Heat shock protein 90 (Hsp90) is emerging as an important target in cancer therapeutics and several other diseases.1,2 Hsp90 is an abundant and ubiquitously expressed molecular chaperone that is involved in the maturation of multiple client proteins, many of which are involved in regulating cell signaling, proliferation and survival.3,4 Client proteins of Hsp90 include Her-2, Akt, Src, Abl, c-Met and Raf-1, which are currently being targeted for intervention within oncology drug discovery or in clinical development. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors display remarkable selectivity for cancer cells as compared with normal cells.5,6 Hsp90 is effectively inhibited by benzoquinone ansamycins such as geldanamycin (GM, 1), herbimycin, macbecin and many of their derivatives, which bind to the ATP-binding site in the N-terminal domain.7,8 However, the development of 1 as a clinical agent has so far been limited by its toxicity, especially liver toxicity.9 However, the promising antitumor properties of 1 have spurred initiatives to develop biologically active derivatives.10,11 17-Allylamino-17demethoxyGM (17-AAG) has reduced toxicity compared with 1 and is the most advanced Hsp90 inhibitor in clinical development (Phase II/III).12 17-AAG and its benzoquinone analogs conjugate with glutathione, leading to cellular depletion. This conjugation with sulfur-containing nucleophiles may contribute to the dose-limiting hepatotoxicity of these quinone-containing compounds.13,14 For these reasons, new non-quinone 1 analogs with improved pharmacological profiles are needed.15,16 Recently, we reported the development of non-quinone 1 analogs by a mutasynthetic approach and directed biosynthetic method.16,17 Of these non-quinone 1 analogs, DHQ3 (3), a 15-hydroxyl-17demethoxy non-quinone analog, was found to inhibit Hsp90 ATPase activity more than 1.16 Moreover, during these studies, novel tricyclic 1 analogs were prepared from a genetically engineered strain (AC15) of Streptomyces hygroscopicus.18 Presently, we describe the fermentation of mutant AC15, and the isolation, structural determination and bioactivity of new non-quinone 1 analogs produced by the mutant: DHQ7 (4) and DHQ8 (5). AC15 was constructed by a combinational mutation with site-directed mutagenesis of the first dehydratase domain of the geldanamycin polyketide synthase (PKS) gene (gelA) and a post-PKS modification gene (gel7) of S. hygroscopicus JCM4427, as previously reported.16 The seed medium and production medium (YEME) consisted of sucrose 103.0 g, yeast extract 3.0 g, peptone (Difco, Sparks, MD, USA) 5.0 g, malt extract (Difco) 3.0 g, glucose 10.0 g and MgCl2 6H2O 1.0 g in 1.0 l of distilled water. Spores that developed during growth on ISP4 medium were harvested and inoculated into 300 ml of YEME in a 1.0 l baffled flask and cultured for 3 days at 28 1C. Equal volumes of the seed culture were inoculated into 25 baffled flasks containing 300 ml of YEME medium. Fermentation was subsequently carried out for 7 days at 28 1C and 160 r.p.m. The resulting culture (B8 l) was extracted twice with an equal volume of ethyl acetate (EtOAc). The extract was filtered through a fritted funnel and the resulting filtrate was evaporated in vacuo to yield the EtOAc extract. This extract was partitioned between EtOAc and water. The EtOAc-soluble material (2.7 g) was subjected to silica gel chromatography using a stepwise gradient elution of mixtures of CH2Cl2 and MeOH. The fraction eluted with CH2Cl2:MeOH of 85:15 was further purified by reverse-phase HPLC using YMC-J’sphere ODS-H80 (10 250 mm, 3 ml min 1; YMC, Kyoto, Japan) with a linear gradient from 30 to 100% acetonitrile (MeCN) containing 0.05% trifluoroacetic acid to yield DHQ7 (4; 31.8 mg) and DHQ8 (5; 17.6 mg). Compound 3 (317 mg) was purified from the fraction eluted with CH2Cl2:MeOH (90:10), subjected to passage over a Sephadex LH-20 (GE Healthcare, Buckinghamshire, UK) column using MeOH as eluant and further purified by HPLC (YMC J’sphere ODS-H80, 10 250 mm, MeOH-H2O (0.05% trifluoroacetic acid) gradient, 3 ml min 1).