COMPOSITIONS OF 1,2,4-OXADIAZOLE BENZOIC ACID COMPOUNDS AND METHODS FOR THEIR USE

Abstract

Novel 1,2,4-oxadiazole benzoic acid compounds, methods of using and pharmaceutical compositions comprising an 1,2,4-oxadiazole benzoic acid derivative are disclosed. The methods include methods of treating or preventing a disease ameliorated by modulation of premature translation termination or nonsense-mediated mRNA decay, or ameliorating one or more symptoms associated therewith.

Claims

1-20. (canceled)

21. A method for treating retinitis pigmentosa associated with a premature stop codon in a patient having retinitis pigmentosa associated with a premature stop codon, comprising administering to the patient a therapeutically effective amount of a compound having the formula: ##STR00210## or a pharmaceutically acceptable salt thereof, wherein: Z is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heterocycle, substituted or unsubstituted arylalkyl; R.sup.1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, (CH.sub.2CH.sub.2O).sub.nR.sup.6 or any biohydrolyzable group; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen, CF.sub.3, OCF.sub.3, OCHF.sub.2, CN, COOH, COOR.sup.7, SO.sub.2R.sup.7, NO.sub.2, NH.sub.2, or N(R.sup.7).sub.2; each occurrence of R.sup.7 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen or CF.sub.3; and n is an integer from 1 to 7.

22. The method of claim 21, wherein the compound has the formula: ##STR00211## or a pharmaceutically acceptable salt thereof, wherein: Z is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heterocycle, substituted or unsubstituted arylalkyl; and R is hydrogen or halogen.

23. The method of claim 21, wherein the compound has the formula: ##STR00212## or a pharmaceutically acceptable salt thereof, wherein: Z is substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted arylalkyl; R.sup.1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, or (CH.sub.2CH.sub.2O).sub.nR.sup.6; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, alkoxy, aryloxy, halogen, CF.sub.3, OCF.sub.3, OCHF.sub.2, CN, COOH, COOR.sup.7, SO.sub.2R.sup.7, NO.sub.2, NH.sub.2, or N(R.sup.7).sub.2; each occurrence of R.sup.7 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, alkoxy, aryloxy, halogen or CF.sub.3; and n is an integer from 1 to 7.

24. The method of claim 21, wherein the compound has the formula: ##STR00213## or a pharmaceutically acceptable salt thereof, wherein: Z is substituted aryl, substituted or unsubstituted cycloalkyl, substituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted arylalkyl; R.sup.1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, (CH.sub.2CH.sub.2O).sub.nR.sup.6 or any biohydrolyzable group; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, alkoxy, aryloxy, halogen, CF.sub.3, OCF.sub.3, OCHF.sub.2, CN, COOH, COOR.sup.7, SO.sub.2R.sup.7, NO.sub.2, NH.sub.2, or N(R.sup.7).sub.2; each occurrence of R.sup.7 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, alkoxy, aryloxy, halogen or CF.sub.3; and n is an integer from 1 to 7.

25. The method of claim 21, wherein the compound has the formula: ##STR00214## or a pharmaceutically acceptable salt thereof, wherein: X is halogen, substituted alkyl, alkoxy or hydroxy; R.sup.1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, (CH.sub.2CH.sub.2O).sub.nR.sup.6 or any biohydrolyzable group; R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen, CF.sub.3, OCF.sub.3, OCHF.sub.2, CN, COOH, COOR.sup.7, SO.sub.2R.sup.7, NO.sub.2, NH.sub.2, or N(R.sup.7).sub.2; each occurrence of R.sup.7 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, alkoxy, aryloxy, heteroaryloxy, halogen or CF.sub.3; and n is an integer from 1 to 7.

26. The method of claim 21, wherein the compound is 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or a pharmaceutically acceptable salt thereof.

27. The method of claim 26, wherein the premature stop codon is caused by a mutation comprising a point mutation, a missense mutation, a frameshift mutation, an insertion mutation, a deletion mutation, a transition mutation or a transversion mutation.

28. The method of claim 26, wherein the patient has undergone a screening process to determine the presence of the premature stop codon.

29. The method of claim 26, wherein the patient is a human.

30. The method of claim 26, wherein the compound is administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, or orally.

31. The method of claim 26, wherein the therapeutically effective amount is from about 1 mg to about 2000 mg per day.

32. The method of claim 21, wherein the compound is 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.

33. The method of claim 32, wherein the premature stop codon is caused by a mutation comprising a point mutation, a missense mutation, a frameshift mutation, an insertion mutation, a deletion mutation, a transition mutation or a transversion mutation.

34. The method of claim 32, wherein the patient has undergone a screening process to determine the presence of a premature stop codon.

35. The method of claim 32, wherein the patient is a human.

36. The method of claim 32, wherein the compound is administered parenterally, transdermally, mucosally, nasally, buccally, sublingually, or orally.

37. The method of claim 36, wherein the compound is administered orally in a tablet, liquid or capsule form.

38. The method of claim 32, wherein the therapeutically effective amount is from about 1 mg to about 2000 mg per day.

Description

5. EXAMPLES

[0290] The following examples employ methodology which can be used to prepare all of the compounds embodied in this invention, provided the appropriate reagents and substrates are utilized, and minor variations of the described conditions are maintained. Such variations would be easily performed by one of skill in the art without undue experimentation given the description below.

5.1 Example 1: Preparation of 3-[5-(4-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid

[0291] ##STR00103##

[0292] 40 g of 2-chlorotrityl chloride resin (Rapp polymere, Germany), was suspended in dry dimethylformamide (200 mL) for 10 min and the solvent was drained. To the resin was added a solution of 3-cyanobenzoic acid (12.71 g, 96.4 mmol) in 300 mL of dimethylformamide and agitated 4 h at room temperature. The solvents were drained and the resin was washed with dichloromethane (3200 mL1 min), dimethylformamide (3200 mL1 min), methanol (3200 mL1 min), and dichloromethane (3200 mL1 min). The resin was vacuum dried for 4 h. The desired product was analyzed by cleavage of a small amount of the reacted resin with triethyl silane/trifluoroacetic acid/dichloromethane(10/50/40). LC/MS (ESI) m/z 148 [M+H].sup.+ and 97% purity.

[0293] The 3-cyanobenzoic trityl resin in ethanol (300 mL) was agitated for 10 min at room temperature, and then the solvent was drained. To a solution of hydroxy amine hydrochloride (35.81 g, 516 mmol) in ethanol (200 mL) was added diisopropylethylamine (89.3 mL, 516 mmol) and stirred 5 min at room temperature. To the resin was added the reaction mixture and agitated 24 h at 40 C. The solvents were drained, and the resin was washed with dichloromethane (3200 mL10 min), dimethylformamide (3200 mL10 min), methanol (3200 mL10 min), and dichloromethane (3200 mL10 min). The resin was vacuum dried for 4 h. The desired product was analyzed by cleavage of a small amount of the reacted resin with triethylsilane/trifluoroacetic acid/dichloromethane(10/50/40). LC/MS (ESI) m/z 181 [M+H].sup.+ and 90% purity.

[0294] To a suspension of hydoxyamidine resin (500 mg, 0.4 mmol) in anhydrous dichloromethane (3 mL) was added 4-Fluorobenzoyl chloride (95 uL, 0.8 mmol) and diisopropylethylamine (138 uL, 0.8 mmol). The reaction mixture was agitated overnight at room temperature. The solvents were drained, and the resin was washed with dichloromethane (310 mL10 min), dimethylformamide (310 mL10 min), methanol (310 mL10 min), and dichloromethane (310 mL10 min). The resin was vacuum dried for 4 h. The desired product was analyzed by cleavage of a small amount of the reacted resin with triethyl silane/trifluoroacetic acid/dichloromethane(10/50/40). LC/MS (ESI) m/z 303 [M+H].sup.+ and 65% purity.

[0295] To a suspension of acylated resin in anhydrous dichloromethane (1.5 mL) was added 50% trifluoroacetic acid in dichloromethane (1.5 mL). The reaction mixture was agitated 2 h at room temperature. The resin was removed and the filtrate was concentrated under reduced pressure. The residue was dissolved in 10% dimethylformaide in toluene (4 mL) and then stirred for 2 h at 130 C. The solvents were removed and the desired product was purified by preparative LC/MS. LC/MS (ESI) m/z 285 [M+H].sup.+ and 98% purity.

[0296] The following compounds are prepared using the procedures described above. Compounds are analyzed by a LC/MS using Electrospray ionization (ESI).

TABLE-US-00003 TABLE 2 Compound Compound Name [M + H].sup.+ [00104] 3-[5-(3-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 301.7 [00105] 3-[5-(4-Pentyloxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 353.4 [00106] 3-(5-Naphthalen-1-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 317.3 [00107] 3-(5-p-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3 [00108] 3-(5-Biphenyl-4-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 343.3 [00109] 3-[5-(4-Isobutyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 323.4 [00110] 3-(5-Phenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 267.3 [00111] 3-(5-Cyclohexyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 273.3 [00112] 3-[5-(3,4,5-Trimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 357.3 [00113] 3-[5-(4-Nitro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 312.2 [00114] 3-[5-(4-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3 [00115] 3-[5-(o-tolyl)-[1,2,4]oxadiazol-3- yl]-benzoic acid 281.3 [00116] 3-(5-Benzo[1,3]dioxol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 311.3 [00117] 3-(5-Isopropyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 233.2 [00118] 3-[5-(3,5-Bis-trifluoromethyl- phenyl)-[1,2,4]oxadiazol-3-yl]- benzoic acid 403.2 [00119] 3-[5-(4-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 335.2 [00120] 3-[5-(4-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 310.3 [00121] 3-[5-(2-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3 [00122] 3-[5-(3-Methoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 297.3 [00123] 3-(5-Furan-2-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 257.2 [00124] 3-(5-tert-Butyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 247.3 [00125] 3-(5-Benzo[1,2,5]oxadiazol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 309.2 [00126] 3-[5-(4-Chloromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 315.7 [00127] 3-[5-(4-tert-Butyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 323.4 [00128] 3-(5-Butyl-[1,2,4]oxadiazol-3-yl)- benzoic acid 247.3 [00129] 3-(5-Cyclopropyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 231.2 [00130] 3-(5-Thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 273.3 [00131] 3-(5-Propenyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 231.2 [00132] 3-(5-Cyclopentyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 259.3 [00133] 3-(5-Thiophen-2-ylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 287.3 [00134] 3-[5-(4-Chloro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 315.7 [00135] 3-[5-(4-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 331.7 [00136] 3-[5-(2-Trifluoromethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 335.3 [00137] 3-[5-(2,6-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00138] 3-[5-(4-Ethyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 296.3 [00139] 3-[5-(3,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 304.2 [00140] 3-(5-m-Tolyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3 [00141] 3-[5-(4-Pyrrol-1-yl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 332.0 [00142] 3-(5-Benzyl-[1,2,4]oxadiazol-3- yl)-benzoic acid 281.3 [00143] 3-(5-Methoxymethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 235.2 [00144] 3-[5-(2,5-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00145] 3-[5-(1-Phenyl-propyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 309.3 [00146] 3-[5-(2-Chloro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 301.7 [00147] 3-[5-(3-Trifluoromethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 351.2 [00148] 3-[5-(4-Fluoro-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 299.3 [00149] 3-[5-(2,5-Dimethyl-furan-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.3 [00150] 3-[5-(3-Methyl-thiophen-2-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 287.3 [00151] 3-[5-(3-Chloro-phenoxymethyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 331.7 [00152] 3-(5-Isoxazol-5-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 258.2 [00153] 3-[5-(6-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 302.7 [00154] 3-{5-[3-(2-Chloro-phenyl)-5- methyl-isoxazol-4-yl]- [1,2,4]oxadiazol-3-yl}-benzoic acid 382.8 [00155] 3-{5-[3-(2-Chloro-6-fluoro- phenyl)-5-methyl-isoxazol-4-yl]- [1,2,4]oxadiazol-3-yl}-benzoic acid 400.0 [00156] 3-(5-Cyclopentylmethyl- [1,2,4]oxadiazol-3-yl)-benzoic acid 273.3 [00157] 3-[5-(2,4-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00158] 3-(5-Pyridin-3-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 268.2 [00159] 3-(5-Pyridin-4-yl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 268.2 [00160] 3-(5-Cyclobutyl-[1,2,4]oxadiazol- 3-yl)-benzoic acid 245.2 [00161] 3-[5-(4-Methoxy-benzyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 311.3 [00162] 3-[5-(3,4-Dimethoxy-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 327.3 [00163] 3-[5-(2-Chloro-pyridin-3-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 302.7 [00164] 3-[5-(1-Acetyl-piperidin-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 316.3 [00165] 3-(5-Benzo[b]thiophen-2-yl- [1,2,4]oxadiazol-3-yl)-benzoic acid 323.3 [00166] 3-[5-(3-Dimethylamino-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 310.3 [00167] 3-[5-(2,3-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00168] 3-[5-(2-Fluoro-5-methyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 299.3 [00169] 3-[5-(2-Methylsulfanyl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid 314.3 [00170] 3-[5-(2,2-Difluoro- benzo[1,3]dioxol-5-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 347.2 [00171] 2-Fluoro-5-[5-(4-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00172] 3-[5-(4-Bromo-2-fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 364.1 [00173] 3-[5-(3-Fluoro-biphenyl-4-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 361.3 [00174] 3-[5-(3-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.2 [00175] 3-[5-(2,3-Difluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 303.2 [00176] 3-[5-(2-Fluoro-5-methyl-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 299.3 [00177] 3-[5-(2-Methylsulfanyl-pyridin-3- yl)-[1,2,4]oxadiazol-3-yl]-benzoic acid 314.3 [00178] 3-[5-(2,2-Difluoro- benzo[1,3]dioxol-5-yl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 347.2 [00179] 3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.1 [00180] 3-[5-(4-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 285.2 [00181] 3-[5-(4-Cyano-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid 292.08 [00182] 3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid sodium salt 306.04 [00183] 3-[5-(2-Fluoro-phenyl)- [1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester 299.08

5.2 Example 2: Preparation of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid

[0297] ##STR00184##

[0298] To a solution of 3-Cyanobenzoic acid (44.14 g, 300 mmol) in DMF (0.6 L) was added K.sub.2CO.sub.3 (62.19 g, 450 mmol) and then stirred for 30 min at room temperature. To the suspension was added methyl iodide (28 mL, 450 mmol) over 20 min, and the reaction mixture was stirred further 4 h at room temperature. The reaction mixture was poured to 1.2 L of ice water and stirred for 30 min, and the precipitate was filtered off. The white cake was dissolved in methanol (70 mL), and then re-precipitated in cold water. The desired product was obtained as a white powder with 79% yield (38 g, 99% purity by LC/UV). .sup.1H-NMR (CDCl.sub.3) 8.85 (2H), 8.28 (1H), 8.02 (1H), 4.17 (3H).

[0299] To a solution of 3-Cyanobenzoic acid methyl ester (50 g, 310 mmol) in ethanol (500 mL) was added 50% aqueous hydroxylamine (41 mL, 620 mmol) at room temperature. The reaction mixture was stirred for 1 h at 100 C. and the solvents were removed under reduced pressure. The oily residue was dissolved in 20/80 ethanol/toluene (50 mL2) and then concentrated again. The desired ester (61 g, quan. yield) was obtained as a white powder with 98% purity (LC/UV). .sup.1H-NMR (CDCl.sub.3) 9.76 (1H), 8.24 (1H), 7.82 (2H), 7.51 (1H), 5.92 (2H), 3.82 (3H).

[0300] To a solution of 3-(N-Hydroxycarbamimidoyl)-benzoic acid methyl ester (60 g, 310 mmol) in anhydrous THF (200 mL) was added diisopropylethylamine (75 mL, 434 mmol) at 5 C., and then to the mixture was added 2-fluorobenzoyl chloride (48.1 mL, 403 mmol) over 20 min. The reaction mixture was stirred for 1 h at room temperature. The precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethylacetate (400 mL) and then washed with water (200 mL2). The solvent was removed under reduced pressure and the desired product was crystallized in 60% ethylacetate in hexane to yield the desired product (81 g, 83% yield) as a white solid. .sup.1H-NMR (CDCl.sub.3) 8.18 (1H), 8.03 (2H), 7.48 (2H), 7.18 (2H), 5.61 (2H), 3.82 (3H).

[0301] 44 g of 3-(N-2-Fluorobenzoylcarbamimidoyl)-benzoic acid methyl ester in toluene (500 mL) was refluxed for 4 h at 130 C. using Dean-Stark apparatus. The reaction mixture was stirred at 5 C. for 18 h. The white precipitate was filtered off and the filtrate was concentrated, crystallized again in toluene. The desired oxadiazole (38 g, 92% yield) was obtained as a white solid with 99% purity (LC/UV). .sup.1H-NMR (CDCl.sub.3) 8.91 (1H), 8.38 (1H), 8.15 (2H), 7.62 (2H), 7.35 (2H), 3.95 (3H).

[0302] To a solution of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester (33 g, 111 mol) in THF (400 mL) was added 1.5M aqueous NaOH (100 mL, 144 mmol). The reaction mixture was refluxed for 2 h at 100 C. The organic solvent was removed under reduced pressure and the aqueous solution was stirred 2 h at 5 C. The white precipitate was filtered off and the filtrate was concentrated and precipitated again in water. The white cake was washed with cold water and then dried using lyophilizer. The desired salt (33 g, 96% yield) was obtained as a white powder with 98.6% purity (LC/UV).

[0303] To a solution of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester (3.3 g, 11 mmol) in THF (40 mL) was added 1.5M aqueous NaOH (10 mL, 14 mmol). The reaction mixture was refluxed for 2 h at 100 C. The organic solvent was removed and the aqueous solution was diluted with water (50 mL), and then acidified with aqueous HCl. The white precipitate was filtered off and the white cake was washed with cold water and then dried using lyophilizer. The desired acid (3.0 g, 96% yield) was obtained as a white powder with 98% purity (LC/UV). Melting point 242 C.; IR 3000 (Aromatic CH), 1710 (CO); .sup.1H-NMR (D6-DMSO) 8.31 (1H), 8.18 (2H), 8.08 (1H), 7.88 (2H), 7.51 (2H); .sup.13C-NMR (D6-DMSO) 172.71, 167.38, 166.48, 161.25, 135.80, 132.24, 131.79, 131.79, 131.08, 130.91, 129.81, 127.76, 125.48, 117.38, 111.70; .sup.19F-NMR (D6-DMSO) 109.7.

[0304] The following compounds are prepared using the procedures described above.

TABLE-US-00004 TABLE 3 Compound Compound Name [M + H].sup.+ [00185] 3-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 285.2 [00186] 3-[5-(2-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 285.1 [00187] 3-[5-(3-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 285.2 [00188] 4-Fluoro-3- [5-(4- fluoro-phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 303.2 [00189] 5-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]-2-methoxy- benzoic acid 315.3 [00190] 3-[5-(4-Chloro- 2-fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 319.7 [00191] 3-[5-(3-Fluoro- biphenyl-4-yl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 361.3 [00192] 3-[5-(4-Bromo- 2-fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 364.1 [00193] 3-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid methyl ester 299.08 [00194] 5-[5-(4-Fluoro- phenyl)- [1,2,4]oxadiazol- 3-yl]-2- methoxy- benzoic acid 339.13 [00195] 3-[5-(4-Bromo- 2-fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 365.05 [00196] 3-[5-(3-Fluoro- biphenyl-4-yl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 361.16 [00197] 3-[5-(6- Pyrrolidin-1- yl-pyridin-3-yl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 337.20 [00198] 3-[5-(6- Morpholin-4- yl-pyridin-3-yl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 353.18 [00199] 3-[5-(3,4,5,6- Tetrahydro-2H- [1,2]bipyridinyl- 5-yl)-[1,2,4] oxadiazol- 3-yl]-benzoic acid 351.18 [00200] 3-[5-(2- Fluoro-6- hydroxy-phenyl)- [1,2,4]oxadiazol- 3-yl]- benzoic acid 301.18 [00201] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 2-methoxy- ethyl ester 343.16 [00202] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol- 3-yl]- benzoic acid 2- (2-methoxy- ethoxy)-ethyl ester 387.49 [00203] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 2-[2-(2- methoxy- ethoxy)-ethoxy]- ethyl ester 431.31 [00204] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 2-{2-[2-(2- methoxy- ethoxy)-ethoxy]- ethoxy}-ethyl ester 475.26 [00205] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 2-(2-{2-[2- (2-methoxy- ethoxy)-ethoxy]- ethoxy}-ethoxy)- ethyl ester 519.33 [00206] 3-[5-(2-Fluoro- phenyl)-[1,2,4] oxadiazol-3-yl]- benzoic acid 2- [2-(2-{2-[2-(2- hydroxy-ethoxy)- ethoxy]-ethoxy}- ethoxy)-ethoxy]- ethyl ester 549.35 [00207] 3-[5-(4-Amino- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 282.20 [00208] 3-[5-(4-Azido- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 309.20 [00209] 3-[5-(4- Benzyloxy- phenyl)-[1,2,4] oxadiazol-3- yl]-benzoic acid 373.16

5.3 Example 3: Identification and Characterization of Compounds that Promote Nonsense Suppression and/or Modulate Translation Termination

[0305] The assays described above in Section 4.2 were used in two high throughput screens. Compounds were screened in the cell-based and biochemical assays. Compounds were tested, resynthesized and tested again to confirm chemical structures. 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt was characterized further with the in vitro luciferase nonsense suppression assay. To ensure that the observed nonsense suppression activity of the selected compounds was not limited to the rabbit reticulocyte assay system, HeLa cell extract was prepared and optimized (Lie & Macdonald, 1999, Development 126(22):4989-4996 and Lie & Macdonald, 2000, Biochem. Biophys. Res. Commun. 270(2):473-481).

5.4 Example 4: Characterization of Compounds that Increase Nonsense Suppression and Produce Functional Protein

[0306] It was previously demonstrated that compounds of the invention increase the level of nonsense suppression in the biochemical assay three to four fold over untreated extracts. To determine whether compounds also function in vivo, a stable cell line harboring the UGA nonsense-containing luciferase gene was treated with selected compounds. Cells were grown in standard medium supplemented with 1% penicillin-streptomycin (P/S) and 10% fetal bovine serum (FBS) to 70% confluency and split 1:1 the day before treatment. On the following day, cells were trypsinized and 40,000 cells were added to each well of a 96-well tissue culture dish. Serial dilutions of each compound were prepared to generate a six-point dose response curve spanning 2 logs (30 M to 0.3 M). The final concentration of the DMSO solvent remained constant at 1% in each well. Cells treated with 1% DMSO served as the background standard, and cells treated with gentamicin served as a positive control.

5.5 Example 5: 3-[2-(4-isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt alters the accessibility of the chemical modifying agents to specific nucleotides in the 28 S rRNA

[0307] Previous studies have demonstrated that gentamicin and other members of the aminoglycoside family that decrease the fidelity of translation bind to the A site of the 16S rRNA. By chemical footprinting, UV cross-linking and NMR, gentamicin has been shown to bind at the A site (comprised of nucleotides 1400-1410 and 1490-1500, E. coli numbering) of the rRNA at nucleotides 1406, 1407, 1494, and 1496 (Moazed & Noller, 1987, Nature 327(6121):389-394; Woodcock et al., 1991, EMBO J. 10(10):3099-3103; and Schroeder et al., 2000, EMBO J. 19:1-9

[0308] Ribosomes prepared from HeLa cells were incubated with the small molecules (at a concentration of 100 M), followed by treatment with chemical modifying agents (dimethyl sulfate [DMS] and kethoxal [KE]). Following chemical modification, rRNA was phenol-chloroform extracted, ethanol precipitated, analyzed in primer extension reactions using end-labeled oligonucleotides hybridizing to different regions of the three rRNAs and resolved on 6% polyacrylamide gels. The probes used for primer extension cover the entire 18S (7 oligonucleotide primers), 28S (24 oligonucleotide primers), and 5S (one primer) rRNAs. Controls in these experiments include DMSO (a control for changes in rRNA accessibility induced by DMSO), paromomycin (a marker for 18S rRNA binding), and anisomycin (a marker for 28S rRNA binding).

[0309] The results of these foot-printing experiments indicated that 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt alters the accessibility of the chemical modifying agents to specific nucleotides in the 28S rRNA. More specifically, the regions protected by 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt include: (1) a conserved region in the vicinity of the peptidyl transferase center (domain V) implicated in peptide bond formation and (2) a conserved region in domain II that may interact with the peptidyl transferase center based on binding of vernamycinin B to both these areas.

5.6 Example 6: Readthrough of Premature Termination Codons in Cell-Based Disease Models

[0310] To address the effects of the nonsense-suppressing compounds on mRNAs altered in specific inherited diseases, a bronchial epithelial cell line harboring a nonsense codon at amino acid 1282 (W1282X) was treated with 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt (20M) and CFTR function was monitored as a cAMP-activated chloride channel using the SPQ assay (Yang et al., 1993, Hum Mol Genet. 2(8):1253-1261 and Howard et al., 1996, Nat Med. 2(4):467-469). These experiments showed that cAMP treatment of these cells resulted in an increase in SPQ fluorescence, consistent with stimulation of CFTR-mediated halide efflux. No increase in fluorescence was observed when cells were not treated with compound or if the cells were not stimulated with cAMP. These results indicate that the full-length CFTR expressed from this nonsense-containing allele following compound treatment also functions as a cAMP-stimulated anion channel, thus demonstrating that cystic fibrosis cell lines increase chloride channel activity when treated with 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt.

5.7 Example 7: Primary cells from the mdx nonsense-containing mouse express full-length dystrophin protein when treated with 3-[2-(4-isopropyl-3-methyl-phenozy)-acetylamino] benzoic acid, sodium salt

[0311] The mutation in the mdx mouse that premature termination of the 427 kDa dystrophin polypeptide has been shown to be a C to T transition at position 3185 in exon 23 (Sicinski et al., 1989, Science. 244(4912):1578-1580). Mouse primary skeletal muscle cultures derived from 1-day old mdx mice were prepared as described previously (Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381). Cells were cultured for 10 days in the presence of 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt (20 M). Culture medium was replaced every four days and the presence of dystrophin in myoblast cultures was detected by immunostaining as described previously (Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381). A primary monoclonal antibody to the C-terminus of the dystrophin protein (F19A12) was used undiluted and rhodamine conjugated anti-mouse IgG was used as the secondary antibody. The F19A12 antibody will detect the full-length protein produced by suppression of the nonsense codon. Staining was viewed using a Leica DMR micropscope, digital camera, and associated imaging software at the University of Pennsylvania.

5.8 Example 8: Readthrough of Premature Termination Codons in the Mdx Mouse

[0312] As previously described (Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381), compound was delivered by Alzet osmotic pumps implanted under the skin of anesthetized mice. Two doses of 3-[2-(4-Isopropyl-3-methyl-phenoxy)-acetylamino]-benzoic acid, sodium salt were administered. Gentamicin served as a positive control and pumps filled with solvent only served as the negative control. Pumps were loaded with appropriate compound such that the calculated doses to which tissue was exposed were 10 M and 20 M. The gentamicin concentration was calculated to achieve tissue exposure of approximately 200 M. In the initial experiment, mice were treated for 14 days, after which animals were anesthetized with ketamine and exsanguinated. The tibialis anterior (TA) muscle of the experimental animals was then excised, frozen, and used for immunofluorescence analysis of dystrophin incorporation into striated muscle. The presence of dystrophin in TA muscles was detected by immunostaining, as described previously (Barton-Davis et al., 1999, J Clin Invest. 104(4):375-381).

5.9 Example 9: 200 Mg Dosage Capsule

[0313] Table 3 illustrates a batch formulation and single dosage formulation for a 200 mg single dose unit, i.e., about 40 percent by weight.

TABLE-US-00005 TABLE 3 Formulation for 200 mg capsule Percent Quantity Quantity Material By Weight (mg/tablet) (kg/batch) Compound of the 40.0% 200 mg 16.80 kg invention Pregelatinized Corn 9.5% 297.5 mg 24.99 kg Starch, NF5 Magnesium Stearate 0.5% 2.5 mg 0.21 kg Total 100.0% 500 mg 42.00 kg

[0314] The pregelatinized corn starch (SPRESS B-820) and compound of the invention components are passed through a 710 m screen and then are loaded into a Diffusion Mixer with a baffle insert and blended for 15 minutes. The magnesium stearate is passed through a 210 m screen and is added to the Diffusion Mixer. The blend is then encapsulated in a size #0 capsule, 500 mg per capsule (8400 capsule batch size) using a Dosator type capsule filling machine.

5.10 Example 10: 100 Mg Oral Dosage Form

[0315] Table 4 illustrates a batch formulation and a single dose unit formulation containing 100 mg of a compound of the invention.

TABLE-US-00006 TABLE 4 Formulation for 100 mg tablet Percent by Quantity Material Weight (mg/tablet) Quantity (kg/batch) compound of the 40% 100.00 20.00 invention Microcrystalline 53.5% 133.75 26.75 Cellulose, NF Pluronic F-68 4.0% 10.00 2.00 Surfactant Croscarmellose 2.0% 5.00 1.00 Sodium Type A, NF Magnesium Stearate, 0.5% 1.25 0.25 NF Total 100.0% 250.00 mg 50.00 kg

[0316] The microcrystalline cellulose, croscarmellose sodium, and compound of the invention components are passed through a #30 mesh screen (about 430 to about 655). The Pluronic F-68 (manufactured by JRH Biosciences, Inc. of Lenexa, Kans.) surfactant is passed through a #20 mesh screen (about 457a to about 1041). The Pluronic F-68 surfactant and 0.5 kgs of croscarmellose sodium are loaded into a 16 qt. twin shell tumble blender and are mixed for about 5 minutes. The mix is then transferred to a 3 cubic foot twin shell tumble blender where the microcrystalline cellulose is added and blended for about 5 minutes. The compound is added and blended for an additional 25 minutes. This pre-blend is passed through a roller compactor with a hammer mill attached at the discharge of the roller compactor and moved back to the tumble blender. The remaining croscarmellose sodium and magnesium stearate is added to the tumble blender and blended for about 3 minutes. The final mixture is compressed on a rotary tablet press with 250 mg per tablet (200,000 tablet batch size).

5.11 Example 11: Aerosal Dosage Form

[0317] A concentrate is prepared by combining a compound of the invention, and a 12.6 kg portion of the trichloromonofluoromethane in a sealed stainless steel vessel equipped with a high shear mixer. Mixing is carried out for about 20 minutes. The bulk suspension is then prepared in the sealed vessel by combining the concentrate with the balance of the propellants in a bulk product tank that is temperature controlled to 210 to 27 C. and pressure controlled to 2.8 to 4.0 BAR. 17 ml aerosol containers that have a metered valve which is designed to provide 100 inhalations of the composition of the invention. Each container is provided with the following:

TABLE-US-00007 compound of the invention 0.0141 g trichloromonofluoromethane 1.6939 g dichlorodifluoromethane 3.7028 g dichlorotetrafluoroethane 1.5766 g total 7.0000 g