The structure of neamphine (1), a cytotoxic metabolite of the marine sponge Neamphius huxleyi, has been solved by single crystal x-ray diffraction analysis. Neamphine (1) represents the first example, either from nature or synthesis, of the imidazo[4,5-e]-1,2-thiazine ring system. A number of polycyclic aromatic alkaloids have recently been isolated from marine invertebrates and many of them have been found to be potently cytotoxic and antineoplastic. One small subset of these invertebrate alkaloids contain sulfur atoms either as part of the heterocyclic ring systems or as a component of attached ring substituents. In our continuing search for cytotoxic and antineoplastic metabolites from marine invertebrates, we have discovered that crude extracts of the sponge Neamphius huxleyi (Sollas, 1888) exhibit significant in vitro cytotoxicity. Bioassay guided fractionation of the crude extracts of N. huxleyi has resulted in the isolation of neamphine (1), a novel cytotoxic sulfur heterocycle. Specimens of N. huxleyi were collected by hand using SCUBA on nearshore reefs off Sek Point, Madang, Papua New Guinea. Freshly collected sponge (320g dry wt.) was frozen on site and transported to UBC on dry ice. Thawed samples of the sponge were extracted with 1:1 MeOH/CH2Cl2 and the crude extract was concentrated in vacuo to give a gummy residue that was partitioned between 6:4 H2O/MeOH and chloroform. The more polar phase was concentrated in vacuo to an aqueous suspension that was extracted exhaustively with EtOAc. Fractionation of the EtOAc soluble materials by repeated isocratic silica-gel column chromatography eluting in the first instance with 96:4 CH2Cl2/MeOH and in the second instance with 1:2 hexane/EtOAc gave pure neamphine (1) (0.6mg, colorless needles from 2:1 hexane/CHCl3). Neamphine (1) gave an intense parent ion in the EI-MS at m/z 167.0153 Da appropriate for a molecular formula of C6H6N3OS (ΔM -0.1 mmu). The 1H NMR spectrum (400 MHz, CDCl3) of 1 contained three singlets at δ 4.18 (3H), 7.84 (1H) and 8.63 (1H) that could be assigned to a methyl group attached to a heteroatom and two heterocyclic aromatic protons, respectively. A strong absorption band at 1625 cm-1 in the IR spectrum of neamphine (1) was tentatively assigned to a carbonyl stretching vibration. 13C NMR spectra recorded on the limited quantity of neamphine that was available failed to show resonances for all six carbon atoms in the molecule. The information available from the spectral data was insufficient to identify a unique constitution for neamphine. Therefore, the structure was solved by single crystal x-ray diffraction analysis. A flat needle (0.1 × 0.2 × 0.7 mm) was selected and preliminary X-ray examination revealed monoclinic symmetry with lattice constants of a = 7.837(5), b = 3.880(3), c = 11.518(9) Å, and β = 97.09(6)°. Systematic extinctions and density considerations led to space group P21 with one molecule of composition C6H6N3OS in the asymmetric unit. All diffraction maxima with 2θ < 45° were collected with a computer controlled four-circle diffractometer using θ:2θ scans and graphite monochromated Mo Kα radiation. A total of 527 unique reflections were measured and after correction for Lorentz, polarization, and background effects, 460 (87%) were judged observed (|F0| ≥ 3σ(|F0|)). A phasing model was found easily using direct methods. Full-matrix least-squares refinements with anisotropic heavy atoms and isotropic riding hydrogen atoms converged to a final crystallographic residual of 3.61%. Several different assignments of atom types were investigated, but all gave substantially worse agreement. A computer generated perspective drawing of the final X-ray model of neamphine (1) is given in Figure 1. The molecule is flat - as would be expected from its aromatic nature - with a rms deviation of 0.017 Å. Since this is the first example of this ring system, the bond lengths - especially the S-N bond - are interesting. In neamphine (1) the S-N bond is 1.654(6) Å, and in related compounds, S-N bonds of 1.652(15) Å are reported. Neamphine (1) represents the first example, either from nature or synthesis, of the imidazo[4,5-e]-1,2-thiazine ring system. It bears an interesting biogenetic resemblance to ovothiol A (2), a metabolite found in the eggs of a number of marine asteroids. Neamphine (1) exhibited in vitro cytotoxicity (L1210 ED50 < 10 μg/mL), however, the limited amount of material available prevented in vivo testing.