CN101518654A - Method for synthesizing novel preparation capable of carrying glucocorticoid to be used as inflammation targeted drug - Google Patents

Abstract

The invention discloses a method for synthesizing novel preparation capable of carrying glucocorticoid to be used as inflammation targeted drug. The method uses water-soluble N-(2-hydroxypropyl) methacrylamide polymer as the carrier for carrying the glucocorticoid and comprises the following five steps of synthesizing the inflammation targeted drug formed through the joint of the polymer N-(2-hydroxypropyl) methacrylamide polymer and prednisolone (1): synthesizing polymer N-(2-hydroxypropyl) methacrylamide polymer; (2) synthesizing short peptide chains; (3) synthesizing polymer-peptide chain multipolymer; (4) synthesizing leading molecules of the polymer; and (5) synthesizing polymer-peptide chain prednisolone. The novel inflammation targeted drug with excellent performance can orientationally carry the prednisolone to inflammatory tissues, particularly the arthritis tissues so that the prednisolone releasing in the arthritis tissues exerts favorable anti-inflammatory effect without causing adverse reaction to normal tissues.

Description

A kind of portability glucocorticoid is as the synthetic method of inflammation targeted drug novel formulation

Technical field

The present invention relates to the synthetic method of a kind of portability glucocorticoid, belong to the technical field that medicine is made as the inflammation targeted drug novel formulation.

Background technology

Prednisolone is a glucocorticoid medicine, and such medicine is clinical anti-inflammatory drug commonly used, uses very extensive.Owing to exist steroid receptor in the body, these receptors extensively distribute in many in vivo effector lymphocytes, the physiological effect of glucocorticoid is diversified, comprise and change saccharide, protein and lipid metabolism, keep body fluid and electrolyte balance, safeguard cardiovascular system, immune system, kidney, skeletal muscle, hormonal system and neural normal function, in addition, corticosteroid can be given the ability of body one opposing tense situation.The effect and the dosage of glucocorticoid are closely related, heavy dose of, when to be glucocorticoid as medicine surpass physiological dose, its pharmacotoxicological effect is also very extensive, particularly has important pharmacodynamics effects such as antiinflammatory, immunosuppressant and shock, but can produce many untoward reaction simultaneously, some untoward reaction such as hyperglycemia, meetings such as negative nitrogen balance and osteoporosis cause than severe impairment body, and particularly osteoporosis and femur head necrosis have limited steroid medicine application clinically.Glucocorticoid is very responsive to the influence of skeleton, and the glucocorticoid of physiological dose and low dosage is the necessary promoting factor of osteocyte growth promoter, and heavy dose particularly can suppress osteoblastic growth during pharmacology dosage, causes osteoporosis.The osteoporosis that glucocorticoid causes is called the steroid osteoporosis clinically, seniority among brothers and sisters the 3rd in osteoporotic sickness rate, account for prolonged application glucocorticoid treatment patient's 30%～50%, the steroid osteoporosis does not also have good Therapeutic Method at present, how to put prevention first, in case find drug withdrawal at once.Symptomatic treatment does not also have ideal medicine at present.

Water solublity N-(2-hydroxypropyl) Methacrylamide [N-(2-hydroxypropyl) methacrylamide, HPMA] polymer is as the history in existing more than 10 year of the research of anti-cancer medicament carrier, it has excellent biological compatibility, non-immunogenic, avirulence and can advantage such as modify to structure according to application target, be applied to engage with the micromolecule cancer therapy drug, exploitation becomes novel cancer therapy drug.These high molecular weight water soluble polymers engage with the micromolecule cancer therapy drug, the cancer therapy drug that the new pharmaceutics of forming is transformed, compare with the micromolecule cancer therapy drug, have two big advantages: the one, can avoid being removed fast, thereby prolong the half-life of medicine in blood circulation by kidney; The 2nd, solid tumor is had the tendentiousness deposition, be called " enhanced seeing through and retention effect " (EPR effect).The reason that this effect produces mainly is because the tumor tissues rich blood vessel, and tumor vessel can produce materials such as the angionecrosis factor, permeability factor increases its permeability, polymer is easy to arrive the solid tumor tissue by blood circulation, simultaneously, the permeability that the lymph circulation of tumor locus is more normally organized is low, thereby make that polymer is difficult to discharge by lymph circulation, finally enter tumor cell.These jointers enter tumor cell by pinocytosis after entering the solid tumor tissue by the EPR effect again, are engulfed by lysosome after lysosomal enzyme decomposes polypeptide interval base and discharges medicine ^[5～6]Studies show that this type of jointer can significantly improve small-molecule drug distribution and pharmacokinetic properties in vivo ^[7], HPMA polymer-HPMA polymer-cancer therapy drug jointers such as amycin jointer have entered clinical I, II phase and have tested.

The HPMA polymer is not also studied at present fully as glucocorticoid medicine carrier and delivery system, the present invention is a carrier with the HPMA polymer, engage prednisolone, form a kind of new targeting preparation, result of study shows, this novel targeting preparation, can directionally be transported to the inflammation tissue to prednisolone, in the particularly arthritic Inflamed tissue, discharge in the inflammation tissue again, bring into play good antiinflammatory action, and the prednisolone that this novel formulation is carried does not discharge in normal structure, not producing pharmacological action, also do not have untoward reaction, is a very good novel inflammation targeted preparation.

Summary of the invention

A kind of portability glucocorticoid is as the synthetic method of the new formulation of inflammation targeted drug, it is characterized in that with N-(2-hydroxypropyl) methacrylamide polymer as pharmaceutical carrier, by following five steps, the inflammation targeted preparation that synthetic N-(2-hydroxypropyl) methacrylamide polymer engages with prednisolone:

(1) synthetic high polymer N-(2-hydroxypropyl) methacrylamide polymer (HPMA)

Method: methacrylic chloride 100ml (1.01mol) is added in the acetonitrile solution that contains 1-amino-2-propanol 160ml (2.02mol) stirring at room 30～40min, elimination precipitation, filtrate is at-50 ℃ of insulation 0.5～1h, separate out fully until precipitation, filter, the precipitation acetone recrystallization.

(2) synthetic short peptide chain Ma-Gly-Phe-Leu-Gly-ONp

Method: get methacrylic chloride Ma.Cl 10ml (0.1mol), slowly be added drop-wise in the DMF solvent of glycyl phenylalanine Gly-Phe-OH, maintain the temperature at 0 ℃ of stirring reaction 8～10h, reaction finishes and removes solvent under reduced pressure and get crude product Ma-Gly-Phe-OH.

(2.0g 11mmol) is dissolved in the 20ml methanol, and (1.39g 11.7mmol), maintains the temperature at 0 ℃ of stirring reaction 4～6h, and reaction finishes and removes solvent under reduced pressure and get crude product H-Leu-Gly-OMe.HCl to dissolve thionyl chloride in the methanol in advance to get leucine Leu.

Get H-Leu-Gly-OMe.HCl (254mg, 1.07mmol) being dissolved in 2mlN, dinethylformamide adds 150mlTEA, mixture low temperature stirs down, add the 2mlDMF solvent, in DMF, dissolve in advance Ma-Gly-Phe-OH (307mg, 1.06mmol), in reactant liquor, add I-hydroxybenzotriazole (175mg, 1.15mmol) and DCCI (232mg 1.15mmol), is dissolved among the 4mlDMF among the DCCI in advance.Keep reaction temperature to stir 1h at 0 ℃.Reactant liquor places refrigerator overnight, filter next day, get the filtrate decompression distillation and remove DMF, residue is dissolved in ethyl acetate, with 5% sodium bicarbonate aqueous solution, aqueous citric acid solution and sodium-chloride water solution extraction, keep organic facies successively, dewater with anhydrous sodium sulfate drying, evaporate to dryness then, product is with ethyl acetate-ether mixed solvent recrystallization.

Above-mentioned crystallized product is dissolved in 50% methanol aqueous solution, add the 0.1M sodium hydrate aqueous solution and be adjusted to pH12, reflux hydrolysis 4h, steaming desolventizes methanol, solution adds the 0.1M hcl acidifying to pH2, polymerization small peptide ethyl acetate extraction, organic addition dried over sodium sulfate dehydration, recrystallization promptly gets product Ma-Gly-Phe-Leu-Gly-OH in ethyl acetate-ether mixed solvent.

Methacryl tetrapeptide and the esterification of 4-nitrophenol prepare methacryl tetrapeptide nitrate.Get Ma-Gly-Phe-Leu-Gly-OH and be dissolved among the THF, slowly drip 4-nitrophenol THF solution, keep room temperature stirring reaction 24h, filter, get filtrate decompression and steam to desolventize and promptly get Ma-Gly-Phe-Leu-Gly-ONp.

(3) synthetic polymer-peptide chain copolymer (polymer-oligopeptidic sequence copolymer) HPMA/Ma-Gly-Phe-Leu-Gly-ONp

Method: with 2,2-azodiisobutyronitrile (AIBN) is an initiator, and (monomer-AIBN-solvent 12:3:85 wt%) prepares target product to HPMA and 10% (mol/mol) MA-Gly-Phe-Leu-Gly by the reaction of free radical precipitation polymerization.Concrete operations are HPMA (812.5mg, 6.9mmol), (120mg 0.7mmol) is dissolved in the 7.2ml acetone MA-Gly-Phe-Leu-Gly-ONp, add AIBN (8.6mg) as initiator, add 3-mercaptopropionic acid (11.2mg) as chain-transferring agent.Mixture solution changes nitrogen bubble 5min in the ampoule over to, drains oxygen, and sealing places 50 ℃ of constant temperature polyreaction 24h.Remove acetone under reduced pressure after reaction finishes, solid residue is dissolved in the methanol, column chromatography purification, and Sephadex LH-60 column chromatography is used methanol-eluted fractions.Low-molecular-weight and heavy polymer discard, and collecting molecular weight is the part of MW10000, collect the liquid pressurization and concentrate, and product drops in the absolute ether, separates out precipitation and is end-product.Productive rate 60%, MW10000, dispersion 1.3.

(4) synthetic polymer guide molecule (polymer precursor) HPMA/Ma-Gly-Phe-Leu-Gly-NHNH2 4

Get polymer (3) (317mg, 170 μ mol ONp) be dissolved among the 1.2ml DMSO, keep under the stirring at room, dropwise add excess hydrazine hydrate (1700mg, 34mmol), hydrazine hydrate is dissolved among the 4mlDMF in advance, continue to stir 30min, thin up is dialysed to cumulative volume 50ml, use 20% ethanol water, dilute hydrochloric acid (pH3), water dialysis 3d successively, polymer lyophilized.Product further activates with excessive N-Hydroxysuccinimide (HOSu), obtains having the copolymer of diazanyl.

(5) synthetic polymer-peptide chain-prednisolone (P-Prs conjugate) HPMA/Ma-Gly-Phe-Leu-Gly-Prs

(0.33g 0.92mmol) mixes, and mixes and adopts NN-dimethyl formamide (1ml) as solvent, and drip an acetic acid as catalyst for copolymer and excessive prednisolone.Stirring reaction spends the night under the room temperature, and column chromatography LH-20 separation is removed unreacted low molecular weight compound 2 times.Collecting molecular weight is the part of MW10000, collects the liquid concentrating under reduced pressure, obtains end product P-prednisolone (P-Prs) after the lyophilizing.The content of prednisolone product is the 50mg/g copolymer.

Above method, synthetic inflammation targeted prednisolone new product proves through zoopery, can detect more prednisolone and discharge in the joint capsule of inflammation part, does not but observe the release of medicine at the natural joint capsule.Toxicologic study does not find that this product has tangible toxicity.

This synthetic method can also engage other steroid medicines, as cortisone, hydrocortisone, prednisone, prednisolone acetate, prednisolone, prednisolone acetate, preparations such as dexamethasone sodium phosphate and betamethasone.

Claims (2)

1, a kind of portability glucocorticoid is as the synthetic method of the new formulation of inflammation targeted drug, it is characterized in that with N-(2-hydroxypropyl) methacrylamide polymer as pharmaceutical carrier, by following five steps, the inflammation targeted preparation that synthetic N-(2-hydroxypropyl) methacrylamide polymer engages with prednisolone:

(1) synthetic high polymer N-(2-hydroxypropyl) methacrylamide polymer

Method: methacrylic chloride 100ml (1.01mol) is added in the acetonitrile solution that contains 1-amino-2-propanol 160ml (2.02mol) stirring at room 30～40min, elimination precipitation, filtrate is at-50 ℃ of insulation 0.5～1h, separate out fully until precipitation, filter, the precipitation acetone recrystallization;

(2) synthetic short peptide chain

Method: get methacrylic chloride 10ml (0.1mol), slowly be added drop-wise in the DMF solvent of glycyl phenylalanine Gly-Phe-OH, maintain the temperature at 0 ℃ of stirring reaction 8～10h, reaction finishes and removes solvent under reduced pressure and get crude product Ma-Gly-Phe-OH;

(2.0g 11mmol) is dissolved in the 20ml methanol, and (1.39g 11.7mmol), maintains the temperature at 0 ℃ of stirring reaction 4～6h, and reaction finishes and removes solvent under reduced pressure and get crude product H-Leu-Gly-OMe.HCl to dissolve thionyl chloride in the methanol in advance to get leucine Leu;

Get H-Leu-Gly-OMe.HCl (254mg, 1.07mmol) being dissolved in 2mlN, dinethylformamide adds 150mlTEA, mixture low temperature stirs down, add the 2mlDMF solvent, in DMF, dissolve in advance Ma-Gly-Phe-OH (307mg, 1.06mmol), in reactant liquor, add I-hydroxybenzotriazole (175mg, 1.15mmol) and DCCI (232mg 1.15mmol), is dissolved among the 4mlDMF among the DCCI in advance.Keep reaction temperature to stir 1h at 0 ℃, reactant liquor places refrigerator overnight, filter next day, get the filtrate decompression distillation and remove DMF, residue is dissolved in ethyl acetate, successively with 5% sodium bicarbonate aqueous solution, aqueous citric acid solution and sodium-chloride water solution extraction, keep organic facies, with anhydrous sodium sulfate drying dehydration, evaporate to dryness then, product is with ethyl acetate-ether mixed solvent recrystallization;

Above-mentioned crystallized product is dissolved in 50% methanol aqueous solution, add the 0.1M sodium hydrate aqueous solution and be adjusted to pH12, reflux hydrolysis 4h, steaming desolventizes methanol, solution adds the 0.1M hcl acidifying to pH2, polymerization small peptide ethyl acetate extraction, organic addition dried over sodium sulfate dehydration, recrystallization promptly gets product Ma-Gly-Phe-Leu-Gly-OH in ethyl acetate-ether mixed solvent;

Methacryl tetrapeptide and the esterification of 4-nitrophenol prepare methacryl tetrapeptide nitrate.Get Ma-Gly-Phe-Leu-Gly-OH and be dissolved among the THF, slowly drip 4-nitrophenol THF solution, keep room temperature stirring reaction 24h, filter, get filtrate decompression and steam to desolventize and promptly get Ma-Gly-Phe-Leu-Gly-Onp;

(3) synthetic polymer-peptide chain copolymer

Method: with 2,2-azodiisobutyronitrile (AIBN) is an initiator, and (monomer-AIBN-solvent 12:3:85 wt%) prepares target product to HPMA and 10% (mol/mol) MA-Gly-Phe-Leu-Gly by the reaction of free radical precipitation polymerization.Concrete operations are HPMA (812.5mg, 6.9mmol), (120mg 0.7mmol) is dissolved in the 7.2ml acetone MA-Gly-Phe-Leu-Gly-ONp, add AIBN (8.6mg) as initiator, add 3-mercaptopropionic acid (11.2mg) as chain-transferring agent.Mixture solution changes nitrogen bubble 5min in the ampoule over to, drains oxygen, and sealing places 50 ℃ of constant temperature polyreaction 24h, after finishing, reaction removes acetone under reduced pressure, solid residue is dissolved in the methanol, column chromatography purification, Sephadex LH-60 column chromatography, use methanol-eluted fractions, low-molecular-weight and heavy polymer discard, and collecting molecular weight is the part of MW10000, collect the liquid pressurization and concentrate, product drops in the absolute ether, separates out precipitation and is end-product.Productive rate 60%, MW10000, dispersion 1.3;

(4) synthetic polymer guide molecule

Get polymer (3) (317mg, 170 μ mol ONp) and be dissolved among the 1.2ml DMSO, keep under the stirring at room, dropwise add excess hydrazine hydrate (1700mg, 34mmol), hydrazine hydrate is dissolved among the 4mlDMF in advance, continues to stir 30min, thin up is to cumulative volume 50ml, 20% ethanol water, dilute hydrochloric acid (pH3), water dialysis 3d are used in dialysis successively, and be polymer lyophilized, product further activates with excessive N-Hydroxysuccinimide (HOSu), obtains having the copolymer of diazanyl;

(5) synthetic polymer-peptide chain-prednisolone

(0.33g 0.92mmol) mixes, and mixes and adopts NN-dimethyl formamide (1ml) as solvent, and drip an acetic acid as catalyst for copolymer and excessive prednisolone.Stirring reaction spends the night under the room temperature, and column chromatography LH-20 separation is removed unreacted low molecular weight compound 2 times.Collecting molecular weight is the part of MW10000, collects the liquid concentrating under reduced pressure, obtains end product P-prednisolone (P-Prs) after the lyophilizing.The content of prednisolone product is the 50mg/g copolymer.

2, a kind of portability glucocorticoid as claimed in claim 1 is as the synthetic method of the new formulation of inflammation targeted drug, it is characterized in that this synthetic method can also engage other steroid medicines, as cortisone, hydrocortisone, prednisone, prednisolone acetate, prednisolone, prednisolone acetate, dexamethasone sodium phosphate and betamethasone.