US008318728B2
(12) United States Patent (10) Patent No.: US 8,318,728 B2 Chong (45) Date of Patent: Nov. 27, 2012
(54) COMPOUNDS AND COMPOSITIONS FOR (56) References Cited TREATING CHEMICAL WARFARE AGENT INDUCED INURES U.S. PATENT DOCUMENTS 4,297,494 A 10/1981 Groman et al. (75) Inventor: Jayhong A. Chong, Brookline, MA (US) 4,871,742 A 10, 1989 Bonne et al. 4,960,773. A 10, 1990 KorbonitSet al. 5,068,236 A 11/1991 Suzuki et al. (73) Assignee: Hydra Biosciences, Inc., Cambridge, 6,545,002 B1 4/2003 Linden et al. MA (US) 6,608,069 B1 8/2003 Daluge et al. 7,135,475 B2 11/2006 Dunten et al. (*) Notice: Subject to any disclaimer, the term of this 2004.0053973 A1 3/2004 Ohkawa et al. 2006, O247226 A1 11/2006 Himmelsbach et al. patent is extended or adjusted under 35 2007/OO72851 A1 3/2007 Buchanan et al. U.S.C. 154(b) by 371 days. 2007/0219.222 A1 9, 2007 Moran et al. (21) Appl. No.: 12/466,187 FOREIGN PATENT DOCUMENTS WO 2007073505 A2 6, 2007 (22) Filed: May 14, 2009 OTHER PUBLICATIONS (65) Prior Publication Data Cassillas et al., J. Appl. Toxicol. 20, S145-S151 (2000).* US 2012/O108613 A1 May 3, 2012 International Search Report dated Jun. 14, 2010 for related applica tion PCT/US 10/35005. European Agency for the Evaluation of Medicinal Products. EMEA Related U.S. Application Data CPMP Guidance Document on the Use of Medicinal Products for the (60) Provisional application No. 61/127,666, filed on May Treatment of Patients Exposed to Terrorist Attacks with Chemical 14, 2008, provisional application No. 61/127,664, Agents. 2003, pp. 2-17.
Formula (II) R3 wherein, r 45 R" is C-C alkyl, C-C alkenyl, or C-C alkynyl, each of O iii. L which is optionally substituted with 1-4 R: X is N or CR R is H. C-C alkyl, C-C alkenyl, or C-C alkynyl, each of which is optionally substituted with 1-4 R: 50 L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) J-2 NR, NRC(O), C(O)NR, O, C(O), S, S(O), S(O), NR CH2 cyclyl, aryl, heterocyclyl, or heteroaryl; wherein, R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is R" is C-C alkyl, C-C alkenyl, or C-C alkynyl, each of optionally substituted with 1-4 R7: which is optionally substituted with 1-4 R: 55 each R is independently halo, hydroxyl, alkoxy, amino, alky X is N or CR lamino, dialkylamino, cyano, nitro, amido, alkylamido, R is H. C-C alkyl, C-C alkenyl, or C-C alkynyl, each of dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or which is optionally substituted with 1-4 R: heteroaryl; L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) each Risindependently H, C-C alkyl, arylalkyl, S(O)alkyl, NR, NRC(O), C(O)NR, C(O), O, S, S(O), S(O), NR 60 acetyl, amidyl, or S(O)H; CH, cyclyl, aryl, heterocyclyl, or heteroaryl; each R" is independently C-C alkyl, C-C alkenyl, or R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is C-C alkynyl, halo, hydroxyl, alkoxy, aryl, heteroaryl, optionally substituted with 1-4 R7: cyclyl, aryloxy, arylalkoxy, amino, alkylamino, dialky each R is independently halo, hydroxyl, alkoxy, amino, alky lamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, amido, lamino, dialkylamino, cyano, nitro, amido, alkylamido, 65 hydroxylalkoxyl, alkoxyalkoxyl, urea, Sulfonylurea, acyl, dialkylamido, thionyl, Sulfonyl, cyclyl, heterocyclyl, aryl, nitro, cyano, each of which is independently Substituted or heteroaryl; with 1-3 R: US 8,318,728 B2 3 4 each R is independently C-C alkyl, C-C alkenyl, or agent in a subject, Such as a human, the method comprising C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, administering to the Subject a compound of Formula (V) or a alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, salt thereof amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero cyclyl, aryl, or heteroaryl; each R is independently H, C-C alkyl, C-C alkenyl, or Formula (V) C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy,
amino, alkylamino, dialkylamino, thioyl, alkylthioyl, Sul fonyl, Sulfonamidyl, amido, urea, Sulfonylurea, acyl, nitro, cyano, each of which is independently substituted with 1-3 10 R; each m and n are independently 0, 1, 2, 3, 4, 5, or 6. In another aspect, the invention features a method of treat ing an injury resulting from exposure to a chemical warfare agent in a Subject, such as a human, the method comprising 15 administering to the Subject a compound of Formula (IV) or a salt thereof wherein, RandR are each independently C-C alkyl, C-C alkenyl, or C-C alkynyl, each of which is optionally substituted Formula (IV) with 1-4 R: L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) NR, NR°C(O), C(O)NR', O, C(O), S, S(O), S(O), NR CH2 cyclyl, aryl, heterocyclyl, or heteroaryl; R N 25 R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is NN M optionally substituted with 1-4 R7: each R is independently halo, hydroxyl, alkoxy, amino, alky Ousul N lamino, dialkylamino, cyano, nitro, amido, alkylamido, 9 dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or R 30 heteroaryl; each R is independently H, C1-C-6 alkyl, arylalkyl, S(O) wherein, alkyl, acetyl, amidyl, or S(O)H: R" and Rare eachindependently C-C alkyl, C-C alkenyl, each R" is independently C-C alkyl, C-C alkenyl, C-C, or C-C alkynyl, each of which is optionally substituted alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halo, with 1-4 R: 35 hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino, L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) dialkylamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, NR, NRC(O), C(O)NR, O, C(O), S, S(O), S(O), NR amido, urea, Sulfonylurea, hydroxyl alkoxyl, alkoxy CH2 cyclyl, aryl, heterocyclyl, or heteroaryl; alkoxyl, acyl, nitro, cyano, each of which is independently R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is substituted with 1-3 R: optionally substituted with 1-4 R7: 40 each R is independently C-C alkyl, C-C alkenyl, or each R is independently halo, hydroxyl, alkoxy, amino, alky C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, lamino, dialkylamino, cyano, nitro, amido, alkylamido, alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero heteroaryl; cyclyl, aryl, or heteroaryl; each Risindependently H, C-C alkyl, arylalkyl, S(O)alkyl, 45 R is H. C-C alkyl, or arylalkyl; acetyl, amidyl, or S(O)H; each m and n are independently 0, 1, 2, 3, 4, 5, or 6. each R" is independently C-C alkyl, C-C alkenyl, C-C, In another aspect, the invention features a method of treat alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halo, ing an injury resulting from exposure to a chemical warfare hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino, agent in a Subject, such as a human, the method comprising dialkylamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, 50 administering to the Subject a compound of Formula (VI) amido, urea, Sulfonylurea, hydroxyl alkoxyl, alkoxy or a salt thereof alkoxyl, acyl, nitro, cyano, each of which is independently substituted with 1-3 R: each R is independently C-C alkyl, C-C alkenyl, or Formula (VI) C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, 55 R3 alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, (? amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero L cyclyl, aryl, or heteroaryl; O (?? each m and n are independently 0, 1, 2, 3, 4, 5, or 6: N each R is independently H, C-C alkyl, C-C alkenyl, or 60 R1 X- R9 C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy, N N amino, alkylamino, dialkylamino, thioyl, alkylthioyl, Sul A fonyl, Sulfonamidyl, amido, urea, Sulfonylurea, acyl, nitro, R2 cyano, each of which is independently substituted with 1-3 R. 65 wherein, In another aspect, the invention features a method of treat R" is C-C alkyl, C-C alkenyl, C-C alkynyl, each of ing an injury resulting from exposure to a chemical warfare which is optionally substituted with 1-4 R: US 8,318,728 B2 5 6 R is C-C alkyl, C-C alkenyl, C-C alkynyl, or cyclyl, each R" is independently C-C alkyl, C-C alkenyl, or each of which is optionally substituted with 1-4 R: C-C alkynyl, halo, hydroxyl, alkoxy, aryl, heteroaryl, L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) cyclyl, aryloxy, arylalkoxy, amino, alkylamino, dialky NR, NRC(O), C(O)NR, O, C(O), S, S(O), S(O), NR lamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, amido, CH, cyclyl, aryl, heterocyclyl, or heteroaryl; hydroxylalkoxyl, alkoxyalkoxyl, urea, Sulfonylurea, acyl, R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is nitro, cyano, each of which is independently Substituted optionally substituted with 1-4 R: with 1-3 R: each Risindependently halo, hydroxyl, alkoxy, amino, alky each R is independently C-C alkyl, C-C alkenyl, or lamino, dialkylamino, cyano, nitro, amido, alkylamido, C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or 10 alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, heteroaryl; amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero each R is independently H, C1-C6 alkyl, arylalkyl, S(O) cyclyl, aryl, or heteroaryl; alkyl, acetyl, amidyl, or S(O)H; each m and n are independently 0, 1, 2, 3, 4, 5, or 6. each R" is independently C-C alkyl, C-C alkenyl, C-C, In another aspect, the invention features a method of treat alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halo, 15 ing an injury resulting from exposure to a chemical warfare hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino, agent in a subject, Such as a human, the method comprising dialkylamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, administering to the Subject a compound of Formula (IX) or amido, urea, Sulfonylurea, hydroxyl alkoxyl, alkoxy a salt thereof alkoxyl, acyl, nitro, cyano, each of which is independently substituted with 1-3 R: Formula (DX) each R is independently C-C alkyl, C-C alkenyl, or R3 C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, RI Lr amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero 25 ?? cyclyl, aryl, or heteroaryl; N21 N each R is independently H, C-C alkyl, C-C alkenyl, or C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy, 1. X- R9 amino, alkylamino, dialkylamino, thioyl, alkylthioyl, Sul O N 30 fonyl, Sulfonamidyl, amido, urea, Sulfonylurea, acyl, nitro, R2 cyano, each of which is independently substituted with 1-3 R8; each m and n are independently 0, 1, 2, 3, 4, 5, or 6. wherein, In another aspect, the invention features a method of treat R" and R are each independently H, C-C alkyl, C-C, 35 alkenyl, or C-C alkynyl, each of which is optionally Sub ing an injury resulting from exposure to a chemical warfare stituted with 1-4 R: agent in a Subject, such as a human, the method comprising L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) administering to the subject a compound of Formula (VII) or NR, NRC(O), C(O)NR, O, S, S(O), S(O)NR, or CH, a salt thereof R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is 40 optionally substituted with 1-4 R7: each R is independently halo, hydroxyl, alkoxy, amino, alky Formula (VII) lamino, dialkylamino, cyano, nitro, amido, alkylamido, dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or heteroaryl; 45 each Risindependently H, C-C alkyl, arylalkyl, S(O)alkyl, R n N acetyl, amidyl, or S(O)H; N Y each R" is independently C-C alkyl, C-C alkenyl, C-C, alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, halo, O1. N NM hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, alkylamino, 50 dialkylamino, thioyl, alkylthioyl, Sulfonyl, Sulfonamidyl, k amido, urea, Sulfonylurea, hydroxyl alkoxyl, alkoxy alkoxyl, acyl, nitro, cyano, each of which is independently wherein, substituted with 1-3 R: R" and Rare eachindependently C-C alkyl, C-C alkenyl, each R is independently C-C alkyl, C-C alkenyl, or or C-C alkynyl, each of which is optionally substituted 55 C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, with 1-4 R: alkylamino, dialkylamino, thioyl, Sulfonyl, Sulfonamidyl, L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC(O) amido, urea, Sulfonylurea acyl, nitro, cyano, cyclyl, hetero NR, NRC(O), C(O)NR, O, C(O), S, S(O), S(O), NR cyclyl, aryl, or heteroaryl; CH2 cyclyl, aryl, heterocyclyl, or heteroaryl; each R is independently H, C-C alkyl, C-C alkenyl, or R is cyclyl, heterocyclyl, aryl, heteroaryl, each of which is 60 C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, arylalkoxy, optionally substituted with 1-4 R7: amino, alkylamino, dialkylamino, thioyl, alkylthioyl, Sul each R is independently halo, hydroxyl, alkoxy, amino, alky fonyl, Sulfonamidyl, amido, urea, Sulfonylurea, acyl, nitro, lamino, dialkylamino, cyano, nitro, amido, alkylamido, cyano, each of which is independently substituted with 1-3 dialkylamido, thioyl, Sulfonyl, cyclyl, heterocyclyl, aryl, or R; heteroaryl; 65 m is 1, 2, 3, 4, 5, or 6. each Risindependently H, C-C alkyl, arylalkyl, S(O)alkyl, In another aspect, the invention features a method of treat acetyl, amidyl, or S(O)H; ing an injury resulting from exposure to a chemical warfare US 8,318,728 B2 7 8 agent in a Subject, such as a human, the method comprising chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-sub administering to the Subject a compound of Formula (X) or a stituted cycloalkyl groups, and cycloalkyl-substituted alkyl salt thereof 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 Formula (X) branched chains), and more preferably 20 or fewer, and most E preferably 10 or fewer. Likewise, preferred cycloalkyls have R Air from 3-10 carbon atoms in their ring structure, and more 21 preferably have 5, 6 or 7 carbons in the ring structure. 10 RN NR The term “alkynyl', as used herein, refers to an aliphatic group containing at least one triple bond. The term “alkylthio’ refers to a hydrocarbyl group having W a sulfur radical attached thereto. In some embodiments, the “alkylthio’ moiety is represented by one of S-alkyl, —S- wherein 15 alkenyl, or —S-alkynyl. Representative alkylthio groups W represents O or S; include methylthio, ethylthio, and the like. R, independently for each occurrence, represents H or lower The terms "amine” and “amino” refer to both unsubstituted alkyl: and Substituted amines, e.g., a moiety that can be represented R" represents substituted or unsubstituted alkyl or substituted by the general formula: or unsubstituted aryl; E represents carboxylic acid (COH), ester or amide; and Ar represents a Substituted or unsubstituted aryl ring. R10 R10 Exemplary compounds of Formula X are described in WO A . 2007/073505. -N or -N-Rio 25 V In one aspect, the invention features a method of treating a R9 R9 Subject who has been exposed to a chemical warfare agent, the method comprising administering to a Subject an effective amount of a compound of Formula (II), (III), (IV), (V), (VI), wherein R, Ro and R'o each independently represent a (VII), (IX) or (X), or a salt thereof. In some embodiments the hydrogen, an alkyl, an alkenyl, -(CH2), Rs, or Ro and Rio treatment reduces the severity of injury resulting from the 30 taken together with the Natom to which they are attached exposure to the chemical warfare agent. complete a heterocycle having from 4 to 8 atoms in the ring In some embodiments, the compound is administered structure: Rs represents an aryl, a cycloalkyl, a cycloalkenyl, orally, via intramuscular injection, by topical ocular admin an alkoxy, a heterocycle or a polycycle; and m is Zero or an istration, topically, or by inhalation. In some embodiments, integer in the range of 1 to 8. the Subject being treated is a human. 35 The term "amido” refers to a moiety that can be represented by the general formula: DETAILED DESCRIPTION OF THE INVENTION
Definitions O The term “acyl refers to a group represented by the gen 40 eral formula hydrocarbylC(O)—, preferably alkylC(O)—. The term “acylamino” refers to a moiety that can be rep - I . resented by the general formula: R10 45 wherein Ro, Ro 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 heteroaro matic group). 50 The term “aryl as used herein includes 5-, 6-, and 7-mem bered single-ring aromatic groups that may include from Zero wherein R is as defined below, and R'11 represents a to four heteroatoms, for example, benzene, pyrrole, furan, hydrogen, an alkyl, analkenyl or—(CH2)m-R8, where mand thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, R8 are as defined herein. pyridine, pyrazine, pyridazine and pyrimidine, and the like. The term “aliphatic group' refers to a straight-chain, 55 Those aryl groups having heteroatoms in the ring structure branched-chain, or cyclic aliphatic hydrocarbon group and may also be referred to as “arylheterocycles' or "heteroaro includes saturated and unsaturated aliphatic groups, such as matics. The aromatic ring can be substituted at one or more an alkyl group, an alkenyl group, and an alkynyl group. ring positions with Such substituents as described above, for The term “alkenyl, as used herein, refers to an aliphatic example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, group containing at least one double bond. 60 cycloalkyl, polycyclyl, hydroxyl, alkoxyl, amino, nitro, Sulf The terms “alkoxyl or “alkoxy” as used herein refers to an hydryl, imino, amido, phosphate, phosphonate, phosphinate, alkyl group, as defined below, having an oxygen radical carbonyl, carboxyl, silyl ether, alkylthio, Sulfonyl, Sulfona attached thereto. Representative alkoxyl groups include mido, ketone, aldehyde, ester, heterocyclyl, aromatic or het methoxy, ethoxy, propyloxy, tert-butoxy and the like. An eroaromatic moieties, —CF3, —CN, or the like. The term “ether is two hydrocarbons covalently linked by an oxygen. 65 “aryl also includes polycyclic ring systems having two or The term “alkyl refers to the radical of saturated aliphatic more cyclic rings in which two or more carbons are common groups, including straight-chain alkyl groups, branched to two adjoining rings (the rings are “fused rings’) wherein at US 8,318,728 B2 10 least one of the rings is aromatic, e.g., the other cyclic rings azine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or piperazine, morpholine, lactones, lactams such as azetidino heterocyclyls. nes and pyrrolidinones, Sultams, Sultones, and the like. The The term “carbocycle orcyclyl as used herein, refers to an heterocyclic ring can be substituted at one or more positions aromatic or non-aromatic ring in which each atom of the ring with Such substituents as described above, as for example, is carbon. halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, Sulfhydryl, imino, amido, phosphate, The term “carbonyl refers to moieties represented by the phosphonate, phosphinate, carbonyl, carboxyl, silyl ether, general formula: alkylthio. Sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an 10 aromatic or heteroaromatic moiety, —CF, —CN, or the like. O O The term "heterocyclylalkyl, as used herein, refers to an alkyl group Substituted with a heterocycle group. --x-R, O -x-I-R The term “hydrocarbyl, as used herein, refers to a group that is bonded through a carbonatom that does not have a =O 15 or =S Substituent, and typically has at least one carbon wherein X is a bond or represents an oxygen or a Sulfur, and hydrogen bond and a primarily carbon backbone, but may R represents a hydrogen, an alkyl, an alkenyl, -(CH) - optionally include heteroatoms. Thus, groups like methyl, Rs or a pharmaceutically acceptable counter-ion, R' repre ethoxyethyl 2-pyridyl, and trifluoromethyl are considered to sents a hydrogen, an alkyl, an alkenyl or —(CH), Rs. be hydrocarbyl for the purposes of this application, but sub where m and Rs are as defined above. Where X is an oxygen stituents such as acetyl (which has a =O substituent on the and R or R' is not hydrogen, the formula represents an linking carbon) and ethoxy (which is linked through oxygen, “ester'. Where X is an oxygen, and R is as defined above, not carbon) are not. Hydrocarbyl groups include, but are not the moiety is referred to herein as a carboxyl group, and limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, particularly when R is a hydrogen, the formula represents a alkenyl, alkynyl, and combinations thereof. “carboxylic acid”. Where X is an oxygen, and R' is hydro 25 As used herein, the term "nitro” means —NO2; the term gen, the formula represents a “formate'. In general, where the “halogen designates —F. —Cl, Br or —I; the term “sulf oxygen atom of the above formula is replaced by sulfur, the hydryl' means —SH; the term “hydroxyl means —OH; and formula represents a “thiocarbonyl group. Where X is a the term “sulfonyl means —SO2-. Sulfur and R or R' is not hydrogen, the formula represents The terms “polycyclyl or “polycyclic group' refer to two a “thioester.” Where X is a sulfur and R is hydrogen, the 30 or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalky formula represents a “thiocarboxylic acid.” Where X is a nyls, aryls and/or heterocyclyls) in which two or more car sulfur and R' is hydrogen, the formula represents a “thio bons are common to two adjoining rings, e.g., the rings are formate.” On the other hand, where X is a bond, and R is not “fused rings'. Rings that are joined through non-adjacent hydrogen, the above formula represents a “ketone' group. atoms are termed “bridged rings. Each of the rings of the Where X is a bond, and R is hydrogen, the above formula 35 polycycle can be substituted with such substituents as represents an 'aldehyde' group. described above, as for example, halogen, alkyl, aralkyl, alk The term “ester, as used herein, refers to a group —C(O) enyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, Sulfhydryl, OR wherein R represents a hydrocarbyl group. imino, amido, phosphate, phosphonate, phosphinate, carbo The terms “halo' and “halogen as used herein means nyl, carboxyl, silyl ether, alkylthio. Sulfonyl, ketone, alde halogen and includes chloro, fluoro, bromo, and iodo. 40 hyde, ester, a heterocyclyl, an aromatic or heteroaromatic The terms “hetaralkyl and "heteroaralkyl, as used herein, moiety, —CF3, —CN, or the like. refers to an alkyl group Substituted with a hetaryl group. Exemplary monocyclic rings include furan, thiophene, The terms "heterocyclyl or "heterocyclic group' refer to pyrrole, pyrroline, pyrrolidine, oxazole, thiazole, imidazole, 3- to 10-membered ring structures, more preferably 3- to imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, 7-membered rings, whose ring structures include one to four 45 isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, heteroatoms. Heterocycles can also be polycycles. The term piperidine, dioxane, morpholine, dithiane, thiomorpholine, "heterocyclyl or "heterocyclic group' includes "heteroaryl pyridazine, pyrimidine, pyrazine, piperazine, triazine, and and “saturated or partially saturated heterocyclyl structures. trithiane. The term "heteroaryl” refers to an aromatic 5-8 membered Exemplary bicyclic rings include indolizinyl, indolyl, monocyclic, 8-12 membered bicyclic, or 11-14 membered 50 isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, inda tricyclic ring system having 1-3 heteroatoms if monocyclic, Zolyl, benzimidazolyl, benthiazolyl, purinyl, quinolizinyl, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazoli said heteroatoms selected from O, N, or S (e.g., carbonatoms nyl, quinoxalinyl, naphthyridinyl, pteridinyl, indenyl, naph and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, thalenyl, aZulenyl, imidazopyridazionyl, pyrazolopyrim bicyclic, or tricyclic, respectively). Any ring atom can be 55 idinedionyl, or pyrrolopyrimidinedionyl moieties. Substituted (e.g., by one or more Substituents). The term Exemplary tricyclic rings include carbazole, acridine, “saturated or partially saturated heterocyclyl refers to a non phenazine, phenothiazine, phenoxazine, fluorine, and aromatic cyclic structure that includes at least one heteroa anthracene. tom. Heterocyclyl groups include, for example, thiophene, The phrase “protecting group’ as used herein means tem thianthrene, furan, pyran, isobenzofuran, chromene, Xan 60 porary Substituents which protect a potentially reactive func thene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiaz tional group from undesired chemical transformations. ole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, Examples of Such protecting groups include esters of car indolizine, isoindole, indole, indazole, purine, quinolizine, boxylic acids, silyl ethers of alcohols, and acetals and ketals isoquinoline, quinoline, phthalazine, naphthyridine, qui of aldehydes and ketones, respectively. The field of protecting noxaline, quinazoline, cinnoline, pteridine, carbazole, carbo 65 group chemistry has been reviewed (Greene, T. W.; Wuts, P. line, phenanthridine, acridine, pyrimidine, phenanthroline, G. M. Protective Groups in Organic Synthesis, 2" ed., Wiley: phenazine, phenarsazine, phenothiazine, furazan, phenox New York, 1991). US 8,318,728 B2 11 12 The term “substituted” refers to moieties having substitu The term “sulfonate' refers to a moiety that can be repre ents replacing a hydrogen on one or more carbons of the sented by the general formula: backbone. It will be understood that “substitution' or “sub stituted with includes the implicit proviso that such substi tution is in accordance with permitted valence of the substi O tuted atom and the substituent, and that the substitution -S-OR results in a stable compound, e.g., which does not spontane ously undergo transformation Such as by rearrangement, O cyclization, elimination, etc. As used herein, the term "sub stituted” is contemplated to include all permissible substitu 10 in which R41 is an electron pair, hydrogen, alkyl, ents of organic compounds. In a broad aspect, the permissible cycloalkyl, or aryl. Substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non The terms “sulfoxido’ or “sulfinyl, as used herein, refers aromatic Substituents of organic compounds. The permissible to a moiety that can be represented by the general formula substituents can be one or more and the same or different for 15 —S(=O)—R44, in which R44 is selected from the group appropriate organic compounds. For purposes of this inven consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, tion, the heteroatoms such as nitrogen may have hydrogen heterocyclyl, aralkyl, or aryl. Substituents and/or any permissible Substituents of organic The term “thioester, as used herein, refers to a group compounds described herein which satisfy the valences of the —C(O)SR or SC(O)R’ wherein R represents a hydrocar heteroatoms. Substituents can include any Substituents byl. described herein, for example, a halogen, a hydroxyl, a car As used herein, the definition of each expression, e.g., bonyl (Such as a carboxyl, an alkoxycarbonyl, a formyl, oran alkyl, m, n, etc., when it occurs more than once in any struc acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a ture, is intended to be independent of its definition elsewhere thioformate), an alkoxyl, a phosphoryl, a phosphate, a phos in the same structure. phonate, a phosphinate, an amino, an amido, an amidine, an 25 Exemplary monocyclic rings include furan, thiophene, imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a pyrrole, pyrroline, pyrrolodine, oxazole, thiazole, imidazole, Sulfate, a Sulfonate, a sulfamoyl, a Sulfonamido, a Sulfonyl, a imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, heterocyclyl, an aralkyl, or an aromatic or heteroaromatic isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, moiety. It will be understood by those skilled in the art that the piperidine, dioxane, morpholine, dithiane, thiomorpholine, moieties Substituted on the hydrocarbon chain can them 30 pyridazine, pyrimidine, pyrazine, piperazine, triazine, and selves be substituted, if appropriate. For instance, the sub trithiane. stituents of a substituted alkyl may include substituted and Exemplary bicyclic rings include indolizinyl, indolyl, unsubstituted forms of amino, azido, imino, amido, phospho isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, inda ryl (including phosphonate and phosphinate), Sulfonyl (in Zolyl, benzimidazolyl, benthiazolyl, purinyl, quinolizinyl, cluding Sulfate, Sulfonamido, Sulfamoyl and Sulfonate), and 35 silyl groups, as well as ethers, alkylthios, carbonyls (includ quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazoli ing ketones, aldehydes, carboxylates, and esters), —CF, nyl, quinoxalinyl, naphthyridinyl, pteridinyl, indenyl, naph —CN and the like. Exemplary substituted alkyls are thalenyl, aZulenyl, imidazopyridazionyl, pyrazolopyrim described below. Cycloalkyls can be further substituted with idinedionyl, or pyrrolopyrimidinedionyl moieties. alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl 40 Exemplary tricyclic rings include carbazole, acridine, Substituted alkyls, —CF, —CN, and the like. Analogous phenazine, phenothiazine, phenoxazine, fluorine, and Substitutions can be made to alkenyl and alkynyl groups to anthracene. produce, for example, aminoalkenyls, aminoalkynyls, ami The terms “antagonist' and “inhibitor are used inter doalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, changeably to refer to an agent that decreases or suppresses a thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or 45 biological activity. Such as to repress an activity of an ion alkynyls. channel, such as TRPA1. TRPA1 inhibitors include inhibitors The term “sulfate' refers to a moiety that can be repre having any combination of the structural and/or functional sented by the general formula: properties disclosed herein. An “effective amount of, e.g., a TRPA1 antagonist, with 50 respect to the subject methods of treatment, refers to an O amount of the antagonist in a preparation which, when applied as part of a desired dosage regimen brings about a -O-S-OR desired clinical or functional result. Without being bound by O theory, an effective amount of a TRPA1 antagonist for use in 55 the methods of the present invention, includes an amount of a TRPA1 antagonist effective to decrease one or more in vitro in which R41 is as defined herein. or in vivo functions of a TRPA1 channel. Exemplary func The term "sulfonamido” refers to a moiety that can be tions include, but are not limited to, membrane polarization represented by the general formula: (e.g., an antagonist may promote hyperpolarization of a cell), 60 ion flux, ion concentration in a cell, outward current, and O inward current. Compounds that antagonize TRPA1 function include compounds that antagonize an in vitro or in vivo N R - N| - S|| - K functional activity of TRPA1. When a particular functional Ro O activity is only readily observable in an in vitro assay, the 65 ability of a compound to inhibit TRPA1 function in that in vitro assay serves as a reasonable proxy for the activity of that in which R9 and R11 are as defined above. compound. In certain embodiments, an effective amount is an US 8,318,728 B2 13 14 amount sufficient to inhibit a TRPA1-mediated current and/or number of diagnoses of the infection in a treated population the amount sufficient to inhibit TRPA1 mediated ion flux. Versus an untreated control population, and/or delaying the The term “preventing,” when used in relation to a condi onset of symptoms of the infection in a treated population tion, Such as a local recurrence (e.g., pain), a disease Such as Versus an untreated control population. Prevention of pain cancer, a syndrome complex Such as heart failure or any other 5 includes, for example, reducing the magnitude of, or alterna medical condition, is well understood in the art, and includes tively delaying, pain sensations experienced by Subjects in a administration of a composition which reduces the frequency treated population versus an untreated control population. of or delays the onset of symptoms of a medical condition or The term “prodrug is intended to encompass compounds injury in a subject relative to a Subject which does not receive that, under physiological conditions, are converted into the the composition. Thus, prevention of cancer includes, for 10 therapeutically active agents of the present invention. A com example, reducing the number of detectable cancerous mon method for making a prodrug is to include selected growths in a population of patients receiving a prophylactic moieties that are hydrolyzed under physiological conditions treatment relative to an untreated control population, and/or to reveal the desired molecule. In other embodiments, the delaying the appearance of detectable cancerous growths in a prodrug is converted by an enzymatic activity in the host treated population versus an untreated control population, 15 animal. e.g., by a statistically and/or clinically significant amount. Exemplary compounds are provided in Table 1-7. Prevention of an infection includes, for example, reducing the Compounds of Formula II include those shown in Table 1. TABLE 1 N-(4-(3,4- dichlorophenyl)thiazol-2-yl)-2- (1-methyl-6-oxo-1H-purin 7(6H)-yl)acetamide Me - H - N ) is n N N C
ls Nl/ N
N-(4-(3,4- S Me dimethylphenyl)thiazol-2-yl)-2- O \ (1-methyl-6-oxo-1H-purin N N 7(6H)-yl)acetamidede Me H N n N X Me
ls Nl/ N
N-(5-(3,4-difluorophenyl)-3H F pyrrol-2-yl)-2-(1-methyl-6-oxo O \ 1H-purin-7(6H)-yl)acetamide
Me N\, n NN-1- X F 2-(1-methyl-6-oxo-1H-purin S 7(6H)-yl)-N-(4-p-tolylthiazol O \ 2-yl)acetamide
Me N\, n N X Me
ls N l/N
N-(4-(4-fluorophenyl)thiazol-2- S yl)-2-(1-methyl-6-oxo-1H O \ purin-7(6H)-yl)acetamide N-N Me n N H N F
US 8,318,728 B2 101 102 Certain compounds disclosed herein may exist in particu all physical forms are equivalent for the uses contemplated by lar geometric or stereoisomeric forms. The present invention the present invention and are intended to be within the scope contemplates all such compounds, including cis- and trans of the present invention. isomers, R- and S-enantiomers, diastereomers, (d)-isomers, As set out above, certain embodiments of the present com (1)-isomers, the racemic mixtures thereof, and other mixtures pounds may contain a basic functional group, Such as amino thereof, as falling within the scope of the invention. For or alkylamino, and are, thus, capable of forming pharmaceu example, if one chiral center is present in a molecule, the tically acceptable salts with pharmaceutically acceptable invention includes racemic mixtures, enantiomerically acids. The term “pharmaceutically acceptable salts' in this enriched mixtures, and Substantially enantiomerically pure respect, refers to the relatively non-toxic, inorganic and 10 organic acid addition salts of compounds disclosed herein. compounds. The composition can contain, e.g., more than These salts can be prepared in situ during the final isolation 50%, more than 60%, more than 70%, more than 80%, more and purification of the compounds of the invention, or by than 90%, more than 95%, or more than 99% of a single separately reactingapurified compound of the invention in its enantiomer. The “enantiomeric excess' or “% enantiomeric excess' of free base form with a Suitable organic or inorganic acid, and 15 isolating the salt thus formed. Representative salts include the a composition can be calculated using the equation shown hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, below. In the example shown below a composition contains nitrate, acetate, Valerate, oleate, palmitate, Stearate, laurate, 90% of one enantiomer, e.g., the Senantiomer, and 10% of the benzoate, lactate, phosphate, tosylate, citrate, maleate, fuma other enantiomer, i.e., the Renantiomer. rate. Succinate, tartrate, naphthylate, mesylate, glucohepto nate, lactobionate, and laurylsulphonate salts and the like. ee=(90–10)/100=80%. (See, for example, Berge et al. (1977) “Pharmaceutical Salts', Thus, a composition containing 90% of one enantiomer and J. Pharm. Sci. 66:1-19.) 10% of the other enantiomer is said to have an enantiomeric In other cases, the compounds disclosed herein may con excess of 80%. tain one or more acidic functional groups and, thus, are 25 capable of forming pharmaceutically acceptable salts with Methods of preparing Substantially isomerically pure com pharmaceutically acceptable bases. The term “pharmaceuti pounds are known in the art. If, for instance, a particular cally acceptable salts' in these instances refers to the rela enantiomer of a compound disclosed herein is desired, it may tively non-toxic, inorganic and organic base addition salts of be prepared by asymmetric synthesis, or by derivation with a compounds disclosed herein. These salts can likewise be chiral auxiliary, where the resulting diastereomeric mixture is 30 prepared in situ during the final isolation and purification of separated and the auxiliary group cleaved to provide the pure the compounds, or by separately reacting the purified com desired enantiomers. Alternatively, where the molecule con pound in its free acid form with a suitable base, such as the tains a basic functional group, such as amino, or an acidic hydroxide, carbonate or bicarbonate of a pharmaceutically functional group, such as carboxyl, diastereomeric salts may acceptable metal cation, with ammonia, or with a pharmaceu be formed with an appropriate optically active acid or base, 35 tically acceptable organic primary, secondary or tertiary followed by resolution of the diastereomers thus formed by amine. Representative alkalioralkaline earth salts include the fractional crystallization or chromatographic means well lithium, Sodium, potassium, calcium, magnesium, and alumi known in the art, and Subsequent recovery of the pure enan num salts and the like. Representative organic amines useful tiomers. Alternatively, enantiomerically enriched mixtures for the formation of base addition salts include ethylamine, and pure enantiomeric compounds can be prepared by using 40 diethylamine, ethylenediamine, ethanolamine, diethanola synthetic intermediates that are enantiomerically pure in mine, piperazine and the like. (See, for example, Berge et al., combination with reactions that either leave the stereochem Supra). istry at a chiral center unchanged or result in its complete An antagonist of TRPA1 function may inhibit the outward inversion. Techniques for inverting or leaving unchanged a current, the inward current, or both currents. Compounds that particular stereocenter, and those for resolving mixtures of 45 inhibit both currents may do so with the same or with differ stereoisomers are well known in the art, and it is well within ing ICso values. The inhibition of a particular current is mea the ability of one of skill in the art to choose an appropriate sured by the ability to inhibit or reduce such current (e.g., method for a particular situation. See, generally, Furniss etal. inward and/or outward) in an in vitro or an in Vivo assay. (eds.), Vogel's Encyclopedia of Practical Organic Chemistry Compounds that inhibit any of the foregoing currents in an in 5" Ed., Longman Scientific and Technical Ltd., Essex, 1991, 50 vitro or in vivo assay are characterized as compounds that pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990). inhibit a function of TRPA1. Additionally or alternatively, a The compounds described herein may also contain unnatu further exemplary function of TRPA1 that may be inhibited ral proportions of atomic isotopes at one or more of the atoms by the present compounds is ion flux mediated by TRPA1. that constitute such compounds. For example, the compounds A compound described herein (e.g., a TRPA1 antagonist) may be radiolabeled with radioactive isotopes, such as for 55 can be chosen for a use described herein may be chosen example tritium (H), iodine-125 ('I) or carbon-14 (''C). because it inhibits a TRPA1 function with an ICs less than or All isotopic variations of the compounds disclosed herein, equal to 10 uM, 5uM, 1 uM, or less than or equal to 700, 600, whether radioactive or not, are intended to be encompassed 500, 400, 300, 250, 200, or 100 nM. In other embodiments, within the scope of the present invention. For example, deu the compound described herein inhibits a TRPA1 function terated compounds and compounds incorporated 'C are 60 with an ICso less than or equal to 75 nM, less than or equal to intended to be encompassed within the scope of the invention. 50 nM, or less than or equal to 25, 10, 5, or 1 nM. Certain compounds disclosed herein can exist in unsol In certain embodiments, inhibition of TRPA1 function vated forms as well as Solvated forms, including hydrated means that a function, for example a TRPA1 mediated cur forms. In general, the Solvated forms are equivalent to unsol rent, is decreased by greater than 50% in the presence of an vated forms and are encompassed within the Scope of the 65 effective amount of a compound in comparison to in the present invention. Certain compounds disclosed herein may absence of the compound or in comparison to an ineffective exist in multiple crystalline or amorphous forms. In general, amount of a compound. In certain other embodiments, the US 8,318,728 B2 103 104 inhibition of a TRPA1 function means that a function, for The ICso values are measured in vitro using, for example, example a TRPA1 mediated current or TRPA1 mediated ion patch clamp analysis or standard measurements of calcium flux, is decreased by at least 50%, 60%, 70%, 75%, 80%, flux. Exemplary in vitro methods for calcium flux-based ICso 85%, or 90% in the presence of an effective amount of a estimation are described in Example 2. Methods used to compound in comparison to in the absence of the compound. obtain more definitive ICso measurements are described in In still other embodiments, the inhibition of a TRPA1 func Example 3. Alternatively, estimates of% inhibition of current tion means that a function, for example a TRPA1 mediated or ion flux can also be calculated and used to assess efficacy current, is decreased by at least 92%. 95%, 97%, 98%, 99%, of a compound as an inhibitor. or 100% in the presence of an effective amount of a com Indications pound in comparison to in the absence of the compound. 10 Some embodiments, a compound described herein (e.g., a Movement of ions across cellular membranes is carried out TRPA1 antagonist) can be characterized by some level of by specialized proteins. TRP channels are one large family of activity versus other ion channels (e.g., certain compounds non-selective cation channels that function to help regulate are selective for inhibiting TRPA1 and other compounds 15 ion flux and membrane potential. Non-selective cation chan exhibit a level of cross reactivity againstone or more otherion nels such as TRPA1 modulate the flux of calcium and sodium channel). When a compound described herein is character ions across cellular membranes. Sodium and calcium influx ized by its activity against another ion channel, inhibition of leads to depolarization of the cell. This increases the prob a function or activity of the other ion channel is defined ability that voltage-gated ion channels will reach the thresh analogously to the way in which a function of a TRPA1 old required for activation. As a result, activation of non channel is defined. Thus, inhibiting the function of another ion channel means, for example, inhibiting ion flux mediated selective cation channels can increase electrical excitability by that other ion channel or inhibiting the current mediated by and increase the frequency of voltage-dependent events. Volt that other ion channel. age-dependent events include, but are not limited to, neuronal In some embodiments, a compound described herein (e.g., 25 action potentials, cardiac action potentials, Smooth muscle a TRPA1 antagonist) is chosen for use because it is more contraction, cardiac muscle contraction, and skeletal muscle selective for one TRP isoform than others, e.g., 10-fold, and contraction. more preferably at least 20, 40, 50, 60, 70, 80, or at least 100 or 1000-fold more selective for TRPA1 over one or more of Calcium influx caused by the activation of non-selective TRPC6, TRPV5, TRPV6, TRPM8, TRPV1, TRPV2, 30 cation channels such as TRPA1 also alters the intracellular TRPV4, and/or TRPV3. In other embodiments, the differen free calcium concentration. Calcium is a ubiquitous second tial is smaller, e.g., it more strongly inhibits TRPA1 than messenger molecule within the cell, so alterations in intrac TRPM8, TRPV1, TRPV2, TRPV3, and/or TRPV4, prefer ellular calcium levels have profound effects on signal trans ably at least twice, three times, five times, or ten times more duction and gene expression. As a result, activation of non strongly. Such comparisons may be made, for example, by 35 comparing ICso values. selective cation channels such as TRPA1 can lead to changes In other embodiments, a compound described herein (e.g., in gene expression and cellular phenotype. Gene expression a TRPA1 antagonist) is chosen for use because it is more events include, but are not limited to, production of mRNAs selective for TRPA1 than for other non-TRP ion channels, encoding cell Surface receptors, ion channels, and kinases. e.g., 10-fold, and more preferably at least 20, 40, 50, 60, 70. 40 These changes in gene expression can lead to hyperexcitabil 80, or at least 100- or 1000-fold more selective for TRPA1 ity in that cell. over one or more of NaV1.2, Cav1.2, Cav3.1, HERG, and/or mitochondrial uniporter. In other embodiments, the differen Modulating the function of TRPA1 proteins provides a tial is smaller, e.g., it more strongly inhibits TRPA1 than means of modulating calcium homeostasis, Sodium homeo NaV1.2, Cav1.2, Cav3.1, HERG, and/or mitochondrial uni 45 Stasis, membrane polarization, and/or intracellular calcium porter, preferably at least twice, three times, five times, or ten levels, and compounds that can modulate TRPA1 function are times more strongly. useful in many aspects, including, but not limited to, main In certain embodiments, the compound described herein taining calcium homeostasis, modulating intracellular cal (e.g., a TRPA1 antagonist) inhibits TRPA1 with an ICs at cium levels, modulating membrane polarization, and treating least one order of magnitude more potent than its Ki for the 50 or preventing diseases, disorders, or conditions associated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic with calcium and/or sodium homeostasis or dyshomeostasis. acid (AMPA) receptor. In certain other embodiments, the Bautista et al. have reported that TRPA1 mediates the compound described herein inhibits TRPA1 with an ICs at inflammatory actions of agents such as acrolein, which causes least two orders of magnitude, three orders of magnitude, or the inflammatory and toxic actions of teargas. (Bautista et al., four orders of magnitude more potent than its Ki for the 55 AMPA receptor. In certain embodiments, the compound Cell (2006) 124:1269-82.) Bessac et al. have shown that described herein does not appreciably bind the AMPA recep hypochlorite—the main mediator of chlorine irritancy—ac tor. In other words, the compound described herein inhibits tivates Ca" influx and membrane currents in sensory neurons TRPA1 with a particular ICs and, when administered at that cultured from Trpal" mice, while the influx of Ca" was concentration, the antagonist does not appreciably bind the 60 absent in neurons cultured from Trpal mice. In addition, AMPA receptor. Bessac et al. compared the responses of Trpal" mice and In certain embodiment, a compound is chosen because it Trpal mice to NaOCl exposure. The Trpal" mice showed antagonizes the function of TRPA1 and the function of much more respiratory depression than did the Trpal mice. TRPM8, TRPV1 and/or TRPV3. Although such compounds (Bessac et al., Journal of Clinical Investigation (2008) selectively antagonize the function of more than one ion 65 18:1899-1910) These data suggest that TRPA1 antagonists channel, the ICso values for the different ion channels need could be useful to treat or prevent the effects of chemical not be identical. warfare agents including tear gas and chlorine. US 8,318,728 B2 105 106 Chemical Warfare Agents thylamine (HN2), and tris(2-chloroethyl)amine (HN3). Sul A subject may be exposed to a chemical warfare agent, e.g., fur mustards include, for example, 1,2-Bis(2-chloroeth by inhalation or by contact with the skin. If a compound ylthio)ethane (Sesquimustard; Q), 1,3-Bis(2- described herein (e.g., a TRPA1 antagonist) is administered, chloroethylthio)-n-propane, 1,4-Bis(2-chloroethylthio)-n- the symptoms or injuries resulting from the exposure to the butane, 1.5-Bis(2-chloroethylthio)-n-pentane, chemical warfare agents can be reduced, prevented, or both. 2-Chloroethylchloromethylsulfide, bis(2-chloroethyl) sul The compound described herein can be administered to a fide (Mustard gas; HD), bis(2-chloroethylthio) methane, bis Subject before, during, or following Such exposure and is (2-chloroethylthiomethyl)ether, and bis(2-chloroethylthioet therefore administered within 24 hours, 18 hours, 6 hours, 4 hyl)ether (0 Mustard). hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 10 Pulmonary agents. A pulmonary agent (or choking agent) minutes, 5 minutes, one minute, or thirty seconds before or is a chemical weapon agent designed to impede a subjects after such exposure. The TRPA1 anta compound described ability to breathe, resulting in Suffocation. Exemplary agents herein gonist can be administered prophylactically, when include adamsite (DM), acrolein, bis(chloromethyl)ether exposure to an agent is anticipated. It can also be adminis (BCME), chlorine (Cl), chloropicrin (PS), diphosgene (DP), tered after exposure to the chemical warfare agent (e.g., 15 methyl chlorosulfonate, phosgene (CG), and Stannic chlo before or after symptoms of injury present in a Subject). ride. Injuries resulting from the exposure to chemical warfare Incapacitating agents. Incapacitating agents or riot-control agents are known in the art and include any physical injuries, agents typically produce temporary physiological or mental Such as injuries to the skin, eyes, respiratory tract, musculo effects, or both, such that individuals who are exposed to them skeletal system, circulatory system, gastrointestinal tract, are incapable of concerted effort. Upon their exposure, lach central nervous system, peripheral nervous system, heart, rymatory agents (or lachrymators) for example, irritate the liver, lungs, and kidneys. Exemplary symptoms or injuries eyes to cause tearing, pain, and even temporary blindness. resulting from the exposure to chemical warfare agents The most common lachrymatory agents are tear gas and pep include inflammation, burn, itch, pain, rash, blisters, Sweat per spray and include, for example, a-Chlorotoluene, benzyl ing, muscle twitching, nausea, vomiting, diarrhea, weakness, 25 bromide, bromoacetone (BA), bromobenzylcyanide (CA) loss of consciousness, convulsions, muscular twitching, bromomethyl ethyl ketone, capsaicin (OC), chloracetophe paralysis, secretions (from the mouth, nose, or lung for none (Teargas; CN), chloromethyl chloroformate, dibenzox example), difficulty breathing, blurred vision, eye pain, lac azepine (CR), ethyl iodoacetate, ortho-chlorobenzylidene rimation, red eyes, shortness of breath, coughing, phlegm malononitrile (Super tear gas; CS), trichloromethyl chloro production and narrowing of the airways, headaches, tremors, 30 formate, and Xylyl bromide. Other incapacitating agents dizziness, numbness or tingling, anxiety, insomnia, depres include, for example, 3-Quinuclidinyl benzilate (psyche sion, emotional instability, and even death. The term "chemi delic: BZ), hydrocyanic acid (paralytic), diphenylchloroars cal warfare agent' includes all of those agents classified as ine (sternutatory; DA), diphenylcyanoarsine (DC), and schedule 1, 2, and 3 agents under the Chemical Weapons KOLOKOL-1 (tranquilizer). Convention of 1993 and may be in liquid form, gas form, solid 35 Toxins. Exemplary toxins are abrin, ricin, and saxitoxin. form, or combinations thereof. Exemplary agents are Pharmaceutical Compositions described in further detail below and include, for example, While it is possible for a compound disclosed herein to be nerve agents, blood agents, blister agents, pulmonary agents, administered alone, it is preferable to administer the com incapacitating agents, and toxins. Other agents include pound as a pharmaceutical formulation, where the compound methyl isocyanate, hexamethylene diisocyanate, 2,4-toluene 40 is combined with one or more pharmaceutically acceptable diisocyanate, and diphenylmethane-4,4-diisocyanate (MDI). excipients or carriers. The compounds disclosed herein may Nerve agents. Nerve agent poisoning typically leads to be formulated for administration in any convenient way for contraction of pupils, profuse salivation, convulsions, invol use in human or veterinary medicine. In certain embodi untary urination and defecation, and eventual death by ments, the compound included in the pharmaceutical prepa asphyxiation as control is lost over respiratory muscles. These 45 ration may be active itself, or may be a prodrug, e.g., capable symptoms are reduced or prevented by the administration of of being converted to an active compound in a physiological the TRPA1 antagonists. Exemplary agents include G agents Setting. Such as tabun (GA), Sarin (GB). Soman (GD), cyclosarin The phrase “pharmaceutically acceptable' is employed (GF), and GV; V agents such as VE, VG, VM, VX, and herein to refer to those compounds, materials, compositions, Novichok agents. 50 and/or dosage forms which are, within the scope of Sound Blood agents. A blood agent (or cyanogen agent) is a com medical judgment, Suitable for use in contact with the tissues pound containing a cyanide group that prevents the body from of human beings and animals without excessive toxicity, irri utilizing oxygen. These agents exert their toxic effect at the tation, allergic response, or other problem or complication, cellular level by directly interrupting cellular respiration. commensurate with a reasonable benefit/risk ratio. Exemplary agents include cyanogen chloride, hydrogen cya 55 Examples of pharmaceutically acceptable carriers include: nide, and hydrogen sulfide. (1) Sugars, such as lactose, glucose and Sucrose; (2) starches, Blister agents. Blister agents or vesicants typically cause Such as corn starch and potato starch; (3) cellulose, and its severe skin, eye and mucosal pain and irritation. These agents derivatives. Such as Sodium carboxymethyl cellulose, ethyl also have the ability to cause large, painful water blisters. cellulose and cellulose acetate; (4) powdered tragacanth; (5) Blister agents include, for example, lewisites, nitrogen mus 60 malt, (6) gelatin; (7) talc.; (8) excipients, such as cocoa butter tard, Sulfur mustard, ethyldichloroarsine (a lewisite analog: and Suppository waxes; (9) oils, such as peanut oil, cottonseed ED), methyldichloroarsine (MD), phenyldichloroarsine oil, safflower oil, Sesame oil, olive oil, corn oil and Soybean (PD), and phosgene oxime (CX). Lewisites include, for oil: (10) glycols, such as propylene glycol; (11) polyols. Such example, 2-Chlorovinyldichloroarsine (Lewisite 1), Bis(2- as glycerin, Sorbitol, mannitol and polyethylene glycol; (12) chlorovinyl)chloroarsine (Lewisite 2), and Tris(2-chlorovi 65 esters, such as ethyl oleate and ethyl laurate; (13) agar, (14) nyl)arsine (Lewisite 3). Exemplary nitrogen mustards are buffering agents, such as magnesium hydroxide and alumi bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)me num hydroxide: (15) alginic acid, (16) pyrogen-free water; US 8,318,728 B2 107 108 (17) isotonic saline: (18) Ringer's solution; (19) ethyl alco to produce a single dosage form will generally be that amount hol; (20) phosphate buffer solutions; (21) cyclodextrins such of the compound which produces a therapeutic effect. Gen as Captisol(R); and (22) other non-toxic compatible Substances erally, out of one hundred percent, this amount will range employed in pharmaceutical formulations. from about 1 percent to about ninety-nine percent of active Examples of pharmaceutically acceptable antioxidants ingredient, preferably from about 5 percent to about 70 per include: (1) water Soluble antioxidants, such as ascorbic acid, cent, most preferably from about 10 percent to about 30 cysteine hydrochloride, Sodium bisulfate, sodium met percent. abisulfite, sodium sulfite and the like; (2) oil-soluble antioxi The tablets, and other solid dosage forms of the pharma dants, such as ascorbyl palmitate, butylated hydroxyanisole ceutical compositions disclosed herein, such as dragees, cap (BHA), butylated hydroxytoluene (BHT), lecithin, propyl 10 gallate, alpha-tocopherol, and the like; and (3) metal chelat Sules, pills and granules, may optionally be scored or pre ing agents, such as citric acid, ethylenediamine tetraacetic pared with coatings and shells, such as enteric coatings and acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the other coatings well known in the pharmaceutical-formulating like. art. They may also be formulated so as to provide slow or Solid dosage forms (e.g., capsules, tablets, pills, dragees, 15 controlled release of the active ingredient therein using, for powders, granules and the like) can include one or more example, hydroxypropylmethyl cellulose in varying propor pharmaceutically acceptable carriers, such as Sodium citrate tions to provide the desired release profile, other polymer or dicalcium phosphate, and/or any of the following: (1) matrices, liposomes and/or microspheres. They may be ster fillers or extenders, such as starches, lactose, Sucrose, glu ilized by, for example, filtration through a bacteria-retaining cose, mannitol, and/or silicic acid; (2) binders, such as, for filter, or by incorporating sterilizing agents in the form of example, carboxymethylcellulose, alginates, gelatin, polyvi sterile solid compositions that can be dissolved in sterile nyl pyrrolidone. Sucrose and/or acacia; (3) humectants, such water, or some other sterile injectable medium immediately as glycerol; (4) disintegrating agents. Such as agar-agar, cal before use. These compositions may also optionally contain cium carbonate, potato or tapioca starch, alginic acid, certain opacifying agents and may be of a composition that they silicates, and Sodium carbonate; (5) Solution retarding agents, 25 release the active ingredient(s) only, or preferentially, in a Such as paraffin; (6) absorption accelerators, such as quater certain portion of the gastrointestinal tract, optionally, in a nary ammonium compounds; (7) wetting agents, such as, for delayed manner. Examples of embedding compositions that example, cetyl alcohol and glycerol monostearate; (8) absor can be used include polymeric Substances and waxes. The bents, such as kaolin and bentonite clay; (9) lubricants, such active ingredient can also be in micro-encapsulated form, if a talc, calcium Stearate, magnesium Stearate, Solid polyethyl 30 appropriate, with one or more of the above-described excipi ene glycols, sodium lauryl Sulfate, and mixtures thereof, and entS. (10) coloring agents. Dosage forms for the topical or transdermal administration Liquid dosage forms can include pharmaceutically accept of a compound of this invention include powders, sprays, able emulsions, microemulsions, Solutions, Suspensions, Syr ointments, pastes, creams, lotions, gels, Solutions, patches ups and elixirs. In addition to the active ingredient, the liquid 35 and inhalants. The active compound may be mixed under dosage forms may contain inert diluents commonly used in sterile conditions with a pharmaceutically acceptable carrier, the art, such as, for example, water or other solvents, solubi and with any preservatives, buffers, or propellants that may be lizing agents and emulsifiers. Such as ethyl alcohol, isopropyl required. alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl The formulations disclosed herein can be delivered via a benzoate, propylene glycol, 1,3-butylene glycol, oils (in par 40 device. Exemplary devices include, but are not limited to, a ticular, cottonseed, groundnut, corn, germ, olive, castor and catheter, wire, stent, or other intraluminal device. Further sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene exemplary delivery devices also include a patch, bandage, glycols and fatty acid esters of sorbitan, and mixtures thereof. mouthguard, or dental apparatus. Transdermal patches have Suspensions, in addition to the active compounds, may the added advantage of providing controlled delivery of a contain Suspending agents as, for example, ethoxylated isos 45 compound disclosed herein to the body. Such dosage forms tearyl alcohols, polyoxyethylene Sorbitol and Sorbitan esters, can be made by dissolving or dispersing the compound in the microcrystalline cellulose, aluminum metahydroxide, bento proper medium. Absorption enhancers can also be used to nite, agar-agar and tragacanth, and mixtures thereof. increase the flux of the compound across the skin. The rate of Ointments, pastes, creams and gels may contain, in addi such flux can be controlled by either providing a rate control tion to an active compound, excipients, such as animal and 50 ling membrane or dispersing the compound in a polymer Vegetable fats, oils, waxes, paraffins, starch, tragacanth, cel matrix or gel. lulose derivatives, polyethylene glycols, silicones, bento Another example of a device is a metered dose aerosol nites, silicic acid, talc and Zinc oxide, or mixtures thereof. dispenser containing an aerosol pharmaceutical composition Powders and sprays can contain, in addition to an active for pulmonary or nasal delivery comprising an agent that compound, excipients such as lactose, talc, silicic acid, alu 55 inhibits a TRPA1-mediated current with an ICs of 1 micro minum hydroxide, calcium silicates and polyamide powder, molar or less. For instance, it can be a metered dose inhaler, a or mixtures of these Substances. Sprays can additionally con dry powder inhaler or an air-jet nebulizer. tain customary propellants, such as chlorofluorohydrocar Ophthalmic formulations, eye ointments, drops, solutions bons and volatile unsubstituted hydrocarbons, such as butane and the like, are also contemplated as being within the scope and propane. 60 of this invention. The formulations may conveniently be presented in unit In some cases, in order to prolong the effect of a drug, it is dosage form and may be prepared by any methods well desirable to slow the absorption of the drug from subcutane known in the art of pharmacy. The amount of active ingredient ous or intramuscular injection. This may be accomplished by which can be combined with a carrier material to produce a the use of a liquid Suspension of crystalline or amorphous single dosage form will vary depending upon the host being 65 material having poor water solubility. The rate of absorption treated, the particular mode of administration. The amount of of the drug then depends upon its rate of dissolution, which, in active ingredient that can be combined with a carrier material turn, may depend upon crystal size and crystalline form. US 8,318,728 B2 109 110 Alternatively, delayed absorption of a parenterally adminis In certain other embodiments, a compound of the invention tered drug form is accomplished by dissolving or Suspending is conjointly administered with one or more additional agents the drug in an oil vehicle. or therapeutic regimens appropriate for the particular injury Injectable depot forms are made by forming microencap being treated. For example, current treatments for injuries Sule matrices of the Subject compounds in biodegradable caused by exposure to nerve agents include treatment with polymers such as polylactide-polyglycolide. Depending on atropine and Pralidoxime chloride (2-PAM). Injuries caused the ratio of drug to polymer, and the nature of the particular by exposure to cyanides are currently treated with. Scaven polymer employed, the rate of drug release can be controlled. gers such as human plasma-derived butyrylcholinesterase Examples of other biodegradable polymers include poly (HuBuOhE), anti-epileptic drugs and neutralizing decon (orthoesters) and poly(anhydrides). Depot injectable formu 10 tamination Solutions such as 0.5% hypochloriate are also used lations are also prepared by entrapping the drug in liposomes to treat such injuries. Treatments for cyanide poisoning or microemulsions that are compatible with body tissue. include hydroxocobalamin, Sodium nitrite, and rhodanase. When the compounds disclosed herein are administered as M291 resinkits are also currently used to treat topical injuries pharmaceuticals, to humans and animals, they can be given resulting from exposure to chemical warfare agents. Any of per se or as a pharmaceutical composition containing, for 15 these agents can be combined with the TRPA1 antagonists example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active described herein. ingredient in combination with a pharmaceutically accept Dosages able carrier. Actual dosage levels of the active ingredients in the phar The formulations can be administered topically, orally, maceutical compositions of this invention may be varied so as transdermally, rectally, vaginally, parentally, intranasally, to obtain an amount of the active ingredient that is effective to intrapulmonary, intraocularly, intravenously, intramuscu achieve the desired therapeutic response for a particular larly, intraarterially, intrathecally, intracapsularly, intraorbit patient, composition, and mode of administration, without ally, intracardiacly, intradermally, intraperitoneally, transtra being toxic to the patient. cheally, Subcutaneously, Subcuticularly, intraarticularly, The selected dosage level will depend upon a variety of Subcapsularly, Subarachnoidly, intraspinally, intrasternally or 25 factors including the activity of the particular compound dis by inhalation. closed herein employed, or the ester, salt oramide thereof, the Particularly useful modes of administration include topical route of administration, the time of administration, the rate of administration, intramuscular injection, inhalation, topical excretion of the particular compound being employed, the ocular administration (e.g., via eye drops), or oral adminis duration of the treatment, other drugs, compounds and/or tration. 30 materials used in combination with the particular compound Combination Therapy employed, the age, sex, weight, condition, general health and Another aspect of the invention provides a conjoint therapy prior medical history of the patient being treated, and like wherein one or more other therapeutic agents are adminis factors well known in the medical arts. tered with a compound described herein (e.g., a TRPA1 A physician or veterinarian having ordinary skill in the art modulator Such as an antagonist). Such conjoint treatment 35 can readily determine and prescribe the effective amount of may be achieved by way of the simultaneous, sequential, or the pharmaceutical composition required. For example, the separate dosing of the individual components of the treat physician or veterinarian could start doses of the compounds ment. of the invention employed in the pharmaceutical composition In certain embodiments, two or more compounds of the at levels lower than that required in order to achieve the invention are conjointly administered. When two or more 40 desired therapeutic effect and gradually increase the dosage compounds of the invention are conjointly administered, the until the desired effect is achieved. two or more compounds may have a similar selectivity profile In general, a Suitable daily dose of a compound of the and functional activity, or the two or more compounds may invention will be that amount of the compound that is the have a different selectivity profile and functional activity. By lowest dose effective to produce atherapeutic effect. Such an way of example, the two or more compounds may both be 45 effective dose will generally depend upon the factors approximately 10, 100, or 1000 fold selective for antagoniz described above. Generally, intravenous, intracerebroven ing a function of TRPA1 over TRPV1, TRPV5, and TRPV6 tricular and Subcutaneous doses of the compounds of this (e.g., the two or more compounds have a similar selectivity invention for a patient will range from about 0.0001 to about profile), and further may inhibit a function of TRPA1 with a 100 mg per kilogram of body weight per day. For example, the similar ICs (e.g., a similar functional activity). Alternatively, 50 dose can be 1-50, 1-25, or 5-10 mg/kg. the one of the two or more compounds may selectively inhibit Disease and Injury Models TRPA1 while the other of the two or more compounds inhib Compounds that antagonize TRPA1 function may be use its both TRPA1 and TRPV1 (e.g., the two or more compounds ful in the prophylaxis and treatment of any of the foregoing have differing selectivity profiles). Administration of combi injuries, diseases, disorders, or conditions. In addition to in nations of two or more compounds of the invention having 55 vitro assays of the activity of these compounds, their efficacy similar or differing properties are contemplated. can be readily tested in one or more animal models. Com In certain embodiments, a compound of the invention is pounds that may reduce pain or other undesirable symptoms conjointly administered with one or more additional com in the animals can be readily tested by observing behavioral pounds that antagonize the function of a different channel. By and/or physical characteristics of challenged animals in the way of example, a compound of the invention may be con 60 presence versus the absence of the test compound(s) or pro jointly administered with one or more compounds that cedure. antagonize TRPV1, TRPM8, and/or TRPV3. The compound Mouse Ear Edema Model. The mouse ear edema model (s) that antagonize TRPV1, TPRM8, or TRPV3 may be selec provides a quantitative edema response as well as histopatho tive for TRPV1, TRPM8 or TRPV3 (e.g., inhibit TRPV1 or logical and biochemical endpoints as measurements of TRPV3 10, 100, or 1000 fold more strongly than TRPA1). 65 inflammation and tissue damage following exposure to the Alternatively, the compound(s) that antagonize TRPV1 or chemical warfare agent half mustard. The model is performed TRPV3 may cross react with other TRP channels. using CD-1 mice. Twenty-four hours following IP adminis US 8,318,728 B2 111 112 tration of test compound or vehicle, a solution of half mustard Fura-2 experiments, or at 485 nM for Fluo4 experiments. in organic solvent is applied to the inner Surface of the right Frames were acquired at a rate of 0.2 Hz. For the screening ear of each mouse. The left ear is not treated with half mustard assay, a diluted stock (at 50 uM) of compounds to be tested and instead is maintained as a control. Animals are sacrificed was added to each well for 2 minutes following the collection at 24 hours, 72 hours, and 7 days post exposure to half mus of a short (4 frame) baseline. AITC (allylisothiocyanate) was tard. The ear weights, draize scoring, and pathology are then added to each well, achieving a final concentration of 10 recorded at each time point. Half mustard injury in the mouse uMeach compound and 7.5 uM AITC. Data were collected ear is measured by both edema response (fluid accumulation) for at least 3 minutes following addition of AITC, and evalu and histopathological damage (necrosis, epidermal-dermal ated for the Ca", which is proportional to the fluorescent separation). 10 intensity (for Fluo4) or the F340/F380 ratio (for Fura-2). In an alternate model, the test compound is administered therapeutically, e.g., immediately after, or one hour after Negative controls consisted of HEK293/TREXTRPA1 cells exposure to half mustard. The models can also be performed exposed to AITC, but no compound. Positive control cells using Sulfur mustard or nitrogen mustard instead of half mus were usually HEK293/TREX (“parental') cells exposed to tard. 15 AITC but no compound, but sometimes normal HEK/293 Guinea Pig Model for Nerve Agent Challenge. A Guinea TREXTRPA1 cells were also used, but not exposed to AITC pig model can be used to study the protective effects of the or compound. These controls defined a screening window, compounds against mortality induced by a nerve agent. The and “hits” were defined as those compounds inhibiting the animals are pretreated with two IP doses of test compound, fluorescence response by at least 40%. ICs values were one IM dose of atropine, one IM dose of 2-PAM, or two IP determined for compounds defined as “hits.” The Fluo4 cell doses of vehicle before challenge with 2xLD50 (s.c.) of a based fluorescence assay was used to determine the intracel nerve agent. The animals are observed at 6 observation times lular Ca" concentration in the presence of varying drug con post challenge. The number of animals in each group Surviv centration. To determine ICso values, concentrations tested ing 24 hours post challenge is then recorded. were 40 uM, 20 uM, 10 uM, 5 uM, 2.5 uM, 1.25 uM, and In an alternate model, the test compound, atropine, and 25 0.625 uM. Compounds were tested in triplicate at all concen 2-PAM are administered therapeutically, e.g., immediately trations. Standard software was used to fit ICso curves. after, or one hour after exposure to the nerve agent. Additionally or alternatively, potency can be represented The following examples are meant to be illustrative and are as % inhibition of a response in the presence (of a given not meant to be limiting in any way. concentration of compound) versus the absence of compound 30 or in comparison to a control compound. For example, effi Example 1 cacy can be represented as % inhibition of ion flux in the presence versus the absence of compound. High Throughput Screening Assay Example 2 The assay depended on detection of the rise in intracellular 35 Ca" concentration (Cal.) following channel activation in Patch Clamp Experiments cells inducibly expressing the TRPA1 channel Ca" rise was quantified with the use of fluorescent Ca" indicators that Patch clamp experiments permit the detection of currents were loaded into cells and thereafter indicated the Ca". through the TRPA1 channel in the cell line described above. Ca" influx followed activation of the TRPA1 channel. Com 40 The whole-cell configuration of the patch clamp technique pounds inhibiting the Ca"), rise were considered hits for was used to test the compounds described herein. In normal further investigation. whole-cell patch clamp recordings, a glass electrode is The commercially available HEK293/TREx line (Invitro brought into contact with a single cell and a high-resistance gen) was stably transfected with a TRPA1 construct (specifi (gigaohm) seal is established with the cell membrane. The cally a construct encoding a TRPA1 protein with an amino 45 membrane is then ruptured to achieve the whole-cell configu acid sequence depicted in SEQ ID NO: 1 of WO 2007/ ration, permitting control of the Voltage of the cell membrane 073505) and screened by conventional calcium imaging to and measurement of currents flowing across the membrane find clones with TRPA1 expression following stimulation using the amplifier attached to the electrode and resulting in with 1 lug/ml tetracycline. These cells were maintained in the the replacement of cytoplasm with the pipette Solution. growth medium recommended by the manufacturer Supple 50 HEK293/TREXTRPA1 cells were induced in the presence mented with 100 g/ml hygromycin to promote retention of of 1 lug/ml tetracycline for 20–48 hours, removed from growth the TRPA1 construct. After growing to near confluency, cells plates, and replated at low density (to attain good single-cell were plated at a density of ~25,000 cells/well in 384 well physical separation) on glass coverslips for measurement. In CellBind plates (Corning) in the presence of 1 Jug/ml tetracy Some cases, cells were grown in low density overnight on cline, and allowed to grow for 20-30 hrs. A nearly confluent 55 glass coverslips. Potential blockers were tested for ability to monolayer resulted. Cells were then loaded with Ca' dye: block current in the continued presence of AITC. Fura-2/AM or Fluo4/AM was added to the wells to a final Incorporation. By Reference concentration of 2 uM or 1 uM, respectively, and incubated All publications and patents mentioned herein are hereby for ~60 min at room temperature. Supernatant was then incorporated by reference in their entirety as if each indi removed from the cells by inverting plates with a sharp flick, 60 vidual publication or patent was specifically and individually and Hank's Balanced Salt Solution (HBSS; 0.185 g/l D-glu indicated to be incorporated by reference. cose, 0.9767 g/l MgSO (anhydrous), 0.4 g/1 KC1, 0.06 g/1 Equivalents KHPO (anhydrous), 0.35 g/l NaHCO, 8.0 g/l NaCl, and Those skilled in the art will recognize, or be able to ascer 0.04788 g/l NaHPO (anhydrous); pH 7.4) was then added to tain using no more than routine experimentation, many each well. Following recovery from loading, cells were 65 equivalents to the specific embodiments of the invention assayed using the Hamamatsu FDSS 6000 system, which described herein. Such equivalents are intended to be encom permitted illumination alternately at 340 nM and 380 nM for passed by the following claims. US 8,318,728 B2 113 114 I claim: each R is independently H, C-C alkyl, arylalkyl, S(O) 1. A method of treating an injury to the skin or respiratory alkyl, acetyl, amidyl, or S(O)H; tract on a subject in need thereof resulting from exposure to a each R is independently C-C alkyl, C-C alkenyl, chemical warfare agent, the method comprising administer C-C alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, ing to a subject an effective amount of a compound of For halo, hydroxyl, alkoxy, aryloxy, arylalkoxy, amino, aky mula (IX), or a salt thereof lamino, dialkylamino, thioyl, alkylthioyl, Sulfonyl, Sul fonamidyl, amido, urea, Sulfonylurea, hydroxylalkoxyl, alkoxyalkoxyl, acyl, nitro, cyano, each of which is inde Formula (IX) pendently substituted with 1-3 R: 10 each R is independently C-C alkyl, C-C alkenyl, or r C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, amino, akylamino, dialkylamino, thioyl, Sulfonyl, Sulfona R1 r midyl, amido, urea, Sulfonylurea, acyl, nitro, cyano, N21 N cyclyl, heterocyclyl, aryl, or heteroaryl; 15 each R is independently H, C-C alkyl, C-C alkenyl, or 1. X-R C-C alkynyl, halo, hydroxyl, alkoxy, aryloxy, aryla O N N lkoxy, amino, akylamino, dialkylamino, thioyl, alkylth k ioyl, Sulfonyl, Sulfonamidyl, amido, urea, Sulfonylurea, acyl, nitro, cyano, each of which is independently Sub wherein, stituted with 1-3 R: R" and Rare each independently H, C-C alkyl, C-C, m is 1, 2, 3, 4, 5, or 6 and alkenyl, or C-C alkynyl, each of which is optionally n is 0, 1 or 2. substituted with 1-4 R: 2. The method of claim 1, wherein the compound is admin L is NRSO, SONR, OC(O)NR, NRC(O)O, NRC istered after exposure to the chemical warfare agent. (O)NR, NR°C(O), C(O)NR', O, S, S(O), S(O), NR, 25 3. The method of claim 1, wherein the subject is a human. or CH, 4. The method of claim 1, wherein the injury is inflamma R is cyclyl, heterocyclyl, aryl, or heteroaryl, each of which tion, burn, itch, pain, rash, blisters, Sweating, secretions from is optionally substituted with 1-4R': the mouth, nose, or lung, difficulty breathing, shortness of each R is independently halo, hydroxyl, alkoxy, amino, breath, coughing, phlegm production and narrowing of the alkylamino, dialkylamino, cyano, nitro, amido, alkyla 30 airways. mido, dialkylamido, thioyl, Sulfonyl, cyclyl, heterocy clyl, aryl, or heteroaryl;