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Competition Reactions between Glucosyl Donors and Galactosyl Donors - A Study of Glycosidation Reactions
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Ph.D.dissertation submitted by: Anne Blow Department of Chemistry University of Aarhus August 2004
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Table of Contents
Table of Contents........................................................................................................... iii Preface ............................................................................................................................vii Acknowledgements...................................................................................................... viii List of Appendices ..........................................................................................................ix List of Abbreviations.......................................................................................................x Summary ........................................................................................................................xii
INTRODUCTION ...........................................................................................................1 1.1 1.2 1.3 COCAINE A STIMULANT OF THE CENTRAL NERVOUS SYSTEM .............................1 DEVELOPMENT OF MEDICATIONS FOR TREATMENT OF COCAINE ABUSE ...............3 POTENTIAL DOPAMINE TRANSPORTER LIGANDS ....................................................4 1.3.1 1.3.2 1.4 Phenyltropanes ..........................................................................................4 Various Structural Classes of Potential Dopamine Transporter Ligands 10
COMBINATORIAL CHEMISTRY............................................................................14 2.1 2.2 2.3 INTRODUCTION .....................................................................................................14 IDENTIFICATION OF ACTIVE COMPOUNDS IN A LIBRARY ......................................15 SOLID PHASE VERSUS SOLUTION PHASE APPROACHES.........................................17
THE GRIGNARD REACTION...................................................................................19 3.1 3.2 THE GRIGNARD REAGENT ....................................................................................19 3.1.1 Grignard Reagents in Conjugate Addition ..............................................20 THE GRIGNARD REACTION IN COMBINATORIAL CHEMISTRY ...............................21
SYNTHESIS OF THE TROPANE SKELETON .......................................................22 4.1 SYNTHESES OF COCAINE AND OTHER TROPANES ..................................................22 iii
[3+4] Cycloaddition of Pyrroles and ,-Dibromoketones .................. 24 Tandem Cyclopropanation/Cope Rearrangement of Vinylcarbenoids with Pyrroles ................................................................................................... 26 Tropanes from Pyrrolidine Derivatives .................................................. 27
TWO- AND THREE-DIMENSIONAL SOLUTION PHASE COMBINATORIAL LIBRARIES OF 3- AND 8-SUBSTITUTED TROPANES FROM MULTICOMPONENT GRIGNARD REAGENTS.......................................................................... 31 5.1 GENERATION OF A TWO-DIMENSIONAL LIBRARY FROM MULTICOMPONENT GRIGNARD REAGENTS ......................................................................................... 31 5.1.1 5.1.2 5.1.3 5.1.4 5.2 Designing the Library ............................................................................. 31 Initial model studies................................................................................ 33 Synthesis and Analysis of the Two-Dimensional Library ...................... 34 Biological Results for the Two-Dimensional Library ............................ 37
GENERATION OF A THREE-DIMENSIONAL LIBRARY FROM MULTICOMPONENT GRIGNARD REAGENTS ......................................................................................... 40 5.2.1 5.2.2 5.2.3 Initial model studies................................................................................ 40 Synthesis and Analysis of the Three-Dimensional Library .................... 42 Biological Results for the Three-Dimensional Library .......................... 44
5.3 5.4 6
APPLYING TWO- AND THREE-DIMENSIONAL LIBRARIES TO OTHER SYSTEMS ...... 46 SUMMARY AND CONCLUDING REMARKS ............................................................. 47
BICYCLO[3.2.1]OCTANE ANALOGUES OF PHENYLTROPANES.................. 49 6.1 INTRODUCTION .................................................................................................... 49 6.1.1 6.1.2 6.1.3 6.2 6.2.1 8-Oxa Analogues .................................................................................... 49 8-Carba Analogues.................................................................................. 50 Biological Activity of Non-Amines........................................................ 51 Attempts to Synthesise Methyl 3-(4-iodophenyl)-bicyclo[3.2.1]octane carboxylate and its 8-methyl and 8,8-dimethyl Analogues ................... 52
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6.2.2
Attempts to Perform Conjugate Additions to Methyl bicyclo[3.2.1]octa2,6-diene-2-carboxylate and Methyl bicyclo[3.2.1]oct-2-ene-2carboxylate ..............................................................................................53
CONCLUSIONS ......................................................................................................62
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors - A Study of Glycosidation Reactions
1 INTRODUCTION .........................................................................................................65 1.1 1.2 1.3 CARBOHYDRATES UBIQUITIOUS MOLECULES ...................................................65 ACID-CATALYSED HYDROLYSIS OF GLYCOSIDES.................................................66 GLYCOSIDATION REACTIONS ...............................................................................69 1.3.1 2 The Trichloroacetimidate Method...........................................................73
RESULTS AND DISCUSSION....................................................................................76 2.1 COMPETITION REACTIONS USING TRICHLOROACETIMIDATES...............................76 2.1.1 2.1.3 2.2 2.3 2.4 2.5 Synthesis of Trichloroacetimidate Donors ..............................................76 Competition Reactions between Perbenzylated Gluco and Galacto Trichloroacetimidates..............................................................................78 COMPETITION REACTIONS USING GLYCALS AS DONORS ......................................82 RELATIVE REACTION RATES AMONG N-PENTENYL GLYCOSIDES .........................83 INVESTIGATION OF SUPPOSED SN2-TYPE REACTIONS ..........................................84 MECHANISTIC CONSIDERATIONS ..........................................................................86 2.1.2 Synthesis of the Competition Reaction Products ....................................77
CONCLUSIONS............................................................................................................88
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
1 INTRODUCTION......................................................................................................... 91 1.1 1.2 1.3 1.4 2 AZASUGARS AS GLYCOSIDASE INHIBITORS .......................................................... 91 MECHANISM OF GLYCOSIDASE CATALYSED HYDROLYSIS................................... 92 SLOW INHIBITION................................................................................................. 93 1.3.1 The -Method ......................................................................................... 95 DETERMINATION OF THERMODYNAMIC PARAMETERS ......................................... 96
RESULTS AND DISCUSSION ................................................................................... 97 2.1 DETERMINATION OF THERMODYNAMIC PARAMETERS FOR BINDING OF AZASUGARS TO -GLUCOSIDASE ......................................................................... 97 2.1.1 2.2 2.3 2-Hydroxyl Analogues of Azasugars.................................................... 101 DISCREPANCY BETWEEN THERMODYNAMIC RESULTS OF BINDING OF ISOFAGOMINE AND 1-DEOXYNOJIRIMYCIN TO -GLUCOSIDASE ........................ 102 DETERMINATION OF THERMODYNAMIC PARAMETERS BY NUMERICAL SOLUTION
OF DIFFERENTIAL EQUATIONS ........................................................................... 103
2.3.1 3
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Preface
This Ph.D.-dissertation is based on work performed almost exclusively by the author, under supervision of Professor Mikael Bols at the Department of Chemistry, Aarhus University over the past four years. However, the work on enzyme kinetics was initiated in the spring 2000, but not finished until fall 2000 and has therefore been included and discussed briefly. The research has resulted in a number of scientific publications, which are attached as appendices. The results in appendix 4 have only been discussed briefly since most work was performed by Huizhen Liu and Xifu Liang, and the authors contribution was only associated with biological testing of the synthesised compounds. The authors contribution to appendix 7 was also minor and mainly associated with know-how related to the multicomponent Grignard reactions and the format of the synthesised libraries. The dissertation is divided into three separate and very different chapters. The first chapter is dealing with developing a combinatorial synthesis of cocaine analogues and has been conducted in the period August 2001 till present date. Chapter II presents a mechanistic study of glycosidation reactions and was mainly performed from November 2000 till August 2001. After that date the project was continued by master student Tine Meyer and fellow student Tomasz K. Olszewski. The last chapter consists of enzyme kinetic experiments for determination of thermodynamic parameters for the reaction of -glucosidase with various inhibitors.
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Acknowledgements
First of all, I would like to thank my supervisor Professor Mikael Bols for giving med the opportunity to become a Ph.D. student in his group and for his inspiring ideas and enthusiasm. In addition, I thank ass. prof. Igor W. Plesner for a fruitful collaboration on the physical chemistry concerning the enzyme kinetic experiments. Tine Meyer and Tomasz K. Olszewski are thanked for finishing the glycosidation project. Biological testing of cocaine analogues was done in collaboration with molecular biologists at Psychiatric University Hospital, Risskov, and therefore, Ph.D. student Steffen Sinning and ass. prof. Ove Wiborg are kindly acknowledged for testing compounds and for their willingness to discus the biological part of the project. Laboratory technician Ib Thomsen is also thanked for his enthusiasm, chemistrytricks, and for providing starting materials when necessary. I would also like to thank all present and former co-workers from the bioorganic chemistry group for creating a magnificent atmosphere in the laboratory. Especially, Vinni Hyer Lillelund, Henrik Helligs Jensen, Brian S. Rasmussen, and Kathrine Bjerre are thanked for numerous discussions on chemistry and other matters. All proofreaders are kindly acknowledged for their help on creation of this thesis. For financial support I thank Novo Nordisk A/S and the Lundbeck Foundation. Last but not least, I would like to thank my family and friends for their trust, love, and support. Especially, Marcus Simonsen, Tina Thorslund, Magdalena Pyrz, and Rikke Se are thanked for cheering me up during creation of this thesis.
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List of Appendices
Blow, A.; Plesner, I. W.; Bols, M. J. Am. Chem. Soc. 2000, 122, 8567-8568.
Appendix 1:
Appendix 2:
Blow, A.; Plesner, I. W.; Bols, M. Biochim. Biophys. Acta 2001, 1545, 207-215.
Appendix 3:
Plesner, I. W.; Blow, A.; Bols, M. Anal. Biochem. 2001, 295, 186-193.
Appendix 4:
Liu, H.; Liang, X.; Shoel, H.; Blow, A.; Bols, M. J. Am. Chem. Soc. 2001, 123, 5116-5117.
Appendix 5:
Blow, A.; Meyer, T.; Olszewski, T. K.; Bols, M. Eur. J. Org. Chem. 2004, 323-329.
Appendix 6:
Blow, A.; Sinning, S.; Wiborg, O.; Bols, M. J. Comb. Chem. 2004, 6, 509-519.
Appendix 7:
Pedersen, H.; Sinning, S.; Blow, A.; Wiborg, O.; Bols, M. Org. Biomol. Chem. 2004 accepted for publication
Appendix 8: Appendix 9:
Experimental section List of ligands used for evaluation of IC50 and Ki values for potential cocaine antagonists
NMR spectra of compounds 111 and 112 Derivation of the Integrated Rate Equation for Slow-Binding Inhibitors Described by Model 1.
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List of Abbreviations
Acetamidobenzenesulfonyl azide Acetyl 1-Chloroethyl chloroformate Attention-deficit hyperactivity disorder 2,2-Azobisisobutyronitrile Aryl Angstrom Benzyl tert-Butoxycarbonyl Butyl Benzoyl Catalyst Benzyloxycarbonyl Cyclooctadiene Correlation spectroscopy Cyclohexyl Dopamine Dopamine transporter trans,trans-Dibezylideneacetone 1,8-diazabicyclo[5.4.0]undec-7-ene Differential Equation N,N-Diisopropylethylamine N,N-Dimethylformamide (dimethylthio)methylsulfonium trifluoromethanesulfonate Deoxyribonucleic acid 2,6-di-tert-butyl-4-methylpyridine Enzyme Enzyme-Inhibitor complex Equivalent Enzyme-Substrate complex Electronspray mass spectrometry Ethyl 9-Fluorenylmethoxycarbonyl Fucose Galactose Gist-Brocades Gas chromatography Glucose Hour(s) or human 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Hexamethylphosphoramide High Performance Liquid Chromatography High Resolution Mass Spectrometry Inhibitor Inhibition concentration, 50 % Iodonium dicollidine perchlorate
ABSA Ac ACE-Cl ADHD AIBN Ar Bn Boc Bu Bz cat Cbz cod COSY Cy DA DAT Dba DBU DE DIEA DMF DMTST DNA DTBMP E EI Equiv. ES ESMS Et Fmoc Fuc Gal GBR GC-MS Glc h HBTU HMPA HPLC HRMS I IC50 IDPC x
iPr Ki LDA LG MBHA Me Mes Min MMP-1 Ms MS n NBS NE NET NIS NMR Nu Oct P Pent Ph ppm PS QSAR rds RNA rt RTI S SAR SER SERT TBACN TBAF TBDMS Tf TFA THF TLC TMS Tol Troc WIN
isopropyl Inhibition constant Lithium diisopropyl amide Leaving group 4-Methylbenzhydrylamine Methyl Mesityl (2,4,6-trimethylphenyl) Minute(s) Matrix metalloproteinase-1 Methanesulfonyl Molecular sieves Normal N-Bromosuccinimide Norepinephrine Norepinephrine transporter N-Iodosuccinimide Nuclear Magnetic Resonance Nucleophile Octyl Product n-Pentenyl Phenyl parts per million Polystyrene Quantitative structure-activity relationship Rate-determining step Ribonucleic acid Room temperature Research Triangle Institute Substrate Structure-activity relationship Serotonin Serotonin transporter Tetrabutylammonium cyanide Tetrabutylammonium fluoride tert-Butyldimethylsilyl Trifluoromethanesulfonyl Trifluoroacetic acid Tetrahydrofuran Thin layer chromatograhpy Trimethylsilyl p-Methylphenyl 2,2,2-Trichloroethoxycarbonyl Sterling-Wintrop Institute
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Summary
As cocaine abuse has become a serious social and economic burden in the Western world the need for a potential medication that can facilitate withdrawal has grown. A suitable therapeutic agent is thought to be obtained via interaction with the dopamine transporter and we therefore set out to develop a combinatorial synthesis of tropane-based compounds that could be possible dopamine transporter ligands. Initially, several de novo approaches to the tropane skeleton were suggested, but they were all discarded because of synthetic difficulties. Instead, we set out to develop a combinatorial synthesis based on an existing tropane skeleton using anhydroecgonine methyl ester as starting material. By addition of multicomponent Grignard reagents to the ,-unsaturated ester, 10 sublibraries of 5 3-substituted tropanes each were constructed. By variable mixing of the Grignard reagents 25 different compounds were obtained in a two-dimensional format, where each library member was contained in 2 sublibraries. This was done to facilitate identification of biologically active compounds in the mixtures. Screening of the library led to identification of two new compounds that bind to monoamine transporters with high affinity and inhibit reuptake. In addition, it was shown that 3-alkyltropanes were poor monoamine transporter ligands. To extend the gain associated with the combinatorial synthesis, a third dimension was added to the library. This was done via a multicomponent N-alkylation resulting in a library of 5 anhydroecgonine methyl ester analogues that was subsequently reacted with multicomponent Grignard reagents. In that way, 125 compounds were synthesised in 15 sublibraries of 25 compounds each. Three high affinity compounds were synthesised individually and showed similar affinity to the dopamine transporter as 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
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conjugate addition whereupon a ring closing metathesis resulted in reconstruction of the bicyclic skeleton. In another project the difference in electron-withdrawing properties of equatorial and axial C4-OBn substituents were used to investigate glycosidation reactions. For that reason several glucosyl and galactosyl donors were synthesised and their reactivity compared in direct competition experiments where the donors were forced to compete for an acceptor under various reaction conditions. In general, the reactivity of the galactosyl donors was four to five times higher than the corresponding glucosyl donors indicating that the orientation of the C4 substituent affected the reactivity of the donors. The observation suggests that the transition state of the reaction has considerable positive charge (SN1-like reaction) and that this positive charge is less destabilised for galacto stereochemistry (axial C4 substituent) compared 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 -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.
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CO2CH3
1 4 2
O
3
Ph
O R-Cocaine, 1
Only the naturally occurring R-isomer (referring to stereochemistry at C-1) of cocaine is addictive and has many physiological effects e.g. it is a local anaesthetic, a vasoconstrictant, and is known to increase heart rate and blood pressure. However, concerning drug abuse the most relevant effect is its euphoria producing ability and its reinforcing properties (i.e. the increase in the probability of repeated use of cocaine).3 Along with other rewarding effects such as reduced fatigue and psychomotorial stimulation these effects finally lead to abuse and addiction.4
NH2 OH HO HO Dopamine (DA), 2 NH2 HO N H Serotonin (SER), 3 HO HO Norepinephrine (NE), 4 NH2
Primarily, the pharmacological effects of cocaine arise due to inhibition of reuptake of monoamines (Figure 1.2) at the serotonin, norepinephrine, and dopamine transporters (SERT, NET, and DAT, respectively) in the mammalian brain. Affinities for binding and inhibition of reuptake are shown in Table 1.1.5, I
IC50 (nM) DAT [3H]WIN35428d 10212 24118a SERT [3H]paroxetine 104589 1122b NET [3H]nisoxetine 3298293 16015c
R-Cocaine
Binding Uptake
Table 1.1 Binding potencies and inhibition of reuptake by cocaine at the three monoamine transporters. a Ki value for displacement of [3H]DA uptake. b Ki value for displacement of [3H]SER uptake. c Ki value for displacement of [3H]NE uptake. d Structures of displaced ligands are shown in appendix 9.
However, the primary mechanism of action of cocaine has been ascribed to its ability to inhibit the dopamine transporter (known as the dopamine hypothesis).3 The dopamine transporter consists of 12 transmembrane -helices and is found in dopaminergic neurons. The primary structure of the protein is known but no three-dimensional structure is available at present. The biochemical action of cocaine on the dopaminergic nervous system is outlined in Figure 1.3.6
Figure 1.3 Cocaine's action on the dopaminergic nervous system - the dopamine hypothesis.
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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.
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
dopamine transporter. Studies have suggested that cocaine binds to the dopamine transporter at a different site than dopamine.12 This observation suggest that it is possible to design therapeutic agents that bind to the cocaine recognition site either without inhibiting dopamine transport (i.e. cocaine antagonists) or inhibiting it weakly (i.e. cocaine partial agonists). A selective dopamine transporter ligand can also serve to be useful as a diagnostic tool when used as a marker for deficits in the density of receptor population e.g. with respect to Parkinsons disease which is characterised by the degeneration of dopaminergic neurons.14 Selective dopamine transporter inhibitors are already used as a drug today. An example is methylphenidate (5, Ritalin, Figure 1.4). It is used as a stimulant in the treatment of attentionOCH3 O HN
Methylphenidate, 5 IC50 83 nM
Figure 1.4 Methylphenidate - a selective dopamine transporter inhibitor. Inhibition data is obtained from displacement of [3H]WIN35428 binding to rat striatal membranes.13
deficit hyperactivity disorder (ADHD) in children and for depression in adults.15 Nevertheless, clinical studies using methylphenidate showed no efficacy for the treatment of cocaine dependence.16
1.3.1 Phenyltropanes
Compared to cocaine the main difference of phenyltropanes is that they have an aryl group directly attached to the 3-position of the tropane ring instead of through a 3-benzoyl ester as is present in cocaine. This group of compounds have been known since 1973, where the first synthesis of a phenyltropane was published by Clarke et al.17 The synthesis was carried out 4
from (1R, 5S)-anhydroecgonine methyl ester 7 prepared from R-cocaine, which was reacted with an aryl Grignard reagent at low temperature to give the 1,4-addition products 8 and 9 (Scheme 1.1). The vast majority of phenyltropanes have been synthesised by the same route.
CO2CH3 N
1N aq. HCl
COOH N
1. POCl3
O O R-cocaine, 1
Ph 6
OH
CO2CH3
2. MeOH, H+
7
ArMgBr low temp, Et2O
CO2CH3 N Ar + N CO2CH3 Ar
Variations have been carried out in other positions than the 3-position. Especially, changing the ester functionality in the 2-position and the substituent at the nitrogen. In addition, a few C-6/C-7-substituted analogues have been synthesised. 1.3.1.1 Structure-Activity Relationship Studies Based on the large number of biological data available for phenyltropanes, structure-activity relationships (SAR) and quantitative SAR studies have provided information about important interaction sites between the dopamine transporter and substrates.3-5 It is suggested that the most important factor for activity of a phenyltropane to the DAT is its configuration the preferred being the R-configuration.18 This feature is also seen for cocaine itself, where the Risomer is about 150 times more potent than the S-isomer (IC50 for inhibition of [3H]WIN35428 binding to rat striatal membranes: 0.102 M and 15.8 M respectively).18 It is also evident from several other analogues e.g. for WIN35065 the R-enantiomer (WIN350652) has been noted to be approximately 800 fold more active than the S-enantiomer (WIN35065-3, Figure 1.5).19
CO2CH3 N Ph N CO2CH3 Ph
Figure 1.5 Difference in inhibition of binding of [3H]cocaine to mouse striatal membranes of enantiomers.
Significant and important effects on activity are obtained by substitution at C-3. Replacement of the aromatic ring of the benzoyl group in cocaine by an aryl group as in the phenyltropane series, have shown to enhance activity by a factor of up to 50.3 The stereochemistry at C-3 seems to be of less importance, since a 3-substituent causes to 6-membered ring to flip to the boat conformation, which will position the 3-substituent in a pseudoequatorial position that is approximately the same position as for the 3-substituent.20 The necessity of a 3-aryl substituent has been mentioned throughout literature to be of great importance for obtaining affinity for the DAT. But no tropanes with simple 3-alkyl substituents have been reported! The aryl group is thought to interact via hydrophobic bonding to a lipophilic pocket in the protein. In Table 1.2 binding affinities for a selection of phenyltropanes are presented. As seen halogen substituents increase the binding affinities where 3,4-Cl2>4-Cl>4-I>4-Br>4-F but also other electron withdrawing or donating groups tend to increase affinity compared to the unsubtituted phenyltropane.21 Furthermore a decrease in affinity is observed for large para substituents such as isopropyl and butyl, which is supported by QSAR studies ascribing it to sterical hindrance.22 Contrary to that observation, compounds having a second aryl group attached in the para position of a phenyl group via a linker have also shown to bind strongly to DAT. This have been ascribed to the presence of a remote phenyl binding domain.23 It 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.
R WIN35065-2
NH2
IC50/nM IC50/nM [3H]WIN35428 [3H]DA uptake 23.05.021 24.81.321 10.10.1021 8.141.321 2.120.121 49.82.3a,24 5577925 6168425 230.5a,24 3.680.09a,24 1.960.09a,24 7.00.3a,24 -
IC50/nM IC50/nM [3H]WIN35428 [3H]DA uptake 55223 68.57.123 >500b,26 5975227 1.20.123 15.60.623 0.490.0429 3.70.1623 29.43.828 3.530.0928 10205228 70.51.028
NO2
OCH3
N3
WIN35428
Cl
Br
RTI-55
CH2Ph
Cl Cl
CH2CH2Ph
CH2CH2CH2Ph
CF3
13.12.221 466b,26
Table 1.2 Binding affinities and inhibition of reuptake at the DAT for selected phenyltropanes. a Ki value instead of IC50. b Ki values for inhibition of binding of [3H]GBR12935 instead of [3H]WIN35428. c Inhibition of binding of [3H]cocaine instead of [3H]WIN35428.
A 2-carbomethoxy group has been thought to be crucial for binding of cocaine to the DAT, since replacing it by hydrogen, a carboxy group, or an N-methylcarboxamido group decreased activity by 25-2000 fold.3 The interaction has been suggested to happen through hydrogen bonds. 2-substituted phenyltropanes have been designed to explore whether this is also the case for phenyltropanes. Changing the methyl ester for isopropyl or phenyl esters (13) does not affect the binding affinity for the DAT, but the selectivity for DAT over NET and SERT is increased.30 Neither changing the 2-carbomethoxy group for an alkyl group as in 11 and 12 affects the binding affinity for the DAT, since other compounds bearing alkyl or arylvinyl groups at the C-2 7
position were found to exhibit nanomolar and subnanomolar affinities for binding to the dopamine transporter (Figure 1.6).31 2-heterocyclic analogues have also been synthesised and shown good binding affinity at the DAT and at present the heterocyclic isoxazole analogue 14 is claimed to be the strongest compound binding to the DAT.32 Taken together these results show that the substituent in the 2-position is of minor importance in the phenyltropane series and a large degree of flexibility is allowed.
Ph CO2Ph N Cl N Cl N Cl N N O Cl
11 Ki 1.46 nM
12 Ki 1.21 nM
13 IC50 1.99 nM
14 IC50 0.59 nM
Figure 1.6 Examples of binding affinities to DAT for 2-substituted phenyltropanes. Ki values are obtained from displacement of [3H]mazindol from rat striatal membranes whereas IC50 values are obtained from displacement of [3H]WIN35428.30-32
The presence of a nitrogen in position 8 that can participate in either an ionic bond or a hydrogen bond to the transporter have also been proposed to be necessary for binding.3 Several N-substituted phenyltropanes have been synthesised and from these it has been demonstrated that N-substituents do not affect DAT affinity significantly compared to their N-methyl analogues exemplified by similar IC50 values for 15 and 16 (Figure 1.7).33 Moreover, it has been observed that N-substitutions could increase the specificity for DAT over SERT and NET.34
CO2CH3 N F N CO2CH3 F
15 IC50 22.6 nM
Figure 1.7 Examples of N-substituted phenyltropane analogues. IC50 values for binding to the DAT are obtained from displacement of [3H]cocaine from monkey caudate-putamen membranes.33
Most N-substituted analogues have been synthesised from their N-methyl analogues by demethylation followed by N-alkylation.35 It turns out that the presence of a nitrogen in position 8 is not strictly necessary, since exchanging the nitrogen for an oxygen or a carbon
can be done without severe loss in binding potency.36,37 This will be subjected to further discussion in section 6.2. Only a few phenyltropanes bearing substituents at C-6 or C-7 have been synthesised. Among these, -oriented hydroxyl groups have been introduced with the rationale of being capable of making intramolecular hydrogen bonds to the 8-nitrogen (Figure 1.8). In that way, the effect of reducing the nucleophilicity of the nitrogen was explored.38
CO2CH3 N N HO HO WIN35065-2, 10 IC50 65 nM 17 IC50 235 nM 18 IC50 6150 nM CO2CH3 N CO2CH3
Figure 1.8 Examples of C-6/C-7-hydroxylated phenyltropanes. Inhibition data are obtained from displacement of [3H]WIN35428 binding to the DAT in monkey caudate-putamen.
From the studies it was shown that the 7-hydroxylated compound 17 is more potent at the DAT than the 6-hydroxylated counterpart 18.38 A small increase in selectivity of DAT over SERT is also observed for the hydroxylated compounds. As a conclusion to the SAR studies a pharmacophore model can be suggested (Figure 1.9).
Figure 1.9 A general accepted pharmacophore model for binding of phenyltropanes to the DAT.
From the huge amount of phenyltropanes synthesized, it appears that a pharmacophore model cannot be deduced unambiguously and several deviations remains unexplained by the model. Therefore further explorations of this class of compounds are of great interest in the search for dopamine transporter ligands that could be used as a potential cocaine abuse treatment.
Arecoline, 19
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 3-diphenylmethoxy substituent. It was first synthesised in 1952 and was subsequently demonstrated to be useful as an anticholinergic drug in the treatment of Parkinsons disease.40 It is a stimulant of the central nervous system, where it acts through inhibition of dopamine reuptake just as cocaine and the phenyltropanes, but since benztropine does not self-administer in rhesus monkeys, it is thought to bind to a different site on the dopamine transporter than cocaine.41
10
Figure 1.11 Structure of benztropine (23) and selected analogues. IC50 and Ki values are obtained from inhibition of [3H]WIN35428 binding to the DAT (monkey caudate-putamen).
A wide variety of benztropine analogues have been synthesised, especially phenyl ring substituted analogues where the difluoro compound 24 has turned out to be the most potent benztropine analogue against the DAT at present (Figure 1.11).42 It is also interesting to note that for the hybrid compound 25 (difluoropine), the S-isomer is more than 150 times more potent than the R-isomer.40 This is the opposite stereochemistry than required for cocaine binding and is again suggesting different binding sites for cocaine and benztropines.
1.3.2.3 GBR compounds Another important group of potential cocaine antagonists is the GBR compounds. In 1980 the first synthesis of an aryl 1,4-diaryl piperazine as potential DAT ligand was reported a class of compounds now knowns as the GBR compounds.43 Until date one of the most interesting compounds is GBR12909 (26), which binds tightly to the dopamine transporter and inhibit the action of dopamine uptake (Figure 1.12).44 In addition it is very selective against the dopamine transporter.
PhCH2CH2CH2N N O F
A difference in the action of GBR12909 and cocaine is seen. GBR12909 produces a relatively modest and long-lasting increase in the dopamine concentration, which does not cause the
11
same degree of euphoria compared to cocaines burst of pleasure. In addition GBR12909 has been shown to decrease cocaine-seeking behavior.45 1.3.2.4 Bivalent Ligand Approach Recently, it was proposed to employ a bivalent ligand approach being capable of bridging neighbouring recognition sites on the transporters.46 By linking two binding moieties differing the length of the linker connecting them, it was assumed to obtain transporter selectivity based on a difference in location of neighbouring sites at the respective monoamine transporters. Piperidine-based bivalent inhibitors linked by varying methylene chains at C-2 turned out to be inhibitors of the DAT and the SERT or just the SERT depending on linker length.47 A similar study was reported by linking 3-aryl tropanes through amide linkages at the 2-carbomethoxy groups resulting in compounds 27-30 (Figure 1.13).48
N O N H ( )n N H O N 27, n = 1: 28, n = 2: 29, n = 4: 30, n = 6: Ki 65.1 nM Ki 21.7 nM Ki 18.4 nM Ki 6.7 nM
Cl Cl
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.
12
Section 4 describes the effort put into trying to construct the tropane skeleton in a way that could be useful for generation of combinatorial libraries. Not many positive results are presented in this section, but it has been included because of the considerable time spend on it. Section 5 describes the synthesis of two- and three-dimensional combinatorial libraries consisting of 25 and 125 compounds, respectively (Figure 1.14). Most of these results are also found in appendix 6 in a published article.
CO2CH3 R2 N R1
Figure 1.14 General structure of tropanes synthesised in two- and three-dimensional libraries.
The last section (section 6) deals with the attempts to synthesise carbon analogues of the above-mentioned tropanes. This turned out to be considerable more difficult than expected and ended up being a study of conjugate additions to ,-unsaturated esters. In addition, a carbon analogue was synthesised by changing the synthesis route.
13
2 Combinatorial Chemistry
2.1 Introduction
Compared to traditional synthetic chemistry, combinatorial chemistry consists of a range of techniques allowing rapid synthesis of a large number of compounds in few reactions through combination of different building blocks as shown in Scheme 2.1.49
Traditional synthesis: A + B AB 1 compound
Combinatorial synthesis:
A1 A2 A3
B1 B2 B3 AnBm n x m compounds
An
Bm
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 Merrifields solid phase tetrapeptide synthesis in 1963,52 peptides and peptide-like molecules have been the target of numerous combinatorial syntheses, primarily because or their easy preparation on solid phase. Also with the introduction of split and mix synthesis in the early 1990s the number of peptides that could be generated in a few reaction steps exploded.53 But from a pharmaceutical point of view, peptides are not very interesting molecules, since they have limited use as drugs because of their poor oral absorption and their rapid clearing times. Thus, extensions to the existing methods were needed and in the beginning of the 1990s several publications for solid phase synthesis of more drug-like molecules such as benzodiazepines appeared. E.g. Bunin et al. constructed a library of 192
14
1,4-benzodiazepine derivatives from 2-aminobenzophenones, amino acids, and various alkylating agents according to Scheme 2.2. 54,55
RC O RB NH2 O Support
N-Fmoc-amino acid fluoride CH2Cl2
RB
NHFmoc NH O
1. Piperidine, DMF 2. 5% AcOH, DMF, 60 C
o
RB
H N N
O RC
Support
Support
RA
RA
RA
1. lithiated 5-phenyl2-oxazolidinone 2. Alkylating agents, DMF
RB 192 Benzodiazepines
RD N N
O RC
TFA/H2O/Me2S (95:5:10)
RB
RD N N
O RC
Support
RA
RA
they obtained more than 34 million different compounds in 108 pools that were tested for biological activity. From the biological results, the amino acid giving rise to the most potent peptide can be determined for each position. By synthesising the combination of defined amino acids in the most active mixture in each position, the most active compound was identified.
1. A1 X X X X X Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 OXXXXX XOXXXX XXOXXX XXXOXX XXXXOX XXXXXO 18 mixtures " " " " " Total: 108 mixtures 18. A18 X X X X X
In 1995 Pirrung and Chen introduced a technique, that is essentially the same as positional scanning, where indexing permits the preparation and identification of active non-oligomeric compounds.58 The library was represented by a matrix, where each axis has as many elements as are in each set of building blocks (m and n in Figure 2.2). This method is applicable to any molecule that can be assembled in a simple chemical process from multiple subunits. Using this technique a library of carbamates, suggested to be acetylcholinesterase inhibitors, was prepared. By reacting 9 alcohols with 6 isocyanates they obtained 54 compounds in 15 sublibraries. From the biological screening of sublibraries the most potent compound was
R R' 1 R X (m) + n Y R' m R R' n R' R' Y (n) + mX R n R' Rm R1
n sublibraries of m compounds m sublibraries of n compounds
identified directly from the two sublibraries showing inhibition. Indexed libraries and positional scanning are not limited to two dimensions but can be extended by using multicomponent reactions such as the Ugi four-component reaction59 and the Biginelli reaction60 or by introducing more reaction sites or polymeric chains.
16
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
17
40
R'OH or R'NH2 R 80 R
O OR' O NHR'
Scheme 2.3 Smith's approach to 1600 esters/amides via mixture based solution phase combinatorial synthesis.
The library was constructed in an indexed manner generating 80 sublibraries of 40 compounds each giving a total of 1600 different compounds. From the library 31 was identified as a lead compound for the NK3 receptorII and 32 showed affinity for matrix metalloproteinase-1 (MMP-1)III was identified (Figure 2.3).
O Ph N N NC Cl O N H
Figure 2.3 Lead compounds identified from Smith's ester/amide library by indexed libraries.
With respect to discovering dopamine transporter ligands by combinatorial chemistry, only one study has been reported. This involved screening of Houghtens positional scanning combinatorial hexapeptide library build from D-amino acids containing 186 peptides.65 Twelve hexapeptides were resynthesised individually and turned out to bind to the DAT (IC50 1.7-9 M). A variety of organic reactions have been employed for generation of solution phase combinatorial libraries, among these the Grignard reaction.66,67
II
NK3 receptor antagonists are thought to have a potential role in anxiety-related and psychotic disorders such as schizophrenia. III Inhibition of MMP-1 is beneficial in the treatment of arthritis and corneal ulceration.
18
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.
nitriles, sulfones, imines), highly strained ringsystems (e.g. epoxides, cyclohexenes), acidic hydrogens (e.g. alkynes), and some highly polar single bonds (e.g. carbon-halogen, metalhalogen).
Figure 3.1 ,-unsaturated methyl esters that undergo uncatalysed 1,4-addition upon treatment with phenyl magnesium bromide at low temperature.
Interestingly, these examples all afford the possibility of conformational fixation of an intermediate through coordination to the nitrogen. This will be discussed further in section 6.2.2 The reaction of Grignard reagents with electrophiles is considered to be complex, and to vary depending on the given reaction. Two mechanistic possibilities are generally proposed for addition of Grignard reagents to electrophiles i.e. through a single-electron transfer or a polar mechanism.72 When adding Grignard reagents in a conjugate manner it has been suggested to 20
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
OH R
CH 3 OO RC N Ph
3
OR
Mg 2 eq Et2O
Rn
Rn
3 compounds
6 compounds
OH R Rn
HO RC
OR
Rn Ph OR
3 compounds
3 compounds
Scheme 3.2 Preparation of multicomponent Grignard reagents and their use in synthesis of libraries of secondary and tertiary alcohols, ethers, and 2-substituted quinolines. Generation of stereocenters have not been taken into account in this scheme.
Bearing in mind that 2-alkyl- and 2-alkenylquinolines have shown promising activity against leishmanian protozoas, libraries of 2-substituted quinolines were generated from mixtures of Grignard reagents and quinolinium salts.77 In addition, model studies employing multicomponent Grignard reagents have been conducted on -azidobenzyl ethers, aldehydes, and esters.66,67
21
R N O
Na/Hg
CO2CH3 N OH
Bz2O
CO2CH3 N OBz
O COOH R = H, 35 R = CH3, 37
R = H, Tropinone, 36 R = CO2CH3, 38
39
Cocaine, 1
A new interesting approach to cocaine was developed by Tufariello and co-workers in 1978.82,83 By a nitrone-based entry to the tropane skeleton, they were able to control the 22
stereochemistry of the ester function in cocaine, which is often a problem. Their key compound was the hydroxylamine 40 that upon dehydration was converted into nitrone 41. 41 underwent a 1,3-dipolar cycloaddition to give the tricyclic compound 42. Methylation and cleavage of the nitrogen oxygen bond afforded ecgonine methyl ester (39) that was easily benzoylated to provide racemic cocaine (1) (Scheme 4.2). Even though this represents an elegant way to the tropanes, the yield of the cycloaddition step was rather low.
O NHOH
-H2O
N O O OCH3
O
4 - 11 %
CO2CH3
CO2CH3 N OH
OCH3 40
42 41
39
BzCl, Na2CO3 benzene, 37 %
CO2CH3 N O O 1 Ph
Most approaches to the synthesis of cocaine built on construction of tropinone (36) that is further derivatised to cocaine. Tropinone has been obtained from 2,6-cycloheptadiene by Michael addition with methanolic methylamine.84 Other examples for generation of the tropane skeleton employs reaction of pyrroles with cyclopropanones,85 addition of oxyallyl cations to pyrroles,86,87 and tandem cyclopropanation/Cope rearrangement of vinylcarbenoids with pyrroles (Scheme 4.3).88
O OCH3 O + RNH2 Br Br N2 + R N O + R N
Tropanes
O +
R N
N O
23
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 Robinsons pathway by reacting a resin-bound -amine of lysine with succinic dialdehyde and acetonedicarboxylic acid.91 In another study a tropane scaffold was attached to a dihydropyran linker and subjected to further transformations in the C-3 position.92 The last solid phase approach is based on a 1,3-dipolar cycloaddition of a 3-oxidopyridinium betaine to activated resin-bound olefins.93
4.1.1
Oxyallyl cations can be generated from ,-dibromoketones and it is well known from literature that they can react as dienophiles in [3+4] cycloadditions with dienes such as cyclopentadiene, furan and pyrrole.94,95 By using a pyrrole in such a reaction one would obtain a tropane scaffold in a very simple way. This reaction seems to be an attractive short route to the tropanes and it also offers possibilities for introduction of combinatiorial chemistry by using different ,-dibromoketones and pyrroles. In addition, a double bond (C-6/C-7) is formed, which can be used as a handle for introduction of further substituents. It is interesting to obtain C-6/C-7-substituted cocaine analogues, since relatively few compounds of this type are reported.5 Therefore, experiments on generating tropanes from a [3+4] cycloaddition were initiated. 4.1.1.1 Synthesis of ,-dibromoketones Paparin et al. have synthesized several tropane scaffolds by [3+4] cycloadditions from ,dibromoketones using Et2Zn to generate the oxyallyl cations.96,97 However, the synthesis of ,-dibromoketones is not straightforward and in addition they decompose easily. According to a literature procedure, a synthesis of 1,3-dibromo-1-phenyl-2-propanone (44) was done by bromination of phenylacetone (43) in acetic acid.98 This was followed by cycloaddition of the ,-dibromoketone 44 to Boc-pyrrole generating the 8-azabicyclo[3.2.1]octene 45 in 53 % yield (Scheme 4.4). 24
Boc N Ph Boc N
Et2Zn, toluene 53 %
Ph O 45
Br 43
Br 44
From these experiments it was expected that by synthesizing the corresponding ,dibromoketone 47 from methyl acetoacetate (46) and reacting it with Boc-pyrrole, the 2carbomethoxy analogue of 45 would be generated directly i.e. compound 48 (Scheme 4.5).
O O OCH3 Br 46 Br 47 48 O O OCH3 Boc N Boc N
Et2Zn
CO2CH3 O
Several attempts were made to synthesize methyl-2,4-dibromo-acetoacetate (47) (Scheme 4.6). First a bromination of methyl acetoacetate was tried under the same conditions as the bromination of phenylacetone, though without success. Then the addition of bromine was carried out in CH2Cl2 but again no product formation was observed.99 Further attempts were made by bromination of 1,3-bis(trimethylsiloxy)-1-methoxybuta-1,3-diene (49) (Scheme 4.6).
O O OCH3
Br2 AcOH Br2 CH2Cl2
O OCH3
NBS THF, RT
TMSO
OTMS OCH3
Br
Br 47
Br2 CH2Cl2
46
49
Scheme 4.6 Attempts to synthesise methyl 2,4-dibromoacetoacetate. All turned out to be unsuccessful.
First the bis-TMS enol ether 49 was synthesized by a standard method from methyl acetoacetate (46) in two steps by first protecting the keto functionality using TMSCl and triethylamine in pentane followed by treatment with LDA and TMSCl in THF in an overall yield of 62 %.100 Attempts toward bromination of 49 were then carried out by using Br2 in CH2Cl2, but again without generation of the desired product. No better was the attempt using NBS as brominating agent. Due to problems associated with the synthesis of the starting material this method was eliminated from further investigations.
25
Boc N
CO2CH3
Scheme 4.7 Synthesis of 8-azabicyclo[3.2.1]octadiene 51 through a tandem cyclopropanation/Cope rearrangement done by Davies et al.
51 was then thought to undergo 1,4-addition of Grignard reagents in a combinatorial fashion using a method developed in our lab.66 This route seems very attracting, since not only a handle for introduction of substituents in the C-6/C-7 double bond is obtained, in addition it offers the possibility of making both 8-carba, 8-oxa, and 8-thia bicyclic analogues by employing cyclopentadienes, furans, and thiophenes instead of the pyrrole. 4.1.2.1 Synthesis of Methyl 2-Diazobut-3-enoate (50) According to Davies procedure, Et3N was used as base for diazo transfer from p-acetamidobenzenesulfonyl azide (p-ABSA) to methyl acetoacetate (46) in the preparation of methyl diazoacetoacetate (52).103 Reduction of 52 with sodium borohydride in methanol proceeded to give the desired alcohol 53 in 82 % yield. The last step in the synthesis of 2-diazobut-3-enoate (50) was dehydration of alcohol 53 by phosphorous oxychloride, reported to be done in 38 % yield.104 This procedure was tried several times with no positive outcome. Instead the elimination reaction was successfully carried out using MsCl and base, which turned out to give the desired vinyl diazo compound 50 in 37 % yield (Scheme 4.8).
26
O OCH3 N2 52
OH O OCH3 N2 53
O OCH3 N2 50
Alternatively, methyl 2-diazobut-3-enoate (50) was synthesised from 3-butenoic acid according to Bulugahapitiyas procedure.105 Esterification of 3-butenoic acid (54) using AcCl in MeOH gave the ,-unsaturated ester 55. Subsequently, a diazo transfer from p-ABSA using DBU as base was carried out from 55, resulting in the desired vinyl diazo compound 50 in 48 % yield (Scheme 4.9). The relative low yields of 50 are probably due to its easy decomposition.
O OH 54
AcCl MeOH 49 %
O OCH3 55
O OCH3 N2 50
The reaction of 50 with Boc-pyrrole catalysed by rhodium catalysts, described by Davies et al., was performed yielding the desired 8-azabicyclo[3.2.1]octadiene 51 in 52 % yield (Scheme 4.10). The following attempts to perform at 1,4-conjugate addition of phenyl magnesium bromide to form 56 did not succeed this will be discussed further in section 6.2. Because of the problems considering the Grignard reaction, it was decided to ignore this route to the tropanes.
Boc N
O OCH3 N2 50
Rh2(O2CC7H15)4 pentane
Boc N
CO2CH3
PhMgBr Et2O, -40oC
CO2CH3 Boc N Ph 56
51, 52 %
Scheme 4.10 Synthesis of 51 and attempts to add PhMgBr in a conjugate manner to form 56.
the bicyclic skeleton. 58 was readily reduced by sodium borohydride to the hydroxy compound 59, which upon benzoylation gave the 8-oxa analogue of cocaine, 60 (Scheme 4.11).
TMSO OTMS OCH3 49 57 + H3CO O OCH3
TiCl4 CH2Cl2, -78oC, 3h
CO2CH3 O 58, 79 %
NaBH4
CO2CH3 O O O 60
BzCl
CO2CH3 O OH Ph
pyridine
59
As proposed in Scheme 4.12, a similar methodology using a Boc-protected pyrrolidine instead of 2,5-dimethoxy-tetrahydrofuran, might be a way to obtain a tropane skeleton having a 2-carbomethoxy group.
Boc N Boc N OCH3 61
TMSO OTMS
H3CO
H3CO
Boc N OCH3 62
TMSO OTMS
49
OCH3 OCH3
49 TiCl4, CH2Cl2
TiCl4, CH2Cl2
Boc N
CO2CH3 O
Boc N
CO2CH3 O
63
64
The first challenge was to synthesize N-Boc-2,5-dimethoxypyrrolidine (62). Some 2,5-dimethoxylated pyrrolidines have been prepared by anodic oxidation of the corresponding protected pyrrolidines in methanol.107 However, it was decided to use the same procedure, which was used for dimethoxylation of furan (Scheme 4.12).108 This procedure was also successful for dimethoxylation of Boc-pyrrole to give 61 in 52 % yield. 61 underwent hydrogenation using Raney Nickel as catalyst to give the desired dimethoxylated pyrrolidine 28
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.
N H 66
N O
65
Scheme 4.13 Pyrrole attached to a MBHA resin through a linker derived from succinic anhydride.
By using the resin bound pyrrole 66, the tropane skeleton was supposed to be generated on solid phase. The [3+4] cycloaddition of 1,3-dibromo-1-phenyl-2-propanone (44) and resinbound pyrrole 66 was tried. It is not known for sure whether the reaction works or not, but after cleaving the expected product from the resin, using TfOH and TFA, no product was isolated. A reason might be that the product has decomposed because of the harsh cleaving conditions and could probably have been solve by changing the resin. Because of the above mentioned methods for generation of tropanes were discarded, no further studies were carried out trying to extend it to solid phase synthesis.
29
4.3 Conclusion
Three possible ways that were thought to be used for a combinatorial approach to the tropane skeleton, have been presented. First via a [3+4] cycloaddition of pyrroles and ,dibromoketones. From this tropane synthesis, it was suggested to introduce a 2-carbomethoxy group on the tropane by employing the ,-dibromoketone of acetoacetate. Because of problems associated with synthesising the ,-dibromoketone this approach was rejected for further investigations. Thereupon attempts to the tropanes were made by a tandem cyclopropanation/Cope rearrangement of a vinylcarbenoid and Boc-pyrrole. The constructed tropane was subjected to reaction with phenyl magnesium bromide, which turned out to be unsuccessful. This will be discussed further in section 6.2.2. Third, a new way to construct a tropane from pyrrolidines was proposed. By further optimisation, it might be possible to generate the desired tropane from this procedure. But due to a very unclean reaction, it was not investigated further. In addition, considerations on how to extend these methods to solid phase chemistry were made.
30
5 Two- and Three-Dimensional Solution Phase Combinatorial Libraries of 3- and 8-Substituted tropanes from Multicomponent Grignard Reagents
5.1 Generation of a Two-Dimensional Library from Multicomponent Grignard Reagents
Due to the difficulties associated with a de novo construction of the tropane skeleton, it was decided to start a combinatorial approach of potential dopamine transporter ligands from a tropane that was already constructed. For this purpose anhydroecgonine methyl ester (7) was chosen as starting material, since 3-substituted phenyltropanes are known to be synthesised from this compound. As described in the introduction (see Scheme 1.1), phenyltropanes can be obtained by a 1,4-conjugate addition of aryl Grignard reagents to the electrophile 7. Therefore, it was expected that libraries could be obtained by reaction of 7 with a mixture of different Grignard reagents. In addition, 3-phenyl substituted tropanes are known to be potent dopamine transporter ligands and in the development of a cocaine abuse treatment, libraries of such analogues would be beneficial. Furthermore, the use of anhydroecgonine methyl ester (7) raised the possibility of introducing more combinatorial steps by introducing substituents at other reaction sites such as substituting the N-methyl group and changing the ester functionality.
reagents necessary for synthesis of compounds in column 1,2n in the vertical dimension) would give the desired requirements for the library design. This is illustrated in Scheme 5.1. In this way, if a library in the horizontal dimension contains an active compound, the compound will be identified from the vertical library containing the same compound. After synthesising the 2n libraries, no further deconvolution is needed and active compounds can be synthesised individually.
R1,4MgBr R2,4MgBr R3,4MgBr R4,4MgBr R5,4MgBr E
Scheme 5.1 The synthesis of 25 compounds in two dimensions using variable mixing of Grignard reagents. Reaction of 10 different five-component Grignard reagents with the electrophile (E) results in 10 sublibraries of 5 compounds each. If compound E-R2,4 turns out to be active it will be found in both the horizontal and vertical sublibrary and can be identified directly.
Generally, synthesis of n2 compounds will require n libraries of n compounds. For the abovementioned method, it is necessary to synthesise 2n libraries of n compounds each and in that way, all library members will be present in 2 sublibraries. The advantage of the method is the direct identificaton of an active compound, which eliminates the need for resynthesis of sublibraries or a larger number of individually compounds. The major drawback of the method is that if all library members have approximately the same biological activity, it might be difficult to get any usable information from the biological assays. The addition of aryl Grignard reagents to anhydroecgonine methyl ester have been used widely to synthesise 3-phenyl tropanes.5 But even though it has been emphasised that simple 3-alkyl substituted tropanes are poor ligands to the DAT no synthesis or occurrence of this type of compounds have been found in literature that supports this statement. Therefore, it was decided to include 3-alkyl substituted tropanes in the library. In addition, cycloalkyl Grignard reagents were included in the mixture of Grignard reagents, since the resulting 32
products might be interesting compounds, which due to their size and lipophilicity, might show some similarities with the benzene ring present in phenyl tropanes. Four aryl Grignard reagents were also included of which two resulted in unknown products (3-methylphenyl and 3,4-dimethylphenyl magnesium bromide). 4-tert-Butyl phenyl magnesium bromide has previously been employed for synthesis of the corresponding phenyl tropane, but since no biological data has been published for that compound, it was also chosen to become a member of the library.110 In addition, it is known that the corresponding p-iodo phenyl tropane (RTI-55) is a very potent compound against all three monoamine transporters and since the size of an iodine is approximately the same as for a t-butyl group, it was proposed that the t-butyl might be a good substitution for an iodine. The last known compound, which was chosen as a constituent of the library, was 10.24 This compound has shown to bind to DAT, NET, and SERT (Table 5.1) and was included as a positive control to confirm the validity of the biological assay.
IC50 binding (nM) Compound DAT SERT NET DAT [3H]DA [3H]WIN35428 [3H]paroxetine [3H]nisoxetine
CO2CH3 N
196261
92073
49.22.2
17313
37.25.2
Table 5.1 Biological data for binding and reuptake of WIN35065-2 (10) to DAT, SERT, and NET all obtained from rat tissue.24
33
CO2CH3 N
67
Scheme 5.2 Conjugate addition of n-butyl magnesium bromide to anhydroecgonine methyl ester.
After proving that a non-aromatic Grignard reagent can participate in the 1,4-conjugate addition as well as aryl Grignard reagents, a selection of alkyl- and aryl magnesium bromides were screened for their ability to undergo 1,4-conjugate addition. This was done by preparing mixtures of Grignard reagents by sequential addition of the selected bromides to excess magnesium in Et2O. In this way, observation of the exothermic reaction of each halide with magnesium was used to ensure that each Grignard reagent had been formed. A number of Grignard reagents failed to undergo addition to 7, among these methyl magnesium iodide, allyl-, propargyl-, cyclopropanyl- and o-isopropyl phenyl magnesium bromide. This might be explained by high reactivity leading to homocoupling products or by sterical hindrance. From these screening experiments, it was also shown that knowledge of the precise concentration of the Grignard reagent was crucial for the reaction to complete and for that reason all Grignard reagents were titrated according to a known procedure.113
34
a
CO2CH3 N
b
CO2CH3 N
N
c
CO2CH3
d
CO2CH3 N
N
e
CO2CH3
67
CO2CH3 N
CO2CH3 N N
CO2CH3 N
CO2CH3 N
CO2CH3
II
CO2CH3
CO2CH3 N N 68
CO2CH3 N
CO2CH3
CO2CH3 N
t
III
Bu
69
CO2CH3
CO2CH3 N N
CO2CH3
CO2CH3 N
CO2CH3 N
IV
CO2CH3 N
CO2CH3 N
CO2CH3 N
CO2CH3 N
N
CO2CH3
V
10
70
71
Figure 5.1 Matrix representation of the two-dimensional library contained in sublibraries I-V (rows) and a-e (columns).
For sublibrary synthesis, the reaction was performed in a similar way as described for addition of n-butyl magnesium bromide, with the exception of adding the freshly prepared mixture of 5 Grignard reagents slowly to the electrophile via a syringe pump. This was done to ensure equal formation of products, even though a difference in the reactivity of the used nucleophiles might occur. As an example, the synthesis of library II is shown in Scheme 5.3. Even though an excess of Grignard reagents were employed, it did not have any consequences for the purity of the libraries. The amine functionality allowed a successful acid/base extraction and no further purification was necessary.
35
CO2CH3 +
CO2CH3 N N
CO2CH3
7 N
CO2CH3
Library II
Scheme 5.3 Conjugate addition of a five component Grignard reagent to anhydroecgonine methyl ester (7) synthesis of library II.
A lot of effort was addressed to analysis of the libraries. All libraries were analysed by
1
H-NMR, ESMS, and GC-MS to ensure that all library members were present in
approximately equal amounts. 1H-NMR spectra were only useful in libraries containing separated peaks e.g. library V containing both aromatic and alkyl protons. An example of a GC chromatogram and ESMS spectrum are shown in Figure 5.2, which clearly show generation of all 5 desired products in approximately equal amounts in library II.
Figure 5.2 GC-MS chromatogram (A) and ESMS (B) of library II showing formation of all 5 expected products in approximately equal amounts.
36
5.2.
Ki binding (nM) Library I II III IV V a b c d e hDAT 9850 15800 11300 9300 43 1880 8750 6250 64 265 hSERT 8800 4900 9700 5400 38 4700 5200 11800 55 610 hNET 10200 9100 6600 11500 113 1900 9700 10200 130 370 hDAT 5000 10500 4800 4900 53 650 4400 3500 83 170 Ki uptake (nM) HSERT 10000 12400 10700 9200 52 4400 11900 11800 57 750 hNET 4850 3000 6600 5800 40 350 4550 7700 65 165
Table 5.2 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET for 2D libraries I-V and a-e. Each library contains 5 compounds.
37
Graphical displays in Figure 5.3 are a visual form of the biological data in Table 5.2. In order to obtain a meaningful value of the height of a column in the diagrams, the two sublibraries containing a given compound were analysed. The sublibrary having the highest Ki value was chosen among the two. In principle, the lowest possible Ki value of the given compound is Ki/5, if only that compound contributes to the overall affinity of the sublibrary. To obtain high columns for high affinity compounds the reciprocal of Ki/5 was plotted in the diagrams and is a value of the highest possible association constant for that compound.
hDAT binding hSERT binding hNET binding
0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e 5/K i(max) (nM )
-1
II III IV
hDAT uptake
hSERT uptake
hNET uptake
0.07 0.06 5/K i(max) (nM ) 5/K i(max) (nM-1) 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I
-1
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 a b c V d e II III IV I 5/K i(max) (nM-1)
Figure 5.3 Graphical displays showing for each library member the smallest value of 5/Ki for each of the two sublibraries in which it appears.
As seen from the diagrams, the two library members 70 (d,V) and 71 (e,V) show high activity in all six assays. It is also seen that the positive control 10 (a,V), shows activity against the transporters, especially against hDAT and hNET as expected from Table 5.1, but the activity is considerably lower than for 70 (d,V) and 71 (e,V). Not surprisingly, the two high affinity compounds were bearing a 3-aryl substituent, but to our surprise 69 (e,III) bearing a t-butyl phenyl substituent, did not turn out to bind to the transporters at all. A reason for this might be sterical hindrance from the t-butyl group. As suggested in literature, 3-alkyl substituted tropanes did not turn out to bind appreciable to the three transporters.
38
To ensure that the two dimensional screening procedure was a useful way to identify possible leads, a variety of the library members were resynthesised as single compounds. The biological data for binding to and uptake of monoamine at hDAT, hNET, and hSERT are listed in Table 5.3.
Ki binding (nM) Compound 10 67 68 69 70 71 hDAT 22095 6900650 210004500 380005250 1910 11545 hSERT 750680 231007300 270007500 3700620 156 25050 hNET 555455 4700650 105003000 299501300 207 190110 hDAT 11268 1900750 53002600 117001850 147 6540 Ki uptake (nM) hSERT 614208 249003100 3600023000 41502950 1811 8320 hNET 11530 2650750 41002500 157003300 135 4028
Table 5.3 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.
These data clearly shows the validity of the two-dimensional display. Of the two high affinity compounds 70 and 71, 70 turns out to be the most potent compound binding to all three transporters, which was also seen from the diagrams in Figure 5.3. In addition, the compounds 68 and 67 that did not seem to bind according to the matrix representation of the libraries, did not show binding affinities as single compounds either. With respect to the reference compound 10, the data in Table 5.3 do not correspond well to the literature values presented in Table 5.1. The reason for this might first of all be that Table 5.3 present Ki and not IC50 values. In addition, the Ki values are obtained from assays using the cloned human transporters, whereas most other published results (as in Table 5.1) are obtained by employing transporters from rat, mouse, or monkey brain tissue.
39
HN
CO2CH3
72
Scheme 5.4 Synthesis of (1R, 5S)-8-azabicyclo[3.2.1]oct-2-ene-2-carboxylic acid methyl ester (72) by demethylation of 7 using ACE-Cl and MeOH.
This was followed by alkylation of the nitrogen using 1.1 equivalent of an alkyl halide or a mixture of alkyl halides. Initial studies of the multicomponent N-alkylation turned out successfully. As a model experiment, equi-molar amounts of allyl-, benzyl-, and n-butyl bromide were refluxed overnight with the demethylated compound 72 in acetonitrile using KI as nucleophilic catalyst and K2CO3 as proton sponge (Scheme 5.5). An equi-molar mixture of 3 products was clearly obtained, witnessed by TLC, 1H-NMR (Figure 5.4), and GC-MS, in 75 % yield (obtained from an average of molecular masses).
40
Figure 5.4 1H-NMR spectrum of a mixture of N-substitued anhydroecgonine methyl ester analogues 73 showing approximately equal formation of each product.
CO2CH3
HN
CO2CH3 + Ph 72
Br Br Br
CO2CH3
CO2CH3
73
1.
MgBr MgBr MgBr
CO2CH3 N N
CO2CH3
Ph
CO2CH3 N
CO2CH3 N N
CO2CH3
Ph
CO2CH3 N
CO2CH3 N Ph N
CO2CH3 Ph
Ph
CO2CH3 N Ph
74
This mixture of N-alkylated analogues (73) was subjected to the same Grignard conditions as used for the two-dimensional library using a mixture of PhMgBr, iPrMgBr, and EtMgBr (Scheme 5.5). Of the expected nine products (74), only 6 were obtained. The missing library 41
members were all consistent with having an N-benzyl substituent. By synthesising the Nbenzyl constituent of mixture 73 and subjecting it to reaction with PhMgBr, it was realised that no reaction occurred this will be discussed further in section 6.2. Therefore, it was concluded that the N-substituent had to be more identical for the Grignard reaction to take place.
HN
CO2CH3 +
Br Br Br
Br CH3CN, reflux Br
R1
CO2CH3 +
CO2CH3
1. Et2O, -40oC 2. TFA, -78oC
R1
R2
72
75-79 5 compounds
Library Y1 25 compounds
The ESMS of library Y1 showed all the 25 expected masses with a Gaussian-like form of the peaks. This is consistent with having several compounds with identical masses for the peaks in the middle, decreasing on going to both sides. In addition, the GC-MS showed many (>15) of the expected product peaks. It was therefore assumed that the probability of having all 25 compounds present in approximately equal amounts was high (Figure 5.5).
42
Figure 5.5 GC-MS chromathogram (A) and ESMS spectrum (B) of library Y1 showing a high probability of having all 25 products present.
In the same way, the 9 libraries X1-X5 (vertical cross sections) and Y2-Y5 (horizontal cross sections) of 25 compounds each were prepared. When preparing layers Z1-Z5, the given N-substituted anhydroecgonine methyl ester analogue, was reacted with a 25-component Grignard reagent. All 125 compounds are represented by the cube of Figure 5.6. Each compound is contained in 3 sublibraries.
X1 Z1
CO2CH3 N N
X2
CO2CH3 N
X3
CO2CH3 N
X4
CO2CH3 N
X5
CO2CH3
X1 Z2
N CO2CH3 CH CO2 3 N N
X2
CO2CHCO CH 3 2 3 N N
X3
CO2CH3 CH CO2 3 N N
X4
CO2CH3 CH CO2 3 N N
X5
CO2CHCO CH 3 2 3 N
X1 Z3
N CO2CHCO2CH3 2CH3 3 CO N N N
X2
CO2CHCO2CHCO2CH3 3 3 N N N
X3
CO2CHCO2CH3 2CH3 3 CO N N N
X4
CO2CHCO2CH3 2CH3 3 CO N N N
X5
CO2CHCO2CHCO CH 3 2 3 3 N N
t
X1 Z4
N
X2
X3
X4
X5
CO2CHCO2CHCO2CHCO CH 3 3 2 3 3 N N N t Bu
Bu
CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N
CO2CHCO2CHCO2CHCO CH 3 3 2 3 3 N N N N
CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N
CO2CHCO2CHCO2CH3 CH 3 3 CO2 3 N N N N
X1 Y1 Z5
N
X2
X3
X4
X5
CO2CH3 2CHCO CHCO CH CO2CH3 2CHCO CHCO CH CO CH3 CO CH3 CO CO CH3 CO 3 2 3 3 2 3 2 3 3 CO2CH3 3 3 3 3 2 3 3 CO2CH2 CO2CHCO2CHCO2CHCO2CH2 CO2CHCO2CHCO2CH3 2CH2 CO2CHCO2CHCO2CH3 2CH3 CO 3 CO 3 3 3 N N N N N N N N N N N N N N N N N N N N t N N N N Bu
Y2
Y3
CO2CH3 2CHCO CH CO 3 2 3 N N
CO2CH3 2CHCO CH CO 3 2 3 N N
CO2CH3 2CHCO CH CO 3 2 3 N N
CO2CH3 2CHCO CH CO 3 2 3 N N
CO2CH3 2CH3 CO
CO2CH3 2CH3 CO N N N
Y4
CO2CH3 2CH3 CO N
CO2CH3 2CH3 CO N N
CO2CH3 CH CO2 3 N
CO2CH3
CO2CH3 N N
CO2CH3 N
CO2CH3 N
CO2CH3
Y5
43
Table 5.4 Ki values for three-dimensional libraries X1-X5, Y1-Y5, and Z1-Z5 for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.
From Table 5.4 diagrams for direct identification of hits were constructed in the same way as for the two-dimensional libraries (Figure 5.7). The only modification being that columns in the diagrams are obtained for each compound from the lowest value of 25/Ki from the three libraries in which the library member appears.
44
0.16
0.025
0.08
0.07
0.06
0.015
0.01
Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1
0.005
0.02 0
X1 X2 X3 X4 X5 Y3,Z1 Y1,Z1
Y2,Z2 Y5,Z1
0.01 0
X1 X2 X3 X4 X5 Y3,Z1 Y1,Z1
Y2,Z2 Y5,Z1
0
X1 X2 X3 X4 X5
Y3,Z1 Y1,Z1
hDAT uptake
hSERT uptake
hNET uptake
0.14
0.012
0.1 0.09
0.12
0.01 0.08
0.08
Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1
0.006
Y5,Z5 Y3,Z5 Y1,Z5 Y4,Z4 Y2,Z4 Y5,Z3 Y3,Z3 Y1,Z3 Y4,Z2 Y2,Z2 Y5,Z1
0.06
0.004
0.04
0.02
Y3,Z1 X1 X2 Y1,Z1 X3 X4 X5
0.002
0
X1 X2 X3 X4 X5
Y3,Z1 Y1,Z1
Figure 5.7 25/Ki (nM-1) for binding and reuptake at hDAT, hSERT, and hNET for each library member. These values are obtained as the smallest value of 25/Ki for the three libraries in which the library member appears.
From the above diagrams, the most potent compounds against the 3 transporters can be read directly. With respect to binding to hDAT, it is seen that compound (x,y,z) = 4, 5, 4 corresponding to N-pentyl-3-dimethyl-phenyl analogue 81, appears to be the strongest inhibitor of hDAT binding. The N-hexyl-3-dimethyl-phenyl analogue 82 ((x,y,z) = 4, 5, 5) appears to be the most potent compound binding to hSERT, while the strongest inhibitor of hNET binding, according to the diagram, is the N-ethyl-3-dimethyl-phenyl analogue 80 ((x,y,z) = 4, 5, 1). Again it is seen that the most potent compounds are those containing a 3 aromatic substituent and as in the two-dimensional library, having a 3,4-dimethyl substituted phenyl group results in the most potent compounds binding to all three monoamine transporters. The most potent compound, with respect to binding to each transporter, was selected for individual synthesis (i.e. 80-82). The binding affinity and inhibition of reuptake are shown in Table 5.5. 45
CO2CH3 N N
CO2CH3 N
CO2CH3
80
81
82
Ki binding (nM) Compound 80 81 82 hDAT 185 143 256 hSERT 13350 22015 29782 hNET 16589 345230 580140 hDAT 4213 3410 14044
Ki uptake (nM) hSERT 7831 6933 31064 hNET 5926 7238 28564
Table 5.5 Ki values for binding (displacement of [125I]RTI-55) and uptake of [3H]DA and [3H]SER at hDAT, hSERT or hNET.
It is seen that all three compounds 80-82 bind very strongly to the hDAT with a Ki value in the same range as the N-methyl analogue 70. As suggested from Figure 5.7, 81 turns out to be the most potent compound with respect to hDAT binding, but the magnitude of the Ki value compared to 80 and 82 seems to be much smaller than it appears in Figure 5.7. The prediction that the 80 is the most potent compound binding to hNET is also seen to be the case, whereas the prediction that 82 was the strongest binding compound to hSERT, do not turn out to be true. This might be because of the relatively large standard error of the Ki determinations from the libraries X1-X5, Y1-Y5, and Z1-Z5 (not shown in Table 5.4). It is also seen from Table 5.5 that both binding and uptake at hSERT and hNET are decreasing with the length of the N-substituent and that selectivity for hDAT over hSERT and hNET binding affinity is induced. This is consistent with previous results showing increased selectivity for DAT with increasing size of the N-substituent.34
46
As mentioned in the introduction, benztropines are another class of interesting compounds showing affinity to the dopamine transporter. Starting from tropine, benzyl ether 83 was obtained in four steps. The key step in the synthesis was radical azidonation of 83 resulting in azide 84 that was now ready to undergo substitution with Grignard reagents. This new synthesis route to the benztropines is particular interesting, since so far only 3-diarylmethoxy substituted benztropines have been investigated, whereas the present method also allows introduction of alkyl groups. By reacting 84 with 10 different five-component Grignard reagents followed by Bocdeprotection, 25 benztropine analogues were synthesised in two dimensions (Scheme 5.7).IV Extension to a third dimension was done by reaction of the two-dimensional library with five alkyl bromides to form a three-dimensional library of 125 benztropine analogues in 15 sublibraries.
BocN O Ph 83 84
IN3 10 % DMF in CH2Cl2 reflux, 73 % 1. RMgBr toluene, 2h, rt
BocN O Ph N3
HN
R'Br, K2CO3
O Ph 56 - 70 %
Scheme 5.7 Synthesis of benztropine analogues in two- and three-dimensional libraries. R = alkyl and aryl, R = alkyl.
Screening of the libraries for binding and inhibition of uptake at hDAT, hSERT, and hNET did not result in identification of compounds that were stronger than benztropine. See appendix 7 for further details.
IV
47
new high affinity compounds were identified. The two compounds and several other library members were resynthesised as single compounds to validate the method. In a similar way, a three dimensional library was constructed by adding an extra dimension via varying substituents at the tropane nitrogen. For this purpose a multicomponent N-alkylation was employed followed by a multicomponent Grignard reaction. In this way 125 compounds were generated in 15 sublibraries of 25 compounds each. From the biological screening, three compounds were selected for individual synthesis. As expected, they all turned out to have high affinity for the three monoamine transporters both in binding and reuptake assays. The same method was also applied for construction of two- and three-dimensional libraries of benztropine analogues. The presented method turned out to be a good way for generation of libraries and for identification of possible hits. By extending the method, larger libraries can be synthesised and in that way the gain associated with the use of combinatorial chemistry will increase. In that case, it is important to consider the size of the libraries related to the difference in biological activity expected for biologically active versus biologically inactive compounds, since the method cannot be used for hit generation when all library members show approximately equal activity.
48
have been incorporated at present. In this way, both the 3- (87) and 3- (88) products were obtained by reduction of the octenes 86 with samarium diiodide (Scheme 6.1). The 8-oxa analogues cannot be involved in binding to the transporter through an ionic bond, but the possibility of attaining binding affinity via hydrogen bonding is still present.
CO2CH3 O 58
Na(TMS)2N, Ph(Tf)2N THF, -78oC - RT overnight, 79 %
CO2CH3 OTf 85
CO2CH3 Ar 86
I2 h Sm H, 2 % O Me 0 -65 F/ TH o C, 5 -78
CO2CH3 O Ar + O
Ar CO2CH3
87
88
Cl 89
77 %
O 90
O 91
50
comparable yields (Scheme 6.1). A serious drawback of this synthetic pathway is that for the 8-carba analogues all four possible stereoisomers are obtained. Because of their considerable unpolar properties, separation of the products appeared to be very difficult, resulting in very low yields of the pure compounds.
CO2CH3 X
Cl
CO2CH3
Cl Cl
Cl
Table 6.1 Inhibition of [3H]WIN35428 binding to the DAT in cynomolgus monkey caudate-putamen. a Inhibition of [3H]WIN35428 binding to the DAT in rat striatal membranes. b mixture of isomers.
It is seen that even among the 8-oxa and 8-carba analogues, extremely potent inhibitors of DAT are found. Thus, the effect of changing the 8-nitrogen in phenyltropanes does not affect the magnitude of inhibition dramatically. It is also observed that the potency of the ligands is largely dependent on the aryl group, since compounds having a 3,4-dichlorosubstituted phenyl ring are by far the most active. Another notable observation is that the 2,3-unsaturated compounds show a striking selectivity of 460-720 fold for inhibition of DAT over SERT (IC50 values for inhibition at SERT are not shown). The studies of non-amine analogues of phenyltropanes conclude that formation of neither an ionic bond nor hydrogen bonding is crucial for binding of phenyltropanes to the DAT, when the aryl group is carefully chosen.
51
The
initial
proposal
was
to
synthesise
methyl
3-(4-iodophenyl)-
92
93
94
These compounds were thought to be reasonably easy available from Davies bicyclo[3.2.1]octene 95119 by a 1,4-conjugate addition of a Grignard reagent, in the same way as describe for the tropanes in section 5 followed by iodination. Since the bicyclic system can be obtained from a tandem cyclopropanation/Cope rearrangement of vinyldiazomethane 50 and cyclopentadiene, substituents in the 8-position was thought to be introduces by employing 1-methyl cyclopentadiene and 1,1-dimethyl cyclopentadiene. In addition, an establishment of such a method would also be beneficial for developing a combinatorial synthesis of 8-carba analogues of phenyltropanes and further expand the usefulness of the method described in section 5.
O OCH3 N2 50 +
1. Rh(II) 2. H2, Wilkinson's
CO2CH3
PhMgBr
CO2CH3 Ph
95
96
Scheme 6.3 Construction of Davies' bicyclo[3.2.1]octene 95 via a tandem cyclopropanation/Cope rearrangement followed by selective hydrogenation of the unconjugated double bond. This was suggested to be followed by a 1,4-conjugate addition to yield the desired compound 96.
52
Methyl 2-diazobut-3-enoate (50) was synthesised from methyl acetoacetate according to procedure outlined in section 4.1.2.1.104 This was followed by construction of the [3.2.1]bicyclic system through a rhodium-catalysed tandem cyclopropanation/Cope rearrangement with cyclopentadiene to give 97. The bicyclic octadiene 97 could now be used for studies of the conjugate addition, as well as its C-6/C-7-saturated analogue 95, obtained from selective hydrogenation of the unconjugated double bond by Wilkinsons catalyst (Scheme 6.4). The use of 97 was preferred, since the presence of the C-6/C-7 double bond might allow introduction of substituents in the 6 and/or 7 position.
O OCH3 N2 50 +
3 % Rh2(O2CC7H15)4 pentane, reflux 1h 90 %
CO2CH3
CO2CH3
97
95
6.2.2 Attempts to Perform Conjugate Additions to Methyl bicyclo[3.2.1]octa-2,6diene-2-carboxylate and Methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate
Initially, the conjugate addition of a Grignard reagent to methyl bicyclo[3.2.1]octa-2,6-diene2-carboxylate 97 was attempted (in the same way as was done with the anhydroecgonine methyl ester in section 5), but to our surprise no reaction occurred. Several reaction conditions were investigated (Table 6.2, all reaction mixtures were analysed by TLC and GC-MS). As entries 1 - 3 were all unsuccessful, no effect resulted from adding more Grignard reagent not even the 1,2-addition product was observed. Neither did it have any effect to add Cu(I) nor TMSCl or HMPA (entries 4 - 6).120 A last attempt was made using a higher order cuprate, but this also turned out to be unsuccessful. Contrary to the expectation, it turned out to be difficult to perform a conjugate addition on the ,-unsaturated bicyclic octadiene 97 and even though it seemed unreasonable that the C-6/C-7-double bond could cause the difficulties, it was decided to try to see if the conjugate addition could be performed on the saturated bicyclic ester 95.
53
CO2CH3
97
98
Entry 1 2 3 4 5 6 7 8
Reagents PhMgBr (1 eq) PhMgBr (2 eq) PhMgBr (6 eq) PhMgBr (1 eq), CuBrMe2S (1 eq) PhMgBr (2 eq), LiCl (1 eq), TMSCl (1 eq), CuBrMe2S (1 eq) PhMgBr (1.5 eq), TMSCl (2 eq), HMPA (3 eq), 10 % CuBrMe2S PhLi (2 eq), CuCN (1 eq) PhLi (3 eq), CuCN (1.5 eq)
Reaction conditions -40 C, 4h -40 C, 5h then rt overnight -40 C, 5h then rt overnight -40 C, 1h, then PhMgBr (4 eq) 3h, then rt overnight -78 C -78 C, 6h -40 C rt overnight -78 C -30 C, 4h
Table 6.2 Unsuccessful attempts to add phenyl nucleophiles to the ,-unsaturated ester 97.
A few attempts to do the conjugate addition on the C-6/C-7-saturated ester 95 were performed according to Table 6.3, but still no addition reaction occurred.
CO2CH3
Reagents Solvent, connditions
CO2CH3
95
96
Entry 1 2 3
Reagents PhMgBr (1 eq) PhMgBr (2 eq) PhLi (26 eq), CuCN (13 eq), BF3Et2O (15 eq)
Table 6.3 Unsuccessful attempts to add phenyl nucleophiles to ,-unsaturated ester 95.
In addition, it was also tried to do the conjugate addition of PhMgBr to the t-butyl ester of 95 and the temperature was raised to -20C, but again no reaction occurred. Since the conjugate addition of a Grignard reagent was easily performed on the 8-aza analogue 7 and the only difference from 7 to 95 is the presence of the 8-nitrogen in 7, this nitrogen must be crucial for the reaction to occur when generating phenyltropanes. An 54
explanation can be proposed by taking a careful look at the conjugate addition of a nucleophile to 2-cyclohexenones, which preferentially happens by axial attack of the nucleophile.121 Axial attack of a nucleophile leads to a chair-like intermediate, whereas equatorial attack leads to a boat-like intermediate. Therefore, attack from the axial position is thought to be favoured because of a lower energy of the transition state and intermediate of a chair compared to a boat conformation. This is exemplified by the copper-catalysed addition of iodomethylmagnesium to 5-methyl-2-cyclohexenone (98) where the major product 99 was presumed to arise via a chair-like intermediate (Scheme 6.5).122
O
MeMgI, CuI Et2O, 55-66 %
O +
98
99, 94-96 %
100, 4-6 %
Scheme 6.5 Copper-catalysed conjugate addition of a Grignard reagent to 5-methyl-2-cyclohexenone preferentially by axial attack of the nucleophile.
In case of addition to a 2,3-unsaturated bicyclo[3.2.1]octane as 95, axial attack is probably too sterically hindered because of the ethylene bridge causing unfavourable 1,3-diaxial interactions. The only possibility is then equatorial attack, which might be energetically unfavoured because the energy of the transition state via a boat-like conformation is too high (Figure 6.2).
Nu O CO2CH3 OCH3 CO2CH3 O OCH3 Nu
But why is the Grignard reaction possible when a nitrogen is present in the 8-position? An explanation might be that the presence of a nitrogen in position 8 afford a coordination site for the Grignard reagent, which offers stabilisation of the boat-like transition state and thereby lowering of the transition state energy is obtained (Figure 6.3). In this way a six-membered transition state is generated. In addition, this can explain why only the 3-isomer is obtained when generating phenyltropanes through a 1,4-conjugate Grignard reaction to anhydroecgonine methyl ester (7). 55
Mg
OCH3
Figure 6.3 Possible stabilisation of a boat-like transition state by coordination of magnesium to the tropane nitrogen.
A similar explanation have been proposed as a rationale for equatorial attack of magnesium containing thiol nucleophiles to anhydroecgonine methyl ester in the synthesis of cocaine benzoyl thioester.123 It has also been observed that the reactivity of Grignard reagents in general increases in the presence of aliphatic amines, possibly through coordination to the Lewis acidic magnesium.124 The nitrogen in anhydroecgonine methyl ester could therefore account for an increased reactivity of the Grignard reagent. Another explanation could be that in the 8-azabicyclic system, the double bond of the ,-unsaturated ester is more electrophilic, because of the electron-withdrawing effect of the 8-nitrogen compared to the 8-carba bicyclic system. To further explore the influence of the atom in the 8-position, a series of methyl bicyclo[3.2.1]oct-2-ene carboxylates were prepared varying the atom or substituent in the 8-position. 101, 102, and 103 were prepared as previously described from vinyldiazomethane 50 (see section 4.1.2),125,126 whereas the remaining nitrogen containing compounds 104, 105, and 106 were prepared by alkylation of 72 as in the synthesis of the three-dimensional library in section 5.1.2 (Figure 6.4).V
O O CO2CH3 RO N CO2CH3 R N CO2CH3
101
Figure 6.4 101-106 were all reacted with 2 eq. PhMgBr in Et2O at -40C for 4h before quenching with TFA at -78C. No reaction was observed for any of the compounds.
101, 102, 104, and 106 were synthesised by bachelor students Naja Nielsen, Sara Kobbelgaard and Mette Nymand under supervision of the author.
56
101-106 were all reacted with 2 equivalents of freshly prepared PhMgBr in Et2O at 40C for 4h. TLC analysis revealed that no reaction had occurred and after quenching with TFA at -78C and workup this was confirmed from GC-MS analysis. With the presence of an oxygen in 101 and thereby the possible existence of a coordination of the Grignard reagent, in the same way as described above, it was expected that the reaction would work smoothly. Since the reaction does not take place, the theory of coordination cannot be further rendered probable. By having a carbamate functionality, as in 102 and 103, the basicity of the nitrogen is decreased. And since the reaction does not take place in this case, it can be explained by the poor coordination possibilities of the carbamate nitrogen compared to the corresponding amine. Thus, it is spectacular that a similar Grignard reaction have been successfully conducted on a methyl carbamate 7-azabicyclo[2.2.1] analogue 33 (Figure 3.1).71 In the case of having a primary alkyl substituted nitrogen it is known to be possible to do the 1,4-conjugate addition of a Grignard reagents to the ,-unsaturated ester.127 By introducing an N-isopropyl group the electronic properties was assumed to be approximately the same as for a primary alkyl substituted analogue. The presence of the isopropyl group in 104 was therefore suggested give insight into the importance of the steric factor of the nitrogen substituent. As seen in Figure 6.4 no reaction occurred when having an isopropyl group at the nitrogen. Neither when having an N-benzyl (105) nor a p-methoxybenzyl (106) group at the nitrogen causes the reaction to occur. Again it might be because of steric interactions or the explanation could be the reduced basicity of the nitrogen. It is therefore concluded that the 1,4-addition of Grignard reagents to the bicyclic system is very sensitive and depends largely on the nitrogen substituent and the atom in the 8-position.
57
Nu OCH3
O OCH3
I
107
Reagents Conditions
CO2CH3
CO2CH3 Nu
II
95
Entry 1 2 3 4
Reagents PhI (1 eq), Bu3SnH (1.3 eq), AIBN (cat) Bu3PhSn (1.1 eq), 5 % [RhCl(cod)]2 PhB(OH)2 (1.5 eq), 5 % [RhCl(cod)]2
OTMS Ph
(1 eq), TiCl4 (1 eq), CH3CN THF Et2O/THF Reflux overnight 70 C overnight -40 C, 1h 1,4-add. 1,4-add. 1,4-add. no reaction no reaction no reaction
Ti(OiPr)4 (0.4 eq) 5 6 7 Acetone cyanohydrin (1.2 eq), KCN (1.6 eq), 5 % TBACN MeNO2 (1.3 eq), TBAF (1 eq) PhMgBr (1.5 eq), TMSCl (3 eq), HMPA (3 eq), 5 % CuBr
As seen from Table 6.4, it was not possible to do a radical addition of a phenyl radical to methyl crotonate. But this is not surprising, since it is known to be difficult to add an electrophilic radical (as a phenyl radical) to an electrophilic double bond as an ,-unsaturated ester.128 Newer methods for addition of aryl-stannanes to unsaturated carbonyl compounds by rhodium catalysis have been introduced by Oi et al.129 As seen in entry 2 this turned out to be a very convenient way to perform a 1,4-addition on 107 and the desired product was obtained in quantitative yield. Also rhodium-catalysed Suzuki-type coupling of PhB(OH)2 to 107 was very successful (entry 3).130 Another way of making a conjugate addition is by employing a Mukaiyama-Michael addition of silyl enol ethers to an unsaturated carbonyl system by Lewis acid catalysis. In this way a 1,5-dicarbonyl compound is obtained. According to entry 4, an addition of the silyl enol ether derived from acetophenone to 107 was performed, which afforded the desired 1,4-addition product.131 It was also tried to add more simple nucleophiles such as cyanide (entry 5) and CH3NO2 (entry 58
6) to methyl crotonate (107).132 These reactions afforded the desired products without any problems. At last a Cu(I) catalysed conjugate addition of PhMgBr using TMSCl and HMPA as additives was carried out. In contrary, as what was seen for the bicyclic system, it had a positive outcome when performed on the simple ,-unsaturated ester 107 yielding the 1,4addition product. However, methyl crotonate is also a more accessible ,-unsaturated ester than the bicyclic conjugate ester 95 and therefore sterical and electronic factors must control the conjugate addition of a phenyl nucleophile to ,-unsaturated methyl esters. As shown in Table 6.4, all conjugate additions performed on the bicyclic unsaturated ester 95 failed. It seems to be very difficult to do a conjugate addition on the bicyclic system no matter what nucleophile was used, which might be due to sterical hindrance, electronic properties of the double bond or to the absence of coordination possibilities to a nitrogen.
of the more sterically hindered five-membered ring, was more difficult and raising the temperature and dilution was necessary to obtain the ring closed compound (Scheme 6.6B).
TBDMSO TBDMSO
5 % Cl2(PCy3)2RuCHPh c = 0.050 M CH2Cl2, rt, 87 %
TBDMSO
OTBDMS
In another example, a tropane skeleton was generated from a ring closing metathesis strategy.136 This employed reaction of the Cbz-protected divinyl compound 108 with Grubbs first generation catalyst, which afforded the desired product 109 in high yield (Scheme 6.7). This ring closure is probably facilitated by the presence of a carbamate, as 1,3-allylic strain between the carbonyl and the substituents to the nitrogen that favours the diaxial conformer. This conformer is the species being ring closed.
Cbz N
Cbz N O 109
O 108
60
6.2.4.2 Synthesis of Methyl 3-Phenethyl-bicyclo[3.2.1]oct-6-ene Carboxylate (112) Methyl 4,6-divinyl-cyclohexene carboxylate (110) was easily prepared by ring opening metathesis of methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate (97) using Grubbs first generation catalyst in an ethylene atmosphere (Scheme 6.8).
CO2CH3 CO2CH3
97
MesN NMes Ph Cl Ru Cl PCy3
110
CO2CH3 CH2CH2Ph
111
112
Scheme 6.8 Synthesis of methyl 3-phenethyl-bicyclo[3.2.1]oct-6-ene carboxylate (112) via an olefin metathesis strategy.
The cyclic ,-unsaturated ester 110 was now ready for the crucial step the conjugate addition. Initially, it was tried to use PhMgBr as nucleophile the same reaction that was performed for synthesis of the tropane libraries. This reaction is also known to be successfully conducted on arecoline (19), which also contains a nitrogen that could possibly coordinate to the Grignard reagent (see Figure 3.1). Coordination possibilities are not present in the divinylcyclohexene 110 and the attempt to do conjugate addition of PhMgBr did fail. This was also the case by copper catalysed addition of PhMgBr including additives as TMSCl and HMPA.137 By changing the nucleophile from phenyl magnesium bromide to phenethyl magnesium bromide, the latter being a better nucleophile, it was possible to add the nucleophile by using copper catalysis, TMSCl and HMPA resulting in isolation of the addition product 111 in high yield. But because of small impurities having almost identical polarity as the product, only 31 % pure 111 was isolated by careful column chromatography. The rationale for choosing a phenethyl nucleophile was based on the fact that in the tropane series it appears that by having a 3-phenethyl group, the binding affinity is increased compared to having a 3-phenyl group (Table 1.2). It appears that only one addition product arises from the Grignard reaction. Based on the NMR spectra (1H and COSY), it was assigned to be the compound 111 arising from axial attack of the organocopper reagent. This assignment was especially based on the 61
double doublet from H-1 having a large and a small coupling constant (11.6 Hz and 4.4 Hz, respectively). In addition assignment of the final product 112 could confirm these results. It is also worth mentioning that addition of phenethyl magnesium bromide is not possible to perform on 95 when the bicyclic system is intact. To complete the synthesis, a cyclisation of 111 had to be performed. Several attempt were made using Grubbs first generation catalyst in CH2Cl2 and in ClCH2CH2Cl. Indeed formation of the ring-closed compound was observed, but it was not possible to drive the reaction to completion, which was necessary for avoiding purification problems, because of similar polarity of starting material and product. By changing to Grubbs second generation catalyst, which is known to have a higher reactivity towards olefins, it was possible to obtain the ring closed compound 112 in 58 % yield.138 In order to close the ring, it was necessary to use ClCH2CH2Cl as the solvent to be able to reach a higher reflux temperature. The difficulties regarding the ring closure are probably due to the conformation of 111. Having two equatorial vinyl groups, a ring flip is necessary in order to adapt to the ring closing conformer having two axial vinyl groups. The 8-carba analogue 112 is sent for biological testing against hDAT, hSERT, and hNET, and the results are waited in great suspense.
6.3 Conclusions
In order to synthesise bicyclo[3.2.1]octane analogues of phenyltropanes as potential dopamine transporter ligands, it was proposed to employ a synthesis route based on a 1,4-conjugate addition of Grignard reagents to a known ,-unsaturated ester. For that reason a variety of conjugate additions were performed on methyl bicyclo[3.2.1]octa-2,6-diene-2carboxylate and methyl bicyclo[3.2.1]oct-2-ene-2-carboxylate. All attempts were unsuccessful and did not result in the desired addition products. The lack of reaction was ascribed to sterical hindrance from the ethylene bridge, which impaired the possibility of axial attack from the nucleophile. In addition, it was proposed that a nitrogen was essential for stabilisation of a boat-like intermediate and perhaps for increasing the reactivity of the Grignard reagent. As model studies, a number of different conjugate additions were performed on methyl crotonate. Even though several reactions resulted in quantitative yields when performed on the model compound, they all turned out to be unsuccessful when conducted on the bicyclic ester. 62
By changing the synthesis strategy, methyl 3-phenethyl-bicyclo[3.2.1]oct-6-ene carboxylate was synthesis by first ring opening of the bicyclic system followed by successful addition of phenethyl magnesium bromide to the ,-unsaturated ester. A ring closing metathesis, to yield the desired compound, completed the synthesis. This route to 8-carba analogues of phenyltropanes might also be useful for generation of analogues containing methyl and dimethyl substitutions at C-8. Such compounds could be interesting for evaluation of the effect of having substituents in the 8-position, which maybe resulting in induction of selectivity for one monoamine transporter over another.
63
64
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions 1 Introduction
1.1 Carbohydrates Ubiquitious Molecules
Traditionally, compounds occurring in living organisms are termed biomolecules and include proteins, lipids, nucleic acids, and carbohydrates. Of these four classes of biomolecules, carbohydrates are by far the most abundant and make up most of the organic matter on earth. This is mainly due to the existence of cellulose (a polymer made of -1,4 linked glucose units, 112), which plays a major role as a structural element in the cell walls of bacteria and plants.139 Monosaccharide units as ribose and deoxyribose also serve as structural elements, as being a part of the phosphate-sugar backbone of RNA and DNA (115). Another very important role of carbohydrates is associated with energy storage. Starch and glycogen are polymers build from glucose units, that serve as readily mobilisable stores of energy in plants and animals, respectively. In addition, as a component of e.g. potatoes, rice, pasta, and bread, starch has a great nutritional value when ingested by humans.139
O O P O O OH
OH O HO O
HO HO
OH O OH
Base
OH
Galactose, 114
Although glucose is the most abundant monosaccharide, many other monosaccharide units exists (especially galactose (114), mannose, and fructose) and they can all be a part of disaccharides or oligosacchardes. Carbohydrates have a high degree of diversity since there are numerous ways in which monosaccharide units can combine to form di- and oligosaccharides. This property makes them very useful for carrying biological information a thing they indeed do in nature. Being a part of glycoproteins, carbohydrates are involved in many specific biological processes e.g. in recognition processes the sugar part of a glycoprotein can serve as a signal, which directs it to be recognised by a specific cell type or 65
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
receptor.139 They are also involved in other important processes such as cell growth, inflammation, viral replication, and metastasis.140,141 For these reasons, it is of great importance to understand the chemistry of carbohydrates.
OH HO HO O HO OR H
-ROH slow
OH HO HO O HO
fast
OH HO HO O HO OH
H2O, -H+ fast
OH HO HO O HO
Since 1908 it has been known that the rate of acid-catalysed hydrolysis of glycosides depends on the configuration of the hydroxyl groups on the sugar unit the more axial hydroxyl groups, the faster hydrolysis.142,144 E.g. altropyranonside 118 hydrolyses 3-4 times faster than galactoside 117, which hydrolyses 5 times faster than glucoside 116 (Figure 1.2).143
OH HO HO O HO HO OCH3 116 krel 1 HO OCH3 117 5 HO OH O HO HO 118 18 OH OH O OCH3
66
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
In 1955 Edward proposed an explanation for this general observation based on relief of steric strain.145 He reasoned that detachment of the leaving group is accompanied by a conformational change of the pyranose ring from a chair to a half-chair in the transition state. Small rotations around the C-2/C-3 and C-4/C-5 bonds are required for entering this halfchair. This transformation decreases the strain connected with axial substituents and therefore makes a positive contribution to the total reaction rate, when axial substituents are present. This explanation has recently been reconsidered. In a study of the acetolysis of methyl 2,3,6-tri-O-methyl--D-galacto- and glucopyranosides varying the substituent at C-4, Miljkovi et al. found that the rate of acetolysis depends strongly on the electronegativity and orientation of the C-4 substituent.146 Having different C-4 equatorial substituent (methoxy, acetoxy or acetamido substituent, gluco series), the rate of acetolysis did not change significantly. Small changes were explained by a through bond interaction (inductive effect) of the C-4 substituent and the ring oxygen. But in the galacto series (axial C-4 substituent) a large dependence of the reaction rate with respect to electronegativity of the C-4 substituent was observed (4-methoxy > 4-acetoxy > 4-acetamido). The enhanced rate of acetolysis by having electronegative substituents in the axial position was explained by a through-space electron donation from the axially oriented substituent at C-4 of the galactopyranoside into the oxocarbenium ion under formation (Scheme 1.2). This through-space electrostatic stabilisation is not possible for the corresponding gluco series and acetolysis of glucosides are therefore not affected to the same extend as the galacto series (Scheme 1.2, 119 is cleaved approximately 14 times faster than the corresponding gluco configured (equatorial 4-OCH3) methyl pyranoside). The experimental observations were supported by ab initio calculations.
OCH3 O CH3O CH3O OCH3
H+
CH3O
CH3O CH3O
CH3O CH3O
OCH3 O CH3O
119
Scheme 1.2 Proposed stabilisation of the transition state under formation by the axial electronegative substituent suggested by Miljkovi et al.
Another explanation was recently proposed by Jensen et al.147,148 In a study of the basicity of azasugars, it was observed that protonated azasugars having axial 4-OH groups were slightly more basic than their equatorial counterparts (Figure 1.3). From a large amount of pKa values 67
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
of epimeric piperidines, the average effect of having a given substituent in a given position was determined.149 Based on these data, s values were obtained and from Hammett plots a free-energy relationship was established with the rate of acidic hydrolysis of glycosides.150
OH HO HO NH2 pKa 8.4 HO HO OH NH2 pKa 8.8 HO HO OH NH2 OH pKa 6.7 HO HO OH NH2 OH pKa 7.5
Figure 1.3 The difference in pKa values of epimeric piperidinium ions is explained by axial polar groups being less electron-withdrawing than their equatorial counterparts.
The existence of a free-energy relationship suggested that the same effect determining the difference in azasugar basicity also accounts for the difference in the rate of hydrolysis of stereoisomeric glycosides. The effect was explained by a difference in electron-withdrawing power between an axial and an equatorial hydroxyl group the equatorial being more electron-withdrawing through space than the axial (i.e. a field effect). This means that in the A-1 mechanism of glycosidic hydrolysis, the stability of the transition state (approximated by the intermediate carbocation/oxocarbenium ion) depends on the configuration of the hydroxyl groups. Thus, on hydrolysis of a galactoside, the transition state is less destabilised than on hydrolysis of a glucoside and therefore the rate of hydrolysis of a galactoside is faster than that of the corresponding glucoside (Figure 1.4).
OH HO HO NH2 HO HO OH NH2 HO HO OH O HO HO less stable more stable forms slower HO forms faster HO OH O
Figure 1.4 Relation between stability of epimeric azasugars and the reactivity of glycoside hydrolysis. Arrows showing the difference in charge-dipol interaction with respect to stereochemistry at C-4.
In addition, Jensen and Bols have synthesised model compounds 120 and 121, which were exposed to acidic hydrolysis (Figure 1.5).148 Their results showed that 121 was hydrolysed 1.6 times faster than 120 and therefore steric effects cannot be the controlling factor of the rate of hydrolysis, because then one would expect 120 to be hydrolysed faster than 121.
68
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
OH O HO HO OCH3 HO HO OCH3 OH O
120
krel
1
121
1.6
Namchuk et al. have suggested that during hydrolysis of glycosides 97 % of the charge is positioned at the endocyclic oxygen and that through-space field effects control the rate of hydrolysis.151 This theory contradicts with Jensen et al., who argues that if 97 % of the charge is positioned at the endocyclic oxygen, one would expect the rate of hydrolysis of 2-deoxyxyloglycosides and 4-deoxyxyloglycosides to be approximately the same because of a high degree of symmetry of the transition states.147 However, the rate of hydrolysis of 2-deoxyxyloglycosides is approximately 50 times higher than that of the 4-deoxyxyloglycosides.142 In addition, good correlation is observed in Hammett plots of stereochemical substituent constants and -glucoside hydrolysis rates whether 100 % charge is placed on the ring oxygen or the anomeric carbon.150 Therefore, it is reasonable to conclude that considerable development of positive charge must be present on both the ring oxygen and the anomeric carbon in the transition state.
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
SN1-type mechanism, an oxocarbenium ion intermediate, similar to the one generated in the A-1 mechanism for acid-catalysed glycoside hydrolysis, will be generated (Scheme 1.3).
OBn BnO BnO O BnO LG
activating reagent rds
OBn O BnO
Nu -H+
Scheme 1.3 SN1-type mechanism for glycosidation of a benzyl protected glucosyl donor.
Assuming a similar substituent effect for OBn as for OH and that fission of the C-1-LG bond to generate the oxocarbenium ion intermediate is the rate-determining step, the expectation according to the stereochemical substituent effect, would be that a galactosyl donor (axial 4OBn) would react faster than a glucosyl donor (equatorial OBn). If the positive charge is present exclusively on the ring-oxygen, one would expect a difference in reactivity between the galactosyl- and glucosyl donor to be 6-7 times, whereas fully charge on the anomeric carbon would result in a rate difference of 2-3 times. These values are based on the difference in pKa values of piperidinium ions having an axial or equatorial substituent in or position relative to the positive charge (see Figure 1.3). On the other hand, if a glycosidation reaction happens through an SN2-type mechanism, one would expect that almost no difference in the rate of glucosidation versus galactosidation would be observed. The suggestion is based on the absence of accumulation of charge at the anomeric position in the transition state and therefore the substituent effect would be negligible (Scheme 1.4). However, it is still possible that a small effect would arise from electrostatic destabilisation of the galactosyl donor ground state as explained by Miljkovi.146
OBn BnO BnO O BnO LG
Nu
-
OBn
-LG
-
Nu
BnO BnO
O BnO
Nu
BnO LG
Scheme 1.4 SN2-type mechanism of glycosidation for a benzyl protected glucosyl donor.
Differences in the reactivity of glycosyl donors have been observed for donors bearing different protecting groups. With the observation that acetyl protected n-pentenyl glucosides hydrolyse slower than the corresponding benzyl protected compound, Fraser-Reid and co-workers introduced the concept of armed-disarmed glycosides.156 With respect to 70
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
glycosidation, this can be useful since two glycosyl donors bearing different protecting groups have different reactivity. The concept is based on a difference in electron-withdrawing power of various protecting groups e.g. ester protected glycosides are more disarmed because of the greater electron-withdrawing abilities of an ester functionality compared to an ether protected glycoside (armed). As seen in Scheme 1.5, no self-coupling product (disarmeddisarmed) was observed on reaction of the armed benzyl protected n-pentenyl glucoside 122 with the disarmed acetyl protected donor 123.157
OBn OBn BnO BnO O BnO OPent + AcO AcO OH O AcO OPent
IDPC CH2Cl2, 62 %
BnO BnO
122, armed
123, disarmed
The concept of armed-disarmed effects paved the way for tuning the reactivity of glycosides resulting in oligosaccharide syntheses without the need for tedious protection group manipulations.158,159 Wong and co-workers developed a method for facile one-pot oligosaccharide synthesis.159 Their method was based on a thorough study of reactivities of p-methylphenyl thioglycosides towards glycosidation with MeOH and NIS/TfOH activation. By making competition experiments between a given thioglucoside donor and a reference compound, they obtained a large table of relative reactivities and by constructing a database from these data, they were able to predict a one-pot synthesis of oligosaccharides starting from the most reactive donor decreasing the reactivity on going towards the reducing end of the oligosaccharide. In that way they were able to construct complex oligosaccharides in an easy and one-pot manner, without protection group manipulation as seen in Scheme 1.6.
71
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
BzO BzO
OH O OBz
(13.1) NIS
BnO BnO
(1.7x104)
BnO BnO
Scheme 1.6 One-pot synthesis of a tetrasaccharide. Relative reactivities are given in parentheses.
What was even more interesting for the present project, was the table of relative reactivities. By comparing pairs of C-4 epimeric thioglycosides bearing the same pattern of protection groups, it was seen that the rate of glycosidation was higher when the C-4 substituent was axial compared to a C-4 equatorial substituent (Figure 1.6). With a ratio of the relative reactivity values between C-4 epimers ranging from 4.5 to 6.5, it is obvious that the stereochemistry at C-4 influence the rate of glycosidation, which might be explained by an SN1-type mechanism as described above.
OAc gluco series: AcO AcO O AcO (2.7) STol BzO BzO OBz O BzO (1.3) STol BnO BnO OBn O BnO (2656) STol
BzO BzO
BnO BnO
(1.7x104)
Figure 1.6 Examples of relative reactivity values of protected p-methylphenyl thioglycosides are given in parentheses.
In another study conducted by Lahmann and Oscarson, the varying reactivity of thioglycosides donors with respect to the aglycon was investigated.160 By performing 72
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
DMTST-promoted thioglycoside couplings, they found that the rate of glycosidation of the donors increased with the electron-donating abilities of the aglycon. In addition, they also observed that galactosyl donors were three to seven times more reactive than the corresponding glucosyl derivative. Gervay and Hadd have also observed a difference in the reactivity of C-4 epimeric glycosyl donors.161 The reaction of 2,3,4,6-tetra-O-benzyl--Dgalactosyl iodide with sodium diethyl malonate was completed in 1h, whereas a reaction time of 5h was necessary for the reaction to complete in case of the corresponding glucosyl iodide. Again this suggests that the galactosyl donor is more reactive than the glucosyl donor.
OR RO RO O OH RO
Base
OR RO RO O RO O
CCl3CN
OR RO RO O O RO
CCl3CN
OR O O RO NH CCl3
Kinetic product
Scheme 1.7 Mechanism for formation of thermodynamic versus kinetic product formation of trichloroacetimidate donors.
73
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
On addition of base, the -alkoxide is form initially, which upon addition with trichloroacetonitrile, produces the -trichloroacetimidate product. But since this is a reversible reaction, the thermodynamically more stable -product will eventually form if strong base is used. Traditionally, NaH is used for obtaining the -anomer, whereas K2CO3 is employed for generation of excess -O-glycosyl trichloroacetimidate.166 In the glycosidation step, trichloroacetimidate donors are activated with an acidic catalyst. Regarding the mechanism of reaction, it is believed to be largely dependent on several factors (Scheme 1.8). In nonpolar solvents using a nonparticipating protected donor, SN2-type reactions are often suggested, typically using BF3Et2O as catalyst. By employing more polar solvents and strong acidic catalysts (e.g. TfOH), an SN1-type pathway is believed to be followed and thereby formation of the thermodynamically more stable -glycoside. However, under SN1-type conditions, the influence of the solvent is important for the outcome of the reaction. If nitriles are chosen as of solvent, -nitrilium-nitrile conjugates are formed, resulting in -glycosidation, because of screening of the -face by the solvent.167,168 On the other hand, participation of ethers directs generation of equatorial oxonium ions, which for thermodynamic reasons favour formation of the -product. This is suggested to be due to the reverse anomeric effect.169
OR RO RO O RO
nonpolar solvents SN2-type
OR + LG
polar solvents SN1-type
RO RO
O RO
LG
O RO
S S
A-H, S =Et2O
LGC-
-product
LGH + C slow OR O RO N N
= CH3CN
74
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
Among other things, the stability of O-glycosyl trichloroacetimidates and the possibility of using mild reaction conditions have made them popular glycosyl donors and they have been employed numerous times in glycosidation reactions.170 Both for synthesis of simple disaccharides and for more complicated purposed as synthesis of glycosyl phosphatidyl inositol anchors that possess great importance in anchoring proteins and glycoproteins to membranes.171
75
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
As described in section 1.3.1, 1-O-glycosyl trichloroacetimidates are synthesised from protected glucopyranosyl-1-O-trichloroacetimidate (126) perbenzylated -D-glucose (125) was employed as starting material. As described in literature, K2CO3 resulted in an excess of the -anomer 126b (/ 1:6.6), but only 34 % overall yield.172 Even though DBU is known to be unselective, the best results were obtained using this base.173 After stirring at -20C for 4 hours both anomers were formed in a yield of 85 % favouring the -anomer 126a (/ 5.5:1) (Scheme 2.1).
OBn BnO BnO O BnO OH
CCl3CN, DBU CH2Cl2, -20oC, 4h
125
126a, 72 %
126b, 13 %
Scheme 2.1 Synthesis of 2,3,4,6-tetra-O-benzyl-/-D-glucopyranosyl-1-O-trichloroacetimidates 126a and 126b using DBU as base.
76
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
2,3,4,6-tetra-O-benzyl--D-galactopyranosyl-1-O-trichloroacetimidate (130) was synthesised in a similar manner starting from methyl -D-galactopyranoside (127). First, the galactopyranoside was protected by perbenzylation of the free hydroxyl groups using NaH and BnBr in DMF to give 128 (Scheme 2.2). This was followed by hydrolysis of the acetal to generate the hemiacetal 129, though in a rather low yield.174,175 Even though temperature, reaction time, acid concentration, and acid were varied, no increase in the yield was seen and it was decided to continue without further optimisation. Again the trichloroacetimidate 130 was obtained using DBU as base (Scheme 2.2). No -anomer was observed.
OH HO HO OCH3 OH O
NaH, BnBr DMF, rt, 20h 97 %
BnO BnO
BnO BnO
OBn O BnO OH
CCl3CN, DBU CH2Cl2, -20oC, 2h 75 %
BnO BnO
127
128
129
130
77
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
BnO
BnO BnO
OBn O BnO R2 R1
Scheme 2.4 Competition reactions were performed according to the above scheme using 1-octanol as limiting species.
Equi-molar amounts of glucosyl donor 126a and galactosyl donor 130 were dissolved in the given solvent and 1-octanol added. After cooling to 0C, the activator was added and the reaction mixture stirred for 1h before work up. After removing excess glycosyl donors by filtration on silica gel, the relative ratio of formation of octyl glucoside 131 versus octyl galactoside 132 was determined. For the first reactions, the glucoside/galactoside ratio was determined both by HPLC and
13 13
C-NMR. In the
C-NMR spectrum, the chemical shifts for C-1 for the 4 octyl glycosides were clearly
distinguishable showing peaks at 97.0 ppm for the -glucoside 131a, 97.6 ppm for the -galactoside 132a, 103.8 ppm for the -glucoside 131b, and 104.1 ppm for the -galactoside 132b. Determination of the ratio of galactoside versus glucoside was based on the intensity of the C-1 signals, which is reasonable because of the great resemblance of the products. Results from the competition reactions are shown in Table 2.1.
78
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
Entry 1 2
a
Catalyst/promoter BF3Et2O BF3Et2O BF3Et2O BF3Et2O TfOH TfOH TMSOTf TMSOTf AgOTf BF3Et2O/ AgOTf CsOTf BF3Et2O/ CsOTf TfOH
Solvent CH2Cl2 CH2Cl2 CH3CN Et2O CH2Cl2 CH3CN CH2Cl2 CH3CN CH3CN CH3CN CH3CN CH3CN CH2Cl2
Equiv. catalyst 0.14 0.14 0.14 0.14 0.1 0.1 0.17 0.17 2.9 0.14 + 0.4 2 0.14 + 2c 0.1
b
Gal(132)/Glc(131) 2:1 5:1 5:1 3:1 1:1 1:1 1:1 1:1 1:1 2:1 3:2 5:1
: 1:5 0:1 0:1 0:1 0:1 1:4 1:5 1:3 1:4 0:1 0:1 0:1
3 4 5 6 7 8 9 10 11 12 13
d
Table 2.1 Results of competition experiments between glucosyl and galactosyl imidates, 126a and 130, with 1-octanol. Unless otherwise noted, the experiment was with 1 equivalent of 126a, 130 and alcohol at 0C. a 2 equivalents of 126a and 130 were used. b 0.4 equivalents of AgOTf. c 2 equivalents of CsOTf. d At -78C.
A general observation was seen for all competition experiments, namely that octyl glycosides 131 and 132 were obtained primarily as -anomers no matter what catalyst or solvent was used. The anomeric ratio indicates that inversion has happened to a great extent on the anomeric center, suggesting an SN2-type mechanism. But inversion does not necessarily exclude an SN1-type mechanism. A possible explanation could be that formation of a nonsolvent-equilibrated oxocarbenium ion shields for attack from the -face resulting in glycosidation, i.e. the nucleophilic attack occurs prior to the complete dissociation of the intermediate ion:molecule pair. This has been reported for hydrolysis of glycopyranosides containing neutral leaving groups.178 From entry 1, it is seen that a 2:1 ratio favouring the galactoside 132 is obtained when BF3Et2O was the catalyst in CH2Cl2 and one equivalent of each donor is used. This indicated that the rate of formation of galactoside/glucoside must be higher than 2:1, because 130 becomes depleted when only one equivalent of each donor is employed and therefore appreciable amounts of the glucoside is also obtained. To obtain a ratio that can describe the true difference in rate constants between the two reactions, more equivalents of each donor has to be employed. Therefore, according to entry 2, an experiment where 2 equivalents of 79
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
each donor were employed was performed resulting in a 5:1 ratio. These findings suggest that considerable charge is built up on the endocyclic oxygen and that the reaction has SN1-character. Entries 3 and 4 were made to see the effect of changing the solvent. Using a more polar solvent CH3CN, the reaction was expected to proceed through an SN1-type mechanism. Because of its higher polarity when compared to CH2Cl2, and thereby higher tendency to stabilise a transition state having positive charge, even more excess of the galactoside product was expected. Indeed this was observed. Here according to the nitrile effect, no -glycosides were observed. By using Et2O, generation of the -anomers was expected. Et2O is known to participate by generation of equatorial oxonium ions to the anomeric carbon. This oxonium ion is more stable from the -face, owing to the reverse anomeric effect179, and therefore attack is more likely to come from the -face. However, this is not observed and kinetic effects must control the reaction. Nevertheless, the reactivity of the galactosyl donor was higher than of the glucosyl donor. It was decided to try an alternative activator and triflic acid was chosen (entries 5 and 6). Here a dramatic and surprising effect was observed. No matter what solvent was used a 1:1 galacto/gluco ratio was seen, while the stereochemistry was unchanged still favouring the -anomer. Further experiments were conducted using other triflate containing activators in form of TMSOTf and AgOTf (entries 7 9). Since they all resulted in a 1:1 product ratio, it appeared that the effect was associated with the presence of triflate ions. To check that, experiments catalysed by BF3Et2O in the presence of triflates in form of AgOTf and CsOTf were made (entries 10 and 12, CsOTf does not catalyse the reaction, entry 11). For both reactions the outcome of the competition reaction was altered compared to addition of BF3Et2O alone (entry 3), with a decreased formation of galactoside 132 compared to glucoside 131. The difference in reactivity between BF3 and triflate catalysed glycosidation reactions might be explained by generation of glycosyl triflate intermediates, when triflate containing catalysts are employed. If the presence of a triflate increases the rate of reaction, the differences in reactivity of the two donors will not be observable. This will result in a 1:1 relationship between the products in the competition experiments. To test this hypothesis, a TfOH-catalysed competition experiment was carried out at 78 C (entry 13). By decreasing the temperature a product ratio of 5:1 was obtained favouring the galactoside 132. This suggest that a positively charged intermediate is also formed in the triflate catalysed reactions 80
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
and that decreasing the temperature to 78 C will slow down the reaction to a degree where rate differences are observable. The existence of triflate intermediates have indeed been observed for glycosidation using glycosyl sulfoxides as donors.180 Crich and Sun observed that the anomeric stereoselectivity on coupling sulfoxide donor 133 to the acceptor 134 in the presence of triflic anhydride and DTBMP was reversed if the order of addition of reactants was reversed (Scheme 2.5).
OTBDMS O O AcO AcO O Ph O O BnO OTBDMS O O AcO AcO O AcO OCH3
136
Ph
O O BnO
OTBDMS O + O
133
Ph
O O BnO
Et
AcO OCH3
135
A: 133 + 134 + DTBMP, then Tf2O, 135:136 = 6:58 B: 133 + DTBMP + Tf2O, then 134, 135:136 = 85:8
They suggested that Tf2O serves to activate the donor, generating a sulfonium salt, which immediately collapses to an oxocarbenium ion intermediate. If route A (see Scheme 2.5) is followed, the activation is carried out in the presence of the acceptor and the oxocarbenium ion will be trapped directly generating the -mannoside 136, since this is favoured in Et2O especially for a mannoside. On the other hand, if the mannosyl donor 133 is activated prior to addition of the acceptor, the oxocarbenium ion is trapped by a triflate anion resulting in an -mannosyl triflate (route B, Scheme 2.5). On addition of the glycosyl acceptor 134, they suggested that the triflate intermediate participates in an SN2-like reaction forming the -mannoside 135. From a synthetic point of view this is interesting, since -mannosidic bonds are often difficult to obtain. The effect of changing the glycosyl acceptor to a more complex carbohydrate alcohol was investigated by performing a competition reaction using carbohydrate alcohol 137 as acceptor instead of 1-octanol. When catalysing the reaction by TMSOTf, disaccharide 138 and 139 were formed in a 1:1 ratio (Scheme 2.6).VI
VI
Experiments on the triflate effect in disaccharide synthesis were performed by master student Tine Meyer.
81
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
OBn OH BnO BnO O BnO OCH3
126a (1 equiv.) 130 (1 equiv.)
TMSOTf (0.17 equiv.) CH3CN, 0oC
BnO BnO
OBn O
BnO BnO
O O BnO OCH3
137
138, : 1:19
139, : 1:5
60 %, ratio 1:1
Scheme 2.6 Competition reaction with trichloroacetimidates and methyl 2,3,4-tri-O-benzyl--Dglucopyranoside (137).
C-NMR spectrum at 101.5 ppm and 103.3 ppm, respectively. From the corresponding 2-deoxy-2-iodo--D-talopyranoside (102.3 ppm), 2-deoxy-2-iodo--D-
reaction of the perbenzylated galactal 141, the outcome of the reaction was 143 (the perbenzylated
galactopyranoside (100.0 ppm), and 2-deoxy-2-iodo--D-galactopyranoside (103.9 ppm). Thus, the products from the competition experiment were distinguishable. The competition reaction was performed according to Scheme 2.7. Catalysing the reaction with Ph3PHBr was done in a similar way and the outcome of the reaction was the perbenzylated 2-deoxy glycosides (gluco (144): C-1 97.5 ppm and 100.0 ppm; galacto (145): C-1 97.8 ppm and 100.5 ppm). Results from the competition reactions with 1-octanol are shown in Table 2.2.
82
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
OBn BnO BnO OBn BnO BnO O + OBn O BnO 141
ol ctan , 1-o rt NIS 3, MS CN, CH 3 Ph 3P HBr , 1-o CH ctan 2 Cl ol 2 , rt
O I 142
BnO
140
BnO BnO
Scheme 2.7 Competition reactions of tri-O-benzyl-glucal (140) and tri-O-benzyl-galactal (141) with 1-octanol using NIS or Ph3PHBr as promoters.
Yield 82 % 87 %
Table 2.2 Results from competition reactions using glycals as glycosyl donors.
Both competition experiments showed that the reactivity of galactal 141 was somewhat higher than that of the glucal 140. This is in accordance with having positive charge in the transition state, but the effect was not as distinct as seen for the trichloroacetimidates.
VII
The experiments on n-pentenyl glycosides were performed by fellow student Tomasz Krzysztof Olszewski.
83
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
OBn gluco BnO BnO O O BnO 146 BnO galacto BnO BnO 147 OBn O O
1-octanol NIS, Et3SiOTf CH2Cl2, 22oC 1-octanol NIS, Et3SiOTf CH2Cl2, 22oC
fuco BnO
O OBn 148
O OBn
O BnO OBn
OOct OBn
Scheme 2.8 Reactions of n-pentenyl glycosides with 1-octanol in pseudo-first order reactions. Rate constants were determined by 1H-NMR studies.
As expected n-pentenyl fucoside 148 reacted faster than the galactoside 147, which reacted faster than the glucoside 146. The ratio of relative reactivity differences was determined to be 1:1.3:3 (glc/gal/fuc), which is relatively small compared to previous results of acid catalysed hydrolysis of methyl glucosides, galactosides, and fucosides determined to be 1:5:30.143 In Wongs study of thioglycoside reactivity, they also obtained a larger reactivity difference between benzyl protected glucosyl, galactosyl, and fucosyl donors (1:6.4:27).159 The relatively low difference in reactivity observed for n-pentenyl glycosides 146-148 can be caused by the activation method using NIS/Et3SiOTf. This is in accordance with the observation that relatively small differences in the rate of reactivity of armed-disarmed n-pentenyl glycosides was observed by Fraser-Reid et al., when n-pentenyl glycosides were activated with NIS/Et3SiOTf.157
84
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
was only soluble in CH3CN. Reactions were performed according to Scheme 2.9 yielding the -glycosyl xanthates 152 and 153.
Cl
BnO
BnO
Because of a relative slow reaction rate it was decided to perform the reactions separately in NMR tubes in CD3CN and follow the rate of reaction by recording 1H-NMR spectra at different times. By adding excess of potassium xanthogenate (4-5 equivalents), the decay of glycosyl chloride was measured and by assuming a pseudo-first order reaction, the recorded data were fitted to the general relationship [A] = [A]oe-kt. From plots of concentration versus time (Figure 2.1), the rate constants were determined to 1.610-4 s-1 for the glucosyl chloride and 6.710-4 s-1 for the galactosyl chloride.
D e term in a tio n of ra te co n sta n ts, ga lacto syl ch lo rid e + xan th a te 60
60 D ete rm in a tio n of rate co n sta n ts, glu cosyl d o n or + xa n th a te
y= a e xp (-kx), r =0 .9 6 7 a = 4 5.1 , k= 1 .5 7 E -4 a = 1 .3 2 , k =1 .3 5E -5
20
20
1000
2000 t/s
3000
40 0 0
50 0 0 t/s
10000
1 5 00 0
Figure 2.1 Determination of rate constants by 1H-NMR experiments for reaction of 150 and 151 with excess potassium xanthogenate.
However, the results were surprising, since the rate constants were expected to be similar and not to differ by a factor of 4 as observed. In addition, similar experiments were performed
85
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
using sodium butyl thiolate as nucleophile.VIII The experiments suggested that the galactosyl chloride reacted faster than the glucosyl chloride, but since the elimination products were observed as side products, it was not possible to determine the rate constants. Even though both pairs of experiments were suggested to happen though an SN2-pathway, it is evident that a substituent effect from the orientation of the C-4 substituent affects the rate of reaction. This suggests that positive charge accumulate in the transition state and that even reactions expected to be SN2-like, can have considerable SN1 character. Having the glycosyl donors 150 and 151 in hand, a direct competition experiment with 1-octanol using AgOTf (3.1 equiv.) as promoter in CH2Cl2 at room temperature was performed in a similar way as described for the trichloroacetimidates. By using 1.2 equivalent of each donor competing for 1 equivalent 1-octanol a 1:1 ratio of galacto (132) versus gluco (131) product was seen (Scheme 2.10) this time yielding an equal ratio of - versus -anomers.
OBn 150 + 151
1-octanol AgOTf CH2Cl2, 0oC 97 %
BnO BnO
O BnO
131, / 1:1
glc/gal 1:1
132, / 1:1
Scheme 2.10 Competition reaction between glycosyl chlorides using AgOTf as promoter.
The result of the competition reaction is not that surprising, bearing the previous results on activation of trichloroacetimidates with triflates in mind.
VIII
Experiments employing sodium butyl thiolate were performed by master student Tine Meyer.
86
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
When trichloroacetimidates are activated by BF3, generation of the oxocarbenium ion (step 2, Scheme 2.11a) must as expected be rate-determining and since the oxocarbenium ion is formed faster for the galacto species, the rate of reaction is higher than for the corresponding gluco species. This is believed to be caused by stereoelectronic differences between electronwithdrawing axial and equatorial C-4 substituents. The same explanation is suggested to be the cause of different reactivities among thioglycoside donors activated by NIS/TfOH in the study performed by Wong and co-workers.159 Even though the substitution experiments on glycosyl chlorides using potassium xanthogenate as nucleophile were believed to be SN2 processes, it is evident from the rate constants, that the transition state of the reaction must have considerable carbocation character. Thereby, the electronic effects arising from having an axial or equatorial electron-withdrawing group in the C-4 position also affect the rate constants. To obtain a 1:1 or close to 1:1 relationship in the competition experiments, generation of the oxocarbenium ion cannot be rate-determining and a shift in the rate-determining step must occur. If step 1 or 3 in Scheme 2.11a is rate-determining, it is likely that a 1:1 ratio of the overall glycosidation reaction is obtained, since the activation process and the reaction of the nucleophile with the oxocarbenium ion is probably not affected by the orientation of the C-4 substituent. The observation of 1:1 relationships between galactose and glucose configured products when triflates are used to activate the glycosyl donors, might be explained by the first step in Scheme 2.11a becoming rate-determining. It is likely to be caused by an increase of the rate of step 2, resulting in the first step becoming rate-determining. This increased reactivity can be explained by generation of reactive glycosyl triflate intermediates. When the temperature is lowered to 78C and a 5:1 gal/glc relationship is obtained. It might indicate that generation of the oxocarbenium ion (step 2, Scheme 2.11a) is slowed down and becomes rate-determining again. Reaction of n-pentenyl glycosides are believed to happen through similar mechanism except that it involves two preequilibration steps as shown in Scheme 2.11b. First, the pentenyl double bond is activated, whereupon the cationic intermediate is trapped in an intramolecular fashion by the anomeric oxygen to form a tetrahydrofuranyl oxonium ion. The oxocarbenium ion is then generated from this species and can react with the nucleophile. Since only small electronic effects are observed on activation of n-pentenyl glycosides with NIS/Et3SiOTf, it is suggested that one of the preequilibration steps is rate-determining. This might be the 87
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
generation of the tetrahydrofuranyl oxonium ion, since a positive charge in the vicinity of the anomeric center might to some extent be affected by the electron-withdrawing properties of the C-4 substituents, resulting in a small difference in reactivity.
O a) E + X O XE O Products
O b) E + E O O O
O O E
O Products
O c) I +
O Products I
O d) H +
O Products H
The reaction of glycals probably takes place via direct formation of an iodonium ion or oxocarbenium ion depending on the method of activation. On activation with NIS the low gal/glc ratio can be explained by a smaller electronic effect from the C-4 substituent because of the remoteness of the positive charge in the iodonium ion intermediate in the same way as suggested for the n-pentenyl glycosides (Scheme 2.11c). An identical ratio is obtained on activation of glycals with Ph3PHBr (Scheme 2.11d). Here the relatively small ratio cannot be explained by a remote positive charge. An explanation to the small difference in reactivity on activation with Ph3PHBr, can be that generation of the positively charged intermediate is fast and therefore, it is not possible to observe any difference in reactivity. Another reason could be that the mechanism is not as straight forward as described in Scheme 2.11d.
3 Conclusions
The effect on glycosidation reactions of having an equatorial or axial electron-withdrawing substituent in the C-4 position on various glycosyl donors has been examined. The results show that galactosyl donors react faster than glucosyl donors when activated with non-triflate 88
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
promoters, which imply that formation of the oxocarbenium is the rate-determining step of the reaction and that the reaction has considerable SN1-character. On the other hand, when activation is carried out with triflate activators, an equal reactivity of donors is observed in competition experiments, suggesting a change in the rate-determining step of the reaction. This could be obtained via an increase in the rate of leaving group departure possibly caused by a stable glycosyl triflate ion pair. In addition, attempts were made to show that the reaction of glycosyl halides with a strong nucleophile would react via an SN2 pathway resulting in equal formation of products, because of charge dispersion in the transition state. However, an increased reactivity of the galactosyl donor compared to the glycosyl donor was observed, suggesting that even a supposed SN2 reaction must be affected by the electron-withdrawing properties of the C-4 substituent. Therefore, it must be associated with built up of positive charge in the transition state and have considerable SN1 character. Altogether the experiments show that glycosidation reactions are complex and that in order to benefit from armed/disarmed effects in glycoside synthesis, reaction conditions must be carefully chosen.
89
Chapter II: Competition Reactions between Glucosyl Donors and Galactosyl Donors A Study of Glycosidation Reactions
90
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase 1 Introduction
OH N HO OH
OH NH HO HO
OH NH NH
Figure 1.1 Examples of biological active azasugars. Ki values obtained from ref 190 and 191.
91
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
O H OH HO HO O HO O O O
O OH HO HO
O H O R
+ O + HO O O
O OH HO HO O HO H O O
O H OH HO HO O HO O O OH
O OH HO HO
O H O H
O O
+ O + HO O O
Two carboxylates in the protein, being 5.5 apart, is directly involved in the cleavage. The upper carboxylic acid is acting as a general acid activating the aglycon, which upon attack of the nucleophilic carboxylate at the anomeric center, leaves the glycon. This is believed to happen through an oxocarbenium ion like transition state, which leads to formation of an enzyme-glycosyl intermediate. In the second step of the mechanism, the deprotonated upper carboxylate act as a general base activating a water molecule. This water molecule then attacks the anomeric center cleaving the enzyme-glycosyl intermediate resulting in overall hydrolysis of the glycosidic bond with retention of configuration. Both transition states are believed to have considerable oxocarbenium ion character. A similar mechanism is believed
92
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
to exist for retaining -glycosidases, but in this case the nucleophilic carboxylate is placed above the glycosidic bond to be cleaved.195 The position of the nucleophilic carboxylate above the sugar moiety in -glycosidases, has been used to explain the reason why 1-deoxynojirimycin (156) is more potent against -glucosidase inhibitor, whereas -glucosidase is less affected by the presence of the inhibitor (Figure 1.1).196 On the other hand, moving the nitrogen to the anomeric position as in isofagomine (157) creates potent -glucosidase inhibitors with poorer affinity towards -glucosidases. This is also in accordance with results published by Bols et al. which show that azafagomine (158), having a nitrogen in both positions, binds to both - and -glucosidases with high affinity.197
k1S k2 konI
ES
k3
E +
93
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
On the other hand, if formation of the EI-complex is fast, but followed by a slow isomerisation to a locked complex EI* (kcf being the rate of conformational change) the inhibition follows model 2 (Scheme 1.2).
E koff EI konI
k1S k2
ES
k3
E +
kcf k-cf
EI*
The last model is described by a necessary slow conformational change of the enzyme before binding of the inhibitor in a fast step (Scheme 1.3).
E k-cf E
*
ES
k3
E + P
EI*
Until recently it has been assumed that the most common mechanism for slow inhibition was model 2.199 But several more recent studies have suggested that the more simple model 1 involving one slow step is also quite common.200-202 This was also observed in a study on slow binding of isofagomine (157), azafagomine (158) and some analogues to almond -glucosidase and yeast isomaltase performed in our group.203 In addition, it was observed that association of inhibitor and enzyme (kon) was relatively slow compared a to a diffusioncontrolled rate. Dissociation of enzyme and inhibitor (koff) also turned out to be slow and almost constant for the different inhibitors investigated. In that way, the differences in Ki values (Ki = koff/kon) among the inhibitors were entirely dependent on the rate of association, kon. In order to try to explain these observations, it was decided to take a closer look at the
94
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
slow binding of a range of azasugars to almond -glucosidase at different temperatures to be able to evaluate the thermodynamic parameters for the enzyme-inhibitor process.
(1)
A, , , and C are dependent on the given model followed by the inhibition process as
Model 2
Vmax s 1+ s + i
Model 3
Vmax s 1+ s
k on
[I] 1+ s
k cf
i 1+ s + i
k on
[I] 1+ s
+ k off (ei) t =0
+ k -cf (ei) t =0
k cf k + cf 1+ s 1+ i (ei) t =0
[S]
KM
, i=
Ki
[I] ,
(ei) =
By fitting the kinetic progress curves to equation 1, the apparent rate constant , can be determined. From Table 1.1 it follows that if turns out to be a linear function of the inhibitor concentration, [I], (a -plot) model 1 can describe the enzyme-inhibitor interaction. In case of being a increasing hyperbolic function of [I], model 2 is followed and if is a decreasing hyperbolic function of [I] the interaction is described by model 3. If model 1 is followed, the rate constants kon and koff can easily be obtained from the -plot (slope and intercept, respectively) and the Ki value of the inhibitor of interest can be calculated by Ki = koff/kon.
95
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
where k is a rate constant, A is the pre-exponential factor, Ea the activation energy, R the gas constant, and T the temperature. In the case of reactions in solution this can be written as
k= G k bT exp RT h S k T = e b exp R h E exp a (3) RT
where kb is the Boltzmann constant and h is Plancks constant. If kon and koff for the reaction of a slow inhibitor with an enzyme are determined at various temperatures the activation parameters can be found from Arrhenius plots (ln k versus 1/T) and the enthalpy of activation, H, can be determined from the activation energy (Ea = H + RT), and S is calculated from the equation (3). Eventually, G can be found from the general relationship G = H - TS. From the activation parameters of association and dissociation, the standard thermodynamic parameters can be found from the equation P = Pon - Poff, where P denotes any thermodynamic parameter. If an inhibitor is not of the slow binding type, the thermodynamic parameters can be obtained by determination of the Ki values at different temperatures in a similar way from the vant Hoff equation (4): ln K i = H o S o + RT R (4)
H and S are determined from vant Hoff plots and G is subsequently calculated from G = -RT ln Ki.
96
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
Isofagomine, 157
Isogalactofagomine, 159
Azafagomine, 158
3-Amino-3-deoxy1-azafagomine, 160
Progress curves were obtained for each inhibitor at 8 different inhibitor concentrations by spectrophotometric monitoring of the enzymatic cleavage of 4-nitrophenyl--D-glucopyranoside by almond -glucosidase. An example of a set of progress curves is shown in Figure 2.2. From each progress curve the apparent rate constant , was obtained by fitting the
Progress curves 1.0
0.0075
-plot
0.8
0.0060
Absorbance
0.6
/(s )
-1
0.0045
0.4
0.0030
0.2
250 time/(s)
500
0.0015
0.4
0.8 I/(M)
1.2
1.6
Figure 2.2 Example of progress curves and -plot for inhibition of -glucosidase by isogalactofagomine (159) at 15C.
97
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
curves to equation 1 using the freeware programme INRATE in the software package Simfit (W.G. Bardsley, university of Manchester, UK). By plotting versus [I] straight lines were obtained (example in Figure 2.2), which showed that for all inhibitors model 1 described the slow binding interaction with the enzyme. From the -plots the rate constants for dissociation and association (koff and kon) of inhibitor and enzyme were determined and Ki could be calculated. By recording progress curves at 3-5 different temperatures for inhibitors 157-160, Ki, kon, and koff were determined and the thermodynamic parameters and activation energies were determined from Arrhenius plots as described in the introduction. 1-Deoxynojirimycin (156), turned out not to be a slow inhibitor of -glucosidase, thus only standard thermodynamic parameters were obtained. This was done by measuring Ki values at 5 different temperatures according to normal Michaelis-Menten kinetics. From the Ki values a vant Hoff plot was constructed and from that the thermodynamic parameters were deduced. All thermodynamic parameters are summarised in Table 2.1. For values of Ki, kon, and koff see tables in appendix 2.
G -37.6 -39.9 -38.2 -24.8 -26.1 Gon 48.4 46.2 44.6 58.4 Goff 86.0 86.1 82.8 83.2 H 58.6 58.7 -1.5 4.3 -25.7 Hon 56.1 56.2 39.3 49.4 Hoff -2.5 -2.5 40.8 45.1 S 323.8 331.0 123.1 97.3 1.3 Son 25.8 33.6 -17.9 -30.4 Soff -297.0 -297.4 -141.0 -127.7 -
156
Table 2.1 Thermodynamic activation parameters and standard parameters for binding of azasugars to almond -glucosidase at pH 6.8. G and H in kJ/mol, S in J/molK. * Inhibitor is racemic.
The thermodynamic parameters for isofagomines 157 and 159 were almost similar resulting in similar energy profiles (Figure 2.3). Binding of 157 and 159 to the enzyme to formation of the transition state (EI), was found to be associated with a large positive change in enthalpy and almost no change in entropy, thus it is an endothermic process. From the transition state to formation of the EI-complex a large change in entropy was seen and almost no change in enthalpy. Thus, the overall process is driven by entropy and therefore not favourable in terms of binding energies.
98
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
G
120 100
H
400
EI
EI
80
EI
EI
300
80
energy, kJ/mol
energy, kJ/mol
e.u., J/molK
E+I
60
200
E+I
40
40
100 E+I EI
20 EI
Figure 2.3 Energy diagrams showing the change in free energy (G), enthalpy (H), and entropy (S) on binding of isofagomine (157) to almond -glucosidase.
The corresponding thermodynamic data for azafagomines 158 and 160 were also found to be quite similar. From the energy profiles (Figure 2.4) it is seen that binding is associated with a positive change in enthalpy going from reactants to transition state, while almost no change in entropy is seen. Moving further along the reaction coordinate from the transition state to EI-complex a negative change in enthalpy is observed resulting in no appreciable change in the overall process. Again a large change in entropy was seen when going from the transition state to EI-complex and again the overall process is driven by entropy, but not with a smaller degree than observed for the isofagomines 157 and 159.
G
120 80
150 EI
EI
60 80
EI
energy, kJ/mol
energy, kJ/mol
40
E+I
40 E+I 20 EI
e.u., J/(molK)
100
50
E+I EI 0 0 0 EI
Figure 2.4 Energy diagrams showing the change in free energy (G), enthalpy (H), and entropy (S) on binding of azafagomine (158) to almond -glucosidase.
99
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
The thermodynamic data for interaction of 1-deoxynojirimycin (156) with -glucosidase was found to be much different from especially the isofagomines. Binding was now entirely driven by enthalpy and almost no change in entropy i.e. the binding process is now favourable in terms of binding energies. This is in accordance with the observation that binding of various oligosaccharides to lectins is often driven by negative enthalpy whereas the entropy seems to be less important.205-207 The only difference between isofagomine (157) and azafagomine (158) is that 158 has an additional ring nitrogen. Thus, this nitrogen must be the reason for the altered thermodynamic parameters, i.e. approximately 60 kJ/mol of enthalpy is gained on having an extra nitrogen in this ring. From this we suggested that the extra ring nitrogen must be involved in binding of the inhibitors through hydrogen bonds or an ionic interaction via a salt bridge to a carboxylate in the enzyme. It is also evident that the anomeric nitrogen present in both isofagomines and azafagomines does not contribute to binding through bond energy, but instead a large gain of entropy is paid of during the binding process. The large change in entropy on having an anomeric nitrogen is to some extent suggested to be caused by the release of highly ordered water molecules from the active site to bulk solution on binding of the inhibitor, generating a more disordered system. The most important difference between 156 and isofagomines and azafagomines is that 1-deoxynojirimycin (156) does not have a nitrogen in the anomeric position but has an additional hydroxyl group in the 2-position (carbohydrate numbering). Because of the absence of an anomeric nitrogen, no change in entropy is observed. Compared to the azafagomines, 156 has approximately 25 kJ/mol lower binding enthalpy, which mainly must be described to the presence of the 2-OH group capable of making hydrogen bonds to the enzyme. This is in accordance with previous studies of the importance of the 2-OH group, in which the interaction of the 2-hydroxyl group with the enzyme is believed to stabilise the transition state by 30-40 kJ/mol.208 From X-ray crystallographic studies it has been proposed that the 2-OH group makes a short, strong hydrogen bond to the nucleophilic carboxylate in the enzyme active site and thereby a stabilisation is obtained (Figure 2.5).209 In addition, kinetic studies on hydrolysis of other -D-glucosides (4-methylumbelliferyl--D-glucosides) by -glucosidase have shown that hydrolysis of the glucoside was considerably faster than hydrolysis of its 2-deoxy congener.210 These findings support the importance of having a 2-hydroxyl group present in a potent inhibitor. 100
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
O OH HO HO H O O + O O + H O R
Figure 2.5 Proposed stabilisation through hydrogen bond formation from the enzyme to a 2-hydroxyl group.
OH HO
Noeuromycin, 161
Galacto-noeuromycin, 162
Fuco-noeuromycin, 163
Figure 2.6 Structures of 2-hydroxyl analogues of isofagomine and its galacto- and fuco-isomers.
Biological testing was performed according to normal Michaelis-Menten kinetics and Ki values determined from Hanes plots. All three 2-hydroxyl analogues 161, 162, and 163 turned out to be extremely potent inhibitors of glycosidases with lower Ki values than their 2-deoxy analogues 157, 159, and 164 (Table 2.2).211,212-214
IX
The authors contribution to this work is only associated with the biological testing of the products.
101
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
OH HO HO 157 NH HO HO
OH NH HO 161
HO HO
OH NH 159
HO HO
OH
NH
NH HO 162
NH OH OH HO 163
OH HO 164
22 25 69 -
742 91 35 397 -
4000 6400l
4.7 3.2
Table 2.2 Inhibition constants Ki in nM for binding of inhibitors to various enzymes. a from yeast. b from almond. c from Green Coffee Bean. d from Saccharomyces fragilis. e from Aspargillus oryzae. f from E. coli. g from bovine kidney. h from human placenta. i From ref 111. j From ref 212. k inhibitor is racemic. From ref 214. l From ref 213.
It is seen that by introduction of a 2-hydroxy group in isofagomine to form 161, the affinity of the inhibitor to the enzyme increases by 2-4000 fold. With respect to the galactonoeuromycin (162) and fuco-noeuromycin (163) the inibition also increased significantly. In addition, the affinity of 161, 162, and 163 increase significantly more for -glycosidases compared their 2-deoxy analogues. Therefore, the anomeric nitrogen is presumably involved in forming an interaction with the enzyme possibly through a salt bridge. It is evident from the inhibition data that the 2-hydroxyl group indeed is involved in creating very strong binding glycosidase inhibitors.
2.2 Discrepancy
between
Thermodynamic
Results
of
Binding
of
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
different methods for determination of thermodynamic parameters that show different results. This type of contradiction between vant Hoff analysis and calorimetric data is not unique. Among others, discrepancies have also been observed for binding of ligands to Acyl-Coenzyme A Binding Protein.216 It was suggested that the discrepancies indicate that the binding reaction is more complex than a simple one-to-one binding model used to describe the data. Other researchers have performed experimental and simulation studies of enzyme-ligand binding that do not indicate any significant discrepancies between the calorimetric determined enthalpies and those deduced from vant Hoff plots.217 From this study they conclude that where such discrepancies are observed, they are likely to arise from either uncertainties due to extrapolation or inadequate calibration of the instrument. At present, the cause of the contradiction between our results and Zechel et al. remain unclear.
First we analysed the mode of binding according to the -method in the same way as described for other azasugars in section 2.1. The -plots turned out to be perfectly straight lines consistent with the inhibition process following model 1 (Scheme 1.1). We obtained Ki values of 14531 nM at 15 C and 3512 nM at 25C and not 1.2 nM as previously suggested. But the reliability of the method did not seem overwhelming (large standard deviations) and in addition simulation of the model using the rate constants from the 103
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
-method gave a poor fit of experimental data and the simulated curve (Figure 2.8). It turns out that the -method is not a good method for describing enzyme-inhibitor interaction when the rate constants turn out to be very small. Therefore, if a given slow binding inhibitor is also tight binding i.e. if the inhibitor has high affinity and the Ki value is comparable to the enzyme concentration, the -method is unreliable. This is due to the fact that koff is determined as an intercept in a graph and this becomes inaccurate when koff is too close to zero. We continued analysing the results by deviation of the differential equations describing model 1 (see appendix 3 for further details). The kinetic data were fitted to these differential equations using the program GEPASI.219,
X
regardless of the initial enzyme concentration, the rate constants were identical. It is therefore possible to use this method in experiments where the exact enzyme concentration is not known. In addition, when simulating a curve from the data obtained from the differential equation method (DE method), a perfect fit with the experimental data were obtained (Figure 2.8).
25 C
0.03
0.02
P, mM
0.01
200
400
600
800
1000
time, s
Figure 2.8 Comparison of two different fitting procedures. Data obtained at 25C, pH 6.8, and [I]=2.8 mM.
By using the DE-method a Ki value of 2.01.2 nM was obtained at 35 C, which is comparable to the inhibitor constant obtained by Panday et al.218
All computations using GEPASI were carried out by ass. prof. Igor W. Plesner.
104
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
By using the DE method kinetic data obtained at 4 different temperatures were fitted and rate constants obtained. In addition to the kon and koff rate constants, the substrate binding rate constant, k1, was also determined. Surprisingly, the magnitude of this constant was in the same order as kon (104 M-1s-1). This suggests that not only binding of inhibitors are slow, but association of enzyme and substrate is also a slow process. From values of kon, koff, and Ki (see appendix 3 for values of kon, koff, and Ki) thermodynamic parameters were determined as described earlier from Arrhenius plots (Table 2.3).
G On Off Standard 25C 46.9 94.1 -47.2 H 57.5 -2.5 60.0 S 35.7 -324 359.7
Table 2.3 Thermodynamic parameters for binding of 165 to -glucosidase. G and H in kJ/mol, S in J/molK.
The thermodynamic parameters are very similar to those presented for isofagomine resulting in almost similar energy profiles (Figure 2.3). It is highly surprising, since for isofagomines the large positive entropy was suggested to be associated with the presence of an anomeric nitrogen. Since the inhibitor 165 does not have a nitrogen in the same position as isofagomine, the positive change in enthalpy and entropy are therefore believed to be caused by the presence of the phenethylimidazole moiety. A possible interaction could be obtained between the imidazole moiety and a carboxylic acid in the enzyme.
105
Chapter III: Determination of Thermodynamic Parameters for Binding of Azasugars to Almond -Glucosidase
suggesting that slow association of enzyme-inhibitor is caused by resemblance with the substrate. By determining thermodynamic parameters for interaction between enzyme and inhibitors, it was shown that slow binding of 1-azasugars is caused by a large enthalpy barrier that has to be overcome before binding can occur. The overall process is driven by entropy, which seems to be associated with the presence of an anomeric nitrogen. Binding of 1-deoxynojirimycin, which is not a slow binding inhibitor, was also investigated. In contrast to the slow inhibitors, the binding process was driven by enthalpy, which was attributed to the existence of a favourable interaction between the 2-hydroxylgroup and the enzyme. This led to the design of three new potent inhibitors having 2-hydroxyl groups, which all turned out to be more potent than their 2-deoxy analogues.
106
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