CN113444069B - 2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC double-target inhibitor - Google Patents
2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC double-target inhibitor Download PDFInfo
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Abstract
The invention relates to a 2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC (high Density lipoprotein lipase) double-target inhibitor, a preparation method thereof and application thereof in preparing antitumor drugsApplication, belonging to the technical field of pharmaceutical chemistry. The compounds have the following general formula:
wherein R is1Preferably CH3、OCH3And H; r2F and H are preferred. The compound has strong inhibitory activity on LSD1 and HDAC, and has strong inhibitory activity on IC of HDAC150The value is less than 5nM, which is obviously superior to the positive medicine SAHA. The compound shows better in-vitro anti-tumor activity on human acute myelogenous leukemia THP-1 cell strain, is better than a positive medicine SAHA, provides a foundation for the research and development of LSD1/HDAC double-target-point inhibitor medicines, and can be used as a candidate or lead compound for further development to develop anti-tumor treatment medicines.
Description
Technical Field
The invention specifically relates to a 2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC (high Density lipoprotein lipase) double-target inhibitor, a preparation method thereof and application thereof in preparation of antitumor drugs, and belongs to the technical field of pharmaceutical chemistry.
Background
Epigenetic dysregulation is closely related to the occurrence, development and poor prognosis of tumors, and the research and development of anticancer drugs targeting epigenetic regulatory proteins has attracted great interest and made great progress.
Histone lysine-specific demethylase 1(LSD1), also known as KDM1A, CPRF, BHC110 and AOF2, was discovered in 2004 by the team of yang of harvard university. LSD1 is a flavin adenine dinucleotide dependent amino oxidase whose primary function is to specifically remove the monomethylation and dimethylation modifications on histone H3K 4. LSD1 inhibits the transcription of gene by removing the methylation mark of H3K4me1/2, thereby regulating a plurality of key cellular processes such as cell cycle, proliferation, differentiation and embryo pluripotency. LSD1 can also remove single or double methylation modification on H3K9 by interacting with estrogen receptor or androgen receptor, so that it can be converted from transcription inhibitor to transcription activator to activate transcription of downstream genes. In addition, LSD1 can remove methylation modification of non-histone such as p53, DNMT1, STAT3, E2F1, MYPT1 and HIF-1, and further regulate stability and activity of downstream genes. The expression level of LSD1 in various tumors such as acute myeloid leukemia, lung cancer, gastric cancer, breast cancer, colon cancer, prostate cancer, synovial sarcoma, neuroblastoma and the like is remarkably increased, and the LSD1 is closely related to the poor prognosis of various malignant tumors such as lung cancer, acute myeloid leukemia, colon cancer, breast cancer and the like. The small molecular compound is adopted to inhibit the activity of LSD1, so that the proliferation, the metastasis and the invasion of tumor cells can be effectively inhibited. The development of antitumor drugs aiming at LSD1 is one of the attractive strategies in the field of medicinal chemistry at present, seven LSD1 small-molecule inhibitors such as IMG-7289, GSK2879552, TAK-418, ORY-2001, INCB059872, CC-90011 and SP-2577 enter a phase I/II clinical test and are used for treating acute myelogenous leukemia and non-small cell lung cancer. In addition, the study shows that the LSD1 is closely related to other diseases, such as metabolic disorder, mental disease, virus infection and the like.
Histone Deacetylases (HDACs) primarily remove acetylation modifications on histone N-terminal tail lysine residues, leading to gene transcriptional silencing. HDACs not only deacetylate histones, but also act on various non-histone proteins, including RUNX3, p53, E2F, STAT, NF-. kappa.F, HIF-1. alpha., ER. alpha., AR and HSP90, among others. In various malignant tumors such as leukemia, lymph cancer, cervical cancer, colorectal cancer, breast cancer and the like, the expression level and activity of HDAC family members are obviously up-regulated, and the adverse prognosis of various malignant tumors such as leukemia, lymph cancer, cervical cancer, colorectal cancer and the like is positively correlated with the expression level of HDAC. At present, 5 HDAC small molecule inhibitors such as Vorinostat, Romidepsin, Belinostat, Panobinostat and Chidamide are approved by FDA and CFDA to be marketed and used for treating various tumors such as cutaneous T cell lymphoma and myeloma. In addition, there are several HDAC inhibitors in clinical trials at various stages. In addition, HDAC is closely related to the occurrence of various diseases such as neurodegenerative diseases, autoimmune diseases, acute graft-versus-host disease, etc., and is considered as a potential therapeutic target for these diseases.
While LSD1 inhibitors and HDAC inhibitors have achieved good results in the treatment of a variety of hematological tumors, LSD1 and HDAC inhibitors have had poor therapeutic results on solid tumors. As two important histone epigenetic regulatory proteins, LSD1 and HDAC are closely related to the occurrence and development of cancer, and there is a close cross-talk relationship between the two. LSD1 and HDAC1/2 are jointly present in NuRD, CoREST and Sin3A co-suppression composite proteins and are involved in regulating the transcription of various genes. HDAC1 can deacetylate K374 in the substrate binding region of LSD1, promote effective binding of LSD1 and histone H3, enhance demethylase activity of LSD1, and inhibit expression of downstream target genes. In breast cancer, the expression levels of LSD1 and HDAC5 are both obviously up-regulated, and HDAC5 can improve the stability and demethylation activity of LSD1 by up-regulating the expression of the deubiquitinase USP28 of LSD1, thereby promoting the occurrence and development of breast cancer. On the contrary, inhibiting the activity of HDAC5 or knocking out HDAC5 promotes the ubiquitination degradation of LSD1, inhibiting the proliferation and invasion of breast cancer cells. The combination of LSD1 and HDAC inhibitor showed good synergistic antitumor effect on various malignancies including rhabdomyosarcoma, glioblastoma, breast cancer, ewing's sarcoma and acute myeloid leukemia cells. Therefore, the discovery of a novel and high-activity LSD1/HDAC double-target inhibitor is expected to discover a novel and high-efficiency antitumor lead compound and improve the treatment effect on solid tumors by simultaneously inhibiting LSD1, HDAC and the signal path transduction of mutual crosstalk, and has very important significance for researching the physiological functions of LSD1 and HDAC and developing novel and high-efficiency antitumor drugs. In order to find a novel LSD1/HDAC double-target inhibitor, a class of 2-aryl-4- (1H-pyrazol-3-yl) pyridine compounds is explored and synthesized, and the LSD1, HDAC double-target inhibition activity and in-vitro anti-tumor activity of the compounds are verified as the starting points of the application, so that no report of the synthesis, LSD1/HDAC inhibition activity and anti-tumor activity of the compounds is found at present.
Disclosure of Invention
From the above, an object of the present invention is to provide a class of 2-aryl-4- (1H-pyrazol-3-yl) pyridines, which provides the possibility of new drug screening.
It is another object of the present invention to provide a process for the preparation of such 2-aryl-4- (1H-pyrazol-3-yl) pyridines.
The invention further aims to provide application of the 2-aryl-4- (1H-pyrazol-3-yl) pyridine compound in preparation of antitumor drugs by taking LSD1/HDAC as a target.
In order to achieve the purpose, the 2-aryl-4- (1H-pyrazol-3-yl) pyridine compound has the structural general formula:
in the general formula I, R1Is H, CH3、OCH3Any one of the above; r2F, H is used.
Preferably: in the general formula I, R1、R2The substituents or atoms represented are shown in the following table:
to achieve the second object, the reaction scheme for synthesizing the compound of the present invention is as follows:
synthetic routes to compounds of general formula I:
a method for preparing compound 3: the compound 2-bromo-4-iodopyridine (compound 1) and pyrazole-3-boric acid (compound 2) are heated and stirred to react in toluene in the presence of an alkaline compound and a palladium catalyst, after the reaction is finished, water and ethyl acetate are added into a reaction system for extraction, ethyl acetate layers are combined, water and a saturated NaCl aqueous solution are respectively used for washing, anhydrous sodium sulfate is used for drying, filtering is carried out, the filtrate is decompressed and concentrated, and the concentrate is separated by silica gel column chromatography to obtain the compound 3. Wherein the alkaline compound is selected from one of potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and sodium hydride, and the palladium catalyst is selected from one of tetrakis (triphenylphosphine) palladium, palladium acetate, bis (dibenzylideneacetone) palladium and palladium dichloride;
a method for preparing compound 4: in DMF, in the presence of an alkaline compound, reacting the compound 3 with methyl 4-bromomethylbenzoate at room temperature under stirring, after the reaction is finished, adding water and ethyl acetate into a reaction system for extraction, combining ethyl acetate layers, washing with water and a saturated NaCl aqueous solution respectively, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating the concentrate by silica gel column chromatography to obtain a compound 4. Wherein the alkaline compound is selected from one of potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide;
a method for preparing compound 5: and (2) heating and stirring the compound 4 and various substituted phenylboronic acids or substituted phenylboronic acid pinacol esters in toluene in the presence of an alkaline compound and a palladium catalyst for reaction, adding water and ethyl acetate into a reaction system after the reaction is finished, extracting, combining ethyl acetate layers, washing with water and a saturated NaCl aqueous solution respectively, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the concentrate by silica gel column chromatography to obtain a compound 5. Wherein the alkaline compound is selected from one of potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and sodium hydride, and the palladium catalyst is selected from one of tetrakis (triphenylphosphine) palladium, palladium acetate, bis (dibenzylideneacetone) palladium and palladium dichloride;
a process for the preparation of compound I: in dichloromethane solution, Compound 5 and NH2OK/NH2And (3) reacting with an OH methanol solution, after the reaction is finished, concentrating the reaction system in vacuum, dissolving the concentrate with water, adjusting the pH value to 5-6 with dilute hydrochloric acid, performing suction filtration, washing, collecting solids, and recrystallizing with methanol to obtain the compound I.
The invention has the advantages that: the 2-aryl-4- (1H-pyrazol-3-yl) pyridine compound synthesized by the invention has stronger inhibitory activity on LSD1 and HDAC, and has good selectivity on LSD 1. IC of all Compounds on HDAC150The value is less than 5nM, which is obviously superior to the positive medicine SAHA. The LSD1/HDAC double-target preparation shows better in-vitro anti-tumor activity on human leukemia THP-1 cell strains, is superior to a positive medicament SAHA, and particularly the inhibiting activity of the compound I-2 on THP-1 is 13 times of that of SAHA. The compounds of the invention represent a new class of compounds having a novel structureThe highly active LSD1/HDAC double-target inhibitor provides a foundation for the research and development of LSD1/HDAC double-target inhibitor drugs, and provides an effective tool for the biological function research of LSD1 and HDAC. Can be used as a further candidate or lead compound for developing anti-tumor, anti-virus, anti-AIDS and other disease treatment medicines, has simple synthesis method, and is beneficial to popularization and application.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to examples.
EXAMPLE 12 Synthesis of bromo-4- (1H-pyrazol-3-yl) pyridine (3)
A50 mL two-necked flask was charged with the compound 2-bromo-4-iodopyridine (1212.1mg,6.0mmol), toluene (20mL), ethanol (25mL), H2O(7.5mL),K2CO3(1.68g,11.1mmol),Pd(PPh3)4(694.0mg,0.6mmol) and pyrazole-3-boronic acid (1451.9mg,7.2mmol) are stirred and reacted for 4 hours at 92 ℃ under the protection of nitrogen, after the reaction is finished, the reaction system is cooled to room temperature, then water and ethyl acetate are added into the reaction system for extraction, the ethyl acetate layers are combined, water and saturated aqueous NaCl solution are respectively used for washing, anhydrous sodium sulfate is used for drying, filtering is carried out, the filtrate is decompressed and concentrated, and the concentrate is separated and purified by silica gel column chromatography (petroleum ether: acetone ═ 5:1) to obtain a compound 3a (850.4mg) which is a white solid, the yield is 65.5 percent, and Mp:140 and 141 ℃.1H NMR(400MHz,DMSO-d6)δ13.36(s,1H),8.39(d,1H,J＝5.2Hz),8.03(d,1H,J＝1.6Hz),7.88(s,1H),7.86(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.03(d,1H,J＝2.0Hz).HRMS(ESI)calcd for C8H6BrN3Na[M+Na]+:245.9637,Found:245.9639.
Example Synthesis of methyl- ((3- (2-bromo-pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzoate (4)
In a 50ml two-neck flaskAdding compound 3(1244.4mg,6.0mmol), methyl 4-bromomethylbenzoate (1451.9mg,7.2mmol) and cesium carbonate (2932.2mg,9.0mmol), adding DMF (8mL), stirring at room temperature under the protection of nitrogen for reaction for 5h, then adding water and ethyl acetate into the reaction system for extraction, combining ethyl acetate layers, washing with water and saturated aqueous NaCl solution respectively, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the concentrate by a silica gel column chromatography (petroleum ether: acetone ═ 2:1) to obtain compound 4(1755.4mg), a white solid, the yield: 78.6%, and Mp: 127-.1H NMR(400MHz,CDCl3)δ8.36(d,1H,J＝5.2Hz),8.03(d,2H,J＝8.0Hz),7.90(d,1H,J＝1.2Hz),7.64(dd,1H,J1＝1.6Hz,J1＝5.2Hz),7.47(d,1H,J＝2.4Hz),7.29(d,2H,J＝8.4Hz),6.69(d,1H,J＝2.4Hz),5.43(s,2H),3.92(s,3H).13C NMR(101MHz,CDCl3)δ166.57,150.41,147.94,143.43,142.86,140.92,131.57,130.23,130.14,127.45,124.04,119.13,104.82,56.06,52.27.HRMS(ESI)calcd for C17H14BrN3NaO2[M+Na]+:394.0162,Found:394.0167.
Example Synthesis of methyl- ((3- (2- (4-methylphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzoate (5a)
In a 50mL two-necked round-bottomed flask, Compound 4(521.1mg,1.4mmol), toluene (5mL), ethanol (5mL), H2O(1.3mL),K2CO3(359.3mg,2.6mmol),Pd(PPh3)4(162.0mg,0.14mmol) and 4-methylbenzeneboronic acid (231.1mg,1.7mmol) were reacted under nitrogen at 92 ℃ for 4 hours with stirring, after the reaction was completed, the reaction system was cooled to room temperature, extracted with water and ethyl acetate, the ethyl acetate layers were combined, washed with water and a saturated aqueous NaCl solution, respectively, dried over anhydrous sodium sulfate, dried, suction-filtered, the filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography (petroleum ether: ethyl acetate: 2:1) to obtain compound 5a (483.7mg) with a yield of 90.1% and a white solid Mp:92-93 ℃.1H NMR(400MHz,CDCl3)δ8.68(dd,1H,J1＝0.8Hz,J2＝4.8Hz),8.13(dd,1H,J1＝0.8Hz,J2＝1.6Hz),8.03(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.4Hz),7.59(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.46(d,1H,J＝2.4Hz),7.30-7.27(m,4H),6.75(d,1H,J＝2.4Hz),5.44(s,2H),3.90(s,3H),2.41(s,3H).13C NMR(101MHz,CDCl3)δ166.63,158.00,150.03,149.67,141.34,141.29,138.93,136.70,131.33,130.20,130.03,129.44,127.42,126.89,118.32,116.57,104.57,55.94,52.25,21.32.HRMS(ESI)calcd for C24H22N3O2[M+H]+:384.1707,Found:384.1705.
Example Synthesis of methyl 44- ((3- (2- (4-methoxyphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzoate (5b)
The procedure of example 3 was followed, substituting 4-methoxyphenylboronic acid (258.33mg,1.7mmol) for 4-methylphenylboronic acid, to give compound 5b (201.9mg) as a white solid in 36.1% yield, Mp: 102-.1H NMR(400MHz,CDCl3)δ8.65(dd,1H,J1＝0.8Hz,J2＝5.2Hz),8.10(dd,1H,J1＝0.8Hz,J2＝1.6Hz),8.03-8.01(m,4H),7.56(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.47(d,1H,J＝2.4Hz),7.29(d,2H,J＝8.0Hz),7.00(d,2H,J＝8.8Hz),6.74(d,1H,J＝2.4Hz),5.44(s,2H),3.91(s,3H),3.86(s,3H).13C NMR(101MHz,CDCl3)δ166.62,160.45,157.65,149.97,149.70,141.31,141.29,132.14,131.33,130.19,130.03,128.30,127.41,117.97,116.12,114.05,104.55,55.94,55.37,52.24.HRMS(ESI)calcd for C24H22N3O3[M+H]+:400.1656,Found:400.1658.
Example Synthesis of methyl 54- ((3- (2- (2-fluoro-4-methylphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzoate (5c)
The procedure of example 3 was followed, substituting 2-fluoro-4-methylphenylboronic acid (261.7mg,1.7mmol) for 4-methylphenylboronic acid, to give compound 5c (363.1mg) as a white solid in yield of 64.6% and Mp:129-130 ℃.1H NMR(400MHz,CDCl3)δ8.71(dd,1H,J1＝1.2Hz,J2＝5.2Hz),8.15(t,1H,J＝2.0Hz),8.02(d,2H,J＝8.4Hz),7.87(t,1H,J＝8.4Hz),7.66(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.46(d,1H,J＝2.4Hz),7.29(d,2H,J＝8.4Hz),7.08(dt,1H,J1＝1.6Hz,J2＝7.6Hz),6.99(dt,1H,J1＝1.2Hz,J2＝12.4Hz),6.75(d,1H,J＝2.4Hz),5.44(s,2H),3.91(s,3H),2.40(s,3H).13C NMR(101MHz,CDCl3)δ166.64,160.34(d,JC-F＝250.3Hz),154.16(d,JC-F＝2.3Hz),150.07,149.52,141.28,141.14(d,JC-F＝8.4Hz),141.05,131.30,130.68(d,JC-F＝3.4Hz),130.19,130.00,127.43,125.29(d,JC-F＝2.9Hz),124.69(d,JC-F＝11.7Hz),120.66(d,JC-F＝8.7Hz),118.52,116.66(d,JC-F＝22.3Hz),104.65,55.95,52.25,21.20.HRMS(ESI)calcd for C24H21FN3O2[M+H]+:402.1612,Found:402.1614.
Example 6 Synthesis of N-hydroxy-4- ((3- (2- (4-methylphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzamide (I-1)
Slowly dripping anhydrous methanol solution (7.0g,45mL methanol) of potassium hydroxide into anhydrous methanol solution (5.84g,20mL methanol) of hydroxylamine hydrochloride under ice bath and stirring, stirring for 5min, and filtering to obtain NH2OK-NH2OH methanol solution, sealed and reserved, in a 50mL two-neck round-bottom flask, compound 5a (383.4mg,1.0mmol) is added, dissolved by anhydrous dichloromethane (5mL), and added with NH prepared in the step slowly and dropwise under the protection of nitrogen and stirring in ice bath2OK-NH2OH solution (16.5mL), stirring at room temperature for 1.0h, concentrating the reaction system under reduced pressure, and concentratingThe resulting extract was dissolved in distilled water (20mL), the pH was adjusted to 5-6 with dilute HCl, and a solid precipitated, which was collected by filtration and recrystallized from methanol to give Compound I-1(324.8mg) as a white solid in a yield of 84.7% and Mp: 185-.1H NMR(400MHz,DMSO-d6)δ11.21(s,1H),9.05(s,1H),8.63(d,1H,J＝5.2Hz),8.23(t,1H,J＝1.2Hz),8.06-8.02(m,3H),7.73(d,2H,J＝8.4Hz),7.69(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.34(d,2H,J＝8.4Hz),7.32(d,2H,J＝8.8Hz),7.13(d,1H,J＝2.0Hz),5.50(s,2H),2.37(s,3H).13C NMR(101MHz,DMSO-d6)δ164.35,157.11,150.47,148.96,141.76,140.84,139.10,136.38,133.30,132.67,129.80,127.89,127.68,126.97,118.45,115.84,105.08,55.22,21.31.HRMS(ESI)calcd for C23H21N4O2[M+H]+:385.1659,Found:385.1659.
Example 7 Synthesis of N-hydroxy-4- ((3- (2- (4-methoxyphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzamide (I-2)
The procedure of example 6 was followed, substituting compound 5b (399.45mg,1.0mmol) for 5a, to give compound I-2(338.4mg) as a yellow solid in 84.5% yield, Mp:187-188 ℃.1H NMR(400MHz,DMSO-d6)δ11.21(s,1H),9.06(s,1H),8.60(d,1H,J＝4.8Hz),8.20(t,1H,J＝1.2Hz),8.11(d,2H,J＝8.8Hz),8.03(d,1H,J＝2.4Hz),7.73(d,2H,J＝8.4Hz),7.65(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.34(d,2H,J＝8.0Hz),7.12(d,1H,J＝2.4Hz),7.06(d,2H,J＝8.8Hz),5.50(s,2H),3.83(s,3H).13C NMR(101MHz,DMSO-d6)δ164.31,160.64,156.89,150.38,149.02,141.70,140.84,133.27,132.66,131.61,128.45,127.90,127.68,117.99,115.41,114.54,105.05,55.68,55.22.HRMS(ESI)calcd for C23H21N4O3[M+H]+:401.1608,Found:401.1608.
Example 8 Synthesis of N-hydroxy-4- ((3- (2- (2-fluoro-4-methylphenyl) pyridin-4-yl) -1H-pyrazol-1-yl) methyl) benzamide (I-3)
The procedure of example 6 was followed, replacing 5a with compound 5c (401.4mg,1.0mmol) to give compound I-3(318.7mg) as a white solid in 79.2% yield and Mp: 114-.1H NMR(400MHz,DMSO-d6)δ11.23(s,1H),9.08(s,1H),8.69(d,1H,J＝5.2Hz),8.13(t,1H,J＝2.0Hz),8.04(d,1H,J＝2.4Hz),7.86(t,1H,J＝8.0Hz),7.75-7.73(m,3H),7.34(d,2H,J＝8.4Hz),7.20-7.15(m,2H),7.02(d,1H,J＝2.4Hz),5.51(s,2H),2.38(s,3H).13C NMR(101MHz,DMSO-d6)δ164.33,160.16(d,JC-F＝248.9Hz),153.66(d,JC-F＝2.3Hz),150.70,148.67,141.76(d,JC-F＝8.6Hz),141.37,140.80,133.41,132.67,131.03(d,JC-F＝3.4Hz),127.89,127.68,125.93(d,JC-F＝2.8Hz),124.58(d,JC-F＝11.5Hz),119.80(d,JC-F＝8.7Hz),118.91,117.03(d,JC-F＝22.7Hz),104.93,55.22,21.08.HRMS(ESI)calcd for C23H20FN4O2[M+H]+:403.1565,Found:403.1569.
Evaluation of LSD1, HDAC and THP-1 inhibitory Activity of the synthesized Compounds of the present invention:
evaluation of inhibitory Activity of LSD1, HDAC and THP-1:
1. LSD1 inhibitory activity evaluation experimental method
The sample was obtained by purifying the above compound synthesized in the examples; sample stock solution: 3-5mg of the sample was weighed into a 1.5mL EP tube and then prepared into a 20mM solution with DMSO, stored at-20 ℃ in the dark and diluted with DMSO according to the desired concentration during the experiment. After a sample to be detected and LSD1 protein are incubated at room temperature, adding LSD1 reaction substrate H3K4me2 for incubation reaction, finally adding fluorescent dye Amplex and horseradish peroxidase HRP for incubation at room temperature, exciting light on a microplate reader at 530nm, and emitting light at 590nm to detect a fluorescence value.
Test results the IC was calculated using SPSS software50The value is obtained.
2. HDAC inhibitory activity evaluation experiment method
3-5mg of the sample was weighed into a 1.5mL EP tube and then prepared into a 20mM solution with DMSO, stored at-20 ℃ in the dark and diluted with DMSO according to the desired concentration during the experiment. Prepare 1 Xbuffer (modified Tris buffer). HDAC enzymes were diluted with 1 × buffer to 1.67 × final concentration. Trypsin and acetyl peptide substrates were mixed to make a substrate solution, diluted with 1X buffer to 2.5X final concentration. 250nL of test compound was transferred to 384-well plates using Echo 550. Then, 15. mu.L of the enzyme solution was added to a 384-well plate, and preincubated with the test compound at room temperature for 15 min. As a negative control, 15. mu.L of 1 Xbuffer was used. Then 10. mu.L of substrate solution was added to 384 wells to start the reaction. Fluorescence intensity was measured at excitation 355nm and emission 460nm with EnVision.
3. Evaluation of recombinant protein level MAO-A/B inhibitory Activity
The sample was the compound synthesized in the examples and purified; sample stock solution: 3-5mg of the sample was weighed into a 1.5mL EP tube and then prepared into a 20mM solution with DMSO, stored at-20 ℃ in the dark and diluted with DMSO according to the desired concentration during the experiment. The inhibitory activity against MAO-A and MAO-B was determined using A commercial MAO-Glo assay kit from PromegA, according to the manufacturer's protocol.
4. In vitro anti-tumor Activity assay
The samples were the compounds synthesized in the examples; weighing 3-5mg of sample, placing the sample in a 1.5mL EP tube, preparing the solution with the concentration of 10mmol/L by DMSO, storing the solution at the temperature of-20 ℃ in a dark place, and diluting the solution by using a culture medium according to the required concentration during the experiment.
Taking cells in logarithmic phase, digesting and counting, adjusting cell density by using a culture medium, inoculating the cells into a 96-well plate at the rate of 4000-. After the drug acts for 72 hours, 40 mu L of CellTiter-Glo Reagent is added into each hole, the well is evenly mixed for 2 minutes by oscillation, after the incubation is continued for 10 minutes at room temperature, the fluorescence intensity is detected by an enzyme-labeling instrument, and the inhibition rate is calculated, wherein the calculation formula is as follows:
the inhibition ratio (%) × (maximum fluorescence intensity-fluorescence intensity of administered group)/(maximum fluorescence intensity-minimum fluorescence intensity) × 100%.
5. Results of the experiment
TABLE 1 evaluation results of LSD1, HDAC1 and THP-1 inhibitory Activity
aN.D. not determined.
bThe inhibition at 30nM concentration was 83%.
From the experimental results shown in table 1 above, it can be seen that the compounds of the present invention have better inhibitory activity against both LSD1 and HDAC 1. IC of all Compounds on HDAC150Less than 5nM, significantly better than the SAHA, positive drug, especially IC of compound I-2 on HDAC1501.88nM, 7 times higher than the SAHA of the positive drug. The evaluation of in vitro antitumor activity shows that the inhibitory activity of all compounds on THP-1 acute myeloid leukemia cells is superior to that of SAHA, in particular the IC of compound I-2 on THP-1500.44. mu.M, 13 times that of SAHA.
The compound I-2 also has strong inhibiting activity on other subtypes of HDAC, and has stronger inhibiting activity on HDAC2, HDAC5, HDAC6 and HDAC8 than SAHA. Compound I-2 had no inhibitory activity against the homologous proteins of LSD1, MAO-A and MAO-B, and very good selectivity for LSD1 (Table 2).
TABLE 2 evaluation of HDAC subtype and MAO-A/B inhibitory Activity of Compound I-2
aND: not detected
Experimental results show that the compound represents a LSD1/HDAC double-target inhibitor with a brand-new structure, has good in-vitro anti-tumor activity, can be used as a candidate or lead compound for further development, is applied to preparation of anti-cancer drugs, provides a foundation for research and development of LSD1/HDAC double-target inhibitor drugs, and provides an effective tool for biological function research of LSD1 and HDAC.
Claims (5)
1. A2-aryl-4- (1H-pyrazol-3-yl) pyridine compound is characterized by having a structure shown in a general formula (I):
in the general formula I, R1Is OCH3、CH3Any one of H and H; r2Is any one of F and H.
2. A class of 2-aryl-4- (1H-pyrazol-3-yl) pyridines according to claim 1, selected from one of the following:
3. a process for the preparation of a class of 2-aryl-4- (1H-pyrazol-3-yl) pyridines according to claim 1 or 2, by:
(1) a method for preparing compound 3: heating and stirring a compound 2-bromo-4-iodopyridine (compound 1) and pyrazole-3-boric acid (compound 2) in toluene in the presence of an alkaline compound and a palladium catalyst for reaction, extracting, washing, drying, filtering, concentrating and separating by column chromatography after the reaction is finished to obtain a compound 3; wherein the alkaline compound is selected from one of potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and sodium hydride, and the palladium catalyst is selected from one of tetrakis (triphenylphosphine) palladium, palladium acetate, bis (dibenzylideneacetone) palladium and palladium dichloride;
(2) a method for preparing compound 4: in DMF, in the presence of an alkaline compound, stirring the compound 3 and 4-bromomethyl benzoate at room temperature for reaction, and after the reaction is finished, extracting, washing, drying, filtering, concentrating and carrying out column chromatography separation on a reaction system to obtain a compound 4; wherein the alkaline compound is selected from one of potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide;
(3) a method for preparing compound 5: heating and stirring the compound 4 and substituted phenylboronic acid or substituted phenylboronic acid pinacol ester with the structural formula shown in the specification in toluene in the presence of an alkaline compound and a palladium catalyst for reaction, and after the reaction is finished, extracting, washing, drying, filtering, concentrating, and performing column chromatography separation and purification on a reaction system to obtain a compound 5; wherein the alkaline compound is selected from one of potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and sodium hydride, and the palladium catalyst is selected from one of tetrakis (triphenylphosphine) palladium, palladium acetate, bis (dibenzylideneacetone) palladium and palladium dichloride;
R1is OCH3，CH3，H；R2Is F or H;
(4) the preparation method of the compound I comprises the following steps: in dichloromethane solution, Compound 5 and NH2OK/NH2And (3) reacting with an OH methanol solution, concentrating the reaction system under reduced pressure after the reaction is finished, dissolving the concentrate with water, adjusting the pH value to 5-6, performing suction filtration, washing, collecting solids, and recrystallizing to obtain a compound I.
4. Use of a class of 2-aryl-4- (1H-pyrazol-3-yl) pyridines according to any of claims 1 to 2 for the preparation of medicaments as active ingredients for the preparation of LSD1/HDAC dual-target inhibitor drugs.
5. The application of 2-aryl-4- (1H-pyrazol-3-yl) pyridine compounds in the preparation of medicines according to claim 4, which are used as active ingredients for preparing medicines for treating acute myeloid leukemia.
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