CN1840201A - Polyethylene glycol prodrug of gambogicacid, its preparation method, formulation and use - Google Patents
Polyethylene glycol prodrug of gambogicacid, its preparation method, formulation and use Download PDFInfo
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Abstract
This invention relates to the field of nature medicament and high molecular material, specifically relates to a kind of former medicine (1)of polietilenglicol. This invention also discloses the method for preparing the former medicine and the medicine composition that taking (1) as active constituent and their application in tumour threatment.
Description
Technical Field
The invention relates to the field of natural medicines and high polymer materials, in particular to a polyethylene glycol gambogic acid prodrug, and also discloses a preparation method thereof, a pharmaceutical composition taking the prodrug as an active ingredient, and application of the prodrug in treating tumors.
Background
Total gambogic acid comprises gambogic acid, neogambogic acid, and allogambogic acid, and is an acidic effective component extracted from dried resin secreted by resina Garciniae. The literature reports that the compound has good antitumor effect, but the clinical application of the compound is limited due to the defects of poor water solubility and high toxicity.
CN01108049.3 discloses that gambogic acid compounds form water-soluble complexes with different bases or ions, increasing the effectiveness of gambogic acid compounds and reducing irritation and toxicity. Intravenous injection LD of total gambogic acid sodium mouse50It was 28.56 mg/kg. CN0212455. X discloses that gambogic acid compound and glucosamine compound are formed into compound, so that its solubility in water is raised, and the solubility of gambogic acid-meglumine compound in water is up to 0.05g/ml, at the same time its medicinal effect is also raised, and in the test of growth of liver cancer SMMC7721 cell the IC50 of gambogic acid meglumine, neogambogic acid glucosamine and neogambogic acid meglumine are respectively 3.21(ug/ml), 2.17(ug/ml) and 2.00 (ug/ml). There is a clinical need for drugs that have both good water solubility and better potency with less toxicity.
Disclosure of Invention
The invention discloses a polyethylene glycol prodrug of gambogic acid compounds, and the gambogic acid compounds refer to total gambogic acid, neogambogic acid, allogambogic acid and the like. The total gambogic acid contains gambogic acid, neogambogic acid, allogambogic acid, etc. The structure of the gambogic acid compound is as follows:
gambogic acid allogambogic acid
Neogambogic acid
Polyethylene glycol (PEG) plays an important role in the synthesis and preparation of prodrugs with incomparable advantages of good water solubility, no toxicity, good biocompatibility, no immunogenicity and other macromolecules. The FDA in the United states has approved PEG to be used in human body, and the Chinese pharmacopoeia 2005 edition also collects polyethylene glycol as an auxiliary material. The invention prepares gambogic acid (including total gambogic acid, neogambogic acid and allogambogic acid) into the pegylated prodrug, and the obtained prodrug has good water solubility, improved drug effect and reduced toxic and side effects, and is more suitable for clinical use.
The pegylated gambogic acid prodrugs of the present invention can be represented by the following formula:
a compound of the general formula:
(m is 1 or 2)
Wherein: g is total gambogic acid, neogambogic acid or allogambogic acid;
p is polyethylene glycol, polyethylene glycol diamine, polyethylene glycol monomethyl ether, polyethylene glycol amine monomethyl ether or other polyethylene glycol derivatives;
a is a connecting bond, or an amino acid, oligopeptide, a disubstituted p-aminobenzyl alcohol derivative or a disubstituted p-hydroxybenzoic acid derivative, wherein the substituent is C1-4 alkyl or C1-4 alkoxy.
When A is a connecting bond, the structural formula is II.
The invention also includes pharmaceutically acceptable salts of general formula I or II in addition to the prodrugs described above.
The structural formula of PEG is HOCH2CH2(OCH2CH2O)nCH2CH2OH or HOCH2(CH2OCH2)nCH2And (5) OH. n is the degree of substitution, and n is more than or equal to 1. The average molecular weight of PEG in the present invention is preferably 400-40,000 (40K). More preferred is PEG2000(2K), PEG4000(4K), PEG6000(6K), PEG10,000(10K), PEG15,000(15K), PEG20,000(20K) or PEG40,000 (40K).
A is a linking arm which can be a bond only, namely a structure shown in a general formula II, and can also be a compound which is formed by connecting P and G by modifying or chemically reacting α -amino acid, β -amino acid, oligopeptide with 2-4 amino acids or other compounds with one carboxyl group and one amino group (or hydroxyl group), wherein the compounds with one carboxyl group and one amino group (or hydroxyl group) are preferably disubstituted P-aminobenzyl alcohol derivatives or disubstituted P-hydroxybenzoic acid derivatives, wherein the substituent is C1-4 alkyl or C1-4 alkoxy, more preferably 3, 5-dimethyl-4-amino-benzyl alcohol, 3, 5-dimethoxy-4-amino-benzyl alcohol, 3, 5-dimethyl-4-hydroxy-benzoic acid or 3, 5-dimethoxy-4-hydroxy-benzoic acid.
The amino acid is preferably glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine or arginine, etc.
Further preferred amino acids are: glycine, leucine, lysine, tryptophan, valine.
The preparation process of the pegylated gambogic acid prodrug is simple, and the preparation of the gambogic acid compound can be prepared by reference documents (Chenbaozhu: the research of gambogic anticancer components, Jiangxi medical college, 1980, 37 (2): 1-7; Lvgui Bao: the separation and structure of neogambogic acid in gamboge, pharmaceutical science, 1984, 19 (8): 636-639.).
The prodrug is prepared as follows:
when A is a bond, i.e., formula II:
dissolving gambogic acid compounds in a solvent a under the action of a dehydrating agent and a catalyst on dimethylaminopyridine, stirring for 0.5-5 h, adding polyethylene glycol, stirring at room temperature, filtering, concentrating the filtrate to dryness, recrystallizing with a solvent b, purifying and drying by a sephadex column to obtain the compound I. Wherein the dehydrating agent is Dicyclohexylcarbodiimide (DCC), EDC or DIPC, preferably DCC; solvent a is dichloromethane, chloroform, DMF or dioxane, preferably dichloromethane; solvent b is isopropanol, chloroform-diethyl ether, dichloromethane-diethyl ether or ethanol-diethyl ether, preferably isopropanol.
The better method is as follows: dissolving gambogic acid compounds (total gambogic acid, neogambogic acid or allogambogic acid) in a certain amount of dichloromethane under the action of a dehydrating agent Dicyclohexylcarbodiimide (DCC) and a catalyst p-Dimethylaminopyridine (DMAP), stirring at 0 ℃ for 0.5-5 h, preferably 2h, adding PEG (the molar ratio of amino or hydroxyl of PEG or PEG derivatives to drug carboxyl is 1: 1-2.5), heating to room temperature (15-30 ℃), preferably stirring at 25 ℃ overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, lyophilizing or vacuum concentrating, and drying to obtain gambogic acid polyethylene glycol prodrug represented by formula II.
The reaction route is exemplified by gambogic acid:
when A is amino acid or oligopeptide, the preparation method is as follows:
dissolving polyethylene glycol, N-tert-butyloxycarbonylamino acid (Boc-A), a dehydrating agent and a catalyst into a solvent a, stirring, filtering or centrifuging, concentrating the filtrate to dryness, dissolving the residue into the solvent, dropwise adding into glacial ethyl ether, standing, filtering after solid is separated out, dissolving into dichloromethane-trifluoroacetic acid or ethyl acetate-trifluoroacetic acid after drying, concentrating to dryness, pouring the residue into ethyl ether, filtering, and recrystallizing the crude product with ethanol-ethyl ether to obtain polyethylene glycol-amino acid trifluoroacetate; dissolving polyethylene glycol-amino acid trifluoroacetate, gambogic acid compound, DCC and solvent c in dichloromethane, stirring at room temperature, filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with sephadex column, and drying to obtain I; wherein the dehydrating agent is Dicyclohexylcarbodiimide (DCC), [ 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or Diisopropylcarbodiimide (DIPC), preferably DCC; solvent a is dichloromethane, chloroform, DMF or dioxane, preferably dichloromethane; solvent b is isopropanol, chloroform-diethyl ether, dichloromethane-diethyl ether or ethanol-diethyl ether, preferably isopropanol; the solvent c is pyridine, diethylamine, N-methylmorpholine or triethylamine, preferably triethylamine.
The better preparation method comprises the following steps:
dissolving PEG derivative and N-tert-butyloxycarbonyl amino acid in a dehydrating agent (DCC or EDC, DIPC, preferably DCC) and a catalyst (DMAP) in a certain amount of dichloromethane (or chloroform, dioxane, DMF, preferably dichloromethane), and stirring overnight at 25 deg.C (10-30 deg.C,preferably 25 deg.C). Filtering or centrifuging, concentrating the filtrate to dryness, adding cold acetone (or methanol, ethanol, dichloromethane, chloroform, preferably acetone), filtering again to remove a small amount of insoluble substances, concentrating the filtrate to dryness again, dissolving the residue in a certain amount of dichloromethane (or chloroform, dioxane, DMF, preferably dichloromethane), dropwise adding into glacial ethyl ether, standing, filtering after the solid is completely separated out, vacuum drying, dissolving in dichloromethane-trifluoroacetic acid (1: 1) (or ethyl acetate-trifluoroacetic acid (1: 1)) solution, stirring at room temperature, concentrating to dryness, pouring the residue into diethyl ether, filtering, and recrystallizing the crude product with ethanol-diethyl ether to obtain polyethylene glycol-glycine trifluoroacetate. Dissolving gambogic acid compound and DCC and triethylamine (pyridine, diethylamine, N-methylmorpholine, etc., preferably triethylamine) 2ml in a certain amount of dichloromethane at a molar ratio of 1: 1-8 of amino group and drug carboxyl group, and stirring at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain gambogic acid compound polyethylene glycol prodrug represented by formula I. The reaction formula is as follows:
a is oligopeptide, which is similar to amino acid, but several amino acids are linked together by amido bond, or one amino end is a carboxyl end, and can be represented by the formula.
When A is aminobenzol derivative, the preparation method is as follows:
dissolving PEG derivative T-PEG (PEG-Thiazolidine-2-thione) (preparation method reference Green et al: PEGThiazolidine-2-thon. a Novel Reagent for a facility Protein Modification of Bovine Hemoglobin. Bioconugate chem.1996, 7: 638-; wherein solvent a is dichloromethane, chloroform, DMF or dioxane, preferably dichloromethane; solvent b is isopropanol, chloroform-diethyl ether, dichloromethane-diethyl ether or ethanol-diethyl ether, preferably isopropanol.
The better preparation method comprises the following steps:
dissolving T-PEG, p-aminobenzyl alcohol and catalyst p-dimethylaminopyridine in a certain amount of dichloromethane (or chloroform, dioxane and DMF are preferably dichloromethane), and stirring overnight at 10-30 ℃ (preferably 25 ℃). Concentrating the reaction solution, pouring the residue into ethyl ether, filtering, standing, filtering after the solid is completely separated out, and recrystallizing by using isopropanol. Dissolving the obtained solid and gambogic acid compound with DCC and DMAP in a certain amount of dichloromethane (or chloroform, dioxane and DMF preferably dichloromethane) according to the ratio (molar ratio) of amino to drug carboxyl of 1: 1-8, and stirring overnight at 10-30 ℃ (preferably 25 ℃). Filtering or centrifuging, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain gambogic acid compound polyethylene glycol prodrug represented by formula I. The reaction formula is as follows:
the inventor finds in research that the gambogic acid (including total gambogic acid, neogambogic acid and allogambogic acid) polyethylene glycol prodrug has excellent water solubility and reduced toxicity, andcompared with sodium gambogic acid, the sodium gambogic acid prodrug has LD (laser shock resistance) injected intravenously in mice5029.01mg/kg, the toxicity of the gambogic acid polyethylene glycol prodrug is lower than that of sodium gambogic acid, and the LD of partial prodrug is50Comprises the following steps: LD of polyethylene glycol 10K-lysine-gambogic acid, polyethylene glycol 20K-glycine-gambogic acid and polyethylene glycol 6K-lysine-gambogic acid injected into mouse vein5048.31, 42.15 and 50.12mg/kg (calculated by gambogic acid) respectively.
The present study shows that: the gambogic acid (including total gambogic acid, neogambogic acid and allogambogic acid) polyethylene glycol prodrug has no vascular irritation (equivalent to 1mg gambogic acid/20 ml/kg for rabbit ear intravenous drip). A hemolysis test of the gambogic acid polyethylene glycol prodrug of the invention is carried out by using 2% rabbit erythrocyte suspension, and shows that the hemolysis and aggregation of erythrocytes are not found within 2h when the dosage of below 0.5ml (which is equivalent to 25mg of gambogic acid dissolved in 500ml of 0.9% sodium chloride injection).
The research of the in vitro growth inhibition effect of the tumor cells by the inventor shows that the absorbance values (A) of cell culture solutions and a control group with different prodrug concentrations are measured at the wavelength of 570nm of an enzyme labeling instrument by using a tetramethyl azoazolate colorimetric method, and the cell inhibition rate is calculated.IC50According to the calculation of the improved kouzhi method, after the gambogic acid polyethylene glycol prodrug acts on human liver cancer cells 7721, the cell growth is obviously inhibited, the cell proliferation rate is reduced, the cell inhibition rate is increased, and part of prodrugs (polyethylene glycol10K-lysine-gambogic acid, polyethylene glycol 20K-glycine-gambogic acid and polyethylene glycol 6K-lysine-gambogic acid) are IC50Respectively 1.78 mug/ml, 1.82 mug/ml and 1.90 mug/ml (calculated by gambogic acid).
According to the invention, the gambogic acid compound is prepared into the polyethylene glycol prodrug, so that the water solubility is greatly improved to be about 100-800 mg/ml, and the preparation method is more favorable for preparing various injection preparations for clinical use. Moreover, the gambogic acid and the polyethylene glycol are combined through covalent bonds, and the quality is more stable compared with the complex.
After the traditional Chinese medicine enters a human body, under the action of in-vivo enzymes, amide bonds and ester bonds are hydrolyzed firstly, and the original medicine, namely gambogic acid compounds, is released to play a pharmacological action. The polyethylene glycol is further broken into smaller polyethylene glycol fragments, filtered through the glomeruli, and excreted from the kidney. The amino acid as the connecting arm is the basic unit of protein composing human body, and has no toxic and side effect to human body. PEG prodrugs can extend the half-life of the drug, enabling it to maintain therapeutic levels for longer periods of time. The stability of the medicine is increased, the medicine intake of cells through endocytosis is improved, and the passive targeting of the medicine is enhanced by utilizing the permeation and retention enhancing Effect (EPR) of tumor cells.
The invention also discloses a pharmaceutical composition, which contains therapeutically effective dose of the gambogic acid polyethylene glycol prodrug taking the general formula (I) or (II) as an active ingredient or pharmaceutically acceptable salt thereof, and also contains one or more pharmaceutically acceptable carriers.
The pharmaceutically acceptable salts of the present invention may be: the phenolic hydroxyl group of gambogic acid, allogambogic acid or neogambogic acid has chemical structure of sodium salt, magnesium salt, calcium salt or other salts.
The pharmaceutically acceptable carrier refers to diluents, pH regulators and other pharmaceutically common auxiliary materials, and comprises various monosaccharides, disaccharides, polysaccharides, various electrolytes, various solvents and the like and mixtures thereof. Such as mannitol, sorbitol, glucose, sucrose, lactose, trehalose, dextran, hydroxyethyl starch, sodium chloride, hydrochloric acid, sodium hydroxide, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium acetate, sodium citrate, water for injection, propylene glycol, ethanol, glycerol, polyethylene glycol, povidone, and the like. The optimal composition is active ingredient, mannitol, glucose, lactose or sucrose, sodium chloride, water for injection, and hydrochloric acid.
The dosage form of the pharmaceutical composition comprises: injection or powder for injection. Prepared according to conventional methods in the pharmaceutical field, for example, by mixing the active ingredient with one or more carriers and then formulating it into the desired dosage form.
The pharmaceutical composition of the present invention may contain 0.1 to 100% by weight of the active ingredient of the general formula (I) or (II).
The amount of the polyethylene glycol prodrug of gambogic acid administered in accordance with the present invention may vary depending on the route of administration, the age, weight, body surface area of the patient, the type and severity of the condition being treated, etc., and the daily dosage may be from 0.05 to 100mg/m (in terms of gambogic acid)2Body surface area, preferably 5-100mg/m2Andcan be used for one or more times of intravenous drip after being diluted by isotonic glucose injection or sodium chloride injection.
The pegylated gambogic acid prodrug or the pharmaceutically acceptable salt thereof can be used for treating tumor diseases.
Detailed Description
Example 1
Preparation of polyethylene glycol 4K-gambogic acid
Gambogic acid (0.346g, 0.55mmol), Dicyclohexylcarbodiimide (DCC) (1.23g, 0.6mmol), and Dimethylaminopyridine (DMAP) (0.03g, 0.25mmol) were dissolved in 30ml of dichloromethane, stirred at 0 ℃ for 2 hours, added PEG4K (1g, 0.25mmol), warmed to 25 ℃, and stirred overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder polyethylene glycol 4K-gambogic acid 1.0G, with yield: 76.1 percent. mp: 52-54 ℃. Content of gambogic acid: 21.93% (theory: 23.9%). Solubility in water at room temperature is about: 650mg/ml
1HNMR(500MHz，CDCl3): the delta 3.6 is the strong peak of hydrogen on PEG, the rest peaks are the peaks of hydrogen on gambogic acid, delta 1.28-1.45(C24-H, C25-H, 12H), delta 1.55-2.06(C29-H, C34-H, C35-H, C39-H, C40-H, 30H), delta 2.31-2.62(C21-H, C22-H, 6H), delta 2.95(C26-H, 4H), delta 3.10(C31-H, 4H), delta 3.56-3.87(PEG-H, C11-H, 1H, CO-O-CH)2-，4H)，δ5.05-5.17(C32-H，C37-H，4H)，δ5.41-5.45(C3-H(C4-H)，2H)，δ6.30(C27-H，2H)，δ6.64-6.67(C4-H(C3-H)，2H)，δ7.50-7.55(C10-H，2H)。
IR: besides the characteristic peak of PEG, 1745cm is added in the infrared spectrum-1The ester carbonyl peak of (a), indicates that gambogic acid has been attached to PEG via an ester linkage.
Example 2
Preparation of polyethylene glycol 20K-glycine-gambogic acid
1. Preparation of polyethylene glycol 20K-Glycine trifluoroacetate (PEG 20K-Gly-FA):
PEG20K (40g, 2mmol), N-t-butyloxycarbonylglycine (Boc-Gly) (0.77g, 4.4mmol), and p-Dimethylaminopyridine (DMAP) (0.12g, 1mmol) were weighed out and dissolved in 100ml of dichloromethane, and Dicyclohexylcarbodiimide (DCC) (1.2g, 4.8mmol) was weighed out and dissolved in 4ml of dichloromethane and added dropwise to the reaction mixture. Stir at room temperature overnight. Centrifuging, removing precipitate, concentrating the clear solution to dryness, adding 40ml of cold acetone, filtering again to remove a small amount of insoluble substances, concentrating the filtrate to dryness again, dissolving the residue in 20ml of dichloromethane, dropwise adding 300ml of vigorously stirred glacial ethyl ether, standing, filtering after the solid is completely separated out, and vacuum drying to obtain 36.4g of white solid. Dissolving the white solid in 60ml dichloromethane-trifluoroacetic acid (1: 1) solution, stirring at room temperature for 30min, concentrating to dryness, pouring 300ml ether into the residue, filtering to obtain crude product, and recrystallizing with ethanol-ether to obtain 33.1g white solid polyethylene glycol-glycine trifluoroacetate (PEG20K-Gly-FA), with yield: 82.5 percent.
2. Preparation of polyethylene glycol 20K-glycine-gambogic acid:
PEG20K-Gly-FA (2.5g, 0.125mmol), gambogic acid (0.189g, 0.3mmol), DCC (0.062g, 0.3mmol) and triethylamine (2 ml) were weighed out and dissolved in 20ml dichloromethane and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex gel column, and lyophilizing to obtain yellow powder of polyethyleneglycol-glycine-gambogic acid (PEG-Gly-GA)2.2G, with yield: 81 percent. Content of gambogic acid: 5.18% (theory: 5.88%). Solubility in water at room temperature is about: 120 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest peaks are the peaks of hydrogen on gambogic acid and glycine, delta 1.26-1.45 (C)24-H，C25-H，12H)，δ1.54-2.06(C29-H，C34-H，C35-H，C39-H，C40-H，30H)，δ2.30-2.62(C21-H，C22-H，6H)，δ2.88(C26-H，4H)，δ3.18-3.27(C31-H，4H)，δ3.56-3.77(PEG-H，C11-H)，δ3.89(CO-O-CH2-，4H)，δ4.32(CO-N-CH2-CO，4H)，δ5.05-5.17(C32-H，C37-H，4H)，δ5.41-5.45(C3-H(C4-H)，2H)，δ6.30(C27-H，2H)，δ6.64-6.67(C4-H(C3-H)，2H)，δ7.50-7.55(C10-H，2H)。
FTIR: except the characteristic peak of PEG in the infrared spectrum, the molecular weight of PEG is largeThe carbonyl peak was weaker but still seen to increase 1745cm-1And the ester carbonyl peak of 1658cm-1The amide carbonyl peak of (A) shows that amino acid is used as a connecting arm on PEG, one end of the amino acid is ester-forming, and the other end of the amino acid is amide-connected with gambogic acid
Example 3
Preparation of polyethylene glycol 10K-lysine-gambogic acid
1. Preparation of polyethylene glycol-lysine trifluoroacetate (PEG 10K-Lys-FA):
the operation method is the same as example 2, and the yield is as follows: 78.7 percent.
2. Preparation of polyethylene glycol 10K-lysine-gambogic acid:
PEG-Lys-FA (1.25g,0.125mmol), gambogic acid (0.47g, 0.75mmol), DCC (0.16g, 0.8mmol), triethylamine or 2ml was dissolved in 20ml dichloromethane and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder polyethylene glycol 20K-lysine-gambogic acid (PEG-Lys-GA)1.2G, with yield: 68.3 percent. Content of gambogic acid: 16.46% (theory: 19.79%). Solubility in water at room temperature is about: 440 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest are the peaks of hydrogen on gambogic acid and lysine, delta 1.23-1.45(C24-H, C25-H, 24H, lysine-CH)2-, 8H), delta 1.55-2.12(C29-H, C34-H, C35-H, C39-H, C40-H, 60H, on-CH of lysine2-，4H)，δ2.31-2.62(C21-H，C22-H，12H)，δ2.88-3.00(C26-H，8H，CO-N-CH2，4H)，δ3.10(C31-H，8H)，δ3.56-3.77(PEG-H，C11-H)，δ3.91(CO-O-CH2-, 4H), delta 4.36 (CO-CH-N, 2H on lysine), delta 5.05-5.17(C32-H, C37-H, 8H), delta 5.41-5.45(C3-H (C4-H), 4H), delta 6.30(C27-H, 4H), delta 6.64-6.67(C4-H (C3-H), 4H), delta 7.50-7.55(C10-H, 4H).
IR: besides the characteristic peak of PEG, 1740cm is added in the infrared spectrum-1And the ester carbonyl peak of 1651cm-1The amide carbonyl peak of (A), indicating that PEG has been attached to itAmino acid is a connecting arm, one end of the amino acid is ester, and the other end of the amino acid is amide connected with gambogic acid.
Example 4
Preparation of polyethylene glycol 6K-tryptophan-gambogic acid
1. Preparation of polyethylene glycol-tryptophan trifluoroacetate (PEG 6K-Trp-FA):
the operation method is the same as example 2, and the yield is as follows: 81.3 percent.
2. Preparation of polyethylene glycol 6K-tryptophan-gambogic acid:
2ml of PEG6K-Trp-FA (0.75g, 0.125mmol), gambogic acid (0.189g, 0.3mmol), DCC (0.062g, 0.3mmol), triethylamine or (pyridine, diethylamine, N-methylmorpholine, etc., preferably triethylamine) was weighed out and dissolved in 20ml of dichloromethane, and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder of polyethylene glycol 10K-tryptophan-gambogic acid 0.68G, with yield: 75.8 percent. Content of gambogic acid: 18.91% (theory: 20.97%). Solubility in water at room temperature is about: 500 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest peaks are the peaks of hydrogen on gambogic acid and tryptophan, delta 1.26-1.45 (C)24-H，C25-H，12H)，δ1.52-2.06(C29-H，C34-H，C35-H，C39-H，C40-H，30H)，δ2.30-2.62(C21-H，C22-H，6H)，δ2.88(C26-H，4H)，δ3.15-3.28(C31-H, 4H, -CH on Tryptophan2-，4H)，δ3.56-3.77(PEG-H，C11-H)，δ3.92(CO-O-CH2-，4H)，δ4.36(CO-N-CH-CO，2H)，δ5.05-5.17(C32-H，C37-H，4H)，δ5.41-5.45(C3-H(C4-H)，2H)，δ6.30(C27-H，2H)，δ6.64-6.67(C4-H(C3-H), 2H), δ 6.78 (pyrrole-on-tryptophan H, 2H), δ 7.20 (ArH, 8H on tryptophan) δ 7.50-7.55 (C)10-H，2H)。
IR: in the infrared spectrumBesides the characteristic peak of PEG, 1741cm is increased-1And the ester carbonyl peak of 1655cm-1The amide carbonyl peak of (1) shows that amino acid is already used as a connecting arm on PEG, one endof the amino acid forms ester, and the other end forms amide to which gambogic acid is connected.
Example 5
Preparation of polyethylene glycol 12K-leucine-neogambogic acid
1. Preparation of polyethylene glycol 12K-leucine trifluoroacetate (PEG 12K-Leu-FA):
the operation method is the same as example 2, and the yield is as follows: 80.5 percent.
2. Preparation of polyethylene glycol 12K-leucine-neogambogic acid
PEG12K-Leu-FA (1.5g, 0.125mmol), neogambogic acid (0.194g, 0.3mmol), DCC (0.062g, 0.3mmol), and triethylamine (2 ml) were weighed out and dissolved in 20ml dichloromethane and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder of polyethylene glycol 12K-leucine-neogambogic acid 1.28G, with yield: 77.6 percent. Content of neogambogic acid: 8.44% (theory: 9.34%), a solubility in water at room temperature of about: 350 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest are the peaks of hydrogen on neogambogic acid and leucine, delta 1.05 (on leucine-CH)3，12H)，δ1.29-1.45(C24-H，C25-H，12H)，δ1.54-2.06(C29-H，C34-H，C35-H，C39-H，C40-H, 30H, -CH-, -CH on leucine2-，8H)，δ2.30-2.62(C21-H，C22-H，6H)，δ2.93(C26-H，4H)，δ3.20-3.38(C31-H，4H，C36-H，4H)，δ3.56-3.77(PEG-H，C11-H)，δ3.89(CO-O-CH2-，4H)，δ4.32(CO-N-CH2-CO，4H)，δ5.11-5.24(C32-H，C37-H，4H)，δ6.09(C27-H，2H)，δ7.58(C10-H，2H)。
IR: in the infrared spectrumIn addition to the characteristic peak of PEG, the carbonyl peak is weaker due to the large molecular weight of PEG, but the peak is still seen to be increased by 17421cm-1Ester carbonyl peak of (1), and 1660cm-1The amido carbonyl peak of (A) shows that amino acid is already used as a connecting arm on PEG, one end of the amino acid forms ester, and the other end forms amide to connect neogambogic acid.
Example 6
Preparation of polyethylene glycol 8K-valine-allogambogic acid
1. Preparation of polyethylene glycol 8K-valine trifluoroacetate (PEG 8K-Val-FA):
the operation method is the same as example 2, and the yield is as follows: 79.3 percent.
2. Preparation of polyethylene glycol 8K-valine-allogambogic acid
PEG8K-Val-FA (1.0g, 0.125mmol), allogambogic acid (0.189g, 0.3mmol), DCC (0.062g, 0.3mmol) and triethylamine (2 ml) were weighed out and dissolved in 20ml dichloromethane and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder polyethylene glycol 8K-valine-allogambogic acid 1.25G, with yield: 76.8 percent. The content of the allogambogic acid is as follows: 11.97% (theory: 13.33%), solubility in water at room temperature was about: 450 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest are the peaks of hydrogen on neogambogic acid and leucine, delta 1.07 (on valine-CH)3，12H)，δ1.29-1.43(C24-H，C25-H，12H)，δ1.53-2.09(C29-H，C34-H，C35-H，C39-H，C40-H，30H)，δ2.29-2.60(C21-H，C22-H，6H)，δ2.88(C26-H, 4H), δ 2.94-3.11 (on valine-CH-) δ 3.19-3.30 (C)31-H，4H，C36-H，4H)，δ3.56-3.79(PEG-H，C11-H)，δ3.91(CO-O-CH2-，4H)，δ4.41(CO-N-CH2-CO，4H)，δ5.08-5.19(C32-H，C37-H，4H)，δ5.43(C3-H(C4-H)，2H)δ6.28(C27-H，2H)，δ6.65(C4-H(C3-H)，2H)，δ7.47-7.53(C10-H，2H)。
IR: except the characteristic peak of PEG in the infrared spectrum, the carbonyl peak is weaker due to the large molecular weight of PEG, but 1740cm is increased simultaneously-1And the ester carbonyl peak of 1657cm-1The amide carbonyl peak of (a) shows that amino acid is already used as a connecting arm on PEG, one end of the amino acid is formed into ester, and the other end of the amino acid is formed into amide to which the allogambogic acid is connected.
Example 7
Preparation of polyethylene glycol 2K-glycine-valine-gambogic acid
1. Preparation of polyethylene glycol 2K-Glycine-valine trifluoroacetate (PEG 2K-Gly-Val-FA):
the operation method is the same as example 2, and the yield is as follows: 78.6 percent.
2. Preparation of polyethylene glycol 2K-glycine-valine-gambogic acid
PEG2K-Gly-Val-FA (0.27g, 0.125mmol), gambogic acid (0.189g, 0.3mmol), DCC (0.062g, 0.3mmol) and triethylamine (2 ml) were weighed out and dissolved in 20ml dichloromethane and stirred at room temperature overnight. Filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with Sephadex G-25 Sephadex column, and lyophilizing to obtain yellow powder 0.34G of polyethylene glycol 2K-glycine-valine-gambogic acid, with yield: 78.2 percent. Content of gambogic acid: 33.74% (theory: 36.83%), solubility in water at room temperature of about: 800 mg/ml.
1HNMR(500MHz，CDCl3): delta 3.6 is the strong peak of hydrogen on PEG, the rest peaks are the peaks of hydrogen on gambogic acid and oligopeptide (Gly-Ala), delta 1.10 (on valine-CH)3，12H)，δ1.23-1.46(C24-H，C25-H，12H)，δ1.56-2.09(C29-H，C34-H，C35-H，C39-H，C40-H，30H)，δ2.33-2.64(C21-H，C22-H，6H)，δ2.91(C26-H, 4H), δ 2.97-3.14 (on-CH-valine), δ 3.18-3.27 (C)31-H，4H)，δ3.56-3.77(PEG-H，C11-H)，δ3.92(CO-O-CH2-，4H)，Delta.4.34 (Glycine-CH)2-，4H)，δ5.05-5.17(C32-H，C37-H，4H)，δ5.41-5.45(C3-H(C4-H)，2H)，δ6.30(C27-H，2H)，δ6.64-6.67(C4-H(C3-H)，2H)，δ7.50-7.55(C10-H，2H)。
IR: besides the characteristic peak of PEG, 1743cm is also added in the infrared spectrum-1Ester carbonyl peak of (1), and 1665cm-1The amido carbonyl peak of (A) shows that oligopeptide (Gly-Ala) is used as a connecting arm on PEG, one end of the oligopeptide is formed into ester, and the other end of the oligopeptide is formed into amide to be connected with gambogic acid.
Example 8
Preparation of polyethylene glycol total gambogic acid freeze-dried powder injection
Dissolving 25g of pegylated total gambogic acid equivalent to the total gambogic acid in a proper amount of water for injection, adding 20g of mannitol, uniformly stirring, adjusting the pH value to 5 by hydrochloric acid, adding 0.5% of activated carbon for injection, preserving the heat at 60 ℃ for 20 minutes, decarburizing,adding sterile water for injection into the filtrate to 1000ml, filtering by using a 0.22 mu m microporous filter membrane under an aseptic condition, subpackaging in 7ml of penicillin bottles, freeze-drying 1ml of each bottle, taking out the bottles, and rolling covers.
Example 9
Preparation of pegylated gambogic acid injection
Dissolving 25g of pegylated gambogic acid equivalent to gambogic acid in appropriate amount of water for injection, adding 4.5g of sodium chloride, stirring uniformly, adjusting pH to 5 with hydrochloric acid, adding 0.5% of activated carbon for injection, keeping the temperature at 60 ℃ for 20 minutes, removing the carbon, adding water for injection into the filtrate to 2000ml, filtering with a 0.22 mu m microporous filter membrane, subpackaging in 2ml per bottle, sealing by melting, and sterilizing with 100 ℃ circulating steam for 30 minutes.
Example 10
Preparation of pegylated neogambogic acid powder injection
Adding polyethylene glycol neogambogic acid crystal powder corresponding to 25g neogambogic acid into glucose powder for injection 20g, mixing, aseptically packaging in penicillin bottles, each bottle corresponding to 50mg neogambogic acid, sealing, and rolling.
Claims (10)
1. A pegylated gambogic acid prodrug of general formula (I) or a pharmaceutically acceptable salt thereof:
wherein G is total gambogic acid, neogambogic acid or allogambogic acid;
p is polyethylene glycol, polyethylene glycol monomethyl ether, polyethylene glycol amine monomethyl ether, polyethylene glycol diamine;
a is a connecting bond, or an amino acid, oligopeptide, a disubstituted p-aminobenzyl alcohol derivative or a disubstituted p-hydroxybenzoic acid derivative, wherein the substituent is C1-4 alkyl or C1-4 alkoxy;
and m is 1 or 2.
2. The pegylated gambogic acid prodrug of claim 1, wherein the average molecular weight of the polyethylene glycol is 400-.
3. The pegylated gambogic acid prodrug of claim 2, wherein the polyethylene glycol is polyethylene glycol 2000, 4000, 6000, 10,000, 15,000, 20,000 or 40,000.
4. The pegylated gambogic acid prodrug of claim 1, wherein the amino acid is glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine or arginine.
5. The pegylated gambogic acid prodrug of claim 4, wherein the amino acid is glycine, leucine, tryptophan, valine or lysine.
6. The pegylated gambogic acid prodrug of claim 1, wherein a is 3, 5-dimethyl-4-amino-benzyl alcohol, 3, 5-dimethoxy-4-amino-benzyl alcohol, 3, 5-dimethyl-4-hydroxy-benzoic acid, or 3, 5-dimethoxy-4-hydroxy-benzoic acid.
7. A process for the preparation of a compound of formula (I) comprising:
when A is a connecting bond:
dissolving gambogic acid compounds in a solvent a under the action of a dehydrating agent and a catalyst on dimethylaminopyridine, stirring for 0.5-5 h, adding polyethylene glycol, stirring at room temperature, filtering, concentrating the filtrate to dryness, recrystallizing with a solvent b, and purifying and drying by a sephadex column to obtain I;
when A is an amino acid:
dissolving polyethylene glycol, N-tert-butyloxycarbonyl amino acid, a dehydrating agent and a catalyst into a solvent a, stirring, filtering or centrifuging, concentrating the filtrate to dryness, dissolving the residue into the solvent, dropwise adding into glacial ethyl ether, standing, filtering after solid is separated out, dissolving into dichloromethane-trifluoroacetic acid or ethyl acetate-trifluoroacetic acid after drying, concentrating to dryness, pouring the residue into ethyl ether, filtering, and recrystallizing the crude product with ethanol-ethyl ether to obtain polyethylene glycol-amino acid trifluoroacetate; dissolving polyethylene glycol-amino acid trifluoroacetate, gambogic acid compound, dicyclohexylcarbodiimide and a solvent c in dichloromethane, stirring at room temperature, filtering, concentrating the filtrate to dryness, recrystallizing with isopropanol, purifying with a sephadex column, and drying to obtain I; wherein the dehydrating agent is dicyclohexylcarbodiimide, [ 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or diisopropylcarbodiimide; the solvent a is dichloromethane, chloroform, DMF or dioxane, and the solvent b is isopropanol, chloroform-ether, dichloromethane-ether or ethanol-ether; the solvent c is pyridine, diethylamine, N-methylmorpholine or triethylamine.
8. The production method according to claim 6, wherein the dehydrating agent is dicyclohexylcarbodiimide; solvent a is dichloromethane; solvent b is isopropanol; solvent c is triethylamine; the Sephadex G-25 type Sephadex column is used.
9. Use of a pegylated gambogic acid prodrug of general formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of a neoplastic disease.
10. A pharmaceutical composition comprising a therapeutically effective amount of a pegylated gambogic acid prodrug of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
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| WO2014114262A1 (en) * | 2013-01-28 | 2014-07-31 | 天津键凯科技有限公司 | Water soluble polymer-amino acid oligopeptide-medicine combination, preparation method therefore, and use thereof |
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| WO2014114262A1 (en) * | 2013-01-28 | 2014-07-31 | 天津键凯科技有限公司 | Water soluble polymer-amino acid oligopeptide-medicine combination, preparation method therefore, and use thereof |
| US9700633B2 (en) | 2013-01-28 | 2017-07-11 | Jenkem Technology Co., Ltd., Tianjin Branch | Conjugates of water soluble polymer-amino acid oligopeptide-drug, preparation method and use thereof |
| CN105616351A (en) * | 2014-11-07 | 2016-06-01 | 安徽中医药大学 | Neogambogic acid mixed micelle preparation, and preparation method thereof |
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