Quinolinone derivative and application thereof

Abstract

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

Description

Technical Field

[0003] The invention belongs to the pharmaceutical field and provides a quinolinone compound and a preparation method and a pharmaceutical use thereof. The compound can be used to treat irritable bowel syndrome. Background Art

[0005] Irritable bowel syndrome (IBS) is a gastrointestinal disorder. It has clinical symptoms such as abdominal pain, bloating, changes in bowel habits, and abnormal stool shape or mucus in stool. The symptoms persist or occur intermittently, but there is no definite evidence such as morphological and laboratory abnormalities. Although IBS does not endanger people's lives, it can affect work and life to varying degrees and reduce the quality of life.

[0006] The cause of IBS is still unknown, and it involves many aspects such as motility disorder, visceral hypersensitivity, inflammation and immune dysfunction. Some people with acute intestinal infection (about 20%) develop IBS-like symptoms after recovery, suggesting that there may be a relationship between intestinal infection and IBS. Due to the increase in stress factors in the current family, work, and social environment, the incidence of IBS is gradually increasing. The incidence of IBS is about 10-20%. In most countries, there are more women than men, with a male-to-female ratio of about 1:2. The incidence of IBS is higher in young people, and it is rare for people to develop the disease for the first time after the age of 50.

[0007] Due to the complex etiology of IBS, there are currently very few effective therapies for this chronic pain disorder. Given the negative impact of IBS on quality of life and its economic burden on society, there is a clear need for more treatments to relieve IBS-related abdominal pain.

[0008] Calcitonin gene-related peptide (CGRP) is a 37-amino acid member of the calcitonin peptide family, produced in both peripheral and central neurons, acting as a potent vasodilator and playing a role in the transmission of nociception. CGRP is considered an important factor in the transmission of sensory and pain stimuli and many other aspects of gastrointestinal physiology, including motility, secretion, and neuroprotection. In the gastrointestinal tract, CGRP is widely distributed throughout the enteric nervous system from the esophagus to the rectum, and is distributed in both intrinsic and extrinsic primary afferent neurons in a variety of mammals.

[0009] One study showed that intraperitoneal injection of CGRP in mice can induce diarrhea, while administration of anti-CGRP monoclonal antibodies can antagonize CGRP-induced diarrhea. Another clinical study showed that intravenous infusion of hCGRP can induce gastrointestinal hyperfunction.

[0010] Another study showed that targeting peripheral calcitonin gene-related peptide (CGRP) with a monoclonal anti-CGRP bivalent antigen-binding fragment antibody reduced central sensitization and attenuated colonic hypersensitivity induced by chronic adult stress or early life stress, and targeting CGRP may provide a new treatment strategy for irritable bowel syndrome. Summary of the invention

[0012] Technical problem to be solved: This type of compound is a CGRP receptor antagonist, which can antagonize CGRP-induced diarrhea and can be used to prepare drugs for treating irritable bowel syndrome.

[0013] Technical solution: A quinolinone compound as shown in formula I or a pharmaceutically acceptable salt thereof,

[0014]

[0015] Formula I

[0016] in,

[0017] R1 is selected from H, F, Cl, Br;

[0018] R2 is selected from H, or C1-C8 alkyl;

[0019] R3, R4 are independently selected from H, F, Cl;

[0020] n is selected from the numbers 1 to 8.

[0021] Preferably, wherein

[0022] R1 is selected from H, F, Cl;

[0023] R2 is selected from C1-C5 alkyl;

[0024] R3, R4 are independently selected from H, F, Cl;

[0025] n is selected from the numbers 1 to 3.

[0026] Preferably, wherein

[0027] R1 is selected from F, Cl;

[0028] R2 is selected from C1-C3 alkyl;

[0029] R3, R4 are independently selected from H, F;

[0030] n is selected from the number 1 or 2.

[0031] More preferably, the compound is:

[0032] S1

[0033] S2

[0034] S3

[0035] Beneficial effects:

[0036] This patent describes a class of quinolinone compounds, which are CGRP receptor antagonists that can antagonize CGRP-induced diarrhea and can be used to prepare drugs for treating irritable bowel syndrome. BRIEF DESCRIPTION OF THE DRAWINGS Instructions attached Figure 1 :Inhibition curve of CGRP antagonist activity of compound S1 Instructions attached Figure 2 :Study on the efficacy of compound S1 in CGRP-induced diarrhea model in mice DETAILED DESCRIPTION

[0038] The following examples will enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0039] Example 1: Synthesis of Compound S1

[0040] Synthesis route:

[0041]

[0042] Synthesis process:

[0043] first step:

[0044]

[0045] To a solution of compound 1 (40.0 g, 279 mmol, 1.00 eq.) in DCM (400 mL) at 0°C (plus 3 drops of DMF) was added (COCl)2 (42.5 g, 334.8 mmol, 1.20 eq.), and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. This acid 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 hours. 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 obtain a residue. The crude product was used in the next step without further purification. Compound 2 (5.00 g, 26.8 mmol, 9.61% yield) was obtained as a brown oil.

[0046] Step 2:

[0047]

[0048] 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.) and stirred at -78°C for 1 hour, then compound 2 (10.0 g, 53.7 mmol, 1.00 eq.) was added and stirred at -78°C for 1 hour. LC-MS showed that ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, and ~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×3). The combined organic layers were washed with brine 500 mL, dried over Na 2 SO4, filtered and concentrated to obtain 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, as confirmed by HNMR.

[0049] ESI-MS: 282.9[M+H]+

[0050] 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)

[0051] Step 3:

[0052]

[0053] Compound 3 (10.0 g, 35.3 mmol, 1.00 eq.), hydrochloride 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 to toluene (100 mL), the mixture was N2 protected, and then the mixture was stirred at 100 ° C for 4 hours. LC-MS showed that compound 3 was completely consumed, and a main peak of m / z (306.2) was detected. H 2 O 100 mL was added to quench the reaction mixture at 0 ° C, and extracted with EA 30.0 mL (30.0 mL × 5). The combined organic layer was washed with 30.0 mL of brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH 3 • H 2 O). Compound 4 (2.00 g, 6.55 mmol, yield 18.5%) was a brown oil.

[0054] ESI-MS: 306.2 [M+H] +

[0055] Step 4:

[0056]

[0057] To a solution of compound 4A (1.39 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (with 3 drops of DMF) at 0°C, (COCl)2 (0.99 g, 7.86 mmol, 1.20 eq.) was added, and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then, it was added dropwise to DCM (20 mL) with compound 4 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 hours. LC-MS showed that compound 4 was completely consumed, and a main peak of m / z (464.1) was detected. The reaction mixture was quenched at 0°C by adding H2O 70.0 mL, and extracted with EA 30.0 mL (30.0 mL × 3). The combined organic layers were washed with brine 50.0 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The crude product was used in the next step without further purification. Compound 5 (4.00 g, ) was a brown oil.

[0058] ESI-MS: 464.1 [M+H] +

[0059] Step 5:

[0060]

[0061] LiOH.H 2 O (1.09 g, 25.9 mmol, 3.00 equiv) was added to a solution of compound 5 (4.00 g, 8.63 mmol, 1.00 equiv) in THF (24.0 mL) and H 2 O (8.00 mL). The mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 5 was completely consumed and a major peak of m / z (450.1) was detected. The reaction mixture was quenched by adding H 2 O 120 mL at 0°C and extracted with EA 20.0 mL (20.0 mL×2). The combined aqueous phase was adjusted to pH=6 and then extracted with EA 50.0 mL (50.0 mL×3), and the combined organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was carried into the next step without purification. Compound 6 (450 mg, 1.00 mmol, yield 11.6%) was obtained as a brown oil.

[0062] ESI-MS: 450.1 [M+H] +

[0063] Step 6:

[0064]

[0065] 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., HCl) in DMF (4.50 mL) were 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 hours. LC-MS showed that compound 6 was completely consumed, and one major peak of m / z (660.3) was detected. The reaction mixture was quenched by adding H 2 O 50.0 mL at 0 °C, and extracted with DCM 50.0 mL (50.0 mL×3). The combined organic layer was washed with brine 50.0 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column (DCM: MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was an off-white solid.

[0066] ESI-MS: 660.3 [M+H] +

[0067] 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 = 7.2Hz, 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)

[0068] Example 2: Synthesis of Compound S2

[0069] Synthesis route:

[0070]

[0071] first step:

[0072]

[0073] To a solution of compound 1 (40.0 g, 279 mmol, 1.00 eq.) in DCM (400 mL) at 0°C (plus 3 drops of DMF) was added (COCl)2 (42.5 g, 334.8 mmol, 1.20 eq.), and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. This acid 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 hours. 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 obtain a residue. The crude product was used in the next step without further purification. Compound 2 (5.00 g, 26.8 mmol, 9.61% yield) was obtained as a brown oil.

[0074] Step 2:

[0075]

[0076] 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.) and stirred at -78°C for 1 hour, followed by the addition of compound 2 (10.0 g, 53.7 mmol, 1.00 eq.) and stirred at -78°C for 1 hour. LC-MS showed that ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, and ~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×3). The combined organic layers were washed with brine 500 mL, dried over Na 2 SO4, filtered and concentrated under reduced pressure to obtain 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, as confirmed by HNMR.

[0077] ESI-MS: 282.9[M+H]+

[0078] 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)

[0079] Step 3:

[0080]

[0081] Compound 3 (10.0 g, 35.3 mmol, 1.00 eq.), hydrochloride 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 to toluene (100 mL), the mixture was N2 protected, and then the mixture was stirred at 100 ° C for 4 hours. LC-MS showed that compound 3 was completely consumed, and a main peak of m / z (292.2) was detected. H 2 O 100 mL was added to quench the reaction mixture at 0 ° C, and extracted with EA 30.0 mL (30.0 mL × 5). The combined organic layer was washed with 30.0 mL of brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH 3 • H 2 O). Compound 4 (1.91 g, 6.55 mmol, yield 18.5%) was a brown oil.

[0082] ESI-MS: 292.2 [M+H] +

[0083] Step 4:

[0084]

[0085] To a solution of compound 4A (1.23 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (with 3 drops of DMF) was added (COCl)2 (0.99 g, 7.86 mmol, 1.20 eq.) at 0°C, and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then, it was added dropwise to DCM (20 mL) with compound 4 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 hours, and LC-MS showed that compound 4 was completely consumed, 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 × 3). The combined organic layers were washed with brine 50.0 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The crude product was used in the next step without further purification. Compound 5 (3.7 g, ) was a brown oil.

[0086] ESI-MS: 430.1 [M+H] +

[0087] Step 5:

[0088]

[0089] LiOH.H 2 O (1.09 g, 25.9 mmol, 3.00 equiv) was added to a solution of compound 5 (3.7 g, 8.63 mmol, 1.00 equiv) in THF (24.0 mL) and H 2 O (8.00 mL). The mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 5 was completely consumed and a main peak of m / z (416.1) was detected. The reaction mixture was quenched by adding H 2 O 120 mL at 0°C and extracted with EA 20.0 mL (20.0 mL×2). The combined aqueous phase was adjusted to pH=6 and then extracted with EA 50.0 mL (50.0 mL×3), and the combined organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was carried into the next step without purification. Compound 6 (415 mg, 1.00 mmol, yield 11.6%) was obtained as a brown oil.

[0090] ESI-MS: 416.1 [M+H]++

[0091] Step 6:

[0092]

[0093] 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) were 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 hours. LC-MS showed that compound 6 was completely consumed, and one major peak of m / z (626.3) was detected. The reaction mixture was quenched by adding H 2 O 50.0 mL at 0 °C and extracted with DCM 50.0 mL (50.0 mL×3). The combined organic layer was washed with brine 50.0 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column (DCM: MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was an off-white solid.

[0094] ESI-MS: 626.3 [M+H] +

[0095] 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)

[0096] Example 3: Synthesis of Compound S3

[0097] Synthesis route:

[0098]

[0099] first step:

[0100]

[0101] To a solution of compound 1 (47.9 g, 279 mmol, 1.00 eq.) in DCM (400 mL) at 0°C (plus 3 drops of DMF) was added (COCl)2 (42.5 g, 334.8 mmol, 1.20 eq.), and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. This acid 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 hours. 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 obtain a residue. The crude product was used in the next step without further purification. Compound 2 (5.7 g, 26.8 mmol, 9.61% yield) was obtained as a brown oil.

[0102] Step 2:

[0103]

[0104] 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.) and stirred at -78°C for 1 hour, then compound 2 (11.4 g, 53.7 mmol, 1.00 eq.) was added and stirred at -78°C for 1 hour. LC-MS showed that ~39.5% of compound 2 remained. Several new peaks were shown on LC-MS, and ~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×3). The combined organic layers were washed with brine 500 mL, dried over Na 2 SO4, filtered and concentrated under reduced pressure to obtain 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 a brown oil, as confirmed by HNMR.

[0105] ESI-MS: 311.1[M+H]+

[0106] 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)

[0107] Step 3:

[0108]

[0109] 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 to toluene (100 mL), the mixture was N2 protected, and then the mixture was stirred at 100 ° C for 4 hours. LC-MS showed that compound 3 was completely consumed, and a main peak of m / z (334.2) was detected. H 2 O 100 mL was added to quench the reaction mixture at 0 ° C, and extracted with EA 30.0 mL (30.0 mL × 5). The combined organic layer was washed with 30.0 mL of brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase high performance liquid chromatography (0.1% NH 3 • H 2 O). Compound 4 (2.19 g, 6.55 mmol, yield 18.5%) was a brown oil.

[0110] ESI-MS: 334.2 [M+H]+

[0111] Step 4:

[0112]

[0113] To a solution of compound 4A (1.24 g, 7.86 mmol, 1.20 eq) in DCM (20 mL) (with 3 drops of DMF) was added (COCl)2 (0.99 g, 7.86 mmol, 1.20 eq.) at 0°C, and the mixture was stirred at 25°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove (COCl)2. Then, it was added dropwise to DCM (20 mL) with compound 4 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 hours, and LC-MS showed that compound 4 was completely consumed, and a main peak of m / z (474.1) 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 × 3). The combined organic layers were washed with brine (50.0 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The crude product was used in the next step without further purification. Compound 5 (4.1 g, ) was a brown oil.

[0114] ESI-MS: 474.1 [M+H] +

[0115] Step 5:

[0116]

[0117] LiOH.H 2 O (1.09 g, 25.9 mmol, 3.00 equiv) was added to a solution of compound 5 (4.1 g, 8.63 mmol, 1.00 equiv) in THF (24.0 mL) and H 2 O (8.00 mL). The mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 5 was completely consumed and a main peak of m / z (460.1) was detected. The reaction mixture was quenched by adding H 2 O 120 mL at 0°C and extracted with EA 20.0 mL (20.0 mL×2). The combined aqueous phase was adjusted to pH=6 and then extracted with EA 50.0 mL (50.0 mL×3), and the combined organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was carried into the next step without purification. Compound 6 (459 mg, 1.00 mmol, yield 11.6%) was obtained as a brown oil.

[0118] ESI-MS: 460.1 [M+H] +

[0119] Step 6:

[0120]

[0121] 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., HCl) in DMF (4.50 mL) were 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 hours. LC-MS showed that compound 6 was completely consumed, and one major peak of m / z (670.3) was detected. The reaction mixture was quenched by adding H 2 O 50.0 mL at 0 °C and extracted with DCM 50.0 mL (50.0 mL×3). The combined organic layer was washed with brine 50.0 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column (DCM: MeOH system). The target compound S1 (215.67 mg, 325 umol, yield 32.5%, purity 99.5%) was an off-white solid.

[0122] ESI-MS: 670.3 [M+H]+

[0123] 1H NMR (400 MHz, DMSO-d6) δ 11.78 (br s, 1H), 8.23 ​​(s, 1H), 8.02 (brt, 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 = 9.4Hz, 1H), 3.11 (br t, J = 12.3 Hz, 1H), 2.98 (br d, J = 11.2 Hz, 3H), 2.84 (brs, 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)

[0124] Example 4 Study on CGRP target activity of compound S1

[0125] 1 Materials and methods

[0126] 1.1 Reagents and consumables

[0127] CGRP cells (HEK293 cells)

[0128] Fluo-4 Direct Calcium Assay Kit (Invitrogen#F10471)

[0129] Bovine Serum Albumine(B265993)

[0130] Cell plate (Greiner#781946)

[0131] Compound plate (Greiner#781280)

[0132] 1.2 Testing instruments

[0133] Liquid handler: Echo 555 (Labcyte)

[0134] Reader: FLIPR (Molecular Device)

[0135] 1.3 Experimental methods

[0136] 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 culture overnight.

[0137] 1.3.2 Preparation of assay buffer: Fluo-4 Direct calcium assay buffer containing 0.5% BSA.

[0138] 1.3.3 Serially dilute the agonist reference compound (CGRP) with DMSO to 10 concentration gradients, use Echo as the plate map, and spot 750nL of the compound on a DRC plate (Greiner #781280). Add 30uL of detection buffer to each well of the DRC plate (Greiner #781280) and centrifuge at 1000rpm for 10 seconds.

[0139] 1.3.4 Remove the EC80 test plate from the incubator and gently dispense 20ul of 2X Fluo-4 DirectTM No-Wash Loading Buffer into the 384-well cell culture plate using a pipette. The final volume in the cell plate is 40ul.

[0140] 1.3.5 Incubate at 37°C, 5% CO2 for 50 minutes and at room temperature for 10 minutes.

[0141] 1.3.6 Remove the EC80 test plate from the incubator and place it in the FLIPR. Place the DRC plate and tip box in the FLIPR.

[0142] 1.3.7 Run the protocol on FLIPRTETRA and calculate EC80.

[0143] 1.3.8 Antagonist compounds were diluted 10 times in DMSO at 1:5 starting from 1000uM. 900nl of compound was spotted onto the compound plate (Greiner#781280) using Echo. 30uL of assay buffer was added to each well of the compound plate (Greiner#781280) and centrifuged at 1000rpm for 10 seconds.

[0144] 1.3.9 Remove the cell plate from the incubator and gently dispense 20ul of 2X Fluo-4 DirectTM No-Wash Loading Buffer into the 384-well cell culture plate using a pipette. The final volume in the cell plate is 40ul.

[0145] 1.3.10 Incubate at 37°C, 5% CO2 for 50 minutes and at room temperature for 10 minutes.

[0146] 1.3.11 Dilute CGRP in assay buffer to obtain 6X EC80 and transfer 30uL to each well of the EC80 compound plate.

[0147] 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 FLIPRTETRA and transfer 10 uL of the agonist reference compound from the compound plate to the cell plate. Read the fluorescence signal. Transfer 10 uL of the agonist reference compound from the EC80 plate to the cell plate. Read the fluorescence signal. Calculate "max-min" from readings 1 to 90 to generate a final signal for calculating percent inhibition.

[0148] 2 The results are as attached Figure 1 The CGRP antagonist activity inhibition curve of compound S1 is shown.

[0151] Example 5 Study on the efficacy of compound S1 in CGRP-induced diarrhea model in mice

[0152] 1 Materials and methods

[0153] 1.1 Experimental animals

[0154] BALB / c mice, male, weight: 20-25g, SPF grade

[0155] 1.2 Experimental methods

[0156] 1.2.1 Preparation of CGRP-induced diarrhea model. Main steps: 0.1 mg / kg CGRP was intraperitoneally injected into mice, and the mice were placed in an isolation box. The looseness of feces was observed after 30 minutes.

[0157] 1.2.2 Animal Grouping and Dosing The experimental animals were randomly divided into 5 groups, namely, control group, model group, low-dose group (0.35 mg / kg) of compound S1, medium-dose group (1 mg / kg) of compound S1, and high-dose group (3.5 mg / kg) of compound S1, with 10 animals in each group. The dosing volume of each group was 10 μL / g.

[0158] 1.2.3 Assessment of loose stool index

[0159] If the feces is too watery to maintain its pellet shape, the score is 3 points; if the feces is dry and can maintain its pellet shape, the score is 1 point; if the feces is between the first two situations and can maintain its pellet shape, the score is 2.

[0160] 2 The results are as attached Figure 2 The pharmacological efficacy of compound S1 shown in the CGRP-induced diarrhea model in mice.

Claims

1. A quinolinone compound as shown in formula I or a pharmaceutically acceptable salt thereof, Formula I in, 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.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: in, 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.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: in, 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.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is: S1 S2 S3。 5. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the treatment of irritable bowel syndrome.

6. A pharmaceutical composition, characterized in that It comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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