阅读说明：本技术 芒柄花素衍生物在制备治疗或预防围绝经期综合征的药物中的应用 (Application of formononetin derivative in preparation of medicine for treating or preventing perimenopausal syndrome ) 是由 罗晓东 赵云丽 王兆杰 闫晓君 代智 何英杰 于 2021-09-13 设计创作，主要内容包括：本发明涉及药物制备技术领域,提供了芒柄花素衍生物在制备治疗或预防围绝经期综合征的药物中的应用。芒柄花素衍生物可以调节雌激素失衡个体的体内雌激素平衡,减轻围绝经期综合征,增加子宫萎缩个体中的子宫内膜厚度或子宫重量,调节骨组织中破骨细胞异常个体的成骨和破骨的平衡,进而改善围绝经期综合症引起的骨质疏松等症状。同时,芒柄花素衍生物具备稳定性好、适于成药、生物活性好、低毒性的优点,本发明将其应用于抗围绝经期综合征药物的开发中,或用于制备研究围绝经期综合征病理机制以及评价围绝经期综合征疗效的试剂中,具有广阔的应用前景。(The invention relates to the technical field of medicine preparation, and provides application of formononetin derivatives in preparation of medicines for treating or preventing perimenopausal syndromes. The formononetin derivative can regulate the in vivo estrogen balance of individuals with estrogen imbalance, relieve perimenopausal syndrome, increase the thickness of endometrium or the weight of uterus of individuals with uterine atrophy, regulate the balance of osteogenesis and osteoclast of individuals with osteoclast abnormality in bone tissues, and further improve the symptoms of osteoporosis and the like caused by the perimenopausal syndrome. Meanwhile, the formononetin derivative has the advantages of good stability, suitability for patent medicine, good biological activity and low toxicity, and has wide application prospect when being applied to the development of perimenopausal syndrome resistant medicines or being applied to the preparation of reagents for researching the pathological mechanism of the perimenopausal syndrome and evaluating the curative effect of the perimenopausal syndrome.)

1. Use of an formononetin derivative for the manufacture of a medicament for the treatment or prevention of perimenopausal syndrome, said formononetin derivative having the structure shown in formula I:

in formula I:

R₁is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group;

R₂is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group;

R₃is 1-pyrrolidinyl, (3-methyl) pyridylalkyl, C_1-3Alkyl-pyridyl, (4-acetyl) -piperazinyl, C_1-3Alkyl-piperazinyl, C_1-3Alkyl-4- (acetyl) piperazinyl, 2- (indol-3-yl) ethylamino, formyl, C_1-3Alkyl-phenethylamino, C_1-3Alkyl-benzylamino group, C_1-3Alkyl-amino acid residues, N' -bis (3-aminopropyl) -1, 4-butanediamine groups, 3, 4-dihydroxybenzylamino, anilino groups, benzylamino, 4-fluoro-benzylamino, 4-trifluoromethylanilino groups, 2- (3, 4-dihydroxyphenyl) ethylamino groups, isopropylamino groups, cyclohexylamino groups, N-hexylamino groups, 2-furanmethylamino groups, N-butylamino groups, 2-hydroxyethylamino groups, 3-carboxypropylamino groups, 2-amino-4-carboxylic acid anilino groups, 2-chloro-4-nitroanilino groups, 1, 4-butanediamine groups, ureido groups, N- (3-aminopropyl) -1, 4-butanediamine groups or C- (3-aminopropyl) -1, 4-butanediamine groups_1-3Alkyl-di (C)_1-12Alkyl) -amino; wherein said C_1-3The amino acid residue in the alkyl-amino acid residue is connected with the mother nucleus through an N-C bond;

or, R₂And R₃The linkage forms an oxazine ring structure, when the formononetin derivative has a structure represented by formula II:

in formula II: r₄Is C_1-3Alkyl-benzyl, phenyl, C_1-3Alkyl-phenethyl, 4-fluorobenzyl, 3, 4-dihydroxybenzyl, 2- (indol-3-yl) ethyl, 2- (3, 4-dihydroxyphenyl) ethyl, cyclohexyl, 2-furylmethyl, isopropyl, 2-amino-4-carboxylic acid phenyl, 2-pyridyl, 4-nitro-2-chlorophenyl, 4- (trifluoromethyl) phenyl, 3-carboxypropyl, n-propyl, n-butyl, 2-hydroxyethyl, C_1-3Alkyl-di (C)_1-12Alkyl), 1-amino-4, 9-diazododecyl or 1-amino-5-azooctane.

2. The use of claim 1, wherein the medicament comprises an formononetin derivative and at least one pharmaceutically acceptable carrier or adjuvant.

3. The use of claim 1, wherein the medicament is a tablet, powder, capsule, granule, suspension, soft extract, syrup, elixir, liniment or injection.

4. The use according to claim 1, 2 or 3, wherein the daily dose of the formononetin derivative is 0.2-150 mg/kg body weight.

5. Use according to claim 1, wherein R is₁Is hydrogen, hydroxyl, methoxy or acetoxy.

6. Use according to claim 1, wherein R is₂Is hydrogen, hydroxyl, methoxy or acetoxy.

7. The use of claim 1, wherein said formononetin derivative has the structure of formula I-1, I-2, II-1 or II-2:

in the formulae I-1 and I-2, R₃The same as in claim 1; in the formulae II-1 and II-2, R₄The same as in claim 1.

8. The use according to claim 1, wherein when the formononetin derivative has the structure of formula I, the preparation method of the formononetin derivative comprises the following steps:

mixing a compound with a structure shown in a formula a, formaldehyde and a first amino compound for reaction to obtain a compound with a structure shown in a formula I; the molar ratio of the compound with the structure shown in the formula a, formaldehyde and the first amine compound is 1:1: 1; the structure of the first amine compound comprises a primary amine group or a secondary amine group and R₃A group;

when the formononetin derivative has a structure shown as a formula II, the preparation method of the formononetin derivative comprises the following steps:

mixing a compound with a structure shown in a formula b, formaldehyde and a second amino compound for reaction to obtain an formononetin derivative with a structure shown in a formula II; the molar ratio of the compound having the structure shown in the formula b, formaldehyde and the second amino compound is 1:2: 1; the second amino compound has a structure comprising a primary amine group and R₄A group;

r in the formulas a and b₁、R₂Group and formulaAnd I is consistent.

9. Use of an formononetin derivative having a structure represented by formula I or formula II of claim 1 for the preparation of a reagent for studying the mechanism of perimenopausal syndrome, or for the preparation of a reagent for evaluating perimenopausal syndrome.

Technical Field

The invention relates to the technical field of drug synthesis, in particular to application of an formononetin derivative in preparation of a drug for treating or preventing perimenopausal syndrome.

Background

Perimenopause syndrome (Perimenopausal syndrome) refers to a series of syndromes, mainly metabolic disorders, of vegetative nerve functional disturbance caused by the gradual decline or loss of ovarian function and the reduction of estrogen level in women during or after perimenopause. After a female enters menopause, the estrogen level in the body is obviously reduced, thereby causing the imbalance of systems such as nerves, spirit, psychology, internal secretion, metabolism and the like, and perimenopausal syndrome appears, which is specifically manifested as hot flashes, insomnia, dysphoria, osteoporosis, endometrial atrophy, cardiovascular and cerebrovascular diseases, metabolic syndrome, breast cancer, endometrial cancer and the like.

Medical research finds that estrogen replacement therapy can effectively relieve menopausal symptoms and urogenital symptoms caused by low estrogen, and improve the life quality of postmenopausal women. However, long-term application of estrogen can easily cause side effects such as high blood coagulation state, hypertension, edema, dementia and the like, and increase the risk of the onset of gynecological tumors such as breast cancer, endometrial cancer and the like. In view of the risk of estrogen replacement therapy, efforts have been directed to the discovery of estrogen substitutes that both alleviate the climacteric syndrome and exert the estrogen's protective effect on the respective system, while avoiding the above-mentioned side effects. Soybean isoflavone compounds are typical phytoestrogens, including glycitein, formononetin, ipriflavone, genistein, and glycitin. At present, the phytoestrogens compounds are applied less in the medicines for resisting the perimenopausal syndrome, so that the discovery of new compounds has important significance for the diagnosis and treatment of the perimenopausal syndrome based on the existing medicines.

Disclosure of Invention

In view of the above, the invention provides an application of formononetin derivatives in preparing a medicine for treating or preventing perimenopausal syndrome. The invention provides a derivative taking formononetin (formononetin) as a mother nucleus based on natural phytoestrogen, and the derivative has wide prospect in treating or preventing perimenopausal syndrome.

In order to achieve the above object, the present invention provides the following technical solutions:

use of an formononetin derivative for the manufacture of a medicament for the treatment or prevention of perimenopausal syndrome, said formononetin derivative having the structure shown in formula I:

in formula I:

R₁is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group;

R₂is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group;

R₃is 1-pyrrolidinyl, (3-methyl) pyridylalkyl, C_1-3Alkyl-pyridyl, (4-acetyl) -piperazinyl, C_1-3Alkyl-piperazinyl, C_1-3Alkyl-4- (acetyl) piperazinyl, 2- (indol-3-yl) ethylamino, formyl, C_1-3Alkyl-phenethylamino, C_1-3Alkyl-benzylamino group, C_1-3Alkyl-amino acid residues, N' -bis (3-aminopropyl) -1, 4-butanediamine groups, 3, 4-dihydroxybenzylamino, anilino groups, benzylamino, 4-fluoro-benzylamino, 4-trifluoromethylanilino groups, 2- (3, 4-dihydroxyphenyl) ethylamino groups, isopropylamino groups, cyclohexylamino groups, N-hexylamino groups, 2-furanmethylamino groups, N-butylamino groups, 2-hydroxyethylamino groups, 3-carboxypropylamino groups, 2-amino-4-carboxylic acid anilino groups, 2-chloro-4-nitroanilino groups, 1, 4-butanediamine groups, ureido groups, N- (3-aminopropyl) -1, 4-butanediamine groups or C- (3-aminopropyl) -1, 4-butanediamine groups_1-3Alkyl-di (C)_1-12Alkyl) -amino; wherein said C_1-3The amino acid residue in the alkyl-amino acid residue is connected with the mother nucleus through an N-C bond;

or, R₂And R₃The linkage forms an oxazine ring structure, when the formononetin derivative has a structure represented by formula II:

in formula II: r₄Is C_1-3Alkyl-benzyl, phenyl, C_1-3Alkyl-phenethyl, 4-fluorobenzyl, 3, 4-dihydroxybenzyl, 2- (indol-3-yl) ethyl, 2- (3, 4-dihydroxyphenyl) ethyl, cyclohexyl, 2-furylmethyl, isopropyl, 2-amino-4-carboxylic acid phenyl, 2-pyridyl, 4-nitro-2-chlorophenyl, 4- (trifluoromethyl) phenyl, 3-carboxypropyl, n-propyl, n-butyl, 2-hydroxyethyl, C_1-3Alkyl-di (C)_1-12Alkyl), 1-amino-4, 9-diazododecyl or 1-amino-5-azooctane.

Preferably, the medicament comprises an formononetin derivative and at least one medicinal carrier or auxiliary agent.

Preferably, the medicament is a tablet, powder, capsule, granule, suspension, soft extract, syrup, elixir, liniment or injection.

Preferably, the daily dosage of the formononetin derivative is 0.2-150 mg/kg of body weight.

Preferably, said R is₁Is hydrogen, hydroxyl, methoxy or acetoxy.

Preferably, said R is₂Is hydrogen, hydroxyl, methoxy or acetoxy.

Preferably, the formononetin derivative has a structure represented by formula I-1, I-2, II-1 or II-2:

in the formulae I-1 and I-2, R₃The same as in claim 1; in the formulae II-1 and II-2, R₄The same as in claim 1.

Preferably, when the formononetin derivative has a structure shown in formula I, the preparation method of the formononetin derivative comprises the following steps:

mixing a compound with a structure shown in a formula a, formaldehyde and a first amino compound for reaction to obtain a compound with a structure shown in a formula I; the molar ratio of the compound with the structure shown in the formula a, formaldehyde and the first amine compound is 1:1: 1; the structure of the first amine compound comprises a primary amine group or a secondary amine group and R₃A group;

when the formononetin derivative has a structure shown as a formula II, the preparation method of the formononetin derivative comprises the following steps:

mixing a compound with a structure shown in a formula b, formaldehyde and a second amino compound for reaction to obtain an formononetin derivative with a structure shown in a formula II; the molar ratio of the compound having the structure shown in the formula b, formaldehyde and the second amino compound is 1:2: 1; the second amino compound has a structure comprising a primary amine group and R₄A group;

r in the formulas a and b₁、R₂The radicals are identical to those in formula I.

The invention also provides application of the formononetin derivative in preparing a reagent for researching a perimenopausal syndrome mechanism and a reagent for evaluating the perimenopausal syndrome, wherein the formononetin derivative has a structure shown in a formula I or a formula II in the scheme.

The invention provides an application of an formononetin derivative in preparing a medicine for treating or preventing perimenopausal syndrome, wherein the formononetin derivative has a structure shown in a formula I or a formula II. The formononetin derivative can regulate the in vivo estrogen balance of individuals with estrogen imbalance, relieve perimenopausal syndrome, increase the thickness of endometrium or the weight of uterus of individuals with uterine atrophy, regulate the balance of osteogenesis and osteoclast of individuals with osteoclast abnormality in bone tissues, and further improve the symptoms of osteoporosis and the like caused by the perimenopausal syndrome. Meanwhile, the formononetin derivative has the advantages of good stability, suitability for patent medicine, good biological activity and low toxicity, is applied to the development of perimenopausal syndrome resistant medicines or is used for preparing reagents for researching the pathological mechanism of the perimenopausal syndrome and evaluating the curative effect of the perimenopausal syndrome, and has wide application prospect.

According to the invention, an oophorectomy osteoporosis model mouse is selected to observe the improvement condition of the formononetin derivative on osteoporosis, and the results show that F11 and F13 can both obviously increase bone density, increase the ratio of bone surface area to tissue volume, and increase relative bone volume and bone volume fraction; the invention also observes the influence of the formononetin derivative on a mouse hormone imbalance model caused by ovary removal, and the result shows that F11 and F13 can obviously correct the hormone deficiency of a mouse; further pathological histological observation of a mouse uterine atrophy model caused by ovary removal shows that F11 and F13 can obviously resist endometrial atrophy of a model mouse and increase the thickness of an endometrium. The experimental results show that the formononetin derivative can be safely used for preparing medicines for treating perimenopausal syndrome and intervening osteoporosis and uterine atrophy caused by the perimenopausal syndrome.

Drawings

FIG. 1 is a graph showing the effect of formononetin derivatives on bone density in ovariectomized mice in example 7;

FIG. 2 is a graph of the effect of formononetin derivatives on bone volume fraction in ovariectomized mice of example 8;

FIG. 3 is a graph of the effect of formononetin derivatives on the ratio of bone surface area to shaft volume in ovariectomized mice in example 9;

FIG. 4 is a graph showing the effect of formononetin derivatives on uterine coefficients in ovariectomized mice in example 10;

FIG. 5 is a graph showing the effect of the formononetin derivative of example 11 on intrauterine thickness in ovariectomized mice;

FIG. 6 is a graph showing the effect of formononetin derivatives on serum estrogen levels in ovariectomized mice of example 12.

Detailed Description

The invention provides an application of an formononetin derivative in preparing a medicine for treating or preventing perimenopausal syndrome, wherein the formononetin derivative has a structure shown in a formula I:

in formula II:

R₁is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group; said C is_1-12Alkoxy is preferably methoxy, C_1-12The alkyl group is preferably isopropyl;

R₂is hydrogen, hydroxy, acetoxy, C_1-12Alkyl or C_1-12An alkoxy group; said C is_1-12Alkoxy is preferably methoxy, C_1-12The alkyl group is preferably isopropyl;

R₃is 1-pyrrolidinyl, (3-methyl) pyridylalkyl, C_1-3Alkyl-pyridyl, (4-acetyl) -piperazinyl, C_1-3Alkyl-piperazinyl,C_1-3Alkyl-4- (acetyl) piperazinyl, 2- (indol-3-yl) ethylamino, formyl, C_1-3Alkyl-phenethylamino, C_1-3Alkyl-benzylamino group, C_1-3Alkyl-amino acid residue, N' -bis (3-aminopropyl) -1, 4-butanediamine group, 3, 4-dihydroxybenzylamine group, anilino group, benzylamine group, 4-fluoro-benzylamine group, 4-trifluoromethylanilino group, 2- (3, 4-dihydroxyphenyl) ethylamino group, isopropylamino group, cyclohexylamino group, 2-furanmethylamino group, N-butylamino group, 2-hydroxyethylamino group, 3-carboxypropylamino group, 2-amino-4-carboxylic acid anilino group, 2-chloro-4-nitroanilino group, 1, 4-butanediamine group, ureido group, N- (3-aminopropyl) -1, 4-butanediamine group or C_1-3Alkyl-di (C)_1-12Alkyl) -amino; wherein said C_1-3The amino acid residue in the alkyl-amino acid residue is linked to the parent nucleus by an N-C bond;

or, R₂And R₃The linkage forms an oxazine ring structure, when the formononetin derivative has a structure represented by formula II:

in formula II: r₄Is C_1-3Alkyl-benzyl, phenyl, C_1-3Alkyl-phenethyl, 4-fluorobenzyl, 3, 4-dihydroxybenzyl, 2- (indol-3-yl) ethyl, 2- (3, 4-dihydroxyphenyl) ethyl, cyclohexyl, 2-furylmethyl, isopropyl, 2-amino-4-carboxylic acid phenyl, 2-pyridyl, 4-nitro-2-chlorophenyl, 4- (trifluoromethyl) phenyl, 3-carboxypropyl, n-propyl, n-butyl, 2-hydroxyethyl, C_1-3Alkyl-di (C)_1-12Alkyl), 1-amino-4, 9-diazododecyl or 1-amino-5-azooctane.

In the present invention, the formononetin derivative preferably has a structure represented by formula I-1, I-2, II-1 or II-2:

formula I-1, formula I-2, formula II-1 and formula II-2: r₃And R₄The kind of the above-mentioned solution is the same.

In the present invention, the derivatives of formula I-1, formula I-2, formula II-1 or formula II-2 are numbered according to different substituents, as shown in Table 1:

TABLE 1 Formononetin derivative Structure and numbering

In the present invention, the preparation method of the formononetin derivative preferably comprises the steps of:

when the formononetin derivative has a structure shown in a formula I, the preparation method comprises the following steps:

mixing a compound with a structure shown in a formula a, formaldehyde and a first amino compound for reaction to obtain a compound with a structure shown in a formula I; the molar ratio of the compound with the structure shown in the formula a, formaldehyde and the first amine compound is 1:1: 1; the structure of the first amine compound comprises a primary amine group orSecondary amine group and R₃A group;

r in the formula a₁、R₂The radicals are identical to those in formula I.

In the invention, the compound with the structure shown in the formula a is specifically and preferably formononetin (the structural formula is shown in a formula a-1) or glycitein (the structural formula is shown in a formula a-2).

In the present invention, the first amino compound is specifically according to R₃The kind of the group is selected, specifically, when the R is₃When formamide, the first amino compound is hexamethylenetetramine, and when R is₃In the case of 1-pyrrolidinyl, the first amino compound is pyrrolidine; when said R is₃(ii) when it is (3-methyl) -pyridylalkyl, the first amino compound is (3-methyl) -pyridylalkyl; when said R is₃(4-acetyl) -piperazine, the first amino compound is (4-acetyl) -piperazine; when said R is₃In the case of 2-furylmethyl, the first amino compound is 2-furanmethanamine; when said R is₃When the amino group is n-butylamino, the first amino compound is n-butylamine; the others are not listed and may be selected according to the common general knowledge of those skilled in the art.

In the present invention, the solvent for reaction is preferably glacial acetic acid or a dimethylsulfoxide-methanol mixed solvent, and the volume ratio of dimethylsulfoxide to methanol in the dimethylsulfoxide-methanol mixed solvent is preferably 1: 4; the formaldehyde is preferably used in the form of an aqueous formaldehyde solution, and the mass fraction of the aqueous formaldehyde solution is preferably 36%; the invention has no special requirement on the source of the formaldehyde aqueous solution, and can adopt a commercial formaldehyde aqueous solution which is well known by the technical personnel in the field; in the specific embodiment of the present invention, it is preferable to use glacial acetic acid as a solvent in the preparation of F1 and a dimethylsulfoxide-methanol mixed solvent as a solvent in the preparation of other compounds.

In the invention, the reaction is preferably carried out under the condition of heating reflux, and the reaction time is preferably 6-12 h; in the embodiment of the present invention, when a dimethylsulfoxide-methanol mixed solvent is used as the reaction solvent, it is preferable that the compound having the structure represented by formula a is first dissolved in dimethylsulfoxide, a mixed solution of methanol, an aqueous formaldehyde solution and the first amino compound is added dropwise to the solution of the compound of formula a, stirred and mixed uniformly at room temperature, and then heated to reflux temperature to carry out the reaction.

In the present invention, the reaction formula for preparing an formononetin derivative having a structure represented by formula I is as follows:

after the reaction is finished, the invention preferably carries out post-treatment on the obtained product liquid to obtain the compound with the structure shown in the formula I. In the present invention, when glacial acetic acid is used as a solvent, the post-treatment preferably comprises the following steps: adding hydrochloric acid into the obtained product liquid, stirring for 5min, cooling to room temperature, adding water to obtain a precipitate, collecting the precipitate to obtain a crude product, and carrying out silica gel column chromatography separation on the crude product to obtain the compound with the structure shown in the formula I.

When a mixed solvent of dimethyl sulfoxide and methanol is used as a solvent, the post-treatment preferably comprises the following steps: evaporating the obtained product liquid under reduced pressure to remove the solvent, separating out solids, filtering the residual product to obtain a crude product, and carrying out silica gel column chromatography separation on the crude product to obtain a compound with a structure shown in a formula I; the reagent adopted by the silica gel column chromatography separation is a petroleum ether-ethyl acetate mixed reagent or a petroleum ether-methanol-ethyl acetate mixed reagent, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed reagent is preferably 2:1, and the volume ratio of petroleum ether, methanol and ethyl acetate in the petroleum ether-methanol-ethyl acetate mixed reagent is preferably 2:1: 1.

In the present invention, when the formononetin derivative has a structure represented by formula II, the preparation method includes the steps of:

mixing a compound with a structure shown in a formula b, formaldehyde and a second amino compound for reaction to obtain an formononetin derivative with a structure shown in a formula II; the molar ratio of the compound having the structure shown in the formula b, formaldehyde and the second amino compound is 1:2: 1; the second amino compound has a structure comprising a primary amine group and R₄A group;

r in the formula b₁The radicals are identical to those in formula I.

In the invention, the compound with the structure shown in the formula b is particularly preferably formononetin or daidzein, and the structural formula is shown as above.

In the present invention, the second amino compound is specifically according to R₄The kind of the group is selected, specifically, when the R is₄When it is 2-furylmethyl, said second amino compound is 2-furylmethylamine, when said R is₄When the alkyl group is n-butyl, the second amino compound is n-butylamine; when said R is₄When benzyl, the second amino compound is benzylamine; when said R is₄In the case of 2- (indol-3-yl) ethyl, the second amino compound is tryptamine; the others are not listed and may be selected according to the common general knowledge of those skilled in the art.

In the present invention, the reaction formula for preparing an formononetin derivative having a structure represented by formula II is as follows:

in the invention, when the formononetin derivative has the structure shown in the formula II, the solvent for reaction, the specific operation method and the reaction conditions are the same as those of the preparation of the formononetin derivative with the structure shown in the formula I, and the specific conditions are not repeated.

The reaction principle of the invention is illustrated below by way of example with F11 and F13:

in the preparation of F11 and F13, the compound with the structure shown in the formula b is formononetin with the structural formula shown in a formula a-1, and F11 and F13 have the following structures:

in the reaction process, Mannich reaction is carried out on the active H at the 8-C position of the ring A of the formononetin and aliphatic primary/secondary amine or aromatic primary/secondary amine in a formaldehyde water solution, and a Mannich alkali derivative is synthesized; in the Mannich reaction of primary amine, for example, when the molar ratio of benzylamine or p-methylaniline to formaldehyde is 1:2, the reaction product Mannich base further reacts with formaldehyde to generate an N-substituted hydroxymethyl compound, and then the N-substituted hydroxymethyl compound is dehydrated and cyclized to obtain the dihydrobenzoxazine derivative containing a six-membered ring. Generating the formononetin derivative with linear alkyl or substituted linear alkyl, cycloalkyl, five-membered or six-membered heterocyclic ring, benzene ring or substituted benzene ring and amino according to different substituent groups.

In the present invention, the medicament comprises an formononetin derivative and at least one pharmaceutically acceptable carrier or auxiliary agent. In the present invention, the pharmaceutically acceptable carrier specifically refers to a pharmaceutically acceptable carrier, a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier and lubricant; in particular, the excipient is preferably water; the filler is preferably starch and/or sucrose; the adhesive is preferably one or more of cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone; the humectant is preferably glycerin; the disintegrating agent is preferably one or more of agar, calcium carbonate and sodium bicarbonate; the absorption enhancer is preferably a quaternary ammonium compound; the surfactant is preferably cetyl alcohol; the adsorption carrier is preferably kaolin and/or bentonite; the lubricant is preferably one or more of talcum powder, calcium stearate, magnesium stearate and polyethylene glycol.

The auxiliary agent is not particularly required in the invention, and medicinal auxiliary agents well known to those skilled in the art can be adopted, such as flavoring agents and sweetening agents.

The formononetin derivative medicine can be applied to a patient needing treatment by means of oral administration, nasal inhalation, rectal administration or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, capsule, granule, suspension, soft extract, liniment or injection, and liquid preparations such as water, oil suspension, syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.; in a particular embodiment of the invention, the form of the medicament is preferably tablets, capsules and injections.

The invention has no special requirements on the preparation method of the preparation of the various derivatives, and the preparation method is prepared according to the conventional production method in the pharmaceutical field, for example, the active ingredient is mixed with one or more carriers and then is prepared into the required preparation.

In a specific embodiment of the present invention, the amount of the formononetin derivative to be administered may be determined according to the route of administration, the age, weight, type and severity of the disease to be treated, etc., and specifically, the daily dose of the formononetin derivative is preferably 0.2 to 150mg/kg body weight, more preferably 0.5 to 100mg/kg body weight, and may be administered once or more.

The invention also provides application of the formononetin derivative in the scheme in preparing a reagent for researching a perimenopausal syndrome mechanism and a reagent for evaluating the perimenopausal syndrome, wherein the reagent is specifically used for:

a) researching the mechanism and the drug activity mechanism of the soybean isoflavone Mannich reaction;

b) studying the pathological mechanism of perimenopausal syndrome;

c) the effect against perimenopausal syndrome was evaluated.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1: preparation of F1

Dissolving formononetin 134.130mg (0.5mmol) and hexamethylenetetramine 70mg (0.5mmol) in 6mL of glacial acetic acid, stirring at room temperature until completely dissolved, heating and refluxing for 6h, keeping the temperature at 100 ℃, rapidly adding 2mL of 20% hydrochloric acid, stirring for 5min, cooling to room temperature, adding 10mL of water to obtain a brown yellow precipitate, collecting the brown yellow precipitate to obtain a crude product, separating by silica gel column chromatography, wherein the adopted reagent is petroleum ether-ethyl acetate, the volume ratio of the petroleum ether to the ethyl acetate is 2:1, and R is_f0.11, giving the product F163.5 mg, 47.31% yield.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，DMSO-D6)δ10.48(s，1H)，8.48(s，1H)，8.23(d，1H)，7.52(dd，2H)，7.12(dd，2H)，7.01(d，1H)，3.88(s，3H).

¹³C-NMR(400MHz，DMSO-D6)δ190.32，174.39，166.36，159.66，157.26，153.51，134.05，130.55，124.60，123.95，116.95，116.38，114.15，112.00，55.46.

mass spectrum (M + H)⁺)：297.1

The molecular formula is as follows: c₁₇H₁₃O₅

Example 2: preparation of F2

Completely dissolving formononetin 67.065mg (0.25mmol) in 1mL DMSO, slowly adding dropwise the prepared mixture [4mL methanol, 22.5. mu.L 36% formaldehyde (0.25mmol) and 0.042mL pyrrolidine (0.25mmol)]Stirring at room temperature for 2h, refluxing for 10h, distilling off solvent under reduced pressure to obtain light red solid, collecting light red solid to obtain crude product, separating with silica gel column chromatography, wherein the adopted reagent is petroleum ether-methanol-ethyl acetate, the volume ratio of petroleum ether, methanol and ethyl acetate is 2:1:1, R_f0.108, product F2 was obtained in 7.25% yield.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，DMSO-D6)δ8.35(s，1H)，7.91(d，1H)，7.49(d，2H)，7.00(d，2H)，6.85(d，1H)，4.11(s，3H)，3.78(s，2H)，2.71(m，4H)，1.80(m，4H).

¹³C-NMR(400MHz，DMSO-D6)δ23.65，49.86，53.51，55.61，109.16，114.06，115.84，116.13，123.40，124.73，126.14，130.56，153.18，155.32，159.41，164.51，175.16.

mass spectrum (M + H)⁺)：352.1579

The molecular formula is as follows: c₂₂H₂₄NO₄

Example 3: preparation of F3

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1mL DMSO, slowly dropwise adding the prepared mixed solution [4mL methanol, 22.5 mu L36% formaldehyde (0.25mmol) and 24.75mg 3-methylpyridine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, evaporating the solvent under reduced pressure to obtain a light yellow solid, collecting the light yellow solid to obtain a crude product, and carrying out silica gel column chromatography to obtain a product F3 with the yield of 23.58%.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，DMSO-D6)δ8.37(s，1H)，7.90(d，1H)，7.52(d，2H)，7.01(d，1H)，6.88(d，2H)，3.79(s，3H)，3.62(s，2H)，2.19-2.51(dm，4H)，1.69(dm，4H)，1.53(m，1H)，0.87(d，3H).

¹³C-NMR(400MHz，DMSO-D6)δ19.65，25.01，49.06，53.09，55.61，60.50，108.24，109.59，114.07，115.71，123.46，124.65，126.20，130.56，153.29，159.41，164.25，164.92，175.20.

mass spectrum (M + H)⁺)：380.1346

The molecular formula is as follows: c₂₃H₂₆NO₄

Example 4: preparation of F4

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1mL DMSO, slowly dropwise adding the prepared mixed solution [4mL methanol, 22.5. mu.l 36% formaldehyde (0.25mmol) and 32mg (4-acetyl) -piperazine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, evaporating the solvent under reduced pressure to obtain a light yellow solid, collecting the light yellow solid to obtain a crude product, and separating by silica gel column chromatography to obtain the product F4 with the yield of 28.37%.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，DMSO-D6)δ8.38(s，1H)，7.92(d，1H)，7.52(d，2H)，6.99(d，1H)，6.97(d，2H)，3.88(s，2H)，3.78(s，3H)，3.44(m，4H)，2.47-2.54(m，4H)，1.98(s，3H).

¹³C-NMR(400MHz，DMSO-D6)δ21.61，50.91，52.52，52.91，55.60，109.71，114.07，115.33，116.88，123.40，124.64，126.34，130.53，153.45，156.06，159.42，162.70，168.61，175.28.

mass spectrum (M + H)⁺)：409.1720

The molecular formula is as follows: c₂₃H₂₅N₂O₅

Example 5: preparation of F11

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1mL DMSO, slowly dropwise adding the prepared mixed solution [4mL methanol, 45 mu L36% formaldehyde (0.5mmol) and 24.25mg 2-furanmethanamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, evaporating the solvent under reduced pressure to obtain a brown solid, collecting the brown solid to obtain a crude product, and carrying out silica gel column chromatography to obtain a product F11 with the yield of 18.55%.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，CDCl₃)δ8.05(s，1H)，7.53(d，1H)，7.13(dd，2H)，6.64(d，2H)，6.60(d，1H)，6.06(m，1H)，6.01(d，1H)，4.66(s，2H)，3.78(s，2H)，3.54(s，2H)，3.42(s，3H).

¹³C-NMR(400MHz，DMSO-D6)δ43.87，48.22，55.61，82.86，108.17，109.47，110.96，114.10，115.55，117.77，123.91，124.47，124.88，130.56，143.35，151.88，153.58，154.62，158.56，159.50，175.30.

mass spectrum (M + H)⁺)：390.0816

The molecular formula is as follows: c₂₃H₁₉NO₅

Example 6: preparation of F13

Completely dissolving 67.065mg (0.25mmol) of formononetin in 1mL of DMSO, slowly dropwise adding the prepared mixed solution [4mL of methanol, 45 mu L of 36% formaldehyde (0.5mmol) and 18.25mg of n-butylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, and evaporating the solvent under reduced pressure to obtain a light brown solid, collecting the light brown solid to obtain a crude product, and carrying out silica gel column chromatography to obtain a product F13 with the yield of 36.82%.

The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data of the product are as follows:

¹H-NMR(400MHz，CDCl₃)δ8.08(s，1H)，7.91(d，1H)，7.50(d，2H)，6.97(d，2H)，6.87(d，1H)，4.96(s，2H)，4.17(s，2H)，3.83(s，3H)，2.75(t，2H)，1.58(m，2H)，1.38(m，2H)，0.94(t，3H).

¹³C-NMR(400MHz，CDCl₃)δ13.96，20.30，30.23，45.01，51.51，55.34，83.21，107.73，113.97，115.21，117.96，124.21，124.93，125.24，130.15，151.60，154.69，158.67，159.59.

mass spectrum (M + H)⁺)：366.1420

The molecular formula is as follows: c₂₂H₂₃NO₄

Example 7: effect on bone Density (BMD/BV) in ovariectomized mice

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. Postoperative recovery breeding is carried out for 7 days, and animals with good states are selected for grouping; f11 and F13 were suspended in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and gavage, the dosage for intraperitoneal injection of F11 and F13 was 10mg/kg, and the dosage for gavage was 20mg/kg, the mice were sacrificed after 12 weeks, and femurs were taken for microscopic CT scan analysis.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The effect of the formononetin derivative of the present invention on the bone density of ovariectomized mice is shown in FIG. 1, wherein Sham in FIG. 1 represents a Sham group, Model represents a Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 1 show that: after intraperitoneal injection or intragastric administration for 12 consecutive weeks of F11 and F13, the bone density of mice is obviously increased, and the effect is superior to that of ipriflavone and estradiol, which shows that the formononetin derivative can inhibit the formation of osteoporosis in the perimenopausal syndrome by increasing the bone density of the mice, so as to improve the perimenopausal syndrome.

Example 8: effect on bone volume fraction (BV/TV) in ovariectomized mice

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. And (3) recovering and breeding for 7 days after operation, selecting animals with good states for grouping, dissolving F11 and F13 in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and intragastric administration, wherein the dosage of intraperitoneal injection F11 and F13 is 10mg/kg, the dosage of intragastric administration is 20mg/kg, continuously carrying out 12 weeks, killing the mice after the expiration, and taking thighbones for carrying out micro CT scanning to analyze bone density.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The influence of the formononetin derivatives on the bone volume fraction of ovariectomized mice is shown in FIG. 2, wherein Sham in FIG. 2 represents a Sham surgery group, Model represents a Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 2 show that: after intraperitoneal injection and gastric lavage for 12 consecutive weeks of F11 and F13, the bone body number of the mice is obviously increased, which shows that the formononetin derivative can inhibit the formation of osteoporosis in the perimenopausal syndrome through the bone volume fraction, thereby improving the perimenopausal syndrome.

Example 9: effect on the bone surface area to bone volume ratio (BS/BV) in ovariectomized mice

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. And (3) recovering and breeding for 7 days after operation, selecting animals with good states for grouping, dissolving F11 and F13 in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and intragastric administration, wherein the dosage of intraperitoneal injection F11 and F13 is 10mg/kg, the dosage of intragastric administration is 20mg/kg, continuously carrying out 12 weeks, killing the mice after the expiration, and taking thighbones for carrying out micro CT scanning to analyze bone density.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The effect of formononetin derivatives of the present invention on the ratio of bone surface area to bone volume of ovariectomized mice is shown in FIG. 3, where Sham in FIG. 3 represents a Sham group, Model represents a Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 3 show that: after intraperitoneal injection and gastric lavage for 12 consecutive weeks of F11 and F13, the ratio of the bone surface area to the bone volume of the mice is obviously increased, which shows that the formononetin derivative can relieve the formation of osteoporosis by increasing the ratio of the bone surface area to the bone volume of the mice, thereby improving the perimenopausal syndrome.

Example 10: influence on uterine coefficient of ovariectomized mice

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. And (3) recovering and breeding for 7 days after operation, selecting animals with good states for grouping, dissolving F11 and F13 in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and intragastric administration, wherein the dosage of intraperitoneal injection F11 and F13 is 10mg/kg, the dosage of intragastric administration is 20mg/kg, continuously carrying out 12 weeks, killing the mice after the expiration, and weighing uterus.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The effect of the formononetin derivatives of the present invention on uterine coefficients of ovariectomized mice is shown in FIG. 4, where Sham in FIG. 4 represents a Sham group, Model represents a Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 4 show that: after intraperitoneal injection and gastric lavage for 12 consecutive weeks of F11 and F13, the coefficient of uterus of mice is obviously increased, which shows that the formononetin derivative can improve perimenopausal syndrome by increasing the weight of the uterus of the mice to shrink the uterus in the perimenopausal period.

Example 11: effect on ovariectomized mouse endometrium

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. And (3) recovering and breeding for 7 days after operation, selecting animals with good states for grouping, dissolving F11 and F13 in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and intragastric administration, wherein the dosage of intraperitoneal injection F11 and F13 is 10mg/kg, the dosage of intragastric administration is 20mg/kg, continuously carrying out 12 weeks, killing the mice after the expiration, and taking the uterus for histopathological section analysis.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The effect of the formononetin derivatives of the present invention on intrauterine thickness removal in mice with ovaries removed is shown in FIG. 5, Sham in FIG. 4 represents a Sham group, Model represents a Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 5 show that: after intraperitoneal injection and gastric perfusion administration for 12 consecutive weeks of F11 and F13, the number of endometrial glands of a mouse is obviously increased, the cavity of the gland is larger, the endometrium is obviously thickened and recovered to a normal level, the number of glands is increased, the cavity of the gland is enlarged, the intercellular space is enlarged, interstitial cells mainly take a round shape, a small part of the interstitial cells are fusiform, the blood vessels of the interstitial cells are rich, and glandular epithelial cells are in a single-layer columnar shape.

Example 12: effect on serum Estrogen (E) levels in ovariectomized mice

Selecting SPF female ICR mouse from Kunming medical university, anesthetizing, fixing on operating table, sterilizing abdominal skin, making an incision on the side of the leucorrhea to fully expose uterus, cutting ovaries on both sides, and suturing layer by layer with suture. The sham group removed adipose tissue around the ovaries of the same size as the ovaries. And (3) recovering and breeding for 7 days after operation, selecting animals with good states for grouping, dissolving F11 and F13 in 0.5% sodium carboxymethylcellulose for intraperitoneal injection and intragastric administration, wherein the dosage of intraperitoneal injection F11 and F13 is 10mg/kg, the dosage of intragastric administration is 20mg/kg, continuously carrying out 12 weeks, taking blood of the mice after the expiration period, separating serum, and determining the content of estrogen in the serum.

And simultaneously setting a model group, estradiol group and ipriflavone group for comparison, wherein administration is carried out on the mice with ovariectomized, the model group is that 0.5 percent of sodium carboxymethylcellulose is administrated by intragastric administration, the intragastric administration volume is 10mL/kg, the intragastric administration is adopted for the estradiol group, the administration amount is 1.5mg/kg, the ipriflavone is administrated by intraperitoneal injection and intragastric administration, and the administration amount is consistent with the administration amounts of F11 and F13.

The effect of formononetin derivatives of the present invention on the level of estrogen in serum of ovariectomized mice is shown in FIG. 6, where Sham in FIG. 6 represents the Sham group, Model represents the Model, E2 represents estradiol, IP represents ipriflavone, IP represents intraperitoneal injection, and ig represents intragastric administration.

The experimental results in fig. 6 show that: after intraperitoneal injection and gastric lavage for 12 consecutive weeks of F11 and F13, the content of estrogen in the serum of the mice is obviously increased, which shows that the formononetin derivative can improve the hormone imbalance caused by the perimenopause by regulating the estrogen level in the mice, thereby improving the perimenopause syndrome.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.