WO2007084868A2 - Treatment of disorders by activation of the unfolded protein response - Google Patents

Abstract

The present invention is directed to a novel method for the treatment of diseases by activating the unfolded protein response (UPR), or of inhibiting the expression of the inducible isoform of the nitric oxide synthase (iNOS) enzyme, or both. New methylpyrazole compounds which activate the UPR, downregulate iNOS, and reduce inflammatory markers are disclose herein, as are compositions comprising said compounds and their applications as pharmaceuticals for the treatment of disease.

Description

TREATMENT OF INFLAMMATION AND RELATED DISORDERS BY ACTIVATION OF

THE UNFOLDED PROTEIN RESPONSE

This application claims the benefit of priority of United States provisional application No. 60/761,043, filed January 17, 2006, the disclosure of which is hereby incorporated by reference as if written herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to a novel method for the treatment of diseases by activating the unfolded protein response (UPR), or of inhibiting the expression of the inducible isoform of the nitric oxide synthase (iNOS) enzyme, or both. New methylpyrazole compounds which activate the UPR, downregulate iNOS, and reduce inflammatory markers are disclosed herein, as are compositions comprising said compounds and their applications as pharmaceuticals for the treatment of disease.

BACKGROUND OF THE INVENTION

The endoplasmic reticulum (ER) is an intracellular organelle responsible for the proper synthesis and folding of secreted and membrane-bound proteins. Many disturbances, including aberrant Ca2+ regulation, altered redox status, or interference with protein glycosylation, can result in the accumulation of unfolded ER client proteins in a phenomenon referred to as ER stress. To compensate for this increased burden, a highly specific trans-organelle signaling pathway, known as the unfolded protein response (UPR) is activated (Harding et al. 2002). Lumenal signals generated during ER stress activate at least three ER transmembrane protein sensors including ATF-6, IRE-I, and PERK. Under normal conditions, all three proteins are bound in an inactive conformation by Grp78, an HSP-70-family chaperone. Increased unfolded protein load results in release of Grp78 and subsequent activation of signal transduction. Proteases release the bZIP transcription factor domain of ATF-6 into the cytosol which then translocates into the nucleus to activate transcription of downstream UPR genes. Both IRE-I and PERK are kinases that self-oligomerize upon release of Grp78. In the case of IRE-I, autophosphorylation activates an internal ribonuclease domain that triggers alternative splicing of its mRNA substrate, XBP-I. The XBP-I gene encodes a bZIP transcription factor which is then translated to activate one or more downstream UPR genes. Oligomerized PERK phosphorylates two substrates, the bZIP transcription factor NRF2 and elongation factor 2α (eIF2α). Phosphorylation of eIF2α results in attenuation of protein translation and selective translation of specific downstream UPR genes.

Together, these three signaling pathways activate a compensatory gene program, thereby increasing the levels of specific transcription factors, protein folding chaparones, and protein degradation machinery, in an attempt to reestablish ER homeostasis. Some of the transcription factors upregulated in the UPR pathway are cAMP-response element binding proteins (CREB) ATF3/ATF4, as well as XBP-I. Also upregulated are chaparones such as Grp78 and ERdj4 representing ER-localized HSP70 and HSP40 family members, respectively. Finally, components of the endoplasmic reticulum-associated protein degradation (ERAD) machinery, which govern the destruction of misfolded proteins in a proteosome- dependent fashion, are upregulated. However, if ER dysfunction is prolonged or unresolved, one or more apoptotic pathways may be initiated. CHOP, a CEBP family transcription factor is upregulated following ER stress and has been implicated in UPR-dependent apoptosis. Natural products, including thapsigargin, tunicamycin, and brefeldin A, have been instrumental in elucidating the components of the UPR. Thapsigargin inhibits the resident Ca2+-ATPase within the ER, causing protein misfolding indirectly by disrupting the function of Ca2+-dependent chaparones Grp78, Grp94, and calreticulin. Tunicamycin is a nucleoside analog that inhibits N-linked glycosylation causing a disruption in protein traffic from the ER to the golgi. Brefeldin A inhibits a nucleotide exchange activity in the ARF complex, thereby disrupting ER-to-golgi vesicle transport. More recently, the synthetic compound salubrinal, was reported to inhibit dephosphorylation of eIF2α and inhibit ER stress-induced apoptosis.

ER stress has been suggested to induce an inflammatory response, in part via the upregulation of the transcription factor NFKB (Schroder and Kaufman 5-36). However, identified herein is a synthetic small molecule inducer of ER stress from within a group of compounds which are also inhibitors of the inflammatory mediator inducible nitric oxide synthase (iNOS). NOS enzymes synthesize nitric oxide in order to regulate numerous essential physiologic functions, including maintainence of blood pressure, platelet adhesion, gastrointestinal motility, bronchomotor tone and neurotransmission. However, excess nitric oxide generated by the inducible form of NOS has been implicated in the pathogenesis of inflammatory and other related diseases. Once induced, iNOS synthesizes 100-1000 times more NO than the constitutive isoforms endothelial NOS (eNOS) and neuronal NOS (nNOS) enzymes synthesize and does so for prolonged periods. This excessive production of NO and resulting NO-derived metabolites {e.g., peroxynitrite) elicit cellular toxicity and tissue damage which contribute to the pathophysiology of a number of diseases, disorders and conditions. These deleterious effects can be mediated by selective inhibition of iNOS, which itself can be accomplished by various means. For example, the enzyme can be inactivated by antagonism at the active site, by inhibition of dimerization which is required for activity, or by inhibition of expression of the gene or protein. In particular, inhibition of the expression of iNOS and hence NO production is understood to have beneficial effects against iNOS and NO mediated diseases . This suggests a surprising and heretofore undescribed anti-inflammatory role for UPR activation. Disclosed herein is a class of compounds having downregulatory effects on iNOS, at least one of which is also a potent UPR activator. Additionally, these molecules show reduced iNOS activity and inflammatory phenotypes in vivo. Taken together, these disparate mechanisms represent a novel and potentially synergistic approach to treating inflammation disorders, and an understanding of the specific nature of these overlapping cellular pathways could be applied to future therapeutic approaches. SUMMARY OF THE INVENTION

Novel compounds and pharmaceutical compositions that ameliorate inflammatory and related disorders by activating the UPR or inhibiting expression of inducible NOS have been found together with methods of synthesizing and using the compounds, including methods for inhibiting or modulating nitric oxide synthesis and/or lowering nitric oxide levels in a patient by administering the compounds.

A class of compounds useful both in activating the UPR and inhibiting expression of iNOS has been discovered, and is defined by Formula I:

or a salt, ester, or prodrug thereof, wherein: A is selected from the group consisting of acyl, alkyl, alkoxy, alkoxyalkyl, alkylene, alkynyl, amido, amino, aminosulfonyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrazinyl, imino, thio, sulfonate and sulfonylamino, any of which may be optionally substituted;

B is selected from the group consisting of acyl, alkoxy, alkoxyalkyl, alkyl, alkylene, alkynyl, alkylimino, amido, aryl, carboxy, cyano, cycloalkyl, ester, halo, haloalkyl, heteoaryl, heterocycloalkyl, hydroxyalkyl, hydrogen, imino and iminohydroxy, any of which may be optionally substituted;

C is selected from the group consisting of -OR¹, -SR¹, -S(O)R¹, -S(O)₂R¹, -NR²R³, acyl, alkyl, alkylene, alkynyl, amido, carboxy, cycloalkyl, ester, halo, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and imino, any of which may be optionally substituted; X is selected from the group consisting of acyl, alkoxyalkyl, alkyl, alkylamino, amino, amido, alkylamino, arylalkyl, aryl, arylalkyl, carboxy, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydrogen, sulfonate and sulfonyl, any of which may be optionally substituted;

R¹ is selected from the group consisting of acyl, alkoxy, arylalkoxyalkyl, alkyl, amino, amido, aminoalkyl, aryl, arylalkyl, carboxy, cycloalkyl, haloalkyl, heteroarylalkyl, heterocycloalkyl, hydrogen, hydroxy and hydroxyalkyl, any of which may be optionally substituted; and

R² and R³ are each independently selected from the group consisting of alkyl, amino, arylalkyl, aryl, cycloalkyl, haloalkyl, heteroarylalkyl, heterocycloalkyl and hydrogen, R² and R³ may combine to form a heterocycloalkyl, any of which may be optionally substituted. DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides a method for achieving an effect in a patient, comprising the administration of an effective amount of a compound which activates the unfolded protein response (UPR), wherein said effect is selected from the group consisting of: the downregulation of an inflammation-related gene or protein, the reduction of an inflammatory marker, the stimulation of apoptosis in cells, the alteration of splicing in a gene, and the increase of phosphorylation ofEIF-2α.

In further embodiments, said effect is the downregulation of an inflammation-related gene or protein.

In yet further embodiments, said inflammation-related gene or protein is a NOS gene or protein. In yet further embodiments, said NOS gene or protein is iNOS.

In yet further embodiments, said inflammation-related gene or protein is the TNF-α gene or protein. In other embodiments, said effect is the reduction of an inflammatory marker.

In yet further embodiments, said inflammatory marker is nitric oxide. In further embodiments, said effect is the stimulation of apoptosis in cells. In yet further embodiments, said stimulation of apoptosis in cells occurs more in those cells which are in a state of inflammation compared to normal cells. In certain embodiments, the present invention provides a method for the treatment of a hyperproliferative, autoimmune, cardiovascular, respiratory, neurologic, inflammatory, chronic pain, neuropathic pain, or iNOS-related disease, comprising the administration of a therapeutically effective amount of a compound which activates the unfolded protein response (UPR) to a patient in need of such treatment. In further embodiments, said compound also inhibits the expression if iNOS.

In yet further embodiments, said disease is an inflammatory disease.

In yet further embodiments, said inflammatory disease is selected from the group consisting of inflammation, neuropathy, neuropathic pain, inflammatory pain, postherpetic neuralgia, post-surgical pain, arthritis, arthritic pain, inflammatory bowel disease, Crohn's disease, irritable bowel syndrome, ulcerative colitis allodynia, fibromyalgia, complex regional pain syndrome, and hyperalgesia. In other embodiments, said disease is a hyperproliferative disease.

In further embodiments, said hyperproliferative disease is selected from the group consisting of cancer, myelodysplastic syndrome and psoriasis.

In yet further embodiments, said cancer is selected from the group consisting of hematologic cancers, nonhematologic cancers, and solid tumors. In certain embodiments, the invention provides for compounds of Formula 1 wherein:

A is selected from the group consisting of alkyl, aryl, haloalkyl and heteroaryl, any of which may be optionally substituted;

B is selected from the group consisting of acyl, amido, carboxy, cyano, ester, hydroxyalkyl, hydrogen, imino and iminohydroxy, any of which may be optionally substituted; and

C is selected from the group consisting of hydroxy., -OR¹, -SR¹, -S(O)R¹, -S(O)₂R¹, and - NR²R³.

In further embodiments,

A is CF₃; and C is selected from the group consisting of -SR¹, -S(O)₂R¹, -NR²R³ and -OR¹, any of which may be optionally substituted.

In yet further embodiments, X is alkyl or heteroaryl, either of which may be optionally substituted.

In yet further embodiments, B is acyl, hydrogen, amido, carboxy, ester, nitrile or imino, any of which may be optionally substituted.

In yet further embodiments, X is optionally substituted alkyl.

In yet further embodiments, C is -SR¹.

In yet further embodiments, A is alkyl or heteroaryl, either of which may be optionally substituted. In yet further embodiments, the compound is selected from the group consisting of Examples

28-57.

The present invention also provides for the use of a compound which activated the UPR in the manufacture of a medicament for the treatment of (iNOS) related diseases.

The present invention also provides for a pharmaceutical composition comprising as disclosed herein or a pharmaceutically acceptable salt thereof and together with pharmaceutically acceptable carrier.

As used in the present specification the following terms have the meanings indicated:

The term "acyl," as used herein, alone or in combination, refers to a carbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety were the atom attached to the carbonyl is carbon. An "acetyl" group refers to a -C(O)CH₃ group. An "alkylcarbonyl" or

"alkanoyl" group refers to an alkyl group attached to the parent molecular moiety through a carbonyl group. Examples of such groups include methylcarbonyl and ethylcarbonyl. Examples of acyl groups include formyl, alkanoyl and aroyl.

The term "alkenyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon radical having one or more double bonds and containing from 2 to 20, preferably 2 to 6, carbon atoms. Alkenylene refers to a carbon-carbon double bond system attached at two or more positions such as ethenylene [(-CH=CH-),(-C::C-)]. Examples of suitable alkenyl radicals include ethenyl, propenyl, 2-methylpropenyl, 1,4-butadienyl and the like. The term "alkoxy," as used herein, alone or in combination, refers to an alkyl ether radical, wherein the term alkyl is as defined below. Examples of suitable alkyl ether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like.

The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain alkyl radical containing from 1 to and including 20, preferably 1 to 10,. and more preferably 1 to 6, carbon atoms. Alkyl groups may be optionally substituted as defined herein. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, noyl and the like. The term "alkylene," as used herein, alone or in combination, refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene (-CH₂-).

The term "alkylamino," as used herein, alone or in combination, refers to an alkyl group attached to the parent molecular moiety through an amino group. Suitable alkylamino groups may be mono- or dialkylated, forming groups such as, for example, N-methylamino, N-ethylamino, N,N- dimethylamino, N,N-ethylmethylamino and the like. The term "alkylidcnc," as used herein, alone or in combination, refers to an alkcnyl group in which one carbon atom of the carbon-carbon double bond belongs to the moiety to which the alkenyl group is attached.

The term "alkylthio," as used herein, alone or in combination, refers to an alkyl thioether (R-S-) radical wherein the term alkyl is as defined above and wherein the sulfur may be singly or doubly oxidized. Examples of suitable alkyl thioether radicals include methylthio, ethylthio, n- propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio, tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term "alkynyl," as used herein, alone or in combination, refers to a straight-chain or branched chain hydrocarbon radical having one or more triple bonds and containing from 2 to 20, preferably from 2 to 6, more preferably from 2 to 4, carbon atoms. "Alkynylene" refers to a carbon- carbon triple bond attached at two positions such as ethynylene (-C:::C-, -C≡C-). Examples of alkynyl radicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, 3- methylbutyn-1-yl, hexyn-2-yl, and the like.

The terms "amido" and "carbamoyl,"as used herein, alone or in combination, refer to an amino group as described below attached to the parent molecular moiety through a carbonyl group, or vice versa. The term "C-amido" as used herein, alone or in combination, refers to a -C(=O)-NR₂ group with R as defined herein. The term "N-amido" as used herein, alone or in combination, refers to a RC(=O)NH- group, with R as defined herein. The term "acylamino" as used herein, alone or in combination, embraces an acyl group attached to the parent moiety through an amino group. An example of an "acylamino" group is acetylamino (CH₃C(O)NH-).

The term "amino," as used herein, alone or in combination, refers to — MRR , wherein R and R are independently selected from the group consisting of hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. The term "aryl," as used herein, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term "aryl" embraces aromatic radicals such as benzyl, phenyl, naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl, and biphenyl. The term "arylalkenyl" or "arallcenyl," as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an allcenyl group.

The term "arylalkoxy" or "aralkoxy," as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an alkoxy group.

The term "arylalkyl" or "aralkyl," as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an alkyl group.

The term "arylalkynyl" or "aralkynyl," as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an alkynyl group.

The term "arylalkanoyl" or "aralkanoyl" or "aroyl,"as used herein, alone or in combination, refers to an acyl radical derived from an aryl-substituted alkanecarboxylic acid such as benzoyl, napthoyl, phcnylacctyl, 3-phcnylpropionyl (hydrocinnamoyl), 4-phcnylbutyryl, (2-naphthyl)acctyl, 4- chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an oxy.

The terms "benzo" and "benz," as used herein, alone or in combination, refer to the divalent radical CsBL_t= derived from benzene. Examples include benzothiophene and benzimidazole.

The term "carbamate," as used herein, alone or in combination, refers to an ester of carbamic acid (-NHCOO-) which may be attached to the parent molecular moiety from either the nitrogen or acid end, and which may be optionally substituted as defined herein.

The term "O-carbamyl" as used herein, alone or in combination, refers to a -OC(O)NRR', group-with R and R' as defined herein.

The term "N-carbamyl" as used herein, alone or in combination, refers to a ROC(O)NR'- group, with R and R' as defined herein.

The term "carbonyl," as used herein, when alone includes formyl [-C(O)H] and in combination is a -C(O)- group. The term "carboxy," as used herein, refers to -C(O)OH or the corresponding "carboxylate" anion, such as is in a carboxylic acid salt. An "O-carboxy" group refers to a RC(O)O- group, where R is as defined herein. A "C-carboxy" group refers to a -C(O)OR groups where R is as defined herein.

The term "cyano," as used herein, alone or in combination, refers to -CN.

The term "cycloalkyl," or, alternatively, "carbocycle,"as used herein, alone or in combination, refers to a saturated or partially saturated monocyclic, bicyclic or tricyclic alkyl radical wherein each cyclic moiety contains from 3 to 12, preferably five to seven, carbon atom ring members and which may optionally be a benzo fused ring system which is optionally substituted as defined herein. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, octahydronaphthyl, 2,3-dihydro-lH-indenyl, adamantyl and the like. "Bicyclic" and "tricyclic" as used herein are intended to include both fused ring systems, such as decahydonapthalene, octahydronapthalene as well as the multicyclic (multicentered) saturated or partially unsaturated type. The latter type of isomer is exemplified in general by, bicyclo[l,l,l]pentane, camphor, adamantane, and bicyclo[3,2,l]octane.

The term "ester," as used herein, alone or in combination, refers to a carboxy group bridging two moieties linked at carbon atoms.

The term "ether," as used herein, alone or in combination, refers to an oxy group bridging two moieties linked at carbon atoms. The term "halo," or "halogen," as used herein, alone or in combination, refers to fluorine, chlorine, bromine, or iodine.

The term "haloalkoxy," as used herein, alone or in combination, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.

The term "haloalkyl," as used herein, alone or in combination, refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens arc replaced with a halogen. Specifically embraced are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkyl radical, for one example, may have an iodo, bromo, chloro or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. "Haloalkylene" refers to a haloalkyl group attached at two or more positions. Examples include fluoromethylene (-CFH-), difluoromethylene (-CF₂ -), chloromethylene (-CHC1-) and the like.

The term "heteroalkyl," as used herein, alone or in combination, refers to a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃.

The term "heteroaryl," as used herein, alone or in combination, refers to 3 to 7 membered, preferably 5 to 7 membered, unsaturated heteromonocyclic rings, or fused polycyclic rings in which at least one of the fused rings is unsaturated, wherein at least one atom is selected from the group consisting of O, S, and N. The term also embraces fused polycyclic groups wherein heterocyclic radicals are fused with aryl radicals, wherein heteroaryl radicals are fused with other heteroaryl radicals, or wherein heteroaryl radicals are fused with cycloalkyl radicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoiπdolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl, benzoxadiazolyl, benzotlαiazolyl, benzothiadiazolyl, benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclic heterocyclic groupsinclude carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The terms "heterocycloalkyl" and, interchangeably, "heterocycle," as used herein, alone or in combination, each refer to a saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or tricyclic heterocyclic radical containing at least one, preferably 1 to 4, and more preferably 1 to 2 heteroatoms as ring members, wherein each said heteroatom may be independently selected from the group consisting of nitrogen, oxygen, and sulfur, and wherein there are preferably 3 to 8 ring members in each ring, more preferably 3 to 7 ring members in each ring, and most preferably 5 to 6 ring members in each ring. "Heterocycloalkyl" and "heterocycle" are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclic fused and benzo fused ring systems; additionally, both terms also include systems where a heterocycle ring is fused to an aryl group, as defined herein, or an additional heterocycle group. Heterocycle groups of the invention are exemplified by aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, dihydro[l,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl, dihy- dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. The heterocycle groups may be optionally substituted unless specifically prohibited.

The term "hydrazinyl" as used herein, alone or in combination, refers to two amino groups joined by a single bond, i.e., -N-N-.

The term "hydroxy," as used herein, alone or in combination, refers to -OH. The term "hydroxyalkyl," as used herein, alone or in combination, refers to a hydroxy group attached to the parent molecular moiety through an alkyl group.

The term "imino," as used herein, alone or in combination, refers to =N-.

The term "iminohydroxy," as used herein, alone or in combination, refers to =N(0H) and =N-O-. The phrase "in the main chain" refers to the longest contiguous or adjacent chain of carbon atoms starting at the point of attachment of a group to the compounds of this invention.

The term "isocyanato" refers to a -NCO group.

The term "isothiocyanato" refers to a -NCS group.

The phrase "linear chain of atoms" refers to the longest straight chain of atoms independently selected from carbon, nitrogen, oxygen and sulfur.

The term "lower," as used herein, alone or in combination, means containing from 1 to and including 6 carbon atoms. The term "mercaptyl" as used herein, alone or in combination, refers to an RS- group, where R is as defined herein.

The term "nitro," as used herein, alone or in combination, refers to -NO₂.

The terms "oxy" or "oxa," as used herein, alone or in combination, refer to -O-. The term "oxo," as used herein, alone or in combination, refers to =0.

The term "perhaloalkoxy" refers to an alkoxy group where all of the hydrogen atoms are replaced by halogen atoms.

The term "perhaloalkyl" as used herein, alone or in combination, refers to an alkyl group where all of the hydrogen atoms are replaced by halogen atoms. The terms "sulfonate," "sulfonic acid," and "sulfonic," as used herein, alone or in combination, refer the -SO₃H group and its anion as the sulfonic acid is used in salt formation.

The term "sulfanyl," as used herein, alone or in combination, refers to -S-.

The term "sulfinyl," as used herein, alone or in combination, refers to -S(O)-.

The term "sulfonyl," as used herein, alone or in combination, refers to -S(O)₂-. The term "N-sulfonamido" refers to a RS(=O)₂NR'- group with R and R' as defined herein.

The term "S-sulfonamido" refers to a -S(=O)₂NRR', group, with R and R' as defined herein.

The terms "thia" and "thio," as used herein, alone or in combination, refer to a -S- group or an ether wherein the oxygen is replaced with sulfur. The oxidized derivatives of the thio group, namely sulfinyl and sulfonyl, are included in the definition of thia and thio. The term "thiol," as used herein, alone or in combination, refers to an -SH group.

The term "thiocarbonyl," as used herein, when alone includes thioformyl -C(S)H and in combination is a -C(S)- group.

The term "N-thiocarbamyl" refers to an ROC(S)NR'- group, with R and R' as defined herein.

The term "0-thiocarbamyl" refers to a -OC(S)NRR', group with R and R' as defined herein. The term "thiocyanato" refers to a -CNS group.

The term "trihalomethanesulfonamido" refers to a X₃CS(O)₂NR- group with X is a halogen and R as defined herein.

The term "trihalomethanesulfonyl" refers to a X₃CS(O)₂- group where X is a halogen.

The term "trihalomethoxy" refers to a X₃CO- group where X is a halogen. The term "trisubstituted silyl," as used herein, alone or in combination, refers to a silicone group substituted at its three free valences with groups as listed herein under the definition of substituted amino. Examples include trimethysilyl, tert-butyldimethylsilyl, triphenylsilyl and the like.

Any definition herein may be used in combination with any other definition to describe a composite structural group. By convention, the trailing element of any such definition is that which attaches to the parent moiety. For example, the composite group alkylamido would represent an alkyl group attached to the parent molecule through an amido group, and the term alkoxyalkyl would represent an alkoxy group attached to the parent molecule through an alkyl group.

When a group is defined to be "null," what is meant is that said group is absent. The term "optionally substituted" means the anteceding group may be substituted or unsubstituted. When substituted, the substituents of an "optionally substituted" group may include, without limitation, one or more substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower allcyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower aBcylcarbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower allcylthio, arylthio, lower alkylsulfϊnyl, lower alkylsulfonyl, arylsulfinyl, arylsulfonyl, arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Two substituents may be joined together to form a fused five-, six-, or seven-menbered carbocyclic or heterocyclic ring consisting of zero to three heteroatoms, for example forming methylenedioxy or ethylenedioxy. An optionally substituted group may be unsubstituted (e.g., -CH₂CH₃), fully substituted (e.g., -CF₂CF₃), monosubstitutcd (e.g., -CH₂CH₂F) or substituted at a level anywhere in-bctwccn fully substituted and monosubstituted (e.g., -CH₂CF₃). Where substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. Where a substituent is qualified as "substituted," the substituted form is specifically intended. Additionally, different sets of optional substituents to a particuar moiety may be defined as needed; in these cases, the optional substitution will be as defined, often immediately following the phrase, "optionally substituted with."

The term R or the term R', appearing by itself and without a number designation, unless otherwise defined, refers to a moiety selected from the group consisting of hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl and heterocycloalkyl, any of which may be optionally substituted. Such R and R' groups should be understood to be optionally substituted as defined herein. Whether an R group has a number designation or not, every R group, including R, R' and Rⁿ where n=(l, 2, 3, ...n), every substituent, and every term should be understood to be independent of every other in terms of selection from a group. Should any variable, substituent, or term (e.g. aryl, heterocycle, R, etc.) occur more than one time in a formula or generic structure, its definition at each occurrence is independent of the definition at every other occurrence. Those of skill in the art will further recognize that certain groups may be attached to a parent molecule or may occupy a position in a chain of elements from either end as written. Thus, by way of example only, an unsymmetrical group such as -C(O)N(R)- may be attached to the parent moiety at either the carbon or the nitrogen.

Asymmetric centers exist in the compounds of the present invention. These centers are designated by the symbols "R" or "S," depending on the configuration of substituents around the chiral carbon atom. It should be understood that the invention encompasses all stereochemical isomeric forms, including diastereomeric, enantiomeric, and epimeric forms,as well as d-isomers and 1 -isomers, and mixtures thereof. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art. Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art. Additionally, the compounds of the present invention may exist as geometric isomers. The present invention includes all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. Additionally, compounds may exist as tautomers; all tautomeric isomers are provided by this invention. Additionally, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

The term "bond" refers to a covalent linkage between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. A bond may be single, double, or triple unless otherwise specified. A dashed line between two atoms in a drawing of a molecule indicates that an additional bond may be present or absent at that position.

The term "combination therapy" means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple or separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

The term "iNOS expression inhibitor" is used herein to refer to a compound which decreases the amount of protein present in or produced by cellular machinery, an effect which may take place at the transcriptional, translational, or post-translational level. Such a compound would also be said to be a "downregulator" of iNOS. Such a compound would have an IC₅₀ with respect to iNOS expression of no more than about 100 μM and more typically not more than about 50 μM, as measured in the iNOS RAW Cytoblot Assay described generally hereinbelow. "IC₅₀" is that concentration of inhibitor which reduces the activity of an enzyme (e.g., iNOS) to half-maximal level. Representative compounds of the present invention have been discovered to exhibit inhibitory activity against iNOS in cell-based assays. Compounds of the present invention preferably exhibit an IC₅₀ with respect to iNOS of no more than about 10 μM, more preferably, no more than about 5 μM, even more preferably not more than about 1 μM, and most preferably, not more than about 200 nM, as measured in the iNOS assay(s) described herein. The phrase "therapeutically effective" is intended to qualify the amount of active ingredients used in the treatment of a particular disease or condition, for example, inflammation or aberrant iNOS activity. This amount will achieve the goal of reducing or eliminating the condition. The term "prodrug" refers to a compound that is made more active in vivo. Certain compounds of the present invention may also exist as prodrugs, as described in Hydrolysis in Dmg and Prodrug Metabolism : Chemistry, Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M. Wiley- VHCA, Zurich, Switzerland 2003). Prodrugs of the compounds described herein are structurally modified forms of the compound that readily undergo chemical changes under physiological conditions to provide the compound. Additionally, prodrugs can be converted to the compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to a compound when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the compound, or parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound which is administered as an ester (the "prodrug"), but then is metabolically hydrolyzcd to the carboxylic acid, the active entity. Additional examples include pcptidyl derivatives of a compound.

The compounds of the present invention can exist as therapeutically acceptable salts. The present invention includes compounds listed above in the form of salts, in particular acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question. Basic addition salts may also be formed and be pharmaceutically acceptable. For a more complete discussion of the preparation and selection of salts, refer to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002). The term "therapeutically acceptable salt," as used herein, represents salts or zwitterionic forms of the compounds of the present invention which are water or oil-soluble or dispersible and therapeutically acceptable as defined herein. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound in the form of the free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L- ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethansulfonate (isethionate), lactate, maleate, malonate, DL-mandelate, mesirylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para- toluenesulfonate (p-tosylate), and undecanoate. Also, basic groups in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauiyl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Salts can also be formed by coordination of the compounds with an alkali metal or alkaline earth ion. Hence, the present invention contemplates sodium, potassium, magnesium, and calcium salts of the compounds of the compounds of the present invention and the like.

Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxy group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine. The cations of therapeutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N.N-dimethylaniline, 7V-methylpiperidine, N-methylmorpholine, dicyclohcxylaminc, procaine, dibcnzylaminc, N.TV-dibcnzylphcncthylaminc, 1-cphcnaminc, and N₁N- dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

The compounds of the present invention can exist as therapeutically acceptable salts. The present invention includes compounds listed above in the form of salts, in particular acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question.

While it may be possible for the compounds of the subject invention to be administered as the raw chemical, it is also possible to present them as a pharmaceutical formulation. Accordingly, the subject invention provides a pharmaceutical formulation comprising a compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the subject invention or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. AU formulations for oral administration should be in dosages suitable for such administration. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutancously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner. Such compositions may comprise the active ingredient in a flavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides. Compounds of the present invention may be administered topically, that is by non-systemic administration. This includes the application of a compound of the present invention externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. The active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It may however comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1% to 1% w/w of the formulation.

Gels for topical or transdermal administration of compounds of the subject invention may comprise, generally, a mixture of volatile solvents, nonvolatile solvents, and water. The volatile solvent component of the buffered solvent system may preferably include lower (C1-C6) alkyl alcohols, lower alkyl glycols and lower glycol polymers. More preferably, the volatile solvent is ethanol. The volatile solvent component is thought to act as a penetration enhancer, while also producing a cooling effect on the skin as it evaporates. The nonvolatile solvent portion of the buffered solvent system is selected from lower alkylene glycols and lower glycol polymers. Preferably, propylene glycol is used. The nonvolatile solvent slows the evaporation of the volatile solvent and reduces the vapor pressure of the buffered solvent system. The amount of this nonvolatile solvent component, as with the volatile solvent, is determined by the pharmaceutical compound or drug being used. When too little of the nonvolatile solvent is in the system, the pharmaceutical compound may crystallize due to evaporation of volatile solvent, while an excess will result in a lack of bioavailability due to poor release of drug from solvent mixture. The buffer component of the buffered solvent system may be selected from any buffer commonly used in the art; preferably, water is used. The preferred ratio of ingredients is about 20% of the nonvolatile solvent, about 40% of the volatile solvent, and about 40% water. There are several optional ingredients which can be added to the topical composition. These include, but are not limited to, chelators and gelling agents. Appropriate gelling agents can include, but are not limited to, semisynthetic cellulose derivatives (such as hydroxypropylmcthylccllulosc) and synthetic polymers, and cosmetic agents.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100⁰C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol. Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

For administration by inhalation the compounds according to the invention are conveniently delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluorometliane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effective dose, as herein below recited, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The compounds of the invention may be administered orally or via injection at a dose of from

0.1 to 500 mg/kg per day. The dose range for adult humans is generally from 5 mg to 2 g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5 mg to 500 mg, usually around 10 mg to 200 mg. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

The compounds of the subject invention can be administered in various modes, e.g. orally, topically, or by injection. The precise amount of compound administered to a patient will be the responsibility of the attendant physician. The specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the indication or condition being treated. Also, the route of administration may vary depending on the condition and its severity. In certain instances, it may be appropriate to administer at least one of the compounds described herein (or a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate) in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient upon receiving one of the compounds herein is hypertension, then it may be appropriate to administer an anti-hypertensive agent in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effectiveness of one of the compounds described herein maybe enhanced by administration of an adjuvant (i.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit of experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit. By way of example only, in a treatment for diabetes involving administration of one of the compounds described herein, increased therapeutic benefit may result by also providing the patient with another therapeutic agent for diabetes. In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient may simply be additive of the two therapeutic agents or the patient may experience a synergistic benefit.

Specific, non-limiting examples of possible combination therapies include use of the compounds of the invention with: a) corticosteroids including betamethasone dipropionate (augmented and nonaugemnted), betamethasone valerate, clobetasol propionate, diflorasone diacetate, halobetasol propionate, amcinonide, dexosimethasone, fluocinolone acetononide, fiuocinonide, halocinonide, clocortalone pivalate, dexosimetasone, and flurandrenalide; b) non-steroidal anti-inflammatory drugs including ibuprofen, diclofenac, ketoprofen, and piroxicam; c) muscle relaxants and combinations thereof with other agents, including cyclobenzaprine, baclofen, cyclobenzaprine/lidocaine, baclofen/cyclobenzaprine, and cyclobenzaprine/lidocaine/ketoprofen; d) anaesthetics and combinations thereof with other agents, including lidocaine, lidocaine/deoxy-D-glucose (an antiviral), prilocaine, and EMLA Cream [Eutectic Mixture of Local Anesthetics (lidocaine 2.5% and prilocaine 2.5%; an emulsion in which the oil phase is a eutectic mixture of lidocaine and prilocaine in a ratio of 1 : 1 by weight. This eutectic mixture has a melting point below room temperature and therefore both local anesthetics exist as a liquid oil rather then as crystals)]; e) expectorants and combinations thereof with other agents, including guaifenesin and guaifenesin/ketoprofen/cyclobenzaprine; f) antidepressants including tricyclic antidepressants (e.g., amitryptiline, doxepin, desipramine, imipramine, amoxapine, clomipramine, nortriptyline, and protriptyline), selective serotonin/norepinephrine reuptake inhibitors including (e.g, duloxetine and mirtazepine), and selective norepinephrine reuptake inhibitors (e.g., nisoxetine, maprotiline, and reboxetine), selective serotonin reuptake inhibitors (e.g., fluoxetine and fluvoxamine); g) anticonvulsants and combinations thereof, including gabapentin, carbamazepine, felbamate, lamotrigine, topiramate, tiagabine, oxcarbazepine, carbamezipine, zonisamide, mexiletine, gabapentin/clonidine, gabapentin/carbamazepine, and carbamazepine/cyclobenzaprine; h) antihypertensives including clonidine; i) opioids including loperamide, tramadol, morphine, fentanyl, oxycodone, levorphanol, and butorphanol; j) topical counter-irritants including menthol, oil of wintergreen, camphor, eucalyptus oil and turpentine oil; k) topical cannabinoids including selective and non-selective CB1/CB2 ligands; and other agents, such as capsaicin. These agents would be particularly suited for use in combination for the treatment of inflammatory diseases, iNOS-mediated diseases, and pain. Other examples of possible combination therapies include use of the compounds of the invention with one or more chemotherapeutic agents such as aromatase inhibitors, antiestrogen, anti- androgen, or a gonadorelin agonists, topoisomerase land 2 inhibitors, microtubule active agents, alkylating agents, antimeoplastic antimetabolite, or platin containing compound, lipid or protein kinase targeting agents, protein or lipid phosphatase targeting agents, anti-angiogentic agents, agents that induce cell differentiation, bradykinin 1 receptor and angiotensin II antagonists, cyclooxygenase inhibitors, heparanase inhibitors, lymphokines or cytokine inhibitors, bisphosphanates, rapamycin derivatives, anti-apoptotic pathway inhibitors, apoptotic pathway agonists, PPAR agonists, inhibitors of Ras isoforms, telomerase inhibitors, protease inhibitors, metalloproteiαase inhibitors, aminopeptidase inhibitors, and biologic drugs including but not limited to antibodies, cytokines, HDAC inhibitors, selective and broad-spectrum kinase inhibitors, and growth factors.

In some aspects of the invention, the chemotherapeutic agents that are useful for the treatment of multiple myeloma include, but are not limited to, alkylating agents (eg, melphalan), anthracyclines (eg. doxorubicin), corticosteroids (eg. dexamethasome), IMiDs (eg. thalidomide, lenalidomide), protease inhibitors (eg. bortezomib, NPI0052), IGF-I inhibitors, CD40 antibodies, Smac mimetics (eg. telomestatin), FGF3 modulator (eg. CHIR258), mTOR inhibitor (Rad 001), HDAC inhibitors (eg. SAHA, Tubacin), IKK inhibitors, P38MAPK inhibitors, HSP90 inhibitors (eg 17-AAG), and akt inhibitors (eg. Perifosine).

In any case, the multiple therapeutic agents (at least one of which is a compound of Formula I, described herein or an alternative activator of the UPR) may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may be any duration of time ranging from a few minutes to four weeks. Compounds of the subject invention are useful in treating NOS-mediated diseases, disorders and conditions, and particularly iNOS-mediated diseases. The compounds of the present invention are useful to treat patients with neuropathy or inflammatory pain such as reflex sympathetic dystrophy/causalgia (nerve injury), peripheral neuropathy (including diabetic neuropathy), intractable cancer pain, complex regional pain syndrome, and entrapment neuropathy (carpel tunnel syndrome). The compounds are also useful in the treatment of pain associated with acute herpes zoster (shingles), postherpetic neuralgia (PHN), and associated pain syndromes such as ocular pain. The compounds are further useful as analgesics in the treatment of pain such as surgical analgesia, or as an antipyretic for the treatment of fever. Pain indications include, but are not limited to, post-surgical pain for various surgical procedures including post-cardiac surgery, dental pain/dental extraction, pain resulting from cancer, muscular pain, mastalgia, pain resulting from dermal injuries, lower back pain, headaches of various etiologies, including migraine, and the like. The compounds are also useful for the treatment of pain- related disorders such as tactile allodynia and hyperalgesia. The pain may be somatogenic (either nociceptive or neuropathic), acute and/or chronic. The nitric oxide expression inhibitors of the subject invention are also useful in conditions where NSAIDs, morphine or fentanyl opiates and/or other opioid analgesics would traditionally be administered.

Furthermore, the compounds of the subject invention can be used in the treatment or prevention of opiate tolerance in patients needing protracted opiate analgesics, and benzodiazepine tolerance in patients taking benzodiazepines, and other addictive behavior, for example, nicotine addiction, alcoholism, and eating disorders. Moreover, the compounds and methods of the present invention are useful in the treatment or prevention of drug withdrawal symptoms, for example treatment or prevention of symptoms of withdrawal from opiate, alcohol, or tobacco addiction.

In addition, the compounds of the subject invention can be used to treat insulin resistance and other metabolic disorders such as atherosclerosis that arc typically associated with an exaggerated inflammatory signaling.

The present invention encompasses therapeutic methods using activators of the UPR and novel selective iNOS inhibitors to treat or prevent respiratory disease or conditions, including therapeutic methods of use in medicine for preventing and treating a respiratory disease or condition including: asthmatic conditions including allergen-induced asthma, exercise-induced asthma, pollution-induced asthma, cold-induced asthma, and viral-induced-asthma; chronic obstructive pulmonary diseases including chronic bronchitis with normal airflow, chronic bronchitis with airway obstruction (chronic obstructive bronchitis), emphysema, asthmatic bronchitis, and bullous disease; and other pulmonary diseases involving inflammation including bronchioectasis cystic fibrosis, pigeon fancier's disease, farmer's lung, acute respiratory distress syndrome, pneumonia, aspiration or inhalation injury, fat embolism in the lung, acidosis inflammation of the lung, acute pulmonary edema, acute mountain sickness, acute pulmonary hypertension, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, hyaline membrane disease, acute pulmonary thromboembolism, heparin-protamine reactions, sepsis, status asthamticus and hypoxia. Other disorders or conditions which can be advantageously treated by the compounds of the present invention include inflammation. The compounds of the present invention are useful as antiinflammatory agents with the additional benefit of having significantly less harmful side effects. The compounds are useful to treat autoimmune disorders and arthritis, including but not limited to rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, juvenile arthritis, acute rheumatic arthritis, enteropathic arthritis, neuropathic arthritis, psoriatic arthritis, and pyogenic arthritis. The compounds are also useful in treating osteoporosis and other related bone disorders. These compounds can also be used to treat gastrointestinal conditions such as reflux esophagitis, diarrhea, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome and ulcerative colitis. The compounds may also be used in the treatment of pulmonary inflammation, such as that associated with viral infections and cystic fibrosis. In addition, compounds of invention are also useful in organ transplant patients either alone or in combination with conventional immunomodulators. Yet further, the compounds of the invention are useful in the treatment of pruritis and vitaligo.

The compounds of the present invention are also useful in treating tissue damage in such diseases as vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes, neuromuscular junction disease including myasthenia gravis, white matter disease including multiple sclerosis, sarcoidosis, nephritis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, periodontis, hypersensitivity, swelling occurring after injury, ischemias including myocardial ischemia, cardiovascular ischemia, and ischemia secondary to cardiac arrest, and the like.

The compounds of the subject invention are also useful for the treatment of certain diseases and disorders of the nervous system. Central nervous system disorders in which nitric oxide inhibition is useful include cortical dementias including Alzheimer's disease, central nervous system damage resulting from stroke, ischemias including cerebral ischemia (both focal ischemia, thrombotic stroke and global ischemia (for example, secondary to cardiac arrest), and trauma. Neurodegenerative disorders in which nitric oxide inhibition is useful include nerve degeneration or nerve necrosis in disorders such as hypoxia, hypoglycemia, epilepsy, and in cases of central nervous system (CNS) trauma (such as spinal cord and head injury), hyperbaric oxygen convulsions and toxicity, dementia e.g. pre-senile dementia, and AIDS-related dementia, cachexia, Sydenham's chorea, Huntington's disease, Parkinson's Disease, amyotrophic lateral sclerosis (ALS), Korsakoffs disease, imbecility relating to a cerebral vessel disorder, sleeping disorders, schizophrenia, depression, depression or other symptoms associated with Premenstrual Syndrome (PMS), and anxiety. Furthermore, the compounds of the present invention are also useful in inhibiting NO production from L-arginine including systemic hypotension associated with septic and/or toxic hemorrhagic shock induced by a wide variety of agents; therapy with cytokines such as TNF, IL-I and IL-2; and as an adjuvant to short term immunosuppression in transplant therapy. These compounds can also be used to treat allergic rhinitis, respiratory distress syndrome, endotoxin shock syndrome, and atherosclerosis.

In further embodiments, said hyperproliferative condition is selected from the group consisting of hematologic and nonhematologic cancers. In yet further embodiments, said hematologic cancer is selected from the group consisting of multiple myeloma, leukemias, and lymphomas. In yet further embodiments, said leukemia is selected from the group consisting of acute and chronic leukemias. In yet further embodiments, said acute leukemia is selected from the group consisting of acute lymphocytic leukemia (ALL) and acute nonlymphocytic leukemia (ANLL). In yet further embodiments, said chronic leukemia is selected from the group consisting of chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML). In further embodiments, said lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma. In further embodiments, said lymophoma is selected from the group consisting of cutaneous t-cell lymphoma (CTCL) and mantle cell lymphoma (MCL). In further embodiments, said hematologic cancer is multiple myeloma. In other embodiments, said hematologic cancer is of low, intermediate, or high grade. In other embodiments, said nonhematologic cancer is selected from the group consisting of: brain cancer, cancers of the head and neck, lung cancer, breast cancer, cancers of the reproductive system, cancers of the digestive system, pancreatic cancer, and cancers of the urinary system. In further embodiments, said cancer of the digestive system is a cancer of the upper digestive tract or colorectal cancer. In further embodiments, said cancer of the urinary system is bladder cancer or renal cell carcinoma. In further embodiments, said cancer of the reproductive system is prostate cancer.

Additional types of cancers which may be treated using the compounds and methods described herein include: cancers of oral cavity and pharynx, cancers of the respiratory system, cancers of bones and joints, cancers of soft tissue, skin cancers, cancers of the genital system, cancers of the eye and orbit, cancers of the nervous system, cancers of the lymphatic system, and cancers of the endocrine system. In certain embodiments, these cancer s may bcsclcctcd from the group consisting of: cancer of the tongue, mouth, pharynx, or other oral cavity; esophageal cancer, stomach cancer, or cancer of the small intestine; colon cancer or rectal, anal, or anorectal cancer; cancer of the liver, intrahepatic bile duct, gallbladder, pancreas, or other biliary or digestive organs; laryngeal, bronchial, and otiier cancers of the respiratory organs; heart cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, other non-epithelial skin cancer; uterine or cervical cancer; uterine corpus cancer; ovarian, vulvar, vaginal, or other female genital cancer; prostate, testicular, penile or other male genital cancer; urinary bladder cancer; cancer of the kidney; renal, pelvic, or urethral cancer or other cancer of the genito-urinary organs; thyroid cancer or other endocrine cancer; chronic lymphocytic leukemia; and cutaneous T-cell lymphoma, both granulocytic and monocytic. Yet other types of cancers which may be treated using the compounds and methods described herein include: adenocarcinoma, angiosarcoma, astrocytoma, acoustic neuroma, anaplastic astrocytoma, basal cell carcinoma, blastoglioma, chondrosarcoma, choriocarcinoma, chordoma, craniopharyngioma, cutaneous melanoma, cystadenocarcinoma, endotheliosarcoma, embryonal carcinoma, ependymoma, Ewing's tumor, epithelial carcinoma, fibrosarcoma, gastric cancer, genitourinary tract cancers, glioblastoma multiforme, hemangioblastoma, hepatocellular carcinoma, hepatoma, Kaposi's sarcoma, large cell carcinoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphangioendomeliosarcoma, medullary thyroid carcinoma, medulloblastoma, meningioma mesothelioma, myelomas, myxosarcoma neuroblastoma, neurofibrosarcoma, oligodendroglioma, osteogenic sarcoma, epithelial ovarian cancer, papillary carcinoma, papillary adenocarcinomas, parathyroid tumors, pheochromocytoma, pinealoma, plasmacytomas, retinoblastoma, rhabdomyosarcoma, sebaceous gland carcinoma, seminoma, skin cancers, melanoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, thyroid cancer, uveal melanoma, and Wilm's tumor. In some aspects of the invention, the disease to be treated by the methods of the present invention may be a hematologic disorder. In certain embodiments, said hematologic disorder is selected from the group consisting of sickle cell anemia, myelodysplastic disorders (MDS), and myeloproliferative disorders. In further embodiments, said myeloproliferative disorder is selected from the group consisting of polycythemia vera, myelofibrosis and essential thrombocythemia.

In some aspects of the invention, the disease to be treated by the methods of the present invention may be a neurological disorder. In further embodiments, said neurological disorder is selected from the group consisting of epilepsy, neuropathic pain, depression and bipolar disorders.

In some aspects of the invention, the disease to be treated by the methods of the present i invention may be a cardiovascular condition. In certain embodiments, said cardiovascular condition is selected from the group consisting of cardiac hypertrophy, idiopathic cardiomyopathies, and heart failure.

In some aspects of the invention, the disease to be treated by the methods of the present invention may be an autoimmune disease. In certain embodiments, said autoimmune disease is selected from the group consisting of systemic lupus crythromatosus (SLE), multiple sclerosis (MS), and systemic lupus nephritis.

In some aspects of the invention, the disease to be treated by the methods of the present invention may be a dermatologic disorder. In certain embodiments, said dermatologic disorder is selected from the group consisting of psoriasis, melanoma, basal cell carcinoma, squamous cell carcinoma, and other non-epithelial skin cancers.

The compounds of the subject invention can be used in the treatment of ophthalmic diseases, such as dry eye, glaucoma (both closed and open angle), retinal ganglion degeneration, occular ischemia, retinitis, retinopathies, uveitis, ocular photophobia, and of inflammation and pain associated with acute injury to the eye tissue. Specifically, the compounds can be used to treat glaucomatous retinopathy and/or diabetic retinopathy. The compounds can also be used to treat post-operative inflammation or pain as from ophthalmic surgery such as cataract surgery and refractive surgery.

Moreover, compounds of the subject invention may be used in the treatment of menstrual cramps, dysmenorrhea, premature labor, tendonitis, bursitis, skin-related conditions such as psoriasis, eczema, burns, sunburn, dermatitis, pancreatitis, hepatitis, and the like. Other conditions in which the compounds of the subject invention provide an advantage in inhibiting nitric oxide inhibition include diabetes (type I or type II), congestive heart failure, myocarditis, atherosclerosis, and aortic aneurysm. The present compounds may also be used in co-therapies, partially or completely, in place of other conventional anti-inflammatory therapies, such as together with steroids, NSAIDs, COX-2 selective inhibitors, 5-lipoxygenase inhibitors, LTB₄ antagonists and LTA₄ hydrolase inhibitors. The compounds of the subject invention may also be used to prevent tissue damage when therapeutically combined with antibacterial or antiviral agents. Besides being useful for human treatment, these compounds are also useful for veterinary treatment of companion animals, exotic animals and farm animals, including mammals, rodents, and the like. More preferred animals include horses, dogs, and cats.

All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein.

GENERAL SYNTHETIC METHODS FOR PREPARING COMPOUNDS The following schemes can be used to practice the present invention.

Examples 1-57 can be synthesized using the following general synthetic procedure set forth in Scheme I. Scheme I

The invention is further illustrated by the following examples.

EXAMPLE 1 l-MethyI-5-phenylsulfanyI-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

This compound is commercially available from Key Organics, Ltd.

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 3 5-(2-chloro-ben2yIsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

3c

Steo l

Preparation of compound 3a: l-Methyl-3-trifluoromethyl-lH-pyrazol-5-ol. Methylhydrazine (3.42 mL, 65.1 mmol) was added all at once to a stirred solution of ethyl 4,4,4- trifluoromethylacetoacetate (9.53 mL, 65.1 mmol) and ethanol (261 mL) at rt under nitrogen. The reaction mixture was heated to 80 ⁰C for 15 h then cooled to rt. NaOH (5.22 g) was added all at once and the mixture stirred for 1 h at rt. The reaction mixture was concentrated under vacuum, diluted with ethyl acetate (2 x 300 mL) and washed with IM HCl (300 mL). The combined organics were dried over MgSO₄, filtered and concentrated to afford 10.5 g (97%) of l-methyl-3-trifluoromethyl-lH-pyrazol-5-ol as a yellow solid. Step 2

Preparation of compound 3b: S-Chloro-l-methyl-S-trifluoromethyM-pyrazolecarboxaldehyde. To a well stirred 0⁰C solution of DMF (8.78 mL, 120 mmol) and 1,2-dichlorocthanc (60 mL) was added phosphorus oxychloride (11.2 mL, 120 mmol) under nitrogen. The resulting suspension was warmed to rt and l-methyl-3-(trifluoromethyl)-lH-pyrazol-5-ol (5.00 g, 30.0 mmol) was added. The mixture was heated to 80 ⁰C for 4 h then cooled to rt. The solution was poured directly into ice-water and extracted with ethyl acetate (2 x 200 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum to afford an oily residue. The material was purified by column chromatography (DCM to 9:1 DCM/MeOH) to afford 3.23 g (51%) of 5-chloro-l-methyl-3- trifluoromethyl-4-pyrazolecarboxaldehyde as a white solid. Step 3 Preparation of compound 3c: 5-(2-ChlorobenzyIsulfanyl)-l~methyl-3-trifluoromethyl-lH-pyrazoIe-4- carboxaldehyde.

To a well stirred solution of 5-chloro-l-methyl-3-trifluoromethyl-4-pyrazolecarboxaldehyde (3.20 g, 15.1 mmol) and 2-chlorobenzenemethanethiol (3.58 g, 22.6 mmol) in 1,2-dichloroethane (64 mL) was added triethylamine (4.20 mL, 30.1 mmol) at rt under nitrogen. The reaction mixture was heated to 80 ⁰C for 14 h then cooled to rt. The reaction mixture as concentrated under vacuum to an oil and purified by column chromatography (hexanes to 1:1 hexanes/EtOAc) to afford 4.82 g (96%) of 5-(2- chloroben2ylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxaldehyde as a white solid. Step 4 Preparation of compound 3: 5-(2-Chloro-benzylsuIfanyl)-l-methyl-3-trifluoromethyI-lH-pyrazole-4- carbaldehyde oxime.

To a well stirred solution of 5-(2-chlorobenzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4- carboxaldehyde (531 mg, 1.59 mmol) in 2,2,2-trifluoroethanol (7 mL) was added hydroxylamine hydrochloride (165 mg, 2.38 mmol) at rt under nitrogen. The reaction mixture was heated to 77 ⁰C for 2 h then cooled to rt. The mixture was washed with sat. aq. NaHCθ₃ and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were dried over MgSO/_t, filtered and concentrated under vacuum to a residue. The material was purified by column chromatography (hexanes to 1:1 hexanes/EtOAc) to afford 487 mg (88%) of 5-(2-chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4- carbaldehyde oxime as a white solid. [M+H]⁺ 350.41; ¹H-NMR (400 MHz, CDCl₃) δ 8.43 (s, IH), 8.11 (s, IH), 7.36 (d, IH), 7.20 (dt, IH), 7.08 (t, IH), 6.76 (dd, IH), 4.06 (s, 2H), 3.53 (s, 3H).

EXAMPLE 4

5-(2-Chloro-phenylsuIfanyI)-l-methyI-3-trifluoromethyl-lH-pyrazole-4-carbonitriIe

This compound is commercially available from Key Organics, Ltd. EXAMPLE 5 l-Methyl-S-phenylsulfanyl-S-trifluoromethyl-lH-pyrazole^-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 6 2-(4-Cyano-2-methyl-5-trifluoromethyl-2H-pyrazole-3-sulfonyl)-benzoic acid methyl ester

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 7 5-Benzenesulfonyl-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 8 l-Methyl-S-o-tolylmethanesulfonyl-S-trifluoromethyl-lH-pyrazole^-carbonitrile

This compound is commercially available from Key Organics, Ltd. F.y AMPLE 9 2-(4-Cyano~2-methyI-5-trifluoromethyl-2H-pyrazoI-3-ylsulfanyl)-benzoic acid methyl ester

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 10 5-BenzyIamino-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 11 l-MethyI-5-phenylmethanesulfonyl-3-trifluoromethyI-lH-pyrazole-4-carbonitriIe

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 12 5-(4-Benzhydryl-piperazin-l-yI)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd. EXAMPLE 13 l-Methyl-5-morpholin-4-yl-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 14 5-(3-Methoxy-benzyIamino)-l-methyl-3-trifluoromethyl-lH-pyrazoIe-4-carbonitriIe

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 15 l-Methyl-5-[(pyridin-3-ylmethyl)-amino]-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 16 l-Methyl-5-(4-methyl-piperazin-l-yI)-3-trifluoromethyl-lH-pyrazoIe-4-carbonitrile

This compound is commercially available from Key Organics, Ltd. EXAMPLEIZ l-Methyl-5-(4-phenyl-piperazin-l-yl)-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 18 5-[4-(4-Fluoro-phenyl)-piperazin-l-yl]-l-methyI-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 19 2-(4-Cyano-2-methyl-5-phenyl-2H-pyrazol-3-yIsulfanyl)-benzoic acid methyl ester

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 20 l-Methyl-S-phenyl-δ-phenylsulfanyl-lH-pyrazole^-carbaldehyde oxime

This compound is commercially available from Key Organics, Ltd. EXAMPLE 21

2,4-Dichloro-benzoic acid N'-(4-cyano-2-methyl-5-trifluoromethyl-2H-pyrazol-3-yl)-N'-methyl- hydrazide

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 22 N'-(4-cyano-l-methyI-3-(trifluoromethyI)-lH-pyrazol-5-yl)-N'-methylbenzenesulfonohydrazide

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 23

N'-(4-cyano-l-methyl-3-(trifluoromethyl)-lH-pyrazoI-5-yl)-N'-methyl-N- (methylsulfonyl)methanesulfonohydrazide

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 24 (4-Cyano-2-methyI-5-trifluoromethyl-2H-pyrazol-3-ylsulfanyl)-acetic acid ethyl ester

This compound is commercially available from Key Organics, Ltd. KXAMPLE 25

2-(4-Cyano-2-methyl-5-trifluoromethyl-2H-pyrazol-3-ylsulfanyl)-propionic acid ethyl ester

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 26 5-Ethoxy-l-methyI-3-trifluoromethyl-lH-pyrazole-4-carbonitrile

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 27 l-MethyI-5-phenoxy-3-trifluoromethyl-lH-pyrazoIe-4-carbonitriIe

This compound is commercially available from Key Organics, Ltd.

EXAMPLE 28 5-(Furan-2-yImethylsulfanyl)-l-methyl-3-trifluoromethyI-lH-pyrazole-4-carbaIdehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. EXAMPLE 29 5-(Furan-2-ylmethylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 30 2-Methyl-5-pyridin-4-yl-2H-pyrazol-3-ol

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 31 2,5-DimethyI-2H-pyrazol-3-ol

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 32

S-Cl-Chloro-β-fluoro-benzylsuIfanylJ-l-methyl-S-trifluoromethyl-lH-pyrazoI^-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 33 S-Benzylsulfanyl-l-methyl-S-trifluoromethyl-lH-pyrazole-^-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. EXAMPLE 34 l-MethyI-5-(2-methyl-benzylsulfanyl)-3-trifluoromethyl-lH-pyrazoIe-4-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 35 l-Methyl-S-phenethylsulfanyl-S-trifluoromethyl-lH-pyrazole^-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 36 5-BenzyIsulfanyl-l-methyl-3-trifluoromethyI-lH-pyrazoIe~4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 37

5-(2-Chloro-6-fluoro-benzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. EXAMPLE 38 l-Methyl-5-(2-o-toIyl-ethyIsulfanyl)-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 39 l-MethyI-5-phenethylsulfanyl-3-trifluoromethyl-lH-pyrazole-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 40

(E)-5-(2-chlorobenzylthio)-l-methyI-3-(trifluoromethyl)-lH-pyrazole-4-carbaldehyde O-benzyl oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 41

(EJ-S-Cl-chlorobenzylthioJ-l-methyl-S-^rifluoromethy^-lH-pyrazole^-carbaldehyde O-ethyl oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. RXAMPLE 42 5-(2-Chloro-benzylsulfanyl)-l,3-dimethyl-lH-pyrazole-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 43 5-(2-Chloro-benzylsulfanyI)-l,3-dimethyl-lH-pyrazole-4-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 44 5-(2-Chloro-benzylsulfanyl)-l-methyl-3-ρyridin-4-yl-lH-pyra2θle-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 45 S-(2-ChIoro-benzylsulfanyl)-l-methyl-3-pyridin-4-yl-lH-pyrazole-4-carbaldehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. EXAMPLE 46 [5-(2-chloro-benzylsulfanyl)-l-methyI-3-trifluoromethyl-lH-pyrazol-4-yl]-methanol

Preparation of compound 46: [5-(2-Chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyHH-pyrazol- 4-yl]-methanoI.

To a well stirred, vented solution of 5-(2-chlorobenzylsulfanyl)-l-methyl-3-(trifiuoromethyl)-lH- pyrazole-4-carboxaldehyde (100 mg, 0.298 mmol) in MeOH (500 μL) was added sodium borohydride (11.3 mg) at rt. The reaction mixture was stirred for 2 h then quenched with water (5 niL) and extracted with ethyl acetate (2 x 10 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum to afford a residue. The material was purified by column chromatography (hexanes to 9:1 EtOAc/hexanes) to afford 94 mg (94%) of [5-(2-chloro-benzylsulfanyl)-l-methyl-3- trifluoromethyl-lH-pyrazol-4-yl] -methanol as a clear oil. [M+H]⁺ 336.88.

EXAMPLE 47

[5-(2-chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyI-lH-pyrazol-4-ylmethylenc]-hydrazine

Preparation of compound 47: [S-(2-Chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyI-lH-pyrazol- 4-ylmethylene]-hydrazine.

To a well stirred solution of 5-(2-chloroben2ylsulfanyl)-l-methyl-3-(trifluoromethyl)-lH-pyrazole-4- carboxaldehyde (115 mg, 0.340 mmol) in ethanol (1.5 mL) was added hydrazine (17.0 μL, 0.520 mmol) at rt under nitrogen. The reaction mixture was heated to 80 ⁰C for 16 h then cooled to rt. The mixture was concentrated under vacuum to afford 115 mg (96%) of [5-(2-chloro-benzylsulfanyl)-l-methyl-3- trifluoromethyl-lH-pyrazol-4-ylmethylene]-hydrazine as a white solid. [M+H]⁺ 348.90. EXAMPLE 48

N-[5-(2-Chloro-benzylsulfanyI)-l-methyl-3-trifluoromethyl-lH-pyrazol-4-ylmethyIene]-N'-methyl- hydrazine

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 49 5-(2-ChlorobenzyIsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid

Preparation of compound 49a: 5-Chloro-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid.

To awell stirred solution of 5-chloro-l-methyl-3-(trifiuoromethyl)-4-pyrazolecarboxaldehyde (1.01 g, 4.75 mmol) and sodium hydroxide (500 mg, 12.5 mmol) in water (5 mL) was added hydrogen peroxide (3.44 g of a 30 wt. % solution in water) at rt. The reaction mixture was stirred for 16 minutes then pH was adjusted to 1 with cone. HCl causing precipitation of the product. The product was collected via vacuum filtration, washed with water and dissolved in diethyl ether. The organic solution was dried over Na₂SO₄, filtered and concentrated under vacuum to afford 627 mg (58%) of 5-chloro-l-methyl-3- trifluoromethyl-lH-pyrazole-4-carboxylic acid as a white solid.

Step 2

Preparation of compound 49: 5-(2-Chlorobenzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4- carboxylic acid.

To a well stirred solution of 5-chloro-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid (600 mg, 2.63 mmol) and 2-chlorobenzenemethanethiol (850 mg, 5.25 mmol) in DMF (10 mL) was added triethylamine (1.10 mL, 15.0 mmol) at rt under nitrogen. The reaction mixture was heated to 80 ⁰C for 4 h then cooled to rt. The reaction mixture as concentrated under vacuum to an oil and purified by column chromatography (DCM to 9:1 DCM/MeOH) to afford 710 mg (87%) of 5-(2-chlorobenzylsulfanyl)-l- methyl-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid as a white solid. [M+H]⁺ 351.05. EXAMPLE 50 5-(2-chloro-benzylsulfanyl)-l-methyI-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid

49 50

To a well stirred solution of 5-(2-chlorobenzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4- carboxylic acid (35 mg, 0.10 mmol), THF (200 μL) and methanol (200 μL) was added (trimethylsilyl)diazomethane (115 μL of a 2.0 M solution in Et₂O, 0.23 mmol) at rt under nitrogen. The reaction mixture was stirred for 16 h then concentrated under vacuum to afford a residue. The product was purified by column chromatography (hexanes to 1:1 hexanes/DCM) to afford 17 mg (46%) of 5-(2- chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazole-4-carboxylic acid methyl ester as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.37-7.35 (m, IH), 7.22-7.20 (m, IH), 7.08-7.05 (m, IH), 6.81-6.79 (m, IH), 4.24 (s, 2H), 3.91 (s, 3H), 3.49 (s, 3H).

EXAMPLE 51

5-(2-Chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyl-lH-pyrazoIe-4-carboxylic acid methylamide

Step l

Preparation of compound 50a: 5-(2-Chloro-benzylsulfanyI)-l-methyl-3-trifluoromethyI-lH-pyrazole-

4-carboxylic acid chloride.

To a well stirred, vented solution of 5-(2-chlorobenzylsulfanyl)-l-methyl-3-trifluoromethyl-lH- pyrazole-4-carboxylic acid (490 mg, 1.40 mmol), DMF (1 drop) and DCM (6 mL) was added oxalyl chloride (180 μL, 2.10 mmol) at rt under nitrogen. The reaction mixture was stirred for 2 h then concentrated under vacuum to afford 515 mg (quant.) of 5-(2-chloro-benzylsulfanyl)-l_:methyl-3- trifluoromethyl-lH-pyrazole-4-carboxylic acid chloride as a white solid.

Step 2

Preparation of compound 50: 5-(2-Chloro-benzylsulfanyl)-l-methyI-3-trifluoromethyl-lH-pyrazoIe-

4-carboxylic acid methylamide. To a well stirred solution of 5-(2-cMoro-benzylsulfanyl)-l-metliyl-3-trifluoromethyl-lH-pyrazole-4- carboxylic acid chloride (76 mg, 0.21 mmol) and methylamine (3.0 mL of a 2.0 M solution in THF, 6.0 mmol) was added triethylamine (50 μL, 0.36 mmol) at rt under nitrogen. The reaction mixture was stirred for 72 h then concentrated under vacuum to an oil. The residue was dissolved in DCM (5 mL) and washed with sat. aq. NaHCO₃. The organic layer was dried over Na₂SO₄, filtered and concentrated under vacuum to a residue. The material was purified by column chromatography (DCM to 9: 1 DCM/MeOH) to afford 42 mg (56%) of 5-(2-chloro-benzylsulfanyl)-l-methyl-3-trifluoromethyl-lH- pyrazole-4-carboxylic acid methylamide as a white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.38-7.36 (m, IH), 7.25-7.20 (m, IH), 7.10-7.06 (m, IH), 6.77-6.75 (m, IH), 6.50 (bs, IH), 4.13 (s, 2H), 3.56 (s, 3H), 2.91-2.90 (d, 3H).

EXAMPLE 52 5-(2-chIorobenzyIthio)-N, N,l-trimethyl-3-(trifluoromethyl)-lH-pyrazole-4-carboxamide

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 53 N-benzyl-5-(2-chlorobenzylthio)-l-methyI-3-(trifluoromethyl)-lH-pyrazole-4-carboxamide

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 54 5-(2-chlorobenzylthio)-l-methyl-3-(triiluoromethyl)-lH-pyrazole-4-carboxamide

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51. EXAMPLE 55 l-(pyridin-2-yl)-3-(trifluoromethyl)-lH-pyrazol-5-ol

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 56 5-(2-chlorobenzyIthio)-l-(pyridin-2-yl)-3-(trifluoromethyl)-lH-pyrazole-4-carbaIdehyde

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

EXAMPLE 57 (E)-5-(2-chlorobenzylthio)-l-(pyridin-2-yl)-3-(trifluoromethyl)-lH-pyrazoIe-4-carbaldehyde oxime

This compound was prepared by analogous procedures to that of Example 3, 46, 47, 49, 50, and 51.

The compounds of Examples 1 to 57 were characterized by proton nuclear magnetic resonance spectral analysis (HNMR) and/or high resolution mass spectrometry (HRMS). The results are shown below in Table 1, with the exception of Example 3, for which such data can be found above. Table 1: Compound Characterization

All chemical IUPAC names were generated using CambridgeSoft's ChemDraw 10.0.

The invention is further illustrated by the following examples, which can generally be made using the methods described above. It is expected that these compounds when made will have activity similar to those that have been made in the examples above. The following compounds are represented herein using the Simplified Molecular Input Line Entry System, or SMILES. SMILES is a modern chemical notation system, developed by David Weininger and Daylight Chemical Information Systems, Inc., that is built into all major commercial chemical structure drawing software packages. Software is not needed to interpret SMILES text strings, and an explanation of how to translate SMILES into structures can be found in Weininger, D., /. Chem. Inf. Comput. Set 1988, 28, 31-36.

CN1N=C(C(F)(F)F)C(/C=N/O)=C1SCC2=C(OC)C=CC=C2

CN1N=C(C(F)(F)F)C(/C=N/O)=C1SCC2=CC=CC=C2F

CN1N=C(C(F)(F)F)C(/C=N/O)=C1SCC2=C(C)C=C(C)C=C2 CN1N=C(C(F)(F)F)C(/C=N/O)=C1SCC2=CC=CS2

O/N=C/C1=C(SCC2=C(C1)C=CC=C2)N(CC)N=C1C(F)(F)F

CN1N=C(C(F)(F)F)C(/C=N/OCC2=CC=CC=C2)=C 1 SCC3=CC(C=CC=C4)=C4C=C3

CN1N=C(C(F)(F)F)C(/C=N/N)=C1SCC2=CC=NC=C2

CN1N=C(C(F)(F)F)C(/C=N/OCC)=C1SCC2=NC=CC=C2 CN1N=C(C(F)(F)F)C(/C=N/O)=C1 SC2=C(C1)C=CC=C2

CN1N=C(C(F)(F)F)C(/C=N/O)=C1S(CC2=CC=CC=C2C)(=O)=O CN1N=C(C(F)(F)F)C(/C=N/O)=C1SCC(OCC)=O CN1N=C(C(F)(F)F)C(/C=N/O)=C1SC2=C(C(OC)=O)C=CC=C2 CN1N=C(C(F)(F)F)C(/C=N/O)=C1S(C2=C(C(OC)=O)C=CC=C2)(=O)=O CN1N=C(C(F)(F)F)C(/C=N/O)=C1S(C2=CC=CC=C2)(=O)=O CN1N=C(C(F)(F)F)C(/C=N/O)=C1 S(CC2=CC=CC=C2)(=O)=O CN1N=C(C(F)(F)F)C(C=O)=C1SCC2=CC=CC=C2F CN1N=C(C(F)(F)F)C(C=O)=C1SCC2=CC=CS2 CNlN=C(C(F)(F)F)C(C=O)=C 1 SCC2=C(C)C=C(C)C=C2 CN1N=C(C(F)(F)F)C(C(NC)=O)=C1SCC2=NC=CC=C2 CN1N=C(C(F)(F)F)C(C=O)=C1SCC2=C(OC)C=CC=C2 O=CC1=C(SCCC2=CC=CC=C2)N(CC)N=C1C(F)(F)F CN1N=C(C(F)(F)F)C(C(O)=O)=C1SCC2=CC(C=CC=C3)=C3C=C2 CN1N=C(C(F)(F)F)C(C(N(C)C)=O)=C1SCC2=CC=NC=C2 CN1N=C(C2=CC=NC=C2)C(C=O)=C1SCC3=C(C1)C=CC=C3F CN1N=C(C2=CC=NC=C2)C(C=O)=C1 SCC3=CC=CO3

CN1N=C(C2=CC=NC=C2)C(C=O)=C1SCC3=CC=CC=C3 CN1N=C(C2=CC=NC=C2)C(C(NC)=O)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=NC=C2)C(C=O)=C1SCC3=C(C)C=CC=C3 CN1N=C(C2=CC=NC=C2)C(C=O)=C1SCCC3=CC=CC=C3 CN1N=C(C2=CC=CC=N2)C(C(N(C)C)=O)=C1 SCC3=C(C1)C=CC=C3

CN1N=C(C2=CC=CC=N2)C(C(NCC3=COCC=C3)=0)=C1SCC4=C(C1)C=CC=C4 CN1N=C(C2=CC=CC=N2)C(C(N)=O)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CC=N2)C(C(O)=O)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CC=N2)C(C(OC)=O)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CC=C2)C(/C=N/O)=C1SCC3=CC=CC=C3

CN1N=C(C2=CC=CC=C2)C(/C=N/O)=C1SCC3=C(C1)C=CC=C3F CN1N=C(C2=CC=CC=N2)C(/C=N/O)=C1SCC3=C(C)C=CC=C3 CN1N=C(C2=CC=CC=N2)C(/C=N/O)=C1SCC3=CC=CO3 CN1N=C(C2=CC=NC=C2)C(/C=N/O)=C1SCCC3=CC=CC=C3 CN1N=C(C2=CC=NC=C2)C(/C=N/OCC3=CC=CC=C3)=C1 SCC4=C(C1)C=CC=C4 CN1N=C(C2=CC=NC=C2)C(/C=N/N)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=NC=C2)C(/C=N/OCC)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CN=C2)C(/C=N/NC)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CN=C2)C(/C=N/OC(C(C1)C1)=O)=C1SCC3=C(C1)C=CC=C3 CN1N=C(C2=CC=CN=C2)C(/C=N/O)=C1 SCC3=C(C1)C=CC=C3 BIOLOGICAL ASSAYS OF THE COMPOUNDS OF THE INVENTION

Cell Culture Protocol A-172 human glioblastoma cells, RAW 264.7 mouse macrophages, and HeIa human adenocarcinoma cells (ATCC) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Upon stimulation, serum concentration was reduced to 5%. A-172 cells were stimulated with a cytokine cocktail containing MFN-γ (4,000 U/ml), hTNF-α (40 ng/ml), ML-I β (4ng/ml). RAW 264.7 cells were stimulated in media with LPS (1 μg/ml) (Sigma) and mIFN-γ (100 U/ml) (Roche Diagnostics).

Compounds of the present invention as activators of the UPR

To investigate the biological pathways impacted by the compounds disclosed herein, we analyzed RNA extracts from A-172 cells simultaneously treated with cytokine stimulation media and Example 3, Example 42, or DMSO (vehicle) using Affymctrix oligonuclcotidc-bascd microarray technology. We conducted an analysis of total RNA on an array of 23,000 probe sets representing 18,400 transcripts including 14,500 genes. No significant changes in gene expression were observed following 30 minutes of compound treatment. However, after 6 hours of incubation with Example 3, many hallmark genes of the UPR showed increased expression. This effect was not seen when cells were treated with Example 42 or vehicle. The data are represented as a ratio of cells treated with either Example 3 or Example 42 versus vehicle in the presence of proinflammatory cytokines. XBP-I, ATF3, ATF4, and CHOP are transcription factors known to play critical roles in activating the ER stress response and are significantly upregulated in Example 3 -treated cells independent of cytokine induction. The molecular chaperones ERdj4 and BiP were upregulated by 24-fold and 2.6-fold, respectively. SEC23B and SEC3 ILl, both significantly upregulated by Example 3, are involved in ER to Golgi protein transport. Herpudl/ Herp, a membrane-associated protein which functions in ER associated degradation (ERAD) was upregulated 9.7-fold by treatment with Example 3. These results indicate that compounds disclosed herein can affect the expression of genes involved in many aspects of the UPR, either directly or indirectly, including those involved in signal transduction of ER stress, transcription of UPR mediators, chaparones, protein degradation machinery, and inducers of ER stress apoptosis. Closely related gene expression signatures have been described for known ER stressors including tunicamycin and thapsigargin . Taken together, these findings strongly suggest that compounds have been discovered which act in the ER stress pathway. Results are shown below in Table 2.

Quantitative real time PCR and Microarray Analysis Assay Protocol

After incubation with lOμM compound and proinflammatory cytokine stimulation media, cells were lysed in 350 μl buffer RLT, homogenized over QiaShredder columns, and total RNA was purified using the Rneasy kit with on-column Dnase digestion (Qiagen). RNA quality and concentration was determined using the Bioanalyzer RNA6000 assay (Agilent). 2 μg of RNA was used in cDNA synthesis reactions (invitrogen). Each cDNA sample was amplified using SYBRgreen-based qPCR and a melting curve was determined. 5 μg of the remaining RNA was analyzed on U133A 2.0 Gene chips (Afϊymetrix). Analysis was conducted using gcRMA normalization with GeneSpring and Spotfire software platforms.

Table 2

Compounds of the present invention activate the UPR broadly and via apstream targets To confirm that the UPR gene expression signature induced by Example 3 is mediated by action on an upstream target, we measured the phosphorylation status of eIF-2α (Ser 51) which is directly phosphorylated by PERK in an early event of UPR activation. eIF-2α phosphorylation results in an attenuation of protein translation in cells and, due to its abundance, a small change in the phosphorylation status can have a significant effect on cells. Immunoblot analysis of protein extracts isolated from cells treated with Example 3 for 30 minutes showed significant eIF-2α phosphorylation (Table 3). This effect was also seen in cells treated with thapsigargin,, but not in cells treated with Example 42 or vehicle alone. Total eIF-2α protein levels were unchanged by drug treatment. (Table 3).

EIF2α phosphorylation Assay RAW 264.7 cells were incubated for the indicated times with stimulation media and Example 3 or Example 42 (lOμM), thapsigargin (lμM), or DMSO 0.1%. Whole cell extracts were prepared in the presence of protease inhibitors (Roche) and phosphatase inhibitors (Pierce). Samples were sonicated for 6 seconds and centrifuged at 13k rpm for 10 minutes. Total protein concentration was measured using the Advanced Protein Assay (cytoskeleton, inc) and protein quantity was normalized for loading. Samples were run on 6-20% Tris Glycine gels (Invitrogen) and electro-transferred (Biorad) to nitrocellulose membrane (osmonics). Membranes were blocked overnight in Starting Block (Pierce) containing 0.1% Tween-20 and incubated in a 1:500 dilution of either EIF2α or EIF2α-phospho S51 for 2 hours, followed by 1:2000 dilution of HRP-conjugated secondary antibodies for 1 hour. All of the westerns were developed with the WestDura substrate (Pierce) and imaged on the FluorChem imager. To ascertain whether erstressin acts solely ttixough the PERK-eIF2α pathway or more broadly in ER stress signaling, we tested whether the splicing of XBP-I mRNA, regulated by the ER transmembrane ribonuclease IRE-I, was altered by treatment with Example 3. RT-PCR of RNA samples isolated from cells incubated with either 20μM Example 3, 20μM Example 42 or 5OnM thapsigargin for 1 hour revealed the presence of alternativly spliced XBP-I . Once again, neither vehicle nor Example 42 induced alternative splicing of XBP-I (Table 3). These findings suggest that compounds of the invention activate multiple ER stress signaling pathways within 30 minutes to 1 hour of compound treatment.

XBPl Splicing Assay

HeIa cells (ATCC) were seeded in 6 well dishes and treated with 20μM Example 3, 20μM Example 42, 5OnM Thapsigargin or 0.2% DMSO for 1 hour. Cells were then washed twice with PBS and RNA was isolated using the RNeasy kit including on-column DNase treatment (Qiagen). cDNA was transcribed using SuperscriptIII (Invitrogen) and PCR of a 597bp fragment, including the alternately spliced intron, was performed.

Table 3

Compounds of the present invention as iNOS expression inhibitors To investigate the mechanism by which the compounds disclosed herein inhibit NO production, we employed a microplate-based in-cell western or "cytoblof'assay to assess compound activity in dose- response in RAW 264.7 cells. As shown in Table 3, compounds disclosed herein abrogate expression of iNOS protein in activated macrophages.

RAW Cvtoblot Assay Protocol

This assay is performed in a 384-well plate. RAW 264.7 (Abelson murine leukemia virus-induced tumor) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) / 10% fetal bovine serum (FBS) / IX penicillin- streptomycin solution (P/S; final concentration in the media is 100U/ml penicillin and 1 OOug/ml streptomycin). Cells were either trypsinized or scraped to subculture and resuspended at 400,000 cells/mL (-16,000 cells/well) in DMEM/ 5% FBS/ IX P/S. 40μL cells were added to each well of a white TC-treated 384-well plate allowed to adhere for 4-6 hours at 37°C under 10% CO₂. Compound in dimethyl sulfoxide (DMSO) was then added, 500nL/well in dose response (half-log serial dilution). Immediately after adding compound, lOμL per well of an induction cocktail, consisting of lOμg/mL lipopolysaccharidc (LPS, bacterial endotoxin) and 0.25μg/mL mouse interferon gamma (IFN- γ) in DMEM/ 5% FBS/ IX P/S, was added. Plate was then incubated for 12-18 hours at 37°C under 10% CO₂. Afterward, media was shaken out of plate and cells were fixed with a fixative made by mixing 142.5mL 98% ethanol and 7.5mL glacial acetic acid. This was immediately washed by shaking the reagent out of the plate and adding 85μL phosphate-buffered saline (PBS) with no magnesium salt; wash was repeated twice more. 30μL/well of 4% bovine serum albumin (BSA) in PBS was added and incubated 1 hour at room temperature, then washed once as above. After this solution was shaken out, 35μL/well primary mouse iNOS antibody (diluted 1:250 in PBS containing 1% BSA) was added, incubated 1 hour at room temperarture, and washed as above. When this solution was shaken out, 35μL/well secondary goat anti-mouse horseradish peroxidase (HRP) antibody (diluted 1:2500 in PBS) was added, incubated 1 hour at room temperature, washed three times as above, and the solution was shaken out. Finally, 40μL/well Luminol reagent (Santa Cruz Biotechnology) was added and chemiluminescence read on an Analyst GT multimode microplate reader (Molecular Devices). Data are shown below in Table 4.

Table 4

Compounds of the present invention as regulators of genes linked to inflammation

Further inspection of the effects of the compounds disclosed herein on global gene expression revealed that in addition to affects on UPR genes and the downregulation of iNOS expression, other inflammation-related genes show altered transcription. As expected, cytokine stimulation induced many gene expression changes. The mRNA levels of approximately 732 genes were altered 2-fold or more by cytokine stimulation at 6 hours; this is compared to about 285 genes affected by Example 3 treatment. Of the genes modulated by Example 3 , about one quarter were also affected by cytokine, indicating that compounds disclosed herein broadly impact inflammatory pathways. Most of these genes are downregulated by Example 3, including the canonical cytokines IL-I β and TNFα, the chemokmes MCP-2 and I-TAC, and downstream regulators of IFN-γ signaling such as INDO, SOCSl, and SOCS3 (Table #). Interestingly, a subset of inflammation-related genes are upregulated. Perhaps most interesting is a modest increase in the expression of NFκBl/plO5, consistent with a reported role for this transcription factor in the UPR. In addition to NFkB, the proinflammatory transcription factor, ESE-I, the cytokine IL-15, and receptors E-selectin and RDC-I were also upregulated (Table 5). Because none of these effects are observed with Example 42, which does not robustly activate the UPR nor inhibit iNOS expression , it is likely that they are mediated via an ER stress mechanism.

Quantitative real time PCR and Microarray Analysis Assay Protocol

After incubation with lOμM compound and proinflammatory cytokine stimulation media, cells were lysed in 350 μl buffer RLT, homogenized over QiaShredder columns, and total RNA was purified using the Rneasy kit with on-column Dnase digestion (Qiagen). RNA quality and concentration was determined using the Bioanalyzer RNA6000 assay (Agilent). 2 μg of RNA was used in cDNA synthesis reactions (invitrogen). Each cDNA sample was amplified using SYBRgreen-based qPCR and a melting curve was determined. 5 μg of the remaining RNA was analyzed on U133A 2.0 Gene chips (Affymetrix). Analysis was conducted using gcRMA normalization with GeneSpring and Spotfire software platforms.

Table 5.

Compounds of the present invention mediatelevels of inflammatory markers, inflammation, and pain in vivo Rat Endotoxemia LPS Model

Male Lewis rats (Charles River, Portage MI) 150-200 g were administered a dose of 0.3 mg/kg of lipopolysacharride - LPS (Sigma, L2880) dissolved in 0.9% sodium chloride. The intravenous injection was given though the penal vein under anesthesia with 5% isoflurane in medical grade pure oxygen and maintained at 2-3% isoflurane. Example 3 plus vehicle (30mg/kg in Encapsin, 5% DMSO) or vehicle alone was administered 3 to 5 minutes prior to LPS challenge via intraperitoneal injection. Rats were returned to their home cages and sacrificed 6 hours later. Blood samples were collected for analysis of nitrates (Cayman Chemical Nitrate/Nitrite Flourometric Assay Kit catalog #780051). First, the nitrates are converted to nitrite using nitrate reductase. Then diaminonapthalene (DAN) is added followed by NaOH which results in the conversion of DAN into a fluorescent compound. Fluorescence was measured using the Aquest plate reader (Molecular Probes). Results are shown in Table 6, and values reported are +/- 20μM.

Table 6.

Rat Carrageenan Inflammation & Pain Model

Male Sprague-Dawley rats (Charles River, Portage MI, 170-200 g) were used. A 1% w/v suspension of carrageenan (Sigma, lot#l 0k0776) was prepared by dispersing into saline overnight in 4OC. Paw edema and hyperalgesia were induced by the injection of 0.1 ml of a θ.1% carrageenan solution into the hind footpad. Drug (30mg/kg in Encapsin, 5% DMSO) or vehicle alone was administered 30 minutes prior to carrageenan via intraperitoneal injection. Paw edema was measured at 6hr and thermal hyperalgesia was measured at 3hr and 5hr as indicated in the figure legends. Paw volume was measured with a water displacement plethysmometer before and 6hr after the carrageenan injection. The differences of paw volume in the same rat before or after carrageenan injection are expressed as paw edema. Hyperalgesic response was measured in the same animal. Animals were placed in chambers with a glass bottom and allowed to acclimate. Hind paws were exposed to radiant heat emitted from a high intensity projection bulb at selected times after carrageenan injection. The latency to withdraw the paw from the glass plate was determined. The differences of paw withdrawal latencies between the normal or carrageenan injected paws indicate hyperalgesic response. Results are shown below as mean in Table 7.

Table 7.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

CLAIMS We claim:

1. A method for the treatment of a hyperproliferative, autoimmune, cardiovascular, respiratory, neurologic, inflammatory, chronic pain, neuropathic pain, or iNOS-related disease, comprising the administration of a therapeutically effective amount of a compound which activates the unfolded protein response (UPR) to a patient in need of such treatment.

2. The method as recited in Claim 1, wherein said compound also inhibits the expression of iNOS.

3. The method as recited in Claim 2, wherein said disease is an inflammatory disease.

4. The method as recited in Claim 3 , wherein said inflammatory disease is selected from the group consisting of inflammation, neuropathy, neuropathic pain, inflammatory pain, herpes zoster, postherpetic neuralgia, post-surgical pain, arthritis, arthritic pain, inflammatory bowel disease, Crohn's disease, irritable bowel syndrome, ulcerative colitis allodynia, fibromyalgia, complex regional pain syndrome, and hyperalgesia.

5. The method as recited in Claim 2, wherein said disease is a hyperproliferative disease.

6. The method as recited in Claim 5, wherein said hyperproliferative disease is selected from the group consisting of cancer, myelodysplastic syndrome, and psoriasis.

7. The method as recited in Claim 6, wherein said cancer is selected from the group consisting of hematologic cancers, nonhematologic cancers, and solid tumors.

8. The method as recited in Claim 1, wherein said compound is a compound of Formula I:

or a salt, ester, or prodrug thereof, wherein:

A is selected from the group consisting of acyl, alkyl, alkoxy, alkoxyalkyl, alkylene, alkynyl, amido, amino, aminosulfonyl, aryl, arylalkoxy, arylamino, arylthio, carboxy, cycloalkyl, ester, ether, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydrazinyl, imino, thio, sulfonate and sulfonylamino, any of which may be optionally substituted;

B is selected from the group consisting of acyl, alkoxy, alkoxyalkyl, alkyl, alkylene, alkynyl, alkylimino, amido, aryl, carboxy, cyano, cycloalkyl, ester, halo, haloalkyl, heteoaryl, heterocycloalkyl, hydroxyalkyl, hydrogen, imino and iminohydroxy, any of which may be optionally substituted;

C is selected from the group consisting of -OR¹, -SR¹, -S(O)R¹, -S(O)₂R¹, -NR²R³, acyl, alkyl, alkylene, alkynyl, amido, carboxy, cycloalkyl, ester, halo, haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and imino, any of which may be optionally substituted; X is selected from the group consisting of acyl, alkoxyalkyl, alkyl, alkylamino, amino, amido, alkylamino, arylalkyl, aryl, arylalkyl, carboxy, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydrogen, sulfonate and sulfonyl, any of which may be optionally substituted; R¹ is selected from the group consisting of acyl, alkoxy, arylalkoxyalkyl, alkyl, amino, amido, aminoalkyl, aryl, arylalkyl, carboxy, cycloalkyl, haloalkyl, heteroarylalkyl, heterocycloalkyl, hydrogen, hydroxy and hydroxyalkyl, any of which may be optionally substituted; and

R² and R³ are each independently selected from the group consisting of alkyl, amino, arylalkyl, aryl, cycloalkyl, haloalkyl, heteroarylalkyl,.heterocycloalkyl and hydrogen, R² and R³ may combine to form a heterocycloalkyl, any of which may be optionally substituted.

9. The method as recited in Claim 8 wherein:

A is selected from the group consisting of alkyl, aryl, haloalkyl and heteroaryl, any of which may be optionally substituted; B is selected from the group consisting of acyl, amido, carboxy, cyano, ester, hydroxyalkyl, hydrogen, imino and iminohydroxy, any of which may be optionally substituted; and

C is selected from the group consisting of hydroxyl, -OR¹, -SR¹, -S(O)R¹, -S(O)₂R¹, and - NR²R³.

10. The method as recited in Claim 9 wherein: A is CF₃; and

C is selected from the group consisting of -SR¹, -S(O)₂R¹, -NR²R³ and -OR¹, any of which may be optionally substituted.

11. The method as recited in Claim 10 wherein X is alkyl or heteroaryl, either of which may be optionally substituted.

12. The method as recited in Claim 11 wherein B is acyl, hydrogen, amido, carboxy, ester, nitrile or imino, any of which may be optionally substituted.

13. The method as recited in Claim 12 wherein X is optionally substituted alkyl.

14. The method as recited in Claim 13 wherein C is -SR¹.

15. The method as recited in Claim 14, wherein A is alkyl or heteroaryl, either of which may be optionally substituted.

16. A compound selected from the group consisting of Examples 28, 29, and 32-57.

17. Use of a compound as recited in Claim 16 in the manufacture of a medicament for the treatment of (iNOS) related diseases.

18. A pharmaceutical composition comprising a compound according to Claim 16 or a pharmaceutically acceptable salt thereof and together with pharmaceutically acceptable carrier.