CN110407824B - Aryl formamide compound and preparation method, pharmaceutical composition and application thereof - Google Patents

Aryl formamide compound and preparation method, pharmaceutical composition and application thereof Download PDF

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CN110407824B
CN110407824B CN201910730990.7A CN201910730990A CN110407824B CN 110407824 B CN110407824 B CN 110407824B CN 201910730990 A CN201910730990 A CN 201910730990A CN 110407824 B CN110407824 B CN 110407824B
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刘明明
王洋
刘新华
梁玉茹
朱仲珍
李�荣
石静波
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Anhui Medical University
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Abstract

The invention relates to the field of medicinal chemistry and pharmacotherapeutics, and more particularly discloses aryl formamide compounds and a preparation method thereof, and also relates to application of the compounds in preparation of medicaments for preventing or treating diseases caused by upregulation of hypoxia inducible factors, application in preparation of medicaments for preventing or treating anti-tumor or anti-retinal vascular diseases, and a pharmaceutical composition containing the compounds.

Description

Aryl formamide compound and preparation method, pharmaceutical composition and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to an aryl formamide compound, and a preparation method, a pharmaceutical composition and application thereof.
Background
Tumor metastasis is the leading cause of clinical tumor treatment failure and patient death, and about 90% of cancer patients do not die from the primary tumor, but rather the metastasis of the tumor. Therefore, inhibiting tumor metastasis is a serious challenge for cancer therapy. Aiming at key targets in the process of tumor cell invasion and metastasis, research and development of effective tumor metastasis inhibiting drugs become a new direction for research and development of antitumor drugs, and have very important theoretical research significance and clinical application prospect.
Diseases associated with the pathological growth of retinal vessels, including retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, retinal vein occlusion, hypertensive retinopathy, retinal artery occlusion, and the like, are the main causes of vision loss.
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is ubiquitous in human and mammalian cells and consists of two subunits, HIF-1. alpha. and HIF-1. beta. HIF-1 β is in aerobic Environment (21% O)2) And hypoxia, HIF-1 alpha is used as active subunit, and its stability is regulated by oxygen. Under an normoxic environment, HIF-1 alpha protein is quickly degraded by an intracellular oxygen-dependent ubiquitin protease degradation pathway and can be stably expressed only under an anoxic condition. The alpha and beta subunits of HIF-1 form heterodimers, enter the nucleus, bind to Hypoxia Response Elements (HREs) of downstream target gene promoters, and activate gene transcription.
The target genes downstream of HIF-1 are quite extensive and involved in biological processes including angiogenesis and the ability of malignant cells to acquire migratory invasion (epithelial-mesenchymal transition, EMT). Malignant tumor grows vigorously and is in an anoxic state, HIF-1 alpha can be activated, the adhesion capability of tumor cells is reduced, the migration and movement capability is increased, the occurrence of tumor EMT is promoted, and finally tumor metastasis is realized. Progressive damage to the retinal microvascular network leads to tissue ischemia, HIF-1 upregulation, stimulation of Vascular Endothelial Growth Factor (VEGF) secretion, and ultimately to pathological retinal vascular growth and various related diseases.
Therefore, HIF-1 can be used as the drug action target for treating malignant tumor, especially metastatic malignant tumor, and diseases related to the pathological growth of retinal blood vessels. HIF-1 inhibitor can inhibit VEGF level, inhibit new blood vessel growth, inhibit cell invasion and migration ability, thus has effects of preventing and treating malignant tumor, especially metastatic malignant tumor, and retina blood vessel pathological growth related diseases. At present, clinical antitumor drugs are mainly cytotoxic drugs and inhibit cell growth, and the market lacks drugs which take HIF-1 as a drug action target to inhibit tumor metastasis so as to prevent and treat metastatic malignant tumors and diseases related to pathological growth of retinal blood vessels.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the market lacks a medicine which takes HIF-1 as a medicine action target to inhibit tumor metastasis so as to prevent and treat metastatic malignant tumor and diseases related to pathological growth of retinal blood vessels, and provides an aryl formamide compound, a preparation method, a pharmaceutical composition and application thereof.
The invention solves the technical problems through the following technical scheme, and the aryl formamide compound shown as the following general formula (I) or pharmaceutically acceptable salt thereof is prepared by the following steps:
Figure BDA0002160574090000021
wherein:
Ar1selected from the following aromatic rings or aromatic ring systems:
Figure BDA0002160574090000022
Ar2selected from any one of phenyl, substituted phenyl, pyridine-3-yl and pyridine-4-yl.
The substituted position of the substituted benzene is selected from any one of 2-position, 3-position and 4-position of the benzene ring, and the number of the substituted groups is 1;
the substituents at the 2-position and the 3-position are selected from fluorine or chlorine, and the substituent at the 4-position is selected from any one of fluorine, chlorine, hydroxyl, amino, methyl and nitro.
Ar is1Selected from any one of the groups represented by the following general formula (II):
Figure BDA0002160574090000031
wherein:
Ar3any one selected from furan-2-yl, furan-3-yl, thiophen-2-yl, thiophen-3-yl, pyridin-4-yl, 1H-pyrazol-4-yl, and substituted phenyl;
the substituent of the substituted benzene is selected from any one of fluorine, hydroxyl, methyl and cyano, and the number of the substituents is 1.
The aryl formamide compound shown in the general formula (I) or pharmaceutically acceptable salt thereof has a structure shown in the following general formula (III):
Figure BDA0002160574090000032
the aryl formamide compound shown in the general formula (I) or pharmaceutically acceptable salt thereof is selected from any one of the following compounds:
Figure BDA0002160574090000033
the aryl formamide compound shown in the general formula (I) or pharmaceutically acceptable salt thereof is applied to preparation of medicines for preventing or treating diseases caused by the upregulation of hypoxia inducible factor-1.
The disease comprises at least one of breast cancer, ovarian cancer, lung cancer, liver cancer, cervical cancer, gastric cancer and intestinal cancer.
The aryl formamide compound shown in the general formula (I) or pharmaceutically acceptable salt thereof is applied to preparation of medicines for preventing or treating pathological growth diseases of retinal blood vessels.
A pharmaceutical composition comprises one or more aryl formamide compounds shown in the general formula (I) or pharmaceutically acceptable salts thereof in a therapeutically effective amount, and pharmaceutically acceptable auxiliary materials.
The retinal vascular pathological growth related diseases comprise at least one of vascular retinopathy, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, retinopathy of prematurity, retinal vein and artery obstruction.
The positive progress effect of the invention is to provide and prove that the aryl formamide compound or the pharmaceutically acceptable salt thereof has a better inhibiting effect on an HIF-1 signal path under an anoxic condition in an in vitro experiment, can inhibit the accumulation of HIF-1 alpha under the anoxic condition, can obviously inhibit the migration and invasion of tumor cells and the tumor metastasis in a nude mouse, and can inhibit the generation of hypoxia-induced VEGF, the formation of an in vitro Human Umbilical Vein Endothelial Cell (HUVEC) vascular network and the formation of zebra fish microvasculature. The pharmacological experiments show that the aryl formamide compounds or the pharmaceutically acceptable salts thereof have good prevention or treatment effects when being applied to the preparation of the medicines for preventing or treating the anti-tumor or anti-retinal vascular diseases.
The novelty of the invention lies in that the chemical structure of the aryl formamide compound disclosed by the invention is different from that of the existing HIF-1 inhibitor, and the aryl formamide compound is a HIF-1 inhibitor with a brand-new structure. In addition, compared with the recognized HIF-1 inhibitor LW6, the representative compounds disclosed by the invention have stronger inhibitory effect and have good anti-angiogenesis and tumor metastasis effects in vivo and in vitro.
Drawings
FIG. 1 is a graph of the inhibition of hypoxia-induced HIF-1 α aggregation by arylcarboxamides of examples 40, 44 and 51;
FIG. 2 is the inhibitory effect of the compound of example 51 on VEGF mRNA;
FIG. 3 is the effect of the compound of example 51 on the formation of HUVEC capillary-like microtubules in vitro;
FIG. 4 is the effect of the compound of example 51 on the neovascularization of zebrafish;
FIG. 5 is a scratch test to examine the effect of the compound of example 51 on the ability of breast cancer cells to migrate;
FIG. 6 is the effect of the compound of example 51 on breast cancer cell invasion and migration;
FIG. 7 is a graph of the reduction of metastasis formation in the lung of nude mice by the compound of example 51;
FIG. 8 is a lung histological section of nude mice showing that the compound of example 51 reduces metastasis formed by breast cancer cells.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
EXAMPLE 1 Synthesis of N- (2-chlorophenylethyl) -2-methyl-thiazole-4-carboxamide
The reaction formula is as follows:
the synthesis steps are as follows:
(1) dissolving 2-methylthiazole-4-carboxylic acid (1mmol) in 5mL of dichloromethane solution at the temperature of 0 ℃, slowly dropwise adding oxalyl chloride (0.17mL) and N, N-dimethylformamide (1 drop), reacting at room temperature for 1 hour after the completion of the addition, and removing the solvent by reduced pressure distillation after the reaction to obtain residue;
(2) the residue was dissolved in 2mL of dichloromethane, and a mixed solution of 2- (2-chlorophenyl) ethylamine (1mmol), dichloromethane (5mL) and triethylamine (0.16mL) was added thereto, followed by stirring at room temperature for 24 hours. After confirming completion of the reaction by TLC analysis, the solvent was removed by distillation under the reduced pressure, and the residue was purified by flash column chromatography to give the objective compound as a colorless oil in 84.3% yield.
1HNMR(600MHz，CDCl3)δ7.94(s，1H)，7.42(brs，1H)，7.36(dd，J＝7.4，1.4Hz，1H)，7.28-7.26(m，1H)，7.20-7.17(m，2H)，3.70(q，J＝7.4Hz，2H)，3.06(t，J＝7.4Hz，2H)，2.68(s，3H)；13CNMR(151MHz，CDCl3)δ165.85，161.17，149.70，136.52，134.15，130.92，129.57，127.97，126.92，122.77，38.97，33.66，19.04；ESI-HRMS(m/z)calcdC13H13ClN2OS(M+H+)281.0510，found 281.0512。
EXAMPLE 2 Synthesis of N- (2-chlorophenylethyl) -5-methyl-furan-2-carboxamide
The same procedure as in example 1 was repeated except that 5-methylfuran-2-carboxylic acid was used as a starting material.
The compound was prepared as a colorless oil in 67.0% yield.
1HNMR(600MHz，CDCl3)δ7.37(dd，J＝7.5，1.5Hz，1H)，7.28-7.26(m，1H)，7.22-7.17(m，2H)，6.99(d，J＝3.3Hz，1H)，6.37(brs，1H)，6.08(d，J＝3.3Hz，1H)，3.68(td，J＝7.0Hz，2H)，3.05(t，J＝7.0Hz，2H)，2.32(s，3H)；13CNMR(151MHz，CDCl3)δ158.59，154.32，146.38，136.51，134.13，131.00，129.59，128.02，126.96，115.28，108.43，38.77，33.62，13.74；ESI-HRMS(m/z)calcdC14H14ClNO2(M+H+)264.0786，found 264.0789。
EXAMPLE 3 Synthesis of N- (2-Chloroethyl) -2- (pyridin-3-yl) -thiazole-4-carboxamide
The procedure of example 1 was otherwise the same as for the selection of 2- (pyridin-3-yl) -thiazole-4-carboxylic acid as the starting material.
The compound was prepared as a white solid in 74.1% yield.
1HNMR(600MHz，CDCl3)δ9.16(s，1H)，8.70(d，J＝5.4Hz，1H)，8.19(d，J＝7.8Hz，1H)，8.15(s，1H)，7.42-7.38(m，2H)，7.30(d J＝7.8Hz，1H)，7.24-7.19(m，2H)，3.77(t，J＝7.0Hz，2H)，3.12(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ164.08，160.22，150.72，150.65，147.08，135.84，133.59，133.11，130.43，129.08，128.26，127.55，126.39，123.16，122.88，38.54，32.90；ESI-HRMS(m/z)calcd C17H14ClN3OS(M+H+)344.0619，found 344.0618。
EXAMPLE 4 Synthesis of N- (2-Chloroethyl) -2- (pyridin-4-yl) -thiazole-4-carboxamide
The procedure of example 1 was otherwise the same as for the selection of 2- (pyridin-4-yl) -thiazole-4-carboxylic acid as the starting material.
The compound was prepared as a white solid in 75.6% yield.
1H NMR(600MHz，CDCl3)δ8.74(dd，J＝4.6，1.5Hz，2H)，8.21(s，1H)，7.78(dd，J＝4.6，1.5Hz，2H)，7.39(dd，J＝7.3，1.7Hz，1H)，7.30(dd，J＝7.1，1.9Hz，1H)，7.24-7.19(m，2H)，3.76(t，J＝7.2Hz，2H)，3.12(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.54，160.02，150.90，150.19，138.89，135.82，133.59，130.43，129.08，127.55，126.40，123.80，119.68，38.46，32.88；ESI-HRMS(m/z)calcd C17H14ClN3OS(M+H+)344.0619，found 344.0619。
EXAMPLE 5 Synthesis of N- (2-chlorophenylethyl) -5-phenyl-1, 3, 4-oxadiazole-2-carboxamide
The same procedure as in example 1 was repeated except that 5-phenyl-1, 3, 4-oxadiazole-2-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 65.7% yield.
1H NMR(600MHz，CDCl3)δ8.15(d，J＝8.3Hz，2H)，7.59(t，J＝8.3Hz，1H)，7.53(t，J＝8.3Hz，2H)，7.38-7.37(m，1H)，7.34(brs，1H)，7.27-7.26(m，1H)，7.23-7.19(m，2H)，3.80(q，J＝7.0Hz，2H)，3.12(t，J＝7.0Hz，2H)；13C NMR(151MHz，DMSO-d6)δ165.41，158.90，153.48，136.83，133.65，133.12，131.59，130.01，129.76，128.83，127.80，127.51，123.26，39.33，32.88；ESI-HRMS(m/z)calcd C17H14ClN3O2(M+H+)328.0847；found 328.0846。
EXAMPLE 6 Synthesis of N- (2-chlorophenylethyl) -2-phenyl-oxazole-4-carboxamide
The other embodiments are the same as in example 1, using 2-phenyl-oxazole-4-carboxylic acid as a starting material.
The compound was prepared as a white solid in 60.1% yield.
1H NMR(600MHz，CDCl3)δ8.23(s，1H)，8.03-8.02(m，2H)，7.50-7.47(m，3H)，7.39(dd，J＝7.5，1.5Hz，1H)，7.30(dd，J＝7.3，1.7Hz，1H)，7.24-7.18(m，2H)，7.16(brs，1H)，3.73(q，J＝7.2Hz，2H)，3.10(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ161.37，160.74，140.69，137.26，136.41，134.17，131.00，130.98，129.62，128.87，128.07，126.97，126.63，126.59，38.77，33.58；ESI-HRMS(m/z)calcd C18H15ClN2O2(M+H+)349.0714；found 349.0702。
EXAMPLE 7 Synthesis of N- (2-chlorophenylethyl) -5-phenyl-oxazole-2-carboxamide
The same procedure as in example 1 was repeated except for using 5-phenyl-oxazole-2-carboxylic acid as a starting material.
The compound was prepared as a white solid in 63.5% yield.
1H NMR(600MHz，CDCl3)δ7.76-7.74(m，2H)，7.46-7.43(m，2H)，7.40-7.37(m，3H)，7.28-7.27(m，1H)，7.23-7.18(m，2H)，7.14(brs，1H)，3.75(q，J＝6.8Hz，2H)，3.09(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ155.27，154.21，153.95，136.09，134.13，130.95，129.68，129.48，128.97，128.21，127.04，126.88，124.93，122.54，39.26，33.34；ESI-HRMS(m/z)calcd C18H15ClN2O2(M+H+)349.0714；found 349.0716。
EXAMPLE 8 Synthesis of N- (2-chlorophenylethyl) -5-phenyl-1, 2, 4-oxadiazole-3-carboxamide
The same procedure as in example 1 was repeated except for using 5-phenyl-1, 2, 4-oxadiazole-3-carboxylic acid as a starting material.
The compound was prepared as a white solid in 69.4% yield.
1H NMR(400MHz，CDCl3)δ8.57(d，J＝6.0Hz，2H)，8.17-8.15(m，2H)，7.67-7.63(m，1H)，7.58-7.54(m，2H)，7.21(d，J＝6.0Hz，2H)，7.12(brs，1H)，3.81(q，J＝7.0Hz，2H)，3.00(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ176.20，163.28，155.86，135.36，133.54，132.84，130.40，129.13，128.64，127.73，127.70，126.51，122.75，38.78，32.56；ESI-HRMS(m/z)calcd C17H14ClN3O2(M+H+)350.0667；found 350.0655。
EXAMPLE 9 Synthesis of N- (2-chlorophenylethyl) -3-phenyl-1, 2, 4-oxadiazole-5-carboxamide
The same procedure as in example 1 was repeated except that 3-phenyl-1, 2, 4-oxadiazole-5-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 75.6% yield.
1H NMR(600MHz，CDCl3)δ8.09-8.08(m，2H)，7.56-7.54(m，1H)，7.52-7.49(m，2H)，7.41-7.39(m，1H)，7.29-7.26(m，1H)，7.25-7.22(m，3H)，3.80(q，J＝7.1Hz，2H)，3.13(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ168.68，168.56，153.11，135.66，134.13，131.74，130.95，129.80，128.97，128.44，127.53，127.18，125.78，39.71，33.07；ESI-HRMS(m/z)calcd C17H14ClN3O2(M+H+)350.0667；found 350.0664。
EXAMPLE 10 Synthesis of N- (2-chlorophenylethyl) -biphenyl-4-carboxamide
The same procedure as in example 1 was repeated except that biphenyl-4-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 73.7% yield.
1H NMR(600MHz，CDCl3)δ7.79(d，J＝8.4Hz，2H)，7.64(d，J＝8.4Hz，2H)，7.61-7.59(m，2H)，7.47-7.44(m，2H)，7.40-7.37(m，2H)，7.29(dd，J＝7.3，1.9Hz，1H)，7.24-7.18(m，2H)，6.29(brs，1H)，3.77(q，J＝6.8Hz，2H)，3.12(t，J＝6.8Hz，2H)；13C NMR(151MHz，CDCl3)δ166.64，143.62，139.39，136.05，133.52，132.60，130.51，129.08，128.28，127.53，127.35，126.75，126.60，126.56，126.47，39.20，32.72；ESI-HRMS(m/z)calcd C21H18ClNO(M+H+)336.1150；found 336.1151。
EXAMPLE 11 Synthesis of N- (2-chlorophenylethyl) -6-phenylnicotinamide
The same procedure as in example 1 was repeated except that 6-phenylbicotinic acid was used as a starting material.
The compound was prepared as a white solid in 71.6% yield.
1H NMR(600MHz，CDCl3)δ8.95(s，1H)，8.14(d，J＝8.3Hz，1H)，8.02(d，J＝7.6Hz，2H)，7.81(d，J＝8.3Hz，1H)，7.50(t，J＝7.6Hz，2H)，7.47-7.46(m，1H)，7.40(d，J＝7.7Hz，1H)，7.29(d，J＝7.3Hz，1H)，7.25-7.19(m，2H)，6.24(brs，1H)，3.80(q，J＝6.8Hz，2H)，3.13(t，J＝6.8Hz，2H)；13C NMR(151MHz，CDCl3)δ165.10，159.31，147.04，137.65，135.79，135.42，135.02，133.50，132.99，130.50，129.17，128.29，127.70，127.36，126.58，119.60，39.32，32.57；ESI-HRMS(m/z)calcd C20H17ClN2O(M+H+)337.1102；found 337.1102。
EXAMPLE 12 Synthesis of N- (2-chlorophenylethyl) -6-phenyl-pyrimidine-4-carboxamide
Synthesized in the same manner as in example 1.1 using 6-phenyl-pyrimidine-4-carboxylic acid as a starting material, and was a white solid with a yield of 71.2%.1H NMR(600MHz，CDCl3)δ9.20(d，J＝1.0Hz，1H)，8.54(d，J＝1.0Hz，1H)，8.20-8.18(m，2H)，8.15(brs，1H)，7.54-7.52(m，3H)，7.38(dd，J＝7.2，1.6Hz，1H)，7.28-7.27(m，1H)，7.23-7.18(m，2H)，3.79(q，J＝7.0Hz，2H)，3.12(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ165.77，162.48，157.27，155.99，149.43，146.95，135.36，131.04，128.52，126.80，123.46，113.33，39.12，34.45；ESI-HRMS(m/z)calcd C18H16N4O(M+H+)305.1397；found 305.1401。
EXAMPLE 13 Synthesis of N- (2-chlorophenylethyl) -5-phenyl-pyridine-2-carboxamide
The same procedure as in example 1 was repeated except for using 5-phenyl-pyridine-2-carboxylic acid as a starting material.
The compound was prepared as a white solid in 76.5% yield.
1H NMR(600MHz，CDCl3)δ8.75(d，J＝1.9Hz，1H)，8.28(d，J＝8.1Hz，1H)，8.22(brs，1H)，8.05(dd，J＝8.1，1.9Hz，1H)，7.62(d，J＝7.3Hz，2H)，7.51(t，J＝7.3Hz，2H)，7.45(t，J＝7.3Hz，1H)，7.38(dd，J＝7.5，1.6Hz，1H)，7.30(dd，J＝7.2，1.8Hz，1H)，7.22-7.17(m，2H)，3.78(q，J＝7.1Hz，2H)，3.12(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ160.38，144.61，142.64，135.09，133.06，132.65，131.53，130.25，127.01，125.67，125.24，124.70，124.05，123.28，122.99，118.23，35.18，29.72；ESI-HRMS(m/z)calcd C20H17ClN2O(M+H+)341.1260；found 341.1263。
EXAMPLE 14 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -2-methyl-thiazole-4-carboxamide
The reaction formula is as follows:
2-methylthiazole-4-carboxylic acid (1mmol) was dissolved in 5mL of dichloromethane at 0 ℃ and oxalyl chloride (0.17mL) and N, N-dimethylformamide (1 drop) were slowly added dropwise thereto, after completion of the addition, the reaction was carried out at room temperature for 1 hour, and after the completion of the reaction, the solvent was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 2mL of methylene chloride, and a mixed solution of 2- (pyridin-4-yl) ethylamine (1mmol) in methylene chloride (5mL) and triethylamine (0.16mL) was added thereto, followed by stirring at room temperature for 24 hours. After confirming completion of the reaction by TLC analysis, the solvent was removed by distillation under the reduced pressure, and the residue was purified by flash column chromatography to give the objective compound as a white solid in a yield of 77.3%.
1H NMR(600MHz，CDCl3)δ8.52(d，J＝5.8Hz，2H)，7.93(s，1H)，7.38(brs，1H)，7.17(d，J＝5.8Hz，2H)，3.71(q，J＝7.2Hz，2H)，2.93(t，J＝7.2Hz，2H)，2.67(s，3H)；13C NMR(151MHz，CDCl3)δ165.45，160.59，149.33，148.78，147.27，123.51，122.41，38.89，34.63，18.45；ESI-HRMS(m/z)calcd C12H13N3OS(M+H+)248.0852，found 248.0855。
EXAMPLE 15 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -5-methyl-furan-2-carboxamide
The same procedures as in example 14 were repeated except that 5-methylfuran-2-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 74.1% yield.
1H NMR(600MHz，CDCl3)δ8.51(d，J＝5.8，2H)，7.15(d，J＝5.8Hz，2H)，6.99(d，J＝3.3Hz，1H)，6.43(brs，1H)，6.07(d，J＝3.3，1H)，3.68(q，J＝7.0Hz，2H)，2.91(t，J＝7.0Hz，2H)，2.29(s，3H)；13C NMR(151MHz，CDCl3)δ158.00，153.94，149.31，147.32，145.50，123.56，114.99，107.95，38.68，34.67，13.18；ESI-HRMS(m/z)calcd C13H14N2O2(M+H+)231.1128，found 231.1128。
EXAMPLE 16 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -2- (pyridin-3-yl) -thiazole-4-carboxamide
The procedure of example 14 was otherwise the same as in the previous example, except that 2- (pyridin-3-yl) -thiazole-4-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 74.1% yield.
1H NMR(600MHz，CDCl3)δ9.13(d，J＝1.5Hz，1H)，8.68(dd，J＝4.8，1.5Hz，1H)，8.54(d，J＝6.0Hz，2H)，8.20-8.18(m，1H)，8.19(s，1H)，7.53(brs，1H)，7.40(dd，J＝8.0，4.8Hz，1H)，7.19(d，J＝6.0Hz，2H)，3.76(q，J＝7.2Hz，2H)，2.98(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.27，160.24，150.81，150.39，149.40，147.15，147.06，133.08，128.16，123.53，123.19，123.13，39.04，34.65；ESI-HRMS(m/z)calcd C16H14N4OS(M+H+)311.0961，found 311.0966。
EXAMPLE 17 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -2- (pyridin-4-yl) -thiazole-4-carboxamide
The procedure of example 14 was otherwise the same as in the previous example, except that 2- (pyridin-4-yl) -thiazole-4-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 75.6% yield.
1H NMR(600MHz，CDCl3)δ8.73(d，J＝6.0Hz，2H)，8.54(d，J＝5.8Hz，2H)，8.21(s，1H)，7.75(d，J＝6.0Hz，2H)，7.49(brs，1H)，7.19(d，J＝5.8Hz，2H)，3.77(q，J＝7.2Hz，2H)，2.98(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.74，160.09，150.65，150.23，149.41，147.11，138.76，124.03，123.54，119.64，39.05，34.65；ESI-HRMS(m/z)calcd C16H14N4OS(M+H+)311.0961，found 311.0965。
EXAMPLE 18 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -5-phenyl-1, 3, 4-oxadiazole-2-carboxamide
The same procedures as in example 14 were carried out except for using 5-phenyl-1, 3, 4-oxadiazole-2-carboxylic acid as a starting material.
The compound was prepared as a white solid in 65.7% yield.
1H NMR(600MHz，CDCl3)δ8.56(dd，J＝4.5，1.4Hz，2H)，8.16-8.14(m，2H)，7.61-7.59(m，1H)，7.56-7.53(m，2H)，7.28(brt，1H)，7.21(dd，J＝4.5，1.4Hz，2H)，3.81(q，J＝7.0Hz，2H)，2.99(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ166.60，158.28，153.41，150.10，147.03，132.69，129.20，127.49，124.00，122.79，40.01，34.84；ESI-HRMS(m/z)calcd C16H14N4O2(M+H+)317.1009；found 317.1014。
EXAMPLE 19 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -2-phenyl-oxazole-4-carboxamide
The same procedure as in example 14 was repeated except that 2-phenyl-oxazole-4-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 60.1% yield.
1H NMR(600MHz，CDCl3)δ8.56(d，J＝5.9Hz，2H)，8.24(s，1H)，8.03-7.99(m，2H)，7.50-7.46(m，3H)，7.22(d，J＝5.9Hz，2H)，7.13(brs，1H)，3.74(q，J＝7.2Hz，2H)，2.97(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ160.88，160.14，149.38，147.15，140.17，136.43，130.51，128.30，125.99，125.91，123.53，38.65，34.65；ESI-HRMS(m/z)calcd C17H15N3O2(M+H+)294.1237；found 294.1237。
EXAMPLE 20 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -5-phenyl-oxazole-2-carboxamide
The same procedure as in example 14 was repeated except for using 5-phenyl-oxazole-2-carboxylic acid as a starting material.
The compound was prepared as a white solid in 63.5% yield.
1H NMR(600MHz，CDCl3)δ8.56(d，J＝4.6Hz，2H)，7.76-7.74(m，2H)，7.46-7.44(m，2H)，7.41-7.39(m，1H)，7.38(s，1H)，7.23(d，J＝4.6Hz，2H)，7.12(brs，1H)，3.77(q，J＝7.0Hz，2H)，2.98(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ154.67，153.58，153.37，149.48，146.75，128.99，128.41，126.19，124.37，123.45，121.95，39.12，34.39；ESI-HRMS(m/z)calcd C17H15N3O2(M+H+)294.1237；found 294.1237。
EXAMPLE 21 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -5-phenyl-1, 2, 4-oxadiazole-3-carboxamide
The same procedures as in example 14 were carried out except for using 5-phenyl-1, 2, 4-oxadiazole-3-carboxylic acid as a starting material.
The compound was prepared as a white solid in 69.4% yield.
1H NMR(600MHz，DMSO-d6)δ9.15(t，J＝7.2Hz，1H)，8.44(d，J＝5.9Hz，2H)，8.12(d，J＝7.0Hz，2H)，7.72(t，J＝7.0Hz，1H)，7.64(t，J＝7.0Hz，2H)，7.25(d，J＝5.9Hz，2H)，3.55(q，J＝7.2Hz，2H)，2.88(t，J＝7.2Hz，2H)；13C NMR(151MHz，DMSO-d6)δ176.41，164.62，156.57，149.99，148.45，134.20，130.13，128.52，124.71，123.42，40.49，34.21；ESI-HRMS(m/z)calcd C16H14N4O2(M+H+)317.1009；found 317.1006。
EXAMPLE 22 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -3-phenyl-1, 2, 4-oxadiazole-5-carboxamide
The same procedures as in example 14 were carried out except for using 3-phenyl-1, 2, 4-oxadiazole-5-carboxylic acid as a starting material.
The compound was prepared as a white solid in 75.6% yield.
1H NMR(600MHz，CDCl3)δ8.55(dd，J＝4.5，1.4Hz，2H)，8.06-8.05(m，2H)，7.55-7.52(m，1H)，7.50-7.48(m，2H)，7.44(brs，1H)，7.19(dd，J＝4.5，1.4Hz，2H)，3.80(q，J＝7.0Hz，2H)，3.00(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ168.71，168.44，153.24，150.04，147.07，131.80，128.97，127.48，125.64，124.04，40.18，34.67；ESI-HRMS(m/z)calcd C16H14N4O2(M+H+)317.1009；found 317.1006。
EXAMPLE 23 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -biphenyl-4-carboxamide
The same procedure as in example 14 was repeated except that biphenyl-4-carboxylic acid was used as a starting material.
The compound was prepared as a white solid in 73.7% yield.
1H NMR(600MHz，CDCl3)δ8.53(d，J＝4.6Hz，2H)，7.78(d，J＝8.3Hz，2H)，7.64(d，J＝8.3Hz，2H)，7.60(d，J＝7.2Hz，2H)，7.47-7.44(m，2H)，7.39-7.37(m，1H)，7.17(d，J＝4.6Hz，2H)，6.34(brs，1H)，3.76(q，J＝7.0Hz，2H)，2.97(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ166.71，149.39，147.43，143.86，139.29，132.31，128.31，127.43，126.73，126.67，126.56，123.59，39.69，34.53；ESI-HRMS(m/z)calcd C20H18N2O(M+H+)303.1492；found 303.1487。
EXAMPLE 24 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -6-phenylnicotinamide (2-11).
The same procedure as in example 14 was repeated except that 6-phenylnicotinic acid was used as a starting material.
The compound was prepared as a white solid, yield: 71.6 percent.
1H NMR(600MHz，CDCl3)δ8.97(d，J＝2.0Hz，1H)，8.56(d，J＝5.9Hz，2H)，8.15(dd，J＝8.3，2.0Hz，1H)，8.05-8.04(m，2H)，7.83(d，J＝8.3Hz，1H)，7.53-7.48(m，3H)，7.21(d，J＝5.9Hz，2H)，6.33(brs，1H)，3.81(q，J＝7.0Hz，2H)，3.01(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ165.74，160.00，149.95，147.87，147.71，138.20，135.91，129.79，128.86，128.09，127.15，124.14，120.07，40.34，35.06；ESI-HRMS(m/z)calcd C19H17N3O(M+H+)304.1444；found 304.1445。
EXAMPLE 25 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -6-phenyl-pyrimidine-4-carboxamide (2-12).
The procedure is as in example 14 except that 6-phenyl-pyrimidine-4-carboxylic acid is used as a starting material.
The compound was prepared as a white solid in 71.2% yield.
1H NMR(600MHz，CDCl3)δ9.20(s，1H)，8.54(d，J＝4.6Hz，2H)，8.52(s，1H)，8.19-8.18(m，2H)，8.13(brs，1H)，7.54-7.53(m，3H)，7.18(d，J＝4.6Hz，2H)，3.79(q，J＝7.2Hz，2H)，2.97(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ165.67，162.45，157.25，156.27，135.64，135.46，133.59，130.98，130.33，129.11，128.51，127.58，126.80，126.42，113.35，38.66，32.83；ESI-HRMS(m/z)calcd C19H16ClN3O(M+H+)360.0874；found 360.0874。
EXAMPLE 26 Synthesis of N- [2- (pyridin-4-yl) ethyl ] -5-phenyl-pyridine-2-carboxamide (2-13).
The procedure is as in example 14 except that 5-phenyl-pyridine-2-carboxylic acid is used as a starting material.
The compound was prepared as a white solid in 76.5% yield.
1H NMR(600MHz，CDCl3)δ8.73(d，J＝2.0Hz，1H)，8.54(d，J＝5.8Hz，2H)，8.24(d，J＝8.1Hz，1H)，8.13(brs，1H)，8.02(dd，J＝8.1，2.0Hz，1H)，7.60(d，J＝7.3Hz，2H)，7.50(t，J＝7.3Hz，2H)，7.44(t，J＝7.3Hz，2H)，7.19(d，J＝5.8Hz，2H)，3.78(q，J＝7.1Hz，2H)，2.97(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ163.74，149.36，147.66，147.33，146.04，138.61，136.31，134.95，128.62，128.11，126.63，123.54，121.61，39.03，34.67；ESI-HRMS(m/z)calcd C19H17N3O(M+H+)304.1444；found 304.1448。
EXAMPLE 27 Synthesis of N- (2-phenylethyl) -5-phenyl-pyridine-2-carboxamide
The reaction formula is as follows:
5-phenyl-pyridine-2-carboxylic acid (1mmol) was dissolved in 5mL of dichloromethane at 0 deg.C, oxalyl chloride (0.17mL) and N, N-dimethylformamide (1 drop) were slowly added dropwise, reaction was carried out at room temperature for 1 hour, and after completion of the reaction, the solvent was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 2mL of methylene chloride, and a mixed solution of phenethylamine (1mmol) in methylene chloride (5mL) and triethylamine (0.16mL) was added thereto, followed by stirring at room temperature for 24 hours. After confirming completion of the reaction by TLC analysis, the solvent was removed by distillation under the reduced pressure, and the residue was purified by flash column chromatography to give the objective compound as a white solid in a yield of 71.4%
1H NMR(600MHz，DMSO-d6)δ8.91(d，J＝2.0Hz，1H)，8.81(brs，1H)，8.25(dd，J＝8.1，2.0Hz，1H)，8.08(d，J＝8.1Hz，1H)，7.78(d，J＝7.6Hz，2H)，7.52(t，J＝7.6Hz，2H)，7.45(t，J＝7.6Hz，1H)，7.29-7.27(m，2H)，7.24-7.23(m，2H)，7.20-7.17(m，1H)，3.56(q，J＝7.1Hz，2H)，2.87(t，J＝7.1Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.95，149.27，146.88，139.83，138.31，136.66，135.97，129.66，129.16，129.04，128.80，127.59，126.55，122.38，40.80，35.59；ESI-HRMS(m/z)calcd C20H18N2O(M+H+)303.1492；found 303.1496。
EXAMPLE 28 Synthesis of N- (3-chlorophenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (3-chlorophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.74(d，J＝2.2Hz，1H)，8.25(d，J＝8.1Hz，1H)，8.15(brt，1H)，8.02(dd，J＝8.1，2.2Hz，1H)，7.61-7.60(m，2H)，7.50(t，J＝7.5Hz，2H)，7.45-7.43(m，1H)，7.26-7.21(m，3H)，7.15(d，J＝7.2Hz，1H)，3.74(q，J＝7.2Hz，2H)，2.94(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.28，148.44，146.60，140.98，139.10，136.96，135.51，134.35，129.84，129.19，128.94，128.67，127.22，126.96，126.70，122.20，40.49，35.65；ESI-HRMS(m/z)calcd C20H17ClN2O(M+H+)337.1102；found 337.1108。
EXAMPLE 29 Synthesis of N- (4-chlorophenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-chlorophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.73(d，J＝1.8Hz，1H)，8.25(d，J＝8.1Hz，1H)，8.13(brs，1H)，8.01(dd，J＝8.1Hz，1.8Hz，1H)，7.60(d，J＝7.6Hz，2H)，7.50(t，J＝7.6Hz，2H)，7.44(t，J＝7.6Hz，1H)，7.28(d，J＝8.3Hz，2H)，7.19(d，J＝8.3Hz，2H)，3.73(q，J＝7.2Hz，2H)，2.93(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.22，148.48，146.59，139.06，137.42，136.95，135.49，132.28，130.12，129.20，128.71，128.67，127.22，122.19，40.57，35.32；ESI-HRMS(m/z)calcd C20H17ClN2O(M+H+)337.1102；found 337.1102。
EXAMPLE 30 Synthesis of N- (2-fluorophenethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (2-fluorophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.73(d，J＝1.5Hz，1H)，8.24(d，J＝8.0Hz，1H)，8.16(brs，1H)，8.00(dd，J＝8.0，1.5Hz，1H)，7.59(d，J＝7.6Hz，2H)，7.49(t，J＝7.6Hz，2H)，7.43(t，J＝7.6Hz，1H)，7.25-7.22(m，1H)，7.21-7.19(m，1H)，7.08-7.06(m，1H)，7.05-7.02(m，1H)，3.75(q，J＝7.1Hz，2H)，3.00(t，J＝7.1Hz，2H)；13C NMR(151MHz，DMSO-d6)δ164.08，161.23，149.25，146.93，138.34，136.69，136.02，131.63，129.72，129.22，128.77，127.65，126.49，124.84，122.45，115.60，40.49，28.98；ESI-HRMS(m/z)calcd C20H17FN2O(M+H+)321.1398；found321.1399。
EXAMPLE 31 Synthesis of N- (3-fluorophenethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as defined in example 27 except that 2- (3-fluorophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.76(d，J＝1.8Hz，1H)，8.28(d，J＝8.1，1H)，8.17(brt，1H)，8.05(dd，J＝8.1，1.8Hz，1H)，7.63(d，J＝7.3Hz，2H)，7.53(t，J＝7.3Hz，2H)，7.48-7.47(m，1H)，7.32-7.29(m，1H)，7.07(d，J＝7.6Hz，1H)，7.01-6.99(m，1H)，6.97-6.95(m，1H)，3.78(q，J＝7.2Hz，2H)，2.99(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ163.67，162.10，147.81，146.02，140.89，138.50，136.35，134.95，129.45，128.62，128.09，126.64，123.85，121.62，115.07，112.81，39.89，35.10；ESI-HRMS(m/z)calcd C20H17FN2O(M+H+)321.1398；found 321.1399。
EXAMPLE 32 Synthesis of N- (4-fluorophenethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-fluorophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.74(d，J＝2.3Hz，1H)，8.25(d，J＝8.1Hz，1H)，8.11(brs，1H)，8.02(dd，J＝8.1，2.3Hz，1H)，7.62-7.60(m，2H)，7.52-7.49(m，2H)，7.46-7.43(m，1H)，7.23-7.21(m，2H)，7.02-6.99(m，2H)，3.73(q，J＝7.2Hz，2H)，2.94(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ163.62，160.77，147.91，146.01，138.46，136.38，134.90，134.00，129.58，128.60，128.07，126.63，121.58，114.78，40.18，34.56；ESI-HRMS(m/z)calcd C20H17FN2O(M+H+)321.1398；found 321.14。
EXAMPLE 33 Synthesis of N- [2- (pyridin-2-yl) ethyl ] -5-phenyl-pyridine-2-carboxamide
The procedure is as defined in example 27 except that 2- (pyridin-2-yl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.74(d，J＝2.0Hz，1H)，8.59(d，J＝4.7Hz，1H)，8.51(brt，1H)，8.24(d，J＝8.1Hz，1H)，7.99(dd，J＝8.1，2.0Hz，1H)，7.62-7.58(m，3H)，7.48(t，J＝7.6Hz，2H)，7.42(t，J＝7.6Hz，1H)，7.21(d，J＝7.6Hz，1H)，7.16-7.13(m，1H)，3.91(q，J＝6.5Hz，2H)，3.14(t，J＝6.5Hz，2H)；13C NMR(151MHz，CDCl3)δ164.28，159.28，149.34，148.76，146.66，138.86，137.07，136.53，135.36，129.16，128.58，127.20，123.37，122.14，121.52，38.84，37.57；ESI-HRMS(m/z)calcd C19H17N3O(M+H+)304.1444；found 304.1448。
EXAMPLE 34 Synthesis of N- [2- (pyridin-3-yl) ethyl ] -5-phenyl-pyridine-2-carboxamide
The procedure is as defined in example 27 except that 2- (pyridin-3-yl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.73(dd，J＝2.3，0.8Hz，1H)，8.53(d，J＝1.8Hz，1H)，8.50(dd，J＝4.8，1.8Hz，1H)，8.24(dd，J＝8.1，0.8Hz，1H)，8.15(brs，1H)，8.02(dd，J＝8.1，2.3Hz，1H)，7.61-7.60(m，3H)，7.51-7.49(m，2H)，7.46-7.43(m，1H)，7.25-7.24(m，1H)，3.77(q，J＝7.0Hz，2H)，2.98(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ164.32，150.19，148.37，148.04，146.62，139.14，136.94，136.17，135.49，134.35，129.18，128.66，127.21，123.45，122.16，40.35，33.15；ESI-HRMS(m/z)calcd C19H17N3O(M+H+)304.1444；found 304.1449。
EXAMPLE 35 Synthesis of N- (4-Hydroxyphenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-hydroxyphenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.75(d，J＝1.7Hz，1H)，8.28(d，J＝8.1Hz，1H)，8.21(brs，1H)，8.05(dd，J＝8.1，1.7Hz，1H)，7.62-7.60(m，2H)，7.51(t，J＝7.5Hz，2H)，7.45(t，J＝7.5Hz，1H)，7.13(d，J＝8.4Hz，2H)，6.80(d，J＝8.4Hz，2H)，3.72(q，J＝7.0Hz，2H)，2.90(t，J＝7.0Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.28，155.47，148.67，146.29，137.68，136.06，135.39，131.35，129.31，129.08，128.57，127.00，121.78，114.99，40.53，34.19；ESI-HRMS(m/z)calcd C20H18N2O2(M+H+)337.1102；found 337.1102。
EXAMPLE 36 Synthesis of N- (4-aminophenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-aminophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.74(d，J＝2.1Hz，1H)，8.26(d，J＝8.0Hz，1H)，8.12(brs，1H)，8.01(dd，J＝8.0，2.1Hz，1H)，7.60(d，J＝7.7Hz，2H)，7.50(t，J＝7.7Hz，2H)，7.44(t，J＝7.7Hz，1H)，7.06(d，J＝8.2Hz，2H)，6.66(d，J＝8.2Hz，2H)，3.70(q，J＝7.2Hz，2H)，3.63(brs，2H)，2.85(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ164.14，148.74，146.57，144.78，138.90，137.06，135.42，129.58，129.17，128.89，128.59，127.21，122.14，115.42，41.03，35.08；ESI-HRMS(m/z)calcd C20H19N3O(M+H+)318.1601；found 318.1603。
EXAMPLE 37 Synthesis of N- (4-methylphenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-methylphenyl) ethylamine is used as the starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.75(d，J＝2.1Hz，1H)，8.27(d，J＝8.1Hz，1H)，8.16(brs，1H)，8.02(dd，J＝8.1，2.1Hz，1H)，7.61(d，J＝7.3Hz，2H)，7.51(t，J＝7.3Hz，2H)，7.45(t，J＝7.3Hz，1H)，7.18-7.14(m，4H)，3.75(q，J＝7.2Hz，2H)，2.94(t，J＝7.2Hz，2H)，2.34(s，3H)；13C NMR(151MHz，CDCl3)δ164.21，148.63，146.58，138.97，137.01，135.94，135.84，135.62，135.47，129.29，129.19，128.65，127.22，122.19，40.89，35.52，21.02；ESI-HRMS(m/z)calcd C21H20N2O(M+H+)317.1648；found 317.1648。
EXAMPLE 38 Synthesis of N- (4-Nitrophenylethyl) -5-phenyl-pyridine-2-carboxamide
The procedure is as in example 27 except that 2- (4-nitrophenyl) ethylamine is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.72(d，J＝1.6Hz，1H)，8.23(d，J＝8.0Hz，1H)，8.17-8.15(m，3H)，8.02(dd，J＝8.0，1.6Hz，1H)，7.59(d，J＝7.3Hz，2H)，7.49(t，J＝7.3Hz，2H)，7.46-7.41(m，3H)，3.79(q，J＝7.1Hz，2H)，3.08(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ164.39，148.22，146.85，146.75，146.64，139.24，136.84，135.53，129.63，129.21，128.73，127.20，123.80，122.21，40.14，35.85；ESI-HRMS(m/z)calcd C20H17N3O3(M+H+)348.1343；found 348.1339。
EXAMPLE 39 Synthesis of N- (2-fluorophenethyl) -5- (furan-2-yl) -pyridine-2-carboxamide
The reaction formula is as follows:
the compound 5-bromo-N- (2-fluorophenethyl) picolinamide (0.5mmol), (furan-2-yl) boronic acid (0.66mmol), potassium carbonate (0.9mmol) and bis (triphenylphosphine) palladium dichloride (0.05mmol) were weighed into a sealed tube, dioxane and water (v: v ═ 3:1, 20mL) were added, nitrogen was bubbled through, and the sealed tube was heated in an oil bath at 110 ℃ for 10 hours. The reaction solution was cooled to room temperature, and extracted with ethyl acetate (3X 30 mL). The organic layer was dried and concentrated over anhydrous sodium sulfate, and the desired product was obtained as a white solid by flash column chromatography in 67.8% yield.
1H NMR(600MHz，CDCl3)δ8.84(d，J＝2.2Hz，1H)，8.08(dd，J＝8.3，2.2Hz，1H)，7.73(d，J＝8.3Hz，1H)，7.57(d，J＝1.7Hz，1H)，7.25-7.22(m，2H)，7.14(d，J＝3.4Hz，1H)，7.11-7.10(m，1H)，7.09-7.06(m，1H)，6.56(dd，J＝3.4，1.7Hz，1H)，6.26(brs，1H)，3.74(q，J＝6.6Hz，2H)，3.01(t，J＝6.6Hz，2H)；13C NMR(151MHz，CDCl3)δ164.79，160.50，152.20，150.78，147.11，143.65，135.26，130.60，128.00，127.26，125.00，123.82，117.50，114.89，111.81，109.89，39.61，28.50；ESI-HRMS(m/z)calcd C18H15FN2O2(M+H+)311.1190；found 311.1190。
EXAMPLE 40 Synthesis of N- (2-fluorophenethyl) -5- (furan-3-yl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (furan-3-yl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ8.91(d，J＝2.1Hz，1H)，8.85(t，J＝6.8Hz，1H)，8.45(s，1H)，8.20(dd，J＝8.1，2.1Hz，1H)，8.02(d，J＝8.1Hz，1H)，7.85(t，J＝1.7Hz，1H)，7.32(td，J＝7.6，1.7Hz，1H)，7.28-7.25(m，1H)，7.15-7.13(m，3H)，3.56(q，J＝6.8Hz，2H)，2.92(t，J＝6.8Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.45，160.44，148.03，145.23，144.76，140.94，133.72，130.97，130.24，128.12，125.83，124.19，122.08，121.80，114.96，108.35，39.87，28.34；ESI-HRMS(m/z)calcd C18H15FN2O2(M+H+)311.1190；found 311.1191。
EXAMPLE 41 Synthesis of N- (2-fluorophenethyl) -5- (thiophen-2-yl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (thien-2-yl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.77(d，J＝2.3Hz，1H)，8.19(d，J＝8.1Hz，1H)，8.09(brs，1H)，8.00(dd，J＝8.1，2.3Hz，1H)，7.44(dd，J＝3.6，1.0Hz，1H)，7.42(dd，J＝5.1，1.0Hz，1H)，7.25-7.21(m，2H)，7.15(dd，J＝5.1，3.6Hz，1H)，7.08(t，J＝7.4，1H)，7.07-7.02(m，1H)，3.75(q，J＝7.0Hz，2H)，3.01(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ164.07，161.32，148.40，145.12，139.59，133.79，132.68，131.06，128.46，128.26，126.92，125.84，125.05，124.14，122.29，115.34，39.53，29.34；ESI-HRMS(m/z)calcd C18H15FN2OS(M+H+)327.0962；found 327.0962。
EXAMPLE 42 Synthesis of N- (2-fluorophenethyl) -5- (thiophen-3-yl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (thien-3-yl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ8.98(d，J＝1.6Hz，1H)，8.84(t，J＝6.0Hz，1H)，8.28(dd，J＝8.1，2.2Hz，1H)，8.17-8.16(m，1H)，8.01(d，J＝8.1Hz，1H)，7.72(dd，J＝5.0，2.9Hz，1H)，7.70(dd，J＝5.0，1.3Hz，1H)，7.32-7.28(m，1H)，7.26-7.21(m，1H)，7.15–7.08(m，2H)，3.54(q，J＝6.9Hz，2H)，2.90(t，J＝7.3Hz，2H)；13C NMR(151MHz，CDCl3)δ163.64，160.41，147.59，145.31，137.42，133.91，133.19，131.48，130.51，127.90，127.69，126.73，125.21，123.58，121.77，114.76，38.92，28.76；ESI-HRMS(m/z)calcd C18H15FN2OS(M+H+)327.0962；found 327.0963。
EXAMPLE 43 Synthesis of N- (2-fluorophenethyl) -3, 4' -bipyridine-6-carboxamide
The procedure of example 39 was repeated except that (pyridin-4-yl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.78(d，J＝1.8Hz，1H)，8.74(d，J＝5.9Hz，2H)，8.30(d，J＝8.1Hz，1H)，8.16(brs，1H)，8.07(dd，J＝8.1，2.2Hz，1H)，7.52(dd，J＝4.6，1.5Hz，2H)，7.26-7.23(m，1H)，7.23-7.19(m，1H)，7.08(t，J＝7.2Hz，1H)，7.06-7.02(m，1H)，3.76(q，J＝6.9Hz，2H)，3.01(t，J＝7.1Hz，2H)；13C NMR(151MHz，CDCl3)δ163.78，162.12，150.67，150.12，146.47，144.36，136.11，135.64，132.10，131.04，128.30，124.18，122.39，121.55，115.30，39.60，29.32；ESI-HRMS(m/z)calcd C19H16FN3O(M+H+)322.1350；found 322.1354。
EXAMPLE 44 Synthesis of N- (2-fluorophenethyl) -5- (1H-pyrazol-4-yl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (1H-pyrazol-4-yl) -boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.69(d，J＝1.8Hz，1H)，8.20(d，J＝8.1Hz，1H)，8.12(brs，1H)，7.96(s，2H)，7.92(dd，J＝8.1，1.8Hz，1H)，7.25-7.21(m，2H)，7.09-7.03(m，2H)，3.75(q，J＝7.1Hz，2H)，3.01(t，J＝7.1Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.58，160.38，147.02，144.76，136.63，132.92，131.26，130.99，128.09，126.61，125.87，124.19，121.81，117.07，114.96，38.83，28.36；ESI-HRMS(m/z)calcd C17H15FN4O(M+H+)311.1303；found 311.1300。
EXAMPLE 45 Synthesis of N- (2-fluorophenethyl) -5- (2-hydroxyphenyl) -pyridine-2-carboxamide
The same procedure as in example 39 was repeated except that (2-hydroxyphenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ9.82(s，1H)，8.83(t，J＝7.0Hz，1H)，8.80(d，J＝2.0Hz，1H)，8.14(dd，J＝8.1，2.0Hz，1H)，8.05(d，J＝8.1Hz，1H)，7.38(d，J＝7.6Hz，1H)，7.33(t，J＝7.6Hz，1H)，7.27-7.25(m，2H)，7.16-7.12(m，2H)，7.01(d，J＝8.1Hz，1H)，6.95(t，J＝7.6Hz，1H)，3.59(q，J＝7.0Hz，2H)，2.94(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ163.55，160.61，159.04，148.15，147.41，136.82，133.48，130.49，129.92，129.84，127.68，125.23，124.29，124.20，123.56，121.28，115.82，114.76，38.93，28.76；ESI-HRMS(m/z)calcd C20H16F2N2O(M+H+)339.1303；found 339.1309。
EXAMPLE 46 Synthesis of N- (2-fluorophenethyl) -5- (3-hydroxyphenyl) -pyridine-2-carboxamide
The same procedure as in example 39 was repeated except that (3-hydroxyphenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.72(d，J＝1.8Hz，1H)，8.23-8.20(m，2H)，7.93(dd，J＝8.1，2.2Hz，1H)，7.36(t，J＝7.9Hz，1H)，7.26-7.24(m，1H)，7.23-7.19(m，1H)，7.14(d，J＝7.7Hz，1H)，7.10-7.06(m，2H)，7.06-7.02(m，1H)，6.94(dd，J＝8.1，1.8Hz，1H)，3.76(q，J＝6.9Hz，2H)，3.01(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ163.67，162.81，160.73，148.26，145.98，138.53，137.25，135.02，130.51，130.24，127.76，125.12，123.61，122.34，121.67，115.00，114.79，113.61，39.03，28.71；ESI-HRMS(m/z)calcd C20H16F2N2O(M+H+)339.1303；found 339.1306。
EXAMPLE 47 Synthesis of N- (2-fluorophenethyl) -5- (4-hydroxyphenyl) -pyridine-2-carboxamide
The same procedure as in example 39 was repeated except that (4-hydroxyphenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.70(d，J＝2.2Hz，1H)，8.25(d，J＝8.1Hz，1H)，8.14(brs，1H)，7.97(dd，J＝8.1，2.2Hz，1H)，7.59-7.56(m，2H)，7.25-7.23(m，1H)，7.21-7.18(m，3H)，7.09-7.03(m，2H)，3.76(q，J＝7.0Hz，2H)，3.02(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ163.58，162.44，160.39，147.98，145.81，137.44，134.73，132.55，130.49，128.36，127.69，125.22，123.56，121.60，115.65，114.76，38.94，28.75；ESI-HRMS(m/z)calcd C20H16F2N2O(M+H+)339.1303；found 339.1304。
EXAMPLE 48 Synthesis of N- (2-fluorophenethyl) -5- (2-fluorophenyl) -pyridine-2-carboxamide
The procedure is as defined in example 39, except that (2-fluorophenyl) boronic acid is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ9.82(s，1H)，8.83(t，J＝7.0Hz，1H)，8.80(d，J＝2.0Hz，1H)，8.14(dd，J＝8.1，2.0Hz，1H)，8.05(d，J＝8.1Hz，1H)，7.38(d，J＝7.6Hz，1H)，7.33(t，J＝7.6Hz，1H)，7.27-7.25(m，2H)，7.16-7.12(m，2H)，7.01(d，J＝8.1Hz，1H)，6.95(t，J＝7.6Hz，1H)，3.59(q，J＝7.0Hz，2H)，2.94(t，J＝7.0Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.64，160.26，154.50，148.10，147.74，137.40，136.51，130.96，130.16，129.73，128.09，125.83，124.16，123.42，121.07，119.60，116.09，114.93，38.85，28.36；ESI-HRMS(m/z)calcd C20H17FN2O2(M+H+)337.1347；found 337.1348。
EXAMPLE 49 Synthesis of N- (2-fluorophenethyl) -5- (3-fluorophenyl) -pyridine-2-carboxamide
The procedure is as defined in example 39, except that (3-fluorophenyl) boronic acid is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ9.72(s，1H)，8.93(t，J＝7.0Hz，1H)，8.86(d，J＝2.0Hz，1H)，8.19(dd，J＝8.1，2.0Hz，1H)，8.07(d，J＝8.1Hz，1H)，7.63-7.61(m，1H)，7.34(t，J＝7.8Hz，2H)，7.33-7.32(m，1H)，7.20-7.19(m，1H)，7.15-7.11(m，2H)，6.89-6.87(m，1H)，3.58(q，J＝7.0Hz，2H)，2.93(t，J＝7.0Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.50，160.50，157.90，148.67，146.14，137.88，137.43，135.24，130.95，130.13，128.51，128.05，124.17，121.79，117.69，115.59，114.93，113.70，38.88，28.34；ESI-HRMS(m/z)calcd C20H17FN2O2(M+H+)337.1347；found 337.1350。
EXAMPLE 50 Synthesis of N- (2-fluorophenethyl) -5- (4-fluorophenyl) -pyridine-2-carboxamide
The procedure is as defined in example 39, except that (4-fluorophenyl) boronic acid is used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，DMSO-d6)δ9.73(s，1H)，8.85(d，J＝2.0Hz，1H)，8.79(t，J＝7.0Hz，1H)，8.16(dd，J＝8.2，2.0Hz，1H)，8.03(d，J＝8.2Hz，1H)，7.64(d，J＝8.6Hz，2H)，7.34-7.31(m，1H)，7.27-7.26(m，1H)，7.16-7.11(m，2H)，6.92(d，J＝8.6Hz，2H)，3.58(q，J＝7.0Hz，2H)，2.93(t，J＝7.0Hz，2H)；13C NMR(151MHz，DMSO-d6)δ163.60，160.53，158.14，147.67，145.51，137.73，134.18，130.95，128.17，128.09，126.61，125.89，124.16，121.71，115.94，114.93，38.85，28.36；ESI-HRMS(m/z)calcd C20H17FN2O2(M+H+)337.1347；found 337.1345。
EXAMPLE 51 Synthesis of N- (2-fluorophenethyl) -5- (2-methoxyphenyl) -pyridine-2-carboxamide
The same procedures used in example 39 were repeated except for using (2-methoxyphenyl) boronic acid as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.68(d，J＝2.1Hz，1H)，8.22(d，J＝8.1Hz，1H)，8.18(brs，1H)，8.00(dd，J＝8.1，2.1Hz，1H)，7.41-7.38(m，1H)，7.33(dd，J＝7.5，1.5Hz，1H)，7.26-7.25(m，1H)，7.22-7.21(m，1H)，7.09-7.06(m，2H)，7.04-7.01(m，2H)，3.83(s，3H)，3.75(q，J＝7.0Hz，2H)，3.01(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ164.45，162.13，156.60，148.60，147.97，137.87，136.81，131.06，130.58，130.02，128.18，126.32，125.96，124.11，121.48，121.13，115.40，111.39，55.52，39.49，29.39；ESI-HRMS(m/z)calcd C21H19FN2O2(M+H+)351.1503；found 351.1508。
EXAMPLE 52 Synthesis of N- (2-fluorophenethyl) -5- (3-methoxyphenyl) -pyridine-2-carboxamide
The same procedures used in example 39 were repeated except for using (3-methoxyphenyl) boronic acid as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.72(s，1H)，8.23(d，J＝8.1Hz，1H)，8.16(brs，1H)，7.99(d，J＝8.1Hz，1H)，7.41-7.38(m，1H)，7.26-7.24(m，1H)，7.22-7.20(m，1H)，7.19-7.16(m，1H)，7.11(d，J＝1.8Hz，1H)，7.07(t，J＝7.5Hz，1H)，7.05-7.02(m，1H)，6.97(d，J＝7.5Hz，1H)，3.86(d，J＝1.8Hz，3H)，3.75(q，J＝7.2Hz，2H)，3.00(t，J＝7.2Hz，2H)；13C NMR(151MHz，DMSO-d6)δ164.06，161.22，160.35，149.33，147.03，138.17，136.13，131.62，130.81，128.76，126.48，124.82，122.36，119.87，115.66，115.52，114.82，113.08，55.73，39.51，28.97.ESI-HRMS(m/z)calcd C21H19FN2O2(M+H+)351.1503；found 351.1508。
EXAMPLE 53 Synthesis of N- (2-fluorophenethyl) -5- (4-methoxyphenyl) -pyridine-2-carboxamide
The same procedures used in example 39 were repeated except for using (4-methoxyphenyl) boronic acid as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.73(dd，J＝2.2，0.6Hz，1H)，8.24(dd，J＝8.1，0.6Hz，1H)，8.17(brs，1H)，7.99(dd，J＝8.1，2.2Hz，1H)，7.58-7.57(m，2H)，7.30-7.28(m，1H)，7.26-7.22(m，1H)，7.11-7.07(m，2H)，7.06-7.04(m，2H)，3.89(s，3H)，3.78(q，J＝7.0Hz，2H)，3.04(t，J＝7.2Hz，2H)；13C NMR(151MHz，CDCl3)δ163.79，160.74，159.61，147.32，145.56，138.02，134.20，130.50，128.74，127.75，127.66，125.27，123.55，121.55，114.75，114.09，54.79，38.91，28.77；ESI-HRMS(m/z)calcd C21H19FN2O2(M+H+)351.1503；found 351.1505。
EXAMPLE 54 Synthesis of N- (2-fluorophenethyl) -5- (2-cyanophenyl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (2-cyanophenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1H NMR(600MHz，CDCl3)δ8.59(d，J＝2.2Hz，1H)，8.22(d，J＝8.0Hz，1H)，8.16(t，J＝7.0Hz，1H)，7.92(dd，J＝8.0，2.2Hz，1H)，7.70(d，J＝7.6Hz，1H)，7.56(td，J＝7.5，1.2Hz，1H)，7.50(td，J＝7.6，1.0Hz，1H)，7.39(d，J＝7.5Hz，1H)，7.26-7.25(m，1H)，7.22-7.20(m，1H)，7.09-7.07(m，1H)，7.06-7.02(m，1H)，3.74(q，J＝7.0Hz，2H)，3.00(t，J＝7.0Hz，2H)；13C NMR(151MHz，CDCl3)δ170.06，163.52，160.45，148.31，147.05，137.78，136.63，135.16，134.47，130.48，130.27，129.98，128.11，127.87，127.70，125.18，123.60，121.25，114.77，38.93，28.75；ESI-HRMS(m/z)calcd C21H16FN3O(M+H+)346.1350；found 346.1350。
EXAMPLE 55 Synthesis of N- (2-fluorophenethyl) -5- (3-cyanophenyl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (3-cyanophenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1HNMR(600MHz，CDCl3)δ8.75(d，J＝2.0Hz，1H)，8.32(d，J＝8.1Hz，1H)，8.17(brs，1H)，8.04(dd，J＝8.1，2.0Hz，1H)，7.91(s，1H)，7.86(d，J＝7.8Hz，1H)，7.76(d，J＝7.8Hz，1H)，7.66(t，J＝7.8Hz，1H)，7.27-7.25(m，1H)，7.24-7.23(m，1H)，7.12-7.11(m，1H)，7.10-7.06(m，1H)，3.79(q，J＝7.0Hz，2H)，3.04(t，J＝7.0Hz，2H)；13CNMR(151MHz，CDCl3)δ163.25，160.31，148.96，145.86，137.79，136.15，135.08，131.41，130.93，130.49，130.14，129.52，127.75，125.14，123.58，121.80，117.65，114.79，112.99，39.00，28.73；ESI-HRMS(m/z)calcd C21H16FN3O(M+H+)346.1350；found 346.1350。
EXAMPLE 56 Synthesis of N- (2-fluorophenethyl) -5- (4-cyanophenyl) -pyridine-2-carboxamide
The procedure of example 39 was repeated except that (4-cyanophenyl) boronic acid was used as a starting material.
The compound was prepared as a white solid in 74.3% yield.
1HNMR(600MHz，CDCl3)δ8.75(d，J＝1.5Hz，1H)，8.30(d，J＝8.1Hz，1H)，8.15(brs，1H)，8.04(dd，J＝8.1，1.5Hz，1H)，7.80(d，J＝8.1Hz，2H)，7.72(d，J＝8.1Hz，2H)，7.24-7.21(m，2H)，7.09(t，J＝7.4Hz，1H)，7.05(t，J＝8.0Hz，1H)，3.76(q，J＝7.0Hz，2H)，3.02(t，J＝7.0Hz，2H)；13CNMR(151MHz，CDCl3)δ163.23，160.51，149.09，145.99，140.88，136.44，135.19，132.37，130.48，127.76，127.31，125.13，123.60，121.78，117.77，114.79，111.88，39.00，28.72；ESI-HRMS(m/z)calcdC21H16FN3O(M+H+)346.1350；found 346.1352。
The properties of the compounds prepared in examples 1 to 56 were examined as follows:
1. the dual-luciferase reporter method was used to detect the inhibitory effect of the compounds prepared in examples 1-56 on HIF-1 α.
Human hepatoma cells LM3 were seeded into 24-well plates. Firefly luciferase reporter plasmid (pGL3-HRE-Luc) containing 5 copies of HIF-1 binding element (HRE) and pRL-SV40 plasmid encoding Renilla luciferase were co-transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen, Inc.) according to the instructions. Then in the presence of atmospheric oxygen (21% O)2) Incubate for 6 hours. Subsequently, the medium in each well was changed to fresh medium containing DMSO (negative control) or 10. mu.M of the test compound, and the plate was incubated under normoxic conditions for 1 hour, then under hypoxia (1% O)2) Incubate for another 24 hours. After washing the cells with PBS buffer, luciferase activity was measured using dual luciferase reporter assay kit (Promega) and microplate reader (SpectraMax M4, Molecular Device) according to the instructions. FireflyLuciferase activity values were normalized to Renilla luciferase activity values (equation 1). The inhibition rate of the test compound on luciferase was calculated with the inhibition rate of the DMSO group (negative control) as 0% (formula 2).
Equation 1:
Figure BDA0002160574090000251
equation 2:
Figure BDA0002160574090000252
the experiment was repeated three times and expressed as mean ± standard deviation (table 1). Compounds with inhibition rates over 60%, detecting inhibition rates at different concentrations, and calculating half-inhibitory concentration IC50And detecting cytotoxicity (CC) by MTT assay50) (the results are shown in Table 2). The normoxic condition means that the proportion of oxygen in the cell culture box is about 21 percent, and the anoxic condition means that the proportion of oxygen in the cell culture box is 1 percent.
TABLE 1 inhibition of HIF-1 by arylcarboxamides prepared in examples 1-56 at a concentration of 10. mu.M
Figure BDA0002160574090000261
LW6 was a positive control, HIF-1 inhibitor containing adamantane structures.
TABLE 2 half maximal inhibitory concentration and cytotoxicity of representative compounds to inhibit HIF-1
Figure BDA0002160574090000262
LW6 was a positive control, HIF-1 inhibitor containing adamantane structures.
2. Detection of inhibitory Effect of Compounds on hypoxia-induced HIF-1 alpha by Western blotting
Human liver cancer cell LM3, breast cancer cell MDA-MB-231, ovarian cancer cell SKOV3 and cervical cancer cell Hela were inoculated in a 60mm culture dish and cultured under an normoxic condition. To 70-80% confluence, 10 μ M of the compound of examples 40, 44, 51, LW6 (positive control) or DMSO (negative control) was added to LM3 cells, 2, 5, 10 μ M of example 51 or DMSO (negative control) was added to MDA-MB-231, SKOV3, Hela cells, respectively, the cells were treated under normoxic conditions for 1 hour and then treated under hypoxia for 24 hours. Subsequently, cells were harvested and lysed with RIPA lysis buffer. The proteins were separated by SDA-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp.). Then, WB blocking solution (Shanghai Biyuntian Co., Ltd.) was blocked, reacted with a primary antibody (Proteitech Co., Ltd.), and then incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology Co., Ltd.). The protein bands were developed with a chemiluminescent reagent (Millipore Co.) and photographed. As a result, as shown in FIG. 1A, hypoxia conditions induced a significant increase in HIF-1 α expression levels in LM3 cells, but the compounds of examples 40, 44, 51, LW6, inhibited HIF-1 α expression to varying degrees. As shown in FIG. 1B, hypoxia induced a significant increase in HIF-1 α in MDA-MB-231, SKOV3, Hela cells, but the compound of example 51 inhibited HIF-1 α expression in the 3 cells described above in a concentration-dependent manner. The normoxic condition means that the proportion of oxygen in the cell culture box is about 21 percent, and the anoxic condition means that the proportion of oxygen in the cell culture box is 1 percent.
3. Detection of Effect of the Compounds of example 51 on hypoxia-induced Vascular Epidermal Growth Factor (VEGF) messenger RNA (mRNA) by real-time PCR
VEGF is a downstream gene of HIF-1 regulation. HIF-1. alpha. and dimers of HIF-1. beta. bind to the promoter sequence of the VEGF gene, driving its transcription, and we therefore examined whether the HIF-1 inhibitor, example 51, inhibits hypoxia-induced transcription of the VEGF gene, i.e., an increase in VEGF mRNA. The specific operation method comprises the following steps: human Umbilical Vein Endothelial Cells (HUVECs) were treated with the compound of example 51 or DMSO (negative control) under normoxic conditions for 1 hour and then under hypoxic conditions for 24 hours. Total RNA was extracted using TRIzol reagent (product of Invitrogen corporation), and prime script RT kit (product of Takara corporation) was usedPin) was subjected to reverse transcription to obtain cDNA, which was then amplified by real-time PCR using SYBR Premix Ex Taq II (product of Takara). Through 2-ΔΔCtThe method calculates gene expression and calculates relative expression level with GAPDH as internal reference. As a result, as shown in fig. 2, hypoxia conditions can induce significant increase in mRNA of VEGF, and the compound of example 51 can inhibit increase in VEGF concentration-dependently. Results were obtained from three independent assays and are expressed as mean ± standard deviation. The normoxic condition means that the proportion of oxygen in the cell culture box is about 21 percent, and the anoxic condition means that the proportion of oxygen in the cell culture box is 1 percent.
4. Human Umbilical Vein Endothelial Cells (HUVEC) in vitro capillary-like microtubule formation assay to examine the effect of the compound of example 51 on in vitro vascularization
HUVECs are able to form a microtubule network on matrigel, which is believed to mimic the growth of new blood vessels in vitro, and therefore we used this experiment to examine the effect of the compound of example 51 on angiogenesis in vitro. The specific operation method comprises the following steps: the 96-well plate was coated with matrigel (product of Corning Corp.) and the matrigel was solidified by incubating at 37 ℃ for 1 hour. HUVEC (3X 10 per well) mixed with the compound of example 51 or DMSO (negative control)4) Seeded on top of the gel and incubated under hypoxic conditions for 12 hours. The formation of capillary-like microtubules was observed and photographed with an inverted microscope containing a CCD camera (Olympus). As shown in FIG. 3, the hypoxia culture induced HUVEC to form complete microtubule network, and the compound of example 51 inhibited the formation of microtubule network, indicating that the compound of example 51 has a potential effect of inhibiting new blood vessels.
5. The zebrafish model measures the effect of the compound of example 51 on neovascularisation.
The specific operation method comprises the following steps: transgenic zebrafish (fil 1: EGFP) embryos of 48hpf were placed in 6-well plates (n ═ 10 per well), added to water containing the compound of example 51, apatinib (positive control) or DMSO (negative control), incubated at 28 ℃ for 24 hours, then broken after anesthesia, and the effect of the test compound on the sub-intestinal vessels (SIV) of zebrafish was observed with a confocal microscope. As shown in fig. 4, the compound of example 51 can disrupt the formation of blood vessels in the lower intestine of zebrafish, with an effect comparable to apatinib. The experiment was independently repeated three times.
6. Cell scratch healing assay the effect of the compound of example 51 on tumor cell migration was examined
After scribing a scratch in an adherently growing monolayer of cells, the cells at the edge of the scratch can heal the scratch to different degrees, i.e., with a reduced width, by migrating into the scratch area. After 24 hours, scratch width is inversely proportional to cell migration ability. The specific operation method comprises the following steps: MDA-MB-231 cells were seeded in six-well plates and cultured under normoxic conditions until about 90% confluence. A scratch was scribed on the cell monolayer using a sterile pipette tip. Subsequently, cells were incubated with the compound of example 51 or DMSO (negative control) under normoxic conditions for 1 hour and hypoxic for another 24 hours. The healing of the scratch was observed and imaged with an inverted microscope including a CCD camera (product of Olympus Co.). Results as shown in fig. 5, the DMSO-treated group had a smaller scratch width than the compound-treated group of example 51 24 hours after scratching, indicating that the concentration of the compound of example 51 dependently inhibited the ability of tumor cells to migrate in vitro.
7. Transwell experiment to examine the influence of the Compound of example 51 on the invasion and migration of tumor cells
The Transwell chamber has a membrane with pores at the bottom, the pore size is 8 μ M, when cell migration is observed, cells suspended in serum-free medium are added to the chamber, and the chamber is placed in serum-containing medium, and due to chemotaxis of cytokines in serum, the cells migrate through the pores from the upper chamber to the back of the membrane, and the cell migration ability is characterized by the number of cells that migrate through the pores. When observing cell invasion, a layer of matrigel is coated on the surface of the cell membrane in advance to simulate the basement membrane in vivo. The cells suspended in the serum-free medium are inoculated on the surface of the cell matrigel, and the cells penetrate through the matrigel by invasion and then penetrate through a membrane containing small holes at the bottom of the chamber to the back surface of the membrane, so that the invasion capacity of the cells is characterized by the number of the cells which firstly invade and then migrate through the small holes to the back surface of the membrane. The specific operation is as follows: 2 x 10 to5MDA-MB-231 cell suspension0.3mL of serum-free medium per well was inoculated into a Transwell chamber (Corning Co.). For the invasion assay, the chamber was pre-coated with matrigel (matrigel, product of Corning). The chamber was placed in a 24-well plate and complete medium containing 10% FBS and the compound of example 51 or DMSO (negative control) was added to the lower well plate. The well plates were incubated under normoxic conditions for 1 hour and under hypoxic conditions for a further 24 hours. Cells that did not migrate through the wells at the upper surface of the chamber were wiped off with a cotton swab, cells that migrated through the wells to the back of the membrane were fixed with methanol, stained with crystal violet, and then counted under an inverted microscope from 5 different areas of each well. Results were obtained from three independent assays and are expressed as mean ± standard deviation. As a result, as shown in fig. 6, the compound of example 51 significantly reduced the number of cells invading and migrating through the transwell chamber, indicating that the compound of example 51 can inhibit the invasion and migration ability of tumor cells.
8. In vivo tumor metastasis assay in nude mice the effect of the compound of example 51 on in vivo tumor metastasis was examined
The concrete construction is as follows: female BALB/c nude mice (purchased from Slac laboratory animals Co., Ltd., Shanghai) at an age of 4-5 weeks were housed in Individual Ventilated Cages (IVC) under specific pathogen-free conditions (SPF). Stably luciferase-expressing MDA-MB-231 cells (2X 10 cells per mouse) were injected intravenously via the tail vein of nude mice5). 24 hours after injection, mice were randomly grouped (n-4), intraperitoneally injected with 15 and 30mg/kg of the compound of example 51 (injection solvent was PBS containing 5% ethanol and 5% cremophor EL, 0.2mL per mouse) or blank control (0.2 mL injection solvent) once every 2 days for 20 days. Metastatic tumors were detected and quantified using bioluminescent imaging. After imaging, mice were weighed and sacrificed, and their lung tissue was collected, fixed with 4% paraformaldehyde and embedded in paraffin. Histochemical analysis was carried out by H&E staining identifies metastatic nodules. The results are shown in fig. 7, where the compound of example 51 significantly reduced the luminescence of the lung metastases and was dose-dependent compared to the blank control. The results of sectioning are shown in FIG. 8, the size of the lung metastasis in the compound-treated group of example 51Significantly less than the placebo group, indicating that the compound of example 51 can inhibit the metastasis of breast cancer tumors to the lung in nude mice.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (4)

1. An arylcarboxamide compound or a pharmaceutically acceptable salt thereof, characterized in that it is selected from any one of the following compounds:
Figure 225643DEST_PATH_IMAGE002
2. use of an arylcarboxamide compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prophylaxis or treatment of diseases which are caused by the upregulation of hypoxia inducible factor-1.
3. The use according to claim 2, wherein the disease is at least one of breast cancer, ovarian cancer, lung cancer, liver cancer, cervical cancer, gastric cancer, and intestinal cancer.
4. The use of an arylcarboxamide compound as claimed in claim 1 or of a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prophylaxis or treatment of diseases of the pathological growth of retinal blood vessels.
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