Discovery of (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3- (phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662), a farnesyltransferase inhibitor with potent preclinical antitumor activity.

Continuing structure-activity studies were performed on the 2,3,4, 5-tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepine farnesyltransferase (FT) inhibitors. These studies demonstrated that a 3(R)-phenylmethyl group, a hydrophilic 7-cyano group, and a 4-sulfonyl group bearing a variety of substituents provide low-nanomolar FT inhibitors with cellular activity at concentrations below 100 nM. Maximal in vivo activity in the mutated K-Ras bearing HCT-116 human colon tumor model was achieved with analogues carrying hydrophobic side chains such as propyl, phenyl, or thienyl attached to the N-4 sulfonyl group. Several such compounds achieved curative efficacy when given orally in this model. On the basis of its excellent preclinical antitumor activity and promising pharmacokinetics, compound 20 (BMS-214662, (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thie nyl sulfonyl)-1H-1,4-benzodiazepine) has been advanced into human clinical trials.

3-Imidazolylmethylaminophenylsulfonyltetrahydroquinolines, a novel series of farnesyltransferase inhibitors.

Design, synthesis and structure-activity relationship of a series of 3-imidazolylmethylaminophenylsulfonyltetrahydroquinolines as farnesyltransferase inhibitors are presented. A working pharmacophore of inhibiting farnesyltransferase by this series of inhibitors is proposed.

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase.

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.

Ascosteroside, a new antifungal agent from Ascotricha amphitricha. I. Taxonomy, fermentation and biological activities.

Ascosteroside, a novel antifungal compound, was isolated from the culture broth of Ascotricha amphitricha. This compound is an alpha-linked glycoside of a lanostane type triterpenoid. It is active against yeasts such as Candida albicans and Saccharomyces cerevisiae and against filamentous fungi but shows no activity against bacteria. It is not toxic to mammalian cells at concentrations up to 150 microM. In a mouse model, the compound afforded protection comparable to that of ketoconazole.

Potent, cell active, non-thiol tetrapeptide inhibitors of farnesyltransferase.

All previously reported CAAX-based farnesyltransferase inhibitors contain a thiol functionality. We report that attachment of the 4-imidazolyl group, via 1-, 2-, or 3-carbon alkyl or alkanoyl spacers, to Val-Tic-Met or tLeu-Tic-Gln provides potent FT inhibitors. (R*)-N-[[1,2,3,4-Tetrahydro-2-[N-[2-(1H-imidazol-4-yl)ethyl] -L-valyl]-3-isoquinolinyl]carbonyl]-L-methionine ([imidazol- 4-yl-ethyl]-Val-Tic-Met), with FT IC50 = 0.79 nM, displayed potent cell activity in the absence of prodrug formation (SAG EC50 = 3.8 muM).

Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity.

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 = 1 nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methyleneamine isostere and the Tic replacement were incorporated. This derivative inhibited FT with an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-3T3 fibroblast cells by 50% at 5 microM. Replacing the A1 side chain of this derivative with a tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50 of 2.8 nM and an EC50 of 0.19 microM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-valyl]-1,2,3,4- tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This derivative, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4 - tetrahydro-3-isoquinolinyl]-carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3- mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3- isoquinolinyl]carbonyl]-L-methionine methyl ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive days, both compounds showed prolonged survival time (T/C = 142-145%), thus demonstrating efficacy against ras oncogene-containing tumors in vivo.

Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype.

Neurofibromatosis type I (NF1) is a hereditary tumor and developmental disorder whose defective gene was cloned previously. The protein product of the NF1 gene, neurofibromin, contains a domain that shows significant sequence homology to the known catalytic domains of mammalian Ras GTPase-activating proteins (GAP) and the yeast IRA1 and IRA2 proteins. This homologous region of neurofibromin has been shown to exhibit GAP activity toward Ras proteins. Malignant schwannoma cell lines from NF1 patients contain normal levels of GAP and nonmutated Ras proteins but barely detectable levels of neurofibromin, based on genetic mutations in the NF1 gene. Because these cells contain constitutively activated Ras.GTP, it has been proposed that neurofibromin may be the sole negative regulator of Ras in these cells. Overall, these results have implied an important role of the Ras signaling pathway in NF1 malignant schwannomas. Recently, several laboratories have developed small molecule inhibitors of Ras function that inhibit the enzyme farnesyltransferase (FT). FT-mediated post-translational farnesylation of Ras proteins is absolutely necessary for Ras function since this modification is required for the anchoring of Ras proteins to the plasma cell membrane. Although previous studies have shown that FT inhibitors can block the growth of tumor cells carrying mutant Ras proteins, it remained unclear how this class of inhibitors would affect tumor cells such as in NF1, whose malignant growth appears to be mediated by up-regulation of wild-type Ras activity. Thus, in the current study, we investigated whether BMS-186511, a bisubstrate analogue inhibitor of FT, would inhibit the malignant growth properties of a cell line established from malignant schwannoma of an NF1 patient. Our results indicate that the malignant growth properties of ST88-14 cells, the most malignant cell line among several well-characterized NF1 cells, are inhibited by BMS-186511 in a concentration-dependent manner. Following treatment with BMS-186511, ST88-14 cells became flat, nonrefractile, were contact-inhibited, and lost their ability to grow in soft agar. In the drug-exposed cells, Ras proteins were prevented from FT-mediated membrane association. BMS-186511 was found to specifically inhibit FT, but not geranylgeranyltransferase I, a closely related enzyme. Thus, it is conceivable that FT inhibitors may ultimately become the first generation of drugs against the malignant phenotype in NF1 based on rational insights into the mechanism of action of neurofibromin.

Bisubstrate inhibitors of farnesyltransferase: a novel class of specific inhibitors of ras transformed cells.

We describe the biological properties of a new class of potent farnesyltransferase (FT) inhibitors designed as bisubstrate analog inhibitors. These inhibitors incorporate the structural motifs of both farnesyl pyrophosphate and the CAAX tetrapeptide, the two substrates of the reaction catalyzed by FT. Both the phosphinate inhibitor, BMS-185878, and the phosphonate inhibitor, BMS-184467, exhibited higher in vitro FT selectivity than some of the previously reported CVFM peptidomimentics and benzodiazepine analogs. Xenopus oocyte maturation induced by microinjected oncogenic Ras proteins was blocked by coinjected BMS-184467 and BMS-185878. However, both inhibitors showed poor cell activity presumably because of the doubly charged nature of the compounds. Thus, masking the charge on the carboxylate ion markedly improved the cell permeability of BMS-185878, leading to BMS-186511, the methyl carboxyl ester prodrug. BMS-186511 inhibited FT activity in whole cells as determined by inhibition of p21 Ras protein processing, inhibition of farnesylation of proteins including Ras and the accumulation of unfarnesylated Ras proteins in the cytosolic fraction. While the cellular effects of these bisubstrate analog inhibitors had no significant effect on growth of untransformed NIH3T3 cells, they produced pronounced inhibition of Ras transformed cell growth. Both the anchorage dependent and independent growth of ras transformed cells were severely curtailed by micromolar concentrations of BMS-186511. We also found that both H-ras and K-ras transformed cells are affected by this bisubstrate inhibitor. However, K-ras transformed cells appear to be less sensitive. The inhibition of FT activity in cells and the ensuing inhibition of ras transformed cell growth is further manifested in distinct morphological changes in cells. Cells flattened, became less refractile and grew in contact inhibited monolayer. Moreover, the highly diffused character of the actin cytoskeleton in the ras transformed cells was dramatically reverted to an organized network of stress cables crisscrossing the entire cells upon treatment with BMS-186511. All of these effects of BMS-186511 are limited to ras transformed cells that utilize farnesylated Ras, but are not seen in transformed cells that use geranylgeranyl Ras or myristoyl Ras. Significantly, these FT inhibitors did not produce any signs of gross cytotoxicity in untransformed, ras transformed cells or other oncogene transformed cells.

Distribution of beta-lactam and beta-lactone producing bacteria in nature.

Over one million bacteria were isolated from a large variety of soil, plant and water samples collected from different environments and examined in an extremely sensitive and highly specific screen for beta-lactam production. A group of seven related monocyclic beta-lactams (monobactams) were isolated from strains representing four genera-Agrobacterium, Chromobacterium, Gluconobacter and Pseudomonas. Monobactam-producing strains of Agrobacterium and Pseudomonas were isolated only rarely. Producing strains of Chromobacterium were isolated from a relatively limited number of habitats while the Gluconobacter strains appeared to be widespread in nature. In addition, three closely related beta-lactone-containing molecules were isolated from strains representing three genera-Arthrobacter, Bacillus and Pseudomonas. The Bacillus and Pseudomonas strains were isolated infrequently but from a variety of samples. The producing strain of Arthrobacter was isolated only once.

Siderochelin, a new ferrous-ion chelating agent produced by Nocardia.

A new ferrous-ion chelating agent, siderochelin, was isolated from fermentation broths of Nocardia sp. SC 11,340. Siderochelin was produced by conventional submerged culture and purified by solvent extraction and recrystallization. The antibiotic was crystallized from acetonitrile as a mixture of diastereoisomers. The molecular formula of siderochelin was determined as C11H13N3O3 on the basis of elemental analysis and mass spectrometry, and the structure was elucidated by X-ray crystallography. The compound shows a broad spectrum of antimicrobial activity, being active against bacteria, fungi and protozoa.