Polyamine Drug Discovery: Rsc: Volume 17 (Drug Discovery Series) - Hardcover

Hardcover

ISBN 10: 184973190X ISBN 13: 9781849731904

Publisher: Royal Society of Chemistry, 2011

Synopsis

This is the first comprehensive description of the discovery and therapeutic potential of polyamine drugs.

About this title

About the Author

Patrick M. Woster, Ph.D. is Professor and Center for Economic Excellence Endowed Chair in the Department of Pharmaceutical and Biomedical Sciences at the Medical University of South Carolina. He is a medicinal chemist with an interest in the synthesis of molecules that modulate polyamine metabolism or chromatin remodeling as potential antitumor agents. Dr. Woster also maintains a program in antiparasitic drug discovery with a particular emphasis on malaria and trypanosomiasis. He has produced a number of inhibitors that target enzymes in the polyamine biosynthetic pathway, and synthesized the first unsymmetrically substituted alkylpolyamine analogues. Molecules developed in the Woster laboratory have been shown to produce dramatic effects on a variety of tumor cells by initiating apoptosis, binding to DNA and by producing epigenetic changes in gene expression. Robert A. Casero, Jr., Ph.D. is a Professor of Oncology in the Johns Hopkins University School of Medicine. Dr. Casero is a molecular pharmacologist who has spent most of the last 30 years studying the role of polyamines in normal and tumour cell growth, and devising strategies to target polyamine function and metabolism for therapeutic benefit. His laboratory was responsible for cloning several genes involved in human polyamine catabolism; genes whose expression are thought to play a role in determining cellular responses to specific polyamine analogues.

From the Back Cover

Polyamines are ubiquitous molecules that are involved in a number of important cellular processes. Aberrations in their function or metabolism play a role in diseases such as cancer and parasitic infection. A number of validated drug targets have been identified, including enzymes in the polyamine biosynthetic and catabolic pathways and the S-adenosylmethionine synthetic and salvage pathways. Polyamine Drug Discovery is the first comprehensive volume to cover all aspects of the design and development of potential therapeutics targeting polyamine metabolism. The book details research progress from 1975 to the present date and discusses the design and use of polyamine metabolism inhibitors as therapeutic agents. Various polyamine-containing drugs are described that can be used in chemotherapy, and as treatments for infections including trypanosomiasis, leishmaniasis and malaria. Finally, the roles of polyamine analogues in chemoprevention, polyamine-containing vectors for gene delivery, and the design of polyamine-based epigenetic modulators are detailed. Each chapter addresses a different aspect of polyamine drug discovery and all are written by medicinal and biological chemists with particular expertise in developing agents that modulate polyamine metabolism or function. The book will increase the visibility of polyamine drug discovery among pharmaceutical researchers and provide a valuable reference for everyone working in the field.

Excerpt. © Reprinted by permission. All rights reserved.

Polyamine Drug Discovery

By Patrick M. Woster, Robert A. Casero Jr.

The Royal Society of Chemistry

Copyright © 2012 Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-190-4

Contents

Chapter 1 Polyamine Drug Discovery: Synthetic Approaches to Therapeutic Modulators of Polyamine Metabolism Patrick M. Woster, 1, 
Chapter 2 Structural Biology in Polyamine Drug Discovery Shridhar Bale and Steven E. Ealick, 28, 
Chapter 3 Antiparasitic Drug Discovery for the Polyamine Pathway Nigel Yarlett and Mary Morada, 50, 
Chapter 4 Inhibitors of Polyamine Biosynthetic Enzymes Anthony E. Pegg, 78, 
Chapter 5 Symmetrical-and Unsymmetrical Terminally Alkylated Polyamines Patrick M. Woster and Robert A. Casero, Jr, 104, 
Chapter 6 Targeting the Polyamine Catabolic Enzymes Spermine Oxidase, N1-Acetylpolyamine Oxidase and Spermidine/Spermine N1-Acetyltransferase Andrew C. Goodwin, Tracy R. Murray-Stewart and Robert A. Casero, Jr, 135, 
Chapter 7 Design of Polyamine Transport Inhibitors as Therapeutics Otto Phanstiel IV and Jennifer Julian Archer, 162, 
Chapter 8 Non-Covalent Polynuclear Platinum Compounds as Polyamine Analogs Yun Qu, Joseph J. Moniodis, Amanda L. Harris, Xiaohong Yang, Alex Hegmans, Lawrence F. Povirk, Susan J. Berners-Price and Nicholas P. Farrell, 191, 
Chapter 9 Polyamine-Based Agents for Gene and siRNA Transfer Ian S. Blagbrough, Abdelkader A. Metwally and Osama A. A. Ahmed, 205, 
Chapter 10 The Design and Development of Polyamine-Based Analogs with Epigenetic Targets Yi Huang, Laurence J. Marton and Patrick M. Woster, 238, 
Chapter 11 Clinical Applications of Polyamine-Based Therapeutics André S. Bachmann and Victor A. Levin, 257, 
Subject Index, 277, 

CHAPTER 1

Polyamine Drug Discovery: Synthetic Approaches to Therapeutic Modulators of Polyamine Metabolism

PATRICK M. WOSTER

Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, 70 President St., Charleston, SC 29425, USA

1.1 Introduction

In the following chapters, a complete description of the design, bioevaluation and development of modulators of polyamine metabolism is presented. There are numerous synthetic approaches to these inhibitors, and as such a comprehensive review of the chemical literature in this area is beyond the scope of this book. In this chapter, specific examples of synthetic approaches to nucleosides, analogs of the natural polyamines and other agents that affect polyamine metabolism are described. The reader should bear in mind that the literature is replete with alternative strategies for the synthesis of compounds described herein. However, the examples provided will allow the reader to appreciate the vast chemical diversity that is available to medicinal chemists working in the polyamine field.

1.2 Polyamine Metabolism as a Drug Target

The mammalian polyamine biosynthetic pathway is shown in Figure 1.1. Ornithine is converted to putrescine by the action of the enzyme ornithine decarboxylase (ODC). Mammalian ODC, a dimeric enzyme with a molecular weight of about 80 000, is a typical pyridoxal phosphate-requiring amino acid decarboxylase that has been studied quite extensively. ODC is known to be one of the control points in the polyamine biosynthetic pathway, producing a product that is committed to polyamine biosynthesis. The synthesis and degradation of ODC are controlled by a number of factors including degradation assisted by a specific ODC antizyme, a polyamine-induced protein that binds to ODC and promotes ubiquitin-independent degradation by the 26S proteasome. As a result, ODC has a functional half-life of about 10 min. Putrescine is next converted to spermidine via an aminopropyltransferase known as spermidine synthase, which requires decarboxylated S-adenosylmethionine as a co-substrate. A second closely related but distinct aminopropyltransferase, spermine synthase, then adds an additional aminopropyl group to spermidine to yield spermine, the longest polyamine occurring in mammalian systems. The by-product for the spermidine and spermine synthase reactions is 5'-methyl-thioadenosine (MTA), a potent product inhibitor for the aminopropyl transfer process. In mammalian systems, MTA is rapidly hydrolyzed by the enzyme MTA-phosphorylase, and the components are converted to adenosine and methionine via salvage pathways. The aminopropyl donor for both amino- propyltransferases is decarboxylated S-adenosylmethionine (dc-AdoMet), produced from S-adenosylmethionine (AdoMet) by S-adenosylmethionine decarboxylase (AdoMet-DC). AdoMet-DC, like ODC, is a highly regulated enzyme in mammalian cells, and also serves as a regulatory point in the path- way. However, unlike ODC, AdoMet-DC belongs to a class of pyruvoyl enzymes that do not require pyridoxal phosphate as a cofactor (see below).

Polyamine metabolism is tightly controlled by a combination of inducible enzymes and the import/export of cellular polyamines. In addition to the enzymes mentioned above, intracellular polyamine content is modulated by a pair of acetyltransferases. Spermidine in the cell nucleus is acetylated on the four-carbon end by spermidine-N8-acetyltransferase, possibly altering the compound's binding affinity for DNA. A specific deacetylase can then reverse this enzymatic acetylation. Cytoplasmic spermidine and spermine serve as substrates for spermidine/spermine-N1-acetyltransferase (SSAT), resulting in acetylation on the three-carbon end of each molecule (Figure 1.1). The acetylated spermidine or spermine then acts as a substrate for acetylpolyamine oxidase (APAO), which catalyzes the formation of 3-acetamidopropionaldehyde and either putrescine or spermidine, respectively. Excess acetylated polyamines can also be exported from the cell via the polyamine transport system. More recently, a second polyamine oxidase, the inducible spermine oxidase (SMO) was discovered and characterized. Thus, SSAT, APAO and SMO together serve as a reverse route for the interconversion of polyamines. An additional mechanism for control of cellular polyamines is provided by the polyamine transport system, which has been well characterized in some organisms (bacteria, yeast), but has not been well characterized in mammalian organisms. The function of enzymes in polyamine metabolism and the polyamine transport system, and the consequences of modulating their activity, are described in more detail elsewhere in this book.

1.3 Synthetic Approaches to Modulators of Polyamine Metabolism and Function

1.3.1 Ornithine Decarboxylase (ODC)

Mammalian ODC is a highly unstable protein, and cellular levels of ODC depend on rates of synthesis and degradation as outlined above. For this reason, reversible and irreversible inhibitors of ODC have proven to be of limited value, since the synthesis of new protein occurs very rapidly in response to reduced polyamine levels in the cell. The catalytic mechanism of ODC involves the formation of a Schiff's base between the amino group of ornithine and the pyridoxal phosphate cofactor which is tightly bound to ODC. The most useful inhibitor of ODC to date, α-difluoromethylornithine (DFMO, 1, Scheme 1.1), takes advantage of this aspect of the mechanism, and belongs to a group of rationally designed mechanism-based inactivators specifically targeted to individual amino acid decarboxylases. The chemical synthesis of DFMO is shown in Scheme 1.1. The (bis)benzylidene-protected amino ester 2 is treated with lithium diisopropylamide (LDA) followed by exposure to 1-chloro-2,2-difluoroethane to form the alkylated product 3. Removal of the benzylidene protecting groups and cleavage of the methyl ester are accomplished simultaneously to afford DFMO 1 in a 60% overall yield. It is noteworthy that the pathway shown in Scheme 1.1 is not used at the industrial scale, and the large-scale production of DFMO is an expensive undertaking. Thus, until recently, the drug has been produced almost exclusively in sufficient quantities for inclusion in commercial preparations such as the the lifestyle drug Vaniqa®. Although DFMO is available commercially in small quantities for research, the cost is prohibitive.

The mechanism of inactivation of ODC by DFMO is shown in Scheme 1.2. As a substrate analog, DFMO forms a Schiff's base with the pyridoxal phosphate cofactor bound to ODC. The subsequent decarboxylation step results in the generation of a latent electrophile, and ODC is rapidly and irreversibly deactivated by forming a covalent bond with CYS360. The discovery of DFMO provided an enormous stimulus to the field of mammalian polyamine biology. Historically, DFMO has been marketed as a treatment for Pneumocystis carinii secondary infections in immunocompromised patients, and has been shown to be effective in curing infections of Trypanosoma brucei gambiense (but not T. brucei rhodesiense) in limited clinical trials.

A relatively small number of ODC inhibitors related to 1 have been described, mostly in the mid- to late 1980s, by the highly productive Merrell Dow research group, but none were as successful as 1 in either in vitro or in vivo studies. The ODC inactivator (2R,5R)-6-heptyne-2,5-diamine (R,R-MAP), was shown to possess a Ki of 3 µM, and to penetrate mammalian cells relatively well. This compound was the subject of human clinical trials but was never marketed. Based on the promising activity of 1 and R,R-MAP, a number of fluorine-containing mechanism based inactivators of ODC were developed, the most potent of which was 2,2-difluoro-5-hexyne-1,4-diamine, 4. The synthesis of 4 is outlined in Scheme 1.3, and illustrates that reasonably complex syntheses are often required to access simple but specifically designed target molecules. The requisite ester ethyl 2,2-difluoro-4-pentenoate 5 was reduced in quantitative yield to afford the corresponding alcohol 6. Formation of the triflate followed by addition of phthalimide then gave the protected aminoolefin 7. Ozonolysis was then used to convert the olefinic linkage to the aldehyde 8. The acetylene group was added via a Grignard reagent (HCC-MgBr), followed by addition of phthalimide using a Mitsunobu reaction to produce 9. The phthalimide protecting groups were removed (hydrazine) to afford 10, followed by conversion to the dihydrochloride 4. Despite early successes, 4 was not developed as a drug following the dissolution of the Merrell Dow research effort in polyamine research. More recently, Gehring et al. have described transition-state analog inhibitors of ODC involving the structures of ornithine and pyridoxal phosphate.

1.3.2 S-Adenosylmethionine Decarboxylase (AdoMet-DC)

Mammalian AdoMet-DC is a pyruvoyl enzyme which has two subunits of 32 000 Mr. The pyruvate residue at the N-terminus of one of the subunits serves the function of pyridoxal phosphate, forming a Schiff's base with the primary amino group in AdoMet. AdoMet-DC is an inducible enzyme, and responds dramatically to either polyamine depletion or elevation of spermidine or spermine. AdoMet-DC requires putrescine for activation, which proceeds autocatalytically, as shown in Figure 1.2. In the case of human AdoMet-DC, cleavage occurs between residues Glu67 and Ser68. The Ser68 oxygen attacks the adjacent carbonyl carbon of Glu67 to generate a five-membered oxyoxazolidine intermediate that rapidly rearranges to form an ester intermediate. His243 then abstracts a proton from the alpha carbon of Ser68, thus cleaving the ester and forming two chains. The N-terminal end of the cleavage product becomes the b-chain, and the C-terminal portion becomes the α-chain. The N-terminal dehydroalanine residue resulting from cleavage of the ester tautomerizes to form an imine, which is then hydrolyzed to a pyruvate.

The antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG, 11, Figure 1.3) is a potent competitive inhibitor of the putrescine-activated mammalian AdoMet-DC, with a Ki value of less than 1 [microM. However, MGBG is of limited use as an inhibitor due to a wide variety of other effects on cells, including induction of severe mitochondrial damage, interference with polyamine transport and induction of SSAT. In an attempt to abrogate these off-target effects, a series of restricted rotation MGBG analogs was synthesized and evaluated. The most promising of these agents were the Ciba-Geigy compounds CBG 48664 (12) and CGP 39937 (13). Both compounds proved to be nanomolar inhibitors of mammalian AdoMet-DC, but CGP 48664 13 was the more effective antitumor agent. This compound produced growth inhibition in a panel of tumor cell lines including one multidrug-resistant line, and was 1000 times less potent against Chinese hamster ovary (CHO) cells in vitro. The synthesis of 12 is shown in Scheme 1.4. The starting material 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile 14 was treated with hydrogen sulfide in pyridine, followed by triethyloxonium tetrafluoroborate to afford the carbimidothioate 15. Compound 15 was converted to the corresponding amidine 16 in the presence of ammonium chloride, followed by the addition of N-aminoguanidine to form 12. The synthesis of CGP 39937 is detailed in Scheme 1.5. Compound 17 was stirred in methanol with a catalytic amount of sodium methoxide, yielding 18, which was then converted to 13 via treatment with ammonium chloride and ammonia in ethanol.

There are a multitude of known AdoMet-DC inhibitors, the most effective of which are analogs of S-adenosylmethionine. Initially, analogs of AdoMet containing a sulfonium center were studied. The a-difluoromethyl analog of AdoMet (analogous to DFMO 1) has been synthesized but has no activity against AdoMet-DC. S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA) has been shown to act as an irreversible inhibitor of AdoMet-DC in L1210 cells with an IC50 of 100 µm. Subsequently, a series of restricted rotation analogs of AdoMet were produced and evaluated as irreversible, enzyme-activated inhibitors of AdoMet-DC (19–23, Figure 1.4). The first of these analogs, AdoMac 19, was synthesized as shown in Scheme 1.6. The mixed ester/alcohol 26 was converted to the corresponding phthalimide 27 under Mitsunobu conditions (phthalimide, DEAD, triphenylphosphine), followed by removal of the phthalimide protecting group (hydrazine) to give amine 28. The free amine 28 was N-Boc protected to yield 29, the methyl ester was cleaved (LiOH) to form 30, and this alcohol was mesylated in the presence of lithium chloride to afford the requisite alkyl chloride 31. Compound 31 was then coupled to thioacetyladenosine 32 (NaOCH3), and the N-Boc and isopropylidene moieties in the protected adduct 33 were removed under acid conditions. Methylation in the presence of silver perchlorate then afforded AdoMac, 19, as a mixture of two diastereomers. Subsequently, the synthesis of the four pure diastereomers of AdoMac was undertaken, as shown in Scheme 1.7.36 The (meso)diacetate 35 was selectively de-esterified at the pro-(S) carbon to produce enantiomerically pure 1S, 4R-26. Compound 26 was elaborated as described in Scheme 1.6 to form 33. The isopropylidene and N-Boc protecting groups were removed, and the mixture of diastereomers was resolved by careful flash chromatography to afford the pure 1S, 4S- and 1S, 4R--diastereomers of 34, followed by methylation to produce the corresponding 1S, 4S- and 1S, 4R--diastereomers of 19. Compound 19 was shown to act as a mechanism-based, enzyme-activated inactivator of both the E. coli and human forms of S-adenosylmethionine decarboxylase, and optimal inhibitory activity was produced by different pure diastereomers in the case of each isozyme. The dihydro form of AdoMac, 20, and norAdoMac 21 (Figure 1.4), were also synthesized, and each lacked the driving force for generation of the latent electrophile produced by 19. Compounds 20 and 21 were both weak, reversible inhibitors of the enzyme. Similar synthetic routes were used to produce the homologs AdoMao 22 and AdoHyz 23 (Figure 1.4) both of which were mechanism-based inhibitors of the enzyme. The α-cyano derivatives of dc AdoMet 24 and 25 (Figure 1.4) were synthesized as shown in Scheme 1.8. The TBDMS-protected iodoalcohol 35 was coupled with the glycine equivalent 36 (LDA) under phase transfer conditions to afford 37. The TBDMS group was then removed, and the resultant alcohol was mesylated to form 38, which was coupled to 5'-thioacetyl-2',3'-isopropylideneadenosine 32. The coupled product was deprotected and methylated (CH3I, AgClO4) to afford 24 and 25. Compounds 24 and 25 also acted as mechanism-based inhibitors of AdoMet-DC, with Ki values of 9 and 50 µM, respectively, against the E. coli form of the enzyme. Interestingly, the specificity of the two compounds was reversed when evaluated against the human form of AdoMet-DC, with 24 and 25 exhibiting Ki values of 246.6 and 7.2 µM, respectively, against human AdoMet-DC.

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2011
• Language: English
• ISBN 10: 184973190X
• ISBN 13: 9781849731904
• Binding: Hardcover
• Number of pages: 302
• Editor: Patrick Woster