Chalcone derivatives, processes for their preparation and use in the manufacture of anti-tumor medicaments

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and specifically discloses a chalcone derivative and a preparation method thereof. 2,4-dihydroxy formaldehyde is used as an initial raw material, isopentenyl is introduced into 2,4-dihydroxybenzaldehyde through a Friedel-Crafts reaction, and then a heterocycle is introduced through a hydroxy aldehyde condensation reaction of the heterocycle-substituted formaldehyde, so that a chalcone structure is formed. The prepared chalcone derivative has a structural formula as shown in Formula I, and the physicochemical properties thereof are studied. It is proved that the chalcone derivatives I-4, I-5, I-7, I-8, I-9, I-10, I-11, I-12 and I-14 all show a strong inhibitory effect on the proliferation of four tumor cells (PC9, MDA-MB-231, SMMC-7721 and SGC-7901). Among them, the chalcone derivative I-4 can effectively inhibit the proliferation of human lung cancer PC9 and H1975 cells in a concentration-dependent manner, and shows good in-vivo anti-tumor activity and can induce iron death of human lung cancer PC9 and H1975 cells, and has a good application prospect as an anticancer drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically to chalcone derivatives and their preparation methods, as well as the application of pharmaceutical compositions with chalcone derivatives as active ingredients in the treatment of malignant tumors. Background Technology

[0002] Chalcones possess α,β-unsaturated ketone structures, and the presence of double bonds conjugated with carbonyl functional groups is considered the reason for their biological activity, exhibiting pharmacological activities such as anticancer, anti-inflammatory, antibacterial, and antiviral effects. Chalcones belong to the flavonoid family and are intermediates in flavonoid biosynthesis. They exhibit structural specificity, allowing them to act on various drug targets. Furthermore, chalcones are important structures for many bioactive molecules. Studies have shown that 4,4'-dimethoxychalcone (DMC) can inhibit the activity of human ferrous chelate synthase (FECH) and induce Keap1 degradation through the ubiquitin-proteasome system, leading to nuclear translocation of Nrf2 and upregulation of the Nrf2-targeting gene HMOX1, thereby inducing ferroptosis in cancer cells. However, the mechanism by which chalcone compounds induce ferroptosis in lung cancer cells has not yet been reported.

[0003] Summary of the Invention

[0004] The purpose of this invention is to provide a series of chalcone derivatives, using inexpensive and readily available 2,4-dihydroxybenzaldehyde as the starting material. First, isopentenyl groups are introduced into 2,4-dihydroxybenzaldehyde through Friedel-Crafts reaction, and then heterocycles are introduced through aldol condensation reaction to form a chalcone structure, thereby improving the anticancer activity of chalcone derivatives.

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

[0006]

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

[0008] When R1 and R2 are both At that time, R3 is

[0009] The preparation method of the chalcone derivative shown in Formula I is as follows: dissolve the raw material compound in ethanol, add potassium hydroxide and heterocyclic substituted formaldehyde derivative, stir at room temperature until the reaction is completed, quench with water, extract, dry and purify to obtain the chalcone derivative shown in Formula I.

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

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

[0012] When R1 and R2 are both In this case, 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 Formula I above use 2,4-dihydroxybenzaldehyde as the starting material. The starting material compound is prepared first, and then the chalcone derivative is synthesized. The starting material is inexpensive and readily available, the preparation process is simple and feasible, pollution-free, yields are stable, and reproducibility is good. Nine of the prepared chalcone derivatives showed strong inhibitory effects on the proliferation of four types of tumor cells (PC9, MDA-MB-231, SMMC-7721, and SGC-7901). Among them, derivative I-4 effectively inhibited the proliferation of human lung cancer PC9 and H1975 cells in a concentration-dependent manner, showing good in vivo antitumor activity, and could induce ferroptosis in human lung cancer PC9 and H1975 cells.

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

[0016] The chalcone derivatives of this invention exhibit strong cytotoxicity against four types of tumor cells. Among them, derivative I-4 can effectively inhibit the proliferation of human lung cancer PC9 and H1975 cells in a concentration-dependent manner, showing good in vivo antitumor activity, and can induce ferroptosis in human lung cancer PC9 and H1975 cells, showing great promise for anticancer drug applications.

[0017] The chalcone derivatives are used to formulate dosage forms of intestinal or parenteral combination drugs. Dosage forms include liquid preparations, tablets, granules, powders, pills, capsules, sustained-release preparations, drop pills, or injections. The administration route is oral or injectable. Attached Figure Description

[0018] Figure 1 This study investigated the effects 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 viability.

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

[0021] Figure 4 This study investigated the effect of chalcone derivative I-4 on glutathione levels in PC9 and H1975 lung cancer cells.

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

[0023] Figure 6 The effect of chalcone derivative I-4 on the intracellular LPO and MDA content of PC9 and H1975 lung cancer cells.

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

[0025] Figure 8 The effect of chalcone derivative I-4 on the expression of ferroptosis-related proteins in lung cancer PC9 and H1975 cells. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to embodiments. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations can be made by those skilled in the art, all of which fall within the scope of protection of the present invention.

[0027] The preparation of the starting compounds (compounds 2, 3, and 4) is based on existing techniques, and their synthetic routes are as follows; the specific processes will not be detailed 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), and potassium hydroxide (0.9 mM) and 3-thiophenecarboxaldehyde (0.4 mM) were added. The mixture was stirred at room temperature for 4–6 h. After the reaction was completed by TLC monitoring, the reaction mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and then evaporated to dryness. The petroleum ether:ethyl acetate (20:1) eluent was used for column chromatography to give 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 C10 NMR (CDCl3, 101MHz): δ 191.0 (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 C10 20 H 22 O3S, m / z 343.13629 [M+H] + .

[0033] The preparation methods of chalcone derivatives I-2 to I-12 are the same as those of I-1, except that the formaldehyde derivatives are substituted with different heterocycles. The amount of potassium hydroxide added is 0.9 to 1.8 mM, and the amount of heterocyclic substituted formaldehyde derivatives added is 0.4 to 0.8 mM. These details will not be elaborated here.

[0034] Chalcone derivative I-2 (yellow solid, 70% yield, 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); 13C10 NMR (CDCl3, 101MHz): δ 190.7 (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 C10 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 C10 NMR (CDCl3, 101MHz): δ 190.5 (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 C10 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); 13C10 NMR (CDCl3, 101MHz): δ 189.0 (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 C10. 19 H 21 NO3S, m / z 344.13181 [M+H] + .

[0038] Chalcone derivative I-6 (yellow oil, 80% yield, 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 C10 NMR (CDCl3, 101MHz): δ 190.1 (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 C10 21 N 25 NO3, m / z 340.19025 [M+H] + .

[0039] Chalcone derivative I-7 (yellow oil, 90% yield, 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 C10 NMR (CDCl3, 101MHz): δ 189.4 (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 C10. 20 H 24 N₂O₃, m / z 341.18524 ​​[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); 13C10 NMR (CDCl3, 101MHz): δ 188.9 (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 C10 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 C10 NMR (CDCl3, 101MHz): δ 190.5 (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 C10. 21 H 22 FNO3, m / z 356.16489 [M+H] + .

[0042] Chalcone derivative I-10 (yellow solid, 20% yield, 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 C10 NMR (CDCl3, 101MHz): δ 190.4 (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 C10. 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); 13C10 NMR (CDCl3, 101MHz): δ 190.4 (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 C10 21 H 22 BrNO3, m / z 416.08609 [M+H] + .

[0044] Chalcone derivative I-12 (yellow solid, 20% yield, 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), and potassium hydroxide (1.8 mM) and pyridine-2-carboxaldehyde (0.6 mM) were added. The mixture was stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, the reaction mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and then evaporated to dryness. The petroleum ether:ethyl acetate (20:1) eluent was used for column chromatography to give the 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), and potassium hydroxide (1.8 mM) and pyridine-2-carboxaldehyde (0.6 mM) were added. The mixture was stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, the reaction mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and then evaporated to dryness. The petroleum ether:ethyl acetate (20:1) eluent was used for column chromatography to give the 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 assay to detect the effects of chalcone derivatives I-1 to I-14 prepared in Examples 1 and 2 on the proliferation of four types of tumor cells:

[0054] This experimental section 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 in China.

[0057] Reagents and materials: 5-fluorouracil (5-FU) and MTT were purchased from Sigma-Aldrich, USA; DMEM and RPMI 1640 culture medium were purchased from GIBCO Biotechnology Co., Ltd.; DMSO was purchased from Titan Biotechnology Co., Ltd.; 0.25% trypsin, penicillin and streptomycin were purchased from Beyotime, Shanghai Co., Ltd.; 96-well culture plates were purchased from Corning Biotechnology Co., Ltd.; fetal bovine serum was purchased from Ecosai Biotechnology Co., Ltd.

[0058] Instruments: SP-DJ series vertical clean bench (Shanghai General Physical Optical Instrument Factory), CO2 incubator (Thermo Scientific), multi-functional microplate reader (BioTek, USA), inverted microscope (Olympus, Japan).

[0059] (2) Method:

[0060] The four tumor cell lines were seeded in culture flasks containing DMEM or RPMI 1640 (containing 10% inactivated fetal bovine serum, 100 IU / L penicillin, and 100 μg / mL streptomycin) and cultured at 37°C with saturated humidity and 5% CO2. Tumor cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, centrifuged, and the cells were collected. They were then seeded at a density of 5000 cells per well in 96-well plates and cultured in an incubator. After 24 hours of culture, the cells were treated with different concentrations of chalcone derivatives and 5-fluorouracil (positive control group) and cultured for another 72 hours (chalcone derivatives were used to treat human lung cancer PC9 and H1975 cells for 24, 48, and 72 hours, respectively). After culturing, 10 μL of 5 g / L MTT solution was added to each well and incubated for another 4 hours. The culture medium was then discarded, and 100 μL of DMSO was added. The mixture was incubated at 37°C for 30 minutes, followed by micro-oscillation for 10 minutes to fully dissolve the crystals. The absorbance (A) of each well was measured using a microplate reader at a wavelength of 490 nm. Cell viability was calculated as follows: Cell viability / % = Experimental group A 490 nm / Control Group A 490nm ×100%, plot the dose-response curve.

[0061] (3) Experimental Results: As shown in Table 1, chalcone derivatives I-4, I-5, I-7, I-8, I-9, I-10, I-11, I-12, and I-14 exhibited strong in vitro inhibitory effects on the proliferation of four types of tumor cells, with their half-maximal inhibitory concentrations (IC50) being [missing data]. 50 The value range is 2.11 to 9.48 μmol / L. Figure 1 The results showed that with the increase of the concentration of chalcone derivative I-4 and the extension of the treatment time, the survival rate of human lung cancer PC9 cells gradually decreased, showing a concentration- and time-dependent effect.

[0062] Table 1. Effects of chalcone derivatives I-1 to I-14 on the proliferation of four types of tumor cells (72h, IC50) 50 (μM)

[0063]

[0064]

[0065] Example 5. Effect of chalcone derivative I-4 on 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 the Shanghai Cell Bank in China.

[0068] Reagents and materials: Fer-1 and DFO were purchased from MCE; MTT was purchased from Sigma-Aldrich; DMEM and RPMI 1640 culture medium were purchased from GIBCO Biotechnology Co., Ltd.; DMSO was purchased from Titan Biotechnology Co., Ltd.; 0.25% trypsin, penicillin and streptomycin were purchased from Beyotime (Shanghai); 96-well culture plates were purchased from Corning; fetal bovine serum was purchased from Ecosai Biotechnology Co., Ltd.

[0069] Instruments: SP-DJ series vertical clean bench (Shanghai General Physical Optical Instrument Factory), CO2 incubator (Thermo Scientific), multi-functional microplate reader (BioTek, USA), inverted microscope (Olympus, Japan).

[0070] (2) Method:

[0071] PC9 and H1975 cells in logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension. After centrifugation, the cells were collected and seeded into 96-well plates at a density of 5000 cells per well. The plates were then incubated for 24 hours. Afterward, the cells were pretreated with the ferroptosis inhibitor Fer-1 (30 μM) and DFO (30 μM), respectively. Two hours later, chalcone derivative I-4 (20 μM) was added, and the cells were cultured for another 24 hours. After the initial culture, 10 μL of 5 g / L MTT solution was added to each well, and the cells were incubated for another 4 hours. The culture medium was discarded, and 100 μL of DMSO was added. The plates were incubated at 37°C for 30 minutes, followed by 10 minutes of micro-oscillation to dissolve any crystals. The absorbance (A) of each well was measured at 490 nm using a microplate reader, and the cell viability was calculated as follows: Cell viability / % = Experimental group A. 490 nm / Control Group A 490nm ×100%, and draw a bar chart.

[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, and after 24 h of incubation, the cells were digested, centrifuged, and collected. The cells were then transferred to EP tubes, and the cell pellet was fixed with electron microscopy fixative (2.5% glutaraldehyde) and stored at 4 °C. The cells were then sectioned and photographed by Wuhan Sewell Biotechnology Co., Ltd.

[0073] (3) Experimental results:

[0074] By examining the cell viability after combining chalcone derivative I-4 with ferroptosis inhibitors, the results showed that cell viability increased by approximately 20% after combining with the inhibitor compared to treatment alone. Figure 2 As shown.

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

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

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

[0078] (1) Experimental materials:

[0079] Reagents and materials: Large plates were purchased from Corning; the reduced glutathione (GSH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute.

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

[0081] (2) Method:

[0082] Lung cancer PC9 and H1975 cells in logarithmic growth phase were digested and centrifuged, then seeded in large dishes and cultured in an incubator until the cell density reached 80%. Different concentrations of chalcone derivative I-4 (5, 10, 20, 40 μM) were added to six-well plates, and after 24 h of incubation, cells were collected and centrifuged at 2500 rpm for 10 minutes, discarding the supernatant. 50 μL of RIPA lysis buffer containing a protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes, followed by centrifugation at 12000 rpm for 30 minutes. The protein concentration in the supernatant was determined using the BCA quantification method. The cell pellet was sonicated on ice, and the lysed cell suspension was collected for later use. A 20 μmol / L GSH standard solution was prepared according to the manufacturer's instructions. The reagents were added to the cell suspension, mixed, and allowed to stand for 5 min. The absorbance was measured at 405 nm using a microplate reader, and the GSH content in the cells was calculated according to the formula in the manufacturer's instructions.

[0083] (3) Experimental results:

[0084] Depend on Figure 4 It can be seen that as the concentration of 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: Large dishes were purchased from Corning; tissue iron test kits were purchased from Nanjing Jiancheng Biotechnology Institute.

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

[0089] (2) Method:

[0090] PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then seeded in large dishes and cultured in an incubator until the cell concentration reached 80%. Different concentrations of chalcone derivative I-4 (5, 10, 20, and 40 μM) were added to six-well plates, and after 24 hours of incubation, cells were collected and centrifuged at 2500 rpm for 10 minutes, discarding the supernatant. 50 μL of RIPA lysis buffer containing a protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes, followed by centrifugation at 12000 rpm for 30 minutes. The protein concentration in the supernatant was determined using the BCA quantification method. The cell pellet was sonicated on ice, and the lysed cell suspension was collected for later use. According to the instructions, prepare a 2 mg / L iron standard solution and iron chromogenic reagent. Add the relevant reagents to the cell suspension, mix well, and place in a boiling water bath at 95°C or above for 5 minutes. After cooling, centrifuge at 3500 r / min for 10 minutes, take 1 mL of the supernatant, and detect the absorbance at 520 nm using an ELISA reader. Calculate the iron content in the cells according to the formula in the instructions.

[0091] (3) Experimental results:

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

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

[0094] (1) Experimental materials:

[0095] Reagents and materials: Large plates were purchased from Corning; lipid peroxide (LPO) test kits and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute.

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

[0097] (2) Method:

[0098] PC9 and H1975 cells in the logarithmic growth phase were digested and centrifuged, then seeded in large dishes and cultured in an incubator until the cell concentration reached 80%. Different concentrations of chalcone derivative I-4 (5, 10, 20, and 40 μM) were added to six-well plates, and after 24 hours of incubation, cells were collected and centrifuged at 2500 rpm for 10 minutes, discarding the supernatant. 50 μL of RIPA lysis buffer containing a protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes, followed by centrifugation at 12000 rpm for 30 minutes. The protein concentration in the supernatant was determined using the BCA quantification method. The cell pellet was sonicated on ice, and the lysed cell suspension was collected for later use. Following the instructions, prepare the standard application solution, add the relevant reagents to the cell suspension, mix well, and incubate in a boiling water bath at 45°C or higher for 60 minutes. After cooling, centrifuge at 4000 rpm for 10 minutes, collect 200 μL of the supernatant, and measure the absorbance at 586 nm using a microplate reader. Calculate the LPO content in the cells according to the formula in the instructions. Similarly, add the MDA detection reagent, mix well, incubate in a boiling water bath at 90°C for 40 minutes, centrifuge at 4000 rpm for 10 minutes, collect 200 μL of the supernatant, and measure the absorbance at 532 nm using a microplate reader. Calculate the MDA content in the cells according to the formula in the instructions.

[0099] (3) Experimental results:

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

[0101] Example 9. Effects 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 Beyotime Biotechnology Co., Ltd.

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

[0105] (2) Method:

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

[0107] (3) Experimental results:

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

[0109] Example 10. Detection of 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 by Western blotting:

[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, USA; Nfr2 antibody was purchased from Affinity, USA.

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

[0113] (2) Method:

[0114] Lung cancer PC9 and H975 cells in logarithmic growth phase were seeded in 10×10 dishes. When the cell density reached 80%, chalcone derivative I-4 was added to the dishes at different concentrations (0, 5, 10, 20, 40 μM). After 24 h of drug treatment, the cells were collected and centrifuged at 2500 rpm for 15 min. The supernatant was discarded, and the cell pellet was collected. The liquid in the tube wall was aspirated, lysis buffer was added, and the cells were lysed on ice for an appropriate time. After centrifugation at 4 °C and 12000 rpm for 30 min, the supernatant was collected, and the protein was quantified using the BCA method. 40 μg of protein from each group was taken for SDS-PAGE electrophoresis (stacking gel constant voltage 75 V, separating gel constant voltage 110 V, electrophoresis was stopped when the bromophenol blue dye reached the gel front).

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

[0116] (Sealing): The PVDF membrane was washed three times (5 min each time) with 1×TBST, then placed in 5% skim milk and sealed at 4°C for 4 h. The PVDF membrane was removed from the skim milk, washed three times (5 min each time), then placed in primary antibody and incubated at 4°C for 12-16 h. It was washed three times (5 min each time), and secondary antibody dilution was added. The membrane was then incubated on a shaker at 4°C for 2 h. The PVDF membrane was washed three times (5 min each time) with 1×TBST, then immersed in exposure solution (solution A:solution B = 1:1) for an appropriate time and developed using a gel imaging system.

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

Claims

1. A chalcone derivative characterized in that, It has the structural formula shown in Equation I: ； In formula I, when R1and R2are both -CH3, R3is , , , , , , or ; when R1and R2are both R3is .

2. The method of preparing the chalcone derivative according to claim 1, characterized by, Specifically: The starting compound was dissolved in ethanol, potassium hydroxide and heterocyclic substituted formaldehyde derivative were added, and the mixture was stirred at room temperature until the reaction was completed. The mixture was then quenched with water, extracted, dried and purified to obtain the chalcone derivative shown in Formula I. The structural formula of the raw material compound is: ; wherein, when R1and R2are both -CH3, the heterocyclic substituted formaldehyde derivative is , , , , , , or ; When R1and R2are both the heterocycle-substituted formaldehyde derivative is .

3. The method of preparing a chalcone derivative according to claim 2, wherein: The molar ratio of the starting 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, wherein: The molar ratio of the raw material compound to potassium hydroxide is 1:(4.5-9).

5. The use of the chalcone derivative according to claim 1 in the preparation of clinical antitumor drugs, characterized in that: The tumor in question is lung cancer, breast cancer, liver cancer, or stomach cancer.

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