A method for preparing a 5-ALA conjugate and its use

Abstract

The present invention provides a 5-ALA conjugate preparation method and use thereof. The preparation method comprises the steps of synthesizing 5-((2-aminoethyl)amino)-4-oxopentanoic acid ethyl ester and synthesizing a 5-ALA conjugate. The obtained 5-ALA conjugate is applied to medical purposes, including the preparation of anti-tumor drugs. The present invention synthesizes leonurine, which is originally used for promoting blood circulation and removing blood stasis, promoting diuresis and reducing swelling, with 5-ALA to prepare the 5-ALA conjugate. The results of preliminary pharmacological activity tests show that the tumor clearance rate of the conjugate at the cellular level is improved by more than 20% compared with the tumor clearance rate of 5-ALA and its esters.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthetic pharmaceuticals, and more specifically, to a method for preparing a 5-ALA conjugate and a use thereof. Background Art

[0002] 5-aminolevulinic acid (5-ALA) is an endogenous substance and a precursor for the biosynthesis of animal hemoglobin and plant chlorophyll. It is widely used in the medical field and can be used as a photosensitizer for the treatment of various cancers. It can also be used to make pesticides and herbicides. Since the 1990s, a variety of esterified drugs have been launched on the market, such as methyl ester and ethyl ester, due to the unstable structure and limited permeability of 5-aminolevulinic acid.

[0003] Leonurine, whose chemical name is 3,5-dimethoxy-4-hydroxy-benzoic acid (4-guanidino)-1-butyl ester, is widely present in the leaves of Leonurus tenuifolia or the whole herb of Leonurus argyi and Leonurus mugwort. It has many functions such as promoting blood circulation, removing blood stasis, promoting diuresis and reducing swelling. Specifically, the pharmacological effects of motherwort alkaloids are mainly in five aspects: 1) It has an excitatory effect on the uterus of many animals. The uterine contraction effect of motherwort alkaloids can last for several hours, but it can be restored after flushing; 2) The effect on the circulatory system: a small dose of motherwort alkaloids has an enhanced contraction effect on the isolated frog heart, but when used in large quantities, it is inhibited. Motherwort alkaloids have a significant dilation effect on the blood vessels of warm-blooded animals and have an anti-adrenaline effect; 3) Motherwort alkaloids have a direct excitatory effect on the respiratory center; 4) Motherwort alkaloids can relax the tension of the isolated intestinal tract of rabbits and expand the amplitude. The amplitude becomes smaller and the frequency increases when the amount is large; 5) Motherwort is a traditional gynecological medicine that improves blood circulation and promotes blood circulation and removes blood stasis. Relevant studies have shown that the effects of motherwort alkaloids in reducing total cholesterol and triglycerides are better than or close to statins, but the toxicity reaction is much lower than statins. In addition, motherwort alkaloids also have antioxidant and bactericidal and anti-inflammatory effects, and can quickly remove free radicals and resist fungi and molds.

[0004] In the prior art, there are various solutions to the problems of 5-ALA. For example, patent CN 111840574 reports the preparation and application of a 5-ALA and camptothecin small molecule prodrug to enhance the anti-cancer effect of 5-ALA. However, since camptothecin has significant side effects and can easily cause symptoms such as hematuria, the application of this conjugate is greatly limited. Summary of the invention

[0005] In view of the above-mentioned defects and problems in the prior art, the present invention provides a method for preparing a 5-ALA conjugate and its use.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a 5-ALA conjugate, comprising the following steps:

[0008] Step A, synthesis of ethyl 5-((2-aminoethyl)amino)-4-oxopentanoate, protecting the amino portion of 2-(methylthio)ethylamine with Boc, followed by condensation with 5-ALA or its ester, and removing the amino protection with TFA to obtain ethyl 5-((2-aminoethyl)amino)-4-oxopentanoate;

[0009] Step B, synthesis of 5-ALA conjugate, condensing leonurine with malonic acid, and then condensing with 5-((2-aminoethyl)amino)-4-oxopentanoic acid ethyl ester to obtain 5-ALA conjugate.

[0010] In the above technical scheme, the condensation in step B is completed by dehydration under heating conditions with a dehydrating agent, and the dehydrating agent includes phosphorus pentoxide and acetic anhydride.

[0011] In the above technical solution, the 5-ALA includes 5-ALA, 5-ALA methyl ester and 5-ALA ethyl ester.

[0012] The 5-ALA conjugate obtained by the present invention is used for medical purposes, including the preparation of anti-tumor drugs.

[0013] The present invention synthesizes leonurine, which is originally used for promoting blood circulation and removing blood stasis, promoting diuresis and reducing swelling, with 5-ALA to prepare a 5-ALA conjugate. The results of preliminary pharmacological activity tests show that its tumor clearance rate at the cellular level is more than 20% higher than that of 5-ALA and its esters. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the experimental data of the effects of leonurine, compound A, compound B and 5-ALA on cell activity after 24 hours of action on HepG2 and MCF-7 cells. DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] As a 5-ALA conjugate shown in the embodiment, according to the different 5-ALA, 5-ALA methyl ester, and 5-ALA ethyl ester, compound A, compound B, and compound C are synthesized. Taking the synthesis of compound A as an example, the specific synthesis route is:

[0017] A novel 5-ALA conjugate (Compound A) has the following structure:

[0018]

[0019] The compound is obtained by condensing 5-ALA ethyl ester and leonurine through 2-(methylthio)ethylamine and propylene glycol. The compound is not the final structure, wherein 5-ALA methyl ester or 5-ALA can also be prepared into compound A analogs, compound B and compound C by condensing with leonurine in the above manner, and produce the same or similar pharmacological effects. In addition, the present invention conjugates leonurine, which is originally used for promoting blood circulation and removing blood stasis, promoting diuresis and reducing swelling, and has no correlation with the photodynamic therapy of 5-ALA and its esters, into a compound, which will improve the efficacy of 5-ALA photosensitizer. This technical solution is a solution that cannot be foreseen by those skilled in the art.

[0020] Specific synthesis preparation method:

[0021] Step A, Synthesis of 5-((2-aminoethyl)amino)-4-oxopentanoic acid ethyl ester

[0022]

[0023] Dissolve 10 g of 2-(methylthio)ethylamine in 60 ml of water, add 15 ml of triethylamine, stir to dissolve, slowly dropwise add (Boc)2O solution (dissolve 30 g of (Boc)2O in 15 ml of tetrahydrofuran and stir to obtain the solution), add 0.1 g of DMAP, stir evenly, raise the temperature to 40°C, keep warm for 90 hours, monitor the progress of the reaction by TLC, filter, concentrate the filtrate to a small volume, add 30 ml of DCM to dissolve, extract with water twice, collect the DCM layer, concentrate, purify by silica gel column chromatography, and elute with petroleum ether:ethyl acetate (36:1) to obtain Boc-protected S-2-(methylthio)ethylamine (5.8 g).

[0024]

[0025] Dissolve 16 g of Boc-protected S-2-(methylthio)ethylamine in 50 ml of DMF, stir and dissolve, cool to -10°C, add 8 g of 5-ALA ethyl ester, stir for 30 min, heat to 20°C, keep stirring for 3 h, monitor the reaction progress by TLC, and after the reaction is completed, concentrate the reaction solution to a small volume, add 30 ml of DCM, stir and dissolve, extract twice with water, add 15 g of anhydrous magnesium chloride, stir for half an hour, let stand overnight, filter, concentrate the filtrate to a small volume, add 20 ml of dichloromethane to dissolve, cool to -5°C, add 15 ml of TFA dropwise with stirring, stir at low temperature overnight, monitor the reaction to the end point by TLC, concentrate under reduced pressure to a small volume, add 40 ml of ether (room temperature), stir evenly, cool to -5°C, and precipitate a solid (12 g).

[0026] Step B, Synthesis of 5-ALA Conjugate Compound A

[0027]

[0028] In a reaction container, add 30 ml of dichloromethane, add 4 g of malonic acid, 8 g of DPTs and 1.4 g of DIC, stir for 1 hour, then add 8 g of leonurine, stir and react overnight, monitor the reaction endpoint by TLC, filter, concentrate the filtrate to a small volume, add 20 ml of methanol, heat and stir to dissolve, and let stand to crystallize to obtain the product.

[0029]

[0030] Place 40 ml of dichloromethane in a reaction container, add 10 g of the product obtained in the previous step and 8 g of ethyl 5-((2-aminoethyl)amino)-4-oxopentanoate, stir to dissolve, then slowly drop 20 ml of acetic anhydride, control the reaction endpoint by TLC, after the reaction is completed, extract three times with water, concentrate the organic phase to a small volume, add 20 ml of methanol, heat and stir evenly, then cool and crystallize to obtain 8.6 g of compound A.

[0031] Similarly, the 5-ALA ethyl ester in the above synthetic route is replaced by 5-ALA or 5-ALA methyl ester to obtain compound B and compound C.

[0032]

[0033]

[0034] HepG2 human liver cancer cells and MCF-7 human breast cancer cells, which are commonly used in pharmacological research, were used for cell activity test. The cell culture medium was DMEM high-glucose medium, which was prepared by adding antibiotics and 10% fetal bovine serum. The culture medium was cultured in a cell culture incubator with appropriate light during the test.

[0035] Figure 1 This is the effect of leonurine, compound A, compound B and 5-ALA on the cell activity of HepG2 and MCF-7 cells after 24 hours of action.

[0036] The results show that the cell survival rate of the leonurine treatment group is higher than 90%, indicating that leonurine has no significant inhibitory effect on the two tumor cells. When the sample concentration is 1.0ug / mL, the survival rates of HepG2 cells in the compound A, compound B and 5-ALA treatment groups are 85.48%, 55.39% and 7.24%, respectively; the survival rates of MCF-7 cells are 88.82%, 72.11% and 67.01%, respectively. The inhibitory effects of compound A, compound B and 5-ALA on HepG2 cells are higher than their inhibitory effects on MCF-7 cells, indicating that compound A, compound B and 5-ALA have selective inhibitory effects on the two tumor cells. As the concentration of the test sample increases, the survival rate of the two cancer cells also decreases significantly, indicating that compound A and compound B have a dose-dependent inhibition on the proliferation of the two cancer cells. In addition, the anticancer effects of compound A and compound B on the two cancer cells were significantly higher than that of 5-ALA, which can prove that the inhibitory proliferation effect of 5-ALA conjugate on the two cancer cells was significantly higher than that of 5-ALA alone. At the same time, the anti-tumor activity of compound A was higher than that of compound B.

[0037] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a 5-ALA conjugate, characterized in that: The steps are: Step A: The amino portion of 2-(methylthio)ethylamine is protected with Boc, then condensed with 5-ALA ethyl ester, and the amino protection is removed with TFA to obtain 5-((2-aminoethyl)amino)-4-oxopentanoic acid ethyl ester; Step B: condensing leonurine with malonic acid, and then condensing with ethyl 5-((2-aminoethyl)amino)-4-oxopentanoate to obtain 5-ALA conjugate, i.e., compound A; Wherein, the 5-ALA ethyl ester in step A can be replaced by 5-ALA or 5-ALA methyl ester, and the intermediate generated in step A is 5-((2-aminoethyl)amino)-4-oxopentanoic acid or 5-((2-aminoethyl)amino)-4-oxopentanoic acid methyl ester; the final products are compound B and compound C; Wherein, the dehydrating agent for the condensation of leonurine malonate and ethyl 5-((2-aminoethyl)amino)-4-oxopentanoate in step B may be acetic anhydride or phosphorus pentoxide; 。 2. Use of the 5-ALA conjugate according to claim 1 in the preparation of anti-tumor drugs, wherein the tumor is a tumor caused by human liver cancer cells or human breast cancer cells.

Images