Three new cytotoxic alkaloids, 6,7-dihydro-11-oxo-onnamide A [2], 11-oxo-onnamide A [3], and 4Z-onnamide A [4], have been isolated from an Okinawan marine sponge of the genus Theonella and their structures elucidated on the basis of spectroscopic data. Sponges of the genus Theonella have produced a variety of bioactive secondary metabolites with unique chemical structures such as cyclic peptides (1-3) or macrocyclic bislactones (4-6). We have also reported the isolation and structural elucidation of the cyclic peptides konbamide (7) and keramamides A-D (8,9) and F (10) from the genus Theonella. Onnamide A [1], a potent antiviral compound, was first isolated from a sponge of the genus Theonella (11). During our investigations on bioactive substances from marine organisms (12-14), we further examined the same Theonella sponge from which keramamides B-D were isolated and obtained three new onnamide congeners, 6,7-dihydro-11-oxo-onnamide A [2], 11-oxo-onnamide A [3], and 4Z-onnamide A [4], together with onnamide A [1] and dihydroonnamide A [5] (15). The sponge Theonella sp., collected off the Kerama Islands, Okinawa, was extracted with toluene-MeOH (1:3), and the extract was partitioned between toluene and H₂O. The CHCl₃-soluble material of the aqueous phase was subjected to flash chromatography on a Si gel column with CHCl₃-MeOH (1:1), followed by chromatography on an ODS column, preparative Si gel TLC, and reversed-phase HPLC on an ODS column to afford 6,7-dihydro-11-oxo-onnamide A (2) (5.6×10⁻³% wet wt of the sponge), 11-oxo-onnamide A (3) (3.0×10⁻³%), and 4Z-onnamide A (4) (7.9×10⁻³%), together with onnamide A (1) (8.8×10⁻³%) and 6,7-dihydro-onnamide A (5) (1.3×10⁻³%). Compound 2 was shown to have molecular formula C₃₉H₆₃N₅O₁₂ by HRFABMS (m/z 794.4565, [M+H]⁺, Δ +1.3 mmu). IR absorptions at 3400 and 1650 cm⁻¹ indicated the presence of hydroxy and/or amino group(s) and amide group(s), respectively. The UV spectrum (λ max 263 nm) was indicative of the presence of a dienone moiety. The ¹H NMR (Table 1) spectrum revealed that 2 possessed an exo-methylene (δH 4.63, s and 4.79, s), a conjugated diene (δ 6.01, 6.08, 6.20, and 7.10), two methoxy groups (δ 3.24, s and 3.55, s), and four methyl groups (δ 0.86, 0.95, 1.03, and 1.18). In the ¹³C NMR spectrum of 2 (Table 2), a quaternary carbon signal at δ 158.6 was assigned as a guanidine carbon of arginine. These spectral data resembled those of onnamide A [1] except for ¹H and ¹³C resonances in the olefinic region. The blue shift of the UV spectra (λ max 298 nm in 1 to 263 nm in 2) suggested differences in a chromophore (C-1-C-7) between 1 and 2. The ¹H-¹H COSY spectrum revealed the presence of a conjugated diene (C-2-C-5) which had all E geometry judging from the ¹H-¹H coupling constants (J₂₋₃=15.3 Hz and J₄₋₅=14.7 Hz). The carbon chemical shift (δ 71.0 in 1 to δ 210.8 in 2) of C-11 indicated that the hydroxyl group at C-11 in 1 was oxidized to be a ketone group. As a result, compound 2 was elucidated to be 6,7-dihydro-11-oxo-onnamide A. The molecular formula of compound 3 was determined to be C₃₉H₆₁N₅O₁₂ by HRFABMS (m/z 792.4431 [M+H]⁺, Δ +3.6 mmu) which was less than that of 1 by 2 daltons. The ¹H NMR (Table 1) spectrum was very similar to that of compound 1. The presence of a ketone group (C-11) was revealed by comparison of the chemical shifts of H-12 (δ 2.49) and H-13 (δ 3.95) in 3 with those in 1 and by lack of an H-11 signal for 3. Thus compound 3 was assigned as 11-oxo-onnamide A. Compound 4 was shown to have the molecular formula C₃₉H₆₃N₅O₁₂ by HRFABMS (m/z 794.4593 [M+H]⁺, Δ +4.1 mmu) which was the same as that of onnamide A [1]. Six olefinic protons were observed at δ 6.06 (H-2), 7.66 (H-3), 5.99 (H-4), 6.27 (H-5), 6.70 (H-6), and 5.96 (H-7), which were assignable to a conjugated triene group on the basis of the ¹H-¹H COSY spectrum. The coupling constant (J=10.7 Hz) between H-4 and H-5 revealed a Z configuration of the C-4 double bond, while an E configuration was indicated for the C-2 double bond (J₂₋₃=14.4 Hz).