Steroid derivative FXR agonist
11667667 · 2023-06-06
Assignee
Inventors
- Haiying He (Pudong New Area Shanghai, CN)
- Hualing Xiao (Pudong New Area Shanghai, CN)
- Peng Li (Pudong New Area Shanghai, CN)
- Chunyan Du (Pudong New Area Shanghai, CN)
- Zhi Luo (Pudong New Area Shanghai, CN)
- Shuhui Chen (Pudong New Area Shanghai, CN)
Cpc classification
C07J41/0094
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
C07J17/00
CHEMISTRY; METALLURGY
A61K31/575
HUMAN NECESSITIES
C07J41/0061
CHEMISTRY; METALLURGY
C07J9/005
CHEMISTRY; METALLURGY
A61P13/02
HUMAN NECESSITIES
C07J9/00
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
A61K31/58
HUMAN NECESSITIES
International classification
C07J43/00
CHEMISTRY; METALLURGY
C07J33/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a compound represented by formula (I), a tautomer thereof or a pharmaceutically acceptable salt thereof, and relates to applications thereof in the preparation of drugs for treating FXR related diseases. ##STR00001##
Claims
1. A compound represented by formula (III), ##STR00169## wherein, ring A is selected from 5- to 12-membered aryl, 5- to 12-membered heteroaryl, 5_to 6-membered non-aromatic heterocyclyl, or 5- to 6-membered cycloalkyl; and said ring A is optionally substituted with 1, 2 or 3 of R; L is selected from C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, or C.sub.2-6 alkenyl, and said L is optionally substituted with 1, 2 or 3 of R; R is selected from F, Cl, Br, I, OH, CN, NO.sub.2, NH.sub.2, C.sub.1-3 all ylamino; N,N-di(C.sub.1-3 alkyl)amino, C.sub.1-3 alkyl, C.sub.1-3 alkyloxy, or C.sub.1-3 alkylthio, and said R is optionally substituted with 1, 2 or 3 R′; R′ is selected from F, Cl, Br, I, OH, NH.sub.2, NO.sub.2, CN, COOH, Me, Et, CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.3O, CH.sub.3S, NH(CH.sub.3), or N(CH.sub.3).sub.2; said “hetero” represents heteroatom or heteroatomic group, selected from —C(═O)NH—, N, —NH—, —C(═NH)—, —S(═O).sub.2NH—, —S(═O)NH—, —O—, —S—, ═O, ═S, —C(═O)C—, —C(═O)—, —C(═S)—, —S(═O)—, —S(═O).sub.2—, or —NHC(═O)NH—; in any of the above cases, the number of the heteroatom or heteroatomic groups is independently selected from 1, 2 or 3; or a pharmaceutically acceptable salt thereof.
2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R is selected from F, Cl, Br, I, Me, CF.sub.3, CHF.sub.2, CH.sub.2F, Et, OMe, NH(CH.sub.3), or N(CH.sub.3).sub.2.
3. The compound or the pharmaceutically acceptable salt thereof according to claim wherein ring A is selected from phenyl, pyridyl, pyridin-2(1H)-onyl, pyrimidyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, thienyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, isoxazolyl, isothiazolyl, bicyclo[1.1.1]pentyl, benzoxazolyl, benzo[d]isoxazolyl, indazolyl, indolyl, quinolinyl, isoquinolinyl, quinazolinyl, 1H-pyrrolo[2,3-B]pyridyl, indolizinyl benzothiazoyl or benzothienyl, and said ring A is optionally substituted with 1, 2 or 3 of R.
4. The compound or the pharmaceutically acceptable salt thereof according to claim 3, wherein ring A is selected from ##STR00170## ##STR00171## and said ring A is optionally substituted with 1, 2 or 3 R.
5. The compound or the pharmaceutically acceptable salt thereof according to claim 4, wherein ring A is selected from ##STR00172## ##STR00173##
6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is selected from 6- to 10-membered aryl, 5- to 12-membered heteroaryl containing 1 to 2 heteroatoms, 5- to 6-membered non-aromatic heterocyclyl containing 1 to 2 heteroatoms, or 5- to 6-membered cycloalkyl, and said heteroatom is selected from —NH—, N, —O— or —S—, and said ring A is optionally substituted with 1, 2 or 3 of R.
7. The compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein ring A is selected from 6- to 10-membered aryl, 6- to 10-membered heteroaryl containing 1 to 2 heteroatoms, 5- to 6-membered non-aromatic heterocyclyl containing 1 to 2 heteroatoms, or 5- to 6-membered cycloalkyl, and said heteroatom is selected from —NH—N, O or S, and said ring A is optionally substituted with 1, 2 or 3 of R.
8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is selected from ##STR00174## and said ring A is optionally substituted with 1, 2 or 3 R.
9. The compound or the pharmaceutically acceptable salt thereof according to claim 8, wherein ring A is selected from ##STR00175##
10. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein L is selected from C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, C.sub.1-4 alkylamino, C.sub.1-4 alkyl-C(═O)NH—, or C.sub.2-4 alkenyl, and said L is optionally substituted with 1, 2 or 3 R.
11. The compound or the pharmaceutically acceptable salt thereof according to claim 10, wherein L is selected from ##STR00176## and said L is optionally substituted with 1, 2 or 3 R.
12. The compound or the pharmaceutically acceptable salt thereof according to claim 11, wherein ring A is selected from ##STR00177##
13. The compound or the pharmaceutically acceptable salt thereof according to claim 12, wherein L is selected from ##STR00178##
14. The compound or the pharmaceutically acceptable salt according to claim 1, wherein ring A is selected from ##STR00179## and L is selected from ##STR00180##
15. The compound or the pharmaceutically acceptable salt thereof thereof according to claim 1, wherein ring A is selected from ##STR00181## and L is selected from ##STR00182##
16. A pharmaceutical composition comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.
17. A method of treating Farnesoid X Receptor related diseases, comprising administering a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1.
18. The method according to claim 17, wherein the diseases are selected from non-alcoholic fatty liver disease (NAFLD), cholestatic hepatopathy, cholestatic liver diseases, hepatitis C infection, alcoholic liver disease, hepatic fibrosis, primary sclerosing cholangitis (PSC), gallstone, biliary atresia, lower urinary tract symptom and benign prostatic hyperplasia (BPH), ureteral calculi, obesity, type 2 diabetes, arteriosclerosis, hepatic function injury resulting from hypercholesterolemia or hyperlipidemia.
19. The method according to claim 18, wherein the Farnesoid X Receptor related diseases are selected from the group consisting of chronic liver disease, fibrotic diseases, hypercholesterol diseases, hypertriglyceride diseases or cardiovascular diseases.
20. The method according to claim 19, wherein the diseases are selected from the group consisting of non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC) or atherosclerosis.
Description
DETAILED DESCRIPTION OF THE INVENTION
(1) In order to illustrate the present invention in more details, the following examples are given, but the scope of the present invention is not limited thereto.
Reference Example 1: Preparation of INT-747
(2) ##STR00058## ##STR00059##
Reference Example 1A
(3) ##STR00060##
(4) To a solution of chenodeoxycholic acid (60.0 g, 152.8 mmol) in methanol/acetic acid/water/ethyl acetate (360/120/30/780 mL), tetrabutylammonium bromide (81.0 g, 251.3 mmol) and sodium bromide (9.0 g, 87.5 mmol) were added in portions, and then sodium hypochlorite (210 mL, 3.4 mol) was added dropwise at 0° C. within 30 min. After being stirred at 28° C. for 16 hours, saturated sodium bisulfite solution (500 mL) was added for quenching. The aqueous layer was extracted with ethyl acetate (1000 mL×2), and the combined organic layer was washed with water (1000 mL×5). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was recrystallized (dichloromethane, 200 mL) to give Reference Example 1A (yellow solid, 41 g, 69% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 3.44-3.60 (m, 1H), 2.99 (dd, J=5.77, 12.30 Hz, 1H), 2.54 (t, J=11.29 Hz, 1H), 2.08-2.41 (m, 3H), 2.00-2.08 (m, 1H), 1.75-1.96 (m, 6H), 1.28-1.69 (m, 9H), 1.09-1.27 (m, 8H), 0.97 (d, J=6.53 Hz, 3H), 0.71 (s, 3H).
Reference Example 1B
(5) ##STR00061##
(6) To a solution of Reference Example 1A (246.0 g, 629.9 mmol) in methanol (2 L), p-toluenesulfonic acid (10.9 g, 63.0 mmol) was added in one portion, and they were reacted at 80° C. for 4 hours. After being cooled to room temperature, they were evaporated to dryness, and quenched with saturated sodium bicarbonate solution (1500 mL). Then, the aqueous layer was extracted with ethyl acetate (1500 mL×3). The combined organic layer was washed with brine (1000 mL×3), dried over sodium sulfate, filtered and evaporated to dryness. The residue was recrystallized (ethyl acetate, 500 mL) to give Reference Example 1 B (white solid, 202 g, 79% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 3.66 (s, 3H), 3.55-3.62 (m, 1H), 2.85 (dd, J=6.02, 12.55 Hz, 1H), 2.28-2.43 (m, 2H), 2.13-2.26 (m, 2H), 1.64-2.04 (m, 10H), 1.19-1.51 (m, 11H), 1.00-1.17 (m, 3H), 0.86-0.97 (m, 3H), 0.65 (s, 3H).
Reference Example 1C
(7) ##STR00062##
(8) To a solution of chlorotrimethylsilane (107.5 g, 989.5 mmol) in tetrahydrofuran (500 mL), lithium diisopropylamide (87.4 g, 815.6 mmol) was added dropwise at −78° C. under nitrogen atmosphere, and after being stirred for 40 min, a solution of Reference Example 1B (50 g, 123.6 mmol) in tetrahydrofuran (300 mL) was further added dropwise. After completion of the dropwise addition, they were stirred at −78° C. for another 40 min, and then triethylamine (182.5 g, 1.8 mol) was added. They were quenched with saturated sodium hydrogencarbonate (1000 mL) after 1 hour, and the aqueous layer was extracted with ethyl acetate (1000 mL×3). The combined organic layer was further washed with water (100 mL×6) and saturated saline solution (1000 mL×2). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness, so as to give Reference Example 1C (brown-yellow oil, 68 g, 100% yield), which can be directly used in the next step without further purification. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 4.75 (dd, J=1.38, 5.90 Hz, 1H), 3.69 (s, 3H), 3.48-3.59 (m, 1H), 2.13-2.42 (m, 2H), 1.52-2.04 (m, 10H), 1.29-1.48 (m, 7H), 0.99-1.23 (m, 5H), 0.95 (d, J=6.53 Hz, 3H), 0.85 (s, 3H), 0.70 (s, 3H), 0.17-0.20 (m, 9H), 0.13 (s, 9H).
Reference Example 1D
(9) ##STR00063##
(10) To a solution of Reference Example 1C (68.0 g, 123.9 mmol) in dichloromethane (500 mL), anhydrous acetaldehyde (10.1 g, 229.2 mmol) was added. A solution of boron trifluoride-diethyl ether (64.4 g, 453.4 mmol) in dichloromethane (300 mL) was added dropwise at −78° C. under nitrogen atmosphere. The dropping was required to be kept at an internal temperature of −78° C. After being stirred for 1 hour, it was warmed up to 30° C. and stirred for another 2 hours. The above solution was quenched with saturated sodium hydrogencarbonate (1000 mL). The aqueous layer was extracted with dichloromethane (1000 mL×3). The combined organic layer was washed with saturated saline solution (1000 mL×2), dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by column chromatography to give Reference Example 1D (yellow solid, 43 g, 81% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 6.12 (q, J=7.03 Hz, 1H), 3.52-3.66 (m, 4H), 2.54 (dd, J=4.02, 13.05 Hz, 1H), 2.13-2.40 (m, 5H), 1.68-1.98 (m, 7H), 1.65 (d, J=7.03 Hz, 3H), 1.00-1.52 (m, 11H), 0.97 (s, 3H), 0.89 (d, J=6.53 Hz, 3H), 0.61 (s, 3H).
Reference Example 1E
(11) ##STR00064##
(12) To a solution of Reference Example 1D (212.0 g, 492.3 mmol) in methanol (500 mL), a solution of NaOH (39.4 g, 984.6 mmol) in water (50 mL) was added, and they were stirred at 50° C. for 2 hours. After the solvent was evaporated to dryness, water (500 mL) was added, and ethyl acetate (500 mL×2) was employed for extracting. The aqueous phase was adjusted to pH 3 with dilute HCl, and extracted with dichloromethane (600 mL×2). The combined organic layer was concentrated. The residue was purified by recrystallization (ethanol, 200 mL) to give Reference Example 1E (yellow solid, 147 g, 72% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 6.19 (q, J=7.36 Hz, 1H), 3.60-3.74 (m, 1H), 2.58 (dd, J=4.02, 13.05 Hz, 1H), 2.40 (tt, J=5.02, 10.29 Hz, 3H), 2.19-2.32 (m, 2H), 1.61-2.06 (m, 10H), 1.04-1.54 (m, 14H), 1.01 (s, 3H), 0.95 (d, J=6.53 Hz, 3H), 0.65 (s, 3H).
Reference Example 1F
(13) ##STR00065##
(14) To a solution of Reference Example 1E (140.0 g, 336.1 mmol) in a solution of NaOH (0.5 mol) in water (600 mL), 10% Pd—C(19.9 g, 134.4 mmol) was added in one portion, and 15 psi of hydrogen was introduced, and they were reacted at 100° C. for 16 hours. After suction filtration, the filtrate was adjusted to pH 3 with dilute hydrochloric acid. The aqueous layer was extracted with dichloromethane (1500 mL×3). The combined organic layer was washed with brine (1000 mL×3), and dried over sodium sulfate, filtered and evaporated to dryness, so as to give Reference Example 1F (white solid, 101 g, 72% yield), which can be directly used in the next step without further purification. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 3.49-3.60 (m, 1H), 2.70 (q, J=6.02 Hz, 1H), 2.12-2.45 (m, 4H), 1.65-2.02 (m, 9H), 1.29-1.52 (m, 6H), 1.05-1.24 (m, 8H), 0.93 (d, J=6.53 Hz, 5H), 0.81 (t, J=7.53 Hz, 3H), 0.66 (s, 3H).
Reference Compound INT-747
(15) ##STR00066##
(16) To a solution of Reference Example 1F (16.0 g, 38.2 mmol) in sodium hydroxide (2 mol, 100 mL), sodium borohydride (8.7 g, 229.3 mmol) was added in portions, and they were stirred at 100° C. for 2 hours. After being cooled to room temperature, saturated aqueous ammonium chloride solution (150 mL) was added. The pH thereof was adjusted to 3 with dilute hydrochloric acid. The aqueous layer was extracted with dichloromethane (300 mL×3). The combined organic layer were washed with brine (200 mL×3), dried over sodium sulfate, filtered and evaporated. The residue was purified by column chromatography, so as to give the reference compound INT-747 (white solid, 14.5 g, 90% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 3.71 (br. s, 1H), 3.36-3.48 (m, 1H), 2.18-2.47 (m, 2H), 1.56-2.01 (m, 10H), 1.06-1.54 (m, 15H), 0.86-0.97 (m, 9H), 0.66 (s, 3H).
(17) Route 1
(18) ##STR00067## ##STR00068##
Example 1
(19) ##STR00069##
Example 1A
(20) ##STR00070##
(21) To a solution of the reference compound INT-747 (2.7 g, 6.4 mmol) and formic acid (0.3 g, 6.4 mmol) in tetrahydrofuran (40 mL), perchloric acid (6.0 g, 60.0 mmol) was added under nitrogen atmosphere. After being stirred at 55° C. for 6 hours and concentrated under vacuum, the above solution was diluted with water (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL×2). The combined organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was purified by column chromatography, so as to give Example 1 A as a white solid (2.8 g, 92% yield). NMR (400 MHz, CHLOROFORM-d) δ 8.16 (s, 1H), 8.05 (s, 1H), 5.20 (br. s., 1H), 4.77-4.65 (m, 1H), 2.45-2.34 (m, 1H), 2.31-2.21 (m, 1H), 2.01-1.58 (m, 11H), 1.55-1.30 (m, 8H), 1.22-1.05 (m, 6H), 0.97-0.88 (m, 9H), 0.66 (s, 3H).
Example 1B
(22) ##STR00071##
(23) To a solution of Example 1A (100.0 mg, 0.2 mmol) and lead acetate (186.0 mg, 0.4 mmol) in carbon tetrachloride (2 mL), iodine (106.0 mg, 0.4 mmol) was added, and the reaction system was reacted for 12 hours under light irradiation. The reaction of the above solution was quenched by adding a solution of sodium thiosulfate (1 mL). The aqueous layer was extracted with dichloromethane (10 mL×3), dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was purified by a preparative thin layer plate (petroleum ether:ethyl acetate=5:1), so as to give Example 1B (colorless solid, 50.0 mg, 38% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ 8.14 (s, 1H), 8.03 (s, 1H), 5.19 (br. s., 1H), 4.62-4.77 (m, 1H), 3.23-3.32 (m, 1H), 3.00-3.12 (m, 1H), 1.96-2.02 (m, 2H), 1.85-1.92 (m, 2H), 1.70-1.82 (m, 7H), 1.66-1.72 (m, 2H), 1.39-1.45 (m, 2H), 1.23-1.32 (m, 5H), 1.09-1.19 (m, 6H), 0.96 (s, 3H), 0.90-0.93 (m, 6H), 0.67 (s, 3H).
Example 1
(24) ##STR00072##
(25) To a solution of methyl 6-oxopiperidine-3-carboxylate (21.0 mg, 134 μmol) in N,N-dimethylformamide (1 mL), sodium hydrogen (7.0 mg, 179 μmol, 60%) was added. After half an hour, a solution of Example 1B (50.0 mg, 89.5 μmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise at 0° C. After the dropwise addition was completed, the reaction system was slowly warmed up to room temperature, and reacted for 12 hours. Water (3 mL) and lithium hydroxide monohydrate (4 mg, 89.5 mmol) were added to the reaction system, and it was stirred for another 3 hours. Water (10 mL) was added, and the reaction system was adjusted to pH=6 with hydrochloric acid (1M), extracted with dichloromethane:methanol=10:1 (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by preparative thin layer plate (dichloromethane:methanol=10:1), so as to give Example 1 (15 mg, 32.4% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 3.67 (br. s., 1H), 3.58-3.48 (m, 2H), 3.45-3.34 (m, 2H), 3.31-3.27 (m, 1H), 2.87 (d, J=13.1 Hz, 1H), 2.49-2.29 (m, 2H), 2.11 (br. s., 2H), 2.00-1.35 (m, 19H), 1.30-1.10 (m, 6H), 1.04 (d, J=6.5 Hz, 3H), 0.95-0.90 (m, 6H), 0.72 (s, 3H).
(26) Example compounds 2-16 were synthesized via the same procedure as Example 1, and shown as follows:
(27) TABLE-US-00001 Compound Yield No. % Compound sturcture .sup.1H NMR Example 2 6%
(28) Route 2
(29) ##STR00088##
Example 17 and Example 18
(30) ##STR00089##
Example 2A
(31) ##STR00090##
(32) To a solution of Example 1A (500.0 mg, 1 mmol) in tetrahydrofuran (5 mL), triethylamine (153.0 mg, 1.5 mmol) and ethyl chloroformate (167.0 mg, 1.5 mmol) were added, and the reaction system was reacted at 25° C. for 2 hours. The system was cooled to 0° C., and a solution of sodium borohydride (210.0 mg, 5.6 mmol) in methanol (5 mL) was slowly added to the reaction system, and they were reacted at 0° C. for 15 min, and further reacted at 25° C. for 15 min. The reaction was quenched with 0.2 M dilute hydrochloric acid (1 mL). The aqueous layer was extracted with ethyl acetate (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was purified by column chromatography (petroleum ether:ethyl acetate=4:1), so as to give Example 2A (250.0 mg, 70%). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.15 (s, 1H), 8.04 (s, 1H), 5.19 (br. s., 1H), 4.77-4.66 (m, 1H), 3.66-3.57 (m, 2H), 2.06-1.77 (m, 7H), 1.45-1.06 (m, 21H), 0.97-0.88 (m, 9H), 0.66 (s, 3H).
Example 2B
(33) ##STR00091##
(34) To a mixed solution of Example 2A (280.0 mg, 605 μmol), triphenylphosphine (476.0 mg, 1.8 mmol) and imidazole (124.0 mg, 1.8 mmol) in toluene (4 mL) and acetonitrile (1 mL), iodine (461.0 mg, 1.8 mmol) was added, and the reaction system was reacted at 25° C. for 3 hours. A solution of saturated sodium sulfite (10 mL) was added to the reaction system, and the aqueous layer was extracted with ethyl acetate (10 mL×3). The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by column chromatography (petroleum ether:ethyl acetate=20:1), so as to give Example 2B (250.0 mg, 70%). .sup.1H-NMR (CDCl.sub.3, 400 MHz) □δ 8.16 (s, 1H), 8.07-8.02 (m, 1H), 5.20 (br. s., 1H), 4.76-4.66 (m, 1H), 3.24-3.08 (m, 2H), 2.01-1.72 (m, 10H), 1.46-1.06 (m, 17H), 0.97-0.89 (m, 9H), 0.66 (s, 3H).
Example 17 and Example 18
(35) ##STR00092##
(36) To a solution of methyl 6-hydroxynicotinate (80.0 mg, 523 μmop in N,N-dimethylformamide (3 mL), sodium hydrogen (21.0 mg, 523 mmol, 60%) was added at 0° C. After half an hour, a solution of Example 2B (150.0 mg, 262 μmop in N,N-dimethylformamide (5 mL) was added dropwise at 0° C. After the dropwise addition was completed, the reaction system was slowly warmed up to room temperature, and reacted for 12 hours. Water (3 mL) and lithium hydroxide monohydrate (55 mg, 1.31 mmol) were added to the reaction system, and stirred for another 3 hours. Water (10 mL) was added, and the system was adjusted to pH=6 with hydrochloric acid (1M), extracted with dichloromethane:methanol=10:1 (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was isolated and purified by a preparative thin layer plate (dichloromethane:methanol=10:1), so as to give Example 17 (40 mg, 29%), .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.44 (d, J=2.5 Hz, 1H), 7.97 (dd, J=2.5, 9.5 Hz, 1H), 6.55 (d, J=9.5 Hz, 1H), 4.10-3.96 (m, 2H), 3.70-3.63 (m, 1H), 3.37-3.34 (m, 1H), 2.02-1.11 (m, 27H), 0.98 (d, J=6.0 Hz, 3H), 0.95-0.88 (m, 6H), 0.71 (s, 3H); and Example 18 (20 mg, 14%), .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.77 (s, 1H), 8.21 (dd, J=1.8, 8.8 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 4.34 (d, J=2.5 Hz, 2H), 3.78-3.58 (m, 1H), 3.38-3.33 (m, 1H), 2.02-1.22 (m, 27H), 1.01 (d, J=6.5 Hz, 3H), 0.95-0.90 (m, 6H), 0.72 (s, 3H).
(37) Route 3
(38) ##STR00093##
Example 19
(39) ##STR00094##
Example 3A
(40) ##STR00095##
(41) To a solution of Example 2A (500.0 mg, 1.1 mmol), copper acetate (99.0 mg, 0.6 mmol) and lead acetate (2.4 g, 11.0 mmol) in toluene (5 mL), pyridine (980.0 mg, 12.4 mmol) was added, and the reaction system was reacted at 110° C. for 12 hours. The reaction system was filtered, and the filtrate was concentrated. The residue was purified by column chromatography (petroleum ether:ethyl acetate=20:1), so as to give the title compound (120.0 mg, 27.0%). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.15 (s, 1H), 8.05 (s, 1H), 5.71-5.59 (m, 1H), 5.19 (br. s., 1H), 4.93-4.80 (m, 2H), 4.76-4.67 (m, 1H), 2.12-1.61 (m, 11H), 1.52-1.09 (m, 13H), 1.04 (d, J=6.8 Hz, 3H), 0.97 (s, 3H), 0.91 (t, J=7.4 Hz, 3H), 0.68 (s, 3H).
Example 313
(42) ##STR00096##
(43) To a solution of Example 3A (100.0 mg, 0.2 mmol), sodium carbonate (74.0 mg, 0.7 mmol) and tetrabutylammonium bromide (66.0 mg, 0.2 mmol) in N,N-dimethylformamide (2 mL), palladium acetate (5.0 mg, 23.0 μmop and methyl 2-iodobenzoate (60.8 mg, 0.2 mmol) were added under nitrogen, and the reaction system was reacted at 85° C. for 12 hours. The solvent was evaporated to dryness and water (10 mL) was added to the reaction system. The aqueous layer was extracted with dichloromethane (20 mL×3). The organic layers were combined. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was purified by preparative thin layer chromatography (petroleum ether:ethyl acetate=10:1), so as to give the title compound (80.0 mg, 61.0%).
(44) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.16 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.90-7.84 (m, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.41-7.33 (m, 1H), 6.40-6.31 (m, 1H), 6.16 (dd, J=8.8, 15.8 Hz, 1H), 5.21 (br. s., 1H), 4.79-4.63 (m, 1H), 3.94 (s, 3H), 2.29 (d, J=6.8 Hz, 1H), 2.08-1.62 (m, 11H), 1.52-1.20 (m, 11H), 1.15 (d, J=6.5 Hz, 3H), 1.00 (s, 3H), 0.95-0.90 (m, 3H), 0.74 (s, 3H).
Example 3C
(45) ##STR00097##
(46) To a solution of Example 3B (100.0 mg, 0.2 mmol) in methanol (3 mL), wet palladium on carbon (50 mg, 10%) was added under nitrogen, and the reaction system was reacted under hydrogen condition (15 psi) at 25° C. for 12 hours. The reaction system was filtered and evaporated to dryness, so as to give Example 3C (90.0 mg, 90.0%). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.18-8.11 (m, 1H), 8.06-8.00 (m, 1H), 7.85-7.81 (m, 2H), 7.35-7.31 (m, 2H), 5.18 (br. s., 1H), 4.76-4.51 (m, 1H), 2.77-2.64 (m, 1H), 2.54-2.44 (m, 1H), 1.95-1.20 (m, 25H), 1.01 (d, J=6.5 Hz, 3H), 0.95-0.87 (m, 6H), 0.64 (s, 3H).
Example 19
(47) ##STR00098##
(48) To a solution of Example 3C (160.0 mg, 282.0 μmol) in tetrahydrofuran (1 mL), methanol (1 mL) and water (1 mL), lithium hydroxide (59.0 mg, 1.4 mmol) was added. The reaction system was reacted at 20° C. for 12 hours. Water (5 mL) was added to the reaction system, and the system was adjusted to pH=1-2 with hydrochloric acid (1 M). The aqueous phase was extracted with ethyl acetate (10 mL×3). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was isolated and purified by preparative isolation (HCl), so as to give Example 19 (80 mg, 57%). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 7.93-7.76 (m, 2H), 7.45-7.35 (m, 2H), 3.67 (br. s., 1H), 3.37-3.35 (m, 1H), 2.84-2.73 (m, 1H), 2.60 (d, J=10.5 Hz, 1H), 2.06-1.27 (m, 25H), 1.09 (d, J=6.3 Hz, 3H), 0.94-0.91 (m, 6H), 0.70 (s, 3H).
(49) Route 4
(50) ##STR00099##
Example 20
(51) ##STR00100##
(52) To a solution of Example 3B (40.0 mg, 70.8 μmop in tetrahydrofuran (0.5 mL), methanol (0.5 mL) and water (0.5 mL), lithium hydroxide (15 mg, 0.35 mmol) was added. The reaction system was reacted at 20° C. for 12 hours. Water (5 mL) was added to the reaction system, and the system was adjusted to pH=1-2 with hydrochloric acid (1 M). The aqueous phase was extracted with ethyl acetate (10 mL×3). The organic layers were combined and dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was purified by preparative thin layer chromatography (dichloromethane:methanol=10:1), so as to give Example 20 (25.0 mg, 71.0%). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.00 (br. s., 1H), 7.84 (d, J=7.0 Hz, 1H), 7.55 (d, J=7.5 Hz, 1H), 7.42-7.34 (m, 1H), 6.46-6.32 (m, 1H), 6.19 (dd, J=8.8, 15.6 Hz, 1H), 3.72-3.62 (m, 1H), 3.39-3.37 (m, 1H), 2.32 (d, J=6.0 Hz, 1H), 2.07-1.29 (m, 22H), 1.17 (d, J=6.3 Hz, 3H), 0.95-0.89 (m, 6H), 0.78 (s, 3H).
(53) Route 5
(54) ##STR00101##
Example 21
(55) ##STR00102##
Example 5A
(56) ##STR00103##
(57) After a mixture of Reference Example 1 (100.0 mg, 0.2 mmol), triethylamine (48.0 mg, 0.5 mmol) and O,N-dimethylhydroxylamine hydrochloride (23.0 mg, 0.2 mmol) in acetonitrile (2 mL) were stirred at 25° C. for 0.5 hour, O-benzotriazol-N,N,N′,N′-tetramethyluronium tetrafluoroborate (95.0 mg, 0.3 mmol) was added thereto. The resulting mixture was stirred at 25° C. for 12 hours. After the solvent was removed by evaporation under vacuum, the residue was purified by column chromatography, so as to give Example 5A as a white solid (90 mg, 82% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 3.73-3.67 (m, 4H), 3.45-3.36 (m, 1H), 3.18 (s, 3H), 2.51-2.40 (m, 1H), 2.38-2.27 (m, 1H), 2.00-1.89 (m, 2H), 1.87-1.74 (m, 5H), 1.71-1.56 (m, 5H), 1.53-1.31 (m, 11H), 1.23-1.14 (m, 3H), 1.06-0.99 (m, 1H), 0.96 (d, J=6.3 Hz, 3H), 0.93-0.88 (m, 6H), 0.67 (s, 3H).
Example 5B
(58) ##STR00104##
(59) To a solution of Example 5A (100.0 mg, 0.2 mmol) in tetrahydrofuran (5 mL), a solution of methylmagnesium bromide (0.4 mL, 1.1 mmol, 3N) in diethyl ether was added at 0° C., and they were stirred at 0° C. for another 30 min, and then warmed up to room temperature and stirred for 12 hours. The reaction was quenched by adding ice water, and then they were extracted with ethyl acetate (60 mL×2). After being washed with water, the organic phase was dried over anhydrous sodium sulfate, and filtered. After the solvent was removed under vacuum, the residue was purified by preparative TLC, so as to give Example 5B as a white solid (6 mg, 66% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 3.71 (br. s., 1H), 3.48-3.35 (m, 1H), 2.51-2.41 (m, 1H), 2.39-2.29 (m, 1H), 2.14 (s, 3H), 1.99-1.79 (m, 5H), 1.76-1.56 (m, 5H), 1.51-1.30 (m, 10H), 1.22-1.11 (m, 3H), 1.00 (dt, J=3.3, 14.2 Hz, 1H), 0.94-0.87 (m, 9H), 0.66 (s, 3H).
Example 5C
(60) ##STR00105##
(61) Example 5B (1.0 g, 2.4 mmol) was dissolved in 1,4-dioxane (20.0 mL), and 1,4-dihydropyran (2.0 g, 23.9 mmol, 2.2 mL) and p-toluenesulfonic acid (90.9 mg, 478.0 μmop were added thereto. The reaction solution was stirred at 30° C. for 36 hours. The solvent was removed by concentration, and water (5 mL) was added to the reaction solution, which was extracted with ethyl acetate (10 mL×3). The organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated, so as to give a crude product. The crude product was isolated by column chromatography, so as to give Example 5C (450.0 mg, 32.1% yield, colorless oil). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 4.73 (d, J=3.3 Hz, 1H), 4.56 (d, J=4.3 Hz, 1H), 3.95-3.81 (m, 2H), 3.76-3.72 (m, 1H), 3.55-3.31 (m, 4H), 2.52-2.41 (m, 111), 2.34 (ddd, J=5.8, 9.9, 16.0 Hz, 1H), 1.97-1.80 (m, 7H), 1.74-1.67 (m, 4H), 1.55 (br. s., 4H), 1.41 (d, J=7.3 Hz, 3H), 1.33-1.27 (m, 3H), 1.17-1.08 (m, 3H), 0.90 (s, 311), 0.88 (s, 3H), 0.69-0.59 (m, 3H).
Example 5D
(62) ##STR00106##
(63) Metal sodium (68.2 mg, 3.0 mmol) was dissolved in anhydrous ethanol (5 mL), cooled to 0° C., to which Example 5C (170.0 mg, 296.8 μmol, dissolved in 2 mL of anhydrous ethanol) was slowly added, and they were stirred for another half an hour at 0° C. Then, diethyl oxalate (65.0 mg, 445.1 μmol, 60.8 μL, dissolved in 1 mL of anhydrous ethanol) was added dropwise. After being stirred at 0° C. for half an hour, the reaction solution was warmed up to 25° C. and stirred for another 35 hours. Ethanol (6 mL) was added to the reaction solution, and the solvent was removed by concentration. Ethyl acetate (40 mL) was added, and 2 mol of citric acid aqueous solution was added under stirring in an ice bath (to pH=5-6). The aqueous phase was extracted with ethyl acetate (10 mL×2). The organic layer was washed with water (10 mL), saline solution (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated, so as to give a crude product. The crude product was isolated by column chromatography, so as to give Example 5D (90.0 mg, 44.2% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 4.73 (d, J=3.3 Hz, 1H), 4.57 (d, J=4.0 Hz, 1H), 4.36 (q, J=7.0 Hz, 2H), 3.97-3.80 (m, 2H), 3.70 (br. s., 1H), 3.52-3.34 (m, 3H), 2.54 (ddd, J=5.1, 10.3, 15.4 Hz, 1H), 2.44-2.29 (m, 1H), 1.98-1.90 (m, 2H), 1.82 (br. s., 3H), 1.74-1.67 (m, 4H), 1.53 (br. s., 7H), 1.38 (s, 3H), 1.26 (t, J=7.0 Hz, 2H), 1.16 (d, J=9.8 Hz, 2H), 0.95 (d, J=6.5 Hz, 2H), 0.89 (s, 3H), 0.71-0.59 (m, 3H).
Example 5E
(64) ##STR00107##
(65) Example 5D (90.0 mg, 131.0 μmol) was dissolved in ethanol (3.0 mL), and hydroxylamine hydrochloride (27.3 mg, 393.0 μmol) was added thereto, and the reaction solution was stirred at 80° C. for 5 hours. The solvent was removed by concentration, and water (5 mL) was added to the reaction solution. The aqueous phase was extracted with ethyl acetate (10 mL×3). The organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under vacuum. Example 5E was obtained without purifying the crude product (86.0 mg, crude product, yellow oil). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 6.39 (s, 1H), 4.43 (q, J=7.3 Hz, 2H), 3.72 (br. s., 1H), 3.43-3.40 (m, 1H), 2.90-2.64 (m, 2H), 2.42 (t, J=6.9 Hz, 2H), 2.00-1.95 (m, 2H), 1.75 (d, J=6.8 Hz, 3H), 1.69 (br. s., 4H), 1.60 (d, J=3.3 Hz, 3H), 1.48 (d, J=7.5 Hz, 4H), 1.41 (s, 3H), 1.28 (br. s., 4H), 1.20 (d, J=8.5 Hz, 3H), 0.90 (s, 3H), 0.89-0.88 (m, 1H), 0.65 (s, 3H).
Example 21
(66) ##STR00108##
(67) Example 5E (115.0 mg, 222.9 μmol) was dissolved in tetrahydrofuran (1.0 mL), water (1.0 mL) and methanol (1.0 mL), and lithium hydroxide monohydrate (93.6 mg, 2.2 mmol) were added thereto. The reaction solution was stirred at 25° C. for 12 hours. The reaction solution was acidified with 1 mol of hydrochloric acid (to pH=5-6), and extracted with ethyl acetate (10 mL×3). The organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness under vacuum. The crude product was isolated by thin layer chromatography, so as to give Example 21 (28.0 mg, 25.8% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 6.41 (s, 1H), 3.67 (br. s., 1H), 3.31 (br. s., 1H), 2.94-2.83 (m, 1H), 2.80-2.70 (m, 1H), 2.03 (d, J=12.0 Hz, 1H), 1.96-1.83 (m, 5H), 1.80-1.73 (m, 2H), 1.64 (br. s., 1H), 1.58-1.50 (m, 5H), 1.48-1.42 (m, 2H), 1.42-1.34 (m, 3H), 1.32-1.27 (m, 3H), 1.24 (d, J=9.5 Hz, 211), 1.05 (d, J=6.0 Hz, 3H), 0.93 (s, 3H), 0.92-0.89 (m, 3H), 0.71 (s, 3H).
(68) Route 6
(69) R.sub.b═H or X, for example:
(70) ##STR00109##
Example 22
(71) ##STR00110##
Example 6A
(72) ##STR00111##
(73) To a solution of methyl 6-hydroxynicotinate (438.7 mg, 2.9 mmol), Example 1B (800 mg, 1.4 mmol) in chloroform (20 mL), silver carbonate (790 mg, 2.9 mmol) was added. The reaction system was reacted at 60° C. for 72 hours, filtered, and concentrated. The residue was isolated by column (petroleum ether:ethyl acetate=10:1), so as to give the title compound 6A (400 mg, 47.9%). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.82 (d, J=2.3 Hz, 1H), 8.16 (s, 1H), 8.16-8.12 (m, 1H), 8.05 (s, 1H), 6.74 (d, J=8.5 Hz, 1H), 5.21 (br. s., 1H), 4.80-4.61 (m, 1H), 4.48-4.27 (m, 2H), 3.91 (s, 3H), 2.02-1.63 (m, 11H), 1.46-1.09 (m, 14H), 1.02 (d, J=6.5 Hz, 3H), 0.97 (s, 3H), 0.94-0.89 (m, 3H), 0.68 (s, 3H).
Example 22
(74) ##STR00112##
(75) To a solution of Example 6A (800 mg, 1.4 mmol) in methanol (10 mL), a solution of 10% sodium hydroxide (volume ratio of methanol to water was 1:1) (600 mg, 15 mmol, 6 mL) was added, and the reaction system was reacted at 70° C. for 2 hours. The solvent was evaporated to dryness. The system was adjusted to pH=1-2 with dilute hydrochloric acid (1M) and extracted with dichloromethane:methanol=10:1 (20 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative chromatography, so as to give the title compound 22 (460 mg, 64% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.78 (d, J=1.8 Hz, 1H), 8.21 (dd, J=2.3, 8.5 Hz, 1H), 6.84 (d, J=8.8 Hz, 1H), 4.56-4.23 (m, 2H), 3.68 (br. s., 1H), 3.35-3.34 (m, 1H), 2.04-1.17 (m, 25H), 1.07 (d, J=6.5 Hz, 3H), 0.96-0.89 (m, 6H), 0.74 (s, 3H).
(76) The preparation of Examples 23-26 was referred to the procedure of the title compound 22.
(77) TABLE-US-00002 Compound Yield No. % Compound sturcture .sup.1H NMR Example 23 47.2%
(78) Route 7
(79) ##STR00117##
Example 27
(80) ##STR00118##
(81) To a solution of Example 22 (100.0 mg, 194.7 μmop, DCC (60.3 mg, 292 μmol) and DMAP (23.8 mg, 194.7 μmol) in dichloromethane (5 mL), methanesulfonamide (37 mg, 389.3 μmol) was added. The reaction system was reacted at 25° C. for 12 hours. Water (5 mL) was added to the system. The system was adjusted to pH=2 with dilute hydrochloric acid (1 M), and extracted with dichloromethane (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative chromatography, so as to give Example 27 (25 mg, 21.7% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.74 (d, J=2.3 Hz, 1H), 8.23 (dd, J=2.3, 8.8 Hz, 1H), 7.06-6.87 (m, 1H), 4.57-4.40 (m, 2H), 3.67 (br s, 1H), 3.39 (s, 3H), 2.03-1.27 (m, 25H), 1.07 (br d, J=6.5 Hz, 3H), 0.96-0.90 (m, 6H), 0.73 (s, 3H).
(82) The preparation of Examples 28-34 was referred to the procedure of the title compound 27.
(83) TABLE-US-00003 Compound Yield No. % Compound sturcture .sup.1H NMR Example 28 50.0
(84) Route 8
(85) ##STR00126##
Example 35
(86) ##STR00127##
(87) Example 35 was synthesized by using Example 11 as a raw material, and the procedure was referred to the synthesis of Example 27. The yield was 17.4%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.95-8.70 (m, 2H), 8.48 (s, 1H), 4.41-4.27 (m, 2H), 3.67 (br s, 1H), 3.41 (s, 3H), 3.33-3.32 (m, 1H), 2.02-1.16 (m, 25H), 1.08 (d, J=6.3 Hz, 3H), 0.94-0.88 (m, 6H), 0.74 (s, 3H).
(88) Route 9
(89) ##STR00128##
Example 36
(90) ##STR00129##
(91) Example 36 was synthesized by using Example 23 as a raw material, and the procedure was referred to the synthesis of Example 27. The yield was 17.5%. .sup.1H NMR (400 MHz, CHLOROFORM-d) 6=8.60 (d, J=2.3 Hz, 1H), 8.18 (d, J=2.3 Hz, 1H), 4.56-4.33 (m, 2H), 3.69 (br s, 1H), 3.45 (s, 3H), 1.96-1.15 (m, 25H), 1.01 (d, J=6.5 Hz, 3H), 0.89-0.81 (m, 6H), 0.66 (s, 3H).
(92) Route 10
(93) ##STR00130##
Example 37
(94) ##STR00131##
(95) Example 37 was synthesized by using Example 23 as a raw material, and the procedure was referred to the synthesis of Example 27. The yield was 60.8%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.55 (d, J=1.8 Hz, 1H), 8.17 (d, J=1.8 Hz, 1H), 4.57-4.38 (m, 2H), 3.87-3.75 (m, 2H), 3.66 (br s, 1H), 3.32-3.20 (m, 1H), 3.11 (t, J=6.9 Hz, 2H), 2.06-1.23 (m, 25H), 1.06 (br d, J=6.5 Hz, 3H), 0.96-0.86 (m, 6H), 0.71 (s, 3H).
(96) Route 11
(97) ##STR00132##
Example 38
(98) ##STR00133##
(99) Example 38 was synthesized by using Example 22 as a raw material, and the procedure was referred to the synthesis of Example 27. The yield was 53%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) 8.63-8.62 (m, 1H), 8.09-8.06 (m, 1H), 6.84-6.82 (m, 1H), 4.39-4.28 (m, 2H), 3.82-3.74 (m, 311), 3.40-3.30 (m, 1H), 3.12-3.08 (m, 2H), 1.99-1.20 (m, 25H), 1.06 (d, J=6.5 Hz, 3H), 0.94-0.90 (m, 6H), 0.73 (s, 3H).
(100) Route 12
(101) ##STR00134##
Example 39
(102) ##STR00135##
Example 12A
(103) ##STR00136##
(104) Glycine hydrochloride (14 mg, 112 μmol) was reacted with a solution of triethylamine (20.0 μL, 146 μmol) in ethyl acetate (5 mL) at 20° C. for 0.5 hour. Example 22 (50 mg, 97 μmop and 2-ethoxy-1-ethoxycarbonyl-1.2-dihydroquinoline (36 mg, 146 μmol) were added to the system. The reaction system was reacted at 65° C. for 10 hours. Water (5 mL) was added to the system, and the aqueous phase was extracted with ethyl acetate (15 mL×2). The organic phases were combined and sequentially washed with 0.5 N of aqueous sodium hydroxide solution (15 mL), water (15 mL), 0.5 N of hydrochloric acid (15 mL) and water (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was isolated and purified by preparative isolation (TFA), so as to give Example 12A (25 mg, 43.9%). .sup.1H NMR (400 MHz, CDCl.sub.3) 8.66-8.65 (m, 1H), 8.08-8.05 (m, 1H), 6.81-6.67 (m, 1H), 6.68 (s, 1H), 4.42-4.24 (m, 2H), 4.35-4.24 (m, 2H), 3.81 (s, 3H), 3.72-3.71 (m, 1H), 3.46-3.44 (m, 1H), 1.99-1.20 (m, 25H), 1.03 (d, J=6.5 Hz, 3H), 0.92-0.88 (m, 6H), 0.68 (s, 3H).
Example 39
(105) ##STR00137##
(106) To a solution of Example 12A (25.0 mg, 42.7 μmol) in tetrahydrofuran (3 mL), methanol (1 mL) and water (2 mL), lithium hydroxide (8.9 mg, 213.7 μmol) was added. The reaction system was reacted at 30° C. for 4 hours. The system was adjusted to pH=5 with hydrochloric acid (1M), and the aqueous phase was extracted with ethyl acetate (20 mL×2). The organic layers were combined, and dried over sodium sulfate, filtered and evaporated to dryness. The residue was isolated by preparative TLC, so as to give Example 39 (24 mg, 98%). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) 8.66-8.65 (m, 1H), 8.11-8.02 (m, 1H), 6.84-6.82 (m, 1H), 4.48-4.29 (m, 2H), 4.09-4.08 (m, 3H), 3.66-3.65 (m, 1H), 1.99-1.20 (m, 25H), 1.06 (d, J=6.5 Hz, 311), 0.92-0.88 (m, 6H), 0.72 (s, 3H).
(107) Route 13
(108) ##STR00138##
Example 40
(109) ##STR00139##
Example 13A
(110) ##STR00140##
(111) Using Example 2B as a raw material, the procedure of Example 13A was referred to the synthesis of Example 6A. The yield was 16.3%. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.69 (d, J=2.0 Hz, 1H), 8.21 (d, J=2.0 Hz, 1H), 8.16 (s, 1H), 8.05 (s, 1H), 5.20 (br s, 1H), 4.72 (br s, 1H), 4.46-4.36 (m, 2H), 3.92 (s, 3H), 2.06-1.66 (m, 15H), 1.31-1.04 (m, 10H), 0.97-0.81 (m, 9H), 0.67 (s, 3H).
Example 40
(112) ##STR00141##
(113) The procedure of Example 40 was referred to the synthesis of Example 22. The yield was 96.4%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ=8.74 (d, J=2.0 Hz, 1H), 8.29 (d, J=1.8 Hz, 1H), 4.51 (t, J=6.4 Hz, 2H), 3.72 (br s, 1H), 3.43-3.39 (m, 1H), 2.11-1.23 (m, 27H), 1.07 (d, J=6.5 Hz, 3H), 1.00-0.93 (m, 6H), 0.77 (s, 3H).
(114) Route 14
(115) ##STR00142## ##STR00143##
Example 14A
(116) ##STR00144##
(117) To a solution of Reference Example 1 (40.0 g, 95.1 mmol) in methanol (500 mL), p-toluenesulfonic acid (4.0 g, 21.0 mmol) was added. The reaction system was reacted at 70° C. for 3 hours. The solvent was evaporated to dryness, and the system was diluted with dichloromethane (500 mL). The organic phase was sequentially washed with saturated aqueous solution of sodium carbonate (3×100 mL), aqueous solution (100 mL) and saturated saline solution (100 mL), and dried over anhydrous sodium sulfate, filtered and concentrated. Example 14A (41 g, 100% yield) was obtained without purification, and can be directly used in the next step.
Example 14B
(118) ##STR00145##
(119) Example 14A (41 g, 95.1 mmol) was dissolved in anhydrous tetrahydrofuran solution (300 mL), and warmed up to 50° C. under nitrogen atmosphere, and a solution of methylmagnesium bromide (800 mL, 1 M) in tetrahydrofuran was slowly added dropwise. After the dropwise addition was completed, the reaction solution was cooled to room temperature, and stirred overnight. After the reaction was completed, cyclohexane (25 mL) was added and filtered. The filter residue was dissolved in a mixed solution of 3N aqueous hydrochloric acid solution (800 mL) and dichloromethane (200 mL). They were stirred for another 30 min. After the reaction was completed, the organic phase was separated from the aqueous phase. The aqueous phase was further extracted with dichloromethane (2×200 mL). The organic phases were combined and sequentially washed with water (100 mL) and saturated saline solution (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. Example 14B (51 g, 100% yield) was obtained without purification, and can be directly used in the next step.
Example 14C
(120) ##STR00146##
(121) Acetic anhydride (9.9 mL, 105.1 mmol), pyridine (1.6 mL, 19.8 mmol) and 4-dimethylaminopyridine (0.8 g, 6.6 mmol) were added to a solution of Example 14B (51 g, 95.1 mmol) in anhydrous tetrahydrofuran (300 mL). The reaction solution was stirred overnight at room temperature. After the reaction was completed, it was diluted with aqueous solution (100 mL). The aqueous phase was extracted with dichloromethane (3×150 mL). The organic phases were combined, and washed with saturated saline solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Example 14C (55 g, 100% yield) was obtained without purification, and can be directly used in the next step.
(122) .sup.1H-NMR (CDCl.sub.3) δ 0.66 (3H, s, CH.sub.3—I8); 0.77 (3H, s, CH.sub.3-26); 1.00 (3H, d, CH.sub.3-21); 1.20 (3H, s, CH.sub.3-19); 1.96 (3H, s, AcO), 2.18-2.31 (1H, m, CH-22); 3.70 (1H, m, CH-7); 4.55 (1H, m, CH-3); 6.11 (1H, dd, =6.2 Hz, J 2=8.3 Hz; CH-23); 7.14-7.36 (10H, m, Ph).
Example 14D
(123) ##STR00147##
(124) Sodium periodate (13.2 g, 61.9 mmol) was dissolved in water (13 mL) and 2N aqueous sulfuric acid solution (1.7 mL). After being stirred for 15 mins, the reaction solution was cooled to 0° C., and ruthenium trichloride (71.3 mg, 0.4 mmol) was added thereto. The reaction solution was stirred continuously until the color thereof turned bright yellow. Ethyl acetate (27 mL) and acetonitrile (20 mL) were added to the reaction solution, and they were stirred for another 5 min. Example 14C (4 g, 6.9 mmol) was added to the above reaction solution at 0° C., and it was filtered after the reaction was completed. The filtrate was poured into water and extracted with ethyl acetate (3×100 mL). The organic phases were combined and washed with saturated sodium thiosulfate solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness, and then isolated by silica gel column chromatography, so as to give Example 14D (2.7 g, 89% yield).
(125) .sup.1H-NMR (CDCl.sub.3) δ 0.71 (3H, s, CH.sub.3-I8); 0.86-1.07 (9H, m, CH.sub.3-19, CH.sub.3-21, C-24); 2.03 (3H, s, AcO); 4.48-4.61 (1H, m, CH-3).
Example 14E
(126) ##STR00148##
(127) Triethylamine (6.7 mL, 3.4 mmol) was added to a solution of Example 14D (1 g, 2.2 mmol) and isobutyl chloroformate (3.5 mL, 2.7 mmol) in tetrahydrofuran (20 mL), and the reaction was completed after 1 hour. The reaction solution was filtered, and sodium borohydride (847 mg, 22.4 mmol) was added in portions to the filtrate at 0° C. After the reaction was completed, water (3 mL) was added to quench the reaction, and they were stirred at room temperature for another 2 hours, followed by acidified with 3N dilute aqueous hydrochloric acid, and extracted with ethyl acetate (3×15 mL). The organic phases were combined and washed with saturated saline solution (15 mL), dried over anhydrous sodium sulfate, and filtered. After the filtrate was evaporated to dryness, Example 14E (800 mg, 85% yield) was obtained by separation via silica gel column chromatography. .sup.1H-NMR (CDCl.sub.3) δ 0.67 (3H, s, CH.sub.3-I8); 0.86-0.97 (9H, m, CH.sub.3-19, CH.sub.3-21, CH.sub.3-24); 2.03 (3H, s, AcO); 3.72 (3H, m, (2H, m, CH-7, CH-23); 4.48-4.61 (1H, m, CH-3).
Example 41
(128) ##STR00149##
(129) Sodium hydrogen (18.4 mg, 460.1 μmol, 60%) was added to a solution of Example 14E (100 mg, 230.1 μmop in N,N-dimethylformamide (1 mL) at 0° C. After half an hour, a solution of methyl 4-chloropicolinate (78.9 mg, 460.1 μmop in N,N-dimethylformamide (2 mL) was slowly added dropwise at 0° C. After the dropwise addition was completed, the reaction system was slowly warmed up to room temperature and reacted for 12 hours. Water (10 mL) was added, and the reaction system was adjusted to pH=6 with hydrochloric acid (1M), and extracted with dichloromethane (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. 10% sodium hydroxide solution (volume ratio of methanol to water was 1:1) (50 mg, 1.3 mmol, 0.5 mL) was added to a solution of the residue in methanol (1 mL), and the reaction system was reacted at 70° C. for 2 hours. The solvent was evaporated to dryness. The system was adjusted to pH=1-2 with dilute hydrochloric acid (1 M), and extracted with dichloromethane:methanol=10:1 (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative chromatography, so as to give Example 41 (5 mg, 4% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.67 (br d, J=6.5 Hz, 1H), 7.99 (d, J=2.5 Hz, 1H), 7.69 (br d, J=6.5 Hz, 1H), 4.50 (br d, J=6.5 Hz, 2H), 3.78-3.59 (m, 1H), 3.32-3.30 (m, 1H), 2.18-1.17 (m, 25H), 1.10 (d, J=6.5 Hz, 3H), 0.95-0.89 (m, 6H), 0.76 (s, 3H).
(130) Route 15
(131) ##STR00150##
Example 42
(132) ##STR00151##
(133) To a solution of Example 16D (100.0 mg, 245.9 μmop and 5-aminopyridine-3-carboxylic acid (50.0 mg, 295 μmol) in DMF (5 mL), 1,3-dicyclohexylcarbodiimide (101.5 mg, 492) and 4-dimethylaminopyridine (1.5 mg, 12.3 μmol) were added. The reaction system was reacted at 30° C. for 16 hours. A solution of lithium hydroxide (10.3 mg, 246 μmol) in water (2.0 mL) was added to the system. The reaction system was reacted at 30° C. for 2 hours. The system was adjusted to pH=4 with hydrochloric acid (1M), and the aqueous phase was extracted with dichloromethane/methanol (10:1) (20 mL×3). The organic layers were combined, and dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was isolated and purified by preparative isolation (HCl), so as to give Example 42 (20 mg, 15.4%). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) 9.14-9.13 (m, 1H), 8.90-8.89 (m, 1H), 8.71-8.70 (m, 1H), 3.68 (s, 1H), 3.40-3.30 (m, 1H), 2.64-2.61 (m, 1H), 2.15-2.06 (m, 2H), 1.99-1.20 (m, 25H), 1.07 (d, J=6.5 Hz, 3H), 0.95-0.91 (m, 6H), 0.78 (s, 3H).
(134) Route 16
(135) ##STR00152## ##STR00153##
Example 43
(136) ##STR00154##
Example 16A
(137) ##STR00155##
(138) Reference Example 1F (10.0 g, 23.9 mmol) was dissolved in tetrahydrofuran (60.0 mL). Perchloric acid (240.0 mg, 2.4 mmol, 144.6 μL) (about 10 drops) was added thereto. Formic acid (40.3 g, 874.7 mmol, 33.0 mL) was added dropwise within half an hour at 30° C., and the reaction solution was stirred at 50° C. for 11.5 hours. The solvent was removed by concentration. Water (35.0 mL) was added to the reaction solution, which was extracted with ethyl acetate (30.0 mL×3). The organic layer was washed with water (10.0 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated, so as to give a crude product. The crude product was isolated by column chromatography, so as to give Example 16A (7.0 g, 15.7 mmol, 65.6% yield, white solid). .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=8.01 (s, 1H), 4.86-4.73 (m, 1H), 2.82-2.67 (m, 1H), 2.47-2.35 (m, 2H), 2.33-2.16 (m, 2H), 2.05-1.93 (m, 2H), 1.89 (d, J=13.1 Hz, 2H), 1.82 (dd, J=5.5, 16.8 Hz, 2H), 1.75 (dd, J=6.5, 14.1 Hz, 3H), 1.71 (br. s., 1H), 1.58-1.30 (m, 7H), 1.26 (s, 3H), 1.23-1.02 (m, 4H), 0.95 (d, J=6.5 Hz, 3H), 0.83 (t, J=7.4 Hz, 3H), 0.68 (s, 3H).
Example 16B
(139) ##STR00156##
(140) Example 16A (5.8 g, 13.0 mmol) was dissolved in trifluoroacetic acid (40.0 mL) and trifluoroacetic anhydride (20.5 g, 97.4 mmol) at 0° C. After the solid was dissolved, sodium nitrite (2.7 g, 39.0 mmol) was added in portions, which was stirred at 0° C. for another 1 hour, and warmed up to 40° C. and stirred for another 1 h. The reaction solution was cooled to 30° C., and neutralized with 0.5 mol of aqueous sodium hydroxide solution (pH=7-8) at 0° C. The reaction solution was extracted with ethyl acetate (40 mL×3), and the organic layer was washed with water (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was isolated by chromatographic column (silica gel), so as to give Example 16B (3.5 g, 8.5 mmol, 93.0% yield, pale yellow oil). .sup.1H NMR (400 MHz, CHLOROFORM-d) 8=3.59-3.47 (m, 1H), 2.69 (q, J=6.2 Hz, 1H), 2.42-2.31 (m, 2H), 2.29-2.15 (m, 2H), 2.01-1.88 (m, 2H), 1.86-1.68 (m, 7H), 1.61-1.45 (m, 6H), 1.26 (t, J=7.2 Hz, 5H), 1.19-1.12 (m, 5H), 1.02-0.91 (m, 1H), 0.80 (t, J=7.4 Hz, 3H), 0.71-0.64 (m, 3H).
Example 16C
(141) ##STR00157##
(142) Example 16B (3.5 g, 8.5 mmol) was dissolved in methanol (100.0 mL). Aqueous potassium hydroxide solution (70.0 g, 1.3 mol, dissolved in 100.0 mL of water) was added thereto, and the reaction solution was stirred at 100° C. for 12 hours. A part of the solvent was removed by concentration, and extracted with dichloromethane (30 mL×3). The aqueous phase was acidified with 1 mol of hydrochloric acid (pH=3-4), and extracted with ethyl acetate (30 mL×3). The organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated, so as to give a crude product. Example 16C (3.2 g, 7.9 mmol, 93.5% yield, yellow oil) was obtained without purification. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=3.65-3.50 (m, 1H), 2.71 (d, J=5.8 Hz, 1H), 2.48 (dd, J=2.6, 14.9 Hz, 1H), 2.43-2.31 (m, 2H), 2.22-2.15 (m, 1H), 2.07-1.98 (m, 2H), 1.95-1.86 (m, 3H), 1.82-1.70 (m, 6H), 1.53-1.46 (m, 3H), 1.19-1.10 (m, 6H), 1.02 (d, J=6.3 Hz, 3H), 0.86 (d, J=10.3 Hz, 5H), 0.69 (s, 3H).
Example 16D
(143) ##STR00158##
(144) To a solution of aqueous sodium hydroxide (949.2 mg, 23.7 mmol, dissolved in 10.00 mL of water), Example 16C (3.2 g, 7.9 mmol) was added. The reaction solution was warmed up to 80° C., and sodium borohydride (1.8 g, 47.5 mmol) was added in portions. The reaction solution was stirred at 100° C. for 12 hours. Methanol (6 mL) was added dropwise, and concentrated to remove a part of the solvent. The reaction solution was acidified with 1 mol of hydrochloric acid (pH=5-6), extracted with ethyl acetate (40 mL×3). The organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated. Example 16D (3.1 g, 7.6 mmol, 96.4% yield, white solid) was obtained without isolating the crude product. .sup.1H NMR (400 MHz, CHLOROFORM-d) 6=3.70 (br. s., 1H), 3.46-3.36 (m, 1H), 2.52-2.39 (m, 1H), 2.02-1.88 (m, 3H), 1.85-1.77 (m, 4H), 1.71-1.59 (m, 3H), 1.53-1.44 (m, 4H), 1.41-1.37 (m, 1H), 1.36-1.27 (m, 4H), 1.24-1.13 (m, 4H), 1.04 (d, J=6.5 Hz, 3H), 0.92-0.88 (m, 6H), 0.73-0.69 (m, 3H).
Example 16E
(145) ##STR00159##
(146) To a solution of Example 16D (100 mg, 245.8 μmol) in acetonitrile (2 mL), triethylamine (49.8 mg, 491.9 mmol, 68.2 μL) and N,O-dimethylhydroxylamine hydrochloride (24.0 mg, 245.9 μmop were added under nitrogen atmosphere. After being stirred at 25° C. for 30 min, O-benzotriazol-N,N,N′,N′-tetramethyluronium tetrafluoroborate (98.7 mg, 307.4 μmop was then added and stirred for another 11.5 hours. The reaction solution was poured into cold water (30 mL), and extracted with ethyl acetate (40 mL×3). The organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated. Example 16E (90 mg, 91.4% yield, white solid) was obtained without isolating the crude product. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=3.76 (s, 3H), 3.70 (br. s., 1H), 3.46-3.36 (m, 1H), 2.79 (s, 3H), 2.43-2.39 (m, 1H), 2.24-2.18 (m, 1H), 2.00-1.97 (m, 41H), 1.03-0.88 (m, 10H), 0.73 (s, 3H).
Example 16F
(147) ##STR00160##
(148) To a solution of lithium aluminum hydride (6.3 mg, 168.9 mop in tetrahydrofuran (2 mL), Example 16E (50 mg, 111.2 μmol) was added at −78° C., and stirred at −78° C. for 2 hours. After the reaction was completed, water (0.006 mL) was added and the reaction solution was filtered. The filter cake was oven-dried to give Example 16F (50 mg, 100%, white solid).
(149) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=9.76 (s., 1H), 3.70 (br. s., 1H), 3.46-3.36 (m, 1H), 2.43-2.39 (m, 1H), 2.24-2.18 (m, 1H), 2.00-1.97 (m, 41H), 1.03-0.88 (m, 10H), 0.73 (s, 3H).
Example 16G
(150) ##STR00161##
(151) To a solution of Example 16F (150.0 mg, 0.3 mmol) and methyl 5-aminopyridine-3-carboxylate (64.0 mg, 0.4 mmol) in ethyl acetate (5 mL), trifluoroacetic acid (57 μL, 0.8 mmol) and sodium triacetoxyborohydride (146.0 mg, 0.7 mmol) were added. The reaction system was reacted at 20° C. for 16 hours. Ethyl acetate (30 mL) was added to the reaction system. The system was washed with saturated sodium bicarbonate solution (20 mL×2) and saturated saline solution (20 mL×1). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness, so as to give the crude product. The crude product was isolated by preparative isolation (TFA) to give Example 16G (60.0 mg, 29.7%). .sup.1H NMR (400 MHz, CHLOROFORM-d) 8.44-8.43 (m, 1H), 8.43-8.42 (m, 1H), 7.92-7.91 (m, 1H), 4.01 (s, 3H), 3.72-3.71 (m, 1H), 3.49-3.48 (m, 1H), 3.28-3.13 (m, 2H), 1.99-1.20 (m, 25H), 1.01 (d, J=6.5 Hz, 3H), 0.95-0.87 (m, 6H), 0.67 (s, 3H).
Example 43
(152) ##STR00162##
(153) To a solution of Example 16G (60.0 mg, 113.9 μmol) in tetrahydrofuran (2 mL), methanol (1 mL) and water (2 mL), lithium hydroxide (60.0 mg, 1.43 mmol) was added. The reaction system was reacted at 40° C. for 1 hour. The system was adjusted to pH=4 with hydrochloric acid (1M), and the aqueous phase was extracted with dichloromethane/methanol (10:1) (20 mL×3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The residue was isolated and purified by preparative isolation (HCl) to give Example 43 (40 mg, 68%). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) 8.30-8.29 (m, 1H), 8.03-8.02 (m, 1H), 7.59-7.58 (m, 1H), 3.70-3.65 (m, 1H), 3.40-3.30 (m, 1H), 3.12-3.07 (m, 2H), 1.99-1.20 (m, 25H), 1.05 (d, J=6.5 Hz, 3H), 0.95-0.87 (m, 6H), 0.72 (s, 3H).
(154) Route 17
(155) ##STR00163##
Example 44
(156) ##STR00164##
(157) Concentrated sulfuric acid (368.0 mg, 3.8 mmol) was added to a solution of Example 22 (50.0 mg, 97.3 μmol) in methanol (5 mL). The reaction system was reacted at 70° C. for 12 hours. Water (5 mL) was added to the system. The system was adjusted to pH=7 with sodium hydroxide (1M), and extracted with dichloromethane (10 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by preparative chromatography, so as to give the title compound 44 (15 mg, 29.2% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.76 (d, J=2.0 Hz, 1H), 8.18 (dd, J=2.5, 8.8 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 4.49-4.28 (m, 2H), 3.89 (s, 3H), 3.65 (br s, 1H), 3.28 (br s, 1H), 2.03-1.15 (m, 25H), 1.04 (d, J=6.5 Hz, 3H), 0.93-0.86 (m, 6H), 0.71 (s, 3H).
(158) Route 18
(159) ##STR00165##
Example 45
(160) ##STR00166##
Example 18A
(161) ##STR00167##
(162) Example 18A was synthesized by using Example 23 as a raw material, and the procedure was referred to the synthesis of Example 12A. The yield was 79.7%. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.48 (d, J=2.3 Hz, 1H), 8.08 (d, J=2.3 Hz, 1H), 7.26 (s, 1H), 6.55 (t, J=4.8 Hz, 1H), 4.57-4.38 (m, 2H), 4.24 (d, J=5.0 Hz, 2H), 3.81 (s, 3H), 3.71 (s, 1H), 3.46-3.36 (m, 1H), 2.05-1.12 (m, 33H), 1.06-0.84 (m, 14H), 0.68 (s, 3H).
Example 45
(163) ##STR00168##
(164) The procedure of Example 45 was referred to the synthesis of Example 39. The yield was 56.8%. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ 8.80-8.72 (m, 1H), 8.48 (d, J=2.0 Hz, 1H), 8.10 (d, J=2.0 Hz, 1H), 4.50-4.33 (m, 2H), 3.99 (d, J=4.5 Hz, 2H), 3.56 (s, 1H), 1.98-1.03 (m, 31H), 1.01-0.87 (m, 5H), 0.84-0.74 (m, 10H), 0.62 (s, 3H).
Example 1: In Vitro Evaluation
(165) FXR Biochemical Experiment
(166) Experimental Purpose:
(167) The activation effect of the compound on FXR binding reaction was detected by AlphaScreen.
(168) Experimental Materials:
(169) 1. Protein: Glutathione-S-transferase-labeled FXR human protein (Invitrogen)
(170) 2. Co-activator: Biotin-labeled steroid receptor coactivator (Anaspec)
(171) 3. Detection reagent: AlphaScreen Detection Kit (PerkinElmer)
(172) Experimental Method:
(173) 1. Compound Dilution: The compound to be tested was prepared as a 40 μM DMSO solution, and then diluted 3-fold to 10 concentration points. The reference compound was prepared as a 400 μM DMSO solution, and then diluted 1.5-fold to 10 concentration points. The diluted DMSO solution was added to the wells of a 384-well plate in a volume of 150 nL per well.
(174) 2. The glutathione-S-transferase-labeled FXR human protein and the biotin-labeled steroid receptor coactivator were formulated as a mixed solution with concentrations of 0.4 nM and 30 nM, respectively, added to the wells of the 384-well plate in a volume of 15 μL per well, and incubated for 1 hour at room temperature.
(175) 4. The mixed solution of acceptor beads in the AlphaScreen Detection Kit was diluted 125-fold, and added to the wells of the 384-well plate in a volume of 7.54 μL per well. The operation during the experimental process was protected from light. The incubation was performed for 1 hour at room temperature.
(176) 5. The mixed solution of donor beads in the AlphaScreen Detection Kit was diluted 125-fold, and added to the wells of the 384 well-plate in a volume of 7.5 μL per well. The operation during the experimental process was protected from light. The incubation was performed for 1 hour at room temperature.
(177) 6. EC50 test: Envision was used with excitation at 680 nm to read the absorbance signals at 520-620 nm.
(178) 7. Analytical data: The data were analyzed via using Prism 5.0, and the EC50 values of the activation effects of the compounds were calculated. The ratio of the highest signal value of the compound to that of the reference compound was then used to give the percentage of activation efficacy of the compound.
(179) FXR Cell Experiment
(180) Experimental Purpose:
(181) The effect of the compound on the cellular functional activity was detected by β-lactamase reporter gene technique.
(182) Experimental Materials:
(183) 1. Cell line: FXR HEK 293T DA
(184) 2. Cell culture medium: DMEM medium supplemented with 10% serum and Penicillin/Streptomycin (1×)
(185) 3. Detection reagent: GeneBLAzer® Reporter Gene Detection Kit (Invitrogen)
(186) Experimental Method:
(187) 1. Compound Dilution: The compound to be tested was prepared as a 100 μM DMSO solution, and then the compound was diluted 3-fold to 10 concentration points. The reference compound was prepared as a 100 μM DMSO solution, and then diluted 1.3-fold to 10 concentration points. The diluted DMSO solution was added to the wells of a 384-well plate in a volume of 200 nL per well.
(188) 2. Cell inoculation: FXR HEK 293T DA cells were resuscitated, resuspended in a culture medium, diluted to a density of 5×10.sup.5 cells/mL, and added to the wells of the 384-well plate in a volume of 40 μL per well.
(189) 3. The 384-well microplate was incubated at 37° C., 5% CO.sub.2 for 16 hours.
(190) 4. 6 μL of 1 mM LiveBLAzer™-FRET B/G (CCF4-AM) substrate was mixed with 60 μL of B solution and 934 μL of C solution, and added to the wells of the 384-well plate in a volume of 8 μL per well.
(191) 5. The 384-well microplate was incubated in dark for 2 hours at room temperature.
(192) 6. EC50 test: Envision was used with excitation at 409 nm to read the absorbance signals at 460 and 530 nm.
(193) 7. Analytical data: The data was analyzed via using Prism 5.0, and the EC50 values of the activation effects of the compounds were calculated. The ratio of the highest signal value of the test compound to that of the reference compound (chenodeoxycholic acid, CDCA) was then used to give the percentage of activation efficacy of the compound.
(194) TABLE-US-00004 TABLE 1 Test results of EC.sub.50 for the biochemical experiment and cell experiment: Test Samples FXR enzyme activity FXR cell activity (Title compound) EC.sub.50 (μM) Efficacy EC.sub.50 (μM) Efficacy Chenodeoxycholic 12.14 100% 10.22 100% acid, CDCA Example 1 0.9 320% 17.9 134% Example 2 1.46 114% Example 3 2.62 106% Example 4 0.72 334% 1.87 131% Example 5 0.09 134% 0.57 190% Example 6 0.36 375% 5.16 132% Example 7 0.03 360% 0.06 140% Example 8 0.11 264% Example 9 0.16 407% 0.14 130% Example 10 0.06 188% 0.2 138% Example 11 0.03 241% 0.1 132% Example 12 0.07 240% Example 13 0.39 270% Example 14 0.73 195% Example 16 0.31 247% Example 17 0.60 313% 2.50 131% Example 18 0.07 187% Example 19 0.15 251% 0.37 143% Example 20 0.06 287% Example 22 0.006 249% Example 23 0.0025 248% 0.003 150% Example 24 0.0025 138% Example 25 0.011 233% Example 26 0.13 280% Example 27 0.006 212% Example 28 0.007 219% Example 29 0.012 190% Example 30 0.056 150% Example 31 0.027 204% Example 32 0.650 140% Example 33 0.141 194% Example 34 0.093 191% Example 38 0.02 210% Example 39 0.02 205% Example 40 0.045 197% Example 41 0.08 127% Example 42 1.43 121% Example 43 0.37 193% Example 44 0.330 140%
(195) Conclusion: The agonistic effect of the compound of the present invention on FXR receptor is significant, and the agonistic effect on FXR receptor at the cellular level is also significant.
Experimental Example 2: In Vivo Study
(196) Pharmacokinetics in Mice Administrated with Single Compound:
(197) 12 male mice (C.sub.57BL/6J) were randomly divided into two groups, i.e., 6 mice per group. The first group was the intravenous administration group, involving administration at a dose of 2 mg/kg, 2 mL/kg by injecting via tail vein (the vehicle was 10% HPbCD aqueous solution, and if the drug solubility was not satisfactory, the cosolvent was added); the second group was the oral administration group, involving intragastrical administration at a dose of 10 mg/kg, 10 mL/kg (the vehicle was 0.5% HPMC aqueous solution). Plasma (using K.sub.2-EDTA as anticoagulant) samples were taken at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours in the intravenous administration group after administration; and plasma samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours in the oral administration group after administration. For 6 animals in each group, blood samples were collected for 3 animals at one time point. The first batch of 3 animals was alternately sampled with the second batch of 3 animals. Plasma sample analysis was performed by using LC-MS/MS. The resultant plasma concentrations were plotted with repect to time, and PK parameters were calculated by using Phoenix WinNonlin 6.3.
(198) TABLE-US-00005 TABLE 2 Compound INT-747 Example 11 Example 22 Example 23 Dosage (mg/kg) 10 10 10 10 PK Cmax 1013 1433 936 1777 parameters (nM) in plasma Tmax 0.3 2 0.5 0.5 (h) AUC 993 5173 2337 1109 (nM .Math. h) F% 13% — 34% 20%
(199) Conclusion: As shown in Table 2, after oral administration at the same dosage, the peak concentration and the drug exposure of compound 11 were higher than those of the control compound INT-747. After oral administration at the same dosage, the peak concentration of compound 22 was close to that of the control compound INT-747, and the drug exposure of compound 22 was higher than that of the control compound INT-747. After oral administration at the same dosage, the peak concentration of compound 23 was higher than that of the control compound INT-747, and the drug exposure of compound 23 was also higher than that of the control compound INT-747.
(200) Liver-Blood Ratio Experiment of Mice Via Cassette Dosing
(201) 6 male mice (C57BL/6J) were grouped as an oral administration group. 5 kinds of the developed drugs were contained in the formulation, and intragastrical administration was performed at a dose of 2 mg/kg/compound (the vehicle was 0.5% HPMC aqueous solution). The five compounds were firstly and respectively dissolved in the vehicle, and sonicated or whirled to form a 1 mg/mL solution (clear solution or suspension), respectively; and then the solutions of the five compound were mixed in equal volumes (1:1:1:1:1, v:v:v:v:v) into a glass bottle. After intragastrical oral administration, plasma and liver tissue samples were collected from 3 animals at 0.5 h after administration; corresponding samples were collected from the other 3 animals at 3 h after administration. After collection, the liver tissue was homogenized by using ice-cold homogenization buffer (methanol:15 mM PBS buffer (pH 7.4)=1:2, v:v) based on the ratio of liver weight:homogenization buffer volume=1:3. Plasma and liver tissue samples were analyzed by using a five-in-one LC-MS/MS analysis method developed in advance. The concentrations of plasma and liver tissue homogenate were obtained, and the concentration ratios of liver tissue to plasma were calculated by using Excel.
(202) TABLE-US-00006 TABLE 3 Example Example Compound INT-747 22 26 Dosage (mg/kg) 2 2 2 PK Concentration in liver 711/625 1959/701 3904/358 parameters (nM) 0.5 h/3 h Concentration in plasma 151/63 83/45 387/18 (nM) 0.5 h/3 h Concentration ratio of liver 5/10 24/16 10/20 to plasma 0.5 h/3 h Note: ND indicates “not detected”.
(203) Conclusion: As shown in Table 3, after oral administration of the compound in the present invention at the same dosage, the drug concentrations of Example 22 in the liver at 0.5 h and 3 h were higher than those of the control compound, and the liver/blood concentration ratios were also higher than those of the control compound at 0.5 h and 3 h. The drug concentration of Example 26 in the liver at 0.5 h was higher than that of the control compound, and the liver/blood concentration ratios of Example 26 were higher than those of the control compound at 0.5 h and 3 h.