A hydroxamic acid Mer / HDAC dual-target inhibitor and its preparation method and application

Abstract

The present invention relates to a hydroxamic acid 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 few 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 hydroxamic acid 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+The key pharmacophore is the chelation complex; 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] 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 new 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. However, there are no reports of dual-target inhibitors targeting Mer and HDAC. Summary of the Invention

[0010] The purpose of the present invention is to provide a hydroxamic acid 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.

[0011] The present invention adopts the following technical solutions:

[0012] A hydroxamic acid Mer / HDAC dual-target inhibitor, the general formula is:

[0013]

[0014] R 1 Selected from alkyl, alkoxyethyl, cycloalkyl, substituted cycloalkyl;

[0015] R 2 Selected from H, aryl, aromatic heterocyclic group, substituted aromatic heterocyclic group.

[0016] Furthermore, R 1 Selected from trans-hydroxycyclohexyl, cyclohexyl, methoxyethyl, trans-methylcyclohexyl; R 2 Selected from H, phenyl, 5-pyrimidinyl, 3-methoxy-2-pyridinyl.

[0017] Furthermore, R 1 Selected from the following groups: R 2 The groups are: H,

[0018] Specifically, the following specific compounds are preferred from the compounds of formula (I):

[0019] I-1:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidin]-2-yl)amino)phenyl)octanediamide;

[0020] I-2:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)octanediamide;

[0021] I-3:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)subanediamide;

[0022] I-4:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide;

[0023] I-5:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-4-methylcyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide;

[0024] I-6:N 1 -(4-((4-(cyclohexylamino)pyrimidin-2-yl)amino)phenyl)-N 8 -Hydroxyoctanamide;

[0025] I-7:N 1 -Hydroxy-N 8 -(4-((4-(((trans)-2-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide;

[0026] I-8:N 1 -Hydroxy-N 8-(4-((4-((2-methoxyethyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide.

[0027] The structural formulas of the above compounds are shown in Table 1.

[0028] Table 1 Preferred compound numbers and corresponding structural formulas

[0029]

[0030] A method for preparing the above-mentioned hydroxamic acid Mer / HDAC dual-target inhibitor,

[0031] (1) Synthesis routes of target compounds I-1 to I-3

[0032]

[0033] The specific steps include:

[0034] 1) 2,4-dichloro-5-bromopyrimidine reacts with compound II in the presence of an acid-binding agent through a nucleophilic substitution reaction to obtain intermediate III;

[0035] 2) Intermediate III reacts with methyl 8-((4-aminophenyl)amino)-8-oxooctanoate in the presence of an acid catalyst via nucleophilic substitution to obtain intermediate IV;

[0036] 3) Intermediate IV and compound V are reacted with Suzuki coupling reaction in the presence of a catalyst to obtain intermediate VI;

[0037] 4) intermediate VI is hydrolyzed in the presence of a base catalyst to obtain intermediate VII;

[0038] 5) Intermediate VII is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate VIII;

[0039] 6) Intermediate VIII is deprotected to obtain target compounds I-1 to I-3.

[0040] (2) Synthesis routes of target compounds I-4 to I-8

[0041]

[0042] The specific steps include:

[0043] a) 2,4-dichloropyrimidine reacts with compound II in the presence of an acid-binding agent through a nucleophilic substitution reaction to obtain intermediate IX;

[0044] b) reacting methyl 8-((4-aminophenyl)amino)-8-oxooctanoate with intermediate IX in the presence of an acid catalyst to obtain intermediate X through nucleophilic substitution reaction;

[0045] c) hydrolyzing the intermediate X in the presence of a base catalyst to obtain the intermediate XI;

[0046] d) intermediate XI is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate XII;

[0047] e) Intermediate XII is deprotected to obtain target compounds I-4 to I-8.

[0048] In the above preparation method, in steps 1) and a), the acid-binding agent is a commonly used organic base, such as triethylamine, DIPEA, etc.; and the reaction temperature is 10-40°C.

[0049] In the above preparation method, in steps 2) and b), the acid is a commonly used organic acid, such as p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, etc.; the reaction temperature is 60-100°C.

[0050] In the above preparation method, in step 3), 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.

[0051] In the above preparation method, in steps 4) and c), 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.

[0052] In the above preparation method, in steps 5) and d), 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.

[0053] In the above preparation method, in steps 6) and e), 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.

[0054] A use of the above-mentioned hydroxamic acid Mer / HDAC dual-target inhibitor in the preparation of anti-tumor drugs.

[0055] In the application, the Mer / HDAC dual-target inhibitor is specifically a Mer / HDAC1 dual-target inhibitor.

[0056] In the application, the indications of the anti-tumor drug include one or more of chronic myeloid leukemia, myelomonocytic leukemia, non-small cell lung cancer, colorectal cancer, liver cancer and prostate cancer.

[0057] 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.

[0058] The anti-tumor drug comprises a therapeutically effective dose of a compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0059] The carrier refers to a common carrier in the pharmaceutical field, for example: diluents, excipients such as water, etc.; binders such as cellulose derivatives, gelatin, polyvinyl pyrrolidone, etc., fillers such as starch, etc.; disintegrants such as calcium carbonate, sodium bicarbonate; in addition, other auxiliary agents such as flavoring agents and sweeteners can also be added to the composition.

[0060] The anti-tumor pharmaceutical composition of the present invention can be prepared into conventional solid preparations, such as tablets and capsules, for oral administration, or into injectable formulations 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.

[0061] The beneficial effects of the present invention are:

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 fewer toxic side effects, and are easier to use as anti-tumor drugs. DETAILED DESCRIPTION

[0066] 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.

[0067] Example 1 Synthesis of the intermediate trans-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexanol

[0068]

[0069] 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.

[0070] Example 2 Synthesis of Intermediate 8-((4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester

[0071]

[0072] 8-((4-aminophenyl)amino)-8-oxooctanoic acid methyl ester (2.0g, 7.19mmol), intermediate trans-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexanol (2.63g, 8.63mmol), and PTSA (1.86g, 10.79mmol) were dissolved in N,N-dimethylformamide (DMF, 20mL), heated to 90°C, and reacted for 3h. After the reaction, the reaction solution was naturally cooled to room temperature, slowly added dropwise to 200mL of ice water, and the pH was adjusted to about 9.0 with 2N Na2CO3 aqueous solution. Solid precipitated, filtered, and the filter cake was dried. The filter cake was purified by column chromatography (DCM:MeOH=80:1~20:1) to obtain 2.12g of white solid with a yield of 53.9%. HRMS:m / zC 25 H 35 BrN5O4[M+H] + 548.1794,Found 548.1812.

[0073] Example 3 Synthesis of Intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester

[0074]

[0075] The intermediate 8-((4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester (1.00 g, 1.82 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)pyrimidine (1.51 g, 7.32 mmol), NaI (82.18 mg, 0.54 mmol), and 2N Na2CO3 solution (10 mL) were dissolved in DMF (15 mL) under N2 protection. After 10 minutes, Pd(PPh3)2Cl2 (0.64 g, 0.92 mmol) was added, and the temperature was raised to 90°C and the reaction was allowed to react for 2 hours. After the reaction, the reaction solution was naturally cooled to room temperature, filtered through celite, concentrated, and solid precipitated. The solid was filtered and the filter cake was dried. The filter cake was purified by column chromatography (DCM:MeOH=80:1-20:1) to obtain 513.0 mg of a yellow solid with a yield of 50.4%. HRMS: m / zC 29 H 38 N7O4[M+H] + 548.2907,Found 548.2929.

[0076] Example 4 Synthesis of Intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)amino)-8-oxooctanoic acid

[0077]

[0078] The intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester (513.0 mg, 0.93 mmol) was dissolved in CH3OH (16 mL) / THF (16 mL), and 1M aq LiOH (8 mL) was added and stirred at room temperature for 3 h. After the reaction, the reaction solution was concentrated and slowly added dropwise to 40 mL of ice water. The pH was adjusted to about 3.0 with 2M HCl aqueous solution. Solid precipitated and was filtered. The filter cake was dried to obtain 447.2 mg of a white solid with a yield of 90.1%. HRMS: m / z C 28 H 36 N7O4[M+H] + 534.2751,Found 534.2782.

[0079] Example 5 Intermediate N 1-(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)-N 8 Synthesis of -((tetrahydro-2H-pyran-2-yl)oxy) suberamide

[0080]

[0081] The intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidin]-2-yl)amino)phenyl)amino)-8-oxooctanoic acid (180.00 mg, 0.34 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (43.47 mg, 0.37 mmol), HATU (154.01 mg, 0.41 mmol), and triethylamine (170.78 mg, 1.69 mmol) were dissolved in DMF (6 mL) under N2 protection and stirred at room temperature for 7 h. After the reaction, the reaction solution was concentrated and slowly added dropwise to 30 mL of ice water. Solid precipitated and was filtered and the filter cake was dried. The filter cake was purified by P-TLC (DCM:MeOH=10:1) to obtain 71.4 mg of a white solid with a yield of 33.5%. HRMS: m / z C 33 H 45 N8O5[M+H] + 633.3435,Found 633.3473.

[0082] Example 6N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)suberamide (target compound I-1)

[0083]

[0084] The intermediate N 1 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)-N 8 -((Tetrahydro-2H-pyran-2-yl)oxy)octanediamide (71.4 mg, 0.11 mmol) was dissolved in MeCN (24 mL) / H₂O (6 mL), and TFA (1 mL) was added. The mixture was stirred at room temperature for 6 h. After completion of the reaction, the reaction solution was concentrated and slowly dripped into 30 mL of ice water. The pH was adjusted to approximately 9.0 with 2N aqueous Na₂CO₃. A solid precipitated, which was filtered and the filter cake dried to yield 58.5 mg of a light yellow solid (94.5% yield). HRMS: m / z C 28 H 37N8O4[M+H] + 549.2860,Found 549.2928.

[0085] 1 H NMR (500MHz, DMSO-d6) δ10.35(s,1H),9.76(s,1H),9.19(s,1H),9.13(s,1H),8.77(s,2H),7.79(s,1H),7.72(d,J=8.5Hz,2H),7.48(d,J=8.5Hz, 2H),6.65(d,J=6.4Hz,1H),4.64(s,1H),3.94(s,1H),3.39(s,1H),2.26 (s,2H),1.98-1.84(m,6H),1.57(s,2H),1.49(s,2H),1.35-1.24(m,8H).

[0086] Example 7N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-phenylpyrimidin-2-yl)amino)phenyl)octanediamide (target compound I-2)

[0087]

[0088] 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 18.6%. HRMS: m / z C 30 H 39 N6O4[M+H] + 547.2955,Found 547.3027.

[0089] 1H NMR(500MHz,DMSO-d6)δ10.35(s,1H),9.75(d,J=3.6Hz,1H),9.07(s,1H),8.66(s,1H),7.75-7.7 0(m,3H),7.49-7.43(m,4H),7.38(d,J=7.3Hz,2H),7.35(t,J=7.2Hz,1H),5.83(d,J=7.5Hz,1H),4 .62(s,1H),3.94(s,1H),3.40(s,1H),2.26(t,J=7.2Hz,2H),1.97-1.90(m,4H),1.86(d,J=10.0Hz ,2H),1.57(t,J=4.1Hz,2H),1.50(t,J=6.4Hz,2H),1.34(d,J=12.8Hz,2H),1.30(d,J=9.0Hz,6H).

[0090] Example 8N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-5-(6-methoxypyridin-2-yl)pyrimidin-2-yl)amino)phenyl)suberamide (target compound I-3)

[0091]

[0092] 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 55.7%. HRMS: m / z C 30 H 40 N7O5[M+H] + 578.3013,Found 578.3103.

[0093] 1 H NMR(500MHz,DMSO-d6)δ10.35(s,1H),10.20(s,1H),9.89(s,2H),8.58(s,1H),7.82 (s,1H),7.65(d,J=7.6Hz,2H),7.59(d,J=7.0Hz,2H),7.55(d,J=6.7Hz,1H),6.80(d ,J=7.4Hz,1H),4.70(s,1H),3.93(s,4H),3.62(s,1H),2.29(s,2H),2.09(d,J=7.7H z, 2H), 1.94 (s, 4H), 1.58 (s, 2H), 1.50 (s, 2H), 1.39 (d, J = 11.4Hz, 2H), 1.29 (s, 6H).

[0094] Example 9 Synthesis of Intermediate (trans)-4-((2-chloropyrimidin-4-yl)amino)cyclohexane-1-ol)

[0095]

[0096] The intermediate (trans)-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexanol was prepared according to the method for preparing the intermediate (trans)-4-((5-bromo-2-chloropyrimidin-4-yl)amino)cyclohexan-1-ol in Example 1 with a yield of 70.8%. HRMS: m / z C 10 H 15 ClN3O[M+H] + 228.0825,Found 228.0831.

[0097] Example 10 Synthesis of Intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester

[0098]

[0099] According to the preparation method of the intermediate 8-((4-((5-bromo-4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester in Example 2, the intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid methyl ester was prepared in a yield of 30.9%. HRMS: m / z C 25 H 36 N5O4[M+H] + 470.2689,Found 470.2700.

[0100] Example 11 Synthesis of Intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid

[0101]

[0102] According to the preparation method of the intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidin]-2-yl)amino)phenyl)amino)-8-oxooctanoic acid in Example 4, the intermediate 8-((4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid was prepared in a yield of 95.9%. HRMS: m / z C 24 H 34N5O4[M+H] + 456.2533,Found 456.2551.

[0103] Example 12 Intermediate N 1 Synthesis of -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N8-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide

[0104]

[0105] According to Example 5, intermediate N 1 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)-[5,5'-dipyrimidinyl]-2-yl)amino)phenyl)-N 8 -((tetrahydro-2H-pyran-2-yl)oxy) suberic acid amide preparation method, can prepare intermediate N 1 -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)-N 8 -((tetrahydro-2H-pyran-2-yl)oxy)octanediamide, yield 39.1%. HRMS: m / z C 29 H 43 N6O5[M+H] + 555.3217,Found555.3265.

[0106] Example 13N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-4-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide (target compound I-4)

[0107]

[0108] According to the preparation method of target compound I-1 in Example 6, target compound I-4 was prepared with a yield of 29.6%. HRMS: m / z C 24 H 35 N6O4[M+H] + 471.2642,Found 471.2716.

[0109] 1H NMR(500MHz,DMSO-d6)δ10.34(s,1H),9.68(s,1H),8.83(s,1H),8.66(s,1H ),7.74(s,1H),7.68(d,J=7.3Hz,2H),7.43(d,J=7.8Hz,2H),7.01(s,1H),5 .86(s,1H),4.61(s,1H),3.74(s,1H),3.43(s,1H),2.25(s,2H),1.95(s,2H ), 1.88 (d, J = 9.9Hz, 2H), 1.57 (s, 2H), 1.49 (s, 2H), 1.25 (d, J = 11.5Hz, 10H).

[0110] Example 14N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-4-methylcyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide (target compound I-5)

[0111]

[0112] According to the preparation method of target compound I-4 in Examples 9 to 13, target compound I-5 was prepared with a yield of 48.8%. HRMS: m / z C 25 H 37 N6O3[M+H] + 469.2849,Found 469.2921.

[0113] 1 H NMR(500MHz,DMSO-d6)δ10.35(s,1H),10.12(s,1H),9.89(s,1H),8.67(s,1H),8.51(s,1H),7.7 4(d,J=4.7Hz,1H),7.58(d,J=8.6Hz,2H),7.53(d,J=8.2Hz,2H),6.09(d,J=5.8Hz,1H),3.73(s,1 H),2.29(t,J=7.3Hz,2H),2.04-1.89(m,4H),1.58(d,J=12.2Hz,2H),1.49(t,J=6.4Hz,2H),2.29 (t,J=6.8Hz,2H),1.38(s,1H),1.32-1.23(m,6H),1.00(q,J=11.7Hz,2H),0.91(d,J=6.4Hz,3H).

[0114] Example 15N 1-(4-((4-(cyclohexylamino)pyrimidin-2-yl)amino)phenyl)-N 8 Synthesis of -hydroxyoctanamide (target compound I-6)

[0115]

[0116] According to the preparation method of target compound I-4 in Examples 9 to 13, target compound I-6 was prepared with a yield of 67.9%. HRMS: m / z C 24 H 35 N6O3[M+H] + 455.2692,Found 455.2754.

[0117] 1 H NMR (500MHz, DMSO-d6) δ10.35(s,1H),10.07(s,1H),9.87(s,1H),8.67(s,1H),8.45(s,1H),7.74(s,1H),7.55(d,J=9.6H z, 4H), 6.10 (s, 1H), 3.79 (s, 1H), 2.28 (s, 2H), 1.99-1.90 (m, 4H), 1.76 (d, J = 9.3Hz, 2H), 1.66-1.45 (m, 6H), 1.28 (s, 8H).

[0118] Example 16N 1 -Hydroxy-N 8 Synthesis of -(4-((4-(((trans)-2-hydroxycyclohexyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide (target compound I-7)

[0119]

[0120] According to the preparation method of target compound I-4 in Examples 9 to 13, target compound I-7 was prepared with a yield of 25.7%. HRMS: m / z C 24 H 35 N6O4[M+H] + 471.2642,Found 471.2704.

[0121] 1H NMR (500MHz, DMSO-d6) δ10.35(s,1H),9.67(s,1H),8.80(s,1H),8.71(s,1H),7.74(s,1H),7.68(d,J=7.6Hz,2H),7.43(d,J=7.5Hz,2H),6.99(s ,1H),5.92(s,1H),4.63(s,1H),3.69(s,1H),3.50(s,1H),2.25(s,2H), 2.09-1.90(m,4H),1.67(s,2H),1.57(s,2H),1.49(s,2H),1.27(s,8H).

[0122] Example 17N 1 -Hydroxy-N 8 Synthesis of -(4-((4-((2-methoxyethyl)amino)pyrimidin-2-yl)amino)phenyl)suberamide (target compound I-8)

[0123]

[0124] According to the preparation method of target compound I-4 in Examples 9 to 13, target compound I-8 was prepared with a yield of 48.7%. HRMS: m / z C 21 H 31 N6O4[M+H] + 431.2329,Found 431.2415.

[0125] 1 H NMR (400MHz, DMSO-d6) δ10.32(s,1H),9.64(s,1H),8.81(s,1H),8.67(s,1H),7.77(s,1H),7.65(d,J=7.0Hz,2H),7.42(d,J=7 .2Hz,2H),7.15(s,1H),5.93(s,1H),3.48(s,3H),3.28(s,2H),2.25(s,2H),1.94(s,2H),1.53(d,J=28.6Hz,4H),1.28(s,4H).

[0126] Example 18 In vitro inhibitory activity test of the compounds of the present invention on Mer and HDAC1

[0127] (I) Use a kit to test the inhibitory activity of the compound on Mer, and perform the test according to the instructions of the kit. 50 Test, the test operation is as follows:

[0128] (1) Experimental process

[0129] 1. Prepare 1× buffer: HEPES 50 mM, MgCl2 10 mM, EGTA 1 mM, NP-40 0.0001%, DTT 2 mM.

[0130] 2. Dilute the compound with DMSO. For each test compound, prepare a 100× final concentration solution.

[0131] 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%.

[0132] 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.

[0133] 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, add to the assay plate, spin at 1000 rpm, and centrifuge for 30 seconds.

[0134] 6. After 60 min at 30°C, add 10 μL of detection solution to each detection well.

[0135] 7. Mix briefly and centrifuge for 60 minutes.

[0136] 8. Record the luminescence conditions.

[0137] (2) Data Analysis

[0138]

[0139] Conversion%-sample: conversion% value of the sample;

[0140] Conversion%-min: average conversion% value of negative control;

[0141] Conversion%-max: average conversion rate% value of positive control;

[0142] Conversion%-maxDose-response curves were obtained using PraphPadPrism5 and IC 50 Perform fitting;

[0143] The log(inhibitor) vs. response-Variable slope program is calculated as follows:

[0144] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0145] The test results are shown in Table 2.

[0146] (II) Use the kit to test the inhibitory activity of the compound on HDAC1, and the test operation is carried out according to the kit instructions. 50 Test, the test operation is as follows:

[0147] (1) Compound preparation

[0148] The compound was prepared into a DMSO solution with a stock concentration of 10 mM and stored in the dark for future use.

[0149] (2) HDAC1 reaction process

[0150] a. Prepare 1× reaction solution.

[0151] b. Compound Preparation: Test compounds starting at a final concentration of 10 μM, diluted 3-fold to 8 concentrations, in single-well assays. Test the positive control compound, Vorinostat, at a final concentration of 3 μM, diluted 3-fold to 10 concentrations, in replicate assays. Serial dilutions were performed in a 384-well Source plate to the corresponding 100-fold final concentrations. Then, using an Echo550, 250 nL was transferred to a 384-well reaction plate for assay. 250 nL of 100% DMSO was transferred to both the Max and Min wells.

[0152] c. Prepare 1.67× enzyme solution using 1× reaction solution.

[0153] 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.

[0154] e. Prepare 2.5× substrate mixed solution using 1× reaction solution.

[0155] f. Add 10 μL of 2.5× substrate mixture solution to each well of the reaction plate to start the reaction.

[0156] g. Use Synergy to continuously read the fluorescence signal.

[0157] (3) Data Analysis

[0158] The linear response segment was selected to obtain the slope and the percentage inhibition rate was calculated using the following formula:

[0159]

[0160] 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.

[0161] 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.

[0162] Table 2 Inhibitory activity of compounds against Mer and HDAC1 (IC 50 / nM)

[0163]

[0164] 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 nanomolar level. Among them, I-4 has the best activity and good inhibitory activity against both targets. The IC 50 The IC value for HDAC1 is 164.2 nM. 50 The value was 54.8 nM.

[0165] Example 19 Tumor cell inhibitory activity test of the compounds of the present invention

[0166] The activity of the test compounds against human chronic myeloid leukemia cells K562, human liver cancer cell line HepG2, human colorectal cancer cell line HCT116, non-small cell lung cancer cell line A549, human prostate cancer cell line PC-3 and human myelomonocytic leukemia cell line MV4-11 was measured. The IC 50 The value was measured by CCK-8 method, and the test operation was as follows:

[0167] (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.

[0168] (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.

[0169] (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.

[0170] (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.

[0171] (5) Result statistics: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0172] 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).

[0173] (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).

[0174] Table 3 In vitro inhibitory activity of compounds on tumor cells (IC 50 / μM)

[0175]

[0176] As can be seen from Table 3 above, the present invention has a good inhibitory effect on the above 6 types of tumor cells, among which the anti-proliferation activity IC 50 The value is 3.4~10.5μM; the anti-proliferative activity IC of HepG2 cells 50 The value is 2.6~12.4μM; the IC value of antiproliferative activity against HCT116 cells 50 The value is 2.2~11.3μM; the IC value of antiproliferative activity against MV4-11 cells 50 The value is 2.8~13.2μM; the antiproliferative activity IC of PC-3 cells 50 The value is 2.7~12.5μM; the antiproliferative activity IC of A549 cells 50 The values ​​are 3.3~14.6μM.

[0177] Example 20 In vitro inhibitory activity test of the compounds of the present invention on normal cells

[0178] The activity of the compounds of the present invention on MRC-5 human embryonic lung fibroblasts was determined, and IC50 The value was measured by CCK-8 method, and the test operation was as follows:

[0179] (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.

[0180] (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.

[0181] (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.

[0182] (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.

[0183] (5) Result statistics: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0184] 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).

[0185] (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).

[0186] Table 4 In vitro inhibitory activity of compounds on normal cells

[0187]

[0188] 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 value is 20.4-34.1 μM, and the inhibitory effect is weak, indicating that the compound of the present invention has good selectivity and less toxic and side effects on normal cells.

[0189] Example 21 hERG potassium channel effect test of the compounds of the present invention

[0190] 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:

[0191] (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.

[0192] (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).

[0193] (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.

[0194] (4) Data analysis: The experimental data were analyzed using GraphPad Prism 5.0 software.

[0195] Table 5 hERG potassium channel effect test results of compounds (IC 50 / μM)

[0196]

[0197] The hERG test results showed that the inhibitory activity of the compounds of the present invention on hERG potassium ion channels was greater than 35 μM, indicating that the compounds of the present invention had low potential cardiac toxicity.

[0198] 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 as equivalent replacement methods and are included in the scope of the present invention.

Claims

1. A hydroxamic acid Mer / HDAC dual-target inhibitor, characterized in that: The general formula is: R 1 Selected from trans-hydroxycyclohexyl, cyclohexyl, methoxyethyl, trans-methylcyclohexyl; R 2 Selected from H, phenyl, 5-pyrimidinyl, 3-methoxy-2-pyridinyl.

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

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

4. A method for preparing the hydroxamic acid Mer / HDAC dual-target inhibitor according to claim 2, characterized in that: Its synthetic route is: The specific steps include: 1) 2,4-dichloro-5-bromopyrimidine reacts with compound II in the presence of an acid-binding agent through a nucleophilic substitution reaction to obtain intermediate III; 2) intermediate III reacts with methyl 8-((4-aminophenyl)amino)-8-oxooctanoate 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 target compounds I-1 to I-3.

5. The preparation method according to claim 4, characterized in that The condensing agent used in step 5) is HBTU, HATU, HOBt or TFCH; the catalyst in step 6) is trifluoroacetic acid, hydrochloric acid or sulfuric acid.

6. A method for preparing the hydroxamic acid Mer / HDAC dual-target inhibitor according to claim 3, characterized in that: Its synthetic route is: The specific steps include: a) 2,4-dichloropyrimidine reacts with compound II in the presence of an acid-binding agent through a nucleophilic substitution reaction to obtain intermediate IX; b) reacting methyl 8-((4-aminophenyl)amino)-8-oxooctanoate with intermediate IX in the presence of an acid catalyst to obtain intermediate X through nucleophilic substitution reaction; c) hydrolyzing the intermediate X in the presence of a base catalyst to obtain the intermediate XI; d) intermediate XI is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate XII; e) Intermediate XII is deprotected to obtain target compounds I-4 to I-8.

7. The preparation method according to claim 6, characterized in that The condensing agent used in step d) is HBTU, HATU, HOBt or TFCH; the catalyst in step e) is trifluoroacetic acid, hydrochloric acid or sulfuric acid.

8. Use of the hydroxamic acid Mer / HDAC dual-target inhibitor according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

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

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