A new epipolythiodioxopiperazine, designated chetracin A, exhibiting remarkable cytotoxicity, was isolated from Chaetomium spp. The compound was proved to have the 11α, 11'α-dihydroxychaetocin nucleus by NMR and correlation reactions and found to have the tetrasulfide bridge by X-ray analysis. The epipolythiodioxopiperazines are the mold secondary metabolites exhibiting antimicrobial and cytotoxic properties. Production of the group of compounds such as chetomin (I) and chaetocin (II) by the genus Chaetomium was reported. 12) In our course of studies on mycotoxin production by the fungi belonging to the genus and related fungi, 3) the extracts of the culture on rice of three species, C. abuense Lodha (a), C. retardatum Carter & Khan (b), and C. tenuissimum Sergejeva (c), attracted our attention by the production of the mycotoxins detected by the remarkable cytotoxicity to HeLa cells and by the positive spots on TLC by Ehrlich's and silver nitrate reagents. Now the causative agents of the three (a - c) were isolated and identified respectively as (a) a new tetrasulfide type epipolythiodioxopiperazine named chetracin A (IVa), (b) dihydroxychaetocin (IIIa) and IVa, and (c) chetomin (I)? All these compounds showed nearly the same order of strong cytotoxicity to HeLa cells (IC50 ca 0.03 μg/ml). IIIa had been isolated from Verticillium tenerum 4) and Acrostalagmus cinnabarinus var. melinacidinus. 5) Colorless precipitate formed by concentration of the ethyl acetate extract of molded rice of C. retardatum was mainly composed of chetracin A (IVa). Due to its small solubility, it was converted to the acetate with acetic anhydride and pyridine and purified by HPLC using Develosil 60-3. The main fraction was hydrolyzed with NH4OH-MeOH to the original compound, chetracin A (IVa) (identified by TLC and HPLC), amorphous powder, mp 248-251° (decomp), [α]D²⁵ +723.5° (CHCl3), UV λmax^MeOH nm (log ε): 242, 306 (4.33, 3.70), IR νmax^KBr cm⁻¹: 3410, 1660, 1635, 1480, 1415, 1380, 1310, 1205, 1140, 1095, 1065, 750. The same compound was obtained from C. abuense by the same procedure. Reacetylation of IVa gave the triacetate (IVb), mp 260° (decomp), [α]D²⁵ +830° (CHCl3). M+ ions were not detectable in IVa and IVb by any modes of mass spectrometry. However the elemental analyses suggested that the molecular formula, C36H34N6O11S8, is most probable for IVb. ¹H- and ¹³C-NMR spectra of IVb showed similarity to those of the triacetate (IIIb) of IIIa as shown in Table 1. All the data suggested that IVa might be a tetrathio homolog of IIIa. In order to confirm the assumption, IVa was treated with boiling potassium hydroxide to give bi-indol-3-yl, 7) while the treatment with triphenylphosphine-NH4OH-MeOH (a modified method of that reported for sporidesmins 8)) of IVa gave IIIa. The CD spectra of the acetates (IIIb and IVb) also showed the same sign to indicate the same absolute configurations. In order to confirm the number of S atoms, the absolute configuration, and the conformation, X-ray analysis of IVb was performed using a single crystal obtained by recrystallization by vapor diffusion method from benzene saturated with water and xylene. A single crystal of approximate dimensions 0.1 × 0.08 × 0.12 mm was cut out from the aggregate and sealed in a thin walled glass capillary tube to prevent the loss of water of crystallization. The diffraction data were obtained using CuKα radiation monochromated by a graphite plate. The crystal data are shown in Table 2. Intensities of 3062 reflections were measured in 2θ range of 6° through 178°. The structure was solved by the direct method using MULTAN 9) and refined by the block-diagonal least-squares method to an R value of 0.084. The unit cell dimensions are a = b = 15.337(α), c = 19.922(10) Å, crystal system: tetragonal, W = 1019.2. The difference map indicated three water oxygen atoms. The occupancy factors of these oxygen atoms were estimated by the difference electron-density map. Absolute configuration was determined by the anomalous dispersion method. |F|² values were calculated for 392 Friedel pairs introducing the dispersion corrections for atomic scattering factors of C, O, N and S atoms for CuKα radiation and compared with the observed values. Of the total of 64 pairs for which the difference of structure factors between h, k, l and ḣ, k̄, l̄ exceeds 2σ(|Fo|), 55 pairs showed clearly the absolute configuration shown in Fig.1. The final refinement was carried out introducing the dispersion correction for C, N, O and S atoms and anisotropic thermal parameters for all atoms. Hydrogen atoms were not included. The R value was reduced to 0.081 for 3062 observed structure factors. Fig. 1 shows the molecular structure of chetracin A triacetate and the chemical structure of chetracin A has now been established as shown in the formula IVa. The absolute configuration of the compound was proved to be the same as IIIa. The dioxopiperazine rings are in the boat conformations but the ring is more planar than in the case of a disulfide bridge 10). The four terminal S-S bonds (1.996, 2.005, 2.002, 2.011 Å) are shorter than the two central S-S bonds (2.066 and 2.092 Å) as in the case of sporidesmin G having the same tetrasulfide bridge 11). There exist four possible conformers in the cyclic tetrasulfide. The NMR spectra of IVb indicated the presence of only one conformer. X-Ray analysis showed that the central pairs of sulfur atoms were directed towards the nitrogen atoms of the dioxopiperazine rings (Fig. 2). The interatomic distances between the central sulfur atoms and the peptide nitrogen atoms (3.14, 3.17, 3.19, 3.21 Å) were shorter than the sum of their van der Waal's radii (3.35 Å). This might arise from donor-acceptor interactions and may be the reason for the stability of the conformation 11). Chetracin A (IVa) is a symmetric dimer but forms triacetate by acetic anhydride-pyridine as in the case of IIIa 7). Such phenomenon was also reported in the case of verticillins. Examination of the crystallographic data revealed that the free hydroxyl group at C11α is sterically hindered and forms a hydrogen bond with the acetyl carbonyl group introduced at the 11α-hydroxyl group. The final atomic parameters will be compiled in Cambridge Crystallographic Database. The list of Fo and Fc bond lengths and angles may be obtained from one of the authors (Y. I.) upon request.