阅读说明：本技术 一种抗骨质疏松的金合欢素衍生物及其制备方法 (Anti-osteoporosis acacetin derivative and preparation method thereof ) 是由 张洪胜 于 2021-01-19 设计创作，主要内容包括：本发明公开了一种抗骨质疏松的金合欢素衍生物及其制备方法,属于药物化学合成技术领域；包括以下步骤：在氮气保护下,将金合欢素与2,4-二甲氧基苄基氯加入到有机溶剂中,在碱的作用下,反应6～8h,经硅胶柱层析分离,重结晶,最终得到金合欢素衍生物；本发明通过病理实验发现,所述的金合欢素衍生物能够抑制缺乏雌性激素引起的骨吸收增加和骨组织破坏,同时还能够提高血钙浓度,有利于骨沉积,由此说明,新化合物对于由雌性激素缺乏导致的骨质疏松症具有一定的防治作用。(The invention discloses an anti-osteoporosis acacetin derivative and a preparation method thereof, belonging to the technical field of chemical synthesis of medicines; the method comprises the following steps: under the protection of nitrogen, adding farnesine and 2, 4-dimethoxy benzyl chloride into an organic solvent, reacting for 6-8 h under the action of alkali, separating by silica gel column chromatography, and recrystallizing to finally obtain the farnesine derivative; according to the invention, pathological experiments show that the acacetin derivative can inhibit increase of bone resorption and bone tissue damage caused by estrogen deficiency, and can improve the blood calcium concentration and is beneficial to bone deposition, so that the novel compound has a certain prevention and treatment effect on osteoporosis caused by estrogen deficiency.)

1. An anti-osteoporotic acacetin derivative, which has a molecular structure of formula (I):

2. a preparation method of an anti-osteoporosis acacetin derivative is characterized by comprising the following steps:

under the protection of nitrogen, dissolving farnesin and 2, 4-dimethoxy benzyl chloride in an anhydrous organic solvent, heating to 80-120 ℃, carrying out reflux reaction for 6-8 h under the action of alkali, cooling to room temperature after the reaction is finished, adding 30-40 mL of ethyl acetate into a reaction solution for extraction and liquid separation, combining organic phases, drying with anhydrous sodium sulfate, filtering, concentrating a filtrate under reduced pressure to obtain a crude product, separating the crude product through silica gel column chromatography, combining the same components, and recrystallizing with ethanol to obtain a target product;

the feeding molar ratio of the farnesoid to the 2, 4-dimethoxy benzyl chloride is 1: 1.5-4.5;

the organic solvent is tetrahydrofuran, dichloromethane, diethyl ether, acetone, toluene, benzotrifluoride, methanol, ethanol or isopropanol;

the alkali is triethylamine, pyridine, N-diisopropylethylamine, sodium hydroxide or potassium carbonate;

the silica gel column chromatography separation is carried out by adopting petroleum ether-ethyl acetate for gradient elution, and the volume ratio of the petroleum ether to the ethyl acetate is 60:1, 40:1, 20:1, 10:1, 5:1 and 1:1.

3. The method of preparing an anti-osteoporosis acacetin derivative according to claim 2, comprising the steps of:

under the protection of nitrogen, dissolving 15g of farnesene and 14.8g of 2, 4-dimethoxybenzyl chloride in anhydrous dichloromethane, heating to 110-120 ℃, carrying out reflux reaction for 8 hours under the action of triethylamine, cooling to room temperature after the reaction is finished, adding 30-40 mL of ethyl acetate into reaction liquid for extraction and liquid separation, combining organic phases, drying with anhydrous sodium sulfate, filtering, carrying out silica gel column chromatography separation on a crude product obtained by decompressing and concentrating filtrate, carrying out gradient elution by adopting petroleum ether-ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 60:1, 40:1, 20:1, 10:1, 5:1 and 1:1, combining the same components, and recrystallizing by using ethanol to obtain the target product.

Technical Field

The invention belongs to the technical field of chemical drug synthesis, and relates to an anti-osteoporosis acacetin derivative and a preparation method thereof.

Background

Acacetin is a natural flavonoid compound widely present in acacia, chrysanthemum, locust tree, and the like. The acacetin has the traditional effects of resolving stagnation, soothing nerves, regulating qi and dredging collaterals, and also has the effect of resisting osteoporosis. However, acacetin has poor solubility in water, so that acacetin cannot be sufficiently dissolved in gastrointestinal tract aqueous solution, the dissolution rate is reduced, and finally the oral bioavailability of acacetin is reduced. Therefore, it is necessary to modify the structure of farnesoid to improve its water solubility so that it can sufficiently exert its biological activity against osteoporosis.

Disclosure of Invention

In order to solve the technical problems, the invention provides an anti-osteoporosis acacetin derivative, which has a molecular structure of a formula (I):

another object of the present invention is to provide a method for preparing an anti-osteoporosis acacetin derivative, comprising the steps of:

under the protection of nitrogen, dissolving farnesin and 2, 4-dimethoxy benzyl chloride in an anhydrous organic solvent, heating to 80-120 ℃, carrying out reflux reaction for 6-8 h under the action of alkali, cooling to room temperature after the reaction is finished, adding 30-40 mL of ethyl acetate into a reaction solution for extraction and liquid separation, combining organic phases, drying with anhydrous sodium sulfate, filtering, concentrating a filtrate under reduced pressure to obtain a crude product, separating the crude product through silica gel column chromatography, combining the same components, and recrystallizing with ethanol to obtain a target product;

the feeding molar ratio of the farnesoid to the 2, 4-dimethoxy benzyl chloride is 1: 1.5-4.5;

the organic solvent is tetrahydrofuran, dichloromethane, diethyl ether, acetone, toluene, benzotrifluoride, methanol, ethanol or isopropanol;

the alkali is triethylamine, pyridine, N-diisopropylethylamine, sodium hydroxide or potassium carbonate;

the silica gel column chromatography separation is carried out by adopting petroleum ether-ethyl acetate for gradient elution, and the volume ratio of the petroleum ether to the ethyl acetate is 60:1, 40:1, 20:1, 10:1, 5:1 and 1:1.

According to a preferred embodiment of the above preparation method, the method comprises the following steps:

under the protection of nitrogen, dissolving 15g of farnesene and 14.8g of 2, 4-dimethoxybenzyl chloride in anhydrous dichloromethane, heating to 110-120 ℃, carrying out reflux reaction for 8 hours under the action of triethylamine, cooling to room temperature after the reaction is finished, adding 30-40 mL of ethyl acetate into reaction liquid for extraction and liquid separation, combining organic phases, drying with anhydrous sodium sulfate, filtering, carrying out silica gel column chromatography separation on a crude product obtained by decompressing and concentrating filtrate, carrying out gradient elution by adopting petroleum ether-ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 60:1, 40:1, 20:1, 10:1, 5:1 and 1:1, combining the same components, and recrystallizing by using ethanol to obtain the target product.

Compared with the prior art, the invention has the beneficial effects that:

the farnesoid derivative with a novel structure is prepared through a alkylation reaction, and compared with the farnesoid, the solubility of the new compound in water is increased, so that the preparation method is beneficial to preparing an oral preparation; pharmacological experiments show that the new compound can inhibit the increase of bone absorption and the damage of bone tissues caused by ovarian deficiency, can also adjust the mineral content in an animal body, increase the bone density and is beneficial to the deposition of bones, so that the new compound has a certain prevention and treatment effect on osteoporosis caused by estrogen deficiency; the preparation method is simple, the reaction condition is mild, the used solvent is a common organic solvent, special treatment is not needed, the environmental pollution is small, only silica gel column chromatography is needed for separation, the post-treatment is simple, the method is economical and cheap, and the method is suitable for industrial production.

Drawings

FIG. 1: nuclear magnetic resonance hydrogen spectra of anti-osteoporosis farnesoid derivatives of example 1.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1

Under the protection of nitrogen, 15g of farnesine and 14.8g of 2, 4-dimethoxy benzyl chloride are dissolved in 78mL of anhydrous dichloromethane, the temperature is raised to 110-120 ℃, carrying out reflux reaction for 8 hours under the action of triethylamine, cooling to room temperature after the reaction is finished, adding 30-40 mL of ethyl acetate into reaction liquid for extraction and liquid separation, combining organic phases, drying with anhydrous sodium sulfate, filtering, carrying out silica gel column chromatography separation on a crude product obtained by decompressing and concentrating filtrate, carrying out gradient elution by adopting petroleum ether-ethyl acetate, wherein the volume ratio of the petroleum ether to the ethyl acetate is 60:1, 40:1, 20:1, 10:1, 5:1 and 1:1, carrying out TLC detection, combining the same components, obtaining 10 components in total, carrying out silica gel column chromatography on the 4 th component, carrying out gradient elution by adopting petroleum ether-ethyl acetate 10:1 and 5:1, and carrying out ethanol recrystallization on the collected eluent to obtain a target product. The yield was 82.07%.

And (3) nuclear magnetic resonance hydrogen spectrum detection:

the sample was placed in a sample tube, and 0.5ml of DCL3 (deuterated chloroform) was injected into the sample tube with a syringe to dissolve the sample sufficiently. The sample and the reagent are required to be fully mixed, the solution is clear and transparent, and has no suspended matters or other impurities, and a nuclear magnetic resonance hydrogen spectrogram is obtained through nuclear magnetic resonance identification, and the result is shown in figure 1.

Example 2 Water solubility testing of Compounds of the invention

0.1g of the acacetin derivative obtained in example 1 was weighed in a test tube, 10.0mL of purified water was added, the tube was shaken every 5min at room temperature for 30 seconds, the dissolution was observed after 30min, the amount of the solvent was recorded, the experimental results were converted to standard solubility (25 ℃), and the test results are shown in Table 1.

Table 1 solubility and yield of the compound of example 1

The results show that the water solubility of the acacetin derivative is significantly improved compared to acacetin, and thus it is possible to obtain an acacetin derivative having a good water solubility by the method of the present invention.

Experimental example 3 pharmaceutical study of the Compound of the present invention for anti-osteoporosis

The experimental method comprises the following steps: selecting healthy 5-month-old female SD rats with weight of 280 +/-50 g, randomly grouping the rats into a normal control group (a control group 1), an ovariectomy model group (a control group 2), an acacetin group (a control group 3) and an acacetin derivative (an experimental group), culturing the rats of each group in a proper environment for 2 weeks, injecting 2% sodium pentobarbital into the abdominal cavity of each group of female experimental rats according to the dose of 40mg/kg of body weight for anesthesia, opening the abdominal cavity of the normal control group and then suturing the rats, the other groups of rats are subjected to bilateral ovariectomy, and from 3 weeks after operation, the rats in each group eat feed and water every day, at the same time, farnesin and farnesin derivative were administered to rats of the farnesin group and the farnesin derivative group, respectively, at a dose of 10mg/kg for 3 months, and rats of the model group were weighed 1 time per week without any drug. After the experiment is finished, the rat is injected with 2% sodium pentobarbital for anesthesia, the eyeball of the rat is bled, centrifuged, and serum is reserved and stored at-20 ℃ for detection indexes. The rat uterus was enucleated and weighed. The bilateral femurs and the bilateral tibias of the rats are taken out, all muscles and connective tissues are removed, the two tibias are subjected to muffle ashing at 800 ℃ for 3 hours to enable the bones to be white blocks, and the bones are weighed after cooling. Grinding the ashed tibia into powder, taking 0.1g of bone ash to dissolve in hydrochloric acid solution, measuring 0.2mL of bone ash to dilute to 1mL of bone ash with water, and using a full-automatic biochemical analyzer to determine the contents of calcium and phosphorus in the bone. Bone density (BMD) was measured using bone densitometer and serum biochemical analysis detected for serum (ALP), calcium (Ca) and phosphorus (P).

Results of the experiment

(1) Effect of Compounds of the invention on serum ALP, serum Ca and serum P in bilateral ovariectomized rats

The data in table 2 show that the serum alkaline phosphatase in the model group rats was significantly increased after ovariectomy due to rapid estrogen level decrease, increased bone resorption, rapid bone loss and bone tissue destruction after ovariectomy. After the rat is administrated with the medicine, the alkaline phosphatase in serum is reduced to different degrees, wherein the reduction amplitude of an experimental group is more remarkable, and the acacetin derivative can reduce bone tissue damage and reduce the risk of osteoporosis. The data in the table also show that compared with the model group, the serum calcium and phosphorus contents of rats in each administration group are increased, particularly the experimental group is most obvious, which indicates that the farnesoid derivative can regulate the mineral content in animals, increase the bone density and has certain prevention and treatment effect on postmenopausal osteoporosis.

TABLE 2 Effect of Compounds of the present invention on serum ALP, serum Ca and serum P in bilateral ovariectomized rats

Group of	Serum ALP (U/L)	Serum Ca (mmol/L)	Serum P (mmol/L)
				Control group 1	56.22	2.42	1.83
Control group 2	267.50	1.73	1.62
				Control group 3	179.00	2.15	1.85
Experimental group	113.54	3.08	2.31

(2) Effect of Compounds of the invention on left femoral BMD in bilateral ovariectomized rats

TABLE 3 Effect of Compounds of the invention on left femoral BMD in bilateral ovariectomized rats

Group of	Left femoral BMD (g/cm)2)
		Control group 1	0.345
Control group 2	0.168
		Control group 3	0.204
Experimental group	0.285

The data in table 3 show that the bone density of the left femur of the model group rats after ovariectomy is obviously reduced compared with that of the normal group, and the bone density is obviously increased after the administration of the medicine, wherein the increase of the acacetin derivative is more obvious.

(3) Effect of Compounds of the invention on bilateral tibial parameters in bilateral ovariectomized rats

The data in table 4 show that, compared with the model group, after the drug is administered, the bone calcium and bone phosphorite substances of the rat with the osteoporosis model are obviously increased, wherein the increase effect of the farnesoid derivative is more obvious, and the farnesoid derivative has the effect of preventing and treating the osteoporosis.

TABLE 4 Effect of Compounds of the invention on bilateral tibial parameters in bilateral ovariectomized rats

Group of	Double tibia bone calcium (g/g)	Double tibia phosphorus (g/g)	Double-shank bone ash weight (g)
				Control group 1	0.351	0.340	0.90
Control group 2	0.179	0.163	0.58
				Control group 3	0.225	0.218	0.67
Experimental group	0.273	0.282	0.81

In conclusion, the farnesoid derivative disclosed by the invention can inhibit the increase of bone resorption and bone tissue damage caused by ovarian deficiency, can regulate the mineral content in an animal body, increases the bone density and is beneficial to bone deposition, so that the farnesoid derivative has a certain effect of preventing and treating osteoporosis caused by estrogen deficiency, and can provide a little inspiration for the research of anti-osteoporosis medicines.