CN113444038A - 2-aryl isonicotinic acid amide LSD1/HDAC double-target inhibitor, and preparation method and application thereof - Google Patents

2-aryl isonicotinic acid amide LSD1/HDAC double-target inhibitor, and preparation method and application thereof Download PDF

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CN113444038A
CN113444038A CN202110766466.2A CN202110766466A CN113444038A CN 113444038 A CN113444038 A CN 113444038A CN 202110766466 A CN202110766466 A CN 202110766466A CN 113444038 A CN113444038 A CN 113444038A
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CN113444038B (en
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段迎超
张少杰
关圆圆
于童
靳林峰
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Xinxiang Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C07D213/82—Amides; Imides in position 3
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Abstract

The invention relates to a 2-aryl isonicotinic acid amide LSD1/HDAC double-target inhibitor, a preparation method thereof and application thereof in preparing antitumor drugs, belonging to the technical field of pharmaceutical chemistry. The compounds have the following general formula:
Figure DDA0003151843380000011
wherein R is1OH and H are preferred; r2OH and OCH are preferred3、F、H、CH3；R3Preferably H, OH, Cl, OCH3、NH2、
Figure DDA0003151843380000012
R4H, OH, CF are preferred3、OCH3、CH3、
Figure DDA0003151843380000013

Description

2-aryl isonicotinic acid amide LSD1/HDAC double-target inhibitor, and preparation method and application thereof
Technical Field
The invention particularly relates to a 2-aryl isonicotinic acid amide LSD1/HDAC double-target inhibitor, a preparation method thereof and application thereof in preparing antitumor drugs, belonging to the technical field of pharmaceutical chemistry.
Background
Histone lysine specific demethylase 1(LSD1) is a flavin adenine dinucleotide dependent amino oxidase, and its main function is to specifically remove single and double methylation modification on histone H3K4, and inhibit transcription of gene. LSD1 may also remove single and double methylation modifications on H3K9 by interacting with estrogen receptors or androgen receptors, thereby activating transcription of downstream genes. In addition, LSD1 can remove methylation modification of non-histone such as p53, DNMT1, STAT3, E2F1, MYPT1, ERA and HIF-1, and further regulate stability and activity of downstream genes. The expression level of LSD1 in various tumors such as acute myelogenous leukemia, prostatic cancer, lung cancer, gastric cancer, estrogen receptor negative breast cancer, colon cancer, synovial sarcoma and neuroblastoma is obviously increased, and the LSD1 is closely related to the poor prognosis of various malignant tumors such as lung cancer, leukemia, colon cancer and breast cancer. The RNA interference technology is adopted to knock out the expression of LSD1 or a small molecular compound is used to inhibit the activity of LSD1, so that the LSD1 can inhibit the proliferation, metastasis and invasion of tumor cells, is one of hot targets for the development of the current antitumor drugs, and seven LSD1 small molecular inhibitors such as IMG-7289, GSK2879552, TAK-418, ORY-2001, INCB059872, CC-90011 and SP-2577 enter phase I and phase II clinical tests to treat acute myelogenous leukemia and non-small cell lung cancer.
Histone Deacetylases (HDACs) are an important class of histone deacetylases, primarily responsible for removing acetylation modifications from histone tail lysine residues, silencing gene transcription. 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 malignant lymphoma and myeloma. In addition, there are a number of HDAC inhibitor candidates undergoing clinical trials.
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. LSD1 deacetylates H4K16 in an interdependent manner with HDAC1 via the CoREST complex, thereby modulating pluripotency of embryonic and cancer cells. LSD1 and Sin3A/HDAC complex can synergistically inhibit a series of pro-apoptotic genes and maintain sensitivity of breast cancer to chemotherapy. HDAC1 can remove acetylation modification of a substrate binding region K374 of LSD1, further promote effective combination 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 combined use 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. Therefore, the discovery of a novel and high-activity LSD1/HDAC double-target inhibitor can play a synergistic antitumor role of '1 +1> 2' by simultaneously inhibiting LSD1, HDAC and mutual cross-talk signal pathway transduction, is expected to discover a novel and high-efficiency antitumor lead compound, 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 isonicotinic acid amide 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 on 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 isonicotinic acid amide compounds, which provides possibility for new drug screening.
The invention also aims to provide a preparation method of the 2-aryl isonicotinic acid amide compound.
The invention further aims to provide the application of the 2-aryl isonicotinic acid amide compound in preparing antitumor drugs by taking LSD1/HDAC as a target.
In order to achieve the purpose, the 2-aryl isonicotinic acid amide compound has the structural general formula as follows:
Figure BDA0003151843370000021
in the general formula I, R1Is any one of OH and H; r2Is OH, OCH3、F、H、CH3Any one of the above; r3Is H, OH, Cl, OCH3、NH2、
Figure BDA0003151843370000022
Any one of the above; r4Is H, OH, CF3、OCH3、CH3、
Figure BDA0003151843370000023
Figure BDA0003151843370000024
Any one of the above; x is N or CH.
Preferably: in the general formula I, R1、R2、R3、R4And substituents or atoms represented by X are shown in the following table:
Figure BDA0003151843370000025
Figure BDA0003151843370000031
to achieve the second object, the reaction scheme for synthesizing the compound of the present invention is as follows:
Figure BDA0003151843370000032
synthetic routes to compounds of general formula I:
the preparation method of the compound 3 comprises the steps of stirring and reacting a compound 1 and 4-aminomethyl methyl benzoate hydrochloride (a compound 2) in N, N-Dimethylformamide (DMF) at room temperature in the presence of O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) and an alkaline compound, adding water and ethyl acetate into a reaction system for extraction after the reaction is finished, combining organic phases, washing with water and a saturated NaCl aqueous solution, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate in vacuum, and separating the concentrate by column chromatography to obtain the compound 3. Wherein the strong alkaline compound is selected from one of N, N-diisopropylethylamine and triethylamine.
The preparation method of the compound 4 comprises the steps of heating and stirring the compound 3 and various substituted phenylboronic acids or substituted phenylboronic acid pinacol esters or substituted pyridine boric acids in toluene in the presence of an alkaline compound and a palladium catalyst for reaction, adding water and ethyl acetate into a reaction system for extraction after the reaction is finished, combining organic phases, washing with water and a saturated NaCl aqueous solution, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating the concentrate through column chromatography to obtain the compound 4. 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.
Preparation method of Compound 5 in Compound 4, when R is4When the hydroxyl group is the hydroxyl group, the compound 4 and 4- (bromomethyl) piperidine-1-formic acid tert-butyl ester or 4- (2-bromoethyl) morpholine hydrobromide are heated and stirred to react in DMF in the presence of an alkaline compound, after the reaction is finished, water and ethyl acetate are added into a reaction system for extraction, organic phases are combined, water and a saturated NaCl aqueous solution are 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 column chromatography to obtain the compound 5. Wherein the alkaline compound is selected from one of potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide.
The compound I is prepared by dissolving compound 4 or compound 5 and NH in dichloromethane2OK/NH2And (3) reacting with an OH methanol solution, after the reaction is finished, concentrating the reaction system in vacuum, adding water to dissolve the concentrate, adjusting the pH value to 5-6 by using dilute hydrochloric acid, performing suction filtration, washing, collecting a solid, and recrystallizing by using methanol to obtain the compound I.
The invention has the advantages that: the 2-aryl isonicotinic acid amide compound synthesized by the invention has stronger LSD1/HDAC double inhibition activity and in-vitro anti-tumor activity, and has good selectivity on LSD 1. IC to HDAC1 for multiple compounds reported in the present invention50The value is less than 2nM, which is 7 times that of the positive drug SAHA. The LSD1/HDAC double-target inhibitor shows good in-vitro anti-tumor activity on human leukemia THP-1, MOLT-4 and MV4:11 cell strains, the in-vitro anti-tumor activity of a plurality of compounds is obviously superior to that of a positive medicament SAHA, and particularly the activity of the compounds I-5 and I-8 on THP-1 is 12 times and 18 times of that of SAHA respectively. The compound represents a high-activity LSD1/HDAC double-target inhibitor with a brand-new structure, provides a foundation for the research and development of LSD1/HDAC double-target inhibitor medicines, provides an effective tool for the research on the biological functions of LSD1 and HDAC, can be used as a candidate or lead compound for further development to develop anti-tumor, anti-virus, anti-AIDS and other disease treatment medicines, has a simple synthesis method, and is beneficial to the development of anti-tumor, anti-virus, anti-AIDS and other disease treatment medicinesAnd (5) popularization and application.
Detailed Description
The technical scheme of the invention is explained in detail by taking examples in combination with the reaction scheme.
Example 14 Synthesis of methyl- ((2-bromoisonicotinamide) methyl) benzoate (3a)
Figure BDA0003151843370000041
In a 50mL two-neck flask were added compound 1a (1212.1mg,6.0mmol), compound 2(1451.9mg, 7.2mmol), HBTU (2502.9mg,6.6mmol), dissolved in anhydrous DMF (8mL), protected with nitrogen, and then N, N-diisopropylethylamine (1705.9mg,13.2mmol), after the addition was completed, the reaction was stirred at room temperature for 1.5 hours, then water and ethyl acetate were added to the reaction system for extraction, the ethyl acetate layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the concentrate was separated and purified by silica gel column chromatography (petroleum ether: acetone ═ 5:1) to give compound 3a (850.4mg), a white solid, yield: 40.6%, Mp: 114-.1H NMR(400MHz, DMSO-d6)δ9.51(t,1H,J＝6.0Hz),8.57(d,1H,J＝5.2Hz),8.06(s,1H), 7.94(d,2H,J＝8.4Hz),7.85(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.48(d,2H, J＝8.4Hz),4.57(d,2H,J＝6.0Hz),3.85(s,3H).13C NMR(101MHz,DMSO-d6) δ166.53,163.77,151.75,144.86,144.65,142.37,129.78,128.79,127.99, 126.11,121.72,52.56,43.10.HRMS(ESI)calcd for C15H13BrN2NaO3[M+Na]+: 371.0002,Found:371.0004.
Example Synthesis of methyl- ((2-bromo-5-fluoroisonicotinamide) methyl) benzoate (3b)
Figure BDA0003151843370000051
The procedure of example 1 was followed, substituting compound 1b (660.0mg,3.0mmol) for 1a, to give the objective compound 3b (900.1mg) as a white solid in 81.7% yield, Mp: 131-.1H NMR(400MHz,DMSO-d6)δ 9.39(t,1H,J＝5.6Hz),8.61(s,1H),7.96(d,2H,J＝8.0Hz),7.90(d,1H, J＝5.2Hz),7.49(d,2H,J＝8.0Hz),4.56(d,2H,J＝5.6Hz),3.85(s,3H). 13C NMR(101MHz,DMSO-d6)δ166.53,161.38,155.84(d,JC-F＝259.6Hz),144.51, 140.32(d,JC-F＝27.1Hz),136.12(d,JC-F＝2.9Hz),134.71(d,JC-F＝15.2Hz), 129.80,128.83,127.91,127.87,52.58,42.94.HRMS(ESI)calcd for C15H12BrFN2NaO3[M+Na]+:388.9908,Found:388.9904.
Example 34 Synthesis of methyl- ((2- (3-hydroxyphenyl) isonicotinamide) methyl) benzoate (4a)
Figure BDA0003151843370000052
In a 50mL two-necked round bottom flask, compound 3a (500.0mg,1.4mmol), toluene (5mL), ethanol (5mL), H was added2O(1.3mL),K2CO3(359.3mg,2.6mmol),Pd(PPh3)4(162.0mg,0.14mmol) and 3-hydroxyphenylboronic acid (235.0mg,1.7mmol) are stirred and reacted for 4 hours at the temperature of 92 ℃ under the protection of nitrogen, after the reaction is finished, the reaction system is cooled to the room temperature, water and ethyl acetate are used for extraction, an ethyl acetate layer is combined, then water and saturated sodium chloride are respectively used for washing, anhydrous sodium sulfate is used for drying, suction filtration is carried out after drying is finished, filtrate is decompressed and concentrated, and a concentrate is separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate: 2:1) to obtain a compound 4a (312.7mg), the yield is 61.5%, and a white solid is Mp: 185-.1H NMR(400MHz,DMSO-d6)δ9.62(s,1H),9.53(t,1H,J＝ 6.0Hz),8.80(d,1H,J＝4.8Hz),8.31(d,1H,J＝1.6Hz),7.95(d,2H,J＝ 8.4Hz),7.35(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.57-7.55(m,2H),7.50(d,2H, J＝8.0Hz),7.32(t,1H,J＝8.0),6.87(dd,1H,J1＝1.6Hz,J2＝7.2Hz), 4.62(d,2H,J＝5.6Hz),3.84(s,3H).13C NMR(101MHz,DMSO-d6)δ166.56, 165.25,158.32,157.30,150.73,145.22,142.48,140.01,130.32,129.81,128.73, 127.95,120.68,117.89,116.93,113.95,52.56,43.02.HRMS(ESI)calcd for C21H19N2O4[M+H]+:363.1339,Found:363.1341.
Example Synthesis of methyl 44- ((2- (4-hydroxyphenyl) isonicotinamide) methyl) benzoate (4b)
Figure BDA0003151843370000061
The procedure of example 3 was followed, substituting 4-hydroxyphenylboronic acid (235.0mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give compound 4b (417.4mg) as a white solid in 81.3% yield, Mp: 169-.1H NMR(400MHz, DMSO-d6)δ9.85(s,1H),9.50(t,1H,J＝6.0Hz),8.74(d,1H,J＝5.2Hz), 8.27(s,1H),8.02(d,2H,J＝8.8Hz),7.96(d,2H,J＝8.0Hz),7.67(dd,1H, J1＝1.2Hz,J2＝4.8Hz),7.51(d,2H,J＝8.0Hz),6.91(d,2H,J＝8.4Hz), 4.63(d,2H,J＝6.0Hz),3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ166.56, 165.45,159.36,157.42,150.53,145.26,142.40,129.81,129.58,128.73,128.62, 127.94,119.48,116.79,116.04,52.55,43.00.HRMS(ESI)calcd for C21H18N2NaO4 [M+Na]+:385.1159,Found:385.1158.
Example Synthesis of methyl 54- ((2- (2-hydroxyphenyl) isonicotinamide) methyl) benzoate (4c)
Figure BDA0003151843370000062
The procedure of example 3 was followed, substituting 2-hydroxyphenylboronic acid (235.0mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give compound 4c (313mg) as a pale yellow solid in 61.7% yield, Mp: 180-.1H NMR(400MHz, DMSO-d6)δ13.69(s,1H),9.62(t,1H,J＝6.0Hz),8.79(dd,1H,J1＝0.8Hz, J2＝5.2Hz),8.61(t,1H,J＝5.2Hz),8.10(dd,1H,J1＝1.6Hz,J2＝8.4Hz), 7.96(d,2H,J＝8.4Hz),7.84(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.52(d,2H, J＝8.4Hz),7.38-7.33(m,1H),7.00-6.96(m,2H),4.65(d,2H,J＝6.0Hz), 3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ166.55,164.90,159.38,157.97, 147.83,145.07,143.36,132.22,129.82,128.77,128.00,127.83,120.38,119.48, 119.43,118.41,117.94,52.57,43.11.HRMS(ESI)calcd for C21H17N2O4[M-H]-: 361.1194,Found:361.1190.
Example Synthesis of methyl 64- ((2- (2-methoxyphenyl) isonicotinamide) methyl) benzoate (4d)
Figure BDA0003151843370000071
The procedure of example 3 was followed, using 2-methoxyphenylboronic acid (258.3mg,1.7mmol) in place of 3-hydroxyphenylboronic acid, to give compound 4d (379.9mg) as a colorless oil, yield: 72.1%.1H NMR(400MHz,DMSO-d6)δ 9.46(t,1H,J＝6.0Hz),8.82(d,1H,J＝5.2Hz),8.26(t,1H,J＝1.2Hz), 7.96(d,2H,J＝8.0Hz),7.76-7.71(m,2H),7.49(d,2H,J＝8.0Hz),7.47- 7.42(m,1H),7.19(d,1H,J＝8.0Hz),7.09(t,1H,J＝7.6Hz),4.60(d,2H, J＝6.0Hz),3.85(s,6H).13C NMR(101MHz,DMSO-d6)δ166.56,165.55,157.19, 156.65,150.41,145.29,141.48,131.20,130.93,129.79,128.70,128.38,127.90, 122.79,121.12,119.81,112.37,56.11,52.55,43.01.HRMS(ESI)calcd for C22H20N2NaO4[M+Na]+:399.1315,Found:399.1312.
Example Synthesis of methyl 74- ((2- (4-methoxyphenyl) isonicotinamide) methyl) benzoate (4e)
Figure BDA0003151843370000072
The procedure of example 3 was followed, substituting the compound 4-methoxyphenylboronic acid (258.3mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the objective compound 4e (320.5mg) as a white solid in a yield of 60.8%, Mp: 142-.1H NMR(400MHz,CDCl3)δ8.66(d,1H,J＝5.2Hz),8.02(s,1H),7.95(d,2H,J ＝8.4Hz),7.92(d,2H,J＝8.8Hz),7.44(dd,1H,J1＝1.2Hz,J2＝4.8Hz), 7.35(d,2H,J＝8.0Hz),7.23(t,1H,J＝5.2Hz),6.94(d,2H,J＝8.8Hz), 4.65(d,2H,J＝6.0Hz),3.88(s,3H),3.83(s,3H).13C NMR(101MHz,CDCl3)δ 166.81,166.10,160.87,158.20,150.21,142.86,142.01,130.99,130.08,129.49, 128.32,127.66,118.25,117.29,114.22,55.38,52.22,43.79.HRMS(ESI)calcd for C22H21N2O4[M+H]+:377.1496,Found:377.1496.
Example Synthesis of methyl 84- ((2- (4-methylphenyl) isonicotinamide) methyl) benzoate (4f)
Figure BDA0003151843370000073
The procedure of example 3 was followed, substituting the compound 4-methylphenylboronic acid (231.1mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the objective compound 4f (321.4mg) as a white solid in a yield of 63.7% and Mp: 152-.1H NMR (400MHz,CDCl3)δ9.54(d,1H,J＝6.0Hz),8.80(d,1H,J＝4.8Hz),8.36(s, 1H),8.06(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.0Hz),7.75(dd,1H,J1＝1.6 Hz,J2＝5.2Hz),7.51(d,2H,J＝8.0Hz),7.34(d,2H,J＝8.0Hz),4.63(d, 2H,J＝6.0Hz),2.38(s,3H).HRMS(ESI)calcd for C22H21N2O3[M+H]+:361.1547, Found:361.1544.
Example Synthesis of methyl 94- ((2- (4-trifluoromethylphenyl) isonicotinamide) methyl) benzoate (4g)
Figure BDA0003151843370000081
The procedure of example 3 was followed, using 4-trifluoromethylphenylboronic acid (332.1mg,1.7mmol) in place of 3-hydroxyphenylboronic acid, to give 4g (356.0mg) of the compound in a yield of 63.3% as a white solid, Mp: 154-.1H NMR(400 MHz,CDCl3)δ8.81(d,1H,J＝4.8Hz),8.16(t,1H,J＝1.2Hz),8.13(d,2H, J＝8.4Hz),7.99(d,2H,J＝8.4Hz),7.72(d,2H,J＝8.4Hz),7.58(dd,1H, J1＝1.2Hz,J2＝4.8Hz),7.39(d,2H,J＝8.4Hz),6.97(t,1H,J＝6.0Hz), 4.71(d,2H,J＝6.0Hz),3.90(s,3H).13C NMR(101MHz,CDCl3)δ166.78, 165.59,157.02,150.65,142.64,142.31,141.67,131.33(q,JC-F＝32.7Hz), 130.15,129.63,127.72,127.31,125.82(q,JC-F＝3.7Hz),124.05(q,JC-F＝273.3 Hz),119.71,118.48,52.26,43.91.HRMS(ESI)calcd for C22H18F3N2O3[M+H]+: 415.1264,Found:415.1265.
Example Synthesis of methyl 104- ((2- (4-morpholinylphenyl) isonicotinamide) methyl) benzoate (4h)
Figure BDA0003151843370000082
The procedure of example 3 was followed, substituting the compound 4-morpholinophenylboronic acid pinacol ester (491.6mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the compound 4h (199.3mg) as a white solid in yield of 33.0%, Mp: 189-.1H NMR(400MHz,CDCl3)δ8.73(dd,1H,J1＝1.2Hz,J2＝5.2Hz),8.05-8.01(m, 3H),7.98(d,2H,J＝9.2Hz),7.43-7.39(m,3H),6.98(d,2H,J＝8.8Hz), 6.71(t,1H,J＝6.0Hz),4.73(d,2H,J＝6.0Hz),3.91(s,3H),3.88(t,4H, J＝5.2Hz),3.25(t,4H,J＝5.2Hz).13C NMR(101MHz,CDCl3)δ166.72, 166.07,158.38,152.16,150.27,142.78,141.92,130.18,129.71,129.49,127.96, 127.73,117.72,116.94,115.10,66.77,52.19,48.54,43.87.HRMS(ESI)calcd for C25H26N3O4[M+H]+:432.1918,Found:432.1917.
Example Synthesis of methyl 114- ((2- (3-morpholinophenyl) isonicotinamide) methyl) benzoate (4i)
Figure BDA0003151843370000091
The procedure of example 3 was followed, substituting the compound 3-morpholinophenylboronic acid pinacol ester (491.6mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the compound 4i (211.4mg) as a white solid in a yield of 35.0% and Mp: 155-.1H NMR(400MHz,CDCl3)δ8.74(d,1H,J＝5.2Hz),8.10(s,1H),7.98(d,2H,J ＝8.4Hz),7.62(t,1H,J＝2.0Hz),7.51(dd,1H,J1＝1.6Hz,J2＝5.2Hz), 7.44(d,1H,J＝7.6Hz),7.38(d,2H,J＝8.0Hz),7.34(d,1H,J＝7.6Hz), 7.04(d,1H,J＝5.2Hz),6.98(dd,1H,J1＝2.0Hz,J2＝8.0Hz),4.69(d,2H, J＝6.0Hz),3.89(s,3H),3.86(t,4H,J＝4.8Hz),3.22(t,4H,J＝4.8Hz). 13C NMR(101MHz,CDCl3)δ166.79,165.93,158.84,151.85,150.28,142.80, 142.04,139.40,130.12,129.65,129.56,127.71,118.99,118.58,118.21,116.86, 114.21,66.91,52.24,49.27,43.85.HRMS(ESI)calcd for C25H26N3O4[M+H]+: 432.1918,Found:432.1918.
Example Synthesis of methyl 124- ((2- (4- (4-methyl-1-piperazinyl) phenyl) isonicotinamide) methyl) benzoate (4j)
Figure BDA0003151843370000092
The procedure of example 3 was followed, using the compound 4- (4-methyl-1-piperazinyl) phenylboronic acid pinacol ester (531.8mg, 1.7mmol) in place of 3-hydroxyphenylboronic acid, to give the compound 4j (94.6mg) as a white solid in a yield of 15.2% and Mp: 167-.1H NMR(400MHz,CDCl3)δ9.51(t,1H,J＝6.0Hz),8.73(d,1H,J ＝5.2Hz),8.27(s,1H),8.04(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.0Hz), 7.64(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.50(d,2H,J＝8.0Hz),7.05(d,2H, J＝8.4Hz),4.62(d,2H,J＝6.0Hz),3.85(s,3H),3.25(t,4H,J＝5.2Hz), 2.46(t,4H,J＝5.2Hz),2.23(s,3H).13C NMR(101MHz,CDCl3)δ166.56, 165.46,157.33,152.19,150.54,145.29,142.30,129.81,128.71,128.40,127.94, 119.29,116.53,115.14,54.95,52.57,47.75,46.25,42.99.HRMS(ESI)calcd for C26H29N4O3[M+H]+:445.2234,Found:445.2236.
Example Synthesis of methyl 134- ((2- (4-morpholinosulfonyl) phenyl) isonicotinamide) methyl) benzoate (4k)
Figure BDA0003151843370000101
The procedure of example 3 was followed, substituting the compound 4-morpholinosulfonylphenylboronic acid (460.9mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the compound 4k (189.3mg) as a white solid in 38.2% yield, Mp:216-217 ℃.1H NMR(400MHz,DMSO-d6))δ9.59(t,1H,J＝6.0Hz),8.91(d,1H,J＝5.2Hz), 8.51(t,1H,J＝1.2Hz),8.43(d,2H,J＝8.8Hz),7.96(d,2H,J＝8.4Hz), 7.91-7.87(m,3H),7.52(d,2H,J＝8.4Hz),4.65(d,2H,J＝6.0Hz),3.85(s, 3H),3.65(t,4H,J＝4.4Hz),2.92(t,4H,J＝4.4Hz).13C NMR(101MHz,DMSO- d6)δ166.55,165.07,155.49,151.17,145.11,143.02,142.94,135.43,129.81, 128.79,128.74,128.10,127.99,121.83,119.05,65.76,52.56,46.38,43.08. HRMS(ESI)calcd for C25H24N3O6S[M-H]-:494.1391,Found:494.1392.
Example Synthesis of methyl 144- ((2- (3-chloro-4-methoxyphenyl) isonicotinamide) methyl) benzoate (4l)
Figure BDA0003151843370000102
The procedure of example 3 was followed, using the compound 3-chloro-4-methoxyphenylboronic acid (316.9mg,1.7mmol) in place of 3-hydroxyphenylboronic acid, to give 4l (207.1mg) of the compound as a white solid in a yield of 36.0% and Mp of 154-.1H NMR (400MHz,CDCl3)δ8.75(dd,1H,J1＝1.2Hz,J2＝5.2Hz),8.10(d,1H,J＝2.0 Hz),8.04-8.01(m,3H),7.92(dd,1H,J1＝2.0Hz,J2＝8.4Hz),7.48(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.42(d,2H,J＝8.4Hz),7.01(d,1H,J＝8.8Hz), 6.75(t,1H,J＝5.6Hz),4.73(d,2H,J＝6.0Hz),3.96(s,3H),3.92(s,3H). 13C NMR(101MHz,CDCl3)δ166.80,165.87,156.84,156.07,150.34,142.76, 142.13,131.76,130.10,129.54,128.80,127.71,126.40,123.01,118.78,117.30, 112.00,56.26,52.24,43.86.HRMS(ESI)calcd for C22H20ClN2O4[M+H]+:411.1106, Found:411.1105.
EXAMPLE 154 Synthesis of methyl- ((2- (3, 4-dimethoxyphenyl) isonicotinamide) methyl) benzoate (4m)
Figure BDA0003151843370000103
The procedure of example 3 was followed, substituting the compound 3, 4-dimethoxyphenylboronic acid (309.4mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the compound 4m (275.4mg) as a yellow solid in 48.4% yield, Mp: 132-.1H NMR (400MHz,CDCl3)δ8.69(d,1H,J＝5.2Hz),8.09(t,1H,J＝1.2Hz),7.95(d, 2H,J＝8.4Hz),7.65(d,1H,J＝2.0Hz),7.52(d,1H,J＝2.0Hz),7.49(dd, 1H,J1＝1.2Hz,J2＝5.2Hz),7.40-7.34(m,3H),6.88(d,1H,J＝8.4Hz),4.67 (d,2H,J＝5.2Hz),3.93(s,3H),3.89(s,3H),3.88(s,3H).13C NMR(101MHz, CDCl3)δ166.80,166.06,158.09,150.33,150.14,149.26,142.91,142.01, 131.34,130.06,129.46,127.66,119.65,118.37,117.50,111.02,109.81,55.96, 55.93,52.22,43.80.HRMS(ESI)calcd for C23H21N2O5[M-H]-:405.1456,Found: 405.1456.
Example Synthesis of methyl 164- ((2- (2, 4-dimethylphenyl) isonicotinamide) methyl) benzoate (4n)
Figure BDA0003151843370000111
The procedure of example 3 was followed, substituting the compound 2, 4-dimethylbenzeneboronic acid (258.5mg,1.7mmol) for 3-hydroxybenzeneboronic acid, to give the objective compound 4n (400.4mg) as a white solid in a yield of 80.4% and Mp at 88-89 ℃.1H NMR(400MHz,CDCl3)δ8.76(d,1H,J＝5.2Hz),8.00(d,2H,J＝8.4Hz), 7.73(dd,1H,J1＝1.2Hz,J2＝2.0Hz),7.54(dd,1H,J1＝1.6Hz,J2＝5.2Hz), 7.38(d,2H,J＝8.4Hz),7.26-7.25(m,1H),7.09(s,2H,J＝7.6Hz),7.09(s, 1H),7.07(d,1H,J＝8.0Hz),6.84(t,1H,J＝5.6Hz),4.69(d,2H,J＝5.6 Hz),3.90(s,3H),2.36(s,3H),2.32(s,3H).13C NMR(101MHz,CDCl3)δ 166.76,165.87,161.35,149.93,142.77,141.50,138.61,136.71,135.68, 131.72130.14,129.61,129.57,127.69,126.73,121.33,118.52,52.22,43.82, 21.18,20.25.HRMS(ESI)calcd for C23H23N2O3[M+H]+:375.1703,Found:375.1701.
Example Synthesis of methyl 174- ((2- (2-fluoro-4-methylphenyl) isonicotinamide) methyl) benzoate (4o)
Figure BDA0003151843370000112
The procedure of example 3 was followed, substituting 2-fluoro-4-methylphenylboronic acid (269.4mg,1.8mmol) for 3-hydroxyphenylboronic acid, to give the objective compound 4o (490.1mg) as a white solid in a yield of 89.3% and Mp:126-127 ℃.1H NMR (400MHz,CDCl3)δ8.76(d,1H,J＝5.2Hz),8.06(dd,1H,J1＝1.2Hz,J2＝2.4 Hz),7.98(d,2H,J＝8.0Hz),7.84(t,1H,J＝8.4Hz),7.54(dd,1H,J1＝ 1.6Hz,J2＝4.8Hz),7.37(d,2H,J＝8.0Hz),7.07-7.01(m,1H),6.95(d,1H,J ＝12.8Hz),4.68(d,2H,J＝5.6Hz),3.90(s,3H),2.39(s,3H).13C NMR(101 MHz,DMSO-d6)δ162.05,161.15,155.56(d,JC-F＝250.7Hz),149.85(d,JC-F＝2.5 Hz),145.63,138.10,137.24(d,JC-F＝8.6Hz),137.05,125.84(d,JC-F＝3.1Hz), 125.33 124.75 122.88 120.75(d JC-F＝3.0Hz)118.86(d JC-F＝11.3Hz) 116.52(d,JC-F＝9.7Hz),114.76,112.00(d,JC-F＝22.8Hz)47.45,39.04,16.46. HRMS(ESI)calcd for C22H20FN2O3[M+H]+:379.1451,Found:379.1451.
Example Synthesis of methyl 184- ((2- (3-chloro-4-morpholinophenyl) isonicotinamide) methyl) benzoate (4p)
Figure BDA0003151843370000121
By the method of example 3, using 3-chloro-4-Morpholinobenzeneboronic acid (410.5mg,1.7mmol) was substituted for 3-hydroxyphenylboronic acid to give the desired compound 4p (355mg) as a white solid in 76.2% yield, Mp:147-148 ℃.1H NMR(400 MHz,CDCl3)δ8.70(dd,1H,J1＝0.8Hz,J2＝5.2Hz),8.06(d,2H,J＝2.0Hz), 7.97(d,2H,J＝8.4Hz),7.85(dd,1H,J1＝2.4Hz,J2＝8.4Hz),7.51(dd,1H, J1＝1.6Hz,J2＝4.8Hz),7.37(d,2H,J＝8.4Hz),7.25(t,1H,J＝5.6Hz), 7.05(d,1H,J＝8.8Hz),4.68(d,2H,J＝6.0Hz),3.90(s,3H),3.88(t,4H, J＝4.8Hz),3.10(t,4H,J＝4.8Hz),1.82(s,1H).13C NMR(101MHz,CDCl3)δ 166.79,165.83,156.81,150.37,150.03,142.82,142.14,133.93,130.10,129.52, 129.32,128.94,127.70,126.12,120.21,118.97,117.54,67.05,52.25,51.46, 43.84.HRMS(ESI)calcd for C25H23ClN3O4[M-H]-:464.1383,Found:464.1380.
Example 194 Synthesis of methyl- ((2- (2-methyl-4-aminophenyl) isonicotinamide) methyl) benzoate (4q)
Figure BDA0003151843370000122
The procedure of example 3 was followed, using 2-methyl-3-aminophenylboronic acid (256.6mg,1.7mmol) in place of 3-hydroxyphenylboronic acid, to give compound 4q (420.5mg) as a colorless oil in 80% yield.1H NMR(400MHz,CDCl3)δ 8.63(dd,1H,J1＝0.8Hz,J2＝5.2Hz),7.89(d,2H,J＝8.4Hz),7.63(dd,1H, J1＝0.8Hz,J2＝1.6Hz),7.48(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.28(d,2H, J＝8.4Hz),7.17(t,1H,J＝6.0Hz),6.98(t,1H,J＝8.0Hz),6.65(d,2H,J ＝7.6Hz),4.56(d,2H,J＝6.0Hz),3.81(s,3H),1.96(s,3H),1.16(s,2H). 13C NMR(101MHz,CDCl3)δ166.84,165.91,161.63,149.73,145.34,142.94, 141.61,140.64,130.05,129.43,127.61,126.44,121.63,120.14,120.09,118.95, 115.50,52.23,43.72,14.08.HRMS(ESI)calcd for C22H22N3O3[M+H]+:376.1656, Found:376.1656.
EXAMPLE synthesis of methyl 204- ((6 '-methoxy- [2,3' -pyridine ] -4-carboxamido) methyl) benzoate (4r)
Figure BDA0003151843370000131
The procedure of example 3 was followed, using 2-methoxy-5-pyridineboronic acid (236.5mg,1.6mmol) in place of 3-hydroxyphenylboronic acid, to give compound 4r (353.2mg) as a yellow solid in a yield of 72.6% and Mp:156-157 ℃.1H NMR(400 MHz,DMSO-d6)δ9.50(t,1H,J＝5.2Hz),8.95(s,1H),8.81(d,1H,J＝4.8 Hz),8.43(d,1H,J＝8.4Hz),8.36(s,1H),7.96(d,2H,J＝7.6Hz),7.76(d, 1H,J＝4.8Hz),7.51(d,2H,J＝7.6Hz),6.98(d,1H,J＝8.4Hz),4.63(d, 2H,J＝5.6Hz),3.93(s,3H),3.85(s,3H).13C NMR(101MHz,DMSO-d6)δ 166.54,165.23,164.75,155.15,150.88,146.11,145.16,142.67,137.86,129.80, 128.74,128.10,127.96,120.50,117.55,111.09,53.95,52.56,43.02.HRMS(ESI) calcd for C21H18N3O4[M-H]-:376.1303,Found:376.1302.
Example Synthesis of methyl 214- ((5-fluoro-2- (p-tolyl) isonicotinamide) methyl) benzoate (4s)
Figure BDA0003151843370000132
The procedure of example 3 was followed, using 3b (514.0mg,1.4mmol) in place of 3a and 4-methylphenylboronic acid (244.4mg,1.7mmol) in place of 3-hydroxyphenylboronic acid, to give compound 4s (450.29mg) as a white solid in 85.0% yield, Mp:135-136 ℃.1H NMR(400MHz,CDCl3)δ8.61(d,1H,J＝2.0Hz),8.36 (d,1H,J＝6.0Hz),8.04(d,2H,J＝8.4Hz),7.91(d,2H,J＝8.0Hz),7.43(d, 2H,J＝8.4Hz),7.29(d,2H,J＝8.0Hz),7.19-7.12(m,1H),4.76(d,2H,J＝ 6.4Hz),3.92(s,3H),2.41(s,3H).13C NMR(101MHz,CDCl3)δ166.73,161.73 (d,JC-F＝2.6Hz),155.45(d,JC-F＝256.5Hz),155.13(d,JC-F＝4.4Hz),142.55, 139.56,138.90,138.62,134.80,130.18,129.66,127.79(d,JC-F＝10.4Hz), 127.59,126.76,121.09,52.20,43.93,21.30.HRMS(ESI)calcd for C22H20FN2O3[M+ H]+:379.1452,Found:379.1450.
Example Synthesis of methyl 224- ((5-fluoro-2- (4-methoxyphenyl) isonicotinamide) methyl) benzoate (4t)
Figure BDA0003151843370000133
The procedure used in example 21 was followed, substituting the compound 4-methoxyphenylboronic acid (260.0mg,1.7mmol) for 3-hydroxyphenylboronic acid, to give the compound 4t (387.1mg) as a white solid in 70.1% yield, Mp:128-129 ℃.1H NMR (400MHz,CDCl3)δ8.58(d,1H,J＝2.4Hz),8.32(d,1H,J＝6.0Hz),8.04(d, 2H,J＝8.0Hz),7.96(d,2H,J＝8.8Hz),7.43(d,2H,J＝8.4Hz),7.20-7.13 (m,1H),7.00(d,2H,J＝8.8Hz),4.76(d,2H,J＝6.4Hz),3.92(s,3H),3.87 (s,3H).13C NMR(101MHz,CDCl3)δ166.74,161.78(d,JC-F＝2.7Hz),160.80, 155.22(d,JC-F＝256.1Hz),154.81(d,JC-F＝4.0Hz),142.56,138.65(d,JC-F＝ 28.0Hz),130.23,130.17,129.66,128.23,127.79(d,JC-F＝10.3Hz),127.58, 120.58,114.28,55.39,52.19,43.92.HRMS(ESI)calcd for C22H20FN2O4[M+H]+: 395.1407,Found:395.1400.
Example Synthesis of methyl 234- ((2- (4- (2-morpholinoethoxy) phenyl) isonicotinamide) methyl) benzoate (5a)
Figure BDA0003151843370000141
Adding cesium carbonate (358.4mg,1.1mmol) into a 50mL two-necked round-bottomed flask, adding anhydrous N, N-dimethylformamide (5mL), adding compound 4b (362.4mg,1.0mmol) and compound 4- (2-bromoethyl) morpholine hydrobromide (330.0mg,1.2mmol) with stirring, heating at 70 ℃ and stirring for reaction for 1 hour, after the reaction is finished, adding water and ethyl acetate into the reaction system for extraction, combining ethyl acetate layers, adding water, adding ethyl acetate, adding water, stirring, cooling, and packaging,Washing with saturated sodium chloride, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the concentrate by silica gel column chromatography (petroleum ether: acetone 4:1) to obtain compound 5a (385.7mg) as a white solid with a yield of 81.1% and Mp: 132-.1HNMR(400MHz,CDCl3) δ8.70(dd,1H,J1＝0.4Hz,J2＝5.2Hz),8.04(dd,1H,J1＝0.8Hz,J2＝1.6 Hz),7.98(d,2H,J＝8.4Hz),7.95(d,2H,J＝8.8Hz),7.45(dd,1H,J1＝1.6 Hz,J2＝9.2Hz),7.38(d,2H,J＝8.0Hz),7.10(t,1H,J＝5.6Hz),6.97(d, 2H,J＝6.8Hz),4.68(d,2H,J＝5.6Hz),4.15(t,2H,J＝5.6Hz),3.90(s, 3H),3.73(t,4H,J＝4.4Hz),2.82(t,2H,J＝5.6Hz),2.59(t,4H,J＝4.4 Hz).13C NMR(101MHz,CDCl3)δ166.79,166.06,159.99,158.13,150.23,142.87, 142.03,131.18,130.09,129.51,128.33,127.67,118.28,117.33,114.84,66.89, 65.81,57.57,54.08,52.23,43.80.HRMS(ESI)calcd for C27H30N3O5[M+H]+: 476.2180,Found:476.2175.
EXAMPLE 24 Synthesis of tert-butyl 4- ((4- (4- ((4- (methoxycarbonyl) phenyl) carbamoyl) -2-pyridinyl) phenoxy) methyl) -1-piperidinecarboxylic acid ester (5b)
Figure BDA0003151843370000142
The procedure of example 23 was followed, using the compound tert-butyl 4- (bromomethyl) piperidine-1-carboxylate (140.2mg,1.2 mmol) in place of 4- (2-bromoethyl) morpholine hydrobromide, to give compound 5b (253.0mg) as a white solid in 45.2% yield, Mp: 105-.1H NMR(400MHz,CDCl3)δ8.71(d,1H,J＝4.8Hz),8.05 (t,1H,J＝1.2Hz),8.00(d,2H,J＝8.4Hz),7.96(d,2H,J＝8.8Hz),7.46 (dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.40(d,2H,J＝8.4Hz),7.07(t,1H,J＝ 6.0Hz),6.95(d,2H,J＝8.8Hz),4.70(d,2H,J＝6.0Hz),4.15(brs,2H), 3.90(s,3H),3.83(d,2H,J＝6.4Hz),2.73(brs,2H),2.03-1.91(m,1H), 1.84-1.79(m,2H),1.46(s,9H),1.31-1.20(m,2H).13C NMR(101MHz,CDCl3)δ 166.75,166.03,160.33,158.22,154.90,150.25,142.88,142.05,131.08,130.11, 129.60,128.34,127.69,118.19,117.26,114.74,79.48,72.38,52.18,43.82, 36.20,28.86,28.47.HRMS(ESI)calcd for C32H36N3O6[M-H]-:558.2610,Found: 558.2609.
EXAMPLE 25 Synthesis of N- (4- (hydroxycarbamoyl) benzyl) -2- (3-hydroxyphenyl) isonicotinic acid amide (I-1)
Figure BDA0003151843370000151
Under ice bath and stirring, an anhydrous methanol solution of potassium hydroxide (7.0g,45mL of methanol) was slowly added dropwise to an anhydrous methanol solution of hydroxylamine hydrochloride (5.84g,20mL of methanol), and after the addition, stirring was carried out for 5 minutes, and NH was obtained by filtration2OK-NH2And (5) sealing and storing the OH methanol solution for later use. In a 50mL two-necked round bottom flask, compound 4a (362.4mg,1.0mmol) was added, dissolved in anhydrous dichloromethane (5mL), and the NH prepared in the previous step was slowly dropped under stirring in a nitrogen blanket and ice bath2OK-NH2OH solution (16.5mL), stirring at room temperature for 1.0 h after the addition, concentrating the reaction system under reduced pressure, dissolving the concentrate with distilled water (20mL), adjusting the pH to 5-6 with dilute HCl, precipitating a solid, filtering, collecting the solid, recrystallizing the solid with methanol to obtain the compound I-1 (171.5mg) as a white solid with a yield of 47.2% and Mp: 145-.1H NMR(400MHz,DMSO-d6) δ11.22(s,1H),9.64(s,1H),9.50(t,1H,J＝6.0Hz),9.04(s,1H),8.80(d, 1H,J＝4.8Hz),8.31(t,1H,J＝1.2Hz),7.76-7.73(m,3H),7.59-7.56(m,2H), 7.43(d,2H,J＝8.4Hz),7.32(t,1H,J＝8.0Hz),6.88(dd,1H,J1＝1.6Hz, J2＝8.0Hz),4.58(d,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.20, 158.32,157.28,150.71,142.78,142.56,140.02,131.91,130.33,127.67,127.47, 120.68,117.89,116.93,113.94,43.01.HRMS(ESI)calcd for C20H17N3NaO4[M+ Na]+:386.1111,Found:386.1112.
EXAMPLE 26 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4-hydroxyphenyl) isonicotinamide (I-2)
Figure BDA0003151843370000152
The procedure of example 25 was followed, replacing 4a with 4b (362.4mg,1.0mmol) to give compound I-2(223.1 mg) as a white solid in 61.4% yield, Mp:161 and 162 ℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.85(s,1H),9.46(t,1H,J＝5.6Hz),9.05(s,1H),8.74(d,1H,J＝ 8.4Hz),8.25(s,1H),8.01(d,2H,J＝8.8Hz),7.74(d,2H,J＝8.0Hz),7.66 (dd,1H,J1＝0.8Hz,J2＝4.8Hz),7.43(d,2H,J＝8.4Hz),6.90(d,2H,J＝ 8.8Hz),4.58(d,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.39,164.50, 159.35,157.40,150.52,142.83,142.47,131.90,129.58,128.62,127.67,127.47, 119.49,116.78,116.04,42.99.HRMS(ESI)calcd for C20H16N3O4[M-H]-:362.1146, Found:362.1147.
EXAMPLE 27 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (2-hydroxyphenyl) isonicotinamide (I-3)
Figure BDA0003151843370000161
The procedure of example 25 was followed, replacing 4a with 4c (362.4mg,1.0mmol) to give compound I-3(252.9 mg) in 69.6% yield as a yellow solid, Mp:192-193 ℃.1H NMR(400MHz,DMSO-d6)δ13.68 (s,1H),11.22(s,1H),9.56(t,1H,J＝6.0Hz),9.04(s,1H),8.78(d,1H,J＝ 5.2Hz),8.59(s,1H),8.09(dd,1H,J1＝1.6Hz,J2＝8.4Hz),7.82(dd,1H,J1＝1.2Hz,J2＝5.2Hz),7.75(d,2H,J＝8.4Hz),7.44(d,2H,J＝8.4Hz),7.35 (td,1H,J1＝1.6Hz,J2＝7.6Hz),6.99-6.95(m,2H),4.60(d,2H,J＝5.6Hz). 13C NMR(101MHz,DMSO-d6)δ164.85,159.37,157.97,147.82,143.47,142.62, 132.21,131.99,127.84,127.72,127.48,120.38,119.49,119.46,118.40,117.95, 43.12.HRMS(ESI)calcd for C20H16N3O4[M-H]-:362.1146,Found:362.3349.
EXAMPLE 28 Synthesis of N- (4- (hydroxycarbamoyl) benzyl) -2- (2-methoxyphenyl) isonicotinamide (I-4)
Figure BDA0003151843370000162
The procedure of example 25 was followed, replacing 4a with 4d (376.4mg,1.0mmol) to give compound I-4(232.8 mg) in 61.7% yield as a white solid with Mp: 189-.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.40(t,1H,J＝6.0Hz),9.03(d,1H,J＝2.0Hz),8.80(d,1H,J＝ 4.8Hz),8.24(t,1H,J＝1.6Hz),7.74-7.71(m,4H),7.47-7.39(m,3H),7.18 (d,1H,J＝7.6Hz),7.09(td,1H,J1＝1.2Hz,J2＝7.6Hz),4.55(d,2H,J＝ 5.6Hz),3.84(s,3H).13C NMR(101MHz,DMSO-d6)δ165.49,164.52,157.19, 156.62,150.40,142.88,141.57,131.89,131.20,130.93,128.39,127.61,127.46, 122.79,121.13,119.81,112.39,56.13,42.98.HRMS(ESI)calcd for C21H19N3NaO4[M +Na]+:400.1268,Found:400.1269.
EXAMPLE 29 Synthesis of N- (4- (hydroxycarbamoyl) benzyl) -2- (4-methoxyphenyl) isonicotinic acid amide (I-5)
Figure BDA0003151843370000171
The procedure of example 25 was followed, replacing 4a with 4e (376.4mg,1.0mmol) to give compound I-5(201.1 mg) as a white solid in 53.3% yield, Mp:203-204 ℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.52(t,1H,J＝5.6Hz),9.04(s,1H),8.77(d,1H,J＝5.2Hz),8.32 (s,1H),8.13(d,2H,J＝8.8Hz),7.74(d,2H,J＝8.0Hz),7.70(d,1H,J＝ 4.4Hz),7.43(d,2H,J＝8.4Hz),7.08(d,2H,J＝8.8Hz),4.58(d,2H,J＝ 5.6Hz),3.83(s,3H).13C NMR(101MHz,DMSO-d6)δ165.32,164.53,160.91, 157.04,150.62,142.83,142.57,131.92,131.12,128.55,127.68,127.49,119.86, 117.12,114.67,55.74,43.00.HRMS(ESI)calcd for C21H20N3O4[M+H]+:378.1448, Found:378.1446.
EXAMPLE 30 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4-methylphenyl) isonicotinamide (I-6)
Figure BDA0003151843370000172
The procedure of example 25 was followed, replacing 4a with 4f (360.4mg,1.0mmol) to give Compound I-6 (215.39mg) as a white solid in 59.6% yield and Mp 218-219 ℃.1H NMR(400MHz,DMSO-d6) δ11.22(s,1H),9.49(t,1H,J＝6.0Hz),9.04(s,1H),8.80(d,1H,J＝ 5.2Hz),8.35(s,1H),8.06(d,2H,J＝8.0Hz),7.76-7.73(m,3H),7.43(d, 2H,J＝8.0Hz),7.34(d,2H,J＝8.0Hz),4.59(d,2H,J＝5.6Hz),2.38(s, 3H).HRMS(ESI)calcd for C21H20N3O3[M+H]+:362.1499,Found:362.1494.
EXAMPLE 31 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4-trifluoromethylphenyl) isonicotinamide (I-7)
Figure BDA0003151843370000173
The procedure of example 25 was followed, replacing 4a with 4g (414.4mg,1.0mmol) to give compound I-7(196.1 mg) as a white solid in 47.2% yield, Mp: 233-.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.54(t,1H,J＝6.0Hz),9.05(s,1H),8.89(d,1H,J＝4.8Hz), 8.48(t,1H,J＝1.2Hz),8.38(d,2H,J＝8.4Hz),7.91(d,2H,J＝8.4Hz), 7.86(dd,1H,J1＝1.2Hz,J2＝4.8Hz),7.74(d,2H,J＝8.4Hz),7.44(d,2H,J ＝8.4Hz),4.60(d,2H,J＝6.0Hz).13C NMR(101MHz,DMSO-d6)δ165.01, 155.62,151.09,142.96,142.69,142.44,131.95,129.97(q,JC-F＝31.9Hz), 127.92,127.70,127.48,126.24(q,JC-F＝3.4Hz),124.72(q,JC-F＝273.2Hz), 123.36,121.70,118.76,43.05.HRMS(ESI)calcd for C21H16F3N3NaO3[M+Na]+: 438.1036,Found:438.1037.
EXAMPLE 32 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4-morpholinophenyl) isonicotinamide (I-8)
Figure BDA0003151843370000181
The procedure of example 25 was followed, replacing 4a with 4h (431.5mg,1.0mmol) to give compound I-8(281.9 mg) as a yellow solid in 65.2% yield and Mp:158 and 159 ℃.1H NMR(400MHz,DMSO-d6)δ11.26 (s,1H),9.51(t,1H,J＝6.0Hz),9.08(s,1H),8.78(d,1H,J＝4.8Hz),8.32 (s,1H),8.10(d,2H,J＝8.4Hz),7.79(d,2H,J＝8.4Hz),7.69(d,1H,J＝ 4.8Hz),7.48(d,2H,J＝8.0Hz),7.12(d,2H,J＝4.8Hz),4.63(d,2H,J＝ 5.6Hz),3.81(t,4H,J＝4.4Hz),3.27(t,4H,J＝4.4Hz).13C NMR(101MHz, DMSO-d6)δ165.42,164.55,157.30,152.30,150.52,142.85,142.47,131.94, 128.91,127.97,127.66,127.48,119.37,116.65,114.99,66.48,48.17,43.01. HRMS(ESI)calcd for C24H23N4O4[M-H]-:431.1725,Found:431.1725.
Example 33N- (4- (Hydroxyaminocarboxamide) benzyl) -2- (3-morpholinylphenyl) isonicotinamide (I-9)
Figure BDA0003151843370000182
The procedure of example 25 was followed, replacing 4a with 4I (431.5mg,1.0mmol) to give compound I-9(346.8 mg) as a yellow solid in 80.2% yield and Mp:107-108 ℃.1H NMR(400MHz,DMSO-d6)δ11.14 (s,1H),9.56(s,1H),9.11(s,1H),8.80(d,1H,J＝4.8Hz),8.35(s,1H), 7.75-7.71(m,4H),7.58(d,1H,J＝7.2Hz),7.43-7.36(m,3H),7.07(d,1H,J ＝8.0Hz),4.91(s,2H),3.77(t,4H,J＝4.0Hz),3.19(t,4H,J＝4.0Hz).13C NMR(101MHz,Pyr-d5)δ166.04,158.29,152.12,150.40,143.27,142.71,139.83, 132.97,129.65,127.94,127.68,120.44,118.42,116.69,114.20,66.68,49.03, 43.53.HRMS(ESI)calcd for C24H23N4O4[M-H]-:431.1725,Found:431.1725.
EXAMPLE 34 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4- (4-methylpiperazin-1-yl) phenyl) isonicotinamide (I-10)
Figure BDA0003151843370000191
The procedure of example 25 was followed, replacing 4a with 4j (444.5mg,1.0mmol) to give compound I-10(129.3 mg) as a red solid in 29.1% yield and Mp:197 and 198 ℃.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.46(t,1H,J＝6.0Hz),9.04(s,1H),8.72(d,1H,J＝4.8Hz),8.26 (s,1H),8.03(d,2H,J＝8.8Hz),7.73(d,2H,J＝8.4Hz),7.63(dd,1H,J1＝ 1.2Hz,J2＝4.8Hz),7.42(d,2H,J＝8.0Hz),7.05(d,2H,J＝8.8Hz),4.57 (d,2H,J＝6.0Hz),3.25(t,4H,J＝4.8Hz),2.46(t,4H,J＝4.8Hz),2.23 (s,3H).13C NMR(101MHz,DMSO-d6)δ165.40,164.47,157.32,152.18,150.52, 142.85,142.39,131.90,128.42,127.94,127.66,127.46,119.29,116.53,115.14, 54.94,47.75,46.24,42.98.HRMS(ESI)calcd for C25H28N5O3[M+H]+:446.2187, Found:446.2184.
EXAMPLE 35 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4- (4-morpholinosulfonyl) phenyl) isonicotinamide (I-11)
Figure BDA0003151843370000192
The procedure of example 25 was followed, replacing 4a with 4k (495.6mg,1.0mmol) to give compound I-11(268.6 mg) as a yellow solid in 54.1% yield, Mp:160-161 ℃.1H NMR(400MHz,DMSO-d6))δ11.23 (s,1H),9.55(t,1H,J＝5.2Hz),9.05(s,1H),8.90(d,1H,J＝4.8Hz),8.50 (s,1H),8.43(d,2H,J＝8.4Hz),7.91-7.86(m,3H),7.75(d,2H,J＝8.0Hz), 7.44(d,2H,J＝8.0Hz),4.60(d,2H,J＝5.2Hz),3.65(t,4H,J＝4.0Hz), 2.92(t,4H,J＝4.0Hz).13C NMR(101MHz,DMSO-d6)δ165.00,164.49,155.46, 151.17,143.03,142.99,142.70,135.32,131.95,128.75,128.11,127.71,127.49, 121.84,119.05,65.75,46.37,43.06.HRMS(ESI)calcd for C24H24N4NaO6S[M+Na]+: 519.1309,Found:519.1307.
EXAMPLE 36 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (3-chloro-4-methoxyphenyl) isonicotinamide (I-12)
Figure BDA0003151843370000193
The procedure of example 25 was followed, replacing 4a with 4l (410.9mg,1.0mmol) to give compound I-12(264.4 mg) as a yellow solid in 64.2% yield and Mp:177-178 ℃.1H NMR(400MHz,DMSO-d6)11.22(s, 1H),9.54(t,1H,J＝6.0Hz),9.01(s,1H),8.79(d,1H,J＝5.2Hz),8.38(s, 1H),8.23(d,1H,J＝2.4Hz),8.15(dd,1H,J1＝2.0Hz,J2＝8.4Hz),7.75- 7.72(m,3H),7.43(d,2H,J＝8.0Hz),7.31(d,1H,J＝8.8Hz),4.58(d,2H, J＝6.0Hz),3.94(s,3H).13C NMR(101MHz,DMSO-d6)δ165.14,164.36,155.97, 155.60,150.74,142.75,142.71,131.99,128.36,127.67,127.46,127.19,122.06, 120.56,117.36,113.43,56.77,43.01.HRMS(ESI)calcd for C21H17ClN3O4[M-H]-: 410.0913,Found:410.0914.
EXAMPLE 37 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (3, 4-dimethoxyphenyl) isonicotinamide (I-13)
Figure BDA0003151843370000201
The procedure of example 25 was followed, replacing 4a with 4m (406.4mg,1.0mmol) to give compound I-13(306.8 mg) as a yellow solid in 75.3% yield, Mp:210 and 211 ℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.49(t,1H,J＝5.2Hz),9.06(s,1H),8.78(d,1H,J＝4.8Hz), 8.31(s,1H),7.76-7.69(m,5H),7.43(d,2H,J＝8.0Hz),7.10(d,1H,J＝8.4 Hz),4.59(d,2H,J＝5.2Hz),3.87(s,3H),3.83(s,3H).13C NMR(101MHz, DMSO-d6)δ165.17,164.75,164.50,155.14,150.87,146.11,142.75,137.88, 131.93,128.11,127.69,127.47,120.51,117.56,111.09,53.96,43.02.HRMS(ESI) calcd for C22H21N3NaO5[M+Na]+:430.1373,Found:430.1373.
EXAMPLE 38 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (2, 4-dimethylphenyl) isonicotinamide (I-14)
Figure BDA0003151843370000202
The procedure of example 25 was followed, replacing 4a with 4n (374.4mg,1.0mmol) to give Compound I-14(82.9 mg) as a white solid in 22.0% yield and Mp:196-197 ℃.1H NMR(400MHz,DMSO-d6)δ11.20 (s,1H),9.43(t,1H,J＝6.0Hz),9.03(s,1H),8.79(d,1H,J＝5.2Hz),7.90 (s,1H),7.75(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.72(d,2H,J＝8.0Hz),7.40 (d,2H,J＝8.0Hz),7.35(d,1H,J＝8.0Hz),7.15-7.11(m,2H),4.54(d,2H, J＝6.0Hz),2.33(s,3H),2.31(s,3H).13C NMR(101MHz,DMSO-d6)δ165.22, 160.45,150.12,142.73,142.06,138.25,137.27,135.82,131.97,131.87,130.15, 127.65,127.42,127.01,121.60,119.79,43.00,21.19,20.58.HRMS(ESI)calcd for C22H20N3O3[M-H]:374.1510,Found:374.1509.
EXAMPLE 39 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (2-fluoro-4-methylphenyl) isonicotinamide (I-15)
Figure BDA0003151843370000211
The procedure of example 25 was followed, replacing 4a with 4o (378.4mg,1.0mmol) to give compound I-15(281.8 mg) as a white solid in 74.3% yield, Mp: 189-.1H NMR(400MHz,DMSO-d6)δ11.21 (s,1H),9.48(t,1H,J＝6.0Hz),9.09(s,1H),8.85(d,1H,J＝5.2Hz),8.18 (s,1H),7.87(t,1H,J＝8.0Hz),7.81(dd,1H,J1＝1.6Hz,J2＝5.2Hz),7.73 (d,2H,J＝8.0Hz),7.41(d,2H,J＝8.0Hz),7.23-7.17(m,2H),4.56(d,2H, J＝5.6Hz),2.39(s,3H).13C NMR(101MHz,DMSO-d6)δ165.12,164.43,160.18 (d,JC-F＝249.4Hz),153.92(d,JC-F＝2.4Hz),150.85,142.73,142.36,142.24(d, JC-F＝8.6Hz),131.96,131.05(d,JC-F＝3.2Hz),127.66,127.44,126.05(d,JC-F＝ 2.8Hz),124.09(d,JC-F＝11.4Hz),121.79(d,JC-F＝8.7Hz),120.51,117.12(d, JC-F＝21.8Hz),43.03,21.11.HRMS(ESI)calcd for C21H17FN3O3[M-H]-:378.1259, Found:378.1257.
EXAMPLE 40 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (3-chloro-4-morpholinophenyl) isonicotinamide (I-16)
Figure BDA0003151843370000212
The procedure of example 25 was followed using, instead of 4a, 4p (465.9mg,1.0mmol) to give compound I-16 (318.4mg) as a yellow solid in 68.2% yield and Mp:185-186 ℃.1H NMR(400MHz,DMSO-d6)) δ11.20(s,1H),9.51(t,2H,J＝5.2Hz),8.80(d,1H,J＝5.2Hz),8.37(s, 1H),8.21(d,1H,J＝1.6Hz),8.11(dd,1H,J1＝1.6Hz,J2＝8.4Hz),7.76- 7.73(m,3H),7.43(d,2H,J＝8.4Hz),7.29(d,1H,J＝8.4Hz),4.59(d,2H, J＝5.2Hz),3.77(t,4H,J＝4.4Hz),3.07(t,4H,J＝4.4Hz).13C NMR(101 MHz,DMSO-d6)δ165.13,164.37,155.59,150.80,150.10,142.74,142.68, 134.20,132.02,128.90,128.21,127.66,127.44,126.79,121.35,120.71, 117.55,66.73,51.58,43.03.HRMS(ESI)calcd for C24H22ClN4O4 -[M-H]:465.1335, Found:465.1335.
EXAMPLE 41N- (4- (Hydroxyaminocarboxamide) benzyl) -2- (3-amino-2-methylphenyl) isonicotinamide (I-17)
Figure BDA0003151843370000221
Following the procedure of example 25, substituting 4q (375.4mg,1.0mmol) for 4a, Compound I-17(335.0 mg) was obtained as a yellow solidThe solid content, yield 89.0%, Mp 143-144 ℃.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.44(t,1H,J＝6.0Hz),9.04(s,1H),8.79(d,1H,J＝4.0Hz),7.84 (s,1H),7.76-7.71(m,3H),7.40(d,2H,J＝7.2Hz),7.00(t,1H,J＝6.8Hz), 6.73(d,1H,J＝7.6Hz),6.60(d,1H,J＝7.2Hz),5.02(brs,2H),4.54(d,2H, J＝5.6Hz),1.98(s,3H).13C NMR(101MHz,DMSO-d6)δ165.23,164.53,161.60, 150.00,147.67,142.83,141.78,141.00,131.89,127.66,127.46,126.22,121.83, 119.70,119.26,118.28,114.70,42.99,14.66.HRMS(ESI)calcd for C21H21N4O3[M +H]+:377.1608,Found:377.1608.
EXAMPLE 42 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -6 '-methoxy- [2,3' -bipyridine ] -4-carboxamide (I-18)
Figure BDA0003151843370000222
The procedure of example 25 was followed, replacing 4a with 4r (377.4mg,1.0mmol) to give compound I-21(312.9 mg) as a white solid in 82.7% yield, Mp:196-197 ℃.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.47(t,1H,J＝5.6Hz),9.06(s,1H),8.95(d,1H,J＝2.0Hz),8.81 (d,1H,J＝4.8Hz),8.43(dd,1H,J1＝2.4Hz,J2＝8.8Hz),8.36(s,1H),7.76- 7.74(m,3H),7.44(d,2H,J＝8.0Hz),6.98(d,1H,J＝8.8Hz),4.59(d,2H, J＝4.6Hz),3.93(s,3H).13C NMR(101MHz,DMSO-d6)δ165.17,164.75,164.50, 155.14,150.87,146.11,142.75,137.88,131.93,128.11,127.69,127.47,120.51, 117.56,111.09,53.96,43.02.HRMS(ESI)calcd for C20H17N4O4[M-H]-:377.1255, Found:377.1257.
EXAMPLE 435 Synthesis of hydroxy-N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4-methylphenyl) isonicotinamide (I-19)
Figure BDA0003151843370000223
Pressing to realThe procedure of example 25 was used, replacing 4a with 4s (378.4mg,1.0mmol), to give compound I-19(118.0 mg) as a yellow solid in 31.1% yield and Mp: 235-.1H NMR(400MHz,DMSO-d6)δ12.11 (s,1H),11.21(s,1H),9.69(t,1H,J＝6.0Hz),9.03(s,1H),8.41(s,1H), 8.29(s,1H),7.92(d,2H,J＝8.0Hz),7.74(d,2H,J＝8.0Hz),7.44(d,2H, J＝8.4Hz),7.28(d,2H,J＝8.0Hz),4.61(d,2H,J＝6.0Hz),2.35(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.38,164.49,153.44,147.66,142.30,140.87, 138.08,135.90,132.03,129.78,127.70,127.53,126.11,123.45,117.35,42.79, 21.23.HRMS(ESI)calcd for C21H18N3O4[M-H]-:376.1303,Found:376.1303.
EXAMPLE 445 Synthesis of hydroxy-N- (4-hydroxyaminocarboxamide) benzyl) -2- (4-methoxyphenyl) isonicotinamide (I-20)
Figure BDA0003151843370000231
The procedure of example 25 was followed, replacing 4a with 4t (394.4mg,1.0mmol) to give compound I-20(186.6 mg) as a yellow solid in 47.2% yield, Mp: 224-.1H NMR(400MHz,DMSO-d6)δ12.02 (s,1H),11.23(s,1H),9.64(t,1H,J＝4.8Hz),9.05(s,1H),8.41(s,1H), 8.26(s,1H),7.97(d,2H,J＝8.8Hz),7.75(d,2H,J＝8.4Hz),7.45(d,2H, J＝8.0Hz),7.04(d,2H,J＝8.8Hz),4.62(d,2H,J＝5.6Hz),3.81(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.38,164.50,159.95,153.04,147.59,142.33, 140.73,132.01,131.24,127.70,127.53,123.55,116.93,114.54,55.65,42.78. HRMS(ESI)calcd for C21H18N3O5[M-H]-:392.1252,Found:392.1251.
EXAMPLE 45 Synthesis of N- (4- (hydroxyaminocarboxamide) benzyl) -2- (4- (2-morpholinoethoxy) phenyl) isonicotinamide (I-21)
Figure BDA0003151843370000232
According to example 25 by replacing 4a with 5a (475.5mg,1.0mmol), Compound I-21(401.2 mg) was obtained as a white solid in 84.2% yield, Mp: 110-.1H NMR(400MHz,DMSO-d6)δ11.22 (s,1H),9.48(t,1H,J＝6.0Hz),9.05(s,1H),8.70(d,1H,J＝4.8Hz),8.31 (s,1H),8.11(d,2H,J＝8.4Hz),7.74(d,2H,J＝7.6Hz),7.69(d,1H,J＝ 4.4Hz),7.43(d,2H,J＝8.0Hz),7.09(d,2H,J＝8.4Hz),4.58(d,2H,J＝ 4.8Hz),4.16(t,2H,J＝5.6Hz),3.59(t,4H,J＝4.8Hz),3.38(brs,4H), 2.72(t,2H,J＝5.6Hz).13C NMR(101MHz,DMSO-d6)δ165.31,164.52,160.13, 157.02,150.63,142.82,142.55,131.91,131.10,128.54,127.67,127.48,119.87, 117.08,115.21,66.65,65.89,57.47,54.11,43.00.HRMS(ESI)calcd for C26H27N4O5[M-H]-:475.1987,Found:475.1990.
EXAMPLE 46 Synthesis of tert-butyl 4- ((4- (4- ((4- (hydroxycarbamimidoyl) benzyl) carbamoyl) -2-pyridinyl) phenoxy) methyl) piperidine-1-carboxylic acid ester (I-22)
Figure BDA0003151843370000241
The procedure of example 25 was followed, replacing 4a with 5b (559.7mg,1.0mmol) to give compound I-22(455.8 mg) as a white solid in 81.3% yield and Mp: 122-.1H NMR(400MHz,DMSO-d6)δ11.23 (s,1H),9.52(t,1H,J＝6.0Hz),9.06(s,1H),8.76(d,1H,J＝4.0Hz),8.32 (s,1H),8.11(d,2H,J＝8.0Hz),7.75(d,2H,J＝7.6Hz),7.70(d,1H,J＝ 4.8Hz),7.43(d,2H,J＝7.6Hz),7.07(d,2H,J＝8.0Hz),4.58(d,2H,J＝ 5.6Hz),4.00-3.97(m,2H),3.91(d,2H,J＝5.2Hz),2.81-2.68(m,2H),1.97- 1.91(m,1H),1.77(d,2H,J＝12.4Hz),1.41(s,9H),1.22-1.10(m,2H).13C NMR(101MHz,DMSO-d6)δ165.30,164.52,160.33,157.02,154.36,150.62, 142.83,142.54,131.90,131.06,128.54,127.68,127.48,119.86,117.08,115.16, 78.99,72.27,43.00,35.83,28.77,28.57.HRMS(ESI)calcd for C31H35N4O6[M-H]-: 559.2562,Found:559.2561.
Evaluation of inhibitory Activity of LSD1, HDAC1 and THP-1 of the synthesized Compound of the present invention (I) evaluation of inhibitory Activity of LSD1, HDAC1 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, LSD1 reaction substrate H3K4me2 is added for incubation reaction, and finally fluorescent dye Amplex and horseradish peroxidase HRP are added for incubation at room temperature, excitation light is 530nm on an enzyme labeling instrument, and emission light is 590nm to detect a fluorescence value.
Figure BDA0003151843370000242
Test results the IC was calculated using SPSS software50The value is obtained.
2. HDAC1 inhibitory activity evaluation test method
3-5mg of the sample was weighed into a 1.5mL EP tube and then prepared into a 10mM 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 1X buffer 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. In vitro anti-tumor Activity assay
The samples were the compounds synthesized in the examples; sample stock solution: weighing 3-5mg of sample, placing the sample in a 1.5mL EP tube, preparing the sample into a 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 growth 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 2min by oscillation, after the incubation is continued for 10min 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%.
4. Results of the experiment
TABLE 1 results of evaluation of LSD1, HDAC1 and THP-1 inhibitory Activity
Figure BDA0003151843370000251
Figure BDA0003151843370000261
aN.D. not determined.
bThe inhibition at 30nM concentration was 83%.
As can be seen from the experimental results in the table above, the compounds of the present invention have good inhibitory activity, IC, on both LSD1 and HDAC150Values are at nanomolar to micromolar levels. IC of most Compounds on HDAC150Less than 10nM, superior to the positive control SAHA, particularly the IC of compounds I-2, I-8, I-10, I-16 and I-21 for HDAC150Less than 2nM, 7 times higher than the SAHA of the positive drug. In vitro antitumor activity evaluation shows that a plurality of compounds have good inhibitory activity on THP-1 leukemia cells, wherein the IC of the compounds I-5, I-8 and I-15 on THP-150Less than 1 μ M, significantly better than SAHA. In particular, compounds I-5 and I-8 were 12-fold and 18-fold more active on THP-1 than SAHA, respectively.
The THP-1 inhibitory activity of the compounds I-5, I-8 and I-15 is obviously better than that of a positive control SAHA, and the in vitro anti-tumor activity of the compounds I-5, I-8 and I-15 is further evaluated on two leukemia cell strains (MOLT-4, MV4: 11).
TABLE 2 evaluation results of in vitro antitumor Activity of Compounds I-5 and I-8
Figure BDA0003151843370000262
The experimental results show that the compounds I-5, I-8 and I-15 have good inhibitory activity on MOLT-4 and MV4:11 leukemia cell strains and IC50The values are all less than 0.5. mu.M. The compound represents a LSD1/HDAC double-target inhibitor with a brand-new structure, a plurality of compounds have good in-vitro anti-tumor activity, can be used as a candidate or lead compound for further development, can be 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.
(II) evaluation of recombinant protein level MAO-A/B inhibitory Activity
1. The experimental method comprises the following steps: 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. Clorgyline and R- (-) -deprenyl were used as positive control drugs for MAO-A and MAO-B activity evaluation, respectively. 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.
2. The experimental results are as follows:
TABLE 3 evaluation of MAO-A/B inhibitory Activity of Compounds I-5 and I-8
Figure BDA0003151843370000271
aN.D. not detecting
The experimental result shows that the compounds I-5 and I-8 have no inhibitory activity on homologous proteins MAO-A and MAO-B of LSD1 and have good selectivity on LSD 1.

Claims (5)

1. A2-aryl isonicotinic acid amide compound is characterized by having a structure shown in a general formula (I):
Figure FDA0003151843360000011
in the general formula I, R1Is any one of OH and H; r2Is OH, OCH3、F、H、CH3Any one of the above; r3Is H, OH, Cl, OCH3、NH2、
Figure FDA0003151843360000012
Any one of the above; r4Is H, OH, CF3、OCH3、CH3、
Figure FDA0003151843360000013
Any one of the above; x is N or CH.
2. The 2-arylisonicotinic acid amide compound of claim 1, selected from one of the following compounds:
Figure FDA0003151843360000014
Figure FDA0003151843360000021
3. a process for the preparation of a class of 2-arylisonicotinic acid amides of claim 1 or 2, characterized in that it is carried out by:
Figure FDA0003151843360000022
(1) a method for preparing compound 3: stirring a compound 1 and 4-aminomethyl methyl benzoate hydrochloride (a compound 2) in N, N-Dimethylformamide (DMF) under the existence of O-benzotriazole-4-methyl urea Hexafluorophosphate (HBTU) and an alkaline compound at room temperature for reaction, and after the reaction is finished, extracting, washing, drying, filtering, concentrating and separating by column chromatography to obtain a compound 3; wherein the strong basic compound is selected from one of N, N-diisopropylethylamine and triethylamine;
(2) a method for preparing compound 4: heating and stirring the compound 3 and substituted phenylboronic acid or substituted phenylboronic acid pinacol ester or substituted pyridine boric acid 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 and separating by column chromatography to obtain a compound 4; 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;
(3) a method for preparing compound 5: in the compound 4, when R4When the hydroxyl group is adopted, heating and stirring the compound 4 and 4- (bromomethyl) piperidine-1-tert-butyl formate or 4- (2-bromoethyl) morpholine hydrobromide in DMF in the presence of an alkaline compound 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 5; wherein the alkaline compound is selected from one of potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide;
(4) a process for the preparation of compound I: in dichloromethane solution, Compound 4 or Compound 5 and NH2OK/NH2And (3) reacting with an OH methanol solution, and after the reaction is finished, concentrating, filtering, washing and recrystallizing a reaction system to obtain a compound I.
4. Use of a class of 2-arylisonicotinic acid amides according to any of claims 1-2 for the preparation of a medicament, as active ingredient for the preparation of a medicament of the LSD1/HDAC dual target inhibitor class.
5. The application of the 2-aryl isonicotinic acid amide compounds in the preparation of the medicaments as claimed in claim 4, characterized in that the 2-aryl isonicotinic acid amide compounds are used as active ingredients for preparing the medicaments for treating acute myeloid leukemia.
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