Imidazolo[1,2-c]pyrimidine compounds and preparation methods and applications thereof

Abstract

The present invention discloses an imidazo[1,2-c]pyrimidine compound, a preparation method and an application thereof. The compound is shown as general formula VIII. The compound has the effect of inhibiting LSD1, thereby opening up a new way for finding a new class of innovative drugs based on the LSD1 target. In addition, the combination of the imidazo[1,2-c]pyrimidine compound and 1,25(OH)2D3 can be used for the synergistic treatment of cancer, especially for the treatment of leukemia.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to an imidazo[1,2-c]pyrimidine compound and a preparation method and application thereof. Background Art

[0002] Lysine-specific demethylase 1 (LSD1) is the first histone demethylase discovered. It specifically removes methyl groups from monomethylated and dimethylated H3K4 and H3K9 sites through a flavin adenine dinucleotide (FAD)-dependent mechanism, thereby regulating gene expression and transcriptional activity. Studies have shown that LSD1 is overexpressed in a variety of tumor cells, and can activate or inhibit chromatin domains through histone demethylation to regulate gene expression, and regulate the occurrence and development of tumors by affecting the expression of factors necessary for cell proliferation and differentiation. In addition, LSD1 is also closely related to the occurrence and development of other diseases such as viral infections, central nervous system diseases, cardiovascular and cerebrovascular diseases, etc.

[0003] At present, there are many reports of LSD1 inhibitors, which are divided into covalent inhibitors and reversible inhibitors according to their mode of action. Most of the LSD1 inhibitors in the clinical research stage are phenylcyclopropylamine covalent inhibitors. This type of LSD1 covalent inhibitors irreversibly covalently bind to FAD, show high affinity to various targets with FAD as a cofactor, and have certain potential toxicity. Reversible inhibitors bind to FAD in a non-covalent manner and have certain advantages in safety. Among them, CC-90011 and SP-2577 have entered Phase I / II clinical use for tumor treatment. In addition, the combination of LSD1 inhibitors with other drugs to treat tumors has also entered the clinical research stage. Although reversible inhibitors have potential advantages in safety, their structural types are relatively limited. Therefore, the development of safe, effective and novel LSD1 inhibitors has good clinical application prospects, providing a material basis for the development of innovative drugs targeting LSD1, and also providing new strategies for the combination of drugs to treat tumors. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an imidazo[1,2-c]pyrimidine compound and a preparation method and application thereof.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The first object of the present invention is to provide an imidazo[1,2-c]pyrimidine compound, the general structural formula of which is as follows:

[0007]

[0008] The above compound may exist in the form of a pharmaceutically acceptable salt, such as trifluoroacetate, sulfate, hydrochloride, methanesulfonate, and tartaric acid. Taking trifluoroacetate as an example, its general structural formula is as follows:

[0009]

[0010] In the above general formula, R 1 Amino, fatty chain amino, C 3～8 Cyclic amino group;

[0011] R 2 is aryl or substituted aryl;

[0012] R 3 is an aryl group or a substituted aryl group.

[0013] Furthermore, R 2 It is phenyl, naphthyl, furan, thiophene, pyrrole, imidazole, pyrazole, pyridine, quinoline, benzothiophene, indole, benzofuran or substituted phenyl, naphthyl, furan, thiophene, pyrrole, imidazole, pyrazole, pyridine, quinoline, benzothiophene, indole, benzofuran; substituted groups such as halogen, methyl, methoxy, ethyl, propyl, tert-butyl, cyclopropyl, cyclohexyl, trifluoromethyl, cyano, nitro, methyl formate, phenyl.

[0014] Furthermore, R 3 It is phenyl, naphthyl, furan, thiophene, pyrrole, imidazole, pyrazole, pyridine, quinoline, benzothiophene, indole, benzofuran or substituted phenyl, naphthyl, furan, thiophene, pyrrole, imidazole, pyrazole, pyridine, quinoline, benzothiophene, indole, benzofuran; substituted groups such as halogen, methyl, methoxy, ethyl, propyl, tert-butyl, cyclopropyl, cyclohexyl, trifluoromethyl, cyano, nitro, methyl formate, phenyl.

[0015] Furthermore, R 1 For amino,

[0016] Furthermore, R 2 It is p-tolyl, p-chlorophenyl, p-fluorophenyl, p-nitrophenyl, p-trifluoromethylphenyl, p-cyanophenyl, 3-fluoro-4-methoxyphenyl, 3-fluoro-4-cyanophenyl, 2-fluoro-5-nitrophenyl.

[0017] Furthermore, R 3 It is p-tolyl, p-chlorophenyl, p-fluorophenyl, p-nitrophenyl, p-trifluoromethylphenyl, p-cyanophenyl, 3-fluoro-4-methoxyphenyl, 3-fluoro-4-cyanophenyl, 2-fluoro-5-nitrophenyl.

[0018] Furthermore, the specific structure of the imidazo[1,2-c]pyrimidine compound is as follows:

[0019] VIII-1:

[0020] VIII-2:

[0021] VIII-3:

[0022] VIII-4:

[0023] VIII-5:

[0024] VIII-6:

[0025] VIII-7:

[0026] VIII-8:

[0027] VIII-9:

[0028] VIII-10:

[0029] VIII-11:

[0030] VIII-12:

[0031] VIII-13:

[0032] The second object of the present invention is to provide a method for preparing an imidazo[1,2-c]pyrimidine compound, the method comprising the following steps:

[0033] S1, dissolving 2,4,5,6-tetrachloropyrimidine (I) and triethylamine in a solvent, adding aminoacetaldehyde dimethyl acetal (II) under ice bath conditions to react, to obtain intermediate III;

[0034] S2, heating the intermediate III to reflux, and then adjusting the pH of the reaction system with a base to obtain the intermediate IV;

[0035] S3, dissolving intermediate IV in phosphorus oxychloride, adding an organic base, and heating under reflux to obtain intermediate V;

[0036] S4, intermediate V, amine compound R 1 H and an organic base are dissolved in a solvent and reacted at room temperature to obtain intermediate VI;

[0037] S5, intermediate VI, boronic acid compound R 2 B(OH)2, palladium catalyst and inorganic base are dissolved in a solvent and heated under reflux to obtain intermediate VII or compound VIII (if R 2 and R 3 When they are the same substituents, the target compound can be obtained directly by step S5. 2 and R 3 When the substituents are different, the target compound needs to be prepared through two steps of reaction, steps S5 and S6);

[0038] S6, intermediate VII, boronic acid compound R 3 B(OH)2, palladium catalyst and inorganic base are dissolved in a solvent and heated under reflux to obtain compound VIII, i.e., an imidazo[1,2-c]pyrimidine compound.

[0039] Furthermore, the solvents in the above reactions are all reagents that can dissolve the raw materials, such as ethanol, DMF (N,N-dimethylformamide), ethyl acetate, DCM (dichloromethane), chloroform, toluene, xylene, methanol, etc.

[0040] Furthermore, in the above reaction, the organic bases are DIPEA (N,N-diisopropylethylamine), triethylamine, etc.; the inorganic bases are sodium carbonate, potassium carbonate, etc.

[0041] Furthermore, the palladium catalyst is tetrakistriphenylphosphine palladium, dichlorobistriphenylphosphine palladium, diphenylphosphinocene palladium dichloride or palladium acetate.

[0042] Furthermore, in step S1, the molar ratio of 2,4,5,6-tetrachloropyrimidine, triethylamine and aminoacetaldehyde dimethyl acetal is 1:1:1 to 1:3:3; and the reaction is stirred in an ice bath for 4 to 6 hours.

[0043] In step S1, after the reaction is completed under ice bath conditions, the reaction product can be purified. The purification process includes: removing the solvent in the system by reduced pressure distillation, washing the obtained solid, and filtering to obtain the intermediate III.

[0044] Furthermore, in step S2, the heating reflux time is 4 to 6 hours.

[0045] In step S2, after the heating reflux reaction is completed, the pH can be adjusted with sodium hydroxide solution, and then purification is performed. The purification process is as follows: extraction with dichloromethane and brine, extraction of the aqueous phase with dichloromethane three times, combining the organic solvents, distilling under reduced pressure, and drying to obtain intermediate IV.

[0046] Furthermore, in step S3, the molar ratio of intermediate IV to the organic base is 1:1 to 1:2, and the reflux reaction time is 8 to 10 hours.

[0047] In step S3, after the heating reflux reaction is completed, the reaction product is cooled to room temperature, the phosphorus oxychloride in the system is distilled off under reduced pressure, poured into ice water, the pH is adjusted to 8-9 with a saturated sodium bicarbonate solution, extracted three times with ethyl acetate, the organic layers are combined, the organic layers are dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography to obtain intermediate V.

[0048] Further, in step S4, the intermediate V and the amine compound R 1 The molar ratio of H and the organic base is 1:1:2 to 1:1:3; the reaction is stirred at room temperature for 1 to 3 hours.

[0049] In step S4, after the reaction is completed, the reaction product is purified. The purification process is as follows: equal amounts of water and ethyl acetate are added to the reaction product for extraction, the aqueous phase is extracted three times with ethyl acetate, the organic layers are combined, the organic layers are dried over anhydrous magnesium sulfate, filtered, and evaporated to obtain intermediate VI.

[0050] Further, in step S5, the intermediate VI and the boronic acid compound R 2 The molar ratio of B(OH)2, palladium catalyst and inorganic base is 1:2-2.5:0.05-1:2; the reflux reaction is carried out for 10-18 hours under protective gas conditions; the protective gas can be nitrogen or inert gas.

[0051] In step S5, after heating and reflux, the reaction product is purified. The purification process is as follows: equal amounts of water and ethyl acetate are added to the reaction product for extraction, the organic phase is extracted three times with water, the organic layers are combined, the organic layers are dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography to obtain intermediate VII.

[0052] Further, in step S6, intermediate VII, boronic acid compound R 3 The molar ratio of B(OH)2, palladium catalyst and inorganic base is 1:1-1.5:0.05-1:2; the reflux reaction is carried out for 10-18 hours under protective gas conditions; the protective gas can be nitrogen or inert gas.

[0053] In step S6, after the heating and reflux are completed, the reaction product is purified. The purification process is as follows: equal amounts of water and ethyl acetate are added to the reaction product for extraction, the organic phase is extracted three times with water, the organic layers are combined, the organic layers are dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography to obtain the final product VIII.

[0054] If the salt of product VIII is to be prepared, taking trifluoroacetate as an example, the preparation process is as follows:

[0055] At room temperature, compound VIII is dissolved in a solvent, and then TFA (trifluoroacetic acid) is added and stirred for reaction to obtain compound IX. The specific process is as follows:

[0056] Compound VIII is dissolved in dichloromethane and stirred at room temperature to form a VIII solution with a molar concentration of 0.1 to 10 mmol / mL; trifluoroacetic acid is added dropwise to the VIII solution and stirring is continued for 1 to 2 hours; after the reaction is complete as monitored by TLC, the solvent is removed by vacuum distillation, and the product is washed with ether to obtain a trifluoroacetate product of the general formula IX.

[0057] The reaction formula of the above reaction is as follows:

[0058]

[0059]

[0060] The third object of the present invention is to provide an application of an imidazo[1,2-c]pyrimidine compound, wherein the compound is used as a LSD1 inhibitor or for preparing a LSD1 inhibitor.

[0061] The fourth object of the present invention is to provide an application of an imidazo[1,2-c]pyrimidine compound for preparing a drug for treating cancer.

[0062] The above cancers are hematological cancers, such as leukemia and the like.

[0063] The fifth object of the present invention is to provide a combined drug comprising the above-mentioned imidazo[1,2-c]pyrimidine compound and 1,25(OH)2D3.

[0064] The sixth object of the present invention is to provide the use of the above-mentioned combined drug in the preparation of a drug for treating cancer; the cancer is a hematological cancer, such as leukemia.

[0065] The present invention has the following beneficial effects:

[0066] The present invention provides a compound with imidazo[1,2-c]pyrimidine as a skeleton, which has the function of inhibiting LSD1, so that the present invention opens up a new way for finding a new class of innovative drugs based on LSD1 target.

[0067] In addition, the combination of imidazo[1,2-c]pyrimidine compounds and 1,25(OH)2D3 can synergistically treat cancer, especially for the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The results show the effects of compounds IX-8 and CC-90011 on THP-1 cell viability.

[0069] Figure 2 The results show the effects of compounds IX-8 and CC-90011 on CD11b expression in THP-1 cells.

[0070] Figure 3 This is the result of the combined application of IX-8 and 1,25(OH)2D3 on the differentiation of leukemia cells THP-1. DETAILED DESCRIPTION

[0071] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0072] Embodiment 1:

[0073] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0074]

[0075] The preparation method of the above compound comprises the following steps:

[0076] Preparation of intermediate III: Add 15 mL of anhydrous ethanol to 2,4,5,6-tetrachloropyrimidine (4.8 g, 22.03 mmol) to dissolve it, then add triethylamine (3 mL, 21.43 mmol) and stir under ice bath conditions. Dissolve aminoacetaldehyde dimethyl acetal (2.4 mL, 22.03 mmol) in anhydrous ethanol, add dropwise to the system, and react for 6 hours. Monitor the reaction by thin layer chromatography. After the reaction is completed, evaporate the ethanol, add an appropriate amount of ether to the system, filter, and dry to obtain 5.02 g of white intermediate III, with a yield of about 80%;

[0077] Preparation of intermediate IV: Add intermediate III (5.0 g, 7.02 mmol) to a dry round-bottom flask, add 5 mL of concentrated sulfuric acid, and stir under reflux for 4 hours. Monitor the reaction by thin layer chromatography. After the reaction is completed, transfer the system to a beaker, adjust the pH of the system to 5-6 with sodium hydroxide solution, extract with dichloromethane and saturated brine, add anhydrous magnesium sulfate to dry, and concentrate under vacuum. Dry to obtain 2.69 g of white solid intermediate IV, with a yield of about 45%; 1 H NMR (400MHz, DMSO-d6) δ7.81 (d, J = 1.9 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H);

[0078] Preparation of intermediate V: Add intermediate IV (2 g, 10.8 mmol) and 10 mL of phosphorus oxychloride to a round-bottom flask, slowly drip DIPEA (1.88 mL, 21.5 mmol) into the system, and then heat and reflux for 9 hours. After the reaction is completed, pour into ice water, adjust pH 8 to 9 with saturated NaHCO3 solution, extract with ethyl acetate and saturated brine, add anhydrous magnesium sulfate to dry, concentrate under vacuum, and separate and purify by column chromatography to obtain 1.29 g of yellow intermediate V, with a yield of 50%. 1 H NMR (400MHz, DMSO-d6) δ8.20 (d, J = 1.5 Hz, 1H), 7.85 (d, J = 1.5 Hz, 1H);

[0079] Preparation of intermediate VI-1: Add intermediate V (500 mg, 2.25 mmol), 4-tert-butyloxycarbonylaminopiperidine (450 mg, 2.25 mmol) and DIPEA (1.56 mL, 9 mmol) to a round-bottom flask, add 10 ml of DMF to dissolve it, and stir at room temperature for 2 to 3 hours. Monitor the reaction by thin layer chromatography. After the reaction is complete, add the same volume of water and ethyl acetate, extract the aqueous phase with ethyl acetate three times, and dry the organic layer over anhydrous magnesium sulfate, filter it, and dry it to obtain 690 mg of intermediate VI-1, with a yield of 80%;

[0080] The structural formula of intermediate VI-1 is: 1 H NMR (400MHz, DMSO-d6) δ7.85(d,J=1.5Hz,1H),7.64(d,J=1.5Hz,1H),7.00(d,J=7.6Hz,1H),3.93-3. 80(m,2H),3.63-3.48(m,1H),3.17-2.98(m,2H),1.93-1.82(m,2H),1.67-1.50(m,2H),1.40(s,9H);

[0081] Preparation of intermediate VIII-1: Dissolve intermediate VI-1 (500 mg, 1.3 mmol), phenylboronic acid (434 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate and react under reflux for 10 hours under nitrogen protection. Monitor the reaction by TLC. After the reaction is complete, add the same volume of water and ethyl acetate, extract the organic phase three times with water, dry the organic layer over anhydrous magnesium sulfate, filter it, and separate it by column chromatography to obtain 154 mg of the intermediate VIII-1, with a yield of about 25%; 1H NMR (400MHz, DMSO-d6) δ7.79(d,J=1.5Hz,1H),7.60(d,J=1.5Hz,1H),7.40-7.35(m,2H),7.31(s,5H),7.26-7.19(m,3H),6.99(d,J =7.7Hz,1H),3.91(d,J=12.4Hz,2H),3.57(s,1H),3.10(t,J=11.9Hz,2H),1.92(d,J=12.4Hz,2H),1.76-1.61(m,2H),1.41(s,9H);

[0082] Preparation of product IX-1: 150 mg (0.3 mmol) of intermediate VIII-1 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the white solid product IX-1 was obtained by suction filtration. The yield was 70%; 1 H NMR (400MHz, DMSO-d6) δ8.21(d,J=5.5Hz,3H),8.10(d,J=1.9Hz,1H),7.93(d,J=1.9Hz,1H),7.54-7.49(m,1H),7.43-7.38(m,4H),7.3 6-7.31(m,2H),7.31-7.24(m,3H),4.05(t,J=18.5Hz,2H),3.51-3.35(m,1H),3.29-3.19(m,2H),2.14-2.03(m,2H),1.96-1.78(m,2H).

[0083] Embodiment 2:

[0084] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0085]

[0086] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0087] Preparation of intermediate VIII-2: Intermediate VI-1 (1.3 mmol), p-chlorophenylboronic acid (506 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) were dissolved in ethyl acetate and heated under reflux for 10 hours under nitrogen protection. The reaction was monitored by TLC. After the reaction was complete, the same volume of water and ethyl acetate were added, and the organic phase was extracted with water three times. The organic layer was dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography to obtain 252 mg of light yellow intermediate VIII-2 with a yield of 36%; 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=1.5Hz,1H),7.79(d,J=8.3Hz,1H),7.62(d,J=1.5Hz,1H),7.44-7.38(m,3H),7.38-7.33(m,4H),7.01 (d,J=7.5Hz,1H),3.91(d,J=12.3Hz,2H),3.57(s,1H),3.10(t,J=11.8Hz,2H),1.91(d,J=12.3Hz,2H),1.76-1.61(m,2H),1.41(s,9H);

[0088] Preparation of product IX-2: 215 mg (0.4 mmol) of intermediate VIII-2 was dissolved in DCM, and 1.5 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the solid was precipitated by ultrasound for 5 minutes. The product IX-2 was obtained by suction filtration with a yield of 80%. 1 H NMR (400MHz, DMSO-d6) δ8.09(s,3H),8.03(s,1H),7.84(s,1H),7.79(d,J=8.0Hz,1H),7.49(d,J=8.4Hz,2H),7.43-7.3 3(m,5H),4.06(d,J=13.4Hz,2H),3.48-3.32(m,1H),3.20(t,J=12.4Hz,2H),2.06(d,J=13.1Hz,2H),1.92-1.74(m,2H).

[0089] Embodiment 3:

[0090] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0091]

[0092] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0093] Preparation of intermediate VIII-3: Dissolve intermediate VI-1 (1.3 mmol), p-fluorophenylboric acid (454 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate and heat under reflux for 11 hours under nitrogen protection. Monitor the reaction by TLC. After the reaction is complete, add the same volume of water and ethyl acetate, extract the organic phase three times with water, dry the organic layer over anhydrous magnesium sulfate, filter it, and separate it by column chromatography to obtain 262 mg of the intermediate VIII-3, with a yield of about 30%; 1 H NMR (400MHz, DMSO-d6) δ7.80(d,J=1.5Hz,1H),7.60(d,J=1.5Hz,1H),7.44-7.32(m,4H),7.23-7.07(m,4H),7.01(d,J=7.9Hz, 1H), 3.90 (d, J = 12.4Hz, 2H), 3.57 (s, 1H), 3.10 (t, J = 11.5Hz, 2H), 1.91 (d, J = 12.4Hz, 2H), 1.68 (q, J = 11.5Hz, 2H), 1.41 (s, 9H);

[0094] Preparation of product IX-3: 250 mg (0.4 mmol) of intermediate VIII-3 was dissolved in DCM, and 1.8 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-3 was obtained by suction filtration. The yield was about 78%; 1 HNMR(400MHz, DMSO-d6)δ8.26-8.06(m,3H),8.04(d,J=1.7Hz,1H),7.85(s,1H),7.46-7.39(m,2H),7.39-7.33(m,2H),7.26(t,J=8.8Hz, 2H), 7.15 (t, J = 8.8Hz, 2H), 4.06 (d, J = 13.6Hz, 2H), 3.51-3.34 (m, 1H), 3.21 (t, J = 12.8Hz, 1H), 2.07 (d, J = 10.9Hz, 1H), 1.94-1.78 (m, 2H).

[0095] Embodiment 4:

[0096] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0097]

[0098] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0099] Preparation of intermediate VIII-4: Dissolve intermediate VI-1 (1.3 mmol), p-trifluoromethylphenylboronic acid (616 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate and heat under reflux for 10 hours under nitrogen protection. Monitor the reaction by TLC. After the reaction is complete, add the same volume of water and ethyl acetate, extract the organic phase three times with water, dry the organic layer over anhydrous magnesium sulfate, filter it, and separate it by column chromatography to obtain 198 mg of the intermediate VIII-4, with a yield of 25%; 1 H NMR (400MHz, DMSO-d6) δ7.86(d,J=1.5Hz,1H),7.73(s,1H),7.71(s,1H),7.65(d,J=7.6Hz,3H),7.61-7.53(m,4H),6.99(d,J=7. 7Hz,1H),3.95(d,J=14.0Hz,2H),3.44-3.34(m,1H),3.21-3.02(m,2H),1.93(d,J=14.0Hz,2H),1.82-1.66(m,2H),1.41(s,9H);

[0100] Preparation of product IX-4: 190 mg (0.3 mmol) of intermediate VIII-4 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-4 was filtered to obtain a yellow solid with a yield of 80%. 1 H NMR (400MHz, DMSO-d6) δ8.08(d,J=4.8Hz,3H),8.05(d,J=1.7Hz,1H),7.81(d,J=1.7Hz,1H),7.77(d,J=8.1Hz,2H),7.68(d,J=8.2Hz,2H ), 7.58 (dd, J = 8.3, 2.3Hz, 4H), 4.09 (d, J = 13.4Hz, 2H), 3.53-3.35 (m, 1H), 3.21 (t, J = 13.4Hz, 2H), 2.16-2.03 (m, 2H), 1.92-1.74 (m, 2H).

[0101] Embodiment 5:

[0102] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0103]

[0104] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0105] Preparation of intermediate VIII-5: Dissolve intermediate VI-1 (1.3 mmol), p-nitrophenylboronic acid (541 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate and heat under reflux for 12 hours under nitrogen protection. Monitor the reaction by TLC. After the reaction is complete, add the same volume of water and ethyl acetate, extract the organic phase three times with water, dry the organic layer over anhydrous magnesium sulfate, filter it, and separate it by column chromatography to obtain 255 mg of the intermediate VIII-5 as a yellow solid, with a yield of about 30%; 1 H NMR (400MHz, DMSO-d6) δ8.21(d,J=8.7Hz,2H),8.18-8.10(m,2H),7.91(d,J=1.6Hz,1H),7.70-7.59(m,5H),7.01(d,J= 7.8Hz,1H),4.04-3.84(m,2H),3.59(s,1H),3.21-3.02(m,2H),1.91(t,J=16.4Hz,2H),1.75-1.57(m,2H),1.41(s,9H);

[0106] Preparation of product IX-5: 250 mg (0.4 mmol) of intermediate VIII-5 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-5 was obtained by suction filtration. The yield was about 70%. 1 HNMR (400MHz, DMSO-d6) δ8.27-8.22(m,2H),8.18-8.14(m,2H),8.04(d,J=5.3Hz,3H),8.02(d,J=1.5Hz,1H),7.76(d,J=1.5Hz,1 H),7.66v7.59(m,4H),4.10(d,J=13.3Hz,2H),3.45-3.38(m,1H),3.20(t,J=12.6Hz,2H),2.12-2.01(m,2H),1.90-1.78(m,2H).

[0107] Embodiment 6:

[0108] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0109]

[0110] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0111] Preparation of intermediate VIII-6: The intermediate VI-1 (1.3 mmol), 2-fluoro-5-nitrobenzeneboronic acid (590 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) were dissolved in ethyl acetate, heated under reflux for 12 hours under nitrogen protection, and the reaction was monitored by TLC. After the reaction was complete, the same volume of water and ethyl acetate were added, and the organic phase was extracted three times with water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and silica gel was added for frying. Column chromatography was separated to obtain 180 mg of the intermediate VIII-6 with a yield of 20%; 1 H NMR (400MHz, DMSO-d6) δ8.45v8.40(m,1H),8.37-8.24(m,3H),7.95(d,J=1.5Hz,1H),7.69(d,J=1.5Hz,1H),7.52(t,J=8.9Hz,1H),7.43(t,J=9.2Hz, 1H),7.02(d,J=7.8Hz,1H),3.99(d,J=11.9Hz,2H),3.60(s,1H),3.17(t,J =12.5Hz,2H),1.94(d,J=12.5Hz,2H),1.67(d,J=11.9Hz,2H),1.41(s,9H).

[0112] Preparation of product IX-6: 150 mg (0.2 mmol) of intermediate VIII-6 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the brown solid product IX-6 was obtained by suction filtration. The yield was 78%; 1H NMR (400MHz, DMSO-d6) δ8.44(dd,J=6.1,3.1Hz,1H),8.37-8.26(m,3H),8.15-8.03(m,2H),7.76(s,1H),7.55(d,J=9.2Hz ,1H),7.44(t,J=9.2Hz,1H),4.11(d,J=13.1Hz,2H),3.41(s,1H),3.21(t,J=12.5Hz,2H),2.22-2.00(m,2H),1.83(s,2H).

[0113] Embodiment 7:

[0114] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0115]

[0116] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0117] Preparation of intermediate VIII-7: Dissolve intermediate VI-1 (1.3 mmol), 4-cyanophenylboronic acid (476 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 12 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 20 mg of the intermediate VIII-7, with a yield of 20%; 1 H NMR (400MHz, DMSO-d6) δ8.07-7.97(m,1H),7.90-7.87(m,1H),7.83(d,J=1.9Hz,2H),7.77(d,J=1.9Hz,2H),7.65(d,J=1.4Hz,1H),7.57-7.49( m,3H),6.99(d,J=7.8Hz,1H),3.95(d,J=12.8Hz,2H),3.58(s,1H),3.14(t,J=12.8Hz,2H),1.96-1.85(m,2H),1.72-1.60(m,2H),1.40(s,9H).

[0118] Preparation of product IX-7: 180 mg (0.3 mmol) of intermediate VIII-7 was dissolved in DCM, and 2 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-7 was obtained by suction filtration with a yield of 80%. 1 H NMR(400MHz, DMSO-d6)δ8.08(d,J=4.9Hz,3H),8.01(d,J=1.7Hz,1H),7.90-7.83(m,2H),7.81-7.75(m,3H), 7.59-7.46(m,4H),4.15-3.95(m,2H),3.39(s,2H),3.23-3.02(m,1H),2.15-1.98(m,2H),1.90-1.75(m,2H).

[0119] Embodiment 8:

[0120] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0121]

[0122] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0123] Preparation of intermediate VIII-8: Dissolve intermediate VI-1 (1.3 mmol), 4-cyano-3-fluorophenylboronic acid (535 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 11 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, add silica gel to stir-fry, and separate by column chromatography to obtain 186 mg of the intermediate VIII-8, with a yield of 25%; 1 H NMR (400MHz, DMSO-d6) δ7.97-7.79(m,3H),7.76-7.54(m,3H),7.34-7.19(m,1H),7.00(d,J=8.0Hz,0H),3.94(d d,J=33.7,13.7Hz,2H),3.59(s,1H),3.22-3.06(m,2H),1.92(d,J=13.7Hz,2H),1.75-1.59(m,2H),1.41(s,9H).

[0124] Preparation of product IX-8: 150 mg (0.3 mmol) of intermediate VIII-8 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-8 was obtained by suction filtration with a yield of 80%. 1 H NMR (400MHz, DMSO-d6) δ8.24-8.06(m,3H),8.02(d,J=7.9Hz,1H),7.97-7.81(m,2H),7.78(d,J=7.9Hz,1H),7.62(dd,J=16.0,10.4Hz,2H),7.27(d d,J=16.0,8.1Hz,2H),4.06(dd,J=36.6,13.0Hz,2H),3.51-3.33(m,1H), 3.28-3.09(m,2H),2.08(d,J=13.0Hz,2H),1.84(qd,J=12.5,3.9Hz,2H).

[0125] Embodiment 9:

[0126] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0127]

[0128] In the preparation method of the above compound, the preparation process of intermediate III to intermediate V is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0129] Preparation of intermediate VI-2: Add intermediate V (500 mg, 2.25 mmol), 1-Boc-3-aminopyrrolidine (419 mg, 2.25 mmol) and DIPEA (1.56 mL, 9 mmol) to a round-bottom flask, add 10 ml of DMF to dissolve the raw materials, and stir at room temperature for 2 to 3 hours. Monitor the reaction by thin layer chromatography. After the reaction is completed, add an appropriate amount of water to the system, ultrasonicate for five minutes, and solid precipitates. Filter the residue and wash it with water three times. After drying, 696 mg of intermediate VI-2 is obtained, with a yield of 85%; wherein the structure of intermediate VI-2 is 1H NMR (400MHz, DMSO-d6) δ8.31-8.04(m,2H),7.57(d,J=1.5Hz,1H),4.64-4.43(m,1H),3.74-3.62(m,1H),3.49 -3.37(m,2H),3.30(d,J=4.4Hz,1H),2.22(dd,J=13.1,6.6Hz,1H),2.02(dd,J=13.3,6.3Hz,1H),1.42(s,9H);

[0130] Preparation of intermediate VIII-9: Dissolve intermediate VI-2 (482 mg, 1.3 mmol), 4-cyano-3-fluorophenylboronic acid (535 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 12 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 211 mg of intermediate VIII-9, with a yield of 30%; 1 H NMR(400MHz,DMSO-d6)δ8.25(s,1H),8.15(s,1H),7.96-7.75(m,2H),7.66-7.51(m,3H),7.29-7.13(m,2H),4.73(s,1H ),3.84-3.65(m,1H),3.47(d,J=7.2Hz,1H),3.39(t,J=7.3Hz,2H),2.31-2.20(m,1H),2.18-2.04(m,1H),1.42(s,9H);

[0131] Preparation of product IX-9: 200 mg (0.3 mmol) of intermediate VIII-9 was dissolved in DCM, 1 ml of TFA was added under stirring at room temperature, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the solid was precipitated by ultrasound for 5 minutes. The product IX-9 was obtained by suction filtration with a yield of 70%. 1H NMR (400MHz, DMSO-d6) δ9.10 (s, 1H), 8.93 (s, 1H), 8.41 (d, J = 5.5Hz, 1H), 8.25 (d ,J=1.6Hz,1H),7.93-7.80(m,2H),7.73(d,J=1.6Hz,1H),7.68-7.56(m,2H),7.32 -7.17(m,2H),4.88-4.74(m,1H),3.72-3.59(m,1H),3.54-3.40(m,1H),3.35(dt ,J=11.8,6.0Hz,2H),2.36(dt,J=14.3,6.9Hz,1H),2.22(dt,J=13.1,6.6Hz,1H).

[0132] Embodiment 10:

[0133] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0134]

[0135] In the preparation method of the above compound, the preparation process of intermediate III to intermediate V is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0136] Preparation of intermediate VI-3: Add intermediate V (500 mg, 2.25 mmol), 1-Boc-piperazine (419 mg, 2.25 mmol) and DIPEA (1.56 mL, 9 mmol) to a round-bottom flask, add 10 ml of DMF to dissolve the raw materials, and stir at room temperature for 2 to 3 hours. Monitor the reaction by thin layer chromatography. After the reaction is completed, add an appropriate amount of water to the system, ultrasonicate for five minutes, and solid precipitates. Filter the residue and wash it with water three times. After drying, obtain intermediate VI-3 (690 mg, 85%); wherein the structure of the intermediate VI-3 is 1 H NMR (400MHz, DMSO-d6) δ7.97(d,J=1.6Hz,1H),7.67(d,J=1.6Hz,1H),3.55(dd,J=6.8,3.4Hz,4H),3.45(dd,J=6.8,3.4Hz,4H),1.43(s,9H);

[0137] Preparation of intermediate VIII-10: Dissolve intermediate VI-3 (1.3 mmol), 4-cyano-3-fluorophenylboronic acid (535 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 12 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 180 mg of intermediate VIII-10, with a yield of 20%; 1 H NMR(400MHz, DMSO-d6)δ8.05(d,J=1.5Hz,1H),7.95-7.80(m,2H),7.72(d,J=1.5Hz,1H),7. 68-7.55(m,2H),7.34-7.19(m,2H),3.60(d,J=5.4Hz,4H),3.56-3.47(m,4H),1.44(s,9H).

[0138] Preparation of product IX-10: 200 mg (0.3 mmol) of intermediate VIII-10 was dissolved in DCM, 1 ml of TFA was added under stirring at room temperature, and stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-10 was obtained by suction filtration with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ9.07(s,1H),8.17(s,1H),7.93(t,J=7.5Hz,1H),7.85(t,J=7.5Hz,1 H),7.80(s,1H),7.68-7.58(m,2H),7.34-7.21(m,2H),3.79-3.71(m,4H),3.44-3.36(m,4H).

[0139] Embodiment 11:

[0140] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0141]

[0142] In the preparation method of the above compound, the preparation process of intermediate III to intermediate V is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0143] Preparation of intermediate VI-4: Add intermediate V (500 mg, 2.25 mmol) and N-tert-butyloxycarbonyl-1,2-ethylenediamine (360 mg, 2.25 mmol) and DIPEA (1.56 mL, 9 mmol) to a round-bottom flask, add 10 ml of DMF to dissolve the raw materials, and stir at room temperature for 2 to 3 hours. Monitor the reaction by thin layer chromatography. After the reaction is completed, add equal amounts of water and ethyl acetate to the product mixture for extraction. The aqueous phase is extracted three times with ethyl acetate, and the organic layers are combined; the organic layer is dried over anhydrous magnesium sulfate, filtered, and the organic solvent is evaporated to obtain 621 mg of intermediate VI-4, with a yield of 80%; wherein the structure of intermediate VI-4 is 1 H NMR (400MHz, DMSO-d6) δ8.33(t,J=5.5Hz,1H),8.04(d,J=1.5Hz,1H),7.55(d,J=1.5Hz ,1H),6.96(t,J=6.0Hz,1H),3.48(q,J=6.0Hz,2H),3.24(d,J=6.0Hz,2H),1.34(s,9H);

[0144] Preparation of intermediate VIII-11: Dissolve intermediate VI-4 (1.3 mmol), 4-cyano-3-fluorophenylboronic acid (535 mg, 3.24 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 12 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 151 mg of intermediate VIII-11, with a yield of 22%; 1 H NMR (400MHz, DMSO-d6) δ8.29(t,J=5.7Hz,1H),8.09(s,1H),7.90-7.71(m,2H),7.71-7.46(m,3H),7.22( dd,J=8.1,1.6Hz,2H),7.01(t,J=5.9Hz,1H),3.62(q,J=6.2Hz,2H),3.30(q,J=6.2Hz,2H),1.35(s,9H);

[0145] Preparation of product IX-11: 140 mg (0.3 mmol) of intermediate VIII-11 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-11 was obtained by suction filtration with a yield of 70%. 1H NMR (400MHz, DMSO-d6) δ8.61(t,J=5.5Hz,1H),8.13(d,J=1.6Hz,1H),7.94(s,3H),7.91-7.80(m,2H),7. 73(d,J=1.6Hz,1H),7.66-7.57(m,2H),7.25-7.19(m,2H),3.85(q,J=5.9Hz,2H),3.20(q,J=5.9Hz,2H).

[0146] Embodiment 12:

[0147] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0148]

[0149] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0150] Preparation of intermediate VIII-12: Intermediate VI-1 (500 mg, 1.05 mmol), 3-fluoro-4-methoxyphenylboronic acid (268 mg, 1.57 mmol), potassium carbonate (107 mg, 2.10 mmol) and palladium catalyst (0.06 mmol) were dissolved in ethyl acetate and heated under reflux for 12 hours under nitrogen protection. The reaction was monitored by TLC. After the reaction was complete, the same volume of water and ethyl acetate were added, and the organic phase was extracted three times with water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and separated by column chromatography to obtain 100 mg of the intermediate VIII-120, with a yield of 10%; 1 H NMR (400MHz, DMSO-d6) δ7.78(d,J=1.5Hz,1H),7.59(d,J=1.5Hz,1H),7.28-7.20(m,2H),7.19-7.11(m,2H),7.06(t,J=8.8Hz,2H),7.00(d,J=7.7Hz ,1H),3.90(d,J=13.0Hz,2H),3.84(d,J=16.2Hz,6H),3.57(s,1H),3.10( t,J=12.0Hz,2H),1.91(d,J=12.0Hz,2H),1.75-1.60(m,2H),1.41(s,9H);

[0151] Preparation of product IX-12: 100 mg (0.2 mmol) of intermediate VIII-12 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-12 was obtained by suction filtration with a yield of 70%. 1 H NMR (400MHz, DMSO-d6) δ8.17(d,J=5.0Hz,3H),8.08(d,J=1.9Hz,1H),7.93(d,J=1.9Hz,1H),7.37-7.21(m,3H),7.19-7.03(m,3H),4 .07(d,J=13.1Hz,2H),3.87(d,J=22.3Hz,6H),3.49-3.36(m,1H),3.24(t,J=12.5Hz,2H),2.08(d,J=12.5Hz,2H),1.95-1.80(m,2H).

[0152] Embodiment 13:

[0153] An imidazo[1,2-c]pyrimidine compound, the structural formula of the compound is as follows:

[0154]

[0155] In the preparation method of the above compound, the preparation process of intermediate III to intermediate VI is the same as that in Example 1, and the subsequent preparation is changed due to different groups, as follows:

[0156] Preparation of intermediate VII-1: Dissolve intermediate VI-1 (500 mg, 1.3 mmol), 3-fluoro-4-methoxyphenylboronic acid (332 mg, 1.95 mmol), potassium carbonate (138 mg, 2.59 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 10 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 150 mg of intermediate VII-1, with a yield of 20%; the structural formula of intermediate VII-1 is:

[0157] 1H NMR (400MHz, DMSO-d6) δ7.80-7.75(m,1H),7.62-7.56(m,1H),7.28-7.20(m,1H),7.13(d,J=8.7Hz,1H),7.09-7.02(m,1H),6.99(t,J=6.8Hz ,1H),3.92(d,J=4.0Hz,2H),3.88(d,J=7.4Hz,3H),3.57(s,1H),3.18-3.04(m,2H),1.92(d,J=12.6Hz,2H),1.77-1.61(m,2H),1.41(s,9H);

[0158] Preparation of intermediate VIII-13: Dissolve intermediate VII-1 (500 mg, 1.05 mmol), 4-cyano-3-fluorophenylboronic acid (300 mg, 1.57 mmol), potassium carbonate (107 mg, 2.10 mmol) and palladium catalyst (0.06 mmol) in ethyl acetate, and heat under reflux for 11 hours under nitrogen protection. Monitor the reaction by TLC, add the same volume of water and ethyl acetate after the reaction is complete, extract the organic phase with water three times, dry the organic layer over anhydrous magnesium sulfate, filter, and separate by column chromatography to obtain 120 mg of the intermediate VIII-13, with a yield of 15%; 1 H NMR(400MHz,DMSO-d6)δ7.96-7.76(m,2H),7.70-7.48(m,2H),7.44-7.25(m,2H),7.22-7.10(m,1H),7.09-6.94(m,2H ),4.02-3.83(m,5H),3.58(s,1H),3.10(dd,J=24.4,13.3Hz,2H),2.01-1.84(m,2H),1.83-1.62(m,2H),1.41(s,9H);

[0159] Preparation of product IX-13: 110 mg (0.2 mmol) of intermediate VIII-13 was dissolved in DCM, and 1 ml of TFA was added under stirring at room temperature. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, an appropriate amount of ether was added, and the product IX-13 was obtained by suction filtration. The yield was 70%; 1H NMR(400MHz,DMSO-d6)δ8.11(d,J=13.5Hz,3H),8.04(d,J=11.4Hz,1H),7.8 6(d,J=5.6Hz,1H),7.60-7.53(m,1H),7.43-7.34(m,1H),7.30-7.17(m,3H), 7.03(d,J=8.6Hz,1H),4.14-4.05(m,2H),3.86(d,J=15.7Hz,3H),3.47-3.3 7(m,1H),3.20(t,J=12.4Hz,2H),2.08(d,J=12.4Hz,2H),1.92-1.78(m,2H).

[0160] Test Example 1: LSD1 inhibitory activity assay

[0161] Weigh the compounds IX1-13 (samples) prepared in Examples 1-13 respectively, dissolve them in DMSO (dimethyl sulfoxide) to a mother solution with a concentration of 10 mM, and dilute them with DMSO to the required concentration for the experiment. The samples were incubated with the human complex protein LSD1 / CoREST purified from the Escherichia coli expression system, and then the H3K4me2 polypeptide synthesized by Gill Biochemical (Shanghai) Co., Ltd. was added and incubated for 30 minutes. After the incubation, the fluorescent dye Amplex Red and horseradish peroxidase HRP were added for 5 minutes, and then the fluorescence signal (E X =535nm, E m =595nm), and calculate its inhibition rate. The inhibition rate calculation formula is as follows:

[0162]

[0163] Among them, the "fluorescence intensity of the sample group", "fluorescence intensity of the standard group" and "fluorescence intensity of the blank group" in the above inhibition rate calculation formula are all fluorescence intensity values ​​measured by referring to the above experimental method. The difference is that: under the same conditions, the object of measurement of the "fluorescence intensity of the sample group" is the sample solution containing the above compound to be tested, and the object of measurement of the "fluorescence intensity of the standard group" is the standard solution, and the standard solution does not contain the sample compared with the sample solution. The object of measurement of the "fluorescence intensity of the blank group" is a blank sample without LSD1 / CoREST complex protein and H3K4me2 polypeptide. The IC values ​​of the above sample compounds were processed using Graphpad Prism 8.0 50 The data and results are shown in Table 1.

[0164] Table 1 Data table of the inhibitory activity of the compounds provided in the examples of the present invention on LSD1 / CoREST

[0165] Compound <![CDATA[IC 50 (nM)]]> Compound <![CDATA[IC 50 (nM)]]> IX-1 4483 IX-8 11.5 IX-2 410.9 IX-9 306.2 IX-3 4861 IX-10 1307 IX-4 1573 IX-11 411.3 IX-5 9.05 IX-12 117.4 IX-6 2018 IX-13 8.474 IX-7 11.74

[0166] As can be seen from Table 1, different substituents of a compound have a great influence on the function of the compound, that is, when the rest of the structure is the same, but the difference is different groups in the same substituent, it will also have a great influence on the function of the compound. In addition, the function of the compound is also determined by different substituents on the skeleton structure, including the position and type of the substituent, which will affect the function of the compound.

[0167] Experimental Example 2: Cell Viability Assay

[0168] It can be seen from Experimental Example 1 that the effects of compounds IX-5 and IX-8 are both excellent. Considering the safety of the compounds, IX-8 was selected as a sample for the cell viability experiment. The specific process is as follows:

[0169] The sample, IX-8, was dissolved in DMSO (dimethyl sulfoxide) into a mother solution with a concentration of 10mM. Leukemia THP-1 cells in the logarithmic growth phase were collected, the cell suspension density was adjusted to 10,000 cells / well, 100 μL of cell suspension was inoculated in a 96-well plate, and the drug concentration gradient was added. The edge wells were filled with sterile PBS or water and cultured in an incubator (37°C, 5% CO2). After the cells were cultured for 5 days, 7 days, and 10 days, 10 μL of CCK-8 reagent was added to each well, and the cells were placed in an incubator for further incubation for 1-4 hours. The absorbance at 450nm was measured with an enzyme marker, and the inhibition rate was calculated. The inhibition rate calculation formula is as follows:

[0170] Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100%

[0171] A (drug added): absorbance of wells with cells, CCK-8 solution and drug solution.

[0172] A (0 drug addition): absorbance of the wells with cells, CCK-8 solution but no drug solution.

[0173] A (blank): absorbance of the well with culture medium, CCK-8 solution but no cells.

[0174] According to the calculated inhibition rate, the dose-effect curve was fitted using the analysis software GraphPad to obtain the IC value of the compound on cell inhibition. 50 Value, the result is Figure 1 shown.

[0175] Depend on Figure 1It can be seen that compound IX-8 can inhibit the proliferation of THP-1 cells in a time- and dose-dependent manner, and its anti-proliferation effect is better than that of CC-90011.

[0176] Experimental Example 3: Detection of differentiation of leukemia cells THP-1 by flow cytometry

[0177] The sample, IX-8, was dissolved in DMSO (dimethyl sulfoxide) to a stock solution with a concentration of 10 mM. Leukemia THP-1 cells in the logarithmic growth phase were collected and the cell suspension density was adjusted to 1×10 5 1 mL of cell suspension was inoculated into a 12-well plate and a concentration gradient (10-fold gradient, 10 0 ~10 5 ) to 2 mL of drug-containing culture medium and culture in an incubator (37°C, 5% CO2). After the cells were cultured for 96 hours, the cells were collected by centrifugation at 4°C, 500g for 5 minutes; the supernatant was discarded, and 500 μL of pre-cooled PBS was added, and the cells were centrifuged at 4°C, 500g for 5 minutes, and washed twice; after washing, the cells were resuspended with 100 μL of PBS, and 1 μL of anti-human CD11b-PE (12-0118-41) flow cytometry antibody was added under light-proof conditions, and stained at 4°C for 1 hour; after staining, the cells were centrifuged at 4°C, 500g for 5 minutes, and the supernatant was discarded; 500 μL of pre-cooled PBS was added, and the cells were centrifuged at 4°C, 500g for 5 minutes, and the supernatant was discarded, and the cells were washed twice; after washing, 500 μL of PBS was added to each tube to resuspend the cells, and the cells were transferred to a flow tube, and the expression of CD11b was detected by flow cytometry, and the detection results were analyzed by FlowJo software, and the dose-effect curve was fitted using the analysis software GraphPad, so as to obtain the EC of IX-8 and CC-90011 on the expression of CD11b in THP-1 cells. 50 Value, see the result Figure 2 .

[0178] Depend on Figure 2 It can be seen that compound IX-8 can effectively induce the differentiation of THP-1 cells and increase the expression of CD11b, and its effect is improved compared with CC-90011.

[0179] Experimental Example 4: Effect of combined use of IX-8 and 1,25(OH)2D3 on differentiation of leukemia cells THP-1

[0180] Dilute the 10 mM IX-8 stock solution prepared above to a 10 μM stock solution with DMSO. Prepare 1,25(OH)2D3 to a 10 μM stock solution with DMSO. Collect THP-1 cells in the logarithmic growth phase and adjust the cell suspension density to 1×10 5 1 mL of cell suspension was inoculated into a 12-well plate and a concentration gradient (10-fold gradient, 100 ~10 5 ) to 2mL of drug-containing culture medium and culture in an incubator (37°C, 5% CO2). After the cells have been cultured for 96 hours, centrifuge at 4°C, 500g for 5 minutes to collect the cells; discard the supernatant, add 500μL of pre-cooled PBS, centrifuge at 4°C, 500g for 5 minutes, and wash twice; after washing, resuspend the cells with 100μL PBS, add 1μL anti-human CD11b-PE (12-0118-41) flow cytometry antibody under light-proof conditions, and stain at 4°C for 1h; after staining, centrifuge at 4°C, 500g for 5 minutes, discard the supernatant; add 500μL of pre-cooled PBS, centrifuge at 4°C, 500g for 5 minutes, discard the supernatant, and wash twice; after washing, add 500μL of PBS to each tube to resuspend the cells and transfer them to flow tubes, use flow cytometry to detect the expression of CD11b on the cell surface, and use FlowJo software to analyze the test results. The results are shown in Figure 3 .

[0181] Depend on Figure 3 It can be seen that compared with untreated cells, after IX-8 (5nM) alone treated for 4 days, CD11b + The proportion of cells with CD11b increased by about 14%; after 4 days of treatment with 1,25(OH)2D3 (1nM) and 1,25(OH)2D3 (5nM) alone, compared with untreated cells, + When IX-8 (5 nM) was used in combination with 1,25(OH)2D3 (1 nM) and 1,25(OH)2D3 (5 nM), the CD11b + The proportion of cells increased by about 30% and 52%, respectively. In summary, the combination of compound IX-8 and 1,25(OH)2D3 can synergistically induce the differentiation of leukemia cells THP-1.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an imidazo[1,2-c]pyrimidine compound, characterized in that: The reaction formula is as follows: Among them, R in VII 1 = , R 2 = ; or R 1 = , R 2 = ; or R 1 = , R 2 = ; The specific reaction process includes the following steps: S1, dissolving 2,4,5,6-tetrachloropyrimidine and triethylamine in a solvent, adding aminoacetaldehyde dimethyl acetal under ice bath conditions to react, to obtain intermediate III; S2, heating the intermediate III to reflux, and then adjusting the pH of the reaction system with a base to obtain the intermediate IV; S3, dissolving intermediate IV in phosphorus oxychloride, adding an organic base, and heating under reflux to obtain intermediate V; S4, intermediate V, amine compound R 1 H and an organic base are dissolved in a solvent and reacted at room temperature to obtain intermediate VI; S5, intermediate VI, boronic acid compound R 2 B(OH)2, palladium catalyst and inorganic base are dissolved in a solvent and heated under reflux to obtain intermediate VII, i.e., imidazo[1,2-c]pyrimidine compound.

2. The preparation method according to claim 1, characterized in that In step S1, the molar ratio of 2,4,5,6-tetrachloropyrimidine, triethylamine and aminoacetaldehyde dimethyl acetal is 1:1:1 to 1:3:3; the reaction is stirred under ice bath conditions for 4 to 6 hours; In step S2, the heating reflux time is 4 to 6 h; In step S3, the molar ratio of intermediate IV to the organic base is 1:1-1:2, and the reflux reaction time is 8-10 h; In step S4, intermediate V and amine compound R 1 The molar ratio of H and organic base is 1:1:2 to 1:1:3; the reaction is stirred at room temperature for 1 to 3 hours; In step S5, intermediate VI and boronic acid compound R 2 The molar ratio of B(OH)2, palladium catalyst and inorganic base is 1:2~2.5:0.05~1:2; and the reflux reaction is carried out under protective gas conditions for 10~18 hours.

3. A method for preparing an imidazo[1,2-c]pyrimidine compound, characterized in that: The reaction formula is as follows: Among them, R in VIII 1 = , R 2 = , R 3 = ; The specific reaction process includes the following steps: S1, dissolving 2,4,5,6-tetrachloropyrimidine and triethylamine in a solvent, adding aminoacetaldehyde dimethyl acetal under ice bath conditions to react, to obtain intermediate III; S2, heating the intermediate III to reflux, and then adjusting the pH of the reaction system with a base to obtain the intermediate IV; S3, dissolving intermediate IV in phosphorus oxychloride, adding an organic base, and heating under reflux to obtain intermediate V; S4, intermediate V, amine compound R 1 H and an organic base are dissolved in a solvent and reacted at room temperature to obtain intermediate VI; S5, intermediate VI, boronic acid compound R 2 B(OH)2, palladium catalyst and inorganic base are dissolved in a solvent and heated under reflux to obtain intermediate VII; S6, intermediate VII, boronic acid compound R 3 B(OH)2, palladium catalyst and inorganic base are dissolved in a solvent and heated under reflux to obtain compound VIII, i.e., an imidazo[1,2-c]pyrimidine compound.

4. The preparation method according to claim 3, characterized in that In step S1, the molar ratio of 2,4,5,6-tetrachloropyrimidine, triethylamine and aminoacetaldehyde dimethyl acetal is 1:1:1 to 1:3:3; the reaction is stirred under ice bath conditions for 4 to 6 hours; In step S2, the heating reflux time is 4 to 6 h; In step S3, the molar ratio of intermediate IV to the organic base is 1:1-1:2, and the reflux reaction time is 8-10 h; In step S4, intermediate V and amine compound R 1 The molar ratio of H and organic base is 1:1:2 to 1:1:3; the reaction is stirred at room temperature for 1 to 3 hours; In step S5, intermediate VI and boronic acid compound R 2 The molar ratio of B(OH)2, palladium catalyst and inorganic base is 1:2~2.5:0.05~1:2; reflux reaction is carried out under protective gas conditions for 10~18 hours; In step S6, intermediate VII, boronic acid compound R 3 The molar ratio of B(OH)2, palladium catalyst and inorganic base is 1:1~1.5:0.05~1:2; and the reflux reaction is carried out under protective gas conditions for 10~18 hours.

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