阅读说明：本技术 一种5-ala缀合物制备方法及其用途 (Preparation method and application of 5-ALA conjugate ) 是由 鲁光英 于 2021-08-09 设计创作，主要内容包括：本发明提供了一种5-ALA缀合物制备方法及其用途,制备方法包括5-((2-氨基乙基)氨基)-4-氧代戊酸乙酯合成和5-ALA缀合物合成步骤,所得到5-ALA缀合物应用于医药用途,包括抗肿瘤药物制备,本发明将原本用于活血化瘀、利水消肿益母草碱与5-ALA合成,制备得到5-ALA缀合物,通过初步药理活性测试,结果表明,其在细胞水平上,对肿瘤的清除率,比5-ALA及其酯化物肿瘤清除率提高了20％以上。(The invention provides a preparation method of 5-ALA conjugate and application thereof, wherein the preparation method comprises the steps of synthesizing 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester and synthesizing 5-ALA conjugate, the obtained 5-ALA conjugate is applied to medical application, and the preparation method comprises the preparation of antitumor drugs.)

1. A process for the preparation of a 5-ALA conjugate, which process comprises: the method comprises the following steps:

step A, synthesizing 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester, protecting an amino group of 2- (methylthio) ethylamine by using Boc, condensing with 5-ALA or an esterified product thereof, and removing the amino protection by using TFA to obtain 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester;

and step B, synthesizing a 5-ALA conjugate, namely condensing leonurine and malonic acid, and condensing with 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester to obtain the 5-ALA conjugate.

2. A process for the preparation of a 5-ALA conjugate as claimed in claim 1, wherein: and C, completing the condensation in the step B by dehydrating under the heating condition by adopting a dehydrating agent, wherein the dehydrating agent comprises phosphorus pentoxide and acetic anhydride.

3. A process for the preparation of a 5-ALA conjugate as claimed in claim 2, wherein: the 5-ALA comprises 5-ALA, 5-ALA methyl ester and 5-ALA ethyl ester.

4. Use of a 5-ALA conjugate, characterized in that: 5-ALA conjugate obtained by the method for preparing 5-ALA conjugate as claimed in claims 1-3, which is used in medical application, including preparation of antineoplastic medicine.

Technical Field

The invention relates to the technical field of organic matter synthesis pharmacy, in particular to a preparation method and application of a 5-ALA conjugate in the aspect of pesticides.

Background

5-aminolevulinic acid (5-ALA) is a biological endogenous substance, is a precursor substance for biosynthesis of animal hemoglobin and plant chlorophyll, is widely applied to the field of medicines, can be used as a photosensitizer for treating various cancers, and can also be used for preparing pesticides, herbicides and the like. Due to the unstable structure and limited permeability of 5-aminolevulinic acid, various esterified medicaments such as methyl ester, ethyl ester and the like are continuously on the market in the past 90 s.

Leonurine, with the chemical name of 3, 5-dimethoxy-4-hydroxy-benzoic acid (4-guanidino) -1-butyl ester, widely exists in leaves of Leonurus sibiricus or in Leonurus sibiricus and whole herb of Artemisia argyi and Leonurus heterophyllus, and has various effects of promoting blood circulation, removing blood stasis, inducing diuresis, relieving swelling and the like. Specifically, the pharmacological action of leonurine mainly has five aspects: 1) has exciting effect on uterus of various animals, and the uterine contraction effect of leonurine can last for several hours but can be recovered after being washed; 2) the leonurine has effect in promoting contraction of isolated frog heart, inhibiting blood vessel expansion of warm-blooded animal, and resisting epinephrine; 3) leonurine has direct excitation effect on respiratory center; 4) leonurine can relax the tension of the isolated intestinal canal of the rabbit, expand the amplitude, reduce the amplitude when the quantity is large, and increase the frequency; 5) motherwort is a traditional gynecological medicine with the effects of improving blood circulation, activating blood and removing stasis, and related researches show that the effects of reducing total cholesterol and triglyceride of leonurine are superior to or close to statin drugs, but the toxic reaction is far lower than that of the statin drugs. Moreover, the leonurine also has the effects of oxidation resistance, sterilization and inflammation resistance, can quickly remove free radicals and resist fungi and mould.

In view of the problems of 5-ALA, various solutions have been proposed in the prior art, for example, in the patent CN 111840574, the preparation and application of a small molecule prodrug of 5-ALA and camptothecin are reported to enhance the anticancer effect of 5-ALA, but the application of the conjugate is greatly limited because camptothecin has larger side effects and is easy to cause symptoms such as hematuria and the like.

Disclosure of Invention

In view of the above-mentioned drawbacks and problems of the prior art, the present invention provides a method for preparing a 5-ALA conjugate and its use.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of 5-ALA conjugate comprises the following steps:

step A, synthesizing 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester, protecting an amino group of 2- (methylthio) ethylamine by using Boc, condensing with 5-ALA or an esterified product thereof, and removing the amino protection by using TFA to obtain 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester;

and step B, synthesizing a 5-ALA conjugate, namely condensing leonurine and malonic acid, and condensing with 5- ((2-aminoethyl) amino) -4-oxopentanoic acid ethyl ester to obtain the 5-ALA conjugate.

In the above technical scheme, the condensation in the step B is completed by dehydrating with a dehydrating agent under heating, wherein the dehydrating agent comprises phosphorus pentoxide and acetic anhydride.

In the technical scheme, the 5-ALA comprises 5-ALA, 5-ALA methyl ester and 5-ALA ethyl ester.

The 5-ALA conjugate obtained by the invention is applied to medical application, including preparation of antitumor drugs.

The 5-ALA conjugate is prepared by synthesizing the leonurine originally used for promoting blood circulation to remove blood stasis and inducing diuresis to alleviate edema and 5-ALA, and the result of a preliminary pharmacological activity test shows that the clearance rate of the leonurine on the cell level to tumors is improved by more than 20 percent compared with the clearance rate of 5-ALA and esterified substances thereof.

Drawings

FIG. 1 is a graph of experimental data showing the effect of leonurine, compound A, compound B and 5-ALA on cell viability 24 hours after interaction with HepG2 and MCF-7 cells.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments and the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As a 5-ALA conjugate shown in an example, a compound A, a compound B and a compound C are synthesized according to the difference that 5-ALA adopts 5-ALA, 5-ALA methyl ester and 5-ALA ethyl ester, and a specific synthetic route taking the compound A as an example is as follows:

a novel 5-ALA conjugate (Compound A) of the structure:

the compound is prepared by condensing 5-ALA ethyl ester and leonurine with propylene glycol through 2- (methylthio) ethylamine, the compound is not a final structure, and 5-ALA methyl ester or 5-ALA can also be prepared into compound A analogue compound B and compound C through condensation with leonurine in the above mode, so that the same or similar pharmacological effects can be generated. The invention combines leonurine which is originally used for promoting blood circulation to remove blood stasis and inducing diuresis to alleviate edema and has no relevance to the photodynamic therapy effect of 5-ALA and esters thereof into a compound, and the curative effect of the 5-ALA photosensitizer can be improved.

The specific synthesis preparation method comprises the following steps:

step A Synthesis of ethyl 5- ((2-aminoethyl) amino) -4-oxopentanoate

Dissolving 10g of 2- (methylthio) ethylamine in 60ml of water, adding 15ml of triethylamine, stirring to dissolve, and slowly dropwise adding (Boc)₂O solution (Boc)₂Dissolving 30g of O in 15ml of tetrahydrofuran, stirring to obtain the solution), adding 0.1g of DMAP after dripping, uniformly stirring, heating to 40 ℃, keeping the temperature for 90 hours, monitoring the reaction progress by TLC (thin layer chromatography), after the reaction is finished, filtering, concentrating the filtrate to a small volume, adding 30ml of DCM (DCM) for dissolving, extracting for 2 times by using water, collecting a DCM layer, concentrating, and dissolving silica gelColumn chromatography purification, petroleum ether: ethyl acetate (36:1) was eluted to give Boc protected S-2- (methylthio) ethylamine (5.8 g).

Dissolving 16g of Boc protected S-2- (methylthio) ethylamine in 50ml of DMF, stirring for dissolving, cooling to-10 ℃, adding 8g of 5-ALA ethyl ester, stirring for 30min, heating to 20 ℃, keeping the temperature and stirring for 3h, detecting the reaction progress by TLC, concentrating the reaction solution to a small volume after the reaction is finished, adding 30ml of DCM for stirring for dissolving, extracting twice with water, adding 15g of anhydrous magnesium chloride, stirring for half an hour, standing overnight, filtering, concentrating the filtrate to a small volume, adding dichloromethane for dissolving 20ml, cooling to-5 ℃, dropwise adding 15ml of TFA under the stirring state, stirring at low temperature for overnight, detecting the reaction by TLC to an end point, concentrating under reduced pressure to a small volume, adding 40ml of diethyl ether (room temperature), stirring uniformly, cooling to-5 ℃, and separating out a solid (12 g).

Step B, Synthesis of 5-ALA conjugate Compound A

Adding 30ml of dichloromethane into a reaction vessel, adding 4g of malonic acid, 4g of DPTs8g and 1.4g of DIC, stirring for 1h, adding 8g of leonurine, reacting overnight under stirring, monitoring the reaction end point by TLC, filtering, concentrating the filtrate to a small volume, adding 20ml of methanol, heating, stirring for dissolving, and standing for crystallization to obtain the final product.

Putting 40ml of dichloromethane into a reaction vessel, adding 10g of the product obtained in the previous step and 8g of ethyl 5- ((2-aminoethyl) amino) -4-oxopentanoate, stirring and dissolving, slowly dropwise adding 20ml of acetic anhydride, controlling the reaction end point by TLC, after the reaction is finished, adding water for extraction for three times, concentrating an organic phase to a small volume, adding 20ml of methanol, heating and stirring uniformly, and cooling and crystallizing to obtain 8.6g of a compound A.

In the same way, 5-ALA ethyl ester in the synthetic route is replaced by 5-ALA or 5-ALA methyl ester to obtain a compound B and a compound C.

HepG2 human liver cancer cells and MCF-7 human breast cancer cells which are commonly used in pharmacological research are adopted to carry out cell activity test research. The cell culture medium is prepared by selecting a DMEM high-sugar culture medium and adding antibiotics and 10% fetal calf serum, the culture medium is cultured in a cell culture box, and proper light is matched during the test.

FIG. 1 shows the effect of leonurine, compound A, compound B and 5-ALA on cell viability 24 hours after interaction with HepG2 and MCF-7 cells.

The results show that the cell survival rate of the leonurine-treated group is higher than 90%, which indicates that the leonurine has no obvious inhibition effect on two tumor cells. The survival rates of HepG2 cells of the compound A, the compound B and the 5-ALA treated groups were 85.48%, 55.39% and 7.24% respectively at a sample concentration of 1.0 ug/mL; the survival rates of MCF-7 cells were 88.82%, 72.11% and 67.01%, respectively. The proliferation inhibiting effect of the compound A, the compound B and the 5-ALA on HepG2 cells is higher than that of MCF-7 cells, which shows that the compound A, the compound B and the 5-ALA have selective inhibiting effect on two tumor cells. The survival rate of the two cancer cells also decreased significantly with increasing concentrations of the test sample, indicating that compound a and compound B are dose-dependent on inhibition of proliferation of the two cancer cells. In addition, the anticancer effect of the compound A and the compound B on two cancer cells is obviously higher than that of 5-ALA, so that the effect of the 5-ALA conjugate on the proliferation inhibition of the two cancer cells is obviously higher than that of the 5-ALA conjugate which is singly used, and meanwhile, the antitumor activity of the compound A is higher than that of the compound B.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.