A small molecule targeting the MAGI2 GK domain and its use

Abstract

The present invention provides a small molecule compound targeting the MAGI2GK domain or a pharmaceutically acceptable salt thereof, and uses thereof. The compound can be used as a small molecule inhibitor of the MAGI2 protein GK domain and has tumor-suppressing activity.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical pharmacy, and in particular to a small molecule targeting the MAGI2 GK domain and a use thereof. Background Art

[0002] The MAGUK protein family is a member of many scaffold protein families, mainly distributed in cells or synaptic junctions, and has complex and diverse biological functions, such as cell apoptosis, cell adhesion, mitotic spindle orientation, maintaining the formation and plasticity of neuronal synapses, etc. The protein structure of this family is conservative, mainly composed of PDZ domain, L27 domain, WW domain, SH3 domain and GK domain without enzyme activity.

[0003] Membrane-bound guanylate kinase inverse protein (MAGI) is a subfamily of the MAGUKs protein family, consisting of 6 PDZ domains, 2 WW domains and 1 GK domain. Unlike the other members of the MAGUK family, the GK domain of MAGI protein is at the nitrogen end. This subfamily of proteins is divided into 3 subtypes, namely MAGI1, MAGI2 and MAGI3. Although these three protein subtypes have similar structures, their tissue distribution shows certain differences. For example, MAGI1 is mainly expressed in epithelial cells and neurons, MAGI2 is mainly expressed in neuronal synapses, and MAGI3 is expressed in epithelial cells and endothelial cells. MAGI2 protein (also known as Synaptic scaffolding molecule, S-SCAM) contains 3 protein subtypes, namely MAGI2α, MAGI2β and MAGI2γ, of which MAGI2α has 6 PDZ domains, and MAGI2β and MAGI2γ have 5 PDZ domains. MAGI2 protein is highly expressed in brain tissue, acting as a scaffold molecule with neurotransmitter receptors and cell adhesion proteins in synapses, and plays a role in nerve-mediated signal transduction. Studies have shown that mutations in the MAGI2 gene can lead to severe congenital nephrotic syndrome and Parkinson's syndrome. The GK domain evolved from guanylate kinase, which is highly conserved in structure and sequence. It plays an important role in the GMP / cGMP cycle and purine metabolism. At present, the structure and function of the GK domain of the MAGI2 protein are still unclear, and the development of small molecule inhibitors of the GK domain will help study the function of the GK domain.

[0004] The MAGI2 protein was first discovered in a yeast two-hybrid system with PTEN as a bait plasmid. MAGI2 can interact with the tumor suppressor protein PTEN. Among them, MAGI2 can bind to the PDZ domain at the carbon end of the PTEN protein through the PDZ domain. PTEN protein is currently known to be a tumor suppressor protein. The non-phosphorylated PTEN protein is unstable. MAGI2 can bind to the phosphorylated PTEN to stabilize the PTEN protein. Therefore, some studies currently believe that MAGI2 protein functions as a tumor suppressor protein by stabilizing the PTEN protein. In addition, studies have shown that MAGI2 can participate in the regulation of multiple signaling pathways, such as the Wnt / β-catenin pathway, the TGF-β pathway, and the Notch pathway, and can regulate the endocytosis of membrane proteins. MAGI2 protein may participate in the Wnt / β-catenin signaling pathway to play the role of a tumor suppressor protein by binding to PDZ5 and β-catenin. Studies have found that in human prostate cancer, MAGI2 gene expression loss can be detected, and the mRNA level of MAGI2 is significantly downregulated in prostate cancer cell lines and tissues. Down-regulation of MAGI2 protein expression can also be observed in liver cancer, and overexpression of MAGI2 can increase the level of cancer cell apoptosis. Loss of MAGI2 expression can also be detected in cervical cancer. The amino acid sequence of the GK domain of MAGI2 protein is: MSKSLKKKSHWTSKVHESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGKVAYESGSKLVSEELLLEVNETPVAGLTIRDVLAVIKHCKDPLRLKCVKQGGIVDKDLRHYLNLRFQKGSVDHELQQIRDNLYLRTVPCTTRPHKEGEVPGVDYIFITVEEFMELEKSGALLESGTYEDNYYGTPKPPAEPAPLLNVTDQILPGATPSAEGKRKRNKSVTNMEKASIEPPEEEEEERPVVNGNGVVITPESSEHDKSAGASGETPSQPYPAPVYSQPEELKDQMDDTKPTKPEENEDS.

[0005] However, there are also reports that MAGI2 protein may have a pro-oncogenic function in cancer. Jeffery's team found through immunohistochemistry that compared with normal tissues, MAGI2 immunoreactivity was significantly increased in prostate cancer and high-grade prostatic intraepithelial neoplasia, suggesting that MAGI2 protein may contribute to prostate cancer. In addition, although the MAGUKs family protein DLG1 has a tumor suppressor function in combination with PTEN, studies have found that DLG1 is highly expressed in the cytoplasm in the early stages of cervical cancer, colon cancer, and breast cancer. In some cell lines, DLG1 has a carcinogenic effect. By silencing DLG1 protein expression, the invasive ability of tumor cells is inhibited. Liu Ji's team reported that DLG3 protein expression is positively correlated with the pathological stage of breast cancer patients, and the patient survival rate is reduced. These also reveal that MAGUK protein may have the function of proto-oncoprotein.

[0006] Gastric cancer is the most common digestive tract malignancy in my country, with the highest incidence and mortality rates among all malignant tumors, and has become the third leading cause of cancer death in China. The number of new cases in my country each year accounts for 42% of the world's total number of cases. Although there are multiple treatment options in clinical practice, the 5-year survival rate is still low. The 5-year survival rate of stage I gastric cancer with standardized treatment is 82%-95%, stage II is 50%, stage III is 15%-30%, and stage IV is only 2%. Data show that 90% of gastric cancer deaths are caused by the malignant metastasis of gastric cancer cells. At present, the gastric cancer drug treatment options recommended by the CSCO gastric cancer diagnosis and treatment guidelines include traditional chemotherapy and HER2 / VEGF monoclonal antibody targeted drugs, etc. However, in the treatment of metastatic gastric cancer, there are generally disadvantages such as low response rate, poor selectivity, and easy to produce drug resistance, which have failed to significantly prolong the survival of patients. For this reason, new therapies for metastatic gastric cancer are urgently needed in the clinic. Summary of the invention

[0007] The present invention provides a small molecule targeting the MAGI2 GK domain with oxidized indole as a skeleton, which can target the molecule of the GK domain of the MAGI2 protein and can inhibit the migration and invasion ability of tumors.

[0008] The present invention first provides a compound targeting the MAGI2 GK domain, or a pharmaceutically acceptable salt thereof, which has the structural formula shown in the formula:

[0009]

[0010] in,

[0011] X is N or -CH-; R1, R5 and R6 are each independently hydrogen, C1-4 alkyl, halogen, or C1-4 alkoxy, trifluoromethyl, heteroaryl;

[0012] R2 is hydroxy, amino, C1-4 alkylamide, arylamino, heteroarylamino, phenyl C1-4 alkylamide, C1-4 alkoxyamide, oxime, trifluoromethylamino, aminocyano, halogen, sulfonamide, urea or sulfonylurea, and disubstituted amino groups of the above amino substituents, and any disubstituted combination of the above amino substituents on the amino group;

[0013] R3 is H, C1-4 alkyl, aryl, heteroaryl, 3-7 membered cycloalkyl, benzyl, acyl or sulfonyl;

[0014] R4 is C1-4 alkyl, cycloalkyl, acyl, sulfonyl, benzyl, or a 5-10 membered aryl or heteroaryl substituted by 1-4 substituents independently selected from C1-4 alkyl, aryl, heteroaryl, cycloalkyl, benzyl, acyl or sulfonyl;

[0015] Preferably, R4 is a biaryl group consisting of two aryl groups connected via a single bond, and the aryl groups are each independently selected from phenyl and naphthyl;

[0016] Preferably, R4 is a biheteroaryl group consisting of two heteroaryl groups, each of which is independently selected from a monocyclic 5-6-membered heteroaryl group containing one or two heteroatoms, each of which is independently selected from N, O and S;

[0017] Preferably, R4 is an arylheteroaryl group composed of an aryl group and a heteroaryl group connected via a single bond, wherein the aryl group is selected from phenyl and naphthyl, the heteroaryl group is selected from a monocyclic 5-6 membered heteroaryl group containing 1 or 2 heteroatoms, each heteroatom is independently selected from N, O and S, and the heteroaryl moiety is bonded to the parent molecular group;

[0018] Preferably, R4 is a heteroarylaryl group consisting of a heteroaryl group and an aryl group connected via a single bond, the heteroaryl group being a monocyclic 5-6 membered heteroaryl group containing one or two heteroatoms, each heteroatom being independently selected from N, O and S, the aryl groups being independently selected from phenyl and naphthyl, whereby the heteroaryl and aryl groups are together, and whereby Hal is bonded to the parent molecular group via the aryl portion,

[0019] Preferably, R4 is a heteroaryl-aryl group consisting of a heteroaryl group and an aryl group, the heteroaryl group being selected from the group consisting of a fused bicyclic 9-10 membered heteroaryl group containing one, two or three heteroatoms, each heteroatom being selected from the group consisting of nitrogen, oxygen and sulfur, the aryl group being selected from the group consisting of phenyl and phthalyl, whereby the heteroaryl and aryl groups are linked together via a single bond, and whereby Ha2 is bonded to the parent molecular group via the aryl moiety,

[0020] Preferably, R4 is a heteroaryl-aryl group consisting of a heteroaryl group and an aryl group connected via a single bond, the heteroaryl group is selected from a monocyclic 5-membered heteroaryl group containing 3-4 heteroatoms, each heteroatom is independently selected from N, O and S, the aryl group is selected from phenyl and naphthyl, and the heteroaryl-aryl group is bonded to the parent molecular group via the aryl portion;

[0021] Preferably, R4 is a heteroaryl-aryl group, the heteroaryl aryl group consisting of a heteroaryl group and an aryl group connected via a single bond, the heteroaryl group being selected from a 9-10 membered heteroaryl group consisting of a benzene ring and a partially saturated fused bicyclic ring containing 1-2 heteroatoms, the heteroatoms being each independently selected from N, O and S, the aryl group being selected from phenyl and phthalyl, and the aryl moiety being bonded to the parent molecular group.

[0022] In an embodiment according to the present invention, the compound is selected from:

[0023]

[0024] The present invention also provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.

[0025] In an embodiment according to the present invention, it further comprises one or more of a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or vehicle.

[0026] In an embodiment according to the present invention, the pharmaceutical composition is an injection, an oral agent or a mucosal administration agent.

[0027] Another aspect of the present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable salt, or pharmaceutical composition in the preparation of a drug for treating or preventing a MAGI2-related disease. Preferably, the MAGI2-related disease is a tumor. Preferably, the tumor is gastric cancer.

[0028] The beneficial effects of the above technical solution of the present invention are as follows:

[0029] The compound provided by the present invention can target the molecule of the GK domain of the MAGI2 protein, and the molecule can inhibit the migration and invasion ability of tumors, and can be used for cancer treatment, especially the treatment of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the synthesis route of the aza-oxyindole compounds of the present invention;

[0031] Figure 2The structural formula of compound 1 prepared according to Example 2 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0032] Figure 3 The structural formula of compound 2 prepared according to Example 3 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0033] Figure 4 The structural formula of compound 19 prepared according to Example 4 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0034] Figure 5 The structural formula and hydrogen nuclear magnetic resonance spectrum of compound 24 prepared according to Example 5 of the present invention;

[0035] Figure 6 The structural formula and hydrogen nuclear magnetic resonance spectrum of compound 27 prepared according to Example 6 of the present invention;

[0036] Figure 7 The structural formula of compound 26 prepared according to Example 7 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0037] Figure 8 The structural formula of compound 22 prepared according to Example 8 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0038] Fig. 9 The structural formula of compound 25 prepared according to Example 9 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0039] Fig.10 The structural formula of compound 36 prepared according to Example 10 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0040] Fig.11 The structural formula of compound 37 prepared according to Example 11 of the present invention and its hydrogen nuclear magnetic resonance spectrum;

[0041] Fig.12 The stability of the screening system of MAGI2 GK (MAGI2-GK / FITC-SAPAP binding curve at different time points).

[0042] Fig.13 is the inhibition curve of compound 24 according to the present invention and MAGI2-GK / p-SAPAP;

[0043] Fig.14 To detect the effect of different concentrations of compound 24 on the migration ability of MGC803 cells;

[0044] Fig.15 To detect the effect of different concentrations of compound 24 on the invasion ability of MGC803 cells. DETAILED DESCRIPTION

[0045] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0046] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was detected by thin layer chromatography.

[0047] Example 1 Synthesis formula of the compound

[0048] The synthesis process of the aza-oxindole compound according to the present invention is as follows Figure 1 As shown, 1-methyl-1H-pyrrolo[2,3-b]pyridine-2,3-dione and differently substituted maleimides undergo a Morita-Baylis-Hillman reaction under the catalysis of a tertiary amine catalyst to generate target compounds with different substitutions at R4, and a base and dimethyl sulfate are added to obtain the target compound with hydroxymethylation protection at R2.

[0049] Dissolve 7-azaindole derivative a (0.1 mmol), maleimide derivative b (0.6 mmol), DABCO (20 mol%) (to prepare racemic compound) in 1 mL toluene, stir at 50°C for 24 h until the reaction of raw material a is complete, cool the reaction solution to room temperature, remove the solvent and purify by silica gel column chromatography to obtain racemic compounds 1-27 and 31-44, respectively. (Chiral compounds can be obtained using β-ICD (20 mol%) as a catalyst) Figure 1 (eq 1).

[0050] The 3-NBoc-substituted compound 27 (0.05 mmol) and trifluoroacetic acid (10 equiv.) were dissolved in 0.5 ml of toluene and reacted at room temperature for 3 h until the conversion of the raw materials was complete. The reaction solution was purified by silica gel column chromatography to obtain compound 28 ( Figure 1 (eq 2).

[0051] The 3-NH2-substituted compound 28 (0.05 mmol), propargyl bromide (1.5 equiv.), and DIPEA (3.0 equiv.) were dissolved in 0.5 ml DMF and reacted at room temperature for 24 h in an argon atmosphere until the raw materials were completely converted. The reaction solution was purified by silica gel column chromatography to obtain compound 29 ( Figure 1 (eq3).

[0052] The 3-NH2-substituted compound 28 (0.05 mmol) was dissolved in 0.5 mL of dichloromethane and stirred at 0°C for 5 min., followed by the addition of triethylamine (10 equiv.) and methyl 3-chlorosulfonyl-2-thiophenecarboxylate (1.0 equiv.), and the reaction was carried out at room temperature for 24 h. After the conversion of the raw materials was complete, the reaction solution was quenched with a saturated ammonium chloride solution, and then extracted three times with dichloromethane (1 mL). The organic phases were combined and dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain compound 30 ( Figure 1 (eq 3).

[0053] The list of some aza-oxindole compounds synthesized by the present invention is shown in Table 1:

[0054] Table 1 shows some of the aza-oxindole compounds synthesized by the present invention

[0055]

[0056]

[0057] Table 1 Synthesis of Compound 1 in Example 2

[0058]

[0059] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a1 (16.2mg, 0.1mmol), maleimide derivative b1 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a1 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 1 (30mg, yield 86%), white solid. The structural formula of compound 1 and its H NMR spectrum are shown as follows Figure 2 shown.

[0060] 1 H NMR (600MHz, CDCl3): δ8.35–8.21(m,1H),7.72–7.62(m,1H),7.20(d,J=8.2Hz,2H),7.11(d,J= 8.2Hz,2H),7.03(dd,J=7.0,5.6Hz,1H),6.93(s,1H),4.55(s,1H),3.34(s,3H),2.34(s,3H)ppm

[0061] Example 3 Synthesis of Compound 2

[0062]

[0063] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a1 (16.2mg, 0.1mmol), maleimide derivative b2 (124mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a1 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 2 (29mg, yield 79%) as a white solid. The structural formula of compound 2 and its H NMR spectrum are shown as follows Figure 3 shown.

[0064] 1 H NMR (600MHz, CDCl3): δ8.31 (dd, J=5.3, 1.4Hz, 1H), 7.64 (dd, J=7.3, 1.4Hz, 1H), 7.38 (d, J=8.8Hz, 2H),7.23(d,J=8.8Hz,2H),7.04(dd,J=7.2,5.4Hz,1H),6.95(s,1H),4.24(s,1H),3.35(s,3H)ppm

[0065] Example 4 Synthesis of Compound 19

[0066]

[0067] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a1 (16.2mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a1 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 19 (30mg, yield 85%), white solid. The structural formula of compound 19 and its H NMR spectrum are shown as follows Figure 4 shown.

[0068] 1H NMR (600MHz, CDCl3): δ8.29(d,J=5.1Hz,1H),7.61(d,J=7.3Hz,1H),7.32-7.26( m,5H),7.03-7.00(m,1H),6.69(s,1H),4.59(s,2H),3.93(s,1H),3.34(s,3H)ppm

[0069] Example 5 Synthesis of Compound 24

[0070]

[0071] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a2 (18.6mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a2 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 24 (25.7mg, yield 69%), white solid. The structural formula of compound 24 and its H NMR spectrum are shown as follows Figure 5 shown.

[0072] 1 H NMR (600MHz, CDCl3): δ8.34(dd,J=5.3,1.5Hz,1H),7.66(dd,J=7.4,1.5Hz,1H),7.31–7.25(m,5H),7.0 5(dd,J=7.3,5.3Hz,1H),6.66(s,1H),4.62(d,J=2.5Hz,2H),4.59(s,2H),3.94(s,1H),2.24(s,1H)ppm

[0073] Example 6 Synthesis of Compound 27

[0074]

[0075] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a3 (26.1mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a3 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=4:1) to separate the target product to obtain product 27 (18mg, yield 40%) as a light yellow solid. The structural formula of compound 27 and its H NMR spectrum are shown as follows: Figure 6 shown.

[0076] 1 H NMR (600MHz, CDCl3): δ8.25(dd,J=5.3,1.4Hz,1H),7.83(d,J=7.2Hz,1H),7.31–7.25(m,5H),7.00 (dd,J=7.3,5.3Hz,1H),6.41(s,1H),5.91(s,1H),4.63–4.56(m,2H),3.38(s,3H),1.31(s,9H)ppm

[0077] Example 7 Synthesis of Compound 26

[0078]

[0079] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a4 (23.8mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a4 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=3:1) to separate the target product to obtain product 26 (30.5mg, yield 58%), white solid. The structural formula of compound 26 and its H NMR spectrum are shown as follows Figure 7 shown.

[0080] 1H NMR (600MHz, CDCl3): δ8.26(dd,J=5.2,1.4Hz,1H),7.63–7.58(m,1H),7.44(d,J=7.2Hz,2H),7.27(ddd,J= 11.5,9.8,4.6Hz,8H),6.98(dd,J=7.3,5.4Hz,1H),6.63(s,1H),5.01(s,2H),4.57(s,2H),4.13(s,1H)ppm

[0081] Example 8 Synthesis of Compound 22

[0082]

[0083] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a5 (19mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a5 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=3:1) to separate the target product to obtain product 22 (16mg, yield 43%), white solid. The structural formula of compound 22 and its H NMR spectrum are shown as follows Figure 8 shown.

[0084] 1 H NMR (600MHz, CDCl3): δ8.29–8.24(m,1H),7.59(dd,J=7.3,1.1Hz,1H),7.31–7.25(m,5H),6.99(dd,J=7.2,5.4Hz,1H) ,6.71(s,1H),4.57(s,2H),4.03(s,1H),3.80(t,J=7.4Hz,2H),1.80(dd,J=14.8,7.4Hz,2H),0.98(t,J=7.4Hz,3H)ppm

[0085] Example 9 Synthesis of Compound 25

[0086]

[0087] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a6 (21.6mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a6 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=3:1) to separate the target product to obtain product 25 (22mg, yield 55%), white solid. The structural formula of compound 25 and its H NMR spectrum are shown as follows: Fig. 9 shown.

[0088] 1 H NMR (600MHz, CDCl3): δ8.25(dd,J=5.2,1.4Hz,1H),7.55(dd,J=7.3,1.4Hz,1H),7.30–7.22(m,5H),6.96(dd,J=7.2,5.3Hz,1H ),6.72(s,1H),4.81(p,J=8.4Hz,1H),4.56(s,2H),2.28–2.17(m,2H),1.96(t,J=13.0Hz,4H),1.64(dd,J=6.9,3.4Hz,2H)ppm

[0089] Example 10 Synthesis of Compound 36

[0090]

[0091] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a7 (17.6mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a7 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 36 (20mg, yield 54%), white solid. The structural formula of compound 36 and its H NMR spectrum are shown as follows Fig.10 shown.

[0092] 1H NMR (600MHz, CDCl3): δ8.04(s,1H),7.40(s,1H),7.30–7.21(m,5H),6.75(d,J=3.5Hz,1H),4.55(s,2H),3.28(s,3H),2.26(s,3H)ppm

[0093] Example 11 Synthesis of Compound 37

[0094]

[0095] Take a 10mL reaction tube, add a magnetic stirrer, weigh 7-azaindole derivative a8 (19.2mg, 0.1mmol), maleimide derivative b19 (112mg, 0.6mmol), β-ICD (6.2mg, 20mol%), then add 1mL of dry toluene and add a rubber stopper, stir at 50°C for 24h, use thin layer chromatography (Thin Layer Chromatography, TLC) to determine that the raw material a8 is completely converted and then stop the reaction, remove toluene under vacuum, and use column chromatography (PE / EA=2:1) ​​to separate the target product to obtain product 37 (27.6mg, yield 73%), light yellow solid. The structural formula of compound 37 and its H NMR spectrum are shown as follows Fig.11 shown.

[0096] 1 H NMR (600MHz, CDCl3): δ7.91(d,J=2.7Hz,1H),7.29–7.21(m,6H),6.74(s,1H),4.71(s,1H),4.56(s,2H),3.80(s,3H),3.28(s,3H)ppm

[0097] Example 12: Purification and expression of the GK domain of MAGI2 protein

[0098] The MAGI2 GK domain gene sequence was cloned into the pET-15b vector containing a His×6 tag at the N-terminus. After sequencing, it was transformed into BL21 (DE3) Escherichia coli for expression. After immunoblotting, it was amplified and expressed. 0.2 M IPTG (isopropyl-β-D-thiogalactoside) was used to induce prokaryotic expression of the protein at 16°C for 18 h. The His×6-tagged protein was expressed using Ni 2+ The protein was first purified by -NTA agarose affinity chromatography and then purified by size exclusion chromatography using a protein eluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).

[0099] Example 13: Evaluation of Binding Activity of the Compounds Synthesized in Example 1 Using Polarized Fluorescence Method

[0100] (1) Establishment of polarized fluorescence experimental method:

[0101] (A) Screening method: Use a black 96-well plate for sample loading, the experimental temperature is room temperature, the reaction system is 80μL, the buffer is PH7.5 Tris-HCl buffer (50mM Tris, 100mM NaCl), the FITC fluorescence excitation wavelength is set to 485nm, the emission wavelength is set to 530nm, and the reading is read using a multifunctional microplate reader. In the screening system, the MAGI2 GK protein is 1μM, the FITC-SAPAP concentration is set to 10nM, and the screening experiment process is as follows: first, a certain concentration of MAGI2 GK protein solution and FITC-SAPAP solution are added to the system to allow them to fully combine, and then the small molecule compound to be screened is added. After oscillation for 10 minutes, it is allowed to stand at room temperature away from light for two hours, and the system polarization value is read. Depending on whether the system polarization value decreases, it is determined whether it is a positive compound. The results are as follows Fig.12 shown.

[0102] (2) Calculate the half inhibitory concentration IC50 and affinity constant Ki of the compound

[0103] (A) Calculation of compound half-inhibitory concentration IC50 value: Compounds were set with a series of concentrations of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 250 μM and 500 μM, 20 μL of the above compound solutions of different concentrations were added to 40 μL of protein solution and 20 μL of FITC-SAPAP system, and the polarization value was read after incubation for 2 hours. IC50 was calculated using GraphPad Prism8 software using the nonlinear regression least squares method. 50 value.

[0104] (B) Calculation of affinity constant Ki value of compound: Since the Cheng–Prusoff formula for calculating Ki value traditionally is not applicable to competitive binding experimental conditions of polarized fluorescence, the polarized fluorescence experimental Ki value calculation method is used in the present invention (see formula), wherein P represents protein molecule, L represents fluorescently labeled ligand molecule, I represents inhibitor, PL represents protein-fluorescent ligand complex, PI represents protein inhibitor complex, [P] T and [L] T Represent the total concentration of protein and ligand respectively, [P]0 and [PL]0 represent the concentration of protein P and protein-ligand complex PL when the inhibition rate is 0, [I] 50 , [L] 50 and [PL] 50They represent the concentrations of inhibitor, fluorescently labeled ligand and protein fluorescently labeled ligand complex when the inhibition rate is 50%, respectively. According to formula 1-3, [P]0, [L] 50 and[I] 50 The Ki value was calculated by combining the IC50 and Kd values ​​using Formula 4.

[0105] The inhibition constant K of compound 24 was calculated. i The inhibition constant was 0.477 μM.

[0106]

[0107] [L] 50 =[L] T -[PL]0 / 2 (2)

[0108] [I] 50 =IC 50 -[P] T +K d ×[PL] 50 / [L] 50 +[PL] 50 (3)

[0109] Ki=[I] 50 / ([L] 50 / K d +[P]0 / K d +1) (4)

[0110] The inhibition curve of compound 24 prepared according to the present invention and MAGI2-GK / p-SAPAP is as follows: Fig.13 shown.

[0111] Example 14: Cell migration and invasion assay of compound 24 in the example

[0112] Scratch test:

[0113] Principle of assay: The cell scratch test is a common method for analyzing cell migration ability. When cells grow to a fused monolayer state, a blank area is artificially created on the fused monolayer of cells, called a "scratch". The cells at the edge of the scratch will gradually enter the blank area to heal the "scratch", simulating the process of cell migration in vivo to a certain extent. The stronger the cell migration ability, the smaller the remaining area of ​​the scratch area.

[0114] Experimental steps:

[0115] (1) Cell plating: Invert a six-well plate and mark three equally spaced horizontal lines on the bottom of each well with a marker. Digest and resuspend MGC803 cells in logarithmic growth phase, count the cells, and plate at a density of 800,000 cells per well, and continue culturing in an incubator.

[0116] (2) Cell scratching and drug treatment: When the cell confluence of the six-well plate reaches more than 90%, use a 10μL pipette tip to scratch, set three vertical and equidistant scratches in each well, and use PBS buffer to wash and remove cell debris. The compound is set to 0μM, 10μM, and 50μM, and the serum-free culture medium is used for gradient dilution. The volume is 2.5ml, and 2ml of compound solution of different concentrations is slowly added to each well along the wall. After the compound solution and the adherent cells are fully mixed, an inverted microscope is used to take pictures at 0h. The field of view of 6 different positions at the intersection of the scratch and the bottom line of each well is sampled. After taking pictures, the well plate is placed in the incubator for continued culture, and pictures are taken and recorded at 12h, 24h, and 48h.

[0117] Transwell experiment:

[0118] Experimental principle: Place the Transwell chamber in the culture plate. The chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper chamber contains the upper culture medium, and the lower chamber contains the lower culture medium. The upper and lower culture mediums are separated by a polycarbonate membrane. We planted cells in the upper chamber. Since the polycarbonate membrane is permeable, the components in the lower culture medium can affect the cells in the upper chamber, so that we can study the effects of the components in the lower culture medium on cell growth, movement, etc. The Matrigel used in the invasion experiment is a matrix component extracted from mouse EHS sarcoma, containing LN, type IV collagen, contact protein and heparin sulfate polysaccharide, which is spread on a polycarbonate filter membrane without polyvinylpyrrolidone and can reconstruct a membrane structure in DMEM culture medium. This membrane structure is very similar to the natural matrix membrane structure. The pore size of the filter membrane of the Transwell chamber is generally 8μm. In the invasion experiment, the membrane pores are covered with Matrigel, and the cells cannot pass through freely. They must secrete hydrolases and deform to pass through the filter membrane covered with Matrigel, which is similar to the situation in the body. If cells want to enter the lower chamber, they must first secrete matrix metalloproteinases (MMPs) to degrade the matrix gel before they can pass through the polycarbonate membrane. Counting the number of cells entering the lower chamber can reflect the invasive ability of tumor cells.

[0119] Experimental steps:

[0120] Take cells in good condition, digest them, resuspend them and count them, then spread them on a six-well plate so that there are 200,000 cells in each well, mark them, and place them in an incubator for 12 hours. After the cells adhere, use complete culture medium to prepare compound solutions, with concentrations set to 0μM, 10μM and 50μM, remove the old culture medium, and add culture medium containing different concentrations of compounds for pretreatment for 12 hours.

[0121] Take a 24-well plate and a transwell chamber, prepare working matrix gel at a matrix gel: air culture ratio of 1:4, evenly add 30 μL of matrix gel to each well, and place it in a 37°C incubator for 2 hours. Use DMEM medium (serum-free) to prepare compound solutions of different concentrations of 0 μM, 10 μM and 50 μM. After digestion and centrifugation of the cells pretreated with the compounds in the previous step, resuspend the cells in DMEM medium (serum-free) containing different concentrations of compounds, and adjust the cell density to 1.25×10 5 / mL, add 200μL of cell suspension, that is, 25,000 cells per well, and set 3 replicates for each compound concentration. Add 500μL of DMEM complete medium containing 10% serum to the lower chamber and put it in the incubator. After 24h, carefully remove the transwell chamber, fix it with 4% paraformaldehyde for 20min, and then stain the cells with 0.1% crystal violet for 20min. Use PBS buffer to gently rinse the stained chamber 3-5 times to remove excess crystal violet solution, gently wipe off the remaining cells and matrix glue in the upper chamber with a cotton swab, dry it, use an inverted microscope to randomly select different fields of view for photography and record, and use ImagJ software to count cells.

[0122] Experimental results:

[0123] As a skeleton protein, MAGI2 protein is closely related to the migration and invasion of tumor cells. Based on the fact that compound 24 can target MAGI2 protein, the inventors investigated the effects of the compound on the migration and invasion of gastric cancer cell line MGC803. Fig.14 As shown in Figure 2, compound 24 can significantly inhibit the migration of MGC803. Fig.15 As shown, compound 24 can also inhibit the invasion ability of MGC803 cell line. The above results indicate that the compound can inhibit the migration and invasion of tumor cell line MGC803.

[0124] Therefore, in summary, the present invention purifies the GK domain protein of MAGI2 and uses polarized fluorescence experiments to confirm that aza-oxindole compounds can target the GK domain of MAGI2 protein. Further, scratch experiments and transwell experiments confirm that such compounds can inhibit tumor cell migration and invasion.

[0125] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A compound targeting the MAGI2 GK domain, or a pharmaceutically acceptable salt thereof, having the following structural formula:

2. A pharmaceutical composition, characterized in that Comprising the compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, characterized in that It also contains a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 3, characterized in that The excipient is one or more of a diluent, an adjuvant or a vehicle.

5. The pharmaceutical composition according to claim 3, characterized in that The composition is for injection, oral administration or mucosal administration.

6. Use of the compound or pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to any one of claims 2 to 5, in the preparation of a medicament for treating or preventing a MAGI2 GK-related disease.

7. The use according to claim 6, characterized in that The MAGI2 GK-related disease is a tumor.

8. The use according to claim 7, characterized in that The tumor is gastric cancer.

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