Prodrug modification method for improving intestinal absorption rate of drug

Abstract

The invention discloses a prodrug modification method for improving the intestinal absorption rate of a drug, belongs to the field of biochemical engineering and enzyme catalysis, and particularly relates to a prodrug synthesized step by step from an active drug with groups such as (primary) carboxyl and (primary) amino through chemical-enzyme catalysis. Aiming at the problems of high reaction temperature and addition of a chemical catalyst in the existing full-chemical process, the invention provides a green and environment-friendly chemical-enzymatic synthesis process, which comprises the following steps: firstly, synthesizing an intermediate from an active drug and a linker by using a chemical method or an enzymatic method; and then synthesizing an active drug-cholic acid derivative by using an enzyme method, and separating and purifying to obtain the prodrug. Compared with the current traditional total chemical synthesis method, the method has the advantages of mild reaction conditions, small pollution, simple process and high yield, and has the potential of wide industrial popularization and application.

Description

Technical fields:

[0001] This invention belongs to the fields of biochemical engineering and enzyme catalysis, and particularly relates to a method for modifying prodrugs to improve the intestinal absorption rate of drugs. Technical background:

[0002] Currently, most drugs suffer from poor water solubility, low oral absorption, poor drug distribution, and adverse reactions with long-term administration. Therefore, extensive research is focused on developing new dosage forms with high bioavailability. Current administration methods include tablets, suspensions, liposomes, chitosan nanospheres, and solid dispersions, all of which improve drug absorption to some extent. Regarding drug modification, some studies have shown that bile acid derivatives have significant solubilizing effects, but these often involve a two-step chemical synthesis of prodrugs, which carries risks due to high reaction temperatures and the addition of chemical catalysts.

[0003] Lipases are widely found in plants, animals, and microorganisms, making them readily available. They possess diverse catalytic abilities and, as an industrial enzyme preparation, can catalyze reactions such as esterification, transesterification, and ester synthesis, finding wide application in oil processing, food, and pharmaceuticals. Since lipases can catalyze esterification and transesterification reactions in organic phases, and most drugs possess active groups such as primary carboxyl and primary amino groups, and bile acid molecules also contain carboxyl groups, selecting a suitable linker can satisfy the enzyme's catalytic reaction type, ensuring synthetic feasibility.

[0004] Research on prodrug modification using enzymatic catalysis is limited, yet enzymatic synthesis offers advantages such as mild reaction conditions, high efficiency, environmental friendliness, and low impurity levels. Developing a novel, green, and efficient prodrug modification method to improve intestinal absorption, based on existing chemical synthesis methods, is a noteworthy issue.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention:

[0006] This invention proposes a chemical-biological enzymatic catalytic method for preparing modified prodrugs. This method provides a green and environmentally friendly chemical-biological enzymatic synthesis process. The active drug, linker, and bile acid are linked in two steps to produce an active drug-choleic acid derivative, thus preparing prodrugs in a mild, environmentally friendly, and efficient manner.

[0007] This invention includes the following steps: First, reacting the (primary)carboxyl group, (primary)amino group, etc., in the active drug that can form ester or amide bonds with a linker to obtain an active drug-linker intermediate; Second, esterifying the intermediate obtained in the first step with bile acid to produce an active drug-choleic acid derivative.

[0008] The linker is a long multi-carbon chain with an amino group at one end and a hydroxyl group at the other. The catalysts used in the following examples are lipases.

[0009] Optionally, the method further includes, in the first step of catalysis, the reaction process is carried out using chemical methods and biological enzymatic methods respectively, and in the second step of catalysis, enzyme catalysis is used.

[0010] Preferably, the first step of the reaction uses DMF as a solvent and is heated to complete the chemical synthesis.

[0011] Preferably, the first step reaction uses chloroform as a solvent, and various lipases catalyze the enzymatic synthesis at a suitable temperature.

[0012] Preferably, in the first step of the chemical method, the high temperature conditions are not met and no catalyst is added separately; in the first step of enzyme catalysis, lipase Novozym435 is used as the catalyst; in the second step, lipase RM is used as the catalyst.

[0013] Optionally, the method further includes the selection of enzyme catalysts and the adjustment of substrate ratio, reaction temperature, and enzyme amount. Based on the catalytic differences of different enzyme catalytic conditions, appropriate reaction conditions should be selected.

[0014] Preferably, the enzyme-catalyzed reaction temperature range is 45°C to 60°C.

[0015] Preferably, the lipases are Novozym435, RM, or TLIM.

[0016] Preferably, the amount of enzyme used is in the range of 0.025g to 0.3g.

[0017] Preferably, the substrate ratio is 1:1, 1:2, 1:3, 1:4, or 1:5.

[0018] In this embodiment of the invention, the amount of enzyme catalyst used is 0.1g, the substrate ratio is 1:5, the reaction temperature is 50°C, and the enzyme type is selected as lipase Novozym435 in the first step and lipase RM in the second step.

[0019] Beneficial effects:

[0020] This invention addresses the problems of pollution, numerous byproducts, and many impurities associated with chemical synthesis by providing an enzyme-catalyzed method for prodrug modification. This method uses biological enzymes to catalyze esterification reactions for intermolecular linkages, and has the advantages of being pollution-free, easy to operate, and having low energy consumption.

[0021] This invention proposes a novel chemical-bioenzymatic synthesis method for prodrugs, with a conversion rate of 51% in the first step of chemical synthesis, 2% in the first step of bioenzymatic synthesis, and 28% in the second step of bioenzymatic synthesis. Attached image description:

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a reaction flow diagram. Detailed implementation method:

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] It should be noted that the catalysts involved in the following examples can be any type of lipase.

[0026] Example 1

[0027] According to the method for synthesizing doxorubicin-cholic acid derivatives provided by the present invention, a chemical-enzymatic method is used to synthesize doxorubicin-cholic acid derivatives. Doxorubicin and a linker (taking 8-amino-1-octanol as an example) are synthesized by chemical and enzymatic catalysis, respectively, to synthesize doxorubicin-linker. Then, the doxorubicin-cholic acid derivatives are synthesized by enzymatic catalysis. The specific steps are as follows:

[0028] S1: In the first step of the chemical synthesis, doxorubicin and 8-amino-1-octanol (molar ratio 1:3) were dissolved in DMF, heated in a metal bath at 95°C and 700 rpm for 24 h, and then extracted at low temperature after adding an appropriate amount of water to obtain the crude product. In the bioenzymatic synthesis, doxorubicin and cholic acid were dissolved in chloroform, Novozym 4350.1 g of enzyme was added, the metal bath temperature was 50°C, the rotation speed was 700 rpm, and the reaction time was 48 h. The reaction solution was then rotary evaporated to obtain the crude product.

[0029] S2: The crude product obtained in the first step was separated by silica gel column chromatography using dichloromethane:methanol 17:1 (v / v) as the eluent. The new product was obtained by rotary evaporation in conjunction with TLC analysis.

[0030] S3: The conversion rate was determined by liquid chromatography, and the relative molecular mass of the product was determined by mass spectrometry, confirming it as doxorubicin-linker.

[0031] The conversion rates of doxorubicin-linker in this embodiment were calculated to be 52.61% and 2.64%.

Claims

1. A method for modifying prodrugs to improve intestinal absorption of drugs, characterized in that, include: The active drug is linked to a linker via chemical or enzymatic methods, then linked to bile acids via an enzyme catalyst, and finally separated and purified to obtain the prodrug. The active drug structure should contain active groups such as (primary)carboxyl and (primary)amino groups that can form ester or amide bonds. The enzyme catalyst is a lipase preparation.

2. The method according to claim 1, characterized in that, The bio-enzyme preparations are immobilized lipases Novozym435 and RM.

3. The method according to claim 1, characterized in that, The reaction is carried out in an organic solvent. The organic solvent used in the chemical synthesis is DMF, and the organic solvent used in the enzymatic synthesis includes either chloroform or acetone.

4. The method according to claim 1, characterized in that, The reaction is carried out under normal pressure; the reaction is carried out in a metal bath, a magnetically stirred oil bath, at a speed of 400 rpm-800 rpm.

5. The method according to claim 1, characterized in that, The amount of catalyst used is 0.1g.

6. The method according to claim 1, characterized in that, The chemical reaction temperature is 95℃ and the reaction time is 24h; the biological enzymatic reaction temperature is 50℃ and the reaction time is 48h. Preferably, the temperature of the bio-enzymatic reaction is 45–60°C.

7. The method according to claim 1, characterized in that, The method further includes separation and purification after the reaction, wherein the active drug-linker and the active drug-cholic acid derivative are separated and purified by column chromatography. Preferably, the eluent ratio used in the column chromatography separation and purification is dichloromethane:methanol 17:

1.

8. The method according to claim 1, characterized in that, The method further includes post-processing the product after the reaction is completed; Preferably, the post-processing includes adding water to precipitate and filtering to obtain a crude product, and freeze-drying to obtain a dried crude product.

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