Bicylic compound acting as an inhibitor
11466024 · 2022-10-11
Assignee
- Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Lianyungang, CN)
- Medshine Discovery Inc. (Jiangsu, CN)
Inventors
- Kevin X Chen (Lianyungang, CN)
- Kai Zhou (Lianyungang, CN)
- Yanxin Yu (Lianyungang, CN)
- Boyu Hu (Lianyungang, CN)
- Linghui Wu (Lianyungang, CN)
- Xiaoyuan Chen (Lianyungang, CN)
- Jiangfeng Lin (Lianyungang, CN)
- Haomin Chen (Lianyungang, CN)
- Xiangjian Wang (Lianyungang, CN)
- Xiquan Zhang (Lianyungang, CN)
- Xin Tian (Lianyungang, CN)
- Hongjiang Xu (Lianyungang, CN)
Cpc classification
International classification
Abstract
A bicyclic compound acting as a RORγ inhibitor. Provided are a compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound having a structure as represented by formula (I-A) or formula (I-B). The provided compound of formula (I-A) or formula (I-B) or pharmaceutically acceptable salt thereof has good RORγ inhibitory activities, being expected to be used for treating diseases mediated by RORγ receptors in a mammal. ##STR00001##
Claims
1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has a structure represented by formula (I-A) ##STR00130## wherein, X is CR.sup.7R.sup.8 or NR.sup.9; Y is CR.sup.10; Z is CR.sup.11R.sup.12, NR.sup.13, —O— or —S—; U is ##STR00131## W is CH or N; Cy.sup.1 is selected from 3- to 6-membered cycloalkyl, 6- to 10-membered aryl or 5- to 10-membered heteroaryl, which is optionally substituted by one or more R.sup.16; R.sup.1, R.sup.4, R.sup.9, and R.sup.13 are independently selected from hydrogen or C.sub.1-6 alkyl; R.sup.2 and R.sup.3 are independently selected from hydrogen, halogen, amino, hydroxyl, nitro, cyano, or C.sub.1-6 alkyl; or R.sup.2 and R.sup.3 form ##STR00132## together; or R.sup.2 and R.sup.3 together with the C atom to which they are attached form a 3- to 6-membered cycloalkyl; R.sup.5 is selected from hydrogen or C.sub.1-6 alkyl; the C.sub.1-6 alkyl is optionally substituted by one or more hydroxyl, amino, nitro, cyano, halogen, C.sub.1-4 alkoxy, C.sub.1-4 alkylamino, or di(C.sub.1-4 alkyl)amino; n is 0, 1, 2, 3, 4, or 5; R.sup.6 is selected from hydroxyl, amino, nitro, cyano, halogen, or ##STR00133## R.sup.7 and R.sup.8 are independently selected from hydrogen, halogen, amino, hydroxyl, nitro, cyano, or C.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl is optionally substituted by halogen, amino, hydroxyl, nitro, cyano, or 3- to 6-membered cycloalkyl; or R.sup.7 and R.sup.8 form ##STR00134## together; or R.sup.7 and R.sup.8 together with the C atom to which they are attached form a 3- to 6-membered cycloalkyl; R.sup.10 is selected from hydrogen, C.sub.1-6 alkyl, halogen, amino, hydroxyl, nitro, cyano, or C.sub.1-6 haloalkyl; R.sup.11 and R.sup.12 are independently selected from hydrogen, halogen, amino, hydroxyl, nitro, cyano, or C.sub.1-6 alkyl; or R.sup.11 and R.sup.12 form ##STR00135## together; or R.sup.11 and R.sup.12 together with the C atom to which they are attached form a 3- to 6-membered cycloalkyl; R.sup.14 is present or not present; when R.sup.14 is present, ##STR00136## is selected from ##STR00137## or R.sup.15 is selected from C.sub.1-6 alkyl or C.sub.1-6 alkoxy; R.sup.16 is selected from hydroxyl, amino, nitro, cyano, halogen, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, C.sub.1-4 alkoxy, and C.sub.1-4 haloalkoxy; and when the compound of formula (I) has a structure represented by formula (I-A), X is CR.sup.7R.sup.8, U is ##STR00138## Y is CR.sup.10, and W is CH, then Cy.sup.1 is not a benzene ring.
2. The compound of claim 1, wherein U is ##STR00139## and W is CH; or U is ##STR00140## and W is N.
3. The compound of claim 1, wherein X is CR.sup.7R.sup.8 or NR.sup.9; wherein R.sup.7 and R.sup.8 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, hydroxyl, nitro, cyano, or C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by fluorine, chlorine, bromine, iodine, amino, hydroxyl, nitro, cyano, or 3- to 6-membered cycloalkyl; or R.sup.7 and R.sup.8 form ##STR00141## together; or R.sup.7 and R.sup.8 together with the C atom to which they are attached form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; R.sup.9 is selected from hydrogen or C.sub.1-3 alkyl.
4. The compound of claim 1, wherein Y is CR.sup.10; wherein R.sup.10 is selected from hydrogen, C.sub.1-3 alkyl, fluorine, chlorine, bromine, iodine, amino, hydroxyl, nitro, cyano, or C.sub.1-3 fluoroalkyl.
5. The compound of claim 1, wherein Z is CR.sup.11R.sup.12, NR.sup.13, —O—, or —S—; wherein R.sup.11 and R.sup.12 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, hydroxyl, nitro, cyano, or C.sub.1-3 alkyl; or R.sup.11 and R.sup.12 form ##STR00142## together; or R.sup.11 and R.sup.12 together with the C atom to which they are attached form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; R.sup.13 is selected from hydrogen or C.sub.1-3 alkyl.
6. The compound of claim 1, wherein Cy.sup.1 is selected from cyclohexyl or phenyl, which is optionally substituted by one or more R.sup.16; wherein R.sup.16 is selected from hydroxyl, amino, nitro, cyano, fluorine, chlorine, bromine, iodine, C.sub.1-2 alkyl, C.sub.1-2 haloalkyl, C.sub.1-2 alkoxy, C.sub.1-2 haloalkoxy.
7. The compound of claim 1, wherein R.sup.1 and R.sup.4 are selected from hydrogen or C.sub.1-3 alkyl.
8. The compound of claim 1, wherein R.sup.2 and R.sup.3 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, hydroxyl, nitro, cyano, or C.sub.1-3 alkyl; or R.sup.2 and R.sup.3 form ##STR00143## together; or R.sup.2 and R.sup.3 together with the C atom to which they are attached form cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
9. The compound of claim 1, wherein R.sup.5 is selected from hydrogen or C.sub.1-3 alkyl; said C.sub.1-3 alkyl is optionally substituted by one or more hydroxyl, amino, nitro, cyano, fluorine, chlorine, bromine, iodine, C.sub.1-2 alkoxy, C.sub.1-2 alkylamino, or di(C.sub.1-2 alkyl)amino.
10. The compound of claim 1, wherein n is 0, 1, or 2.
11. The compound of claim 1, wherein R.sup.6 is selected from hydroxyl, amino, nitro, cyano, fluorine, chlorine, bromine, iodine, ##STR00144## wherein R.sup.15 is selected from C.sub.1-3 alkyl or C.sub.1-3 alkoxy.
12. The compound of claim 1, wherein the compound of formula (I) has a structure represented by formula (I-A-1): ##STR00145## wherein W, Z, Cy.sup.1, R.sup.1, R.sup.4, R.sup.5, R.sup.7, R.sup.8, R.sup.10 and R.sup.14 are as defined in claim 1.
13. The compound of claim 1, wherein X is CH.sub.2, C(CH.sub.3).sub.2, C═O, CHCH(CH.sub.3).sub.2, ##STR00146##
14. The compound of claim 1, wherein Y is CH.
15. The compound of claim 1, wherein Cy.sup.1 is selected from cyclohexyl or phenyl, which is optionally substituted by one CF.sub.3.
16. The compound of claim 1, wherein R.sup.6 is selected from ##STR00147##
17. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is: ##STR00148##
18. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is: ##STR00149## ##STR00150## ##STR00151##
19. A pharmaceutical composition, comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 with a pharmaceutically acceptable excipient.
20. A method for inhibiting or alleviating a RORγ receptor-mediated disease in a mammal, comprising administering the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, in a therapeutically effective amount to a mammal, wherein the RORγ receptor-mediated disease is selected from multiple sclerosis, rhematiod arthritis, inflammatory disease, irritable bowel syndrome or psoriasis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION
(7) The present disclosure is described in detail below by Examples, but it does not imply any disadvantageous limitation on the present disclosure. The present disclosure has been described in detail herein, and its specific embodiments are also disclosed therein. Various changes and improvements made to the specific embodiments of the present disclosure will be apparent to a person skilled in the art without departing from the spirit and scope of the present disclosure.
Example 1 and Example 2
(8) ##STR00053## ##STR00054## ##STR00055## ##STR00056##
Synthesis of Compound 1-2
(9) ##STR00057##
(10) To a solution of Compound 1-1 (25 g) and N-methoxymethylamine hydrochloride (14.9 g) in 500 mL dichloromethane, TEA (51.6 g, 71.0 mL), EDCI (36.7 g) and HOBt (25.8 g) were added. The reaction solution was reacted at 10 to 20° C. for 16 hours. TLC (PE:EtOAc=1:1) showed the completion of the reaction. The reaction solution was dispersed in 500 mL dichloromethane and 500 mL water. The resultant was subjected to liquid-liquid separation. The organic phase was washed once with 500 mL saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to obtain a crude product. The crude product was allowed to pass through a flash column (SepaFlash® silica gel flash column (220 g), eluent: ethyl acetate/petroleum ether; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 100%; flow rate: 100 mL/min) and Compound 1-2 was obtained after the purification.
(11) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 3.71 (s, 3H), 3.19 (s, 3H), 2.62-2.74 (m, 1H), 1.99-2.07 (m, 3H), 1.92 (br d, J=13.6 Hz, 2H), 1.48-1.61 (m, 2H), 1.32-1.44 (m, 2H);
(12) .sup.19F NMR (400 MHz, CHLOROFORM-d): δ=−73.8.
Synthesis of Compound 1-3
(13) ##STR00058##
(14) At −65° C., a solution of Compound 1-2 (10 g) in 50 mL tetrahydrofuran was added dropwise to a suspension of lithium aluminum hydride (1.90 g) in 50 mL tetrahydrofuran. After the completion of the dropwise addition, the reaction solution was reacted at −70 to −65° C. for 4 hours. TLC (PE:EtOAc=5:1) showed that Compound 1-2 was reacted completely. The reaction was quenched with methanol (6.43 g) at −65° C. When the reaction mixture was heated to −10° C., 1 M citric acid aqueous solution was added dropwise, the pH was adjusted to approximately 4, and the temperature was controlled at −10 to −5° C. during this process. The resulting mixture was extracted with ethyl acetate (300 mL×3). The organic phases were combined, washed with 300 mL saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to obtain crude Compound 1-3, which was used directly in the next step without further purification.
(15) .sup.1HNMR (400 MHz, CHLOROFORM-d): δ 9.65 (s, 1H), 2.09-2.16 (m, 1H), 2.03-2.05 (m, 4H), 1.92-1.99 (m 1H), 1.23-1.32 (m, 4H).
Synthesis of Compound 1-5
(16) ##STR00059##
(17) Compound 1-4 (50.0 g) was dissolved in N-methylpyrrolidone (500 mL), potassium carbonate (49.1 g) and sodium ethylthiolate (31.0 g) were added to the reaction solution, and the mixture was then stirred at 20° C. for 12 hours. TLC (PE:EtOAc=5:1) showed the disappearance of the starting materials. The reaction solution was poured into 1500 mL water, and a solid precipitated. The solid was filtered, and the filter cake was dried under vacuum to obtain Compound 1-5.
(18) MS: m/z 165.1 [M+H].sup.+.
Synthesis of Compound 1-6
(19) ##STR00060##
(20) Compound 1-5 (58.0 g) was dissolved in methanol (600 mL), and a suspension of potassium peroxymonosulfate (434.2 g) in 900 mL water was added dropwise to the reaction solution at 0° C. Thereafter, the reaction solution was stirred at 0 to 20° C. for 16 hours. TLC (PE/EtOAc=3:1) showed that the starting materials were reacted completely. The reaction solution was filtered, and the excess potassium peroxymonosulfate in the reaction solution was quenched with sodium thiosulfate. After methanol was removed under reduced pressure, the resulting mixture was extracted with ethyl acetate (500 mL×2). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product of Compound 1-6. The crude product was purified by column chromatography (silica, 100 to 200 mesh silica gel, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether:ethyl acetate (V:V)=10:1 to 2:1), thereby obtaining Compound 1-6.
(21) MS: m/z 197.0 [M+H].sup.+;
(22) .sup.1H NMR (400 MHz, CDCl.sub.3-d): δ 9.13 (d, J=1.6 Hz, 1H), 8.38 (dd, J=8.4, 2.4 Hz, 1H), 7.94 (d, J=8.4 Hz, 1H), 3.21 (Q, J=7.2 Hz, 2H), 1.35 (d, J=7.6 Hz, 3H).
Synthesis of Compound 1-7
(23) ##STR00061##
(24) Compound 1-6 (10 g) was dissolved in anhydrous methanol (100.00 mL), and dry palladium on carbon (10%) was added under nitrogen protection. The mixture was purged with nitrogen gas for 3 times, and then purged with hydrogen gas for 3 times. The reaction solution was stirred at 25° C. for 3 hours in an atmosphere of H.sub.2 (50 Psi). When LC-MS showed the completion of the reaction, the reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain Compound 1-17.
(25) MS: m/z 201.0 [M+H].sup.+;
(26) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.04 (d, J=2.0 Hz, 1H), 8.15 (dd, J=2.4, 8.4 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 4.12 (s, 2H), 3.16 (q, J=7.6 Hz, 2H), 1.32 (t, J=7.6 Hz, 3H).
Synthesis of Compound 1-10
(27) ##STR00062##
(28) At 20° C., an aqueous solution (188 mL) of KOH (105.93 g, purity: 85%) was added to a suspension of Compound 1-8 (188 g) in water (188 mL). The resultant was stirred at 20° C. until Compound 1-8 was completely dissolved. The solution was cooled to 0 to 5° C., Compound 1-9 (91.2 g, 114.00 mL) was added dropwise under nitrogen protection, and the temperature was kept constant. After the dropwise addition was complete, the reaction solution was stirred at 0 to 5° C. for 3 hours. After the completion of the reaction was monitored by LC-MS, water (470 mL) was added to the reaction solution, and the pH of the resultant was adjusted to approximately 5 with concentrated hydrochloric acid (approx. 130 mL). A large amount of solid was generated, and as a result, a suspension was produced. The suspension was stirred continuously at 0 to 5° C. for 1 hour. A solid was obtained by filtration and washed with water (47 mL). The solid was collected and dried under vacuum at 60° C. to obtain Compound 1-10.
(29) MS: m/z 170.9 [M+H].sup.+;
(30) .sup.1H NMR (400 MHz, DEUTERIUM OXIDE): δ 3.46 (d, J=4.0 Hz, 1H), 3.32 (br t, J=6.8 Hz, 2H), 2.92 (br t, J=6.8 Hz, 2H), 2.05-2.26 (m, 1H), 0.94 (br dd, J=6.8, 17.6 Hz, 6H).
Synthesis of Compound 1-11
(31) ##STR00063##
(32) Compound 1-10 (94 g) was added to a prepared solution of absolute ethanol (450 mL) in concentrated H.sub.2SO.sub.4 (150 mL). Under the protection of nitrogen, the reaction solution was stirred at 90° C. for 16 hours. After the completion of the reaction was monitored by LC-MS, the reaction solution was poured into a saturated sodium bicarbonate solution until pH was approximately 8, and the mixture was extracted three times with 300 mL dichloromethane (100 mL×3). The organic phases were combined and washed with 100 mL (100 mL×1) of saturated saline. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product of Compound 1-11. The crude product was directly used in the next step without further purification.
(33) MS: m/z 186.0 [M+H].sup.+;
(34) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 3.74 (s, 3H), 3.70 (s, 3H), 2.90-3.01 (m, 2H), 2.58-2.74 (m, 2H), 2.48-2.52 (m, 2H), 1.91 (qd, J=6.8, 13.2 Hz, 1H), 0.94 (dd, J=2.0, 6.8 Hz, 6H).
Synthesis of Compound 1-12
(35) ##STR00064##
(36) To a solution of Compound 1-11 (96.0 g) in tetrahydrofuran (50 mL) was added a 1 M solution of potassium tert-butoxide in tetrahydrofuran (645 mL). Under the protection of nitrogen, the reaction solution was stirred at 20° C. for 20 minutes. After the completion of the reaction was monitored by LC-MS, the reaction solution was concentrated to dryness to obtain a crude product of Compound 1-12. The crude product was directly used in the next step without further purification.
(37) MS: m/z 186.0 [M+H].sup.+.
Synthesis of Compound 1-13
(38) ##STR00065##
(39) To a solution of Compound 1-12 (80.0 g) in ethanol (640 mL) was added concentrated hydrochloric acid (640 mL). Under the protection of nitrogen, the reaction solution was stirred at 90° C. for 0.5 hours. After the completion of the reaction was monitored by LC-MS, the reaction solution was concentrated to dryness to obtain the hydrochloride of Compound 1-13 as a crude product. The crude product was directly used in the next step without further purification.
(40) MS: m/z 127.9 [M+H].sup.+.
Synthesis of Compound 1-14
(41) ##STR00066##
(42) To a solution of the hydrochloride of Compound 1-13 (70.0 g) in dichloromethane (500 mL), triethylamine (129.8 g, 178.62 mL) and Boc.sub.2O (112.0 g, 117.92 mL) were added. Under the protection of nitrogen, the reaction solution was stirred at 20° C. for 2 hours. After the completion of the reaction was monitored by LC-MS, water (300 mL) was added to the reaction solution, and the mixture was extracted three times with 900 mL (300 mL×3) of dichloromethane. The combined organic phases were washed with 200 mL (200 mL×1) of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by using a flash purification system (SepaFlash® silica gel flash column (330 g), eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V)=0% to 30%; flow rate: 100 mL/min) to obtain Compound 1-14.
(43) MS: m/z 171.9 [M−56+H].sup.+;
(44) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 3.93-4.10 (m, 1H), 3.75-3.87 (m, 1H), 3.56 (br d, J=8.8 Hz, 1H), 2.52-2.66 (m, 1H), 2.40-2.51 (m, 1H), 2.16-2.38 (m, 1H), 1.50 (s, 9H), 1.02 (d, J=6.8 Hz, 3H), 0.96 (d, J=6.8 Hz, 3H).
Synthesis of Compound 1-15
(45) ##STR00067##
(46) POCl.sub.3 (3.96 g, 2.40 mL) was added dropwise to DMF (14.3 g, 15.00 mL) at 0° C., and the reaction solution was stirred at 25° C. for 0.5 hours. Then, a solution of Compound 1-14 (3 g) in DMF (30 mL) was added dropwise to the reaction solution, and the reaction solution was stirred at 25° C. for 3.5 hours. After the completion of the reaction was monitored by LC-MS, eight parallel reactions were combined, ice and a saturated aqueous sodium acetate solution (800 mL) were added to the reaction solution at 0° C., and the reaction solution was stirred at 0° C. for 1 hour. The reaction solution was extracted three times with ethyl acetate (500 mL). The combined organic phases were washed once with saturated brine (500 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by using a flash purification system (SepaFlash® silica gel flash column (80 g), eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V)=0% to 30%; flow rate: 60 mL/min) to obtain Compound 1-15 as the target compound.
(47) MS (ESI) m/z: 217.8 [M−56+H].sup.+;
(48) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 9.93-10.04 (m, 1H), 4.62-4.83 (m, 1H), 4.37-4.59 (m, 1H), 4.04-4.16 (m, 1H), 2.25-2.48 (m, 1H), 1.49 (s, 9H), 1.02 (br s, 6H).
Synthesis of Compound 1-17
(49) ##STR00068##
(50) Triethylamine (10.54 g, 14.50 mL) was added to a solution of Compound 1-15 (14.5 g) and Compound 1-16 (7.02 g, 6 mL) in dichloromethane (100 mL). The reaction solution was stirred at 25° C. for 9 hours, and then the reaction solution was stirred at 50° C. for 3 hours. After the completion of the reaction was monitored by LC-MS, the reaction solution was concentrated, 100 mL water was added to dilute the residue, and the aqueous phase was extracted three times with ethyl acetate (100 mL×3). The combined organic phases were washed once with saturated brine (100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by using a flash purification system (SepaFlash® silica gel flash column (80 g), eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V)=0% to 30%; flow rate: 60 mL/min) to obtain Compound 1-17.
(51) MS (ESI) m/z: 269.9 [M−56+H].sup.+;
(52) .sup.1H NMR (400 MHz, CHLOROFORM-d): btain Compo J=14.8 Hz, 1H), 4.85-4.99 (m, 1H), 4.44-4.61 (m, 1H), 4.29-4.37 (m, 1H), 3.81 (s, 3H), 2.29-2.60 (m, 1H), 1.44 (s, 9H), 0.99 (t, J=7.2 Hz, 3H), 0.55 (t, J=7.2, 8.4 Hz, 3H).
Synthesis of Compound 1-18
(53) ##STR00069##
(54) To a solution of Compound 1-17 (13 g) in methanol (100 mL) was added a solution of sodium hydroxide (2.3 g) in water (10 mL), and the reaction solution was stirred at 50° C. for 45 minutes. After the completion of the reaction was monitored by LC-MS, the reaction solution was directly concentrated and dried by rotary evaporation to obtain a sodium salt of Compound 1-18 as a crude product. The crude product was directly used in the next step without further purification.
(55) MS (ESI) m/z: 256 [M−56+H].sup.+.
Synthesis of Compound 1-19
(56) ##STR00070##
(57) To a suspension of the sodium salt of Compound 1-18 (25 g) and Compound 1-7 (9 g) in dichloromethane (100 mL), triethylamine (10.91 g, 15 mL), EDCI (10 g), and HOBt (10 g) were added. The reaction solution was stirred at 25° C. for 16 hours, and then the reaction solution was stirred at 50° C. for 3 hours. After the completion of the reaction was monitored by thin-layer chromatography and LC-MS, the reaction solution was concentrated, 150 mL water was added to dilute the residue, and the aqueous phase was extracted three times with ethyl acetate (100 mL×3). The combined organic phases were washed once with saturated brine (100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by using a flash purification system (SepaFlash® silica gel flash column (40 g), eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether:ethyl acetate (V/V)=0% to 80%; flow rate: 40 mL/min) to obtain Compound 1-19.
(58) MS (ESI) m/z: 494.2 [M+H].sup.+;
(59) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 8.99 (d, J=2.00 Hz, 1H), 8.11 (dd, J=2.2, 8.2 Hz, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.32 (d, J=9.60 Hz, 1H), 4.86-5.00 (m, 1H), 4.77 (d, J=4.0 Hz, 2H), 4.53-4.65 (m, 1H), 4.34 (dd, J=4.0, 13.80 Hz, 1H), 3.06-3.13 (m, 3H), 2.34-2.56 (m, 1H), 1.44 (s, 9H), 1.24-1.28 (m, 3H), 1.00 (t, J=7.04 Hz, 3H), 0.56 (t, J=7.04 Hz, 3H).
Synthesis of Compound 1-20
(60) ##STR00071##
(61) To a solution of Compound 1-19 (6.3 g) in ethyl acetate (10 mL) was added a solution of hydrogen chloride in ethyl acetate (10 mL). The reaction solution was stirred at 25° C. for 0.5 hours. After the completion of the reaction was monitored by LC-MS, the reaction solution was directly concentrated and dried by rotary evaporation to obtain the hydrochloride of Compound 1-20 as a crude product. The crude product was directly used in the next step without further purification.
(62) MS (ESI) m/z: 394 [M+H].sup.+.
Synthesis of Example 1 and Example 2
(63) ##STR00072##
(64) Triethylamine (2.04 g, 2.8 mL) was added to a suspension of the hydrochloride of Compound 1-20 (5.5 g) in 1,2-dichloroethane (80 mL). The reaction solution was stirred at 25° C. for 0.5 hours. Thereafter, acetic acid (2.63 g, 2.5 mL), Compound 1-3 (2.6 g) and sodium borohydride acetate (5.50 g) were added to the reaction solution. After the addition, the reaction solution was stirred at 25° C. for 0.5 hours. After the completion of the reaction was monitored by thin-layer chromatography and LC-MS, the reaction solution was concentrated, and 150 mL water was added to dilute the residue. The aqueous phase was extracted three times with ethyl acetate (100 mL×3). The combined organic phases were washed once with saturated brine (100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by using a flash purification system (SepaFlash® silica gel flash column (40 g), eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V)=0% to 80%; flow rate: 40 mL/min) to give a chemically pure product (i.e., a mixture of the compound of Example 1 in its free state and the compound of Example 2 in its free state). The chemically pure product was allowed to pass through SFC (column: AD (250 mm×50 mm, 10 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was neutral ethanol, wherein the volume ratio of phase B was 40%; column temperature: 40° C.). The compound of Example 1 in its free state and the compound of Example 2 in its free state were obtained by separation, respectively. These compounds in their free state were purified by preparative high performance liquid chromatography (HCl system) to obtain the hydrochlorides of the compound of Example 1 and the compound of Example 2.
(65) The compound of Example 1 was analyzed by the SFC method, and the analysis conditions were as follows: column: Chiralpak AD-3 50×4.6 mm I.D., 3 μm; mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was neutral ethanol, wherein the volume ratio of phase B was 40%; flow rate: 4 mL/min; column temperature: 40° C.; retention time Rt=1.89 min.
(66) MS (ESI) m/z: 558 [M+H].sup.+;
(67) .sup.1H NMR (400 MHz, DMSO-d6): δ 10.74 (br s, 1H), 9.52 (br s, 1H), 8.96 (d, J=2.0 Hz, 1H), 8.26 (dd, J=2.4, 8.4 Hz, 1H), 7.80 (s, 1H), 7.60 (d, J=8.0 Hz, 1H), 4.90 (br s, 2H), 4.56-4.75 (m, 2H), 4.42 (br d, J=10.4 Hz, 1H), 3.47-3.55 (m, 2H), 3.28 (br s, 3H), 1.80-2.46 (m, 7H), 1.21-1.36 (m, 2H), 0.97-1.18 (m, 10H).
(68) The compound of Example 2 was analyzed by the SFC method, and the analysis conditions were as follows: Chiralpak AD-3 50×4.6 mm I.D., 3 jam; mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was neutral ethanol, wherein the volume ratio of phase B was 40%; flow rate: 4 mL/min; column temperature: 40° C.; retention time Rt=1.18 min.
(69) MS (ESI) m/z: 558 [M+H].sup.+;
(70) 1H NMR (CHLOROFORM-d): δ 8.98 (s, 1H), 8.10 (dd, J=8.2, 2.1 Hz, 1H), 7.49 (d, J=8.2 Hz, 1H), 7.26 (s, 1H), 7.00-7.11 (m, 1H), 4.72-4.82 (m, 2H), 4.13 (dd, J=12.4, 3.6 Hz, 1H), 3.78-3.86 (m, 1H), 3.42 (dd, J=12.4, 3.1 Hz, 1H), 3.09 (q, J=7.3 Hz, 2H), 2.41-2.58 (m, 1H), 2.10-2.20 (m, 1H), 1.85-1.99 (m, 3H), 1.23-1.31 (m, 6H), 1.15-1.22 (m, 3H), 1.00 (d, J=7.0 Hz, 3H), 0.88 (br d, J=11.5 Hz, 1H), 0.79-0.85 (m, 1H), 0.75 (d, J=6.8 Hz, 3H).
Example 3 and Example 4
(71) ##STR00073##
(72) In Examples 1 and 2, a mixture of the compound of Example 1 in its free state and the compound of Example 2 in its free state, which was not separated by SFC, was placed in the air. An oxygenated product appeared and was purified by preparative high performance liquid chromatography (HCl system) to obtain a mixture of Example 3 and Example 4.
(73) The mixture of Example 3 and Example 4 was allowed to pass through SFC (column: AD (250 mm×30 mm, 10 μm). Mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was ethanol containing 0.1% aqueous ammonia, wherein the volume ratio of phase B was 50%. Example 3 and Example 4 were obtained by separation, respectively.
(74) The compound of Example 3 was analyzed by the SFC method, and the analysis conditions were as follows: column: Chiralpak AD-3 50×4.6 mm I.D., 3 jam; mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was ethanol containing 0.05% diethylamine, wherein the volume ratio of phase B was 40%; flow rate: 4 mL/min; column temperature: 40° C.; retention time Rt=1.086 min.
(75) MS (ESI) m/z: 572.1 [M+H].sup.+;
(76) .sup.1H NMR (400 MHz, DMSO-d6): δ 9.50 (s, 1H), 8.97 (d, J=2.00 Hz, 1H), 8.26 (dd, J=2.4, 8.4 Hz, 1H), 8.02 (s, 1H), 7.64 (d, J=8.0 Hz, 1H), 4.80 (d, J=3.6 Hz, 1H), 4.62-4.71 (m, 2H), 3.52-3.58 (m, 1H), 3.45 (m, 2H), 2.89-3.01 (m, 1H), 2.23 (m, 1H), 1.74-1.96 (m, 4H), 1.55-1.71 (m, 2H), 1.17 (m, 10H), 0.38 (d, J=6.4 Hz, 3H).
(77) The compound of Example 4 was analyzed by the SFC method, and the analysis conditions were as follows: column: Chiralpak AD-3 50×4.6 mm I.D., 3 m; mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was ethanol containing 0.05% diethylamine; elution gradient: a volume ratio of 40%; flow rate: 4 mL/min; column temperature: 40° C.; retention time Rt=0.692 min.
(78) MS (ESI) m/z 572.1 [M+H].sup.+;
(79) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.43 (t, J=5.6 Hz, 1H), 8.97 (d, J=2.0 Hz, 1H), 8.26 (dd, J=2.4, 8.40 Hz, 1H), 8.00 (s, 1H), 7.64 (d, J=8.4 Hz, 1H), 4.80 (d, J=3.6 Hz, 1H), 4.68 (m, 2H), 3.57-3.63 (m, 1H), 3.40 (m, 2H), 2.96 (br dd, J=4.0, 14.0 Hz, 1H), 2.20 (m, 1H), 1.76-1.94 (m, 4H), 1.53-1.72 (m, 2H), 1.07-1.23 (m, 10H), 0.39 (d, J=6.0 Hz, 3H).
Example 5 and Example 6
(80) ##STR00074## ##STR00075## ##STR00076##
Synthesis of Compound 5-3
(81) ##STR00077##
(82) LDA (2 M, 206.10 mL, 2.31 eq) was added dropwise to a solution of Compound 5-2 (20 g, 178.44 mmol, 1 eq) in anhydrous tetrahydrofuran (100 mL) at 0° C. under the protection of nitrogen. The reaction solution was stirred at 0° C. for 0.5 hours, and then a solution of Compound 5-1 in anhydrous tetrahydrofuran (100 mL) was slowly added to this reaction solution within 1 hour. Upon the completion of the dropwise addition, the reaction solution was heated to 20° C. and stirred for 1 hour. When LC-MS showed the completion of the reaction, water (100 mL) was added to quench the reaction, and the reaction solution was separated into different layers. The separated aqueous phase was washed with ethyl acetate (200 mL), and then the pH was adjusted to 3 using hydrochloric acid (1 N, 50 mL), followed by the extraction with ethyl acetate (250 mL×3). The combined organic layers were washed with saturated brine (180 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give Compound 5-3 (racemate, the R/S ratio was about 1:1).
(83) MS (ESI) m/z: 287.9 [M+H].sup.+
(84) .sup.1H NMR (CHLOROFORM-d): δ 7.27 (d, J=2.0 Hz, 1H), 7.21 (d, J=2.0 Hz, 1H), 6.63 (d, J=2.0 Hz, 1H), 6.60 (d, J=2.0 Hz, 1H), 4.71-4.79 (m, 1H), 4.45 (dd, J=10.4, 8.4 Hz, 1H), 2.12-2.23 (m, 1H), 2.05-2.12 (m, 1H), 1.17-1.21 (m, 18H), 1.02 (d, J=6.8 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H), 0.76 (d, J=6.4 Hz, 3H), 0.72 (d, J=6.8 Hz, 3H).
Synthesis of Compound 5-4
(85) ##STR00078##
(86) Compound 5-3 (30.26 g, 105.30 mmol, 1 eq) was dissolved in hydrochloric acid/ethyl acetate (60 mL), and the mixture was stirred and reacted at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was filtered, and the residue was washed with ethyl acetate to obtain Compound 5-4.
(87) MS (ESI) m/z: 166.9 [M+H].sup.+
(88) .sup.1HNMR (DMSO-d.sub.6): δ 8.80 (br s, 3H), 7.87 (d, J=1.6 Hz, 1H), 6.76 (d, J=1.6 Hz, 1H), 4.72 (br s, 1H), 2.21-2.37 (m, 1H), 1.01 (d, J=6.4 Hz, 3H), 0.78 (d, J=6.8 Hz, 3H).
Synthesis of Compound 5-5
(89) ##STR00079##
(90) Compound 5-4 (4.4 g, 20.03 mmol, 1 eq, HCl) was dissolved in anhydrous DMF (450 mL), and triethylamine (6.08 g, 60.09 mmol, 8.36 mL, 3 eq) was added thereto. After the mixture was stirred at 20° C. for 0.5 hours, HATU (11.42 g, 30.05 mmol, 1.5 eq) was added, and the mixture was stirred for another 0.5 hours. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove DMF, ethyl acetate (80 mL) was added thereto, and then the resulting mixture was washed with water (50 mL×2). The organic layer was washed with saturated brine (60 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a flash column (SepaFlash® silica gel flash column (40 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 60%; flow rate: 35 mL/min) to obtain Compound 5-5.
(91) MS (ESI) m/z: 166.0 [M+H].sup.+
(92) .sup.1H NMR (CHLOROFORM-d): δ 7.50 (d, J=1.6 Hz, 1H), 6.81 (br s, 1H), 6.62 (d, J=2.0 Hz, 1H), 4.36 (d, J=5.6 Hz, 1H), 1.99-2.16 (m, 1H), 1.04 (d, J=6.8 Hz, 3H), 1.00 (d, J=6.8 Hz, 3H).
Synthesis of Compound 5-6
(93) ##STR00080##
(94) Compound 5-5 (2.4 g, 14.53 mmol, 1 eq) was dissolved in anhydrous dichloromethane (30 mL), DMAP (177.50 mg, 1.45 mmol, 0.1 eq), triethylamine (2.94 g, 29.06 mmol, 4.04 mL, 2 eq) and Boc anhydride (3.49 g, 15.98 mmol, 3.67 mL, 1.1 eq) were added thereto, and the mixture was stirred and reacted at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, water (20 mL) was added to dilute the resulting mixture, and the mixture was extracted with dichloromethane (20 mL×2). The combined organic layers were washed with water (15 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by a flash column (SepaFlash® silica gel flash column (40 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 30%; flow rate: 35 mL/min) to obtain Compound 5-6.
(95) MS (ESI) m/z: 210.0 [M+H].sup.+
(96) .sup.1H NMR (CHLOROFORM-d): δ 7.44 (d, J=1.6 Hz, 1H), 6.56 (d, J=2.0 Hz, 1H), 4.79 (d, J=3.6 Hz, 1H), 2.48-2.66 (m, 1H), 1.50 (s, 9H), 1.15 (d, J=7.2 Hz, 3H), 0.49 (d, J=6.8 Hz, 3H).
Synthesis of Compound 5-7
(97) ##STR00081##
(98) Borane-dimethylsulfide (10 M, 5.01 mL, 4 eq) was added slowly to a solution of Compound 5-6 (3.32 g, 12.51 mmol, 1 eq) in anhydrous tetrahydrofuran (60 mL), and the mixture was stirred and reacted at 60° C. for 1 hour. When LC-MS showed the completion of the reaction, methanol (20 mL) was slowly added at 20° C. to quench the reaction. The resulting mixture was then concentrated under reduced pressure to remove methanol and tetrahydrofuran, water (20 mL) was then added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL×2). The combined organic layers were washed with saturated brine (30 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (20 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 20%; flow rate: 30 mL/min) to obtain Compound 5-7.
(99) MS (ESI) m/z: 196.0 [M+H].sup.+
(100) .sup.1H NMR (CHLOROFORM-d): δ 7.30 (d, J=1.2 Hz, 1H), 6.24 (dd, J=17.2, 1.6 Hz, 1H), 4.56-4.77 (m, 1H), 4.26-4.44 (m, 1H), 4.12-4.23 (m, 1H), 2.21-2.57 (m, 1H), 1.43 (s, 9H), 1.00 (t, J=7.6 Hz, 3H), 0.53 (dd, J=6.8, 3.6 Hz, 3H).
Synthesis of Compound 5-8
(101) ##STR00082##
(102) At 0° C., NBS (1.56 g, 8.75 mmol, 1.1 eq) was added to a solution of Compound 5-7 (2 g, 7.96 mmol, 1 eq) in acetonitrile (20 mL), and the mixture was stirred and reacted at 0° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove acetonitrile, water (10 mL) was then added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL×2). The combined organic layers were washed with saturated brine (15 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (20 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 20%; flow rate: 30 mL/min) to obtain Compound 5-8.
(103) MS (ESI) m/z: 273.9 [M+H].sup.+;
(104) .sup.1H NMR (CHLOROFORM-d): δ 6.19 (d, J=16.4 Hz, 1H), 4.56-4.75 (m, 1H), 4.26-4.41 (m, 1H), 4.16 (dd, J=13.2, 4.4 Hz, 1H), 2.21-2.53 (m, 1H), 1.42 (s, 9H), 0.99 (t, J=7.6 Hz, 3H), 0.56 (dd, J 6.8, 3.6 Hz, 3H).
Synthesis of Compound 5-9
(105) ##STR00083##
(106) Compound 5-8 (0.57 g, 1.73 mmol, 1 eq) was dissolved in anhydrous methanol (4 mL) and anhydrous DMF (1 mL), and triethylamine (192.13 mg, 1.90 mmol, 264.28 μL, 1.1 eq) and Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (140.96 mg, 172.61 μmol, 0.1 eq) were added thereto under the protection of nitrogen. After the reaction solution was purged with carbon monoxide for three times, the reaction solution was stirred and reacted at 60° C. and 50 psi for 16 hours. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove methanol and DMF, water (5 mL) was added to dilute the reaction solution, and the resulting mixture was extracted with ethyl acetate (6 mL×3). The combined organic layers were washed with saturated brine (8 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (4 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 20%; flow rate: 18 mL/min) to obtain Compound 5-9.
(107) MS (ESI) m/z: 254.0 [M+H].sup.+;
(108) .sup.1H NMR (CHLOROFORM-d): δ 7.02 (d, J=11.2 Hz, 1H), 4.58-4.84 (m, 1H), 4.28-4.48 (m, 1H), 4.19 (dd, J=13.6, 3.6 Hz, 1H), 3.82 (s, 3H), 2.23-2.58 (m, 1H), 1.43 (s, 9H), 0.95-1.11 (m, 3H), 0.57 (dd, J=6.4, 4.4 Hz, 3H).
Synthesis of Compound 5-10
(109) ##STR00084##
(110) To a solution of Compound 5-9 (0.51 g, 1.65 mmol, 1 eq) in methanol (5 mL) and water (1.25 mL) was added sodium hydroxide (131.88 mg, 3.30 mmol, 2 eq). The reaction solution was stirred and reacted at 50° C. for 1 hour. When LC-MS showed the completion of the reaction, MeOH (6 mL) was added at 50° C. to quench the reaction. After the mixture was concentrated under reduced pressure to remove methanol and water, a sodium salt of Compound 5-10 was obtained.
(111) MS (ESI) m/z: 240.0 [M+H].sup.+
Synthesis of Compound 5-11
(112) ##STR00085##
(113) The sodium salt of Compound 5-10 (0.49 g, 1.66 mmol, 1 eq) was dissolved in anhydrous DMF (6 mL), HATU (946.29 mg, 2.49 mmol, 1.5 eq), Compound 1-7 (398.71 mg, 1.99 mmol, 1.2 eq) and DIPEA (643.30 mg, 4.98 mmol, 866.99 μL, 3 eq) were added, and the mixture was stirred and reacted at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove DMF, and then dissolved in ethyl acetate (20 mL), washed with water (10 mL×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a flash column (SepaFlash® silica gel flash column (4 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 70%; flow rate: 18 mL/min) to obtain Compound 5-11.
(114) MS (ESI) m/z: 478.1 [M+H].sup.+;
(115) .sup.1H NMR (CHLOROFORM-d): δ 8.99 (d, J=2.0 Hz, 1H), 8.10 (dd, J=8.0, 2.4 Hz, 1H), 7.95 (s, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.28-7.37 (m, 1H), 7.03 (d, J=11.6 Hz, 1H), 4.72-4.88 (m, 2H), 4.30-4.48 (m, 1H), 4.14-4.27 (m, 1H), 3.05-3.13 (m, 2H), 1.43 (s, 8H), 1.39-1.41 (m, 1H), 1.23-1.27 (m, 3H), 1.00-1.08 (m, 3H), 0.59 (t, J=6.0 Hz, 3H).
Synthesis of Compound 5-12
(116) ##STR00086##
(117) A solution of Compound 5-11 (0.3 g, 628.18 μmol, 1 eq) in HCl/EtOAc (5 mL) was stirred and reacted at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove ethyl acetate, so as to obtain the hydrochloride of Compound 5-12.
(118) MS (ESI) m/z: 378.0 [M+H].sup.+.
Example 5 and Example 6
(119) ##STR00087##
(120) Triethylamine (67.23 mg, 664.38 μmol, 92.47 b L, 1.1 eq) was added to a solution of Compound 5-12 (0.25 g, 603.98 μmol, 1 eq, HCl) in anhydrous dichloroethane (5 mL), and the resulting mixture was stirred at 20° C. for 1 hour. Subsequently, acetic acid (36.27 mg, 603.98 mol, 34.54 μL, 1 eq) was added to adjust the pH to 5, followed by the addition of Compound 1-3 and NaBH(OAc).sub.3 (384.03 mg, 1.81 mmol, 3 eq). The reaction solution was stirred for an additional hour at 20° C. When LC-MS showed the completion of the reaction, a saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with dichloromethane (5 mL×3). The combined organic layers were washed with saturated brine (6 mL) and then dried over anhydrous sodium sulfate to obtain a crude product. The crude product was purified by a flash column (SepaFlash® silica gel flash column (4 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 30%; flow rate: 18 mL/min) to obtain a chemically pure product (a mixture of Example 5 and Example 6). The chemically pure product was separated by SFC to give Example 5 and Example 6. The specific separation conditions of SFC were as follows: SFC (column: AD (250 mm×30 mm, 5 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was isopropanol containing 0.1% aqueous ammonia, wherein the volume ratio of phase B was 35%.
Example 5
(121) The retention time Rt of the SFC analysis was 3.772 min. The SFC analysis conditions were as follows: (column: Chiralpak AD-3 100 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was isopropanol containing 0.05% diethylamine; gradient: the volume ratio of the mobile phase B was increase from 5% to 40% within 4.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1 minute; flow rate: 2.8 mL/min; column temperature: 40° C.
(122) MS (ESI) m/z: 542.3 [M+H].sup.+;
(123) .sup.1H NMR (CHLOROFORM-d): δ 8.98 (br s, 1H), 8.09 (br d, J=7.2 Hz, 1H), 7.49 (br d, J 7.6 Hz, 1H), 6.96 (br s, 1H), 4.56-4.95 (m, 2H), 3.97 (br d, J=9.2 Hz, 1H), 3.62 (br s, 1H), 3.27 (br d, J=11.6 Hz, 1H), 3.09 (br d, J=7.2 Hz, 2H), 2.58 (br d, J=8.8 Hz, 1H), 2.43 (br t, J=10.4 Hz, 1H), 2.15 (br d, J=11.2 Hz, 1H), 1.91 (br d, J=8.0 Hz, 2H), 1.76 (br d, J=12.4 Hz, 3H), 1.38 (br s, 1H), 1.25 (br s, 6H), 1.00 (br d, J=6.4 Hz, 3H), 0.77 (br d, J=5.6 Hz, 4H).
Example 6
(124) The retention time Rt of the SFC analysis was 4.337 min. The SFC analysis conditions were as follows: (column: Chiralpak AD-3 100 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was isopropanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was increased from 5% to 40% within 4.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1 minute; flow rate: 2.8 mL/min; column temperature: 40° C.
(125) MS (ESI) m/z: 542.3 [M+H].sup.+;
(126) .sup.1H NMR (CHLOROFORM-d): δ 8.98 (br s, 1H), 8.09 (br d, J=8.0 Hz, 1H), 7.49 (br d, J=8.0 Hz, 1H), 7.22 (br s, 1H), 6.96 (s, 1H), 4.55-4.97 (m, 2H), 3.97 (br d, J=11.2 Hz, 1H), 3.62 (br s, 1H), 3.27 (br d, J=11.6 Hz, 1H), 3.09 (q, J=7.2 Hz, 2H), 2.50-2.70 (m, 1H), 2.43 (br t, J=10.8 Hz, 1H), 2.15 (br d, J=12.0 Hz, 1H), 1.91 (br d, J=10.0 Hz, 3H), 1.75 (br d, J=13.6 Hz, 2H), 1.09-1.48 (m, 7H), 1.00 (br d, J=6.8 Hz, 3H), 0.82-0.94 (m, 1H), 0.77 (br d, J=6.4 Hz, 3H).
Example 7 and Example 8
(127) ##STR00088## ##STR00089##
Synthesis of Compound 7-1
(128) ##STR00090##
(129) A mixed solution of Compound 1-17 (5 g, 15.36 mmol, 1 eq) and HCl/EtOAc (4 M, 3.84 mL, 1 eq) was stirred at 20 to 30° C. for one hour. LC-MS showed that Compound 1-17 was completely consumed and the formation of the target product was detected. After the reaction solution was concentrated under reduced pressure to remove the solvent, the hydrochloride of Compound 7-1 was obtained. The crude product was used directly in the next step without further purification.
(130) MS (ESI) m/z: 225.9 [M+H].sup.+;
Synthesis of Compound 7-2
(131) ##STR00091##
(132) Et.sub.3N (1.53 g, 15.13 mmol, 2.11 mL, 1.2 eq) was added to a solution of Compound 7-1 (3.3 g, 12.61 mmol, hydrochloride) in 40 mL DCE, and then the mixture was stirred at 20 to 30° C. for 30 minutes. Thereafter, AcOH (2.27 g, 37.82 mmol, 2.16 mL, 3 eq) was added to adjust the pH to 5, and Compound 1-3 (4.54 g, 25.21 mmol, 2 eq) and NaBH(OAc).sub.3 (5.34 g, 25.21 mmol, 2 eq) were added in sequence. The reaction solution was stirred at 20 to 30° C. for 15.5 hours. LC-MS showed that Compound 7-1 was completely consumed and the formation of the target product was detected. 100 mL saturated sodium bicarbonate solution was added slowly to the reaction solution to quench the reaction, and then the reaction solution was extracted twice with 200 mL dichloromethane. The combined organic phases were washed with 200 mL saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was allowed to pass through a flash purification system (SepaFlash® silica gel flash column (40 g), eluent: petroleum ether/ethyl acetate; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 50%; flow rate: 40 mL/min), and Compound 7-2 was obtained after the purification.
(133) MS (ESI) m/z: 415.0 [M+H].sup.+.
Synthesis of Compound 7-3
(134) ##STR00092##
(135) To a solution of Compound 7-2 (1 g, 2.57 mmol, 1 eq) in 8 mL THF and 2 mL H.sub.2O was added LiOH.H.sub.2O (323.23 mg, 7.70 mmol, 3 eq). The reaction solution was stirred at 35° C. for 16 hours. LC-MS showed that Compound 7-2 was completely consumed and the formation of the target product was detected. The pH of the reaction solution was adjusted to 3 with an aqueous hydrochloric acid solution, and then the reaction solution was extracted twice with 100 mL ethyl acetate. The combined organic phases were washed with 50 mL saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give Compound 7-3, which was used directly in the next step without further purification.
(136) MS (ESI) m/z: 376.1 [M+H].sup.+.
Synthesis of Compound 7-4
(137) ##STR00093##
(138) To a solution of Compound 7-3 (1 g, 1.41 mmol, 1 eq) in 10 mL DMF, HATU (800 mg, 2.10 mmol, 1.49 eq) and DIPEA (593.60 mg, 4.59 mmol, 0.8 mL, 3.26 eq) were added. The reaction solution was stirred at 20 to 30° C. for 30 minutes, then Compound 7-3A (500 mg, 1.67 mmol, 1.18 eq) was added, and the resulting mixture was stirred at 20 to 30° C. for 15.5 hours. LC-MS showed that Compound 7-4 was completely consumed and the target product was formed. The reaction solution was dispersed in 50 mL water and 50 mL ethyl acetate. The organic phase was separated, washed with 50 mL saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was allowed to pass through a flash purification system (SepaFlash® silica gel flash column (12 g), eluent: petroleum ether/ethyl acetate; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 100%; flow rate: 30 mL/min) to be purified, and Compound 7-4 was obtained.
(139) MS (ESI) m/z: 657.2 [M+H].sup.+.
Synthesis of Example 7 and Example 8
(140) ##STR00094##
(141) A mixed solution of Compound 7-4 (500.00 mg, 403.46 μmol, 1 eq) and HCl/dioxane (4 M, 33.13 mL, 328.41 eq) was reacted at 20 to 30° C. for one hour. LC-MS showed that the starting materials were completely consumed and the formation of the target product was detected. The reaction solution was concentrated under reduced pressure to give a crude product. The crude product was prepared by HPLC (column model: YMC-Actus Triart C18 100×30 mm×5 μm; mobile phase: [phase A was water containing 0.05% HCl; and phase B was acetonitrile]; elution gradient: content of phase B: 20% to 90%, 10 min) to obtain the target product, which was further resolved by SFC (separation conditions: column model: C2 250 mm×30 mm, 10 μm; mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was ethanol; elution gradient (v/v): B %=45% to 45%) to obtain Example 7 and Example 8.
Example 7
(142) The retention time Rt of the SFC analysis was 6.588 min. The SFC analysis conditions were as follows: (column: Lux Cellulose-2 150 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine, wherein the volume ratio of phase B was 40%; flow rate: 2.5 mL/min; column temperature: 40° C.
(143) MS (ESI) m/z: 557.1 [M+H].sup.+;
(144) .sup.1H NMR (400 MHz, METHANOL-d4) δ ppm 9.18 (s, 1H), 8.51 (br d, J=6.6 Hz, 1H), 7.70-7.91 (m, 2H), 4.99 (br s, 1H), 4.83 (s, 2H), 4.53 (br d, J=12.0 Hz, 1H), 4.12 (br d, J=7.0 Hz, 2H), 3.41 (br s, 2H), 2.45 (br s, 1H), 1.76-2.34 (m, 7H), 1.37-1.50 (m, 5H), 1.17-1.31 (m, 5H), 1.08 (br d, J=5.2 Hz, 3H).
Example 8
(145) The retention time Rt of the SFC analysis was 7.542 min. The SFC analysis conditions were as follows: (column: Lux Cellulose-2 150 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine, wherein the volume ratio of phase B was 40%; flow rate: 2.5 mL/min; column temperature: 40° C.
(146) MS (ESI) m/z: 557.1 [M+H].sup.+;
(147) .sup.1H NMR (400 MHz, METHANOL-d4) δ ppm 9.19 (s, 1H), 8.52 (br d, J=6.0 Hz, 1H), 7.73-7.89 (m, 2H), 4.99-5.01 (m, 1H), 5.00 (br s, 1H), 4.84 (s, 2H), 4.54 (br d, J=11.2 Hz, 1H), 4.15 (br d, J=7.0 Hz, 2H), 3.42 (br s, 2H), 2.46 (br s, 1H), 1.76-2.35 (m, 7H), 1.42 (br t, J=6.4 Hz, 5H), 1.26 (br d, J=5.2 Hz, 5H), 1.09 (br d, J=5.0 Hz, 3H).
Synthesis of Intermediate 7-3A
(148) ##STR00095##
Synthesis of Compound 7-3A-1
(149) ##STR00096##
(150) At 0° C., periodic acid (18.00 g, 78.97 mmol, 1.08 eq) was added in portions to a suspension of Compound 1-5 (12 g, 73.07 mmol, 1 eq) and ferric trichloride (1.20 g, 7.40 mmol, 0.1 eq) in acetonitrile (100 mL). The reaction solution was stirred at 15° C. for 2 hours. When LC-MS showed the completion of the reaction, a saturated aqueous sodium sulfite solution (500 mL) was added to quench the reaction. The resulting aqueous phase was extracted three times with ethyl acetate (250 mL). The combined organic layers were washed with saturated brine (250 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain Compound 7-3A-1.
(151) MS (ESI) m/z: 180.9 [M+H].sup.+;
(152) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 8.82 (d, J=1.6 Hz, 1H), 8.21 (dd, J=2.4, 8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 3.04-3.13 (m, 1H), 2.83-2.88 (m, 1H), 1.28 (t, J=7.2 Hz, 3H).
Synthesis of Compound 7-3A-2
(153) ##STR00097##
(154) At 20° C., to a solution of Compound 7-3A-1 (6.5 g, 36.07 mmol, 1 eq) in methanol (50 mL), iodobenzene diacetate (35.21 g, 109.31 mmol, 3.03 eq) and ammonium carbamate (11.38 g, 145.70 mmol, 4.04 eq) were added in portions, and the reaction solution was stirred at 15° C. for 2 hours. When thin-layer chromatography and LC-MS showed the completion of the reaction, the reaction solution was directly concentrated and dried by rotary evaporation. The residue was subjected to column separation using a flash purification system (SepaFlash® silica gel flash column (40 g), eluent: petroleum ether/ethyl acetate; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 100%; flow rate: 50 mL/min) and purified to obtain Compound 7-3A-2.
(155) MS (ESI) m/z: 195.9 [M+H].sup.+;
(156) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 9.22 (d, J=2.0 Hz, 1H), 8.41 (dd, J=2.4, 8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 3.21-3.27 (m, 2H), 1.30 (t, J=7.2 Hz, 3H).
Synthesis of Compound 7-3A-3
(157) ##STR00098##
(158) At 15° C., to a solution of Compound 7-3A-2 (2 g, 10.24 mmol, 1 eq) in tetrahydrofuran (20 mL), sodium hydride (600.00 mg, 15.00 mmol, purity: 60%, 1.46 eq) was added in portions, and the reaction solution was stirred at 15° C. for 0.5 hours. Boc.sub.2O (4.47 g, 20.46 mmol, 4.70 mL, 2 eq) was added to the reaction solution in portions, and the reaction solution was stirred at 15° C. for 16 hours. When the thin-layer chromatography and LC-MS showed the completion of the reaction, water (150 mL) was added to the reaction solution to quench the reaction. The resulting aqueous phase was extracted three times with ethyl acetate (100 mL). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to column separation and purification using a flash purification system (SepaFlash® silica gel flash column (40 g), eluent: petroleum ether/ethyl acetate; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 30%; flow rate: 50 mL/min), and Compound 7-3A-3 was obtained.
(159) MS (ESI) m/z: 195.9 [M−100+H].sup.+, 239.9 [M−56+H].sup.+;
(160) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 9.17 (d, J=2.0 Hz, 1H), 8.41 (dd, J=2.4, 8.0 Hz, 1H), 7.95 (d, J=8.0 Hz, 1H), 3.35-3.53 (m, 2H), 1.40 (s, 9H) 1.35 (t, J=7.2 Hz, 3H).
Synthesis of Intermediate 7-3A
(161) ##STR00099##
(162) In a nitrogen atmosphere, Pd/C (10%, 200 mg) was added to a solution of Compound 7-3A-3 (500 mg, 1.70 mmol, 1 eq) in 15 mL methanol. The reaction solution was purged with hydrogen gas for three times, and then stirred at 20 to 30° C. for 5 hours in a hydrogen atmosphere (hydrogen balloon). LC-MS showed that Compound 7-3A-3 was completely consumed and the formation of the product was detected. The reaction solution was filtered through celite and then concentrated under reduced pressure to give a crude product of Compound 7-3A. The crude product was used directly in the next step without purification.
(163) MS (ESI) m/z: 243.9 [M−56+H].sup.+.
Example 9
(164) ##STR00100##
(165) The preparation method of Example 9 was the same as that of Example 1, except that Compound 1-3 was replaced with 4-trifluoromethylbenzaldehyde in the last step of Example 9 to obtain Example 9.
(166) MS (ESI) m/z: 552.3 [M+H].sup.+;
(167) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 8.95 (d, J=1.2 Hz, 1H), 8.08 (dd, J=2.2, 8.2 Hz, 1H), 7.50-7.56 (m, 2H), 7.40-7.49 (m, 3H), 7.22 (s, 1H), 7.12 (br t, J=5.0 Hz, 1H), 4.75 (d, J=5.2 Hz, 2H), 4.14 (br d, J=14.0 Hz, 1H), 3.90-4.08 (m, 2H), 3.75 (d, J=14.0 Hz, 1H), 3.42 (dd, J=2.8, 12.42 Hz, 1H), 3.08 (q, J=7.2 Hz, 2H), 1.95 (qd, J=6.8, 10.8 Hz, 1H), 1.24 (t, J=7.4 Hz, 3H), 1.04 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.8 Hz, 3H).
Example 10 and Example 11
(168) ##STR00101##
(169) The preparation methods of Example 10 and Example 11 were the same as that of Example 1, except that Example 10 and Example 11 employed Compound 10-1, i.e., (R)-1-(4-(ethylsulfonyl)phenyl)-2-methoxyethylamine in lieu of Compound 1-7 in the synthetic step of Compound 1-19 in Example 1, while the remaining steps were the same. Example 10 and Example 11 were obtained by SFC separation.
Example 10
(170) The retention time Rt of the SFC analysis was 1.479 min. The SFC analysis conditions were as follows: (column: Chiralcel OD-3 50 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was isopropanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was kept at 5% for 0.2 minutes, increased from 5% to 40% within 1.4 minutes, kept at 40% for 1.05 minutes, and then kept at 5% for 0.35 minutes; flow rate: 4 mL/min; column temperature: 40° C.
(171) MS (ESI) m/z: 601.0 [M+H].sup.+;
(172) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 7.87 (d, J=8.0 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 7.32 (s, 1H), 6.76 (d, J=7.0 Hz, 1H), 5.30-5.37 (m, 1H), 4.18 (dd, J=3.8, 12.4 Hz, 1H), 3.93 (br d, J=3.6 Hz, 1H), 3.75-3.80 (m, 1H), 3.69-3.74 (m, 1H), 3.47 (dd, J=3.6, 12.0 Hz, 1H), 3.38 (s, 3H), 3.09 (q, J=7.6 Hz, 2H), 2.92 (t, J=11.8 Hz, 1H), 2.44-2.51 (m, 1H), 2.10 (br s, 1H), 1.87-1.98 (m, 4H), 1.69 (br s, 2H), 1.59 (br s, 2H), 1.48-1.53 (m, 2H), 1.28 (t, J=7.6 Hz, 3H), 1.10 (d, J=7.0 Hz, 3H), 0.82 (d, J=7.0 Hz, 3H).
Example 11
(173) The retention time Rt of the SFC analysis was 1.748 min. The SFC analysis conditions were as follows: (column: Chiralcel OD-3 50 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was isopropanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was kept at 5% for 0.2 minutes, increased from 5% to 40% within 1.4 minutes, kept at 40% for 1.05 minutes, and then kept at 5% for 0.35 minutes; flow rate: 4 mL/min; column temperature: 40° C.
(174) MS (ESI) m/z: 601.0 [M+H].sup.+;
(175) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 7.86 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.0 Hz, 2H), 7.31 (s, 1H), 6.77 (d, J=7.0 Hz, 1H), 5.29-5.39 (m, 1H), 4.20 (br d, J=12.0 Hz, 1H), 3.89 (br d, J=3.0 Hz, 1H), 3.75-3.81 (m, 1H), 3.68-3.74 (m, 1H), 3.49 (dd, J=3.0, 12.6 Hz, 1H), 3.38 (s, 3H), 3.09 (q, J=7.6 Hz, 2H), 2.50-2.64 (m, 2H), 2.22 (br d, J=14.6 Hz, 1H), 1.77-2.06 (m, 5H), 1.44 (br s, 1H), 1.29-1.38 (m, 2H), 1.24-1.29 (m, 3H), 1.06 (d, J=7.0 Hz, 3H), 0.85-1.02 (m, 2H), 0.82 (d, J=7.0 Hz, 3H).
Preparation of Intermediate 10-1 (i.e., (R)-1-(4-(ethylsulfonyl)phenyl)-2-methoxyethylamine)
(176) ##STR00102## ##STR00103##
Synthesis of Intermediate 10-1-B
(177) ##STR00104##
(178) At 25 to 30° C., triethylamine (195.63 g, 1.93 mol, 267.99 mL, 1.20 eq) was added to a solution of Compound 10-1-A (100.00 g, 1.61 mol, 90.09 mL, 1.00 eq) in 1 L anhydrous dichloromethane. After the resulting mixture was cooled down to 0° C., TBSCl (242.82 g, 1.61 mol, 197.41 mL, 1.00 eq) dissolved in 300 mL dichloromethane was added dropwise within one hour. The reaction solution was stirred at 25 to 35° C. for 18 hours. The reaction solution was added with 400 mL saturated ammonium chloride solution to quench the reaction, and then subjected to liquid-liquid separation. The aqueous phase was extracted twice with 800 mL methyl tert-butyl ether, and the combined organic phases were concentrated under reduced pressure and then dissolved with 400 mL methyl tert-butyl ether. The methyl tert-butyl ether phase was washed twice with 1,000 mL water, and then washed with 500 mL saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a yellow liquid. The resulting Compound 10-1-B was directly used in the next step. .sup.1H NMR (400 MHz, CHLOROFORM-d) 6=3.61-3.64 (m, 2H), 3.56 (br d, J=4.4 Hz, 2H), 2.17 (br s, 1H), 0.82-0.83 (m, 9H), 0.00 (s, 6H).
Synthesis of Intermediate 10-1-C
(179) ##STR00105##
(180) After 500 mL solution of dichloromethane was cooled down to −30° C., (COCl).sub.2 (39.59 g, 311.92 mmol, 27.30 mL, 1.10 eq) was added dropwise. After the completion of the dropwise addition, the reaction solution was cooled down to −70° C., and DMSO (48.74 g, 623.83 mmol, 44.31 mL, 2.20 eq) was added dropwise. After the completion of the addition, the reaction solution was stirred at the same temperature for 30 min. At the same temperature, 100 mL dichloromethane in which Compound 10-1-B (50.00 g, 283.56 mmol, 1.00 eq) was dissolved was slowly added dropwise to the reaction system. After the reaction solution was stirred at −70° C. for one hour, Et.sub.3N (143.47 g, 1.42 mol, 196.53 mL, 5.00 eq) was added dropwise. The reaction solution was stirred at −70° C. for 30 minutes, and then heated to 25 to 35° C. and reacted for 16 hours. TLC showed that Compound 10-1-B was completely consumed and a new spot was formed. The reaction solution was washed with 500 mL water, then washed twice with 1000 mL of 1 mol/L hydrochloric acid, and washed again with 500 mL of saturated sodium bicarbonate solution and 500 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give Compound 10-1-C, which was used directly in the next step.
(181) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 9.64 (s, 1H), 4.15 (s, 2H), 0.87 (s, 9H), 0.04 (s, 6H).
Synthesis of Intermediate 10-1-E
(182) ##STR00106##
(183) A solution (1.50 L) of Compound 10-1-C (102.00 g, 585.16 mmol, 1.00 eq), Compound 10-1-D (70.92 g, 585.16 mmol, 1.00 eq) and CuSO.sub.4 (233.50 g, 1.46 mol, 224.52 mL, 2.50 eq) in anhydrous dichloromethane was stirred at 25 to 35° C. for 16 hours. TLC showed that Compound 10-1-C was completely consumed and a major spot appeared. The reaction solution was quenched with 1000 mL water and subjected to liquid-liquid separation. The aqueous phase was extracted twice with 2000 mL dichloromethane. The combined organic phases were washed once with 1000 mL water and 1000 mL saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was allowed to pass through a flash column (ISCO®; SepaFlash® silica gel flash column (220 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 20%; flow rate: 100 mL/min) in three portions and was purified to give Compound 10-1-E.
(184) MS (ESI) m/z: 278.1 [M+H].sup.+;
(185) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.96 (t, J=3.2 Hz, 1H), 4.44 (d, J=3.0 Hz, 2H), 1.11 (s, 9H), 0.82 (s, 9H), 0.00 (s, 6H).
Synthesis of Intermediate 10-1-G
(186) ##STR00107##
(187) At −65° C., n-BuLi (2.5 M, 63.42 mL, 2.20 eq) was added dropwise to a solution of Compound 10-1-F (31.30 g, 144.14 mmol, 2.00 eq) in 150 mL anhydrous tetrahydrofuran. After the completion of the dropwise addition, the reaction solution was stirred at the same temperature for half an hour. A solution of Compound 10-1-E (20.00 g, 72.07 mmol, 1.00 eq) in 50 mL anhydrous tetrahydrofuran was added dropwise to the reaction solution at −65° C. Upon the completion of the dropwise addition, the reaction solution was stirred at the same temperature for 2 hours, and then heated to 30° C. and stirred for 2 hours. LC-MS showed that Compound 10-1-E was completely consumed and there was a main peak showing the MS of the product. The reaction solution was quenched with 200 mL saturated ammonium chloride solution and then extracted twice with 200 mL ethyl acetate. The combined organic phases were washed sequentially with 200 mL of water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by a flash column (ISCO®; SepaFlash® silica gel flash column (120 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 20%; flow rate: 80 mL/min) to obtain Compound 10-1-G.
(188) MS (ESI) m/z: 438.2 [M+Na].sup.+;
(189) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.15-7.23 (m, 4H), 4.42 (m, 1H), 4.19 (s, 1H), 3.67-3.71 (m, 1H), 3.49-3.55 (m, 1H), 2.88 (q, J=7.4 Hz, 2H), 1.25 (t, J=7.4 Hz, 3H), 1.14-1.17 (m, 9H), 0.83 (s, 9H), −0.01 (d, J=7.6 Hz, 6H).
Synthesis of Intermediate 10-1-H
(190) ##STR00108##
(191) To a solution of Compound 10-1-G (2.00 g, 4.81 mmol, 1.00 eq) in 30 mL tetrahydrofuran was added TBAF (2.52 g, 9.62 mmol, 2.00 eq). The reaction solution was stirred for 1 hour at 20 to 25° C. TLC showed that Compound 10-1-G was completely consumed and a new spot was generated. The reaction solution was washed with 15 mL of a saturated sodium bicarbonate solution and 15 mL of a saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product as an oil. The crude product was purified by a flash column (ISCO®; SepaFlash® silica gel flash column (20 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 100%; flow rate: 35 mL/min) to obtain Compound 10-1-H.
(192) MS (ESI) m/z: 324.0 [M+Na].sup.+;
(193) .sup.1H NMR (400 MHz, DMSO-d6) δ=7.21-7.28 (m, 4H), 5.04 (t, J=5.8 Hz, 1H), 4.95 (d, J=4.0 Hz, 1H), 4.20-4.25 (m, 1H), 3.52 (t, J=6.2 Hz, 2H), 2.94 (q, J=7.4 Hz, 2H), 1.21 (t, J=7.4 Hz, 3H), 1.10 (s, 9H).
Synthesis of Intermediate 10-1-I
(194) ##STR00109##
(195) At 0° C., NaH (199.02 mg, 4.98 mmol, purity: 60%, 1.50 eq) was added to a solution of Compound 10-1-H (1.00 g, 3.32 mmol, 1.00 eq) in 30 mL tetrahydrofuran, and then the resulting mixture was stirred for half an hour. Iodomethane (990.00 mg, 6.97 mmol, 434.21 μL, 2.10 eq) was added dropwise, and then the reaction solution was stirred at 20 to 25° C. for 15.5 h. LC-MS showed that Compound 10-1-H was completely consumed. The reaction solution was dispersed in 30 mL water and 30 mL ethyl acetate, and was subjected to liquid-liquid separation. The organic phases were washed with 30 mL saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by a flash column (ISCO®; SepaFlash® silica gel flash column (60 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 100%; flow rate: 40 mL/min) to obtain Compound 10-1-I.
(196) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.16-7.23 (m, 4H), 4.51-4.60 (m, 1H), 4.02 (s, 1H), 3.42-3.46 (m, 2H), 3.32 (s, 3H), 2.88 (q, J=7.45 Hz, 2H), 1.25 (t, J=7.28 Hz, 3H), 1.15 (s, 9H)
Synthesis of Intermediate 10-1-J
(197) ##STR00110##
(198) A solution of Compound 10-1-I (800.00 mg, 2.54 mmol, 1.00 eq) in HCl/dioxane (2.54 mmol, 4.00 mL, 1.00 eq) was stirred at 20 to 25° C. for 2 hours. LC-MS showed that Compound 10-1-I was completely consumed. The reaction solution was concentrated under reduced pressure to remove the solvent, so as to obtain a crude product of Compound 10-1-J, which was directly used in the next step without purification.
(199) MS (ESI) m/z: 194.9 [M-NH.sub.2].sup.+;
Synthesis of Intermediate 10-1
(200) ##STR00111##
(201) Oxone (4.07 g, 6.63 mmol, 2.00 eq) was added to 30 mL aqueous solution of Compound 10-1-J (700.00 mg, 3.31 mmol, 1.00 eq). The reaction solution was stirred at 20 to 25° C. for 2 hours. LC-MS showed that Compound 10-1-J was completely consumed and MS of Compound 10-1 appeared. The reaction solution was directly lyophilized, and the crude product was purified by a flash column (ISCO®; SepaFlash® silica gel flash column (4 g), eluent: dichloromethane and methanol; elution gradient: 0% to 10%; flow rate: 18 mL/min) to obtain Compound 10-1-I.
(202) MS (ESI) m/z: 244.0 [M+H].sup.+;
(203) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.91 (d, J=7.6 Hz, 2H), 7.69 (d, J=7.8 Hz, 2H), 4.57 (br s, 1H), 3.75 (br d, J=5.8 Hz, 2H), 3.35-3.45 (m, 5H), 3.12 (q, J=7.2 Hz, 2H), 1.25 (t, J=7.4 Hz, 3H).
Example 12 and Example 13
(204) ##STR00112##
(205) The preparation methods of Example 12 and Example 13 were the same as that of Example 1, except that Example 12 and Example 13 employed Compound 12-1, i.e., (R)-2-amino-(4-(ethylsulfonyl)phenyl)ethanol in lieu of Compound 1-7 in the synthetic step of Compound 1-19 in Example 1, while the remaining steps were the same. Example 12 and Example 13 were obtained by SFC separation.
Example 12
(206) The retention time Rt of the SFC analysis was 2.963 min. The SFC analysis conditions were as follows: (column: Chiralpak AS-H 150 mm×4.6 mm, I.D. 5 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was kept at 5% for 0.5 minutes, increased from 5% to 40% within 3.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1.5 minutes; flow rate: 3 mL/min; column temperature: 40° C.
(207) MS (ESI) m/z: 587.0 [M+H].sup.+;
(208) .sup.1HNMR (400 MHz, CHLOROFORM-d): δ 7.82 (br d, J=8.6 Hz, 2H), 7.51 (d, J=8.6 Hz, 2H), 7.27 (s, 1H), 6.74 (br s, 1H), 5.20 (br s, 1H), 4.12 (br d, J=12.6 Hz, 1H), 3.96 (br d, J=15.0 Hz, 2H), 3.82 (br s, 1H), 3.42 (br d, J=12.0 Hz, 1H), 3.03 (q, J=7.6 Hz, 2H), 2.43-2.59 (m, 2H), 2.15 (br d, J=14.0 Hz, 1H), 1.92 (br d, J=10.6 Hz, 3H), 1.72-1.87 (m, 2H), 1.24-1.43 (m, 1H), 1.22 (t, J=7.6 Hz, 4H), 1.00 (d, J=7.0 Hz, 3H), 0.78-0.94 (m, 2H), 0.76 (d, J=6.6 Hz, 3H).
Example 13
(209) The retention time Rt of the SFC analysis was 3.342 min. The SFC analysis conditions were as follows: (column: Chiralpak AS-H 150 mm×4.6 mm, I.D. 5 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was kept at 5% for 0.5 minutes, increased from 5% to 40% within 3.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1.5 minutes; flow rate: 3 mL/min; column temperature: 40° C.
(210) MS (ESI) m/z: 587.0 [M+H].sup.+;
(211) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 7.89 (br d, J=7.6 Hz, 2H), 7.58 (br d, J=8.0 Hz, 2H), 7.34 (s, 1H), 6.82 (br s, 1H), 5.28 (br s, 1H), 4.20 (br d, J=14.6 Hz, 1H), 4.03 (br d, J=19.0 Hz, 2H), 3.89 (br s, 1H), 3.49 (br d, J=10.6 Hz, 1H), 3.10 (br d, J=7.0 Hz, 2H), 2.47-2.66 (m, 2H), 2.22 (br d, J=13.6 Hz, 1H), 1.98 (br d, J=10.6 Hz, 3H), 1.77-1.92 (m, 2H), 1.25-1.30 (m, 6H), 1.06 (br d, J=7.0 Hz, 3H), 0.82 (br d, J=6.6 Hz, 4H).
Preparation of the Intermediate (R)-2-amino-(4-(ethylsulfonyl)phenyl)ethanol
(212) ##STR00113##
Synthesis of Intermediate 12-1-1A
(213) ##STR00114##
(214) At 0° C., to a solution of Compound 10-1-G (400.00 mg, 962.16 μmol, 1.00 eq) in 70 mL dichloromethane was added HCl/dioxane (4 M, 481.08 μL, 2.00 eq). The reaction solution was stirred at 20 to 25° C. for 1.5 hours. LC-MS showed that Compound 10-1-G was completely consumed and MS of Compound 12-1-A was obtained. After the solvent was removed from the reaction solution under reduced pressure, Compound 12-1-A was obtained and was directly used in the next step without further purification.
(215) MS (ESI) m/z: 180.8 [M+H].sup.+;
Synthesis of Intermediate 12-1
(216) ##STR00115##
(217) At 0° C., an aqueous solution of Oxone (1.18 g, 1.92 mmol, 2.00 eq) in 2 mL H.sub.2O (2.00 mL) was added dropwise to a solution of Compound 12-1-1A (224.91 mg, 962.14 μmol, 1.00 eq, HCl) in 2 mL MeOH. After the mixture was stirred at 20 to 25° C. for one hour, the completion of the reaction was monitored by LC-MS. The reaction solution was filtered. The filtrate was quenched with 30 mL saturated sodium thiosulfate solution, and then concentrated under reduced pressure to give a crude product. After the crude product was dissolved in a solution of CH.sub.2Cl.sub.2:MeOH (3:1, 20 mL), the mixture was stirred for 30 min, filtered, and concentrated under reduced pressure to obtain Compound 12-1, which was directly used in the next step without further purification.
(218) MS (ESI) m/z: 213.0 [M-NH.sub.2].sup.+;
(219) .sup.1HNMR (400 MHz, DEUTERIUM OXIDE) δ=7.93 (d, J=8.6 Hz, 2H), 7.65 (d, J=8.6 Hz, 2H), 4.56 (dd, J=7.0, 4.8 Hz, 1H), 3.81-3.97 (m, 2H), 3.30 (q, J=7.2 Hz, 2H), 1.13 (t, J=7.4 Hz, 3H).
Example 14 and Example 15
(220) ##STR00116##
Example 14 or Example 15 Example 14 or Example 15
(221) ##STR00117## ##STR00118##
Synthesis of Compound 15-3
(222) ##STR00119##
(223) Compound 15-2 (20 g, 165.02 mmol, 1 eq) was dissolved in anhydrous dichloromethane (300.00 mL), MgSO.sub.4 (99.91 g, 830.03 mmol, 5.03 eq) and PPTS (4 g, 15.92 mmol, 0.096 eq) were sequentially added thereto, Compound 15-1 was added finally, and the reaction solution was stirred at 20° C. for 16 hours. When TLC showed that a spot was formed under UV light, the reaction solution was filtered through celite. After the filter cake was washed with dichloromethane, the resultant was combined with the filtrate, and the mixture was concentrated under reduced pressure to give a crude product. The crude product was purified by a flash column (SepaFlash® silica gel flash column (220 g), eluent: ethyl acetate and petroleum ether; elution gradient: ethyl acetate/petroleum ether (V/V)=0% to 100%, 0% to 30%; flow rate: 100 mL/min) to obtain Compound 15-3.
(224) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 7.92 (d, J=4.2 Hz, 1H), 1.12 (s, 9H), 1.11 (d, J=2.0 Hz, 3H), 1.10-1.10 (m, 1H), 1.09 (d, J=2.0 Hz, 3H).
Synthesis of Compound 15-5
(225) ##STR00120##
(226) Compound 15-4 (5 g, 39.02 mmol, 1 eq) was dissolved in anhydrous THF (25 mL), to which LDA (2 M, 45.06 mL, 2.31 eq) was slowly added dropwise at 0° C. under the protection of nitrogen, and the mixture was stirred for 0.5 hours while the temperature was kept at −60° C. Then, a solution of Compound 15-3 (8.2 g, 46.8 mmol, 1.2 eq) in anhydrous THF (25 mL) was added dropwise to the above reaction solution within 1 hour. After the completion of the dropwise addition, the reaction solution was heated to 20° C. and then stirred for 1 hour. When LC-MS showed the completion of the reaction, water (100 mL) was added to quench the reaction, and an aqueous phase was obtained by separation. The resulting mixture was washed with ethyl acetate (100 mL), and then its pH was adjusted to 3 with hydrochloric acid (1 N, 20 mL), followed by extraction with ethyl acetate (150 mL×3). The combined organic layers were washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain 10.3 g of a crude product of Compound 15-5. The crude product was diluted with 10 mL ethyl acetate. 70 mL petroleum ether was slowly added to the resulting suspension, and the suspension was stirred for half an hour and then filtered. The filter cake was slurried with a mixed solvent of 3 mL ethyl acetate and 20 mL petroleum ether, and the resultant was filtered and dried to obtain Compound 15-5.
(227) MS (ESI) m/z: 303.9 [M+H].sup.+
(228) .sup.1H NMR (400 MHz, CHLOROFORM-d): δ 7.48 (d, J=5.2 Hz, 1H), 7.13 (d, J=5.2 Hz, 1H), 2.24-2.36 (m, 1H), 1.22 (s, 9H), 1.09 (br d, J=6.8 Hz, 3H), 0.91 (br d, J=6.2 Hz, 3H)
Synthesis of Compound 15-6
(229) ##STR00121##
(230) A solution of Compound 15-5 (5.1 g, 16.97 mmol, 1 eq) in hydrochloric acid-ethyl acetate (30 mL) was stirred at 28° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was filtered to obtain Compound 15-6. The filter cake was slurried twice with 10 mL ethyl acetate. The resultant was filtered, dried, and directly used in the next step.
(231) MS (ESI) m/z: 183 [M-NH.sub.2].sup.+
(232) .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.69 (br s, 3H), 7.68 (d, J=5.0 Hz, 1H), 7.40 (d, J=5.6 Hz, 1H), 5.21 (br dd, J=5.6, 9.6 Hz, 1H), 2.14-2.28 (m, 1H), 1.09 (d, J=6.6 Hz, 3H), 0.80 (d, J=7.0 Hz, 3H)
Synthesis of Compound 15-7
(233) ##STR00122##
(234) To a solution of Compound 15-6 (3.15 g, 13.36 mmol, 1 eq, HCl) in anhydrous dichloromethane (600.00 mL), HATU (7.62 g, 20.04 mmol, 1.5 eq) and TEA (4.06 g, 40.09 mmol, 5.58 mL, 3 eq) were added, and the mixture was stirred at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, water (50.00 mL) was added for dilution and the resulting mixture was extracted with dichloromethane (200.00 mL×3). The combined organic layers were washed with saturated brine (100.00 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (40 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 60%; flow rate: 35 mL/min) to obtain Compound 15-7.
(235) MS (ESI) m/z: 182.0 [M+H].sup.+
(236) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.27 (d, J=5.0 Hz, 1H), 7.16-7.21 (m, 1H), 6.73 (br s, 1H), 4.45 (d, J=5.2 Hz, 1H), 1.92-2.07 (m, 1H), 0.96 (d, J=6.8 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H).
Synthesis of Compound 15-8
(237) ##STR00123##
(238) BH.sub.3-Me.sub.2S (10 M, 13.24 mL, 10 eq) was slowly added dropwise to a solution of Compound 15-7 (2.4 g, 13.24 mmol, 1 eq) in anhydrous tetrahydrofuran (30 mL) at room temperature, and the resulting mixture was stirred until the bubbles disappeared. Afterwards, the mixture was heated to 65° C. and reacted for 96 hours. When LC-MS showed the completion of the reaction, the reaction solution was cooled down to room temperature, and hydrochloric acid (2 N, 10 mL) was slowly added dropwise to quench the reaction. Thereafter, the reaction solution was washed with ethyl acetate (10 mL) and the aqueous phase was then separated. After the pH of the aqueous phase was adjusted to 8 with a saturated sodium bicarbonate solution, the mixture was extracted with ethyl acetate (40 mL×2). The combined organic layers were washed with water (10 mL), and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a crude product. After the crude product was combined with another batch, they were purified by a flash column (SepaFlash® silica gel flash column (80 g), eluent: ethyl acetate and petroleum ether; elution gradient: 0% to 30%; flow rate: 50 mL/min) to obtain Compound 15-8.
(239) MS (ESI) m/z: 167.9 [M+H].sup.+
(240) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.08-7.25 (m, 1H), 6.73 (d, J=4.8 Hz, 1H), 4.22 (td, J=2.8, 5.4 Hz, 1H), 4.03 (dd, J=3.2, 5.8 Hz, 2H), 1.81 (sxtd, J=6.6, 12.8 Hz, 1H), 0.92 (t, J=6.6 Hz, 6H).
Synthesis of Compound 15-9
(241) ##STR00124##
(242) To a solution of Compound 15-8 (5 g, 29.89 mmol, 1 eq) in anhydrous dichloromethane (50 mL), TEA (6.05 g, 59.78 mmol, 8.32 mL, 2 eq) and Boc anhydride (7.83 g, 35.87 mmol, 8.24 mL, 1.2 eq) were added, and the reaction solution was reacted at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (40 g), eluent: ethyl acetate and petroleum ether, elution gradient: ethyl acetate/petroleum ether (v/v)=0% to 20%; flow rate: 35 mL/min) to obtain Compound 15-9.
(243) MS (ESI) m/z: 211.9 [M-tert-butyl+H].sup.+
(244) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.17 (d, J=5.0 Hz, 1H), 6.78 (dd, J=4.8, 16.44 Hz, 1H), 4.81-4.98 (m, 1H), 4.43-4.60 (m, 1H), 4.33 (dd, J=3.8, 13.8 Hz, 1H), 2.27-2.59 (m, 1H), 1.43 (s, 9H), 0.98 (t, J=7.8 Hz, 3H), 0.54 (t, J=7.0 Hz, 3H).
Synthesis of Compound 15-10
(245) ##STR00125##
(246) To a solution of Compound 15-9 (1 g, 3.74 mmol, 1 eq) in acetonitrile (10 mL) was added NBS (732.21 mg, 4.11 mmol, 1.1 eq), and the reaction solution was stirred at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by a flash column (SepaFlash® silica gel flash column (4 g), eluent: ethyl acetate and petroleum ether, elution gradient: 0% to 10%; flow rate: 18 mL/min) to obtain Compound 15-10.
(247) MS (ESI) m/z: 291.9 [M-tert-butyl+H].sup.+
(248) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 6.79 (d, J=18.8 Hz, 1H), 4.76-4.95 (m, 1H), 4.25-4.60 (m, 2H), 2.21-2.57 (m, 1H), 1.43 (s, 9H), 0.94 (t, J=7.0 Hz, 3H), 0.56 (t, J=7.2 Hz, 3H).
Synthesis of Compound 15-12
(249) ##STR00126##
(250) A mixed solution of Compound 15-10 (0.3 g, 1.32 mmol, 1 eq), Compound 15-11 (548.49 mg, 1.58 mmol, 1.2 eq), CuI (251.39 mg, 1.32 mmol, 1 eq) and K.sub.3PO.sub.4 (560.37 mg, 2.64 mmol, 2 eq) in anhydrous dioxane (10 mL) was purged three times with nitrogen, N,N-diisopropylethylamine (127.99 mg, 1.45 mmol, 156.28 μL, 1.1 eq) was then added thereto under nitrogen protection, and the reaction solution was stirred at 100° C. for 1.5 hours. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove dioxane, and then diluted with water (10 mL). The resulting mixture was extracted with dichloromethane (15 mL×3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was prepared and purified by thin-layer chromatography (SiO.sub.2, PE:EA=1:1) to obtain Compound 15-12.
(251) MS (ESI) m/z: 393.0 [M-Boc+H].sup.+
(252) .sup.1H NMR (CHLOROFORM-d) δ: 7.86 (d, J=8.0 Hz, 2H), 7.53 (br d, J=8.0 Hz, 2H), 6.45 (d, J=16.4 Hz, 1H), 4.85-5.01 (m, 1H), 4.50 (t, J=14.8 Hz, 1H), 4.33 (td, J=13.2, 3.6 Hz, 1H), 3.80-3.84 (m, 2H), 3.69-3.77 (m, 2H), 3.12 (q, J=7.6 Hz, 2H), 2.29-2.62 (m, 1H), 1.50 (d, J=3.6 Hz, 9H), 1.28-1.32 (m, 3H), 0.96-1.05 (m, 3H), 0.52-0.65 (m, 3H).
Synthesis of Intermediate 15-11
(253) ##STR00127##
(254) A solution of Compound 15-11A (1 g, 4.38 mmol, 1 eq) in SOCl.sub.2 (16.40 g, 137.85 mmol, 10 mL, 31.47 eq) was stirred at 20° C. for 2 hours, and then concentrated under reduced pressure to remove SOCl.sub.2 and dissolved in anhydrous dichloromethane (20 mL), aqueous ammonia (9.10 g, 64.92 mmol, 10 mL, purity: 25%, 14.82 eq) was added thereto, and the reaction solution was stirred at 20° C. for 3 hours. When LC-MS showed the completion of the reaction, the mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL×3). The combined organic layers were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give Intermediate 15-11.
(255) MS (ESI) m/z: 228.0 [M-Boc+H].sup.+
(256) .sup.1H NMR (DMSO-d.sub.6) δ: 7.81 (d, J=8.4 Hz, 2H), 7.59 (br s, 1H), 7.53 (d, J 8.4 Hz, 2H), 7.00 (br s, 1H), 3.52 (s, 2H), 3.27 (q, J=7.2 Hz, 2H), 1.10 (t, J=7.6 Hz, 3H).
Synthesis of Compound 15-13
(257) ##STR00128##
(258) A solution of Compound 15-12 (0.09 g, 182.69 μmol, 1 eq) in hydrochloric acid-dioxane (3 mL) was stirred at room temperature for 16 hours. When LC-MS showed the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove dioxane, so as to give the hydrochloride of Compound 15-13.
(259) MS (ESI) m/z: 393.2 [M+H].sup.+
Synthesis of Example 14 and Example 15
(260) ##STR00129##
(261) Triethylamine (20.76 mg, 205.13 μmol, 28.55 μL, 1.1 eq) was added to a solution of Compound 15-13 (0.08 g, 186.48 μmol, 1 eq, HCl) in anhydrous dichloroethane (3 mL), the resulting mixture was stirred at 20° C. for 0.5 hours, and acetic acid (11.20 mg, 186.48 μmol, 10.67 μL, 1 eq) was then added to adjust the pH to 5, followed by the addition of Compound 1-3 (101.36 mg, 559.45 μmol, 3 eq) and NaBH(OAc).sub.3 (118.57 mg, 559.45 μmol, 3 eq). The reaction solution was stirred at 20° C. for 1 hour. When LC-MS showed the completion of the reaction, a saturated ammonium chloride solution was added to quench the reaction, and the resulting mixture was then extracted with dichloromethane (6 mL×2). The combined organic layers were washed with saturated brine (8 mL) and then dried over anhydrous sodium sulfate to give a crude product. The crude product was purified by preparative thin-layer chromatography and separated by SFC (SFC separation conditions: (column: DAICEL CHIRALCEL OD-H 250 mm×30 mm, 5 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.1% aqueous ammonia, wherein the volume ratio of phase B was 40%) to obtain Example 14 and Example 15.
Example 14
(262) The retention time Rt of the SFC analysis was 3.983 min. The SFC analysis conditions were as follows: (column: Chiralcel OD-3 150 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was increased from 5% to 40% within 4.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1 minute; flow rate: 2.8 mL/min; column temperature: 40° C.
(263) MS (ESI) m/z: 557.1 [M+H].sup.+;
(264) .sup.1H NMR (CHLOROFORM-d) δ: 7.82 (d, J 8.4 Hz, 2H), 7.49 (br d, J=7.6 Hz, 2H), 6.36 (s, 1H), 4.14 (br d, J=6.4 Hz, 1H), 3.77 (s, 2H), 3.40 (br s, 1H), 3.05 (q, J=7.4 Hz, 2H), 2.55 (br s, 1H), 2.14 (br d, J=12.4 Hz, 1H), 1.85-1.98 (m, 3H), 1.78 (br d, J=11.2 Hz, 2H), 1.17-1.29 (m, 7H), 0.97 (br d, J=7.2 Hz, 3H), 0.80-0.93 (m, 3H), 0.76 (br d, J=5.6 Hz, 4H).
Example 15
(265) The retention time Rt of the SFC analysis was 4.277 min. The SFC analysis conditions were as follows: (column: Chiralcel OD-3 150 mm×4.6 mm, I.D. 3 μm); mobile phase: phase A was supercritical fluid carbon dioxide, and phase B was methanol containing 0.05% diethylamine; gradient: the volume ratio of mobile phase B was increased from 5% to 40% within 4.5 minutes, kept at 40% for 2.5 minutes, and then kept at 5% for 1 minute; flow rate: 2.8 mL/min; column temperature: 40° C.
(266) MS (ESI) m/z: 557.1 [M+H].sup.+;
(267) .sup.1H NMR (CHLOROFORM-d): δ 7.79 (br d, J=8.4 Hz, 2H), 7.55 (br s, 2H), 6.44 (br s, 1H), 3.84 (br s, 2H), 3.04 (q, J=7.2 Hz, 2H), 2.17 (br s, 1H), 1.74-2.00 (m, 4H), 1.13-1.34 (m, 9H), 1.02 (br s, 5H), 0.83 (br s, 6H).
Bioactivity Experiment 1-A: In Vitro Test for Inhibitory Activity on RORγ
(268) 1. Test Method
(269) Reporter cells: Reporter cells expressing a chimeric RORγ receptor were used in this experiment. The chimeric RORγ receptor was a RORγ receptor formed by replacing the N-terminal DNA-binding domain of the natural RORγ protein with the DNA-binding domain of the yeast Gal4 protein. The luciferase reporter gene was located at the downstream of the Gal4 activating sequence (UAS). The details were as shown in the table below:
(270) TABLE-US-00001 Receptor Form Receptor --- Host cell (gene symbol) Reporter gene line Reference RORγ Gal4 hybrid receptor HEK293 Inverse agonist, --- i.e., ursolic acid Gal4 upstream activating sequence -- luciferase
(271) Step 1: Reporter cells were prepared into a cell suspension using INDIGO's Cell Recovery Medium (CRM, containing 5% fetal bovine serum treated by activated carbon). The cell suspension was dispensed into a white 96-well culture plate in a volume of 100 μl/well.
(272) Step 2: Eight concentration gradients were set for each test compound, and the test was conducted twice at each concentration. Prior to the experiment, the main stock solution of the test compound was serially diluted with DMSO, so as to formulate solutions with “1000×-concentration” relative to each of the final test concentrations. Subsequently, the compound was further diluted with the cell recovery medium (CRM, containing 5% fetal bovine serum treated by activated carbon), so as to formulate test working solutions with “2×-concentration”. As per a volume of 100 μl/well, the test working solutions were respectively added to the test wells in which the reporter cells were added in advance, so as to obtain the desired final test concentrations. The residual concentrations of DMSO in all test wells were 0.1%. The test plate was incubated in a cell incubator for 24 hours. Conditions of the incubator were set as follows: temperature: 37° C., 5% CO.sub.2, humidity: ˜85%.
(273) Step 3: After 24 hours of incubation, the liquid in the culture plate was discarded, 100 μl of luciferase detection reagent was added to each test well, and then, the fluorescence intensity (Relative Luminescence Units, RLUs) in each well was read.
(274) Inspection of the Test:
(275) In this test, the reference compound ursolic acid was used as an internal standard of a positive compound to verify the inhibitory effect of the test compound on RORγ in reporter cells of a specific batch. The tests for the reference compound and the test compound were performed simultaneously, and the reference compound and the test compound were thus exposed to the same test reagent and environment. The reference compound group containing 0.1% DMSO as solvent was used as an internal standard of a positive compound, so as to determine the effect of the solvent DMSO on the test results and calculate the percentage reduction in RORγ activity.
(276) 2. Data Processing
(277) The test data was managed and archived by Microsoft Excel, and mean±standard deviation (SD) of RLU, fold of reduction, inhibition rate, coefficient of variation (% CV) and Z factor were calculated.
Coefficient of variation (% CV): 100×(SD/Ave. RLU);
Fold of reduction for inverse agonist: [Ave. RLU.sup.vehicle/Ave. RLU.sup.test compound].
Percentage reduction for inverse agonist: 100×(1−[Ave. RLU.sup.test compound/Ave. RLU.sup.vehicle]);
(278) Theoretically, the minimum reduction (0% reduction) was shown in the solvent control group free of compound;
Z factor: 1−[(3×[SD.sup.vehicle+SD.sup.test compound])/(RLU.sup.vehicle−RLU.sup.test compound)].
(279) 3. Graphic Data Processing Method
(280) The dose-response curves (DRCs) of the reference compound and the test compound in the tests aiming at ROR γ were obtained by non-linearly fitting the inhibition rate of ROR γ activity and the logarithmic values of the concentrations of the compounds using the GraphPad Prism software.
(281) 4. Test Results
(282) The results of the in vitro screening test for the compounds of the Examples were shown in Table 1 in detail.
(283) TABLE-US-00002 TABLE 1 Results of the In Vitro Screening Test for the Compounds of the Examples Specific value of the Value of the concentration concentration required for required for 50% Inhibition 50% inhibition (EC50) of Test Samples (EC.sub.50) of RORγ RORγ Example 1 A 15.8 nM (in free state) Example 3 B 147 nM (in free state) Definition of bioactivity: A: EC.sub.50 ≤ 100 nM; B: 100 nM < EC.sub.50 ≤ 500 nM; C: 500 nM < EC.sub.50 ≤ 1000 nM; D: 1000 nM < EC.sub.50 ≤ 5000 nM.
Bioactivity Experiment 1-B: In Vitro Test for Inhibitory Activity on RORγ: TR-FRET Screening
(284) The compounds of the present application was able to regulate (inhibit) the bioactivity of the nuclear receptor RORγ, and the strength of this regulatory (inhibitory) effect could be evaluated by a TR-FRET (time-resolved fluorescence resonance energy transfer) screening system. Nuclear receptor cofactors (co-activators and co-inhibitors) could regulate the transcription of a target gene by the interaction with nuclear receptors. If a ligand (test compound) affected the interaction between the nuclear receptors and the cofactors, this ligand (test compound) was able to regulate the transcription of the corresponding gene.
(285) This method adopted the TR-FRET (time-resolved fluorescence resonance energy transfer) technique to determine the ability of a compound to regulate the interaction between the polypeptide indirectly bound to APC (phycocyanin) (namely, APC was indirectly bound to the polypeptide by binding to Streptavidin and biotin) and RORC2-LBD linked to a europium (Eu)-labeled anti-His antibody (Perkin Elmer #AD0111). In the absence of the compound, RORC2 could bind to an APC-modified polypeptide. If the compound was an agonist, it could enhance the interaction between RORC2 and the APC-modified polypeptide after binding to RORC2. On the contrary, if the compound was an inverse agonist, it could inhibit the interaction between RORC2 and the APC-modified polypeptide after binding to RORC2. When the APC-modified polypeptide and the Eu-labeled anti-His antibody-RORC2-LBD complex approached to each other and the distance therebetween was within a certain distance, energy transfer could occur and a TR-FRET signal was generated.
(286) (1) The final concentrations and reaction conditions of the reaction system of this method were as shown in the following table.
(287) TABLE-US-00003 Concen- Concentration tration Biotin- of the of labeled APC- Eu-labeled Total RORc- SRC1 labeled anti-His volume Reaction LBD peptide Streptavidin antibody (μL) conditions 15 nM 200 nM 50 nM 1 nM 25 4° C., overnight SRC1 peptide: a polypeptide containing LXXLL-motif and capable of binding to RORC2-LBD; wherein LXXLL-motif referred to a structural sequence containing LXXLL, in which L referred to leucine and X referred to any amino acid.
(288) 2. Experimental Method
(289) 2.1 Formulation of Buffer Solution
(290) A buffer solution comprising 50 mM Tris (pH 7.0), 50 mM KCl, 1 mM Na-EDTA, 0.01% BSA (0.1 mg/ml), and 0.1 mM DTT was freshly formulated prior to the experiment.
(291) 2.2 Formulation of a RORγ-Free Working Solution
(292) Prior to the test, a working solution containing 1.67 nM Eu/anti-His and 0.75 μl/well of SF9 cell lysate was freshly formulated, and was placed on ice prior to use.
(293) 2.3 Formulation of a RORγ-containing working solution. Prior to the test, a working solution containing 1.67 nM Eu/anti-His and 0.75 μl/well of SF9 cell lysate was freshly formulated, and was placed on ice before use. 25.05 nM of RORγ was added to the working solution.
(294) 2.4 Formulation of a Mixed Solution of Peptide-Streptomycin/APC
(295) A buffer solution containing 500 nM polypeptide and 125 nM Streptavidin/APC was freshly formulated prior to the test, and was placed on ice prior to use.
(296) 2.5 Ten concentration gradients were set for each test compound, and duplicate wells were set for each concentration. The formulating method of the test compounds was as follows:
(297) 1) aspirating 2 μl solution from the source plate in which a 10 mM solution of the compound in DMSO was added, and adding said solution to columns 1 and 11 of coming 3656;
(298) 2) adding 38 μl DMSO to columns 1 and 11 via Bravo, and diluting the compound to 500 μM;
(299) 3) transferring 10 μl of a 500 μM solution of the compound via Bravo to columns 1 and 11 of the LDV plate;
(300) 4) adding 7.5 μl DMSO to columns 2 to 10 and 12 to 22 of the LDV plate via Bravo;
(301) 5) serially diluting the compound via Bravo (4 folds, 10 doses, 2.5 μl compound+7.5 μl DMSO); and
(302) 6) transferring 250 nL solution of the compound from the dose plate to the test plate (Greiner 781076) via Echo
(303) 2.6 Operating Process of the Experiment
(304) 1) 15 μl of the RORγ-free working solution was added to column 22 of the tested 384-well plate (the final concentration of Eu/anti-His was 1 nM);
(305) 2) 15 μl of the RORγ-containing working solution was added to columns 1 to 21 of the tested 384-well plate (the final concentration of Eu/anti-His was 1 nM, and the final concentration of RORg was 15 nM); and
(306) 3) 10 μl of the mixed solution of peptide-streptomycin/APC was added to columns 1 to 22 of the tested 384-well plate (the final concentration of SA-APC was 50 nM, and the final concentration of SRC1 peptide was 200 nM);
(307) 4) the mixture in the plate was mixed thoroughly on a shaker for 2 minutes;
(308) 5) the test plate was incubated at 4° C. overnight;
(309) 6) the test plate was left at room temperature for 1 hour to reach an equilibration with the room temperature;
(310) 7) the plate was centrifuged at 1000 rpm for 1 minute;
(311) 8) the plate was read on an Envision reader; and
(312) 9) data analysis was performed using the ratio of the numerical value detected at an emission wavelength of 665 nm to the numerical value detected at an emission wavelength of 615 nm.
(313) 3. Data Analysis
(314) GraphPad Prism software was used to plot a logarithmic curve of the TR-FRET ratio F665/F615 vs the concentration of the compound and to calculate the EC50 value. A smaller value indicated that the compound had a stronger regulatory (inhibitory) effect on the receptor RORγ.
(315) The EC.sub.50 values of the compounds of the present application against RORγ were listed in the following table (Table 2).
(316) TABLE-US-00004 Value of the concentration required for Test Samples 50% inhibition (EC.sub.50) of RORγ Example 1 (hydrochloride) A Example 3 (free state) B Example 4 (free state) D Example 5 (hydrochloride) D Example 6 (hydrochloride) A Example 7 (hydrochloride) A Example 8 (hydrochloride) A Example 9 (hydrochloride) A Example 11 (hydrochloride) A Example 12 (hydrochloride) A Example 13 (hydrochloride) D Definition of bioactivity: A: EC.sub.50 ≤ 100 nM; B: 100 nM < EC.sub.50 ≤ 500 nM; C: 500 nM < EC.sub.50 ≤ 1000 nM; D: 1000 nM < EC.sub.50 ≤ 5000 nM.
Bioactivity Experiment 2: Pharmacokinetic Evaluation
(317) 1. Experimental Method
(318) Balb/c mice (female, 15 to 30 g, 7- to 9-week old, Shanghai Lingchang) were used to test in vivo pharmacokinetics of compounds. The experimental method was as follows.
(319) The pharmacokinetic characteristics of the compounds were tested by standard protocols after the compounds were administered to rodents by intravenous injection and oral administration. In the experiment, candidate compounds were formulated into clear solutions and administered to mice by a single intravenous injection and a single oral administration. The vehicle for intravenous injection (IV) was a mixed solvent of 5% of DMSO and 95% of 10% Cremophor EL, and the vehicle for oral administration (PO) was a mixed solvent of 1% tween 80, 9% PEG400 and 90% water. Whole blood samples within 48 hours were collected, and centrifuged at 3000 g at 4° C. for 15 minutes. The supernatant was separated to obtain the plasma sample, to which an acetonitrile solution containing an internal standard was added to precipitate the proteins, and the volume of the acetonitrile solution was 20 times that of the plasma sample. The supernatant was obtained by centrifugation, an equal volume of water was added thereto, and the mixture was centrifuged again to get the supernatant for sample injection. Plasma concentrations were quantitatively analyzed by an LC-MS/MS analytical method, and pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the concentration-time curve, and bioavailability, were calculated.
(320) Of these, “10% Cremophor EL” referred to a deionized aqueous solution of Cremophor EL with a volume concentration of 10%. For example, taking the formulation of a 100 ml solution as an example, 10 ml Cremophor EL was taken and deionized water was added thereto, the mixture was well stirred, and then deionized water was added until the total volume was 100 ml. “Mixed solvent of 5% of DMSO and 95% of 10% Cremophor EL” referred to a mixed solvent comprised of DMSO and 10% Cremophor EL, in which DMSO 10% Cremophor EL, and 10% Cremophor EL accounted for 95% of the volume of the mixed solvent. “Mixed solvent of 1% tween 80, 9% PEG400 and 90% water” meant that the volumes of tween 80, PEG400 and water accounted for 1%, 9% and 90% of the volume of the mixed solvent, respectively.
(321) 2. Test Results
(322) The test results were as shown in Table 2.
(323) TABLE-US-00005 TABLE 2 PK Paramters of the Compound of the Example in Plasma PK Paramters of the compound (hydrochloride) of Example 1 in Plasma PK parameters IV PO Rsq_adj 0.998 0.978 No. points used for T.sub.1/2 3.00 4.00 C.sub.0 (nM) 955 — C.sub.max (nM) — 3520 T.sub.max (h) — 1.00 T.sub.1/2 (h) 6.84 8.70 Vd.sub.ss (L/kg) 3.50 — Cl (mL/min/kg) 7.62 — T.sub.last (h) 24.0 24.0 AUC.sub.0-last (nM .Math. h) 3670 30872 AUC.sub.0-inf (nM .Math. h) 3916 35142 MRT.sub.0-last (h) 5.91 7.19 MRT.sub.0-inf (h) 7.66 10.8 AUC.sub.Extra (%) 6.28 12.1 AUMC.sub.Extra (%) 27.8 41.3 Bioavailability (%).sup.a — 89.7 “—” indicated that the test was not performed or the data was unavailable.
Bioactivity Experiment 3: Inhibition Test of hERG Potassium Channel
(324) 1. Experimental Method
(325) (1) Preparation of Cells
(326) CHO-hERG cells (from Shanghai Institute of Materia Medica, Chinese Academy of Sciences) were cultured in a 175 cm.sup.2 culture flask. When the cell density reached 60% to 80%, the culture medium was removed, the cells were washed once with 7 mL of PBS, and then 3 mL of Detachin was added for cell detachment. After complete cell detachment, 7 mL of culture medium were added for neutralization, the mixture was then centrifuged, the supernatant was aspirated, and another 5 mL of culture medium was added for re-suspension, so as to ensure that the cell density was 2×10.sup.6/mL to 5×10.sup.6/mL.
(327) (2) Formulation of Intracellular and Extracellular Fluids
(328) TABLE-US-00006 TABLE 3 Compositions of Intracellular and Extracellular Fluids Reagents Extracellular Fluid (mM) Intracellular Fluid (mM) CaCl.sub.2 2 5.374 MgCl.sub.2 1 1.75 KCl 4 120 NaCl 145 — Glucose 10 — HEPES 10 10 EGTA — 5 Na.sub.2ATP — 4 pH 7.40 7.25 (adjusted with NaOH), (adjusted with KOH), Osmotic pressure ~305 mOsm ~290 mOsm
(329) (3) Recording Process of Electrophysiology
(330) The single-cell giga-seal and the formation of whole-cell mode were both completed automatically by a Q patch instrument. After a whole-cell recording mode was achieved, the cells were clamped at −80 mV, exposed to a front voltage of −50 mV for 50 ms prior to a depolarizing stimulus (5 s, +40 mV), and then repolarized to −50 mV for 5 s, followed by a recovery to −80 mV. This voltage stimulation was applied every 15 s and recorded for 2 minutes, and the extracellular fluid was then administered, followed by a record lasting for 5 minutes. Thereafter, the administration process was started. Starting from the lowest test concentration, the compounds were administered at each test concentration and the process lasted for 2.5 minutes. After the continuous administration of all concentrations of the compounds, 3 μM Cisapride was administered as a positive control compound. At least 3 cells (n≥3) were tested at each concentration.
(331) (4) Preparation of the Compounds
(332) 20 mM stock solution of the compound was diluted with the extracellular fluid, and 5 μL of the 20 mM stock solution of the compound was taken, added to 2495 μL of the extracellular fluid, and diluted by 500 times to 40 μM. Thereafter, the resulting mixture was serially and successively diluted by 3 times in an extracellular fluid containing 0.2% DMSO, so as to obtain a final concentration to be tested. The highest test concentration was 40 μM, and there were 6 concentrations in total: 40 μM, 13.33 μM, 4.44 μM, 1.48 μM, 0.49 μM, and 0.16 M. The content of DMSO in the final test concentration did not exceed 0.2%, and such concentration of DMSO had no effect on hERG potassium channel.
(333) (5) Data Analysis
(334) The experimental data was analyzed by XLFit software.
(335) (6) Quality Control
(336) Environment: humidity 20% to 50%, temperature 22 to 25° C.
(337) Reagents: The experimental reagents used herein were purchased from Sigma, purity >98%
(338) The experimental data in the report must meet the following standards:
(339) whole-cell sealing impedance >100 MΩ
(340) tail current amplitude >400 pA
(341) Pharmacological Parameters:
(342) Inhibitory effects of multiple concentrations of Cisapride on hERG channel were set as the positive control.
(343) (7) Test Results
(344) Results of IC50 value of the compounds of Examples against hERG were as shown in Table 4.
(345) TABLE-US-00007 TABLE 4 Results of IC50 value of the compounds of Examples against hERG Inhibition rate (%) at the maximum Test Samples hERG IC50 (μM) concentration Example 1 (hydrochloride) >40 26.33 Cisapride 0.05 89.61
Bioactivity Experiment 4: In Vivo Pharmacodynamic Research on MOG35-55-Induced Experimental Autoimmune Encephalomyelitis (EAE) in Mice
(346) Experimental Method:
(347) 1. Preparation of MOG/CFA Emulsion
(348) Preparation of CAF: M. tuberculosis H37Ra was weighed, placed in an agate mortar, and ground into fine powder. IFA was added thereto, and the mixture was formulated into a 4 mg/ml suspension.
(349) Preparation of MOG35-55 solution: MOG35-55 was weighed, dissolved in physiological saline, and formulated into a 2 mg/ml solution, which was used immediately after it was formulated.
(350) 2 mg/ml MOG35-55 and 4 mg/ml CFA were mixed in equal volume in a 40-ml glass bottle and homogenized with a homogenizer for 1 h (homogenization was performed for 45 s and then paused for 15 s), so as to prepare a MOG/CFA emulsion, which was kept on ice for later use.
(351) 2. Preparation of PTX Solution
(352) Preparation of PTX solution: 1 ml PBS was added to PTX powder (50 μg for each vial) to formulate into a 50 μg/ml stock solution. The stock solution was diluted with PBS to 1 μg/ml before use, and was used immediately after it was formulated.
(353) 3. Induction of EAE
(354) The day of immunization was recorded as day 0, and the subsequent days were marked sequentially. Mice were anesthetized with isoflurane, and 100 μl of the emulsion was administered via subcutaneous injection at three sites (the middle part between shoulder and back, and two sides of the tail that were close to the groin), namely, 33 μl of the emulsion for each site. 0 hour and 48 hours after the injection of the emulsion, 200 ng (i.e. 200 μl) of the PTX solution was intraperitoneal injected. Mice in the normal group were not required to be immunized.
(355) 4. Designs for Administration and Dose
(356) After the immunization on day 0, the immunized mice were randomly divided into 3 groups with 10 mice in each group according to the body weights except for the mice in the normal group, the details were shown in Table 1. FTY720 served as a positive drug, and a dose of 0.05 mg/ml was an effective dose commonly used in the EAE model.
(357) The first group was normal mice without any treatment; vehicle was administered to the second group; FTY720 was administered to the third group at a dose of 0.5 mg/kg once a day; and the compound of Example 1 was administered to the fourth group at a dose of 75 mg/kg twice a day. The administration lasted for 25 days in total. The volume of intragastric administration was 10 ml/kg.
(358) TABLE-US-00008 TABLE 5 Grouping and Dose Design Adminis- Concen- tration tration Dose Dosing Group Test Drugs QTY. Route mg/mL mg/kg Frequency G1 Normal 5 N/A N/A N/A NA (blank control) G2 Vehicle 10 p.o. N/A N/A b.i.d, (solvent 25 days control)* G3 FTY720** 10 p.o. 0.05 0.5 qd, 25 days G4 Example 1 10 p.o. 7.5 75 b.i.d, (hydro- 25 days chloride)* *vehicle: a mixed solvent of DMSO/PEG400/H.sub.2O, wherein the volume ratio of DMSO/PEG400/H.sub.2O was 5:20:75. **vehicle (solvent): DDH.sub.2O.
(359) 5. Surveillance of the Indicators of the Onset of Disease
(360) The day when animals were immunized was day 0. Animal body weights were recorded three times a week from day 0 to day 10, and the body weights and clinical scores of the animals were closely observed and recorded daily from day 11. The scoring criteria were as shown in the following table.
(361) TABLE-US-00009 TABLE 6 Clinical Scoring Criteria Score Clinical Symptoms 0 Normal performance, no obvious sign of disease 1 Drooping tail and weakness in single hind limb 2 Drooping tail, weakness in both hind limbs, and staggering gait 3 Weakness and paralysis in single hind limb 4 Weakness and paralysis in both hind limbs
(362) 6. Statistical Process
(363) The experimental data was expressed as mean±standard error of mean (Mean±SEM). AUC of clinical scores was analyzed by one-way analysis of variance (One-way ANOVA), and the difference was considered as significant when p<0.05.
(364) 7. Experimental Results
(365) (1) Clinical Scores
(366) On day 13 after immunization, mice began to develop clinical symptoms of EAE. The average clinical score of G2 (solvent control group) gradually increased and reached 3.6 points on day 18, indicating the successful establishment of the model (see
(367) (2) Results of Inhibition Rate
(368) The area under the curve of clinical scores over time (i.e. “AUC of clinical scores” or “area under the curve of clinical scores”) was calculated by analyzing the curve of clinical scores for each animal in each group, and the inhibition rates of the remaining groups (G1, G3, and G4) relative to G2 (solvent control group) were calculated by the mean AUC of clinical scores among groups. The results were shown in
(369) (3) Results of Morbidity
(370) Results of the morbidity of EAE were as shown in
(371) (4) Results of Body Weight Changes
(372) As compared with G1 (normal group), the body weights of the mice in G2 (solvent control group) decreased on day 12, decreased sharply from day 12 to day 16, and recovered slowly from day 17. The body weights in G3 (positive control FTY720 group) and G4 (the compound of Example 1) kept increasing with a same tendency, and were significantly different from those of the solvent control group. Moreover, the body weights in G4 (the compound of Example 1) were the highest from the beginning to end (
Bioactivity Experiment 5: In Vivo Pharmacodynamic Study of the Therapeutic Efficacy and Mechanism Studies in Mouse Psoriasis (IMQ) Model
(373) Experimental Method:
(374) 5.1 Animal Grouping and the Establishment of an Imiquimod-Induced Psoriasis Model in Mice
(375) 6- to 8-week old female C57BL/6 mice were selected, depilated on the backs, and sensitized two days later except for the sham operation group. On the day of sensitization, the mice were randomly divided into the following 6 groups (8 mice per group). Group I was a sham operation group; Group II was a vehicle control group and PBS was administered; Group III was a positive control group and 5 mg/kg methotrexate was administered; Groups IV, V and VI were ip treatment group, ig treatment group, and topical treatment group of the compound of Example 1, and 75 mg/kg of the compound of Example 1 was administered via intraperitoneal injection, intragastric administration, and dorsal application, respectively. Administration was started on the day of sensitization, once daily for Group III and twice daily for Groups IV to VI. On the day of sensitization, the mice in Groups II to VI were given 60 to 80 mg of imiquimod cream (5%) on the dorsal skin for 4 consecutive days. (Please refer to Bouchaud, G., et al., Epidermal IL-15Ralpha acts as an endogenous antagonist of psoriasiform inflammation in mouse and man. J. Exp. Med., 2013. 210(10): p. 2105-17.)
(376) The mice were weighed daily from the day of sensitization, and the skin scales, induration, and erythema were observed and scored according to a four-grade scoring method: 0 point, no disease; 1 point, mild; 2 points, moderate; 3 points, severe; and 4 points, very severe. (Please refer to Qin, S., et al., Endogenous n-3 polyunsaturated fatty acids protect against imiquimod-induced psoriasis-like inflammation via the IL-17/IL-23 axis. Mol Med Rep, 2014. 9(6): p. 2097-104.)
(377) 5.2 Research Results: Effect of Example 1 on the Clinical Scores of the Mice Suffering from Imiquimod-Induced Psoriasis
(378) As shown in
(379) As shown in
(380) TABLE-US-00010 TABLE 7 Effects on the Clinical Scores of Mice Suffering from Imiquinomot-Induced Psoriasis After Daily Administration of Example 1 (Mean ± SEM, n = 8) Sham Operation Vehicle Control Methotrexate Day Group Group (5 mg/kg, ig) 1 0 ± 0 0 ± 0 0 ± 0 2 0 ± 0 0.43 ± 0.17 0.19 ± 0.13 3 0 ± 0 1.36 ± 0.18 0.50 ± 0.13 4 0 ± 0 2.29 ± 0.29 0.88 ± 0.25 5 0 ± 0 2.86 ± 0.34 0.93 ± 0.23 Example 1 Example 1 Example 1 (hydrochloride) (hydrochloride) (hydrochloride) Day (75 mg/kg, ip) (75 mg/kg, ig) (75 mg/kg, topical) 1 0 ± 0 0 ± 0 0 ± 0 2 0.25 ± 0.13 0.31 ± 0.13 0.25 ± 0.13 3 0.50 ± 0.16 0.50 ± 0.19 0.44 ± 0.15 4 1.25 ± 0.19 1.29 ± 0.21 0.94 ± 0.24 5 1.81 ± 0.25 1.64 ± 0.18 1.43 ± 0.20