NZ754169B2 - Apoptosis signal-regulating kinase inhibitor - Google Patents

Abstract

Disclosed is a dosage unit comprising selonsertib (5-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-fluoro-4-methylbenzamide) and its use as an apoptosis signal-regulating kinase inhibitor for the treatment of kidney fibrosis, liver fibrosis, lung fibrosis, diabetic nephropathy or diabetic kidney disease. Also disclosed are the two intermediates 5-(4-cyclopropy1-1H-imidazol-1-y1)-2-fluoro-4-methylbenzoic acid and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine used in the preparation of said compound Selonsertib. diabetic nephropathy or diabetic kidney disease. Also disclosed are the two intermediates 5-(4-cyclopropy1-1H-imidazol-1-y1)-2-fluoro-4-methylbenzoic acid and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine used in the preparation of said compound Selonsertib.

Description

APOPTOSIS SIGNAL-REGULATING KINASE INHIBITOR CROSS NCE TO D APPLICATION This application is a divisional of NZ 739339, which is a divisional of NZ 722945, which is a divisional of NZ 627947, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to a novel compound for use in the treatment of ASK1- mediated diseases. The invention also relates to intermediates for its preparation and to ceutical compositions ning said novel compound.

BACKGROUND Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated n kinase kinase kinase (“MAP3K”) family that activates the c-Jun N-terminal protein kinase ) 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 , reactive oxygen species (ROS), LPS, TNF-, FasL, ER stress, and increased intracellular calcium concentrations (Hattori, K., Naguro, I., Runchel, C., and , H. (2009) Cell Comm. . 7:1-10; Takeda, K., Noguchi, T., Naguro, I., and Ichijo, H. (2007) Annu. Rev. Pharmacol. l. 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 have been reported to play an important role in a broad range of diseases including neurodegenerative, cardiovascular, inflammatory, autoimmune, and metabolic ers. 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 7443; Terada et al. (2007) BBRC 364: 1043-49).

ROS are reported be associated with increases of inflammatory cytokine production, fibrosis, apoptosis, and necrosis in the kidney. (Singh DK, Winocour P, Farrington K. ive stress in early diabetic nephropathy: fueling the fire. Nat Rev Endocrinol 2011 Mar;7(3):176- 184; Brownlee M. Biochemistry and molecular cell biology of diabetic cations. Nature 2001 Dec 13; 414(6865):813-820; Mimura I, Nangaku M. The suffocating kidney: tubulointerstitial hypoxia in end-stage renal e. Nat Rev Nephrol 2010 Nov; 6(11):667- 678).

Moreover, oxidative stress facilitates the formation of advanced ion end-products (AGEs) that cause further renal injury and production of ROS. (Hung KY, et al. N- [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm acetylcysteine-mediated antioxidation prevents hyperglycemia-induced apoptcsis and collagen sis in rat mesangial cells. Am ol 2009;29(3):192—202).

Tubulointerstitial fibrosis in the kidney is a strong tor of progression to renal failure in ts with chronic kidney diseases (Schainuck LI, et a1. Structural-functional correlations in renal disease. Part II: The c0rrelations. Hum Pathol 1970; 1: 631—641.).

Unilateral al obstruction (UUO) in rats is a Widely used model of tubulointerstitial fibrosis.

UUO causes tubulointerstital inflammation, increased expression of transforming growth factor beta (TGF—B), 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 renal fibrosis (Chevalier et al., Ureteral obstruction as a model of renal titial fibrosis and obstructive nephropathy, Kidney International (2009) 75, 1 145—1 1 52.

Thus, therapeutic agents that function as inhibitors of ASK1 signaling have 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, ASKl inhibitors have the potential to treat cardio—rena1 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 c liver diseases. 2O U.S. Publication No. 2007/0276050 bes methods for identifying ASKl inhibitors useful for preventing and/or treating vascular disease and methods for preventing and/or treating cardiovascular disease in an animal.

W02009027283 ses triazolopyridine compounds, methods for preparation thereof and methods for treating mune disorders, inflammatory diseases, cardiovascular diseases and egenerative es.

US. Patent Publication No. 2001/00095410A1, pub1ished January 13, 2011, discloses compounds useful as ASK-l inhibitors. US. Patent Publication No. 2001/00095410A1 relates to compounds of Formula (I): [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m ation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm R x R3 x 8\ \ x N X2 1\ l H N /N\// X7\X// X5 wherein: R1 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or cyclyl, all of which are optionally substituted with l, 2, or 3 tuents selected from halo, oxo, alkyl, cycloalkyl, heterocyclyl, aryl, aryloxy, —N02, R6, —C(O)—R6, ~OC(O)—R6 —C(O)—O-R6, — C<O>-N(R6><R7), -OC<O>-N<R6)<R7>, —S-R6, —R6, -s<=0>2R6, -S(=0)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, cycloalkyl, heterocyclyl, phenyl, and phenoxy are optionally substituted by l, 2, or 3 substituents selected from alkyl, cycloalkyl, alkoxy, hydroxyl, and halo; wherein R6 and R7 are independently selected from the group consisting of hydrogen, C1- C15 alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, all of which are optionally substituted with 1—3 substituents selected from halo, alkyl, mono— or dialkylamino, alkyl or aryl or aryl 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, all of which are optionally tuted with one or more substituents selected from alkyl, , cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl,heterocyclylalkyl, halo, OX0, —N02, haloalkyl, haloalkoxy, —CN, -O—R6, —'O-C(O)—R6, -O—C(O)—N(R6)(R7), -S—R6, — N<R6)<R7), -S<=O>-R6, —S(=0)2R6, 2—N<R6)<R7>, —S<=0)2R6, -N(R")-C<O>- R7, -N(R6)—C(O)-O—R7, —N(R6)—C(O)—N(R6)(R7), R6, -C(O)-O-R6, —C(O)— R7), and —N(R6)—S(=O)2—R7, wherein the alkyl, alkoxy, cycloalkyl, aryl, heteroaryl or heterocyclyl is further optionally substituted with one or more substituents selected from halo, oxo, -N02, alkyl, haloalkyl, haloalkoxy, -N(R6)(R7), —C(O)—R6, — [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm ation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm C(O)-O—R6, —C(O)-N(R6)(R7), -CN, —O-R6, cycloalkyl, aryl, heteroaryl and cyclyl; 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, — N02, 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, -C(O)—R7, —N(R6)—C(O)—O-R7, —N(R6)—C(O)- N(R")(R7), —C(O)—R6, -C(O)-O—R6, —C(O)—N(R6)(R7), or -N(R6)—S(=O)2-R7, n the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is further optionally substituted with one or more substituents selected from halo, oxo, -N02, -CF3, -O—CF3, —N(R6)(R7), -C(O)-R6, -C(O)—O-R7, N(R6)(R7), —CN, —O—R6; or X5 and X6 or X6 and X7 are joined to provide optionally substituted fused aryl or optionally tuted 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 osz, 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 pharmacodynamic s for the ent of diseases related to ASKl activation. singly, applicants have discovered a novel compound within the scope of US. patent publication USZOl 1/000941 0A exhibiting good potency, improved pharmacokinetie and/or pharmacodynamic profiles, on aggregate, compared to compounds disclosed n.

SUMMARY OF THE INVENTION The present invention relates to a compound of the formula: WN\ O Q/ \ Nfix \ N N N / ‘ F \< (I), or a pharmaceutically acceptable salt thereof.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by m [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm In one ment, the invention relates to the use of a compound of formula (I) in the treatment of a disease in a patient in need of ent with an ASKI tor.

In another embodiment, the invention s to a ceutical composition comprising a compound of formula (I) or a ceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

In another ment, the invention is a method of treating ic nephropathy, or complications of diabetes, comprising stering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, to a patient in need thereof.

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

In another embodiment, the invention relates to a method of treating kidney fibrosis, lung fibrosis, or idiopathic pulmonary fibrosis (IPF) comprising administering a therapeutically effective amount of a compound of a (I) or a pharmaceutically acceptable salt 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 nd or salt 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 salt thereof for the treatment of chronic kidney disease.

In another embodiment, the invention s to the use of a compound of formula (I) or a pharmaceutically acceptable salt 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 salt thereof, in the manufacture of a medicament for the treatment of chronic kidney disease.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by m [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 1001531696 In yet r embodiment, the ion 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 unilateral ureteral obstruction and treated with either vehicle, or nd of formula (I) at 1, 3, 10, or 30 mg/kg b.i.d. per day.

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

Definitions and General Parameters As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not ed to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in New Zealand or any other jurisdiction.

As used , the following words and phrases are intended to have the meanings set forth below, except to the extent that the context in which they are used indicates otherwise.

Where no indication or tion is given, the ordinary g 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. c kidney disease includes by implication kidney disease.

The term “diabetic kidney disease” as used herein refers to kidney disease caused by diabetes, exacerbated by diabetes, or co-presenting with diabetes. It is a form of chronic kidney disease occurring in approximately 30% of patients with diabetes. It is defined as diabetes with the presence of albuminuria and/or impaired renal 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 . JAMA 2011 Jun 22; 305(24):2532-2539).

The term “pharmaceutically able salt” refers to salts of pharmaceutical compounds e.g. compound of formula (I) that retain the biological effectiveness and ties of the underlying nd, and which are not biologically or otherwise undesirable. There are acid [Annotation] cassarm None set by m [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] m None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm 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. rly, 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 maceutical Science, Jan. ‘1977 V01. 66, No.1, 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, ic acid, p- toluene—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 salt of the compound of formula (I) is the hydrochloride salt of the compound of formula (I).

As used , “pharmaceutically able carrier” includes excipients or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic 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 prepare compositions of ceutically active substances is well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS. Banker & C.T. Rhodes, Eds.) The term “cardio-renal diseases” as used herein refers to diseases, d to the on of the kidney, that are caused or exacerbated by cardiovascular problems such as, for example, high blood re 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 (PP).

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 e being treated, the weight and/or age of the patient, the severity of the disease, or the manner of administration, as determined by a ied prescriber or care giver.

[Annotation] m None set by cassarm ation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm The term “treatment” or “treating” means administering a compound or ceutically acceptable salt 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 regression of al symptoms or the severity thereof.

In a preferred ment, the invention relates to the use of the compound of formula (I) in treating chronic kidney disease sing 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 r 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 tory tration (ICSO) 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 desirable goal to discover a therapeutic agent, for example a compound that inhibits apoptosis signal—regulating kinase (ASKl) with a low leo. In this manner, undesirable side effects are minimized by the ability to use a lower dose of the therapeutic agent to t the ASKl enzyme.

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

Similarly, it is a desirable goal to discover a compound having a low Eng. EC50 is the concentration of a drug that achieves 50% maximal efficacy in the cell. The EC50 value translates to the tration of a compound in the assay medium necessary to achieve 50% of the maximum efficacy. Thus, a lower EC50 is lly red for drug development.

[Annotation] cassarm None set by cassarm [Annotation] m MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm A useful unit of measure associated with EC50 is the protein binding adjusted EC50 (FEW-£050 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 es the efficacy of the drug corrected for or correlated to the amount of drug that is available at the target site of action.

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

Another desirable goal is to discover a drug that exhibits minimal off-target inhibition.

That is, a drug that minimally inhibits the Cyp450 hrome 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 nd 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 ered a weak inhibitor.

A measure useful for comparing cyp3A4 inhibition among drug candidates is the ratio of Cyp3A4 inhibition and the protein binding adjusted ECso. This value gives an indication of the relative potential for cyp inhibition corrected for the protein binding adjusted EC50 which is c to each drug. A higher ratio in this e is preferred as indicative of lower potential for cyp3A4 inhibition.

Unexpectedly and advantageously, applicants have ered a compound (of formula (1) herein) within the generic scope of U.S. Patent publication No. 2001/00095410A1 that provides advantages compared to structurally close compounds (herein ated as compounds A and B) disclosed in U.S. Patent publication No. 2001/00095410Al WN\ 0 KEY \ NUKH \ N N N / ‘N \(NJ Compound A ation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] m Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by m wN\ 0 NQV/N‘/ H NJN Compound B.

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

Combination Therapy Patients being treated for cardio-renal diseases such as chronic kidney disease may benefit from combination drug treatment. For example the nd of the present 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 benefit of combination may be sed efficacy and/or reduced side effects for a ent as the dose of that ent may be ed down to reduce its side effects While benefiting from its efficacy ted 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 benefit from co—administration (as directed by a qualified caregiver) of a therapeutic agent or agents that are antibiotic, analgesic, antidepressant and/or anti—anxiety agents in combination with compound of formula (1).

Combination treatments may be administered simultaneously or one after the other within intervals as directed by a qualified caregiver or via a fixed dose (all active ingreditents are combined into the a single dosage form e.g. tablet) presentation of two or more active .

Pharmaceutical Compositions and Administration The compound 0f the present invention may be administered in the form of a pharmaceutical composition. The present invention therefore provides pharmaceutical compositions that contain, as the active ingredient, the compound of a (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients and/or carriers, including inert solid diluents and fillers, diluents, including sterile aqueonolution and s organic solvents, permeation enhancers, solubilizers and adjuvants.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] m Unmarked set by cassarm The pharmaceutical compositions may be administered alone or in combination with other therapeutic agents. Compositions may be prepared for delivery as solid tablets, capsules, s, ointments, skin patches, sustained release, fast disintegrating tablets, tion preparations, etc. Typical pharmaceutical compositions are prepared and/or stered 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 Modern 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 ted as fixed dose formulations e. g. tablets, elixirs, liquids, ointments, inhalants, gels, etc., using ures 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 arterial ion, intravenously, intraperitoneally, parenterally, intramuscularly, aneously, orally, topically, as an inhalant, or Via an impregnated or coated device such as a stent, for e, or an artery—inserted cylindrical polymer. Most preferred routes of administration include oral, parental and intravenous stration.

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 ly 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 nd if applicable, the age, weight, response of the individual patient, the severity of the patient’s symptoms, and the like.

Nomenclature The name of the compound of the present ion as generated using ChemBioDraw Ultra 11.

WN\ O / \ N \ N F \(N [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm ed set by cassarm is 5-(4—cyclopropyl- l H—imidazol— l —yl)-N—(6—(4—isopropyl—4H—l ,2,4—triazol—3 ridin—2—yl)—2- fluoro—4—methylbenzamide also known as 5—((4-cyclopropyl-lH—imdazol— l -yl)~2—fluoro—N—(6—(4- isopropyl-4H—1,2,4-triazole-3 —yl)pyridine—2-yl)—4—methylbenzamide.

SJ/nthesis of the Compound of Formulafll The compound of the invention may be prepared using s sed 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. Alternatively, reagents may be prepared using reaction schemes and methods known to one of skill in the art.

Synthetic Reaction Parameters The terms “solven 79 ‘6'inert organic solven ” or “inert solven ” refer to a solvent inert under the conditions of the reaction being described in conjunction therewith (including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (Tl-IF), dimethylformamide (DMF), chloroform, methylene de (or dichloromethane), l ether, petroleum ether (PE), methanol, pyridine, ethyl e (EA) and the like. Unless ed to the contrary, the solvents used in the reactions of the present invention are inert organic solvents, and the reactions are carried out under an inert gas, preferably en.

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

Scheme 1 Bye\ \ H2NNH2 l DMF-DMA __““‘““*__ / o H2N N H2N N 0\ HN.NHZ OYN/ \ I AcOH CH CN ’ 3 O HN2 N / ~ \NQN N/ N | \FNHZ N\// B HNiNAlTl/ C \< Preparation of Compound A To a solution of methyl 6-aminopicolinate (432 g, 2.84 mol) in MeOH (5 L) was added NHZWZO (284 g, 5.68 mol, 2.0 eq.). The reaction mixture was heated under reflux for 3 hr [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm and then cooled to room temperature. The precipitate formed in the mixture was ted by filtration, washed with EA (2 L><2) and then dried in vacuo to give compound A (405 g, 94% yield) as White solid.

Preparation 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 temperature 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 temperature, the solid was collected by ion and dried in vacuo to give compound B (572 g, 82% yield) as white solid.

Preparation of C To a solution of compound B (572 g, 2.18 mol) in a mixture of CH3CN-AcOH (3.6 L, 4:1) was added propanamine (646 g, 5.0 eq.). The resulting mixture was heated under reflux for 24 hr and then cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in water (2.8 L) and l N aqueous NaOH was added to a pH of 8.0 H. The precipitate was collected by filtration and the filtrate was extracted with EA (500 mLX3). The combined organic layers were dried over ous Na2804, and then trated to a volume of 150 mL. To this mixture at 0°C was slowly added PE (400 mL) and the ing suspension was filtered. The combined solid was stallized from EA—PE to give compound C (253 g, 57% yield) as off-white solid. 1H-NMR (400 MHZ, CDC13): 5 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 (1381+) m/z: 204 (M+1)+.

Compound C is a key ediate for the synthesis of the compound of formula (1). Thus, an object of the present invention is also the ion of the intermediate compound C, its salts or protected forms thereof, for the preparation of the compound of formula (I). An example of a salt of the compound C is the HCl addition salt. An e of a protected form of compound C is the carbamate compound such as obtained with Cbz—Cl. Protective groups, their ation and uses are taught in Peter G.M. Wuts and Theodora W. Greene, Protective Groups in Organic Chemistry, 2nd edition, 1991, Wiley and Sons, Publishers.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm Scheme 2 Preparation of the nd of formula (I) continued: wager Mtg“! fikBr V/LK/HN CN 1 2 3 O \7 U: W\NUCN SH OH .___ ‘ D /\\j/N\ CN Formula (I) Compound 6 is a key ediate for the synthesis of the compound of a (1). Thus an object of the present invention is also the provision of intermediate nd 6, salts or protected forms thereof, for the preparation of the compound of formula (I). An example of a salt of the compound 6 is the HCl addition salt. An example of a protected form of the compound 6 is an ester (6. g. methyl, ethyl or benzyl esters) or the carbamate compound such as obtained with Cbz—Cl. tive groups, their preparations and uses are taught in Peter G.M.

Wuts and Theodora W. Greene, Protective Groups in Organic Chemistry, 2nd edition, l99l, Wiley and Sons, Publishers.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm Step 1 —— Preparation of 5—amino—2-fluoro—4-methylbenzonitrile — Compound (21 The starting 5—bromo—4—fluoro-Z—methylaniline (l) (20g, 98 mmol) was dissolved in anhydrous 1—methylpyrrolidinone (100 mL), and copper (l) cyanide , 196 mmol) was added. The reaction was heated to 180°C for 3 hours, cooled to room temperature, and water (300 mL) and concentrated ammonium hydroxide (300 mL) added. The mixture was stirred for minutes and extracted with EA (3 x 200 mL). The combined extracts were dried over magnesium 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 romethane and loaded onto a silica gel . Eluting with 0 to 25% EA in hexanes gradient provided S—amino-Z—fluoro— 4—methy1benzonitrile (10.06g, 67.1, mmol). LC/MS (m/zzlSl M“).

Step 2 — Pre aration of 5— 2-0 clo ro ~2-fluoro—4—meth lbenzonitrile — Compound (3) ~Amino—2-fluoro~4-methylbenzonitrile (12g, 80mmol) was dissolved in anhydrous N,N— dimethylformamide (160 mL) under nitrogen, and potassium ate (13.27g, 96 mmol) and ium iodide (14.61g were added as solids with stirring. The on was stirred for 5 minutes at room ature 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 re. The residue was dissolved in EA (400 mL) and washed with 400 mL of water. The organic layer was dried over magnesium 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 on to 3:1 hexanes: EA by . The product precipitated out of solution and was collected by filtration to e 5-(2—cyclopropyl oxoethylamino)—2-fluoro~4—methylbenzonitrile (14.19g, 61.2 mmol). LC/MS (m/z : 233, M“) Step 3 - Pre aration of 5— 4—c clo ro 1—2—merca to—lH—imidazol-l- l—2—fluoro methylbenzonitrile’ — Compound {41 —(2—Cyclopropyl—2~oxoethylamino)—2—fluoro—4—methylbenzonitrile (14.19g, 61.2mm01) 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 s extract was extracted with (b 200 mL) additional dichloromethane, the organic extracts combined and dried over [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm magnesium sulfate. The solvent was removed under reduced pressure and the oily residue was re—dissolved in BA (50 mL) and 150 mL hexanes was added. A dark layer formed and a stir bar was added to the flask. Vigorous ng caused the t to precipitate as a peach colored solid. The product was collected by filtration, to yield 5—(4—cyclopropyl—2-mercapto—lH- imidazol—l—y1)-2—fluoro—4—methylbenzonitrile, (14.26g, 52.23 mmol). Anal. LC/MS (m/Z : 274, M+1) Step 4 — Preparation of 5-(4—cyclopropyl—1H—imidazol—l—yl)—2-fluoro—4—methylbenzonitrile - Compound 15) In a 500 mL three neck round bottom flask was placed acetic acid (96 mL), water (19 mL) and hydrogen de (30%, 7.47 mL, 6588 mmol). The mixture was heated to 45°C with stirring under nitrogen while monitoring the al temperature. 5—(4—Cyclopropyl—2— mercapto-lH—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 internal temperature below 55°C. When addition of the thioimidazole was complete the reaction was stirred for 30 s at a temperature of 45°C, and then cooled to room temperature, 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 magnesium sulfate, and the solvent was removed under d re. The residue was ved 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, yclopropy1—1H— imidazol—l—yl)—2—fluoro—4—methy1benzonitrile was obtained as a Viscous oil (5.14 g, 21.33 mmol) Anal. LC/MS (m/z: 242, M“) Step 5 — Preparation of 5—! 4-cyclopropyl—1H—imidazol—1—yl_)_—2—fluoro—4—methylbenzoic acid hydrochloride 1 6 ) —(4-Cyclopropyl-1H-imidazol—1—yl)-2—fluoro—4-methylbenzonitri1e (l 1.21 g, 46.50mmol) 3O 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 temperature. Solvent was removed under reduced pressure to give a pink solid, to which was a 100ml of EA. The solid product was collected by filtration and washed with 3 x100 [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm mL EA. To the solid t 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 filtrates were combined and solvent was removed under reduced pressure, to provide crude 5—(4—cyclopropyl—1H—imidazol—l—yl)-2—fluoro-4— methylbenzoic acid hydrochloride. No further purification was carried out (11.13g, 37.54mm61). Anal. LC/MS (m/z: 261, M“) Step 6 — Preparation of 5-14—cyclopropyl-lH—imidazol—l—yl1—2~fluoro—N—(6-(4-isoprgpyl~4H— 1,2J4—triazol-3~yllp3gridin—2—yl)—4—methvlbenzamide — formula (I) 5-(4—Cyclopropyl—1H—imidazol—l~yl)-2—fluoro—4-methylbenzoic acid hydrochloride (1.5g, .07mmol) was suspended in anhydrous 1,2—dichloromethane (25 mL) at room temperature.

Oxalyl chloride ml, 6.59mmol) was added with stirring under nitrogen, followed by N,N— dimethylformamide (0.044ml, 0.507mmol). The ;mixture was stirred for 4 hr at room temperature, 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—2— amine (1.13 g, 5.58mmol) (compound C) and 4—dimethylaminopyridine (0.62g, 5.07 mmol) were rapidly added with stirring under nitrogen. The on was stirred for 2 hours at room ature and aqueous ted NaHC03 (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 romethane. The ed organics were dried (MgSO4), d and concentrated. The residue was dissolved in a m amount of CH3CN and water was slowly added until solids precipitated from the mixture. The solid was collected by ion and dried to give 5—(4- cyclopropyl— 1 H—imidazol— l —yl)fluoro—N—(6—(4—isopropyl-4H— l ,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“). The material was further purified by RP-HPLC (reverse phase HPLC) to obtain an analytically pure sample as the HCl salt. wflkN/ENEYN‘NN\ O / F \(NJ/ C24H24FN7O-HC1. 446.2 (M+1). 1H—Nl\/IR(Dl\/ISO): 8 11.12 (s, 1H), 9.41 (s, 1H), 9.32 (s, 1Hb820 (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 = [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 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), .06 (m, 2H), 0.85—0.89 (m, 2H).

Biological Assays ASK1 (Apoptosis Signal-Regulating Kinase 1) TR—FRET Kinase Assay emical IC50) The abilityof compounds to inhibit ASKl kinase activity was determined using a time resolved fluorescence resonance 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 2uL/well of compounds in 2.44% s DMSO into low volume 384-well polypropylene plates [Nunc, #267460] to give a final concentration ofbetween 100uM and 0.5nM compound in the kinase assay. A Deerac Fluidics Equator was used to dispense ll of 0.667ng/uL [Upstate Biotechnologies, #14—606, or the equivalent protein prepared in-house] and 0.1665ng/mL biotin-MBP [Upstate Biotechnologies, 1] 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 nds.

The enzyme was allowed to pre-incubate with compound for 20 minutes prior to initiating the kinase reaction with the addition of SuL/well 300MM ATP in buffer (50mM MOPS, pH 7.0, 5mM Mg-acetate, lmM DTT, 5% DMSO) using the Deerac Fluidics Equator.

The kinase reactions were allowed to proceed for 20 s at ambient ature and were subsequently d with the addition of 5uL/well 25mM EDTA using the Deerac Fluidics Equator. The Biomek FX was then used to transfer 1 uL/well of each completed kinase on to the wells of an OptiPlate-1536 white polystyrene plate [PerkinElmer, 99] that contained SuL/well detection reagents (1.11nM Eu-W1024 labeled hosphothreonine antibody [PerkinElmer, #AD0094] and 55.56nM streptavidin ycocyanin [PerkinElmer, #CR130—100] in 1X LANCE detection buffer [PerkinElmer, #CR97-100]). The TR—FRET signal was then read on a Perkin Elmer Envision plate reader after incubating the plates at ambient temperature for 2 hours.

The 100% inhibition positive control wells were generated by ing 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.

Result [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm ed set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 1001569449 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 itive ligand ATP.

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

Materials and Methods Reagents Dephosphorylated recombinant human ASK1 kinase was from Gilead Sciences. Small molecule kinase inhibitor staurosporine (Catalogue # S6942) and threitol (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). All other reagents were of the highest grade cially available.

Assays The assay measures the phosphorylation level of a biotinylated peptide substrate by the ASK1 kinase using HTRF detection (6.1). This is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on HTRF® KinEASE™-STK manual from Cisbio (6.1). Test nd, 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 µM ATP is added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with 1X Eu3+ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 are added to stop the on and phosphorylated peptide substrate is detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence is measured at 615 nm (Cryptate) and 665 nm ) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET level (a ratio of 665 [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm nm/615 nm) is proportional to the phosphorylation level. Under these assay ions, the degree of phosphorylation of peptide substrate was linear with time and concentration for the . The assay system yielded consistent results with regard to Km and specific activities for the enzyme. For inhibition experiments (IC50 values), activities were med with constant concentrations of ATP, peptide and several fixed concentrations of inhibitors. Staurosporine, the nonselective kinase inhibitor, was used as the positive control. All enzyme activity data are reported as an average of plicate ination.

Data Analysis The IC50 values were calculated ing equation: 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 (1) exhibited an ICSO 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 or.

ASKl (Apoptosis -Regulating Kinase 1) 293 ased assay (Cellular ECSO) The cellular potency of compounds was assayed in cells stably expressing an AP- 1:luciferase reporter construct (293/AP1—Luc cells - Panomics Inc, 6519 Dumbarton Circle, Fremont, CA). Cells were infected with an adenovirus expressing kinase active ASKl (631— 1381 of rat ASKl cDNA), which will activate the AP—l transcription factor and increase the sion of luciferase. Inhibitors of ASK1 will decrease the enzyme activity ofASK1 and therefore decrease the activity of AP-l ription factor and the expression of luciferase.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm 1. MATERIALS REQUIRED FOR THIS PROTOCOL Media and Reagents Source Company g No.

AP—l er 293 Stable Cell Line Panomics Unknown DMEM (W/ high e, W/o L— MediaTech 15—018—CM glutamine, w/ pyruvate, W/ HEPES DMEM (W/ high glucose, w/o L— Invitrogen 028 glutamine, w/o pyruvate, w/o HEPES, w/o phenol red HEPES, 1M Invitrogen 15630—080 Sodium Pyruvate, 100 mM Invitrogen 11360-070 Fetal Bovine Serum, “FBS” e SH30088.03 rep—Glut., “PSG” lnvitrogen 10378—016 HygromycinB Calbiochern 400052 Dulbecco’s PBS (sterile) MediaTech 21—030-CM Trypsin—EDTA (0.25%) Invitrogen 25200—056 Steady—G10 rase Assay Promega E2550 System Labware Source Catalog No.

Flasks (poly—D—Lysine coated, 150 BD Biosciences 356538 cmz, vented cap) Plates (poly—D—Lysine coated, 3 84— Greiner (through VWR 781944 (82051 -3 54) well, clear, sterile TCT) Scientific) White Backing Tape PerkinElmer 6005199 Cell Strainers (40 um nylon, blue VWR Scientific 21008—949 ring, fits 50 mL conical Vials) 2. REFERENCE MATERIALS Panomics 293/APl-Luc stable cell—line product insert.

Promega Steady-G10 Luciferase Assay System product insert.

[Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] m None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm 3. MEDIA ED Complete Growth Medium, “CGM” DMEM (MediaTech) % PBS 1% PSG 100 ug/mL HygromycinB Assay Medium, “AM” DMEM (Invitrogen) mM HEPES 1 mM Sodium Pyruvate 1% PSG 4. METHODS Maintenance: 293/APl—Luc Maintain ells per vendor’s instructions; harvest cells at ~80% confluence in T150 flasks as follows: Aspirate media, wash gently with ~12 mL e 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 r poly—D-Lysine coated 384—well plates at 1.2 x 104 cells per well using BioTek uFill (80 uL per well).

Immediately dose plates with 0.4 uL of compound dose series (in 100% DMSO) incubate 24 hours in humidified incubator (37°C, 5% C02).

Assay Day 2: Process plates (per manufacturer’s instructions) as follows: [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m ation] cassarm Unmarked set by cassarm Set plates in laminar flow hood & uncover for 30 minutes at room temperature to cool.

Remove 60 uL ofAM from assay wells Add 20 uL per well Steady-G10 Firefly substrate, let sit for l0-20 minutes at room temperature Cover bottom of assay plates with white backing tape.

Acquire data on a fluorescence plate reader The 100% inhibition positive control wells were ted by infecting cells with an adenovirus expressing catalytically inactive ASKl mutant with lysine to argine mutation at residue 709.

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

Determination of Kd Kinase assays Kinase—tagged T7 phage s 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 lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in 3 cells and subsequently tagged with DNA for qPCR detection. Streptavidin—coated magnetic beads were treated with biotinylated small le ligands for 30 minutes at room ature to te affinity resins for kinase The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% (bovine serum albumin), 0.05% Tween 20, 1 mM DTT(dithiothreitol)) to remove unbound ligand and to reduce non—specific binding. Binding reactions were assembled by ing kinases, liganded affinity beads, and test compounds in 1x binding buffer (20% SeaBlock, 0.17X PBS, 0.05% Tween 20, 6 mM DTT). All ons were med in polystyrene 96—well plates in a final volume of 0.135 mL. The assay plates were incubated at room temperature 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 (1X PBS, 0.05% Tween 20, 0.5 uM non—biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.

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

[Annotation] m None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm Result The nd of formula (1) exhibited a Kd of 0.24 nM. This data ts that the compound of formula (I) binds ly to ASKl receptor in the e of ATP.

Determination of Percent of Compound bound to Plasma Experimental : lmL Teflon dialysis cells from Harvard tus (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 uM of compound was spiked into lmL of plasma or lmL of cell culture media. The total volume of liquid on each side ' of the cell was 1mL. After 3 hours equilibration in a 370C water bath, samples from each side of the cell were aliquoted into the appropriate vials containing either 1mL of human plasma (cell culture media), or buffer. Sample'vials were weighed and recorded. A 100 uL aliquot was removed and added to 400 uL quenching solution (50% methanol, 25% acetonitrile, 25% water and internal standard). Samples were vortexed and centrifuged for 15 minutes at 12000 G. 200 uL of the supernatant was removed and placed into a new 96 well plate. An additional 200 uL of 1:1 ACNzwater was added. The plate was then vortexed and subjected to LC—MS is.

The percent unbound for an analyte in plasma was calculated using the following equations % Unbound = 100(Cf /Ct) where Cf and Ct are the post-dialysis buffer and plasma concentrations, respectively.

Results The percent unbound ed in human plasma for the compound of formula (1) is 11.94% ination of CACO-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% NEAA and 10% fetal bovine serum in an incubator set at 37°C, 90% humidity and 5% C02. Caco-2 cells between [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] m MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm passage 62 and 72 were seeded at 2100 cells/well and were grown to confluence over at least 21— days on 24 well PET (polyethylene—terephthalate) plates (BD Biosciences). The receiver well contained HBSS buffer (lOmM HEPES, lSmM Glucose with pH adjusted to pH 6.5) supplemented with 1% BSA pH adjusted to pH 7.4. After an initial equilibration with ort buffer, TEER values were read to test membrane integrity. Buffers containing test nds were added and 100 Lil of on was taken at l and 2 hrs from the receiver compartment. d buffer was replaced with fresh buffer and a correction is applied to all calculations for the removed material. The experiment was carried out in replicate. All samples were immediately collected into 400 pl 100% acetonitrile acid to precipitate protein and stabilize test nds. Cells were dosed on the apical or basolateral side to determine 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 bility 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, Papp, and % recovery were calculated as follows: Pa”, = (dR/dt) X Vr/(A X D0) % Recovery = 100 X ((Vr x R120) + (Vd x D120))/ (Vd X D0) where, dR/dt is the slope of the cumulative concentration in the receiver compartment versus time in uM/s based on er concentrations measured at 60 and 120 minutes.

V, and Vd is the volume in the receiver and donor compartment in cm3, respectively.

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

D0 and D120 is the ed donor concentration at the beginning and end of the experiment, respectively.

R120 is the receiver concentration at the end of the ment (120 s).

Absorption and Efflux Classification: Papp (A to B) 2 1.0 x 10‘6 cm/s High 1.0 x 10~6 cm/s > Pam, (A to B) 2 0.5 x 10'6 cm/s Medium [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm Papp (A to B) < 0.5 x 10'6 cm/s Low Papp (B to A)/ Papp (A to B) Z 3 Significant Efflux % recovery < 20% May affect measured permeability Cell Free Papp < 15 May affect measured permeability Result The nd of formula (I) was observed to have a CACO A—eB value of 27; and a CACO B—>A value of 35 ing in a efflux ratio (B—eA)/(A——>B) of 1.3.

Determination of Metabolic Stability in Hepatic omal Fraction: Experimental: Metabolic stability was assessed using cofactors for both oxidative metabolism (NADPH) and conjugation (UDP glucuronic acid D. ate aliquots of the compound of formula (I) (3 uL of 0.5 mM DMSO stock) or metabolic stability standards (Buspirone) were added to microsome stock diluted with ium 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 ofNADPH rating system and UDPGA cofactor. The final composition of each reaction mixture was: 3 uM test compound, 1 mg microsomal protein/mL, 5 mM UDPGA, 23.4 ug/mL alamethicin, 1.25 mM NADP, 3.3 mM glucose—6-phosphate, 0.4 U/mL glucose—6—phosphate dehydrogenase and 3.3 mM Mng in 50 mM potassium ate , pH 7.4. At 0, 2, 5, 10, 15, 30, 45, and 60 min, ML aliquots of the reaction mixture were transferred to plates containing 250 ul of 18/Q (quenching solution containing internal standard). After quenching, the plates were centrifuged at 3000 X g for 30 minutes, and 10 uL aliquots of the supernatant were analyzed using LC/MS to obtain analyte/internal standard peak area ratios. lic 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: [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] m Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm Ct = C0 oe“K" and TU2 =ln2/Kwhere Ct % of parent remaining at time = t C0 % of parent remaining at time = 0 t time (hr) K First order elimination rate constant (hr’l) T1/2 In Vitro half—life (hr) The predicted hepatic clearance was calculated as s {reference 1}: CL. __ KOV-YI/ 01‘ CLint __ K-VOYH ~ __ int P H CLh : (CLint . Q11)/(CL + Qh) Where int 9 IO CLh Predicted hepatic clearance (L/hr/kg body weight) CLim Intrinsic hepatic clearance (L/hr/kg body weight) V Incubation volume (L) Yp Microsome protein yield (mg protein/kg body weight) YH Hepatocyte yield (millions of cells/kg body weight) P Mass of protein in the incubation (mg) H Number of cytes in the incubation (million) Qh Hepatic blood flow (L/hr/kg body weight) Predicted hepatic extraction was then calculated by comparison of predicted hepatic nce to hepatic blood flow. A nd was considered stable if the reduction of substrate concentration was < l0% over the course of the incubation (corresponding to an extrapolated half—life of > 395 min in microsomal fractions and > 39.5 hr in hepatocytes).

Values used for calculation of the predicted hepatic clearance are shown in the tables below: [Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by m [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm Table 1. Values Used for Calculation of the Predicted Hepatic Clearance from Microsomal Stability Hepatic Microsomes V P Y (L/kg) Spedes (L) (mg) (mg/kg) Rat 0.001 1.0 1520 4.2 Cynomolgus Monkey 0.001 1.0 684 1.6 Rhesus Monkey 0.001 1.0 1170 2.3 Dog 0.001 1.0 1216 1.8 Human 0.001 1.0 977 1.3 The predicted hepatic clearance in human as determined from in vitro experiments in microsomal fractions is 0.1 L/h/kg.

Determination of Rat CL and Vss for Test Compounds Pharmacokinetics of Test Compounds following a 1 mg/kg 1V infusion and 5.0 mg/kg PO dose in rats Test e and formulation For N administration the test compound was formulated in 60:40 PEG 400:water with 1 equivalent HCl at 0.5 mg/mL. The formulation was a solution.

For PO (oral) administration, the test compound was formulated in 0/10 ethanol/PG/solutol/water at 2.5 mg/mL. The formulation was a solution.

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

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 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 inerTM tubes (Becton—Disckinson Corp, New Jersey, USA) containing EDTA as the anti—coagulant and were immediately placed on wet ice pending centrifugation for plasma.

Determination of the trations of the Compound of Formula fl) in plasma An LC/MS/MS method was used to measure the concentration of test compound in plasma. ations Non—compartmental cokinetic analysis was performed on the plasma concentration-time data.

Results The compound of formula (1) ted a CL of 0.09 L/hr/kg; an oral bioavailability of 75 %; tug 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, CYPlA, CYP1A2, CYP2B6, CYP2C8, CYP2C9, 9, CYP2D6 and CYP3A4 (2 substrates).

Cytochrome P450 Inhibition ICso Determination (8 lsoform, 9 ates) Protocol Summary Test compound (0.1 uM —— 25 MM) is ted with human liver omes and NADPH in the presence of a cytochrome P450 m—specific probe substrate. For the , CYP2C8, CYP2C9, CYP2Cl9, CYP2D6 and CYP3A4 specific reactions, the metabolites are monitored by mass spectrometry. CYPlA activity is monitored by measuring the [Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm formation of a fluorescent metabolite. A decrease in the formation of the metabolite compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).

Assay ements 500uL of a 10mM test compound solution in DMSO. mental Procedure CYP lA tion Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 uM in DMSO; final DMSO tration = 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 nM) for 5 min at 37°C. The selective CYPlA inhibitor, alpha—naphthoflavone, is screened alongside the test compounds as a positive control.

CYP2B6 Inhibition Six test compound concentrations (0.1, 0.25, l, 2.5, 10, 25 nM 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 uM) for 5 min at 37°C. The selective CYP2B6 tor, 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 uM 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 ate paclitaxel (7.5 pM) for 30 min at 37°C. The selective CYP2C8 inhibitor, montelukast, is ed alongside the test compounds as a positive control.

CYP2C9 Inhibition Six test compound concentrations (0.1, 0.25, l, 2.5, 10, 25 nM 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 nM) for 60 min at 37 °C. The ive CYP2C9 inhibitor, sulphaphenazole, is screened alongside the test compounds as a positive control.

[Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm ation] cassarm ionNone set by cassarm [Annotation] cassarm Unmarked set by cassarm CYP2C19 tion Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 uM 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 mephenytoin (25 MM) for 60 min at 37 OC. The selective CYP2C19 inhibitor, tranylcypromine, is ed alongside the test compounds as a positive l.

CYP2D6 Inhibition Six test compound concentrations (0.1, 0.25, l, 2.5, 10, 25 uM 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 ate dextromethorphan (5 uM) for 5 min at 37 °C. The selective 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 uM in DMSO; final DMSO concentration = 0.26%) are incubated with human liver microsomes (0.1 ing/mL) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 uM) for 5 min at 37°C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.

CYP3A4 tion (Testosterone) Six test compound concentrations (0.1, 0.25, l, 2.5, 10, 25 uM in DMSO; final DMSO tration = 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 uM) for 5 min at 37°C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.

For the CYPlA incubations, the reactions are terminated by methanol, and the formation of the metabolite, resorufin, is monitored by fluorescence (excitation wavelength = 535 nm, emission wavelength = 595 nm). For the CYP2B6, CYP2C9, 9, CYP2D6, and CYP3A4 incubations, the reactions are terminated by methanol. The samples are then centrifuged, and the atants are combined, for the simultaneous analysis of 4— hydroxytolbutamide, 4—hydroxymephenytoin, dextrorphan, and 1—hydroxymidazolam by LC— ation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 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 internal standard is added to the final sample prior to analysis. A decrease in the formation of the metabolites compared to e control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).

Results CYP450 Calculated IC50 Substrate Metabolite Isoform (HM) lA Ethoxyresorufin Resorufin > 25 uM 1A2 etin Acetaminophen > 25 uM 2B6 Bupropion Hydroxybupropion 19.2 uM 2C8 Paclitaxel 60t—Hydroxypaclitaxel 21.6 uM 2C9 Tolbutamide oxytolbutamide >25 uM 2C19 S~mephenytoin 4—Hydroxymephenytoin > 25 uM 2D6 Dextromethorphan Iphan 17.7 uM 3A4 Midazolam Hydroxymidazolam 2.7 uM 3A4 Testosterone 6 l3 ytestosterone 10.5 nM General study design for the rat unilateral ureter obstruction (UUO) model of kidney 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 ion 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 ane anesthesia on a nosecone, and laparotomy was performed. Rats underwent complete obstruction of the right ureter (UUO) using heat sterilized instruments and aseptic surgical technique. Rats were administered 50 ul Penicillin G (i.m.) immediately post—operatively. Rats [Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm ation] cassarm Unmarked set by cassarm ation] cassarm None set by cassarm [Annotation] cassarm ionNone set by cassarm [Annotation] cassarm ed set by cassarm were d to recover in a clean, heated cage before being returned to normal Vivarium conditions. Rats were administered compounds at the dose described above twice daily (at 12 hour intervals) for the subsequent 7 days. On day 7 following surgery, rats were anesthetized with isoflurane and serum, plasma, and urine collected. Animals were then euthanized, the kidneys harvested, and renal cortical biopsies collected for morphological, histological, and biochemical analysis. All tissues for biochemical is are flash—frozen in liquid nitrogen and stored at ~80°C, tissues for histological analysis were fixed in 10% neutral buffered formalin Renal fibrosis was ted 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 enized in RIPA buffer then centrifuged at 14000 x g for s at at 4 OC. The supernatant was ted into pro-chilled tubes and the protein concentration was determined. Equivalent amount of total protein were subjected to a C01 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 Role of p38 n—Activated Protein Kinase Activation in Renal Fibrosis JAm Soc Nephrol 15: 370—3 79, 2004).

Results: The compound of formula (I) was found to significantly reduce kidney Collagen lV ion (figure 1) and accumulation of alpha—smooth muscle positive myofibroblasts (figure 2) at doses of3 to 30 mg/kg.

Comparative Data for Compound of Formula 11) and Reference Compounds The following table provides comparative results for the nd of formula (I) and the reference compounds A and B disclosed in US. Patent publication No. 2001/00095410Al, published January 13, 2011. Applicants note that experiments for which results are compared below were performed under similar conditions but not necessarily simultaneously.

[Annotation] cassarm None set by cassarm [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by cassarm ation] cassarm MigrationNone set by m [Annotation] cassarm Unmarked set by m Table Compound of nd A Compound B formula (I) -———Cyp3A4ICsoTestesterone(TST)) l. l (10X) 4 (2. 8X) (11M) ———— ———-* t 1/2 in rats (hr) . 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 onal Ing that is comparable to leos for compounds A and B.

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

The compound of formula (I) is a weaker Cyp3A4 inhibitor compared to compounds A and B.

The nd of Formula (I) has a CYP3A4 ICso/ PBAdj.EC50 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.

[Annotation] cassarm None set by m [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm [Annotation] cassarm None set by m [Annotation] cassarm MigrationNone set by cassarm [Annotation] cassarm Unmarked set by cassarm 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 nd 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 ties compared to compounds of formula A and B; and that the compound of a (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. 1003306246

Claims (6)

What is claimed is:

1. Use of a compound of formula (I) (I), or a pharmaceutically acceptable salt f, and a pharmaceutically acceptable carrier, in the preparation of a tablet for treating diabetic kidney disease and chronic kidney disease.

2. The use of claim 1, wherein the tablet comprises 1mg to 500 mg of the compound of formula (I).

3. The use of claim 1, wherein the tablet comprises 1mg to 250 mg of the compound of formula (I).

4. Use of a compound of formula (I) (I), or a pharmaceutically acceptable salt thereof, in the preparation of a dosage unit for treating ic kidney disease and c kidney disease.

5. The use of claim 4, wherein the dosage unit comprises 1mg to 500 mg of the compound of formula (I).

6. The use of claim 4, n the dosage unit comprises 1mg to 250 mg of the compound of formula (I).