A pyrimidine compound, its preparation method and application

Abstract

The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a pyrimidine compound, a preparation method thereof and an application. The pyrimidine compound provided by the present invention has a structure shown in Formula I, and the pyrimidine compound provided by the present invention has the function of dual-targeting inhibition of VEGFR and PARP.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a pyrimidine compound, a preparation method thereof, and an application thereof. Background Art

[0002] During the continuous growth, invasion, and metastasis of tumors, the nutritional supply of tumor neovascularization is indispensable. Research shows that neovascularization is not only closely related to the growth, infiltration, and metastasis of solid tumors, but also significantly related to prognosis. Different angiogenic factors are preferentially expressed at different stages of this process, and vascular endothelial growth factors (VEGFs), as the key factor with the strongest pro-tumor angiogenesis activity, are expressed throughout the entire process of tumor development. Moreover, vascular endothelial growth factor receptors (VEGFRs) are one of the important molecules mediating tumor-related angiogenesis and lymphangiogenesis, and are abnormally expressed in breast cancer. Anti-angiogenic drugs represented by VEGF monoclonal antibodies and VEGFR tyrosine kinase inhibitors act on tumor blood vessels or lymphatic endothelial cells to inhibit the formation of new blood vessels or lymphatics, thereby blocking the nutritional supply of tumors and curbing the malignant progression of tumors. All along, the discovery of drugs targeting VEGFR has attracted the attention of major pharmaceutical companies and researchers, and pan-VEGFR inhibitors have also played a good role in the treatment of many solid tumors. In the clinical treatment of different types of tumors, VEGFR inhibitors show varying degrees of clinical benefits by inhibiting tumor-related angiogenesis. However, the disadvantages of pan-VEGFR inhibitors are also very prominent, including serious adverse reactions caused by inhibiting physiological angiogenesis (such as bleeding, delayed wound healing, gastrointestinal perforation, hypertension, thromboembolic complications, proteinuria, etc.), short duration of antitumor effect, and therapeutic resistance caused by the activation of angiogenesis compensatory signaling pathways, which greatly limit the clinical application of this type of inhibitor.

[0003] To increase the efficacy of VEGFR inhibitors and reduce toxic side effects, VEGFR inhibitors are often combined with chemotherapy, radiotherapy, surgery, endocrine therapy and other regimens in clinical practice to achieve the maximum synergistic effect. Among them, a lot of preclinical and clinical data have proved that the combination of VEGFR inhibitors and inhibitors of many tumor-related targets has a synergistic effect, including epigenetic drugs (HDAC inhibitors), immunotherapy drugs (PD-1 and PD-L1 inhibitors and monoclonal antibodies), and other RTK inhibitors (EGFR inhibitors, FGFR inhibitors, BRAF inhibitors and c-Met inhibitors), etc. Although combination therapy is a clinically effective strategy for tumor treatment, the combination dose of drugs, the complex pharmacokinetic characteristics of the two drugs and potential toxic side effects also need to be considered. At present, dual-target inhibitors with synergistic effects have received increasing attention in the development of anti-tumor drugs. As an alternative strategy to combination therapy, dual-target drugs have certain advantages. First of all, dual-target drugs not only largely retain the advantages of combination therapy, but also partially overcome the disadvantages of combination therapy. Since it is an integrated molecule, the risk of drug-drug adverse interactions of dual-target drugs is low, the pharmacokinetic parameters are stable, the probability of target drug resistance is low, and the patient compliance is high. Therefore, the design and discovery of novel and highly effective VEGFR dual-target inhibitors are expected to provide new methods and ideas for the treatment of TNBC with VEGFR-related inhibitors.

[0004] In the related combination strategy of VEGFR inhibitors, the pan-VEGFR inhibitor cediranib is also combined with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib to treat patients with recurrent ovarian cancer and breast cancer (NCT02484404). Mechanistic studies have shown that VEGFR inhibitors form a hypoxic environment by inhibiting angiogenesis, resulting in downregulation of the expression of key homologous recombination repair HRR factors (RAD51, BRCA1 / 2) and reduced DNA repair ability. Developing small molecule inhibitors that dual-target VEGFR / PARP may be an effective strategy for treating non-BRCA mutant breast cancer, and there is currently no report on small molecule inhibitors that dual-target VEGFR / PARP. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a pyrimidine compound having the structure shown in Formula I:

[0006]

[0007] In Formula I, R1 is CH3 or H; R 2 is H, CF3, CH3 or OCH3;

[0008] R3 is

[0009] Rx, Rn, Rm, Ry and Rz are independently F, Cl, H, OCH3, CF3 or CH3; R4 is or H; R5 is F or H; R6 is F or H. Preferably, it has the structures shown in Formulas 1 to 20:

[0010]

[0011]

[0012] The present invention also provides a preparation method of the pyrimidine compounds described above, including the following steps:

[0013] Dissolve the 2,4-dichloropyrimidine derivative with the structure shown in Formula II and 2,3-dimethyl-6-amino-2H-indazole first, and then carry out a first substitution reaction under alkaline conditions to obtain the intermediate product III with Formula III;

[0014] Dissolve the intermediate product III and the methylation reagent second, and then carry out a methylation reaction under a protective atmosphere and alkaline conditions to obtain a methylated compound with the structure of Formula IV;

[0015] Dissolve the methylated compound and the aminobenzoic acid derivative third, and then carry out a second substitution reaction under acidic conditions to obtain an aminated product; the aminobenzoic acid derivative includes a p-aminobenzoic acid derivative with the structure of Formula V-1 and an m-aminobenzoic acid derivative with the structure of Formula V-2;

[0016] When the aminobenzoic acid derivative is a p-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-1, and when the aminobenzoic acid derivative is an m-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-2;

[0017] Dissolve the aminated product with the structure of Formula VI-1, the first condensing agent and the benzimidazole compound with the structure of Formula VII fourth, and then carry out a first amide condensation reaction under alkaline conditions to obtain a first pyrimidine compound;

[0018] The first pyrimidine compound has the structure shown in Formula I and R3 is

[0019] Dissolve the aminated product with the structure of Formula VI-1 or the aminated product with the structure of Formula VI-2, the second condensing agent and the aniline derivative with the structure of Formula VIII fifth, and then carry out a second amide condensation reaction. Mix the obtained second condensation product with acetic acid and carry out a first cyclization reaction to obtain a second pyrimidine compound and a third pyrimidine compound respectively;

[0020] The second pyrimidine compound has the structure shown in formula I, and R3 is

[0021] The third pyrimidine compound has the structure shown in formula I, and R3 is

[0022]

[0023] Preferably, the molar ratio of the 2,4-dichloropyrimidine derivative to 2,3-dimethyl-6-amino-2H-indazole is 1:1.2 to 2; the temperature of the first substitution reaction is 80 to 90 °C, and the time is 12 to 15 h.

[0024] Preferably, the methylation reagent is CH3I; the molar ratio of the intermediate III to the methylation reagent is 1:1.1 to 1.5; the temperature of the methylation reaction is 25 to 30 °C, and the time is 6 to 18 h.

[0025] Preferably, the molar ratio of the methylated compound to the aminobenzoic acid derivative is 1:1.2 to 1.5; the temperature of the second substitution reaction is 80 to 90 °C, and the time is 15 to 20 h.

[0026] Preferably, the first condensing agent is 1-hydroxybenzotriazole and 1-ethyl-3(3-dimethylpropylamine)carbodiimide; the temperature of the first amide condensation reaction is -5 to 0 °C, and the time is 15 to 20 h.

[0027] Preferably, the second condensing agent is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the temperature of the second amide condensation reaction is 25 to 30 °C, and the time is 24 to 30 h; the temperature of the first cyclization reaction is 110 to 130 °C, and the time is 15 to 20 h.

[0028] Preferably, the temperature of the third amide condensation reaction is 25 to 30 °C, and the time is 24 to 30 h.

[0029] The present invention also provides the use of the pyrimidine compound or the preparation method as described above in the preparation of a dual-target VEGFR / PARP inhibitor.

[0030] The present invention provides a pyrimidine compound having the structure shown in Formula I. The 2,3-dimethyl-6-amino-2H-indazole fragment in Formula I occupies the key hydrophobic pocket of VEGFR2. The 2-aminopyrimidine moiety forms two key hydrogen bonds with the Cys917 residue in the hinge region. The indazole and aminopyrimidine fragments are the key pharmacophores for the binding of the small molecule to VEGFR2. In addition, the 1H-benzo[d]imidazole fragment in Formula I forms key hydrogen bonds with the Gly863 and Ser904 residues in the nicotinamide binding pocket of the target PARP1, and forms a π-π stacking interaction with Tyr907. This fragment is the key pharmacophore for the binding of Formula I to PARP1. Therefore, the pyrimidine compound provided by the present invention has the effect of dual-targeting inhibition of VEGFR and PARP. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a test result diagram for exploring the DNA damage mechanism of compound 17, positive control Olaparib (Ola), Pazopanib (Paz), and their combination by immunoblotting assay in BRCA wild-type breast cancer cells;

[0032] Figure 2 It is a diagram showing the change in the tumor volume of nude mice in the MDA-MB-231 xenograft tumor model treated with compound 17, Olaparib, Pazopanib, the combination of Olaparib and Pazopanib for 15 days;

[0033] Figure 3 It is a diagram showing the lung metastasis foci of nude mice after 15 days of treatment with compound 17, Olaparib, Pazopanib, the combination of Olaparib and Pazopanib in the MDA-MB-231 lung metastasis model of nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides a pyrimidine compound having the structure shown in Formula I:

[0035]

[0036] In the said Formula I, R1 is CH3 or H; R 2 is H, CF3, CH3 or OCH3;

[0037] R3 is

[0038] Rx, Rn, Rm, Ry and Rz are independently F, Cl, H, OCH3, CF3 or CH3;

[0039] R4 is or H; R5 is F or H; R6 is F or H.

[0040] In the present invention, the pyrimidine compound is specifically preferably a compound having the structures shown in Formula 1 to Formula 20:

[0041]

[0042]

[0043]

[0044] The present invention also provides a method for preparing the above-mentioned pyrimidine compound, comprising the following steps:

[0045] Dissolve a 2,4-dichloropyrimidine derivative having the structure shown in Formula II and 2,3-dimethyl-6-amino-2H-indazole first, and then carry out a first substitution reaction under alkaline conditions to obtain an intermediate product III having the structure of Formula III;

[0046] Dissolve the intermediate product III and a methylation reagent second, and then carry out a methylation reaction under a protective atmosphere and alkaline conditions to obtain a methylated compound having the structure of Formula IV;

[0047] Dissolve the methylated compound and an aminobenzoic acid derivative third, and then carry out a second substitution reaction under acidic conditions to obtain an aminated product; the aminobenzoic acid derivative includes a p-aminobenzoic acid derivative having the structure of Formula V-1 and an m-aminobenzoic acid derivative having the structure of Formula V-2;

[0048] When the aminobenzoic acid derivative is a p-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-1, and when the aminobenzoic acid derivative is an m-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-2;

[0049] Dissolve the aminated product having the structure of Formula VI-1, a first condensing agent and a benzimidazole compound having the structure of Formula VII fourth, and then carry out a first amide condensation reaction under alkaline conditions to obtain a first pyrimidine compound;

[0050] The first pyrimidine compound has the structure shown in Formula I and R3 is

[0051] Dissolve the aminated product having the structure of Formula VI-1 or the aminated product having the structure of Formula VI-2, a second condensing agent and an aniline derivative having the structure of Formula VIII fifth, and then carry out a second amide condensation reaction. Mix the obtained second condensation product with acetic acid and carry out a first cyclization reaction to obtain a second pyrimidine compound and a third pyrimidine compound respectively;

[0052] The second pyrimidine compound has the structure shown in Formula I, and R3 is

[0053] The third pyrimidine compound has the structure shown in Formula I, and R3 is

[0054] The first pyrimidine compound, the second pyrimidine compound and the third pyrimidine compound constitute the pyrimidine compounds described in the above technical solution;

[0055]

[0056] In the present invention, the 2,4-dichloropyrimidine derivative having the structure shown in Formula II and 2,3-dimethyl-6-amino-2H-indazole are first dissolved and then subjected to a first substitution reaction under alkaline conditions to obtain an intermediate product III having Formula III.

[0057] In the present invention, the first solvent for the first dissolution is preferably C2H5OH and THF; the dosage ratio of the 2,4-dichloropyrimidine derivative to the first solvent is preferably 1 mmol: 16 - 20 mL, more preferably 1 mmol: 17 - 18 mL. In the present invention, the alkaline condition is preferably adjusted to 10 by NaHCO3. In the present invention, the molar ratio of the 2,4-dichloropyrimidine derivative to 2,3-dimethyl-6-amino-2H-indazole is preferably 1: 1.2 - 2, more preferably 1: 1.5.

[0058] In the present invention, the temperature of the first substitution reaction is preferably 80 - 90 °C, more preferably 85 °C; the time is preferably 12 - 15 h, more preferably 13 - 14 h.

[0059] After obtaining the intermediate product III, the present invention dissolves the intermediate product III and a methylation reagent for the second time and then conducts a methylation reaction under a protective atmosphere and alkaline conditions to obtain a methylated compound having the structure of Formula IV.

[0060] In the present invention, the methylation reagent is preferably CH3I. In the present invention, the second solvent for the second dissolution is preferably DMF. In the present invention, the alkaline condition for the methylation reaction is preferably adjusted to pH 10 by CsCO3. In the present invention, the protective atmosphere is preferably nitrogen.

[0061] In the present invention, the dosage ratio of the intermediate product III to the second solvent is preferably 1 mmol: 8 - 10 mL, more preferably 1 mmol: 9 mL. In the present invention, the molar ratio of the intermediate product III to the methylation reagent is preferably 1: 1.1 - 1.5, more preferably 1: 1.2. In the present invention, the temperature of the methylation reaction is preferably 25 - 30 °C, more preferably 25 °C, and the time is preferably 6 - 18 h, more preferably 7 - 12 h.

[0062] After obtaining the methylated compound, the present invention dissolves the methylated compound and the aminobenzoic acid derivative for the third time, and then conducts a second substitution reaction under acidic conditions to obtain an aminated product.

[0063] In the present invention, the third solvent for the third dissolution is preferably isopropyl alcohol. In the present invention, the acidic condition is preferably adjusted to a pH value of 2 with hydrochloric acid. In the present invention, the aminobenzoic acid derivative includes a p-aminobenzoic acid derivative having a structure of formula V-1 and a m-aminobenzoic acid derivative having a structure of formula V-2.

[0064] In the present invention, the dosage ratio of the methylated compound to the second solvent is preferably 1-1.2 mmol: 15-20 mL, more preferably 1.1 mmol: 17-18 mL. In the present invention, the molar ratio of the methylated compound to the aminobenzoic acid derivative is preferably 1: 1.2-1.5, more preferably 1: 1.3-1.4.

[0065] In the present invention, the temperature of the second substitution reaction is preferably 80-90 °C, more preferably 85 °C, and the time is preferably 15-20 h, more preferably 17-18 h.

[0066] In the present invention, when the aminobenzoic acid derivative is a p-aminobenzoic acid derivative, the aminated product has a structure shown in formula VI-1, and when the aminobenzoic acid derivative is a m-aminobenzoic acid derivative, the aminated product has a structure shown in formula VI-2.

[0067] After obtaining the aminated product having a structure shown in formula VI-1, the present invention dissolves the aminated product having a structure shown in VI-1, the first condensing agent, and the benzimidazole compound having a structure of formula VII for the fourth time, and then conducts a first amide condensation reaction under basic conditions to obtain a first pyrimidine compound.

[0068] In the present invention, the fourth solvent for the fourth dissolution is preferably DMF. In the present invention, the first condensing agent is 1-hydroxybenzotriazole (HOBT) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI); the molar ratio of HOBT to EDCI is preferably 1: 1.1-1.5, more preferably 1: 1.2-1.3. In the present invention, the basic condition for the first amide condensation reaction is preferably provided by Et3N, and the pH value of the basic condition is preferably 9-10.

[0069] In the present invention, the dosage ratio of the amidated product having the structure of Formula VI-1 to the fourth solvent is preferably 1 mmol: 10-15 mL, more preferably 1: 12 mL. In the present invention, the molar ratio of the amidated product having the structure of Formula VI-1 to the benzimidazole compound is preferably 1: 1.2-1.5, more preferably 1: 1.3. In the present invention, the molar ratio of the amidated product having the structure of Formula V-1 to the first condensing agent is preferably 1: 1-1.2, more preferably 1: 1.1.

[0070] In the present invention, the temperature of the first amide condensation reaction is preferably -5 to 0 °C, more preferably -1 to -3 °C, and the time is preferably 15 to 20 h, more preferably 17 to 18 h.

[0071] In the present invention, after the first amide condensation reaction, it is preferably further included to extract the first amide condensation reaction system, and the obtained organic phase is successively dried with a desiccant, concentrated under reduced pressure, and subjected to silica gel column chromatography.

[0072] In the present invention, the extraction reagent is preferably ethyl acetate, the desiccant is preferably anhydrous sodium sulfate, and the developing agent for the silica gel column chromatography is preferably a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 15:1.

[0073] In the present invention, the first pyrimidine compound has the structure shown in Formula I and R3 is Specifically: Compounds having the structures of Formula 2-3 and Formula 12-15 belong to the first pyrimidine compounds.

[0074] After obtaining the amidated product, in the present invention, the amidated product having the structure of Formula VI-1 or the amidated product having the structure of Formula VI-2, the second condensing agent, and the aniline derivative having the structure of Formula VIII are dissolved fifth, and then the second amide condensation reaction is carried out. The obtained second condensation product is mixed with acetic acid to carry out the first cyclization reaction, respectively obtaining the second pyrimidine compound and the third pyrimidine compound.

[0075] In the present invention, the fifth solvent for the fifth dissolution is preferably DMF. In the present invention, the second condensing agent is preferably 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0076] In the present invention, the molar ratio of the amidated product having the structure of formula V-1 to the second condensing agent is preferably 1:2 to 5, more preferably 1:2 to 4. In the present invention, the molar ratio of the amidated product having the structure of formula V-1 to the aniline derivative is preferably 1:1.2 to 1.5, more preferably 1:1.3. In the present invention, the dosage ratio of the amidated product having the structure of formula V-1 to the fifth solvent is preferably 1 mmol:10 to 15 mL, more preferably 1 mol:12 to 13 mL.

[0077] In the present invention, the temperature of the second amide condensation reaction is preferably 25 to 30 °C, more preferably 25 °C; the time is preferably 24 to 30 h, more preferably 25 h.

[0078] In the present invention, the molar ratio of the second condensation product to acetic acid is preferably 1 mmol:18 to 20 mL, more preferably 1:19 mL. In the present invention, the temperature of the first cyclization reaction is preferably 110 to 130 °C, more preferably 120 °C; the time is preferably 15 to 20 h, more preferably 17 to 18 h.

[0079] In the present invention, after the first cyclization reaction, it is preferably further included to extract the first cyclization reaction, and the obtained organic phase of the extraction is dried with a desiccant, concentrated and separated by a silica gel column.

[0080] In the present invention, the extraction reagent is preferably ethyl acetate, the desiccant is preferably anhydrous sodium sulfate, and the developing agent for the silica gel column chromatography is preferably a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 10:1.

[0081] In the present invention, the second pyrimidine compound has the structure shown in formula I, and R3 is Specifically: Compounds having the structures of formula 1, 4 to 11 belong to the second pyrimidine compounds.

[0082] In the present invention, the dosage ratio of the amidated product having the structure of formula V-2 to the fifth solvent is preferably 1 mmol:12 to 15 mL, more preferably 1 mmol:13 to 14 mL. In the present invention, the molar ratio of the amidated product having the structure of formula V-2 to the aniline compound is preferably 1:1 to 1.5, more preferably 1:1.3 to 1.4.

[0083] In the present invention, the third pyrimidine compound has the structure shown in formula I, and R3 is Specifically, compounds having the structures of formula 16 to 20 belong to the third pyrimidine compounds.

[0084] The present invention also provides the use of the pyrimidine compounds described above or the pyrimidine compounds prepared by the preparation method described in any one of the above in the preparation of a dual-target VEGFR / PARP inhibitor.

[0085] In the present invention, the dual-target VEGFR / PARP inhibitor includes the pyrimidine compounds described above and pharmaceutically acceptable salts. In the present invention, the pharmaceutically acceptable salts preferably include nitrates, hydrochlorides, sulfates or phosphates.

[0086] In the present invention, the form of the dual-target VEGFR / PARP inhibitor preferably includes tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, suppositories, powders or aerosols.

[0087] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0088] Example 1

[0089] Compound 1 has the following structure:

[0090]

[0091] The preparation process is as follows:

[0092] (1) Dissolve 1 mmol of 2,4-dichloropyrimidine and 1.5 mmol of 2,3-dimethyl-6-amino-2H-indazole in 20 mL of C2H5OH and THF (the volume ratio of C2H5OH to THF is 4:1), then adjust the pH to 10 with NaHCO3, and carry out the first substitution reaction (temperature: 85 °C, time: 12 h) to obtain an intermediate product.

[0093] (2) Dissolve 1 mmol of the intermediate product obtained in step (1) and 1.1 mmol of CH3I in 10 mL of DMF, adjust the pH to 10 with CsCO3, and carry out the methylation reaction under nitrogen protection. The temperature of the methylation reaction is 25 °C and the time is 18 h to obtain a methylated compound.

[0094] (3) Dissolve 0.5 mmol of the methylated product obtained in step (2) and 0.75 mmol of p-aminobenzoic acid in 25 mL of isopropanol, then adjust the pH to 2 with hydrochloric acid, and carry out the second substitution reaction at a temperature of 85 °C to obtain an aminated product.

[0095] (4) Dissolve 0.5 mmol of the amidated product obtained in step (3), 1 mmol of HATU, and 0.51 mmol of 2,3-diaminobenzamide in DMF, adjust the pH to 10 with DIPE, and then carry out an amide condensation reaction at 25 °C. Dissolve the obtained product in 30 mL of acetic acid, and then reflux (cyclize) at 120 °C for 12 h. Pour the system obtained from the cyclization reaction into 200 mL of water, extract with ethyl acetate (3 × 40 mL), add anhydrous sodium sulfate to dry the obtained organic phase, concentrate under reduced pressure, and then separate by silica gel column. The eluent is a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol is 10:1), and finally compound 1 is obtained.

[0096] The high-resolution mass spectrometry detection results of compound 1 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 28 H 25 N9NaO 526.2074,found 526.2074。

[0097] The spectral information of the 1H NMR of compound 1 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.00(s,1H),8.82(s,1H),8.58–8.27(m,1H),8.13–7.99(m,1H),7.97–7.55(m,5H),7.45(d,J=12.1Hz,1H),7.22(t,J=7.7Hz,1H),6.88(t,J=9.1Hz,1H),5.92–7.84(m,1H),4.06(s,3H),3.47(s,3H),2.63(s,3H)。

[0098] The spectral information of the 13C NMR of compound 1 is as follows: 13 C NMR(151MHz,DMSO-d6)δ172.81,166.80,162.91,159.46,156.33,150.63,147.49,144.42,142.33,132.65,132.38,132.15,123.12,122.58(2),122.35(2),120.32(2),120.09(2),119.33,117.36,114.48,97.89,38.58,37.84,9.88。

[0099] Example 2

[0100] Compound 2 has the following structure:

[0101]

[0102] Steps (1)-(3) are the same as in Example 1.

[0103] (4) Dissolve 0.5 mmol of the amidated product obtained in step (3), 1 mmol of HOBT, 1.01 mmol of EDCI, and 0.75 mmol of 2-amino-1H-benzimidazole-4-carboxamide in DMF, adjust the pH to 10 with Et3N, then carry out an amide condensation reaction at 0 °C. Pour the system obtained from the amide condensation reaction into 200 mL of water, extract with ethyl acetate (3 × 40 mL), add anhydrous sodium sulfate to dry the obtained organic phase, concentrate under reduced pressure, and then separate by silica gel column. The eluent is a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol is 15:1), and finally Compound 2 is obtained.

[0104] The high-resolution mass spectrometry detection results of Compound 2 are as follows: HRMS(ESI)(m / z):[M+H] + calcd forC 29 H 27 N 10 O2547.2313,found 547.2323.

[0105] The spectral information of the 1H NMR of Compound 2 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.44(s,1H),8.63(s,1H),7.98(dd,J=8.2,1.3Hz,1H),7.89(d,J=6.0Hz,1H),7.82–7.64(m,5H),7.47(d,J=1.0Hz,1H),7.37(t,J=7.9Hz,1H),7.22(t,J=7.8Hz,1H),6.90(dd,J=8.8,1.6Hz,1H),5.82(d,J=6.0Hz,1H),4.06(s,3H),3.53(s,3H),2.62(s,3H).

[0106] The spectral information of the 13C NMR of Compound 2 is as follows: 13 C NMR(151MHz,DMSO-d6)δ167.22,162.87,159.89,156.28,147.45,142.37,141.96,132.65(2),128.87(2),122.97(2),122.25(2),120.66(2),120.13(2),119.95(2),119.07(2),114.35(2),97.27,38.49,37.82,9.86.

[0107] Example 3

[0108] Compound 3 has the following structure:

[0109]

[0110] The difference from Example 2 is that 2,4-dichloropyrimidine is replaced by 5-trifluoromethyl-2,4-dichloropyrimidine.

[0111] The high-resolution mass spectrometry detection results of Compound 3 are as follows: HRMS(ESI)(m / z):[M+H] + calcd forC 30 H 26 F3N 10 O2615.2187,found 615.2194.

[0112] The spectral information of the 1H NMR of Compound 3 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.68(s,1H),9.62(s,1H),7.98–7.80(m,5H),7.68(d,J=8.7Hz,1H),7.51(d,J=8.2Hz,1H),7.36–7.24(m,1H),7.14(s,1H),6.88(dd,J=8.8,1.6Hz,1H),4.03(s,3H),3.48(s,3H),2.60(s,3H),1.41(s,3H).

[0113] The spectral information of the 13C NMR of Compound 3 is as follows: 13 C NMR(151MHz,DMSO-d6)δ167.34,166.59,163.13,158.42,147.21,146.45,146.04,143.34,132.57,130.72(2),130.60,128.16,126.09,124.13,122.48,121.79,121.19,119.80,119.18,117.63,117.54,111.85(2),110.60,52.08,41.66,37.76,16.68,9.88.

[0114] Example 4

[0115] Compound 4 has the following structure:

[0116]

[0117] The difference from Example 1 is only that 2,4-dichloropyrimidine is replaced by 5-methyl-2,4-dichloropyrimidine.

[0118] The high-resolution mass spectrometry detection results of Compound 4 are as follows:

[0119] HRMS(ESI)(m / z):[M+Na] + calcd for C 29 H 27 N9NaO 540.2231,found 540.2227。

[0120] The spectral information of the 1H NMR of Compound 4 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.68(s,1H),9.62(s,1H),7.98–7.80(m,5H),7.68(d,J=8.7Hz,1H),7.51(d,J=8.2Hz,1H),7.36–7.24(m,1H),7.14(s,1H),6.88(dd,J=8.8,1.6Hz,1H),4.03(s,3H),3.48(s,3H),2.60(s,3H),1.41(s,3H).

[0121] The spectral information of the 13C NMR of Compound 4 is as follows: 13 C NMR(151MHz,DMSO-d6)δ167.34,166.59,163.13,158.42,147.21,146.45,146.04,143.34,132.57,130.72,130.60,128.16,126.09,124.13,122.48,121.79,121.19(2),119.80,119.18,117.63,117.54,111.85,110.60,52.08,41.66,37.76,16.68,9.88.

[0122] Example 5

[0123]

[0124] The only difference from Example 1 is that 2,4-dichloropyrimidine is replaced by 4-methyl-2,4-dichloropyrimidine.

[0125] The high-resolution mass spectrometry detection results of Compound 5 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd for C 29 H 27 N9NaO 540.2231,found 540.2227.

[0126] The spectral information of the 1H NMR of Compound 5 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.54(s,1H),9.49(s,1H),9.17(s,1H),8.17(d,J=8.7Hz,1H),8.00(d,J=8.5Hz,1H),7.91–7.60(m,6H),7.27–7.19(m,1H),7.12(d,J=16.5Hz,1H),6.92–6.83(m,1H),4.03(s,3H),3.51(s,3H),3.46(s,3H),2.60(s,3H).

[0127] The spectral information of the 13C NMR of Compound 5 is as follows: 13 C NMR(151MHz,DMSO-d6)δ167.14,163.15,158.53,150.48,147.47,144.93,144.81,136.48,132.56(2),130.00(2),127.98(2),121.79(2),119.19(2),118.44(2),111.85(2),102.52(2),99.68,37.76,24.80,16.67,9.89.

[0128] Example 6

[0129]

[0130] The difference from Example 1 is that 2,4-dichloropyrimidine is replaced by 4-methoxy-2,4-dichloropyrimidine.

[0131] The high-resolution mass spectrometry detection results of Compound 6 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 29 H 27 N9NaO2556.2180,found 556.2179.

[0132] The spectral information of the 1H NMR of Compound 6 is as follows: 11H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 9.28 (s, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.96–7.86 (m, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.74–7.65 (m, 3H), 7.59 (dd, J = 8.7, 5.7 Hz, 1H), 7.32–7.24 (m, 1H), 7.19 (d, J = 7.9 Hz, 1H), 6.81 (t, J = 9.4 Hz, 1H), 4.02 (s, 3H), 3.51 (s, 3H), 3.48 (s, 3H), 2.60 (s, 3H).

[0133] The spectral information of the carbon nuclear magnetic resonance of Compound 6 is as follows: 13 13C NMR (151 MHz, DMSO-d6) δ 169.14, 167.01, 162.84, 159.65, 156.56, 153.23, 153.01, 147.49, 143.80, 142.69, 141.69, 128.10 (2), 128.03 (2), 127.83 (2), 127.35, 122.81 (2), 121.96 (2), 119.45, 118.70, 97.02, 38.39, 33.59, 24.58, 24.37.

[0134] Example 7

[0135]

[0136] The difference from Example 1 is that p-aminobenzoic acid is replaced by 4-amino-3-fluorobenzoic acid.

[0137] The high-resolution mass spectrometry detection result of Compound 7 is as follows: HRMS (ESI) (m / z): [M+Na] + calcd for C 28 H 24 FN9NaO 544.1980, found 544.1980.

[0138] The spectral information of the hydrogen nuclear magnetic resonance of Compound 7 is as follows: 1 1H NMR (400 MHz, CDCl3) δ 10.95 (s, 1H), 9.87 (s, 1H), 8.55 (s, 1H), 8.30–8.06 (m, 1H), 7.95–7.45 (m, 5H), 7.39–7.28 (m, 1H), 6.88 (d, J = 8.6 Hz, 1H), 5.92 (d, J = 8.1 Hz, 1H), 4.13 (s, 3H), 3.54 (s, 3H), 2.65 (s, 3H).

[0139] The spectral information of the carbon-13 nuclear magnetic resonance of Compound 7 is as follows: 13 C NMR(151MHz,DMSO-d6)δ167.76,167.71,162.90,159.55,156.34,153.60,147.46,142.27,132.62(2),125.57,122.23(2),121.86(2),120.12(2),119.97,115.77,114.44(2),97.87,97.72,38.29,38.20,37.84,21.78,9.88.

[0140] Example 8

[0141] Compound 8 has the following structure:

[0142] It is only different from Example 1 in that p-aminobenzoic acid is replaced by 4-amino-2-chlorobenzoic acid.

[0143] The spectral information of the proton nuclear magnetic resonance of Compound 8 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.74(s,1H),8.27(s,1H),8.03–7.66(m,7H),7.49(d,J=0.6Hz,1H),7.34(t,J=7.8Hz,1H),6.92(dd,J=8.8,1.5Hz,1H),5.91(d,J=6.0Hz,1H),4.06(s,3H),3.52(s,3H),2.63(s,3H).

[0144] The spectral information of the carbon-13 nuclear magnetic resonance of Compound 8 is as follows: 13 C NMR(151MHz,DMSO-d6)δ162.94,159.50,156.32,150.63,147.50,144.46,142.34,132.65(2),132.39(2),122.34(2),120.35(2),120.15(2),119.35(2),117.35(2),114.47(2),97.86,38.58,37.85,9.89.

[0145] The high-resolution mass spectrometry detection result of Compound 8 is as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 28 H 24 ClN9NaO 560.1685,found 560.1684.

[0146] Example 9

[0147] Compound 9 has the following structure:

[0148] The difference from Example 1 is that p-aminobenzoic acid is replaced by 4-amino-2-trifluoromethylbenzoic acid.

[0149] The spectral information of the 1H NMR of Compound 9 is as follows: 1 H NMR(600MHz,DMSO-d6)δ9.87(s,1H),9.37(s,1H),8.66(s,1H),8.25–7.59(m,7H),7.50(s,1H),7.32(s,1H),6.92(d,J=6.5Hz,1H),5.87(s,1H),4.06(s,3H),3.53(s,3H),2.63(s,3H).

[0150] The spectral information of the 13C NMR of Compound 9 is as follows: 13 C NMR(151MHz,DMSO-d6)δ173.60,168.47,166.90,164.62,162.89,159.55,156.29,151.58,151.53,147.44,143.38,142.19,132.92(2),132.69,122.74(2),122.40,122.21,121.12,120.05,116.49(2),114.54,97.97,38.41,37.85,23.52,9.88.

[0151] The high-resolution mass spectrometry detection result of Compound 9 is as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 29 H 24 F3N9NaO 594.1948,found 594.1950.

[0152] Example 10

[0153] Compound 10 has the following structure:

[0154] The difference from Example 1 is that p-aminobenzoic acid is replaced by 4-amino-2,6-difluorobenzoic acid.

[0155] The spectral information of the 1H NMR of Compound 10 is as follows: 11H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 8.20 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 6.0 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.55–7.41 (m, 2H), 7.35 (d, J = 2.3 Hz, 1H), 7.17–6.96 (m, 3H), 6.89 (d, J = 8.3 Hz, 1H), 5.95 (d, J = 6.2 Hz, 1H), 4.12 (s, 2H), 3.53 (s, 3H), 2.64 (s, 3H).

[0156] The spectral information of the carbon nuclear magnetic resonance of Compound 10 is as follows: 13 13C NMR (151 MHz, DMSO-d6) δ 166.79, 162.86, 160.07, 156.31, 155.08, 153.45, 152.26, 150.60, 147.41, 142.24, 133.52, 132.67, 127.54, 125.73, 123.18, 122.23, 121.67, 120.09, 119.92, 115.75 (2), 114.35, 111.45, 111.31, 97.48, 38.09, 37.82, 9.86.

[0157] The high-resolution mass spectrometry detection results of Compound 10 are as follows: HRMS (ESI) (m / z): [M+Na] + calcd for C 28 H 23 F2N9NaO 562.1886, found 562.1885.

[0158] Example 11

[0159] Compound 11 has the following structure:

[0160] The difference from Example 1 is that 4-aminobenzoic acid is replaced with 4-amino-2-methoxybenzoic acid.

[0161] The spectral information of the nuclear magnetic resonance hydrogen spectrum of Compound 11 is as follows: 11H NMR(400MHz,DMSO-d6)δ9.44(s,1H),8.55(d,J=8.4Hz,1H),7.92(d,J=5.8Hz,2H),7.84–7.66(m,6H),7.50–7.32(m,2H),7.26(t,J=7.7Hz,1H),6.91(d,J=8.9Hz,1H),5.90(d,J=5.9Hz,1H),4.07(s,3H),4.04(s,3H),3.51(s,3H),2.65(s,3H).

[0162] The spectral information of the carbon nuclear magnetic resonance of Compound 11 is as follows: 13 13C NMR(151MHz,DMSO-d6)δ167.08,162.94,159.19,156.38,153.45,147.89,142.47,132.69,131.86,129.64,129.58,122.75,122.63,122.24,121.83,120.39,120.16,119.99,118.23,117.49,117.34,114.41,101.19,103.94,97.78,56.62,38.55,37.87,9.91.

[0163] The high-resolution mass spectrometry detection results of Compound 11 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd for C 29 H 27 N9NaO2556.2180,found 556.2182.

[0164] Example 12

[0165] Compound 12 has the following structure:

[0166] The difference from Example 2 is that 4-aminobenzoic acid is replaced with 4-amino-3-fluorobenzoic acid.

[0167] The spectral information of the nuclear magnetic resonance hydrogen spectrum of Compound 12 is as follows: 11H NMR(400MHz,DMSO-d6)δ8.83(s,1H),8.37(t,J=8.4Hz,1H),8.07–7.64(m,7H),7.47(d,J=0.9Hz,1H),7.23(t,J=7.8Hz,1H),6.90(dd,J=8.8,1.6Hz,1H),5.92(d,J=6.0Hz,1H),4.07(s,3H),3.48(s,3H),2.64(s,3H).

[0168] The spectral information of the carbon nuclear magnetic resonance of Compound 12 is as follows: 13 13C NMR(151MHz,DMSO-d6)δ168.40,167.29,162.89,159.61,156.33,153.70,153.17,152.08,151.55,147.43(2),142.28,142.20,132.71,122.30,122.22,120.12(2),120.08,120.00,114.42(2),114.38,98.21,97.61,38.31,38.17,37.84,9.87.

[0169] The high-resolution mass spectrometry detection results of Compound 12 are as follows: HRMS(ESI)(m / z):[M+H] + calcd forC 29 H 26 FN 10 O2565.2219,found 565.2230.

[0170] Example 13

[0171] Compound 13 has the following structure:

[0172] The difference from Example 2 is only that: p-aminobenzoic acid is replaced by: 4-amino-2-chlorobenzoic acid.

[0173] The spectral information of the nuclear magnetic resonance hydrogen spectrum of Compound 13 is as follows:

[0174] 11H NMR(400MHz,DMSO-d6)δ9.65(d,J=25.1Hz,1H),8.01–7.78(m,6H),7.68(d,J=8.9Hz,1H),7.54(d,J=8.3Hz,1H),7.41–7.24(m,2H),7.14(d,J=1.9Hz,1H),6.87(dt,J=8.8,1.7Hz,1H),4.03(s,3H),2.60(s,3H),1.41(s,3H).

[0175] The spectral information of the carbon nuclear magnetic resonance spectrum of Compound 13 is as follows:

[0176] 13 13C NMR(151MHz,DMSO-d6)δ162.89,159.91,156.28,147.47,142.40,141.97,133.82,132.64,128.86,123.00(2),122.55,122.24(2),121.22,120.68(2),120.13,119.95,119.10,118.25,114.35(2),112.34,97.28,49.08,38.48,37.81,9.85.

[0177] The high-resolution mass spectrometry detection results of Compound 13 are as follows:

[0178] HRMS(ESI)(m / z):[M+H] + calcd for C 29 H 26 ClN 10 O2581.1923,found581.1935.

[0179] Example 14

[0180] Compound 14 has the following structure:

[0181] The difference from Example 2 is that 4-aminobenzoic acid is replaced by 4-amino-2-(trifluoromethyl)benzoic acid.

[0182] The spectral information of the nuclear magnetic resonance hydrogen spectrum of Compound 14 is as follows: 11H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.84 (s, 1H), 8.50 (d, J = 10.2 Hz, 1H), 8.08–7.87 (m, 2H), 7.83–7.66 (m, 2H), 7.62 (d, J = 8.3 Hz, 1H), 7.52–7.27 (m, 2H), 6.90 (d, J = 8.8 Hz, 1H), 5.88 (t, J = 6.3 Hz, 1H), 4.07 (s, 3H), 3.50 (s, 3H), 2.63 (s, 3H).

[0183] The spectral information of the carbon nuclear magnetic resonance of Compound 14 is as follows: 13 13C NMR (151 MHz, DMSO-d6) δ 167.42, 166.24, 162.87, 159.33, 156.28, 147.44 (2), 145.17, 142.17, 132.68 (2), 132.45 (2), 124.75 (2), 122.38 (2), 120.51 (2), 120.05 (2), 114.58 (2), 110.21 (2), 98.19, 52.83, 38.41, 37.85, 9.86.

[0184] The high-resolution mass spectrometry detection results of Compound 14 are as follows: HRMS (ESI) (m / z): [M+Na] + calcd for C 30 H 25 F3ClN 10 NaO 2637.2006, found 637.2002.

[0185] Example 15

[0186] Compound 15 has the following structure:

[0187] The difference from Example 2 is that p-aminobenzoic acid is replaced by 4-amino-2,6-difluorobenzoic acid.

[0188] The spectral information of the nuclear magnetic resonance hydrogen spectrum of Compound 15 is as follows: 1 1H NMR (600 MHz, DMSO-d6) δ 12.77 (s, 1H), 8.96 (s, 1H), 8.80 (s, 1H), 8.01 (s, 1H), 7.88–7.57 (m, 5H), 7.44 (s, 1H), 7.26 (s, 1H), 7.13 (s, 1H), 6.88 (s, 1H), 5.81 (s, 1H), 4.06 (s, 3H), 3.42 (s, 3H), 2.63 (s, 3H).

[0189] The spectral information of the carbon-13 nuclear magnetic resonance of Compound 15 is as follows: 13 C NMR(151MHz,DMSO-d6)δ162.87(2),160.09(2),156.31,155.06,153.48,152.24,150.57,147.42(2),142.25(2),132.65(2),125.65,122.22,120.09(2),119.93(2),114.36(2),111.42,111.27,97.47,38.09,37.83,9.87.

[0190] The high-resolution mass spectrometry detection results of Compound 15 are as follows: HRMS(ESI)(m / z):[M+Na] +

[0191] calcd for C 29 H 24 F2N 10 NaO2605.1944,found 605.1942.

[0192] Example 16

[0193] Compound 16 has the following structure:

[0194] The difference from Example 1 is only that: p-aminobenzoic acid is replaced by m-aminobenzoic acid.

[0195] The spectral information of the proton nuclear magnetic resonance of Compound 16 is as follows: 1 H NMR(400MHz,CDCl3)δ9.86(s,1H),8.46(s,1H),8.12(d,J=6.9Hz,1H),7.85(d,J=5.3Hz,1H),7.71(d,J=7.7Hz,1H),7.59–7.28(m,6H),6.90(d,J=8.8Hz,1H),6.06(s,1H),5.92(d,J=4.4Hz,1H),4.05(s,3H),3.53(s,3H),2.51(s,3H).

[0196] The spectral information of the carbon-13 nuclear magnetic resonance of Compound 16 is as follows: 1313C NMR(151MHz,DMSO-d6)δ169.14,167.01,162.84,159.65,156.56,153.23,153.01,147.49,143.80,142.69,141.69,128.10(2),128.03(2),127.83,127.35,122.81(2),121.96(2),119.45(2),118.70,97.02,38.39,33.59,24.37.

[0197] The high-resolution mass spectrometry detection results of Compound 16 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd for C 28 H 25 N9NaO 526.2074,found 526.2068.

[0198] Example 17

[0199] Compound 17 has the following structure:

[0200] The difference from Example 1 is only that: p-aminobenzoic acid is replaced by 3-amino-6-methylbenzoic acid.

[0201] The spectral information of the 1H NMR of Compound 17 is as follows: 1 1H NMR(400MHz,CDCl3)δ9.84(s,1H),8.23–8.09(m,2H),7.80(d,J=5.9Hz,1H),7.56(dd,J=19.5,8.2Hz,2H),7.48–7.30(m,4H),7.24(d,J=8.4Hz,1H),6.88(d,J=8.9Hz,1H),6.00–5.84(m,2H),4.07(s,3H),3.50(s,3H),2.63(s,3H),2.55(s,3H).

[0202] The spectral information of the 13C NMR of Compound 17 is as follows: 1313C NMR(151MHz,DMSO-d6)δ173.41,166.86,162.86,159.99,156.30,153.68,147.44,142.38,139.76,132.58,131.69,129.72,129.16,122.98,122.43,122.12,120.74,120.38,120.15(2),119.90,114.31(2),96.90,38.38,37.81,22.85,20.49,9.83.

[0203] The high-resolution mass spectrometry detection results of Compound 17 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 29 H 27 N9NaO 540.2231,found 540.2227.

[0204] Example 18

[0205] Compound 18 has the following structure:

[0206] The difference from Example 1 is only that: p-aminobenzoic acid is replaced by 3-amino-4-methoxybenzoic acid.

[0207] The spectral information of the 1H NMR of Compound 18 is as follows: 1 1H NMR(400MHz,CDCl3)δ9.44(s,1H),7.82(dd,J=28.1,16.7Hz,2H),7.70(dd,J=8.5,2.0Hz,1H),7.62(d,J=8.7Hz,1H),7.56–7.47(m,1H),7.38–7.27(m,1H),7.14–7.05(m,1H),7.03–6.95(m,1H),6.90(d,J=8.6Hz,2H),5.83(d,J=6.1Hz,1H),4.12(s,3H),3.96(s,3H),3.87(s,3H),2.64(s,3H).

[0208] The spectral information of the 13C NMR of Compound 18 is as follows: 1313C NMR (151 MHz, DMSO-d6) δ 172.73, 162.94, 159.63, 156.33, 153.23, 150.43, 147.44, 142.18, 142.08, 132.63 (2), 130.23 (2), 122.21 (2), 121.07, 120.07 (2), 119.98, 118.18, 114.47 (2), 111.21, 97.56, 56.67, 38.41, 37.85, 21.94, 9.85.

[0209] The high-resolution mass spectrometry detection results of Compound 18 are as follows: HRMS (ESI) (m / z): [M+Na] + calcd for C 29 H 27 N9NaO2 556.2180, found 556.2187.

[0210] Example 19

[0211] Compound 19 has the following structure:

[0212] The only difference in the preparation process from Example 17 is that 2,3-diaminobenzamide is replaced with "4,5-difluorobenzene-1,2-diamine".

[0213] The spectral information of the 1H NMR of Compound 19 is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.28 (s, 1H), 7.95–7.54 (m, 5H), 7.44 (s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 6.86 (dd, J = 8.7, 1.4 Hz, 1H), 5.78 (d, J = 5.9 Hz, 1H), 4.04 (s, 3H), 3.47 (s, 3H), 2.58 (s, 3H), 2.47 (s, 3H).

[0214] The spectral information of the 13C NMR of Compound 19 is as follows: 13 13C NMR (151 MHz, DMSO-d6) δ 172.79, 162.86, 160.01, 156.21, 155.00, 147.46, 142.40, 139.64, 132.55, 131.51, 130.13, 129.07, 122.09, 120.47, 120.32 (2), 120.13 (2), 119.90 (2), 114.30 (2), 96.92, 38.34, 37.77, 21.81, 20.35, 9.80.

[0215] The high-resolution mass spectrometry detection results of Compound 19 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 28 H 24 F2N8Na 533.1984,found 533.1977.

[0216] Example 20

[0217] Compound 20 has the following structure:

[0218] The difference from Example 17 is only that: 2,3-diaminobenzamide is replaced by "o-phenylenediamine".

[0219] The spectral information of the 1H NMR of Compound 20 is as follows: 1 H NMR(400MHz,DMSO-d6)δ9.27(s,1H),8.25(s,1H),7.94–7.38(m,6H),7.20(dd,J=5.9,3.0Hz,3H),6.99–6.78(m,1H),5.91–5.67(m,1H),4.06(s,3H),3.47(s,3H),2.61(s,3H),2.48(s,3H).

[0220] The spectral information of the 13C NMR of Compound 20 is as follows: 13 C NMR(151MHz,DMSO-d6)δ172.91,162.88,160.05,156.25,152.98,147.46,142.42,139.56,132.59,131.40,130.76,129.10,122.16,120.46,120.22,120.16(2),119.92(2),114.32(2),96.89,38.35,37.81,23.97,22.15,20.39,9.85.

[0221] The high-resolution mass spectrometry detection results of Compound 20 are as follows: HRMS(ESI)(m / z):[M+Na] + calcd forC 28 H 26 N8Na 497.2173,found 497.2177.

[0222] The present invention tested the enzymatic inhibitory activities, anti-proliferation activities against BRCA wild-type breast cancer cells MDA-MB-231 and MCF-7 cells of Compounds 1-20. The test method is as follows:

[0223] Enzymatic inhibitory activity: The technical service support for the enzymatic inhibitory activities of VEGFR2 and PARP1 was provided by Shanghai ChemPartner Co., Ltd., and the assays were performed using enzyme-linked immunosorbent assay (ELISA) kits.

[0224] Anti-proliferative activity test against BRCA wild-type breast cancer cells MDA-MB-231 and MCF-7 cells: Compounds 1 - 20 and positive controls (PARP inhibitor - Olaparib, VEGFR inhibitor - Pazopanib) were prepared into DMSO solutions with a concentration of 1 mM / L. After digesting BRCA wild-type breast cancer cells MDA-MB-231 and MCF-7 with trypsin to form cell suspensions, the cell density was counted using a cell counting chamber and seeded in 96-well plates at a density of 6000 cells per well. After incubating at 37 °C for 24 h, the cells were treated with test compounds at various concentrations for 48 h. Then, 20 μL of MTT solution (concentration 5 mg / L) was added to each well and incubated in a cell culture incubator. After 4 h, the 96-well plates were taken out, the culture medium in each well was aspirated, and then 200 μL of DMSO was added to each well to dissolve the purple formazan crystals at the bottom. The plates were placed on a shaker and shaken at low speed for 15 min to promote crystal dissolution, and the absorbance (OD value) at 570 nm was measured using a microplate reader.

[0225] Using the formula: Inhibition rate = (OD value of negative control group - OD value of experimental group) × 100% / (OD value of negative control group - OD value of blank group), calculate the inhibition rate of the test compounds on tumor cells, and then use GraphPad Prism 8.0 software to calculate the IC 50 value.

[0226]

[0227]

[0228] a IC 50 values were obtained through 3 tests (± standard deviation).

[0229] b N.D. = Not detected.

[0230] As can be seen from Table 1: The compounds of the present invention have inhibitory activities against VEGFR2 and PARP1 and anti-proliferative activities against BRCA wild-type breast cancer cells. Among them, compound 17 has the strongest inhibitory activities against PARP1 and VEGFR2 and BRCA wild-type breast cancer inhibitory activity, and its cell activity is significantly better than that of the positive control.

[0231] Experimental Example 2

[0232] To investigate the mechanism of DNA damage induced by compound 17, positive control Olaparib (Ola), Pazopanib (Paz), and their combination in BRCA wild-type breast cancer cells using Western blot assay.

[0233] The Western blot assay was as follows: MDA-MB-231 and MCF-7 cells were seeded into six-well plates (3×10 5 cells per well) and incubated overnight in a 37°C incubator. After treatment with different concentrations of compound 17, positive control Olaparib (abbreviated as Ola), Pazopanib (abbreviated as Paz), and their combination (Ola + Paz) for 48 h, the cells were washed twice with PBS and then made into cell suspensions with cell lysis buffer. After centrifugation at 13,000 rpm for 20 min, the protein concentration was quantified using a BCA protein assay kit. After separation by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), an equivalent concentration of total protein was transferred onto a nitrocellulose (PVDF) membrane. After blocking with 5% skim milk-TBST solution at room temperature for 1 h, the membrane was incubated with the corresponding primary antibody overnight at 4°C. After washing twice with TBST solution, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody, and ECL was used as the HRP substrate for observation.

[0234] The test results are shown in Figure 1 , and it can be seen from Figure 1 that both compound 17 and the combination of Pazopanib + Olaparib can positively regulate the expression of hypoxia-inducible factor-1α (HIF-1α), a key molecule mediating the hypoxic effect. In addition, compared with the combination treatment group, 17 induced enhanced expression of HIF-1α. 17 significantly inhibited the expression of homologous recombination repair pathway factors (FOXM1, RAD51, and BRCA1) in MDA-MB-231 and MCF-7 cells at a concentration of 8 μM, thereby inducing DNA damage in tumor cells.

[0235] Experimental Example 3

[0236] Compound 17 (50 mg / kg or 25 mg / kg), Olaparib (50 mg / kg), Pazopanib (50 mg / kg), or the combination group of Olaparib + Pazopanib (50 mg / kg + 50 mg / kg) were used to treat the MDA-MB-231 xenograft tumor model in nude mice by oral administration for 15 days, and the changes in the tumor volume of the nude mice were observed.

[0237] The test results are shown in Figure 2 , and it can be seen from Figure 2It can be seen that Compound 17 can significantly inhibit the size and weight of tumors. Compared with the combination group, Compound 17 (50 mg / kg) has a more significant inhibitory effect on tumors, and its tumor growth inhibition rate (TGI) is 72.1%.

[0238] Test Example 4

[0239] Compound 17 (50 mg / kg or 25 mg / kg), Olaparib (50 mg / kg), Pazopanib (50 mg / kg) or the Olaparib + Pazopanib combination group (50 mg / kg + 50 mg / kg) were used to treat the nude mouse MDA-MB-231 lung metastasis model by oral administration. After 15 days, the figure of lung metastasis lesions in nude mice is shown in Figure 3 , where the arrows represent lung metastasis nodules.

[0240] From Figure 3 it can be seen that by visually observing the number of metastasis nodules on the lung metastasis lesion tissue, the combination administration group and Compound 17 can effectively inhibit the formation of lung metastasis nodules.

[0241] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A pyrimidine compound, characterized in that, It has the structures shown in Formula 1 to Formula 20:

2. The method for preparing the pyrimidine compound according to claim 1, characterized in that, It includes the following steps: After dissolving the 2,4-dichloropyrimidine derivative having the structure shown in Formula II and 2,3-dimethyl-6-amino-2H-indazole first, a first substitution reaction is carried out under alkaline conditions to obtain the intermediate product III having the structure of Formula III; After dissolving the intermediate product III and the methylation reagent second, a methylation reaction is carried out under a protective atmosphere and alkaline conditions to obtain a methylated compound having the structure of Formula IV; After dissolving the methylated compound and the aminobenzoic acid derivative third, a second substitution reaction is carried out under acidic conditions to obtain an aminated product; the aminobenzoic acid derivative includes a p-aminobenzoic acid derivative having the structure of Formula V-1 and an m-aminobenzoic acid derivative having the structure of Formula V-2; When the aminobenzoic acid derivative is a p-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-1, and when the aminobenzoic acid derivative is an m-aminobenzoic acid derivative, the aminated product has the structure shown in Formula VI-2; After dissolving the aminated product having the structure of Formula VI-1, the first condensing agent and the benzimidazole compound having the structure of Formula VII fourth, a first amide condensation reaction is carried out under alkaline conditions to obtain a first pyrimidine compound; When R1 in formula II is H, R2 is H, R in formula V-1 n , R m and R x are H, R5 and R6 in formula VII are H, and R4 is , the structure of the first pyrimidine compound is formula 2; When R1 in Formula II is H, R2 is CF3, R in Formula V-1 n , R m and R x are H, R5 and R6 in Formula VII are H, and R4 is , the structure of the first pyrimidine compound is Formula 3; When R1 in Formula II is H, R2 is H, R in Formula V-1 n is F, R m and R x are H, R5 and R6 in Formula VII are H, and R4 is at this time, the structure of the first pyrimidine compound is Formula 12; When R1 in formula II is H, R2 is H, R in formula V-1 x is Cl, R m and R n are H or R m is Cl, R x and R n are H, R5 and R6 in formula VII are H, R4 is when, the structure of the first pyrimidine compound is formula 13; When R1 in Formula II is H, R2 is H, R in Formula V-1 x is CF3, R m and R n are H or R m is CF3, R x and R n are H, R5 and R6 in Formula VII are H, R4 is when, the structure of the first pyrimidine compound is Formula 14; When R1 in Formula II is H, R2 is H, R and R in Formula V-1 x and R m are F, R n is H or R m is CF3, R x and R n are H, R5 and R6 in Formula VII are H, R4 is when, the structure of the first pyrimidine compound is Formula 15; After dissolving the aminated product having the structure of Formula VI-1 or the aminated product having the structure of Formula VI-2, the second condensing agent and the aniline derivative having the structure of Formula VIII fifth, a second amide condensation reaction is carried out, and the obtained second condensation product is mixed with acetic acid to carry out a first cyclization reaction to obtain a second pyrimidine compound and a third pyrimidine compound respectively; When R1 and R2 in formula II are H, R x , Rn and R m in formula V-1 are H, R5 and R6 in formula VIII are H, and R4 is , the structure of the first pyrimidine compound is formula 1; When R1 in formula II is CH3, R2 is H, R x , Rn and R m are H, R5 and R6 in formula VIII are H, and R4 is , the structure of the second pyrimidine compound is formula 4; When R1 in formula II is H, R2 is CH3, R in formula V-1 x , Rn and R m are H, R5 and R6 in formula VIII are H, and R4 is , the structure of the second pyrimidine compound is formula 5; When R1 in formula II is H, R2 is OCH3, R x , Rn and R m are H, R5 and R6 in formula VIII are H, and R4 is , the structure of the second pyrimidine compound is formula 6; When R1 and R2 in formula II are H, R x and R m are H, R n is F, R5 and R6 in formula VIII are H, and R4 is the structure of the second pyrimidine compound is formula 7; When R1 and R2 in Formula II are H, R in Formula V-1 x is Cl, R m and R n are H or R m is Cl, R x and R n are H, R5 and R6 in Formula VIII are H, R4 is When, the structure of the second pyrimidine compound is Formula 8; When R1 and R2 in Formula II are H, R in Formula V-1 x is CF3, R m and R n are H or R m is CF3, R x and R n are H, R5 and R6 in Formula VIII are H, and R4 is When this is the case, the structure of the second pyrimidine compound is Formula 9; When R1 and R2 in formula II are H, R x and R m are F, R n is H or R m is CF3, R n is H, R5 and R6 in formula VIII are H, and R4 is the structure of the second pyrimidine compound is formula 10; When R1 and R2 in Formula II are H, R y and R z in Formula V-2 are H, R5 and R6 in Formula VIII are H, and R4 is the structure of the third pyrimidine compound is Formula 16; When R1 and R2 in formula II are H, R in formula V-2 y is H, R z is CH3, R5 and R6 in formula VIII are H, and R4 is then the structure of the third pyrimidine compound is formula 17; When R1 and R2 in formula II are H, R y in formula V-2 is OCH3, R z is H, R5 and R6 in formula VIII are H, and R4 is the structure of the third pyrimidine compound is formula 18; When R1 and R2 in Formula II are H, R in Formula V-2 y is H, R z is CH3, R5 and R6 in Formula VIII are F, and R4 is H, the structure of the third pyrimidine compound is Formula 19; When R1 and R2 in formula II are H, R in formula V-2 y is H, R z is CH3, and R4, R5 and R6 in formula VIII are H, the structure of the third pyrimidine compound is formula 20; 3. The preparation method according to claim 2, wherein The molar ratio of the 2,4-dichloropyrimidine derivative to 2,3-dimethyl-6-amino-2H-indazole is 1:1.2 to 2; the temperature of the first substitution reaction is 80 to 90 °C, and the time is 12 to 15 h.

4. The preparation method according to claim 2, characterized in that, The methylation reagent is CH3I; the molar ratio of the intermediate product III to the methylation reagent is 1:1.1 to 1.5; the temperature of the methylation reaction is 25 to 30 °C, and the time is 6 to 18 h.

5. The preparation method according to claim 2, wherein The molar ratio of the methylated compound to the aminobenzoic acid derivative is 1:1.2 to 1.5; the temperature of the second substitution reaction is 80 to 90 °C, and the time is 15 to 20 h.

6. The preparation method according to claim 2, wherein The first condensing agent is 1-hydroxybenzotriazole and 1-ethyl-3(3-dimethylpropylamine)carbodiimide; the temperature of the first amide condensation reaction is -5 to 0 °C, and the time is 15 to 20 h.

7. The preparation method according to claim 2, characterized in that, The second condensing agent is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the temperature of the second amide condensation reaction is 25 to 30 °C, and the time is 24 to 30 h; the temperature of the first cyclization reaction is 110 to 130 °C, and the time is 15 to 20 h.

8. The preparation method according to claim 2, characterized in that, The temperature of the second amide condensation reaction is 25 to 30 °C, and the time is 24 to 30 h.

9. Use of the pyrimidine compound according to claim 1 or the pyrimidine compound obtained by the preparation method according to any one of claims 2 to 8 in the preparation of an inhibitor that dual-targets VEGFR and PARP.

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