CN103816548A - Target hydrophilic polymer-triptolide conjugate - Google Patents

Abstract

Targeting hydrophilic polymer-triptolide conjugate (I), wherein, P is a water-soluble polymer; D is triptolide; T is a targeting molecule; L and Z are linking groups. The combination improves the water solubility of triptolide, reduces its toxicity and prolongs its circulating half-life in organisms. (I)

Description

Targeted hydrophilic polymer-triptolide conjugate

technical field

The invention relates to a targeted water-soluble polymer-triptolide conjugate and a preparation method thereof, in particular to a folic acid compound-polyglutamic acid-triptolide prodrug and a preparation method thereof.

Background technique

Tripeterygium wilfordii (Tripeterygium wilfordii) is a plant belonging to the family Maseratiaceae, also known as yellow vine, yellow vine wood, classic wax vine, and heartbroken grass. This genus is mainly distributed in Zhejiang, Hunan, Anhui, Fujian, Guangdong, Guangxi, Yunnan and other provinces south of the Yangtze River Basin in China and the Changbai Mountain area in Northeast China. This product has been circulated among the people as a herbal medicine for a long time. The main medicinal part is the root, which has the functions of promoting blood circulation and eliminating addiction, clearing away heat and detoxification, reducing swelling and dissipating stagnation, killing insects and stopping bleeding, etc., but it is poisonous. In recent years, studies have shown that Tripterygium wilfordii has anti-inflammatory, antibacterial, immunosuppressive, anti-fertility, and anti-tumor effects. varying degrees of efficacy.

Among the various components of Tripterygium wilfordii, diterpenoids have the strongest medicinal effect. Triptolide, also known as triptolide, is the most studied ingredient. It is one of the main effective ingredients for treating tumors, and it is also the main toxic ingredient. It has obvious inhibitory or killing effect on isolated white blood cells of leukemia patients. 0.1 and 0.25 mg/kg have obvious activity on mouse L1210 and P388, and the life extension rate is above 159%, and some animals can survive for a long time. Triptolide showed obvious growth inhibitory effect on three domestic gastric cancer cell lines tested.

The pure product of triptolide is white columnar crystal, almost insoluble in water, and has great toxic and side effects. These shortcomings have greatly hindered its further promotion in clinical application. Therefore, while improving the water solubility of triptolide, reduce its toxicity, increase the pharmacological half-life of the drug, enhance its stability and the probability of reaching the target site, change the route of administration and improve bioavailability, and water-soluble polymer polymerization Physical bonding is the object of the present invention.

Polyglutamic acid (PGA) was first discovered in 1937. The researchers found PGA in the cell capsules of Bacillus anthracis and Saccharification bacteria, and it is one of the main components of certain microbial capsules. PGA was later also found in Bacillus subtilis and Bacillus natto. PGA is a kind of polypeptide molecule formed by condensation of glutamic acid units in the form of peptide bonds.

PGA has the following outstanding advantages: (1) biodegradability, PGA can be degraded into endogenous glutamic acid by lysosome in vivo and directly absorbed by organisms; The carboxyl group is easy to modify and combine with drugs; (3) Solubilization, PGA can increase the solubility and stability of drugs. These properties of PGA make it an ideal drug carrier.

Folate receptors are overexpressed on the cell surface of some human tumors (ovarian cancer, cervical cancer, endometrial cancer, breast cancer, colon cancer, lung cancer, choroidal cancer, ependymal cell carcinoma, etc.), while normal tissues rarely have Folate receptor overexpression. They have the potential to serve as anti-cancer therapeutic targets and can be utilized by various approaches such as chemotherapy, immunotherapy, radiation therapy, and gene therapy. Compared with tumor-specific monoclonal antibodies, folic acid compounds have the advantages of low cost, high chemical and biological stability, and small size, and the binding of folic acid compound ligands to receptors is specific, selective, and saturable. characteristics such as sex. Therefore, the folic acid compound has great research and development prospects as a tumor diagnosis and treatment drug. The related drug Vintafolide (a small-molecule conjugate targeting folic acid receptors, which is currently used in platinum-resistant ovarian cancer and non-small cell lung cancer) has entered phase 2 clinical research.

Therefore, the object of the present invention is to combine polyglutamic acid water-soluble polymers with folic acid compounds and triptolide by chemical methods, thereby improving the solubility of triptolide, making it targeted, and reducing Its toxic and side effects prolong the circulating half-life of triptolide in organisms, so as to ensure proper drug concentration and provide sustained release function.

Contents of the invention

According to one aspect of the present invention, the targeted water-soluble polymer-triptolide conjugate of general formula (I) is provided:

(I)

in:

P is a water-soluble polymer selected from the group consisting of α-polyglutamic acid, γ-polyglutamic acid, etc.;

n is an integer, the maximum number of active functional groups on the terminal group does not exceed P;

L is a linking group selected from O(CH ₂ ) _k CO, O(CH ₂ ) _k OCO, O(CH ₂ ) _k KNHCO, NH(CH ₂ ) _k OCO, NH(CH ₂ ) _k NHCO, NH( CH ₂ ) group consisting of _k CO, wherein k is an integer from 0 to 6;

T is a targeting molecule selected from folic acid compounds;

Z is a linking group selected from the group consisting of ester groups, carbonate esters and amino acid ester groups;

D is triptolide.

In a preferred embodiment of the invention, the polyglutamic acid is preferably γ-polyglutamic acid.

In a preferred embodiment of the present invention, the conjugate is a folic acid-water-soluble polymer-triptolide conjugate represented by the following general formula:

in:

P is a water-soluble polymer, preferably γ-polyglutamic acid;

n is an integer, the maximum number of active functional groups on the terminal group does not exceed P;

L is a linking group selected from O(CH ₂ ) _k CO, O(CH ₂ ) _k OCO, O(CH ₂ ) _k KNHCO, NH(CH ₂ ) _k OCO, NH(CH ₂ ) _k NHCO, NH( CH ₂ ) group consisting of _k CO, wherein k is an integer from 0 to 6;

T is a targeting molecule, preferably folic acid;

Z is a linking group selected from the group consisting of ester groups, carbonate esters and amino acid ester groups;

According to the present invention, there is provided a pharmaceutical composition comprising the above-mentioned combination.

The advantage of the present invention is that the combination drug of triptolide can be protected through the modification of the water-soluble polymer, its stability and water solubility are improved, and the circulation half-life of the active extract of triptolide in organisms is prolonged, It ensures proper drug concentration and provides sustained release function; in addition, folic acid molecules realize the active targeting of drugs and reduce toxicity.

Detailed ways

The conjugate of the present invention can be prepared as follows: the hydroxyl group on triptolide is directly combined with the carboxyl group on polyglutamic acid; or the triptolide is first modified (such as with amino acids), and then combined with polyglutamic acid combined.

Now, γ-polyglutamic acid will be described as an example of polyglutamic acid. However, it should be understood that the polyglutamic acid of the present invention is not limited to γ-polyglutamic acid, and α-polyglutamic acid and the like can also be used.

The structural formulas of α-polyglutamic acid and γ-polyglutamic acid can be as shown in (II), (III):

  

(II)

(III)

n is any integer, characterizing its degree of polymerization.

For polyglutamic acid, it is generally expressed by molecular weight, as long as the molecular weight of the polyglutamic acid forming the conjugate is 10,000-60,000 Daltons, which corresponds to n being about 75-385. More preferably, n is 75, 153, 230, 308 and 385, which correspond to molecular weights of 10,000, 20,000, 30,000, 40,000 and 50,000, respectively. Due to the potential heterogeneity of the starting polyglutamic acid compound, usually defined by its average molecular weight rather than from repeating units, it is preferred to characterize polyglutamic acid polymers by molecular weight rather than by the integer n representing the polyglutamic acid polymer. self-repeating unit. Starting polyglutamate compounds of various molecular weights can be prepared by methods known in the art or can be obtained from commercial sources.

The chemical structure of triptolide (triptolede) is shown below:

Triptolide (triptolede)

The above structure contains hydroxyl groups, which can be combined with polyglutamic acid through ester groups to achieve effective protection and rational utilization of drug molecules. The ester group can release the active ingredient of the drug through biodegradation in the organism.

The chemical structure of folic acid is shown below:

Folic acid

Its structure contains a carboxyl group, which can be combined with the terminal amino group of polyglutamic acid through an ester group to achieve the active targeting effect of the prodrug molecule.

Example 1

Synthesis of folic acid-α-polyglutamic acid-triptolide conjugate (1): 50 mg triptolide, 5 mg folic acid, 0.2 g α-polyglutamic acid (Mw 30,000) were dissolved in 10 mL anhydrous In N,N-dimethylformamide, add 30 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 30 mg N-hydroxysuccinimide, 5 mg 4- Dimethylaminopyridine, stirred at room temperature, reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. Folate-α-polyglutamic acid-triptolide (1) was obtained.

(1).

Example 2

Synthesis of folic acid-γ-polyglutamic acid-triptolide conjugate (2): 50 mg triptolide, 5 mg folic acid, 0.2 g γ-polyglutamic acid (Mw 30,000) were dissolved in 10 ml anhydrous In N,N-dimethylformamide, add 30 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 30 mg N-hydroxysuccinimide, 5 mg 4- Dimethylaminopyridine, stirred at room temperature, reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. Get folic acid-γ-polyglutamic acid-triptolide (2)

 

 

(2).

Example 3

Synthesis of folic acid-γ-polyglutamic acid-glycine-triptolide conjugate (3): 50 mg of triptolide, 50 mg of Boc-glycine were dissolved in 10 ml of anhydrous dichloromethane at 0 °C Add 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 10 mg of 4-dimethylaminopyridine, stir and dissolve for 2 hours, and wash the product with 10 ml of 0.5% sodium bicarbonate successively Twice, washed once with 10 ml of water, washed twice with 10 ml of 0.1N hydrochloric acid solution, washed once with 10 ml of saturated saline, and finally dried with anhydrous magnesium sulfate, filtered to remove insoluble matter, and the solution was vacuum-dried, and the obtained solid was dissolved in 5 ml 4M dioxane hydrochloric acid solution, stirred at room temperature for 1 hour, added 50 ml of ether, filtered the solid and dissolved it in 10 ml of dichloromethane, adjusted the pH to 2.5 with saturated sodium bicarbonate, and washed the organic phase with anhydrous carbonic acid After drying over magnesium, the product was dissolved in 5 mL of dichloromethane and washed out with 50 mL of ether to give a solid (TPL-Gly). Dissolve the resulting solid with 5 mg of folic acid, 0.2 g of γ-polyglutamic acid (Mw 30,000) in 10 ml of anhydrous N,N-dimethylformamide, and add 30 mg of 1-(3-dimethylaminopropyl Base)-3-ethylcarbodiimide, 30 mg of N-hydroxysuccinimide, 5 mg of 4-dimethylaminopyridine, stirred at room temperature, and reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. The folic acid-γ-polyglutamic acid-glycine-triptolide conjugate (3) was obtained.

(3).

Example 4

Synthesis of folate-γ-polyglutamic acid-methionine-triptolide conjugate (4): 50 mg triptolide, 70 mg Bsmoc-methionine were dissolved in 10 ml anhydrous dichloromethane at 0 °C Add 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 10 mg of 4-dimethylaminopyridine, stir and dissolve for 2 hours, and wash the product with 10 ml of 0.5% sodium bicarbonate successively Twice, wash once with 10 ml of water, wash twice with 10 ml of 0.1N hydrochloric acid solution, wash once with 10 ml of saturated saline, and finally dry with anhydrous magnesium sulfate, filter to remove insoluble matter, and dry the solution in vacuum, dissolve the solid in 10 ml In anhydrous dichloromethane, add 30 mg of 4-piperidinyl piperidine, stir at room temperature for 5 hours, then wash twice with 10 ml of 0.1N hydrochloric acid solution, dry and filter the organic phase with anhydrous magnesium sulfate, and vacuum dry , to give a solid (TPL-Met). Dissolve the resulting solid with 5 mg of folic acid, 0.2 g of γ-polyglutamic acid (Mw 30,000) in 10 ml of anhydrous N,N-dimethylformamide, and add 30 mg of 1-(3-dimethylaminopropyl Base)-3-ethylcarbodiimide, 30 mg of N-hydroxysuccinimide, 5 mg of 4-dimethylaminopyridine, stirred at room temperature, and reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. The folic acid-γ-polyglutamic acid-methionine-triptolide conjugate (4) was obtained.

 

 

(4).

Example 5

Synthesis of folic acid-γ-polyglutamic acid-sarcosine-triptolide conjugate (5): 50 mg of triptolide, 45 mg of Boc-sarcosine were dissolved in 10 ml of anhydrous dichloromethane, Add 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mg of 4-dimethylaminopyridine, stir and dissolve at 0°C for 1 hour, then rise to room temperature and continue the reaction for 1 hour. The product was washed twice with 10 ml of 0.5% sodium bicarbonate, once with 10 ml of water, twice with 10 ml of 0.1N hydrochloric acid solution, once with 10 ml of saturated saline, and finally dried with anhydrous magnesium sulfate, and filtered to remove insoluble matter , the solution was dried in vacuo to obtain a solid. The obtained solid was dissolved in 10 ml of dichloromethane, 6 ml of trifluoroacetic acid was added, stirred at room temperature for 1 hour, then washed twice with 10 ml of 0.1N hydrochloric acid solution, the organic phase was dried and filtered with anhydrous magnesium sulfate, vacuum Drying gave a solid (TPL-Sar). Dissolve the resulting solid with 5 mg of folic acid, 0.2 g of γ-polyglutamic acid (Mw 30,000) in 10 ml of anhydrous N,N-dimethylformamide, and add 30 mg of 1-(3-dimethylaminopropyl Base)-3-ethylcarbodiimide, 30 mg of N-hydroxysuccinimide, 5 mg of 4-dimethylaminopyridine, stirred at room temperature, and reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. The folic acid-γ-polyglutamic acid-sarcosine-triptolide conjugate (5) was obtained.

 

(5).

Example 6

Synthesis of folic acid-γ-polyglutamic acid-alanine-triptolide conjugate (6): 50 mg of triptolide, 50 mg of Boc-alanine were dissolved in 10 ml of anhydrous dichloromethane, 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mg of 4-dimethylaminopyridine were added, stirred and dissolved at 0°C for 1 hour, and raised to room temperature for 1 hour of reaction. The product was washed successively twice with 10 ml of 0.5% sodium bicarbonate, once with 10 ml of water, twice with 10 ml of 0.1N hydrochloric acid solution, once with 10 ml of saturated saline, and finally dried with anhydrous magnesium sulfate, and filtered to remove insoluble matter , the solution was vacuum-dried, the solid was dissolved in 10 milliliters of anhydrous dichloromethane, 30 milligrams of 4-piperidinyl piperidine was added, stirred at room temperature for 5 hours, and then washed twice with 10 milliliters of 0.1N hydrochloric acid solution, the The organic phase was dried and filtered over anhydrous magnesium sulfate, and dried in vacuo to obtain a solid (TPL-Ala). Dissolve the resulting solid with 5 mg of folic acid, 0.2 g of γ-polyglutamic acid (Mw 30,000) in 10 ml of anhydrous N,N-dimethylformamide, and add 30 mg of 1-(3-dimethylaminopropyl Base)-3-ethylcarbodiimide, 30 mg of N-hydroxysuccinimide, 5 mg of 4-dimethylaminopyridine, stirred at room temperature, and reacted overnight. The solution was added to 30 ml of chloroform, filtered to obtain a solid, dissolved in NaHCO ₃ saturated solution, dialyzed, and the product was lyophilized. The folic acid-γ-polyglutamic acid-alanine-triptolide conjugate (6) was obtained.

(6).

Claims (4)

1. the targeted hydrophilic polymer-triptolide conjugate of general formula (I): (I) in: P is a water-soluble polymer selected from the group consisting of α-polyglutamic acid, γ-polyglutamic acid, etc.; n is an integer, the maximum number of active functional groups on the terminal group does not exceed P; L is a linking group selected from O(CH ₂ ) _k CO, O(CH ₂ ) _k OCO, O(CH ₂ ) _k KNHCO, NH(CH ₂ ) _k OCO, NH(CH ₂ ) _k NHCO, NH( CH ₂ ) group consisting of _k CO, wherein k is an integer from 0 to 6; T is a targeting molecule selected from folic acid compounds; Z is a linking group selected from the group consisting of ester groups, carbonate esters and amino acid ester groups; D is triptolide. 2. The conjugate according to claim 1, wherein the polyglutamic acid has a molecular weight between 10,000-60,000 Daltons. 3. The combination as claimed in claim 1, wherein the folic acid compound comprises folic acid, leucovorin, dihydrofolate, tetrahydrofolate, tetrahydropterine, dipryl polyglutarate, 2-deamino-hydroxyfolate, 1 - one of desamino-hydroxyfolate, 1-deazofolate, 3-deazofolate or 8-deazofolate. 4. The conjugate of claim 1, wherein the conjugate is selected from the group consisting of: Folic acid-α-polyglutamic acid-triptolide conjugate (1); Folic acid-γ-polyglutamic acid-triptolide conjugate (2); Folic acid-γ-polyglutamic acid-glycine-triptolide conjugate (3); Folic acid-γ-polyglutamic acid-methionine-triptolide conjugate (4); Folic acid-γ-polyglutamic acid-sarcosine-triptolide conjugate (5); Folic acid-γ-polyglutamic acid-alanine-triptolide conjugate (6); composed of groups.