Polyethylene glycol modified cardiac glycoside compound prodrug and anti-tumor application thereof
阅读说明：本技术 聚乙二醇修饰的强心苷类化合物前药及其抗肿瘤用途 (Polyethylene glycol modified cardiac glycoside compound prodrug and anti-tumor application thereof ) 是由 殷军 韩娜 李怡雯 叶纯 刘志惠 翟健秀 李嗣凯 于 2019-09-25 设计创作，主要内容包括：本发明属于医药技术领域,涉及聚乙二醇修饰的强心苷类化合物前药及其制备方法,包含所述的化合物前药的药物组合物,及其它们在制备抗肿瘤药物中的应用。所述的前药显著提高了原形药物的水溶性,解决了其给药困难的问题。体外细胞实验显示,该类前体药物具有良好的抑制肿瘤细胞生长的作用。体内药代动力学性质考察显示,该类前药能延长其体内半衰期。裸鼠体内药效评价显示,该类前体药物对裸鼠接种的人肺癌A549细胞株移植瘤具有良好的生长抑制作用,其抑制强度显著优于原形药物,具有更好的抗肿瘤效果。所述的前体药物的结构如下,其中,R<Sub>1</Sub>、R<Sub>2</Sub>、R<Sub>3</Sub>、R<Sub>4</Sub>如权利要求和说明书所述。<Image he="282" wi="700" file="DDA0002213907250000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the technical field of medicines, and relates to a polyethylene glycol modified cardiac glycoside compound prodrug and a preparation method thereof, a pharmaceutical composition containing the compound prodrug, and application of the compound prodrug and the pharmaceutical composition in preparation of antitumor drugs. The above-mentionedThe prodrug remarkably improves the water solubility of the prototype drug and solves the problem of difficult administration. In vitro cell experiments show that the prodrug has good effect of inhibiting the growth of tumor cells. In vivo pharmacokinetic property investigation shows that the prodrug can prolong the in vivo half-life period. The evaluation of the drug effect in the body of a nude mouse shows that the prodrug has good growth inhibition effect on the transplanted tumor of a human lung cancer A549 cell strain inoculated by the nude mouse, the inhibition strength of the prodrug is obviously superior to that of an original drug, and the prodrug has better anti-tumor effect. The structure of the prodrug is shown in the specification, wherein R 1 、R 2 、R 3 、R 4 As described in the claims and specification.)
1. The polyethylene glycol modified cardiac glycoside compound prodrug is characterized in that: comprising at least one of the following general formulae:
wherein the content of the first and second substances,
R 1、R 2is H or OH;
R 3is A-X,
R 4is H or OH or OAc;
R 5is A-X;
a is linear chain or branched polyethylene glycol, and the molecular weight of the polyethylene glycol is 2000-40000;
x is a linker arm comprising- (CH) 2) 2-O-CO(CH 2) 2-CO-，-CH 2-CO-，-(CH 2) 2-O-CO-or- (CH) 2) 2-O-aa-, aa is an amino acid.
2. The polyethylene glycol-modified cardiac glycoside prodrug of claim 1, wherein:
R 1is OH;
R 2is H;
R 3is A-X,
R 5Is A-X;
a is linear chain or branched chain monomethoxy polyethylene glycol, the molecular weight of the polyethylene glycol is 2000-20000;
x is a linker arm comprising- (CH) 2) 2-O-CO(CH 2) 2-CO-，-CH 2-CO-，-(CH 2) 2-O-CO-or- (CH) 2) 2-O-aa-, aa is glycine, alanine, phenylalanine, leucine, proline.
3. The polyethylene glycol-modified cardiac glycoside prodrug of claim 1, wherein:
R 1is OH;
R 2is H;
R 3is composed of
R 5Is A-X;
a is linear chain or branched chain monomethoxy polyethylene glycol, the molecular weight of the polyethylene glycol is 2000-20000;
x is a linker arm comprising- (CH) 2) 2-O-CO(CH 2) 2-CO-，-CH 2-CO-，-(CH 2) 2-O-CO-or- (CH) 2) 2-O-aa-, aa is glycine, alanine, phenylalanine, leucine, proline.
4. The polyethylene glycol-modified cardiac glycoside prodrug of any one of claims 1 to 3, wherein: the connecting arm is a succinic anhydride connecting arm, an ester bond connecting arm, a carbonic ester connecting arm or an amino acid connecting arm.
5. The polyethylene glycol modified cardiac glycoside compound prodrug has the following structure:
6. the method of claim 1, wherein a hydroxyl terminus of monomethoxy polyethylene glycol is activated via a linker and chemically linked to the cardiac glycoside.
7. A pharmaceutical composition comprising the polyethylene glycol-modified cardiac glycoside compound prodrug of any one of claims 1 to 4 and a pharmaceutically acceptable carrier or excipient.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is formulated with a pharmaceutically acceptable carrier into a clinically acceptable nanosuspension, micelle, nanoparticle, nanoemulsion or liposome.
9. Use of the polyethylene glycol-modified cardiac glycoside prodrug of any one of claims 1 to 3 or the pharmaceutical composition of any one of claims 7 to 8 for the preparation of an anti-tumor medicament.
10. The use of claim 9, wherein the neoplasm is lung cancer, gastric cancer, liver cancer, cervical cancer, acute leukemia, colon cancer, breast cancer, sarcoma, nasopharyngeal cancer, ovarian cancer, skin cancer, prostate cancer, bladder cancer, chorioepithelial cancer, kidney cancer, rectal cancer, oral cancer, esophageal cancer, biliary tract cancer, pancreatic cancer, bone cancer, laryngeal cancer, tongue cancer, thymus cancer, lymphoid cancer, malignant thyroid cancer, brain tumor, central nervous system tumor, mediastinal tumor, melanoma.
Technical Field
The invention belongs to the technical field of medicines, and relates to a polyethylene glycol modified cardiac glycoside compound prodrug and preparation and application thereof. In particular to a polyethylene glycol prodrug of cardiac glycoside compounds separated from streptocaulon juventas, a preparation method and anti-tumor application thereof.
Background
Cardiac glycosides (cardioglycosides) are Cardiac steroidal glycosides present in plants. At present, it is known that several hundreds of plants of more than ten families mainly contain cardiac glycosides, especially the Scrophulariaceae and Apocynaceae plants are the most common, and others such as Liliaceae, Asclepiadaceae, Cruciferae, Celastraceae, Leguminosae and Moraceae are also common. Statistically, only the cardiac glycoside component newly found in 1976 to 1995 is more than 250 (Liselotte K, et al. phytochemistry,1998,48(1):1-29), and is mainly present in fruits, leaves or roots of plants. Cardiac glycosides are structurally complex and consist of an aglycone (cardioaglycone) and a sugar. The side chain at C17 position of cardiac aglycone is unsaturated lactone ring, and is delta of five-membered ring αβ-gamma-lactone, known as cardiotonic aglycone a; also having a.DELTA.of a six-membered ring αβ,γδThe-delta-lactones, called cardiogenin b, all belong to the β -configuration (individually α -type) (wu-riarmy, eds natural medicinal chemistry (4 th edition) people health press 2003, 316).
At present, two or thirty cardiac glycosides are used in clinical treatment, mainly for treating heart diseases such as congestive heart failure and dysrhythmia, such as cedilanid, digoxin and digitoxin. However, with the progress of studies on cardiac glycosides, since the 60 s of the 20 th century, reports on cardiac glycoside therapy of tumors began to appear successively (Shiratiri O. Gann,1967,58(6): 521-. Since then, many studies have demonstrated that cardiac glycosides have antiproliferative and apoptosis-inducing effects on a variety of tumor cells, such as breast cancer, prostate cancer, melanoma, pancreatic cancer, non-small cell lung cancer, leukemia, neuroblastoma, renal adenocarcinoma, and the like.
Dark degummed rattan (streptaculon juventas (Lour.) Merr.) is a plant belonging to Asclepiadaceae (Asclepiadaceae) Ma Nelumbus (streptaculon) and is mainly produced in southeast Asia, China is mainly distributed in Yunnan and Guangxi, according to the record in the medicinal plant dictionary, the dark degummed rattan is a folk medicine with few stems, the roots play a role in tonifying the kidney and strengthening the body, the roots and the stems can strengthen the spleen and the stomach, milk has the effect of removing nebula, and is used for treating conjunctivitis.
So far, more than 40 cardiac glycosides have been isolated and identified from streptocaulon juventas at home and abroad and all have antitumor activities of different degrees. In the earlier research, a series of cardiac glycoside anticancer lead compounds which have stronger in vitro anticancer activity than a positive control medicament taxol, basically have no toxic or side effect on organisms and have relatively simple chemical structures are separated from the streptocaulon juventas, and although the compounds have obvious antitumor activity, the clinical application of the compounds is greatly limited due to the characteristics of the medicaments. Firstly, the water solubility and the fat solubility are poor, the acid-base dependence is avoided, and water-soluble salts cannot be prepared for clinical application; secondly, in order to realize intravenous administration, some cosolvents are added in the process of dissolving the medicine, but the cosolvents have strong irritation to blood vessels and certain toxic and side effects on normal tissues and organs; finally, the elimination in vivo is too fast, the half-life period is only about 5-10min, and the drug effect cannot be well exerted. At present, the pH value of the injection such as the regulator is tried firstly; replacing the cosolvent; adding a solubilizer or a miscible agent; preparing cyclodextrin inclusion compound, emulsion or nano suspension. Because of the limitations of the physical properties of the compounds, the preparation has the problems of poor stability, precipitation of crystals during dilution, and too fast metabolism, and cannot replace the existing administration mode of preparing solution by using a small amount of organic solvent as a cosolvent. Secondly, the preparation of small molecule prodrugs such as phosphate, carbonate and benzoate has been tried, but because the toxicity of cardiac glycoside carbonate prodrugs itself is too high, the phosphate prodrugs only increase the water solubility, and the method does not achieve the ideal effect. Therefore, in order to further improve the clinical treatment value of the medicines, numerous solutions are proposed around improving the solubility, the in vivo half-life period and the targeting property of the medicines. Especially, the strategy of combining a macromolecule carrier with high water solubility, in vivo long circulation and tumor targeting with a prototype compound to prepare a macromolecule prodrug which is researched in recent years draws attention.
The polymer prodrug connects drug molecules to a polymer carrier chain through chemical bonds, does not have drug activity, but has specific functionality, such as solubilization, protection, targeted delivery, cell uptake enhancement, release control and the like, and is one of the currently advanced drug property improvement modes of drugs. Currently, the polymer carriers widely used are polyethylene glycol (PEG) and its derivatives. Polyethylene glycol (PEG) has the characteristics of high water solubility, low immunogenicity and high biocompatibility, is widely used for plasma substitutes, freeze-drying protective agents, long-circulating modification materials and the like in clinic, and has been successfully applied to artificially synthesized macromolecules modified by protein polypeptide drugs.
After the cardiac glycoside and its derivative are coupled with PEG via easy-to-dissociate covalent bond, they form prodrug, and after entering into body or reaching target tissue, the combined cardiac glycoside is released again due to metabolism or hydrolysis in body, so that it can play the role of anticancer. When PEG is coupled to drug molecules, their solubility in aqueous solutions can be improved due to the introduction of hydrophilic groups; because the polyethylene glycol has long molecules, a space barrier is generated around the modified drug, so that the enzymolysis of the drug can be reduced, the half-life period is improved, and the aim of drug delivery called Passive targeting (Passive targeting) is achieved while the drug is prevented from metabolizing in the kidney.
At present, reports about preparing prodrugs by modifying small molecular compounds such as paclitaxel, camptothecin or scutellarin and the like with PEG can be seen, but the application of the method to cardiac glycoside compounds is not seen. Therefore, the structural modification of the cardiac glycoside compound by using PEG and derivatives thereof to develop a medicament with good water solubility and high drug effect is an effective method which has wide application prospect and can effectively improve the poor properties of the original medicament.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a series of polyethylene glycol modified cardiac glycoside compound prodrugs, and the prodrugs can be used for preparing antitumor drugs.
Specifically, the invention is realized by the following technical scheme:
the structure of the polyethylene glycol modified cardiac glycoside compound prodrug at least comprises one of the following general formulas:
wherein the content of the first and second substances,
R 1、R 2is H or OH;
R 3is A-X,
R 4is H or OH or OAc;
R 5is A-X;
a is linear chain or branched chain polyethylene glycol, preferably linear chain polyethylene glycol fragment, the polyethylene glycol is monomethoxy polyethylene glycol, the end of the polyethylene glycol is hydroxyl, the molecular weight is 2000-40000, preferably 2000-20000, can be 2000, 5000, 20000, most preferably 5000;
x is a linker arm comprising- (CH) 2) 2-O-CO(CH 2) 2-CO-，-CH 2-CO-，-(CH 2) 2-O-CO-or- (CH) 2) 2-O-aa-, aa is an amino acid including glycine, alanine, phenylalanine, leucine and proline.
Specifically, the structure of the polyethylene glycol modified cardiac glycoside compound prodrug of the invention is as follows:
the invention also provides a preparation method of the polyethylene glycol modified cardiac glycoside compound prodrug, wherein the terminal hydroxyl of the monomethoxy polyethylene glycol is activated through a connecting arm and then chemically connected with cardiac glycoside. The preparation methods of several typical polyethylene glycol modified cardiac glycoside compound prodrugs are as follows:
A. the hydroxyl at the end of the monomethoxy polyethylene glycol is directly oxidized into carboxyl, and then the carboxyl and cardiac glycoside are subjected to coupling reaction under the catalysis of a condensing agent and an organic base, and the synthetic route is as follows:
B. introducing succinic anhydride into terminal hydroxyl of monomethoxy polyethylene glycol, and then carrying out coupling reaction with cardiac glycoside under the catalysis of a condensing agent and an organic base, wherein the synthetic route is as follows:
C. active carbonate is introduced into terminal hydroxyl of monomethoxy polyethylene glycol, and then the active carbonate and cardiac glycoside are subjected to ester exchange reaction under the catalysis of organic base, and the synthetic route is as follows:
D. amino acid is introduced into the terminal hydroxyl of the monomethoxy polyethylene glycol, and then the amino acid and cardiac glycoside are subjected to esterification reaction under the catalysis of a condensing agent and an organic base, and the synthetic route is as follows:
wherein the condensing agent is DCC, DIC, HBTU or EDC, and the most preferable condensing agent is DCC and EDCI.
The organic base is DMAP, pyridine and triethylamine, preferably triethylamine or DMAP.
The reaction solvent is a mixed solvent of one of pyridine, DMF or DMAO and dichloromethane, and the best mixed solvent is dichloromethane and DMF.
The reaction temperature is 0-40 ℃, and the optimal temperature is 0-25 ℃; the reaction time is 2-72 hours, and the optimal reaction time is 8-16 hours.
Compounds I, II, III and IV (wherein the cardiac glycoside is acovogenin A- β -glucoside, hereinafter TXA 9; the compound I is-CH) 2-CO-a cardiac glycoside prodrug as linker; the compound II is represented by- (CH) 2) 2-O-CO(CH 2) 2-CO-a cardiac glycoside prodrug as linker; the compound III is represented by- (CH) 2) 2-O-CO-a cardiac glycoside prodrug as linker arm; the compound IV is represented by- (CH) 2) 2O-aa-a cardiac glycoside prodrug with a linker arm, wherein aa is a common amino acid, such as glycine, alanine, phenylalanine, leucine, proline, etc.) as an example, further illustrates a preparation method of each polyethylene glycol modified cardiac glycoside prodrug.
(1) Process for the preparation of compounds I
After oxidizing the terminal hydroxyl of monomethoxy polyethylene glycol (mPEG) into carboxyl, the carboxyl reacts with TXA9 in a reaction solvent of DCM/DMF under the catalysis of DCC and triethylamine to prepare the compound I.
(2) Process for preparing compounds II
Monomethoxy polyethylene glycol (mPEG) reacts with succinic anhydride, and then reacts with TXA9 in DCM/DMF solvent under the catalysis of DCC and triethylamine to obtain a compound II.
(3) Process for preparing compound III
Monomethoxy polyethylene glycol (mPEG) reacts with p-nitro phenyl chloroformate, and then the obtained product reacts with TXA9 in DCM/DMF solvent under the catalysis of DMAP to obtain a compound III.
(4) Preparation method of compound IV
Enabling monomethoxy polyethylene glycol (mPEG) to react with p-nitrophenyl chloroformate to prepare mPEG-pNP with the tail end being active carbonate, enabling the mPEG-pNP to perform ester exchange reaction with amino acid under an alkaline condition to prepare mPEG-aa, and finally enabling the tail end carboxyl of the amino acid to perform esterification reaction with active hydroxyl of a cardiac glycoside compound in a DCM/DMF solvent under the catalysis of EDCI and DMAP to prepare a compound IV.
The invention further inspects the water solubility of the compounds I-IV according to a method under a solubility experimental item in Chinese pharmacopoeia. The determination result shows that the compounds I-IV can obviously increase the water solubility of cardiac glycoside compounds, and are easier to prepare into various pharmaceutical preparations, and the result is shown in table 1.
In-vitro anti-tumor activity experiments are carried out on the compounds I-IV, and the results show that the prodrug has good inhibitory activity on the growth of human prostate cancer PC-3 cells, human cervical cancer Hela cells, human gastric cancer SGC7901 cells, human lung cancer A549 cells and human liver cancer SMMC-7721 cells, and is equivalent to the inhibitory activity of the original drug on the growth of tumor cells, and the results are shown in Table 2.
The in vivo pharmacokinetic properties of the compounds I-IV were examined. The results show that compared with the prototype drug, the prodrug can increase the blood concentration of the original drug and prolong the in vivo half-life period, wherein the compound II shows the longest in vivo half-life period and the highest area under the time curve, which is more beneficial to enhancing the in vivo anti-tumor effect of the drug, and the results are shown in figure 1 and table 3.
Since compound II showed the best water solubility, the strongest tumor cell inhibitory effect and the longest half-life in vivo among the above experimental results, the in vivo antitumor effect experiment of nude mice was performed on compound II. The experimental results (table 4) show that compared with the TXA9 group, the compound ii can significantly improve the in vivo antitumor effect of the original drug, and the inhibition strength of the high-dose group of the compound ii is equivalent to that of the positive drug control drug paclitaxel, which indicates that the compound ii has a good tumor growth inhibition effect on the human lung adenocarcinoma cell line a549 transplantation tumor inoculated to the nude mouse.
The invention also provides a pharmaceutical composition which comprises the polyethylene glycol modified cardiac glycoside prodrug and a pharmaceutically acceptable carrier or excipient.
The invention also provides application of the polyethylene glycol modified cardiac glycoside compound prodrug and a pharmaceutical composition in preparation of antitumor drugs. The tumor is lung cancer, gastric cancer, liver cancer, cervical cancer, acute leukemia, colon cancer, breast cancer, sarcoma, nasopharyngeal cancer, ovarian cancer, skin cancer, prostatic cancer, bladder cancer, chorioepithelioma, kidney tumor, rectal cancer, oral cancer, esophageal cancer, gallbladder cancer, biliary tract cancer, bile duct cancer, pancreatic cancer, bone cancer, laryngeal cancer, tongue cancer, thymus cancer, lymph cancer, malignant thyroid tumor, brain tumor, central nervous system tumor, mediastinal tumor, and melanoma.
According to the invention, PEG has a strong hydrophilic group and excellent characteristics of improving the half-life period of the medicine, the PEG is used for carrying out structural modification on a series of cardiac glycoside anticancer active compounds from streptocaulon juventas, a series of cardiac glycoside compound prodrugs are prepared by a chemical synthesis method, so that the water solubility of the cardiac glycoside compounds is increased, the half-life period in vivo of the cardiac glycoside compounds is improved, the pharmacokinetic property of the cardiac glycoside compounds is improved, and the antitumor effect in vivo of the cardiac glycoside compounds is further enhanced.
Drawings
FIG. 1 is a graph of the in vivo pharmacokinetics of compounds I-IV.
Detailed Description
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