CN111303175B - A kind of 10-hydroxycamptothecin derivatives and its preparation method, medicine and application - Google Patents

Abstract

The present invention provides a 10-hydroxycamptothecin derivative, and the 10-hydroxycamptothecin derivative has the structure shown in formula (I). The present invention designs a 10-hydroxycamptothecin prodrug. The 10-hydroxycamptothecin derivative of the structure is modified by guanidylation of 10-hydroxycamptothecin to make it easier to be taken up by cells, thereby enhancing the cellular Toxic, more effective to kill tumor cells; at the same time, it effectively solves the problems of poor water solubility and low drug availability of 10-hydroxycamptothecin, and has broad development prospects in the field of tumor treatment. The 10-hydroxycamptothecin derivatives provided by the present invention can not only achieve enhanced water solubility and cellular uptake ability, but also have a simple preparation method, mild conditions, wide raw material sources, can realize batch production, and are suitable for industrialization and large-scale applications and applications. Subsequent expansion.

Description

A kind of 10-hydroxycamptothecin derivatives and its preparation method, medicine and application

technical field

The invention belongs to the technical field of preparation of 10-hydroxycamptothecin derivatives, in particular to a 10-hydroxycamptothecin derivative and its preparation method, medicine and application, in particular to a 10-hydroxycamptothecin prodrug The preparation method, medicine and anti-tumor application thereof.

Background technique

Tumor refers to a new organism (neogrowth) formed by the proliferation of local tissue cells under the action of various tumorigenic factors, because this new organism is mostly space-occupying lumpy protrusions, also known as neoplasms. . According to the cell characteristics of new organisms and the degree of harm to the body, tumors are divided into two categories: benign tumors and malignant tumors, and cancer is the general term for malignant tumors. There are many types of cancer, which seriously threaten human health. They have different types, involved tissues and organs, different disease stages, and different responses to various treatments. Therefore, it is difficult to treat and has always been a researcher in the field. One of the topics of continuous attention and research.

Camptothecin is a plant anti-cancer drug, extracted from Camptotheca, which is distributed in central and southwestern China. In 1976, Chinese chemist Gao Yisheng and others successfully synthesized racemic camptothecin. Camptothecin has a good short-term curative effect on gastrointestinal cancer and head and neck cancer, but it has the side effect of hematuria in a few patients. To this end, the researchers found 10-hydroxycamptothecin, namely ((S)-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indone Azino[1,2-B]quinoline-3,14-(4H,12H), HCPT. It is a hydroxyl-derived active compound of camptothecin, and its structural formula is as follows:

10-Hydroxycamptothecin is another alkaloid extracted from camptothecin, so it can be considered as a natural derivative of camptothecin. The mechanism of action of 10-hydroxycamptothecin is similar to that of camptothecin. It inhibits DNA topoisomerase I mainly in the DNA synthesis phase (S phase). It has stronger anti-tumor activity than camptothecin, and has less urinary toxicity and side effects than camptothecin. It is a broad-spectrum cytotoxic chemotherapeutic drug, which is widely used in clinical anti-cancer field, and also has obvious curative effect on liver cancer and head and neck cancer. However, because 10-hydroxycamptothecin needs to enter the cell to play its role, the availability of the drug is greatly reduced. At the same time, as a hydrophobic drug, 10-hydroxycamptothecin is difficult to be administered intravenously directly. These all limit its wider clinical application.

Therefore, how to obtain a more suitable 10-hydroxycamptothecin prodrug and solve the above-mentioned problems in the application of 10-hydroxycamptothecin has become one of the focuses of many prospective researchers.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a 10-hydroxycamptothecin derivative and its preparation method, medicine and application, especially a 10-hydroxycamptothecin prodrug. The 10-hydroxycamptothecin prodrug provided by the present invention can kill tumor cells more effectively, and at the same time effectively solve the problems of poor water solubility and low drug availability of 10-hydroxycamptothecin, and has broad development prospects in the field of tumor treatment . Moreover, the preparation method provided by the present invention is simple, the conditions are mild, the raw material sources are wide, the batch production can be realized, and it is suitable for industrialization and large-scale application and subsequent expansion.

The present invention provides a 10-hydroxycamptothecin derivative, the 10-hydroxycamptothecin derivative has the structure shown in formula (I),

in,

R ₁ is selected from one or more of straight-chain alkyl groups of C1-C10, branched-chain alkyl groups of C3-C10 and aryl groups of C6-C20;

R ₂ is selected from hydrogen atoms or cations;

R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -.

Preferably, the 10-hydroxycamptothecin derivatives are 10-hydroxycamptothecins prodrugs;

The R ₁ is one or more of a C1-C8 straight-chain alkyl group, a C5-C8 branched-chain alkyl group, and a C8-C15 aryl group;

The cations include metal cations and/or organic cations.

Preferably, the metal cations include sodium ions and/or potassium ions;

The organic cations include guanidine ions and/or ammonium ions;

The 10-hydroxycamptothecins prodrugs are small molecule prodrugs.

The invention provides a preparation method of 10-hydroxycamptothecin derivatives, comprising the following steps:

1) under the action of the condensing agent, after the small molecule with the structure of formula (II), the small molecule with the structure of formula (III) and the solvent are reacted, a reaction system is obtained;

2) after removing the solvent from the reaction system obtained in the above steps, obtain a solid, then dissolve the solid in an organic solvent, and then separate after the mixed solvent settling to obtain the 10-hydroxycamptothecin derivative with the structure of formula (I) ;

in,

R ₁ is selected from one or more of straight-chain alkyl groups of C1-C10, branched-chain alkyl groups of C3-C10 and aryl groups of C6-C20;

R ₂ is selected from hydrogen atoms or cations;

R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -.

Preferably, the molar ratio of the small molecule with the structure of formula (III) to the small molecule with the structure of formula (II) is (1-1.2): (0.8-1);

The molar ratio of the condensing agent to the small molecule having the structure of formula (III) is (1-1.5): (1-1.2);

The condensing agent comprises one or one of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, N,N-diisopropylcarbodiimide and dicyclohexylcarbodiimide. variety;

The solvent includes one or more of pyridine, N,N-dimethylformamide, dimethylsulfoxide, chloroform and dichloromethane.

Preferably, the temperature of the reaction is 10-60°C;

The time of described reaction is 2～90 hours;

The method of removing the solvent comprises negative pressure evaporation;

The organic solvent includes one or more of methanol, N,N-dimethylformamide, dimethylsulfoxide, chloroform and dichloromethane;

The reaction is a closed reaction.

Preferably, the mixed solvent includes solvent A and solvent B;

Described solvent A comprises one or more in methylene chloride, methanol and ethyl acetate;

Described solvent B comprises one or more in ether, petroleum ether and n-hexane;

The volume ratio of the solvent A and the solvent B is 1: (1-2);

After the separation, it also includes a post-processing step;

The post-processing steps include one or more of washing, filtration and vacuum evaporation.

The present invention provides a medicine, which comprises the 10-hydroxycamptothecin derivatives described in any one of the above technical solutions or 10-hydroxycamptothecin prepared by the preparation method described in any one of the above technical solutions Base derivatives, and pharmaceutically acceptable excipients.

Preferably, the dosage form of the medicine includes oral preparations, injections, suppositories, inhalants or dosage forms that can be directly applied to tumors;

The dosage of the 10-hydroxycamptothecin derivatives is 5-20 mg/kg;

In the medicine, the mass content of the 10-hydroxycamptothecin derivatives is 1% to 100%.

The present invention also provides the 10-hydroxycamptothecin derivatives according to any one of the above technical solutions, the 10-hydroxycamptothecin derivatives prepared by the preparation method according to any one of the above technical solutions, or the above technology Application of the medicine described in any one of the schemes in the field of tumor treatment.

The present invention provides a 10-hydroxycamptothecin derivative, and the 10-hydroxycamptothecin derivative has a structure represented by formula (I). Compared with the prior art, the present invention aims at the fact that the existing 10-hydroxycamptothecin needs to enter the cell to play its role, and the research shows that it has poor cellular uptake ability, which greatly reduces the drug availability. At the same time, it is difficult to directly administer intravenously, which limits its wider clinical application. The present invention creatively designs a 10-hydroxycamptothecin derivative, that is, a 10-hydroxycamptothecin prodrug. The 10-hydroxycamptothecin derivative of this structure is modified by guanidylation of 10-hydroxycamptothecin to make it easier to be taken up by cells, thereby enhancing cytotoxicity and killing tumor cells more effectively; at the same time, it effectively solves the problem of 10 -Hydroxycamptothecin has the problems of poor water solubility and low drug availability, and has broad development prospects in the field of tumor therapy.

The 10-hydroxycamptothecin derivatives provided by the present invention can not only achieve enhanced water solubility and cellular uptake ability, but also have simple preparation methods, mild conditions, wide raw material sources, can realize batch production, and are suitable for industrialization and large-scale applications and Subsequent expansion.

The experimental results show that the cell uptake capacity of the 10-hydroxycamptothecin derivatives prepared by the present invention is 1.57 times that of 10-hydroxycamptothecin after co-incubating with cells for 20 minutes. The cellular uptake capacity of camptothecin derivatives is 1.35 times that of 10-hydroxycamptothecin, and due to the better cellular uptake of 10-hydroxycamptothecin derivatives, its cytotoxicity to CT26 cell line is increased to 10- 1.52 times the cytotoxicity of hydroxycamptothecin.

Description of drawings

Fig. 1 is the ¹ H NMR chart of the carboxylated 10-hydroxycamptothecin intermediate prepared in Example 1 of the present invention;

Fig. 2 is the ¹³ C NMR chart of the carboxylated 10-hydroxycamptothecin intermediate prepared in Example 1 of the present invention;

Figure 3 is the ¹ H NMR chart of the 10-hydroxycamptothecin prodrug prepared in Example 2 of the present invention;

Fig. 4 is the ESI-MS diagram of the 10-hydroxycamptothecin prodrug prepared in Example 2 of the present invention;

Figure 5 is the cellular uptake result of 10-hydroxycamptothecin and its prodrugs in Example 3 of the present invention;

Fig. 6 is the cytotoxicity test result of 10-hydroxycamptothecin in Example 4 of the present invention;

Fig. 7 is the cytotoxicity test result of 10-hydroxycamptothecin prodrug in Example 4 of the present invention.

Detailed ways

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

All the raw materials of the present invention, their sources are not particularly limited, can be purchased in the market or prepared according to conventional methods well known to those skilled in the art. All the raw materials in the present invention are not particularly limited in their purity, and the present invention preferably adopts analytical purity or meets the standard of pharmaceutical purity.

All the raw materials of the present invention, their trade marks or abbreviations belong to the conventional trade marks or abbreviations in this field, and each trade mark or abbreviation is clear and definite in the field of its related use, and those skilled in the art can It can be purchased from the market or prepared by conventional methods.

In the present invention, those skilled in the art can correctly understand that the meanings represented by the two expressions of formula (*) and formula * are equivalent, and the presence or absence of parentheses does not affect their actual meanings.

The present invention provides a 10-hydroxycamptothecin derivative, and the 10-hydroxycamptothecin derivative has the structure shown in formula (I):

in,

R ₁ is selected from one or more of straight-chain alkyl groups of C1-C10, branched-chain alkyl groups of C3-C10 and aryl groups of C6-C20;

R ₂ is selected from hydrogen atoms or cations;

R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -.

The 10-hydroxycamptothecin derivative of the present invention is preferably a 10-hydroxycamptothecin prodrug, having the structure of formula (I).

In the present invention, R ₁ is selected from one or more of C1-C10 straight-chain alkyl, C3-C10 branched-chain alkyl and C6-C20 aryl, more preferably C1-C10 straight-chain Alkyl, C3-C10 branched-chain alkyl or C6-C20 aryl. Wherein, the straight-chain alkyl group of C1-C10 may be a straight-chain alkyl group of C2-C9, a straight-chain alkyl group of C3-C8, a straight-chain alkyl group of C4-C7, or a straight-chain alkyl group of C4-C7. C5-C6 straight-chain alkyl. The C3-C10 branched chain alkyl group may be a C4-C9 branched chain alkyl group, a C5-C8 branched chain alkyl group, or a C6-C7 branched chain alkyl group. The C6-C20 aryl group may be a C8-C18 aryl group, a C10-C16 aryl group, or a C12-C14 aryl group. Specifically, the R ₁ can be selected from C1-C5 straight-chain alkyl groups, C5-C8 branched-chain alkyl groups, or C8-C15 aryl groups. More specifically, the R ₁ preferably includes methyl, ethyl , n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl and One or more of anthracenes. In the present invention, the straight-chain alkyl group, branched-chain alkyl group and C6-C20 aryl group may also include substituted straight-chain alkyl or unsubstituted straight-chain alkyl, substituted branched-chain alkyl or unsubstituted branched-chain alkyl and substituted or unsubstituted aryl groups.

In the present invention, R ₂ is selected from hydrogen atoms or cations. Among them, the cations preferably include metal cations and/or organic cations, more preferably metal cations or organic cations. Specifically, the metal cation preferably includes sodium ion and/or potassium ion, more preferably sodium ion or potassium ion. The organic cations preferably include guanidine ions and/or ammonium ions, more preferably guanidine ions or ammonium ions.

In the present invention, R ₃ and R ₄ are each independently selected from -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ - or more, more preferably each independently selected from -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - or -CH ₂ CH ₂ CH ₂ CH ₂ -.

The invention provides a preparation method of 10-hydroxycamptothecin derivatives, comprising the following steps:

1) under the action of the condensing agent, after the small molecule with the structure of formula (II), the small molecule with the structure of formula (III) and the solvent are reacted, a reaction system is obtained;

2) after removing the solvent from the reaction system obtained in the above steps, obtain a solid, then dissolve the solid in an organic solvent, and then separate after the mixed solvent settling to obtain the 10-hydroxycamptothecin derivative with the structure of formula (I) ;

in,

R ₁ is selected from one or more of straight-chain alkyl groups of C1-C10, branched-chain alkyl groups of C3-C10 and aryl groups of C6-C20;

R ₂ is selected from hydrogen atoms or cations;

R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -.

In the present invention, the structure, group and specific selection of the 10-hydroxycamptothecin derivatives with the structure of formula (I) in the above preparation method, as well as the corresponding preferred principles, are in line with the aforementioned structures with the structure of formula (I), The groups and specific selections, as well as the corresponding preference principles, can preferably be corresponding, and will not be repeated here.

In the present invention, the reaction system is obtained by reacting the small molecule with the structure of formula (II), the small molecule with the structure of formula (III) and a solvent under the action of a condensing agent.

In principle, the present invention has no particular limitation on the sources of the small molecules (compounds) with the structure of formula (II) and the small molecules (compounds) with the structure of formula (III), and the preparation or Commercially available ones can be purchased, and those skilled in the art can select and adjust them according to actual conditions, performance requirements and product requirements. The present invention does not make special limitations, and can also be prepared by referring to the methods described in the embodiments of the present invention. In the present invention, the substituents in the small molecule with the structure of formula (II) and the small molecule with the structure of formula (III) and the 10-hydroxycamptothecin derivatives with the structure of formula (I) are preferred. One-to-one correspondence is possible, and details are not repeated here.

In principle, the present invention does not limit the specific dosage of the small molecule having the structure of formula (III), and those skilled in the art can select and adjust it according to the production situation, product structure and product performance requirements. The present invention is a better enhancement. Water solubility and cellular uptake ability of 10-hydroxycamptothecin derivatives, enhanced cytotoxicity, and improved drug availability, and the molar ratio of the small molecule with the structure of formula (III) to the small molecule with the structure of formula (II) (1-1.2): (0.8-1) is preferable, (1.02-1.18): (0.8-1) is more preferable, (1.05-1.15): (0.8-1) is more preferable, (1.08-1) is more preferable 1.12): (0.8～1), or (1～1.2): (0.82～0.98), or (1～1.2): (0.85～0.95), or (1～1.2): (0.87 ~0.97).

In principle, the present invention does not limit the specific amount of the condensing agent. Those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptothecins. The water-solubility and cellular uptake ability of the derivative can enhance cytotoxicity and improve drug availability. The molar ratio of the condensing agent to the small molecule having the structure of formula (III) is preferably (1-1.5): (1-1.2 ), more preferably (1.1-1.4): (1-1.2), more preferably (1.2-1.3): (1-1.2), may be (1-1.5): (1.02-1.18), or may be (1-1.5): (1.05-1.15), (1-1.5): (1.08-1.12) may be sufficient.

In principle, the present invention has no particular restrictions on the specific selection of the condensing agent. Those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptothecins. Water solubility and cellular uptake ability of the derivative, enhance cytotoxicity, and improve drug availability, and the condensing agent preferably includes benzotriazol-1-yl-oxytripyrrolidinohexafluorophosphate, N,N-difluorophosphate One or more of isopropylcarbodiimide and dicyclohexylcarbodiimide, more preferably benzotriazol-1-yl-oxytripyrrolidinohexafluorophosphate, N,N- Diisopropylcarbodiimide or dicyclohexylcarbodiimide, more preferably benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate.

In principle, the present invention has no particular restrictions on the specific selection of the solvent, and those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptothecin derivatives. water solubility and cellular uptake ability of the substance, enhance cytotoxicity, and improve drug availability, and the solvent preferably includes one of pyridine, N,N-dimethylformamide, dimethyl sulfoxide, chloroform and dichloromethane or more, more preferably pyridine, N,N-dimethylformamide, dimethylsulfoxide, chloroform or dichloromethane, more preferably anhydrous pyridine.

In principle, the specific parameters of the reaction are not particularly limited in the present invention, and those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance the derivatization of 10-hydroxycamptothecin The water solubility and cellular uptake ability of the substance are enhanced, the cytotoxicity is enhanced, and the drug availability is improved. The reaction time is preferably 2-90 hours, more preferably 12-85 hours, more preferably 32-80 hours, more preferably 52-70 hours, specifically 50-80 hours. In the present invention, the reaction is preferably a closed reaction.

The present invention subsequently removes the solvent from the reaction system obtained in the above-mentioned steps to obtain a solid, and then dissolves the solid in an organic solvent, and then uses a mixed solvent to settle and separate to obtain a 10-hydroxycamptothecin derivative having the structure of formula (I). thing.

In principle, the method of removing the solvent is not particularly limited in the present invention, and those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. To improve the water solubility and cellular uptake ability of the substance, enhance cytotoxicity, and improve drug availability, the solvent removal method preferably includes negative pressure evaporation. Specifically, a vacuum pump can be used for negative pressure evaporation, and then a cold trap is used to solidify to remove the solvent.

In principle, the present invention has no particular restrictions on the specific selection of the organic solvent, and those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptothecins. The water-solubility and cellular uptake ability of the derivative can enhance cytotoxicity and improve drug availability, and the organic solvent preferably includes methanol, N,N-dimethylformamide, dimethyl sulfoxide, chloroform and dichloromethane. One or more, more preferably methanol, N,N-dimethylformamide, dimethylsulfoxide, chloroform or dichloromethane.

In principle, the present invention has no particular restrictions on the specific selection and parameters of the mixed solvent. Those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptotheca. The water-solubility and cellular uptake ability of the base derivatives can enhance cytotoxicity and improve drug availability. The mixed solvent preferably includes solvent A and solvent B. Specifically, the solvent A preferably includes one or more of dichloromethane, methanol and ethyl acetate, more preferably dichloromethane, methanol or ethyl acetate. The solvent B preferably includes one or more of diethyl ether, petroleum ether and n-hexane, more preferably diethyl ether, petroleum ether or n-hexane. The volume ratio of the solvent A and the solvent B is preferably 1:(1-2), more preferably 1:(1.2-1.8), and more preferably 1:(1.4-1.6).

In principle, the present invention has no particular limitation on the specific method of separation, and those skilled in the art can select and adjust according to production conditions, product structure and product performance requirements. The present invention is to better enhance 10-hydroxycamptothecin derivatives. To enhance the water solubility and cellular uptake ability of the substance, enhance cytotoxicity, and improve drug availability, the separation preferably includes centrifugation.

The present invention is a complete and refined production method, in order to better enhance the water solubility and cellular uptake ability of 10-hydroxycamptothecin derivatives, enhance cytotoxicity, and improve drug availability, the separation preferably also includes post-processing step. Wherein, the post-processing step includes one or more steps of washing, filtration and vacuum evaporation, more preferably washing, filtration and vacuum evaporation.

The present invention is a complete and refined production method, in order to better enhance the water solubility and cell uptake ability of 10-hydroxycamptothecin derivatives, enhance cytotoxicity, and improve drug availability, the above 10-hydroxycamptothecin derivatives The preparation method of the compound can be specifically the following steps:

Dissolve the prepared small molecule with the structure of formula (III) and the small molecule with the structure of formula (II) in a solvent (anhydrous pyridine), slowly add the solvent in which the condensing agent is dissolved, and seal it at a temperature of 25°C Reaction 72h. After that, the obtained reaction solution was drained, the precipitated solid was dissolved in an organic solvent, and sedimented with a mixed solvent, the resulting precipitate was washed three times with diethyl ether after centrifugation, and the remaining solvent was drained with a vacuum pump and a cold trap at room temperature to obtain 10- Hydroxycamptothecin prodrug.

The present invention provides a medicine, which comprises the 10-hydroxycamptothecin derivatives described in any one of the above technical solutions or 10-hydroxycamptothecin prepared by the preparation method described in any one of the above technical solutions Base derivatives, and pharmaceutically acceptable excipients.

The structure, group and specific selection of the 10-hydroxycamptothecin derivatives in the present invention, as well as the corresponding preference principles, are related to the structures in the aforementioned 10-hydroxycamptothecin derivatives and their preparation methods. , groups and specific selections, as well as the corresponding preferred principles, can preferably be corresponding, and will not be repeated here.

In principle, the present invention has no particular limitation on the dosage form of the drug, and those skilled in the art can select and adjust according to the application situation, product structure and product performance requirements. The present invention is to better enhance the 10-hydroxycamptothecin derivatives. The water-solubility and cellular uptake ability of the drug are enhanced, the cytotoxicity is enhanced, and the drug availability is improved. The dosage form of the drug preferably includes oral preparations, injections, suppositories, inhalants or dosage forms that can be directly applied to tumors, specifically capsules, microcapsules, Tablets, granules, pills, dispersible powders, liquid preparations, decoctions, suspensions, syrups, gels, aerosols, patches, liposomes, oral liquids, intravenous injections or intramuscular injections.

In principle, the present invention does not limit the content of 10-hydroxycamptothecin derivatives in the medicine, and those skilled in the art can select and adjust according to application conditions, product structure and product performance requirements. To enhance the water solubility and cellular uptake ability of 10-hydroxycamptothecin derivatives, enhance cytotoxicity, and improve drug availability, in the drug, the mass content of the 10-hydroxycamptothecin derivatives is preferably 1 % to 100%, more preferably 10% to 90%, more preferably 20% to 80%, more preferably 30% to 70%, more preferably 40% to 60%.

In principle, the present invention does not limit the dosage of 10-hydroxycamptothecin derivatives in the medicine. Those skilled in the art can select and adjust according to the application situation, product structure and product performance requirements. The present invention is better To enhance the water solubility and cellular uptake ability of 10-hydroxycamptothecin derivatives, enhance cytotoxicity, and improve drug availability, the dosage of 10-hydroxycamptothecin derivatives is preferably 5-20 mg/kg, more Preferably it is 7-18 mg/kg, More preferably, it is 10-15 mg/kg.

The present invention also provides the 10-hydroxycamptothecin derivatives according to any one of the above technical solutions, the 10-hydroxycamptothecin derivatives prepared by the preparation method according to any one of the above technical solutions, or the above technology Application of the medicine described in any one of the schemes in the field of tumor treatment.

The structure, group and specific selection of the 10-hydroxycamptothecin derivatives or drugs in the application, as well as the corresponding preference principles, are related to the aforementioned 10-hydroxycamptothecin derivatives and their preparation methods or The structures, groups, and specific selections in the drug, as well as the corresponding preference principles, can preferably be corresponding, and will not be repeated here.

The present invention does not specifically limit the types of the tumors, and the types of conventional tumors well known to those skilled in the art can be used. Those skilled in the art can select and adjust according to the actual situation, effect data and specific etiology. It can include one or more of gastrointestinal cancer, cervical cancer, lung cancer, liver cancer, bladder cancer, breast cancer, esophageal cancer, head and neck cancer, kidney cancer, prostate cancer and ovarian cancer, as well as cervical cancer, lung cancer , liver cancer, bladder cancer, breast cancer, esophageal cancer, head and neck cancer, kidney cancer, prostate cancer or ovarian cancer, specifically one or more of gastrointestinal cancer, head and neck cancer and liver cancer.

The above steps of the present invention provide a 10-hydroxycamptothecin-based prodrug and its preparation method, medicine and application in anti-tumor. The present invention designs a 10-hydroxycamptothecin derivative, that is, a 10-hydroxycamptothecin prodrug. The 10-hydroxycamptothecin derivative of this structure is modified by guanidylation of 10-hydroxycamptothecin to make it easier to be taken up by cells, thereby enhancing cytotoxicity and killing tumor cells more effectively; at the same time, it effectively solves the problem of 10 -Hydroxycamptothecin has the problems of poor water solubility and low drug availability, and has broad development prospects in the field of tumor therapy.

The 10-hydroxycamptothecin derivatives provided by the present invention can not only achieve enhanced water solubility and cell uptake ability, but also specially adopt the method of condensation of raw materials and the method of co-settling of mixed solvents in the preparation process, and the preparation method is simple, The mild conditions and wide range of raw material sources are more conducive to mass production, and are suitable for industrialization and large-scale applications and subsequent expansion.

The experimental results show that the cell uptake capacity of the 10-hydroxycamptothecin derivatives prepared by the present invention is 1.57 times that of 10-hydroxycamptothecin after co-incubating with cells for 20 minutes. The cellular uptake capacity of camptothecin derivatives is 1.35 times that of 10-hydroxycamptothecin, and due to the better cellular uptake of 10-hydroxycamptothecin derivatives, its cytotoxicity to CT26 cell line is increased to 10- 1.52 times the cytotoxicity of hydroxycamptothecin.

In order to further illustrate the present invention, a 10-hydroxycamptothecin derivative provided by the present invention and its preparation method, medicine and application are described in detail below with reference to the examples, but it should be understood that these examples are based on the technology of the present invention. The scheme is implemented on the premise, and the detailed embodiment and specific operation process are given, only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the present invention, and the protection scope of the present invention is not limited to the following example.

Example 1

1.02 g (2.7 mmol) of 10-hydroxycamptothecin and 1.06 g (9.3 mmol) of glutaric anhydride were dissolved in 500 mL of anhydrous pyridine with stirring, and the reaction was stirred at 25° C. for 72 h. After the reaction, the pyridine in the obtained reaction solution was drained, washed with 0.01M hydrochloric acid solution and filtered in turn, and dried with a vacuum pump and cold trap for 24 hours at room temperature to obtain the intermediate product carboxylated 10-hydroxycamptothecin.

The prepared carboxylated 10-hydroxycamptothecin intermediate was analyzed by nuclear magnetic resonance, and deuterated dimethyl sulfoxide was used as the deuterated reagent.

Referring to FIG. 1 , FIG. 1 is a ¹ H NMR chart of the carboxylated 10-hydroxycamptothecin intermediate prepared in Example 1 of the present invention.

Referring to FIG. 2 , FIG. 2 is a ¹³ C NMR chart of the carboxylated 10-hydroxycamptothecin intermediate prepared in Example 1 of the present invention.

The analysis results show that the chemical shift of 1.94ppm in the H NMR spectrum is the signal peak of the carboxyl β-methylene group formed by the ring-opening bond of glutaric anhydride, and the chemical shifts of 2.40ppm and 2.73ppm are formed by glutaric anhydride, respectively. The methylene signal peaks at the α-position and γ-position of the carboxyl group; the chemical shift positions of 32.13ppm, 32.02ppm and 29.71ppm in the C NMR spectrum are the γ-position, α-position and β-position of the carboxyl moiety formed by the ring-opening of glutaric anhydride, respectively. The signal peak of the methylene group, the methyl signal peak of the ortho-substituted ethyl group of the 10-hydroxycamptothecin lactone ring carbonyl group with a chemical shift of 7.20ppm, the signal intensity ratio of the above four groups of peaks is 1:1:1: 1, indicating that glutaric anhydride has been successfully ring-opened to 10-hydroxycamptothecin.

The combined yield was 97.0%.

Example 2

1.15g (2.4mmol) of the carboxylated 10-hydroxycamptothecin prepared above and 0.0.64g (2.4mmol) of L-arginine methyl ester dihydrochloride were stirred and dissolved in 500mL of anhydrous pyridine, and 10mL was slowly added to dissolve the solution. 1.38g (2.6mmol) of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate in anhydrous pyridine was sealed and reacted for 72h at a temperature of 25°C. After that, the obtained reaction solution was drained, and the precipitated solid was dissolved in 10 mL of methanol, and settled with 500 mL of a mixed solution of dichloromethane and diethyl ether with a volume ratio of 1:1. The remaining solvent was drained by vacuum pump and cold trap to obtain 10-hydroxycamptothecin prodrug.

The 10-hydroxycamptothecin prodrug prepared by the present invention is subjected to nuclear magnetic resonance analysis, and deuterated dimethyl sulfoxide is used as the deuterated reagent.

Referring to FIG. 3 , FIG. 3 is a ¹ H NMR chart of the 10-hydroxycamptothecin prodrug prepared in Example 2 of the present invention.

Referring to Figure 4, Figure 4 is an ESI-MS image of the 10-hydroxycamptothecin prodrug prepared in Example 2 of the present invention.

The results showed that the chemical shift of 4.27 ppm was the amide signal peak formed after the reaction, and the chemical shift of 3.64 ppm was the methoxy signal peak of the arginine methyl ester part after the reaction. Characterization by ESI-MS (ESI+) showed that [M+H] ⁺ peak appeared at m/z=649.5, and m/z=650.6 was the product isotopic peak.

All the above results show that the 10-hydroxycamptothecin prodrug has been successfully prepared, the comprehensive yield is 73.6%, and the obtained product has good water solubility.

Example 3

Comparison of differences in cellular uptake of 10-hydroxycamptothecin and its prodrugs

CT26 cells (4.0×10 ⁵ cells/well) were seeded in a 6-well plate with a cover slip, and 1.8 mL of DMEM medium containing 10% fetal bovine serum was added, and cultured for 20 hours to make the cells adhere to the cover glass. a. 10-Hydroxycamptothecin or 10-Hydroxycamptothecin prodrug were added to the corresponding wells respectively, and incubated after mixing. Fixed for 15 min at room temperature, and the coverslips were washed three times with PBS buffer at 4°C and then sealed with glycerol. Cellular uptake was photographed with fluorescence confocal microscopy over 24 hours and quantitative results were obtained with Image-J.

Referring to FIG. 5, FIG. 5 is the cellular uptake result of 10-hydroxycamptothecin and its prodrugs in Example 3 of the present invention.

As shown in Figure 5, the cellular uptake of 10-hydroxycamptothecin prodrug was significantly higher than that of 10-hydroxycamptothecin when the drug was co-incubated with the cells for 20 minutes. Differences in cellular uptake of drugs remained evident over time. This result shows that the 10-hydroxycamptothecin prodrug is more readily taken up by cells.

Example 4

Comparison of Cytotoxicity Difference Between 10-Hydroxycamptothecin and Its Prodrugs

CT26 colon cancer cells were seeded on a 96-well culture plate, 5×10 ³ cells per well, and 1.8 mL of DMEM medium containing 10% fetal bovine serum was added, and cultured for 20 hours to allow the cells to adhere. 10-Hydroxycamptothecin and its prodrug were dissolved in PBS buffer containing 1.5% DMSO, pH=7.4, respectively, to obtain a 10.0 mM high concentration stock solution. After two-fold dilution of the stock solution, 20 μL of dilution or blank medium was added to each well and incubated for 24 hours. Next, 20 μL of 5.0 mg/mL MTT solution was added to each well and incubated for 3 hours in the dark at 37°C. Subsequently, all the liquid in the well was emptied, 200 μL of dimethyl sulfoxide was added, and the cells were shaken for 5 min in the dark. Measure the absorbance at 490 nm on a microplate reader.

The relative cell viability was obtained by comparing the absorbance of the cells in the drug-treated group and the cells in the blank group.

Referring to FIG. 6, FIG. 6 is the cytotoxicity test result of 10-hydroxycamptothecin in Example 4 of the present invention.

Referring to FIG. 7, FIG. 7 is the cytotoxicity test result of the 10-hydroxycamptothecin prodrug in Example 4 of the present invention.

It can be seen from the comparison between Figure 6 and Figure 7 that in Figure 6, the IC ₅₀ of 10-hydroxycamptothecin on CT26 cells is 13.2 μM, while the IC ₅₀ of 10-hydroxycamptothecin prodrug on CT26 cells in Figure 7 is 8.7 μM, this result shows that the 10-hydroxycamptothecin prodrug has stronger cytotoxicity to tumor cells, which fully proves the antitumor superiority of the 10-hydroxycamptothecin prodrug designed in the present invention.

A 10-hydroxycamptothecin prodrug provided by the present invention and its preparation method, drug, and application in anti-tumor have been introduced in detail above. Specific examples are used in this paper to describe the principle and implementation of the present invention. Explanation, the description of the above embodiment is only used to help understand the method of the present invention and its core idea, including the best mode, and also to enable any person skilled in the art to practice the present invention, including making and using any device or system, and implement any combined method. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal expressions of the claims.

Claims (10)

1. A 10-hydroxycamptothecin derivative, wherein the 10-hydroxycamptothecin derivative has the structure shown in formula (I):

(I); in, R ₁ is selected from C1～C10 straight-chain alkyl, C3～C10 branched alkyl; R ₂ is selected from guanidine ions; R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -. 2. The 10-hydroxycamptothecin derivative according to claim 1, wherein the 10-hydroxycamptothecin derivative is a 10-hydroxycamptothecin prodrug; The R ₁ is a C1-C8 straight-chain alkyl group or a C5-C8 branched-chain alkyl group. 3. The 10-hydroxycamptothecin derivative according to claim 2, wherein the 10-hydroxycamptothecin prodrug is a small molecule prodrug. 4. a preparation method of 10-hydroxycamptothecin derivatives, is characterized in that, comprises the following steps: 1) Under the action of the condensing agent, the reaction system is obtained after the small molecule with the structure of formula (II), the small molecule with the structure of formula (III) and the solvent are reacted;

(II);

(III); 2) After removing the solvent from the reaction system obtained in the above steps, a solid is obtained, and then the solid is dissolved in an organic solvent, and then a mixed solvent is used for sedimentation and separation to obtain a 10-hydroxycamptothecin derivative having the structure of formula (I) ;

(I); in, R ₁ is selected from C1～C10 straight-chain alkyl, C3～C10 branched alkyl R ₂ is selected from guanidine ion; R ₃ and R ₄ are each independently selected from one or more of -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ -. 5. The preparation method according to claim 4, wherein the molar ratio of the small molecule with the structure of formula (III) to the small molecule with the structure of formula (II) is (1~1.2): (0.8~ 1); The molar ratio of the condensing agent to the small molecule having the structure of formula (III) is (1~1.5): (1~1.2); The condensing agent comprises one or one of benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, N,N-diisopropylcarbodiimide and dicyclohexylcarbodiimide. variety; The solvent includes one or more of pyridine, N,N-dimethylformamide, dimethylsulfoxide, chloroform and dichloromethane. 6. preparation method according to claim 4, is characterized in that, the temperature of described reaction is 10～60 ℃; The time of described reaction is 2～90 hours; The method of removing the solvent comprises negative pressure evaporation; The organic solvent includes one or more of methanol, N,N-dimethylformamide, dimethylsulfoxide, chloroform and dichloromethane; The reaction is a closed reaction. 7. The preparation method according to any one of claims 4 to 6, wherein the mixed solvent comprises solvent A and solvent B; Described solvent A comprises one or more in methylene chloride, methanol and ethyl acetate; Described solvent B comprises one or more in ether, petroleum ether and n-hexane; The volume ratio of the solvent A and the solvent B is 1: (1~2); After the separation, it also includes a post-processing step; The post-processing steps include one or more of washing, filtration and vacuum evaporation. 8. A medicine, characterized in that the medicine comprises the 10-hydroxycamptothecin derivatives described in any one of claims 1 to 3 or prepared by the preparation method described in any one of claims 4 to 7 10-hydroxycamptothecin derivatives, and pharmaceutically acceptable excipients. 9. The medicine according to claim 8, wherein the dosage form of the medicine comprises an oral preparation, an injection, a suppository, an inhalation or a dosage form that can be directly applied to tumors; The dosage of the 10-hydroxycamptothecin derivatives is 5-20 mg/kg; In the medicine, the mass content of the 10-hydroxycamptothecin derivatives is 1% to 100%. 10. The 10-hydroxycamptothecin derivative according to any one of claims 1 to 3, the 10-hydroxycamptothecin derivative prepared by the preparation method according to any one of claims 4 to 7 or the right Application of the medicine described in any one of requirements 8 to 9 in the field of preparing a tumor treatment medicine.

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