Small molecule inhibitors and preparation methods and their use in the preparation of anticancer drugs

Abstract

The present invention belongs to the field of biomedicine technology, and relates to small molecule inhibitors and preparation methods and their applications in the preparation of anticancer drugs. Its chemical structural formula is shown in formula (I), and the small molecule inhibitors provided by the present invention have good competitive affinity for the β-catenin / BCL9 target, and can be tightly bound to the target, and have a strong ability to competitively bind to destroy the β-catenin / BCL9 complex. Among them, some small molecule inhibitors have excellent inhibitory effects on the expression of the target gene Axin and better biological activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a small molecule inhibitor and a preparation method thereof and application thereof in the preparation of anticancer drugs. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Colorectal cancer (CRC) is the third most common cancer in the world. Currently, targeted drugs used to treat colon cancer mainly include anti-vascular endothelial growth factor (VEGF) (bevacizumab, aflibercept, ramucirumab), anti-epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab) and multikinase inhibitors (regorafenib). The combination of traditional chemotherapy and the use of these new targeted drugs has significantly improved the overall survival of patients with colorectal cancer. However, the rapid development of drug resistance and cancer recurrence may change the effectiveness of certain drugs. Therefore, it is necessary to design and develop new alternative drugs to avoid the reduction in the effectiveness of existing drugs, thereby reducing the mortality of clinical colorectal cancer patients. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the purpose is to provide a small molecule inhibitor and a preparation method and its use in the preparation of anticancer drugs. Studies have shown that the small molecule inhibitor provided by the present invention has good competitive affinity for the β-catenin / BCL9 target, can bind tightly to the target, and has a strong ability to competitively bind and destroy the β-catenin / BCL9 complex. Among them, some small molecule inhibitors have excellent inhibitory effects on the expression of the target gene Axin and better biological activity.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In one aspect, a small molecule inhibitor, whose chemical structure is shown in formula (I),

[0007]

[0008] Wherein, X, Y are independently selected from C, N, and R is

[0009]

[0010] In some embodiments, when X is C, Y is C or N; when Y is C, X is C or N. Further, X is C, and Y is C.

[0011] In some embodiments, R is

[0012] Furthermore, R is Furthermore, R is

[0013] In some embodiments, X is C, Y is C, and R is Studies have shown that the compounds under this condition not only have better competitive affinity for the β-catenin target protein, but also have a good inhibitory effect on the target gene Axin of the Wnt signaling pathway.

[0014] On the other hand, a method for preparing the above-mentioned small molecule inhibitor comprises a preparation process according to the following reaction route;

[0015]

[0016] Wherein, X, Y, and R are as described above.

[0017] In some embodiments, the synthetic routes of compounds 15-19 are as follows:

[0018]

[0019] Specifically, compound 1 is reacted with cyclopropylamine via reductive amination to generate compound 3; compound 3 is reacted with triphosgene to generate intermediate 4; compound 5 and compound 6 are subjected to Williamson etherification reaction to generate intermediate compound 7; compound 9 is generated by Buchwald coupling reaction of compound 7 and compound 8; compound 9 is reacted with compound 4 to generate compound 11; compound 13 is obtained by Suzuki coupling reaction of compound 11 and compound 12; compound 14 is obtained by removing the tert-butyl group of compound 13; target compounds 15-19 are obtained by amide condensation of compound 14 with different amines.

[0020] More specifically, in the synthetic routes of compounds 15-19: (a) cyclopropylamine, MeOH, room temperature, overnight; (b) NaBH 4 , MeOH, room temperature, 2h, two steps, yield 80%; (c) triphosgene, DIPEA, DCM, room temperature, overnight, yield 100%. (d) K 2 CO 3 ,ACN,80℃,reflux,24h,yield 90%,(e)Pd 2 (dba) 3 ,Ruphos,Cs 2 CO 3, Tol, 80℃, 72h, yield 65%; (f) TFA, DCM, room temperature, 2h, yield 55%; (g) DIPEA, THF, 50℃, overnight, yield 60%; (h) K 3 PO 4 ,Pd(dppf)Cl 2 , 1,2-dioxane / EtOH / H 2 O, 80℃, 48h, yield 70%; (i) TFA, DCM, room temperature, 3h, yield 100%; (j) HBTU, DIPEA, DCM, room temperature 6h, yield 65-75%.

[0021] In some embodiments, the synthetic routes of compounds 20 to 39 are as follows:

[0022]

[0023] Among them, the corresponding relationship between the structures of compounds 20 and 22 to 32 and R is shown in the following table.

[0024]

[0025] Specifically, compound 20 is obtained by amide condensation of compound 14 and glycine methyl ester; compound 21 is obtained by hydrolysis of compound 20; and target compounds 22-32 are obtained by amide condensation of compound 21 and different amine derivatives.

[0026] More specifically, in the synthetic routes of compounds 20-39: (a) HATU, DIPEA, THF, room temperature, overnight, yield 100%. (b) LiOH, THF, H 2 O, room temperature, 5h, yield 100%; (c) HATU, DIPEA, THF, room temperature, overnight, yield 85%. (d). TFA, DCM, room temperature, yield 100%

[0027] Specifically, the synthetic routes of compounds 48 to 52 are as follows:

[0028]

[0029] Among them, the corresponding relationship between the compound structures of compounds 48-52 and R is shown in the following table.

[0030]

[0031] Specifically, compound 40a-b is reacted with cyclopropylamine through reductive amination reaction to generate compound 42a-b; compound 10 and compound 42a-b are condensed through CDI to generate compound 43a-b; compound 43a-b is obtained by Suzuki coupling reaction of compound 42a-b and raw material 12; compound 445a-b is obtained by removing the tert-butyl group of compound 44a-b; compound 46a-b is obtained by amide condensation of compound 45a-b and glycine; compound 47a-b is obtained by hydrolysis reaction of compound 46a-b; target compound 48-52 is obtained by amide condensation of compound 47a-b and an amine derivative.

[0032] More specifically, in the synthetic routes of compounds 48-52: (a) cyclopropylamine, MeOH, room temperature, overnight; (b) NaBH 4 , MeOH, rt, 2h, room temperature, 2h, two steps, yield 80%; (c) CDI, DIPEA, THF, room temperature, overnight, yield 60%; (d) K 3 PO 4 ,Pd(dppf)Cl 2 , 1,2-dioxane / EtOH / H 2 O, 80°C, 48h, yield 70%; (e) TFA, DCM, rt, 3h, yield 100%;

[0033] (f) HATU, DIPEA, THF, room temperature, overnight, yield 100%. (g) LiOH, THF, H 2 O, room temperature, 5h, yield 100%;

[0034] (h) HATU, DIPEA, THF, room temperature, overnight, yield 85%; (i). TFA, DCM, room temperature, yield 100%

[0035] A third aspect provides a pharmaceutical composition comprising the above-mentioned small molecule inhibitor or a pharmaceutically acceptable salt thereof.

[0036] The pharmaceutically acceptable salts described in the present invention include sulfates, hydrochlorides, benzenesulfonates, citrates and the like.

[0037] In a fourth aspect, a pharmaceutical preparation comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the above-mentioned small molecule inhibitor or pharmaceutical composition.

[0038] The dosage form of the pharmaceutical preparation of the present invention can be tablets, capsules, granules, injections, etc.

[0039] The pharmaceutically acceptable excipients described in the present invention include excipients and / or carriers.

[0040] The excipients of the present invention include, but are not limited to, binders, penetration enhancers, disintegrants, plasticizers, preservatives, chelating agents, viscosity enhancers, wetting agents, fillers, emulsifiers, and the like.

[0041] The carrier of the present invention includes but is not limited to aluminum stearate, lecithin, glycerol, serum protein and the like.

[0042] A fifth aspect provides a use of the above-mentioned small molecule inhibitor, pharmaceutical composition or pharmaceutical preparation in the preparation of a β-catenin protein target gene inhibitor drug and / or a target gene Axin inhibitor drug.

[0043] In a sixth aspect, a use of the above-mentioned small molecule inhibitor, pharmaceutical composition or pharmaceutical preparation in the preparation of an anticancer drug. The anticancer drug is used to treat colorectal cancer.

[0044] The beneficial effects of the present invention are:

[0045] Studies have shown that the small molecule inhibitor provided by the present invention has good competitive affinity for β-catenin protein. In order to increase the biological activity, an amide bond was introduced at the right end of the parent nucleus as a backbone-derived linker. The results showed that when an amide bond was introduced at the right end of the parent nucleus In particular At the same time, the present invention has shown through experiments on the inhibition of the expression of the target gene Axin that when the right end of the mother nucleus is introduced In particular When the target gene Axin of the Wnt signaling pathway is inhibited, the inhibitory effect is good. In addition, molecular docking studies show that the compound 26 provided by the present invention can form more hydrogen bond interactions with the β-catenin protein, thereby enhancing its competitive affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0047] Figure 1 This is a diagram of the molecular docking simulation results of compound 22 and ZW4864 in the examples of the present invention (PDB: 2GL7), A is ZW4864, and B is compound 22. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0049] Example

[0050] 1. Synthesis of compounds:

[0051] Preparation of N-(4-bromobenzyl)cyclopropylamine (Compound 3):

[0052]

[0053] Dissolve 4-bromobenzaldehyde (10 g, 54 mmol, 1 eq) and cyclopropylamine (18.5 g, 324 mmol, 6 eq) in 100 ml methanol and add to a 250 ml three-necked flask. Protect the reaction apparatus with nitrogen and stir overnight at room temperature. Then lower the temperature to 0 °C and add sodium borohydride (4.1 g, 108 mmol, 2 eq) to keep the temperature below 10 °C. After stirring the reaction for 2 h, add saturated ammonium chloride solution to quench the reaction and monitor the reaction using a TLC plate (petroleum ether: ethyl acetate = 10:1). After the reaction was basically completed, the solvent was removed under vacuum, water and ethyl acetate (100 ml × 3 each) were added to the residue, extracted three times, the organic phases were combined, washed three times with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column chromatography (V (petroleum ether): V (ethyl acetate) = 20:1 as the developing solvent, 2.3 L) to obtain the target compound 3 (milky white oil, 9.78 g, yield 82.21%). 1 H NMR (400MHz, DMSO-d6) δ7.47–7.41(m,2H),7.21(dt,J=7.9,1.1Hz,2H),4.12(dt,J=6.2,3.9Hz,1H),3.93(ddt,J=13.9,4.0,1.1Hz,1H ), 3.86(ddt,J=13.9,3.8,0.9Hz,1H),2.52(dp,J=6.2,4.3Hz,1H),0.65(tddd,J=11.7,5.7,3.2,1.5Hz,4H).MS(ESI)[M+H]+:225.42.

[0054] Preparation of (4-bromobenzyl)(cyclopropyl)carbamoyl chloride (Compound 4):

[0055]

[0056] Compound 3 (2 g, 1 eq) and triphosgene (1.31 g, 0.5 eq) were dissolved in 40 ml of tetrahydrofuran and added to a 100 ml three-necked flask. DIPEA (0.56 g, 4 eq) was added to the reaction flask and stirred overnight at room temperature. After the reaction was completed, the solvent was dried by spin drying to directly obtain compound 4 (brown oily solid, 2.55 g, yield 100%).1 H NMR (400MHz, DMSO-d6) δ7.52–7.46(m,2H),7.23(dt,J=8.2,1.0Hz,2H),4.47(dt,J=13.0,0.9Hz,1H ),4.38(dt,J=13.0,0.9Hz,1H),3.30(p,J=6.0Hz,1H),0.75–0.56(m,4H).MS(ESI)[M+H]+:287.42.

[0057] Preparation of tert-butyl 2-(3-bromophenoxy)-2-methylpropanoate (Compound 7):

[0058]

[0059] Dissolve 3-bromophenol (compound 5, 1.0 g, 4.48 mmol, 1 eq), tert-butyl bromide (compound 6, 1.55 g, 8.96 mmol, 2 eq), potassium carbonate (2.47 g, 17.9 mmol, 4 eq), and magnesium sulfate (0.54 g, 4.48 mmol, 1 eq) in 50 ml of acetonitrile and add to a 100 ml three-necked flask. Stir and reflux at 80 °C overnight, and monitor the reaction using a TLC plate (petroleum ether:

[0060] Ethyl acetate = 10:1), the solvent was removed under reduced pressure, extracted three times with water and ethyl acetate (100 ml × 3 each), the organic phases were combined and washed with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated the filtrate, and purified by silica gel column chromatography (V (petroleum ether): V (ethyl acetate) = 40:1 as the developing solvent, 0.7 L) to obtain the target compound 7 (colorless transparent oily liquid, 1.27 g, yield 95.62%). 1 H NMR (400MHz, DMSO-d6) δ7.30 (ddd, J=8.0, 2.2, 1.3Hz, 1H), 7.25 (dd, J=8.1, 7.0Hz, 1H), 7.0 7(t,J=2.2Hz,1H),6.88(ddd,J=7.0,2.3,1.4Hz,1H),1.43(s,7H).MS(ESI)[M+H]+:314.65.

[0061] Preparation of tert-butyl (S)-2-(3-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)phenoxy)-2-methylpropanoate (Compound 9):

[0062]

[0063] Compound 7 (3 g, 41.4 mmol, 3 eq), compound 8 (2 g, 13.8 mmol, 1 eq), cesium carbonate (13.46 g, 441.4 mmol, 3 eq), Pd 2 (dbu) 3 1.26g, 1.4mmol.0.1eq), Ruphos (1.28g, 27.6mmol, 0.2eq) were dissolved in 60ml toluene and added to a 100ml three-necked flask. The mixed solvent was reacted overnight at 80°C under nitrogen protection. After thin layer chromatography monitoring (petroleum ether: ethyl acetate = 3:1), after the reaction was basically completed, the filtrate was concentrated by suction and the solvent was removed under reduced pressure, and directly purified by silica gel column chromatography (V (petroleum ether): V (ethyl acetate) = 10:1 as the developing solvent, 3.2L), to obtain the target compound 9 (2.28g, milky white oil, yield 77.21%).

[0064] Preparation of (S)-tert-butyl 2-(3-(3-aminopiperidin-1-yl)phenoxy)-2-methylpropanoate (Compound 10):

[0065]

[0066] Compound 9 (1 g, 2.23 mmol, 1 eq) was dissolved in 20 ml of DCM and added to a 50 ml three-necked flask. 3 ml of TFA was added to the reaction solution, and the reaction was carried out at room temperature for 3-4 hours, and then monitored by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). After the reaction was basically completed, the solvent dichloromethane was dried, 100 ml of water was added to the dried product, and saturated Na 2 CO 3 After the pH of the aqueous solution was adjusted to 3-4, the aqueous phase was extracted three times with dichloromethane (100 ml×3), and the organic phase was washed three times with saturated NaCl aqueous solution (100 ml×3). After the solvent dichloromethane was dried, compound 10 (0.6 g, reddish brown oily liquid, yield 63.72%) was obtained. 1H NMR(400MHz, DMSO-d6)δ7.15(t,J=7.3Hz,1H),6.65(ddd,J=7.3,2.2,1.1Hz,1H),6.49( ddd,J=7.3,2.3,1.2Hz,1H),6.44(t,J=2.2Hz,1H),3.43–3.27(m,4H),3.03(tdddd,J=7 .1,5.7,4.2,2.9,1.5Hz,1H),2.41(t,J=7.0Hz,1H),2.26(t,J=7.0Hz,1H),1.88(ddt,J =11.8,8.8,5.8Hz,1H),1.83–1.64(m,2H),1.61–1.52(m,6H).MS(ESI)[M+H]+:334.26.

[0067] Preparation of (S)-tert-butyl 2-(3-(3-(4-bromobenzyl)-3-cyclopropylurea)piperidin-1-yl)phenoxy)-2-methylpropanoate (Compound 13):

[0068]

[0069] Compound 10 (0.87 g, 1 eq) was dissolved in 50 ml tetrahydrofuran, and then compound 4 (0.90 g, 1.2 eq) and DIPEA (1.34 g, 4 eq) were added. The mixture was added to a 100 ml three-necked flask, and the reaction was stirred at 50 °C overnight. After TLC monitoring (petroleum ether: ethyl acetate = 1:1), the solvent was removed under reduced pressure, and water and DCM (100 ml × 3 each) were added to extract three times, and the organic phase was washed three times with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, and the filtrate was concentrated by suction and concentrated under vacuum. The target compound 11 (0.91 g, white solid, yield 61.54%) was purified by silica gel column chromatography (V (petroleum ether): V (ethyl acetate) = 3:1 as the developing solvent, 1 L). 1H NMR (400MHz, DMSO-d6) δ7.46–7.40(m,2H),7.24(dt,J=8.0,1.0Hz,2H),7.15(t,J=7.3Hz,1H),6.65(ddd,J=7.3,2.2,1.1Hz,1H) ,6.49(ddd,J=7.3,2.2,1.1Hz,1H),6.44(t,J=2.2Hz,1H),5.89(t,J=5.2Hz,1H),5.57(d,J=9.3Hz,1H),4.31(dq,J=5.2,0.9Hz,2 H),3.85–3.76(m,1H),3.49–3.39(m,2H),3.38(ddd,J=12.4,5.9,3.5Hz,1H),3.30(ddd,J=12.4,5.9,3.5Hz,1H),1.93(ddt,J=12 .1,8.8,6.1Hz,1H),1.88–1.78(m,1H),1.78–1.68(m,1H),1.68–1.58(m,1H),1.54(s,2H),1.43(s,7H).MS(ESI)[M+H]+:545.30.

[0070] Preparation of (S)-2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureido)piperidin-1-yl)phenoxy)-2-methylpropanoate tert-butyl ester (Compound 13):

[0071]

[0072] Compound 11 (0.2 g, 1 eq), boric acid pyranoside (compound 12, 0.1 g, 1.2 eq), Pd(dppf)Cl 2 (0.021 g, 0.1 eq), K 3 PO 4 (0.177 g, 3 eq) was dissolved in dioxane: ethanol: water (16 ml, V = 5:2:1) and added to a 50 ml three-necked flask. The mixed solvent was heated at 80 °C, N 2 The reaction was allowed to proceed overnight under protection.

[0073] (petroleum ether: ethyl acetate = 1:1), the filtrate was concentrated by suction and the solvent was removed under reduced pressure, and then directly purified by silica gel column chromatography

[0074] (V (petroleum ether): V (ethyl acetate) = 3:1 as developing solvent, 0.3 L) to obtain the target compound 13 (white sticky solid, 0.12 g, yield 63.85%). 1H NMR (400MHz, DMSO-d6) δ7.88–7.82(m,2H),7.51(dd,J=3.0,2.1Hz,1H),7.37(dt,J=8.9,1.0Hz,2H),7.15(d,J=14.5Hz,0H),6.65(ddd,J=7.3 ,2.2,1.1Hz,1H),6.61(d,J=2.2Hz,1H),6.49(ddd,J=7.3,2.2,1.1Hz,1H),6.44(t,J=2.2Hz,1H),5.85(t,J=5.1Hz,1H),5.57(d,J=9.3Hz,1H) ,4.31(dt,J=5.2,1.1Hz,2H),3.85–3.76(m,1H),3.49–3.39(m,2H),3.38(ddd,J=12.4,5.9,3.5Hz,1H),3.30(ddd,J=12.4,5.9,3.5Hz,1H),1. 93(ddt,J=12.1,8.8,6.1Hz,1H),1.88–1.78(m,1H),1.78–1.68(m,1H),1.68–1.58(m,1H),1.54(s,2H),1.43(s,6H).MS(ESI)[M+H]+:533.35.

[0075] Preparation of (S)-2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylurea)piperidin-1-yl)phenoxy)-2-methylpropanoic acid (Compound 14):

[0076]

[0077] Compound 13 (0.45 g, 1 eq) was dissolved in 10 ml of dichloromethane and added to a 50 ml three-necked flask and stirred. Then, the same volume of 10 ml of trifluoroacetic acid was added dropwise to the reaction flask and stirred at room temperature for 6 h. After monitoring the reaction with a thin layer chromatography plate (petroleum ether: ethyl acetate = 1:1), the solvent was concentrated and the trifluoroacetic acid was removed under vacuum to obtain compound 14 (white sticky solid, 0.42 g, yield 100%). 1H NMR (400MHz, DMSO-d6) δ7.88–7.82(m,2H),7.51(dd,J=3.0,2.1Hz,1H),7.37(dt,J=8.9,1.0Hz,2H),7.15(d,J=14.5Hz,0H),6.65(ddd ,J=7.3,2.2,1.1Hz,1H),6.61(d,J=2.2Hz,1H),6.49(ddd,J=7.3,2.2,1.1Hz,1H),6.44(t,J=2.2Hz,1H),5.85(t,J=5.1Hz,1H),5.57( d,J=9.3Hz,1H),4.31(dt,J=5.2,1.1Hz,2H),3.85–3.76(m,1H),3.49–3.39(m,2H),3.38(ddd,J=12.4,5.9,3.5Hz,1H),3.30(ddd,J=1 2.4,5.9,3.5Hz,1H),1.93(ddt,J=12.1,8.8,6.1Hz,1H),1.88–1.68(m,2H),1.68–1.58(m,1H),1.51(s,2H).MS(ESI)[M+H]+:477.50.

[0078] Preparation of compound 15-19:

[0079]

[0080] Compound 14 (1.44 g, 1 eq) was dissolved in dichloromethane. HBTU (1.37 g, 2 eq) and piperazine were added.

[0081] (0.24g, 1eq), and added to a 50ml three-necked flask with stirring, and then gradually added DIPEA (1.87g, 4eq). Stir the reaction at room temperature for 6h, and monitor the reaction on a thin layer chromatography plate (petroleum ether: ethyl acetate = 1:2). After the reaction is basically completed, add water and dichloromethane to extract 3 times (100ml×3 each), then combine the organic phases, wash with saturated brine three times (100ml×3), dry with anhydrous sodium sulfate, filter and concentrate the filtrate, and vacuum reduce the solvent, and purify with silica gel column chromatography (V (petroleum ether): V (ethyl acetate) = 1:3 as the developing solvent, 1.2L), to obtain the target compound 15 (white sticky solid, 1.64g, yield 82.76%).

[0082] 1 H NMR (400 MHz, DMSO-d 6)δ8.99(s,1H),8.02(s,2H),7.55(d,J=7.8Hz,2H),7.20(d,J=7.9Hz,2H),7.11(t,J=8.2Hz,1H),6.61(dd,J=8.3,2.3Hz,1H),6. 43–6.34(m,1H),6.19(dd,J=8.1,2.3Hz,1H),6.05(d,J=7.8Hz,1H),4.47(d,J=15.4Hz,1H),4.40(d,J=15.4Hz,1H),3.97(s,2H) ,3.78–3.61(m,3H),3.54(dd,J=12.0,3.7Hz,1H),3.45–3.37(m,1H),2.92–2.68(m,4H),2.34(dq,J=6.7,3.4Hz,1H),1.89–1.79 (m,1H),1.72(dq,J=11.8,7.1,6.4Hz,1H),1.55(s,7H),1.42(s,1H),0.75(dt,J=6.9,3.4Hz,2H),0.64(p,J=3.7Hz,2H).MS(ESI)

[0083] [M+H]+:585.62.

[0084] Compound 15:

[0085] The above raw material piperazine was replaced by ethylenediamine for reaction. The synthesis and purification methods were based on compound 15, and the target compound 16 was obtained as a white solid with a yield of 80.31%. 1 H NMR (400 MHz, Methanol-d 4)δ8.50(t,J=5.8Hz,1H),7.95(s,2H),7.58–7.51(m,2H),7.30–7.24(m,2H),7.17(t,J=8.2Hz,1H),6.78(dd,J =7.9,2.3Hz,1H),6.65(t,J=2.3Hz,1H),6.47(dd,J=7.8,2.2Hz,1H),4.56(s,2H),3.98(dt,J=8.1,4.2Hz,1H) ,3.52(p,J=6.3Hz,3H),3.11–2.96(m,4H),2.44(tt,J=6.7,3.8Hz,1H),1.98–1.85(m,2H),1.80–1.65(m,2H), 1.52(d,J=1.8Hz,6H),1.32(s,1H),0.84(dt,J=10.0,3.4Hz,2H),0.79–0.72(m,2H).MS(ESI)[M+H]+:559.67.

[0086] Compound 16:

[0087] The above raw material piperazine was replaced by 1,3-propylenediamine and the reaction was carried out. The synthesis and purification methods were based on compound 15, and the target compound 17 was obtained as a white solid with a yield of 72.88%. 1 H NMR (400 MHz, DMSO-d 6 )δ12.95(s,1H),8.26(t,J=6.0Hz,1H),7.92(s,2H),7.61–7.52(m,2H),7.19(d,J=8.0Hz,2H),7.09(t,J=8.2Hz,1H),6.62(dd,J=8.3,2. 3Hz,1H),6.45(t,J=2.3Hz,1H),6.25(dd,J=8.0,2.2Hz,1H),6.07(d,J=7.8Hz,1H),4.43(q,J=15.4Hz,2H),3.75(t,J=6.7Hz,1H),3.52( dd,J=11.8,3.7Hz,1H),3.39(d,J=12.4Hz,2H),3.17(q,J=6.5Hz,2H),2.89–2.67(m,4H),2.33(dq,J=6.8,3.5Hz,1H),1.86–1.79(m,1H) ,1.72(p,J=6.9Hz,3H),1.58(t,J=8.5Hz,2H),1.42(s,6H),0.75(dq,J=6.9,3.6Hz,2H),0.64(p,J=3.8Hz,2H).MS(ESI)[M+H]+:573.86.

[0088] Compound 17:

[0089]

[0090] The above raw material piperazine was replaced by morpholine for reaction. The synthesis and purification methods were based on compound 15, and the target compound 18 was obtained as a white solid with a yield of 55.41%. 1 H NMR (400 MHz, DMSO-d 6 )δ12.87(s,1H),8.02(s,1H),7.55(d,J=7.9Hz,2H),7.19(d,J=7.9Hz,2H),7.09(t,J=8.2Hz,1H),6.59(dd,J=8.3,2.2Hz,1H),6. 36(t,J=2.3Hz,1H),6.20(dd,J=8.1,2.3Hz,1H),6.03(d,J=7.9Hz,1H),4.46(d,J=15.4Hz,1H),4.39(d,J=15.3Hz,1H),3.86–3.65 (m,3H),3.57–3.39(m,5H),3.19(s,2H),2.82(ddd,J=20.5,12.0,8.8Hz,2H),2.32(dq,J=6.8,3.4Hz,1H),1.83(s,1H),1.77–1.69 (m,1H),1.64–1.55(m,2H),1.53(s,6H),1.24(s,1H),0.74(dt,J=6.9,3.5Hz,2H),0.63(p,J=3.6Hz,2H).MS(ESI)[M+H]+:586.84.

[0091] Compound 18:

[0092]

[0093] The raw material piperazine was replaced by 2-hydroxyethylamine for reaction. The synthesis and purification methods were similar to those of compound 15, and the target compound 19 was obtained as a white solid with a yield of 86.15%. NMR (400MHz, DMSO-d6) δ8.02(s,1H),7.95(t,J=5.7Hz,1H),7.55(d,J=7.9Hz,2H),7.19(d,J=8.0Hz,2H),7.07(t,J=8.1Hz,1H),6.62(dd,J= 8.3, 2.3Hz, 1H), 6.48 (d, J=2.3Hz, 1H), 6.29 (dd, J=8.0, 2.2Hz, 1H), 6.06 (d, J=7.8Hz, 1H), 4.75 (s, 1H), 4.50–4.35 (m, 2H), 3.77 (tt, J=8.7, 4.5Hz,1H),3.55–3.45(m,2H),3.41(d,J=12.9Hz,3H),3.20(q,J=6.2Hz,2H),2.95–2.74(m,3H),2.68(s,2H),2.33(td,J=6.7,3.4Hz,1H),1 .87–1.66(m,2H),1.65–1.46(m,3H),1.40(s,6H),1.23(s,2H),0.74(dq,J=6.6,3.9Hz,2H),0.63(p,J=4.2Hz,2H).MS(ESI)[M+H]+:560.55.

[0094] Compound 19:

[0095] Preparation of (S)-(2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureido)piperidin-1-yl)phenoxy)-2-methylpropanoyl)glycine methyl ester (Compound 20):

[0096]

[0097] Compound 14 (0.33 g, 1 eq) was dissolved in 30 ml tetrahydrofuran. HATU (0.24 g, 1 eq) and DIPEA (0.64 g, 8 eq) were added to the mixture and added to a 50 ml three-necked flask. After stirring for 20 min, glycine methyl ester (0.09 g, 1.2 eq) was added to the solvent. The mixture was stirred at room temperature overnight. After the reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 1:1), the solvent was dried after the reaction was complete. Water and DCM were added for extraction 3 times (100 ml × 3). The organic phases were then combined, washed with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated, and the filtrate was concentrated under vacuum to obtain compound 20 (viscous brown-yellow liquid, 0.34 g, yield 94.58%). 1 H NMR (400MHz, DMSO-d6) δ7.89–7.82(m,3H),7.51(dd,J=3.0,2.1Hz,1H),7.37(dt,J=8.9,1.0Hz,2H),7.15(d,J=14.6Hz,0H),6.66(ddd,J=7.3,2. 2,1.1Hz,1H),6.61(d,J=2.2Hz,1H),6.49(ddd,J=7.3,2.2,1.1Hz,1H),6.44(t,J=2.2Hz,1H),5.85(t,J=5.1Hz,1H),5.57(d,J=9.3Hz,1H),4.31 (dt,J=5.2,1.1Hz,2H),3.93(dd,J=5.6,1.7Hz,2H),3.85–3.76(m,1H),3.69(s,2H),3.49–3.39(m,2H),3.38(ddd,J=12.4,5.9,3.5Hz,1H),3.30 (ddd,J=12.4,5.9,3.5Hz,1H),1.93(ddt,J=12.1,8.8,6.1Hz,1H),1.88–1.78(m,1H),1.78–1.68(m,1H),1.68–1.58(m,1H),1.47(s,2H).MS(ESI)

[0098] [M+H]+:548.92.

[0099] Preparation of (S)-(2-(3-(3-(4-(1H-pyrazol-4-yl)benzyl)-3-cyclopropylureido)piperidin-1-yl)phenoxy)-2-methylpropanoyl)glycine (Compound 21):

[0100]

[0101] Saturated lithium hydroxide (0.15g, 2eq) aqueous solution (2ml) was added to a solution of compound 20 (1g, 1eq) and tetrahydrofuran (8ml), and then added to a 50ml three-necked flask. The mixture was stirred at room temperature for 3h. After monitoring the reaction by thin layer chromatography (petroleum ether: ethyl acetate = 1:1), the solvent was removed by decompression. The residue was redissolved with 50ml of water, and then the pH of the aqueous solution was adjusted to 3-4 with 1mol / L hydrochloric acid, and then 50ml of ethyl acetate was added for extraction. The organic phase obtained after washing three times with saturated brine (50ml×3) was filtered and concentrated, and the solvent was removed by vacuum decompression to obtain compound 21 (yellow solid, 1.2g, yield 100%). 1 H NMR (400MHz, DMSO-d6) δ8.09(t,J=5.9Hz,1H),7.88–7.82(m,2H),7.51(dd,J=3.0,2.1Hz,1H),7.37(dt,J=8.9,1.0Hz,2H),7.15(d,J=14.6Hz,0 H),6.66(ddd,J=7.3,2.2,1.1Hz,1H),6.61(d,J=2.2Hz,1H),6.49(ddd,J=7.3,2.2,1.1Hz,1H),6.44(t,J=2.2Hz,1H),5.85(t,J=5.1Hz,1H),5.5 7(d,J=9.3Hz,1H),4.31(dt,J=5.2,1.0Hz,2H),4.00–3.87(m,2H),3.85 –3.76(m,1H),3.49–3.39(m,2H),3.38(ddd,J=12.4,5.9,3.5Hz,1H),3.3 0(ddd,J=12.4,5.9,3.5Hz,1H),1.93(ddt,J=12.1,8.8,6.1Hz,1H),1.88–1.68(m,2H),1.68–1.58(m,1H),1.47(s,2H).MS(ESI)[M+H]+:534.55.

[0102] Preparation of compound 22-32:

[0103]

[0104] Compound 21 (0.33 g, 1 eq) was dissolved in 30 ml THF and added to a 50 ml three-necked flask. HATU (0.24 g, 1 eq) and DIPEA (0.64 g, 8 eq) were then added thereto. After stirring for 20 min, the raw material S-pyrrolidin-3-ol (1.2 eq) was added to the solvent. The mixture was stirred at room temperature overnight. After the reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:2), the solvent was dried after the reaction was complete, and water and dichloromethane were added for extraction 3 times (100 ml × 3 each). The organic phases were then combined, washed with saturated brine (100 ml × 3), dried over anhydrous sodium sulfate, filtered and concentrated, and the filtrate was concentrated under vacuum to obtain compound 22 (viscous brown-yellow liquid 0.34 g, yield 94.33%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.46(t,J=5.9Hz,1H),7.55(d,J=7.8Hz,2H),7.19(d,J=7.9Hz,2H),7.09(t,J=8.2Hz,1H),6.63(dd,J=8.2,2.3Hz,1H),6.53(t,J=2.3H z,1H),6.38(dd,J=8.0,2.2Hz,1H),6.03(d,J=7.8Hz,1H),4.43(d,J=11.5Hz,2H),3.86(d,J=5.9Hz,2H),3.76(td,J=7.9,3.7Hz,1H),3.6 2(s,3H),3.51(dd,J=12.0,3.7Hz,1H),3.38(d,J=13.2Hz,2H),2.85–2.80(m,1H),2.33(tt,J=6.9,3.7Hz,1H),1.82(d,J=11.4Hz,1H),1. 79–1.72(m,1H),1.60–1.56(m,1H),1.41(s,6H),1.26(s,1H),0.74(dq,J=6.6,3.5Hz,2H),0.64(p,J=3.9Hz,2H).MS(ESI)[M+H]+:588.96.

[0105] Compound 22:

[0106] The above raw material S-pyrrolidin-3-ol was replaced with compound (S)-3-(dimethylamino)pyrrolidine for reaction. The synthesis and purification methods were based on compound 22 to obtain the target product 23 as a viscous white liquid with a yield of 90.20%. 1 H NMR (400 MHz, DMSO-d 6)δ8.07–7.95(m,3H),7.55(d,J=8.2Hz,2H),7.19(d,J=7.9Hz,2H),7.11(t,J=8.2Hz,1H),6.69(d,J=8.4Hz,1H),6.63(s,1H), 6.46(d,J=8.1Hz,1H),6.06(d,J=7.8Hz,1H),4.48–4.32(m,3H),4.24(tt,J=4.2,2.6Hz,1H),3.90(dd,J=8.2,5.3Hz,2H),3.85 (d,J=5.3Hz,2H),3.49(d,J=4.8Hz,2H),3.36–3.24(m,3H),2.97–2.86(m,2H),2.33(tt,J=6.8,3.8Hz,1H),1.91–1.68(m,4H), 1.60(q,J=8.8,7.8Hz,2H),1.41(d,J=1.9Hz,6H),0.74(dq,J=6.6,3.8Hz,2H),0.64(p,J=4.0Hz,2H).MS(ESI)[M+H]+:643.44.

[0107] Compound 23:

[0108] The above raw material S-pyrrolidin-3-ol was replaced with (S)-2-(dimethylaminomethyl)pyrrolidine for reaction. The synthesis and purification methods were based on compound 22 to obtain the target product 24 as a sticky yellow-white solid with a yield of 80.24%. 1 H NMR (400 MHz, DMSO-d 6)δ8.04–7.96(m,2H),7.58–7.48(m,2H),7.19(d,J=8.0Hz,2H),7.08(t,J=8. 2Hz, 1H), 6.63 (dd, J=8.3, 2.3Hz, 1H), 6.55 (q, J=2.6Hz, 1H), 6.40 (ddd, J=8. 1,4.0,2.1Hz,1H),6.05(d,J=7.8Hz,1H),4.49–4.36(m,2H),3.95–3.81(m,2 H),3.76(dq,J=9.4,5.0Hz,1H),3.74–3.47(m,5H),3.41–3.34(m,5H),3.24–3 .10(m,2H),3.00(dd,J=11.3,8.1Hz,1H),2.92–2.77(m,2H),2.69(s,1H),2. 50–2.38(m,2H),2.32(td,J=6.6,3.4Hz,1H),2.17(d,J=3.3Hz,7H),2.08–1.9 4(m,2H),1.86–1.69(m,3H),1.57(t,J=9.1Hz,3H),1.41(s,7H),1.24(s,1H) ,0.73(dd,J=6.9,4.1Hz,2H),0.63(p,J=4.2Hz,2H).MS(ESI)[M+H]+:670.56.

[0109] Compound 24:

[0110] The above raw material S-pyrrolidin-3-ol was replaced with L-prolinol for reaction. The synthesis and purification methods were based on compound 22, and the target product 25 was obtained as a yellow-white solid with a yield of 83.42%. 1 H NMR (400 MHz, DMSO-d 6)δ8.04(s,3H),7.55(d,J=8.1Hz,2H),7.19(d,J=7.9Hz,3H),6.96–6.73(m,2H),6.58(d,J=8.1Hz,1H),6.17(d,J=7.8Hz,1H),4 .43(d,J=2.7Hz,2H),4.33(tt,J=8.5,4.3Hz,1H),4.04–3.85(m,3H),3.55(dd,J=11.8,3.8Hz,1H),3.46(t,J=6.1Hz,3H),3.18( dt,J=11.8,5.7Hz,1H),3.12–2.94(m,3H),2.87(d,J=4.5Hz,3H),2.80(d,J=4.7Hz,3H),2.35(tt,J=6.7,3.7Hz,1H),1.98–1.8 0(m,6H),1.75–1.55(m,2H),1.44(d,J=1.9Hz,6H),0.76(dt,J=6.5,3.1Hz,2H),0.64(p,J=4.3Hz,2H).MS(ESI)[M+H]+:684.82.

[0111] Compound 25:

[0112] The above-mentioned raw material S-pyrrolidin-3-ol was replaced with S-2-Boc-aminomethylpyrrolidine for reaction. The synthesis and purification methods were based on compound 22, and intermediate 33 was obtained as a sticky brown solid with a yield of 94.55%. Compound 33 was dissolved in 10 ml of methanol, and 2 ml of 4M hydrochloric acid and dioxane solution was added. The mixture was stirred at room temperature for 6 h. After the reaction was completed as monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a white solid, namely compound 26, with a yield of 100%. 1 H NMR (400 MHz, DMSO-d 6)δ8.02(d,J=9.5Hz,3H),7.83(s,1H),7.55(d,J=8.1Hz,2H),7.19(d,J=8.0Hz,2H ),7.12(t,J=8.2Hz,1H),6.71(dd,J=8.3,2.3Hz,1H),6.63(t,J=2.3Hz,1H),6.46( dd,J=8.1,2.1Hz,1H),6.09(d,J=7.7Hz,1H),4.47(d,J=15.4Hz,1H),4.39(d,J=15 .4Hz,1H),4.15(dq,J=7.3,4.1,3.6Hz,1H),3.96(dd,J=16.9,5.3Hz,1H),3.89(dd ,J=16.9,5.3Hz,1H),3.83–3.76(m,1H),3.55(dd,J=12.0,3.7Hz,1H),3.43(td,J =9.0,7.9,4.1Hz,3H),3.00–2.83(m,4H),2.33(td,J=6.7,3.5Hz,1H),1.97–1.81( m,4H),1.78(dt,J=12.0,5.2Hz,2H),1.60(q,J=10.0,9.5Hz,2H),1.42(d,J=5.0Hz ,6H),0.75(dq,J=6.7,4.1Hz,2H),0.64(p,J=4.1Hz,2H).MS(ESI)[M+H]+:656.76.

[0113] Compound 26:

[0114] The above raw material S-pyrrolidin-3-ol was replaced with R-2-Boc-aminomethylpyrrolidine for reaction. The synthesis and purification methods were based on compound 26 to obtain the target product 27 as a white solid with a yield of 82.34%. 1 H NMR (400 MHz, DMSO-d 6)δ8.03(s,2H),8.03–7.94(m,2H),7.88(s,2H),7.55(d,J=7.9Hz,2H),7. 19(d,J=8.0Hz,2H),7.14(td,J=8.2,1.5Hz,1H),6.73(dd,J=8.3,2.2Hz,1 H),6.65(dt,J=12.3,2.3Hz,1H),6.49(dt,J=8.1,2.7Hz,1H),6.10(t,J= 8.6Hz,1H),4.41(t,J=14.8Hz,2H),4.16(qd,J=6.6,3.1Hz,1H),4.02–3.8 3(m,2H),3.81(dt,J=8.7,4.6Hz,1H),3.54(dt,J=11.4,4.7Hz,1H),3.44 (tdt,J=13.1,8.6,4.1Hz,3H),3.08–2.79(m,4H),2.34(tt,J=6.9,3.8Hz, 1H),1.96–1.70(m,6H),1.61(q,J=10.5Hz,2H),1.43(q,J=1.7Hz,6H),0. 75(dq,J=6.6,3.9Hz,2H),0.64(p,J=4.1Hz,2H).MS(ESI)[M+H]+:656.69.

[0115] Compound 27:

[0116] The above raw material S-pyrrolidin-3-ol was replaced with S-3-N-Boc-aminomethylpyrrolidine hydrochloride for reaction. The synthesis and purification methods were based on compound 26 to obtain the target product 28 as a sticky white solid with a yield of 86.71%. 1 H NMR (400 MHz, DMSO-d 6)δ8.04(s,2H),7.99–7.95(m,2H),7.55(d,J=8.0Hz,2H),7.19(d,J=8.0Hz,2H),7.15(dd,J=8.2,4.0Hz,1H),6.80–6.70(m,2H),6.55(d,J=8. 1Hz,1H),6.11(dd,J=7.9,2.2Hz,1H),4.46(d,J=15.4Hz,1H),4.39(d,J=15.4Hz,1H),3.90(t,J=5.2Hz,2H),3.88–3.81(m,1H),3.65–3.55(m ,2H),3.54(d,J=4.4Hz,1H),3.52–3.42(m,2H),3.25(ddt,J=17.4,7.2,4.8Hz,1H),3.10–2.92(m,3H),2.88(t,J=6.5Hz,2H),2.43–2.26(m,2 H),2.08(dq,J=11.4,6.4Hz,1H),1.90–1.55(m,5H),1.43(s,6H),0.74(dd,J=6.9,4.4Hz,2H),0.64(p,J=4.2Hz,2H).MS(ESI)[M+H]+:656.33.

[0117] Compound 28:

[0118] The above raw material S-pyrrolidin-3-ol was replaced with R-3-N-Boc-aminomethylpyrrolidine hydrochloride for reaction. The synthesis and purification methods were based on compound 26 to obtain the target product 29 as a viscous brown-yellow liquid with a yield of 88.13%. 1 H NMR (400 MHz, DMSO-d 6)δ8.04(s,2H),7.99–7.95(m,2H),7.55(d,J=8.0Hz,2H),7.19(d,J=8.0Hz,2H),7.15(dd,J=8.2,4.0Hz,1H),6.80–6.70(m,2H),6.55(d,J=8. 1Hz,1H),6.11(dd,J=7.9,2.2Hz,1H),4.46(d,J=15.4Hz,1H),4.39(d,J=15.4Hz,1H),3.90(t,J=5.2Hz,2H),3.88–3.81(m,1H),3.65–3.55(m ,2H),3.54(d,J=4.4Hz,1H),3.52–3.42(m,2H),3.25(ddt,J=17.4,7.2,4.8Hz,1H),3.10–2.92(m,3H),2.88(t,J=6.5Hz,2H),2.43–2.26(m,2 H),2.08(dq,J=11.4,6.4Hz,1H),1.90–1.55(m,5H),1.43(s,6H),0.74(dd,J=6.9,4.4Hz,2H),0.64(p,J=4.2Hz,2H).MS(ESI)[M+H]+:656.59.

[0119] Compound 29:

[0120] The above raw material S-pyrrolidin-3-ol was replaced with S-2-BOC-aminomethyl-piperidine for reaction. The synthesis and purification methods were based on compound 26 to obtain the target product 30 as a viscous white liquid with a yield of 86.11%. 1 H NMR (400 MHz, DMSO-d 6)δ8.01(d,J=12.5Hz,3H),7.87(s,2H),7.55(d,J=7.9Hz,2H),7.19(d,J= 7.8Hz,2H),7.15–7.07(m,1H),6.73–6.58(m,2H),6.46(dd,J=8.1,2.2Hz, 1H),6.07(d,J=7.8Hz,1H),4.44(s,2H),3.88(d,J=5.4Hz,2H),3.79(s,1H ),3.56(ddt,J=16.9,12.4,5.2Hz,3H),3.41(d,J=10.0Hz,2H),3.25(ddd, J=17.3,12.9,7.2Hz,1H),3.05(dd,J=11.8,7.6Hz,1H),2.88(td,J=7.6, 3.7Hz,4H),2.33(td,J=6.6,2.9Hz,2H),2.13–2.01(m,1H),1.97(dt,J=12 .6,6.2Hz,1H),1.87–1.67(m,3H),1.67–1.53(m,3H),1.42(s,6H),0.74(d d, J=6.9, 4.2Hz, 2H), 0.64 (p, J=4.4, 4.0Hz, 2H). MS (ESI) [M+H]+: 656.81.

[0121] Compound 30:

[0122] The above raw material S-pyrrolidin-3-ol was replaced with R-2-BOC-aminomethyl-piperidine for reaction. The synthesis and purification methods were based on compound 26 to obtain the target product 31 as a white solid with a yield of 80.13%. 1 H NMR (400 MHz, DMSO-d 6)δ8.01(d,J=12.5Hz,3H),7.87(s,2H),7.55(d,J=7.9Hz,2H),7.19(d,J= 7.8Hz,2H),7.15–7.07(m,1H),6.73–6.58(m,2H),6.46(dd,J=8.1,2.2Hz, 1H),6.07(d,J=7.8Hz,1H),4.44(s,2H),3.88(d,J=5.4Hz,2H),3.79(s,1H ),3.56(ddt,J=16.9,12.4,5.2Hz,3H),3.41(d,J=10.0Hz,2H),3.25(ddd, J=17.3,12.9,7.2Hz,1H),3.05(dd,J=11.8,7.6Hz,1H),2.88(td,J=7.6, 3.7Hz,4H),2.33(td,J=6.6,2.9Hz,2H),2.13–2.01(m,1H),1.97(dt,J=12 .6,6.2Hz,1H),1.87–1.67(m,3H),1.67–1.53(m,3H),1.42(s,6H),0.74(d d, J=6.9, 4.2Hz, 2H), 0.64 (p, J=4.4, 4.0Hz, 2H). MS (ESI) [M+H]+: 670.67.

[0123] Compound 31:

[0124] The above raw material S-pyrrolidin-3-ol was replaced with tert-butyl 2-(2-pyrrolidinethyl)carbamate and the reaction was carried out. The synthesis and purification methods were based on compound 26 to obtain the target product 32 as a white solid with a yield of 80.19%. 1 H NMR (400 MHz, DMSO-d 6)δ8.26–8.19(m,2H),7.99(dd,J=3.4,1.7Hz,1H),7.61–7.52(m,2H),7.45–7.37(m,2H),7.16(t,J=7.3Hz,1H),6 .78(dddd,J=25.2,7.3,1.9,1.2Hz,2H),6.46(t,J=2.0Hz,1H),4.51–4.35(m,2H),4.01–3.72(m,4H),3.66–3.57( m,2H),3.51(d,J=2.2Hz,1H),3.44–3.19(m,4H),2.93–2.72(m,2H),1.95–1.70(m,11H),1.63(dq,J=13.8,5.6Hz, 1H),1.53(s,3H),1.48(s,3H),0.82(dd,J=5.7,5.0Hz,2H),0.64(dd,J=5.7,5.0Hz,2H).MS(ESI)[M+H]+:670.52.

[0125] Compound 32:

[0126] Preparation of compounds 48-52:

[0127] The synthesis and purification methods were similar to those of compound 22; compound 11 in the synthesis process was replaced by compound 43a, and the target compound 48 was a white solid with a yield of 54.67%. 1 H NMR (400 MHz, DMSO-d 6)δ8.51(dd,J=1.6,0.7Hz,1H),8.38(d,J=1.8Hz,1H),8.22(t,J=4.9Hz,1H),8.08(dd,J=3.8,1.8Hz,1H),7.77–7.68(m,2H),7.16(t ,J=7.3Hz,1H),6.82(ddd,J=7.3,2.3,1.2Hz,1H),6.75(ddd,J=7.3,2.2,1.2Hz,1H),6.46(t,J=2.2Hz,1H),6.16(d,J=9.1Hz,1H),4. 45(d,J=14.7Hz,1H),4.42–4.29(m,3H),3.93–3.78(m,3H),3.66–3.43(m,5H),3.43–3.35(m,1H),3.35–3.19(m,3H),2.06–1.96(m, 1H),1.93–1.70(m,5H),1.53(s,3H),1.48(s,3H),0.82(dd,J=5.7,5.0Hz,2H),0.64(dd,J=5.7,5.0Hz,2H).MS(ESI)[M+H]+:644.52.

[0128] Compound 48:

[0129] The synthesis and purification methods were based on compound 26; compound 11 was replaced by compound 43b, and the target compound 49 was a white solid with a yield of 81.22%. 1 H NMR (400 MHz, DMSO-d 6)δ8.59(d,J=2.2Hz,1H),8.35(d,J=1.8Hz,1H),8.22(t,J=5.3Hz,1H),8.04(dd,J=3.4,1.7Hz,1H),7.96(dd,J=8.3,2.3Hz,1H),7.39(d,J=8.2H z,1H),7.16(t,J=7.3Hz,1H),6.82(ddd,J=7.3,2.0,1.2Hz,1H),6.75(ddd,J=7.3,1.9,1.2Hz,1H),6.46(t,J=1.9Hz,1H),6.16(d,J=9.2Hz,1H), 4.66–4.51(m,2H),3.95–3.82(m,3H),3.77(dd,J=15.8,5.3Hz,1H),3.6 8–3.59(m,2H),3.51(d,J=2.2Hz,1H),3.44–3.28(m,4H),2.93–2.70(m, 3H),2.64(q,J=6.7Hz,1H),2.00–1.65(m,8H),1.53(s,3H),1.48(s,3H),0.82(dd,J=5.6,5.0Hz,2H),0.64(dd,J=5.6,5.0Hz,2H).MS(ESI)[M+H] + :657.52.

[0130] Compound 49:

[0131] The synthesis and purification methods were similar to those of compound 26; compound 11 was replaced by compound 43a, and the target compound 50 was a white solid with a yield of 32.41%. 1 H NMR (400 MHz, DMSO-d 6)δ8.51(dd,J=1.8,0.8Hz,1H),8.38(d,J=1.8Hz,1H),8.22(t,J=5.3Hz,1H), 8.08(dd,J=3.8,1.8Hz,1H),7.77–7.68(m,2H),7.16(t,J=7.3Hz,1H),6.82( ddd,J=7.3,2.0,1.2Hz,1H),6.75(ddd,J=7.3,1.9,1.2Hz,1H),6.46(t,J=1. 9Hz,1H),6.16(d,J=9.1Hz,1H),4.45(d,J=14.7Hz,1H),4.34(d,J=14.8Hz,1 H),3.95–3.82(m,3H),3.77(dd,J=15.8,5.3Hz,1H),3.68–3.59(m,2H),3.51 (d,J=2.2Hz,1H),3.44–3.28(m,3H),3.24(p,J=5.7Hz,1H),2.93–2.70(m,3H ),2.64(q,J=6.7Hz,1H),2.00–1.65(m,8H),1.53(s,3H),1.48(s,3H),0.82( dd,J=5.7,5.0Hz,2H),0.64(dd,J=5.7,5.0Hz,2H).MS(ESI)[M+H]+:657.52.

[0132] Compound 50:

[0133] The synthesis and purification methods were based on compound 26; compound 11 was replaced by compound 43b, and the target compound 51 was a white solid with a yield of 54.31%. 1 H NMR (400 MHz, DMSO-d 6)δ8.59(d,J=2.2Hz,1H),8.35(d,J=1.8Hz,1H),8.22(t,J=5.3Hz,1H),8.0 4(dd,J=3.4,1.7Hz,1H),7.96(dd,J=8.3,2.3Hz,1H),7.39(d,J=8.2Hz,1H) ,7.16(t,J=7.3Hz,1H),6.82(ddd,J=7.3,2.0,1.2Hz,1H),6.75(ddd,J=7.3 ,1.9,1.2Hz,1H),6.46(t,J=1.9Hz,1H),6.16(d,J=9.2Hz,1H),4.66–4.51( m,2H),3.94–3.85(m,2H),3.84(s,1H),3.77(dd,J=15.8,5.3Hz,1H),3.67 –3.58(m,2H),3.51(d,J=2.2Hz,1H),3.44–3.28(m,4H),2.93–2.70(m,3H), 2.64(q,J=6.7Hz,1H),1.98–1.69(m,8H),1.53(s,3H),1.48(s,3H),0.82(d d,J=5.6,5.0Hz,2H),0.64(dd,J=5.6,5.0Hz,2H).MS(ESI)[M+H]+:657.87.

[0134] Compound 51:

[0135] The synthesis and purification methods were based on compound 26; compound 11 was replaced by compound 43a, and the target compound 52 was a white solid with a yield of 87.99%. 1 H NMR (400 MHz, DMSO-d 6)δ8.51(dd,J=1.8,0.8Hz,1H),8.38(d,J=1.8Hz,1H),8.22(t,J=5.3Hz,1H), 8.08(dd,J=3.8,1.8Hz,1H),7.77–7.68(m,2H),7.16(t,J=7.3Hz,1H),6.82( ddd,J=7.3,2.0,1.2Hz,1H),6.75(ddd,J=7.3,1.9,1.2Hz,1H),6.46(t,J=1. 9Hz,1H),6.16(d,J=9.1Hz,1H),4.45(d,J=14.7Hz,1H),4.34(d,J=14.8Hz,1 H),3.94–3.82(m,3H),3.77(dd,J=15.8,5.3Hz,1H),3.67–3.58(m,2H),3.51 (d,J=2.2Hz,1H),3.44–3.28(m,3H),3.24(p,J=5.7Hz,1H),2.93–2.70(m,3H ),2.64(q,J=6.7Hz,1H),1.98–1.69(m,8H),1.53(s,3H),1.48(s,3H),0.82( dd,J=5.7,5.0Hz,2H),0.64(dd,J=5.7,5.0Hz,2H).MS(ESI)[M+H]+:657.65.

[0136] Compound 52:

[0137] The synthesis and purification methods of compound 43a refer to the preparation process of compound 3, and compound 1 in the preparation process of compound 3 replaces 2-bromo-5-formylpyridine to prepare compound 43a.

[0138] The synthesis and purification methods of compound 43b refer to the preparation process of compound 3, except that compound 1 in the preparation process of compound 3 is replaced by 5-bromo-2-pyridinecarboxaldehyde to prepare compound 43b.

[0139] 2. Pharmacological testing experiments:

[0140] Fluorescence polarization experiment:

[0141] Prepare fluorescence polarization (FP) buffer with 25mM HEPES, 100mM NaCl, 0.01% TritonX-100, 0.1% BSA, and add 180μl buffer and 20nM BCL9-FAM tracer to each well of a black bottom opaque 96-well plate. At the beginning of the experiment, 1μM β-catenin protein and different concentrations of compound were added to the wells, with a concentration gradient of 0.625, 1.25, 2.5, 5 and 10μM, and 3 replicates were tested under each concentration condition. The positive control wells representing 100% inhibition contained only tracer, while the negative control wells representing 0% inhibition contained tracer and β-catenin protein. Use a spray gun to gently blow all the wells to mix, and shake on a horizontal shaker at room temperature for 2h to allow the compound to fully contact the β-catenin protein and compete with the BCL9-FAM tracer for binding to the β-catenin protein. After the shaking, the fluorescence polarization analysis was performed immediately in the range of 428nm-528nm using an ELISA reader. The result data were analyzed using Graphpad software to draw the concentration gradient-competitive inhibition curves of different compounds, thereby screening compounds with excellent affinity and strong ability to competitively bind and destroy the β-catenin / BCL9 complex.

[0142] Real-time fluorescence quantitative PCR analysis:

[0143] HCT116 and CT26 cells were treated with different concentrations of ZW4864 and compounds for 24 hours. After drug treatment, the supernatant in the wells was discarded and 500 μL Trizol (Accurate Biology, A3A2199) was added to each well. After mixing by pipetting, the mixture was lysed at room temperature for 10 minutes, and the mixture in the well was transferred to a 1.5 mL EP tube. 100 μL chloroform was added to each tube, and the mixture was allowed to stand for stratification after thorough mixing, and then centrifuged at 12000 rpm and 4°C for 10 minutes. The upper layer of liquid was carefully aspirated with a 200 μL pipette tip and transferred to a new 1.5 mL EP tube. An equal amount of isopropanol was added to the new tube, and the mixture was centrifuged at 2000 rpm and 4°C for 10 minutes after thorough mixing. The supernatant was carefully removed and the precipitate was washed with 500 μL 75% ethanol. Centrifuged at 12000 rpm and 4°C for 10 minutes, the ethanol was carefully aspirated, and after the precipitate was dried, 20 μL DEPC water was added to dissolve the RNA precipitate, and the RNA concentration was measured with an ELISA reader. Total RNA (500 ng) was isolated from the cells and reverse transcribed into cDNA using Evo-mmlv reverse transcription premix. cDNA was diluted and quantified using Green premixed Pro Taq (Accurate Biology, AG11706, AG11701) and StepOne-Plus real-time PCR system (Applied Biosystems).

[0144] In vitro biological evaluation results:

[0145] Competitive affinity assay:

[0146] In this example, full-length β-catenin and BCL9 HD2 polypeptides were used to establish a competitive fluorescence polarization (FP) analysis method to evaluate the inhibitory activity of urea-structured small molecule inhibitors on β-catenin / BCL9 PPI. This method can directly reflect the competitive affinity of the inhibitor for the target protein β-catenin.

[0147] The competitive affinity test results of compounds 15-20, 22-32, 48-52 are shown in Tables 1-3.

[0148]

[0149] Table 1 Competitive affinity of compounds 17 to 21

[0150]

[0151] Among them, the general formula of compounds 22 to 32 is as follows:

[0152]

[0153] Table 2 Competitive affinity of compounds 25 to 36

[0154]

[0155]

[0156] Among them, the general formula of compounds 48 to 52 is as follows:

[0157]

[0158] Table 3 Competitive affinity of compounds 58 to 62

[0159]

[0160] Among them, the general formula of compounds 48 to 52 is as follows:

[0161]

[0162] In this example, based on the core structure of compounds 15 to 20, piperazine structure, amide ethylenediamine, amide propylenediamine, amide morpholine, amide ethanol and amide acetic acid structure were introduced at the right end of the core to obtain compounds 15 to 20, respectively. In the comparison of these 6 compounds, the FP result of compound 16 was better, and the FP IC50 reached 2.217 μM, indicating that the introduction of the propylenediamine structure may help enhance the affinity of the compound.

[0163] Considering the help of amide bond to biological activity, this embodiment introduces amide bond as a skeleton-derived Linker at the right end of the above-mentioned mother core structure, and connects it to the pyrrole meta-hydroxyl and methylene hydroxyl structures on the terminal carboxylic acid group to synthesize compounds 22 and 25, among which the competitive affinity of compound 22 is twice as fast as that of compound 16, showing good competitive affinity, and its FP IC50 is 1.633μM. However, neither compounds 20 nor 22 have improved in affinity with the target. On the contrary, the target affinity of compound 27 is more than four times lower than that of 22. In this embodiment, based on 22, the meta-hydroxyl is changed and replaced with ortho- and meta-methylene primary amine structures, and its reverse isomers are synthesized to obtain compounds 26-29, and it is found that the competitive binding with the target is not significantly improved. Later, we demethylated the primary amine on the modifying group and removed the methylene structure to synthesize compound 24, which performed well in competitive binding to the target. This example also draws on the design idea of ​​compound 16, replaces its similar structural group on the pyrrole ring, and designs compound 32, which is found to have improved affinity, with an FP IC50 of 1.954 μM. This example also changes the pyrrole ring structure to pyridine, retains the methylene primary amine structure, and finds that it does not help much in improving the biological activity.

[0164] The introduction of nitrogen atoms may successfully improve the competitive binding ability of the compound target, so based on compound 22, this example introduced a nitrogen atom on the benzene ring connected to the pyrazole ring at the other end of the mother core to synthesize compound 48, but it was found that the competitive binding ability of the compound target was 3 times lower than that of compound 22. The same attempt was made on the basis of retaining the primary methylene amine, and compounds 49-52 were synthesized, and it was found that the competitive affinity of compound 50 was significantly improved.

[0165] Detection of the inhibitory activity of target gene Axin:

[0166] After competitive fluorescence polarization detection, 8 representative compounds were selected for reverse transcription and real-time fluorescence quantitative PCR experiments to further explore the inhibition of the expression of the target gene Axin by the compounds. The results are shown in Table 4.

[0167] Table 4 Expression of target gene Axin by qPCR of eight compounds

[0168]

[0169]

[0170] The results in Table 4 show that the IC50 value of compound 22 in the qPCR experiment reached the nanomolar level, which was 0.746 μM, indicating that it had a good inhibitory effect on the target gene Axin of the Wnt signaling pathway. However, other compounds that performed well in the competitive fluorescence polarization experiment did not show a good inhibitory effect on the target gene Axin.

[0171] Comparison of molecular docking studies between ZW4864 and compound 22

[0172] In this example, the co-crystal structure of β-catenin / BCL9 protein (PDB: 2GL7) was selected to define the binding site, and ZW4864A and compound 22 with the best biological activity in FP and qPCR experiments were selected to explore the similarities and differences in the binding modes of the two with the β-catenin target protein, thereby verifying the improvement of their competitive affinity. Figure 1 shown.

[0173] The amide piperazine ring on the right end of compound ZW4864 forms hydrogen bonds with D-145 and K-181 of β-catenin protein, and the amino group on the pyrazole ring forms hydrogen bond interactions with E-155 ( Figure 1 A), compound 22 forms hydrogen bond interactions with residues D-145 and K-181 of β-catenin protein ( Figure 1 B). The molecular docking results showed that compound 22 occupied the hydrophobic pocket of the target protein better than ZW4864, which also indirectly explained the reason for its improved competitive affinity.

[0174] The chemical structure of ZW4864 is:

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A small molecule inhibitor, characterized in that: Its chemical structure is shown below: 。 2. A method for preparing the small molecule inhibitor according to claim 1, characterized in that: The method comprises the following steps of preparing the product: Wherein, X is CH, Y is CH, and R is .

3. A pharmaceutical composition, characterized in that: Containing the small molecule inhibitor according to claim 1 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical preparation comprising an active ingredient and a pharmaceutically acceptable excipient, characterized in that: The active ingredient is the small molecule inhibitor according to claim 1 or the pharmaceutical composition according to claim 3.

5. The pharmaceutical preparation according to claim 4, characterized in that: The pharmaceutically acceptable excipients include excipients and / or carriers.

6. Use of the small molecule inhibitor according to claim 1, the pharmaceutical composition according to claim 3, or the pharmaceutical preparation according to claim 4 or 5 in the preparation of a β-catenin protein target gene inhibitor drug.

7. Use of the small molecule inhibitor according to claim 1, the pharmaceutical composition according to claim 3, or the pharmaceutical preparation according to claim 4 or 5 in the preparation of a drug for treating colorectal cancer.

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