Three new cytotoxic bromotyrosine-derived secondary metabolites, aplysamines 3 [2], 4 [3], and 5 [4], were isolated from the sponge Psammaplysilla purpurea. Marine sponges of the order Verongida are often characterized by a wide range of bioactive bromotyrosine constituents (1,2). The large number of biosynthetically related compounds is due to chemical variations occurring in the side chain and/or aromatic ring of the tyrosine moiety. We now report the structures and in vitro bioactivity of aplysamines 3 [2], 4 [3], and 5 [4], which are related to aplysamine 2 [1] (3). A CH₂Cl₂-iPrOH (1:1) extract of the sponge Psammaplysilla purpurea Carter (family Aplysinellidae), collected by scuba on the south shore of Maui, Hawaii, at ~40 m (sample ID 12-DD-91) displayed antimicrobial activity and cytotoxicity against human epidermoid carcinoma KB cells. Bioassay-guided fractionation of the extract resulted in the isolation of three new compounds 2-4. The hrfabms of the major metabolite, aplysamine 3 [2], indicated a molecular formula of C₂₁H₂₂Br₃N₃O₄. The ¹H-nmr spectrum of 2 (Table 1) showed the characteristic pattern of a 1,2,4-trisubstituted benzene (1H doublets at 7.42 and 6.88 and a doublet of doublets at 7.17 ppm) and a two-proton singlet at 7.43 ppm for a symmetrically tetrasubstituted benzene. Amide and oxime functionalities were indicated by ir absorptions at 3350, 1655, and 1620 cm⁻¹ and confirmed by two ¹³C-nmr signals at 165.8 and 153.0 ppm (Table 2). The upfield ¹³C-nmr shift of C-7 (28.7 ppm) suggested E configuration of the oxime, as the corresponding value for a (Z) oxime is >35 ppm (4). Comparison with the nmr data for aplysamine 2 [1] (3) (Tables 2 and 3, numbering system same as in 2) showed that the dimethylammonium group in 1 was replaced by ammonium, which shifted the H₂-20 and H₂-19 signals to 3.29 and 2.19 ppm, the C-20 resonance upfield by 17.9 ppm and the C-19 signal downfield by 2.6 ppm. Treatment of 2 dissolved in CD₃OD with aqueous KOH and ¹H-nmr analysis of the resulting free amine confirmed that 2 was a salt. The ¹H-nmr signals of the methylenes α and β to the amino group were shifted upfield by 0.38 and 0.23 ppm. No attempt was made to determine the nature of the counter ion. The multiplicity of the shifted signals also proved the linkage mode of the two bromotyrosine-derived segments, in which the three-carbon chain C-18 to C-20 was bearing the terminal amine rather than the internal amide nitrogen. An alternative mode is represented by debromoprearaplysillin I [5] (5). The molecular formula of the second metabolite, aplysamine 4 [3], was C₂₁H₂₂Br₄N₃O₄ by hrfabms. ¹H- and ¹³C-nmr spectra of 3 resembled those of 2 except for ring A signals (Tables 1 and 2), which unambiguously showed that the additional bromine atom was at C-4. The hrfabms of the third metabolite, aplysamine 5 [4], indicated a molecular formula of C₃₆H₃₂Br₃N₃O₃. The ir spectrum displayed two strong bands in the amide region at 1660 and 1645 cm⁻¹. The ¹H-nmr spectrum of 4 exhibited the same characteristic resonances as 2 (Table 1), except for a 2H triplet (H-20) that was shifted downfield (3.41 ppm). In addition, the spectrum showed signals typical for a terminal iso-branched saturated fatty acid chain: protons α and β to an acyl group (2.17 and 1.60 ppm), a 6H doublet of two methyls (0.86 ppm), a multiplet of a methine (1.51 ppm), and two signals for methylenes (1.28 ppm, 16H and 1.16 ppm, 2H). These data, when compared with those for 2, suggested that the ammonium group in 2 was replaced by a terminal iso-branched saturated fatty acid chain in 4.