Synthesis and Evaluation of Microtubule Assembly Inhibition and Cytotoxicity of Prenylated Derivatives of cyclo-L-Trp-L-Pro Juan F. Sanz-Cervera, a Emily M. Stocking, b Takeo Usui, c Hiroyuki Osada c and Robert M. Williams b, * a Departamento de QuõÂmica Orga Ânica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain b Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA c Laboratory of Antibiotics, RIKEN, 2-1 Wako-shi, Saitama 351-0198, Japan Received 10 January 2000; accepted 16 May 2000 AbstractÐThe synthesis of three isoprenylated derivatives of cyclo-l-Trp-l-Pro is described. These substances have been evaluated for cytotoxic activity in rat normal ®broblast 3Y1 cells and have also been evaluated in vitro for the inhibition of microtubule assembly. # 2000 Elsevier Science Ltd. All rights reserved. Introduction The tryprostatins, 1 cyclotryprostatins 2 and spirotrypro- statins 3 (Fig. 1) are a class of prenylated fungal metabo- lites produced from Aspergillus fumigatus. Osada et al. have demonstrated that these substances are cell cycle inhibitors interfering with the G2/M phase progression in G2/M synchronous cultures of tsFT210 cells. 1 5 The primary target of tryprostatin A and cyclotryprostatins A and B are microtubules which induce M-phase spe- ci®c inhibition and microtubule disassembly. Cell cycle inhibitors are considered to be promising candidates as anticancer drugs and have also received a considerable amount of attention recently as probes of the cell cycle. 4 The tryprostatin family of secondary metabolites are the consequence of several modes of isoprenylation of the tryptophan moiety of the simple cyclic dipeptide pro- genitor cyclo-l-Trp-l-Pro. The structurally most inter- esting and complex members of this family are the spirotryprostatins which, curiously, display among the weakest biological activity of this family of cell cycle inhibitors. 5,6 On the other hand, the very simple sub- stance tryprostatin A has been shown to inhibit tau or MAP2-dependent microtubule assembly. The presence of the methoxy group on the aromatic ring reduces the cytotoxicity while enhancing the speci®city against microtubule disruptive activities. For example, the IC 50 of tryprostatin A was determined by MTT assay to be 400 mM whereas tryprostatin B exhibited an IC 50 value of 4 mM. The interesting pro®le of cell cycle inhi- bitory activity displayed by the tryprostatin family has prompted us to investigate the synthesis of some very simple and readily prepared analogues of tryprostatin B. Results and Discussion Synthesis of tryprostatin B analogues The disposition of the dimethylallyl moiety at the 2- position of tryprostatins A and B that is essential for bio- logical activity, suggested that the display of the isoprene group at either the indole nitrogen or the tryptophyl amide nitrogen, might closely mimic the display of this side-chain in the natural products. We therefore designed and synthesized compounds 5, 7 and 10, which can be viewed as analogues of tryprostatin B and eval- uated their biological activities. The simple tryprostatin B analogues targeted in this study (compounds 5, 7 and 10) were prepared as shown in Schemes 1 and 2. The simple indole N-prenylated compound 5 has been previously reported by Sammes et al. by a ®ve-step procedure starting with N-CBz-l-tryptophan. 7 We have devised an alternate, four-step route from N-Boc-l- tryptophan (1) as shown in Scheme 1. Dimethylallyl- ation of 1 with prenyl bromide in the presence of sodium hydride in DMF yielded the desired N-prenylated sub- stance 2 in 68% yield. Peptide coupling of l-proline methyl ester to 2 gave the dipeptide 3 in 69% yield. 0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(00)00171-1 Bioorganic & Medicinal Chemistry 8 (2000) 2407±2415 *Corresponding author. Tel.: +1-970-491-6747; fax: +1-970-491- 3944; e-mail: rmw@chem.colostate.edu