CEP-32496: a novel orally active BRAF(V600E) inhibitor with selective cellular and in vivo antitumor activity.

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non-small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAF(V600E) (K(d) BRAF(V600E) = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal-regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC(50) = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAF(V600E) versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10-55 mg/kg) of both pMEK and pERK in BRAF(V600E) colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30-100 mg/kg twice daily) against BRAF(V600E) melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development.

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.

Novel series of pyrrolotriazine analogs as highly potent pan-Aurora kinase inhibitors.

The synthesis and SAR for a novel series of pyrrolotriazines as pan-Aurora kinase inhibitors are described. Optimization of the cyclopropane carboxamide terminus of lead compound 1 resulted in analogs with high cellular activity and improved rat PK profiles. Notably, compound 17l demonstrated tumor growth inhibition in a mouse xenograft model.

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.

Identification of N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (AC220), a uniquely potent, selective, and efficacious FMS-like tyrosine kinase-3 (FLT3) inhibitor.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML).

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.

In vitro metabolic studies on the selective metabotropic glutamate receptor sub-type 5 (mGluR5) antagonist 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl]-pyridine (MTEP).

Metabotropic glutamate receptors (mGluR) are G-protein-coupled receptors that play a major role in modulatory pathways in the CNS and have been suggested to have pharmacological implications in pain, psychiatric disorders and other neurological states. 3-[(2-Methyl-1,3-thiazol-4-yl) ethynyl]-pyridine (MTEP) is a specific and selective antagonist for the mGluR sub-type 5. Previous studies using rat liver microsomes showed that the major oxidative metabolites of MTEP are a hydroxymethyl metabolite (M1), two oxides (M2 and M4), a thiazole-ring opened metabolite (M3) and CO(2) (M5). In the present study, we examined the metabolism of MTEP in liver microsomes and expressed rat and human CYP isoforms. In rat liver microsomes, metabolic stability studies accurately predicted the in vivo clearance for MTEP. Incubation of MTEP with expressed rat and human CYP isoforms showed that CYP1A and CYP2C isoforms are primarily responsible for the metabolism of this compound. The results suggest that species differences in MTEP metabolism is possible and could contribute to specie-differences in biological effects of the compound.

3-[Substituted]-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitriles: identification of highly potent and selective metabotropic glutamate subtype 5 receptor antagonists.

Structure-activity relationship studies on the phenyl ring of 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile 2 led to the discovery that small, non-hydrogen bond donor substituents at the 3-position led to a substantial increase in in vitro potency. In particular, 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (7) is a highly potent and selective mGlu5 receptor antagonist with good rat pharmacokinetics, brain penetration, and in vivo receptor occupancy.

Cyclohexenyl- and dehydropiperidinyl-alkynyl pyridines as potent metabotropic glutamate subtype 5 (mGlu5) receptor antagonists.

Structure-activity relationship studies leading to the discovery of novel mGlu5 receptor antagonists are described. These compounds show high in vitro potency, have good in vivo receptor occupancy, and a reasonable intravenous pharmacokinetic profile.

Influence of acid surrogates toward potency of VLA-4 antagonist.

A series of VLA-4 antagonist were synthesized wherein carboxylic acid was replaced by various acid surrogates. The effect of these acid surrogates toward potency was evaluated in a binding assay. A number of acid surrogates were potent antagonist of VLA-4, albeit significantly less potent than the corresponding carboxylic acid. Heterocyclic acid surrogate, oxadiazolidinone 3, demonstrated an improved pharmacokinetic property when dosed intravenously.

Biphenyl-indanones: allosteric potentiators of the metabotropic glutamate subtype 2 receptor.

We have identified and synthesized a series of biphenyl-carboxylic acid indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency and the brain to plasma ratio of the initial lead led to the discovery of 5 and 23 (EC50=111 and 5 nM, respectively).

Metabolism and disposition of a potent group II metabotropic glutamate receptor agonist, in rats, dogs, and monkeys.

Metabolism and disposition of MGS0028 [(1R,2S,5S,6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate], a potent group II metabotropic glutamate receptor agonist, were examined in three preclinical species (Sprague-Dawley rats, beagle dogs, and rhesus monkeys). In rats, MGS0028 was widely distributed and primarily excreted in urine as parent and as a single reductive metabolite, identified as the 4R-isomer MGS0034 [(1R,2S,4R,5S,6S)-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]-hexane-2,6-dicarboxylic acid]. MGS0028 had a low brain to plasma ratio at efficacious doses in rats and was eliminated more slowly in rat brain than in plasma. Exposure increased proportionally (1--10 mg/kg p.o.) in rats, with bioavailability>60% at all doses. However, bioavailability was only approximately 20% in monkeys, and MGS0034 was found in relatively high abundance in plasma. In dogs, oral bioavailability was >60%, and the metabolite was not detected. In vitro metabolism was examined in liver subcellular fractions (microsomes and cytosol) from rat, dog, monkey, and human. Reductive metabolism was observed in rat, monkey, and human liver cytosol incubations, but not in dog liver cytosol incubations. No metabolism of MGS0028 was detected in incubations with liver microsomes from any species. Similar to in vivo results, MGS0028 was reduced in cytosol stereospecifically to MGS0034. The rank order of in vitro metabolite formation (monkey >> rat approximately human >> dog) was in agreement with in vivo observations in rats, dogs, and monkeys. Based on the observation of species difference in reductive metabolism, rat and monkey were recommended to be the preclinical species for further characterization prior to testing in humans. Finally, allometric scaling predicts that human pharmacokinetic parameters would be acceptable for further development.

Water soluble prodrug of a COX-2 selective inhibitor suitable for intravenous administration in models of cerebral ischemia.

A water soluble choline prodrug (17) of a COX-2 selective inhibitor (16) suitable for intravenous dosing in models of cerebral ischemia has been developed. Constant infusion studies using 17 demonstrate that extrapolated brain levels of 16 may be maintained for over 24h in rats.

2-(2-[3-(pyridin-3-yloxy)phenyl]-2H-tetrazol-5-yl) pyridine: a highly potent, orally active, metabotropic glutamate subtype 5 (mGlu5) receptor antagonist.

Structure-activity relationship studies on 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile 2 led to the discovery of 2-(2-[3-(pyridin-3-yloxy)phenyl]-2H-tetrazol-5-yl)pyridine (10)-a highly potent and selective mGlu5 receptor antagonist with good brain penetration and in vivo receptor occupancy in rat and cross-species oral bioavailability.

3-[3-Fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine: a highly potent and orally bioavailable metabotropic glutamate subtype 5 (mGlu5) receptor antagonist.

Structure-activity relationship studies performed around 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile for the purpose of developing novel mGlu5 receptor antagonists are described. Synthesis of a series of four-ring tetrazoles led to the discovery of 3-[3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine, a highly potent, brain penetrant, azole-based mGlu5 receptor antagonist.

Discovery of highly potent, selective, orally bioavailable, metabotropic glutamate subtype 5 (mGlu5) receptor antagonists devoid of cytochrome P450 1A2 inhibitory activity.

Structure-activity relationship studies focused on bio-isosteric replacements of 2-pyridyl resulted in mGlu5 receptor antagonists with reduced inhibition of cytochrome P450 1A2. This led to highly potent, selective and orally bioavailable 2-imidazolyl tetrazoles such as (10) that are devoid of cytochrome P450 inhibitory activity.

5-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-2,3'-bipyridine: a highly potent, orally active metabotropic glutamate subtype 5 (mGlu5) receptor antagonist with anxiolytic activity.

Structure-activity relationship studies leading to the discovery of a new, orally active mGlu5 receptor antagonist are described. The title compound, 5-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-2,3'-bipyridine, is highly potent in vitro, has good in vivo receptor occupancy, and is efficacious in the rat fear-potentiated startle model of anxiety following oral dosing.

Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity ligands to the alpha 2 delta subunit of voltage gated calcium channels.

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the alpha2delta subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished binding to the alpha2delta subunit. Modifications to the pyridazine ring revealed numerous permissive substitutions, and detailed SAR studies were carried out in this portion of the molecule. Replacement of the pyridazine ring methyl group with an aminomethyl functionality provided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and was shown to be efficacious in the Chung model for neuropathic pain in rats.