In the course of screening for antitumor antimetabolites,l) L.J. HANKA of these laboratories submitted lyophilized beer from a control culture lacking antimetabolite activity, for in vivo assay against PS-3 leukemia in mice. The lyophilized beer solids significantly prolonged the life span of the leukemic mice and our efforts were enlisted to isolate the active principle. The lipophilic active principle was readily extracted with solvents; mycelial cake was the richest source of active extract. Solvent extracts had reasonable in vitro activity and bioautography distinguished the activity from all the antibiotics in the Upjohn collection of known antibiotics. Mycelial extracts were defatted with petroleum ether and purified by silica gel chromatography, preparative thin-layer chromatography, and counter current distribution yielding a homogenous crystalline antibiotic, U-48, 160. Infrared mull spectra suggested a very close relationship with quinomycin A (echinomycin)3) from which U-48, 160 had already been differentiated by bioautographyleaving the probability that the antibiotic was a member of the quinoxaline4) family. Although the ultraviolet spectrum and optical rotation of U-48, 160 were consistent with the quinoxaline antibiotics,5,6) NMR spectra, both proton and 13C, apparently differentiated the antibiotic from the reported structures shown (Fig. 1). An apparent S-CH3 (s, 5 2.1) was inconsistent with published* quinomycin and triostin structures. Since structures of the minor components of the quinoxaline family were based on comparison with quinomycin A, the structure determination of which was accomplished without the benefit of NMR, we investigated the proton and 13C spectra of that antibiotic. To our surprise, the spectra betrayed the presence of the S-CH3 in a sample which had been found identical to authentic echinomycin** by bioautography on 3 systems as well as comparison of infrared spectra. The 100 MHZ proton spectrum*** (Fig. 2) of quinomycin A shows the presence of 64 protons (4 exchangeable) and agrees well with the expected resonances for quinoxaline, alanine, serine, and N-methylvaline fragments of the molecule. The 2 N-CH3's adjacent to the bridge section are present but the 2 expected isolated bridge methylenes are absent. Instead a methine (d, J,g=11.0, JBx=O, 5 6.14) coupled to an S-CH2 (AB of an ABX, JAB=-17, centered at 5 3.16) and a CH (d, J=9.0, 5 6.5) coupled to a CH (under a 9 proton multiplet between 8 4.6 and 5.1) was established by spin decoupling. In addition a signal for an S-CH3 (s, 5 2.1) is evident. The 13C spectrum* of quinomycin A (Fig. 3) shows the presence of 51 carbons, including 10 carbonyls, and a lack of C2 symmetry. The absorptions (Table 1) of the quinoxaline, alanine and serine carbons were assigned by off-resonance decoupling experiments, standard chemical shift data, and comparison to model compounds.7-10) The absorptions of N-methylvaline were assigned by comparison with the spectrum (Fig. 3) of U-48,160 which we feel is the same as quinomycin C in which the N-methylvaline is replaced by a N, 8-dimethylleucine enantiomer* and by the expected downfield shift of the a CH carbon when a N-CH3 is present. The bridge N-CH3's (2Q, 5 31.0, 31.6), S-CH2 (T, 6 27.2), and S-CH, (Q, 815.3) were assigned by off-resonance decoupling. The methine carbons at 8 53.6 and 60.0 were assigned to the proton doublets at 8 6.14 and 6.5 respectively by single frequency proton decoupling. The remaining carbon at 6 53.4 was assigned by default. A count of protons from the off-resonance carbon splitting patterns confirms 60 non-exchangeable protons. Based on these NMR studies and the excellent earlier work of others, a revised structure (Fig. 4) is proposed for quinomycin A. Field desorption mass spectrometry afforded further support for this revised structure. A weak M+ at 1100 was observed (C51H64N12O12S2 requires 1100.421). It is noteworthy that a strong mercaptan odor had been observed during acid hydrolysis of echinomycin3'. Proton (Fig. 2) and 13C NMR spectra (Fig. 3) of U-48,160 were very similar to those from quinomycin A; the only apparent difference was the substitution of N, Q-dimethylleucine residues for the 2 N-methylvaline residues. This difference is the same as the reported difference5) between quinomycin C and quinomycin A where 2 N, r-dimethylalloisoleucine residues in quinomycin C replace the N-methylvaline residues of quinomycin A. Although we were unable to secure an authentic sample of quinomycin C for direct comparison, we feel that U-48, 160 is actually quinomycin C based on (a) the NMR comparison already cited, (b) comparison of the infrared solution spectrum* of U-48, 160 (Fig. 5) with quinomycin C5), (c) optical rotation ([a]D -268; lit. [6] [a]D -250), and (d) the relative mobilities of U-48,160 and quinomycin A are consistent with the reported6) relative mobilities of quinomycins A and C. Accordingly a revised structure (Fig. 4) is proposed for quinomycin C. Field desorption mass spectrometry afforded a weak M+ at 1156 in support of this structure (C55H72N12O12S2 requires 1156.4833).