OA19542A

OA19542A - Apoptosis Signal-Regulating Kinase Inhibitor.

Info

Publication number: OA19542A
Application number: OA1201400328
Authority: OA
Inventors: Gregory Notte
Original assignee: Gilead Sciences Inc.
Priority date: 2012-01-27
Filing date: 2013-01-24
Publication date: 2020-12-11

Abstract

The present invention relates to a compound of formula (I): The compound has apoptosis signal-regulating kinase ("ASK 1") inhibitory activity, and is thus useful in the treatment of diseases such as kidney disease, diabetic nephropathy and kidney fibrosis.

Description

APOPTOSIS SIGNAL-REGULATING KINASE INHIBITOR

FIELD OF THE INVENTION

The présent invention relates to a novel compound for use in the treatment of ASK1mediated diseases. The invention also relates to intermediates for its préparation and to pharmaceutical compositions containing said novel compound.

BACKGROUND

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase (“MAP3K”) family that activâtes the c-Jun N-terminal protein kinase (“JNK”) and p38 MAP kinase (Ichijo, H., Nishida, E., Irie, K., Dijke, P. T., Saitoh, M., Moriguchi, T., Matsumoto, K., Miyazono, K., and Gotoh, Y. (1997) Science, 275, 90-94). ASK1 is activated by a variety of stimuli including oxidative stress, reactive oxygen species (ROS), LPS, TNF-a, FasL, ER stress, and increased intracellular calcium concentrations (Hattori, K., Naguro, L, Runchel, C., and Ichijo, H. (2009) CellComm. Signal. 7:1-10; Takeda, K., Noguchi, T., Naguro, L, and Ichijo, H. (2007) Annu. Rev. Pharmacol. Toxicol. 48: 1-8.27; Nagai, H., Noguchi, T., Takeda, K., and Ichijo, I. (2007) J. Biochem. Mol. Biol. 40:1-6). Phosphorylation of ASK1 protein can lead to apoptosis or other cellular responses depending on the cell type. ASK1 activation and signaling hâve been reported to play an important rôle in a broad range of diseases including neurodegenerative, cardiovascular, inflammatory, auto immune, and metabolic disorders. In addition, ASK1 has been implicated in mediating organ damage following ischemia and reperfusion of the heart, brain, and kidney (Watanabe et al. (2005) BBRC 333, 562-567; Zhang et al., (2003) Life Sci 74-37-43; Terada et al. (2007) BBRC364; 1043-49).

ROS are reported be associated with increases of inflammatory cytokine production, fîbrosis, apoptosis, and necrosis in the kidney. (Singh DK, Winocour P, Farrington K. Oxidative stress in early diabetic nephropathy: fueling the fïre. Nat Rev Endocrinol 2011 Mar;7(3): 176184; Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001 Dec 13; 414(6865):813-820; Mimura I, Nangaku M. The suffocating kidney: tubulointerstitial hypoxia in end-stage rénal disease. Nat Rev Nephrol 2010 Nov; 6(11):667678).

Moreover, oxidative stress facilitâtes the formation of advanced glycation end-products (AGEs) that cause further rénal injury and production of ROS. (Hung KY, et al. N acetylcysteine-mediated antioxidation prevents hyperglycemia-induced apoptosis and collagen synthesis in rat mesangial cells. Am JNephrol 2009;29(3): 192-202).

Tubulointerstitial fibrosis in the kidney is a strong predictor of progression to rénal failure in patients with chronic kidney diseases (Schainuck LI, et al. Structural-functional corrélations in rénal disease. Part II: The corrélations. Hum Pathol 1970; 1: 631-641.). Unilatéral urétéral obstruction (UUO) in rats is a widely used model of tubulointerstitial fibrosis. UUO causes tubulointerstital inflammation, increased expression of transforming growth factor beta (TGF-β), and accumulation of myofibroblasts, which secrete matrix proteins such as collagen and fibronectin. The UUO model can be used to test for a drug’s potential to treat chronic kidney disease by inhibiting rénal fibrosis (Chevalier et al., Urétéral obstruction as a model of rénal interstitial fibrosis and obstructive nephropathy, Kidney International (2009) 75, 1145-1152.

Thus, therapeutic agents that function as inhibitors of ASK1 signaling hâve the potential to remedy or improve the lives of patients in need of treatment for diseases or conditions such as neurodegenerative, cardiovascular, inflammatory, autoimmune, and metabolic disorders. In particular, ASK1 inhibitors hâve the potential to treat cardio-renal diseases, including kidney disease, diabetic kidney disease, chronic kidney disease, fibrotic diseases (including lung and kidney fibrosis), respiratory diseases (including chronic obstructive pulmonary disease (COPD) and acute lung injury), acute and chronic liver diseases.

U.S. Publication No. 2007/0276050 describes methods for identifying ASK1 inhibitors useful for preventing and/or treating cardiovascular disease and methods for preventing and/or treating cardiovascular disease in an animal.

WO2009027283 discloses triazolopyridine compounds, methods for préparation thereof and methods for treating autoimmune disorders, inflammatory diseases, cardiovascular diseases and neurodegenerative diseases.

U.S. Patent Publication No. 2001/00095410A1, published January 13, 2011, discloses compounds useful as ÀSK-1 inhibitors. U.S. Patent Publication No. 2001/00095410Â1 relates to compounds of Formula (I):

(I) wherein:

R¹ is alkyl, alkenyl, alkynyl, cycloaikyl, aryl, heteroaryl, or heterocyclyl, ail of which are optionally substituted with 1, 2, or 3 substituents selected from halo, oxo, alkyl, cycloaikyl, heterocyclyl, aryl, aryloxy, -NO2, R⁶, -C(O)-R⁶, -OC(O)-R⁶ -C(O)-O-R⁶, C(O)-N(R⁶)(R⁷), -OC(O)-N(R⁶)(R⁷), -S-R⁶, -S(=O)-R⁶, -S(=O)2R⁶, -S(=O)2-N(R⁶)(R⁷), -S(=O)2-O-R⁶, -N(R⁶)(R⁷), -N(R⁶)-C(O)-R⁷, -N(R⁶)-C(O)-O-R⁷, -N(R⁶)-C(O)N(R⁶)(R⁷), -N(R⁶)-S(=O)₂-R⁶, -CN, and-O-R⁶, wherein alkyl, cycloaikyl, heterocyclyl, phenyl, and phenoxy are optionally substituted by 1, 2, or 3 substituents selected from alkyl, cycloaikyl, alkoxy, hydroxyl, and halo; wherein R⁶ and R⁷ are independently selected from the group consisting of hydrogen, CiC15 alkyl, cycloaikyl, heterocyclyl, aryl, and heteroaryl, ail of which are optionally substituted with 1 -3 substituents selected from halo, alkyl, mono- or dialkylamino, alkyl or aryl or heteroaryl amide, -CN, lower alkoxy, -CF3, aryl, and heteroaryl; or R⁶ and R⁷ when taken together with the nitrogen to which they are attached form a heterocycle;

R² is hydrogen, halo, cyano, alkoxy, or alkyl optionally substituted by halo;

R³ is aryl, heteroaryl, or heterocyclyl, ail of which are optionally substituted with one or more substituents selected from alkyl, alkoxy, cycloaikyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, halo, oxo, -NO2, haloalkyl, haloalkoxy, -CN, -O-R⁶, -O-C(O)-R⁶, -O-C(O)-N(R⁶)(R⁷), -S-R⁶, N(R⁶)(R⁷), -S(=O)-R⁶, -S(=O)2R⁶, -S(=O)2-N(R⁶)(R⁷), -S(=O)2-O-R⁶, -N(R⁶)-C(O)R⁷, -N(R⁶)-C(O)-O-R⁷, -N(R⁶)-C(O)-N(R⁶)(R⁷), -C(O)-R⁶, -C(O)-O-R⁶, -C(O)N(R⁶)(R⁷), and -N(R⁶)-S(=O)2-R⁷, wherein the alkyl, alkoxy, cycloaikyl, aryl, heteroaryl or heterocyclyl is further optionally substituted with one or more substituents selected from halo, oxo, -NO2, alkyl, haloalkyl, haloalkoxy, -N(R⁶)(R⁷), -C(O)-R⁶, 3

C(O)-O-R⁶, -C(O)-N(R⁶)(R⁷), -CN, -O-R⁶, cycloalkyl, aryl, heteroaryl and heterocyclyl;

with the proviso that the heteroaryl or heterocyclyl moiety includes at least one ring nitrogen atom;

X¹, X², X³, X⁴, X⁵, X⁶, X⁷ and X⁸ are independently C(R⁴) or N, in which each R⁴ is independently hydrogen, alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, NO2, haloalkyl, haloalkoxy, -CN, -O-R⁶, -S-R⁶, -N(R⁶)(R⁷), -S(=O)-R⁶, -S(=O)2R⁶, -S(=O)2-N(R⁶)(R⁷), -S(=O)2-O-R⁶, -N(R⁶)-C(O)-R⁷, -N(R⁶)-C(O)-O-R⁷, -N(R⁶)-C(O)N(R⁶)(R⁷), -C(O)-R⁶, -C(O)-O-R⁶, -C(O)-N(R⁶)(R⁷), or -N(R⁶)-S(=O)2-R⁷, wherein the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is further optionally substituted with one or more substituents selected from halo, oxo, -NO2, -CF3, -O-CF₃, -N(R⁶)(R⁷), -C(O)-R⁶, -C(O)-O-R⁷, -C(O)-N(R⁶)(R⁷), -CN, -O-R⁶; or

X and X or X and X are joined to provide optionally substituted fused aryl or optionally substituted fused heteroaryl; and with the proviso that at least one of X², X³, and X⁴ is C(R⁴);

at least two of X⁵, X⁶, X⁷, and X⁸ are C(R⁴); and at least one of X², X³, X⁴, X⁵, X⁶, X⁷ and X⁸ is N.

The above disclosures notwithstanding, there is a need for compounds that are potent and exhibit improved pharmacokinetic and/or pharmacodynamie profiles for the treatment of diseases related to ASK1 activation.

Surprisingly, applicants hâve discovered a novel compound within the scope of U.S. patent publication US2011/0009410A exhibiting good potency, improved pharmacokinetic and/or pharmacodynamie profiles, on aggregate, compared to compounds disclosed therein.

SUMMARY OF THE INVENTION

The présent invention relates to a compound of the formula:

(I), or a pharmaceutically acceptable sait thereof^

In one embodiment, the invention relates to the use of a compound of formula (I) in the treatment of a disease in a patient in need of treatment with an ASK1 inhibitor.

In another embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable sait thereof, and one or more pharmaceutically acceptable carriers.

In another embodiment, the invention is a method of treating diabetic nephropathy, or complications of diabètes, comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable sait thereof, to a patient in need thereof.

In another embodiment, the invention relates to a method of treating kidney disease, or diabetic kidney disease comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable sait thereof, to a patient in need thereof.

In another embodiment, the invention relates to a method of treating kidney fîbrosis, lung fibrosis, or idiopathic pulmonary fîbrosis (IPF) comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable sait thereof, to a patient in need thereof.

In another embodiment, the invention relates to a method of treating diabetic kidney disease, diabetic nephropathy, kidney fibrosis, liver fibrosis, or lung fibrosis comprising administering a therapeutically effective amount of a compound or sait of forumia (I), to a patient in need thereof.

In another embodiment, the invention relates to intermediates useful for the synthesis of the compound of formula (I).

In another embodiment, the invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable sait thereof for the treatment of chronic kidney disease.

In another embodiment, the invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable sait thereof for the treatment of diabetic kidney disease.

In another embodiment, the invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable sait thereof, in the manufacture of a médicament for the treatment of chronic kidney disease. <7^

In yet another embodiment, the invention relates to the compound of formula (I) for use in therapy.

DETAILED DESCRIPTION OF THE INVENTION

Figures

Figure 1 is a bar graph showing the levels of Collagen IV in the kidney cortex of rats subjected to seven days of unilatéral urétéral obstruction and treated with either vehicle, or compound of formula (I) at 1, 3, 10, or 30 mg/kg b.i.d. per day.

Figure 2 shows représentative images of kidney cortex sections stained with alphasmooth muscle actin (a marker of activated myofibroblasts) from rats subjected to seven days of unilatéral urétéral obstruction and treated with either vehicle, or compound of formula (I) at 1, 3, 10, or 30 mg/kg b.i.d. per day.

Définitions and General Parameters

As used herein, the following words and phrases are intended to hâve the meanings set forth below, except to the extent that the context in which they are used indicates otherwise. Where no indication or définition is given, the ordinary meaning of the word or phrase as found in a relevant dictionary or in common usage known to one of skill in the art is implied.

The term “chronic kidney disease” as used herein refers to progressive loss of kidney function over time typically months or even years. Chronic kidney disease (CKD) is diagnosed by a competent care giver using appropriate information, tests or markers known to one of skill in the art. Chronic kidney disease includes by implication kidney disease.

The term “diabetic kidney disease” as used herein refers to kidney disease caused by diabètes, exacerbated by diabètes, or co-presenting with diabètes. It is a form of chronic kidney disease occurring in approximately 30% of patients with diabètes. It is defined as diabètes with the presence of albuminuria and/or impaired rénal function (i.e. decreased glomerular filtration rate ( See. de B, I, et al. Temporal trends in the prevalence of diabetic kidney disease in the United States. JAMA 2011 Jun 22; 305(24):2532-2539).

The term “pharmaceutically acceptable sait” refers to salts of pharmaceutical compounds e.g. compound of formula (I) that retain the biological effectiveness and properties of the underlying compound, and which are not biologically or otherwise undesirable. There are acict>

addition salts and base addition salts. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids.

Acids and bases useful for reaction with an underlying compound to form pharmaceutically acceptable salts (acid addition or base addition salts respectively) are known to one of skill in the art. Similarly, methods of preparing pharmaceutically acceptable salts from an underlying compound (upon disclosure) are known to one of skill in the art and are disclosed in for example, Berge, at al. Journal of Pharmaceutical Science, Jan. 1977 vol. 66, No.l, and other sources. Salts derived from inorganic acids include but are not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include but are not limited to maleic acid, fumaric acid, tartaric acid, ptoluene-sulfonic acid, and the like. Bases useful for forming base addition salts are known to one of skill in the art. An example of a pharmaceutically acceptable sait of the compound of formula (I) is the hydrochloride sait of the compound of formula (I).

As used herein, “pharmaceutically acceptable carrier” includes excipients or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonie and absorption delaying agents and the like that are not deleterious to the compound of the invention or use thereof. The use of such carriers and agents to préparé compositions of pharmaceutically active substances is well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modem Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker & C.T. Rhodes, Eds.)

The term “cardio-renal diseases” as used herein refers to diseases, related to the function of the kidney, that are caused or exacerbated by cardiovascular problems such as, for example, high blood pressure or hypertension. It is believed that hypertension is a major contributor to kidney disease.

The term “respiratory diseases” as used herein refers to diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

The term “therapeutically effective amount” refers to an amount of the compound of formula (I) that is sufficient to effect treatment as defined below, when administered to a patient (particularly a human) in need of such treatment in one or more doses. The therapeutically effective amount will vary, depending upon the patient, the disease being treated, the weight and/or âge of the patient, the severity of the disease, or the manner of administration, as determined by a qualified prescriber or care giver.

The term “treatment” or “treating” means administering a compound or pharmaceutically acceptable sait of formula (I) for the purpose of:

(i) delaying the onset of a disease, that is, causing the clinical symptoms of the disease not to develop or delaying the development thereof;

(ii) inhibiting the disease, that is, arresting the development of clinical symptoms; and/or (iii) relieving the disease, that is, causing the régression of clinical symptoms or the severity thereof.

In a preferred embodiment, the invention relates to the use of the compound of formula (I) in treating chronic kidney disease comprising administering a therapeutically effective amount to a patient in need thereof.

In another preferred embodiment the invention relates to the use of the compound of formula (I) in treating diabetic kidney disease comprising administering a therapeutically effective amount to a patient in need thereof.

In another preferred embodiment the invention relates to the use of the compound of formula (I) in treating lung or kidney fibrosis comprising administering a therapeutically effective amount to a patient in need thereof.

The half maximal inhibitory concentration (IC50) of a therapeutic agent is the concentration of a therapeutic agent necessary to produce 50% of the maximum inhibition against a target enzyme. It is a désirable goal to discover a therapeutic agent, for example a compound that inhibits apoptosis signal-regulating kinase (ASK1) with a low IC50. In this manner, undesirable side effects are minimized by the ability to use a lower dose of the therapeutic agent to inhibit the ASK1 enzyme.

Similarly, it is a désirable goal to discover a therapeutic agent that has a low dissociation constant (Ka). Kj is used to describe the affinity between a ligand (such as a therapeutic agent) and the corresponding kinase or receptor; i.e. a measure of how tightly a therapeutic agent binds to a particular kinase, for example the apoptosis signal-regulating kinase ASK1. Thus, a lower Ka is generally preferred in drug development.

Similarly, it is a désirable goal to discover a compound having a low EC₅o· EC50 is the concentration of a drug that achieves 50% maximal effîcacy in the cell. The EC50 value translates to the concentration of a compound in the assay medium necessary to achieve 50% of the maximum effîcacy. Thus, a lower EC50 is generally preferred for drug development.

A useful unit of measure associated with EC50 is the protein binding adjusted EC50 (PBadj.ECso as used herein). This value measures the amount of a drug e.g. compound of formula (I) correlated to the fraction of the drug that is unbound to protein which provides 50% maximal efficacy. This value measures the efficacy of the drug corrected for or correlated to the amount of drug that is available at the target site of action.

Another désirable property is having a compound with a low cell membrane efflux ratio as determined by CACO cell permeability studies. An efflux ratio ((B/A) / (A/B)) less than 3.0 is preferred. A compound with a ratio greater than 3 is expected to undergo active rapid efflux from the cell and may not hâve suffïcient duration in the cell to achieve maximal efficacy.

Another désirable goal is to discover a drug that exhibits minimal off-target inhibition. That is, a drug that minimally inhibits the Cyp450 (cytochrome p450) enzymes. More particularly, a drug that is a weak inhibitor of cyp3A4, the most important of the P450 enzymes, is desired. A weak inhibitor is a compound that causes at least 1.25-fold but less than 2-fold increase in the plasma AUC values, or 20-50% decrease in clearance (wikipedia.org/wiki/cyp3A4, visited 11/12/11). Generally, a compound exhibiting a Cyp3A4 IC50 of greater than lOuM is considered a weak inhibitor.

A measure useful for comparing cyp3A4 inhibition among drug candidates is the ratio of Cyp3 A4 inhibition and the protein binding adjusted EC50. This value gives an indication of the relative potential for cyp inhibition corrected for the protein binding adjusted EC50 which is spécifie to each drug. A higher ratio in this measure is preferred as indicative of lower potential for cyp3A4 inhibition.

Unexpectedly and advantageously, applicants hâve discovered a compound (of formula (I) herein) within the generic scope of U.S. Patent publication No. 2001/00095410A1 that provides advantages compared to structurally close compounds (herein designated as compounds A and B) disclosed in U.S. Patent publication No. 2001/00095410A1

Compound B.

Therefore, objects of the présent invention include but are not limited to the provision of a compound of formula (I) or pharmaceutically acceptable sait thereof, and methods of using the compound of formula (I) for the treatment of kidney disease, chronic kidney disease, diabetic kidney disease, diabetic nephropathy, kidney fibrosis or lung fibrosis.

Combination Therapy

Patients being treated for cardio-renal diseases such as chronic kidney disease may benefît from combination drug treatment. For example the compound of the présent invention may be combined with one or more of angiotensin converting enzyme (ACE) inhibitors such as enalapril, captopril, ramipril, lisinopril, and quinapril; or angiontesin II receptor blockers (ARBs) such as losartan, olmesartan, and irbesartan; or antihypertensive agents such as amlodipine, nifedipine, and felodipine. The benefît of combination may be increased efficacy and/or reduced side effects for a component as the dose of that component may be adjusted down to reduce its side effects while benefiting from its efficacy augmented by the efficacy of the compound of formula (I) and/or other active component(s).

Patients presenting with chronic kidney disease treatable with ASKI inhibitors such as compound of formula (I), may also exhibit conditions that benefît from co-administration (as directed by a qualified caregiver) of a therapeutic agent or agents that are antibiotic, analgésie, antidepressant and/or anti-anxiety agents in combination with compound of formula (I). Combination treatments may be administered simultaneously or one after the other within intervals as directed by a qualified caregiver or via a fixed dose (ail active ingreditents are combined into the a single dosage form e.g. tablet) présentation of two or more active agents. Pharmaceutical Compositions and Administration

The compound of the présent invention may be administered in the form of a pharmaceutical composition. The présent invention therefore provides pharmaceutical compositions that contain, as the active ingrédient, the compound of formula (I), or a pharmaceutically acceptable sait thereof, and one or more pharmaceutically acceptable excipients and/or carriers, including inert solid diluents and fillers, diluents, including stérile aqueous solution and various organic solvents, perméation enhancers, solubilizers and adjuvants.^

The pharmaceutical compositions may be administered alone or in combination with other therapeutic agents. Compositions may be prepared for delivery as solid tablets, capsules, caplets, ointments, skin patches, sustained release, fast disintegrating tablets, inhalation préparations, etc. Typical pharmaceutical compositions are prepared and/or administered using methods and/or processes well known in the pharmaceutical art (see, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modem Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker & C.T. Rhodes, Eds.).

Formulations for combination treatments comprising the compound of formula (I) may be presented as fîxed dose formulations e.g. tablets, élixirs, liquids, ointments, inhalants, gels, etc., using procedures known to one of skill in the art.

Pharmaceutical compositions of the compound of formula (I) may be administered in either single or multiple doses by routes including, for example, rectal, buccal, intranasal and transdermal routes; by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer. Most preferred routes of administration include oral, parental and intravenous administration.

The compound of formula (I) may be administered in a pharmaceutically effective amount. For oral administration, each dosage unit preferably contains from 1 mg to 500 mg of the compound of formula (I). A more preferred dose is from 1 mg to 250 mg of the compound of formula (I). Particularly preferred is a dose of the compound of formula (I) ranging from about 20 mg twice a day to about 50 mg twice a day. It will be understood, however, that the amount of the compound actually administered usually will be determined by a physician in light of the relevant circumstances including the condition to be treated, the chosen route of administration, co-administration compound if applicable, the âge, weight, response of the individual patient, the severity ofthe patient’s symptoms, and the like. Nomenclature

The name of the compound of the présent invention as generated using ChemBioDraw Ultra 11.

is 5-(4-cyclopropyl- IH-imidazol-1 -yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2fluoro-4-methylbenzamide also known as 5-((4-cyclopropyl-lH-imdazol-l-yl)-2-fluoro-N-(6-(4isopropyl-4H-1,2,4-triazole-3 -yl)pyridine-2-yl)-4-methylbenzamide.

Synthesis of the Compound of Formula (I)

The compound of the invention may be prepared using methods disclosed herein or modifications thereof which will be apparent given the disclosure herein. The synthesis of the compound of the invention may be accomplished as described in the following example. If available, reagents may be purchased commercially, e.g. from Sigma Aldrich or other Chemical suppliers. Altematively, reagents may be prepared using reaction schemes and methods known to one of skill in the art.

Synthetic Reaction Parameters

The ternis “solvent,” “inert organic solvent” or “inert solvent” refer to a solvent inert under the conditions of the reaction being described in conjunction therewith (including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chioroform, methylene chloride (or dichloromethane), diethyl ether, petroleum ether (PE), methanol, pyridine, ethyl acetate (EA) and the like. Unless specified to the contrary, the solvents used in the reactions of the présent invention are inert organic solvents, and the reactions are carried out under an inert gas, preferably nitrogen.

One method of preparing compounds of formula (I) is shown in Reaction Schemes 1 and 2 below.

DMF-DMA

Préparation of Compound A

To a solution of methyl 6-aminopicolinate (432 g, 2.84 mol) in MeOH (5 L) was added NH2NH2.H2O (284 g, 5.68 mol, 2.0 eq.). The reaction mixture was heated under reflux for 3 hr and then cooled to room température. The precipitate formed in the mixture was collected by filtration, washed with EA (2 Lx2) and then dried in vacuo to give compound A (405 g, 94% yield) as white solid.

Préparation of compound B

A mixture of compound A (405 g, 2.66 mol) in dimethylformamide-dimethylacetal (DMF-DMA) (3.54 L) was heated under reflux for 18 hr, cooled to room température and then concentrated under reduced pressure. The residue was taken up in EA (700 mL) and heated at 50°C for 20 min. After being cooled to room température, the solid was collected by filtration and dried in vacuo to give compound B (572 g, 82% yield) as white solid. Préparation of C

To a solution of compound B (572 g, 2.18 mol) in a mixture of CHsCN-AcOH (3.6 L, 4:1) was added propan-2-amine (646 g, 5.0 eq.). The resulting mixture was heated under reflux for 24 hr and then cooled to room température, and the solvent was removed under reduced pressure. The residue was dissolved in water (2.8 L) and 1 N aqueous NaOH was added to a pH of 8.0 H. The precipitate was collected by filtration and the fîltrate was extracted with EA (500 mL><3). The combined organic layers were dried over anhydrous Na₂SO4, and then concentrated to a volume of 150 mL. To this mixture at 0°C was slowly added PE (400 mL) and the resulting suspension was filtered. The combined solid was re-crystallized from EA-PE to give compound C (253 g, 57% yield) as off-white solid.

^!H-NMR (400 MHz, CDC13): δ 8.24 (s, 1 H), 7.52 (m, 2 H), 6.51 (dd, J = 1.6, 7.2 Hz, 1 H), 5.55 (m, 1 H), 4.46 (bs, 2 H), 1.45 (d, J = 6.8 Hz, 6 H). MS (ESI+) m/z: 204 (M+l)⁺. Compound C is a key intermediate for the synthesis of the compound of formula (I). Thus, an object of the présent invention is also the provision of the intermediate compound C,

its salts or protected forms thereof, for the préparation of the compound of formula (I). An example of a sait of the compound C is the HCl addition sait. An example of a protected form of compound C is the carbamate compound such as obtained with Cbz-Cl. Protective groups, their préparation and uses are taught in Peter G.M. Wuts and Theodora W. Greene, Protective Groups in Organic Chemistry, 2^nd édition, 1991, Wiley and Sons, Publishers. a

Scheme 2

Préparation of the Compound of formula (I) continued:

Compound 6 is a key intermediate for the synthesis of the compound of formula (I). Thus an object of the présent invention is also the provision of intermediate compound 6,

salts or protected forms thereof, for the préparation of the compound of formula (I). An example of a sait of the compound 6 is the HCl addition sait. An example of a protected form of 10 the compound 6 is an ester (e.g. methyl, ethyi or benzyl esters) or the carbamate compound such as obtained with Cbz-Cl. Protective groups, their préparations and uses are taught in Peter G.M.

Wuts and Theodora W. Greene, Protective Groups in Organic Chemistry, 2^nd édition, 1991, Wiley and Sons, Publishers.

Step 1 - Préparation of 5-amino-2-fluoro-4-methylbenzonitrile - Compound (2)

The starting 5-bromo-4-fluoro-2-methylaniline (1) (20g, 98 mmol) was dissolved in anhydrous 1-methylpyrrolidinone (100 mL), and copper (I) cyanide (17.6g, 196 mmol) was added. The reaction was heated to 180°C for 3 hours, cooled to room température, and water (300 mL) and concentrated ammonium hydroxide (300 mL) added. The mixture was stirred for 30 minutes and extracted with EA (3 x 200 mL). The combined extracts were dried over magnésium sulfate, and the solvent was removed under reduced pressure. The oily residue was washed with hexanes (2 x 100 mL), and the solid dissolved in dichloromethane and loaded onto a silica gel column. Eluting with 0 to 25% EA in hexanes gradient provided 5-amino-2-fluoro4-methylbenzonitrile (10.06g, 67.1 mmol). LC/MS (m/z:151 M⁴⁴).

Step 2 - Préparation of 5-(2-cvclopropyl-2-oxoethylamino)-2-fluoro-4-methvlbenzonitrile Compound (3)

5-Amino-2-fluoro-4-methylbenzonitrile (12g, 80mmol) was dissolved in anhydrous N,Ndimethylformamide (160 mL) under nitrogen, and potassium carbonate (13.27g, 96 mmol) and potassium iodide (14.61g , 88mmol) were added as solids with stirring. The reaction was stirred for 5 minutes at room température and then bromomethyl cyclopropylketone (20.24 mL, 180 mmol) was added. The reaction mixture was heated to 60°C for 3 hours, and then the solvents removed under reduced pressure. The residue was dissolved in EA (400 mL) and washed with 400 mL of water. The organic layer was dried over magnésium sulfate, and solvent was removed under reduced pressure. The residue was re-dissolved in a minimum amount of EA, and hexanes were added to bring the solution to 3:1 hexanes: EA by volume. The product precipitated out of solution and was collected by filtration to provide 5-(2-cyclopropyl-2oxoethylamino)-2-fluoro-4-methylbenzonitrile (14.19g, 61.2 mmol). LC/MS (m/z : 233, MT⁴)

Step 3 - Préparation of 5-(4-cyclopropyl-2-mercapto-lH-imidazol-l-yl)-2-fluoro-4methylbenzonitrile - Compound (4)

5-(2-Cyclopropyl-2-oxoethylamino)-2-fluoro-4-methylbenzonitrile (14.19g, 61.2mmol) was dissolved in glacial acetic acid (300 mL). Potassium thiocyanate (11.9g, 122.4mmol) was added as a solid with stirring. The reaction mixture was heated to 110°C for 4 hours at which time the solvent was removed under reduced pressure. The residue was taken up in dichloromethane (200 mL) and washed with 200 mL water. The aqueous extract was extracted with (2 x 200 mL) additional dichloromethane, the organic extracts combined and dried over_e magnésium sulfate. The solvent was removed under reduced pressure and the oily residue was re-dissolved in EA (50 mL) and 150 mL hexanes was added. A dark layer formed and a stir bar was added to the flask. Vigorous stirring caused the product to precipitate as a peach colored solid. The product was collected by filtration, to yield 5-(4-cyclopropyl-2-mercapto-lHimidazol-l-yl)-2-fluoro-4-methylbenzonitrile, (14.26g, 52.23 mmol). Anal. LC/MS (m/z : 274, M⁺¹)

Step 4 - Préparation of 5 -(4-cyclopropyl-1 H-imidazol-1 -yl)-2-fluoro-4-methylbenzonitrile Compound (5)

In a 500 mL three neck round bottom flask was placed acetic acid (96 mL), water (19 mL) and hydrogen peroxide (30%, 7.47 mL, 65.88 mmol). The mixture was heated to 45°C with stirring under nitrogen while monitoring the internai température. 5-(4-Cyclopropyl-2mercapto-1 H-imidazol-l-yl)-2-fluoro-4-methylbenzonitrile (6.00g, 21.96 mmol) was then added as a solid in small portions over 30 minutes while maintaining an internai température below 55°C. When addition of the thioimidazole was complété the reaction was stirred for 30 minutes at a température of 45 C, and then cooled to room température, and a solution of 20% wt/wt sodium sulfite in water (6 mL) was slowly added. The mixture was stirred for 30 minutes and solvents were removed under reduced pressure. The residue was suspended in 250 mL of water and 4N aqueous ammonium hydroxide was added to bring the pH to -10. The mixture was extracted with dichloromethane (3 x 200ml), the organics combined, dried over magnésium sulfate, and the solvent was removed under reduced pressure. The residue was dissolved in 20 mL EA, and 80 mL of hexanes were added with stirring. The solvents were decanted off and an oily residue was left behind. This process was repeated and the product, 5-(4-cyclopropyl-lHimidazol-l-yl)-2-fluoro-4-methylbenzonitrile was obtained as a viscous oil (5.14 g, 21.33 mmol) Anal. LC/MS (m/z: 242, M⁺¹)

Step 5 - Préparation of 5-(4-cyclopropyl-l H-imidazol-l-yl)-2-fluoro-4-methylbenzoic acid hydrochloride (6)

5-(4-Cyclopropyl-lH-imidazol-l-yl)-2-fluoro-4-methylbenzonitrile (11.21g, 46.50mmol) was placed in a round bottom flask fitted with a reflux condenser, and suspended in 38% hydrochloric acid (200 mL). The mixture was heated to 100°C for 4.5 hours, and then cooled to room température. Solvent was removed under reduced pressure to give a pink solid, to which was added 100ml of EA. The solid product was collected by filtration and washed with 3 xlOO.

mL EA. To the solid product was added 100 mL 10% methanol in dichloromethane, the mixture stirred, and the filtrate collected. This was repeated with 2 more 100ml portions of 10% methanol in dichloromethane. The filtrâtes were combined and solvent was removed under reduced pressure, to provide crude 5-(4-cyclopropyl-lH-imidazol-l-yl)-2-fluoro-4methylbenzoic acid hydrochloride. No further purification was carried out (11.13g, 37.54mmol). Anal. LC/MS (m/z: 261, M*¹)

Step 6 - Préparation of 5-(4-cyclopropyl-lH-imidazol-l-yl)-2-fluoro-N-(6-(4-isopropyl-4Hl,2,4-triazol-3-yl)pyridin-2-yl)-4-methylbenzamide - formula (I)

5-(4-Cyclopropyl-lH-imidazol-l-yl)-2-fluoro-4-methylbenzoic acid hydrochloride (1.5g, 5.07mmol) was suspended in anhydrous 1,2-dichloromethane (25 mL) at room température. Oxalyl chloride (0.575ml, 6.59mmol) was added with stirring under nitrogen, followed by N,Ndimethylformamide (0.044ml, 0.507mmol). The mixture was stirred for 4 hr at room température, and then the solvent was removed under reduced pressure. The residue was dissolved in 25 mL anhydrous dichloromethane. 6-(4-isopropyl-4H-l,2,4-triazol-3-yl)pyridin-2amine (1.13g, 5.58mmol) (compound C) and 4-dimethylaminopyridine (0.62g, 5.07 mmol) were rapidly added with stirring under nitrogen. The reaction was stirred for 2 hours at room température and aqueous saturated NaHCO₃ (15 mL) was added. The mixture was stirred for 10 minutes, and the layers were separated, and the aqueous layer was washed 1 x 20 mL dichloromethane. The combined organics were dried (MgSO₄), filtered and concentrated. The residue was dissolved in a minimum amount of CH₃CN and water was slowly added until solids precipitated from the mixture. The solid was collected by filtration and dried to give 5-(4cyclopropyl-1 H-imidazol-1 -yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3 -yl)pyridin-2-yl)4-methylbenzamide in -96% purity (1.28g, 2.88 mmol). Anal. LC/MS (m/z: 446, M⁺¹). The material was further purified by RP-HPLC (reverse phase HPLC) to obtain an analytically pure sample as the HCl sait.

C₂₄H24FN₇O-HC1. 446.2 (M+1). ’H-NMR (DMSO): δ 11.12 (s, 1H), 9.41 (s, 1H), 9.32 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.07 (t, J = 8.4 Hz, 1H), 7.95 (d, J = 6.4 Hz, 1H), 7.92 (d, J =

7.6 Hz, 1H), 7.79 (s, 1H), 7.59 (d, J = 10.4 Hz, 1H), 5.72 (sept, J = 6.8 Hz, 1H), 2.29 (s, 3H), 2.00-2.05 (m, 1H), 1.44 (d, J = 6.8 Hz, 6H), 1.01-1.06 (m, 2H), 0.85-0.89 (m, 2H).

Biological Assays

ASK1 (Apoptosis Signal-Regulating Kinase 1) TR-FRET Kinase Assay (Biochemical IC₅o)

The ability of compounds to inhibit ASK1 kinase activity was determined using a time resolved fluorescence résonance energy transfer [TR-FRET] assay utilizing biotinylated myelin basic protein [biotin-MBP] as the protein substrate. A Beckman Biomek FX liquid handling robot was utilized to spot 2pL/well of compounds in 2.44% aqueous DMSO into low volume 384-well polypropylene plates [Nunc, #267460] to give a final concentration of between 100μΜ and 0.5nM compound in the kinase assay. A Deerac Fluidics Equator was used to dispense 3pL/well of 0.667ng/pL [Upstate Biotechnologies, #14-606, or the équivalent protein prepared in-house] and 0.1665ng/mL biotin-MBP [Upstate Biotechnologies, #13-111] in buffer (85mM MOPS, pH 7.0, 8.5mM Mg-acetate, 5% glycerol, 0.085% NP-40, 1.7mM DTT and 1.7mg/mL BSA) into the plates containing the spotted compounds.

The enzyme was allowed to pre-incubate with compound for 20 minutes prior to initiating the kinase reaction with the addition of 5pL/well 300μΜ ATP in buffer (50mM MOPS, pH 7.0, 5mM Mg-acetate, ImM DTT, 5% DMSO) using the Deerac Fluidics Equator. The kinase reactions were allowed to proceed for 20 minutes at ambient température and were subsequently stopped with the addition of 5pL/well 25mM EDTA using the Deerac Fluidics Equator. The Biomek FX was then used to transfer 1 μΕ/well of each completed kinase reaction to the wells of an OptiPlate-1536 white polystyrène plate [PerkinElmer, #6004299] that contained 5pL/well détection reagents (1.1 InM Eu-W1024 labeled anti-phosphothreonine antibody [PerkinElmer, #AD0094] and 55.56nM streptavidin allophycocyanin [PerkinElmer, #CR130-100] in lx LANCE détection buffer [PerkinElmer, #CR97-100]). The TR-FRET signal was then read on a Perkin Elmer Envision plate reader after incubating the plates at ambient température for 2 hours.

The 100% inhibition positive control wells were generated by switching the order of addition of the EDTA and ATP solutions described above. These wells and 0% inhibition wells containing spots of 2.44% DMSO at the beginning of the assay were used in calculating the % inhibition for the test compounds.

Resuit

The compound of formula (I) inhibited ASK1 with an IC50 of 3.0 nM. This data suggests that the compound of formula (I) is a potent inhibitor of ASK1 in the presence of the compétitive ligand ATP.

In an updated version of the assay above, the inhibitory activity of compound of the invention against ASK1 was examined using a TR-FRET ASK1 assay which determined the amount of phosphate transferred to a peptide substrate from ATP.

Materials and Methods

Reagents

Dephosphorylated recombinant human ASK1 kinase was from Gilead Sciences. Small molécule kinase inhibitor staurosporine (Catalogue # S6942) and dithiothreitol (DTT, catalogue # 43815-5G) were obtained from Sigma Chemicals (St. Louis, MO). ATP (catalogue # 7724) was from Affymetrix (Santa Clara, CA) and the compound of formula (I) was from Gilead Sciences. HTRF KinEASE™-STK S3 kit was obtained from Cisbio (Bedford, Mass). Ail other reagents were of the highest grade commercially available.

Assays

The assay measures the phosphorylation level of a biotinylated peptide substrate by the

ASK1 kinase using HTRF détection (6.1). This is a compétitive, time-resolved fluorescence résonance energy transfer (TR-FRET) immunoassay, based on HTRF® KinEASE™-STK manual from Cisbio (6.1). Test compound, 1 μΜ STK3 peptide substrate, 4 nM of ASK1 kinase are incubated with 10 mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025 % NP-40, 1 mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100 DM ATP is added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with IX Eu³⁺ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 are added to stop the reaction and phosphorylated peptide substrate is detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET level (a ratio of 665^ nm/615 nm) is proportional to the phosphorylation level. Under these assay conditions, the degree of phosphorylation of peptide substrate was linear with time and concentration for the enzyme. The assay System yielded consistent results with regard to K_m and spécifie activities for the enzyme. For inhibition experiments (IC50 values), activities were performed with constant concentrations of ATP, peptide and several fixed concentrations of inhibitors. Staurosporine, the nonselective kinase inhibitor, was used as the positive control. Ail enzyme activity data are reported as an average of quadruplicate détermination.

Data Analysis

The IC50 values were calculated following équation:

y = Range /{1 + (x / ICso)^s } + Background

Where x and y represent the concentration of inhibitors and enzyme activity, respectively. Enzyme activity is expressed as the amount of Phosphate incorporated into substrate peptide from ATP. Range is the maximum y range (no inhibitor, DMSO control) and s is a slope factor (6-2).

Results

The compound of formula (I) exhibited an IC50 of 3.2nM under this test condition. The data demonstrates that the compound of formula (I) is a potent inhibitor of the ASK-1 1 receptor.

ASK1 (Apoptosis Signal-Regulating Kinase 1) 293 cell-based assay (Cellular EC50)

The cellular potency of compounds was assayed in cells stably expressing an APlüuciferase reporter construct (293/APl-Luc cells - Panomics Inc., 6519 Dumbarton Circle, Fremont, CA). Cells were infected with an adenovirus expressing kinase active ASK1 (6311381 of rat ASK1 cDNA), which will activate the AP-1 transcription factor and increase the expression of luciferase. Inhibitors of ASK1 will decrease the enzyme activity of ASK1 and therefore decrease the activity of AP-1 transcription factor and the expression of luciferase.

1. MATERIALS REQUIRED FOR THIS PROTOCOL ⁵

Media and Reagents	Source Company	Catalog No.
AP-1 Reporter 293 Stable Cell Line	Panomics	Unknown
DMEM (w/ high glucose, w/o Lglutamine, w/ pyruvate, w/ HEPES	MediaTech	15-018-CM
DMEM (w/ high glucose, w/o Lglutamine, w/o pyruvate, w/o HEPES, w/o phénol red	Invitrogen	31053-028
HEPES, IM	Invitrogen	15630-080
Sodium Pyruvate, 100 mM	Invitrogen	11360-070
Fêtai Bovine Sérum, “FBS”	Hyclone	SH30088.03
Pen-Strep-Glut., “PSG”	Invitrogen	10378-016
HygromycinB	Calbiochem	400052
Dulbecco’s PBS (stérile)	MediaTech	21-030-CM
Trypsin-EDTA (0.25%)	Invitrogen	25200-056
Steady-Glo Luciferase Assay System	Promega	E2550
Labware	Source	Catalog No.

Flasks (poly-D-Lysine coated, 150 cm², vented cap)	BD Biosciences	356538
Plates (poly-D-Lysine coated, 384well, white/clear, stérile TCT)	Greiner (through VWR Scientific)	781944 (82051-354)
White Backing Tape	PerkinElmer	6005199
Cell Strainers (40 um nylon, blue__ ring, fîts 50 mL conical vials)	VWR Scientific	21008-949

2. REFERENCE MATERIALS

Panomics 293/APl-Luc stable cell-line product insert.

Promega Steady-Glo Luciferase Assay System product insert.

3. MEDIA REQUIRED

Complété Growth Medium, “CGM”

DMEM (MediaTech)

10%FBS

1% PSG

100 ug/mL HygromycinB

Assay Medium, “AM”

DMEM (Invitrogen) mM HEPES mM Sodium Pyruvate

1%PSG

4. METHODS

Maintenance:

293/APl-Luc Maintain 293/Acells per vendor’s instructions; harvest cells at -80% confluence in Tl 50 flasks as follows:

Aspirate media, wash gently with -12 mL stérile D-PBS, aspirate.

Add 5 mL Trypsin-EDTA, tilt gently to coat flask, and incubate -5 min. at 37°C.

Do not tap flask; add 5 mL CGM, wash flask 4X with cell suspension, transfer to 50 mL conical vial, centrifuge 5 min. at 1200 rpm.

Aspirate media from cell pellet, add 20 to 30 mL CGM, resuspend pellet by pipeting 6X, pass through cell strainer to disperse clumps (if necessary), and count cells with hemocytometer.

Assay Day 1 :

Harvest cells as above, except resuspend cell pellet.

Count cells and dilute to 1.5 x 10⁵ cells per mL; add adenovirus such that there are 5 __infectious forming units per cell____________

Prime (20 to 30 mL) and plate cells in Greiner poly-D-Lysine coated 384-well plates at 1.2 x 10⁴cells per well using BioTek uFill (80 uL per well).

Immediately dose plates with 0.4 uL of compound dose sériés (in 100% DMSO) incubate 24 hours in humidified incubator (37°C, 5% CO2).

Assay Day 2:

Process plates (per manufacturer’s instructions) as follows:

Set plates in laminar flow hood & uncover for 30 minutes at room température to cool. Remove 60 uL of AM from assay wells

Add 20 uL per well Steady-Glo Firefly substrate, let sit for 10-20 minutes at room température Cover bottom of assay plates with white backing tape.

Acquire data on a fluorescence plate reader

The 100% inhibition positive control wells were generated by infecting cells with an adenovirus expressing catalytically inactive ASK1 mutant with lysine to argine mutation at residue 709. Resuit

The compound of formula (I) exhibits an EC50 of 2.0 nM.

Détermination of Kd

Kinase assays

Kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32°C until lysis. The lysâtes were centrifuged and filtered to remove cell débris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR détection. Streptavidin-coated magnetic beads were treated with biotinylated small molécule ligands for 30 minutes at room température to generate affinity resins for kinase assays.

The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% (bovine sérum albumin), 0.05% Tween 20, 1 mM DTT(dithiothreitol)) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in lx binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). Ail reactions were performed in polystyrène 96-well plates in a final volume of 0.135 mL. The assay plates were incubated at room température with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05% Tween 20, 0.5 μΜ non-biotinylated affinity ligand) and incubated at room température with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.

Binding constants (Kds) were calculated with a standard dose-response curve using the Hill équation.

Resuit

The compound of formula (I) exhibited a Ka of 0.24 nM. This data suggests that the compound of formula (I) binds potently to ASK1 receptor in the absence of ATP.

Détermination of Percent of Compound bound to Plasma

Experimental Design:

ImL Teflon dialysis cells from Harvard Apparatus (Holliston, Mass, USA) were used in these experiments. Prior to the study, dialysis membrane was soaked for approximately one hour in 0.133 M phosphate buffer, pH 7.4. A nominal concentration of 2 μΜ of compound was spiked into ImL of plasma or ImL of cell culture media. The total volume of liquid on each side of the cell was ImL. After 3 hours équilibration in a 37°C water bath, samples from each side of the cell were aliquoted into the appropriate vials containing either ImL of human plasma (cell culture media), or buffer. Sample vials were weighed and recorded. A 100 pL aliquot was removed and added to 400 pL quenching solution (50% methanol, 25% acetonitrile, 25% water and internai standard). Samples were vortexed and centrifuged for 15 minutes at 12000 G. 200 pL of the supematant was removed and placed into a new 96 well plate. An additional 200 pL of 1:1 ACN:water was added. The plate was then vortexed and subjected to LC-MS analysis. The percent unbound for an analyte in plasma was calculated using the following équations % Unbound = 100(C_f /C_t) where Cf and C_t are the post-dialysis buffer and plasma concentrations, respectively.

Results

The percent unbound measured in human plasma for the compound of formula (I) is 11.94%

Détermination of CÀCO-2 Efflux Ratio

Experimental:

Caco-2 cells were maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM) with sodium pyruvate, Glutmax supplemented with 1% Pen/Strep, 1% NE AA and 10% fêtai bovine sérum in an incubator set at 37°C, 90% humidity and 5% CO2. Caco-2 cells between 5 passage 62 and 72 were seeded at 2100 cells/well and were grown to confluence over at least 21days on 24 well PET (polyethylene-terephthalate) plates (BD Biosciences). The receiver well contained HBSS buffer (lOmM HEPES, 15mM Glucose with pH adjusted to pH 6.5) supplemented with 1% BSA pH adjusted to pH 7.4. After an initial équilibration with transport buffer, TEER values were read to test membrane integrity. Buffers containing test compounds were added and 100 μΐ of solution was taken at 1 and 2 hrs from the receiver compartment.

Removed buffer was replaced with fresh buffer and a correction is applied to ail calculations for the removed material. The experiment was carried out in replicate. Ail samples were immediately collected into 400 μΐ 100% acetonitrile acid to precipitate protein and stabilize test compounds. Cells were dosed on the apical or basolateral side to détermine forward (A to B) and reverse (B to A) permeability. Permeability through a cell free trans-well is also determined as a measure of cellular permeability through the membrane and non-specific binding. To test for non-specific binding and compound instability percent recovery is determined. Samples were analyzed by LC/MS/MS.

The apparent permeability, P_app, and % recovery were calculated as foliows:

P_app = (dR/dt) x V_r/(A x Do) % Recovery = 100 x ((V_r x R120) + ( Vj x D120))/ (Va x Do) where, dRJdt is the slope of the cumulative concentration in the receiver compartment versus time in μΜ/s based on receiver concentrations measured at 60 and 120 minutes.

V_r and Va is the volume in the receiver and donor compartment in cm , respectively.

A is the area of the cell monolayer (0.33 cm ).

Do and D120 is the measured donor concentration at the beginning and end of the experiment, ïëspe'ctively.

R120 is the receiver concentration at the end of the experiment (120 minutes).

Absorption and Efflux Classification:

Papp (A to B) > 1.0 x 10'⁶ cm/s	Eligh
1.0 x 10‘⁶ cm/s > P_app (Ato B)> 0.5 x 10'⁶ cm/s	Medium

Papp (A to B) < 0.5 x 10'⁶ cm/s	Low
P_app (B to A)/ P_app (A to B) > 3	Significant Efflux
% recovery < 20%	May affect measured permeability
Cell Free P_app <15	May affect measured permeability

Resuit

The compound of formula (I) was observed to hâve a CACO A—>B value of 27; and a CACO B—>A value of 35 resulting in a efflux ratio (B—>A)/(A—>B) of 1.3.

Détermination of Metabolic Stability in Hepatic Microsomal Fraction:

Experimental:

Metabolic stability was assessed using cofactors for both oxidative metabolism (NADPH) and conjugation (UDP glucuronic acid (UDPGA)). Duplicate aliquots of the compound of formula (I) (3 pL of 0.5 mM DMSO stock) or metabolic stability standards (Buspirone) were added to microsome stock diiuted with potassium phosphate buffer, pH 7.4, to obtain a protein concentration of 1.0 mg/mL and containing alamethicin as a permeabilizing agent. Metabolic reactions were initiated by the addition of NADPH regenerating System and UDPGA cofactor. The final composition of each reaction mixture was: 3 μΜ test compound, 1 mg microsomal protein/mL, 5 mM UDPGA, 23.4 pg/mL alamethicin, 1.25 mM NADP, 3.3 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase and 3.3 mM MgCl₂ in 50 mM potassium phosphate buffer, pH 7.4. At 0, 2, 5, 10, 15, 30, 45, and 60 min, 25 pL aliquots of the reaction mixture were transferred to plates containing 250 pl of IS/Q (quenching solution containing internai standard). After quenching, the plates were centrifuged at 3000 x g for 30 minutes, and 10 pL aliquots of the supematant were analyzed using LC/MS to obtain analyte/intemal standard peak area ratios.

Metabolic stability in microsomal fractions were determined by measuring the rate of disappearance of the compound of formula (I). Data (% of parent remaining) were plotted on a semi logarithmic scale and fitted using an exponential fit:

C, = C_o · e ^K'‘ and T_l/2 = ln2/K where

C_t % of parent remaining at time = t

Co % of parent remaining at time = 0 t time (hr)

K First order élimination rate constant (hr'¹)

T% In vitro half-life (hr)

The predicted hepatic clearance was calculated as follows {reference 1} :

CT _ K*V*Y_F/ _CT _ K.V.Y_H/ ^- /P ^{OT -} /H

CL_h = (CL·^ · Q^KCL·^ + Q_h), where

CLh Predicted hepatic clearance (L/hr/kg body weight)

CLi_nt Intrinsic hepatic clearance (L/hr/kg body weight)

V Incubation volume (L)

Y_P Microsome protein yield (mg protein/kg body weight)

Yh Hépatocyte yield (millions of cells/kg body weight)

P Mass of protein in the incubation (mg)

H Number of hépatocytes in the incubation (million)

Qh Hepatic blood flow (L/hr/kg body weight)

Predicted hepatic extraction was then calculated by comparison of predicted hepatic clearance to hepatic blood flow. A compound was considered stable if the réduction of substrate concentration was < 10% over the course of the incubation (corresponding to an extrapolated half-life of > 395 min in microsomal fractions and > 39.5 hr in hépatocytes).

Values used for calculation of the predicted hepatic clearance are shown in the tables below: λ

Table 1. Values Used for Calculation of the Predicted Hepatic Clearance from Microsomal Stability

Species	Hepatic Microsomes	Qh (L/kg)
V (L)	P (mg)	Y (mg/kg)
Rat	0.001	1.0	1520	4.2
Cynomolgus Monkey	0.001	1.0	684	1.6
Rhésus Monkey	0.001	1.0	1170	2.3
Dog	0.001	1.0	1216	1.8
Human	0.001	1.0	977	1.3

Resuit:

The predicted hepatic clearance in human as determined from in vitro experiments in microsomal fractions is 0.1 L/h/kg.

Détermination of Rat CL and Vss for Test Compounds

Pharmacokinetics of Test Compounds following a 1 mg/kg IV infusion and 5.0 mg/kg PO dose in rats

Test article and formulation

For IV administration the test compound was formulated in 60:40 PEG 400:water with 1 équivalent HCl at 0.5 mg/mL. The formulation was a solution.

For PO (oral) administration, the test compound was formulated in 5/75/10/10 ethanol/PG/solutol/water at 2.5 mg/mL. The formulation was a solution.

Animais Used - - - —

IV and PO dosing groups each consisted of 3 male SD rats. At dosing, the animais generally weighed between 0.317 and 0.355 kg. The animais were fasted overnight prior to dose administration and up to 4 hr after dosing.

Dosing

For the IV infusion group, the test compound was administered by intravenous infusion over 30 minutes. The rate of infusion was adjusted according to the body weight of each animal to deliver a dose of 1 mg/kg at 2 mL/kg. For the oral dosing group, the test article was administered by oral gavage at 2 mL/kg for a dose of 5.0 mg/kg.

Sample collection

Serial venous blood samples (approximately 0.4 mL each) were taken at specified time points after dosing from each animal. The blood samples were collected into Vacutainer™ tubes (Becton-Disckinson Corp, New Jersey, USA) containing EDTA as the anti-coagulant and were immediately placed on wet ice pending centrifugation for plasma.

Détermination of the concentrations of the Compound of Formula (I) in plasma

An LC/MS/MS method was used to measure the concentration of test compound in plasma.

Calculations

Non-compartmental pharmacokinetic analysis was performed on the plasma concentration-time data.

Results

The compound of formula (I) exhibited a CL of 0.09 L/hr/kg; an oral bioavailability of 75 %; ti/2 of 5.07 hr and a Vss of 0.55 L/kg in rats.

Cyp Inhibition Assay

Objective *

To assess the potential of the test compound to inhibit the main cytochrome P450 isoforms, CYP1A, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (2 substrates).

Cytochrome P450 Inhibition IC50 'Détermination (8 Isoform, 9 Sübstrates)

Protocol Summary

Test compound (0.1 μΜ — 25 μΜ) is incubated with human liver microsomes and NADPH in the presence of a cytochrome P450 isoform-specific probe substrate. For the CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 spécifie reactions, the métabolites are monitored by mass spectrometry. CYP1A activity is monitored by measuring the formation of a fluorescent métabolite. A decrease in the formation of the métabolite compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).

Assay Requirements

500pL of a lOmM test compound solution in DMSO.

Experimental Procedure

CYP1A Inhibition

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.3%) are incubated with human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the presence of the probe substrate ethoxyresorufin (0.5 μΜ) for 5 min at 37°C. The sélective CYP1A inhibitor, alpha-naphthoflavone, is screened alongside the test compounds as a positive control.

CYP2B6 Inhibition

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.3%) are incubated with human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate bupropion (110 μΜ) for 5 min at 37°C. The sélective CYP2B6 inhibitor, ticlopidine, is screened alongside the test compounds as a positive control.

CYP2C8 Inhibition

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.3%) are incubated with human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in the presence of the probe substrate paclitaxel (7.5 μΜ) for 30 min at 37°C. The sélective CYP2C8 inhibitor, montelukast, is screened alongside the test compounds as a positive control.

CYP2C9 Inhibition ----Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate tolbutamide (120 μΜ) for 60 min at 37 °C. The sélective CYP2C9 inhibitor, sulphaphenazole, is screened alongside the test compounds as a, positive control.

CYP2C19 Inhibition

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 rnM) in the presence of the probe substrate mephenytoin (25 μΜ) for 60 min at 37 °C. The sélective CYP2C19 inhibitor, tranylcypromine, is screened alongside the test compounds as a positive control.

CYP2D6 Inhibition

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.25%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate dextromethorphan (5 μΜ) for 5 min at 37 °C. The sélective CYP2D6 inhibitor, quinidine, is screened alongside the test compounds as a positive control.

CYP3A4 Inhibition (Midazolam)

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.26%) are incubated with human liver microsomes (0.1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 μΜ) for 5 min at 37°C. The sélective CYP3A4 inhibitor, kétoconazole, is screened alongside the test compounds as a positive control.

CYP3A4 Inhibition (Testosterone)

Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μΜ in DMSO; final DMSO concentration = 0.275%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate testosterone (50 μΜ) for 5 min at 37°C. The sélective CYP3A4 inhibitor, kétoconazole, is screened alongside the test compounds as a positive control.

For the CYP1A incubations, the reactions are terminated by methanol, and the formation of the métabolite, resorufin, is monitored by fluorescence (excitation wavelength = 535 nm, émission wavelength = 595 nm). For the CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions are terminated by methanol. The samples are then centrifuged, and the supematants are combined, for the simultaneous analysis of 4hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan, and 1-hydroxymidazolam by LC31

MS/MS. Hydroxybupropion, 6a-hydroxypaclitaxel and 6B-hydroxytestosterone are analysed separately by LC-MS/MS. Formic acid in deionised water (final concentration = 0.1%) containing internai standard is added to the final sample prior to analysis. A decrease in the formation of the métabolites compared to vehicle control is used to calculate an IC50 value (test 5 compound concentration which produces 50% inhibition).

Results

CYP450 Isoform	Substrate	Métabolite	Calculated IC₅₀(μΜ)
IA	Ethoxyresorufin	Resorufin	>25 μΜ
1A2	Phenacetin	Acetaminophen	>25 μΜ
2B6	Bupropion	Hydroxybupropion	19.2 μΜ
2C8	Paclitaxel	6a-Hydroxypaclitaxel	21.6 μΜ
2C9	Tolbutamide	4-Hydroxytolbutamide	>25 μΜ
2C19	S-mephenytoin	4-Hydroxymephenytoin	>25 μΜ
2D6	Dextromethorphan	Dextrorphan	17.7 μΜ
3A4	Midazolam	Hydroxymidazolam	2.7 μΜ
3A4	Testosterone	6 B Hydroxytestosterone	10.5 μΜ

General study design for the rat unilatéral ureter obstruction (UUO) model of kidney 10 fibrosis.

Male Sprague-Dawley rats were fed normal chow, housed under standard conditions, and allowed to acclimate for at least 7 days before surgery. At the inception of study, rats were placed into weight-matched groups, and administered (2 ml/kg p.o. bid) via oral gavage vehicle, one of four dose levels of compounds (1, 3, 10, or 30 mg/kg). Rats were anesthetized with isoflurane anesthésia on a nosecone, and laparotomy was performed. Rats underwent complété obstruction of the right ureter (UUO) using heat sterilized instruments and aseptie surgical technique. Rats were administered 50 μΐ Penicillin G (i.m.) immediately post-operatively. Rats „ were allowed to recover in a clean, heated cage before being retumed to normal vivarium conditions. Rats were administered compounds at the dose described above twice daily (at 12 hour intervals) for the subséquent 7 days. On day 7 following surgery, rats were anesthetized with isoflurane and sérum, plasma, and urine collected. Animais were then euthanized, the kidneys harvested, and rénal cortical biopsies collected for morphological, histological, and biochemical analysis. Ail tissues for biochemical analysis are flash-frozen in liquid nitrogen and stored at -80°C, tissues for histological analysis were fixed in 10% neutral buffered formalin

Rénal fibrosis was evaluated by measuring the amount of collagen IV in the kidney by an

ELISA method and by examining the accumulation of alpha-smooth muscle actin positive myofibroblasts in the kidney by immunohistochemistry. For the former, a small piece of frozen kidney cortex was transferred homengenized in RIPA buffer then centrifuged at 14000 x g for minutes at at 4 °C. The supematant was collected into pre-chilled tubes and the protein concentration was determined. Equivalent amount of total protein were subjected to a Col IV

ELISA assay (Exocell) according to the manufacturers instructions.

Formalin fixed and paraffin embedded kidney tissue was stained with an alpha-smooth muscle actin as previously described (Stambe et al., The Rôle of p38 Mitogen-Activated Protein Kinase

Activation in Rénal Fibrosis J Am Soc Nephrol 15: 370-379, 2004).

Results:

The compound of formula (I) was found to significantly reduce kidney Collagen IV induction (figure 1) and accumulation of alpha-smooth muscle positive myofibroblasts (figure 2) at doses of 3 to 30 mg/kg.

Comparative Data for Compound of Formula (I) and Reference Compounds

The following table provides comparative results for the compound of formula (I) and the reference compounds A and B disclosed in U.S. Patent publication No. 2001/00095410A1, published January 13, 2011. Applicants note that experiments for which results are compared below were performed under similar conditions but not necessarily simultaneously.

Table

	Compound of formula (I)	Compound A	Compound B
IC₅o (nM)	3	5	6.5
EC₅₀(nM)	2	3.4	18 (9X)
PBadj EC₅₀ (nM)	17	71 (4X)	563 (33X)
CACO (A/B, B/A)	27/35	3.1 /18.5	0.26/4
Efflux ratio (B/A)/(A/B)	1.3	6.0	15.4
fu	12	4.8	3.2
Cyp3A4 ICsoTestesterone (TST)) (uM)	11	1.1 (10X)	4 (2.8X)
Cyp3A4 IC₅₀ /PBAdj.ECso	647	15 (43X)	7 (92X)
Vss (L/Kg) in rats	0.55	0.17 (3.2X)	0.54
CL (L/hr/kg) in rats	0.11	0.30 (2.7X)	0.39 (3.6X)
%F in rats	75	11 (6.8X)	50(1.5X)
t % in rats (hr)	5.07	0.59 (8.6X)	1.3 (3.9X)

( ) values in parenthesis represent the number of times the compound of formula (I) shows an improvement over the indicated compound for the indicated parameter.

The following can be deduced from the above comparative data:

The compound of formula (I) has an EC50 that is comparable to that of Compound A.

The compound of formula (I) has a functional IC50 that is comparable to IC50S for compounds A and B.

The compound of formula (I) has a protein binding adjusted EC50 that is 4 times lower 10 than that of compound A and 33 times lower than that of compound B.

The compound of formula (I) is a weaker Cyp3 A4 inhibitor compared to compounds A and B.

The compound of Formula (I) has a CYP3A4 IC50/ PBAdj.ECso value that is 43 times higher than that for compound of formula A, and 92 times higher than for the compound of formula B.

The compound of formula (I) has a Rat CL value that is 2.7 times lower than that for compound of formula A, and 3.6 times lower than that for the compound of formula B. Λ

The compound of formula (I) has a percent bioavailability in rats that is 6.8 times higher than compound A and 1.5 times higher than compound B.

The compound of formula (I) has a half life in rats that is 8.6 times longer than that of compound A and 3.9 times longer than that of compound B.

The above data fairly suggest that the compound of formula (I) has unexpected and advantageous properties compared to compounds of formula A and B; and that the compound of formula (I) is likely a better candidate for further development for the treatment of chronic kidney disease, lung and/or kidney fibrosis, and/or cardio-renal diseases.

Claims

What is claimed is:

1. A compound of formula (I)

Namely, 5-(4-cyclopropyl-lH-imidazol-l-yl)-N-(6-(4-isopropyl-4H-l,2,4-triazol-3-yl)pyridin-2yl)-2-fhioro-4-methylbenzamide, or a pharmaceutically acceptable sait thereof.
2. The compound of formula (I)

namely, 5 -((4-cyclopropyl-1 H-imdazol-1 -yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazole-3 yl)pyridine-2-yl)-4-methylbenzamide.
3. A pharmaceutical composition comprising a therapeutically effective amount of the compound or sait according to Claim 1 and a pharmaceutically acceptable carrier.
4. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to Claim 2 and a pharmaceutically acceptable carrier.
5. A method of treating chronic kidney disease comprising administering a therapeutically effective amount of a compound according to Claim 2, or pharmaceutically acceptable sait thereof, to a patient in need thereof.
6. A method of treating diabetic nephropathy comprising administering a therapeutically effective amount of a compound according to Claim 2 or a pharmaceutically acceptable sait thereof, to a patient in need thereof, Λ
7. A method of treating kidney fibrosis, liver fibrosis, or lung fïbrosis comprising administering a therapeutically effective amount of a compound or sait according to Claim 1, to a patient in need thereof.
8. A method of treating diabetic kidney disease, comprising administering a therapeutically effective amount of a compound or sait according to Claim 1, to a patient in need thereof.
9. Use of a compound or sait according to Claim 1 in the manufacture of a médicament for the treatment of chronic kidney disease.
10. Use of a compound or sait according to Claim 1 in therapy.
11. An intermediate of formula:

H₂N N namely, 2-amino, 5-(4-isopropyl-4H-l,2,4-triazol-3-yl) pyridine, or a sait or protected form thereof.
12. An intermediate of formula:

namely, 5-(4-cyclopropyl-lH-imidazol-l-yl)-2-fluoro-4-methylbenzoic acid, a sait or protected form thereof. Y