15-substituted derivatives of baicalein and their antitumor applications

Abstract

This invention discloses 15-substituted derivatives of allophyllin and their antitumor applications, belonging to the field of pharmaceutical technology. This invention discovers a group of 15-substituted derivative compounds of allophyllin, AH-1 to AH-29, and their antitumor applications, which can be used to prepare drugs for treating leukemia and / or gastric cancer, prostate cancer, and ovarian cancer; among them, AH-26 exhibits an effect of promoting tumor cell apoptosis, and compared with existing antitumor drugs, it has stronger activity and higher selectivity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the 15-substituted derivatives of allophyllin and their antitumor applications. Background Technology

[0002] Cancer is a major public health problem worldwide, the second leading cause of death globally, with low survival rates, thus creating an urgent need for effective anti-cancer drugs. Searching for highly effective and low-toxicity anti-cancer active chemical components from natural products has been a major focus of modern cancer drug research. Allophyllin is a plant-derived, bioactive indolithidine alkaloid. Currently, there are few research reports related to allophyllin, with only a limited number of total synthesis studies. Summary of the Invention

[0003] To address the shortcomings and deficiencies of existing technologies, a 15-position substituted derivative of baicalein is provided.

[0004] Another object of the present invention is to provide the application of the above-mentioned 15-substituted derivative of the alkaloid.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] The 15-substituted derivative of *Alternaria solani* is a compound having the structure shown in Formula I:

[0007]

[0008] In the formula, R represents any one of the following: ortho-, meta-, para-substituted halogens, disubstituted halogens, disubstituted methoxy groups, acetyl groups, nitro groups, methoxyformyl groups, trifluoromethyl groups, methoxy groups, ethyl groups, tert-butyl groups, isopropyl groups, phenyl groups, and benzyl groups.

[0009] Further, the 15-position substituted derivative of the alkaloid is any one of compounds AH-1, AH-2, AH-3, AH-4, AH-5, AH-6, AH-7, AH-8, AH-9, AH-10, AH-11, AH-12, AH-13, AH-14, AH-15, AH-16, AH-17, AH-18, AH-19, AH-20, AH-21, AH-22, AH-23, AH-24, AH-25, AH-26, AH-27, AH-28, and AH-29; preferably AH-26; compounds AH-1 to AH-29 are respectively compounds having the following structures:

[0010]

[0011] The above-mentioned 15-substituted derivatives of eurythrin are pharmaceutically acceptable salts.

[0012] Furthermore, the pharmaceutically acceptable salt has an anion that is either inorganic or organic; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate, or hydrogen phosphate, etc.; the organic anion is acetate, propionate, cinnamate, benzosulfonate, citrate, lactate, or gluconate, etc.

[0013] The use of the above-mentioned 15-substituted derivatives of eurythrin or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0014] Furthermore, the tumors mentioned include any one or more of leukemia, ovarian cancer, gastric cancer, and prostate cancer.

[0015] Furthermore, the aforementioned antitumor drug is a drug that promotes the activity of Bax protein.

[0016] Furthermore, the antitumor drug is a drug that antagonizes the malignant proliferation of tumors caused by the expression of Bcl-XL, Bcl-2, TAZ, GLI1, MCL1, XIAP, YAP1, β-catenin, c-Myc, p-AKT, JAK1 / 2, STAT3 and / or p-STAT3 proteins.

[0017] Furthermore, in the aforementioned applications, the 15-substituted derivatives of allopurinol can be formulated with pharmaceutically acceptable excipients using common techniques to create various dosage forms, including solid, semi-solid, liquid, and aerosol dosage forms. Specific dosage forms within these categories include tablets, pills, granules, lozenges, ointments, solutions, suppositories, injections, inhalers, and sprays. Sprays are administered via a pressurizer and a propellant or a dry powder inhaler. Suitable propellants used in the propellant include dichlorodifluoromethane, chlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and dimethyl ether. The dosage of the spray can be adjusted via a valve on the propellant. These dosage forms can be used for both local and systemic administration, as well as for immediate or sustained-release administration. There are many routes of administration for these drugs, including, in addition to the methods mentioned above, oral administration, buccal administration, rectal administration, peritoneal administration, topical administration, subcutaneous injection, and intratracheal administration.

[0018] When administered subcutaneously, these compounds can be formulated into solutions, suspensions, and emulsions using water-soluble or lipid-soluble solvents. Lipid-soluble solvents specifically include vegetable oils and similar oils, synthetic fatty acid glycerides, higher fatty acid esters, and glycol esters.

[0019] For oral administration, commonly used techniques can be employed to create complexes with pharmaceutically acceptable excipients. These excipients can then be formulated into various patient-acceptable dosage forms, such as tablets, pills, suspensions, and gels. There are several methods for preparing oral formulations, such as first mixing the compound and solid excipients, thoroughly grinding the mixture, adding appropriate excipients, and then processing it into granules. Excipients that can be used to formulate oral dosage forms include: sugars such as lactose, sucrose, mannitol, or sorbitol; and celluloses such as corn starch, wheat starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone.

[0020] The method for preparing the 15-substituted derivative of the above-mentioned allophyllotoxin involves reacting allophyllotoxin with a substituted iodobenzene containing an R group via a Heck reaction to obtain the target product; specifically, it includes the following steps:

[0021] S1. Under an inert gas atmosphere, heat and dissolve the alkaloid in an organic solvent; add a catalyst, substituted iodobenzene containing an R group, and potassium carbonate, stir the reaction, and obtain a mixed solution;

[0022] S2. Filter the mixed solution under vacuum, wash the filter residue, dry the collected filtrate and then evaporate it under reduced pressure to obtain the crude product; purify it by silica gel column chromatography to obtain the target product.

[0023] Furthermore, the organic solvent mentioned in step S1 is either anhydrous 1,4-dioxane or methanol.

[0024] Furthermore, the catalyst mentioned in step S1 is a Pd(OAc)2 and dppp system; preferably a 5 mol% Pd(OAc)2 and 5 mol% dppp system.

[0025] Furthermore, the molar ratio of the alkaloid and the iodobenzene containing the R group mentioned in step S1 is 1-2:1-2.

[0026] Furthermore, the heating temperature described in step S1 is 80±5℃.

[0027] Furthermore, the stirring reaction time described in step S1 is 8 ± 1 hours.

[0028] Furthermore, the rinsing agent used in step S2 is dichloromethane.

[0029] Furthermore, the drying reagent used in step S2 is anhydrous sodium sulfate.

[0030] The present invention has the following advantages and effects compared with the prior art:

[0031] This invention utilizes natural products as lead compounds and employs organic chemistry and medicinal chemistry techniques for structural modification studies to identify new drug lead compounds, representing an innovative model for innovative drug research. This invention uses natural allegorine as a precursor compound for chemical modification, yielding a series of novel allegorine derivatives. Pharmacodynamic evaluation using in vitro models of leukemia cells, gastric cancer cells, prostate cancer cells, and ovarian cancer cells revealed that the novel allegorine derivatives exhibit anti-proliferative effects against various cancer cells and can promote apoptosis in cancer cells. Compared with the existing antitumor drug camptothecin, they demonstrate stronger activity and higher selectivity.

[0032] This invention provides a compound with a novel structure, and the synthetic route employed utilizes green chemistry reactions, making it more environmentally friendly and economical in terms of raw materials. These compounds can be used to develop new drugs for the treatment of leukemia, gastric cancer, prostate cancer, and ovarian cancer. Attached Figure Description

[0033] Figure 1 Synthetic route for the 15-position substituted derivative of schizophyllum commune.

[0034] Figure 2 The image shows the apoptosis detection results of NB4 (a~d), K562 (e~h), HL-60 (i~l), and U937 (m~p) cells after 48 hours of treatment with compound AH-26.

[0035] Figure 3 The image shows the apoptosis detection results of HGC-27 (a~d) and SKOV3 (e~h) cells after 48 hours of treatment with compound AH-26.

[0036] Figure 4 The image shows the mitochondrial membrane potential of NB4 (a~d), K562 (e~h), HL-60 (i~l), and U937 (m~p) cells after 48 hours of treatment with compound AH-26.

[0037] Figure 5 The image shows the mitochondrial membrane potential detection results after 48 hours of treatment of HGC-27 (a~d) and SKOV3 (e~h) cells with compound AH-26.

[0038] Figure 6 The image shows the cell cycle detection results after NB4 (a~d) and HL-60 (e~h) cells were treated with compound AH-26 for 48 hours.

[0039] Figure 7 The image shows the cell cycle detection results after 48 hours of treatment of HGC-27 (a~d) and SKOV3 (e~h) cells with compound AH-26.

[0040] Figure 8The figure shows the protein expression changes detected after AH-26 treatment of NB4(a) and HL-60(b) cells for 48 hours.

[0041] Figure 9 The figure shows the protein expression changes detected after AH-26 was applied to SKOV3(a) and HGC-27(b) cells for 48 hours. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0043] Example 1: Preparation of AH-1 to AH-29

[0044]

[0045] The Heck reaction of *Alternaria alternifolia* alkaloid and substituted iodobenzene was performed as follows: *Alternaria alternifolia* alkaloid (150 mg, 0.69 mmol) was weighed and added to a double-necked flask filled with nitrogen and containing 5 mL of ultra-dry 1,4-dioxane. The mixture was heated to 80 °C, stirred to dissolve, and then Pd(OAc)₂ and dppp were added. Substituted iodobenzene (1.04 mmol) and potassium carbonate (1.04 mmol) were then added, and the mixture was stirred for 8 hours. The reaction was monitored by TLC; after approximately 8 hours, the reaction ceased, and the reaction was stopped. The mixture was then filtered, and the residue was washed with dichloromethane. The collected filtrate was dried over anhydrous sodium sulfate for 5 minutes and then evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the target products AH-1 to AH-29, with yields ranging from 30% to 80%.

[0046] Characterization of compound AH-1 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.36(q,J=7.2Hz,2H),7.22-7.10(m,2H),6.81(s,1H),5.78(s,1H),4.38(d,J=5.8Hz,1H),3.67(dd,J=13.0,3.3Hz,1H ),2.81(dd,J=9.5,5.0Hz,1H),2.69(td,J=10.2,5.1Hz,1H),2.59-2.53(m,1H),2.13(d,J=9.8Hz,1H),1.61-1.58(m,4H),1.46-1.38(m,2H). 13C NMR (101MHz, CDCl3) δ172.84,167.24,131.04,130.95,128.86,124.73,124.70,120.94,1 16.73,116.50,109.12,91.12,62.26,60.83,44.27,42.95,22.34,20.76,18.86.HR-ESIMS m / z 312.1411[M+H] + calcd for C 19 H 19 FNO2 + 312.1400.

[0047] Characterization of compound AH-2 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.45-7.32(m,2H),7.27-7.23(m,1H),7.10(dt,J=8.0,3.9Hz,1H),6.90(d,J=1.9Hz,1H),5.82(s,1H),4.48(d,J=4.6Hz,1H), 3.69(dd,J=13.1,3.2Hz,1H),2.83(dd,J=9.9,4.6Hz,2H),2.74-2.54(m,2 H),2.06(d,J=9.9Hz,1H),1.72-1.58(m,3H),1.41(dd,J=8.3,4.1Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.68,166.95,164.26,161.80,156.40,140.42,130.68,121.83,117.76,1 16.75,113.10,109.37,90.98,61.54,60.64,43.93,42.36,29.68,21.84,21.13,18.47.HR-ESIMS m / z 312.1375[M+H] + calcd for C 19 H 19 FNO2 + 312.1400.

[0048] Characterization of compound AH-3 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ7.55(dd,J=8.9,5.2Hz,2H),7.11(t,J=8.6Hz,2H),6.83(d,J=1.9Hz,1H),5.77(s,1H),4.45(d,J=2.6Hz,1H),3.6 8(dd,J=12.9,3.3Hz,1H),2.82(dd,J=9.9,4.6Hz,1H),2.65-2.60(m,2H),2.05(d,J=9.8Hz,1H),1.71-1.57(m,4H),1.44-1.37(m,2H). 13 CNMR(101MHz,CDCl3)δ172.91,167.39,156.99,133.87,129.03,128.08,127.99,126.08,11 6.25,116.03,109.12,91.02,61.74,60.74,44.05,42.49,22.06,21.16,18.54.HR-ESIMSm / z 310.1274[MH] + calcd for C 19 H 18 FNO2 + 310.1244.

[0049] Characterization of compound AH-4 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.53(d,J=2.6Hz,1H),7.45-7.41(m,1H),7.37(dd,J=4.9,1.8Hz,2H),6.89(d,J=1.9Hz,1H),5.81(s,1H),4.45(d,J=4.5Hz, 1H),3.69(dd,J=13.0,3.1Hz,1H),2.84(dd,J=9.9,4.8Hz,1H),2.69-2.5 7(m,2H),2.06(d,J=9.8Hz,1H),1.70-1.62(m,4H),1.41(d,J=8.3Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.72,167.01,156.66,140.15,135.15,130.29,129.65,126.17,1 24.25,117.82,109.35,91.03,61.64,60.73,44.10,41.62,22.04,21.22,18.54.HR-ESIMS m / z 328.1107[M+H] +calcd for C 19 H 19 ClNO2 + 328.1104.

[0050] Characterization of compound AH-5 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.40-7.35(m,2H),7.29-7.25(m,2H),6.75(d,J=1.9Hz,1H),5.67(s,1H),4.34(d,J=2.6Hz,1H),3.56(dd, J=13.1,3.2Hz,1H),2.70(dd,J=9.9,4.8Hz,1H),2.53-2.44(m,2H),1.93(d,J=9.9Hz,1H),1.59-1.44(m,4H),1.32-1.25(m,2H). 13 C NMR (101MHz, CDCl3) δ172.83,167.19,156.70,136.67,135.89,129.28,127.33,1 17.05,108.89,91.00,61.52,60.68,43.98,42.40,21.96,21.12,18.45.HR-ESIMS m / z 328.1070[M+H] + ,calcd forC 19 H 19 ClNO2 + 328.1104.

[0051] Characterization of compound AH-6 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.59-7.51(m,2H),7.46-7.38(m,2H),6.88(d,J=1.8Hz,1H),5.79(s,1H),4.44(d,J=2.4Hz,1H),3.68(dd,J =13.0,3.1Hz,1H),2.82(dd,J=10.3,5.1Hz,1H),2.67-2.54(m,2H),2.05(d,J=9.8Hz,1H),1.84-1.49(m,4H),1.47-1.35(m,2H). 13C NMR (101MHz, CDCl3) δ172.86,167.24,156.94,137.20,132.27,127.59,124.21,1 17.08,108.43,91.05,61.55,60.74,44.07,42.47,22.07,21.19,18.55.HR-ESIMS m / z 372.0611[M+H] + ,calcd forC 19 H 19 BrNO2 + 372.0599.

[0052] Characterization of compound AH-7 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ8.29(d,J=8.9Hz,2H),7.72(d,J=9.0Hz,2H),7.00(d,J=1.9Hz,1H),5.91(s,1H),4.48(d,J=2.4Hz,1H),3.71(dd,J=13.0,3. 1Hz,1H),2.88(dd,J=9.6,5.1Hz,1H),2.71-2.57(m,2H),2.11(d,J=9.9Hz,1H),1.67(ddd,J=15.0,12.0,6.1Hz,4H),1.44(dd,J=8.8,4.1Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.38,166.36,155.96,148.10,144.47,127.62,124.32,1 19.89,110.72,90.98,62.03,60.51,43.74,43.23,22.82,21.43,18.02.HR-ESIMS m / z 339.1417[M+H] + calcd for C 19 H 19 N2O4 + 339.1345.

[0053] Characterization of compound AH-8 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ8.15 (t, J=1.8Hz, 1H), 7.96 (dt, J=7.8, 1.4Hz, 1H), 7.75 (ddd ,J=7.9,2.1,1.1Hz,1H),7.54(t,J=7.8Hz,1H),6.95(d,J=1.8Hz,1H),5.82(s,1H), 4.54(d,J=3.6Hz,1H), 3.69(dd,J=13.1,3.2Hz,1H), 2.84(dd,J=10.2,4.3Hz,1H), 2.64(s,5H),2.08(d,J=9.9Hz,1H),1.74-1.55(m,4H),1.41(dd,J=9.1,4.1Hz,2H). 13 C NMR (101MHz, CDCl3) δ197.50,172.79,167.11,157.05,138.95,137.73,130.36,129.55,129.43,1 25.61,117.90,109.30,91.07,61.66,60.73,44.05,42.47,26.77,21.98,21.18,18.47.HR-ESIMS m / z336.1605[M+H] + calcd for C 21 H 22 NO3 + 336.1600.

[0054] Characterization of compound AH-9 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.98(d,J=8.6Hz,2H),7.63(d,J=8.6Hz,2H),6.96(d,J=1.9Hz,1H),5.83(s,1H),4.50(d,J=6.5Hz,1H),3.67(dd,J=12.9, 3.2Hz,1H),2.83(dd,J=9.9,4.6Hz,1H),2.61(s,5H),2.06(d,J=9.8Hz,1H),1.69-1.54(m,3H),1.44-1.34(m,2H),1.18(dd,J=12.9,5.9Hz,1H). 13C NMR (101MHz, CDCl3) δ197.17,172.63,166.92,156.83,142.47,137.41,128.93,126.21,1 18.51,109.60,91.00,61.48,60.68,44.04,42.40,26.65,22.00,21.20,18.50.HR-ESIMS m / z336.1568[M+H] + calcd for C 21 H 22 NO3 + 336.1600.

[0055] Characterization of compound AH-10 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.10-7.02(m,2H),6.91-6.80(m,2H),5.84(s,1H),4.37(d,J=2.6Hz,1H),3.67(dd,J=13.0,3.0Hz,1H),2 .83(dd,J=9.9,4.8Hz,1H),2.70-2.55(m,2H),2.04(d,J=9.8Hz,1H),1.74-1.57(m,3H),1.48-1.36(m,2H),1.23-1.12(m,1H). 13 C NMR (101MHz, CDCl3) δ172.54,166.60,164.59,162.11,155.77,141.52,118.53,110.06,1 09.22,108.96,104.91,91.02,61.61,60.76,44.18,42.47,22.14,21.30,18.61.HR-ESIMS m / z 330.1335[M+H] + calcd for C 19 H 18 FNO2 + 330.1306.

[0056] Characterization of compound AH-11 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ7.37(q,J=8.5Hz,1H),6.96-6.89(m,2H),6.79(s,1H),5.79(s,1H),4.33(s,1H),3.68(d,J=12.6Hz,1H),2.8 1(dd,J=9.9,4.8Hz,1H),2.66(d,J=5.0Hz,1H),2.58(d,J=5.6Hz,1H),2.12(d,J=9.9Hz,1H),1.68-1.64(m,4H),1.46-1.39(m,2H). 13 C NMR (101MHz, CDCl3) δ172.72,167.05,159.92,153.83,140.76,129.87,122.70,120.96,1 12.29,109.43,105.10,91.03,62.79,60.78,43.91,42.30,22.20,21.34,18.75.HR-ESIMS m / z 330.1335[M+H] + calcd for C 19 H 18 FNO2 + 330.1306.

[0057] Characterization of compound AH-12 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.24-7.11(m,2H),6.84(s,1H),5.84(s,1H),4.33(d,J=4.3Hz,1H),3.67(d,J=13.1Hz,1H),2. 83(dd,J=9.9,4.8Hz,1H),2.73-2.52(m,2H),2.03(d,J=9.9Hz,1H),1.72-1.58(m,4H),1.41(dd,J=9.9,3.0Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.45,166.36,154.92,152.67,142.76,139.10,134.34,118.29,1 10.51,110.29,110.09,90.90,61.50,60.75,44.22,42.37,22.13,21.27,18.59.HR-ESIMS m / z 348.1223[M+H] + ,calcd forC 19 H 17 F3NO2 +348.1211.

[0058] Characterization of compound AH-13 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR(400MHz, CDCl3) δ8.06(d,J=8.6Hz,2H),7.67(d,J=8.6Hz,2H),7.19(s,1H) ,5.96(s,1H),5.11(t,J=3.0Hz,1H),3.93(s,3H),3.20(d,J=9.9Hz,1H),2.88( dd,J=11.3,2.7Hz,1H),2.71-2.59(m,2H),2.14(dd,J=11.5,2.4Hz,1H),2.04( s,1H),1.82(ddd,J=13.0,3.8,2.6Hz,1H),1.57(s,4H),1.25(d,J=2.3Hz,2H). 13 C NMR (101MHz, CDCl3) δ172.11,166.57,164.36,143.52,142.29,130.41,130.06,126.15,122. 46,113.50,82.65,73.71,70.83,60.19,56.09,52.29,40.69,25.06,23.81,23.13.HR-ESIMS m / z 352.1572[M+H] + ,calcd forC 21 H 22 NO4 + 352.1549.

[0059] Characterization of compound AH-14 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.72-7.62(m,4H),6.93(d,J=1.9Hz,1H),5.85(s,1H),4.47(d,J=2.5Hz,1H),3.70(dd,J=13.0,3.1Hz,1H ),2.86(dd,J=9.8,4.7Hz,1H),2.62(qd,J=10.3,5.0Hz,2H),2.08(d,J=9.8Hz,1H),1.69-1.59(m,4H),1.43(d,J=11.0Hz,2H). 13C NMR (101MHz, CDCl3) δ171.92,167.71,157.27,141.78,131.37,126.39,126.05,126.01,125.0 8,122.60,118.60,109.82,91.05,62.80,60.79,45.66,42.97,24.02,20.52,16.44.HR-ESIMS m / z362.1400[M+H] + calcd for C 20 H 19 F3NO2 + 362.1368.

[0060] Characterization of compound AH-15 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.47(q,J=7.9Hz,2H),7.39(s,1H),7.26(s,1H),6.91(d,J=2.0Hz,1H),5.83(s,1H),4.44(d,J=2.6Hz,1H),3.69(dd,J= 13.0,3.1Hz,1H),2.85(dd,J=9.9,4.6Hz,1H),2.72-2.57(m,2H),2.07(d,J=9.9Hz,1H),1.64(dq,J=10.3,5.4,4.6Hz,4H),1.49-1.36(m,2H). 13 C NMR (101MHz, CDCl3) δ172.71,166.95,156.54,149.80,140.39,130.48,124.37,121.85,1 18.64,118.01,109.51,91.05,61.68,60.78,44.13,42.55,22.10,21.24,18.57.HR-ESIMS m / z 378.1314[M+H] + calcd for C 20 H 19 F3NO2 + 378.1317.

[0061] Characterization of compound AH-16 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ7.56(s,2H),7.42(d,J=4.1Hz,3H),6.90(s,1H),5.78(d,J=3.4Hz,1H),4.53(s,1H),3.70(d,J=13.0Hz, 1H), 2.84 (d, J=9.8Hz, 1H), 2.65 (d, J=3.4Hz, 2H), 2.07 (dd, J=9.9, 3.3Hz, 1H), 1.65 (d, J=12.9Hz, 4H), 1.41 (d, J=9.6Hz, 2H). 13 C NMR (101MHz, CDCl3) δ173.05,167.63,158.12,138.30,129.81,129.06,126.11,1 16.79,108.46,91.15,61.65,60.75,44.00,42.58,22.02,21.15,18.52.HR-ESIMS m / z 294.1508[M+H] + ,calcd forC 19 H 20 NO2 + 294.1494.

[0062] Characterization of compound AH-17 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR(400MHz, CDCl3)δ7.36(d,J=6.9Hz,2H),7.33-7.28(m,1H),7.21(d,J=7.4H z,1H),6.88(d,J=1.8Hz,1H),5.76(s,1H),4.51(d,J=4.5Hz,1H),3.68(dd,J=1 3.0,3.3Hz,1H),2.82(dd,J=9.4,5.0Hz,1H),2.66-2.60(m,2H),2.39(s,3H),2 .05(d,J=9.8Hz,1H),1.69-1.58(m,3H),1.44-1.35(m,2H),1.26-1.18(m,1H). 13 C NMR (101MHz, CDCl3) δ173.07,167.71,158.28,138.71,138.27,130.60,128.90,126.69,123. 26,116.59,108.21,91.13,61.60,60.71,43.93,42.54,21.98,21.52,21.10,18.48.HR-ESIMS m / z 308.1634[M+H] +,calcdfor C 20 H 22 NO2 + 308.1651.

[0063] Characterization of compound AH-18 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.47(d,J=8.4Hz,2H),7.27-7.20(m,2H),6.88(dd,J=8.9,1.8Hz,1H),5.76(d,J=12.9Hz,1H),4.53(d,J=4.4Hz,1H),3.68 (dd,J=12.9,3.3Hz,1H),2.81(dd,J=10.3,5.1Hz,1H),2.66-2.58(m,2H ),2.38(s,3H),2.07-2.02(m,1H),1.72-1.54(m,4H),1.43-1.35(m,2H). 13 C NMR (101MHz, CDCl3) δ173.10,167.75,157.71,140.25,135.27,129.75,125.99,115. 80,107.89,91.06,61.35,60.64,43.79,42.39,21.83,21.28,20.99,18.37.HR-ESIMS m / z308.1634[M+H] + calcd for C 20 H 22 NO2 + 308.1651.

[0064] Characterization of compound AH-19 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.34(t,J=8.1Hz,1H),7.14(d,J=7.9Hz,1H),7.06(t,J=2.2Hz,1 H),6.94(dd,J=8.6,2.1Hz,1H),6.88(d,J=1.9Hz,1H),5.78(s,1H),4.50(d,J=4.5Hz, 1H),3.84(s,3H),3.68(dd,J=12.9,3.2Hz,1H),3.48(s,1H),2.82(dd,J=9.9,4.6Hz,1 H),2.65(d,J=3.9Hz,1H),2.06(d,J=9.9Hz,1H),1.71-1.57(m,4H),1.44-1.36(m,2H). 13C NMR (101MHz, CDCl3) δ173.00,167.52,159.97,157.82,139.66,130.08,129.07,126.11,118.61,117. 00,114.95,111.98,108.60,91.13,61.68,60.70,55.41,43.94,42.51,21.93,21.13,18.46.HR-ESIMS m / z 324.1611[M+H] + calcd for C 20 H 22 NO3 + 324.1600.

[0065] Characterization of compound AH-20 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.36-7.28(m,2H),7.18(d,J=8.0Hz,1H),6.87(d,J=1.8Hz,1H),5.73(s,1H),4.53(d,J=6.5Hz,1H),3.68(dd,J=12.9,3.3Hz ,1H),2.81(dd,J=9.9,4.6Hz,1H),2.63(dd,J=4.1,2.1Hz,2H),2.30(d,J =3.9Hz,6H),2.05(d,J=9.8Hz,1H),1.69-1.60(m,4H),1.45-1.35(m,2H). 13 C NMR (101MHz, CDCl3) δ173.69,167.95,158.07,139.05,137.35,135.82,130.30,127.91,123.64, 115.67,107.74,91.63,61.40,60.73,43.88,42.50,21.98,21.04,20.02,19.71,18.47.HR-ESIMS m / z 322.1824[M+H] + calcd for C 21 H 24 NO2 + 322.1807.

[0066] Characterization of compound AH-21 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ7.54(d,J=8.4Hz,2H),7.24(d,J=8.3Hz,2H),7.10(s,1 H),5.87(s,1H),5.12(t,J=3.0Hz,1H),3.19(d,J=11.6Hz,1H),2.85(dd,J=1 1.4,2.8Hz,1H),2.71-2.58(m,4H),2.13(dd,J=11.4,2.4Hz,1H),1.81(dt,J =13.0,3.1Hz,1H),1.64(q,J=2.7Hz,2H),1.56(s,4H),1.25(d,J=2.9Hz,3H). 13 C NMR (101MHz, CDCl3) δ172.58,165.18,145.86,144.68,135.27,128.40,126.22,120.07,111.98 ,82.72,74.15,70.82,56.09,40.90,31.52,29.72,28.69,25.08,23.81,23.20,16.35.HR-ESIMS m / z 322.1824[M+H] + calcd for C 21 H 24 NO2 + 322.1807.

[0067] Characterization of compound AH-22 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.52(d,J=7.1Hz,2H),7.43(d,J=7.2Hz,2H),6.89(s,1H),5.75(s,1H),4.55(d,J=4.6Hz,1H),3.73-3.65(m,1H), 2.82(dd,J=9.9,4.7Hz,1H),2.72-2.61(m,2H),2.04(d,J=9.9Hz,1H),1.73-1.56(m,4H),1.38(d,J=17.3Hz,2H),1.33(d,J=1.2Hz,9H). 13C NMR (101MHz, CDCl3) δ173.12,167.79,157.72,153.39,135.14,126.01,125.87,115.91,10 7.94,91.08,61.38,60.75,43.88,42.53,34.81,31.13,21.88,21.01,18.38.HR-ESIMSm / z 350.2134[M+H] + calcd for C 23 H 28 NO2 + 350.2120.

[0068] Characterization of compound AH-23 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.35(dd,J=3.6,1.3Hz,2H),7.23-7.13(m,2H),6.47(s,1H),5.74(s,1H),4.03(d,J=5.4Hz,1H),3.66(dd,J=12.7,2.9Hz,1H ),2.89-2.78(m,3H),2.61-2.51(m,1H),2.18(d,J=9.6Hz,1H),1.69-1.6 0(m,4H),1.49-1.40(m,2H),1.34(d,J=5.8Hz,3H),1.13(d,J=6.8Hz,3H). 13 C NMR (101MHz, CDCl3) δ172.95,167.40,145.90,139.19,134.76,129.06,127.77,126.18,126.05,120 .10,108.41,91.04,65.05,61.19,44.49,43.06,30.95,24.78,23.92,22.85,21.78,19.36.HR-ESIMS m / z 336.1980[M+H] + calcd for C 22 H 26 NO2 + 336.1964.

[0069] Characterization of compound AH-24 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ8.05 (s, 1H), 7.90-7.83 (m, 3H), 7.67 (dd, J = 8.8, 2.0Hz, 1H),7.54(dt,J=6.4,3.6Hz,2H),7.04(d,J=2.0Hz,1H),5.81(s,1H),4.72(d,J =4.0Hz,1H),3.73(dd,J=13.0,3.3Hz,1H),2.89(dd,J=9.9,4.6Hz,1H),2.72- 2.62(m,2H),2.13(d,J=9.9Hz,1H),1.62-1.59(m,4H),1.44(d,J=10.1Hz,2H). 13 C NMR (101MHz, CDCl3) δ173.63,158.50,135.14,133.85,133.58,129.10,128.86,127.82,127.5 5,127.09,126.31,123.32,117.17,61.62,60.93,44.18,42.60,21.79,21.30,18.57.HR-ESIMS m / z 344.1674[M+H] + ,calcd forC 23 H 22 NO2 + 344.1651.

[0070] Characterization of compound AH-25 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.21(d,J=8.4Hz,1H),6.64(s,1H),6.55-6.47(m,2H),5.66(s,1H),4.45(s,1H),3.84(d,J=3.6Hz,6H),3.65( d,J=13.3Hz,1H),2.71(ddd,J=28.3,9.6,5.1Hz,2H),2.55-2.47(m,1H),2.11(d,J=9.8Hz,1H),1.59(s,4H),1.41(d,J=15.0Hz,2H). 13C NMR (101MHz, CDCl3) δ176.90,173.37,161.87,158.14,129.70,118.78,109.45,107.00,104.94 ,98.96,95.60,91.46,62.66,61.36,55.52,55.48,43.64,42.25,22.22,20.98,18.83.HR-ESIMS m / z 354.1719[M+H] + calcd for C 21 H 24 NO4 + 354.1705.

[0071] Characterization of compound AH-26 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ6.88(d,J=1.9Hz,1H),6.67(d,J=2.1Hz,2H),6.50(t,J=2.2Hz,1H),5.78(s,1H),4.51(d,J=2.8Hz,1H),3.83(s ,6H),2.82(dd,J=9.6,4.3Hz,1H),2.73-2.57(m,3H),2.05(d,J=9.9Hz,1H),1.87-1.74(m,1H),1.72-1.57(m,4H),1.44-1.41(m,1H). 13 C NMR (101MHz, CDCl3) δ172.99,167.51,161.11,158.08,140.29,117.05,108.64,104. 49,101.31,90.78,61.75,60.71,55.51,43.98,42.58,22.05,21.17,18.54.HR-ESIMS m / z354.1758[M+H] + calcd for C 21 H 24 NO4 + 354.1705.

[0072] Characterization of compound AH-27 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1H NMR (400MHz, CDCl3) δ7.45-7.31(m,6H),7.15(d,J=8.5Hz,2H),7.01(d,J=8 .0Hz,1H),6.86(s,1H),5.77(s,1H),5.10(s,2H),4.46(d,J=4.8Hz,1H),3.6 7(d,J=13.0Hz,1H),2.81(dd,J=9.9,4.7Hz,1H),2.69-2.55(m,2H),2.04(d, J=9.9Hz,1H),1.69-1.55(m,3H),1.38(d,J=12.4Hz,2H),1.28-1.12(m,1H). 13 C NMR (101MHz, CDCl3) δ172.95,167.50,159.06,157.91,139.68,136.46,130.03,128.63,128.12,127.41,118. 78,116.91,115.81,112.94,108.46,91.09,70.12,61.63,60.67,43.88,42.50,21.97,21.09,18.47.HR-ESIMS m / z 400.1938[M+H] + calcd for C 26 H 26 NO3 + 400.1913.

[0073] Characterization of compound AH-28 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR(400MHz, CDCl3)δ7.54(d,J=8.9Hz,2H),7.45-7.34(m,5H),7.01(d,J=9.0Hz ,2H),6.82(s,1H),5.72(s,1H),5.11(s,2H),4.52(d,J=4.6Hz,1H),3.68(dd,J=1 2.9,3.3Hz,1H),2.81(dd,J=9.8,4.7Hz,1H),2.63(td,J=11.6,11.1,5.3Hz,2H) ,2.04(d,J=9.8Hz,1H),1.70-1.57(m,3H),1.47-1.35(m,2H),1.26-1.17(m,1H). 13C NMR (101MHz, CDCl3) δ173.22,167.90,160.19,157.26,136.33,130.77,128.69,128.21,127.64,127. 45,115.30,114.76,107.38,91.02,70.12,61.31,60.75,43.85,42.41,22.37,20.98,18.42.HR-ESIMS m / z 400.1938[M+H] + calcd for C 26 H 26 NO3 + 400.1913.

[0074] Characterization of compound AH-29 by 1H NMR, 1C NMR, and high-resolution mass spectrometry: 1 H NMR (400MHz, CDCl3) δ7.33-7.27(m,2H),7.19(t,J=8.8Hz,2H),6.47(s,1H),5.73(s,1H),4.07(d,J=5.1Hz,1H),3.68-3.63(m,1H),2. 82(dq,J=10.1,5.5Hz,2H),2.63-2.53(m,3H),2.18(d,J=9.6Hz,1H),1.60-1.58(m,4H),1.44(d,J=7.9Hz,2H),1.23(t,J=7.6Hz,3H). 13 C NMR (101MHz, CDCl3) δ172.68,164.40,141.53,140.36,138.18,129.01,128.88,127.74,126.12, 120.26,108.08,90.34,65.47,61.95,45.92,42.97,26.57,22.90,21.74,19.33,15.18.HR-ESIMS m / z322.1824[M+H] + calcd for C 21 H 24 NO2 + 322.1807.

[0075] Example 2: Assay for antitumor activity

[0076] All cells were cultured in a 37°C, 5% CO2 incubator. The culture medium for human acute promyelocytic leukemia cells (NB4), human histiocytic lymphoma cells (U937), human chronic myeloid leukemia cells (K562), and gastric cancer cells (MNK45) consisted of RPMI-1640 medium + 10% FBS + 1% antibiotics. The culture medium for human promyelocytic leukemia cells (HL-60) consisted of IMDM medium + 20% FBS + 1% antibiotics. The culture medium for human ovarian cancer cells (SKOV3) consisted of high-glucose DMEM medium + 10% FBS + 1% antibiotics. The culture medium for human undifferentiated gastric cancer cells (HGC-27) and human pancreatic cancer cells (PANC-1) consisted of DMEM medium + 10% FBS + 1% antibiotics.

[0077] (1) MTT assay procedure: SKOV3, MNK45, HGC-27, and PANC-1 cells in logarithmic growth phase were digested and centrifuged, the supernatant was discarded, the cells were resuspended and counted, and the cells were counted at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of / mL in 96-well plates and cultured overnight until adherence. The next day, the old culture medium was aspirated, and 100μL of medium containing the drug solution was added. After culturing for a specific time of 72h in an incubator, 20μL of MTT solution (5mg / mL) was added to each well, and the cells were incubated for 4h. The supernatant was aspirated, and 120μL of LDMSO was added to each well. The cells were shaken for 5 minutes, and the absorbance was measured at 490nm using a microplate reader. The inhibition rate was calculated as [=(OD 空白 -OD 实验 ) / OD 空白 ×100% and IC 50 (Calculated using GraphPad software). MTT results showed that allegorical alkaloids and most of their derivatives could inhibit tumor cell growth. Among them, the most active compound, AH-26, showed the highest IC50 activity in tumor cells. 50 The concentration is 0.01-0.49 μM, which is stronger than that of the positive control drug camptothecin.

[0078] (2) CCK8 experimental procedure: Centrifuge NB4, HL-60, U937, and K562 cells in logarithmic growth phase, discard the supernatant, resuspend the cells and count them, according to 1×10⁻⁶. 5 Cells were seeded at a density of 50 μL / mL in 96-well plates, followed by 50 μL of culture medium containing the drug solution per well. After incubation for 72 h, 10 μL of CCK8 solution was added to each well, and the plates were incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader. The inhibition rate was calculated as follows: [=(OD] 空白 -OD 实验 ) / (OD 空白 -OD 培养基 [100%] and IC 50(Calculated using GraphPad software). CCK8 results showed that allegorical baicalein and its derivatives exhibited excellent antitumor activity, with the most active compound, AH-26, showing the best activity against IC50 in myeloid leukemia cells. 50 The concentration was 0.16-0.42 μM, which was stronger than that of the positive control camptothecin.

[0079] Table 1. Antitumor activity of AH-1 to AH-29

[0080]

[0081]

[0082] Example 3: Study on inducing tumor cell apoptosis

[0083] Cell (NB4, HL-60, U937, K562, SKOV3, HGC-27) culture method follows Example 3. Cells in logarithmic growth phase are seeded in 6-well plates at 1×10⁶ cells / well. 5 Cells / well were incubated with the compound for 48 hours, then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were collected. 100 μL of Annexin V Binding Buffer was added to resuspend the cells, followed by 2.5 μL of Annexin V-FITC Reagent and 2.5 μL of PI Reagent. The mixture was gently vortexed and incubated at room temperature in the dark for 20 minutes. Then, 400 μL of Annexin V Binding Buffer was added, and the sample was thoroughly mixed. Appropriate flow cytometry parameters were set, and the cells were analyzed. The results showed that AH-26 significantly induced apoptosis in leukemia cells in a concentration-dependent manner. Figure 2 , Figure 3 ).

[0084] Example 4: Study on the effect on mitochondrial membrane potential of tumor cells

[0085] Cell (NB4, HL-60, U937, K562, SKOV3, HGC-27) culture method follows Example 3. Cells in logarithmic growth phase are seeded in 6-well plates at 1×10⁶ cells / well. 5Cells / well were treated with the compound for 48 hours, then centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and cells were collected. 0.5 mL of JC-1 staining working solution was added, and the cells were mixed by inverting several times. Cells were incubated at 37°C for 20 minutes, then centrifuged at 600 × g at 4°C for 4 minutes and collected. Cells were washed twice with JC-1 staining buffer, and finally resuspended in 200 μL of JC-1 staining buffer. Appropriate flow cytometry parameters were set, and the cells were analyzed. The results showed that the mitochondrial membrane potential of NB4, HL-60, U937, K562, SKOV3, and HGC-27 cells treated with AH-26 underwent a significant concentration-dependent inversion, indicating that AH-26 promoted mitochondrial-mediated apoptosis in tumor cells. Figure 4 , Figure 5 ).

[0086] Example 5: Study on the effect on tumor cell cycle

[0087] The cell (NB4, HL-60, SKOV3, HGC-27) culture method was as described in Example 3. Cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. 5 Cells were collected per well, treated with the compound for 48 hours, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were fixed overnight at 4°C with 500 μL of pre-cooled 70% ethanol. After centrifugation, the cells were collected again, and 100 μL of RNase A solution was added to resuspend the cells. The cells were then incubated at 37°C for 30 minutes. 400 μL of PI staining solution was added and mixed thoroughly, and the cells were incubated at 4°C in the dark for 30 minutes. Appropriate flow cytometry parameters were set, and the cells were analyzed. The results showed that NB4, HL-60, SKOV3, and HGC-27 cells were arrested in the G1 phase after treatment with AH-26, indicating that the compound AH-26 inhibits the tumor cell cycle and thus inhibits tumor cell proliferation. Figure 6 , Figure 7 ).

[0088] Example 6: Study on the effect on tumor cell protein expression

[0089] The cell (NB4, HL-60, SKOV3, HGC-27) culture method was as described in Example 3. Cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1 × 10⁶ cells / well. 5Cells / well were added and incubated with the compound for 48 hours. Cells were then collected, protein samples were extracted, protein concentration was determined by BCA method, protein samples were denatured, electrophoresed, transferred to a membrane, blocked with 5% skim milk powder, incubated with primary and secondary antibodies, developed, and detected by ultrasensitive chemiluminescence analyzer to obtain bands. The results showed that AH-26 promoted the expression of the tumor suppressor protein Bax in tumor cells in a significant concentration-dependent manner. AH-26 activated Caspase 3 / 9, decreased the expression of the original Caspase 3 / 9, and increased the expression of activated Caspase 3 / 9. AH-26 also inhibited the expression of oncogenic proteins Bcl-XL, Bcl-2, TAZ, GLI1, MCL1, XIAP, YAP1, β-catenin, c-Myc, p-AKT, JAK1 / 2, STAT3, and p-STAT3 in tumor cells, also in a concentration-dependent manner. Figure 8 , Figure 9 In summary, AH-26 exerts its anti-tumor effect by inhibiting the JAK / STAT3 pathway.

[0090] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A 15-substituted derivative of cypermethrin, characterized in that: The 15-position substituted derivative of the aforementioned allophylline is any one of compounds AH-2, AH-4, AH-10, AH-15, AH-17, AH-19, AH-21, AH-22, AH-23, AH-25, AH-26, AH-27, AH-28, and AH-29; compounds AH-2, AH-4, AH-10, AH-15, AH-17, AH-19, AH-21, AH-22, AH-23, AH-25, AH-26, AH-27, AH-28, and AH-29 are compounds having the following structures: 。 2. A pharmaceutically acceptable salt of the 15-substituted derivative of the allophylline as described in claim 1.

3. The pharmaceutically acceptable salt of the 15-substituted derivative of allophyllin according to claim 2, characterized in that: The pharmaceutically acceptable salt has an anion that is either inorganic or organic; the inorganic anion is chloride, bromide, iodide, sulfate, nitrate, nitrite, phosphate, or hydrogen phosphate; the organic anion is acetate, propionate, cinnamate, benzosulfonate, citrate, lactate, or gluconate.

4. The use of the 15-substituted derivative of allophyllin as described in claim 1 or the salt described in any one of claims 2-3 in the preparation of antitumor drugs, characterized in that: The tumors mentioned include any one or more of leukemia, ovarian cancer, and gastric cancer.

5. The application according to claim 4, characterized in that: The tumor in question is stomach cancer.

6. The application according to claim 5, characterized in that: The 15-substituted derivative of the aforementioned allophylline is compound AH-26.

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