L-696474

The microbiological transformation of L-696,474 [1], a novel cytochalasin that is an inhibitor of HIV-1 protease, was investigated using Actinoplanes sp. ATCC 53771. Six hydroxylated metabolites 2-7 of 1 were isolated and purified using reversed-phase hplc. All six metabolites were found to have undergone hydroxylation at the C-16 methyl group (C-22) of 1. Three of the compounds, 3, 4, and 5, were further hydroxylated at the para (C-29), the meta (C-28), and both the para and the meta, positions of the phenyl ring, respectively. Metabolites 6 and 7 were shown to result from vicinal dihydroxylation on both C-16 and its attached Me (C-22). The metabolite 7 was further hydroxylated on the meta position of the phenyl ring. The structures of the metabolites were established using spectroscopic techniques including ms, 1H nmr, 13C nmr, and various 2D nmr spectroscopy experiments.

L-696,474, an inhibitor of the HIV-1 protease, was discovered in extracts of the fungal culture Hypoxylon fragiforme (MF5511; ATCC 20995). L-696,474 is a novel cytochalasin with a molecular weight of 477 and an empirical formula of C30H39NO4. L-696,474 inhibited HIV-1 protease activity with an IC50 of 3 microM and the mode of inhibition was competitive with respect to substrate (apparent Ki = 1 microM). Furthermore, L-696,474 was not a slow-binding inhibitor. The inhibition due to L-696,474 was also independent of the HIV-1 protease concentration. L-696,474 was inactive against pepsin, another aspartyl protease; stromelysin, a zinc-metalloproteinase; papain, a cysteine-specific protease or human leucocyte elastase, a serine-specific protease. Two other novel cytochalasins (L-697,318 and L-696,475) isolated from the same culture were inactive against the HIV-1 protease. Commercially available cytochalasins B, C, D, E, F, H and J were inactive while cytochalasin A was as active as L-696,474 against the HIV-1 protease.

The cytochalasins are structurally complex natural products with a broad range of apparently unrelated effects in different biological systems. Different members of the family have variously demonstrated inhibitory activity toward the formation of actin filaments, toward the functioning of HIV protease, and toward the process of angiogenesis. The structural series is defined by a largely conserved, rigid bicyclic isoindolone core that is fused to a macrocyclic appendage. The latter structural component varies widely within the cytochalasins and seems to play an important role in the determination of biological activity. In this work, we describe the development of a convergent and enantioselective synthetic route to the cytochalasins that allows for the late-stage introduction of macrocyclic appendages of different sizes and constitutions. We illustrate the route with the synthesis of the 14-membered macrolactone cytochalasin B (1, an inhibitor of the formation of actin filaments) and the 11-membered macrocarbocyclic cytochalasin L-696,474 (2, an inhibitor of HIV protease) by using common precursors.