Tri-substituted heteroaryls and methods of making and using the same

Compounds of formula (I) possess unexpectedly high affinity for Alk 5 and/or Alk 4, and can be useful as antagonists thereof for preventing and/or treating numerous diseases, including fibrotic disorders.In one embodiment, the invention features a compound of the general formula (I).

BACKGROUND OF THE INVENTION

TGFβ (Transforming Growth Factor β) is a member of a large family of dimeric polypeptide growth factors that includes, for example, activins, inhibins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and mullerian inhibiting substance (MIS). TGFβ exists in three isoforms (TGFβ1, TGFβ2, and TGFβ3) and is present in most cells, along with its receptors. Each isoform is expressed in both a tissue-specific and developmentally regulated fashion. Each TGFβ isoform is synthesized as a precursor protein that is cleaved intracellularly into a C-terminal region (latency associated peptide (LAP)) and an N-terminal region known as mature or active TGFβ. LAP is typically non-covalently associated with mature TGFβ prior to secretion from the cell. The LAP-TGFβ complex cannot bind to the TGFβ receptors and is not biologically active. TGFβ is generally released (and activated) from the complex by a variety of mechanisms including, for example, interaction with thrombospondin-1 or plasmin.

Following activation, TGFβ binds at high affinity to the type II receptor (TGFβRII), a constitutively active serine/threonine kinase. The ligand-bound type II receptor phosphorylates the TGFβ type I receptor (Alk 5) in a glycine/serine rich domain, which allows the type I receptor to recruit and phosphorylate downstream signaling molecules, Smad2 or Smad3. See, e.g., Huse, M. et al.,Mol. Cell.8: 671-682 (2001). Phosphorylated Smad2 or Smad3 can then complex with Smad4, and the entire hetero-Smad complex translocates to the nucleus and regulates transcription of various TGFβ-responsive genes. See, e.g., Massagué, J.Ann. Rev. Biochem. Med.67: 773 (1998).

Activins are also members of the TGFβ superfamily, which are distinct from TGFβ in that they are homo- or heterodimers of activin βa or βb. Activins signal in a manner similar to TGFβ, that is, by binding to a constitutive serine-threonine receptor kinase, activin type II receptor (ActRIIB), and activating a type I serine-threonine receptor, Alk 4, to phosphorylate Smad2 or Smad3. The consequent formation of a hetero-Smad complex with Smad4 also results in the activin-induced regulation of gene transcription.

SUMMARY OF THE INVENTION

The invention is based on the discovery that compounds of formula (I) are unexpectedly potent antagonists of the TGFβ family type I receptors, Alk5 and/or Alk 4. Thus, compounds of formula (I) can be employed in the prevention and/or treatment of diseases such as fibrosis (e.g., renal fibrosis, pulmonary fibrosis, and hepatic fibrosis), progressive cancers, or other diseases for which reduction of TGFβ family signaling activity is desirable.

In one aspect, the invention features a compound of formula I:

In an embodiment, X can be a 4- to 8-membered monocyclic cycloalkyl or heterocycloalkyl, or X can be a 4- to 8-membered bicyclic cycloalkyl or heterocycloalkyl. For example, X can be cyclohexyl, cyclopentyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrofuran, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.1]octane, 2-oxa-bicyclo[2.2.2]octane, 2-aza-bicyclo[2.2.2]octane, 3-aza-bicyclo[3.2.1]octane, or 1-aza-bicyclo[2.2.2]octane.

In an embodiment, X can be piperidinyl, piperazinyl, or pyrrolidinyl; each of which can be bonded to moiety Y via its nitrogen ring atom; and Y can be a bond, —C(O)O—, —C(O)—N(Rb)—, —S(O)2—, or —S(O)2—N(Rb)—, wherein Rbcan be hydrogen or C1-4alkyl.

In an embodiment, m can be 0-2.

In an embodiment, A1can be N and A2can be NRb, or A1can be NRband A2can be N; wherein Rbcan be hydrogen or C1-4alkyl.

In an embodiment, m can be 0-2; R1can be aryl (e.g., substituted phenyl) or heteroaryl; R2can be hydrogen, C1-6alkyl (e.g., C1-4alkyl), aryl, heteroaryl, —C1-4alkyl-aryl (e.g., benzyl), or —C1-4alkyl-heteroaryl (e.g., pyridylmethyl); X can be cyclohexyl, cyclopentyl, or bicyclo[2.2.2]octane; and Y can be —N(Rb)—C(O)—, —N(Rb)—S(O)2—, —C(O)—, —C(O)—O—, —O—C(O)—, —C(O)—N(Rb)—, —S(O)p—, —O—, —S(O)2—N(Rb)—, —N(Rb)—, —N(Rb)—C(O)—O—, or —N(Rb)—C(O)—N(Rc)—, —C(O)—N(Rb)—S(O)p—N(Rc)—, or —C(O)—O—S(O)p—N(Rb)—, wherein each of Rband Rccan independently be hydrogen or C1-4alkyl; A1can be N and A2can be NH, or A1can be NH and A2can be N; m can be 1; and Racan be substituted at the 6-position. For compounds of formula (I) wherein m is 1, Racan be generally substituted at the 6-position.

A1can be N and A2can be NH, or A1can be NH and A2can be N; m can be 1; and Racan be substituted at the 6-position.

Some examples of a compound of formula (I) are shown in Examples 5-215 below.

An N-oxide derivative or a pharmaceutically acceptable salt of each of the compounds of formula (I) is also within the scope of this invention. For example, a nitrogen ring atom of the imidazole core ring or a nitrogen-containing heterocyclyl substituent can form an oxide in the presence of a suitable oxidizing agent such as m-chloroperbenzoic acid or H2O2.

A compound of formula (I) that is acidic in nature (e.g., having a carboxyl or phenolic hydroxyl group) can form a pharmaceutically acceptable salt such as a sodium, potassium, calcium, or gold salt. Also within the scope of the invention are salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, and N-methylglycamine. A compound of formula (I) can be treated with an acid to form acid addition salts. Examples of such acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, methanesulfonic acid, phosphoric acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, ascorbic acid, maleic acid, acetic acid, and other mineral and organic acids well known to those skilled in the art. The acid addition salts can be prepared by treating a compound of formula (I) in its free base form with a sufficient amount of an acid (e.g., hydrochloric acid) to produce an acid addition salt (e.g., a hydrochloride salt). The acid addition salt can be converted back to its free base form by treating the salt with a suitable dilute aqueous basic solution (e.g., sodium hydroxide, sodium bicarbonate, potassium carbonate, or ammonia). Compounds of formula (I) can also be, e.g., in a form of achiral compounds, racemic mixtures, optically active compounds, pure diastereomers, or a mixture of diastereomers.

Compounds of formula (I) exhibit surprisingly high affinity to the TGFβ family type I receptors, Alk 5 and/or Alk 4, e.g., with IC50and Kivalues of less than 10 μM under conditions as described below in Examples 215 and 217, respectively. Some compounds of formula (I) exhibit IC50and Kivalues of less than 1 μM (such as below 50 nM).

Compounds of formula (I) can also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and/or alter rate of excretion. Examples of these modifications include, but are not limited to, esterification with polyethylene glycols, derivatization with pivolates or fatty acid substituents, conversion to carbamates, hydroxylation of aromatic rings, and heteroatom-substitution in aromatic rings.

The present invention also features a pharmaceutical composition comprising a compound of formula (I) (or a combination of two or more compounds of formula (I)) and at least one pharmaceutically acceptable carrier. Also included in the present invention is a medicament composition including any of the compounds of formula (I), alone or in a combination, together with a suitable excipient.

The invention also features a method of inhibiting the TGFβ family type I receptors, Alk 5 and/or Alk 4 (e.g., with an IC50value of less than 10 μM; such as, less than 1 μM; and for example, less than 5 nM) in a cell, including the step of contacting the cell with an effective amount of one or more compounds of formula (I). Also within the scope of the invention is a method of inhibiting the TGFβ and/or activin signaling pathway in a cell or in a subject (e.g., a mammal such as a human), including the step of contacting the cell with or administering to the subject an effective amount of one or more of the compounds of formula (I).

Also within the scope of the present invention is a method of treating a subject or preventing a subject from suffering a condition characterized by or resulting from an elevated level of TGFβ and/or activin activity. The method includes the step of administering to the subject an effective amount of one or more of the compounds of formula (I). The conditions include, for example, an accumulation of excess extracellular matrix; a fibrotic condition (e.g., glomerulonephritis, diabetic nephropathy, hypertensive nephropathy, lupus nephropathy or nephritis, hepatitis-induced cirrhosis, biliary fibrosis, scleroderma, pulmonary fibrosis, post-infarction cardiac fibrosis, fibrosclerosis, fibrotic cancers, fibroids, fibroma, fibroadenomas, or fibrosarcomas); TGFβ-induced metastasis of tumor cells; and carcinomas (e.g, carcinomas of the lung, breast, liver, biliary tract, gastrointestinal tract, head and neck, pancreas, prostate, cervix as well as multiple myeloma, melanoma, glioma, or glioblastomas).

As used herein, an “amino” group refers to —NRXRYwherein each of RXand RYis independently hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, aryl, aralkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, heteroaryl, or heteroaralkyl. When the term “amino” is not the terminal group (e.g., alkylcarbonylamino), it is represented by —NRX—. RXhas the same meaning as defined above.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., a C1-4alkyl group) that is substituted with an aryl group. Both “alkyl” and “aryl” have been defined above. An example of an aralkyl group is benzyl.

As used herein, “cyclic moiety” includes cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which has been defined previously.

As used herein, an “acyl” group refers to a formyl group or alkyl-C(═O)— where “alkyl” has been defined previously. Acetyl and pivaloyl are examples of acyl groups.

As used herein, a “carbamoyl” group refers to a group having the structure —O—CO—NRxRyor —NRx—CO—O—Rzwherein Rxand Ryhave been defined above and Rzcan be alkyl, aryl, aralkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl.

As used herein, a “carboxy” and a “sulfo” group refer to —COOH and —SO3H, respectively.

As used herein, an “alkoxy” group refers to an alkyl-O— group where “alkyl” has been defined previously.

As used herein, a “sulfoxy” group refers to —O—SO—RXor —SO—O—RX, where RXhas been defined above.

As used herein, a “halogen” or “halo” group refers to fluorine, chlorine, bromine or iodine.

As used herein, a “sulfamoyl” group refers to the structure —S(O)2—NRxRyor —NRx—S(O)2—Rzwherein Rx, Ry, and Rzhave been defined above.

As used herein, a “sulfamide” group refers to the structure —NRX—S(O)2—NRYRZwherein RX, RY, and RZhave been defined above.

As used herein, a “urea” group refers to the structure —NRX—CO—NRYRZand a “thiourea” group refers to the structure —NRX—CS—NRYRZ. RX, RY, and RZhave been defined above.

As used herein, an effective amount is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al.,Cancer Chemother. Rep.,50: 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970). As used herein, “patient” refers to a mammal, including a human.

An antagonist, as used herein, is a molecule that binds to the receptor without activating the receptor. It competes with the endogenous ligand(s) or substrate(s) for binding site(s) on the receptor and, thus inhibits the ability of the receptor to transduce an intracellular signal in response to endogenous ligand binding.

As compounds of formula (I) are antagonists of TGFβ receptor type I (Alk5) and/or activin receptor type I (Alk4), these compounds are useful in inhibiting the consequences of TGFβ and/or activin signal transduction such as the production of extracellular matrix (e.g., collagen and fibronectin), the differentiation of stromal cells to myofibroblasts, and the stimulation of and migration of inflammatory cells. Thus, compounds of formula (I) inhibit pathological inflammatory and fibrotic responses and possess the therapeutic utility of treating and/or preventing disorders or diseases for which reduction of TGFβ and/or activin activity is desirable (e.g., various types of fibrosis or progressive cancers). In addition, the compounds of formula (I) are useful for studying and researching the role of TGFβ receptor type I (Alk5) and/or activin receptor type I (Alk4), such as their role in cellular processes, for example, signal transduction, production of extracellular matrix, the differentiation of stromal cells to myofibroblasts, and the stimulation of and migration of inflammatory cells.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention features compounds of formula (I), which exhibit surprisingly high affinitiy for the TGFβ family type I receptors, Alk 5 and/or Alk 4.

Synthesis of the Compounds of Formula (I)

Compounds of formula (I) may be prepared by a number of known methods from commercially available or known starting materials. In one method, compounds of formula (I) wherein A1is N and A2is NH, or A1is NH and A2is N are prepared according to Scheme 1a or Scheme 1b below. Specifically, in Scheme 1a, optionally substituted 2-methylpyridine (II) is deprotonated by LDA before reacting with R1-substituted carboxylic acid methoxy-methyl-amide (V) to form an R1-(6-methylpyridyl)-ketone (III). R1has been defined above. See Example 3B below. The methoxy-methyl-amide can be prepared by reacting a corresponding acid chloride (i.e., R1—CO—Cl) with N,O-dimethylhydroxylamine hydrochloride. See Example 2 below. The R1-(6-methylpyridyl)-ketone (III) can then be treated with sodium nitrite in acetic acid to afford an α-keto-oxime (IV), which can undergo further reaction with an appropriate substituted (and optionally protected) aldehyde (VI) in the presence of ammonium acetate to yield a compound of formula (I).

In another method, the above-described compounds of formula (I) can be prepared according to Scheme 1b below. Specifically, R1-substituted pyridine-2-carbaldehyde (IIa) is first reacted with aniline and diphenyl phosphite to form a resulting N,P-acetal, which can further couple with an R1-substituted aldehyde to produced an (R1-methyl)-pyridyl-ketone (IIIa). See, e.g., Journet et al.,Tetrahedron Lett.39:1717-1720 (1998) and Example 3C below. Treatment of the (R1-methyl)-pyridyl-ketone (IIIa) with sodium nitrite in acetic acid produces an α-keto-oxime (IVa), which can undergo reaction with an appropriate substituted (and optionally protected) aldehyde (VI) to yield a compound of formula (I) as described in Scheme 1a above.

If compound (VI) is in its protected form, appropriate deprotecting agents can be applied to the resulting compound after the coupling reaction of compound (IV) or (IVa) and compound (VI) to yield a compound of formula (I). See, e.g., T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., New York (1981), for suitable protecting groups.

Alternatively, a compound of formula (I) can be prepared by reacting intermediate (IV) or (IVa) with an aldehyde (VII) to yield a further intermediate (VIII), which can then react with compound (IX) to yield a compound of formula (I). Note that moieties Y′ and Y″ are precursors of moiety Y. See Scheme 2 below. In addition, desired substitutions at Racan be obtained by selecting, for example, the appropriate compound (IIa) intermediate. See, e.g., Example 3A below.

In some embodiments, moiety X in compound (VII) is a nitrogen-containing heterocycloalkyl (e.g., piperidine). The nitrogen ring atom can be protected by a nitrogen protecting group (e.g., Cbz, Boc, or FMOC) before coupling to compound (IV) or (IVa) and deprotected afterwards (see first step of Scheme 3) to yield compound (VIIIa). This compound can further react with various compounds (IX) to produce a compound of formula (I). See second steps of Scheme 3 below. It should be noted that compound (VIII) or compound (VIIIa) can be a compound of formula (I) as well.

Similarly, when moiety X in compound (VII) is a cycloalkyl (e.g., cyclopentyl, cyclohexyl, or bicyclo[2.2.2]octane), it can be further functionalized to form a compound of formula (I) as depicted in Schemes 4, 5a, 5b, and 5c below.

Compound of formula (I) wherein A1is N and A2is NRb(or A1is NRband A2is N) can be prepared by known methods. For example, compounds of formula (I) with an unsubstituted imidazolyl core ring can be treated with RbI and CsCO3to produce a compound of formula (I) having a substituted imidazolyl core ring. See, e.g., Liverton, et al.,J. Med. Chem.,42: 2180-2190 (1999).

Compounds of formula (I) wherein A1is O and A2is NH (or A1is NH and A2is O) or wherein A1is S and A2is NH (or A1is NH and A2is S), can be prepared according to known methods. One of these methods employs the same intermediate (III) or (IIIa) as described above. See, e.g., Revesz, et al.,Bioorg. & Med. Chem. Lett.10: 1261-1264 (2000) and Scheme 6 below.

As is well known to a skilled person in chemistry, desired substitutions can be placed on the 2-pyridyl ring in the last step of the synthesis. See, e.g., Example 24 below.

Uses of Compounds of Formula (I)

As discussed above, hyperactivity of the TGFβ family signaling pathways can result in excess deposition of extracellular matrix and increased inflammatory responses, which can then lead to fibrosis in tissues and organs (e.g., lung, kidney, and liver) and ultimately result in organ failure. See, e.g., Border, W. A. and Ruoslahti E.J. Clin. Invest.90: 1-7 (1992) and Border, W. A. and Noble, N. A.N. Engl. J. Med.331: 1286-1292 (1994). Studies have been shown that the expression of TGFβ and/or activin mRNA and the level of TGFβ and/or activin are increased in patients suffering from various fibrotic disorders, e.g., fibrotic kidney diseases, alcohol-induced and autoimmune hepatic fibrosis, myelofibrosis, bleomycin-induced pulmonary fibrosis, and idiopathic pulmonary fibrosis.

Compounds of formula (I), which are antagonists of the TGFβ family type I receptors Alk 5 and/or Alk 4, and inhibit TGFβ and/or activin signaling pathway, are therefore useful for treating and/or preventing fibrotic disorders or diseases mediated by an increased level of TGFβ and/or activin activity. As used herein, a compound inhibits the TGFβ family signaling pathway when it binds (e.g., with an IC50value of less than 10 μM; such as, less than 1 μM; and for example, less than 5 nM) to a receptor of the pathway (e.g., Alk 5 and/or Alk 4), thereby competing with the endogenous ligand(s) or substrate(s) for binding site(s) on the receptor and reducing the ability of the receptor to transduce an intracellular signal in response to the endogenous ligand or substrate binding. The aforementioned disorders or diseases include any condition (a) marked by the presence of an abnormally high level of TGFβ and/or activin; and/or (b) an excess accumulation of extracellular matrix; and/or (c) an increased number and synthetic activity of myofibroblasts. These disorders or diseases include, but are not limited to, fibrotic conditions such as scleroderma, idiopathic pulmonary fibrosis, glomerulonephritis, diabetic nephropathy, lupus nephritis, hypertension-induced nephropathy, ocular or corneal scarring, hepatic or biliary fibrosis, acute lung injury, pulmonary fibrosis, post-infarction cardiac fibrosis, fibrosclerosis, fibrotic cancers, fibroids, fibroma, fibroadenomas, and fibrosarcomas. Other fibrotic conditions for which preventive treatment with compounds of formula (I) can have therapeutic utility include radiation therapy-induced fibrosis, chemotherapy-induced fibrosis, and surgically induced scarring including surgical adhesions, laminectomy, and coronary restenosis.

Increased TGFβ activity is also found to manifest in patients with progressive cancers. Studies have shown that in late stages of various cancers, both the tumor cells and the stromal cells within the tumors generally overexpress TGFβ. This leads to stimulation of angiogenesis and cell motility, suppression of the immune system, and increased interaction of tumor cells with the extracellular matrix. See, e.g., Hojo, M. et al.,Nature397: 530-534 (1999). As a result, the tumor cells become more invasive and metastasize to distant organs. See, e.g., Maehara, Y. et al.,J. Clin. Oncol.17: 607-614 (1999) and Picon, A. et al.,Cancer Epidemiol. Biomarkers Prev.7: 497-504 (1998). Thus, compounds of formula (I), which are antagonists of the TGFβ type I receptor and inhibit TGFβ signaling pathways, are also useful for treating and/or preventing various late stage cancers which overexpress TGFβ. Such late stage cancers include carcinomas of the lung, breast, liver, biliary tract, gastrointestinal tract, head and neck, pancreas, prostate, cervix as well as multiple myeloma, melanoma, glioma, and glioblastomas.

Importantly, it should be pointed out that because of the chronic, and in some cases localized, nature of disorders or diseases mediated by overexpression of TGFβ and/or activin (e.g., fibrosis or cancers), small molecule treatments (such as treatment disclosed in the present invention) are favored for long-term treatment.

Not only are compounds of formula (I) useful in treating disorders or diseases mediated by high levels of TGFβ and/or activin activity, these compounds can also be used to prevent the same disorders or diseases. It is known that polymorphisms leading to increased TGFβ and/or activin production have been associated with fibrosis and hypertension. Indeed, high serum TGFβ levels are correlated with the development of fibrosis in patients with breast cancer who have received radiation therapy, chronic graft-versus-host-disease, idiopathic interstitial pneumonitis, veno-occlusive disease in transplant recipients, and peritoneal fibrosis in patients undergoing continuous ambulatory peritoneal dialysis. Thus, the levels of TGFβ and/or activin in serum and of TGFβ and/or activin mRNA in tissue can be measured and used as diagnostic or prognostic markers for disorders or diseases mediated by overexpression of TGFβ and/or activin, and polymorphisms in the gene for TGFβ that determine the production of TGFβ and/or activin can also be used in predicting susceptibility to disorders or diseases. See, e.g., Blobe, G. C. et al.,N. Engl. J. Med.342(18): 1350-1358 (2000); Matsuse, T. et al.,Am. J. Respir. Cell Mol. Biol.13: 17-24 (1995); Inoue, S. et al.,Biochem. Biophys. Res. Comm.205: 441-448 (1994); Matsuse, T. et al,Am. J. Pathol.148: 707-713 (1996); De Bleser et al.,Hepatology26: 905-912 (1997); Pawlowski, J. E., et al.,J. Clin. Invest.100: 639-648 (1997); and Sugiyama, M. et al.,Gastroenterology114: 550-558 (1998).

Administration of Compounds of Formula (I)

As defined above, an effective amount is the amount required to confer a therapeutic effect on the treated patient. For a compound of formula (I), an effective amount can range, for example, from about 1 mg/kg to about 150 mg/kg (e.g., from about 1 mg/kg to about 100 mg/kg). Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments including use of other therapeutic agents and/or radiation therapy.

Compounds of formula (I) can be administered in any manner suitable for the administration of pharmaceutical compounds, including, but not limited to, pills, tablets, capsules, aerosols, suppositories, liquid formulations for ingestion or injection or for use as eye or ear drops, dietary supplements, and topical preparations. The pharmaceutically acceptable compositions include aqueous solutions of the active agent, in an isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient. Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds. As to route of administration, the compositions can be administered orally, intranasally, transdermally, intradermally, vaginally, intraaurally, intraocularly, buccally, rectally, transmucosally, or via inhalation, implantation (e.g., surgically), or intravenous administration. The compositions can be administered to an animal (e.g., a mammal such as a human, non-human primate, horse, dog, cow, pig, sheep, goat, cat, mouse, rat, guinea pig, rabbit, hamster, gerbil, or ferret, or a bird, or a reptile, such as a lizard).

Optionally, compounds of formula (I) can be administered in conjunction with one or more other agents that inhibit the TGFβ signaling pathway or treat the corresponding pathological disorders (e.g., fibrosis or progressive cancers) by way of a different mechanism of action. Examples of these agents include angiotensin converting enzyme inhibitors, nonsteroid and steroid anti-inflammatory agents, as well as agents that antagonize ligand binding or activation of the TGFβ receptors, e.g., anti-TGFβ, anti-TGFβ receptor antibodies, or antagonists of the TGFβ type II receptors.

Synthesis of the title compound is described in parts (a)-(c) below.

Diisobutylaluminum hydride (1.0 M solution, 6.24 mL, 6.24 mmol) was added slowly to a solution of (4-(methoxy-methyl-carbamoyl)-cyclohexyl)-carbamic acid benzyl ester

Synthesis of the title compound is described in parts (a)-(c) below.

A mixture of 6-bromo-pyridine-2-carbaldehyde (2.0 g, 10.75 mmol), ethylene glycol (3 mL, 53.75 mmol), and a catalytic amount of TsOH in toluene (50 mL) was heated to reflux with a Dean-Stark trap for 1.5 hours and cooled down to room temperature and concentrated in vacuo. The residue was purified on silica gel column with 2% EtOAc in CH2Cl2to yield 2-bromo-6-[1,3]dioxolan-2-yl-pyridine as a colorless liquid (1.97 g, 80%).

To a solution of ZnCl2in THF (0.5 M, 25 mL) was added dropwise a solution of cyclopropylmagnesium bromide (0.5 M, 25 mL) at −78° C. under nitrogen. The reaction mixture was then allowed to warm up to room temperature and stirred for an hour. The above mixture was then transferred to a sealed tube with 2-bromo-6-[1,3]dioxolan-2-yl-pyridine (1.9 g, 8.25 mmole, see subpart (a) above) and Pd(PPh3)4(0.4 g, 0.35 mmole). TLC showed major formation of the product and some starting material. The mixture was then heated to 120° C. for 2 hours and cooled down to room temperature and then worked up with EtOAc and saturated ammonium chloride and dried over MgSO4. The residue from concentration was purified on silica gel column with 5% EtOAc in CH2Cl2to yield 2-cyclopropyl-6-[1,3]dioxolan-2-yl-pyridine as a bright yellow liquid (0.96 g, 61%).

A mixture of 2-cyclopropyl-6-[1,3]dioxolan-2-yl-pyridine (0.9 g, see subpart (b) above) and a catalytic amount of TsOH hydrate in a mixture of acetone (10 mL) and water (2 mL) was heated to reflux overnight until most of the starting materials were consumed according to TLC. It was then cooled down to room temperature and concentrated. The residue was dissolved in diethyl ether and washed with saturated sodium carbonate, and then water, and then dried over MgSO4and concentrated. The concentrate was purified on silica gel column with 100% CH2Cl2to yield 6-cyclopropyl-pyridine-2-carbaldehyde as a bright liquid (0.65 g, 94%).1H NMR (CDCl3, 300 MHz), δ 9.90 (s, 1H), 7.58(m, 2H), 7.23 (m, 1H), 2.01 (m, 1H), 1.02-0.92 (m, 4H).

The titled aldehyde was converted to the corresponding N,P-acetal for ketone preparation according to Scheme 1b above.

Synthesis of the title compound is described in parts (a) and (b) below.

To a solution of 6-iodo-[1,2,4]triazolo[1,5-a]pyridine (5.0 g, 20 mmol; prepared from 2-amino-5-iodopyridine (Aldrich-Sigma, St. Louis, Mo.) according to WO 01/62756) in anhydrous THF (300 mL) at 0° C. was slowly added a solution of isopropylmagnesium bromide in THF (1 M, 31 mL, 31 mmol). The resulting milky suspension was stirred at 0° C. After an hour, DMF (6 mL, 50 mmol) was added to the suspension at 0° C. and the suspension was allowed to warm up to room temperature and stirred for 4 additional hours. 100 mL of water was then added at room temperature and stirred for 1 hour. The resulting mixture was extracted with diethylether and washed with saturated Na2CO3. The extracts were dried over MgSO4and concentrated. The residue was purified on a short silica gel cake with EtOAc to give [1,2,4]triazolo[1,5-a]pyridine-6-carbaldehyde as a light yellow solid (3 g, 100%). ESP+ m/e 148.0.1H NMR (CDCl3, 300 MHz), δ 10.03 (s, 1H), 9.10 (s, 1H), 8.49 (s, 1H), 8.02 (d, 1H), 7.82 (d, 1H).

Sodium nitrite (0.405 g, 5.88 mmol) was added to a solution of 1-benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)-ethanone (1.0 g, 3.92 mmol; see Example 3B above) in a mixture of HOAc/THF/H2O (6:4:1, 22 mL). The mixture was stirred at 0° C. for 1 hour and then at room temperature for 1 hour. Solvent was removed under reduced pressure. Residue was dissolved in water and NaOH (3N) was added until the pH value was more than 8. The aqueous solution was then extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated to give 0.90 g (81%) of 1-Benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)ethane-1,2-dione 2-oxime as a yellow foam. MS (ESP+) m/z 285.1 (M+1).1H NMR (300 MHz, CDCl3) δ 7.49 (m, 4H), 7.09 (d, 1H, J=7.5 Hz), 6.81 (d, 1H, J=7.8 Hz), 6.04 (s, 2H), 2.43 (s, 3H).

Synthesis of exemplary compounds of formula (I) are described in Examples 5-24 below.

4-Formyl-N-Cbz-piperidine (0.297 g, 1.2 mmol) was added to a solution of 1-benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)-ethane-1,2-dione 2-oxime (0.280 g, 1.0 mmol, see Example 4) and ammonium acetate (1.54 g, 20.0 mmol) in acetic acid (10 mL). The mixture was reflux for 2 hours. Solvent was removed under reduced pressure. The reaction mixture was then quenched with an ammonia/ice mixture. The aqueous solution was extracted with ethyl acetate. The extract was washed with brine, dried over sodium sulfate, filtered, and concentrated. HPLC purification eluting with acetonitrile:water gave 0.12 g (23%) of the hydroxyimidazole as a yellow solid. MS (ESP+) m/z 513.2 (M+1)

(4-Formyl-cyclohexyl)-carbamic acid benzyl ester (0.133 g, 0.507 mmol; see Example 1 above) was added to a solution of 1-benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)-ethane-1,2-dione 2-oxime (0.120 g, 0.422 mmol; see Example 4) and ammonium acetate (0.651 g, 8.44 mmol) in acetic acid (5 mL). The mixture was refluxed for 2 hours and solvent was removed under reduced pressure. The reaction mixture was then quenched with an ammonia/ice mixture. The aqueous solution was extracted with ethyl acetate. Ethyl acetate extract was washed with brine, dried over sodium sulfate, filtered, and concentrated. HPLC purification eluting with acetonitrile:water gave 0.035 g (16%) of the hydroxyimidazole as a yellow solid. MS (ESP+) m/z 527.2 (M+1).

4-Formyl-bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (0.284 g, 1.0 mmol) was added to a solution of 1-benzo[1,3]dioxol-5-yl-2-(6-methyl-pyridin-2-yl)-ethane-1,2-dione 2-oxime (see Example 4; 0.215 g, 1.1 mmol) and ammonium acetate (1.54 g, 20 mmol) in acetic acid (5 mL). The mixture was refluxed for 2 hours. Solvent was removed under reduced pressure. The reaction mixture was then quenched with ammonia/ice mixture. The aqueous solution was extracted with ethyl acetate. Ethyl acetate extract was washed with brine, dried over sodium sulfate, filtered, and concentrated to give 0.300 g (65%) of the hydroxyimidazole as a yellow solid. MS (ESP+) m/z 462.3 (M+1).

To a solution of 4-[4-benzo[1,3]dioxol-5-yl-5-(6-bromo-pyridin-2-yl)-1H-imidazol-2-yl]-piperidine-1-carboxylic acid benzyl ester (prepared in accordance with Scheme 1b with 6-bromo-piperidine-2-carbaldehyde as the starting material; 100 mg, 0.18 mmol) in DMF (1 mL) and triethylamine (2 mL) under nitrogen, was added PdCl2(PPh3)2(2 mg, 0.005 mmol) and CuI (2 mg, 0.01 mmol), then followed with trimethylsilylacetylene (30 uL, 0.20 mmol). The mixture was stirred at room temperature for 4 hours until LC-MS showed complete coupling. Diethyl ether (30 mL) was added and the precipitate was filtered off. The clear solution was washed with saturated aqueous NH4Cl, then 0.5M EDTA solution, and water, and then dried (MgSO4). Concentration gave a yellow syrup that was dissolved in THF (20 mL). The solution was cooled to 0° C. and tetrabutylammonium fluoride (2 mL, 1 M in THF) was added. The mixture was stirred at room temperature for 30 minutes until LC-MS indicated complete removal of the silyl group. The reaction mixture was then concentrated in vacuum and passed through a short silica gel column with ethyl acetate/dichloromethane (1:1). The purified material was dissolved in ethanol (20 mL) and PtO2(50 mg) was added. The mixture was stirred under hydrogen (1 atm) at room temperature for 3 days until LC-MS showed major conversion of the alkyne to the correponding alkane. The solids were filtered off and the filtrates were concentrated and purified on preparative HPLC to give the title compound (3 mg, 3%) as a TFA salt. MS (EPS+: 511.3 (MH+)).1H NMR (400 MHz, MeOH-d4) δ 7.60 (t, 1H), 7.39-7.29 (m, 5H), 7.23 (d, 1H), 7.13 (d, 1H), 6.93 (dd, 1H), 6.91 (dd, 1H), 6.81 (d, 1H), 5.95 (s, 2H), 5.14 (s, 2H), 4.28 (d(br), 2H), 3.04 (m, 1H), 3.02 (br, 2H), 2.79 (q, 2H), 2.00 (d(br), 2H), 1.85 (ddd, 2H), 1.28 (t, 3H)

The compounds listed in the following Table were prepared in an analogous manner to those described in the methods and examples above. The mass spectroscopy data of these compounds are included in the Table.

The TGFβ inhibitory activity of compounds of formula (I) can be assessed by methods described in the following examples.

Cell-Free Assay for Evaluating Inhibition of Autophosphorylation of TGFβ Type I Receptor

The serine-threonine kinase activity of TGFβ type I receptor was measured as the autophosphorylation activity of the cytoplasmic domain of the receptor containing an N-terminal poly histidine, TEV cleavage site-tag, e.g., His-TGFβRI. The His-tagged receptor cytoplasmic kinase domains were purified from infected insect cell cultures using the Gibco-BRL FastBac HTb baculovirus expression system.

To a 96-well Nickel FlashPlate (NEN Life Science, Perkin Elmer) was added 20 μl of 1.25 μCi33P-ATP/25 μM ATP in assay buffer (50 mM Hepes, 60 mM NaCl, 1 mM MgCl2, 2 mM DTT, 5 mM MnCl2, 2% glycerol, and 0.015% Brij® 35). 10 μl of each test compound of formula (I) prepared in 5% DMSO solution were added to the FlashPlate. The assay was then initiated with the addition of 20 ul of assay buffer containing 12.5 ρmol of His-TGFβRI to each well. Plates were incubated for 30 minutes at room temperature and the reactions were then terminated by a single rinse with TBS. Radiation from each well of the plates was read on a TopCount (Packard). Total binding (no inhibition) was defined as counts measured in the presence of DMSO solution containing no test compound and non-specific binding was defined as counts measured in the presence of EDTA or no-kinase control.

Alternatively, the reaction performed using the above reagents and incubation conditions but in a microcentrifuge tube was analyzed by separation on a 4-20% SDS-PAGE gel and the incorporation of radiolabel into the 40 kDa His-TGFβRI SDS-PAGE band was quantitated on a Storm Phosphoimager (Molecular Dynamics).

Compounds of formula (I) typically exhibited IC50values of less than 10 μM; some exhibited IC50values of less than 1 μM; and some even exhibited IC50values of less than 50 nM.

Cell-Free Assay for Evaluating Inhibition of Activin Type I Receptor Kinase Activity

Inhibition of the Activin type I receptor (Alk 4) kinase autophosphorylation activity by test compounds of formula (I) can be determined in a similar manner to that described above in Example 215 except that a similarly His-tagged form of Alk 4 (His-Alk 4) is used in place of the His-TGFβRI.

TGFβ Type I Receptor Ligand Displacement FlashPlate Assay

50 nM of tritiated 4-(3-pyridin-2-yl-1H-pyrazol-4-yl)-quinoline (custom-ordered from PerkinElmer Life Science, Inc., Boston, Mass.) in assay buffer (50 mM Hepes, 60 mM NaCl2, 1 MM MgCl2, 5 mM MnCl2, 2 mM 1,4-dithiothreitol (DTT), 2% Brij® 35; pH 7.5) was premixed with a test compound of formula (I) in 1% DMSO solution in a v-bottom plate. Control wells containing either DMSO without any test compound or control compound in DMSO were used. To initiate the assay, His-TGFβ Type I receptor in the same assay buffer (Hepes, NaCl2, MgCl2, MnCl2, DTT, and 30% Brij® added fresh) was added to a nickel coated FlashPlate (PE, NEN catalog number: SMP107), while the control wells contained only buffer (i.e., no His-TGFβ Type I receptor). The premixed solution of tritiated 4-(3-pyridin-2-yl-1H-pyrazol-4-yl)-quinoline and test compound of formula (I) was then added to the wells. The wells were aspirated after an hour at room temperature and radioactivity in wells (emitted from the tritiated compound) was measured using TopCount (PerkinElmer Lifesciences, Inc., Boston Mass.).

Compounds of formula (D) typically exhibited Kivalues of less than 10 μM; some exhibited Kivalues of less than 1 μM; and some even exhibited Kivalues of less than 50 nM.

Assay for Evaluating Cellular Inhibition of TGFβ Signaling and Cytotoxicity

Biological activity of the compounds of formula (I) was determined by measuring their ability to inhibit TGFβ-induced PAI-Luciferase reporter activity in HepG2 cells.

HepG2 cells were stably transfected with the PAI-luciferase reporter grown in DMEM medium containing 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and non-essential amino acids (1×). The transfected cells were then plated at a concentration of 2.5×104cells/well in 96 well plates and starved for 3-6 hours in media with 0.5% FBS at 37° C. in a 5% CO2incubator. The cells were then stimulated with 2.5 ng/ml TGFβ ligand in the starvation media containing 1% DMSO either in the presence or absence of a test compound of formula (I) and incubated as described above for 24 hours. The media was washed out the following day and the luciferase reporter activity was detected using the LucLite Luciferase Reporter Gene Assay kit (Packard, cat. no. 6016911) as recommended. The plates were read on a Wallac Microbeta plate reader, the reading of which was used to determine the IC50values of compounds of formula (I) for inhibiting TGFβ-induced PAI-Luciferase reporter activity in HepG2 cells. Compounds of formula (I) typically exhibited IC50values of less 10 uM.

Cytotoxicity was determined using the same cell culture conditions as described above. Specifically, cell viability was determined after overnight incubation with the CytoLite cell viability kit (Packard, cat. no. 6016901). Compounds of formula (I) typically exhibited LD25values greater than 10 μM.

Assay for Evaluating Inhibition of TGFβ Type I Receptor Kinase Activity in Cells

The cellular inhibition of activin signaling activity by the test compounds of formula (I) is determined in a similar manner as described above in Example 218 except that 100 ng/ml of activin is added to serum starved cells in place of the 2.5 ng/ml TGFβ.

Assay for TGFβ-Induced Collagen Expression

Preparation of Immortalized Collagen Promotor—Green Fluorescent Protein Cells

Fibroblasts are derived from the skin of adult transgenic mice expressing Green Fluorescent Protein (GFP) under the control of the collagen 1A1 promoter (see Krempen, K. et al., Gene Exp. 8: 151-163 (1999)). Cells are immortalized with a temperature sensitive large T antigen that is in an active stage at 33° C. Cells are expanded at 33° C. and then transferred to 37° C. at which temperature the large T antigen becomes inactive (see Xu, S. et al., Exp. Cell Res. 220: 407-414 (1995)). Over the course of about 4 days and one split, the cells cease proliferating. Cells are then frozen in aliquots sufficient for a single 96 well plate.

Assay of TGFβ-induced Collagen—GFP Expression

Cells are thawed, plated in complete DMEM (contains non-essential amino acids, 1 mM sodium pyruvate and 2 mM L-glutamine) with 10% fetal calf serum, and then incubated for overnight at 37° C., 5% CO2. The cells are trypsinized in the following day and transferred into 96 well format with 30,000 cells per well in 50 μl complete DMEM containing 2% fetal calf serum, but without phenol red. The cells are incubated at 37° C. for 3 to 4 hours to allow them to adhere to the plate. Solutions containing a test compound of formula (I) are then added to wells with no TGFβ (in triplicates), as well as wells with 1 ng/ml TGFβ (in triplicates). DMSO is also added to all of the wells at a final concentration of 0.1%. GFP fluorescence emission at 530 nm following excitation at 485 nm is measured at 48 hours after the addition of solutions containing a test compound on a CytoFluor microplate reader (PerSeptive Biosystems). The data are then expressed as the ratio of TGFβ-induced to non-induced for each test sample.

Other Embodiments