CN103772696B - A kind of polyglycerol fatty acid ester derivative of hydroxycamptothecine - Google Patents

Abstract

The present invention relates to a kind of polyglycerol fatty acid ester derivative of hydroxycamptothecine, it is characterized in that polyglycerol fatty acid and hydroxycamptothecine 10 hydroxyls form the structure of ester.Invention describes its constitutional features, customary preparation methods and the purposes in antitumor drug.

Description

A kind of polyglycerol fatty acid ester derivative of hydroxycamptothecin

technical field

The invention relates to the field of medicines, in particular to a polyglycerol fatty acid ester derivative of hydroxycamptothecin, a preparation method thereof, and an application thereof in the preparation of antitumor drugs.

Background technique

Camptothecin (CPT), whose structure is shown in formula (I), was first isolated from the Chinese endemic plant Camptothecin by Wall, an American scientist, and is a toxic alkaloid. Camptothecin has excellent antitumor activity, but it also has great toxic and side effects. The excellent anti-cancer effect of camptothecin has aroused people's great interest, and its naturally occurring or synthetic analogues have been studied one after another to find new anti-tumor drugs. Among them, compared with camptothecin, hydroxycamptothecin (HCPT) has the advantages of higher anticancer efficacy and less toxic side effects. It is used in the treatment of gastric cancer, liver cancer, leukemia, breast cancer, head and neck tumors It has a strong application value in my country and has been widely used in clinical practice in China; derivatives of hydroxycamptothecin, water-soluble prodrugs of 10-hydroxy-7-ethylcamptothecin (SN-38) (Iriontecan, irifor Kang), 10-hydroxy-9-dimethylaminomethyl camptothecin (Topotecan, Topotecan) has also been listed in 1994 and 1999, respectively, for the treatment of colorectal cancer, ovarian cancer and small cell lung cancer treat. In addition, a large number of camptothecin analogs or derivatives are still under development, such as Chinese patent document I (authorized announcement number CN100432076/C) introduces camptothecin derivatives containing a dihydrofuran ring, Chinese patent document Ⅱ (application publication number CN102574866A) introduced 14-nitro, 14-amino and 14-substituted amino camptothecin derivatives, Chinese patent document Ⅲ (application publication number CN102731772A) introduced the polymerization of camptothecin compounds Ethylene glycol derivatives.

The chemical structure of formula (I) camptothecin

Studies have shown that the anti-tumor activity of camptothecin and its analogs is mainly exerted by inhibiting the activity of topoisomerase I (TopoI) in tumor cells. During the DNA replication process of cell division, TopoI combines with DNA to form a binary complex, and makes the DNA have a temporary single-strand gap during the replication process, so that the semi-conservative replication can proceed smoothly. If camptothecin or its derivatives are given during this process, it can specifically bind to the TopoI-DNA complex in the S phase of cell division to form an irreversible TopoI-CPT-DNA ternary complex, enabling DNA replication Termination, resulting in a killing effect on cells.

The unique inhibitory effect of camptothecin and its analogs on topoisomerase I is closely related to the lactone structure present in its E ring. Because studies have shown that only camptothecin in the form of lactone can form a stable complex with TopoI-DNA, which may be an important part of its anticancer effect. However, camptothecin and its analogues in the form of lactones exist in the form of carboxylic acids due to the instability of the lactones, which are easy to reversibly open the ring, so there are two forms of equilibrium in human plasma. Since only an acidic environment is conducive to the existence of the lactone form, and the carboxylic acid form has a higher binding force to plasma proteins, so after camptothecin and its analogs enter the blood, the concentration of the lactone form is much lower than that of the carboxylic acid form. form concentration. What's more unfavorable is that the anti-tumor activity of camptothecin and its analogs in the form of carboxylic acid is significantly lower than that of the lactone form, and the adverse reactions are enhanced, while the camptothecin in the form of lactone is almost insoluble in water, and has a short biological half-life. Unfavorable characteristics such as poor combination. Therefore, improving the water solubility of camptothecin analogs, prolonging their residence time in vivo, and increasing the stability of metabolism have become the focus of research on such compounds and their preparations.

The shortcomings of camptothecin compounds, such as short biological half-life and almost insoluble in water, have not been well resolved in the currently marketed dosage forms. For example, "Hydroxycamptothecin Sodium Chloride Injection" and "Hydroxycamptothecin for Injection" listed in China, in order to solve the problem of their solubility in water, use the reaction with alkali to promote the opening of the lactone ring to form carboxylate. Although the water-solubility has been improved, due to the too low concentration of hydroxycamptothecin in the form of lactone, there are still defects in that the anti-tumor activity is obviously reduced and the toxic and side effects are increased.

In order to improve the clinical efficacy and improve many unfavorable characteristics of camptothecin analogues, people's research directions mainly focus on two aspects. First, various modifications are made to the chemical structure of camptothecin, such as adding hydrophilic groups, using prodrug technology, etc. There are a large number of domestic and foreign reports on this aspect (such as the aforementioned Chinese patent documents Ⅰ~Ⅲ) , and also achieved certain applied results, such as the listing of drugs irinotecan and topotecan. However, there are still some difficult problems in these applied achievements, such as the distribution and action time of drugs in the body are not satisfactory, and the use is inconvenient. In order to solve this kind of problems, people have made efforts in the second aspect, that is, using some new drug delivery systems (such as nanoparticles, liposomes, microemulsions, micelles, etc.) in order to achieve the desired effect. These new drug delivery systems have changed the pharmacokinetic parameters and biodistribution characteristics of camptothecin and its analogues in the body, and have the effects of sustained release, prolonged residence time in the body, and increased drug delivery, which can improve the curative effect , to reduce the purpose of toxic and side effects.

In these new drug delivery systems, in order to enhance the water solubility of the drug and prolong the circulation time in the body, it is a very common choice to use polyethylene glycol (PEG) to modify the delivered drug or the drug delivery system itself. . For example, the first liposomal drug doxorubicin approved by the U.S. FDA is formulated with polyethylene glycol-modified phospholipids. HongMinghuang et al. The combination of protein, polyethylene glycol and hydroxycamptothecin to enhance its water solubility and targeting has achieved good results. The Chinese patent of Liu Keliang et al. (Chinese Patent Document III) also provides camptothecins For the preparation method and application of PEGylated derivatives of the compound, for more literature on the modification of drugs with polyethylene glycol, please refer to the review by Zhang Yue et al. (Non-Patent Document II, Journal of Hebei University of Science and Technology, 2013, 34, 4 : 355-365).

However, PEG-modified drugs and drug delivery systems also have certain problems, the most obvious of which is that after the first administration of the PEGylated drug delivery system, the circulation time of subsequent doses of drugs in the blood becomes shorter, and in the liver. This is called "accelerated blood clearance phenomenon (ABC)". Regarding the "ABC phenomenon", it has been discovered very early in many PEG-modified drugs or drug delivery systems, and there are many related reports. Anti-PEG IgM is rapidly cleared (Non-Patent Document III, Bioconjugate Chemistry, 1999, 10:520-528). For the "ABC phenomenon" caused by PEG, there is no clear conclusion on its specific cause. It is reported (Non-Patent Document IV, Journal of Controlled Release, 2006, 115:243-250) that the "ABC phenomenon" is the result of the innate immune system of the human body. Its mechanism may be the same as that in PEG repeating unit, when the PEG-modified drug or drug delivery system reaches the immune organ spleen for the first time, The combination of the chain and the immunoglobulin on the active B cells in the marginal zone of the spleen produces a special stimulus to the B cells, causing them to secrete anti-PEG IgM, which leads to the accumulation of the drug or its delivery system in the liver and spleen, and the drug in the blood The decrease of the concentration results in "accelerated blood flow clearance phenomenon", which shortens the action time of the drug.

Polyglycerol (PG) is an important fine chemical product and synthetic intermediate. Its synthesis research has been reported as early as the beginning of the 20th century. However, due to product quality problems, its application in food, medicine and daily chemicals is limited. . In recent years, with people's in-depth research and improvement of polyglycerol, its quality has been greatly improved. At present, some large companies (such as Solvay Chemicals of Belgium Solvay Group, Japan Sakamoto Pharmaceutical Co., Ltd., Italy SPIGA Company and Jinan Dongrun Fine Chemical Technology Co., Ltd., etc.) provide commercialized high-quality polyglycerol, which can be directly used in cosmetics and pharmaceutical products.

Many other beneficial attempts have been made to avoid or mitigate the "ABC phenomenon" of drugs or drug delivery systems. Among them, there are documents (non-patent literature V, International Journal of Pharmaceuticals, 2013, 456:235-242) that polyglycerol-modified phosphatidylethanolamine (PG-DSPE) is used instead of polyethylene glycol-modified phosphatidylethanolamine (PEG-DSPE) The preparation of liposomes effectively avoids the "ABC phenomenon". The mechanism may lie in lipids modified with branched polyglycerols, The existence of the -CH ₂ OH side chain in the repeating unit constitutes a steric hindrance, which hinders the interaction and (or) effective combination of the B cell immunoglobulin and the repeating unit, so that the B cell lacks effective special stimulation and cannot secrete or secrete extremely Less anti-PG IgM, thereby eliminating or reducing the "ABC phenomenon". However, this study was limited to the phospholipid materials that make up liposomes, and did not mention the effect of modifying the drug itself with polyglycerol.

Therefore, it is more practical to develop a new non-polyethylene glycol-modified drug derivative aiming at the shortcomings of camptothecin compounds, such as low solubility in water, poor distribution in vivo, short action time, and strong toxic and side effects.

Contents of the invention

The present inventors have found through research that after camptothecin is hydroxylated at the 10-position to generate hydroxycamptothecin, its antitumor activity does not change significantly after being modified with polyglycerol fatty acid ester, its toxicity and side effects are reduced, and its solubility in water is greatly improved. The circulation time in the body is prolonged, and there is no obvious "ABC phenomenon".

The polyglycerol fatty acid ester derivative of hydroxycamptothecin involved in the present invention has a chemical structure as shown in formula (II). This structure is the ester structure formed by the straight-chain fatty acid of polyglycerol and the 10-hydroxyl group of hydroxycamptothecin.

Chemical structure of polyglycerol fatty acid ester derivatives of formula (II) hydroxycamptothecin

The polyglycerol fatty acid ester derivative of hydroxycamptothecin of the present invention is characterized in that the polyglycerol part is polyglycerol with side chains, and the degree of polymerization n of the polyglycerol is 3-50, preferably 10 or 20.

The polyglycerol fatty acid ester derivative of hydroxycamptothecin of the present invention is characterized in that m is 1-4, preferably 2 or 3.

The polyglycerol fatty acid ester derivative of hydroxycamptothecin of the present invention is used for preparing antitumor drugs.

In the polyglycerol fatty acid ester derivative of hydroxycamptothecin of the present invention, the m value is a definite value, and the polyglycerol part can be a single compound with a unique definite n value, or it can be centered on a definite n value, with certain A polymer mixture formed by mixing compounds with a molecular weight distribution range. The preferred compound of the polyglycerol fatty acid ester derivative of hydroxycamptothecin in the present invention is hydroxycamptothecin polyglycerol 1500 succinate (code-named PG1500-HCPT).

The polyglycerol fatty acid ester derivative of hydroxycamptothecin of the present invention can be synthesized by a simple chemical method. Its general synthetic route is shown in formula (Ⅲ).

General synthetic route of polyglycerol fatty acid ester derivatives of formula (Ⅲ) hydroxycamptothecin

The invention uses ethoxy ethyl glycidyl ether as raw material to synthesize polyglycerol fatty acid ester derivatives of hydroxycamptothecin. Each compound was analyzed by HPLC and TLC, and the structure was confirmed to be correct by mass spectrometry, hydrogen spectrum and carbon spectrum NMR analysis.

According to the present invention, the polyglycerol fatty acid ester derivatives of hydroxycamptothecin of the present invention can be used for the preparation of anti-tumor drugs and these anti-tumor drugs in various new drug delivery systems, including but not limited to nanoparticles, lipids Applications in the preparation of bodies, microemulsions, micelles, etc.

Detailed ways

The above content of the present invention will be further described in detail in the form of specific implementation examples below, but the scope of the present invention is not limited to the following examples. All technologies realized based on the above contents of the present invention are included in the scope of the present invention.

Example 1: Synthesis of hydroxycamptothecin polyglycerol 1500 succinate (PG1500-HCPT)

1. Synthesis of polyglycerol 1500 (refer to Non-Patent Document IV, Journal of American Chemical Society 2002, 124:9698-9699)

Potassium tert-butoxide (0.7g, 6mmol) and ethoxyethyl glycidyl ether (5.3g, 36mmol) were placed in a flask, and tert-butanol was evaporated under reduced pressure at 120°C. Transfer the reactant into a three-necked flask, heat to 95°C, and drop ethoxyethyl glycidyl ether (136.8g, 0.96mol) into the above reactant at a rate of 4mL/min under constant stirring and nitrogen flow , After the dropwise addition was completed, the reaction was continued for 6 hours. After the reaction was completed, tetrahydrofuran and hydrochloric acid were added to the reaction mixture, and then tetrahydrofuran was removed under reduced pressure. A small amount of methanol was added to dissolve the reactants, then acetone was added to shake, and the upper layer solution was discarded after standing. This operation was repeated twice. The solution of the lower layer obtained by the reaction was removed under reduced pressure at 80°C to obtain polyglycerol (48.1 g, about 33% yield) with an average molecular weight of 1500.

2. Synthesis of Polyglycerol 1500 Succinic Acid

Polyglycerol 1500 (3.0 g, 2 mmol) and succinic anhydride (0.2 g, 2 mmol) were dissolved in 100 mL of dioxane, and a small amount of pyridine was added as a catalyst. The above solution was heated to reflux for more than 24 hours until the reaction reached equilibrium. After the reactant was cooled to room temperature, diethyl ether was added to precipitate a precipitate, the mixture was stirred in an ice bath for 30 minutes, and the crude product of polyglycerol 1500 succinic acid was obtained by filtration. The crude product was dissolved by adding an appropriate amount of dichloromethane, and then diethyl ether was added to precipitate a precipitate. The mixture was stirred in an ice bath for 15 minutes, and the precipitate was filtered off and dried to obtain polyglycerol 1500 succinic acid (2.3 g, yield 44%).

3. Synthesis of Hydroxycamptothecin Polyglycerol 1500 Succinate

Hydroxycamptothecin (0.29g, 0.8mmol, polyglycerol 1500 succinic acid (0.64g, 0.4mmol) was dissolved in 50mL of anhydrous N,N-dimethylformamide, and 1-ethyl-3-(3 -Dimethylaminopropyl) carbodiimide hydrochloride 280mg and 1,4-dimethylaminopyridine 12mg.The mixture was stirred and reacted in an ice bath under nitrogen protection for 2 hours, and then placed at room temperature for 24 hours.Reaction mixture 5% NaHCO ₃ was added to remove excess hydroxycamptothecin, the residue was extracted with dichloromethane (100mL×3), and then 5% NaHCO ₃ solution, water, 0.1mol/L hydrochloric acid and saturated chlorination The sodium solution was washed and concentrated under reduced pressure to obtain the crude product. The crude product was a light yellow solid, which was recrystallized with ether to obtain a white-like hydroxycamptothecin polyglycerol 1500 succinate solid. ¹ HNMR (DMSO): δ0.82 (t, 3H, H–18), δ1.91 (m, 2H, H–19), δ3.23–4.57 (–CH2OH), δ5.26 (s, 2H, H–5), δ5.46 (s, 2H , H–17), δ7.37 (s, 1H, H–14), δ7.60 (dd, 1H, H–11), δ7.88 (d, 1H, H–9), δ8.20 (d , 1H, H–12), δ8.64 (s, 1H, H–7).

Example 2: Evaluation of the antitumor activity of hydroxycamptothecin polyglycerol 1500 succinate

Select two cell lines of human liver cancer cells HepG2 and human erythroleukemia K562 cells, inoculate the cells in DMEM cell culture medium containing 10% fetal bovine serum, and place them in a cell incubator at 37°C containing 5% CO ₂ , every 2 days The medium was changed once, the dispersed cells were digested with 0.25% trypsin, and the cell viability was tested with trypan blue. The tumor cell viability was required to reach more than 95%. Inoculate 10,000 cells per well in a 96-well culture plate, culture in a cell incubator for 4 hours, add 20 μL of drug solution to each well, and test drug concentrations are 10.0, 5.0, 2.5, 1.25, 0.625, 0.3125, 0.15625 μg /mL, 5 replicate wells were set up for each drug concentration, and the control group was added with DMEM medium, and cultured in a cell incubator containing 5% CO ₂ at 37°C for 72 hours. Add freshly prepared 0.5 mg/mL tetrazolium blue tetrazolium-free serum-free culture solution to each well, incubate at 37°C for another 4 hours, and discard the supernatant. Add 200 μL of dimethyl sulfoxide to each well to fully dissolve, set the detection wavelength to 570 nm, and the reference wavelength to 450 nm, and detect the absorbance (OD value) with a microplate reader. The tumor inhibition rate was calculated according to the formula (1), and the half concentration (IC ₅₀ ) was calculated by using Origin software to draw a graph, and the measured maximum maximum inhibition rate (I _max ) was given. The specific results are shown in Table 1. It can be seen from the data in the table that hydroxycamptothecin polyglycerol 1500 succinate and hydroxycamptothecin have similar inhibitory effects on the two tumor cells, and there is no significant difference between the two.

Formula 1)

Table 1 Inhibitory effect of hydroxycamptothecin and hydroxycamptothecin polyglycerol 1500 succinate on tumor cell growth

Claims (5)

1. a polyglycerol fatty acid ester derivative for hydroxycamptothecine, it is characterized in that polyglycerol fatty acid and hydroxycamptothecine 10 hydroxyls form the structure of ester, its structure is such as formula shown in I:

Formula I

Wherein, n value is 3 ~ 50, m value is 1 ~ 5.

2. the polyglycerol fatty acid ester derivative of a kind of hydroxycamptothecine as claimed in claim 1, is characterized in that Polyglycerine part is the Polyglycerine with side chain.

3. the polyglycerol fatty acid ester derivative of a kind of hydroxycamptothecine as claimed in claim 1, is characterized in that the polymerization degree n of Polyglycerine part is preferably 10 or 20.

4. the polyglycerol fatty acid ester derivative of a kind of hydroxycamptothecine as claimed in claim 1, is characterized in that the m value of fatty acid part is preferably 2 or 3.

5. the polyglycerol fatty acid ester derivative of a kind of hydroxycamptothecine as claimed in claim 1, its purposes is for the preparation of antitumor drug.