H 2 28%) from 5-iodobenzo[ b]furan-2- 4 I 4''' carboxylic acid [44] (322 mg, 1.10 mmol), HOBt (150 mg, 1.10 mmol), EDC (265 mg, 1.32 mmol), 6-(3,4-dihydro-6,7- dimethoxy-1-phenylisoquinolin-2(1H)-yl)hexan-1-amine [129] (402 mg, 1.10 mmol) and

DIPEA (0.55 mL, 2.20 mmol) in anhydrous DMF (10 mL) using coupling Method B, mp

o 133-136 C. HPLC analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time 17.0

1 min). H NMR (CDCl 3) δ 1.13-1.17 (m, 4H, H3 ′, H4 ′), 1.35-1.52 (m, 4H, H2 ′, H5 ′), 2.15-

2.21 (m, 1H, H4 a′′), 2.36-2.48 (m, 2H, H6 ′), 2.62-2.68 (m, 1H, H4 b′′), 2.83-2.90 (m, 1H,

H3a′′), 3.00-3.06 (m, 1H, H3 b′′), 3.28-3.33 (m, 2H, H1 ′), 3.48 (s, 3H, OCH 3), 3.73 (s, 3H,

OCH 3), 4.36 (s, 1H, H1 ′′), 6.06 (s, 1H, H5 ′′), 6.09 (s, 1H, H8 ′′), 6.41 (bs, 1H, NH), 7.11-

7.17 (m, 7H, Ph, H3, H7), 7.56 (dd, 1H, J = 8.4, 1.6 Hz, H6), 7.90 (d, 1H, J = 2.0 Hz, H4).

13 C NMR (CDCl 3) δ 26.8 (C3 ′), 26.9 (C4 ′), 27.0 (C4 ′′), 28.4 (C2 ′), 29.7 (C5 ′), 39.6 (C1 ′),

47.1 (C3 ′′), 54.1 (C6 ′), 55.9 (OCH 3), 56.0 (OCH 3), 68.2 (C1 ′′ ), 87.3 (CI), 109.2 (C3), 111.0

(C5 ′′), 112.0 (C8 ′′), 113.8 (C7), 127.1 (C8a ′′), 127.2 (C4), 128.2 (C4 ′′′ ), 129.7 (C4a ′′),

130.40 (C3 ′′′ , C5 ′′′ ), 130.43 (C2 ′′′ , C6 ′′′ ), 131.6 (C3a), 135.5 (C6), 144.6 (C1 ′′′ ), 147.2

(C6 ′′), 147.5 (C7 ′′), 149.8 (C2), 154.1 (C7a), 158.4 (CO). MS-ES + m/z 639 (MH +, 100%);

+ HRMS-ES calculated for C 32 H36 N2O4I: 639.1720, found 639.1715.

Mark Ashford, PhD Thesis 2010 182 Chapter 8 - Experimental

(4-(3-(3,4-Dihydro-6-methoxyisoquinolin-2(1H)-yl)propyl)piperidin-1-yl)(5- iodobenzofuran-2-yl)methanone [139]

The title compound [139] was O 4'' prepared as off-white wax (150 mg, H CO 1' O 3 N N 4' 3 6 17%) from 5-iodobenzo[ b]furan-2- 8'' 1'' 4 I carboxylic acid [44] (450 mg, 1.57 mmol), Et 3N (317 mg, 3.14 mmol), DMAP (40 mg, 0.31 mmol), NsCl (383 mg, 1.73 mmol) and 1,2,3,4-tetrahydro-6-methoxy-2-(3-(piperidin-4-yl)propyl)isoquinoline [132]

(503 mg, 1.73 mmol) in anhydrous ACN (20 mL) using coupling Method C. HPLC

1 analysis (0.1M PO 4 buffer pH 7.5: MeOH, 25:75, retention time 11.9 min). H NMR

(CDCl 3) δ 1.25-1.27 (m, 4H, H3a′, H5a′, NCH 2CH2CH 2CH), 1.53-1.54 (m, 3H, H4 ′,

NCH 2CH 2CH2CH), 1.77 (bd, 2H, J = 11.2 Hz, H3e′, H5e′), 2.39 (t, 2H, J = 6.8 Hz,

NC H2CH 2CH 2CH), 2.58 (t, 2H, J = 5.2 Hz, H4 ′′), 2.75 (t, 2H, J = 4.4 Hz, H3 ′′), 3.16 (bs,

2H, H2a′, H6a′), 3.43 (s, 2H, H1 ′′), 3.68 (s, 3H, OCH 3), 4.13-4.40 (m, 2H, H2e′, H6e′), 6.66

(m, 2H, H5 ′′, H7 ′′), 6.93 (d, J = 8.4 Hz, H8 ′′ ) 7.28 (s, 1H, H3), 7.52 (d, 1H, J = 8.8 Hz, H7),

13 7.70 (d, 1H, J = 8.8 Hz, H6), 8.11 (s, 1H, H4). C NMR (CDCl 3) δ 23.6 (C4 ′), 29.0

(NCH 2CH2CH 2CH), 33.6 (C3 ′, C5 ′), 35.2 (C4 ′′), 36.9 (NCH 2CH 2CH2CH), 48.5 (C2 ′, C6 ′),

50.5 (C3 ′′), 54.8 (N CH2CH 2CH 2CH), 57.9 (C1 ′′ ), 87.7 (CI), 109.0 (C3), 111.8 (C7 ′′), 112.7

(C8 ′′ ), 114.1 (C7), 127.0 (C5 ′′), 127.2 (C4a ′′), 129.6 (C4), 130.7 (C3a), 134.4 (C6), 135.3

(C8a ′′), 149.3 (C2), 153.1 (C7a), 157.3 (C6 ′′), 158.3 (CO). MS-EI m/z 558 (M +, 2), 162

(100%); HRMS-EI calculated for C 27 H31 N3O2I: 558.1379, found 558.1371.

Mark Ashford, PhD Thesis 2010 183 Chapter 8 - Experimental

8.5. Experimental procedures for the stannylation reactions and radioiodination reactions

(4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)(5-

(trimethylstannyl)benzofuran-2-yl)methanone [144]

O To a solution of the bromo derivative [60] 5'' 4'' H CO 1' O 3 N 7 (100 mg, 0.20 mmol) in anhydrous toluene N 3 H3CO 1'' 4' 4 Sn (10 mL) was added hexamethylditin (80 mg, 0.24 mmol), and a catalytic amount of freshly prepared Pd(0)(PPh 3)4 (~10 mg). The reaction mixture was heated at reflux for 7 h under N 2, adding 2-5 mg of catalyst throughout the time period. The reaction mixture was then cooled to rt, filtered and washed through celite with EtOAc (50 mL) to remove the palladium, and the solvent was evaporated. The residue was subjected to silica gel column chromatography using (EtOAc:

NH 4 solution, 100%:3 mL) and RP HPLC (ACN:20 mM ammonium bicarbonate,

90%:10%, retention time 28 min) and dried in vacuo to yield [144] (45 mg, 43%) as a clear

1 wax. H NMR (CDCl 3) δ 0.32 (s, 9H, Sn(CH 3)3), 1.25-1.32 (m, 2H, H3a′, H5a′), 1.94 (m,

3H, H3e′, H5e′, H4 ′), 2.41 (d, 2H, J = 6.6 Hz, NC H2CH), 2.72 (t, 2H, J = 5.2 Hz, H4 ′′),

2.82 (t, 2H, J = 5.6 Hz, H3 ′′), 3.18 (bs, 2H, H2a′, H6a′), 3.57 (s, 2H, H1 ′′), 3.837 (s, 3H,

OCH 3), 3.844 (s, 3H, OCH 3), 4.52 (bs, 2H, H2e′, H6e′), 6.52 (s, 1H, H5 ′′), 6.60 (s, 1H,

13 H8 ′′), 7.21 (s, 1H, H3), 7.48-7.52 (m, 2H, H6, H7), 7.76 (s, 1H, H4). C NMR (CDCl 3) δ -

9.17 (Sn(CH 3)3), 28.7 (C4 ′′), 32.8 (C3 ′, C5 ′), 34.3 (C4 ′), 44.6 (C2 ′, C6 ′), 51.5 (C3 ′′), 56.10

(OCH 3), 56.11 (OCH 3), 56.4 (NCH 2CH), 64.1 (C1 ′′ ), 109.6 (C3), 111.0 (C5 ′′), 111.6 (C8 ′′),

111.8 (C7), 126.8 (C4a ′′), 127.5 (C8a ′′), 129.7 (C4), 133.7 (C6), 136.5 (C3a), 147.5 (C6 ′′),

147.8 (C7 ′′), 149.1 (C2), 155.1 (C7a), 160.2 (CO). MS-ES + m/z 594 ( 116 SnMH +, 63), 596

118 + 120 + + 118 ( SnMH , 72), 598 ( SnMH , 100%); HRMS-ES calculated for C 29 H38 N2O4 Sn:

598.1865, found 598.1893.

Mark Ashford, PhD Thesis 2010 184 Chapter 8 - Experimental

N-(6-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexyl)-5-

(trimethylstannyl)benzofuran-2-carboxamide [145]

To a solution of the bromo 5'' 4'' H CO 3 O 1' derivative [75] (100 mg, 0.12 N 2 O H3CO1'' N 4' H 3 mmol) in anhydrous toluene (10 4 Sn mL) was added hexamethylditin

(46 mg, 0.14 mmol), and a catalytic amount of freshly prepared Pd(0)(PPh 3)4 (~10 mg).

The reaction mixture was heated at reflux for 7 h under N 2, adding 2-5 mg of catalyst throughout the time period. The reaction mixture was then cooled to rt, filtered and washed through celite with EtOAc (50 mL) to remove the palladium, and the solvent was evaporated. The residue was subjected to silica gel column chromatography using (EtOAc:

NH 4 solution, 100%:3 mL) and RP HPLC (ACN:MeOH:20 mM ammonium bicarbonate,

80%:10%:10%, retention time 35 min) and dried in vacuo to yield [145] (38 mg, 49%) as a

1 clear wax. H NMR (CDCl 3) δ 0.33 (s, 9H, Sn(CH 3)3), 1.43-1.48 (m, 4H, H3 ′, H4 ′), 1.58-

1.70 (m, 4H, H2 ′, H5 ′), 2.50 (t, 2H, J = 8.0 Hz, H6 ′), 2.70 (t, 2H, J = 6.0 Hz, H4 ′′), 2.82 (t,

2H, J = 6.0 Hz, H3 ′′), 3.49 (m, 2H, H1 ′), 3.55 (s, 2H, H1 ′′), 3.83 (s, 3H, OCH 3), 3.84 (s,

3H, OCH 3), 6.52 (s, 1H, H5 ′′), 6.59 (s, 1H, H8 ′′), 6.30 (m, 1H, NH), 7.43-7.54 (m, 3H, H3,

13 H6, H7), 7.79 (s, 1H, H4). C NMR (CDCl 3) δ -9.14 (Sn(CH 3)3), 27.1 (C4′), 27.3 (C3 ′),

27.4 (C4 ′′), 28.9 (C2 ′), 29.8 (C5 ′), 39.5 (H1 ′), 51.2 (C3 ′), 55.9 (C6 ′), 56.08 (OCH 3), 56.10

(OCH 3), 58.7 (C1 ′′ ), 109.7 (C3), 110.0 (C5 ′′), 111.56 (C8 ′′ ), 111.59 (C7), 126.4 (C8a ′′),

127.0 (C4), 128.1 (C4a ′′), 130.3 (C3a), 133.8 (C4a ′′ ), 136.8 (C6), 147.4 (C6 ′′), 147.7 (C7 ′′ ),

148.7 (C2), 155.3 (C7a), 159.1 (CO). MS-ES + m/z 597 ( 116 SnMH +, 78), 599 ( 118 SnMH +,

120 + + 118 88), 601 ( SnMH , 100%); HRMS-ES calculated for C 29 H41 N2O4 Sn: 599.1721, found

599.1718.

Mark Ashford, PhD Thesis 2010 185 Chapter 8 - Experimental

[123 I](4-((3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)methyl)piperidin-1-yl)( benzofuran-2-yl)methanone [ 123 I][65]

To a solution of stannane [144] (100 µg) in O H CO O 123 3 N EtOH (100 µL) was added Na I (3.06 mCi), N H3CO CAT (1 mg/ mL, 100 µL) and HCl (0.1M, 123I 100 µL). After 5 min at rt, the reaction was quenched with Na 2S2O5 (50 mg/mL, 100 µL) and NaHCO 3 (50 mg/mL, 100 µL). Mobile phase (ACN:H 2O (0.1% TFA), 50:50 v/v, 250 µL) was added and the solution was injected onto a semipreperative C-18 RP HPLC column. The peak at a retention time of 8.9 min at a flow rate of 3 mL/ min was collected to give [123 I] [65] (1.90 mCi, 87% RCY).

[123 I] N-(6-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1 H)-yl)hexyl)-1-benzofuran-2- carboxamide [ 123 I][78]

To a solution of stannane [145] (100 H CO 3 O N O µg) in EtOH (150 µL) was added H3CO N H Na 123 I (4.55 mCi), CAT (1 mg/ mL,

123I 100 µL), HCl (0.1M, 100 µL). After 5 min at rt, the reaction was quenched with Na 2S2O5 (50 mg/mL, 100 µL) and NaHCO 3 (50 mg/mL, 100 µL). Mobile phase (ACN:H 2O (0.1% TFA), 40:60 v/v, 150 µL) was added and the solution was injected onto a semipreperative C-18 RP HPLC column. The peak at a retention time of 18.5 min at a flow rate of 5 mL/ min was collected to give [123 I] [78] (3.13 mCi, 78% RCY).

Mark Ashford, PhD Thesis 2010 186 Chapter 8 - Experimental

8.6. Pharmacological Methods

8.6.1. Membrane Preparation

Male Sprague-Dawley rats were sacrificed by CO 2 administration followed by cervical dislocation. The whole brain plus cerebella was removed and placed in 10 volumes (w/v) of ice cold 0.32 M sucrose buffer and homogenised with a polytron (PCU-Kinematica,

Bioblock, Switzerland) with one 20 sec burst at setting 6 on ice. The homogenate was then centrifuged at 900g for 20 min at 4 oC. After centrifuging, the top layer is removed, and the pellets are resuspended in 10 volumes (w/v) of ice cold buffer (50 mM Tris-HCl, pH 7.4) and incubated at 37 oC for 30 mins. The homogenate was then centrifuged at 22 000g for

20 mins at 4 oC. The pellets were resuspended in buffer to yield a protein concentration of

5 mg/mL. Membranes were stored at -80 oC until required. Protein measurements were made by Lowry’s method using bovine serum albumin as standard.

8.6.2. In Vitro binding Assay for Sigma-1 Receptor

The test compounds were dissolved up in DMSO to make up a stock solution of 1000 µM.

Six concentrations of test compound were made up with buffer in six test tubes by serial dilution of the stock solution. A drop of Tween 80 was used in the first solution to help dissolve the compound if required. The brain membranes were thawed, placed in 10 volumes (w/v) of buffer (50 mM Tris-HCl, pH 7.4), homogenised and centrifuged at 22

000g. The supernatant was discarded and the membranes homogenised in fresh buffer. The inhibition constant of the test compound (IC 50 ) was determined by incubating aliquots (0.1 mL), in triplicate, of diluted brain membranes at 37 oC for 150 min with six or more concentrations of test compound (usually 10 -5 to 10 -10 M), together with a trace amount (3 nM) of [ 3H](+)-pentazocine in a final volume of 0.8 mL. Non-specific binding was determined by competition with non-radioactive Haloperidol (10 µM). Incubation was

Mark Ashford, PhD Thesis 2010 187 Chapter 8 - Experimental terminated by rapid filtration through a Whatman GF/B glass fibre filter pre-soaked in

0.5% polyethenemine solution. Each sample tube and filter was immediately washed with

3 aliquots of 5 mL ice-cold 50 mM Tris-HCl at pH 7.4. The filters were placed in scintillation vials along with scintillation fluid (2 mL) and counted with a β-scintillation counter (Packard) to measure the amount of bound radioactivity. IC 50 values were calculated by an iterative non-linear least squares curve fitting computer program called

Prism. The whole method was performed in triplicate to obtain the average and standard deviation of at least three IC 50 determinations.

8.6.3. In Vitro binding Assay for Sigma-2 Receptor

The inhibition constant of the test compound (IC 50 ) was determined by incubating aliquots

(0.1 mL), in triplicate, of diluted brain membranes preparation (as above) at 25 oC for 90 min with six concentrations of test compound (10 -5 to 10 -10 M), together with a trace amount of [ 3H]DTG (10 nM) and non-radioactive (+)-pentazocine (1 µM). Non-specific binding was determined by competition with non-radioactive haloperidol (10 µM).

Incubation was terminated by rapid filtration through Whatman GF/C glass filter. Each sample tube and filter was immediately washed with 3 aliquots of 5 mL ice-cold 50 nM

Tris-HCl at pH 7.4. Filters were counted with a β-scintillation counter (Packard) to measure the amount of bound radioactivity. IC 50 values were calculated by the program

Prism. The whole method was performed in triplicate to obtain the average and standard deviation of at least three IC 50 determinations.

8.6.4. In Vivo Biodistribution Studies for [ 123 I][65] and [ 123 I][78]

Directly after radiolabelling, [ 123 I] [65] was diluted in a solution of saline and EtOH (2%) to achieve a concentration of 0.74 Mbq/100 µL. The Biodistribution of [ 123 I] [65] was studied

Mark Ashford, PhD Thesis 2010 188 Chapter 8 - Experimental in normal male Sprague-Dawley rate, weighing between 230 g and 280 g. Twenty four rats were injected with the radiolabelled compound (0.74 Mbq/100 µL) via the tail vein (after warming the tail in warm water). After 15 min, 30 min, 1, 3, 6 and 24 h post injection, 4 rats per time point were sacrificed by CO 2 adminsitration. The rats were weighed, and 17 organs were removed rom each rat and weighed. The organs in the study include the liver, spleen, kidney, muscle, heart, blood, luncgs, stomach, GIT, thyroid, pancreas, thymus, tail, bone, testes, and brain. The amount of radioactivity in each organ was measured using an automated gamma counter and the percent injected dose (% ID) for each organ was calculated by comparison with a diluted standard solution of the initial injected dose. The density of radioactivity of each organ (% ID/g) was found by dividing the % ID for each tissue by the weight of the tissue.

The in vivo biodistribution of [ 123 I] [78] was performed using the same procedure as for

[123 I] [65] .

8.6.5. In Vivo Competition Studies for [ 123 I][78]

The radiotracer [ 123 I] [78] was synthesised and formulated to 185 Mbq/100 µL of saline.

The competition drugs used for this study were unlabelled non-radioactive [78] , (+)- pentazocine and haloperidol. Male Sprague-Dawley rats were grouped into four groups of four (four rats per competition drug plus four rats for control), with each group having similar weights. Competition drugs were dissolved in saline to a concentration of 1 mg/kg rat. Rats in each group were injected via the tail vein with drugs (as listed below) and sacrificed 1 h later by CO 2 administration and cervical dislocation. Rats were injected with the competition drug 5 min prior to injection of [ 123 I] [78] .

Group 1 [123 I] [78] (control)

Group 2 non radioactive [78] and [ 123 I] [78]

Mark Ashford, PhD Thesis 2010 189 Chapter 8 - Experimental

Group 3 (+)-pentazocine and [ 123 I] [78]

Group 4 haloperidol and [ 123 I] [78]

Blood was collected immediately after sacrifice from the heart cavity. Organs dissected were liver, spleen, kidney, lungs, heart, bladder, stomach, GIT, thyroid, pancreas, testis, thymus, and brain. Organs were placed into pre-weighed tubes and weighed and counted using a Wallac Gamma Counter 1470. From weights and counts acquired, a percent injected dose per gram (% ID/g) was calculated against standards of the injection solution.

8.6.6. In Vivo Stability Studies for [ 123 I][78]

Three male Sprague-Dawley rats were each administered [ 123 I] [78] (37 Mbq/100 µL saline per rat) into the tail vein. A rat each was sacrificed at 15, 30 and 60 min post injection. The cortex, lung and spleen were removed and the blood was collected. The blood was added to a heparinised tube and centrifuged at 2000 g for 5 min to collect the plasma (200-300 µL).

The samples were weighed and the activity counted. The organs were minced, ACN (1 mL) was added and the tissues were sonicated using an unltrasonic probe for 2 min. The smple was then centrifuged at 2000 rpm for 5 min and the supernatant was collected. A second extraction was performed and the combined ACN fraction evaporated to dryness. The activity was reconstituted in MeOH (50 µL) and applied onto an aluminium backed silica

TLC plate (20 x 20 cm), along wth the radioactive and non-radioactive standards. After the

TLC was run in EtOAc:MeOH (9:1), the radioactivity on the plate was counted using a

Berthold radio-TLC scanner using the Bertold TLABE software. The radioactivity in the remaining tissue was counted by the Wallac counter and compared with the counted activity before the extraction to calculate the extraction efficiency.

The radioactive CPM (counts per minute) after extraction is corrected for decay (13 h half life of 123 I) using the following formula:

Corrected CPM = CPM before extraction x e (ln2 / 780 min x min between counts)

Mark Ashford, PhD Thesis 2010 190 Chapter 8 - Experimental

The extraction efficiency formula is:

Extraction efficiency = 100 – (corrected CPM after extraction x 100 / CPM before extraction).

Using the Bertold TLABE software, the radioactive peaks were integrated.

8.6.7. Lipophilicity Estimations

The lipohilicity of each lignad was examined by determination of the log P7.5 value using a

HPLC method.77 Samples were analysed using a C18 column (X-Terra, 5 µ, 4.6 x 250 mm) and a mobile phase of MeOH and 0.1 M phosphate buffer at a flow rate of 1 mL/min. The log P values were estimated by comparing HPLC retention times of test compounds with retention times of standards having known log P values. The standards used were aniline, benzene, bromobenzene, ethylbenzene and trimethylbenzene. A calibration curve of log P versus ln retention time was generated. The equation was linera with an r2 of 0.9954.

Mark Ashford, PhD Thesis 2010 191 Chapter 9 - References

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