阅读说明：本技术 一种紫杉醇前药及其制备方法和应用 (Paclitaxel prodrug and preparation method and application thereof ) 是由 姚庆鑫 黄振涛 郝好 高远 于 2019-03-05 设计创作，主要内容包括：本发明提供了一种紫杉醇前药及其制备方法和应用,本发明提供的紫杉醇前药(TCO-PTX)相比于化疗药物紫杉醇(PTX),通过反式环辛烯醇(TCO)对紫杉醇的药物活性位点进行改性,能够显著降低药物对正常细胞或组织的毒性；改性单元为反式环辛烯醇,结构明确；且当肿瘤细胞预先富集含有四嗪的纳米组装体时,紫杉醇前药会在肿瘤细胞中发生快速特异性的前药激活反应,释放活性药物分子,或偶联到纳米组装体后作为缓释纳米药物,最终实现杀灭肿瘤细胞而不引起药物副作用的目的；此外,紫杉醇前药的制备方法简单、收率及化学纯度高。(Compared with a chemotherapeutic drug Paclitaxel (PTX), the paclitaxel prodrug (TCO-PTX) provided by the invention has the advantages that the drug active site of the paclitaxel is modified through trans-cyclooctenol (TCO), so that the toxicity of the drug to normal cells or tissues can be obviously reduced; the modified unit is trans-cyclooctenol with definite structure; when tumor cells are enriched in the tetrazine-containing nano-assembly in advance, the paclitaxel prodrug can generate rapid and specific prodrug activation reaction in the tumor cells to release active drug molecules, or is coupled to the nano-assembly to be used as a slow-release nano-drug, and finally the purpose of killing the tumor cells without causing drug side effects is achieved; in addition, the taxol prodrug has simple preparation method and high yield and chemical purity.)

1. A paclitaxel prodrug, comprising any one of scissoring TCO axial isomer-paclitaxel, scissoring TCO planar isomer-paclitaxel, conjugated TCO axial isomer-paclitaxel, or conjugated TCO planar isomer-paclitaxel, having the structure:

2. the method of making a paclitaxel prodrug according to claim 1, comprising the steps of: and carrying out a light-shielding reaction on the trans-cyclooctenol p-nitrophenylcarbonate and the paclitaxel in a solvent to obtain the paclitaxel prodrug.

3. The production method according to claim 2, wherein the trans-cyclooctenol comprises any one of a cleaved trans-cyclooctenol axial isomer, a cleaved trans-cyclooctenol plane isomer, a coupled trans-cyclooctenol axial isomer, or a coupled trans-cyclooctenol plane isomer.

4. The preparation method according to claim 2 or 3, characterized in that the reaction is carried out in a manner of avoiding light and further comprises a catalyst;

preferably, the catalyst is any one or a combination of at least two of N, N-diisopropylethylamine, 4-dimethylaminopyridine or triethanolamine.

5. The production method according to any one of claims 2 to 4, wherein the solvent is an anhydrous solvent;

preferably, the solvent is any one of dichloromethane, trichloromethane or carbon tetrachloride or a combination of at least two of them.

6. The preparation method according to any one of claims 2 to 5, wherein the molar ratio of trans-cyclooctenol p-nitrophenylcarbonate to paclitaxel is less than 1:1, preferably (0.2-0.9): 1;

preferably, the molar ratio of the catalyst to the trans-cyclooctenol p-nitrophenylcarbonate is less than 1:1, preferably (0.1-0.9): 1.

7. The preparation method according to any one of claims 2 to 6, wherein the temperature of the light-shielding reaction is 20 to 35 ℃;

preferably, the reaction time is 12-48 h.

8. The method according to any one of claims 2 to 7, wherein the trans-cyclooctenol p-nitrophenylcarbonate is prepared by reacting 4-nitrophenol chloromethyl ester with trans-cyclooctenol.

9. The method of claim 8, wherein the reaction further comprises a base;

preferably, the base is triethylamine and/or pyridine;

preferably, the molar ratio of the alkali to the 4-nitrophenol chloromethyl ester is (1-2) to 1, preferably 1: 1;

preferably, the molar ratio of the trans-cyclooctenol to the 4-nitrophenol chloromethyl ester is (1-2) to 1, preferably 1: 1;

preferably, the solvent for the reaction is any one or a combination of at least two of tetrahydrofuran, diethyl ether or methyl tert-butyl ether;

preferably, the reaction temperature is 25-35 ℃;

preferably, the reaction time is 12-48 h.

10. The use of the paclitaxel prodrug according to claim 1 for the preparation of an antitumor drug.

Technical Field

The invention belongs to the field of biological medicines, relates to a paclitaxel prodrug and a preparation method and application thereof, and particularly relates to a paclitaxel prodrug capable of being specifically activated in tumors and a preparation method and application thereof.

Background

Chemotherapy drugs often have major toxic side effects, which limit the amount of drug and its range of use, and may cause cancer recurrence or metastasis. Paclitaxel (paclitaxel, PTX) is a natural secondary metabolite separated and purified from the bark of yew, and has good anti-tumor effect clinically, especially good curative effect on ovarian cancer, uterine cancer, breast cancer and the like with high incidence of cancer. Paclitaxel is one of the hottest anticancer drugs in the international market in recent years. However, paclitaxel has adverse reactions such as anaphylaxis, bone marrow suppression, neurotoxicity, cardiovascular toxicity and the like, and even endangers the life of patients, so the side effects of paclitaxel should be regarded as a great deal of attention. The taxol prodrug with good anticancer effect and large toxic and side effects is prepared by adopting a prodrug activation strategy through chemical structure modification, has low chemical toxicity, and is decomposed to release the original medicine to exert the drug effect through the action of chemicals or enzymes in vivo. Compared with the original medicine, the prodrug keeps or enhances the efficacy of the original medicine and can overcome the toxic and side effects of the original medicine.

Bioorthogonal reactions (biorthogonal reactions) refer to a class of chemical reactions that can be performed in living cells and do not affect the living body itself. At present, Diels-Alder reactions (Diels-Alder reactions) based on inverse electron requirements of Trans-cyclooctene (TCO) and Tetrazine (Tetrazine) have high reaction speed and strong specificity, are bio-orthogonal reactions with rapid development, and can be better suitable for in-vivo prodrug activation strategies.

CN100361985A discloses a paclitaxel prodrug of biodegradable polymer and a synthesis method thereof, wherein the prodrug is formed by bonding polyethylene glycol-aliphatic polyester segmented copolymer and paclitaxel. In the presence of polyethylene glycol (PEG), solvent and catalyst, the ring-opening polymerization of aliphatic cyclic ester is carried out to obtain polyethylene glycol-aliphatic polyester segmented copolymer, the terminal hydroxyl is converted into terminal carboxyl, and then the esterification reaction is carried out with paclitaxel in the presence of condensing agent to obtain paclitaxel prodrug. The prodrug has amphipathy, so that the prodrug can be prepared into a water-based preparation, and the defects of poor water solubility, serious anaphylactic reaction and the like of the existing taxol preparation are overcome, but the taxol prodrug modified by the method cannot obviously reduce the side effect of taxol.

At present, the report of preparing taxol prodrug by using trans-cyclooctenol modified taxol is not available.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a taxol prodrug, a preparation method and application thereof, so as to achieve the aim of reducing the toxic and side effects of the medicine and finally realize safe and effective anti-tumor effect.

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

in a first aspect, the present invention provides a paclitaxel prodrug, wherein the paclitaxel prodrug comprises paclitaxel (TCO), a scissoring TCO axial isomer_daxPTX), a shear type TCO planar isomer paclitaxel (TCO)_deq-PTX), coupled TCO axial isomer-paclitaxel (TCO)_laxPTX) or coupled TCO planar isomer paclitaxel (TCO)_leq-PTX), the structure is as follows:

the taxol prodrug modification unit provided by the invention is trans-cyclooctenol, and the structure of the taxol prodrug modification unit is clear. When tumor cells are pre-enriched with tetrazine-containing nano-assemblies, the paclitaxel prodrug undergoes a rapid specific bio-orthogonal shearing or coupling reaction in the tumor cells. For the taxol prodrug obtained by modifying the shearing TCO, the TCO is quickly removed to release raw taxol; for the paclitaxel prodrug obtained by modifying the coupling TCO, the TCO can be connected to the nano assembly to obtain nano paclitaxel, and the nano drug is degraded by cells to release original drug paclitaxel along with time and can be used as a slow-release nano drug. More importantly, the taxol prodrug provided by the invention can obviously reduce the toxicity of the original medicine taxol on cells, thereby relieving the toxic and side effects of chemotherapeutic drugs.

In a second aspect, the present invention provides a method for preparing a paclitaxel prodrug, comprising the steps of: and carrying out a light-shielding reaction on the trans-cyclooctenol p-nitrophenylcarbonate and the paclitaxel in a solvent to obtain the paclitaxel prodrug.

The preparation method provided by the invention is simple to operate, and the prepared taxol prodrug has high chemical purity and high yield. The modified unit trans-cyclooctenol used in the invention can passivate the active site of paclitaxel on the spatial structure, and can realize high-efficiency specific activation of paclitaxel prodrug or obtain nano sustained-release drug in tumors, thereby realizing safe and effective anti-tumor effect.

Preferably, the trans-cyclooctenol comprises a trans-cyclooctenol axial isomer in cleaved form (TCO)_dax) Trans-cyclooctenol plane isomer (TCO) in shearing form_deq) Coupled trans-cyclooctenol axial isomer (TCO)_lax) Or coupled trans-cyclooctenol plane isomer (TCO)_leq) Any one of them. The structure is shown as the following formula:

preferably, a catalyst is further included in the light-shielding reaction.

Preferably, the catalyst is any one or a combination of at least two of N, N-diisopropylethylamine, 4-dimethylaminopyridine or triethanolamine.

Preferably, the solvent is a dry solvent.

Preferably, the solvent is any one of dichloromethane, trichloromethane or carbon tetrachloride or a combination of at least two of them.

Preferably, the molar ratio of trans-cyclooctenol p-nitrophenylcarbonate to paclitaxel is less than 1:1, preferably (0.2-0.9): 1, and may be, for example, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, or 0.9:1, and the like.

Preferably, the molar ratio of the catalyst to the trans-cyclooctenol p-nitrophenylcarbonate is less than 1:1, preferably (0.1-0.9): 1, and may be, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, or 0.9: 1.

Preferably, the temperature of the light-shielding reaction is 20 to 35 ℃, and for example, the temperature can be 20 ℃, 25 ℃, 30 ℃, 35 ℃ and the like.

Preferably, the reaction time is 12-48 h, such as 12h, 15h, 18h, 20h, 25h, 30h, 35h, 40h, 45h or 48 h.

Preferably, the trans-cyclooctenol p-nitrophenyl carbonate is prepared by reacting 4-nitrophenol chloromethyl ester with trans-cyclooctenol.

Preferably, the reaction of chloromethyl 4-nitrophenol with trans-cyclooctenol further comprises a base.

Preferably, the base is triethylamine and/or pyridine.

Preferably, the molar ratio of the base to 4-nitrophenol chloromethyl ester is (1-2: 1), and may be, for example, 1:1, 1.2:1, 1.3:1, 1.5:1, 1.6:1, 1.8:1, or 2:1, preferably 1: 1.

Preferably, the molar ratio of the trans-cyclooctenol to 4-nitrophenol chloromethyl ester is (1-2): 1, and may be, for example, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1, and preferably 1: 1.

Preferably, the solvent for the reaction of 4-nitrophenol chloromethyl ester and trans-cyclooctenol is any one or a combination of at least two of tetrahydrofuran, diethyl ether or methyl tert-butyl ether.

Preferably, the temperature for the reaction of the 4-nitrophenol chloromethyl ester and trans-cyclooctenol is 25-35 ℃, and can be 25 ℃, 28 ℃, 30 ℃, 32 ℃ or 35 ℃ and the like.

Preferably, the reaction time of the 4-nitrophenol chloromethyl ester and trans-cyclooctenol is 12-48 h, for example, 12h, 15h, 18h, 20h, 25h, 30h, 35h, 40h, 45h or 48h and the like.

As a preferred technical scheme, the preparation method of the paclitaxel prodrug provided by the invention specifically comprises the following steps:

(1) reacting trans-cyclooctenol and 4-nitrophenol chloromethyl ester in a molar ratio of (1-2): 1 in a solvent in the presence of alkali, wherein the molar ratio of the alkali to the 4-nitrophenol chloromethyl ester is (1-2): 1, and the reaction is carried out at 25-35 ℃ for 12-48 h to obtain trans-cyclooctenol p-nitrophenylcarbonate;

(2) the trans-cyclooctenol p-nitrophenyl carbonate and paclitaxel with the molar ratio of less than 1:1 are reacted in a solvent in the presence of a catalyst, wherein the molar ratio of the catalyst to the trans-cyclooctenol p-nitrophenyl carbonate is less than 1:1, and the paclitaxel prodrug is obtained after the reaction is carried out for 12-48 hours at the temperature of 20-35 ℃ in a dark place.

In a third aspect, the present invention provides a paclitaxel prodrug as described in the first aspect, for use in preparing an anti-tumor drug.

Specifically, when the paclitaxel prodrug is applied, the paclitaxel prodrug obtained by modifying the shearing TCO is used for a prodrug activation strategy, and the paclitaxel prodrug is quickly and specifically released in vivo; the paclitaxel prodrug obtained by modifying the coupling TCO can be used for preparing sustained-release nano paclitaxel medicaments in tumor in situ.

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

(1) compared with taxol bulk drugs, the taxol prodrug provided by the invention can passivate the active site of taxol on a spatial structure by using trans-cyclooctenol, and the EC of the taxol prodrug₅₀About 0.34 μ M, significantly reducing the toxicity to cells.

(2) The paclitaxel prodrug provided by the invention has definite modification units, and after entering tumor cells enriched with tetrazine functional nano assemblies in advance, the paclitaxel prodrug can generate rapid and specific bio-orthogonal shearing or coupling reaction, and can be rapidly and specifically activated in cancer cells or used as a slow-release nano medicament.

(3) The preparation method provided by the invention is simple to operate, and the prepared taxol prodrug has high chemical purity and high yield which can reach over 80%.

Drawings

FIG. 1 is a scissoring TCO axial isomer-paclitaxel prodrug (TCO) prepared in example 1 of the present invention_daxPTX) and paclitaxel prodrug and preaccharified tetrazine nanoassembly activated paclitaxel prodrug on cervical cancer cells (helacels) for 72 hours.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.