Compounds B, C, D and E were highly active in vitro against Gram-positive and selected Gram-negative bacteria. When administered subcutaneously or orally, at sublethal doses, B, C, D and E protected mice infected with Staphylococcus aureus Smith and Streptococcus pyogenes C-203. Compounds D and E were also active in mice against transplanted mammary adenocarcinoma (72j). Compound D was effective against leptospirosis in the chick embryo test. Antibiotics B, C, D and E showed considerable toxicity in these tests. The D component was the least toxic. Compound AB crystallized in red-purple needles, melting at 124-126'. Calculated for C16H19N3O6: C, 55.0; H, 5.5; N, 12.0; O, 27.5; mol. wt., 349; 4.3 for 1 CH3; active H, 0.289 for 1, 0.578 for 2; found: C, 54.8; H, 5.7; N, 11.8; O, 27.2; mol.wt.,346±7; OMe,4.6; NMe,2.6; CMe,3.1; active H, 0.37. It is named mitiromycin A to indicate the relationship with the mitomycins and porfiromycin. Compound B crystallized in purple needles with m.p. 159-160'. Calculated for C16H19N3O6: C, 55.0; H, 5.5; N, 12.0; O, 27.5; mol. wt., 349; OMe, 8.5 for 2 CH3; CMe, 4.3 for 1 CH3; active H, 0.86 for 3; found: C, 54.7; H, 5.8; N, 11.8; O, 27.9; mol. wt., 366±10; OMe, 8.4; NMe, none; CMe, 4.0; active H, 0.93; [α]26D -143°, (C 0.107 in methanol); 212 mp (ε 500), 319 mp (ε 295), 515 mp (ε 35). A sample of mitomycin A made available to us was found to be identical with our B component by paper chromatography, ultraviolet and infrared absorption. Pigments D and E were also isolated as purple crystalline compounds. Direct comparison of the chemical and physical properties, including paper chromatographic mobilities, of pigment D with those of porfiromycin proved them to be identical. Similarly, crystalline pigment E was compared directly with mitomycin C and found to be identical by paper chromatography, ultraviolet and infrared absorption and by X-ray powder diffraction. When compound B was treated with aqueous ammonium carbonate, a crystalline product was obtained with characteristics corresponding to compound E (mitomycin C). The above compounds were reduced readily with sodium hydrosulfite and reoxidized in air. This suggests the possibility of a quinoid structure. On the basis of chemical studies and spectrophotometric data we have concluded that mitomycins A, B and C and porfiromycin all have the common structure II differing only in minor substituents (Table A). These antibiotics are the first members of a new and unusual structural type, representing the first naturally occurring examples of an aziridine, the pyrrolo [1,2-a]indole ring system, an aminobenzoquinone and a pyrrolizine elaborated by a microorganism. We have studied a group of four antibiotics which were isolated by Lefemine, et al., from three soil isolates of Streptomyces verticillatus. These compounds were later recognized as mitomycins A, B and C and porfiromycin. Most of the present investigation was carried out on mitomycin A (I-A), which was correlated with the other three antibiotics as follows. Reaction of I-A with methanolic ammonia replaced the quinone methoxyl at C-7 by an amino group, producing mitomycin C (I-C). Similarly N-methylmitomycin A (I-E) reacted with methanolic ammonia to give porfiromycin (I-D). The chromophore of I-A and I-B [218 mp (ε 17,400), 320 mp (ε 10,400), 520 mp (ε 1,400)] was identified by its similarity to the spectrum of 2-dimethylamino-5-methoxybenzoquinone [218 mp (ε 18,500), 305 mp (ε 13,900), 490 mp (ε 3,900)]. Likewise the chromophore of I-C and I-D [217 mp (ε 24,600), 360 mp (ε 23,000), 555 mp (ε 209)] was similar to that of 2,5-bis-dimethylaminobenzoquinone [222 mp (ε 24,000), 365 mp (ε 21,400), 513 mp (ε 407)]. Furthermore, I-A in 1 N NaOH was converted to a product with λ max NaOH 323-330 (doublet) (ε 24,500) which was nearly identical spectrally with 2,5-dihydroxy-p-xyloquinone [λ max NaOH 332 (ε 26,300)]. In 0.1 N HCl at 25° for a few hours I-A underwent a marked change forming one mole of methanol and a new product, apo-mitomycin A (II-A), C16H17N3O6, golden plates from dimethylformamide-water, m.p. 180-200° dec., [α]26D -10° (3.3% 0.1 N HCl), containing one C-methyl, one O-methyl, and one amino group. The methanol formed did not come from hydrolysis of the quinone methoxyl group, as indicated by the continued presence of the 6.0 τ peak in the n.m.r. spectrum of II-A (D6 dimethyl sulfoxide). The chromophore of II-A [232 mp (ε 20,700), 285 mp (ε 14,400), 346 mp (ε 3,620), 430 mp (ε 1,200)] was unaltered by short term pH changes, but hydrolysis in 0.1 N NaOH at 26° liberated one mole of methanol resulting in a new product (II-F), C14H17N3O6, purple needles from dimethylformamide-water, [α]26D +14.2° (1% 0.1 N HCl) which was an indicator [λ max HCl 235 mp (ε 21,700), 294 mp (ε 15,900), 346 mp (ε 3,920), 460 (ε 1,050); λ max NaOH 254 mp (ε 19,200), 312 mp (ε 11,900), 560 mp (ε 1,220)], a characteristic of 2-hydroxyquinones. When oxidized with KMnO4 in alkali II-F showed spectral changes typical of the Hooker oxidation of a 3-alkyl-2-hydroxy-1,4-naphthoquinone. An n.m.r. peak (8.1-8.2 τ, unsplit) likewise indicated the presence of a methyl group on the quinone ring (no adjacent proton) in both I-A and II-A. The foregoing data suffice to establish the interrelations and chromophores of the antibiotics in this series; the following communication presents additional data sufficient to prove the total structures. In the preceding communication we report the interrelations and chromophores of the mitomycins and porfiromycin. In the determination of the rest of the skeletal structure and the arrangement of functional groups, the reactions described below were of major significance. apo-Mitomycin A (II-A) formed a diacetyl derivative (II-G) m.p. 244-246.5° dec., with carbonyl bands at 6.44 μ (amide II) and 5.75 μ (alkylacetate) suggesting the presence in II-A of alkyl NH2 and OH, neither of which was present in I-A. One of the two remaining nitrogen atoms and both unassigned oxygen atoms in II-A and II-F were part of a -OCONH2 group: hydrolysis of either II-A or II-F (and even I-A) in 6 N HCl at 25° produced one mole each of CO2 and NH4+ and a new compound (II-L) [C14H14N2O6; [α]26D 6907 +26±5° (1% 0.1 N HCl); tetraacetyl derivative (II-M) m.p. 225-230°], but in strong base neither NH4+ nor CO32- were formed from II-A and II-F until after brief acidification. The band at 5.5 μ, absent in II-L but present in I-A, -B, -C and -D; II-A, -B, -F and -J, was attributed to a carbamate carbonyl. By exclusion the remaining nitrogen atom in II-A must have been the original nitrogen of the aminobenzoquinone in I-A; in II-A it was neither hydrolyzable nor basic; there remained the possibility that it was heterocyclic. In fact the ultraviolet spectrum of 5,6,7,8-tetrahydro-3-hydroxy-2-methyl-1,4-carbazoledione (III) [λ max HCl 237 mp (ε 20,200), 293 mp (ε 19,000), 370 mp (ε 4,330), 510 mp (ε 1,390); λ max NaOH 246 mp (ε 24,500), 306 mp (ε 12,500), 365 mp (ε 4,620), 595 mp (ε 1,610)] was nearly identical with that of II-F. The arrangement of the groups (-OH, -NH2, -CH2OCONH2) on the skeleton of II-A was established by treatment of it with HNO2 to produce a compound (IV) C16H14N2O6, which contained a new carbonyl group, λ max 5.77 μ, was optically inactive, and lacked the NH2 and OH groups of IIA. The ultraviolet spectrum of IV [λ max EtOH 280 mp (ε 41,400)] indicated probable conjugation of the new carbonyl with the indoloquinone chromophore, but was unlike that of ethyl 5-hydroxy-2,6-dimethyl-4,7-dioxo-3-indolecarboxylate; therefore, the new carbonyl probably was not at the 3-indole position and hence must be at 2-. The n.m.r. spectrum of IV (D6-dimethyl sulfoxide) displayed two widely separated triplets characteristic of an A2X2 pattern not shown by II-A. This suggested the presence of the moiety Y-CH2-CH2-CO- at the 2-indole position. Y had to be the indole nitrogen: the -CH2OCONH2 group in IV must be at the 3- rather than 1-indole position since strong acid hydrolysis of IV produced no formaldehyde, and acid permanganate oxidation of IV yielded β-alanine, identified by paper chromatography.