Inhibitors of Plasmin that Extend into Both the S and S′ Binding Sites: Cooperative Interactions between S1 and S2 Paul Abato, Courtney M. Yuen, Jeanne Y. Cubanski, and Christopher T. Seto* Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island 02912 Christopher_Seto@brown.edu Received August 24, 2001 A new procedure for the synthesis of cyclohexanone-based inhibitors of serine proteases is reported. In this procedure the reactive ketone functionality is carried through the synthesis in masked form as a TBDMS-protected alcohol. Deprotection followed by oxidation of the alcohol generates the final form of the inhibitor. Two new inhibitors, which interact with the S1-S3 and S2′ subsites of plasmin, are synthesized using this procedure. Inhibitors 1 and 2 have IC 50 values against plasmin of 20 and 24 μM, respectively. The inhibition studies show that cooperative binding of inhibitors in the S1 and S2 subsites of plasmin is important for determining inhibitor selectivity. Introduction Plasmin is a serine protease that is central to a number of normal physiological processes such as lysis of fibrin clots, tissue remodeling, and cell migration. 1 There is now mounting evidence that also implicates plasmin in the processes of angiogenesis and metastasis during the progression of cancer. 2 Plasmin acts by hydrolyzing components of the basement membrane including fibrin, type IV collagen, fibronectin, and laminin. It also has an indirect mode of action through hydrolytic activation of matrix metalloproteases. 2 During angiogenesis, degrada- tion of the basement membrane allows epithelial cells to migrate into the extracellular matrix and form new blood vessels. Lesions in the basement membrane also promote metastasis by allowing cancer cells to penetrate into the underlying tissues and form secondary tumors. 3 Thus, plasmin is a potential target for the development of new chemotherapeutic agents that could act by inhibiting degradation of the basement membrane, and as a result, inhibiting angiogenesis and metastasis. 2 Most of the current pharmaceutical agents that inhibit plasmin, such as -aminocaproic acid and trans-4-amino- methylcyclohexanecarboxylic acid, are targeted to the lysine binding site. 4 This binding site anchors plasmino- gen, which is the inactive precursor to plasmin, to fibrin. 5 Therefore, the compounds are good inhibitors of fibrin- olysis. However, they do not affect the active site of the protease, which is separate from the lysine binding site. Inhibitors that are targeted directly to the active site of plasmin may be useful as potential anticancer agents. In addition, they may lead to a better understanding of the diverse roles that are played by this enzyme in both normal and pathological processes. 6 In this paper we report on two new inhibitors (compounds 1 and 2) that are designed to make a variety of specific contacts with the active site of plasmin. The aminohexyl, R 1 , and Cbz groups are designed to bind in the S1-S3 subsites, respectively, while the C-terminal Trp residue will bind in the S2′ subsite. These multiple noncovalent interac- tions position the ketone moiety of the inhibitors so that it can react with the active site serine nucleophile. The inhibitors show reasonable activity and moderate speci- ficity for plasmin when compared to other related serine proteases including trypsin, thrombin, and kallikrein. Design of Inhibitors. Over the past several years we have been investigating 4-heterocyclohexanone deriva- tives as inhibitors for serine and cysteine proteases. 7 These compounds are reversible inhibitors of the pro- teases and are designed to give a reversibly formed covalent bond between the enzyme active site nucleophile and the electrophilic ketone functionality of the inhibitor. 8 In previous studies we have synthesized inhibitors 3-5 (Table 1) that interact with the S1-S3 subsites of plasmin. 9 The aminohexyl side chains in compounds 3 and 4 are designed to form an electrostatic interaction (1) (a) Dano, K.; Andreasen, P. A.; Grondahl-Hansen, J.; Kristensen, P. L.; Nielsen, L. S.; Skriver, L. Adv. Cancer Res. 1985, 44, 139. (b) Tryggvason, K.; Hoyhtya, M.; Salo, T. Biochim. Biophys. Acta 1987, 907, 191. (2) (a) Pepper, J. S.; Montesano, R.; Mandriots, S. J.; Orci, L.; Vassalli, J. Enzyme Protein 1996, 49, 138. (b) Kobayashi, H.; Shino- hara, H.; Takeuchi, K.; Itoh, M.; Fujie, M.; Saitoh, M.; Terao, T. Cancer Res. 1994, 54, 844. (3) Liotta, L. A. Sci. Am. 1992, Feb., 54. (4) Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Bohgaki, M.; Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Chem. Pharm. Bull. 1988, 36, 1289. (5) Sherry, S. Fibrinolysis, Thrombosis, and Hemostasis; Lea & Febiger: Philadelphia, 1992; chapter 1. (6) For other active site directed inhibitors of plasmin, see: (a) Teno, N.; Wanaka, K.; Okada, Y.; Tsuda, Y.; Okamoto, U.; Hijikata- Okunomiya, A.; Naito, T.; Okamoto, S. Chem. Pharm. Bull. 1991, 39, 2340. (b) Teno, N.; Wanaka, K.; Okada, Y.; Taguchi, H.; Okamoto, U.; Hijikata-Okunomiya, A.; Okamoto, S. Chem. Pharm. Bull. 1993, 41, 1079. (c) Wanaka, K.; Okamoto, S.; Horie, N.; Hijikata-Okunomiya, A.; Okamoto, U.; Naito, T.; Ohno, N.; Bohgaki, M.; Tsuda, Y.; Okada, Y. Thrombosis Res. 1996, 82, 79. (d) Tamura, S. Y.; Goldman, E. A.; Brunck, T. K.; Ripka, W. C.; Semple, J. E. Bioorg. Med. Chem. Lett. 1997, 7, 331. See also ref 4. (7) Conroy, J. L.; Sanders, T. C.; Seto, C. T. J. Am. Chem. Soc. 1997, 119, 4285. (8) Conroy, J. L.; Seto, C. T. J. Org. Chem. 1998, 63, 2367. (9) Sanders, C. T.; Seto, C. T. J. Med. Chem. 1999, 42, 2969. 1184 J. Org. Chem. 2002, 67, 1184-1191 10.1021/jo0160569 CCC: $22.00 © 2002 American Chemical Society Published on Web 01/22/2002