CN101953826B - Application of flavanolignan to preparation of medicament for treating viral hepatitis B - Google Patents

Abstract

The invention relates to application of flavanolignan to preparation of a medicament for treating viral hepatitis B, in particular to application of angular flavanolignan or a pharmaceutical salt thereof to preparation of a medicament for reducing the surface antigen HBsAg of hepatitis B virus, inhibiting HBV DNA duplication and treating hepatitis B virus infection diseases. The flavanolignan caninhibit the HBsAg activity obviously, and the strength of clearing the HBsAg at the lower concentration exceeds five and half times that of positive control medicaments (alpha-interferon); and simultaneously, the compound shows the inhibition rate of over 45 percent on HBV DNA at 20 micrograms/milliliter of concentration. The pharmacodynamic result indicates that the flavanolignan or the pharmaceutical salt thereof can be used for preparing the medicament for clearing the surface antigen of the hepatitis B virus, inhibiting the HBV DNA duplication or treating the hepatitis B virus infection diseases expectably.

Description

A flavanolignan is used to prepare the purposes of treatment viral hepatitis B medicine

Technical field

The present invention relates to medical technical field; Particularly; The present invention relates to a kind of angle type flavanolignan or its officinal salt and be used to prepare the purposes that reduces HBsAg HBsAg medicine, inhibition HBV dna replication dna or treat hepatitis b virus infected disease medicament; This flavanolignan has and suppresses the HBsAg activity very significantly, and it is removed HBsAg intensity and surpasses positive control medicine (alpha-interferons of 10000 units per ml) 5 sesquialters down at low concentration (20 mcg/ml); Simultaneously, this chemical compound demonstrates the suppression ratio greater than 45% to HBV DNA when 20 mcg/ml concentration.Above pharmacodynamic result shows that this flavanolignan or its officinal salt can expect to be used to prepare and removes HBsAg, suppresses the HBV dna replication dna or treat the purposes of hepatitis b virus infected disease medicament.

Technical background

Hepatitis B (hepatitis B) is the infectious disease that is caused by hepatitis b virus hbv, and HBV is a member of Hepadnaviridae hepadnaviridae, and it is shaped as the spheroidal particle of diameter 42 nanometers.HBV is peculiar virus, and less being infectious in other animal has only in human body or primate chimpanzee body and just can duplicate.This virus is propagated through hepatitis B virus carriers and hepatitis B patients'blood, saliva, seminal fluid, vaginal secretions, has chronic carrier state.Hepatitis B is widely current in China, and because of it is divided in vertical transmission, horizontal transmission, the family propagation, iatrogenic infection and multiple mode such as spread through sex intercourse, high to the crowd infection rate, infection rate reaches more than 35% in certain areas.According to interrelated data, hepatitis detects male patient and has reached 1.89 hundred million, and the number (carrier) nearly 400,000,000 of should not going to a doctor is one of the most serious infectious disease of current harm people ' s health.Hepatitis B clinical manifestation variation is prone to develop into chronic hepatitis B (CHB) and liver cirrhosis, and few patients can change primary hepatocarcinoma into.

Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus, and the HBsAg positive is a goldstandard of judging that HBV infects.HBsAg is positive but do not have the hepatitis symptom person of appearance and become HBV virus carrier; The HBsAg titre is big more, and it is just big more that it merges the active probability that raises of HBcAg HBeAg, the HBV DNA positive and DNA polymerase, thereby infectiousness is strong more.Therefore, suppress the secretion of HBsAg and duplicate be in the research and development anti-hepatic-B virus medicine important target with detect target.Report: hepatitis B HBsAg such as the refined cloud of the Wu of Beijing Ditan Hospital remove and there is certain dependency in hepatitis B closed loop covalency DNA (cccDNA), and removing HBsAg is the significantly reduced sign of cccDNA level.2002, deliver the researcher of result of study at " New England Journal of Medicine " and think: for CHB patient, remove as before liver cirrhosis, obtaining HBsAg, then its liver cirrhosis and hepatocarcinoma incidence rate will reduce by 60 times.All HBsAg serum is removed as one of treatment terminal point determining standard in the guide of the AASLD of U.S. hepatopathy research association, the Asia-Pacific liver APASL of research association and the EASL of EASL.

According to European liver EASD in 2008 annual meeting: Polyethylene Glycol Intederon Alpha-2a treatment CHB patient 48 week drug withdrawals afterwards; Followed up a case by regular visits to 1,2,3,4 year; Its HBsAg clearance rate is respectively 3%, 6%, 8% and 11%; And with the lamivudine person HBsAg clearance rate was merely 0%, 0%, 0% and 3% in 1,2,3,4 year after drug withdrawal separately, thereby this interferon is considered to treat at present the negative CHB patient's optimal treatment of HBeAg and selects.

Medication to hepatitis B patient mainly is divided into several big type of the liver protecting and ALT lowering, antiviral, anti-hepatic fibrosis and adjusting immunity etc.Antiviral is a basic method, and the liver protecting and ALT lowering is an auxiliary treatment, takes stopgap measures and rarely seen effecting a permanent cure more, the research that still is the antiviral treatment aspect that made progress in the last few years.Yet; Can only reach for viral hepatitis B therapeutic scheme clinically at present and suppress hbv replication and secondary infection; Main medicine is still nucleoside medicine such as lamivudine (3-TC), Entecavir, adefovirdipivoxil (ADV), Sebivo etc., is in the interim emtricitabine of clinical trial, tenofovir, clevuding etc. in addition.In China, lamivudine has become the medical insurance medication, is applied to large quantities of HBV patients.Nucleoside medicine part advantage is: bioavailability is high, and is oral safer.Yet, though their disease controlling, fetch long prices first effectively; Second life-time service all drug resistance can occur, and the bounce-back in various degree that occurs HBV DNA, ALT and liver histological after the drug withdrawal; The 3rd, the comparatively tangible well-known ill effect that the life-time service nucleoside medicine occurs, for example kidney injury, baby's teratogenesis etc.Headache is the most: virus is drug-fast cure rate to occur greatly reducing; Because nucleoside medicine is reversible to virus replication, thus to most of patient if desire to reach greatest treatment efficacy, the course of treatment must be more than 1 year; So its drug resistance occurs thereupon, does not just reach the effect of expection; And nucleoside medicine be difficult in addition remove cccDNA, treatment after 1 year HBsAg be difficult to weak points such as cloudy commentaries on classics.

The biological engineering class antiviral drugs that interferon (α, beta-interferon) and recombinant interferon class etc. derive from the HL becomes research and treatment CHB focus medicine in the recent period, and it has antiviral and immunomodulating dual function.Thereby it both can suppress virus replication through antivirus action and alleviate the liver cell inflammatory reaction, reduces hepatocellular damage, plays and improves patients clinical symptom and liver physiological function thereby delay PD; Again can the enhance immunity effect, through the effect of natural killer cell in the intensive aspect and helper T lymphocyte, especially can promote killer T cell to go to kill and wound by virus infected cell, therefore play antivirus action indirectly.The HBsAg serology conversion that interferon therapy CHB patient obtains is more more lasting than lamivudine, and " digestive tract " magazine the next item up research in 2003 shows: 3 years relapse rates of interferon therapy group HBsAg significantly are lower than the lamivudine group; And long-acting interferon can use weekly once, and is comparatively convenient.Therefore, interferon day by day becomes one of choice drug that is used to treat chronic viral hepatitis B virus clinically, but that its side effect and untoward reaction are reported is more, and total effective rate is not high, costs an arm and a leg, and patient economy burden is big, thereby causes and be difficult to clinically be widely used; And do not use the decompensated cirrhosis patient is suitable.

Along with the research of hepatopathy, developed the analysis of standardized HBV DNA in recent years, advanced understanding greatly the hepatitis B patient state of an illness.The quantitative analysis of HBV DNA can be predicted the seriousness and the prognosis thereof of hepatitis B, because HBV DNA lasting masculin (promptly continuing viremia) makes the hepatitis B disease progression easily and increases the weight of; High hepatitis B virus (HBV DNA) content promotes the formation of liver cirrhosis easily; The lasting existence of HBV DNA is that the high risk factor, particularly viral level of hepatocarcinoma (HCC) generation is higher, the course of disease is long, older or merge other liver patient; The HBV DNA that continues high concentration exists, and the mortality rate that can cause losing compensatory liver cirrhosis and constitutional liver serious symptom obviously increases.Simultaneously must recognize that the relation of HBV dna level and liver histological is extremely close: bibliographical information is through antiviral therapy, and the improvement of hepatic fibrosis and elimination are obviously; Recent international hepatopathy meeting report is imitated by force and hang down chemical sproof antiviral therapy, along with the reduction of HBV DNA with turn out cloudy, reverse in various degree can occur and observe liver cirrhosis, and therefore the opinion liver cirrhosis also should carry out antiviral therapy now.

Therefore, the application of HBV DNA index in antiviral therapy also plays a part very important: the level of HBV DNA is the important indicator whether the decision chronic hepatitis B needs antiviral therapy; Make the HBeAg positive or negative difference treatment standard and the requirement of HBeAg respectively according to the different situations of HBV DNA; In antiviral therapy, according to the therapeutic response of HBVDNA, judge whether that virusology replys in early days and then determine the strategy of long-term prescription to reply with the virusology that obtains persistence, reach and continue the purpose that virus suppresses; According to the virological situation of replying of HBVDNA, create serological switching foundation of HBeAg and condition, to reach its good middle therapeutic goal; Continue the inhibition situation according to HBV DNA and strive for that virus continues feminine gender, to strive for reaching the final therapeutic goal of antiviral; Continue to be suppressed fully according to HBV DNA, also demonstrated improvement in various degree and the disappearance of cccDNA; In antiviral therapy, assess and prevent the risk of caused virus variation of antiviral drugs and drug resistance generation with the variation of HBV DNA; In case when virus variation or drug resistance took place, the variation of HBV DNA was unique signal at first and diagnosis basis, also be treatment drug resistance and the guidance and the foundation that change therapeutic strategy.In sum, the inhibition degree of HBV DNA there is new important meaning in the further diagnosis of hepatitis B and treatment,, assessment hepatitis B prognosis and drug resistance danger is all had bigger directive function the observation of curative effect.

Can be known by above-mentioned factor: the basic link of another one that the inhibition hepatitis B virus is bred in vivo is to suppress duplicating of HBV DNA.The reduction of HBV dna level or be lower than detection level be the check antiviral drugs other one JINYAOSHI.So, Asia-Pacific liver EASD and European liver EASD all with the HBV DNA detection less than as one of hepatitis B virus patient treatment terminal point; Also tested compounds is regarded as one of the test event that to accomplish in China's new drug development guide for the inhibition strength of HBV DNA.Why lamivudine can become first-selected nucleoside medicine is because it has the activity of the inhibition HBV dna replication dna of strong effect.Therefore can suppress the novelty medicine that chemical compound that the HBV dna replication dna can suppress HBsAg again will more promise to be the treatment hepatitis B patient.

Mandatory declaration be: the antiviral drugs of using at present is the inhibitor of virus replication just in fact, directly kill virus and break virus body, otherwise will damage host cell.These antiviral drugs (being mostly nucleoside medicine) also exist above-mentioned toxic and side effects big, be prone to cause and be prone to shortcoming such as knock-on after viral gene sudden change, the drug withdrawal; Therefore the development of new antiviral drugs is the task of top priority in current medicament research and development field, and it all has extremely important social meaning and economic implications for treatment China a large amount of hepatitis B patient and virus carrier, the control source of infection etc.So new non-nucleoside hepatitis B virus inhibitor and this type of lead compound that can reduce HBsAg or inhibition HBV dna replication dna of discovery has very big guiding significance from the natural drug of national folk life-time service, and vast development prospect is arranged.

Based on this purpose; The inventor once accomplished the patent and the article of multinomial anti-hepatitis virus natural product and structure of modification derivant thereof in the past; Having found that multiple inhibition HBsAg HBsAg or hepatitis B virus e antigen HBeAg are active or suppress the chemical compound of HBV dna replication dna, is feasible [referring to " medical usage of one type of mapping eucalyptus globulus alkanol type sesquiterpene for inhibiting hepatitis virus " (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo etc. thereby explanation filters out the novelty medicine that can reduce HBsAg, control hepatitis B virus infection from natural product and synthesis of derivatives thereof; ZL 200610053827.4); " medical usages of 2 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Lee school Kun, Zhao Yu, Huang Kexin, Li Haibo etc.; ZL200610053749.8); " 2 α, the medical usage of 3 beta-dihydroxies-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Zhang Li and, Dong Sun Han, Li Haibo etc.; ZL200610053601.4); The purposes and the pharmaceutical composition thereof of hepatitis B virus " the eremophilane lactone suppress " (Zhao Yu, Li Haibo, Yang Leixiang, girth are new etc.; ZL 03153691.3); " a kind of Eremophilone lactones acid natural product and application thereof " (Zhao Yu, girth are new, Shi Shuyun, Wang Xiaoyu etc.; ZL 200610053575.5); " a kind of eudesmane type sesquiterpenes acid and uses thereof " (Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei etc.; ZL 200610053579.3); " purposes of laggera plant abstract in inhibiting herpes simplex virus and hepatitis B virus " (Zhao Yu, girth are new, Yu Rongmin, Bai Hua; CN 1989989 A); " medical usage of 1 β-oxo-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Li Haibo etc.; CN 1927197A); " medical usage of 1 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Wu Xiumei etc.; CN 1935131A); " medical usage of acid of mapping eremophilane and inhibition hepatitis B surface antigen thereof " (Huang Kexin, Lee school Kun, Wang Xiaoyu, Zhao Yu etc.; CN 101239054); " 1-oxygen-substituted benzene formyl quinic acid chemical compound and inhibition hepatitis B virus purposes thereof " (Lee school Kun, Hu Lihong, Wu Xiumei, Zhao Yu etc.; CN101293836); Inhibition HBsAg that the inventor has delivered and HBV DNA isoreactivity article are referring to " In Vitro Antiviral Activity of Three Enantiomeric SesquiterpeneLactones from Senecio Species Against Hepatitis B Virus ", Haibo Li (Li Haibo), Changxin Zhou (girth is new); Xiumei Wu (Wu Xiumei); Yu Zhao* (Zhao Yu) etc., Antiviral Chemistry Chemotherapy, 2005; 16,277-282; " Evaluation of Antiviral Activity of Compounds Isolated fromRanunculus sieboldii Miq.and Ranunculus sceleratus L ", Haibo Li (Li Haibo), Changxin Zhou (girth is new); Xiumei Wu (Wu Xiumei); Yu Zhao* (Zhao Yu) etc., Planta Medica, 2005; 71 (12), 1128-1133; " Application ofhigh-speed counter-current chromatography for the isolation of antiviraleremophilenolides from Ligularia atroviolacea ", Shi, Shu-Yun (Shi Shuyun); Hai-Bo (Li Haibo), Zhao, Yu (Zhao Yu) etc.; Biomedical Chromatography; 2008,22 (9), 985-991; " Purification and identification of antiviralcomponents from Laggera pterodonta by high-speed counter-currentchromatography "; Shuyun Shi (Shi Shuyun); Yu Zhao (Zhao Yu) etc., JournalofChromatographyB, 2007; 859,119-124].Undoubtedly, continuing from natural product and structure of modification derivant thereof, to seek the lead compound that can remove HBsAg or inhibition HBV dna replication dna is that very therefore being necessary property is also classified as one of great special project of new drug development by the Ministry of Science and Technology with urgent.

In above-mentioned treatment CHB medicine, also have one type to be type of protecting the liver medicine, its clinical a large amount of uses be typically the silymarin in the seed that is present in the feverfew Herba Silybi mariani.Herba Silybi mariani is extensive use clinically, its commodity Legalon by name on market ^TMGrand or the Flavobion of sharp liver ^TM, its representative compound surely belongs to flavanolignan's silibinin.Flavone lignin compound belongs to weedtree quality class, is one type of natural product of and a part flavone be combined into plain by a part phenylpropyl alcohol.Silibinin content is maximum in the Herba Silybi mariani, and activity is also the highest.This medicine effect mainly contain following some: (one) free radical resisting is active: silymarin has protective effect for the hepatic injury that is caused by carbon tetrachloride, galactosamine, alcohols and other hepatotoxin.People such as nineteen ninety Lotteron have reported in the Mouse Liver microsome; Silymarin can reduce external lipid peroxidation that is caused by the carbon tetrachloride metabolism and the peroxidation that is caused separately by reduced coenzyme, and these show that all silymarin is the chain interruption antioxidant or is free radical scavenger.(2) protection liver plasma membrane: keep flowability of cell membranes through the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special receptor on mycotoxin phalloidine and α-amanitin etc. and the hepatocyte, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistance of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with ER after getting into cell; And make it to activate; Activated receptors can enhance hepatocyte nuclear RNA polymerase 1 activity, rna transcription is strengthened, promote enzyme and proteinic synthetic; And promote the synthetic of DNA indirectly, help hepatocellular reparation and regeneration.(4) antitumor action: various active oxygens can form 8-hydroxyl guanine by the oxidation guanine, cause DNA damage, and then cause tumor, and silibinin has also shown the effect of prevention and treatment tumor as an effective free radical resisting material.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult Flora, K. etc., Am.J.Gastroenterol, 1998,93,139-143; Saller R. etc., Drugs, 2001,61 (14), 2035-2063;

; R. etc.; Curr.Med.Chem.2007,14,315-338; Z. etc.; Phytother.Res.2003; 17,524-530;

R. etc.; Bioorg.Med.Chem.2004; 12,5677-5687; Varga, Z. etc.; Phytothe.Res.2001,15,608-612.Singh, R.P. etc., Curr.Cancer Drug Tar.2004,4,1-11).Therefore; The flavone lignin chemical compound that with the silibinin is representative has caused increasing concern; In the period of 2006-2009, prepare and a plurality of serial silibinin analog derivative reported also has obvious antioxidation activity (Yang Leixiang, Zhao Yu etc., " Design, synthesis andexamination of neuron protective properties of alkenylated and amidateddehydro-silybin derivatives " like the inventor; Journal of Medicinal Chemistry; 2009,52 (23), 7732-7752; Wang Feng; Zhao Yu etc.; " Preparation of C-23esterified silybinderivatives and evaluation of their lipid peroxidation inhibitory and DNAprotective properties ", Bioorganic & Medicinal Chemistry, 2009; 17 (17), 6380-6389; Yang Leixiang, Zhao Yu etc., " Synthesis and Antioxidant PropertiesEvaluation of Novel Silybin Analogues "; Journal of Enzyme Inhibitionand Medicinal Chemistry; 2006,21 (4), 399-404; Or the like).In the above-mentioned article of inventor report, A ring, B ring, E ring, 23 substituted flavanolignan compounds of a plurality of series that design and synthesize out through the inventor all demonstrate strong effect and catch the activity of DPPH free radical and ultra-oxygen anion free radical, antioxidant activity and protection PC12 cell activity.But obvious: above-mentioned research only concentrates on antioxidation and the cytoprotection of studying the silibinin flavonolignan.

Though be the antioxidation curative effect that flavanolignan's chemical compound of representative has the above with the silibinin, yet that it appears in the newspapers is less relatively in the document of antiviral therapy aspect.(2006) in the recent period; Xie Jun has reported that Combined application has the interferon of antiviral and immunomodulating dual function and the silibinin phosphatide complexes of hepatoprotective is treated the result of chronic viral hepatitis B; It is more obvious with the interferon effect than independent to find that drug combination reduces ALT, AST value in patient's body, explains that the silibinin phosphatide complexes can strengthen the effect of the treatment chronic viral hepatitis B of interferon.But similarly therapeutic scheme is still flavanolignans such as silibinin as adjuvant drug, and mainly is to be used to protect liver plasma membrane but not directly to implement antivirus action with it.

The compounds for treating DNA of flavanolignan viroid infects especially its new purposes that is used for anti-hepatitis virus aspect (comprise and suppress HBsAg or suppress the HBV dna replication dna) is effectively developed as yet, so from flavanolignan, seek the reactive compound in anti-hepatitis virus field, also being about to flavanolignan's structure of modification, to make it have anti-DNA viroid activity be a brand-new field.From the lead compound challenge that forefathers did not attempt especially of wherein finding to suppress HBsAg or suppressing the HBV dna replication dna.In order to explore this field; Our design has also prepared and the silibinin structure a kind of new angle type flavanolignan derivant of difference to some extent, also promptly is coupled to dioxane at B ring prosposition; Form C ring/B ring/D ring and be upset angie type ring system space structure; And silibinin is because of on the B ring being the synthetic dioxane modes of 3,4 digit pairs, so its C ring/B ring/D ring is linear stretch ring system space structure.So design can generate the plain flavanone alcohol compound (also being one type of novel flavanolignan chemical compound) of one type of novel wooden that the new spatial structure is different from silibinin fully.In the hope of finding the activity and even the lead compound of unusual inhibition hepatitis B HBsAg or inhibition HBV dna replication dna.

Description of drawings the structural difference of angle type flavanolignan and silibinin class linear stretch flavanolignan in the design concept of the present invention.

(left side is state-run, Liu Shuling, Xu Guili for the state-run grade in a left side; World Chinese digests magazine; 2006 14 13 phases of volume, the 1241-1246 page or leaf) summarized the active progress of the external anti-HBV of medicinal plants composition over nearly 20 years, the multiple natural product of touching upon in the literary composition; Do not report that wherein any flavanolignan compounds has the active relative recording of anti-HBV; Especially silibinin class natural product and derivant thereof, domestic almost nobody relates to, and only by inventor team it has been carried out structure of modification and the anti-HBV activity research that forefathers do not add attention.One of our purpose is: flavanolignan's lead compound of hoping to find to reduce HBsAg or inhibition HBV dna replication dna; Thereby it is developed further into to have can remove HBsAg or suppress the novelty medicine that HBVDNA duplicates treatment CHB, accomplishes the present invention in view of the above.

Summary of the invention

The purpose of this invention is to provide the angle type flavanolignan shown in the formula (1) or its officinal salt and be used for the new purposes that preparation reduces hepatitis B HBsAg, inhibition HBV dna replication dna, treatment hepatitis B medicine.

The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.

The present invention also provides the method for the flavanonol lignin chemical compound of B epidioxy six rings shown in a kind of preparation formula (1); It is characterized in that: (±)-2-(2; The 3-dihydroxy phenyl)-2,3-dihydro-3,5; 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone and carries out coupling reaction to the hydroxyl cinnamyl alcohol and gets under silver catalyst;

Wherein, silver salt can be Disilver carbonate or silver nitrate etc., has reported the example that uses Disilver carbonate among the present invention; Anhydrous solvent can make anhydrous aprotic solvent or their mixture such as anhydrous benzene, anhydrous propanone, anhydrous tetrahydro furan, reported the mixed solvent of anhydrous benzene, anhydrous propanone among the present invention.

Another object of the present invention provides a kind of pharmaceutical composition that is used to reduce hepatitis B HBsAg or suppresses HBV dna replication dna, treatment hepatitis B, it is characterized by the perhaps mixture formed of its officinal salt and pharmaceutically acceptable auxiliaries of the formula as active component (1) chemical compound of treat effective dose by containing.Its pharmaceutical dosage form can be that tablet, capsule, injection, aerosol, suppository, membrane, drop pill, paster agent, subcutaneous planting bury agent, externally-applied liniment, oral liquid or ointment, can also adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.

B ring 2 of the present invention; The angle type flavanolignan chemical compound (1) of 3 dioxane couplings is compared with natural flavone Lignanoids compounds silibinin; Characteristic with differentiation on many structures and the physico-chemical property comprises that speciality such as its hydrophobicity, armaticity, Gibbs free energy, hydrogen bond receptor, electrical, intermolecular Van der Waals force and 3D conformation, direction of extension, molecule center of gravity, electrical distribution center all have obviously different with silibinin; And chemical compound (1) molecular weight ratio silibinin has reduced 30 mass units.The ligand-receptor that above-mentioned characteristic has all determined the three-dimensional conformation of chemical compound shown in the formula (1) to combine with the 3d space structure of HBsAg or HBV DNA combines complex form and combination all possibly produce bigger difference; Its binding site and binding pattern, its combination free energy etc. all can produce bigger change, thereby possibly suppress HBsAg or suppress aspect the HBV dna replication dna beyond thought effect arranged.

We have tested the growth inhibited effect of this chemical compound to the HepG2.2.15 cell, have tested its HBsAg to HepG2.2.15 emiocytosis simultaneously and have reached the inhibition of HBV dna replication dna active.Result of the test is found: the synthetic angle type lignin flavanone alcohol compound that obtains is (±)-2-[2 among the present invention; 3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1; 4 benzodioxanes-5]-2; 3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone has certain inhibition activity to the hepatitis B HBsAg of HepG2.2.15 emiocytosis; It is removed HBsAg intensity and under low concentration (20 mcg/ml), just is higher than 5.5 times of positive controls (alpha-interferons of 10000 units per ml), and is farther far above another positive control one line medication lamivudine; Simultaneously, this chemical compound demonstrates the suppression ratio greater than 45% to HBV DNA when 20 mcg/ml concentration.Explain all that below formula (1) chemical compound has beyond thought anti-HBV effect, thereby can expect that it can be used as the active lead compound continuation exploitation of removing hepatitis B HBsAg or suppressing HBV dna replication dna, treatment hepatitis B.

Through the detailed Literature Consult of the inventor, up to the present, still there is not report about this compounds for treating hepatitis B virus infection property disease and preparation anti-hepatic-B virus medicine.Flavanolignan's formula (1) chemical compound is imitated inhibition by force for HBsAg and HBV DNA and is all belonged to beyond thought discovery, and definite originality is arranged.In sum; Uniqueness on this flavanolignan's existing structure of our de novo synthesis; The novelty that the research of antivirus action aspect is arranged again; And in anti-hepatitis B activity test, found to be expected to the activity of uncommon inhibition HBsAg become and to reduce HBsAg or suppress the HBV dna replication dna and the lead compound of treatment CHB.

Usefulness of the present invention is: the angle type flavanolignan shown in the discoverable type (1) has the effect that reduces hepatitis B HBsAg, suppresses the HBV dna replication dna first; And the patent medicine potentiality aspect the control hepatitis B virus; The original new drug that becomes treatment hepatitis B virus original new drug, exploitation reduction hbs antigen HBsAg or inhibition HBV dna replication dna for exploitation provides new material base, has potential huge social benefit and economic benefit.The present invention's characteristics again is: the present invention's synthetic starting material convenient sources, and synthetic convenient.Its preparation method is simple, and raw material sources conveniently are easy to get, and cost is low, pollutes for a short time, is beneficial to the large-scale production under the energy-saving and emission-reduction condition, and industrialization prospect is very clear and definite.

The specific embodiment

The inventor is simply synthetic through multistep; And obtain this through the chromatography means and can imitate the secretion that suppresses hepatitis B HBsAg by force and can effectively suppress the active flavanolignan's class reactive compound of HBV dna replication dna again, derive its chemical constitution through integration analysis such as mass spectrum and NMR spectrums again.The inventor finds; Formula (1) chemical compound does not have obvious inhibitory action to the growth of HepG2.2.15 cell; And secretion and the duplicating of HBV DNA of the hepatitis B HBsAg of HepG2.2.15 emiocytosis had significant inhibitory effect, point out this chemical compound to have drug safety, the strong effect reduced HBsAg, and suppressed HBV dna replication dna characteristics of high efficiency.Therefore, according to the inventor's research, the inventor design and synthetic formula (1) shown in the angle type flavanolignan chemical compound medicine that can be used to prepare treatment hepatitis B virus infection property disease be used to treat hepatitis B virus infection property disease.

In order to understand essence of the present invention better; Use the preparation of formula (1) chemical compound below respectively and, its new purposes in pharmaceutical field is described to the HepG2.2.15 cell growth inhibition and to the result of the inhibitory action test of the HBsAg of HepG2.2.15 emiocytosis and HBV dna replication dna.Embodiment has provided partial synthesis, the structure of formula (1) chemical compound and has identified and activity data that wherein OMe is meant that methoxyl group, OMOM are meant the methoxy methoxy base.Mandatory declaration, embodiments of the invention are to be used to explain the present invention rather than limitation of the present invention, and the simple modifications that essence according to the present invention is carried out the present invention all belongs to the present invention and requires the scope protected.

Embodiment 1:Formula (1) (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5, the preparation of 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone

Instrument and reagent:

Ultraviolet spectra is measured with Shimadzu UV-240 ultraviolet spectrophotometer; Proton nmr spectra ¹H-NMR measures (tetramethylsilane ether TMS is interior mark) by INOVA type NMR spectrometer with superconducting magnet (VARIAN INOVA-400MHz); Electrospray Mass Spectrometry ESI-MS is measured by BrukerEsquire 3000+ mass spectrograph, and column chromatography is produced by Haiyang Chemical Plant, Qingdao with silica GF254 (10-40 order) with silica gel (100-200,200-300 and 300-400 order) and thin layer chromatography; Agents useful for same is analytical pure, and wherein the petroleum ether boiling range is 60-90 ℃; Thin layer preparative chromatography (PTLC) is with the aluminium foil silica gel plate of Merck company; Column chromatography adopts Sweden Amersham Pharmacia Biotech AB Company products with polydextran gel SephadexLH-20; Reverse phase silica gel RP-18 adopts the Chromatorex product of Japanese Fuji Silysia Chemical company; MCI is a Mitsubishi chemical company product, and thin plate (TLC) detects the uviol lamp with 254nm and 365nm; Developer is with iodine vapor, 10% sulphuric acid-ethanol and phosphorus molybdenum acid solution.

1.1 the preparation of starting material A:

starting material A

2,3-4-dihydroxy benzaldehyde 4.8 grams are dissolved in 30 milliliters of acetone, stir to add potassium carbonate 17.5 grams after 10 minutes, drip 6 milliliters of chloromethyl ethers again, and reflux 1 hour is filtered.Filtrating concentrating obtains yellow oil 7.0 grams.

1.2 the preparation of starting material B:

starting material B

40 milliliters of DMF solution ice-water baths cooling with the sodium hydride of 2.6 grams; Dropwise 5 .6 gram 2,4 under the nitrogen protection state, 60 milliliters of benzene of 6-trihydroxy-acetophenone and the mixed solution of 7.0 milliliters of DMF; The ice bath cooling drips 9.0 milliliters of chloromethyl ether solution down, stirs 24 hours under the room temperature.In 100 milliliter of 10% sodium hydrate aqueous solution of impouring, ether extraction 3 times, each 50 milliliters; The saturated sodium bicarbonate washing, anhydrous sodium sulfate drying filters; Concentrate 40 gram 200-300 order silica gel column chromatographies, petroleum ether/ethyl acetate (4: 1) eluting; Obtain 7.0 gram starting material B (2,4,6-trimethoxy methoxyacetophenone).Yellow oil; R _f(petroleum ether/ethyl acetate=3/1): 0.30; Proton nmr spectra (400MHz, deuterochloroform): δ 2.52 (unimodal, 3H, CH ₃), 3.50 (unimodal, 9H, OCH ₃), 5.17 (unimodal, 6H, OCH ₂O), 6.52 (unimodal, 2H, H-3,5).

1.3 the preparation of intermediate chalcone derivative:

intermediate chalcone derivative

2.8 the gram potassium hydroxide dissolves in 30 ml methanol, stirring drips down 10 milliliters of the mixing methanol solutions of 1.4 gram starting material A and 1.3 gram starting material B, stirs 8 hours under the room temperature; Remove solvent under reduced pressure, in residue, add 20 ml waters, ethyl acetate extraction (3 times; Each 20 milliliters), merge organic layer, remove solvent under reduced pressure after; Residue is through 30 gram 200-300 order silica gel column chromatographies, and petroleum ether/ethyl acetate (3: 1) eluting obtains 1.76 gram intermediate chalcone derivative.Yellow oil; R _f(petroleum ether/ethyl acetate=2/1): 0.38; UV: (methanol) λ max:209,300nm.Be used for next step reaction.

1.4 the preparation of intermediate epoxy chalcone derivative:

intermediate epoxy chalcone derivative

1.4 gram intermediate chalcone derivative is dissolved in 25 ml methanol, adds 1.6 milliliters of 2N potassium hydroxide aqueous solutions, adds 1.6 milliliter of 30% hydrogen peroxide solution again, stirring at room 2 hours.Add 25 ml waters, concentrating under reduced pressure is with ethyl acetate extraction (3 times; Each 20 milliliters), merge organic layer, anhydrous sodium sulfate drying; Filter, remove solvent under reduced pressure after, residue is through 20 grams, 200～300 order silica gel column chromatographies; Petroleum ether/ethyl acetate (3: 1) eluting obtains 1.1 gram intermediate epoxy chalcone derivative.Yellow oil; R _f(petroleum ether/ethyl acetate=2/1): 0.33; UV: (methanol) λ max:210,285nm.Be used for next step reaction.

1.5 important intermediate (±)-2-(2, the 3-dihydroxy phenyl)-2,3-dihydro-3,5, the preparation of 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone:

1.0 gram intermediate epoxy chalcone derivative is dissolved in 15 ml methanol, adds under the stirring in the 10 ml methanol solution that are dissolved with 1.5 milliliters of concentrated hydrochloric acid, heats up 60 ℃ and reacts half an hour, removes heating; Remove solvent after the cooling under reduced pressure, in residue, add 50 ml waters, with ethyl acetate extraction (3 times, each 20 milliliters); Merge organic layer, saturated common salt washing 2 times, anhydrous sodium sulfate drying filters; After removing solvent under reduced pressure, residue is through 20 gram 200-300 order silica gel column chromatographies, and petroleum ether/ethyl acetate (3: 1) eluting obtains 97 milligrams (±)-2-(2; The 3-dihydroxy phenyl)-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.Yellow oil: R _f(chloroform/methanol=3/1): 0.23; Electrospray Mass Spectrometry ESI-MS:m/z 303 [M-H] ⁺

1.6 chemical compound (1) i.e. is (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5, the preparation of 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone

In exsiccant reaction bulb, drop into Disilver carbonate 0.22 gram; Add 20 milliliters of anhydrous benzene and 5 milliliters of anhydrous propanones, drip 90 milligrams (±)-2-(2, the 3-dihydroxy phenyl)-2 under the room temperature; 3-dihydro-3; 5,3 milliliters of the anhydrous propanone solution of 5 milliliters and the 76 milligrams 4-hydroxyl cinnamyl alcohol of anhydrous benzene solution of 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone, insulation reaction is 12 hours in the time of 55 ℃.Leave standstill after being chilled to room temperature, the elimination insoluble matter, the mother solution concentrating under reduced pressure gets yellow oil, and warp 20 gram 200-300 order silica gel are column chromatography repeatedly, and chloroform/methanol (10: 1) eluting through the gel filtration chromatography purification, finally obtains 18 milligrams of chemical compounds (1).R _f(chloroform/methanol/ethyl acetate/acetone/acetic acid=11: 0.5: 1: 1: 0.1): 0.15; Proton nmr spectra ¹H NMR (400MHz, deuterated acetone) δ: 3.95 (multiplet, 1H, H-23a), 4.23 (multiplet, 1H, H-23b), 4.87 (bimodal, J=8.0Hz, 1H; H-11), 4.99 (bimodal, J=11.2Hz, 1H, H-3), 5.50 (multiplet, 1H, H-12), 5.67 (bimodal, J=11.2Hz; 1H, H-2), 5.84 (bimodal, J=1.2Hz, 1H, H-6), 5.87 (bimodal, J=1.2Hz, 1H, H-8); (6.69 bimodal, J=8.0Hz, 2H, H-19,21), 6.98 (multiplet, 1H, H-15), 7.02 (bimodal, J=8.0Hz; 1H, H-14), 7.11 (bimodal, J=8.0Hz, 1H, H-16), 7.22 (bimodal, J=8.0Hz, 2H, H-18; 22), 10.20 (wide unimodal, 1H, OH-20), 10.56 (wide unimodal, 1H, OH-7), 12.19 (unimodal, 1H, 5-OH); Electrospray Mass Spectrometry ESI-MS m/z:451 [M-H] ⁺

Embodiment 2:Chemical compound (1) is to the inhibitory action of the hbs antigen (HBsAg) of HepG2.2.15 emiocytosis

2.1 cell culture:

In containing 10% inactivated fetal bovine serum, 100U/ ml penicillin and 100U/ milliliter streptomycin in the DMEM culture medium of 100 mcg/ml G418, are put 37 ℃, 5%CO with the HepG2.2.15 cell culture ₂, cultivate in the incubator of 100% relative humidity.

Measure the inhibitory action of formula (1) chemical compound 2.2 adopt mtt assay to the growth of HepG2.2.15 cell:

The take the logarithm HepG2.2.15 cell of trophophase becomes 1 * 10 with culture medium with cell dilution ⁵Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO ₂, cultivate the chemical compound (1) that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 1000 mcg/ml; 200 mcg/ml, 40 mcg/ml and 8 mcg/ml, every hole 200 microlitres; Each concentration is established three multiple holes, places 37 ℃, 5%CO ₂, cultivate in the incubator of 100% relative humidity, to cultivate after 72 hours, every hole adds MTT reagent 10 microlitres, continues to cultivate 4 hours, discards culture medium, and every hole adds the DMSO200 microlitre, with agitator vibration 20 minutes, under the 570nm wavelength, measures the OD value with ELIASA.With the culture hole that only adds culture medium is control wells.Suppression ratio (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.The experiment triplicate.

Measure the inhibitory action of chemical compound to hbs antigen (HBsAg): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution ⁵/ milliliter is inoculated in 96 porocyte culture plates, 100 milliliters in every hole, and at 37 ℃, 5%CO ₂, cultivate the sample that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 20 mcg/ml, 4 mcg/ml and 0.8 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO ₂, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.With HBsAg concentration in the ELISA kit measurement culture medium, represent with P/N; With the positive contrast 1 of lamivudine (3-TC) (annotate: the lamivudine test concentrations is 100 mcg/ml, 20 mcg/ml and 4 mcg/ml); With the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).

2.3 experimental result:

Experimental result is as shown in table 1, and formula (1) chemical compound has the effect of significant inhibition hbs antigen (HBsAg).Its growth to the HepG2.2.15 cell does not have obvious inhibitory action, all is higher than lamivudine but the HBsAg of HepG2.2.15 emiocytosis was suppressed activity under the high, medium and low dosage the 8th day the time.

Table 1. sample is to the excretory hbs antigen suppression ratio of HepG2.2.15 (%)

This embodiment presentation of results: the flavanolignan's chemical compound (±) shown in the formula (1)-2-[2; 3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1; 4 benzodioxanes-5]-2; 3-dihydro-3; 5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone has significant inhibitory effect to the hbs antigen (HBsAg) of HepG2.2.15 emiocytosis, and it suppresses the active lamivudine that surpasses far away to HBsAg when extremely low concentration (4 mcg/ml); And when low concentration (20 mcg/ml), HBsAg is suppressed activity nearly is the octuple of positive control medicine 1 lamivudine (100 mcg/ml), suppresses active high 5.5 times than corresponding HBsAg under the highest test concentrations 10000 units per ml concentration of alpha-interferon; Under this low concentration (20 mcg/ml), have 88.6% HBsAg activity to be suppressed by formula (1) chemical compound, there is the activity of very strong inhibition viral secretory hepatitis B surface antigen in visible this flavanolignan.

It is clinically near the state of curing that HBsAg removes, and for hepatitis B patient, its HBsAg removes becomes very valuable CHB treatment terminal point.Thereby the flavanolignan's chemical compound shown in the formula (1) can be expected and developed into the medicine of removing hbs antigen, control Type B viral hepatitis symptom.

Embodiment 3:The inhibitory action that formula (1) chemical compound duplicates the hepatitis B virus DNA (HBV DNA) of HepG2.2.15 emiocytosis

3.1 cell culture:

Method is with embodiment 2.

Measure the inhibitory action of the flavanolignan's chemical compound shown in the formula (1) 3.2 adopt mtt assay to the growth of HepG2.2.15 cell:

Method is with embodiment 2.

3.3 the inhibitory action that the flavanolignan's chemical compound shown in the mensuration formula (1) duplicates hepatitis B virus DNA (HBV DNA):

The take the logarithm HepG2.2.15 cell of trophophase becomes 1 * 10 with culture medium with cell dilution ⁵Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO ₂, cultivate the flavanolignan's chemical compound that adds after 24 hours with shown in the formula (1) of culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 20 mcg/ml; 4 mcg/ml and 0.8 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes; Place 37 ℃, 5%CO ₂, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.Measure HBVDNA concentration in the culture medium with HBV DNA quantitative PCR kit in the time of the 8th day.(annotate: the lamivudine test concentrations is 100 mcg/ml with the positive contrast 1 of lamivudine (3-TC); 20 mcg/ml and 4 mcg/ml); With the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).

3.4 experimental result:

Experimental result illustrates as shown in table 2, and flavanolignan's formula (1) chemical compound of de novo synthesis has the effect of the inhibition hepatitis B virus DNA replication of strong effect.

Table 2 sample in the time of the 8th day to the suppression ratio of the HBV dna replication dna of HepG2.2.15 cell

This embodiment presentation of results: the flavanolignan's chemical compound shown in the formula (1) has more intense inhibitory action to duplicating of hepatitis B virus DNA (HBV DNA), and it suppresses active to duplicating of HBV DNA and just surpasses 45% than low dosage (20 mcg/ml) time.And the positive control alpha-interferon only has 38.2% inhibition active to HBV DNA the 8th day test concentrations the highest when (10000 units per ml).Therefore this chemical compound belongs to significantly effectively non-nucleoside inhibition hepatitis B virus natural product, is worth further paying close attention to and further investigation, and can expects that further optimized development is to suppress the medicine of HBV dna replication dna.

When above-mentioned description elaboration was of the present invention, the purpose that embodiment is provided simultaneously was to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of in getting into claim of the present invention and its equivalent scope, practical application of the present invention comprises all general variations, cooperates, or improves.

Claims (1)

1. have the angle type flavanolignan of structure shown in the formula (1) or the purposes that its officinal salt is used for preparation treatment hepatitis B medicine;

The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.

2. have the angle type flavanolignan of structure shown in the formula (1) or the purposes that its officinal salt is used to prepare the medicine that reduces hepatitis B virus surface antigen HBsAg;

The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.

3. have the angle type flavanolignan of structure shown in the formula (1) or the purposes that its officinal salt is used to prepare the medicine that suppresses hepatitis B virus DNA HBV dna replication dna;

The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(4-hydroxy phenyl)-2-methylol-1,4 benzodioxane-5]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.

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