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Purpose: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. Methods: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. Results: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. Conclusions: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

We examined the chemoprotective effects of KF41399, a novel derivative of carbazole compounds, on severe thrombocytopenia induced by nimustine (ACNU, 45 mg/kg administered for 2 consecutive days intravenously) in mice. Administration schedule studies revealed that pretreatment of mice with KF41399 was necessary to improve thrombocytopenia. Oral administration of KF41399 ameliorated thrombocytopenia induced by ACNU and accelerated the rate of platelet recovery in a dose-dependent fashion. In addition, KF41399 pretreatment improved the decrease in body weight and spleen weight and in the colony-forming activity of bone marrow mononuclear cells (MNC). Oral administration of KF41399 to normal mice induced G0/G1-phase accumulation of MNC as well as hematopoietic progenitor cells (lineage negative cells [Lin−]) and reduced the colony-forming activity of MNC. In Lin− cells derived from KF41399-treated mice, up-regulation of Bcl-2 and down-regulation of cyclin E and cyclin A proteins were observed. In the same cells, a decrease in the phosphorylated form of Rb protein and an increase in the p130 protein were observed without changes in the protein level of cell cycle-dependent kinase 2 (Cdk2), Cdk4, and Cdk6. More important, KF41399 did not affect the antitumor activity of ACNU against mouse Sarcoma180 and human lung cancer LC-6. However, 25-mg/kg KF41399 treatment reduced the antitumor activity of ACNU against human lung cancer Lu-65, and 5 mg/kg KF41399 caused a slight reduction of the antitumor activity of ACNU without inducing thrombocytopenia. These results suggest that KF41399 might be useful as a chemoprotective agent to improve chemotherapy-induced thrombocytopenia and types of other toxicity.

Novel compounds MS-681a, b, c and d were isolated from the culture broth of a fungal strain KY6568. The strain was identified as Myrothecium sp. from its morphological characteristics. MS-681a, b, c and d inhibited the activity of purified smooth muscle myosin light chain kinase with IC50 values of 0.11, 0.29, 0.095 and 0.26 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited at 100 microM by MS-681 compounds.

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.

The mechanism of the BF 3 •etherate-catalyzed rearrangement of fucosterol benzoate 24,28-epoxide leading to desmosterol benzoate and two carbonyl compounds was investigated

Radicicol (1), a macrocyclic antifungal antibiotic, is the lead compound of a novel class of heat shock protein 90 (Hsp90) inhibitors that result in the inhibition or degradation of Hsp90-associated proteins, such as v-src and Raf-1 kinases. New O-carbamoylmethyloxime derivatives of 1 were synthesized and evaluated for their in vitro antiproliferative activities against v-src- and K-ras-transformed cells and for their inhibitory activity against v-src tyrosine kinase. O-(Piperidinocarbonyl)methyloxime 9b, one of the most potent of these derivatives, exhibited more potent antiproliferative activity than 1 and its hydroxime KF25706 (2) and had an IC(50) of 25 nM for the inhibition of v-src kinase activity. Compound 9b was also found to decrease the Raf-1 protein level of KNRK5.2 cells. Furthermore, compound 9b exhibited significant antitumor activity when tested against MX-1 and A431 xenografts in nude mice.

MS-681a, b, c and d are new inhibitors of myosin light chain kinase produced by Myrothecium sp. They are novel octapeptides containing alpha-alkyl-alpha-amino acids and a polyamine moiety. The structures were determined by NMR and FAB-MS/MS spectral analyses of the intact peptides. Their absolute configurations were elucidated by GC analysis on a chiral column of the constituent amino acids and by chemical synthesis of the polyamine moieties.

The C-25 stereochemistry of the reaction catalysed by sterol-Δ24-reductase from the insect, B. mori, has been investigated by the use of 13C NMR spectroscopy; incubation of isopropylidene (E)-methyl-13C labelled desmosterol afforded cholesterol with the label at pro-R-methyl of the isopropyl group, and the results indicated the C-25 hydrogen was introduced stereospecifically from the si-face of the double bond.

The fate of the diastereotopic methyl groups on the prochiral C-25 centre of fucosterol epoxide and isofucosterol epoxide during enzymatic conversion into desmosterol has been investigated. Incubation of stereospecifically 13C-labelled fucosterol (24R, 28R)-epoxides and isofucosterol (24R, 28S)-epoxides with a cell-free preparation obtained from the guts of larvae of the silkworm, Bombyx mori, followed by 13C NMR analysis of the product has shown that the reaction is stereospecific, where the pro-S and pro-R methyl groups of the epoxides turn stereospecifically into (Z)- and (E)-methyl groups of desmosterol, respectively.

We report a) a modified synthesis of diketo selenides, b) preparation of 2,5-diacylselenophenes by condensation of diketo selenides with glyoxal, c) preparation of poly-substituted selenophenes by intramolecular reductive coupling reaction of diketo selenides with low-valent titanium reagent under controlled conditions (0°C) followed by dehyration of the resulting 3,4-dihydroxyselenolanes, and d) formation of 2,5-dihydroselenophenes and 1,3-dienes by treatment of diketo selenides with the low-valent titanium reagent in refluxing tetrahydrofuran.

Reduction of a series of diketo selenides with a low-valent titanium reagent affords 2,5-dihydroselenophenes and 1,3-dienes in comparable yields.

We report a) a modified synthesis of diketo selenides, b) preparation of 2,5-diacylselenophenes by condensation of diketo selenides with glyoxal, c) preparation of poly-substituted selenophenes by intramolecular reductive coupling reaction of diketo selenides with low-valent titanium reagent under controlled conditions (0°C) followed by dehyration of the resulting 3,4-dihydroxyselenolanes, and d) formation of 2,5-dihydroselenophenes and 1,3-dienes by treatment of diketo selenides with the low-valent titanium reagent in refluxing tetrahydrofuran.

Reduction of a series of diketo selenides with a low-valent titanium reagent affords 2,5-dihydroselenophenes and 1,3-dienes in comparable yields.

The conversion of fucosterol epoxide (1) into desmosterol (2), by the cell-free preparation obtained from the guts of larvae of the silkworm, Bombyx mori, is stereospecific, where the isopropyl pro-R and pro-S methyl groups of (1) turn stereospecifically into (E)- and (Z)-methyl groups, respectively, of (2).

The mechanism of the BF3-catalysed rearrangement of an acyclic trisubstituted epoxide to a carbonyl compound has been studied using (24S,25S)-[26-13C] and (24R,25R)-[26-13C]-desmosterol benzoate 24,25-epoxides (2) and (3), demonstrating that C-24 hydrogen migration leading to the 24-oxo compound (4) occurs with retention of configuration at the migration terminus (C-25), whereas C-23 alkyl group migration leading to aldehyde (5) proceeds with inversion of configuration at C-25.