CN103739575B - 14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application - Google Patents
14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application Download PDFInfo
- Publication number
- CN103739575B CN103739575B CN201410010214.7A CN201410010214A CN103739575B CN 103739575 B CN103739575 B CN 103739575B CN 201410010214 A CN201410010214 A CN 201410010214A CN 103739575 B CN103739575 B CN 103739575B
- Authority
- CN
- China
- Prior art keywords
- andrographolide
- deoxidation
- bis
- acid
- yang
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
Abstract
The invention belongs to technical field of pharmaceuticals, be specifically related to the 14-deoxidation-11,12-bis-andrographolide (1-4) shown in general formula (I), its pharmaceutical composition, its preparation method, and this analog derivative and pharmaceutical composition thereof are preparing the application in anti-hepatitis B medicine.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, specifically, relate to 14-deoxidation-11,12-bis-andrographolide (1-4), with the pharmaceutical composition that it is activeconstituents, its preparation method, and this compounds and pharmaceutical composition thereof are preparing the application in anti-hepatitis B medicine.
Background technology
Hepatitis B be that hepatitis B virus (Hepatitis B virus, HBV) causes, cause a kind of communicable disease of multiple organ injury based on hepatic disease, about there are 3.6 hundred million chronic infection in the whole world, every year because HBV infection causes about 600,000 people dead.At present, hepatitis B has become one of nine large diseases affecting human longevity.Show according to hepatitis B seroepidemiology data in 2006, China's hepatitis B virus surface antigen carrying rate 7.18%, i.e. hepatitis B virus surface antigen carrier 0.93 hundred million.The Anti-HBV drugs of current Clinical practice is mainly hepatitis B vaccine, immunomodulator, nucleoside medicine.
Hepatitis B vaccine, as a kind of special medicine, be the most important means of prevention hepatitis B infection, but vaccine is to the infected is invalid.Be used for the treatment of chronic viral hepatitis B medicine clinically and mainly contain immunomodulator and nucleoside medicine.Immunomodulator can cause the untoward reactions such as influenza-like symptom, depression, Neutrophilic granulocytopenia, thrombopenia.Nucleoside medicine mainly contains lamivudine (lamivudine, 3-TC), adefovir ester (adefovir dipivoxil, ADV), Entecavir (entecavir, ETV), Telbivudine (telbivudine, LdT), tenofovir (tenofovir, TDF) etc.It is strong that nucleoside medicine has Anti-HBV activity effect, the advantages such as patient tolerability is good, but easily recur after drug withdrawal, causes that sb.'s illness took a turn for the worse, and liver even occurs not normal, long-term treatment easily produces resistance.Therefore, the non-nucleoside medicine of searching novel structure, new mechanism of action becomes study hotspot.14-deoxidation-11,12-bis-dehydrogenation rographolide is Acanthaceae punching Nelumbo (Andrographis) plant Herba Andrographis (Andrographis panicuata) main component, have anti-inflammatory, antibacterial, antiviral, protect the liver, effect [new medical college in Jiangsu such as analgesia, Chinese medicine voluminous dictionary, Shanghai science tech publishing house, 1995,1728-1729].The derivative obtained is modified to it: the amber injection for curing that 14-deoxidation-11,12-bis-dehydrogenation andrographolide succinic acid half-ester natrium potassium salt (a) is prepared as is for upper respiratory tract infection and virus pneumonia; 19-O-ethanoyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (b), 19-O-succinic acid list acyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (c) has anti-inflammatory activity.C-11, C-12 double bond additive derivative has HIV (human immunodeficiency virus)-resistant activity [yellow civilian imperial CN101829110A].Report before this, to 14-deoxidation-11,12-bis-dehydrogenation rographolide C-3, C-15, C-19 modify and obtain derivative (general formula d), and have restraining effect to HBsAg, HBV DNA replication dna, selectivity index (SI) is less than 8; It is active that dhbv dna model measurement shows that this derivative has anti-dhbv dna, but its structure completely different from derivative required by the application [Dai Guifu, WO2013/004171A1].
At present, the type does not have 14-deoxidation-11,12-bis-andrographolide (I) provided by the invention and is applied in the report in preparation or treatment hepatitis B medicament as the pharmaceutical composition of effective constituent.
Summary of the invention
The present invention aims to provide a kind of 14-deoxidation-11 with pharmaceutical use newly, 12-bis-andrographolide (I), and the compound 14-deoxidation-11 containing treatment hepatitis B significant quantity, 12-bis-andrographolide (I) and pharmaceutical carrier or vehicle be used for the treatment of hepatitis B pharmaceutical composition, its preparation method, this compound or its pharmaceutical composition are preparing the application in anti-hepatitis B medicine.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Shown in lamps structure formula (I) 14-deoxidation-11,12-bis-andrographolide (1-4)
Pharmaceutical composition, the 14-deoxidation-11,12-bis-andrographolide (1-4) containing general formula according to claim 1 (I) and pharmaceutically acceptable carrier and/or vehicle.
The present invention's preparation is containing 14-deoxidation-11, the method of 12-bis-andrographolide (1-4) and medicine thereof is: to 14-deoxidation-11, 12-bis-dehydrogenation rographolide, carboxylic acid (TRANSCINNAMIC ACID, 3, 4, 5-trimethoxy-TRANSCINNAMIC ACID, 2-furancarboxylic acid, 2-thiophenic acid), the N of 4-dimethylamino pyridine, N is added in dinethylformamide solution, N-dicyclohexylcarbodiimide, stirring at room temperature 10 hours, the gained reaction solution diluted ethyl acetate of 3 times of volumes, use dilute hydrochloric acid successively, saturated sodium bicarbonate aqueous solution and saturated common salt water washing, after anhydrous sodium sulfate drying, decompression and solvent recovery obtains crude product, crude product, through silica gel column chromatography, obtains 14-deoxidation-11,12-bis-andrographolide (1-4) with ethyl acetate-light petrol wash-out.
The invention provides the application of 14-deoxidation-11,12-bis-andrographolide (I) in preparation treatment hepatitis B medicament.
The invention provides the application of pharmaceutical composition in preparation treatment hepatitis B medicament containing 14-deoxidation-11,12-bis-andrographolide (I).
Anti-HBV activity based on the present inventor screens, and finds that 14-deoxidation-11,12-bis-andrographolide has Anti-HBV activity effect.14-deoxidation-11,12-bis-andrographolide is the 14-deoxidation-11 to exist in plant, 12-bis-dehydrogenation rographolide (14-deoxy-11,12-didehydroandrographolide) is raw material, adopts 4 compounds that chemical synthesis process obtains.Report before the application's derivative required for protection is different from; by to 14-deoxidation-11; chemically modified is carried out in the C-19 position of 12-bis-dehydrogenation rographolide; obtain 4 derivatives and there is better Anti-HBV effect; comparatively high inhibition effect is had especially to HBV DNA replication dna; all have higher selectivity index, especially compound 2 selectivity index is greater than 165.1, has better security.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.This pharmaceutical composition contains 0.1 – 99%, and be preferably the compounds of this invention of 0.5 – 90%, all the other are acceptable on pharmacology, pharmaceutically acceptable carrier of and inertia nontoxic to humans and animals and/or vehicle.
Described pharmaceutical carrier or vehicle are one or more solids, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention can through injection (quiet note, intramuscular injection) and oral two kinds of form administrations.
Embodiment
In order to understand essentiality content of the present invention better, preparation method and the pharmacological action result of the compounds of this invention 14-deoxidation-11,12-bis-andrographolide (I) being described with embodiments of the invention below, but not limiting the present invention with this.
Compound prepares embodiment 1
The preparation of raw material 14-deoxidation-11,12-bis-dehydrogenation rographolide (14-deoxy-11,12-dehydroandrographolide):
10 kilograms of Herba Andrographis plant herbs are pulverized, with 30 kilogram of 90% extraction using alcohol three times, each 3 hours, merge ethanol extract, be concentrated into volume 10 liters, concentrated solution is extracted with ethyl acetate three times, each 18 liters, combined ethyl acetate extraction liquid, reclaim under reduced pressure ethyl acetate, obtains 336 grams of ethyl acetate extraction part.
By 336 grams of ethyl acetate portion with acetone solution, be adsorbed on 600 grams of silica gel, room temperature evaporates into dry, crosses 60-100 mesh sieve, for subsequent use.Get 3000 grams of column chromatography silica gel (200-300 orders, Qingdao Mei Gao company), wet method is filled in glass column with ethyl acetate-light petrol (40:60) mixed solvent that (post is directly through 18cm, filling gel height 28cm), after loading, with ethyl acetate-light petrol (40:60-60:40, volume ratio) mixed solvent wash-out, flow velocity 100mL/min, every 3 are upgraded to first-class part, (Qingdao Mei Gao company is checked through TLC thin plate, developping agent: ethyl acetate-light petrol, 60:40), will containing 14-deoxidation-11, stream part of 12-bis-dehydrogenation rographolide merges, recycling design obtains 14-deoxidation-11, 12-bis-dehydrogenation rographolide crude product 70 grams.
By 14-deoxidation-11,12-bis-dehydrogenation rographolide crude product 70 grams, with 80% dissolve with ethanol, adds 600 milligrams of gacs, refluxes one hour, leach gac, reclaim solution and obtain 62 grams of crude products, then dissolve by 60 ml methanol, crystallization under room temperature, filter out crystal, 50 ml methanol dissolve rear recrystallization, obtain 14-deoxidation-11,12-bis-dehydrogenation rographolide 45 grams.Through spectral data (
1h NMR,
13c NMR, MS) qualification, and and standard control, confirm that gained is 14-deoxidation-11,12-bis-dehydrogenation rographolide, the raw material as following preparation 14-deoxidation-11,12-bis-andrographolide uses.
The preparation of 19-O-cinnamyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (compound 1):
0.57mmol(200mg is added in 10mL round-bottomed flask) 14-deoxidation-11,12-bis-dehydrogenation rographolide, 0.28mmol(14mg) 4-dimethylamino pyridine (DMAP) and 0.7mmol(103mg) TRANSCINNAMIC ACID, then 5 milliliters of anhydrous N are added, dinethylformamide (DMF), after stirring and dissolving, 0.7mmol(144mg is added again under condition of ice bath) N, N-dicyclohexylcarbodiimide (DCC) stirring, temperature of reaction rises to room temperature.TLC detects 14-deoxidation-11; after 12-bis-dehydrogenation rographolide has reacted; reaction solution adds 30mL acetic acid ethyl dissolution; use dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, saturated common salt solution washing three times successively; after anhydrous sodium sulfate drying, decompression and solvent recovery obtains crude product; through silica gel column chromatography, obtain compound 19-O-cinnamyl-14-deoxidation-11,12-bis-dehydrogenation rographolide 170 milligrams with ethyl acetate-light petrol (30:70) wash-out.
HRESIMS above measures at LCMS-IT-TOF mass spectrograph (Shimadzu, Kyoto, Japan), nuclear magnetic resonance spectrum (
1h and
13c NMR) at Bruker AM400(
1h/
13c, 400M Hz/100M Hz) NMR spectrometer with superconducting magnet (Bruker, Bremerhaven, Germany) mensuration, with TMS(tetramethylsilane) be interior mark.Column chromatography silica gel (200 ~ 300 order) and thin-layer chromatography silica GF254 are Qingdao Makall Group Co., Ltd. and produce.Reaction reagent is purchased from Alfa Aesar, lark prestige and Acros company.
19-O-cinnamyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (1) structured data:
Molecular formula: C
29h
34o
5
Molecular weight: 462
Proterties: white amorphous powder
Spectrum data:
1h NMR (CDCl
3, 400MHz): δ 0.76 (3H, s, H-20), 1.08 (3H, s, H-18), 3.21 (1H, dd, J=10.4, 5.1Hz, H-3), 4.22 (1H, d, J=11.8Hz, H-19a), 4.27 (1H, d, J=11.8Hz, H-19b), 4.40 (1H, brs, H-17a), 4.65 (1H, brs, H-17b), 4.72 (2H, s, H-15), 6.00 (1H, d, J=15.8Hz, H-12), 6.31 (1H, d, J=15.7Hz, H-2), 6.74 (1H, dd, J=15.8, 10.1Hz, H-11), 7.14 (1H, s, H-14), 7.41 (2H, d, J=7.1Hz, H-5and H-9), 7.53 (1H, d, J=15.7Hz, H-3).
13c NMR (CDCl
3, 100MHz): δ 14.9 (C-20), 22.3 (C-18), 23.6 (C-6), 27.0 (C-2), 36.4 (C-7), 38.3 (C-1), 38.5 (C-10), 42.3 (C-4), 54.3 (C-5), 61.4 (C-9), 65.2 (C-19), 69.8 (C-15), 78.3 (C-3), 108.6 (C-17), 117.3 (C-2), 120.8 (C-12), 127.8 (2C, C-6, 8), 128.6 (2C, C-5, 9), 128.7 (C-13), 130.2 (C-7), 133.9 (C-4), 135.4 (C-11), 143.6 (C-3), 144.9 (C-14), 147.9 (C-8), 167.2 (C-1), 172.8 (C-16) .HRESIMS: calculated value C
29h
35o
5[M+H]
+463.2479, experimental value 463.2503.
Compound prepares embodiment 2-4
In 10mL round-bottomed flask, 0.4mmol(200mg is added according to embodiment 1) 14-deoxidation-11,12-bis-dehydrogenation rographolide, 0.2mmol(24mg) 4-dimethylamino pyridine (DMAP) and 0.7mmol respective acids, then 5 milliliters of anhydrous N are added, dinethylformamide, after stirring and dissolving, 0.7mmol(144mg is added again under condition of ice bath) N, N-dicyclohexylcarbodiimide (DCC) stirring, temperature of reaction rises to room temperature.TLC detects 14-deoxidation-11, after 12-bis-dehydrogenation rographolide has reacted, reacting liquid filtering, use dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, saturated common salt solution washing three times successively, after anhydrous sodium sulfate drying, decompression and solvent recovery obtains crude product, through silica gel column chromatography, obtain compound with ethyl acetate-light petrol (30:70 – 40:60) wash-out, prepare following structure 14-deoxidation-11,12-bis-andrographolide (2-4):
Compound 2,3,4 structure determination data:
19-O-(3,4,5-trimethoxy) cinnamyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (2)
Molecular formula: C
32h
40o
8
Molecular weight: 552
Proterties: white amorphous powder
Spectrum data:
1h NMR (CDCl
3, 400MHz): δ 0.76 (3H, s, H-20), 1.08 (3H, s, H-18), 3.79 (3H, s, H-OMe), 3.81 (6H, s, H-OMe), 4.46 (1H, brs, H-17a), 4.71 (1H, brs, H-17b), 4.74 (2H, s, H-15), 6.05 (1H, d, J=15.7Hz, H-12), 6.26 (1H, d, J=15.9Hz, H-2), 6.67 (2H, s, H-5and H-9), 6.81 (1H, dd, J=15.7,10.1Hz, H-11), 7.14 (1H, s, H-14), 7.49 (1H, d, J=15.9Hz, H-3).
13c NMR (CDCl
3, 100MHz): δ 15.5 (C-20), 22.5 (C-18), 23.5 (C-6), 27.6 (C-2), 36.6 (C-7), 38.4 (C-1), 38.7 (C-10), 42.7 (C-4), 54.7 (C-5), 56.1 (2 × OMe), 60.9 (OMe), 61.5 (C-9), 65.0 (C-19), 69.7 (C-15), 79.0 (C-3), 105.1 (2C, C-5, 9), 109.1 (C-17), 117.0 (C-2), 121.2 (C-12), 129.0 (C-13), 129.7 (C-4), 135.6 (C-11), 139.9 (C-7), 143.4 (C-14), 145.2 (C-3), 148.0 (C-8), 153.3 (2C, C-6, 8), 167.0 (C-1), 172.4 (C-16) .HRESIMS: calculated value C
32h
41o
8[M+H]
+553.2796, experimental value 553.2820.
19-O-(2-furancarbonyl)-14-deoxidation-11,12-bis-dehydrogenation rographolide (3)
Molecular formula: C
25h
30o
6
Molecular weight: 426
Proterties: white amorphous powder
Spectrum data:
1h NMR (CDCl
3, 400MHz): δ 0.84 (3H, s, H-20), 1.19 (3H, s, H-18), 2.31 (1H, d, J=9.8Hz, H-9), 3.33 (1H, dd, J=11.4, 4.6Hz, H-3), 4.34 (1H, d, J=11.1, H-19a), 4.51 (1H, brs, H-17a), 4.74 (1H, brs, H-17b), 4.76 (2H, s, H-15), 6.09 (1H, d, J=15.7Hz, H-12), 6.47 (1H, dd, J=3.5, 1.7Hz, H-4), 6.84 (1H, dd, J=15.7, 10.1Hz, H-11), 7.18 (1H, s, H-14), 7.11 (1H, d, J=3.5Hz, H-3), 7.54 (1H, dd, J=1.7, 0.8Hz, H-5).
13c NMR (CDCl
3, 100MHz): δ 15.4 (C-20), 22.6 (C-18), 23.6 (C-6), 27.6 (C-2), 36.6 (C-7), 38.5 (C-1), 38.7 (C-10), 42.7 (C-4), 54.6 (C-5), 61.6 (C-9), 65.5 (C-19), 69.7 (C-15), 78.9 (C-3), 109.1 (C-17), 111.9 (C-4), 118.0 (C-3), 121.2 (C-12), 129.0 (C-13), 135.6 (C-11), 143.3 (C-14), 144.5 (C-5), 146.5 (C-2), 148.0 (C-8), 158.6 (C-1), 172.4 (C-16) .HRESIMS: calculated value C
25h
30o
6na [M+Na]
+449.1935, experimental value 449.1951.
19-O-(2-Thenoyl)-14-deoxidation-11,12-bis-dehydrogenation rographolide (4)
Molecular formula: C
25h
30o
5s
Molecular weight: 442
Proterties: white amorphous powder
Spectrum data:
1h NMR (CDCl
3, 400MHz): δ 0.94 (3H, s, H-20), 1.17 (3H, s, H-18), 4.80 (2H, s, H-15), 6.14 (1H, dd, J=15.7,9.9Hz, H-11), 6.30 (1H, d, J=15.7Hz, H-12), 7.57 (1H, m, H-4).
13c NMR (CDCl
3, 100MHz): δ 15.4 (C-20), 22.8 (C-18), 23.8 (C-6), 24.4 (C-2), 36.6 (C-7), 38.2 (C-1), 38.6 (C-10), 41.8 (C-4), 55.9 (C-5), 61.6 (C-9), 64.8 (C-19), 69.6 (C-15), 78.7 (C-3), 109.4 (C-17), 117.3 (C-4), 121.4 (C-12), 129.0 (C-13), 129.5 (C-3), 135.5 (C-11), 143.4 (C-14), 144.9 (C-5), 147.8 (C-8), 153.3 (C-2), 166.9 (C-1), 172.2 (C-16) .HRESIMS: calculated value C
25h
29o
5s [M-H]
-441.1741, experimental value 441.1753.
Hepatitis B virus resisting pharmacological test example is below used for illustrating the pharmacological action result of 14-deoxidation-11,12-bis-andrographolide of the present invention:
Test example 1:
The above-mentioned compound of the present invention prepares compound 14-deoxidation-11,12-bis-andrographolide (I) (compound 1,2,3,4) that embodiment obtains on HepG2.2.15 cell model to the restraining effect of hepatitis B virus surface antigen (HBsAg), e antigen (HBeAg) and HBV DNA replication dna and it is to the toxicity of cell:
1 materials and methods
1.1 materials: the present invention prepares compound 14-deoxidation-11,12-bis-andrographolide (compound 1-4) that embodiment obtains; Tenofovir (Jiangxi Chen Yang pharmaceutcal corporation, Ltd) makes positive control drug; The Hep G2.2.15 cell (voluntarily Secondary Culture) of HBV transfection; Cell culture fluid: high sugared nutrient solution adds 10% foetal calf serum, 0.03%L – glutamine, the blue or green enzyme element of 100mg/L G418,105IU/L, 100mg/L Streptomycin sulphate, uses 5%Na
2cO
3adjust pH to 6.8 – 7.0; DMEM in high glucose (GIBICO); G418(GIBICO); Foetal calf serum (Tianjin blood grinds institute); L – glutamine (AMRESCO); Penicillin, Streptomycin sulphate (Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., Shiyao Group); HBsAg and HBeAg test kit (Antu bio-engineering corporation).
1.2Hep G2.2.15 cell, cultivate with DMEM in high glucose liquid, nutrient solution adds 10% foetal calf serum, 0.03%L – glutamine, 100mg/L G418,10
5the blue or green enzyme element of IU/L, 100mg/L Streptomycin sulphate, 5%NaHCO
3adjust pH to 6.8 – 7.0.
1.3 instruments: the microplate reader Bio-RAD680(U.S.); CO
2the incubator Thermo Forma3310(U.S.); Inverted biologic microscope XD-101 type (Nanjing) etc.
1.4 experimentation
1.4.1HBsAg with the mensuration of HBeAg inhibit activities:
Utilize ELISA(enzyme linked immunosorbent assay) method, working sample is to HBsAg and HBeAg inhibit activities.The inoculation of Hep G2.2.15 cell and 48 orifice plates, 3 × 10
4cell per well, adds growth medium, in 5%CO
2, cultivate 72h in 37 DEG C of incubators, absorb former substratum, add different concns testing sample solution, in 5%CO
2, in 37 DEG C of incubators, cultivate 72h.Get supernatant liquor, utilize HBsAg and HBeAg test kit to detect respectively.Utilize microplate reader measure solution absorbance (490nm).
Inhibiting rate η
inhibitory=(A
cell controls group– A
test sample group)/(A
cell controls group– A
blank group) × 100
IC
50=Anti lg [(50 – <50% inhibiting rate)/(>50% suppression Shuai – <50% inhibiting rate) × lg(extension rate) concentration of+lg(<50% inhibiting rate)]
SI=CC
50/IC
50
1.4.2HBV the mensuration of DNA replication dna inhibit activities:
Hep G2.2.15 cell is inoculated in 24 porocyte plates, and 5 × 10
5individual every hole, in 5%CO
2, in 37 DEG C of incubators, cultivate 72h.Be replaced by pastille substratum, after cultivating 48h, again change a pastille substratum, continue to cultivate 48h.Blood/cell/tissue genome DNA extracting reagent kit (TIANamp Gemomic DNA Kit, TIANGEN, China) is used to extract DNA.By the method detection by quantitative HBV DNA carrying capacity of quantitative fluorescent PCR.1 μ L DNA sample, 20 μ L2 × SYBR Green PCR Master Mix(Applied Biosystems, USA) and the special primer of 2 HBV as PCR amplification system: front primer (5'GGA ACCTCT ATG TAT CCC TCC3'), rear primer (5'TCC GTC CGA AGG TTTGGT AC3'). amplification and detection Mastercycler Ep Realplex System quantitative PCR apparatus (Eppendorf on probation, Masteraycler Eprealplex, German) 95 DEG C of preheatings 2 minutes, and by 95 DEG C (20 seconds), 58 DEG C (15 seconds), the condition of 72 DEG C (20 seconds) repeats the reaction of 40 circulations.Inhibition percentage and the IC of medicine is calculated according to result
50value, method of calculation are identical with 1.4.1.
1.4.3 cytotoxic assay:
According to the cytotoxicity of the mtt assay detection of drugs that Mosmann sets up.Concrete grammar is: the inoculation of Hep G2.2.15 cell and 48 orifice plates, 3 × 10
4cell per well, adds growth medium, in 5%CO
2, cultivate 72h in 37 DEG C of incubators, absorb former substratum, add different concns testing sample solution, in 5%CO
2, in 37 DEG C of incubators, cultivate 72h.After absorption supernatant (for two anti-mensuration), every hole adds the MTT that concentration is 0.4mg/mL, and 0.3mL, in 5%CO
2, hatch 4h for 37 DEG C.Absorb supernatant liquor, add 0.3mL dimethyl sulfoxide (DMSO) in every hole, hatch 10min in 37 DEG C, microplate reader measures solution absorbance (490nm).Medicine is calculated to the destruction percentage of cell according to result:
Destructive rate η
destroy=(A
cell controls group– A
test sample group)/(A
cell controls group– A
blank group) × 100
CC
50=Anti lg [(50 – <50% destructive rate)/(>50% Po Huai Shuai – <50% destructive rate) × lg(extension rate) concentration of+lg(<50% destructive rate)]
2. result: to the restraining effect result of HBsAg, HBeAg with selectivity index (SectiveIndex, SI, SI=CC
50/ IC
50, for evaluating the parameter of clinical drug application prospect) evaluate, wherein SI>2 is effectively nontoxic, and 1<SI<2 is effectively poisonous, and SI<1 is poisonous invalid.Concrete outcome is in table 2:
Show 214-deoxidation-11,12-bis-andrographolide (I) to the toxic action of Hep G2.2.15 cell, to the inhibit activities of HBsAg, HBeAg and HBV DNA
3. conclusion: experimental result shows, compound 14-deoxidation-11,12-bis-andrographolide is in vitro in Hep G2.2.15 cell, stronger restraining effect is copied to HBV DNA, special 19-O-(3,4,5-trimethoxy) cinnamyl-14-deoxidation-11,12-bis-dehydrogenation rographolide (2) SI value is greater than 165.1; In addition, 19-O-(2-furancarbonyl)-14-deoxidation-11,12-bis-dehydrogenation rographolide (3) suppression HBsAg; Compound 19-O-(2-Thenoyl)-14-deoxidation-11,12-bis-dehydrogenation rographolide (4) has good restraining effect to HBeAg.
Example of formulations
Example of formulations 1:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, after dissolving with a small amount of DMSO, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Example of formulations 2:
By the method first obtained 14-deoxidation-11 preparing embodiment 1,12-bis-andrographolide, after dissolving with a small amount of DMSO, be dissolved in sterile water for injection, be stirred to dissolve, filter with aseptic suction funnel, more aseptic essence filter, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
Example of formulations 3:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, add vehicle in itself and excipient weight than the ratio for 9:1, make pulvis.
Example of formulations 4:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, add vehicle, pelletizing press sheet in itself and excipient weight than the ratio for 5:1.
Example of formulations 5:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, oral liquid method for making makes oral liquid routinely.
Example of formulations 6:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, add vehicle in itself and excipient weight than the ratio for 5:1, make capsule.
Example of formulations 7:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, add vehicle in itself and excipient weight than the ratio for 3:1, make capsule.
Example of formulations 8:
By method first obtained 14-deoxidation-11,12-bis-andrographolide preparing embodiment 1, add vehicle in itself and excipient weight than the ratio for 5:1, make granule.
Claims (5)
1. 14 shown in structural formula (I) ?Tuo Yang ?11,12 ?two andrographolide (1 ?4),
In formula, R is
time be compound 1 ?4.
2. pharmaceutical composition, containing general formula according to claim 1 (I) 14 ?Tuo Yang ?11,12 ?two andrographolide (1 ?4) and pharmaceutically acceptable carrier or vehicle.
3. prepare general formula according to claim 1 (I) 14 ?Tuo Yang ?11, 12 ?the method of two andrographolide (1 ?4), it is characterized in that 14 ?Tuo Yang ?11, 12 ?two dehydrogenation rographolides, carboxylic acid, and 4 ?the N of dimethylamino pyridine, N ?add N in dimethyl formamide solution, N ?dicyclohexylcarbodiimide, described carboxylic acid is TRANSCINNAMIC ACID or 3, 4, 5 ?San Jia Yang Ji ?TRANSCINNAMIC ACID or 2 ?furancarboxylic acid or 2 ?thiophenic acid, after reacting completely, the reaction solution diluted ethyl acetate of 3 times of volumes, use dilute hydrochloric acid successively, saturated sodium bicarbonate aqueous solution and saturated common salt water washing, after anhydrous sodium sulfate drying, decompression and solvent recovery obtains crude product, crude product through silica gel column chromatography, Yong Yi Suan Yi Zhi ?sherwood oil wash-out obtain 14 ?Tuo Yang ?11,12 ?two andrographolide (1 ?4).
4. according to claim 1 14 ?Tuo Yang ?11,12 ?two andrographolide (1 ?4) preparing the application in anti-hepatitis B medicine.
5. pharmaceutical composition according to claim 2 is preparing the application in anti-hepatitis B medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410010214.7A CN103739575B (en) | 2014-01-09 | 2014-01-09 | 14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410010214.7A CN103739575B (en) | 2014-01-09 | 2014-01-09 | 14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103739575A CN103739575A (en) | 2014-04-23 |
| CN103739575B true CN103739575B (en) | 2015-09-16 |
Family
ID=50496663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410010214.7A Expired - Fee Related CN103739575B (en) | 2014-01-09 | 2014-01-09 | 14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103739575B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057676A (en) * | 2021-10-21 | 2022-02-18 | 香港中文大学（深圳） | Andrographolide compound and application and pharmaceutical composition thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101098691B (en) * | 2004-11-08 | 2012-09-05 | 康乃尔研究基金会有限公司 | Andrographolide derivatives to treat viral infections |
| CN101190912A (en) * | 2006-12-01 | 2008-06-04 | 黄振华 | Antiviral compound and preparation method thereof |
| CN101416958B (en) * | 2008-12-15 | 2010-12-22 | 郑州大学 | Use of 15-methano-substituted-andrographolide derivative in preparing anti-hepatitis B medicine |
| CN102250142B (en) * | 2010-05-21 | 2015-07-15 | 广东东阳光药业有限公司 | Andrographolide compound and application of andrographolide compound in medicaments |
-
2014
- 2014-01-09 CN CN201410010214.7A patent/CN103739575B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103739575A (en) | 2014-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102219725B (en) | Benzohetercyclic compound as well as preparation method and applications thereof | |
| CN106176716B (en) | The new application of daphane diterpene compound pimelotide C | |
| CN109265504B (en) | 4-amino acid substituted pyrimidine nucleoside compound and pharmaceutical application thereof | |
| EP3345896A1 (en) | Derivative of inula lineariifolia lactone a | |
| CN103880856B (en) | Bisabolane sesquiterpene derivant and pharmaceutical composition thereof and its application in pharmacy | |
| CN103739575B (en) | 14-deoxidation-11,12-bis-andrographolide and pharmaceutical composition thereof and application | |
| CN111333694B (en) | Application of hederagenin derivative in medicine for resisting myocardial anoxia reoxygenation injury | |
| CN104945361B (en) | Germacrane Sesquiterpenoids derivative and preparation method and application | |
| CN103739597B (en) | 14-deoxidation-14,15-bis-andrographolide and pharmaceutical composition thereof and purposes | |
| CN102796157B (en) | Caudatin derivative and medicinal composition and application thereof | |
| CN114315855A (en) | Curcumenol derivatives, preparation method and application thereof in preparation of anti-inflammatory drugs | |
| CN101774921A (en) | Method for preparing dicaffeoylquinic acid methyl compound and composition thereof | |
| CN102584846B (en) | Curcumenol derivatives resisting influenza A(H1N1) virus | |
| CN1900046B (en) | Kaurane diterpine compound and its preparing method and use | |
| CN101774920A (en) | Preparation method of 3,5-cynarin methyl ester and medicament composition thereof | |
| CN102603522B (en) | Phenol derivatives and application thereof in preparation of anti-HBV (hepatitis B virus) medicines | |
| CN101829090B (en) | Application of diamine formyl dehydrogenated silybin serving as medicament for curing viral hepatitis B | |
| CN101255184B (en) | Hepatitis B resistant compound, medicament composition and use in medicament preparation | |
| CN111518157A (en) | Triptolide derivative and preparation method and application thereof | |
| CN101851196B (en) | 2-(1H) quinolinone derivative, medical composition, preparation method and application thereof | |
| CN101602787B (en) | Alisol A derivative, medicinal composition, preparation method and application thereof | |
| CN101829083B (en) | Application of substituted silybin ester in preparing medicament for treating virus hepatitis B | |
| CN102101864B (en) | Trigonolactone A-D and medicinal composition, preparation method and application thereof | |
| CN101912387B (en) | Application of benzyl-containing flavonoid lignan in preparation of medicament for treating hepatitis B | |
| CN101659629A (en) | Houttuynin high water-solubility and stability prodrug and preparing method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150916 Termination date: 20210109 |