CN101766632A - Preparation method and applications of lamivudine twin drug - Google Patents
Preparation method and applications of lamivudine twin drug Download PDFInfo
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- CN101766632A CN101766632A CN201010018228A CN201010018228A CN101766632A CN 101766632 A CN101766632 A CN 101766632A CN 201010018228 A CN201010018228 A CN 201010018228A CN 201010018228 A CN201010018228 A CN 201010018228A CN 101766632 A CN101766632 A CN 101766632A
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Abstract
The invention provides a preparation method and applications of a lamivudine twin drug. Lamivudine, and ursolic acid or oleanolic acid are condensed into the lamivudine-ursolic acid or lamivudine-oleanolic acid twin drug at a low temperature by using ethyl chloroacetate (ethyl bromoacetate) or ethyl chloropropionate (ethyl bromopropionate) as the linking group. The twin drug possibly has a dual-action mechanism; and the twin drug has the action of antivirus, and also has the actions of resisting inflammations, protecting the liver cell membranes, improving the liver function and resisting fibrosis. Thus, the invention organically combines the antivirus therapy and the liver protection treatment and provides a new concept for the research and development of drugs for treating hepatitis.
Description
Technical field
The invention belongs to pharmaceutical field, relate to a kind of preparation method of lamivudine twin drug, and this twin medicine is as the application for the treatment of hepatitis medicine.
Background technology
The whole world has nearly 2,000,000,000 people to be infected by hepatitis B virus (HBV), and it is HBV chronic infection person that 3.5 hundred million people are wherein arranged approximately, and the chronic asymptomatic HBV carrier of China may surpass 1.2 hundred million people.Because hepatitis B is very harmful to the mankind's, has every year 1200000 HBV infected patients to die from the HBV relevant disease approximately.China is directly used in the medical expense of viral hepatitis every year up to hundred million yuan of 300-500, therefore research and development to HBV the infected safely and effectively medicine particularly seem particularly urgent in the whole world in China.
What anti-clinically HBV adopted mainly is Drug therapy and gene therapy, and Drug therapy is the emphasis of clinical practice, mainly is divided into biological species medicine, chemical classes medicine and Chinese herbal medicine and effective ingredient three classes.But real worldwide the have only interferon and the lamivudine of extensive use are the ucleosides antiviral drugs of representative.What clinical practice at present was more is lamivudine, and the curative effect of its treatment hepatitis B is the main medicine of a line of antiviral therapy to obtain global approval.
(LAM II) by competitive inhibition hepatitis B virus DNA polymerase, suppresses the synthetic of hepatitis B virus DNA to lamivudine, can continue to suppress duplicating of hepatitis B virus, and the chronic hepatitis B patient liver histological is obviously improved.Because lamivudine can not be killed hepatitis B virus, the state of an illness is bounced easily after the drug withdrawal.In the continuous use process, hepatitis B virus is to the easy drug resistance of lamivudine.Clinical data shows that single lamivudine therapy chronic hepatitis B liver function recovery of using is slow, recovers normally to the transaminase from the beginning medication, and one section long inflammatory phase is arranged therebetween.Be reduced to a certain degree because have only when viral load, inflammation just can stop, and this process needs 2-3 month at least.Studies confirm that liver function recovery delay meeting causes to a certain degree chronic lesion to liver.Lamivudine can not dissolve the fibrocyte in the matter between liver, can only delay the hepatic fibrosis process indirectly, thereby it can not alleviate patient's hepatic fibrosis effectively, when the lamivudine therapy chronic hepatitis B, must strengthen the treatment of fibrosis.
(UA III) belongs to nonpolar pentacyclic triterpenoid to ursolic acid, is a kind of natural activity medicine that widely distributes at nature, has physiologically active widely.The ursolic acid clinical manifestation have significantly and rapidly reduce glutamate pyruvate transaminase, serum transaminase, the effect of the yellow cellulitis that disappears, appetite stimulator, fibrosis and recovery liver function, have the characteristics of instant effect, short treating period, effect stability.India scholar Saraswat etc. find ursolic acid, and (5～20mg/kg) to CCl
4The rats'liver toxicity that causes has protective effect.Make the survival rate that pretreatment can obviously improve rat hepatocytes with ursolic acid, and observe the effect that ursolic acid has anti-cholestasis in experiment, bile flow and content thereof all have increase.By CCl
4Mechanism of action infer that ursolic acid may be similar to its isomers oleanolic acid to damaging hepatocellular mechanisms of therapeutic action; be to protect and the biofilm system of stable liver plasma membrane and organelle; make its change penetrating and the Active transport functional rehabilitation is normal; moving and distributing of inside and outside ion of cell and water also restored thereupon; regeneration capacity is recovered in succession, promotes the downright bad hepatocyte of lobules of liver central area to repair.No matter Li Kaiquan etc. discover ursolic acid and use separately in preparation treatment viral hepatitis medicine, still be used with other drug, all have the effect of remarkable treatment viral hepatitis, effect is better than the curative effect of oleanolic acid matched group.Clinical trial proves that ursolic acid can significantly and rapidly reduce glutamate pyruvate transaminase, and the jaundice that disappears is recovered liver function, and hepatitis B is also had certain therapeutical effect.
(OA is a kind of oleanane type pentacyclic triterpenoid IV) to oleanolic acid, is distributed widely in about 60 section's 190 kind of plant of occurring in nature.Oleanolic acid has antiinflammatory, enhance immunity, inhibition platelet and falls many-sided clinical pharmacology effects such as collection, blood sugar lowering, antioxidation and diuresis, also has biological activitys such as hepatoprotective, lipidemia, atherosclerosis and antitumor.Oleanolic acid is treatment acute icterohepatitis type hepatitis and the more satisfactory medicine of chronic viral hepatitis, and toxicity is low, and untoward reaction is few.The seventies in 20th century, people just began the research to the oleanolic acid hepatoprotective effect; glutamate pyruvate transaminase obviously descends in the carbon tetrachloride poisoning rat blood serum of discovery after the oleanolic acid treatment; triacylglycerol is accumulated obvious minimizing in the liver; histology and histochemistry show hepatocellular degeneration; necrosis obviously alleviates, and glycogen is accumulated increase, RNA granule recovery in the endochylema; observe mitochondrial swelling under the Electronic Speculum, it is normal that the rough endoplasmic reticulum vesiculation recovers substantially.Show that oleanolic acid can be to CCl
4The acute, chronic hepatitis damage that causes has obvious protective effect, can prevent the generation of experimental liver cirrhosis.Its mechanism may be the oleanolic acid protection and stablize the biofilm system of liver plasma membrane and organelle; make its change penetrating and the Active transport functional rehabilitation is normal; moving and distributing of inside and outside ion of cell and water also restored thereupon; so hepatocyte loose and that the balloon sample becomes is converted into normally kytoplasm; regeneration capacity is recovered in succession, promotes the downright bad hepatocyte of lobules of liver central area to repair.The eighties in 20th century, people further confirmed the liver protective effect of oleanolic acid again by histopathologic inspection.Residual its karyokinesis number showed increased of liver under the effect of research report oleanolic acid.Oleanolic acid to the protective effect of liver mainly owing to its antioxidation, antiinflammatory action and to the influence of drug metabolism enzyme.Han Dewei finds in experimentation, oleanolic acid can promote liver cell regeneration, the necrotic area is repaired rapidly, the vigor of hepatic tissue recovery normally the alleviating alanine of degeneration necrosis is descended, make the liver tissues inflammatory habituation, gamma globulin descends in the blood, thereby suppresses hair jelly fibril hypertrophy, makes liver cirrhosis be difficult to form.Its pharmacological action is relevant with dosage, and the oleanolic acid of low dosage has hepatoprotective effect.
Structural formula is:
Summary of the invention
Technical problem:
The present invention proposes at causing to a certain degree chronic lesion to liver in the lamivudine therapy chronic hepatitis B process; and can not dissolve the fibrocyte in the matter between liver; can only delay the hepatic fibrosis process indirectly; can not alleviate patient's weak points such as hepatic fibrosis effectively; a kind of lamivudine-ursolic acid or the twin medicine of lamivudine-oleanolic acid and preparation method thereof are provided; this twin medicine has stronger antiinflammatory again when having antivirus action; the protection liver plasma membrane; improve liver function and anti-fibrosis effect; having remedied the deficiency of lamivudine clinical treatment, is the combination of antiviral therapy and liver protection treatment.
Technical scheme:
The present invention proposes a kind of twin medicine that the antiviral regulating liver-QI is protected dual curative effect that has, its general structure (I) is:
Wherein: R
1=H or CH
3, R
2=H or CH
3, R
3=H or CH
3, n=1,2.
Discharge former medicine lamivudine and ursolic acid or oleanolic acid after such degradation.Be hydroxyacetic acid or hydracrylic acid after its linking group hydrolysis, hydroxyacetic acid or hydracrylic acid are the intermediate metabolitess of tricarboxylic acid cycle in the body, and its absorption and metabolic mechanism are clear and definite, have reliable biological safety.
Beneficial effect:
Can cause to a certain degree chronic lesion to liver in order to remedy in the lamivudine therapy chronic hepatitis B process; and can not dissolve the fibrocyte in the matter between liver; can only delay the hepatic fibrosis process indirectly; can not alleviate patient's weak points such as hepatic fibrosis effectively; the imagination of lamivudine-ursolic acid or the twin medicine of lamivudine-oleanolic acid has been proposed; on the molecule of anti-hbv drug lamivudine, connect and have the fibrosis regulating liver-QI and protect active ursolic acid or oleanolic acid to make twin medicine; discharge former medicine lamivudine and ursolic acid or oleanolic acid after entering in the body, when having antivirus action, have stronger antiinflammatory again; the protection liver plasma membrane; improve liver function and anti-fibrosis effect.This design has remedied the deficiency of lamivudine clinical treatment, is the combination of antiviral therapy and liver protection treatment.
Twin medicine of the present invention is, and to be linking group with chlorine (bromine) ethyl acetate or chlorine (bromine) ethyl propionate go into a molecule with lamivudine and ursolic acid or oleanolic acid with the covalent bond amalgamation, makes the structure of twin medicine not too complicated, is easy to preparation and quality control, is easy to use.
Twin medicine of the present invention Central Plains medicine is connected with the form of ester bond and amido link, helps the hydrolysis in vivo of twin medicine.
The specific embodiment
The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.
Synthetic route of the present invention (is example as the twin medicine of the lamivudine-ursolic acid that is linking group with chlorine (bromine) ethyl acetate):
Embodiment 1 2-Folium Vaccinii vitis-idaeae acyloxy acetic acid
Ursolic acid 5g is dissolved among the DMF125ml, adds ethyl chloroacetate 1.17ml (or etc. mol bromoacetate), K
2CO
34.5g, stirring at room reaction 5 hours.Remove by filter insoluble solids, filtrate decompression concentrates, and toluene 30ml * 3 bands are DMF to the greatest extent, gets faint yellow edible vegetable oil shape liquid and is esterification products, and are not purified, are directly used in next step reaction.
Will be in the above-mentioned oily liquid add 4N NaOH aq 30ml, methanol 50ml, THF 75ml, stir faint yellow clear liquid, stirring at room reaction 18 hours.Concentrating under reduced pressure gets the pale pink solid, adds entry 90ml, transfer pH=3 with 4N NaOH aq, solid bleaches, and stiff more, add chloroform 75ml, be stirred to the solid dissolving, separatory, with chloroform 70ml aqueous layer extracted, merge the chloroform layer, be concentrated into dried, the off-white color solid be hydrolyzate, 5.35g, two step total recoverys 94.9%.MS-ESI(-)：513.3[M-H]
-，MS-ESI(+)：515.3[M+H]
+。
Embodiment 2 is the twin medicine of lamivudine-ursolic acid (LMX) of linking group with chlorine (bromine) ethyl acetate
Said hydrolyzed product 3g is added among the DMF 30ml stirring and dissolving ,-15 ℃～-20 ℃ of the outer temperature of control, add triethylamine 0.81ml, slowly drip isobutyl chlorocarbonate 1.2g, dropwise, insulated and stirred reaction 20min, the adularescent solid is separated out, and not treatedly is directly used in next step.
Preparation lamivudine 1.337g, the solution of triethylamine 1.2ml in DMF 15ml slowly are added drop-wise in the above-mentioned reactant liquor under warm-15 ℃～-20 ℃ the condition outside.Dropwise, insulated and stirred reaction 1 hour, after rise to room temperature, continue to stir 1 hour.The elimination insoluble matter, filtrate decompression concentrates, and toluene 30ml * 4 bands are DMF to the greatest extent, gets white paste and is crude product.In above-mentioned crude product, add chloroform 75ml, 5min vibrates in 60 ℃ of water-baths, the filtering insoluble matter, filtrate is washed with 1%HCl aq 75ml * 2, separatory, chloroform layer concentrating under reduced pressure gets the light brown yellow oily liquid, and (eluant: ethyl acetate/petroleum ether (60-90 ℃)=2: 1) purification gets white solid 1.3g, two step total recoverys 30.7% through silica gel column chromatography.MS-ESI(-)：724.3[M-H]
-，MS-ESI(+)：726.4[M+H]
+。
1H?NMR(d，ppm，CDCl
3)：0.709-2.277(m，45H)，3.190-3.242(m，2H)，3.628-3.687(dd，1H)，3.956-4.007(dd，1H)，4.089-4.161(m，1H)，4.172-4.215(m，1H)，4.564-4.619(m，2H)，4.741-4.794(m，1H)，5.375(s，2H)，6.340(s，1H)，7.393(d，1H)，8.555-8.567(d，1H)。
Embodiment 3 is the twin medicine of lamivudine-ursolic acid (LMX-2) of linking group with 2-chlorine (bromine) ethyl propionate
Operation is with embodiment 1,2;
Embodiment 4 is the twin medicine of lamivudine-ursolic acid (LMX-3) of linking group with 3-chlorine (bromine) ethyl propionate
Operation is with embodiment 1,2;
Embodiment 5 is the twin medicine of lamivudine-oleanolic acid (LMX-4) of linking group with chlorine (bromine) ethyl acetate
Operation is with embodiment 1,2;
Embodiment 6 is the twin medicine of lamivudine-oleanolic acid (LMX-5) of linking group with 2-chlorine (bromine) ethyl propionate
Operation is with embodiment 1,2;
Embodiment 7 is the twin medicine of lamivudine-oleanolic acid (LMX-6) of linking group with 3-chlorine (bromine) ethyl propionate
Operation is with embodiment 1,2;
Embodiment 8 anti-DHB effect experiments
A male age in days sheldrake, conventional raising, feeding environment: 24 ℃～26 ℃ of temperature, relative humidity 60～80%, regularly ventilation, illumination about 12 hours of every day.About 70 grams of body weight during experiment/only, got blood in the 5th day through the shin vein, behind the separation of serum, with the method detection DHBV of PCR, the duck of selecting congenital infection experimentizes.
Be subjected to reagent thing LMX with 0.5% sodium carboxymethyl cellulose by 250mg/kg/d, 100mg/kg/d and the preparation of 50mg/kg/d dosage.By every experiment duck 70g, each administration 1ml calculates, and every experiment duck is weighed, and determines the administration volume by actual weight.So analogize, after experiment duck body weight P＜0.05 increases, adjust concentration in proportion.
Sample divides big or middle and small dose group (is respectively 250mg/kg/d; 100mg/kg/d; 50mg/kg/d), dosage is 1ml//day; Other establishes virus control (DHBV), replaces medicine with 0.5% sodium carboxymethyl cellulose; Positive drug is with lamivudine (3TC) 1ml//day.The equal gastric infusion of above medicine, 10 days one courses of treatment.(T before medication
0), the 5th day (T of medication
5), the 10th day (T
10) and drug withdrawal after the 5th day (P
5), get blood from duck shin vein, separation of serum ,-70 ℃ are frozen to be checked.Wherein, big and small dose group Sanguis Anas domestica is measured DHBV-DNA with quantitative PCR method clearly, and middle dosage group Sanguis Anas domestica is measured DHBV-DNA with spot hybridization clearly.
The result shows: experiment virus control treated animal serum DHBV-DNA horizontal stable, be subjected to reagent thing LMX 250mg/kg/d, 100mg/kg/d and 50mg/kg/d each time point animal serum DHBV-DNA 10 day course of treatment significantly less than positive drug lamivudine group (P＜0.05), be subjected to reagent thing LMX antiviral activity to be better than the positive drug lamivudine.
DMF is meant N herein, dinethylformamide, K
2CO
3Be meant potassium carbonate, NaOH is meant sodium hydroxide, and THF is meant oxolane, and HCl is meant hydrochloric acid.
Claims (9)
1. a twin medicine that has the liver protective effect when having antivirus action again is characterized in that lamivudine regulating liver-QI protection medicine is put together, and this chemical compound general formula is as described in (I):
Wherein, R
1=CH
3, R
2=H, R
3=H or methyl; R
1=H, R
2=CH
3, R
3=H or methyl; N=1,2.
2. the lamivudine twin drug described in the claim 1 is characterized in that liver protection medicine comprises ursolic acid and oleanolic acid.
3. the preparation method of the lamivudine twin drug described in the claim 1, it is characterized in that lamivudine and ursolic acid or oleanolic acid are linking group with chlorine (bromine) ethyl acetate or chlorine (bromine) ethyl propionate, earlier under cryogenic conditions, prepare mixed acid anhydride, be condensed into lamivudine-ursolic acid or the twin medicine of lamivudine-oleanolic acid with lamivudine again.
4. according to the preparation method described in the claim 3, it is characterized in that its linking group is ethyl chloroacetate, bromoacetate, 2-chloropropionate, 2 bromopropionic acid ethyl ester, 3-chloropropionate, 3-ethyl bromide or their other alkyl esters derivant; Preferred ethyl chloroacetate.
5. preparation method according to claim 3 is characterized in that cryogenic conditions refers to 0 ℃～-78 ℃.
6. preparation method according to claim 3 is characterized in that mixed acid anhydride is meant that ursolic acid or its corresponding linking group of oleanolic acid connect the mixed acid anhydride of back and isopropyl chlorocarbonate, isobutyl chlorocarbonate or the preparation of other alkyl chloroformate derivant; Preferred isobutyl chlorocarbonate.
7. preparation method according to claim 3 is characterized in that the deacidification agent that condensation is adopted is triethylamine or pyridine; Preferred triethylamine.
8. preparation method according to claim 3 is characterized in that selected solvent is chloroform, N, dinethylformamide, dichloromethane.
9. according to the lamivudine twin drug described in the claim 1, the application in the clinical hepatitis treatment.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948615A (en) * | 2014-04-30 | 2014-07-30 | 陕西奥星制药有限公司 | Medicinal preparation for treating hepatitis B and preparation method thereof |
| CN104311622A (en) * | 2014-10-08 | 2015-01-28 | 四川大学 | Asiatic acid monoamide derivative |
| CN109810063A (en) * | 2017-11-21 | 2019-05-28 | 中国科学院上海药物研究所 | A kind of novel resisiting influenza virus " twin medicine ", preparation method and the usage |
| CN114835767A (en) * | 2022-04-29 | 2022-08-02 | 华侨大学 | Arbutin conjugate and application thereof |
-
2010
- 2010-01-20 CN CN201010018228A patent/CN101766632A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103948615A (en) * | 2014-04-30 | 2014-07-30 | 陕西奥星制药有限公司 | Medicinal preparation for treating hepatitis B and preparation method thereof |
| CN104311622A (en) * | 2014-10-08 | 2015-01-28 | 四川大学 | Asiatic acid monoamide derivative |
| CN109810063A (en) * | 2017-11-21 | 2019-05-28 | 中国科学院上海药物研究所 | A kind of novel resisiting influenza virus " twin medicine ", preparation method and the usage |
| CN114835767A (en) * | 2022-04-29 | 2022-08-02 | 华侨大学 | Arbutin conjugate and application thereof |
| CN114835767B (en) * | 2022-04-29 | 2023-09-29 | 华侨大学 | Arbutin conjugate and application thereof |
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Open date: 20100707 |