anne bülow- combinatorial synthesis of cocaine analogues

8/3/2019 Anne Blow- Combinatorial Synthesis of Cocaine Analogues

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Combinatorial Synthesis of Cocaine Analogues

and

Competition Reactions between Glucosyl Donors andGalactosyl Donors - A Study of Glycosidation Reactions

and

Determination of Thermodynamic Parameters for Binding ofAzasugars to Almond-Glucosidase

Ph.D.dissertation submitted by:

Anne BlowDepartment of Chemistry

University of AarhusAugust 2004


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i Table of Contents

i Table of Contents........................................................................................................... iii

ii Preface ............................................................................................................................vii

iii Acknowledgements...................................................................................................... viii

iv List of Appendices ..........................................................................................................ix

v List of Abbreviations.......................................................................................................x

vi Summary ........................................................................................................................xii

Chapter I: Combinatorial Synthesis of Cocaine Analogues

1 INTRODUCTION...........................................................................................................1

1.1 COCAINEA STIMULANT OF THE CENTRALNERVOUS SYSTEM .............................1

1.2 DEVELOPMENT OF MEDICATIONS FORTREATMENT OF COCAINE ABUSE ...............3

1.3 POTENTIAL DOPAMINE TRANSPORTERLIGANDS ....................................................4

1.3.1 Phenyltropanes ..........................................................................................4

1.3.2 Various Structural Classes of Potential Dopamine Transporter Ligands10

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

2 COMBINATORIAL CHEMISTRY............................................................................14

2.1 INTRODUCTION.....................................................................................................14

2.2 IDENTIFICATION OF ACTIVE COMPOUNDS IN A LIBRARY ......................................15

2.3 SOLID PHASE VERSUS SOLUTION PHASE APPROACHES.........................................17

3 THE GRIGNARD REACTION...................................................................................19

3.1 THE GRIGNARD REAGENT ....................................................................................19

3.1.1 Grignard Reagents in Conjugate Addition ..............................................20

3.2 THE GRIGNARD REACTION IN COMBINATORIAL CHEMISTRY ...............................21

4 SYNTHESIS OF THE TROPANE SKELETON.......................................................22

4.1 SYNTHESES OF COCAINE AND OTHERTROPANES..................................................22


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4.1.1 [3+4] Cycloaddition of Pyrroles and ,-Dibromoketones .................. 24

4.1.2 Tandem Cyclopropanation/Cope Rearrangement of Vinylcarbenoids with

Pyrroles ................................................................................................... 26

4.1.3 Tropanes from Pyrrolidine Derivatives .................................................. 27

4.2 SOLID PHASE CONSIDERATIONS........................................................................... 29

4.3 CONCLUSION........................................................................................................ 30

5 TWO- AND THREE-DIMENSIONAL SOLUTION PHASE COMBINATORIAL

LIBRARIES OF 3- AND 8-SUBSTITUTED TROPANES FROM MULTICOM-

PONENT GRIGNARD REAGENTS.......................................................................... 31

5.1 GENERATION OF A TWO-DIMENSIONAL LIBRARY FROM MULTICOMPONENTGRIGNARD REAGENTS .........................................................................................31

5.1.1 Designing the Library ............................................................................. 31

5.1.2 Initial model studies................................................................................ 33

5.1.3 Synthesis and Analysis of the Two-Dimensional Library ...................... 34

5.1.4 Biological Results for the Two-Dimensional Library ............................ 37

5.2 GENERATION OF A THREE-DIMENSIONAL LIBRARY FROM MULTICOMPONENT

GRIGNARD REAGENTS .........................................................................................40

5.2.1 Initial model studies................................................................................ 40

5.2.2 Synthesis and Analysis of the Three-Dimensional Library .................... 42

5.2.3 Biological Results for the Three-Dimensional Library .......................... 44

5.3 APPLYING TWO- AND THREE-DIMENSIONAL LIBRARIES TO OTHERSYSTEMS ...... 46

5.4 SUMMARY AND CONCLUDING REMARKS ............................................................. 47

6 BICYCLO[3.2.1]OCTANE ANALOGUES OF PHENYLTROPANES.................. 49

6.1 INTRODUCTION .................................................................................................... 496.1.1 8-Oxa Analogues .................................................................................... 49

6.1.2 8-Carba Analogues.................................................................................. 50

6.1.3 Biological Activity of Non-Amines........................................................ 51

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

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 ................... 52


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6.2.2 Attempts to Perform Conjugate Additions to Methyl bicyclo[3.2.1]octa-

2,6-diene-2-carboxylate and Methyl bicyclo[3.2.1]oct-2-ene-2-

carboxylate ..............................................................................................53

6.2.3 Model Studies on Methyl Crotonate .......................................................57

6.2.4 Synthesis of an 8-Carbon Analogue........................................................59

6.3 CONCLUSIONS ......................................................................................................62

Chapter II:Competition Reactions between Glucosyl Donors andGalactosyl Donors - A Study of Glycosidation Reactions

1 INTRODUCTION.........................................................................................................65

1.1 CARBOHYDRATESUBIQUITIOUS MOLECULES ...................................................65

1.2 ACID-CATALYSED HYDROLYSIS OF GLYCOSIDES.................................................66

1.3 GLYCOSIDATION REACTIONS ...............................................................................69

1.3.1 The Trichloroacetimidate Method...........................................................73

2 RESULTS AND DISCUSSION....................................................................................76

2.1 COMPETITION REACTIONS USING TRICHLOROACETIMIDATES...............................76

2.1.1 Synthesis of Trichloroacetimidate Donors ..............................................76

2.1.2 Synthesis of the Competition Reaction Products ....................................77

2.1.3 Competition Reactions between Perbenzylated Gluco and Galacto

Trichloroacetimidates..............................................................................78

2.2 COMPETITION REACTIONS USING GLYCALS AS DONORS ......................................82

2.3 RELATIVE REACTION RATES AMONGN-PENTENYL GLYCOSIDES .........................83

2.4 INVESTIGATION OF SUPPOSED SN2-TYPE REACTIONS ..........................................84

2.5 MECHANISTIC CONSIDERATIONS..........................................................................86

3 CONCLUSIONS............................................................................................................88


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Chapter III: Determination of Thermodynamic Parameters forBinding of Azasugars to Almond -Glucosidase1 INTRODUCTION......................................................................................................... 91

1.1 AZASUGARS AS GLYCOSIDASE INHIBITORS.......................................................... 91

1.2 MECHANISM OF GLYCOSIDASE CATALYSED HYDROLYSIS................................... 92

1.3 SLOW INHIBITION................................................................................................. 93

1.3.1 The -Method ......................................................................................... 95

1.4 DETERMINATION OF THERMODYNAMIC PARAMETERS ......................................... 96

2 RESULTS AND DISCUSSION ................................................................................... 97

2.1 DETERMINATION OF THERMODYNAMIC PARAMETERS FORBINDING OF

AZASUGARS TO -GLUCOSIDASE ......................................................................... 97

2.1.1 2-Hydroxyl Analogues of Azasugars.................................................... 101

2.2 DISCREPANCY BETWEEN THERMODYNAMIC RESULTS OF BINDING OF

ISOFAGOMINE AND 1-DEOXYNOJIRIMYCIN TO -GLUCOSIDASE ........................ 102

2.3 DETERMINATION OF THERMODYNAMIC PARAMETERS BYNUMERICAL SOLUTION

OF DIFFERENTIAL EQUATIONS ........................................................................... 103

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

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

References....................................................................................................................... 107

Appendix 1-11


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vii

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 -glucosidase with various

inhibitors.

Anne Blow, August 2004


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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, chemistry-

tricks, 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 Hyer

Lillelund, Henrik Helligs 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 Se are

thanked for cheering me up during creation of this thesis.


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iv List of Appendices

Appendix 1: Blow, A.; Plesner, I. W.; Bols, M.J. Am. Chem. Soc.2000, 122,

8567-8568.

Appendix 2: Blow, A.; Plesner, I. W.; Bols, M.Biochim. Biophys. Acta2001,

1545, 207-215.

Appendix 3: Plesner, I. W.; Blow, A.; Bols, M. Anal. Biochem. 2001, 295,

186-193.

Appendix 4: Liu, H.; Liang, X.; Shoel, H.; Blow, A.; Bols, M.J. Am. Chem.

Soc.2001, 123, 5116-5117.

Appendix 5: Blow, A.; Meyer, T.; Olszewski, T. K.; Bols, M. Eur. J. Org.

Chem. 2004, 323-329.

Appendix 6: Blow, A.; Sinning, S.; Wiborg, O.; Bols, M. J. Comb. Chem.

2004, 6, 509-519.

Appendix 7: Pedersen, H.; Sinning, S.; Blow, A.; Wiborg, O.; Bols, M. Org.

Biomol. Chem. 2004 accepted for publication

Appendix 8: Experimental section

Appendix 9: List of ligands used for evaluation of IC50 and Ki values for

potential cocaine antagonists

Appendix 10: NMR spectra of compounds 111 and 112

Appendix 11: Derivation of the Integrated Rate Equation for Slow-Binding

Inhibitors Described by Model 1.


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v List of Abbreviations

ABSA Acetamidobenzenesulfonyl azideAc Acetyl

ACE-Cl 1-Chloroethyl chloroformateADHD Attention-deficit hyperactivity disorderAIBN 2,2-AzobisisobutyronitrileAr Aryl AngstromBn BenzylBoc tert-ButoxycarbonylBu ButylBz Benzoylcat CatalystCbz Benzyloxycarbonyl

cod CyclooctadieneCOSY Correlation spectroscopyCy CyclohexylDA DopamineDAT Dopamine transporterDba trans,trans-DibezylideneacetoneDBU 1,8-diazabicyclo[5.4.0]undec-7-eneDE Differential EquationDIEA N,N-DiisopropylethylamineDMF N,N-DimethylformamideDMTST (dimethylthio)methylsulfonium trifluoromethanesulfonateDNA Deoxyribonucleic acidDTBMP 2,6-di-tert-butyl-4-methylpyridineE EnzymeEI Enzyme-Inhibitor complexEquiv. EquivalentES Enzyme-Substrate complexESMS Electronspray mass spectrometryEt EthylFmoc 9-FluorenylmethoxycarbonylFuc Fucose

Gal GalactoseGBR Gist-BrocadesGC-MS Gas chromatographyGlc Glucoseh Hour(s) or humanHBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateHMPA HexamethylphosphoramideHPLC High Performance Liquid ChromatographyHRMS High Resolution Mass SpectrometryI InhibitorIC50 Inhibition concentration, 50 %

IDPC Iodonium dicollidine perchlorate


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iPr isopropylKi Inhibition constantLDA Lithium diisopropyl amideLG Leaving group

MBHA 4-MethylbenzhydrylamineMe MethylMes Mesityl (2,4,6-trimethylphenyl)Min Minute(s)MMP-1 Matrix metalloproteinase-1Ms MethanesulfonylMS Molecular sievesn Normal

NBS N-Bromosuccinimide NE Norepinephrine NET Norepinephrine transporter

NIS N-Iodosuccinimide NMR Nuclear Magnetic Resonance Nu NucleophileOct OctylP ProductPent n-PentenylPh Phenyl

ppm parts per millionPS PolystyreneQSAR Quantitative structure-activity relationshiprds Rate-determining stepRNA Ribonucleic acidrt Room temperatureRTI Research Triangle InstituteS SubstrateSAR Structure-activity relationshipSER SerotoninSERT Serotonin transporterTBACN Tetrabutylammonium cyanideTBAF Tetrabutylammonium fluorideTBDMS tert-Butyldimethylsilyl

Tf TrifluoromethanesulfonylTFA Trifluoroacetic acidTHF TetrahydrofuranTLC Thin layer chromatograhpyTMS TrimethylsilylTol p-MethylphenylTroc 2,2,2-TrichloroethoxycarbonylWIN Sterling-Wintrop Institute


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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 ,-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 theirN-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-carbaanalogues 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


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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 togluco 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 -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 astabilising 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.


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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 cocaines 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 Coca-

Cola 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.

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 effectssuch as reduced fatigue and psychomotorial stimulation these effects finally lead to abuse and

addiction.4

Figure 1.1R-Cocaine - the naturally occurring stereoisomer.

Figure 1.2 Structure of the three natural monoamine neurotransmitters.

N

CO2CH3

OPh

O

1 2

345

67

8

R-Cocaine, 1

HO

HO

HO HO

HO

NH2

OH

NH

NH2

NH2

Dopamine (DA), 2 Serotonin (SER), 3 Norepinephrine (NE), 4


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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

SERT[3H]paroxetine

NET[3H]nisoxetine

Binding 10212 104589 3298293R-Cocaine

Uptake 24118a 1122b 16015c

Table 1.1 Binding potencies and inhibition of reuptake by cocaine at the three monoamine transporters. aKivalue for displacement of [3H]DA uptake. bKi value for displacement of [

3H]SER uptake. cKi value for

displacement of [3

H]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 -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

I

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

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


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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, sinceM5-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 cocaineabuse 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


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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

Parkinsons 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 attention-

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 3-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

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

HN

OCH3

O

Methylphenidate, 5

IC50 83 nM


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from (1R, 5S)-anhydroecgonine methyl ester7 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.

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 theR-configuration.18 This feature is also seen for cocaine itself, where theR-

isomer is about 150 times more potent than the S-isomer (IC50 for inhibition of

[3H]WIN35428 binding to rat striatal membranes: 0.102 M and 15.8 M respectively).18 It is

also evident from several other analogues e.g. for WIN35065 theR-enantiomer (WIN35065-

2) has been noted to be approximately 800 fold more active than the S-enantiomer

(WIN35065-3, Figure 1.5).19

Scheme 1.1 Synthesis of phenyltropanes fromR-cocaine.

Figure 1.5 Difference in inhibition of binding of [3

H]cocaine to mouse striatal membranes of enantiomers.

N

CO2CH3

PhN

Ph

CO2CH3

WIN35065-2, R-10

IC50 40.7 nM

WIN35065-3, S-10

IC50 32400 nM

N

CO2CH3

OPh

O

1N aq. HCl N

COOH

OH1. POCl3

2. MeOH, H+N

CO2CH3

ArMgBr

low temp, Et2O

N

CO2CH3

ArN

Ar+ CO2CH3

R-cocaine, 1 6 7

8 9


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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 3-substituent causes to 6-membered ring to flip to

the boat conformation, which will position the 3-substituent in a pseudoequatorial position

that is approximately the same position as for the 3-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 isalso 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.


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Table 1.2 Binding affinities and inhibition of reuptake at the DAT for selected phenyltropanes. aKi value insteadof IC50.

bKi values for inhibition of binding of [3H]GBR12935 instead of [3H]WIN35428. c Inhibition of binding

of [3H]cocaine instead of [3H]WIN35428.

A 2-carbomethoxy group has been thought to be crucial for binding of cocaine to the DAT,

since replacing it by hydrogen, a carboxy group, or anN-methylcarboxamido group decreasedactivity by 25-2000 fold.3 The interaction has been suggested to happen through hydrogen

bonds. 2-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 2-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

N

CO2CH3

R

RIC50/nM

[3

H]WIN35428

IC50/nM

[3

H]DA uptake

R

IC50/nM

[3

H]WIN35428

IC50/nM

[3

H]DA uptakeWIN35065-2 23.05.021 49.82.3a,24 55223 -NH2 24.81.3

21 5577925 68.57.123 -

NO2 10.10.1021 6168425 >500b,26 -

OCH3 8.141.321 - 5975227 -

N3 2.120.121 - 1.20.123 -

FWIN35428 15.71.421 230.5a,24 15.60.623 29.43.828

Cl 1.170.121 3.680.09a,24 0.490.0429 3.530.0928

Br 1.810.3021 - 3.70.1623 -

IRTI-55 1.260.04

21 1.960.09a,24 CH2Ph 5266523 -Cl

Cl 1.090.0229 -

CH2CH2Ph 5.140.6323 -1.710.321 7.00.3a,24 CH2CH2CH2Ph 3515223 -

CF3 13.12.221 - 88518c,28 10205228

I 466b,26 - 9.940.33c,28 70.51.028


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8

position were found to exhibit nanomolar and subnanomolar affinities for binding to the

dopamine transporter (Figure 1.6).31 2-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.

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

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

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

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

N

CO2CH3

N

CO2CH3F F

15

IC50 22.6 nM

WIN35428, 16

IC50 17.5 nM

N N N

CO2Ph

Cl Cl Cl

Ph

11

Ki 1.46 nM

12

Ki 1.21 nM

13

IC50 1.99 nM

N Cl

14

IC50 0.59 nM

N

O


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9

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, -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

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).

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.

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

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

N

CO2CH3

N

CO2CH3

N

CO2CH3

HOHO

WIN35065-2, 10

IC50 65 nM

17

IC50 235 nM

18

IC50 6150 nM


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10

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 weresuggested 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).

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 ap-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 3-diphenylmethoxy substituent. It was

first synthesised in 1952 and was subsequently demonstrated to be useful as an

anticholinergic drug in the treatment of Parkinsons 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

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

NCO2CH3

NCO2CH3

Cl N ClE

(-)-20

IC50 76919 nM

(-)-21

IC50 24.81.6 nM

()-22

IC50 1978 nM

NCO2CH3

Arecoline, 19


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11

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 theR-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.

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

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

Figure 1.12 Inhibition of binding of [125I]RTI-55 by GBR12909 to rat caudate.

N

O

Benztropine, 23

IC50 312 nM

N

O

F

F

24

Ki 11.8 nM

N

O

F

F

CO2CH3

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

PhCH2CH2CH2N

N

O

F

F

GBR12909, 26

IC50 3.7 nM


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12

same degree of euphoria compared to cocaines 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 atthe 2-carbomethoxy groups resulting in compounds 27-30 (Figure 1.13).48

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.

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

N

Cl

N

Cl

NH

O

NH

O

( )n

27, n = 1: Ki 65.1 nM

28, n = 2: Ki 21.7 nM

29, n = 4: Ki 18.4 nM

30, n = 6: Ki 6.7 nM


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13

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.

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 ,-unsaturated esters. In addition, a

carbon analogue was synthesised by changing the synthesis route.

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

N

CO2CH3

R1R2


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14

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

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 allowsthe 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 Merrifields 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

Scheme 2.1 Comparison of traditional and combinatorial synthesis.

A + B

A1 B1

A2 B2

A3 B3

An Bm

Traditional synthesis:

Combinatorial synthesis:

AnBm

AB 1 compound

n x m compounds


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15

1,4-benzodiazepine derivatives from 2-aminobenzophenones, amino acids, and various

alkylating agents according to Scheme 2.2. 54,55

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 tofacilitate 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,

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

NH2

O

RB

Support

RA

N-Fmoc-amino acid fluoride

CH2Cl2

NH

O

RB

Support

RA

ONHFmoc

RC

HN

RB

Support

1. Piperidine, DMF

2. 5% AcOH, DMF, 60 oCN

RA

O

RC

NRB

SupportN

RA

O

RC

1. lithiated 5-phenyl-2-oxazolidinone

2. Alkylating agents,DMF

RD

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

NRB

N

RA

O

RC

RD

192 Benzodiazepines


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16

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.

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

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.

Figure 2.1 Concept of positional scanning of a hexapeptide library.

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

R X + n Y R'(m)

R' Y + m X R(n)

m sublibraries ofn compounds

n sublibraries ofm compounds

R R'1

R R'n

m

n

R' R1

R' Rm

Position 1

Position 2

Position 3

Position 4

Position 5

Position 6

O X X X X X

X O X X X X

X X O X X X

X X X O X X

X X X X O X

X X X X X O

18 mixtures

"

"

"

"

"

1. A1 X X X X X

18. A18 X X X X X

Total: 108 mixtures


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17

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 additionthe 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 outin 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


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18

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).

With respect to discovering dopamine transporter ligands by combinatorial chemistry, only

one study has been reported. This involved screening of Houghtens 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 M). 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.

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

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

R Cl

O

(40)

+R'OH

R'NH2or40

R'OHR'NH2

or

(40)

+ 40R Cl

O

80

R OR'

O

R NHR'

O

80 sublibraries of 40compounds each

N

N

Ph

O

Cl

31, NK3 inhibitor

NH

O

NC

32, MMP-1 inhibitor


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19

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).

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 ReagentGeneration 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 > secondaryalkyl, 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,

Scheme 3.1 Generation of Grignard reagents.

Mg + R X RMgX (R2Mg + MgX2)


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20

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, metal-

halogen).

3.1.1 Grignard Reagents in Conjugate Addition

Grignard reagents can also add to conjugated carbon-carbon multiple bonds present in e.g.

,-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 obtain1,4-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 ,-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 ,-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 withPhMgBr.17,39,71

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

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

NCO2CH3 N

CO2CH3N

H3CO2C

CO2CH3

19 7 33


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21

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).

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 -azidobenzyl ethers, aldehydes,

and esters.66,67

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

R1Br

R2Br

R3Br

R1MgBr

R2MgBr

R3MgBr

Mg 2 eqEt2O

three componentGrignard reagent

PhOR

N3

NO

O

ORN Rn

RCOO

CH3

RCHO

R Rn

OH

Ph OR

Rn

R Rn

OH

Rn

3 compounds 6 compounds

3 compounds3 compounds


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22

4 Synthesis of the Tropane Skeleton

Initially, it was suggested to develop an efficient method usable for a combinatorial de novoapproach 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 8-

azabicyclo[3.2.1]octane moiety containing a seven-membered ring with a bridgehead. Seven-

membered 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 Willsttter 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 one-

pot 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). Willsttter modified Robinsons 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).

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

Scheme 4.1 Willsttter's synthesis of cocaine from 1923.

O

O

+

O

COOH

OR

O

CH3NH2 NR

O

Na/Hg NCO2CH3

OHBz2O N

CO2CH3

OBz

34 R = H, 35

R = CH3, 37

R = H, Tropinone, 36

R = CO2CH3, 38

39 Cocaine, 1


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23

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.

Most approaches to the synthesis of cocaine built on construction of tropinone (36) that isfurther 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

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

Scheme 4.3 Different routes to the tropanes.

O+ N

R

O

+ RNH2

OCH3

N2

O

+ N

R

O

BrBr

+ N

R

N

O

Tropanes

-H2ONHOH

OCH3O

O

N

OCH3O

O N

OCO2CH3 1. MeI, CH2Cl2

2. Zn, AcOH, 47 %N

CO2CH3

OH

BzCl, Na2CO3

benzene, 37 %

N

CO2CH3

OPh

O

1

42 39

40 41

4 - 11 %


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24

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

naturalR-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 Robinsons pathway by reacting a

resin-bound -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 ,-DibromoketonesOxyallyl cations can be generated from ,-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 combinatiorialchemistry by using different ,-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,-dibromoketonesPaparin et al. have synthesized several tropane scaffolds by [3+4] cycloadditions from ,-

dibromoketones using Et2Zn to generate the oxyallyl cations.96,97 However, the synthesis of

,-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 ,-dibromoketone 44 to Boc-pyrrole generating the

8-azabicyclo[3.2.1]octene45 in 53 % yield (Scheme 4.4).


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26

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

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, Et3 N 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 of52 with sodium borohydride in methanol

proceeded to give the desired alcohol 53 in 82 % yield. The last step in the synthesis of2-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).

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

OCH3N2

O

+N

Boc

Rh2(O2CC7H15)4

hexane, 63 %

NCO

2CH

3Boc

5150


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27

Alternatively, methyl 2-diazobut-3-enoate (50) was synthesised from 3-butenoic acid

according to Bulugahapitiyas procedure.105 Esterification of 3-butenoic acid (54) using AcCl

in MeOH gave the ,-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.

The reaction of50 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.

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-1,3-

diene (49) with 2,5-dimethoxytetrahydrofuran (57) and TiCl4 as activator for generation of

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

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

Scheme 4.10 Synthesis of51 and attempts to add PhMgBr in a conjugate manner to form 56.

OH

OAcCl

OCH3

ODBU, p-ABSA

OCH3

O

N2

55 5054

MeOH49 %

CH3CN, rt overnight

48 %

OCH3

O O p-ABSA, Et3N

CH3CN, 19h, rt

85 %OCH3

O O

N2

MeOH, 20 min,

0oC, 82 %OCH3

OH O

N2

CH2Cl2, 0oC to rt

overnight, 37 %

MsCl, Et3N

52 53

OCH3

O

N2

50

NaBH4

46

OCH3N2

ON

Boc

pentane

NCO2CH3

Boc

51, 52 %50

PhMgBr

Et2O, -40oC

N

CO2CH3

PhBoc

56

Rh2(O2CC7H15)4


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28

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).

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.

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

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

Scheme 4.12 Proposed method for generation of tropanes.

N

Boc

CH3COOK, Br2

MeOH, 52 %

N

Boc

OCH3H3CO

61

H2, Raney Ni

MeOH, 88 %

N

Boc

OCH3H3CO

62

OCH3

TMSO OTMS

TiCl4, CH2Cl2

OCH3

TMSO OTMS

TiCl4, CH2Cl2

N

O

CO2CH3Boc N

O

CO2CH3Boc

63 64

49 49

OCH3

TMSO OTMS+

O OCH3H3COTiCl4

CH2Cl2, -78oC, 3h

O

O

CO2CH3

58, 79 %

NaBH4

O

CO2CH3

OHBzCl

pyridine

O

CO2CH3

OPh

O

5749

5960


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29

62 in 88 % yield. Now both 61 and 62 could be used in a condensation reaction with the enol

silyl ether49. 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 of64

(+ 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

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 resin-

bound 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.

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

HN 1. K, EtOH

2.OO O

, THF

N

O

COOH

65

NH2 , HBTU, DIEA

DMF/CH2Cl2NH

N

O

O

6662 %


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30

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 ,-

dibromoketones. From this tropane synthesis, it was suggested to introduce a 2-carbomethoxy

group on the tropane by employing the ,-dibromoketone of acetoacetate. Because of

problems associated with synthesising the ,-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.


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5 Two- and Three-Dimensional Solution Phase Combinatorial

Libraries of 3- and 8-Substituted tropanes from Multicom-

ponent 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) waschosen 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 1,2n in the

anne bülow- combinatorial synthesis of cocaine analogues

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