Combinatorial Synthesis of Cocaine Analogues

and

Competition Reactions between Glucosyl Donors and Galactosyl Donors - A Study of Glycosidation Reactions
and

Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

Ph.D.dissertation submitted by: Anne Bülow Department of Chemistry University of Aarhus August 2004

i
i ii iii iv v vi

Table of Contents
Table of Contents........................................................................................................... iii Preface ............................................................................................................................vii Acknowledgements...................................................................................................... viii List of Appendices ..........................................................................................................ix List of Abbreviations.......................................................................................................x Summary ........................................................................................................................xii

Chapter I: Combinatorial Synthesis of Cocaine Analogues

1

INTRODUCTION ...........................................................................................................1 1.1 1.2 1.3 COCAINE – A STIMULANT OF THE CENTRAL NERVOUS SYSTEM .............................1 DEVELOPMENT OF MEDICATIONS FOR TREATMENT OF COCAINE ABUSE ...............3 POTENTIAL DOPAMINE TRANSPORTER LIGANDS ....................................................4 1.3.1 1.3.2 1.4 Phenyltropanes ..........................................................................................4 Various Structural Classes of Potential Dopamine Transporter Ligands 10

PURPOSE OF THIS WORK .......................................................................................12

2

COMBINATORIAL CHEMISTRY............................................................................14 2.1 2.2 2.3 INTRODUCTION .....................................................................................................14 IDENTIFICATION OF ACTIVE COMPOUNDS IN A LIBRARY ......................................15 SOLID PHASE VERSUS SOLUTION PHASE APPROACHES.........................................17

3

THE GRIGNARD REACTION...................................................................................19 3.1 3.2 THE GRIGNARD REAGENT ....................................................................................19 3.1.1 Grignard Reagents in Conjugate Addition ..............................................20 THE GRIGNARD REACTION IN COMBINATORIAL CHEMISTRY ...............................21

4

SYNTHESIS OF THE TROPANE SKELETON .......................................................22 4.1 SYNTHESES OF COCAINE AND OTHER TROPANES ..................................................22 iii

4.1.1 4.1.2 4.1.3 4.2 4.3 5

[3+4] Cycloaddition of Pyrroles and α,α’-Dibromoketones .................. 24 Tandem Cyclopropanation/Cope Rearrangement of Vinylcarbenoids with Pyrroles ................................................................................................... 26 Tropanes from Pyrrolidine Derivatives .................................................. 27

SOLID PHASE CONSIDERATIONS ........................................................................... 29 CONCLUSION........................................................................................................ 30

TWO- AND THREE-DIMENSIONAL SOLUTION PHASE COMBINATORIAL LIBRARIES OF 3- AND 8-SUBSTITUTED TROPANES FROM MULTICOMPONENT GRIGNARD REAGENTS.......................................................................... 31 5.1 GENERATION OF A TWO-DIMENSIONAL LIBRARY FROM MULTICOMPONENT GRIGNARD REAGENTS ......................................................................................... 31 5.1.1 5.1.2 5.1.3 5.1.4 5.2 Designing the Library ............................................................................. 31 Initial model studies................................................................................ 33 Synthesis and Analysis of the Two-Dimensional Library ...................... 34 Biological Results for the Two-Dimensional Library ............................ 37

GENERATION OF A THREE-DIMENSIONAL LIBRARY FROM MULTICOMPONENT GRIGNARD REAGENTS ......................................................................................... 40 5.2.1 5.2.2 5.2.3 Initial model studies................................................................................ 40 Synthesis and Analysis of the Three-Dimensional Library .................... 42 Biological Results for the Three-Dimensional Library .......................... 44

5.3 5.4 6

APPLYING TWO- AND THREE-DIMENSIONAL LIBRARIES TO OTHER SYSTEMS ...... 46 SUMMARY AND CONCLUDING REMARKS ............................................................. 47

BICYCLO[3.2.1]OCTANE ANALOGUES OF PHENYLTROPANES.................. 49 6.1 INTRODUCTION .................................................................................................... 49 6.1.1 6.1.2 6.1.3 6.2 6.2.1 8-Oxa Analogues .................................................................................... 49 8-Carba Analogues.................................................................................. 50 Biological Activity of Non-Amines........................................................ 51 Attempts to Synthesise Methyl 3-(4-iodophenyl)-bicyclo[3.2.1]octane carboxylate and its 8-methyl and 8,8-dimethyl Analogues ................... 52

RESULTS AND DISCUSSION................................................................................... 52

iv

6.2.2

Attempts to Perform Conjugate Additions to Methyl bicyclo[3.2.1]octa2,6-diene-2-carboxylate and Methyl bicyclo[3.2.1]oct-2-ene-2carboxylate ..............................................................................................53

6.2.3 6.2.4 6.3

Model Studies on Methyl Crotonate .......................................................57 Synthesis of an 8-Carbon Analogue........................................................59

CONCLUSIONS ......................................................................................................62

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors - A Study of Glycosidation Reactions
1 INTRODUCTION .........................................................................................................65 1.1 1.2 1.3 CARBOHYDRATES – UBIQUITIOUS MOLECULES ...................................................65 ACID-CATALYSED HYDROLYSIS OF GLYCOSIDES.................................................66 GLYCOSIDATION REACTIONS ...............................................................................69 1.3.1 2 The Trichloroacetimidate Method...........................................................73

RESULTS AND DISCUSSION....................................................................................76 2.1 COMPETITION REACTIONS USING TRICHLOROACETIMIDATES...............................76 2.1.1 2.1.3 2.2 2.3 2.4 2.5 Synthesis of Trichloroacetimidate Donors ..............................................76 Competition Reactions between Perbenzylated Gluco and Galacto Trichloroacetimidates..............................................................................78 COMPETITION REACTIONS USING GLYCALS AS DONORS ......................................82 RELATIVE REACTION RATES AMONG N-PENTENYL GLYCOSIDES .........................83 INVESTIGATION OF SUPPOSED SN2-TYPE REACTIONS ..........................................84 MECHANISTIC CONSIDERATIONS ..........................................................................86 2.1.2 Synthesis of the Competition Reaction Products ....................................77

3

CONCLUSIONS............................................................................................................88

v

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase
1 INTRODUCTION......................................................................................................... 91 1.1 1.2 1.3 1.4 2 AZASUGARS AS GLYCOSIDASE INHIBITORS .......................................................... 91 MECHANISM OF GLYCOSIDASE CATALYSED HYDROLYSIS................................... 92 SLOW INHIBITION................................................................................................. 93 1.3.1 The β-Method ......................................................................................... 95 DETERMINATION OF THERMODYNAMIC PARAMETERS ......................................... 96

RESULTS AND DISCUSSION ................................................................................... 97 2.1 DETERMINATION OF THERMODYNAMIC PARAMETERS FOR BINDING OF AZASUGARS TO β-GLUCOSIDASE ......................................................................... 97 2.1.1 2.2 2.3 2-Hydroxyl Analogues of Azasugars.................................................... 101 DISCREPANCY BETWEEN THERMODYNAMIC RESULTS OF BINDING OF ISOFAGOMINE AND 1-DEOXYNOJIRIMYCIN TO β-GLUCOSIDASE ........................ 102 DETERMINATION OF THERMODYNAMIC PARAMETERS BY NUMERICAL SOLUTION
OF DIFFERENTIAL EQUATIONS ........................................................................... 103

2.3.1 3

The Differential Equation Method........................................................ 103

SUMMARY AND CONCLUSIONS ......................................................................... 105

References....................................................................................................................... 107 Appendix 1-11

vi

ii

Preface

This Ph.D.-dissertation is based on work performed almost exclusively by the author, under supervision of Professor Mikael Bols at the Department of Chemistry, Aarhus University over the past four years. However, the work on enzyme kinetics was initiated in the spring 2000, but not finished until fall 2000 and has therefore been included and discussed briefly. The research has resulted in a number of scientific publications, which are attached as appendices. The results in appendix 4 have only been discussed briefly since most work was performed by Huizhen Liu and Xifu Liang, and the authors contribution was only associated with biological testing of the synthesised compounds. The authors contribution to appendix 7 was also minor and mainly associated with know-how related to the multicomponent Grignard reactions and the format of the synthesised libraries. The dissertation is divided into three separate and very different chapters. The first chapter is dealing with developing a combinatorial synthesis of cocaine analogues and has been conducted in the period August 2001 till present date. Chapter II presents a mechanistic study of glycosidation reactions and was mainly performed from November 2000 till August 2001. After that date the project was continued by master student Tine Meyer and fellow student Tomasz K. Olszewski. The last chapter consists of enzyme kinetic experiments for determination of thermodynamic parameters for the reaction of β-glucosidase with various inhibitors.

Anne Bülow, August 2004

vii

iii

Acknowledgements

First of all, I would like to thank my supervisor Professor Mikael Bols for giving med the opportunity to become a Ph.D. student in his group and for his inspiring ideas and enthusiasm. In addition, I thank ass. prof. Igor W. Plesner for a fruitful collaboration on the physical chemistry concerning the enzyme kinetic experiments. Tine Meyer and Tomasz K. Olszewski are thanked for finishing the glycosidation project. Biological testing of cocaine analogues was done in collaboration with molecular biologists at Psychiatric University Hospital, Risskov, and therefore, Ph.D. student Steffen Sinning and ass. prof. Ove Wiborg are kindly acknowledged for testing compounds and for their willingness to discus the biological part of the project. Laboratory technician Ib Thomsen is also thanked for his enthusiasm, chemistrytricks, and for providing starting materials when necessary. I would also like to thank all present and former co-workers from the bioorganic chemistry group for creating a magnificent atmosphere in the laboratory. Especially, Vinni Høyer Lillelund, Henrik Helligsø Jensen, Brian S. Rasmussen, and Kathrine Bjerre are thanked for numerous discussions on chemistry and other matters. All proofreaders are kindly acknowledged for their help on creation of this thesis. For financial support I thank Novo Nordisk A/S and the Lundbeck Foundation. Last but not least, I would like to thank my family and friends for their trust, love, and support. Especially, Marcus Simonsen, Tina Thorslund, Magdalena Pyrz, and Rikke Søe are thanked for cheering me up during creation of this thesis.

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iv

List of Appendices
Bülow, A.; Plesner, I. W.; Bols, M. J. Am. Chem. Soc. 2000, 122, 8567-8568.

Appendix 1:

Appendix 2:

Bülow, A.; Plesner, I. W.; Bols, M. Biochim. Biophys. Acta 2001, 1545, 207-215.

Appendix 3:

Plesner, I. W.; Bülow, A.; Bols, M. Anal. Biochem. 2001, 295, 186-193.

Appendix 4:

Liu, H.; Liang, X.; Søhoel, H.; Bülow, A.; Bols, M. J. Am. Chem. Soc. 2001, 123, 5116-5117.

Appendix 5:

Bülow, A.; Meyer, T.; Olszewski, T. K.; Bols, M. Eur. J. Org. Chem. 2004, 323-329.

Appendix 6:

Bülow, A.; Sinning, S.; Wiborg, O.; Bols, M. J. Comb. Chem. 2004, 6, 509-519.

Appendix 7:

Pedersen, H.; Sinning, S.; Bülow, A.; Wiborg, O.; Bols, M. Org. Biomol. Chem. 2004 accepted for publication

Appendix 8: Appendix 9:

Experimental section List of ligands used for evaluation of IC50 and Ki values for potential cocaine antagonists

Appendix 10: Appendix 11:

NMR spectra of compounds 111 and 112 Derivation of the Integrated Rate Equation for Slow-Binding Inhibitors Described by Model 1.

ix

v

List of Abbreviations
Acetamidobenzenesulfonyl azide Acetyl 1-Chloroethyl chloroformate Attention-deficit hyperactivity disorder 2,2’-Azobisisobutyronitrile Aryl Angstrom Benzyl tert-Butoxycarbonyl Butyl Benzoyl Catalyst Benzyloxycarbonyl Cyclooctadiene Correlation spectroscopy Cyclohexyl Dopamine Dopamine transporter trans,trans-Dibezylideneacetone 1,8-diazabicyclo[5.4.0]undec-7-ene Differential Equation N,N-Diisopropylethylamine N,N-Dimethylformamide (dimethylthio)methylsulfonium trifluoromethanesulfonate Deoxyribonucleic acid 2,6-di-tert-butyl-4-methylpyridine Enzyme Enzyme-Inhibitor complex Equivalent Enzyme-Substrate complex Electronspray mass spectrometry Ethyl 9-Fluorenylmethoxycarbonyl Fucose Galactose Gist-Brocades Gas chromatography Glucose Hour(s) or human 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Hexamethylphosphoramide High Performance Liquid Chromatography High Resolution Mass Spectrometry Inhibitor Inhibition concentration, 50 % Iodonium dicollidine perchlorate

ABSA Ac ACE-Cl ADHD AIBN Ar Å Bn Boc Bu Bz cat Cbz cod COSY Cy DA DAT Dba DBU DE DIEA DMF DMTST DNA DTBMP E EI Equiv. ES ESMS Et Fmoc Fuc Gal GBR GC-MS Glc h HBTU HMPA HPLC HRMS I IC50 IDPC x

iPr Ki LDA LG MBHA Me Mes Min MMP-1 Ms MS n NBS NE NET NIS NMR Nu Oct P Pent Ph ppm PS QSAR rds RNA rt RTI S SAR SER SERT TBACN TBAF TBDMS Tf TFA THF TLC TMS Tol Troc WIN

isopropyl Inhibition constant Lithium diisopropyl amide Leaving group 4-Methylbenzhydrylamine Methyl Mesityl (2,4,6-trimethylphenyl) Minute(s) Matrix metalloproteinase-1 Methanesulfonyl Molecular sieves Normal N-Bromosuccinimide Norepinephrine Norepinephrine transporter N-Iodosuccinimide Nuclear Magnetic Resonance Nucleophile Octyl Product n-Pentenyl Phenyl parts per million Polystyrene Quantitative structure-activity relationship Rate-determining step Ribonucleic acid Room temperature Research Triangle Institute Substrate Structure-activity relationship Serotonin Serotonin transporter Tetrabutylammonium cyanide Tetrabutylammonium fluoride tert-Butyldimethylsilyl Trifluoromethanesulfonyl Trifluoroacetic acid Tetrahydrofuran Thin layer chromatograhpy Trimethylsilyl p-Methylphenyl 2,2,2-Trichloroethoxycarbonyl Sterling-Wintrop Institute

xi

vi

Summary

As cocaine abuse has become a serious social and economic burden in the Western world the need for a potential medication that can facilitate withdrawal has grown. A suitable therapeutic agent is thought to be obtained via interaction with the dopamine transporter and we therefore set out to develop a combinatorial synthesis of tropane-based compounds that could be possible dopamine transporter ligands. Initially, several de novo approaches to the tropane skeleton were suggested, but they were all discarded because of synthetic difficulties. Instead, we set out to develop a combinatorial synthesis based on an existing tropane skeleton using anhydroecgonine methyl ester as starting material. By addition of multicomponent Grignard reagents to the α,β-unsaturated ester, 10 sublibraries of 5 3-substituted tropanes each were constructed. By variable mixing of the Grignard reagents 25 different compounds were obtained in a two-dimensional format, where each library member was contained in 2 sublibraries. This was done to facilitate identification of biologically active compounds in the mixtures. Screening of the library led to identification of two new compounds that bind to monoamine transporters with high affinity and inhibit reuptake. In addition, it was shown that 3-alkyltropanes were poor monoamine transporter ligands. To extend the gain associated with the combinatorial synthesis, a third dimension was added to the library. This was done via a multicomponent N-alkylation resulting in a library of 5 anhydroecgonine methyl ester analogues that was subsequently reacted with multicomponent Grignard reagents. In that way, 125 compounds were synthesised in 15 sublibraries of 25 compounds each. Three high affinity compounds were synthesised individually and showed similar affinity to the dopamine transporter as their N-methyl analogue. Since a nitrogen is not prerequisite for interaction of a cocaine analogue with the dopamine transporter, 8-carba analogues were suggested as potential cocaine antagonists. These 8-carba analogues of phenyltropanes were thought to be obtained through a similar conjugate addition of Grignard reagents to the 8-carbon analogue of anhydroecgonine methyl ester. It turned out to be impossible to perform the conjugate addition in absence of a nitrogen in the ringsystem. Thus, the ring nitrogen was crucial for the reaction to occur perhaps through stabilisation of a boat-like transition state via coordination of the Grignard reagent to the nitrogen. Instead an 8-carba analogue was synthesised by first ring opening of the bicyclic system followed by

xii

conjugate addition whereupon a ring closing metathesis resulted in reconstruction of the bicyclic skeleton. In another project the difference in electron-withdrawing properties of equatorial and axial C4-OBn substituents were used to investigate glycosidation reactions. For that reason several glucosyl and galactosyl donors were synthesised and their reactivity compared in direct competition experiments where the donors were forced to compete for an acceptor under various reaction conditions. In general, the reactivity of the galactosyl donors was four to five times higher than the corresponding glucosyl donors indicating that the orientation of the C4 substituent affected the reactivity of the donors. The observation suggests that the transition state of the reaction has considerable positive charge (SN1-like reaction) and that this positive charge is less destabilised for galacto stereochemistry (axial C4 substituent) compared to gluco stereochemistry (equatorial C4 substituent). However, when triflates were used to catalyse the reaction the difference in reactivity of galactosyl and glucosyl donors was equalised. As an explanation for this observation it was suggested that the presence of a triflate increases the rate of oxocarbenium ion formation to a rate where it is no longer rate-determining and therefore a difference in reactivity is not observed. This was supported by a triflate catalysed experiment performed at low temperature, where a 5:1 ratio of galactoside versus glucoside product was obtained. The last project presented in this thesis deals with determination of thermodynamic parameters for the interaction between various azasugars and β-glucosidase. It was shown that the slow binding of isofagomines and azafagomines was driven by entropy whereas binding of 1-deoxynojirimycin was driven by enthalpy. The gain of entropy for isofagomines and azafagomines was addressed to the presence of a nitrogen in the anomeric position and to some extent explained by the release of water molecules, resulting in a more disordered state. The enthalpy gain associated with binding of 1-deoxynojirimycin is probably obtained by a stabilising effect from the 2-hydroxyl group via a strong hydrogen bond to the enzyme. Based on these results, 2-hydroxyl analogues of isofagomine were designed and turned out to be more potent inhibitors of various glycosidases than their 2-deoxy analogues.

xiii

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Chapter I: Combinatorial Synthesis of Cocaine Analogues 1 Introduction
1.1 Cocaine – a Stimulant of the Central Nervous System
Cocaine is an alkaloid isolated from the leaves of Erythroxylon coca a shrub growing primarily in South America. Its stimulating effects has been known since ancient times by the Incas, who regarded chewing coca leaves as a gift from the Gods.1 Cocaine was not isolated until the 1850s and its addictive properties was not realised until the end of that century. As an example of the ignorance of cocaine’s addictive properties, Sigmund Freud used it in the 1880s as treatment against other kind of addictive compounds such as morphine and alcohol, resulting in addiction to cocaine as well.1 In addition, cocaine was not omitted from CocaCola until 1903.2 Today cocaine is seen as one of the most addictive drugs of abuse and the economic and social costs associated with cocaine abuse is a growing problem in the US and Western Europe.
N
5 7 6 8

CO2CH3
1 4 2

O
3

Ph

O R-Cocaine, 1

Figure 1.1 R-Cocaine - the naturally occurring stereoisomer.

Only the naturally occurring R-isomer (referring to stereochemistry at C-1) of cocaine is addictive and has many physiological effects e.g. it is a local anaesthetic, a vasoconstrictant, and is known to increase heart rate and blood pressure. However, concerning drug abuse the most relevant effect is its euphoria producing ability and its reinforcing properties (i.e. the increase in the probability of repeated use of cocaine).3 Along with other rewarding effects such as reduced fatigue and psychomotorial stimulation these effects finally lead to abuse and addiction.4
NH2 OH HO HO Dopamine (DA), 2 NH2 HO N H Serotonin (SER), 3 HO HO Norepinephrine (NE), 4 NH2

Figure 1.2 Structure of the three natural monoamine neurotransmitters.

1

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Primarily, the pharmacological effects of cocaine arise due to inhibition of reuptake of monoamines (Figure 1.2) at the serotonin, norepinephrine, and dopamine transporters (SERT, NET, and DAT, respectively) in the mammalian brain. Affinities for binding and inhibition of reuptake are shown in Table 1.1.5, I
IC50 (nM) DAT [3H]WIN35428d 102±12 241±18a SERT [3H]paroxetine 1045±89 112±2b NET [3H]nisoxetine 3298±293 160±15c

R-Cocaine

Binding Uptake

Table 1.1 Binding potencies and inhibition of reuptake by cocaine at the three monoamine transporters. a Ki value for displacement of [3H]DA uptake. b Ki value for displacement of [3H]SER uptake. c Ki value for displacement of [3H]NE uptake. d Structures of displaced ligands are shown in appendix 9.

However, the primary mechanism of action of cocaine has been ascribed to its ability to inhibit the dopamine transporter (known as the dopamine hypothesis).3 The dopamine transporter consists of 12 transmembrane α-helices and is found in dopaminergic neurons. The primary structure of the protein is known but no three-dimensional structure is available at present. The biochemical action of cocaine on the dopaminergic nervous system is outlined in Figure 1.3.6

Figure 1.3 Cocaine's action on the dopaminergic nervous system - the dopamine hypothesis.
I

It is important mention that IC50 values are only comparable within the same series of experiments, since they depend on the assay conditions. Therefore, if possible Ki values are presented.

2

Chapter I: Combinatorial Synthesis of Cocaine Analogues

When a nerve terminal in the normal state (Figure 1.3A) is stimulated, dopamine ( ) is released from vesicles in the presynaptic neuron and diffuses across the synaptic cleft where dopamine receptors ( ) on the postsynaptic neuron are stimulated to mediate a response. The stimulating action of dopamine ends by its reuptake by the dopamine transporter ( ) into the presynaptic neuron, where it is partly enzymatically inactivated and partly stored in vesicles. When cocaine ( ) is present (Figure 1.3B), it binds to the dopamine transporters and thereby blocks the transporter function acting as an indirect dopamine agonist. The result is a flooding of the synapse with excess dopamine, which prolongs signalling at key brain synapses. This build up of dopamine in the synaptic cleft is thought to be responsible for the reinforcing properties of cocaine and perhaps for some of the euphorigenic effects as well. The dopamine hypothesis has been further emphasised from experiments involving knock-out mice, genetically lacking the dopamine transporter, in which cocaine had no stimulant effect.7 However, other experiments involving DAT knock-out mice have shown an effect of cocaine suggesting that other systems e.g. the serotonergic or norepinephrinergic, are involved as well.8 Recently, it has also been suggested that glutamate, a well-known participant in memory and learning, plays an important role with respect to cocaine addiction.9 And also the muscarinic M5 receptor has turned out to be important for self-administration of cocaine, since M5-deficient mice self-administer cocaine to a much lower level than wild-type controls.10

1.2 Development of Medications for Treatment of Cocaine Abuse
At present there are no suitable medications for the treatment of cocaine abuse. Thus it is highly desired to find a compound that could facilitate withdrawal as is available e.g. for treatment of heroin abuse (methadone) and alcohol abuse (antabuse). A variety of medicinal chemistry approaches for development of medications for cocaine abuse are possible. Among these are the use of cocaine-specific monoclonal antibodies for rapid and effective reduction of toxic substances in the blood serum.11 Using this strategy cocaine cannot enter the brain and is prevented from interacting with its target. Another point of intervention is through the dopamine receptors where both agonists (direct or indirect) and antagonists have been suggested as partial abuse treatment candidates.6 This approach is being complicated by the existence of different subtypes of dopamine receptors (D1-D5). The most plausible way to interfere with the dopaminergic nervous system must be through the 3

Chapter I: Combinatorial Synthesis of Cocaine Analogues

dopamine transporter. Studies have suggested that cocaine binds to the dopamine transporter at a different site than dopamine.12 This observation suggest that it is possible to design therapeutic agents that bind to the cocaine recognition site either without inhibiting dopamine transport (i.e. cocaine antagonists) or inhibiting it weakly (i.e. cocaine partial agonists). A selective dopamine transporter ligand can also serve to be useful as a diagnostic tool when used as a marker for deficits in the density of receptor population e.g. with respect to Parkinson’s disease which is characterised by the degeneration of dopaminergic neurons.14 Selective dopamine transporter inhibitors are already used as a drug today. An example is methylphenidate (5, Ritalin, Figure 1.4). It is used as a stimulant in the treatment of attentionOCH3 O HN

Methylphenidate, 5 IC50 83 nM

Figure 1.4 Methylphenidate - a selective dopamine transporter inhibitor. Inhibition data is obtained from displacement of [3H]WIN35428 binding to rat striatal membranes.13

deficit hyperactivity disorder (ADHD) in children and for depression in adults.15 Nevertheless, clinical studies using methylphenidate showed no efficacy for the treatment of cocaine dependence.16

1.3 Potential Dopamine Transporter Ligands
Throughout the years, a large amount of potential dopamine transporter ligands have been synthesized, the largest class being the phenyltropanes.5 But several other groups of compounds have been developed as well. An introduction to the phenyltropanes will be given along with a short examination of other classes of important compounds binding to the dopamine transporter.

1.3.1 Phenyltropanes
Compared to cocaine the main difference of phenyltropanes is that they have an aryl group directly attached to the 3-position of the tropane ring instead of through a 3β-benzoyl ester as is present in cocaine. This group of compounds have been known since 1973, where the first synthesis of a phenyltropane was published by Clarke et al.17 The synthesis was carried out 4

Chapter I: Combinatorial Synthesis of Cocaine Analogues

from (1R, 5S)-anhydroecgonine methyl ester 7 prepared from R-cocaine, which was reacted with an aryl Grignard reagent at low temperature to give the 1,4-addition products 8 and 9 (Scheme 1.1). The vast majority of phenyltropanes have been synthesised by the same route.
CO2CH3 N
1N aq. HCl

COOH N
1. POCl3

O O R-cocaine, 1

Ph 6

OH

N

CO2CH3

2. MeOH, H+

7
ArMgBr low temp, Et2O

CO2CH3 N Ar + N CO2CH3 Ar

8

9

Scheme 1.1 Synthesis of phenyltropanes from R-cocaine.

Variations have been carried out in other positions than the 3-position. Especially, changing the ester functionality in the 2-position and the substituent at the nitrogen. In addition, a few C-6/C-7-substituted analogues have been synthesised. 1.3.1.1 Structure-Activity Relationship Studies Based on the large number of biological data available for phenyltropanes, structure-activity relationships (SAR) and quantitative SAR studies have provided information about important interaction sites between the dopamine transporter and substrates.3-5 It is suggested that the most important factor for activity of a phenyltropane to the DAT is its configuration – the preferred being the R-configuration.18 This feature is also seen for cocaine itself, where the Risomer is about 150 times more potent than the S-isomer (IC50 for inhibition of [3H]WIN35428 binding to rat striatal membranes: 0.102 µM and 15.8 µM respectively).18 It is also evident from several other analogues e.g. for WIN35065 the R-enantiomer (WIN350652) has been noted to be approximately 800 fold more active than the S-enantiomer (WIN35065-3, Figure 1.5).19
CO2CH3 N Ph N CO2CH3 Ph

WIN35065-2, R-10 IC50 40.7 nM

WIN35065-3, S-10 IC50 32400 nM

Figure 1.5 Difference in inhibition of binding of [3H]cocaine to mouse striatal membranes of enantiomers.

5

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Significant and important effects on activity are obtained by substitution at C-3. Replacement of the aromatic ring of the benzoyl group in cocaine by an aryl group as in the phenyltropane series, have shown to enhance activity by a factor of up to 50.3 The stereochemistry at C-3 seems to be of less importance, since a 3α-substituent causes to 6-membered ring to flip to the boat conformation, which will position the 3α-substituent in a pseudoequatorial position that is approximately the same position as for the 3β-substituent.20 The necessity of a 3-aryl substituent has been mentioned throughout literature to be of great importance for obtaining affinity for the DAT. But no tropanes with simple 3-alkyl substituents have been reported! The aryl group is thought to interact via hydrophobic bonding to a lipophilic pocket in the protein. In Table 1.2 binding affinities for a selection of phenyltropanes are presented. As seen halogen substituents increase the binding affinities where 3,4-Cl2>4-Cl>4-I>4-Br>4-F but also other electron withdrawing or donating groups tend to increase affinity compared to the unsubtituted phenyltropane.21 Furthermore a decrease in affinity is observed for large para substituents such as isopropyl and butyl, which is supported by QSAR studies ascribing it to sterical hindrance.22 Contrary to that observation, compounds having a second aryl group attached in the para position of a phenyl group via a linker have also shown to bind strongly to DAT. This have been ascribed to the presence of a remote phenyl binding domain.23 It is also interesting to note that R = benzyl has poor affinity for the DAT, while extending the chain by one carbon to R = phenethyl increases the affinity approximately 100 times.

6

Chapter I: Combinatorial Synthesis of Cocaine Analogues
CO2CH3 N R

R WIN35065-2
NH2

IC50/nM IC50/nM [3H]WIN35428 [3H]DA uptake 23.0±5.021 24.8±1.321 10.1±0.1021 8.14±1.321 2.12±0.121 49.8±2.3a,24 557±7925 616±8425 23±0.5a,24 3.68±0.09a,24 1.96±0.09a,24 7.0±0.3a,24 -

R

IC50/nM IC50/nM [3H]WIN35428 [3H]DA uptake 55±223 68.5±7.123 >500b,26 597±5227 1.2±0.123 15.6±0.623 0.49±0.0429 3.7±0.1623 29.4±3.828 3.53±0.0928 1020±5228 70.5±1.028

NO2

OCH3

N3

F

WIN35428

15.7±1.421 1.17±0.121 1.81±0.3021 1.26±0.0421 1.09±0.0229 1.71±0.321

Cl

Br

I

RTI-55

CH2Ph

526±6523 5.14±0.6323 351±5223 885±18c,28 9.94±0.33c,28

Cl Cl

CH2CH2Ph

CH2CH2CH2Ph

CF3

13.1±2.221 46±6b,26

I

Table 1.2 Binding affinities and inhibition of reuptake at the DAT for selected phenyltropanes. a Ki value instead of IC50. b Ki values for inhibition of binding of [3H]GBR12935 instead of [3H]WIN35428. c Inhibition of binding of [3H]cocaine instead of [3H]WIN35428.

A 2β-carbomethoxy group has been thought to be crucial for binding of cocaine to the DAT, since replacing it by hydrogen, a carboxy group, or an N-methylcarboxamido group decreased activity by 25-2000 fold.3 The interaction has been suggested to happen through hydrogen bonds. 2β-substituted phenyltropanes have been designed to explore whether this is also the case for phenyltropanes. Changing the methyl ester for isopropyl or phenyl esters (13) does not affect the binding affinity for the DAT, but the selectivity for DAT over NET and SERT is increased.30 Neither changing the 2β-carbomethoxy group for an alkyl group as in 11 and 12 affects the binding affinity for the DAT, since other compounds bearing alkyl or arylvinyl groups at the C-2 7

Chapter I: Combinatorial Synthesis of Cocaine Analogues

position were found to exhibit nanomolar and subnanomolar affinities for binding to the dopamine transporter (Figure 1.6).31 2β-heterocyclic analogues have also been synthesised and shown good binding affinity at the DAT and at present the heterocyclic isoxazole analogue 14 is claimed to be the strongest compound binding to the DAT.32 Taken together these results show that the substituent in the 2-position is of minor importance in the phenyltropane series and a large degree of flexibility is allowed.
Ph CO2Ph N Cl N Cl N Cl N N O Cl

11 Ki 1.46 nM

12 Ki 1.21 nM

13 IC50 1.99 nM

14 IC50 0.59 nM

Figure 1.6 Examples of binding affinities to DAT for 2β-substituted phenyltropanes. Ki values are obtained from displacement of [3H]mazindol from rat striatal membranes whereas IC50 values are obtained from displacement of [3H]WIN35428.30-32

The presence of a nitrogen in position 8 that can participate in either an ionic bond or a hydrogen bond to the transporter have also been proposed to be necessary for binding.3 Several N-substituted phenyltropanes have been synthesised and from these it has been demonstrated that N-substituents do not affect DAT affinity significantly compared to their N-methyl analogues exemplified by similar IC50 values for 15 and 16 (Figure 1.7).33 Moreover, it has been observed that N-substitutions could increase the specificity for DAT over SERT and NET.34
CO2CH3 N F N CO2CH3 F

15 IC50 22.6 nM

WIN35428, 16 IC50 17.5 nM

Figure 1.7 Examples of N-substituted phenyltropane analogues. IC50 values for binding to the DAT are obtained from displacement of [3H]cocaine from monkey caudate-putamen membranes.33

Most N-substituted analogues have been synthesised from their N-methyl analogues by demethylation followed by N-alkylation.35 It turns out that the presence of a nitrogen in position 8 is not strictly necessary, since exchanging the nitrogen for an oxygen or a carbon

8

Chapter I: Combinatorial Synthesis of Cocaine Analogues

can be done without severe loss in binding potency.36,37 This will be subjected to further discussion in section 6.2. Only a few phenyltropanes bearing substituents at C-6 or C-7 have been synthesised. Among these, β-oriented hydroxyl groups have been introduced with the rationale of being capable of making intramolecular hydrogen bonds to the 8-nitrogen (Figure 1.8). In that way, the effect of reducing the nucleophilicity of the nitrogen was explored.38
CO2CH3 N N HO HO WIN35065-2, 10 IC50 65 nM 17 IC50 235 nM 18 IC50 6150 nM CO2CH3 N CO2CH3

Figure 1.8 Examples of C-6/C-7-hydroxylated phenyltropanes. Inhibition data are obtained from displacement of [3H]WIN35428 binding to the DAT in monkey caudate-putamen.

From the studies it was shown that the 7-hydroxylated compound 17 is more potent at the DAT than the 6-hydroxylated counterpart 18.38 A small increase in selectivity of DAT over SERT is also observed for the hydroxylated compounds. As a conclusion to the SAR studies a pharmacophore model can be suggested (Figure 1.9).

Figure 1.9 A general accepted pharmacophore model for binding of phenyltropanes to the DAT.

From the huge amount of phenyltropanes synthesized, it appears that a pharmacophore model cannot be deduced unambiguously and several deviations remains unexplained by the model. Therefore further explorations of this class of compounds are of great interest in the search for dopamine transporter ligands that could be used as a potential cocaine abuse treatment.

9

Chapter I: Combinatorial Synthesis of Cocaine Analogues

1.3.2 Various Structural Classes of Potential Dopamine Transporter Ligands
1.3.2.1 Piperidine Analogues of Phenyltropanes As truncated analogues of phenyltropanes lacking the ethylene bridge, piperidines were suggested to be of interest as dopamine transporter ligands.39 Given the reduced molecular size relative to the tropanes, less conformational restriction, and the fact that they still contain the suggested pharmacophores, made the piperidines interesting new analogues. Their synthesis has been carried out from arecoline (19) similar to the synthesis of phenyltropanes by a 1,4-conjugate addition of Grignard reagents and has resulted in several interesting compounds (Figure 1.10).
N CO2CH3 N CO2CH3 N CO2CH3 Cl N E Cl

Arecoline, 19

(-)-20 IC50 769±19 nM

(-)-21 IC50 24.8±1.6 nM

(±)-22 IC50 197±8 nM

Figure 1.10 Structure of selected piperidine analogues of phenyltropanes. E = CO2CH3.

It is of great interest to see that introduction of a p-chloro substituent as in 21 increases the binding affinity by 31 fold compared to 20. A similar effect is seen for the corresponding phenyltropanes, where the potency by introduction of a p-chloro substituent is increased by 20 fold, suggesting that the piperidine analogues and phenyltropanes bind to the same site at the DAT.39

1.3.2.2 Benztropines Benztropine (23) consists of a tropane ring having a 3α-diphenylmethoxy substituent. It was first synthesised in 1952 and was subsequently demonstrated to be useful as an anticholinergic drug in the treatment of Parkinson’s disease.40 It is a stimulant of the central nervous system, where it acts through inhibition of dopamine reuptake just as cocaine and the phenyltropanes, but since benztropine does not self-administer in rhesus monkeys, it is thought to bind to a different site on the dopamine transporter than cocaine.41

10

Chapter I: Combinatorial Synthesis of Cocaine Analogues
CO2CH3 N O N O F N O F

F Benztropine, 23 IC50 312 nM 24 Ki 11.8 nM

F (R)-25, IC50 2040 nM (S)-25, IC50 10.9 nM

Figure 1.11 Structure of benztropine (23) and selected analogues. IC50 and Ki values are obtained from inhibition of [3H]WIN35428 binding to the DAT (monkey caudate-putamen).

A wide variety of benztropine analogues have been synthesised, especially phenyl ring substituted analogues where the difluoro compound 24 has turned out to be the most potent benztropine analogue against the DAT at present (Figure 1.11).42 It is also interesting to note that for the hybrid compound 25 (difluoropine), the S-isomer is more than 150 times more potent than the R-isomer.40 This is the opposite stereochemistry than required for cocaine binding and is again suggesting different binding sites for cocaine and benztropines.

1.3.2.3 GBR compounds Another important group of potential cocaine antagonists is the GBR compounds. In 1980 the first synthesis of an aryl 1,4-diaryl piperazine as potential DAT ligand was reported – a class of compounds now knowns as the GBR compounds.43 Until date one of the most interesting compounds is GBR12909 (26), which binds tightly to the dopamine transporter and inhibit the action of dopamine uptake (Figure 1.12).44 In addition it is very selective against the dopamine transporter.
PhCH2CH2CH2N N O F

GBR12909, 26 IC50 3.7 nM
125

F

Figure 1.12 Inhibition of binding of [ I]RTI-55 by GBR12909 to rat caudate.

A difference in the action of GBR12909 and cocaine is seen. GBR12909 produces a relatively modest and long-lasting increase in the dopamine concentration, which does not cause the

11

Chapter I: Combinatorial Synthesis of Cocaine Analogues

same degree of euphoria compared to cocaine’s burst of pleasure. In addition GBR12909 has been shown to decrease cocaine-seeking behavior.45 1.3.2.4 Bivalent Ligand Approach Recently, it was proposed to employ a bivalent ligand approach being capable of bridging neighbouring recognition sites on the transporters.46 By linking two binding moieties differing the length of the linker connecting them, it was assumed to obtain transporter selectivity based on a difference in location of neighbouring sites at the respective monoamine transporters. Piperidine-based bivalent inhibitors linked by varying methylene chains at C-2 turned out to be inhibitors of the DAT and the SERT or just the SERT depending on linker length.47 A similar study was reported by linking 3-aryl tropanes through amide linkages at the 2-carbomethoxy groups resulting in compounds 27-30 (Figure 1.13).48
N O N H ( )n N H O N 27, n = 1: 28, n = 2: 29, n = 4: 30, n = 6: Ki 65.1 nM Ki 21.7 nM Ki 18.4 nM Ki 6.7 nM

Cl Cl

Figure 1.13 Example of bivalent tropane-based ligands inhibiting [125I]RTI-55 at hDAT.

Some of the bivalent tropanes attained good binding affinities and turned out to have high discrimination ratios (IC50(uptake)/Ki(binding)), which suggest that the ligand binding site and the dopamine binding site are not identical.

1.4 Purpose of this Work
The work described in this chapter of the thesis will deal with the development of methods for generation of combinatorial libraries of tropane-based compounds. Having established such a method, a large amount of potential cocaine antagonists can be synthesised and in that way it is possible to gain more insight into mode of binding of ligands to the dopamine transporter. Section 2 will give an introduction to combinatorial chemistry mainly concerning solution phase combinatorial approaches. This is followed by a short introduction to the Grignard reaction that has been employed numerous times in the combinatorial synthesis of tropanes describes in section 5.

12

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Section 4 describes the effort put into trying to construct the tropane skeleton in a way that could be useful for generation of combinatorial libraries. Not many positive results are presented in this section, but it has been included because of the considerable time spend on it. Section 5 describes the synthesis of two- and three-dimensional combinatorial libraries consisting of 25 and 125 compounds, respectively (Figure 1.14). Most of these results are also found in appendix 6 in a published article.
CO2CH3 R2 N R1

Figure 1.14 General structure of tropanes synthesised in two- and three-dimensional libraries.

The last section (section 6) deals with the attempts to synthesise carbon analogues of the above-mentioned tropanes. This turned out to be considerable more difficult than expected and ended up being a study of conjugate additions to α,β-unsaturated esters. In addition, a carbon analogue was synthesised by changing the synthesis route.

13

Chapter I: Combinatorial Synthesis of Cocaine Analogues

2 Combinatorial Chemistry
2.1 Introduction
Compared to traditional synthetic chemistry, combinatorial chemistry consists of a range of techniques allowing rapid synthesis of a large number of compounds in few reactions through combination of different building blocks as shown in Scheme 2.1.49
Traditional synthesis: A + B AB 1 compound

Combinatorial synthesis:

A1 A2 A3

B1 B2 B3 AnBm n x m compounds

An

Bm

Scheme 2.1 Comparison of traditional and combinatorial synthesis.

Combinatorial chemistry can be used for systematically generation of compound libraries either as mixtures or as single compounds in arrays. It has developed into a very powerful tool in the drug discovery process, because linked with high throughput screening, it allows the pharmaceutical industry to screen a large amount of compounds either for lead generation or lead optimisation. Combinatorial chemistry dates back to the mid-1980s where parallel synthetic approaches for solid phase synthesis of peptides using pins50 and tea-bags51 were introduced. Originating back from Merrifield’s solid phase tetrapeptide synthesis in 1963,52 peptides and peptide-like molecules have been the target of numerous combinatorial syntheses, primarily because or their easy preparation on solid phase. Also with the introduction of split and mix synthesis in the early 1990s the number of peptides that could be generated in a few reaction steps exploded.53 But from a pharmaceutical point of view, peptides are not very interesting molecules, since they have limited use as drugs because of their poor oral absorption and their rapid clearing times. Thus, extensions to the existing methods were needed and in the beginning of the 1990s several publications for solid phase synthesis of more drug-like molecules such as benzodiazepines appeared. E.g. Bunin et al. constructed a library of 192

14

Chapter I: Combinatorial Synthesis of Cocaine Analogues

1,4-benzodiazepine derivatives from 2-aminobenzophenones, amino acids, and various alkylating agents according to Scheme 2.2. 54,55
RC O RB NH2 O Support
N-Fmoc-amino acid fluoride CH2Cl2

RB

NHFmoc NH O
1. Piperidine, DMF 2. 5% AcOH, DMF, 60 C
o

RB

H N N

O RC

Support

Support

RA

RA

RA
1. lithiated 5-phenyl2-oxazolidinone 2. Alkylating agents, DMF

RB 192 Benzodiazepines

RD N N

O RC
TFA/H2O/Me2S (95:5:10)

RB

RD N N

O RC

Support

RA

RA

Scheme 2.2 Synthesis of 192 1,4-benzodiazepines on solid support.

2.2 Identification of Active Compounds in a Library
Since the essential part of combinatorial chemistry is primarily to discover a biological active compound, an important part is to identify this compound. When employing arrays of single compounds high throughput screening is necessary for having an efficient lead identification and active compounds are directly identified. When employing mixtures, the deconvolution process is more complex and therefore a number of different methods have been developed to facilitate identification of possible lead compounds. Most of these methods are based on synthesis of sublibraries as for iterative methods, which involves resynthesis of several sublibraries.56 Positional scanning and indexed libraries are other approaches, where one building block is held constant in a specified position at a time. Positional scanning was introduced by Houghten et al. in a synthesis of a hexapeptide library.57 The method involves synthesis of hexapeptide pools prepared with one position fixed (O) and the rest randomised (X) (Figure 2.1). By applying 18 different amino acids, 15

Chapter I: Combinatorial Synthesis of Cocaine Analogues

they obtained more than 34 million different compounds in 108 pools that were tested for biological activity. From the biological results, the amino acid giving rise to the most potent peptide can be determined for each position. By synthesising the combination of defined amino acids in the most active mixture in each position, the most active compound was identified.
1. A1 X X X X X Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 OXXXXX XOXXXX XXOXXX XXXOXX XXXXOX XXXXXO 18 mixtures " " " " " Total: 108 mixtures 18. A18 X X X X X

Figure 2.1 Concept of positional scanning of a hexapeptide library.

In 1995 Pirrung and Chen introduced a technique, that is essentially the same as positional scanning, where indexing permits the preparation and identification of active non-oligomeric compounds.58 The library was represented by a matrix, where each axis has as many elements as are in each set of building blocks (m and n in Figure 2.2). This method is applicable to any molecule that can be assembled in a simple chemical process from multiple subunits. Using this technique a library of carbamates, suggested to be acetylcholinesterase inhibitors, was prepared. By reacting 9 alcohols with 6 isocyanates they obtained 54 compounds in 15 sublibraries. From the biological screening of sublibraries the most potent compound was
R R' 1 R X (m) + n Y R' m R R' n R' R' Y (n) + mX R n R' Rm R1
n sublibraries of m compounds m sublibraries of n compounds

Figure 2.2 Two-dimensional indexed libraries resulting in n x m compounds in m + n sublibraries.

identified directly from the two sublibraries showing inhibition. Indexed libraries and positional scanning are not limited to two dimensions but can be extended by using multicomponent reactions such as the Ugi four-component reaction59 and the Biginelli reaction60 or by introducing more reaction sites or polymeric chains.

16

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Additional procedures for facilitating deconvolution have been applied for solid phase library synthesis e.g. encoding by tagging either by binary codes61 or encoding with a sequence.62

2.3 Solid Phase versus Solution Phase Approaches
Most combinatorial approaches have been conducted by solid phase synthesis, but within the last decade, methodologies for generation of solution phase combinatorial libraries have also attracted great interest as an alternative route for drug discovery and lead optimisation.63 Advantages and disadvantages are associated with both solid phase and solution phase approaches. It is clear that solid phase combinatorial chemistry benefits from its easy handling and the possibility of using excess reagents to drive reactions to completion. In spite of that, solution phase approaches have obtained considerable interest and include several advantages over solid phase synthesis such as 1) a shorter reaction sequence, since there is no need for linker manipulation, attachment, and detachment from a resin, 2) an unlimited number of reactions are directly applicable to solution phase combinatorial chemistry, whereas solid phase approaches often need extensive development and optimisation of reactions, 3) reactions can be monitored by several methods (TLC, GC-MS, HPLC etc), 4) large excesses of reagents are not needed, and 5) the scale of reaction is not limited by loading capacity and generation of sufficient quantities of libraries are allowed. In addition the development of solid phase reagents and scavenger resins have found widespread use in solution phase combinatorial chemistry. The major drawback of solution phase combinatorial synthesis is a requirement of similar reactivities among building blocks when mixtures are involved. This can often be controlled by slow addition of reagents or by employing no more than stoichiometric amounts of reagents. In addition, it is highly desirable to use high yielding reactions, since purification is often difficult to perform on mixtures. These drawbacks are probably the reason, why most solution phase combinatorial approaches have been carried out in a parallel fashion i.e. synthesis of single compounds in arrays. Making single compounds also offers the possibility of easier automation of the syntheses, which is employed by many pharmaceutical companies by the use of robots. As an example of a solution phase mixture based library Smith and co-workers reported a synthesis of 1600 amides/esters obtained from reaction of 40 acid chlorides with 40 nucleophiles (amines and alcohols, Scheme 2.3).64

17

Chapter I: Combinatorial Synthesis of Cocaine Analogues
O + R Cl (40) O + 40 R Cl
80 sublibraries of 40 compounds each

40

R'OH or R'NH2 R 80 R

O OR' O NHR'

R'OH or R'NH2 (40)

Scheme 2.3 Smith's approach to 1600 esters/amides via mixture based solution phase combinatorial synthesis.

The library was constructed in an indexed manner generating 80 sublibraries of 40 compounds each giving a total of 1600 different compounds. From the library 31 was identified as a lead compound for the NK3 receptorII and 32 showed affinity for matrix metalloproteinase-1 (MMP-1)III was identified (Figure 2.3).
O Ph N N NC Cl O N H

31, NK3 inhibitor

32, MMP-1 inhibitor

Figure 2.3 Lead compounds identified from Smith's ester/amide library by indexed libraries.

With respect to discovering dopamine transporter ligands by combinatorial chemistry, only one study has been reported. This involved screening of Houghten’s positional scanning combinatorial hexapeptide library build from D-amino acids containing 186 peptides.65 Twelve hexapeptides were resynthesised individually and turned out to bind to the DAT (IC50 1.7-9 µM). A variety of organic reactions have been employed for generation of solution phase combinatorial libraries, among these the Grignard reaction.66,67

II

NK3 receptor antagonists are thought to have a potential role in anxiety-related and psychotic disorders such as schizophrenia. III Inhibition of MMP-1 is beneficial in the treatment of arthritis and corneal ulceration.

18

Chapter I: Combinatorial Synthesis of Cocaine Analogues

3 The Grignard Reaction
The Grignard reaction is without doubt one of the most classical name reactions in organic chemistry.68 Dating back from the work of Barbier and Grignard around the year 1900, the utility of the reaction has grown with the years. Today the synthetic chemist takes advantage of the generality of the Grignard reaction as a building block for an impressive range of structures and functional groups. In general a Grignard reaction consists of two discrete steps. First, the Grignard reagent is generated from magnesium and an organic halide (R-X) usually in ethereal solvents such as Et2O or THF (Scheme 3.1).
Mg + R X RMgX ½ (R2Mg + MgX2)

Scheme 3.1 Generation of Grignard reagents.

Subsequently, the freshly prepared reagent can act both as a carbon nucleophile that undergoes addition or substitution reactions and as a strong base deprotonating acidic substrates, giving conjugate bases or elimination products.

3.1 The Grignard Reagent
Generation of the Grignard reagent (RMgX) is a complex process, which depend on several factors. In general the rate of insertion is faster when the halide is an iodide with the decreasing rate of insertion being dependent on the halide in the order I>Br>Cl.69 On the other hand, the reactivity of the reagent is dependent on the halide in the opposite order (Cl ≥ Br >> I), which is caused by the increased polarity of the carbon-magnesium bond due to higher electronegativity of earlier halides.70 The reactivity of the reagent is also highly dependent on the R group. The general reactivity is allyl, benzyl > primary alkyl > secondary alkyl, cycloalkyl ≥ tertiary alkyl, aromatic > vinyl. Since the insertion process is an oxidative addition the reduction potential of R-X can be used as a guideline for the reactivity of a given halide. Hence, it is important to realise that the more reactive a Grignard reagent is, the higher is the probability of generation of Wurtz-type homocoupling products (R-R). Other factors altering the reactivity of a Grignard reagent are e.g. the solvent and the Schlenk equilibrium. The great utility of Grignard reagents are associated with the fact that they can react with most organic functional groups containing polar multiple bonds (e.g. carbonyl groups, 19

Chapter I: Combinatorial Synthesis of Cocaine Analogues

nitriles, sulfones, imines), highly strained ringsystems (e.g. epoxides, cyclohexenes), acidic hydrogens (e.g. alkynes), and some highly polar single bonds (e.g. carbon-halogen, metalhalogen).

3.1.1 Grignard Reagents in Conjugate Addition
Grignard reagents can also add to conjugated carbon-carbon multiple bonds present in e.g. α,β-unsaturated carbonyl compounds. Especially, the conjugate addition to enones have been subjected to intense studies. Enones can react with Grignard reagents either through carbonyl addition giving 1,2-addition or as olefins resulting in generation of the 1,4-addition product. The degree of 1,4- versus 1,2-addition can to some extent be controlled by the sterical hindrance of either the Grignard reagent or the electrophile. Another typical way to obtain 1,4-addition products is by catalysing the reaction with Cu(I) species generating more soft organocopper nucleophiles, which have larger tendency to undergo 1,4-addition. The conjugate addition to α,β-unsaturated ester take place less efficiently than to enones due to the less electron poor double bond. Again the yields of the conjugate addition product can be increased by the presence of Cu(I). In fact, only a few examples of uncatalysed 1,4-additions of Grignard reagents to α,β-unsaturated methyl esters exist (Figure 3.1). The unsaturated esters 7, 19, and 33 all undergo 1,4-addition without Cu(I) catalysis upon treatment with PhMgBr.17,39,71
N CO2CH3 N CO2CH3 H3CO2C N CO2CH3 19 7 33

Figure 3.1 α,β-unsaturated methyl esters that undergo uncatalysed 1,4-addition upon treatment with phenyl magnesium bromide at low temperature.

Interestingly, these examples all afford the possibility of conformational fixation of an intermediate through coordination to the nitrogen. This will be discussed further in section 6.2.2 The reaction of Grignard reagents with electrophiles is considered to be complex, and to vary depending on the given reaction. Two mechanistic possibilities are generally proposed for addition of Grignard reagents to electrophiles i.e. through a single-electron transfer or a polar mechanism.72 When adding Grignard reagents in a conjugate manner it has been suggested to 20

Chapter I: Combinatorial Synthesis of Cocaine Analogues

happen through a cyclic mechanism.73 This has been questioned by several authors – one of the reasons being that generation of the proposed six-membered transition state is hardly possible for cyclic conjugated systems such as 2-cyclohexenone.74,75

3.2 The Grignard Reaction in Combinatorial Chemistry
As a tool for generating combinatorial libraries, the Grignard reaction has also found great importance. Most approaches have been conducted in solid phase syntheses,76 but a few examples of solution phase combinatorial synthesis using Grignard reagents exists (Scheme 3.2).
R1Br R2Br R3Br N Rn N O O R1MgBr R2MgBr R3MgBr
three component Grignard reagent

OH R
CH 3 OO RC N Ph
3

OR

Mg 2 eq Et2O

Rn

Rn

3 compounds

6 compounds

OH R Rn

HO RC

OR

Rn Ph OR

3 compounds

3 compounds

Scheme 3.2 Preparation of multicomponent Grignard reagents and their use in synthesis of libraries of secondary and tertiary alcohols, ethers, and 2-substituted quinolines. Generation of stereocenters have not been taken into account in this scheme.

Bearing in mind that 2-alkyl- and 2-alkenylquinolines have shown promising activity against leishmanian protozoas, libraries of 2-substituted quinolines were generated from mixtures of Grignard reagents and quinolinium salts.77 In addition, model studies employing multicomponent Grignard reagents have been conducted on α-azidobenzyl ethers, aldehydes, and esters.66,67

21

Chapter I: Combinatorial Synthesis of Cocaine Analogues

4 Synthesis of the Tropane Skeleton
Initially, it was suggested to develop an efficient method usable for a combinatorial de novo approach to the tropane skeleton. For that reason previous literature syntheses of the tropane skeleton were of great interest, since it might be possible to find methods developed for traditional organic synthesis of one compound, that could further developed into a suitable method for a combinatorial approach to the tropanes. The tropane skeleton consists of an 8azabicyclo[3.2.1]octane moiety containing a seven-membered ring with a bridgehead. Sevenmembered carbocycles are an important class of organic compounds but they have been less studied than their lower homologues, which might be due to synthetic difficulties. However, several attempts have been made to synthesise the tropane skeleton.

4.1 Syntheses of Cocaine and other Tropanes
The pioneering work on tropane syntheses was done especially by Willstätter starting in the late 19th century. Among other things, he developed a synthesis of tropinone (36) and was the first to synthesise cocaine from tropinone in 1903.78 However, the tropinone synthesis required 16 steps from cycloheptanone and was therefore overshadowed by an elegant onepot synthesis of tropinone reported by Robinson in 1917.79,80 This reaction was based on a double Mannich-type reaction of succinic aldehyde (34), methylamine, and the calcium salt of acetonedicarboxylic acid (35). Willstätter modified Robinson’s tropinone synthesis and employed the mono methyl ester of acetonedicarboxylic acid (37) for direct generation of the 2-carbomethoxy group present in cocaine (1).81 Via this improved route, cocaine could be synthesised in only three steps (Scheme 4.1).
O + O 34 O OR
CH3NH2

R N O
Na/Hg

CO2CH3 N OH
Bz2O

CO2CH3 N OBz

O COOH R = H, 35 R = CH3, 37

R = H, Tropinone, 36 R = CO2CH3, 38

39

Cocaine, 1

Scheme 4.1 Willstätter's synthesis of cocaine from 1923.

A new interesting approach to cocaine was developed by Tufariello and co-workers in 1978.82,83 By a nitrone-based entry to the tropane skeleton, they were able to control the 22

Chapter I: Combinatorial Synthesis of Cocaine Analogues

stereochemistry of the ester function in cocaine, which is often a problem. Their key compound was the hydroxylamine 40 that upon dehydration was converted into nitrone 41. 41 underwent a 1,3-dipolar cycloaddition to give the tricyclic compound 42. Methylation and cleavage of the nitrogen oxygen bond afforded ecgonine methyl ester (39) that was easily benzoylated to provide racemic cocaine (1) (Scheme 4.2). Even though this represents an elegant way to the tropanes, the yield of the cycloaddition step was rather low.
O NHOH

-H2O

N O O OCH3

O
∆ 4 - 11 %

N

CO2CH3

1. MeI, CH2Cl2 2. Zn, AcOH, 47 %

CO2CH3 N OH

O

OCH3 40

42 41

39
BzCl, Na2CO3 benzene, 37 %

CO2CH3 N O O 1 Ph

Scheme 4.2 Tufariello's approach to racemic cocaine via a nitrone-based cycloaddition.

Most approaches to the synthesis of cocaine built on construction of tropinone (36) that is further derivatised to cocaine. Tropinone has been obtained from 2,6-cycloheptadiene by Michael addition with methanolic methylamine.84 Other examples for generation of the tropane skeleton employs reaction of pyrroles with cyclopropanones,85 addition of oxyallyl cations to pyrroles,86,87 and tandem cyclopropanation/Cope rearrangement of vinylcarbenoids with pyrroles (Scheme 4.3).88
O OCH3 O + RNH2 Br Br N2 + R N O + R N

Tropanes

O +

R N

N O

Scheme 4.3 Different routes to the tropanes.

23

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Newer enantioselective approaches to cocaine involve selective deprotonation of tropinone using a chiral lithium amide resulting in S-cocaine achieved in 5 steps from tropinone (36) with an overall yield of 78 %.89 A procedure that could probably be used for obtaining the natural R-enantiomer by changing the chiral base. In addition Lin et al. proposed a route to enantiomerically pure natural cocaine from D-glutamic acid.90 At present no combinatorial approaches to the tropane skeleton have been reported, but a literature search revealed three examples of solid phase syntheses of tropanes. One using Robinson’s pathway by reacting a resin-bound ε-amine of lysine with succinic dialdehyde and acetonedicarboxylic acid.91 In another study a tropane scaffold was attached to a dihydropyran linker and subjected to further transformations in the C-3 position.92 The last solid phase approach is based on a 1,3-dipolar cycloaddition of a 3-oxidopyridinium betaine to activated resin-bound olefins.93

4.1.1

[3+4] Cycloaddition of Pyrroles and α,α’-Dibromoketones

Oxyallyl cations can be generated from α,α’-dibromoketones and it is well known from literature that they can react as dienophiles in [3+4] cycloadditions with dienes such as cyclopentadiene, furan and pyrrole.94,95 By using a pyrrole in such a reaction one would obtain a tropane scaffold in a very simple way. This reaction seems to be an attractive short route to the tropanes and it also offers possibilities for introduction of combinatiorial chemistry by using different α,α’-dibromoketones and pyrroles. In addition, a double bond (C-6/C-7) is formed, which can be used as a handle for introduction of further substituents. It is interesting to obtain C-6/C-7-substituted cocaine analogues, since relatively few compounds of this type are reported.5 Therefore, experiments on generating tropanes from a [3+4] cycloaddition were initiated. 4.1.1.1 Synthesis of α,α’-dibromoketones Paparin et al. have synthesized several tropane scaffolds by [3+4] cycloadditions from α,α’dibromoketones using Et2Zn to generate the oxyallyl cations.96,97 However, the synthesis of α,α’-dibromoketones is not straightforward and in addition they decompose easily. According to a literature procedure, a synthesis of 1,3-dibromo-1-phenyl-2-propanone (44) was done by bromination of phenylacetone (43) in acetic acid.98 This was followed by cycloaddition of the α,α’-dibromoketone 44 to Boc-pyrrole generating the 8-azabicyclo[3.2.1]octene 45 in 53 % yield (Scheme 4.4). 24

Chapter I: Combinatorial Synthesis of Cocaine Analogues
O Ph
Br2 AcOH, 76 %

O

Boc N Ph Boc N
Et2Zn, toluene 53 %

Ph O 45

Br 43

Br 44

Scheme 4.4 Synthesis of 8-azabicyclo[3.2.1]octene 45 according to Paparin's procedure.

From these experiments it was expected that by synthesizing the corresponding α,α’dibromoketone 47 from methyl acetoacetate (46) and reacting it with Boc-pyrrole, the 2carbomethoxy analogue of 45 would be generated directly i.e. compound 48 (Scheme 4.5).
O O OCH3 Br 46 Br 47 48 O O OCH3 Boc N Boc N
Et2Zn

CO2CH3 O

Scheme 4.5 Proposed synthesis of 2-carbomethoxy analogue 48.

Several attempts were made to synthesize methyl-2,4-dibromo-acetoacetate (47) (Scheme 4.6). First a bromination of methyl acetoacetate was tried under the same conditions as the bromination of phenylacetone, though without success. Then the addition of bromine was carried out in CH2Cl2 but again no product formation was observed.99 Further attempts were made by bromination of 1,3-bis(trimethylsiloxy)-1-methoxybuta-1,3-diene (49) (Scheme 4.6).
O O OCH3
Br2 AcOH Br2 CH2Cl2

O

O OCH3

NBS THF, RT

TMSO

OTMS OCH3

Br

Br 47

Br2 CH2Cl2

46

49

Scheme 4.6 Attempts to synthesise methyl 2,4-dibromoacetoacetate. All turned out to be unsuccessful.

First the bis-TMS enol ether 49 was synthesized by a standard method from methyl acetoacetate (46) in two steps by first protecting the keto functionality using TMSCl and triethylamine in pentane followed by treatment with LDA and TMSCl in THF in an overall yield of 62 %.100 Attempts toward bromination of 49 were then carried out by using Br2 in CH2Cl2, but again without generation of the desired product. No better was the attempt using NBS as brominating agent. Due to problems associated with the synthesis of the starting material this method was eliminated from further investigations.

25

Chapter I: Combinatorial Synthesis of Cocaine Analogues

4.1.2 Tandem Cyclopropanation/Cope Rearrangement of Vinylcarbenoids with Pyrroles
Another way to approach the tropane skeleton is by reacting rhodium-stabilized vinylcarbenoids with pyrroles,88 a reaction that can be done enantioselectively either by using chiral auxiliaries at the vinylcarbenoid or by employing chiral proline derived catalysts.101 An obvious choice for generation of a cocaine analogue, is by using vinyldiazomethane 50, which upon reaction with Boc-pyrrole result in formation of 51 as shown by Davies et al. (Scheme 4.7).102 The reaction proceeds through generation of a vinylcarbenoid that undergo a tandem cyclopropanation/Cope rearrangement.88
O OCH3 N2 50 51 + Boc N
Rh2(O2CC7H15)4 hexane, 63 %

Boc N

CO2CH3

Scheme 4.7 Synthesis of 8-azabicyclo[3.2.1]octadiene 51 through a tandem cyclopropanation/Cope rearrangement done by Davies et al.

51 was then thought to undergo 1,4-addition of Grignard reagents in a combinatorial fashion using a method developed in our lab.66 This route seems very attracting, since not only a handle for introduction of substituents in the C-6/C-7 double bond is obtained, in addition it offers the possibility of making both 8-carba, 8-oxa, and 8-thia bicyclic analogues by employing cyclopentadienes, furans, and thiophenes instead of the pyrrole. 4.1.2.1 Synthesis of Methyl 2-Diazobut-3-enoate (50) According to Davies’ procedure, Et3N was used as base for diazo transfer from p-acetamidobenzenesulfonyl azide (p-ABSA) to methyl acetoacetate (46) in the preparation of methyl diazoacetoacetate (52).103 Reduction of 52 with sodium borohydride in methanol proceeded to give the desired alcohol 53 in 82 % yield. The last step in the synthesis of 2-diazobut-3-enoate (50) was dehydration of alcohol 53 by phosphorous oxychloride, reported to be done in 38 % yield.104 This procedure was tried several times with no positive outcome. Instead the elimination reaction was successfully carried out using MsCl and base, which turned out to give the desired vinyl diazo compound 50 in 37 % yield (Scheme 4.8).

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Chapter I: Combinatorial Synthesis of Cocaine Analogues
O O OCH3 46
p-ABSA, Et3N CH3CN, 19h, rt 85 %

O

O OCH3 N2 52

NaBH4 MeOH, 20 min, 0oC, 82 %

OH O OCH3 N2 53

MsCl, Et3N CH2Cl2, 0oC to rt overnight, 37 %

O OCH3 N2 50

Scheme 4.8 Synthesis of methyl 2-diazobut-3-enoate (50).

Alternatively, methyl 2-diazobut-3-enoate (50) was synthesised from 3-butenoic acid according to Bulugahapitiya’s procedure.105 Esterification of 3-butenoic acid (54) using AcCl in MeOH gave the β,γ-unsaturated ester 55. Subsequently, a diazo transfer from p-ABSA using DBU as base was carried out from 55, resulting in the desired vinyl diazo compound 50 in 48 % yield (Scheme 4.9). The relative low yields of 50 are probably due to its easy decomposition.
O OH 54
AcCl MeOH 49 %

O OCH3 55

DBU, p-ABSA CH3CN, rt overnight 48 %

O OCH3 N2 50

Scheme 4.9 Another route to methyl 2-diazibut-3-enoate (50).

The reaction of 50 with Boc-pyrrole catalysed by rhodium catalysts, described by Davies et al., was performed yielding the desired 8-azabicyclo[3.2.1]octadiene 51 in 52 % yield (Scheme 4.10). The following attempts to perform at 1,4-conjugate addition of phenyl magnesium bromide to form 56 did not succeed – this will be discussed further in section 6.2. Because of the problems considering the Grignard reaction, it was decided to ignore this route to the tropanes.
Boc N

O OCH3 N2 50
Rh2(O2CC7H15)4 pentane

Boc N

CO2CH3
PhMgBr Et2O, -40oC

CO2CH3 Boc N Ph 56

51, 52 %

Scheme 4.10 Synthesis of 51 and attempts to add PhMgBr in a conjugate manner to form 56.

4.1.3 Tropanes from Pyrrolidine Derivatives
In 1979 Brownbridge et al. published a simple synthetic route to the 8-oxa analogue of cocaine.106 It was based on a [3+4] annulation of 1,3-bis(trimethylsiloxy)-1-methoxybuta-1,3diene (49) with 2,5-dimethoxytetrahydrofuran (57) and TiCl4 as activator for generation of 27

Chapter I: Combinatorial Synthesis of Cocaine Analogues

the bicyclic skeleton. 58 was readily reduced by sodium borohydride to the hydroxy compound 59, which upon benzoylation gave the 8-oxa analogue of cocaine, 60 (Scheme 4.11).
TMSO OTMS OCH3 49 57 + H3CO O OCH3
TiCl4 CH2Cl2, -78oC, 3h

O

CO2CH3 O 58, 79 %
NaBH4

CO2CH3 O O O 60
BzCl

CO2CH3 O OH Ph
pyridine

59

Scheme 4.11 Brownbridge's route to the 8-oxa analogue of cocaine, 60.

As proposed in Scheme 4.12, a similar methodology using a Boc-protected pyrrolidine instead of 2,5-dimethoxy-tetrahydrofuran, might be a way to obtain a tropane skeleton having a 2-carbomethoxy group.
Boc N Boc N OCH3 61
TMSO OTMS

CH3COOK, Br2 MeOH, 52 %

H3CO

H2, Raney Ni MeOH, 88 %

H3CO

Boc N OCH3 62
TMSO OTMS

49
OCH3 OCH3

49 TiCl4, CH2Cl2

TiCl4, CH2Cl2

Boc N

CO2CH3 O

Boc N

CO2CH3 O

63

64

Scheme 4.12 Proposed method for generation of tropanes.

The first challenge was to synthesize N-Boc-2,5-dimethoxypyrrolidine (62). Some 2,5-dimethoxylated pyrrolidines have been prepared by anodic oxidation of the corresponding protected pyrrolidines in methanol.107 However, it was decided to use the same procedure, which was used for dimethoxylation of furan (Scheme 4.12).108 This procedure was also successful for dimethoxylation of Boc-pyrrole to give 61 in 52 % yield. 61 underwent hydrogenation using Raney Nickel as catalyst to give the desired dimethoxylated pyrrolidine 28

Chapter I: Combinatorial Synthesis of Cocaine Analogues

62 in 88 % yield. Now both 61 and 62 could be used in a condensation reaction with the enol silyl ether 49. The condensation reaction was tried for the saturated compound 62 using TiCl4 as activator, but formation of the desired product was not seen. TLC analysis showed formation of at least 6 compounds, which have not been separated. A mass spectrum, however, showed a peak at m/z 206, which corresponds to the Boc-deprotected product of 64 (+ Na). Due to a very unclean reaction this approach was also discarded.

4.2 Solid Phase Considerations
Doing combinatorial chemistry on solid phase supports offers some advantages as described in section 2.3. Therefore, it was considered how to extend the above methods for generation of the tropane skeleton on solid phase. All three methods involve a pyrrole in which the nitrogen could be used as point of attachment to the solid support. A suitable linker was thought to be derived from succinic anhydride attached to pyrrole, which turned out to give 65 in 62 % yield. Using an amino-terminated PS resin (MBHA), pyrrole was attached to the solid phase through the linker by a simple amide bond, as used in peptide chemistry (Scheme 4.13).109
COOH O H N O
1. K, EtOH 2. O O 62 % O , THF

N

NH2 , HBTU, DIEA DMF/CH2Cl2

N H 66

N O

65

Scheme 4.13 Pyrrole attached to a MBHA resin through a linker derived from succinic anhydride.

By using the resin bound pyrrole 66, the tropane skeleton was supposed to be generated on solid phase. The [3+4] cycloaddition of 1,3-dibromo-1-phenyl-2-propanone (44) and resinbound pyrrole 66 was tried. It is not known for sure whether the reaction works or not, but after cleaving the expected product from the resin, using TfOH and TFA, no product was isolated. A reason might be that the product has decomposed because of the harsh cleaving conditions and could probably have been solve by changing the resin. Because of the above mentioned methods for generation of tropanes were discarded, no further studies were carried out trying to extend it to solid phase synthesis.

29

Chapter I: Combinatorial Synthesis of Cocaine Analogues

4.3 Conclusion
Three possible ways that were thought to be used for a combinatorial approach to the tropane skeleton, have been presented. First via a [3+4] cycloaddition of pyrroles and α,α’dibromoketones. From this tropane synthesis, it was suggested to introduce a 2-carbomethoxy group on the tropane by employing the α,α’-dibromoketone of acetoacetate. Because of problems associated with synthesising the α,α’-dibromoketone this approach was rejected for further investigations. Thereupon attempts to the tropanes were made by a tandem cyclopropanation/Cope rearrangement of a vinylcarbenoid and Boc-pyrrole. The constructed tropane was subjected to reaction with phenyl magnesium bromide, which turned out to be unsuccessful. This will be discussed further in section 6.2.2. Third, a new way to construct a tropane from pyrrolidines was proposed. By further optimisation, it might be possible to generate the desired tropane from this procedure. But due to a very unclean reaction, it was not investigated further. In addition, considerations on how to extend these methods to solid phase chemistry were made.

30

Chapter I: Combinatorial Synthesis of Cocaine Analogues

5 Two- and Three-Dimensional Solution Phase Combinatorial Libraries of 3- and 8-Substituted tropanes from Multicomponent Grignard Reagents
5.1 Generation of a Two-Dimensional Library from Multicomponent Grignard Reagents
Due to the difficulties associated with a de novo construction of the tropane skeleton, it was decided to start a combinatorial approach of potential dopamine transporter ligands from a tropane that was already constructed. For this purpose anhydroecgonine methyl ester (7) was chosen as starting material, since 3-substituted phenyltropanes are known to be synthesised from this compound. As described in the introduction (see Scheme 1.1), phenyltropanes can be obtained by a 1,4-conjugate addition of aryl Grignard reagents to the electrophile 7. Therefore, it was expected that libraries could be obtained by reaction of 7 with a mixture of different Grignard reagents. In addition, 3-phenyl substituted tropanes are known to be potent dopamine transporter ligands and in the development of a cocaine abuse treatment, libraries of such analogues would be beneficial. Furthermore, the use of anhydroecgonine methyl ester (7) raised the possibility of introducing more combinatorial steps by introducing substituents at other reaction sites such as substituting the N-methyl group and changing the ester functionality.

5.1.1 Designing the Library
An important point with respect to the combinatorial library, was to make a library design that allowed facile identification of a possible hit compound. We came up with a solution, where the compound set was resolved into dimensions, in a similar way as for positional scanning or indexed libraries. However, these methods cannot be used for synthesis of a library made from reacting one compound with a mixture of many reagents, since it would be meaningless to vary both reaction partners resulting in more reactions than necessary for individual synthesis of each library member. By resolving the library into a matrix representation, it was suggested that variable mixing of the Grignard reagents, first in the horizontal dimension (i.e. prepare a mixture of Grignard reagents necessary for synthesising row 1,2…n in the horizontal dimension) followed by the vertical dimension (i.e. prepare a mixture of Grignard 31

Chapter I: Combinatorial Synthesis of Cocaine Analogues

reagents necessary for synthesis of compounds in column 1,2…n in the vertical dimension) would give the desired requirements for the library design. This is illustrated in Scheme 5.1. In this way, if a library in the horizontal dimension contains an active compound, the compound will be identified from the vertical library containing the same compound. After synthesising the 2n libraries, no further deconvolution is needed and active compounds can be synthesised individually.
R1,4MgBr R2,4MgBr R3,4MgBr R4,4MgBr R5,4MgBr E

R2,1MgBr R2,2MgBr R2,3MgBr R2,4MgBr R2,5MgBr

E-R1,1 E E-R2,1 E-R3,1 E-R4,1 E-R5,1

E-R1,2 E-R2,2 E-R3,2 E-R4,2 E-R5,2

E-R1,3 E-R2,3 E-R3,3 E-R4,3 E-R5,3

E-R1,4 E-R2,4 E-R3,4 E-R4,4 E-R5,4

E-R1,5 E-R2,5 E-R3,5 E-R4,5 E-R5,5

Scheme 5.1 The synthesis of 25 compounds in two dimensions using variable mixing of Grignard reagents. Reaction of 10 different five-component Grignard reagents with the electrophile (E) results in 10 sublibraries of 5 compounds each. If compound E-R2,4 turns out to be active it will be found in both the horizontal and vertical sublibrary and can be identified directly.

Generally, synthesis of n2 compounds will require n libraries of n compounds. For the abovementioned method, it is necessary to synthesise 2n libraries of n compounds each and in that way, all library members will be present in 2 sublibraries. The advantage of the method is the direct identificaton of an active compound, which eliminates the need for resynthesis of sublibraries or a larger number of individually compounds. The major drawback of the method is that if all library members have approximately the same biological activity, it might be difficult to get any usable information from the biological assays. The addition of aryl Grignard reagents to anhydroecgonine methyl ester have been used widely to synthesise 3-phenyl tropanes.5 But even though it has been emphasised that simple 3-alkyl substituted tropanes are poor ligands to the DAT no synthesis or occurrence of this type of compounds have been found in literature that supports this statement. Therefore, it was decided to include 3-alkyl substituted tropanes in the library. In addition, cycloalkyl Grignard reagents were included in the mixture of Grignard reagents, since the resulting 32

Chapter I: Combinatorial Synthesis of Cocaine Analogues

products might be interesting compounds, which due to their size and lipophilicity, might show some similarities with the benzene ring present in phenyl tropanes. Four aryl Grignard reagents were also included of which two resulted in unknown products (3-methylphenyl and 3,4-dimethylphenyl magnesium bromide). 4-tert-Butyl phenyl magnesium bromide has previously been employed for synthesis of the corresponding phenyl tropane, but since no biological data has been published for that compound, it was also chosen to become a member of the library.110 In addition, it is known that the corresponding p-iodo phenyl tropane (RTI-55) is a very potent compound against all three monoamine transporters and since the size of an iodine is approximately the same as for a t-butyl group, it was proposed that the t-butyl might be a good substitution for an iodine. The last known compound, which was chosen as a constituent of the library, was 10.24 This compound has shown to bind to DAT, NET, and SERT (Table 5.1) and was included as a positive control to confirm the validity of the biological assay.
IC50 binding (nM) Compound DAT SERT NET DAT [3H]DA [3H]WIN35428 [3H]paroxetine [3H]nisoxetine
CO2CH3 N

Ki uptake (nM) SERT [3H]SER NET [3H]DA

23±5 WIN35065-2 (10)

1962±61

920±73

49.2±2.2

173±13

37.2±5.2

Table 5.1 Biological data for binding and reuptake of WIN35065-2 (10) to DAT, SERT, and NET all obtained from rat tissue.24

5.1.2 Initial model studies
Initially, to demonstrate the usability of non-aromatic Grignard reagents in the addition reaction to 7, the 1,4-conjugate addition of n-butyl magnesium bromide to 7 was studied. Anhydroecgonine methyl ester (7) was obtained from R-cocaine as previously described (see Scheme 1.1).111 By adding the freshly prepared n-butyl magnesium bromide to the electrophile 7 in Et2O at –40 °C, the desired stereoisomer, being the 2β,3β-isomer 67, was obtained in high yield by quenching the reaction at –78°C with TFA, allowing for kinetic protonation of the ester enolate (Scheme 5.2). Only traces of the 2α,3β-isomer was observed but not isolated. It is a well known phenomenon that the selectivity of the stereoisomeric relationship can to some extend be controlled by the quenching conditions.112

33

Chapter I: Combinatorial Synthesis of Cocaine Analogues
N CO2CH3
1. n-BuMgBr (2eq), Et2O, -40oC, 2h 2. TFA, -78oC-0oC 76 %

CO2CH3 N

7

67

Scheme 5.2 Conjugate addition of n-butyl magnesium bromide to anhydroecgonine methyl ester.

After proving that a non-aromatic Grignard reagent can participate in the 1,4-conjugate addition as well as aryl Grignard reagents, a selection of alkyl- and aryl magnesium bromides were screened for their ability to undergo 1,4-conjugate addition. This was done by preparing mixtures of Grignard reagents by sequential addition of the selected bromides to excess magnesium in Et2O. In this way, observation of the exothermic reaction of each halide with magnesium was used to ensure that each Grignard reagent had been formed. A number of Grignard reagents failed to undergo addition to 7, among these methyl magnesium iodide, allyl-, propargyl-, cyclopropanyl- and o-isopropyl phenyl magnesium bromide. This might be explained by high reactivity leading to homocoupling products or by sterical hindrance. From these screening experiments, it was also shown that knowledge of the precise concentration of the Grignard reagent was crucial for the reaction to complete and for that reason all Grignard reagents were titrated according to a known procedure.113

5.1.3 Synthesis and Analysis of the Two-Dimensional Library
Following these initial experiments, the library synthesis was initiated. A 5x5 format of the library was chosen resulting in 2x5 sublibraries of 5 compounds each i.e. a total of 25 compounds each contained in exactly 2 sublibraries. The horizontal libraries are denoted by IV whereas the vertical libraries are denoted by a-e (Figure 5.1).

34

Chapter I: Combinatorial Synthesis of Cocaine Analogues

a
CO2CH3 N

b
CO2CH3 N
N

c
CO2CH3

d
CO2CH3 N
N

e
CO2CH3

I

67

CO2CH3 N

CO2CH3 N N

CO2CH3 N

CO2CH3 N

CO2CH3

II

CO2CH3

CO2CH3 N N 68

CO2CH3 N

CO2CH3

CO2CH3 N
t

III

N

Bu

69

CO2CH3

CO2CH3 N N

CO2CH3

CO2CH3 N

CO2CH3 N

IV

N

CO2CH3 N

CO2CH3 N

CO2CH3 N

CO2CH3 N
N

CO2CH3

V
10

70

71

Figure 5.1 Matrix representation of the two-dimensional library contained in sublibraries I-V (rows) and a-e (columns).

For sublibrary synthesis, the reaction was performed in a similar way as described for addition of n-butyl magnesium bromide, with the exception of adding the freshly prepared mixture of 5 Grignard reagents slowly to the electrophile via a syringe pump. This was done to ensure equal formation of products, even though a difference in the reactivity of the used nucleophiles might occur. As an example, the synthesis of library II is shown in Scheme 5.3. Even though an excess of Grignard reagents were employed, it did not have any consequences for the purity of the libraries. The amine functionality allowed a successful acid/base extraction and no further purification was necessary.

35

Chapter I: Combinatorial Synthesis of Cocaine Analogues
CO2CH3 N N CO2CH3

N

CO2CH3 +

CH3(CH2)6MgBr CH3(CH2)7MgBr CH3(CH2)8MgBr CH3(CH2)9MgBr CH3(CH2)11MgBr

1. Et2O, -40oC, 2h 2. TFA, -78oC-0oC

CO2CH3 N N

CO2CH3

7 N

CO2CH3

Library II

Scheme 5.3 Conjugate addition of a five component Grignard reagent to anhydroecgonine methyl ester (7) synthesis of library II.

A lot of effort was addressed to analysis of the libraries. All libraries were analysed by
1

H-NMR, ESMS, and GC-MS to ensure that all library members were present in

approximately equal amounts. 1H-NMR spectra were only useful in libraries containing separated peaks e.g. library V containing both aromatic and alkyl protons. An example of a GC chromatogram and ESMS spectrum are shown in Figure 5.2, which clearly show generation of all 5 desired products in approximately equal amounts in library II.

A

B

Figure 5.2 GC-MS chromatogram (A) and ESMS (B) of library II showing formation of all 5 expected products in approximately equal amounts.

36

Chapter I: Combinatorial Synthesis of Cocaine Analogues

5.1.4 Biological Results for the Two-Dimensional Library
All 10 sublibraries were screened for binding to the monoamine transporters hDAT, hNET, and hSERT in a competitive assay with 125I-labeled RTI-55 and also for inhibition of reuptake of [3H]-dopamine (hDAT and hNET) and [3H]-serotonin (hSERT) in cells expressing one of the three transporters, respectively. For both assays, IC50-values were determined from doseresponse curves and an average of three experiments were used to evaluate IC50 values that
⎛ IC 50 were converted to Ki values ⎜ K i = 125 ⎜ 1 + [ I − RTI − 55] / K d ⎝ ⎞ ⎟ . These are presented in Table ⎟ ⎠

5.2.
Ki binding (nM) Library I II III IV V a b c d e hDAT 9850 15800 11300 9300 43 1880 8750 6250 64 265 hSERT 8800 4900 9700 5400 38 4700 5200 11800 55 610 hNET 10200 9100 6600 11500 113 1900 9700 10200 130 370 hDAT 5000 10500 4800 4900 53 650 4400 3500 83 170 Ki uptake (nM) HSERT 10000 12400 10700 9200 52 4400 11900 11800 57 750 hNET 4850 3000 6600 5800 40 350 4550 7700 65 165

Table 5.2 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET for 2D libraries I-V and a-e. Each library contains 5 compounds.

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Chapter I: Combinatorial Synthesis of Cocaine Analogues

Graphical displays in Figure 5.3 are a visual form of the biological data in Table 5.2. In order to obtain a meaningful value of the height of a column in the diagrams, the two sublibraries containing a given compound were analysed. The sublibrary having the highest Ki value was chosen among the two. In principle, the lowest possible Ki value of the given compound is Ki/5, if only that compound contributes to the overall affinity of the sublibrary. To obtain high columns for high affinity compounds the reciprocal of Ki/5 was plotted in the diagrams and is a value of the highest possible association constant for that compound.
hDAT binding hSERT binding hNET binding

0.08 0.07 5/K i(max) (nM ) 5/K i(max) (nM )
-1 -1

0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e 5/K i(max) (nM )
-1

0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 a b c V d e II III IV I

0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I

II III IV

I

hDAT uptake

hSERT uptake

hNET uptake

0.07 0.06 5/K i(max) (nM ) 5/K i(max) (nM-1) 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I
-1

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I 5/K i(max) (nM-1)

0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I

Figure 5.3 Graphical displays showing for each library member the smallest value of 5/Ki for each of the two sublibraries in which it appears.

As seen from the diagrams, the two library members 70 (d,V) and 71 (e,V) show high activity in all six assays. It is also seen that the positive control 10 (a,V), shows activity against the transporters, especially against hDAT and hNET as expected from Table 5.1, but the activity is considerably lower than for 70 (d,V) and 71 (e,V). Not surprisingly, the two high affinity compounds were bearing a 3-aryl substituent, but to our surprise 69 (e,III) bearing a t-butyl phenyl substituent, did not turn out to bind to the transporters at all. A reason for this might be sterical hindrance from the t-butyl group. As suggested in literature, 3-alkyl substituted tropanes did not turn out to bind appreciable to the three transporters.

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Chapter I: Combinatorial Synthesis of Cocaine Analogues

To ensure that the two dimensional screening procedure was a useful way to identify possible leads, a variety of the library members were resynthesised as single compounds. The biological data for binding to and uptake of monoamine at hDAT, hNET, and hSERT are listed in Table 5.3.
Ki binding (nM) Compound 10 67 68 69 70 71 hDAT 220±95 6900±650 21000±4500 38000±5250 19±10 115±45 hSERT 750±680 23100±7300 27000±7500 3700±620 15±6 250±50 hNET 555±455 4700±650 10500±3000 29950±1300 20±7 190±110 hDAT 112±68 1900±750 5300±2600 11700±1850 14±7 65±40 Ki uptake (nM) hSERT 614±208 24900±3100 36000±23000 4150±2950 18±11 83±20 hNET 115±30 2650±750 4100±2500 15700±3300 13±5 40±28

Table 5.3 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.

These data clearly shows the validity of the two-dimensional display. Of the two high affinity compounds 70 and 71, 70 turns out to be the most potent compound binding to all three transporters, which was also seen from the diagrams in Figure 5.3. In addition, the compounds 68 and 67 that did not seem to bind according to the matrix representation of the libraries, did not show binding affinities as single compounds either. With respect to the reference compound 10, the data in Table 5.3 do not correspond well to the literature values presented in Table 5.1. The reason for this might first of all be that Table 5.3 present Ki and not IC50 values. In addition, the Ki values are obtained from assays using the cloned human transporters, whereas most other published results (as in Table 5.1) are obtained by employing transporters from rat, mouse, or monkey brain tissue.

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Chapter I: Combinatorial Synthesis of Cocaine Analogues

5.2 Generation of a Three-Dimensional Library from Multicomponent Grignard Reagents
One of the goals using combinatorial chemistry is to save time by synthesising many compounds in few reactions. One might argue that the experiments in the two-dimensional format do not fulfil these requirements, since only 25 compounds are synthesised in 10 reactions. To increase the gain obtained from the combinatorial approach two possibilities exists: 1) increasing the size of the matrix (e.g. n = 10 would result in 100 compounds in 20 libraries) or 2) another dimension could be introduced through variation of substituents at another reaction site. It was decided to expand the method to a third dimension by varying the substituent on the nitrogen and in that way to synthesise a three-dimensional library consisting of tropanes substituted in both the 3- and 8-position.

5.2.1 Initial model studies
It turned out that the easiest way to introduce N-substituents at the nitrogen, was by first demethylating anhydroecgonine methyl ester (7) by reacting with 1-chloroethyl chloroformate (ACE-Cl) followed by methanol to give the demethylated analogue 72 in 75 % yield (Scheme 5.4).114
N CO2CH3
1. ACE-Cl, Na2CO3 ClCH2CH2Cl, reflux, 5h 2. MeOH, overnight, 75 %

HN

CO2CH3

7

72

Scheme 5.4 Synthesis of (1R, 5S)-8-azabicyclo[3.2.1]oct-2-ene-2-carboxylic acid methyl ester (72) by demethylation of 7 using ACE-Cl and MeOH.

This was followed by alkylation of the nitrogen using 1.1 equivalent of an alkyl halide or a mixture of alkyl halides. Initial studies of the multicomponent N-alkylation turned out successfully. As a model experiment, equi-molar amounts of allyl-, benzyl-, and n-butyl bromide were refluxed overnight with the demethylated compound 72 in acetonitrile using KI as nucleophilic catalyst and K2CO3 as proton sponge (Scheme 5.5). An equi-molar mixture of 3 products was clearly obtained, witnessed by TLC, 1H-NMR (Figure 5.4), and GC-MS, in 75 % yield (obtained from an average of molecular masses).

40

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Figure 5.4 1H-NMR spectrum of a mixture of N-substitued anhydroecgonine methyl ester analogues 73 showing approximately equal formation of each product.

N

CO2CH3

HN

CO2CH3 + Ph 72

Br Br Br

KI, K2CO3 CH3CN, reflux 18h Ph N

CO2CH3

N

CO2CH3

73
1.
MgBr MgBr MgBr

Et2O, -40oC 2. TFA, -78oC

CO2CH3 N N

CO2CH3

Ph

CO2CH3 N

CO2CH3 N N

CO2CH3

Ph

CO2CH3 N

CO2CH3 N Ph N

CO2CH3 Ph

Ph

CO2CH3 N Ph

74

Scheme 5.5 Model studies of N-alkylation followed by Grignard reaction.

This mixture of N-alkylated analogues (73) was subjected to the same Grignard conditions as used for the two-dimensional library using a mixture of PhMgBr, iPrMgBr, and EtMgBr (Scheme 5.5). Of the expected nine products (74), only 6 were obtained. The missing library 41

Chapter I: Combinatorial Synthesis of Cocaine Analogues

members were all consistent with having an N-benzyl substituent. By synthesising the Nbenzyl constituent of mixture 73 and subjecting it to reaction with PhMgBr, it was realised that no reaction occurred – this will be discussed further in section 6.2. Therefore, it was concluded that the N-substituent had to be more identical for the Grignard reaction to take place.

5.2.2 Synthesis and Analysis of the Three-Dimensional Library
From these initial studies, it was decided to use a series of N-alkyl substituted analogues for synthesis of the three-dimensional library in order not to obtain problems with the Grignard reaction. Therefore, it was decided to react 72 with five homologous alkyl bromides (ethylhexyl) in the presence of KI and K2CO3 obtaining an equi-molar mixture of 75-79. Subsequently, the mixture 75-79 was reacted with a five component Grignard reagent resulting in a mixture of 25 products (Scheme 5.6).

HN

CO2CH3 +

Br Br Br

KI, K2CO3 18h

Br CH3CN, reflux Br

R1

N

CO2CH3 +

MgBr MgBr MgBr MgBr MgBr

CO2CH3
1. Et2O, -40oC 2. TFA, -78oC

R1

N

R2

72

75-79 5 compounds

Library Y1 25 compounds

Scheme 5.6 Example of combinatorial synthesis of three-dimensional library Y1

The ESMS of library Y1 showed all the 25 expected masses with a Gaussian-like form of the peaks. This is consistent with having several compounds with identical masses for the peaks in the middle, decreasing on going to both sides. In addition, the GC-MS showed many (>15) of the expected product peaks. It was therefore assumed that the probability of having all 25 compounds present in approximately equal amounts was high (Figure 5.5).

42

Chapter I: Combinatorial Synthesis of Cocaine Analogues

A

B

Figure 5.5 GC-MS chromathogram (A) and ESMS spectrum (B) of library Y1 showing a high probability of having all 25 products present.

In the same way, the 9 libraries X1-X5 (vertical cross sections) and Y2-Y5 (horizontal cross sections) of 25 compounds each were prepared. When preparing layers Z1-Z5, the given N-substituted anhydroecgonine methyl ester analogue, was reacted with a 25-component Grignard reagent. All 125 compounds are represented by the cube of Figure 5.6. Each compound is contained in 3 sublibraries.
X1 Z1
CO2CH3 N N

X2
CO2CH3 N

X3
CO2CH3 N

X4
CO2CH3 N

X5
CO2CH3

X1 Z2
N CO2CH3 CH CO2 3 N N

X2
CO2CHCO CH 3 2 3 N N

X3
CO2CH3 CH CO2 3 N N

X4
CO2CH3 CH CO2 3 N N

X5
CO2CHCO CH 3 2 3 N

X1 Z3
N CO2CHCO2CH3 2CH3 3 CO N N N

X2
CO2CHCO2CHCO2CH3 3 3 N N N

X3
CO2CHCO2CH3 2CH3 3 CO N N N

X4
CO2CHCO2CH3 2CH3 3 CO N N N

X5
CO2CHCO2CHCO CH 3 2 3 3 N N
t

X1 Z4
N

X2

X3

X4

X5
CO2CHCO2CHCO2CHCO CH 3 3 2 3 3 N N N t Bu

Bu

CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N

CO2CHCO2CHCO2CHCO CH 3 3 2 3 3 N N N N

CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N

CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N

X1 Y1 Z5
N

X2

X3

X4

X5

CO2CH3 2CHCO CHCO CH CO2CH3 2CHCO CHCO CH CO CH3 CO CH3 CO CO CH3 CO 3 2 3 3 2 3 2 3 3 CO2CH3 3 3 3 3 2 3 3 CO2CH2 CO2CHCO2CHCO2CHCO2CH2 CO2CHCO2CHCO2CH3 2CH2 CO2CHCO2CHCO2CH3 2CH3 CO 3 CO 3 3 3 N N N N N N N N N N N N N N N N N N N N t N N N N Bu

Y2

N

CO2CH3 2CHCO CHCO CH CO 3 2 3 3 2 N N N N

CO2CH3 2CHCO CHCO CH CO 3 2 3 3 2 N N N N

CO2CH3 2CHCO CHCO CH CO 3 2 3 3 2 N N N N

CO2CH3 2CHCO CHCO CH CO 3 2 3 3 2 N N N N

CO2CH3 2CHCO CHCO CH CO 3 2 3 3 2 N N N t Bu

Y3

N

CO2CH3 2CHCO CH CO 3 2 3 N N

N

CO2CH3 2CHCO CH CO 3 2 3 N N

N

CO2CH3 2CHCO CH CO 3 2 3 N N

N

CO2CH3 2CHCO CH CO 3 2 3 N N

N

CO2CH3 CHCO CH CO2 3 2 3 N N t Bu

CO2CH3 2CH3 CO

CO2CH3 2CH3 CO N N N

Y4

N

CO2CH3 2CH3 CO N

CO2CH3 2CH3 CO N N

N

N

CO2CH3 CH CO2 3 N

CO2CH3

CO2CH3 N N

CO2CH3 N

CO2CH3 N

CO2CH3

Y5

N

Figure 5.6 The three-dimensional library.

43

Chapter I: Combinatorial Synthesis of Cocaine Analogues

5.2.3 Biological Results for the Three-Dimensional Library
All 15 libraries X1-X5, Y1-Y5, and Z1-Z5 were screened for binding to hDAT, hSERT, and hNET and for reuptake of monoamine at the three transporters. In the same way as for the two-dimensional library, the Ki values were obtained from dose-response curves from each library (Table 5.4).
Ki binding (nM) Library X1 X2 X3 X4 X5 Y1 Y2 Y3 Y4 Y5 Z1 Z2 Z3 Z4 Z5 hDAT 6230 28000 9000 12 430 5800 36800 1620 2800 170 335 377 513 20 300 hSERT 31810 40000 40000 215 7500 15500 25553 11471 17050 365 1912 7185 5040 2500 1200 hNET 8500 34000 22660 333 640 12700 16120 5450 3900 150 350 650 1000 500 700 hDAT 3910 9350 8080 110 490 5350 40500 415 2205 200 484 485 1040 100 80 Ki uptake (nM) hSERT 40000 40000 40000 490 14600 29700 35060 27500 41000 580 3810 15275 12540 2480 3680 hNET 13810 23000 17370 140 790 16310 37900 5400 6880 250 630 470 1190 30 200

Table 5.4 Ki values for three-dimensional libraries X1-X5, Y1-Y5, and Z1-Z5 for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.

From Table 5.4 diagrams for direct identification of hits were constructed in the same way as for the two-dimensional libraries (Figure 5.7). The only modification being that columns in the diagrams are obtained for each compound from the lowest value of 25/Ki from the three libraries in which the library member appears.

44

Chapter I: Combinatorial Synthesis of Cocaine Analogues
hDAT binding hSERT binding hNET binding

0.16

0.025

0.08

0.14 0.02 0.12 25/Ki(max) (nM-1) 25/Ki(max) (nM-1) 25/Ki(max) (nM-1)

0.07

0.06

0.1 0.08 0.06 0.04
Y4,Z2 Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3

0.015

0.05 0.04 0.03 0.02
Y4,Z2 Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3

0.01

Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1

0.005

0.02 0
X1 X2 X3 X4 X5 Y3,Z1 Y1,Z1

Y2,Z2 Y5,Z1

0.01 0
X1 X2 X3 X4 X5 Y3,Z1 Y1,Z1

Y2,Z2 Y5,Z1

0
X1 X2 X3 X4 X5

Y3,Z1 Y1,Z1

hDAT uptake

hSERT uptake

hNET uptake

0.14

0.012

0.1 0.09

0.12

0.01 0.08

0.1 25/Ki(max) (nM-1) 25/Ki(max) (nM-1) 25/Ki(max) (nM-1) 0.008

0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
X1 X2 X3 X4 X5 Y3,Z1 Y1,Z1 Y2,Z2 Y5,Z1 Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2

0.08
Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1

0.006
Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1

0.06

0.004

0.04

0.02
Y3,Z1 X1 X2 Y1,Z1 X3 X4 X5

0.002

0

0
X1 X2 X3 X4 X5

Y3,Z1 Y1,Z1

Figure 5.7 25/Ki (nM-1) for binding and reuptake at hDAT, hSERT, and hNET for each library member. These values are obtained as the smallest value of 25/Ki for the three libraries in which the library member appears.

From the above diagrams, the most potent compounds against the 3 transporters can be read directly. With respect to binding to hDAT, it is seen that compound (x,y,z) = 4, 5, 4 corresponding to N-pentyl-3-dimethyl-phenyl analogue 81, appears to be the strongest inhibitor of hDAT binding. The N-hexyl-3-dimethyl-phenyl analogue 82 ((x,y,z) = 4, 5, 5) appears to be the most potent compound binding to hSERT, while the strongest inhibitor of hNET binding, according to the diagram, is the N-ethyl-3-dimethyl-phenyl analogue 80 ((x,y,z) = 4, 5, 1). Again it is seen that the most potent compounds are those containing a 3β aromatic substituent and as in the two-dimensional library, having a 3,4-dimethyl substituted phenyl group results in the most potent compounds binding to all three monoamine transporters. The most potent compound, with respect to binding to each transporter, was selected for individual synthesis (i.e. 80-82). The binding affinity and inhibition of reuptake are shown in Table 5.5. 45

Chapter I: Combinatorial Synthesis of Cocaine Analogues

CO2CH3 N N

CO2CH3 N

CO2CH3

80

81

82

Ki binding (nM) Compound 80 81 82 hDAT 18±5 14±3 25±6 hSERT 133±50 220±15 297±82 hNET 165±89 345±230 580±140 hDAT 42±13 34±10 140±44

Ki uptake (nM) hSERT 78±31 69±33 310±64 hNET 59±26 72±38 285±64

Table 5.5 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.

It is seen that all three compounds 80-82 bind very strongly to the hDAT with a Ki value in the same range as the N-methyl analogue 70. As suggested from Figure 5.7, 81 turns out to be the most potent compound with respect to hDAT binding, but the magnitude of the Ki value compared to 80 and 82 seems to be much smaller than it appears in Figure 5.7. The prediction that the 80 is the most potent compound binding to hNET is also seen to be the case, whereas the prediction that 82 was the strongest binding compound to hSERT, do not turn out to be true. This might be because of the relatively large standard error of the Ki determinations from the libraries X1-X5, Y1-Y5, and Z1-Z5 (not shown in Table 5.4). It is also seen from Table 5.5 that both binding and uptake at hSERT and hNET are decreasing with the length of the N-substituent and that selectivity for hDAT over hSERT and hNET binding affinity is induced. This is consistent with previous results showing increased selectivity for DAT with increasing size of the N-substituent.34

5.3 Applying Two- and Three-Dimensional Libraries to other Systems
The method of employing two- and three-dimensional libraries in the search for biologically active compounds have also been applied to other compound classes than for the 3- and 8-substituted tropanes as just described. In a similar study, two- and three-dimensional libraries of benztropine analogues, were synthesised as putative dopamine transporter ligands.

46

Chapter I: Combinatorial Synthesis of Cocaine Analogues

As mentioned in the introduction, benztropines are another class of interesting compounds showing affinity to the dopamine transporter. Starting from tropine, benzyl ether 83 was obtained in four steps. The key step in the synthesis was radical azidonation of 83 resulting in azide 84 that was now ready to undergo substitution with Grignard reagents. This new synthesis route to the benztropines is particular interesting, since so far only 3-diarylmethoxy substituted benztropines have been investigated, whereas the present method also allows introduction of alkyl groups. By reacting 84 with 10 different five-component Grignard reagents followed by Bocdeprotection, 25 benztropine analogues were synthesised in two dimensions (Scheme 5.7).IV Extension to a third dimension was done by reaction of the two-dimensional library with five alkyl bromides to form a three-dimensional library of 125 benztropine analogues in 15 sublibraries.
BocN O Ph 83 84
IN3 10 % DMF in CH2Cl2 reflux, 73 % 1. RMgBr toluene, 2h, rt

BocN O Ph N3

HN
R'Br, K2CO3

R' N O R Ph 70 - 90 % 3D library (125 compounds)

2. 2,6-lutidine, TMSOTf CH2Cl2, 0oC-20oC, 1h

O Ph 56 - 70 %

R DMF, rt, 40h

2D library (25 compounds)

Scheme 5.7 Synthesis of benztropine analogues in two- and three-dimensional libraries. R = alkyl and aryl, R’ = alkyl.

Screening of the libraries for binding and inhibition of uptake at hDAT, hSERT, and hNET did not result in identification of compounds that were stronger than benztropine. See appendix 7 for further details.

5.4 Summary and Concluding Remarks
As a contribution to the search for potential cocaine antagonists, a solution phase combinatorial approach to 3-substituted tropanes was developed. The conjugate addition of multicomponent Grignard reagents to anhydroecgonine methyl ester was employed to construct a two dimensional library of 25 2β-carbomethoxy-3β-substituted tropanes. All 25 compounds were each contained in two sublibraries, which facilitated the identification of active species. By screening for biological activity against the hDAT, hSERT, and hNET, two

IV

This study was carried out by master student Hanne Pedersen.

47

Chapter I: Combinatorial Synthesis of Cocaine Analogues

new high affinity compounds were identified. The two compounds and several other library members were resynthesised as single compounds to validate the method. In a similar way, a three dimensional library was constructed by adding an extra dimension via varying substituents at the tropane nitrogen. For this purpose a multicomponent N-alkylation was employed followed by a multicomponent Grignard reaction. In this way 125 compounds were generated in 15 sublibraries of 25 compounds each. From the biological screening, three compounds were selected for individual synthesis. As expected, they all turned out to have high affinity for the three monoamine transporters both in binding and reuptake assays. The same method was also applied for construction of two- and three-dimensional libraries of benztropine analogues. The presented method turned out to be a good way for generation of libraries and for identification of possible hits. By extending the method, larger libraries can be synthesised and in that way the gain associated with the use of combinatorial chemistry will increase. In that case, it is important to consider the size of the libraries related to the difference in biological activity expected for biologically active versus biologically inactive compounds, since the method cannot be used for hit generation when all library members show approximately equal activity.

48

Chapter I: Combinatorial Synthesis of Cocaine Analogues

6 Bicyclo[3.2.1]octane Analogues of Phenyltropanes
6.1 Introduction
The vast majority of therapeutic agents and drugs targeting brain receptors are amine-based, which is also the case for numerous natural neurotransmitters. In general a model of ligandreceptor interactions have been postulated to happen via formation of an ionic bond between the protonated amine nitrogen of the ligand and a carboxylic acid residue on the target protein. The interactions between the DAT, SERT, and NET with ligands are no exception. The amine functionality has been proposed to make an ionic bond between the protonated amine of the ligand and an aspartate on the DAT, SERT, and NET.3 This hypothesis was supported by the fact that mutation of a highly conserved aspartate residue on the monoamine transporters to neutral amino acids (dopamine: D79A, norepinephrine: D75A, serotonin: D98A) reduced binding affinities for both substrates and inhibitors.115,116 Another possibility for interaction with the amine functionality is through a hydrogen bond. The necessity for the presence of an amine in putative cocaine antagonists has been challenged and several non-amine analogues of phenyltropanes have been synthesised. A number of these turned out to bind strongly to the monoamine transporters, which imply that other structural features than the amine are of greater importance to obtain binding affinity. It was therefore concluded that the amine is not prerequisite for binding to the dopamine transporter.

6.1.1 8-Oxa Analogues
Substitution of the nitrogen of a cocaine analogue by an oxygen a new class of analogues arise – the 8-oxa analogues. In 1979 the first synthesis of 8-oxacocaine was reported by Brownbridge et al. as mentioned in section 4.1.3.106 The 8-oxa analogue of cocaine 60 (Scheme 4.11) turned out to have reduced affinity for the DAT (Ki([3H]Mazindol) = 4.2 µM) compared to cocaine (Ki([3H]Mazindol) = 0.28 µM), but still retains activity in the binding studies.117 In 1997 the first synthesis of 3-aryl-8-oxa analogues of the phenyltropane series was reported by Meltzer et al.36 The synthesis was based on Brownbridge’s procedure for making the bicyclic system (see Scheme 4.11). Ketone 58 was converted to the enol triflate 85, which was coupled to various boronic acids by a Suzuki coupling. Less than 10 different aryl groups 49

Chapter I: Combinatorial Synthesis of Cocaine Analogues

have been incorporated at present. In this way, both the 3-β (87) and 3-α (88) products were obtained by reduction of the octenes 86 with samarium diiodide (Scheme 6.1). The 8-oxa analogues cannot be involved in binding to the transporter through an ionic bond, but the possibility of attaining binding affinity via hydrogen bonding is still present.
CO2CH3 O 58
Na(TMS)2N, Ph(Tf)2N THF, -78oC - RT overnight, 79 %

O

O

CO2CH3 OTf 85

ArB(OH)2, Pd2dba3, LiCl, Na2CO3 CH2(OEt)2, reflux 1h 82 - 97 %

O

CO2CH3 Ar 86

I2 h Sm H, 2 % O Me 0 -65 F/ TH o C, 5 -78

CO2CH3 O Ar + O

Ar CO2CH3

87

88

Scheme 6.1 Synthesis of 3-aryl-8-oxabicyclo[3.2.1]octanes.

6.1.2 8-Carba Analogues
In order to evaluate the necessity of an 8-heteroatom further, Meltzer et al. prepared a series of bicyclo[3.2.1]octane analogues of phenyl tropane.37,118 Since carbon is not a bioisostere for oxygen or nitrogen, exchange of an amine or ether functionality with a methylene is not generally considered. By replacing the heteroatom in position 8 of the tropane with a methylene, the possibility of making ionic or hydrogen bonding is omitted, i.e. the methylene provides nothing else than maintenance of the overall topological shape of the bicyclic system. Starting from the commercially available 3-chlorobicyclo[3.2.1]oct-2-ene (89), the βketoester 91 was obtained in 2 steps as shown in Scheme 6.2. The remaining part of the synthesis was carried out by a similar route as the synthesis of the 8-oxa analogues and with
CO2CH3
H2SO4 CNCO2CH3, LDA

Cl 89

77 %

O 90

THF, -78oC - rt, 2h 83 %

O 91

Scheme 6.2 First steps in the synthesis of 3-aryl-bicyclo[3.2.1]octanes.

50

Chapter I: Combinatorial Synthesis of Cocaine Analogues

comparable yields (Scheme 6.1). A serious drawback of this synthetic pathway is that for the 8-carba analogues all four possible stereoisomers are obtained. Because of their considerable unpolar properties, separation of the products appeared to be very difficult, resulting in very low yields of the pure compounds.

6.1.3 Biological Activity of Non-Amines
Despite the lack of an amine functionality some of the above mentioned non-amines turned out to have affinity for the dopamine transporter. Examples of binding affinities for bicyclo[3.2.1]analogues having nitrogen, oxygen or carbon in the 8-position are shown in Table 6.1.21,36,37,118
CO2CH3

X

CO2CH3 X
Cl

CO2CH3

Cl Cl

X

Cl

X (R) (R) (R/S) NCH3 O CH2

IC50/nM 1.2 4.2 7.1

IC50/nM 1.1 3.9 9.6

IC50/nM 23a >1000 3820b

Table 6.1 Inhibition of [3H]WIN35428 binding to the DAT in cynomolgus monkey caudate-putamen. a Inhibition of [3H]WIN35428 binding to the DAT in rat striatal membranes. b mixture of isomers.

It is seen that even among the 8-oxa and 8-carba analogues, extremely potent inhibitors of DAT are found. Thus, the effect of changing the 8-nitrogen in phenyltropanes does not affect the magnitude of inhibition dramatically. It is also observed that the potency of the ligands is largely dependent on the aryl group, since compounds having a 3,4-dichlorosubstituted phenyl ring are by far the most active. Another notable observation is that the 2,3-unsaturated compounds show a striking selectivity of 460-720 fold for inhibition of DAT over SERT (IC50 values for inhibition at SERT are not shown). The studies of non-amine analogues of phenyltropanes conclude that formation of neither an ionic bond nor hydrogen bonding is crucial for binding of phenyltropanes to the DAT, when the aryl group is carefully chosen.

51

Chapter I: Combinatorial Synthesis of Cocaine Analogues

6.2 Results and Discussion
6.2.1 Attempts to Synthesise Methyl 3-(4-iodophenyl)-bicyclo[3.2.1]octane carboxylate and its 8-methyl and 8,8-dimethyl Analogues
Bearing the above results in mind, it was suggested to evaluate the presence of substituents at C-8 in 8-carba analogues in combination with exploring the importance of having a 4-iodophenyl at C-3. Even though the 4-iodophenyl substituted phenyltropane RTI-55 proved to be a very potent DAT ligand, the corresponding 8-carba analogue has never been synthesised. (Figure 6.1).
CO2CH3 I CO2CH3 I CO2CH3 I

The

initial

proposal

was

to

synthesise

methyl

3-(4-iodophenyl)-

bicyclo[3.2.1]octane carboxylate (92), its 8-methyl and 8,8-dimethyl analogues 93 and 94

92

93

94

Figure 6.1 The initially proposed synthesis products.

These compounds were thought to be reasonably easy available from Davies’ bicyclo[3.2.1]octene 95119 by a 1,4-conjugate addition of a Grignard reagent, in the same way as describe for the tropanes in section 5 followed by iodination. Since the bicyclic system can be obtained from a tandem cyclopropanation/Cope rearrangement of vinyldiazomethane 50 and cyclopentadiene, substituents in the 8-position was thought to be introduces by employing 1-methyl cyclopentadiene and 1,1-dimethyl cyclopentadiene. In addition, an establishment of such a method would also be beneficial for developing a combinatorial synthesis of 8-carba analogues of phenyltropanes and further expand the usefulness of the method described in section 5.
O OCH3 N2 50 +
1. Rh(II) 2. H2, Wilkinson's

CO2CH3
PhMgBr

CO2CH3 Ph

95

96

Scheme 6.3 Construction of Davies' bicyclo[3.2.1]octene 95 via a tandem cyclopropanation/Cope rearrangement followed by selective hydrogenation of the unconjugated double bond. This was suggested to be followed by a 1,4-conjugate addition to yield the desired compound 96.

52

Chapter I: Combinatorial Synthesis of Cocaine Analogues

Methyl 2-diazobut-3-enoate (50) was synthesised from methyl acetoacetate according to procedure outlined in section 4.1.2.1.104 This was followed by construction of the [3.2.1]bicyclic system through a rhodium-catalysed tandem cyclopropanation/Cope rearrangement with cyclopentadiene to give 97. The bicyclic octadiene 97 could now be used for studies of the conjugate addition, as well as its C-6/C-7-saturated analogue 95, obtained from selective hydrogenation of the unconjugated double bond by Wilkinson’s catalyst (Scheme 6.4). The use of 97 was preferred, since the presence of the C-6/C-7 double bond might allow introduction of substituents in the 6 and/or 7 position.
O OCH3 N2 50 +
3 % Rh2(O2CC7H15)4 pentane, reflux 1h 90 %

CO2CH3

2 % (Ph3P)3RhCl, H2 10 bar EtOH, overnight 77 %

CO2CH3

97

95

Scheme 6.4 Synthesis of methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate (95).

6.2.2 Attempts to Perform Conjugate Additions to Methyl bicyclo[3.2.1]octa-2,6diene-2-carboxylate and Methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate
Initially, the conjugate addition of a Grignard reagent to methyl bicyclo[3.2.1]octa-2,6-diene2-carboxylate 97 was attempted (in the same way as was done with the anhydroecgonine methyl ester in section 5), but to our surprise no reaction occurred. Several reaction conditions were investigated (Table 6.2, all reaction mixtures were analysed by TLC and GC-MS). As entries 1 - 3 were all unsuccessful, no effect resulted from adding more Grignard reagent – not even the 1,2-addition product was observed. Neither did it have any effect to add Cu(I) nor TMSCl or HMPA (entries 4 - 6).120 A last attempt was made using a higher order cuprate, but this also turned out to be unsuccessful. Contrary to the expectation, it turned out to be difficult to perform a conjugate addition on the α,β-unsaturated bicyclic octadiene 97 and even though it seemed unreasonable that the C-6/C-7-double bond could cause the difficulties, it was decided to try to see if the conjugate addition could be performed on the saturated bicyclic ester 95.

53

Chapter I: Combinatorial Synthesis of Cocaine Analogues
CO2CH3
Reagents Solvent, conditions

CO2CH3

97

98

Entry 1 2 3 4 5 6 7 8

Reagents PhMgBr (1 eq) PhMgBr (2 eq) PhMgBr (6 eq) PhMgBr (1 eq), CuBr⋅Me2S (1 eq) PhMgBr (2 eq), LiCl (1 eq), TMSCl (1 eq), CuBr⋅Me2S (1 eq) PhMgBr (1.5 eq), TMSCl (2 eq), HMPA (3 eq), 10 % CuBr⋅Me2S PhLi (2 eq), CuCN (1 eq) PhLi (3 eq), CuCN (1.5 eq)

Solvent Et2O Et2O Et2O THF THF THF Et2O Et2O

Reaction conditions -40 °C, 4h -40 °C, 5h then rt overnight -40 °C, 5h then rt overnight -40 °C, 1h, then PhMgBr (4 eq) 3h, then rt overnight -78 °C -78 °C, 6h -40 °C → rt overnight -78 °C → -30 °C, 4h

Table 6.2 Unsuccessful attempts to add phenyl nucleophiles to the α,β-unsaturated ester 97.

A few attempts to do the conjugate addition on the C-6/C-7-saturated ester 95 were performed according to Table 6.3, but still no addition reaction occurred.
CO2CH3
Reagents Solvent, connditions

CO2CH3

95

96

Entry 1 2 3

Reagents PhMgBr (1 eq) PhMgBr (2 eq) PhLi (26 eq), CuCN (13 eq), BF3⋅Et2O (15 eq)

Solvent Et2O Et2O Et2O

Reaction conditions -40 °C, 5h -40 °C, 5h -78° → -30 °C, 6h

Table 6.3 Unsuccessful attempts to add phenyl nucleophiles to α,β-unsaturated ester 95.

In addition, it was also tried to do the conjugate addition of PhMgBr to the t-butyl ester of 95 and the temperature was raised to -20°C, but again no reaction occurred. Since the conjugate addition of a Grignard reagent was easily performed on the 8-aza analogue 7 and the only difference from 7 to 95 is the presence of the 8-nitrogen in 7, this nitrogen must be crucial for the reaction to occur when generating phenyltropanes. An 54

Chapter I: Combinatorial Synthesis of Cocaine Analogues

explanation can be proposed by taking a careful look at the conjugate addition of a nucleophile to 2-cyclohexenones, which preferentially happens by axial attack of the nucleophile.121 Axial attack of a nucleophile leads to a chair-like intermediate, whereas equatorial attack leads to a boat-like intermediate. Therefore, attack from the axial position is thought to be favoured because of a lower energy of the transition state and intermediate of a chair compared to a boat conformation. This is exemplified by the copper-catalysed addition of iodomethylmagnesium to 5-methyl-2-cyclohexenone (98) where the major product 99 was presumed to arise via a chair-like intermediate (Scheme 6.5).122
O
MeMgI, CuI Et2O, 55-66 %

O +

O

98

99, 94-96 %

100, 4-6 %

Scheme 6.5 Copper-catalysed conjugate addition of a Grignard reagent to 5-methyl-2-cyclohexenone preferentially by axial attack of the nucleophile.

In case of addition to a 2,3-unsaturated bicyclo[3.2.1]octane as 95, axial attack is probably too sterically hindered because of the ethylene bridge causing unfavourable 1,3-diaxial interactions. The only possibility is then equatorial attack, which might be energetically unfavoured because the energy of the transition state via a boat-like conformation is too high (Figure 6.2).
Nu O CO2CH3 OCH3 CO2CH3 O OCH3 Nu

Nu Nu Axial attack Chair-like Equatorial attack Boat-like

Figure 6.2 Intermediates for axial versus equatorial attack of a nucleophile.

But why is the Grignard reaction possible when a nitrogen is present in the 8-position? An explanation might be that the presence of a nitrogen in position 8 afford a coordination site for the Grignard reagent, which offers stabilisation of the boat-like transition state and thereby lowering of the transition state energy is obtained (Figure 6.3). In this way a six-membered transition state is generated. In addition, this can explain why only the 3β-isomer is obtained when generating phenyltropanes through a 1,4-conjugate Grignard reaction to anhydroecgonine methyl ester (7). 55

Chapter I: Combinatorial Synthesis of Cocaine Analogues
Br N
δ+

Mg

R

OCH3

Figure 6.3 Possible stabilisation of a boat-like transition state by coordination of magnesium to the tropane nitrogen.

A similar explanation have been proposed as a rationale for equatorial attack of magnesium containing thiol nucleophiles to anhydroecgonine methyl ester in the synthesis of cocaine benzoyl thioester.123 It has also been observed that the reactivity of Grignard reagents in general increases in the presence of aliphatic amines, possibly through coordination to the Lewis acidic magnesium.124 The nitrogen in anhydroecgonine methyl ester could therefore account for an increased reactivity of the Grignard reagent. Another explanation could be that in the 8-azabicyclic system, the double bond of the α,β-unsaturated ester is more electrophilic, because of the electron-withdrawing effect of the 8-nitrogen compared to the 8-carba bicyclic system. To further explore the influence of the atom in the 8-position, a series of methyl bicyclo[3.2.1]oct-2-ene carboxylates were prepared varying the atom or substituent in the 8-position. 101, 102, and 103 were prepared as previously described from vinyldiazomethane 50 (see section 4.1.2),125,126 whereas the remaining nitrogen containing compounds 104, 105, and 106 were prepared by alkylation of 72 as in the synthesis of the three-dimensional library in section 5.1.2 (Figure 6.4).V
O O CO2CH3 RO N CO2CH3 R N CO2CH3

101

102, R=Me 103, R=t-Bu

104, R=i-Pr 105, R=Bn 106, R=p-methoxybenzyl

Figure 6.4 101-106 were all reacted with 2 eq. PhMgBr in Et2O at -40°C for 4h before quenching with TFA at -78°C. No reaction was observed for any of the compounds.

101, 102, 104, and 106 were synthesised by bachelor students Naja Nielsen, Sara Kobbelgaard and Mette Nymand under supervision of the author.

V

56

Chapter I: Combinatorial Synthesis of Cocaine Analogues

101-106 were all reacted with 2 equivalents of freshly prepared PhMgBr in Et2O at –40°C for 4h. TLC analysis revealed that no reaction had occurred and after quenching with TFA at -78°C and workup this was confirmed from GC-MS analysis. With the presence of an oxygen in 101 and thereby the possible existence of a coordination of the Grignard reagent, in the same way as described above, it was expected that the reaction would work smoothly. Since the reaction does not take place, the theory of coordination cannot be further rendered probable. By having a carbamate functionality, as in 102 and 103, the basicity of the nitrogen is decreased. And since the reaction does not take place in this case, it can be explained by the poor coordination possibilities of the carbamate nitrogen compared to the corresponding amine. Thus, it is spectacular that a similar Grignard reaction have been successfully conducted on a methyl carbamate 7-azabicyclo[2.2.1] analogue 33 (Figure 3.1).71 In the case of having a primary alkyl substituted nitrogen it is known to be possible to do the 1,4-conjugate addition of a Grignard reagents to the α,β-unsaturated ester.127 By introducing an N-isopropyl group the electronic properties was assumed to be approximately the same as for a primary alkyl substituted analogue. The presence of the isopropyl group in 104 was therefore suggested give insight into the importance of the steric factor of the nitrogen substituent. As seen in Figure 6.4 no reaction occurred when having an isopropyl group at the nitrogen. Neither when having an N-benzyl (105) nor a p-methoxybenzyl (106) group at the nitrogen causes the reaction to occur. Again it might be because of steric interactions or the explanation could be the reduced basicity of the nitrogen. It is therefore concluded that the 1,4-addition of Grignard reagents to the bicyclic system is very sensitive and depends largely on the nitrogen substituent and the atom in the 8-position.

6.2.3 Model Studies on Methyl Crotonate
Bearing the above results in mind, it was decided to try the possibility of making other types of conjugate additions to the α,β-unsaturated system. Before carrying out the reactions on the bicyclic system, some model studies were conducted on methyl crotonate (107) – a readily available α,β-unsaturated ester. All successful attempts were also conducted on the bicyclic unsaturated ester 95. The reactions were performed according to Table 6.4 and analysed by TLC and GC-MS of the reaction mixtures.

57

Chapter I: Combinatorial Synthesis of Cocaine Analogues

O

Nu OCH3

O OCH3

I
107

Reagents Conditions

CO2CH3

CO2CH3 Nu

II
95

Entry 1 2 3 4

Reagents PhI (1 eq), Bu3SnH (1.3 eq), AIBN (cat) Bu3PhSn (1.1 eq), 5 % [RhCl(cod)]2 PhB(OH)2 (1.5 eq), 5 % [RhCl(cod)]2
OTMS Ph

Solvent Benzene H2O Dioxane/H2O CH2Cl2

Reaction conditions 50 °C, 19h 90 °C, 15h -78 °C, 1h

Observation by GC-MS I 1,4-add. 1,4-add. 1,4-add. II no reaction no reaction no reaction

Reflux overnight no reaction

(1 eq), TiCl4 (1 eq), CH3CN THF Et2O/THF Reflux overnight 70 °C overnight -40 °C, 1h 1,4-add. 1,4-add. 1,4-add. no reaction no reaction no reaction

Ti(OiPr)4 (0.4 eq) 5 6 7 Acetone cyanohydrin (1.2 eq), KCN (1.6 eq), 5 % TBACN MeNO2 (1.3 eq), TBAF (1 eq) PhMgBr (1.5 eq), TMSCl (3 eq), HMPA (3 eq), 5 % CuBr

Table 6.4 Studies of various types of conjugate additions.

As seen from Table 6.4, it was not possible to do a radical addition of a phenyl radical to methyl crotonate. But this is not surprising, since it is known to be difficult to add an electrophilic radical (as a phenyl radical) to an electrophilic double bond as an α,β-unsaturated ester.128 Newer methods for addition of aryl-stannanes to unsaturated carbonyl compounds by rhodium catalysis have been introduced by Oi et al.129 As seen in entry 2 this turned out to be a very convenient way to perform a 1,4-addition on 107 and the desired product was obtained in quantitative yield. Also rhodium-catalysed Suzuki-type coupling of PhB(OH)2 to 107 was very successful (entry 3).130 Another way of making a conjugate addition is by employing a Mukaiyama-Michael addition of silyl enol ethers to an unsaturated carbonyl system by Lewis acid catalysis. In this way a 1,5-dicarbonyl compound is obtained. According to entry 4, an addition of the silyl enol ether derived from acetophenone to 107 was performed, which afforded the desired 1,4-addition product.131 It was also tried to add more simple nucleophiles such as cyanide (entry 5) and CH3NO2 (entry 58

Chapter I: Combinatorial Synthesis of Cocaine Analogues

6) to methyl crotonate (107).132 These reactions afforded the desired products without any problems. At last a Cu(I) catalysed conjugate addition of PhMgBr using TMSCl and HMPA as additives was carried out. In contrary, as what was seen for the bicyclic system, it had a positive outcome when performed on the simple α,β-unsaturated ester 107 yielding the 1,4addition product. However, methyl crotonate is also a more accessible α,β-unsaturated ester than the bicyclic conjugate ester 95 and therefore sterical and electronic factors must control the conjugate addition of a phenyl nucleophile to α,β-unsaturated methyl esters. As shown in Table 6.4, all conjugate additions performed on the bicyclic unsaturated ester 95 failed. It seems to be very difficult to do a conjugate addition on the bicyclic system no matter what nucleophile was used, which might be due to sterical hindrance, electronic properties of the double bond or to the absence of coordination possibilities to a nitrogen.

6.2.4 Synthesis of an 8-Carbon Analogue
It was realised that in order to synthesise an 8-carba analogue, the synthetic strategy had to be changed, since it was not possible to conduct a conjugate addition on the bicyclic system. If the difficulties arise from sterical hindrance from the ethylene bridge during axial attack, a conjugate addition would have to be carried out on a compound that did not contain this bridge. This led us to propose that a solution might be to open the five-membered ring of 97, conduct the conjugate addition, and close the ring once again. 6.2.4.1 Ring Opening and Closing Metathesis for Generation of an 8-Carba Analogue Olefin metathesis reactions have become a very versatile synthetic method since the invention of various Ru carbene complexes by Grubbs et al.133 Ring opening metathesis reactions can be carried out by cross metathesis with ethylene affording di-olefinic compounds.134 This reaction was thought to be useful for opening of the five-membered ring in 97. With respect to ring closing metathesis, most reported examples involve macrocyclisations, but in 1998 Morehead and Grubbs published an article on the formation of small ring bicycloalkenes from monocyclic dienes.135 From the results, it was concluded that ring closures of five-, six-, and seven-membered rings to form [3.x.1], [4.x.1], and [5.x.1] bicycloalkenes are relatively facile processes. Formation of a bicyclo[3.2.1]octene system was easiest when the six-membered ring was being closed (Scheme 6.6A). Generation of the bicyclo[3.2.1]system via ring closing 59

Chapter I: Combinatorial Synthesis of Cocaine Analogues

of the more sterically hindered five-membered ring, was more difficult and raising the temperature and dilution was necessary to obtain the ring closed compound (Scheme 6.6B).
TBDMSO TBDMSO
5 % Cl2(PCy3)2RuCHPh c = 0.050 M CH2Cl2, rt, 87 %

A

TBDMSO

B

10 % Cl2(PCy3)2RuCHPh c = 0.005 M CH2Cl2, reflux, 84 %

OTBDMS

Scheme 6.6 Formation of bridged bicycloalkenes via ring closing metathesis.

In another example, a tropane skeleton was generated from a ring closing metathesis strategy.136 This employed reaction of the Cbz-protected divinyl compound 108 with Grubbs’ first generation catalyst, which afforded the desired product 109 in high yield (Scheme 6.7). This ring closure is probably facilitated by the presence of a carbamate, as 1,3-allylic strain between the carbonyl and the substituents α to the nitrogen that favours the diaxial conformer. This conformer is the species being ring closed.
Cbz N

10 % Cl2(PCy3)2RuCHPh c = 0.1 M CH2Cl2, rt, 15h 86 %

Cbz N O 109

O 108

Scheme 6.7 Formation of a tropane skeleton by ring closing metathesis.

60

Chapter I: Combinatorial Synthesis of Cocaine Analogues

6.2.4.2 Synthesis of Methyl 3-Phenethyl-bicyclo[3.2.1]oct-6-ene Carboxylate (112) Methyl 4,6-divinyl-cyclohexene carboxylate (110) was easily prepared by ring opening metathesis of methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate (97) using Grubbs’ first generation catalyst in an ethylene atmosphere (Scheme 6.8).
CO2CH3 CO2CH3

Cl2(PCy3)2RuCHPh c = 0.04 M, CH2CH2 CH2Cl2, overnight 85 %

PhCH2CH2MgBr, CuBr (cat), TMSCl, HMPA Et2O/THF, -40oC, 4½h 31 %

CO2CH3 CH2CH2Ph 111

97
MesN NMes Ph Cl Ru Cl PCy3

110

CO2CH3 CH2CH2Ph

111

c = 0.009 M ClCH2CH2Cl reflux overnight 58 %

112

Scheme 6.8 Synthesis of methyl 3-phenethyl-bicyclo[3.2.1]oct-6-ene carboxylate (112) via an olefin metathesis strategy.

The cyclic α,β-unsaturated ester 110 was now ready for the crucial step – the conjugate addition. Initially, it was tried to use PhMgBr as nucleophile – the same reaction that was performed for synthesis of the tropane libraries. This reaction is also known to be successfully conducted on arecoline (19), which also contains a nitrogen that could possibly coordinate to the Grignard reagent (see Figure 3.1). Coordination possibilities are not present in the divinylcyclohexene 110 and the attempt to do conjugate addition of PhMgBr did fail. This was also the case by copper catalysed addition of PhMgBr including additives as TMSCl and HMPA.137 By changing the nucleophile from phenyl magnesium bromide to phenethyl magnesium bromide, the latter being a better nucleophile, it was possible to add the nucleophile by using copper catalysis, TMSCl and HMPA resulting in isolation of the addition product 111 in high yield. But because of small impurities having almost identical polarity as the product, only 31 % pure 111 was isolated by careful column chromatography. The rationale for choosing a phenethyl nucleophile was based on the fact that in the tropane series it appears that by having a 3-phenethyl group, the binding affinity is increased compared to having a 3-phenyl group (Table 1.2). It appears that only one addition product arises from the Grignard reaction. Based on the NMR spectra (1H and COSY), it was assigned to be the compound 111 arising from axial attack of the organocopper reagent. This assignment was especially based on the 61

Chapter I: Combinatorial Synthesis of Cocaine Analogues

double doublet from H-1 having a large and a small coupling constant (11.6 Hz and 4.4 Hz, respectively). In addition assignment of the final product 112 could confirm these results. It is also worth mentioning that addition of phenethyl magnesium bromide is not possible to perform on 95 when the bicyclic system is intact. To complete the synthesis, a cyclisation of 111 had to be performed. Several attempt were made using Grubbs’ first generation catalyst in CH2Cl2 and in ClCH2CH2Cl. Indeed formation of the ring-closed compound was observed, but it was not possible to drive the reaction to completion, which was necessary for avoiding purification problems, because of similar polarity of starting material and product. By changing to Grubbs’ second generation catalyst, which is known to have a higher reactivity towards olefins, it was possible to obtain the ring closed compound 112 in 58 % yield.138 In order to close the ring, it was necessary to use ClCH2CH2Cl as the solvent to be able to reach a higher reflux temperature. The difficulties regarding the ring closure are probably due to the conformation of 111. Having two equatorial vinyl groups, a ring flip is necessary in order to adapt to the ring closing conformer having two axial vinyl groups. The 8-carba analogue 112 is sent for biological testing against hDAT, hSERT, and hNET, and the results are waited in great suspense.

6.3 Conclusions
In order to synthesise bicyclo[3.2.1]octane analogues of phenyltropanes as potential dopamine transporter ligands, it was proposed to employ a synthesis route based on a 1,4-conjugate addition of Grignard reagents to a known α,β-unsaturated ester. For that reason a variety of conjugate additions were performed on methyl bicyclo[3.2.1]octa-2,6-diene-2carboxylate and methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate. All attempts were unsuccessful and did not result in the desired addition products. The lack of reaction was ascribed to sterical hindrance from the ethylene bridge, which impaired the possibility of axial attack from the nucleophile. In addition, it was proposed that a nitrogen was essential for stabilisation of a boat-like intermediate and perhaps for increasing the reactivity of the Grignard reagent. As model studies, a number of different conjugate additions were performed on methyl crotonate. Even though several reactions resulted in quantitative yields when performed on the model compound, they all turned out to be unsuccessful when conducted on the bicyclic ester. 62

Chapter I: Combinatorial Synthesis of Cocaine Analogues

By changing the synthesis strategy, methyl 3-phenethyl-bicyclo[3.2.1]oct-6-ene carboxylate was synthesis by first ring opening of the bicyclic system followed by successful addition of phenethyl magnesium bromide to the α,β-unsaturated ester. A ring closing metathesis, to yield the desired compound, completed the synthesis. This route to 8-carba analogues of phenyltropanes might also be useful for generation of analogues containing methyl and dimethyl substitutions at C-8. Such compounds could be interesting for evaluation of the effect of having substituents in the 8-position, which maybe resulting in induction of selectivity for one monoamine transporter over another.

63

Chapter I: Combinatorial Synthesis of Cocaine Analogues

64

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions 1 Introduction
1.1 Carbohydrates – Ubiquitious Molecules
Traditionally, compounds occurring in living organisms are termed biomolecules and include proteins, lipids, nucleic acids, and carbohydrates. Of these four classes of biomolecules, carbohydrates are by far the most abundant and make up most of the organic matter on earth. This is mainly due to the existence of cellulose (a polymer made of β-1,4 linked glucose units, 112), which plays a major role as a structural element in the cell walls of bacteria and plants.139 Monosaccharide units as ribose and deoxyribose also serve as structural elements, as being a part of the phosphate-sugar backbone of RNA and DNA (115). Another very important role of carbohydrates is associated with energy storage. Starch and glycogen are polymers build from glucose units, that serve as readily mobilisable stores of energy in plants and animals, respectively. In addition, as a component of e.g. potatoes, rice, pasta, and bread, starch has a great nutritional value when ingested by humans.139
O O P O O OH

OH O HO O

HO HO

OH O OH

O

Base

OH

Glucose in cellulose, 113

Galactose, 114

Deoxyribose in DNA, 115

Figure 1.1 Examples of carbohydrates.

Although glucose is the most abundant monosaccharide, many other monosaccharide units exists (especially galactose (114), mannose, and fructose) and they can all be a part of disaccharides or oligosacchardes. Carbohydrates have a high degree of diversity since there are numerous ways in which monosaccharide units can combine to form di- and oligosaccharides. This property makes them very useful for carrying biological information – a thing they indeed do in nature. Being a part of glycoproteins, carbohydrates are involved in many specific biological processes e.g. in recognition processes the sugar part of a glycoprotein can serve as a signal, which directs it to be recognised by a specific cell type or 65

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

receptor.139 They are also involved in other important processes such as cell growth, inflammation, viral replication, and metastasis.140,141 For these reasons, it is of great importance to understand the chemistry of carbohydrates.

1.2 Acid-Catalysed Hydrolysis of Glycosides
When exposed to acidic aqueous media, glycosidic bonds are hydrolysed. Acidic hydrolysis of glycosides is generally believed to happen through an A-1 mechanism,142,143 i.e. a fast protonation of the exocyclic oxygen followed by detachment of the aglycon in the ratedetermining step. The resulting oxocarbenium ion intermediate, stabilised through resonance, is then attacked by water to produce the hemiacetal product (Scheme 1.1).
OH HO HO O HO OR
H
+

OH HO HO O HO OR H
-ROH slow

OH HO HO O HO

fast

OH HO HO O HO OH
H2O, -H+ fast

OH HO HO O HO

Scheme 1.1 A-1 mechanism for acidic hydrolysis of a glucoside.

Since 1908 it has been known that the rate of acid-catalysed hydrolysis of glycosides depends on the configuration of the hydroxyl groups on the sugar unit – the more axial hydroxyl groups, the faster hydrolysis.142,144 E.g. altropyranonside 118 hydrolyses 3-4 times faster than galactoside 117, which hydrolyses 5 times faster than glucoside 116 (Figure 1.2).143
OH HO HO O HO HO OCH3 116 krel 1 HO OCH3 117 5 HO OH O HO HO 118 18 OH OH O OCH3

Figure 1.2 Relative rate of hydrolysis at 60°C in 2.0 N HCl.

66

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

In 1955 Edward proposed an explanation for this general observation based on relief of steric strain.145 He reasoned that detachment of the leaving group is accompanied by a conformational change of the pyranose ring from a chair to a half-chair in the transition state. Small rotations around the C-2/C-3 and C-4/C-5 bonds are required for entering this halfchair. This transformation decreases the strain connected with axial substituents and therefore makes a positive contribution to the total reaction rate, when axial substituents are present. This explanation has recently been reconsidered. In a study of the acetolysis of methyl 2,3,6-tri-O-methyl-α-D-galacto- and glucopyranosides varying the substituent at C-4, Miljković et al. found that the rate of acetolysis depends strongly on the electronegativity and orientation of the C-4 substituent.146 Having different C-4 equatorial substituent (methoxy, acetoxy or acetamido substituent, gluco series), the rate of acetolysis did not change significantly. Small changes were explained by a through bond interaction (inductive effect) of the C-4 substituent and the ring oxygen. But in the galacto series (axial C-4 substituent) a large dependence of the reaction rate with respect to electronegativity of the C-4 substituent was observed (4-methoxy > 4-acetoxy > 4-acetamido). The enhanced rate of acetolysis by having electronegative substituents in the axial position was explained by a through-space electron donation from the axially oriented substituent at C-4 of the galactopyranoside into the oxocarbenium ion under formation (Scheme 1.2). This through-space electrostatic stabilisation is not possible for the corresponding gluco series and acetolysis of glucosides are therefore not affected to the same extend as the galacto series (Scheme 1.2, 119 is cleaved approximately 14 times faster than the corresponding gluco configured (equatorial 4-OCH3) methyl pyranoside). The experimental observations were supported by ab initio calculations.
OCH3 O CH3O CH3O OCH3
H+

CH3O

CH3O CH3O

OCH3 O CH3O OCH3 H TS
-CH3OH

CH3O CH3O

OCH3 O CH3O

119

Scheme 1.2 Proposed stabilisation of the transition state under formation by the axial electronegative substituent suggested by Miljković et al.

Another explanation was recently proposed by Jensen et al.147,148 In a study of the basicity of azasugars, it was observed that protonated azasugars having axial 4-OH groups were slightly more basic than their equatorial counterparts (Figure 1.3). From a large amount of pKa values 67

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

of epimeric piperidines, the average effect of having a given substituent in a given position was determined.149 Based on these data, σs values were obtained and from Hammett plots a free-energy relationship was established with the rate of acidic hydrolysis of glycosides.150
OH HO HO NH2 pKa 8.4 HO HO OH NH2 pKa 8.8 HO HO OH NH2 OH pKa 6.7 HO HO OH NH2 OH pKa 7.5

Figure 1.3 The difference in pKa values of epimeric piperidinium ions is explained by axial polar groups being less electron-withdrawing than their equatorial counterparts.

The existence of a free-energy relationship suggested that the same effect determining the difference in azasugar basicity also accounts for the difference in the rate of hydrolysis of stereoisomeric glycosides. The effect was explained by a difference in electron-withdrawing power between an axial and an equatorial hydroxyl group – the equatorial being more electron-withdrawing through space than the axial (i.e. a field effect). This means that in the A-1 mechanism of glycosidic hydrolysis, the stability of the transition state (approximated by the intermediate carbocation/oxocarbenium ion) depends on the configuration of the hydroxyl groups. Thus, on hydrolysis of a galactoside, the transition state is less destabilised than on hydrolysis of a glucoside and therefore the rate of hydrolysis of a galactoside is faster than that of the corresponding glucoside (Figure 1.4).
OH HO HO NH2 HO HO OH NH2 HO HO OH O HO HO less stable more stable forms slower HO forms faster HO OH O

Figure 1.4 Relation between stability of epimeric azasugars and the reactivity of glycoside hydrolysis. Arrows showing the difference in charge-dipol interaction with respect to stereochemistry at C-4.

In addition, Jensen and Bols have synthesised model compounds 120 and 121, which were exposed to acidic hydrolysis (Figure 1.5).148 Their results showed that 121 was hydrolysed 1.6 times faster than 120 and therefore steric effects cannot be the controlling factor of the rate of hydrolysis, because then one would expect 120 to be hydrolysed faster than 121.

68

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions
OH O HO HO OCH3 HO HO OCH3 OH O

120
krel
1

121
1.6

Figure 1.5 Relative rates of hydrolysis at 74°C in 2.0 N HCl.

Namchuk et al. have suggested that during hydrolysis of glycosides 97 % of the charge is positioned at the endocyclic oxygen and that through-space field effects control the rate of hydrolysis.151 This theory contradicts with Jensen et al., who argues that if 97 % of the charge is positioned at the endocyclic oxygen, one would expect the rate of hydrolysis of 2-deoxyxyloglycosides and 4-deoxyxyloglycosides to be approximately the same because of a high degree of symmetry of the transition states.147 However, the rate of hydrolysis of 2-deoxyxyloglycosides is approximately 50 times higher than that of the 4-deoxyxyloglycosides.142 In addition, good correlation is observed in Hammett plots of stereochemical substituent constants and α-glucoside hydrolysis rates whether 100 % charge is placed on the ring oxygen or the anomeric carbon.150 Therefore, it is reasonable to conclude that considerable development of positive charge must be present on both the ring oxygen and the anomeric carbon in the transition state.

1.3 Glycosidation Reactions
To see whether the stereochemical substituent effect, which has been observed for acid-catalysed glycoside hydrolysis, has an influence on the rate of glycosidation, it was decided to take a closer look at this type of reaction. A general way of forming a glycosidic bond is by reacting a protected glycosyl donor, bearing a leaving group at the anomeric center, with a glycosyl acceptor being another suitably protected carbohydrate or organic alcohol.152 A variety of methods of glycosidation reactions have been published throughout the years, starting with Fischer’s glycosidation153 and the Koenigs-Knorr method.154 Other methods involve glycosyl donors such as sulfoxides, glycals, n-pentenyl glycosides, and trichloroacetimidates.155 The mechanism of glycosidation has been discussed in the past and is regarded to be influenced by several factors such as temperature, solvent, type of promoter, and type of leaving group.152 If a general glycosidation reaction is thought to proceed through an 69

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

SN1-type mechanism, an oxocarbenium ion intermediate, similar to the one generated in the A-1 mechanism for acid-catalysed glycoside hydrolysis, will be generated (Scheme 1.3).
OBn BnO BnO O BnO LG
activating reagent rds

OBn BnO BnO O BnO BnO BnO

OBn O BnO
Nu -H+

OBn BnO BnO O BnO Nu

Scheme 1.3 SN1-type mechanism for glycosidation of a benzyl protected glucosyl donor.

Assuming a similar substituent effect for OBn as for OH and that fission of the C-1-LG bond to generate the oxocarbenium ion intermediate is the rate-determining step, the expectation according to the stereochemical substituent effect, would be that a galactosyl donor (axial 4OBn) would react faster than a glucosyl donor (equatorial OBn). If the positive charge is present exclusively on the ring-oxygen, one would expect a difference in reactivity between the galactosyl- and glucosyl donor to be 6-7 times, whereas fully charge on the anomeric carbon would result in a rate difference of 2-3 times. These values are based on the difference in pKa values of piperidinium ions having an axial or equatorial substituent in β or γ position relative to the positive charge (see Figure 1.3). On the other hand, if a glycosidation reaction happens through an SN2-type mechanism, one would expect that almost no difference in the rate of glucosidation versus galactosidation would be observed. The suggestion is based on the absence of accumulation of charge at the anomeric position in the transition state and therefore the substituent effect would be negligible (Scheme 1.4). However, it is still possible that a small effect would arise from electrostatic destabilisation of the galactosyl donor ground state as explained by Miljković.146
OBn BnO BnO O BnO LG
Nu
-

OBn BnO BnO O

δ−

OBn
-LG
-

Nu

BnO BnO

O BnO

Nu

BnO LG
δ−

Scheme 1.4 SN2-type mechanism of glycosidation for a benzyl protected glucosyl donor.

Differences in the reactivity of glycosyl donors have been observed for donors bearing different protecting groups. With the observation that acetyl protected n-pentenyl glucosides hydrolyse slower than the corresponding benzyl protected compound, Fraser-Reid and co-workers introduced the concept of armed-disarmed glycosides.156 With respect to 70

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

glycosidation, this can be useful since two glycosyl donors bearing different protecting groups have different reactivity. The concept is based on a difference in electron-withdrawing power of various protecting groups e.g. ester protected glycosides are more disarmed because of the greater electron-withdrawing abilities of an ester functionality compared to an ether protected glycoside (armed). As seen in Scheme 1.5, no self-coupling product (disarmeddisarmed) was observed on reaction of the armed benzyl protected n-pentenyl glucoside 122 with the disarmed acetyl protected donor 123.157
OBn OBn BnO BnO O BnO OPent + AcO AcO OH O AcO OPent
IDPC CH2Cl2, 62 %

BnO BnO

O BnO AcO AcO 124 O O AcO OPent

122, armed

123, disarmed

Scheme 1.5 The concept of armed/disarmed effects in glycosyl donors.

The concept of armed-disarmed effects paved the way for tuning the reactivity of glycosides resulting in oligosaccharide syntheses without the need for tedious protection group manipulations.158,159 Wong and co-workers developed a method for facile one-pot oligosaccharide synthesis.159 Their method was based on a thorough study of reactivities of p-methylphenyl thioglycosides towards glycosidation with MeOH and NIS/TfOH activation. By making competition experiments between a given thioglucoside donor and a reference compound, they obtained a large table of relative reactivities and by constructing a database from these data, they were able to predict a one-pot synthesis of oligosaccharides starting from the most reactive donor decreasing the reactivity on going towards the reducing end of the oligosaccharide. In that way they were able to construct complex oligosaccharides in an easy and one-pot manner, without protection group manipulation as seen in Scheme 1.6.

71

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

OBz HO BzO O STol NHTroc

BzO BzO

OH O OBz
(13.1) NIS

OBn STol HO AcO O AcO OCH3
NIS

BnO BnO

OBn O BnO STol

(162.9) CH2Cl2, NIS,TfOH

(1.7x104)

BnO BnO

OBn O BnO O BzO OBz O TrocHN BzO O BzO OBz 40 % O AcO O

OBn O AcO OCH3

Scheme 1.6 One-pot synthesis of a tetrasaccharide. Relative reactivities are given in parentheses.

What was even more interesting for the present project, was the table of relative reactivities. By comparing pairs of C-4 epimeric thioglycosides bearing the same pattern of protection groups, it was seen that the rate of glycosidation was higher when the C-4 substituent was axial compared to a C-4 equatorial substituent (Figure 1.6). With a ratio of the relative reactivity values between C-4 epimers ranging from 4.5 to 6.5, it is obvious that the stereochemistry at C-4 influence the rate of glycosidation, which might be explained by an SN1-type mechanism as described above.
OAc gluco series: AcO AcO O AcO (2.7) STol BzO BzO OBz O BzO (1.3) STol BnO BnO OBn O BnO (2656) STol

AcO galacto series: AcO

OAc O AcO (14.3) STol

BzO BzO

OBz O BzO (5.7) STol

BnO BnO

OBn O BnO STol

(1.7x104)

Figure 1.6 Examples of relative reactivity values of protected p-methylphenyl thioglycosides are given in parentheses.

In another study conducted by Lahmann and Oscarson, the varying reactivity of thioglycosides donors with respect to the aglycon was investigated.160 By performing 72

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

DMTST-promoted thioglycoside couplings, they found that the rate of glycosidation of the donors increased with the electron-donating abilities of the aglycon. In addition, they also observed that galactosyl donors were three to seven times more reactive than the corresponding glucosyl derivative. Gervay and Hadd have also observed a difference in the reactivity of C-4 epimeric glycosyl donors.161 The reaction of 2,3,4,6-tetra-O-benzyl-α-Dgalactosyl iodide with sodium diethyl malonate was completed in 1h, whereas a reaction time of 5h was necessary for the reaction to complete in case of the corresponding glucosyl iodide. Again this suggests that the galactosyl donor is more reactive than the glucosyl donor.

1.3.1 The Trichloroacetimidate Method
The goal with the present study was to determine whether a protected galactosyl donor reacts faster than the corresponding glucosyl donor upon glycosidation with an acceptor. For this purpose O-glycosyl-trichloroacetimidates were employed for most experiments, since it is a well-established method for activation of a glycosyl donor through retention of the anomeric oxygen.162,163 In the activation step, O-glycosyl trichloroacetimidates are easily formed by a reversible base-catalysed addition of the anomeric hydroxyl group to the electron-deficient nitrile, trichloroacetonitrile. They can be synthesised in a stereocontrolled way by careful choice of base (Scheme 1.7).164,165
OR RO RO O RO OH
Base

OR RO RO O OH RO
Base

OR RO RO O RO O
CCl3CN

OR RO RO O O RO

CCl3CN

OR RO RO O RO O CCl3 NH Thermodynamic product RO RO

OR O O RO NH CCl3

Kinetic product

Scheme 1.7 Mechanism for formation of thermodynamic versus kinetic product formation of trichloroacetimidate donors.

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Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

On addition of base, the β-alkoxide is form initially, which upon addition with trichloroacetonitrile, produces the β-trichloroacetimidate product. But since this is a reversible reaction, the thermodynamically more stable α-product will eventually form if strong base is used. Traditionally, NaH is used for obtaining the α-anomer, whereas K2CO3 is employed for generation of excess β-O-glycosyl trichloroacetimidate.166 In the glycosidation step, trichloroacetimidate donors are activated with an acidic catalyst. Regarding the mechanism of reaction, it is believed to be largely dependent on several factors (Scheme 1.8). In nonpolar solvents using a nonparticipating protected donor, SN2-type reactions are often suggested, typically using BF3⋅Et2O as catalyst. By employing more polar solvents and strong acidic catalysts (e.g. TfOH), an SN1-type pathway is believed to be followed and thereby formation of the thermodynamically more stable α-glycoside. However, under SN1-type conditions, the influence of the solvent is important for the outcome of the reaction. If nitriles are chosen as of solvent, α-nitrilium-nitrile conjugates are formed, resulting in β-glycosidation, because of screening of the α-face by the solvent.167,168 On the other hand, participation of ethers directs generation of equatorial oxonium ions, which for thermodynamic reasons favour formation of the α-product. This is suggested to be due to the reverse anomeric effect.169
OR RO RO O RO
nonpolar solvents SN2-type

OR + LG
polar solvents SN1-type

catalyst: C acceptor: A-H

+

RO RO

O RO

LG

nonpolar solvents SN2-type

OR β-product A-H RO RO LGH + C fast OR RO RO O RO N N RO RO
S

O RO

S S

A-H, S =Et2O
LGC-

α-product

LGH + C slow OR O RO N N

= CH3CN

Scheme 1.8 Generally accepted mechanism of glycosidation.

74

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

Among other things, the stability of O-glycosyl trichloroacetimidates and the possibility of using mild reaction conditions have made them popular glycosyl donors and they have been employed numerous times in glycosidation reactions.170 Both for synthesis of simple disaccharides and for more complicated purposed as synthesis of glycosyl phosphatidyl inositol anchors that possess great importance in anchoring proteins and glycoproteins to membranes.171

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Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

2 Results and Discussion
In order to investigate if a difference in reactivity between galactosyl and glucosyl donors existed, direct competition reactions between the two types of donors competing for an acceptor were made. For the majority of reactions performed, trichloroacetimidates were employed as glycosyl donors, but experiments with glycosyl chlorides, glycals, and n-pentenyl glycosides were also performed. On question of protecting groups on the donors, it was decided to use benzyl groups to protect the hydroxyl groups in order to avoid any influence from neighbouring group participation. As glycosyl acceptor, 1-octanol was chosen, since by introducing an aliphatic chain, separation of the product from the starting material was thought to be facilitated. In this section, the synthesis of glycosyl donors will be described and the results of competition reactions performed under various conditions will be presented and discussed.

2.1 Competition Reactions using Trichloroacetimidates
2.1.1 Synthesis of Trichloroacetimidate Donors
hemiacetal sugar moieties. For synthesis of 2,3,4,6-tetra-O-benzyl-D-

As described in section 1.3.1, 1-O-glycosyl trichloroacetimidates are synthesised from protected glucopyranosyl-1-O-trichloroacetimidate (126) perbenzylated α-D-glucose (125) was employed as starting material. As described in literature, K2CO3 resulted in an excess of the β-anomer 126b (α/β 1:6.6), but only 34 % overall yield.172 Even though DBU is known to be unselective, the best results were obtained using this base.173 After stirring at -20°C for 4 hours both anomers were formed in a yield of 85 % favouring the α-anomer 126a (α/β 5.5:1) (Scheme 2.1).
OBn BnO BnO O BnO OH
CCl3CN, DBU CH2Cl2, -20oC, 4h

OBn BnO BnO O BnO O CCl3 NH + BnO BnO

OBn O BnO O CCl3 NH

125

126a, 72 %

126b, 13 %

Scheme 2.1 Synthesis of 2,3,4,6-tetra-O-benzyl-α/β-D-glucopyranosyl-1-O-trichloroacetimidates 126a and 126b using DBU as base.

76

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl-1-O-trichloroacetimidate (130) was synthesised in a similar manner starting from methyl α-D-galactopyranoside (127). First, the galactopyranoside was protected by perbenzylation of the free hydroxyl groups using NaH and BnBr in DMF to give 128 (Scheme 2.2). This was followed by hydrolysis of the acetal to generate the hemiacetal 129, though in a rather low yield.174,175 Even though temperature, reaction time, acid concentration, and acid were varied, no increase in the yield was seen and it was decided to continue without further optimisation. Again the trichloroacetimidate 130 was obtained using DBU as base (Scheme 2.2). No β-anomer was observed.
OH HO HO OCH3 OH O
NaH, BnBr DMF, rt, 20h 97 %

BnO BnO

OBn O BnO OCH3
2M TfOH AcOH, 80oC, 11h 47 %

BnO BnO

OBn O BnO OH
CCl3CN, DBU CH2Cl2, -20oC, 2½h 75 %

BnO BnO

OBn O BnO O CCl3 NH

127

128

129

130

Scheme 2.2 Synthesis of 2,3,4,6-tetra-O-benzyl-α-D-galactosyl-1-O-trichloroacetimidate (130) from methyl α-D-galactopyranoside (127).

2.1.2 Synthesis of the Competition Reaction Products
Before initiating the competition reactions, 3 of the 4 possible products from reacting 126a and 130 with 1-octanol were synthesised. This was done in order to be able to detect the products in a mixture obtained from the competition reactions. Both β-products (131b and 132b) were synthesised as described in Scheme 2.4 using 1-octanol and BF3⋅Et2O in CH2Cl2, but since only trace amounts of the octyl α-glycosides were isolated under these reaction conditions, another procedure was employed for synthesis of the α-glucoside 131a.176,177 Excess octyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (131a) was synthesised under thermodynamic conditions according to Scheme 2.3.
OBn BnO BnO O BnO OH 125
TMSOTf, LiClO4 CaSO4, 1-octanol benzene, rt, 12h

OBn BnO BnO O BnO OOct 131a, 24 % + BnO BnO

OBn O BnO 131b, 14 % OOct

Scheme 2.3 Synthetic approach for formation of excess α-glucoside.

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Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

2.1.3 Competition Reactions between Perbenzylated Gluco and Galacto Trichloroacetimidates
Quantification of glycosyl donor reactivity was obtained by making direct competition reactions between excess of the two glycosyl donors with respect to the acceptor. In this way the two glycosyl donors were forced to compete for the acceptor. If the reaction follows an SN1-type pathway and the rate-determining step in the glycosidation is generation of the oxocarbenium ion as described in section 1.3, the rate of glucosidation versus galactosidation is dependent of the stability of the transition states relative to their respectable ground-states. A series of competition reactions were performed between 126a and 130 using different activators, solvents, and equivalents of the donors according to Scheme 2.4 and Table 2.1.
OBn BnO BnO O BnO O + CCl3 NH 126a 130 OBn O BnO BnO O CCl3 NH 131a: R1=H, R2=OOct 131b: R1=OOct, R2=H 132a: R1=H, R2=OOct 132b: R1=OOct, R2=H
1-octanol, cat. solvent, 0oC, 1h

BnO

OBn BnO BnO O BnO R2 R1 +

BnO BnO

OBn O BnO R2 R1

Scheme 2.4 Competition reactions were performed according to the above scheme using 1-octanol as limiting species.

Equi-molar amounts of glucosyl donor 126a and galactosyl donor 130 were dissolved in the given solvent and 1-octanol added. After cooling to 0°C, the activator was added and the reaction mixture stirred for 1h before work up. After removing excess glycosyl donors by filtration on silica gel, the relative ratio of formation of octyl glucoside 131 versus octyl galactoside 132 was determined. For the first reactions, the glucoside/galactoside ratio was determined both by HPLC and
13 13

C-NMR, but since the results from both detection methods
13

were in agreement, the remaining reactions were analysed solely by

C-NMR. In the

C-NMR spectrum, the chemical shifts for C-1 for the 4 octyl glycosides were clearly

distinguishable showing peaks at 97.0 ppm for the α-glucoside 131a, 97.6 ppm for the α-galactoside 132a, 103.8 ppm for the β-glucoside 131b, and 104.1 ppm for the β-galactoside 132b. Determination of the ratio of galactoside versus glucoside was based on the intensity of the C-1 signals, which is reasonable because of the great resemblance of the products. Results from the competition reactions are shown in Table 2.1.

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Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

Entry 1 2
a

Catalyst/promoter BF3⋅Et2O BF3⋅Et2O BF3⋅Et2O BF3⋅Et2O TfOH TfOH TMSOTf TMSOTf AgOTf BF3⋅Et2O/ AgOTf CsOTf BF3⋅Et2O/ CsOTf TfOH

Solvent CH2Cl2 CH2Cl2 CH3CN Et2O CH2Cl2 CH3CN CH2Cl2 CH3CN CH3CN CH3CN CH3CN CH3CN CH2Cl2

Equiv. catalyst 0.14 0.14 0.14 0.14 0.1 0.1 0.17 0.17 2.9 0.14 + 0.4 2 0.14 + 2c 0.1
b

Yield 97 % 100 % 37 % 100 % 82 % 71 % 45 % 76 % 60 % 50 % ÷ 37 % 87 %

Gal(132)/Glc(131) 2:1 5:1 5:1 3:1 1:1 1:1 1:1 1:1 1:1 2:1 ÷ 3:2 5:1

α:β 1:5 0:1 0:1 0:1 0:1 1:4 1:5 1:3 1:4 0:1 ÷ 0:1 0:1

3 4 5 6 7 8 9 10 11 12 13
d

Table 2.1 Results of competition experiments between glucosyl and galactosyl imidates, 126a and 130, with 1-octanol. Unless otherwise noted, the experiment was with 1 equivalent of 126a, 130 and alcohol at 0°C. a 2 equivalents of 126a and 130 were used. b 0.4 equivalents of AgOTf. c 2 equivalents of CsOTf. d At -78°C.

A general observation was seen for all competition experiments, namely that octyl glycosides 131 and 132 were obtained primarily as β-anomers no matter what catalyst or solvent was used. The anomeric ratio indicates that inversion has happened to a great extent on the anomeric center, suggesting an SN2-type mechanism. But inversion does not necessarily exclude an SN1-type mechanism. A possible explanation could be that formation of a nonsolvent-equilibrated oxocarbenium ion shields for attack from the α-face resulting in βglycosidation, i.e. the nucleophilic attack occurs prior to the complete dissociation of the intermediate ion:molecule pair. This has been reported for hydrolysis of glycopyranosides containing neutral leaving groups.178 From entry 1, it is seen that a 2:1 ratio favouring the galactoside 132 is obtained when BF3⋅Et2O was the catalyst in CH2Cl2 and one equivalent of each donor is used. This indicated that the rate of formation of galactoside/glucoside must be higher than 2:1, because 130 becomes depleted when only one equivalent of each donor is employed and therefore appreciable amounts of the glucoside is also obtained. To obtain a ratio that can describe the true difference in rate constants between the two reactions, more equivalents of each donor has to be employed. Therefore, according to entry 2, an experiment where 2 equivalents of 79

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

each donor were employed was performed resulting in a 5:1 ratio. These findings suggest that considerable charge is built up on the endocyclic oxygen and that the reaction has SN1-character. Entries 3 and 4 were made to see the effect of changing the solvent. Using a more polar solvent CH3CN, the reaction was expected to proceed through an SN1-type mechanism. Because of its higher polarity when compared to CH2Cl2, and thereby higher tendency to stabilise a transition state having positive charge, even more excess of the galactoside product was expected. Indeed this was observed. Here according to the nitrile effect, no α-glycosides were observed. By using Et2O, generation of the α-anomers was expected. Et2O is known to participate by generation of equatorial oxonium ions to the anomeric carbon. This oxonium ion is more stable from the β-face, owing to the reverse anomeric effect179, and therefore attack is more likely to come from the α-face. However, this is not observed and kinetic effects must control the reaction. Nevertheless, the reactivity of the galactosyl donor was higher than of the glucosyl donor. It was decided to try an alternative activator and triflic acid was chosen (entries 5 and 6). Here a dramatic and surprising effect was observed. No matter what solvent was used a 1:1 galacto/gluco ratio was seen, while the stereochemistry was unchanged still favouring the β-anomer. Further experiments were conducted using other triflate containing activators in form of TMSOTf and AgOTf (entries 7 – 9). Since they all resulted in a 1:1 product ratio, it appeared that the effect was associated with the presence of triflate ions. To check that, experiments catalysed by BF3⋅Et2O in the presence of triflates in form of AgOTf and CsOTf were made (entries 10 and 12, CsOTf does not catalyse the reaction, entry 11). For both reactions the outcome of the competition reaction was altered compared to addition of BF3⋅Et2O alone (entry 3), with a decreased formation of galactoside 132 compared to glucoside 131. The difference in reactivity between BF3 and triflate catalysed glycosidation reactions might be explained by generation of glycosyl triflate intermediates, when triflate containing catalysts are employed. If the presence of a triflate increases the rate of reaction, the differences in reactivity of the two donors will not be observable. This will result in a 1:1 relationship between the products in the competition experiments. To test this hypothesis, a TfOH-catalysed competition experiment was carried out at –78 °C (entry 13). By decreasing the temperature a product ratio of 5:1 was obtained favouring the galactoside 132. This suggest that a positively charged intermediate is also formed in the triflate catalysed reactions 80

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

and that decreasing the temperature to –78 °C will slow down the reaction to a degree where rate differences are observable. The existence of triflate intermediates have indeed been observed for glycosidation using glycosyl sulfoxides as donors.180 Crich and Sun observed that the anomeric stereoselectivity on coupling sulfoxide donor 133 to the acceptor 134 in the presence of triflic anhydride and DTBMP was reversed if the order of addition of reactants was reversed (Scheme 2.5).
OTBDMS O O AcO AcO O Ph O O BnO OTBDMS O O AcO AcO O AcO OCH3
136

Ph

O O BnO

OTBDMS O + O
133

OH AcO AcO O AcO OCH3
134
Tf2O, DTBMP -78oC, Et2O

Ph

O O BnO

+

S

Et

AcO OCH3
135

A: 133 + 134 + DTBMP, then Tf2O, 135:136 = 6:58 B: 133 + DTBMP + Tf2O, then 134, 135:136 = 85:8

Scheme 2.5 Reversal of anomeric stereoselectivity on reversal of addition of reagents.

They suggested that Tf2O serves to activate the donor, generating a sulfonium salt, which immediately collapses to an oxocarbenium ion intermediate. If route A (see Scheme 2.5) is followed, the activation is carried out in the presence of the acceptor and the oxocarbenium ion will be trapped directly generating the α-mannoside 136, since this is favoured in Et2O especially for a mannoside. On the other hand, if the mannosyl donor 133 is activated prior to addition of the acceptor, the oxocarbenium ion is trapped by a triflate anion resulting in an α-mannosyl triflate (route B, Scheme 2.5). On addition of the glycosyl acceptor 134, they suggested that the triflate intermediate participates in an SN2-like reaction forming the β-mannoside 135. From a synthetic point of view this is interesting, since β-mannosidic bonds are often difficult to obtain. The effect of changing the glycosyl acceptor to a more complex carbohydrate alcohol was investigated by performing a competition reaction using carbohydrate alcohol 137 as acceptor instead of 1-octanol. When catalysing the reaction by TMSOTf, disaccharide 138 and 139 were formed in a 1:1 ratio (Scheme 2.6).VI

VI

Experiments on the triflate effect in disaccharide synthesis were performed by master student Tine Meyer.

81

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions
OBn OH BnO BnO O BnO OCH3
126a (1 equiv.) 130 (1 equiv.)
TMSOTf (0.17 equiv.) CH3CN, 0oC

BnO BnO

OBn O

BnO BnO

O BnO BnO BnO O O BnO OCH3 +

BnO BnO BnO

O O BnO OCH3

137

138, α:β 1:19

139, α:β 1:5

60 %, ratio 1:1

Scheme 2.6 Competition reaction with trichloroacetimidates and methyl 2,3,4-tri-O-benzyl-α-Dglucopyranoside (137).

2.2 Competition Reactions using Glycals as Donors
Another type of glycosyl donors are the commercially available perbenzylated glycals 140 and 141. Glycosidation using glycals is also believed to involve development of positive charge a C-1 in the transition state and therefore competition experiments were performed using the protected glycals.157 The glycals were activated in two different ways: 1) using NIS, generating the 2-deoxy-2-iodo glycosides181 and 2) using Ph3P⋅HBr, forming 2-deoxy glycosides.182 By promoting the reaction with NIS, the possibility of formation of several products again arose. Therefore, the products had to be synthesised individually to make analysis of the mixture from the competition reaction possible. Reaction of perbenzylated glucal 140 with NIS and 1-octanol resulted in formation of perbenzylated octyl 2-deoxy-2-iodo-α-Dmannopyranoside and octyl 2-deoxy-2-iodo-β-D-glucopyranoside (142) showing C-1 in the
13

C-NMR spectrum at 101.5 ppm and 103.3 ppm, respectively. From the corresponding 2-deoxy-2-iodo-α-D-talopyranoside (102.3 ppm), 2-deoxy-2-iodo-α-D-

reaction of the perbenzylated galactal 141, the outcome of the reaction was 143 (the perbenzylated

galactopyranoside (100.0 ppm), and 2-deoxy-2-iodo-β-D-galactopyranoside (103.9 ppm). Thus, the products from the competition experiment were distinguishable. The competition reaction was performed according to Scheme 2.7. Catalysing the reaction with Ph3P⋅HBr was done in a similar way and the outcome of the reaction was the perbenzylated 2-deoxy glycosides (gluco (144): δC-1 97.5 ppm and 100.0 ppm; galacto (145): δC-1 97.8 ppm and 100.5 ppm). Results from the competition reactions with 1-octanol are shown in Table 2.2.

82

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions
OBn BnO BnO OBn BnO BnO O + OBn O BnO 141
ol ctan , 1-o rt NIS 3Å, MS CN, CH 3 Ph 3P HBr , 1-o CH ctan 2 Cl ol 2 , rt

BnO + OOct BnO

OBn O I 143 OOct

O I 142

BnO

140

OBn BnO BnO O OOct 144 +

BnO BnO

OBn O OOct 145

Scheme 2.7 Competition reactions of tri-O-benzyl-glucal (140) and tri-O-benzyl-galactal (141) with 1-octanol using NIS or Ph3P⋅HBr as promoters.

Catalyst/promoter NIS Ph3P⋅HBr

Solvent CH3CN CH2Cl2

Equiv. promoter 2.9 0.06

Yield 82 % 87 %

Gal/Glc 1.4:1 1.3:1

Table 2.2 Results from competition reactions using glycals as glycosyl donors.

Both competition experiments showed that the reactivity of galactal 141 was somewhat higher than that of the glucal 140. This is in accordance with having positive charge in the transition state, but the effect was not as distinct as seen for the trichloroacetimidates.

2.3 Relative Reaction Rates among n-Pentenyl GlycosidesVII
The relative reactivity of three n-pentenyl glycosides upon glycosidation was also investigated. Again benzyl protected glycosides (146 and 147) were employed.183 Furthermore the benzyl protected n-pentenyl fucoside 148 was included in the study.184 Being a 6-deoxy analogues of the galactoside 147, the fucoside 148 was expected to react even faster than the galactoside because of the absence of a 6-hydroxyl group. When activating with NIS/Et3SiOTf in the presence of excess 1-octanol, a pseudo-first order reaction was assumed. Because of the relative slow rate of reaction, it was possible to follow the reaction directly by analysing reaction samples at defined times and determine the conversion by 1HNMR spectroscopy. The observed rate constants are shown in Scheme 2.8.

VII

The experiments on n-pentenyl glycosides were performed by fellow student Tomasz Krzysztof Olszewski.

83

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions
OBn gluco BnO BnO O O BnO 146 BnO galacto BnO BnO 147 OBn O O
1-octanol NIS, Et3SiOTf CH2Cl2, 22oC 1-octanol NIS, Et3SiOTf CH2Cl2, 22oC

131 kobs 4.25 x 10-4 s-1

132 kobs 5.5 x 10-4 s-1

fuco BnO

O OBn 148

O OBn

1-octanol NIS, Et3SiOTf CH2Cl2, 22oC

O BnO OBn

OOct OBn

149, kobs 12.8 x 10-4 s-1

Scheme 2.8 Reactions of n-pentenyl glycosides with 1-octanol in pseudo-first order reactions. Rate constants were determined by 1H-NMR studies.

As expected n-pentenyl fucoside 148 reacted faster than the galactoside 147, which reacted faster than the glucoside 146. The ratio of relative reactivity differences was determined to be 1:1.3:3 (glc/gal/fuc), which is relatively small compared to previous results of acid catalysed hydrolysis of methyl glucosides, galactosides, and fucosides determined to be 1:5:30.143 In Wong’s study of thioglycoside reactivity, they also obtained a larger reactivity difference between benzyl protected glucosyl, galactosyl, and fucosyl donors (1:6.4:27).159 The relatively low difference in reactivity observed for n-pentenyl glycosides 146-148 can be caused by the activation method using NIS/Et3SiOTf. This is in accordance with the observation that relatively small differences in the rate of reactivity of armed-disarmed n-pentenyl glycosides was observed by Fraser-Reid et al., when n-pentenyl glycosides were activated with NIS/Et3SiOTf.157

2.4 Investigation of Supposed SN2-Type Reactions
In order to show that an SN2-type mechanism of glycosidation would result in a 1:1 ratio of galactoside versus glucoside product in a competition experiment, it was decided to employ perbenzylated glycopyranosyl chlorides. 2,3,4,6-tetra-O-benzyl-α-D-glucosyl chloride (150) and the corresponding galactosyl chloride (151) were synthesised by standard procedure from the perbenzylated glycosyl pyranoses with oxalylchloride and DMF in CH2Cl2.185-187 By using a good nucleophile, the glycosyl chlorides were expected to react in an SN2-type pathway. The choice of nucleophile fell on potassium xanthogenate, which dictated the solvent, since it

84

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

was only soluble in CH3CN. Reactions were performed according to Scheme 2.9 yielding the β-glycosyl xanthates 152 and 153.

OBn BnO BnO O BnO 150 OBn O BnO BnO 151 Cl
KSC=S(OEt) CD3CN KSC=S(OEt) CD3CN

OBn BnO BnO O BnO S S OEt

Cl

152, kobs 1.6 x 10-4 s-1 OBn O BnO BnO S S OEt

BnO

BnO

153, kobs 6.7 x 10-4 s-1

Scheme 2.9 Substitution of perbenzylated glycosyl chlorides with potassium xanthogenate.

Because of a relative slow reaction rate it was decided to perform the reactions separately in NMR tubes in CD3CN and follow the rate of reaction by recording 1H-NMR spectra at different times. By adding excess of potassium xanthogenate (4-5 equivalents), the decay of glycosyl chloride was measured and by assuming a pseudo-first order reaction, the recorded data were fitted to the general relationship [A] = [A]o⋅e-kt. From plots of concentration versus time (Figure 2.1), the rate constants were determined to 1.6⋅10-4 s-1 for the glucosyl chloride and 6.7⋅10-4 s-1 for the galactosyl chloride.
D e term in a tio n of ra te co n sta n ts, ga lacto syl ch lo rid e + xan th a te 60
60 D ete rm in a tio n of rate co n sta n ts, glu cosyl d o n or + xa n th a te

y= a e xp (-kx), r =0 .99 6 a =5 6 .0 , k= 6 .6 4 E -4 σ a = 1.0 8 , σ k = 1.8 3 E -5 40
[A]/mM

2

y= a e xp (-kx), r = 0 .9 9 7 a = 64 .3, k= 1.6 8E -4 σ a = 0.6 4 6, σ k =4 .6 6 E -6 40
[A]/mM

2

y= a e xp (-kx), r = 0 .9 8 7 a =4 7 .7 , k= 6 .6 8 E -4 σ a = 1.3 3 , σ k = 2.7 7 E -5

2

y= a e xp (-kx), r =0 .9 6 7 a = 4 5.1 , k= 1 .5 7 E -4 σ a = 1 .3 2 , σ k =1 .3 5E -5

2

20

20

0

0

1000

2000 t/s

3000

40 0 0

0

0

50 0 0 t/s

10000

1 5 00 0

Figure 2.1 Determination of rate constants by 1H-NMR experiments for reaction of 150 and 151 with excess potassium xanthogenate.

However, the results were surprising, since the rate constants were expected to be similar and not to differ by a factor of 4 as observed. In addition, similar experiments were performed

85

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

using sodium butyl thiolate as nucleophile.VIII The experiments suggested that the galactosyl chloride reacted faster than the glucosyl chloride, but since the elimination products were observed as side products, it was not possible to determine the rate constants. Even though both pairs of experiments were suggested to happen though an SN2-pathway, it is evident that a substituent effect from the orientation of the C-4 substituent affects the rate of reaction. This suggests that positive charge accumulate in the transition state and that even reactions expected to be SN2-like, can have considerable SN1 character. Having the glycosyl donors 150 and 151 in hand, a direct competition experiment with 1-octanol using AgOTf (3.1 equiv.) as promoter in CH2Cl2 at room temperature was performed in a similar way as described for the trichloroacetimidates. By using 1.2 equivalent of each donor competing for 1 equivalent 1-octanol a 1:1 ratio of galacto (132) versus gluco (131) product was seen (Scheme 2.10) this time yielding an equal ratio of α- versus β-anomers.
OBn 150 + 151
1-octanol AgOTf CH2Cl2, 0oC 97 %

BnO OOct + BnO

OBn O BnO OOct

BnO BnO

O BnO

131, α/β 1:1

glc/gal 1:1

132, α/β 1:1

Scheme 2.10 Competition reaction between glycosyl chlorides using AgOTf as promoter.

The result of the competition reaction is not that surprising, bearing the previous results on activation of trichloroacetimidates with triflates in mind.

2.5 Mechanistic Considerations
From the described results, it is obvious that it is difficult to predict the outcome of a glycosidation reaction and that the mechanistic aspects of glycosidation reactions are rather complex. In order to come up with possible explanations to the above results, the reaction mechanisms suggested by Fraser-Reid et al., can serve as a useful tool (Scheme 2.11).157 Activation of imidates, halides, and thioglycosides followed by reaction with a glycosyl acceptor, is believed to follow a pathway described by Scheme 2.11a. Initially, the promoter activates the leaving group. This is followed by detachment of the leaving group to form the oxocarbenium ion, which can react with the acceptor.

VIII

Experiments employing sodium butyl thiolate were performed by master student Tine Meyer.

86

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

When trichloroacetimidates are activated by BF3, generation of the oxocarbenium ion (step 2, Scheme 2.11a) must as expected be rate-determining and since the oxocarbenium ion is formed faster for the galacto species, the rate of reaction is higher than for the corresponding gluco species. This is believed to be caused by stereoelectronic differences between electronwithdrawing axial and equatorial C-4 substituents. The same explanation is suggested to be the cause of different reactivities among thioglycoside donors activated by NIS/TfOH in the study performed by Wong and co-workers.159 Even though the substitution experiments on glycosyl chlorides using potassium xanthogenate as nucleophile were believed to be SN2 processes, it is evident from the rate constants, that the transition state of the reaction must have considerable carbocation character. Thereby, the electronic effects arising from having an axial or equatorial electron-withdrawing group in the C-4 position also affect the rate constants. To obtain a 1:1 or close to 1:1 relationship in the competition experiments, generation of the oxocarbenium ion cannot be rate-determining and a shift in the rate-determining step must occur. If step 1 or 3 in Scheme 2.11a is rate-determining, it is likely that a 1:1 ratio of the overall glycosidation reaction is obtained, since the activation process and the reaction of the nucleophile with the oxocarbenium ion is probably not affected by the orientation of the C-4 substituent. The observation of 1:1 relationships between galactose and glucose configured products when triflates are used to activate the glycosyl donors, might be explained by the first step in Scheme 2.11a becoming rate-determining. It is likely to be caused by an increase of the rate of step 2, resulting in the first step becoming rate-determining. This increased reactivity can be explained by generation of reactive glycosyl triflate intermediates. When the temperature is lowered to –78°C and a 5:1 gal/glc relationship is obtained. It might indicate that generation of the oxocarbenium ion (step 2, Scheme 2.11a) is slowed down and becomes rate-determining again. Reaction of n-pentenyl glycosides are believed to happen through similar mechanism except that it involves two preequilibration steps as shown in Scheme 2.11b. First, the pentenyl double bond is activated, whereupon the cationic intermediate is trapped in an intramolecular fashion by the anomeric oxygen to form a tetrahydrofuranyl oxonium ion. The oxocarbenium ion is then generated from this species and can react with the nucleophile. Since only small electronic effects are observed on activation of n-pentenyl glycosides with NIS/Et3SiOTf, it is suggested that one of the preequilibration steps is rate-determining. This might be the 87

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

generation of the tetrahydrofuranyl oxonium ion, since a positive charge in the vicinity of the anomeric center might to some extent be affected by the electron-withdrawing properties of the C-4 substituents, resulting in a small difference in reactivity.
O a) E + X O XE O Products

O b) E + E O O O

O O E

O Products

O c) I +

O Products I

O d) H +

O Products H

Scheme 2.11 Possible mechanisms for activation of various glycosyl donors.

The reaction of glycals probably takes place via direct formation of an iodonium ion or oxocarbenium ion depending on the method of activation. On activation with NIS the low gal/glc ratio can be explained by a smaller electronic effect from the C-4 substituent because of the remoteness of the positive charge in the iodonium ion intermediate in the same way as suggested for the n-pentenyl glycosides (Scheme 2.11c). An identical ratio is obtained on activation of glycals with Ph3P⋅HBr (Scheme 2.11d). Here the relatively small ratio cannot be explained by a remote positive charge. An explanation to the small difference in reactivity on activation with Ph3P⋅HBr, can be that generation of the positively charged intermediate is fast and therefore, it is not possible to observe any difference in reactivity. Another reason could be that the mechanism is not as straight forward as described in Scheme 2.11d.

3 Conclusions
The effect on glycosidation reactions of having an equatorial or axial electron-withdrawing substituent in the C-4 position on various glycosyl donors has been examined. The results show that galactosyl donors react faster than glucosyl donors when activated with non-triflate 88

Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

promoters, which imply that formation of the oxocarbenium is the rate-determining step of the reaction and that the reaction has considerable SN1-character. On the other hand, when activation is carried out with triflate activators, an equal reactivity of donors is observed in competition experiments, suggesting a change in the rate-determining step of the reaction. This could be obtained via an increase in the rate of leaving group departure possibly caused by a stable glycosyl triflate ion pair. In addition, attempts were made to show that the reaction of glycosyl halides with a strong nucleophile would react via an SN2 pathway resulting in equal formation of products, because of charge dispersion in the transition state. However, an increased reactivity of the galactosyl donor compared to the glycosyl donor was observed, suggesting that even a supposed SN2 reaction must be affected by the electron-withdrawing properties of the C-4 substituent. Therefore, it must be associated with built up of positive charge in the transition state and have considerable SN1 character. Altogether the experiments show that glycosidation reactions are complex and that in order to benefit from armed/disarmed effects in glycoside synthesis, reaction conditions must be carefully chosen.

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Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors – A Study of Glycosidation Reactions

90

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase 1 Introduction

1.1 Azasugars as Glycosidase Inhibitors
Glycosidases are a class of enzymes hydrolysing glycosidic bonds. They are widespread in organisms and have many important biological functions e.g. associated with breakdown of energy storage and modification of glycoproteins. Therefore, it is of great medical interest to be able to control such enzymatic processes in development of new therapeutics. One way of doing this is by using glycosidase inhibitors and indeed a large effort has been put into synthesising such compounds. Especially azasugars have attracted much attention, since many potent inhibitors are found among this type of compounds.188,189 Azasugars are nitrogen-containing mimics of carbohydrates. Numerous examples of azasugars are found in nature and interesting analogues have been synthesised (examples of azasugars are shown in Figure 1.1).190,191 Among these a few compound have been developed into medical agents e.g. acarbose (154) and miglitol (155) are used in the treatment of non-insulin dependent diabetes.192,193 Through α-glucosidase inhibitory activity, both compounds retard the breakdown of starch to glucose after ingestion of food.
OH HO HO HO HN HO O HO O HO O HO O HO OH O HO Acarbose, 154 OH Miglitol, 155 HO HO

OH N HO OH

OH HO HO NH HO β-glucosidase (almond) α-glucosidase (yeast) 1-Deoxynojirimycin, 156 Ki 47 µM Ki 25 µM HO HO

OH NH HO HO

OH NH NH

Isofagomine, 157 Ki 0.11 µM Ki 86 µM

Azafagomine, 158 Ki 0.32 µM Ki 6.9 µM

Figure 1.1 Examples of biological active azasugars. Ki values obtained from ref 190 and 191.

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Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

1.2 Mechanism of Glycosidase Catalysed Hydrolysis
Glycosidases are divided into different groups referring to whether they hydrolyse α- or β-glycosidic bonds (α- and β-glycosidases, respectively) and whether hydrolysis occurs with net inversion or retention of configuration. Almond β-glucosidase belongs to the group of retaining glycosidases believed to hydrolyse glycosidic bonds through the mechanism outlined in Figure 1.2 proposed by Koshland.194

O H OH HO HO O HO O O O

O OH HO HO

δ− O H O R

O

δ+ O δ+ HO O O

R

δ− O OH HO HO O HO H O O

H

O H OH HO HO O HO O O OH

O OH HO HO

δ− O H O H

O O

O

δ+ O δ+ HO O O

δ−

Figure 1.2 Catalytic mechanism of retaining β-glycosidase proposed by Koshland.

Two carboxylates in the protein, being 5.5 Å apart, is directly involved in the cleavage. The upper carboxylic acid is acting as a general acid activating the aglycon, which upon attack of the nucleophilic carboxylate at the anomeric center, leaves the glycon. This is believed to happen through an oxocarbenium ion like transition state, which leads to formation of an enzyme-glycosyl intermediate. In the second step of the mechanism, the deprotonated upper carboxylate act as a general base activating a water molecule. This water molecule then attacks the anomeric center cleaving the enzyme-glycosyl intermediate resulting in overall hydrolysis of the glycosidic bond with retention of configuration. Both transition states are believed to have considerable oxocarbenium ion character. A similar mechanism is believed

92

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

to exist for retaining α-glycosidases, but in this case the nucleophilic carboxylate is placed above the glycosidic bond to be cleaved.195 The position of the nucleophilic carboxylate above the sugar moiety in α-glycosidases, has been used to explain the reason why 1-deoxynojirimycin (156) is more potent against α-glucosidase inhibitor, whereas β-glucosidase is less affected by the presence of the inhibitor (Figure 1.1).196 On the other hand, moving the nitrogen to the anomeric position as in isofagomine (157) creates potent β-glucosidase inhibitors with poorer affinity towards α-glucosidases. This is also in accordance with results published by Bols et al. which show that azafagomine (158), having a nitrogen in both positions, binds to both α- and β-glucosidases with high affinity.197

1.3 Slow Inhibition
Some azasugars have turned out to be so-called slow inhibitors of glycosidases. Slow inhibition is described as a phenomenon where the establishment of the equilibria between enzyme, inhibitor, and enzyme-inhibitor complexes, occurs slowly on the steady-state time scale of seconds to minutes.198 The slow binding inhibitors are of great importance, because it is possible to follow the progress of the inhibition process. This can provide useful information on the mechanism of binding of an inhibitor to an enzyme, which is important when designing new and stronger inhibitors. Three different models for describing the phenomenon of slow inhibition have been suggested.198,199 If the rate of binding (kon) and dissociation (koff) of inhibitor to the enzyme are small compared to the rate of formation of the enzyme-substrate complex (ES), the slow step is formation of the enzyme-inhibitor complex (EI) and the simple model 1 is followed (Scheme 1.1).
E koff EI
Scheme 1.1 Slow inhibition according to model 1 - the 'simple model'.

k1S k2 konI

ES

k3

E +

P

93

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

On the other hand, if formation of the EI-complex is fast, but followed by a slow isomerisation to a locked complex EI* (kcf being the rate of conformational change) the inhibition follows model 2 (Scheme 1.2).

E koff EI konI

k1S k2

ES

k3

E +

P

kcf k-cf

EI*

Scheme 1.2 Slow inhibition according to model 2.

The last model is described by a necessary slow conformational change of the enzyme before binding of the inhibitor in a fast step (Scheme 1.3).
E k-cf E
*

k1 S k2 kcf konI koff

ES

k3

E + P

EI*

Scheme 1.3 Slow inhibition according to model 3.

Until recently it has been assumed that the most common mechanism for slow inhibition was model 2.199 But several more recent studies have suggested that the more simple model 1 involving one slow step is also quite common.200-202 This was also observed in a study on slow binding of isofagomine (157), azafagomine (158) and some analogues to almond β-glucosidase and yeast isomaltase performed in our group.203 In addition, it was observed that association of inhibitor and enzyme (kon) was relatively slow compared a to a diffusioncontrolled rate. Dissociation of enzyme and inhibitor (koff) also turned out to be slow and almost constant for the different inhibitors investigated. In that way, the differences in Ki values (Ki = koff/kon) among the inhibitors were entirely dependent on the rate of association, kon. In order to try to explain these observations, it was decided to take a closer look at the

94

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

slow binding of a range of azasugars to almond β-glucosidase at different temperatures to be able to evaluate the thermodynamic parameters for the enzyme-inhibitor process.

1.3.1 The β-Method
Whether a slow inhibition process can be described by model 1, 2, or 3, can be determined by performing kinetic experiments under steady-state conditions with respect to substrate. In that way, progress curves (product concentration versus time) described by equation 1 (for derivation see appendix 11) can be obtained.198,199 ⎛ α ⎞ A⋅C P ( t ) = A ⋅ t ⎜1 − ⎟ + (exp(−β ⋅ t ) − 1) ⎜ β⎟ β ⎝ ⎠ outlined in Table 1.1.
Model 1 A
Vmax ⋅ s 1+ s

(1)

A, α, β, and C are dependent on the given model followed by the inhibition process as

Model 2
Vmax ⋅ s 1+ s + i

Model 3
Vmax ⋅ s 1+ s

α

k on ⋅

[I] 1+ s

k cf ⋅

i 1+ s + i

k on ⋅

[I] 1+ s

β C

α + k off (ei) t =0 − α β

α + k -cf (ei) t =0 − α β

k cf k + −cf 1+ s 1+ i (ei) t =0 − α β

Table 1.1 A, α, β, and C. s =

[S]
KM

, i=

Ki

[I] ,

(ei) =

[EI] , [E ] = [E] + [ES] + [EI] tot [E]tot

By fitting the kinetic progress curves to equation 1, the apparent rate constant β, can be determined. From Table 1.1 it follows that if β turns out to be a linear function of the inhibitor concentration, [I], (a β-plot) model 1 can describe the enzyme-inhibitor interaction. In case of β being a increasing hyperbolic function of [I], model 2 is followed and if β is a decreasing hyperbolic function of [I] the interaction is described by model 3. If model 1 is followed, the rate constants kon and koff can easily be obtained from the β-plot (slope and intercept, respectively) and the Ki value of the inhibitor of interest can be calculated by Ki = koff/kon.

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Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

1.4 Determination of Thermodynamic Parameters
Thermodynamic parameters and activation energies for given reaction can be determined from the rate constants.204 The dependence of the rate constants on temperature are represented by the Arrhenius equation (2): ⎛ E ⎞ k = A ⋅ exp⎜ − a ⎟ ⎝ RT ⎠ (2)

where k is a rate constant, A is the pre-exponential factor, Ea the activation energy, R the gas constant, and T the temperature. In the case of reactions in solution this can be written as
k= ⎛ − ∆G ≠ k bT exp⎜ ⎜ RT h ⎝ ⎞ ⎛ ∆S ≠ k T ⎟ = e ⋅ b exp⎜ ⎟ ⎜ R h ⎠ ⎝ ⎞ ⎛ E ⎞ ⎟ exp⎜ − a ⎟ (3) ⎟ ⎝ RT ⎠ ⎠

where kb is the Boltzmann constant and h is Planck’s constant. If kon and koff for the reaction of a slow inhibitor with an enzyme are determined at various temperatures the activation parameters can be found from Arrhenius plots (ln k versus 1/T) and the enthalpy of activation, ∆H≠, can be determined from the activation energy (Ea = ∆H≠ + RT), and ∆S≠ is calculated from the equation (3). Eventually, ∆G≠ can be found from the general relationship ∆G≠ = ∆H≠ - T⋅∆S≠. From the activation parameters of association and dissociation, the standard thermodynamic parameters can be found from the equation ∆P° = ∆P≠on - ∆P≠off, where P denotes any thermodynamic parameter. If an inhibitor is not of the slow binding type, the thermodynamic parameters can be obtained by determination of the Ki values at different temperatures in a similar way from the van’t Hoff equation (4): ln K i = − ∆H o ∆S o + RT R (4)

∆H° and ∆S° are determined from van’t Hoff plots and ∆G° is subsequently calculated from ∆G° = -RT ln Ki.

96

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

2 Results and Discussion
2.1 Determination of Thermodynamic Parameters for Binding of Azasugars to β-Glucosidase
In order to obtain further insight into the mode of binding of azasugars to almond β-glucosidase kinetic experiments were performed for the slow binding inhibitors isofagomine (157), isogalactofagomine (159), azafagomine (158), and 3-amino-3-deoxy-1azafagomine (160) (Figure 2.1).
OH HO HO NH HO HO OH NH HO HO OH NH NH HO H2N OH NH NH HO HO OH NH OH 1-Deoxynojirimycin, 156

Isofagomine, 157

Isogalactofagomine, 159

Azafagomine, 158

3-Amino-3-deoxy1-azafagomine, 160

Figure 2.1 Structure of the 5 azasugars employed in this study.

Progress curves were obtained for each inhibitor at 8 different inhibitor concentrations by spectrophotometric monitoring of the enzymatic cleavage of 4-nitrophenyl-β-D-glucopyranoside by almond β-glucosidase. An example of a set of progress curves is shown in Figure 2.2. From each progress curve the apparent rate constant β, was obtained by fitting the
Progress curves 1.0
0.0075
β-plot

0.8
0.0060

Absorbance

0.6
β /(s )
-1

0.0045

0.4

0.0030

0.2

0

0

250 time/(s)

500

0.0015

0

0.4

0.8 I/(µM)

1.2

1.6

Figure 2.2 Example of progress curves and β-plot for inhibition of β-glucosidase by isogalactofagomine (159) at 15°C.

97

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

curves to equation 1 using the freeware programme INRATE in the software package Simfit (W.G. Bardsley, university of Manchester, UK). By plotting β versus [I] straight lines were obtained (example in Figure 2.2), which showed that for all inhibitors model 1 described the slow binding interaction with the enzyme. From the β-plots the rate constants for dissociation and association (koff and kon) of inhibitor and enzyme were determined and Ki could be calculated. By recording progress curves at 3-5 different temperatures for inhibitors 157-160, Ki, kon, and koff were determined and the thermodynamic parameters and activation energies were determined from Arrhenius plots as described in the introduction. 1-Deoxynojirimycin (156), turned out not to be a slow inhibitor of β-glucosidase, thus only standard thermodynamic parameters were obtained. This was done by measuring Ki values at 5 different temperatures according to normal Michaelis-Menten kinetics. From the Ki values a van’t Hoff plot was constructed and from that the thermodynamic parameters were deduced. All thermodynamic parameters are summarised in Table 2.1. For values of Ki, kon, and koff see tables in appendix 2.
∆G° -37.6 -39.9 -38.2 -24.8 -26.1 ∆G≠on 48.4 46.2 44.6 58.4 ∆G≠off 86.0 86.1 82.8 83.2 ∆H° 58.6 58.7 -1.5 4.3 -25.7 ∆H≠on 56.1 56.2 39.3 49.4 ∆H≠off -2.5 -2.5 40.8 45.1 ∆S° 323.8 331.0 123.1 97.3 1.3 ∆S≠on 25.8 33.6 -17.9 -30.4 ∆S≠off -297.0 -297.4 -141.0 -127.7 -

Inhibitor 157* 159 158 160
*

156

Table 2.1 Thermodynamic activation parameters and standard parameters for binding of azasugars to almond β-glucosidase at pH 6.8. ∆G and ∆H in kJ/mol, ∆S in J/mol⋅K. * Inhibitor is racemic.

The thermodynamic parameters for isofagomines 157 and 159 were almost similar resulting in similar energy profiles (Figure 2.3). Binding of 157 and 159 to the enzyme to formation of the transition state (EI≠), was found to be associated with a large positive change in enthalpy and almost no change in entropy, thus it is an endothermic process. From the transition state to formation of the EI-complex a large change in entropy was seen and almost no change in enthalpy. Thus, the overall process is driven by entropy and therefore not favourable in terms of binding energies.

98

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase
∆G
120 100

∆H
400

∆S

EI

EI

80

EI

EI
300

80

energy, kJ/mol

energy, kJ/mol

e.u., J/mol⋅K
E+I

60

200

E+I

40

40
100 E+I EI

20 EI

0

0

0

reaction coordinate (arbitrary)

reaction coordinate (arbitrary)

reaction coordinate (arbitrary)

Figure 2.3 Energy diagrams showing the change in free energy (∆G), enthalpy (∆H), and entropy (∆S) on binding of isofagomine (157) to almond β-glucosidase.

The corresponding thermodynamic data for azafagomines 158 and 160 were also found to be quite similar. From the energy profiles (Figure 2.4) it is seen that binding is associated with a positive change in enthalpy going from reactants to transition state, while almost no change in entropy is seen. Moving further along the reaction coordinate from the transition state to EI-complex a negative change in enthalpy is observed resulting in no appreciable change in the overall process. Again a large change in entropy was seen when going from the transition state to EI-complex and again the overall process is driven by entropy, but not with a smaller degree than observed for the isofagomines 157 and 159.
∆G
120 80

∆H

∆S

150 EI

EI

60 80

EI

energy, kJ/mol

energy, kJ/mol

40

E+I

40 E+I 20 EI

e.u., J/(mol⋅K)

100

50

E+I EI 0 0 0 EI

reaction coordinate (arbitrary)

reaction coordinate (arbitrary)

reaction coordinate (arbitrary)

Figure 2.4 Energy diagrams showing the change in free energy (∆G), enthalpy (∆H), and entropy (∆S) on binding of azafagomine (158) to almond β-glucosidase.

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Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

The thermodynamic data for interaction of 1-deoxynojirimycin (156) with β-glucosidase was found to be much different from especially the isofagomines. Binding was now entirely driven by enthalpy and almost no change in entropy i.e. the binding process is now favourable in terms of binding energies. This is in accordance with the observation that binding of various oligosaccharides to lectins is often driven by negative enthalpy whereas the entropy seems to be less important.205-207 The only difference between isofagomine (157) and azafagomine (158) is that 158 has an additional ring nitrogen. Thus, this nitrogen must be the reason for the altered thermodynamic parameters, i.e. approximately 60 kJ/mol of enthalpy is gained on having an extra nitrogen in this ring. From this we suggested that the extra ring nitrogen must be involved in binding of the inhibitors through hydrogen bonds or an ionic interaction via a salt bridge to a carboxylate in the enzyme. It is also evident that the anomeric nitrogen present in both isofagomines and azafagomines does not contribute to binding through bond energy, but instead a large gain of entropy is paid of during the binding process. The large change in entropy on having an anomeric nitrogen is to some extent suggested to be caused by the release of highly ordered water molecules from the active site to bulk solution on binding of the inhibitor, generating a more disordered system. The most important difference between 156 and isofagomines and azafagomines is that 1-deoxynojirimycin (156) does not have a nitrogen in the anomeric position but has an additional hydroxyl group in the 2-position (carbohydrate numbering). Because of the absence of an anomeric nitrogen, no change in entropy is observed. Compared to the azafagomines, 156 has approximately 25 kJ/mol lower binding enthalpy, which mainly must be described to the presence of the 2-OH group capable of making hydrogen bonds to the enzyme. This is in accordance with previous studies of the importance of the 2-OH group, in which the interaction of the 2-hydroxyl group with the enzyme is believed to stabilise the transition state by 30-40 kJ/mol.208 From X-ray crystallographic studies it has been proposed that the 2-OH group makes a short, strong hydrogen bond to the nucleophilic carboxylate in the enzyme active site and thereby a stabilisation is obtained (Figure 2.5).209 In addition, kinetic studies on hydrolysis of other β-D-glucosides (4-methylumbelliferyl-β-D-glucosides) by β-glucosidase have shown that hydrolysis of the glucoside was considerably faster than hydrolysis of its 2-deoxy congener.210 These findings support the importance of having a 2-hydroxyl group present in a potent inhibitor. 100

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

δ− O OH HO HO H O O δ+ O δ− O δ+ H O R

O

Figure 2.5 Proposed stabilisation through hydrogen bond formation from the enzyme to a 2-hydroxyl group.

2.1.1 2-Hydroxyl Analogues of Azasugars
Based on the above observations, it is evident that the mode of binding of 157 and 156 is very different. This suggests that the 2-OH group in 156 and the nitrogen in 157 are not participating in identical interactions with the enzyme. It was therefore anticipated that the 2-hydroxy analogue of isofagomine, noeuromycin (161), would be a more potent inhibitor of β-glucosidase, since a gain in enthalpy was expected. Work was therefore initiated with the aim of synthesising this compound and its galacto and fuco analogues 162 and 163 and evaluating their biological activity.IX The syntheses were performed according to reaction schemes in appendix 4.
OH HO HO HO NH HO HO HO OH NH NH OH

OH HO

Noeuromycin, 161

Galacto-noeuromycin, 162

Fuco-noeuromycin, 163

Figure 2.6 Structures of 2-hydroxyl analogues of isofagomine and its galacto- and fuco-isomers.

Biological testing was performed according to normal Michaelis-Menten kinetics and Ki values determined from Hanes plots. All three 2-hydroxyl analogues 161, 162, and 163 turned out to be extremely potent inhibitors of glycosidases with lower Ki values than their 2-deoxy analogues 157, 159, and 164 (Table 2.2).211,212-214

IX

The authors contribution to this work is only associated with the biological testing of the products.

101

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

OH HO HO 157 NH HO HO

OH NH HO 161

HO HO

OH NH 159

HO HO

OH
NH

NH HO 162

NH OH OH HO 163

OH HO 164

α-glucosidase Isomaltasea β-glucosidase

a

86000 7200i 110 d i

i

22 25 69 -

50000 328 4j 200 k

742 91 35 397 -

4000 6400l

4.7 3.2

b

α-galactosidasec β-galactosidase β-galactosidasee β-galactosidase α-fucosidaseg α-fucosidaseh
f

Table 2.2 Inhibition constants Ki in nM for binding of inhibitors to various enzymes. a from yeast. b from almond. c from Green Coffee Bean. d from Saccharomyces fragilis. e from Aspargillus oryzae. f from E. coli. g from bovine kidney. h from human placenta. i From ref 111. j From ref 212. k inhibitor is racemic. From ref 214. l From ref 213.

It is seen that by introduction of a 2-hydroxy group in isofagomine to form 161, the affinity of the inhibitor to the enzyme increases by 2-4000 fold. With respect to the galactonoeuromycin (162) and fuco-noeuromycin (163) the inibition also increased significantly. In addition, the affinity of 161, 162, and 163 increase significantly more for α-glycosidases compared their 2-deoxy analogues. Therefore, the anomeric nitrogen is presumably involved in forming an interaction with the enzyme possibly through a salt bridge. It is evident from the inhibition data that the 2-hydroxyl group indeed is involved in creating very strong binding glycosidase inhibitors.

2.2 Discrepancy

between

Thermodynamic

Results

of

Binding

of

Isofagomine and 1-Deoxynojirimycin to β-Glucosidase
Zechel et al. recently published a paper on determination of thermodynamic parameters of binding of isofagomine (157) and 1-deoxynojirimycin (156) to β-glucosidases, i.e. a study of the same thermodynamic parameters as performed from our lab and described above.215 In contrast to our results showing a difference in the mode of binding of isofagomine and 1-deoxynojirimycin, they found from isothermal titration calorimetry studies that binding of both inhibitors were driven by a large and favourable enthalpy. Thus, we are faced with two 102

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

different methods for determination of thermodynamic parameters that show different results. This type of contradiction between van’t Hoff analysis and calorimetric data is not unique. Among others, discrepancies have also been observed for binding of ligands to Acyl-Coenzyme A Binding Protein.216 It was suggested that the discrepancies indicate that the binding reaction is more complex than a simple one-to-one binding model used to describe the data. Other researchers have performed experimental and simulation studies of enzyme-ligand binding that do not indicate any significant discrepancies between the calorimetric determined enthalpies and those deduced from van’t Hoff plots.217 From this study they conclude that where such discrepancies are observed, they are likely to arise from either uncertainties due to extrapolation or inadequate calibration of the instrument. At present, the cause of the contradiction between our results and Zechel et al. remain unclear.

2.3 Determination of Thermodynamic Parameters by Numerical Solution of Differential Equations
2.3.1 The Differential Equation Method
In order to investigate thermodynamics for binding of a tight binding inhibitor, we set out to take a closer look at the interaction of the slow binding 2-phenethylglucoimidazole inhibitor 165 with almond β-glucosidase. The slow interaction of enzyme and inhibitor has previously been described to follow model 2 (Scheme 1.2) and a Ki value of 1.2 nM at 37 °C has been published.218 At present, 165 is the strongest known β-glucosidase inhibitor.
OH HO HO N OH 165 N Ph

Figure 2.7 Structure of the very slow binding inhibitor 165.

First we analysed the mode of binding according to the β-method in the same way as described for other azasugars in section 2.1. The β-plots turned out to be perfectly straight lines consistent with the inhibition process following model 1 (Scheme 1.1). We obtained Ki values of 145±31 nM at 15 °C and 35±12 nM at 25°C and not 1.2 nM as previously suggested. But the reliability of the method did not seem overwhelming (large standard deviations) and in addition simulation of the model using the rate constants from the 103

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

β-method gave a poor fit of experimental data and the simulated curve (Figure 2.8). It turns out that the β-method is not a good method for describing enzyme-inhibitor interaction when the rate constants turn out to be very small. Therefore, if a given slow binding inhibitor is also tight binding i.e. if the inhibitor has high affinity and the Ki value is comparable to the enzyme concentration, the β-method is unreliable. This is due to the fact that koff is determined as an intercept in a graph and this becomes inaccurate when koff is too close to zero. We continued analysing the results by deviation of the differential equations describing model 1 (see appendix 3 for further details). The kinetic data were fitted to these differential equations using the program GEPASI.219,
X

The obtained rate constants showed that

regardless of the initial enzyme concentration, the rate constants were identical. It is therefore possible to use this method in experiments where the exact enzyme concentration is not known. In addition, when simulating a curve from the data obtained from the differential equation method (DE method), a perfect fit with the experimental data were obtained (Figure 2.8).
25 °C
0.03

0.02

P, mM

0.01

'β-method' fit diff.eqs.fit exp. data

0

0

200

400

600

800

1000

time, s

Figure 2.8 Comparison of two different fitting procedures. Data obtained at 25°C, pH 6.8, and [I]=2.8 mM.

By using the DE-method a Ki value of 2.0±1.2 nM was obtained at 35 °C, which is comparable to the inhibitor constant obtained by Panday et al.218

X

All computations using GEPASI were carried out by ass. prof. Igor W. Plesner.

104

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

By using the DE method kinetic data obtained at 4 different temperatures were fitted and rate constants obtained. In addition to the kon and koff rate constants, the substrate binding rate constant, k1, was also determined. Surprisingly, the magnitude of this constant was in the same order as kon (104 M-1s-1). This suggests that not only binding of inhibitors are slow, but association of enzyme and substrate is also a slow process. From values of kon, koff, and Ki (see appendix 3 for values of kon, koff, and Ki) thermodynamic parameters were determined as described earlier from Arrhenius’ plots (Table 2.3).
∆G On Off Standard 25°C 46.9 94.1 -47.2 ∆H 57.5 -2.5 60.0 ∆S 35.7 -324 359.7

Table 2.3 Thermodynamic parameters for binding of 165 to β-glucosidase. ∆G and ∆H in kJ/mol, ∆S in J/mol⋅K.

The thermodynamic parameters are very similar to those presented for isofagomine resulting in almost similar energy profiles (Figure 2.3). It is highly surprising, since for isofagomines the large positive entropy was suggested to be associated with the presence of an anomeric nitrogen. Since the inhibitor 165 does not have a nitrogen in the same position as isofagomine, the positive change in enthalpy and entropy are therefore believed to be caused by the presence of the phenethylimidazole moiety. A possible interaction could be obtained between the imidazole moiety and a carboxylic acid in the enzyme.

3 Summary and Conclusions
Through kinetic experiments slow inhibition of almond β-glucosidase by 5 different azasugars was shown to follow a direct binding model, which involve a single step having slow on and off rates. Normally, the β-method can be used for determination of reaction rates, but in the case of tight binding inhibitors these reaction rates become inaccurate. Therefore, a method involving numerical solution of differential equations was presented that could be applied when the β-method was inadequate. By numerical solution of the differential equations, rate constants for association of enzyme and substrate were also shown to be slow,

105

Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond β-Glucosidase

suggesting that slow association of enzyme-inhibitor is caused by resemblance with the substrate. By determining thermodynamic parameters for interaction between enzyme and inhibitors, it was shown that slow binding of 1-azasugars is caused by a large enthalpy barrier that has to be overcome before binding can occur. The overall process is driven by entropy, which seems to be associated with the presence of an anomeric nitrogen. Binding of 1-deoxynojirimycin, which is not a slow binding inhibitor, was also investigated. In contrast to the slow inhibitors, the binding process was driven by enthalpy, which was attributed to the existence of a favourable interaction between the 2-hydroxylgroup and the enzyme. This led to the design of three new potent inhibitors having 2-hydroxyl groups, which all turned out to be more potent than their 2-deoxy analogues.

106

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