METHODS QF MODULATING NEUROTROPHIN-MEDIATED ACTIVITY

Related Applications

This application claims priority to U.S. Provisional P itent Application No. 60/974,666, Attorney Docket No. PCI-063-1, filed Septembt 124, 2007. This application also claims priority to U.S. Provisional Patent Application No. 60/980,091 ₁ Attorney Docket No. PCϊ-063-2, filed October 15, 2007. Both of these applications are incorporated herein by reference in their entirety. The ccntents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties. j

Technical Field

The present invention relates to compositions which iiodulate the interaction of nerve growth factor, and precursors thereof, with the reα ep tor TrkA, as well as the common neurotrophin receptor p75 NTR, and methods of use thereof.

Background

The neurotrophic are a family of structurally and functionally related proteins, including Nerve Growth Factor (NGF), Brain-Derh ed Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin- 4/5 (NT-4/5) and Neurotrophm-6 (NT-6). These proteins promote the survival and differential on of diverse neuronal populations in both the peripheral and central nervous systems and are involved in the pathogenesis of diverse neurological disorders (Hefti, J. Neurosci, 6:2155-2162

(1986); Hefli and Weiner, Annals of Neurology 20:275-281 (|1986); Levi-Montalcini, EMBO J. 6:1145-1154 (1987); Barde, Neuron 2:1525-1534 (11989); Leibrock et al., Nature 341:14^152 (1989); Maisonpierre er α/., Science 247J: 1446-1451 (1990); Rosenthal etal, Neuron 4:767-773 (1990); Hohn et al., Natulre 344:339-341 (1990); Gotz et al,, Nature 372:266-269 (1994); Maness et al, Neurcsci. Biobehav. Rev.

18: 143- 159 (1994); Dechant et al., Nature Neurosci. 5:1131- 1136 (2002)). This broad spectrum of biological activities exerted by the neurotrophin! results from their ability to bind and activate two structurally unrelated receptor types, the p75 neurotrophin receptor (p75 ) and the three members of the Trk receptor amily of tyrosine kinases (Kaplan et al, Curr. Opin. Cell Biol. 9:213-221 (19$j); Friedman et al, Exp.

A. et a!., Exp. Neurol. 2005; 191(2):337-43).

NGF is synthesized as a larger precursor form (referred to herein as "proNGF," also known as "preproNGF' or "pro-peptide NGJF") which is then processed by proteolytic cleavages to produce the mature neurotrophic Factor. This prepro region is located at the amino terminus of the precurs&r molecule and is needed _^ for proper folding and secretion of the NGF protein. The primary structure of proNGF has been deduces! from the nucleotide sequence of the mouse NGF cDNA (Scott et at Nature 302:538 (1983); Ullrich et at Nature 303:821 (1983)).

The common neurotrophin receptor p75 ^rπR is a transmembrane glycoprotein structurally related to the tumor necrosis factor and CD-40 receptors (Meakin and Shooter, Trends Neurosci. 15:323-331 (1992), Ryden and Iblanez, J. Biol. Chem. 271.5623-5627 (1996)). As all neurotrophins bind to p75 ^NT | ^R with similar affinities (Rodrigues-Tebar et at, Neuron 4:487-492 (1990); Hallbooi et al., Neuron 6:845-858 (1991); Rodrigues-Tέbar et al., EMBO J. 11:917-922 (1992); Ibafie^ Trends Biotech. 13:217-227 (1995)), neurotrophin specificity is conventionally thought to be conferred by the binding selectivity for Trk receptors which are differentially expressed in different neuronal populations (Ibaftez, Trends Biotech. 13:217-227 (1995)). While initially studied primarily in neurons, p75 ^NTR has also been found to play critical rolea in vascular biology (von Schack et at, Nat. Neurosci. 4:977-978, 2001; Wan et at, Am. J. Pathol. 157: 1247-1258, 2001), glial biology (Bentley et al., J. Neurosci.

20:7706-7715, 2000; Syroid et al., J. Neurosci. 20:5741-5747, 2000), the immune system (Tokuoka et al, Br. J. Pharmacol. 134:1580-1586, 2001), and tumor biology

(Sakamoti et al, Oncol. Rep. 8:973-980, 2001; Desoamps et ιL, J. Biol. Chem.

276:17864017870, 2001). For example, p75 N ^N T ^l R ^K , has been demonstrated to participate in human melanoma progression (Hemnaπn et al, MoI. Biol 4: 1205-1216 (1993); Marchetti eι #l., Cancer Res. 56:2856-2863 (1996)).

Unlike p75 ^N , the Trk receptors (TrkA, TrkB andTrkC) exhibit selectivity for specific neurotrophic. (Kaplan et al, Science 252:554-558 (1991); Klein et al.. Cell

65:189-197 (1991) ; Klein et al., Neuron 8:947-956 (1992); E oppet et al., Cell 65:895- 903 (1991); Squinto et al., Cell 65:885-893 (1991); Berkeme er et al., Neuron 7:857-

866 (1991); Escandon et al, Neurosci. Res. 34:601-613 (1993); Lamballe et al, Cell

66:967-970 (1991)). For example, TrkA primarily binds NG ⁷ (Kaplan et al., 1991; Klein et al., 1991) and has been reported to bind NT-3 (J. Bi 1. Chem.271(10):5623-7,

1996); TrkB binds BDNF and NT-4/5 (Soppet et al., 1991 ; Støiiπtø et al., 1991; Berkemeier et al., ]99ϊ ; Escandon et al. , 1993 ; Lamballe et a I 1., 1991; Klein et al., 1992; Vale and Shooter, Methods Enzymol. 109:21-39 (19851 _: ; Barbacid, Oncogene 8:2033-2042 (1993)); and TrkC exclusively binds NT-3 (Laniballe et al '.., 1991; Vale and Shooter, 1985). This is particularly evident when the TrI : receptors are coexpressed with the common neurotrophin receptor p75 NTR (For review see Meakin and Shooter, 1992; Barbacid, 1993; Chao, 1994; Bradshaw et al, 1994; Ibafiez, 1995).

Due to the implication of NGF, and its precursor proNG F, binding to homomeric and heteromeric neurotrophin receptor complexes i in various dise ase states, especially pain, inflammation, neurological disorders and disorders of the res] >iratory ' _: , genitourinary and gastrointestinal systems, a need exists for pharmaceutical a jents and methods of use thereof for modulating the interactions of NGF with the common neurotrophin receptor p75 , and the Trk receptor TrkA.

Summary of the Invention

There remains a need for new treatments and therapie for neurotophin- mediated activity, and conditions, diseases and disorders relatjsd to neurotophin- iϊiediated activity. There is also a need for compounds useful in the treatment or prevention or amelioration of one or more symptoms of pain, inflammatory disorders,

neurological disorders, respiratory disorders, genitourinary disorders, and/or gastrointestinal disorders. Furthermore, there is a need for methods for modulating the activity of NGFj proNGF, pTS ¹ " ¹ ™, aad/or TrkA, using the compounds provided herein,

In one aspect, the invention provides a compound of the invention of the Formula I, Formula II, Formula HI, Formula HIA, Formula iy, Formula V, Formula VI, Formula VII, or Formula VIII ₃ as well as the species liste|d in Tables A-J.

In one aspect, the invention provides a method of modulating the interaction of a neurotrophin and a neurotrophin receptor, comprising contacting cells expressing a neurotrophiπ receptor with an effective amount of a compound of the invention. In one embodiment, the neurotrophin is nerve growthj factor and/or precursors thereof. In another embodiment, the neurotrophin receptor isj selected from the group consisting of pTS^ ¹¹ and TrkA. In yet another embodiment, the neurotrophin receptor is pTS"™. In still another embodiment, the neurotrophin receptor is TrkA. In another embodiment the compound further modulates the interaction |of NGF and/or proNGF with TrkA.

In another embodiment, the method is used to modulate a neurotrophin- mediated activity in a subject in need thereof. In one embodiment, the neurotrophtn- mediated activity is associated with pain. In still another embodiment, the neurotrophin-mediated activity is associated with an inflammatory disorder. In another embodiment, the neurotrophin-mediated activity is associated with a neurological disorder. i in another embodiment, the pain treated by the compounds of the invention is selected from the group consisting of cutaneous pain, somatic, pain, visceral pain and neuropathic pain. In another embodiment, the pain is acute pain or chronic pain. In still another embodiment, the cutaneous pain is associated with injury, disease, disorder or neoplasms of the skin, subcutaneous tissules and related organs. In another embodiment, the injury, disease or disorder of the skin, subcutaneous tissues and related organs is selected from the group consisting of trabmas, cuts, lacerations, punctures, bums, surgical incisions, infections, psoriasis, eczema, and inflammation (e.g., acute inflammation). ^' ,

In another embodiment, the somatic pain is associated with an injury, disease, disorder or neoplasms of the musculoskeletal and connective system. In another

embodiment, the injury, disease or disorder of the musculoskeletal and connective system is selected from the group consisting of sprains, broken bones, arthritis, arthralgia, myalgia, chronic lower back pain, cancer-associated pain, dental pain, Fibromyalgia, idiopathic pain disorder, chronic non-specific pain, post-operative paiα and referred pain. !

In another embodiment, the visceral pain is associated with an injury, disease, disorder or neoplasms of the circulatory system, the respiratory system, the gastrointestinal system, or the genitourinary system. La one embodiment, the disease or disorder of the circulatory system Created by the compounds of the invention is selected from the group consisting of ischaemic heart disease , angina, acute myocardial infarction, cardiac arrhythmia, phlebitis, intermittent claudication, varicose veins and hemorrhoids. In one embodiment, the disease or di sorder of the respiratory system treated by the compounds of the invention is selected from the group consisting of asthma, Chronic Obstructive Pulmonary Disease (COPD), respiratory infection, chronic bronchitis and emphysema. In one embodiment, the iisease or disorder of the gastrointestinal system treated by the compounds of the inveitionis selected from the group consisting of gastritis, duodenitis, irritable bowel syndiome, colitis, Crohn's disease, ulcers and diverticulitis. In one embodiment, the dis sase or disorder of the genitourinary system treated by the compounds of the inventbn is selected from the group consisting of cystitis, urinary tract infections, glomueπ lonephritis, polycystic kidney disease, and kidney stones.

In another embodiment, the neuropathic pain is asso< iated with an injury, disease, disorder or neoplasms of the nervous system. In still another embodiment, the injury, disease or disorder of the nervous system is selected fiom the group consisting of neuralgia, neuropathy, headache, chronic cephalic pain, ph intotn limb pain and spinal cord injury.

In one embodiment, the inflammatory disorder treated by the compounds of the ¹ invention is selected from an inflammatory disorder of the ski n and subcutaneous tissues, the musculoskeletal and connective tissue system, the respiratory system, the circulatory system, the genitourinary system, the gastrointestinal system or the nervous system. In one embodiment, the inflammatory disorder of the skin and subcutaneous tissues is selected from the group consisting of psoriasis, dermatitis and eczema. In

- S -

one embodiment, the inflammatory disorder of the musculosi celetal and connective tissue system is selected from the group consisting of arthriti I, gout, myositis, bursitis and synovitis. In one embodiment, the inflammatory disorde r of the respiratory system treated by the compounds of the invention is selected from the group consisting of asthma, bronchitis, sinusitis, pharyngitis, rhinitis and respi ratory infections. In another embodiment, the inflammatory disorder of the circuli itory system is selected from the group consisting of vasculitis, artherosclerosis, phle bitiiss, carditis and coronary heart disease. In one embodiment, the inflammato 3ry disorder of the gastrointestinal system treated by the compounds of the invent iioonn is selected from the group consisting of inflammatory bowel disorder, ulcerative colitis _; , Crohn's disease, diverticulitis, viral infection, bacterial infection, chronic hepa titis :,, gingivitis, stomatitis, and gastritis. In one embodiment, the inflammato y dis soorrddeerr ooff tthhei genitourinary system treated by the compounds of the inventi on is i selected from the group consisting of cystitis, nephritic syndrome, glomerulonephritis s., urinary tract infection, prostatitis, salpingitis, endometriosis and cystinosis

In another embodiment, the neur< ological disorder treated by the compounds of die invention is selected from the group consisting of schizophrenia, bipolar disorder, depression, Alzheimer's disease, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, stroke, cerebral ischemia, neuropathy, retinal pignw nt degeneration, glaucoma, cardiac aπhythmia, shingles, Huntington's chorea, and Parkinson's disease. In another aspect, the invention provides a method of reating pain in a subject in need thereof, comprising administering to the subject an ef iective amount of a compound of the invention, In one embodiment, the pain is s sleeted from the group consisting of cutaneous pain, somatic pain, visceral pain and : ieuropathic pain. In another embodiment, the pain is acute pain, breakthrough paii i or chronic pain.

In another aspect, the invention provides a method of reatiπg an inflammatory disorder in a subject in need thereof, comprising administerin j to the subject an effective amount of a compound of the invention. In one emt odimeπt, the inflammatory disorder is inflammatory disorder of the tnusctt oskeletal and connective tissue system, the respiratory system, the circulatory system, the genitourinary system, the gastrointestinal system or the nervous system.

In another aspect, the invention provides a method of treating a neurological disorder in a subject in need thereof, comprising adminis ¹ an effective amount of ' a compound of the invention. In one embodiment, the neurological disorder is selected from the group consisting of schizophrenia, bipolar disorder, depression, Alzheimer's disease, epilepsy, multiple sclerosis, amyotrophic lateral [ sole: osiiss,, stroke, cerebral ischemia, neuropathy, retinal pigment degeneration, glaucom|a, cardiac arrhythmia, Huntington's chorea, and Parkinson's disease.

In another aspect, , the invention provides a method of treating a disease or disorder associated with the genitourinary and/or gastrointest nal systems of a subject in need thereof, comprising administering to the subject an effective amount of a compound of the invention. In one embodiment, the disease Jγ disorder of the gastrointestinal system is selected from the group consisting < f gastritis, duodenitis, irritable bowel syndrome, colitis, Crohn's disease, ulcers and diverticulitis. In another embodiment the disease or disorder of the genitourinary systdm is selected from the group consisting of cystitis, urinary tract infections, glomueπ lonephri ittiis, polycystic kidney disease, kidney stones and cancers of the geπitourinar r system i,.

In another aspect, the invention provides a method coinpr iissii;ng administering to the subject an additional therapeutic agent. In one embodime nt :, the additional therapeutic agent is selected from the group consisting of an i φπalgesic, an anti- inflammatory agent, an anesthetic, a corticosteroid, an anti-cqnvulsani, an antidepressant, an anti-nausea/anti-emetic agent, an anti-psychiatric agent, a cardiovascular agent and a cancer therapeutic.

Brief Description of the Drawings

Figure IA and IB, illustrate the dose-dependent effedt of Compound 2E on chemically-induced spontaneous pain evoked by intraplantar njection of formalin in the rat (Formalin model in example 5).

Figures 2A and 2B, illustrate the dose-dependent effe ct of Compound IG on chemically-induced spontaneous pain evoked by intraptantar mjection of formalin in the rat (Formalin model in example Si- Figure 3A and 3B, illustrate the effect of Compound G on the mechanical

(Randall-Sellito) (A) and thermal (Hargreaves' assay) (B) hyperalgesia resulting from

an acute paw inflammation caused by the intraplantar injectii in of λ-carrageenan (Carrageenan model in example 6).

Figure 4A and 4B, illustrate the effect of Compound IG on the mechanical (Randafl-Sellito) (A) and thermal (Hargreaves' assay) (B) hyperalgei sia resulting from paw treatment with capsaicin (Capsaicin model in example

Figure 5A and SB, illustrate the effect of Compound IG on the mechanical allodynia (Von-Frey hair) resulting from sciatic nerve injuries (spinal nerve ligation - SNL Model (A) and spared nerve injury - SNI Model (B), d scribed in examples 9 and 10, respectively).

Figures 6A and 6B, illustrate the dose-dependent effect of Compound 47G on chemically-induced spontaneous pain evoked by intraplantar injection of formalin in the rat (Formalin model in example 5).

Figures 7A and 7B ₇ , jillustrate the dose-dependent effi ;ct of Compound 50G on chemically-induced spontaneous pain evoked by intraplantar injection of formalin in the rat (Formalin model in example 5).

Figure 8A and 8B, illustrate the effect of Compound 50G on the thermal (observed in the Hargreaves' assay) (A), and mechanical (RaJidaU I-Sellito) (B) hyperalgesia resulting from an acute paw inflammation caused by th e. intraplantar injection of λ-carrageeπan (Carrageenan model in example 6)

Detailed Description of the Invents a

The present invention relates to the discovery of comr ounds which modulate the interaction of a neurotrophin - either in its mature {e.g., NGF) or precursor (e.g., proNGF) form - with a neurotrophin receptor, for example, the common neurotrophin receptor p75 ^{N1 R} and/or a Trk receptor. Such compounds are ( f use ;., for example, for modulating the interaction of NGF and/or a precursor thereof {e..gg.., proNGF) to p75 ^NTR , and the compounds within the invention can also hav i the ability to modulate the interaction of NGF and/or proNGF with TrkA. For example, a compound that modulates the binding of NGF or proNGF to p75 ^NTR can furtrjer modulate the binding of the neurotrophin to TrkA Such compounds can also be us< id to treat a subject having a condition with at least one symptom that is directly i r indirectly mediated, at

I eliminating damaged cells by activating the apoptotic machinery via p75 ^NTR after spinal cord injury.

Based on the above, there is a need for compositions which modulate the interaction of nerve growth factor, and precursors thereof, with the receptor TrkA, as well as the common neurotrophiπ receptor p75 ^NTR , and methods of use thereof.

Definitions

As used herein, the term "acid" refers to any gubstitudnt that can readily donate a hydrogen ion to another compound. Particularly preferred acid functional gioups include carboxylic acid, sulfonic acid, sulfinic acid, sulfamic Sacid, phosphonic acid and boronic acid functional groups.

The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups (e.g , methyl, ethyl, propyl, butyl, pentyl, hexyl, Iheptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cycloihexyl, cycloheptyl, cycloocty]), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups The term "alkyl" also includes alkenyl groups and alkynyl groups. Furthermore, the expression "C _x -C _y -alkyl", wherein x is 1-5 and y is 2-10 indicates a particular alkyl group (straight- or branched-chain) of a particular range of carbons. For example, the expression Ci-CValkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

The term alkyl further includes alkyl groups which caji further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more tarbons of the hydrocarbon backbone. In an embodiment, a straight chain o^ branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., C ₁ -Cio foi! straight chain, C ₃ -C]O for branched chain), and more preferably 6 or fewer carbons. Likewise, preferred Cycloalkyls have from 4-7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. j

Moreover, alkyl {e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc ) include both "ungubstitttted alkyl" and "substituted alkyl", the latter ojf which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, which allow the molecule to perform its intended function.

The term "substituted" is intended to describe moieties having substituents replacing a hydrogen on one or more atoms, e.g. C, O or N, of a molecυle. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxy!, al cylcarbonyloxy, arylcarboπyloxy, alkoxyoarbonyloxy, aryloxyoarbonyloxy, carboxylate, alkyloarbonyl, arylcarbonyl, alkoxycarboπyl, βminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate!, phosphonato, phosphinato, amino (including alkyl amino, dialkylaimno, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthioj arylthio, thiocarboxylate, Sulfates, alkylsulfϊnyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoroniethyl, cyano, azido, heterocyolyl, alkylaryl, morpholino, phenol, benzyl, phenyl, piperizine, cyclopentane, cyclohexane, pyridine, SH-tetrazoIe, triazole, piperidine, or an aromatic or heteroaromatic moiety.

Further examples of substituents of the invention, which are not intended to be limiting, include moieties selected from straight or branched ilkyl (preferably C ₁ -C ₅ ), cycloalkyl (preferably C ₃ -C ₈ ), alkoxy (preferably C ₁ -C ₆ ), thioaJkyl (preferably C ₁ -C ₆ ), atkenyl (preferably C ₂ -C ₆ ), alkynyl (preferably C ₂ -C ₆ ), heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy {e.g., phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaraltyl, alkylcarbonyl and arylcarbonyl or other such acyl group, heteroarylcarbonyl, or heteroaryl group, (CR'R") _0-3 NR'R" (e.g., -NH ₂ ), (CR'R") _0-3 CN (e.g., -CN), -NO ₂ , halogen (e.g., -F, -CI, -Br, or-I), (CR'R") _0-3 C(halθgen)3 (e.g., -CF ₃ ), (CR'R") _0-3 CH (halogen) ₂ , (CR'R") _0-3 CH ₂ (halogen), (CR'R") _0-3 CONR'R", (CR'R") _0-3 (CNH)NR'R", (CR'R") _{0- 3} S(O) _1-2 NR'R", (CR'R") _0-3 CHO, (CR-R") _0-3 O(CR'R") _0-3 H, (CR'R") _0-3 S(O) _0-3 R' {e.g., -SO ₃ H, -OSO ₃ H), (CR'R") _0-3 O(CR'R") _0-3 H(e. g., -CH ₂ OCH ₃ and -OCH ₃ ), (CR'R") _0-3 S(CR'R") _0-3 H (e.g., -SH and -SCH ₃ ), (CR'R") _0-3 OH (e.g., -OH), (CR'R") _0-3 COR', (CR'R") _0-3 (substituted or unsubstitutβd phenyl), (CR'R") _0-3 (C ₃ -C ₈ cycloalkyl), (CR'R") _0-3 CO ₂ R' (e.g., -CO ₂ H), or (CR'R") _0-3 OR' group, or the side chain of any naturally occurring amino acid; wherein R' and R" are each independently hydrogen, a C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, or aryl group. Such substituents can include, for example, halogen, hydroxy., alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, arylolxycarbonyloxy,

carboxylate, alkylcarbonyl, alkoxycaibonyl, aminocarbonyJ, alkylthiocarbony], alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylainino, arylaraino, diaiylamino, and alkylaiylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and uxeido), amidino, imino, 5 oxime, sulfhydryl, alkylthio, arylthio, thiooarboxylate, sulfates, sulfonate sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or heteroarQmatic moiety. In certain embodiments, a carbonyl moiety (C=O) may be further derivatized with an oxime moiety, e.g., an aldehyde moiety may be derivatized as its oxime (-C=N-OH) analog. It will be understood by those skilled in the art that

] O the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "aralkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmeύiyl (Le., benzyl)).

The term "alkenyl" includes unsaturated aliphatic groups analogous in length is and possible substitution to the alkyls described above, but which contain at least one double bond.

For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethenyJ, propenyl, buteny], pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chaiπ alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl,0 cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cydoalkenyl substituted alkenyl groups. The term alkenyl further includes alkenyl groups that include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer5 carbon atoms in its backbone (e.g., C _ϊ -Q for straight chain, C ₃ -C ₀ for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C ₁ -C _& includes alkenyl groups containing 2 to 6 carbon atoms

Moreover, the term alkenyl includes both "unsubstituted alkenyls" and0 "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups,

halogens, hydroxy!, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, allcylthio, aryltbio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety, The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.

For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptyny], octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups that include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., Cj-Q for straight chain, C ₃ -Cj for branched chain). The term C _∑ -Cβ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both "unsubstituted alJcynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, aryloarbonyl alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkyllhiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate,

sulfamoyl, sulfonamide), nitro, trifluoromethyl, cyano, azido, heterocycly], alkylaiyl, or an aromatic or heteroaromatic moiety.

The term "amine" or "amino" should be understood as being broadly applied to both a molecule, or a moiety or functional group, as generally understood in the art, and may be primary, secondary, or tertiary. The term "amine" or "amino" includes compounds where a nitrogen atom is covalently bonded to at least one carbon, hydrogen or heteroatom. The terms include, for example, but are not limited to, "alky! amino," "arylaminα," "diarylamino," "alkylarylamiπo," "alkylaminoaryl," "arylaminoalkyl," "alkaminoalkyl," "amide," "amido," and "aminocarbonyl." The term "alkyl amino" comprises groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term "dialkyl amino" includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term "arylamino" and "diarylamino" include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. The term "alkylarylamino," "alkylaminoaryl" or "arylaminoalkyl" refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term "alkaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.

The term "amide," "amido" or "aminocarbonyl" includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes "alkaaiinocarbonyl" or "alkylammocarbonyl" groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylammocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms

"alkylaminocarbonyl," "aJkenylarπinocarbonyl," "alkynylaminocarbonyl," "arylaminocarbonyl," "alkylcarbonylainino," "alkenylcarbonylamino," "alkynylcarbonylamiπo," and "arylcarbonylamino" are included in term ''amide."

Amides also include urea groups (amiπocarbonylamino) and carbamates (oxycarbonylamiπo).

In a particular embodiment of the invention, the terra "amine" or "amino" refers to substituents of the formulas N(R ⁸ )R ⁹ , CH ₂ N(R ⁹ JR ⁹ and CH(CH ₃ )N(R ^a )R ⁹ , wherein R ³ and R ⁹ are each, independently, selected from the group consisting of H and (Cι- ₄ -alky])o.|G, wherein G is selected from the group consisting of COOH, H, PO ₃ H, SO ₃ H, Br, Cl, F, O-C _1-4 -alkyl, S-C _1-4 -alkyl, aryl, C(O)OC, -Cβ-alkyl, C(O)C _1-4 - alkyl-COOH, C(O)Ci-Q-alkyl and C(O)-aryl; or N(R ⁸ )R ⁹ is pyrrolyl, tetrazole, pyrrolidinyl, pyrrolidiπyl-2-one, dimethylpyrrolyl, imidazolyl, moipholino or

The term "aryl" includes groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, phenyl, pyrrole, furaπ, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, the term "aryl" includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodi oxazole, benzothiazote, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, anthryl, phenanthryl, napthridiπe, indole, benzofuran, purine, benzofiiran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterooyclea", "heterocycles," "heteroaryls" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, alkyl _> halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarboπyloxy, carboxylate, alkylcarbonyl, alkylaminoaoarbonyl, aralkylaminocarbony], alkenylaminocarbonyl, alkylcarbonyl, arylcarbony], aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyaπo, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylaiylamiπo), acylamino (including alkylcaibonylamino, aiylcarboπylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or

heteroaromatic moiety. Aiyl groups can also be fused or bridged with alioyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g , tetralin),

It will be noted that the structures of some of the compounds of this invention include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemical^ controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Compounds described herein may be obtained through art recognized synthesis strategies.

As used herein, the term "pharmaceutically acceptable salts" refers to salts of the invention prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. Suitable non-toxic acids include inorganic and organic acids such as acetic, benzenesulfoπic, benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethioπic, lactic, maleic, malic, maπdelic, methanesulfonic, mucic, nitric, pamotc, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic and the like, Particularly preferred salts are sodium, lysine and argentine salts of the compounds of the invention. As used herein, the term "neurotrophic factor" or "neurotrophin" (also referred to herein as "NT') refers to members of a family of proteins, usually in the form of dimers, which are structurally homologous to NGF. The term includes the precursors (pro-neurotrophins, e.g., pro-NGF) and the mature proteins which include three surface 3-hairpin loops, a p-strand, an internal reverse turn region, and N- and C- termini. Neurotrophic promote at least one of the biological activities related to vertebrate neuron survival, differentiation, and function, as determined using assays described, for example, in US 2002/0169182Al and RiopeUe et al., Can J. of Phys, and Pharm. 60:707 (1982); Harrington et al PNAS USA 101(16):6226-6230, (2004)). Neurotrophic factors include, for example, brain-derived neurotrophic factor (BDNF), NGF, neurotrophin 3 (NT-3), neurϋtiophin 4/5 (NT-4/5), and neurotrophin 6 (NT-6) (R. M. Lindsay et al.: TINS, vol. 17, p. 182 (1994) and R. M. Lindsay: Phil. Trans. R. Soc. Lond. B. vol. 351, p. 365-373 (1996)). In addition, ciliary neurotrophic factor

(CNTF), glia-derived neurotrophic factor (GDNF) ₁ glia growth factor (GGF2), central nerve growth factor (AF-I), hepatocyte growth factor (HGF) (A. Ebens et a!,, Neuron, vol. 17, p. 1157-1172 (1996)) can also be considered as neurotrophic factors. Moreover, biotechnologically engineered products of the above neurotrophic factors, which are derived by a partial substitution, an addition, a deletion or a removal by conventional genetic engineering techniques, are also included within the scope of the neurotrophic factors of the present invention as far as such product shows biological activities of the- naturally-occurred neurotrophic factors.

As used herein, the term "neurotrophin receptor" (also referred to herein as "NTR") is meant to refer to a receptor which binds a neurotrophin. In certain embodiments, the neurotrophin receptor is a member of the tyrosine kinase family of receptors, generally referred to as the "Trk" receptors or "Trks", which are expressed on cellular surfaces. The Trk family includes, but is not limited to, TrkA, TrkB, and TrkC In a particular embodiment the neurotrophin receptor is TrkA. In other embodiments, the neurotrophin receptor is p75 ^{N rR} , also called p75 or low-affinity nerve growth factor receptor or common neurtotrophin receptor. These receptors may be from any animal species that expresses neurotrophin receptors (e.g. human, murine, rabbit, porcine, equine, etc.), and include full length receptors, their truncated and variant forms, such as those arising by alternate splicing and/or insertion, and naturally-occurring allelic variants, as well as functional derivatives of such receptors.

"Neurotrophin-mediated activity" is a biological activity that is normally modulated (e.g., inhibited or promoted), either directly or indirectly, in the presence of a neurotrophin. Neurotrophin-mediated activities include, for example, neurotrophin binding to the p75 ^NTR receptor or neurotrophin binding to one of the TTk receptors (e.g. , TrkA), the ability to promote neurotrophin receptor dimerization and/or phosphorylation, neuron survival, neuron differentiation including neuron process formation and neurite outgrowth, neurotransmission and biochemical changes such as enzyme induction. A biological activity that is mediated by a particular neurotrophin, e.g. NGF or pro-NGF, is referred to herein by reference to that neurotrophin, e.g. NGF-mediated activity. (It is noted that "NGF-mediated activity" also includes "proNGF-mediated activity.") To determine the ability of a compound to inhibit a neurotrophin-mediated activity, conventional in vitro and in vivo assays can be used.

For example, a receptor binding assay, such as the assay described in US 2002/0169182 Al, can be used to assess the extent to which a compound inhibits neurotnophiπ/receptor binding. Inhibition of neurite survival and outgrowth can be determined using the in vitro assay described by Riopelle et al. in the Can. J. of Phys. and Pharm., 1982, 60: 707. Other examples of in vitro and in vivo assays for use in determining the ability of a compound to inhibit a neurotrophin-mediated activity are described in the "Exemplification of the Invention" section of the application.

"Neurotransmission," as used herein, is a process by which small signaling molecules, termed neurotransmitters, are rapidly passed in a regulated fashion from a neuron to another cell. Typically, following depolarization associated with an incoming action potential, a neurotransmitter is secreted from the presynaptic neuronal terminal. The neurotransmitter then diffuses across the synaptic cleft to act on specific receptors on the postsynaptic cell, which is most often a neuron but can also be another cell type (such as muscle fibers at the neuromuscular junction). The action of neurotransmitters can either be excitatory, depolarizing the postsynaptic cell, or inhibitory, resulting in hyperpolarization. Neurotransmission Can be rapidly increased or decreased by neuromodulators, which typically act either pre-synaptically or post- synaptically. The neurotrophin family (notably NGF and BDNF) have been shown to have prominent neuromodulator^ effects on diverse neuronal types (Lohof el al, Nature. 363(6427):350-3 (1993); Li et al. J Neurosci. 18(24):10231-40. (1998)).

BDNF has also been shown to behave like a neurotransmitter, acting directly on target cells to alter their excitability by rapidly and directly gating ion certain ion channels (Rose et al., Bioessays. 26(11): I l 85-94. (2004)).

There are several simple fashions in which neurotransmission can be studied. The release of neurotransmitters from cultured neurons can be directly quantified using HPLC, radiolabled neurotransmitters or other methodologies. Neurotransmission can be estimated by dyes such as FM 1-43, a fluorescent marker of synaptic vesicle cycling. Moreover, neurotransmission between neurons can be directly monitored using standard electrophysiological techniques, as can any direct neurotransmitter-like effects of neurotrophins on ion channel currents. These various methodologies have been used to study the effects of neurotrophins, such as BDNF and NGF, on neurotransmitter release and neurotransmission (Lohof ei al.; Li et al. ; Ros« et al.)

The term "contacting" as used herein refers to bringing a compound of the invention and a target, e.g., NGF, p75 ^NTR and/or TrkA together in such a manner that the compound can affeci the activity of the target, either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another target on which the catalytic activity of the target is dependent. For example, a compound of the invention may effect the activity of TrkA by contacting (e.g., binding to) TrkA directly, or by contacting (e.g., binding to) plS ¹ ™*, which may effect the activity of TrkA. Such "contacting"" can be accomplished "in vitro " i.e., in a test tube, a petri dish or the like, or "in vivo " i.e., administered to a subject such as a mouse, rat or human. In a test tube, contacting may involve only a compound and a target of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment "Contacting" can refer to a compound of the invention directly binding to a target, or being in the vicinity of a target. Examples of neurotrophin-mediated activities include, but are not limited to, pain (e.g., inflammatory pain, acute pain, chronic malignant pain, chronic nonrnaϋgπant pain and neuropathic pain), inflammatory disorders, diseases and disorders of the genitourinary and gastrointestinal systems, and neurological disorders (e.g., neurodegenerative or neuropsychiatry disorders). "Pain" is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage (International Association for the Study of Pain - IASP). Pain is classified most often based on duration (i.e., acute vs. chronic pain) and/or the underlying pathophysiology (i.e., nociceptive vs. neuropathic pain). Acute pain can be described as an unpleasant experience with emotional and cognitive, as well as sensory, features that occur in response to tissue trauma and disease and serves as a defensive mechanism. Acute pain is usually accompanied by a pathology (e.g., trauma, surgery, labor, medical procedures, acute disease states) and the pain resolves with healing of the underlying injury. Acute pain is mainly nociceptive, but may also be neuropathic.

Chronic pain is pain that extends beyond the period of healing, with levels of identified pathology that often are low and insufficient to explain the presence,

intensity and/or extent of the pain (American Pain Society — APS). Unlike acute pain, chronic pain serves no adaptive purpose. Chronic pain may be nociceptive, neuropathic, or both and caused by injury {e.g., trauma or surgery), malignant conditions, or a variety of chronic conditions (e.g., arthritis, fibromyalgia and neuropathy). In some cases, chronic pain exists de novo with no apparent cause. "Nociceptive pain" is pain that results from damage to tissues and organs. Nociceptive pain is caused by the ongoing activation of pain receptors in either the surperficial or deep tissues of the body. Nociceptive pain is further characterized as "somatic pain", including "cutaneous pain" and "deep somatic pain", and "visceral pain".

"Somatic pain" includes "cutaneous pain" and "deep somatic pain." Cutaneous pain is caused by injury, diseases, disorders or neoplasms of the skin, subcutaneous tissues and related organs. Examples of conditions associated with cutaneous pain include, but are not limited to, cuts, bums, infections, lacerations, as well as traumatic injury and post-operative or surgical pain (e.g. , at the site of incision).

"Deep somatic pain" results from injuries, diseases, disorders or neoplasms of the musculoskeletal tissues, including ligaments, tendons, bones, blood vessels and connective tissues. Examples of deep somatic pain or conditions associated with deep somatic pain include, but are not limited to, sprains, broken bones, arthralgia, vasculitis, myalgia and myofascial pain. Arthralgia refers to pain caused by a joint that has been injured (such as a contusion, break or dislocation) and/or inflamed (e.g., arthritis). Vaculitis refers to inflammation of blood vessels with pain. Myalgia refers to pain originating from the muscles. Myofascial pain refers to pain Stemming from injury or inflammation of the fascia and/or muscles.

"Visceral" pain is associated with injury, inflammation, disease or neoplasms of the body organs and internal cavities, including but not limited to, the circulatory system, respiratory System, gastrointestinal system, genitourinary system, immune system, as well as the ear, nose and throat. Visceral pain can also be associated with infectious and parasitic diseases that affect the body organs and tissues. Visceral pain is extremely difficult to localize, and several injuries to visceral tissue exhibit "referred" pain, where the sensation is localized to an area completely unrelated to the

site of injury- For example, myocardial ischaemia (the loss of blood flow to a part of the heart muscle tissue) is possibly the best known example of referred pain; the sensation can occur in the upper chest as a restricted feeling, or as an ache in the left shoulder, arm or even hand. Phantom limb pain is the sensation of pain from a limb that one no longer has or no longer gets physical signals from - an experience almost universally reported by amputees and quadriplegics.

"Neuropathic pain" or "neurogenic pain" is pain initiated or caused by a primary lesion, dysfunction or perturbation in the nervous system. "Neuropathic pain" can occur as a result of trauma, inflammation, disease or neoplasms of the peripheral nervous system ("peripheral neuropathic pain") and/or the central nervous system ("central pain"). For example, neuropathic pain can be caused by a nerve or nerves that are irritated, trapped, pinched, severed or inflamed (neuritis). There are many neuropathic pain syndromes, such as diabetic neuropathy, trigeminal neuralgia, postherpetic neuralgia ("shingles"), post-stroke pain, and complex regional pain syndromes (also called reflex sympathetic dystrophy or "RSD" and causalgia).

As used herein, the term "inflammatory disease or disorder" includes diseases or disorders which are caused, at least in part, or exacerbated by, inflammation, which is generally characterized by increased blood flow, edema, activation of immune cells (e.g. ( proliferation, cytokine production, or enhanced phagocytosis), heat, redness, swelling, pain and loss of function in the affected tissue and organ. The cause of inflammation may be due to physical damage, chemical substances, micro-organisms, tissue necrosis, cancer or other agents. Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders are generally of relatively short duration, and last for from about a few minutes to about one to two days, although they may last several weeks. The main characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils. Chronic inflammatory disorders, generally, are of longer duration, e.g., weeks to months to years or longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders

include disorders which recur after a period of time or which have periodic episodes. Some disorders may fall within one or more categories.

The terms "neurological disorder" and "neurodegenerative disorder" refer to injuries, diseases and dysfunctions of the nervous system, including the peripheral nervous system and central nervous system. Neurological disorders and neurodegenerative disorders include, but are not limited to, diseases and disorders that are associated with neυrotrophin-mediated biological activity. Examples of neurological disorders include, but are not limited to, Alzheimer's disease, epilepsy, cancer, neuromuscular diseases, multiple sclerosis, amyotrophic lateral sclerosis, stroke, cerebral ischemia, neuropathy (e.g. , chemotherapy-induced neuropathy, diabetic neuropathy), retinal pigment degeneration, Huntington's chorea, and Parkinson's disease, and ataxia-telangiectasia.

As used herein, "neuropathy" is defined as a failure of the nerves that cany information to and from the brain and spinal cord resulting in one or more of pain, loss of sensation, and inability to control muscles. In some cases, the failure of nerves that control blood vessels, intestines, and other organs results in abnormal blood pressure, digestion problems, and loss of other basic body processes. Peripheral neuropathy may involve damage to a single nerve or nerve group (mononeuropathy) or may affect multiple nerves (polyneuropathy). The term "treated," "treating" or "treatment" includes the diminishment or alleviation of at least one symptom associated with the pain, inflammatory disorder, neurological disorder, genitourinary disorder or gastrointestinal disorder (e,g, , associated with or caused by neurotrophin mediated activity) being treated. In certain embodiments, the treatment comprises the modulation of the interaction of a neurotrophin (e.g. , monomer or dimer) and its receptor by an NT/NTR modulating compound, for example an NGF/NTR modulating compound, which would in turn diminish or alleviate at least one symptom directly or indirectly associated with or caused by the neurotrophin-mediated activity being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.

As used herein, the phrase "therapeutically effective amount" of the compound is the amount necessary or sufficient to treat or prevent pain, an inflammatory disorder,

a neurological disorder, a gastrointestinal disorder or a genitourinary disorder, (e.g., to prevent the various morphological and somatic symptoms of a neurotrophin-mediated activity). In an example, an effective amount of the compound is the amount sufficient to alleviate at least one symptom of the disorder, e.g., pain, inflammation, a neurological disorder, a gastrointestinal disorder or a genitourinary disorder, in a subject.

The term "subject" is intended to include animals, which are capable of suffering from or afflicted with a neurotrophin-associated state or neurσtrophύv associated disorder, or any disorder involving, directly or indirectly, neurotrophin signaling, In another embodiement, a subject is also intended to include animals, which are capable of suffering from pain, an inflammatory disorder, a neurological disorder, a respiratory disorder, a gastrointestinal disorder or a genitourinary disorder. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from pain, inflammation, a neurological disorder, a gastrointestinal disorder or a genitourinary disorder (e.g. associated with neurotrophin-associated activity).

The language "NT/NTR modulator" refers to compounds that modulate, i.e., inhibit, promote or otherwise alter the interaction of a neurotrophin with a neurotrophin receptor. For example, "NGF/NTR modulator" refers to compounds that modulate, e.g., inhibit, promote, or otherwise alter, the interaction of NGF (orproNGF) with P ₇₅ ^NiE _{1 TrkA^ or p75} ^NTR _{and TrkA} _ E _xam p[ _es of NGF/NTR modulators include compounds of Formulas I, II, III, 1HA, IV, V, VI, VII, and VIII, as well as the compounds shown in Tables A-J, including salts thereof, e.g., a pharmaceutically acceptable salt. Additional examples of NGF/NTR modulators include compounds of Tables A-J, or derivatives and fragments thereof, including salts thereof, e.g., a pharmaceutically acceptable salt. Compounds of Formulas I, II, III, 1HA, IV, V, VI, VII, and VIII, as well as the compounds shown in Tables A-J, i.e., the NT/NTR modulators or NGF/NTR modulators of the invention, are also referred to herein as

"compounds of the invention." In a particular embodiment, the NGF/NTR modulators of the invention, including the compounds shown in Tables A-J, can be used to treat a

disease or disorder associated with pain, inflammation, neurological disorders, respiratoiy disorders, gastrointestinal disorders or genitourinary disorders in a subject in need thereof. In another embodiment, the compounds of the invention, including the compounds of shown in Tables A-J, can be used to treat an inflammatory disorder in a subject in need thereof.

Modulators ofNeurotrophin/Neurotroπhin Receptor Interaction

In one aspect, the present invention provides compounds which modulate the interaction of a neurotrophin with a neurotrophin receptor. In certain embodiments, the compounds modulate the interaction of nerve growth factor (NGF) and/or a precursor thereof with a neurotrophin receptor (NTR). In other embodiments the compound modulates the interaction of NGF and/or a precursor thereof witfi the $15*^ receptor. In still other embodiments, the compound also modulates the interaction of NGF (or proNGF) with the TrkA receptor. In further embodiments, the compound modulates the interaction of NGF (or proNGF) with both the p75 ^mk and TrkA receptor

In another aspect, the compounds of the invention treat pain, inflammatory disorders, neurological disorders, respiratory disorders, gastrointestinal disorders or genitourinary disorders in a subject in need thereof, comprising administering to the subject an effective amount of a compound of the invention. In one aspect, the compound of the invention is of the general Formula I:

(D and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof; wherein

R ¹ is selected from the group consisting of alkyl, aryl, heteroaryl, C ₃ ..6- cycloalkyl, and Cj.g-heterocycloalkyl, all of which can be independently substituted one or more times with amino, halogen, hydroxyl, acid, cyano, C _| _ ₆ -alkyl- sulfonamide, C _1-e >-alkyl-amide, C _1-6 -alkyl-ester, O-C^-alkyl, S-C|.<i-alkyl, C,. ₆ -alkene,

furanyl, thiophenyl, thiazolyl, nitro, tetrazole, SO ₂ -C _1-6 -alkyl, SO ₃ -C _1-6 -alkyl, C _1-6 alkyl-urea, C _1-6 -alkyl-thiourea, morpholino, piperidinyl, piperazinyl, or azepanyl; R ² and R ⁴ are each, independently, selected from the group consisting of a hydrogen atom, C _1-6 -alkyl, C _1-6 -alkoxy, halogen, hydroxy!, CO ₂ H, cyano, sulfonamide, nitro, tetrazole, methyl-substituted tetrazole, pyrrolyl, SO ₃ H, COPh, N(R ⁸ )R ⁹ , C(O)N(R ⁸ )R ⁹ , CH ₂ N(R ⁸ )R ⁹ and CH(C _1-4 -alkyl)N(R ⁸ )R ⁹ (e.g., CH(CH ₃ )N(R ⁸ R ⁹ ); or R ² and R ⁴ can together form a fused ring of the Formula A;

wherein the ring of Formula A can be substituted, independently, one or more times with a substituent selected from the group consisting of C _1-6 -alkyl, amino, halogen, hydroxyl, CO ₂ H, cyano, sulfonamide, nitro, tetrazole, methyl-substituted tetrazole, pyrrolyl, SO ₃ H, COPh, N(R ⁸ )R ⁹ , CH ₂ N(R ⁸ )R ⁹ and CH(C _1-4 -alkyl)N(R ⁸ )R ⁹ (e.g.,CH(CH ₃ )N(R ⁸ )R ⁹ ); wherein R ⁸ and R ⁹ are each, independently, selected from the group consisting of H and (C _1-4 -alkyI) _0-1 G, wherein G is selected from the group consisting of COOH, H, PO ₃ H, SO ₃ H, Br, Cl, F, O-C _1-4 -alkyl, S-C _1-4 -alkyl, aryl, C(O)OC ₁ -C ₆ -alkyl, C(O) C _1-4 -alkyl-COOH, C(O)C _1-4 -alkyl -COOH, C(O)C ₁ -C ₄ -alkyl and C(O)-aryl; and R ³ is H or =0. In one embodiment of Formula I, R ³ is =0. In another embodiment, R ¹ is C _1-6 - alkyl, phenyl, CH ₂ -phenyl or naphthyl, all of which can be independently substituted one or more times with the substituents listed above for R ¹ .

In another embodiment of Formula I, R ¹ is independently substituted one or more times with C _1-6 -alkyl, C _1-6 -alkyl-ester, amino, halogen, acid, OH or cyano. In still another embodiment of Formula I, R ¹ is (CH ₂ ) _n CO ₂ H, (CH ₂ ) _n CO ₂ C _1-4 -alkyl (e.g., (CH ₂ ) _n CO ₂ CH ₃ or (CH ₂ ) _n CO ₂ Et), phenyl, CH ₂ - phenyl or naphthyl, wherein the phenyl and naphthyl groups can be independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)C _1-4 -alkyl {e.g., N(H)C(O)CH ₃ ), CO ₂ C _1-4 -alkyl (e.g., CO ₂ CH ₂ CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OrNH ₂ , wherein n is 1, 2, 3, 4 or 5. ϊn still another embodiment, n is 3 or 4.

In another embodiment of Formula I, R ² and R ⁴ are each, independently, selected from the group consisting of a hydrogen atom, COjH, NO2, Cl, F, Br, OH, NH ₂₇ CN, CONH ₂ , tstrazole, Ph-CO ₂ H, C(O)N(H)(CHj) _n CO ₂ H, and C(O)N(H)Ph- CO ₂ H, wherein n is 1 , 2, 3, 4 or 5; or R ² and R ⁴ can together form a fused ring of the Formula A:

(A) wherein the ring of Formula A can be optionally substituted one or more times with NO2. In other embodiments, one of R ² and R ⁴ is H; R ⁴ is H, and R ² is not H or R ² and R ⁴ are Cl.

In another embodiment, Formula I is a compound represented by the Formula Ia (wherein R ³ of Formula I is =O);

(Ia). In one embodiment, a compound of Formula Ia is represented by the compounds in Table A (NT = not tested):

-2S-

In another embodiment, Formula I is a compound represented by the Formula

Ib:

(Ib).

In one embodiment, a compound of Foimula Ib is represented by the compounds Ln Table B:

In another aspect, the compound of the invention is of the general Formula II;

and pharmaceutically acceptable salts, enantiomers, stereoisomers, retainers, tautomers, diastereomers, or racemates thereof; wherein

R ¹ is selected from the group consisting of alkyl, aryl, heteroaryl, C ₃ 4- cycloalkyl, and Qs-g-heterocycloalkyl, all of which can be independently substituted one or more times with C^-alky!, amino, halogen, hydroxy], acid, cyano, C] _-6 -alkyl- sulfonamidc, d-s-alkyl-amide, Ci- ₆ -alkyl-ester, O-C _1-6 -alkyl, S-C _1-6 -alkyl, C _1-6 -alkene, furanyl, thiophenyl, thiazolyl, nitro, tetrazole, SO ₂ -C (.j-alkyl, SOrCi- ₆ -alkyl, C ₁ ^,- alkyl-urea, C _1-6 -alkyl-thiourea, morpholino, piperidinyl, piperazinyl, or azepanyl.

In another embodiment of Formula II, R ¹ is Ci.g-alkyl, phenyl, CH ₂ -phenyl or naphthyl, all of which can be independently substituted one or more times with the substituents listed above for R ¹ . In another embodiment, R ¹ is independently

substituted one or more times with Cus-alkyl-ester, amino, halogen, acid, OH or cyano.

In still another embodiment of Formula II, R ¹ is (CHz) _n CO ₂ H, (CHa) _n CO ₂ C _{1 A} - alkyl (e.g., (CHj) _n CO ₂ CH ₃ or (C^) _n CO ₂ Et), phenyl, CH _∑ -phenyl or naphthyl, wherein the phenyl or naphthyl groups can be independently substituted one or more times with COiH, CN, Cl ₁ N(H)C(0)Ci ₄ -alkyl (e.g.,N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , or NH _ϊ , wherein n is 1, 2, 3, 4 or 5,

In yet another embodiment of Formula 11, R ¹ is (CHs) _n COjH or phenyl, wherein the phenyl group can be independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)C , - ₄ -alkyl (e.g. ,N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , or NHj, wherein n is 1 , 2, 3, 4 or 5. ϊn another embodiment, n is 2, 3, or 4.

In one embodiment, a compound of Formula II is represented by the compounds in Table C:

In another aspect, the compound of the invention is of the Formula III:

(III) and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof; wherein

X is a bond, C(O), (CH ₂ J _n , or O;

R and R are each, independently, selected from the group consisting of alfcyl, aryl, heteroaryl, C ₃ -6-cycloalkyl, and Ca-s-heterocycloalkyl, all of which can be independently substituted One or more times with C _1- s-alkyl, amino, halogen, hydroxy ^] , acid, cyano, O-C|_ 6-alkyi, S-C _1- 5-alky], Cu-alkene, furanyl, thiophenyl, thiazolyl, nitro, tetrazole, SO ₂ - C _1-6 -alkyl, C|. ₆ -alkyl-urea, C _1-6 -alkyl-thiourea, morphoJino, piperidinyl, piperazinyl, or azepanyl;

R ³ is selected from the group consisting of hydrogen, halogen, and O-C| ₄ -alkyl; and n is 1, 2, 3, or 4.

In one embodiment of Formula III, R ¹ and R ² are each, independently, Ci-u- alkyl, phenyl, CH ₂ -phenyl or naphthyl, all of which can be independently substituted one or more times with the substituents listed above for R ¹ . in another embodiment, R'and R ² are each, independently, substituted one or more times with Cμg- alkyl-ester, amino, halogen, acid, OH, or cyano.

In still another embodiment of Formula III, R ¹ and R ² are each, independently, (CH ₂ J _n CO ₂ H ₅ (CH ₂ ) _B Cθ ₂ C _1-4 -alkyl (e.g., (CHa) _n CO ₂ CH ₃ , (CH^) _n CO ₂ Et), phenyl, CH ₂ - phenyl or naphthyl, wherein the phenyl or naphthyl groups can be optionally independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)Ci ^a lkyl (e.g., N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , or NH ₂ , wherein n is 1, 2, 3, 4 or 5.

In another embodiment of Formula III, R ¹ and R ² are each, independently, (CH ₂ ) _n Cθ2H, (CH ₂ ) _π Cθ2C _1-4 -alkyl, (CH ₂ ) _n SO ₃ H _? (CHjVtetrazole, (CH ₂ ) _n CN or phenyl, wherein the phenyl group can be independently substituted one or more times with CO ₂ H, CN ₇ Cl, N(H)C(O)C _1-4 alkyl (e.g. ,N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , or NH ₂ , wherein n is 1, 2, 3, 4 or 5, and wherein each CH ₂ group can be optionally substituted with OH. In still another embodiment, R ³ is H, F, Cl, Br, CH ₃ or O-Cμ- alkyl.

In one embodiment, a compound of Formula III is represented by the compounds in Table D:

In another aspect, the compound of the invention is of the Formula III A:

( 1HA) and pharmaceutically acceptable SaItS ₁ enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof; wherein

10 X is a bond, C _1-6 -alkyl or aryl, wherein the Q-e-alkyl or aryl groups can be optionally substituted with OH, CO ₂ H or SOjH; and

R ¹ is independently selected from the group consisting of acid, halogen, nitro, alkyl, axyl, heteroaryl, C ₃ _a-cycloalkyt, and wherein the alkyl, aryl, heteroaryl, C ₃ - _$ -cycloalkyl, and Cs^-heterocycloalkyl groups can be

15 independently substituted one or more times with Cns-altyl. amino, halogen, hydroxyl, acid, cyano, C _1-6 -alkyl-sulfonamide, C _1-6 -alkyl-amide, Cns-alkyl-ester, O-C|. s-alkyl, S-C|^-alkyl, Ci.6-alkeπe, furanyl, ϊhiophenyl, thiazolyl, nitro, tetrazole, SO ₂ - Ci-fi-alkyl, SOj-Ci-g-alkyl, Cns-alkyl-urea, Ci-g-alkyl-thiourea, morpholino, piperidinyl, piperazinyl, or azepanyl.

20 In one embodiment of Fomϊula HIA, R ¹ is, independently, CO2H or nitro. In another embodiment, X is (CH ₂ ),, or phenyl, wherein the CH ₂ or phenyl groups can be optionally independently substituted one or more times with OH, CO ₂ H or SO ₃ H, wherein n is 1 , 2, 3, 4, or 5,

In one embodiment, a compound of Formula HIA is represented by the compounds in Table E (NT = not tested):

In another aspect, the compound of the invention is of the Formula IV:

(IV)

and pharmaceutically acceptable salts, eπantiomers, stereoisomers, retainers, tautomers, diastereomers, or racemates thereof; wherein

R' and R ² are each, independently, selected from the group consisting of (CH ₂ J _n R ³ or Ph ₃ wherein R ³ is Ph, CO ₂ H, or COϊCWalkyl-Ph, wherein n is 1, 2, 3, 4 or 5, wherein each CHj can be further substituted with CO2H, and wherein each Ph can independently be substituted one or more times with Ci-g-alkyl-ester, amino, halogen, acid, or cyano.

In one embodiment of Formula IV ₁ R ¹ is (CHi) _n CChH or (CHj) _n Ph, wherein n is 1, 2, 3, 4 or 5, and R ² is Ph, wherein Ph is independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)C _M alkyl (e.g. ,N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OH or NH ₂ , and wherein each CH2 group can be optionally substituted with COjH. In another embodiment, R ¹ is CH(CO ₂ H)(CHi) _n R ³ , wherein R ¹ is CO ₂ H ₇ CO2CH1PI1 or Ph, wherein n is 1, 2, 3, or 4, and R ² is Ph, wherein each Ph can be independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)Ci. ₄ alkyl (e.g. , N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OH or NH ₂ , In another embodiment, n is 1, 2, or 3.

In another embodiment of Formula IV, R ² is Ph, which is independently substituted one or more times with CO ₂ H, CN, CL, N(H)C(O)Q ^alkyl (e.g., N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OH or NH ₂ . In still another embodiment, R ¹ is (CHj) _n COOH, wherein n is 1, 2, 3, 4, or 5, wherein each CHj group can be optionally substituted with COjH, and R ² is Ph, wherein Ph is independently substituted one or more times with COjH, CN, Cl, N(H)C(O)C] ^alkyl (e.g., N(H)C(O)CH ₃ ), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OH or NHj. In yet another embodiment, R ¹ and R ² are each, independently, Ph, wherein Ph is independently substituted one or more times with CO ₂ H, CN, Cl, N(H)C(O)Ci. _λ alkyl (e.g. ,N(H)C(O)CHj), SO ₃ H, CH ₂ CO ₂ H, CF ₃ , OH Qr NH ₂ , wherein each CH ₂ group can be optionally substituted with CO ₂ H. In another embodiment, R ¹ and R ² are each, independently (CHj) _n COOH or (CHj) _n CO ₂ (CH) _In Ph, wherein n and m are each, independently, 1 , 2, 3, 4, or S, and wherein each CHj group can be optionally substituted with CO∑H. In a particular embodiment, rπ is 1.

In o«e embodiment, a compound of Formula IV is represented by the compounds in Table F:

In another aspect, the compound of the invention is of the Formula V:

(V) and pharmaceutically acceptable salts, enaπtiomers, stereoisomers, retainers, tautomers, diastereomers, or racemates thereof; wherein

R ¹ is selected from the group consisting of (CHO ₈ CXhH, or Ph, wherein n is 1, 2, 3, 4 or 5, wherein at least one CH ₃ is substituted with CCfcH, and Ph can be substituted one or more times with C _1-6 -alkyl-ester, amino, halogen, acid, or cyano; and

X is H, CChU, C^alkyl-ester, halogen, C _1-6 alkoxy, or NOj In one embodiment of Formula V, R ¹ is CH(CO ₂ H)(CH ₂ X _n COjH, wherein m is 1, 2, or 3 (.e.g., 2). In another embodiment, R ¹ is Ph that is independently substituted one or more times with COOH or halogen, e.g., Cl.

In one embodiment, a compound of Formula V is represented by the compounds in Table G:

In one embodiment, Compound 2E can be used to treat pain in a subject (e.g., human) in need thereof. In another embodiment, Compound 2E can be used to treat inflammation in a subject (e.g., a human) in need thereof. In another aspect, the invention provides a compound of the Formula Vl:

(VI) and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, lautomers, diastereomers, or racemates thereof; wherein

R ¹ is selected from the group consisting of C _1-6 -alkyl, C|-s-alkoxy _> aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, all of which can be further independently substituted one or more times with C|. ₆ -alkyl, amino, halogen, hydroxyl, acid, cyano, Ci- ₆ -alkyl-sulfonaπiide, aryl, heteroatyl, C _3-s -cycloaIkyl, Qj-ή-heterocycloalkyl, C|.g- alkyl-amide, Cnj-alkene, furanyl, thiophenyl, thiazolyl, nitro, tctrazole, sulfone, urea, thiourea, morpholino, piperidinyl, piperazinyl or azepanyl; and

R ² and R ³ are each H, or together form a fused ring of the Formula B or C:

(B) (C) wherein the rings of Formulae B or C can be substituted one or more times with C| _-s -alkyl, amino, halogen, hydroxyl, CO ₂ H, cyajw, sulfonamide, tetrazole or nitro.

In one embodiment of Formula VI, R ¹ is selected from the group consisting of (CHj) _n CO ₂ H or Ph, wherein A is 1, 2, 3, 4 or 5, wherein one or more CH2 groups can be substituted with CO ₂ H, and Ph can be independently substituted one or more times with Ci- ₆ -alkyl, Cu-alkyl-ester, amino, halogen, acid, or cyano. ϊn another embodiment, R ¹ is Ph that is Independently substituted one or more times with COOH or halogen, e.g., chloro. In another embodiment, R ² and R ³ together form the fused rings of the Formulae B or C, wherein the Formula B can be substituted by COOH. In one embodiment, a compound of Formula VT is represented by the compounds in Table H:

In another aspect, the invention provides a compound of the Formula VII:

and pharmaceutically acceptable salts, eπantipmers, stereoisomers, retainers, tautomers, diastereomers, or racemates thereof; wherein

R ¹ is of the Formula M, N or P:

(M) (N) (P) _{; aQd} R ² and R ³ are each, independently, selected from the group consisting of

NHCOMe, NH ₂ , NO ₂₅ NHCX ⁾ CH ₁ NBu, NHCOCH ₂ CH ₂ COOH ₅ NMe ₂ , NHCHMe ₂ , CN, Pyrrol, Br ₁ NHCO'Bu, NHCH ₂ COCH ₂ CH ₂ COOH ₅ COOH, and tetrazole.

In one embodiment, a compound of Formula VII is represented by the compounds in Table I:

In one embodiment, Compound IG can be used to treat pain in a subject (e.g., a human) in need thereof. In another embodiment, Compound I G can be used io treat inflammation in a subject (e.g., a human) in need thereof. In another aspect, the invention provides a compound of the Formula VIII:

(VϊIϊ) and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is CO ₂ H or tetrazole, wherein the tetrazole group can be further substituted with C^-alkyl (e.g., CH ₃ ); and

R ² is in the 3 or 4 position, and is selected from the group consisting of: hydrogen, NOj ₃ NH ₂ , N(H)C(O)C ₁ ^alkyl (e.g., NHCOCH ₃ ), C0NH _z> SO ₂ NMe ₂ , CN, halogen, SO ₃ H, COPh, CO ₂ H, halogen (e.g., Br) and aryl.

In one embodiment of Formula VIII, R ¹ is

^■ wherein R ⁴ Is H or C^-alkyl (e.g., CH ₃ ).

In one embodiment of Formula VIII ₁ aryl is a tetrazole or phenyl that is optionally substituted with Ci-i-alkyl, O-C _1-6 -alkyl, or C _1- ή-alkyl substituted by OH. In another embodiment, the aryl is tetrazole, which is optionally substituted with Cw alkyl (e.g., CH ₃ ).

In another embodiment of Formula VIII, R ² is pyrazole or triazole.

In a particular embodiment of Formula VIII, R ¹ is CO2H, and R ² is tetrazole, wherein the letrazole is optionally substituted with Ci-4-alkyl (e.g., CH ₃ ). This embodiment can be used to treat pain in a subject (e.g., a human) in need thereof. This embodiment can be also used to treat inflammation in a subject (e.g., a human) in need thereof.

In one embodiment, a compound of Formula VIII is represented by the compounds in Table J:

In one embodiment, Compound AlG can be used to treat pain in a subject (e g., a human) in need thereof. In another embodiment, Compound 47G can be used to treat inflammation in a subject (e.g., a human) in need thereof.

In one embodiment, Compound 5OG can be used to treat pain in a subject (e.g. , a human) in need thereof. In another embodiment, Compound 5OG can be used to treat inflammation in a subject {e.g., & human) in need thereof.

In still another embodiment, die compound of Formula VIII is selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein R ¹ is H or CM alkyl, e.g., CH ₃ . In a particular aspect of the invention, R ¹ is H or CHj.

The generic structures described herein should be construed in congruity with the laws and principals of chemical bonding. For example, it may be necessary to remove a hydrogen atom in order accommodate a substitutent at any given location. Furthermore, it is to be understood that definitions of the variables (f.e., "R groups"), as well as the bond locations of the generic formulae of the invention, will be consistent with the laws of chemical bonding known in the art. It is also to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, bonds and/or hydrogen atoms are added to provide the following number of total bonds to each of the following types of atoms; carbon: four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur: two-six bonds.

It should be understood that the all tautomeric forms (e.g., tautomers of tetrazole), insofar as they can exist, are included within the invention. The term

"tautomer" refers to compounds of the invention that may exist in their tautomeric form, in which hydrogen atoms are transposed to other parts of the molecules and the chemical bonds between the atoms of the molecules are consequently rearranged. In a particular embodiment of the invention, the NGF/NTR modulator of Formulas I, II, III, IIIA, IV, V, VI, VII, and VIII are any one of the compounds shown in Tables A-J ₁ or derivatives and fragments thereof, including salts thereof, e.g., pharmaceutically acceptable salts.

In another embodiment, the invention pertains to the NGF/NTR modulators of Formulas I, II, III, IIIA, IV, V, VI ₅ VII, and VIII including salts thereof, e.g., pharmaceutically acceptable salts. Particular embodiments of the invention pertain to the modulating compounds shown in Tables A-J, or derivatives thereof, including salts thereof, e.g., pharmaceutically acceptable salts.

In yei another embodiment, the invention pertains to pharmaceutical compositions comprising NT/NTR modulating compounds described herein and a pharmaceutical acceptable carrier.

In another embodiment, the invention includes any novel compound or pharmaceutical compositions containing compounds of the invention described herein. For example, compounds and pharmaceutical compositions containing compounds set forth herein are part of this invention, including salts thereof, e.g., pharmaceutically acceptable salts.

In one embodiment of the invention, the modulating compounds of the invention are capable of chemically interacting with NGF, pTS ^1* ™, and/or TrkA, The language "chemical interaction" is intended to include, but is not limited to, reversible interactions such as hydrophobic/hydrophtlic, ionic (e.g., coulombic attraction/ repulsion, ion-dipole, charge-transfer), covalent bonding, Van der Waals, and hydrogen bonding. In certain embodiments, the chemical interaction is a reversible Michael addition. In a specific embodiment, the Michael addition involves, at least in part, the formation of a covalent bond.

Compounds of the invention can be synthesized according to standard organic synthesis procedures that are known in the art. Additionally, compounds similar to the compounds of the invention can be found in U.S. Patent No. 6,492,380, U.S. Patent

No. 6,468,990, and U.S. Patent Application Nos. 09/758,917, 11/521,582, and 1 1/521,592, each of which are incorporated herein by reference.

Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, said include for example those formed with inorganic acids, e.g., hydrochloric, sulphuric or phosphoric acids and organic acids e.g- succinic, maleic, acetic or ftαmaric acid. Other non-phaππaceutically acceptable salts, e.g. , oxalates, may be used for example in the isolation of the invention, and the compounds of the invention for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt U treated with a solution of base, e.g., sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt Particularly preferred salts are sodium, lysine and argentine salts of the compounds of the invention.

In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of the invention having a free carboxy group may be esterified using standard conditions which may include activation followed by treatment with the desired alcohol in the presence of a suitable base.

Assays

The present invention also relates to a method of modulating the interaction of NGF (or proNGF) with a neurotrophin receptor, e.g., p75 ^NTR and/or TrkA. In certain embodiments, the method comprises contacting NGF and/or a precursor thereof ( proNGF) in the presence of pTS* ¹ ™ ¹ and/or TrkA with a NQF/NTR modulating amount of a NGF/NTR modulator compound (i.e., a compound of the invention), thereby modulating the interaction of NGF (and/or proNGF) with p75 ^NTR and/or TrkA.

The methods of the invention can be practiced in vitro, for example, in a cell culture screening assay to screen compounds which potentially modulate, directly or indirectly, receptor function. In such a method, the modulating compound can function by interacting with and eliminating any Junction or activity (e,g, , receptor binding) of NGF and/or a precursor thereof in the sample or culture. The modulating compounds can also be used to control NGF activity in neuronal cell culture. In vitro cross-Unking assays for determining the ability of a compound within the scope of the invention to modulate the interaction of NGF with p75 ^NTR and/or TrkA, are well known in the art and described in the examples herein. Cross-linking data for compounds similar to the compounds of the invention can be found in U.S. Patent No, 6,492,380, U.S. Patent No. 6,468,990, and U.S. Patent Application No. 09/758,917, each of which are incorporated herein by reference. Other assays for determining the ability of a compound to modulate the activity of NGF with its respective receptors are also readily available to the skilled artisan (see, Barker et al., Neuron 13(1): 203-215; (1994), Dehant et al., Development 1 19: 545-558 (1993); and US 2002/016982). Recombinant and native neurotrophin polypeptides from different species, including humans, are commercially available from several sources (e.g., Promega Corporation and R&D Systems). In addition, neurotrophin polypeptides for use in the assays described herein can be readily produced by standard biological techniques or by chemical synthesis- For example, a host cell transfected with an expression vector containing a nucleotide sequence encoding the desired neurotrophin can be cultured under appropriate conditions to allow expression of the peptide to occur. The secreted peptide can then be isolated according to standard techniques. Coding polynucleotides, precursors and promoters for a number of neurotrophins are known, including coding sequences for neurotrophins of some mammalian species, For example, GenBank M61 176 sets for the coding sequence for BDNF (see also, XM.006027); BDNF precursor is set forth at BF439589; and a BDNF specific promoter is set forth at Eo5933. A similar range of coding sequences for other neurotrophins, including proNGF and mature NGF (e.g., NCBI ACCESSION NO POl 138 and CAA37703), NT-4/S and NT-3, are also available through GenBank and odier publicly accessible nucleotide and amino acid sequence databases. Alternatively, the neurotrophin, e.g., NGF can be obtained by culturing a primary cell

culture or an established cell line that can produce the neurotrophic and isolating from the culture broth thereof (e.g., culture supernatant, cultured cells).

The method can also be practiced in vivo, for example, to modulate one or more processes mediated by the interaction of NOF (and/ot proNGF) with p75 ^NTR , and/or the interaction of NGF with TrkA. Animal models For determining the ability of a compound of the invention to treat a disorder associated with or caused by a neurotrophm-mediated biological activity (e.g., pain, inflammatory disorders, respiratory disorders, neurological disorders, genitourinary disorders and gastrointestinal disorders) are well known and readily available to the skilled artisan. For example, animal models of neuropathic pain based on injury inflicted to a nerve (mostly the sciatic nerve) are described in Zeltser et al , 2000, Pain 89: 19-24; Bennett et al., 1988, Paiπ 33;87-lθ7; Seltzer et al., 1990, Pain 43:205-218; Kim et al,, 1992, Pain 50:355-363; Decosterd et al., 2000, Pain 87: 149-158 and DeLeo St al, 1994, Pain 56:9-16. There are also models of diabetic neuropathy (STZ induced diabetic neuropathy - Courteix et al., 1994, Pain 57: 153-160) and drug induced neuropathies (vincristine induced neuropathy - Aley et al, 1996, Neuroscience 73: 259-265; oncology-related immunotherapy, anti-GD2 antibodies - Slart et al., 1997, Pain 60:119-125), Acute pain in humans can be reproduced using in murine animals chemical stimulation; Martinez et al., Pain 81: 179-186; 1999 (the writhing test - intraperitoneal acetic acid in mice), Coderre et al. _t Pain. 1993, 54:43-50 (intraplantar injection of formalin). Other types of acute pain models are described in Whiteside et al., 2004, Br J Pharmacol 141 :85-91 (the incisional model, a post-surgery model of pain) and Johanek and Simone, 2004, Pain 109:432-442 (a heat injury model). An animal model of inflammatory pain using complete Freund's adjuvant (intraplantar injection) is described in Jasmin et al., 1998, Pain 75: 367-382. Intracapsular injection of irritant agents (complete Freund's adjuvant, ϊodoacetate, capsaicins, urate crystals, etc.) is used to develop arthritis models in animals (Fernihough et al., 2004, Pain 112:83-93; Coderre and Wall, 1987, Pain 28:379-393; Otsuki et a!,, 1986, Brain Res. 365:235-240). A stress-induced hyperalgesia model is described in Quintero et al. ₄ 2000, Pharmacology, Biochemistry and Behavior 67:449-458. Further animal models for pain are considered in an article of Walker et al 1999 Molecular Medicine Today 5:319-321 , comparing models for different types of pain, which are acule pain,

chronic/inflammatory pain and chronic/neuropathic pain, on the basis of behavioral signs. Animal models for depression are described by E. Tatarczynska et al, Br. J. Pharmacol. 132(7): 1423-1430 (2001) and P. J. M. Will et al., Trends in Pharmacological Sciences 22(7):331-37 (2001)); models for anxiety are described by D. Treit, "Animal Models for the Study of Anti-anxiety Agents: A Review,"

Neurosciencc & Biobehavioral Reviews 9(2):203-222 (1985). Additional animal models for pain are also described herein in the Exemplification section.

Genitourinary models include methods for reducing the bladder capacity of test animals by infusing either protamine sulfate and potassium chloride (See, Chuang, Y. C. et al. , Urology 61(3): 664-670, 2003) into the bladder. These methods also include the use of a well accepted model of for urinary tract disorders involving the bladder using intravesically administered acetic acid as described in Sasaki et al. (2002) J. Ural. 168: 1259-64, Efficacy for treating spinal cord injured patients can be tested using methods as described in Yoshiyama et al. (1999) Urology 54: 929-33. Gastrointestinal models can be found in: Gawad, K. A., et al., Ambulatory long-teim pH monitoring in pigs, Surg Endosc, (2003); Johnson, S. E. et al., Esophageal Acid Clearance Test in Healthy Dogs, Can. J. Vet. Res. 53(2): 244-7 (1989); and Cicente, Y. et al., Esophageal Acid Clearance: More Volume-dependent Than Motility Dependent in Healthy Piglets, J. Pediatr. Gastroenterol. Nutr 35(2): 173 -9 (2002). Models for a variety of assays can be used to assess visceromotor and pain responses to rectal distension. See, for example, Gunter et al., Physiol. Behav,, 69(3): 379-82 (2000), Depoortere et al., J. Pharmacol, and Exp, Ther., 294(3): 583-990 (2000), Morteau et al., Fund. Clin. Pharmacol., 8(6): 553-62 (1994), Gibson et al., Gastroenterology (Suppl. 1), 120(5): A19-A20 (2001) and Gschossmann et al, Eur. J. Gastro, Hepat., 14(10); 1067-72 (2002) the entire contents of which are each incorporated herein by reference.

Gastrointestinal motility can be assessed based on either the in vivo recording of mechanical or electrical events associated intestinal muscle contractions in whole animals or the activity of isolated gastrointestinal intestinal muscle preparations recorded in vitro in organ baths (see, for example, Yaun et al. , Br. J. Pharmacol., 112(4): 1095-1100 (1994), Jin et al., J. Pharm. Exp. Ther,, 288(1): 93-97 (1999) and Venkova et al., J. Pharm. Exp. Ther. _s 300(3): 1046-1052 (2002)). Tatersall et al and

Bountra et al, European Journal of Pharmacology, 250: (1993) R5 and 249:(1993) R3- R4 and Milano et al.J. Phaπnacol. Exp. Ther, 274(2): 951-961 (1995),

Animal models for investigating neurological disorders include, but are not limited to, those described in Moms et al, (Learn. Motiv. 1981; 12: 239-60) and Abeliovitch et (tl,, Cell 1993; 75: 1263-71). For example, neurological models for studying spinal cord injury, are described in Yoshiyama, M. et al., Urology 54(5): 929-933 (1999).

Further examples of animal models for pain and inflammation include, but are not limited to the models listed in Table 1.

TABLE l

Accordingly, an agent identified as described herein (e.g., an NGF/NTR modulator) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent, Accordingly, this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of a NGF/NTR modulator, and preferably one or more compounds of the invention described above, and a pharmaceutically acceptable carrier or excipient. Suitable pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.

The phrase "pharmaceutically acceptable carrier" is an recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol, esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl pahnitate, butylated hydroxyanisole (BHA) ₁ butylated hydroxytoluene (BHT) ₁ lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g. , NaCl), alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, cyclodcxtrin, magnesium stearate, talo, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyroϋdoπe, etc The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as trilycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollϊdone, sodium saccharine, cellulose, magnesium carbonate, etc.

The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubϊlizing agent and a local anesthetic to ease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions of the invention can also include an agent which controls release of the compound of the invention, thereby providing a timed or sustained relase composition.

The present invention also relates to prodrugs of the compounds disclosed herein, as well as pharmaceutical compositions comprising such prodrugs. For example, compounds of the invention which include acid functional groups or hydroxyl groups can also be prepared and administered as a corresponding ester with a suitable alcohol or acid. The ester can then be cleaved by endogenous enzymes within the subject to produce the active agent.

Formulations of the present invention include those suitable for oral, nasal, topical, transmucosal, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent,

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then.

if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, Qγ as an σil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethykellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and beπtom ^' te clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such exoipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disiniegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be

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made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent,

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl beπzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrøfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyt alcohols, polyoxyethyleπe sorbitol and

sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable ncmiπϊtating exoipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion qf various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the diug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-adininistered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the

subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions lhat are compatible with body tissue.

Methods of Administration

The invention provides a method of treating a condition mediated by an NGF/NTR interaction in a subject, including, but not limited to, pain, inflammatory disorders, respiratory disorders, neurological disorders, gastrointestinal disorders and genitourinary disorders. The method comprises the step of administering to the subject a therapeutically effective amount of a NGF/NTR modulator. The condition to be treated can be any condition which is mediated, at least in part, by the interaction of a neurotrophin (eg. NGF) with a neurotrophin receptor {e.g. , plS ¹ *™ and TrkA).

The quantity of a given compound to be administered will be determined on an individual basis and will be determined, at least in part, by consideration of the individual's size, the severity of symptoms to be treated and the result sought- The NGF/NTR modulators described herein can be administered alone or in a pharmaceutical composition comprising the modulator, an acceptable carrier or diluent and, optionally, one or more additional drugs.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration. The NGF/NTR modulator can be administered subcutaneously, intravenously, parenterally, intraperitoneally, intradermal^, intramuscularly, topically, enterally (e.g., orally), rectally, nasally, buccally, sublingually, syslemically, vaginally, by inhalation spray, by drug pump or via an implanted reservoir in dosage formulations containing conventional nςm-toxic, physiologically acceptable carriers or vehicles. The preferred method of administration is by oral delivery. The form in which it is administered (e.g., syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sol) will depend in part on the route by which it is administered. For example, for mucosal (e.g., oral mucosa, rectal mucosa, intestinal mucosa, bronchial mucosa) administration, nose drops, aerosols,

inhalants, nebulizers, eye drops or suppositories can be used. The compounds and agents of this invention can be administered together with other biologically active agents, such as analgesics, e.g., opiates, anti-inftammatoty agents, e.g. , NS AJDs ₁ anesthetics and other agents which can control one or more symptoms or causes of an NTR-mediated condition.

In a specific embodiment, it may be desirable to administer the agents of the invention locally to a localized area in need of treatment; this maybe achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, transdermal patches, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes or fibers. For example, the agent can be injected into the joints or the urinary bladder.

The compounds of the. invention can, optionally, be administered in combination with one or more additional drugs which, for example, are known for treating and/or alleviating symptoms of the condition mediated by NGF p75 ^NTR or

TrkA. The additional drug can be administered simultaneously with the compound of the invention, or sequentially. Fof example, the compounds of the invention can be administered in combination with at least one of an analgesic, an anti-inflammatory agent, an anesthetic, a corticosteroid (e.g.) dexamethasone, beclomethasone diproprionate (BDP) treatment), an anti-convulsant, an antidepressant, an anti-nausea agent, an anli-psycbotic agent, a cardiovascular agent (e.g>, a beta-blocker) or a cancer therapeutic, In certain embodiments, the compounds of the invention are administered in combination with a pain drug. As used herein the phrase, "pain drugs" is intended to refer to analgesics, anti-inflammatory agents, anesthetics, corticosteroids, antiepileptics, barbiturates, antidepressants, and marijuana.

The combination treatments mentioned above can be started prior to, concurrent with, or after the administration of the compositions of the present invention. Accordingly, the methods of the invention can further include the step of administering a second treatment, such as a second treatment for the disease or disorder or to ameliorate side effects of other treatments. Such second treatment can include, e.g., anti-inflammatory medication and any treatment directed toward creating pain. Additionally or alternatively, further treatment can include administration of

drugs to further treat the disease or to treat a side effect of the disease or other treatments (e.g., anti-nausea drugs, anti-inflammatory drugSj anti-depressants, anti- psychiatric drugs, anticonvulsants, steroids, cardiovascular drags, and cancer chemotherapeutics). As used herein, an "analgesic" is an agent that relieves pain without significant impairment of consciousness or sense perception and may result in the reduction of inflammation as do corticosteroids, e.g.. an anti-inflammatory agent. Analgesics can be subdivided into NSAIDs (non-steroidal-anti-inflammatory agents), narcotic analgesics, and non-narcotic analgesics. NSAIDs can be further subdivided into non- selective COX (cyclooxygenase) inhibitors, and selective C0X2 inhibitors. Opioid analgesics can be natural, synthetic or semi-synthetic opioid (narcotic) analgesics, and include for example, morphine, codeine, meperidine, propxyphen, oxycodone, hydromorphone, heroine, tramadol, and fentanyl. Non-opϊoid analgesics (nonnarcotic) analgesics include, for example, acetaminophen, clonidine, NMDA antagonists, and cannabinoids. Non-selective COX inhibitors include, but are not limited to acetylsalicylic acid (ASA), ibuprofen, naproxen, lcetoprofen, piroxicam, etodolac, and bromfeπac. Selective COX2 inhibitors include, but are not limited to celecoxib, valdecoxib, parecoxib, and etoricoxib.

As used herein an "anesthetic" is an agent that interferes with sense perception near the site of administration, a local anesthetic, or result in alteration or loss of consciousness, e.g., systemic anesthetic agents. Local anesthetics include but are not limited to lidocaiπe and buvicaine.

Non-limiting examples of antiepileptic agents are carbamazepine, phenytoin and gabapentin. Non-limiting examples of antidepressants are amitriptyline and desmethylimiprimine.

Non-limiting examples of anti-inflammatory drugs include corticosteroids (e.g., hydrocortisone, cortisone, prednisone, prednisolone, methyl prednisone, triamcinolone, fluprednisolone, betamethasone and dexamethasone), salicylates, antihistamines and H ₂ receptor antagonists. The phrases "parenteral administration" and "administered parenteral];/' as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous,

intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, dosages of a compound of the invention may be determined by deriving dose-response curves using an animal model for the condition to be treated. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the

dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a subject, when used for the indicated analgesic effects, wjjl range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 100 mg per kg per day, and still more preferably from about 1.0 to about 50 mg per kg per day. An effective amount is that amount treats a neurotrophin- associated state or neurotrophiπ disorder.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, il is preferable to administer the compound as a pharmaceutical composition.

Methods of Treatment

The above compounds can be used for administration to a subject for the modulation of a neurotrophin-mediated activity, involved in, but not limited to, pain, inflammatory disorders, neurological disorders, and any abnormal function of cells, organs, or physiological systems that are modulated, at least in part, directly or indirectly by a neurotrophin-mediated activity. Additionally, it is understood that the compounds may also alleviate or treat one or more additional symptoms of a disease or disorder discussed herein.

Accordingly, in one aspect, the compounds of the invention may be used to treat pain, including acute, chronic, malignant and non-malignant somatic pain (including cutaneous pain and deep somatic pain), visceral pain, and neuropathic pain. Il is further understood that the compounds may also alleviate or treat one or more additional signs or symptoms of pain and sensory deficits (e.g., hyperalgesia, allodynia, dysesthesia, hyperesthesia, hypeipathia, paresthesia). ϊn some embodiments of this aspect of the invention, the compounds of the

invention may be used to treat somatic or cutaneous pain associated with injuries, inflammation, diseases and disorders of the skin, subcutaneous tissues and related OTgans including, but not limited to, cuts, burns, lacerations, punctures, incisions, surgical pain, post-operative pain, orodental surgery, psoriasis, eczema, dermatitis, and allergies. The compounds of the invention may also be used to treat somatic pain associated with malignant and non-malignant neoplasm of the skin, subcutaneous tissues and related organs (e.g., melanoma, basal cell carcinoma).

In other embodiments of this aspect of the invention, the compounds of the invention may be used to treat deep somatic pain associated with injuries, inflammation, diseases and disorders of the musculoskeletal and connective tissues including, but not limited to, arthralgias, myalgias, fibromyalgias, myofascial pain syndrome, dental pain, lower back pain, pain during labor and delivery, surgical pain, post-operative pain, headaches, idiopathic pain disorder, sprains, bone fractures, bone injury, osteoporosis, severe bums, gout, arthiritis, osteoarfhithis, myositis, and dorsopathies (e.g., spondylolysis, subluxation, sciatica, and torticollis). The compounds of the invention may also be used to treat deep somatic pain associated with malignant and non-malignant neoplasm of the musculoskeletal and connective tissues (e.g., sarcomas, rhabdomyosarcomas, and bone cancer).

In other embodiments of this aspect of the invention, compounds of the invention may be used to treat visceral pain associated with injuries, inflammation, diseases or disorders of the circulatory system, the respiratory system, the genitourinary system, the gastrointestinal system and the eye, car, nose and throat.

For example, the compounds of the invention may be used to treat visceral pain associated with injuries, inflammation and disorders of the circulatory system including, but are not limited to, ischaemic diseases, ischaemic heart diseases (e.g. , angina pectoris, acute myocardial infarction, coronary thrombosis, coronary insufficiency), diseases of the blood and lymphatic vessels (e.g., peripheral vascular disease, intermittent claudication, varicose veins, haemorrhoids, embolism or thrombosis of the veins, phlebitis, thrombophlebitis lymphadenitis, lymphangitis), and visceral pain associated with malignant and non-malignant neoplasm of the circulatory system (e.g., lymphomas, myelomas, Hodgkin's disease).

In another example, the compounds of the invention may be used to treat

visceral pain associated with injuries, inflammation, diseases and disorders of the respiratory system including, but are not limited to, upper respiratory infections (e.g., nasopharyngitis, sinusitis, and rhinitis), influenza, pneumoniae (e.g., bacterial, viral, parasitic and fungal), lower respiratory infections (e.g., bronchitis, bronchiolitis, tracheobronchitis),, interstitial lung disease, emphysema, bronchiectasis, status aslhmaticus, asthma, pulmonary fibrosis, chronic obstructive pulmonary diseases (COPD), diseases of the pleura, and visceral pain associated with malignant and πon- malignant neoplasm of the respiratory system (e.g., small cell carcinoma, lung cancer, neoplasm of the trachea, of the larynx). In another example, the compounds of the invention may be used to treat visceral pain associated with injuries, inflammation and disorders of the gastrointestinal system including, but are not limited to, injuries, inflammation and disorders of the tooth and oral mucosa (e.g., impacted teeth, dental caries, periodontal disease, oral aphthae, pulpitis, gingivitis, periodontitis, and stomatitis), of the oesophagus, stomach and duodenum (e.g. , ulcers, dyspepsia, oesophagitis, gastritis, duodenitis, diverticulitis and appendicitis), of the intestines (e.g., Crohn's disease, paralytic ileus, intestinal obstruction, irritable bowel syndrome, neurogenic bowel, megacolon, inflammatory bowel disease, ulcerative colitis, and gastroenteritis), of the peritoneum (e.g. peritonitis), of the liver (e.g., hepatitis, liver necrosis, infarction of liver, hepatic veno-occlusive diseases), of the gallbladder, biliary tract and pancreas (e.g., cholelithiasis, cholecystolithiasis, choledocholithiasis, cholecystitis, and pancreatitis), functional abdominal pain syndrome (FAPS), gastrointestinal motility disorders, as well as visceral pain associated with malignant and non-malignant neoplasm of the gastrointestinal system (e.g., neoplasm of the oesophagus, stomach, small intestine, colon, liver and pancreas).

In another example, the compounds of the invention may be used to treat visceral pain associated with injuries, inflammation, diseases, and disorders of the genitourinary system including, but are not limited to, injuries, inflammation and disorders of the kidneys (e.g., nephrolithiasis, glomerulonephritis, nephritis, interstitial nephritis, pyelitis, pyelonephritis), of the urinay tract (e.g. include urolithiasis, urethritis, urinary tract infections), of the bladder (e.g. cystitis, neuropathic bladder, neurogenic bladder dysfunction, overactive bladder, bladder-neck obstruction), of the

male genital organs (e.g., prostatitis, orchitis and epididymitis), of the female genital organs (e.g., inflammatory pelvic disease, endometriosis, dysmenorrhea, ovarian cysts), as well as pain associated with malignant and non-malignant neoplasm of the genitourinary system (e.g., neoplasm of the bladder, the prostate, the breast, the ovaries).

In further embodiments of this aspect of the invention, compounds of the invention may be used to treat neuropathic pain associated with injuries, inflammation, diseases and disorders of the nervous system, including the central nervous system and the peripheral nervous systems. Examples of such injuries, inflammation, diseases or disorders associated with neuropathic pain include, but are not limited to, neuropathy (e.g., diabetic neuropathy, drug-induced neuropathy, radiotherapy-induced neuropathy), neuritis, radiculopathy, radiculitis, neurodegenerative diseases (e.g,, muscular dystrophy), spinal cord injury, peripheral nerve injury, nerve injury associated with cancer, Morton's neuroma, headache (e.g., nonorganic chronic headache, tension-type headache, cluster headache and migraine), multiple somatization syndrome, postherpetic neuralgia (shingles), trigeminal neuralgia complex regional pain syndrome (also known as causalgia or Reflex Sympathetic Dystrophy), radiculalgia, phantom limb pain, chronic cephalic pain, nerve trunk pain, somatoform pain disorder, central pain, non-cardiac chest pain, central post-stroke pain.

In another aspect, the compounds of the invention may be used to treat inflammation associated with injuries, diseases or disorders of the skin, subcutaneous tissues and related organs, the musculoskeletal and connective tissue system, the respiratory system, the circulatory system, the genitourinary system and the gastrointestinal system.

In some embodiments of this aspect of the invention, examples of inflammatory conditions, diseases or disorders of the skin, subcutaneous tissues and related organs that may be treated with the compounds of the invention include, but are not limited to allergies, atopic dermatitis, psoriasis, eczema and dermatitis. In other embodiments of this aspect of the invention, inflammatory conditions, diseases or disorders of the musculoskeletal and connective tissue system that may be treated with the compounds of the invention include, but are not limited to arthritis,

osteoarthritis, and myositis.

In other embodiments of this aspect of the invention, inflammatory conditions, diseases or disorders of the respiratory system that may be treated with the compounds of the invention include, but are not limited to allergies, asthma, rhinitis, neurogenic inflammation, pulmonary fibrosis, chronic obstructive pulmonaiy disease (COPD), adult respiratory distress syndrome, nasopharyngitis, sinusitis, and bronchitis.

In still other embodiments of this aspect of the invention, inflammatory conditions, disease or disorders of the circulatory system that may be treated with the compounds of the invention include, but are not limited to, endocarditis, pericarditis, myocarditis, phlebitis, lymphadenitis and artherosclerosis,

In further embodiments of this aspect of the invention, inflammatory conditions, diseases or disorders of thegenitOurinary system that may be treated with the compounds of the invention include, but are not limited to, inflammation of the kidney (e.g., nephritis, interstitial nephritis), of the bladder (e.g., cystitis), of the urethra (e.g..urethritis), of the male genital organs (e.g. , prostatitis), and of the female genital organs (e.g., inflammatory pelvic disease).

In further embodiments of this aspect of the invention, inflammatory conditions, diseases or disorders of the gastrointestinal system that may be treated with the compounds of the invention include, but are not limited to, gastritis, gastroenteritis, colitis (e.g., ulcerative colitis), inflammatory bowel syndrome, Crohn's disease, cholecystitis, pancreatitis and appendicitis.

In still further embodiments of this aspect of the invention, inflammatory conditions, diseases or disorders that may be treated with the compounds of the invention, but are not limited to inflammation associated with microbial infections (e.g., bacterial, viral and fungal infections), physical agents (e,g., burns, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis and various types of immunologic reactions and autoimmune diseases (e.g., Lupus erythematosus).

In another aspect, the compounds of the invention may be used to treat injuries, diseases or disorders of the nervous system including, but not limited to neurodegenerative diseases (e.g., Alzheimer's disease, Duchenne's disease), epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, stroke, cerebral ischemia,

neuropathies (e.g., chemotherapy-induced neuropathy, diabetic neuropathy), retinal pigment degeneration, trauma of the central nervous system (e.g., spinal cord injury), and cancer of the nervous system (e.g., neuroblastoma, retinoblastoma, brain cancer, and glioma), and other certain cancers (e.g., melanoma, pancreatic cancer). In further aspects of the invention, the compounds of the invention may also be used to treat other disorders of the skin, subcutaneous tissues and related organs {e.g. , hair loss), of the respiratory system (e g., asthma), of the circulatory system, (e.g., cardiac arrhythmias and fibrillation and sympathetic hyper-innervation), and of the genitourinary system (e.g., neurogenic bladder dysfunction and overactive bladder). The present invention provides a method for treating a subject that would benefit from administration of a composition of the present invention. Any therapeutic indication that would benefit from a NGF/NTR modulator (i.e., a compound of the invention) can be treated by the methods of the invention. The method includes the step of administering to the subject a composition of the invention, such that the disease or disorder is treated.

The invention further provides a method for preventing in a subject, a disease or disorder which can be treated with administration of the compositions of the invention. Subjects "at risk" may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. "At risk" denotes that an individual who is determined to be more likely to develop a symptom based on conventional risk assessment methods or has one or more risk factors that correlate with development of a disease or disorder that may be treated according the methods of the invention. For example, risk factors include family history, medication history, and history of exposure to an environmental substance which is known or suspected to increase the risk of disease. Subjects at risk for a disease or condition which can be treated with the agents mentioned herein can also be identified by, for example, any or a combination of diagnostic or prognostic assays known to those skilled in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of tfie disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

EXEMPLIFICATION OF THE INVENTION:

The invention is further illustrated by the following example, which could be used to examine the neurotrophin/πeurotrophin precursor binding inhibition of the compounds of the invention. The example should not be construed as further limiting. The animal models used throughout the Examples are accepted animal models and the demonstration of efficacy in these animal models is predictive of efficacy in humans.

Biological Activity Materials and Methods

Cell Culture

All cells were incubated at 37°C in 5% CO2. PC 12 cells were maintained in RPM3-1640 medium supplemented with 10% fetal bovine serum (FBS). Neuroscreen- 1 Cells were maintained in RPMI- 1640 medium supplemented with 10% horse serum (HS) and 5% fetal bovine serum (FBS). A875 human melanoma cells were maintained in DMEM with 10% FBS. HEK 293 or CHO-Kl cells were stably transfected with human or rat TrkA plasmid (see below) and maintained in DMEM with 10% FBS and G418 (600 μg/ml) or Zeocin (200 μg/ml) for selection.

Cell Transfections:

Expression of human or rat TrkA in HEK or CHO-Kl cells was achieved by transfecting the cells with the TrkA expression vector using Lipofectamine (Invitrogen) reagent. HEK or CHO cells were plated in 100 mm Petri dishes at a concentration of 10 ⁶ cells per dish. The next day, a solution of DNA diluted in I mL OptiMEM (Invitrogen) per dish was prepared and incubated for 15 min at room temperature. Concurrently, 42 μl of Lipofectamine reagent was prepared in OPtiMEM (1 mL per dish) and incubated for 15 min at room temperature. The DNA and lipofectamine reagent solutions were then mixed together and incubated for a further 15 minutes. During this 30 min of incubation, the cells were rinsed twice with OptiMEM. The DNA-lipofectamine solution in OptiMEM was then added to the dish, which was then placed in the incubator (37°C; 5% COj) for 3 h. This solution was then aspirated and the cells were rinsed with DMEM. From this point, cells were

grown in DMEM + FBS (10%), their normal growth medium. For stable cell lines, the culture medium contained G4IS (600 μg/mL) or Zeocin (200 μg/mL) a selection agent for maintenance of human or rat TrkA expression in the cells. The presence of TrkA was confirmed with ¹²⁵ I-NGF binding (see below) and Western blots (see below) labeled with TrkA specific antisera.

Example I: NGF Binding

NGF binding was evaluated using methods familiar to those who are skilled in the art. Briefly, cells expressing one or both NGF receptors (PCl 2: TrkA + p75; A875: p75 alone; HEK/CHO_trkA: TrkA alone) were harvested by replacing the medium with the cell dissociation buffer (Amersham) and incubating at 37°C for 15 min. For NGF binding, cells were resuspended at a concentration of 2 x 10* cells / mL in HEPES-Krebs-Ringer (HKR) buffer (1 OmM HEPES; 12SmM NaCl, 4.8mM KCl; I .3mM CaCl ₂ ,- I .2mM MgSO ₄ ; 1.2mM KH ₂ PO ₄ ; 1 mg/ml BSA; 1 mg/ml glucose; pH 7,4) and exposed to ¹²⁵ I-NGF (--0.4-0.6 πM) in the presence or absence of varying concentrations of the compound. Non-specific binding was determined for reference by incubating ¹²⁵ I-NGF with an excess of non-radioactive NGF in the absence of compound. Following a two-hour incubation period at 4°C, ¹²⁵ I-NGF bound to the cells was quantified in a gamma radiation counter following separation from unbound NGF by filtration or centrifugation through glycerol (10% in HKR). Inhibition of binding was calculated as a percent of the specific binding (calculated as the differential between ¹²⁵ I-NGF binding in the absence and presence of an excess of nonradioactive NGF without compound). Dose-response inhibition curves were typically generated with seven concentrations of a given compound, with three replicates for each concentration. For most compounds, multiple dose-response curves were generated.

The IC ₅₀ data shown in Tables A-J were acquired using the procedure described herein using PC 12 cells.

Example 2: NGF Crosslinkine to Receptors

NGF binding to TrkA and p75 is qualitatively evaluated following chemical cross-linking, and separation of proteins according to molecular weight with SDS-

PAGE. PC 12 (for p75 and TrkA binding), HEK/CHO_trkA (for TrIcA only) and A875 (for p75 only) cells are recovered using Grey's solution, pelleted by centrifugation, and suspended in HBIR. In a total volume of 1 πiL, 2 x 10 ⁶ cells are incubated, rotating, with 0,4-0,6 pM ¹²⁵ I-NGF, with or without compound, for 2 h at 4°C, At the conclusion of the binding reaction, a 20 μL volume of BS ³ (5z.j[sulfosucciniinidyl] suberate) crosslinker is added for a final concentration 0.4 mM and incubated, rocking, for an additional 30 min at room temperature. Cells are washed twice in HKR. Following centrifugation, the pellets are solubilized directly in SDS sample buffer and heated for 10 min at 95°C. All samples are electrophoresed on a 6% SDS-PAGE gel, which is then dried and autoradiographed. Bands at the appropriate molecular weights for p75-NGF conjugates and TrkA--NGF conjugates are visualized by exposing the dried gel to film (BioMax, Kodak) overnight. Modulatory effects of the compounds on NGF binding are determined by variations in band intensity. Reduced binding of NGF to its receptors is represented by lighter bands,

Example 3; Erk Phosphorylation

This assay is useful for establishing that the compounds of the invention are functional NGF antagonists, not receptor agonists (an agonist could conceivably block NGF binding but actually activate the receptor). Erk 1/2 is a kinase activated down stream of TrkA and is a well studied member of the NGF-induced signal transduction cascade.

PCl 2 cells expressing TrkA and p75 are acutely exposed to 1 ng/mL NGF (15 min; 37°C; 5% CO ₂ ) that is pre-incubated (30 min; room temperature) with or without the compounds. Cells are lysed in Laemmli sample buffer (for SDS-PAGE) or a lysis buffer containing Triton X-100 (for ELISA), Following SDS-PAGE, proteins are electroblotted onto nitrocellulose and immuπoprobed for phosphorylatcd Erk 1 and 2. Blocking and primary antibody incubations of immunoblots are performed in Tris- bufferedsaline-Tweeπ (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.2% Tween20) supplemented with 5% (w/v) bovine serum albumin (BSA); secondary antibody incubations are performed in 5% (w/v) dried skim milk powder. Immunoreactivc bands are detected by chemiluminescence

A quantitative method to detect phosphorylated Erk 1/2 by immunofluorescence is also used. Neuroscreen-1 cells are grown in a 96 well plate. NGF preincubated with or without the compounds for 30 minutes, is added to the cells for 5 minutes at 37 ^σ C. Cells are then fixed and stained with an antibody against phosphor-ERK 1/2. A fluorescently labeled secondaiy antibody is used to show the presence of phosphorylated ERK 1/2. Quantitative results are generated by photographing the wells with light of the appropriate wavelength, followed by software based image analysis

Table 2. Inhibition of pERK Phosphorylation and NGF Binding

There is an excellent correlation (R ² = 0.90) between the inhibition of NGF- induced ERK phosphorylation and the NGF binding potency. The effect of a compound on the pERK tends to be less pronounced than its potency in binding {linear regression analysis done on pICjo values of each set of data: pICjø _π ERK = 0.8 * pIC ₅ o binding)-

Example 4; Neurite Outgrowth

This assay is run as a further functional marker of NGF antagonism and takes advantage of the differentiation of PCl 2 cells (neurite outgrowth) induced by NGF. Cultures of PC 12 cells are grown on Terasaki plates pre-coated with poly-D-lysine. Cells are exposed to NGF (1-50 (preferably 5) πg/ml) to induce neurite outgrowth as

described elsewhere [LA Greene &. AS Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc Natl Acad Sci USA. 1976 July; 73(7): 2424-2426]. In addition to NQF, cells are exposed to varying concentration of the compounds or vehicle. Following 4 days of exposure to NGF in the presence or absence of compound, neurite outgrowth is quantified. A neurite is scored if its caliber from origin to terminal is approximately the same and the length is equal to or greater than 1.5 the cell body diameter. The number of neurite bearing cells per total number of viable cells is calculated for each condition; the number of cells with neurites in the presence of NGF (without compound exposure) is considered to represent maximal (100%) outgrowth, to which the inhibitory effect of compounds of the invention on NGF-induced outgrowth is compared.

Table 3. Inhibition of Neurite Outgrowth and NGF Binding

There is an excellent correlation (R ² = 0.81) between the neurite outgrowth and the binding potency. The effect of a compound on the neurite outgrowth is generally less pronounced (linear regression analysis done on pICso values of each set of data: pICso neuriie = 0.6 * pICso biπdin _E )-

Exatnϋle 5: Formalin model — model of acute tonic pain

This example describes a procedure for the in vivo assessment of the inhibitory activity of the compounds αf the present invention.

A number of well-established models of pain are described in the literature and are known to the skilled in the art (see, for example, Table 1). This example describes the use of the Formalin test.

Male Sprague-Dawley rats Eire housed together in groups of three animals under standard conditions with unrestricted access to food and water. All experiments are conducted according to the ethical guidelines for investigations of experimental pain in conscious animals (Zimmerman, 1983).

Assessment of formalin-induced flinching behavior in normal, uninjured rats (body weight 200-300 g) is made after formalin (2.5% in saline, 50-100 μl, s.c.) is injected into the plantar surface of the hindpaw using a 27G needle. Rats are pretreated with test compounds either administered intravenously (TV), by subcutaneous injection (SC) or by orally (PO).

Nociceptive behavior is determined manually every 5 minby measuring the amount of time spent in each of four behavioral categories: 0, treatment of the injected hiπdpaw is indistinguishable from that of the contralateral paw; \ , the injected paw has little or no weight placed on it; 2, the injected paw is elevated and is not in contact with any surface; 3, the injected paw is licked, bitten, or shaken. A weighted nociceptive score, ranging from 0 to 3 is calculated by multiplying the time spent in each category by the category weight, summing these products, and dividing by the total time for each 5 min block of time. (Coderre et ai, Pain 1993; 54: 43). On the basis of the resulting response patterns, 2 phases of nociceptive behavior are identified and scored: first phase (Pl; 0-5 min), interphase (Int; 6-15 min), second phase (P2; 60 min), phase 2A (P2A; 16-40 min) and phase 2B (P2B; 41-60 min).

Statistical analysis is performed using the Prism™ 4.01 software package (GraphPad, San Diego, CA, USA). The difference in response levels between treatment groups and control vehicle group is analyzed using an ANOVA followed by Bonferroni's method for post-hoc pair-wise comparisons. Ap value < 0.05 is considered to be significant.

Figure IA and IB, illustrate the dose-dependent effect of Compound 2E on chemically-induced spontaneous pain evoked by intraplantar injection of formalin in the rat (Formalin model in example 5). These results indicate that compound 2E caused a dose-dependent reduction of the pain intensity as evaluated by the licking

behavior (Figure IA). Compound 2E (0.1-10 mg/kg s.c.) was given 30 miπ prior to formalin injection. Figure IB depicts the dose-response relationship of Compound 2E on the number of licking and biting episodes in phase Ha of the formalin test. The ED ₅₀ for the effect of Compound 2E is about 3mg/kg. **p<0.01 vs vehicle (two-way ANOVA).

Figures 2A and 2B, illustrate the dose-dependent effect of Compound IG on chemically-induced spontaneous pain evoked by intraplantar injection of formalin in the rat (Formalin model in example 5). These results indicate that compound IG caused a dose-dependent reduction of the pain intensity as evaluated by the licking behavior (Figure 2A). Compound 1 G (0.4-20 mg/kg s.c.) was given 30 min prior to formalin injection. Figure 2B depicts the dose-response relationship of Compound IG on the number of licking and biting episodes in phase Ha of the formalin test. The ED ₅₀ for the effect of Compound IG is about 10mg/kg. */><Q.05 vs vehicle, **p<0.0l vs vehicle (two-way ANOVA)- Figures 6A and 6B, illustrate the dose-dependent effect of Compound 47G on chemically-induced spontaneous pain evoked by intraplantar injection of formalin in the rat (Formalin model in example 5). These results indicate that compound 47G caused a dose-dependent reduction of the pain intensity as evaluated by the licking behavior (Figure IA). Compound 47G (10, 30, 100, 300 μmol/kg s.c.) was given 30 min prior to formalin injection. Figure IB depicts the dose-response relationship of Compound 47G on the number of licking and biting episodes in phase Ha of the formalin test The ED ₅₀ for the effect of Compound 47G is about 100μmol/kg. *p<0.05 vs vehicle, ** _j θ<0.01 vs vehicle, ***p<Q.Q01 vs vehicle (Two-way ANOVA). Figures 7A and 7B, illustrate the effect of Compound 5OG on chemically- induced spontaneous pain evoked by intraplantar injection of formalin in the rat

(Formalin model in example 5). These results indicate that compound 5OG caused a dose-dependent reduction of the pain intensity as evaluated by the licking behavior (Figure IA). Compound 5OG (30 and 100 and μmol/kg s.c.) was given 30 min prior to formalin injection. Figure 7B depicts the dose-response relationship of Compound 50G on the number of licking and biting episodes in phase IIa of the formalin test. * p<0.05 vs vehicle (two-way ANOVA).

Example 6: Carra _& eenan model- model of chronic nociceptive ( inflammatory pain) Sain

Acute inflammatory hyperalgesia is induced in rats by unilateral injection of 150μ! of a 3% solution of λ-carrageenan into the plantar surface of the left hind paw 3- 5 hours prior to testing. Thermal hyperalgesia, mechanical allodynia, mechanical hyperalgesia, weight bearing asymmetry and paw inflammation are determined. Rats are pretreated with test compounds either administered intravenously (IV), by subcutaneous injection (SC) or by oral gavage (PO).

Thermal nociceptive thresholds are determined according to the method described elsewhere (Hargreaves et al., 1988). Briefly, through the glass surface, a radiant heat source (SV, 5OW projector bulb) is focused onto the plantar surface of the hind paw. The rat's paw-withdrawal latency to this stimulus is recorded to the nearest 0.1 s. Each latency score is an average of three trials, which are separated by at least 5 min. In all rats, both the injured and uninjured hind paws are similarly tested, allowing direct comparisons between inflamed and non- inflamed paws.

For mechanical allodynia assessment, the hindpaw withdrawal threshold (PWT) is determined using a calibrated series of von Frey hairs (Stoelting, IL, USA) ranging from 1 to 26 g. Animals are placed individually into Plexiglass chambers with customized platform that contains 1.5 mm diameter holes in a 5 mm grid of perpendicular rows throughout the entire area of the platform (Pitcher et al,, 1999), The protocol used in this study is a variation of that described by Takaishi et al. (1996)- After acclimation to the test chamber, a series of height calibrated von Frey hairs are applied to the central region of the plantar surface of one hindpaw in ascending order (1, 2, 4, 6, 8, 10, 15, and 26 g). A particular hair is applied until buckling of the hair occurred. This is maintained for approximately 2 s. The hair is applied only when the rat is stationary and standing on all four paws. A withdrawal response is considered valid only if the hindpaw is completely removed from the customized platform. Each hair is applied five times at 5 s intervals. If withdrawal responses do not occur more than twice during five applications of a particular hair, the next ascending hair in the series is applied in a similar manner. Once the hindpaw is withdrawn from a particular hair three out of the five consecutive applications, the paw is re-tested with the next descending hair until less than three withdrawal

responses occurs in five applications. The paw withdrawal threshold (PWT) is defined as the lowest hair force in grams that produced at least three withdrawal responses in five tests. After the threshold is determined for one hindpaw, the same testing procedure is repeated on the other hindpaw at 5-min interval. The decrease in PWT between ipsi and contralateral paw reflects the level of mechanical allodynia. Mechanical hyperalgesia is determined by measuring the difference of withdrawal thresholds in response to increasing pressure in the inflamed vs contralateral paw using the Randall-Sellito Paw pressure meter (ETC Life Science) (Randall LO and Sellito JJ., 1957). Briefly, rats are held in a contention jacket suspended by a stand and allowed to acclimate for 10 min. Then the tips of the paw pressure applicator are positioned close to the middle of the plantar and the dorsal area of the paw, avoiding the saphenous nerve innervations and an increasing pressure is applied until the rat removes it. Measures are performed in triplicate for each paw by alternating each paw with an interval of at least 1 min to avoid sensitization. To determine the weight bearing asymmetry, rats are put in the box of an incapacitance meter (ϊITC Life Science) for 5 min of acclimation. Rats are gently positioned on the 2 hind paws for lOsec to measure the difference between the weight bearing on the left and the right hind paw. The Test is repeated 3 times with a minimum of 5 min between tests in the same rat. Carrageenan induced paw oedema is measured with a plethysmometer (HTC

Life Science). Briefly, a mark is made on the ankles of the rat and the ipsi and contralateral paw are submerged 3 times into water up to the mark in order to determine the paw volume by calculation of water displacement.

Figure 3A and 3B, illustrate the effect of 20mg/kg of Compound IG administered SC on the mechanical (Randall-Sellito) (A) and thermal (Hargreaves' assay) (B) hyperalgesia resulting from an acute paw inflammation caused by the incraplantar injection of 150μI of a 3% solution of λ-carrageenan (Carrageenan model in example 6). Compound IG was given 10 min pre- and 3h post-carrageenan injection. Hyperalgesia was tested 4h post carrageenan injection. Results show that 20mg/kg of Compound IG significantly reversed the thermal hyperalgesia while marginally improving the mechanical hyperalgesia {not different from contralateral) 4h

hours post-carr&geenan. *p<0.05 vs vehicle, ⁺ p<0.05 vs contralateral, **p<0.01 vs contralateral paw (two-way ANOVA).

Figure 8A, and SB, illustrate the effect of 2x105 rag/kg of Compound 5OG administered SC on the thermal (observed in the Hargreaves' assay) (A), and mechanical (Randall-Sellito) (B) hyperalgesia resulting from an acute paw inflammation caused by the intraplantar injection of 150μl of a 3% solution of λ- carrageenan (Carrageenan model in example 6). Compound 5OG was given 10 min pre- and 3h post-cairageenan injection. Hyperalgesia was tested 4h post carrageenan injection. Results show that 2x105 mg/kg of Compound 5OG significantly reversed the thermal hyperalgesia while marginally improving the mechanical hyperalgesia 4h hours posi-carrageenan. */?<0.05 vs vehicle (unpaired 2-tait t-test).

Example 7: Capsaicin model - model of NGF mediated nociceptive (inflammatory

Topical application of capsaicin (8-methyl-N-vanillyl 6-nonamide) is known to cause a concentration-dependent increase in the NGF content of treated skin which peaks after 4h (Amann R and Schuligoi R, Pain 1 12: 76-82 (2004). In this model, capsaicin (Sigma, ONT) is dissolved in ethanol and diluted 1:1 in saline to a final concentration of 10 mg/ml. The left hindpaw of male sprague-dawley rats (200-250g) is immersed in capsaicin solution three times for 10 s in 10 s intervals. The other hindpaw is immersed in ethanol 50%, NaCl 0.045% and used as control. Secondary mechanical allodynia and thermal hyperalgesia was measured at 4h and 5h after capsaicin immersion. Thermal hyperalgesia is assessed according to the method of Hargreaves described in example 6 and mechanical allodynia is determined by the von Frcy test described in example 6.

Statistical analysis is performed using the Prism™ 4.01 software package (GraphPad, San Diego, CA, USA). The difference in response levels between treatment groups and control vehicle group is analyzed using an ANOVA followed by Bonferroni's method for post-hoc pair-wise comparisons. Ap value < 0.05 is considered to be significant.

Figure 4A and 4B, illustrate the effect of 2x lOmgλcg of Compound IG administered SC on the mechanical (Randall-Sellito) (A) and thermal (Hargreaves ¹

assay) (B) hyperalgesia resulting from paw treatment with capsaicin (Capsaicin model in example 7). Compound IG was given 15 min pre- and 2h post-capsaicϊn. Hyperalgesia was tested 4h post capsaicin. Results show that 2xl0πig/kg of Compound IG significantly reversed the mechanical but not thermal hyperalgesia 4h hours post-capsaicin. */K0.05 vs vehicle, (unpaired 2 tailed t-test).

Example 8: CFA model — model of chronic nociceptive (inflammatory pain) pain Injection of complete Freunds adjuvant (CFA) in the hindpaw of the rat has been shown to produce a long-lasting inflammatory condition, which is associated with behavioural hyperalgesia and allodynia at the injection site (Hylden el a!., Pain 1989; 37: 229). Rats (body weight 200 - 250 g) receive an injection of CFA (50% in saline, 100 μl, Sigma) into the plantar surface of the hindpaw under brief halothane anaesthesia. After 24 h, animals are treated with vehicle or compound (J.C. or i.p.) and, after different time-points following the treatment (e.g., 30, 60 or 90 min.), they are tested for hindpaw weight bearing responses, as assessed using an Incapacitancc Tester (e.g., Linton Instrumentation, UK), (Zhu et al., 2005). The instrument incorporates a dual channel scale that separately measures the weight of the animal distributed to each hindpaw. While normal rats distribute their body weight equally between the two hindpaws (50-50), the discrepancy of weight distribution between an injured and non-injured paw is a natural reflection of the discomfort level in the injured paw (nocifensive behavior). The rats are placed in a plastic chamber designed so that each hindpaw rests on a separate transducer pad. The averager is set to record the load on the transducer over 5 s time period and two numbers displayed represent the distribution of the rat's body weight on each paw in grams (g). For each rat, three readings from each paw are taken and then averaged. Side-to-side weight bearing difference is calculated as the average of the absolute value of the difference between two hindpaws from three trials (right paw reading— left paw reading).

Example 9: Chune or Spinal Nerve Ligation (SNLi mode (neuropathic pain model) The Spinal Nerve Ligation (SNL) model (Kim and Chung, Pain 1992; 50: 355) is used to induce chronic neuropathic pain. Male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) are anesthetized with isoflurane, the left L5 transverse process

is removed, and the L5 and L6 spinal nerves are tightly ligated with 6-0 silk suture. The wound is then closed with interna] sutures and external staples.

Mechanical allodynia testing: Pre- and post-injury baselines as well as post- treatment values (vehicle or compound-treated animals) for non-noxious mechanical sensitivity are evaluated using 8 Semmcs-Weinstein filaments (Stoelting, Wood Dale, IL, USA) with varying stiffness according to the up-down method (Chaplan et al., J Neurosci Methods 1994; 53: 55). Animals are placed on a perforated metallic platform and allowed to acclimate to their surroundings for a minimum of 30 minutes before testing. The mean and standard error of the mean (SEM) are determined for each paw in each treatment group. Since this stimulus is normally not considered painful, significant injury-induced increases in responsiveness (i.e., lower response thresholds) in this test are interpreted as a measure of mechanical allodynia. Effects of compounds are evaluated two weeks after the injury. Compound or vehicle is administered (i.p. or s.c) and effects on mechanical allodynia are measured at different time points after dosing {e.g., 30, 60 and 90 min.). Injections are performed by a separate experimenter who was not involved in testing the animals.

Data analysis: Statistical analyses is conducted using Prism™ 4.01 (GraphPad, San Diego, CA, USA). Mechanical hypersensitivity of the injured paw is determined by comparing contralateral to ipsilateral paw values within the vehicle group at each time point. Effect of vehicle (VEH) and compound on the ipsi and contralateral paw are determined by comparing the post-injury baseline (BL) to post treatment values using a two-way ANOVA followed by Bonferroni's method for post-hoc pair-wise comparisons [e.g. vehicle vs. compound).

Figure SA and SB, illustrate the effect of Compound IG administered SC on the mechanical allodynia (Voπ-Frey hair) resulting from sciatic nerve injuries (spinal nerve ligation - SNL Model (A) and spared nerve injury - SNI Model (B), example 9 and 10, respectively). Nerve injury was performed 14 days before testing to allow for the allodynia to develop. Compound IG was given 90 min before testing. Gabapentin (100 and 65 mg/kg p.o., respectively for the SNL and SNI models) was used as a positive control in these models. Results show that 3-30mg/kg of Compound IG significantly and dose-dependently reversed the mechanical allodynia in each

neuropathic pain model. *p<0.05 vs baseline (BL), **p<O.Ol vs baseline, •••pO.OOl vs baseline (two-way ANOVA).

Example 10: Oecosterd model or Spared Nerve Injury model fSNI> {neuropathic πain model)

The Spared Nerve Injury (SNI) model (Decosterd et al., Pain 2000; 87: 149) is used to induce chronic neuropathic pain. Male Sprague-Dawley rats are anesthetized with isoflurane, and two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) are transected, leaving the remaining sural nerve intact. Animals develop thermal and mechanical hyperalgesia and allodynia, as well as a long _' lasting spontaneous pain or dysesthesia the last month after the nerve injury.

Mechanical allodynia testing: Pre- and post-injury baselines as well as post- treatment values (vehicle or compound-treated animals) for mechanical allodynia are evaluated using Von Frey filaments (Stocking, Wood Dale, IL, USA) with varying stiffness. Animals are placed on a perforated metallic platform and allowed to acclimate to their surroundings for a minimum of 30 minutes before testing. The mean and standard error of the mean (SEM) are determined for each paw in each treatment group (ipsilateral/injured paw and contralateral/non-injured paw). Since this stimulus is normally not considered painful, significant injury-induced increases in responsiveness (i.e. lower response thresholds) in this test are Interpreted as a measure of mechanical allodynia. Effects of compounds are evaluated two weeks after the injury. Compound or vehicle is administered (Lp. or s.c.) and effects on mechanical allodynia were measured at different time points after dosing (e.g _ψ> 30, 60 and 90 min.). Data analysis: Statistical analyses is conducted using Prism™ 4.01 (GraphPad,

San Diego, CA, USA). Mechanical hypersensitivity of the injured paw is determined by comparing contralateral to ipsilateral paw values within the vehicle group at each time point. Effect of vehicle (VEH) and compound on the ipsi and contralateral paw are determined by comparing the post-injury baseline (BL) to post treatment values using a two-way ANOVA followed by Bonferroni's method for post-hoc pair-wise comparisons (e.g. vehicle vs compound).

Figure SA and 5B, illustrate the effect of Compound IG administered SC on the mechanical allodyπia (Von-Frey hair) resulting from sciatic nerve injuries (spinal nerve ligation- SNL Model (A) and spared nerve injury- SNI Model (B), example 9 and 10, respectively). Nerve injury was performed 14 days before testing to allow for the allodynia to develop. Compound IG was given 90 min before testing. Gabapeπtiπ (100 and 65 mg/kg p.o., respectively for the SNL and SNI models) was used as a positive control in these models. Results show that 3-30mg/kg of Compound IG significantly and dose-dependently reversed the mechanical allodynia in each neuropathic pain model, "/xθ.05 vs baseline (BL), **ρ<0 01 vs baseline, ***/x0.0Ql vs baseline (two-way ANOVA).

Example 11; General Procedure for Physical Characterisation of Compounds of the Invention

The materials were obtained from commercial suppliers and used without purification. THF, CH ₂ CI ₂ , and DMF were passed through activated alumina columns to remove impurities prior to use. Unless otherwise stated, all non-aqueous reactions were perfoimed under an atmosphere of dry nitrogen or argon in oven-dried glassware. Standard inert atmosphere techniques were used in handling all air and moisture sensitive reagents and products. Reactions were monitored by thin layer chromatography (TLC) using Merck

60 F254 0.25 mm silica gel plates. The TLC spots were viewed under ultraviolet light and by heating the TLC plate after treatment with a solution of ammonium molybdate in 10% aqueous Conventional flash column chromatography, using Silicyde Ultra Pure Silica Gel (230-400 mesh), was performed to purify all compounds. Automated flash chromatography was performed of a Biotage system equipped with a Flash collector and Horizon detector and recorder. Removal of organic solvents was performed by roto-evaporation on a Bϋchi R-205/R215 Rotovapor using a Buchi V-700 vacuum system. Trace solvents were removed on a high vacuum pump.

All NMR experiments were recorded on an AC-Bruker instrument (400 MHz). Unless otherwise noted, proton and carbon chemical shifts are reported in parts per million using deuterated DMSO ((CDj) ₂ CO), as an internal standard at 2.50 and 39.43 ppm, respectively. Other solvents like deuteraled benzene (QDή), deuterated

chloroform (CDCIs) or deuterated acetone were used. The multiplicity, coupling constants (J in Hz), and number of protons were indicated in parentheses after each chemical shift. The HPLCMS spectra were recorded on a Waters 2795 separation module (LC), equipped with a ZQ 2000 ES+ MS and uv absorption is standardized at 254nm and 235nm.

The PREP HPLC purifications were performed and recorded on a Gilson apparatus equipped with automatic injection and fraction collection and a Waters apparatus with manual injection and fraction collection.

Example 12: General Synthetic Procedure for Formula Ia

The stirred mixture of substituted phthaUc anhydride(lmmol), appropriate amine( 1.1 mmol) in acetic acid ( 10 mL) was refluxed for overnight. The reaction mixture was brought to room temperature. The solid was filtered, washed with acetic acid (ImI) and dried under vacuum to give the desired compound.

Example ISi General Synthetic Procedure for Formula Ib Procedure for the Synthesis of Compound 12A

Methyl 4-bromo-2-methylbenzoate (1)

4-Bromo-2-methylbeπzoic acid (5 g) was dissolved in methanol (20 mL). Concentrated HjSCu (1 mL) was added to the solution dropwise. The reaction was refluxed overnight, The reaction was cooled to room temperature. The solvent was removed under the reduced pressure, The residue was diluted with ether and washed with water, sat. NaHCOj solution and brine. The organic was dried over anhydrous Na2SO ₄ and concentrated to give compound I as colorless oil (4.77 g).

Methyl 4-cyano-2-metb.ylbenzoate (2) A mixture of the compound I (427 g, 18.6 mmol) and CuCN (2.04 g, 23 mmol) in NMP (30 mL) was heated at 180 °C for 5 hours. The reaction was poured into ince water. A solid was collected by filtration to give the compound 2,

Methyl 2-(bromomethyI)-4-cyanobenzoate (3)

The compound 2 (525 ing, 3 mmol) and NBS (594 mg, 3.3 mmol) in CCI ₄ (20 mL) were refluxed for 5 hours. The reaction was cooled to room temperature. Hexane was added into the reaction and the solid obtained was removed by filtration. The filtrate was concentrated under the reduced pressure to give the crude compound 3, which was used directly in the next step.

3-(5-Cyai)0-1-oxoisoindolin-2-yJ) benzoic acid (4)

A mixture of the compound 3 (310 mg) and 3 -amino benzoic acid (217 mg) in DMF (3 mL) was heated under microwave irradiation at 150 ⁰ C for 5 minutes. The reaction was cooled to room temperature. White precipitates were collected by filtration to give (he compound 4.

2-(3-CarboxyphenyI)-1-oxoisoindoline-5-carboxylic acid (12A) The compound 4 (30 mg) in H:SO _A (75%, 5 mL) was heated at 160 °C for 2 hours, and then at 190 °C for 1 hour. The reaction was poured into ice. A solid was collected by filtration, washed with water, and dried in vaccum to give the final product 12A (10 mg).

Compound 12A; MS (ES-) m/z 296 (M-I), (ES+) m/z 298 (M+l); ¹ H NMR (400MHz, DMSO-di) δ ppm 8.52 (s, 1H), 8.23 (s, 1H), 8.18 (d, J = 8.0 Hz ₁ 1H), 8.11

(d, J - 8.0 Hz ₇ 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0

Hz ₁ 1H), 5 17 (s, 2H) .

Example 14; General Synthetic Procedure for Formula II C ^O *" AMtIo acid Reflux

A stirred mixture of 1,2,4,5-benzenetetracarboxyIic anhydride (lmmol), appropriate amine(l .lmmol) in acetic acid (10 mL) was refluxed for overnight. After

the reaction mixture was brought to r.t. The solid separated was filtered, washed with acetic acid (1mI) and dried under vacuum to give the desired compound.

Example 15: General Synthetic Procedure for Formula III Procedure for the Synthesis of Compounds 28C and 29C

MeCk TBA-Br ₃ __

oven

l-Bramo-2-methoxy-4,5-dimethylbenzene (1); To the solution of 4-methoxy-l ₁ 2-dimethylbenzene(0.544g, 4 mmol) in dichloromethane(30mL) and methanol(20mL) was added tetrabutylammonium- tribromide(l ,93g, 4mmol). The reaction mixture was stirred overnight at r.t. The solvent was removed under reduced pressure & extracted with ether The ether layer was dried over sodium sulphate and evaporation of solvent gave title compound (75% yield, >90% purity).

2-(2-Methoxy-4,5-dimethytphenyl)4,4,S _t S-tetramethyl-l,S,2-dioxaborolane(2).'

To the solution of 1 -bromo-2-methoxy-4,5-dimethylbeπzene (0.43Og, 2mmol) in dioxane(l5mL), Bis(pinacolato)diboron(0.556g, 2,2mmol), potassium acetate (0.686g, 7.0mmol) and dppf(O.055g, 0, lmmol) were added and mixture was degassed

under nitrogen for 20 minutes.Then 1,1 '-Bis(diphenylphosphino)ferτoceπe- dichloropalladium (O.O82g, 0.1mmol)was added to the reaction mixture and again degassed for further 5 minutes. The reaction mixture was then heated overnight at 120° C . After cooling, the reaction mixture was diluted with ethyl acetate and passed thro' celite. The filtrate was washed with sodium carbonate and dried. The curde solid obtained after removing the solvent was purified through biotage (using 15%EtOAc:Hexane as eluent) to give compound 2.

2, 2'-Dimethoxy-4,4',5,5'-tetramethylbiphenyl (3): A mixture of the compound 2 (670 mg, 3.1 mmol), 1 -bromo-2-methox y-4,5- dimethylbenzene (900 mg, 3-4 mmol), Pd(OAc) ₂ (36 mg, 5 mol%), SPhos (151 mg, 10 mol%), and K ₃ PO ₄ η ₂ O (1.32 g, 6.2 mmol) in toluene (35 mL) and water (3.5 mL) was heated under nitrogen at 100 °C overnight. The reaction was allowed to cool to room temperature. The solvent was removed and diluted with CHaC^. The organic layer was washed with water, dried over anhydrous NaϊSCλi, and concentrated under reduced pressure. The crude material was purified through Biotage (15% EtOAc:Hexanes as eluent) to give compound 3.

6, ό'-Dimethoxybiphenyt-SJ'^'-tetracarboxylicβcid (4): The compound 3 (500 mg, 1.85 mmol) in NaOH solution (10%, 20 mL) was heated to 100 °C Then KMnO ₄ (3.6 g, 12 mmol) in water (40 mL) was added to the solution slowly. The reaction was refluxed overnight. The hot solution was filtered.

The filtrate was acidified with 1N HCl. A white solid precipitated from the solution.

The solid was collected by filtration to give compound 4 (165 mg).

Dimethoxy-3,3',4,4'-biphenyl tetracarboxylic dianhydride (5);

The tetra acid 4 (40 mg) was heated at 160 °C in vacuum oven overnight to give dianhydride S (30 mg).

General procedure for compound δfComπounds 28C and 29C)

Mixture of substituted 33',4,4'-Biphenyltetracarboxylic dianhydride (lmmol), appropriate amine(3mmol) in acetic acid (5 mL) was heated under microwave

irradiation at 200° C for 30 minutes. The reaction mixture was brought to r.t. filtered, washed with acetic acid (ImI) and dried under vacuum to give compound 6 in good yield,

Procedure for the Synthesis of Compound IQC and its analogues General Procedure:

The stirred mixture of 3,3',4,4' biphenyltetracarboxylic dianhydride (1mmo3) ₇ appropriate amϊne(3mrnol) in acetic acid (10 mL) was refluxed for overnight. The reaction mixture wag brought to r,t, The solid obtained was Filtered, washed with acetic acid (1mI) and dried under vacuum to give the desired compound.

Microwave procedure: Mixture of S^'^^'-Biphenyltccracarboxylic dianhydride (1mniol), appropriate amine(3mmol) in acetic acid (5 mL) is heated under microwave irradiation at 200° C for 15 minutes. The reaction mixture was brought to r.t, The solid obtained was filtered, washed with acetic acid (1mI) and dried under vacuum to give the desired compound.

Specific Synthetic Schemes

Specific Synthetic Scheme for Compound JOC:

Compound 1OC: MS (ES-) m/z 491 (M-1); ¹ H NMR (400MHz, DMSOd ₆ ) 6 ppm 12.05 (bs, 2H), S.29 (s, 2H) ₁ 8.27 (d, J - 7.6 Hz ₁ 2H) ₁ 7.99 (d, J - 7.6 Hz, 2H), 3 61 (t, J = 6.8 Hz, 4H), 2.26 (t, J - 7.2 Hz ₁ 4H), 1.63 (m, 4H), 1.53 (m, 4H).

Specific Synthetic Scheme for Compound 1C:

Compound 1C: MS (ES-) m/z 463 (M-I), ¹ H NMR (400MHz ₁ DMSO-d ₆ ) δ ppm 12.09 (bs, 2H), 8.27 (s, 2H), 8.26 (d, J = 7.2 Hz, 2H), 7 97 (d, J = 8.4 Hz, 2H), 3.64 (t, J = 6.8 Hz, 4H), 2.30 (t, J = 6.8 Hz, 4H), 1.85 (quintet, J = 6.8 Hz, 4H).

Specific Synthetic Scheme for Compound 19C:

Compound 19C: MS (ES-) m/z 519 (M-1); ¹ H NMR (400MHz, DMSO-d ₆ ) δ ppm 11.99 (s, 2H), 8.28 (s, 2H) ₁ 8.27 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 3.60 (t, J = 7.2 Hz, 4H), 2.20 (t, J - 7.2 Hz, 4H), 1.64 (m, 4H), 1.53 (m, 4H), 1.30 (m, 4H).

Specific Synthetic Scheme for Compound 9C:

Compound 9C: MS (ES-) m/z 435 (M-1); ¹ H NMR {400MHz, DMSO-d _β ) δ ppm 12.40 (bs, 2H), 8.30 (a, 2H), 8.29 (d, J = S Hz, 2H), 7.99 (d, J = 8 Hz, 2H), 3.84 (t, J = 7.2 Hz ₁ 4H), 2.64 (t, J - 7.2 Hz, 4H).

Specific Synthetic Scheme for Compound UC:

Compound HC: MS (ES-) mlz 535 (M-1); ¹ H NMR (400MHz, DMSO-ds) S ppm 8.29 (s, 2H), 8.27 (d, J = 7.6 Hz, 2H), 7,99 (d, J = 7.6 Hz, 2H), 3.66 (t, J = 7.2 Hz, 4H), 2.44 (t, J = 7.6 Hz, 4H), 1.90 (m, 4H).

Example 16: General Synthetic Procedure for Formula UIA

Formula KIA

A mixture of a substituted phthalic anhydride (2 equiv.) and a diamine derivative (1 equiv.) in glacial acetic acid (15 mL) was heated under microwave irradiation at 200 °C for 15 minutes. The reaction mixture was cooled to room

temperature and precipitates were collected by filtration. The solids were washed with acetic acid and dried in vacuum to get the product.

Example 17; General Synthetic Procedure for Formulae (IV) and (V) Procedure fyt the Synthesis of Compounds 9D and 8E

DMF, TEA

A mixture of 1 ,4,5,8-naphthalenetetracarbαxylic anhydride (268 mg, 1 mmol) and ^-glutamic acid (147 mg, 1 mmol) in DMF (1O mL) and TEA (0.1 mL) was stirred at room temperature for 10 minutes, and was heated under microwave irradiation at 140 ⁰ C for 5 minutes. The reaction was cooled to room temperature. 3,4- Diaminobenzoic acid (152 mg, 1 mmol) and TEA (0.1 mL) were added to the reaction. The reaction was heated under microwave irradiation at 140 ⁰ C for another 5 minutes. The reaction was cooled to room temperature again. DMF was removed under the reduced pressure. IN HCl solution (about 100 mL) was added to the residue to form pale yellow precipitate. The solid was filtered and dried in vacuum to give compound 9D, which was used for next step.

The compound 9D in acetic acid (5 mL) was heated under microwave irradiation at 200 ⁰ C for 10 mtnuies. The reaction was cooled to room temperature. A brown solid precipitated. The solid was removed by filtration. The filtrate had stayed at room temperature for 2 days. A yellow solid was formed and collected by filtration to give the product 8E.

Example IS: General Synthetic procedure for Formula Vl

NaOftc PflC. H ₃ . OAf __ 15 mln

R* aiid R ^a « H. O^ CS^ _■ 0*.

A mixture of 4, 5-diiiitro-l,S-naphthalic anhydride (1 equiv.), amino derivative

(1.2 equiv.), and NaOAc (1.5 equiv,) in acetic acid was heated under microwave irradiation at 200 ⁰ C for 15 minutes. The reaction was cooled to room temperature and diluted with water. A solid precipitated from the solution and was collected by filtration. A mixture of this intermediate, palladium on charcol 10% (40% w/w) in dimethylfoimamide was hydrogenated under hydrogen balloon at room temperature with vigorous stirring. After completion, the reaction was filtered through celites. The filtrate was evaporated under reduced pressure and dried in vacuum to get a diamine intermediate. A mixture of the diamine and an anhydride derivative (1 equiv.) in acetic acid was heated under microwave irradiation at 200 ⁰ C for 15 minutes. The reaction was cooled to room temperature. Solid precipitates were collected by filtration to afford the product.

Example 19: General Synthetic procedure fyr Formula VIt Procedure for the Synthesis of Compound 19C

NH ₂ (C Hz) ₃ CO OQHH Pd/C

NaOAc/AcOH H ₂

19G

3-Bromo-l,S-naphthalic anhydride (1):

Bromine (13,2mL, 25όmmol) was added to a stirred solution of 1, 8-naphthalic anhydride (5Og, 252mmol) in 70% nitric acid (100OmL) at 25 ⁰ C over 3 period of 10 min. The resulting brown solution was stirred at 70 ⁰ C for 2 hrs and then brought it to r.t. and left it overnight. The cream colored precipitate was collected by filtration and washed with water (4 x 150 mL). The isolated product was dried in vacuum for 24 hrs, resulting in off-white crystalline solid (12.Og, 20%yϊeId)).

(For the preparation of 3-Bromo-1,8-Naphthalic anhydride & removal of Copper: see Jonathan D, Moseley et al. Organic Process Research & Development 2003, 7, 58-66)

3~Cyanø-l,8-naphthaIic anhydride (2):

A mixture of 3-bromo-1,8-naphthalic anhydride (17.Og, όlmmol), copper cyanide ($.4g) and DMF (250 mL) was stirred at reflux temperature overnight (TLC showed no starting material left). The reaction mixture was cooled to r.t. and then quenched by pouring over crushed ice. The solid was filtered, washed with water and dried under vacuum for 24 hrs. The residue was passed through silica gel using CH ₂ CIi as eluent. The organic solvent was washed with 0.15 M of EDTA-disodium salt until the aqueous layer no longer retained the characteristic blue color of solvatβd copper ions (4-5 times, aliquot was checked with NH ₄ OH) and washed with brine. The solvent was dried over sodium sulphate and evaporated, resulting in a yellow solid (yield 40%). NMP may also serve as a suitable solvent for this reaction.

S'Mtrø _' l, 3-dioxo-lH,3H-benzo(de]isochromene-5-carboxylic acid β);

To the suspension of 3-cyano-l ,8-naphthalic anhydride (1.6gm, 7.1 mmol) in concentrated sulphuric acid(30 ml) at 0-5° C was added nitric acid(70%, 8mL) dropwise. After the addition is complete, reaction mixture was heated at 60° C for ] ,5 hr. The reaction mixture was cooled to r.t. and then quenched by pouring over crushed ice. The solid separated was collected by filtration, washed with water & dried under vacuum to give title compound. The compound used as such for the next step.

2-(3-Carboxyprøpyl)-8-nitrø-l, 3-dioxo-2,3-dihydro-lH-benzofde]isoquinoline-5- carboxylic acid (4);

S-Nitro-1,3-dioxo-1H,3H-benzo[rfe]isc>chroπiene-5-car boxylic acid( 0.586 mg,

2 mmol), 4-amiπobutyri« acid (0.41 Ogm, 4mmol) and sodium acetate (0.250 mg, 3mmol) in 1QmL glacial acetic acid was added in 25ml microwave vial. The mixture was heated Under microwave irradiation at 200° C for 15 minutes at high absorption. The reaction mixture was cooled to room temperature and the solid obtained was filtered, and washed with acetic acid and then dried in vacuum for 24 hours to give a desired product as yellow solid (0.60Og ₅ 93% yield).

S-Amino-2-(S-carboxypropyl)-l, 3-dioxo-2,3-dihydTO-JH-benzofdef{soquinoHne-5- car boxy tic add (5):

A mixture isoquinoline-5-carboxylic acid (0.5g) _; palladium on charcoal 10% (0.075g) and dimethylformamide ((50 mL) was hydrogenated with hydrogen balloon at room temperature overnight with vigorous stirring. After the completion of reaction, the catalyst was filtered off through celite and the filtrate obtained was evaporated under reduced pressure and dried in vacuum for 24 hrs to give compound 5 in quantitative yield.

&-(Acetylamino)-2-β-carboxypropyl)-l,$-diQ?co-2,3-dihyd rQ-IH- benzo[de]isøquinoUne-5~carboxyIic acid (compound 19G); A mixture of 8-amino-2-{3-carboxypropyl)-l ,3-dioxo-2,3-dihydτo-l H- ber]zo[-fe]isoquinoline-5-carboxyHc acid(O.Sg), acetic acid(lOmL) and acetic anhydride(lOmL) was stirred at room temperature overnight. The solid obtained was collected by filtration and dried in vacuum for 24hrs to give a desired compound (yield >75%). Compound 19G: ¹ H NMR (DMSO-d6, 400MHz): l.S8(m, 2H), 2.1(s _s 3H), 2.3 l(t, J=7.2Hz, 2H), 4.07(t, J=6.8Hz, 2H), 8.64(d, J=1.8Hz,1H), 8,67(d, J=1 3Hz, 1H), 8.75(d, J=1.6Hz, 1H), 8.80(d, J=1JHz ₅ 1H) ₁ 10.59(s, 1H).

Example 20: General Synthetic procedure for Formula VIII Procedure for the Synthesis of Compound 47G

3-Cyano-1,8-naphthalic anhydride ( 1 ):

Compound is prepared as described for the synthesis of compound 19G.

tert- Butyl 4-(5-cyano-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)--yl)butanoate (2):

To the stirred suspension of tert-butyl aminobutanoate HCI salt (1.18g, 6.0mmol) in ethanol (50ml), triethylaminc (0.6g, 0.84mL, 6,0mmol) was added followed by 3- cyano1,8-naphthalic anhydride (1.1g, 5,0mmol). The mixture was heated to reflux for 6 his and reaction was monitored by TLC. After the completion, the reaction mixture was cooled to room temperature and solid was filtered and dried in vacuum for 24 hours to give desired compound as off white solid, 85% yield.

¹ H NMR((DMSO-d6, 400MHz): l.34(s, 9H), l ,88(m, 2H), 2.31(1, J=7.2Hz, 2H), 4.07(t, J=6.9Hz, 2H), S.OOft, J=7.8H?, 1H), 8.53(d, J=8.1Hz, 1H), 8.6(m, 2H), 9.00(d, J=1.2Hz, ]H)

tert-Butyl 4-fl,3-dioxo-5-(]H-tetrazal-5-yI)-lH-benzo[de]isoquinolin-20 H)- yljbutanoatύ (3):

A mixture of tert-huty\ 4-(5-cyano-1,3-dioxo-1H-benzo[rfe]isoquinolin'-2(3//)- yl)butanoate (1.46g ₅ 4.0mmol), NaN ₃ (0.36g, 5.6mmol), NH ₄ Cl (0.28g, 5.2mmol) in DMF( 20 ml) was heated at 120 ⁰ C for overnight. Completion of reaction was confirmed by TLC (additional NaNj and NH ₄ Cl could be added, if needed, for completion of reaction). The reaction was brought to r.t. and then quenched by pouring over ice water. The solid traces were removed by filtration. The filtrate was acidified with 1M HCl and light yellow solid obtained was filtered, washed with water SL dried to get required compound 3 {1.5g, 94% yield, 97% purity).

¹ H NMR((DMSO-d6, 400MHz): U4(s, 9H), 1.87(m, 2H), 2.32(1, J=7.1Hz, 2H), 4.1 Ht. J=6.9Hz, 2H), 7.97(t, J=7.9Hz, 1H), 8.57(<J, J=7.3Hz, 1H), 8,63(α\ J=8.3Hz, 1H), 9.06(3, 1H), 9.16(s, 1H)

tørt-Butyl 4-lS-(l-methyl-lH-tetrazol-S-yl)-l,3-dioxo-lH-benιo[de]isoq uinolin- 2(3H)-ylJbutanoate (4):

To the solution of tert-hutγ\ 4-[1,3-dioxo-5-(lff-tetrazol-5-yl)-l#- beiizo[de]isoquinolin-2(3/-yyl]butanoate(3) ( 1 5g, 3.7mmol) in DMF( 1 SmL) ₁ was added K ₂ CO ₃ ( 2.Og, 1S.Qmmol) and MeI(0.64g, 0.28inL,4.5mmol) respectively and stirred at room temperature for 3-4 hrs. The reaction was quenched by pouring over ice cold water and left at room temperature for lhr. The light yellow solid precipitated out was filtered, washed with water and dried in vacuum to yield compound 4. This compound was used as such for next step.

¹ H NMR((DMSO-d6, 400MHz): 1.34{s, 9H) ₁ 1.9(m, 2H) ₁ 2.32(t, J=7.2Hz, 2H), 4.11(t, J=6.9Hz, 2H), 4.50(s, 3H), 7.95(t, J=7.8Hz, 1H), 8 5(d, J=7.3Hz, 1H), 8.6(d, J=7.8Hz, 1H), 9.0(d, J=1.6Hz, 1H), 9.1(d, J=1.5Hz, 1H).

4-[5-(1-Methyl-1H-tetrazol-5-yl)-1,3-dioxo-1H-benzo[de]is oquinolin-2(3H)- yl]butanoic acid (41G):

To the solution of N-Me derivative 4 (1,4g) in CH ₂ Cl ₂ (50 ml) was added triethyl silane (3.0mL) followed by triflυoroacetic acid (10 ml). The reaction mixture is stirred at room temperature for 1 hr and then at 40°C for 2hr. The solvent was removed and dried under vacuum. The obtained oily solid was crystallized from AcOH to give desired compound 47G, as yellow crystalline solid (0.9g, >98 purity)

Compound 47G: ¹ H NMR((DMSO-d6, 400MHz): 1.91(m, 2H), 2.33(t, J=7.4Hz, 2H), 4.12(t, J=6.8Hz, 2H), 4.52(s, 3H), 7.96(t, J=7.8Hz, 1H), 8.56(d, J=6.5Hz, 1H), 8.67(d, J=S.2Hz, 1H), 9.00(d, J=1.5Hz, 1H), 9.10(d, J=1.5Hz, 1H), 12.03(s, 1H).

Procedure foe the Synthesis of Compound SOG

4-(5-Nitro-l,3-diøxo-lH-benzo[de]isσquinolin-2(3H)-yt)b utanøic acid (1)

A reaction mixture of 3-nitro-1,8-naphthalic anhydride (2.43g, lOmraol), 4- aminobutyric acid (2.1g ₃ 20mmol) and sodium acetate (0.8g) in 70ml glacial acetic acid was reflux overnight. The mixture was brought to room temperature. The solid obtained was filtered, washed with ether and finally dried in vacuum for 24 hrs to give compound 1 (3g).

¹ H NMR (DMSO-dό, 40O ⁾ V1Hz): l.90(m, 2H), 2.32(t, J ⁽ =7.3Hz, 2H) ₁ 4.00(t, J=ό.9Hz, 2H), 8.00(1, J=7.8Hz, JH), 8.60(d, J=6.9Hz, 1H) ₃ 8.71(d, J=8,1Hz, 1H), S.SO(d, J=2.2Hz, 1H) ₅ 9.40(d, J=2.2Hz, 1H) 4-(5-Amϊno-l,3-dioxo-lH-beπzo[de}LsoquinoUn-2(3H)-yl)butan oic ucid (2)

A mixture of 4-(5-nitro-l ,3-dioxo-l//-beπzo[rf£]isoquinolin-2(3/f)-yl)butaπoic acid (2.3 g, 7mmol), palladium on charcoal 10% (0.25g) and DMF (150 mL) was hydrαgβnated with hydrogen balloon at room temperature overnight with vigorous stirring. After the completion of reaction, the catalyst was filtered off through celite

and the filtrate obtained was evaporated under reduced pressure and dried in vacuum for 24 hrs to give yellow compound 2 in quantitative yield.

¹ H NMR (DMSO-dό, 400MHz): 1,86(m, 2H), 2.29(1. J«7.6Hz, 2H), 4.06(t, J=6.2Hz, 2H) ₅ 7.28(d, J=1.3Hz ₁ 1H), 7.60(t, J=7.9Hz, 1H), 7.9ό(d, J=2.4Hz, 1H), 8.03(d, J=7.4Hz, 1H), 8.07(d, J=ό.9Hz, 1H).

WlJ-Dwxo-S^lH^etrazol-lyQ-lH-benzolfajisoquinϋlm-ZβHϊ _' ylJbitianoic acid (50G);

A mixture of 4-(5-amino-1,3-dioxo-l//-benzo[ufe]isoquinolin-2(3W)- yl)butanoic acid (1 Qg, 3.3mmol), sodium azide(0.7g, lOmmol), himethylorthoformate(1.06g, lOmmol) , in 100 ml of acetic acid was stirred at reflux temperature overnight. Disappreance of starting material was confirmed by LCMS. The traces of solid were removed by filtration and then the filtrate was concentrated to 15 ml and brought to room temperature. The precipitated yellow solid was collected by filtration (0.55Og ₁ 92% purity). The solid was crystallized from acetic acid to give compound 5OG, as light yellow solid (98% purity). Compound 5OG: ¹ H NMR (DMSO-dό, 400MHz): 1.82-1.89(m, 2H) ₁ 2.13(1, J=7.5Hz,

2H), 4.08(t, J=7.2Hz, 2H), 7.98(t, J=7.7Hz, 1H), 8.55-S.5S(m, 2H), 8,91(d, J=2.1Hz, 1H), 9.04(d, J=2.1Hz, 1H), 10.4l(s, 1H)

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Incorporation bv Reference The entire contents of all patents, published patent applications and other references cited herein are hereby expressly incorporated herein in their entireties by reference.