Indoleamine 2,3-dioxygenase (IDO) is an extrahepatic heme-containing dioxygenase that catalyzes the addition of oxygen across the C-2/C-3 bond of indole ring of tryptophan (Trp).1, 2 This is the initial and rate-limiting step in the catabolism of the essential amino acid Trp to N-formylkynurenine along the kynurenine pathway, the de novo biosynthetic route leading to NAD.3, 4 T-cell lymphocytes are extremely sensitive to Trp shortage, which cause them to undergo cell cycle arrest in G1, and leads to apoptosis and immunosuppression.5 Degradation of Trp by the placenta inhibits T-cell proliferation and, as a result, prevents immunological rejection of tumor or fetus. IDO is expressed ubiquitously but predominately in cells within the immune system, where it is specifically induced in dendritic cells and macrophages at the site of inflammation by cytokines.1 It is known that IDO is overexpressed in a variety of diseases, including cancer,6 alzheimer’s disease,7 age-related cataract8 and HIV encephalitis.9 Recent studies have shown that IDO inhibition might enhance the efficacy of cancer treatment. Indeed result from in vitro and in vivo experiment have suggested an improvement of the efficacy of therapeutic vaccination or chemotherapy by concomitant administration of an IDO inhibitor thus highlighting IDO as an attractive target.6, 10, 11, 12 Although there have been a number of reports on the development of IDO inhibitors,13, 14, 15, 16 new types of IDO inhibitors having improved pharmacological properties remain to be discovered.

In the course of our screening of the extracts of fungus for IDO inhibitors, we found activity in the culture broth of the soil fungus FN070315. Bioassay-guided fractionation of the extract led us to isolate a new benzodiazepine alkaloid, named benzomalvin E (1), together with two known benzomalvins B (2) and C (3). In this paper, we describe the fermentation, isolation, structure determination and biological activity of benzomalvins. Strain of a fungus, FN070315 was isolated from the soil sample collected from Daejeon in the Korea and was identified on the basis of the ribosomal RNA (rRNA) sequences and morphological evaluation. A GenBank search with the 26S rRNA gene of FN070315 indicated Penicillium jensenii (AY443470), Penicillium canescens (AY484896) as the closest matches, showing sequence identities of 100% and 99.98%, respectively. Therefore, the fungal strain FN070315 was identified and named as a Penicillium sp. FN070315 (deposited as KCTC1818P at the Korean Collection for Type Culture). Penicillium sp. FN070315 was grown on the PD agar medium for 7 days and was then inoculated into a 500-ml Erlenmeyer flask containing 75 ml of seed culture medium PD broth (24 g l−1 potato dextrose; BD Bioscience, San Jose, CA, USA). Incubation was carried out at 28 °C for 3 days on a rotary shaker operating at 135 r.p.m. This seed medium (150 ml) was transferred to 8 l of the same production medium in a two 14-l jar fermentor. The fermentation was carried out at 28 °C for 6 days with agitation at 165 r.p.m. and an air flow of 10 l min−1. The culture broth (16 l) was filtered and extracted three times with an equal volume of EtOAc and the EtOAc layer was concentrated in vacuo. The EtOAc extract (1.3 g) was subjected to reversed-phase C18 flash column chromatography using a stepwise gradient of MeOH/H2O (from 20/80, 40/60, 60/40, 80/20 to 100/0; 700 ml for each step), to yield five fractions (fractions 1–5). The active fraction 3 (68.0 mg) eluted with MeOH/H2O (60/40) was purified by semi-preparative reverse phase HPLC using an isocratic solvent system of MeCN/H2O (50/50), to yield compound 1 (tR 16.3 min, 3.5 mg), 2 (tR 23.5 min, 5.5 mg) and 3 (tR 26.8 min, 5.2 mg). Compounds 2 and 3 were identified as benzomalvins B and C by the comparison of NMR and MS data (Supplementary Figures S8–S11) with those in the literature, respectively.17

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