Mer / HDAC dual-targeting inhibitors, and preparation method and use thereof

Abstract

The present application relates to a Mer / HDAC dual-target inhibitor, which has strong inhibitory activity on Mer and HDAC, shows specificity and selectivity on tumor cells and normal cells, has low potential cardiotoxicity and small toxic side effects, and the application of the compound as an antitumor drug is also disclosed.

Description

TECHNICAL FIELD

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

[0002] Mer receptor tyrosine kinase belongs to one of the TAM (Tyro3, Axl, Mer) family members, which was first isolated from chicken retrovirus RLP30. MerTK is widely present in human normal tissues and cells, such as testis, ovary, lung, liver tissue, peripheral blood leukocytes, macrophages, natural killer cells and platelets, etc. Under normal physiological conditions, MerTK can mediate the second stage of platelet aggregation, macrophages and epithelial cells to clear phagocytosis of apoptotic cells, regulate macrophage cytokine synthesis, and participate in various complex physiological processes such as cell differentiation, survival, migration, etc. The abnormal activation or overexpression of MerTK is closely related to the development of various cancers, and the poor prognosis is also related to MerTK. Acute lymphoblastic leukemia (ALL) is a common malignant tumor disease in the blood system, and is 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 of normal mouse and human B cells and T cells. MerTK has been identified as a specific therapeutic target for ALL, and inhibition of Mer can reduce the survival of leukemia cells, making tumor cells more likely to die. At the same time, studies have shown that MerTK is overexpressed in various tumors such as non-small cell lung cancer, breast cancer, renal cancer and pancreatic cancer. Therefore, inhibition of MerTK can effectively treat the occurrence and development of tumors.

[0003] At the same time, the occurrence of cancer is related to 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 method in cancer.

[0004] Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from the ε-N-acetylsine residues of histones, playing crucial roles in gene regulation, transcription, cell proliferation, metastasis, and angiogenesis. In the cell nucleus, histone acetylation and deacetylation are in dynamic equilibrium, regulated by both histone acetyltransferases (HATs) and histone deacetylases. HATs transfer the acetyl group of acetyl-CoA to the terminal lysine residue of histones, relaxing the chromosome structure and facilitating the specific binding of various transcription factors and co-transcription factors to DNA binding sites, thus activating gene transcription. HDACs, on the other hand, deacetylate histones, causing them to bind tightly to negatively charged DNA, hindering DNA polymerase binding and inhibiting gene transcription. Under pathological conditions, overexpression of HDACs disrupts this dynamic equilibrium, reducing the expression of certain anti-tumor and tumor suppressor genes, ultimately leading to tumorigenesis.

[0005] Based on their catalytic mechanism, location within 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; and Class IV: HDAC11. Among these, classes I, II, and IV belong to Zn... 2+ This family of proteins requires Zn to catalyze histone deacetylation. 2+ Class III belongs to the nicotinamide adenine dinucleotide dependent family, and its catalytic histone deacetylation requires nicotinamide adenine dinucleotide.

[0006] HDAC inhibitors inhibit HDAC activity, enhance histone acetylation, promote transcription factor binding to DNA strands, and initiate the expression of specific genes such as tumor suppressor genes, thereby inhibiting tumor cell proliferation and inducing tumor cell apoptosis. The structure of HDAC inhibitors generally consists of a Cap group, a Linker, and a Zn group. 2+ The chelating group (ZBG) is involved. The Cap group is typically a hydrophobic and aromatic group that interacts with the surface recognition region of the HDAC active pocket; the ZBG interacts with the Zn at the bottom of the HDAC active pocket. 2+The chelate-forming complex is the key pharmacophore; the linker consists of a linear or cyclic hydrophobic structure linking the Cap group and ZBG. Currently, five HDAC inhibitors are on the market, among which Voronostat, Romidepsin, Belinostat, and Panobinostat have been approved by the US FDA for the treatment of cutaneous T-cell lymphoma, multiple myeloma, and peripheral T-cell lymphoma. Chidamide has been 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 hematologic malignancies and have poor efficacy in treating solid tumors; (3) Drug resistance is easily developed during use.

[0009] Clinical studies have shown that some novel multi-target inhibitors are significantly more effective than single-target inhibitors. By inhibiting tumors through multiple pathways, resistance to single-target drugs can be overcome, and the toxic side effects of combination therapy can be avoided. Currently, some HDAC-based dual-target inhibitors have been reported, such as PI3K / HDAC inhibitors, c-Met / HDAC inhibitors, and EGFR / HDAC inhibitors. No Mer / HDAC dual-target inhibitors have been reported in the literature. Summary of the Invention

[0010] The purpose of this invention is to provide a Mer / HDAC dual-target inhibitor and its application in the preparation of anti-tumor drugs, so as to discover Mer / HDAC dual-target inhibitors with novel structural types and meet the needs of clinical applications.

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

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

[0013]

[0014] R 1 Selected from alkyl and alkylamide groups;

[0015] R 2 Selected from heterocyclic groups.

[0016] Furthermore, R 1 Selected from substituted octamides and substituted prop-1-enes; R 2 Selected from the following groups: N-(4-fluorophenyl)cyclopropylcarbamate, 1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazole-3-one.

[0017] Furthermore, R 1 Selected from the following groups: Among them, R 3 Selected from: R 2 The functional groups are:

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

[0019] I-1: N-(4-fluorophenyl)-N-(4-((2-((4-(8-(hydroxyamino)-8-oxooctylamine)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide;

[0020] I-2: N 1 -(4-((4-(4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)-N 8 -Hydroxyoctadiamide;

[0021] I-3: N-(4-((2-((4-(8-(((2-aminophenyl)amino)-8-oxooctylamine)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide;

[0022] I-4: N 1 -(2-Aminophenyl)-N 8 -(4-((4-(4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamido)phenoxy)pyrimidin-2-yl)amino)phenyl)octadiamide;

[0023] I-5: (E)-N-(4-fluorophenyl)-N-(4-((2-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide;

[0024] I-6: (E)-N-(4-((2-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide;

[0025] I-7: (E)-N-(4-((2-((4-(3-(((2-aminophenyl)amino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide;

[0026] I-8: (E)-N-(4-((2-((4-(3-(((2-aminophenyl)amino)-3-oxopropyl-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide.

[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 Mer / HDAC dual-target inhibitor,

[0031] (1) Synthetic routes of target compounds I-1 to I-2

[0032]

[0033] (2) Synthetic routes of target compounds I-3 to I-4:

[0034]

[0035] Specifically, the steps include the following:

[0036] 1) Methyl 8-((4-aminophenyl)amino)-8-oxooctanoate reacts with intermediate II via nucleophilic substitution in the presence of an acid catalyst to give intermediate III;

[0037] 2) Intermediate III is hydrolyzed in the presence of an alkaline catalyst to obtain intermediate IV;

[0038] 3) Intermediate IV reacts with O-(tetrahydro-2H-pyran-2-yl)hydroxyamine in the presence of a base catalyst to give intermediate V;

[0039] 4) Intermediate V was deprotected to obtain target compounds I-1 to I-2;

[0040] 5) Intermediate IV and o-phenylenediamine undergo a condensation reaction in the presence of a base catalyst to obtain target compounds I-3 to I-4.

[0041] (3) Synthetic routes of target compounds I-5 to I-6

[0042]

[0043] (4) Synthetic routes of target compounds I-7 to I-8

[0044]

[0045] Specifically, the steps include the following:

[0046] a)(E)-3-(4-aminophenyl)acrylate reacts with intermediate II via a nucleophilic substitution reaction in the presence of an acid catalyst to give intermediate VI;

[0047] b) Intermediate VI is hydrolyzed in the presence of an alkaline catalyst to obtain intermediate VII;

[0048] c) Intermediate VII reacts with O-(tetrahydro-2H-pyran-2-yl)hydroxyamine via a condensation reaction in the presence of a base catalyst to give intermediate VIII;

[0049] d) Intermediate VIII was deprotected to obtain target compounds I-5 to I-6;

[0050] e) Intermediate VII reacts with o-phenylenediamine in the presence of a base catalyst via a condensation reaction to yield target compounds I-7 to I-8.

[0051] In the above preparation method, in steps 1) and a), the acid is a commonly used organic acid, such as p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, etc.; the reaction temperature is 60-100℃.

[0052] In the above preparation method, in steps 2) and b), the alkali is a commonly used inorganic alkali, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, etc.; the reaction temperature is 10-40℃.

[0053] In the above preparation method, in steps 3), 5), c), and e), 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.

[0054] In the above preparation method, in steps 4) and d), 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℃.

[0055] Application of the above-mentioned Mer / HDAC dual-target inhibitor in the preparation of antitumor drugs.

[0056] In the aforementioned application, the Mer / HDAC dual-target inhibitor is specifically a Mer / HDAC1 dual-target inhibitor.

[0057] In the aforementioned applications, the indications for the antitumor drugs include one or more of the following: chronic myeloid leukemia, myeloid monocytic leukemia, non-small cell lung cancer, colorectal cancer, liver cancer, and prostate cancer.

[0058] The compounds described in this invention can be used in combination via oral, injectable, or other routes for the treatment of tumors in mammals (including humans), with oral administration being the most effective. The dosage ranges from 0.0001 mg / kg to 200 mg / kg body weight daily. The optimal dosage depends on the individual, and typically a lower initial dose is followed by a gradual increase.

[0059] The antitumor drug comprises a therapeutically effective dose of the compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0060] The carrier mentioned refers to a carrier commonly used in the pharmaceutical field, such as: diluents, excipients such as water; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch; disintegrants such as calcium carbonate and sodium bicarbonate; in addition, other excipients such as flavoring agents and sweeteners may be added to the composition.

[0061] The antitumor pharmaceutical composition of the present invention can be prepared into conventional solid dosage forms, such as tablets and capsules, for oral administration, or into injectable formulations for injection. Various dosage forms can be prepared using conventional methods in the pharmaceutical field, wherein the content of active ingredient I is 0.1% to 99.5% (by weight) of the compound.

[0062] The beneficial effects of this invention are as follows:

[0063] 1) The compounds described in this invention have novel structures, exhibit excellent Mer and HDAC1 inhibitory activities, and demonstrate strong inhibitory effects on various human tumor cells, exhibiting broad-spectrum and high-efficiency characteristics. They may also show good therapeutic effects for diseases caused by abnormal gene expression, such as endocrine disorders, immune system diseases, genetic diseases, and nervous system diseases.

[0064] 2) The compounds described in this invention effectively inhibit tumor cells while exhibiting weak inhibitory effects on normal cells, demonstrating good selective inhibitory activity and showing promising prospects for clinical application in oncology.

[0065] 3) The compound described in this invention has a weak inhibitory effect on hERG potassium channels, low potential cardiotoxicity, good safety profile, and is worthy of further research.

[0066] In summary, the novel structural compounds of this invention exhibit strong enzyme inhibitory activity, as well as specificity and selectivity against both tumor cells and normal cells. Furthermore, these novel structural compounds demonstrate high safety, minimal toxicity, and are more readily used as anti-tumor drugs. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to embodiments, but the implementation of the invention is not limited thereto.

[0068] Example 1: Synthesis of intermediate methyl 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate

[0069]

[0070] Methyl 8-((4-aminophenyl)amino)-8-oxooctanoate (1.0 g, 3.60 mmol), N-(4-((2-chloropyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (1.84 g, 4.32 mmol), and PTSA (0.93 g, 5.40 mmol) were dissolved in N,N-dimethylformamide (DMF, 15 mL), and the mixture was heated to 90 °C and reacted for 1.5 h. After the reaction was complete, the reaction solution was allowed to cool naturally to room temperature and slowly added dropwise to 75 mL of ice water. The pH was adjusted to approximately 9.0 with 2N Na₂CO₃ aqueous solution, resulting in the precipitation of a solid. The solid was filtered, and the filter cake was dried. The filter cake was purified by column chromatography (DCM:MeOH = 100:1–20:1) to give 447.5 mg of a white solid, with a yield of 18.6%. HRMS: m / z C 36 H 38 FN6O6[M+H] + 669.2759, Found 669.2783.

[0071] Example 2: Synthesis of intermediate 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid

[0072]

[0073] The intermediate methyl 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoate (447.00 mg, 0.67 mmol) was dissolved in CH3OH (20 mL) / THF (22 mL), and 1 M aq LiOH (8 mL) was added. The mixture was stirred at room temperature for 7 h. After the reaction was completed, the reaction solution was concentrated and slowly added dropwise to 50 mL of ice water. The pH was adjusted to approximately 3.0 with 2 M HCl aqueous solution, filtered, and the filter cake was dried to give 374.0 mg of white solid, with a yield of 85.3%. HRMS: m / z C 35 H 36 FN6O6[M+H] + 655.2602, Found 655.2637.

[0074] Example 3: Synthesis of intermediate N-(4-fluorophenyl)-N-(4-((2-((4-(8-oxo-8-(((tetrahydro-2H-pyran-2-yl)oxy)amino)octanoamino)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide

[0075]

[0076] Intermediates 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)-8-oxooctanoic acid (268.00 mg, 0.41 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (53.85 mg, 0.46 mmol), HATU (186.31 mg, 0.49 mmol), and triethylamine (207.44 mg, 2.05 mmol) were dissolved in DMF (10 mL). The mixture was stirred at room temperature for 6 h under N2 protection. After the reaction was complete, the reaction solution was concentrated and slowly added dropwise to 30 mL of ice water. A solid precipitated out; the solution was filtered, and the filter cake was dried. The filter cake was purified by P-TLC (DCM:MeOH = 20:1) to give 134.1 mg of a white solid, yield 43.4%. HRMS: m / z C 40 H 45 FN7O7[M+H] + 754.3286, Found 754.3301.

[0077] Example 4: Synthesis of N-(4-fluorophenyl)-N-(4-((2-((4-(8-(hydroxyamino)-8-oxooctylamine)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (target compound I-1)

[0078]

[0079] The intermediate N-(4-fluorophenyl)-N-(4-((2-((4-(8-oxo-8-(((tetrahydro-2H-pyran-2-yl)oxy)amino)octanoic acid amino)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (67 mg, 0.09 mmol) was dissolved in MeCN (24 mL) / H2O (6 mL), and TFA (1 mL) was added. The mixture was stirred at room temperature for 4.5 h. After the reaction was completed, the reaction solution was concentrated and slowly added dropwise to 20 mL of ice water. The pH was adjusted to approximately 9.0 with 2N Na2CO3 aqueous solution, and a solid precipitated out. The solid was filtered, and the filter cake was dried to give 48.7 mg of white solid, with a yield of 81.8%. HRMS: m / z C 35 H 37 FN7O6[M+H] + 670.2711, Found 670.2777.

[0080] 1 H NMR(500MHz,DMSO-d6)δ10.34(s,1H),10.15(s,1H),10.08(s,1H),9.69(s, 1H),9.55(s,1H),8.31(d,J=4.9Hz,1H),7.72(d,J=7.9Hz,2H),7.64(s,2H) ,7.44(d,J=7.4Hz,2H),7.36(d,J=7.1Hz,2H),7.22-7.11(m,4H),6.38(d,J =4.8Hz,1H),2.23(s,2H),1.94(s,2H),1.51(d,J=13.1Hz,8H),1.26(s,4H).

[0081] Example 5N 1 -(4-((4-(4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)-N 8 Synthesis of 1-hydroxyoctanediamide (target compound I-2)

[0082]

[0083] Following the preparation methods for target compound I-1 in Examples 1-4, target compound I-2 can be prepared with a yield of 73.5%. HRMS: m / z C 36 H 39 N8O6[M+H] +679.2914, Found 679.2985.

[0084] 1 H NMR(500MHz,DMSO-d6)δ10.82(s,1H),10.34(s,1H),9.71(s,1H),9.62(s,1H),8.32(d,J=5.7Hz, 1H),7.69(d,J=8.5Hz,2H),7.60(t,J=7.6Hz,2H),7.52(t,J=7.4Hz,1H),7.44(d,J=7.0Hz,4H),7. 37(d,J=8.6Hz,2H),7.21(d,J=8.5Hz,2H),6.41(d,J=5.7Hz,1H),3.37(s,3H),2.72(s,3H),2.23( t,J=7.5Hz,2H),1.94(t,J=7.4Hz,2H),1.54(t,J=6.5Hz,2H),1.48(t,J=6.6Hz,2H),1.26(s,4H).

[0085] Example 6 Synthesis of N-(4-((2-((4-(8-(((2-aminophenyl)amino)-8-oxooctylamine)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (target compound I-3)

[0086]

[0087] Intermediate 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)8-oxooctanoic acid (106.00 mg, 0.16 mmol), o-phenylenediamine (34.60 mg, 0.32 mmol), HBTU (91.02 mg, 0.24 mmol), and DIPEA (31.02 mg, 0.24 mmol) were dissolved in DMF (6 mL). The mixture was stirred at room temperature for 6 h under N2 protection. After the reaction was complete, the reaction solution was slowly added dropwise to 30 mL of ice water, resulting in the precipitation of a solid. The solid was filtered, and the filter cake was dried. The filter cake was slurried with dichloromethane and methanol, dried, and yielded 12.8 mg of a white solid, with a yield of 10.7%. HRMS: m / z C 41 H 42 FN8O5[M+H] + 745.3184, Found 745.3246.

[0088] 1H NMR(500MHz,DMSO-d6)δ10.14(s,1H),10.08(s,1H),9.68(s,1H),9.45(s,1H),9.09(s,1H),8.32(d, J=4.6Hz,1H),7.72(d,J=7.8Hz,2H),7.64(s,2H),7.45(s,2H),7.36(d,J=7.1Hz,2H),7.19(s,1H),7 .16(d,J=9.8Hz,4H),6.89(t,J=6.3Hz,1H),6.72(d,J=7.4Hz,1H),6.54(t,J=6.7Hz,1H),6.35(d,J= 4.4Hz,1H),4.81(s,2H),2.31(s,2H),2.24(s,2H),1.59(s,4H),1.51(d,J=13.9Hz,4H),1.33(s,4H).

[0089] Example 7N 1 -(2-Aminophenyl)-N 8 Synthesis of -(4-((4-(4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamido)phenoxy)pyrimidin-2-yl)amino)phenyl)octadiamide (target compound I-4)

[0090]

[0091] Following the preparation method for target compound I-3 in Example 6, target compound I-4 was prepared with a yield of 42.4%. HRMS: m / z C 42 H 44 N9O5[M+H] + 754.3387, Found 754.3458.

[0092] 1H NMR (500MHz, DMSO-d6) δ10.81(s,1H),9.69(s,1H),9.46(s,1H),9.09(s,1H),8.32(d,J=5.5Hz,1H),7.69(d,J=8.6Hz,2H ),7.60(t,J=7.6Hz,2H),7.52(d,J=7.3Hz,1H),7.48(d,J=8.1Hz,2H),7.44(d,J=7.5Hz,2H),7.36(d,J=8.2Hz,2H),7.20 (d,J=8.6Hz,2H),7.15(d,J=7.6Hz,1H),6.89(t,J=7.3Hz,1H),6.72(d,J=7.7Hz,1H),6.54(t,J=7.2Hz,1H),6.36(d,J=5 .5Hz,1H),4.82(s,2H),3.36(s,3H),2.72(s,3H),2.31(t,J=7.2Hz,2H),2.24(t,J=7.0Hz,2H),1.59(s,4H),1.33(s,4H).

[0093] Example 8 Synthesis of intermediate (E)-3-(4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)methyl acrylate

[0094]

[0095] Following the method described in Example 1 for preparing intermediate methyl 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-formamido)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)8-oxooctanoate, intermediate (E)-3-(4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-formamido)phenoxy)pyrimidin-2-yl)amino)phenyl)acrylate was prepared, with a yield of 58.4%. HRMS: m / z C 31 H 27 FN5O5[M+H] + 568.1918, Found 568.1974.

[0096] Example 9: Synthesis of intermediate (E)-3-(4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-carbamoylamino)phenoxy)pyrimidin-2-yl)amino)phenyl)acrylic acid

[0097]

[0098] Following the method described in Example 2 for preparing intermediate 8-((4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-formamido)phenoxy)pyrimidin-2-yl)amino)phenyl)amino)8-oxooctanoic acid, intermediate (E)-3-(4-((4-(4-(1-(((4-fluorophenyl)carbamoyl)cyclopropane-1-formamido)phenoxy)pyrimidin-2-yl)amino)phenyl)acrylic acid was prepared with a yield of 74.4%. HRMS: m / z C 30 H 25 FN5O5[M+H] + 554.1761, Found 554.1791.

[0099] Example 10 Synthesis of intermediate (E)-N-(4-((2-((4-(3-(((cyclohexyloxy)amino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide)

[0100]

[0101] Following the method described in Example 3 for preparing the intermediate N-(4-fluorophenyl)-N-(4-((2-((4-(8-oxo-8-(((tetrahydro-2H-pyran-2-yl)oxy)amino)octanoic acid amino)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide, the intermediate (E)-N-(4-((2-((4-(3-(((cyclohexyloxy)amino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide was prepared with a yield of 16.8%. HRMS: m / z C 36 H 36 FN6O5[M+H] + 651.2653, Found 651.2689.

[0102] Example 11 Synthesis of (E)-N-(4-fluorophenyl)-N-(4-((2-((4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (target compound I-5)

[0103]

[0104] Following the preparation method for target compound I-1 in Example 4, target compound I-5 was prepared with a yield of 40.9%. HRMS: m / z C 30 H 26FN6O5[M+H] + 569.1870, Found 569.1944.

[0105] 1 H NMR(500MHz,DMSO-d6)δ10.66(s,1H),10.17(s,1H),10.10(s,1H),9.84(s, 1H),8.38(d,J=5.6Hz,1H),7.72(d,J=8.8Hz,2H),7.67(dd,J=8.7,5.1Hz,2 H),7.59(d,J=7.9Hz,2H),7.40-7.33(m,3H),7.21(d,J=8.8Hz,2H),7.16(t ,J=8.8Hz,2H),6.47(d,J=5.6Hz,1H),6.31(d,J=15.8Hz,1H),1.50(s,4H).

[0106] Example 12 Synthesis of (E)-N-(4-((2-((4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (target compound I-6)

[0107]

[0108] Following the preparation methods for target compound I-5 in Examples 8-11, target compound I-6 can be prepared with a yield of 57.2%. HRMS: m / z C 31 H 28 N7O5[M+H] + 578.2074, Found 578.2092.

[0109] 1 H NMR(500MHz,DMSO-d6)δ10.85(s,1H),10.10(s,1H),10.02(s,1H),9.53(s,1H ),8.62(d,J=5.6Hz,1H),7.73(d,J=8.6Hz,2H),7.64(dd,J=8.5,5.0Hz,2H),7. 52(d,J=7.6Hz,2H),7.40-7.36(m,4H),7.25(d,J=8.0Hz,2H),7.21(t,J=6.5Hz ,2H),6.53(d,J=5.2Hz,1H),6.26(d,J=15.8Hz,1H),3.35(s,3H),2.70(s,3H).

[0110] Example 13 Synthesis of (E)-N-(4-((2-((4-(3-(((2-aminophenyl)amino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (target compound I-7)

[0111]

[0112] Following the preparation method for target compound I-3 in Example 6, target compound I-7 was prepared with a yield of 49.7%. HRMS: m / z C 36 H 31 FN7O4[M+H] + 644.2343, Found 644.2429.

[0113] 1 H NMR (500MHz, DMSO-d6) δ10.19(s,1H),10.09(s,1H),9.84(s,1H),9.32(s,1H),8.39(d,J=5. 4Hz,1H),7.73(d,J=8.4Hz,2H),7.69-7.60(m,4H),7.47(d,J=15.6Hz,1H),7.42(d,J=7.8Hz, 2H),7.34(d,J=7.4Hz,1H),7.22(d,J=8.5Hz,2H),7.14(t,J=8.6Hz,2H),6.92(t,J=7.2Hz,1H ),6.79-6.69(m,2H),6.59(t,J=7.3Hz,1H),6.47(d,J=5.4Hz,1H),4.97(s,2H),1.50(s,4H).

[0114] Example 14 Synthesis of (E)-N-(4-((2-((4-(3-(((2-aminophenyl)amino)-3-oxoprop-1-en-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (target compound I-8)

[0115]

[0116] Following the preparation method for target compound I-3 in Example 6, target compound I-8 was prepared in 37.1% yield. HRMS: m / z C 37 H 33 N8O4[M+H] + 653.2547, Found 653.2586.

[0117] 1 H NMR(500MHz,DMSO-d6)δ10.62(s,1H),10.17(s,1H),9.58(s,1H),8.63(d,J=5.4Hz ,1H),7.75(d,J=7.6Hz,2H),7.69(dd,J=8.2,5.2Hz,2H),7.60-7.54(m,4H),7.50(d ,J=7.2Hz,2H),7.44-7.32(m,4H),7.22(d,J=6.5Hz,2H),7.23(t,J=6.5Hz,2H),6. 50(d,J=5.4Hz,1H),6.28(d,J=15.6Hz,1H),4.93(s,2H),3.85(s,3H),2.53(s,3H).

[0118] Example 15: In vitro inhibitory activity test of the compounds of the present invention against Mer and HDAC1.

[0119] (I) Select a reagent kit to test the inhibitory activity of the compound against Mer. Follow the kit instructions for the experimental procedure. Test the IC50 of the compound's inhibitory activity. 50 The test and experimental procedures are as follows:

[0120] (1) Experimental Procedure

[0121] 1. Prepare 1× buffer: HEPES 50mM, magnesium chloride 10mM, EGTA 1mM, NP-40 0.0001%, DTT 2mM.

[0122] 2. Perform a complex dilution using DMSO. Prepare a final concentration solution of 100× for the test compound.

[0123] 3. Using an automated liquid processor, transfer 100 nL of the compound to a 384-well plate. The final DMSO content in the experiment was 1%.

[0124] 4. Dilute the enzyme stock solution to a concentration of 0.5 nM with 1× assay buffer to prepare a 2× working solution. Manually add 5 μL to the assay plate (final concentration 0.25 nM) using a multichannel pipette, centrifuge at 1000 rpm for 30 seconds, and incubate at 25°C for 30 min.

[0125] 5. Dilute the substrate solution with 1× assay buffer. Manually add 5 μL of the mixture or buffer (30 μM ATP and 2 μM TK-Sub-Biotin) using a multichannel pipette, add to the assay plate, and centrifuge at 1000 rpm for 30 seconds.

[0126] 6. After 60 min at 30℃, add 10 μL of detection solution to each detection well.

[0127] 7. Briefly mix with centrifuge and equilibrate for 60 minutes.

[0128] 8. Record the luminescence.

[0129] (2) Data Analysis

[0130]

[0131] Conversion%-sample: The conversion % value of the sample;

[0132] Conversion%_min: Average conversion rate % value for negative control;

[0133] Conversion%_max: The average conversion rate (%) of the positive control;

[0134] Conversion %-max Dose-response curve using PraphPad Prism 5 and IC 50 Perform fitting;

[0135] The log(inhibitor) vs. response-variable slope program is calculated using the following formula:

[0136] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0137] The test results are shown in Table 2.

[0138] (II) Select a reagent kit to test the inhibitory activity of the compound against HDAC1. The experimental procedure should be performed according to the kit instructions. The IC50 of the compound's inhibitory activity should be measured. 50 The test and experimental procedures are as follows:

[0139] (1) Compound preparation

[0140] Prepare a 10 mM DMSO solution of the compound and store it in the dark for later use.

[0141] (2) HDAC1 reaction process

[0142] a. Prepare a 1× reaction solution.

[0143] b. Compound Preparation: The final concentration of the compound to be tested was initially 10 μM, diluted 3-fold, resulting in 8 concentrations, and a single-well assay was set up. The positive control compound, Voronostat, was tested at a final concentration of 3 μM, diluted 3-fold, resulting in 10 concentrations, and a replicate assay was set up. Solutions were serially diluted to the corresponding 100-fold final concentration in a 384-well Source plate, and then 250 nmol was transferred to the 384-well reaction plate using an Echo 550. 250 nmol of 100% DMSO was transferred to both the Max and Min wells.

[0144] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0145] 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.

[0146] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0147] f. Add 10 μL of 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0148] g. Use Synergy to continuously read fluorescence signals.

[0149] (3) Data Analysis

[0150] The slope is obtained by selecting the linear response segment, and the percentage inhibition rate is calculated using the following formula:

[0151]

[0152] Where: Mean(Max) is the mean slope value of each Max well (containing DMSO and enzyme); Mean(Min) is the mean slope value of each Min well (without enzyme); Sample Signal is the slope value of the compound well.

[0153] Fitting dose-response curves: Using the log value of compound concentration as the X-axis and the corresponding percentage inhibition rate as the Y-axis, the dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function of GraphPad Prism 5 to obtain the IC50 of each compound inhibiting enzyme activity. 50 Values. The experimental results are shown in Table 2.

[0154] Table 2 shows the inhibitory activity of the compounds against Mer and HDAC1 (IC50). 50 / nM)

[0155]

[0156] As shown in Table 2 above, the compounds of this invention exhibit good inhibitory activity against both Mer and HDAC1, with results in the nM range. In particular, compound I-2 shows a significantly higher IC50 value against both Mer and HDAC1. 50 The values ​​were 39.7 nM and 4.3 nM, respectively; the IC50 values ​​of compound I-7 for Mer and HDAC1 were... 50 The values ​​were 32.9 nM and 72.1 nM, respectively. The results indicate that the compounds of this invention all possess strong Mer / HDAC dual-target inhibitory activity.

[0157] Example 16: Assay for tumor cell inhibitory activity of the compounds of the present invention

[0158] Compounds with good inhibitory activity against Mer and HDAC1 were selected, and their activities against human chronic myeloid leukemia cells K562, human liver cancer cells HepG2, human prostate cancer cells PC-3, human myeloid monocytic leukemia cells MV4-11, human colorectal cancer cells HCT116, and non-small cell lung cancer cells A549 were tested. Their IC50 values ​​were... 50 The value was measured using the CCK-8 method, and the experimental procedure is as follows:

[0159] (1) Cell seeding: Take cells in the logarithmic growth phase and seed them into 96-well plates, 10,000 cells per well. Incubate for 12 hours to allow the cells to adhere properly.

[0160] (2) Drug preparation: Weigh the drug and dissolve it in the appropriate solvent to prepare a stock solution with a concentration of 1 mM. For testing, perform serial dilutions of 2-fold using complete culture medium.

[0161] (3) Drug treatment: The culture medium in the 96-well plate was removed, and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with 3 replicates for each concentration; DMSO control wells and blank culture medium control wells were also set up. The drug was allowed to act for 12 hours.

[0162] (4) CCK8 detection: Add 10 μL of CCK8 to each well and continue culturing the cells for 1-2 hours. Use dual wavelengths for detection: 450 nm for detection and 600-650 nm for reference.

[0163] (5) Results statistics: Cell viability: [(As-Ab) / (Ac-Ab)] x 100%

[0164] Where: As: absorbance of experimental wells (containing cell culture medium, CCK8, and test drug); Ab: absorbance of blank wells (containing no cells and test drug, and CCK8); Ac: absorbance of control wells (containing cell culture medium, CCK8, and no test drug).

[0165] (6) Graphpad Prism 9.5.1 software was used to calculate the IC50 values ​​for the three different cell types mentioned above. 50 Values. The experimental results are shown in Table 3 (unit: μM).

[0166] Table 3. In vitro inhibitory activity of compounds against tumor cells (IC50) 50 / μM)

[0167]

[0168] As shown in Table 3 above, the present invention has a good inhibitory effect on all six types of tumor cells, among which the antiproliferative activity IC50 against K562 cells is particularly high. 50 The value ranged from 0.36 to 3.52 μM; the IC50 value for anti-proliferative activity against HepG2 cells was 0.36–3.52 μM. 50 The value ranged from 0.68 to 4.51 μM; the IC50 value for anti-proliferative activity against PC-3 cells was 0.68–4.51 μM. 50 The value ranged from 0.67 to 3.61 μM; the IC50 value for antiproliferative activity against MV4-11 cells was 0.67–3.61 μM. 50 The value ranged from 0.55 to 3.97 μM; the antiproliferative activity against HCT116 cells was IC50. 50 The value ranged from 0.72 to 3.73 μM; the IC50 value for anti-proliferative activity against A549 cells was 0.72–3.73 μM. 50 The values ​​range from 0.78 to 2.75 μM.

[0169] Example 17: In vitro inhibitory activity assay of the compound of the present invention against normal cells.

[0170] The activity of the compound of this invention against MRC-5 human embryonic lung fibroblasts was determined, IC50. 50 The value was measured using the CCK-8 method, and the experimental procedure is as follows:

[0171] (1) Cell seeding: Take cells in the logarithmic growth phase and seed them into 96-well plates, 10,000 cells per well. Incubate for 12 hours to allow the cells to adhere properly.

[0172] (2) Drug preparation: Weigh the drug and dissolve it in the appropriate solvent to prepare a stock solution with a concentration of 1 mM. For testing, perform serial dilutions of 2-fold using complete culture medium.

[0173] (3) Drug treatment: The culture medium in the 96-well plate was removed, and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to each well, with 3 replicates for each concentration; DMSO control wells and blank culture medium control wells were also set up. The drug was allowed to act for 12 hours.

[0174] (4) CCK8 detection: Add 10 μL of CCK8 to each well and continue culturing the cells for 1-2 hours. Use dual wavelengths for detection: 450 nm for detection and 600-650 nm for reference.

[0175] (5) Results statistics: Cell viability: [(As-Ab) / (Ac-Ab)] x 100%

[0176] Where: As: absorbance of experimental wells (containing cell culture medium, CCK8, and test drug); Ab: absorbance of blank wells (containing no cells and test drug, and CCK8); Ac: absorbance of control wells (containing cell culture medium, CCK8, and no test drug).

[0177] (6) The IC50 of different drugs on MRC-5 human embryonic lung fibroblasts was calculated using Graphpad Prism 9.5.1 software. 50 Values. The experimental results are shown in Table 4 (unit: μM).

[0178] Table 4. In vitro inhibitory activity of compounds and control drugs on normal cells.

[0179]

[0180] As shown in Table 4 above, the IC50 of the compounds of this invention against normal MRC-5 cells is [not specified]. 50 The concentration range was 8.5–14.1 μM, indicating a relatively weak inhibitory effect. The inhibitory activity against tumor cells was all in the nM range, indicating that the compounds of this invention have good selectivity and minimal toxic side effects on normal cells.

[0181] Example 18: hERG potassium channel activity assay of the compounds of the present invention

[0182] The potential cardiotoxic side effects of the compounds of this invention were preliminarily investigated in vitro using an hERG potassium channel inhibition assay. The experimental procedure is as follows:

[0183] (1) Cell preparation: CHO-hERG cells were cultured at 175 cm⁻¹ 2 In the culture flask, once the cell density 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 complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and resuspend in 5 mL of culture medium to ensure a cell density of 2-5 × 10⁶ cells / year. 6 / mL.

[0184] (2) Electrophysiological recording process: The single-cell high-impedance sealing and whole-cell pattern formation were all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +40 mV depolarization stimulus, 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 fluid was applied for 5 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2.5 minutes. After all concentrations were administered, the positive control compound 3 μM Cisapride was administered. At least 3 cells (n≥3) were tested for each concentration.

[0185] (3) Compound preparation: The stock solution of the compound was diluted with DMSO. 10 μL of the stock solution was added to 20 μL of DMSO solution and serially diluted 3-fold to 6 DMSO concentrations. 4 μL of each of the 6 DMSO concentrations was added to 396 μL of extracellular fluid and diluted 100-fold to 6 intermediate concentrations. Then, 80 μL of each of the 6 intermediate concentrations was added to 320 μL of extracellular fluid and diluted 5-fold to the final concentration to be tested. The highest test concentration was 40.00 μM, with the following concentrations: 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16 μM. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration of DMSO had no effect on the hERG potassium channel. The entire dilution process was performed using a Bravo instrument.

[0186] (4) Data analysis: Experimental data were analyzed using GraphPad Prism 5.0 software.

[0187] Table 5 Results of hERG potassium channel activity assays for the compounds (IC50). 50 / μM)

[0188]

[0189] hERG assay results showed that the compounds of this invention exhibited inhibitory activity against hERG potassium ion channels greater than 40 μM, indicating that the compounds of this invention have low potential cardiotoxicity.

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of the present invention.

Claims

1. A Mer / HDAC dual-target inhibitor, characterized in that, The general formula is: Specifically selected from: I-1： I-2： I-3： I-4： I-5： I-6： I-7： I-8： 2. A method for preparing the Mer / HDAC dual-target inhibitor as described in claim 1, characterized in that: The synthetic routes for I-1 to I-4 are as follows: Specifically, the steps include the following: 1) Methyl 8-((4-aminophenyl)amino)-8-oxooctanoate reacts with intermediate II via nucleophilic substitution in the presence of an acid catalyst to give intermediate III; 2) Intermediate III is hydrolyzed in the presence of an alkaline catalyst to obtain intermediate IV; 3) Intermediate IV reacts with O-(tetrahydro-2H-pyran-2-yl)hydroxyamine in the presence of a base catalyst to give intermediate V; 4) Intermediate V was deprotected to obtain target compounds I-1 to I-2; 5) Intermediate IV and o-phenylenediamine undergo a condensation reaction in the presence of a base catalyst to obtain target compounds I-3 to I-4.

3. The preparation method according to claim 2, characterized in that, The condensing agent used in steps 3) and 5) is HBTU, HATU, HOBt or TFCH; the catalyst used in step 4) is trifluoroacetic acid, hydrochloric acid or sulfuric acid.

4. A method for preparing the Mer / HDAC dual-target inhibitor as described in claim 1, characterized in that: The synthetic routes for I-5 to I-8 are as follows: Specifically, the steps include the following: a)(E)-3-(4-aminophenyl)acrylate reacts with intermediate II via a nucleophilic substitution reaction in the presence of an acid catalyst to give intermediate VI; b) Intermediate VI is hydrolyzed in the presence of an alkaline catalyst to obtain intermediate VII; c) Intermediate VII reacts with O-(tetrahydro-2H-pyran-2-yl)hydroxyamine via a condensation reaction in the presence of a base catalyst to give intermediate VIII; d) Intermediate VIII was deprotected to obtain target compounds I-5 to I-6; e) Intermediate VII reacts with o-phenylenediamine in the presence of a base catalyst via a condensation reaction to yield target compounds I-7 to I-8.

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

6. The use of the Mer / HDAC dual-target inhibitor as described in claim 1 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The indications for the antitumor drugs include one or more of the following: chronic myeloid leukemia, myeloid monocytic leukemia, non-small cell lung cancer, colorectal cancer, liver cancer, and prostate cancer.

Images