阅读说明：本技术 一种芒柄花素衍生物及其制备方法和应用 (Formononetin derivative and preparation method and application thereof ) 是由 罗晓东 王兆杰 赵云丽 闫晓君 代智 何英杰 于 2021-09-13 设计创作，主要内容包括：本发明涉及药物合成技术领域,提供了一种芒柄花素衍生物及其制备方法和应用。本发明基于具有抗骨质疏松活性天然植物雌激素,提供了一类以芒柄花素为母核的衍生物；该衍生物对破骨细胞具有抑制活性,同时能促进骨髓间充质干细胞分化为成骨细胞,并激活成骨细胞骨形成能力；可以促进成骨细胞相关基因的表达,进而改善破骨和成骨的平衡；还能增加骨密度异常个体中的骨密度的水平,升高骨体积分数异常个体中的骨体积分数,调整骨表面积与骨体积比值异常个体中的骨表面积与骨体积比值,在制备预防或治疗成骨细胞异常引起的疾病或病症的药物中具有广阔的应用前景。(The invention relates to the technical field of drug synthesis, and provides an formononetin derivative and a preparation method and application thereof. The invention provides a derivative taking formononetin as a mother nucleus based on natural phytoestrogen with anti-osteoporosis activity; the derivative has inhibitory activity on osteoclast, and can promote bone marrow mesenchymal stem cells to differentiate into osteoblast and activate bone formation ability of the osteoblast; can promote the expression of osteoblast related genes, thereby improving the balance of osteoclast and osteogenesis; the compound preparation can also increase the level of bone density in individuals with abnormal bone density, increase the number of bone volume in individuals with abnormal bone volume number, and adjust the ratio of the bone surface area to the bone volume in individuals with abnormal ratio of the bone surface area to the bone volume, and has wide application prospect in preparing medicaments for preventing or treating diseases or symptoms caused by abnormal osteoblasts.)

1. An formononetin derivative, characterized by 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 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, amino acid, 2- (4-2- (3, 4-bis (4-dihydroxybenzylamino acid, 2-4-dihydroxybenzylamino, 2-4-trifluoromethylanilino-benzylamino, 2-4-benzylamino, 2-1, 4-benzylamino, 2-dihydroxybenzylamino, 2-benzylamino, 4-benzylamino, 2-benzylamino, 4-benzylamino, 2-benzylamino, or-4-1, or-benzylamino, 2-4-benzylamino, or-4-1, or-1-4-1-dihydroxybenzylamino,2-furanmethylamino, N-butylamino, 2-hydroxyethylamino, 3-carboxypropylamino, 2-amino-4-carboxylic acid anilino, 2-chloro-4-nitroanilino, 1, 4-butanediamine, ureido, N- (3-aminopropyl) -1, 4-butanediamine or C_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 formononetin derivative of claim 1, wherein R is₁Is hydrogen, hydroxyl, methoxy or acetoxy.

3. The formononetin derivative of claim 1, wherein R is₂Is hydrogen, hydroxyl, methoxy or acetoxy.

4. An formononetin derivative according to any of claims 1 to 3, characterized in that it has the structure represented by formula I-1, I-2, II-1 or II-2:

in the formulae I-1 and I-2, R₃Of the formula (I) as in claim 1, formula (II-1) and formula (II-2): r₄The same as in claim 1.

5. A method of preparing an formononetin derivative according to any one of claims 1 to 4, characterized in that when the formononetin derivative has a structure represented by formula I, the 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 or a secondary amine group and R₃A group;

when the formononetin derivative has a structure shown in a formula II, the preparation method 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.

6. Use of the formononetin derivative of any one of claims 1 to 4 or the formononetin derivative prepared by the preparation method of claim 5 in preparation of a medicament for preventing or treating diseases or disorders caused by osteoblast abnormality, preparation of a reagent for studying pathological mechanism of osteoporosis, and preparation of a reagent for evaluating curative effect of osteoporosis.

7. The use according to claim 6, wherein the medicament comprises an formononetin derivative according to any one of claims 1 to 4 or prepared by the preparation method of claim 5 and at least one pharmaceutically acceptable carrier or auxiliary agent.

8. The use according to claim 6 or 7, wherein the medicament is a tablet, powder, capsule, granule, suspension, ointment, syrup, elixir, liniment or injection.

9. The use of claim 8, wherein the daily dose of the formononetin derivative is 0.01 to 100mg/kg body weight.

Technical Field

The invention relates to the technical field of drug synthesis, in particular to an formononetin derivative and a preparation method and application thereof.

Background

Osteoporosis (osteoporotis) is a systemic bone disease in which bone fracture is easily caused by a decrease in bone density and bone mass due to various causes, a destruction of bone microarchitecture, and an increase in bone fragility. Osteoporosis is divided into primary and secondary categories. Primary osteoporosis is mostly seen in the elderly and postmenopausal women, idiopathic osteoporosis has not yet been clearly defined at present, and secondary osteoporosis is caused by other diseases or medicines and the like. Normal bone metabolism is in balance between bone formation (osteoblasts) and bone loss (osteoclasts), which complement and cooperate to play an important role in the development and formation of bone. Age, estrogen deprivation and drug intervention, leading to decreased osteoblast activity, increased osteoclast activity, and a much slower rate of bone formation than bone loss, thus leading to decreased bone density, decreased bone mass and altered bone mass (including changes in both macro and micro structure and altered bone properties), leading to systemic metabolic bone diseases with increased bone fragility and susceptibility to fracture.

Osteoporosis is one of the common chronic diseases, and is also the most common bone disease of the middle-aged and elderly people. Investigation has shown that about 2.1 million people in our country have low bone mass, and nearly 7000 million patients with osteoporosis. Approximately 1/3 women and 1/5 men worldwide suffer from osteoporotic fractures, which occur every 3 seconds. Thus, osteoporosis is known as an invisible killer.

Therapeutic agents for osteoporosis are mainly of 3 types: drugs for inhibiting bone resorption (bisphosphonates, calcitonin compounds, estrogens and selective estrogen receptor modulators) singly inhibit osteoclast activity and reduce bone resorption, but have strong toxic and side effects on metabolic organs (such as kidney); medicines (parathyroid hormones) for promoting bone formation, promoting bone calcium to enter blood, increasing blood calcium content, promoting bone calcium absorption and formation, but causing the side effect of hypercalcemia; the multiple action mechanism medicine (strontium salt, active vitamin D and analogues thereof, vitamin K2) can promote bone formation and mineralization, inhibit bone absorption, and is beneficial to increase bone density.

There is a continuing effort to find estrogen substitutes that both alleviate osteoporosis and provide estrogen protection to the individual system while avoiding the side effects described above. Soybean isoflavone compounds are typical phytoestrogens, including daidzein, formononetin, ipriflavone, genistein, and glycitin. At present, the application of phytoestrogen compounds in osteoporosis medicines is less, and the discovery of new compounds has important significance for the diagnosis and treatment of osteoporosis based on the existing medicines.

Disclosure of Invention

In view of the above, the invention provides an formononetin derivative, a preparation method and an application thereof. The invention provides a derivative taking formononetin (formononetin) as a mother nucleus based on natural phytoestrogen with anti-osteoporosis activity, and the derivative has wide prospect in treating diseases or symptoms caused by osteoblast abnormality.

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

an formononetin derivative having the structure of 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) pyridylalkaneBase, 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 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, 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, R₃Of the formula (I) as in claim 1, formula (II-1) and formula (II-2): r₄The same as in claim 1.

The invention also provides a preparation method of the formononetin derivative in the scheme, and 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 or a secondary amine group and R₃A group;

when the formononetin derivative has a structure shown in a formula II, the preparation method 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 the scheme or the application of the formononetin derivative prepared by the preparation method in the scheme in preparing a medicine for preventing or treating diseases or symptoms caused by osteoblast abnormality, a reagent for researching a pathological mechanism of osteoporosis and a reagent for evaluating the curative effect of the osteoporosis.

Preferably, the medicament comprises the formononetin derivative in the scheme 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 0.01-100 mg/kg of body weight.

The invention provides an formononetin derivative which has a structure shown in a formula I or a formula II. The design idea of the invention is as follows: the natural phytoestrogen is used as a mother nucleus, derivatives with different substituents are obtained through the reaction of different groups according to the structure-activity relationship of active sites, active groups and the activity of osteoporosis resistance, the biological activity test of the cell level (inhibiting the formation of osteoclasts and promoting the differentiation of mesenchymal stem cells into osteoblasts) is carried out, and the derivatives are screened to obtain a series of active compounds.

The formononetin derivative provided by the invention has inhibitory activity on osteoclast, can promote mesenchymal stem cells to differentiate into osteoblast and activate bone forming ability of the osteoblast; can also promote the expression of osteoblast related genes, such as alkaline phosphatase (ALP), early factor (sp7), Osteocalcin (OP) and insulin-like growth factor (IGF-1), so as to improve the balance between osteoclasts and osteoblasts; the formononetin derivative provided by the invention can also increase the level of bone density in individuals with abnormal bone density, increase the number of bone body numbers in individuals with abnormal bone body numbers, and adjust the ratio of the bone surface area to the bone body volume in individuals with abnormal bone surface area to bone body volume ratio.

The invention also provides a preparation method of the formononetin derivative, the preparation method is characterized in that a branched chain is introduced on a mother nucleus through a Mannich reaction of the mother nucleus active site to obtain the formononetin derivative, and the synthesis steps are simple and easy to operate.

The invention also provides application of the formononetin derivative in the scheme in preparing a medicine for preventing or treating diseases or symptoms caused by osteoblast abnormality, a reagent for researching a pathological mechanism of osteoporosis and a reagent for evaluating the curative effect of osteoporosis. The formononetin derivative provided by the invention can be used for preventing or treating diseases or symptoms caused by osteoblast abnormality such as osteoporosis and hyperosteogeny, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the results of a test that an formononetin derivative promotes differentiation of rat bone marrow Mesenchymal Stem Cells (MSCs) into osteoblasts in example 23;

FIG. 2 is a graph showing the effect of the formononetin derivative of example 24 on the osteogenesis promoting effect of Human Mesenchymal Stem Cells (HMSC);

FIG. 3 is a graph comparing the effect of F11, F13 and a positive control compound on the osteogenesis of Human Mesenchymal Stem Cells (HMSC) in example 24;

FIG. 4 shows the structures of glycitein, formononetin, ipriflavone, genistein, glycitin and estradiol;

FIG. 5 is a graph showing the results of the test for the inhibition of osteoclast differentiation by an formononetin derivative in example 25;

FIG. 6 is a graph showing the results of tests in example 26 in which F11 and F13 promote osteoblast alkaline phosphatase (ALP) gene expression;

FIG. 7 is a graph showing the results of tests in example 27 in which F11 and F13 promote the expression of osteoblast early factor (sp7) genes;

FIG. 8 is a graph showing the results of tests in example 28 in which F11 and F13 promote osteoblastic Osteocalcin (OP) gene expression;

FIG. 9 is a graph showing the results of tests in example 29 in which F11 and F13 promote the expression of the osteoblast insulin-like growth factor (IGF-1) gene.

Detailed Description

The invention provides an formononetin derivative which 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 of 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

The phytoestrogen has estrogen-like action, is a compound which naturally exists in some plants and has estrogen efficacy, and can be combined with estrogen receptors in vivo to simulate and interfere the physiological and biochemical actions of estrogen due to the similar structure of the estrogen. The invention adopts safe natural phytoestrogen with certain anti-osteoporosis activity as a mother nucleus, obtains the structure-activity relationship of active sites, active groups and anti-osteoporosis activity according to the related activity and the structure-activity relationship of the groups, obtains derivatives of different substituents through the reaction of different groups, and performs biological activity tests of cell level and animal level to obtain the derivatives of the types. The derivative provided by the invention can regulate the metabolic abnormality of osteoblasts and osteoclasts, regulate the balance of osteoblasts and osteoclasts of an abnormal individual of the osteoclasts in bone tissues, inhibit the differentiation of the osteoclasts or promote the differentiation of the osteoblasts, and has wide application prospects in diseases caused by the abnormal osteoblasts such as osteoporosis and the like.

The invention also provides a preparation method of the formononetin derivative in the scheme, which comprises the following steps:

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 or a secondary 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 the structure of formula II, the preparation method 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 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.

The invention also provides application of the formononetin derivative in the scheme or the application of the formononetin derivative prepared by the preparation method in the scheme in preparing a medicine for preventing or treating diseases or symptoms caused by osteoblast abnormality, a reagent for researching a pathological mechanism of osteoporosis and a reagent for evaluating the curative effect of the osteoporosis. In the present invention, the disease or disorder caused by osteoblast abnormality is specifically osteoporosis or hyperosteogeny, specifically hyperosteogeny caused by bone loss, hyperosteogeny caused by abnormal metabolism of osteoblasts and osteoclasts, and osteoporosis caused by abnormal metabolism of osteoclasts and imbalance of osteoblasts and osteoclasts.

In the invention, the medicine comprises the formononetin derivative in the scheme or the formononetin derivative prepared by the preparation method in the scheme and at least one medicinal 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 0.01 to 100mg/kg of body weight, more preferably 0.01 to 10mg/kg of 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 pathological mechanism of osteoporosis and a reagent for evaluating the curative effect of the osteoporosis, wherein the reagent is specifically used for:

a) researching the mechanism of the phytoestrogen Mannich reaction and the drug modification design;

b) studying abnormal bone metabolism pathological mechanisms;

c) the anti-osteoporosis effect was evaluated.

Specifically, the agent includes an agent that inhibits differentiation of RAW264.7 cells into osteoclasts; an agent for promoting differentiation of Mesenchymal Stem Cells (MSCs) into osteoblasts.

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 prepared mixture [4ml methanol, 22.5. mu.l 36% formaldehyde (0.25mmol) and 0.042ml pyrrolidine (0.25mmol)]Stirring at room temperature for 2h, reflux reacting for 10h, distilling off solvent under reduced pressure to obtain light red solid, collecting light red solid to obtain crude product, separating by silica gel column chromatography using petroleum ether-methanol-ethyl acetate, petroleum ether, methanol and ethyl acetate as reagentsThe volume ratio 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 67.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 24.75mg of 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 67.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 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 carrying out silica gel column chromatography to obtain a 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.l 36% 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: preparation of Fa1

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu.l 36% formaldehyde (0.5mmol) and 26.62mg benzylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, 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 Fa1 with the yield of 13.43%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 1.

Example 8: preparation of Fa2

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu.l 36% formaldehyde (0.5mmol) and 31.25mg 4-fluoro-benzylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, evaporating the solvent under reduced pressure to obtain a light brown solid, collecting the light brown solid to obtain a crude product, and separating by silica gel column chromatography to obtain a product Fa2 with the yield of 23.73%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 2.

Example 9: preparation of Fa3

Completely dissolving the formononetin 67.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu.l 36% formaldehyde (0.5mmol) and 40mg tryptamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, 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 the product Fa3 with the yield of 9.13%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 3.

Example 10: preparation of D1

Doremainol (daidzein )63.56mg (0.25mmol) was completely dissolved in 1ml DMSO, and the prepared mixture [4ml methanol, 22.5. mu.l 36% formaldehyde (0.25mmol) and 24.25mg 2-furanmethanamine (0.25mmol) ] was slowly added dropwise and stirred at room temperature for 2h, reflux reaction was carried out for 10h, the solvent was evaporated under reduced pressure to give a pale brown solid, which was collected to give a crude product, which was chromatographed on silica gel to give product D1 in 33.76% yield.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in D1.

Example 11: preparation of D2

Completely dissolving 63.56mg (0.25mmol) of daidzein in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 18.25mg of n-butylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, and distilling off 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 separate to obtain a product D2 with the yield of 49.11%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in D2.

Example 12: preparation of Da1

Completely dissolving 63.56mg (0.25mmol) of daidzein in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 45 mu.l of 36% formaldehyde (0.5mmol) and 24.25mg of 2-furanmethanamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, and distilling off the solvent under reduced pressure to obtain a yellow solid, collecting the yellow solid to obtain a crude product, and carrying out silica gel column chromatography to obtain a product Da1 with the yield of 16.38%.

The product was confirmed to have indeed the structure shown in Da1 by nuclear magnetic hydrogen, carbon and mass spectroscopy.

Example 13: preparation of Da2

Completely dissolving 63.56mg (0.25mmol) of daidzein 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 yellow solid, collecting the light yellow solid to obtain a crude product, and carrying out silica gel column chromatography to obtain a product Da2 with the yield of 32.84%.

The product was confirmed to have indeed the structure shown in Da2 by nuclear magnetic hydrogen, carbon and mass spectroscopy.

Example 14: preparation of Fa8

Completely dissolving the formononetin 66.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu l 36% formaldehyde (0.5mmol) and 40.25mg 4- (trifluoromethyl) aniline (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 Fa8 with the yield of 3.6%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 8.

Example 15 preparation of Fa9

Completely dissolving the formononetin 66.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu.l 36% formaldehyde (0.5mmol) and 24.75mg cyclohexylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, decompressing and distilling off the solvent 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 the product Fa9 with the yield of 12.8%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 9.

Example 16 preparation of F7

Completely dissolving 66.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 24.25mg of 2-furanmethanamine (0.25mmol) ] and stirring at room temperature for 2h, carrying out reflux reaction for 10h, distilling off 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 F7 with the yield of 9.3%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in F7.

Example 17 preparation of F8

Completely dissolving 66.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 18.25mg of n-butylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, and decompressing and distilling off the solvent 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 F8 with the yield of 5.4%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in F8.

Example 18: preparation of Fa4

Completely dissolving formononetin 66.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu l 36% formaldehyde (0.5mmol) and 34.12mg 3, 4-dihydroxy benzylamine (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 Fa4 with the yield of 3.1%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 4.

Example 19: preparation of Fa2

Completely dissolving formononetin 66.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 45 mu l 36% formaldehyde (0.5mmol) and 31.25mg 4-fluorobenzylamine (0.25mmol) ] and stirring at room temperature for 2h, refluxing for 10h, decompressing and evaporating the solvent 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 Fa2 with the yield of 2.0%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in Fa 2.

Example 20: preparation of F6

Completely dissolving 66.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 28.25mg of 4-fluoro-aniline (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 F8 with the yield of 1.1%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in F6.

Example 21 preparation of F5

Completely dissolving 66.065mg (0.25mmol) of formononetin in 1ml of DMSO, slowly dropwise adding the prepared mixed solution [4ml of methanol, 22.5 mu l of 36% formaldehyde (0.25mmol) and 38.25mg of 3, 4-dihydroxy benzylamine (0.25mmol) ] and stirring at room temperature for 2h, carrying out reflux reaction 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 separation to obtain a product F8 with the yield of 0.9%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in F5.

Example 22: preparation of F9

Completely dissolving the formononetin 66.065mg (0.25mmol) in 1ml DMSO, slowly dropwise adding the prepared mixed solution [4ml methanol, 22.5 mu l 36% formaldehyde (0.25mmol) and 22.5mg benzylamine (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 F9 with the yield of 15.7%.

It was confirmed by nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and mass spectrometry that the product indeed had the structure shown in F9.

Example 23: testing the influence of formononetin derivatives Da1, Da2, F4, Fa4, F6, F9, F5, Fa8, Fa9, Fa1, F8, F7, D1, D2, F11, F13, F2, Fa2 and F3 on the osteogenesis of rat bone marrow Mesenchymal Stem Cells (MSC)

Wistar rat bone marrow mesenchymal stem cells (MSC, generation II) and MSC complete culture medium are derived from Shanghai stem cell bank of Chinese academy of sciences, well-grown MSC cells are selected and cultured in osteogenic differentiation medium (ODM, wherein 50 mu M ascorbic acid 2-phosphate, 10mM beta-glycerophosphate and 10nM dexamethasone are supplemented in the MSC culture medium) when the cells grow to 70%, and the cells are treated by using formononetin derivatives with the concentration of 1 mu M, and fresh osteogenic differentiation medium ODM containing the derivatives (1 mu M) is replaced every 2-3 days. After 14 days, the cells were fixed in 10% formalin for 1h and stained with 40mM alizarin red S (pH 4.1) to observe calcium deposition in the extracellular matrix. Images were acquired at 4 x magnification on Eclipse TE200 using Digital Camera DXM1200F using ACT-1 software. To quantify alizarin red staining, cells were destained for 30min with 10% cetylpyridinium chloride monohydrate (Sigma) and the amount of alizarin red was determined by measuring the OD of the solution at 590 nm. Results were normalized to the protein content of the sample and compared to cells exposed to DMSO control only.

The same experiment was performed with daidzein (daidzein), formononetin (formononetin) as a control and DMSO as a solvent control (vehide).

The effect of the formononetin derivatives of the present invention on osteogenesis of rat bone marrow Mesenchymal Stem Cells (MSC) is shown in fig. 1.

The experimental result shows that Fa8, Fa9, Fa1, F8, F7, D1, D2, F11, F13, F2, Fa2, F3 and the like at the concentration of 1 mu M can remarkably promote MSC to differentiate into osteoblasts and generate calcium nodes on osteoblast membranes, and the capacity of the MSC is equivalent to that of mother riboflavin and formononetin (p is less than 0.05); and F11, F13, F2, Fa2 and F3 with the concentration of 1 mu M can greatly promote the differentiation of MSC into osteoblasts, and the effect of generating calcium nodes (p is less than 0.001) on osteoblast membranes is obviously better than that of the mother riboflavin and the formononetin.

Example 24: effect on human bone marrow mesenchymal Stem cell (HMSC) osteogenesis

Human primary mesenchymal stem cells (HMSC) were selected from the experimental centre of the university of kunming medical science. And (4) recovering, passaging and freezing the primary HMSC cells. Selecting HMSC cells with good growth for plating, wherein the experiment is divided into 5 groups, and the first group is an HMSC cell control group; the second, third, fourth and fifth groups are positive control daidzein (concentration 0.4. mu.M and 2. mu.M), formononetin (concentration 0.4. mu.M and 2. mu.M), ipriflavone (concentration 0.4. mu.M and 2. mu.M), and estradiol (concentration 0.1. mu.M), respectively; the sixth and seventh groups are the formononetin derivatives F11 (concentration 0.4. mu.M and 2. mu.M) and F13 (concentration 0.4. mu.M and 2. mu.M) to be tested, respectively. HMSC cells were cultured as described in example 23, stained, and quantitatively analyzed to observe osteoblast formation.

The effect of F11 and F13 on osteogenesis of Human Mesenchymal Stem Cells (HMSC) is shown in fig. 2; where CON represents HMSC cell control group (not dosed), the left side of fig. 2 is the OD value test results of different experimental groups, and the right side is the actual photograph of different experimental groups.

A graph comparing the effect of F11, F13, and a positive control compound on human bone marrow mesenchymal stem cell (HMSC) osteogenesis is shown in fig. 3; wherein CK represents HMSC cells without osteogenic differentiation induction medium (not dosed), CON represents HMSC cell control with osteogenic differentiation induction medium (not dosed), daidz represents glycitein, formo represents formononetin, and iprif represents ipriflavone; FIG. 3 shows the quantitative bone calcium node diagram on the left side and alizarin red-calcium node OD on the ordinate₅₉₀The absorbance values, in which CK absorbance is 0, are not shown in the figure, and the right side of figure 3 is a photograph of alizarin red-calcium nodes of different experimental groups.

The structural formulas of glycitein (daidz), formononetin (formo), ipriflavone (iprif) and estradiol are shown in figure 4.

The experimental results in fig. 2-3 show that F11 and F13 can significantly promote differentiation of HMSC into osteoblasts and generate calcium nodules (p < 0.001) on osteoblast membranes, the actions of the compounds are superior to those of daidzein (daidzein) and formononetin (daidzein) which are prototype compounds, estradiol (estradiol) which is a positive drug and ipriflavone (ipriflavone), and the capability of promoting osteogenic differentiation of MSC cells to generate calcium nodules is superior to that of ipriflavone which is a first-line anti-osteoporosis drug.

Example 25: influence on RANKL to induce RAW.264.7 cells to generate osteoclasts

Selecting RAW.264.7 cells from Kunming cell bank of China academy of sciences, observing cell state under microscope, and culturing at density of 2 × 10³Wells/well were seeded in a 96-well plate, and cell control wells, RANKL control wells, and F11 and F13 compound treatment wells at different concentrations (1 μ M, 5 μ M, and 10 μ M), three in parallel per treatment group, were simultaneously set, and 30min after compound contact with cells, RAW264.7 cells were induced with RANKL at 50ng/mL for 5 days, allowing the osteoclast precursor cells RAW264.7 to completely differentiate into multinucleated osteoclasts. After the expiration, osteoclast differentiation was detected by TRAP staining kit, and the number of osteoclast formation per group was observed and counted under a microscope.

The results of the experiment are shown in FIG. 5, and the number of osteoclasts is plotted on the ordinate of FIG. 5.

According to the figure 5, F11 and F13 can obviously inhibit the differentiation of RAW264.7 cells to osteoclasts, and the fact that the compounds can effectively prevent the generation of osteoclasts and improve osteoporosis is shown.

Example 26: effect on secretion of alkaline phosphatase (ALP) by Human Mesenchymal Stem Cells (HMSC)

Human primary mesenchymal stem cells (HMSC) were selected from the experimental centre of the university of kunming medical science. And (4) recovering, passaging and freezing the primary HMSC cells. The experiment was divided into 5 groups, the first group being HMSC cell control group; the second and third groups are positive control daidzein (1 μ M) and formononetin (1 μ M); the fourth and fifth groups are F11(1 μ M) and F13(1 μ M) to be tested, respectively. Well-grown HMSC cells were selected for plating and when 70% of the cells had grown, they were replaced with osteogenic differentiation medium (ODM, HMSC medium supplemented with 50. mu.M ascorbic acid 2-phosphate, 10 mM. beta. -glycerophosphate and 10nM dexamethasone), 1. mu.M soybean isoflavone derivative-treated cells, and fresh osteogenic differentiation medium ODM containing soybean isoflavone derivative (1. mu.M) was replaced every 2 to 3 days. Cells were collected after 14 days, and the expression of alkaline phosphatase (ALP) gene was detected by real-time quantitative PCR (qRT-PCR).

The effect of the compound of the present invention on the expression of ALP gene of HMSC cells is shown in FIG. 6, wherein daidz represents daidzein experimental group, formononetin experimental group, and vehicle represents HMSC cell control group in FIG. 6.

The experimental results in fig. 6 show that F11 and F13 can significantly increase the expression of ALP gene of HMSC cells, and are superior to daidzein and formononetin, which are prototype compounds, and show that the two compounds promote the formation of osteoblasts by promoting the expression of ALP, which is a bone differentiation gene, so as to interfere with the formation of osteoporosis.

Example 27: effect on secretion of osteocyte early factor (sp7) by Human Mesenchymal Stem Cells (HMSC)

Human primary mesenchymal stem cells (HMSC) were selected from the experimental centre of the university of kunming medical science. And (4) recovering, passaging and freezing the primary HMSC cells. Selecting HMSC cells with good growth for plating, wherein the experiment is divided into 5 groups, and the first group is an HMSC cell control group; the second and third groups are positive control daidzein (1 μ M) and formononetin (1 μ M); the fourth and fifth groups are F11(1 μ M) and F13(1 μ M) to be tested, respectively. HMSC cells were cultured as described in example 1, and after the cells were collected, the expression of the early factor (sp7) gene was detected by real-time quantitative PCR (qRT-PCR).

The effect of the compound of the present invention on the expression of sp7 gene in HMSC cells is shown in FIG. 7, wherein daidz in FIG. 7 represents daidzein test group, formononetin test group, and vehicle represents HMSC cell control group.

The experimental results in fig. 7 show that F11 and F13 can significantly increase the expression of sp7 gene in HMSC cells, and are superior to daidzein (daidzein) and formononetin (formononetin), which are prototype compounds. The two compounds are shown to promote the formation of osteoblasts by promoting the expression of an early bone cell factor sp7 gene, so as to interfere the formation of osteoporosis.

Example 28: effect on secretion of Osteocalcin (OP) by Human Mesenchymal Stem Cells (HMSC)

Human primary mesenchymal stem cells (HMSC) were selected from the experimental centre of the university of kunming medical science. And (4) recovering, passaging and freezing the primary HMSC cells. Selecting HMSC cells with good growth for plating, wherein the experiment is divided into 5 groups, and the first group is an HMSC cell control group; the second and third groups are positive control daidzein (1 μ M) and formononetin (1 μ M); the fourth and fifth groups are F11(1 μ M) and F13(1 μ M) to be tested, respectively. HMSC cells are cultured according to the method described in example 1, and after the cells are collected, the expression of the Osteopontin (OP) gene is detected by a real-time quantitative PCR (qRT-PCR) method.

The effect of the compound of the present invention on OP gene expression of HMSC cells is shown in fig. 8, in fig. 8 daidz represents daidzein experimental group, formononetin experimental group, and vehicle represents HMSC cell control group.

The experimental results in fig. 8 show that F11 and F13 can significantly increase the expression of OP gene in HMSC cells, and are superior to the prototype compounds daidzein (daidzein) and formononetin (formononetin). The two compounds are shown to promote the formation of osteoblasts by promoting the secretion of osteopontin OP by osteocytes, so as to interfere the formation of osteoporosis.

Example 29: effect on secretion of insulin-like growth factor (IGF-1) by Human Mesenchymal Stem Cells (HMSCs)

Human primary mesenchymal stem cells (HMSC) were selected from the experimental centre of the university of kunming medical science. And (4) recovering, passaging and freezing the primary HMSC cells. Selecting HMSC cells with good growth for plating, wherein the experiment is divided into 5 groups, and the first group is an HMSC cell control group; the second and third groups are positive control daidzein (1 μ M) and formononetin (1 μ M); the fourth and fifth groups are F11(1 μ M) and F13(1 μ M) to be tested, respectively. HMSC cells were cultured as described in example 1, collected at the end of the culture, and the expression of insulin-like growth factor (IGF-1) gene was detected by real-time quantitative PCR (qRT-PCR).

The effect of the compound of the present invention on the expression of HMSC cell insulin-like growth factor (IGF-1) gene is shown in FIG. 9, daidz in FIG. 9 represents daidzein experimental group, formononetin experimental group, and vehicle represents HMSC cell control group.

The experimental results in fig. 9 show that F11 and F13 can significantly increase the expression of OP gene in HMSC cells, and are superior to the prototype compounds daidzein (daidzein) and formononetin (formononetin). The two compounds are shown to promote the formation of osteoblasts by promoting the expression of insulin-like growth factor (IGF-1), thereby interfering the formation of osteoporosis.

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.