Design and Synthesis of Novel Inhibitors of Gelatinase B Xueqing Wang, a Youngchool Choe, b Charles S. Craik b and Jonathan A. Ellman a, * a Center for New Directions in Organic Synthesis, Department of Chemistry, University of California, Berkeley, CA 94720, USA b Pharmaceutical Chemistry, Program in Chemistry and Chemical Biology, University of California, San Francisco, CA 94143, USA Received 19 February 2002; accepted 23 April 2002 Abstract—A new method was developed to identify nonpeptidic metalloproteinase inhibitors with novel zinc binding groups. Application of this method to matrix metalloproteinase-9 resulted in the identification of aminomethyl benzimidazole analogue 7a with an IC 50 =13 mM. # 2002 Elsevier Science Ltd. All rights reserved. Matrix metalloproteinases (MMPs) are a family of zinc- containing endopeptidases that are involved in the degradation and remodeling of connective tissues. The activities of MMPs under normal physiological conditions are modulated by endogenous inhibitors, such as the TIMPs (tissue inhibitor of metalloproteinases). 1 Disruption of this balance leads to overactivation of the MMPs, which causes excessive tissue degradation. 2 This has, in turn, been associated with a variety of disease states including arthritis, 3 tumor metastasis, 4 and periodontal disease. 5 Due to the clear therapeutic potential for inhi- biting MMPs, the development of MMP inhibitors has been under intensive investigation. 6 Virtually all MMP inhibitors have been designed upon a key zinc binding pharmacophore to which is appended additional, typically peptidomimetic functionality to provide enhanced affinity and selectivity. Hydroxamic acid is by far the most commonly used zinc binding pharmacophore and has provided the highest affinity MMP inhibitors. However, inhibitors incorporating the hydroxamic acid pharmacophore are often rapidly metabolized and show poor selectivity profiles across the MMPs. 7 Herein, we report a new method to identify completely nonpeptidic MMP inhibitors that incorpo- rate novel zinc binding pharmacophores. In the first step of the method a collection of low molecular weight compounds ( < 150 daltons) containing potential zinc-binding functional groups are screened against a metalloprotease of interest. Assays are performed at high concentrations to identify even weakly inhibitory pharmacophores. In the second step analogues of the identified pharmacophores are assayed to provide SARs. In the final step, focused libraries based upon the preliminarys SARs are synthesized to identify more active compounds. We have chosen gelatinase B (MMP-9) as a prototypical MMP to evaluate this method since it is one of the most highly expressed MMPs in and around a wide variety of tumors and has been implicated in tumor aggressiveness and increased metastatic potential. 8 A collection of potential zinc binders was assayed at 5 mM against murine gelatinase B. 9 The inhibitory activities of a representative set of these compounds are listed in Table 1. Acetohydroxamic acid, which was included in this study as a bench mark, is a very weak inhibitor ( < 15% inhibition at 20 mM). This weak inhibitory activity is consistent with the 17 mM IC 50 value of this compound against stromelysin (MMP-3) as reported by Fesik. 10 Among the compounds tested, aminomethyl benzimidazole showed particularly promising activity. MMP inhibitors have not previously been reported that incorporate an imidazole as the zinc- binding group. This is surprising considering that in MMPs three conserved active site histidine residues coordinate to zinc. The aminomethyl benzimidazole is also advantageous for further optimization because diversity can be introduced at multiple positions on this scaffold using efficient synthetic chemistry. Evaluation of a series of aminomethyl benzimidazole analogues indicated that substitution on the aromatic ring (Fig. 1) results in compounds with improved activity (data not shown). These results strongly suggested that amino- methyl benzimidazole binds to the enzyme active site rather than inhibiting simply by sequestering zinc, since substitution on the aromatic ring is distant from the metal-binding functionality. Furthermore, these results indicated that the preparation of focused libraries of 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(02)00365-7 Bioorganic & Medicinal Chemistry Letters 12 (2002) 2201–2204 *Corresponding author. Tel.: +1-510-642-9818; fax: +1-510-642- 8369; e-mail: jellman@uclink.berkeley.edu