A bifunctional compound targeting degradation of atr protein and preparation method and use thereof

Abstract

The application discloses a bifunctional compound for targeted degradation of ATR protein and a preparation method and application thereof. The compound is a compound with a chemical formula as shown in general formula (I) or a pharmaceutically acceptable salt thereof. The bifunctional compound can be applied to preparation of an antitumor drug. The compound prepared by the application has novel action mechanism, high activity and small side effect. The compounds can be combined with other antitumor drugs to improve the curative effect of the existing antitumor drugs, reduce the dosage and toxicity. Pharmacological experiments show that the compound of the application can produce degradation effect on ATR kinase and can be used for preparation of a drug for treating diseases such as cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to a bifunctional compound and its preparation and use, in particular to a bifunctional compound for targeted degradation of ATR protein and its preparation and use. BACKGROUND

[0002] At present, cancer has become one of the main causes of human death, and it is urgent to develop effective cancer treatment methods.

[0003] Ataxia telangiectasia and Rad3-related kinase (ATR) is a key enzyme acting on homologous recombination repair, belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and is expressed in various tumor cells. ATR inhibitors prevent ATR kinase-mediated signaling in the ATR-Chkl signaling pathway, prevent DNA damage checkpoint activation, disrupt DNA damage repair, and induce tumor cell apoptosis. ATR inhibitors can be used for clinical development of various solid tumors, such as small cell carcinoma, uroepithelial carcinoma and ovarian cancer, etc.

[0004] Proteolysis targeting chimeras (PROTACs) is a hybrid bifunctional compound that can recruit target proteins and E3 ligases, and specifically degrade target proteins using the ubiquitin-proteasome system. PROTACs are a new drug development strategy and a research field that has been developing rapidly and attracting attention from the pharmaceutical industry in recent years. Compared with traditional small molecule inhibitors, PROTACs have obvious advantages. First, PROTACs can turn some currently considered "undruggable" targets into "druggable" targets. In addition, PROTACs also have the advantages of improving selectivity for target proteins, overcoming drug resistance and reducing toxicity. At present, the research on ATR kinase mainly focuses on small molecule inhibitors, and how to use PROTACs technology to design and synthesize bifunctional small molecules to solve the problems of ATR inhibitor drug resistance and toxicity and side effects still needs further in-depth study. SUMMARY

[0005] The present application aims to provide a bifunctional compound for targeted degradation of ATR protein; another object of the present application is to provide a preparation method of the compound; another object of the present application is to provide an application of the compound in the preparation of an antitumor drug.

[0006] Technical solution: The bifunctional compound for targeted degradation of ATR protein according to the present application is a compound of the chemical formula as shown in general formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] wherein:

[0009] A is selected from

[0010] wherein:

[0011] R 1 , R 2 represent H or C1-C4 alkyl, respectively;

[0012] R 3 represents H, F, Cl, Br, I, CN, C1-C3 alkyl or halogenated alkyl;

[0013] L is a linker, representing any of a fatty chain or an aromatic chain.

[0014] Preferably, R 1 represents H or C(CH3)3; R 2 , R 3 represent H or CH3, respectively;

[0015] L is any of the following structures:

[0016]

[0017] wherein X, Y are independently CH or N; m, n are independently any integer between 1 and 10.

[0018] More preferably, L is preferably wherein m, n are independently any integer between 1 and 10.

[0019] The pharmaceutically acceptable salts of the above compounds are acid addition salts of general formula (I), wherein the acid used for the salt formation is: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

[0020] Preferred compounds of the present application are the following:

[0021]

[0022]

[0023]

[0024] When A is L is the compound (la) of the present application can be prepared by the following methods:

[0025]

[0026] wherein R 1 , R 2 and R 3 are as defined above; m, n are independently any integer between 1 and 10.

[0027] Compound IIIa is prepared from compound Ha using a chlorinating agent selected from the group consisting of thionyl chloride or oxalyl chloride, preferably oxalyl chloride; a reaction solvent selected from the group consisting of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, toluene, ethylene glycol dimethyl ether, dioxane, N,N-dimethylformamide or dimethylsulfoxide, preferably dichloromethane; and a reaction temperature of -20 to 50 °C, preferably -5 to 30 °C.

[0028] Compound Va is prepared from compound IIIa and compound IVa using a base selected from the group consisting of triethylamine, N,N-diisopropylethylamine, sodium hydride, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium acetate or potassium acetate, preferably triethylamine; a reaction solvent selected from the group consisting of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, toluene, ethylene glycol dimethyl ether, dioxane, N,N-dimethylformamide or dimethylsulfoxide, preferably dichloromethane; and a reaction temperature of -20 to 50 °C, preferably -5 to 30 °C.

[0029] Compound Ia is prepared from compound VIa and compound Va using a solvent selected from the group consisting of a mixture of tetrahydrofuran, acetonitrile, ethylene glycol monomethyl ether, N-methylpyrrolidone, N,N-dimethylformamide or N,N-dimethylacetamide with water, preferably a mixture of tetrahydrofuran and water; a catalyst selected from the group consisting of cuprous iodide, cuprous bromide, a mixture of copper sulfate pentahydrate and sodium ascorbate, preferably a mixture of copper sulfate pentahydrate and sodium ascorbate; and a reaction temperature of -5 to 65 °C, preferably 20 to 35 °C.

[0030] When A is L is then compound (Ib) of the present application can be prepared by the following method:

[0031]

[0032] wherein m, n are independently any integer between 1 and 10.

[0033] Compound Ib is prepared from the reaction of compound lib with compound Va, using a mixture of one of tetrahydrofuran, acetonitrile, ethylene glycol monomethyl ether, N-methylpyrrolidone, N,N-dimethylformamide or N,N-dimethylacetamide and water, preferably a mixture of tetrahydrofuran and water as the solvent, using one of cuprous iodide, cuprous bromide, a mixture of copper sulfate pentahydrate and sodium ascorbate as the catalyst, preferably a mixture of copper sulfate pentahydrate and sodium ascorbate, at a temperature of -5 to 65°C, preferably 20 to 35°C.

[0034] When A is L is Compound (Ic) of the present application can be prepared by the following method:

[0035]

[0036] Compound Ic is prepared from the reaction of compound lie with compound Va, using a mixture of one of tetrahydrofuran, acetonitrile, ethylene glycol monomethyl ether, N-methylpyrrolidone, N,N-dimethylformamide or N,N-dimethylacetamide and water, preferably a mixture of tetrahydrofuran and water as the solvent, using one of cuprous iodide, cuprous bromide, a mixture of copper sulfate pentahydrate and sodium ascorbate as the catalyst, preferably a mixture of copper sulfate pentahydrate and sodium ascorbate, at a temperature of -5 to 65°C, preferably 20 to 35°C.

[0037] The present application also discloses a pharmaceutical composition containing the above-mentioned compound of general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The compound can be added with a pharmaceutically acceptable carrier to form a common pharmaceutical preparation, such as tablets, capsules, syrup, suspension, injection, and common pharmaceutical adjuvants such as flavoring agents, sweeteners, liquid or solid fillers or diluents, etc. can be added.

[0038] The compound can be applied to the preparation of an antitumor drug; the antitumor drug is an ATR protein degradation agent drug.

[0039] Advantages: Compared with the prior art, the present application has the following remarkable advantages: novel mechanism of action, high activity and small side effects. These compounds can be used in combination with other antitumor drugs to improve the efficacy of existing antitumor drugs and reduce the dose and toxicity. Pharmacological experiments show that the compounds of the present application can degrade ATR kinase and can be used for the preparation of drugs for treating cancer and other diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 2 shows that compound Ib-4 degrades ATR protein in a concentration-dependent manner;

[0041] Figure 2Figure showing the time-dependent degradation of ATR protein by compound Ib-4;

[0042] Figure 3 Figure showing the in vivo anti-tumor effect of compound Ib-4;

[0043] Figure 4 Figure showing the dose-dependent degradation of ATR protein by compound Ib-4. DETAILED DESCRIPTION

[0044] The technical solutions of the present application are further described below in conjunction with examples.

[0045] Example 1

[0046] (2S,4R)-1-((S)-3,3-dimethyl-2-(5-(4-(3-(((R)-methyl-(1-(6-((R)-3-methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 -yl)amino)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)pentanamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Ia-1: R 1 = CH3, m = 2, n = 4) was synthesized 2 = H, R 3 = CH3, m = 2, n = 4) was synthesized

[0047] N-((R)-methyl(1-(6-((R)-3-methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 -sulfinyl)pent-4-ynoic amide (Va-1) was synthesized

[0048] 4-Ynvaleric acid (IIa-1, 130 mg, 1.45 mmol) was dissolved in 10 mL of dichloromethane, and a catalytic amount of DMF was added, and oxalyl chloride (180 mg, 2.42 mmol) was added dropwise at 0°C. After the dropwise addition was completed, it was stirred at room temperature for 1 h, and concentrated under reduced pressure to obtain a yellow oily liquid IIIa-1, which was used directly in the next step without purification.

[0049] IIIa-1 was dissolved in 10 mL of dichloromethane, and (R)-imino(methyl)(1-(6-((R)-3-methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclopropyl)-λ 6- Sulfonamide ketone (IVa-1, 500 mg, 1.21 mmol) and triethylamine (180 mg, 1.82 mmol) after 6 h at 0 °C. TLC showed that the starting material IVa-1 was completely reacted, 10 mL of water was added to the reaction solution, extracted with dichloromethane (3 x 10 mL), the combined organic phase was dried over anhydrous Na2SO4, purified by column chromatography (PE:EA = 10:1-1:2) to give yellow solid Va-1, 430 mg, yield 72%, m.p.: 96-98 °C. 1 HNMR (300 MHz, CDC13) δ (ppm): 10.75 (s, 1H), 8.35 (s, 1H), 8.09 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 6.82 (s, 1H), 4.51 (s, 1H), 4.19 (d, J = 14.5 Hz, 1H), 4.10 (d, J = 11.0 Hz, 1H), 3.87 (d, J = 11.1 Hz, 1H), 3.78 (d, J = 13.0 Hz, 1H), 3.63 (t, J = 12.0 Hz, 1H), 3.53-3.33 (m, 4H), 2.74-2.57 (m, 2H), 2.54-2.45 (m, 1H), 2.42 (s, 1H), 2.28-2.15 (m, 1H), 2.09-1.99 (m, 1H), 1.94-1.83 (m, 1H), 1.76-1.66 (m, 1H), 1.64-1.53 (m, 1H), 1.40 (d, J = 6.8 Hz, 3H). MS (ESI): m / z 493.2 [M+H] + ; found: 493.2.

[0050] (2S,4R)-1-((S)-3,3-dimethyl-2-(5-(4-(3-(((R)-methyl-(1-(6-((R)-3-methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-lambda 6 - Sulfonamide ketone (IVa-1, 500 mg, 1.21 mmol) and triethylamine (180 mg, 1.82 mmol) after 6 h at 0 °C. TLC showed that the starting material IVa-1 was completely reacted, 10 mL of water was added to the reaction solution, extracted with dichloromethane (3 x 10 mL), the combined organic phase was dried over anhydrous Na2SO4, purified by column chromatography (PE:EA = 10:1-1:2) to give yellow solid Va-1, 430 mg, yield 72%, m.p.: 96-98 °C.

[0051] Va-1 (100 mg, 0.20 mmol) and VIa-1 (136 mg, 0.24 mmol) were dissolved in 10 mL THF, and anhydrous copper sulfate (25 mg, 0.10 mmol) and sodium ascorbate (97 mg, 0.49 mmol) were mixed and added to the reaction solution. The reaction was carried out at room temperature for 2 h. After TLC detection showed that the starting material Va-1 was completely reacted, the reaction solution was concentrated under reduced pressure, and the residue was separated and purified by column chromatography (silica gel, EA:MeOH 200:1-50:1) to obtain 103 mg of a yellowish solid, with a yield of 48.4%, m.p.: 104-106 °C. 1 H NMR (300 MHz, DMSO-d6) δ (ppm): 11.89 (s, 1H), 9.02 (s, 1H), 8.63 (s, 1H), 8.39 (d, J = 5.4 Hz, 1H), 8.00-7.95 (m, 2H), 7.67-7.64 (m, 2H), 7.47-7.40 (m, 4H), 7.27 (s, 1H), 6.95 (s, 1H), 5.19 (s, 1H), 4.58-4.55 (m, 2H), 4.50-4.44 (m, 2H), 4.39-4.38 (m, 1H), 4.26-4.22 (m, 4H), 4.05-4.02 (m, 1H), 3.84-3.80 (m, 1H), 3.76-3.65 (m, 2H), 3.59-3.45 (m, 6H), 2.80-2.65 (m, 2H), 2.48 (s, 3H), 2.41-2.29 (m, 2H), 2.19-2.08 (m, 2H), 2.03-1.81 (m, 4H), 1.79-1.62 (m, 2H), 1.66-1.53 (m, 1H), 1.52-1.35 (m, 2H), 1.30-1.18 (m, 4H), 0.96 (s, 9H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 180.22, 172.47, 172.19, 170.18, 163.31, 162.44, 161.53, 151.98, 150.68, 148.22, 146.33, 142.80, 140.01, 136.89, 131.68, 130.14, 129.14, 127.92, 122.02, 118.22, 115.19, 103.42, 102.05, 70.63, 69.39, 66.48, 59.20, 56.84, 49.30, 47.12, 46.19, 42.14, 38.45, 35.70, 34.54, 31.22, 29.81, 26.87, 22.82, 21.91, 16.45, 13.92, 13.15, 12.90. HRMS (ESI): m / z calcd for C 52 H 65 N 13 O7S2Na: 1070.4469 [M+Na] + ; found: 1070.4434. HPLC purity = 98.30%, t R = 6.651 min.

[0052] Example 2

[0053] (2S,4R)-1-((S)-3,3-dimethyl-2-(8-(4-(3-(((R)-methyl-(1-(6-((R)-3-methylmorpholin-2-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 - sulfenyl)amino)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)octanamido)butanoyl)-4-hydroxy-N-(4- (4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (la-2: R 1 = C(CH3)3, R 2 = H, R 3 = CH3, m = 2, n = 7)

[0054] The specific operation steps were the same as the synthesis of compound la-1, taking VIa-2 (146 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol) as raw materials. Yellow solid 120 mg was obtained, yield 54.2%, m.p.: 132-134 °C. 1HNMR (300 MHz, DMSO-d6) δ (ppm): 11.84 (s, 1H), 8.98 (s, 1H), 8.58 (s, 1H), 8.36 (s, 1H), 7.96 (s, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.58 (d, J = 7.6 Hz, 2H), 7.47 - 7.33 (m, 4H), 7.23 (s, 1H), 6.90 (d, J = 3.5 Hz, 1H), 5.13 (s, 1H), 4.54 (d, J = 9.4 Hz, 2H), 4.47 - 4.40 (m, 2H), 4.35 (s, 1H), 4.25 - 4.11 (m, 4H), 3.99 (d, J = 9.5 Hz, 1H), 3.78 (d, J = 11.2 Hz, 1H), 3.65 (s, 3H), 3.55 (d, J = 4.0 Hz, 3H), 3.51 - 3.42 (m, 2H), 2.74 - 2.67 (m, 2H), 2.44 (s, 3H), 2.41 - 2.17 (m, 2H), 2.14 - 2.00 (m, 2H), 1.95 - 1.80 (m, 4H), 1.68 - 1.62 (m, 2H), 1.58 - 1.52 (m, 1H), 1.48 - 1.40 (m, 2H), 1.29 - 1.14 (m, 10H), 0.93 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ (ppm): 180.15, 172.55, 172.42, 170.19, 163.28, 162.38, 161.48, 151.95, 150.65, 148.22, 146.33, 142.72, 139.98, 136.82, 130.11, 129.31, 129.11, 127.96, 127.89, 121.84, 118.31, 115.32, 103.39, 102.01, 70.59, 69.34, 66.43, 59.16, 56.74, 49.52, 47.02, 46.21, 42.12, 39.02, 38.43, 35.68, 35.28, 30.12, 28.91, 28.53, 26.84, 26.16, 25.78, 21.79, 19.03, 16.42, 13.86, 13.07, 12.82. HRMS (ESI): m / z calcd for C 55 H 71 N 13 O7S2Na: 1112.4939 [M + Na] + ; found: 1112.4932. HPLC purity = 98.25%, t R = 7.016 min.

[0055] Example 3

[0056] (2S,4R)-1-((S)-3,3-dimethyl-2-(11-(4-(3-(((R)-methyl-(1-(6-((R)-3-methylmorpholin-2-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-lambda 6 -mercapto)amino)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)undecanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (la-3: R 1 = C(CH3)3, R 2 = H, R 3 = CH3, m = 2, n = 10)

[0057] The specific operation steps were the same as the synthesis of compound la-1, taking VIa-3 (156 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol) as raw materials. Yellow solid 110 mg, yield 47.9%, m.p.: 122-124 °C. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.84 (s, 1H), 9.00 (s, 1H), 8.59 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 7.96 (d, J = 3.6 Hz, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.59 (s, 2H), 7.48 - 7.35 (m, 4H), 7.24 (s, 1H), 6.91 (d, J = 3.3 Hz, 1H), 5.15 (s, 1H), 4.56 (d, J = 9.0 Hz, 2H), 4.52 - 4.39 (m, 2H), 4.36 (s, 1H), 4.27 - 4.13 (m, 4H), 4.00 (d, J = 7.4 Hz, 1H), 3.79 (d, J = 11.6 Hz, 1H), 3.67 (s, 3H), 3.56 (d, J = 4.2 Hz, 3H), 3.53 - 3.43 (m, 2H), 2.79 - 2.65 (m, 2H), 2.46 (s, 3H), 2.42 - 2.21 (m, 2H), 2.16 - 2.01 (m, 2H), 1.97 - 1.82 (m, 4H), 1.66 (s, 2H), 1.59 - 1.45 (m, 3H), 1.31 - 1.16 (m, 16H), 0.94 (s, 9H). 13CNMR (101 MHz, DMSO-d6) δ (ppm): 180.15, 172.57, 172.42, 170.19, 163.27, 162.38, 161.48, 151.92, 150.67, 148.16, 146.31, 142.75, 139.98, 136.82, 131.56, 130.11, 129.11, 127.94, 127.89, 121.84, 118.18, 115.13, 103.39, 101.99, 70.59, 69.33, 66.43, 59.16, 56.74, 49.54, 47.02, 46.21, 43.53, 42.12, 38.91, 38.43, 35.68, 35.34, 30.55, 30.13, 29.33, 29.27, 29.18, 29.13, 28.82, 26.85, 26.26, 25.91, 21.79, 19.03, 16.42, 14.01, 13.86, 12.82. HRMS (ESI): m / z calcd for C 58 H 77 N 13 O7S2Na: 1154.5408 [M+Na] + ; found: 1154.5392. HPLC purity = 95.17%, t R = 7.726 min.

[0058] Example 4

[0059] N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)-5-(4-(3-(((R)-methyl(l- (6-((R)-3-methylmorpholino)-2-(lH-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)- lambda 6 sulfanyl)amino)-3-oxopropyl)-lH-l,2,3-triazol-l-yl)pentanamide (lb-1: m = 2, n = 4)

[0060] Starting from lib-1 (97 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound la-1. Yellow solid 115 mg, yield 51.3%, m.p.: 136-138 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.83 (s, 1H), 11.16 (s, 1H), 9.69 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 8.34 (d, J = 4.7 Hz, 1H), 7.94 (d, J = 5.1 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.65 (s, 1H), 7.62 - 7.55 (m, 2H), 7.22 (s, 1H), 6.89 (s, 1H), 5.14 (dd, J = 12.8, 5.1 Hz, 1H), 4.54 (s, 1H), 4.24 (t, J = 7.1 Hz, 3H), 4.09 - 3.94 (m, 1H), 3.83 - 3.69 (m, 1H), 3.62 (d, J = 10.4 Hz, 1H), 3.55 (d, J = 3.5 Hz, 3H), 3.52 - 3.41 (m, 1H), 3.28 - 3.18 (m, 1H), 2.97 - 2.83 (m, 1H), 2.73 - 2.63 (m, 2H), 2.47 - 2.30 (m, 4H), 2.19 - 2.01 (m, 1H), 1.91 - 21.88 (m, 1H), 1.86 - 1.71 (m, 4H), 1.61 - 1.45 (m, 3H), 1.28 - 1.21 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) d (ppm): 180.16, 173.23, 172.09, 170.26, 168.09, 167.13, 163.30, 162.39, 161.49, 150.65, 146.35, 142.76, 136.91, 136.83, 136.53, 131.92, 127.95, 126.85, 122.01, 118.82, 118.18, 117.55, 115.15, 103.39, 102.00, 70.59, 66.43, 56.50, 55.38, 49.24, 47.08, 46.18, 38.98, 36.02, 31.40, 29.57, 22.46, 22.06, 21.79, 19.03, 14.44, 13.89, 12.83. HRMS (ESI): m / z calcd for C 43 H 46 N 12 O8SNa: 913.3180 [M+Na] + ; found: 913.3163. HPLC purity = 95.33%, t R = 6.638 min.

[0061] Example 5

[0062] N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)-6-(4-(3-(((R)-methyl(l-(6-((R)-3- methylmorpholino)-2-(lH-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)- lambda 6 - sulfido)amino)-3-oxopropyl)-lH-l,2,3-triazol-l-yl)hexanamide (Ib-2: m = 2, n = 5)

[0063] Starting from lib-2 (101 mg, 0.24 mmol) and Va-l (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound la-l. Yellow solid 98 mg, yield 53.3%, m.p.: 132-134 °C. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.85 (s, 1H), 11.18 (s, 1H), 9.69 (s, 1H), 8.46 (d, J = 8.7 Hz, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.97 (d, J = 5.4 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.68 - 7.55 (m, 3H), 7.24 (s, 1H), 6.91 (s, 1H), 5.22 - 5.10 (m, 1H), 4.57 (s, 1H), 4.20 (t, J = 7.1 Hz, 3H), 4.11 - 3.96 (m, 1H), 3.81 - 3.77 (m, 1H), 3.65 - 3.43 (m, 5H), 3.26 - 3.19 (m, 1H), 2.95 - 2.87 (m, 1H), 2.71-2.59 (m, 3H), 2.49 - 2.32 (m, 4H), 2.10 - 2.00 (m, 1H), 2.00 - 1.94 (m, 1H), 1.89 - 1.81 (m, 2H), 1.79 - 1.68 (m, 2H), 1.64 - 1.53 (m, 3H), 1.25 - 1.18 (m, 6H). 13CNMR (75 MHz, DMSO-d6) δ (ppm): 180.03, 173.19, 172.26, 170.22, 168.06, 167.07, 163.20, 162.31, 161.42, 150.58, 146.24, 142.70, 136.89, 136.48, 131.84, 127.91, 126.71, 121.85, 118.71, 118.10, 117.38, 115.07, 103.31, 101.93, 70.51, 66.35, 60.17, 49.31, 46.98, 46.11, 38.80, 36.55, 31.32, 29.82, 25.70, 24.49, 22.38, 21.81, 14.48, 13.80, 13.02, 12.76. HRMS (ESI): m / z calcd for C 44 H 48 N 12 O8SNa: 927.3336 [M+Na] + ; found: 927.3319. HPLC purity = 95.68%, t R = 6.767 min.

[0064] Example 6

[0065] N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)-6-(4-(3-(((R)-methyl(l- (6-((R)-3-methylmorpholino)-2-(lH-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)- lambda 6 sulfanyl)amino)-3-oxopropyl)-lH-l,2,3-triazol-l-yl)heptanamide (Ib-3)

[0066] Using IIb-3 (104 mg, 0.24 mmol) and Va-l (100 mg, 0.20 mmol) as the starting material, the specific operation steps are the same as the synthesis of compound Ia-l. Yellow solid 93 mg, yield 49.8%, m.p.: 114-116 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.85 (s, 1H), 11.18 (s, 1H), 9.69 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 8.36 (s, 1H), 7.97 (s, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.65 - 7.55 (m, 3H), 7.23 (s, 1H), 6.91 (s, 1H), 5.22 - 5.10 (m, 1H), 4.56 (s, 1H), 4.19 (t, J = 7.1 Hz, 3H), 4.04 - 3.98 (m, 1H), 3.78 - 3.76 (m, 1H), 3.64 - 3.48 (m, 5H), 3.01 - 2.84 (m, 1H), 2.74 - 2.57 (m, 3H), 2.45 - 2.37 (m, 4H), 2.14 - 2.01 (m, 1H), 2.00 - 1.89 (m, 1H), 1.89 - 1.83 (m, 2H), 1.73 - 1.66 (m, 2H), 1.57 - 1.55 (m, 3H), 1.28 - 1.13 (m, 8H). 13 CNMR (75 MHz, DMSO-d6) d (ppm): 180.22, 173.30, 172.45, 170.32, 168.20, 167.18, 163.30, 162.41, 161.53, 150.68, 146.37, 142.80, 137.03, 136.85, 136.59, 131.95, 128.02, 126.80, 121.90, 118.81, 118.22, 117.48, 115.19, 103.43, 102.04, 70.62, 66.48, 60.32, 49.53, 49.41, 47.12, 46.22, 36.87, 31.44, 30.03, 28.34, 26.04, 25.04, 22.49, 21.93, 14.59, 13.90, 13.07, 12.86. HRMS (ESI): m / z calcd for C 45 H 50 N 12 O8SNa: 941.3493 [M + Na] + ; found: 941.3483. HPLC purity = 98.69%, t R = 6.976 min.

[0067] Example 7

[0068] N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-8-(4-(3-(((R)-methyl(1-(6-((R)-3- methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 - sulfido)amino)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)octanamide (Ib-4: m = 2, n = 7)

[0069] Using IIb-4 (107 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol) as the starting material, the specific operation steps were the same as the synthesis of compound Ia-1. Yellow solid 102 mg, yield 53.8%, m.p.: 130-132 °C was obtained. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.85 (s, 1H), 11.18 (s, 1H), 9.69 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 8.36 (s, 1H), 7.97 (s, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.65 - 7.55 (m, 3H), 7.23 (s, 1H), 6.91 (s, 1H), 5.40 - 5.34 (m, 1H), 4.78 (s, 1H), 4.41 (t, J = 7.7 Hz, 3H), 4.24 - 4.20 (m, 1H), 4.07 - 3.97 (m, 1H), 3.85 - 3.66 (m, 5H), 3.52 - 3.38 (m, 1H), 3.17 - 3.08 (m, 1H), 2.91 - 2.80 (m, 3H), 2.68 - 2.59 (m, 4H), 2.30 - 2.21 (m, 1H), 2.20 - 2.14 (m, 1H), 2.11 - 2.06 (m, 2H), 1.97 - 1.74 (m, 5H), 1.49 - 1.38 (m, 10H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 180.22, 173.30, 172.51, 170.32, 168.21, 167.18, 163.32, 162.40, 161.52, 150.69, 146.35, 142.79, 137.04, 136.84, 136.60, 131.94, 128.01, 126.75, 121.88, 118.78, 117.42, 115.19, 103.43, 102.04, 70.62, 66.48, 49.55, 49.40, 47.08, 46.18, 38.93, 36.93, 31.44, 30.12, 28.78, 28.56, 26.15, 25.15, 22.48, 21.94, 21.84, 13.88, 12.85. HRMS (ESI): m / z calcd for C 46 H 52 N 12 O8SNa: 955.3649 [M + Na] + ; found: 955.3632. HPLC purity = 98.30%, t R = 6.651 min.

[0070] Example 8

[0071] N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)-9-(4-(3-(((R)-methyl(l-(6- ((R)-3-methylmorpholino)-2-(lH-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)- lambda 6 sulfanyl)amino)-3-oxopropyl)-lH-l,2,3-triazol-l-yl)nonanamide (lb-5: m = 2, n = 8)

[0072] Starting from lib-5 (111 mg, 0.24 mmol) and Va-l (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound la-l. Yellow solid 95 mg, yield 49.4%, m.p.: 100-102 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.84 (s, 1H), 11.18 (s, 1H), 9.69 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.96 (d, J = 5.0 Hz, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.70 - 7.55 (m, 3H), 7.23 (s, 1H), 6.90 (s, 1H), 5.22 - 5.10 (m, 1H), 4.56 (s, 1H), 4.21 - 4.10 (m, 3H), 4.10 - 3.95 (m, 1H), 3.80 - 3.76 (m, 1H), 3.64 - 3.44 (m, 5H), 3.32-3.18 (m, 1H), 3.00 - 2.82 (m, 1H), 2.77 - 2.57 (m, 3H), 2.47 - 2.37 (m, 4H), 2.40 - 2.27 (m, 1H), 2.13 - 2.02 (m, 1H), 2.00 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.71 - 1.52 (m, 5H), 1.33 - 1.07 (m, 12H). 13 C NMR (75 MHz, DMSO-d6) d (ppm): 180.21, 173.29, 172.53, 170.32, 168.21, 167.18, 163.32, 162.41, 161.53, 150.69, 146.35, 142.79, 137.05, 136.78, 136.60, 131.94, 128.00, 126.74, 121.87, 118.78, 118.21, 117.42, 115.17, 103.43, 102.04, 70.63, 66.47, 49.56, 49.40, 47.10, 46.22, 36.99, 31.45, 30.15, 29.03, 28.91, 28.72, 26.25, 25.23, 22.49, 21.83, 14.60, 13.89, 13.11, 12.84. HRMS (ESI): m / z calcd for C 47 H 54 N 12 O8SNa: 969.3806 [M+Na] + ; found: 969.3801. HPLC purity = 98.98%, t R = 7.580 min.

[0073] Example 9

[0074] N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-10-(4-(3-(((R)-methyl(1-(6-((R)-3- methylmorpholino)-2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 - sulfido)amino)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)decanoic amide (Ib-6: m = 2, n = 9)

[0075] Starting from IIb-6 (114 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound Ia-1. Yellow solid, 105 mg, yield 53.8%, m.p.: 108-110 °C. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.84 (s, 1H), 11.18 (s, 1H), 9.69 (s, 1H), 8.48 (d, J = 8.4 Hz, 1H), 8.35 (d, J = 5.0 Hz, 1H), 7.96 (dd, J = 5.0, 1.7 Hz, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.65 - 7.55 (m, 3H), 7.24 (s, 1H), 6.91 (s, 1H), 5.16 (dd, J = 12.9, 5.4 Hz, 1H), 4.56 (s, 1H), 4.23 - 4.07 (m, 3H), 4.02 - 3.97 (m, 1H), 3.80 - 3.76 (m, 1H), 3.64 - 3.44 (m, 5H), 3.33 - 3.18 (m, 1H), 2.95 - 2.82 (m, 1H), 2.79 - 2.55 (m, 3H), 2.46 - 2.29 (m, 4H), 2.12 - 2.02 (m, 1H), 2.02 - 1.88 (m, 1H), 1.86 - 1.81 (m, 2H), 1.73 - 1.50 (m, 5H), 1.32 - 1.06 (m, 14H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 180.22, 173.30, 172.54, 170.32, 168.22, 167.18, 163.32, 162.40, 161.52, 150.69, 146.35, 142.79, 137.05, 136.86, 136.60, 131.93, 128.00, 126.73, 121.88, 118.77, 118.21, 117.41, 115.17, 103.43, 102.04, 70.62, 66.47, 49.56, 49.40, 47.07, 46.21, 37.01, 31.45, 30.16, 29.14, 28.96, 28.80, 26.26, 25.25, 22.49, 21.93, 13.89, 13.12, 12.85. HRMS (ESI): m / z calcd for C 48 H 56 N 12 O8SNa: 983.3962 [M+Na] + ; found: 983.3962. HPLC purity = 99.56%, t R = 7.998 min.

[0076] Example 10

[0077] N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)-l l-(4-(3-(((R)-methyl(l- (6-((R)-3-methylmorpholino)-2-(lH-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)- lambda 6 sulfanyl)amino)-3-oxopropyl)-lH-l,2,3-triazol-l-yl)undecanamide (Ib-7: m = 2, n = 10)

[0078] Starting from IIb-7 (118 mg, 0.24 mmol) and Va-l (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound Ia-l. Yellow solid 100 mg, yield 50.5%, m.p.: 124-126 °C. 1HNMR (300 MHz, DMSO-d6) δ (ppm): 11.85 (s, 1H), 11.78 (s, 1H), 9.69 (s, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.36 (s, 1H), 7.97 (s, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.62 - 7.58 (m, 3H), 7.23 (s, 1H), 6.91 (s, 1H), 5.22 - 5.09 (m, 1H), 4.56 (s, 1H), 4.16 (s, 3H), 4.02 - 3.98 (m, 1H), 3.80-3.76 (m, 1H), 3.69 - 3.44 (m, 5H), 3.29 - 3.21 (m, 1H), 2.99 - 2.86 (m, 1H), 2.73 - 2.65 (m, 3H), 2.44 - 2.37 (m, 4H), 2.09 - 2.08 (m, 1H), 2.00 - 1.94 (m, 1H), 1.88 - 1.76 (m, 2H), 1.74 - 1.42 (m, 7H), 1.42 - 1.00 (m, 14H). 13 C NMR (75 MHz, DMSO-d6) δ (ppm): 180.20, 173.30, 172.37, 170.32, 168.20, 167.18, 163.31, 162.39, 161.54, 150.69, 146.35, 142.81, 136.99, 136.84, 136.60, 131.96, 128.01, 126.84, 121.96, 118.82, 118.20, 117.48, 115.18, 103.41, 102.02, 70.62, 66.46, 49.40, 47.09, 46.22, 41.45, 36.66, 31.44, 30.15, 29.92, 29.28, 29.19, 28.99, 25.80, 24.60, 22.48, 21.82, 13.90, 12.84. HRMS (ESI): m / z calcd for C 49 H 58 N 12 O8SNa: 997.4119 [M + Na] + ; found: 997.4111. HPLC purity = 99.65%, t R = 8.552 min.

[0079] Example 11

[0080] 3-(1-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2- oxoethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((R)-methyl(1-(6-((R)-3-methylmorpholinyl)- 2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-lambda 6 -propylidene)sulfanyl)propanamide (Ic-1: m = 2, n = 1)

[0081] Starting from IIc-1 (98 mg, 0.244 mmol) and Va-1 (100 mg, 0.203 mmol), the specific reaction steps are the same as the synthesis of compound Ia-1. Yellow solid 104 mg, yield 57.3%, m.p.: 94-96 °C. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.84 (s, 1H), 11.18 (s, 1H), 10.24 (s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H), 7.94 (d, J = 5.2 Hz, 1H), 7.90 - 7.76 (m, 2H), 7.67 - 7.56 (m, 2H), 7.21 (s, 1H), 6.87 (s, 1H), 5.24 - 5.12 (m, 1H), 4.58 (s, 3H), 4.16 (s, 3H), 4.01 - 3.91 (m, 3H), 3.76 (d, J = 10.8 Hz, 1H), 3.66 - 3.57 (m, 1H), 3.53 (d, J = 2.3 Hz, 3H), 3.50 - 3.40 (m, 1H), 3.32 - 3.17 (m, 1H), 2.98 - 2.83 (m, 1H), 2.80 - 2.56 (m, 5H), 2.48 - 2.32 (m, 1H), 2.14 - 2.02 (m, 1H), 1.99 - 1.74 (m, 3H), 1.63 - 1.48 (m, 1H), 1.22 (t, J = 7.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 180.28, 173.26, 170.24, 169.22, 168.82, 167.18, 163.32, 162.41, 161.48, 150.67, 146.48, 142.79, 137.03, 136.86, 136.37, 131.74, 127.99, 125.04, 122.88, 118.93, 118.20, 116.75, 115.17, 103.37, 102.02, 70.61, 70.26, 70.13, 66.47, 49.50, 47.04, 46.19, 38.78, 31.45, 22.46, 21.79, 20.50, 13.88, 12.92. HRMS (ESI): m / z calcd for C 42 H 44 N 12 O9SNa: 915.2973 [M+Na] + ; found: 915.2956. HPLC purity = 95.16%, t R = 6.584 min.

[0082] Example 12

[0083] 3-(1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2- oxoethoxy)ethoxy)-1H-1,2,3-triazol-4-yl)-N-((R)-methyl(1-(6-((R)-3-methylmorpholino)- 2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-lambda 6 -sulfidyl)propanamide (Ic-2: m = 2, n = 2)

[0084] Starting from IIc-2 (108 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol), the specific operation steps are the same as the synthesis of compound Ia-1. Yellow solid 110 mg, yield 57.8%, m.p.: 118-120 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 11.86 (s, 1H), 11.19 (s, 1H), 10.32 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 5.0 Hz, 1H), 7.95 (d, J = 5.0 Hz, 1H), 7.90 - 7.78 (m, 1H), 7.69 - 7.56 (m, 3H), 7.22 (s, 1H), 6.89 (d, J = 2.9 Hz, 1H), 5.16 (dd, J = 13.0, 5.6 Hz, 1H), 4.58 (s, 1H), 4.40 (t, J = 5.3 Hz, 2H), 4.26 - 4.11 (m, 3H), 4.04 - 3.95 (m, 1H), 3.83 - 3.74 (m, 3H), 3.73 - 3.67 (m, 2H), 3.66 - 3.58 (m, 3H), 3.55 (d, J = 3.5 Hz, 3H), 3.51 - 3.42 (m, 1H), 3.34 - 3.17 (m, 1H), 2.98 - 2.80 (m, 1H), 2.75 - 2.55 (m, 4H), 2.49 - 2.29 (m, 2H), 2.12 - 2.02 (m, 1H), 1.97 - 1.76 (m, 3H), 1.61 - 1.49 (m, 1H), 1.23 (t, J = 7.5 Hz, 3H). 13 C NMR (75 MHz, CDCl3) d (ppm): 181.44, 172.33, 172.18, 169.16, 168.61, 166.88, 163.45, 162.28, 160.96, 149.63, 146.88, 141.76, 138.09, 136.64, 136.52, 136.35, 131.41, 126.70, 125.17, 122.04, 118.88, 116.17, 115.53, 103.40, 102.77, 77.30, 71.53, 70.88, 70.28, 69.82, 66.68, 49.86, 49.35, 47.35, 46.39, 39.90, 39.48, 38.68, 31.51, 22.80, 21.65, 13.86, 13.77, 13.04, 12.47. HRMS (ESI): m / z calcd for C 44 H 48 N 12 O 10 SNa: 959.3235 [M+Na] + ; found: 959.3213. HPLC purity = 98.57%, t R = 6.634 min.

[0085] Example 13

[0086] 3-(1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2- oxoethoxy)ethoxy)-1H-1,2,3-triazol-4-yl)-N-((R)-methyl(1-(6-((R)-3-methylmorpholinyl)- 2-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyrimidin-4-yl)cyclopropyl)(oxo)-λ 6 -propylsulfinyl)propanamide (Ic-3: m = 2, n = 3)

[0087] The specific operation steps were the same as the synthesis of compound Ia-1, taking IIc-3 (119 mg, 0.24 mmol) and Va-1 (100 mg, 0.20 mmol) as raw materials. Yellow solid 98 mg, yield 49.2%, m.p.: 98-100 °C was obtained. 1 H NMR (300 MHz, DMSO-d6) d (ppm): 11.83 (s, 1H), 11.18 (s, 1H), 10.33 (s, 1H), 8.71 (d, J = 8.6 Hz, 1H), 8.34 (d, J = 5.1 Hz, 1H), 7.95 (d, J = 5.2 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.73 - 7.56 (m, 3H), 7.23 (s, 1H), 6.89 (s, 1H), 5.19 - 5.13 (m, 1H), 4.57 (s, 1H), 4.36 (t, J = 5.1 Hz, 2H), 4.25 - 4.09 (m, 3H), 4.00 (d, J = 7.8 Hz, 1H), 3.80 - 3.76 (m, 1H), 3.74 - 3.66 (m, 4H), 3.66 - 3.58 (m, 3H), 3.56 - 3.54 (m, 3H), 3.51-3.41 (m, 5H), 3.28 (s, 1H), 3.28 - 3.22 (m, 1H), 2.95 - 2.59 (m, 4H), 2.46 - 2.35 (m, 2H), 2.09 - 2.06 (m, 1H), 2.00 - 1.90 (m, 1H), 1.88 - 1.78 (m, 2H), 1.63 - 1.49 (m, 1H), 1.24 (t, J = 7.7 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) d (ppm): 180.20, 173.29, 170.29, 169.86, 168.74, 167.19, 163.33, 162.42, 161.51, 150.68, 146.30, 142.80, 137.00, 136.86, 136.44, 131.78, 128.00, 124.85, 122.51, 118.82, 118.20, 116.52, 115.18, 103.37, 102.04, 71.25, 70.62, 70.17, 70.03, 69.22, 66.47, 55.44, 49.64, 49.45, 47.08, 46.16, 38.85, 31.43, 26.00, 22.44, 21.85, 21.75, 13.90, 12.93. HRMS (ESI): m / z calcd for C 46 H 52 N 12 O 11 SNa: 1003.3497 [M+Na] + ; found: 1003.3486. HPLC purity = 99.77%, t R = 7.624 min.

[0088] The pharmacological experiments and results of some compounds of the present application are as follows:

[0089] (1) Tumor cell source and culture: HCT116 and LoVo cells were provided by the Chinese Academy of Sciences Stem Cell Bank. The HCT116 cell line was cultured in McCoy's 5A containing 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% glutamine and 1% penicillin-streptomycin. The LoVo cell line was cultured in FK12 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.

[0090] (2) Anti-proliferative activity determination: The anti-proliferative activity of the compounds on tumor cells was determined by CCK-8 assay. Cells (2000-5000 / well) were seeded in 96-well plates (100 μL of culture medium / well) and incubated for 24 hours. Then the cells were treated with a series of concentrations of the compounds and incubated for 72 hours, 10 μL of CCK-8 solution was added to each well and incubated for 1-2 h. The optical density of each well was determined by a microplate reader at 490 nm or 570 nm wavelength.

[0091] Experimental results:

[0092] The anti-proliferative activity of the test compounds and the positive drug AZD6738 on tumor cells is shown in Table 1.

[0093] Table 1. Anti-proliferative activity of some compounds on HCT-116 and LoVo cells

[0094]

[0095] Note: A: 0.01-1.0 μM, B 1.0-10 μM, C 10-50 μM, D 50-100 μM, E 100-1000 μM. The anti-proliferative activity of the compounds on tumor cells was determined by CCK-8 assay.

[0096] The results in Table 1 show that some compounds maintain the proliferative activity on HCT116 and LoVo, and compound Ib-4 has better proliferative activity on both cells, which is comparable to the positive drug AZD6738. Like the positive drug, compound Ib-4 has better anti-proliferative activity on ATM mutant LoVo cells than on ATM wild-type HCT116 cells.

[0097] (3) In vitro protein degradation activity determination (WB experiment): Cells (1-2 x 10 6 were seeded in 6-well plates and incubated for 24 hours. Cells were treated with different concentrations of compounds for a specified time or treated with a specified concentration of compound for different times, and then lysed with lysis buffer (Beyotime) with protease and phosphatase inhibitors. The suspension was centrifuged at 12000 rpm for 20 minutes, and the insoluble material was removed. The proteins were separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millpore). After incubation with primary and secondary antibodies (Bi Yun Tian), the membrane was imaged using an imaging system (BIO-RAD). Anti-ATR was purchased from Cell Signaling Technology.

[0098] Experimental results: Compound Ib-4 can significantly degrade ATR protein in HCT116 and LoVo cells in a concentration-dependent manner after 72 hours of culture. Among them, compound Ib-4 is more sensitive to LoVo cells and has the strongest degradation ability. Results are shown in Figure 1 .

[0099] Experimental results: Compound Ib-4 significantly degrades ATR protein in HCT116 and LoVo cells in a time-dependent manner, and the protein degradation rate of ATR is faster after treatment of LoVo cells with compound Ib-4 (2.5 μM). Results are shown in Figure 2 .

[0100] (4) In vivo anti-tumor efficacy evaluation: BALB / c nude mice, female, 6-8 weeks, 18-22 g. All experimental mice were raised in a barrier system and acclimated for at least 3 days in advance. On the day of inoculation, 1x10 7 LoVo cells were resuspended in 1:1 PBS and Matrigel, and tumor growth was observed regularly. When the tumors grew to an average of 80-100 mm 3 When the tumors grew to an average of 80-100 mm

[0101] Experimental results: Ib-4 at a dose of 12.5 mg / kg showed a tumor growth inhibition rate (TGI) comparable to that of the positive drug AZD6738 (50 mg / kg, po). In addition, the TGI value of Ib-4 (25 mg / kg, ip) was 51.8%, which was more effective than that of AZD6738 (50 mg / kg, po) (TGI: 35.9%), indicating that compound Ib-4 can significantly inhibit tumor growth in a dose-dependent manner, and the anti-tumor activity is significantly better than that of AZD6738. The results are shown in Figure 3 .

[0102] (5) In vivo protein degradation activity assay (WB experiment): 2 tumor tissues were selected from each group for homogenization, and then lysed with lysis buffer (Beyotime) containing protease and phosphatase inhibitors. The suspension was centrifuged at 12000 rpm for 20 minutes, and the insoluble material was removed. The proteins were separated by 8% SDS-PAGE and transferred to PVDF membrane (Millpore). After incubation with primary and secondary antibodies (Bi Yun Tian), the membrane was imaged using an imaging system (BIO-RAD). Anti-ATR was purchased from Cell Signaling Technology.

[0103] Experimental results: Animal experiments further showed that compound Ib-4 can significantly degrade ATR protein in LoVo cells in a dose-dependent manner, thereby exhibiting significant anti-tumor activity. The results are shown in Figure 4 .

Claims

1. A bifunctional compound targeting degradation of ATR protein, characterized in that, It is a compound of the chemical formula as general formula (I) or a pharmaceutically acceptable salt thereof: wherein A is selected from R 1 , R 2 each represent H or C1-C4 alkyl, R 3 represents H, F, Cl, Br, I, CN, C1-C3 alkyl or C1-C3 haloalkyl; L is a connecting arm, being any one of the following structures: wherein m, n are independently any integer between 1-10.

2. The compound for targeted degradation of ATR protein according to claim 1, characterized in that, The pharmaceutically acceptable salt is an acid addition salt of the compound with hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

3. A process for the preparation of a compound according to claim 1, characterized in that, When A is L is The reaction formula of the preparation method is as follows: Wherein m, n are independently any one integer between 1-10.

4. A process for the preparation of a compound according to claim 1, characterized in that, When A is L is The reaction scheme for the preparation method when A is Wherein m, n are independently any one integer between 1-10.

5. A process for the preparation of a compound according to claim 1, characterized in that, When A is L is The reaction scheme for the preparation method is as follows: Wherein m, n are independently any one integer between 1-10.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 2, wherein, The pharmaceutical composition is added with a pharmaceutically acceptable carrier to form a pharmaceutical preparation.

7. Use of the compound of any one of claims 1-2 or the pharmaceutical composition of claim 6 in the preparation of an antitumor drug.

8. Use according to claim 7, characterized in that, The antitumor drug is an ATR protein degradation agent drug.

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