HRI ACTIVATORS USEFUL FOR THE TREATMENT OF CARDIOMETABOLIC DISEASES

Abstract

Compounds of formula (I) include: X is CH or N, preferably CH; n is 1-5, preferably 1-2; m is 0-5, preferably 1-2; and, when m is 2-5, two of the R.sup.2 radicals taken together with two adjacent carbons of the benzene ring can form a 5- or 6-membered heterocyclic ring fused with the benzene ring. Compounds of formula (I) are heme-regulated inhibitor (HRI) activators and useful for prevention or treatment of cardiometabolic diseases such as metabolic syndrome, obesity, insulin resistance, type 2 diabetes mellitus, non-alcoholic fatty liver disease, steatosis, non-alcoholic steatohepatitis, hypertension, dyslipidemia, atherosclerosis, and heart disease.

Claims

1. A compound of formula (I) ##STR00008## and pharmaceutically acceptable salts and solvates thereof, wherein: X is CH or N; R.sup.1 is a radical independently selected from the group consisting of H, SF.sub.5, halogen, CF.sub.3, NO.sub.2, CN, and OCF.sub.3; n being an integer from 1 to 5; halogen being F, Cl, Br or I; R.sup.2 is a radical independently selected from the group consisting of SFs, halogen, CF.sub.3, NO.sub.2, CN, OCF.sub.3, hydroxyl, (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, di-(C.sub.1-C.sub.4)alkylaminoethoxy, 2-(piperidin-1-yl)ethoxy, 2-(pyrrolidin-1-yl)ethoxy, 2-(azepan-1-yl)ethoxy, 2-morpholinoethoxy, and 2-(piperazin-1-yl)ethoxy; m being an integer from 0 to 5; alternatively, when X is CH and m is an integer from 2 to 5, two R.sup.2 radicals taken together with two adjacent carbons of the benzene ring to which they are joined form a 5- or 6-membered heterocyclic ring having from 1 to 3 heteroatoms independently selected from the group consisting of O, N, and S, the heterocyclic ring being fused with the benzene ring, and the benzene ring being optionally substituted with one or more radicals independently selected from the group consisting of SFs, halogen, CF.sub.3, NO.sub.2, CN, and OCF.sub.3; provided that the compound of formula (I) does not have any of the following formulas: ##STR00009##

2. The compound according to claim 1, wherein X is CH.

3. The compound according to claim 1, wherein n is 1 or 2, and m is 1 or 2.

4. The compound according to claim 1, wherein: m is an integer from 2 to 5, and two R.sup.2 radicals taken together with two adjacent carbons of the benzene ring to which they are joined form a 5- or 6-membered heterocyclic ring having from 1 to 3 heteroatoms independently selected from the group consisting of O, N, and S, the heterocyclic ring being fused with the benzene ring, and the benzene ring being optionally substituted with one or more radicals independently selected from the group consisting of SF.sub.5, halogen, CF.sub.3, NO.sub.2, CN, and OCF.sub.3.

5. The compound according to claim 4, wherein two R.sup.2 radicals are forming a heterocyclic ring which has one of the following formulas: ##STR00010##

6. The compound according to claim 1, wherein R.sup.2 is independently selected from the group consisting of SF.sub.5, halogen, CF.sub.3, NO.sub.2, CN, OCF.sub.3, hydroxyl, (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, di-(C.sub.1-C.sub.4)alkylaminoethoxy, 2-(piperidin-1-yl)ethoxy, 2-(pyrrolidin-1-yl)ethoxy, 2-(azepan-1-yl)ethoxy, 2-morpholinoethoxy, and 2-(piperazin-1-yl)ethoxy.

7. The compound according to claim 1, which has an SF.sub.5 radical attached to the 3 position of the phenyl ring that has the R.sup.1 radicals.

8. The compound according to claim 1, wherein halogen is F or Cl.

9. The compound according to claim 5, which has one of the following formulas: ##STR00011##

10. The compound according to claim 6, which has one of the following formulas: ##STR00012##

11. A compound according to claim 1, for use as active pharmaceutical ingredient.

12. A compound according to claim 1, for use in the prevention or treatment of a cardiometabolic disease selected from the group consisting of metabolic syndrome, obesity, insulin resistance, type 2 diabetes mellitus, non-alcoholic fatty liver disease, steatosis, non-alcoholic steatohepatitis, hypertension, dyslipidemia, atherosclerosis, and heart disease.

13. The compound for use according to claim 12, wherein the cardiometabolic disease is metabolic syndrome.

14. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1, together with appropriate amounts of pharmaceutically acceptable excipients or carriers.

15. A pharmaceutical composition according to claim 14, for use in the prevention or treatment of a cardiometabolic disease selected from the group consisting of metabolic syndrome, obesity, insulin resistance, type 2 diabetes mellitus, non-alcoholic fatty liver disease, steatosis, non-alcoholic steatohepatitis, hypertension, dyslipidemia, atherosclerosis, and heart disease.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] FIG. 1 shows immunoblot analyses and the quantification of total and phospho-HRI in human Huh-7 hepatic cells incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of compounds I-25, I-26, I-27, I-28, and I-29. Data are presented as the meanSD (n=4). *p<0.05 vs. control (CT) cells. .sup.#p<0.05 vs. BTdCPU-treated cells. BTdCPU=1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea (compound (2) in Chen T. et al., ibid.).

[0027] FIG. 2 shows immunoblot analyses and the quantification of total and phospho-HRI in human Huh-7 hepatic cells incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of compounds I-30, I-2, and I-33. Data are presented as the meanSD (n=4). ***p<0.001 and *p<0.05 vs. control (CT) cells. .sup.#p<0.05 vs. BTdCPU-treated cells. Compound I-2 (1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea is not part of the present invention, but it has been introduced in some figures for comparative purposes.

[0028] FIG. 3 shows immunoblot analyses and the quantification of total and phospho-eIF2 in human Huh-7 hepatic cells incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of compounds I-25, I-26, I-27, I-28 and I-29. Data are presented as the meanSD (n=4). **p<0.01 and *p<0.05 vs. control (CT) cells. .sup.##p<0.01 and .sup.#p<0.05 vs. BTdCPU-treated cells.

[0029] FIG. 4 shows immunoblot analyses and the quantification of total and phospho-eIF2 in human Huh-7 hepatic cells incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of compounds I-30 and I-33. Data are presented as the meanSD (n=4). *p<0.05 vs. control (CT) cells.

[0030] FIG. 5 shows the macroscopic (upper image) and microscopic images of Huh-7 cells stained with Oil Red O. Huh-7 cells were previously incubated for 24 h with BSA (Control, CT), 0.75 mmol/L palmitate (Pal) conjugated with BSA, or 0.75 mmol/L BSA-palmitate plus 10 mol/L BTdCPU (Pal+BTdCPU).

[0031] FIG. 6 shows the immunoblot analyses of total and phosphorylated Akt (A), and total Akt. (B). When indicated (+), cells were incubated with 100 nmol/L insulin (I) for the last 10 min. Data are presented as the meanSD (n=4 per group). ***p<0.001 and *p<0.05 vs. control cells not exposed to insulin. .sup.###p<0.001 and .sup.##p<0.01 vs. insulin-stimulated control cells. .sup.554 \\p<0.001 vs. insulin-stimulated cells incubated with palmitate. Ins: insulin.

[0032] FIG. 7 shows the results of the glucose tolerance test and area under the curve (AUC) of mice fed a standard chow (CT), a HFD for three weeks (HFD), or a HFD for three weeks plus BTdCPU during the last week (HFD+BTdCPU). Data are presented as the meanSD (n=6 per group). *p<0.05 vs. mice fed a standard diet (CT). #p<0.05 vs. mice fed a HFD. G: glucose.

[0033] FIG. 8. (A) Oil Red O and eosin-hematoxylin (H&E) staining of livers of mice fed a standard chow (CT), a HFD for three weeks (HFD) or a HFD for three weeks plus BTdCPU during the last week (HFD+BTdCPU). (B) Liver triglyceride levels. Data are presented as the meanSD (n=6 per group). *p<0.05 vs. mice fed a standard diet (CT). .sup.#p<0.05 vs. mice fed a HFD. G: glucose. TG: triglyceride.

[0034] FIG. 9 shows the effects of different HRI activators on FGF21 expression in hepatocytes. Human Huh-7 hepatocytes were incubated for 24 h in the absence (Control, CT) or in the presence of compounds (10 M). Assessment by quantitative real-time RT-PCR of FGF21. Data are presented as the meanSD (n=4 per group). ***p<0.001 and **p<0.01 vs CT. .sup.###p<0.001, .sup.##p<0.01 and .sup.#p<0.05 vs BTCtFPU. .sup.p<0.01 and .sup.p<0.05 vs BTdCPU. BTCtFPU=1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (compound (3) in Chen T. et al., ibid.).

DESCRIPTION OF EMBODIMENTS

Preparative Example 1. 1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-25). Step 1

[0035] A solution of 4-(pentafluoro-.sup.6-sulfanyl)aniline (259 mg, 1.18 mmol) in toluene (5 mL) was treated with triphosgene (175 mg, 0.59 mmol). Immediately, triethylamine (0.16 mL, 1.18 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 4-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. To a solution of the isocyanate from the previous step a solution of benzo[d][1,2,3]thiadiazol-6-amine (178 mg, 1.18 mmol) in THF (6 mL) was added. The suspension was stirred at room temperature overnight. Evaporation in vacuo of the organics gave an orange solid. Column chromatography (hexane/ethyl acetate mixtures) gave compound I-25 as a white solid (203 mg, 43% overall yield). The analytical sample was obtained by washing with cooled dichloromethane (126 mg). m.p.: 274-275 C. IR (ATR) v: 612, 641, 659, 716, 801, 832, 1059, 1101, 1134, 1191, 1245, 1271, 1297, 1325, 1351, 1413, 1467, 1510, 1528, 1595, 1721, 3100, 3136, 3297, 3338 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.9F.sub.5N.sub.4OS.sub.2H].sup.: 395.0065; Found: 395.0064.

Preparative Example 2. 1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(3-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-26). Step 1

[0036] A solution of 3-(pentafluoro-.sup.6-sulfanyl)aniline (259 mg, 1.18 mmol) in toluene (5.8 mL) was treated with triphosgene (175 mg, 0.59 mmol). Immediately, triethylamine (0.16 mL, 1.18 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. To a solution of the isocyanate from the previous step a solution of benzo[d][1,2,3]thiadiazol-6-amine (178 mg, 1.18 mmol) in THF (6 mL) was added. The suspension was stirred at room temperature overnight. Evaporation in vacuo of the organics gave an orange solid (440 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-26 as a pale white solid (243 mg, 52% overall yield). The analytical sample was obtained by washing with pentane (199 mg). m.p.: 229-230 OC. IR (ATR) v: 614, 680, 718, 780, 801, 819, 830, 878, 912, 922, 1057, 1009, 1113, 1134, 1196, 1219, 1294, 1312, 1349, 1412, 1434, 1466, 1529, 1540, 1568, 1602, 1718, 2852, 2919, 3090, 3126, 3271, 3302, 3338 cm.sup.1. Elemental analysis: Calculated for C.sub.13H.sub.9F.sub.5N.sub.4OS.sub.2. 0.05C.sub.4H.sub.8O.sub.2.0.5C.sub.5H.sub.12: C, 43.17%, H, 3.55%, N, 12.83%, S, 14.68%; Found: C, 43.51%, H, 3.17%, N, 12.96%, S, 14.61%.

Preparative Example 3. 1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-chloro-5-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-27). Step 1

[0037] A solution of 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)aniline (300 mg, 1.18 mmol) in toluene (5 mL) was treated with triphosgene (175 mg, 0.59 mmol). Immediately, triethylamine (0.16 mL, 1.18 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was in vacuo at room temperature to give 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. To a solution of the isocyanate from the previous step a solution of benzo[d][1,2,3]thiadiazol-6-amine (178 mg, 1.18 mmol) in THF (6 mL) was added. The suspension was stirred at room temperature overnight. Evaporation in vacuo of the organics gave an orange solid (378 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-27 as a beige solid (106 mg, 21% overall yield). The analytical sample was obtained by washing with cooled dichloromethane (80 mg). m.p.: 227 C. IR (ATR) v: 615, 664, 729, 805, 816, 837, 884, 922, 964, 1033, 1052, 1134, 1222, 1287, 1349, 1413, 1467, 1536, 1563, 1666, 1721, 3333 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.8ClF.sub.5N.sub.4OS.sub.2H].sup.: 428.9675; Found: 428.9676.

Preparative Example 4. 1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-chloro-3-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-28)

[0038] Step 1. A solution of 2-chloro-3-(pentafluoro-.sup.6-sulfanyl)aniline (300 mg, 1.18 mmol) in toluene (5 mL) was treated with triphosgene (175 mg, 0.59 mmol). Immediately, triethylamine (0.16 mL, 1.18 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 2-chloro-3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. To a solution of the isocyanate from the previous step a solution of benzo[d][1,2,3]thiadiazol-6-amine (178 mg, 1.18 mmol) in THF (6 mL) was added. The suspension was stirred at room temperature overnight. Evaporation in vacuo of the organics gave an orange solid (478 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-28 as a pale white solid (149 mg, 30% overall yield). m.p.: 225 C. IR (ATR) v: 628, 649, 673, 705, 729, 760, 783, 814, 847, 915, 1054, 1126, 1155, 1217, 1245, 1279, 1318, 1346, 1411, 1462, 1527, 1571, 1664, 1718, 2852, 2919, 2956, 3317 cm.sup.1. Elemental analysis: Calculated for C.sub.13H.sub.8ClF.sub.5N.sub.4OS.sub.2.0.05C.sub.4H.sub.8O.sub.2.0.5C.sub.6H.sub.14: C, 40.68%, H, 3.25%, N, 11.71%, S, 13.41%; Found: C, 40.88%, H, 3.00%, N, 11.51%, S, 13.17%.

Preparative Example 5. 1-(Benzo[d][1,2,3]thiadiazol-6-yl)-3-(4-chloro-3-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-29). Step 1

[0039] A solution of 4-chloro-3-(pentafluoro-.sup.6-sulfanyl)aniline (300 mg, 1.18 mmol) in toluene (5 mL) was treated with triphosgene (175 mg, 0.59 mmol). Immediately, triethylamine (0.16 mL, 1.18 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 4-chloro-3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. To a solution of the isocyanate from the previous step a solution of benzo[d][1,2,3]thiadiazol-6-amine (178 mg, 1.18 mmol) in THF (6 mL) was added. The suspension was stirred at room temperature overnight. Evaporation in vacuo of the organics gave an orange solid (325 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-29 as a pale brown solid (237 mg, 47% overall yield). The analytical sample was obtained by washing with pentane (180 mg). m.p.: 213-214 C. IR (ATR) v: 647, 669, 726, 801, 819, 845, 894, 923, 960, 1033, 1126, 1152, 1207, 1245, 1276, 1315, 1349, 1387, 1413, 1465, 1527, 1571, 1594, 1723, 2919, 3091, 3126, 3178, 3271, 3297, 3338 cm.sup.1. Elemental analysis: Calculated for C.sub.13H.sub.8ClF.sub.5N.sub.4OS.sub.2. 0.5C.sub.6H.sub.14.0.25C.sub.4H.sub.8O.sub.2: C, 41.17%, H, 3.46%, N, 11.30%, S, 12.93%; Found: C, 41.30%, H, 3.22%, N, 11.43%, S, 12.72%.

Preparative Example 6. 1,3-Bis(2-chloro-5-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea, (Compound I-32). Step 1

[0040] A solution of 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)aniline (350 mg, 1.38 mmol) in toluene (4 mL) was treated with triphosgene (204 mg, 0.69 mmol). Immediately, triethylamine (0.19 mL, 1.38 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. 2-chloro-5-(pentafluorosulfanyl)aniline (384 mg, 1.51 mmol) was dissolved in anh. THF (12 mL) under argon and cooled to 78 C. on a dry ice in acetone bath. Then, 2.5 M n-butyllithium in hexanes (0.73 mL, 1.78 mmol) was added dropwise during 20 min. Afterwards, the reaction mixture was removed from the dry ice in acetone bath and tempered to 0 C. with an ice bath. Meanwhile, the isocyanate (387 mg, 1.38 mmol) in toluene solution from the previous step was stirred under argon and was continuously added to the reaction mixture. The mixture was stirred at room temperature overnight. Methanol (4.5 mL) was added to quench any unreacted n-butyllithium and evaporation of the solvents provided a pale brown solid (670 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-32 as a beige solid (283 mg, 39% overall yield). m.p.: 227-228 C. IR (ATR) v: 663, 703, 736, 752, 798, 805, 820, 887, 920, 961, 1037, 1075, 1108, 1157, 1233, 1256, 1281, 1294, 1414, 1460, 1533, 1587, 1661, 1696, 1717, 1890, 1908, 2843, 2920, 2956, 3294, 3304, 3329 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.8Cl.sub.2F.sub.10N.sub.2OS.sub.2H].sup.: 530.9223; Found: 530.9222.

Preparative Example 7. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-chloro-5-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-33). Step 1

[0041] A solution of 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)aniline (350 mg, 1.38 mmol) in toluene (4 mL) was treated with triphosgene (204 mg, 0.69 mmol) under argon. Immediately, triethylamine (0.19 mL, 1.38 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 2-chloro-5-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. 4-chloro-3-(trifluoromethyl)aniline (296 mg, 1.51 mmol) was dissolved in anh. THF (12 mL) under argon and cooled to 78 C. on a dry ice in acetone bath. Then, 2.5 M n-butyllithium in hexanes (0.73 mL, 1.78 mmol) was added dropwise during 20 min. Afterwards, the reaction mixture was removed from the dry ice in acetone bath and tempered to 0 C. with an ice bath. Meanwhile, the isocyanate (387 mg, 1.38 mmol) in toluene solution from the previous step was stirred under argon and was continuously added to the reaction mixture. The mixture was stirred at room temperature overnight. Methanol (5 mL) was added to quench any unreacted n-butyllithium and evaporation of the solvents provided a brown oil (767 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-33 as a pale brown solid (138 mg, 22% overall yield). m.p.: 156-157 C. IR (ATR) v: 632, 666, 684, 701, 727, 742, 760, 801, 812, 840, 855, 863, 906, 950, 963, 1034, 1065, 1111, 1126, 1175, 1216, 1229, 1260, 1283, 1301, 1329, 1372, 1408, 1459, 1485, 1513, 1546, 1582, 1592, 1608, 1654, 1695, 1715, 1769, 1905, 1925, 2025, 2179, 2323, 2369, 2851, 2917, 2953, 3276, 3328, 3671, 3733, 3795, 3815 cm.sup.1. Accurate mass: Calculated for [C.sub.14H.sub.8Cl.sub.2F.sub.8N.sub.2OSH].sup.: 475.9534; Found: 475.9538.

Preparative Example 8. 1,3-Bis(3-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-36). Step 1

[0042] A solution of 3-(pentafluoro-.sup.6-sulfanyl)aniline (350 mg, 1.60 mmol) in toluene (6.8 mL) was treated with triphosgene (237 mg, 0.80 mmol). Immediately, triethylamine (0.22 mL, 1.60 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. After, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. 3-(Pentafluoro-.sup.6-sulfanyl)aniline (351 mg, 1.60 mmol) was dissolved in anh. THF (3 mL) under argon and cooled to 78 C. on a dry ice in acetone bath. Then, 2.5 M n-butyllithium in hexanes (0.86 mL, 2.08 mmol) was added dropwise during 20 min. Afterwards, the reaction mixture was removed from the dry ice in acetone bath and tempered to 0 C. with an ice bath. Meanwhile, the isocyanate (392 mg, 1.60 mmol) from previous step was stirred under argon and was continuously added to the reaction mixture. The mixture was stirred at room temperature overnight. Methanol (4.5 mL) was added to quench any unreacted n-butyllithium, and evaporation of solvents provided a beige solid (710 mg). Column chromatography (hexane/ethyl acetate mixtures) gave I-36 as a pale white solid (362 mg, 49% overall yield). The analytical sample was obtained as a white solid (183 mg) by crystallization from hot ethyl acetate. m.p.: 267-268 C. IR (ATR) v: 1117, 1242, 1314, 1418, 1485, 1599, 1663, 3102, 3202, 3310 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.10F.sub.10N.sub.2OS.sub.2H].sup.: 463.0002; Found: 463.0022.

Preparative Example 9. 1-(3-(Pentafluoro-.SUP.6.-sulfanyl)phenyl)-3-(4-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (compound I-37). Step 1

[0043] A solution of 3-(pentafluoro-.sup.6-sulfanyl)aniline (350 mg, 1.60 mmol) in toluene (5 mL) was treated with triphosgene (237 mg, 0.80 mmol). Immediately, triethylamine (0.22 mL, 1.60 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. 4-(Pentafluoro-.sup.6-sulfanyl)aniline (246 mg, 1.12 mmol) was dissolved in anh. THF (5 mL) under argon and cooled to 78 C. on a dry ice in acetone bath. Then, 2.5 M n-butyllithium in hexanes (0.6 mL, 1.46 mmol) was added dropwise during 20 min. Afterwards, the reaction mixture was removed from the dry ice in acetone bath and tempered to 0 C. with an ice bath. Meanwhile, the isocyanate (275 mg, 1.12 mmol) from the previous step was stirred under argon and was continuously added to the reaction mixture. The mixture was stirred at room temperature overnight. Methanol (4.5 mL) was added to quench any unreacted n-butyllithium, and evaporation of the solvents provided a brown gum (742 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-37 as a pale white solid (102 mg, 20% overall yield). m.p.: 216-217 C. IR (ATR) v: 1103, 1196, 1229, 1304, 1410, 1487, 1549, 1597, 1665, 3088, 3134, 3204, 3321 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.10F.sub.10N.sub.2OS.sub.2H].sup.: 463.0002. Found: 463.0017.

Preparative Example 10. 1,3-Bis(4-chloro-3-(pentafluoro-.SUP.6.-sulfanyl)phenyl)urea (Compound I-38). Step 1

[0044] A solution of 4-chloro-3-(pentafluoro-.sup.6-sulfanyl)aniline (350 mg, 1.37 mmol) in toluene (5 mL) was treated with triphosgene (204 mg, 0.69 mmol). Immediately, triethylamine (0.20 mL, 1.37 mmol) was added and the reaction mixture was stirred at 70 C. for 2 h. Afterwards, pentane (1 mL) was added and a white precipitate was formed. The mixture was filtered and pentane was evaporated in vacuo at room temperature to give 4-chloro-3-(pentafluoro-.sup.6-sulfanyl)phenyl isocyanate in toluene solution that was used in the next step without further purification. Step 2. 4-chloro-3-(pentafluoro-.sup.6-sulfanyl)aniline (278 mg, 1.09 mmol) was dissolved in anh. THF (6 mL) under argon and cooled to 78 C. on a dry ice in acetone bath. Then, 2.5 M n-butyllithium in hexanes (0.60 mL, 1.42 mmol) was added dropwise during 20 min. Afterwards, the reaction mixture was removed from the dry ice in acetone bath and tempered to 0 C. with an ice bath. Meanwhile, the isocyanate (307 mg, 1.09 mmol) from the previous step was stirred under argon and was continuously added to the reaction mixture. The mixture was stirred at room temperature overnight. Methanol (4.5 mL) was added to quench any unreacted n-butyllithium, and evaporation of the solvents provided an orange gum (675 mg). Column chromatography (hexane/ethyl acetate mixtures) gave compound I-38 as a white solid (136 mg, 23% overall yield). m.p.: 237-238 C. IR (ATR) v: 1042, 1130, 1227, 1290, 1396, 1477, 1545, 1587, 1645, 1699, 3030, 3138, 3306 cm.sup.1. Accurate mass: Calculated for [C.sub.13H.sub.8Cl.sub.2F.sub.10N.sub.2OS.sub.2H].sup.: 530.9223. Found: 530.9236.

[0045] Identification and Activity Examples of HRI Activators

[0046] The following abbreviations are used: Akt: Protein kinase B; ORO: Oil Red O; BSA: bovine serum albumin; DMSO: dimethylsulfoxide; GTT: glucose tolerance test; AUC: area under the curve; HFD: high-fat diet; CT: control.

[0047] HRI Activators Increase HRI Phosphorylation in Human Huh-7 Hepatic Cells.

[0048] HRI is activated by autophosphorylation and this has been used for evaluating its activation. In fact, the hyperphosphorylation of HRI is accompanied by an increase of its eIF2 kinase activity (cf. Lu L. et al., Translation initiation control by heme-regulated eukaryotic initiation factor 2a kinase in erythroid cells under cytoplasmic stresses, Mol. Cell. Biol., 2001, vol. 21, pp. 7971-7980). Therefore, HRI protein constitutively exists as the native nonphosphorylated 76-kDa species and/or the autoactivated/autophosphorylated 92-kDa, although the relative abundance of each HRI species tends to vary depending on the cells examined (cf. Acharya P. et al., Hepatic heme-regulated inhibitor (HRI) eukaryotic initiation factor 2a kinase: A protagonist of heme-mediated translational control of CYP2B enzymes and a modulator of basal endoplasmic reticulum stress tone, Mol. Pharmacol, 2010, vol. 77, pp. 575-592). To evaluate whether the new synthesized compounds were able to activate HRI, inventors examined by Western-blot whether Huh-7 cells exposed to the new compounds showed an increase in the ratio phosphorylated/total HRI, indicating the activation of this enzyme by these compounds. Thus Huh-7 cells were incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of the assayed compound. The effects of these compounds were compared to the well-known HRI activator BTdCPU. Compounds I-25, I-26, I-27, I-28, and I-29 increased the ratio phosphorylated/total HRI (FIG. 1), especially compounds I-28 and I-29, indicating activation of this kinase. Compound I-30 (FIG. 2) also increased the levels of phosphorylated HRI similarly to those observed for BTdCPU, whereas the increase caused by I-33 was also statistically significant, mainly because of the reduction in total HRI caused by this compound.

[0049] HRI Activators Increase eIF2 Phosphorylation in Human Huh-7 Hepatic Cells.

[0050] HRI is a kinase that phosphorylates eukaryotic Initiation Factor 2 (eIF-2a) at its Ser 51 residue to execute protein synthesis regulation and thereby HRI activators cause the phosphorylation of eIF2 as previously described (Chen T, et al., ibid). To confirm whether the new synthesized compounds were able to activate the HRI eIF2 kinase inventors examined the eIF2 phosphorylation caused by these compounds compared to the well-known HRI eIF2 kinase activator BTdCPU. Human Huh-7 hepatocytes were incubated for 24 h with vehicle (DMSO, CT cells) or 10 M of each of the assayed compounds. eIF2 phosphorylation was determined by Western-blot. Compounds I-26, I-27, I-28, and I-29 caused a marked increase in eIF2 phosphorylation, higher than the observed with BTdCPU, whereas phosphorylation induced by compound I-25 was similar to that observed for BTdCPU (FIG. 3). Compounds I-30 and I-33 enhanced eIF2 phosphorylation to values similar to those achieved by BTdCPU exposure (FIG. 4).

[0051] Triglyceride Accumulation in Hepatocytes.

[0052] To examine the effects of HRI activators on triglyceride accumulation and insulin signaling in hepatocytes, the N,N-diarylurea BTdCPU was used. HRI is a kinase that phosphorylates eIF2 at its Ser 51 residue to execute protein synthesis regulation and thereby HRI activators cause the phosphorylation of eIF2 as previously described (Chen T. et al., ibid). First, the effect of BTdCPU on human Huh-7 hepatocytes exposed to the saturated fatty acid palmitate was explored. Huh-7 cells were incubated for 24 h with BSA (Control, CT), 0.75 mmol/L palmitate (Pal) conjugated with BSA or 0.75 mmol/L palmitate plus 10 mol/L BTdCPU (Pal+BTdCPU). Then, they were stained with Oil Red O that allows selective detection of neutral lipids (primarily triglyceride and cholesterol esters) within Huh-7 cells. Huh-7 cells exposed to palmitate showed a high accumulation of triglycerides, as demonstrated by Oil Red O (ORO) staining, but this accumulation was prevented in the presence of the BTdCPU compound (see FIG. 5). Thus, this assay demonstrates that HRI activation prevents palmitate-induced triglyceride accumulation in human Huh-7 hepatic cells.

[0053] Insulin Signalling in Hepatocytes.

[0054] Inventors examined the insulin signalling pathway by measuring insulin-stimulated Akt phosphorylation. Huh-7 cells were incubated for 24 h with BSA (Control, CT), 0.75 mmol/L palmitate (Pal) conjugated with BSA, or 0.75 mmol/L palmitate plus 10 mol/L BTdCPU (Pal+BTdCPU). Immunoblot analyses of total and phosphorylated Akt was carried out. FIG. 6 shows how, when cells were stimulated with insulin (positive control, CT+) for 10 min, this hormone increased the levels of phosphorylated Akt compared to control cells not exposed to insulin (negative control CT). However, in cells exposed to the saturated fatty acid palmitate, insulin did not increase the phosphorylation of Akt, indicating that the insulin signalling pathway was attenuated. Interestingly, the BTdCPU compound partially restored the reduction in insulin-stimulated Akt phosphorylation caused by palmitate (see, FIG. 6), showing that this drug treatment prevents palmitate-induced insulin resistance. Thus, this assay demonstrates that HRI activation prevents palmitate-induced insulin resistance in human Huh-7 hepatic cells.

[0055] Glucose Tolerance.

[0056] T2DM is characterized by glucose intolerance, which is contributed to by peripheral (muscle, fat, and liver) insulin resistance as well as islet 1-cell dysfunction (cf. Andrikopoulos S. et al., Evaluating the glucose tolerance test in mice, Am. J. Physiol. Endocrinol. Metab., 2008, vol. 295, pp. E1323-32). The glucose tolerance test (GTT) is used in clinical practice and research to identify individuals with impaired glucose tolerance by assessing the disposal of a glucose load. It is important to acknowledge that the GTT is the only means of identifying impaired glucose tolerance, which is considered a prediabetic state (cf. Andrikopoulos S. et al., ibid). The standard presentation of results from GTTs is a description of blood glucose levels over time after the glucose administration. Generally, a time course of absolute glucose levels is presented. When using diabetic or insulin-resistant models (such as the HFD-fed mouse), a time course of absolute glucose levels should still be presented along with a calculation of the AUC above baseline glucose. In this assay, the effect of the BTdCPU compound on glucose tolerance in mice fed a high-fat diet (HFD) was examined. For the assay, mice were fed a standard chow (CT), a HFD for three weeks (HFD), or a HFD for three weeks plus BTdCPU during the last week (HFD+BTdCPU). Mice fed a standard chow and half of the mice fed the HFD received one daily i.p. administration of DMSO (vehicle) for the last week. The rest of the mice fed the HFD received one daily i.p. administration of BTdCPU (70 mg kg.sup.1 day.sup.1) for the last week. As expected, the HFD significantly increased the AUC, indicating the presence of glucose intolerance (see FIG. 7). Of note, BTdCPU administration to mice fed the HFD prevented the increase in the AUC above baseline glucose and these mice showed an AUC similar to that observed in mice fed a standard diet (CT), demonstrating that BTdCPU prevents HFD-induced glucose intolerance. Thus, it is concluded that HRI activation prevents HFD-induced glucose intolerance.

[0057] Steatosis.

[0058] Inventors examined whether the treatment with an HRI activator (BTdCPU) prevented the development of steatosis induced by a HFD. Histological analysis of a liver biopsy remains the gold standard for assessing the degree of steatosis. ORO staining and eosin-hematoxylin (H&E) staining of liver sections were performed. Mice fed a standard chow and half of the mice fed the HFD received one daily i.p. administration of DMSO (vehicle) for the last week. The rest of the mice fed the HFD received one daily i.p. administration of BTdCPU (70 mg kg.sup.1 day.sup.1) for the last week. Compared to mice fed a standard diet, livers of mice fed a HFD showed the presence of fat droplets stained of red in the assessment of ORO sections (FIG. 8A, upper row). Similarly, analysis of H&E sections showed the presence of macrovesicular steatosis (FIG. 8A, lower row). When mice were fed with the HFD and treated with the HRI activator hepatic steatosis was reversed as demonstrated by ORO and H&E staining. Finally, hepatic lipids were extracted and hepatic triglyceride levels were assessed by using a commercially available kit (TR0100, Sigma). Livers of mice fed the HFD showed a significant increase in the levels of hepatic triglyceride and this increase was completely blunted when mice were treated with BTdCPU (FIG. 8B). Overall, these findings indicate that HRI activation prevents HFD-induced steatosis.

[0059] FGF21 Expression in Hepatocytes.

[0060] When inventors examined the effects of HRI activators on FGF21 expression in human Huh-7 hepatocytes they observed that compounds BTdCPU, I-36, I-37 and I-38 showed a significant higher increase than compound BTCtFPU. In addition, compounds I-36, I-37 and I-38 induced a significant higher increase in FGF21 expression than compound BTdCPU (cf. FIG. 9A). Moreover, treatment with compound I-26 led to a higher expression in FGF21 than BTCtFPU, whereas compounds I-26 and I-29 showed a higher increase in FGF21 expression than that observed for compound BTdCPU (cf. FIG. 9B).

REFERENCES CITED IN THE APPLICATION

Patent Documents

[0061] U.S. Pat. No. 3,073,861 [0062] U.S. Pat. No. 7,932,416 [0063] U.S. Pat. No. 8,937,088

Non-Patent Documents

[0064] Acharya P. et al., Hepatic heme-regulated inhibitor eukaryotic initiation factor 2a kinase: A protagonist of heme-mediated translational control of CYP2B enzymes and a modulator of basal endoplasmic reticulum stress tone, Mol. Pharmacol, 2010, vol. 77, pp. 575-592. [0065] Andrikopoulos S. et al., Evaluating the glucose tolerance test in mice, Am. J. Physiol. Endocrinol. Metab., 2008, vol. 295, pp. E1323-32. [0066] Chen T. et al., Chemical genetics identify eIF2 kinase heme-regulated inhibitor as an anticancer target, Nature Chem. Biol., 2011, vol. 7, pp. 610-616, and Supp. Information. [0067] Denoyelle S. et al., In vitro inhibition of translation initiation by N,N-diarylureas potential anti-cancer agents, Bioorg. Med. Chem. Lett., 2012, vol. 22, pp. 402-409. [0068] Karagiannidis L. E. et al., Highly effective yet simple transmembrane anion transporter based upon ortho-phenylenediamine bis-ureas, Chem. Commun., 2014. vol. 50, pp. 12050-12053, Electronic Supplementary Information. [0069] Lu L. et al., Translation initiation control by heme-regulated eukaryotic initiation factor 2a kinase in erythroid cells under cytoplasmic stresses, Mol. Cell. Biol., 2001, vol. 21, pp. 7971-7980. [0070] Marchesini G. et al., Nonalcoholic fatty liver disease: a feature of the metabolic syndrome, Diabetes, 2001, vol. 50, pp. 1844-1850. [0071] Musso G. et al., Metaanalysis: natural history of non-alcoholic fatty liver disease and diagnostic accuracy of non-invasive tests for liver disease severity, Ann. Med., 2011 vol. 43, pp. 617-649.