J. Org. Chem. 2000, 65, 4509-4514
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A Novel Class of Zinc-Binding Inhibitors for the Phosphatidylcholine-Preferring Phospholipase C from Bacillus cereus Stephen F. Martin,* Bruce C. Follows, Paul J. Hergenrother, and Christopher L. Franklin Department of Chemistry and Biochemistry, The University of Texas, Austin, Texas 78712 Received October 8, 1999
The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic N,N′-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLCBc). This class of inhibitors is believed to function by the bidentate chelation of the N,N′dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the affinities of these compounds correlate with the pKas of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions. It is also apparent that there may be rather strict steric requirements for these inhibitors. Introduction Phospholipase C isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup (Scheme 1). The products from the action of mammalian PLCs have been implicated in the cellular signal transduction cascade.1 In particular, DAG and inositol triphosphate are important for their respective activation of protein kinase C and release of intracellular calcium.2 The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a bacterial enzyme with a known antigenic similarity to its mammalian counterpart.3 This similarity has made studies of the PLCBc mechanism of action4-6 and its selective inhibition7 important biological pursuits.
which either the bridging or the nonbridging oxygens have been replaced.7 For example, modest inhibitors of PLCBc activity have been identified wherein the phosphate oxygen of the scissile P-O bond was replaced with CH2, CF2, or NR groups to provide nonhydrolyzable phosphonate or phosphoramidate analogues as generally shown in 1. Replacement of the nonbridging phosphate oxygens with sulfur also leads to inhibitors. Although a number of several monovalent anions and Tris have been found to be weak inhibitors of PLCBc,8 the only potent inhibitor of PLCBc that is not a substrate analogue is the xanthate derivative D609 (2).9,10
Scheme 1
In the context of developing inhibitors of PLCBc, it is noteworthy that most have been substrate analogues in (1) Exton, J. H. Eur. J. Biochem. 1997, 243, 10-20. (2) Bell, R. Cell 1986, 45, 631-632. (3) Clark, M. A.; Shorr, R. G. L.; Bomalaski, J. S. Biochem. Biophys. Res. Comm. 1986, 140, 114-119. (4) Martin, S. F.; Spaller, M. R.; Hergenrother, P. J. Biochemistry 1996, 35, 12970-12977. (5) Martin, S. F.; Hergenrother, P. J. Biochemistry 1998, 37, 57555760. (6) Martin, S. F.; Hergenrother, P. J. Biochemistry 1999, 38, 44034408. (7) Martin, S. F.; Wong, Y.-L.; Wagman, A. S. J. Org. Chem. 1994, 59, 4821-4831.
To design novel, nonsubstrate analogues as inhibitors of PLCBc, it is helpful to examine the X-ray crystal structures of PLCBc in its native state11 as well as in its complexes with various ions12,13 and a phosphonate inhibitor.14 For example, the structure of the PLCBcinhibitor complex reveals PLCBc to be a monomeric, highly R-helical enzyme that contains three zinc ions in its active site (Figure 1). The nonbridging phosphate (8) Aakre, S. E.; Little, C. Biochem. J. 1982, 203, 799-801. (9) Muller-Decker, K. Biochem. Biophys. Res. Commun. 1989, 162, 198-205. (10) Amtmann, E. Drugs Exp. Clin. Res. 1996, 22, 287-294. (11) Hough, E.; Hansen, L. K.; Birkness, B.; Jynge, K.; Hansen, S.; Hordvik, A.; Little, C.; Dodson, E.; Derewenda, Z. Nature 1989, 338, 357-360. (12) Hansen, S.; Hansen, L. K.; Hough, E. J. Mol. Biol. 1992, 225, 543-549. (13) Hansen, S.; Hansen, L. K.; Hough, E. J. Mol. Biol. 1993, 231, 870-876. (14) Hansen, S.; Hough, E.; Svensson, L. A.; Wong, Y.-L.; Martin, S. F. J. Mol. Biol. 1993, 234, 179-187.
10.1021/jo9915731 CCC: $19.00 © 2000 American Chemical Society Published on Web 05/24/2000
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J. Org. Chem., Vol. 65, No. 15, 2000
Martin et al.
having the general structure of 4 might be interesting candidates as possible inhibitors of PLCBc. These compounds contain an N,N′-dihydroxyurea functional group in place of the polyhydroxylated framework of the tropolone ring. While the dihydroxyurea moiety of 4 is technically not a hydroxamic acid, it seemed likely that it would retain the known zinc-chelating ability of hydroxamic acids. In a fashion similar to that proposed for 3, the carbonyl group and the two N-hydroxyl groups on 4 would serve as Lewis basic sites for coordination to two of the zinc ions in the active site of PLCBc; this possibility was supported by preliminary docking studies using SYBYL. The synthesis and evaluation of a series of compounds for the general structure 4 as inhibitors of PLCBc is detailed herein. Figure 1. Interaction of phosphonate inhibitor with three zinc ions in the active site of PLCBc (adapted from ref 14).
oxygens of the substrate analogue are tightly coordinated with the three metal ions, suggesting a catalytic role for all three zincs. Moreover, Zn1 and Zn3 of PLCBc appear to comprise a ‘bimetallic center’ because the internuclear distance is only 3.5 Å, and they are bridged by a carboxylate residue.15 It occurred to us that a promising strategy for the discovery of inhibitors would be to identify bidentate ligands that would bind to this bimetallic site. For example, alkaline phosphatase, dopamine β-monooxygenase, and inositol monophosphatase, each of which contain similar bimetallic sites, are strongly inhibited by polyhydroxy tropolones containing three contiguous oxygen groups on the seven membered ring.16,17 In these cases, the polyhydroxy tropolone presumably chelates to both metal ions at the active site.16 When we tested the most active inhibitor, 2,7-dihydroxytropolone (3) against PLCBc, we found that it exhibited an inhibition constant (Ki) of 16 µM at pH 7.3, a potency comparable to the best of the previously described substrate analogues.7 Although this was an interesting discovery, we recognized the problems associated with optimizing enzyme affinity by derivatizing the tropolone ring, which is known to have limited stability under certain conditions.17-20 A stable zinc binding entity that could be easily derivatized into a variety of structures for optimal inhibition was needed.
Because hydroxamic acids are well-known zinc chelators,21 we reasoned that heterocyclic analogues of 3 (15) Wilcox, D. Chem. Rev. 1996, 96, 2435-2458. (16) Piettre, S. R.; Ganzhorn, A.; Hoflack, J.; Islam, K.; Hornsperger, J.-M. J. Am. Chem. Soc. 1997, 119, 3201-3204. (17) Piettre, S. R.; Andre, C.; Chanel, M.-C.; Ducep, J.-B.; Lesur, B.; Piriou, F.; Raboisson, P.; Rondeau, J.-M.; Schelcher, C.; Zimmermann, P.; Ganzhorn, A. J. Med. Chem. 1997, 40, 4208-4221. (18) Takeshita, H.; Mori, A. Synthesis 1986, 579-580. (19) Takeshita, H.; Mori, A.; Kusaba, T.; Watanabe, H. Bull. Chem. Soc. Jpn. 1987, 60, 4325-4333. (20) Davies, H.; Clark, T. J. Tetrahedron 1994, 50, 9883-9892.
Results and Discussion Design and Synthesis of Cyclic N,N′-Dihydroxyureas. In deciding what analogues of 4 should be evaluated as possible inhibitors of PLCBc, we wanted especially to probe two factors that might affect binding: (1) What role do the pKas of the two hydroxyl groups play in determining inhibitor potency? and (2) Are there possible steric interactions that might interfere with binding? Toward addressing these questions, we focused upon the series of N,N′-dihydroxyurea derivatives 5-10. Upon the basis of electronic effects, we predicted that the pKas of 5 and 9 would be comparable to 3, but 10 and 6-8 should be less acidic. The N,N′-dihydroxybarbituric acid 5 held the greatest potential for incurring unfavorable steric interactions on binding, whereas 9, 10, and 6-8 presenting correspondingly less likelihood for such interactions at the active site of PLCBc.
The barbituric acid 5 was known,22 but it was necessary to devise tactics for synthesizing 6-10 (Schemes 2-4). Compounds 6 and 7 were prepared in a straightforward fashion by a sequence that commenced with the alkylation of O-benzylhydroxylurea with 1,2-dibromoethane or 1,3-dibromopropane in the presence of 2 equiv of KH in tetrahydrofuran (THF) at 0 °C to produce the cyclized products 12 and 13 (Scheme 2).23 Initial attempts to remove the O-benzyl groups by hydrogenolysis using Pearlman’s catalyst, Pd/C, and PtO2 were unselective, and cleavage of the N-O bond was a significant side (21) Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1420-1436. (22) Cowden, W.; Jacobsen, N. W. Aust. J. Chem. 1982, 35, 795797. (23) Sulsky, R.; Demers, J. P. Synth. Commun. 1989, 19, 18711874.
Zinc-Binding Inhibitors for Phospholipase C Scheme 2
J. Org. Chem., Vol. 65, No. 15, 2000 4511 Scheme 4
Scheme 3
reaction. However, the O-benzyl groups were cleanly removed using Pd/BaSO4 as the catalyst in MeOH (40 psi H2) to afford the desired N,N′-dihydroxyureas 6 and 7 in good yields. In initial experiments, we found that the reaction of 11 with 1,4-dibromobutane (15) in the presence of KH gave only low yields (10-fold relative to a typical assay) was added to all the kinetic assays. Control experiments indicated that under these conditions of high APase concentration phosphorylcholine was rapidly and quantitatively converted to Pi and choline, even in the presence of 3.
Acknowledgment. We thank the National Institutes of Health and the Robert A. Welch Foundation for their generous support of this research. We also thank Ms. Nina M. Antikainen and Hilary R. Plake for assistance in preparing the cover graphic art. Supporting Information Available: Copies of 1H NMR spectra of all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org. JO9915731
