Quinolinone derivatives and uses thereof

Abstract

The application discloses a quinolinone derivative compound, a pharmaceutical composition and application thereof. The quinolinone derivative compound can antagonize diarrhea induced by CGRP, and the compound has a good application prospect in preparation of a drug for treating irritable bowel syndrome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and provides a quinolinone compound, a preparation method therefor, and a pharmaceutical use thereof. The compound can be used for treating irritable bowel syndrome. BACKGROUND

[0002] Irritable bowel syndrome (IBS) belongs to a gastrointestinal functional disorder. It has a clinical syndrome of abdominal pain, abdominal distension, change in bowel habits, and abnormal stool shape or mucous stool. The symptoms persist or intermittently occur, but lack of definite evidence of morphological and laboratory abnormal changes. IBS does not endanger human life, but can affect work and life to different degrees and reduce the quality of life.

[0003] The cause of IBS is unknown so far, and involves multiple aspects such as motility disorder, visceral hypersensitivity, inflammation and immune function disorder. Some acute intestinal infection patients (about 20%) have IBS-like symptoms after recovery, suggesting that there may be some relationship between intestinal infection and IBS. Due to the increasing pressure factors of family, work and social environment, the incidence of IBS is gradually increasing. The incidence of IBS is about 10-20%. In most countries, women are more than men, and the ratio of men to women is about 1:2. The incidence of IBS in young people is high, and it is rare for the first onset after 50 years old.

[0004] Due to the abnormal complexity of the cause of IBS, there are very few effective therapies for treating this chronic pain disease at present. In view of the negative impact of IBS on the quality of life and the economic burden on society, it is obviously necessary to have more treatment methods to relieve IBS-related abdominal pain.

[0005] Calcitonin gene-related peptide (CGRP) is a member of the calcitonin peptide family consisting of 37 amino acids, which is produced in peripheral neurons and central neurons, and can act as a potent vasodilator and play a role in the transmission of nociception. CGRP is considered to be an important factor in transmitting sensory and pain stimuli and many other aspects of digestive physiology, including movement, secretion and neuroprotection. In the gastrointestinal tract, CGRP is widely distributed in the entire enteric nervous system from the esophagus to the rectum, and is distributed in the intrinsic and extrinsic primary afferent neurons of various mammals.

[0006] A study shows that intraperitoneal injection of CGRP in mice can induce diarrhea, and administration of anti-CGRP monoclonal antibody can antagonize the diarrhea induced by CGRP. Another clinical study shows that intravenous infusion of hCGRP can induce gastrointestinal hyperfunction.

[0007] Another study showed that targeting peripheral calcitonin gene-related peptide (CGRP) with monoclonal antibodies against a bivalent antigen-binding fragment of calcitonin gene-related peptide (CGRP) reduced central sensitization and attenuated colonic hypersensitivity induced by chronic adult stress or early life stress, and targeting CGRP can provide a new treatment strategy for irritable bowel syndrome. SUMMARY

[0008] The technical problem solved by the application: the compound is a CGRP receptor antagonist, which can antagonize the diarrhea induced by CGRP, and can be used for preparing a drug for treating irritable bowel syndrome.

[0009] Technical solution: a quinolinone compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0010]

[0011] Formula I

[0012] wherein,

[0013] R1 is selected from H, F, Cl, Br;

[0014] R2 is selected from H, or C1-C8 alkyl;

[0015] R3, R4 are independently selected from H, F, Cl;

[0016] n is selected from the numbers 1 to 8.

[0017] Preferably, wherein,

[0018] R1 is selected from H, F, Cl;

[0019] R2 is selected from C1-C5 alkyl;

[0020] R3, R4 are independently selected from H, F, Cl;

[0021] n is selected from the numbers 1 to 3.

[0022] Preferably, wherein,

[0023] R1 is selected from F, Cl;

[0024] R2 is selected from C1-C3 alkyl;

[0025] R3, R4 are independently selected from H, F;

[0026] n is selected from the numbers 1 or 2.

[0027] More preferably, the compound is:

[0028] S1

[0029] S2

[0030] S3

[0031] Advantages:

[0032] The quinolinone compounds described in the patent are CGRP receptor antagonists, which can antagonize the diarrhea induced by CGRP, and can be used for preparing drugs for treating irritable bowel syndrome. BRIEF DESCRIPTION OF DRAWINGS

[0033] The accompanying drawings are included to provide a further understanding of the application, and are incorporated herein and constitute a part of this application. Figure 1 : CGRP antagonistic activity inhibition curve of compound S1

[0034] The accompanying drawings are included to provide a further understanding of the application, and are incorporated herein and constitute a part of this application. Figure 2 : Pharmacodynamic study of compound S1 in CGRP-induced mouse diarrhea model DETAILED DESCRIPTION

[0035] The following examples enable those skilled in the art to more completely understand the present application, but in no way limit the present application.

[0036] Example 1: Synthesis of compound S1

[0037] Synthetic route:

[0038]

[0039] Synthetic process:

[0040] First step:

[0041]

[0042] To a solution of compound 1 (40.0 g, 279 mmol, 1.00 eq.) in DCM (400 mL) (add 3 drops of DMF) was added (COCl)2(42.5 g, 334.8 mmol, 1.20 eq.) at 0 °C, the mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then this acyl chloride was added to a solution of compound 1A (30.0 g, 307 mmol, 1.10 eq.) in DCM (400 mL) at 0 °C, and TEA (84.8 g, 838 mmol, 117 mL, 3.00 eq.) was added dropwise, the mixture was stirred at 25 °C for 2 h. TLC (dichloromethane:methanol = 10:1, compound 1 Rf = 0.00, compound 2 Rf = 0.05) indicated that compound 1 was completely consumed and a new spot was formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was used for the next step without further purification. Compound 2 (5.00 g, 26.8 mmol, 9.61% yield) as a brown oil.

[0043] Second Step:

[0044]

[0045] To a solution of compound 2A (26.7 g, 112 mmol, 2.10 eq.) in DCM (100 mL) was added n-BuLi (2.50 M, 43.0 mL, 2.00 eq.) at -78 °C and stirred for 1 h, then compound 2 (10.0 g, 53.7 mmol, 1.00 eq.) was added at -78 °C and stirred for 1 h. LC-MS showed ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, ~47.5% of the target compound was detected. The reaction mixture was quenched by adding saturated NH4Cl 500 mL at 0 °C, and extracted with DCM 500 mL (500 mL x 3). The combined organic layers were washed with brine 500 mL, dried over Na2SO4, filtered and concentrated to give a residue. The crude product was purified by reverse-phase high-performance liquid chromatography (0.1% NH3•H2O). Compound 3 (10.0 g, 35.3 mmol, 65.7% yield) as a brown oil, confirmed by HNMR.

[0046] ESI-MS: 282.9 [M+H]+

[0047] 1H NMR (400 MHz, DMSO-d6) δ 8.00 - 7.87 (m, 3H), 3.62 - 3.50 (m, 1H), 2.84 - 2.74 (m, 2H), 2.16 (s, 3H), 2.03 - 1.91 (m, 2H), 1.83 - 1.72 (m, 2H), 1.64 - 1.46 (m, 2H)

[0048] Third Step:

[0049]

[0050] Compound 3 (10.0 g, 35.3 mmol, 1.00 eq.), hydrochloride salt of compound 3A (5.92 g, 42.4 mmol, 1.20 eq.), Cs2CO3(34.5 g, 106 mmol, 3.00 eq.), Pd(OAc)2(793 mg, 3.53 mmol, 0.100 eq.), BINAP (2.2 g, 3.53 mmol, 0.1 eq) were added into toluene (100 mL), the mixture was protected by N2, then the mixture was stirred at 100 °C for 4 h. LC-MS showed that compound 3 was completely consumed, and a main peak of m / z (306.2) was detected. The reaction mixture was quenched with H2O 100 mL at 0 °C, and extracted with EA 30.0 mL (30.0 mL x 5). The combined organic layer was washed with brine 30.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH3•H2O). Compound 4 (2.00 g, 6.55 mmol, yield 18.5%) was a brown oil.

[0051] ESI-MS: 306.2 [M+H]+

[0052] Fourth Step:

[0053]

[0054] To a solution of compound 4A (1.39 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (add 3 drops of DMF) was added (COCl)2(0.99 g, 7.86 mmol, 1.20 eq.) at 0 °C, the mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then added to compound 4 in DCM (20 mL) dropwise at 0 °C, and TEA (1.33 g, 13.1 mmol, 1.82 mL, 2.00 eq.) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 4 h, LC-MS showed compound 4 was consumed completely, and a main peak of m / z (464.1) was detected. The reaction mixture was quenched by adding H2O 70.0 mL at 0 °C, and extracted with EA 30.0 mL (30.0 mL x 3). The combined organic layer was washed with brine 50.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was used for the next step without further purification. Compound 5 (4.00 g,) was brown oil.

[0055] ESI-MS: 464.1 [M+H] +

[0056] Fifth step:

[0057]

[0058] To a solution of compound 5 (4.00 g, 8.63 mmol, 1.00 eq) in THF (24.0 mL) and H2O (8.00 mL) was added LiOH.H2O (1.09 g, 25.9 mmol, 3.00 eq). The mixture was stirred at 25 °C for 12 h. LC-MS showed compound 5 was consumed completely, and a main peak of m / z (450.1) was detected. The reaction mixture was quenched by adding H2O 120 mL at 0 °C, and extracted with EA 20.0 mL (20.0 mL x 2). The combined aqueous phase was adjusted pH = 6, then extracted with EA 50.0 mL (50.0 mL x 3), the combined organic phase was dried over Na2SO4, filtered, concentrated under reduced pressure to give a crude product. The crude product was used for the next step without purification. Compound 6 (450 mg, 1.00 mmol, yield 11.6%) was brown oil.

[0059] ESI-MS: 450.1 [M+H] +

[0060] Sixth step:

[0061]

[0062] To a solution of compound 6 (450 mg, 1.00 mmol, 1.00 eq.) and compound 5A (291 mg, 1.10 mmol, 1.10 eq., HC1) in DMF (4.50 mL) was added DIEA (388 mg, 3.00 mmol, 523 uL, 3.00 eq.) and HATU (457 mg, 1.20 mmol, 1.20 eq.). The mixture was stirred at 25 °C for 6 h. LC-MS showed compound 6 was consumed completely and one main peak at m / z (660.3) was detected. The reaction mixture was quenched by adding H2O 50.0 mL at 0 °C and extracted with DCM 50.0 mL (50.0 mL x 3). The combined organic layer was washed with brine 50.0 mL, dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column on silica gel (DCM:MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was an off-white solid.

[0063] ESI-MS: 660.3 [M+H] +

[0064] 1H NMR (400 MHz, DMSO-d6) d 11.78 (br s, 1H), 8.23 (s, 1H), 8.02 (br t, J = 7.8 Hz, 1H), 7.79 (br d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.48 - 7.37 (m, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.24 - 7.08 (m, 3H), 4.50 (br d, J = 12.6 Hz, 1H), 4.29 - 4.18 (m, 2H), 3.95 (br d, J = 9.4 Hz, 1H), 3.11 (br t, J = 12.3 Hz, 1H), 2.98 (br d, J = 11.2 Hz, 3H), 2.84 (br s, 2H), 2.62 - 2.53 (m, 1H), 2.32 (s, 3H), 2.26 (br t, J = 11.1 Hz, 2H), 1.92 - 1.79 (m, 2H), 1.69 (br s, 2H), 1.62 - 1.29 (m, 5H)

[0065] Example 2: Synthesis of compound S2

[0066] Synthetic route:

[0067]

[0068] First step:

[0069]

[0070] To a solution of compound 1 (40.0 g, 279 mmol, 1.00 eq.) in DCM (400 mL) (add 3 drops of DMF) was added (COCl)2(42.5 g, 334.8 mmol, 1.20 eq.) at 0 °C and the mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. This acyl chloride was then added dropwise to a solution of compound 1A (30.0 g, 307 mmol, 1.10 eq.) in DCM (400 mL) at 0 °C and TEA (84.8 g, 838 mmol, 117 mL, 3.00 eq.) was added dropwise, and the mixture was stirred at 25 °C for 2 h. TLC (dichloromethane:methanol = 10:1, compound 1 Rf = 0.00, compound 2 Rf = 0.05) indicated that compound 1 was completely consumed and a new spot was formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was used for the next step without further purification. Compound 2 (5.00 g, 26.8 mmol, 9.61% yield) was a brown oil.

[0071] Second step:

[0072]

[0073] To a solution of compound 2A (26.7 g, 112 mmol, 2.10 eq.) in DCM (100 mL) was added n-BuLi (2.50 M, 43.0 mL, 2.00 eq.) at -78 °C and stirred for 1 h, then compound 2 (10.0 g, 53.7 mmol, 1.00 eq.) was added at -78 °C and stirred for 1 h. LC-MS showed ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, ~47.5% of the target compound was detected. The reaction mixture was quenched with the addition of saturated NH4Cl 500 mL at 0 °C and extracted with DCM 500 mL (500 mL x 3). The combined organic layers were washed with brine 500 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH3•H2O). Compound 3 (10.0 g, 35.3 mmol, 65.7% yield) was a brown oil, confirmed by HNMR.

[0074] ESI-MS: 282.9 [M+H]+

[0075] 1H NMR (400 MHz, DMSO-d6) δ 8.00 - 7.87 (m, 3H), 3.62 - 3.50 (m, 1H), 2.84 - 2.74 (m, 2H), 2.16 (s, 3H), 2.03 - 1.91 (m, 2H), 1.83 - 1.72 (m, 2H), 1.64 - 1.46 (m, 2H)

[0076] Third step:

[0077]

[0078] Compound 3 (10.0 g, 35.3 mmol, 1.00 eq.), hydrochloride salt of compound 3A (5.3 g, 42.4 mmol, 1.20 eq.), Cs2CO3(34.5 g, 106 mmol, 3.00 eq.), Pd(OAc)2(793 mg, 3.53 mmol, 0.100 eq.), BINAP (2.2 g, 3.53 mmol, 0.1 eq) were added into toluene (100 mL), the mixture was protected by N2, then the mixture was stirred at 100 °C for 4 h. LC-MS showed compound 3 was consumed completely, and a main peak of m / z (292.2) was detected. The reaction mixture was quenched with H2O 100 mL at 0 °C, and extracted with EA 30.0 mL (30.0 mL x 5). The combined organic layer was washed with brine 30.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH3•H2O). Compound 4 (1.91 g, 6.55 mmol, yield 18.5%) was brown oil.

[0079] ESI-MS: 292.2 [M+H]+

[0080] Fourth step:

[0081]

[0082] To a solution of compound 4A (1.23 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (add 3 drops of DMF), (COCl)2(0.99 g, 7.86 mmol, 1.20 eq.) was added at 0 °C, the mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then added to compound 4 in DCM (20 mL) dropwise at 0 °C, and TEA (1.33 g, 13.1 mmol, 1.82 mL, 2.00 eq.) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 4 h, LC-MS showed compound 4 was consumed completely, and a main peak of m / z (430.1) was detected. The reaction mixture was quenched by adding H2O 70.0 mL at 0 °C, and extracted with EA 30.0 mL (30.0 mL x 3). The combined organic layer was washed with brine 50.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the residue. The crude product was used for the next step without further purification. Compound 5 (3.7 g,) was brown oil.

[0083] ESI-MS: 430.1 [M+H]+

[0084] Step 5:

[0085]

[0086] To a solution of compound 5 (3.7 g, 8.63 mmol, 1.00 eq.) in THF (24.0 mL) and H2O (8.00 mL) was added LiOH.H2O (1.09 g, 25.9 mmol, 3.00 eq.). The mixture was stirred at 25 °C for 12 h. LC-MS showed compound 5 was consumed completely and one main peak at m / z (416.1) was detected. The reaction mixture was quenched by adding H2O 120 mL at 0 °C and extracted with EA 20.0 mL (20.0 mL x 2). The combined aqueous phase was adjusted to pH = 6 and then extracted with EA 50.0 mL (50.0 mL x 3), the combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was used in the next step without purification. Compound 6 (415 mg, 1.00 mmol, yield 11.6%) was brown oil.

[0087] ESI-MS: 416.1 [M+H]++

[0088] Step 6:

[0089]

[0090] To a solution of compound 6 (415 mg, 1.00 mmol, 1.00 eq.) and compound 5A (291 mg, 1.10 mmol, 1.10 eq., HCl) in DMF (4.50 mL) was added DIEA (388 mg, 3.00 mmol, 523 uL, 3.00 eq.) and HATU (457 mg, 1.20 mmol, 1.20 eq.). The mixture was stirred at 25 °C for 6 h. LC-MS showed compound 6 was consumed completely and one main peak at m / z (626.3) was detected. The reaction mixture was quenched by adding H2O 50.0 mL at 0 °C and extracted with DCM 50.0 mL (50.0 mL x 3). The combined organic layer was washed with brine 50.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the residue. The residue was purified by column on silica gel (DCM:MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was off-white solid.

[0091] ESI-MS: 626.3 [M+H] +

[0092] 1H NMR (400 MHz, DMSO-d6) δ 11.78 (br s, 1H), 8.02 (d, J = 7.8 Hz, 2H), 7.79 (br d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.48 - 7.37 (m, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.24 - 7.08 (m, 3H), 4.29 - 4.18 (m, 2H), 3.11 (br t, J = 12.3 Hz, 1H), 2.98 (br d, J = 11.2 Hz, 3H), 2.84 (br s, 2H), 2.62 - 2.53 (m, 1H), 2.32 (s, 3H), 2.26 (br t, J = 11.1 Hz, 2H), 1.92 - 1.79 (m, 2H), 1.69 (br s, 2H), 1.62 - 1.29 (m, 5H)

[0093] Example 3: Synthesis of compound S3

[0094] Synthetic route:

[0095]

[0096] First step:

[0097]

[0098] To a solution of compound 1 (47.9 g, 279 mmol, 1.00 eq.) in DCM (400 mL) (add 3 drops of DMF) was added (COCl)2(42.5 g, 334.8 mmol, 1.20 eq.) at 0 °C, the mixture was stirred at 25 °C for 4 h. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then this acyl chloride was added to a solution of compound 1A (30.0 g, 307 mmol, 1.10 eq.) in DCM (400 mL) at 0 °C, and TEA (84.8 g, 838 mmol, 117 mL, 3.00 eq.) was added dropwise, the mixture was stirred at 25 °C for 2 h. TLC (dichloromethane:methanol = 10:1, compound 1 Rf = 0.00, compound 2 Rf = 0.05) indicated that compound 1 was completely consumed and a new spot was formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was used for the next step without further purification. Compound 2 (5.7 g, 26.8 mmol, 9.61% yield) was brown oil.

[0099] Second Step:

[0100]

[0101] To a solution of compound 2A (26.7 g, 112 mmol, 2.10 eq.) in DCM (100 mL) was added n-BuLi (2.50 M, 43.0 mL, 2.00 eq.) at -78 °C and stirred for 1 h, then compound 2 (11.4 g, 53.7 mmol, 1.00 eq.) was added at -78 °C and stirred for 1 h. LC-MS showed ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, ~47.5% of the target compound was detected. The reaction mixture was quenched by the addition of saturated NH4Cl 500 mL at 0 °C, and extracted with DCM 500 mL (500 mL x 3). The combined organic layer was washed with brine 500 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH3•H2O). Compound 3 (10.9 g, 35.3 mmol, 65.7% yield) was brown oil, confirmed by HNMR.

[0102] ESI-MS: 311.1 [M+H]+

[0103] 1H NMR (400 MHz, DMSO-d6) δ 8.00 - 7.87 (m, 3H), 3.62 - 3.50 (m, 1H), 2.84 - 2.74 (m, 2H), 2.16 (m, 1H), 2.03 - 1.91 (m, 2H), 1.83 - 1.72 (m, 2H), 1.64 - 1.46 (m, 2H), 1.2 - 0.9 (m, 6H)

[0104] Third Step:

[0105]

[0106] Compound 3 (10.9 g, 35.3 mmol, 1.00 eq.), Compound 3A hydrochloride (5.92 g, 42.4 mmol, 1.20 eq.), Cs2CO3(34.5 g, 106 mmol, 3.00 eq.), Pd(OAc)2(793 mg, 3.53 mmol, 0.100 eq.), BINAP (2.2 g, 3.53 mmol, 0.1 eq) were added into toluene (100 mL), the mixture was protected by N2, then the mixture was stirred at 100 °C for 4 h. LC-MS showed that compound 3 was completely consumed, and a main peak of m / z (334.2) was detected. The reaction mixture was quenched with H2O 100 mL at 0 °C, and extracted with EA 30.0 mL (30.0 mL x 5). The combined organic layer was washed with brine 30.0 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH3•H2O). Compound 4 (2.19 g, 6.55 mmol, yield 18.5%) was a brown oil.

[0107] ESI-MS: 334.2 [M+H]+

[0108] Fourth Step:

[0109]

[0110] To a solution of compound 4A (1.24 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (add 3 drops of DMF) add (COCl)2(0.99 g, 7.86 mmol, 1.20 eq.) at 0 °C, stir the mixture at 25 °C for 4 h. Concentrate the reaction mixture under reduced pressure to remove (COCl)2. Then add to the DCM (20 mL) of compound 4 at 0 °C, and add TEA (1.33 g, 13.1 mmol, 1.82 mL, 2.00 eq.) dropwise at 0 °C. Stir the mixture at 25 °C for 4 h, LC-MS shows compound 4 is consumed completely, and a major peak of m / z (474.1) is detected. Quench the reaction mixture by adding H2O 70.0 mL at 0 °C, and extract with EA 30.0 mL (30.0 mL x 3). Dry the combined organic layer with Na2SO4, filter, and concentrate under reduced pressure to get a residue. The crude product is used for the next step without further purification. Compound 5 (4.1 g,) is a brown oil.

[0111] ESI-MS: 474.1 [M+H] +

[0112] Fifth step:

[0113]

[0114] To a solution of compound 5 (4.1 g, 8.63 mmol, 1.00 eq) in THF (24.0 mL) and H2O (8.00 mL) add LiOH.H2O (1.09 g, 25.9 mmol, 3.00 eq). Stir the mixture at 25 °C for 12 h. LC-MS shows compound 5 is consumed completely, and a major peak of m / z (460.1) is detected. Quench the reaction mixture by adding H2O 120 mL at 0 °C, and extract with EA 20.0 mL (20.0 mL x 2). Adjust the pH of the combined aqueous phase to 6, then extract with EA 50.0 mL (50.0 mL x 3), dry the combined organic phase with Na2SO4, filter, concentrate under reduced pressure to get the crude product. The crude product goes to the next step without purification. Compound 6 (459 mg, 1.00 mmol, yield 11.6%) is a brown oil.

[0115] ESI-MS: 460.1 [M+H] +

[0116] Sixth step:

[0117]

[0118] To a solution of compound 6 (459 mg, 1.00 mmol, 1.00 eq.) and compound 5A (291 mg, 1.10 mmol, 1.10 eq., HC1) in DMF (4.50 mL) was added DIEA (388 mg, 3.00 mmol, 523 uL, 3.00 eq.) and HATU (457 mg, 1.20 mmol, 1.20 eq.). The mixture was stirred at 25 °C for 6 h. LC-MS showed compound 6 was consumed completely and one main peak at m / z (670.3) was detected. The reaction mixture was quenched by adding H2O 50.0 mL at 0 °C and extracted with DCM 50.0 mL (50.0 mL x 3). The combined organic layer was washed with brine 50.0 mL, dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column on silica gel (DCM:MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was an off-white solid.

[0119] ESI-MS: 670.3 [M+H]+

[0120] 1H NMR (400 MHz, DMSO-d6) δ 11.78 (br s, 1H), 8.23 (s, 1H), 8.02 (br t, J = 7.8 Hz, 1H), 7.79 (br d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.48 - 7.37 (m, 1H), 7.28 (m, J = 8.1 Hz, 2H), 7.24 - 7.08 (m, 3H), 4.50 (br d, J = 12.6 Hz, 1H), 4.29 - 4.18 (m, 2H), 3.95 (br d, J = 9.4 Hz, 1H), 3.11 (br t, J = 12.3 Hz, 1H), 2.98 (br d, J = 11.2 Hz, 3H), 2.84 (br s, 2H), 2.62 - 2.53 (m, 2H), 2.26 (br t, J = 11.1 Hz, 2H), 1.92 - 1.79 (m, 2H), 1.69 (br s, 2H), 1.62 - 1.29 (m, 5H), 1.2 - 0.9 (m, 6H)

[0121] Example 4 Study of CGRP target activity of compound S1

[0122] 1. Materials and methods

[0123] 1.1 Reagents and consumables

[0124] CGRP cell (HEK293 cell)

[0125] Fluo-4 Direct Calcium Assay Kit (Invitrogen #F10471)

[0126] Bovine Serum Albumine (B265993)

[0127] Cell plate (Greiner #781946)

[0128] Compound plate (Greiner #781280)

[0129] 1.2 Detection instrument

[0130] Liquid handler: Echo 555 (Labcyte)

[0131] Reader: FLIPR (Molecular Device)

[0132] 1.3 Experimental method

[0133] 1.3.1 Dilute cells to 10^6 cells / mL with culture medium. Then transfer 20 uL to each well of the cell plate and incubate overnight.

[0134] 1.3.2 Preparation of assay buffer: Fluo-4 Direct calcium assay buffer with 0.5% BSA.

[0135] 1.3.3 Dilute the agonist reference compound (CGRP) 10 concentration gradients with DMSO in series, spot 750 nL of compound on the DRC plate (Greiner #781280) with Echo as the plate map. Add 30 uL of assay buffer to each well of the DRC plate (Greiner #781280) and centrifuge at 1000 rpm for 10 seconds.

[0136] 1.3.4 Take the EC80 test plate out of the incubator, and gently dispense 20 ul of 2X Fluo-4 DirectTM wash-free loading buffer into the 384-well cell culture plate with a pipette. The final volume in the cell plate is 40 microliters.

[0137] 1.3.5 Incubate for 50 minutes at 37°C, 5% C02and 10 minutes at room temperature.

[0138] 1.3.6 Remove the EC80 test plate from the incubator and place it in the FLIPR. Place the DRC plate and tip box into the FLIPR.

[0139] 1.3.7 Run the protocol on the FLIPR TETRA and calculate the EC80.

[0140] 1.3.8 Antagonist compounds are diluted 1 :5 in DMSO 10 times starting from 1000 uM. Spot 900 nl of compound onto the compound plate (Greiner #781280) with the Echo. Add 30 uL of assay buffer to each well of the compound plate (Greiner #781280) and centrifuge at 1000 rpm for 10 seconds.

[0141] 1.3.9 Remove the cell plate from the incubator and gently dispense 20 ul of 2X Fluo-4 Direct™ wash-free loading buffer into the 384 well cell culture plate with a pipette. The final volume in the cell plate is 40 microliters.

[0142] 1.3.10 Incubate for 50 minutes at 37°C, 5% C02and 10 minutes at room temperature.

[0143] 1.3.11 Dilute the CGRP in assay buffer to 6X EC80 and transfer 30 uL to each well of the EC80 compound plate.

[0144] 1.3.12 Remove the cell plate from the incubator and place it in the FLIPR. Place the compound plate and tip box into the FLIPR. Run the protocol on the FLIPR TETRA to transfer 10 uL of agonist reference compound from the compound plate to the cell plate. Read the fluorescence signal. Transfer 10 uL of agonist reference compound from the EC80 plate to the cell plate. Read the fluorescence signal. Calculate the "max-min" from read 1 to 90 to produce a final signal for calculation of percent inhibition.

[0145] 2 Results are shown in Figure Figure 1 Figure 2 shows the CGRP antagonistic activity inhibition curve of compound S1.

[0146] Example 5 Pharmacodynamic study of compound S1 in the CGRP-induced mouse diarrhoea model

[0147] 1 Materials and methods

[0148] 1.1 Experimental animals

[0149] BALB / c mice, male, body weight: 20-25 g, SPF level

[0150] 1.2 Experimental method

[0151] 1.2.1 Preparation of CGRP-induced diarrhea model. Main steps: 0.1 mg / kg CGRP was injected intraperitoneally into mice, and the mice were placed in an isolation box. After 30 min, the stool was observed for diarrhea.

[0152] 1.2.2 Animal grouping and administration The experimental animals were randomly divided into 5 groups, namely the control group, the model group, the compound S1 low-dose group (0.35 mg / kg), the compound S1 medium-dose group (1 mg / kg), and the compound S1 high-dose group (3.5 mg / kg), with 10 animals in each group. The administration volume was 10 μL / g for each group.

[0153] 1.2.3 Evaluation of the diarrhea index

[0154] If the stool was wet and could not maintain the shape of the particles, it was scored as 3 points; if the stool was dry and could maintain the shape of the particles, it was scored as 1 point; if the stool was wet and could maintain the shape of the particles, it was scored as 2 points.

[0155] 2 Results are shown in the following table: Figure 2 Pharmacodynamic study of compound S1 in the CGRP-induced mouse diarrhea model.

Claims

1. A class of quinolinone compounds of Formula I, or a pharmaceutically acceptable salt thereof, ###0001### wherein, R1 is selected from H, F, Cl, Br; R2 is selected from H, or C1-C8 alkyl; R3, R4 are independently selected from H, F, Cl; n is selected from the numbers 1 to 8. wherein, R1 is selected from H, F, Cl; R2 is selected from C1-C5 alkyl; R3, R4 are independently selected from H, F, Cl; n is selected from the numbers 1 to 3. wherein, R1 is selected from F, Cl; R2 is selected from C1-C3 alkyl; R3, R4 are independently selected from H, F; n is selected from the numbers 1 or 2. The compound is: ###0002### 5. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of irritable bowel syndrome. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein, ​ ​ ​ ​ ​ 3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ​ ​ ​ ​ ​ 4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ​ ​ 6. A pharmaceutical composition, characterized by, ​

Images