CN105037429A - Podophyllotoxin phospholipid compound as well as pharmaceutical composition and application of podophyllotoxin phospholipid compound - Google Patents

Abstract

The invention discloses a podophyllotoxin phospholipid compound, its pharmaceutical composition and application. The pharmaceutical composition comprises a podophyllotoxin phospholipid compound or a combined pharmaceutical composition of a podophyllotoxin phospholipid compound and a pharmacodynamically acceptable carrier, and is a liquid preparation, a solid preparation, a semi-solid preparation, a capsule, or a granule , Gels, Injections. The pharmaceutical composition is a podophyllotoxin phospholipid compound or a liposome nanoparticle made of a podophyllotoxin phospholipid compound and an auxiliary agent, and the particle diameter is 10-1000 nanometers. The podophyllotoxin phospholipid compound and its liposome nanoparticle can be used as a liquid preparation, a solid preparation, a semi-solid preparation, a sterilized preparation and an aseptic preparation, have low toxicity, and can be used for efficient treatment of tumors.

Description

A kind of podophyllotoxin phospholipid compound, its pharmaceutical composition and application

technical field

The invention relates to a podophyllotoxin phospholipid compound with antitumor effect, its pharmaceutical composition and application, and relates to the technical field of medicine.

Background technique

Podophyllotoxin (podophyllotoxin), also known as podophyllin and podophyllotoxin, is an anti-tumor natural lignan compound isolated from plants of the genus Podophyllum. It has various biological activities such as anti-tumor, anti-virus and anti-inflammatory. Among them, the remarkable antitumor activity is the most proud advantage of this kind of compound. As early as 1942, Kaplan confirmed that podophyllotoxin had an inhibitory effect on tumor growth, and then King and Sullivna pointed out that podophyllotoxin inhibited tumor growth by preventing the formation of microtubule bundles in the metaphase of cell mitosis.

Podophyllotoxin has significant antitumor activity, but its side effects are relatively large, especially the toxicity to the gastrointestinal tract, so it cannot be directly used clinically. Since the 1950s, podophyllotoxins have been structurally modified at home and abroad, and a large number of derivatives have been synthesized and screened. The Swiss Sandoz Company has semi-synthesized two glycoside compounds of epipodophyllotoxin: Etoposide (VP-16) and Teniposide (VM-26), and found that they have very high antitumor activity. active. They have been approved by the US FDA and are widely used in the clinical treatment of testicular cancer, lymphoma, leukemia, non-small cell lung cancer, etc. However, in the process of use, it is found that these compounds still have disadvantages such as poor water solubility, large toxic and side effects, and easy drug resistance.

Podophyllotoxin itself achieves anti-tumor effects by preventing tubulin from polymerizing to form microtubules; while 4'-desmethyl epipodophyllotoxins such as etoporpol and tenipop are inhibitors of DNA topoisomerase II It is an anti-tumor agent that hinders DNA repair by forming a drug-enzyme-DNA stable reversible complex.

In order to overcome the above defects and find more effective drugs for tumor treatment, medicinal chemists have carried out a lot of research work. The water solubility can be improved by making the 4'-phenolic hydroxyl group of Etopolop into phosphoric acid (Etobolol Phosphate) or introducing dimethylamino group (NK-611) on the sugar group. Changing the 4-position substituent can enhance the anti-tumor multi-drug resistance activity, and GL-331 is a compound that introduces a p-nitroaniline group at the 4β-position of podophyllotoxin. Compared with Etopolop, GL-331 has shown strong anti-tumor activity in both in vitro and in vivo tests, especially high anti-tumor multidrug resistance activity. The stability and biocompatibility of its preparations Better, its pharmacokinetic properties are similar to that of Etopolop, and it is currently in phase 2 clinical trials.

Podophyllotoxin and its analogs can also be chemically synthesized or semi-synthesized. The biggest drawback of podophyllotoxin for tumor therapy is poor water solubility, making it difficult to obtain high-concentration solutions. Therefore, it is of great value to improve the water solubility of such compounds, overcome drug resistance, and reduce toxic and side effects. In order to improve the defects of podophyllotoxin, many research institutes and enterprises have modified the chemical structure of podophyllotoxin. Structural modification of podophyllotoxin or analogues, or the use of highly efficient carrier systems, to achieve the purpose of improving drug solubility, targeting, improving curative effect, and reducing toxicity has always been a research hotspot in the field of medicine.

Liposome is a new type of pharmaceutical preparation with targeted drug delivery function, and it has extremely wide application in the field of anticancer drug research. Phospholipids are the main chemical components that make up liposomes, consisting of a hydrophilic head with phosphate-linked substitution genes and a hydrophobic tail with long hydrocarbon chains. When dispersed in the aqueous phase, the hydrophobic tails of the molecules tend to gather together, avoiding the aqueous phase, while the hydrophilic heads are exposed to the aqueous phase, forming closed vesicles with a bilayer structure, forming liposomes. Since the hydrophobic tails of natural phospholipids often contain unsaturated bonds, which have a great impact on the stability of liposomes, structural modification of the hydrophobic tails of phospholipids to improve their stability has gradually become a research hotspot. As a carrier, hydrophobic drugs are usually in the double lipid layer of liposomes, and hydrophilic drugs are in the aqueous phase of liposomes. Due to the fluidity of the liposome membrane, the drug is easy to leak out, making it difficult for the encapsulated drug to exert a good drug effect.

The present invention uses 2 molecules of podophyllotoxin and its derivative chemical structure as hydrophobic tail and phospholipid hydrophilic head to be connected by covalent bond to prepare podophyllotoxin-like phospholipid compound, and its solubility in water is significantly better than that of podophyllotoxin and its derivatives. Derivatives; the present invention prepares podophyllotoxin phospholipid compounds into nanoparticles, which has the characteristics of liposomes, and has the characteristics of forming liquid preparations, solid preparations, semi-solid preparations, sterilized preparations and sterile preparations, and is used for tumors treat. This podophyllotoxin phospholipid compound nanoparticle is not only a prodrug, but also a brand-new drug release carrier for podophyllotoxin drugs, and has a targeting function; the podophyllotoxin phospholipid compound and its liposome nanoparticle of the present invention It is also a brand-new prodrug of podophyllotoxin drugs, which exerts its drug effect through esterase hydrolysis in the body, and has low toxic and side effects.

Contents of the invention

Technical problem: The object of the present invention is to provide a podophyllotoxin phospholipid compound that improves the solubility of podophyllotoxin or its analogs and reduces toxic and side effects, and at the same time provides a pharmaceutical composition based on the podophyllotoxin phospholipid compound, And the application of the podophyllotoxin phospholipid compound in the preparation of antitumor drugs.

Technical solution: In order to name and locate the compound, the position of each atom of podophyllotoxin is defined as follows:

The parent rings of all podophyllotoxins and their analogs or their phospholipid compounds involved in the present invention are defined according to the positions in formula (A).

The podophyllotoxin phospholipid compound of the present invention is a pharmaceutically acceptable salt formed by the compound of the following formula (1) or the compound of the formula (1) and a counter ion:

In formula (1), Ry is a linear or branched alkylene/alkene group with 1 to 10 carbon atoms, or an aromatic hydrocarbon group with 6 to 10 carbon atoms; L represents 2-amino-2- Carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl;

Rx is the 4-position substituent of the podophyllotoxin analogue of the following formula (2) or the 4'-position substituent of the podophyllotoxin analogue of the formula (3),

In formulas (2), (3), R ₁ is any one of the following groups: hydrogen, hydroxyl, methoxy, C ₁ -C ₆ linear or branched alkyl ester groups; R ₂ is Any of the following groups: hydrogen, hydroxyl, amino, C ₁ -C ₆ linear or branched alkylamine, anilino, C ₁ -C ₆ linear or branched alkyl ester group, C ₁ -C ₆ linear or branched alkoxy group, p-nitrophenylamine group, phenylacetate group,

In the podophyllotoxin phospholipid compound of the present invention, Rx is preferably a substituent at the 4-position of podophyllotoxin, a substituent at the 4-position of demethyl epipodophyllotoxin, and a substituent at the 4' position of demethyl epipodophyllotoxin.

In the preferred version of the podophyllotoxin-like phospholipid compound of the present invention, the counter ion is hydrogen ion, sodium ion, potassium ion, chloride ion, nitrate ion, carbonate ion, bromide ion, phosphate ion, acetate, citrate, lactic acid Any one or a combination of any two of fumarate, tartrate, and gluconate ions.

The podophyllotoxin phospholipid compound of the present invention can be prepared according to the following method. First, react the hydroxyl group of podophyllotoxin or podophyllotoxin analog with dianhydride or carboxylic acid to prepare an intermediate containing carboxyl group, and then further prepare the intermediate The body and the hydrophilic part undergo a condensation reaction under the action of a condensation reagent to obtain a podophyllotoxin phospholipid compound.

The pharmaceutical composition of the present invention comprises the above-mentioned podophyllotoxin phospholipid compound or the podophyllotoxin phospholipid compound and a pharmacodynamically acceptable carrier.

The pharmaceutical composition of the present invention is a liquid preparation, a solid preparation, a semi-solid preparation, a capsule, a granule, a gel, an injection, a sustained-release preparation or a controlled-release preparation.

In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is a liposome nanoparticle with a particle diameter of 10-1000 nanometers.

In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is a liposome nanoparticle with a particle diameter of 10-1000 nanometers, and the pharmaceutical composition also includes an auxiliary agent.

In the above preferred version of the pharmaceutical composition of the present invention, the auxiliary agent is one or a combination of phospholipids and cholesterol.

The application of the podophyllotoxin phospholipid compound of the present invention in the preparation of antitumor drugs is to prepare the above podophyllotoxin phospholipid compound or a pharmaceutically acceptable salt thereof and a pharmacodynamically acceptable carrier into a medicament.

The compound of the present invention may exist in the form of isomers, and generally speaking "the compound of the present invention" includes isomers of the compound.

The compound of the present invention may have an asymmetric center with S configuration or R configuration, and the present invention includes all possible stereoisomers and mixtures of two or more isomers.

The present invention also relates to pharmaceutical compositions comprising, as active ingredient, the compound according to the invention or the compound according to the invention and conventional pharmaceutical auxiliaries. Usually the pharmaceutical composition of the invention contains 0.1-100% by weight of the compound of the invention.

Pharmaceutical compositions of compounds of the present invention can be prepared according to methods well known in the art. When used for this purpose, the compound of the present invention or the compound of the present invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if necessary, into a suitable administration form for use as human medicine or dosage form.

The compound of the present invention or the pharmaceutical composition containing it can be administered in the form of unit dosage, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc. The route of administration of the compound of the present invention or the pharmaceutical composition containing it may be injection. The dosage forms for administration may be liquid dosage forms or solid dosage forms.

The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

To form a dosage unit into a capsule, the compound of the present invention as an active ingredient is mixed with the various carriers mentioned above, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredient compound of the present invention can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application.

The compound of the present invention is made into injection preparations, such as solutions, suspension solutions, emulsions, and lyophilized powder injections. This preparation can be aqueous or non-aqueous, and can contain one and/or more pharmacodynamic agents. Acceptable carrier, diluent, binder, lubricant, preservative, surface active agent or dispersant. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the art.

Liposome nanoparticles are prepared from the compound of the present invention, and the particle size is 10-1000 nanometers.

Liposome nanoparticles are made from the compound of the present invention and auxiliary agents, and the particle size is 10-1000 nanometers. The auxiliary agents used are phospholipids, preferably distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and distearoylphosphatidylethanolamine DSPE. Fatty-acylphosphatidyl polyethylene glycol monomethyl ether (polyethylene glycol molecular weight 400-3000), distearoylphosphatidyl polyethylene glycol-targeting group (folate, galactose, antibody, biotin or peptide) , or cholesterol, or one or more of cholesterol derivatives containing targeting groups.

The pharmaceutical composition liposome nanoparticle of the present invention is a liquid preparation, a solid preparation, a semi-solid preparation, a capsule, a granule, a gel, an injection, a sustained-release preparation or a controlled-release preparation.

From the screening of anti-tumor activity in vitro, the compound of the present invention exhibits good anti-tumor activity. Tests show that the toxicity of the compound of the present invention is less than that of podophyllotoxin and demethyl epipodophyllotoxin. Therefore, it can be used as an antitumor drug for animals, preferably for mammals, especially for humans.

From the in vitro anti-tumor activity screening, the compound liposome nanoparticles of the present invention exhibit good anti-tumor activity. Tests show that the in vivo toxicity of the compound liposome nanoparticle of the present invention is far less than that of podophyllotoxin and demethyl epipodophyllotoxin. Therefore, it can be used as an antitumor drug for animals, preferably for mammals, especially for humans.

The preparation method of the podophyllotoxin phospholipid compound liposome nanoparticle of the present invention comprises the mixture of the compound podophyllotoxin phospholipid compound of the present invention or the compound of the present invention and an auxiliary agent, through thin film dispersion method, reverse phase evaporation method, freeze-drying method, ultrasonic dispersion method, spray drying method, film extrusion method, or high pressure homogenization method.

The present invention utilizes 2 molecules of podophyllotoxin or podophyllotoxin analogs as hydrophobic tails and phospholipid hydrophilic heads connected by covalent bonds to prepare podophyllotoxin-like phospholipid compounds, the solubility in water is significantly better than that of podophyllotoxin or podophyllotoxin Analogs; the present invention prepares podophyllotoxin phospholipid compounds into nanoparticles, which has the characteristics of liposomes, and has the characteristics of forming liquid preparations, solid preparations, semi-solid preparations, sterilized preparations and sterile preparations, and is used for tumors treat. This podophyllotoxin phospholipid compound nanoparticle is not only a prodrug, but also a brand-new drug release carrier for podophyllotoxin drugs, and has a targeting function; the podophyllotoxin phospholipid compound and its liposome nanoparticle of the present invention It is also a brand-new prodrug of podophyllotoxin-like drugs, and it is also a drug carrier. It exerts its drug effect through hydrolysis in the body, and has low toxicity and side effects.

The podophyllotoxin phospholipid compounds of the formula (1) of the present invention and the pharmaceutically acceptable salts containing counter ions formed by these compounds have good water solubility, have anti-tumor effects, and can prolong the half-life of drug release without obvious toxic side effect.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

The podophyllotoxin phospholipid compound of the present invention has the following structural formula (1):

In formula (1), Ry is a linear or branched alkylene/alkene group with 1 to 10 carbon atoms, or an aromatic hydrocarbon group with 6 to 10 carbon atoms; L represents 2-amino-2- Carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl; Rx is a podophyllotoxin analog substituent, see technical scheme for details.

The structure of the formula (1) contains a hydrophilic head part, which makes the water solubility of the podophyllotoxin phospholipid compound significantly better than that of podophyllotoxin and demethyl epipodophyllotoxin, and has low toxicity and excellent antitumor activity.

In the formula (1), R _x is a substituent of podophyllotoxin or podophyllotoxin analogue, which is connected to the phospholipid hydrophilic head through an ester bond. In the human body, the bond is degraded by esterase, and a molecule of podophyllotoxin-like phospholipid compound is released Two molecules of podophyllotoxin or podophyllotoxin analogs;

The substituents of R _x podophyllotoxin or podophyllotoxin analogs have a hydrophobic effect and cooperate with each other to directly form a stable liposome, which is a part of the liposome structure and overcomes the universal liposome encapsulation of podophyllotoxin or its analogues The disadvantage of easy leakage of the drug when the drug is hydrophobic, and at the same time improve the efficiency of the drug package.

In formula (1), R ₁ and R ₂ are spacer arms, and the spacer arm and the adjacent two ester bonds have the effect of enhancing the hydrophobicity of the hydrophobic head, and the orderly arrangement of podophyllotoxin phospholipid compounds and self-assembly into lipids Plastid nanoparticles are very beneficial, forming stable liposome particles, thereby prolonging the release and anti-tumor effect time of podophyllotoxin or similar drugs, and at the same time making podophyllotoxin phospholipids easy to add into cells through liposomes, Play a medicinal effect.

The structure of the podophyllotoxin phospholipid compound of the present invention contains two molecules of podophyllotoxin or podophyllotoxin analog active molecules;

The podophyllotoxin phospholipid compound of the present invention can be very easily self-assembled into liposome nanoparticles by thin film method, etc., with a particle size of 10-1000 nanometers;

The podophyllotoxin phospholipid compound of the present invention can form a composite system with phospholipids, and can be easily self-assembled into liposome nanoparticles by thin film method, etc., with a particle size of 10-1000 nanometers;

The liposome structure of the podophyllotoxin phospholipid compound liposome nanoparticles of the present invention is similar to the cell membrane structure, and is easy to be phagocytized by cells, and releases active drug molecules podophyllotoxin or podophyllotoxin analogs to exert anti-tumor effects ;

The podophyllotoxin phospholipid compound liposome nanoparticles of the present invention have a passive targeting effect; and podophyllotoxin analogs such as podophyllotoxin and demethylated epipodophyllotoxin do not have this property;

The podophyllotoxin-like phospholipid compound and its liposome nanoparticle of the present invention are a brand-new release carrier of podophyllotoxin-like drugs, and are also a kind of prodrug;

The podophyllotoxin phospholipid compound and its liposome nanoparticles of the present invention are especially suitable for treating soft tissue tumors such as skin and muscle on the body surface;

The compound of the present invention or the pharmaceutical composition of the compound of the present invention and a conventional drug carrier, containing 0.1-100% by weight of the compound of the present invention, has low toxicity and excellent antitumor activity;

The podophyllotoxin phospholipid compound of the present invention and liposome nanoparticles thereof can be used as liquid preparation, solid preparation, semi-solid preparation, sterilized preparation and aseptic preparation, and can be prepared in water, phosphate buffer, citrate buffer Liposomes are formed in the aqueous phase system;

The podophyllotoxin phospholipid compound liposome of the present invention is combined with a lipid assistant containing a targeting group, and has an active targeting effect;

The preparation process of the podophyllotoxin phospholipid compound of formula (1) and its liposome nanoparticles involved in the present invention is simple;

The podophyllotoxin phospholipid compound of formula (1) and its liposomal nanoparticles involved in the present invention are also a brand-new prodrug of podophyllotoxin-like drugs, which can be degraded by esterase in the body to exert the drug effect and prolong drug release Half-life, and has lower toxic and side effects, anti-tumor effect is significantly better than podophyllotoxin, demethyl epipodophyllotoxin and other podophyllotoxin analogs.

Description of drawings

Fig. 1 is a synthetic route diagram of bis(podophyllotoxin-4-succinic acid) phosphatidylcholine.

Figure 2 is the nuclear magnetic spectrum of bis(podophyllotoxin-4-succinic acid)phosphatidylcholine.

Figure 3 is the mass spectrum of bis(podophyllotoxin-4-succinate)phosphatidylcholine.

Figure 4 is a synthetic route diagram of bis(4'-desmethyl epipodophyllotoxin-4-succinic acid) phosphatidylcholine.

Fig. 5 is a synthetic route diagram of bis(podophyllotoxin-4-succinic acid) phosphatidylethanolamine compound.

Fig. 6 is a synthetic route diagram of bis(podophyllotoxin-4-succinic acid) phosphatidylethanolamine polyethylene glycol compound.

Fig. 7 is a synthetic route diagram of bis(podophyllotoxin-4-suberic acid) phosphatidylglycerol compound.

Figure 8 is a synthetic route diagram of bis(podophyllotoxin-4-adipate)phosphatidylserine compound.

Fig. 9 is a particle size distribution diagram of bis(podophyllotoxin-4-succinate) phosphatidylcholine liposomes.

Fig. 10 is a transmission electron micrograph of bis(podophyllotoxin-4-succinic acid) phosphatidylcholine liposomal nanoparticles.

Fig. 11 double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposome cytotoxicity (MCF-7, U87)

The in vivo antitumor effect of Fig. 12 double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposome

The influence of Fig. 13 double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposome on body weight of nude mice

Detailed ways

The technical solutions of the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

A podophyllotoxin phospholipid compound of the present invention is a pharmaceutically acceptable salt formed by the compound of the following formula (1) or the compound of the formula (1) and a counter ion:

In formula (1), Ry is a linear or branched alkylene/alkene group with 1 to 10 carbon atoms, or an aromatic hydrocarbon group with 6 to 10 carbon atoms; L represents 2-amino-2- Carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl;

Rx is the 4-position substituent of the podophyllotoxin analogue of the following formula (2) or the 4'-position substituent of the podophyllotoxin analogue of the formula (3),

In formulas (2), (3), R ₁ is any one of the following groups: hydrogen, hydroxyl, methoxy, C ₁ -C ₆ linear or branched alkyl ester groups; R ₂ is Any of the following groups: hydrogen, hydroxyl, amino, C ₁ -C ₆ linear or branched alkylamine, anilino, C ₁ -C ₆ linear or branched alkyl ester group, C ₁ -C ₆ linear or branched alkoxy group, p-nitrophenylamine group, phenylacetate group,

In the podophyllotoxin phospholipid compound of the present invention, Rx is preferably a substituent at the 4-position of podophyllotoxin, a substituent at the 4-position of demethyl epipodophyllotoxin, and a substituent at the 4' position of demethyl epipodophyllotoxin.

Counter ions are cationic hydrogen ion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion, anion chloride ion, sulfate ion, sulfate ion, nitrate ion, carboxylate ion, carbonate ion ion, bromide ion, phosphate ion, formate, acetate, citrate, lactate, fumarate, tartrate, gluconate ion or any combination of two, preferably proton, sodium ion, chloride ion .

The method for preparing the podophyllotoxin phospholipid compound, the method first reacts the hydroxyl group of the podophyllotoxin or podophyllotoxin analog with an acid anhydride or carboxylic acid to prepare an intermediate containing a carboxyl group. Then, the intermediate is condensed with glycerophosphocholine to obtain the podophyllotoxin-like phosphatidylcholine compound; or, the intermediate is condensed with monohydroxyl-protected glycerol, deprotected, and reacted with trichloro After the oxon reaction, it is condensed with glycerol acetone, and deacetonated to obtain podophyllotoxin-like phosphatidylglycerol; or, the intermediate is condensed with monohydroxyl-protected glycerol, deprotected, and reacted with phosphorus oxychloride Finally, condensation reaction with the hydroxyl group of aminoethanol protected by tert-butoxycarbonyl Boc, after deprotection, to obtain podophyllotoxin-like phosphatidylethanolamine; or, the intermediate is condensed with monohydroxyl protected glycerol, deprotected, and After reacting with phosphorus oxychloride, it reacts with the hydroxyl group of serine protected by both amino and carboxyl groups, and deprotects the group to obtain podophyllotoxin-like phosphatidylserine.

Another method is to condense the hydroxyl group of podophyllotoxin or podophyllotoxin analogs with two carboxyl groups of glyceryl dicarboxylic acid dimonoester protected by a single hydroxyl group, remove the hydroxyl protection group, and further react with phosphorus oxychloride for activation, and then , or condense with the hydroxyl group of amino-protected aminoethanol to obtain podophyllotoxin-like phosphatidylethanolamine after deprotection; or condense with glycerol acetonide to obtain podophyllotoxin-like phosphatidylglycerol through deacetonation; Either react with dimethylaminoethanol and methylate to obtain podophyllotoxin-like phosphatidylcholine compounds; or react with the hydroxyl group of serine protected by both amino and carboxyl groups, and deprotect the group to obtain podophyllotoxins Phosphatidylserine.

Use condensation reagents preferably 1,3-dicyclohexylcarbodiimide, dipyridine carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbimide hydrochloride, 1,3-diiso One of propylcarbimide or N,N-carbonyldiimidazole and a tertiary amine, the tertiary amine is preferably 4-dimethylaminopyridine or 4-pyrrolidinylpyridine.

The pharmaceutical composition of the present invention is a liquid preparation, a solid preparation, a semi-solid preparation, a capsule, a granule, a gel, an injection, a sustained-release preparation or a controlled-release preparation.

The pharmaceutical composition of the present invention is a liposome nanoparticle with a particle diameter of 10-1000 nanometers, which is composed of podophyllotoxin phospholipid compounds with the structure of formula (1) self-assembled.

The liposome nanoparticle with a particle diameter of 10-1000 nanometers is formed by co-self-assembling the podophyllotoxin phospholipid compound with the structure of formula (1) and the phospholipid.

The application of the podophyllotoxin phospholipid compound of the present invention in the preparation of antitumor drugs is characterized in that the method combines the podophyllotoxin phospholipid or its pharmaceutically acceptable salt with a pharmacodynamically acceptable carrier Prepared as medicine. The compound of the present invention or the compound of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to make an appropriate administration form or dosage form that can be used as human medicine. Usually the pharmaceutical composition of the invention contains 0.1-100% by weight of the compound of the invention.

The route of administration of the compound of the present invention or the pharmaceutical composition containing it can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc. It can be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and point injection. The dosage forms for administration may be liquid dosage forms or solid dosage forms. For example, the liquid dosage forms can be true solutions, colloids, particulate dosage forms, emulsion dosage forms, and suspension dosage forms. Other dosage forms such as tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

The compound of the present invention can be made into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

The carriers used in the pharmaceutical composition of the present invention are, for example, diluents, absorbents, wetting agents, binders, disintegration inhibitors, lubricants and the like. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

The compound of the present invention is made into injection preparations, such as solutions, suspension solutions, emulsions, and lyophilized powder injections. This preparation can be aqueous or non-aqueous, and can contain one and/or more pharmacodynamic agents. Acceptable carrier, diluent, binder, lubricant, preservative, surface active agent or dispersant. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the art.

The liposome nanoparticle is prepared from the compound of the present invention and an auxiliary agent, and the particle size is 10-1000 nanometers, and the auxiliary agent used is one or more of phospholipids such as dimyristoylphosphatidylcholine or cholesterol. Adjuvants also contain a targeting group folic acid, galactose, antibodies, biotin or peptides.

From the screening of anti-tumor activity in vitro, the compound of the present invention exhibits good anti-tumor activity. Tests show that the in vivo toxicity of the compound of the present invention is less than that of podophyllotoxin and demethyl epipodophyllotoxin. Therefore, it can be used as an antitumor drug for animals, preferably for mammals, especially for humans.

From the in vitro anti-tumor activity screening, the compound liposome nanoparticles of the present invention exhibit good anti-tumor activity. Tests show that the toxicity of the liposome compound of the present invention in vivo is much lower than that of podophyllotoxin and demethylated epipodophyllotoxin.

The preparation method of the podophyllotoxin phospholipid compound liposome nanoparticle of the present invention comprises the mixture of the compound podophyllotoxin phospholipid compound of the present invention or the compound of the present invention and an auxiliary agent, through thin film dispersion method, reverse phase evaporation method, freeze-drying method, ultrasonic dispersion method, spray drying method, film extrusion method, or high pressure homogenization method.

The present invention utilizes two molecules of podophyllotoxin or podophyllotoxin analogs as hydrophobic tails and phospholipid hydrophilic heads linked by covalent bonds to prepare podophyllotoxin-like phospholipid compounds, which have significantly better solubility than podophyllotoxins or podophyllotoxin-like compounds. The present invention prepares the podophyllotoxin phospholipid compound into nanoparticles, has the characteristics of liposomes, has the characteristics of forming liquid preparations, solid preparations, semi-solid preparations, sterilized preparations and aseptic preparations, and is used for tumor treatment . This podophyllotoxin phospholipid compound nanoparticle is not only a prodrug, but also a brand-new drug release carrier for podophyllotoxin drugs, and has a targeting function; the podophyllotoxin phospholipid compound and its liposome nanoparticle of the present invention It is also a brand-new prodrug of podophyllotoxin-like drugs, and it is also a drug carrier, which exerts its drug effect through esterase degradation in the body, can prolong the half-life of drug release, and has lower toxic and side effects.

The following are part of the reagent codes used in the preparation process:

DMAP4-dimethylaminopyridine

CDIN, N'-carbonyldiimidazole

DMSO Dimethyl Sulfoxide

GPC Glycerophosphorylcholine

DBU1,5-diazabicyclo[5.4.0]undec-5-ene

EDCIN, N'-thiocarbonyldiimidazole

HOBT1-Hydroxybenzotriazole

The following examples further illustrate the present invention, but the present invention is not limited to the following examples.

Example 1:

Synthesis of bis(podophyllotoxin-4-succinic acid) phosphatidylcholine (see Figure 1 for the synthetic route)

Podophyllotoxin 0.5g, succinic anhydride 3g, DMAP 0.1g, triethylamine 3g, add dichloromethane as reaction solvent, reflux reaction for 12h; wash with dilute hydrochloric acid three times, filter dilute hydrochloric acid layer, take filter cake; add methanol to dissolve, Place at 4°C for 12 hours, filter, take the filter cake, and dry to obtain the intermediate product podophyllotoxin-4-succinic acid monoester.

Podophyllotoxin-4-succinic acid monoester 0.6g, add CDI1.2g, dichloromethane is reaction solvent, add GPC0.4g and DBU1g to react at room temperature, the obtained reaction solution is purified by column chromatography, and the product bis(podophyllotoxin - 4-succinic acid) phosphatidylcholine (MW1249.4) 0.28g.

The NMR spectrum of the compound is shown in Figure 2, ¹ HNMR (500MHz, (CD ₃ ) ₂ SO): δ6.86 (2H, s, 8-H), 6.56 (2H, s, 5-H), 6.48 (4H, s ,13-H),5.99(2H,d,2'-H),5.69(2H,d,6'-H),5.06(4H,d,11-H),4.30(4H,m,-O- CH ₂ -),3.73(18H,s,3-O-CH ₃ ),3.65(2H,m,-CH ₂ -N ⁺ -),3.12(9H,s,-N ⁺ -(CH ₃ ) ₃ ) , 2.52 (4H, m, -CH ₂ CH ₂ -).

See Figure 3 for the mass spectrum, [M+H] ⁺ m/z, 1250.34.

Example 2:

Synthesis of bis(4'-desmethyl epipodophyllotoxin-4-succinic acid) phosphatidylcholine (see Figure 4 for the synthetic route)

0.5g of 4'-desmethyl epipodophyllotoxin, 3g of succinic anhydride, 0.1g of DMAP, 3g of triethylamine, add dichloromethane as the reaction solvent, react at 45°C for 48h; wash with dilute hydrochloric acid three times, filter the dilute hydrochloric acid layer, Take the filter cake; add methanol to dissolve, place at 4°C for 12h, filter, take the filter cake, and dry to obtain the intermediate product 4'-desmethyl epipodophyllotoxin-4-succinic acid monoester.

Add 0.5g of 4'-desmethyl epipodophyllotoxin-4-succinic acid monoester, add 1.0g of CDI, dichloromethane as the reaction solvent, add 0.3g of GPC and 0.8g of DBU to react at room temperature, and the resulting reaction solution is passed through column chromatography After purification, 0.24 g of the product bis(4'-desmethylepipodophyllotoxin-4-succinic acid) phosphatidylcholine (MW1222.09) was obtained.

¹ HNMR (500MHz, (CD ₃ ) ₂ SO): δ8.73 (2H, s, OH), 6.86 (2H, s, 8-H), 6.56 (2H, s, 5-H), 6.48 (4H, s,13-H),5.99(2H,d,2'-H),5.70(2H,d,6'-H),5.06(4H,d,11-H),4.30(4H,m,-O -CH ₂ -),3.73(12H,s,3-O-CH3),3.65(2H,m,-CH ₂ -N ⁺ -),3.12(9H,s,-N ⁺ -(CH ₃ ) ₃ ) , 2.52 (4H, m, -CH ₂ CH ₂ -).

[M+H] ⁺ m/z, 1223.35.

Example 3:

Synthesis of bis(podophyllotoxin-4-succinic acid) phosphatidylethanolamine (see Figure 5 for the synthetic route)

Take 0.5g of 3-(4-methoxybenzyloxy)propane-1,2-diol, 0.5g of succinic anhydride, 0.1g of DMAP, 0.5g of triethylamine, add dichloromethane as the reaction solvent, add reflux React, react for 12 hours; wash with dilute hydrochloric acid three times, filter the dilute hydrochloric acid layer, and take the filter cake; add 0.5g of CDI, add dichloromethane as the reaction solvent, and react at room temperature for 3h; add 0.5g of podophyllotoxin, DBU0.5g, and react at room temperature 12h, spin dry the solvent; add dilute acetic acid, react at room temperature for 6h, remove the solvent; add phosphorus oxychloride 0.5g, triethylamine 0.5g, add dichloromethane as the reaction solvent, react at 10°C for 24h, spin dry the solvent; Add 0.5 g of aminoethanol, 0.3 g of triethylamine, add dichloromethane as the reaction solvent, react at 10°C for 24 hours, and evaporate the solvent to dryness; add isopropanol and acetic acid aqueous solution, and heat for 24 hours. The resulting reaction solution was purified by column chromatography to obtain 0.31 g of the product bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine. Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.05M hydrochloric acid aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine solid powder containing hydrogen ion counter ions.

¹ HNMR (500MHz, CD3OD: _CDCl3 1:1):.

¹ HNMR (500MHz, (CD ₃ ) ₂ SO): δ8.31(3H,m,-NH ₃ )6.86(2H,s,8-H),6.56(2H,s,5-H),6.48(4H ,s,13-H),5.99(2H,d,2'-H),5.69(2H,d,6'-H),5.06(4H,d,11-H),4.74(2H,m,- OCH ₂ (CH ₂ ))4.30(4H,m,-O-CH ₂ -),3.73(18H,s,3-O-CH3),3.68(2H,m,-CH ₂ -N ⁺ -),2.52 (4H,m, _{-CH2CH2- )} .

[M+H] ⁺ m/z, 1209.33

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium chloride aqueous solution, and lyophilized to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing chloride ion and sodium ion counter ion Ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M potassium bromide aqueous solution, and lyophilized to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing bromide ion and potassium ion counter ion Ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium acetate aqueous solution, and lyophilized to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing acetate ion and sodium ion counter ion solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium carbonate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing carbonate ion and sodium ion counter ion Ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium phosphate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing phosphate ion and sodium ion counter ion Ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium lactate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing lactate ion and sodium ion counter ion solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine is dissolved in 0.1M sodium citrate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phospholipid containing citrate ion and sodium ion counter ion Acetyl ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium fumarate aqueous solution, and lyophilized to obtain bis(podophyllotoxin-4-succinic acid containing fumarate ion and sodium ion counter ion ) Phosphatidylethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium tartrate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing tartrate ion and sodium ion counter ion Ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium gluconate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phospholipid containing gluconate ion and sodium ion counterion Acetyl ethanolamine solid powder.

Bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine was dissolved in 0.1M sodium nitrate aqueous solution, and freeze-dried to obtain bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine containing nitrate ion and sodium ion counter ion Ethanolamine solid powder.

Example 4:

Synthesis of bis(podophyllotoxin-4-succinic acid) phosphatidylethanolamine polyethylene glycol (see Figure 6 for the synthetic route)

Take 1g of mPEG2000-COOH, 0.2g of CDI, and dichloromethane as the reaction solvent, and react at room temperature for 2h; add 0.2g of the bis(podophyllotoxin-4-succinic acid) phosphatidylethanolamine compound in Example 3, and 0.4g of DBU, and heat the reaction 12h. The resulting reaction solution was purified by column chromatography to obtain 0.16 g of the product bis(podophyllotoxin-4-succinic acid)phosphatidylethanolamine polyethylene glycol compound.

Example 5:

Synthesis of bis(podophyllotoxin-4-suberic acid) phosphatidylglycerol (see Figure 7 for the synthetic route)

Take 0.5g of 3-(4-methoxybenzyloxy)propane-1,2-diol, 0.5g of suberic anhydride, 0.2g of DMAP, 0.5g of triethylamine, add dichloromethane as the reaction solvent, add reflux React, react for 12 hours; wash with dilute hydrochloric acid three times, filter the dilute hydrochloric acid layer, take the filter cake; add CDI1g, add dichloromethane as the reaction solvent, and react at room temperature for 2h; add podophyllotoxin 0.6g, DBU0.6g, react at room temperature for 12h, Dry the solvent by rotary evaporation; add dilute acetic acid, react at room temperature for 2 hours, remove the solvent; add 0.5 g of phosphorus oxychloride, 0.5 g of triethylamine, add dichloromethane as the reaction solvent, react at 10 °C for 24 hours, and dry the solvent by rotary evaporation; add glycerin Add 0.5 g of acetone and 0.5 g of triethylamine, add dichloromethane as the reaction solvent, react at 10°C for 24 hours, and evaporate the solvent to dryness; add isopropanol and acetic acid aqueous solution, and heat for 12 hours. The resulting reaction solution was purified by column chromatography to obtain 0.32 g of bis(podophyllotoxin-4-suberic acid) phosphatidylglycerol.

¹ HNMR (500MHz, (CD ₃ ) ₂ SO): δ6.86(4H,d),6.52(4H,s),6.07(4H,s),5.31(2H,d),4.81-4.76(4H,m ),4.65-4.10(11H,m),3.83(18H,s),3.79-3.60(2H,t),3.41-3.39(3H,m),2.8(2H,m),2.35-2.30(8H,m ), 1.65-1.29(16H,m).

[M+H] ⁺ m/z, 1352.20.

Embodiment 6:

Synthesis of bis(podophyllotoxin-4-adipic acid)phosphatidylserine (see Figure 8 for the synthetic route)

Take 0.2g of podophyllotoxin, 0.3g of adipic anhydride, 0.1g of DMAP, and 0.3g of triethylamine, add dichloromethane as the reaction solvent, heat and reflux for 12 hours; wash with dilute hydrochloric acid three times, filter the dilute hydrochloric acid layer, and take filter cake; separate to obtain podophyllotoxin-4-adipate monoester, then add CDI1g, add dichloromethane as the reaction solvent, and react at room temperature for 2h; add 3-(4-methoxybenzyloxy)propane-1, 2-diol 0.6g, DBU1g, react at room temperature for 8h, and dry the solvent by rotary evaporation; add dilute acetic acid, react at room temperature for 2h, remove the solvent; add the compound bis(diisopropylamino)(benzyloxy)phosphine and benzyloxycarbonyl ( Amino) protected serine benzyl ester, tert-butanol peroxide as reaction solvent, heating reaction; palladium carbon catalytic hydrogenation reduction. The resulting reaction solution was purified by column chromatography to obtain 0.13 g of bis(podophyllotoxin-4-adipic acid)phosphatidylserine.

¹ HNMR (500MHz, (CD ₃ ) ₂ SO): δ6.86(2H,s,8-H),6.56(2H,s,5-H),6.48(4H,s,13-H),5.99( 2H,d,2'-H),5.69(2H,d,6'-H),5.06(4H,d,11-H),4.61-4.38(5H,m,OCH,OCH ₂ )4.30(4H, m,-O-CH ₂ -),3.73(18H,s,3-O-CH ₃ ),3.41(12H,m),2.8(2H,m),2.35-2.30(8H,m,-COCH ₂ ) ,1.65-1.60(8H,m).

[M+H] ⁺ m/z, 1309.38.

Bis(podophyllotoxin-4-adipic acid) phosphatidylserine was dissolved in 0.1M sodium chloride aqueous solution, and lyophilized to obtain bis(podophyllotoxin-4-adipic acid) containing sodium ion and chloride ion counter ion Phosphatidylserine solid powder.

Embodiment 8:

Preparation of bis(podophyllotoxin-4-succinate)phosphatidylcholine liposomes

Add 1 mmol of bis(podophyllotoxin-4-succinic acid) phosphatidylcholine obtained in Example 1, add 20 ml of chloroform, and evaporate the solvent at 60° C.; add 20 ml of PBS (pH=7.4) to form a film at 60° C. to obtain bis (podophyllotoxin-4-succinate) phosphatidylcholine liposomal nanoparticle solution. The particle size analysis results are shown in Figure 9 below. The average particle size is 160nm, the Zeta potential is -12.8mV, and the particle size distribution is uniform. The morphology of nanoparticles measured by transmission electron microscopy is shown in Figure 11.

Embodiment 9:

Preparation of bis(4'-desmethylepipodophyllotoxin-4-succinate)phosphatidylcholine liposomes

Add 1 mmol of bis(4'-desmethylepipodophyllotoxin-4-succinic acid) phosphatidylcholine obtained in Example 2, add 20 ml of chloroform, and evaporate the solvent at 60° C.; add 20 ml of PBS (pH=7.4) The membrane was formed at 60° C., and filtered with a 200 nm filter to obtain a bis(4′-desmethylepipodophyllotoxin-4-succinic acid) phosphatidylcholine liposome nanoparticle solution. The particle size analysis results showed that the average particle size was 130nm.

The bis(4'-desmethyl epipodophyllotoxin-4-succinic acid) phosphatidylcholine liposome nanoparticle solution was lyophilized to obtain powdery nanoparticles.

Example 10:

Preparation of bis(podophyllotoxin-4-succinate)phosphatidylcholine liposome A

Take 0.5mmol of bis(podophyllotoxin-4-succinic acid)phosphatidylcholine and 0.5mmol of distearoylphosphatidylcholine DSPC in Example 1, add 20ml of chloroform, and spin dry the solvent at 60°C; add 20ml of PBS (pH=7.4) filming at 60° C. to obtain bis(podophyllotoxin-4-succinic acid) phosphatidylcholine liposome A nanoparticle solution. Particle size analysis showed that the average particle size was 210nm.

Example 11:

Preparation of bis(podophyllotoxin-4-succinate)phosphatidylcholine liposome B

Get two (podophyllotoxin-4-succinic acid) phosphatidylcholine 0.5mmol, distearoylphosphatidylcholine DSPC0.5mmol, distearoylphosphatidylethanolamine-polyethylene glycol-folic acid (polyethylene glycol Molecular weight 1500) 0.2mmol, add 20ml of chloroform, 60 ℃ rotary evaporation to dryness; add 20ml of PBS (pH=7.4) 60 ℃ to form a film, get targeted bis(podophyllotoxin-4-succinic acid) phosphatidylcholine Liposome B. Particle size analysis showed that the average particle size was 165nm. The solution of podophyllotoxin-4-phospholipid liposome B nanoparticles was freeze-dried to obtain powdered nanoparticles.

Example 12:

Preparation of bis(podophyllotoxin-4-succinate)phosphatidylcholine liposome C

Take 0.5mmol of bis(podophyllotoxin-4-succinic acid)phosphatidylcholine, 0.5mmol of distearoylphosphatidylcholine DSPC, and 0.2mmol of cholesterol, add 20ml of chloroform, and evaporate the solvent at 60°C to dryness; add 20ml of PBS (pH=7.4) was filmed at 60°C to obtain a bis(podophyllotoxin-4-succinic acid)phosphatidylcholine liposome C nanoparticle solution. Particle size analysis showed that the average particle size was 180nm. The bis(podophyllotoxin-4-succinate)phosphatidylcholine liposome C nanoparticle solution was freeze-dried to obtain powdery nanoparticles.

Pharmacological experiment

Experimental example 13:

MTT Corporation Cancer Cell Killing Test

Drugs and reagents: Calf serum is the product of Nanjing Shengxing Biotechnology Co., Ltd.; DMSO is analytically pure; RPMI1640 is the product of GIBCO.

Instrument: BIORAD680 microplate reader.

MTT assay: collect well-growing human breast cancer cells MCF-7 and brain tumor cells U87, prepare 1×10-4/mL cell suspension with RPMI1640 medium containing 10% calf serum, and place in a 96-well culture plate Internal inoculation, every empty 100 μ L (containing 1000 tumor cells), put 37 ℃, 5% CO2 incubator and cultivate for 24 hours, add bis(podophyllotoxin-4-succinic acid) phosphatidylcholine prepared in Example 8 For the liposome, a blank control and a solvent control were set in the experiment, and 4 concentrations were set for the tested samples, and 3 parallel wells were set for each concentration, and they were cultured in a 5% CO2 incubator at 37° C. for 4 days. The culture medium was discarded, and 100 μL of MTT solution (0.4 mg/mL, prepared in RPMI1640) was added to each well, and incubated at 37° C. for 4 hours. The supernatant was discarded, and 150 μL of MTT solution was added to each well to dissolve the particles. After shaking slightly, the OD value was measured with a 680-type microplate reader at a detection wavelength of 540 nm and a reference wavelength of 450 nm.

Result calculation: the drug concentration was plotted against the inhibition rate of the cells (Figure 12). From the antitumor activity screening in vitro, the double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposome of the embodiment of the present invention 1 has antitumor activity, and the half inhibitory concentration IC50 of MCF-7 is 180 μ g/ml, The half inhibitory concentration IC50 of U87 was 225μg/ml, and the cytotoxicity of liposomes was enhanced with the increase of drug concentration, showing obvious concentration dependence.

Experimental example 14:

Toxicity test of podophyllotoxin phospholipids in mice

Animals: ICR mice, male, 18-22 g, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd.

The results of the in vivo toxicity test of bis(podophyllotoxin-4-succinic acid)phosphatidylcholine, the compound of the present invention, show that its maximum tolerated dose is greater than 100 mg/kg, indicating that its toxicity is far less than that of podophyllotoxin.

Table 2 Podophyllotoxin phospholipid compounds in vivo toxicity test results in mice

Experimental example 15:

Antitumor test of podophyllotoxin phospholipid liposome

The cultured MCF-7 cell suspension was collected at a concentration of 1×10 ⁷ cells/ml, and 0.1 ml of each was inoculated subcutaneously in the right axilla of BALB/c nude mice (male). The diameter of transplanted tumors in nude mice was measured with a vernier caliper, and the animals were randomly divided into groups when the tumors grew to 75 mm ³ . At the same time, the nude mice in each group started to be administered. For the dosage regimen, see Group and Dosage Scheme. The antitumor effect of the test samples was dynamically observed by measuring the tumor diameter. Evaluation index of antitumor activity: tumor growth inhibition rate (%), the calculation formula is as follows:

Average tumor weight of blank group-average tumor weight of treatment group

Dosing regimen: administration group: double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposome microsphere solution (0.2mg/kg) prepared in embodiment 8, intravenous injection, once a day, volume is 0.2ml, for 3 consecutive weeks. (n=1); blank group: normal saline, intravenous injection, once a day, with a volume of 0.2 ml, for 3 consecutive weeks. (n=3)

Figure 12 shows the anti-tumor results of bis(podophyllotoxin-4-succinic acid) phosphatidylcholine liposomes. As can be seen from the figure, the tumor volume of double (podophyllotoxin-4-succinic acid) phosphatidylcholine liposomes administered by intravenous injection was observed by observing the tumor volume of nude mice inoculated with MCF-7 breast cancer cells for 21 days. The volume is smaller than that of the negative control group (blank group), and it exhibits a certain tumor inhibition effect, and the tumor inhibition rate is about 37%. Figure 13 is the body weight change of nude mice. As can be seen from the figure, there is no significant reduction in the administration group relative to the blank group, indicating that the bis (podophyllotoxin-4-succinic acid) phosphatidylcholine lipid administered intravenously No obvious toxic side effects.

The foregoing embodiments are only preferred implementations of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and equivalent replacements can be made, which are important to the rights of the present invention. Technical solutions requiring improvement and equivalent replacement all fall within the protection scope of the present invention.

Claims (7)

1. A podophyllotoxin phospholipid compound, characterized in that the phospholipid compound is a pharmaceutically acceptable salt formed by a compound of formula (1) or a compound of formula (1) and a counter ion: In formula (1), Ry is a linear or branched alkylene/alkene group with 1 to 10 carbon atoms, or an aromatic hydrocarbon group with 6 to 10 carbon atoms; L represents 2-amino-2- Carboxyethyl, 2-aminoethyl, 2-trimethylaminoethyl cation or 2,3-dihydroxypropyl; Rx is the 4-position substituent of the podophyllotoxin analogue of the following formula (2) or the 4'-position substituent of the podophyllotoxin analogue of the formula (3), In formulas (2), (3), R ₁ is any one of the following groups: hydrogen, hydroxyl, methoxy, C ₁ -C ₆ linear or branched alkyl ester groups; R ₂ is Any of the following groups: hydrogen, hydroxyl, amino, C ₁ -C ₆ linear or branched alkylamine, anilino, C ₁ -C ₆ linear or branched alkyl ester group, C ₁ -C ₆ linear or branched alkoxy group, p-nitrophenylamine group, phenylacetate group, 2. podophyllotoxin phospholipid compound according to claim 1, is characterized in that, described counter ion is hydrogen ion, sodium ion, potassium ion, chloride ion, nitrate ion, carbonate ion, bromide, phosphate ion, acetate, citrate, lactate, fumarate, tartrate, gluconate, any one or a combination of any two. 3. A pharmaceutical composition, characterized in that the composition comprises the podophyllotoxin phospholipid compound or the podophyllotoxin phospholipid compound and a pharmacodynamically acceptable carrier according to claim 1 or 2. 4. according to the pharmaceutical composition of claim 3, it is characterized in that, described pharmaceutical composition is liquid preparation, solid preparation, semisolid preparation, capsule, granule, gel, injection, sustained release preparation or controlled release preparation. 5. The pharmaceutical composition according to claim 3, characterized in that, the pharmaceutical composition is a liposome nanoparticle with a particle diameter of 10-1000 nanometers, and the pharmaceutical composition also includes an auxiliary agent. 6. The pharmaceutical composition according to claim 5, wherein the auxiliary agent is any one or a combination of phospholipids and cholesterol. 7. The application of a podophyllotoxin phospholipid compound in the preparation of antitumor drugs, characterized in that the application is the podophyllotoxin phospholipid compound or its pharmaceutically acceptable salt according to claim 1 or 2, It is prepared into a medicament with a pharmacodynamically acceptable carrier.