Synthesis and Biological Activity of Analogues of the Antimicrotubule Agent N,,-Trimethyl-L-phenylalanyl-N 1 -[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]- N 1 ,3-dimethyl-L-valinamide (HTI-286) Arie Zask,* Gary Birnberg, Katherine Cheung, Joshua Kaplan, Chuan Niu, Emily Norton, Ronald Suayan, Ayako Yamashita, Derek Cole, Zhilian Tang, Girija Krishnamurthy, Robert Williamson, Gulnaz Khafizova, Sylvia Musto, Richard Hernandez, Tami Annable, Xiaoran Yang, Carolyn Discafani, Carl Beyer, Lee M. Greenberger, Frank Loganzo, and Semiramis Ayral-Kaloustian Chemical and Screening Sciences, and Oncology Research,Wyeth Research, 401 North Middletown Road, Pearl River, New York 10965 Received March 2, 2004 Hemiasterlin (1), a tripeptide isolated from marine sponges, induces microtubule depolymer- ization and mitotic arrest in cells. HTI-286 (2), an analogue from an initial study of the hemiasterlins, is presently in clinical trials. In addition to its potent antitumor effects, 2 has the advantage of circumventing the P-glycoprotein-mediated resistance that hampers the efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models. This paper describes an in-depth study of the structure-activity relationships of analogues of 2, their effects on microtubule polymerization, and their in vitro and in vivo anticancer activity. Regions of the molecule necessary for potent activity are identified. Groups tolerant of modification, leading to novel analogues, are reported. Potent analogues identified through in vivo studies in tumor xenograft models include one superior analogue, HTI-042 (48). Modulation of the dynamics of microtubule formation represents a major therapeutic approach for the treat- ment of cancer. 1 The taxanes and Vinca alkaloids are the two classes of tubulin inhibiting natural products currently in use as anticancer agents. Despite their successes, inherent and acquired resistance by tumors to these agents limits their utility. 2 Hemiasterlin 3 (1) is a member of a recently discovered class of natural products whose potent effects on tubulin make them especially attractive as drug targets (Figure 1). These tripeptides contain highly unusual and sterically con- gested amino acids, which give rise to their stability and in vivo activity. Other peptidic antimicrotubule agents (e.g. dolastatins, cemadotin and cryptophycin), some of which competitively inhibit 4 binding of hemiasterlin to tubulin, have been reported and several are under clinical investigation. 1,5 Clinical trials of compounds interacting with the colchicine domain (e.g. combret- astatins) and the taxane site (e.g. epothilones) are also underway. 1 The relative structural simplicity of the hemiasterlins allows for diverse structural manipulation of the molecule via total synthesis. HTI-286 6 (2), an analogue from an initial study of the hemiasterlins wherein a phenyl group replaces the indole ring, 7 is presently in clinical trials 8 (Figure 1). In addition to its potent antitumor effects, 2 has the advantage of cir- cumventing the P-glycoprotein-mediated resistance which hampers the efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models. 6 This paper describes an in-depth study of the struc- ture-activity relationships of analogues of 2 in which each region of the molecule was systematically inves- tigated. Each analogue was evaluated in terms of its direct effects on extracellular tubulin polymerization, and its cytotoxic effects both in the absence and presence of expression of P-glycoprotein transporters. In vivo studies in tumor xenograft models identified several potent analogues including one superior analogue. Synthesis Several total syntheses of hemiasterlin have been reported in the literature. 9 The synthetic strategy comprises synthesizing the individual amino acids, followed by peptide coupling. This route can also be used for the synthesis of 2 and other analogues (Scheme 1). 7 The most challenging synthesis is that of the A-piece amino acid 3, necessitating construction of the carbon scaffold and establishing the chirality by means of a chiral auxiliary group. 9a Alternative A-piece syntheses utilizing SnCl 4 -mediated ring opening of epoxides by indoles 9b or asymmetric Strecker synthesis 9c have ap- peared in the literature. The chirally pure B-piece (4) was commercially available and the CD-piece (5) was * Corresponding author. Tel: 845-602-2836; Fax: 845-602-5561; e-mail: zaska@wyeth.com. Figure 1. 4774 J. Med. Chem. 2004, 47, 4774-4786 10.1021/jm040056u CCC: $27.50 © 2004 American Chemical Society Published on Web 08/03/2004