A Mer / HDAC dual-target inhibitor and its application in the preparation of anti-tumor drugs

Abstract

The present invention relates to a Mer / HDAC dual-target inhibitor, which has strong inhibitory activity against both Mer and HDAC, shows specificity and selectivity for tumor cells and normal cells, has low potential cardiac toxicity, and has relatively small toxic and side effects. The present invention also discloses the use of the compound as an anti-tumor drug.

Description

Technical Field

[0001] The present invention relates to a Mer / HDAC dual-target inhibitor, a preparation method thereof, and application of the compound in preventing and treating tumors and other related diseases by inhibiting the activities of Mer and HDAC1. Background Art

[0002] Mer receptor tyrosine kinase is a member of the TAM (Tyro3, ​​Axl, Mer) family and was first isolated from the chicken retrovirus RLP30. MerTK is widely present in normal human tissues and cells, such as the testis, ovary, lung, liver tissue, peripheral blood leukocytes, macrophages, natural killer cells, and platelets. Under normal physiological conditions, MerTK mediates phase II platelet aggregation, the clearance and phagocytosis of apoptotic cells by macrophages and epithelial cells, regulates macrophage factor synthesis, and participates in a variety of complex physiological processes, including cell differentiation, survival, and migration. Abnormal activation or overexpression of MerTK is closely associated with the development of various cancers, and poor prognosis is also associated with MerTK. Acute lymphoblastic leukemia (ALL) is a common malignant tumor of the hematological system and the most common malignant tumor in children. Studies have shown that MerTK is abnormally expressed in B-cell and T-cell ALL, but is not expressed at any stage in normal mouse and human B and T cells. MerTK has been identified as a specific therapeutic target for ALL. Inhibiting Mer can reduce leukemia cell survival and increase tumor cell death. Studies have also shown that MerTK is overexpressed in various tumors, including non-small cell lung cancer, breast cancer, renal cancer, and pancreatic cancer. Therefore, inhibiting MerTK may be an effective treatment for tumor development and progression.

[0003] At the same time, the occurrence of cancer is associated with genomic changes and epigenetic modifications, such as DNA methylation and histone modification. Histone modification mainly includes histone acetylation, phosphorylation and methylation, among which histone acetylation is an important epigenetic modification in cancer.

[0004] Histone deacetylases (HDACs) are enzymes that remove acetyl groups from histone ε-N-acetylysine and play a crucial role in gene regulation, transcription, cell proliferation, metastasis, and angiogenesis. In the cell nucleus, histone acetylation and deacetylation exist in a dynamic equilibrium, regulated by histone acetyltransferases (HATs) and histone deacetylases. HATs transfer the acetyl group of acetyl-CoA to lysine residues at the termini of histones, loosening the chromosome structure and facilitating the specific binding of various transcription factors and co-transcription factors to DNA binding sites, thereby activating gene transcription. HDACs, in turn, deacetylate histones, allowing them to bind tightly to negatively charged DNA, hindering the binding of DNA polymerase and inhibiting gene transcription. In pathological conditions, HDACs are overexpressed, disrupting this dynamic equilibrium and reducing the expression of certain anti-cancer genes and tumor suppressor genes, ultimately leading to tumorigenesis.

[0005] Based on the catalytic mechanism, location in the cell, homology with yeast sequences, and peptide chain folding, HDACs can be divided into two families and four categories: Class I: HDAC1, 2, 3, 8; Class II: HDAC4, 5, 6, 7, 9; Class III: SIRT1-7; Class IV: HDAC11. Among them, Class I, II, and IV belong to Zn 2+ The family of histone deacetylation catalyzed by the kinases is Zn-dependent. 2+ ; Class III belongs to the nicotinamide adenine dinucleotide-dependent family, and its catalytic histone deacetylation requires nicotinamide adenine dinucleotide.

[0006] HDAC inhibitors inhibit the activity of HDAC, enhance the acetylation of histones, promote the binding of transcription factors to DNA chains, and activate the expression of specific genes such as tumor suppressor genes, thereby achieving the effects of inhibiting tumor cell proliferation and promoting tumor cell apoptosis. The structure of HDAC inhibitors is generally composed of Cap group, Linker and Zn 2+ The Cap group is generally a hydrophobic and aromatic group that can interact with the surface recognition area of ​​the HDAC active pocket; ZBG interacts with the Zn at the bottom of the HDAC active pocket. 2+Chelation forms a complex and is the key pharmacophore; the linker is composed of a linear or cyclic hydrophobic structure connecting the Cap group and the ZBG group. Currently, five HDAC inhibitors are marketed, including Vorinostat, Romidepsin, Belinostat, and Panobinostat, which are approved by the US FDA for the treatment of cutaneous T-cell lymphoma, multiple myeloma, and peripheral T-cell lymphoma. Chidamide is approved in China for the treatment of relapsed or refractory peripheral T-cell lymphoma.

[0007]

[0008] Currently, HDAC inhibitors still have the following shortcomings: (1) they have significant clinical side effects, such as nausea, vomiting, and bone marrow suppression; (2) they are only effective in treating hematological tumors, but less effective in treating solid tumors; and (3) they are prone to drug resistance during use.

[0009] Clinical studies have shown that some novel multi-target inhibitors are significantly more effective than single-target inhibitors. By suppressing tumors through multiple pathways, they can overcome single-target drug resistance and avoid the toxic side effects of combination therapy. Currently, several HDAC-based dual-target inhibitors have been reported, including PI3K / HDAC inhibitors, c-Met / HDAC inhibitors, and EGFR / HDAC inhibitors.

[0010] While Mer and HDAC dual-targeting inhibitors have not yet been reported, Mer, c-Met, and EGFR belong to the same receptor tyrosine kinase family, making the study of Mer and HDAC dual-targeting inhibitors theoretically feasible. Furthermore, studies have shown that the Axl inhibitor BGB324, combined with the HDAC inhibitor Panobinostat, can inhibit glioma cells, producing a synergistic anti-tumor effect. Mer and Axl belong to the TAM family and share a high degree of homology. Therefore, the study of Mer / HDAC dual-targeting inhibitors is theoretically feasible and could provide new research ideas for anti-tumor drug development. Summary of the Invention

[0011] The purpose of the present invention is to provide a Mer / HDAC dual-target inhibitor and its use in the preparation of anti-tumor drugs, so as to discover Mer / HDAC dual-target inhibitors with new structural types to meet the needs of clinical application.

[0012] The present invention adopts the following technical solutions:

[0013] A Mer / HDAC dual-target inhibitor with the general formula:

[0014]

[0015] R 1 is selected from aryl, alkyl, and cycloalkyl;

[0016] R 2 is selected from H, aryl, and aromatic heterocyclic group;

[0017] R 3 Selected from N-hydroxycarbamoyl and o-aminophenylcarbamoyl.

[0018] Furthermore, R 1 Selected from phenyl, p-hydroxyphenyl, methyl, ethyl, cyclohexyl, trans-hydroxycyclohexyl;

[0019] R 2 Selected from the following groups: H, phenyl, p-tolyl, 3-methoxycarbonyl-2-pyridyl, 4-N-hydroxycarbamoylphenyl, 3-pyridyl, 3-trifluoromethylphenyl, 4-benzoylphenyl, 5-pyrimidinyl, biphenyl, 2-naphthyl, 3-methoxy-2-pyridyl, 4-methoxy-2-pyridyl, m-cyanophenyl;

[0020] R 3 Selected from N-hydroxycarbamoyl and o-aminophenylcarbamoyl.

[0021] More preferably, R 1 Selected from the following groups:

[0022] R 2 Selected from H,

[0023] R 3 Selected from

[0024] More preferably, the following specific compounds are preferred from the compounds of formula (I):

[0025] I-1: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide;

[0026] I-2: methyl 6-(2-((4-((E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-5-yl)picolinate;

[0027] I-3: N-hydroxy-4-(2-((4-((E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-5-yl)benzamide;

[0028] I-4: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide;

[0029] I-5: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(naphthalen-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide;

[0030] I-6: (E)-3-(4-((5-([1,1′-biphenyl]-4-yl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide;

[0031] I-7: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-[5,5'-dipyrimidinyl]-2-2-yl)amino)phenyl)acrylamide;

[0032] I-8: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide;

[0033] I-9: (E)-3-(4-((5-(3-cyanophenyl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide;

[0034] I-10: (E)-3-(4-((5-(4-benzoylphenyl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide;

[0035] I-11: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(3-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)phenyl)acrylamide;

[0036] I-12: (E)-N-hydroxy-3-(4-((4-((4-hydroxyphenyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide;

[0037] I-13: (E)-N-(2-aminophenyl-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide;

[0038] I-14: (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide;

[0039] I-15: (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide

[0040] I-16: (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)acrylamide

[0041] I-17: (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide.

[0042] The structural formulas of the above compounds are shown in Table 1.

[0043] Table 1 Preferred compound numbers and corresponding structural formulas

[0044]

[0045] A method for preparing the above-mentioned Mer / HDAC dual-target inhibitor,

[0046] (1) Synthesis routes of target compounds I-1 to I-12

[0047]

[0048] The steps are:

[0049] 1) 2,4-dichloro-5-bromopyrimidine and compound II are reacted with a nucleophilic substitution reaction in the presence of an acid-binding agent to obtain intermediate III;

[0050] 2) Intermediate III reacts with (E)-3-(4-aminophenyl) methyl acrylate in the presence of an acid catalyst to obtain intermediate IV through nucleophilic substitution reaction;

[0051] 3) Intermediate IV and compound V are reacted with Suzuki coupling reaction in the presence of a catalyst to obtain intermediate VI;

[0052] 4) intermediate VI is hydrolyzed in the presence of a base catalyst to obtain intermediate VII;

[0053] 5) Intermediate VII is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate VIII;

[0054] 6) Intermediate VIII is deprotected to obtain target compounds I-1 to I-12.

[0055] (2) Synthesis routes of target compounds I-13 and I-15 to I-17

[0056]

[0057] The steps are:

[0058] a) Intermediate VII is reacted with o-phenylenediamine in the presence of a base catalyst to give target compounds I-13 and I-15 to I-17.

[0059] (3) Synthesis route of target compound I-14

[0060]

[0061] The steps are:

[0062] i) (trans)-4-((2-chloropyrimidin-4-yl)amino)cyclohexan-1-ol and (E)-3-(4-aminophenyl)acrylate are reacted by nucleophilic substitution in the presence of an acid catalyst to give (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate;

[0063] ii) hydrolyzing methyl (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate in the presence of a base catalyst to obtain (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid;

[0064] iii) condensing (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide;

[0065] iv) (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide was deprotected to give the target compound I-14.

[0066] In step 1) of the preparation method, the acid-binding agent is a commonly used organic base, such as triethylamine, DIPEA, etc.; and the reaction temperature is 10-40°C.

[0067] In steps 2) and i) of the preparation method, the acid is a commonly used organic acid, such as p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, etc.; and the reaction temperature is 60-100°C.

[0068] In step 3) of the preparation method, the catalyst is a commonly used palladium catalyst, such as Pd(PPh3)2Cl2, Pd(PPh3)4, Pd(dppf)2Cl2, etc.; the reaction temperature is 60-100°C.

[0069] In steps 4) and ii) of the preparation method, the base is a commonly used inorganic base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.; and the reaction temperature is 10-40°C.

[0070] In steps 5), a) and iii) of the preparation method, the condensing agent is HBTU, HATU, HOBt or TFCH; the base catalyst is triethylamine, N,N-diisopropylethylamine or pyridine; the reaction solvent is DMF or DMA; and the reaction temperature is 10-40°C.

[0071] In steps 6) and iv) of the preparation method, the catalyst is trifluoroacetic acid, hydrochloric acid or sulfuric acid; the reaction solvent is acetonitrile / water, tetrahydrofuran / water, or 1,4-dioxane / water; and the reaction temperature is 10-40°C.

[0072] A use of the Mer / HDAC dual-target inhibitor in the preparation of anti-tumor drugs.

[0073] In the application, the Mer / HDAC dual-target inhibitor is specifically a Mer / HDAC1 dual-target inhibitor.

[0074] In the application, the indications of the anti-tumor drug include one or more of chronic myeloid leukemia, liver cancer, prostate cancer, myelomonocytic leukemia, colorectal cancer or non-small cell lung cancer.

[0075] The compounds of the present invention can be administered in combination to mammals (including humans) requiring tumor treatment via oral administration, injection, or other routes, with oral administration being particularly preferred. The dosage is 0.0001 mg / kg to 200 mg / kg body weight per day. The optimal dosage depends on the individual, typically starting with a low dose and then gradually increasing.

[0076] The anti-tumor drug comprises a therapeutically effective dose of a compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0077] The carrier refers to a common carrier in the pharmaceutical field, for example: a diluent, an excipient such as water; a binder such as a cellulose derivative, gelatin, polyvinyl pyrrolidone; a filler such as starch; a disintegrant such as calcium carbonate, sodium bicarbonate; in addition, other auxiliary agents such as flavoring agents and sweeteners may also be added to the composition.

[0078] The anti-tumor pharmaceutical composition of the present invention can be prepared as conventional solid preparations, such as tablets and capsules, for oral administration, or as injectables for injection. These various dosage forms can be prepared using conventional pharmaceutical methods, wherein the active ingredient I is present in an amount of 0.1% to 99.5% (by weight) of the compound.

[0079] The beneficial effects of the present invention are:

[0080] 1) The compounds described herein have novel structures, exhibit strong Mer and HDAC1 inhibitory activity, and exhibit broad-spectrum, high-efficiency inhibitory effects against a variety of human tumor cells. They may also be effective in treating diseases caused by abnormal gene expression, such as endocrine disorders, immune system disorders, genetic diseases, and neurological disorders.

[0081] 2) The compounds of the present invention effectively inhibit tumor cells while having a weak inhibitory effect on normal cells, showing good selective inhibitory activity and having a good prospect for clinical application in tumors.

[0082] 3) The compounds of the present invention have weak inhibitory effects on hERG potassium channels, low potential cardiac toxicity, good safety, and are worthy of further research.

[0083] In summary, the novel structural type compounds of the present invention not only have strong enzyme inhibitory activity, but also show specificity and selectivity for tumor cells and normal cells. At the same time, the novel structural compounds are safer, have less toxic side effects, and are easier to use as anti-tumor drugs. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0085] Example 1 Synthesis of Intermediate (trans)-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexanol

[0086]

[0087] 2,4-Dichloro-5-bromopyrimidine (5.00 g, 17.99 mmol), trans-4-aminocyclohexane-1-ol (2.90 g, 25.19 mmol), and triethylamine (2.73 g, 26.99 mmol) were dissolved in isopropyl alcohol (i-PrOH, 170 mL) and stirred at room temperature for 6 h. After the reaction, the reaction solution was concentrated to remove the solvent, slurried with petroleum ether for 1 h, filtered, and the filter cake was washed with water and dried to obtain 5.66 g of a white solid (99.1% yield). HRMS: m / z C 10 H 14 BrClN3O[M+H] + 305.9931,Found 305.9827.

[0088] Example 2 Synthesis of Intermediate (E)-3-(4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate

[0089]

[0090] (E)-3-(4-aminophenyl) methyl acrylate (1.5 g, 8.47 mmol), intermediate (trans)-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexanol (3.10 g, 10.16 mmol), and PTSA (2.19 g, 12.71 mmol) were dissolved in N,N-dimethylformamide (DMF, 15 mL), heated to 90 ° C, and reacted for 2 h. After the reaction, the reaction solution was naturally cooled to room temperature, slowly dripped into 150 mL of ice water, and adjusted to pH 9.0 with 2N Na2CO3 aqueous solution. Solid precipitated, filtered, and the filter cake was dried. The filter cake was slurried with dichloromethane and methanol and dried to obtain 1.93 g of white solid with a yield of 51.1%. HRMS: m / z C 20 H 24 BrN4O3[M+H] + 447.0954,Found 447.1005.

[0091] Example 3 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylate

[0092]

[0093] The intermediate (E)-methyl 3-(4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate (1.28 g, 2.87 mmol), 4,4,5,5-tetramethyl-2-phenyl-1,3,2-dioxaborolane (2.34 g, 11.48 mmol), NaI (128.91 mg, 0.86 mmol), and 2N Na2CO3 solution (8 mL) were dissolved in DMF (15 mL) under N2 protection. After 10 minutes, Pd(PPh3)2Cl2 (1.01 g, 1.44 mmol) was added, and the temperature was raised to 90°C and the reaction was allowed to react for 1 hour. After the reaction, the reaction solution was naturally cooled to room temperature, filtered through celite, and concentrated. Solid precipitated, which was filtered and the filter cake was dried. The filter cake was slurried with dichloromethane and methanol and dried to obtain 791.70 mg of a white solid with a yield of 59.6%. HRMS: m / z C 26 H 29 N4O3[M+H] + 445.2161,Found 445.2187.

[0094] Example 4 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylic acid

[0095]

[0096] The intermediate (E)-methyl 3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylate (718.00 mg, 1.62 mmol) was dissolved in CH3OH (9 mL) / THF (9 mL), and 1M aq LiOH (15 mL) was added, followed by stirring at room temperature for 7 h. After the reaction, the reaction solution was concentrated and slowly added dropwise to 200 mL of ice water. Solid precipitated, which was filtered and the filter cake dried to obtain 677.02 mg of an orange solid, with a yield of 97.1%. HRMS: m / z C 25 H 27 N4O3[M+H] + 431.2005,Found 431.2107.

[0097] Example 5 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-aminopyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide

[0098]

[0099] The intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylic acid (300.00 mg, 0.70 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (86.98 mg, 0.74 mmol), HATU (315.59 mg, 0.83 mmol), triethylamine (349.11 mg, 3.45 mmol) were dissolved in DMF (8 mL), N2 protected, and stirred at room temperature for 9 h. After the reaction, the reaction solution was concentrated and slowly added dropwise to 60 mL of ice water. Solid precipitated, filtered, and the filter cake was dried. The filter cake was slurried with dichloromethane and methanol and dried to give 153.20 mg of a white solid with a yield of 42.0%. HRMS: m / zC 30 H 36 N5O4[M+H] + 530.2689,Found 530.2698.

[0100] Example 6 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-1)

[0101]

[0102] The intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-aminopyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (59.00 mg, 0.11 mmol) was dissolved in MeCN (60 mL) / H2O (15 mL), TFA (0.3 mL) was added, and the reaction was stirred at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated, and a solid precipitated, which was filtered and the filter cake was dried. The filter cake was purified by P-TLC (DCM:MeOH=10:1) to obtain 33.9 mg of a white solid, with a yield of 69.2%. HRMS: m / z C 25 H 28 N5O3[M+H] + 446.2114,Found 446.2223.

[0103] 1H NMR (500MHz, DMSO-d6) δ10.69(s,1H),9.44(s,1H),8.97(s,1H),7.88(d,J=8.3Hz,2H),7.79(s,1H),7. 46(d,J=7.4Hz,4H),7.42-7.45(m,4H),6.34(d,J=15.7Hz,1H),5.96(d,J=7.3Hz,1H),4.61(d,J=2.55Hz 1H), 3.97 (s, 1H), 3.40 (s, 1H), 1.93 (d, J = 11.3Hz, 2H), 1.87 (d, J = 10.6Hz, 2H), 1.40-1.31 (m, 4H).

[0104] Example 7 Synthesis of methyl 6-(2-((4-((E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-5-yl)picolinate (target compound I-2)

[0105]

[0106] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-2 was prepared with a yield of 51.8%. HRMS: m / z C 26 H 29 N6O5[M+H] + 505.2121,Found 505.2187.

[0107] 1 H NMR(500MHz,DMSO-d6)δ11.34(s,1H),10.75(s,1H),10.63(s,1H),8.88(s,1H),8.35( d,J=8.1Hz,1H),8.09(t,J=7.7Hz,1H),7.97(d,J=7.4Hz,1H),7.79(d,J=7.9Hz,2H),7. 64(s,1H),7.56(d,J=7.8Hz,2H),7.45(d,J=15.7Hz,1H),6.42(d,J=15.7Hz,1H),3.95 (s, 4H), 3.53 (s, 1H), 2.15 (d, J = 9.5Hz, 2H), 1.97 (d, J = 10.0Hz, 2H), 1.48-1.34 (m, 4H).

[0108] Example 8 Synthesis of N-hydroxy-4-(2-((4-((E)-3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-5-yl)benzamide (target compound I-3)

[0109]

[0110] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-3 was prepared with a yield of 85.0%. HRMS: m / z C 26 H 29 N6O5[M+H] + 505.2121,Found 505.2201.

[0111] 1 H NMR(500MHz,DMSO-d6)δ11.34(s,1H),10.76(s,1H),10.43(s,1H),9.12(s,1H),7 .88(d,J=7.9Hz,2H),7.84(s,1H),7.74(d,J=8.1Hz,2H),7.59(d,J=8.1Hz,3H),7. 49(d,J=8.0Hz,2H),7.46(d,J=16.2Hz,1H),6.42(d,J=15.8Hz,1H),3.98(s,1H),3 .36 (s, 1H), 1.87 (d, J = 4.5Hz, 4H), 1.43 (q, J = 11.3Hz, 2H), 1.25 (q, J = 10.2Hz, 2H).

[0112] Example 9 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-4)

[0113]

[0114] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-4 was prepared with a yield of 85.4%. HRMS: m / z C 24 H 27 N6O3[M+H] + 447.2066,Found 447.2151.

[0115] 1H NMR(500MHz,DMSO-d6)δ10.67(s,1H),9.47(s,1H),8.97(s,1H),8.58(s,1H),8. 54(d,J=3.1Hz,1H),7.89(d,J=7.9Hz,2H),7.79(d,J=8.9Hz,2H),7.46(d,J=6.8 Hz,3H),7.41(d,J=15.8Hz,1H),6.38(d,J=7.3Hz,1H),6.33(d,J=15.9Hz,1H),4 .59(s,1H),3.99(s,1H),3.39(s,1H),1.89(t,J=11.2Hz,4H),1.40-1.27(m,4H).

[0116] Example 10 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(naphthalen-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-5)

[0117]

[0118] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-5 was prepared with a yield of 54.7%. HRMS: m / z C 29 H 30 N5O3[M+H] + 496.2270,Found 496.2311.

[0119] 1 H NMR (500MHz, DMSO-d6) δ10.67(s,1H),9.47(s,1H),8.95(s,1H),7.99(d,J=8.5Hz,1H),7. 95(d,J=8.4Hz,2H),7.91(d,J=10.2Hz,4H),7.56-7.51(m,3H),7.47(d,J=8.2Hz,2H),7.41 (d,J=15.7Hz,1H),6.34(d,J=15.8Hz,1H),6.18(d,J=7.6Hz,1H),4.59(d,J=3.4Hz,1H),4. 01(s,1H),3.38(s,1H),1.94(d,J=10.7Hz,2H),1.87(d,J=10.3Hz,2H),1.39-1.31(m,4H).

[0120] Example 11 Synthesis of (E)-3-(4-((5-([1,1'-biphenyl]-4-yl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide (target compound I-6)

[0121]

[0122] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-6 was prepared with a yield of 85.3%. HRMS: m / z C 31 H 32 N5O3[M+H] + 522.2427,Found 522.2468.

[0123] 1 H NMR(500MHz,DMSO-d6)δ10.76(s,1H),10.52(s,1H),9.03(s,1H),7.86(s,1H),7.81(d ,J=8.1Hz,2H),7.75(t,J=8.3Hz,4H),7.59(d,J=8.2Hz,3H),7.51(t,J=7.2Hz,4H),7.4 7(d,J=16.0Hz,1H),7.41(t,J=7.3Hz,1H),6.43(d,J=15.8Hz,1H),4.01(s,1H),3.37(s ,1H),1.89(d,J=10.0Hz,4H),1.48(q,J=11.6,11.1Hz,2H),1.26(q,J=9.5,8.3Hz,2H).

[0124] Example 12 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-[5,5'-dipyrimidine]-2-2-yl)amino)phenyl)acrylamide (target compound I-7)

[0125]

[0126] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-7 was prepared with a yield of 71.1%. HRMS: m / z C 23 H 26 N7O3[M+H] + 448.2019,Found 448.2059.

[0127] 1H NMR (500MHz, DMSO-d6) δ9.51(s,1H),9.15(s,1H),8.79(s,2H),7.88(d,J=8.1Hz,2H),7.84(s,1H),7.46(d,J=7.8Hz,2H),7.37(d,J=15.3Hz ,1H),6.73(d,J=7.2Hz,1H),6.34(d,J=15.7Hz,1H),4.61(s,1H),3.97(s,1H),3.40(s,1H),1.90(t,J=11.9Hz,4H),1.32(q,J=11.7Hz,4H).

[0128] Example 13 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-8)

[0129]

[0130] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-8 was prepared with a yield of 80.8%. HRMS: m / z C 25 H 29 N6O4[M+H] + 477.2172,Found 477.2213.

[0131] 1 H NMR(500MHz,DMSO-d6)δ10.75(s,1H),10.54(s,1H),9.98(s,1H),8.62(s,1H),7.8 4(t,J=7.9Hz,1H),7.79(d,J=8.4Hz,2H),7.57(d,J=7.5Hz,4H),7.45(d,J=15.7Hz ,1H),6.83(d,J=8.2Hz,1H),6.42(d,J=15.8Hz,1H),3.97(s,1H),3.94(s,3H),3.5 1-3.44(m,1H),2.09(d,J=13.2Hz,2H),1.93(d,J=11.1Hz,2H),1.43-1.31(m,4H).

[0132] Example 14 Synthesis of (E)-3-(4-((5-(3-cyanophenyl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide (target compound I-9)

[0133]

[0134] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-9 was prepared with a yield of 80.0%. HRMS: m / z C 26 H 27 N6O3[M+H] + 471.2066,Found 471.2114.

[0135] 1 H NMR (500MHz, DMSO-d6) δ10.76(s,1H),10.57(s,1H),9.04(s,1H),7.91(d,J=7. 5Hz,1H),7.89(s,2H),7.74(d,J=8.3Hz,3H),7.70(d,J=7.7Hz,1H),7.68(s,1H) ,7.59(d,J=8.3Hz,2H),7.46(d,J=15.8Hz,1H),6.43(d,J=15.8Hz,1H),3.98(s, 1H), 3.37 (s, 1H), 1.88 (s, 4H), 1.43 (q, J = 11.1, 10.4Hz, 2H), 1.29-1.22 (m, 2H).

[0136] Example 15 Synthesis of (E)-3-(4-((5-(4-benzoylphenyl)-4-(((trans)-4-hydroxycyclopropyl)amino)pyrimidin-2-yl)amino)phenyl)-N-hydroxyacrylamide (target compound I-10)

[0137]

[0138] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-10 was prepared with a yield of 85.2%. HRMS: m / z C 32 H 32 N5O4[M+H] + 550.2376,Found 550.2423.

[0139] 1H NMR(500MHz,DMSO-d6)δ10.76(s,1H),10.54(s,1H),9.08(s,1H),7.92(s,1 H),7.85(d,J=8.1Hz,2H),7.80(d,J=7.3Hz,2H),7.77-7.69(m,4H),7.64-7. 58(m,6H),7.46(d,J=15.8Hz,1H),6.43(d,J=15.8Hz,1H),4.00(s,1H),3.3 8(s,1H),1.89(d,J=10.4Hz,4H),1.46(q,J=11.7Hz,2H),1.30-1.23(m,2H).

[0140] Example 16 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclopropyl)amino)-5-(3-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-11)

[0141]

[0142] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-11 was prepared with a yield of 82.2%. HRMS: m / z C 26 H 27 F3N5O3[M+H] + 514.1988,Found 514.2036.

[0143] 1 H NMR(500MHz,DMSO-d6)δ10.76(s,1H),10.61(s,1H),8.96(s,1H),7.91(s, 1H),7.81(d,J=5.05Hz,1H),7.75(d,J=8.0Hz,3H),7.73(s,3H),7.60(d,J =8.2Hz,2H),7.46(d,J=15.8Hz,1H),6.43(d,J=15.8Hz,1H),3.98(s,1H), 3.36 (s, 1H), 1.88 (s, 4H), 1.42 (q, J = 11.5Hz, 2H), 1.25 (d, J = 13.0Hz, 2H).

[0144] Example 17 Synthesis of (E)-N-hydroxy-3-(4-((4-((4-hydroxyphenyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-12)

[0145]

[0146] According to the preparation method of target compound I-1 in Examples 1 to 6, target compound I-12 was prepared with a yield of 38.7%. HRMS: m / z C 25 H 22 N5O3[M+H] + 440.1644,Found 440.1714.

[0147] 1 H NMR(500MHz,DMSO-d6)δ10.72(s,1H),9.93(s,1H),9.42(s,1H),9.00(s,1H),8.72(s,1H),7.93(s,1H),7.63(d,J=8.1Hz,2H),7.55-7.48(m,4 H), 7.45 (d, J = 5.4Hz, 1H), 7.41 (d, J = 10.4Hz, 1H), 7.37 (d, J = 6.6Hz, 2H), 7.28 (d, J = 8.2Hz, 2H), 6.79 (d, J = 8.1Hz, 2H), 6.34 (d, J = 15.7Hz, 1H).

[0148] Example 18 Synthesis of (E)-N-(2-aminophenyl-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-13)

[0149]

[0150] The intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylic acid (200.00 mg, 0.46 mmol), o-phenylenediamine (99.14 mg, 0.92 mmol), HBTU (261.68 mg, 0.69 mmol), and DIPEA (89.18 mg, 0.69 mmol) were dissolved in DMF (4 mL) under N2 protection and stirred at room temperature for 5.5 h. After the reaction, the reaction solution was concentrated and slowly added dropwise to 50 mL of ice water. Solid precipitated, which was filtered and the filter cake was dried. The filter cake was purified by P-TLC (DCM:MeOH=20:1) to obtain 26.2 mg of a yellow solid with a yield of 11.0%. HRMS: m / z C 31 H 33 N6O2[M+H] + 521.2587,Found 521.2670.

[0151] 1H NMR(500MHz,DMSO-d6)δ9.55(s,1H),9.38(s,1H),7.91(d,J=8.1Hz,2H),7.62(s,1H),7.54( d,J=8.4Hz,2H),7.50(s,1H),7.47(d,J=7.3Hz,2H),7.43-7.35(m,4H),6.92(t,J=7.1Hz,1H ),6.83-6.73(m,2H),6.59(t,J=7.3Hz,1H),6.10(d,J=5.6Hz,1H),5.25(s,2H),4.62(s,1H) ,3.99(s,1H),3.40(s,1H),1.94(d,J=10.3Hz,2H),1.89(d,J=9.9Hz,2H),1.43-1.32(m,4H).

[0152] Example 19 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate

[0153]

[0154] According to the preparation method of the intermediate (E)-3-(4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate in Example 2, the intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate was prepared in a yield of 50.0%. HRMS: m / z C 20 H 25 N4O3[M+H] + 369.1848,Found369.1892.

[0155] Example 20 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid

[0156]

[0157] According to the preparation method of the intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)acrylic acid in Example 4, the intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid was prepared in a yield of 62.3%. HRMS: m / z C 19 H 23N4O3[M+H] + 355.1692,Found 355.1703.

[0158] Example 21 Synthesis of Intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide

[0159]

[0160] According to the method for preparing the intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-aminopyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide in Example 5, the intermediate (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide was prepared in a yield of 42.6%. HRMS: m / z C 24 H 32 N5O4[M+H] + 454.2376,Found454.2390.

[0161] Example 22 Synthesis of (E)-N-hydroxy-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-14)

[0162]

[0163] The intermediates prepared in Examples 19 to 21 were used and the method for preparing the target compound I-1 in Example 6 was followed to prepare the target compound I-14 in a yield of 27.5%. HRMS: m / z C 19 H 24 N5O3[M+H] + 370.1801,Found370.1880.

[0164] 1H NMR(500MHz,DMSO-d6)δ10.75(s,1H),10.64(s,1H),9.03(s,1H),8.86(s,1H),7 .81(d,J=6.7Hz,1H),7.67(d,J=7.5Hz,2H),7.58(d,J=7.9Hz,2H),7.45(d,J=15 .8Hz,1H),6.43(d,J=15.8Hz,1H),6.19(d,J=6.8Hz,1H),4.65(s,1H),3.78(s,1 H), 3.46 (s, 1H), 1.97 (d, J = 8.9Hz, 2H), 1.90 (d, J = 9.6Hz, 2H), 1.37-1.23 (m, 4H).

[0165] Example 23 Synthesis of (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-15)

[0166]

[0167] According to the preparation method of target compound I-13 in Example 18, target compound I-15 was prepared with a yield of 35.8%. HRMS: m / z C 30 H 32 N7O2[M+H] + 522.2539,Found 522.2583.

[0168] 1 H NMR (500MHz, DMSO-d6) δ10.10(s,1H),9.55(s,1H),7.84(d,J=8.0Hz,2H),7.80(s,1H),7. 62(s,1H),7.52(s,1H),7.47(d,J=7.3Hz,2H),7.41-7.33(m,4H),6.98(t,J=7.2Hz,1H),6 .92-6.79(m,2H),6.60(t,J=7.3Hz,1H),6.12(d,J=5.8Hz,1H),5.23(s,2H),4.60(s,1H), 3.85(s,1H),3.42(s,1H),1.98(d,J=8.3Hz,2H),1.86(d,J=8.6Hz,2H),1.40-1.27(m,4H).

[0169] Example 24 Synthesis of (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)acrylamide (target compound I-16)

[0170]

[0171] According to the preparation method of target compound I-13 in Example 18, target compound I-16 was prepared with a yield of 47.6%. HRMS: m / z C 29 H 31 N8O2[M+H] + 523.2492,Found 523.2505.

[0172] 1 H NMR(500MHz,DMSO-d6)δ9.87(s,1H),9.23(s,1H),7.92(s,1H),7.80(d,J=8.0Hz,2H), 7.48(s,1H),7.45(d,J=7.5Hz,2H),7.40-7.32(m,4H),6.97(t,J=7.0Hz,1H),6.85-6.7 0(m,2H),6.52(t,J=6.8Hz,1H),6.12(d,J=5.8Hz,1H),5.23(s,2H),4.64(s,1H),4.00 (s,1H),3.44(s,1H),2.12(d,J=10.3Hz,2H),1.88(d,J=9.6Hz,2H),1.40-1.34(m,4H).

[0173] Example 25 Synthesis of (E)-N-(2-aminophenyl)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (target compound I-17)

[0174]

[0175] According to the preparation method of target compound I-13 in Example 18, target compound I-17 was prepared with a yield of 58.0%. HRMS: m / z C 31 H 34 N7O3[M+H] + 552.2645,Found 521.2670.

[0176] 1H NMR (500MHz, DMSO-d6) δ9.65 (s, 1H), 9.30 (s, 1H), 7.98 (d, J = 8.0Hz, 2H), 7.82 (s, 1H), 7.5 0(d,J=8.2Hz,2H),7.48(s,1H),7.45-7.30(m,4H),6.98(t,J=7.5Hz,1H),6.83-6.73(m,2 H),6.23(t,J=7.8Hz,1H),6.12(d,J=6.0Hz,1H),5.24(s,2H),4.60(s,1H),3.84(s,3H),3 .62(s,1H),3.48(s,1H),1.98(d,J=7.6Hz,2H),1.92(d,J=9.6Hz,2H),1.45-1.38(m,4H).

[0177] Example 26 In vitro inhibitory activity test of the compounds of the present invention on Mer and HDAC1

[0178] (I) Use a kit to test the inhibitory activity of the compound on Mer. The test procedure is carried out according to the instructions of the kit. IC of the test compound on Mer kinase 50 The test operation is as follows:

[0179] (1) Experimental process

[0180] 1. Prepare 1× buffer: HEPES 50 mM, MgCl2 10 mM, EGTA 1 mM, NP-40 0.0001%, DTT 2 mM.

[0181] 2. Dilution of compounds with DMSO: For each test compound, prepare a 100× final concentration solution.

[0182] 3. Use an automated liquid handler to transfer 100 nL of compound to a 384-well plate. The final DMSO% in the experiment was 1%.

[0183] 4. Dilute the enzyme stock solution to a concentration of 0.5 nM in 1× assay buffer to make a 2× working solution. Manually add 5 μL to the assay plate using a multichannel pipette (final concentration 0.25 nM), spin at 1000 rpm, centrifuge for 30 seconds, and incubate at 25°C for 30 minutes.

[0184] 5. Dilute the substrate solution with 1× assay buffer. Manually add 5 μL of the mixture or buffer (final concentration of ATP: 30 μM and TK-Sub-Biotin: 2 μM) using a multichannel pipette to the assay plate. Spin at 1000 rpm and centrifuge for 30 seconds.

[0185] 6. After 60 min at 30°C, add 10 μL of detection solution to each detection well.

[0186] 7. Mix briefly and centrifuge for 60 minutes.

[0187] 8. Record the luminescence.

[0188] (2) Data Analysis

[0189]

[0190] Conversion%_sample: conversion% value of sample;

[0191] Conversion%-min: average conversion% value of negative control;

[0192] Conversion%_max: average conversion rate % value of positive control;

[0193] Conversion%_maxDose-response curves were obtained using PraphPadPrism5 and IC 50 Perform fitting;

[0194] The log(inhibitor) vs. response-Variable slope program is calculated as follows:

[0195] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0196] The test results are shown in Table 2.

[0197] (II) The inhibitory activity of the test compound on HDAC1 was tested using a kit, and the test procedure was carried out according to the kit instructions. 50 The test operation is as follows:

[0198] (1) Compound preparation

[0199] The compound was prepared into a DMSO solution with a stock concentration of 10 mM and stored in the dark for future use.

[0200] (2) HDAC1 reaction process

[0201] a. Prepare 1× reaction solution.

[0202] b. Compound Preparation: Start with a final concentration of 10 μM for each compound and perform a 3-fold dilution across eight concentrations in a single-well assay. Serially dilute the compound in a 384-well Source plate to a corresponding 100-fold final concentration. Transfer 250 nL of the solution to the 384-well reaction plate using an Echo550. Transfer 250 nL of 100% DMSO to both the Max and Min wells.

[0203] c. Prepare 1.67× enzyme solution using 1× reaction solution.

[0204] d. Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to the Min well. Incubate at room temperature for 15 minutes.

[0205] e. Prepare 2.5× substrate mixed solution using 1× reaction solution.

[0206] f. Add 10 μL of 2.5× substrate mixture solution to each well of the reaction plate to start the reaction.

[0207] g. Use Synergy to continuously read the fluorescence signal.

[0208] (3) Data Analysis

[0209] The linear response segment was selected to obtain the slope and the percentage inhibition rate was calculated using the following formula:

[0210]

[0211] Where: Mean(Max) is the mean of the slope values ​​of the Max wells (containing DMSO and enzyme); Mean(Min) is the mean of the slope values ​​of the Min wells (wells without enzyme); Sample Signal is the slope value of the compound well.

[0212] Fitting the dose-effect curve: The log value of the compound concentration is used as the X-axis and the corresponding percentage inhibition rate is used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 is used to fit the dose-effect curve to obtain the IC value of each compound for enzyme inhibition. 50 The test results are shown in Table 2.

[0213] Table 2 In vitro inhibitory activity of compounds on Mer and HDAC1 (IC 50 / nM)

[0214]

[0215] As can be seen from Table 2 above, the compounds of the present invention have good inhibitory activity against Mer and HDAC1, and the results are all at the nM level, especially the IC values ​​of compounds I-1, I-3, I-4, I-7 and I-14 against Mer. 50 The values ​​were 180nM, 57nM, 76nM, 146nM and 73nM; IC for HDAC1 50 The values ​​were 305nM, 76nM, 102nM, 310nM and 105nM. The results showed that the compounds of the present invention all had strong Mer / HDAC dual-target inhibitory activity.

[0216] Example 27 Determination of the inhibitory activity of the compounds of the present invention on tumor cells

[0217] Compounds with good inhibitory activity against Mer and HDAC1 were selected and tested for their activity against human chronic myeloid leukemia cells K562, human liver cancer cell line HepG2, human prostate cancer cell line PC-3, human myelomonocytic leukemia cells MV4-11, human colorectal cancer cell line HCT116 and non-small cell lung cancer A549. 50 The value was measured by CCK-8 method, and the test operation was as follows:

[0218] (1) Cell seeding: Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 10,000 cells per well. Culture for 12 hours to allow the cells to adhere normally.

[0219] (2) Drug preparation: Weigh the drug and dissolve it in the corresponding solvent to prepare a 1 mM stock solution. Perform a 2-fold serial dilution in complete culture medium for testing.

[0220] (3) Drug treatment: The culture medium in the 96-well plate was aspirated and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with three replicates for each measurement. A DMSO-added control well and a blank culture medium control well were also set up. The drug treatment was allowed to proceed for 12 hours.

[0221] (4) CCK8 detection: 10 μL CCK8 was added to each well, and the cells were cultured for 1-2 hours. The cells were assayed using a dual wavelength, with a detection wavelength of 450 nm and a reference wavelength of 600-650 nm.

[0222] (5) Result statistics: Cell survival rate: [(As-Ab) / (Ac-Ab)] x 100%

[0223] Wherein: As: absorbance of the experimental well (culture medium containing cells, CCK8, and test drug); Ab: absorbance of the blank well (culture medium without cells and test drug, CCK8); Ac: absorbance of the control well (culture medium containing cells, CCK8, and no test drug).

[0224] (6) Graphpad Prism 9.5.1 software was used to calculate the IC for the three different cell types mentioned above. 50 The test results are shown in Table 3 (unit: μM).

[0225] Table 3 Antiproliferative activity of compounds against tumor cells (IC 50 / μM)

[0226]

[0227]

[0228] As can be seen from Table 3 above, the present invention has a good inhibitory effect on the above 6 types of tumor cells and can effectively inhibit the proliferation of tumor cells. Among them, the anti-proliferation activity IC 50 The value is 0.84~7.45μM, and the IC value of antiproliferative activity against HepG2 cells is 50 The value is 1.23~6.34μM, and the IC value of antiproliferative activity against PC-3 cells is 50 The value was 0.77~6.23μM, and the IC value of antiproliferative activity against MV4-11 cells was 50 The value was 0.54~5.24μM, and the IC value of antiproliferative activity against HCT116 cells was 50 The value was 0.79~5.27μM, and the IC value of antiproliferative activity against A549 cells was 50 The values ​​are 0.67~6.02μM.

[0229] Example 28 In vitro inhibitory activity test of the compounds of the present invention on normal cells

[0230] The activity of the compounds of the present invention on MRC-5 human embryonic lung fibroblasts was determined, and IC 50 The value was measured by CCK-8 method, and the test operation was as follows:

[0231] (1) Cell seeding: Cells in the logarithmic growth phase were seeded into 96-well plates at a density of 10,000 cells per well. Culture for 12 hours to allow the cells to adhere normally.

[0232] (2) Drug preparation: Weigh the drug and dissolve it in the corresponding solvent to prepare a 1 mM stock solution. Perform a 2-fold serial dilution in complete culture medium for testing.

[0233] (3) Drug treatment: The culture medium in the 96-well plate was aspirated and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with three replicates for each measurement. A DMSO-added control well and a blank culture medium control well were also set up. The drug treatment was allowed to proceed for 12 hours.

[0234] (4) CCK8 detection: 10 μL CCK8 was added to each well, and the cells were cultured for 1-2 hours. The cells were assayed using a dual wavelength, with a detection wavelength of 450 nm and a reference wavelength of 600-650 nm.

[0235] (5) Result statistics: Cell survival rate: [(As-Ab) / (Ac-Ab)] x 100%

[0236] Wherein: As: absorbance of the experimental well (culture medium containing cells, CCK8, and test drug); Ab: absorbance of the blank well (culture medium without cells and test drug, CCK8); Ac: absorbance of the control well (culture medium containing cells, CCK8, and no test drug).

[0237] (6) Graphpad Prism 9.5.1 software was used to calculate the IC values ​​of different drugs on MRC-5 human embryonic lung fibroblasts. 50 The test results are shown in Table 4 (unit: μM).

[0238] Table 4 Inhibitory activity of compounds against normal cells (IC 50 / μM)

[0239]

[0240] As can be seen from Table 4 above, the IC values ​​of the compounds of the present invention for normal MRC-5 cells are 50 The inhibitory activity against tumor cells was at the nM level, indicating that the compounds of the present invention had good selectivity and had little toxicity to normal cells.

[0241] Example 29 hERG potassium channel effect test of the compounds of the present invention

[0242] The hERG potassium channel inhibition test was used to preliminarily investigate the potential cardiac toxicity and side effects of the compounds of the present invention in vitro. The test procedure was as follows:

[0243] (1) Cell preparation: CHO-hERG cells were cultured at 175 cm 2 When the cell density in the culture flask reaches 60-80%, remove the culture medium, wash once with 7 mL of PBS (Phosphate Buffered Saline), and then add 3 mL of Detachin for digestion. After digestion is complete, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and add 5 mL of culture medium to resuspend to ensure that the cell density is 2-5×10 6 / mL.

[0244] (2) Electrophysiological recording process: The single-cell high-impedance sealing and whole-cell pattern formation processes were all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording mode, the cell was clamped at -80 mV. Before a 5-second +40 mV depolarizing stimulus was given, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular solution was added for 5 minutes. Then the drug administration process began. The compound concentration started from the lowest test concentration and was administered for 2.5 minutes at each test concentration. After all concentrations were administered continuously, the positive control compound 3 μM Cisapride was administered. At least 3 cells were tested for each concentration (n ≥ 3).

[0245] (3) Compound preparation: Dilute the compound stock solution with DMSO, take 10 μL of the compound stock solution and add it to 20 μL of DMSO solution, and dilute it 3-fold to 6 DMSO concentrations. Take 4 μL of the compound at 6 DMSO concentrations and add it to 396 μL of extracellular fluid, dilute it 100-fold to 6 intermediate concentrations, then take 80 μL of the 6 intermediate concentrations and add it to 320 μL of extracellular fluid, and dilute it 5-fold to the final concentration to be tested. The highest test concentration is 40.00 μM, and the concentrations are 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16 μM, totaling 6 concentrations. The DMSO content in the final test concentration does not exceed 0.2%, and this concentration of DMSO has no effect on hERG potassium channels. The compound preparation is completed by the Bravo instrument throughout the dilution process.

[0246] (4) Data analysis: The experimental data were analyzed using GraphPad Prism 5.0 software.

[0247] Table 5 hERG potassium channel effect test results of compounds (IC 50 / μM)

[0248]

[0249] The hERG test results showed that the inhibitory activity of the compounds of the present invention on hERG potassium ion channels was greater than 40 μM, indicating that the compounds of the present invention had low potential cardiac toxicity.

[0250] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, replacement combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of the present invention.

Claims

1. A Mer / HDAC dual-target inhibitor, characterized in that: The general formula is: R 1 Selected from the following groups: R 2 Selected from the following groups: R 3 Selected from N-hydroxycarbamoyl and o-aminophenylcarbamoyl.

2. A method for preparing the Mer / HDAC dual-target inhibitor according to claim 1, characterized in that: Its synthetic route is: The specific steps include: 1) reacting 2,4-dichloro-5-bromopyrimidine with compound II in the presence of an acid-binding agent to obtain intermediate III; 2) Intermediate III reacts with (E)-3-(4-aminophenyl) methyl acrylate in the presence of an acid catalyst to obtain intermediate IV through nucleophilic substitution reaction; 3) intermediate IV and compound V are reacted with a Suzuki coupling reaction in the presence of a catalyst to obtain intermediate VI; 4) Intermediate VI is hydrolyzed in the presence of a base catalyst to obtain intermediate VII; 5) intermediate VII is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate VIII; 6) Intermediate VIII is deprotected to obtain the target compound.

3. The Mer / HDAC dual-target inhibitor according to claim 1, characterized in that R 1 The groups are: R 2 Selected from the following groups: R 3 The groups are:

4. A method for preparing the Mer / HDAC dual-target inhibitor according to claim 3, characterized in that: Its synthetic route is: The specific steps include: a) reacting 2,4-dichloro-5-bromopyrimidine with compound II in the presence of an acid-binding agent to obtain intermediate III; b) reacting the intermediate III with (E)-3-(4-aminophenyl) methyl acrylate in the presence of an acid catalyst to obtain the intermediate IV; c) intermediate IV and compound V are reacted by Suzuki coupling reaction in the presence of a catalyst to obtain intermediate VI; d) hydrolyzing intermediate VI in the presence of a base catalyst to obtain intermediate VII; e) The intermediate VII is reacted with o-phenylenediamine in the presence of a base catalyst to obtain the target compound through condensation reaction.

5. The Mer / HDAC dual-target inhibitor according to claim 1, characterized in that R 1 The groups are: R 2 The groups are: H; R 3 The groups are:

6. A method for preparing the Mer / HDAC dual-target inhibitor according to claim 5, characterized in that: Its synthetic route is: The specific steps include: i) (trans)-4-((2-chloropyrimidin-4-yl)amino)cyclohexan-1-ol and (E)-3-(4-aminophenyl)acrylate are reacted by nucleophilic substitution in the presence of an acid catalyst to give (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate; ii) hydrolyzing methyl (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylate in the presence of a base catalyst to obtain (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid; iii) condensing (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)acrylic acid with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide; iv) (E)-3-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide was deprotected to give the target compound.

7. Use of the Mer / HDAC dual-target inhibitor according to any one of claims 1, 3 or 5 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The anti-tumor drug is indicated for at least one of chronic myeloid leukemia, liver cancer, prostate cancer, myelomonocytic leukemia, colorectal cancer, and non-small cell lung cancer.

Images