CN1947796B - Chemical modified adefovir and tynofovir - Google Patents
Chemical modified adefovir and tynofovir Download PDFInfo
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- CN1947796B CN1947796B CN2005100947549A CN200510094754A CN1947796B CN 1947796 B CN1947796 B CN 1947796B CN 2005100947549 A CN2005100947549 A CN 2005100947549A CN 200510094754 A CN200510094754 A CN 200510094754A CN 1947796 B CN1947796 B CN 1947796B
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- tenofovir
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Abstract
A chemically modified adefovir and tenofovir is prepared through the combination of adefovir (or tenofovir) with polyethanediol (or glycosylated polyethanediol). Their preparing process and their antiviral application are also disclosed.
Description
Technical field
The present invention relates to drug molecule is carried out chemical modification to improve the deficiency of existing medicine, be adefovirdipivoxil (Adefovir specifically, PMEA) or tenofovir (tenofovir, PMPA) with the conjugate of Polyethylene Glycol, adefovirdipivoxil or tenofovir and the bonded conjugate of glycosylation Polyethylene Glycol also relate to their preparation method and in the application of anti-virus aspect.
Background technology
Adefovirdipivoxil and tenofovir are the close nucleoside analogs of structure, have the broad-spectrum antiviral activity, and its structural formula is:
Wherein A can select H or CH
3, when A represents H, be adefovirdipivoxil; When A represents CH
3The time, be tenofovir.
Clinical trial finds that adefovirdipivoxil and tenofovir all have AIDS virus resisting (HIV), hepatitis B virus (HBV) activity preferably.Experiment confirm adefovirdipivoxil and tenofovir to hepatitis B virus among the duck liver cell of In vitro culture, the Bel7402 duplicate and expression has significant inhibitory effect.Animal experiment studies show that adefovirdipivoxil and tenofovir also have significant inhibitory effect for duplicating and expressing of intravital hepatitis virus.Surely wait in the therapeutic process as drawing the miaow husband at the prolonged application nucleoside analog, the sudden change of viral gene takes place easily, thereby Drug resistance takes place.Studies show that adefovirdipivoxil and tenofovir not only have significant inhibitory effect for wild strain, and also have significant inhibitory effect (foreign medical science virusology fascicle, 2001,3/8,85-90 for the multidrug resistant disease strain that draws the miaow husband to wait surely; Hepatology 2004,40:1421-25).
But under the physiological pH situation, the phosphonate radical that directly exposes in the molecule has limited adefovirdipivoxil and tenofovir to the penetration power of intestinal, makes their bioavailability in animal and human's body reduce, and has limited it to the treatment of diseases effect.By modification to phosphonic acid functional groups on the adefovirdipivoxil, synthesized adefovir ester (Adefovir dipivoxil) and tenofovir disoproxil (tenofovir disoproxil), structural formula is as follows:
Adefovir ester
Tenofovir disoproxil
The antiviral of adefovir ester and tenofovir disoproxil part is respectively PMEA and PMPA, enters to be converted into PMEA and PMPA in the body fully and to play a role.Prodrug as adefovirdipivoxil and tenofovir, electric charge in adefovir ester and the tenofovir disoproxil on the phosphonate has obtained shielding, increased the fat-soluble of parent, improved the penetration power to the intestinal cell film, its oral administration biaavailability is also increased accordingly.
But, A Defuwei ester main concentrating in human body is distributed in kidney, liver and lung, clinical research finds that the A Defuwei ester has bigger nephrotoxicity, therefore can only adopt 10mg/ days unoptimizable therapeutic dose, and this has limited it to a certain extent to the treatment of diseases effect.The nephrotoxicity of tenofovir disoproxil is littler than A Defuwei ester, but oral dose needs 150-300mg, and bioavailability is lower, therefore, is still seeking better prodrug at present.
At present, the clinical anti-hepatitis virus of being used (HBV) medicament categories is very limited, marketed drug comprise draw that the miaow husband is fixed, A Defuwei ester, entecavir, the water solublity of these medicines is very poor, be difficult to make the dosage form of injection administration, have only oral administration, the anti-HBV medicine of clinical special shortage drug administration by injection approach does not satisfy anxious clinically severe disease human needs.
Current, utilizing polyethyleneglycol modified medicine has been a kind of mature technique, and medicine is after polyethyleneglycol modified, and molecular weight significantly increases on the one hand, and Polyethylene Glycol forms barrier to medicine on the other hand, can reduce the kidney of medicine and remove.Pharmacokinetic property also thereby bigger variation takes place, absorb slow, distribution volume reduces, plasma half-life prolongs.Therefore blood drug level can maintain level more stably for a long time, thereby curative effect is strengthened, and particularly in antiviral therapy, because the antiviral pressure that continues can prevent virus replication, thereby suppresses the generation of drug resistance variant.In addition, dosing interval prolongs can improve the compliance of patient to treatment, improves patients ' life quality, and this also helps to improve clinical efficacy.The tranquilization of blood drug level also has the another one benefit, promptly reduces the caused untoward reaction of fluctuating of plasma drug level peak valley, has improved safety.We combine the pharmacokinetics character that can improve them with adefovirdipivoxil and tenofovir with Polyethylene Glycol, in the hope of obtaining the antiviral drugs of long-acting low toxicity.
Hepatocyte galactosylated acceptor (hepatocyte galactose receptor, H-Gal-R) be present on the mammiferous hepatic parenchymal cells film, can discern specifically and be terminal glycoprotein in conjunction with non-reduced galactose or N-acetyl galactose, by endocytosis it is transported to that lysosome carries out metabolism in the born of the same parents, utilizes orientable the transferring in the hepatocyte of various medicines of H-Gal-R mediation to play a role.Experiment confirm, there is higher affinity in H-Gal-R mediation system to liver, and liver absorbs rapidly.Because hepatitis B virus core antigen is present in the hepatocyte, nucleoside medicine need enter hepatocyte, and through thymidine kinase catalysis phosphorylation rear produce effects, and nucleoside medicine is difficult for seeing through hepatocyte, and the cell of hepatitis B virus infection lacks thymidine kinase, the host cell phosphorylation is slow, do not reach the valid density that suppresses virus replication, heavy dose of administration can improve blood drug level, but can not occurred toxic and side effects (World Chin J Digestol 2003 June such as hematuria and renal dysfunction rapidly through renal excretion by the medicine of phosphorylation in blood; 11 (6): 799-802).The present invention is attached to the glycosylation Polyethylene Glycol on adefovirdipivoxil and the tenofovir, the medicine orientation is transferred in the hepatocyte, thereby improved the therapeutic effect of adefovirdipivoxil and tenofovir.
Summary of the invention
The objective of the invention is to carry out chemical modification so that a kind of antiviral drugs of new long-acting low toxicity to be provided, and a kind ofly have highly liver cell targeted adefovirdipivoxil and a tenofovir prodrug having active nucleoside analog adefovirdipivoxil of broad-spectrum antiviral and tenofovir.
Another object of the present invention provides the method that adefovirdipivoxil and tenofovir are carried out chemical modification.
A further object of the present invention provide above-mentioned adefovirdipivoxil and tenofovir prodrug in the antiviral field, the particularly application in anti-hepatitis virus field.
The polyethylene glycol conjugate of adefovirdipivoxil of the present invention and tenofovir and Polyethylene Glycol or terminal saccharideization has following general formula:
(I) or
Wherein, A is H or CH
3
R is the structure of form such as X-Y, i.e. the combination of radicals X and Y, and wherein Y is H, C
1-6Straight chain, side chain or cyclic alkyl, lactose and/or its derivant, galactose and/or its derivant, wherein X is O, NH, O-CO, CO-O, S-S, SO
2, O-NH, NH-O, O-CO-O, NH-CO-O, O-CO-NH, O-W, NH-W, O-CO-W, CO-O-W, O-W-O, O-W-NH, O-W-CO-O, NH-W-NH, NH-W-O, NH-W-CO-O, CO-O-W-O, CO-O-W-NH or CO-O-W-CO-O, wherein W is low alkyl group or aryl, the effect of X is the end group that connects Polyethylene Glycol, particularly with being connected of lactose or galactose, lactose, galactose and being connected of Polyethylene Glycol generally are coupled with ester bond and glycoside key, peptide bond, imines ester bond;
R ' is CH
2-(CH
2-O-CH
2)
n-CH
2, n=11-450;
R " be O, NH, O-CO, CO-O, S-S, SO
2, O-NH, NH-O, O-CO-O, NH-CO-O, O-CO-NH, O-Z, NH-Z, O-CO-Z, CO-O-Z, O-Z-O, O-Z-NH, O-Z-CO-O, NH-Z-NH, NH-Z-O, NH-Z-CO-O, CO-O-Z-O, CO-O-Z-NH or CO-O-Z-CO-O, wherein Z is lower paraffin hydrocarbon or aromatic hydrocarbons; R " be the group that is used for connecting polyethylene glycol long chain R ' and adefovirdipivoxil or tenofovir phosphate ester, above group links with R ' and phosphate ester respectively from left to right;
Above-mentioned low alkyl group is C
1-6Straight chain, side chain or cyclic alkylene, aromatic hydrocarbons are the subunit of benzene or derivatives thereof or heteroaromatic compound or derivatives thereof.
When A is the adefovirdipivoxil derivant during for H, when A is CH
3The time be the tenofovir derivant;
When Y is C
1-6Straight chain, side chain or cyclic alkyl can be-CH
3,-CH
2CH
3,-CH
2CH
2CH
3,-CH (CH
3)
2,-CH
2CH
2CH
2CH
3,-CH
2CH (CH
3)
2,-CH (CH
3) CH
2CH
3,-CH (CH
3)
3,-CH
2CH
2CH
2CH
2CH
3,-CH (CH
3) CH
2CH
2CH
3,-CH (CH
2CH
3)
2,-C (CH
3)
2CH
2CH
3,-CH (CH
3) CH (CH
3)
2,-CH
2CH
2CH (CH
3)
2,-CH
2CH (CH
3) CH
2CH
3,-CH
2C (CH
3)
3,-CH
2CH
2CH
2CH
2CH
2CH
3,-CH
2CH (CH
3) CH
2CH
2CH
3,-CH
2CH
2CH (CH
3) CH
2CH
3,-CH
2CH
2CH
2CH (CH
3)
2,-CH
2CH (CH
3) CH (CH
3)
2,-CH
2C (CH
3)
2CH
2CH
3,-CH
2CH
2C (CH
3)
3,-CH
2CH (CH
2CH
3)
2,-CH (CH
3) CH
2CH
2CH
2CH
3,-CH (CH
3) CH (CH
3) CH
2CH
3,-CH (CH
3) CH
2CH (CH
3)
2,-CH (CH
2CH
3) (CH
2CH
2CH
3) ,-CH (CH
2CH
3) CH (CH
3)
2,-C (CH
3) (CH
2CH
3)
2,-C (CH
3)
2CH
2CH
2CH
3,-C (CH
3)
2CH (CH
3)
2, cyclopropyl, cyclobutyl, cyclopropyl methyl, Pentamethylene., cyclobutylmethyl, 1-cyclopropyl-1-ethyl, 2-cyclopropyl-1-ethyl, cyclohexyl, cyclopentyl-methyl, 1-cyclobutyl-1-ethyl, 2-cyclobutyl-1-ethyl, 1-cyclopropyl-1-propyl group, 2-cyclopropyl-1-propyl group, 3-cyclopropyl-1-propyl group, 2-cyclopropyl-2-propyl group and 1-cyclopropyl-2-propyl group;
Lactose base and/or its derivant, galactosyl and/or its derivant can be sulfo-galactose, 2-imino group-2-methoxyl group-1-sulfo--β-D-galactose pyrrole glucosides, right-aminophenyl-α-D-breast pyranoside, D (+)-galactosamine;
When Y is H, straight chain, side chain or cyclic alkyl, provide a kind of adefovirdipivoxil and tenofovir of Pegylation, Y is preferably methyl, i.e. the conjugate of mono methoxy polyethylene glycol and adefovirdipivoxil and tenofovir; When Y is lactose and/or its derivant, galactose and/or its derivant, provide a kind of Pegylation adefovirdipivoxil and tenofovir of terminal saccharideization, Y preferably galactose base or sulfo-galactosyl;
Lower paraffin hydrocarbon can be methylene, ethylidene, propylidene, butylidene, pentylidene, hexylidene, the heteroaromatic compound or derivatives thereof includes but not limited to pyridine radicals, thiazolyl, pyrimidine radicals, furyl, thienyl, pyrrole radicals, pyrazolyl, imidazole radicals, tetrazole radical, benzofuranyl, the sulfur naphthyl, indyl, inferior indyl (indolenyl), quinolyl, isoquinolyl, benzimidazolyl, piperidyl, the 4-piperidone base, pyrrolidinyl, the 2-Pyrrolidone base, pyrrolinyl, triazine radical, thianthrene group, pyranose, isobenzofuran-base Fen Evil thienyl (phenoxathinyl), isothiazolyl isoxazolyl, pyrazinyl, pyridazinyl, fast quinoline base, quinoxalinyl, quinazolyl, the cinnoline base, 4aH-card azoles base, card azoles base, the B-carboline base, phenanthridinyl, bifurcation pyridine base, pyrimidine radicals, the phenanthroline base, phenazinyl, phenothiazinyl, our basic phenoxazine group of instep, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, quininuclidinyl, morpholinyl oxazolidinyl, the benzotriazole base, the benzoisoxazole base, hydroxyindole base benzoxazole quinoline base.
The numerical value meaning of described n is to make the molecular weight of Polyethylene Glycol reach 500-20000.
Formula (II) is meant that the Polyethylene Glycol two ends all combine the chemical compound of adefovirdipivoxil or tenofovir, and what provide also is a kind of antiviral prodrug with slow releasing function and good safety energy.
The present invention is to provide conjugate and they and the bonded noval chemical compound of glycosylation Polyethylene Glycol of a kind of adefovirdipivoxil or tenofovir and Polyethylene Glycol with hepatocyte orientation, they can be used in the treatment viral disease, particularly treat hepatitis B, be a kind of efficient, low toxicity and have the antiviral drugs of height liver cell targeting effect.
The compounds of this invention is as active component, can be added with or do not add medicinal carrier, makes the preparation of different way of administration by the production method of pharmaceutical field routine.These preparations can be applied to the patient who needs treatment by oral, rectum or parenteral mode.Be used for when oral, can be made into conventional solid preparation such as tablet, capsule, powder, granule etc., also can be made into liquid preparation such as syrup, oral liquid etc.
Because derivant of the present invention has good water-solubility, can add or not add the medicine carrier and make liquid preparations such as injection, oral liquid.Described injection comprises injection, sterile powder for injection pin and concentrated solution for injection.
The preparation method of injection is, above-claimed cpd is added in the water for injection, be stirred to molten entirely, adding isoosmotic adjusting agent is stirred to molten entirely, regulate acid-base value, if desired, can also comprise adding stabilizing agent, solubilizing agent, get final product through taking off charcoal, coarse filtration, standardize solution, fine straining, embedding, sterilization successively then.
The preparation method of oral liquid is, directly above-claimed cpd joined in the pure water, according to circumstances can add sweeting agent and/or antiseptic.
Chemical compound shown in formula (I) or the formula (II) can prepare as follows:
The preparation method of adefovirdipivoxil (or tenofovir) polyethylene glycol conjugate.
Mono methoxy polyethylene glycol or derivatives thereof and adefovirdipivoxil (or tenofovir) are added in the organic solvent, and in the presence of pyridine and dicyclohexylcarbodiimide (DCC), 20～120 ℃ of reacting by heating 0.5～24 hour obtain chemical compound shown in the formula (I).
Polyethylene Glycol or derivatives thereof and adefovirdipivoxil (or tenofovir) are added in the organic solvent, and in the presence of pyridine and dicyclohexylcarbodiimide (DCC), 40～120 ℃ of reacting by heating 0.5～10 hour obtain chemical compound shown in the formula (II).
The preparation of the polyethylene glycol conjugate of adefovirdipivoxil (or tenofovir) and terminal saccharideization:
Method one: the adefovirdipivoxil (or tenofovir) that will be made by said method is converted into alcoholic extract hydroxyl group with Iodotrimethylsilane with methoxyl group with the conjugate of mono methoxy polyethylene glycol, react with succinic anhydrides again, make adefovirdipivoxil (or tenofovir)-Polyethylene Glycol-succinate monoester, make target product with right-aminophenyl-α-D-breast pyranoside reaction then.
Method two: after the conjugate of adefovirdipivoxil (or tenofovir) and mono methoxy polyethylene glycol (mPEG) is converted into alcoholic extract hydroxyl group with Iodotrimethylsilane with methoxyl group; under alkali condition, with glycol dibromide generation alkylation reaction; then with 2-S-(2; 3,4,6-four-O-acetyl-β-1)-galactopyranose base-2-isothiourea hydrobromate; reaction in the presence of potassium carbonate and sodium sulfite; in methanol, the acetyl protection base with on the Feldalat NM reduction glycosyl promptly gets target product at last.
Adefovirdipivoxil and tenofovir are after process is polyethyleneglycol modified, and PEG forms barrier to former medicine, and molecular weight significantly increases, and have reduced the kidney of medicine and have removed, and have reduced the nephrotoxicity of medicine; Bigger variation also takes place in pharmacokinetic property on the other hand, mainly show as absorb slow, distribution volume reduces, plasma half-life prolongation etc.Because drug half-life prolongs, blood drug level can maintain steadily for a long time, effective level, thereby has improved bioavailability of medicament, curative effect is strengthened, particularly in antiviral therapy, the antiviral pressure that continues can prevent virus replication, suppresses the generation of drug resistance variant.In addition, dosing interval prolongs and can improve the compliance of patient to treatment, improves patients ' life quality, and the tranquilization of blood drug level also has the another one benefit, promptly reduces the caused untoward reaction of fluctuating of plasma drug level peak valley, has improved safety.We have also proved these above conclusions by zoopery.Therefore, compare with the tenofovir disoproxil medicine with the prodrug adefovir ester of existing adefovirdipivoxil and tenofovir, The compounds of this invention has marked improvement.
We are with adefovirdipivoxil and tenofovir and the terminal Polyethylene Glycol combination that is connected lactose or galactose, not only has polyethyleneglycol modified advantage, and can utilize hepatocellular endocytosis, make medicine see through hepatocyte performance antivirus action easily, make medicine have the targeting of height, to reduce the side effect such as nephrotoxicity of medicine, improve its antiviral ability.
The present invention has also solved the deficiency that existing medicine is difficult to make liquid preparation.Former medicine of adefovirdipivoxil and tenofovir and existing prodrug adefovir ester and tenofovir disoproxil all are refractory to water, and are difficult to make liquid preparation.But this derivant has good water-solubility, can add or not add the medicine carrier and make liquid preparations such as injection, oral liquid.At present, the clinical anti-hepatitis virus of being used (HBV) medicine has only oral administration, lacks the anti-HBV medicine of drug administration by injection approach, can not satisfy anxious clinically severe disease human needs.Adefovirdipivoxil of the present invention and tenofovir Pegylation prodrug have overcome this defective of prior art.
The specific embodiment
Following examples are to illustrate of the present invention, but not limitation of the present invention.
Raw material adefovirdipivoxil used in the present invention is by document: Holy A, Roseenberg I, Dvorakova H.Synthesis of N-(2-phosphonylmethoxyethyl) derivatives of heterocyclic base[J] .Collect Czech Chem Commun, 1989,5:2190～2195 methods are synthetic.Tenofovir is synthetic by the described method of patent US4808716, WO9804569.
The preparation of embodiment 1 adefovirdipivoxil-methoxy poly (ethylene glycol) 2000
The mono methoxy polyethylene glycol 2000 (mPEG2000) of 140g (0.07mol) is added in the 500ml toluene, the heating fusion, steam the about 400ml of toluene, the N that adds 250ml, dinethylformamide, the 70ml pyridine, 9.6g (0.035mol) adefovirdipivoxil and 27g dicyclohexylcarbodiimide, heat temperature raising to 100 ℃, and under this temperature, reacted 120 minutes, be cooled to room temperature, add the 1000ml ethyl acetate, stirred 2 hours, filter, mother solution reclaims solvent, adds the 500ml anhydrous alcohol solution in residue, slowly adds then in the 2000ml absolute ether of vigorous stirring, cooling, filter, filter cake reduced pressure at room temperature 24 hours gets 130g.49～52 ℃ of fusing points contain adefovirdipivoxil 6.8%.
The preparation of embodiment 2 adefovirdipivoxils-methoxy poly (ethylene glycol) amine 2000
Polyoxamide 2000 (mPEG-NH after 140g (0.07mol) dewatered
2) add the N of 250ml, in the dinethylformamide, add 9.6g (0.035mol) adefovirdipivoxil and 21g DCM, stirring at room 24 hours adds the 1000ml ethyl acetate, stirs 2 hours, filter, mother solution reclaims solvent, adds the 500ml anhydrous alcohol solution in residue, slowly adds then in the 2000ml absolute ether of vigorous stirring, cooling, filter, filter cake reduced pressure at room temperature 24 hours gets the 126g product.49～53 ℃ of fusing points contain adefovirdipivoxil 6.8%.
The preparation of embodiment 3 adefovirdipivoxils-methoxy poly (ethylene glycol) 1000
According to the preparation of embodiment 1 method, mono methoxy polyethylene glycol 1000 and adefovirdipivoxil molar ratio are 2: 1, get the adefovirdipivoxil cetomacrogol 1000, and fusing point is 34～38 ℃, contains adefovirdipivoxil 13.6%.
The preparation of embodiment 4 adefovirdipivoxils-methoxy poly (ethylene glycol) 5000
According to the preparation of embodiment 1 method, mono methoxy polyethylene glycol 5000 and adefovirdipivoxil molar ratio are 2: 1, get adefovirdipivoxil Polyethylene Glycol 5000, and fusing point is 50～55 ℃, contains adefovirdipivoxil 2.7%.
The preparation of embodiment 5 adefovirdipivoxils-methoxy poly (ethylene glycol) 10000
According to the preparation of embodiment 1 method, mono methoxy polyethylene glycol 10000 and adefovirdipivoxil molar ratio are 2: 1, get adefovirdipivoxil cetomacrogol 1000 0, and fusing point is 56～60 ℃, contains adefovirdipivoxil 1.3%.
The preparation of embodiment 6 adefovirdipivoxils-methoxy poly (ethylene glycol) 20000
According to the preparation of embodiment 1 method, mono methoxy polyethylene glycol 20000 and adefovirdipivoxil molar ratio are 2: 1, get adefovirdipivoxil Macrogol 2000 0, and fusing point is 58～64 ℃, contains adefovirdipivoxil 0.6%.
The preparation of embodiment 7 two adefovirdipivoxils-Macrogol 2000
The PEG2000 (Macrogol 2000) of 140g (0.07mol) is added in the 500ml toluene, the heating fusion, steam the about 400ml of toluene, the N that adds 250ml, dinethylformamide, the 70ml pyridine, 19.2g (0.07mol) adefovirdipivoxil and 54g dicyclohexylcarbodiimide, heat temperature raising to 100 ℃, insulation reaction 120 minutes, be cooled to room temperature, add the 1000ml ethyl acetate, stirred 2 hours, filter, mother solution reclaims solvent, adds the 500ml anhydrous alcohol solution in residue, slowly adds then in the 2000ml absolute ether of vigorous stirring, cooling, filter, filter cake reduced pressure at room temperature 24 hours gets the 132g product.48～52 ℃ of fusing points contain adefovirdipivoxil 13.6%.
The preparation of embodiment 8 two adefovirdipivoxils-cetomacrogol 1000 0
According to the preparation of embodiment 6 methods, cetomacrogol 1000 0 and adefovirdipivoxil molar ratio are 1: 1, get two adefovirdipivoxil cetomacrogol 1000s 0, and fusing point is 56～60 ℃, contains adefovirdipivoxil 2.6%.
The preparation of embodiment 9 tenofovirs-Polyethylene Glycol
Prepare the tenofovir Polyethylene Glycol by embodiment 1 to 6 same method, the Pegylation tenofovir character such as the table 1 that make.
The character of table 1 Pegylation tenofovir
| Fusing point (℃) | Tenofovir content (%) | |
| Tenofovir-methoxy poly (ethylene glycol) 2000 | 49～55 | 2.8 |
| Tenofovir-methoxy poly (ethylene glycol) 10000 | 56～61 | 5.6 |
| Two tenofovirs-Macrogol 2000 | 48～55 | 12.6 |
Embodiment 10 adefovirdipivoxils-Macrogol 2000-lactose
1, adefovirdipivoxil-Macrogol 2000-succinate monoester
149g (0.035mol) adefovirdipivoxil-methoxy poly (ethylene glycol) 2000 is added in the 450ml chloroform, add the 7.0g Iodotrimethylsilane, stirring at room 6 hours adds 25ml water, stirs 10 minutes, standing demix, get chloroform layer, anhydrous sodium sulfate drying spends the night, and filters, the pyridine that adds 35.0g (0.35mol) succinic anhydride and 20ml, 60 ℃ of left and right sides back flow reaction 48 hours, evaporated under reduced pressure adds the sodium bicarbonate aqueous solution of the 0.1M of 150ml in residue, sucking filtration, filtrate is used the hcl acidifying of the 0.1M of 150ml, and regulates pH value to 3-4, then uses ethyl acetate 150ml * 3 extractions, combined ethyl acetate solution, with saturated nacl aqueous solution 100ml * 2 washings, add anhydrous sodium sulfate drying, the rotation solvent evaporated, residue adds the 30ml acetic acid ethyl dissolution, under vigorous stirring, add then in a large amount of absolute ethers, cooling and stirring half an hour, filter, vacuum drying gets 120g adefovirdipivoxil-Macrogol 2000-succinate monoester to constant weight.Fusing point 46.1-47.8 ℃.
2, adefovirdipivoxil-Macrogol 2000-lactose
Get 5g (0.001mol) adefovirdipivoxil-Macrogol 2000-succinate monoester, add the anhydrous N of 20ml, in the dinethylformamide, add the 2ml triethylamine, in ice-water bath, dropwise add and be dissolved in the dry N of 2ml, in the solution of the 2g isopropyl chlorocarbonate of dinethylformamide, stirred the mixture 10 minutes, right-aminophenyl-α-D-breast the pyranoside (Sigma) that adds 0.002mol then, ice bath stirred 30 minutes down, at room temperature stirred again 3 hours, and added in a large amount of absolute ethers precipitated product then.Cross ConA-Sepharose post (Sigma) purification at last, get adefovirdipivoxil-Macrogol 2000-lactose, yield 73%.
Embodiment 11 adefovirdipivoxils-Macrogol 2000-galactose
1, adefovirdipivoxil-Macrogol 2000-acetylation galactose
Get 149g (0.035mol) adefovirdipivoxil-methoxy poly (ethylene glycol) 2000, with Iodotrimethylsilane the methoxyl group on the Polyethylene Glycol is converted into alcohol as stated above, washing, boil off solvent chloroform after the drying, add the 450ml acetic acid ethyl dissolution, add the 15g Anhydrous potassium carbonate, reflux, drip 16g 1, the 2-Bromofume, back flow reaction 4 hours, be chilled to room temperature, filter evaporated under reduced pressure, add 400ml acetone and 150ml water dissolution, add 67g (0.14mol) 2-S-(2,3,4,6-four-O-acetyl-β-1)-galactopyranose base-2-isothiourea hydrobromate, with potassium carbonate 23g (0.167mol), stirring at room 10 minutes adds sodium sulfite 14.6g (0.14mol), lucifuge stirring at room 24 hours, evaporated under reduced pressure is with the dissolving of 500ml chloroform, with saturated aqueous common salt 100ml * 1 washing, concentrating under reduced pressure, the gained residue is soluble in water, with column chromatography purification (resin HPD-300, eluant: water/ethanol=95/5), eluent concentrates, use the 200ml acetic acid ethyl dissolution, anhydrous sodium sulfate drying filters, concentrate ethyl acetate to 50ml, add in a large amount of absolute ethers of vigorous stirring, cooling is filtered, vacuum drying, get the white powder solid, fusing point 42-44 ℃, yield 32%.
2, adefovirdipivoxil-Macrogol 2000-galactose
Getting adefovirdipivoxil-Macrogol 2000-acetylation galactose 20g that said method makes is dissolved in the 100ml absolute methanol; the methanol solution 20ml that adds the Feldalat NM of 0.1N; stirring at room 6 hours; concentrating under reduced pressure; add chloroform 200ml dissolving, with saturated sodium-chloride water solution 100ml * 3 washings, anhydrous sodium sulfate drying; filter; concentrate, use the 20ml acetic acid ethyl dissolution, add in a large amount of absolute ethers of vigorous stirring; cooling; filter, vacuum drying gets the white powder solid; fusing point 40-42 ℃, yield 82%.
Embodiment 12
Tenofovir mPEG2000 and two tenofovir PEG2000 in the duck body to hepatitis b virus infected therapeutic effect
To infect positive duck random packet, 6 every group, be divided into 1. virus control groups, use normal saline.2. tenofovir mPEG2000 small dose group (50mg/kg).3. dosage group (100mg/kg) among the tenofovir mPEG2000.4. tenofovir mPEG2000 heavy dose is organized (200mg/kg).5. two paracetamol De Fuwei PEG2000 small dose group (50mg/kg), 6. dosage group (100mg/kg) among the two tenofovir PEG2000.7. two tenofovir PEG2000 heavy dose is organized (200mg/kg).The oral cavity was irritated and was fed 2 times every day.The experimental drug time is 10 days, and drug withdrawal was observed 3 days.Observation index: (T0), medication (T5), medication (T10), drug withdrawal (P3) external jugular vein blood drawing respectively in 3 days in 10 days in 5 days before the medication, separation of serum is to be checked in-20 ℃ of preservations.Experimental result sees Table 2,3.
Table 2 tenofovir mono methoxy polyethylene glycol 2000 and two tenofovir Macrogol 2000 treatment groups and the clear DHBA-DNA OD of viral infection matched group Sanguis Anas domestica value are relatively
| Group | Dosage mg/kg (Bid * 10) | Duck number (only) | The clear DHBV-DNA OD of Sanguis Anas domestica value | |||
| T0 | T5 | T10 | P3 | |||
| Matched group tenofovir mPEG2000 two tenofovir PEG2000 | 50 100 200 50 100 200 | 6666666 | 1.08±0.20 0.99±0.06 1.01±0.11 1.14±0.09 1.15±0.09 1.10±0.10 1.08±0.14 | 1.00±0.19 0.90±0.23 0.85±0.19 0.89±0.18 1.08±0.17 1.07±0.12 1.00±0.09 * | 1.13±0.16 0.78±0.16 * 0.75±0.11 * 0.81±0.06 ** 0.93±0.18 * 0.76±0.10 * 0.73±0.13 * | 1.22±0.21 0.88±0.15 0.69±0.09 ** 0.74±0.14 ** 1.02±0.13 0.66±0.11 * 0.56±0.10 ** |
Statistical method is to treat front and back self paired t-test.Dna level before medication group different time dna level and the medication is on the same group compared:
*P＜0.05,
*P＜0.01.
The comparison of table 3 tenofovir mono methoxy polyethylene glycol 2000 and two tenofovir Macrogol 2000 treatment groups and the horizontal suppression ratio of the clear DHBA-DNA of viral infection matched group Sanguis Anas domestica
| Medicine | Dosage mg/kg (Bid * 10) | Duck number (only) | Suppression ratio (%) | ||
| T5 | T10 | P3 | |||
| Normal saline | 6 | 7.02 | -4.75 | -13.27 | |
| Tenofovir mPEG2000 | 50 100 200 | 6 6 6 | 9.08 16.15 22.19 | 21.56 25.74 28.94 | 11.11 31.68 35.09 |
| Two tenofovir PEG2000 | 50 100 200 | 6 6 6 | 6.09 2.73 | 19.13 30.91 | 11.30 40.00 |
| 7.41 | 32.41 | 48.15 | |||
Serum DHBA-DNA level does not have significant change after the medication of normal saline group, 10 days serum DHBV-DNA of the low dose of medication of tenofovir mPEG2000 level significantly reduces (P＜0.05), the 3rd day serum DHBV-DNA level raises to some extent after the drug withdrawal, among the tenofovir mPEG2000, all had serum DHBV dna level significantly to reduce (P＜0.05 or P＜0.01 on the 3rd day after heavy dose of medication 10 days and the drug withdrawal; 10 days serum DHBV-DNA of the low dose of medication of two tenofovir PEG2000 level significantly reduces (P＜0.05), the 3rd day serum DHBV-DNA level raises to some extent after the drug withdrawal, among the two tenofovir PEG2000, all had serum DHBV dna level significantly to reduce (P＜0.05 or P＜0.01) on the 3rd day after heavy dose of medication 10 days and the drug withdrawal.
The result shows that tenofovir mPEG2000 and two tenofovir PEG2000 have the effect that DHBV-DNA duplicates in the obvious inhibition liver, and " knock-on " phenomenon after drug withdrawal not occurring under 100mg/kg, the 200mg/kg dosage.
Embodiment 13 tests of pesticide effectiveness
Adefovirdipivoxil mPEG2000 and two adefovirdipivoxil PEG2000 in the duck body to hepatitis b virus infected therapeutic effect
To infect positive duck random packet, 6 every group, be divided into 1. virus control groups, use normal saline.2. adefovirdipivoxil mPEG2000 small dose group, dosage is 50mg/kg.3. dosage group among the adefovirdipivoxil mPEG2000, dosage is 100mg/kg.4. adefovirdipivoxil mPEG2000 is heavy dose of organizes, and dosage is 200mg/kg.5. two adefovirdipivoxil PEG2000 small dose group, dosage is 50mg/kg, 6. dosage group among the two adefovirdipivoxil PEG2000, dosage is 100mg/kg, the 7. heavy dose of group of two adefovirdipivoxil PEG2000, dosage is 200mg/kg.The oral cavity was irritated and was fed 2 times every day.The experimental drug time is 10 days, and drug withdrawal was observed 3 days.Observation index: (T0), medication (T5), medication (T10), drug withdrawal (P3) external jugular vein blood drawing respectively in 3 days in 10 days in 5 days before the medication, separation of serum is to be checked in-20 ℃ of preservations.Experimental result sees Table 4,5.
Table 4 adefovirdipivoxil mono methoxy polyethylene glycol 2000 and two adefovirdipivoxil Macrogol 2000 treatment groups and the clear DHBA-DNA OD of viral infection matched group Sanguis Anas domestica value are relatively
| Group | Dosage mg/kg (Bid * 10) | Duck number (only) | The clear DHBV-DNA OD of Sanguis Anas domestica value | |||
| T0 | T5 | T10 | P3 | |||
| Matched group adefovirdipivoxil mPEG2000 two adefovirdipivoxil EG2000 | 5010020050100200 | 6 6 6 6 6 6 6 | 0.914±0.16 1.266±0.03 1.313±0.27 1.071±0.09 0.962±0.11 1.192±0.12 1.088±0.16 | 0.784±0.151.209±0.081.132±0.20 *0.882±0.11 *0.928±0.091.099±0.110.905±0.13 | 0.799±0.101.119±0.07 **1.084±0.18 *0.594±0.11 **0.940±0.120.994±0.09 **0.830±0.15 ** | 1.017±0.121.059±0.07 **0.923±0.13 **0.4±0.11 **0.972±0.101.005±0.04 *0.737±0.14 ** |
Statistical method is for treating front and back self paired t-test.Dna level before medication group different time dna level and the medication is on the same group compared:
*P＜0.05,
*P＜0.01.
The comparison of table 5 adefovirdipivoxil mono methoxy polyethylene glycol 2000 and two adefovirdipivoxil Macrogol 2000 treatment groups and the horizontal suppression ratio of the clear DHBA-DNA of viral infection matched group Sanguis Anas domestica
| Medicine | Dosage mg/kg (Bid * 10) | Duck number (only) | Suppression ratio (%) | ||
| T5 | T10 | P3 | |||
| Normal saline | 6 | 12.52 | 10.17 | -15.60 | |
| Adefovirdipivoxil mPEG2000 | 50 100 200 | 6 6 6 | 4.47 13.18 17.42 | 11.57 16.27 44.14 | 16.29 27.28 62.65 |
| Two adefovirdipivoxil PEG2000 | 50 100 200 | 6 6 6 | 3.22 7.55 | 1.69 16.10 | -2.08 15.15 |
| 15.52 | 23.43 | 32.36 | |||
Serum DHBA-DNA level does not have significant change after the medication of normal saline group, all there was serum DHBV-DNA level significantly to reduce (P＜0.05) on the 3rd day after low dose of medication of adefovirdipivoxil mPEG2000 10 days and the drug withdrawal, among the adefovirdipivoxil mPEG2000, all had serum DHBVDNA level significantly to reduce (P＜0.05 or P＜0.01) on the 3rd day after heavy dose of medication 5 days, 10 days and the drug withdrawal, among the two adefovirdipivoxil EG2000, all had serum DHBV dna level significantly to reduce (P＜0.05 or P＜0.01) on the 3rd day after heavy dose of medication 10 days and the drug withdrawal.
Results suggest adefovirdipivoxil mPEG2000 and two adefovirdipivoxil PEG2000 have the effect that DHBV-DNA duplicates in the obvious inhibition liver, and the phenomenon of " knock-on " after the drug withdrawal do not occur.
Embodiment 14 anxious poison tests
Adefovirdipivoxil mono methoxy polyethylene glycol 2000 is carried out safety testing, and one time vein is given and mice 5g/kg (being equivalent to adefovirdipivoxil 0.3g/kg) dosage, the dead mouse phenomenon do not occur, shows that the safety of Pegylation adefovirdipivoxil is good.
Claims (11)
1. the polyethylene glycol derivative of adefovirdipivoxil and tenofovir, its general formula is:
Wherein, A is H or CH
3
R is the structure of form such as X-Y, and wherein Y is H, C
1-6Straight chain, side chain or cyclic alkyl, lactose, lactose derivatives, galactose or galactose derivative; Wherein X is O, NH, O-CO, CO-O, S-S, SO
2, O-NH, NH-O, O-CO-O, NH-CO-O, O-CO-NH, O-W, NH-W, O-CO-W, CO-O-W, O-W-O, O-W-NH, O-W-CO-O, NH-W-NH, NH-W-O, NH-W-CO-O, CO-O-W-O, CO-O-W-NH or CO-O-W-CO-O; Wherein W is low alkyl group or aryl;
Wherein, lactose derivatives, galactose derivative are sulfo-galactose, 2-imino group-2-methoxyl group-1-sulfo--β-D-galactose pyrrole glucosides, right-aminophenyl-α-D-breast pyranoside or D (+)-galactosamine;
Wherein, low alkyl group is C
1-6Straight chain, side chain or cyclic alkylene;
R ' is CH
2-(CH
2-O-CH
2)
n-CH
2, n=11-450;
R " be O or NH.
2. the described chemical compound of claim 1 is characterized in that X is O, and Y is methyl, lactose, sulfo-galactosyl or galactose.
3. the described chemical compound of claim 2 is characterized in that X is O, and Y is a methyl.
4. the described chemical compound of claim 2 is characterized in that X is O, and Y is galactose or sulfo-galactosyl.
5. the described chemical compound of claim 2 is characterized in that X is O, and Y is a lactose.
6. the described chemical compound of claim 1 is characterized in that R " be O.
7. the described chemical compound of claim 1 is in the purposes of preparation in the antiviral drugs.
8. the described purposes of claim 7 is the purposes in the preparation anti-hepatic-B virus medicine.
9. a pharmaceutical composition contains the described chemical compound of claim 1 as active component.
10. the compositions of claim 9 is peroral dosage form or injection type.
11. the described compositions of claim 10 is characterized in that peroral dosage form is a tablet, capsule, granule or oral liquid; Injection is injection, sterile powder for injection pin and concentrated solution for injection.
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| WO2013072745A1 (en) * | 2011-11-16 | 2013-05-23 | Laurus Labs Private Limited | Process for the preparation of tenofovir |
| CN107011382A (en) * | 2016-01-28 | 2017-08-04 | 单爱莲 | A kind of tenofovir prodrug and preparation method thereof |
| CN110372765B (en) * | 2019-07-08 | 2020-07-28 | 四川大学华西医院 | Tenofovir derivative and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014032481A1 (en) * | 2012-08-30 | 2014-03-06 | 上海源力生物技术有限公司 | Tenofovir prodrug and pharmaceutical uses thereof |
| US9908908B2 (en) | 2012-08-30 | 2018-03-06 | Jiangsu Hansoh Pharmaceutical Co., Ltd. | Tenofovir prodrug and pharmaceutical uses thereof |
| RU2664534C2 (en) * | 2012-08-30 | 2018-08-20 | Цзянсу Хэнсох Фармасьютикал Груп Ко., Лтд. | Tenofovir prodrug and pharmaceutical uses thereof |
| RU2664534C9 (en) * | 2012-08-30 | 2018-09-28 | Цзянсу Хэнсох Фармасьютикал Груп Ко., Лтд. | Tenofovir prodrug and pharmaceutical uses thereof |
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