阅读说明：本技术 一种前药激活化合物、前药体系及其制备方法和应用 (Prodrug activating compound, prodrug system, preparation method and application thereof ) 是由 姚庆鑫 黄振涛 郝好 高远 于 2019-03-21 设计创作，主要内容包括：本发明提供一种前药激活化合物,其特征在于,所述前药激活化合物包括多肽片段,以及通过化学键连接在所述多肽片段上的四嗪基团和疏水基团；所述多肽片段的氨基酸中至少有一个磷酸化的酪氨酸。本发明提供的前药激活化合物具有靶向性和专一性,能够在肿瘤细胞内快速高效地累积并原位自组装形成纳米组装体,进而高效特异性地激活反式环辛烯改性的抗肿瘤前药；本发明的前药激活化合物及前药体系生物相容性好,无系统毒性。所述前药激活化合物的制备方法中采用固相合成法,操作简单,得到的产物化学纯度高,总产率高。(The invention provides a prodrug activating compound, which is characterized by comprising a polypeptide fragment, and a tetrazine group and a hydrophobic group which are connected on the polypeptide fragment through chemical bonds; the polypeptide fragment has at least one phosphorylated tyrosine in the amino acids. The prodrug activating compound provided by the invention has targeting property and specificity, can be rapidly and efficiently accumulated in tumor cells and can be self-assembled in situ to form a nano assembly, so that the trans-cyclooctene modified antitumor prodrug can be efficiently and specifically activated; the prodrug activating compound and the prodrug system have good biocompatibility and no system toxicity. The preparation method of the prodrug activation compound adopts a solid-phase synthesis method, is simple to operate, and the obtained product has high chemical purity and high total yield.)

1. A prodrug activating compound comprising a polypeptide fragment, and a tetrazine group and a hydrophobic group chemically linked to the polypeptide fragment; the polypeptide fragment has at least one phosphorylated tyrosine in the amino acids.

2. The prodrug activating compound of claim 1 wherein the number of amino acids of the polypeptide fragment is 3 to 6;

preferably, at least one of said amino acids contains 2-NH groups₂The amino acid of (1);

preferably, at least one of the amino acids contains an aryl group;

preferably, the amino acid is selected from any one or a combination of at least two of lysine, phenylalanine, tyrosine, tryptophan, arginine, glycine, alanine, leucine or glutamine;

preferably, the amino acids of the polypeptide fragments are phosphorylated tyrosine, lysine and phenylalanine.

3. The prodrug activating compound of claim 1 wherein a tetrazine group is introduced on a polypeptide fragment by the tetrazine derivative forming an amide bond with the polypeptide fragment;

preferably, the tetrazine derivative contains a carboxyl group;

preferably, the tetrazine derivative is a compound having a structure represented by formula I or formula II:

4. the prodrug activating compound of claim 1, wherein the hydrophobic group is a substituted or unsubstituted aromatic group;

preferably, the substituted or unsubstituted aromatic group is a substituted or unsubstituted naphthyl group, a substituted or unsubstituted phenanthryl group, a substituted or unsubstituted anthryl group, or a substituted or unsubstituted pyrenyl group;

preferably, a hydrophobic group is introduced on the polypeptide fragment by an aromatic compound forming an amide bond with the polypeptide fragment;

preferably, the aromatic compound is a C9-C30 aromatic compound;

preferably, the aromatic compound is an aromatic compound containing a carboxyl group;

preferably, the aromatic compound is 2-naphthylacetic acid, 2-anthracene-9-acetic acid or 1-pyreneacetic acid;

preferably, the prodrug activating compound is a compound having a structure as shown in any one of formula III, formula IV or formula V, or a combination of at least two of the following:

5. a process for the preparation of a prodrug activating compound according to any one of claims 1 to 4, comprising the steps of:

(1) by adopting a solid-phase synthesis method, on carrier resin, amino acid with protected terminal amino group and side chain amino group is taken as raw material, and polypeptide fragments are obtained through condensation and reaction in sequence;

(2) connecting an aromatic compound to the polypeptide fragment obtained in the step (1) to obtain a polypeptide fragment with a hydrophobic group;

(3) removing the carrier resin and the side chain amino protecting group on the polypeptide fragment with the hydrophobic group obtained in the step (2) through reaction; separating and purifying to obtain polypeptide products;

(4) and (4) reacting the polypeptide product obtained in the step (3) with an activated tetrazine derivative, and introducing a tetrazine group into the polypeptide product to obtain the prodrug activated compound.

6. The process for preparing a prodrug activating compound according to claim 5, wherein the protecting group of the terminal amino group in the step (1) is Fmoc protecting group;

preferably, the protecting group of the side chain amino group in the step (1) is a Boc protecting group;

preferably, the carrier resin of step (1) is a carboxyl resin or a 2-chloro-trityl chloride resin;

preferably, the sequential condensation method of step (1) comprises the following steps:

(a) swelling the carrier resin;

(b) mixing the first amino acid with the carrier resin according to the amino acid sequence of the polypeptide fragment, so that the first amino acid and the carrier resin are subjected to coupling reaction and are connected; removing the terminal amino protecting group from the first amino acid;

(c) coupling and ligating the second amino acid to the first amino acid; until the condensation of all amino acids in the polypeptide fragment is completed;

(d) removing the terminal amino protecting group of the last amino acid to obtain the polypeptide fragment;

preferably, the reagent used for swelling in step (a) is dichloromethane;

preferably, the coupling agent used in the coupling reaction is a mixed solution of N, N-diisopropylethylamine, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-dimethylformamide;

preferably, the coupling reaction time is 1.5-3 hours;

preferably, the temperature of the coupling reaction is 20-30 ℃;

preferably, the reaction product is washed after the coupling reaction is completed;

preferably, the cleaning reagent is N, N-dimethylformamide and/or dichloromethane;

preferably, the deprotection agent used for removing the terminal amino protecting group is a mixed solution of piperidine and N, N-dimethylformamide;

preferably, the volume ratio of the piperidine to the N, N-dimethylformamide is 1: 4;

preferably, the duration of removing the terminal amino protecting group is 20-40 min;

preferably, the temperature for removing the terminal amino protecting group is 20-30 ℃;

preferably, the reaction product is washed after the removal of the terminal amino protecting group is completed;

preferably, the cleaning reagent is N, N-dimethylformamide and/or dichloromethane;

preferably, the method for linking the aromatic compound to the polypeptide fragment obtained in step (1) in step (2) is: coupling reaction is carried out on the carboxyl terminal of the aromatic compound and the last amino acid of the polypeptide segment, and then the aromatic compound and the last amino acid of the polypeptide segment are connected;

preferably, the removing agent removed in the reaction in the step (3) is trifluoroacetic acid;

preferably, the reaction removal time in the step (3) is 1.5-3 hours;

preferably, the temperature of the reaction removal in the step (3) is 20-30 ℃;

preferably, the separation and purification method in step (3) is: adding glacial ethyl ether into the filtrate, filtering and washing the precipitate;

preferably, the addition mode of the ethyl glacial ether is dropwise addition;

preferably, the reaction of step (4) is carried out in the presence of an organic base;

preferably, the organic base is N, N-diisopropylethylamine;

preferably, the reaction time in the step (4) is 12-24 hours;

preferably, the temperature of the reaction in the step (4) is 20-30 ℃;

preferably, the activated tetrazine derivative in the step (4) is obtained by activating the tetrazine derivative through an activating agent;

preferably, the activators are N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide;

preferably, the steps (1) to (4) are carried out in the presence of a protective gas; the protective gas is preferably nitrogen.

7. Use of a prodrug activating compound according to any one of claims 1 to 4 as an anti-tumour prodrug activator.

8. A prodrug system comprising the prodrug activating compound of claims 1 to 4 and an anti-tumor prodrug molecule.

9. The prodrug system of claim 7 wherein the anti-tumor prodrug molecule is a trans-cyclooctene modified parent drug molecule;

preferably, the parent drug molecule is selected from any one or a combination of at least two of paclitaxel, doxorubicin, cisplatin or camptothecin.

10. Use of a prodrug system according to claim 8 or 9 for the preparation of a medicament for the treatment of a tumour.

Technical Field

The invention belongs to the technical field of chemical medicine, and particularly relates to a prodrug activating compound, a prodrug system, and preparation methods and applications thereof.

Background

In tumor treatment, small molecule chemotherapy drugs are often used with serious drug side effects (ADR) because small molecule drugs lack selective recognition of tumor cells, often cause toxic and side effects on normal tissues around tumors and even other organs, greatly limit the dose of administration, and may cause failure of chemotherapy. Prodrugs (pro-drugs) are currently important methods for alleviating the side effects of chemotherapeutic drugs, and the design concept is to convert the parent drug into a compound with inactive drug activity by chemical modification or physical entrapment; after entering a designated area in a body, the prodrug is activated by the microenvironment difference (such as over-expressed enzyme, oxide or reductant) or exogenous stimulus (such as light, magnetism or ultrasound) of the area to release an active drug in situ to play a therapeutic role.

CN 108395460A discloses a hypoxia activated adriamycin prodrug and a preparation method thereof, wherein azo bonds with hypoxia activation effect and response functional groups of self-occlusion structures are modified on the amino group at the 3' position of adriamycin to synthesize the adriamycin prodrug, the drug can effectively realize selective activation in a hypoxia environment, and the regulation and control of tumor cell growth inhibition activity are realized by regulating the distribution of subcellular organelles of the adriamycin in a normoxic and hypoxic environment, so that the toxic and side effects of the drug on normal cells are reduced, and the targeting efficiency of tumor therapy is improved. CN 102940651a discloses a method for activating a prodrug by using tumor-targeted bacteria, which uses tumor-targeted bacteria as a natural prodrug activation tool to activate the prodrug. The tumor targeting bacteria can constitutively express endogenous prodrug activating enzyme genes without any gene modification or over-expression of exogenous genes. Santra et al report a folate-modified anti-tumor prodrug in which the targeted molecular folate is covalently linked to the anti-tumor drug doxorubicin via a disulfide bond; because glutathione in the tumor tissue is over-expressed and the surface of the tumor cell has more vitamin receptors than normal cells, the anticancer prodrug can be enriched in the tumor tissue; excess glutathione in the tumor tissue undergoes a reduction reaction with the disulfide bonds in the prodrug, releasing the active drug molecule doxorubicin in the prodrug (Santa Santimukuku, et al. journal of the American chemical society.2011,133(41), 16680). Moses Bio et al, using a photocleavage reaction, designed and synthesized a class of diagnostic therapeutic prodrugs consisting of five parts: photosensitizer, targeted folate functional group, singlet oxygen cleavable aminoacrylate, hydrophilic PEG segment and anticancer parent drug, wherein under the irradiation of 605nm infrared light, the photosensitizer in the prodrug generates singlet oxygen to break aminoacrylate bond, thereby releasing anticancer parent drug (Moses Bio, et al. journal of medicinal chemistry,2013,56(10), 3936).

However, in the prior art, the traditional tumor targeting mainly includes an active targeting tumor strategy relying on antibody protein and a passive targeting delivery strategy relying on a nano-carrier, and although the active targeting of the antibody protein has good targeting property, the active targeting of the antibody protein still faces the difficulties of low drug loading efficiency of the antibody, expression difference of tumor antigens, large difficulty in endocytosis of the antibody and the like, so that the drug targeting efficiency is low; passive targeting by means of nanocarriers faces the problem of low delivery efficiency. The currently developed prodrugs realize the release and activation of parent drugs under the conditions of endogenous activation or exogenous stimulation, wherein endogenous activation conditions such as over-expression enzymes, oxides, reductants and the like exist in normal cells, so that the prodrugs can release drugs at non-focus positions, thereby weakening the treatment effect and causing toxic and side effects; exogenous stimuli such as light, magnetic field, ultrasound and the like are complex to operate and expensive, and inevitably cause damage to normal tissues.

Therefore, the development of a novel prodrug system which has high reactivity, high targeting and specificity to the focus, and no damage to the body caused by reaction substrates and generated byproducts is the research focus in the field.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a prodrug activating compound, a prodrug system, a preparation method and application thereof.

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

in a first aspect, the present invention provides a prodrug activating compound comprising a polypeptide fragment, and a tetrazine group and a hydrophobic group linked to the polypeptide fragment by a chemical bond; the polypeptide fragment has at least one phosphorylated tyrosine in the amino acids.

Illustratively, the amino acids comprising the polypeptide fragment contain 1 phosphorylated tyrosine, 2 phosphorylated tyrosines, 3 phosphorylated tyrosines, 4 phosphorylated tyrosines, or more phosphorylated tyrosines.

The structure of the prodrug activating compound provided by the invention can be divided into three definite functional units, namely a polypeptide fragment, a hydrophobic group and a tetrazine group. Wherein the phosphorylated tyrosine in the polypeptide fragment is a phosphatase response unit, the presence of an overexpressed phosphatase in the tumor cell dephosphorylates the phosphorylated tyrosine in the polypeptide fragment, and the absence of an overexpressed phosphatase in the normal cell, whereby the prodrug activating compound relies on the phosphorylated tyrosine in the polypeptide fragment for targeting to the tumor cell; after the prodrug activating compound reaches tumor cells, hydrophobic groups in the compound gather based on pi-pi interaction, and then self-assemble in situ in the tumor cells to form a nano assembly, so that the prodrug activating compound and the specific enrichment of tetrazine groups in the tumor cells are realized; when the trans-cyclooctene modified anticancer small molecule prodrug has tetrazine groups, inverse electron demand D-A reaction (inverse Diels-Alder reaction) can occur, rearrangement and breakage of chemical bonds occur in the trans-cyclooctene groups, so that a parent drug with pharmaceutical activity in the prodrug is released, and activation of the prodrug is realized.

The design of the prodrug activating compound is to realize targeting based on a specific signal for identifying that a tumor is different from a normal tissue, induce the prodrug activating compound to generate non-covalent supermolecular assembly in the presence of over-expressed phosphatase in a tumor cell to form a nano assembly structure, and further activate a prodrug molecule under the catalysis of a tetrazine group; the mechanism of the prodrug activating compound for activating the prodrug is that tetrazine groups catalyze the bioorthogonal reaction of trans-cyclooctene groups, and the prodrug activating compound has the characteristics of simplicity, high efficiency and high specificity, so that the polypeptide tetrazine prodrug activating compound provided by the invention has good biocompatibility, low immunogenicity and low toxic and side effects.

Preferably, the number of amino acids of the polypeptide fragment is 3-6, such as 4 or 5;

preferably, at least one of said amino acids contains 2-NH groups₂For example at least 1 lysine or arginine;

preferably, at least one of the amino acids contains an aryl group, such as at least 1 phenylalanine or tryptophan;

preferably, the amino acid is selected from any one or a combination of at least two of lysine, phenylalanine, tyrosine, tryptophan, arginine, glycine, alanine, leucine or glutamine;

preferably, the amino acids of the polypeptide fragments are phosphorylated tyrosine, lysine and phenylalanine.

The polypeptide fragment of the invention at least contains three amino acids, one is phosphorylated tyrosine, and the other contains 2-NH₂And the third is amino acid containing aryl, and the amino acid sequence in the polypeptide fragment can be adjusted according to actual conditions. Containing 2-NH₂Amino acids of (2), e.g. lysine, -NH on a chiral carbon₂Participate in condensation reaction with other amino acids to form peptide bond, -NH on side chain₂As a linking unit for a subsequent tetrazine group; the amino acid containing aryl, taking phenylalanine as an example, wherein the aryl and the hydrophobic group in the prodrug activating compound are jointly used as a pi-pi action unit for constructing a nano assembly of the prodrug activating compound on tumor cells; in addition, hydrogen bonding in polypeptide fragments is also prodrug activationThe assembly driving force of the nano-assembly.

Preferably, a tetrazine group is introduced on the polypeptide fragment by forming an amide bond with the polypeptide fragment through a tetrazine derivative;

preferably, the tetrazine derivative contains a carboxyl group;

preferably, the tetrazine derivative is a compound having a structure represented by formula I or formula II:

preferably, the hydrophobic group is a substituted or unsubstituted aromatic group;

preferably, the substituted or unsubstituted aromatic group is a substituted or unsubstituted naphthyl group, a substituted or unsubstituted phenanthryl group, a substituted or unsubstituted anthryl group, or a substituted or unsubstituted pyrenyl group;

preferably, a hydrophobic group is introduced on the polypeptide fragment by an aromatic compound forming an amide bond with the polypeptide fragment;

preferably, the aromatic compound is a C9-C30 aromatic compound, such as a C10, C11, C12, C14, C15, C16, C18, C20, C23, C25, C28 or C30 aromatic compound;

preferably, the aromatic compound is an aromatic compound containing a carboxyl group;

preferably, the aromatic compound is 2-naphthylacetic acid, 2-anthracene-9-acetic acid or 1-pyreneacetic acid;

preferably, the prodrug activating compound is a compound having a structure as shown in any one of formula III, formula IV or formula V, or a combination of at least two of the following:

in another aspect, the present invention provides a process for the preparation of a prodrug activating compound as described above, said process comprising the steps of:

(1) by adopting a solid-phase synthesis method, on carrier resin, amino acid with protected terminal amino group and side chain amino group is taken as raw material, and polypeptide fragments are obtained through condensation and reaction in sequence;

(2) connecting an aromatic compound to the polypeptide fragment obtained in the step (1) to obtain a polypeptide fragment with a hydrophobic group;

(3) removing the carrier resin and the side chain amino protecting group on the polypeptide fragment with the hydrophobic group obtained in the step (2) through reaction; separating and purifying to obtain polypeptide products;

(4) and (4) reacting the polypeptide product obtained in the step (3) with an activated tetrazine derivative, and introducing a tetrazine group into the polypeptide product to obtain the prodrug activated compound.

Preferably, the protecting group of the terminal amino group of step (1) is an Fmoc protecting group;

preferably, the protecting group of the side chain amino group in the step (1) is a Boc protecting group;

preferably, the carrier resin of step (1) is a carboxyl resin or a 2-chloro-trityl chloride resin;

preferably, the sequential condensation method of step (1) comprises the following steps:

(a) swelling the carrier resin;

(b) mixing the first amino acid with the carrier resin according to the amino acid sequence of the polypeptide fragment, so that the first amino acid and the carrier resin are subjected to coupling reaction and are connected; removing the terminal amino protecting group from the first amino acid;

(c) coupling and ligating the second amino acid to the first amino acid; until the condensation of all amino acids in the polypeptide fragment is completed;

(d) removing the terminal amino protecting group of the last amino acid to obtain the polypeptide fragment;

preferably, the reagent used for swelling in step (a) is dichloromethane;

preferably, the coupling agent used in the coupling reaction is a mixed solution of N, N-diisopropylethylamine, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-dimethylformamide;

preferably, the coupling reaction is carried out for a period of time of 1.5 to 3 hours, such as 1.6 hours, 1.8 hours, 2 hours, 2.1 hours, 2.3 hours, 2.5 hours, 2.8 hours, or 3 hours;

preferably, the temperature of the coupling reaction is 20 to 30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃;

preferably, the reaction product is washed after the coupling reaction is completed;

preferably, the cleaning reagent is N, N-dimethylformamide and/or dichloromethane;

preferably, the deprotection agent used for removing the terminal amino protecting group is a mixed solution of piperidine and N, N-dimethylformamide;

preferably, the volume ratio of the piperidine to the N, N-dimethylformamide is 1: 4;

preferably, the duration of the removal of the terminal amino protecting group is 20-40 min, such as 20min, 22min, 25min, 30min, 32min, 35min, 36min, 38min or 40 min;

preferably, the temperature for removing the terminal amino protecting group is 20 to 30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃;

preferably, the reaction product is washed after the removal of the terminal amino protecting group is completed;

preferably, the cleaning reagent is N, N-dimethylformamide and/or dichloromethane;

preferably, the method for linking the aromatic compound to the polypeptide fragment obtained in step (1) in step (2) is: coupling reaction is carried out on the carboxyl terminal of the aromatic compound and the last amino acid of the polypeptide segment, and then the aromatic compound and the last amino acid of the polypeptide segment are connected; and (3) the coupling agent, the coupling reaction time and the coupling reaction temperature used in the coupling reaction in the step (2) are the same as those in the step (1), and the conditions of the cleaning process of the obtained product are the same as those of the cleaning process after the amino acid is condensed in the step (1).

Preferably, the removing agent removed in the reaction in the step (3) is trifluoroacetic acid; both carrier resin and side chain amino protecting groups can be removed under the action of trifluoroacetic acid to obtain polypeptide fragments connected with hydrophobic groups;

preferably, the reaction removal time in step (3) is 1.5 to 3 hours, such as 1.6 hours, 1.8 hours, 2 hours, 2.1 hours, 2.3 hours, 2.5 hours, 2.8 hours or 3 hours;

preferably, the temperature for the reaction removal in step (3) is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃;

preferably, the separation and purification method in step (3) is: adding glacial ethyl ether into the filtrate, filtering and washing the precipitate;

preferably, the addition mode of the ethyl glacial ether is dropwise addition;

preferably, the reaction of step (4) is carried out in the presence of an organic base;

preferably, the organic base is N, N-diisopropylethylamine;

preferably, the reaction time in step (4) is 12-24 hours, such as 13 hours, 14 hours, 15 hours, 17 hours, 19 hours, 20 hours, 21 hours or 23 hours;

preferably, the temperature of the reaction in step (4) is 20-30 ℃, such as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃;

preferably, the activated tetrazine derivative in the step (4) is obtained by activating the tetrazine derivative through an activating agent;

preferably, the activators are N-hydroxysuccinimide and N, N' -diisopropylcarbodiimide;

preferably, the steps (1) to (4) are carried out in the presence of a protective gas; the protective gas is preferably nitrogen.

In another aspect, the present invention provides a prodrug activating compound as described above for use as an anti-tumor prodrug activator.

In another aspect, the present invention provides a prodrug system comprising a prodrug activating compound as described above and an anti-tumor prodrug molecule.

Preferably, the anti-tumor prodrug molecule is a trans-cyclooctene modified parent drug molecule;

preferably, the parent drug molecule is selected from any one or a combination of at least two of paclitaxel, doxorubicin, cisplatin or camptothecin.

The process for the preparation of the trans-cyclooctene-modified parent drug molecule according to the invention is carried out with reference to the prior art, for example Versteegen Ron M, et al, click to Release, instant Doxorubicin Elimination on Tetrazine Ligation [ J ]. Angewandte Chemie,2013,52, 14112.

In another aspect, the present invention provides the use of a prodrug system as described above for the preparation of a medicament for the treatment of a tumour.

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

the prodrug activating compound provided by the invention has targeting property and specificity, can be rapidly and efficiently accumulated in tumor cells and can be self-assembled in situ to form a nano assembly, so that the trans-cyclooctene modified antitumor prodrug can be efficiently and specifically activated; the prodrug activating compound and the prodrug system have good biocompatibility and no system toxicity. The preparation method of the prodrug activation compound adopts a solid-phase synthesis method, is simple to operate, and the obtained product has high chemical purity and high total yield.

Drawings

FIG. 1 is a graph of the cumulative performance of the prodrug activating compound of example 4 of the present invention in HeLa and HUVECs cells;

FIG. 2 is a statistical graph of the activation performance of the prodrug by the prodrug activating compound described in example 5 of the present invention.

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.