The design and synthesis of novel beta-substituted amino acids, bicyclic dipeptide mimetics, and their incorporation into cholecystokinin/opioidchimeric peptides
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Authors Ndungu, John M.
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Link to Item http://hdl.handle.net/10150/290126 THE DESIGN AND SYNTHESIS OF NOVEL P SUBSTITUTED
AMINO ACIDS, BICYCLIC DIPEPTIDE MIMETICS, AND THEIR
INCORPORATION INTO CHOLECYSTOKININ/OPIOID
CHIMERIC PEPTIDES
by
John Maina Ndungu
A dissertation Submitted to the Faculty of the
DEPARTMENT OF CHEMISTRY
In Partial Fulfilment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2004 UMI Number: 3145110
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dissertation prepared by John Malna Ndungu
entitled Design and Synthesis of Novel beta-Substituted Amino Acids,
Blcycllc Dipeptide Mlmetics, and their Incorporation into
Cholecystoklnin/Oplold Chimeric Peptides
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy
Professor Vicyor J. Hruby date
Professor Robxn P date
Profe^or Eug,ene A. Mas
/Trof ofe^r John ,H. Enemark ^date
Professor S. Scott Saavedra date
recpfflr^nd th^ i^^ccepted as fulfillinj
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Brief quotations irom this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: 4
ACKNOWLEDGEMENTS
I would like to extend my utmost gratitudes to my research advisor, Regents Professor
Victor J. Hruby for his mentorship, guidance and advice in my research and in other aspects of life.
Special thanks go to all my committee members; Dr. Scott Saavedra, Dr. John Enemark,
Dr. Eugene Mash, Dr. Robin Polt and the late Dr. David F. O'Brien, for their scientific guidance, encouragement and support.
I would also like to thank all the members of the Hruby group. Dr. Scott Cowell, Dr.
Xuyuan Gu, Dr. Toru Okayama, Dr. Ruben Vardanyan, Dr. Chiyi Xiong, Dr. Zhanna
Zhilina, Dr. Yeon Sun Lee, Dr. Alexander Mayorov, Jinfa Ying, Vinod Kulkami, Byoung
Min, Hank Wooden, Minying Cai, Isabel Alves, Ravil Petrov, Jim Cain, and Magda
Stankova. Special thanks also go to Dustin E. Gross who contributed immensely at the start of my research. Special gratitudes also go Dr. Hruby's assistants, Cheryl McKinley and Margie Colie for their unselfish help, support and encouragement.
I also extend my gratitudes to Dr. Yamamura, Dr. Josephine Lai, and Dr. Frank Porreca laboratories for performing the bioassays and in particular. Peg Davis, Edita Navratilova,
Dr. Balaz Hargattai and Dr. Shou-wu Ma who ran the samples.
I am also indebted to my undergraduate advisor. Prof. Peter M. Gitu whose guidance was instrumental in my success as an undergraduate, and was also instrumental in securing me graduate studies in Arizona.
Thanks to the Department of Chemistry, the University of Arizona for offering me a chance to pursue graduate studies. I also acknowledge NIDA for supporting this research. DEDICATION
To my parents
Peter Ndungu Maina and Lydia Muthoni Ndungu
To my grandparents
Henry Maina Gathoni and Hannah Wanjira Maina
To my brothers
Samuel Kamanga Ndungu and Daniel Muigai Ndungu
For the sacrifices they have made, their love, encouragement and support.
Ashanteni nyote 6
TABLE OF CONTENTS Page
LIST OF FIGURES 10
LIST OF SCHEMES 12
LIST OF TABLES 13
ABSTRACT 14
CHAPTER 1. INTRODUCTION
1.1 Structure-activity relationships of opioids 15
1.1.1 Importance of N-terminal Tyr at position 1 16
1.1.2 Structure-activity relationship studies at position 2 16
1.1.3 Structure-activity relationship studies at position 3 and 4 16
1.1.4 Structure-activity relationship studies at position 5 17
1.1.5 Cyclic analogues of enkephalins 17
1.2 Cholecystokinin 18
1.2.1 Structure-activity relationship studies for cholecystokinin 19
1.2.2 Cyclic analogues of CCK peptides 21
1.3 CCK involvement in pain modulation 21
1.4 Overlapping pharmacophores of opioids and cholecystokinin 23
1.5 CCK/opioid chimeric peptides 23
1.6 Peptides 25
1.6.1 Peptide secondary structures 26
1.6.2 Peptidomimetics 27 7
TABLE OF CONTENTS - continued
CHAPTER 2. DESIGN AND RETROSYTHETIC ANALYSIS OF
BICYCLIC DIPEPTIDE MIMETICS
2.1 Introduction 31
2.2 Design and retrosynthetic analysis of bicyclic dipeptide mimetics 32
2.3 Synthesis of P-substituted y,6- and 5,s-unsaturated amino acids 34
2.3.1 Alkylation of aspartic acid 35
2.3.2 Kazmaier-Claisen rearrangement reaction 39
2.4. Experimental 43
CHAPTER 3. SYNTHESIS OF Nle-GIy BICYCLIC DIPEPTIDE
MIMETICS
3.1 Introduction 48
3.2 Synthesis of Nle-Gly bicyclic dipeptide mimetic from ^-substituted
aspartic acid 48
3.3 Synthesis of Nle-Gly bicyclic dipeptide mimetics via the Kazmaier-Claisen
rearrangement reaction 50
3.4 Characterization of Nle-Gly bicyclic dipeptide mimetics 51
3.5 Experimental 53
CHAPTER 4. SYNTHESIS OF Nle-Asp BICYCLIC DIPEPTIDE
MIMETICS
4.1 Introduction 59
4.2 Synthesis of analogues of proline 61 8
TABLE OF CONTENTS - continued
4.3 Synthesis of bicyclic dipeptide mimetics for Nle-Asp 64
4.4 Characterization of Nle-Asp bicyclic dipeptide mimetics 67
4.5 Synthesis of ^-thiol substituted aspartic acid 68
4.6 Experimental 72
CHAPTER 5. SYNTHESIS OF BICYCLIC DIPEPTIDE MIMETICS FOR
Asp-Gly AND homoPhe-Gly
5.1 Introduction 78
5.2 Synthesis of Asp-Gly bicyclic dipeptide mimetic 78
5.2.1 Characterization of Asp-Gly bicyclic dipeptide mimetics 81
5.3 Synthesis of a homophe-Gly bicyclic dipeptide mimetic 82
5.4 Experimental 85
CHAPTER 6. SYNTHESIS OF BICYCLIC DIPEPTIDE MIMETIC
CONTAINING CCK/OPIOID CHIMERIC PEPTIDES,
BIOLOGICAL ASSAY RESULTS, DISCUSSION AND
FUTURE WORK
6.1 Synthesis of peptides 92
6.2 Experimental 94
6.2.1 Synthesis and purification of peptides 94
6.2.2 Assay Methods 95
6.3 Biological evaluation at the CCK receptors 99
6.4 Biological evaluation at the opioid receptors 101 9
TABLE OF CONTENTS - continued
6.5 Discussion and conclusion 102
6.6 Future work 104
6.6.1 Structure activity relationsliip studies 104
6.6.2 Cyclized chimeric peptides 104
6.6.3 Synthesis of cyclized peptides using |3-allyl substituted aspartic acid and
ring closing metathesis 105
6.6.4 Synthesis of cyclized peptides using 6-allyl substituted proline analogues
and ring closing metathesis 106
Appendix A 108
Appendix B 109
Appendix C 110
REFERENCES Ill
Abbreviations 145 10
LIST OF FIGURES Page
Figure 1.1 General structure of amino acid and peptide 25
Figure 1.2 Definition of (j), xj/, co, dihedral angles 26
Figure 1.3 Different types of P-tum 27
Figure 1.4 External and internal p-tum mimetics 28
Figure 1.5 Examples of bicyclic P-tum mimetics 30
Figure 2.1 Retrosynthetic analysis of bicyclic P-tum dipeptide 31
Figure 2.2 Synthetic strategy for [5,5]- and [6,5]-bicyclic dipeptide mimetics 32
Figure 2.3 Design of indolizidinone bicyclic dipeptide mimetics 32
Figure 2.4 Bicyclic dipeptide mimetics for homoPhe-Gly and Nle-Gly 33
Figure 2.5 Retrosynthetic design for dipeptide mimetics 33
Figure 2.6 Bicyclic dipeptide mimetic for Nle-Asp dipeptide unit 33
Figure 2.7 Retrosynthetic analysis of a Nle-Asp bicyclic dipeptide mimetic 34
Figure 2.8 Examples of Y,5-unsaturated amino acids 35
Figure 2.9 Silyl ketene acetals 36
Figure 2.10 Transition state for alkylation of aspartic acid 37
Figure 2.11 Chelated enolate intermediate 37
Figure 2.12 Mechanism for the formation of ortho esters 39
Figure 2.13 Oxazole and Ireland-Claisen rearrangements 41
Figure 2.14 Transition state in Kazmaier-Claisen rearrangement 42
Figure 3.1 Nle-Asp bicyclic dipeptide mimetics 48
Figure 3.2 nOe data for the Nle-Gly bicyclic dipeptide mimetics 52 11
LIST OF FIGURES - continued Page
Figure 3.3 X-Ray crystal structure of a Nle-Gly bicyclic dipeptide mimetic 52
Figure 4.1 Bicyclic dipeptide mimetic for Nle-Asp dipeptide unit 59
Figure 4.2 Transition state model for the generation of proline analogues 63
Figure 4.3 X-ray crystal structure of 5-allyl substituted proline analogue 63
Figure 4.4 nOe analysis of 5-allyl substituted proline analogues 64
Figure 4.5 Optically active phosphines 65
Figure 4.6 Bicyclic dipeptide mimetic for Nle-Asp 67
Figure 4.7 nOe data for Nle-Asp bicyclic dipeptide mimetics 68
Figure 4.8 Retrosyntheitic analysis for a thiol BTD for Nle-Asp 68
Figure 4.9 Mechanism for the synthesis of disulfides 71
Figure 5.1 nOe observed for Asp-Gly bicyclic dipeptide mimetics 82
Figure 5.2 X-Ray crystal structure of homoPhe-Gly bicyclic dipeptide mimetic 83
Figure 6.1 Novel BTD containing peptides 93
Figure 6.2 Structures of JMNl and JMN2 104
Figure 6.3 Synthesis of cyclized peptides 106
Figure 6.4 Synthesis of highly constrained cyclic peptides 106 12
LIST OF SCHEMES Page
Scheme 2.1 Alkylation of aspartic acid with allyl bromide 38
Scheme 2.2 Alkylation of aspartic acid with benzyl bromide 38
Scheme 2,3 Alkylation of ortho ester protected aspartic acid 39
Scheme 3.1 Synthesis of Nle-Gly bicyclic dipeptide mimetic 49
Scheme 3.2 Hydrogenation and reduction of p-allyl substituted aspartic Acid 50
Scheme 3.3 Synthesis of Nle-Gly BTD via the Kazmaier-Claisen rearrangement
Reaction 51
Scheme 4.1 Synthesis of analogues of proline 60
Scheme 4.2 Synthesis of cw-dicarboxylate proline analogues 62
Scheme 4.3 Synthesiss of ?ra«5'-dicarboxylate proline analogues 63
Scheme 4.4 Synthesis of Nle-Asp bicyclic dipeptide mimetic 66
Scheme 4.5 Synthesis of Nle-Asp bicyclic dipeptide mimetic 67
Scheme 4.6 Sulfenylation of aspartic acid with benzyl disulfide 69
Scheme 4.7 Synthesis of Nyps protected aspartic acid 70
Scheme 5.1 Synthesis of trifluoroacetyl benzotriazole 79
Scheme 5.2 Synthesis of Asp-Gly bicyclic dipeptide mimetics 80
Scheme 5.3 Synthesis of Asp-Gly bicyclic dipeptide mimetic 80
Scheme 5.4 Synthesis of homoPhe-Gly bicyclic dipeptide mimetic 83
Scheme 5.5 Synthesis of analogues of Leu-enkephalin 84
Scheme 5.6 Bromination of p-benzyl homoserine 85
Scheme 6.1 Protocol for the synthesis of peptides 93 13
LIST OF TABLES page
Table 1.1 Gastrin-1 and Caerulein 18
Table 6.1 Biological evaluation at the CCK receptors 100
Table 6.2 Functional analysis at the opioid receptors 101
Table 6.3 Biological evaluation at the opioid receptors 102 14
ABSTRACT
Peptide ligands and protein receptors play critical roles in the regulation of nearly every biological system. However, peptides are characteristically highly flexible and thus identifying the basic conformational elements necessary for recognition between a peptide ligand and it's receptor at the molecular level remains a formidable task. Great emphasis in peptide research has thus focused on the determination of the receptor-bound conformation adopted by bioactive peptides by sjoithesizing constrained analogues of the peptides. Knowledge of the three dimensional interaction between a peptide ligand and a receptor could be invaluable in understanding bioactivity and in the design of therapeutics.
To determine the bioactive conformation of our novel chimeric peptides for the opioid and cholecystokinin receptors, constrained analogues were designed to limit the conformations that the peptides would adopt. In this regard, [5,5]- and [6,5]-bicyclic dipeptide mimetics were designed and synthesized to constrain a dipeptide unit and by extension limit the flexibility of the peptide. The bicyclic dipeptide mimetics were synthesized from precursors obtained by the (3-alkylation of aspartic acid and from the
Kazmaier-Claisen rearrangement reaction. A protocol for the alkylkation of aspartic acid with allyl bromide, benzyl bromide, and benzyl disulfide was developed. The bicyclic dipeptide mimetics were then introduced into the peptides whose biological activity was evaluated at both the opioid and cholecystokinin receptors. The peptides showed good binding and functional activities at the CCK receptors, but low activities at the opioid receptors. 15
CHAPTER 1
INTRODUCTION
The central goal of the research outlined in this thesis is the synthesis of constrained chimeric peptides that are agonists at the 5-opioid receptor and also antagonists at the cholecystokinin (CCK) receptor. The constraint would be effected by the design and syntheses of bicyclic dipeptide mimetics which would then be incorporated into the desired biologically active peptides. Structure activity relationship of the resultant peptides would aid in the elucidation of the biologically active conformation(s) of the peptides, the dream of all peptide chemists. In the introduction, a brief overview of opioid receptors and ligands, cholecystokinin, peptides, CCK/opioid chimeric peptides, and peptidomimetics is provided.
1.1 Structure-activity relationships of opioids
The enkephalin peptides, [Leu^]-enkephalin (Tyr'-Gly^-Gly^-Phe'^-Leu^-OH) and [Met^]- enkephalin (Tyr^-Gly^-Gly^-Phe'*-Met^-OH) were isolated by Hughes and Kosterlitz from porcine brain. ^ The theory of multiple subtypes of opioid receptors was proposed by
Martin et al. and confirmed by Lord et al. who demonstrated the existence of n receptors in the guinea pig ileum (GPI) and 5 receptors in the mouse vas deferens
(MVD). Both Met- and Leu-enkephalin interact with both the 5 and ^ opioid receptors and were found to be more potent at the 5 receptor than morphine based opiates which are more potent )U. agonists. 1.1.1 Importance of N-terminal Tyr at position 1
Early work on the structure-activity relationships of enkephalins showed great losses of
potency when Tyr' was modified.'^ Alkylation of the amino terminal group results in loss
of potency at the receptor and more so at the 5 receptor.^ It has been proposed that the
free NH2 terminus (which is protonated at physiological pH) interacts with an anionic site
on both the 5 and |j, opioid receptors.^ Addition of an amino acid at the zero position was
also found to be detrimental as was replacement of Tyr' with other amino acids.
1.1.2 Structure-activity relationship studies at position 2
Numerous changes at the Gly position have resulted in analogues with increased
potency with all the successful changes involving a D-amino acid." '^ Hambrook et found that the first metabolic step for enkephalins was cleavage at the Tyr-Gly peptide bond. Substitution of D-amino acid at the 2-position was thus expected to increase stability but there was also an increase in potency.'^ Interestingly, D-amino acids were later discovered to be naturally occurring in the amino acid sequence of the frog skin heptapeptide dermorphin, (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2),''* deltorphin I (Tyr-D-
Ala-Phe-Asp-Val-Val-Gly-NHi), and deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-
1.1.3 Structure-activity relationship studies at position 3 and 4
The Gly^ position in enkephalins is very intolerant to substitutions.'^ On the other hand, there has been numerous successful substitutions at Phe'^. Aromatic residues at position 4 are not fully required to interact with opioid receptors. The ring can be methylated, methoxylated, halogenated, nifrated, phenylated, or reduced to a cyclohexyl group.^'^" 17
Replacement of Phe'^ with aliphatic hydrophobic amino acids leads to improved selectivity for }i receptor over 6 receptor.21 ' 23 Position 4 can also be substituted with other aromatic residues such as Trp^"^, but not with Tyr.^
1.1.4 Structure-activity relationship studies at position 5
Leu^ or Met^ can be replaced with alkyl groups, esters, or alcohols which improve potency at |i receptor while decreasing activity at 5 receptors.^^ The carboxyl terminal is not necessary for activity at the )j. receptor. For a series of D-Ala^ analogues, replacement '7^ 97 of the terminal carboxyl group with -NH2 or -CONH2 gave analogues that were more |a, active than CO2H terminal analogues. The C-terminal alcohol analogue FK33824
(T3n"-D-Ala-Phe-Gly-NH2) was found to be more active at the receptor than Met- enkephalin.^'
1.1.5 Cyclic analogues of enkephalins
Cyclization greatly decreases the available degrees of conformational freedom. 28
Appropriate conformational constraints will restrict a residue or group of residues to a sufficiently small region of conformational space that when the peptide interacts with its receptor or some other acceptor molecule (enzyme, carrier protein, etc.), the
conformation seen in solution will remain due to the high activation energy or free energy
needed to change that conformation. Cyclic analogues have recently been employed to help elucidate the conformational requirements, in particular the topographical
arrangement of the side chains, for the opioid receptors. Side chain to C-terminal and side
chain to side chain cyclic lactam analogues of enkephalin have been shown to be potent
and selective for the |i receptor.Potent 5 opioid receptor selective cyclic enkephalin 18 analogues were developed by side chain to side chain disulfide bridges. These analogues were generally substituted with D-Cys and D- or L-Cys residues. More active analogues were obtained by substitution with Pen and/or D-pen residues at positions 2 and Cyclic lathionine analogues have also been shown to have high potency at both
|a and 6 receptors.^^'^^
1.2 Cholecystokinin
Cholecystokinin (CCK) is a gut-brain peptide that exerts a variety of physiological actions in the gastrointestinal tract and central nervous system through cell surface CCK receptors. It was first isolated from the porcine duodenum as a 33 amino acid peptide.^^ A number of biologically active variants were subsequently described and the most abundant peptide present in the brain was shown to be the sulfated C-terminal octapeptide amide [CCK26-33 or CCK-8, H-Asp^''-Tyr(S03H)^^-Met^^-Gly^'^-Trp^''-Met^'-
Asp^^-Phe^^-NH2).^^ CCK-8 has been shown to be involved in numerous physiological functions such as feeding behaviour, central respiratory control, and cardiovascular tonus, vigilance states, memory processes, nociception, emotional and motivational responses.
CCK is closely related to the gastrin family of peptides, and also to the naturally occurring decapeptide caerulein (CRL), which all share a common C-terminal amino acid sequence.40
Gastrin-1 pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp- Phe-NH2 Caerulein Glp-Gln-Asp-Tyr-Thr-Gly-Grp-Met-Asp-Phe-NHa
Table 1.1 Gastrin-1 and Caerulein peptides CCK receptors are divided into two subtypes, namely, CCK-A and CCK-B receptors
(also denoted CCK-1 and CCK-2, respectively) both belonging to the class of G-protein coupled receptors characterized by seven trans membrane I domains."^' CCK-A receptors are located mainly in the periphery but are also found in some regions of the brain,''^"'*^ while CCK-B receptors are predominantly located in the central nervous system (brain and spinal cord), but also are found in the stomach and vagus nerve (the longest of the cranial nerve)."^ Studies with selective CCK receptor antagonists have revealed that although both CCK-A and CCK-B may exist in the brain, CCK-B receptors predominate in the spinal cord of rodents whereas the CCK-A receptor subtype predominates in
primates.CCK-8 is the minimal sequence necessary for full activation of the CCK-A
receptor, whereas for the CCK-B only the C-terminal tetrapeptide is required.
Considerable interest is devoted to the pharmacology of CCK-B receptors since administration of selective agonists produces behavioral changes such as anxiety, pertubation of memory, and hyperalgesia. Dysfuctioning of CCK-B related neural pathways could also be involved in neuropsychiatric disorders. Accordingly, CCK-B antagonists have been shown to block panic attacks induced in humans by systematic administration.'^^
1.2.1 Structure activity relationstiip for choiecystokinin
It has been demonstrated that replacement of both Met^^ and Met^' residues by the isosteric amino acid norleucine (Nle) provides CCK analogues with virtually identical activities as CCK-8.'^^'^^ This modification allows for an efficient synthesis of CCK analogues since side reactions associated with methionine are avoided. These analogues 20
should also be resistant to enzyme degradation. Replacement of Met^' in the C-terminal
tetrapeptide of CCK with Lys(NH'^CONHR) reversed subtype selectivity and led to the
discovery of a highly potent CCK-A agonist.^" Unsulfated analogues containing N-
methylnorleucine (N-MeNle) substitutions at positions 28 and 31 were shown to have
exceptional potency and selectivity for CCK-B receptors/^
Truncation studies have shown that Trp^" is an important residue for interaction with
either the CCK-A or CCK-B receptor.^Substitution of Trp^*^ with D-Trp, Nal(l), N-
MeTrp, and P-methyl tryptophan resulted in compounds with less potency. The N-
terminal amino group of CCK-7 is not important for cholecystokinin activity,^'* and
similarly, the N-acetyl derivative of CCK-7 shows the same potency as CCK-8 in the
pancrease amylase release test.^^ Structure-activity work with Ac-CCK-7 suggests that
requirements for potent agonist activity includes an intact C-terminal carboxamide,^*' an
aryl- or cycloalkylalanine in position Substitution of Asp^^ by alanine,P-
alanine,*'®'^' P-aspartic acid,*'^ or glutamic acid''' leads to decreased CCK-A potency,
whereas substitution by a sulfated serine, threonine, or hydroxyproline gave fully active
analogues.^^ In the latter case, the hydroxyproline may act to stabilize a preferred
conformation. It has also been shown that the Asp^^ plays a conformational role rather than mediating binding to the CCK-A receptor through a charge-charge interaction.^'^
It has been reported that the main metabolic cleavage sites of CCK-8 is between Gly-Trp or Trp-Met dipeptide residues. In order to increase enzymatic resistance, the Trp and Met amino acids were substituted with their D enantiomers while Gly was substituted with D- alanine. These resulted in loss of potency except for the analogue containing D-Ala which retained some activity and led to prolonged in-vivo activity.
1.2.2 Cyclic analogues of CCK peptides
CCK-8 was found by NMR to exist preferentially under folded form in aqueous solution with a proximity between Asp' and Gly"^.^^ This property was used to synthesize cyclic peptides through amide bond (lactam bridges) formation between Asp' or a-, p-Glu' and
Lys'' side chains to give highly potent and selective CCK-B agonists.^^ CCK-4 was also found to adopt a folded conformation and some cyclic analogues have been synthesized.
These CCK-4 analogues contain in place of Trp-Met residue dipeptide a diketopiperazine moiety resulting from a cyclization between Nle and N-substituted (D)Trp residues and coupled with a small linker to Asp-Phe-NHi.*''
1.3 CCK involvement in pain modulation
CCK has been found in regions of the brain known to be associated with pain modulation, for example the cortical grey matter, PAG, ventromedial thalamus and spinal dorsal hom.^^ CCK has also been shown to co-exist with a substance P-like peptide in the ICS 71 spinal ganglia and in the mid brian PAG. " Substance P has been shown to function as a neurotransmitter and/or modulator in synaptic transmission between primary afferent fibres and neurons within the spinal cord.^^ Willets et alP demonstrated that in rat dorsal horn, a majority of neurons within the spinal cord excited by substance P could also be excited by CCK-8, suggesting a common mechanism. In addition, it has been reported that CCK-containing neurons located in the Edinger-Westphal nucleus of the PAG responds with increased firing and, on occasions, depolarization blockade to noxious stimuli/'' The spontaneous firing and noxious stimulation-induced firing of these neurons was suppressed by systematically injected morphine. It has been shown that administration of CCK reduces morphine analgesia which could be attributed to an independent hyperalgesic induction effect for CCK.^^ CCK does not itself show hyperalgesia meaning it is not a pro-nociceptive effect. It has been concluded that exogenous CCK attenuates analgesia induced by morphine or release of endogenous opioids. However conflicting results emerge from these studies. Zetler/^ BarbazJ^ and •yy IQ Id Hill suggest an indirect opiate-mediated analgesic effect of CCK, whereas Paris suggests an opiate antagonistic effect of CCK. The differences could be attributed to the fact that large doses of CCK induce a 'pharmacological' analgesia whereas small doses of the peptide produces a physiological antagonism of opioid analgesia that are specific to the sulfated octapeptide form. Furthermore, endogenous CCK may be a factor in determining the magnitude of the opioid analgesic response, acting in the short-term as a negative feedback modulator of opiate action.
Studies using proglumide (4-Benzamido-N,N-dipropylglutaramic acid), a weak, non selective, CCK antagonist, supports the observation that endogenous and exogenous CCK attenuates morphine analgesia. Proglumide was also shown to attenuate morphine tolerance^^ and potentiated the analgesic effect of P-endorphin.^'^ It has also been demonstrated that concomitant administration of proglumide and transcript, a weak CCK antagonist, with morphine over a period of time enhances the analgesic effects of the opiate. Furthermore, proglumide and benzotript inhibited the development of morphine tolerance, but had no effect on opioid dependence. " MK-329, a potent and selective 23
CCK-antagonist has also been shown to enhance morphine analgesia but does not block it
at high doses as is the case with proglumide.^^ This would suggest that tolerance may, in
part, result from a progressive compensatory increase in the activity of CCK systems in response to prolonged opiate administration. This would imply that blockade of CCK receptors by CCK antagonists may reverse or prevent the development of opiate tolerance in patients.
1.4 Overlapping pharmacophores of opioids and cholecystokinin
Biophysical studies have suggested that unsulfated C-terminal CCK-7 might have biological activity at opioid receptors.^^ Computational studies of CCK-7 and [Met^]- enkephalin showed similarities in structure. The aromatic moiety of Tyr' in [Leu^]- enkephalin can be aligned with the Tyr residue of CCK-7. Similarly, the side chain of
Phe"^ in [Leu^]-enkephalin can be aligned with the indole ring of Trp in CCK-7.Several modeling studies have suggested similarities between the proposed biologically active conformation between CCK and opioid ligands.^^ Examination of these proposed bioactive conformations demonstrated topographical similarities of the surfaces of the aromatic side chain residues suggesting that, at least in part, 5-opioid receptors and CCK-
B receptors have overlapping structural and topographical requirements.^"
1.5 CCK/opioid chimeric peptides
Interactions between opioid peptides and CCK-8 in regulation of pain pathways are supported by evidence from a number of laboratories.^^'^'"^^ However, the mechanism by which CCK-8 affects nociception is not completely understood. CCK-8 has been proposed to be an anti-opioid peptide,^'*'^^ to release endogenous opioids,^^'^^ and to have intrinsic antinociceptive activity.^^ A synthetic CCK-8 analog, SNF-9007 (Asp-Tyr-D-
Phe-Gly-Trp-N-MeNle-Asp-Phe-NH2) was found to be a potent and selective agonist at the CCK-B receptor but also had a weak affinity but robust agonist activity at the delta opioid receptor.^^ Modelling studies of SNF-9007 showed an agreement between the receptor bound conformation of CCK-B ligands with delta opioid receptor template model based on DPDPE.^^ Efforts were then put into synthesizing analogues of SNF-
9007 that have a mixed delta and mu binding affinity and a more balanced binding affinity for the CCK-A and CCK-B receptors.
It is well known that the N-terminal tyrosine is important for potency at the opioid receptors. To improve the delta opioid receptor potency, the Asp*^ was removed to give a nanomolar agonist at the 5 opioid receptor while also retaining high selectivity at the
CCK-B receptor.'"" It has also been established that the second amino acid in opioid ligands should either be a glycine or a D-amino acid residue." '^ When Gly was substituted for D-Phe in SNF-9007, a weak delta and mu opioid receptor agonist, and a very weak CCK agonist was obtained. When the Gly^ was replaced by D-Ala^, bioactivity at the MVD and GPI improved slightly and interestingly, the compound showed a weak antagonist activity in the CCK assay. The analogue containing Dphe^,
DTrp"* and NmeNle^ had good bioactivity in all three bioassays.
Cyclic disulfide analogues with substitutions of D-Cys or D-Pen in position 2 and L-Cys,
D-Cys, or D-Pen in position 5 displayed selectivity for the 5 opioid receptors and antagonist activities against CCK-8 in tissue assays.'"' Substitution of D-Trp'^ in these cyclic disulfide analogs led to significant losses in activity. Lactam analogues obtained 25
by substituting L-Lys or D-Lys and Glu or D-Glu gave analogues that were agonists at [i opioid receptor as well as antagonists at the CCK-8 receptor. The CCK-8 antagonist activity was increased when D-Trp"^ was substituted while the opioid agonist activity was maintained. However, the lactam and disulfide analogues did not have any competitive binding affinities at the human CCK-A and CCK-B receptors.
1.6 Peptides
Proteins and peptides are formed by a series of amino acids linked together by amide bonds (Figure 1.1). They are involved in numerous physiological processes as
neurotransmitters, neuromodulators, hormones, inhibitors, antibiotics, growth factors,
cytokines, antigens, etc.
O a-amino acid i -1 i + 1
Figure 1.1 General structure of amino acid and peptide
Although numerous native peptides have great potential for medical applications, these peptides generally have to be modified to overcome certain problems, such as degradation by peptidases, nonselectivity for different receptors and receptor subtypes, and their limited permeation across biological membranes. Since the biological functions of peptides are determined by their chemical, structural, conformational, topographical and dynamic properties, different approaches have been designed to study these parameters. The conformation of peptides and proteins can be described by their backbone conformation and side chain conformations. The backbone conformations are 26 characterized by three dihedral angles (j), \\i, and co (Figure 1.2). The side chain conformations are characterized by the dihedral• angles %12 , % etc.
C o x'VA H
i-1. i+1 H (j) CO o i-1 i+1
Figure 1.2 Definition of (j), \|/, to, % 1, % 2 dihedral angles
1.6.1 Peptide secondary structures a-Helixes, p-sheets, P-tums and y-tums and extended conformations are the common secondary structures in peptides and proteins. The preference of p-tums and y-tums, 1 09 1 fl"? which have often been implicated as recognition elements, ' has led to an intense search for templates that mimic these stmctures."^'^'''^^ A P-tum consists of four amino 27
acid residues i to i + 3 leading to a reversal in the backbone direction, with amino acid
residues i + 1 and i + 2 found in the two comers of the turn. A C=0 HN hydrogen bond
between the i and i + 3 residues respectively, stabilizes almost all of this turns forming a 1 A-l 10-membered intramolecular hydrogen-bonded ring. Up to eight different subtypes and
their mirror images have been reported based on the dihedral angles, (|) and \\f, of the i + 1
and i + 2 amino acid residues(Figure 1.3). The definition of the most common p-
tums (types I, II and their mirror images I' and IF respectively) requires that the amide
bonds reside in the trans orientation and that the distance between the a-carbon and the i
and i + 3 residues be <7 For a specific turn, the deviation of the dihedral angles
should not be larger than 20°.
R2 Turn
1 -60 -30 -90 0 I' 60 30 90 0 II -60 120 80 0 11' 60 -120 -80 0 III -60 -120 -80 0 III' 60 30 60 30 V -80 80 80 -80 v' 80 -80 -80 80
Figure 1.3 Different types of P-tum
1.6.2 Peptidomimetics
To elucidate the contribution of reverse-tums to the bioactive conformation of a minimal bioactive sequence, which otherwise is highly flexible, and to enhance and select for a specific profile of pharmacological activity, rigidified reverse-tum mimetics (otherwise
called peptidomimetics) have been developed. As defined by Hmby;109 28
A peptidomimetic is an organic molecule, such as a peptide analog or nonpetide ligand,
that interacts with receptor or acceptor in a similar manner as the native peptide/protein
ligand to affect the same biochemical (biological) activities. In the parlance of medicinal
chemistry, both peptidomimetics and native ligands/proteins should share common
pharmacophore elements.
In our laboratories, there is an impetus to design and synthesize P-tum mimetics for the
opioid, CCK, and melanorcotin receptors. In general, P-tum mimetics can be classified as
either external or internal mimetics depending on the support, which can be either outside
or inside the P-tum^^° (Figure 1.4). These peptidomimetics are potential conformational
probes enabling biological activity to be more effectively correlated with stmcture, and
prospective therapeutics overcoming drawbacks such as proteolytic susceptibility and
poor bioavailability, typical of many important bioactive peptides.
Extemal P-tum mimetics Intemal P-tum mimetics
'—(j SupporTlJ—
Ri+1 I Support R'^ o r'+2
Figure 1.4 External and intemal P-tum mimetics
One goal in P-tum peptidomimicry is to stabilize the relevant dihedral angles in the turn so they fall within the ranges appropriate for the sought after tum. A common strategy for
P-tum peptidomimicry is to replace the i + 1 and i + 2 residues of the tum with a scaffold to stabilize the turn. This type of scaffold best serves its purpose if it provides sites for attaching side chain-like appendages, positions an amide bond in the central dipeptide and possesses an amine and carboxylic acid groups so the scaffold may be incorporated into a peptide chain in place of the putative turn residues. Mimetics where the stabilizing hydrogen bonds in |3-tums and y-tums have been substituted with covalent bonds' as well as mimetics enhancing the reverse turn propensity of a peptide have been developed.Residue substitution in the parent peptide for example the use of N- methyl, dehydro-, a-methyl-, or D-amino acids to enhance the turn population has been reported.'^' There are also a variety of methods that incorporate short-range cyclizations, which are called heterodetic cyclizations, into a peptide sequence by connecting amino acid side chains to form amide and ester linkages, or disulfide bridges. Other cyclizations that attach amino acid side chains to the peptide backbone have been used in the synthesis of peptidomimetics.121 " 122 Several cyclic and bicyclic dipeptide analogues intended to stabilize the peptide in a reverse turn have been reported(Figure 1.5).
Among these, the 5,5-bicyclic dipeptide mimetic i was designed as a type II P-tum mimetic'^^ and the 6,5-bicyclic dipeptidomimetic BTD ii as a type IF P-tum mimetic.
125 A [7,5]-bicyclic dipeptide iii has been proposed to represent many different P-tums depending on the different chiralities on the ring and the different amino acids in the sequence.'^*' An S-spirolactam iv was suggested to replace a type II P-tum, while a spirotricycle was thought as the best mimic of a type II P-tum.Other types of bicyclic structures have been designed as P-tum mimetics, such as azobicyclic[4.3.0]- alkane v for type IF and cyclooctapyrrole vi for type VI P-tum'^^"'^'' and tricyclic vii type 30
II P-tum.'^' Bicyclic tripeptide mimetics viii and ix were designed and synthesized for incorporation into angiotensin 11.'^^ Some of the bicycHc dipeptide mimetics have also been introduced into peptides.However, it is interesting to note that most of these
P-tum mimetics do not have the side chain groups found in most natural a-amino acids.
H O boNHCH
Figure 1.5 Examples of bicyclic p-tum mimetics 31
CHAPTER 2
DESIGN AND RETROSYNTHETIC ANALYSIS FOR BICYCLIC DIPEPTIDE
MIMETICS
2.1 Introduction
The development of novel |3-tum mimetics has drawn significant attention in
peptidomimetic design. Two reviews on the design and synthesis of bicyclic dipeptide
mimetics has been provided by Lubell and Hanessian.*'^^"''^^ Our group has focused on the
synthesis of external P-tum mimetics in which both backbone conformation (cj) and vj/
angles) and side chain conformations (x 1 and x 2) are constrained (Figure 2.1). In our
design, n could be 0, 1, and 2 representing [5,5], [6,5] and [7,5]-bicyclic dipeptide
mimetics respectively. X could be CH2, O or S making this approach more versatile from
a synthetic point of view.
oyW*- n = 0,1,2 X = CH2, S, O
Figure 2.1 Retrosynthetic analysis of bicyclic P-tum dipeptide
In our group, asymmetric synthetic methodologies towards the scaffolds for [5,5]- and
[6,5]-bicyclic dipeptides have been developed (Figure 2.2). The [5,5]-bicyclic dipeptides 32 are synthesized from y,S-unsaturated amino acids while the syntheses of the [6,5]- analogues employs 5,8-unsaturated amino acids.
R, H HS. PGHN COpM© HgN^COsH O COpM© HS. PGHN PGHN'^f CO2M© HgN'^^COaH O COaMe
PG = protecting group
Figure 2.2 Synthetic strategy for [5,5]- and [6,5]-bicyclic dipeptide mimetics
A synthetic methodology for the synthesis of the all-carbon backbone (indolizidinone)
[6,5]-bicyclic dipeptides from analogues of 3, 5- or y, 5-substituted prolines also has been developed (Figure 2.3).^147-149
R2 M6O2G
CbzHN N- CbzHN Boc CO9R Boc CO2R CO2R
Figure 2.3 Design of indolizidinone bicyclic dipeptide mimetics
2.2 Design and retrosynthetic analysis of bicyclic dipeptide mimetics
In the synthesis of constrained CCK/Opioid chimeric peptides that incorporate bicyclic dipeptide mimetics two peptides; Tyr-D-Phe-Gly-Trp-Nle-Asp-Phe-NHa and Tyr-Nle-
Gly-Trp-Nle-Asp-Phe-NHa (RSA501) were chosen as the lead compounds. A bicyclic dipeptide mimetic for homoPhe-Gly 1 was designed to be substituted for the D-Phe in
Tyr-D-Phe-Gly-Trp-Nle-Asp-Phe-NH2- A bicyclic dipeptide mimetic for Nle-Gly 2 was 33 also designed to be substituted for Nle-Gly dipeptide unit in Tyr-Nle-Gly-Trp-Nle-Asp-
Phe-NH2 (Figure 2.4).
BocHN BocHN'
1 2
Figure 2.4 Bicyclic dipeptide mimetics for homoPhe-Gly and Nle-Gly.
Retrosynthetically the two compounds can be obtained from p-substituted aspartic acid or
P-substituted y,5-unsaturated amino acids (Figure 2.5).
BocHN BocHN
R = benzyl, n-propyl
Figure 2.5 Retrosynthetic design for dipeptide mimetics
It has been shown that CCK-4 adopts a folded conformation. fsl To introduce such a turn at the C-terminal of our target peptides a Nle-Asp bicyclic dipeptide mimetic 3 was designed to be substituted for the Nle-Asp dipeptide unit in Tyr-Nle-Gly-Trp-Nle-Asp-
Phe-NH2 (Figure 2.6).
CbzHN C02Bn 3
Figure 2.6 Bicyclic dipeptide mimetic for Nle-Asp dipeptide unit 34
This compound can be derived by elaboration of 6-allyl substituted ydroxy analogues
which can be obtained from P-allyl substituted aspartic acids (Figure 2.7).
H MeOoC Vie CbzHN CbzHN
C02Me C> BocHN
CO2CH3
Figure 2.7 Retrosynthetic analysis of a Nle-Asp bicyclic dipeptide mimetic
As has been described above, our design is based on P-substituted Y,6- and 5,8-
unsaturated amino acids. These can be obtained by alkylation of aspartic acid, by the
Kazmaier-Claisen rearrangement'"^'^' and via a Nickel-II chiral auxiliary.In
this chapter, a discussion on alkylation of aspartic acid and the Kazmaier-Claisen rearrangement, the methods employed in this research, are discussed.
2.3 Synthesis of P-substituted y,5- and 6,s-unsaturated amino acid
The synthesis of y,5-unsaturated amino acids has received much attention in recent years not only because they occur naturally but also because they are used in the synthesis of complex amino acids and peptides. Examples of y,5-unsaturated amino acids includes the isoleucine antagonist cyclopentenylglycine x and the antibiotic furanomycine xi'^'^
(Figure 2.8). Unsaturated amino acids are also important intermediates for the synthesis of functionalized amino acids and have been used as asymmetric synthetic building blocks."'" The terminal double bond is a precursor in organic synthesis which can be 35 converted to hydroxy!, oxo, carboxyl, epoxy, and amino a-amino acids.They have also been used in cyclization of peptides via ring closing metathesis.
HgN GO2H HgN UO2H X xi
Figure 2.8 Examples of y,5-unsaturated amino acids
2.3.1 Alkylation of aspartic acid
Alkylation of aspartic acid is a concept that has been examined in detail in the recent literature.Treatment of aspartic acid with lithium di-isopropylamide (LDA), or lithium hexamethyldisilazide (LHMDS) in tetrahydrofuran at -78°C, followed by warming to -30°C, recooling to -78°C and adding the electrophile results in the formation of P-alkylated products in 50-60% yields. No products resulting from a-deprotonation were observed. Rapoport et al. used 9-phenylfluorenyl-9-yl, a large N-protecting group to suppress a-deprotonation. Diastereomeric ratios and yields for these alkylations depend on the ester identity, the reactivity of the electrophile, and the base used.
Alkylation with alkyl halides gives low yields, while alkylation with bis-electrophiles gave no products. On the other hand, alkylation with activated electrophiles like allyl bromide and benzyl bromide proceeded with good yields. The diastereomeric ratios were found to depend on the ester identity at both the a- and |3- carboxyl groups. Potassium hexamethyldisilazide (ICHMDS) and the corresponding lithium antiide (LHMDS) were found to give opposite diastereoselectivity. Trapping studies using TMSCl showed that 36 the potassium and Hthium enolates were different giving two distinct silyl ketene acetals
(Figure 2.9).
OTMS
PhFIBnN PhFIBnN OTMS H H
Figure 2.9 Silyl ketene acetals
From these results KHMDS forms an E-enolate while the lithium analogue forms a Z- enolate. It has been reported that (Z)-lithium ester enolates are significantly more reactive than the corresponding (E)-lithium ester enolates. The facial selectivity of electrophilic attack can be explained by a novel type of enolate-ester chelation that is controlled by enolate geometry. For the LHMDS/THF system, the Z-lithium enolate (Figure 2.10), in which the OM"^ cannot form a cyclic chelate because of its (E)-geometry, assumes a hydrogen-in-plane conformation that is attacked opposite the bulky nitrogen. The (Z)- potassium enolate, although significantly less reactive, selectively provides the opposite stereoselectivity. This can be explained by the fact that for the (Z)-potassium enolate, the alkylation is determined by a preference for a-ester chelation by the counterion of the enolate oxygen atom, giving a cyclic chelate that the electrophile attacks preferentially opposite the bulky protected nitrogen group. North et al. has also proposed the diastereoselectivity observed is due to the formation of a chelated enolate (Figure 37
E- AI .OLi H t ,aOR Z-Lithium ^-potassium ROoA NR2)^o-K r RO E^
Figure 2.10 Transition state for alkylation of aspartic acid
OMe
9^1 ^'~~N^C02'Bu
BnO OLi
Figure 2.11 Chelated enolate intermediate
When the method reported by North et al. was applied to the N'^-Boc aspartic acid 4a, the reaction would not go to completion necessitating the development of a modified procedure. The protected amino acid was dissolved in THF and cooled to -42°C. LHMDS followed by HMPA were then added and the mixture stirred for 30 min before adding the electrophile. When the electrophile was allyl bromide, two products (5a and 6a) in a total yield of 57% and a ratio of 3:1 were obtained after purification by flash column chromatography (Scheme 2.1). The major isomer was found to be the (2S, 3R) isomer 5a on NMR analysis of the final bicyclic dipeptide, in agreement with literature.When
4b was subjected to the same reaction condition, a yield of 62%) and a ratio of 4:1 in favor of the (25", 3R) isomer 6a was obtained. The difference in yield and diastereomeric ratio is as a result of the different ester protection used. 38
BocHN ,2 1.LHDMS/HMPA BocHN BocHN 2. Allyl bromide Y^COjR^ COzR^ COsR^ CO2R'/^n D1
4a R'' = CH3, = Bn 5a R^ = CH3, r2 = Bn 6a R'' = CH3, r2 = Bn 4b = Bn, R^ = CH3 5b R^ = Bn, r2 = CH3 6b R^ = Bn, r2 = CH3
Scheme 2.1 Alkylation of aspartic acid with allyl bromide
In case of alkylation with benzyl bromide, only one p-benzyl substituted aspartic acid (7a or 7b) was isolated after column chromatography (Scheme 2.2). Cbz protected aspartic acid 4c could also be alkylated to give p-benzyl substituted aspartic acid in 70% yield.
However the alkylation is done at -78°C otherwise a lot of a-alkylated aspartic acid is obtained.
BocHN ,2 1.LHDMS/HMPA BocHN
4a R'' = CH3, r2 = Bn, R^ = Boc 7a R'' = CH3, r2 = Bn, R^ = Boc
4b R^ =Bn,R2 = tBu,R^ = Boc 7b R^ = Bn, r2 = tBu, R^ = Boc
4c R^ = Bn, r2 = tBu, R^ = Cbz 7c R'' = Bn, R^ = tBu, R^ = Cbz
Scheme 2.2 Alkylation of aspartic acid with benzyl bromide
The acidity of the a-hydrogen of amino acids can be lowered by protection of the a- carboxyl group as an ortho ester. 171 The ortho ester is stable to strongly basic reagents and is easily regenerated under mild acidic conditions. Ortho esters can be prepared irom nitriles, imido esters, ortho ester exchange or directly from carboxylic acids.The ester of 3-methyl-3-hydroxymethyloxetane rearranges in the presence of BF3.0et2 to give bridged ortho esters (Figure 2.12). Coordination of the oxetane ester xii with lewis acid forms xiii which induces heterolysis of the dioxetane C-0 bond with ester carbonyl 39 participation to give zwitterions xiv that collapses to give the ortho ester xv. The 3- methyl-3-hydroxyl oxetane ester of aspartic acid 10 was easily converted to the ortho ester 11 in 70% yield after exposure to BFs.OEti. Alkylation of the ortho ester 11 with benzyl bromide in the presence of LHMDS and HMPA gave ^-alkylated aspartic acid analogue 12 in 61% yield.
XV
Figure 2.12 Mechanism for the formation of ortho ester
BocHN
DMAP
BocHN 1. LHMDS/HMPA BocHN
11 12
Scheme 2.3 Alkylation of ortho ester protected aspartic acid
2.3.2 Kazmaier-CIaisen rearrangement reaction
In 1975, Steglich et al. described the thermal Claisen rearrangement of N-benzoyl-a- amino acid allyl esters on treatment with a dehydrating agent such as phosgene in which a 5-allyloxazole intermediate was formed.As a result of the fixed olefin geometry 40 in the oxazole ring, and a strong preference for the chair-like transition state, the diastereoselectivity for these reactions is very high (Figure 2.12). However, this methodology is limited to N-benzoyl amino acid esters (and related aromatic or heteroaromatic N-acyl derivatives), because other common N-protecting groups like carbamates do no allow formation of the oxazole. Barlett et al. investigated the Ireland-
Claisen rearrangementof N-acylated glycine allyl esters in detail.'^" This method offers great flexibility with respect to variation of the protecting group. In the rearrangement of crotyl esters syn selectivity, which can be explained by the formation of an (E)-Lithium enolate and its chelation by the anionic acylamide a-group, was observed.
However, the diastereoselectivity of the rearrangement strongly depends on solvent, base,
N-protecting group, and the substitution of the allylic ester moiety.Similar chelate- bridged enolates have also been postulated in sigmatropic rearrangement of a-alkoxy- substituted allyl esters.Corey and Lee have also described an asymmetric boron enolate variant of the Claisen rearrangement.'^^ All these procedures for the Claisen rearrangement of ester enolates of a-amino acid allyl esters make use of the Ireland silylketene acetal variant. 187 Atttempts to rearrange lithiated glycine allyl esters directly leads to decomposition of the lithium enolates, since the rearrangement occurs at high temperatures. 41
N ^ .0 Nr . ,0 , NaOH Pyridine Oxazole rearrangement
R U Ph' N
LDA MeOH
^ '"V-COzSiMea
OSiMeg Ph' Ireland-Claisen rearrangement
Figure 2.13 Oxazole and Ireland-Claisen rearrangements
To improve the stability of the enolates, Kazmaier used metal chelated enolates'^'^"'^^ that also are superior to sillylketene acetals, both in terms of their reactivity and selectivity.
Due to the fixed enolate geometry, as a result of chelate formation, the rearrangement proceeds with high degree of diastereoselectivity, independent of the substitution pattern and the protecting groups on nitrogen. The nitro group must however be monoprotected as a Cbz, Boc or TFA since no rearrangement is observed with tertiary amino group or N- phthaloyl protected amino acids. Many different metal salts (ZnCh, MgCb, EtAlCla,
SnCl2, Al(OiPr)3, C0CI2 etc.) can be used for chelation but the best results are obtained with ZnCla-^'^ When E-allyl ester is used in this reaction, only the ^yw-product is 42
generated in high diastereoselectivity (95% ds), resuhing from a preferential
rearrangement via a chair-like transition state A (Figure 2.12). In the chair-like transition
state, sterical interactions between the pseudoaxial hydrogen and the chelate complex in
the boat-like trasition state are avoided. When Z-allyl esters are used in this
rearrangement, a diastereomeric mixture will be formed since the chair-like transition
state B that is formed is competitive with a boat-like transition state C. The lower
diastereoselectivity may result from the interactions of the cw-oriented side chain and the
chelated enolate destabilizing the chair-like transition state.
R Ov,
N--PG N—PG a ^ Zn CI CI B
R R GPHN GPHN, CO2H CO2H syn-product anti-product
Figure 2.14 Transition state in Kazmaier-Claisen rearrangement
The Kazmaier-Claisen rearrangement described is highly diastereoselective but not
enantioselective. A number of strategies have been studied for an asymmetric rearrangement.153 43
2.4 Experimental
General
and NMR spectra were recorded on Bruker DRX 500 and DRX 600 spectrometers. The chemical shifts were expressed in ppm (5) downfield from tetramethylsilane (TMS) as an internal standard. X-ray crystallography was conducted at the Molecular Structure Laboratory, Department of Chemistry, University of Arizona.
Mass spectrometric analysis were conducted by Mass Spectrometry Laboratory,
Department of Chemistry, University of Arizona. Optical rotations were measured on a
JASCO PI020 polarimeter. Column chromatography was performed with 200-400 silica gel from EM Science. Thin layer chromatography was performed with Kodak F-254 silica gel plates. Dichloromethane was distilled from CaHi and THF from potassium and benzophenone under Argon atmosphere. All chemicals were purchased from Aldrich
Chemical Company and used as received while Aspartic acid was purchased from
Calbiochem-Novabiochem Corporation. Melting points are uncorrected and were obtained in open capillaries. Ozone was generated from an ozone generating machine from Dr. Eugene Mash laboratories, University of Arizona.
General alkylation of aspartic acid
In a 100-mL flask, a-methyl p-benzyl N'^-tert-butoxycarbonylamino-(/S)-aspartate 4a (2.3 g, 6.82 mmol) was dissolved in 10 mL THF and cooled to -42''C. Lithium bis(trimethylsilyl)amide (LHMDS), (IM solution in THF, 15.0 mL, 15.0 mmol) followed by hexamethylphosphoramide (HMPA) (0.7 mL, 4.02 mmol) were added and the mixture stirred at -42°C for 30 min before adding allyl bromide (0.8 mL, 9.24 mmol). The mixture was stirred for a further 4 h, and 20 mL saturated aqueous NH4CI (20 mL) added.
The mixture was warmed up to room temperature and the THF evaporated off. The product was redissolved in DCM (50 mL) and washed by saturated aqueous NH4CI (2 x
50 mL) and brine (60 mL). The organic layer was dried over MgS04, and the solvent evaporated off. After purification on a silica gel column with hexane-ethyl acetate (10:1 v/v), two colorless liquids (1.1 g of 5a and 0.4 g of 6a) were obtained in a total yield of
58%.
Methyl 2-?^r#-butoxycarbonylamino-3-carbobenzyloxy-(25, 3i?)-hex-5-enoate (5a):
[a]^^D = +28.7° (c = 1.52, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.37-7.29 (5H, m), 5.84-5.69 (IH, m), 5.49 (IH, d, J= 9.92 Hz), 5.23-5.06 (4H, m), 4.60 (IH, dd, / =
3.63, 9.92 Hz), 3.56 (3H, s), 3.22-3.13 (IH, m), 2.56-2.45 (IH, m), 2.36-2.26 (IH, m),
1.44 (9H, s); '^C, 125 MHz, CDCI3, 5(ppm): 173.3, 171.2, 155.8, 135.3, 134.1, 128.5,
128.3, 128.2, 118.2, 80.0, 67.2, 53.5, 51.9, 46.4, 32.8, 28.2; HRMS (FAB) MH^ calcd for
378.1917, found 378.1921.
Methyl 2-?^rf-butoxycarbonylamino-3-carbobenzyloxy-(25', 35)-hex-5-enoate (6a):
[a]^^D = +25.5" (c = 1.33, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.39-7.31 (5H, m), 5.74-5.69 (IH, m), 5.29 (IH, d, 9.92 Hz), 5.21-5.13 (4H, m), 4.68 (IH, dd, J =
4.78, 8.6 Hz), 3.62 (3H, s), 2.95-2.92 (IH, m), 2.55-2.49 (IH, m), 2.28-2.25 (IH, m),
1.44 (9H, s); '^C, 125 MHz, CDCI3, 6(ppm): 172.5, 170.5, 155.2, 135.1, 134.5, 128.6,
128.5, 117.8, 80.2, 67.5, 54.1, 52.0, 47.9, 32.2, 28.2; HRMS (FAB) MH^ calcd for
378.1917, found 378.1927. 45
The same protocol was followed for alkylation of 4b to give (2.5 g of 5b and 0.75g of 6b)
in a total yield of 58% and a ratio of 3:1 in favor of 5b.
Methyl l-ter^-butoxycarbonylamino-S-carbobenzyloxy-CZiS, 3/?)-hex-5-enoate (5b):
[a]^^D = +28.7° (c = 1.52, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.37-7.29 (5H,
m), 5.84-5.69 (IH, m), 5.49 (IH, d, /= 9.92 Hz), 5.23-5.06 (4H, m), 4.60 (IH, dd, J =
3.63, 9.92 Hz), 3.56 (3H, s), 3.22-3.13 (IH, m), 2.56-2.45 (IH, m), 2.36-2.26 (IH, m),
1.44 (9H, s); '^C, 125 MHz, CDCI3, 5(ppm): 173.3, 171.2, 155.8, 135.3, 134.1, 128.5,
128.3, 128.2, 118.2, 80.0, 67.2, 53.5, 51.9, 46.4, 32.8, 28.2; HRMS (FAB) MH^
calculated for 378.1917, found 378.1921.
Methyl 2-ter?-butoxycarbonylamino-3-carbobenzyloxy-(25', 3iS)-hex-5-enoate (6b):
[a]^^D = +25.5" (c = 1.33, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.39-7.31 (5H,
m), 5.74-5.69 (IH, m), 5.29 (IH, d, J= 9.92 Hz), 5.21-5.13 (4H, m), 4.68 (IH, dd, / =
4.78, 8.6 Hz), 3.62 (3H, s), 2.95-2.92 (IH, m), 2.55-2.49 (IH, m), 2.28-2.25 (IH, m),
1.44 (9H, s); 125 MHz, CDCI3, 6(ppm): 172.5, 170.5, 155.2, 135.1, 134.5, 128.6,
128.5, 117.8, 80.2, 67.5, 54.1, 52.0, 47.9, 32.2, 28.2; HRMS (FAB) MH+ calculated for
378.1917, found 378.1927.
When benzyl bromide was used as the electrophile in the alkylation of 4a only product 7a was isolated as a clear semi-solid in a yield of 62%. Alkylation of 4b gave 7b in 58% yield while alkylation of 4c at -78"C gave 7c in 70% yield.
Methyl 2-te/*f-butoxycarbonylamino-3-carbobenzyloxy-4-phenyl-(25', 3/f)-butanoate
(7a): [a]^^D = +28.8" (c = 1.42, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.30-7.18
(lOH, m), 5.56 ( IH, d, J = lOHz), 5.06 ( 2H, s), 4.47 (IH, dd, J = 4.0, lOHz), 3.58 (3H, s), 3.42-3.39 (IH, m ), 3.12-3.08 (IH, m), 2.87-2.83 (IH, m), 1.48 (9H, s); '^C, 125 MHz,
CDCI3, 5(ppm): 172.7, 171.6, 155.7, 137.8, 135.2, 129.0, 128.9, 128.54, 128.48, 128.43,
128.40, 128.37, 128.31, 128.25, 126.7, 80.0, 66.8, 53.5, 52.4, 48.7, 34.7, 28.2; HRMS
(FAB) MH"" calculated for 428.2073, found 428.2071.
Benzyl 2-carboxycarbonylainino-3-carbobenzyloxy-4-phenyl-(25', 3/?)-butanoate
(7b): 'H NMR, 500 MHz, CDCI3, 5(ppin): 7.30-7.18 (lOH, m), 5.64 (IH, d, J= 10.0 Hz),
5.17 (IH, d, J= 12.0 Hz), 5.08 (IH, d, J= 12.0 Hz), 4.45-4.42 (IH, dd, J= 4.0, 10.0 Hz),
3.28-3.24 (IH, m), 3.06-3.02 (IH, m), 1.46 (9H, s), 1.30 (9H, s); 125 MHz, CDCI3,
5(ppm): 1723, 171.3, 155.9, 138.1, 135.4, 129.1, 128.5, 128.45, 128.2, 128.1, 126.6, 81.9,
79.9, 67.1, 53.8, 49.0, 35.1, 28.3, 27.8.
Benzyl 2-ter^-butoxycarbonylamino-3-carbobenzyloxy-4-phenyl-(25', 3/?)-butanoate
(7c): 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.42-7.08 (lOH, m), 5.92 (IH, d, J= 10.0 Hz),
5.21-5.0 (4H, m), 4.49 (IH, dd, /= 3.8, 10.0 Hz), 3.32-3.23 (IH, m), 3.04 (IH, dd, J =
7.0, 13.5 Hz), 2.81 (IH, dd, J = 8.5, 14.0 Hz), 1.29 (9H, s); '^C, 125 MHz, CDCI3,
5(ppm): 172.5, 171.4, 157.0, 138.3, 135.7, 129.6, 129.0, 128.9, 128.7, 128.6, 128.5,
127.1, 82.5, 67.7, 67.5, 54.6, 49.4, 35.5, 28.2.
Synthesis and alkylation of ortho ester protected aspartic acid
Boc-Asp(Bzl)-OH (4.0g, 12.4 mmol) was dissolved in a CH2CI2 (40 mL) and cooled to
0"C. DCC (3.1 g, 15.0 mmol) was added and the mixture stirred for 10 min before adding
3-methyl-3-hydroxymethyloxetane (1.9 mL, 19.05 mmol) and stirring for 8 hr. DCHU was filtered off and the filtrate diluted with CH2CI2 (40 mL) and washed by saturated aqueous NH4CI (2 x 40 mL) and brine (40 mL). The product was purified by column to give the oxetane ester 10 (4.7g, 95% yield). The ester was dissolved in CH2CI2 (50 mL) and BF3.0et2 (70 |j,L, 0.55 mmol) was added and the mixture stirred at room temperature
10 hr. TEA (300 |J.L, 2.2 mmol) was added and the mixture stirred for 40 min. The mixture was then diluted by CH2CI2 (30 mL) and washed by saturated aqueous NH4CI (2
X 40 mL) and brine (40 mL). The product was then recrystallized from ethylacetate/hexanes to give the ortho ester 11 (3.9 g, 84% yield). 48
CHAPTER 3
SYNTHESIS OF Nle-Gly BICYCLIC DIPEPTIDE MIMETICS
3.1 Introduction
A Nle-Gly bicyclic dipeptide mimetic was designed to be substituted for the Nle-Gly dipeptide unit in CCK-8 peptide analogues. Different templates can be used to mimic a
Nle-Gly dipeptide unit. The side chain can be incorporated as part of the ring as in xvi
(Figure 3.1) or outside the ring as in xvii. Inorder to fully study the biologically active conformation, xvi was chosen as the template since the chi-space is also constrained
(Figure 3.1). This bicyclic can be obtained from ^-allyl substituted aspartic acid(s) or from P-vinyl substituted norleucine, the latter being a product of the Kazmaier-Claisen rearrangement reaction.
•GO2M© CbzHN CbzHN
xvi xvii X = O, S, CH2 n = 0, 1, 2 Figure 3.1 Nle-Asp bicyclic dipeptide mimetics 3.2 Syntliesis of Nle-Gly bicyclic dipeptide mimetic from P-allyl substituted aspartic
acid
Hydrogenation of compound 5a reduced the alkene and deprotected the benzyl ester to afford a carboxylic acid which was then subjected to reduction. Reduction of the carboxylic acid using borane and sodium perborate^^^ failed to give the desired product. 49
Utilization of a recently reported method that uses BOP, DIPEA and NaBH4'^^ gave the
desired alcohol in a yield of 53%, contaminated with some 5-membered ring lactone
resulting from cyclization. Lactone formation was also found to happen during column purification while use of the crude alcohol gave low yields of the bicyclic dipeptide mimetic. Characterization of alcohol 13 could not be done due to lactone contamination.
1. H2, Pd/C 2. BOP. DIPEA COsMe NaBH4 COoMe 53% 5a
1. Swern 2. L-Cys, Pyr, RT, 4hr 3. Pyr, 50°C, 3 days BocHN- 4. CH2N2 350/^ O CO2M6 2a
Scheme 3.1 Synthesis of Nle-Gly bicyclic dipeptide mimetic
Swem oxidation'^® of the alcohol afforded the aldehyde which without purification was coupled with L-Cys to form an intermediate thiazolidine. Bicyclization was then achieved by heating the reaction mixture at SO^C for 3 days, followed by methylation to afford a bicyclic dipeptide mimetic for Nle-Gly 2a in 35% yield from the alcohol 13 (Scheme
3.1). When the minor product 6a was subjected to hydrogenation followed by
B0P/DIPEA/NaBH4 reduction, only the lactone 14 was obtained in 58% yield (scheme
3.2). Obviously, a ^ran^-relationship of the P-substituent makes this lactone thermodynamically more stable compared to the cw-analogue. 50
J 1. H2, Pd/C > BocHN Y^COaBn 2. BOP, DIPEA .0 CO2M© NaBH4 BOCHN 58% O 6b 14
Scheme 3.2 Hydrogenation and reduction of P-allyl substituted aspartic acid
3.3 Synthesis of Nle-Gly bicyclic dipeptide mimetic via the Kazmaier-Claisen
rearrangement reaction
To fully investigate the stereochemical and topographical requirements of the receptors, other isomers of Nle-Gly bicyclic dipeptide mimetic were synthesized via the Kazmaier-
Claisen rearrangement reaction.Coupling of Boc-Gly with commercially available trans 2-hexen-l-ol gave 17. Compound 17 was then subjected to the Kazmaier-Claisen rearrangement reaction to give racemic p-vinyl substituted Nle 18. The crude product was then subjected to ozonolysis'^^ and the resultant crude aldehyde coupled with L-Cys-
OMe in a two step strategy involving thiazolidine formation and bicyclization. Two bicyclic dipeptide mimetics 2b, and 2c were isolated in a yield of 50% from 17. 51
DCC B0CHNCH2CO2H DMAP 15 16
Pr Pr/., BocHN LDA OH DMS ZnCl2, THF BocHN o"/ O 17 118
OH 1. L-Cys-OMe, EtOH, Sieves /. P 2. EDAC, DIPEA, CH2CI2 BocHN BocHN O 20
H
BocHN-^f ^ + BocHNi" <,
O CO2M© O CO2M© 2b 2c
Scheme 3.3 Synthesis of Nle-Gly BTD via the Kazmaeir-Claisen rearrangement
reaction
3.4 Characterization of Nle-Gly bicyclic dipeptide mimetics
The three Nle-Gly bicyclic dipeptide mimetics were characterized by DQF-COSY and
ID-transient nOe. The nOe's from the N"-Boc-products were not sufficient to assign the chiral centers since not all the nOe relationships were observable particularly for the left- hand ring. As a result, the A'-trifluoroacetamide analogs (21a, 21b, and 21c) were synthesized using trifluoroacetic anhydride and pyridine'^^ and their ID-transient nOe data used to assign the chiral centers. From our earlier work'"^^"'"*^ and also from Scheme
3.1, the 3/?-|3-propyl substituted homoserines should give only one BTD while the 3S- 52 isomers should give two BTD's with the bridgehead-H up or down. Based on this information, the {2R, 3i?)-isomer of aldehyde 19 should give only one BTD (in this case
2c), while the {IS, 35)-isomer should give two BTD's. However, only two products were obtained from the cyclization. Compounds 21a and 21c showed a weak nOe between H'' and (1.12% and 0.83% respectively) compared to a value of 5.43% observed for 21b.
This is an indication of a trans relationship for H'* and in compounds 21a, and 21c and a cis relationship in 21b. A similar trans relationship is observed in 21b and 21c for and while a strong nOe (4.45%) indicative of a cis relationship is observed for 21a.
1.12% 0.83%
4.45% 1.03% O 0.77%
O CO2M6 O CO2M6 O CO2M6 21a 21b 21c
Figure 3.2 nOe data for the Nle-Gly bicyclic dipeptide mimetics
Additionally, the structure of compound 21c was confirmed by an X-ray crystal structure.
'V. f1 I J'
Figure 3.3 X-Ray crystal structure of a Nle-Gly bicyclic dipeptide mimetic 53
3.5 Experimental
General procedure for hydrogenation of the benzyl ester and reduction of the resultant carboxylic acid
Compound 5a (2.88 g, 6.7 mmol) was dissolved in degassed methanol (40mL). Pd/C
(10%) was added and the mixture was exposed to hydrogen gas for 2 h. The catalyst was filtered off through Celite and the solvent evaporated off. The crude product was dried under vacuum and then dissolved in THF (35 mL). Benzotriazol-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (3.2 g, 7.23 mmol) followed by DIPEA (1.3 mL, 7.46 mmol) were added and the mixture stirred for about 5 min until it turned clear. The solution was then cooled to O^C and NaBH4 (400 mg, 10.57 mmol) added slowly. The mixture was stirred for 30 min at 0°C and at room temperature for 18 h. The solvent was evaporated off and the product dissolved in ethylacetate (80 mL) and the organic phase was washed with saturated aqueous NaHC03 (2 x 50 mL), 5% aqueous HCl (2 x 50 mL) and brine (50 mL). The organic phase was then dried over
Na2S04 and the solvent evaporated off. The product was purified by column to give 1.2 g of 13 (3.71 mmol) in 55% yield. The product was then subjected to Swem oxidation.
Oxalyl chloride (2.8 mL, 5.6 mmol) was dissolved in CH2CI2 (40 mL). The solution was cooled to -78°C, DMSO (530 fxL, 7.47 mmol) added and the solution stirred for 10 min before adding 10 1.2 g, 3.71 mmol). The mixture was stirred for 25 min at -78°C before adding DIPEA (2.6 mL, 14.93 mmol) , removing the cold bath and stirring for a fiirther
20 min. Water (20 mL) was added, and the organic phase was separated and washed with saturated aqueous NH4CI (2 x 50 mL) and brine (50 mL). The organic phase was dried 54
over MgS04 and the solvent evaporated off to give 1.17 g of the crude aldehyde after
drying. The crude aldehyde was dissolved in pyridine (30 mL) containing molecular sieves 4 A° and the mixture stirred at room temperature for 4 h. Pyridine (60 mL) was
added and the mixture heated at 50°C for 3 days. The solvent was evaporated off and the
product dissolved in water (20 mL) which was acidified to pH = 2 using IN aqueous
HCl. The aqueous layer was extracted with ether (4 x 20 mL) and the ether layer dried over MgS04. The solvent was evaporated off and the product dried under high vacuum to give 1.03 g of crude product. The crude product was dissolved in CH2CI2 (15 mL) and
cooled to 0°C. DCC (650 mg, 3.15 mmol) was added and the mixture stirred for 10 min before adding methanol (450 \xL, 11.12 mmol) and DMAP (30 mg, 0.25 mmol) and stirring the mixture for 8 h. The mixture was filtered and the filtrate diluted by CH2CI2
(20 mL). The organic phase was washed by saturated aqueous NH4CI (2 x 20 mL) and brine (30 mL). The organic phase was dried over MgS04 and the solvent evaporated off.
The product was then purified by column to give 470 mg of 2a as a yellow oil in 31% yield irom 5a.
Methyl (3S, 4R, 5S, 8if)-4-benzyI-3-terf-butoxycarbonylamino-l-aza-2-oxo-6- thiabicyclic [3.3.0]-octane-8-carboxylate (2a): [a]^^D = -88.7" (c = 0.98, CHCI3); 'H
NMR, 500 MHz, CDCI3, 5(ppm); 7.34-7.2 (5H, m), 5.04-5.0 (2H, m), 4.89(1 H, d, J = 3.5
Hz), 4.69 (IH, t, J = 8.0 Hz), 3.77 (3H, s), 3.38-3.34 (IH, m), 3.25 (IH, dd, J = 4.0, n.5
Hz), 3.04 (IH, dd, J = 5.0, 14.0 Hz), 3.02-2.94 (IH, m), 2.53(1H, m); '^C, 125 MHz,
CDCI3, §(ppm): 173.2, 169.7, 155.5, 138.1, 128.9, 128.7, 126.7, 80.5, 68.4, 57.6, 56.1, 52.9, 44.2, 36.9, 34.0, 28.3; HRMS (FAB) MH^ calculated for 324.1811, found
324.1818.
The same procedure was repeated for the debenzylation and reduction of 6a to only give the lactone 14 as a white solid in 58% yield.
3-terf-Butoxycarbonylamino-4-propyl-dihydro-furan-2-one (14): = -12.1° (c =
1.01, CHCI3), mp = 106-108 °C; NMR, 500 MHz, CDCI3, 5(ppm); 5.0-4.83 (IH, br),
4.40 (IH, t, J = 8.5 Hz), 4.11 (IH, br), 3.86 (IH, t, J = 10.0 Hz), 0.94 (3H, t, J = 7.5 Hz);
125 MHz, CDCI3, 5(ppm): 175.2, 155.5, 80.6, 70.4, 55.7, 43.0, 33.1, 28.4, 20.2,
14.1. HRMS (FAB) MH^ calculated for 244.1549, found 244.1559.
Synthesis of Nle-Gly BTD's via the Kazmaeir-Claisen rearrangement reaction
N"-Boc-Gly (10.0 g, 57.08 mmol) was dissolved in CH2CI2 (120 mL) and DMF (12 mL).
The solution was cooled to 0°C and DCC (15.0 g, 72.7 mmol) was added. The mixture was stirred for 10 min before adding trans 2-hexen-l-ol (8.5 mL, 72.05 mmol) and
DMAP (500 mg, 4.09 mmol) and stirring the mixture at 0°C for 30 min and at room temperature for 8 h. The mixture was filtered and the filtrate washed by saturated aqueous NH4CI (2 x 60 mL) and brine (80 mL). The organic phase was dried over
MgS04 and the solvent evaporated off The product was purified by column to give 13.0 g of 17 in 89% yield. In the Kazmaeir-Claisen rearrangement reaction, 17 (12.0 g, 46.66 mmol) was dissolved in THF (500 mL). ZnCb (0.5 M in THF, 112 mL, 56.0 mmol) was added and the mixture cooled to -78''C. Freshly prepared LDA (136.5 mmol) was slowly added and the mixture stirred for 20 min at -78°C. The cold bath was removed and the mixture stirred for 1 h. THF was evaporated off and the product dissolved in 1 N HCl (100 mL). The aqueous layer was extracted with ether (4 x 50 ml). The ether layer was evaporated to 100 mL and extracted with 1 N NaOH. The NaOH layer was acidified to pH = 2 by 6 N HCl and then extracted with ether (4 x 50 mL). The ether layers were combined and evaporated to 200 mL and washed with brine (40 mL). The ether layer was then dried over MgS04 and the solvent evaporated off. The crude product was dried under high vacuum and then dissolved in DCM (200 mL). The mixture was cooled to -
78°C and subjected to ozonolysis. Dimethyl sulfide (30 mL) was added and the mixture stirred for 70 h at room temperature. The solvent was evaporated off and the product dried under high vacuum. The dried product was dissolved in absolute ethanol (100 mL) containing 4 A° molecular sieves. Z-Cysteine methyl ester hydrochloride and DIPEA were added and the mixture stirred for 10 h. The molecular sieves were filtered out and the solvent evaporated off. The product was dried under high vacuum and dissolved in
CH2CI2 (400 mL). The mixture was cooled to 0°C and l-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride and DIPEA added. The mixture was stirred for 12 h and the solvent evaporated to 200 mL. The organic phase was washed by 1 N HCl (2 x 100 mL) and brine (100 mL). The solvent was dried over MgS04 and then evaporated off.
The products were purified by column to give 4.0 g of 2b and 5.8 g of 2c in a total yield of 59% from 17.
Methyl (Si^, 419, 5S, 8/?)-4-benzyl-3-ter^-butoxycarbonylamino-l-aza-2-oxo-6- thiabicyclic [3.3.0]-octane-8-carboxylate (2b): [a]^^D = -215.4" (c = 0.80, CHCI3); 'H
NMR, 500 MHz, CDCI3, 5(ppm): 5.19 (IH, q, J = 6.0 Hz), 5.07 (IH, q, J = 4.5Hz), 4.88
(IH, d, J = 4.5 Hz), 5.02-5.0 (IH, t, J = 8.0 Hz), 4.06 (IH, m) 3.77 (3H, s), 3.40-3.27 (2H, 57 m), 2.67 (IH, m), 1.84-1.79 (IH, m), 1.60-1.39 (IIH, m), 0.96 (3H, t, 7.5 Hz); '^C, 125
MHz, CDCI3, 5(ppm): 176.4, 170.4, 155.4, 80.3, 69.3, 59.0, 56.4, 52.8, 40.1, 35.3, 33.0,
28.3, 21.0, 14.0; HRMS (FAB) MH^ calculated for 359.1641, found 359.1632.
Methyl (3i?, 4R, 5S, 8i?)-4-benzyl-3-ter#-butoxycarbonylamino-l-aza-2-oxo-6-
thiabicyclic [3.3.0]-octane-8-carboxylate (2c): = -94.3° (c = 1.60, CHCI3); 'H
NMR, 500 MHz, CDCI3, 5(ppm): 5.16 (IH, q, J = 4.0 Hz), 4.95 (IH, d, J = 6.1 Hz), 4.75
(IH, d, J = 7.6 Hz), 4.42 (IH, t, J = 8.0 Hz), 3.38-3.27 (2H, m) 3.78 (3H, s), 3.38-3.27
(2H, m), 2.25-2.23 (IH, m), 1.85-1.79 (2H, m), 1.60-1.33 (IIH, m), 0.96 (3H, t, 7.1 Hz).
125 MHz, CDCI3, 5(ppm): 171.2, 169.5, 155.5, 80.2, 66.8, 59.4, 57.7, 53.4, 52.9,
35.0, 33.7, 28.4, 20.8, 14.1; HRMS (FAB) MH"" calculated for 359.1641, found
359.1637.
General procedure for N"-trifluoroacetaimde protection
The N"-Boc group was deprotected with TFA (20%) and the resultant compounds dissolved in CH2CI2 and treated with 2.0 eq trifluoroacetic anhydride and 3 eq pyridine to give the N"-trifluoroacetamide analogues.
Methyl (3S, 4R, 5S, 8i?)-4-benzyl-3-[2,2,2-trifluoroacetylamino]-l-aza-2-oxo-6- thiabicyclic [3.3.0]-octane-8-carboxylate (21a); white solid, mp = 119-121 °C, =
-151.9° (c = 0.58, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.48 (IH, d, J = 7.0 Hz),
5.08 (IH, q, J = 4.0, 8.0 Hz), 4.96 (IH, d, J = 5.0 Hz), 4.89 (IH, t, J = 8.0 Hz), 3.84 (3H, s), 3.53-3.41 (2H, m), 2.71-2.69 (2H, m), 1.50-1.38 (4H, m), 0.97 (3H, t, J = 7.5 Hz); '^C,
125 MHz, CDCI3, 5(ppm): 171.3, 170.4, 158.1 (q, J = 37.7 Hz), 116.1 (q, J = 286.2 Hz), 69.8, 58.0, 56.0, 53.6, 43.6, 37.2, 30.1, 21.0, 14.4; HRMS (FAB) MH^ calculated for
355.0939, found 355.0933.
Methyl (3S, 4S, 5S, 8if)-4-benzyl-3-[2,2,2-trifluoroacetyIamino]-l-aza-2-oxo-6- thiabicyclic [3.3,0]-octane-8-carboxylate (21b); white solid, mp = 109-111 °C, =
-259.0° (c = 0.69, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.37 (IH, d, J = 8.0 Hz),
5.21 (IH, d, J = 6.5 Hz), 5.01 (IH, q, J = 5.0 Hz, 8.5 Hz), 4.41 (IH, t, J = 9.5 Hz), 3.80
(3H, s), 3.47-3.29 (IH, m), 2.77-2.74 (IH, m), 1.78-1.62 (2H, m), 1.45-1.39 (2H, m),
0.96 (3H, t, J = 7.5 Hz); 5(ppm): 174.6, 170.3, 157.6 ( q, J = 37.6 Hz), 115.6 (q, J =
286.2 Hz), 69.3, 59.0, 55.4, 53.0, 39.6, 35.4, 32.9, 20.9, 14.0; HRMS (FAB) MH"' calculated for 355.0939, found 355.0933.
Methyl (3R, 4R, 55, 8/?)-4-benzyl-3-[2,2,2-trifluoroacetylamino]-l-aza-2-oxo-6- thiabicyclic [3.3.0]-octane-8-carboxylate (21c): white solid, mp = 145-147 °C = -
144.8" (c = 0.54, CHCI3); ^H NMR, 500 MHz, CDCI3, 6(ppm): 7.21 (IH, d, J = 7.5 Hz),
5.13 (IH, q, J = 3.5, 7.0 Hz), 4.81 (IH, d, J = 7.5 Hz), 4.77 (IH, t, J = 8.5 Hz), 3.79 (3H, s), 3.42-3.33 (2H, m), 2.42-2.38 (2H, m), 1.81-1.75 (IH, m), 1.68-1.63 (IH, m), 1.45-
1.39 (2H, m), 0.96 (3H, t, J = 7.5 Hz); 125 MHz, CDCI3, 5(ppm): 169.3, 169.1,
157.7 (q, J = 37.7 Hz), 115.6 (q, J = 286 Hz), 67.0, 58.3, 57.7, 53.1, 52.5, 35.4, 33.5,
20.7, 14.0; HRMS (FAB) MH^ calculated for 355.0939, found 355.0933. 59
CHAPTER 4
SYNTHESIS OF Nle-Asp BICYCLIC DIPEPTIDE MIMETICS
4.1 Introduction
To introduce a turn at the C-terminal of our target peptides, a Nle-Asp bicyclic dipeptide mimetic was designed to be substituted for the Nle-Asp dipeptide unit in Tyr-Nle-Gly-
T rp-Nle-Asp-Phe-NH2.
3
Figure 4.1 Bicyclic dipeptide mimetic for Nle-Asp dipeptide unit
Synthesis of indolizidinone type compounds through dehydroamino acid intermediates has been reported by a number of groups. Revesz employed the Schollkopf bislactam ether methodology to sjoithesize the indolizidinone scaffford without any side chain appendages. Lubell assembled all the isomers of the indolizidinone skeleton by a
Claisen condensation of two identical glutamic acid derivatives.Kahn followed a similar approach to synthesize one stereoisomer of the indolizidinone.'^^ In our group, synthetic methodologies have been developed to introduce side chains on both sides of the ring, starting from analogues of pyroglutamic acid.''^^"'"'^ Based on this work, the synthesis of a bicyclic dipeptide mimetic for Nle-Asp starting from aspartic acid was designed (Figure 2.7). Oxidation of carbamate protected 5,£-unsaturated amino acids leads to the formation of hemiaminals instead of the free aldehyde. The hemiaminal can be converted to 2,4-pyrrolidinedicarboxylate (PDC), an inhibitor of glutamate uptake. 60
L-glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. In this role, glutamate binds to a number of different proteins, transport systems, and enzymes/^^ In addition, abnormal levels of glutamate have been linked to neurological disorders, including, ischemia, anoxia, hypoglycemia, epilepsy, and
Huntingtons, Parkinsons, and Alzheimers diseases.As a result, conformationally restricted glutamate analogues were synthesized to study the interactions between synthetic amino acids and the proteins to which glutamate binds. The L-trans isomer of
PDC gave the most potent and selective inhibitor of glutamate uptake.'^'
BF30Et2 MeO-x ,SiMe-5 ft Boc C02Bn Et20 Boc C02Bn xix xviii RiRsCH^CHLi BF30Et2 TMS = -TMS CuBr.Me2S SnCU/AICU
TMS-
Boc C02Bn Boc C02Bn XX xxi
Scheme 4.1 Synthesis of analogues of proline.
Methylation of the hemiaminals, generation of N-acyliminium ion and addition of vinyl cuprates gives 5-vinylproline analogues (Scheme 4.1). 204-205 Ethynyl groups can also be introduced at the S-position.^"^ The hemiaminals also can be allylated to give analogues of proline that are substituted with an allyl group at Elaboration of both the allyl 61 or the vinylic group can afford dehydroaminoacids which on asymmetric hydrogenation and lactam cyclization would give indolizidinone analogues.
4.2 Synthesis of analogues of proline
When 5b was subjected to ozonolysis, only the cyclized hemiaminal 22 was obtained.
The hemiaminal was then reacted with BF3.0Et2 and allyl trimethyl silane to give compounds 24a and 24b in low yield (Scheme 4.2). The yield was improved by methylating the hydroxyl group and the resultant compound 23a reacted with BF3.0Et2 and allyl trimethyl silane at -78°C RT to give compounds 24a and 24b in 48 % yield and a ratio of 1:1. When BFs.OEti and allyl trimethyl silane were added at -VS^C and the reaction warmed to room temperature, the two products were formed in equal amounts.
North et al. have reported similar reaction in which the hemiaminal was reacted with
TMS-CN or trimethyl silane.'^'' The yields for the reactions were however low, typically
20%. 62
MeOH PTSA
24a 24b
1 1
Scheme 4.2 Synthesis of cis-dicarboxylate prohne analogues
Multzer et al. have shown that neigbouring group participation of the alpha carboxyl group in xviii (Scheme 4.1) leads to stereoselective formation of the cis isomer.This stereoselectivity is attributed to the facial preference with which allylsilane attacks the cyclic acyliminium intermediate generated by BF3 catalyzed Ome-elimination from aminal xviii. The mechanistic pathway would involve the formation of a BF3 complex with the ester in which the fluoride would acquire sufficient nucleophilicity to attack the trimethylsilyl group and, hence, facilitate the concomitant allyl transfer to the iminium function (Figure 4.2). Fa Mea
H Boc
Figure 4.2 Transition state model for the generation of proline analogues
The structure of compound 24a was proved by X-ray Crystal structure (Figure 4.3).
0I7I II V / / \\ i. , - J 0ioi6i) 01121 ^CSJ3! ^ , \ i .o; VfX
Figure 4.3 X-ray crystal structure of 5-allyl substituted proline analogue
When the minor isomer 6b was subjected to osmylation and allylation, product 24c obtained in 51 % yield (Scheme 4.3).
O3 Me0-.^,.^,.uC02Me BocHN. .. ^ \. / MeOH ( ) ^ Y COsMe DMS / CO^Bn Boc CO^Bn goc^ ^O^Bn 6b 22b 23b BF30Et2 .ivvCOoM©
Et20 Boc^ 'C02Bn 24c
Scheme 4.3 Synthesis of trans-dicarboxylate proline analogue 64
The three 5-allyl substituted proUne analogues (24a, 24b, and 24c) were further characterized by NOE experiments. The stereochemistry of compound 24c was confirmed irom the nOe data of the final bicyclic dipeptide mimetic.
1.6%
H COsMe H COaMe \ / ''34.8% ^ \ ''J A.2% \ /^^)1.6% boc'^ C02Bn Boc'^ C02Bn Boo'' C02Bn 24a 24b 24c
Figure 4.4 nOe analysis of 5-allyl substituted proline analogues
4.3 Synthesis of bicyclic dipeptide mimetics for Nle-Asp.
Compound 24a was subjected to ozonolysis and the resultant aldehyde 25a subjected to a
Homer-Emmons olefination^"^ to give the dehydroamino acid 26a in 58% yield. When
Osmylation was used for the oxidation of 24a, followed by olefination, 71% yield of the dehydroamino acid was obtained. Attempts to cyclize 26a failed possibly due to the E- stereochemistry of the alkene that keeps the ester fiinctionality away fi"om the amine.
Asymmetric homogeneous hydrogenation^"^'^^'' of 26a using Burks catalyst [Rh(I) (COD)
(S,S)-Et-DuPHOS]^" gave 27a in high yields. The catalyst high asymmetric induction is attributed to two factors. First, the substrate olefin coordinates in a bidentate manner, with the amide oxygen acting as the second ligand, to form two diastereomeric olefin complexes.^®^ The two diastereomers undergo reaction at substantially different rates and they equilibrate rapidly with the rate determining step being the oxidative addition of hydrogen to the metal olefin complex. The minor diastereomer undergoes this reaction at a rate 10^ greater than the other and provided the two diastereomers can equilibrate 65 rapidly, high asymmetric induction is achieved. However, high hydrogen pressure and lower temperatures decrease the enantioselectivity since both interfere with this equilibration. For the diphosphine catalysts such as DIOP, DIP AMP, CHIRAPHOS,
BPPM and BINAP, asymmetric induction is primarily effective with Z- acetamidoacrylates. The E-isomer undergoes E-Z isomerization that often compromises the enantiselectivity. In contrast, related catalyst containing the phosphole ligands
DUPHOS or BPE ligands reduce both E and Z acetamidoacrylates with equally high enantioselectivity, and, for a given catalyst configuration, the same absolute stereochemistry is obtained regardless of olefin geometry.
Ph PPh2
yV-pph. c'^Pho::
DIOP CHIRAPHOS DIPAMP BPPM
PP.. cjp^pp
R R R R BINAP DuPHOS BPE
Figure 4.5 Optically active phosphines
Asymmetric hydrogenation was followed by deprotection of the Boc group, then the compound was dissolved in pyridine and heated at 50°C for 4 days to give the indolizidinone 3a in 50% yield from 24a. 66
^ /'^x^COoMe osO/i /^\^C02M6 \ / % \ / (Me0)2P0CH(NHCbz)C02Me N—( Nal04 DBU ^ Boc C02Bn Boc C02Bn 68% 24a 25a
Me02C rn MP CbzHN ^ /\^C02Me y //-„./\^C02Me CbzHN (S,S)-COD-EtDUPHOS Rh(l)OTf Boc^ COsBn ^2 c02Bn 26a 27a
1. 20% TFA/DCM^ CO2M6 2. pyridine, 50°C CbzHN C02Bn
Scheme 4.4 Synthesis of Nle-Asp bicyclic dipeptide mimetic
Oxidation of compound 24b by ozonolysis followed by Homer-Emmons olefination gave the desired compound 25b in a yield of 19%. An unidentified compound was also formed in a similar yield. The reaction was optimized by changing the protocol to oxidation by osmylation followed by olefination to give the desired dehydroamino acid in 70% yield.
From these results it is clear osmylation gives better results in the oxidation of the proline analogues. Compound 25b was then subjected to asymmetric hydrogenation, N"-Boc deprotection and cyclization to give the desired bicyclic compound 3b in 65% yield. 67
'COzMe 0s04^ (Me0)2P0CH(NHCbz)C02Me N-C Nal04 N-(^ DBU Boc COjBn Boc COsBn 24b 25b
MeOaC CbzHN W t /•^^/X^COoMe \ / (S,S)-COD-EtDUPHOS Rh(l)OTf \ [ ^ CbzHN N-(^ " MeOaC n—( Boc^ C02Bn 95% Boc^/ COoBn 26b 27b H
1 • 20% TFA/DCM^ I ^ )—COaMe 2. pyridine, 50°C CbzHN Q C02Bn 3b
Scheme 4,5 Synthesis of Nle-Asp bicyclic dipeptide mimetic
Compounds 24c was subjected to the same reaction conditions in Scheme 4.6 to give the bicyclic dipeptide mimetics 3c (Figure 4.4).
H
CbzHN
3c
Figure 4.6 Bicyclic dipeptide mimetic for Nle-Asp
4.4 Characterization of Nle-Asp bicyclic dipeptide mimetics
The three Nle-Asp bicyclic dipeptide mimetics were characterized by IH NMR, DQF
COSY and 2D transient nOe. The three bicyclic dipeptide mimetics were then characterized by nOe measurements. A strong nOe value was observed for and H*' in compounds 3b and 3c (4.2% and 4.0%) respectively) signifying a cis relationship between the two hydrogens. For compound 3a, the nOe value between and was relatively 68
low (1.9%) due to the trans relationship between the hydrogens. The cis relationship
between and in 3a and 3b resulted in a strong nOe value of 4.8% compared with
the weak value of 1.9% observed for compound 3c.
CbzHN CbzHN CbzHN 3a 3b 3c
Figure 4.7 nOe data for Nle-Asp bicyclic dipeptide mimetics
4.5 Synthesis of P-thiol substituted aspartic acid
A Nle-Asp bicyclic dipeptide mimetic can also be synthesized from P-vinyl substituted
Nle (products of Kazmaier-Claisen rearrangemement) and P-thiol substituted aspartic
acid (Figure 4.3). P-Thiol substituted aspartic acid can be synthesized by the P- sulfenylation of aspartic acid with sulfur electrophiles. Sulfenyaltion has been reported by
Enders and Klumpen in which case di-toluoyl disulfide and methyl disulfide were used as electrophiles.^'^
PGHN PGHN O CO2M0 CO2H CO2H
XXII xxiii xxiv
Figure 4.8 Retrosynthetic analysis for a thiol BTD for Nle-Asp 69
When the Cbz protected aspartic acid 28 was subjected to sulfenylation with benzyl disulfide at -42°C, a-alkylated products were preferentially formed. The desired product
29 was obtained when the reaction was carried out at -78°C.
SBn CbzHN-^C02'Bu I-LHDMS/HMPA ^O^Bn 2.BnSSBn ' C02Bn 28 29
Scheme 4.6 Sulfenylation of aspartic acid with benzyl disulfide
Further manipulation of 29 to get the Iree amino acid failed. Sodium in liquid ammonia reduction^^"^ gave products that could not be identified while no reaction was observed with Pd/C in liquid ammonia or transfer hydrogenation.^'^ To circumvent the problem described above, attempts to sulfenylate Boc protected aspartic acid were undertaken.
Matsueda and coworkers have reported the synthesis of 3-nitro-2-pyridinesulfenyl (Nyps) halides that react with amino, hydroxyl and thiol functions to form sulfenamides, sulfenates, and mixed disulfides, respectively. 70
OMe
1. LHDMS/HMPA BocHN CO2M© COz'Bu 2. (p-MeOC6H4CH2)2 BocHN CO2M6 30 COz'Bu 31
SH
NypsCI BocHN cOsMe nBu3P/acetone/H20 COs'Bu BocHN 33
COs'Bu 32
Scheme 4.7 Synthesis of Nyps protected aspartic acid
The Nyps group can also serve as a protecting group and in activation during peptide synthesis. This group is easily removed under neutral conditions using tertiary phosphine and water, but it is sufficiently resistant to acids such as TFA, HCl, and HF. We consequently designed sulfenation of aspartic acid with (p-MeOC6H4CH2)2S which would give a p-Methoxybenzyl protected thiol 31 that is easier to convert to Nyps disulfide 32 (Scheme 4.8). (p-MeOC6H4CH2)2S was synthesized in large scale using a 9 1 Q method reported in literature. It is well established that DMSO in combination with a variety of acidic co-reagents can be used for the conversion of thiols to disulfides.
However DMSO has a low oxidizing power, a problem that can be circumvented by prior treatment of DMSO with oxophilic co-reagents under acidic conditions.^^" 1,1,1,3,3,3-
Hexamethyldisilazane (HMDS), a weak but stable oxophilic base was found to effect the conversion of thiols to disulfides in high yields. Although the precise role of HMDS is 71
not known, it has been postulated that HMDS reacts with the first molecule of the thiol to
give a reactive species xxv and trimethylsilyl amine (Scheme 4.5). Intermediate xxv in
turn reacts with the second thiol molecule in the presence of trimethylsilylamine with
concomitant release of NH3 to afford the corresponding oxonium intermediate xxvi.
Intermediate xxvi collapses to form the disulfide along with the evolution of
dimethylsulfide and hexamethyldisiloxane as by-products. Using this methodology, 4-
methoxy-a-toluenethiol was converted to the disulfide in the presence of 1.2 eq of
HMDS in 76% yield.
MegSi—N-SiMeg + DMSO H RSH
OSiMea + Me3SiNH2 H,C- CH. SR xxv
NHo MegSiNHj
MeaSiO,© OSiMe ©S-R — RS-SR H.C Me3-0-SiMe3 Me-S-Me XXVI
Figure 4.9 Mechanism for the synthesis of disulfides
All attempts of effecting the sulfenation of N"-Boc-protected aspartic acid with (p-
MeOC6H4CH2)2S and benzyl disulfide failed with only the starting material being recovered. Though the actual reason for the failure of this reaction is not clear, solubility of the disulfides at the low temperatures was found to be low. 72
4.6 Experimental
General procedure for the synthesis of 5-allyl substituted proline analogues
In 500-mL flask, 5b was dissolved in dichloromethane (300 mL and cooled to -78''C. The solution was exposed to O3 until a persistent blue color appeared. Dimethylsulfide (50 mL) was added and the mixture stirred at room temperature for 60 hr. The solvent was evaporated off and the product dried under high vacuum. The dried product was dissolved in methanol (100 mL) and para-toluene sulfonic acid (220 mg, 1.16 mmol) added. The mixture was stirred at room temperature for 12 hr and the solvent evaporated off. The product was redissolved in ethyl acetate (150 mL) and the organic layer washed by aqueous NH4CI (2 x 50 mL) and brine (50 mL). The organic phase was dried over
MgS04 and the solvent evaporated. The product was dried under high vacuum to give
8.16 g of crude product, a 91.5 % yield. The crude product was dissolved in ether (120 mL) and cooled to -78°C. BF30Et2 (8.0 mL, 63.13 mmol) and allyl trimethyl silane (10.0 mL, 62.92 mmol) were added and the mixture stirred at -78''C for 20 min. The cold bath was removed and the mixture stirred for a fiarther 40 min before cooling to -78"C and slowly adding concentrated aqueous NaHCOs. The solution was slowly warmed to room temperature and the aqueous phase separated. The organic phase was washed with concentrated aqueous NaHCOs (2 x 50 mL) and brine (50 mL). The organic phase was dried over MgS04 and the solvent evaporated off. The product was purified by column to give 2.45 g of a white solid 24a and 2.59 g of a yellowish Hquid 24b in a total yield of
55%. When the reaction was repeated with 6b, only one 5-allyl proline analogue 24c was obtained in 50% yield. 73
Benzyl (2S, 3R, 5if)-5-allyl-l-(ter/-butoxycarbonyI)-3-niethoxycarbonyl prolinate
(24a): [a]^^D = -0.016° (c = 0.62, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.44-7.27
(5H, br), 5.81-5.67 (IH, m), 5.19-4.99 (4H, m), 4.59 (0.4H, d,J= 8.0 Hz), 4.50 (0.6H, d,
J= 8.5 Hz), 4.22-4.14 (0.6H, td, J= 3.0, 9.0 Hz), 4.10-4.02 (0.4H, td, /= 3.0, 9.0 Hz),
3.54 (2H, s), 3.46 (IH, s), 3.42-3.28 (IH, m), 2.63-2.55 (0.6H, br), 2.54-2.40 (1.4H, m),
2.20-2.11 (lH,m), 1.91 (IH, q, J=6.5 Hz), 1.46 (3.6H, s), 1.32 (5.4H, s); '^C, 125 MHz,
CDCI3, 5(ppm): 170.63, 170.54, 170.40, 170.1, 154.1, 153.3, 135.42, 135.19, 134.48,
134.37, 128.72, 128.51, 128.40, 128.36, 128.12, 117.87, 117.78, 80.51, 80.43, 67.18,
67.07, 61.87, 61.66, 56.86, 56.79, 52.02, 51.97, 45.1, 44.1, 39.3, 38.4, 31.0, 30.0, 28.3,
28.1; HRMS (FAB) MH^ calculated for 404.2073, found 404.2066.
Benzyl (2S, 3R, 55)-5-allyl-l-(ter^-butoxycarbonyI)-3-methoxycarbonyl prolinate
(24b): [a]^^D = +0.17''(c = 1.39, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.44-7.28
(5H, br), 5.80-5.65 (IH, br), 5.22-4.96 (4H, m), 4.83 (0.4H, d, /= 6.5 Hz), 4.68 (0.6H, d,
J= 8.5 Hz), 3.90-3.71 (IH, br), 3.63-3.41 (3H, br), 3.25-3.12 (IH, br), 3.09-2.97 (0.6H, br), 2.87-2.76 (0.4H, br), 2.36-2.11 (3H, m); 125 MHz, CDCI3, 5(ppm): 170.7,
170.3, 153.9, 153.2, 135.3, 134.4, 128.6, 128.45, 128.38, 128.2, 117.3, 80.4, 67.0, 61.7,
61.4, 57.5, 52.0, 45.4, 44.9, 39.7, 38.4, 33.4, 32.7, 28.3, 28.2; HRMS (FAB) MH^ calculated for 404.2073, found 404.2061.
Benzyl {2S, 3S, 55)-5-allyl-l-(terr-butoxycarbonyl)-3-methoxycarbonyl prolinate
(24c): [a]^^D = 0.1165" (c = 1.05, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm); 7.40-7.30
(5H, br), 5.84-5.69 (IH, br), 5.36-4.98 (4H, m), 4.72-4.65 (0.3H, br), 4.57 (0.5H, d, J =
6.5 Hz), 4.07 (0.65H, br), 3.97 (0.35H, br), 3.67 (3H, s), 3.25-3.13 (IH, br), 2.76-2.66 (0.6H, br), 2.63-2.53 (0.4H, br), 2.32-2.13 (2H, br), 2.10-1.96 (IH, br), 1.55-1.28 (9H,
br); 125 MHz, CDCI3, 5(ppm): 172.1, 171.6, 171.3, 170.6, 153.8, 153.2, 135.6,
135.3, 134.6, 134.4, 134.3, 128.4, 128.3, 128.2, 117.0, 80.4, 77.2, 66.8, 62.3, 57.5, 52.3,
46.5, 45.6, 39.1, 38.2, 33.4, 32.5, 28.3, 28.0; HRMS (FAB) MH^ calculated for 404.2073,
found 404.2063.
General procedure for the synthesis of dehydroamino acids
Compound 24a (1.83 g, 4.54 mmol) was dissolved in THF (60 mL)/H20 (30 mL)
mixture. OSO4 (60 mg, 0.24 mmol), was added and the mixture stirred at room
temperature for 10 min before adding Nal04 (2.7 g, 12.62 mmol) and stirring the mixture
for a fiirther 4 hr. The mixture was filtered and the THF evaporated. Ethyl acetate (50
mL) was added and the aqueous layer separated. The organic phase was washed with
saturated NH4CI (2 x 30 mL) and brine (30 mL). The organic phase was dried over
MgS04 and the solvent evaporated off. The crude product was dried under high vacuum
and used in the next step without purification. In another 100-mL flask, N-
(benzyloxycarbonyl)-a-phosphonoglycine trimethyl ester (1.7g, 5.13 mmol) was dissolved in CH2CI2 (30 mL). DBU (750 }iL, 5.02 mmol) was added and the mixture stirred for 10 min. The crude product from above, dissolved in CH2CI2 (10 mL) was added and the mixture stirred at room temperature for 8 hr. The CH2CI2 layer was diluted to 60 mL and washed with aqueous 1 N HCl (2 x 30 mL) and brine (30 mL). The organic phase was dried over MgS04 and evaporated off The product was then purified by column to give 1. 91g of yellowish liquid 26a in 71% yield. 75
General method for the synthesis of Nle-Asp bicyclic dipeptide mimetics
The dehydroamino acid 26a (2.4g, 3.92 mmol) was dissolved in degassed methanol (50 mL). [Rh-(S,S)-EtDuPHOS]-OTf (6 mg, 0.008 mmol) was added and the mixture exposed to hydrogen at 70 psi for 20 hr. The solvent was evaporated and the product purified by passing it through a short silica column using ethyl acetate. The solvent was evaporated and the product, obtained in quantitative yield, dried under high vacuum. The product was then dissolved in CH2CI2 (30 mL) and cooled to 0°C. TFA (10 ml) was added and the mixture stirred at O^C for 10 min and at room temperature for 40 min. The solvent was evaporated off and the product dissolved in ethyl acetate (50 mL). The organic layer was washed with NaHCOs (2 x 30 mL) and brine (30 mL). The organic phase was dried over MgS04 and the solvent evaporated off The crude product was dried under high vacuum and dissolved in pyridine (250 mL). The mixture was then heated at
50°C for 4 days. The solvent was then evaporated off and the product dissolved in ethyl acetate (50 mL). The organic phase was washed by aqueous I N HCl (2 x 20 mL) and brine (30 mL). The product was purified by column to give 1.0 g of 3a in 53 % yield.
(3S, 6R, SR, 95) Benzyl 2-oxo-3-N-(benzyloxycarbonyl)amino-8-methylester-l- azabicyclo[4.3.0]nonane-9-carboxylate (3a): = -15.9" (c = 0.6, CHCI3); 'H NMR,
500 MHz, CDCI3, 5(ppm): 7.58-7.22 (IIH, m), 6.5 (IH, br), 5.11 (4H, d, J = 8.5 Hz),
4.04 (IH, d, 7.0 Hz), 3.78-3.68 (3H, m), 3.46 (3H, s), 3.34-3.26 (IH, m), 2.38-2.22
(3H, m), 1.81-1.71 (IH, m);'^C, 125 MHz, CDCI3, 6(ppm): 172.7, 171.8, 164.9, 154,
136.1, 135.2, 131.4, 128.58, 128.54, 128.48, 128.44, 128.17, 128.14, 128.03, 67.23, 76
67.17, 62.5, 57.3, 52.3, 51.8, 47.3, 35.0; HRMS (FAB) MH^ calculated for 481.1975, found 481.1958.
(3S, 65, 8R, 9S) Benzyl 2-oxo-3-N-(benzyloxycarbonyl)amino-8-methylester-l- azabicyclo[4.3,0]nonane-9-carboxylate (3b): [a]^^D = +5.2" (c = 0.6, CHCI3); 'H NMR,
500 MHz, CDCI3, 5(ppm): 7.40-7.32 (lOH, m), 5.79 (2H, d, J = 5.5 Hz), 5.13 (4H, s),
4.78 (IH, d, /= 8.5 Hz), 4.23-4.15 (IH, m), 3.83-3.71 (IH, m), 3.48 (3H, s), 3.35-3.26
(IH, m), 2.56-2.45 (IH, m), 2.38 (IH, p, J= 6.28 Hz), 2.24-2.05 (2H, m), 1.83-1.71 (IH, m), 1.68-1.56 (IH, m); '^C, 125 MHz, CDCI3, 5(ppm): 169.9, 169.0, 156.0, 136.4, 135.0,
128.6, 128.5, 128.4, 128.02, 127.94, 67.6, 66.8, 60.1, 55.7, 52.1, 50.2, 45.7, 34.9, 26.91,
26.67; HRMS (FAB) MH^ calculated for 481.1975, found 481.1995.
(3S, 6S, 8S, 9S) Benzyl 2-oxo-3-N-(benzyloxycarbonyl)amino-8-methylester-l-
azabicyclo[4.3.0]nonane-9-carboxylate (3c): [a]^^D = +34.9° (c = 1.2, CHCI3); 'h
NMR, 500 MHz, CDCI3, 5(ppm): 7.41-7.27 (lOH, m), 5.77 (IH, d, J= 4.5 Hz), 5.18 (2H, q, /= 12.0 Hz), 5.11 (2H, s), 4.88 (IH, s), 4.26-4.14 (IH, br), 3.90-3.77 (IH, br), 3.73
(3H, s), 3.12 (IH, d,J= 7.5 Hz), 2.58-2.42 (2H, m), 2.14-2.01 (IH, br), 1.91-1.81 (IH, m), 1.70-1.58 (2H, m); '^C, 125 MHz, CDCI3, 6(ppm): 171.8, 170.0, 168.8, 156.0, 136.4,
135.1, 128.6, 128.5, 128.4, 128.2, 128.01, 127.96, 67.5, 66.7, 61.0, 55.1, 52.7, 50.3, 46.1,
34.9, 26.9, 26.6; HRMS (FAB) MH^ calculated for 481.1975, found 481.1958.
Synthesis of p-methoxybenzyl disulfide
4-metlioxy-a-toluene thiol (10.0 mL, 71.8 mmol) was dissolved in CH3CN (90 mL) and dimethyl sulfoxide (16.0 mL, 225.4 mmol) added. To the solution was added hexamethyldisilazane (18.0 mL, 85.3 mmol) and the mixture stirred for an overnight. 77
Aqueous NaOH (10%, 70.0 mL) was added and the organic phase evaporated off. The product was then dissolved in CH2CI2 (50.0 mL) washed by 10%) NaOH (2 x 30 mL) and water ( 2 x 30 mL). The organic phase was dried over MgS04 and the solvent evaporated off. The product was then recrystallized from ethyl acetate/hexanes to give 7.84 g of product in 71.3% yield. 78
CHAPTER 5
SYNTHESIS OF BICYCLIC DIPEPTIDE MIMETICS FOR Asp-Gly AND
homoPhe-GIy
5.1 Introduction
The dipeptide Asp-Phe is found at the C-terminal of CCK-peptides. Deltorphin I (Tyr-D-
Phe-Asp-Val-Val-Gly-NH2) also has a central Asp-Val dipeptide. As was described in
Chapter 3, our protocol for the synthesis of bicyclic dipeptide mimetics employs 5,s- and y,5-unsaturated amino acids and cysteine or its analogues. P-Phenyl substituted cysteine has been synthesized in our group221 ' 222 and if coupled with P-allyl substituted aspartic acid would give a bicyclic dipeptide mimetic for Asp-Phe. In this chapter, the protocol for the synthesis of an Asp-Phe bicyclic dipeptide mimetic and other aspartic acid- containing bicyclic mimetics is developed by synthesizing a bicyclic dipeptide mimetic for Asp-Gly.
5.2.1 Synthesis of Asp-Gly bicyclic dipeptide mimetic
It has been established that the oxidation of the carbamate protected P-allyl amino acids leads to a 5-membered cyclic hemiaminal instead of the free aldehyde. This problem can be avoided by protecting the amino group as a trifluoroacetamide or by bisprotection.
Introduction of a second Boc group was unsuccessful due to steric hinderance of p- substitution. A number of methods for protection of an amino group as a trifluoroacetamide were attempted. Deprotection of the N"-Boc group followed by coupling with ethyl trifluoroacetamide^^^ gave the product in non-reproducible and variable yields (25-45%) while use of trifluoroacetic anhydride^'"' led to the formation of 79 a mixture of products. A recently reported method that uses (trifluoroacetyl) benzotriazole was found to be more efficient. This reagent is easily synthesized in large scale from benzotriazole and trifluoroacetic acid in THF (Scheme 5.1).
(CF3C0)20
Scheme 5.1 Synthesis of trifluoroacetyl benzotriazole
When 5a was treated with TFA followed by reaction with 35, the N"-trifluoroacetyl amino acid 36a was obtained in good yield (Scheme 5.2). Compound 36a was then subjected to oxidation with Osmium tetraoxide and NaI04 to afford aldehyde 37a. In a one pot reaction, the aldehyde was coupled with Z-cysteine to form two bicyclic dipeptide mimetics 38a and 38b, via the formation of an A^ji'-thiazolidine, bicyclization and methylation. 1.20% TFA/CH2CI^ BocHN 2. THF, 35. TEA TFAHN COoBn C02Bn 76% COoM© CO2M© 36a
1. L-Cys, Pyr,RT,4hr; OSO4 Pyr, 50°C,3days TFAHN Nal04 C02Bn 2.o r-uCH2N2 M 480/, CO2M6 37a
Bn02C, Bn02a,^^y^g
TFAHN TFAHN 0 C02Me o C02Me 38a 38b 2 : 1
Scheme 5.2 Synthesis of Asp-Gly bicyclic dipeptide mimetics
Subjecting the minor isomer 6a to the same set of reaction conditions afforded only bicyclic dipeptide 38c (Scheme 5.3).
^ 1-20%TFA/CH2CI^ r BccHN^^O.Bn ^ ™™Y^CO.Bn ^ C02Me C02Me 5b 36b
^ 1. L-Cys, Pyr,RT,4hr: Bn02C^^^^y g Pyr, 50°C,3days TFAHN^\ TFAHN''^,^'^- T 2, CH2N2 52% ¥ COzMe ^ ^ ^ c02Me 37b 38c
Scheme 5.3 Synthesis of Asp-Gly bicyclic dipeptide mimetic 5.2.2 Characterization of Asp-Gly bicyclic dipeptide mimetics
The 'H-spectra were assigned by DQF-COSY while the stereochemistry of the three bicyclics was assigned by ID transient nOe experiments (Figure 5.1). A lot of nOe data was obtained for compound 38c and 38a but there were more overlaps for compound
38b. In all the isomers, H3 was well resolved and was irradiated to determine the stereochemistry at C-4 and C-6. In 38a and 38b, a weak nOe indicative of a trans relationship was observed for H3 and H4. In 38c, a cis relationship between H3 and H4 is proved by the strong nOe value. A cis relationship for the bridge-head He with H3 in 38a and 38c is confirmed by the strong nOe's (1.50 % and 2.52 % respectively) in 38a and
38c. In comparison, 38b shows an nOe value of 0.20 % that would be as a result of a trans relationship. The coupling constants could not be used to assign the stereochemistries due to overlap of the critical hydrogens.
The formation of two products from the i2S,3R) isomer 5a but only one product from the
{2S,3S) isomer 6a agrees with earlier work done in our labs. However, the stereochemical outcome of the bridge head-H in the three bicyclic compounds is "unusual" in that the favored product would have to be formed via the less thermodynamically stable cis- relationship in the formation of thiazolidine. This may indicate an extra effect of the 4- benzyl carboxylate group other than steric hinderance in the formation of the thiazolidine ring. The possible pathway in this cascade three-bond formation process is not known. 82
Bn02Q-^ •6 Bn02Q> Hfi s ,Nn8bH ^wHsb H4 H4 TFAHN TFAHN* ^N^y ^Hga V'Hg CO2M6 0 C02Me 38b proton %nOe proton %nOe
*H3 H4 0.60 *H, H4 0.55 He 1.50 He 0.20 *H8a Hg 0,96 Hsb 1.40 *H8b He 0.60 H3 0.17 Hg 1.81 Hsa 1,75 Hsb 0.60
Bn02Q ' De S .sHsb
TFAHN COoMe ' the in-adiated proton in nOe experiments proton %nOe
*H3 H4 2.32 He 2.52 *H8a He 0.23 Hg 1.18 *H8b Hg 2.12 He 0.83
Figure 5.1 nOe observed for Asp-Gly bicyclic dipeptide mimetics.
5.3 Synthesis of a homoPhe-Gly bicyclic dipeptide mimetic
When 7a was subjected to hydrogenation and BOP, DIPEA and NaBH4 reduction,the alcohol 39, contaminated by the lactone, was obtained in 53% yield. The alcohol was then subjected to the Swem oxidation. The crude aldehyde was then coupled with L- cysteine under the same set of reaction conditions as described in Scheme 5.2 to give 1 in
42% yield. 83
.Ph 1. H2, Pd/C 1 nOH BocHN, BocHN r C02Bn 2.B0P,DIPEA CO2MG NaBH4 CO2M6 53% 7a 39 Ph 1. Swern 2. L-Cys, Pyr, RT, 4 3. Pyr, 50°C, 3 days BocHN— 4. CH2N2 42% O COoM© 1
Scheme 5.4 Synthesis of homoPhe-Gly bicyclic dipeptide mimetic.
The stereo (Figure 5.2). Figure 5.2 X-Ray crystal structure of homoPhe-Gly bicyclic dipeptide mimetic Compound 1 was consequently used in the synthesis of analogues of Leu-enkephalin as illustrated in Scheme 5.5. 84 Ph Ph ( H 0 ^ h KJUS I.TFA/CH2CI2 BocHN BocHN-^ I, > ' 2. Boc-Gly, DCC, DIPEA N O ^ C02Me O CO2M6 41 1.TFA/CH2CI2 1.TFA/CH2CI2 2. Boc-Tyr{Boc)-OH, DCC 2. Boc-Tyr(Boc)-OH, DCC DIPEA DIPEA OBoc O BocHN C02Me BocHN BocO O CO2M6 1. 0.5N LiOH 1. 0.5N LiOH 2. 20%TFA / CH2CI2 2. 20%TFA / CH2CI2 Ph O Vf Ph ^ H V-N O CO2H HO O CO2H JMN4 JMN3 Scheme 5.5 Synthesis of analogues of Leu-enkephalin Different attempts were made to manipulate the p-benzyl substituted aspartic acid. Deprotection of the P-carboxyl group in 7c followed by reduction afforded the alcohol in 70% yield with no lactone formation. This may be explained by the the bulky protection groups that hinder cyclization. Attempts to mesylate the alcohol^^^ led to formation of the lactone, a side reaction which was more severe when PBrs^^'' was used. To lower the acidity of the reaction medium, carbon tetrabromide and phosphine^^^ were used. The bromide 45 was obtained in excellent yield. However reaction of the bromide with NaCN to give the nitrile failed while deprotection of the Cbz and Benzyl esters with hydrogenation also failed. 85 PH Ph CbzHN,.A^OH1 c CBr4 Ho COaBn CH3CN CbzHN^^ 44 0 45 Ph PPhs r CBr4 CbzHN^J^Brr CH2CI2 + CbzHN^^( COaBn 0 48% 45 46 P. PPhg CbzHN.A^Br CBr4 THF COaBn 45 Scheme 5.6 Bromination of P-benzyl substitited homoserine 5.4 Experimental General procedure for N"-deprotection and reprotection as a trifluoro acetamide In a SO-niL flask, 5a (690 mg, 1.83 mmol) was dissolved in DCM (10 mL) and cooled to 0°C. TFA (2.5 mL) was added and the mixture stirred at 0°C for 10 min and at room temperature for 40 min. The solvents were then evaporated off and the residue dried under high vacuum. The product was dissolved in 8 mL THF to which was then added 35 (470 mg, 2.18 mmol) and TEA (510 |liL, 3.66 mmol) and the mixture stirred for 4 h. THF was evaporated off and the product redissolved in EtOAc (30 mL) and washed by saturated aqueous NH4CI (2 x 20 mL) and brine 20 mL. The organic layer was then dried over MgS04, and the solvent evaporated off. After column purification using hexanes- ethyl acetate (4:1 v/v), 520 mg (76% yield) of 36a was obtained as a yellow liquid. The same procedure was employed in the synthesis of 36b (yellow liquid). Methyl 2-(2,2,2-trifluoro-acetylamino)-3-carbobenzyloxy-(25, 3/f)-hex-5-enoate (36a): = +39.1" (c = 1.31, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.27 (IH, d, J= 9.15 Hz), 7.40-7.32 (5H, m), 5.76-5.68 (IH, m), 5.18-5.08 (4H, m), 4.87 (IH, dd, J = 3.24, 9.53 Hz) 3.67 (3H, s), 3.36-3.32 (IH, m), 2.57-2.51 (IH, m), 229-222> (IH, m); 125 MHz, CDCI3, 5(ppm): 173.0, 169.5, 157.4 (q, J= 37.6 Hz), 134.8, 132.9, 128.7, 128.4, 119.2, 115.7 (q, / = 286 Hz), 67.4, 53.0, 51.7, 45.6, 33.0; HRMS (FAB) MH^ calcd for 374.1215, found 374.1214. Methyl 2-(2,2,2-trifluoro-acetylamino)-3-carbobenzyloxy-(2iS', 3iS)-hex-5-enoate (36b): [a]^^D = +22.9° (c = 1.52, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.39-7.34 (5H, m), 7.18 (IH, d, J= 7.25 Hz), 5.83-5.74 (IH, m), 5.18-5.11 (4H, m), 4.88 (IH, dd, J = 4.0, 8.2 Hz), 3.74 (3H, s), 3.06-3.02 (IH, m), 2.69-2.63 (IH, m), 2.49-2.43 (IH, m); 125 MHz, CDCI3, 5(ppm): 171.3, 169.2, 156.6 (q, J= 37.9 Hz), 135.0, 133.8, 128.7, 128.6, 128.5, 118.7, 115.5 (q, J= 285.8 Hz), 67.5, 53.0, 52.5, 47.1, 32.5; HRMS (FAB) MH^ calcd for 374.1215, found 374.1225. General procedure for oxidation, thlazolidine formation and bicyclization In a 25-mL flask, 36a (330 mg, 0.88 mmol) was dissolved in THF (10 mL) and water (5 mL). OSO4 (11 mg, 0.043 mmol) was added and the mixture stirred for 5 min before slowly adding NaI04 (526 mg, 2.46 mmol) and stirring the mixture for a further 4 h. The mixture was then filtered, THF evaporated off and EtOAc (30 mL) added. The organic phase was extracted with saturated aqueous NH4CI (2 x 20 mL), brine (20 mL) and then 87 dried over MgS04. The solvent was evaporated off and the product dried under high vacuum. The crude aldehyde was redissolved in 3 mL pyridine to which was added preactivated molecular sieves, 4A° (450 mg). L-Cys (35 mg, 0.29 mmol) was added and the mixture stirred at RT for 4 h. Pyridine (5 mL) was then added and the mixture stirred at 50°C for 4 days. The solvent was then evaporated off and the product dissolved in water. The aqueous layer was acidified to pH = 2 by 1 HCl and extracted by ether (4 x 5 mL). The organic phase was then dried over MgS04 and the solvent evaporated off. The product was redissolved in DCM (3 mL) and treated with excess diazomethane. The solution was stirred for 30 min and the solvents evaporated off Two compounds (33a, 65 mg; 38b, 140 mg) were obtained as yellow liquids after separation on a silica gel column using hexane-ethyl acetate (2:1 v/v), in 42% total yield. When the reaction was repeated with compound 36b, only one product, 38c, was obtained as a yellow liquid in 48% yield. Methyl [3S, 4R, 6S, 9/?]-l-Aza-3[2,2,2-trifluoroacetylamino]-4- carbobenzyloxy-2-oxo-7-thiabicyclic[4.3.0]-nonane-9-carboxylate (38a): = - 24.8° (c = 1.06, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.49 (IH, d, J= 5.5 Hz), 7.36-7.34 (5H, m), 5.24 (IH, d, J = 12.0 Hz), 5.1(1H, d, /= 12.0 Hz), 5.09 (IH, dd, / = 5.0, 10.0 Hz), 4.98 (IH, d,J= 7.0 Hz), 4.89 (IH, dd, /= 6.5, 8.5 Hz), 3.77 (3H, s), 3.42 (IH, dd, J= 6.5, 12.0 Hz), 3.27 (IH, d, J = 12.0 Hz), 3.08-3.04 (IH, m), 2.40-2.36 (IH, m); '^C, 125 MHz, CDCI3, 5(ppm): 172.7, 169.0, 165.6, 157.4 (q, /= 37.74 Hz), 135.0, 128.64, 128.61, 128.6, 115.5 (q, J= 286 Hz), 68.1, 62.5, 59.4, 53.1, 51.2, 44.8, 33.3, 32.9; HRMS (FAB) MH+ calcd for 461.0994, found 461.0998. 88 Methyl [35, 4if, 65, 9/?]-l-Aza-3[2,2,2-trifluoroacetylamino]-4-carbobenzyloxy-2- oxo-7-thiablcyclic[4.3.0]-nonane-9-carboxylate (38b): = -157.9° (c = 0.57, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm): 7.39-7.32 (5H, m), 7.11 (d, J = 5.0 Hz), 5.20-5.10 (3H, m), 5.03 (IH, dd, /= 2.5, 7.0 Hz), 4.55 (IH, dd, /= 5.0, 7.0 Hz), 3.78 (3H, s), 3.38-3.31 (2H, m), 3.21 (IH, dd, J= 3.0, 7.5 Hz), 2.63 (IH, m, dt, /= 2.5, 8.5 Hz), 2.11 (IH, dt, J= 2.5, 8.5 Hz), 1.36-1.33 (IH, m); '^C, 125 MHz, CDCI3, 5(ppm): 170.6, 169.8, 163.7, 157.4 (q, J = 37.6 Hz), 134.9, 128.71, 128.7, 128.4, 115.5 (q, / = 286.2 Hz), 67.8, 61.1, 60.7, 53.0, 52.2, 43.5, 31.7, 31.6; HRMS (FAB) MH^ calcd for 461.0994, found 461.0988. Methyl [35, 4R, 65, 9if]-l-Aza-3[2,2,2-trifluoroacetylamino]-4- carbobenzyloxy-2-oxo-7-thiabicyclic[4.3.0]-nonane-9-carboxylate (38c): = - 11.6^(0 = 0.34, CHCI3); 'H NMR, 500 MHz, CDCI3, 6(ppm): 7.63 (IH, d, J = 5.0 Hz), 7.38-7.32 (5H, m), 6.35 (IH, d, J = 12.5 Hz), 6.25 (IH, d,J= 12.0 Hz), 5.0 (IH, d,J = 6.5 Hz), 4.96 (IH, dd, J= 4.5, 11.0 Hz), 4.67 (IH, t, J= 7.0Hz), 3.74 (3H, s), 3.72-3.67 (IH, m), 3.42 (IH, dd, J= 7.0, 12.5 Hz), 3.33 (IH, d, J = 12.0 Hz), 2.70-2.64 (IH, m), 2.48-2.43 (IH, m); 125 MHz, CDCI3, 5(ppm): 171.1, 168.9, 165.0, 157.1 (q, J = 37.70 Hz), 135.0, 128.63, 128.57, 128.5, 115.4 (q,/= 285.7 Hz), 67.5,61.6, 59.0, 53.0, 49.6, 41.5, 33.8, 31.7; HRMS (FAB) MH^ calcd for 461.0994, found 461.0998. General procedure for hydrogenation of the benzyl ester and reduction of the resultant carboxylic acid Compound 7a (2.88 g, 6.7 mmol) was dissolved in degassed methanol (40mL). Pd/C (70 mg, 10%) was added and the mixture was exposed to hydrogen gas for 2 h. The catalyst 89 was filtered off through Celite and the solvent evaporated off The crude product was dried under vacuum and then dissolved in THF (35 mL). Benzotriazol-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (3.2 g, 7.23 mmol) followed by DIPEA (1.3 mL, 7.46 mmol) were added and the mixture stirred for about 5 min until it turned clear. The solution was then cooled to 0°C and NaBH4 (400 mg, 10.57 mmol) added slowly. The mixture was stirred for 30 min at 0°C and at room temperature for 18 h. The solvent was evaporated off and the product dissolved in ethylacetate (80 mL) and the organic phase was washed with saturated aqueous NaHC03 (2 x 50 mL), 5% aqueous HCl (2 x 50 mL) and brine (50 mL). The organic phase was then dried over Na2S04 and the solvent evaporated off. The product was purified by column to give 1.2 g of 39 (3.71 mmol) in 55% yield. Methyl 3-benzyI-2-fer^-butoxycarbonylamino-4-hydroxy-(25', 3if)-butanoate (39): [a]^^D = +35.3° (c = 0.58, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm); 'H 5 7.29-7.20 (5H, m), 5.59 (IH, d, J = 9.0Hz), 4.42 (IH, t, J = 5.5Hz), 3.72 (IH, s), 3.64-3.50 (2H, m), 2.78-2.73 (2H, m), 2.28-2.13 (2H, m); 125 MHz, CDCI3, 5(ppm): 173.1,156.1, 139.3, 129.1, 128.5, 126.3, 80.1, 61.2, 55.4, 52.3, 44.7, 34.4, 28.3; HRMS (FAB) MH^ calculated for 324.1811, found 324.1818. Synthesis of homophe-Gly bicyclic dipeptide mimetic Oxalyl chloride (2.8 mL, 5.6 mmol) was dissolved in CH2CI2 (40 mL). The solution was cooled to -78''C, DMSO (530 )j,L, 7.47 mmol) added and the solution stirred for 10 min before adding 39 (1.2 g, 3.71 mmol). The mixture was stirred for 25 min at -78''C before adding DIPEA (2.6 mL, 14.93 mmol), removing the cold bath and stirring for a further 20 min. Water (20 mL) was added, and the organic phase was separated and washed with saturated aqueous NH4CI (2 x 50 mL) and brine (50 mL). The organic phase was dried over MgS04 and the solvent evaporated off to give L17 g of the crude aldehyde after drying. The crude aldehyde was dissolved in pyridine (30 mL) containing molecular sieves 4 A° and the mixture stirred at room temperature for 4 h. Pyridine (60 mL) was added and the mixture heated at 50°C for 3 days. The solvent was evaporated off and the product dissolved in water (20 mL) which was acidified to pH = 2 using IN aqueous HCl. The aqueous layer was extracted with ether (4 x 20 mL) and the ether layer dried over MgS04. The solvent was evaporated off and the product dried under high vacuum to give 1.03 g of crude product. The crude product was dissolved in CH2CI2 (15 mL) and cooled to O^C. DCC (650 mg, 3.15 mmol) was added and the mixture stirred for 10 min before adding methanol (450 |.iL, 11.12 mmol) and DMAP (30 mg, 0.25 mmol) and stirring the mixture for 8 h. The mixture was filtered and the filtrate diluted by CH2CI2 (20 mL). The organic phase was washed by saturated aqueous NH4CI (2 x 20 mL) and brine (30 mL). The organic phase was dried over MgS04 and the solvent evaporated off The product was then purified by column to give 470 mg of 2 as a yellow oil in 31% yield from 34%. Methyl (3S, 4R, 5S, 8i?)-4-benzyI-3-^erf-butoxycarbonylamino-l-aza-2-oxo-6- thiabicyclic [3.3.0]-octane-8-carboxylate (2): = -88.7° (c = 0.98, CHCI3); 'H NMR, 500 MHz, CDCI3, 5(ppm); 7.34-7.2 (5H, m), 5.04-5.0 (2H, m), 4.89(1 H, d, J = 3.5Hz), 4.69 (IH, t, J = 8.0Hz), 3.77 (3H, s), 3.38-3.34 (IH, m), 3.25 (IH, dd, J = 4.0, 11.5Hz), 3.04 (IH, dd, J = 5.0, 14Hz), 3.02-2.94 (IH, m), 2.53(1H, m); '^C, 125 MHz, 91 CDCls, 6(ppm): 173.2, 169.7, 155.5, 138.1, 128.9, 128.7, 126.7, 80.5, 68.4, 57.6, 56.1, 52.9, 44.2, 36.9, 34.0, 28.3; HRMS (FAB) MH^ calculated for 324.1811, found 324.1818. General procedure for the synthesis of diazo methane A mixture of ether (1 mL/mmol) and KOH (40% wt, 0.3 mL/mmol) was added to a 20- mL vial and cooled down to 0°C in ice-water bath. N-nitrosomethyl urea (1.0 eg) was added in small portions and the mixture vigorously stirred. After all the urea was added and dissolved the vial contents were chilled to -78''C to freeze the water and the ether phase separated into a 20-mL vial containing a crystal of solid KOH. The solution was dried in the refrigerator for 2.5 hr before use. 92 chapter 6 SYNTHESIS OF BICYCLIC DIPEPTIDE MIMETIC CONTAINING CCK/OPIOID CHIMERIC PEPTIDES, BIOLOGICAL ASSAY RESULTS, CONCLUSION AND FUTURE WORK 6.1 Synthesis of peptides All the peptides were synthesized using the N"-Fmoc/t-butyl chemistry and Rink-amide AM resin. The N"-Boc protected BTD's were first hydrolyzed using Li0H/H20/Me0H system. The Boc group was then removed using TFA (30%) in CH2CI2. The amino group was then Fmoc protected and the crude BTD used in peptide synthesis. For bicyclic dipeptide 3b, the benzyl carboxylate and carbobenzyloxyamino groups were deprotected by hydrogenation. The amino terminal was then Fmoc protected, and the product used in peptide synthesis without purification. The protocol used for the synthesis of the peptides is illustrated in Scheme 6.1. In case of peptide JMN8 the peptide was synthesized and cleaved Irom the solid support using the same protocol. The crude product was then dissolved in a HaO/MeOH system and 2.0 eq. of LiOH added. The mixture was stirred for 30 min and MeOH evaporated. The crude product was then obtained by lyophilization. Low yields were obtained during the synthesis of JMN7 while all the other peptides were obtained in reasonable yields. Rink amide FmocHN AM resin i) 25%Plpericline ii) AA, HBTU, HOBt, DIPEA iii) repeat Peptide—HN 95% TFA, 2.5% TIS, 2.5% H2O Peptide Scheme 6.1 Protocol for the synthesis of peptides The peptides that were synthesized are shown below; ho, h -n h o h2n' h V O f H I? O "-N- h h o ^COoH ^COzH JMN1 JMN2 o \ h i N- h JMN5 JMN6 oh H2NOIo v h O C02H JMN8 JMN7 Figure 6.1 Novel BTD containing peptides 6.2 Exprimental 6.2,1 Synthesis and purification of peptides Rink amide resin (0.5g) was swelled in DMF in a course sintered filter fitted vessel for an overnight. The resin was deprotected with piperidine (20% v/v in DMF) first for 5 min and then for 20 min. The resin was then washed with DMF (3 times) and DCM (3 times). The first amino acid was then coupled to the resin using HBTU, HOBt and DIPEA. In all the couplings, 3 equivalents of each reagent (amino acid, HBTU, HOBt, and DIPEA) were used. The coupling was allowed to proceed for 1 hr before coupling the next amino acid. This protocol was followed until the last amino acid was coupled and N-terminal deprotected. The completeness of the coupling was monitored by a negative Kaiser test. The resin was then dried and transferred to a 20-mL borosiHcate scintillation vial. The peptide was cleaved fi'om the resin and other side chain protecting groups removed using a cleavage cocktail (10 mL per gram of resin). The cleavage cocktail was made of 95% TFA, 2.5% TIS and 2.5% water. The cleavage was allowed to proceed for 2 hr before the resin was filtered through a cotton plugged glass pipette. The resin was then washed with an additional TFA (2-3 mL). The TFA-peptide solution was then transferred to a propylethylene conical centrifuge tube and the solvent gently evaporated to about 3 mL using argon. The peptide was then precipitated by addition of ether (10 mL). The precipitate was isolated by centrifiigation. The organic layer was then decanted and the precipitate washed two more times with ether. The product was then dried in air to give 50-90% crude peptide. 95 The crude peptides were purified using a Vydac CI8 semi-preparative column. The crude peptides were dissolved in a mixture of aqueous 0.1% TFA, MeOH and acetonitrile, with the percentage of MeOH and acetonitrile not exceeding 20%. The solution was then filtered through a 0.45 micron cellulose acetate filter (Aerodisc). The loading used was 10 mg crude product per injection and the gradient depended on peptide. The purified fractions were combined and the acetonitrile removed by rotary evaporation. The pure peptide was then obtained by lyophilization. 6.2,2 Assay Methods (The assays were perfomed in the department of pharmacology. University of Arizona) In vitro isolated tissue bioassays for opioid agonist Mouse Isolated Vas Deferens (performed by Peg Davis in Dr. Frank Porreca's laboratory) Male ICR mice under ether anesthesia were sacrificed by cervical dislocation and the vasa deferentia removed. The tissues were tied to gold chains with suture silk and mounted between platinum wire electrodes in 20 mL organ baths at a tension of 0.5 g and bathed in oxygenated (95% O2, 5% CO2) magnesium free Kreb's buffer at SV^C. They were then stimulated electrically (0.1 Hz, single pulses, 2.0 msec duration) at supramaximal voltage. Following an equilibrium period, the compounds were added to the bath in volumes of 14-60 |.iL until maximum inhibition was reached. Response to an IC50 dose of DPDPE (10 nM) was measured to determine tissue integrity before testing. 96 Guinea Pig Isolated Ileum (performed by Peg Davis in Dr. Frank Porreca's laboratory) Male Hartley guinea pigs under ether anesthesia were killed by decapitation and a non terminal portion of the ileum removed. The longitudinal muscle with myenteric plexus (LMMP) was carefully separated from the circular muscle and cut into strips as described in literature. These tissues were tied to gold chains with suture silk and mounted between platinum wire electrodes in 20 mL organ baths at a tension of 1.0 g containing 31^C oxygenated (95% O2, 5% CO2) Kreb's buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCla, 1.19 mM KH2PO4, 1.18 mM MgS04 25 mM NaHCOs, and 11.48 mM glucose), and allowed to equilibrate for 15 min. The tissues were stimulated electronically (0.1 Hz, 0.4 msec duration) at supramaximal voltage. Following an equilibration period, compounds were cumulatively added to the bath in volumes of 15-60 |j,L aliquots until maximum inhibition was reached. Response to an IC50 dose of PL-017 (100 nM) was measured to determine tissue integrity before analogue testing. Antagonism test (performed by Peg Davis in Dr. Frank Porreca's laboratory) Drugs were tested for their antagonist activity by adding an IC50 dose of PL-017 in the GPI or DPDPE in the MVD to the bath after the final 1.0 )aL dose of the test drugs. Blunting of the agonist effect of DPDPE or PL-017 indicated antagonist activity ot the test compound CCK studies in the guinea pig isolated ileum (performed by Peg Davis in Dr. Frank Porreca's laboratory) Male Hartley guinea pigs under ether anesthesia were killed by decapitation and a non terminal portion of the ileum removed. The longitudinal muscle with myenteric plexus (LMMP) was carefully separated from the circular muscle and cut into strips as described in literature. These tissues were tied to gold chains with suture silk and mounted between platinum wire electrodes in 20 mL organ baths at a tension of 1.0 g containing 2>1^C oxygenated (95% O2, 5% CO2) Kreb's bicarbonate buffer. The tissues were stimulated electronically (0.1 Hz, 0.4 msec duration) at supramaximal voltage to stabilize base-line force and tissue health. Response to an IC50 dose of PL-017 (100 nM) was measured to determine tissue integrity before analogue testing. Following an equilibration period the tissues were challenged with KCl (67 mM) to determine initial maximal muscle contractility. An initial non-cumulative CCK-8 dose response curve was constructed using concentrations from 1 to 100 nM. The test compound was added to the bath in concentrations from 1 to 100 nM. If no agonist activity was observed after 3 min, a dose of CCK-8 dose response was added to determine the test compound's antagonist activity until a complete CCK-8 dose-response curve was reconstructed using a dose of antagonist which would seem to cause a three fold shift rightward. Tissues were again challenged with KCl to determine tissue changes during the assay.After thorough washing, electrical stimulation was again applied, tissue resiliency tested with 100 nM PL-017, and an opioid dose response curve was constructed with the test compound For CCK studies, contraction height was calculated as a percentage of the maximal KCl contraction and calculated as an A50. For opioid studies, percentage inhibition was calculated using the average tissue contraction height for 1 min preceding the addition of the agonist divided by the contraction height 3 min after exposure to the dose of agonist. 98 ICso and Emax estimates were determined by computerized nonlinear least-squares analysis (MINSQ, Micromath). [^^S]GTP-y-S-binding assays (performed by Dr Shou-ma in Dr. Josephine Lai's laboratory) Cells expressing hDOR (or rMOR for mu-receptor studies) were incubated with increasing concentrations of the test compounds in the presence of 0.1 nM were incubated with increasing [^^S]GTP-y-S (1000-1500 Ci/mmol, NEN, Boston, MA) in assay buffer (total volume of 1 mL, duplicate samples) as a measure of agonist-mediated G protein activation. After incubation (90 min, SO^C), the reaction was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by four washes with ice-cold 25 mM Tris/120 mM NaCl, pH 7.4. Filters were pretreated with assay buffer prior to filtration to reduce nonspecific binding. Bound reactivity was measured by liquid scintillation spectrophotometry after an overnight extraction with EcoLite (ICN, Biomedicals, Costa Mesa, CA) scintillation cocktail. Phosphoinoside (PI) hydrolysis assays (performed by Dr. Balaz Hargattai in Dr. Josephine Lai's laboratory) Transfected cells were incubated with 0.5 mL IMDM 0.2 )iM [^Hjmyoionositol (final concentratrion) for 20-22 hrs at 37'^C in the presence of humidified air (95% O2, 5% CO2). After the removal of media, the cells were incubated further for 1 hr at 37*^0. After removal of the media solution, 0.5 mL of IMDM was added and then LiCl stock solution to make a final concentration of 10 mM. Following a 10 min equilibration time, the test compound was added and the cells were incubated for 1 hr at 37°C in the presence of 99 humidified air (95% O2, 5% CO2). The cells were placed in ice and the test compound and media were removed. The reactions were terminated with the addition of 0.5 mL cold methanol. The cells were scraped and transferred to a chloroform/water mixture (1 mL/0.5 mL). The sample was centrifuged at 2100 rpm for 10-15 min at 4°C. 0.9 mL •1 aliquots of the supernatant containing the water soluble [ H]-ionositol phosphates was diluted with 2 mL of H2O, which was purified into anion exchange columns (CAG1-X8, 100-200 mesh Bio-Rad laboratory). Following the loading of the sample, the columns were washed with 5 mL water to remove [^H]-ionositol precursor, followed by 5 mL of 5 mM sodium tetraborate/60 mM sodium formate and 0.1 M formic acid mixture into scintillation vials. 9 mL of Aquamix was added to each vial and radioactivity was measured by scintillation spectrometry Analysis Percentage inhibition was calculated using the average tissue contraction height for 1 min preceding the addition of the agonist divided by the contraction height 3 min after exposure to the dose of agonist. IC50 and Emax estimates were determined by computerized nonlinear least-squares analysis (MINSQ, Micromath) 6.3 Biological evaluation at the CCK receptors In the PI hydrolysis assay at CCK-A and CCK-B receptors (Table 6.1), peptides JMNl, 2, 5, and 6, showed potent activity at the CCK-A receptors and no activity at the CCK-B receptors. Substitution of Z-Trp for D-Trp in JMN6 to give JMN7 led to 80 fold loss in biological activity at CCK-A. JMN8 on the other hand was not potent at both the CCK- 100 A and CCK-B receptors. The six peptides also showed no agonist activity at CCK-A in the presence of naloxone (Table 6.2). In the competitive binding assays at the cloned human CCK-A and CCK-B receptors (Table 6.3), JMN5 and 6 had good binding affinities while JMNl and 2 had moderate binding affinities. Substitution of Z-Trp for D-Trp in JMN6 to give JMN7 led to loss of affinity at both the CCK receptors. JMN8 had no binding affinity at both receptors. Table 6.1 Biological evaluation at the CCK receptors Functional analysis^ Binding affinity" (ki, nM) CCK-A hCCK-A hCCK-B hCCK-A hCCK-B agonist Drug EC5o(nM) EC5o(nM) CCKs ['"I] CCKs activity® SNF9007 n/d n/d 3270 2.1 n/d RSA501 790 3100 140 14 none JMNl no response 4.1 740 70 none JMN2 no response 6.9 1200 100 none JMN5 no response 2.3 2400 16 none JMN6 no response 1.9 810 11 none JMN7 no response 160 1800 1400 none JMN8 no response no response >10,000 >10,000 none ^Phosphoinositide (PI) hydrolysis assay in hCCK-A and iCCK-B receptors in HEK eel lines. ''Competition against CCKg (sulfated) in hCCK-A and hCCK-B receptors in HEK cell lines in the presence of naloxone. "Contraction of isolated tissue relative to initial contraction with KCl in the presence of naloxone in GPl/LMMP. n/d, not determined. 101 Table 6.2 Functional analysis at the opioid receptors Functional activity at MVD and GPI/LMMP MV]D GPI/LMMP agonist DPDPE agonist PL-017 activity'' antagonism activity'' antagonism RSA501 17.0 % n/d none n/d JMNl 28.5% none 9.4% none JMN2 18.0% none 7.2% none JMN3 18.7% none none none JMN4 1.0% none none none JMN5 12.5% none 6.5% none JMN6 18.0% none 2.3% none JMN7 2.0% none 1.7% none JMN8 5.6% n/d n/d n/d ^ % inhibition of muscle contraction at 1 |iM at electrically stimulated isolated tissues, n/d, not determined. 6.4 Biological evaluation at the opioid receptors In the competitive binding assays at cloned opioid receptors, all the peptides except JMN2 showed micromolar binding affinities (Table 6.3). JMN2 had a slightly high affinity at both the mu and delta receptors. In the GTP-y-S assay, JMNl, 2 and 5 had no activity. JMN7 showed moderate activity at both the receptors which could result from the low binding affinity. The peptides also showed no agonist or antagonist properties at both the receptors (Table 6.2). At the MVD, all the peptides showed low agonist activity but no antagonist properties. However, this low value may be attributed to the low binding affinities. At the GPI/LMMP, JMNl, 2, 5, 6, and 7 showed low agonist properties while the rest were inactive. Interestingly, JMN7 shows low agonist activity at both the MVD and GPI/LMMP compared to the GTP binding assay. 102 Table 6.3 Biological evaluation at the opioid receptors GTP binding' Competition" hDOR rMOR hDOR rMOR Drug ECsoCnM) ECso (nM) [^H]DPDPE [^H]DAMGO SNF9007 n/d n/d 250 5200 RSA501 1000 1800 74 1000 JMNl n/d n/d 1850 9280 JMN2 no response no response 337 225 JMN5 no response no response 1720 3470 JMN6 no response no response 1410 8610 JMN7 460 775 2750 8110 JMN8 no response 876 1480 3120 ^[^^S]GTP-y-S binding assay. ''Competitive assay against radiolabelled [^H] DPDPE at hDOR and [^H]DAMGO at rMOR. hDOR and rMOR were expressed from CHO cell lines. 6.5 Discussion and Conclusion Alkylation of aspartic acid provided a route to access different bicyclic dipeptide mimetics for SAR studies on CCK/opioid chimeric peptides. Both P-allyl and P-benzyl substituted aspartic acids were employed in the synthesis of bicyclic dipeptide mimetics. The bicyclic dipeptide mimetics were then introduced into peptides which were tested at both the CCK and opioid receptors. JMNl-7 showed good activity at the CCK-B receptor which may be attributed to the C-terminal tetrapetide, a CCK pharmacophore. This will corraborate the observation that the tetrapeptide (CCK-4) is the minimal sequence required for activity at CCK-B receptors. Inactivity of JMN8 at the CCK receptors may thus be as a result of interference with this tetrapeptide. However, unlike in most other CCK peptides where substitution of D-Trp leads to antagonistic properties,'^ JMN7 still retained its agonist properties. There was however a considerable loss of affinity at both CCK receptors on substituting D-Trp for Trp in JMN6. The peptides were all inactive at the CCK-B receptor and the lack of the sulfate group on Tyr may be a partial reason. JMN5 was 150 fold more selective for the CCK-B receptor over CCK-A receptor. Nle^ has been shown to provide a balanced activity between CCK-A and CCK- B receptors in related peptides""^ but this does not seem to apply in the series of peptides that were synthesized. Substitution of NMeNle for Nle^ would thus be relevant to explore its effect on activity at the CCK receptors. NMeNle^ in combination with D-Trp also has been shown to have activities at both the opioid and CCK receptors. Further work would also be required to synthesize peptides that have antagonistic properties at the CCK receptors. This would involve SAR studies on the six peptides particularly for the C- terminal tetrapeptide. The peptides were all found to have weak activities at the opioid receptors. Compared to JMNl and JMN3, JMN2 has a better binding affinity at the opioid receptor which contradicts the observation that /?-chirality at the second amino acid (Figure 6.2) is important for opioid activity. Except for JMN7 which had D-Trp in the sequence, all the other peptides were inactive at the opioid receptors. It would thus be important to substitute D-Trp into the other analogues to study its significance in opioid activity. JMN8 retains some opioid activity even though it was basically inactive at the CCK receptors. This would further compliment the argument that the C-terminal tetrapeptide is a CCK pharmacophore with Trp overlapping between the two receptors. 104 ho HO, R h s h JMNl JMN2 Figure 6.2 Structures of JMNl and JMN2 6.6 Future work 6.6.1 Structure activity relationship studies Based on the results that were obtained, structure activity studies of the peptides would be worthwhile in an attempt to develop ligands that are antagonists and have balanced activity at CCK receptors. This would involve the substitution of Trp with Z)-Trp or other biologically equivalent amino acids like Nal. The Nle^ may also be substituted with NMeNle and D-N\e. JMN7 which shows activity at the opioid receptors could also be modified by introducing NMeNle or Z)-Nle in place of Nle. Multiple substitutions could also be investigated even though it was shown not to lead to additive change in activity. 6.6.2 Cyclized chimeric peptides The synthesis of cyclized peptides using ring closing metathesis reaction (RCM) could offer a new path towards constrained peptides. A number of strategies for the synthesis of ring closing metathesis cyclized peptides have been reported.228 " 231 The^"<1 solid phase ring closing metathesis reaction applying Grubbs catalyst has been used for the tethering of preformed secondary structures in peptides such as p-tums^^^ and a-helices.^^^ RCM has also been applied in the synthesis of a rigidified protein-derived homodetic lOmer peptide epitope.^^"^ Research has shown that cyclization is not affected by ring size, experimental 105 conditions, stereochemical orientations, but is more affected by inability to form appropriate secondary structures?^^"^^^ All published synthesis of peptide cycles with more than five amino acids contain proline or N-alkyl residues within their sequence so as to effect formation of the appropriate secondary structure.^^^ Reversible backbone protection,^^^"^"^' for example use of Mutter's pseudoproline Ser(\(/'^^''^'^pro),^'^^ can also be used to overcome the difficulties in cyclization. To synthesize other constrained analogues of our desired peptides, we propose the synthesis of two classes of CCK/Opioid chimeric peptides cyclized on solid support by ring closing metathesis reaction. 6.6.3 Synthesis of cyclized peptides using P-allyl substituted aspartic acid and ring closing metathesis In the first case p-allyl substituted aspartic acid would be substituted for Asp^ in the peptide Tyr'-D-Nle^-Gly^-Trp'^-Nle^-Asp^-Phe^. Nle^ and Gly^ would then be alternately substituted with 2-amino-4-pentenoic acid to set the conditions necessary for ring closing metathesis. The olefin that results after metathesis can also be reduced to give more analogues of these peptides. However, the indole ring of Trp"^ residue is unstable to reduction^^"^ and may thus be substituted by the biologically equivalent amino acids. Nle^ may also have to be substituted with Pro to facilitate the formation of secondary structures. Cyclization at D-Nle^ position would give a 15 membered ring peptide while cyclization at Gly^ position would give a 18 membered ring peptide. Compared to disulfide bond and lactam cyclization that has been used so far, this protocol provides more constrain at the Asp^ position. 106 CO2M© COoM© Trp-Nle-N Tyr-Nle-I Tyr-Nle-i H CO2H Phe-NH, Tyr-Nle-1 Figure 6.3 Synthesis of cyclized peptides 6.6.4 Synthesis of cyclized peptides using 5-aIlyl substituted proline analogues and ring closing metathesis In the second approach, Nle^ and Gly^ would be alternately substituted with 2-amino-4- pentenoic acid while Asp^ will be substituted with 5-allyl substituted proline analogues (Scheme 6.3). In this case the proline analogues may favor the formation of the secondary structure. To synthesize saturated analogues, Trp'^ will be substituted with biologically equivalent analogues. CO2M© • CO2M© .N- Tyr-Nle—N^fT^Trp-Nle/ V-Phe- Tyr-Nle—N^^Trp-Nle/ O O O O .COoH ,N- Tyr-Nle—N^'Nr'Trp-Nie/ ^Phe-NHj O O Figure 6.4 Synthesis of highly constrained cyclic peptides. 107 These approaches may lead to ligands with good biological activities since the pharmacophore in the peptide is maintained. This may also provide a strategy to the synthesis of cyclized analogues to compliment disulfide and lactam cyclizations. 108 Appendix A Rf Values for the CCK/opioid chimeric peptides TLC Peptide A B C JMNl 0.75 0.84 0.57 JMN2 0.67 0.75 0.43 JMN3 0.44 0.63 0.28 JMN4 0.47 0.63 0.31 JMN5 0.72 0.76 0.51 JMN6 0.73 0.80 0.57 JMN7 0.73 0.78 0.55 JMN8 0.52 0.76 0.40 ''Rf values on thin layer chromatograms of silica gel in the following solvents: (A) 1-butanol/water/acetic acid (4:1:1), (B) chloroform/methanol/water (4:4:1), (C) methanol/ethylacetate/hexanes/water (6:4:4:4:1) 109 Appendix B Retention times for the CCK/opioid chimeric peptides Peptide HPLC Retention time" k' JMNl 22.9 5.5 JMN2 21.7 5.4 JMN3 14.3 3.2 JMN4 15.3 3.4 JMN5 22.0 5.3 JMN6 23.6 6.0 JMN7 23.7 6.1 JMN8 17.0 5.7 ®HPLC retention time; k' = [(peptide retention time - solvent retention time)/solvent retention time] in a solvent of 10% ACN in 0.1 TFA and a gradient of 10-90% ACN over 40 min. An analytical Microsorb-MV C18 5 jam 1OOA column was used with a flow rate of 1 mL/min Appendix C Higli resolution mass spectrometry for the CCK/opioid chimeric peptides HR-MS Peptide molecular formula Calculated Observed JMNl C49H61N9O10S 968.4340 968.4348 JMN2 C49H61N9O10S 968.4340 968.4348 JMN3 C25H28N4O6S 513.1808 513.1816 JMN4 C23H25N3O5S 456.1593 456.1601 JMN5 C49H61N9O10S 968.4340 968.4348 JMN6 C53H61N9O10S 1016.4340 1016.4348 JMN7 C53H61N9O10S 1016.4340 1016.4348 JMN8 C47H57N9O10 908.4278 908.4286 Ill REFERENCES 1. Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan, B. A.; Morris, H. R. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature, 1975, 258, 577-580. 2. Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. The effects of morphine- and nalorphine-Iike drugs in the nondependent and morphine- dependent chronic spinal dog. J. Pharcol. Exp Ther., 1976, 197, 517-532. 3. Lord, J. A. H.; Waterfield, A. A.; Hughes, J.; Kosterliz, H. W. Endogenous opioid peptides: multiple agonists and receptors. Nature, 1977, 267, 495-499. 4. Gehrig, C. A.; Hruby, V. J. Recent developments in the design of receptor specific opioid peptides. Med. Res. Rev. 1989, 9, 343-401. 5. Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan, B. A.; Morris, H. R. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature, 1975, 258, 577-580. 6. Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine- dependent chronic spinal dog. J. Pharcol. Exp Ther., 1976, 197, 517-532. 7. Lord, J. A. H.; Waterfield, A. A.; Hughes, J.; Kosterliz, H. W. Endogenous opioid peptides: multiple agonists and receptors. Nature, 1977, 267, 495-499. 8. Summers, M. C.; Hayes, R. J.; The interaction of N alpha-alkylenkephalins with opiate receptors. Tissue-dependent shifts in the opiate activity of methionine- enkephalin following N alpha-alkylation. J. Biol. Chem., 1981, 256, 4951-4956. 112 9. Morgan, B. A.; Smith, C. F. C.; Waterfield, A. A.; Hughes, J.; Kosterlitz, H. W. Structure-activity relations of methionine-enkephalin. J. Pharmac. 1976, 28, 660-661. 10. Gacel, G.; Foumie-Zaluski, M. C.; Fellion, E.; Rogues, B. P.; Senault, B.; Lecomte, J. -M.; Malfrey, B.; Swerts, J. -P.; Schwartz, J. -C.; Conformation and biological activities of hexapeptides related to enkephalins: respective roles of the ammonium and hydroxyl groups of tyrosine. Life. Sci., 1979, 24, 725-731. 11.Beddell, C. R.; Clark, R. B.; Lowe, L. A.; Wilkinson, S.; Chang, K. J.; A conformational analysis for leucine-enkephalin using activity and binding data of synthetic analogs. Brit. J. Pharm. 1977, 61, 351-356. 12. Shaw, J. S.; Tumbull, M. J. In vitro profile of some opioid pentapeptide analogs. Eur. J. Pharmacol. 1978, 49, 313-317. 13. Hambrook, J. M.; Morgan, B. A.; Ranee, M. J.; Smith, C. F. C. Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature, 1976, 262, 782-783. 14. Montecucchi, P. C.; De CastigHone, R.; Piani, S.; Gozzini, L.; Erspamer, V. Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phyllomedusa sauvagei. Int. J. Pept. Protein Res. 1981, 17, 275-283. 15. Erspamer, V. The opioid peptides of the amphibian skin. Int. J. Dev. Neurosci. 1992, 10, 3-30. 16. Lazarus, L. H.; Salvadori, S.; Attila, M.; Grieco, P.; Bundy, D. M.; Wilson, W. E.; Tomatis, R. Interaction of deltorphin with opioid receptors: molecular determinants for affinity and selectivity. Peptides, 1993, 14, 21-28. 113 17. Lazarus, L. H.; Attila, M. The toad, ugly and venomous, wears yet a precious jewel in his skin. Prog. Neurobiol. 1993, 41, 473-507. 18. Kreil, G.; Barra, D.; Simmaco, M.; Erspamer, V.; Falconieri-Erspamer, V.; Negri, L.; Corsi, R.; Melchiorri, P. Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for 5- opioid receptors. Eur. J. Pharm. 1989, 162, 123-128. 19. Casiano, F. M.; Cumiskey, W. R.; Gordon, T. D.; Hansen, P. E.; Mckay, F. C.; Morgan, B. A.; Pierson, A. K.; Rosi, D.; Singh, J.; Terminiello, L.; Ward, S. J.; Wesco, D. M. Structure function studies in di- and tripeptides related to des- [Gly]3enkephalin. Peptides: Structure and Functions, Pro.8'^ Amer. Pept. Symp., eds. Hruby, V. J. and Rich, D. H., Pierce Chemical Co., Rockford, IL, 1983, pp. 311-314. 20. Roques, B. P.; Gacel, G.; Foumie-Zaluski, Marie, C.; Senault, B.; Lecomte, J. M. Demonstration of the crucial role of the phenylalanine moiety in enkephalin analogs for differential recognition of the |j,- and 5-receptors. Eur. J. Pharmacol., 1979, 60, 109-110. 21. Roques, B. P.; Foumie-Zaluski, M. C.; Gacel, G.; David, M.; Meunier, J. C.; Maigret, B.; Morgat, J. L. in Regulatory Peptides: From Molecular Biology to Function, eds. Costa, E. and Trabucchi, M., Raven Press, New York, 1982, 32, pp. 321-331. 22. Gacel, G.; Dauge, V.; Breuze, P.; Delay-Goyet, P.; Dauge, V.; Roques, B. P. Rational design and biological properties of highly specific |a and 6 opioid peptides. J. Med. Chem. 1988a, 31,374-383. 114 23. Gacel, G.; Dauge, V.; Breuze, P.; Delay-Goyet, P.; Roques, B. P. Investigation of the structural parameters involved in the mu and delta opioid receptor discrimination of linear enkephalin-related peptides. J. Med. Chem. 1988b, 31, 1891-1897. 24. Schiller, P. W.; Yam, C. F.; Prosmanne, J. Synthesis, opiate receptor affinity, and conformational parameters of [4-tryptophan]enkephalin analogs. J. Med. Chem. 1978, 21, 1110-1116. 25. Shaw, J. S.; Tumbull, M. J. In vitro profile of some opioid pentapepfide analogs. Eur. J. Pharmacol. 1978, 49, 313-317. 26. Morgan, B. A.; Bower, J. D.; Guest, K. P.; Handa, B. K.; Metcalf, G.; Smith, C. F. C. Structure-activity relationships of enkephalin analogs. Peptides, Proc. 5'^ American Pept. Symp., eds. Goodman, G. and Meienhofer, J. Wiley, New York, 1977, pp. 111- 113. 27. Frederickson, R. C. A.; Nickander, R.; Smithwick, E. L. Pharmacological activity of met-enkephalin and analogs in vitro and in vivo - depression of single neuronal activity in specified brain regions. Opiates and Endogenous Opioid Peptides, ed. Kosterlitz, H. W., Elsevier/North Holland Biomedical Press, Amsterdam, 1976, pp. 239-246. 28. Hruby V. J. Conformational restrictions of biologically active peptides via amino acid side chain groups. Life sci. 1982, 31, 189-199. 29. DiMaio, J.; Lemieux, C.; Schiller, P. W. Structure-activity relationships of cyclic enkephalin analogs. Life. Sci. 1982, 31, 2253-2256. 115 30. DiMaio, J.; Nguyen, T. M.; Lemieux, C.; Schiller, P. W. Synthesis and pharmacological characterization in vitro of cyclic enkephalin analogs: effect of conformational constraints on opiate receptor selectivity. J. Med. Chem. 1982, 25, 1432-1438. 31. Schiller, P. W.; Eggimann, B. Dimaio, J.; Lemieux, C.; Nguyen, T. M. Cyclic enkephalin analogs containing a cystine bridge. Biochem. Biophys. Res. Commun. 1981, 101,337-343. 32. Hruby, V. J.; Bartosz-Bechowski, H.; Davis, P.; Slaninova, J.; Zalewska, T.; Stropova, D.; Porreca, F.; Yamamura, H. I. Cyclic enkephalin analogs with exceptional potency and selectivity for 5-opioid receptors. J. Med. Chem. 1997, 40, 3957-3962. 33. Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Bis-penicillamine enkephalins possess highly improved specificity toward • opioid receptors. Proc. Natl. acad. Sci. U. S. A. 1983, 80, 5871-5874. 34. Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Akiyama, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Cyclic penicillamine containing enkephalin analogs display profound delta receptor selectivities. Life. Sci. 1983, 33 Suppl 1, 447-450. 35. Haaseth, R. C.; Zalewska, T.; Davis, P.; Yamamura, H. I.; Porreca, F.; Hruby, V. J. Para-substituted phenylalanine-4 analogs of [L-Ala3]DPDPE: highly selective 5 opioid receptor ligands. J. Peptide. Res. 1997, 50, 171-177. 116 36. Svensson, C. I.; Rew, Y.; Malkmus, S.; Schiller, P. W.; Taulane, J. P.; Goodman, M.; Yaksh, T. L. Systemic and spinal analgesic activity of a 5-opioid-selective lanthionine enkephalin analog. J. Pharmacol. Exp. Ther. 2003, 304, 827-832. 37. Noble, F.; Wank, S. A.; Crawley, J. N.; Bradwejn, J.; Seroogy, K. B.; Hamon, M.; Roques, B. P. Structure, distribution, and functions of cholecystokinin receptors. Pharmacol, Rev. 1999, 51, 745-781. 38. Mutt, v.; Jorpes, J. E. Structure of porcine cholecystokinin-pancreozymin. 1. Cleavage with thrombin and with trypsin. Eur. J. Biochem. 1968, 6, 156-162. 39. Vanderhaeghen, J.; Signeau, J. C.; Gepts, W. New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature, 1975, 257, 604-605. 40. Baber, N. S.; Dourish, C, T.; Hill, D. R. The role of CCK, caerulein, and CCK antagonists in nociception. Pain 1989, 39, 307-328. 41. Innis, R. B.; Snyder, S. H.; Distinct cholecystokinin receptors in brain and pancreas. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6917-6921. 42. Hill, D. R.; Shaw, T. M.; Woodruff, G. N.; Species differences in the localization of 'peripheral' type cholecystokinin receptors in rodent brain. Neurosci. Lett. 1987, 79, 286-289. 43. Hill, D. R.; Campbell, N. J.; Shaw, T. M.; Woodruff, G. N.; Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists. J. Neurosci. 1987, 7, 2967- 2976. 117 44. Saito, A.; Sankaran, H.; Goldfine, I. D.; Williams, J. A. Cholecystokinin receptors in the brain: characterization and distribution. Science, 1980, 208, 1155-1156. 45. Ghilardi, J. R.; Allen, C. J.; Vigna, S. R.; McVey, D. C.; Mantyh, P. W. Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey; possible site of opiate-CCK analgesic interactions. J. Neurosci. 1992, 12, 4854-4866. 46. Hill, D. R.; Shaw, T. M.; Woodruff, G. N.; Binding sites for 1251-cholecystokinin in primate spinal cord are of the CCK-A subclass. Neurosci. Lett. 1988, 89, 133-139. 47. Bradwejn, J.; Koszycki, D.; Shiriqui, C. Enhanced sensitivity to cholecystokinin tetrapeptide in panic disorder. Clinical and behavioral findings. Arch. Gen. Psychiat. 1991,48, 603-610. 48. Sugg, E.; Shook, J. E.; Serra, M.; Kroc, M.; Yamamura, H. I.; Burks, T. F.; Hruby, V. J. Synthesis and biological evaluation of Na-hydroxysulfonyl-[Nle28,31]-CCK26-33. Life. Sci. 1986, 39, 1623-1629. 49. Hruby, V. J.; Fang, S.; Knapp, R.; Kazmierski, W.; Lui, G. K.; Yamamura, H. I. Cholecystokinin analogs with high affinity and selectivity for brain membrane receptors. Int. J. Pept. Protein Res. 1990, 35, 566-573. 50. Shiosaki, K.; Lin, C. W.; Kopecka, H.; Craig, R.; Wagenaar, F. L.; Bianchi, B.; Miller, T.; Witte, D.; Nadzan, A. M. Development of CCK-tetrapeptide analogs as potent and selective CCK-A receptor agonists. J. Med. Chem. 1990, 33, 2950-2952. 51. Boteju, L. W.; Nikiforovich, G. V.; Haskell-Luevano, C.; Fang, S. N.; Zalewska, T. Stropova, D.; Yamamura, H. I.; Hruby, V. J. The Use of Topographical Constraints 118 In Receptor Mapping; Investigation of the Topographical Requirements of the Tryptophan 30 Residue for Receptor Binding of Asp-Tyr-D-Phe-Gly-Trp-(N- Me)Nle-Asp-Phe-NH2 (SNF 9007), a Cholecystokinin (26-33) Analog That Binds to both CCK-B and 5-Opioid Receptors. J. Med. Chem. 1996, 39, 4120-4124. 52. Danho, W.; Tilley, J. W.; Shiuey, S. -J.; Kulesha, I.; Swistok, J.; Makofske, J.; Michalewsky, J.; Wagner, R.; Triscari, J; Nelson, D.; Chiruzzo, F. Y.; Weatherford, S. Structure-activity studies of tryptophan30 modified analogs of Ac-CCK-7. Int. J. Pept. Protein Res. 1992, 39, 337-347. 53. Rajh, H. M.; Mariman, E. C. M.; Tesser, G. I.; Nivad, R. J. F. Tryptophan replacement in the C-terminal octapeptide of CCK-PZ. Syntheses of six peptide analogues bearing tryptophan congeners. Int. J. Pept. Protein Res. 1980, 15, 200-210. 54. Bodanszky, M.; Martinez, J.; Walker, M.; Gardner, J. D.; Mutt, V. J. Cholecystokinin (pancreozymin). 5. Hormonally active desamino derivative of Tyr(S03H)-Met-Gly- Trp-Met-Asp-Phe-NH2. J. Med. Chem. 1980, 23, 82-85. 55. Bodanszky, M.; Tolle, J. C.; Gardner, J. D.; Walker, M. D.; Mutt, V. Cholecystokinin (pancreozymin). 6. Synthesis and properties of the N"-acetyl-derivative of cholecystokinin 27-33. Int. J. Pept. Protein Res. 1980, 16, 402-411, 56. Baba, S.; Otsuki, M.; Ohki, A.; Okabayashi, Y.; Sakamoto, C.; Yanaihara, N. Structural requirements of COOH-terminal fragments of cholecystokinin for pancreatic juice, amylase and insulin secretion from the isolated perfused rat pancreas. Biomedical Res. 1984, 5, suppl. 33-40. 119 57. Marseigne, I.; Dor, A.; Pelaprat, D.; Reibaud, M.; Zundel, J. L.; Blanchard, J. C.; Roques, B. P. Structure-activity relationships of CCK26-33-related analogs modified in position 33. Int. J. Pept. Protein Res. 1989, 33, 230-236. 58. Tilley, J. W.; Danho, W.; Shiuey, S, -J.; Kulesha, I.; Sarabu, R.; Swistok, J.; Makofske, R.; Olson, G. L.; Chiang, E.; Rusiecki, V. K.; Wagner, R.; Michalewsky, J.; Triscari, J.; Nelson, D.; Chiruzzo, F. Y.; Weatherford, S. Structure activity of C- terminal modified analogs of Ac-CCK-7. J. Pept. Protein Res. 1992, 39, 322-336. 59. Long, B.; Gardner, J. D. Effects of cholecystokinin on adenylate cyclase activity in dispersed pancreatic acinar cells. Gastroenterelogy, 1977, 73, 1008-1014. 60. Gardner, J. D.; Conlon, T. P.; Klaevenman, H. L.; Adams, T. D.; Ondetti, M. A. Action of cholecystokinin and cholinergic agents on calcium transport in isolated pancreatic acinar cells. J. Clin. Invest. 1975, 56, 366-375. 61. Pan, G. -Z.; Martinez, J.; Bodansky, M.; Jensen, R. T.; Gardner, J. D. The importance of the amino acid in position 32 of cholecystokinin in determining its interaction with cholecystokinin receptors on pancreatic acini. Biochem, Biophys. Acta. 1981, 678, 352-357. 62. Martinez, J.; Wintemitz, F.; Bodansky, M.; Gardner, J. D.; Walker, M. D. Synthesis and some pharmacological properties of Z-Tyr(S03H)-Met-Gly-Trp-Met-Asp(Phe- NH2)-0H, a 32-beta-aspartyl analogue of cholecystokinin (pancreozymin) 27-33. J. Med. Chem. 1982, 25, 589-593. 63. Penke, B.; Hajnal, F.; Lonovics, J.; Holzinger, G.; Kadar, T.; Telegdy, G.; Rivier, J. Synthesis of potent heptapeptide analogs of cholecystokinin. J. Med. Chem. 1984, 27, 120 845-849. 64. Tilley, J. W.; Danho, W.; Madison, V.; Fry, D.; Swistok, J.; Makofske, R.; Michalewsky, J.; Schwartz, A.; Weatherford, S.; Triscari, J.; Nelson, D. Analogs of CCK incorporating conformationally constrained replacements for Asp32. J. Med. Chem. 1992, 35, 4249-4252. 65. Foumier-Zaluski, M. C.; Belleney, J.; Lux, B.; Durieux, C.; Gerard, G.; Gacel, G.; Maigret, B.; Roques, B. P. Conformational analysis of cholecystokinin CCK26-33 and related fragments by proton NMR spectroscopy, fluorescence-transfer measurements, and calculations. Biochemistry 1986, 25, 3778-3787. 66. Charpentier, B.; Pelaprat, D.; Durieux, C.; Dor, A.; Reibaud, M.; Blanchard, J. C.; Roques, B. P. Cyclic cholecystokinin analogs with high selectivity for central receptors. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 1968-1972. 67. Weng, J. H.; Bado, A.; Garbay, C.; Roques, B. P. Novel CCK-B receptor agonists: diketopiperazine analogues derived for CCK4 bioactive conformation. Reg. Pept. 1996, 65, 3-9. 68. Baber, N. S.; Dourish, C. T.; Hill, D. R. The role of CCK, caerulein, and CCK antagonists in nociception. Pain, 1989, 39, 307-328. 69. Dalsgaard, C. J.; Vincent, S. R.; Hokfelt, T.; Lundberg, J. M.; Dahlstrom, A.; Schultzberg, M.; Dockray, G. J.; Cuello, A. C. Coexistence of cholecystokinin and substance P-like peptides in the neurons of the dorsal root ganglia of the rat. Neiirosci. Lett. 1982, 33, 159-163. 121 70. Skirboll, L.; Hokfelt, T.; Dockray, G.; Rehfeld, J.; Brownstein, M.; Cuello, A. Evidence for periaqueductal cholecystokinin-substance P neurons projecting to the spinal cord. J. Neurosci. 1983, 3, 1151-1157. 71. Skirboll, L.; Hokfelt, T.; Rehfeld, J.; Cuello, A. C.; Dockray, G. Coexistence of substance P- and cholecystokinin-like immunoreactivity in neurons of the mesencephalic periaqueductal central grey. Neurosci. Lett. 1982, 28, 35-39. 72. Nicoll, R. A.; Schenker, C.; Leeman, S. E. Substance P as a transmitter candidate. Annu. Rev. Neurosci. 1980, 3, 227-268. 73. Willets, J.; Urban, L.; Murase, K.; Randic, M. Actions of cholecystokinin octapeptide on rat spinal dorsal horn neurons. Annu. N. Y. Acad. Sci. 1985, 448, 385-402. 74. Innis, R. B.; Aghajanian, G. K. Cholecystokinin-containing and nociceptive neurons in rat Edinger-Westphal nucleus. Brain Res. 1986, 363, 230-238. 75. Zetler, G. Analgesia and ptosis caused by caerulein and cholecystokinin octapeptide (CCK). Neuropharmacology 1980, 19, 415-422. 76. Barbaz, B. S.; Autry, W. L.; Ambrose, F. G.; Hall, N. R.; Liebman, J. M. Antinociceptive profile of sulfated CCK: comparison with CCK-4, unsulfated CCK and other neuropeptides. Neuropharmacology 1986, 25, 823-829. 77. Hill, R. G.; Hughes, J.; Pittaway, K. M. Antinociceptive action of cholecystokinin octapeptide (CCK) and related peptides in rats and mice; effects of naloxone and peptidase inhibitors. Neuropharmacology 1987, 26, 133-139. 122 78. Fans, P. L.; Komisanak, B. R.; Watkins, L. R.; Mayer, D. J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 1983, 219, 310-312. 79. Faris, P L. Opiate antagonistic function of cholecystokinin in analgesia and energy balance systems. Ann. N. Y. Acad. Sci. 1985, 448, 437-447. 80. Watkins, L. R.; Kinscheck, I. B.; Rosenquist, G.; Miller, J.; Wimberg, S.; Frenk, H.; Kaufinan, E.; Coghill, R.; Suberg, S. N.; Mayer, D. J. The enhacement of opiate analgesia and the possible reversal of morphine tolerance by proglumide. Ann. N. Y. Acad. Sci. 1985, 448, 676-677. 81. Watkins, L. R.; Kinscheck, I. B.; Mayer, D. J. Potentiation of opiate analgesia and apparent reversal of morphine tolerance by proglumide. Science 1984, 224, 395-396. 82. Watkins, L. R.; Kinscheck, I. B.; Kauftnan, E. F. S.; Miller, J.; Frenk, H.; Mayer, D. J. Cholecystokinin antagonists selectively potentiate analgesia induced by endogenous opiates. Brain Res. 1985, 327, 181-190. 83. Tang, J.; Chou, J.; ladarola, M.; Yang, H. Y. T., Costa, E. Proglumide prevents and curtails acute tolerance to morphine in rats. Neuropharmacology 1984, 23, 715-718. 84. Katsuura, G.; Itoh, S. Potentiation of P-endorphin effects by proglumide in rats. Eur. J. Pharmacol. 1985, 107, 363-366. 85. Panerai, A. E.; Rovati, L. C.; Cocco, E.; Sacerdote, P.; Mantegazza, P. Dissociation of tolerance and dependence to morphine: a possible role for cholecystokinin. Brain Res. 1987, 410, 52-60. 123 86. Rovati, L. C.; Sacerdote, P.; Panerai, A. E. Effects of proglumide on morphine analgesia and tolerance. Ann. N. Y. Acad. Sci. 1985, 448, 630-632. 87. Schiller, P. W.; Lipton, A.; Horrobin, D. F.; Bodanszky, M. Unsulfated C-terminal 7- peptide of cholecystokinin: a new ligand of the opiate receptor. Biochem. Biophys. Res. Commun. 1978, 85, 1332-1338. 88. Pincus, M. R.; Carty, R. P.; Chen, J.; Lubowsky, J.; Avitable, M.; Shah. D.; Scheraga, H. A.; Murphy, R. B. On the biologically active structures of cholecystokinin, little gastrin, and enkephalin in the gastrointestinal system. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 4821-4825. 89. Nikiforovich, G. V.; Hruby, V. J. Models for the A- and B-receptor-bound conformations of CCK-8. Biochem, Biophys. Res. Commun. 1993, 194, 9-16. 90. Hruby, V. J.; Fang, S. N.; Kramer, T. H.; Davis, P.; Parkhurst, D. Nikiforovich, G.; Boteju, L. W.; Slaninova, J.; Yamamura, H. I.; Burks, T. F. Analogs of cholecystokinin26-33 selective for B-type CCK receptors possess 5opioid receptor agonist activity in vitro and in vivo: evidence for similarities in CCK-B and 5-opioid receptor requirements. Peptide: Chem., Struct., Biol., Proc. Am. Pept., 13'^; Hodges, R., Smith, A. J. Eds., 1994; pp 669-671. 91. Stanfa, L.; Dickenson, A.; Xu, X. -J.; Wiesenfeld-Hallin, Z. Cholecystokinin and morphine analgesia: variations on a theme. Trends Pharmacol. Sci. 1994, 15, 65-66. 92. Noble, F.; Derrien, M.; Roques, B. P. Modulation of opioid antinociception by CCK at the supraspinal level: Evidence of regulatory mechanisms between CCK and enkephalin systems in the control of pain. Br. J. Pharmacol. 1993, 109, 1064-1070. 124 93. Suh, H. H.; Collins, K. A.; Tseng, L. F. Intrathecal cholecystokinin octapeptide attenuates the antinociception and release of immunoreactive Met-enkephalin induced by intraventricular p-endorphin in the rat. Neuropeptides 1992, 21, 131-137. 94. Faris, P. L.; Komisaruk, B. R.; Watkins, L. R.; Mayer, D. J. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 1983, 219, 310-312. 95. Itoh, S.; Katsuura, G.; Maeda, Y. Caerulein and cholecystokinin suppress beta- endorphin-induced analgesia in the rat. Eur. J. Pharmacol. 1982, 80, 421-425. 96. Cesselin, F.; Bourgoin, S.; Artaud, F.; Hamon, M. Basic and regulatory mechanisms of in vitro release of Met-enkephalin from the dorsal zone of the rat spinal cord. J. Neurochem. 1984, 43,163-11A. 97. Hong, E. K.; Takemori, A. E. Indirect involvement of delta opioid receptors in cholecystokinin octapeptide-induced analgesia in mice. J. Pharmacol. Exp. Ther. 1989, 251,594-598. 98. Barbaz, B. S.; Autry, W. L.; Ambrose, F. G.; Gerber, R.; Liebman, J. M. Antagonism of cholecystokinin by naloxone and proglumide in mice. Ann. New York Acad. Sci. 1985, 448,573-574. 99. Slaninova, J.; Knapp, R. J.; Wu, J.; Fang, S. -N., Kramer, T., Burks, T. F., Hruby, V. J.; Yamamura, H. I. Opioid receptor binding properties of analgesic analogs of cholecystokinin octapeptide. Eur. J. Pharmacol. 1991, 200, 195-198. 125 100. Hraby, V. J.; Agnes, R. S.; Davis, P.; Ma, S. -W.; Lee, Y. S.; Vanderah, T. W.; Lai, J.; Porreca, F. Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain. Life Sci. 2003, 73, 699-704. 101. Richard Sario Agnes thesis 102. Smith J. A.; Pease, L. G.; Reverse turns in peptides and proteins. Crit. Rev. Biochem. 1980, 8, 315-399. 103. Rose, G. D.; Gierasch, L. M.; Smith, J. A. Turns in peptides and proteins. Adv. Protein. Chem. 1985, 37, 1-109. 104. Ball, J. B.; Alewood, P. F. Conformational constraints: nonpeptide P-tum mimics. J. Mol. Recognit. 1990, 3, 55-64. 105. Giannis, A.; Kolter, T. Peptide mimetics for receptor ligands: discovery, development, and medicinal perspectives. Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267. 106. Fink, B. E.; Kym, P. R.; Katzenellenbogen, J. A. Design, Synthesis, and Conformational Analysis of a Proposed Type I P-Tum Mimic. J. Am. Chem. Soc. 1998, 120, 4334-4334. 107. Venkatachalam, C. M.; Ramachandran, G. M. Conformation of polypeptide chains. Ann. Rev. Biochemistry. 1969, 38, 45-82 108. Kabsch, W.; Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983, 22, 2577-2637. 126 109. Hruby, V. J. Prospects of peptidomimetic drug design. Drug Discovery Today 1997, 2, 165-167. 110. Eguchi, M.; Lee, M. S.; Nakanishi, H.; Stasiak, M.; Lovell, S.; Kahn, M. Solid- phase sjmthesis and structural analysis of bicyclic P-tum mimetics incorporating functionality at the i to i + 3 positions. J. Am. Chem. Soc. 1999, 121, 12204-12205. 111. Olson, G. L.; Voss, M. E.; Hill, D. E.; Kahn, M.; Madison, V. S.; Cook, C. M. Design and synthesis of a protein P-tum mimetic. J. Am. Chem. Soc. 1990, 112, 323- 333. 112. Gardner, B.; Nakanishi, H.; Kahn, M. Conformationally constrained nonpeptide P-tum mimetics of enkephalin. Tetrahedron, 1993, 49, 3433-3448. 113. Newlander, K. A.; Callahan, J. F.; Moore, M. L.; Tomaszek, T. A.; Huffhian, W. F. A novel constrained reduced-amide inhibitor of HIV-1 protease derived firom the sequential incorporation of P-tum mimetics into a model substrate. J. Med. Chem. 1993, 36, 2321-2331. 114. Schmidt, B.; Lindman, S.; Tong, W.; Lindeberg, G.; Gogoll, A.; Lai, Z.; Thomwall, M.; Synnergren, B.; Nilsson, A.; Welch, C. J.; Sohtell, M.; Westerlund, C.; Nyberg, F.; Karlen, A.; Hallberg, A. Design, Synthesis, and Biological Activities of Four Angiotensin II Receptor Ligands with P-Tum Mimetics Replacing Amino Acid Residues 3-5. J. Med. Chem. 1997, 40, 903-919. 115. Brickman, K.; Somfai, P.; Kihlberg, J. An approach to enantiomerically pure inverse P-tum mimetics for use in solid-phase synthesis. Tetrahedron Lett. 1997, 38, 3651-3654. 127 116. Freidinger, R. M.; Veber, D. F.; Schwenk, Perlow, D.; Brooks, J. R.; Saperstein, R. Bioactive conformation of luteinizing hormone-releasing hormone: evidence from a conformationally constrained analog. Science 1980, 210, 656-658. 117. Nagai, U.; Sato, K. Synthesis of a bicyclic dipeptide with the shape of P-tum central part. Tetrahedron Lett. 1985, 26, 647-650. 118. Hinds, M. G.; Richards, N. G. J.; Robinson, J. A. Design and synthesis of a novel peptide p-tum mimetic. J. Chem. Soc., Chem. Commun. 1988, 1447-1449. 119. Genin, M. J.; Johnson, R. L.; Design, synthesis, and conformational analysis of a novel spiro-bicyclic system as a type II P-tum peptidomimetic. J. Am.Chem. Soc. 1992, 114,8778-8783. 120. Gramberg, D.; Weber, C.; Beeli, R.; Inglis, J.; Bruns, C.; Robinson, J. A. Synthesis of a type-VI p-tum peptide mimetic and its incorporation into a high- affinity somatostatin receptor ligand. Helv. Chim. Acta 1995, 78, 1588-1606. 121. Hruby, V. J.; Al-Obeidi, J.; Kazmierski, W. Emerging approaches in the molecular design of receptor-selective peptide ligands: conformational, topographical and dynamic considerations. Biochem, J. 1990, 268, 249-262. 122. Toniolo, C. Conformationally restricted peptides through short-range cyclizations. Int. J. Peptide, Protein Res. 1990, 35, 287-300. 123. Subasinghe, N. L.; Bontems, R. J.; Mclntee, E.; Mishra, R. K.; Johnson, R. L.; Bicyclic thiazolidine lactam peptidomimetics of the dopamine receptor modulating peptide Pro-Leu-Gly-NH2. J. Med Chem. 1993, 36, 2356-2361. 128 124. Comille, F.; Slomczynska, U.; Smythe, M. L.; Bensen, D. D.; Moeller, K. D.; Marshall, G. R. Electrochemical Cyclization of Dipeptides toward Novel Bicyclic, Reverse-Turn Peptidomimetics. 1. Synthesis and Conformational Analysis of 7,5- Bicyclic Systems. J. Am. Chem. Soc. 1995, 117, 909-917. 125. Preissner, R.; Goede, A.; Rother, K.; Koert, U.; Froemmel, C. Matching organic libraries with protein-substructures. J. Computer-Aided Molecula Design 2001, 15, 811-817. 126. Takeuchi, Y.; Marshall, G. R.; Conformational Analysis of Reverse-Turn Constraints by N-Methylation and N-Hydroxylation of Amide Bonds in Peptides and Non-Peptide Mimetics./. Am. Chem. Soc. 1998, 120, 5363-5372. 127. Brana, M. F.; Garxanzo, M.; de Pascual-Teresa, B.; perez-Castells, J.; Torres, M. R. Chemoselective Michael reactions on pyroglutamates. Expeditious synthesis of spiro-bis-y-lactams as (3-tum peptidomimetics. Tetrahedron, 2002, 58, 4825-4836. 128. Polyak, F.; Lubell, W. D. Mimicry of Peptide Backbone Geometry and Heteroatomic Side-Chain Functionality: Synthesis of Enantiopure Indolizidin-2-one Amino Acids Possessing Alcohol, Acid, and Azide Functional Groups. J. Org. Chem. 2001,66, 1171-1180. 129. Hoffinan, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Rational molecular design and EPC synthesis of a type VI P-tum inducing peptide mimetic. Angew. Chem. Int. Ed. Engl. 2001, 40, 3361-3364. 130. Cordero, F. M.; Valenza, S.; Machetti, F.; Brandi, A. Design and synthesis of a new bicyclic dipeptide isostere. Chem. Comm. 2001, 1590-1591. 129 131. Genin, M. J.; Johnson, R. L. Design, synthesis, and conformational analysis of a novel spiro-bicyclic system as a type II P-tum peptidomimetic. J. Am. Chem. Soc. 1992, 114,8778-8783. 132. Johannesson, P.; Lindeberg, G.; Tong, W.; Gogoll, A.; Karlen, A.; Hallberg, A. Bicyclic Tripeptide Mimetics with Reverse Turn Inducing Properties. J. Med. Chem. 1999, 42, 601-608. 133. Xiong, C.; Zhang, J.; Davis, P, Wang, W.; Ying, J.; Porreca, F.; Hruby, V. J. Stereoselective synthesis of individual isomers of Leu-enkephalin analogues containing substituted p-tum bicyclic dipeptide mimetics. Chemcomm. 2003, 1598- 1599. 134. Gu, X.; Vagner, J.; Stankova, M.; Hruby, V. J. Preparation of [BTD^'^]- enkephalins by solid phase synthesis. Biopolymers 2003, 71, 307. 135. Roy, S.; Lombart, H.-G.; Lubell, W. D.; Hancock, R. E. W.; Farmer, S. W. Exploring relationships between mimic configuration, peptide conformation and biological activity in indolizidin-2-one amino acid analogs of gramicidin S. J. Pept. Res. 2002, 60, 198-214. 136. Gosselin, F.; Tourwe, D.; Ceusters, M.; Meert, T.; Heylen, L.; Jurzak, M.; Lubell, W. D. Probing opioid receptor-ligand interactions by employment of indolizidin-9- one amino acid as a constrained• Gly23 -Gly surrogate in a leucine-enkephalin mimic. J. Pept. Res. 2001, 57, 337-344. 137. Tamura, S. Y.; Goldman, E. A.; Brunck, T. K.; Ripka, W. C.; Semple, J. E. Rational design, synthesis, and serine protease inhibitory activity of a novel PI- 130 argininal derivative featuring a conformationally constrained P2-P3 bicyclic lactam moiety. Bioorg. & Med. Chem. Lett. 1997, 7, 331-336. 138. Li, W.; Moeller, K. D. Conformationally Restricted TRH Analogs: The Compatibility of a 6,5-Bicyclic Lactam-Based Mimetic with Binding to TRH-R. J. Am. Chem. Soc. 1996, 118, 10106-10112. 139. Hanessian, S.; McNaughton-Smith, G. A versatile synthesis of a P-tum peptidomimetic scaffold: an approach towards a designed model antagonist of the tachykinin NK-2 receptor. Bioorg. & Med. Chem. Lett. 1996, 6, 1567-1572. 140. Nagai, U.; Sato, K.; Nakamura, R.; Kato, R. Bicyclic turned dipeptide (BTD) as a P-tum mimetic: its design, synthesis and incorporation into bioactive peptides. Tetrahedron 1993, 49, 3577-3592. 141. Bach, A. C. II.; Markwalder, J. A.; Ripka, W. C. Synthesis and NMR conformational analysis of a P-tum mimic incorporated into gramicidin S. A general approach to evaluate P-tum peptidomimetics. J. Pept. Pro. Res. 1991, 38, 314-23. 142. Hanessian, S.; McNaughton-Smith, G.; Lombart, H. -G; Lubell, W. D. Design and synthesis of conformationally constrained amino acids as versatile scaffolds and peptide mimetics. Tetrahedron 1997, 53, 12789-12854. 143. Halab, L.; Gosselin, F.; Lubell, W. D. Design, synthesis, and conformational analysis of azacycloalkane amino acids as conformationally constrained probes for mimicry of peptide secondary stmctures. Biopolymers (Peptide Science), 2000, 55, 101-122. 131 144. Qiu, W.; Gu, X.; Soloshonok, V. A.; Carducci, M. D.; Hruby, V. J. Stereoselective synthesis of conformationally constrained reverse turn dipeptide mimetics. Tetrahedron Lett. 2001, 42, 145-148. 145. Gu, X., Tang, X.; Cowell, S.; Ying, J.; Hruby, V. J. A novel strategy toward [6,5]- bicyclic p-tum dipeptide. Tetrahedron Lett. 2002, 43, 6669-6672. 146. Gu, X.; Cowell, S.; Ying, J.; Tang, X.; Hruby, V. J. Synthesis of P-phenyl-5,£- unsaturated amino acids and stereoselective introduction of side chain groups into [4,3,0]-bicyclic P-tum dipeptides. Tetrahedron Lett. 2003, 44, 5863-5866. 147. Zhang, J.; Xiong, C.; Wang, W.; Ying, J. Hruby, V. J. Stereoselective Bromination-Suzuki Cross-Coupling of Dehydroamino Acids To Form Novel Reverse-Turn Peptidomimetics: Substituted Unsaturated and Saturated Indolizidinone Amino Acids. Org. Lett. 2002, 4, 4029-4032. 148. Zhang, J.; Xiong, C.; Ying, J.; Wang, W.; Hruby, V. J. Stereoselective Synthesis of Novel Dipeptide P-Tum Mimetics Targeting Melanocortin Peptide Receptors. Org. Lett. 2003,5,3115-3118. 149. Wang, W.; Yang, J.; Ying, J.; Xiong, C.; Zhang, J.; Cai, C.; Hruby, V. J. Stereoselective Synthesis of Dipeptide P-Tum Mimetics: 7-Benzyl and 8-Phenyl Substituted Azabicyclo[4.3.0]nonane Amino Acid Esters. J. Org. Chem. 2002, 67, 6353-6360. 150. Kazmaier, U. Synthesis of unsaturated amino acids by [3,3]-sigmatropic rearrangement of chelate-bridged glycine ester enolates. Angew. Chem. Int. Ed. Engl. 1994, 33,998-999 132 151. Kazmaier, U. Diastereoselective synthesis of amino acids containing P-quatemary carbon centers via ester enolate Claisen rearrangement. Synlett. 1995, 1138-1140. 152. Kazmaier, U.; Maier, S. Synthesis of sterically high demanding a-alkylated amino acids via Claisen rearrangement of chelated enolates. Tetrahedron 1996, 52, 941-954. 153. Kazmaier, U. Application of the chelate-enolate Claisen rearrangement to the synthesis of y,§-unsaturated amino acids. Liebibs Ann./Recueil 1997, 2, 285-295. 154. Belokon, Yu. N.; Tararov, V. I.; Maleev, V. I.; Savel'eva, T. F.; Ryzhov, M. G. Improved procedures for the synthesis of (5)-2-[7V-(A^i9-benzylprolyI)amino] benzophenone (BPB) and Ni(II) complexes of Schiff s bases derived from BPB and amino acids. Tetrahedron Asymmetry 1999, 9, 4249-4252. 155. Collet, S.; Carreaux, F.; Boucher, J.-L.; Pethe, S.; Lepoivre, M.; Danion-Bougot, R.; Danion, D. Synthesis and evaluation of co-borono-a-amino acids as active-site probes of arginase and nitric oxide synthases. J. Chem. Soc., Perkin Trans. 1 2000, 177-182. 156. Collet, S.; Bauchat, P.; Danion-Bougot, R.; Danion, D. Stereoselective, nonracemic synthesis of co-borono-a-amino acids. Tetrahedron: Asymmetry 1998, 9, 2121-2131. 157. Cramer, U.; Rehfeldt, A. G.; Spencer, F. Isolation and biosynthesis of cyclopentenylglycine, a novel nonproteinogenic amino acid in Flacourtiaceae. Biochemistry 1980, 19, 3074-3080. 133 158. Edelson, J.; Fissekis, J. D.; Skinner, C. G.; Shive, W. 3-Cyclohexene-l-glycine, an isoleucine antagonist. J. Am. Chem. Soc. 1958, 80, 2698-2700. 159. Katagiri, K.; Tori, K.; Kimura, Y.; Yoshida, T.; Nagasaki, T.; Minato, H.; A new antibiotic. Furanomycin, an isoleucine antagonist. J. Med. Chem. 1967, 10, 1149- 1154. 160. Rutjes, F. P. J. T.; Wolf, L. B.; Schoemaker, H. E. Applications of aliphatic unsaturated non-proteinogenic a-H-a-amino acids. J. Chem. Soc., Perkin Trans. 1, 2000,4197-4212. 161. Reetz, M. T.; Synthesis and Diastereoselective Reactions of A^jA'-Dibenzylamino Aldehydes and Related Compounds. Chem, Rev. 1999, 99, 1121-1162. 162. Hoffman, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Rational molecular design and EPC synthesis of a type VI P-tum inducing peptide mimetic. Angew. Chem. Int. Ed. Engl. 2001,40,3361. 163. Fernandez, M. M.; Diez, A.; Rubiralta, M.; Casamitjana, N.; Kogan, M. J.; Giralt, E. Spirolactams as Conformationally Restricted Pseudopeptides: Synthesis and Conformational Analysis. J. Org. Chem. 2002, 67, 7587-7599. 164. Reichwein, J. F.; Liskamp, R. M. J. Synthesis of cychc dipeptides by ring-closing metathesis. Eur. J. Org. Chem. 2000, 2335-2344. 165. Ripka, A. S.; Bohacek, R. S.; Rich, D. H.; Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis. Bioorg. & Med. Chem. Lett. 1998, 8, 357- 360. 134 166. Baldwin, J. E.; Moloney, M. G.; North, M. Asymmetric amino acid synthesis: preparation of the P anion derived from aspartic acid. J. Chem. Soc., Perkin Iran. /, 1989,833-834. 167. Dunn, P. J.; Haner, R.; Rapoport, H. Stereoselective synthesis of 2,3-diamino acids. 2,3-Diamino-4-phenylbutanoic acid. J. Org. Chem. 1990, 55, 5017-5025. 168. Cotton, R.; Johnstone, A. N. C.; North, M. Asymmetric synthesis of (2S,3S)-3- carboxy-proline. Tetrahedron Lett. 1994, 35, 8859-8862. 169. Humprey, J. M.; Bridges, R. J.; Hart, J. A.; Chamberlin, A. R. 2,3- Pyrrolidinedicarboxylates as Neurotransmitter Conformer Mimics: Enantioselective Synthesis via Chelation-Controlled Enolate Alkylation. J. Org. Chem. 1994, 59, 2467-2472. 170. Cotton R.; Johnstone, A. N. C.; North, M.; Asymmetric synthesis of 3- carboxyprohne and derivatives suitable for peptide synthesis. Tetrahedron 1995, 51, 8525-8544. 171. Corey, E. J.; Raju, N. A new synthetic route to bridged carboxylic ortho esters. Tetrahedron Lett. 1983, 24, 5571-5574. 172. Atkins, M. P.; Golding, B. T.; Howes, D. A.; Sellars, P. Masking the carboxy group as a 2,6,7-trioxabicyclo[2.2.2]octane: application to the synthesis of alkylcobaloximes containing ester and carboxy groups. J. Chem. Comm. 1980, 207- 208. 135 173. Corey, E. J.; Shimoji, K. Total synthesis of the major human urinary metabolite of prostaglandin D2, a key diagnostic indicator J. Am. Chem. Soc. 1983, 105, 1662- 1664. 174. Dewolf, R. H. Synthesis of carboxylic and carbonic ortho esters. Synthesis 1974, 153-172. 175. Kubel, B.; Hofle, G.; Steglich, W. Hetero Cope rearrangement in the cyclization of N-acyl amino acid allyl and propargyl esters in 5-oxazolinones. Angew. Chem. 1975, 87, 64-66. 176. Kubel, B.; Hofle, G.; Steglich, W. Hetero Cope Rearrangements in the Cyclization of Allyl and Propargyl Esters of N-Acyl Amino Acids to Oxazolin-5-one. Angew. Chem. Int. Ed. Engl. 1975, 14, 58-59. 177. Engel, N.; Kubel, B.; Steglich, W. C-C Linkage of Carboxylic Acids with Allyl Alcohols Using 2-Phenylglycine as Vehicle. Angew. Chem. Int. Ed. Engl. 1977, 16, 394-396. 178. Engel, N.; Kubel, B.; Steglich, W. Carbon-carbon bonding of carboxylic acids with allyl alcohols using 2-phenylglycine as vehicle. Angew. Chem.. 1977, 89, 408- 410. 179. Ireland, R. E.; Mueller, R. H.; Willard, A. K. The ester enolate Claisen rearrangement. Stereochemical control through stereoselective enolate formation. J. Am. Chem. Soc. 1976, 98, 2868-2877. 180. Bartlett, P. A.; Barstow, J. F. Ester-enolate Claisen rearrangement of a-amino acid derivatives. J. Org. Chem. 1982, 47, 3933-3941. 136 181. Burke, S. D.; Fobare, W. F.; Pacofsky, G. J. Chelation control of enolate geometry. Acyclic diastereoselection via the enolate Claisen rearrangement. J. Org. Chem. 1983, 48, 5221-5228. 182. Uchikawa, M.; Katsuki, T.; Yamaguchi, M. [2,3]Wittig rearrangement of 2- (alkenyloxy)acetic acid esters. Tetrahedron Lett. 1986, 27, 4581-4582. 183. Uchiyama, H.; Kawano, M.; Katsuki, T.; Yamaguchi, M. Ester enolate Claisen rearrangement via boat-like transition state. Chem Lett. 1987, 351-354. 184. Gould, T. J.; Balestra, M.; Wittman, M. D.; Gary, J. A.; Rossano, L. T.; Kallmerten, J. Stereocontrolled synthesis of highly oxygenated acyclic systems via the enolate Claisen rearrangement of O-protected allylic glycolates. J. Org. Chem. 1987, 52,3889-3901. 185. Altenbach, H. -J. Ester enolate Claisen rearrangements. Organic Synthesis Highlights (Eds.: Mulzer, J.; Altenbach, H. -J.; Braun, M.; Krohn, K.; Reissig, H. - U.), VCH, Weinheim, 1991, p. 116-118. 186. Corey, E. J.; Lee, D. An experimental demonstration of the stereochemistry of enzymic cyclization of 2,3-oxidosqualene to the protosterol system, forerurmer of lanosterol and cholesterol. J. Am. Chem. Soc. 1991, 113, 4026-4028. 187. Baumann, H.; Duthaler, R. O. Synthesis of ethyl (27?5',35'/?)-l-tosyl-3- vinylazetidine-2-carboxylate and ethyl (27?5',ii)-3-ethylidene-l-tosylazetidine-2- carboxylate (= rac-ethyl N-tosylpolyoximate). Heh. Chim. Acta. 1988, 71, 1025- 1034. 137 188. Kabalka, G. W.; Shoup, T. M.; Goudgaon, N. M. Sodium perborate: a mild and convenient reagent for efficiently oxidizing organoboranes. J. Org. Chem. 1989, 54, 5930-5933. 189. McGeary, R. P. Facile and chemoselective reduction of carboxylic acids to alcohols using BOP reagent and sodium borohydride. Tetrahedron Lett. 1998, 39, 3319-3322. 190. Tidwell, T. T. Oxidation of alcohols to carbonyl compounds via alkoxysulfonium ylides: the Moffat, Swem, and related oxidations. Organic Reactions (New York) 1990, 39, 297-572. 191. Murray, R. W. Ozonolysis of organic compounds. Techniques and Methods of Organic and Organometallic Chemistry 1969, 1, 1-32. 192. Pyne, S. G. Intramolecular addition of amines to chiral vinyl sulfoxides. Total synthesis of (R)-(+)-canadine. Tetrahedron Lett. 1987, 28, 4737-4740. 193. Muller, R.; Revesz, L. Synthesis of 6,5-fused bicyclic lactams as potential dipeptide P-tum mimetics. Tetrahedron Lett. 1994, 34, 4091-4092. 194. Lombart, H. -G.; Lubell, W. D.; Rigid Dipeptide Mimetics: Efficient Synthesis of Enantiopure Indolizidinone Amino Acids. J. Org. Chem. 1996, 61, 943-946. 195. Gosselin, F.; Lubell, W. D. An olefination entry for the synthesis of enantiopure a,co,-diaminodicarboxylates and azabicyclo[X.Y.O]alkane amino acids. J. Org. Chem. 1998, 63,7463-7471. 138 196. Kim, H. -O.; Kahn, M. Short synthesis of (35,65',95)-2-oxo-3-(N-Boc-amino)-l- azabicyclo[4.3.0]nonane-9-carboxylic acid methyl ester: tandem cyclization protocol. Tetrahedron Lett. 1997, 37, 6483-6484. 197. Bridges, R, J.; Stanley, M. S.; Anderson, M. W.; Cotman, C. W.; Charmberlin, A. R. Conformationally defined neurotransmitter analogues. Selective inhibition of glutamate uptake by one pyrrolidine-2,4-dicarboxylate diastereomer. J. Med. Chem. 1991,34,717-725. 198. Monaghan, D. T.; Bridges, R. J.; Cotman, C. W. The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Annu. Rev. Pharmacol. Toxicol. 1989, 29, 365-402. 199. Choi, D. W. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988, 1,623-634. 200. Meldrum, B. Possible therapeutic applications of antagonists of excitatory amino acid neurotransmitters. Clin. Sci. 1985, 68, 113-122. 201. Wieloch, T. Hypoglycemia-induced neuronal damage prevented by an N-methyl- D-aspartate antagonist. Science, 1985, 230, 681-683. 202. Rothman, S. M.; Olney, J. W. Glutamate and the pathophysiology of hypoxic- ischemic brain damage. Ann. Neurol. 1986, 19, 105-111. 203. Bridges, R. J.; Geddes, J.; Monaghan, D. T.; Cotman, C. W. Excitatory Amino Acids in Health and Diseases Lodge, D., Ed.; Wiley & Sons, Limited: England, 1987, pp 321-335. 139 204. Wistrand, L. -G.; Skrinjar, M. Chirospecific synthesis of trans-2,5-disubstituted pyrrolidines via stereoselective addition of organocopper reagents to N-acyliminium ion. Tetrahedron, 1991, 47, 573-582. 205. Collado, I.; Ezquerra, J.; Pedregal, C. Stereoselective Addition of Grignard- Derived Organocopper Reagents to N-Acyliminium Ions: Synthesis of Enantiopure 5- and 4,5-Substituted Prolinates. J. Org. Chem. 1995, 60, 5011-5015. 206. Manfre, F.; Kern, J-M.; Biellmann, J-F. Syntheses of proline analogs as potential mechanism-based inhibitors of proline dehydrogenase: 4-methylene-L-, (E)- and (Z)- 4-(fluoromethylene)-L-, cis- and trans-5-ethynyl-(D)-, and cis- and trans-5-vinyl-L- proline. J. Org. Chem. 1992, 57, 2060-2065. 207. Mulzer, J.; Schulzchen, F.; Bats, J-W. Rigid dipeptide mimetics. Stereocontrolled synthesis of all eight stereoisomers of 2-Oxo-3-(N-Cbz-amino)-l- azabicyclo[4.3.0]nonane-9-carboxylic acid ester. Tetrahedron, 2000, 56, 4289-4298. 208. Schmidt, U.; Griesser, H.; Leitenberger, V.; Lieberknecht, A.; Mangold, R.; Meyer, R.; Riedl, B. Amino acids and peptides. Diastereoselective formation of (Z)- didehydroamino acid esters. Synthesis 1992, 487-490. 209. Noyori, R. Asymmetric Catalysis in Organic Synthesis, John Wiley and Sons, New York, 1994, pp. 16-94. 210. Ojima, I. Transition Metal Hydrides: Hydrocarboxylation, Hydroformylation and Asymmetric Hydrogenation, in Comprehensive Organometallic Chemistry II, Abel, E. W.; Stone, F. G. A.; Wilkinson, G., Eds., Pergamon, Oxford, UK, 1995, Vol. 12, pp. 9-38. 140 211. Burk, M. J. Modular Phospholane Ligands in Asymmetric Catalysis. Acc. Chem. Res. 2000, 33, 363-372. 212. Hegedus, L. S. Transition Metals in the Synthesis of Complex Organic Molecules., Second Edition, University Science Books, Sausalito, CA. 1999, 39-55. 213. Enders, D.; Klumpen, T. Asymmetric synthesis of planar chiral 2-mono- and 2,2'- disubstituted l,r-bisbenzoylferrocenes. J. Organometallic. Chem. 2001, 637-639, 698-709. 214. Schon, I. Sodium-liquid ammonia reduction in peptide chemistry. Chemical Reviews, \9%4, 84, 287-97. 215. Brieger, G.; Nestrick, T. J. Catalytic transfer hydrogenation. Chemical Reviews, 1974, 74, 567-80. 216. Matsueda, R.; Higashida, S.; Ridge, R. J.; Matsueda, G. R. Activation of conventional S-protecting groups of cysteine by conversion into the 3-nitro-2- pyridinesulfenyl (Npys) group. Chem. Lett. 1982, 921-924. 217. Matsueda, R.; Aiba, K. A stable pyridinesulfenyl halide. Chem. Lett. 1978, 951- 952. 218. Matsueda, R.; Kimura, T.; Kaiser, E. T.; Matsueda, G. R. 3-Nitro-2- pyrinedinesulfenyl group for protection and activation of the thiol function of cysteine. Chem. Lett. 1981,131-1AO. 219. Karimi, B.; Zareyee, D. Hexamethyldisilazane (HMDS) promotes highly efficient oxidative coupling of thiols by DMSO under nearly neutral reaction conditions. Synlett. 2002, 2, 346-348. 141 220. Mancuso, A. J.; Swem, D. Activated dimethyl sulfoxide: useful reagents for synthesis. Synthesis 1981, 165-185. 221. Xiong, C.; Wang, W.; Hruby, V. J. A General Asymmetric Synthesis of syn- and anti-P-Substituted Cysteine and Serine Derivatives. J. Org. Chem. 2002, 67, 3514- 3517. 222. Xiong C.; Wang W.; Cai C.; Hruby V. J. Regioselective and stereoselective nucleophilic ring opening reactions of a phenyl-substituted aziridine; enantioselective synthesis of beta-substituted tryptophan, cysteine, and serine derivatives. J. Org. Chem, 2002, 67, 1399-402. 223. Svirskaya, P. I.; Leznoff, C. C.; Steinman, M. Trifluoroacetylation of amines and amino acids by polymer-bound trifluoroacetylation reagents. J. Org. Chem., 1987, 52, 1361364. 224. Katritzky, A. R.; Yang, B.; Semenzin, D. (Trifluoroacetyl)benzotriazole; A Convenient Trifluoroacetylating Reagent. J. Org. Chem. 1997, 62, 726-728. 225. Furst, A.; Koller, F. Steroids and sex hormones. CXLV. A new way to prepare a- oxides of cholesterol and trans-dehydroandrostane. Helv. Chim. Acta. 1947, 30, 1454- 1460. 226. Whitmore, F.; Olewine, J. H. The primary active amyl halides J. Am. Chem. Soc. 1938, 60, 2570-2571 227. Takahashi, T.; Nemoto, H.; Kanda, Y.; Tsuji, J. Macroring contraction methodology 3. total syntheses of costunolide and Haageanolide using transannular 142 [2,3]-wittig rearrangement of 13-membered diallylic ethers as key reaction. Tetrahedron 1987, 43, 5499-5520. 228. Prabhakaran, E. N.; Rao, I. N.; Boruah, A.; Iqbal, J. Synthesis of Small Cyclic Peptides via Reverse Turn Induced Ring Closing Metathesis of Tripeptides. J. Org. Chem. 2002, 61, 8247-8250. 229. Miller, S. J.; Grubbs, R. H. Synthesis of Conformationally Restricted Amino Acids and Peptides Employing Olefin Metathesis. J. Am. Chem. Soc. 1995, 117, 5855-5856. 230. Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. A series of well-defined metathesis catalysts - synthesis of [RuCl2(:CHR')(PR3)2] and their reactions. Angew. Chem., Int. Ed. 1995, 34, 2039-2041. 231. Miller, S. J.; Blackwell, H. E.; Grubbs, R. H. Application of Ring-Closing Metathesis to the Synthesis of Rigidified Amino Acids and Peptides. /. Am. Chem. Soc. 1996, 118, 9606-9614. 232. Jarvo, E. R.; Copeland, G. T.; Papaioannou, N.; Bonitatebus, P. J.; Miller, S. J. A Biomimetic Approach to Asymmetric Acyl Transfer Catalysis. J. Am. Chem. Soc. 1999, 121,11638-11643. 233. Schafineister, C. E.; Po, J.; Verdine, G. L. An All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc. 2000, 122, 5891-5892. 143 234. Schmiedeberg, N; Kessler, H. Reversible Backbone Protection Enables Combinatorial Solid-Phase Ring-Closing Metathesis Reaction (RCM) in Peptides. Organic. Lett. 2002, 4, 59-62. 235. Rich, D. H.; Tarn. J. P. A method for introducing secondary amide bonds into strained cyclic peptides. Tetrahedron Lett. 1977, 9, 749-750. 236. Goldring, W. P. D.; Hodder, A. S.; Weiler, L. Synthesis of macrocyclic lactams and lactones via ring-closing olefin metathesis. Tetrahedron Lett. 1998, 39, 4955- 4958. 237. Creighton, Christopher J.; Reitz, Allen B. Synthesis of an eight-membered cyclic pseudo-dipeptide using ring closing metathesis. Organic Lett. 2001, 3, 893-895. 238. Reichwein, J. F.; Wels, B.; Kruijtzer, J. A. W.; Versluis, C.; Liskamp, R. M. J. Rolling loop scan: an approach featuring ring-closing metathesis for generating libraries of peptides with molecular shapes mimicking bioactive conformations or local folding of peptides and proteins. Angew. Chem., Int. Ed. 1999, 38, 3684-3687. 239. Haack, Thomas; Mutter, Manfred. Serine derived oxazolidines as secondary structure disrupting, solubilizing building blocks in peptide synthesis. Tetrahedron Zeff. 1992, 33, 1589-1592. 240. Johnson, T.; Quibell, M. The N-(2-hydroxybenzyl) protecting group for amide bond protection in solid phase peptide synthesis. Tetrahedron Lett. 1994, 35, 463- 466. 144 241. Miranda, L. P.; Meutermans, W. D. F.; Smythe, M. L.; Alewood, P. F. An Activated N Acyl Transfer Auxiliary: Efficient Amide-Backbone Substitution of Hindered "Difficult" Peptides. J. Org. Chem. 2000, 65, 5460-5468. 242. Woehr, T,; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter, M. Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis. J. Am. Chem. Soc. 1996, 118, 9218-9227. 145 Abbreviations Abbreviations are used for amino acids and designation of peptides follows the rules of the lUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem 1972, 247, 977-983. Additional abbreviations are used as follows: Boc tert-butoxylcarbonyl BOP Benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro phosphate Fmoc 9-fluorenylmethoxycarbonyl tBu tert-butyl DMF N,N-dimethyl formamide DBU 1,8 -Diazabicy do[5.4.0]undec-7 -ene HBTU 2-( 1 H-benzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate HOBt N-hydroxylbenzotriazole RP-HPLC reversed-phase high performance liquid chromatography TFA trifluoroacetic acid TIS triisopropyl silane TLC thin layer chromatography