CN111303176A - Chrysin spliced 2-amino 3-cyanopyran compound and preparation method and application thereof - Google Patents

Chrysin spliced 2-amino 3-cyanopyran compound and preparation method and application thereof Download PDF

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CN111303176A
CN111303176A CN202010110842.8A CN202010110842A CN111303176A CN 111303176 A CN111303176 A CN 111303176A CN 202010110842 A CN202010110842 A CN 202010110842A CN 111303176 A CN111303176 A CN 111303176A
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田民义
刘雄利
陈爽
张文会
周英
俸婷婷
刘雄伟
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Abstract

The regioselectivity of the synthesized spliced derivative is different from that of a compound reported in a known literature, the spliced derivative is a novel compound generated regioselectively, can provide a compound source for biological activity screening, and has important application value in the screening of medicaments and the pharmaceutical industry. And the skeleton compound has inhibitory activity on human leukemia cells, human lung adenocarcinoma cells and human prostate cancer cells. The method has the advantages of simple and easy operation, cheap and easily obtained raw material synthesis, capability of being carried out in various organic solvents, better air stability, wide applicability and good compatibility for various substituent groups.

Description

Chrysin spliced 2-amino 3-cyanopyran compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemistry and pharmacy, in particular to a chrysin spliced 2-amino 3-cyanopyran compound and a preparation method and application thereof.
Background
According to the active scaffold splicing and migration principle of drug design, splicing two or more scaffolds with biological activity into a multi-scaffold molecule with potential biological activity is an extremely important research field in organic chemistry and medicinal chemistry. (1) Chrysin belongs to a flavone skeleton and is also commonly found in natural products and drug molecules. (2) The 2-amino 3-cyanopyran backbone is also ubiquitous in natural products and drug molecules, as compound MX58151 (shown in figure 6). These compounds play a significant role in relieving pain and in economic development. Given the potential biological activity of pyrans, spiro-oxindoles and chrysin backbones. Therefore, the 2-amino 3-cyano pyran skeleton is spliced to the chrysin skeleton to synthesize a series of novel chrysin spliced 2-amino 3-cyano pyran compounds with potential multi-active functional groups. Particularly, the synthesized chrysin spliced 2-amino 3-cyanopyran compound is different from a compound (Med Chem Res.2015,24,3696) reported in a known literature in regioselectivity, is a novel compound generated in regioselectivity (shown in figure 7), can provide a compound source for biological activity screening, and has important application value in the drug screening and pharmaceutical industry.
Disclosure of Invention
The purpose of the invention is: the chrysin spliced 2-amino 3-cyanopyran compound is an important medical intermediate analogue and a drug molecule analogue, has important application value to drug screening and pharmaceutical industry, and is very economical and simple in synthesis method.
The invention also discloses the application of the compounds in preparing the medicines for preventing and treating tumor diseases.
The invention is realized by the following steps: a chrysin spliced 2-amino 3-cyanopyran compound has a structure shown in the following general formula (I):
Figure BDA0002389939670000021
in the formula, R1Is H or methoxy; r2Is nitro, methoxy, tert-butyl, methyl, fluorine, chlorine, bromine or hydrogen; r3Is methoxy or hydrogen.
A preparation method of a 2-amino 3-cyanopyran compound spliced by chrysin comprises the step of carrying out a regioselective Michael cycloaddition reaction on a Knoevannagel product 2 obtained by condensing various substituted benzaldehydes and malononitrile and various substituted chrysins 1 in an organic solvent under the action of an inorganic basic catalyst to obtain a 2-amino 3-cyanopyran compound spliced by chrysin 3.
The synthetic route is exemplified as follows:
Figure BDA0002389939670000022
wherein the substituents of the compounds in the synthetic route satisfy the formula R1Is H or methoxy; r2Is nitro, methoxy, tert-butyl, methyl, fluorine, chlorine, bromine or hydrogen; r3Is methoxy or hydrogen.
The reaction mechanism is as follows:
Figure BDA0002389939670000023
the organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
The inorganic alkaline catalyst is as follows: sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium carbonate or sodium carbonate.
The Knoevennagel product of the condensation of various substituted benzaldehydes and malononitrile undergoes a regioselective Michael cycloaddition reaction with various substituted chrysins at a temperature of from room temperature to 70 ℃ for a period of from 1 to 10 hours.
The application of the chrysin spliced 2-amino 3-cyanopyran compound in preparing medicaments for preventing and treating tumor diseases.
By adopting the technical scheme, under the action of an inorganic basic catalyst in an organic solvent, the Knoevennagel product 2 obtained by condensing various substituted benzaldehydes and malononitrile and various substituted chrysins 1 undergo a regioselective Michael cycloaddition reaction to obtain the chrysin spliced 2-amino 3-cyanopyran compound 3. Particularly, the synthesized chrysin spliced 2-amino 3-cyanopyran compound disclosed by the patent has regioselectivity different from that of a compound reported in a known literature (Med Chem Res.2015,24,3696), is a novel compound generated regioselectively (as shown in figure 7), can provide a compound source for biological activity screening, and has important application value in the drug screening and pharmaceutical industry. And the skeleton compound has inhibitory activity on human leukemia cell (K562), human lung adenocarcinoma cell (A549), human lung adenocarcinoma cell (NCI-H1299) and human prostate cancer cell (PC-3). The method has the advantages of simple and easy operation, cheap and easily obtained raw material synthesis, capability of being carried out in various organic solvents, better air stability, wide applicability and good compatibility for various substituent groups.
Drawings
FIGS. 1 and 2 are data of the spectra of compound 3a according to the example of the invention;
FIGS. 3 and 4 are data of the spectra of compound 3b according to the example of the present invention;
FIG. 5 is a single crystal diagram of Compound 3b according to an embodiment of the present invention;
FIG. 6 shows the concept of the compound synthesized by the present invention;
FIG. 7 is a comparison of regioselectivity of the synthesized compounds of the present invention with that of the known literature;
FIG. 8 is a graph showing the effect of compound of the present invention on inducing apoptosis in 3 h;
in FIG. 8, K562 cells were treated with different concentrations (0,5,10, 20. mu.M) of compound for 3h for 24 h. (A) AO-EB double dyeing method; (B) apoptotic cells were quantified by flow cytometry and Annexin V-FITC/PI double staining. Data are presented as mean ± standard deviation obtained in at least three independent experiments. P <0.01 compared to control group.
FIG. 9 Compound 3h induces cycle block patterns of G1 phase in K562 cells;
in FIG. 9, the effect of different concentrations (0,5,10, 20. mu.M) of compound 3h on the K562 cell cycle profile was analyzed by flow cytometry. Data are presented as mean ± standard deviation obtained in at least three independent experiments. P <0.01 compared to control group.
FIG. 10 is a graph showing the expression effect of apoptosis-related protein in K562 cells after 3h treatment with a compound;
in FIG. 10, western blot was used to examine the effect of different concentrations (0,5,10, 20. mu.M) of compound 3h on the expression of apoptosis-related proteins Bax and Bcl-2 β -actin as a control.
Detailed Description
The embodiment of the invention comprises the following steps: the Knoevennagel product 2a (0.8mmol), chrysin 1a (0.5mmol), Ca (OH) condensed with benzaldehyde and malononitrile were sequentially added to the reaction tube2(0.8mmol,59.2mg) and 5.0mL of methanol under reflux and stirring for 8 hours, TLC to detect substantial completion of the reaction, and column chromatography over dry solvent [ eluent: v (petroleum ether): v (ethyl acetate) ═ 3:1]Purification gave compound 3a, yellow solid, melting point:>300.0 ℃; the yield was 90%.
The results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.65(s,1H),6.96(s,1H),7.05(s,1H),7.16-7.21(m,5H),7.27-7.30(m,2H),7.55-7.63(m,3H),8.09(d,J＝6.0Hz,2H),13.24(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:35.8,57.2,94.9,105.2,106.9,107.4,120.0,126.6,126.8,127.1,128.5,129.1,130.3,144.6,153.7,155.2,157.7,159.5,164.1,182.4；HRMS(ESI-TOF)m/z:Calcd.for C25H16N2NaO4[M+Na]+:431.1002；Found:431.1005.
table 1 shows the chemical structure of 2-amino 3-cyanopyran compounds spliced by chrysin
Figure BDA0002389939670000041
Table 2 shows the chemical structure of 2-amino-3-cyanopyran compounds spliced by chrysin
Figure BDA0002389939670000051
The compounds 3b to 3r were prepared by the same method as that for the compound 3a and the same charge ratio as that for the compound 3a, the compounds 3b to 3r were obtained, and the reaction yields are shown in tables 1 and 2, but it should be noted that the compounds of the present invention are not limited to those shown in tables 1 and 2.
This example prepares compound 3b as a yellow solid, melting point:>300.0 ℃; yield 91 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.68(s,1H),6.94(s,1H),7.03(s,1H),7.10-7.23(m,6H),7.54-7.63(m,3H),8.07-8.09(m,2H),13.25(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:35.6,57.5,95.4,105.7,107.5(d,JCF＝24.2Hz),115.7(d,JCF＝21.3Hz),120.3,127.1,129.5,129.6,130.8,132.9,141.2,154.0,155.7,158.2,159.9,161.4(d,JCF＝241.4Hz),164.6,182.8；HRMS(ESI-TOF)m/z:Calcd.for C25H15FN2NaO4[M+Na]+:449.0908；Found:449.0909.
this example prepares compound 3c as a yellow solid, melting point:>300.0 ℃; yield 87%; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.72(s,1H),6.96-6.99(m,2H),7.02-7.07(m,3H),7.20(br s,2H),7.33-7.39(m,1H),7.55-7.64(m,3H),8.08-8.10(m,2H),13.28(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:36.0,57.2,95.5,105.7,107.1,107.4,114.1(d,JCF＝21.0Hz),114.3(d,JCF＝21.2Hz),120.2,123.7,127.1,129.6,130.8,130.9,132.9,147.9,154.1,155.8,158.3,160.0,162.6(d,JCF＝242.0Hz),164.6,182.8；HRMS(ESI-TOF)m/z:Calcd.for C25H15FN2NaO4[M+Na]+:449.0908；Found:449.0911.
this example prepares compound 3d as a yellow solid, melting point:>300.0 ℃; yield 82%; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.93(s,1H),6.99(s,1H),7.07(s,1H),7.11-7.18(m,5H),7.26-7.28(m,1H),7.58-7.66(m,3H),8.12(d,J＝7.2Hz,2H),13.21(brs,1H)；13C NMR(DMSO-d6,100MHz)δ:31.1,56.1,95.2,105.7,106.3,107.3,116.0(d,JCF＝21.0Hz),120.2,125.0,127.1,129.5(d,JCF＝23.3Hz),130.5,130.8,131.4,132.8,154.4,155.8,158.3,160.0,160.5(d,JCF＝244.2Hz),164.6,182.7；HRMS(ESI-TOF)m/z:Calcd.forC25H15FN2NaO4[M+Na]+:449.0908；Found:449.0911.
this example prepares compound 3e as a yellow solid, melting point:>300.0 ℃; yield 88 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.52(s,1H),6.19(s,1H),6.67(s,1H),6.89(s,2H),7.06(br s,2H),7.14(s,2H),7.54-7.56(m,3H),8.01-8.03(m,2H)；13C NMR(DMSO-d6,100MHz)δ:36.6,57.9,86.3,106.9,109.4,111.6,121.2,126.5,128.0,129.5,129.7,130.7,131.5,131.9,145.5,154.1,158.5,160.9,161.6,168.7,180.0；HRMS(ESI-TOF)m/z:Calcd.for C25H15ClN2NaO4[M+Na]+:465.0613；Found:465.0617.
this example prepares compound 3f as a yellow solid, melting point:>300.0 ℃; yield 80 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:5.16(s,1H),6.99(s,1H),7.07(s,1H),7.12-7.16(m,3H),7.21-7.27(m,2H),7.38-7.40(m,1H),7.56-7.64(m,3H),8.11(d,J＝6.0Hz,2H),13.18(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:30.7,55.6,94.8,105.3,106.1,106.8,119.5,126.7,129.2,129.6,130.4,132.0,141.4,154.0,155.4,157.9,159.4,164.2,182.4；HRMS(ESI-TOF)m/z:Calcd.for C25H15ClN2NaO4[M+Na]+:465.0613；Found:465.0617.
this example prepares compound 3g as a yellow solid, melting point:>300.0 ℃; yield 90 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.66(s,1H),6.94(s,1H),7.04(s,1H),7.13-7.20(m,4H),7.49-7.61(m,5H),8.08(d,J＝6.8Hz,1H),13.26(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:35.9,57.2,95.4,105.7,107.2,107.4,120.3,127.1,129.6,130.0,130.8,131.8,132.8,144.5,154.1,155.8,158.3,159.9,164.6,182.8；HRMS(ESI-TOF)m/z:Calcd.for C25H15BrN2NaO4[M+Na]+:509.0107；Found:509.0113.
this example prepared compound 3h as a yellow solid, melting point:>300.0 ℃; yield 86 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.53(s,1H),6.19(s,1H),6.66(s,1H),6.93-6.94(m,3H),7.19-7.25(m,3H),7.54-7.56(m,3H),8.01(d,J＝5.6Hz,2H)；13C NMR(DMSO-d6,100MHz)δ:36.8,57.8,86.2,107.0,109.3,111.8,121.2,121.7,126.5,127.2,129.2,129.5,130.2,131.5,131.9,149.5,154.2,158.6,161.0,161.3,168.9,180.0；HRMS(ESI-TOF)m/z:Calcd.for C25H15BrN2NaO4[M+Na]+:509.0107；Found:509.0103.
this example prepares compound 3i as a yellow solid, melting point:>300.0 ℃; yield 85 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,500MHz)δ:5.10(s,1H),6.87(s,1H),6.95(s,1H),6.99-7.15(m,3H),7.22-7.26(m,1H),7.46-7.62(m,4H),8.01-8.07(m,2H),13.09(br s,1H)；13C NMR(DMSO-d6,125MHz)δ:36.0,56.5,95.2,105.7,106.8,107.3,119.9,123.1,127.1,128.8,129.2,129.6,130.8,132.9,133.2,143.8,154.4,155.9,158.6,159.8,164.6,182.7；HRMS(ESI-TOF)m/z:Calcd.for C25H15BrN2NaO4[M+Na]+:509.0107；Found:509.0106.
this implementationExample preparation of compound 3j yellow solid, melting point:>300.0 ℃; yield 78%; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.92(s,1H),7.00(s,1H),7.05(s,1H),7.29(s,2H),7.55-7.68(m,5H),8.01(s,1H),8.08(d,J＝6.0Hz,3H),13.30(br s,1H)；13CNMR(DMSO-d6,100MHz)δ:36.1,56.7,95.6,105.8,113.6,122.1,122.5,126.4,127.1,128.8,129.6,130.6,132.9,134.6,147.2,148.3,154.0,156.0,158.3,160.1,164.7,182.8；HRMS(ESI-TOF)m/z:Calcd.for C25H15N3NaO6[M+Na]+:476.0853；Found:476.0854.
this example prepares compound 3k as a yellow solid, melting point:>300.0 ℃; yield 87%; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:2.23(s,3H),4.60(s,1H),6.95(s,1H),7.03-7.12(m,7H),7.55-7.63(m,3H),8.09(d,J＝6.0Hz,2H),13.23(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:20.6,35.5,57.3,94.9,105.2,106.9,107.6,120.0,126.6,127.0,129.0,129.1,130.4,135.9,141.7,153.7,155.2,157.7,159.4,164.1,182.4；HRMS(ESI-TOF)m/z:Calcd.for C26H18N2NaO4[M+Na]+:445.1159；Found:445.1162.
this example prepares compound 3l as a yellow solid, melting point:>300.0 ℃; yield 85 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:4.63(s,1H),6.95(s,1H),7.04(s,1H),7.09-7.11(m,4H),7.31(d,J＝7.2Hz,2H),7.54-7.61(m,3H),8.08(d,J＝7.2Hz,2H),13.23(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:31.6,34.6,35.8,57.8,95.4,105.7,107.4,108.2,120.5,125.7,127.1,127.2,129.6,130.8,132.8,142.1,149.5,154.3,155.6,158.1,160.1,164.6,182.8；HRMS(ESI-TOF)m/z:Calcd.for C29H24N2NaO4[M+Na]+:487.1628；Found:487.1627.
this example prepares compound 3m as a yellow solid, melting point:>300.0 ℃; yield 84 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:3.59(s,3H),4.54(s,1H),6.17(s,1H),6.58-6.68(m,3H),6.81(s,2H),7.12(s,2H),7.52-7.59(m,3H),8.01-8.03(m,2H)；13C NMR(DMSO-d6,100MHz)δ:36.1,55.3,58.7,86.2,106.9,110.5,111.6,113.6,121.4,126.4,128.8,129.5,131.5,131.9,138.8,154.3,157.9,158.3,161.0,161.3,168.9,180.1；HRMS(ESI-TOF)m/z:Calcd.for C26H18N2NaO5[M+Na]+:461.1108；Found:461.1107.
this example prepares compound 3n as a yellow solid, melting point:>300.0 ℃; yield 81 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:3.71(s,3H),4.91(s,1H),6.82-7.02(m,7H),7.17-7.21(m,1H),7.52-7.62(m,3H),8.03-8.11(m,2H),13.09(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:31.9,56.1,56.6,94.9,105.7,107.2,107.5,112.3,120.6,120.9,127.1,128.5,129.4,129.6,130.9,132.6,132.8,155.1,155.6,157.4,158.0,160.4,164.5,182.8；HRMS(ESI-TOF)m/z:Calcd.for C26H18N2NaO5[M+Na]+:461.1108；Found:461.1108.
this example prepared compound 3o a yellow solid, melting point:>300.0 ℃; yield 79 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:3.64(s,3H),3.78(s,3H),4.89(s,1H),6.64(d,J＝7.2Hz,1H),6.89-7.04(m,5H),7.57-7.64(m,3H),8.11(d,J＝7.2Hz,2H),13.14(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:31.2,32.0,56.0,60.4,95.1,105.7,107.1,107.9,111.9,121.6,124.0,127.1,129.6,130.9,132.9,137.7,146.8,152.8,154.7,155.8,158.2,159.7,164.6,182.7；HRMS(ESI-TOF)m/z:Calcd.for C27H20N2NaO6[M+Na]+:491.1214；Found:491.1211.
this example prepares compound 3p as a yellow solid, melting point:>300.0 ℃; yield 80 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:2.19(s,3H),2.43(s,3H),4.86(s,1H),6.73(d,J＝7.6Hz,1H),6.87-6.99(m,6H),7.55-7.64(m,3H),8.07(d,J＝7.2Hz,2H),13.12(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:19.4,21.0,31.1,58.0,95.2,105.7,107.3,108.5,120.4,127.1,127.6,128.4,129.6,130.9,131.1,132.8,135.0,135.8,141.0,154.3,155.6,158.2,159.5,164.6,182.8；HRMS(ESI-TOF)m/z:Calcd.for C27H20N2NaO4[M+Na]+:459.1315；Found:459.1321.
this example prepares compound 3q as a yellow solid, melting point:>300.0 ℃; yield 77%; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:2.25(s,3H),4.02(s,3H),4.65(s,1H),7.05-7.11(m,5H),7.22(s,2H),7.59-7.65(m,3H),8.07-8.09(m,2H),12.85(br s,1H)；13CNMR(DMSO-d6,100MHz)δ:21.1,36.1,57.8,62.7,105.7,107.0,108.3,120.4,126.9,127.5,128.2,129.5,129.8,131.0,136.4,142.1,147.9,148.3,152.6,159.9,164.4,183.0；HRMS(ESI-TOF)m/z:Calcd.for C27H20N2NaO5[M+Na]+:475.1264；Found:475.1267.
this example prepares compound 3r as a yellow solid, melting point:>300.0 ℃; yield 79 percent; the results of nuclear magnetic resonance and high resolution mass spectrometry are as follows:1H NMR(DMSO-d6,400MHz)δ:3.86(s,3H),3.91(s,3H),4.64(s,1H),6.69-6.71(m,2H),6.82(s,1H),6.88(s,1H),7.09-7.22(m,6H),7.91(d,J＝9.6Hz,1H),13.37(br s,1H)；13C NMR(DMSO-d6,100MHz)δ:35.6,56.1,56.6,57.5,95.1,99.3,106.8,106.9,107.2,108.2,111.6,115.6(d,JCF＝21.2Hz),129.5,129.6,130.9,141.4,153.8,155.6,158.1,159.9,160.4,161.8(d,JCF＝241.4Hz),164.2,182.6；HRMS(ESI-TOF)m/z:Calcd.for C27H19FN2NaO6[M+Na]+:509.1119；Found:509.1121.
the compound of formula (1) of the invention has important biological activity, and cytotoxicity tests on human leukemia cells (K562), human lung adenocarcinoma cells (A549), human lung adenocarcinoma cells (H1299) and human prostate cancer cells (PC-3) in vitro show that: the chrysin spliced 2-amino 3-cyanopyran compound with the structure shown in the formula (1) has an inhibiting effect on the growth of tumor cells, and can be possibly developed into a new tumor prevention and treatment drug. It is to be emphasized, however, that the compounds of the invention are not limited to the cytotoxicity represented by human leukemia cells (K562), human lung adenocarcinoma cells (A549), human lung adenocarcinoma cells (H1299), human prostate cancer cells (PC-3).
Pharmacological example 1: cytotoxicity of Compounds 3a-3p and 3r against human leukemia cells (K562), human Lung adenocarcinoma cells (A549), human Lung adenocarcinoma cells (H1299), human prostate cancer cells (PC-3)
Human leukemia cells (K562), human lung adenocarcinoma cells (A549), human lung adenocarcinoma cells (H1299) and human prostate cancer cells (PC-3) were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin, respectively. Cells were added to 96 wells at a concentration of 5000 cells per well and 5% CO at 37 deg.C2Incubate in a humidified air incubator for 24 hours.
The cell viability was determined by the modified MTT method. After 24 hours incubation of the cells, a solution of the newly formulated compounds 3a-3p and 3r in dimethylsulfoxide was added to each well in a concentration gradient such that the final concentration of the compounds in the wells was 5,10,20, 40 and 80 μmol/L, respectively. After 48 hours, 10. mu.L of MTT (5mg/mL) in phosphate buffer was added to each well, and after further incubation at 37 ℃ for 4 hours, the unconverted MTT was removed by centrifugation for 5 minutes, and 150. mu.L of dimethyl sulfoxide was added to each well. The OD value was measured at 490nm wavelength with a microplate reader by dissolving reduced MTT crystal formazan (formazan). The results are shown in table 3 below.
TABLE 3
Figure BDA0002389939670000101
aIC50:The concentration which results in 50％ of tumour cellproliferation inhibition after 48h of compounds treatment.Data wererepresented obtained in at least three independent experiments
bParent compound chrysin used as a positive control.
cDDP(cisplatin)used as the reference drug.
Pharmacological example 2: AO-EB dyeing method
Exponentially growing K562 cells (5X 105 cells/well) were seeded in 6-well plates (nested Biotechnology, China) and incubated for 12h, different concentrations of compound were added to each well for 3h, co-cultured with the cells for 24h, the cells were taken, washed with ice-cold PBS, and 50. mu.L of freshly prepared AO-EB staining solution (100. mu.g/mL AO and 100. mu.g/mL EB in PBS) was stained in the dark at room temperature for 10 min. Finally, the stained cells were observed under a fluorescent microscope (lycra, germany).
The compound is studied for inducing apoptosis of K562 cells for 3h by using AO-EB double staining method. When the cells stained with AO-EB were observed under a fluorescence microscope, the result is shown in FIG. 8A, the normal group showed uniform green fluorescence, while the treated group showed gradually decreased green fluorescence and corresponding gradually increased orange fluorescence with the increase of the compound concentration for 3 h. Suggesting that the compound can induce K562 cell apoptosis in 3 h.
Pharmacological example 3: flow cytometry analysis of apoptosis
Exponentially growing K562 cells (5X 105 cells/well) were seeded in 6-well culture plates, incubated for 12h, different concentrations of compound were added to each well for 3h, co-cultured with the cells for 24h, the cells were removed, and washed with ice-cold PBS. Finally, Annexin V-FITC and PI were added according to the instructions of the apoptosis detection kit (CoWin Biosciences, China). After 15min of incubation, the samples were analyzed with a flow cytometer (BD Bioscience, USA).
The degree of apoptosis of K562 cells was investigated. And incubating the compound with different concentrations for 3h and K562 cells for 24 hours, and detecting apoptosis by Annexin-V-FITC/PI double staining flow cytometry. As shown in fig. 8B, the rate of apoptosis of K562 cells gradually increased with increasing concentration of compound for 3h, especially at high concentrations. Thus, these data suggest that compound 3h induces apoptosis in K562 cells in a concentration-dependent manner.
Pharmacological example 4: flow cytometry analysis of the cell cycle
Cells at 5X 105 log phase were plated in 6-well plates and incubated for 12h, compounds were added at different concentrations in each well for 3h, co-incubated with cells for 24h, cells were harvested and washed with ice-cold PBS. Subsequently, 70% ice cold ethanol was added and the cells were fixed at 4 ℃ overnight. After washing with frozen PBS, the cells were stained with 500. mu.L of PI/RNase staining buffer (KeygEN BioTECH, USA) in the dark at room temperature for 30min for flow cytometry analysis.
The effect of compound 3h on the K562 cell cycle was evaluated. After 3h treatment with different concentrations of compound for 24h, cells were taken for flow cytometry analysis. As shown in figure 9, compound 3h had a significant effect on the redistribution of the K562 cell cycle. When the concentration was 5. mu.M, the distribution of G1 phase cells was significantly increased and the distribution of S phase cells was significantly decreased, as compared with the control group. The results show that the G1 phase block occurs in cells, and the compound 3h induces the apoptosis of K562 cells by promoting the formation of cancer cell apoptosis DNA.
Pharmacological example 5: western blot analysis
After co-culturing 3h of different concentrations of compound with K562 cells for 24 hours, the cells were collected and washed with ice-cold PBS, after which the collected cells were lysed in 100 μ l of dissolution buffer containing PMSF for 15 minutes on ice, the cell lysates were centrifuged at 12000 × g for 15 minutes and the total protein concentration was determined using BCA protein assay kit (both from chinese bergin.) the samples were boiled at 100 ℃ for 5 minutes with 5 × loading buffer and then used for subsequent experiments or stored at-80 ℃ for a long period of time, next, the equivalent protein (20 μ g) was separated on 8-12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) skim membranes (microwell, usa) which were blocked at room temperature in TBST (Tris buffered saline Tween 20) containing 5% milk for 1h, then washed three times with appropriate primary antibodies at 4 ℃ after washing with TBST, incubated with anti-rabbit secondary antibodies 1h, washed with clarittm-Western-Tween-tm-Tween, and incubated with anti-rabbit signal (# 12) from Western blot (# 12) and visualized by cell-Western blotting (# 12) for signal (# 12, (# 12) by Western blotting for cells (# 12) for three times (# 12) with anti-rabbit secondary antibodies.
The expression levels of the anti-apoptotic key protein Bcl-2 and the pro-apoptotic effector protein Bax in the Bcl-2 family members were examined by western blot analysis. As shown in figure 10, compound 3h treatment significantly down-regulated Bcl-2 and up-regulated Bax protein expression in a dose-dependent manner, enabling the promotion of cytochrome c and other pro-apoptotic factors release from mitochondria a 36. The results show that the compound 3h can induce K562 cell apoptosis through an endogenous apoptosis pathway.
Note that experimental data are expressed as mean. + -. standard deviation obtained in at least three independent experiments. SPSS19.0 statistical software was used for one-way analysis of variance, LSD was used for group-to-group comparisons, and significance testing was performed. P <0.05 and P <0.01 are considered significant and very significant, respectively.
And (4) experimental conclusion: the in vitro anticancer activity evaluation of the newly synthesized compound is carried out, the antiproliferative activity of partial compound on K562, PC-3, A549 and NCI-H1299 is stronger than that of parent compound chrysin, and compared with cisplatin control drug, the compound has equipotential effect. Especially, the compound 3h has the highest cytotoxicity (IC) on K562 cells506.41 μ M). In addition, compound 3h induced apoptosis in K562 cells in a concentration-dependent manner. Mechanistic studies indicate that compound 3h induces apoptosis, a mechanism that may be associated with mitochondrial-mediated caspase activation. The results show that the composite 3h treatment can increase Bax and reduce Bcl-2 protein level, thereby increasing Bax/Bcl-2 ratio, releasing cytochrome c, activating caspase and causing apoptosis. Thus, our results provide in vitro evidence that compound 3h may be a potential candidate for the development of new antitumor drugs.
Therefore, the experiment shows that the chrysin spliced 2-amino 3-cyanopyran compound shown in the formula (1) has stronger cytotoxicity on human leukemia cells (K562), human lung adenocarcinoma cells (A549), human lung adenocarcinoma cells (H1299) and human prostate cancer cells (PC-3), has the same order of magnitude as or better activity than cisplatin as a first-line medicament for tumor treatment, and can be possibly developed into a new medicament with an anti-tumor effect.

Claims (6)

1. A2-amino 3-cyanopyran compound spliced by chrysin is characterized in that: the compound has a structure shown as a general formula (I):
Figure FDA0002389939660000011
in the formula, R1Is H or methoxy; r2Is nitro, methoxy, tert-butyl, methyl, fluorine, chlorine, bromine or hydrogen; r3Is methoxy or hydrogen.
2. A method for preparing the chrysin spliced 2-amino 3-cyanopyran compound as claimed in claim 1, which is characterized by comprising the following steps: in an organic solvent, under the action of an inorganic basic catalyst, performing a regioselective Michael cycloaddition reaction on a Knoevenagel product obtained by condensing various substituted benzaldehydes and malononitrile and various substituted chrysins to obtain the chrysin spliced 2-amino 3-cyanopyran compounds.
3. The method for preparing the chrysin-spliced 2-amino 3-cyanopyran compound as claimed in claim 2, wherein: the organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
4. The method for preparing the chrysin-spliced 2-amino 3-cyanopyran compound as claimed in claim 2, wherein: the inorganic alkaline catalyst is as follows: sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium carbonate or sodium carbonate.
5. The method for preparing the chrysin-spliced 2-amino 3-cyanopyran compound as claimed in claim 2, wherein: the Knoevennagel product of the condensation of various substituted benzaldehydes and malononitrile undergoes a regioselective Michael cycloaddition reaction with various substituted chrysins at a temperature of from room temperature to 70 ℃ for a period of from 1 to 10 hours.
6. The use of the chrysin-spliced 2-amino 3-cyanopyran compound of claim 1 in the preparation of medicaments for preventing and treating tumor diseases.
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