E-Mail: woster@wizard.pharm.wayne.edu
B.S. Pharmacy 1978, University of Nebraska Medical Center
Ph.D. Medicinal Chemistry 1986, University of Nebraska - Lincoln
Postdoctoral Associate, 1986, Department of Chemistry, Rensselaer Polytechnic Institute
Postdoctoral Associate, 1987, Department of Medicinal Chemistry, University of Michigan
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Studies Involving Polyamine MetabolismOur primary research effort has focused on the design, synthesis and evaluation of compounds which inhibit enzymes involved in the polyamine biosynthetic and interconversion pathways. These pathways are outlined in Figure 1.
It has long been known that polyamines are absolutely required for normal cell growth and division, and that the cellular requirements for polyamines are increased in rapidly dividing cell types. Thus, compounds which can modulate the intracellular levels of the individual polyamines have been sought as potential antitumor and/or antiparasitic agents. We have concentrated our efforts on two controlling enzymes in the polyamine system, S-adenosylmethionine decarboxylase (AdoMet-DC) and spermidine/spermine-N1-acetyltransferase (SSAT).
1. Inhibitors of S-Adenosylmethionine Decarboxylase (AdoMet-DC)AdoMet-DC belongs to a small class of proteins known as pyruvoyl enzymes, which contain a covalently linked pyruvate cofactor at the amino terminus of the active site subunit. AdoMet-DC is one of the controlling enzymes in polyamine biosynthesis, and catalyzes the formation of the committed intermediate decarboxylated AdoMet. The substrate, AdoMet, must form an imine linkage with the terminal pyruvate prior to catalysis. Using this mechanistic feature for inhibitor design, we have synthesized a number of AdoMet analogs (Figure 2) which, following formation of the requisite imine, are activated by general base catalysis to a latent electrophile which binds in the catalytic site, thus inactivating the enzyme.
The first of these analogues, AdoMac, was synthesized in each of its four pure diastereomeric forms, and the pure diastereomers were evaluated for inhibitory activity against the human and Escherichia coli forms of AdoMet-DC. Interestingly, in both cases the enzyme showed a distinct preference for one of the four diastereomers, and the human and bacterial forms of the enzyme preferred different diastereomers. Ki values for this inhibition ranged from 3 to 50 micromolar. The dihydro form of AdoMac, which does not have a driving force for formation of the latent electrophile, was a weak competitive inhibitor (Ki = 250 micromolar).
It has been demonstrated that parasitic organisms such as Trypanosoma brucei do not synthesize purines, but rather import them from the host via a recently discovered specific adenosine transporter. We have shown that our analogs are substrates for this transporter, which is not present in mammalian cells, and thus these analogs act as parasite-specific antiparasitic agents. The most active antiparisitic agent among these analogs, AdoMao, inhibits trypanosomal growth with an IC50 of 0.9 micromolar, and is currently undergoing evaluation in vivo.
One of the most active enzyme-activated inhibitors for AdoMet-DC, MDL 27811, is a nucleoside analogue in which the sulfonium moiety has been replaced by a nitrogen. In all likelyhood, such analogues have an enhanced ability to enter mammalian cells, compared to the corresponding sulfur isosteres, resulting in increased potency. We thus sought to design a synthetic route to the amino isosteres (Figure 3) of our previously described enzyme activated inactivators of AdoMet-DC.
A number of methods for incorporating the amino group into these analogues were tried, with little success. For our purposes, the palladium(0)-catalyzed introduction of phthalimide or benzylamine nucleophiles was more suitable as an alternate strategy to introduce the nitrogen equivalent. We determined that nitrogen nucleophiles can be readily used to generate protected allylic amine intermediates which can be later manipulated and/or readily deprotected. Treatment of the appropriate allylic acetate with two equivalents of phthalimide or benzyl amine in the presence of a catalytic amount of Pd2(dba)3 and bis-1,4-(diphenylphospino)butane (dppb) in THF (Scheme 1) afforded the corresponding allylic amine/amide in good to excellent yield.
In all cases, the reaction time was less than 1 h at 75û C. We have used this strategy to produce the AdoMet analogues shown in Figure 3, and these compounds are currently being evaluated as inactivators of AdoMet-DC. In addition, the analogue amino-AdoHept has been shown to inhibit the growth of Trypanosoma brucei with an IC50 of 5 micromolar.
Pegg, A.E., Wechter, R., Poulin, R., Woster, P.M. and Coward, J.K.: Effect of S-Adenosyl-1,12-diamino-3-thio-9-azadodecane, a Multisubstrate Adduct Inhibitor of Spermine Synthase, on Polyamine Metabolism in Mammalian Cells. Biochemistry 28, 8446-8453 (1989).
Douglas, K.A., Zormeier, M.M., Marcolina, L.M. and Woster, P.M.: Restricted Rotation Analogs of Decarboxylated S-Adenosylmethionine as Inhibitors of Polyamine Biosynthesis. Bioorg. Med. Chem. Lett. 1, 267-270 (1991).
Wu, Y.Q. and Woster, P.M.: S-(5'-Deoxy-5'-adenosyl)-1-amino-4-methylthio-2-cyclopentene (AdoMac): A Potent and Irreversible Inhibitor of S-Adenosylmethionine Decarboxylase. J. Med. Chem. 35, 3196-3201 (1992).
Guo, J.Q., Wu, Y.Q., Douglas, K.A., Farmer, W.L., Garofalo, J., Bacchi, C.J. and Woster, P.M.: Restricted Rotation Analog Inhibitors of S-Adenosylmethionine: Synthesis and Evaluation of their Selective Toxicity in Trypanosoma bruceii bruceii. Bioorg. Med. Chem. Lett. 3, 147-152 (1993).
Wu, Y.Q. and Woster, P.M.: Resolution of the Pure Diastereomeric Forms of S-(5'-Deoxy-5'-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene and their Evaluation as Irreversible Inhibitors of S-Adenosylmethionine Decarboxylase from Escherichia coli. Bioorg. Med. Chem. 1, 349-360 (1993).
Wu, Y.Q., Lawrence, T., Guo, J.Q. and Woster, P.M.: a-Cyano-Substituted Analogues of S-Adenosylmethionine as Enzyme Activated, Irreversible Inhibitors of S-Adenosylmethionine Decarboxylase. Bioorg. Med. Chem. Lett. 3, 2811-2816 (1993).
Wu, Y.Q. and Woster, P.M.: Irreversible Inhibition of Human S-Adenosylmethionine Decarboxylase by the Pure Diastereomeric Forms of S-(5'-Deoxy-5'-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene (AdoMac). Biochem. Pharmacol. 49, 1125-1133 (1995).
Guo, J.Q., Wu, Y.Q., Rattendi, D., Bacchi, C.J. and Woster, P.M.: S-(5'-Deoxy-5'-adenosyl)-1-aminoxy-4-methylsulfonio-2-cyclopentene (AdoMao): An Irreversible Inhibitor of S-Adenosylmethionine Decarboxylase With Potent In Vitro Antitrypanosomal Activity. J. Med. Chem. 38, 1770-1777 (1995).
Woster, P.M.: S-Adenosylmethionine Decarboxylase and Spermidine/Spermine-N1-Acetyltransferase - Emerging Targets for Rational Inhibitor Design, In "Polyamines: Regulation and Molecular Interaction", R.A. Casero, Jr., Ph.D., editor, R.G. Landes Co., Austin, TX, 1995, pp 171-204.
Sirisoma, N.S. and Woster, P.M.: Synthesis of Protected Allylic Amines via Palladium(0)-Catalyzed Amination of Allylic Acetates. Tet. Lett. , 39, 1489-1492 (1998).
Woster, P.M.: Sleeping Sickness: Irreversible Inhibitors of S-Adenosylmethionine Decarboxylase as Antitrypanosomal Agents. In "Biomedical Chemistry", Paul Torrence, ed., Wiley and Sons, New York, 1999, in press.
2. Modulators of Spermidine/Spermine-N1-Acetyltransferase (SSAT)Spermidine/spermine acetyltransferase (SSAT) is the rate limiting step in the polyamine back-conversion pathway (Figure 1), and thus allows cells to modulate the levels of the individual polyamines. It has been shown that bis-alkylated polyamine analogs such as bis(ethyl)spermine (BENSpm, Figure 4) can superinduce SSAT in a number of cultured tumor cell lines.
Specifically, BENSpm induces SSAT dramatically in the H157 large cell lung tumor line, resulting in a cytotoxic response. By contrast, BENSpm produces little induction in the H82 small cell line, and the cells respond in a cytostatic manner. In our laboratories, we have designed and synthesized a number of unsymmetrically substituted alkylpolyamine analogs, and have shown that these analogs act as potent antitumor agents in cell culture in a number of tumor cell lines. One of our first generation analogues, CPENSpm, is undergoing evaluation in pre-clinical studies. Most of the analogs we have produced show the same cell-type specific response seen with BESpm, and we thus hypothesized that the cytotoxicity of these analogs correlated with their ability to superinduce SSAT. However, we have recently synthesized an extremely potent analog, CHENSpm, which maintains the cell-type specific response of BENSpm, but which does not induce SSAT in either the H157 or H82 cell line. We have expanded our study of human solid tumors to include prostate cancer model systems, and examined the effect of CPENSpm, CHENSpm, and the parent symmetric analogue, BENSpm in three representative human prostate cancer cell lines. Two lines, PC-3 and DU145 are androgen receptor negative lines and LNCAP is an androgen receptor positive line. Dose response studies were performed within a concentration range from 0.1 to 30 micromolar for 120 hr. All three lines were significantly growth inhibited by the three analogues, with Du145 being the most sensitive of the three lines.
In studies aimed at finding alternate mechanisms of action for these analogues, we have shown that CPENSpm can induce programmed cell death in the H157 cell line, as well as in the MCF-7 cultured breast tumor line and 5 other breast tumor lines. We have demonstrated that H2O2 produced by the two step polyamine catabolic pathway (secondary to SSAT superinduction) may be responsible for initiating the cell death program. Reactive oxygen species have been indicated in several systems as initial insults leading to programmed cell death (PCD). Interestingly, CHENSpm does not superinduce SSAT, yet still induces PCD in approximately the same time frame as does CPENSpm. A second observation underscores the potential for differing mechanisms of action: although CPENSpm rapidly induces cell death, it does so without any obvious alteration of the cell cycle, suggesting the early toxic activity of CPENSpm is cell cycle-independent. By comparison, in the same treatment time frame, a profound G2/M block accompanies the CHENSpm-induced PCD. These data suggest that the two analogues kill H157 lung cancer cells by different mechanisms. We have recently demonstrated that these polyamine analogues induce PCD through both caspase-dependent and -independent pathways.
The finding that CHENSpm produced a profound G2/M block led us to investigate whether this and related analogues affected the cell cycle. In addition to CHENSpm, the analogue IPENSpm (Figure 4), was found to produce a significant G2/M block in the human non-SCLC line H157 after 24-hour exposure at 10 micromolar. Other agents such as paclitaxel, that are known to produce G2 blocks, interfere with or alter tubulin polymerization and microtubule formation. Therefore, we proceeded to compare the ability of CPENSpm, CHENSpm and IPENSpm to alter tubulin polymerization in vitro.
We recently reported that CHENSpm and IPENSpm can differentially effect tubulin polymerization in vitro, as determined by a standard turbidity assay (see above), and by immunohistochemical studies. It has long been known that polyamine analogues can act as cellular poisons when rapidly catabolized by the SSAT/PAO pathway. Previous investigators have suggested that the polyamines, particularly spermine, may play a role in protecting DNA from radiation or oxidative damage. However, most of the proposed mechanisms suggest that the protection is due to polyamine induced conformational changes of DNA. Therefore, we used a Cu(II)/H2O2 dependent ROS generating system to measure the ability of the natural polyamines and their analogues to protect phiX-174 plasmid DNA from damage, and demonstrated that spermine, as well as CPENSpm and related analogues, were capable of protecting phiX-174 plasmid DNA from ROS induced damage at physiological concentrations. Remarkably, this protection was observed with unsymmetrically substituted polyamine analogues, but not with symmetrically substituted compounds such as BENSpm.
To eliminate the possibility that the observed protection by spermine was solely due to DNA conformational changes, DNA was removed from the system. The effects of addition of the natural polyamine to the ROS generating system was then measured by electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. EPR analysis in the absence of spermine demonstrated the 1:2:2:1 quartet indicative of the formation of the DMPO-OH adduct. However, the addition of 1mM spermine effectively inhibited the formation of DMPO-OH formation by >70%, in adose dependent manner. These results suggest that spermine is capable of reducing hydroxyl radical attack without reliance on changes in DNA conformation, suggesting that spermine could react directly with the ROS. To test this directly, 13C-labeled spermine was synthesized and subjected to the same ROS generating system described above (Scheme 2).
The reaction products were then analyzed by NMR and chemical ionization mass spec-troscopy (CI/MS). The NMR results were consistent with the production of at least two adducts of the hydroxyl radical and spermine. The results of the CI/MS identified two products of hydroxy-radical attack of spermine. These data are the first direct evidence that the natural polyamines can act directly as free radical scavengers, and are certainly suggestive that one of the most critical roles of spermine is its function as a free radical scavenger capable of protecting DNA from ROS attack.
In addition to our studies with polyamine analogues having a 3-3-3 carbon backbone arrangement, we have synthesized a series of polyamine analogs with a 3-7-3 carbon backbone (Figure 5).
Interestingly, these analogues have very poor antitumor activity in vitro, but in many cases act as potent trypanocides, while the antiproliferative 3-3-3 analogues have no antiparasitic activity. The analogues shown in Figure 5 are particularly potent trypanocides, with IC50 values in the 200 nM range. These analogues are also being evaluated as antileishmanial agents, and as antimicrosporidial agents.
Woster, P.M.: Spermidine/Spermine-N1-Acetyltransferase - An Emerging Target for the Design of Antitumor Agents. Curr. Opin. Invest. Drugs 2, 1291-1299 (1993).
Casero, R.A., Mank, A.R., Saab, N.H., Wu, R.H. and Woster, P.M.: Growth and Biochemical Effects of Asymmetrically Substituted Polyamine Analogues in Human Lung Tumor Cells. Cancer Chemother. Pharmacol. 36, 69-74 (1995).
McCloskey, D.E., Casero, R.A., Woster, P.M. and Davidson, N.E.: Induction of Programmed Cell Death in Human Breast Cancer Cells by an Unsymmetrically Alkylated Polyamine Analog. Cancer Res. 55, 3233-3236 (1995).
Felschow, D.M., MacDiarmid, J., Bardos, T., Wu, R.H., Woster, P.M. and Porter, C.W.: Identification of a Plasma Membrane Polyamine Binding Protein by Photoaffinity Labeling. J. Biol. Chem. 270(48), 28780 (1995).
Woster, P.M.: S-Adenosylmethionine Decarboxylase and Spermidine/Spermine-N1-Acetyltransferase - Emerging Targets for Rational Inhibitor Design, In "Polyamines: Regulation and Molecular Interaction", R.A. Casero, Jr., Ph.D., editor, R.G. Landes Co., Austin, TX, 1995, pp 171-204.
McClosky, D.E., Yang, J., Woster, P.M., Davidson, N.E. and Casero, R.A.: Polyamine Analogue Induction of Programmed Cell Death in Human Lung Tumor Cells. Clinical Cancer Research, 2, 441-446 (1996).
Wu, R., Saab, N.H., Huang, H., Wiest, L, Pegg, A.E., Casero, Jr., R.A. and Woster, P.M.: Synthesis and Evaluation of Polyamine Phosphinates and Phosphonamidates as Transition State Analogue Inhibitors of Spermidine/Spermine-N1-Acetyltransferase. Bioorg. Med. Chem. 4, 825-836 (1996).
Bellevue, F.H., Boahbedason, M.L., Wu, R.H., Casero, Jr., R.A., Rattendi, D., Lane, S., Bacchi, C.J. and Woster, P.M.: Structural Comparison of Alkylpolyamine Analogues with Potent In Vitro Antitumor or Antiparasitic Activity. Bioorg. Med. Chem. Lett. 6, 2765-2770 (1996).
Ha, H.C., Woster, P.M., Yager, J.D. and Casero, R.A.: The Role of Polyamine Catabolism in Polyamine Analogue Induced Programmed Cell Death. Proc. Nat'l. Acad. Sci. U.S.A. 94, 11557-11562 (1997).
Ha, H.C., Yager, J.D., Woster, P.M. and Casero, R.A.: Structural Specificity of Polyamines and Polyamine Analogues in the Protection of DNA from Strand Breaks Induced by Reactive Oxygen Species. Bioch. Biophys. Res. Comm. , 244, 298-303 (1998).
Ha, H.C., Sirisoma, N.S., Kuppusamy, P., Zweier, J.L., Woster, P.M. and Casero, R.A.: The Natural Polyamine Spermine Functions Directly as a Free Radical Scavenger. Proc. Nat. Acad. Sci. U.S.A. , 95, 11140-11143 (1998).
Ha, H.C., Woster, P.M. and Casero, R.A.: Unsymmetrically Substituted Polyamine Analogue Induces Caspase-Independent Programmed Cell Death in Bcl-2 Overexpressing Cells. Cancer Research 58, 2711-2714 (1998).
Webb, H.K., Wu, Z.Q., Sirisoma, N., Ha, H.C., Casero, Jr., R.A., Woster, P.M.: 1-[N-(Alkyl)amino]-11-[N-(ethyl)amino]-4,8-diazaundecanes: Simple Synthetic Polyamine Analogues Which Differentially Alter Tubulin Polymerization. J. Med. Chem. 42, 1415-1421 (1999).
Davidson, N.E., Hahm, H.A., McCloskey, D.E., Woster, P.M., Casero, R.A.: Clinical aspects of cell death in breast cancer: the polyamine pathway as a new target for treatment. Endocrine-Related Cancer 6, 69-73 (1999).
Studies Involving Cytochrome P450 CYP3A4
We and others have shown that co-administration of grapefruit juice can reduce the observed pre-systemic metabolism of a number of drugs by inhibition of CYP3A4, the major cytochrome P450 drug metabolizing enzyme in the gut. A number of psoralens, coumarins, and flavonoids found in grapefruit have been hypothesized to produce this inhibition. We recently reported the isolation and identification of 6',7'-dihydroxybergamottin (6,7-DHB), a derivative of the previously described furanocoumarin bergamottin, from a methylene chloride extract of grapefruit juice. Although 6,7-DHB had previously been identified as a component of grapefruit (but not orange) juice, our data was the first to suggest that it acts as a potent inhibitor of CYP3A4. 6,7-DHB and its analogues could theoretically find therapeutic use as agents to increase the oral bioavailability of certain drugs, and/or to prevent the activation of carcinogens, by inhibiting CYP3A4-mediated gut metabolism in vivo. For this reason, we sought to develop an efficient synthesis of 6,7-DHB that could be readily adapted to the production of rationally designed analogues. A synthesis leading to bergamottin (1) has been reported, (10% overall yield for two steps), and a preparation of 6,7-DHB (2) from 1 has also appeared in the literature. However, the extremely low yield resulting from these syntheses is not satisfactory for the efficient production of either 1 or 2. The synthetic route used by our group to afford 6,7-DHB is outlined in Scheme 3 below.
The commercially available furanocoumarin bergapten (3) was demethylated using a previously published procedure, producing the corresponding phenolic derivative bergaptol, 4. Geranylation of 4 under phase transfer conditions (geranyl bromide, benzyl tributylammonium bromide, NaOH) then afforded bergamottin 1 in 98% yield. Subsequent treatment of 1 with m-chloroperbenzoic acid then resulted in selective epoxidation of the terminal double bond of the geranyl sidechain, affording bergamottin-6,7-epoxide 5 in 65% yield. Opening of the epoxide (3% perchloric acid in dioxane) then produced the desired 6,7-DHB 2 in 70% yield. We have recently used this synthetic route to produce the proposed irreversible inhibitor 6-vinylbergamottin, and this analogue is now being evaluated as an inhibitor of CYP 3A4.
Bellevue III, F.H., Edwards, D.J., He, K., Hollenberg, P.F. and Patrick M. Woster, P.M.: Synthesis and biological evaluation of 6',7'-dihydroxybergamottin (6,7-DHB), a naturally occurring inhibitor of cytochrome P450 3A4. Bioorg. Med. Chem. Lett. 7, 2593-2598 (1997).
Schmiedlin-Ren, P., Edwards, D.J., Fitzsimmons, M.E., He, K., Lown, K.S., Woster, P.M., Rahman, A., Collins, J.M., Thummel, K.E., Hollenberg, P.F. and Watkins, P.B.: Furanocoumarin grapefruit constituents: Mechanism-based inhibitors that decrease enterocyte CYP3A4 protein expression. Drug. Metab. Disp. 25, 1228-1233 (1997).
Edwards, D.J., Fitzimmons, M.E., Schuetz, E.G., Yasuda, K., Ducharme, M.P., Warbasse, L.H., Woster, P.M., Schuetz, J.D. and Watkins, P.: 6',7'-Dihydroxybergamottin in Grapefruit Juice and Seville Orange Juice: Effects on Cyclosporin Disposition, Enterocyte CYP3A4 and P-glycoprotein. Clin. Pharmacol. Ther. 65, 237-244 (1999).
Postdoctoral Associates:
Dr. Yu Zou
Ph.D. 1999, Department of Chemistry, Rutgers University
505 Shapero Hall
Phone: 313-577-1052
FAX: 313-577-2033
E-Mail: yzou@wiz2.pharm.wayne.edu
Graduate Students:
Xiaohong Ge
505 Shapero Hall
Phone: 313-577-1052
FAX: 313-577-2033
E-Mail: gexh@wiz2.pharm.wayne.edu
Tracey Ward
505 Shapero Hall
Phone: 313-577-1052
FAX: 313-577-2033
E-Mail: tward@wiz2.pharm.wayne.edu
Zhiqian Wu
507 Shapero Hall
Phone: 313-577-1052
FAX: 313-577-2033
E-Mail: zqw@wizard.pharm.wayne.edu
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