CN1245808A - Compound resisting hepatitis virus and its preparation method and its application in pharmaceutical technology - Google Patents

Abstract

The present invention relates to a compound for resisting hepatitis virus, its preparation method and application in pharmaceutic process. It is characterized by that said compound is beta-L-2',3'-didehydrodeoxy-furanadenosine (beta-L-D4A). Said invented compound possesses obvious inhibition action for HBV DNA duplication, has no biological toxic action, and its pharmaceutical application can effectively cure hepatitis B, and can prevent the production of primary carcinoma of liver, and can promote negative transformation of hepatitis B e antigen and hepatitis B surface antigen.

Description

Hepatitis virus resisting compound and preparation method thereof and the application in pharmacy

The present invention relates to treat the viral hepatitis chemicals.

Hepatitis B virus (Hepatitis B Virus; HBV) cause acute and chronic hepatitis.Chronic HBV infection can cause liver cirrhosis and primary hepatocellular carcinoma.Although hepatitis B virus vaccine comes out for many years, yet the whole world still has 300,000,000 populations to infect HBV, and wherein China accounts for 75%.Though it is clinical that Interferon, rabbit has been used for, only have 25% to 40% efficient.And 10% to 40% patient produces severe side effect.In recent years, many scholars were devoted to the research of nucleoside compound.Experimental result shows that some nucleoside compound is in external effect with inhibition HBV dna replication dna.These comprise Ganciclovir, Acyclovir, Dideoxy cytidine (DddC), Fialuridine (FIAU), 2 ' 3 '-dideoxy-3 ' thiacytidine (SddC) etc.Yet, Ganciclovir, the activity of Acyclovir and the anti-HBV of DddC is not remarkable, and DddC still has mitochondrial toxicity.Though FIAU has stronger anti-HBV activity, after the prolonged application,, can produce the delayed toxicity reaction owing to toxic action to Mitochondrial DNA, as bone marrow depression, peripheral neuropathy, in addition dead.Though SddC has the anti-HBV dna replication dna effect of very strong inside and outside, after the drug withdrawal, duplicating again of HBV recovers.Therefore, the development high reactivity, the anti-HBV medicine of no overt toxicity remains a kind of challenge that is difficult to tackle.

The object of the invention is to provide has obvious active compound of anti-HBV dna replication dna and its preparation method, and the technical scheme of the application of this compound in the production for treating medicine resisting viral hepatitis.

It is provided by the invention that to have the active compound of obvious anti-HBV dna replication dna be β-L-2 ', 3 '-two dehydrogenation deoxidation furans adenosines (β-L-D4A), its chemical structural formula is:

β-L-2 ' provided by the invention, 3 ' ,-two dehydrogenation deoxidation furans adenosines (β-L-D4A) produce by the following method:

A. be raw material with D-L-glutamic acid (D-glutamic acid), at room temperature, with Sodium Nitrite (NaNO ₂) diazotization, with borine (BH ₃) reduction, in imidazoles (lmidazole),, obtain product 1 with the TBDPSCI reaction, promptly contain the five-ring lactone of TBDPS;

B. under-78 ℃, after product 1 and pregnancy aldehyde silicon amine alkane (LiHMDS) effect, at room temperature, replace with chlorination tetramethyl silane (TMSCI), act on down at-78 ℃ with bromination benzaldehyde selenium (PhSeBr) again, obtain product 2, promptly have the stereoselective five-ring lactone that contains benzaldehyde selenium;

C. with product 2 and adipic acid (BIBAL-H), in toluene (toluene), under-78 ℃ of conditions, react, again under 0 ℃, with diacetyl oxide (AC ₂O) acetoxylation obtains product 3;

D. product 3 with tetrasilane-6-chloropurine (TMS-6-chloropurine) effect, obtains product 4 under-22 ℃;

E. with product 4 under 80 ℃, through ammonification, under 0 ℃, obtain product 5 after the oxidation, i.e. the adenine derivative;

F. product 5 is in tetrahydrofuran (THF) (THF), and under the room temperature, (TBAF) sloughs TBDPS with tetrabutylammonium fluoride, obtains target product 6, i.e. β-L-2 ', 3 '-two dehydrogenation deoxidation furans adenosines (β-L-D4A).

With β-L-2 ' provided by the invention, (β-L-D4A) make the hepatitis virus resisting medicament for the treatment of viral hepatitis can be used for the treatment of viral hepatitis to 3 ' ,-two dehydrogenation deoxidation furans adenosines.

β-L-2 ' of the present invention, 3 ' ,-two dehydrogenation deoxidation furans adenosines (positively effect of β-L-D4A) is:

Compound β-the L-2 ' that is provided, 3 ' ,-two dehydrogenation deoxidation furans adenosines (β-L-D4A) has the activity of remarkable anti-HBV dna replication dna, and the lifeless matter toxic action, and this positively effect detects by following screening system and confirmed:

1. main agents and material:

Proteinase K, tRNA, the RNA enzyme, HindIII, BamH1, foetal calf serum, WizardPlus Maxi-Preps DNA Purification System, QIAquick GelExtraction Kit, α- ³²P-dCTP, Nylon Membrane, Gene Amp PCRReagent Kit with Taq DNA Polymerase

2. cell cultures

This tests the hepatoma cell strain 2.2.15 of used clone with clone's people HBV gene transfection.Cell is cultivated with the MEME nutrient solution.Nutrient solution contains 10% the foetal calf serum and the kantlex of 100 mcg/ml.Cell is put 37 ℃, contains 5%CO ₂Incubator in cultivate.

3. the drug treating of cell:

2.2.15 cell is with 5 * 10 ⁴The density in/milliliter/hole is seeded in 12 well culture plates.Changed one time nutrient solution in per 3 days.When the 3rd time and the 4th are changed liquid, add the medicine (in the fresh MEME of 1ml) of serial doubling dilution concentration.Stop after 12 days cultivating, collect nutrient solution and cell and extract HBV DNA in extracellular and the cell respectively.

4.HBV the extraction of DNA

(1) extraction of extracellular HBV DNA

Virion in the nutrient solution spends the night with 4 ℃ of precipitations of polyethylene glycol (PEG), centrifugal 30 minutes of 3000g, and throw out digested 3 hours down for 50 ℃ to extract damping fluid, again with the phenol-chloroform extracting, washing with alcohol ,-20 ℃ of refrigerators are stored in the TE80 dissolving.

(2) extraction of HBV DNA in the cell

Cytolysis was hatched 4 hours in 50 ℃ of water-baths at the DNA extraction damping fluid, phenol-chloroform extracting, HindIII digestion ,-20 ℃ of freezer storages.

5.DNA the preparation of probe

(1) preparation of HBV dna probe

After containing the complete segmental plasmid PBR322 of HBV DNA and in intestinal bacteria Ecoli, increasing, cut,, purify with Prep-A-Genc DNA purification test kit again with electrophoretic separation with Bam H1 enzyme.

(2) preparation of Mitochondrial DNA probe:

The Mitochondrial DNA probe prepares with the PCR method.Used primer is:

Primer I: 5 ' CCA TCA TCC ACA ACC TTA 3 '

Primer I I:5 ' CAT TCG GGA GGA TCC TAT 3 '

6.Southern?Blot

The dna sample that extracts is stated from 0.8% sepharose, and in the TAE electrophoretic buffer, electrophoresis is 2 hours under the 180V condition.Through sex change, neutralization, commentaries on classics film, then with α- ³²The HBV dna probe of P-dCTP mark is hybridized under 65 ℃ of conditions and is spent the night.Radioautograph is done in the washing back in-80 ℃ of refrigerators.

7. the determination of activity of anti-HBV dna replication dna:

Earlier with DNA density scan instrument Personal Densitometer scanning quantitatively with the radioautograph result.

(1) the anti-HBV dna replication dna in extracellular: according to compare the ring-type of loosening (Relaxed Circular with non-drug treating; RC) the shared per-cent of the amount of DNA, it is ID that the dosage that every kind of medicine suppresses viral half is obtained in mapping ₅₀

(2) anti-HBV dna replication dna in the cell: according to comparing with non-drug treating, the ratio of free (Episomal) DNA and integration (Integrated) DNA is equivalent to the per-cent of check sample, and ID is obtained in mapping ₅₀

8. toxicity test:

(1) cytotoxicity: with the 2.2.15 cell with 10 ⁴The density in/2ml/ hole is seeded in 24 well culture plates.After 24 hours, with the drug treating of different concns.After 72 hours, with the fixing dyeing of 0.5%Methylene Blue 30 minutes, the optical density(OD) of reading each sample at 595nm was dissolved with 1% Sarkosyl in the washing back.According to comparing with non-drug treating contrast, calculate the cytostatic percentage of each concentration medicine, the concentration that each medicine cell growth inhibiting 50% is obtained in mapping is TC ₅₀

(2) mitochondrial toxicity: on the active basis of detecting of anti-HBV dna replication dna, with the NaOH of 0.4M, washing is 30 minutes in 42 ℃ of water-baths, to remove the HBV dna probe of mark in cell.Same film and α- ³²The Mitochondrial DNA probe hybridization of P-dCTP mark.The radioautograph result does the DNA quantitative analysis.According to comparing with non-drug treating check sample, the percentage ratio that the ratio of the amount of Mitochondrial DNA and integration HBV DNA is equivalent to contrast draws the percentage that each concentration suppresses Mitochondrial DNA, and the dosage that various medicines suppress Mitochondrial DNAs 50% is obtained in mapping.

9. result

(1) The compounds of this invention has the obvious suppression effect to the HBV dna replication dna

The extracellular is anti-HBV DNA detection result show, from 0.016 to 2 μ M concentration demonstrates tangible dose-effect relationship.ID ₅₀Be 0.2 μ M.Anti-HBV DNA detection result further confirms extracellular detected result in the cell, sees the following form.

Different concns β-L-D4A is to the restraining effect of HBV DNA

Drug level is untreated

Contrast

2 μ M, 0.4 μ M, 0.08 μ M, 0.016 μ MHBV DNA 42.4 505.8 1990.3 3079.4 2996.2 suppresses viral percentage 98.6 83.1 33.6 00 (%) (2) toxic action

The cytotoxicity detected result shows that the growth of The compounds of this invention pair cell does not have overt toxicity effect, TC ₅₀Be 200 μ M.According to therapeutic index TI=TC ₅₀/ ID ₆₀, can obtain its TI is 1000.

Toxicity detected result to Mitochondrial DNA shows that Mitochondrial DNA does not have the overt toxicity effect in the The compounds of this invention pair cell.

Embodiment:

β-L-2 ' provided by the invention, the preparation method of 3 ' ,-two dehydrogenation deoxidation furans adenosines is:

At first, (D-glutamic acid) is raw material with D-L-glutamic acid, at room temperature, and with Sodium Nitrite (NaNO ₂) diazotization, with borine (BH ₃) reduction, in imidazoles (Imidazole),, obtain product 1 with the TBDPSCI reaction, the five-ring lactone that promptly contains TBDPS, under-78 ℃, after product 1 acts on pregnancy aldehyde silicon amine alkane (LiHMDS) again, at room temperature, replace with chlorination tetramethyl silane (TMSCI), act on down at-78 ℃ with bromination benzaldehyde selenium (PhSeBr) again, obtain product 2, promptly have the stereoselective five-ring lactone that contains benzaldehyde selenium.The latter and adipic acid (BIBAL-H), in toluene (toluene) ,-78 ℃ of reactions down are again under 0 ℃, with diacetyl oxide (AC ₂O) acetoxylation obtains product 3, and the latter with tetrasilane-6-chloropurine (TMS-6-chloropurine) effect, obtains product 4 under-22 ℃.Product 4 is under 80 ℃, through ammonification, under 0 ℃, obtain product 5 after the oxidation, i.e. the adenine derivative, the latter is in tetrahydrofuran (THF) (THF), under the room temperature, (TBAF) sloughs TBDPS with tetrabutylammonium fluoride, obtains target product 6, be β-L-2 ', and 3 '-two dehydrogenation deoxidation furans adenosines (β-L-D4A).

β-L-2 provided by the invention, 3 ,-two dehydrogenation deoxidation furans adenosines can suppress duplicating of hepatitis B virus effectively, and it is medicinal, and the generation of efficiency ground treatment hepatitis B and prevention primary hepatocarcinoma impels hepatitis B virus e antigen and hepatitis B surface antigen the moon to change.β-L-2 of the present invention, 3,-two dehydrogenation deoxidation furans adenosines are used for the production for treating hepatitis B medicament, can be made into oral tablet, capsule and intravenous injection injection etc. and reaching acceptable multiple formulation in the treatment in the pharmacy, one of feasible program of its application is: make tablet, every contains β-L-2, and 3,25 milligrams in-two dehydrogenation deoxidation furans, consumption are 100-300 milligram every day; Two of scheme is: make injection: every contains β-L-2, and 100 milligrams in 3 ,-two dehydrogenation deoxidation furans, consumption are 100-500 milligram every day.

Claims (3)

1, hepatitis virus resisting compound is characterized in that this compound is β-L-2 ', 3 '-two dehydrogenation deoxidation furans adenosines (β-L-D4A), its chemical structural formula is:

2, hepatitis virus resisting compound according to claim 1 is characterized in that it produces with following method:

A. be raw material with D-L-glutamic acid (D-glutamic acid), at room temperature, with Sodium Nitrite (NaNO ₂) diazotization, with borine (BH ₃) reduction, in imidazoles (Imidazole),, obtain product 1 with the TBDPSCI reaction, promptly contain the five-ring lactone of TBDPS;

B. under-78 ℃, after product 1 and pregnancy aldehyde silicon amine alkane (LiHMDS) effect, at room temperature, replace with chlorination tetramethyl silane (TMSCI), act on down at-78 ℃ with bromination benzaldehyde selenium (PhSeBr) again, obtain product 2, promptly have the stereoselective five-ring lactone that contains benzaldehyde selenium;

C. with product 2 and adipic acid (BIBAL-H), in toluene (toluene), under-78 ℃ of conditions, react, again under 0 ℃, with diacetyl oxide (AC ₂O) acetoxylation obtains product 3;

D. product 3 with tetrasilane-6-chloropurine (TMS-6-chloropurine) effect, obtains product 4 under-22 ℃;

E. with product 4 under 80 ℃, through ammonification, under 0 ℃, obtain product 5 after the oxidation, i.e. the adenine derivative;

F. product 5 is in tetrahydrofuran (THF) (THF), and under the room temperature, (TBAF) sloughs TBDPS with tetrabutylammonium fluoride, obtains target product 6, i.e. β-L-2 ', 3 '-two dehydrogenation deoxidation furans adenosines (β-L-D4A).

3,, it is characterized in that the application of this compound in preparation treatment viral hepatitis medicine according to claim 1,2 described hepatitis virus resisting compounds.