1-Thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives as antitumor agents.

A class of substituted 1-thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives was found to have potent anti-proliferative activity against a broad range of tumor cell lines. A compound from this class (14) was profiled across a broad panel of hematologic and solid tumor cancer cell lines demonstrating cell cycle arrest at the G0/G1 interphase and has potent anti-proliferative activity against a distinct and select set of cancer cell types with no observed effects on normal human cells. An example is the selective inhibition of human B-cell lymphoma cell line (BJAB). Compound 14 was orally bioavailable and tolerated well in mice. Synthesis and structure activity relationships (SAR) in this series of compounds are discussed.

Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo.

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.

Assessment of CYP3A-mediated drug-drug interaction potential for victim drugs using an in vivo rat model.

The present study aims to determine if an in vivo rat model of drug-drug interaction (DDI) could be useful to discriminate a sensitive (buspirone) from a 'non-sensitive' (verapamil) CYP3A substrate, using ketoconazole and ritonavir as perpetrator drugs. Prior to in vivo studies, ketoconazole and ritonavir were shown to inhibit midazolam hydroxylation with IC50 values of 350 ± 60 nm and 11 ± 3 nm, respectively, in rat liver microsomes (RLM). Buspirone and verapamil were also shown to be substrates of recombinant rat CYP3A1/3A2. In the rat model, the mean plasma AUC0-inf of buspirone (10 mg/kg, p.o.) was increased by 7.4-fold and 12.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. The mean plasma AUC0-inf of verapamil (10 mg/kg, p.o.) was increased by 3.0-fold and 4.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. Thus, the rat DDI model correctly identified buspirone as a sensitive CYP3A substrate (>5-fold AUC change) in contrast to verapamil. In addition, for both victim drugs, the extent of DDI when co-administered was greater with ritonavir compared with ketoconazole, in line with their in vitro CYP3A inhibition potency in RLM. In conclusion, our study extended the rat DDI model applicability to two additional victim/perpetrator pairs. In addition, we suggest that use of this model would increase our confidence in estimation of the DDI potential for victim drugs in early discovery.

A strategy to reduce biliary clearance in early drug discovery.

INTRODUCTION: Biliary excretion can modulate the pharmacokinetic profile of drug candidates, and may represent a liability for drug-drug interactions. This study proposes a strategy to reduce biliary clearance using the efflux ratio in Caco-2 cells in parallel to an abbreviated pharmacokinetic study in bile duct-cannulated rats (BDC). METHODS: Apical to basolateral (A to B) and basolateral to apical (B to A) permeability of 20 new chemical entities (NCEs) were determined in a 24-well permeability assay. In parallel, biliary excretion was determined in an abbreviated format in BDC rats. Test compounds were administered via an intravenous dose of 1 mg/kg and the percentage (%) of parent compound excreted in the bile in the first 3 hours after dosing was determined by LC-MS/MS analysis. RESULTS: A reasonably good correlation (r(2)=0.635) between the in vitro efflux ratio from the Caco-2 assay and in vivo biliary excretion of parent compound in BDC rats was observed. All seven compounds with an efflux ratio of <5 had less than 25% of the parent excreted in rat bile. In contrast, 3 out of the 13 compounds with an efflux ratio >5 had less than 25% of the dose excreted in rat bile. DISCUSSION: This suggests that a compound with an efflux ratio of <5 is at lower risk of having significant biliary clearance and that Caco-2 efflux ratio obtained from a high throughput screening assay may be used as an early indicator of biliary excretion. Although, we propose to reduce the occurrence of false positive prediction for biliary clearance (23%) by performing abbreviated PK in BDC rats for compounds with high efflux ratio.

Potent and selective steroidal inhibitors of 17beta-hydroxysteroid dehydrogenase type 7, an enzyme that catalyzes the reduction of the key hormones estrone and dihydrotestosterone.

17beta-Hydroxysteroid dehydrogenase type 7 (17beta-HSD7) catalyzes the reduction of estrone (E(1)) into estradiol (E(2)) and of dihydrotestosterone (DHT) into 5alpha-androstane-3beta,17beta-diol (3beta-diol), therefore modulating the level of mitogenic estrogens and androgens in humans. By classical and parallel chemistry, we generated several 4-methyl-4-aza-5alpha-androstane derivatives differing in their C-17 substituent: 17beta-formamide, 17beta-benzamide, and 17beta-tertiary amine. Best candidates in each category had demonstrated good inhibitory potency toward the conversion of E(1) into E(2) (IC(50) = 189-451 nM) and also toward the conversion of DHT into 3beta-diol (69-91% at 3 microM). Inhibition assays with 17beta-HSD1, 17beta-HSD5, 5alpha-reductase (5alpha-R) 1 and 5alpha-R2 revealed that 17beta-HSD7 inhibitors with a 4-methyl-4-aza nucleus were also able to inhibit 5alpha-Rs but not the other enzymes tested. Two 4-aza-5alpha-androstane inhibitors were, however, selective and still showed good inhibition of 17beta-HSD7. First selective and efficient inhibitors of 17beta-HSD7 are now available for additional mechanistic and therapeutic studies.