CN101077873A - Novel NEO-clerodane type diterpene compound and application thereof - Google Patents

Novel NEO-clerodane type diterpene compound and application thereof Download PDF

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CN101077873A
CN101077873A CN 200710106626 CN200710106626A CN101077873A CN 101077873 A CN101077873 A CN 101077873A CN 200710106626 CN200710106626 CN 200710106626 CN 200710106626 A CN200710106626 A CN 200710106626A CN 101077873 A CN101077873 A CN 101077873A
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compound
chromatographic column
scutebarbatine
sephadex
mixture
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CN101077873B (en
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戴胜军
赵大洲
李振
刘珂
姜永涛
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Shandong Luye Pharmaceutical Co Ltd
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Shandong Luye Pharmaceutical Co Ltd
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Abstract

The present invention provides one new NEO-crotalkane type diterpene compound extracted from barbed skullcap herb. The preparation process includes reflux extracting barbed skullcap herb with methanol or ethanol, decompression concentrating the extracted liquid to obtain extractum, repeated extracting the extractum with petroleum ether, chloroform, ethyl acetate and n-butanol, eluting the chloroform extract in silica gel column with petroleum ether-acetone, and repeated column chromatography with silica gel column, reverse C18 column and Sephadex LH-20 chromatographic column. The present invention also provides medicine composition with the compound as the active component and the tumor cell inhibiting activity and antitumor medicine preparing application of the compound.

Description

New NEO-Crow alkane type diterpenoid and application thereof
Technical field
The present invention relates to new NEO-Crow alkane type diterpenoid, relate to the new NEO-Crow alkane type diterpenoid that from Herba Scutellariae Barbatae, extracts specifically.The invention still further relates to the application of this compound in suppressing tumour cell.
Background technology
Herba Scutellariae Barbatae (Scutellaria barbata D.Don) is a Labiatae Scutellaria plant, calls narrow leaf Indian Skullcap Herb, and head grass, toothbrush grass etc., with dry all herbal medicine.Its name sees " surgery orthodox school " the earliest, and flavor is hot, bitter, cold in nature, returns lung, liver, kidney channel, has the effect of clearing heat and detoxicating, stagnation resolvation, diuresis, is used for the treatment of diseases such as furuncle swelling toxin, swelling and pain in the throat, venomous snake bite, tumbling and swelling and oedema, jaundice.Among the people in China, often compatible Herba Scutellariae Barbatae and other herbal medicine, with treatment hepatitis and various cancer, as ovarian tumor, secondary pleurisy tumour, nasopharyngeal carcinoma, liver cancer, cancer of the stomach etc.
Contain number of chemical compositions such as flavonoid, sterol, terpene, alkaloid, polysaccharide and organic acid in the Herba Scutellariae Barbatae, up to the present, found that from Herba Scutellariae Barbatae [Xiao Haitao, Lee mill 13 NEO-Crow alkane type diterpene, Herba Scutellariae Barbatae chemical ingredients and pharmacology activity research progress, traditional Chinese medicine research and information, 2005.7 (4), 20-22,25].The C of Scutebarbatine A only wherein 7The position is a chiral carbon atom.By to domestic and international patent and literature search, do not find that so far NEO-Crow alkane type diterpene in the Herba Scutellariae Barbatae has the report of pharmacologically active.
Figure A20071010662600071
In view of above reason, the inventor is through further investigation, and extraction separation has obtained a kind of new NEO-Crow alkane type diterpene from Herba Scutellariae Barbatae, and proves that it has the effect that suppresses growth of tumour cell.
Summary of the invention
The invention provides a kind of new NEO-Crow alkane type diterpenoid or its salt, its structural formula is as follows:
Figure A20071010662600081
Formula I
Wherein:
R 1And R 2Independently be selected from: OH-, AcO-,
Figure A20071010662600082
Or
R 3Be selected from: OH-,
Figure A20071010662600084
Or
Figure A20071010662600085
X is selected from
Figure A20071010662600086
Or
Figure A20071010662600087
Formula II
Wherein:
R 4Be selected from OH-,
Figure A20071010662600089
Or
Figure A200710106626000810
R 5, R 6Independently be selected from OH-or
Figure A200710106626000811
Y is selected from
Figure A200710106626000812
Figure A200710106626000814
Figure A200710106626000815
Or
Figure A20071010662600091
Formula III
Wherein:
R7 is selected from-CH2OAc or CH 3-;
R8 be selected from H-, OH-or
Figure A20071010662600092
R9 is selected from H-, OH-or CH 3-;
N is selected from
Figure A20071010662600093
Or
Figure A20071010662600094
M is selected from
Figure A20071010662600095
Or R wherein 10, R 11Be independently selected from-OEt or hydrogen;
AB is singly-bound or two key.
The said compound of the present invention is specifically as follows:
Figure A20071010662600097
Formula I
Compound 1 (barbatinA): R 1=
Figure A20071010662600098
R 2=OH-, R 3=
Figure A20071010662600099
X=
Compound 2 (scutebarbatine F): R 1=AcO-, R 2=AcO-, R 3=
Figure A200710106626000911
X=
Figure A200710106626000912
Compound 3 (scutebarbatine I): R 1=
Figure A200710106626000913
R 2=
Figure A200710106626000914
R 3=
Figure A200710106626000915
X=
Compound 4 (scutebarbatine J): R 1=
Figure A200710106626000917
R 2=AcO-, R 3= X=
Figure A200710106626000919
Compound 5 (scutebarbatine K): R 1=
Figure A200710106626000920
R 2=OH-, R 3=OH-, X=
Figure A200710106626000921
Compound 6 (barbatin B): R 1=OH-, R 2=
Figure A20071010662600101
R 3=
Figure A20071010662600102
X=
Figure A20071010662600103
Figure A20071010662600104
Formula II
Compound 7 (barbatin C): R 4=OH-, R 5=OH-, R 6=OH-, Y=
Figure A20071010662600105
Compound 8 (scutebarbatine B): R 4=
Figure A20071010662600106
R 5=OH-, R 6=
Figure A20071010662600107
Y=
Compound 9 (scutebarbatine N): R 4=
Figure A20071010662600109
R 5=OH-, R 6=
Figure A200710106626001010
Y=
Figure A200710106626001011
Compound 10 (scutebarbatine O): R 4=OH-, R 5=
Figure A200710106626001012
R 6=OH-, Y=
Compound 11 (scutebarbatine C): R 4=
Figure A200710106626001014
R 5=OH-, R 6=
Figure A200710106626001015
Y=
Figure A200710106626001016
Compound 12 (scutebarbatine D): R 4=
Figure A200710106626001017
R 5=OH-, R 6= Y=
Figure A200710106626001019
Compound 13 (scutebarbatine E): R 4= R 5=OH-, R 6= Y=
Formula III
Compound 14 (scutebarbatine G): AB is a singly-bound, R 7=-CH2OAc, R 8=-H, R 9=OH-, M= R 10=OEt, R 11=H-, N=
Figure A200710106626001025
Compound 15 (scutebarbatineH): AB is a singly-bound, R 7=-CH2OAc, R 8=-H, R 9=-CH 3, M=
Figure A20071010662600111
R 10=H-, R 11=-OEt, N=
Figure A20071010662600112
Compound 16 (scutebarbatine L): AB is two keys, R 7=-CH 3, R 8=
Figure A20071010662600113
R 9=-OH, M= N=
Figure A20071010662600115
Compound 17 (scutebarbatine M): AB is two keys, R 7=-CH 3, R 8=-OH, R 9=-OH, M=
Figure A20071010662600116
N=
Figure A20071010662600117
The said salt of the present invention is meant pharmacy acceptable salt, for example the salt that forms with mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid.
The preparation method of compound provided by the present invention is: take by weighing a certain amount of Herba Scutellariae Barbatae herb, and with methyl alcohol or alcohol reflux 3-4 time, each 1-2 hour, united extraction liquid, concentrating under reduced pressure; Medicinal extract after concentrating extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, gets silicagel column on the chloroform extract, carries out gradient elution with sherwood oil acetone, uses silica gel, anti-phase C18 post, Sephadex LH-20 chromatographic column column chromatography repeatedly then, promptly.
Its concrete preparation method is: take by weighing a certain amount of Herba Scutellariae Barbatae herb, and with the methyl alcohol of 60-95% or alcohol reflux 3-4 time, each 1-2 hour, united extraction liquid, concentrating under reduced pressure; Medicinal extract after concentrating extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, gets silicagel column on the chloroform extract, with sherwood oil-acetone (97: 3-50: 50) carry out gradient elution, be divided into 7 parts (Fraction1-7).
Get wherein that Fraction2 crosses Rp C-18 chromatographic column, the methanol-water wash-out, then by Sephadex LH-20 chromatographic column purifying, compound 3 and compound 4;
Fraction3 crosses silica gel chromatographic column, and hexanaphthene-acetone gradient elution gets compound 9 and compound 16;
The last silica gel chromatographic column of Fraction4, hexanaphthene-acetone gradient elution gets a compound 2 and a mixture; Mixture by Rp C-18 chromatographic column, behind the methanol-water wash-out, by Sephadex LH-20 chromatographic column purifying, is got compound 14 and compound 15;
Fraction5 crosses silica gel chromatographic column, hexanaphthene-acetone gradient elution, compound 1, compound 6 and two mixture M 1, M2, mixture M 1 is crossed Rp C-18 chromatographic column, the methanol-water wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 8 and compound 13; Mixture M 2 is crossed Rp C-18 chromatographic column, and the methanol-water wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 5,10 and 17;
Fraction6 crosses silica gel chromatographic column, and hexanaphthene-acetone gradient elution gets a compound 7 and a mixture; Mixture is crossed Rp C-18 chromatographic column, behind the methanol-water wash-out,, get compound 11 and compound 12 by Sephadex LH-20 chromatographic column purifying.
In order to increase the water-soluble of compound provided by the present invention, can form the sodium salt or the sylvite of Succinic anhydried, maleic anhydride, succinyl oxide etc., preferably its butanedioic acid derivative monopotassium salt.Said derivative can make according to ordinary method, also can obtain by the following method:
Get compound and be dissolved in the anhydrous pyridine, add Succinic anhydried then, 80 ℃ were heated 50 minutes, added saleratus, stirred 30 minutes in the time of 50 ℃, promptly got the butanedioic acid derivative monopotassium salt.
The present invention also provides the Herba Scutellariae Barbatae extract that contains one or more NEO-Crow alkane type diterpenoids or its salt, and wherein NEO-Crow alkane type diterpenoid is medicinal significant quantity.
The present invention also provides compound and the Herba Scutellariae Barbatae extract restraining effect to tumour cell, and the application in preparation treatment antitumor drug.
Compound provided by the present invention or contain the Herba Scutellariae Barbatae extract of this compound can oral or non-oral form administration, dosage is had nothing in common with each other because of compound is different, effective dose is 2mg/kg to 100mg/kg.
During the oral administration administration, can mix, be made into form administrations such as granule, capsule, soft capsule, tablet, dripping pill or oral liquid with the pharmaceutical excipient of routine such as weighting agent, disintegrating agent, tackiness agent, lubricant, Drug coating etc.During non-oral form administration, can be prepared into injection liquid, lyophilized injectable powder, infusion solution etc.When preparing above-mentioned preparation, can use conventional preparation technique, but preferred method provided by the present invention.
Wherein said weighting agent can be selected from lactose, N.F,USP MANNITOL, starch, Microcrystalline Cellulose, the calcium sulfate etc. one or more; Wherein said disintegrating agent can be selected from low-substituted hydroxypropyl cellulose, the crosslinked sodium carboxymethylcellulose pyce that contracts, sodium starch glycolate, cross-linked polyvinylpyrrolidone, the Microcrystalline Cellulose etc. one or more; Wherein said tackiness agent is selected from one or more in hypromellose, polyvinylpyrrolidone, starch, methylcellulose gum, dextrin, the Icing Sugar etc.; Wherein said lubricant can be selected from one or more in Magnesium Stearate, calcium stearate, talcum powder, the micropowder silica gel etc.
Wherein said tablet can make in accordance with the following methods: with compound provided by the invention or contain the Herba Scutellariae Barbatae extract of this compound and weighting agent, disintegrating agent thorough mixing even, after sieving, add certain density binder solution and make softwood in right amount, scalping is granulated, behind the dry whole grain, add proper amount of lubricating agent, mixing, compressing tablet are promptly.Also can select dressing behind the compressing tablet.
Wherein said injection liquid can make in accordance with the following methods: with a certain amount of compound provided by the invention or contain the Herba Scutellariae Barbatae extract of this compound, add an amount of water for injection, stirring and dissolving, the pH value of regulator solution is used the activated carbon treatment after-filtration, measures intermediate pH value and content, after qualified, under aseptic condition with 0.22 μ m filtering with microporous membrane, embedding, sealing by fusing; 100 ℃ of flowing steam sterilizations 30 minutes promptly.
Wherein said lyophilized injectable powder can make in accordance with the following methods: with compound provided by the invention or contain the Herba Scutellariae Barbatae extract of this compound, insert in the sterilized container, add an amount of water for injection, stirring and dissolving, add certain density vehicle again and (be selected from lactose, sucrose, glucose, N.F,USP MANNITOL, gelatin hydrolysate, in the dextran etc. one or more) solution, the pH value of regulator solution behind the mixing, use the activated carbon treatment after-filtration, measure intermediate pH value and content, after qualified, under aseptic condition with 0.22 μ m filtering with microporous membrane, packing filtrate, and add butyl rubber plug, put in the Freeze Drying Equipment and carry out lyophilize promptly.
Embodiment
Following examples illustrate in greater detail the present invention, but do not limit the present invention in any form.
Embodiment one: the preparation of chloroform extract
Get 19.0 kilograms of Herba Scutellariae Barbatae herbs, 95% alcohol reflux 3 times, each 1 hour, united extraction liquid, concentrating under reduced pressure got about 1.0 kilograms of medicinal extract.Medicinal extract extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, and combined chloroform extraction liquid, concentrating under reduced pressure get chloroform extract 156.0 grams.
Embodiment two: the preparation of chloroform extract
Get 19.0 kilograms of Herba Scutellariae Barbatae herbs, 60% methanol eddy extracts 4 times, and each 1 hour, united extraction liquid, concentrating under reduced pressure got about 0.9 kilogram of medicinal extract.Medicinal extract extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, and combined chloroform extraction liquid, concentrating under reduced pressure get chloroform extract 148.0 grams.
Embodiment three: the preparation of compound
Get the last silicagel column of chloroform extract 147.8g among the embodiment one, be divided into 7 parts (Fraction 1-7) by sherwood oil-acetone [V/V, 97: 3-94: 6-90: 10-85: 15-80: 20-70: 30-50: 50] gradient elution.
Get 2.0 gram Fraction 2 and cross Rp C-18 chromatographic column, methanol-water (55: 45) wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 3 (15mg) and compound 4 (23mg).
Get 2.9 gram Fraction 3 and cross silica gel chromatographic column, hexanaphthene-acetone gradient elution gets compound 9 (18mg) and compound 16 (23mg).
Get 1.6 gram Fraction 4 and cross silica gel chromatographic column, hexanaphthene-acetone gradient elution gets compound 2 (8mg) and another mixture of 93mg; By Rp C-18 chromatographic column, methanol-water (45: 55) wash-out at last by Sephadex LH-20 chromatographic column purifying, gets compound 14 (11mg) and compound 15 (27mg) with mixture.
Get 10.0 gram Fraction 5 and cross silica gel chromatographic column, hexanaphthene-acetone gradient elution, compound 1 (95mg), compound 6 (38mg) and two mixture M 1, M2, mixture M 1 is crossed Rp C-18 chromatographic column, methanol-water (55: 45) wash-out, by Sephadex LH-20 chromatographic column purifying, get compound 8 (1.2g) and compound 13 (118mg) then; Mixture M 2 is crossed Rp C-18 chromatographic column, and methanol-water (50: 50) wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 5 (53mg), compound 10 (39mg) and compound 17 (40mg).
Get 2.1 gram Fraction 6 and cross silica gel chromatographic column, hexanaphthene-acetone gradient elution gets compound 7 (14mg) and another mixture of 81mg; By Rp C-18 chromatographic column, methanol-water (45: 55) wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 11 (21mg) and compound 12 (13mg) with mixture.
Compound 1: white needle-like crystals, mp150-151 ℃, [α] 29 D-63.6 ° (c 0.12, MeOH).UV(CDCl 3)λ max：219，255nm；IR(KBr)v max：3430，1776，1668，1630，1600，1581，1460，1382，1021，760，720cm -1；FABMS m/z：575.2[M+H] +，HR-FABMS m/z：575.2649[M+H] +(C 34H 39O 8，575.2645)。
Compound 2: white needle-like crystals, mp 159-160 ℃, [α] 29 D-56.9 ° (c 0.14, MeOH).UV(CDCl 3)λ max：217，222，257；IR(KBr)v max cm -1：1788，1731(br)，1642，1256，1229，1023，890，731；FABMS m/z：556.2[M+H] +；HR-FABMS m/z：556.2506[M+H] +(C 30H 38NO 9，556.2547)。
Compound 3: white needle-like crystals, mp 150-152 ℃, [α] 29 D-57.9 ° (c 0.13, MeOH).IR(KBr)v max：1782，1731，1638，1589，1506，1474cm -1；FABMS m/z：682.2[M+H]+；HR-FABMS m/z：682.2743[M+H] +(C38H39N3O9，682.2765)。
Compound 4: white needle-like crystals, mp 149-151 ℃, [α] 29 D-60.3 ° (c 0.12, MeOH).IR(KBr)v max：1781，1733，1642，1592，1500，1467cm -1；FABMS m/z：619.3[M+H] +；HR-FABMS m/z：619.2647[M+H] +(C 34H 38N 2O 9，619.2656)。
Compound 5: white needle-like crystals, mp 156-158 ℃, [α] 29 D-55.7 ° (c 0.14, MeOH).IR(KBr)v max：3450(br)，1771，1635，1609，1583，1467，1361cm -1；FABMS m/z：472.4[M+H] +；HR-FABMSm/z：472.2331[M+H] +(C26H33NO7，472.2335)。
Compound 6: white needle-like crystals, mp148-150 ℃, [α] 29 D-60.4 ° (c 0.13, MeOH).UV(CDCl 3)λ max：220，256nm；IR(KBr)v max：3455，1770，1661，1629，1608，1577，1458，1380，1013，770，721cm -1，FABMS m/z：575.3[M+H] +，HR-FABMS m/z：575.2653[M+H] +(C 34H 39O 8，575.2645)。
Compound 7: white needle-like crystals, mp 156-158 ℃, [α] 29 D-103.8 ° (c 0.14, MeOH).UV(CDCl 3)λ max：220，257nm；IR(KBr)v max：3438(br)，1713，1665，1638，1012cm -1；FABMS m/z：349.4[M+H] +；HR-FABMS m/z：349.2011[M+H] +(C 20H 29O 5，349.2015)。
Compound 8: white needle-like crystals, mp 151-153 ℃, [α] 29 D-109.6 ° (c 0.13, MeOH).UV(CDCl 3)λ max：17，222，257nm；IR(KBr)v max：3342，1780，1743，1727，1643，1591，1501，1451，740，712cm -1；FABMS m/z：558.3[M+H] +；HR-FABMS m/z：558.2487[M+H] +(C 33H 36NO 7，558.2492)。
Compound 9: white needle-like crystals, mp155-156 ℃, [α] 29 D-100.6 ° (c 0.12, MeOH).IR(KBr)v max：3442，1783，1731，1647，1593，1505，1450cm -1；FABMS m/z：573.4[M+H] +；HR-FABMS m/z：573.2241[M+H] +(C32H32N2O8，573.2237)。
Compound 10: white needle-like crystals, mp154-156 ℃, [α] 29 D-98.3 ° (c 0.13, MeOH).IR(KBr)v max：3449，1780，1729，1643，1590，1506，1460cm -1；FABMS m/z：454.3[M+H] +；HR-FABMS m/z：454.2227[M+H] +(C33H34NO8，454.2230)。
Compound 11: white needle-like crystals, mp 156-158 ℃, [α] 29 D-109.6 ° (c 0.13, MeOH).UV(CDCl 3)λ max：220，258；IR(KBr)v max cm -1：3347，1781，1750，1643，1586，1485，1389，887，740，712；FABMS m/z：574.3[M+H] +；HR-FABMS m/z：574.2396[M+H] +(C 33H 36NO 8，574.2441)。
Compound 12: white needle-like crystals, mp 151-153 ℃, [α] 29 D-98.4 ° (c 0.12, MeOH).UV(CDCl 3)λ max：221，260；IR(KBr)v max cm -1：3340，1778，1739，1718，1635，1590，1475，390，883，747，719；FABMS m/z：574.4[M+H] +；HR-FABMS m/z：574.2398[M+H] +(C 33H 36NO 8：574.2441)。
Compound 13: white needle-like crystals, mp 154-156 ℃, [α] 29 D-108.4 ° (c 0.13, MeOH).UV(CDCl 3)λ max：220，259；IR(KBr)v max cm -1：3444，1780，1742，1705，1640，1586，1470，1400，888，733，710；FABMS m/z：572.3[M+H] +；HR-FABMS m/z：572.2239[M+H] +(C 33H 34NO 8，572.2284)。
Compound 14: white needle-like crystals, mp 151-153 ℃, [α] 29 D-3.1 ° (c 0.13, MeOH).IR(KBr)vmax：1725，1710，1591，1477，1439，1248，890，729cm -1；FABMS m/z：544.2[M+H] +；HR-FABMSm/z：544.2923[M+H] +(C 30H 41NO 8，554.2910)。
Compound 15: white needle-like crystals, mp 151-153 ℃, [α] 29 D-16.5 ° (c 0.12, MeOH).IR(KBr)v max：1726，1710，1590，1478，1440，1251，888，730cm -1；FABMS m/z：544.3[M+H] +；HR-FABMSm/z：544.2919[M+H] +(C30H41NO8，554.2910)。
Compound 16: white needle-like crystals, mp 153-154 ℃, [α] 29 D-73.5 ° (c 0.13, MeOH).IR(KBr)v max：3341，1770，1736，1639，1601，1513，1448cm -1；FABMS m/z：575.3[M+H] +；HR-FABMS m/z：575.2387[M+H] +(C32H34N2O8，575.2392)。
Compound 17: white needle-like crystals, mp 157-159 ℃, [α] 29 D-69.8 ° (c 0.14, MeOH).IR(KBr)v max：3450，1783，1740，1641，1588，1512，1459cm -1；FABMS m/z：470.4[M+H] +；HR-FABMS m/z：470.2173[M+H] +(C26H31NO7，470.2179)。
Compound 1-17's 1H-NMR and 13C NMR data see Table 1 to table 6.
Table 1: compound 1-10's 1H-NMR data (400MHz, in CDCl 3) Ab
H 1 2 3 4 5
1 2 3 6 7 10 11 12 14 16 17 18 19 20 3′ 4′ 5′ 6′ 7′ 3″ 4″ 5″ 6″ 7″ 3 5 6 7 OAc OAc b OAc c 1.67(m，H a-1) 2.05(m，H b-1) 2.17(m，2H) 5.31(br s) 5.67(d，9.9) 3.74(d，9.9) 2.86(dd，2.8，2.3) 5.80(dd，3.8，1.7) 1.76(m， a-12) 2.09(m， b-12) 2.54(d，17.6，H a-14) 3.19(d，17.6，H b-14) 4.05(d，8.5，H a-16) 4.20(d，8.5，H b-16) 1.11(s，3H) 1.68(s，3H) 1.39(s，3H) 1.09(s，3H) 7.80(m) 7.39(m) 7.43(br t，7.8) 7.39(m) 7.80(m) 7.79(m，2H) 7.46(m，2H) 7.40(br t，7.7) 7.46(m，2H) 7.79(m，2H) 1.61(m，H a-1) 2.03(m，H b-1) 2.19(m，2H) 5.32(br s) 5.42(d，10.4) 5.25(d，10.4) 2.71(dd，3.0，12.4) 5.80(dd，3.9，11.8) 1.75(m，H a-12) 2.06(m，H b-12) 2.53(d，17.6， a-14) 3.14(d，17.6， b-14) 4.13(d，8.5，H a-16) 4.28(d，8.5，H b-16) 1.12(s，3H) 1.68(s，3H) 1.37(s，3H) 1.10(s，3H) 9.17(br s) 8.83(brd，4.5) 7.45(dd，4.5，7.8) 8.26(brd，7.8) 2.10(s，3H) 2.02(s，3H) 1.71(m，H a-1) 2.09(m，H b-1) 2.80(m，2H) 5.31(br s) 5.89(d，10.3) 5.68(d，10.3) 2.89(dd，2.6，12.2) 5.85(dd，3.7，12.0) 1.78(m，H a-12) 2.14(m，H b-12) 2.68(d，16.6， a-14) 3.13(d，16.6， b-14) 4.13(d，8.8， a-16) 4.20(d，8.8， b-16) 1.24(s，3H) 1.69(s，3H) 1.58(s，3H) 1.22(s，3H) 9.19(br s) 8.83(brd，4.6) 7.47(dd，4.6，7.8) 8.27(br d，7.8) 9.00(br s) 8.70(brd，4.6) 7.30(dd，4.6，7.7) 8.12(brd，7.7) 8.97(br s) 8.67(br d，4.5) 7.23(dd，4.5，7.8) 8.01(brd，7.8) 1.72(m，H a-1) 2.08(m，H b-1) 2.77(m，2H) 5.33(br s) 5.71(d，10.4) 5.44(d，10.4) 2.83(dd，2.7，12.3) 5.84(dd，3.8，11.8) 1.76(m，H a-12) 2.18(m，H b-12) 2.63(d，17.0， a-14) 3.17(d，17.0， b-14) 4.15(d，8.5，H a-16) 4.22(d，8.5，H b-16) 1.16(s，3H) 1.68(s，3H) 1.53(s，3H) 1.17(s，3H) 9.22(br s) 8.82(br d，4.7) 7.45(dd，4.7，7.8) 8.28(br d，7.8) 9.18(br s) 8.79(br d，4.6) 7.42(dd，4.6，7.8) 8.25(br d，7.8) 1.81 1.63(m，H a-1) 1.92(m，H b-1) 2.73(m，2H) 5.31(br s) 3.63(d，10.4) 3.43(d，10.4) 2.51(dd，2.8，12.3) 5.78(dd，3.7，11.7) 1.58(m，H a-12) 2.17(m，H b-12) 2.56(d，16.8，H a-14) 2.78(d，16.8，H b-14) 4.16(d，8.7，H a-16) 4.20(d，8.7，H b-16) 1.42(s，3H) 1.95(s，3H) 1.19(s，3H) 1.05(s，3H) 9.16(br s) 8.83(br d，4.5) 7.45(dd，4.5，7.6) 8.27(br d，7.6)
The a chemical shift represents that with ppm coupling constant J represents with Hz, and is placed in the bracket
The b chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Table 2: compound 6-10's 1H-NMR data (400MHz, in CDCl 3) Ab
H 6 7 8 9 10
1 2 3 6 7 10 11 12 14 16 17 18 19 20 3′ 4′ 5′ 6′ 7′ 3″ 4″ 5″ 6″ 7″ 1.82(m，H a-1) 2.46(m，H b-1) 1.47(m，H a-2) 2.06(m，H b-2) 2.13(m，H a-3) 2.29(m，H b-3) 5.70(d，10.6) 5.61(d，10.6) 2.38(dd，2.0，2.4) 4.34(dd，3.8，2.0) 1.57(m，H a-12) 2.14(m，H b-12) 2.84(d，17.2，H a-14 3.03(d，17.2， a-14) 4.23(d，9.2， a-16) 4.39(d，9.2， b-16) 1.12(s，3H) 4.60(br s，2H) 1.43(s，3H) 1.58(s，3H) 7.91(m) 7.55(m) 7.42(br t，7.7) 7.55(m) 7.91(m) 7.94(m，2H) 7.63(m，2H) 7.48(br t，7.7) 7.63(m，2H) 7.94(m，2H) 1.28(m，H a-1) 1.55(m，H b-1) 2.01(m，2H) 5.22(br s) 3.75(d，9.2) 3.62(d，9.2) 2.09(dd，1.6，2.8) 6.39(d，16.8) 6.30(d，16.8) 5.91(br s) 4.99(dd，1.2，6.4，2H) 1.23(s，3H) 1.42(s，3H) 1.03(s，3H) 1.12(s，3H) 1.35(m， a-1) 1.66(m， b-1) 2.06(m，2H) 5.25(br s) 5.93(d，10.8) 5.72(d，10.8) 2.36(br d，11.6) 6.46(d，16.8) 6.40(d，16.8) 5.94(br s) 5.00(2H，br s) 1.07(3H，s) 1.58(3H，s) 1.45(3H，s) 1.28(3H，s) 8.98(br s) 8.63(br d，4.6) 7.25(dd，4.6，7.8) 8.05(br d，7.8) 7.83(m，2H) 7.33(m，2H) 7.48(br t，7.6) 7.33(m，2H) 7.83(m，2H) 2.21(dd，2.5，17.3，H a-1) 2.50(dd，13.8，17.3，H b-1) 5.76(br s) 6.13(d，10.0) 5.81(d，10.0) 2.95(dd，2.5，13.8) 6.35(d，16.8) 6.49(d，16.8) 5.98(br s) 5.01(br s，2H) 1.13(s，3H) 1.92(s，3H) 1.57(s，3H) 1.39(s，3H) 9.07(br s) 8.71(br d，4.6) 7.31(dd，4.6，7.7) 8.11(br d，7.7) 9.03(br s) 8.69(br d，4.7) 7.28(dd，4.7，7.8) 8.08(br d，7.8) 1.29(m，H a-1) 1.52(m，H b-1) 2.03(m，2H) 5.23(br s) 3.87(d，9.8) 3.65(d，9.8) 2.26(dd，1.9，12.1) 6.34(d，16.8) 6.42(d，16.8) 5.94(br s) 5.02(br s，2H) 1.18(s，3H) 1.57(s，3H) 1.37(s，3H) 1.16(s，3H) 9.30(br s) 8.82(br d，4.5) 7.43(dd，4.5，7.8) 8.36(br d，7.8)
The a chemical shift represents that with ppm coupling constant J represents with Hz, and is placed in the bracket
The b chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Table 3: compound 11-14's 1H-NMR data (400MHz, in CDCl 3) Ab
H 11 12 13 14
1 2 3 6 7 1.81(m，H a-1) 2.03(m， b-1) 5.75(m) 5.41(br d，9.6) 6.57(d，10.4) 5.78(d，10.4) 1.26(m，H a-1) 2.22(m，H b-1) 1.44(m，H a-2) 1.75(m，H b-2) 4.29(dd，4.8，11.4) 6.28(d，10.0) 5.79(d，10.0) 1.74(m，H a-1) 2.02(m，H b-1) 2.40(m，H a-2) 2.69(m，H b-2) 6.32(d，10.3) 5.74(10.3) 1.43(m，H a-1) 1.95(m，H b-1) 2.23(2H，m) 1.13(m，H a-3) 1.71(m，H b-3) 4.74(dd，4.3，11.2) 1.48(m，H a-7)
8 10 11 12 13 14 15 16 17 18 19 20 3′ 5′ 6′ 7′ 3″ 4″ 5″ 6″ 7″ OAc CH 2 CH 3 5.94(br s) 2.76(dd，3.6，2.6) 6.48(d，16.8) 6.42(d，16.8) 5.94(br s) 5.00(br，2H s) 1.11(s，3H) 1.14(s，3H) 1.29(s，3H) 1.39(s，3H) 9.04(br s) 8.66(br d，.6) 7.24(dd，4.6，8) 8.08(br d，7.8) 7.80(m，2H) 7.29(m，2H) 7.49(br t，7.6) 7.29(m，2H) 7.80(m，2H) 2.18(dd，3.0，12.0) 6.38(d，16.8) 6.40(d，16.8) 5.91(br s) 5.08(br s，2H) 1.08(s，3H) 4.95(br s，H a-18) 5.01(br s，H b-18) 1.51(s，3H) 1.26(s，3H) 8.99(br s) 8.66(br d，4.4) 7.29(dd，4.4，7.8) 8.05(br d，7.8) 7.84(m，2H) 7.33(m，2H) 7.49(br t，7.7) 7.33(m，2H) 7.84(m，2H) 2.65(dd，3.2，12.4) 6.44(d，16.8) 6.49(d，16.8) 5.99(br s) 5.03(br s，2H) 1.13(s，3H) 5.19(br s，H a-18) 5.40(br s，H b-18) 1.46(s，3H) 1.36(s，3H) 8.94(br s) 8.66(br d，4.6) 7.24(dd，4.6，7.7) 8.02(br d，7.7) 7.85(m，2H) 7.34(m，2H) 7.50(br t，7.8) 7.34(m，2H) 7.85(m，2H) 1.69(m，H b-7) 1.50(m) 1.73(dd，3.0，11.8) 4.47(dd，5.6，11.2) 1.53(m，H a-12) 1.77(m，H b-12) 2.87(m) 1.61(m，H a-14) 2.20(m，H b-14) 5.79(d，5.5) 5.08(d，5.6) 0.91(d，6.1) 2.57(d，4.2，H a-18) 2.81(d，4.2，H b-18) 4.57(d，12.0，H a-19) 5.29(d，12.0，H b-19) 0.99(3H，s) 9.40(br s) 8.86(br d，4.5) 7.43(dd，4.5，7.7) 8.51(br d，7.7) 1.64(3H，s) 3.46(1H，m) 3.78(1H，m) 1.18(3H，t，7.1)
The a chemical shift represents that with ppm coupling constant J represents with Hz, and is placed in the bracket
The b chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Table 4: compound 15-17's 1H-NMR data (400MHz, in CDCl 3) Ab
H 15 16 17
1 2 3 6 7 8 10 1.41(m，H a-1) 1.90(m，H b-1) 2.20(2H，m) 1.14(m，H a-3) 1.72(m，H b-3) 4.75(dd，4.4，11.3) 1.48(m，H a-7) 1.67(m，H b-7) 1.51(m) 1.74(dd，3.2，11.3) 4.03(dd，5.2，12.0) 1.83(m，H a-1) 2.11(m，H b-1) 2.19(m，2H) 5.30(br s) 5.87(d，10.3) 5.71(d，10.3) 2.54(dd，1.9，12.0) 1.76(m，H a-1) 2.08(m， b-1) 2.11(m，2H) 5.28(br s) 5.36(d，10.4) 3.85(d，10.4) 2.34(dd，1.8，12.5)
11 12 13 14 15 16 17 18 19 20 3′ 5′ 6′ 7′ 3″ 5″ 6″ 7″ OAc CH 2 CH 3 1.56(m，H a-12) 1.78(m，H b-12) 2.88(m) 1.63(m，H a-14) 2.24(m，H b-14) 5.24(d，5.6) 5.72(d，5.4) 0.89(d，6.1) 2.60(d，4.2，H a-18) 2.79(d，4.2，H b-18) 4.58(d，11.8，H a-19) 5.27(d，11.8，H b-19) 1.02(3H，s) 9.40(br s) 8.87(br d，4.6) 7.43(dd，4.6，7.8) 8.54(br d，7.8) 1.64(3H，s) 3.41(1H，m) 3.72(1H，m) 1.16(3H，t，7.0) 5.59(br d，10.4) 2.70(dd，10.4，14.2， H a-12) 3.4(brd，14.2，H b-12) 4.56(d，16.4，H a-16) 4.75(d，16.4，H b-16) 1.34(s，3H) 1.58(s，3H) 1.47(s，3H) 1.02(s，3H) 9.09(br s) 8.69(br d，4.7) 7.30(dd，4.7，7.8) 8.10(br d，7.8) 8.96(br s) 8.68(br d，4.6) 7.28(dd，4.6，7.7) 8.07(br d，7.7) 5.57(br d，10.3) 2.58(dd，10.3，13.9， H a-12) 3.34(br d，13.9，H b-12) 4.56(d，16.4，H a-16) 4.77(d，16.4，H b-16) 1.40(s，3H) 1.56(s，3H) 1.36(s，3H) 0.87(s，3H) 9.25(br s) 8.79(br d，4.6) 7.46(dd，4.6，7.8) 8.35(br d，7.8)
The a chemical shift represents that with ppm coupling constant J represents with Hz, and is placed in the bracket
The b chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Table 5: compound 1-10's 13C NMR data (100MHz, in CDCl 3) a
H 1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10 11 12 28.8t 33.1t 121.1d 143.9s 44.8s 73.0d 69.8d 83.7s 39.1s 43.7d 72.5d 29.6t 28.8t 33.3t 120.2d 143.6s 44.6s 73.3d 74.2d 81.1s 38.9s 43.5d 72.1d 29.5t 28.8 33.3 120.5 143.2 45.4 74.6 75.4 81.2 39.0 43.6 71.8 29.2 28.7 33.3 120.2 143.4 44.9 74.5 74.1 81.1 39.0 43.5 71.8 29.5 28.7 33.0 118.7 146.4 44.3 74.5 76.4 81.5 38.7 43.4 72.9 29.3 22.5t 28.6t 32.9t 154.6s 46.1s 74.1d 67.7d 85.0s 43.8s 43.6d 74.5d 31.2t 19.5t 26.4t 122.6d 142.4s 43.3s 76.5d 74.9d 77.0s 47.9s 42.7d 147.8d 121.5d 19.2t 26.1t 123.3d 140.7s 43.4s 76.3d 75.8d 77.1s 48.3s 42.8d 146.6d 121.9d 35.5 198.0 127.5 167.8 44.9 74.6 76.2 76.7 48.3 42.2 144.2 123.7 19.6 26.4 123.6 141.1 43.2 74.6 76.6 77.4 48.1 42.9 147.5 122.0
13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1 2 3 5 6 7 OAc 76.8s 43.9t 174.2s 76.3t 19.8q 20.1q 16.7q 22.3q 166.7s 128.9s 130.0d 129.2d 133.4d 129.2d 130.0d 168.3s 129.4s 129.8d 128.6d 133.7d 128.6d 129.8d 76.7s 44.5t 173.7s 76.6t 19.8q 20.2q 16.8q 21.3q 164.4s 126.4s 150.7d 153.8d 123.9d 137.5d 76.9 44.8 173.6 76.6 21.4 20.4 16.9 19.8 165.1 126.1 151.2 154.0 123.8 137.4 164.5 125.8 150.8 153.9 123.5 137.2 164.3 125.0 150.7 153.7 123.6 137.0 76.8 44.5 173.6 76.6 20.8 20.4 17.0 19.8 164.6 126.2 151.2 154.1 123.8 137.2 164.4 126.1 150.9 154.0 123.7 137.1 171.0 21.6 76.3 42.4 174.7 79.8 20.9 21.8 15.3 21.7 164.6 126.5 151.1 153.8 123.8 137.7 77.9s 42.4t 174.3s 79.2t 16.5q 104.9t 17.7q 20.2q 166.5s 128.6s 129.9d 128.9d 133.1d 128.9d 129.9d 167.8s 129.6s 129.7d 128.4d 133.6d 128.4d 129.7d 162.3s 114.7d 174.1s 70.7t 22.5q 22.0q 16.1q 15.5q 162.0s 115.0d 173.9s 70.6t 22.5q 20.1q 17.3q 15.4q 164.5s 125.2 150.3d 152.9d 123.2d 137.0d 165.5s 128.6s 129.6d 128.3d 133.4d 128.3d 129.6d 161.5 116.2 173.9 70.8 22.5 20.9 15.9 15.4 167.8 125.5 151.1 154.2 123.7 137.7 164.7 124.8 150.8 154.0 123.4 137.1 162.3 115.2 174.2 70.9 22.9 20.7 17.6 15.8 162.3 126.3 150.8 153.7 123.8 137.5
The a chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Table 6 compound 11-17's 13C NMR data (100MHz, in CDCl 3) a
H 11 12 13 14 15 16 17
1 2 3 4 5 6 7 8 9 10 11 12 24.7t 128.3d 133.7d 73.8s 46.2s 74.1d 75.5d 77.4s 48.0s 37.3d 146.7d 122.1d 37.2t 21.9t 68.9d 156.3s 46.5s 75.5d 75.8d 77.6s 48.7s 46.0d 146.3d 120.5d 21.4t 40.1t 203.1s 153.8s 46.6s 74.5d 75.1d 77.3s 48.8s 43.8d 145.4d 122.8d 25.3t 22.4t 32.9t 65.3s 46.1s 72.2d 33.6t 36.3d 40.2s 48.7d 83.7d 33.1t 25.2t 22.3t 32.4t 65.2s 46.0s 72.1d 33.5t 36.6d 40.3s 48.5d 83.6d 33.0t 19.6 26.0 123.6 141.1 43.2 76.3 77.1 78.3 47.7 40.6 75.6 28.9 19.7 26.1 123.7 141.4 42.8 79.5 74.5 79.1 47.2 40.6 76.1 28.8
13 14 15 16 17 18 19 20 1′ 2′ 3′ 5′ 6′ 7′ 1″ 2″ 4″ 3″ 5″ 6″ 7″ OAc OEt 162.2s 115.5d 174.0s 70.9t 22.9q 25.6q 12.6q 16.0q 165.7s 126.2s 150.9d 153.6d 137.5d 123.7d 166.1s 128.9s 129.8d 128.6d 133.4d 128.6d 129.8d 162.1s 115.4d 174.2s 70.9t 22.6q 101.2t 17.6q 15.4q 164.5s 126.5s 150.5d 153.0d 137.9d 123.9d 166.1s 128.7s 130.1d 133.7d 128.6d 130.1d 162.0s 116.0d 173.8s 70.8t 22.7q 114.0t 16.9q 15.8q 164.8s 125.7s 150.8d 153.6d 123.5d 137.2d 165.9s 128.7s 129.9d 133.8d 128.6d 129.9d 40.7d 39.8t 104.1d 109.3d 16.6q 48.4t 62.6t 14.4q 165.2s 126.7s 151.6d 153.5d 137.9d 123.6d 170.1s 21.3q 63.3t 15.3q 40.4d 38.4t 103.8d 107.4d 16.6q 48.3t 62.5t 14.3q 165.3s 127.1s 151.6d 153.7d 138.2d 123.5d 170.8s 21.5q 63.1t 15.4q 130.3 139.0 171.3 69.9 21.9 20.5 17.5 16.4 164.6 126.2 150.5 153.2 123.7 138.0 164.5 125.3 150.2 153.1 123.6 137.4 130.9 138.8 171.3 70.2 21.8 20.9 17.5 16.5 166.3 127.0 150.5 153.0 124.0 138.8
The a chemical shift determine to utilize HMQC, HMBC, 1H- 1H COSY technology
Test example one: extracorporeal suppression tumor cell activity test
1, suppress the activity of tumor cells experimental technique:
With the RPMI-1640 culture medium culturing human nasopharyngeal carcinoma HONE-1 cell, oral epithelium cancer KB cell, the colorectal carcinoma HT that contain 5% foetal calf serum 29Cell.Tumour cell is in the logarithmic growth after date and changes 24 well culture plates over to, and cell concn is 5000/ml/ hole.Add the soup to be measured of different concns in the culture hole, cultivated 72 hours.Press down the oncocyte activity with what the methylene blue assay method was estimated medicine.With the control group is reference, calculates IC with graphics 50Value.
Herba Scutellariae Barbatae ethanol extraction: make by [Wang Gang, Dong Mei etc., the research of Chinese medicine Herba Scutellariae Barbatae extract anti tumor activity in vitro, traditional Chinese medicine research and information, 2006.28 (9), 701-702] described method.
2, suppress the activity of tumor cells experimental result:
Use the methylene blue assay method,, estimated Herba Scutellariae Barbatae ethanol extraction and 17 kinds of compounds and pressed down the oncocyte activity with Podophyllum emodi var chinense ethylidene and the positive contrast medicine of cis-platinum.As shown in table 7, the result shows: 17 compounds all have the tumour cell effect (P＜0.01) that significantly presses down, and its anti-tumor activity obviously is better than Herba Scutellariae Barbatae extract.
Table 7 compound 1-17 presses down HONE-1, KB and HT 29The cancer cells experimental result
Compounds Growth inhibition constant(IC 50) a[μM]
HONE-1 KB HT 29
Etoposide b Cisplatin b123456789 10 11 12 13 14 15 16 17 Sculellaria barbata ethanol extracts 0.6±0.2 3.7±0.4 4.7±2.0 4.4±1.9 3.7±2.0 5.0±3.2 4.5±1.5 5.0±2.1 4.1±1.5 4.4±1.9 5.0±2.8 4.6±1.5 4.7±2.0 5.0±2.1 4.1±1.5 3.8±2.1 3.6±1.5 4.4±2.7 3.7±2.0 22.3±2.2 0.9±0.3 4.2±0.9 7.7±2.2 6.1±2.7 7.1±2.2 8.1±1.7 6.1±2.6 8.1±1.8 7.1±2.6 6.1±2.7 7.8±1.3 5.9±2.2 7.1±2.6 7.8±1.8 7.6±2.2 7.8±1.8 7.6±2.2 6.1±2.2 6.7±2.6 25.6±1.9 2.3±0.5 5.6±1.8 5.9±1.3 4.6±1.9 6.9±1.3 6.6±2.4 5.3±2.0 6.6±1.5 4.3±2.0 3.5±2.1 4.6±1.8 6.3±2.0 3.9±2.1 4.6±1.5 5.3±2.0 4.6±1.5 5.3±2.0 3.5±3.4 3.4±2.1 27.8±2.5
A, IC 50Representative makes tumor cell number reduce by 50% drug level.The result with 3 times independently mean ± the standard deviation of revision test represent.
B, positive control drug (Etoposide is the Podophyllum emodi var chinense ethylidene, and Cisplatin is a cis-platinum).
Test example two: the medium lethal dose (LD of intravenous administration 50) measure
1, test materials
Sample: the butanedioic acid derivative monopotassium salt of compound 3 (scutebarbatine I), compound 7 (barbatin C), compound 8 (scutebarbatine B), compound 16 (scutebarbatine L);
Animal: cleaning level Kunming kind small white mouse, body weight 18～22g is provided credit number by Shandong Luye Pharmaceutical Co., Ltd.'s animal center: SYXK (Shandong) 20030020.
Software: DAS medical statistics software
2, test method
Preliminary experiment is got 20 of mouse, carries out pilot study by sequential method, records mouse mainline 100% lethality rate and does not have administration maximal dose under the situation of causing death, and observes 7d continuously, result such as table 8.
Table 8 mouse mainline pilot study result
Group 100% deadly minimum dose (mg/kg) 0% deadly maximal dose (mg/kg)
Compound 3 compounds 7 compounds 8 compounds 16 160.0 220.0 265.0 185.0 75.0 90.0 115.0 60.0
Formal experiment determines that by the Bliss method high dosage to dosage ratio between the group of low dosage, is divided into 6 groups, 10 every group.Take by weighing the mouse body weight, press the administration of 0.2ml/10g tail vein injection, each is organized injection liquid concentration and calculates definitely according to mouse 20g mean body weight level, observes 7d continuously, result such as table 9.。
Table 9 mouse mainline LD 50Measurement result
Group Compound 3 Compound 7 Compound 8 Compound 16
Dosage (mg/kg) Number of animals (n) Death toll (only) Dosage (mg/kg) Number of animals (n) Death toll (only) Dosage (mg/kg) Number of animals (n) Death toll (only) Dosage (mg/kg) Number of animals (n) Death toll (only)
1 2 3 4 5 6 160.0 137.5 118.2 101.6 87.3 75.0 10 10 10 10 10 10 10 8 7 4 2 0 220.0 184.0 153.9 128.7 107.6 90.0 10 10 10 10 10 10 10 9 6 3 1 0 265.0 224.3 189.8 160.6 135.9 115.0 10 10 10 10 10 10 10 9 6 4 2 0 185.0 147.7 117.9 94.1 75.2 60.0 10 10 10 10 10 10 10 8 5 3 1 0
3, test-results
Obtain the LD of compound 3 with DAS medical statistics computed in software 50Be 108.04mg/kg, the 95% credible 99.48mg/kg～117.34mg/kg that is limited to; The LD of compound 7 50Be 143.04mg/kg, the 95% credible 131.22mg/kg～155.92mg/kg that is limited to; The LD of compound 8 50Be 171.49mg/kg, the 95% credible 157.17mg/kg～187.12mg/kg that is limited to; The LD of compound 16 50Be 112.48mg/kg, the 95% credible 100.12mg/kg～126.37mg/kg that is limited to.
Test example three: oral administration medium lethal dose (LD 50) measure
1, test materials
Sample: the butanedioic acid derivative monopotassium salt of compound 3 (scutebarbatine I), compound 7 (barbatin C), compound 8 (scutebarbatine B), compound 16 (scutebarbatine L);
Animal: cleaning level Kunming kind small white mouse, body weight 18～22g is provided credit number by Shandong Luye Pharmaceutical Co., Ltd.'s animal center: SYXK (Shandong) 20030020.
Software: DAS medical statistics software
2, test method
Preliminary experiment is got 20 of mouse, carries out pilot study by sequential method, records mouse mainline 100% lethality rate and does not have administration maximal dose under the situation of causing death, and observes 7d continuously, result such as table 1.
Formal experiment determines that by the Bliss method high dosage to dosage ratio between the group of low dosage, is divided into 6 groups, 10 every group.Take by weighing the mouse body weight, press the administration of 0.2ml/10g tail vein injection, each is organized injection liquid concentration and calculates definite according to mouse 20g mean body weight level.Observe 7d continuously.
3, test-results
Obtain the LD of compound 3 with DAS medical statistics computed in software 50Be 406.23mg/kg, the 95% credible 374.02mg/kg～438.44mg/kg that is limited to; The LD of compound 7 50Be 378.52mg/kg, the 95% credible 352.96mg/kg～404.08mg/kg that is limited to; The LD of compound 8 50Be 298.67mg/kg, the 95% credible 283.52mg/kg～313.82mg/kg that is limited to; The LD of compound 16 50Be 305.42mg/kg, the 95% credible 293.30mg/kg～317.54mg/kg that is limited to.
Test example four: drug administration by injection is tested three kinds of mice transplanted tumor Growth Inhibition
1, test materials
Sample: the butanedioic acid derivative monopotassium salt of compound 3 (scutebarbatine I), compound 7 (barbatin C), compound 8 (scutebarbatine B), compound 16 (scutebarbatine L); With behind the injection physiological saline solution under aseptic condition with 0.22 μ m filtering with microporous membrane, obtain the injection soup of desired concn.
Endoxan: be Hualian Pharmaceutical Co., Ltd., Shanghai (lot number 040205), face with preceding with the injection physiological saline solution and be diluted to desired concn.
Animal and knurl strain: cleaning level Kunming kind small white mouse, body weight 18～22g is provided credit number by Shandong Luye Pharmaceutical Co., Ltd.'s animal center: SYXK (Shandong) 20030020.The male and female dual-purpose, same sex mouse is selected in each experiment for use.
Mouse H22 liver cancer, U14 cervical cancer and S180 sarcoma are all drawn from institute of materia medica, Chinese Academy of Medical Sciences Beijing.
2, test method
The preservation of going down to posterity of tumour: H22 liver cancer, U14 cervical cancer and S180 sarcoma are got the ascites preservation of going down to posterity after Kunming mouse abdominal cavity inoculation.
Tumor inoculation: get H22 liver cancer, U14 cervical cancer or S180 sarcoma tumor-bearing mice that ascites went down to posterity the 10th, take off cervical vertebra and put to death mouse, the sterilization skin of abdomen is drawn oyster white ascites with asepsis injector, and adjusting tumour cell concentration with injection physiological saline is 1 * 107 cell/ml.With cotton ball soaked in alcohol sterilization Kunming mouse right side armpit skin, in the above-mentioned tumor cell suspension 0.2ml of subcutaneous vaccination, the conventional raising.
Grouping and administration: 50 of tumor-bearing mices, be divided into 5 groups at random by body weight, 10 every group, be respectively model group, endoxan group, the high, medium and low dosage group of compound group.Each organize mouse press shown in dosed administration, model group tail every day vein waits volume injection physiological saline, endoxan is inoculation once abdominal cavity injection administration in second day, the compound group was from beginning tail vein injection administration every day 1 time on the secondth, continuous 10 days.After the last administration 24 hours, each treated animal is taken off cervical vertebra put to death, weigh, strip tumor tissue and weigh, calculate tumour inhibiting rate.
Figure A20071010662600251
The result represents with x ± s, adopts t check carrying out statistics between group relatively, and experimental result sees Table 10-13 respectively.
2, test-results
The result shows that the butanedioic acid derivative monopotassium salt tail vein injection administration of compound 3, compound 7, compound 8, compound 16 all has the obvious suppression effect to mice transplanted tumor H22 liver cancer, U14 cervical cancer and S180 sarcoma, illustrate that these compounds have stronger antitumor activity in vivo, can obviously suppress above-mentioned various growth of tumor (P＜0.01) during tail vein injection administration 10mg/kg dosage, during 50mg/kg dosage to the tumour inhibiting rate of above-mentioned tumour all greater than 60%.
The butanedioic acid derivative monopotassium salt of table 10 compound 3 suppresses the test-results of three kinds of knurl strain growths
The knurl strain H22 liver cancer The U14 cervical cancer The S180 sarcoma
Group Dosage (mg/kg) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%)
Model group endoxan compound 3 —— 100 2 10 50 3.00±0.88 0.88±0.44 ** 2.39±0.73 1.72±0.64 ** 1.12±0.52 ** 70.6 20.2 42.5 62.7 3.00±0.68 0.59±0.29 ** 2.29±1.07 1.72±0.80 ** 1.20±0.57 ** 80.4 23.6 42.7 60.1 3.56±0.78 0.84±0.35 ** 2.66±0.98 2.09±0.64 ** 1.40±0.29 ** 76.5 25.2 41.2 60.7
The butanedioic acid derivative monopotassium salt of table 11 compound 7 suppresses the test-results of three kinds of knurl strain growths
The knurl strain H22 liver cancer The U14 cervical cancer The S180 sarcoma
Group Dosage (mg/kg) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%)
Model group endoxan compound 7 —— 100 2 10 50 3.00±0.87 0.81±0.30 ** 2.34±0.81 1.72±0.82 ** 1.12±0.52 ** 73.0 22.0 42.8 62.8 3.26±0.73 0.83±0.35 ** 2.59±1.07 1.88±0.80 ** 1.20±0.45 ** 74.4 20.5 42.3 63.3 3.00±0.87 0.79±0.29 ** 2.31±0.65 1.73±0.49 ** 1.17±0.55 ** 73.8 22.8 42.2 61.0
The butanedioic acid derivative monopotassium salt of table 12 compound 8 suppresses the test-results of three kinds of knurl strain growths
The knurl strain H22 liver cancer The U14 cervical cancer The S180 sarcoma
Group Dosage (mg/kg) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%)
Model group endoxan compound 8 —— 100 2 10 50 3.43±0.92 0.85±0.21 * 2.74±1.12 2.11±0.63 ** 1.24±0.35 ** 75.1 20.3 38.6 63.9 3.99±1.1 1.21±0.43 ** 3.18±1.3 2.41±0.80 ** 1.44±0.41 ** 69.7 20.3 39.6 63.9 4.07±1.1 1.23±0.44 ** 3.25±1.33 2.54±0.70 ** 1.23±0.44 ** 69.7 20.3 38.0 63.9
The butanedioic acid derivative monopotassium salt of table 13 compound 16 suppresses the test-results of three kinds of knurl strain growths
The knurl strain H22 liver cancer The U14 cervical cancer The S180 sarcoma
Group Dosage (mg/kg) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%) Knurl heavy (g) Tumour inhibiting rate (%)
Model group endoxan compound 16 —— 100 2 10 50 3.11±0.79 1.11±0.45 ** 2.67±0.65 2.45±0.56 * 1.84±0.65 ** 65.0 8.8 16.9 35.0 3.22±0.84 1.20±0.51 ** 2.75±0.63 2.55±0.52 1.79±0.53 ** 59.8 7.1 13.7 39.0 3.24±0.77 1.23±0.47 ** 2.72±0.67 2.59±0.54 1.85±0.47 ** 59.4 10.9 14.8 38.0
Test example five: oral administration is tested three kinds of mice transplanted tumor Growth Inhibition
1, test materials
With test example four.
2, test method
With test example four.
3, test-results
The result shows that the butanedioic acid derivative monopotassium salt oral administration of compound 3, compound 7, compound 8, compound 16 all has the obvious suppression effect to mice transplanted tumor H22 liver cancer, U14 cervical cancer and S180 sarcoma, illustrate that these compounds have stronger antitumor activity in vivo, can obviously suppress above-mentioned various growth of tumor (P＜0.01) during oral administration 50mg/kg dosage, during 100mg/kg dosage to the tumour inhibiting rate of above-mentioned tumour all greater than 60%.

Claims (7)

1, a kind of new NEO-Crow alkane type diterpenoid or its salt, its structural formula is as follows:
Figure A2007101066260002C1
Formula I
Wherein:
R 1And R 2Independently be selected from: OH-, AcO-,
Figure A2007101066260002C2
Or
Figure A2007101066260002C3
R 3Be selected from: OH-,
Figure A2007101066260002C4
Or
X is selected from
Figure A2007101066260002C6
Or
Figure A2007101066260002C7
Figure A2007101066260002C8
Formula II
Wherein:
R 4Be selected from OH-,
Figure A2007101066260002C9
Or
Figure A2007101066260002C10
R 5, R 6Independently be selected from OH-or
Figure A2007101066260002C11
Y is selected from
Figure A2007101066260002C12
Or
Figure A2007101066260002C13
Figure A2007101066260003C1
Formula III
Wherein:
R7 is selected from-CH2OAc or CH 3-;
R8 be selected from H-, OH-or
Figure A2007101066260003C2
R9 is selected from H-, OH-or CH 3-;
N is selected from
Figure A2007101066260003C3
Or
Figure A2007101066260003C4
M is selected from
Figure A2007101066260003C5
Or
Figure A2007101066260003C6
R wherein 10, R 11Be independently selected from-OEt or hydrogen;
AB forms singly-bound or two key.
2, according to the compound or its salt of claim 1, described compound choosing is white:
Figure A2007101066260003C7
Formula I
Compound 1 (barbatin A):
Figure A2007101066260003C8
R 2=OH-,
Figure A2007101066260003C9
Figure A2007101066260003C10
Compound 2 (scutebarbatine F): R 1=AcO-, R 2=AcO-,
Figure A2007101066260003C11
Figure A2007101066260003C12
Compound 3 (scutebarbatine I):
Figure A2007101066260003C13
Figure A2007101066260003C14
Figure A2007101066260003C16
Compound 4 (scutebarbatine J):
Figure A2007101066260003C17
R 2=AcO-,
Figure A2007101066260003C18
Figure A2007101066260003C19
Compound 5 (scutebarbatine K):
Figure A2007101066260004C1
R 2=OH-, R 3=OH-,
Figure A2007101066260004C2
Compound 6 (barbatin B): R 1=OH-,
Figure A2007101066260004C3
Figure A2007101066260004C4
Figure A2007101066260004C5
Figure A2007101066260004C6
Formula II
Compound 7 (barbatin C): R 4=OH-, R 5=OH-, R 6=OH-,
Figure A2007101066260004C7
Compound 8 (scutebarbatine B):
Figure A2007101066260004C8
R 5=OH-,
Figure A2007101066260004C10
Compound 9 (scutebarbatine N):
Figure A2007101066260004C11
R 5=OH-,
Figure A2007101066260004C12
Figure A2007101066260004C13
Compound 10 (scutebarbatine O): R 4=OH-,
Figure A2007101066260004C14
R 6=OH-,
Compound 11 (scutebarbatine C):
Figure A2007101066260004C16
R 5=OH-,
Figure A2007101066260004C18
Compound 12 (scutebarbatine D):
Figure A2007101066260004C19
R 5=OH-,
Figure A2007101066260004C20
Figure A2007101066260004C21
Compound 13 (scutebarbatine E):
Figure A2007101066260004C22
R 5=OH-,
Figure A2007101066260004C23
Formula III
Compound 14 (scutebarbatine G): AB is a singly-bound, R 7=-CH2OAc, R 8=-H, R 9=OH-,
Figure A2007101066260004C26
R 10=-OEt, R 11=H-,
Figure A2007101066260004C27
Compound 15 (scutebarbatineH): AB is a singly-bound, R 7=-CH2OAc, R 8=-H, R 9=-CH 3,
Figure A2007101066260005C1
R 10=H-, R 11=-OEt,
Figure A2007101066260005C2
Compound 16 (scutebarbatine L): AB is two keys, R 7=-CH 3,
Figure A2007101066260005C3
R 9=-OH,
Figure A2007101066260005C4
Compound 17 (scutebarbatine M): AB is two keys, R 7=-CH 3, R 8=-OH, R 9=-OH,
Figure A2007101066260005C5
3, according to the preparation method of the described compound of claim 1, it is characterized in that comprising the steps: to take by weighing a certain amount of Herba Scutellariae Barbatae herb, with methyl alcohol or alcohol reflux 3-4 time, each 1-2 hour, united extraction liquid, concentrating under reduced pressure; Medicinal extract after concentrating extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, get silicagel column on the chloroform extract, carry out gradient elution with sherwood oil-acetone gradient, use silicagel column, anti-phase C18 post, Sephadex LH-20 chromatographic column column chromatography repeatedly then, promptly.
4, according to the preparation method of the described compound of claim 2, it is characterized in that: take by weighing a certain amount of Herba Scutellariae Barbatae herb, with the methyl alcohol of 60-95% or alcohol reflux 3-4 time, each 1-2 hour, united extraction liquid, concentrating under reduced pressure; Medicinal extract after concentrating extracts repeatedly with sherwood oil, chloroform, ethyl acetate, propyl carbinol, gets silicagel column on the chloroform extract, carries out gradient elution with sherwood oil-acetone (97: 3～50: 50), is divided into 7 parts (Fraction1-7);
Get wherein that Fraction2 crosses Rp C-18 chromatographic column, the methanol-water wash-out, then by Sephadex LH-20 chromatographic column purifying, compound 3 and compound 4;
Fraction3 crosses silica gel chromatographic column, and hexanaphthene-acetone gradient elution gets compound 9 and compound 16;
The last silica gel chromatographic column of Fraction4, hexanaphthene-acetone gradient elution gets a compound 2 and a mixture; Mixture by Rp C-18 chromatographic column, behind the methanol-water wash-out, by Sephadex LH-20 chromatographic column purifying, is got compound 14 and compound 15;
Fraction5 crosses silica gel chromatographic column, hexanaphthene-acetone gradient elution, compound 1, compound 6 and two mixture M 1, M2, mixture M 1 is crossed Rp C-18 chromatographic column, the methanol-water wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 8 and compound 13; Mixture M 2 is crossed Rp C-18 chromatographic column, and the methanol-water wash-out then by Sephadex LH-20 chromatographic column purifying, gets compound 5,10 and 17;
Fraction6 crosses silica gel chromatographic column, and hexanaphthene-acetone gradient elution gets a compound 7 and a mixture; Mixture is crossed the RpC-18 chromatographic column, behind the methanol-water wash-out,, get compound 11 and compound 12 by Sephadex LH-20 chromatographic column purifying.
5, contain one or more NEO-Crow alkane type diterpenoids of claim 1 or the Herba Scutellariae Barbatae extract of its salt.
6, according to claim 1 or 5 compound or Herba Scutellariae Barbatae extract to the restraining effect of tumour cell, and in the application of preparation in the antitumor drug.
7, compound or Herba Scutellariae Barbatae extract according to claim 1 or 5 is characterized in that and can exist with the form of injection liquid, lyophilized injectable powder, tablet, capsule, soft capsule, dripping pill or oral liquid.
CN2007101066260A 2006-05-23 2007-05-22 Novel NEO-clerodane type diterpene compound and application thereof Expired - Fee Related CN101077873B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104644743A (en) * 2014-10-21 2015-05-27 中山大学 Scutellaria barbata extract as well as preparation method and application thereof
CN105476985A (en) * 2015-12-16 2016-04-13 暨南大学 Application of diterpene compound in preparing medicine or health care product having anti-tumor effect
CN105837550A (en) * 2015-11-19 2016-08-10 中国科学院昆明植物研究所 Natural asymmetric cyclobutane derivatives and pharmaceutical composition thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104644743A (en) * 2014-10-21 2015-05-27 中山大学 Scutellaria barbata extract as well as preparation method and application thereof
CN105837550A (en) * 2015-11-19 2016-08-10 中国科学院昆明植物研究所 Natural asymmetric cyclobutane derivatives and pharmaceutical composition thereof
CN105837550B (en) * 2015-11-19 2018-02-16 中国科学院昆明植物研究所 Natural asymmetric New cyclobutane derivative and its pharmaceutical composition
CN105476985A (en) * 2015-12-16 2016-04-13 暨南大学 Application of diterpene compound in preparing medicine or health care product having anti-tumor effect
CN105476985B (en) * 2015-12-16 2018-07-31 暨南大学 A kind of application of diterpene compound in preparing drug or health products with antitumor action

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