Biochanin derivatives, preparation methods thereof, and applications thereof in drugs for alleviating drug-induced renal injury

Abstract

The present invention discloses a biochanin derivative, a preparation method thereof, and its application in a drug for alleviating drug-induced renal injury. The biochanin derivative has the following general formula: wherein R is hydrogen, a linear, branched, or cyclic alkyl group having 1 to 10 carbon atoms, a linear, branched, or cyclic alkyl group having 1 to 10 carbon atoms containing N, O, S, or a halogen, or any one of aromatic hydrocarbons. The biochanin derivative provided by the present invention is non-toxic to renal cells, has strong anti-inflammatory activity, can effectively alleviate the toxic damage of cisplatin to human proximal renal tubular cells HK-2, and significantly improve cisplatin-induced acute kidney injury in mice.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to a biochanin derivative, a preparation method thereof, and an application thereof in a drug for alleviating drug-induced renal injury. Background Art

[0002] Cisplatin (dichlorodiamine platinum) is an inorganic platinum chemotherapy drug that is widely used in the treatment of various malignant tumors such as head and neck, lung, ovarian, testicular, and bladder cancers. Dr. Burnett Rosenberg first discovered its ability to inhibit cell division in 1965, and first synthesized it in 1970, and it was soon approved for use as a chemotherapy drug. As the first platinum anti-tumor drug approved by the FDA, it has led to the development of countless other transition metal-based chemotherapy drugs since then. Specifically, the anti-tumor properties of cisplatin are related to its ability to restrict cell division by embedding into the DNA of cells. The simplicity and effectiveness of its anti-cancer activity make cisplatin an indispensable drug in cancer treatment, helping to improve the survival rate of solid tumors.

[0003] The use of cisplatin is often limited by its severe side effects. Despite its efficacy in treating a variety of solid tumors, particularly pediatric cancers, its toxicity severely limits its clinical use and the long-term health outcomes and quality of life of cancer patients. Cisplatin-induced adverse drug reactions and toxicity lead to the development of several cisplatin-induced toxicities (CITs), which are primarily targeted to specific sites in the body, such as the kidneys, liver, neurons, and inner ear. This is due to preferential accumulation in these areas after repeated intravenous administration during treatment. Due to its low molecular weight and lack of charge, unbound cisplatin in plasma is freely filtered by the glomeruli, with the majority being concentrated in the renal cortex. Concentrations in proximal tubular cells are approximately five times higher than in serum, and accumulation in the kidneys leads to its potent nephrotoxicity. Cisplatin-induced nephrotoxicity progresses to acute kidney injury (AKI) through tubular apoptosis, inflammation, and necrosis. Approximately 15% to 30% of cancer patients receiving cisplatin chemotherapy develop AKI. Currently, there are no effective therapeutics to alleviate cisplatin-induced renal damage. Therefore, there is an urgent need to develop new therapeutic drugs for AKI. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these objects and other advantages according to the present invention, a biochanin derivative is provided, which has the following general formula:

[0006]

[0007] Wherein, R is any one of hydrogen, a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms, a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms containing N, O, S or halogen, and an aromatic hydrocarbon.

[0008] The present invention also provides a preparation method of the biochanin derivative, which comprises using biochanin A as a parent core structure and substituting a nitrogen-containing or nitrogen- and oxygen-containing heterocyclic compound as an active group; adding biochanin A to methanol, and incubating in an oil bath at 50° C. until the biochanin A is completely dissolved; adding the nitrogen-containing or nitrogen- and oxygen-containing heterocyclic compound and formaldehyde; and continuing the reaction at room temperature for 24 hours to obtain a crude product; purifying the crude product by column chromatography, and drying to obtain a target compound; the eluent is dichloromethane:methanol=50:1; and the usage ratio of biochanin A, formaldehyde, and heterocyclic compound is 1.0 mol:1.5 mol:2.0 mol;

[0009] Its synthetic route is as follows:

[0010]

[0011] The present invention also provides a use of the biochanin derivative in preparing a drug for alleviating drug-induced renal injury.

[0012] Preferably, in the above use, the drug causing drug-induced renal injury includes at least an anti-tumor drug.

[0013] Preferably, in the above use, the anti-tumor drug at least includes cisplatin.

[0014] Preferably, the drug further comprises a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier includes a diluent, a solubilizer, a co-solvent, a disintegrant, a dispersant, a lubricant, a flavoring agent, an antioxidant, a binder, an absorbent, a wetting agent, a buffer, and a cross-linking agent.

[0016] Preferably, the drug is prepared into a pharmaceutically acceptable dosage form.

[0017] Preferably, the dosage forms include pills, tablets, powders, capsules, granules, powders, pellets, drops, sprays, injections, suspensions, ointments, gels, and suppositories.

[0018] The present invention has at least the following beneficial effects:

[0019] The present invention found that biochanin derivatives are non-toxic to renal cells, have strong anti-inflammatory activity, can effectively alleviate the toxic damage of cisplatin to human proximal renal tubular cells HK-2 cells, and significantly improve cisplatin-induced acute kidney injury (AKI) in mice. The biochanin derivatives provided by the present invention have the advantages of strong protection against AKI, high efficiency and low toxicity, and are expected to be developed into drugs for alleviating cisplatin-induced nephrotoxicity.

[0020] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of compound YN-8 in Experimental Example 3 of the present invention on improving the survival rate of human normal kidney cells HK-2 by cisplatin;

[0022] Figure 2 This is the effect of compound YN-8 in Experimental Example 4 of the present invention on apoptosis of HK-2 cells damaged by cisplatin;

[0023] Figure 3 Effects of compounds YN-2, YN-3, YN-5, YN-6, YN-7, YN-8, and YN-9 in Experimental Example 5 of the present invention on the proliferation of Hep-G2 and SK-Hep-1 liver cancer cells induced by cisplatin;

[0024] Figure 4 The effect of YN-8 on the histopathological changes of mice with acute kidney injury induced by cisplatin in Experimental Example 6 of the present invention;

[0025] Figure 5 This is the effect of YN-8 in Experimental Example 6 of the present invention on BUN and CRE in the serum of mice with acute kidney injury induced by cisplatin. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0027] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0028] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0029] In the description of the present invention, the terms "horizontal", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like to indicate orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the present invention.

[0030] <Example 1>

[0031] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-(1,4-oxazacyclohexan-4-ylmethyl)-4H-chromen-4-one (Compound 1):

[0032] Weigh 200 mg of biochanin A, add 20 mL of methanol, and incubate in a 50°C oil bath until completely dissolved. Add 131 μL of morpholine and 85 μL of formaldehyde, and continue the reaction at room temperature for 24 hours. The crude product is purified by column chromatography using a 50:1 ratio of dichloromethane to methanol as the eluent. After drying, the title compound is obtained as a pale yellow solid in a 76% yield.

[0033] The structural formula of compound 1 is shown below:

[0034]

[0035] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0036] 1 H NMR(500MHz,Chloroform-d)δ8.53(s,1H),8.02(s,1H),7.51–7.44(m,2H),6.99–6. 93(m,2H),6.28(s,1H),3.81(d,J=5.5Hz,5H),3.70–3.58(m,4H),2.61–2.46(m,4H). 13 C NMR(125MHz,Chloroform-d)δ182.13,162.98,161.21,159.73,156.28,153.40,13 0.85,126.09,124.21,114.00,104.09,103.05,98.94,66.55,55.31,54.25,53.03.

[0037] HRMS (EI-TOF) M383.4011 and calculated C 21 H 21 NO6 383.4000 matches.

[0038] <Example 2>

[0039] Synthesis of 5,7-dihydroxy-8-{[(3R,5S)-3,5-dimethyl-1,4-oxazacyclohexan-4-yl]methyl}-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 2):

[0040] The steps were the same as in Example 1, except that cis-3,5-dimethylmorpholine was used instead of morpholine (equimolar amounts, and the following examples were consistent with this). A colorless solid was obtained with a yield of 65%. The structural formula of Compound 2 is as follows:

[0041]

[0042] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0043] 1 H NMR(500MHz,Chloroform-d)δ8.51(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H),6.31(s,1H),4.17(d,J=14.5Hz,1H),3. 88–3.80(m,4H),3.67(dd,J=10.4,6.4Hz,2H),3.59(dd,J=10.6,4.2Hz,2H),2.83(qdd,J=7.3,6.4,4.0Hz,2H),1.16(d,J=7.3Hz,6H). 13 C NMR (125 MHz, Chloroform-d) δ 181.66, 163.10, 161.30, 159.68, 158.07, 153.48, 130.68, 123.91, 123.49, 113.89, 104.09, 102.67, 99.13, 72.13, 55.34, 53.87, 47.84, 15.53. HRMS (EI-TOF) M 411.46819, consistent with the calculated C 23 H 25 NO6 411.4540 matches.

[0044] <Example 3>

[0045] Synthesis of 5,7-dihydroxy-8-{[(3R,5S)-3,5-dimethyl-1,4-oxazacyclohexan-4-yl]methyl}-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 3):

[0046] The steps were the same as in Example 1, except that pyridin-2-yl-methanol was used instead of morpholine to obtain a colorless solid with a yield of 55%. The structural formula of compound 3 is as follows:

[0047]

[0048] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0049] 1 H NMR(500MHz,Chloroform-d)δ8.91(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m, 2H),6.34(s,1H),4.46(t,J=7.1Hz,1H),4.14(d,J=14.5Hz,1H),3.94(d,J=14.5Hz,1H),3. 82(s,3H),3.59(ddd,J=11.0,7.1,6.2Hz,1H),3.43(ddd,J=11.0,7.1,6.1Hz,1H),2.76(p, J=5.9Hz,1H),2.53–2.45(m,1H),2.37–2.30(m,1H),1.90–1.74(m,3H),1.74–1.66(m,1H). 13 C NMR (125 MHz, Chloroform-d) δ 182.08, 163.06, 161.18, 159.68, 156.24, 153.20, 130.86, 126.04, 124.00, 113.91, 104.07, 102.09, 98.94, 65.93, 62.97, 55.33, 53.64, 50.45, 27.60, 22.98. HRMS (EI-TOF) M 397.4525, consistent with the calculated C 22 H 23 NO6397.4270 matches.

[0050] <Example 4>

[0051] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-(tetrahydro-1H-pyrrol-1-ylmethyl)-4H-chromen-4-one (Compound 4):

[0052] The steps were the same as in Example 1, except that tetrahydropyrrole was used instead of morpholine to obtain a colorless solid with a yield of 71%. The structural formula of compound 4 is as follows:

[0053]

[0054] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0055] 1 H NMR(500MHz,Chloroform-d)δ8.94(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.93( m,2H),6.33(s,1H),4.06(s,2H),3.80(s,3H),2.81–2.72(m,4H),1.83(h,J=2.6Hz,4H). 13 C NMR(125MHz,Chloroform-d)δ182.16,162.96,161.25,159.78,156.28,153.40,13 0.88,126.13,124.21,113.99,104.09,102.61,98.95,55.32,53.80,53.69,24.02.

[0056] HRMS (EI-TOF) M 367.4010, which is consistent with the calculated C 21 H 21 NO5 367.5429 matches.

[0057] <Example 5>

[0058] Synthesis of 5,7-dihydroxy-8-[(3-hydroxyazetidin-1-yl)methyl]-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 5):

[0059] The steps were the same as in Example 1, except that azetidin-3-ol was used instead of morpholine to obtain a colorless solid with a yield of 71%. The structural formula of compound 5 is as follows:

[0060]

[0061] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0062] 1H NMR(500MHz,Chloroform-d)δ8.82(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.93(m,2H),6.33(s,1H),4.15(dp,J= 6.6, 5.3Hz, 1H), 3.79 (d, J = 10.6Hz, 5H), 3.34 (dd, J = 9.7, 5.4Hz, 2H), 3.13 (dd, J = 9.7, 5.3Hz, 2H), 3.01 (d, J = 6.8Hz, 1H). 13 C NMR (125 MHz, Chloroform-d) δ 182.16, 162.98, 161.25, 159.78, 156.28, 153.40, 130.88, 126.13, 124.21, 113.99, 104.09, 102.63, 98.95, 65.23, 55.45, 55.32, 52.60. HRMS (EI-TOF) M 369.1214, consistent with the calculated C 20 H 19 NO6 369.3730 matches.

[0063] <Example 6>

[0064] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-[(4-methylpiperazin-1-yl)methyl]-4H-chromen-4-one (Compound 6):

[0065] The steps were the same as in Example 1, except that N-methylpiperazine was used instead of morpholine to obtain a colorless solid with a yield of 72%. The structural formula of compound 6 is as follows:

[0066]

[0067] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0068] 1 H NMR(500MHz,Chloroform-d)δ8.53(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H) ,6.30(s,1H),3.83(d,J=10.8Hz,5H),2.71–2.59(m,4H),2.43(t,J=5.3Hz,4H),2.31(s,3H). 13C NMR (125 MHz, Chloroform-d) δ 182.08, 163.01, 161.21, 159.68, 156.16, 153.20, 130.86, 126.04, 124.00, 113.91, 104.09, 103.07, 98.94, 55.31, 54.08, 53.41, 52.63, 45.21. HRMS (EI-TOF) M 396.6852, consistent with the calculated C 22 H 24 N2O5 396.4430 is consistent.

[0069] <Example 7>

[0070] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-[(4-methylhexahydropyridin-1-yl)methyl]-4H-chromen-4-one (Compound 7):

[0071] The steps were the same as in Example 1, except that 4-methylpiperidinium was used instead of morpholine to obtain a colorless solid with a yield of 72%. The structural formula of compound 7 is as follows:

[0072]

[0073] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0074] 1 H NMR(500MHz,Chloroform-d)δ8.52(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H),6.27(s,1H),4.07(s,2H),3. 82(s,3H),2.87(ddd,J=12.1,7.9,5.7Hz,2H),2.14(ddd,J=12.0,8.0,5.5Hz,2H),1.66–1.41(m,5H),1.02(d,J=6.7Hz,3H). 13 C NMR (125 MHz, Chloroform-d) δ

[0075] 182.08,162.96,161.21,159.68,156.16,153.20,130.86,126.04,124.00,113.91,104.09,103.06,98.94,55.31,54.16,51.76,33.18,30.42,21.76. HRMS (EI-TOF) M395.3112 and calculated C 23 H25 NO5

[0076] 395.1732 matches.

[0077] <Example 8>

[0078] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-[(3-methylhexahydropyridin-1-yl)methyl]-4H-chromen-4-one (Compound 8):

[0079] The steps were the same as in Example 1, except that 3-methylpiperidine was used instead of morpholine to obtain a colorless solid with a yield of 72%. The structural formula of compound 8 is as follows:

[0080]

[0081] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0082] 1 H NMR(500MHz,Chloroform-d)δ8.52(s,1H),8.02(s,1H),7.50–7.44(m,2H),6. 99–6.93(m,2H),6.30(s,1H),4.09(d,J=14.5Hz,1H),3.82(s,3H),3.74(d,J=1 4.5Hz,1H),2.89(dd,J=10.3,5.4Hz,1H),2.72(ddd,J=11.8,6.9,4.7Hz,1H),2 .61–2.52(m,2H),1.84–1.73(m,1H),1.72–1.47(m,4H),0.93(d,J=7.0Hz,3H). 13 C NMR (125 MHz, Chloroform-d) δ 182.08, 162.96, 161.21, 159.68, 156.16, 153.20, 130.86, 126.04, 124.00, 113.91, 104.09, 103.16, 98.94, 58.93, 55.31, 54.39, 53.76, 32.52, 30.50, 24.25, 19.29. HRMS (EI-TOF) M 395.2327, consistent with the calculated C 23 H 25 NO5 395.1732 matches.

[0083] <Example 9>

[0084] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-[(2-methylpiperidin-1-yl)methyl]-4H-chromen-4-one (Compound 9):

[0085] The steps were the same as in Example 1, except that 2-methylpiperidine was used instead of morpholine to obtain a colorless solid with a yield of 72%. The structural formula of compound 9 is as follows:

[0086]

[0087] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0088] 1 H NMR(500MHz,Chloroform-d)δ8.56(s,1H),8.02(s,1H),7.50–7.44(m,2H),6. 99–6.93(m,2H),6.31(s,1H),4.14(d,J=14.5Hz,1H),3.84–3.76(m,4H),2.79 (ddd,J=11.9,7.0,4.8Hz,1H),2.67(ddd,J=11.9,7.1,4.7Hz,1H),2.56(qt,J =6.6,5.7Hz,1H),1.74–1.45(m,4H),1.42–1.25(m,2H),1.10(d,J=6.6Hz,3H). 13 C NMR (125 MHz, Chloroform-d) δ 182.08, 163.06, 161.18, 159.68, 156.24, 153.20, 130.86, 126.04, 124.00, 113.91, 104.07, 102.57, 98.94, 56.27, 55.31, 52.31, 52.15, 33.49, 25.05, 24.32, 18.19. HRMS (EI-TOF) M 395.1355, consistent with the calculated C 23 H 25 NO5 395.1732 matches.

[0089] <Example 10>

[0090] Synthesis of 5,7-dihydroxy-8-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 10):

[0091] The steps were the same as in Example 1, except that N-hydroxyethylpiperazine was used instead of morpholine to obtain a colorless solid with a yield of 72%. The structural formula of compound 10 is as follows:

[0092]

[0093] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0094] 1 H NMR(500MHz,Chloroform-d)δ8.50(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.92(m,2H),6.3 4(s,1H),4.27(t,J=7.3Hz,1H),3.82(d,J=4.9Hz,5H),3.54(q,J=6.9Hz,2H),2.70–2.53(m,10H). 13 C NMR (125 MHz, Chloroform-d) δ 182.13, 162.95, 161.36, 159.54, 156.07, 153.21, 130.67, 125.93, 123.87, 113.88, 104.11, 103.08, 98.96, 59.41, 58.54, 55.34, 53.97, 53.21, 51.86. HRMS (EI-TOF) M 426.2231, consistent with the calculated C 23 H 26 N2O6426.1790 is consistent.

[0095] <Example 11>

[0096] Synthesis of 8-[(4-benzylhexahydropyridin-1-yl)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 11):

[0097] The steps were the same as in Example 1, except that 4-benzylpiperidine was used instead of morpholine to obtain a colorless solid with a yield of 65%. The structural formula of compound 11 is as follows:

[0098]

[0099] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0100] 1H NMR(500MHz,Chloroform-d)δ8.66(s,1H),8.02(s,1H),7.50–7.44(m,2H),7.29–7.21(m ,2H),7.20–7.09(m,3H),6.99–6.93(m,2H),6.30(s,1H),4.08(s,2H),3.82(s,3H),2.83( ddd,J=11.5,8.1,5.7Hz,2H),2.58(dt,J=8.0,1.0Hz,2H),2.51(ddd,J=11.5,7.9,5.7Hz ,2H),1.82(tp,J=8.0,6.2Hz,1H),1.66(ddt,J=12.1,8.0,5.9Hz,2H),1.61–1.50(m,2H). 13 C NMR (125 MHz, Chloroform-d) δ 182.14, 162.96, 161.37, 159.70, 156.38, 153.21, 139.08, 130.67, 129.00, 128.59, 126.77, 126.07, 123.93, 113.94, 104.10, 103.01, 99.05, 55.33, 54.05, 51.60, 42.54, 36.18, 30.16. HRMS (EI-TOF) M 471.3154, consistent with the calculated C 29 H 29 NO5 471.2045 matches.

[0101] <Example 12>

[0102] Synthesis of 8-[(4-benzylpiperazin-1-yl)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 12):

[0103] The steps were the same as in Example 1, except that 1-benzylpiperazine was used instead of morpholine to obtain a colorless solid with a yield of 54%. The structural formula of compound 12 is as follows:

[0104]

[0105] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0106] 1H NMR(500MHz,Chloroform-d)δ8.63(s,1H),8.02(s,1H),7.50–7.44(m,2H),7.32–7.19(m,5H),6.99–6.93( m,2H),6.32(s,1H),3.97(s,2H),3.82(s,3H),3.68(d,J=0.8Hz,2H),2.93–2.84(m,2H),2.72–2.60(m,6H). 13 C NMR (125 MHz, Chloroform-d) δ 182.26, 162.96, 161.37, 159.70, 156.38, 153.21, 138.44, 130.67, 128.60, 128.42, 127.66, 126.07, 123.93, 113.94, 104.05, 103.02, 99.05, 61.99, 55.33, 53.97, 53.33, 53.29, 53.28. HRMS (EI-TOF) M 472.2189, consistent with the calculated C 28 H 28 N2O5 472.1998 is consistent.

[0107] <Example 13>

[0108] Synthesis of 5,7-dihydroxy-8-{[4-(hydroxymethyl)piperidin-1-yl]methyl}-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 13):

[0109] The steps were the same as in Example 1, except that 4-hydroxymethylpiperidine was used instead of morpholine to obtain a colorless solid with a yield of 55%. The structural formula of compound 13 is as follows:

[0110]

[0111] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0112] 1H NMR(500MHz,Chloroform-d)δ8.52(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H),6.30(s,1H),4.06(s,2H),3.82(s,3H),3.33(t,J= 6.9Hz,2H),2.84(ddd,J=11.4,7.9,5.7Hz,2H),2.74(t,J=7.0Hz,1H),2.51(ddd,J=11.5,8.0,5.6Hz,2H),1.77–1.66(m,2H),1.66–1.48(m,3H). 13 C NMR (125 MHz, Chloroform-d) δ 182.13, 162.93, 161.27, 159.68, 156.25, 153.40, 130.68, 125.93, 124.00, 113.89, 104.00, 103.06, 99.03, 65.52, 55.34, 54.09, 51.72, 36.60, 27.72. HRMS (EI-TOF) M 411.3269, consistent with the calculated C 23 H 25 NO6 411.1681 matches.

[0113] <Example 14>

[0114] Synthesis of 5,7-dihydroxy-8-[(4-hydroxyhexahydropyridin-1-yl)methyl]-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 14):

[0115] The steps were the same as in Example 1, except that 4-hydroxypiperidine was used instead of morpholine to obtain a colorless solid with a yield of 80%. The structural formula of compound 14 is as follows:

[0116]

[0117] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0118] 1H NMR(500MHz,Chloroform-d)δ9.13(s,1H),8.02(s,1H),7.50–7.44(m,2H),6. 99–6.93(m,2H),6.33(s,1H),4.07(s,2H),3.82(s,3H),3.64(dp,J=6.9,6.1H z,1H),3.07(d,J=6.8Hz,1H),2.84(ddd,J=12.1,8.1,5.9Hz,2H),2.41(ddd,J =12.1,8.0,5.7Hz,2H),1.86(ddt,J=13.2,8.1,5.9Hz,2H),1.81–1.72(m,2H). 13 C NMR (125 MHz, Chloroform-d) δ

[0119] 182.08,163.01,161.21,159.68,156.16,153.20,130.86,126.04,124.00,113.91,104.09,103.06,98.94,67.86,55.33,54.16,50.73,33.43. HRMS (EI-TOF) M397.1432, calculated C 22 H 23 NO6 397.1525 matches.

[0120] <Example 15>

[0121] Synthesis of 5,7-dihydroxy-8-[(3-hydroxyhexahydropyridin-1-yl)methyl]-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 15):

[0122] The steps were the same as in Example 1, except that 3-hydroxypiperidine was used instead of morpholine to obtain a colorless solid with a yield of 80%. The structural formula of compound 15 is as follows:

[0123]

[0124] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0125] 1H NMR(500MHz,Chloroform-d)δ8.92(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H),6 .33(s,1H),4.14(d,J=1.8Hz,2H),3.91–3.82(m,3H),3.15(d,J=6.8Hz,1H),2.90(dd,J=10.8,5 .3Hz,1H),2.84(dd,J=10.8,5.3Hz,1H),2.78(ddd,J=11.8,7.0,4.7Hz,1H),2.61(ddd,J=11.8, 6.8,4.8Hz,1H),1.78–1.65(m,2H),1.65–1.57(m,1H),1.50(dddd,J=12.7,7.7,5.9,5.1Hz,1H). 13 C NMR (125 MHz, Chloroform-d) δ 182.08, 163.01, 161.21, 159.68, 156.16, 153.20, 130.86, 126.04, 124.00, 113.91, 104.09, 103.25, 98.94, 67.40, 58.40, 55.33, 54.46, 53.69, 32.55, 23.36. HRMS (EI-TOF) M 397.1342, consistent with the calculated C 22 H 23 NO6 397.1525 matches.

[0126] <Example 16>

[0127] Synthesis of 1-{[5,7-dihydroxy-3-(4-methoxyphenyl)-4-oxyylidenechromen-8-yl]methyl}piperidine-4-carboxylic acid (Compound 16):

[0128] The steps were the same as in Example 1, except that 4-piperidinic acid was used instead of morpholine to obtain a colorless solid with a yield of 20%. The structural formula of compound 16 is as follows:

[0129]

[0130] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0131] 1H NMR(500MHz,Chloroform-d)δ9.04(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.92(m,2H),6.34(s,1 H),4.06(s,2H),3.82(s,3H),2.89(ddd,J=12.1,7.8,5.8Hz,2H),2.56–2.43(m,3H),2.14–1.98(m,4H). 13 C NMR (125 MHz, Chloroform-d) δ 182.13, 180.64, 162.93, 161.36, 159.54, 156.07, 153.21, 130.67, 125.93, 123.87, 113.88, 104.11, 103.07, 98.96, 55.34, 54.05, 51.90, 40.58, 28.07. HRMS (EI-TOF) M 425.1546, consistent with the calculated C 23 H 23 NO7

[0132] 425.1474 matches.

[0133] <Example 17>

[0134] Synthesis of 1-{[5,7-dihydroxy-3-(4-methoxyphenyl)-4-oxyylidenechromen-8-yl]methyl}piperidine-3-carboxylic acid (Compound 17):

[0135] The steps were the same as in Example 1, except that 3-piperidinic acid was used instead of morpholine to obtain a colorless solid with a yield of 20%. The structural formula of compound 17 is as follows:

[0136]

[0137] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0138] 1H NMR(500MHz,Chloroform-d)δ9.13(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.92(m,2H),6.33(s,1H),4.12(d,J=1.1Hz,2H),3.82(s,3H),3.47( dd,J=10.2,5.8Hz,1H),3.03(dd,J=10.2,5.8Hz,1H),2.76–2.68(m,1H),2 .68–2.56(m,2H),1.78–1.70(m,2H),1.70–1.63(m,2H),1.63–1.57(m,1H). 13 C NMR (125 MHz, Chloroform-d) δ 182.13, 179.38, 162.95, 161.36, 159.54, 156.07, 153.21, 130.67, 125.93, 123.87, 113.88, 104.11, 103.10, 98.96, 55.34, 54.35, 53.97, 53.53, 42.03, 27.07, 23.82. HRMS (EI-TOF) M 425.1345, consistent with the calculated C 23 H 23 NO7 425.14745 matches.

[0139] <Example 18>

[0140] Synthesis of 8-(hexahydropyridin-1-ylmethyl)-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 18):

[0141] The steps were the same as in Example 1, except that piperidine was used instead of morpholine to obtain a colorless solid with a yield of 82%. The structural formula of compound 18 is as follows:

[0142]

[0143] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0144] 1H NMR(500MHz,Chloroform-d)δ9.07(s,1H),8.02(s,1H),7.51–7.44(m,2H),6.99–6.93(m,2H),6.33(s,1H) ,4.07(s,2H),3.81(s,3H),2.58–2.51(m,4H),1.59–1.50(m,4H),1.43(dddd,J=12.2,7.0,5.7,1.3Hz,2H). 13 C NMR (125 MHz, Chloroform-d) δ 182.13, 162.96, 161.21, 159.73, 156.28, 153.40, 130.85, 126.09, 124.21, 114.00, 103.84, 103.07, 98.94, 55.31, 54.19, 53.77, 25.34, 24.02. HRMS (EI-TOF) M 381.1524, consistent with the calculated C 22 H 23 NO5

[0145] 381.15762 matches.

[0146] <Example 19>

[0147] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-(piperazin-1-ylmethyl)-4H-chromen-4-one (Compound 19):

[0148] The steps were the same as in Example 1, except that piperazine was used instead of morpholine to obtain a colorless solid with a yield of 32%. The structural formula of compound 19 is as follows:

[0149]

[0150] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0151] 1 H NMR(500MHz,Chloroform-d)δ9.05(s,1H),8.02(s,1H),7.51–7.44(m,2H),6.99–6.93(m,2H) ,6.33(s,1H),3.80(d,J=9.2Hz,5H),2.82–2.75(m,2H),2.75–2.65(m,5H),2.52–2.44(m,2H). 13C NMR (125 MHz, Chloroform-d) δ 182.13, 163.01, 161.21, 159.73, 156.28, 153.40, 130.85, 126.09, 124.21, 114.00, 103.84, 103.07, 98.94, 55.31, 54.08, 52.81, 45.84. HRMS (EI-TOF) M 382.1573, consistent with the calculated C 21 H 22 N2O5 382.15287 is consistent.

[0152] <Example 20>

[0153] Synthesis of 8-[(6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 20):

[0154] The steps were the same as in Example 1, except that 1,2,3,4-tetrahydroisoquinoline-6,7-diphenol was used instead of morpholine to obtain a colorless solid with a yield of 15%. The structural formula of compound 20 is as follows:

[0155]

[0156] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0157] 1 H NMR(500MHz,Chloroform-d)δ8.88(s,1H),8.02(s,1H),7.50–7.44(m,2H),6. 99–6.93(m,2H),6.89(s,1H),6.70(s,1H),6.60(t,J=1.0Hz,1H),6.50(t,J=0. 9Hz,1H),6.33(s,1H),4.54(dd,J=13.5,1.1Hz,1H),4.43(dd,J=13.5,0.9Hz, 1H),4.22(s,2H),3.82(s,3H),3.03–2.89(m,2H),2.76(td,J=5.3,1.0Hz,2H). 13C NMR (125 MHz, Chloroform-d) δ 182.14, 162.95, 161.37, 159.61, 156.38, 153.21, 144.71, 144.56, 130.68, 126.41, 126.07, 125.68, 123.87, 114.64, 113.86, 113.83, 104.16, 103.37, 99.05, 55.33, 54.66, 52.38, 50.53, 28.84. HRMS (EI-TOF) M 461.1545, consistent with the calculated C 26 H 23 NO7 461.1474 matches.

[0158] <Example 21>

[0159] Synthesis of 8-{[4-(2,4-dimethylphenyl)piperazin-1-yl]methyl}-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 21):

[0160] The steps were the same as in Example 1, except that 1-(2,4-dimethylphenyl)piperazine was used instead of morpholine to obtain a colorless solid with a yield of 33%. The structural formula of compound 21 is as follows:

[0161]

[0162] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0163] 1 H NMR(500MHz,Chloroform-d)δ9.16(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.9 9–6.88(m,4H),6.71(d,J=8.4Hz,1H),6.35(s,1H),3.82(d,J=7.5Hz,5H),3.43 (ddd,J=11.9,6.4,4.0Hz,2H),3.28(ddd,J=11.9,6.5,4.2Hz,2H),3.06(ddd,J =11.9,6.6,4.2Hz,2H),2.93(ddd,J=11.7,6.4,4.2Hz,2H),2.27–2.21(m,6H). 13C NMR (125 MHz, Chloroform-d) δ 182.26, 162.96, 161.37, 159.70, 156.25, 153.21, 145.55, 136.81, 131.67, 130.66, 129.72, 127.79, 126.07, 123.93, 119.07, 113.95, 104.05, 103.00, 99.05, 55.33, 54.04, 52.41, 50.26, 20.85, 18.05. HRMS (EI-TOF) M 486.2276, consistent with the calculated C 29 H 30 N2O5486.2154 is consistent.

[0164] <Example 22>

[0165] Synthesis of 8-[(dimethylamino)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 22):

[0166] The steps were the same as those in Example 1, except that dimethylamine was used instead of morpholine to obtain a colorless solid with a yield of 75%.

[0167] The structural formula of compound 22 is as follows:

[0168]

[0169] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0170] 1 H NMR(500MHz,Chloroform-d)δ8.92(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.94(m,2H),6.34(s,1H),3.81(s,3H),3.64(s,2H),2.29(s,6H). 13 C NMR (125 MHz, Chloroform-d) δ 181.97, 162.55, 161.22, 159.77, 155.96, 153.40, 130.64, 126.12, 124.22, 113.97, 104.22, 104.09, 98.96, 55.31, 53.61, 44.28. HRMS (EI-TOF) M 341.1324, consistent with the calculated C 19 H 19 NO5 341.1263 matches.

[0171] <Example 23>

[0172] Synthesis of 8-[(diethylamino)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 23):

[0173] The steps were the same as in Example 1, except that diethylamine was used instead of morpholine to obtain a colorless solid with a yield of 78%. The structural formula of compound 23 is as follows:

[0174]

[0175] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0176] 1 H NMR(500MHz,Chloroform-d)δ9.08(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.94(m ,2H),6.34(s,1H),3.79(d,J=17.0Hz,5H),2.68(q,J=7.2Hz,4H),1.08(t,J=7.2Hz,6H). 13 C NMR(125MHz,Chloroform-d)δ182.16,162.96,161.22,159.79,156.28,153.43,13 0.88,126.12,124.22,113.99,104.09,103.44,98.95,55.32,51.38,46.52,11.55.

[0177] HRMS (EI-TOF) M 369.1525, which is consistent with the calculated C 21 H 23 NO5 369.1576 matches.

[0178] <Example 24>

[0179] Synthesis of 8-[(dipropylamino)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 24):

[0180] The steps were the same as in Example 1, except that dipropylamine was used instead of morpholine to obtain a colorless solid with a yield of 77%. The structural formula of compound 24 is as follows:

[0181]

[0182] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0183] 1 H NMR(500MHz,Chloroform-d)δ9.04(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.93(m,2H),6.33(s,1 H), 4.05 (s, 2H), 3.82 (s, 3H), 2.56 (t, J = 6.3Hz, 4H), 1.53 (qt, J = 7.7, 6.4Hz, 4H), 0.86 (t, J = 7.6Hz, 6H). 13 C NMR (125 MHz, Chloroform-d) δ 182.05, 162.96, 161.21, 159.68, 156.16, 153.20, 130.85, 126.04, 124.00, 113.91, 104.09, 103.21, 98.94, 57.18, 55.33, 52.04, 20.51, 11.85. HRMS (EI-TOF) M 397.1823, consistent with the calculated C 23 H 27 NO5397.1889 matches.

[0184] <Example 25>

[0185] Synthesis of 5,7-dihydroxy-3-(4-methoxyphenyl)-8-{[methyl(propyl)amino]methyl}-4H-chromen-4-one (Compound 25):

[0186] The steps were the same as in Example 1, except that N-methyl-n-propylamine was used instead of morpholine to obtain a colorless solid with a yield of 77%. The structural formula of compound 25 is as follows:

[0187]

[0188] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0189] 1 H NMR(500MHz,Chloroform-d)δ8.98(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.94(m,2H),6.33(s,1H),3. 81(s,3H),3.64(s,2H),2.52(t,J=6.4Hz,2H),2.43(s,3H),1.53(qt,J=7.7,6.4Hz,2H),0.93(t,J=7.7Hz,3H). 13 C NMR (125 MHz, Chloroform-d) δ

[0190] 182.16,162.81,161.20,159.79,156.15,153.43,130.88,126.12,124.22,113.99,104.02,103.38,98.82,59.21,55.32,54.70,44.10,20.36,11.50. HRMS (EI-TOF) M369.1521, calculated C 21 H 23 NO5 369.1576 matches.

[0191] <Example 26>

[0192] Synthesis of 5,7-dihydroxy-8-{[(2-hydroxyethyl)(methyl)amino]methyl}-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 26):

[0193] The steps were the same as in Example 1, except that 2-(methylamino)ethanol was used instead of morpholine to obtain a colorless solid with a yield of 22%. The structural formula of compound 26 is as follows:

[0194]

[0195] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0196] 1 H NMR(500MHz,Chloroform-d)δ8.93(s,1H),8.02(s,1H),7.51–7.44(m,2H),7.00–6.94(m,2H),6.33(s,1H), 4.05(t,J=7.3Hz,1H),3.81(s,3H),3.72(q,J=6.9Hz,2H),3.65(s,2H),2.79(t,J=6.8Hz,2H),2.44(s,3H). 13 C NMR (125 MHz, Chloroform-d) δ 182.16, 162.80, 161.20, 159.75, 156.15, 153.43, 130.85, 126.12, 124.22, 113.99, 103.77, 103.50, 98.82, 59.45, 59.13, 55.32, 54.58, 44.19. HRMS (EI-TOF) M 371.1391, consistent with the calculated C 20 H 21 NO6

[0197] 371.1368 matches.

[0198] <Example 27>

[0199] Synthesis of 8-[(dibutylamino)methyl]-5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one (Compound 27):

[0200] The steps were the same as in Example 1, except that dibutylamine was used instead of morpholine to obtain a colorless solid with a yield of 31%. The structural formula of compound 27 is as follows:

[0201]

[0202] The H NMR spectrum and high-resolution mass spectrometry (HRMS (EI-TOF)) data are as follows:

[0203] 1 H NMR(500MHz,Chloroform-d)δ9.14(s,1H),8.02(s,1H),7.50–7.44(m,2H),6.99–6.92(m,2H),6.33(s,1H),4.04(s,2H ), 3.82 (s, 3H), 2.58 (t, J = 6.4Hz, 4H), 1.54 (p, J = 6.5Hz, 4H), 1.33 (dtd, J = 14.3, 7.6, 6.7Hz, 4H), 0.94 (t, J = 7.6Hz, 6H). 13 C NMR(125MHz,Chloroform-d)δ182.13,162.93,161.36,159.54,156.07,153.21,130.67,125.93,1 23.98,113.88,104.00,103.17,98.96,55.34,54.94,51.82,29.52,20.79,14.21.HRMS(EI-TOF)M

[0204] 425.2254, compared with the calculated C 25 H 31 NO5 425.2202 matches.

[0205] Nine compounds out of the above 27 compounds (compound 1 numbered YN-1, compound 3 numbered YN-2, compound 6 numbered YN-3, compound 7 numbered YN-4, compound 8 numbered YN-5, compound 10 numbered YN-6, compound 12 numbered YN-7, compound 13 numbered YN-8, and compound 15 numbered YN-9) were selected for the following application experiment research.

[0206] <Test Example 1>

[0207] Cytotoxicity studies

[0208] The growth inhibitory effects of the synthesized biochanin A derivatives (compounds YN-1 to YN-9) on two cell types (HK-2 cells and RAW264.7 cells) were determined using the MTT assay. The results are as follows:

[0209] Table 1 Cytotoxic effects of compounds YN-1 to YN-9

[0210]

[0211] Cytotoxicity experiments were conducted on two cell lines using nine target compounds and biochanin A. The IC 50 The concentrations of the compounds were all greater than 100 μM (as shown in Table 1). The experimental results showed that the obtained compounds had low toxicity to the two cell lines.

[0212] <Test Example 2>

[0213] Study on the inhibition of NO production in RAW264.7 cells stimulated by LPS

[0214] Nitric oxide (NO) is a key inflammatory factor, produced by nitric oxide synthase (NOS) during the conversion of L-arginine to L-citrulline. NO plays a crucial role in inflammation by causing vasodilation, increasing leukocyte adhesion, and increasing vascular permeability. Most immune cells and various non-immune cells (such as endothelial cells, fibroblasts, hepatocytes, and keratinocytes) produce NO. Excessive NO secretion contributes to inflammation and tumorigenesis. This study examined the inhibitory effects of compounds on NO release to provide a preliminary understanding of their anti-inflammatory effects.

[0215] Table 2 Effects of compounds YN-1 to YN-9 on LPS-induced NO secretion in macrophages

[0216]

[0217]

[0218] As can be seen from Table 2, the biochanin derivatives prepared by the present invention (compounds YN-1 to YN-9) showed varying degrees of efficacy in inhibiting LPS-induced NO release in RAW264.7 macrophages, especially compounds YN-4, YN-5, YN-6, and YN-8, which showed a good effect in inhibiting cellular NO production. Among them, compound YN-8 was better than biochanin A in inhibiting cellular NO production, especially high concentrations of YN-8, whose anti-inflammatory activity was comparable to that of dexamethasone. This indicates that the protective mechanism of the biochanin derivatives prepared by the present invention may be related to the inhibition of cisplatin-induced inflammatory response.

[0219] <Test Example 3>

[0220] The cytotoxic effect of compound YN-8 on HK-2 cells and its protective effect against cisplatin-induced HK-2 cell damage (Based on the above experimental data, compound YN-8 has strong inhibitory activity against NO release and low toxicity, so compound YN-8 was selected for further mechanistic studies.)

[0221] Human renal tubular epithelial cells (HK-2) were purchased from the cell bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The effect of compound YN-8, a potent antioxidant obtained in an in vitro antioxidant experiment, on cell viability in human renal tubular epithelial cells (HK-2) was determined by the MTT assay. A blank group, a negative control group, and sample groups (100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM) were set up.

[0222] The protective effect of compound YN-8 on cisplatin-treated HK-2 cells was further tested. A blank group, a negative control group, a cisplatin group (20 μM), and a cisplatin (20 μM) + sample group (100 μM, 90 μM, 80 μM, 70 μM, and 60 μM) were set up. The absorbance was detected at a wavelength of 490 nm using a microplate reader, and the cell survival rate was calculated.

[0223] In the cytotoxicity test of the compounds, YN-8 had no cytotoxicity within 48 hours when the administration concentration was as high as 100μmol / L (μM). In the cell protection test of the compounds, the cell survival rate in the cisplatin (CIS) group was significantly reduced. Compared with the cisplatin group, the survival rate of HK-2 cells in the YN-5, YN-8 and YN-98 intervention groups was improved. At a concentration of 60μmol / L, the survival rate of human renal tubular epithelial cells (HK-2) was significantly improved (P<0.05), indicating that these three compounds can significantly improve the damage of cisplatin to renal cells (such as Figure 1 ), among which YN-8 has the most obvious effect.

[0224] <Test Example 4>

[0225] Effect of compound YN-8 on apoptosis of HK-2 cells damaged by cisplatin (Based on the above experimental data, compound YN-8 has strong inhibitory activity against NO release and low toxicity, so compound YN-8 was selected for further mechanism studies.)

[0226] Annexin V is a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylinositol (PSA). During apoptosis, PSA on the inner membrane is externalized to the membrane surface and is bound by the fluorescent dye FITC-labeled Annexin V. Because late apoptotic or necrotic cell membranes lose their integrity, pyridinium iodide (PI) specifically binds to double-stranded DNA and produces strong fluorescence. Its use in combination with Annexin V can distinguish cells at different stages of apoptosis.

[0227] Figure 2 The results of flow cytometry detection of cell apoptosis are as follows: total apoptosis rate = early apoptosis rate + late apoptosis rate. The results showed that compared with the Normal group ( Figure 2 A) compared with cisplatin group ( Figure 2 The apoptosis rate of CIS+YN-8 group (P<0.05) was significantly increased. Figure 2 The apoptosis rate in the CIS group (C YN-8 at 40 μM and D YN-8 at 60 μM) was significantly decreased (P < 0.05). At a 40 μM YN-8 concentration, the apoptosis rate decreased from 38.97% to 24.3%, and at a 60 μM YN-8 concentration, the apoptosis rate decreased to 17.9%. These results demonstrate that compound YN-8 effectively reduces cisplatin-induced apoptosis in HK-2 cells in vitro.

[0228] <Test Example 5>

[0229] Effects of biochanin A compounds (compounds YN-2, YN-3, YN-5, YN-6, YN-7, YN-8, and YN-9 are used to demonstrate the results) on the anticancer activity of cisplatin

[0230] Cisplatin is a first-line clinical anticancer drug. This application should not inhibit cisplatin's inherent anticancer activity while providing renal protection. Compounds YN-2, YN-3, YN-5, YN-6, YN-7, YN-8, and YN-9 have shown promising effects on enhancing the proliferation of HK-2 cells. Two cancer cell lines were selected and tested using the MTT assay to determine whether these compounds affect the anticancer activity of cisplatin.

[0231] The results are as follows Figure 3 As shown, for SK-Hep-1 liver cancer cells, the proliferation activity of SK-Hep-1 cells after compound intervention was significantly reduced compared with the CIS group, and the results were statistically significant (P<0.05). For HepG2 cells, compared with the CIS group, the intervention of compounds YN-2, YN-5, YN-7 and YN-9 increased the proliferation activity of Hep-G2 cells, and the results were statistically significant (P<0.05). The cell proliferation results of the compound YN-3, YN-6 and YN-8 groups showed no statistical difference compared with the CIS group (P>0.05). Based on the above cell proliferation activity and the results of the compound inhibiting LPS-induced NO release, this application selected the compound YN-8 to focus on studying its protective effect on renal damage.

[0232] <Test Example 6>

[0233] Protective effect on cisplatin-induced acute kidney injury in mice (compound YN-8 was selected for this study)

[0234] Animals were provided by the Animal Experimentation Center of Guangxi Medical University (Use Permit Number: SYXK(Gui)2014.0003). All animal experiments in this study were approved by the Experimental Animal Welfare and Ethics Committee of Guangxi Medical University and complied with relevant animal experimentation regulations. Animals were acclimated for 14 days in the laboratory using a standard diet and free access to food and water. The room temperature was maintained at 22–24°C. To establish a cisplatin-induced AKI animal model, 24 C57 / BL6 male mice (18–22 g) from the same batch were randomly divided into a cisplatin group (CIS), a drug intervention group (YN-8+CIS), and a blank control group. The following experimental procedures were used. Animal grouping: 28 C57 / BL6 mice (18–22 g) were housed at a room temperature of 22–24°C and a relative humidity of 40%–70% with free access to food and water. After acclimation for 2 weeks, they were randomly divided according to body weight into a cisplatin group, a drug intervention group, or a blank control group, with 8 mice in each group. The dosing schedule was as follows: Cisplatin group: 50 mg / kg of normal saline was injected intraperitoneally for 3 consecutive days, and 25 mg / kg of cisplatin solution was injected on the 4th day. Drug intervention group: 50 mg / kg of the sample drug was injected intraperitoneally for 3 consecutive days, and 25 mg / kg of cisplatin solution was injected on the 4th day, followed by 50 mg / kg after a 1-h interval. Blank control group: 50 mg / kg of normal saline was injected intraperitoneally for 4 consecutive days. Specimen acquisition: 72 hours after administration on the 4th day, mice in each group were sampled and sacrificed by cervical dislocation. The intact kidney on one side was dissected and removed, washed twice with normal saline, and fixed with 4% paraformaldehyde.

[0235] Renal pathological staining is an essential examination method for diagnosing kidney diseases. HE staining results show (such as Figure 4 In the control group, the glomeruli were intact and well-defined, with no significant tubular dilatation, degeneration, or necrosis. Compared with the control group, the CIS group showed capillary filling with red blood cells, dilated renal tubular epithelial cells, and vacuolar degeneration. Renal damage was ameliorated in the drug intervention group (P < 0.05). These results demonstrate that compound YN-8 has the ability to ameliorate cisplatin-induced acute kidney injury in vivo.

[0236] In order to verify the protective effect of compound YN-8 on the structure and function of acute kidney injury caused by cisplatin, plasma samples were collected from the heart of mice before they were sacrificed for the detection of blood creatinine (Cr) and blood urea nitrogen (BUN), and the kidneys were immersed in tissue fixative for HE staining sections. Cr and BUN are one of the most important methods for clinical detection of renal function. Figure 5The results showed that compared with the Normal group, the Cr and BUN in the CIS group were significantly increased, and the difference was statistically significant (P<0.05); compared with the CIS group, the Cr and BUN in the CIS+YN-8 (60 mg / kg) drug intervention group were significantly decreased, and the results were statistically significant (P<0.01).

[0237] Observation of the survival status of mice after modeling revealed that the cisplatin-treated group exhibited significantly slower responses to external stimuli, with huddled bodies, matted fur, slowed activity, and decreased water and food intake. No significant changes were observed in the drug intervention and control groups. HE staining further revealed that kidneys in the control group displayed normal glomerular and tubular structures, while kidneys in the cisplatin-treated group showed severe degeneration of the tubular epithelial lining, accompanied by the presence of eosinophilic apoptotic bodies. Compared to cisplatin-treated mice, the renal tubular structure of mice in the YN-8 group was essentially normal, with only a small number of degenerated cells, indicating that compound intervention reduced tubular epithelial cell death.

[0238] In the description of this specification, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without any contradiction.

[0239] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be readily apparent to those skilled in the art.

[0240] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A biochanin derivative, characterized in that as follows: 。 2. The method for preparing the biochanin derivative according to claim 1, wherein: It is obtained by using biochanin A as the mother core structure and replacing it with nitrogen-containing compounds as active groups; Its synthetic route is as follows: R is 4-hydroxymethylpiperidine.

3. Use of the biochanin derivative according to claim 1 in the preparation of a drug for alleviating drug-induced renal injury.

4. The use according to claim 3, characterized in that Drugs that cause drug-induced renal injury include at least anti-tumor drugs.

5. The use according to claim 4, characterized in that The anti-tumor drug at least includes cisplatin.

6. The use according to claim 3, characterized in that The drug also includes a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that Pharmaceutically acceptable carriers include diluents, solubilizers, cosolvents, disintegrants, dispersants, lubricants, flavoring agents, antioxidants, binders, absorbents, wetting agents, buffers, and cross-linking agents.

8. The use according to claim 7, characterized in that The drug is prepared into a pharmaceutically acceptable dosage form.

9. The use according to claim 8, characterized in that The dosage forms include pills, tablets, powders, capsules, granules, powders, pellets, drops, sprays, injections, suspensions, ointments, gels, and suppositories.

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