ACC INHIBITOR AND USE THEREOF
20230002405 · 2023-01-05
Assignee
Inventors
Cpc classification
A61P25/14
HUMAN NECESSITIES
A61P25/18
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P5/50
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
International classification
Abstract
The present invention provides a compound suitable for use as an acetyl CoA carboxylase (ACC) inhibitor, specifically, a thienopyridine derivative, and use of the compound in the preparation of drugs for treating metabolic disorders, cancers or other proliferative disorders, and nonalcoholic steatohepatitis (NASH).
Claims
1. A compound of formula c: ##STR00053## or a pharmaceutically acceptable salt thereof, wherein, R.sub.1 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.2 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.3 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.1 and R.sub.3, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl; and R.sub.4 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.4 and R.sub.2, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl.
2. The compound according to claim 1, wherein R.sub.2 and R.sub.4 are each independently H, and R.sub.1 and R.sub.3 form a four-membered ring.
3. The compound according to claim 1, wherein R.sub.1 and R.sub.3 are each independently H, and R.sub.2 and R.sub.4 form a four-membered ring.
4-14. (canceled)
15. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00054## ##STR00055##
16. A composition which comprises the compound of formula c or a pharmaceutically acceptable salt thereof according to claim 1.
17. The composition according to claim 16, wherein the composition is a pharmaceutical composition.
18. The composition according to claim 16, wherein the composition further comprises a pharmaceutically acceptable carrier.
19. The composition according to claim 16, wherein the formulation of the composition is an oral preparation or intravenous preparation.
20. A method for (a) inhibiting acetyl-CoA carboxylase; (b) treating disorders mediated by ACC; (c) treating a metabolic disease; (d) treating a cancer or other proliferative disorders; and/or (e) treating non-alcoholic steatohepatitis; which the method comprises administering the compound of formula c or a pharmaceutically acceptable salt thereof according to claim 1 or the composition according to claim 16 to a patient.
21. The method according to claim 20, wherein the acetyl-CoA carboxylase is selected from group consisting of acetyl-CoA carboxylase 1, acetyl-CoA carboxylase 2, and combinations therefore.
22. The method according to claim 20, wherein the metabolic disease is obesity, dyslipidemia or hyperlipidemia.
23. The method according to claim 20, wherein the cancer is selected from group consisting of liver cancer, cholangiocarcinoma, angiosarcoma, hemangiosarcoma, melanoma, liposarcoma, lung cancer, breast cancer, prostate cancer, leukemia, kidney cancer, esophageal cancer, brain cancer, lymphoma, colon cancer, and combinations therefore.
24. The method according to claim 23, wherein the liver cancer cell is HepG2 cell.
Description
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0123] The embodiments of the present invention will be described in more detail below in conjunction with the schematic diagrams. The advantages and features of the present invention would be clearer based on the following description. It should be noted that the accompanying drawings are provided at non-accurate scale in a very simple form only for the purpose of conveniently and clearly assisting the explanation of the examples of the present invention.
Synthesis of Intermediate (R)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyran 3.61
[0124] ##STR00004##
Method A:
Synthesis of Compound 3.2
[0125] In the presence of nitrogen, DMSO is put in to a round-bottom flask. Then NaH is added. The mixture is stirred at room temperature. At room temperature, trimethylsulfoxide iodide is added to the mixture in batches. A resulting solution is stirred. A compound 3.1 is dropwise added to the mixture, and a reaction mixture is stirred at room temperature, and then quenched by adding NH.sub.4Cl (aqueous solution). A resulting solution is extracted with EtOAc; and organic layers are combined, dried with anhydrous Na.sub.2SO.sub.4 and concentrated in vacuum to obtain 3.2 as a yellow oil.
Synthesis of Compound 3.3
[0126] In the presence of nitrogen, tetrahydropyran-4-ol and FeCl.sub.3 is added to a round-bottom flask. Then, 3.2 is dropwise added. A reaction mixture is stirred at room temperature. A resulting solution is diluted with H.sub.2O, and then extracted with EtOAc. Organic layers are combined and a solvent is removed in vacuum.
[0127] A crude product is purified by column chromatography to obtain 3.3 as a white solid.
Synthesis of Compound 3.41
[0128] 3.3, toluene, CAL-B, and vinyl butyrate are added to a round-bottom flask. A reaction mixture is stirred at room temperature. Filtration is carried out to remove solids, and a filtrate is concentrated in vacuum to obtain 3.41 as a colorless oil.
Synthesis of Compound 3.51
[0129] 3.41, methanol, water, and NaOH are added to a round-bottom flask. A reaction mixture is stirred at room temperature and then quenched by addition of acetic acid. A resulting mixture is concentrated in vacuum, and then extracted with EtOAc. Organic layers are combined and concentrated in vacuum. A crude product is purified by column chromatography to obtain 3.51 as a white solid.
Synthesis of Compound 3.61
[0130] In the presence of nitrogen, 3.51, CBr.sub.4, CH.sub.2Cl.sub.2, and PPh.sub.3 are added to a round-bottom flask. A reaction mixture is stirred at room temperature overnight and then concentrated in vacuum. A crude product is purified by column chromatography to obtain 3.61 as a yellow oil.
Synthesis of Intermediate
(S)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyran 3.62
[0131] ##STR00005##
Method B
Method B:
Synthesis of Compound 3.2
[0132] In the presence of nitrogen, DMSO is put in to a round-bottom flask. Then NaH is added. The mixture is stirred at room temperature. At room temperature, trimethylsulfoxide iodide is added to the mixture in batches. A resulting solution is stirred. A compound 3.1 is dropwise added to the mixture, and a reaction mixture is stirred at room temperature, and then quenched by adding NH.sub.4Cl (aqueous solution). A resulting solution is extracted with EtOAc; and organic layers are combined, dried with anhydrous Na.sub.2SO.sub.4 and concentrated in vacuum to obtain 3.2 as a yellow oil.
Synthesis of Compound 3.3
[0133] In the presence of nitrogen, tetrahydropyran-4-ol and FeCl.sub.3 is added to a round-bottom flask. Then, 3.2 is dropwise added. A reaction mixture is stirred at room temperature. A resulting solution is diluted with H.sub.2O, and then extracted with EtOAc. Organic layers are combined and a solvent is removed in vacuum. A crude product is purified by column chromatography to obtain 3.3 as a white solid.
Synthesis of Compound 3.42
[0134] 3.3, toluene, CAL-B, and vinyl butyrate are added to a round-bottom flask. A reaction mixture is stirred at room temperature. Filtration is carried out to remove solids, and a filtrate is concentrated in vacuum to obtain 3.42 as a colorless oil.
Synthesis of Compound 3.52
[0135] 3.42, methanol, water, and NaOH are added to a round-bottom flask. A reaction mixture is stirred at room temperature and then quenched by addition of acetic acid. A resulting mixture is concentrated in vacuum, and then extracted with EtOAc. Organic layers are combined and concentrated in vacuum. A crude product is purified by column chromatography to obtain 3.52 as a white solid.
Synthesis of Compound 3.62
[0136] In the presence of nitrogen, 3.52, CBr.sub.4, CH.sub.2Cl.sub.2, and PPh.sub.3 are added to a round-bottom flask. A reaction mixture is stirred at room temperature overnight and then concentrated in vacuum. A crude product is purified by column chromatography to obtain 3.62 as a yellow oil.
Synthesis of Compound of Formula e
[0137] ##STR00006##
Method C
Method C:
[0138] Oxazole and a compound d are added to THF, stirred, dried, filtered and purified to obtain a compound of formula e. R.sub.6 is C.sub.3-6 alkyl.
Synthesis of Compound of Formula f
[0139] ##STR00007##
Method F
Method F:
[0140] Oxazole and a compound d′ are added to THF, stirred, dried, filtered and purified to obtain a compound of formula f. R.sub.6 is C.sub.3-6 alkyl.
Synthesis of Compound of Formula c
[0141] ##STR00008## ##STR00009##
Method D:
[0142] An intermediate I-1 can be prepared by using a method known in the prior art, and further reaction is conducted according to method D to obtain a compound of general formula I. R.sub.1 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.2 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.3 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.1 and R.sub.3, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl; and R.sub.4 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.4 and R.sub.2, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl. R.sub.5 is C.sub.1-6 alkyl. R.sub.6 is C.sub.3-6 alkyl.
Method G:
[0143] ##STR00010## ##STR00011##
[0144] An intermediate I-1 can be prepared by using a method known in the prior art, and further reaction is conducted according to method G to obtain compounds of general formulas I-I-1 and I-I-2. R.sub.1 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.2 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.3 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.1 and R.sub.3, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl; and R.sub.4 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.4 and R.sub.2, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl. R.sub.5 is C.sub.1-6 alkyl. R.sub.6 is C.sub.3-6 alkyl.
Method E:
[0145] ##STR00012##
[0146] An intermediate II-1 can be prepared by using a method known in the prior art, and further reaction is conducted according to method E to obtain a compound of general formula II.R.sub.1 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.2 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy; R.sub.3 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.1 and R.sub.3, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl; and R.sub.4 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.1-6 alkoxy, or R.sub.4 and R.sub.2, together with a carbon atom bonded thereto, form 3-6 membered cycloalkyl. R.sub.5 is C.sub.1-6 alkyl. R.sub.6 is C.sub.3-6 alkyl.
##STR00013##
[0147] EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) and NH.sub.4Cl are added to a solution of compound c in DCM. A reaction mixture is stirred. After no residue of starting materials is shown by TLC, water is added, a separated organic layer is dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains are purified by preparative HPLC to obtain a compound c′ as a white solid.
[0148] In certain embodiments, the compound of formula c of the present invention is selected from Table 1.
TABLE-US-00001 TABLE 1 Exemplary compounds of formula c
Example 1
Synthesis of Compound (R)-3-((1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane carboxylic Acid c-1
[0149] ##STR00024##
Synthesis of 3-(aminomethyl)cyclobutane ethyl formate1.2
[0150] ##STR00025##
[0151] At 0° C., H.sub.2SO.sub.4 (0.1 g, 98%) was slowly added to a solution of compound 1.1 (1.0 g, 7.75 mmol) in EtOH (10 mL). A resulting mixture was stirred for 6 hours at 80° C. Then, a reaction mixture was cooled to room temperature, a saturated NaHCO.sub.3 solution was added, and the mixture was extracted to THF. Organic layers were combined, dried over MgSO.sub.4, filtered and concentrated to obtain a compound 1.2 (980 mg) as a yellow oil.
[0152] Mass spectrum: 158 [M+H].sup.+
Synthesis of 2-(3-((3-(ethyoxylcarbonyl)cyclobutyl)methyl)uramido)-4-methylthiophene-3-ethyl carboxylate 1.3
[0153] ##STR00026##
[0154] BTC (622 mg, 2.10 mmol) and pyridine (0.5 mL) were added to a solution of a compound 2-amino-4-methylthiophene-3-ethyl carboxylate (1.30 g, 7.01 mmol) in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 hours, and then the compound 1.2 (1.0 g, 6.37 mmol) was added to the reaction mixture. A resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=4/1) to obtain a compound 1.3 (1.72 g) as a white solid.
[0155] Mass spectrum: 369 [M+H].sup.+
Synthesis of 3-((5-methyl-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane ethyl formate1.4
[0156] ##STR00027##
[0157] NaOEt (449 mg, 6.51 mmol) was added to a solution of the compound 1.3 (800 mg, 2.17 mmol) in EtOH (20 mL). The reaction mixture was stirred at room temperature for 8 hours, then diluted with H.sub.2O (50 mL), and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=5/1) to obtain a compound 1.4 (510 mg) as a white solid.
[0158] Mass spectrum: 323 [M+H].sup.+
Synthesis of 3-((6-bromo-5-methyl-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutaneethyl carboxylate 1.5
[0159] ##STR00028##
[0160] NBS (277 mg, 1.55 mmol) was added to a solution of the compound 1.4 (500 mg, 1.55 mmol) in DCM (10 mL). A reaction mixture was stirred at room temperature for 3 hours. Then, the reaction mixture was diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=5/1) to obtain a compound 1.5 (520 mg) as a white solid.
[0161] Mass spectrum: 401,403 [M+H].sup.+
Synthesis of (R)-ethyl3-((6-bromo-1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane carboxylate 1.61
[0162] ##STR00029##
[0163] (R)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyran3.61 (329 mg, 1.05 mmol) and K.sub.2CO.sub.3 (207 mg, 1.50 mmol) were added to a solution of the compound 1.5 (200 mg, 0.5 mmol) in DMF (3 mL). The reaction was stirred at 150° C. for 3 hours under microwave, then cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=4/1) to obtain a compound 1.61 (262 mg) as a white solid.
[0164] Mass spectrum: 635 [M+H].sup.+
Synthesis of (R)-ethyl3-((1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol)-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane carboxylate 1.71
[0165] ##STR00030##
[0166] 2-(triisopropylsilyl)oxazole (101.6 mg, 0.473 mmol), Pd(OAc).sub.2 (6.9 mg, 0.032 mmol), CuI (23.9 mg, 0.126 mmol), CsF (143.7 mg, 0.945 mmol) and CatCxium A (24.2 mg, 0.065 mmol) were added to a solution of the compound 1.61 (200 mg, 0.315 mmol) in DMF (6 mL). Then, a reaction mixture was stirred at 120° C. for 4 hours in the presence of nitrogen. The reaction mixture was cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=3/1) to obtain a compound 1.71 (106.5 mg) as a white solid.
[0167] Mass spectrum: 624 [M+H].sup.+
Synthesis of (R)-3-((1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane carboxylic Acid c-1
[0168] ##STR00031##
[0169] NaOH (19.3 mg, 0.482 mmol) was added to a solution of the compound 1.71 (100 mg, 0.161 mmol) in MeOH (3 mL) and water (1 mL). A reaction mixture was stirred at 40° C. for 2 hours. The pH of the reaction mixture was adjusted to 7 with 5% HCl, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain the remains which were purified by preparative HPLC to obtain a compound c-1 (52.5 mg) as a white solid.
[0170] Mass spectrum: 596 [M+H].sup.+
[0171] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.95 (br s, 1H), 8.23 (s, 1H), 7.48 (d, J=7.2 Hz, 1H), 7.33-7.29 (m, 1H), 7.31-7.29 (m, 1H), 7.05-6.95 (m, 1H), 6.51 (s, 1H), 5.31 (d, J=5 Hz, 1H), 4.10-4.02 (m, 3H), 3.96 (s, 3H), 3.74-3.49 (m, 2H), 3.34-3.32 (m, 1H), 3.31-3.30 (m, 2H), 2.89 (s, 3H), 2.39-2.37 (m, 2H), 2.32-2.17 (m, 2H), 2.11-2.08 (m, 2H), 1.32-1.23 (m, 4H).
Example 2
Synthesis of (S)-3-((1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)methyl)cyclobutane carboxylic Acid c-2
[0172] ##STR00032##
[0173] For the synthesis of c-2, a reference can be similarly referred to the method for synthesizing c-1.
Example 3
Synthesis of Compound (R)-2-(3-(1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-m ethyl-6-(oxazol-2-yl)-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetic Acid c-3
[0174] ##STR00033##
Synthesis of 2-(3-((t-butyloxycarbonyl)amino)cyclobutylene)ethyl acetate 2.2
[0175] ##STR00034##
[0176] At 0° C., NaH (230.9 mg, 5.77 mmol) was added to a solution of 2-(diethyoxylphosphino)ethyl acetate (1.0 g, 4.81 mmol) in THF (20 mL). The reaction mixture was stirred at room temperature for 1 hour, and then, the compound 2.1 (978.8 mg, 5.29 mmol) was added to the reaction mixture. The resulting mixture was stirred at room temperature for 6 hours, then diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=6/1) to obtain a compound 2.2 (1.05 g) as a white solid.
[0177] Mass spectrum: 256 [M+H].sup.+
Synthesis of 2-(3-((t-butyloxycarbonyl)amino)cyclobutyl)ethyl acetate 2.3
[0178] ##STR00035##
[0179] 10% Pd/C (80 mg) was added to a solution of the compound 2.2 (800 mg, 3.13 mmol) in EtOH (15 mL). A reaction mixture was stirred for 8 hours at room temperature in the presence of H.sub.2 (1 atm). Diisopropyl ether wad added to the reaction mixture, which was then filtered to collect a compound 2.3 (750 mg) as a yellow oil.
[0180] Mass spectrum: 258 [M+H].sup.+
Synthesis of 2-(3-aminocyclobutyl)ethyl acetate 2.4
[0181] ##STR00036##
[0182] A solution of 4N hydrochloric acid in dioxane (10 mL) was added to a solution of the compound 2.3 (650 mg, 2.53 mmol) in 1,4-dioxane (10 mL). A reaction mixture was stirred at room temperature overnight. Diethyl ether was added to the reaction mixture, which was then filtered to harvest a compound 2.4 (385 mg) as a white solid.
[0183] Mass spectrum: 158 [M+H].sup.+
Synthesis of 2-(3-(3-(2-ethyoxyl-2-oxyethyl)cyclobutyl)uramido)-4-methylthiophene-3-ethyl carboxylate 2.5
[0184] ##STR00037##
[0185] BTC (312 mg, 1.05 mmol) and pyridine (0.5 mL) were added to a coluation of the compound 2-amino-4-methylthiophene-3-ethyl carboxylate (647 mg, 3.50 mmol) in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 hours, then the compound 2.4 (500 mg, 3.18 mmol) was added to the reaction mixture. A resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=4/1) to obtain a compound 2.5 (890 mg) as a white solid.
[0186] Mass spectrum: 369 [M+H].sup.+
Synthesis of 2-(3-(5-methyl-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobut yl)ethyl acetate 2.6
[0187] ##STR00038##
[0188] NaOEt (281 mg, 4.07 mmol) was added to a solution of the compound 2.5 (500 mg, 1.36 mmol) in EtOH (10 mL). After being stirred at room temperature for 8 hours, the reactants were diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=5/1) to obtain a compound 2.6 (368 mg) as a white solid.
[0189] Mass spectrum: 323 [M+H].sup.+
Synthesis of 2-(3-(6-bromo-5-methyl-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)ethyl acetate 2.7
[0190] ##STR00039##
[0191] NBS (204.5 mg, 1.14 mmol) was added to a solution of the compound 2.6 (368 mg, 1.14 mmol) in DCM (10 mL). A reaction mixture was stirred at room temperature for 3 hours. Then, the reaction mixture was diluted with water, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=5/1) to obtain a compound 2.7 (410 mg) as a white solid.
[0192] Mass spectrum: 401,403 [M+H].sup.+
Synthesis of (R)-ethyl2-(3-(6-bromo-1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl)ethyl-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetate 2.81
[0193] ##STR00040##
[0194] (R)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyrane 3.61 (688.6 mg, 2.2 mmol) and K.sub.2CO.sub.3 (414 mg, 3.0 mmol) were added to a solution of the compound 2.7 (400 mg, 1.0 mmol) in DMF (5 mL). The reactants were stirred at 150° C. for 3 hours under microwave, then cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=4/1) to obtain a compound 2.81 (412 mg) as a white solid.
[0195] Mass spectrum: 635 [M+H].sup.+
Synthesis of (R)-ethyl2-(3-(1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetate 2.91
[0196] ##STR00041##
[0197] 2-(triisopropylsilyl)oxazole (101.6 mg, 0.473 mmol), Pd(OAc).sub.2 (6.9 mg, 0.032 mmol), CuI (23.9 mg, 0.126 mmol), CsF (143.7 mg, 0.945 mmol) and CatCxium A (24.2 mg, 0.065 mmol) were added to a solution of the compound 2.81 (200 mg, 0.315 mmol) in DMF (6 mL). Then, a reaction mixture was stirred at 120° C. for 4 hours. The reaction mixture was cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=3/1) to obtain a compound 2.91 as a white solid.
[0198] Mass spectrum: 624 [M+H].sup.+
Synthesis of compound (R)-2-(3-(1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-m ethyl-6-(oxazol-2-yl)-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetic acid c-3
[0199] ##STR00042##
[0200] NaOH (17.3 mg, 0.433 mmol) was added to a solution of the compound 2.91 (90 mg, 0.145 mmol) in MeOH (3 mL) and water (1 mL). A reaction mixture was stirred at 40° C. for 2 hours. The pH of the reaction mixture was adjusted to 7 with 5% HCl, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain the remains, which were purified by HPLC to obtain a compound c-3 as a white solid.
[0201] Mass spectrum: 596 [M+H].sup.+
[0202] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.70 (s, 1H), 7.56-7.52 (m, 1H), 7.59-7.57 (m, 1H), 7.27 (d, J=4.0 Hz, 1H), 7.21 (s, 1H), 7.04-7.00 (m, 1H), 6.86 (d, J=8.0 Hz, 1H), 5.40-5.36 (m, 1H), 5.14 (s, 1H), 4.16-4.06 (m, 2H), 3.85 (s, 3H), 3.76-3.68 (m, 2H), 3.41 (d, J=4.0 Hz, 1H), 3.34-3.31 (m, 3H), 2.69-2.58 (m, 4H), 2.13 (s, 1H), 1.82-1.79 (m, 4H), 1.70 (d, J=5.6 Hz, 2H), 1.58 (d, J=9.2 Hz, 2H).
Example 4
Synthesis of Compound c′-3
[0203] ##STR00043##
[0204] EDCI (27 mg, 0.14 mmol) and DMAP (25.8 mg, 0.21 mmol) and NH.sub.4Cl (15 mg, 0.28 mmol) were added to a solution of the compound c-3 (59.6 mg, 0.10 mmol) in 1.2 mL DCM. A reaction mixture was stirred at 40° C. for 2 hours. After no residue of starting materials was shown by TLC, 5 mL of water was added, a separated organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains are purified by preparative HPLC to obtain a compound c′-3 (27.8 mg) as a white solid.
[0205] Mass spectrum: 595 [M+H]+
Example 5
Synthesis of Compound (S)-ethyl 2-(3-(1-(2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxy-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetate c-4
[0206] ##STR00044##
[0207] For the synthesis of c-4, a reference can be similarly referred to the method for synthesizing c-3.
Example 6
Synthesis of 2-(triisopropylsilyl)oxazole
[0208] ##STR00045##
[0209] A solution of n-butyllithium (11.6 mL, 29 mmol, in 2.5 M ethane solution) was dropwise added to a solution of oxazole (1.0 g, 14.5 mmol) in THF (15 mL) at −78° C. in the presence of N.sub.2. After the reaction mixture was stirred for another 45 minutes at −78° C., triisopropylsilyltrifluoromethanesulfonate (6.65 g, 21.8 mmol) was slowly added to the reaction mixture at −78° C. After the addition was completed, the reaction mixture was slowly heated to room temperature, and stirred for 12 hours. The reaction mixture was diluted with water, and extracted with EA. The organic layers were combined, washed with saline, and dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=20/1) to obtain a compound 2-(triisopropylsilyl)oxazol (1.1 g) as a yellow oily matter.
Example 7
Chiral Separation
[0210] The compound 2.7 was chirally separated into 2.7-1 (2.0 g, a trans-isomer) and 2.7-2 (4.2 g, a cis-isomer).
[0211] Mass spectrum: 401, 403 [M+H].sup.+
Synthesis of 2-((1R,3r)-3-(6-bromo-1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)ethyl acetate 2.81-1
[0212] ##STR00046##
[0213] (R)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyrane 3.61 (2.0 g, 6.4 mmol) and K.sub.2CO.sub.3 (1.5 g, 11 mmol) were added to a solution of the compound 2.7-1 (1.4 g, 3.5 mmol) in DMF (20 mL). The reactants were stirred for 6 hours at 138° C. in an airtight tube, then cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=10/1) to obtain a compound 2.81-1 (1.6 g) as a colorless oil.
[0214] Mass spectrum: 637 [M+H].sup.+
Synthesis of 2-((1R,3r)-3-(1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)eth yl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxp-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)ethyl acetate 2.91-1
[0215] ##STR00047##
[0216] 2-(tributylstannyl)oxazol (1.7 g, 4.7 mmol) and Pd(PPh.sub.3).sub.4 (0.5 g, 0.4 mmol) were added to a solution of the compound 2.81-1 (1 g, 1.6 mmol) in toluene (20 mL). Then, the reaction mixture was stirred for 10 hours at 110° C. The reaction mixture was cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=3/1) to obtain a compound 2.91-1 (700 mg) as a yellow solid.
[0217] Mass spectrum: 624 [M+H].sup.+
Synthesis of Compound 2-((1R,3r)-3-(1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)eth yl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetic Acid c-3-1
[0218] ##STR00048##
[0219] LiOH (300 mg, 7.1 mmol) was added to a solution of the compound 2.91-1 (500 mg, 0.8 mmol) in EtOH (15 mL) and water (3 mL). The reaction mixture was stirred at 50° C. for 2 hours. The pH of the reaction mixture was adjusted to 7 with 5% HCl, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain the remains, which were purified by silica-gel flash chromatography (DCM/MeOH=50/1) to obtain a compound c-3-1 (300 mg) as a white solid.
[0220] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ: 11.99 (br s, 1H), 8.22 (s, 1H), 7.50-7.48 (m, 1H), 7.38 (d, J=0.8 Hz, 1H), 7.32-7.27 (m, 1H), 7.05-6.95 (m, 2H), 5.52-5.43 (m, 1H), 5.31 (t, J=6.4 Hz, 1H), 4.10-3.95 (m, 2H), 3.78 (s, 3H), 3.60-3.55 (m, 1H), 3.52-3.49 (m, 1H), 3.41-3.36 (m, 1H), 3.28-3.20 (m, 2H), 2.917-2.88 (m, 2H), 2.78 (s, 3H), 2.71-2.67 (m, 1H), 2.54-2.50 (m, 2H), 2.03-1.97 (m, 2H), 1.65-1.63 (m, 2H), 1.36-1.31 (m, 1H), 1.20-1.16 (m, 1H).
[0221] MS calculated value: 595; MS experimental value: 596 [M+H].sup.+.
Synthesis of 2-((1S,3s)-3-(6-bromo-1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)ethyl)-5-methyl-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)ethyl acetate 2.81-2
[0222] ##STR00049##
[0223] (R)-4-(2-bromo-1-(2-methoxyphenyl)ethyoxyl)tetrahydro-2H-pyrane 3.61 (3.2 g, 10 mmol) and K.sub.2CO.sub.3 (2 g, 14 mmol) were added to a solution of the compound 2.7-2 (2.0 g, 5.0 mmol) in DMF (20 mL). The reactants were stirred for 6 hours at 138° C. in an airtight tube, then cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=10/1) to obtain a compound 2.81-1 (3 g) as a colorless oil.
[0224] Mass spectrum: 637 [M+H]+
Synthesis of 2-((1S,3s)-3-(1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)eth yl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)ethyl acetate 2.91-2
[0225] ##STR00050##
[0226] 2-(tributylstannyl)oxazol (1.7 g, 4.7 mmol) and Pd(PPh.sub.3).sub.4 (0.5 g, 0.4 mmol) were added to a solution of the compound 2.81-2 (1 g, 1.6 mmol) in toluene (20 mL). Then, the reaction mixture was stirred for 10 hours at 110° C. The reaction mixture was cooled to room temperature, diluted with H.sub.2O, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated. The remains were purified by silica-gel flash chromatography (PE/EA=3/1) to obtain a compound 2.91-2 (600 mg) as a yellow solid.
[0227] Mass spectrum: 624 [M+H]+
Synthesis of Compound 2-((1S,3s)-3-(1-((R)-2-(2-methoxyphenyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)eth yl)-5-methyl-6-(oxazol-2-yl)-2,4-dioxo-1,2-dihydrothieno[2,3-d]pyrimidine-3(4H)-yl)cyclobutyl)acetic Acid c-3-2
[0228] ##STR00051##
[0229] LiOH (338 mg, 8.0 mmol) was added to a solution of the compound 2.91-2 (1 g, 1.6 mmol) in EtOH (15 mL) and water (3 mL). The reaction mixture was stirred at 50° C. for 2 hours. The pH of the reaction mixture was adjusted to 7 with 5% HCl, and extracted with EA. Organic layers were combined, washed with saline, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain the remains, which were purified by silica-gel flash chromatography (DCM/MeOH=50/1) to obtain a compound c-3-2 (600 mg) as a white solid.
[0230] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ:11.98 (br s, 1H), 8.22 (s, 1H), 7.50-7.48 (m, 1H), 7.38 (d, J=0.4 Hz, 1H), 7.32-7.27 (m, 1H), 7.05-6.97 (m, 2H), 5.30 (t, J=6.4 Hz, 1H), 5.13-5.04 (m, 1H), 4.11-4.00 (m, 2H), 3.78 (s, 3H), 3.61-3.57 (m, 1H), 3.53-3.48 (m, 1H), 3.41-3.35 (m, 1H), 3.28-3.20 (m, 2H), 2.78 (s, 3H), 2.50-2.45 (m, 3H), 2.43-2.31 (m, 4H), 1.66-1.62 (m, 2H), 1.38-1.31 (m, 1H), 1.23-1.15 (m, 1H).
[0231] MS calculated value: 595; MS experimental value: 596 [M+H].sup.+.
Example 8
Synthesis of 2-(tributylstannyl)oxazole
[0232] 2-(tributylstannyl)oxazole was prepared by a method similar to that of Example 6.
Example 9
In Vitro Assay of Acetyl-CoA Carboxylase (ACC) Inhibition
[0233] An exemplary procedure for the in vitro ACC inhibition assay, which can be used to determine the inhibitory action of the compounds of the invention toward either ACC1 or ACC2, is as follows. The ADP-Glo™ kinase assay kit from Promega was used. The ADP-Glo™ kinase assay is a luminescent ADP detection assay to measure enzymatic activity by quantifying the amount of ADP produced during an enzyme reaction. The assay was performed in two steps; first, after the enzyme reaction, an equal volume of ADP-Glo™ reagent was added to terminate the reaction and deplete the remaining ATP. Then, the kinase detection reagent was added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction.
[0234] Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve. The detailed procedure was as follows. 4.5 μL of a working solution was added to a 384-well plate. The compound was diluted at 1:3 in succession in DMSO. 0.5 μL of diluted compound solution was added to a 384-well white Optiplate assay plate. The plates were incubated at room temperature for 15 minutes. 5 μL of substrate working solution was added to each well to initiate the reaction. A final ACC reaction solution consisted of: 0.5 nM ACC, 10 μM ATP, 5 μM acetyl-CoA, and 15 mM NaHCO.sub.3; and the final concentrations of the compound were measured as follows: 1 μM, 0.333 μM, 0.111 μM, 0.037 μM, 0.0123 μM, 0.00411 μM, 0.00137 μM, 0.000457 μM, 0.000152 μM, and 0.000051 μM. The plates were incubated at room temperature for 30 minutes. 10 μL of ADP-Glo™ reagent was added, and the plates were incubated at room temperature for 40 minutes. 20 μL of kinase detection reagent was added. The plates were incubated at room temperature for 40 minutes, then read on a Perkin Elmer EnVision 2104 plate reader for luminescence in relative light unit (RLU).
[0235] Data for each concentration, as well as the positive and negative controls were averaged, and the standard deviation was calculated. Percent inhibition was calculated by the formula: 100×(average negative control-compound)/(average negative control-average positive control). The IC.sub.50 for each compound was calculated by fitting the data with a non-linear regression equation: Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC.sub.50−X)×HillSlope)), where X is the log of compound concentration and Y is the percent inhibition.
[0236] The exemplary results of the ACC1 and ACC2 inhibition assay in vitro are listed in Table 2. The compound numbers correspond to those listed in Table 1.
TABLE-US-00002 TABLE 2 Results of ACC1 and ACC2 inhibition assay in vitro Enzymatic activity IC.sub.50 (μM) Compound No. ACC1 ACC2 c-1 0.000487 0.010 c-3 0.000688 0.003 c-3-1 0.000836 0.009 c-3-2 0.001124 0.008
[0237] It can be known from the table that the compounds c-1, c-3, c-3-1, and c-3-2 show good inhibitory properties against ACC1 and ACC2. All other compounds of the present invention have a good inhibitory activity against ACC1 and ACC2, showing equivalent IC.sub.50 values against ACC1 and ACC2 as those of the compounds c-1 and c-3.
Example 10
[2-.SUP.14.C]-Acetate Incorporation Assay in HepG2 Cells
Complete Culture Medium:
[0238] DMEM (GIBCO, catalog number: 11965-092) was used as a complete culture medium for HepG2 cells (ATCC, catalog number: HB-8064). 10% fetal bovein serum (FBS, GIBCO, catalog number: 10099-141), penicillin (100 units/mL) and streptomycin (100 μg/mL) (Pen/Strep, GIBCO, catalog number: 15140-122) were added to a DMEM culture medium.
Cell Culture Condition:
[0239] HepG2 cells were cultured in a 37° C. incubator holding 5% carbon dioxide, and passaged every 2-3 days.
[2-.SUP.14.C]-Acetate Binding Assay:
[0240] The HepG2 cells were spread on a 24-well plate (Corning, catalog number: 3524) at a density of 2×10.sup.5 cells-well, 500 μL per well, and cultured in a 37° C. incubator holding 5% carbon dioxide. On the fourth day of culture, 50 μL of a compound diluted at a gradient of 1:3 was added, with the final concentration of DMSO of 0.5% (v/v); and the cells were cultured for 1 hour in the incubator. 1 μCi [2-.sup.14C]-sodium acetate (Perkin Elmer, catalog number: NEC085H001MC) was added to each well, and culture was conducted for another 5 hours. The culture medium in each well was transferred to a 15 mL centrifugal tube (BD, catalog number: 352096); 0.5 mL of 0.1 M NaOH was added; the cells were blown and washed repeatedly, and then transferred to new centrifugal tubes. Then, 1 mL of ethanol and 0.17 mL of 50% KOH solution were added to each centrifugal tube, which was then treated in a water bath at 90° C. for 1 hour. Next, the mixture was cooled to room temperature; 5 mL of petroleum ether was added to each centrifugal tube, which was turned upside down several times for mixing; the resulting mixture was centrifuged for 5 minutes (Eppendorf, Model: 5702, 1000 rpm); and an upper organic phase was discarded. 1 mL of concentrated hydrochloric acid was added to each centrifugal tube and centrifuged for 5 minutes, and 4 mL of petroleum ether was transferred into a glass tube (18×180 mm). The sample in the glass tube was dried at 64° C., and then dissolved and resuspended by addition of 400 μL of spotting solution (chloroform:n-hexane=1:1). 50 μL of the dissolved sample was drawn by a pipette (BIOHIT, Model: Proline Plus), and spotted onto an Isoplate-96-microwell plate (Perkin Elmer, catalog number: 6005040), and 200 μL of ULTIMA GOLD scintillation solution (Perkin Elmer, catalog number: 77-16061) was added, standing for 5 minutes. Finally, MicroBeta (PerkinElmer, Model: 2450) was used to read scintillation signals.
Data Analysis:
[0241] A 4-parameter logistic model or sigmoidal dose-response model in XLFit 5.3.1.3 (2006-2011 I D Business Solutions Limited) was used to fit IC.sub.50: fit=(A+((B−A)/(1+((C/x){circumflex over ( )}D)))); where the x-axis represented the log of compound concentration, and the y-axis represented count per minute (CPM).
[0242] The exemplary results of the [2-.sup.14C]-acetate binding assay are listed in Table 3. The compound numbers correspond to those listed in Table 1.
TABLE-US-00003 TABLE 3 Results of [2-.sup.14C]-acetate binding assay. Compound No. IC.sub.50 (μM) c-1 0.2317 c-3 0.0185 c-3-l 0.0131 c-3-2 0.0180
[0243] It can be known from Table 3 that the compounds, in particular compounds c-3, c-3-1 and c-3-2, listed in the table above, have good inhibitory properties against HepG2 cells. All other compounds of the present invention have a good inhibitory activity against HepG2 cells, showing equivalent IC.sub.50 values as those of the compounds c-3, c-3-1 and c-3-2.
Example 11
Pharmacokinetic Evaluation of Compounds in Rats
[0244] This example was intended to investigate the pharmacokinetics of the compounds of the present invention and ND630 in SD rats under the conditions of intravenous (iv) and intragastric administration (po). Unless otherwise stated, the experimental reagents and instruments are all commercially available. ND630 was Gilead's acetyl CoA carboxylase (ACC) inhibitor, with a structural formula as follows:
##STR00052##
[0245] Formulation of compound: by taking a physiological saline solution of 5% DMSO+5% Solutol HS 15+90% HP-β-CD (20% w/v) as a solvent, a compound with the final concentration of 0.4 mg/mL was formulated for intravenous administration and a compound with the final concentration of 1 mg/mL was formulated for orally intragastric administration.
[0246] Animal feeding and management: male SD rats aged 6-8 weeks and weighing between 180-250 g were selected and raised in transparent resin plastic rate cages. Before administration, the rats were fasted overnight for at least 16 hours, and the feed supply was restored 4 hours after administration.
[0247] For the intravenous injection administration group, the dose was 2 mg/kg, and 0.2 mL of blood samples was collected from the jugular vein 5 min, 15 min, 30 min, 1 h, 3 h, 5 h, and 8 h after dosage. For the orally intragastric administration group, the dose was 10 mg/kg, and 0.2 mL of blood samples was collected from the jugular vein 15 min, 30 min, 1 h, 3 h, 5 h, 8 h, 12 h, and 24 h after dosage; and liver tissues were collected 1 h, 3 hand 24 h after dosage.
[0248] After the blood were sampled, the plasma was quickly separated by centrifugation at 4000 rpm (10 minutes, 4° C.). After the liver tissues were sampled, a mixture of methanol:water (1:1, v/v) three times the volume of the liver tissues was added to prepare a homogenate. 50 μL of plasma or homogenate sample was drawn, and 200 μL of an internal standard (30 ng/mL terfenadine, and acetonitrile) was added for precipitation. Centrifuging was carried out at 4° C. and 15,400 g for 10 minutes, and a supernatant was taken for analysis.
[0249] The concentration of a target compound was detected by LC-MS/MS. According to the plasma concentration-time curve, pharmacokinetic parameters were calculated by using a non-compartmental model (NCA) of software WinNonlin 7.0. The pharmacokinetic properties of representative compounds are listed in Table 4 below. The analysis results show that the compounds the present invention show good pharmacokinetic properties, for example, high bioavailability F (%), high exposure (AUC), high liver-to-blood ratio (LIP), etc.
TABLE-US-00004 TABLE 4 Pharmacokinetic results ND630 c-3-1 c-3-2 F (%) 35.8 99.6 62.3 C.sub.max 5628 (iv) 4100 (iv) 3113) (iv) (ng/mL) 2882 (po) 7780 (po) 3243 (po) T.sub.max 0.083 (iv) 0.0833 (iv) 0.0833 (iv) (h) 0.4 (po) 0.5 (po) 0.4 (po) AUC
2871 (iv) 3119 (iv) 1386 (iv) (ng .Math. h/mL) 5093 (po) 15688 (po) 4353 (po) t.sub.1/2 1.06 (iv) 1.74 (iv) 0.9 (iv) (h) 0.65 (po) 4.65 (po) 4.82 (po) Vss (mL/kg) 480 826 839 Cl (mL/h/g) 735 642 1503 L/P 5.5-41.9 9.46-17.3 20.6-30.0 F (%): bioavailability C.sub.max: maximum plasma concentration after one dosage T.sub.max: time to reach maximum plasma concentration AUC: area below plasma concentration-time curve, drug uptake t.sub.1/2: half-life period Vss: volume of steady-state performance distribution Cl: blood drug clearance
indicates data missing or illegible when filed