3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 2. Lead optimization.

Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K(i) of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC(50) in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.

Optimization of diarylthiazole B-raf inhibitors: identification of a compound endowed with high oral antitumor activity, mitigated hERG inhibition, and low paradoxical effect.

Aberrant activation of the mitogen-activated protein kinase (MAPK)-mediated pathway components, RAF-MEK-ERK, is frequently observed in human cancers and clearly contributes to oncogenesis. As part of a project aimed at finding inhibitors of B-Raf, a key player in the MAPK cascade, we originally identified a thiazole derivative endowed with high potency and selectivity, optimal in vitro ADME properties, and good pharmacokinetic profiles in rodents, but that suffers from elevated hERG inhibitory activity. An optimization program was thus undertaken, focused mainly on the elaboration of the R(1) and R(2) groups of the scaffold. This effort ultimately led to N-(4-{2-(1-cyclopropylpiperidin-4-yl)-4-[3-(2,5-difluorobenzenesulfonylamino)-2-fluorophenyl]thiazol-5-yl}-pyridin-2-yl)acetamide (20), which maintains favorable in vitro and in vivo properties, but lacks hERG liability. Besides exhibiting potent antiproliferative activity against only cell lines bearing B-Raf V600E or V600D mutations, compound 20 also intriguingly shows a weaker "paradoxical" activation of MEK in non-mutant B-Raf cells than other known B-Raf inhibitors. It also demonstrates very good efficacy in vivo against the A375 xenograft melanoma model (tumor volume inhibition >90% at 10 mg kg(-1) ); it is therefore a suitable candidate for preclinical development.

3-Aminopyrazole inhibitors of CDK2/cyclin A as antitumor agents. 1. Lead finding.

Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.

Identification of potent pyrazolo[4,3-h]quinazoline-3-carboxamides as multi-cyclin-dependent kinase inhibitors.

Abnormal proliferation mediated by disruption of the mechanisms that keep the cell cycle under control is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDKs) and cyclins (Cy) and inhibiting their activity are regarded as promising antitumor agents to complement the existing therapies. An expansion of pyrazolo[4,3-h]quinazoline chemical class oriented to the development of three points of variability was undertaken leading to a series of compounds able to inhibit CDKs both in vitro and in vivo. Starting from the CDK selective but poorly soluble hit compound 1, we succeeded in obtaining several compounds showing enhanced inhibitory activity both on CDKs and on tumor cells and displaying improved physical properties and pharmacokinetic behavior. Our study led to the identification of compound 59 as a highly potent, orally bioavailable CDK inhibitor that exhibited significant in vivo efficacy on the A2780 ovarian carcinoma xenograft model. The demonstrated mechanisms of action of compound 59 on cancer cell lines and its ability to inhibit tumor growth in vivo render this compound very interesting as potential antineoplastic agent.

Identification of N,1,4,4-tetramethyl-8-{[4-(4-methylpiperazin-1-yl)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (PHA-848125), a potent, orally available cyclin dependent kinase inhibitor.

The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a panel of serine-threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.