Dissertation

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

iii
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 COCAINE – A STIMULANT OF THE CENTRAL NERVOUS SYSTEM .............................1
1.2 DEVELOPMENT OF MEDICATIONS FOR TREATMENT OF COCAINE ABUSE ...............3
1.3 POTENTIAL DOPAMINE TRANSPORTER LIGANDS ....................................................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 OTHER TROPANES..................................................22

iv
4.1.1 [3+4] Cycloaddition of Pyrroles and α,α’-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 MULTICOMPONENT
GRIGNARD 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 OTHER SYSTEMS...... 46
5.4 SUMMARY AND CONCLUDING REMARKS ............................................................. 47
6 BICYCLO[3.2.1]OCTANE ANALOGUES OF PHENYLTROPANES.................. 49
6.1 INTRODUCTION .................................................................................................... 49
6.1.1 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

v
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 and
Galactosyl Donors - A Study of Glycosidation Reactions
1 INTRODUCTION.........................................................................................................65
1.1 CARBOHYDRATES – UBIQUITIOUS 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 AMONG N-PENTENYL GLYCOSIDES .........................83
2.4 INVESTIGATION OF SUPPOSED SN2-TYPE REACTIONS ..........................................84
2.5 MECHANISTIC CONSIDERATIONS..........................................................................86
3 CONCLUSIONS............................................................................................................88

vi
Chapter III: Determination of Thermodynamic Parameters for
Binding of Azasugars to Almond β-Glucosidase
1 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 β-Method ......................................................................................... 95
1.4 DETERMINATION OF THERMODYNAMIC PARAMETERS......................................... 96
2 RESULTS AND DISCUSSION ................................................................................... 97
2.1 DETERMINATION OF THERMODYNAMIC PARAMETERS FOR BINDING OF
AZASUGARS TO β-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 β-GLUCOSIDASE ........................ 102
2.3 DETERMINATION OF THERMODYNAMIC PARAMETERS BY NUMERICAL SOLUTION
OF DIFFERENTIAL EQUATIONS ........................................................................... 103
2.3.1 The Differential Equation Method........................................................ 103
3 SUMMARY AND CONCLUSIONS ......................................................................... 105
References....................................................................................................................... 107
Appendix 1-11

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 β-glucosidase with various
inhibitors.
Anne Bülow, August 2004

viii
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 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.

ix
iv List of Appendices
Appendix 1: Bülow, A.; Plesner, I. W.; Bols, M. J. Am. Chem. Soc. 2000, 122,
8567-8568.
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: 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.

x
v List of Abbreviations
ABSA Acetamidobenzenesulfonyl azide
Ac Acetyl
ACE-Cl 1-Chloroethyl chloroformate
ADHD Attention-deficit hyperactivity disorder
AIBN 2,2’-Azobisisobutyronitrile
Ar Aryl
Å Angstrom
Bn Benzyl
Boc tert-Butoxycarbonyl
Bu Butyl
Bz Benzoyl
cat Catalyst
Cbz Benzyloxycarbonyl
cod Cyclooctadiene
COSY Correlation spectroscopy
Cy Cyclohexyl
DA Dopamine
DAT Dopamine transporter
Dba trans,trans-Dibezylideneacetone
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DE Differential Equation
DIEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
DMTST (dimethylthio)methylsulfonium trifluoromethanesulfonate
DNA Deoxyribonucleic acid
DTBMP 2,6-di-tert-butyl-4-methylpyridine
E Enzyme
EI Enzyme-Inhibitor complex
Equiv. Equivalent
ES Enzyme-Substrate complex
ESMS Electronspray mass spectrometry
Et Ethyl
Fmoc 9-Fluorenylmethoxycarbonyl
Fuc Fucose
Gal Galactose
GBR Gist-Brocades
GC-MS Gas chromatography
Glc Glucose
h Hour(s) or human
HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HMPA Hexamethylphosphoramide
HPLC High Performance Liquid Chromatography
HRMS High Resolution Mass Spectrometry
I Inhibitor
IC50 Inhibition concentration, 50 %
IDPC Iodonium dicollidine perchlorate

xi
iPr isopropyl
Ki Inhibition constant
LDA Lithium diisopropyl amide
LG Leaving group
MBHA 4-Methylbenzhydrylamine
Me Methyl
Mes Mesityl (2,4,6-trimethylphenyl)
Min Minute(s)
MMP-1 Matrix metalloproteinase-1
Ms Methanesulfonyl
MS Molecular sieves
n Normal
NBS N-Bromosuccinimide
NE Norepinephrine
NET Norepinephrine transporter
NIS N-Iodosuccinimide
NMR Nuclear Magnetic Resonance
Nu Nucleophile
Oct Octyl
P Product
Pent n-Pentenyl
Ph Phenyl
ppm parts per million
PS Polystyrene
QSAR Quantitative structure-activity relationship
rds Rate-determining step
RNA Ribonucleic acid
rt Room temperature
RTI Research Triangle Institute
S Substrate
SAR Structure-activity relationship
SER Serotonin
SERT Serotonin transporter
TBACN Tetrabutylammonium cyanide
TBAF Tetrabutylammonium fluoride
TBDMS tert-Butyldimethylsilyl
Tf Trifluoromethanesulfonyl
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TLC Thin layer chromatograhpy
TMS Trimethylsilyl
Tol p-Methylphenyl
Troc 2,2,2-Trichloroethoxycarbonyl
WIN Sterling-Wintrop Institute

xii
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

xiii
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.

1
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 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 effects
such as reduced fatigue and psychomotorial stimulation these effects finally lead to abuse and
addiction.4
Figure 1.1 R-Cocaine - the naturally occurring stereoisomer.
Figure 1.2 Structure of the three natural monoamine neurotransmitters.
N
CO2CH3
O
Ph
O
1 2
345
6
7
8
R-Cocaine, 1
HO
HO
HO HO
HO
NH2
OH
N
H
NH2
NH2
Dopamine (DA), 2 Serotonin (SER), 3 Norepinephrine (NE), 4

2
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
[3
H]WIN35428d
SERT
[3
H]paroxetine
NET
[3
H]nisoxetine
Binding 102±12 1045±89 3298±293
R-Cocaine
Uptake 241±18a
112±2b
160±15c
Table 1.1 Binding potencies and inhibition of reuptake by cocaine at the three monoamine transporters. a
Ki
value for displacement of [3
H]DA uptake. b
Ki value for displacement of [3
H]SER uptake. c
Ki 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 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
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.
Figure 1.3 Cocaine's action on the dopaminergic nervous system - the dopamine hypothesis.

3
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

4
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 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 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
Figure 1.4 Methylphenidate - a selective dopamine transporter inhibitor. Inhibition data is obtained
from displacement of [3
H]WIN35428 binding to rat striatal membranes.13
HN
OCH3
O
Methylphenidate, 5
IC50 83 nM

5
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.
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 R-
isomer is about 150 times more potent than the S-isomer (IC50 for inhibition of
[3
H]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 (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 from R-cocaine.
Figure 1.5 Difference in inhibition of binding of [3
H]cocaine to mouse striatal membranes of enantiomers.
N
CO2CH3
Ph
N
Ph
CO2CH3
WIN35065-2, R-10
IC50 40.7 nM
WIN35065-3, S-10
IC50 32400 nM
N
CO2CH3
O
Ph
O
1N aq. HCl N
COOH
OH
1. POCl3
2. MeOH, H+
N
CO2CH3
ArMgBr
low temp, Et2O
N
CO2CH3
Ar
N
Ar
+ CO2CH3
R-cocaine, 1 6 7
8 9

6
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.

7
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 [3
H]GBR12935 instead of [3
H]WIN35428. c
Inhibition of binding
of [3
H]cocaine instead of [3
H]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
N
CO2CH3
R
R
IC50/nM
[3
H]WIN35428
IC50/nM
[3
H]DA uptake
R
IC50/nM
[3
H]WIN35428
IC50/nM
[3
H]DA uptake
WIN35065-2 23.0±5.021
49.8±2.3a,24
55±223
-
NH2 24.8±1.321
557±7925
68.5±7.123
-
NO2
10.1±0.1021
616±8425
>500b,26
-
OCH3 8.14±1.321
- 597±5227
-
N3 2.12±0.121
- 1.2±0.123
-
F
WIN35428 15.7±1.421
23±0.5a,24
15.6±0.623
29.4±3.828
Cl 1.17±0.121
3.68±0.09a,24
0.49±0.0429
3.53±0.0928
Br 1.81±0.3021
- 3.7±0.1623
-
I
RTI-55 1.26±0.0421
1.96±0.09a,24 CH2Ph
526±6523
-
Cl
Cl
1.09±0.0229
-
CH2CH2Ph
5.14±0.6323
-
1.71±0.321
7.0±0.3a,24 CH2CH2CH2Ph 351±5223
-
CF3
13.1±2.221
- 885±18c,28
1020±5228
I
46±6b,26
- 9.94±0.33c,28
70.5±1.028

8
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.
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 2β-substituted phenyltropanes. Ki values are obtained from
displacement of [3
H]mazindol from rat striatal membranes whereas IC50 values are obtained from displacement of
[3
H]WIN35428.30-32
Figure 1.7 Examples of N-substituted phenyltropane analogues. IC50 values for binding to the DAT are
obtained from displacement of [3
H]cocaine from monkey caudate-putamen membranes.33
N
CO2CH3
N
CO2CH3
F 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

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, 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
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 from
displacement of [3
H]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
HO
HO
WIN35065-2, 10
IC50 65 nM
17
IC50 235 nM
18
IC50 6150 nM

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 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).
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
Figure 1.10 Structure of selected piperidine analogues of phenyltropanes. E = CO2CH3.
N
CO2CH3
N
CO2CH3
Cl N ClE
(-)-20
IC50 769±19 nM
(-)-21
IC50 24.8±1.6 nM
(±)-22
IC50 197±8 nM
N
CO2CH3
Arecoline, 19

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 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.
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 from
inhibition of [3
H]WIN35428 binding to the DAT (monkey caudate-putamen).
Figure 1.12 Inhibition of binding of [125
I]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

12
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
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 [125
I]RTI-55 at hDAT.
N
Cl
N
Cl
N
H
O
N
H
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

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 α,β-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
R1
R2

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 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
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

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 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,
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
O
NHFmoc
RC
H
N
RB
Support
1. Piperidine, DMF
2. 5% AcOH, DMF, 60 o
C
N
RA
O
RC
N
RB
Support
N
RA
O
RC
1. lithiated 5-phenyl-
2-oxazolidinone
2. Alkylating agents,
DMF
RD
TFA/H2O/Me2S (95:5:10)
N
RB
N
RA
O
RC
RD
192 Benzodiazepines

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 of
n compounds
n sublibraries of
m 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

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 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

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 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.
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'NH2
or40
R'OH
R'NH2
or
(40)
+ 40
R Cl
O
80
R OR'
O
R NHR'
O
80 sublibraries of 40
compounds each
N
N
Ph
O
Cl
31, NK3 inhibitor
N
H
O
NC
32, MMP-1 inhibitor

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 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,
Scheme 3.1 Generation of Grignard reagents.
Mg + R X RMgX ½ (R2Mg + MgX2)

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.
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
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 α,β-unsaturated methyl esters that undergo uncatalysed 1,4-addition upon treatment with phenyl
magnesium bromide at low temperature.
N
CO2CH3 N
CO2CH3
N
H3CO2C
CO2CH3
19 7 33

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 α-azidobenzyl ethers, aldehydes,
and esters.66,67
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.
R1Br
R2Br
R3Br
R1MgBr
R2MgBr
R3MgBr
Mg 2 eq
Et2O
three component
Grignard reagent
Ph
OR
N
3
N
O
O
OR
N Rn
RCOOCH3
RCHO
R Rn
OH
Ph OR
Rn
R Rn
OH
Rn
3 compounds 6 compounds
3 compounds3 compounds

22
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 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 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 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). 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).
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 Willstätter's synthesis of cocaine from 1923.
O
O
+
O
COOH
OR
O
CH3NH2 N
R
O
Na/Hg N
CO2CH3
OH
Bz2O N
CO2CH3
OBz
34 R = H, 35
R = CH3, 37
R = H, Tropinone, 36
R = CO2CH3, 38
39 Cocaine, 1

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 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
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
-H2O
NHOH
OCH3O
O
N
OCH3O
O
∆ N
O
CO2CH3 1. MeI, CH2Cl2
2. Zn, AcOH, 47 %
N
CO2CH3
OH
BzCl, Na2CO3
benzene, 37 %
N
CO2CH3
O
Ph
O
1
42 39
40 41
4 - 11 %

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
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).

25
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 2-
carbomethoxy analogue of 45 would be generated directly i.e. compound 48 (Scheme 4.5).
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).
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.
Scheme 4.4 Synthesis of 8-azabicyclo[3.2.1]octene 45 according to Paparin's procedure.
Scheme 4.5 Proposed synthesis of 2-carbomethoxy analogue 48.
Scheme 4.6 Attempts to synthesise methyl 2,4-dibromoacetoacetate. All turned out to be unsuccessful.
OCH3
O O
OCH3
O O
Br Br
OCH3
TMSO OTMS
Br2
AcOH
NBS
THF, RT
Br2Br2
CH2Cl2CH2Cl2
46 47 49
Ph
O
Br2
AcOH, 76 %
Ph
O
BrBr
44
N
Boc
Et2Zn, toluene
53 %
N
O
Boc Ph
4543
OCH3
O O
OCH3
O O
BrBr
N
Boc
Et2Zn
N
O
CO2CH3
Boc
46 47 48

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, 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).
Scheme 4.7 Synthesis of 8-azabicyclo[3.2.1]octadiene 51 through a tandem cyclopropanation/Cope
rearrangement done by Davies et al.
OCH3
N2
O
+
N
Boc
Rh2(O2CC7H15)4
hexane, 63 %
N
CO2CH3
Boc
5150

27
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.
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.
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 of 51 and attempts to add PhMgBr in a conjugate manner to form 56.
OH
O
AcCl
OCH3
O
DBU, p-ABSA
OCH3
O
N2
55 5054
MeOH
49 %
CH3CN, rt overnight
48 %
OCH3
O O p-ABSA, Et3N
CH3CN, 19h, rt
85 %
OCH3
O O
N2
MeOH, 20 min,
0o
C, 82 %
OCH3
OH O
N2
CH2Cl2, 0o
C to rt
overnight, 37 %
MsCl, Et3N
52 53
OCH3
O
N2
50
NaBH4
46
OCH3
N2
O
N
Boc
pentane
N
CO2CH3
Boc
51, 52 %50
PhMgBr
Et2O, -40o
C
N
CO2CH3
Ph
Boc
56
Rh2(O2CC7H15)4

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
CO2CH3
Boc N
O
CO2CH3
Boc
63 64
49 49
OCH3
TMSO OTMS
+
O OCH3H3CO TiCl4
CH2Cl2, -78o
C, 3h
O
O
CO2CH3
58, 79 %
NaBH4
O
CO2CH3
OH
BzCl
pyridine
O
CO2CH3
O
Ph
O
5749
5960

29
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
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.
H
N 1. K, EtOH
2.
OO O
, THF
N
O
COOH
65
NH2 , HBTU, DIEA
DMF/CH2Cl2
N
H
N
O
O
6662 %

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 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.

31
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) 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

32
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.
Generally, synthesis of n2
compounds will require n libraries of n compounds. For the above-
mentioned 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
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.
E-R1,1 E-R1,2 E-R1,3 E-R1,4 E-R1,5
E-R2,1
E-R3,1
E-R4,1
E-R5,1
E-R2,2
E-R3,2
E-R4,2
E-R5,2
E-R2,3
E-R3,3
E-R4,3
E-R5,3
E-R2,4
E-R3,4
E-R4,4
E-R5,4
E-R2,5
E-R3,5
E-R4,5
E-R5,5
R1,4MgBr
R2,4MgBr
R3,4MgBr
R4,4MgBr
R5,4MgBr
R2,1MgBr
R2,2MgBr
R2,3MgBr
R2,4MgBr
R2,5MgBr
E
E

33
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) Ki uptake (nM)
Compound DAT
[3
H]WIN35428
SERT
[3
H]paroxetine
NET
[3
H]nisoxetine
DAT
[3
H]DA
SERT
[3
H]SER
NET
[3
H]DA
N
CO2CH3
WIN35065-2 (10)
23±5 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

34
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 I-
V whereas the vertical libraries are denoted by a-e (Figure 5.1).
Scheme 5.2 Conjugate addition of n-butyl magnesium bromide to anhydroecgonine methyl ester.
N
CO2CH3
1. n-BuMgBr (2eq),
Et2O, -40o
C, 2h
2. TFA, -78o
C-0o
C
76 %
N
CO2CH3
677

35
a b c d e
I
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
67
N
CO2CH3
II
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
III N
CO2CH3
N
CO2CH3
68
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
69
IV N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
V
N
CO2CH3
10
N
CO2CH3 N
CO2CH3
N
CO2CH3
70
N
CO2CH3
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.

36
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. 1
H-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.
Scheme 5.3 Conjugate addition of a five component Grignard reagent to anhydroecgonine methyl ester (7) -
synthesis of library II.
Figure 5.2 GC-MS chromatogram (A) and ESMS (B) of library II showing formation of all 5 expected
products in approximately equal amounts.
N
CO2CH3
1. Et2O, -40o
C, 2h
2. TFA, -78o
C-0o
C
N
CO2CH3
+
CH3(CH2)6MgBr
CH3(CH2)7MgBr
CH3(CH2)8MgBr
CH3(CH2)9MgBr
CH3(CH2)11MgBr
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
Library II
7
A B

37
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 125
I-labeled RTI-55 and also for inhibition of reuptake
of [3
H]-dopamine (hDAT and hNET) and [3
H]-serotonin (hSERT) in cells expressing one of
the three transporters, respectively. For both assays, IC50-values were determined from dose-
response curves and an average of three experiments were used to evaluate IC50 values that
were converted to Ki values ⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛
−−+
=
d
125
50
i
K/]55RTII[1
IC
K . These are presented in Table
5.2.
Ki binding (nM) Ki uptake (nM)
Library hDAT hSERT hNET hDAT HSERT hNET
I 9850 8800 10200 5000 10000 4850
II 15800 4900 9100 10500 12400 3000
III 11300 9700 6600 4800 10700 6600
IV 9300 5400 11500 4900 9200 5800
V 43 38 113 53 52 40
a 1880 4700 1900 650 4400 350
b 8750 5200 9700 4400 11900 4550
c 6250 11800 10200 3500 11800 7700
d 64 55 130 83 57 65
e 265 610 370 170 750 165
Table 5.2 Ki values for binding (displacement of [125
I]RTI-55) and uptake of [3
H]DA and [3
H]SER at hDAT,
hSERT or hNET for 2D libraries I-V and a-e. Each library contains 5 compounds.

38
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.
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.
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.
a b c d e
V
IV
III
II
I
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
5/Ki(max)(nM
-1
)
hDAT binding
a b c d e
V
IV
III
II
I
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
5/Ki(max)(nM
-1
)
hSERT binding
a b c d e
V
IV
III
II
I
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
5/Ki(max)(nM
-1
)
hNET binding
a b c d e
V
IV
III
II
I
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
5/Ki(max)(nM
-1
)
hDAT uptake
a b c d e
V
IV
III
II
I
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
5/Ki(max)(nM-1
)
hSERT uptake
a b c d e
V
IV
III
II
I
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
5/Ki(max)(nM-1
) hNET uptake

39
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) Ki uptake (nM)
Compound hDAT hSERT hNET hDAT hSERT hNET
10 220±95 750±680 555±455 112±68 614±208 115±30
67 6900±650 23100±7300 4700±650 1900±750 24900±3100 2650±750
68 21000±4500 27000±7500 10500±3000 5300±2600 36000±23000 4100±2500
69 38000±5250 3700±620 29950±1300 11700±1850 4150±2950 15700±3300
70 19±10 15±6 20±7 14±7 18±11 13±5
71 115±45 250±50 190±110 65±40 83±20 40±28
Table 5.3 Ki values for binding (displacement of [125
I]RTI-55) and uptake of [3
H]DA and [3
H]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.

40
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
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, 1
H-NMR (Figure 5.4), and GC-MS, in
75 % yield (obtained from an average of molecular masses).
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.
N
CO2CH3
HN
CO2CH31. ACE-Cl, Na2CO3
ClCH2CH2Cl, reflux, 5h
2. MeOH, overnight, 75 %
727

41
Figure 5.4 1
H-NMR spectrum of a mixture of N-substitued anhydroecgonine methyl ester analogues 73 showing
approximately equal formation of each product.
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
Scheme 5.5 Model studies of N-alkylation followed by Grignard reaction.
HN
CO2CH3 Br
Br
Ph Br
+
KI, K2CO3
CH3CN, reflux
18h
N
CO2CH3
N
CO2CH3
N
CO2CH3Ph
MgBrMgBr MgBr1.
2. TFA, -78o
C
N
CO2CH3
N
CO2CH3
N
CO2CH3
Ph
N
CO2CH3
Ph
N
CO2CH3
N
CO2CH3
N
CO2CH3
Ph
N
CO2CH3
N
CO2CH3
Ph
Ph
Ph
72
73
74
Et2O, -40o
C

42
members were all consistent with having an N-benzyl substituent. By synthesising the N-
benzyl 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 (ethyl-
hexyl) 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).
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).
Scheme 5.6 Example of combinatorial synthesis of three-dimensional library Y1
HN
CO2CH3
+
Br
Br
Br
Br
Br KI, K2CO3
CH3CN, reflux
18h
N
CO2CH3
R1
75-79
5 compounds
MgBr
MgBr
MgBr
MgBr
MgBr
+
N
CO2CH3
R2
R1
Library Y1
25 compounds
1. Et2O, -40o
C
2. TFA, -78o
C
72

43
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
X1 X2 X3 X4 X5
Z5
Y1
Y2
Y3
Y4
Y5
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
X1 X2 X3 X4 X5
Z4
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
X1 X2 X3 X4 X5
Z3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
X1 X2 X3 X4 X5
Z2
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
N
CO2CH3
t
Bu
X1 X2 X3 X4 X5
Z1
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.
Figure 5.5 GC-MS chromathogram (A) and ESMS spectrum (B) of library Y1 showing a high probability of
having all 25 products present.
Figure 5.6 The three-dimensional library.
A B

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