Improved genistein compound as well as preparation method and application thereof

Abstract

The invention discloses an improved genistein compound and a preparation method and application thereof.The structure of the improved genistein compound is shown as a formula (I) or a formula (II), and the improved genistein compound at least has the beneficial effects that the improved genistein compound has a remarkable inhibition effect on cervical cancer HeLa cells at an obviously low concentration; the compound can induce generation of ROS (reactive oxygen species) in cervical cancer HeLa cells, can reduce mitochondrial membrane potential of the cervical cancer HeLa cells, can induce apoptosis of the cervical cancer HeLa cells by activating a mitochondrial pathway, and has good fat solubility and bioavailability.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis, specifically to an improved genistein compound, its preparation method, and its applications. Background Technology

[0002] All neoplastic lesions occurring in the cervix include both benign and malignant tumors. Benign tumors include cervical polyps and Nabothian cysts, while malignant tumors include cervical cancer and other rare types (such as cervical sarcoma). Cervical cancer is a malignant tumor that occurs in the cervix, originating from abnormal proliferation and lesions of cervical epithelial cells. The main cause is persistent infection with high-risk human papillomavirus (HPV).

[0003] Genistein (also known as genistein) is a naturally occurring isoflavone compound, mainly found in legumes (such as soybeans) and red clover. Its name is 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, and its structural formula is [insert structural formula here].

[0004] There have been reports on the inhibitory effect of genistein on cervical cancer cells, but currently, domestic and international literature reports on the effect of genistein on the IC50 of cervical cancer HeLa cells. 50 The values ​​were all around 100 μM. Furthermore, the lipid solubility and bioavailability of genistein still need improvement. Therefore, developing an improved genistein compound would be of great significance for the prevention or treatment of cervical cancer. Summary of the Invention

[0005] Based on the needs of the prior art, the present invention provides an improved genistein compound, its preparation method, and its application.

[0006] A first aspect of the present invention provides a benzyl-substituted α-carboline compound or a pharmaceutical salt thereof, the structure of which is shown in formula (I) or formula (II):

[0007]

[0008] in:

[0009] Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, wherein each hydrogen atom on the aromatic ring or aromatic heterocycle is independently converted to carbon atom. 1-20 Alkyl, C 1-20 At least two groups from the haloalkyl, halogen, and nitro groups are substituted; or

[0010] Ar2 represents a substituted aromatic ring or aromatic heterocycle, which is a monocyclic ring comprising 4-10 carbon atoms, wherein each hydrogen atom on the aromatic ring or aromatic heterocycle is independently converted to carbon atom. 1-20 Alkyl, C 1-20 Halogenated alkyl groups, halogens, C 1-20 Alkyl carbonyl or carbonyl, C 1-20 At least two groups in the alkylamide group and nitro group are substituted;

[0011] The aromatic heterocycle contains at least one of N, P, O or S.

[0012] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms. In some more preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms.

[0013] In some preferred embodiments, the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms. In some more preferred embodiments, the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms.

[0014] In some preferred embodiments, the hydrogens on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted by C. 1-12 Alkyl, C 1-12 At least two groups from the haloalkyl, halogen, and nitro groups are substituted.

[0015] In some preferred embodiments, the hydrogens on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted by C. 1-6 Alkyl, C 1-6 At least two groups from the haloalkyl, halogen, and nitro groups are substituted.

[0016] In some preferred embodiments, the hydrogens on the aromatic ring or aromatic heterocycle of Ar2 are each independently converted by C. 1-12 Alkyl, C 1-12 Halogenated alkyl groups, halogens, C 1-12 Alkyl carbonyl or carbonyl, C 1-12 At least two groups, including alkylamide and nitro, are substituted.

[0017] In some preferred embodiments, the hydrogens on the aromatic ring or aromatic heterocycle of Ar2 are each independently converted by C. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, halogens, C 1-6 Alkyl carbonyl or carbonyl, C 1-6At least two groups in the alkylamide group and nitro group are substituted.

[0018] In some preferred embodiments, the aromatic heterocycle contains at least one of N, P, or O. In some more preferred embodiments, the aromatic heterocycle contains at least one N and / or at least one P. In some even more preferred embodiments, the aromatic heterocycle contains at least one N.

[0019] In some preferred embodiments, the halogen or halogen is selected from at least one of fluorine, chlorine, bromine, and iodine. In some more preferred embodiments, the halogen or halogen is fluorine or chlorine.

[0020] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 At least two groups from the haloalkyl, halogen, and nitro groups are substituted.

[0021] In some preferred embodiments, the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar2 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 Halogenated alkyl groups, halogens, C 1-12 Alkyl carbonyl or carbonyl, C 1-12 At least two groups, including alkylamide and nitro, are substituted.

[0022] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 At least two groups selected from alkyl halogroups, halogens, and nitro groups are substituted; and / or the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogens on the aromatic ring or aromatic heterocycle of the Ar2 are each independently replaced by C. 1-12 Alkyl, C 1-12 Halogenated alkyl groups, halogens, C 1-12 Alkyl carbonyl or carbonyl, C 1-12 At least two groups, including alkylamide and nitro, are substituted; the aromatic heterocycle contains at least one N and / or at least one P.

[0023] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 At least two groups selected from alkyl halogroups, halogens, and nitro groups are substituted; and the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar2 are each independently replaced by C. 1-12 Alkyl, C 1-12 Halogenated alkyl groups, halogens, C 1-12 Alkyl carbonyl or carbonyl, C 1-12 At least two groups, including alkylamide and nitro, are substituted; the aromatic heterocycle contains at least one N or at least one P.

[0024] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted to carbon atoms. 1-6 Alkyl, C 1-6 At least two groups from the haloalkyl, halogen, and nitro groups are substituted.

[0025] In some preferred embodiments, the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar2 are each independently converted to carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, halogens, C 1-6 Alkyl carbonyl or carbonyl, C 1-6 At least two groups in the alkylamide group and nitro group are substituted.

[0026] In some preferred embodiments, Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of Ar1 are each independently converted to carbon atoms. 1-6 Alkyl, C 1-6 At least two groups selected from alkyl halogroups, halogens, and nitro groups are substituted; and / or the Ar2 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogens on the aromatic ring or aromatic heterocycle of the Ar2 are each independently replaced by C. 1-6 Alkyl, C 1-6 The aromatic heterocycle contains at least two of the following groups: alkyl halogroup, halogen, and nitro group; and at least one nitrogen atom is present in the aromatic heterocycle.

[0027] In some further preferred embodiments, the aromatic ring of Ar1 is a monocyclic ring having 6 carbon atoms; and the hydrogen atoms on the aromatic ring of Ar1 are each independently substituted by at least two groups selected from methyl, trifluoromethyl, chlorine, and nitro; and the aromatic heterocyclic ring of Ar2 is a 6-membered monocyclic ring having 1 nitrogen atom; and the hydrogen atoms on the aromatic heterocyclic ring of Ar2 are each independently substituted by methyl, trifluoromethyl, halogen, C 1-6 Alkyl carbonyl or carbonyl, C 1-6 At least two groups, including alkylamide and nitro, are substituted.

[0028] In some further preferred embodiments, Ar1 is Any of the above; and / or the Ar2 is

[0029] Any one of them.

[0030] In some further preferred embodiments, the compound is selected from:

[0031]

[0032]

[0033]

[0034] In some preferred embodiments, the improved genistein compounds are

[0035]

[0036] and / or

[0038] A fourth aspect of the present invention provides an improved method for preparing genistein compounds, comprising the following steps: adding 20-30 mL of dehydrated tetrahydrofuran as a reaction solvent to a reaction vessel, adding 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide, then adding 1-1.2 mmol of a compound containing Ar2 substituted with another halogen, reacting at room temperature (20-30°C) and pH 9-11 with magnetic stirring at 300-500 rpm for approximately 3-5 hours, monitoring the reaction by TLC, and after the reaction is complete or finished, pouring the reaction solution into 30-50 mL of saturated saline solution and adding ethyl acetate for extraction. The organic phase is collected and concentrated, and purified by silica gel column chromatography with a DCM:MeOH ratio of 100-120:1 to obtain the target compound Ya.

[0039] Wherein, Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, and the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to carbon atom C. 1-20 At least two of the groups in the alkyl halogroup, halogen, and nitro group are substituted, and the additional halogen is substituted by an additional hydrogen on the aromatic ring or aromatic heterocycle of Ar2.

[0040] A fifth aspect of the invention provides a pharmaceutical composition comprising an effective amount of the modified genistein compound described above or a pharmaceutical salt thereof, and optionally a pharmaceutical excipient or pharmaceutical carrier.

[0041] The sixth aspect of the invention provides the use of the improved genistein compounds described above or their pharmaceutical salts, or the pharmaceutical compositions described above, in the preparation of a medicament for the prevention or treatment of cervical fibroids or cervical cancer.

[0042] In this invention, cervical tumors include cervical polyps (cervical mucosal hyperplasia) and cervical leiomyomas.

[0043] The beneficial effects of the present invention include at least the following:

[0044] (1) Currently, the effects of genistein on the IC50 of HeLa cervical cancer cells reported in domestic and international literature are not significant. 50 The concentration was around 100 μM, while the improved genistein compounds of this invention showed a significant inhibitory effect on cervical cancer HeLa cells at a significantly lower concentration. In particular, compounds Z1 and Y1 showed a significant inhibitory effect on the IC50 of cervical cancer HeLa cells. 50 The values ​​were 3.17±0.49μM and 4.7±0.15μM, respectively, which were significantly better than those of genistein.

[0045] (2) The improved genistein compounds of the present invention (especially compounds Z1 and Y1) can induce the generation of ROS in cervical cancer HeLa cells and reduce the mitochondrial membrane potential of cervical cancer HeLa cells. They can induce apoptosis of cervical cancer HeLa cells by activating the mitochondrial pathway.

[0046] (3) In the process of preparing the compounds of the present invention, the inventors of the present invention discovered that: compared with the reaction substrate genistein, all the derivatives have better solubility in ethyl acetate and dichloromethane than the substrate (genistein is slightly soluble in ethyl acetate and dichloromethane), and all compounds (especially Z1 and Y1) are well soluble in ethyl acetate and dichloromethane. In cell experiments, it was found that the reaction substrate genistein had a poor inhibitory effect on HeLa cells, and the drug precipitation phenomenon occurred after the drug concentration increased, indicating poor lipid solubility; in particular, compounds Z1 and Y1 could cause HeLa cell death even at very low concentrations.

[0047] The improved genistein compounds of this invention (especially compounds Z1 and Y1) exhibit significantly superior lipophilicity and bioavailability compared to genistein. The compounds of this invention utilize the high lipophilicity, good metabolic stability, and high electronegativity of substituted groups (especially halogenated alkyl groups such as trifluoromethyl) introduced into the aromatic ring or aromatic heterocycle of the molecule. This improves the properties of the drug, enhances its absorption, distribution, and metabolism in vivo, thereby improving the lipophilicity and bioavailability of the improved genistein compounds of this invention, and thus enhancing their pharmacological activity.

[0048] The features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0049] Figure 1 The following spectra are shown: proton NMR, carbon NMR, fluorine NMR, and mass spectra of compound Z1. Figure 1 (a) is the hydrogen spectrum. Figure 1 (b) is the carbon spectrum. Figure 1 (c) is the fluorine spectrum. Figure 1 (d) is mass spectrometry.

[0050] Figure 2 The following spectra are displayed for the proton, carbon, fluorine, and mass spectra of compound Y1. Figure 2 (a) is the hydrogen spectrum. Figure 2 (b) is the carbon spectrum. Figure 2 (c) is the fluorine spectrum. Figure 2 (d) is mass spectrometry.

[0051] Figure 3 Compound Z1 showed that it inhibited the growth of cervical cancer cells in vitro. Figure 3 Figure (A) shows the structure of compound Z1; Figure 3 Figures (B), (C), (D), and (E) show the MTT assay results for HeLa, SiHa, H8, and TC-1 cell viability, respectively. All data were analyzed by ANOVA. Compared with the control group, *p<0.05, **p<0.01, and ***p<0.001.

[0052] Figure 4 Compound Y1 was shown to inhibit the growth of cervical cancer cells in vitro. Figure 4 Figure (A) shows the structure of compound Y1; Figure 4 Figures (B), (C), (D), and (E) show the MTT assay results for HeLa, SiHa, H8, and TC-1 cell viability, respectively. All data were analyzed using ANOVA. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0053] Figure 5 The main feature shown is the effect of the compounds of this invention on the IC50 of cervical cancer HeLa cells. 50 value.

[0054] Figure 6 The image shows cell viability graphs obtained by detecting the compounds and control substances of this invention using the MTT assay.

[0055] Figure 7 Compounds Z1 and Y1 were shown to induce apoptosis in HeLa cells in a dose-dependent manner. Figure 7 Figure (A) shows the effect of flow cytometry on HeLa cell apoptosis detection; Figure 7 Figure (B) shows the statistical values ​​of HeLa cell apoptosis and necrosis. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0056] Figure 8 Compounds Z1 and Y1 showed that they reduced the mitochondrial membrane potential (ΔΨm) in HeLa cells in a dose-dependent manner. Figure 8 Figure (A) shows the changes in red (green) fluorescence intensity of JC-1 observed under an inverted fluorescence microscope; Figure 8 Figure (B) shows the changes in red (green) fluorescence intensity of JC-1 detected by flow cytometry. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0057] Figure 9 Compounds Z1 and Y1 were shown to induce intracellular ROS production in HeLa cells in a dose-dependent manner. Figure 9 Figure (A) shows the ROS fluorescence intensity observed under an inverted fluorescence microscope; Figure 9 Figure (B) shows the intracellular ROS levels detected by flow cytometry. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the high-dose treatment groups of compounds Z1 and Y1 (20μM), #p<0.05, ##p<0.01, ###p<0.001.

[0058] Figures 10(A) and 10(B) show the flow cytometry analysis of different cell cycle stages after HeLa cells were treated with different concentrations of compounds Z1 and Y1 for 24 h. As shown in Figure 10(A), the vertical axis represents cell number, and the horizontal axis represents the fluorescence intensity of PI. Compared with the control group, with increasing concentrations of compounds Z1 and Y1, the proportion of cells in the G0 / G1 phase significantly decreased, while the proportion of cells in the G2 / M phase significantly increased (p<0.001). As shown in Figure 10(B), the cell proportions of HeLa cells at different stages show that, compared with the control group, compounds Z1 and Y1 induced HeLa cells to arrest in the G2 / M phase in a dose-dependent manner. *p<0.05, **p<0.01, ***p<0.001.

[0059] Figure 11 Compounds Z1 and Y1 were shown to inhibit the growth of cervical cancer cells in vivo. Figure 11 Figure (A) shows 5×10 5 TC-1 cells were injected into the right back of mice, and on day 7, the mice were randomly divided into 9 groups. Figure 11 Figure (B) shows the mouse body weight curve. Figure 11 Figure (C) shows the tumor volume in the mouse.

[0060] Figure 11 Figure (D) shows the size of a mouse tumor. Figure 11 Figure (E) shows the tumor weight of mice. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0061] Figures 12(A) to 12(I) This study demonstrates the effects of compounds Z1 and Y1 on organs in TC-1 tumor-bearing mice. Figures 12(A) to 12(C) Showing changes in organs of tumor-bearing mice; Figures 12(D) to 12(I) The changes in organ indices in tumor-bearing mice were shown. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0062] Figure 13 The effects of compounds Z1 and Y1 on the morphology of liver, kidney, and tumor tissue in tumor-bearing mice were observed using H&E staining.

[0063] Figure 14 This study demonstrates the effects of compounds Z1 and Y1 treatment on liver and kidney function in tumor-bearing mice. Figure 14 Figure (A) shows alanine aminotransferase (ALT); Figure 14 Figure (B) shows aspartate aminotransferase (AST); Figure 14 Figure (C) in the diagram represents creatinine (Cr); Figure 14Figure (D) shows blood urea nitrogen (BUN). Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0064] Figure 15 shows the effects of compounds Z1 and Y1 treatment on the number of T, B, and NK cells in the spleen cells of tumor-bearing mice. Figures (A) and (B) in Figure 15 show the cell population distribution in the spleen of tumor-bearing mice; Figures (C) to (I) in Figure 15 show the number of T, B, NK, and CD4 cells in the spleen. + T cells, CD8 + T cells and CD4 + / CD8 + The proportion of T cells. Compared with the control group, *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0065] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0066] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the description of this application, unless otherwise stated, terms such as "multiple / a variety" mean two / a kind or more.

[0067]

Terminology Explanation

[0068] Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

[0069] As used in this article, when referring to a specific listed value, the value or the term “about” means that the value can vary from the listed value by a range of ±5%.

[0070] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed.

[0071] As used herein, definitions of standard chemical terms (such as groups) can be found in the references in this field.

[0072] Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, IR and UV / VIS spectroscopy, and pharmacological methods, are employed. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reactions and purifications can be carried out using the manufacturer's instructions for use of kits, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally carried out according to conventional methods well known in the art, based on descriptions in several summary and more specific documents cited and discussed in this specification. In this specification, groups and their substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0073] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0074] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0075] Except as otherwise specified, when used in the specification and claims of this application, the following terms shall have the following meanings.

[0076] In this invention, "the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently replaced by..." can be understood as a portion of the hydrogen atoms on the aromatic ring or aromatic heterocycle being replaced.

[0077] In this invention, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group. In some preferred embodiments, it contains 1 to 12 carbon atoms (C... 1-12 The alkyl group is monovalent or polyvalent (e.g., monovalent or divalent). In some more preferred embodiments, the alkyl group contains 1 to 6 carbon atoms (C60-C62). 1- The alkyl group (C6 alkyl) has, for example, 1, 2, 3, 4, 5, or 6 carbon atoms. In other embodiments, the alkyl group contains 1 to 3 carbon atoms, for example, 1, 2, or 3 carbon atoms. Some non-limiting examples of alkyl groups include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, and 2,2-dimethylpropyl. A particularly preferred but non-limiting example of an alkyl group is methyl.

[0078] In this invention, the term "halogen" or "halogenated" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Preferably, "halogen" or "halogenated" refers to fluorine (F), chlorine (Cl), or bromine (Br).

[0079] In this invention, the term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom of the alkyl group has been replaced by a halogen atom. Preferably, "haloalkyl" refers to an alkyl group in which one, two, or three hydrogen atoms of the alkyl group have been replaced by a halogen atom, preferably by fluorine, chlorine, or bromine. A particularly preferred, but non-limiting, example of a haloalkyl group is trifluoromethyl (CF3).

[0080] In this invention, "amide group" refers to "-CONH2".

[0081] In this invention, the term "alkylamide" refers to a group containing a carbonyl moiety, wherein the carbonyl carbon atom is bonded to one or more alkyl groups (such as tert-butyl); the carbonyl carbon atom is also bonded to a nitrogen group, which is bonded to one or more aromatic rings or aromatic heterocycles. Examples include, but are not limited to, methylamide, ethylamide, n-propylamide, isopropylamide, n-butylamide, tert-butylamide, isobutylamide, and sec-butylamide.

[0082] In this invention, the term "carbonyl" refers to an organic functional group (-C=O) formed by carbon and oxygen atoms linked by a double bond.

[0083] In this invention, the term "alkyl carbonyl" refers to a carbonyl group bonded to an "alkyl" group. Examples include, but are not limited to, methyl carbonyl, ethyl carbonyl, n-propyl carbonyl, isopropyl carbonyl, n-butyl carbonyl, tert-butyl carbonyl, isobutyl carbonyl, sec-butyl carbonyl, n-pentyl carbonyl, isopentyl carbonyl, and n-hexyl carbonyl. Preferred embodiments of "alkyl carbonyl" are methyl carbonyl, ethyl carbonyl, and n-propyl carbonyl.

[0084] In this invention, the terms "compound of the present invention" or "active ingredient of the present invention" are used interchangeably to refer to stereoisomers, enantiomers, or pharmaceutical salts of the general formula compound. The term also includes racemic mixtures, optical isomers, isotopic compounds (such as deuterated compounds), or leads.

[0085] "Stereoisomers" are compounds composed of identical atoms bonded by the same bonds, but with different three-dimensional structures. This invention will cover various stereoisomers and mixtures thereof.

[0086] When the compounds of the present invention contain alkene double bonds, unless otherwise stated, the compounds of the present invention are intended to contain both E- and Z- geometric isomers.

[0087] "Tautomer" refers to an isomer formed when a proton is transferred from one atom of a molecule to another atom of the same molecule. All tautomer forms of the compounds of this invention are also included within the scope of this invention.

[0088] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may contain one or more chiral carbon atoms, and thus may produce enantiomers, diastereomers, and other stereoisomers. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. The present invention aims to include all possible isomers, as well as their racemic and optically pure forms. The preparation of the compounds of the present invention may select racemic, diastereomer, or enantiomer as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0089] Conventional techniques for preparing / separating individual isomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography.

[0090] This invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. However, it is common practice for one or more atoms to be substituted with atoms of different atomic weights or mass numbers. Examples of isotopes of compounds that can be included in this invention include hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine isotopes. Compounds of this invention, or enantiomers, diastereomers, isomers, or pharmaceutical salts or solvates, containing isotopic or other isotopic atoms of the aforementioned compounds, are all within the scope of this invention. Certain isotopically labeled compounds of this invention, such as radioactive isotopes, are also included and are useful in tissue distribution experiments of drugs and substrates. For example, tritium, i.e. 3 H and carbon-14, i.e. 14 C, their preparation and detection are relatively easy. They are the preferred isotopes. In addition, heavier isotopes such as deuterium are used for substitution. 2 H, due to its excellent metabolic stability, offers advantages in certain therapies, such as increasing half-life or reducing dosage in vivo, and therefore may be preferred in some cases. Isotopically labeled compounds can be prepared using general methods, by replacing the non-isotopic reagent with an readily available isotopically labeled reagent, according to the schemes disclosed in the examples. In this application, the term "pharmaceutical salt" includes pharmaceutically acceptable acid addition salts.

[0091] "Pharmaceutical acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; preferred inorganic acids include hydrochlorides and sulfates. Organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, and gluconates. Preferred organic acids include formates and acetates. These salts can be prepared using methods known in the art.

[0092] As described herein, the compounds of this invention can be expanded with any number of substituents or functional groups. Generally, whether the term "substitution" appears before or after the term "optional," the general formula for substituents in the formulations of this invention refers to replacing a hydrogen radical with a substituent of a specified structure. When multiple positions in a particular structure are replaced by multiple specific substituents, each position of the substituent can be the same or different. The term "substitution" as used herein includes all permissible substitutions in organic compounds. In a broad sense, permissible substituents include acyclic, cyclic, branched-unbranched, carbocyclic, and heterocyclic, aromatic and non-aromatic organic compounds. In this invention, heteroatomic nitrogen may be supplemented with hydrogen substituents or any permissible organic compound described above to complete its valence state. Furthermore, this invention is not intended to limit permissible substituted organic compounds in any way. This invention considers the combination of substituents and variable groups to be beneficial in the treatment of diseases in the form of stable compounds. The term "stable" here refers to a compound that is stable enough to maintain the integrity of its structure when tested over a sufficiently long period of time, preferably remaining effective over a sufficiently long period of time, and is used here for the purposes described above.

[0093] The compounds involved in this application and their pharmaceutically acceptable salt metabolites, as well as prodrugs that can be converted in vivo into structures of the compounds involved in this application and their pharmaceutically acceptable salts, are also included in the claims of this application.

[0094] Pharmaceutical Compositions and Administration

[0095] The pharmaceutical compositions of this invention are intended for the prevention and / or treatment of cervical fibroids or cervical cancer. In this invention, "pharmaceutical composition" refers to a formulation of the compound of this invention with a medium generally accepted in the art for delivering a bioactive compound to a mammal (e.g., a human). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity. As used herein, the term "pharmaceutical" means a substance (such as a carrier or diluent) that does not affect the bioactivity or properties of the compound of this invention and is relatively non-toxic, i.e., that the substance can be administered to an individual without causing an adverse biological response or interacting adversely with any component contained in the composition.

[0096] In this invention, "pharmaceutical excipients" include, but are not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is permitted by the relevant government regulatory authority to be acceptable for human or livestock use.

[0097] The term “prevention” as used in this article includes reducing the likelihood of a patient developing or worsening a disease or condition.

[0098] The term "treatment" and other similar synonyms used in this article include the following meanings:

[0099] (i) To prevent the occurrence of disease or condition in mammals, especially when such mammals are susceptible to the disease or condition but have not yet been diagnosed with it;

[0100] (ii) To suppress a disease or symptom, that is, to curb its development;

[0101] (iii) To alleviate a disease or symptom, that is, to cause the condition of the disease or symptom to subside; or

[0102] (iv) To alleviate the symptoms caused by the disease or condition.

[0103] As used herein, the terms "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" refer to an amount of at least one drug or compound that, when taken, is sufficient to alleviate, to some extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or relief of signs, symptoms, or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition containing the compounds disclosed herein that is clinically necessary to provide significant symptom relief. Effective amounts suitable for any individual case can be determined using techniques such as dose escalation testing.

[0104] As used herein, the terms “administration,” “application,” “dosage,” etc., refer to methods that deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral administration, duodenal administration, parenteral administration (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), local administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used with the compounds and methods described herein. In a preferred embodiment, the compounds and compositions of the present invention are administered orally, and formulations of the compounds disclosed herein suitable for oral administration may be presented as discrete units, such as tablets, capsules, or pouches, each containing a predetermined amount of the active ingredient. In other preferred embodiments, the compounds and compositions of the present invention are injections and powders.

[0105] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0106] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0107] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures.

[0108] In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures of these substances.

[0109] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0110] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0111] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0112] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers.

[0113] The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary. As used herein, the terms “drug combination,” “drug co-administration,” “combination therapy,” “administration of other treatments,” and “administration of other therapeutic agents” refer to pharmaceutical treatments obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term “fixed combination” refers to the simultaneous administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity or a single dosage form. The term “non-fixed combination” refers to the simultaneous, combined, or sequential administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity at variable intervals.

[0114] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the subject requiring treatment (including mammals, such as humans). Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the subject requiring treatment, which are all within the scope of the skills of a skilled physician.

[0115] The present invention also provides a method for preparing a pharmaceutical composition, comprising the steps of: mixing a pharmaceutical carrier with a compound of formula (I) or formula (II) of the present invention or its crystal form, a pharmaceutical salt, a hydrate or a solvate, thereby forming a pharmaceutical composition.

[0116] Synthetic methods of compounds of formula (I)

[0117] 20-30 mL of dehydrated dimethyl sulfoxide (DMSO) was added to the reaction vessel as the reaction solvent, followed by 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide (t-BuOK). Then, 1-1.2 mmol of a compound containing an additional halogenated Ar1 ring (wherein the additional halogenated Ar1 ring is an additional hydrogen atom on the aromatic ring or aromatic heterocycle) was added. The reaction was carried out at room temperature (20-30°C), pH 9-11, and magnetically stirred at 300-500 rpm for approximately 2-5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 30-50 mL of saturated saline solution, and ethyl acetate was added for extraction. The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4-6:1) to obtain the target compound.

[0118] In some preferred embodiments, 20-30 mL of dehydrated dimethyl sulfoxide (DMSO) is added to the reaction vessel as a reaction solvent, followed by the sequential addition of 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide (t-BuOK). Then, 1-1.2 mmol of a compound containing an additional halogenated Ar1 ring (wherein the additional halogenated Ar1 ring is a hydrogen atom on the aromatic ring or aromatic heterocycle of Ar1 ring, and the compound containing the additional halogenated Ar1 ring is...) The reaction was carried out at room temperature (20-30℃) and pH 9-11 with magnetic stirring at 300-500 rpm for approximately 2-5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 30-50 mL of saturated saline solution, and ethyl acetate was added for extraction. The organic phase was collected, concentrated, and mixed. After separation and purification by silica gel column chromatography (PE:EtOAc = 4-6:1), the target compounds Z1, Z2, Z3, Z4, or Z5 were obtained.

[0119] Synthetic method of compound (II)

[0120] (a) Ya compounds (wherein Ar2 in Ya compounds is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, wherein the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to carbon atoms by C2). 1-20 The first synthetic method involving substitution of at least two groups (among alkyl, halogen, and nitro groups) includes:

[0121] Add 20-30 mL of dehydrated DMSO as the reaction solvent to the reaction vessel, then add 1 mmol of genistein and 1.2-1.6 mmol of potassium tert-butoxide (t-BuOK), followed by 1.2-1.6 mmol of a compound containing Ar2 substituted with another halogen (where Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle being a monocyclic ring containing 4-10 carbon atoms, and the hydrogens on the aromatic ring or aromatic heterocycle being independently converted by C2). 1-20 The target compound Ya is obtained by substituting at least two groups from the alkyl, halogen, and nitro groups, wherein the additional halogen is substituted with another hydrogen atom on the aromatic ring or heterocyclic ring of Ar2. The reaction is carried out at room temperature (20-30℃), pH 9-11, with magnetic stirring at 300-500 rpm for approximately 3-5 hours. The reaction is monitored by TLC. After the reaction is complete, the reaction solution is poured into 30-50 mL of saturated saline solution, and ethyl acetate is added for extraction. The organic phase is collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4-6:1) to obtain the target compound Ya.

[0122] (ii) The second synthetic method for compounds Ya includes:

[0123] Add 20-30 mL of dehydrated tetrahydrofuran as the reaction solvent to the reaction vessel, then add 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide (t-BuOK), followed by 1-1.2 mmol of a compound containing Ar2 with additional halogen substitution (Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle being a monocyclic ring containing 4-10 carbon atoms, the hydrogens on the aromatic ring or aromatic heterocycle being independently converted by C2). 1-20 The target compound Ya is obtained by substituting at least two groups from the alkyl, halogen, and nitro groups, wherein the additional halogen is substituted with another hydrogen atom on the aromatic ring or aromatic heterocycle of Ar2. The reaction is carried out at room temperature (20-30℃), pH 9-11, with magnetic stirring at 300-500 rpm for approximately 3-5 hours. The reaction is monitored by TLC. After the reaction is complete, the reaction solution is poured into 30-50 mL of saturated saline solution, and ethyl acetate is added for extraction. The organic phase is collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100-120:1) to obtain the target compound Ya.

[0124] In some preferred embodiments, 20-30 mL of dehydrofuran is added to the reaction vessel as a reaction solvent, followed by 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide (t-BuOK), and then 1-1.2 mmol of a compound containing an additional halogenated Ar2 group (the compound containing the additional halogenated Ar2 group is...). Under room temperature (20-30℃) and pH 9-11 conditions, the reaction was carried out with magnetic stirring at 300-500 rpm for approximately 3-5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 30-50 mL of saturated saline solution, and ethyl acetate was added for extraction. The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100-120:1) to obtain the target compounds Ya1, Ya2, Ya3, Ya4, or Ya5.

[0125] Regarding Part (II) above:

[0126] 1. Before the improvement to the second synthetic method, the Ya compounds in part (II) (such as Ya1-Ya5) were all byproducts of the preparation of compounds of formula (I) (such as Z1-Z5). Therefore, to obtain the Ya compounds in part (II), under the condition of 1 equivalent of substrate genistein, the catalyst (potassium tert-butoxide) and the compound containing Ar2 with other halogen substitutions are 1.2-1.6 equivalents. After the excess of the compound containing Ar2 with other halogen substitutions reacts with the 7-OH of genistein to saturation, it will react with the 4-OH, so as to obtain a small amount of compound (II). After changing the solvent, i.e., changing DMSO to tetrahydrofuran, it was found that the corresponding compounds of formula (II) (such as Ya1-Ya5) became the main products, while the compounds of formula (I) (such as Z1-Z5) became byproducts. Therefore, the inventors changed the amount of potassium tert-butoxide and the compounds containing Ar2 substituted with other halogens to 1-1.2 equivalents, which can obtain the corresponding compounds of formula (II) in large quantities. In other words, the second synthetic method greatly improved the yield of compounds of formula (II) (such as Ya1-Ya5).

[0127] 2. Since these compounds are isomers, initial purification was achieved using silica gel column chromatography with a PE:EtOAc ratio of 4-6:1. However, in practice, it was found that incompletely reacted substrates were also eluted with this eluent ratio. Therefore, two or three silica gel column chromatography cycles were required to obtain relatively pure compounds. Furthermore, the PE:EtOAc 4-6:1 system could not separate isomers. Therefore, the inventors changed the silica gel column eluent to a DCM:MeOH ratio of 100-120:1, which easily separated the reaction substrates and relatively pure isomers. Subsequent recrystallization could then yield compounds with even higher purity. Therefore, changing the silica gel column chromatography eluent system is the reason for the improved compound purity.

[0128] (iii) Yb compounds (wherein the Ar2 in the Yb compound is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, and the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to C2 by C2). 1-20Alkylamide group substitution and C 1-20 Alkyl, C 1-20 Synthetic methods involving substitution of at least one group among haloalkyl, halogen, and nitro groups include:

[0129] (1) Synthesis of amide intermediate Pb: 20-30 mL of anhydrous N,N-dimethylformamide (DMF) was added to a reaction vessel as a solvent. 1 mmol of a compound containing an additional amino-substituted Ar2 (wherein Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, and the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to carbon atoms by C2) was added. 1-20 Alkylamide group substitution and C 1-20 Alkyl, C 1-20 At least one group selected from alkyl, halogen, and nitro groups is substituted, and the additional amino group is substituted with an aromatic ring of Ar2 or an additional hydrogen atom on an aromatic heterocycle. The mixture is dissolved in anhydrous DMF. Subsequently, 1-1.2 mmol of the acyl chloride compound is added dropwise to the reaction vessel, and the reaction is carried out at 70-80 °C with magnetic stirring at 300-500 rpm for approximately 2-3 h. The reaction is monitored by TLC. After the reaction is complete, the reaction solution is poured into 30-50 mL of saturated sodium carbonate aqueous solution, and ethyl acetate is added for extraction. The organic phase is collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 8-10:1) to obtain intermediate Pb.

[0130] (2) Add 20-30 mL of dehydrated DMSO to the reaction vessel, along with 1 mmol of genistein and 1-1.2 mmol of potassium carbonate (K₂CO₃). React at 70-80 °C, pH 8-9, with magnetic stirring at 300-500 rpm for 0.5-1 h. Then add 1-1.2 mmol of intermediate Pb, raise the temperature to 100-110 °C, and react with magnetic stirring at 300-500 rpm for approximately 8-10 h. Monitor the reaction by TLC. After the reaction is complete, pour the reaction solution into 30-50 mL of saturated saline solution and extract with ethyl acetate. Collect the organic phase, concentrate and mix, and purify by silica gel column chromatography (PE:EtOAc = 3-5:1) to obtain the target Yb compound.

[0131] (iv) Yc compounds (wherein the Ar2 in the Yc compound is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocycle of 4-10 carbon atoms, and the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to C2 by C2). 1-20 Halogenated alkyl groups and C-shaped alkyl groups 1-20 Synthetic methods for alkyl carbonyl or carbonyl-substituted groups include:

[0132] 4-Trifluoromethylbenzoic acid ( ) or 6-trifluoromethylnicotinic acid ( The genistein was dissolved in 15-30 mL of dehydrated dichloromethane, followed by the addition of 1-1.5 mmol of oxaloyl chloride and 1 drop of dehydrated DMF. The mixture was stirred magnetically at 300-500 rpm for approximately 0.5-1 h at 20-30°C, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the activated intermediate. 1 mmol of genistein was dissolved in dehydrated DMF, and 3-5 mmol of triethylamine was added. The activated intermediate was added under ice bath conditions, and after 10-20 min, the mixture was cooled to room temperature and stirred magnetically at 300-500 rpm overnight. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated potassium carbonate solution and extracted with ethyl acetate. The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 3-5:1) to obtain the target compound Yc.

[0133] Example

[0134] It should be noted that, unless otherwise specified, the various materials and reagents used in the following examples are commonly used in the art and can be obtained through conventional commercial channels.

[0135] Example 1 Preparation of 7-(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-benzopyran-4-one (compound Z1)

[0136]

[0137] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added as the reaction solvent to a round-bottom flask, followed by the sequential addition of 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 2,4-dichloro-1,3-dinitro-5-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for 1-2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Z1 as a pale yellow solid, with a yield of 50.2% and a purity of 97.730%. 1 HN MR(600MHz,DMSO-d6)δ13.12(s,1H),9.63(s,1H),8.87(s,1H),8.48(s,1H),7.42-7.37(m,2H),7.03(d,1H),6.86-6.81(m,2H),6.74(d,1H). 13CNMR(151MHz,DMSO-d6)δ180.72,162.33,161.00,157.65,157.11,155.15,146.20,142.33,140.8 3,130.21,130.09,127.87(d),126.51(q),123.08,122.02,120.21,115.12,107.86,99.21,95.03. 19 FNMR (565MHz, DMSO-d6) δ 16.34. HRMS (ESI) m / z: [M+H] Calculated value 539.0099; Observed value 539.0096. The proton, carbon, fluorine, and mass spectra of compound Z1 are as follows: Figure 1 (a) to Figure 1 As shown in (d).

[0138] Example 2 Preparation of 3-(4-(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5,7-dihydroxy-4H-benzopyran-4-one (compound Y1, i.e., Ya1)

[0139] Method 1:

[0140]

[0141] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added as the reaction solvent to a round-bottom flask, followed by the sequential addition of 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK), and then 1.5 mmol of 2,4-dichloro-1,3-dinitro-5-trifluoromethylbenzene. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3-4 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Y1 as a pale yellow solid, with a yield of 15.3% and a purity of 95.113%. 1 HNMR (600MHz, DMSO-d6) δ 12.82 (s, 1H), 10.94 (s, 1H), 8.84 (s, 1H), 8.46 (s, 1H), 7.60–7.55 (m, 2H), 7.24–7.21 (m, 2H), 6.41 (d, J = 2.1Hz, 1H), 6.24 (d, J = 2.1Hz, 1H). HRMS (ESI) m / z: [M+H] Calculated value 539.0099; Measured value 539.0097.

[0142] Method 2:

[0143] 30 mL of dehydrated tetrahydrofuran was added as the reaction solvent to a round-bottom flask, followed by 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 2,4-dichloro-1,3-dinitro-5-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetic stirring at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound Y1 (i.e., Ya1), with a yield of 62.1% and a purity of 98.816%. The proton, carbon, fluorine, and mass spectra of compound Y1 are shown below. Figure 2 (a) to Figure 2 As shown in (d).

[0144] Example 3 Preparation of 7-(2,6-dinitro-4-(trifluoromethyl)phenoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-benzopyran-4-one (compound Z2)

[0145] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added to a round-bottom flask as the reaction solvent, followed by the sequential addition of 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 2-chloro-1,3-dinitro-5-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Z2 as a yellow solid, with a yield of 34.6% and a purity of 98.221%. 1HNMR(600MHz,DMSO-d6)δ13.10(s,1H),9.63(s,1H),8.96(s,2H),8.46(s,1H),7.39(d,J=8.3Hz,2H),6.92(d ,J=2.4Hz,1H),6.84(d,J=8.2Hz,2H),6.64(d,J=2.5Hz,1H).13CNMR(151MHz,DMSO-d6)δ180.71,162.27,161. 53,157.64,157.18,155.05,144.24,142.31,130.22,128.37(q,J=3.7Hz),127.60(q,J=35.3Hz),123.04,122 .89,121.07,120.76,115.13,107.54,99.15,94.80.19FNMR(565MHz,DMSO-d6)δ16.56.HRMS(ESI)m / z:[M+H]C 22 H 11 The calculated value of F3N2O9 is 505.0489; the measured value is 509.0485.

[0146] Example 4 Preparation of 7-(2,4-dinitro-6-(trifluoromethyl)phenoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-benzopyran-4-one (compound Z3)

[0147] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added as the reaction solvent to a round-bottom flask, followed by the sequential addition of 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 2-chloro-1,5-dinitro-3-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Z3 as a yellow solid, with a yield of 42.3% and a purity of 98.050%. 1HNMR(600MHz,DMSO-d6)δ13.10(s,1H),9.62(s,1H),9.26-9.23(m,1H),8.94-8.91(m,1H),8.45(s,1H),7.38(d,J= 8.2Hz,2H),6.84(dd,J=16.1,5.3Hz,3H),6.60-6.57(m,1H).13CNMR(151MHz,DMSO-d6)δ180.69,162.33,161.52,15 7.65,157.17,155.07,148.45,144.58,143.02,130.20,128.02(d,J=5.2Hz),126.78,126.13(q,J=33.4Hz),123.0 6,122.16,120.70,120.34,115.12,107.61,99.07,94.80.19FNMR(565MHz,DMSO-d6)δ17.26.HRMS(ESI)m / z:[M+H]C 22 H 11 The calculated value of F3N2O9 is 505.0489; the measured value is 505.0471.

[0148] Example 5 Preparation of 5-hydroxy-3-(4-hydroxyphenyl)-7-(3-nitro-5-(trifluoromethyl)phenoxy)-4H-benzopyran-4-one (compound Z4)

[0149] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added to a round-bottom flask as the reaction solvent, followed by the sequential addition of 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 1-chloro-3-nitro-5-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Z4 as a yellow solid, with a yield of 38.6% and a purity of 98.062%. 1HNMR (600MHz, DMSO-d6) δ12.84(s,1H),8.51(s,1H),8.25(q,J=1.8,1.3Hz,1H),7.98(t,J=2.2Hz,1H),7.93(t,J=2.0Hz,1H),7.72-7.69( m,2H),7.34-7.28(m,2H),6.43(d,J=2.1Hz,1H),6.25(d,J=2.2Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.80,164.48,162.00,158.37,157. 58, 154.98, 154.77, 132.27 (q, J = 32.8 Hz), 131.05, 127.59, 124.69, 123.68, 123.28, 122.69 (q, J = 3.9 Hz), 121.87, 121.42, 119.43, 114.01 (q, J = 3.8 Hz), 104.43, 99.16, 93.82.19 FNMR (565 MHz, DMSO-d6) δ 16.45.HRMS (ESI) m / z: [M+H]C22H12F3NO7 Calculated value 460.0638; measured value 460.0620.

[0150] Example 6 Preparation of 5-hydroxy-3-(4-hydroxyphenyl)-7-(3-nitro-4-(trifluoromethyl)phenoxy)-4H-benzopyran-4-one (compound Z5)

[0151] 30 mL of dehydrated dimethyl sulfoxide (DMSO) was added as the reaction solvent to a round-bottom flask, followed by the sequential addition of 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK). Then, 1.1 mmol of 4-bromo-2-nitro-1-trifluoromethylbenzene was added. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Z5 as a pale yellow solid, with a yield of 50.9% and a purity of 98.202%. 1HNMR(600MHz,DMSO-d6)δ13.07(s,1H),9.63(s,1H),8.49(s,1H),7.80(d,J=8.5Hz,1H),7.71-7.67(m,1H),7.62(d,J=1.8Hz,1H),7.43-7 .38(m,2H),6.86-6.81(m,2H),6.77(d,J=2.3Hz,1H),6.49(d,J=2.3Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.98,164.42,162.02,158.56, 157.62, 154.55, 141.88, 130.50, 124.86, 124.46, 123.55, 122.65, 122.04, 121.80 (d, J = 9.5 Hz), 121.64, 121.44, 118.65, 116.13, 110.73 (q, J = 5.0 Hz), 104.46, 99.11, 93.79.19 FNMR (565 MHz, DMSO-d6) δ -60.27.HRMS (ESI) m / z: [M+H]C22H12F3NO7 Calculated value 460.0638; measured value 460.2391.

[0152] Example 7 Preparation of 3-(4-(2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5,7-dihydroxy-4H-benzopyran-4-one (compound Ya2)

[0153] First synthetic method: 30 mL of dehydrated DMSO was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.5 mmol of 2-chloro-1,3-dinitro-5-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetic stirring at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Ya2 as a yellow solid, with a yield of 21.6% and a purity of 96.183%.

[0154] The second synthetic method: 30 mL of dehydrated tetrahydrofuran was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.1 mmol of 2-chloro-1,3-dinitro-5-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetic stirring at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound Ya2 as a yellow solid, with a yield of 67.8% and a purity of 98.041%. 1 HNMR(600MHz,DMSO-d6)δ12.83(s,1H),10.93(s,1H),8.94(s,2H),8.46(s,1H),7.59-7.53(m,2H),7.17-7.12(m,2 H),6.41(d,J=2.1Hz,1H),6.24(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.81,164.47,161.98,157.59,156. 33,155.03,144.48,143.23,130.71,127.93(q,J=3.8Hz),127.12,126.89,126.81,126.65,126.42,124.73,122.9 2,121.26,121.11,119.30,115.34,104.41,99.14,93.82.19FNMR(565MHz,DMSO-d6)δ16.67.HRMS(ESI)m / z:[M+H]C 22 H 11 The calculated value of F3N2O9 is 505.0489; the measured value is 505.0482.

[0155] Example 8 Preparation of 3-(4-(2,4-dinitro-6-(trifluoromethyl)phenoxy)phenyl)-5,7-dihydroxy-4H-benzopyran-4-one (compound Ya3)

[0156] First synthetic method: 30 mL of dehydrated DMSO was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.5 mmol of 2-chloro-1,5-dinitro-3-trifluoromethylbenzene. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Ya3 as a yellow solid, with a yield of 17.9% and a purity of 95.836%.

[0157] The second synthetic method: 30 mL of dehydrated tetrahydrofuran was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.1 mmol of 2-chloro-1,5-dinitro-3-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound Ya3 as a yellow solid, with a yield of 57.3% and a purity of 98.084%. 1 HNMR(600MHz,DMSO-d6)δ12.81(s,1H),10.93(s,1H),9.21(d,J=2.8Hz,1H),8.91(d,J=2.8Hz,1H),8.46(s,1H),7.60-7 .55(m,2H),7.13-7.07(m,2H),6.41(d,J=2.1Hz,1H),6.24(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.77,164.48, 161.97,157.58,156.41,155.06,149.70,143.82,143.09,130.76,127.69(q,J=4.9Hz),126.99,126.62,126.07(q,J=33 .1Hz),122.29,121.16,120.47,115.37,104.40,99.14,93.82.19FNMR(565MHz,DMSO-d6)δ17.13.HRMS(ESI)m / z:[M+H]C 22 H 11The calculated value of F3N2O9 is 505.0489; the measured value is 505.0479.

[0158] Example 9 Preparation of 5,7-dihydroxy-3-(4-(3-nitro-5-(trifluoromethyl)phenoxy)phenyl)-4H-benzopyran-4-one (compound Ya4)

[0159] First synthetic method: 30 mL of dehydrated DMSO was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.5 mmol of 1-chloro-3-nitro-5-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Ya4 as a yellow solid, with a yield of 19.8% and a purity of 96.374%.

[0160] The second synthetic method: 30 mL of dehydrated tetrahydrofuran was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.1 mmol of 1-chloro-3-nitro-5-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetic stirring at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound Ya4 as a yellow solid, with a yield of 52.6% and a purity of 98.170%. 1HNMR (600MHz, DMSO-d6) δ12.85(s,1H),10.94(s,1H),8.48(s,1H),7.74(td,J=1.7,0.8Hz,1H),7.68-7.65(m,2H),7.52(t,J=2.0H z,1H),7.38(t,J=2.0Hz,1H),7.26-7.21(m,2H),6.42(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.77,1 64.51,161.98,158.42,157.58,155.09,154.36,149.37,131.79(q,J=33.8Hz),131.18,128.06,123.52,121.71,121.34,121.10( q,J=3.5Hz),119.63,115.90,114.63(t,J=4.1Hz),104.41,99.16,93.83.19FNMR(565MHz,DMSO-d6)δ16.36.HRMS(ESI)m / z:[M+H]C 22 H 12 The calculated value of F3NO7 is 460.0638; the measured value is 460.0630.

[0161] Example 10 Preparation of 5,7-dihydroxy-3-(4-(3-nitro-4-(trifluoromethyl)phenoxy)phenyl)-4H-benzopyran-4-one (compound Ya5)

[0162] First synthetic method: 30 mL of dehydrated DMSO was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.5 mmol of 4-bromo-2-nitro-1-trifluoromethylbenzene. The reaction was carried out at room temperature (25 °C) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Ya5 as a yellow solid, with a yield of 20.5% and a purity of 96.915%.

[0163] The second synthetic method: 30 mL of dehydrated tetrahydrofuran was added to a round-bottom flask as the reaction solvent. 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK) were added, followed by 1.1 mmol of 4-bromo-2-nitro-1-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetically stirred at 500 rpm for approximately 3 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain the target compound Ya5 as a yellow solid, with a yield of 57.2% and a purity of 98.163%. 1 HNMR(600MHz,DMSO-d6)δ12.85(s,1H),10.94(s,1H),8.48(s,1H),7.77(d,J=8.4Hz,1H),7.68-7.60(m,2H),7.57(dd,J=8.4,1.8 Hz,1H),7.23(d,J=1.8Hz,1H),7.21-7.14(m,2H),6.42(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.7 7,164.49,162.01,157.57,155.20(d,J=5.3Hz),154.90,131.02,128.98(q,J=5.1Hz),127.50,127.12,126.75,124.05,122.25, 122.17,121.40,119.47,119.26,119.06,118.89,104.43,99.16,93.82.19FNMR(565MHz,DMSO-d6)δ17.22.HRMS(ESI)m / z:[M+H]C 22 H 12 The calculated value of F3NO7 is 460.0639; the measured value is 460.0631.

[0164] Example 11 Preparation of 3-(4-(3-chloro-4-(trifluoromethyl)phenoxy)phenyl)-5,7-dihydroxy-4H-benzopyran-4-one (compound Ya6)

[0165] First synthetic method: 30 mL of dehydrated DMSO was added to a round-bottom flask as the reaction solvent, followed by 1 mmol of genistein and 1.5 mmol of potassium tert-butoxide (t-BuOK), and then 1.5 mmol of 4-bromo-2-chloro-1-trifluoromethylbenzene. The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetic stirring at 500 rpm for approximately 5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Ya6, with a yield of 30.3% and a purity of 98.032%. 1 HNMR(600MHz,DMSO-d6)δ12.85(s,1H),10.95(s,1H),8.49(s,1H),7.87(d,J=8.8Hz,1H),7.71-7.63(m,2H),7.36(d,J=2.5Hz,1 H),7.30-7.22(m,2H),7.09(dd,J=8.8,2.5Hz,1H),6.42(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179. 78, 164.50, 162.00, 160.90, 157.59, 155.00, 154.17, 132.40, 131.06, 129.72 (q, J = 5.3 Hz), 127.95, 123.86, 122.06, 121.39, 121.04 (q, J = 31.4 Hz), 120.19, 119.99, 115.92, 104.44, 99.17, 93.83.19 FNMR (565 MHz, DMSO-d6) δ 17.72. HRMS (ESI) m / z: [M+H] Calculated value C 22 H 12 ClF3O5, 449.0398; measured value 449.0392.

[0166] Example 12 Preparation of 5,7-dihydroxy-3-(4-(4-nitro-2-(trifluoromethyl)phenoxy)phenyl)-4H-benzopyran-4-one (compound Ya7)

[0167] Second synthetic method: 30 mL of dehydrated tetrahydrofuran was added to a round-bottom flask, followed by 1 mmol of genistein and 1.1 mmol of potassium tert-butoxide (t-BuOK), and then 1.1 mmol of 1-bromo-4-nitro-2-trifluoromethylbenzene (t-BuOK). The reaction was carried out at room temperature (25℃) with pH 9-10 and magnetic stirring at 500 rpm for approximately 5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 100-120:1) to obtain the target compound Ya7, with a yield of 78.8% and a purity of 98.478%. 1 HNMR(600MHz,DMSO-d6)δ12.83(s,1H),10.96(s,1H),8.54(d,J=2.8Hz,1H),8.51(d,J=8.2Hz,2H),7.76-7.71(m,2H),7.3 5-7.30(m,2H),7.18(d,J=9.2Hz,1H),6.43(d,J=2.1Hz,1H),6.26(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ180.15,164 .99,162.43,160.38,158.02,155.55,154.21,142.29,131.69,130.57,129.15,123.97-123.68(m),123.64,121.83,121. 67,120.53,119.62(q,J=32.4Hz),118.89,104.85,99.63,94.29.19FNMR(565MHz,DMSO-d6)δ16.47.HRMS(ESI)m / z:[M+H]C 22 H 12 The calculated value of F3NO7 is 460.0637; the measured value is 460.0630.

[0168] Example 13 Preparation of N-(5-(4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenoxy)-2-nitro-4-(trifluoromethyl)phenyl)acetamide (compound Yb1)

[0169] (1) Synthesis of amide intermediate Pb1: 30 mL of anhydrous N,N-dimethylformamide (DMF) was added to a round-bottom flask as a solvent. 1 mmol of 5-chloro-2-nitro-4-trifluoromethylaniline was dissolved in the anhydrous DMF. Then, 1.2 mmol of acetyl chloride was added dropwise to the reaction flask. The reaction was carried out at 75 °C with magnetic stirring at 500 rpm for approximately 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated sodium carbonate aqueous solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 8:1) to obtain intermediate Pb1 N-(5-chloro-2-nitro-4-(trifluoromethyl)phenyl)acetamide. It is a white, flocculent solid with a yield of 90.2%. 1 ¹H NMR (600 MHz, chloroform-d) δ 10.54 (s, 1H), 9.14 (s, 1H), 8.58 (s, 1H), 2.34 (s, 3H). ¹³C NMR (151 MHz, chloroform-d) δ 169.32, 140.62, 138.02, 133.20, 125.63 (q, J = 5.7 Hz), 124.22, 123.10 (q, J = 33.4 Hz), 122.66, 120.85, 25.86.

[0170] (2) 30 mL of dehydrated DMSO was added to a round-bottom flask, along with 1 mmol of genistein and 1.2 mmol of potassium carbonate (K₂CO₃). The mixture was stirred magnetically at 500 rpm for 1 h at 75 °C and pH 8. Then, 1.2 mmol of intermediate Pb₁ was added, and the mixture was heated to 105 °C and stirred magnetically at 500 rpm for approximately 8 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Yb₁ as a yellow solid, with a yield of 43.8% and a purity of 98.249%. 1HNMR(600MHz,DMSO-d6)δ12.84(s,1H),10.96(s,1H),10.53(s,1H),8.50(s,1H),8.34(s,1H),7.76-7.71(m,2H),7.41(s,1H) ),7.39-7.28(m,2H),6.43(d,J=2.1Hz,1H),6.26(d,J=2.1Hz,1H),2.05(s,3H).13CNMR(151MHz,DMSO-d6)δ179.74,169.00, 164.54,162.01,158.79,157.58,155.13,153.60,137.53,134.68,131.16,125.45(d,J=5.3Hz),123.24,121.43,121.20,12 0.20,113.68(q,J=32.7Hz),111.03,104.44,99.20,93.85,23.88.19FNMR(565MHz,DMSO-d6)δ17.22.HRMS(ESI)m / z:[M+H]C 24 H 15 The calculated value of F3N2O8 is 517.0853; the measured value is 517.0839.

[0171] Example 14 Preparation of N-(5-(4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenoxy)-2-nitro-4-(trifluoromethyl)phenyl)propionamide (compound Yb2)

[0172] (1) Synthesis of amide intermediate Pb2: 30 mL of anhydrous N,N-dimethylformamide (DMF) was added to a round-bottom flask as a solvent. 1 mmol of 5-chloro-2-nitro-4-trifluoromethylaniline was dissolved in the anhydrous DMF. Then, 1.2 mmol of propionyl chloride was added dropwise to the reaction flask. The reaction was carried out at 75 °C with magnetic stirring at 500 rpm for approximately 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated sodium carbonate aqueous solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 8:1) to obtain intermediate Pb2 N-(5-chloro-2-nitro-4-(trifluoromethyl)phenyl)propionamide. It is a white solid with a yield of 90.6%. 1¹³C NMR (600MHz, chloroform-d) δ 10.58 (s, 1H), 9.18 (s, 1H), 8.58 (s, 1H), 2.58 (q, J = 7.5Hz, 2H), 1.30 (t, J = 7.5Hz, 3H). ¹³C NMR (151MHz, chloroform-d) δ 173.13, 140.63, 138.19, 133.13, 125.66 (q, J = 5.6Hz), 124.26, 122.95 (q, J = 33.5Hz), 122.70, 120.89, 31.95, 9.18.

[0173] (2) 30 mL of dehydrated DMSO was added to a round-bottom flask, along with 1 mmol of genistein and 1.2 mmol of potassium carbonate (K₂CO₃). The mixture was stirred magnetically at 500 rpm for 1 h at 75 °C and pH 8. Then, 1.2 mmol of intermediate Pb₂ was added, and the mixture was heated to 105 °C and stirred magnetically at 500 rpm for approximately 8 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Yb₂ as a pale yellow solid, with a yield of 37.5% and a purity of 98.049%. 1 HNMR(600MHz,DMSO-d6)δ12.83(s,1H),10.95(s,1H),10.48(s,1H),8.50(s,1H),8.35(s,1H),7.77-7.71(m,2H),7.50(s,1H),7.36-7. 29(m,2H),6.42(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H),2.36(q,J=7.5Hz,2H),1.01(t,J=7.5Hz,3H).13CNMR(151MHz,DMSO-d6)δ179.75 ,172.53,164.53,162.00,158.83,157.58,155.18,153.62,137.72,134.46,131.16,128.64,125.51(d,J=5.5Hz),123.24,121.44,121. 19,120.17,113.56(q,J=32.8Hz),110.86,104.44,99.19,93.85,29.56,8.84.19FNMR(565MHz,DMSO-d6)δ17.17.HRMS(ESI)m / z:[M+H]C 25 H 17The calculated value of F3N2O8 is 531.1009; the measured value is 531.1007.

[0174] Example 15: Preparation of N-(5-(4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenoxy)-2-nitro-4-(trifluoromethyl)phenyl)neopentamide (compound Yb3)

[0175] (1) Synthesis of amide intermediate Pb3: 30 mL of anhydrous N,N-dimethylformamide (DMF) was added to a round-bottom flask as a solvent. 1 mmol of 5-chloro-2-nitro-4-trifluoromethylaniline was dissolved in the anhydrous DMF. Then, 1.2 mmol of pentanoyl chloride was added dropwise to the reaction flask. The reaction was carried out at 75 °C with magnetic stirring at 500 rpm for approximately 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated sodium carbonate aqueous solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 8:1) to obtain intermediate Pb3 N-(5-chloro-2-nitro-4-(trifluoromethyl)phenyl)neopentamide. It is a white solid with a yield of 92.1%. 1 ¹H NMR (600 MHz, chloroform-d) δ 10.95 (s, 1H), 9.22 (s, 1H), 8.59 (s, 1H), 1.37 (s, 10H). ¹³C NMR (151 MHz, chloroform-d) δ 178.31, 140.65, 138.62, 133.26, 125.67 (q, J = 5.6 Hz), 124.25, 123.21–122.44 (m), 120.92, 41.04, 27.41.

[0176] (2) 30 mL of dehydrated DMSO was added to a round-bottom flask, along with 1 mmol of genistein and 1.2 mmol of potassium carbonate (K₂CO₃). The mixture was stirred magnetically at 500 rpm for 1 h at 75 °C and pH 8. Then, 1.2 mmol of intermediate Pb₃ was added, and the mixture was heated to 105 °C and stirred magnetically at 500 rpm for approximately 8 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Yb₃ as a yellow solid, with a yield of 43.8% and a purity of 98.058%. 1HNMR(600MHz,DMSO-d6)δ12.84(s,1H),10.95(s,1H),10.44(s,1H),8.52(s,1H),8.43(s,1H),7.84(s,1H),7.77-7.73( m,2H),7.34-7.29(m,2H),6.43(d,J=2.1Hz,1H),6.26(d,J=2.1Hz,1H),1.18(s,9H).13CNMR(151MHz,DMSO-d6)δ179.78 ,176.96,164.52,162.00,159.07,157.60,155.27,153.69,138.75,133.68,131.16,128.58,125.70,123.23,121.16,1 20.05,113.69,113.47,110.60,104.45,99.20,93.87,26.64.19FNMR(565MHz,DMSO-d6)δ17.19.HRMS(ESI)m / z:[M+H]C 27 H 21 The calculated value of F3N2O8 is 559.1323; the measured value is 559.1314.

[0177] Example 16: Preparation of N-(4-(4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenoxy)-2-nitro-5-(trifluoromethyl)phenyl)neopentamide (compound Yb4)

[0178] (1) Synthesis of amide intermediate Pb4: 30 mL of anhydrous N,N-dimethylformamide (DMF) was added to a round-bottom flask as a solvent. 1 mmol of 4-chloro-2-nitro-5-trifluoromethylaniline was dissolved in the anhydrous DMF. Then, 1.2 mmol of pentanoyl chloride was added dropwise to the reaction flask. The reaction was carried out at 75 °C with magnetic stirring at 500 rpm for approximately 2 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated sodium carbonate aqueous solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 8:1) to obtain intermediate Pb4 N-(4-chloro-2-nitro-5-(trifluoromethyl)phenyl)neopentamide. It is a white solid with a yield of 93.4%. 1¹H NMR (600 MHz, chloroform-d) δ 8.03 (s, 1H), 7.65 (s, 1H), 1.49 (s, 8H). ¹³C NMR (151 MHz, chloroform-d) δ 176.02, 152.68, 140.04, 128.46, 124.85 (q, J = 31.6 Hz), 124.01, 122.20, 119.21 (q, J = 5.9 Hz), 113.83, 34.57, 28.43.

[0179] (2) 30 mL of dehydrated DMSO was added to a round-bottom flask, along with 1 mmol of genistein and 1.2 mmol of potassium carbonate (K₂CO₃). The mixture was stirred magnetically at 500 rpm for 1 h at 75 °C and pH 8. Then, 1.2 mmol of intermediate Pb₄ was added, and the mixture was heated to 105 °C and stirred magnetically at 500 rpm for approximately 8 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 50 mL of saturated saline solution, and ethyl acetate was added for extraction (50 mL × 3 times). The organic phase was collected, concentrated, and purified by silica gel column chromatography (PE:EtOAc = 4:1) to obtain the target compound Yb₄ as a yellow solid, with a yield of 43.8% and a purity of 98.014%. 1 HNMR(600MHz,DMSO-d6)δ12.85(s,1H),10.93(s,1H),9.05(s,1H),8.45(s,1H),8.18(s,1H),7.66-7.61(m,2H),7.23(s,1 H),7.19-7.14(m,2H),6.42(d,J=2.1Hz,1H),6.25(d,J=2.1Hz,1H),1.14(s,9H).13CNMR(151MHz,DMSO-d6)δ179.84,176.7 6,164.47,161.98,157.60,154.92,154.87,152.18,130.83,128.74,127.24,126.98,124.57(d,J=5.9Hz),123.62,121.8 1,121.49,121.29,120.95,118.61,104.43,99.14,93.83,26.93.19FNMR(565MHz,DMSO-d6)δ17.42.HRMS(ESI)m / z:[M+H]C 27 H 21 The calculated value of F3N2O8 is 559.1323; the measured value is 559.1309.

[0180] Example 17 Preparation of 4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenyl-4-(trifluoromethyl)benzoate (compound Yc1)

[0181] 4-Trifluoromethylbenzoic acid was dissolved in 15 mL of dehydrated dichloromethane, followed by the addition of 1.5 mmol of oxaloyl chloride and 1 drop of dehydrated DMF. The reaction was carried out at 25°C with magnetic stirring at 500 rpm for approximately 1 h, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the activated intermediate 2-chloro-2-oxoacetic acid 4-trifluoromethylbenzoic anhydride. 1 mmol of genistein was dissolved in dehydrated DMF, and 3 mmol of triethylamine was added. The activated intermediate 2-chloro-2-oxoacetic acid 4-trifluoromethylbenzoic anhydride was added under ice bath conditions. After 15 min, the mixture was cooled to room temperature and stirred magnetically at 500 rpm overnight, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated potassium carbonate solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and mixed. After separation and purification by silica gel column chromatography (PE:EtOAc = 3:1), the target compound Yc1, which was a white solid, was obtained with a yield of 54.2% and a purity of 98.232%. 1 HNMR (600MHz, DMSO-d6) δ12.86(s,1H),10.95(s,1H),8.51(s,1H),8.38-8.33(m,2H),8.00(d,J=8.2Hz,2H),7.70-7. 67(m,2H),7.43-7.40(m,2H),6.43(d,J=2.2Hz,1H),6.26(d,J=2.1Hz,1H).13CNMR(151MHz,DMSO-d6)δ179.82,164.5 2,163.57,161.99,157.61,155.15,150.24,133.40(d,J=32.3Hz),132.75,130.73,130.24,128.89,125.95(q,J=3.7 Hz),124.59,122.79,121.70,121.50,104.44,99.18,93.86.19FNMR(565MHz,DMSO-d6)δ16.19.HRMS(ESI)m / z:[M+H]C 23 H 13 The calculated value of F3O6 is 443.0737; the measured value is 443.0731.

[0182] Example 18 Preparation of 4-(5,7-dihydroxy-4-oxo-4H-benzopyran-3-yl)phenyl-6-(trifluoromethyl)nicotinic acid ester (compound Yc2)

[0183] 6-Trifluoromethylnicotinic acid was dissolved in 15 mL of dehydrated dichloromethane, followed by the addition of 1.5 mmol of oxaloyl chloride and 1 drop of dehydrated DMF. The reaction was carried out at 25°C with magnetic stirring at 500 rpm for approximately 1 h, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain the activated intermediate 2-chloro-2-oxoacetic acid 6-(trifluoromethyl)nicotinic anhydride. 1 mmol of genistein was dissolved in dehydrated DMF, and 3 mmol of triethylamine was added. The activated intermediate 2-chloro-2-oxoacetic acid 6-(trifluoromethyl)nicotinic anhydride was added under ice bath conditions. After 15 min, the mixture was cooled to room temperature and stirred magnetically at 500 rpm overnight, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated potassium carbonate solution and extracted with ethyl acetate (50 mL × 3 times). The organic phase was collected, concentrated, and mixed. After separation and purification by silica gel column chromatography (PE:EtOAc = 3:1), the target compound Yc2, which was a white solid, was obtained with a yield of 38.5% and a purity of 98.057%. 1 HNMR(600MHz,DMSO-d6)δ12.85(s,1H),10.96(s,1H),9.43(d,J=2.1Hz,1H),8.77(dd,J=8.1,2.1Hz,1H),8.51(s,1H) ,8.17(dd,J=8.2,0.9Hz,1H),7.73-7.67(m,2H),7.46-7.43(m,2H),6.43(d,J=2.1Hz,1H),6.26(d,J=2.1Hz,1H).13CN MR(151MHz,DMSO-d6)δ179.81,164.53,162.55,161.99,157.62,155.21,151.02,150.02,140.16,130.28,129.05,128 .44,121.66,121.49,121.14,120.31,104.45,99.19,93.88.19FNMR(565MHz,DMSO-d6)δ11.04.HRMS(ESI)m / z:[M+H]C 22 H 12 The calculated value of F3NO6 is 444.0689; the measured value is 444.0681.

[0184] In the following test examples, compound M is the compound that has the best inhibitory activity against cervical cancer HeLa cells among the similar isoflavone compounds reported in the existing literature (structure shown in the figure below), and is used as an activity comparison.

[0185] The structure of compound M is as follows:

[0186]

[0187] Unless otherwise specified, all of the following control / control groups are blank controls (i.e., control groups).

[0188] Test Example 1

[0189] 1.1 Compound Z1 inhibits the proliferation of cervical cancer HeLa cells in vitro.

[0190] The testing method included: culturing cervical cancer HeLa cells in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody; observing cell concentration and growth under a microscope; and seeding 100 μL of HeLa cells per well with 2 × 10⁶ cells when the cell concentration reached 80-90%. 3 100 μL of SiHa cells were seeded into 96-well plates; 8 × 10⁸ cells were seeded per well. 3 100 μL of H8 cells were seeded into 96-well plates; 5 × 10⁶ cells were seeded per well. 3 100 μL of TC-1 cells were seeded into 96-well plates; 5 × 10⁶ cells were seeded per well. 3 Cells were cultured in 96-well plates. After 24 hours, cell status and density were observed under a microscope. Different concentrations of the drug were then prepared and added to the corresponding wells for incubation for 24 hours. After centrifugation at 1200 rpm for 7 minutes, the supernatant was discarded. 100 μL of LTT (0.5 mg / mL) was added to each well and incubated in the dark for 4 hours. After centrifugation and discarding the supernatant, 150 μL of DMSO solution was added to each well. After shaking for 10 minutes, the OD value at 490 nm was measured, and cell viability and IC50 were calculated. 50 value.

[0191] The results are as follows Figure 3 As shown, the MTT assay was used to detect the cell viability of human cervical cancer HeLa and SiHa cells, normal human cervical epithelial cells H8, and mouse TC-1 cells after treatment with different concentrations of compound Z1 for 24 hours, and to determine the IC50 of HeLa, SiHa, and TC-1 cells. 50 The concentrations were 3.17±0.49 μM, 14.46±2.92 μM, and 0.67±0.17 μM, respectively. Compound Z1 showed an IC50 concentration of 3.17±0.49 μM, 14.46±2.92 μM, and 0.67±0.17 μM after 24 h of treatment with normal human cervical epithelial H8 cells. 50 The value was 46.91 ± 1.39 μM.

[0192] 1.2 Compound Y1 inhibits the proliferation of cervical cancer HeLa cells in vitro.

[0193] The testing method included: culturing cervical cancer HeLa cells in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody; observing cell concentration and growth under a microscope; and seeding 100 μL of HeLa cells per well at a concentration of 80-90%. 3100 μL of SiHa cells were seeded into 96-well plates; 8 × 10⁸ cells were seeded per well. 3 100 μL of H8 cells were seeded into 96-well plates; 5 × 10⁶ cells were seeded per well. 3 100 μL of TC-1 cells were seeded into 96-well plates; 5 × 10⁶ cells were seeded per well. 3 Cells were cultured in 96-well plates. After 24 hours, cell status and density were observed under a microscope. Different concentrations of the drug were then prepared and added to the corresponding wells for incubation for 24 hours. After centrifugation at 1200 rpm for 7 minutes, the supernatant was discarded. 100 μL of LTT (0.5 mg / mL) was added to each well and incubated in the dark for 4 hours. After centrifugation and discarding the supernatant, 150 μL of DMSO solution was added to each well. After shaking for 10 minutes, the OD value at 490 nm was measured, and cell viability and IC50 were calculated. 50 value.

[0194] The results are as follows Figure 4 As shown, the MTT assay was used to detect the cell viability of human cervical cancer HeLa and SiHa cells, normal human cervical epithelial cells H8, and mouse TC-1 cells after treatment with different concentrations of compound Y1 for 24 hours, and to determine the IC50 of HeLa, SiHa, and TC-1 cells. 50 The concentrations were 4.7±0.15 μM, 19.13±1.28 μM, and 1.37±0.24 μM, respectively (p<0.001). Compound Y1 showed an IC50 value of 4.7±0.15 μM, 19.13±1.28 μM, and 1.37±0.24 μM after 24 h of treatment with normal human cervical epithelial H8 cells. 50 The value was 52.79 ± 2.36 μM.

[0195] 1.3 In vitro inhibitory rate of the compounds of the present invention on HeLa cells

[0196] The MTT assay was used to detect the in vitro inhibitory effect of the compound on HeLa cells. 100 μL of HeLa cells were seeded per well at a concentration of 2 × 10⁶ cells / well. 3 Cells were cultured in 96-well plates at 37°C with 5% CO2 for 24 hours. Cell morphology and growth were observed. All compounds were prepared as 10 mM stock solutions using cell-grade DMSO. Then, the corresponding concentrations of compounds were added to the culture medium to prepare 25 μM medium, which was then added to the corresponding wells for 24 hours. After 24 hours, the 96-well plates were removed, centrifuged at 1200 rpm for 7 minutes, and the supernatant was discarded. 100 μL of LTT (0.5 mg / mL) solution was added to each well, and the plates were incubated in the dark for 4 hours. After centrifugation and discarding the supernatant, 150 μL of DMSO solution was added to each well, and the plates were shaken for 10 minutes. The OD value at 490 nm was measured using a microplate reader, and cell viability and IC50 were calculated. 50 value.

[0197] All compounds of this invention were co-cultured with HeLa cells at a concentration of 25 μM for 24 h, and cell viability was detected by MTT assay. The results are as follows: Figure 5 (same as table below) and Figure 6 As shown.

[0198] The compounds of this invention have an effect on the IC50 of cervical cancer HeLa cells. 50 value

[0199]

[0200]

[0201] It should be noted that the compounds of this invention (especially Z1 and Y1) exhibit significantly better activity than genistein; the compounds Z1, Y1, Z3, and Ya3 of this invention show significantly better activity than compound M; the compounds Z4, Ya6, Yb3, and Yb4 of this invention have similar activity to compound M. Although compound M has better activity than other compounds synthesized in this invention, all compounds of this invention have significantly better lipophilicity and bioavailability than compound M. Furthermore, compound M is insoluble in ethyl acetate and dichloromethane; and compound M exhibits drug precipitation in the above cell experiments, indicating poor lipophilicity.

[0202] Test Example 2: Compounds Z1 and Y1 induce apoptosis in cervical cancer HeLa cells

[0203] The testing method included culturing cervical cancer HeLa cells in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. Cell concentration and growth were observed under a microscope. When the cell concentration reached 80-90%, the HeLa cells were cultured at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded per well in 6-well plates and incubated at a constant temperature for 24 h. Cells were then treated with different concentrations of drugs for 24 h. Cells were digested with EDTA-free trypsin and collected. After centrifugation and washing twice with PBS, the cells were resuspended in 100 μL of 1× binding buffer. An Annenxin V and PI (1:2) mixture was added and incubated in the dark for 10 min. The incubation was terminated with 200 μL of binding buffer. The fluorescence intensity of FITC and PI was detected by flow cytometry and analyzed using CytExpert.

[0204] The results showed that after treating HeLa cells with different concentrations of compounds Z1 and Y1 for 24 hours, apoptosis in cervical cancer HeLa cells was detected by Annexin V-FITC / PI staining and flow cytometry. The apoptosis results are as follows: Figure 7As shown, compared with the control group, compounds Z1 and Y1 significantly increased the apoptosis rate of HeLa cells in a dose-dependent manner (p<0.001).

[0205] Test Example 3: Compounds Z1 and Y1 reduced the mitochondrial membrane potential (Δψm) in HeLa cervical cancer cells.

[0206] The assay method included: Cells were seeded into 6-well plates and collected using the same method as for apoptosis. JC-1 staining solution was diluted with ultrapure water to prepare JC-1 staining working solution (ratio: 8 mL ultrapure water (200×) per 50 μL JC-1). The cells were incubated in a 37°C cell culture incubator for 20 min, centrifuged, and the supernatant was discarded. Cells were washed twice with JC-1 staining buffer (1×), and resuspended in 200 μL PBS. FITC and PE fluorescence intensities were detected by FASC and analyzed.

[0207] The results showed that treating HeLa cells with different concentrations of compounds Z1 and Y1 for 24 hours, and then detecting mitochondrial membrane potential by flow cytometry, yielded the following results: Figure 8 As shown in Figure (A), compared with the control group, the red fluorescence intensity decreased and the green fluorescence intensity increased with increasing treatment doses of compounds Z1 and Y1 (p<0.001). Meanwhile... Figure 8 Figure (B) shows the same results observed under an inverted fluorescence microscope. This demonstrates that compounds Z1 and Y1 may disrupt mitochondrial function in HeLa cells and induce apoptosis.

[0208] Test Example 4: Compounds Z1 and Y1 induce ROS accumulation in cervical cancer HeLa cells.

[0209] The assay method included: cells were seeded into 6-well plates and collected using the same method as for apoptosis. DCFH-DA was diluted 1:5000 with serum-free culture medium. 300 μL of the diluted DCFH-DA probe was added to each treatment group. After incubation at 37°C for 30 min, the cells were washed three times with serum-free medium. Cells were resuspended in 200 μL PBS, and FITC fluorescence intensity was detected and analyzed using FlowJo software.

[0210] The results showed that the changes in ROS levels in HeLa cells after treatment with compounds Z1 and Y1 for 24 hours were further detected using the DCFH-DA fluorescent probe. The results were captured using an inverted fluorescence microscope. Figure 9 As shown in Figure (A), compounds Z1 and Y1 significantly enhanced green fluorescence in a dose-dependent manner. Furthermore, Figure 9Flow cytometry results in Figure (B) showed that compounds Z1 and Y1 significantly increased intracellular ROS levels in a dose-dependent manner (p<0.001). Subsequently, we pretreated the cells with the ROS scavenger N-acetyl-L-cysteine ​​(NAC), and the results showed that NAC significantly (p<0.001) reduced the increase in ROS levels induced by compounds Z1 and Y1. Figure 9 These results indicate that ROS plays an important role in apoptosis induced by compounds Z1 and Y1.

[0211] Test Example 5: Compounds Z1 and Y1 induce G2 / M phase cell cycle arrest in cervical cancer HeLa cells.

[0212] The testing method includes: inoculating HeLa cells at 5 × 10⁻⁶... 5 Cells were seeded per 60 mm culture dish and collected using the same treatment method as for apoptosis. The cells were washed once with pre-chilled PBS, then fixed overnight at 4°C with pre-chilled 70% ethanol. After centrifugation to remove the fixative and washing with PBS, 0.5 mL of staining buffer (containing 10 μL LPI staining solution and 10 μL RNase A solution) was added sequentially. The cells were incubated at 37°C in the dark for 30 min. Fluorescence intensity was detected by flow cytometry after filtration through a copper mesh and analyzed using FlowJo software.

[0213] The results showed that after treating HeLa cells with different concentrations of compounds Z1 and Y1 for 24 h, flow cytometry was used to analyze different stages of the cell cycle. The cell cycle detection results are shown in Figure 10(A). Compared with the control group, with the increase of compound Z1 and Y1 concentrations, the proportion of cells in the G0 / G1 phase decreased significantly, while the proportion of cells in the G2 / M phase increased significantly (p<0.001), indicating that compounds Z1 and Y1 induced HeLa cells to arrest in the G2 / M phase in a dose-dependent manner.

[0214] Test Example 6: Compounds Z1 and Y1 inhibit tumor growth in TC-1 cervical cancer-bearing mice.

[0215] 6.1 Method

[0216] 6.1.1 Method for Modeling Cervical Cancer in TC-1 Mice

[0217] TC-1 cells in good growth condition were digested with trypsin and collected. They were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS, resuspended, and counted. The required cell suspension (5 × 10⁻⁶ cells / mL) was calculated and prepared. 5 / mouse, 100μL / mouse), place the cell suspension in an ice box and subcutaneously inoculate it on the right side of the mouse (complete within 2 hours) to establish a mouse model of cervical cancer.

[0218] 6.1.2 Grouping and Administration

[0219] Approximately one week after TC-1 cell inoculation, tumor size was observed and measured with calipers. When the tumor diameter reached about 3 mm, mice were randomly divided into 9 groups (control, DMSO, cisplatin, low-, medium-, and high-dose groups of compound Z1, and low-, medium-, and high-dose groups of compound Y1), with 5 mice in each group. Intraperitoneal administration was used. The cisplatin group received injections (5 mg / kg) every 5 days at a dose of 100 μL / mouse, for a total of 3 administrations. The other groups received injections every other day at a dose of 100 μL / mouse, for a total of 7 administrations.

[0220] 6.1.3 Monitoring of tumor volume and mouse weight

[0221] The growth of the tumors in mice was observed daily starting from day 7 after tumor inoculation. Every other day, the long diameter (A) and short diameter (B) of the tumor were measured using calipers. The tumor volume was calculated using the formula (V) = AB. 2 / 2, calculate the tumor volume of the mice, and plot the tumor growth curve with the tumor volume. Measure the weight of the mice every other day and plot the weight change curve.

[0222] 6.1.4 Calculation of organ indices in tumor-bearing mice

[0223] After the final treatment, the mice were dissected, and the heart, liver, spleen, lungs, kidneys, and thymus of each mouse were weighed. The organ index of each organ of each mouse was calculated according to the formula: organ weight (mg) / mouse body weight (g). Finally, the organ index of the mice was plotted in Prism software.

[0224] 6.1.5 Detection of Liver and Kidney Function Indicators in Tumor-Bearing Mice

[0225] Liver and kidney function indicators in tumor-bearing mouse models: After the last treatment, blood was collected from the eyeballs before dissection. Blood was collected from each mouse and allowed to stand at 37°C for 1 hour. Then, the mixture was centrifuged at 3000 rpm for 20 minutes at 4°C. The supernatant in the centrifuge tubes was collected and stored at -20°C. The following procedures were followed to measure the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen in the mice.

[0226] Alanine aminotransferase (ALT / GPT)

[0227]

[0228] Aspartate aminotransferase (AST / GOT)

[0229]

[0230]

[0231] Creatinine (Cr)

[0232]

[0233] Blood urea nitrogen (BUN)

[0234]

[0235]

[0236] 6.1.6 Detection of immune cells in the spleen of tumor-bearing mouse models

[0237] After the final treatment, the spleens of the mice were dissected, weighed, and photographed. The cells were then thoroughly ground in serum-free culture medium, filtered through a copper mesh, and centrifuged (1200 rpm, 7 min). The supernatant was discarded, and the cells were resuspended in 5 mL of PBS, diluted, and counted. The required flow cytometry antibodies were prepared, and 200 μL of the cell suspension was used for flow cytometry to detect immune activity. The cells were stained at room temperature in the dark for 15 min, washed once with PBS, resuspended in the spleen cells, filtered through a copper mesh, and finally, the activation status of immune cells in the spleen was detected using flow cytometry.

[0238] 6.2 Results

[0239] 6.2.1 Compounds Z1 and Y1 inhibit tumor growth in TC-1 cervical cancer-bearing mice

[0240] A TC-1 cervical cancer-bearing mouse model was established. Mice were randomly divided into 9 groups. Treatment involved intraperitoneal injection of corresponding doses of compounds Z1 and Y1 every other day, followed by cisplatin injection every five days for a total of 21 days. Mouse body weight, tumor volume, and tumor weight were measured, and corresponding change curves were plotted. Figure 11 The results showed that after seven treatments, compared with the control group, the treatment groups of compounds Z1 and Y1 significantly inhibited tumor growth in mice, and the mice's body weight did not change significantly, indicating that compounds Z1 and Y1 had no significant toxic side effects on cervical cancer TC-1 tumor-bearing mice. While the cisplatin group also significantly inhibited tumor growth in tumor-bearing mice, it significantly (p<0.001) reduced the mice's body weight, indicating that cisplatin treatment induced systemic toxicity in mice. These results demonstrate that compound Z1 at a dose of 2 mg / kg and compound Y1 at a dose of 4 mg / kg had no significant toxic side effects in mice and exhibited good tumor growth inhibition effects.

[0241] 6.2.2 Compounds Z1 and Y1 showed no significant toxicity to mouse organs.

[0242] The heart, liver, spleen, lungs, kidneys, and thymus of the mouse were weighed and photographed (e.g., Figures 12(A) to 12(C) (As shown), organ indices for different organs in mice were calculated. The results are as follows... Figures 12(D) to 12(I) As shown, compared with the control group, the spleen and thymus indices in the cisplatin group were significantly reduced (p<0.01), indicating that cisplatin has significant toxic side effects on mice. Conversely, compared with the control group, the indices of all organs in the compound Z1 and Y1 treatment groups did not change significantly. Based on the above results, it is preliminarily speculated that the compound Z1 and Y1 treatment groups had no obvious toxic side effects on mice during treatment.

[0243] 6.2.3 Effects of compounds Z1 and Y1 on mouse liver, kidney, and tumor tissue

[0244] H&E staining was performed on tumor tissue, liver, and kidney tissue for observation. Results are as follows: Figure 13 As shown, compared with the control group, the liver and kidney tissues in all treatment groups of compounds Z1 and Y1 showed normal structure, clear cell outlines, and no cell degeneration, necrosis, or pathological changes. However, necrotic areas of varying degrees appeared in the tumor tissue. Therefore, it is preliminarily speculated that compounds Z1 and Y1 have no toxic side effects on mice during treatment.

[0245] 6.2.4 Effects of compounds Z1 and Y1 on liver and kidney function in mice

[0246] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are amino acid transferases widely present in the liver and are important indicators of liver function. Changes in these levels help determine the nature and extent of liver damage. Blood urea nitrogen (BUN) and creatinine (CRE) are important indicators of kidney function; their concentrations depend on renal excretion and indirectly reflect the degree of kidney damage. Test results are as follows... Figure 14 As shown, compared with the control group, there was no significant difference in the levels of ALT, AST, BUN, and CRE in the serum of mice treated with compounds Z1 and Y1. These results further suggest that compounds Z1 and Y1 may be potentially safe drugs for the treatment of cervical cancer.

[0247] 6.2.5 Effects of compounds Z1 and Y1 on the number of T, B, and NK cells in the spleen of tumor-bearing mice

[0248] To investigate whether the antitumor effects of compounds Z1 and Y1 in vivo are related to the immune response, flow cytometry was used to detect the number of immune cells in the spleen of tumor-bearing mice. The results are shown in Figure 15. Compared with the control group, the number of T cells and CD8+ cells in the spleen of mice treated with compound Z1 was significantly higher. + T cells and CD4+ The number of T cells showed an increasing trend; compared with the control, the number of T cells, NK cells, and CD8 cells in the spleen of mice treated with compound Y1 increased. + T cells and CD4 + The number of T cells showed an increasing trend. This suggests that compounds Z1 and Y1 can enhance immune activity by partially activating immune cells, thereby strengthening the anti-tumor immune response in the body and inhibiting the proliferation of tumor cells.

[0249] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. An improved genistein compound or its pharmaceutical salt, characterized in that, The structure of the compound is shown in formula (I) or formula (II): in: Ar1 is a substituted aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, wherein each hydrogen atom on the aromatic ring or aromatic heterocycle is independently converted to carbon atom. 1-20 Alkyl, C 1-20 At least two groups from the haloalkyl, halogen, and nitro groups are substituted; and / or Ar2 represents a substituted aromatic ring or aromatic heterocycle, which is a monocyclic ring comprising 4-10 carbon atoms, wherein each hydrogen atom on the aromatic ring or aromatic heterocycle is independently converted to carbon atom. 1-20 Alkyl, C 1-20 Halogenated alkyl groups, halogens, C 1-20 Alkyl carbonyl or carbonyl, C 1-20 At least two groups in the alkylamide group and nitro group are substituted; The aromatic heterocycle contains at least one of N, P, O or S.

2. The improved genistein compound or its pharmaceutical salt according to claim 1, characterized in that, The Ar1 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar1 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 Substitution of at least two groups from alkyl halogroups, halogens, and nitro groups; and / or The Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-8 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar2 are each independently converted to carbon atoms. 1-12 Alkyl, C 1-12 Halogenated alkyl groups, halogens, C 1-12 Alkyl carbonyl or carbonyl, C 1-12 At least two groups in the alkylamide group and nitro group are substituted; The aromatic heterocycle contains at least one N and / or at least one P.

3. The improved genistein compound or its pharmaceutical salt according to claim 1, characterized in that, The Ar1 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar1 are each independently converted to carbon atoms. 1-6 Alkyl, C 1-6 At least two groups from the haloalkyl, halogen, and nitro groups are substituted; and / or The Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 5-6 carbon atoms; and the hydrogen atoms on the aromatic ring or aromatic heterocycle of the Ar2 are each independently converted to carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl groups, halogens, C 1-6 Alkyl carbonyl or carbonyl, C 1-6 At least two groups are substituted in the alkylamide group and the nitro group; The aromatic heterocycle contains at least one nitrogen atom.

4. The improved genistein compound or its pharmaceutical salt according to claim 1, characterized in that, The halogen or halogen is selected from at least one of fluorine, chlorine, bromine and iodine.

5. The improved genistein compound or its pharmaceutical salt according to claim 3, characterized in that, The aromatic ring of Ar1 is a monocyclic ring having 6 carbon atoms; and the hydrogen atoms on the aromatic ring of Ar1 are each independently replaced by at least two groups selected from methyl, trifluoromethyl, chlorine, and nitro; and the aromatic heterocyclic ring of Ar2 is a 6-membered monocyclic ring having 1 nitrogen atom; and the hydrogen atoms on the aromatic heterocyclic ring of Ar2 are each independently replaced by methyl, trifluoromethyl, halogen, C 1-6 Alkyl carbonyl or carbonyl, C 1-6 At least two groups, including alkylamide and nitro, are substituted.

6. The improved genistein compound or its pharmaceutical salt according to claim 1, characterized in that, Ar1 is any of the following; and / or The Ar2 is Any one of them.

7. The improved genistein compound or its pharmaceutical salt according to claim 1, characterized in that, The compound is selected from:

8. An improved method for preparing genistein compounds, characterized in that, Includes the following steps: 20-30 mL of dehydrated tetrahydrofuran was added to the reaction vessel as a solvent. 1 mmol of genistein and 1-1.2 mmol of potassium tert-butoxide were added, followed by 1-1.2 mmol of a compound containing Ar2 substituted with another halogen. The reaction was carried out at room temperature (20-30°C) and pH 9-11, with magnetic stirring at 300-500 rpm for approximately 3-5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into 30-50 mL of saturated saline solution, and ethyl acetate was added for extraction. The organic phase was collected, concentrated, and purified by silica gel column chromatography with a DCM:MeOH ratio of 100-120:1 to obtain the target compound Ya. Wherein, Ar2 is a substituted aromatic ring or aromatic heterocycle, the aromatic ring or aromatic heterocycle comprising a monocyclic ring of 4-10 carbon atoms, and the hydrogen atoms on the aromatic ring or aromatic heterocycle are each independently converted to carbon atom C. 1-20 At least two of the groups in the alkyl halogroup, halogen, and nitro group are substituted, and the additional halogen is substituted by an additional hydrogen on the aromatic ring or aromatic heterocycle of Ar2.

9. A pharmaceutical composition, characterized in that, The product comprises a therapeutically effective amount of the modified genistein compound or its pharmaceutical salt as described in any one of claims 1 to 7, and optionally a pharmaceutical excipient or pharmaceutical carrier.

10. Use of the improved genistein compound or its pharmaceutical salt according to any one of claims 1 to 8, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for the prevention or treatment of cervical fibroids or cervical cancer.

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