A small molecule inhibitor and its preparation method and application

Abstract

The present invention discloses a small molecule inhibitor with a structural general formula (I). Its preparation method is as follows: Using 5-acetyl-2,8-dihydroxyquinoline and benzyl bromide as the original raw materials, a small molecule inhibitor of formula (I) is obtained through eight-step reactions; this small molecule inhibitor synthesizes new compounds by changing R 1 , and has an obvious inhibitory effect on colorectal tumor cells. Acting on the colorectal cancer cell line SW620, its IC 50 is at least 0.965 μM. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a medicine and a preparation method and application thereof, and in particular to a small molecule inhibitor and a preparation method and application thereof. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors in the world and the third leading cause of cancer death in the world. According to the World Cancer Research Fund, more than 1.9 million new cases of colorectal cancer were registered in 2020, and 935,000 people died of the cancer worldwide. The highest risk of colorectal cancer is observed in Europe and North America, and the highest risk of colorectal cancer is also observed in Australia. The incidence of colorectal cancer is related to age, region, gender, and genetics. At present, the treatment options for colorectal cancer are still mainly surgery, radiotherapy, and chemotherapy. In recent years, with the development of endoscopic examinations and various auxiliary technologies, although the mortality rate of patients has decreased, there is still no effective way to prevent the recurrence and metastasis of colorectal cancer. Therefore, targeted therapy for key pathogenic factors of colorectal cancer has become a major breakthrough, currently mainly bevacizumab and cetuximab. Bevacizumab binds to vascular endothelial growth factor (VEGF), inhibits tumor endothelial proliferation and new blood vessel formation, and reduces tumor tissue nutrition; cetuximab inhibits cancer cell proliferation and induces apoptosis by binding to epidermal growth factor (EGF) receptors to block intracellular signaling pathways. However, compared with radiotherapy and cisplatin alone, the combination of cetuximab with radiotherapy and cisplatin increases serious adverse events, such as cardiac events. Therefore, further research is needed on effective therapeutic drugs for colorectal cancer.

[0003] Indacaterol, 5-[(R)-2-(5,6-diethylindan-2-ylamino)-1-hydroxyethyl]-8-hydroxy-1H-quinolin-2-one, also known as QAB149, is a β2-adrenergic receptor agonist with almost complete affinity for human β2-adrenergic receptors. Previous studies have shown that indacaterol can kill SW620 cells in a dose-dependent manner and has similar cytotoxic effects in RKO, HCT116, and HCT8 cells, with IC 50 Between 20 and 30 μM. Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide a small molecule inhibitor that has an inhibitory effect on colorectal tumor cells; the second purpose is to provide a method for preparing the small molecule inhibitor; the third purpose is to provide an application of the small molecule inhibitor.

[0005] Technical solution: The small molecule inhibitor described in the present invention has the chemical structural formula:

[0006]

[0007] Where R 1 for n=0-3, L is a chemical bond or a 5-7 membered aromatic ring or a nitrogen-containing aromatic heterocycle, and the chiral center* is an S configuration or an R configuration.

[0008] Preferably, n=0-1, and L is a chemical bond or a 6-membered aromatic ring or a nitrogen-containing aromatic heterocycle.

[0009] Preferably, the R 1 -CH 2 -CN,

[0010] The method for preparing the small molecule inhibitor of the present invention comprises the following steps:

[0011] (1) After 5-acetyl-2,8-dihydroxyquinoline reacts with benzyl bromide, the hydroxyl group on the benzene ring is protected by the benzyl group, and then a brominating agent is added to brominate the terminal primary carbon to obtain compound II;

[0012] (2) Using (R) / (S)-2-methyl-CBS-oxazaborolidine as a chiral catalyst and borane as a reducing agent, compound II undergoes a reduction reaction, and the carbonyl group on the benzene ring is selectively reduced to a hydroxyl group to obtain compound III;

[0013] (3) Compound III undergoes an intramolecular cyclization reaction under alkaline conditions to obtain compound IV;

[0014] (4) Compound IV reacts with a nucleophile 5,6-diethyl-2,3-dihydro-1H-inden-2-amine hydrochloride to obtain compound V;

[0015] (5) Compound V reacts with Boc anhydride to obtain compound VI;

[0016] (6) Compound VI is debenzylated under palladium carbon / hydrogen conditions to obtain compound VII;

[0017] (7) Compound VII and R 1 X reacts to obtain compound VIII;

[0018] (8) Finally, compound VIII is subjected to acid removal of Boc to obtain the small molecule inhibitor I; the synthetic route is as follows:

[0019]

[0020] Where R 1 for n=0-3, L is a chemical bond or a 5-7 membered aromatic ring or a nitrogen-containing aromatic heterocycle.

[0021] Preferably, in step (1), the reaction solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, acetone, water, tetrahydrofuran, methanol, N,N-dimethylformamide, dimethyl sulfoxide or dioxane; the base is selected from potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide or triethylamine; the brominating agent is tetrabutylammonium tribromide; the reaction temperature is 45-60° C. The reaction process is monitored by TLC, and the developing solvent is dichloromethane:methanol=15:1.

[0022] Preferably, in step (2), the reduction reaction solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; the reaction temperature is -20°C to 25°C; according to the chirality requirements, (R) or (S)-CBS chiral catalysts are selected respectively, and borane is used for asymmetric reduction. The reaction process is monitored by TLC, and the developing solvent is dichloromethane:methanol = 15:1.

[0023] Preferably, in step (3), the reaction solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, acetone, tetrahydrofuran, methanol, N,N-dimethylformamide, dimethyl sulfoxide or dioxane, the base is selected from potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide or triethylamine, and the reaction is refluxed. The reaction process is monitored by TLC, and the developing solvent is dichloromethane:ethyl acetate=1:1.

[0024] Preferably, in step (4), the reaction solvent is selected from acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, n-butanol or diethylene glycol dimethyl ether; the reaction temperature is 60° C. to 150° C. The reaction progress is monitored by TLC, and the developing solvent is dichloromethane:ethyl acetate=1:1.

[0025] Preferably, in step (5), the reaction solvent is selected from one or more of dichloromethane, chloroform, dioxane, water, methanol, ethanol, acetonitrile, N,N-dimethylformamide or tetrahydrofuran; the base can be selected from triethylamine, sodium hydroxide, sodium bicarbonate, potassium carbonate or 4-dimethylaminopyridine, and the reaction is refluxed. The reaction process is monitored by TLC, and the developing solvent is dichloromethane: methanol = 15:1.

[0026] Preferably, in step (6), the reaction solvent is selected from one or more of methanol, ethanol, tetrahydrofuran or ethyl acetate; the reaction temperature is 25° C. to 35° C. The reaction progress is monitored by TLC, and the developing solvent is dichloromethane:methanol=15:1.

[0027] Preferably, in step (7), the reaction solvent is selected from one or more of N,N-dimethylformamide, acetonitrile, acetone, tetrahydrofuran or dimethyl sulfoxide; the base is selected from potassium carbonate, cesium carbonate, sodium carbonate, triethylamine; reflux reaction. The reaction process is monitored by TLC, and the developing solvent is dichloromethane:methanol = 15:1.

[0028] Preferably, in step (8), the reaction solvent is selected from one or more of dichloromethane, dioxane, methanol or ethyl acetate; the acid is selected from trifluoroacetic acid and hydrochloric acid; the reaction temperature is 25° C. to 35° C. The reaction process is monitored by TLC, and the developing solvent is dichloromethane:methanol=15:1.

[0029] The invention relates to the use of the small molecule inhibitor in drugs for treating tumors, wherein the tumor is a colorectal tumor.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The small molecule inhibitor has a significant effect on R 1 The new compound was synthesized by changing the colorectal cancer cell line SW620, which has a significant inhibitory effect on colorectal cancer cells. 50 0.965 μM; (2) The preparation method designed and synthesized a new compound small molecule inhibitor that can significantly inhibit the occurrence and development of colorectal cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The representative compound I of the present invention 1 Blood drug concentration-time curve in rats after intravenous injection (5 mg / kg) (n=3);

[0032] Figure 2 The representative compound I of the present invention 1 Blood drug concentration-time curve in rats after intragastric administration (50 mg / kg) (n=3). DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with embodiments.

[0034] Example 1

[0035] The small molecule inhibitor of the present invention has a chemical name of (S)-2-((5-(2-((5,6-diethyl-2,3-dihydro-1H-inden-2-yl)amino)-1-hydroxyethyl)-2-oxo-1,2-dihydroquinolin-8-yl)oxy)acetonitrile (I 1 ), the synthetic route is as follows:

[0036] The preparation method comprises the following steps:

[0037] (1) Intermediate II 1 Synthesis

[0038] After adding 10 g (49.21 mmol) of 5-acetyl-8-hydroxyquinoline-2(1H)-one and 8.16 g (59.06 mmol) of anhydrous potassium carbonate into the reaction flask, 150 mL of acetone was added and the temperature was raised to reflux. Under reflux conditions, 7.01 mL (59.06 mmol) of benzyl bromide was added, and after sufficient stirring, 13.40 g of filter cake was collected.

[0039] After adding 13.40 g (45.68 mmol) of the filter cake into the reaction flask, 201 mL of anhydrous THF and 80.4 mL of anhydrous methanol were added, the temperature was raised to 40°C, and the mixture was stirred thoroughly. Then, 30.84 g (63.96 mmol) of tetrabutylammonium tribromide was slowly added in batches to brominate the terminal primary carbon to obtain the representative intermediate 8-(benzyloxy)-5-(2-bromoacetyl)quinoline-2(1H)-one (Compound II 1 )12.59g, yield: 74.04%. 1H NMR (400MHz, DMSO-d6) δ11.07 (s, 1H), 8.51 (d, J = 9.9Hz, 1H), 7.87 (d, J = 8.5Hz, 1H), 7.60 (d, J = 7.0Hz, 2H), 7.42-7.37 (m, 2H), 7.34 (dd, J = 6.8, 1.9Hz, 1H), 7.30 (d, J = 8.7Hz, 1H), 6.68 (d, J = 10.1Hz, 1H), 5.44 (s, 2H), 4.92 (s, 2H).

[0040] (2) Intermediate III 1 Synthesis

[0041] Under nitrogen protection, compound II 1 12.58g (33.80mmol) was added to a 500mL double-necked reaction bottle, and anhydrous THF 190mL and a catalytic amount of (S)-2-methyl-CBS-oxazolidinone 1.41g (5.07mmol) were added, and the mixture was placed in a -10°C cold trap and stirred thoroughly. After 10 minutes, 1M borane-tetrahydrofuran complex (41.23mL, 41.23mmol) was measured and slowly dripped into the reaction system within half an hour. Stirring was continued at this temperature for 15 minutes. The reaction was completed after TLC monitoring, and the reaction was quenched with 50mL of methanol. After removing the solvent by concentrating under reduced pressure, 250mL of 1M hydrochloric acid solution was added to the bottle, and the mixture was stirred at room temperature overnight and then filtered. The filter cake was collected and dried in vacuo to obtain white solid III. 111.76 g, yield: 92.98%. 1H NMR (300 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.19 (d, J = 9.9 Hz, 1H), 7.58 (d, J = 6.6 Hz, 2H), 7.42-7.30 (m, 3H), 7.20 (s, 2H), 6.57 (d, J = 9.9 Hz, 1H), 5.96 (d, J = 4.7 Hz, 1H), 5.31 (s, 2H), 5.22 (dt, J = 7.2, 4.8 Hz, 1H), 3.72-3.58 (m, 2H).

[0042] (3) Intermediate IV 1 Synthesis

[0043] Compound III 1 11.70g (31.26mmol) was added to a 500mL single-mouth reaction bottle, and 180mL of acetone, 1.80mL of water and 8.64g (62.53mmol) of anhydrous potassium carbonate were added, and heated to reflux and stirred thoroughly. After 4h, the reaction was completed by TLC monitoring, and the mixture was filtered, the filter cake was washed with acetone, and the filtrate was collected and combined. After the filtrate was concentrated under reduced pressure to remove the solvent, n-heptane was added for recrystallization to obtain a light yellow solid IV 1 8.43 g, yield 91.93%. 1H NMR (300 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.21 (d, J = 9.8 Hz, 1H), 7.57 (d, J = 6.7 Hz, 2H), 7.41-7.28 (m, 3H), 7.19 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.62 (d, J = 9.8 Hz, 1H), 5.31 (s, 2H), 4.41-4.31 (m, 1H), 3.14 (dd, J = 5.5, 4.1 Hz, 1H), 2.78 (dd, J = 5.5, 2.6 Hz, 1H).

[0044] (4) Intermediate V 1 Synthesis

[0045] Compound IV 1 5g (17.05mmol) and 4.19g (22.16mmol) of 5,6-diethyl-2,3-dihydro-1H-inden-2-amine were added to a single-mouth reaction bottle, 10mL of n-butanol was added to dissolve it, and the temperature was raised to 95°C. The mixture was stirred for 10h at this temperature, and the reaction was completed after TLC monitoring. The reaction solution was concentrated and purified by silica gel column chromatography to obtain intermediate V 16.78 g, yield 82.41%. NMR (300MHz, DMSO-d6) δ10.91(s,1H),8.32(d,J=10.1Hz,1H),7.59(d,J=7.4Hz,2 H),7.39(tt,J=14.8,7.2Hz,3H),7.24(s,2H),6.99(s,2H),6.59(d,J=9.8Hz,1H), 5.42(d,J=7.9Hz,1H),5.32(s,2H),3.88(p,J=7.8Hz,1H),3.15(dt,J=16.2,8.2Hz ,2H),2.95(dd,J=24.1,10.5Hz,4H),2.56(t,J=7.6Hz,4H),1.12(t,J=7.5Hz,6H).

[0046] (5) Intermediate VI 1 Synthesis

[0047] Compound V 1 6.78g (14.05mmol) was added to a 250mL single-mouth reaction bottle and dissolved in 102mL of dichloromethane, and 5.86ml (42.14mmol) of triethylamine and 6.13g (28.10mmol) of di-tert-butyl dicarbonate were added under nitrogen protection. After being stirred at room temperature for 5 hours, the reaction was completed by TLC monitoring, and water was added for extraction three times. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate VI. 1 5.82 g, yield 71.09%. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.22 (d, J = 33.0 Hz, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.33 (dd, J = 25.1, 11.0 Hz, 3H), 7.11 (d, J = 48.5 Hz, 2H), 6.90 (d, J = 24.4 Hz, 2H), 6 .49(s,1H),5.59(s,1H),5.29(d,J=15.1Hz,2H),4.40(d,J=19.5Hz,1H),3.34(s,2 H), 3.05 (d, J = 121.0Hz, 4H), 2.55 (d, J = 7.1Hz, 4H), 1.29 (s, 9H), 1.19–1.00 (m, 6H).

[0048] (6) Intermediate VII 1 Synthesis

[0049] Compound VI 13 g (5.15 mmol) was added to a 100 mL single-mouth reaction bottle and dissolved in 35 mL of anhydrous methanol. 345 mg of 10% Pd / C was added, and a pre-filled hydrogen bag was connected to replace the air. Stir at room temperature for 12 hours. The reaction was completed after TLC monitoring. The reaction was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a light yellow solid VII. 1 2.25 g, yield: 88.72%. 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 2H), 8.19 (s, 1H), 7.07 (s, 1H), 6.96–6.86 (m, 3H), 6.46 (s, 1H), 5.54 (s, 1H), 5.14 (d, J = 53.6 Hz, 1H), 4.41 (s, 1H), 3.33 (s, 2H), 3.16–2.78 (m, 4H), 2.60–2.52 (m, 4H), 1.31 (s, 9H), 1.12 (q, J = 7.5 Hz, 6H).

[0050] (7) Intermediate VIII 1 Synthesis

[0051] Compound VII 1 After adding 550mg (1.12mmol) and 231.46mg (1.67mmol) of anhydrous potassium carbonate into a double-necked reaction bottle, 2mL of N,N-dimethylformamide was added and the temperature was raised to 80°C. Under reflux conditions, 147.31mg (1.23mmol) of 2-bromoacetonitrile dissolved in 1ml of N,N-dimethylformamide was added dropwise, and the reaction was fully stirred until the reaction was completed as monitored by TLC. Water was added and extracted with ethyl acetate, the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate VIII 1 380 mg, yield 64.02%. 1H NMR (400MHz, DMSO-d6) δ11.15(s,1H),8.25(d,J=37.5Hz,1H),7.35(d,J=8.3Hz,1H),7.29–7.15(m,1H),6.92(d,J=16.8Hz,2H),6.53(s,1H),5.7 0(d,J=20.8Hz,1H),5.23(s,2H),5.13(s,1H),4.40(s,1H),3.24(s,2H), 2.94(d,J=10.8Hz,4H),2.56(s,4H),1.30(s,9H),1.12(q,J=7.3Hz,6H).

[0052] (8) Target product I 1 Synthesis

[0053] Compound VIII 1380 mg (0.71 mmol) was added to a single-mouth reaction bottle and dissolved in 2 mL of dichloromethane. 0.95 mL (12.87 mmol) of trifluoroacetic acid was added at room temperature and a balloon was inserted to collect the generated gas. After stirring for 30 min, TLC monitored the completion of the reaction. The reaction solution was concentrated under reduced pressure at 70°C, saturated sodium bicarbonate solution was added to adjust to alkalinity, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the target product I 1 53 mg, yield 17.18%. 1HNMR (400MHz, DMSO-d6) δ11.32 (s, 1H), 8.24 (d, J = 10.0 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 2.8 Hz, 2H), 6.58 (d, J = 9.9 Hz, 1H), 5.24 (s, 2H), 5.1 7(t,J=6.2Hz,1H),3.64–3.59(m,1H),3.03(ddd,J=15.6,11.5,7.1Hz,2H),2.83(d,J= 6.0Hz,2H),2.68(dd,J=15.4,6.4Hz,2H),2.58–2.52(m,4H),1.12(t,J=7.5Hz,6H).13C NMR(101MHz,DMSO-d6)δ161.53,142.32,139.62,139.39,139.34,136.98,135.57,130.31,124.68,124.64,122 .90,119.79,116.93,114.06,68.79,59.46,55.53,55.33,39.09,38.97,25.34,16.14.MS(ESI)m / z=432.2[M+H] + (C 26 H 29 N 3 O 3 ).

[0054] Example 2

[0055] The small molecule inhibitor of the present invention has a chemical name of (S)-4-(((5-(2-((5,6-diethyl-2,3-dihydro-1H-inden-2-yl)amino)-1-hydroxyethyl)-2-oxo-1,2-dihydroquinolin-8-yl)oxy)methyl)benzonitrile (I 2 ), and its synthetic route is as follows:

[0056]

[0057] The preparation method comprises the following steps:

[0058] (1) Intermediate VIII 2 Synthesis

[0059] Compound VII 1 After adding 400 mg (0.81 mmol) and 280.55 mg (2.03 mmol) of anhydrous potassium carbonate into a double-necked reaction bottle, 2 mL of N, N-dimethylformamide was added and the temperature was raised to 80°C. Under reflux conditions, 175.11 mg (0.89 mmol) of 4-cyanobenzyl bromide dissolved in 1 ml of N, N-dimethylformamide was added dropwise, and the mixture was stirred until the reaction was completed as monitored by TLC. Water was added and extracted with ethyl acetate, the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate VIII. 2 340 mg, yield: 68.90%.

[0060] 1H NMR (300MHz, DMSO-d6) δ10.95(s,1H),8.23(d,J=25.0Hz,1H),7.83(q,J=8.2H z,4H),7.23–7.00(m,2H),6.90(d,J=18.6Hz,2H),6.52(s,1H),5.62(s,1H),5 .40(s,2H),5.15(d,J=41.9Hz,1H),4.39(s,1H),3.21(s,2H),2.91(d,J=8.0H z,4H),2.56(dt,J=7.6,3.8Hz,4H),1.28(s,9H),1.12(td,J=7.5,3.1Hz,6H).

[0061] (2) Target product I 2 Synthesis

[0062] Compound VIII 2 340 mg (0.56 mmol) was added to a single-mouth reaction bottle and dissolved in 2 ml of dichloromethane. 0.75 ml (10.07 mmol) of trifluoroacetic acid was added at room temperature and a balloon was inserted to collect the generated gas. After stirring for 30 min, TLC was used to monitor the completion of the reaction. The reaction solution was concentrated under reduced pressure at 70°C, saturated sodium bicarbonate solution was added to adjust to alkalinity, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the target product I 2170 mg, yield 59.86%. 1HNMR(300MHz,DMSO-d6)δ10.87(s,1H),8.21(d,J=10.0Hz,1H),7.84(q,J= 8.1Hz,4H),7.18(s,2H),6.94(d,J=2.4Hz,2H),6.56(d,J=9.9Hz,1H),5.39 (s,3H),5.05(t,J=5.9Hz,1H),3.52(q,J=6.7Hz,1H),2.98(dt,J=15.7,7.8 Hz,2H),2.74(d,J=6.1Hz,2H),2.63–2.51(m,6H),1.12(t,J=7.5Hz,6H).13C NMR (75MHz, DMSO-d6) δ161.49,143.43,142.97,139.77,139.72,139.44,137.25,133.94,132.72,129.73,128.8 2,124.69,122.49,119.85,117.39,112.44,69.44,69.35,59.69,56.09,25.34,16.16.MS(ESI)m / z=508.3[M+H] + (C 32 H 33 N 3 O 3 ).

[0063] Example 3

[0064] The small molecule inhibitor of the present invention has a chemical name of (S)-5-((5-(2-((5,6-diethyl-2,3-dihydro-1H-inden-2-yl)amino)-1-hydroxyethyl)-2-oxo-1,2-dihydroquinolin-8-yl)oxy)pyrazine-2-carbonitrile (I 3 ), and its synthetic route is as follows:

[0065]

[0066] (1) Intermediate VIII 3 Synthesis

[0067] Compound VII 1300 mg (0.61 mmol) was added to a single-mouth reaction bottle, and tetrahydrofuran 4.5 mL, triethylamine 0.17 ml (1.22 mmol), 2-chloro-5-cyanopyrazine 101.98 mg (0.73 mmol) were added and the temperature was raised to 70°C. The mixture was stirred thoroughly under reflux until the reaction was completed as monitored by TLC. The reaction solution was concentrated under reduced pressure, water was added and extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate VIII. 3 350mg, the yield is 96.48%. 1H NMR (300MHz, DMSO-d6) δ11.61(s,1H),8.74(dd,J=33.6,1.3Hz,2H),8.30(d,J=31.6Hz,1H),7.51–7.21(m,2H),6.93(d,J=11.4Hz,2H),6.54(s,1H) ,5.78(s,1H),5.28(d,J=39.9Hz,1H),4.44(s,1H),3.43(s,2H),2.97(s,4 H), 2.55 (dd, J=7.5, 3.3Hz, 4H), 1.32 (s, 9H), 1.12 (td, J=7.5, 3.4Hz, 6H).

[0068] (2) Target product I 3 Synthesis

[0069] Compound VIII 3 420 mg (0.71 mmol) was added to a single-mouth reaction bottle and dissolved in 2 ml of dichloromethane. 0.94 ml (12.69 mmol) of trifluoroacetic acid was added at room temperature and a balloon was inserted to collect the generated gas. After stirring for 30 min, TLC was used to monitor the completion of the reaction. The reaction solution was concentrated under reduced pressure at 70°C, saturated sodium bicarbonate solution was added to adjust to alkalinity, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the target product I 370 mg, yield 20.03%. 1HNMR (300MHz, DMSO-d6) δ11.83 (s, 1H), 8.75 (dd, J = 24.0, 1.4 Hz, 2H), 8.28 (d, J = 10.0 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 2.3 Hz, 2H), 6.57 (d, J = 9. 9Hz,1H),5.48(d,J=91.8Hz,1H),5.18(t,J=6.1Hz,1H),3.56(t,J=6.7Hz,1H),3.01(dt, J=15.4,7.6Hz,2H),2.82(d,J=6.0Hz,2H),2.68–2.52(m,6H),1.13(t,J=7.5Hz,6H).13C NMR (75MHz, DMSO-d6) δ161.87,161.28,146.76,140.27,139.84,139.76,139.71,139.47,137.06,132.50,124. 71,124.66,123.55,122.71,122.49,120.16,118.28,69.30,59.71,56.02,25.35,16.17.(ESI)m / z=496.3[M+H] + (C 29 H 29 N 5 O 3 ).

[0070] Inhibitory effect of compound I on colorectal cancer cell lines

[0071] Indacaterol was used as a positive control and a concentration gradient of 10, 20, 30, and 40 μM was prepared. The IC values ​​of indacaterol on SW620 were compared. 50 Fixed between 20-25 μM. Compound I was prepared in the same concentration gradient for primary screening and had a lower IC than indacaterol 50 The compounds were screened for the second time, and the concentration gradient of the compounds was 1, 2, 4, and 8 μM.

[0072] Both rounds of screening were performed on the colorectal cancer cell line SW620. The initial number of cells was 5000 per well. The cell viability at 48 h was directly measured to determine the IC of the compound. 50 .

[0073] Cell viability = [(experimental group reading - blank group reading) / (control group reading - blank group reading)] × 100%.

[0074] Table 1 Inhibitory effect of compound I on SW620

[0075] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Indacaterol 20.83 <![CDATA[I 2 ]]> 3.467 <![CDATA[I 1 ]]> 0.965 <![CDATA[I 3 ]]> 8.717

[0076] Compound I 1 In vivo pharmacokinetic studies in rats

[0077] Investigated I 1 The overall pharmacokinetic characteristics of (LYS-8) in rats were obtained to obtain its main pharmacokinetic parameters in rats, providing a reference for further drug development. Rats (n=3) were given 5 mg / kg intravenous injection and 50 mg / kg oral gavage, and plasma was collected at a given time. HPLC-MS / MS methods were established to determine the concentration of the compound in rat plasma, and WinNonlin software was used to calculate the pharmacokinetic parameters. The data results are as follows:

[0078] Table 2 Compound I 1 Main pharmacokinetic parameters in rats after administration (n=3)

[0079]

[0080] Table 3 Compound I 1 Blood drug concentration in rats after intravenous injection (5 mg / kg) (ng / mL)

[0081]

[0082]

[0083] Table 4 Compound I 1 Blood drug concentration in rats after intragastric administration (50 mg / kg) (ng / mL)

[0084]

[0085] Note: “40.89” is an abnormal point and is excluded during drawing and parameter calculation.

[0086] According to the sample measurement results and parameter analysis results:

[0087] (1) Compound I was administered intravenously (5 mg / kg) 1 After that, AUC (0-t) was 1031.43±25.02h·μg / L, V was 36.57±8.82L / kg, C 2min The average value was 784.05±86.39 ng / mL, indicating that compound I 1 There may be widespread or specific tissue distribution in rats; CL is 4.73±0.18L / h / kg, t 1 / 2The time of the experiment was 5.39±1.51h, indicating that compound I 1 The clearance in rats is slow.

[0088] (2) Administration of Compound I by intragastric administration (50 mg / kg) 1 After that, C max 131.48±26.13μg / L, AUC (0-t) 1056.37±477.96h·μg / L, T max It is 1.53±2.15h, and the bioavailability is 10.24%.

[0089] (3) Compound I 1 After administration, hemolysis was improved and the rats did not have hematuria.

[0090] In summary, based on the inhibitory effect of indacaterol on colorectal cancer, we screened the lead compound LYW-1 and optimized its structure to synthesize a new compound I 1 In vitro studies have found that it can significantly inhibit the occurrence and development of colorectal cancer cells; the results of in vivo pharmacokinetic experiments provide pharmacokinetic guidance for the structural modification of the compound and data support for subsequent efficacy and toxicology studies; it provides the possibility for the development of small molecule anti-tumor drugs for colorectal cancer and has good application prospects.

Claims

1. A small molecule inhibitor, characterized in that: The chemical structure is: Where R 1 for n=0-1, L is a chemical bond or a six-membered aromatic ring or a six-membered nitrogen-containing aromatic heterocycle, and the chiral center* is S configuration or R configuration.

2. The small molecule inhibitor according to claim 1, characterized in that The R 1 -CH2-CN, 3. The method for preparing a small molecule inhibitor according to claim 1, characterized in that: The following steps are involved: (1) After 5-acetyl-2,8-dihydroxyquinoline reacts with benzyl bromide, the hydroxyl group on the benzene ring is protected by the benzyl group, and then a brominating agent is added to brominate the terminal primary carbon to obtain compound II; (2) Using (R) / (S)-2-methyl-CBS-oxazaborolidine as a chiral catalyst and borane as a reducing agent, compound II undergoes a reduction reaction, and the carbonyl group on the benzene ring is selectively reduced to a hydroxyl group to obtain compound III; (3) Compound III undergoes an intramolecular cyclization reaction under alkaline conditions to obtain compound IV; (4) Compound IV reacts with a nucleophilic reagent 5,6-diethyl-2,3-dihydro-1H-inden-2-amine hydrochloride to obtain compound V; (5) Compound V reacts with Boc anhydride to obtain compound VI; (6) Compound VI is debenzylated under palladium carbon / hydrogen conditions to obtain compound VII; (7) Compound VII and R 1 X reacts to obtain compound VIII; (8) Finally, compound VIII is subjected to acid removal of Boc to obtain the small molecule inhibitor I; the synthetic route is as follows: Where R 1 for n=0-1, L is a chemical bond or a six-membered aromatic ring or a six-membered nitrogen-containing aromatic heterocycle, and the chiral center* is S configuration or R configuration.

4. The method for preparing a small molecule inhibitor according to claim 3, characterized in that: In step (4), the reaction solvent is selected from acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, n-butanol or diethylene glycol dimethyl ether; and the reaction temperature is 60° C. to 150° C.

5. The method for preparing a small molecule inhibitor according to claim 3, characterized in that: In step (7), the reaction solvent is selected from one or more of N,N-dimethylformamide, acetonitrile, acetone, tetrahydrofuran or dimethyl sulfoxide; the base is selected from potassium carbonate, cesium carbonate, sodium carbonate, triethylamine; and the reaction is refluxed.

6. The method for preparing a small molecule inhibitor according to claim 3, characterized in that: In step (2), the reduction reaction solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide; and the reaction temperature is -20°C to 25°C.

7. The method for preparing a small molecule inhibitor according to claim 3, characterized in that: In step (3), the reaction solvent is selected from one or more of acetonitrile, dichloromethane, chloroform, acetone, tetrahydrofuran, methanol, N,N-dimethylformamide, dimethyl sulfoxide or dioxane; the base is selected from potassium carbonate, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide or triethylamine; and the reaction is refluxed.

8. Use of the small molecule inhibitor according to claim 1 or 2 in the preparation of drugs for treating tumors.

9. The use according to claim 8, characterized in that: The tumor is a colorectal tumor.

Images