The isolation and structure elucidation of rebeccamycin 1, a new antitumor agent from Nocardia aerocoligenes, is described. The NMR spectra of 1 and its peracetate 2 are discussed. Recently we have isolated a novel antitumor antibiotic rebeccamycin 1 from fermentations of Nocardia aerocoligenes, strain c38383-RK-2 (ATCC 39243). Rebeccamycin was isolated by extraction of the mycelial mat of the fermentation with THF. Evaporation of the extracts followed by trituration of the crude solids with ether yielded crude rebeccamycin which was recrystallized from THF-MeOH to yield pure rebeccamycin as a yellow solid, mp 326-330°C (decomposition), [α] (THF) = + 131°. The IR spectrum of 1 had bands characteristic of hydroxyl groups and a cyclic imide (IR bands at 3418, 3355, 1752 cm⁻¹ (medium), 1704 cm⁻¹ (strong)). The UV spectrum of 1 in methanol showed two maxima at 238 nm (ε=75.75) and 314 nm (ε=90.51) with shoulders at 256, 293, 362 and 390 nm. Addition of dilute base gave bands at 314, 285, and 237 nm. No shifts were observed on addition of dilute acid. The elemental formula C₂₇H₂₁Cl₂N₃O₇ was determined by elemental analysis and mass spectroscopy. The CI mass spectrum of 1 exhibited an ion at m/z 570 (M+1) and a parent ion at m/z 394 (M-C₇H₁₄O₅). Minor ions at m/z 422 and m/z 436 were also observed. The ¹H NMR spectrum of 1 in DMSO-d₆ exhibited resonances at δ 11.37 (s, 1H, N6-H), 10.30 (s, 1H, N13-H), 9.27 (d, 1H, C8-H), 9.09 (d, 1H, C4-H), 7.74 (d, 1H, C10-H), 7.69 (d, 1H, C2-H), 7.45 (t, 2H, C3-H + C9-H), 6.97 (d, 1H, C1'-H), 5.45 (d, 1H, C3'-OH), 5.36 (t, 1H, C6'-OH), 5.03 (d, 1H, C2'-OH), 3.90 (bt, 2H, C6'-CH₂), 3.66 (quin, 1H, C5'-H), 3.56 (dt, 1H, C2'-H), 3.53 (t, 1H, C4'-H), 3.48 (s, 3H, C4'-OCH₃), and 3.45 (obscured, 1H, C3'-H). The ¹³C NMR spectrum of 1 is listed in Table 1. Treatment of 1 with acetic anhydride/pyridine gave the tetracetate 2 in good yield as a yellow solid, mp 145-147°C. The ¹H NMR spectrum of 2 was very similar to that of 1 in the aromatic region of the spectrum. The glycoside protons became first order and readily led, together with the ¹H NMR spectrum of 1, to an assignment of 4-O-methyl glucose as the carbohydrate portion of the molecule linked by a β-glycoside linkage to the aglycone (see Fig. 1). From the chemical shifts of C1'-H in both 1 and 2 and the position of the anomeric carbon in the ¹³C NMR spectra of 1 and 2 (84.2 and 81.3 ppm, respectively), it was evident that the sugar residue was either a C- or N-glycoside attached to an aromatic or heteroaromatic aglycone. The chemical shift of the C2' acetyl function supported this assignment. That the aglycone of 1 had a good deal of symmetry could also be seen from the ¹H NMR of the aglycone protons as well as the ¹³C NMR spectrum of 1 in which many of the signals appeared to be paired. At this junction, crystals of 1 suitable for X-ray analysis became available. Crystals were grown by slow evaporation of a dioxane solution, and a roughly rectangular specimen with dimensions 0.5×0.3×0.2 mm was used. Preliminary X-ray photographs showed monoclinic symmetry, and accurate lattice constants of a=7.616(1), b=20.610(4), c=18.912(2) Å, and β=82.27(2)° were determined from a least-squares fit of fifteen 2θ values. The systematic extinctions, crystal density, and presence of chirality were uniquely accommodated by space group P2₁ with two molecules of composition C₂₇H₂₁Cl₂N₃O₇ and some solvent forming the asymmetric unit. All unique diffraction maxima with 2θ < 114° were collected using variable speed, 1° ω-scans and graphite monochromated Cu Kα radiation (1.54178 Å). Of the 4468 reflections measured in this way, 3304 (74%) were judged observed (|F| > σ(F)). A phasing model was achieved with some difficulty using a multisolution tangent formula approach and extensive tangent formula recycling of plausible molecular fragments. The structure was finally completed with 2F₀-Fc syntheses. The final X-ray model consisted of the 78 anisotropic nonhydrogen atoms of rebeccamycin, 42 isotropic hydrogens, and ten solvent atoms. Block diagonal least-squares refinements with this model have converged to a standard crystallographic residual of 0.058 for the observed reflections. Figure 1 is a computer-generated perspective drawing of the final X-ray model of rebeccamycin. Both molecules comprising the asymmetric unit have the same conformation, and only one is illustrated. Hydrogens are omitted for clarity, and the enantiomer illustrated was selected on the basis of the known absolute configuration of the D-4-methoxyglucose component. Bond distances and angles generally agree well with accepted values. The aromatic portion of the molecule is planar within experimental error. The plane of the sugar fragment is rotated 90° to the plane of the aromatic ring. There is some evidence of steric interactions between the sugar and the chlorine closest to the sugar: atoms Cl11 and Cl' are on opposite sides of the aromatic plane by 0.27 and -0.65 Å, respectively. From the position of the C2'-OH function in 1, the shielding of the acetate methyl function in 2 may be readily understood since the acetyl group would lie under the aromatic chromophore. It is also notable that the aromatic nitrogen is pyramidal. This may explain some of the chemical shift differences noted in the ¹³C NMR spectrum of 1 for C4a vs C7c, C4b vs C7b, and C4c vs C7a. From the X-ray analysis, it was not possible to assign the absolute configuration of 1. All attempts to hydrolyze the glycosidic linkage in 1 and recover the resultant glycoside failed. Consequently, the absolute configuration was determined by total synthesis as reported in the accompanying communication.