Identification of 4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine Derivatives as Potent, Selective, and Orally Efficacious Inhibitors of the Copper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2).

LOXL2 catalyzes the oxidative deamination of ε-amines of lysine and hydroxylysine residues within collagen and elastin, generating reactive aldehydes (allysine). Condensation with other allysines or lysines drives the formation of inter- and intramolecular cross-linkages, a process critical for the remodeling of the ECM. Dysregulation of this process can lead to fibrosis, and LOXL2 is known to be upregulated in fibrotic tissue. Small-molecules that directly inhibit LOXL2 catalytic activity represent a useful option for the treatment of fibrosis. Herein, we describe optimization of an initial hit 2, resulting in identification of racemic-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone 28, a potent irreversible inhibitor of LOXL2 that is highly selective over LOX and other amine oxidases. Oral administration of 28 significantly reduced fibrosis in a 14-day mouse lung bleomycin model. The (R,R)-enantiomer 43 (PAT-1251) was selected as the clinical compound which has progressed into healthy volunteer Phase 1 trials, making it the "first-in-class" small-molecule LOXL2 inhibitor to enter clinical development.

Small Molecule Lysyl Oxidase-like 2 (LOXL2) Inhibitors: The Identification of an Inhibitor Selective for LOXL2 over LOX.

Two series of novel LOXL2 enzyme inhibitors are described: benzylamines substituted with electron withdrawing groups at the para-position and 2-substituted pyridine-4-ylmethanamines. The most potent compound, (2-chloropyridin-4-yl)methanamine 20 (hLOXL2 IC50 = 126 nM), was shown to be selective for LOXL2 over LOX and three other amine oxidases (MAO-A, MAO-B, and SSAO). Compound 20 is the first published small molecule inhibitor selective for LOXL2 over LOX.

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.

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.

Discovery of sodium R-(+)-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

5-Aryluracils as potent GnRH antagonists-Characterization of atropisomers.

Optimization of a series of uracils bearing a 2-fluoro- or 2-chloro-3-methoxyphenyl group at the 5-position resulted in compounds such as 3d and 3f with subnanomolar binding affinity at the human GnRH receptor. While the 2-fluoro-3-methoxyphenyl compound 3a was characterized as a mixture of interchangeable atropisomers, the diastereoisomers of 2-chloro-3-methoxyphenyl analogs were separated. It was found that the aR-atropisomer was much more potent than the aS-isomer based on the X-ray crystal structure of 3h-II.

Potent and orally bioavailable zwitterion GnRH antagonists with low CYP3A4 inhibitory activity.

Incorporation of a carboxylic acid into a series of uracil derivatives as hGnRH-R antagonists resulted in a significant reduction of CYP3A4 inhibitory activity. Highly potent hGnRH antagonists with low CYP3A4 inhibitory liability, such as 8a and 8d, were identified. Thus, 8a had a K(i) of 2.2 nM at GnRH-R and an IC(50) of 36 microM at CYP3A4.

Synthesis and structure-activity relationships of spirohydantoin-derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1).

The design, synthesis, and SAR of a series of substituted spirohydantoins are described. Optimization of an in-house screening hit gave compounds that exhibited potent binding affinity and functional activity at MCH-R1.

Thienopyrimidinone bis-aminopyrrolidine ureas as potent melanin-concentrating hormone receptor-1 (MCH-R1) antagonists.

A series of thienopyrimidinone bis-aminopyrrolidine ureas were designed, synthesized, and evaluated for their ability to bind melanin-concentrating hormone receptor-1. These compounds exhibit potent binding affinity (K(i)=3 nM) and good in vitro metabolic stability.

Synthesis and structure-activity relationships of retro bis-aminopyrrolidine urea (rAPU) derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1). Part 1.

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited potent binding affinity and functional activity at MCH-R1, and good oral bioavailability in rat.

Synthesis and structure-activity relationships of retro bis-aminopyrrolidine urea (rAPU) derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1). Part 2.

The design, synthesis, and SAR of a series of retro bis-aminopyrrolidine ureas are described. Compounds from this series exhibited considerable binding affinity (Ki = 1 nM) and functional activity at MCH-R1, acceptable CYP2D6 inhibition, and good rat brain exposure.

A thienopyridazinone-based melanin-concentrating hormone receptor 1 antagonist with potent in vivo anorectic properties.

Melanin-concentrating hormone receptor antagonists containing thieno- and a benzopyridazinone cores were designed and tested as potential anorectic agents. These ligands showed high affinity for the receptor, potent functional activity in vitro, and good oral bioavailabilty in rats. The thiophene analogue exhibited low iv clearance, long half-life, and high brain penetration. In obese rats, the thienopyridazinone demonstrated a dose-dependent reduction in feeding and body weight with doses between 1 and 10 mg kg-1.

Synthesis and structure-activity relationships of biarylcarboxamide bis-aminopyrrolidine urea derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1).

A novel series of bis-aminopyrrolidine ureas containing either a 4-biphenylcarboxmide or 5-phenyl-2-thiophenecarboxamide group have been identified as potent and functional antagonists of the melanin-concentrating hormone receptor-1. Syntheses and SAR are described, which led to the discovery of compounds with high binding affinity (Ki = 1 nM) for the receptor. Preliminary in vitro metabolic stability data are also reported for key compounds.

Structure-activity relationships of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists.

SAR studies of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists resulted in potent compounds. The best compound from the series had a binding affinity of 2 nM.

Uracils as potent antagonists of the human gonadotropin-releasing hormone receptor without atropisomers.

Uracil derivatives were designed and synthesized to avoid atropisomers observed in the 6-methyluracils as antagonists of the human GnRH receptor. Optimization at the 1- and 5-positions of the uracil resulted in potent compounds such as 24 (Ki=0.45 nM).

Bis(aminopyrrolidine)-derived ureas (APUs) as potent MCH1 receptor antagonists.

Ureas derived from two substituted 3-aminopyrrolidine subunits were prepared as constrained analogs of a linear lead compound and tested as antagonists of the MCH(1) receptor. The series was optimized for substitution and stereochemistry to generate a functional antagonist with a K(i) of 3.3 nM and IC(50) of 12 nM (GTPgammaS).

3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)- 6-methylpyrimidin-2,4-dione (NBI 42902) as a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor. Design, synthesis, and in vitro and in vivo characterization.

Further structure-activity relationship studies of a series of substituted uracils at the 1, 3, and 5 positions resulted in the discovery of several potent antagonists of the human gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylglycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-dione (R-13b, NBI 42902) displayed subnanomolar binding affinity (K(i) = 0.56 nM) and was a potent functional antagonist (IC(50) = 3.0 nM in Ca(2+) flux assay) at the human GnRH receptor. It also bound to the monkey GnRH receptor with high affinity (K(i) = 3.9 nM). In addition, R-13bhad good plasma exposure in cynomolgus monkeys after oral administration, with a C(max) of 737 ng/mL and an AUC of 2392 ng/mL.h at a 10 mg/kg dose. Moreover, oral administration of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum levels of luteinizing hormone. These results demonstrate that compounds from this series of uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman primates.

Synthesis and structure-activity relationships of uracil derived human GnRH receptor antagonists: (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils containing a substituted thiophene or thiazole at C-5.

The synthesis of a series of (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils containing a substituted thiophene or thiazole at C-5 is described. SAR around C-5 of the uracil led to the discovery that a 2-thienyl or (2-phenyl)thiazol-4-yl group is required for optimal receptor binding. The best compound from the series had a binding affinity of 2 nM (K(i)) for the human GnRH receptor. A novel and convenient preparation of N-1-(2,6-difluorobenzyl)-6-methyluracil is also described.

3-(2-aminoalkyl)-1-(2,6-difluorobenzyl)-5- (2-fluoro-3-methoxyphenyl)-6-methyl-uracils as orally bioavailable antagonists of the human gonadotropin releasing hormone receptor.

Uracils possessing N-3 side chains derived from various amino alcohols were designed and synthesized as potent human gonadotropin releasing hormone receptor antagonists. The compounds herein presented displayed superior metabolic stability than their predecessor molecules. Selected compounds from this series featured good oral bioavailability in mice and cynomolgus monkeys.