WO2010132838A1 - Compounds useful for treating disorders related to trpa1 - Google Patents

Abstract

Compounds and compositions for treating disorders related to TRPAl are described herein.

Description

Compounds Useful for Treating Disorders Related to TRPAl

Claim of Priority

This application claims priority to USSN 61/178,349, filed May 14, 2009, which is hereby incorporated by reference in its entirety.

Background

The invention relates to compounds and compositions useful for treating disorders related to TRPAl. A variety of ion channel proteins exist to mediate ion flux across cellular membranes.

The proper expression and function of ion channel proteins is essential for the maintenance of cell function and intracellular communication. Numerous diseases are the result of misregulation of membrane potential or aberrant calcium handling. Given the central importance of ion channels in modulating membrane potential and ion flux in cells, identification of agents that can promote or inhibit particular ion channels are of great interest, both as research tools and as therapeutic agents.

Summary

The present invention provides methods and compositions for treating or preventing conditions such as pain by modulating the activity of the TRPAl channel. The compounds described herein can modulate the function of TRPAl by inhibiting a TRPAl -mediated ion flux or by inhibiting the inward current, the outward current, or both currents mediated by TRPAl.

The disclosure provides compounds of Formula I, or a pharmaceutically acceptable esters, salts and prodrugs thereof that inhibit TRPAl mediated current with an IC₅₀ of less than 10 micromolar:

Formula I

wherein each of R¹ and R² is, independently, H or C_1-4 alkyl; each of R³ and R⁴ is, independently, H or C_1-6 alkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or Ci_₆ alkyl, C₂-₆ alkenyl, or C₂-₆ alkynyl; each R⁸ is, independently, halo, hydroxyl, Ci_₆ alkyl, C₂-6 alkenyl, C₂-₆ alkynyl, C_1-6 alkoxy, C₂-₆ alkenoxy, C_1-6 haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, - CO2H, cyano, or nitro; n is 0-3; R⁹ is halo, hydroxyl, C_1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C_{1- 6} alkoxy, C₂-₆ alkenoxy, Ci_₆ haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or Ci_₄ alkyl.

Compounds of Formula I include compounds that inhibit an inward and/or outward TRPAl mediated current with an IC₅₀ of less than 10 micromolar, less than 1 micromolar, or about 500 nm or less. This IC₅₀ can be calculated, for example, in an in vitro assay. For example, IC₅₀ can be calculated using electrophysiological determinations of current, such as standard patch clamp analysis. IC₅₀ can also be evaluated using changes in concentration or flux of ion indicators, such as the calcium flux methods described herein. Unless otherwise indicated, IC 50 values are measured in vitro using patch clamp analysis or standard measurements of calcium flux (such as, e.g., Example 3). The compounds are useful, for example, in the synthesis or formulation of compounds and/or medicaments for inhibiting TRPAl, the treatment of TRPAl -mediated conditions, methods of treating TRPAl -mediated conditions and/or the synthesis of compounds for inhibiting TRPAl.

Compounds disclosed herein may be used to treat any diseases disclosed herein. In addition, these compounds may be used to inhibit a function of a TRPAl channel in vitro or in vivo. Detailed Description

Definitions

The term "alkenyl," as used herein, refers to an aliphatic group containing at least one double bond.

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined below, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl- substituted cycloalkyl groups, and cycloalkyl- substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer, and most preferably 10 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

The term "alkynyl", as used herein, refers to an aliphatic group containing at least one triple bond. The term "alkylurea" refers to a group having the structure -NHC(=O)NH-alkyl.

The term "alkylcarbamoyl" refers to a group having the structure -NHCθ₂-alkyl. The term "alkylthio" refers to a hydrocarbyl group having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S- alkenyl, or -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like.

The terms "amine" and "amino" refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

^■R ^Λ 1 0

/ ¹⁰ I ⁺

— N _{Q r} -N- R₁₀

^X R₉ K ' ₉

wherein Ro,, R^Q ^an& R' 10 ^eacn independently represent a hydrogen, an alkyl, an alkenyl, -(CI^)_1n-Rg, or Ro. and R JQ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rg represents an aryl, a cycloalkyl, a cycloalkenyl, an alkoxy, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8.

The term "amido" refers to a moiety that can be represented by the general formula:

^R10 wherein Ro,, R \Q are as defined above.

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

The term "aryl" as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, polycyclyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. Each ring can contain, e.g., 5-7 members.

The term "carbocycle or cyclyl," as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

The term "ester", as used herein, refers to a group -C(O)OR⁹ wherein R⁹ represents a hydrocarbyl group.

The terms "halo" and "halogen" as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

The terms "hetaralkyl" and "hetero aralkyl", as used herein, refers to an alkyl group substituted with a heteroaryl group. As used herein, the term "nitro" means -NO2; the term "halogen" or "halo" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO₂-.

Included within the scope of the current disclosure are, for each compound described herein, and the salts, esters and prodrugs thereof. When the compounds are referred to herein, it is understood that salts, esters and prodrugs of the compounds are also included. Tautomers of the compounds disclosed are also included within the scope of the current invention. Also included are methods for treating a TRPAl mediated disorder in a subject, such as the disorders described below, and pharmaceutical compositions including the compounds described herein.

Certain compounds disclosed herein may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. For example, if one chiral center is present in a molecule, the invention includes racemic mixtures, enantiomerically enriched mixtures, and substantially enantiomerically pure compounds. The composition can contain, e.g., more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% of a single enantiomer. The "enantiomeric excess" or "% enantiomeric excess" of a composition can be calculated using the equation shown below. In the example shown below a composition contains 90% of one enantiomer, e.g., the S enantiomer, and 10% of the other enantiomer, i.e., the R enantiomer. ee = (90-10)/100 = 80%. Thus, a composition containing 90% of one enantiomer and 10% of the other enantiomer is said to have an enantiomeric excess of 80%.

Methods of preparing substantially isomerically pure compounds can be prepared, for example, by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. Alternatively, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Techniques for inverting or leaving unchanged a particular stereocenter, and those for resolving mixtures of stereoisomers are well known in the art, and it is well within the ability of one of skill in the art to choose an appropriate method for a particular situation. See, generally, Furniss et al. (eds.), Vogel's Encyclopedia of Practical Organic Chemistry 5^th Ed., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816; and Heller, Ace. Chem. Res. 23: 128 (1990). The compounds described herein may also contain atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are intended to be encompassed within the scope of the present invention. For example, deuterated compounds and compounds incorporated ¹³C are intended to be encompassed within the scope of the invention.

As set out above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds disclosed herein. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", /. Pharm. ScL 66:1-19.) In other cases, the compounds disclosed herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds disclosed herein. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

An "effective amount" of, e.g., a TRPAl antagonist, with respect to the subject methods of inhibition or treatment, refers to an amount of the antagonist in a preparation which, when administered as part of a desired dosage regimen brings about a desired clinical or functional result. Without being bound by theory, an effective amount of a TRPAl antagonist for use in the methods, includes an amount of a TRPAl antagonist effective to decrease one or more in vitro or in vivo functions of a TRPAl channel. Exemplary functions include, but are not limited to, membrane polarization (e.g., an antagonist may promote hyperpolarization of a cell), ion flux, ion concentration in a cell, outward current, and inward current. Compounds that antagonize TRPAl function include compounds that antagonize an in vitro or in vivo functional activity of TRPAl. When a particular functional activity is only readily observable in an in vitro assay, the ability of a compound to inhibit TRPAl function in that in vitro assay serves as a reasonable proxy for the activity of that compound. In certain embodiments, an effective amount is an amount sufficient to inhibit a TRPAl- mediated current and/or the amount sufficient to inhibit TRPAl mediated ion flux.

The term "treating" includes therapeutic treatments. The term "therapeutic" treatment refers to administration to the host of one or more of the subject compositions, wherein the administration is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof.

Exemplary compounds are shown below, and in the Examples: and

In some embodiments, the compound has more than 80% ee, more than 90% ee, more than 95% ee, or more than 99% ee.

Compounds

The disclosure provides compounds of Formula I, or a pharmaceutically acceptable esters, salts and prodrugs thereof that inhibit TRPAl mediated current with an IC₅₀ of less than 10 micromolar (including, e.g., compounds with IC₅₀ values of less than about 1 micromolar, or about 500 nm or less):

Formula I wherein each of R¹ and R² is, independently, H or Ci_₄ alkyl; each of R³ and R⁴ is, independently, H or C_1-6 alkyl, C₂-₆ alkenyl, or C₂-₆ alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or C_1-6 alkyl, C2-6 alkenyl, or C2-6 alkynyl; each R⁸ is, independently, halo, hydroxyl, C_1-6 alkyl, C₂-6 alkenyl, C₂-₆ alkynyl, C_1-6 alkoxy, C₂-₆ alkenoxy, C_1-6 haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, - CO₂H, cyano, or nitro; n is 0-3; R⁹ is halo, hydroxyl, C_1-6 alkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, C_{1- 6} alkoxy, C₂_₆ alkenoxy, Ci_₆ haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or C_1-4 alkyl. In certain examples, R¹ and R² are both Ci-₄ alkyl; each of R³ and R⁴ is independently H or C_1-6 alkyl; each of R⁵ to R⁷ is H; n is 1; each R⁸ is independently halo; and R⁹ is Ci_₆ alkyl. For example, in some compounds, R¹ and R² can both be methyl, R³ can be H or C_1-6 alkyl, (e.g., methyl), R⁴ can be absent, R⁵ can be H, R⁶ can be H, n can be 1, R⁸ can be halo, and/or R⁹ can be methyl (with R, S or RS stereochemistry) .

Compounds of Formula I include compounds that inhibit an inward and/or outward TRPAl mediated current with an IC₅₀ of less than 10 micromolar, less than 1 micromolar, or about 500 nm or less. This IC₅₀ can be calculated, for example, in an in vitro assay. For example, IC₅₀ can be calculated using electrophysiological determinations of current, such as standard patch clamp analysis. IC₅₀ can also be evaluated using changes in concentration or flux of ion indicators, such as the calcium flux methods described herein. Unless otherwise indicated, IC 50 values are measured in vitro using patch clamp analysis or standard measurements of calcium flux (such as, e.g., Example 3). The compounds are useful, for example, in the synthesis or formulation of compounds and/or medicaments for inhibiting TRPAl, the treatment of TRPAl -mediated conditions, methods of treating TRPAl -mediated conditions and/or the synthesis of compounds for inhibiting TRPAl.

Compounds of Formula I include compounds of Formula II:

Formula II; wherein each of R₁' and R₂' is, independently, H or Ci_₄ alkyl (e.g., H or -CH₃); R₃ 'is H or Ci_₆ alkyl (e.g., H or -CH₃); R₈' is halo (e.g., -F, -Cl, -Br); R₉' is Ci_₆ alkyl, or is absent. The compounds are useful, for example in the formulation of medicaments for inhibiting TRPAl and/or the treatment of TRPAl -mediated conditions, as well as methods of treating TRPAl- mediated conditions. Particularly preferred compounds of Formula I and Formula II are compounds of Formula Ilia and Formula HIb:

Formula Ilia;

Forumula HIb.

In some embodiments, the compound has more than 80% ee, more than 90% ee, more than 95% ee, or more than 99% ee.

Certain compounds of Formula I, including compounds of Formula Ilia and Formula HIb, can be synthesized using known techniques. For example, compounds of Formula Ilia and HIb can be synthesized according to the synthesis of route of Scheme 1 below, where R" is as defined for R₃ or R₃', R'" is as defined for R₉ or R₉' above, and X is as defined for R₈ or R₈'.

Scheme I

Specific representative examples of compounds according to Scheme I are provided in Examples 1 and 2.

Indications

The compounds of Formula I, Formula II, Formula Ilia or Formula HIb useful in the modulation of TRPAl can be used in the formulation of analgesic pharmaceuticals suitable for the treatment and/or prophylaxis of nociceptive and neuropathic pain in mammals, especially in humans. In particular, compounds and pharmaceutical compositions of the present invention can be administered to treat disorders, conditions, or diseases treatable by the inhibition of TRPAl. For instance, the compounds and pharmaceutical compositions of the present invention are suitable for treatment or prophylaxis of the following diseases, conditions, and disorders mediated or associated with the activity of TRPAl receptors: Treatment of Pain, Sensitivity to Pain and Touch, or Pain-Related Diseases or Disorders, Respiratory disorders, Dermatological Diseases or Disorders, Neurological or

Neurodegenerative Diseases and Disorders, Inflammatory Diseases and Disorders, Cancer and Other Proliferative Diseases, Incontinence, Cutaneous diseases, Temperature Regulation, Hypertension, Gastrointestinal diseases, Rheumatoid diseases, Allergies, and Injuries from chemical warfare agents. Exemplary classes of sensitivities to pain and touch that can be treated using a TRPAl inhibitor include, but are not limited to conditions associated with activation of C-fibers (e.g., pruritus), nociceptive pain, inflammatory pain, and neuropathic pain. The pain can be chronic (e.g., neuropathic pain) or acute (e.g., post-surgical pain). As outlined above, TRPAl inhibitors may be particularly useful in the treatment of pain associated with cancer, osteoarthritis, rheumatoid arthritis, post-herpetic neuralgia, burns, and touch. Influx of calcium across plasma membrane of skin cells is a critical signaling element involved in cellular differentiation in the skin epidermis (Dotto, 1999 Crit Rev Oral Biol Med 10:442- 457). Regulating or modulating the calcium entry pathway, and thus a critical control point for skin cell growth, can treat or prevent skin diseases or disorders that are characterized by epidermal hyperplasia, a condition in which skin cells both proliferate too rapidly and differentiate poorly. Such diseases include psoriasis, and basal and squamous cell carcinomas. To further illustrate, additional exemplary indications for which compounds disclosed herein can be used include oral pain, pelvic pain, Fabry's disease, complex regional pain syndrome, pancreatitis, endometriosis, and fibromyalgia syndrome. Pain or sensitivity to pain and touch may be indicated in a variety of diseases, disorders or conditions, including, but not limited to, diabetic neuropathy, breast pain, psoriasis, eczema, dermatitis, burn, postherpetic neuralgia (shingles), nociceptive pain, peripheral neuropathic and central neuropathic pain, chronic pain, cancer and tumor pain, spinal cord injury, crush injury and trauma induced pain, migraine, cerebrovascular and vascular pain, sickle cell disease pain, rheumatoid arthritis pain, musculoskeletal pain including treating signs and symptoms of osteoarthritis and rheumatoid arthritis, orofacial and facial pain, including dental, temperomandibular disorder, and cancer related, lower back or pelvic pain, surgical incision related pain, inflammatory and non-inflammatory pain, visceral pain, psychogenic pain and soft tissue inflammatory pain, fibromyalgia-related pain, and reflex sympathetic dystrophy, and pain resulting from kidney stones or urinary tract infection. The compounds described herein are useful for the treatment or prevention of respiratory conditions, including conditions affecting the lung, pleural cavity, bronchial tubes, trachea, upper respiratory tract as well as the nerves and muscles involved in breathing. Respiratory diseases that may be treated with the compounds described herein include obstructive diseases such as chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, asthma (including asthma caused by industrial irritants), cystic fibrosis, bronchiectasis, bronchiolitis, allergic bronchopulmonary aspergillosis, and tuberculosis; restrictive lung disease including asbestosis, radiation fibrosis, hypersensitivity pneumonitis, infant respiratory distress syndrome, idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis, idiopathic interstial pneumonia sarcoidosis, eosinophilic pneumonia, lymphangioleiomyomatosis, pulmonary Langerhan's cell histiocytosis, and pulmonary alveolar proteinosis; respiratory tract infections including upper respiratory tract infections (e.g., common cold, sinusitis, tonsillitis, pharyngitis and laryngitis) and lower respiratory tract infections (e.g., pneumonia); respiratory tumors whether malignant (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell undifferentiated carcinoma, carcinoid, mesothelioma, metastatic cancer of the lung, metastatic germ cell cancer, metastatic renal cell carcinoma) or benign (e.g., pulmonary hamartoma, congenital malformations such as pulmonary sequestration and congenital cystic adenomatoid malformation (CCAM)); pleural cavity diseases (e.g., empyema and mesothelioma); and pulmonary vascular diseases (e.g, pulmonary embolism such as thromboembolism, and air embolism (iatrogenic), pulmonary arterial hypertension, pulmonary edema, pulmonary hemorrhage, inflammation and damage to capillaries in the lung resulting in blood leaking into the alveoli. Other conditions that may be treated include disorders that affect breathing mechanics (e.g., obstructive sleep apnea, central sleep apnea, amyotrophic lateral sclerosis, Guillan-Barre syndrome, and myasthenia gravis). The present compounds can also be useful for treating, reducing, or preventing one or more symptoms associated with respiratory conditions including, for example, shortness of breath or dyspnea, cough (with or without the production of sputum), cough associated with asthma, cough associated with influenza, coughing blood (haemoptysis), chest pain including pleuritic chest pain, noisy breathing, wheezing, and cyanosis.

Mechanisms associated with calcium signaling may be altered in many neurodegenerative diseases and in disorders resulting from brain injury. For example, fibroblasts or T-lymphocytes from patients with AD have consistently displayed an increase in Ca2+ release from intracellular stores compared to controls (Ito et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:534-538; Gibson et al. (1996) Biochem. Biophys. ACTA 1316:71-77; Etchenberrigaray et al. (1998) Neurobiology of Disease, 5:37-45). Consistent with these observations, mutations in presenilin genes (PSl or PS2) associated with familial AD (FAD) have been shown to increase InsP3-mediated Ca2+ release from internal stores (Guo et al. (1996) Neuro Report, 8:379-383; Leissring et al. (1999) J. Neurochemistry, 72:1061-1068; Leissring et al. (1999) J. Biol. Chem. 274(46):32535-32538; Leissring et al. (2000) J. Cell Biol. 149(4):793-797; Leissring et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97(15):8590- 8593). Furthermore, mutations in PSl or PS2 associated with an increase in amyloidogenic amyloid D peptide generation in AD are reported to be associated with a decrease in intracellular calcium level (Yoo et al. (2000) Neuron, 27(3):561-572). Experimental traumatic brain injury has been shown to initiate massive disturbances in Ca2+ concentrations in the brain that may contribute to further neuronal damage. Intracellular Ca2+ may be elevated by many different ion channels. It has been further shown that channel blockers may be beneficial in the treatment of neurological motor dysfunction when administered in the acute posttraumatic period (Cheney et al. (2000) J. Neurotrauma, 17(1):83-91). Accordingly, the TRPAl modulating compounds of Formula I can be formulated and administered in compositions for treating deurodegenerative diseases and disorders including but are not limited to Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and other brain disorders caused by trauma or other insults including aging.

The activation of neutrophils (PMN) by inflammatory mediators is partly achieved by increasing cytosolic calcium concentration ([Ca2+]i). Certain calcium channel-mediated calcium influx in particular is thought to play an important role in PMN activation. It has been shown that trauma increases PMN store-operated calcium influx (Hauser et al. (2000) J. Trauma Injury Infection and Critical Care 48 (4):592-598) and that prolonged elevations of [Ca2+]i due to enhanced store-operated calcium influx may alter stimulus-response coupling to chemotaxins and contribute to PMN dysfunction after injury. Modulation of PMN [Ca2+]i through store-operated calcium channels might therefore be useful in regulating PMN- mediated inflammation and spare cardiovascular function after injury, shock or sepsis (Hauser et al. (2001) J. Leukocyte Biology 69 (l):63-68). Peripheral neuropathy, for example diabetic neuropathy, is a particular condition that involves both a neuronal and an inflammatory component. Without being bound by a mechanistic theory, the TRPAl antagonists of the invention may be useful in treating peripheral neuropathies including, but not limited to, diabetic neuropathy. In addition to their use in the treatment of peripheral neuropathies (e.g., reducing inflammation), the subject inhibitors may also be useful in reducing the pain associated with peripheral neuropathy. Compositions and methods provided herein may also be used in connection with treatment of inflammatory diseases. These diseases include but are not limited to asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, glomerulonephritis, neuroinflammatory diseases such as multiple sclerosis, and disorders of the immune system. Neurogenic inflammation often occurs when neuronal hyperexcitability leads to the release of peptides that trigger inflammation. These peptides include substance P and CGRP. Blocking TRPAl would reduce neuronal activity and thus could block neurogenic inflammation. Pancreatitis is an inflammation of the pancreas. The pancreas is a large gland behind the stomach and close to the duodenum. Normally, digestive enzymes do not become active until they reach the small intestine, where they begin digesting food. But if these enzymes become active inside the pancreas, they start "digesting" the pancreas itself.

Compositions and methods provided herein may also be used in connection with treatment of malignancies, including, but not limited to, malignancies of lymphoreticular origin, bladder cancer, breast cancer, colon cancer, endometrial cancer, head and neck cancer, lung cancer, melanoma, ovarian cancer, prostate cancer and rectal cancer, in addition to skin cancers described above. Intracellular calcium level may play an important role in cell proliferation in cancer cells (Weiss et al. (2001) International Journal of Cancer 92 (6):877- 882). In addition, pain associated with cancer or with cancer treatment is a significant cause of chronic pain. Cancers of the bone, for example, osteosarcoma, are considered exceptionally painful, and patients with advanced bone cancer may require sedation to tolerate the intense and persistent pain. Accordingly, TRPAl antagonists of the invention represent a significant possible therapeutic for the treatment of pain, for example, the pain associated with cancer or with cancer treatment, including chemotherapy induced peripheral neuropathy. Given that TRPAl is differentially expressed in transformed cells, a compound described herein (e.g., a compound that blocks TRPAl) may also affect the proliferation of transformed cells and thus be a useful way to slow the disease (see Jaquemar et al. (1999) JBC 274(11): 7325-33). Thus a compound described herein (e.g., a TRPAl antagonist) could alleviate both the cause and symptoms of cancer pain.

Because of the effects of ion flux on arterial tension and relaxation, the subject compounds can also be used to affect thermal sensitivity. Furthermore, given that TRPAl channels are thermal responsive channels involved in the reception and sensation of cold stimuli, TRPAl antagonists can be used to modulate the sensation of cool, cold and decreased temperatures that often accompany pain.

Blockers of voltage-gated calcium channels belong to a class of medications originally developed to treat hypertension. Such blockers inhibit the movement of calcium into the muscle cells of the heart and arteries. Because calcium is needed for these muscles to contract, such blockers lower blood pressure by decreasing the force of cardiac contractile response and relaxing the muscle walls of the arteries. Although TRPAl is not a voltage- gated calcium channel, it is still instrumental in regulating calcium homeostasis, as well as the balance of other ions, in cells and tissues.

The compounds disclosed herein can be useful for the treatment and prevention of injuries resulting from the exposure to chemical warfare agents. Such injuries include any physical injuries, such as injuries to the skin (e.g., burn, inflammation, burn, and rash), eyes, respiratory tract, musculo- skeletal system, circulatory system, gastrointestinal tract, central nervous system, peripheral nervous system, heart, liver, lungs, and kidneys. The compound is administered to a subject before, during, or following such exposure and is therefore administered within 24 hours, 18 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, one minute, or thirty seconds of such exposure. A subject may be exposed to a chemical warfare agent by inhalation or touching. As a result of such administration, the symptoms or injuries resulting from the exposure of chemical warfare agents are reduced, prevented, or both. Exemplary symptoms or injuries resulting from the exposure to chemical warfare agents include inflammation, burn, itch, pain, rash, blisters, sweating, muscle twitching, nausea, vomiting, diarrhea, weakness, loss of conciousness, convulsions, muscular twitching, paralysis, secretions (from the mouth, nose, or lung for example), difficulty breating, blurred vision, eye pain, lacrimation, red eyes, shortness of breath, coughing, phlegm production and narrowing of the airways, headaches, tremors, dizziness, numbness or tingling, anxiety, insomnia,depression, emotional instability, and even death. These chemical warfare agents include all those classified as schedule 1, 2, and 3 agents under the Chemical Weapons Convention of 1993 and may be in liquid form, gas form, solid form, or combinations thereof. Exemplary agents are described in further detail below and include, for example, nerve agents, blood agents, blister agents, pulmonary agents, incapacitating agents, and toxins.

Nerve agent poisoning typically leads to contraction of pupils, profuse salivation, convulsions, involuntary urination and defecation, and eventual death by asphyxiation as control is lost over respiratory muscles. These symptoms are reduced or prevented by the administration of the TRPAl antagonists. Exemplary agents include G agents such as tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and GV; V agents such as VE, VG, VM, VX, and Novichok agents. For example, in addition to wearing a full body suit and a gas mask, subjects who are likely to be exposed to such agents are administered a TRPAl antagonist.

A blood agent (or cyanogen agent) is a compound containing a cyanide group that prevents the body from utilizing oxygen. These agents exert their toxic effect at the cellular level by directly interrupting cellular respiration. Exemplary agents include cyanogen chloride, hydrogen cyanide, and hydrogen sulfide.

Blister agents or vesicants typically cause severe skin, eye and mucosal pain and irritation. These agents also have the ability to cause large, painful water blisters. Blister agents include, for example, lewisites, nitrogen mustard, sulfur mustard, ethyldichloroarsine (a lewisite analog; ED), methyldichloroarsine (MD), phenyldichloroarsine (PD), and phosgene oxime (CX). Lewisites include, for example, 2-Chlorovinyldichloroarsine (Lewisite 1), Bis(2-chlorovinyl)chloroarsine (Lewisite 2), and Tris(2-chlorovinyl)arsine (Lewisite 3). Exemplary nitrogen mustards are bis(2-chloroethyl)ethylamine (HNl), bis(2- chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3). Sulfur mustards include, for example, l,2-Bis(2-chloroethylthio) ethane (Sesquimustard; Q), 1,3-Bis(2- chloroethylthio)-n-propane, 1 ,4-Bis(2-chloroethylthio)-n-butane, 1 ,5-Bis(2-chloroethylthio)- n-pentane, 2-Chloroethylchloromethylsulfide, bis(2-chloroethyl) sulfide (Mustard gas; HD), bis(2-chloroethylthio) methane, bis(2-chloroethylthiomethyl) ether, and bis(2- chloroethylthioethyl) ether (O Mustard).

A pulmonary agent (or choking agent) is a chemical weapon agent designed to impede a subject's ability to breathe, resulting in suffocation. Exemplary agents include adamsite (DM), acrolein, bis(chloromethyl) ether (BCME), chlorine (C12), chloropicrin (PS), diphosgene (DP), methyl chlorosulfonate, phosgene (CG), and stannic chloride.

Incapacitating agents or riot-control agents typically produce temporary physiological or mental effects, or both, such that individuals who are exposed to them are incapable of concerted effort. Upon their exposure, lachrymatory agents (or lachrymators) for example, irritate the eyes to cause tearing, pain, and even temporary blindness. The most common lachrymatory agents are tear gas and pepper spray and include, for example, a-Chlorotoluene, benzyl bromide, bromoacetone (BA), bromobenzylcyanide (CA) bromomethyl ethyl ketone, capsaicin (OC), chloracetophenone (Tear gas; CN), chloromethyl chloroformate, dibenzoxazepine (CR), ethyl iodoacetate, ortho-chlorobenzylidene malononitrile (Super tear gas; CS), trichloromethyl chloroformate, and xylyl bromide. Other incapacitating agents include, for example, 3-Quinuclidinyl benzilate (psychedelic; BZ), hydrocyanic acid (paralytic), diphenylchloroarsine (sternutatory; DA), diphenylcyanoarsine (DC), and KOLOKOL-I (tranquilizer).

Exemplary toxins include abrin, ricin, and saxitoxin.

In addition to TRPAl, other TRP channels have been implicated in pain reception and/or sensation. For example, certain TRPM channels including TRPM8 have been implicated in the reception and/or sensation of pain. Accordingly, in certain embodiments, the methods of the present invention include treating pain by administering (i) a combination of a selective TRPAl antagonist and a selective TRPM8 antagonist; (ii) a combination of a selective TRPAl antagonist, a selective TRPM8 antagonist, and one or more of a selective TRPVl and/or TRPV3 antagonist; (iii) a cross-TRP inhibitor that antagonizes a function of TRPAl and TRPM8; or (iv) a pan inhibitor that antagonizes a function of TRPAl, TRPM8, and one or more of TRPVl and TRP V3.

In certain embodiments, a compound of the invention is conjointly administered with one or more additional compounds that antagonize the function of a different channel. By way of example, a compound of the invention may be conjointly administered with one or more compounds that antagonize TRPVl, TRPM8, and/or TRPV3. The compound(s) that antagonize TRPVl, TPRM8, or TRPV3 may be selective for TRPVl, TRPM8 or TRPV3 (e.g., inhibit TRPVl or TRPV3 10, 100, or 1000 fold more strongly than TRPAl). Alternatively, the compound(s) that antagonize TRPVl or TRPV3 may cross react with other TRP channels.

Pharmaceutical Compositions

While it is possible for a compound disclosed herein to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation, where the compound is combined with one or more pharmaceutically acceptable excipients or carriers. The compounds disclosed herein may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting. Pharmaceutical preparations can be prepared in accordance with standard procedures and are administered at dosages that are selected to treat infection (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA and Goodman and Gilman's "The Pharmaceutical Basis of Therapeutics," Pergamon Press, New York, NY, the contents of which are incorporated herein by reference, for a general description of the methods for administering various antimicrobial agents for human therapy).

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

A pharmaceutical composition can comprise one or more compounds of Formula I, Formula II, Formula Ilia or Formula HIb in solid dosage forms (e.g., capsules, tablets, pills, dragees, powders, granules and the like) with one or more pharmaceutically acceptable excipients, liquid dosage forms (e.g., pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and the like), suspensions containing suspending agents (e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof), topical dosage forms (e.g., ointments, pastes, creams and gels), powders and sprays optionally containing customary propellants, and ophthalmic formulations (e.g., eye ointments, drops, solutions and the like). In addition to the active ingredient, the liquid dosage forms may contain inert diluents 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 benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

The pharmaceutical compositions may be formulations presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. 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 which 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.

The formulations disclosed herein can be delivered via a device. Exemplary devices include, but are not limited to, a catheter, wire, stent, inhaler, nebulizer, or other device. Further exemplary delivery devices also include a patch, bandage, mouthguard, or dental apparatus. Transdermal patches have the added advantage of providing controlled delivery of a compound disclosed herein 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 compound in a polymer matrix or gel.

The pharmaceutical compositions can be formulated for various routes of delivery, included oral or parenteral delivery (including intravenous, intramuscular, intraperetoneal, subcutaneous, intraocular, intrathecal, intra-articular, intra-synovial, cisternal, intrahepatic, intralesional and intracranial injection, infusion, injectable depot forms and/or inhaled routes of administration for the therapeutic treatment of TRPAl -mediated medical conditions). For example, the pharmaceutical compositions containing TRAPAl mediating compounds disclosed herein can be administered topically, orally, transdermally, rectally, vaginally, parentally, intranasally, intrapulmonary, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intracardiacly, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly, intraspinally, intrasternally or by inhalation.

When the compounds disclosed herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

One specific embodiment is an antitussive composition for peroral administration comprising an agent that inhibits both a TRPAl -mediated current with an IC₅₀ of 1 micromolar or less, and an orally- acceptable pharmaceutical carrier in the form of an aqueous-based liquid, or solid dissolvable in the mouth, selected from the group consisting of syrup, elixer, suspension, spray, lozenge, chewable lozenge, powder, and chewable tablet. Such antitussive compositions can include one or more additional agents for treating cough, allergy or asthma symptom selected from the group consisting of: antihistamines, 5- lipoxygenase inhibitors, leukotriene inhibitors, H3 inhibitors, β-adrenergic receptor agonists, xanthine derivatives, α-adrenergic receptor agonists, mast cell stabilizers, expectorants, NKl, NK2 and NK3 tachykinin receptor antagonists, and GABA_B agonists.

Still another embodiment is a metered dose aerosol dispenser containing an aerosol pharmaceutical composition for pulmonary or nasal delivery comprising an agent that inhibits a TRPAl -mediated current with an IC₅₀ of 1 micromolar or less. For instance, it can be a metered dose inhaler, a dry powder inhaler or an air-jet nebulizer.

Dosages

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 that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound disclosed herein 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 patient 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, 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, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day. For example, the dose can be 1-50, 1-25, or 5-10 mg/kg.

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.

Disease and Injury Models

Compounds that antagonize TRPAl function may be useful in the prophylaxis and treatment of any of the foregoing injuries, diseases, disorders, or conditions. In addition to in vitro assays of the activity of these compounds, their efficacy can be readily tested in one or more animal models. There are numerous animal models for studying pain. The various models use various agents or procedures to simulate pain resulting from injuries, diseases, or other conditions. Blackburn-Munro (2004) Trends in Pharmacological Sciences 25: 299-305 (see, for example, Tables 1, 3, or 4). Behavioral characteristics of challenged animals can then be observed. Compounds or procedures that may reduce pain in the animals can be readily tested by observing behavioral characteristics of challenged animals in the presence versus the absence of the test compound(s) or procedure.

Exemplary behavioral tests used to study chronic pain include tests of spontaneous pain, allodynia, and hyperalgesia. Id. To assess spontaneous pain, posture, gait, nocifensive signs (e.g., paw licking, excessive grooming, excessive exploratory behavior, guarding of the injured body part, and self-mutilation) can be observed. To measure evoked pain, behavioral responses can be examined following exposure to heat (e.g., thermal injury model).

Exemplary animal models of pain include, but are not limited to, the Chung model, the carageenan induced hyperalgesia model, the Freund's complete adjuvant induced hyperalgesia model, the thermal injury model, the formalin model and the Bennett Model. The Chung model of neuropathic pain (without inflammation) involves ligating one or more spinal nerves. Chung et al. (2004) Methods MoI Med 99: 35-45; Kim and Chung (1992) Pain 50: 355-363. Ligation of the spinal nerves results in a variety of behavioral changes in the animals including heat hyperalgesia, cold allodynia, and ongoing pain. Compounds that antagonize TRPAl can be administered to ligated animals to assess whether they diminish these ligation-induced behavioral changes in comparison to that observed in the absence of compound.

Carageenan induced hyperalgesia and Freund's complete adjuvant (FCA) induced hyperalgesia are models of inflammatory pain. Walker et al. (2003) Journal of Pharmacol Exp Ther 304: 56-62; McGaraughty et al. (2003) Br J Pharmacol 140: 1381-1388; Honore et al. (2005) J Pharmacol Exp Ther. Compounds that antagonize TRPAl can be administered to carrageenan or FCA challenged animals to assess whether they diminish thermal hyperalgesia in comparison to that observed in the absence of compound. In addition, the ability of compounds that antagonize TRPAl function to diminish cold and/or mechanical hypersensitivity can also be assessed in these models. Typically, the carrageenan induced hyperalgesia model is believed to mimic acute inflammatory pain and the CFA model is believed to mimic chronic pain and chronic inflammatory pain.

As outlined above, cancer pain may have any of a number of causes, and numerous animal models exist to examine cancer pain related to, for example, chemotherapeutics or tumor infiltration. Exemplary models of toxin-related cancer pain include the vincristine- induced peripheral neuropathy model, the taxol-induced peripheral neuropathy model, and the cisplatin-induced peripheral neuropathy model. Wang and Wang (2003). An exemplary model of cancer pain caused by tumor infiltration is the cancer invasion pain model (CIP). Id. In addition to any of the foregoing models of chronic pain, compounds that antagonize TRPAl function can be tested in one or more models of acute pain. Valenzano et al. (2005) Neuropharmacology 48: 658-672. Regardless of whether compounds are tested in models of chronic pain, acute pain, or both, these studies are typically (though not exclusively) conducted, for example, in mice, rats, or guinea pigs. Additionally, compounds can be tested in various cell lines that provide in vitro assays of pain. Wang and Wang (2003).

For testing the efficacy of TRPAl antagonists for the treatment of cough, experiments using the conscious guinea pig model of cough can be readily conducted. Tanaka and Maruyama (2003) Journal Pharmacol Sci 93: 465-470; McLeod et al. (2001) Br J Pharmacol 132: 1175-1178. Briefly, guinea pigs serve as a useful animal model for cough because, unlike other rodents such as mice and rats, guinea pigs actually cough. Furthermore, guinea pig coughing appears to mimic human coughing in terms of the posture, behavior, and appearance of the coughing animal.

To induce cough, conscious guinea pigs are exposed to an inducing agent such as citric acid or capsaicin. The response of the animal is measured by counting the number of coughs. The effectiveness of a cough suppressing agent, for example a compound that inhibits TRPAl, can be measured by administering the agent and assessing the ability of the agent to decrease the number of coughs elicited by exposure to citric acid, capsaicin, or other similar cough-inducing agent. In this way, TRPAl inhibitors for use in the treatment of cough can be readily evaluated and identified.

Additional models of cough include the unconscious guinea pig model. Rouget et al. (2004) Br J Pharmacol 141: 1077-1083. Either of the foregoing models can be adapted for use with other animals capable of coughing. Exemplary additional animals capable of coughing include cats and dogs.

Any of the foregoing animal models may be used to evaluate the efficacy of a TRPAl inhibitor in treating pain associated with pancreatitis. The efficacy can be compared to a no teatment or placebo control. Additionally or alternatively, efficacy can be evaluated in comparison to one or more known pain relieving medicaments.

The following examples are meant to be illustrative and are not meant to be limiting in any way. Examples Example 1: Preparation of 8-Substituted Theophylline Acetamide

8-chlorocaffeιne

To a suspension of 8-Chlorotheophylline (2.146 g, 10.0 mmol) in DMF (40 mL) was added potassium carbonate (2.07 g, 15.0 mmol) and iodomehtane (1.87 mL, 30.0 mmol). The resulting mixture was stirred at room temperature for 24 hours, diluted with water (80 mL), cooled to 0 ⁰C for 2 hours. The white solid was collected by filtration, washed with water (2 x 20 mL), EtOH (2 x 10 mL) and Et₂O (40 mL), dried in vacuo to give 8-chlorocaffeine as a white solid. (2.15 g, 94% yield) To a solution of diethyl malonate (0.455 mL, 3.0 mmol) in DMF (5.0 mL) at 0 ⁰C was added sodium hydride (112 mg, 60% suspension in mineral oil, 2.8 mmol) in one portion. The mixture was stirred at 0 ⁰C for 30 minutes; 8-chlorocaffeine (457 mg, 2.0 mmol) was then added. The resulting mixture was heated at 60 ⁰C for 2 hours and 90 ⁰C for 1 hour, cooled to room temperature, partitioned between water (50 mL) and Et₂O (50 mL). The aqueous layer was acidified to pH < 3 by adding 2.0 N aq. HCl. The white precipitate generated was collected, washed with water (5.0 mL), dissolved in CH₂Cl₂ (10 mL), dried over anhydrous sodium sulfate, decanted, concentrated to give A as a white solid. (117 mg, 17% yield)

Compound A (110 mg, 0.312 mmol) was suspended in 18% aq. HCl (5.0 mL). The mixture was heated to reflux for 1 hour to give a homogeneous solution, cooled to room temperature. The solvent was removed. The residue was triturated with acetone (3.0 mL), concentrated and dried in vacuo to give 7-methyltheophylline-8-acetic acid B as a white solid. (78 mg, quantitative yield). A RBF was charged with aminothiazole C (79 mg, 0.347 mmol), DMF (2.0 mL), CH₂Cl₂ (3.0 mL), DMAP (7 mg, 0.058 mmol) and 7-methyltheophylline-8-acetic acid B (73 mg, 0.289 mmol). The resulting mixture was stirred at room temperature for 15 minutes; EDC (111 mg, 0.578 mmol) was added in one portion. The reaction mixture was stirred at room temperature for 66 hours, concentrated in vacuo to remove solvents, the resulting residue was purified by silica gel using a mixture of MeOH in CH₂Cl₂ (0 to 2%, v/v) as the eluent. The compound D was obtained as a brown solid. (100 mg, 68% yield) Mass spectrum: 512 (M+l)⁺. H NMR spectroscopy (DMSO-d6): δ 1.10 (3H, d), 1.60 (IH, m), 1.80, (IH, m), 1.90 (IH, m), 2.10, (IH, m), 3.20 (4H, s), 3.40 (3H, s), 3.50 (IH, m), 3.90 (3H, s), 4.05 (IH, m), 4.20 (2H, s), 6.80 (IH, t), 7.40 (IH, s), 7.60 (2H, t), 12.60 (IH, s).CompoundD had a solubility in Ringer' s Solution of 0.011 mM. Compound D had an IC₅₀ of 81 nM. Example 2

To a suspension of 8 -Chloro theophylline (1.073 g, 5.0 mmol) in DMF (20 niL) was added potassium carbonate (1.03 g, 7.5 mmol) and p-methoxybenzyl bromide (1.08 mL, 7.5 mmol). The resulting mixture was stirred at room temperature for 96 hours, diluted with water (40 mL), cooled to 0 ⁰C. The white solid was collected by filtration, washed with water (2 x 10 mL), EtOH (5 mL) and Et₂O (5 mL), dried in vacuo to give compound £ as a white solid. (1.67 g, quantitative yield)

To a solution of diethyl malonate (0.759 mL, 5.0 mmol) in DMF (5.0 mL) at 0 ⁰C was added sodium hydride (160 mg, 60% suspension in mineral oil, 4.0 mmol) in one portion. The mixture was stirred at 0 ⁰C for 10 minutes, then room temperature for 20 minutes; Compound E (670 mg, 2.0 mmol) was then added. The resulting mixture was heated at 90 ⁰C for 1 hour and 100 ⁰C for 1 hour, cooled to room temperature, partitioned between water (50 mL) and Et₂O (50 mL), filtered. The aqueous layer was separated and acidified to pH < 3 by adding 2.0 N aq. HCl. The white precipitate generated was collected, washed with water (10 mL), dried in vacuo to give F as a white solid. (377 mg, 41% yield)

Compound F (196 mg, 0.427 mmol) was suspended in 18% aq. HCl (5.0 mL). The mixture was heated to reflux for 4 hour to give a homogeneous solution, cooled to room temperature. The solvent was removed. The residue was dissolved in acetone (5.0 mL), concentrated and dried in vacuo to give compound G as a yellow solid which was used in the next step without further purification.

A RBF was charged with aminothiazole C (126 mg, 0.555 mmol), DMF (2.0 mL), CH₂Cl₂ (4.0 mL), DMAP (10.4 mg, 0.0854 mmol) and compound G (about 0.427 mmol). The resulting mixture was stirred at room temperature for 15 minutes; EDC (164 mg, 0.854 mmol) was added in one portion. The reaction mixture was stirred at room temperature for 42 hours, concentrated in vacuo to remove solvents, the resulting residue was purified by silica gel using a mixture of MeOH in CH₂Cl₂ (0 to 2%, v/v) as the eluent. The compound H was obtained as a light yellow solid. (85 mg, 40% yield) Mass spectrum: 498 (M+l)⁺. ¹H NMR spectroscopy (DMS0-d6): δ 1.10 (3H, d), 1.60 (IH, m), 1.80, (IH, m), 1.90 (IH, m), 2.10, (IH, m), 3.20 (4H, s), 3.40 (3H, s), 3.50 (IH, q), 4.00 (3H, s), 4.05 (IH, m), 6.50 (IH, t), 7.50 (IH, s), 7.60 (2H, m), 12.60 (IH, s), 13,50 (IH, bs). Compound H had a solubility in Ringer's Solution of 0.009 niM. The IC₅₀ was 310 nM.

Example 3: Patch clamp experiments

Patch clamp experiments permit the detection of currents through the TRPAl channel in the cell line described above. A glass electrode is brought into contact with a single cell and a high-resistance (gigaohm) seal is established with the cell membrane. The membrane is then ruptured to achieve the whole-cell configuration, permitting control of the voltage of the cell membrane and measurement of currents flowing across the membrane using the amplifier attached to the electrode and resulting in the replacement of cytoplasm with the pipette solution.

TRPAl cells were induced 20-48 hours, removed from growth plates, and replated at low density (to attain good single-cell physical separation) on glass coverslips for measurement. In some cases, cells were grown in low density overnight on glass coverslips. Patch clamp recordings were made in the whole-cell mode with a holding potential of -40 mV. Every 5 seconds, a voltage ramp was applied from -120 to +100 mV, 400 ms in duration. Currents elicited were quantified at -80 mV and +80 mV. Upon addition of AITC, TRPAl current was induced only in TRPAl -expressing cells and not in parental HEK293 TREx cells. Removal of the AITC stimulus causes most of the current to go away. Potential blockers were tested for ability to block both inward and outward currents in the continued presence of AITC.

Except where indicated, the IC 50 values presented in Tables 1 and 2 were obtained from patch clamp experiments.

The table below summarizes the human TRPAl binding obtained according to Example 3 for the compounds of Examples 1 and 2.

Example 4: Testing of TRPAl Antagonists in a Thermal Injury Model of Pain The thermal injury model can be used to evaluate the effectiveness of an exemplary TRPAl inhibitor in the treatment of nociceptive pain using the following protocol. Male Holtzman rats (approximately 300 grams) are tested on thermal escape using a Hargreaves type apparatus. Under light anesthesia, a thermal injury (52 ⁰C for 45 seconds) is applied to one heel. The animals are tested for thermal escape latency of the injured and uninjured paw before and at 30, 60, 80, and 120 minutes after injury. Drug (a TRPAl inhibitor) or vehicle (0.5% methylcellulose) is administered after the baseline measurement and approximately 15-20 minutes prior to the thermal injury. In addition to the escape latency measurement, behavioral observations are made throughout the experiment.

Example 5: Testing of TRPAl Antagonists in the Chung Model of Neuropathic Pain

Briefly, male Sprague Dawley rats (approximately 175 grams) are prepared with ligation of the L4/5 nerve roots. After 5-8 days, the animals are tested for tactile allodynia using Von Frey hairs. Thresholds are assessed with the "up-down" method. Drug or vehicle is administered and the animals tested periodically over the next four hours.

Incorporation by Reference All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

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.

Claims

We claim:

1. A compound of Formula I, or a pharmaceutically acceptable salt thereof:

Formula I

wherein each of R¹ and R² is, independently, H or C_1-4 alkyl; each of R³ and R⁴ is, independently, H or C_1-6 alkyl, C₂_₆ alkenyl, or C₂_₆ alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or C_1-6 alkyl, C₂_6 alkenyl, or C₂_6 alkynyl; each R⁸ is, independently, halo, hydroxyl, C_1-6 alkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, C_1-6 alkoxy, C₂_₆ alkenoxy, Ci_₆ haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, or nitro; n is 0-3; R⁹ is halo, hydroxyl, C_1-6 alkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, C_1-6 alkoxy, C₂_₆ alkenoxy, C_1-6 haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or C ₁-₄ alkyl.

2. The compound of claim 1, wherein R¹ and R² are both methyl.

3. The compound of claim 1, wherein R³ is H and R⁴ is absent.

4. The compound of claim 1, wherein R³ is methyl and R⁴ is absent.

5. The compound of claim 1, wherein R⁵ and R⁶ are both H.

6. The compound of claim 1, wherein R 7 i •s H.

7. The compound of claim 1, wherein n is 1.

8. The compound of claim 1, wherein at least one R is halo.

9. The compound of claim 1, wherein R⁹ is methyl.

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula I, or a pharmaceutically acceptable salt thereof:

Formula I wherein each of R¹ and R² is, independently, H or Ci_₄ alkyl; each of R³ and R⁴ is, independently, H or C_1-6 alkyl, C₂_₆ alkenyl, or C₂_₆ alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or C_1-6 alkyl, C₂-6 alkenyl, or C₂-6 alkynyl; each R is, independently, halo, hydroxyl, Ci_₆ alkyl, C₂_₆ alkenyl, C₂_₆ alkynyl, Ci_₆ alkoxy, C₂_₆ alkenoxy, Ci_₆ haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, or nitro; n is 0-3;

R is halo, hydroxyl, C_1-6 alkyl, C₂_6 alkenyl, C₂_6 alkynyl, C_1-6 alkoxy, C₂_6 alkenoxy, C_1-6 hhaallooaallkkyyll,, --NN((RR¹¹⁰⁰))₂₂,, --CCOONN((RR¹¹⁰⁰))₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or C_1-4 alkyl.

11. A method for treating or alleviating a condition for which reduced TRPAl activity can reduce the severity, comprising administering to a subject in need an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof:

Formula I

wherein each of R¹ and R² is, independently, H or C_1-4 alkyl; each of R³ and R⁴ is, independently, H or C_1-6 alkyl, C₂_6 alkenyl, or C₂-6 alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or Ci_6 alkyl, C₂-6 alkenyl, or C₂-6 alkynyl; each R is, independently, halo, hydroxyl, C_1-6 alkyl, C₂-6 alkenyl, C₂-6 alkynyl, C_1-6 alkoxy, C₂-6 alkenoxy, C_1-6 haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, or nitro; n is 0-3;

R is halo, hydroxyl, C_1-6 alkyl, C₂-₆ alkenyl, C₂-₆ alkynyl, C_1-6 alkoxy, C₂-₆ alkenoxy, C_1-6 hhaallooaallkkyyll,, --NN((RR¹¹⁰⁰))₂₂,, --CCOONN((RR¹¹⁰⁰))₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or C ₁-₄ alkyl.

12. The method of claim 11, wherein the condition is chronic pain.

13. The method of claim 11, wherein the condition is acute pain.

14. The method of claim 11, wherein the condition is asthma.

15. The compound of claim 1, selected from a group consisting of the compound having a formula

, and the compound having a formula

16. The compound of claim 1, having an IC50 for TRPAl inhibition of less than 1 micromolar, as measured by a patch clamp assay.

17. Use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or alleviating a condition for which reduced TRPAl activity can reduce the severity:

Formula I wherein each of R¹ and R² is, independently, H or Ci_₄ alkyl; each of R³ and R⁴ is, independently, H or Ci_6 alkyl, C₂_6 alkenyl, or C2-6 alkynyl, wherein whichever of R³ and R⁴ is not doubly bonded to the adjacent carbon, is absent; each of R⁵ to R⁷ is, independently, H or Ci_6 alkyl, C2-6 alkenyl, or C2-6 alkynyl; each R⁸ is, independently, halo, hydroxyl, Ci_₆ alkyl, C₂-₆ alkenyl, C₂-₆ alkynyl, Ci_₆ alkoxy, C₂-6 alkenoxy, C_1-6 haloalkyl, -N(R¹⁰)₂, -CON(R¹⁰)₂, -CO₂H, cyano, or nitro; n is 0-3;

R is halo, hydroxyl, Ci_6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ci_6 alkoxy, C2-6 alkenoxy, Ci_6 hhaallooaallkkyyll,, --NN((RR¹¹⁰⁰))₂₂,, --CCOONN((RR¹¹⁰⁰))₂, -CO₂H, cyano, nitro, or is absent; and each R¹⁰ is, independently, H or Ci .₄ alkyl.