Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E.

The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway plays a central role in the regulation of cell growth, differentiation, and survival. Expression of mutant BRAF(V600E) results in constitutive activation of the MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe an SAR optimization campaign around a series of quinazoline derived BRAF(V600E) inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-butyl group with fluorinated alkyl moieties is described. This effort led directly to the identification of a clinical candidate, compound 40 (CEP-32496). Compound 40 exhibits high potency against several BRAF(V600E)-dependent cell lines and selective cytotoxicity for tumor cell lines expressing mutant BRAF(V600E) versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF(V600E)-dependent human Colo-205 tumor xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

4-Quinazolinyloxy-diaryl ureas as novel BRAFV600E inhibitors.

Aryl phenyl ureas with a 4-quinazolinoxy substituent at the meta-position of the phenyl ring are potent inhibitors of mutant and wild type BRAF kinase. Compound 7 (1-(5-tert-butylisoxazol-3-yl)-3-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)urea hydrochloride) exhibits good pharmacokinetic properties in rat and mouse and is efficacious in a mouse tumor xenograft model following oral dosing.

Design and synthesis of tetracyclic nonpeptidic biaryl nitrile inhibitors of cathepsin K.

The synthesis and biological profile of a novel series of potent and selective inhibitors of cysteine protease cathepsin K (Cat K) are described. Pharmacokinetic evaluation of 12 indicated that some members of this series could be suitable candidates to develop new orally active therapeutic agents for the treatment of osteoporosis.

Beta-substituted cyclohexanecarboxamide: a nonpeptidic framework for the design of potent inhibitors of cathepsin K.

A new series of nonpeptidic cathepsin K inhibitors that are based on a beta-substituted cyclohexanecarboxamide motif has been developed. Lead optimization yielded compounds with sub-nanomolar potency and exceptional selectivity profiles against cathepsins B, L, and S. Use of fluorine atoms to block metabolism on the cyclohexyl ring led to compounds with excellent pharmacokinetic properties. Considering the well-established role of cathepsin K in osteoclast-mediated bone turnover, compounds such as (-)-34a (hrab Cat K IC(50) 0.28 nM; >800-fold selectivity vs Cat B, L, and S; PK data in dogs: F 55%, t(1/2) = 15 h) exhibit great potential for development as an orally bioavailable therapeutic for treatment of diseases that involve bone loss.

Design and synthesis of tri-ring P3 benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin K.

We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

Synthesis and biological evaluation of penam sulfones as inhibitors of beta-lactamases.

The chemical synthesis of a series of new penam sulfone derivatives bearing a 2beta-substituted-oxyimino and -hydrazone substituents, their beta-lactamase inhibitory properties against selected enzymes representing class A and C beta-lactamases are reported. The oxime containing penam sulfones strongly inhibited the Escherichia coli TEM-1 and Klebsiella pneumoniae cefotaximase (CTX-1) enzymes, but moderately inhibited the Pseudomonas aeruginosa 46012 cephalosporinase; while the 2beta-substituted-hydrazone derivatives were generally less active against these enzymes. Furthermore, most of the inhibitors enhanced the antibacterial activities of piperacillin (PIP) and ceftazidime (CAZ) particularly against TEM-1 and CTX-1 producing bacterial strains.

3,4-disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. Exploring P3 elements for potency and selectivity.

The synthesis of a series of highly potent and selective inhibitors of cathepsin K based on the 3,4-disubstituted azetidin-2-one warhead is reported. A high degree of potency and selectivity was achieved by introducing a basic nitrogen into the distal part of the P3 element of the molecule. Data from kinetic and mass spectrometry experiments are consistent with the interpretation that compounds of this series transiently acylate the sulfhydrile of cathepsin K.

A novel class of nonpeptidic biaryl inhibitors of human cathepsin K.

A novel series of nonpeptidic biaryl compounds was identified as potent and reversible inhibitors of cathepsin K. The P2-P3 amide bond of a known amino acetonitrile dipeptide 1 was replaced with a phenyl ring, thereby giving rise to this biaryl series that retained potency vs cathepsin K and showed an improved selectivity profile against other cathepsins. Structural modification within this series resulted in the identification of compound (R)-2, a potent human cathepsin K inhibitor (IC(50) = 3 nM) that is selective versus cathepsins B (IC(50) = 3950 nM), L (IC(50) = 3725 nM), and S (IC(50) = 2010 nM). In an in vitro assay involving rabbit osteoclasts and bovine bone, compound (R)-2 inhibited bone resorption with an IC(50) of 95 nM. It was shown that, unlike some peptidic nitrile inhibitors of cysteine proteases, the nitrile moiety of (R)-2 is not converted to the corresponding amide 3 by cathepsin K. This indicates that this class of nonpeptidic nitrile inhibitors is unlikely to be hydrolyzed by cysteine proteases. Furthermore, the inhibition of cathepsin K by compound (R)-2 was shown to be fully reversible and not observably time-dependent. To demonstrate the efficacy of compound (R)-2 in vivo, it was administered to ovariectomized (OVX) rhesus monkeys at 20 mg/kg, po once daily for 8 days, and a urinary marker of bone turnover, N-telopeptide of type I collagen (uNTx), was measured. During the eight-day dosing period, the mean reduction by compound (R)-2 in uNTx was 80% (p < 0.001). This demonstrates that inhibition of cathepsin K leads to an inhibition of this bone resorption marker in OVX rhesus monkeys and strongly suggests that inhibition of cathepsin K is a viable therapeutic approach for the treatment of osteoporosis.

3,4-disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. Exploring P2 elements for selectivity.

A novel series of 3,4-disubstituted azetidinones based inhibitors of the cysteine protease cathepsin K (Cat K) has been identified. Although not optimized, some of these compounds show at least 100-fold selectivity against other cathepsins. The use of cyclic moieties as P2 elements has proven to be crucial to achieve a high degree of selectivity.