Chalcone derivative, preparation method thereof and application of chalcone derivative in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly discloses a chalcone derivative and a preparation method thereof.The method includes the steps that 2, 4-dihydroxy formaldehyde serves as an initial raw material, isopentenyl is introduced into 2, 4-dihydroxy benzaldehyde through a Friedel-Crafts reaction, then the 2, 4-dihydroxy benzaldehyde and heterocycle substituted formaldehyde are subjected to an aldol condensation reaction, heterocycle is introduced, a chalcone structure is formed, and the chalcone derivative is obtained. The prepared chalcone derivative has a structural formula as shown in a formula I, physicochemical properties of the chalcone derivative are studied, and the chalcone derivatives I-4, I-5, I-7, I-8, I-9, I-10, I-11, I-12 and I-14 all show relatively strong effects of inhibiting proliferation of four tumor cells (PC9, MDA-MB-231, SMMC-7721 and SGC-7901). The chalcone derivative I-4 can effectively inhibit proliferation of human lung cancer PC9 and H1975 cells, shows concentration dependence, shows good in-vivo anti-tumor activity, can induce ferroptosis of the human lung cancer PC9 and H1975 cells, and has a good application prospect of anti-cancer drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a chalcone derivative and a preparation method thereof, and application of a pharmaceutical composition with the chalcone derivative as an active ingredient in treating malignant tumors. Background Art

[0002] Chalcone has an α, β unsaturated ketone structure. The presence of double bonds conjugated with carbonyl functional groups is believed to be the reason for the biological activity of chalcone, which has anti-cancer, anti-inflammatory, antibacterial, and antiviral pharmacological activities. Chalcone belongs to the flavonoid family and is an intermediate in the biosynthesis of flavonoids. It has structural specificity and can act on a variety of drug targets. Chalcone is an important structure of many bioactive molecules. Studies have shown that 4,4'-dimethoxychalcone (DMC) can inhibit the activity of human ferrochelatase (FECH) and induce Keap1 degradation through the ubiquitin-proteasome system, leading to the nuclear translocation of Nrf2 and upregulating the Nrf2 target gene HMOX1, thereby inducing ferroptosis in cancer cells. However, the mechanism of action of chalcone compounds inducing ferroptosis in lung cancer cells has not been reported.

[0003] Summary of the invention

[0004] The purpose of the present invention is to provide a series of chalcone derivatives, which use cheap and readily available 2,4-dihydroxybenzaldehyde as an initial raw material, first introduce an isopentenyl group into 2,4-dihydroxybenzaldehyde through a Friedel-Crafts reaction, and then introduce a heterocycle through an aldol condensation reaction to form a chalcone structure, so as to improve the anticancer activity of the chalcone derivatives.

[0005] To achieve the above object, the present invention provides a series of chalcone derivatives having the structural formula shown in Formula I:

[0006]

[0007] In Formula I, when R1 and R2 are both -CH3, R3 is selected from

[0008] When R1 and R2 are both When R3 is

[0009] The preparation method of the chalcone derivative shown in formula I is: dissolving the raw material compound in ethanol, adding potassium hydroxide and a heterocyclic substituted formaldehyde derivative, stirring at room temperature until the reaction is completed, adding water to quench, extracting, drying, and purifying to obtain the chalcone derivative shown in formula I;

[0010] The structural formula of the raw material compound is:

[0011] Wherein, when R1 and R2 are both -CH3, the heterocyclic substituted formaldehyde derivative is:

[0012] When R1 and R2 are both When the heterocyclic substituted formaldehyde derivative is:

[0013] The molar ratio of the raw material compound to the heterocyclic substituted formaldehyde derivative is 1:(2-4); the molar ratio of the raw material compound to potassium hydroxide is 1:(4.5-9).

[0014] The chalcone derivatives shown in the above formula I use 2,4-dihydroxybenzaldehyde as the initial raw material, first prepare the raw material compound, and then synthesize the chalcone derivatives. The initial raw materials are cheap and easy to obtain, the preparation process is simple and feasible, pollution-free, the yield is stable, and the repeatability is good. Among the prepared chalcone derivatives, 9 kinds show strong inhibition of the proliferation of 4 tumor cells (PC9, MDA-MB-231, SMMC-7721, SGC-7901). Among them, derivative I-4 can effectively inhibit the proliferation of human lung cancer PC9 and H1975 cells, and presents concentration dependence, showing good in vivo anti-tumor activity, and can induce ferroptosis of human lung cancer PC9 and H1975 cells.

[0015] The present invention also provides the use of the chalcone derivatives shown in formula I in the preparation of anti-tumor drugs, wherein the tumor is lung cancer cells, breast cancer cells, liver cancer cells or gastric cancer cells.

[0016] The chalcone derivatives of the present invention have strong cytotoxicity to four types of tumor cells. Among them, the derivative I-4 can effectively inhibit the proliferation of human lung cancer PC9 and H1975 cells, and is concentration-dependent, showing good in vivo anti-tumor activity, and can induce ferroptosis of human lung cancer PC9 and H1975 cells, and has a good application prospect of anticancer drugs.

[0017] The chalcone derivatives are used to prepare the dosage form of intestinal or parenteral combination medicine. The dosage form is liquid preparation, tablet, granule, granule, pill, capsule, sustained release agent, dripping pill or injection. The dosage form is administered orally or by injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of chalcone derivative I-4 on the proliferation of human lung cancer PC9 and H1975 cells.

[0019] Figure 2 The effect of chalcone derivative I-4 alone and in combination with ferroptosis inhibitors Fer-1 and DFO on cell survival rate.

[0020] Figure 3 The changes in the submicroscopic structure of cells after treatment with the chalcone derivative I-4 were observed using transmission electron microscopy.

[0021] Figure 4 The effect of chalcone derivative I-4 on glutathione content in lung cancer PC9 and H1975 cells.

[0022] Figure 5 The effect of chalcone derivative I-4 on iron content in lung cancer PC9 and H1975 cells.

[0023] Figure 6 Effects of chalcone derivative I-4 on LPO and MDA content in lung cancer PC9 and H1975 cells

[0024] Figure 7 Effects of chalcone derivative I-4 on ROS content in lung cancer PC9 and H1975 cells

[0025] Figure 8 Effects of chalcone derivative I-4 on the expression of ferroptosis-related proteins in lung cancer PC9 and H1975 cells DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the embodiments. It should be understood that the examples and implementations described herein are for illustration only, and those skilled in the art may make various modifications or changes based on them, all of which fall within the scope of protection of the present invention.

[0027] The preparation of the raw material compounds (Compound 2, Compound 3 and Compound 4) are all prior art, and the synthesis routes are as follows, and the specific process is not repeated here:

[0028]

[0029] Example 1. Preparation of chalcone derivatives I-1 to I-12:

[0030] Compound 2 (0.2 mM) was dissolved in ethanol (2 mL), potassium hydroxide (0.9 mM) and 3-thiophenecarboxaldehyde (0.4 mM) were added, and stirred at room temperature for 4 to 6 h. After the reaction was completed by TLC monitoring, water was added to the reaction system to quench, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and spin-dried. Petroleum ether: ethyl acetate (20:1) eluent was eluted through column chromatography to obtain chalcone derivative I-1.

[0031]

[0032] Chalcone derivative I-1 (yellow solid, yield 82%, 68 mg), 1H-NMR (CDCl3, 400MHz): δ7.66 (1H, d, J = 15.7Hz), 7.56 (1H, s), 7.52 (1H, dd, J = 2.9Hz), 7.38-7.31 (2H, m), 7.36 (1H, m), 6 .45(1H,s),5.27(1H,m),3.91(3H,s),3.90(3H,s),3.26(2H,d,J=7.3Hz),1.72(3H,d,J=1.4Hz),1.70(3H,d,J=1.3Hz); 13 C NMR (CDCl3, 101MHz): δ191.0 (C=O), 161.7 (C-4), 159.1 (C-2), 138.9 (C-β), 135.4 (C-3”), 132.7 (C-4'), 132.0 (C-1'), 128.0 (C-2'), 127.3 (C-3'), 126.7 (C-6), 125.5 (C-5), 122.9 (C-2”), 122.3 (C-α), 121.2 (C-1), 95.1 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1”), 25.9 (C-4”), 17.9 (C-5”); HR-ESI-MS: molecular formula is C 20 H 22 O3S,m / z343.13629[M+H] + .

[0033] The preparation method of chalcone derivatives I-2 to I-12 is the same as that of I-1, except that the substituted heterocycle of the formaldehyde derivative is different. The amount of potassium hydroxide added is 0.9 to 1.8 mM, and the amount of the heterocycle substituted formaldehyde derivative added is 0.4 to 0.8 mM, which will not be repeated here.

[0034] Chalcone derivative I-2 (yellow solid, yield 70%, 45 mg), 1 H-NMR (CDCl3, 400MHz): δ7.70(1H,s),7.59(1H,d,J=16.5Hz),7.57(1H,s),7.46(1H,d,J=1.8Hz),7.27(1H,d,J=15.5Hz),6.67(1 H,d,J=1.9Hz),6.47(1H,s),5.29(1H,tq,J=5.8Hz),3.93(6H,s,H-7),3.28(2H,d,J=7.3Hz),1.74(3H,q,J=1.4Hz),1.72(3H,m); 13C NMR (CDCl3, 101MHz): δ190.7 (C=O), 161.7 (C-4), 159.1 (C-2), 144.9 (C-β), 144.3 (C-3'), 132.7 (C-4'), 132.0 (C-3', C-α), 127.5 (C-6), 123.7 (C-5), 122.9 (C-1'), 122.3 (C-2"), 121.1 (C-1), 107.8 (C-2'), 95.1 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1"), 25.9 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 20 H 22 O4,m / z327.15897[M+H] + .

[0035] Chalcone derivative I-3 (yellow solid, yield 30%, 46 mg), 1 H-NMR (CDCl3, 400MHz): δ8.19 (1H, s), 8.17 (1H, s), 8.05 (1H, d, J = 15.8Hz), 7.84(1H,d,J=15.8Hz),7.81(1H,d,J=8.3Hz),7.73(1H,m),7.68(1H,d,J=8 .5Hz),7.63(1H,s),7.54(1H,m),6.46(1H,s),5.28(1H,m),3.95(3H,s),3. 91(3H,s),3.26(2H,d,J=7.3Hz),1.74-1.72(3H,m),1.70(3H,q,J=1.3Hz); 13 C NMR(CDCl3,101MHz): δ190.6(C=O),162.2(C-4),159.6(C-2),154.5(C-9'),148.1(C-8'),140 .6(C-β),137.0(C-2'),133.0(C-3”),132.8(C-7),132.1(C-6'),130.2(C-5'),129.7(C-3'), 128.1(C-4'),127.7(C-7'),127.3(C-6),123.0(C-5),122.3(C-α),120.8(C-2"),120.7(C-1),95.0(C-3),56.2(C-7),55.7(C-8),27.8(C-1"),26.0(C-4"),17.9(C-5"); HR-ESI-MS: molecular formula is C 25 H 25NO3,m / z 388.19122[M+H] + .

[0036] Chalcone derivative I-4 (yellow solid, yield 22%, 30 mg), 1 H-NMR (CDCl3, 400MHz): δ8.66 (1H, dd, J = 4.9Hz), 8.02 (1H, d, J = 15.5Hz), 7.7 3(1H,td,J=7.7Hz),7.64(1H,d,J=15.5Hz),7.60(1H,s),7.49(1H,d,J=7.8H z),7.32-7.21(1H,m),6.44(1H,s),5.26(1H,m,J=8.9Hz),3.94(3H,s),3.90 (3H,s),3.25(2H,d,J=7.3Hz),1.72(3H,d,J=1.4Hz),1.69(3H,d,J=1.3Hz); 13 C NMR (CDCl3, 101MHz): δ190.5 (C=O), 162.2 (C-4), 159.7 (C-2), 153.9 (C-1'), 149.7 (C-5'), 139.5 (C-β), 137.3 (C-3'), 132.7 (C-3"), 132.1 (C-α), 124.8 (C-6), 124.0 (C-5), 122.9 (C-2'), 122.3 (C-2"), 120.9 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1"), 25.9 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 21 H 23 NO3,m / z 338.17432[M+H] + .

[0037] Chalcone derivative I-5 (yellow solid, yield 45%, 64 mg), 1 H-NMR (CDCl3, 400MHz): δ8.82(1H,s),8.04(1H,s),7.80(1H,d,J=15.4Hz), 7.59(1H,s),7.41(1H,d,J=15.4Hz), 6.43(1H,s),5.26(1H,m),3.93(3H,s),3.91(3H,s),3.30-3.21(2H,m),1.71(3H,q,J=1.4Hz),1.70-1.68(3H,m); 13C NMR (CDCl3, 101MHz): δ189.0 (C=O), 162.3 (C-4), 159.5 (C-2), 154.0 (C-3'), 145.3 (C-2'), 136.7 (C-β), 132.8 (C-3"), 132.1 (C-α), 130.6 (C-2"), 129.9 (C-6), 123.2 (C-5), 122.2 (C-1'), 120.4 (C-1), 94.8 (C-3), 56.1 (C-7), 55.7 (C-8), 27.8 (C-1"), 25.9 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 19 H 21 NO3S,m / z 344.13181[M+H] + .

[0038] Chalcone derivative I-6 (yellow oil, yield 80%, 56 mg), 1 H-NMR (CDCl3, 400MHz): δ7.72 (1H, d, J = 15.4Hz), 7.58 (1H, s), 7.31 (1H, d, J = 15.4Hz), 6.80-6.76 (1H, m), 6.74 (1H, dd, J = 4.0Hz), 6.44 (1H, s) ,6.19(1H,dd,J=3.9Hz),5.27(1H,m),3.92(3H,s),3.90(3H,s),3.75(3H,s),3.27-3.24(2H,m),1.72(3H,q,J=1.4Hz),1.70(3H,q,J=1.3Hz); 13 C NMR (CDCl3, 101MHz): δ190.1(C=O), 161.6(C-4), 159.1(C-2), 132.7(C-β), 132.0(C-1'), 130.9(C-α), 130.2(C-1'), 127.4(C-6), 122.9(C-5), 122.5(C-4'), 122.4(C-2"), 121.6(C-1), 112.2(C-2'), 109.6(C-3'), 95.2(C-3), 56.3(C-7), 55.7(C-8), 27.8(C-1"), 26.0(C-4"), 17.9(C-5"); HR-ESI-MS: molecular formula is C 21 N 25 NO3,m / z 340.19025[M+H] + .

[0039] Chalcone derivative I-7 (yellow oil, yield 90%, 62 mg), 1 H-NMR (CDCl3, 400MHz): δ7.71(1H,s),7.59(1H,s),7.55(1H,d,J=15.7Hz),7.52(1H,s),7.46(1H,d,J=15.8Hz), 6.43(1H,s), 5.26(1H,m,J=7.4Hz),3.92(3H,s),3.90(3H,s),3.77(3H,s),3.24(2H,d,J=7.3Hz),1.71(3H,d,J=1.5Hz),1.70-1.66(3H,m); 13 C NMR (CDCl3, 101MHz): δ189.4 (C=O), 162.2 (C-4), 159.4 (C-2), 140.3 (C-3'), 132.8 (C-1'), 132.1 (C-β), 130.4 (C-3"), 130.3 (C-α), 126.6 (C-6), 125.8 (C-5), 123.1 (C-2'), 122.2 (C-2"), 120.7 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1"), 25.9 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 20 H 24 N2O3,m / z341.18524[M+H] + .

[0040] Chalcone derivative I-8 (yellow solid, yield 21%, 15 mg), 1 H-NMR (CDCl3, 400MHz): δ9.17(1H,s),8.93(4H,s),7.73(1H,d,J=15.9Hz),7.64(1H,s),7.57(1H,d,J=16.0Hz) ,6.45(1H,s),5.26(1H,m),3.95(3H,s),3.92(3H,s),3.28-3.19(2H,m),1.74-1.71(3H,m),1.71-1.68(3H,m); 13C NMR (CDCl3, 101MHz): δ188.9 (C=O), 162.7 (C-4), 159.7 (C-4'), 158.8 (C-2'), 155.8 (C-2, C-6'), 133.3 (C-β), 132.9 (C-α), 132.3 (C-3"), 131.2 (C-1'), 129.8 (C-6), 123.4 (C-5), 122.1 (C-2"), 120.2 (C-1), 94.7 (C-3), 56.1 (C-7), 55.8 (C-8), 27.8 (C-1"), 25.9 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 20 H 22 N2O3,m / z 339.17035[M+H] + .

[0041] Chalcone derivative I-9 (yellow solid, yield 25%, 18 mg), 1 H-NMR (CDCl3, 400MHz): δ8.50 (1H, d, J = 2.8Hz), 7.91 (1H, dd, J = 15.4Hz), 7.62 (1H, dd, J = 15.4Hz), 7.60 (1H, s), 7.48 (1H, dd,J=8.4Hz),7.40(1H,m),6.44(1H,s),5.27(1H,m),3.93(6H,s),3.25(2H,m),1.72(3H,q,J=1.3Hz),1.71-1.69(3H,m); 13 C NMR (CDCl3, 101MHz): δ190.5 (C=O), 162.2 (C-4'), 159.6 (C-4), 159.4 (C-2), 150.7 (C-1'), 138.9 (C-β), 138.6 (C-α), 132.8 (C-5'), 132.1 (C-3"), 131.1 (C-2"), 125.5 (C-6), 123.3 (C-5), 123.0 (C-3'), 122.3 (C-2'), 120.9 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1"), 26.0 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 21 H 22 FNO3,m / z 356.16489[M+H] + .

[0042] Chalcone derivative I-10 (yellow solid, yield 20%, 15 mg), 1H-NMR (CDCl3, 400MHz): δ8.59 (1H, d, J = 2.4Hz), 7.97 (1H, d, J = 15.5Hz), 7.67 (1H, dd, J = 8.3Hz), 7.60 (1H, m), 7.59 (1H, d, J = 15.5Hz), 7. 41(1H,dd,J=8.4Hz),6.44(1H,s),5.26(1H,m),3.93(3H,s),3.91(3H,s),3.30-3.16(2H,m),1.72(3H,q,J=1.3Hz),1.71-1.68(3H,m); 13 C NMR (CDCl3, 101MHz): δ190.4 (C=O), 162.3 (C-4), 159.7 (C-1'), 152.5 (C-2), 149.2 (C-5'), 138.6 (C-β), 136.4 (C-3'), 132.8 (C-α), 132.1 (C-4', C-3"), 131.7 (C-6), 125.1 (C-5), 123.0 (C-2"), 122.3 (C-2'), 120.8 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.8 (C-1"), 26.0 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 21 H 22 ClNO3,m / z 372.13654[M+H] + .

[0043] Chalcone derivative I-11 (yellow solid, yield 25%, 21 mg), 1 H-NMR (CDCl3, 400MHz): δ8.69 (1H, d, J = 2.3Hz), 7.98 (1H, d, J = 15.4Hz), 7.82 (1H, dd, J = 8.3Hz), 7.61 (1H, d, J = 0.8Hz), 7.57 (1H, d, J = 15. 4Hz),7.35(1H,dd,J=8.3Hz),6.44(1H,s),5.27(1H,m),3.93(3H,s),3.91(3H,s),3.25(2H,m),1.72(3H,q,J=1.3Hz),1.71-1.68(3H,m); 13C NMR (CDCl3, 101MHz): δ190.4 (C=O), 162.3 (C-4), 159.7 (C-2), 152.8 (C-1'), 151.3 (C-5'), 139.3 (C-3'), 138.7 (C-β), 132.8 (C-3"), 132.1 (C-α), 131.8 (C-2"), 125.5 (C-6), 123.0 (C-5), 122.3 (C-2'), 120.9 (C-4'), 120.8 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.9 (C-1"), 26.0 (C-4"), 17.9 (C-5"); HR-ESI-MS: molecular formula is C 21 H 22 BrNO3, m / z 416.08609[M+H] + .

[0044] Chalcone derivative I-12 (yellow solid, yield 20%, 14 mg), 1 H-NMR (CDCl3, 400MHz): δ8.48 (1H, d, J = 2.2Hz), 7.91 (1H, d, J = 15.5Hz), 7.63 (1H, d, J = 15.5Hz), 7.59 (1H, d, J = 0.7Hz), 7.50 (1H, m) ,7.38(1H,d,J=7.9Hz),6.44(1H,s),5.27(1H,m),3.92(3H,s),3.90(3H,s),3.25(2H,m),1.72(3H,q,J=1.3Hz),1.71-1.68(3H,m); 13 C NMR(CDCl3,101MHz): δ190.9(C=O),162.0(C-4),159.5(C-2),151.6(C-1'),150.8 (C-5'),140.5(C-β),137.2(C-3'),133.9(C-α),132.7(C-4'),132.1(C-3”),130.1 (C-6), 124.2 (C-5), 122.8 (C-2”), 122.4 (C-2'), 121.1 (C-1), 94.9 (C-3), 56.2 (C-7), 55.7 (C-8), 27.9 (C-1”), 26.0 (C-4”), 18.6 (C-5”), 17.9 (C-9); HR-ESI-MS: molecular formula is C 22 H 25 NO3,m / z 352.19080[M+H] + .

[0045] Example 2. Preparation of chalcone derivative I-13:

[0046] Compound 3 (0.2 mM) was dissolved in ethanol (2 mL), potassium hydroxide (1.8 mM) and pyridine-2-carboxaldehyde (0.6 mM) were added, and stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, water was added to the reaction system to quench, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and spin-dried. Petroleum ether: ethyl acetate (20:1) eluent was eluted by column chromatography to obtain chalcone derivative I-13.

[0047] Chalcone derivative I-13 (yellow solid, yield 37%, 25 mg), 1 H-NMR (CDCl3, 400MHz): δ8.64 (1H, m, J = 4.8Hz), 8.07 (1H, d, J = 15.5Hz), 7.68 (1H, td, J = 7.7Hz) ,7.65(1H,s),7.62(1H,d,J=15.5Hz),7.48(1H,dt,J=7.9Hz),7.22(1H,m),6.44(1H,s),5.55( 1H,m),5.48(1H,m),5.27(1H,m),4.67-4.52(4H,m,H-7),3.26(2H,d,J=7.4Hz),1.81(3H,q,J= 1.3Hz), 1.77 (3H, q, J = 1.3Hz), 1.73 (6H, q, J = 1.3Hz), 1.72-1.71 (3H, m), 1.69 (3H, d, J = 1.3Hz); 13 CNMR(CDCl3,101MHz): δ190.4(C=O),161.5(C-4),159.0(C-2),154.7(C-1'),150.1(C-5'),139.7(C-β) ,138.3(C-14),138.1(C-9),136.6(C-3'),132.5(C-α),132.1(C-3”),131.8(C-6),124.0(C-5),123.6( C-2'), 123.4 (C-2"), 122.5 (C-4'), 121.1 (C-1), 119.7 (C-8, C-13), 97.5 (C-3), 66.2 (C-7), 65.4 (C-12), 28.1 (C-1"), 26.0 (C-4"), 25.9 (C-10, C-16), 18.5 (C-5"), 18.4 (C-11), 17.9 (C-15); HR-ESI-MS: molecular formula is C 29 H 35 NO3,m / z 446.26807[M+H]+ .

[0048]

[0049] Example 3. Preparation of chalcone derivative I-14:

[0050] Compound 4 (0.2 mM) was dissolved in ethanol (2 mL), potassium hydroxide (1.8 mM) and pyridine-2-carboxaldehyde (0.6 mM) were added, and stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, water was added to the reaction system to quench, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and spin-dried. Petroleum ether: ethyl acetate (20:1) eluent was eluted by column chromatography to obtain chalcone derivative I-14.

[0051]

[0052] Compound I-14 (yellow oil, yield 36%, 40 mg), 1 H-NMR (CDCl3, 400MHz): δ8.64 (1H, m, J = 4.7Hz), 8.02 (1H, d, J = 15.5Hz), 7.69 ( 1H,td,J=7.7Hz),7.61(1H,s),7.64(1H,d,J=15.6Hz),7.47(1H,m),7.23(1H,m ),6.43(1H,s),6.12-6.00(2H,m),5.48-5.38(2H,m),5.34-5.29(2H,m),4.66- 4.57(4H,m),3.29(2H,dd,J=7.3Hz),1.72(3H,q,J=1.3Hz),1.71-1.68(3H,m); 13 C NMR(CDCl3,101MHz): δ190.8(C=O),160.8(C-4),158.4(C-2),154.3(C-1'),150.1(C-5'),14 0.1(C-β),136.7(C-3'),132.9(C-8),132.9(C-11),132.7(C-α),132.1(C-3”),131.4(C-6),1 24.3(C-5),123.8(C-2'),123.5(C-2"),122.3(C-4'),121.6(C-1),117.9(C-9),117.8(C-12),97.6(C-3),70.1(C-7),69.1(C-10),28.0(C-1"),26.0(C-4"),18(C-5"); HR-ESI-MS: molecular formula is C 25 H 27NO3,m / z 390.20566[M+H] + .

[0053] Example 4. MTT method was used to detect the effects of the chalcone derivatives I-1 to I-14 prepared in Example 1 and Example 2 on the proliferation of four tumor cells:

[0054] This experimental part evaluates the killing effect of chalcone derivatives I-1 to I-14 on four types of tumor cells.

[0055] (1) Experimental materials:

[0056] Cell lines: Human lung cancer PC9 cells, human breast cancer MDA-MB-231 cells, human liver cancer SMMC-7721 cells, and human gastric cancer SGC-7901 cells were purchased from the Shanghai Cell Bank, China.

[0057] Reagents and materials: 5-fluorouracil (5-FU) and MTT were purchased from Sigma, USA; DMEM and RPMI1640 culture medium were purchased from GIBCO Biotechnology Co., Ltd.; DMSO was purchased from Titan Technology Co., Ltd.; 0.25% trypsin, penicillin and streptomycin were purchased from Shanghai Beyotime Co., Ltd.; 96-well culture plates were purchased from Corning; fetal bovine serum was purchased from Ekosai Biotechnology Co., Ltd.

[0058] Instruments: SP-DJ series vertical clean bench (Shanghai General Physical Optical Instrument Factory), carbon dioxide incubator (Thermo Scientific), multifunctional microplate reader (BioTek, USA), inverted microscope (Olympus, Japan).

[0059] (2) Methods:

[0060] The above four tumor cells were planted in culture bottles containing DMEM or RPMI1640 (containing 10% inactivated fetal bovine serum, 100IU / l penicillin, 100μg / mL streptomycin) culture medium, and cultured in a 5% CO2, 37°C saturated humidity environment. Take the tumor cells in the logarithmic growth period, digest them with 0.25% trypsin to make a single cell suspension, centrifuge and collect the cells, inoculate them in a 96-well plate at a density of 5000 cells per well, and culture them in an incubator. After 24 hours of culture, treat with different concentrations of chalcone derivatives and 5-fluorouracil (positive control group), place in an incubator and continue to culture for 72 hours (chalcone derivatives act on human lung cancer PC9 and H1975 cells for 24 hours, 48 ​​hours and 72 hours). After the incubation, 10 μL of 5 g / L MTT solution was added to each well and incubated for another 4 hours. The culture medium was discarded and 100 μL of DMSO was added. The cells were incubated at 37°C for 30 minutes and shaken on a micro-oscillator for 10 minutes to fully dissolve the crystals. The absorbance (A) value of each well was detected at a wavelength of 490 nm using an ELISA reader to calculate the cell survival rate: Cell survival rate / % = Experimental group A 490 nm Control group A 490nm ×100%, draw the dose-effect curve.

[0061] (3) Experimental results: From the results in Table 1, it can be seen that chalcone derivatives I-4, I-5, I-7, I-8, I-9, I-10, I-11, I-12, and I-14 have strong in vitro inhibitory effects on the proliferation of the four tumor cells, and their half inhibitory concentrations (IC 50 ) values ​​ranged from 2.11 to 9.48 μmol / L. Figure 1 The results showed that with the increase of the administration concentration of chalcone derivative I-4 and the extension of the action time, the survival rate of human lung cancer PC9 cells gradually decreased, showing concentration and time dependence.

[0062] Table 1. Effects of chalcone derivatives I-1 to I-14 on the proliferation of four tumor cells (72h, IC 50 ,μM)

[0063]

[0064]

[0065] Example 5. Effect of chalcone derivative I-4 on inducing ferroptosis in human lung cancer PC9 and H1975 cells:

[0066] (1) Experimental materials:

[0067] Cell lines: Human lung cancer PC9 and H975 cells were purchased from Shanghai Cell Bank, China.

[0068] Reagents and materials: Fer-1 and DFO were purchased from MCE; MTT was purchased from Sigma, USA; DMEM and RPMI1640 culture medium were purchased from GIBCO Biotechnology Co., Ltd.; DMSO was purchased from Titan Technology Co., Ltd.; 0.25% trypsin, penicillin and streptomycin were purchased from Shanghai Beyotime Co., Ltd.; 96-well culture plates were purchased from Corning; and fetal bovine serum was purchased from Ekosai Biotechnology Co., Ltd.

[0069] Instruments: SP-DJ series vertical clean bench (Shanghai General Physical Optical Instrument Factory), carbon dioxide incubator (Thermo Scientific), multifunctional microplate reader (BioTek, USA), inverted microscope (Olympus, Japan).

[0070] (2) Methods:

[0071] PC9 and H1975 cells in the logarithmic growth phase were taken, digested with 0.25% trypsin to prepare a single cell suspension, centrifuged and collected, inoculated in a 96-well plate at a density of 5000 cells per well, and placed in an incubator for culture. After 24 hours of culture, they were pretreated with ferroptosis inhibitors Fer-1 (30 μM) and DFO (30 μM), respectively, and chalcone derivative I-4 (20 μM) was added after 2 hours, and culture was continued for 24 hours. After the end of the culture, 10 μL of MTT solution with a concentration of 5 g / L was added to each well and incubated for 4 hours, the culture medium was discarded, and 100 μL of DMSO was added, and it was placed at 37 ° C for 30 minutes. The micro-oscillator was shaken for 10 minutes to fully dissolve the crystals, and the absorbance (A) value of each well was detected at a wavelength of 490 nm using an enzyme marker to calculate the cell survival rate: Cell survival rate / % = experimental group A 490 nm Control group A 490nm × 100% and draw a bar graph.

[0072] PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then seeded in six-well plates and cultured in an incubator until the cell density reached 80%. The prepared chalcone derivative I-4 (20 μM) was added to the six-well plates. After 24 hours of action, the cells were collected by digestion and centrifugation and transferred to EP tubes. The cell pellets were fixed with electron microscopy fixative (2.5% glutaraldehyde) and stored at 4°C. Wuhan Sewell Biotechnology Co., Ltd. was entrusted to perform subsequent sectioning and photography.

[0073] (3) Experimental results:

[0074] By testing the cell survival rate after the chalcone derivative I-4 combined with the ferroptosis inhibition, the results showed that the cell survival rate after the combined use of the inhibitor increased by about 20% compared with the single administration. Figure 2 shown.

[0075] The morphological changes of mitochondria in lung cancer PC9 and H1975 cells treated with chalcone derivative I-4 were observed by transmission electron microscopy. Figure 3 As shown, the mitochondrial volume and mitochondrial cristae of lung cancer PC9 and H1975 cells in the drug-treated group were reduced, and the membrane density was increased.

[0076] The above phenomena indicate that the chalcone derivative I-4 induces ferroptosis in lung cancer PC9 and H1975 cells.

[0077] Example 6. Effect of chalcone derivative I-4 on glutathione in human lung cancer PC9 and H1975 cells:

[0078] (1) Experimental materials:

[0079] Reagents and materials: The large dish was purchased from Corning; the reduced glutathione (GSH) determination kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0080] Instrument: Multifunctional microplate reader (BioTek, USA).

[0081] (2) Methods:

[0082] Lung cancer PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then planted in a large dish and placed in an incubator for culture until the cell density reached 80%. The prepared chalcone derivative I-4 (5, 10, 20, 40 μM) of different concentrations was added to the six-well plate. After 24 hours of action, the cells were collected, centrifuged at 2500 rpm for 10 minutes, and the supernatant was discarded. 50 μL of RIPA lysis buffer containing protease inhibitors was added to each well, lysed on ice for 30 minutes, centrifuged at 12000 rpm for 30 minutes, and the supernatant was taken to measure the protein concentration by BCA quantitative method. The cell precipitate was ultrasonically broken under ice bath conditions, and the broken cell suspension was taken for use. According to the operation table of the instruction manual, a 20 μmol / L GSH standard solution was prepared, the relevant reagents were added to the cell suspension, mixed and allowed to stand for 5 minutes, and the absorbance was detected at 405 nm using an enzyme reader, and the GSH content in the cell was calculated according to the formula in the instruction manual.

[0083] (3) Experimental results:

[0084] Depend on Figure 4 It can be seen that as the administration concentration of the chalcone derivative I-4 increases, the glutathione content in lung cancer PC9 and H1975 cells decreases.

[0085] Example 7. Effect of chalcone derivative I-4 on iron content in human lung cancer PC9 and H1975 cells:

[0086] (1) Experimental materials:

[0087] Reagents and materials: The large dish was purchased from Corning; the tissue iron test kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0088] Instrument: Multifunctional microplate reader (BioTek, USA).

[0089] (2) Methods:

[0090] PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then planted in a large dish and placed in an incubator for culture until the cell concentration reached 80%. The prepared chalcone derivative I-4 (5, 10, 20, 40 μM) of different concentrations was added to the six-well plate. After 24 hours of action, the cells were collected and centrifuged at 2500 rpm for 10 minutes, and the supernatant was discarded. 50 μL of RIPA lysis buffer containing protease inhibitors was added to each well, and the cells were lysed on ice for 30 minutes, and then centrifuged at 12000 rpm for 30 minutes. The supernatant was taken to measure the protein concentration using the BCA quantitative method. The cell pellet was ultrasonically broken under ice bath conditions, and the broken cell suspension was taken for use. According to the instruction manual, prepare 2 mg / L iron standard application solution and iron colorimetric reagent, add relevant reagents to the cell suspension, mix well and place in a boiling water bath above 95°C for 5 minutes. After cooling, centrifuge at 3500r / min for 10 minutes, take 1 mL of supernatant, detect the absorbance at 520nm with an enzyme marker, and calculate the iron content in the cells according to the formula in the instruction manual.

[0091] (3) Experimental results:

[0092] like Figure 5 As shown, with the increase of the administration concentration of chalcone derivative I-4, the iron content in lung cancer PC9 and H1975 cells increased.

[0093] Example 8. Effect of chalcone derivative I-4 on lipid peroxides in human lung cancer PC9 and H1975 cells:

[0094] (1) Experimental materials:

[0095] Reagents and materials: The large dish was purchased from Corning; the lipid peroxide (LPO) test kit and the MDA assay kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0096] Instrument: Multifunctional microplate reader (BioTek, USA).

[0097] (2) Methods:

[0098] PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then planted in a large dish and placed in an incubator for culture until the cell concentration reached 80%. The prepared chalcone derivative I-4 (5, 10, 20, 40 μM) of different concentrations was added to the six-well plate. After 24 hours of action, the cells were collected and centrifuged at 2500 rpm for 10 minutes, and the supernatant was discarded. 50 μL of RIPA lysis buffer containing protease inhibitors was added to each well, and the cells were lysed on ice for 30 minutes, and then centrifuged at 12000 rpm for 30 minutes. The supernatant was taken to measure the protein concentration using the BCA quantitative method. The cell pellet was ultrasonically broken under ice bath conditions, and the broken cell suspension was taken for use. According to the operation table in the instruction manual, the standard application solution was prepared, and the relevant reagents were added to the cell suspension. After mixing, it was placed in a boiling water bath above 45°C for 60 minutes. After cooling, it was centrifuged at 4000r / min for 10 minutes. 200μL of the supernatant was taken, and the absorbance was detected at 586nm with an enzyme marker. The LPO content in the cells was calculated according to the formula in the instruction manual. Similarly, the MDA detection reagent was added, mixed, and placed in a boiling water bath at 90°C for 40 minutes. After centrifugation at 4000r / min for 10 minutes, 200μL of the supernatant was taken, and the absorbance was detected at 532nm with an enzyme marker. The MDA content in the cells was calculated according to the formula in the instruction manual.

[0099] (3) Experimental results:

[0100] like Figure 6 As shown, with the increase of administration concentration, the content of lipid peroxides in lung cancer PC9 and H1975 cells increased.

[0101] Example 9. Effect of chalcone derivative I-4 on ROS in human lung cancer PC9 and H1975 cells:

[0102] (1) Experimental materials:

[0103] Reagents and materials: Six-well plates were purchased from Corning; ROS detection kits were purchased from Shanghai Bio-Tech Co., Ltd.

[0104] Instrument: Flow cytometer (BD Company, USA).

[0105] (2) Methods:

[0106] PC9 and H1975 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension, inoculated in a six-well plate at a density of 300,000 cells per well, and cultured in an incubator. After the cell density reached 80%, the cells were treated with different concentrations (0, 5, 10, 20, 40 μM) of the chalcone derivative I-4 for 24 hours, and then the cells were collected and stained with the ROS detection reagent DCFH-DA for 0.5 hours, washed with PBS, and collected, and detected and analyzed by flow cytometry.

[0107] (3) Experimental results:

[0108] according to Figure 7 The results showed that the chalcone derivative I-4 increased the accumulation of ROS in lung cancer PC9 and H1975 cells in a dose-dependent manner.

[0109] Example 10. Immunoblotting to detect the effect of chalcone derivative I-4 on the expression of ferroptosis-related proteins xCT, GPX4, and Nrf2 in lung cancer PC9 and H1975 cells:

[0110] (1) Experimental materials:

[0111] Reagents and materials: PVDF membrane and exposure solution were purchased from Millipore, USA; GPX4 and xCT antibodies were purchased from Affinity; Nfr2 antibody was purchased from Affinity.

[0112] Instrument: Gel imaging system (BIO-RAD, USA).

[0113] (2) Methods:

[0114] PC9 and H975 lung cancer cells in the logarithmic growth phase were taken and inoculated in a 10×10 large dish. When the cell density reached 80%, the chalcone derivative I-4 was added to the large dish at different concentrations (0, 5, 10, 20, 40 μM). After the drug acted for 24 hours, the cells were collected and centrifuged, and the speed was set to 2500 rpm for 15 minutes. The supernatant was discarded and the cell pellet was retained. The liquid in the tube wall was sucked dry, and the lysis solution was added. After the ice bath lysis for an appropriate time, it was centrifuged at 4°C and the speed was 12000 r / min. After 30 minutes, the supernatant was sucked and quantified by the BCA method. 40 μg of protein was taken from each group for SDS-PAGE electrophoresis (the concentration gel constant voltage was 75V, the separation gel constant voltage was 110V, and the electrophoresis was stopped until the bromophenol blue fuel reached the front edge of the gel).

[0115] (Transfer): After electrophoresis, remove the gel and immerse the gel in an appropriate amount of Transfer buffer. At the same time, take a PVDF membrane of appropriate size and 4 pieces of 3M filter paper, soak the PVDF in anhydrous methanol first, and then immerse it in Transfer buffer together with the filter paper, put the membrane on the anode and the gel on the cathode, and pad 2 pieces of 3M filter paper on each side, keep a constant voltage of 80V, and transfer the membrane in a chromatography cabinet at 4℃ for 2 hours.

[0116] (Sealing): Wash the PVDF membrane 3 times (5 min / time) with 1×TBST, then put it in 5% skim milk and block it at 4°C for 4h. Remove the PVDF membrane from the skim milk, wash it 3 times (5 min / time), then put the membrane in the primary antibody, incubate it at 4°C for 12-16h, wash it 3 times (5 min / time), add the secondary antibody diluent, and incubate it at 4°C on a shaker for 2h. Wash the PVDF membrane 3 times (5 min / time) with 1×TBST, then soak the PVDF membrane in the exposure solution (A solution: B solution = 1:1) for an appropriate time and develop it using a gel imager.

[0117] (3) Experimental results: Figure 8 (A) shows that chalcone derivative I-4 significantly inhibited the expression of ferroptosis-related proteins xCT and GPX4 at the protein level. The transcriptional nuclear factor Nrf2 is a key regulator of cellular antioxidant responses, controlling the expression of genes that resist oxidative and electrophilic stress. As an upstream target of membrane proteins xCT and GPX4, Nrf2 is a key regulator of xCT / GPX4 signaling. The level of Nrf2 is directly related to ferroptosis, and increasing the expression of Nrf2 can inhibit ferroptosis. Figure 8 (B) showed that with the increase of the administration concentration of chalcone derivative I-4, the transcriptional nuclear factor Nrf2-related proteins were significantly downregulated, revealing that chalcone derivative I-4 reduced the expression of Nrf2.

Claims

1. A chalcone derivative, characterized in that It has the structural formula shown in Formula I: In Formula I, when R1 and R2 are both -CH3, R3 is When R1 and R2 are both When R3 is 2. The method for preparing a chalcone derivative according to claim 1, characterized in that: Specifically: The raw material compound is dissolved in ethanol, potassium hydroxide and a heterocyclic substituted formaldehyde derivative are added, and the mixture is stirred at room temperature until the reaction is completed, and then water is added to quench, extract, dry, and purify to obtain a chalcone derivative shown in formula I; The structural formula of the raw material compound is: Wherein, when R1 and R2 are both -CH3, the heterocyclic substituted formaldehyde derivative is When R1 and R2 are both When the heterocyclic substituted formaldehyde derivative is 3. The method for preparing a chalcone derivative according to claim 2, characterized in that: The molar ratio of the raw material compound to the heterocyclic substituted formaldehyde derivative is 1:(2-4).

4. The method for preparing a chalcone derivative according to claim 2 or 3, characterized in that: The molar ratio of the raw material compound to potassium hydroxide is 1:(4.5-9).

5. Use of the chalcone derivative according to claim 1 in the preparation of clinical anti-tumor drugs.

6. The use of the chalcone derivative according to claim 5 in the preparation of clinical anti-tumor drugs, characterized in that: The tumor is lung cancer, breast cancer, liver cancer or gastric cancer cell.

7. The use of the chalcone derivative in the preparation of clinical anti-tumor drugs according to claim 5, characterized in that: The chalcone derivatives are used to prepare the dosage form of enteral or parenteral combination medicine.

8. The use of the chalcone derivative in the preparation of clinical anti-tumor drugs according to claim 7, characterized in that: The dosage form is liquid preparation, tablet, granule, granule, pill, capsule, sustained-release preparation, dripping pill or injection.

9. The use of the chalcone derivative in the preparation of clinical anti-tumor drugs according to claim 7, characterized in that: The dosage form is administered orally or by injection.

Images