Polyamine Structure and Synthetic Analogs Patrick M Woster

1. Introduction

The polyamines putrescine (1,4-diaminobutane), spermidine (1,8-diamino-4-aza-octane, 2), and spermine (1,12-diamino-4,9-diazadodecane, 3) (Fig. 1) are ubiquitous polycationic compounds that are found in significant amounts in nearly every prokary-otic and eukaryotic cell type. Spermidine and spermine primarily exist in aqueous solution at pH 7.4 as fully protonated polycations and possess the pKa values indicated in Fig. 1 (1). This high degree of positive charge is an important factor in the biological functions of these molecules, and, as will be discussed later in this chapter, alterations in the pKa of polyamine nitrogens can affect and disrupt their cellular function. Polyamines are widely distributed in nature and are known to be required in micromolar to millimolar concentrations to support a wide variety of cellular functions. However, data that establish the precise role of the polyamines and their analogs in cellular processes are incomplete. The ongoing identification of new functions for the polyamines ensures that new avenues for research are arising continuously in an extremely diverse set of disciplines. The human and mammalian pathways for polyamine metabolism have been extensively studied, and analogous pathways have been elucidated for a relatively small number of organisms. There are important inter-species differences in polyamine metabolism, especially among eukaryotic cells, plants, and some bacteria and protozoa. In some prokaryotes, only putrescine and spermidine are synthesized, whereas in other cases, such as certain thermophilic bacteria, polyamines with chains longer than spermine are found. In some parasitic organisms, there are additional enzymes that are not present in the host cell, and, as such, provide a target for the design of specific antiparasitic agents. The enzymes involved in human and mammalian polyamine metabolism are reasonably similar, and inhibitors targeted to these enzymes rely on the observation that polyamine metabolism is accelerated, and polyamines are required in higher quantities, in target cell types. The diversity of biological research in the polyamine field is the subject of an excellent book (2). Keeping in mind the diverse nature of polyamine distribution and function, it is reasonable

From: Polyamine Cell Signaling: Physiology Pharmacology and Cancer Research Edited by: J.-Y. Wang and R. A. Casero, Jr. © Humana Press Inc., Totowa, NJ

Spermine Pka
Fig. 1. Structures of putrescine, spermidine, and spermine, and pKa values for spermidine and spermine.

to assume that carefully designed polyamine analogs could have the potential to disrupt polyamine metabolism, and thus such agents have been investigated as potential therapeutic agents in vitro and in vivo. The polyamine pathway represents an important target for chemotherapeutic intervention because depletion of polyamines results in the disruption of a variety of cellular functions and may, in specific cases, result in cytotoxicity (3,4). This chapter will summarize the development of synthetic derivatives of the polyamines, and describe their use as potential chemothera-peutic agents. A comprehensive review of polyamine biosynthesis inhibitors (4) and a review of the role of polyamines in normal and tumor cell metabolism (5) have recently been published.

2. Polyamine Biochemistry

The biosynthesis and catabolism of the polyamines putrescine, spermidine, and spermine are carefully controlled processes in all eukaryotic cell types. The mammalian polyamine biosynthetic pathway is shown in Fig. 2. Although definitive mechanisms for the various functions of the polyamines have not been fully elucidated, it is known that they are absolutely required for normal cell homeostasis. Inhibition of the polyamine pathway, therefore, is viewed as a valid target for the design of antitumor or antiparasitic agents. Available compounds that specifically inhibit individual enzymes in the pathway are extremely useful as research tools to elucidate the cellular functions of the naturally occurring polyamines. Specific inhibitors have now been developed for the enzymes in the forward polyamine biosynthetic pathway, ornithine decarboxylase (ODC), 5-adenosylmethionine decarboxylase (AdoMet-DC), and for the aminopropyl-

spermine

Fig. 2. The mammalian polyamine metabolic pathway.

spermine

Fig. 2. The mammalian polyamine metabolic pathway.

transferases spermidine synthase and spermine synthase. These inhibitors produce a variety of responses ranging from cessation of cell growth to overt cytotoxicity (6,7). The range of these activities appears to be both agent- and cell type-specific.

Polyamine metabolism can be viewed as having forward and reverse component pathways, although careful cellular control of these enzymes and the polyamine transporter act in concert to maintain appropriate levels of the individual polyamines. Ornithine is converted to putrescine by ODC, a typical pyridoxal phosphate-requiring amino acid decarboxylase. ODC is one of the control points in the pathway, producing a product that is committed to polyamine biosynthesis. ODC levels are modulated by

Fig. 3. The structure of a-difluoromethylornithine (DFMO).

cf2h cooh

Fig. 3. The structure of a-difluoromethylornithine (DFMO).

synthesis and degradation of ODC protein, with a half-life of about 10 min. In mammalian cells, the degradation of ODC is facilitated by a specific ODC-antizyme (8), a protein that also appears to downregulate polyamine transport. Competitive inhibitors of ODC have proven to be of limited value, and most useful inhibitor of ODC to date, a-difluoromethylornithine (DFMO;Fig. 3) (9), is an irreversible inactivator of the enzyme. The discovery of DFMO provided an enormous stimulus to the field of mammalian polyamine biology, and led to marketing of the drug as a treatment for Pneumocystis carinii secondary infections. DFMO is also quite effective for the treatment of late-stage West African trypanosomiasis.

Putrescine is next converted to spermidine by the aminopropyltransferase spermi-dine synthase. A second closely related but distinct aminopropyltransferase, spermine synthase, then adds an additional aminopropyl group to spermidine to yield spermine, the longest mammalian polyamine. The byproduct for the spermidine and spermine synthase reactions is 5'-methylthioadenosine, generated from the cosubstrate, decar-boxylated S-adenosylmethionine (dc-AdoMet). 5'-Methylthioadenosine is a potent product inhibitor for the aminopropyl transfer process, and must be rapidly hydrolyzed by 5'-methylthioadenosine-phosphorylase to maintain the forward pathway. Selective inhibition of the individual aminopropyltransferases has proven to be a significant problem because of the similarity of the reactions catalyzed by the two enzymes. The transition state analogs S-adenosyl-1,8-diamino-3-thiooctane (10) and 5-adenosyl-1,12-diamino-3-thio-9-azadodecane (11) remain the only known specific inhibitors of the individual aminopropyltransferases.

The aminopropyl donor for both aminopropyltransferases is dc-AdoMet, produced from AdoMet by the action of AdoMet-DC. AdoMet-DC, like ODC, is a highly regulated enzyme in mammalian cells and belongs to a small class of proteins known as pyruvoyl enzymes. All of the known forms of AdoMet-DC contain a covalently bound pyruvate prosthetic group that is required for activity (12), and formation of a Schiff's base between AdoMet and this pyruvate is a prerequisite for the reaction to occur. The antileukemic agent methylglyoxal Ms(guanylhydrazone) (Fig. 4) is a potent competitive inhibitor of the putrescine-activated mammalian enzyme, with a K. value of less than 1 p,M (6), but is of limited use as a chemotherapeutic agent because of excessive toxicity. The AdoMet analog 5'-{[(2)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (AbeAdo;Fig. 4), is a potent enzyme-activated inhibitor of AdoMet-DC from Escherichia coli, and produces a long-lasting, dose-dependent decrease in AdoMet-DC activity in vivo (13). Several additional inactivators of AdoMet-DC have been described (4).

The so-called reverse polyamine metabolic pathway provides further control of cellular polyamine levels through acetylation and subsequent oxidative deamination processes. In the cell nucleus, spermidine is acetylated on the four carbon end by sper-

Fig. 4. Structures of the AdoMet-DC inhibitors of MGBG and AbeAdo.

midine-A^-acetyltransferase, possibly altering the compound's binding affinity for DNA. A specific deacetylase can then reverse this enzymatic acetylation (14). Neither of these enzymes affects the level of histone acetylation. Cytoplasmic spermidine and spermine are acetylated on the three carbon end by spermidine/spermine-A1-acetyl-transferase (SSAT;Fig. 2) (4,15). This enzyme is the first and rate-limiting step in the catabolic interconversion of putrescine, spermidine, and spermine; its kinetics and substrate specificity have been described elsewhere (4). Acetylated spermidine or spermine can be exported from the cell or oxidized by acetylpolyamine oxidase (PAO) to form 3-acetamidopropionaldehyde and either putrescine or spermidine, respectively (Fig. 2). SSAT and PAO together serve to reverse polyamine biosynthesis, facilitating the interconversion of cellular polyamines. It is important to note that the combination of the highly regulated catabolic enzyme SSAT, coupled with the finely controlled synthetic enzymes ODC and AdoMetDC, allow the cell considerable control of intracellu-lar polyamine concentrations.

A final method of controlling intracellular polyamine levels is afforded by one or more specific polyamine transport mechanisms (16,17). To date, the polyamine transport system in E. coli has been the most completely studied, resulting in the isolation of a transporter gene and a series of protein gene products designated PotA-PotF (18). Specific polyamine transporters have been detected in yeast, Trypanosoma cruzi epi-mastigotes, Crithidia fasciculata, and Leishmania donovani. The process of polyamine transport in mammalian cells is poorly understood, and, to date, none of the proteins involved has been isolated and sequenced. Numerous groups are working to elucidate the mechanism(s) of transport, and the effects of regulation of transport, in normal and tumor cell lines. Several factors have been shown to alter the polyamine transport system, and, as a result, cellular homeostasis. It is worth noting that the polyamine system is significantly upregulated in a variety of tumor cells; thus, this system is regarded as a potential target for cancer chemotherapy (19). Efforts to synthesize specific polyamine transport inhibitors have recently begun in several laboratories; a complete discussion of these efforts is beyond the scope of this chapter and has been previously reviewed (17,19).

2.1. Symmetrical, Terminally Alkylated Polyamine Analogs

The development of analogs of spermidine and spermine as potential antitumor agents was initiated in the mid-1980s. Initially, these analogs were structurally similar tetraamine A (R = H) tetraamine B (R = methyl) tetraamine C (R = ethyl)

Fig. 5. The earliest known polyamine analogs with antitumor effects.

Fig. 5. The earliest known polyamine analogs with antitumor effects.

to the natural polyamines in that they had terminal primary amine groups, with variations in the length of the intermediate carbon chains. Edwards and coworkers synthesized a series of diamines and triamines related to spermidine and a series of tetraamines derived from 1,8-diaminooctane; these analogs were evaluated for antitumor activity in cultured L1210 cells (20). In the series of diamines and triamines, substitution of alkyl groups at the terminal nitrogens, or replacing the central nitrogens with other heteroatoms, failed to produce spermidine analogs with antitumor effects superior to norspermidine. However, compounds with eight carbons between the central nitrogens, such as tetraamine A (Fig. 5), generally showed significant antitumor activity. Tetraamine A increased survival time in male mice inoculated with L1210 leukemia from 7.7 to 16.2 d. Coadministration of spermidine was shown to reverse the antitumor activity of this compound, presumably because of a competition for the polyamine transport system, whereas coadministration of a polyamine oxidase inhibitor potentiated the observed antitumor activity, suggesting that these analogs may be metabolized by polyamine oxidase. Tetraamines B and C (R = CH3 or R = CH2C^, respectively) were also active in the L1210 model, but substitution of larger alkyl groups resulted in a reduction of activity. In a related study, a series of Msbenzyl analogs related to MDL 27695 and an additional series of substituted tetraamines were evaluated for the ability to inhibit proliferation of HeLa cells (21). The Ms(benzyl)polyamine analog MDL 27695 (Fig. 5) and tetraamine A were active antiproliferative compounds, exhibiting IC5o values of 5 and 50 |iM, respectively. Interestingly, no correlation between the DNA binding properties and antitumor activity of these analogs was detected.

Subsequent attempts to develop polyamine analogs as potential modulators of polyamine function focused on the synthesis of symmetrical, terminally substituted ,Ws(alkyl)polyamines. These analogs were designed in response to the finding that natural polyamines use several feedback mechanisms that autoregulate their synthesis (22) and that they can be taken into cells by the energy-dependent transport systems described previously. Several symmetrically substituted polyamine analogs have been synthesized that enter the cell using the polyamine transport system. These analogs specifically slow the synthesis of polyamines by downregulation of the biosynthetic

Fig. 6. Structures of Ws(ethyl)polyamine analogs BENSpm, BESpm, BEHSpm, BIPSpm, BE-4444, and 3,12-dihydroxy-BEHSpm.

enzymes ODC and AdoMet-DC, but cannot substitute for the natural polyamines in terms of their cell growth and survival functions (4,23). As will be discussed, some but not all alkylpolyamine analogs are potent inducers of SSAT in cultured tumor cells, an effect that leads to the induction of apoptosis. The most successful of the symmetrically substituted polyamine analogs to date are the A^-Ms^thy^polyamines shown in Fig. 6: 6is(ethyl)norspermine (BENSpm), ,Ws(ethyl)spermine (BESpm), Ms(ethyl)homo-spermine (BEHSpm), and 1,20-(ethylamino)-5,10,15-triazanonadecane (BE-4444). These compounds have been shown to possess a wide variety of therapeutic effects and illustrate that small structural changes in alkylpolyamine analogs can result in surprisingly significant changes in biological activity.

A major advantage of the Ms(ethyl)polyamines lies in the fact that their synthesis is extremely straightforward and depends only on the availability of the appropriate parent polyamine backbone. Functionalization of the terminal nitrogens can be readily accomplished by protecting all of the nitrogens in the parent chain with a tosyl or mesityl protecting group, producing an intermediate that possesses acidic hydrogens at the terminal nitrogen moieties, and unreactive central nitrogens. Alkylation of the terminal nitrogens is then accomplished by a sodium hydride-catalyzed reaction with ethyl bromide followed by deprotection of the nitrogens and recrystallization from ethanol/water (23-25). The A^A^-Ms^thy^polyamines are readily transported into mammalian cells by the same transport mechanism as the natural polyamines (26). Treatment of mammalian cells with these analogs leads to a reduction of putrescine, spermidine, and spermine, downregulation of ODC and AdoMetDC, and, depending on the cell lines used, cytostasis or cytotoxicity (23,26). These effects are accompanied by a tremendous induction of SSAT activity, in some cases as much as 1000-fold. Preliminary structure/activity correlations, based only on data from the symmetrically alkylated polyamine analogs, suggested that monoalkylation at both terminal nitrogens of spermidine or spermine was important for optimal antiproliferative activity, and that alkylation at an internal nitrogen reduced in vitro activity (23). It was further determined that the greatest induction of SSAT was dependent on the presence of "protected" aminopropyl or aminobutyl moieties (27-29). Adding terminal nitrogen Ms(alkyl) substituents larger than ethyl resulted in a dramatic reduction in antitumor activity (23,30,31). Compounds with a 3-3-3 carbon skeleton were more effective than the corresponding 3-4-3 analogs, and spermine-like compounds (3-3-3 or 3-4-3) are more effective that spermidine-like analogs (3-3 or 3-4). However, these data were collected using only symmetrically substituted Ms-alkylpolyamines, and as a result, only Ms(ethyl)-substituted analogs were advanced to clinical trials. Among these analogs, the most promising were A1,A11-6is(ethyl)norspermine (BENSpm;Fig. 6), which has a 3-3-3 backbone; A1,A14-6is(ethyl)norspermine (BEHSpm;Fig. 6), which has a 4-4-4 carbon skeleton; and 1,20-Ms(ethylamino)-5,10,15-triazanonadecane (BE-4444;Fig. 6), which has a 4-4-4-4 architecture. Interestingly, BEHSpm proved to be useful as an antidiarrheal agent (32) and was advanced to clinical trials for this indication.

BENSpm has shown exceptional promise as an antitumor agent in both in vitro and in vivo studies. Early studies indicated that BENSpm was an effective antitumor agent in cultured human pancreatic adenocarcinoma cells and xenografts, human MALME-3 melanoma xenografts, melanocytes, human bladder cancer cells, and ovarian carcinoma tumor cells (4). In CaCO2 colon cancer cells, the analog causes induction of SSAT, downregulation of ODC, and depletion of cellular polyamines, resulting in cyto-toxicity (33). In addition, SSAT induction appears to be the common event leading to cytotoxicity in non-small-cell lung (SCLC) tumor explants (34). Presumably because of the proprietary nature of data concerning BENSpm, no human clinical trials involving the compound have been published, although it is known anecdotally that these trials were initiated. The closely related analog BEHSpm (Fig. 6) does not show similar promise as an antitumor agent (35), but is being developed as an effective treatment for AIDS-related diarrhea (32,35,36). The potent antidiarrheal activity of BEHSpm has been demonstrated in several animal models, and in human clinical trials involving patients with AIDS-related diarrhea. A limited structure/activity study was conducted, and the closely related analog A1,A12-6is(isopropyl)spermine (BIPSpm;Fig. 6) proved to be the most active antidiarrheal in the series (32). The pharmacokinetics of BENSpm (37) and BEHSpm (38) for in vivo metabolism have been described. BENSpm is metabolized by A-de-ethylation and stepwise removal of aminopropyl equivalents by SSAT and PAO, with a half-life of 73 min. BEHSpm was metabolized almost exclusively to homospermine, which cannot serve as a substrate for SSAT and thus persists in tissues for a period of weeks (liver T1/2 = 15.4 d). Chronic administration of BEHSpm results in tissue accumulation of the analog and homospermine, resulting in disruption of normal polyamine metabolism. The metabolically programmed alkylpolyamine 3,12-dihydroxyBEHSpm (Fig. 6) retained the antidiarrheal activity of the parent BEHSpm, and exhibited a significantly diminished tissue half-life, presumably from the metabolic "handles" provided by the hydroxyl groups (39). The alkylpolyamine BE-4444 (Fig. 6) was originally designed based on the hypothesis that analogs with chain lengths different from spermine could exhibit enhanced binding to DNA and thus exert antiproliferative effects (40). BE-4444 has been shown to be effective in cultured U-251, MG, SF-126, and SF-188 brain tumor cells at a concentration of 5 |iM (40,41) and against DU-145, LNCaP, and PC-3 prostate cancer cells in vitro and in vivo (42).

Specific alterations to the polyamine backbone structure of BESpm, BEHSpm, and BE-4444 has resulted in a series of "second-generation" Ms(ethyl)polyamines with impressive antitumor and antiparasitic activity. Restriction of rotation in the central region of the polyamine chain in BESpm by including a cis- and trans-cyclopropyl or cyclobutyl ring, a cis- and trans-double bond, a triple bond, and a 1,2-disubstituted aromatic ring produced analogs (Fig. 7) with varying antitumor activity in a panel of human tumor cell lines (A549, HT-29, U251MG, DU145, PC-3, and MCF7) (43). There was little difference between the cis and trans isomers in the cyclopropyl, cyclobutyl, and double bond-containing analogs, and the triple bond and aromatic sub-stituents rendered the resulting analogs inactive. All of the analogs were imported by the polyamine transport system, suggesting that the lack of activity was because of diminished DNA binding affinity. In like fashion, insertion of a central dimethylsilane group resulted in a significant decrease in growth inhibition when compared with the 6is(ethyl)polyamine analogs (44). It was later found that the 6is(ethyl)spermine analog A1,A12-6is(ethyl)-cis-6,7-dehydrospermine (SL-11047;Fig. 8) was an effective treatment for Cryptosporidium parvum infections, producing cures in a murine model (45). By contrast, the 4-4-4 (homospermine) analog SL-11093 (Fig. 8), which contains a trans cyclopropyl moiety in the central region, was an effective antitumor agent in vitro, and in vivo against DU-145 nude mouse xenografts (46). Compounds with a 4-4-4 or 4-4-4-4-4 backbone that featured trans-cyclopropyl or a trans-cyclobutyl moieties in noncentral regions of the chain were more active in vitro against prostate tumor cell lines (LnCap, DU145, DUPRO, and PC-3), and inclusion of a cis unsaturation in one of the terminal aminobutyl groups also enhanced activity, presumably by enhancing DNA binding (47). The trans-ôis-cyclopropyl analogs 6is(cyclopropane)tetramine A and 6is(cyclopropyl)hexamine B (Fig. 8) were effective antitumor agents against Du-Pro and DU-145 prostate tumor cells in vitro (48). In general, structural modifications to homospermine-like backbones that are analogous to those made to the BESpm backbone (i.e., cis- and trans-cyclopropyl, cyclobutyl, and double-bond moieties) afforded

Acidos Palmitoleico
Fig. 7. Conformationally restricted analogs of BESpm.
Fig. 8. Conformationally restricted Ws(ethyl)polyamine analogs with enhanced antitumor activity against prostate tumor cells in culture.

analogs with enhanced antitumor activity and diminished systemic toxicity. In addition, insertion of a cis double bond into the terminal aminobutyl moieties of BE-4444

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