CN1990484A - Silybin esters derivatives and preparation and use thereof - Google Patents
Silybin esters derivatives and preparation and use thereof Download PDFInfo
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Abstract
The invention relates to a silibinin monoester derivative and its medicine salt or solvates. The invention also relates to its preparation method and its drug combination and medical application. The compound can inhibit activity of hepatitis b virus, so it is expected to be used as drug to treat hepatitis b virus and relevant virus diseases. The compound can protect liver and is expected to be used as drug preventing liver damage. The compound possesses effect anti free radical and is expected to be used as drug treating diseases caused by free radical.
Description
Invention field
The invention belongs to organic chemistry and pharmaceutical chemistry field, particularly, the present invention relates to the preparation method and the purposes of silybin esters derivatives.This compounds obtains by complete synthesis and semisynthetic method.This compounds has the activity that suppresses human body hepatitis B virus thymus nucleic acid (HBVDNA) and reduce hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), thereby can expect as the medicine for the treatment of Type B viral hepatitis and related diseases toxicity disease.In addition, the present invention causes rat suckling mouse primary hepatocyte damage external model with this series compound to hydrogen peroxide and has carried out hepatocellular injury protection screening active ingredients.This compounds is found to have the protection liver cell and promotes the effect that liver cell is repaired.Secondly, liver injury model in the rat acute carbon tetrachloride hepatic injury animal body has been carried out protection liver injury active testing.Find that this compounds has the effect of endogenous protective animal liver damage.The present invention tests this compounds antioxidant radical activity again, finds the activity that its lipoperoxide with external removing ultra-oxygen anion free radical, removing free radical scavenging activity, inhibition free yl induction generates.Above activity shows that this compounds can be expected and is used to prepare prevention or treats acute chronic hepatic injury class disease and caused or other physiological changes relevant with oxyradical or the medicine of disease by oxyradical.This compounds also shows the very strong PC12 cell injury effect that Green Tea Extract is caused, promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage provide protection.And under same concentration, its energy force rate positive control Quercetin of removing free radical protection cell is also high.Illustrate that it is anti-oxidant to the protection cranial nerve, prevent and treat senile dementia active effect is arranged.Above activity shows that this compounds can be expected and is used to prepare prevention or treats acute chronic hepatic injury class disease and caused or other physiological changes relevant with oxyradical or the medicine of disease by oxyradical.
Background of invention
In numerous Green Tea Extract natural products, Silymarin is one of medicine of a few widespread use clinically.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult people such as Flora K., Am.J.Gastroenterol, 1998,93,139-143; People such as Saller R., Drugs, 2001,61 (14), 2035-2063).Silibinin content is maximum in the Silymarin, and activity is also the highest.This medicine effect mainly contain following some.(1) Green Tea Extract activity: silymarin is for by CCl
4, the hepatic injury that GalN, alcohols and other hepatotoxin cause has provide protection.People such as nineteen ninety Lotteron have reported that in the Mouse Liver microsome silymarin can reduce by CCl
4These show that all silymarin is the chain interruption antioxidant or is free-radical scavengers external lipid peroxidation that metabolism causes and the peroxidation that caused separately by reduced coenzyme.(2) protection liver plasma membrane: keep flowability of cell membranes by the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special acceptor on mycotoxins phalloidin and α-amanitine etc. and the liver cell, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistibility of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with estradiol receptor after entering cell, and make it to activate, activated receptors can enhance hepatocyte nuclear RNA polysaccharase 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 tumour, and silibinin has also shown the effect of prevention and treatment tumour as an effective Green Tea Extract material.
The silibinin compounds has definite curative effect, but because the market that the some shortcomings on its water-soluble and bioavailability have limited this medicine.So seek the new derivative of silibinin class, make it can have pharmacologically active higher or that upgrade, in the hope of obtaining the new drug of independent intellectual property right, real genus is necessary.
Summary of the invention
The purpose of this invention is to provide a kind of active compound that suppresses hepatitis B virus, removing free radical activity, protection liver cell and the organic damage of liver, treatment degenerative brain disorder that has.Particularly, the invention provides silybin esters derivatives shown in a kind of formula (I) and pharmacologically acceptable salt thereof or its solvate.
N=0,1 wherein; R
1, R
2, R
3Be hydrogen, alkoxyl group, halogen, nitro, itrile group, amino, amide group, R
4, R
6Be hydrogen, alkoxyl group, halogen etc., R
5Be hydrogen, alkyl, R
7Be hydrogen or alkoxyl group, wherein the carbonatoms of alkyl or alkoxyl group is a 1-8 carbon atom, can be straight or branched alkyl or alkoxyl group.Prosposition 7 in the formula (I) ', 8 ' steric configuration be respectively or be R configuration or S configuration simultaneously.
The preferred formula of the present invention (I) compound comprises:
Compound I-1: anisic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-2: p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-3: phenylformic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2]-methyl ester;
Compound I-4: m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-5:3, the 4-dimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-6:3,4, the 5-trimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-7: piperinic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-8: parafluorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-9: to itrile group phenylformic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dioxy-1,4-benzodioxane-2-]-methyl ester;
Compound I-10: to tolyl acrylic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-11: to chloro-cinnamic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-12: to bromo-cinnamic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-13: paraacetaminobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-14:3, the 5-dinitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-15: anisic acid [3-(4-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-16: p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-17: para-amino benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-18:3-amino-5-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester.
Another object of the present invention provides the preparation method of a kind of silybin esters derivatives formula I, and the preparation method of Compound I is:
R
1, R
2, R
3, R
7Define identical with formula (I), wherein the azoformic acid diester is a dimethyl ester, diethyl ester, propyl ester, isopropyl ester or butyl ester, solvent is a tetrahydrofuran (THF), methyl-sulphoxide, dimethyl formamide, temperature of reaction is that room temperature is to refluxing, silibinin: phenylformic acid: azoformic acid diester: triphenyl phosphorus=1: 1～3: 1～3: 1～3,0.5～20 hour reaction times.
Another object of the present invention has provided the purposes that formula (I) compound is used to prevent and treat liver cell and the organic damage of liver and prevents and treats the Type B viral hepatitis disease.
A further object of the present invention has provided a kind of pharmaceutical composition that is used to prevent and treat liver cell and organic damage of liver and control Type B viral hepatitis disease that contains formula (I) compound.
Another object of the present invention has provided a kind of being used to prepare antiaging agent based on resisting oxidation free radical mechanism, preventing and treating the purposes of degenerative brain disorder of formula (I) compound of containing.
A further object of the present invention has provided a kind of being used to prepare antiaging agent based on resisting oxidation free radical mechanism, preventing and treating the pharmaceutical composition of degenerative brain disorder of formula (I) compound of containing.
Another purpose of the present invention has provided being used to prepare by oxyradical and causing or the physiological change relevant with oxyradical or disease is particularly cardiovascular and the purposes of cerebrovascular disease medicine of formula (I) compound.
A further object of the present invention has provided a kind of being used to prepare by oxyradical and causing or the physiological change relevant with oxyradical or disease is particularly cardiovascular and the pharmaceutical composition of cerebrovascular disease medicine of formula (I) compound of containing.
In order to understand essence of the present invention better, use the process of the formal specification compound of embodiment below respectively, embodiment has provided part physics and the chemistry and the Wave Spectrum data of representative compounds.Mandatory declaration, embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1: Compound I-1, and the preparation of anisic acid [3-(hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester:
In the exsiccant reaction flask, add 1 and digest compound I-a (silibinin), 0.6 gram anisic acid and 1.6 gram triphenyl phosphorus, with the dissolving of 20 milliliters of anhydrous tetrahydro furans, add 1 gram diethyl azodiformate, add the back in stirring at room 10 hours, underpressure distillation removes and desolvates, add 5 milliliters of chloroforms, remove by filter white solid, mother liquor is through column chromatography, get buff powder 0.7 gram, yield 47%.
Compound I-1:Rf (chloroform: methyl alcohol=50: 1)=0.33;
1H NMR (400MHz, deuterochloroform) δ: 3.80 (s, 3H, OCH
3), 3.86 (s, 3H, OCH
3), 4.20 (m, 1H, H-9 ' a), 4.34 (m, 1H, H-8 '), 4.48 (m, 1H, H-9 ' b), 4.53 (m, 1H, H-3), 4.98 (d, J=12.0Hz, 1H, H-2), 5.00 (d, J=8.0Hz, 1H, H-7 '), 5.99 (s, 1H, H-6), 6.05 (s, 1H, H-8), 6.93 (d, J=8.8Hz, 1H, H-3 , 5 ), 6.85-7.20 (m, 6H, Ar-H), (7.94 d, J=8.8Hz, 1H, H-2 , 6 ) 11.21 (s, 1H, 5-OH); ESI-MS:615[M-1]
+
Prepare compound shown in the embodiment 2-16 in the table one according to embodiment 1 identical method:
Table one:
Each compound physicochemical data:
Compound I-2 (p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.18;
1H NMR (400MHz, deuterochloroform) δ: 3.87 (s, 3H, OCH
3), 4.34 (m, 1H, H-9 ' a), 4.52 (m, 1H, H-8 '), 4.56 (m, 2H, H-3, H-9 ' b), 4.97 (d, J=6.8Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 6.04 (s, 1H, H-6), 6.11 (s, 1H, H-8), 7.55 (d, J=8.8Hz, 2H, 2 , 6 ), 6.90-8.16 (m, 6H, Ar-H), 8.30 (d, J=8.8Hz, 2H, 3 , 5 ), 11.17 (s, 1H, 5-OH); ESI-MS:630[M-1]
+
Compound I-3 (phenylformic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.20
1H NMR (400MHz, deuterochloroform) δ: 3.85 (s, 3H, OCH
3), 4.21 (m, 1H, H-9 ' a), 4.28 (m, 1H, H-8 '), 4.52 (m, 2H, H-3, H-9 ' b), 4.96 (d, J=12.0Hz, 1H, H-2), 5.02 (d, J=8.0Hz, 1H, H-7 '), 6.02 (s, 1H, H-6), 6.06 (s, 1H, H-8), 6.92-8.06 (m, 11H, Ar-H), 11.20 (s, 1H, 5-OH); ESI-MS:585[M-1]
+
Compound I-4 (m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.16
1H NMR (400MHz, deuterochloroform) δ: 3.86 (s, 3H, OCH
3), 4.26 (m, 1H, H-9 ' a), 4.38 (m, 1H, H-8 '), 4.53 (m, 2H, H-3, H-9 ' is b), 4.97 (d, J=12.0Hz, 1H, H-2), 5.00 (d, J=8.0Hz, 1H, H-7 '), 6.00 (s, 1H, H-6), 6.07 (s, 1H, H-8), 6.87-7.87 (m, 10H, Ar-H), 11.20 (s, 1H, 5-OH); ESI-MS:619[M-1]
+
Compound I-5 (2,3-dimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.19
1H NMR (400MHz, deuterochloroform) δ: 3.81 (s, 3H, OCH
3), 3.93 (s, 3H, OCH
3), 3.97 (s, 3H, OCH
3), 4.25 (m, 1H, H-9 ' a), 4.36 (m, 1H, H-8 '), 4.55 (m, 2H, H-3, H-9 ' b), 4.99 (d, J=12.0Hz, 1H, H-2), (5.00 d, J=8.0Hz, 1H, H-7 ') 5.98 (s, 1H, H-6), 6.00 (s, 1H, H-8), 6.86-7.65 (m, 9H, Ar-H), 11.20 (s, 1H, 5-OH); ESI-MS:645[M-1]
+
Compound I-6 (3,4,5-trimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.10
1H NMR (400MHz, deuterochloroform) δ: 3.83 (s, 3H, OCH
3), 3.90 (s, 6H, OCH
3), 3.91 (s, 3H, OCH
3), 4.26 (m, 1H, H-9 ' a), 4.37 (m, 1H, H-8 '), 4.52 (m, 2H, H-3, H-9 ' is b), (4.98 d, J=7.8Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 5.99 (s, 1H, H-6), 6.06 (s, 1H, H-8), 6.86-7.26 (m, 8H, Ar-H), 11.19 (s, 1H, 5-OH); ESI-MS:645[M-1]
+
Compound I-7 (piperinic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.12
1H NMR (400MHz, deuterochloroform) δ: 3.83 (s, 3H, OCH
3), 4.24 (m, 1H, H-9 ' a), 4.33 (m, 1H, H-8 '), 4.54 (m, 2H, H-3, H-9 ' b), 4.98 (d, J=7.2Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 5.98 (s, 1H, H-6), 6.05 (s, 3H, OCH
2O, H-8), 6.84-7.67 (m, 9H, Ar-H), 11.20 (s, 1H, 5-OH); ESI-MS:629[M-1]
+
Compound I-8 (parafluorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.14
1H NMR (400MHz, deuterochloroform) δ: 3.85 (s, 3H, OCH
3), 4.29 (m, 1H, H-9 ' a), 4.40 (m, 1H, H-8 '), 4.55 (m, 2H, H-3, H-9 ' is b), 4.97 (d, J=12.0Hz, 1H, H-2), 5.02 (d, J=8.0Hz, 1H, H-7 '), 6.01 (s, 1H, H-6), 6.07 (s, 1H, H-8), 6.87-7.87 (m, 10H, Ar-H), 11.20 (s, 1H, 5-OH); ESI-MS:603[M-1]
+
Compound I-9 (to the itrile group phenylformic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(and 2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.18
1H NMR (400MHz, deuterochloroform) δ: 4.23 (s, 3H, OCH
3), 4.35 (m, 1H, H-9 ' a), 4.52 (m, 2H, H-8 '), 4.55 (m, 2H, H-3, H-9 ' is b), (4.96 d, J=6.8Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 6.02 (s, 1H, H-6), 6.11 (s, 1H, H-8), 6.94-8.12 (m, 10H, Ar-H), 11.17 (s, 1H, 5-OH); ESI-MS:610[M-1]
+
Compound I-10 (to tolyl acrylic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(and 2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (sherwood oil: ethyl acetate :=1: 1)=0.32
1H NMR (400MHz, deuterochloroform) δ: 2.38 (s, 3H, CH
3), 3.87 (s, 3H, OCH
3), 4.12 (m, 1H, H-9 ' a), 4.29 (m, 1H, H-8 '), 4.38 (m, 1H, H-9 ' b), 4.52 (dd, J=11.6,4Hz, 1H, H-3), 4.93 (d, J=8.0Hz, 1H, H-7 '), 4.98 (d, J=11.6Hz, 1H, H-2), 5.97 (s, 1H, H-6), 6.04 (s, 1H, H-8), 6.40 (d, J=16.0Hz, 1H, H-α), 6.87-7.07 (m, 6H, Ar-H), (7.20 d, J=8.0Hz, 2H, H-3 , 5 ), (7.43 d, J=8.0Hz, 2H, H-2 , 6 ) 7.64 (d, J=16.0Hz, 1H, H-β) 11.20 (s, 1H, 5-OH); ESI-MS:625[M-1]
+
Compound I-11 (to chloro-cinnamic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(and 2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.11
1H NMR (400MHz, deuterochloroform) δ: 3.89 (s, 3H, OCH
3), 4.14 (m, 1H, H-9 ' a), 4.30 (m, 1H, H-8 '), 4.40 (m, 1H, H-9 ' b), 4.53 (dd, J=11.6,4.4Hz, 1H, H-3), 4.93 (d, J=8.0Hz, 1H, H-7 '), 4.99 (d, J=12Hz, 1H, H-2), 5.96 (d, J=0.8Hz, 1H, H-6), 5.96 (d, J=0.8Hz, 1H, H-8), 6.42 (d, J=16Hz, 1H, H-α), and 6.87-7.20 (m, 6H, Ar-H), 7.38 (d, J=8.0Hz, 2H, 3 , 5 ), 7.46 (d, J=8.0Hz, 2H, 2 , 6 ), 7.63 (d, J=16.0Hz, 1H, H-β) 11.20 (s, 1H, 5-OH); ESI-MS:645[M-1]
+
Compound I-12 (to bromo-cinnamic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(and 2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.13
1H NMR (400MHz, deuterochloroform) δ: 3.89 (s, 3H, OCH
3), 4.14 (m, 1H, H-9 ' a), 4.30 (m, 1H, H-8 '), 4.40 (m, 1H, H-9 ' b), 4.53 (dd, J=12.0,4.8Hz, 1H, H-3), 4.93 (d, J=8.0Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 5.96 (d, J=0.8Hz, 1H, H-6), 5.96 (d, J=0.8Hz, 1H, H-8), 6.43 (d, J=16.0Hz, 1H, H-α), 6.87-7.07 (m, 6H, Ar-H), 7.20 (d, J=8.0Hz, 2H, 3 , 5 ), 7.41 (d, J=8.0Hz, 2H, 2 , 6 ), 7.59 (d, J=16.0Hz, 1H, H-β), 11.20 (s, 1H, 5-OH); ESI-MS:689[M-1]
+
Compound I-13 is (to acetaminobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.13
1H NMR (400MHz, deuterochloroform) δ: 2.01 (s, 3H, CH
3), 3.73 (s, 3H, OCH
3), 4.22 (m, 1H, H-9 ' a), 4.45 (m, 2H, H-3,8 '), 4.59 (m, 1H, H-9 ' b), 5.03 (d, J=10.8Hz, 1H, H-2), (5.05 d, J=7.6Hz, 1H, H-7 '), 6.00 (d, J=2.0Hz, 1H, H-6), 6.02 (d, J=2.0Hz, 1H, H-8), 7.18 (d, J=8.4Hz, 2H, H-3 , 5 ), 6.85-7.32 (m, 6H, Ar-H), 7.97 (d, J=8.4Hz, 2H, H-2 , 6 ), 11.53 (s, 1H, 5-OH); ESI-MS:642[M-1]
+
Compound I-14 (3,5-dinitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester): Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.18
1H NMR (400MHz, deuterochloroform) δ: 3.87 (s, 3H, OCH
3), 4.34 (m, 1H, H-9 ' a), 4.52 (m, 1H, H-8 '), 4.56 (m, 2H, H-3, H-9 ' is b), (4.97 d, J=6.8Hz, 1H, H-7 '), 4.99 (d, J=12.0Hz, 1H, H-2), 6.04 (s, 1H, H-6), 6.11 (s, 1H, H-8), 6.90-8.16 (m, 9H, Ar-H), 11.17 (s, 1H, 5-OH); ESI-MS:675[M-1]
+
Embodiment 15: the preparation of Compound I-15 (anisic acid [3-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4 benzodioxane-2-]-methyl ester):
Method according to embodiment 1, use 2-[2,3-dihydro-3-(4-hydroxy 3-methoxybenzene base)-2-methylol-8-methoxyl group-1,4-benzodioxane-6-]-2,3-dihydro-3,5,7-trihydroxy--4H-1-chromene-4-ketone replaces silibinin can obtain Compound I-15:Rf (chloroform: methyl alcohol=50: 1)=0.28;
1H NMR (400MHz, deuterochloroform) δ: 3.80 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.22 (m, 1H, H-9 ' a), 4.33. (m, 1H, H-8 '), 4.50 (m, 1H, H-9 ' b), 4.51 (m, 1H, H-3), 4.98 (d, J=12.0Hz, 1H, H-2), (5.02 d, J=8.0Hz, 1H, H-7 '), 6.00 (s, 1H, H-6), 6.03 (s, 1H, H-8), (6.95 d, J=8.8Hz, 1H, H-3 , 5 ), 6.86-7.15 (m, 5H, Ar-H), 7.95 (d, J=8.8Hz, 1H, H-2 , 6 ) 11.24 (s, 1H, 5-OH); ESI-MS:645[M-1]
+
Embodiment 16: the preparation of p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester:
According to the identical method of embodiment 15, can obtain Compound I-16:Rf (chloroform: ethyl acetate: formic acid=50: 1: 0.25)=0.15;
1H NMR (400MHz, deuterochloroform) δ: 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.35 (H-9 ' is a) for m, 1H, (4.50 m, 1H, H-8 '), 4.57 (m, 2H, H-3, H-9 ' b), 4.96 (d, J=6.8Hz, 1H, H-7 '), 5.01 (d, J=12.0Hz, 1H, H-2), 6.01 (s, 1H, H-6), 6.13 (s, 1H, H-8), 7.59 (d, J=8.8Hz, 2H, 2 , 6 ), and 6.93-8.16 (m, 5H, Ar-H), 8.33 (d, J=8.8Hz, 2H, 3 , 5 ), 11.20 (s, 1H, 5-OH); ESI-MS:660[M-1]
+
Embodiment 17: the preparation of Compound I-17 (para-amino benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester):
0.5 gram I-2 is dissolved in 10 milliliters of ethyl acetate, adds 50 milligram of 10% palladium carbon, with behind the hydrogen exchange air, in stirring at room 5 hours, filters, filtrate concentrates, and obtains buff powder 0.3 through column chromatography for separation and restrains yield: 63%.
Compound I-17:Rf (chloroform: ethyl acetate: acetone: methyl alcohol: formic acid=20: 1: 1: 05: 0.25)=0.25;
1H NMR (400MHz, deuterochloroform) δ: 3.73 (s, 3H, OCH
3), 4.12 (m, 1H, H-9 ' a), 4.41 (m, 2H, H-3,8 '), 4.56 (m, 1H, H-9 ' b), 5.02 (d, J=10.8Hz, 1H, H-2), 5.04 (d, J=7.6Hz, 1H, H-7 '), 5.92 (d, J=2.0Hz, 1H, H-6), 5.94 (d, J=2.0Hz, 1H, H-8), 6.63 (d, J=8.4Hz, 2H, H-3 , 5 ), 6.82-7.29 (m, 6H, Ar-H), (7.74 d, J=8.4Hz H-2 , 6 ), 11.53 (s, 1H, 5-OH); ESI-MS:600[M-1]
+
Embodiment 18: can prepare Compound I-18:(3-amino-5-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2 as initiator with I-14 according to embodiment 17 identical methods, 3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester) preparation:
Rf (chloroform: ethyl acetate: acetone: methyl alcohol: formic acid=20: 1: 1: 0.5: 0.25)=0.17;
1H NMR (400MHz, deuterochloroform) δ: 3.73 (s, 3H, OCH
3), 4.25 (m, 1H, H-9 ' a), 4.50 (m, 2H, H-3,8 '), 4.56 (m, 1H, H-9 ' b), 5.01 (d, J=10.8Hz, 1H, H-2), 5.03 (d, J=7.6Hz, 1H, H-7 '), 5.92 (d, J=2.0Hz, 1H, H-6), 5.94 (d, J=2.0Hz, 1H, H-8), 6.82-8.25 (m, 9H, Ar-H), 11.60 (s, 1H, 5-OH); ESI-MS:645[M-1]
+
Formula (I) compound has multiple important biological, the present invention finds that by testing this compounds has the activity that suppresses human body hepatitis B virus thymus nucleic acid (HBVDNA) and reduce hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), thereby can expect as the medicine for the treatment of Type B viral hepatitis and related diseases toxicity disease.In addition, the present invention causes rat suckling mouse primary hepatocyte damage external model with this series compound to hydrogen peroxide and has carried out hepatocellular injury protection screening active ingredients.This compounds is found to have the protection liver cell and promotes the effect that liver cell is repaired.Secondly, liver injury model in the rat acute carbon tetrachloride hepatic injury animal body has been carried out protection liver injury active testing.Find that this compounds has the effect of endogenous protective animal liver damage.The present invention tests this compounds antioxidant radical activity again, finds the activity that its lipoperoxide with external removing ultra-oxygen anion free radical, removing free radical scavenging activity, inhibition free yl induction generates.Above activity shows that this compounds can be expected and is used to prepare prevention or treats acute chronic hepatic injury class disease and caused or other physiological changes relevant with oxyradical or the medicine of disease by oxyradical.This compounds also shows the very strong PC12 cell injury effect that Green Tea Extract is caused, promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage provide protection.And under same concentration, its energy force rate positive control Quercetin of removing free radical protection cell is also high.Illustrate that it is anti-oxidant to the protection cranial nerve, prevent and treat senile dementia active effect is arranged.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof and solvate thereof can combine with spoke material or carrier pharmaceutically commonly used, prepare to have and suppress hepatitis B virus surface antigen and hepatitis B virus e antigen and suppress hepatitis B virus DNA replication activity, thereby can be used to the pharmaceutical composition or the healthcare products that prevent and treat hepatitis B.Above-mentioned various kinds of drug composition or healthcare products can adopt drug forms such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof and solvate thereof can with the treatment hepatitis B medicine that has now gone on the market such as Ah former times's network Wei, lamivudine, Interferon, rabbit, etc. unite use, prepare medicine or healthcare products with treatment hepatitis B.Above-mentioned various kinds of drug composition or healthcare products can adopt drug forms such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof and solvate thereof can combine with spoke material or carrier pharmaceutically commonly used, have the active pharmaceutical composition or the healthcare products that prevent and treat hepatic diseases of can being used to of the acute and chronic injury of protection liver cell thereby prepare.Above-mentioned various kinds of drug composition or healthcare products can adopt drug forms such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof and solvate thereof can also with the liver protecting that has now gone on the market and liver disease medicine medicine such as Biphenylylmethylcarbinol; silymarin; silybin meglumine; Oleanolic Acid; Tensicor; Protoporphrin Disodium (protoporphyrin disodium); Malotilate (malotilate); ursodesoxycholic acids etc. are united use; prepare and have protection liver active composition, can be used for treating acute and chronic hepatitis; chronic hepatitis; early stage liver cirrhosis; fatty liver and toxic liver injury disease medicine or healthcare products.Above-mentioned various kinds of drug composition or healthcare products can adopt drug forms such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.
Formula of the present invention (I) compound or pharmaceutically acceptable salt thereof and solvate thereof can be united use with the Green Tea Extract oxidant drug that has now gone on the market such as superoxide-dismutase (SOD) etc., prepare and have defence the free radical active antioxidant compositions of infringement or the healthcare products that cause, be used for the treatment of above-mentioned disease.Above-mentioned various kinds of drug composition or healthcare products can adopt drug forms such as injection, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.
In order to understand essence of the present invention better; use the inhibition activity of the formalization compound of pharmacology embodiment below respectively to hepatitis B virus surface antigen (HBsAg); inhibition test to hepatitis B virus e antigen (HBeAg); inhibition test to hepatitis B virus thymus nucleic acid (HBVDNA); the hepatocellular injury protection that hydrogen peroxide is caused rat suckling mouse primary hepatocyte damage external model is active active to rat acute carbon tetrachloride hepatic injury (liver injury model in the animal body) protection; active to the protection of rat chronic carbon tetrachloride hepatic injury; the active The pharmacological results of the super oxyradical of external removing illustrates its new purposes in pharmacy field.Pharmacology embodiment has provided the part activity data of representative compounds.Same mandatory declaration, pharmacology embodiment of the present invention is used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Pharmacology embodiment 1: the inhibition activity of Compound I-12 pair hepatitis B virus surface antigen (HBsAg)
1, materials and methods:
1.1 the automatic fluorescent PCR instrument of instrument and reagent: PE7700, U.S. Perkin Elmer company produces; The HBV-DNA fluorescence quantitative detection kit reaches peace gene diagnosis center by Zhongshan Medical Univ. to be provided, and foetal calf serum, DMEM, G418, trypsinase are all available from Gibco company.
1.2 cell in vitro model: the 2.2.15 cell strain of transfection hepatitis B virus (HepG 2.2.15) is cultivated by Zhejiang University's Chinese medicine and natural drug research department and is provided, HepG 2.2.15 cell inoculation (is contained 10% foetal calf serum in the DMEM nutrient solution, 380ug/mL G418), put 5%CO
2Cultivate in 37 ℃ of incubators.
1.3 drug effect: the HepG2.2.15 cell digests with 0.06% tryptic digestive juice, is diluted to 8 * 10
4/ mL suspension, every hole 200uL changes the pastille nutrient solution two days later, and drug level is respectively 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, each concentration is established 6 parallel holes, changes the pastille substratum once in per three days, and it is frozen to be checked in-20 ℃ to draw the culture supernatant that swaps out.With the nutrient solution cultured cells that do not contain medicine under the similarity condition in contrast,, suct clear liquid during test and measure hepatitis B virus surface antigen (HBsAg) with euzymelinked immunosorbent assay (ELISA) (ELISA) as positive control with lamivudine, it is as shown in the table to obtain the result.Remaining cell is measured drug cell toxicity with mtt assay.Nutrient solution blank group four holes are established in test in addition.
2, the toxicity of mtt assay test sample pair cell:
The cytotoxic activity of compd A pair cell: cell DMEM culture medium culturing, contain 10% foetal calf serum in the substratum, 800, the Streptomycin sulphate of 000U/mL penicillin and 1mg/mL.Cell contains 5%CO at 37 ℃
2Cultivate in the incubator of damp atmosphere.The mensuration of cell survival rate is with improveing mtt assay, every hole adds 20 μ L MTT[bromination 3-(4 when hatching end, 5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole] (5mg/mL, with phosphoric acid buffer PBS preparation), continued to hatch 4 hours under 37 ℃ of conditions, every then hole adds 100 μ L stop buffers (10% sodium lauryl sulphate SDS is with 1: 1 isopropylcarbinol and the preparation of 2M hydrochloric acid) with lysing cell and Rong Xie Jia Za (formazan) crystallization.Micro reaction plate is placed under dark moist condition and is spent the night.Formed Jia Za microplate reader colorimetric under the 570nm wavelength, cell survival rate is by the ratio calculation of sample with respect to contrast.
3, ELISA method (standard HBsAg diagnostic kit) test sample is to the restraining effect of HBsAg: standard reagent box detection method: add the cell culture supernatant of drug incubation after 5,7,14 days, sucking-off a little, measure with the ELISA method, detect the OD value at 450nm.Measure HBsAg, the result is with the P/N value representation.With similarity condition with do not contain sample culture medium culturing cell in contrast.After the P/N ratio of 6 parallel holes averaged, calculate inhibiting rate.
Inhibiting rate=(control wells P/N value-experimental port P/N value) ÷ (control wells P/N value-2.1)
4, positive drug contrast: lamivudine (3-TC).
5, test compounds I-12 sees Table two to the test-results of HBsAg inhibiting rate among detected result: the pharmacology embodiment 1.
Table two. the percent inhibition of Compound I-12 pair HepG2.2.15 excretory hepatitis B surface antigen and the inhibiting rate of the 12nd day cell growth
| Sample number into spectrum | Concentration (μ g/mL) | 4 days | 8 days | 12 days | 12 days MTT |
| Compound I-12 | 100 | 43.87 | 43.67 | 52.56 | 9.88 |
| 20 | 25.07 | 25.73 | 38.63 | 5.05 | |
| 4 | 19.72 | 19.24 | 13.43 | 0.82 | |
| Lamivudine | 100 | 10.10 | 12.16 | 11.96 | 8.94 |
| 20 | 1.41 | 3.23 | 11.05 | 6.68 | |
| 4 | 0 | 0 | 8.24 | 6.59 |
Test-results explanation: the most valuable hepatitis b virus infected sign of judgement has at present: B-mode surface antigen and antibody (HBsAg with anti--HBs), hepatitis B e antigen and antibody (HBeAg and resisting-HBe), hepatitis B core antibody and antibody (HbcAg and anti-HBc) etc.Wherein HBsAg is an important symbol of judging that HBV infects, and suppressing HBsAg and HBsAg is turned out cloudy to react is one of direct purpose in the treatment hepatitis B.Compound I-12 the 4th day, the 8th day and the 12 day compd A all demonstrate certain inhibition activity to hepatitis B surface antigen HBsAg when different concns, and active stronger to the inhibition of hepatitis B surface antigen than positive control medicine lamivudine.
Conclusion: Compound I-12 belongs to highly active hepatitis B surface antigen HBsAg inhibitor.Such silibinin monoesters analog derivative (Compound I-12 is an example) belongs to potent HBsAg inhibitor.
The inhibition test of pharmacology embodiment 2. Compound I-12 pair hepatitis B virus e antigens (HBeAg)
Materials and methods: according to the method for describing among the pharmacology embodiment 1, we have detected the restraining effect of sample to HBeAg with ELISA method (standard HBeAg diagnostic kit).With Compound I-12 is the example explanation.Standard reagent box detection method: add the Xi Bao Pei Yang supernatant liquor of drug incubation after 5,7,14 days, sucking-off a little, measure with the ELISA method, detect the OD value at 450nm.Measure HBeAg, the result is with the P/N value representation.With similarity condition with do not contain sample culture medium culturing cell in contrast.After the P/N ratio of 6 parallel holes averaged, calculate inhibiting rate.Positive drug contrast: lamivudine (3-TC).
Inhibiting rate=(control wells P/N value-experimental port P/N value) ÷ (control wells P/N value-2.1)
Test sees Table three to the test-results of HBeAg inhibiting rate among detected result: the pharmacology embodiment 2.
Table three. the percent inhibition of Compound I-12 pair HepG2.2.15 excretory hepatitis B virus e antigen and the inhibiting rate of the 12nd day cell growth
| Sample number into spectrum | Concentration (μ g/mL) | 4 days | 8 days | 12 days | 12 days MTT |
| Compound I-12 | 100 | 11.32 | 30.73 | 31.03 | 9.88 |
| 20 | 7.65 | 16.67 | 16.92 | 5.05 | |
| 4 | 1.56 | 11.62 | 12.96 | 0.82 | |
| Lamivudine | 100 | 8.01 | 4.17 | 7.88 | 8.94 |
| 20 | / | 3.05 | 5.15 | 6.68 | |
| 4 | / | / | / | 6.59 |
Test-results explanation: hepatitis B e antigen and antibody (HBeAg with anti--HBe) be to judge hepatitis b virus infected important symbol, suppressing HBeAg and making the HBeAg reaction of turning out cloudy is to treat one of direct purpose in the hepatitis B.The 4th day, the 8th day and the 12 day Compound I-12 all demonstrate stronger inhibition activity to hepatitis B virus e antigen HBeAg when high density, and than positive control medicine lamivudine to the active strong manyfold of the inhibition of hepatitis B virus e antigen.
Conclusion: compd B belongs to the hepatitis B virus e antigen of greater activity HBeAg inhibitor.Such silibinin monoesters analog derivative (Compound I-12 is an example) belongs to potent HBeAg inhibitor.
The inhibition test of pharmacology embodiment 3. Compound I-12 pair hepatitis B virus thymus nucleic acid (HBV-DNA)
1. the automatic fluorescent PCR instrument of instrument and reagent: PE7700, U.S. Perkin Elmer company produces; The HBV-DNA fluorescence quantitative detection kit reaches peace gene diagnosis center by Zhongshan Medical Univ. to be provided, and foetal calf serum, DMEM, G418, trypsinase are all available from Gibco company.
2. cell in vitro model: HepG 2.2.15 cell strain provides by being cultivated by Zhejiang University's Chinese medicine and natural drug research department, and the HepG2.2.15 cell inoculation (is contained 10% foetal calf serum, 380ug/mLG418), puts 5%CO in the DMEM nutrient solution
2Cultivate in 37 ℃ of incubators.
3. drug effect: HepG 2.2.15 cell digests with tryptic digestive juice, is diluted to 8 * 10
4/ mL suspension is inoculated in 96 porocyte culture plates, and every hole 200uL is after 24 hours, change the pastille nutrient solution, the I-12 drug level is respectively 0.5mg/mL, 0.05mg/mL, 0.005mg/mL, each concentration is established 6 parallel holes, continues to cultivate 24 hours, and it is frozen to be checked in-20 ℃ to leave and take culture supernatant.With the nutrient solution cultured cells that do not contain medicine under the similarity condition in contrast,, use the same method and measure its restraining effect as positive control with lamivudine HBV.
4.HBV-DNA quantitatively adopt conventional alkaline lysis from culture supernatant, to extract HBV-DNA.Press the operation of reagent specification sheets, contain the 30uL reaction buffer in the 50uL reaction volume, 5uL MgCl
2, 5uL primer and probe, 7uL sample preparation supernatant liquor and 3uL Taq enzyme.Each reaction tubes is put into the PCR instrument, by the amplification of following condition: 92 ℃ of pre-sex change in 2 minutes, press then 93 ℃ 45 seconds-55 ℃ 120 seconds, totally 40 circulations.Reaction is calculated the result by the computer automatic analyser after finishing.
5. test-results: the inhibition test result of Compound I-12 pair hepatitis B virus thymus nucleic acid (HBV-DNA) as shown in Table 4
Table four. the percent inhibition of Compound I-12 pair HepG2.2.15 thymus nucleic acid (HBV-DNA) and the inhibiting rate of the 12nd day cell growth
| Sample number into spectrum | Concentration (μ g/mL) | 8 days | 12 days | 12 days MTT |
| Compound I-12 | 100 | 51.73 | 51.72 | 9.88 |
| 20 | 27.43 | 27.17 | 5.05 | |
| 4 | 16.14 | 16.00 | 0.82 | |
| Lamivudine | 100 | 82.22 | 83.51 | 8.94 |
| 20 | 80.07 | 78.68 | 6.68 | |
| 4 | 38.89 | 37.86 | 6.59 |
Test-results explanation: the 4th day, the 8th day and the 12 day Compound I-12 couple HBV-DNA all demonstrate certain inhibition activity, but a little less than the inhibition activity of positive control medicine lamivudine to HBV-DNA, belonging to has certain active HBV-DNA inhibitor.
Pharmacology embodiment 4 Compound I-1 pair rat acute carbon tetrachloride hepatic injury (liver injury model in the animal body) is protected active test
1, after tetracol phenixin enters in the body, in the liver cell endoplasmic reticulum,, generates active trichloromethyl free radical and chlorine radical through the metabolism of cytochrome P-450 dependency mixed-function oxidase.These free radicals can with cell in and the macromole covalent attachment of cytolemma, make the enzyme afunction, the peroxidation of cytolemma lipid, endochylema Ca
2+Concentration raises, and causes hepatocellular injury, and transaminase is infiltrated blood in the endochylema.
2, ICR mouse, complete male, 48.(available from the drug inspection office, Zhejiang Province).Be divided into normal group, model group, positive group, reagent (L) group, reagent (M) group, reagent (H) group, 8 every group.Each treated animal administration situation is as follows: (1) normal group: physiological saline (NS); (2) model group: NS; (3) positive group: Biphenylylmethylcarbinol 100mg/kg; (4) reagent I-1 (L) group: 50mg/kg; (5) reagent I-1 (M) group: 100mg/kg; (6) reagent I-1 (H) group: 200mg/kg; Each is organized medicine and all dissolves with NS.Grouping back gastric infusion (10ml/kg), be administered once every day, continuous 7 days.After the last administration 1 hour, except that normal group, all participate in modeling.Be made into 1% CCl with sweet oil
4Solution is with the preceding mixing that vibrates repeatedly.The CCl of abdominal injection 1%
4Solution (10ml/kg) intoxicating.Overnight fasting behind the injection poisonous substance.After the last administration 24 hours, pluck eyeball and get blood, separation of serum is surveyed AST and ALT.Get a fritter hepatic tissue, it is fixing to put into formalin solution immediately, is used to make pathological section.
3, the detection of the detection of serum glutamic oxalacetic transaminase (AST/GOT) and serum glutamic pyruvic transminase (ALT/GPT): the results are shown in Table five.
Table five. the influence of Compound I-1 couple acute liver damage mice serum AST/GOT and ALT/GPT
| Group | Dosage (mg/kg) | Number of animals | AST/GOT | ALT/GPT |
| Normal group model control group DDB group I-1 (L) group I-1 (M) group I-1 (H) group | - - 100 50 100 200 | 8 8 8 8 8 8 | 28.46±3.95 39.57±4.21 35.12±5.66 35.02±3.24 31.93±4.81 29.10±2.05 | 16.93±2.17 37.98±6.55 15.78±3.76 22.14±5.57 21.01±5.26 20.67±7.27 |
Serum AST and ALT detected result show: reagent I-1 and positive drug Biphenylylmethylcarbinol can obviously reduce liver injury mice serum AST/GOT and ALT/GPT level under test dose; Wherein I-1 (H) group can significantly reduce liver injury mice serum AST/GOT level and ALT/GPT level; Illustrate that such Silymarin ester compound has the effect of protection hepar damnification.
4, histopathology:
(1), pathological section making method:
Draw materials: every animal is got the roughly the same fritter hepatic tissue (containing coating) in same leaf liver position, size: 0.5cm * 0.5cm * 0.2cm, rip cutting or crosscut, according to standard pathology section program fix, wash, dehydration, transparent, waxdip, embedding, section, exhibition sheet, roasting sheet, dewaxing, cleaning, aquation, HE dye, break up, return indigo plant, dehydration and transparent, mounting, observe after the numbering.1, visual inspection: model group mouse volume and color and luster do not have obvious change.2, microscopically is observed: the necrosis of model group Mouse Liver leaflet central area cytopathy, several hepatic necrosis kitchen ranges fusions are tabular.Reagent group and the damage of positive group mouse liver cell all have clear improvement.Conclusion: histopathology detects and shows, reagent and positive drug can be improved the mouse liver cell damage that tetracol phenixin causes in various degree.
The test-results explanation: such Silymarin ester compound has the effect of protection hepar damnification, and can improve the mouse liver cell damage that tetracol phenixin causes.
The oxygen pressure is caused by the imbalance between body cell generation and the removing free radical, can bring out multiple disease.Oxygen is pressed can cause nervous system disorders, as apoplexy, and Parkinson's disease, alzheimer's disease.It is also relevant with the pathology approach of other disease in addition, as heart trouble, autoimmune disorder, tumour, virus disease (as AIDS, hepatitis).Therefore seek new antioxidant and become the various diseases that causes is pressed in treatment by oxygen effective way.The present invention is with the activity test of the super oxyradical of external removing negatively charged ion; External removing free radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl, activity test DPPH); Measure compound to oxydol H
2O
2Due to the provide protection of PC12 (pheochromocytoma on the kidney of rats) cell injury; The anti-oxidant activity of compound in the test explanation claims of the present invention such as lipoperoxide generation of compound inhibition free yl induction.Illustrated with pharmacology embodiment respectively below.
The detection that the activity test Compound I-1 of the external removing ultra-oxygen anion free radical of pharmacology embodiment 5 Compound I-1 is removed the ultra-oxygen anion free radical ability is to use azophenlyene-N metilsulfate-NADH (phenazine methosulfate-NADH) system, with the calibrating of nitroblue tetrazolium (nitroblue tetrazolium) method of reducing.In the pH value is 8.0 16mM Tris-HCl damping fluid, produce ultra-oxygen anion free radical with 3 milliliters of NADH, 50 μ M nitroblue tetrazolium and 10 μ M azophenlyene-N metilsulfates that contain 78 μ M, the Compound I-1 of different concns is detected its activity.The color of ultra-oxygen anion free radical and nitroblue tetrazolium resultant of reaction is monitored under the 560nm wavelength with spectrophotometer, and Quercetin is used as the positive control medicine.Test-results sees Table six.
Table six
| Sample | Test concentrations (μ g/mL) | To the ultra-oxygen anion free radical clearance rate |
| Compound I-1 Quercetin | 40 40 | 29.06％ 63.24％ |
Test-results shows that Compound I-1 has certain ultra-oxygen anion free radical scavenging(action), but under same concentration, its removing ability is less than the Quercetin height.Conclusion: such silymarin monoester class compound belongs to the antioxidant with certain removing ultra-oxygen anion free radical.
The external removing free radical scavenging activity of pharmacology embodiment 6 Compound I-18 (1,1-diphenyl-2-picrylhydrazyl, activity test DPPH)
The methanol solution of DPPH has strong absorption value at 517nm, and when it was reduced by polyphenoils, absorption value descended, and absorbancy is low more, and its antioxygenation is strong more.Compound I-1825 μ the L that contains various different concns in 250 μ L reaction systems, the methanol solution 40 μ L of DPPH (0.4mgmL-1) and methanol solution 185 μ L, 37 ℃ of water-baths measured absorbancy after 30 minutes at the 517nm place.The methanol solution of DPPH and Quercetin as negative, positive control, the results are shown in Table seven respectively.
Table seven
| Sample | Test concentrations (μ g/mL) | To the ultra-oxygen anion free radical clearance rate |
| Compound I-18 Quercetin | 40 40 | 59.88％ 87.42％ |
Test-results shows that Compound I-18 has certain DPPH free radical scavenging effect, but under same concentration, its removing ability is less than the Quercetin height.Conclusion: such silymarin monoester class compound belongs to the antioxidant with certain removing DPPH free radical.
Pharmacology embodiment 7 Compound I-6 pair oxydol H
2O
2Due to the provide protection activity test of PC12 cell injury
H
2O
2It is a kind of precursor of main living radical, it can cause the apoptosis of central nervous system cell, PC12 cell (pheochromocytoma on the kidney of rats) can be simulated cranial nerve cell, therefore use it always and be used as studying the model that concerns between medicine and the neurocyte, with the survival rate of MTT survey cell, if testing compound has removing by H
2O
2The free radical that causes, the effect of neuroprotective cell, the OD value is high, and cell survival rate is just high, otherwise just low.To Tang Xican institute reported method (Xiaoqiu Xiao etc., Neurosci Letter, 1999,275:73-76.) improved the provide protection of measuring compound.PC12 cell DMEM culture medium culturing contains 10% foetal calf serum in the substratum, 100U/mL penicillin and 100U/mL Streptomycin sulphate.Cell is added in 96 orifice plates with the density in 6000 in every hole, at 37 ℃, and 50%CO
2Cultivated 36 hours in the incubator of damp atmosphere.The mtt assay that cell survival rate is observed and improved with inverted microscope.Cell is after 36 hours hatch, and the dimethyl sulfoxide solution that adds the Compound I-6 of newly joining respectively joins in each hole with concentration gradient.Act on the H that adding is newly joined after 2 hours
2O
2(final concentration is 500 μ mol/L) effect 3 hours, the microscopic examination record discards original fluid, adds new nutrient solution 100 μ L, adds MTT10 μ L then, and after 3 hours, the careful suction removed nutrient solution, adds 150 μ LDMSO Rong Xie Jia Za, in 570nm place reading.Make positive control with Quercetin, the results are shown in Table eight
Table eight Compound I-6 and positive control Quercetin are to hydrogen peroxide damage PC12 cytoprotective rate
| Sample | Test concentrations (μ g/mL) | Cell survival rate before the dosing | Cell survival rate after the dosing |
| Compound I-6 | 32 16 | 16.30％ 15.18％ | 89.58％ 84.24％ |
| Quercetin | 8 32 16 8 | 15.49％ 16.30％ 15.18％ 15.49％ | 78.82％ 52.82％ 47.99％ 45.47％ |
Test-results shows, Compound I-6 has the very strong PC12 cell injury effect that Green Tea Extract is caused, promptly the PC12 cell to the simulation cranial nerve cell has the anti-oxidative damage provide protection.And under same concentration, its energy force rate positive control Quercetin of removing free radical protection cell is also high.Conclusion: such silymarin monoester class compound belongs to the antioxidant of the PC12 cytosis with potent protection simulation cranial nerve cell.
Measure compound to H
2O
2Due to the provide protection of PC12 cell injury can be used as the mechanism of action of its protection maincenter cranial nerve cell of preliminary discussion.So this compounds shows the provide protection to PC12 cell due to the hydrogen peroxide damage, illustrates that its treatment to the senile dementia card has active effect.
Pharmacology embodiment 8 Compound I-2 suppress the activity test of the lipoperoxide generation of free yl induction
1. experimental principle: lipid peroxidation is the product that free radical acts on polyunsaturated fatty acid, and the generation positive correlation of content and free radical because body has the provide protection of oxidation resistant enzyme system and non-enzyme system, constantly produces free radical and constantly is eliminated again.With advancing age, intravital antioxidant constantly descends, and the ability of removing free radical weakens gradually, and lipid peroxidation then strengthens, and lipid peroxidation product increases.Therefore, the working sample lipoid peroxidization resistant is that screening is anti-oxidant, one of important indicator of antiaging agent.
The lipoperoxide generation that 2 Compound I-2 suppress free yl induction is to detect by the mouse hepatomicrosome.(1) preparation of hepatomicrosome: the experimental rat sacrificed by decapitation, take out the liver stolen goods rapidly, prepare hepatomicrosome with ultracentrifugation.(2) sample is to the restraining effect of mouse liver lipid peroxidation: add in the 200 μ g/mL microsomes in containing the 1mL damping fluid of FeSO4 (4 μ mol/L) and Vc (50 μ mol/L), to induce the generation lipid peroxidation, the I-2 sample that adds different concns simultaneously, hatch for 37 ℃ and add 1mL Tricholroacetic Acid (20%) stopped reaction adding 15mL thiobarbituricacid (0.76%) after 30 minutes, 100 ℃ of boiling water baths 20 minutes, centrifugal removal albumen precipitation is measured the supernatant absorbancy under the 532nm wavelength.Lipid peroxy is represented with the mda that generates in the reaction, tests with Quercetin as positive control.Test-results sees Table nine.
Table nine. sample is to the restraining effect of rat liver microsomal lipid peroxidation
| Sample number into spectrum | IC 50(mol/L) |
| The I-2 Quercetin | 2.15×10 -6 2.35×10 -6 |
Test-results shows that Compound I-2 has the lipoperoxide generative capacity of very strong inhibition free yl induction, and promptly pair cell has the anti-oxidative damage provide protection.And under same concentration, its pair cell anti-oxidative damage energy force rate positive control Quercetin is also high.Conclusion: such silymarin monoester class compound belongs to and has potent protection cell antioxidant.Point out it to have liver protecting and expection and be used to the effect for preparing the prevention or treat acute chronic hepatic injury class disease.
The provide protection test of pharmacology embodiment 9 Compound I-18 pair SD neonate rat primary cell hydrogen peroxide damage model
Aseptic taking-up SD rat freshman suckling mouse (in 5 ages in days) liver shreds, and the tryptic digestion with 0.25% is made hepatocyte suspension.Hepatocyte suspension is collected in the Erlenmeyer flask, filters, clean with scavenging solution again with 200 order double-layer nylons, centrifugal 3 times, resuspended with nutrient solution, can obtain most of suspension of hepatic parenchymal cells that is.
With the hepatocyte suspension of RPMI-1640 (including 10% calf serum, 105U/L penicillin, 100mg/L Streptomycin sulphate 10mg/L Regular Insulin) dilution purifying, every milliliter contains 1.0 * 10
6Individual liver cell.Above-mentioned hepatocyte suspension is added in the culture plate of 96 holes (every hole 0.1ml), put 5%CO
2In the incubator, after cultivating 12h under 37 ℃, inhale and abandon supernatant, add the H of 0.6mmol/L
2O
2Behind the effect 1h, add the I-18 specimen test soup of high, medium and low 3 kinds of different concns respectively, each concentration is established 3 multiple holes at least, establishes solvent and positive controls simultaneously.After continuing to cultivate 48h, inhale and abandon supernatant, collect the liver cell sample, calculate protection ratio and hyperplasia index with microplate reader under the 570nm wavelength, the results are shown in Table ten
Table ten. the provide protection of SD neonate rat primary hepatocyte hydrogen peroxide damage model
| Sample number into spectrum | Concentration (μ g/mL) | Protection ratio (%) | The hyperplasia index |
| Quercetin I-18 | 100 50 10 1 100 50 10 1 | 34.9 14.8 4.4 2.2 27.2 9.3 2.1 0.4 | 4.36 2.43 1.42 1.21 3.23 2.01 1.19 1.08 |
Test-results shows: Compound I-18 has the ability that stronger protection SD neonate rat primary hepatocyte is avoided the hydrogen peroxide damage model, promptly SD neonate rat primary hepatocyte is had the anti-oxidative damage provide protection.But under same concentration, its pair cell anti-oxidative damage energy force rate positive control Quercetin is lower slightly.Conclusion: such silymarin monoester class compound belongs to and has effective protection cell antioxidant.Point out it to have liver protecting and expection and be used to the effect for preparing the prevention or treat acute chronic hepatic injury class disease.
Claims (8)
1. silybin esters derivatives shown in the formula (I) and pharmacologically acceptable salt thereof or its solvate:
Formula (I)
N=0,1 wherein; R
1, R
2, R
3Be hydrogen, alkoxyl group, halogen, nitro, itrile group, amino, amide group, R
4, R
6Be hydrogen, alkoxyl group etc., R
5Be hydrogen, alkyl, R
7Be hydrogen or alkoxyl group, wherein the carbonatoms of alkyl or alkoxyl group is a 1-8 carbon atom, can be straight or branched alkyl or alkoxyl group, prosposition in the formula (I), 7 ', 8 ' steric configuration be respectively or be R configuration or S configuration simultaneously.
2. have the silybin ester compounds shown in the formula (I) extremely pharmacologically acceptable salt or its solvate, they are:
Compound I-1: anisic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-2: p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-3: phenylformic acid 3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-4: m-chlorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-5:3, the 4-dimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-6:3,4, the 5-trimethoxybenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-7: piperinic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-8: parafluorobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-9: to itrile group phenylformic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-) 2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-10: to tolyl acrylic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-] methyl ester;
Compound I-11: to chloro-cinnamic acid [3-(4-hydroxyl 3-p-methoxy-phenyl)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-12: to bromo-cinnamic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-13: paraacetaminobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-14:3, the 5-dinitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-15: anisic acid [3-(4-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-16: p-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-8-methoxyl group-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-17: para-amino benzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester;
Compound I-18:3-amino-5-nitrobenzoic acid [3-(4-hydroxy 3-methoxybenzene base)-6-(2,3-dihydro-3,5,7-trihydroxy--4-oxo-chromene-2-)-2,3-dihydro-1,4-benzodioxane-2-]-methyl ester.
3. the preparation method of claim 1 Chinese style (I) compound comprises formula I-a and formula I-b are obtained formula (I) compound through over-churning:
R
1, R
2, R
3, R
7Define identical with formula (I), wherein the azoformic acid diester is a dimethyl ester, diethyl ester, propyl ester, isopropyl ester or butyl ester, solvent is a tetrahydrofuran (THF), methyl-sulphoxide, dimethyl formamide, temperature of reaction is that room temperature is to refluxing, silibinin: phenylformic acid: azoformic acid diester: triphenyl phosphorus=1: 1～3: 1～3: 1～3,0.5～20 hour reaction times.
4. being used for preparation according to the described silibinin monoester class compound I of claim 1～2 suppresses human body hepatitis B virus thymus nucleic acid (HBV-DNA) and reduces hepatitis B virus surface antigen (HBsAg) and the purposes of the medicine of hepatitis B virus e antigen (HBeAg), treatment Type B viral hepatitis and related diseases toxicity disease; Preparation prevents or treats the purposes of acute chronic hepatic injury class disease, liver protecting class medicine; And this compounds is used to prepare, and control is caused by super oxyradical or relevant other physiological changes or disease protection cranial nerve comprise purposes anti-oxidant, that prevent and treat disease medicaments such as senile dementia and inflammation, autoimmune disorder, tumour, myocardial ischemia, myocardial hypertrophy, aging, transformation reactions, atherosclerosis.
According to the preparation-obtained liver protecting medicine of the purposes of claim 4, prevent and treat Type B viral hepatitis, prevent and treat the senile dementia medicine, treatment cardiovascular and cerebrovascular disease medicine, antiaging agent and/or antitumor drug or pharmaceutical composition.
6. one kind is used to prepare the pharmaceutical composition of removing super oxyradical, suppressing hepatitis B virus, protection hepatocellular injury and acute and chronic liver injury, and it contains as the claim 1～2 of the treatment significant quantity of activeconstituents described Compound I or their compound or pharmaceutically acceptable salt thereof or solvate or their mixture and pharmaceutically acceptable auxiliaries.
7. according to the pharmaceutical composition of claim 4, it is liver-protecting medicine, prevents and treats Type B viral hepatitis, prevents and treats the senile dementia medicine, treats cardiovascular and cerebrovascular disease medicine, antiaging agent and/or antitumor drug medicine.
8. according to the pharmaceutical composition of claim 4, it can be tablet, capsule, injection, aerosol, suppository, film, pill, externally-applied liniment, oral liquid or ointment, can also adopt known controlled release of modern pharmaceutical circle or slow release formulation.
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| CN101966173A (en) * | 2010-10-27 | 2011-02-09 | 大理学院 | Medicinal use of dinitrobenzoylsilybin for preparing glycosidase inhibitor |
| CN104744447A (en) * | 2013-12-31 | 2015-07-01 | 天士力制药集团股份有限公司 | Silibinin rosmarinate as well as preparation method and application thereof |
| CN104744447B (en) * | 2013-12-31 | 2017-12-26 | 天士力制药集团股份有限公司 | Legalon rosemary acid esters and its production and use |
| CN106317033A (en) * | 2016-08-18 | 2017-01-11 | 杭州市西溪医院 | Silybin 23-substituted derivative and preparation method and application of injection thereof |
| CN110317197A (en) * | 2018-03-28 | 2019-10-11 | 天士力医药集团股份有限公司 | A kind of aminomethyl substituted silibinin derivative and its preparation method and application |
| CN110317197B (en) * | 2018-03-28 | 2023-03-28 | 天士力医药集团股份有限公司 | Aminomethyl substituted silybin derivative and preparation method and application thereof |
| CN111925394A (en) * | 2020-09-25 | 2020-11-13 | 嘉兴金派特生物科技有限公司 | Silybin derivative or pharmaceutically acceptable salt thereof, and preparation method and application thereof |
| CN112402424A (en) * | 2020-11-06 | 2021-02-26 | 杭州医学院 | Small molecule medicine and application thereof in treating intermittent hypoxia |
| CN112402424B (en) * | 2020-11-06 | 2022-03-29 | 杭州医学院 | Small molecule medicine and application thereof in treating intermittent hypoxia |
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