CN101948453A - Novel NEO-clerodane typed diterpene compound and application thereof - Google Patents
Novel NEO-clerodane typed diterpene compound and application thereof Download PDFInfo
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Abstract
The invention provides a novel NEO-clerodane typed diterpene compound which is obtained from the extraction and separation of sculellaria barbata. The preparation method comprises the following steps: weighting the whole plant of the sculellaria barbata, extracting by the reflux of methanol or alcohol, combining the extracting solution and reducing the pressure to concentrate; extracting the concentrated extract by the petroleum ether, the chloroform, the ethyl acetate and the n-butyl alcohol repeatedly, putting the chloroform extract on the silica gel column to perform the gradient elution by the petroleum ether-acetone, and then performing the column chromatography repeatedly through the silica gel, the reversed C18 column, the Sephadex LH-20 chromatographic column to obtain the product. The invention further provides a medicinal composition with the compound as the active component, a cancer cell activity restraining effect of the compounds and an application in the preparation of the antineoplastic medicines.
Description
The present invention is that application number is 200710106626.0, and the applying date is on May 22nd, 2007, and denomination of invention is divided an application for " new NEO-Crow alkane type diterpenoid and application thereof " Chinese patent application.
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.
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:
Formula I
Wherein:
R
1And R
2Independently be selected from: OH-, AcO-,
R
3Be selected from: OH-,
X is selected from
Formula II
Wherein:
R
4Be selected from OH-,
R
5, R
6Independently be selected from OH-or
Y is selected from
The formula III
Wherein:
R7 is selected from-CH2OAc or CH
3-;
R8 be selected from H-, OH-or
R9 is selected from H-, OH-or CH
3-;
N is selected from
M is selected from
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:
Formula I
Compound 1 (barbatin A):
R
2=OH-,
Compound 2 (scutebarbatine F): R
1=AcO-, R
2=AcO-,
Compound 3 (scutebarbatine I):
Compound 4 (scutebarbatine J):
R
2=AcO-,
Compound 5 (scutebarbatine K):
R
2=OH-, R
3=OH-,
Compound 6 (barbatin B): R
1=OH-,
Formula II
Compound 7 (barbatin C): R
4=OH-, R
5=OH-, R
6=OH-,
Compound 8 (scutebarbatine B):
R
5=OH-,
Compound 9 (scutebarbatine N):
R
5=OH-,
Compound 10 (scutebarbatine O): R
4=OH-,
R
6=OH-,
Compound 11 (scutebarbatine C):
R
5=OH-,
Compound 12 (scutebarbatine D):
R
5=OH-,
Compound 13 (scutebarbatine E):
R
5=OH-,
Formula III
Compound 14 (scutebarb atine G): AB is a singly-bound, R
7=-CH2OAc, R
8=-H, R
9=OH-,
R
10=-OEt, R
11=H-,
Compound 15 (scutebarbatineH): AB is a singly-bound, R
7=-CH2OAc, R
8=-H, R
9=-CH
3,
R
10＝H-，R
11＝-OEt，
Compound 16 (scutebarbatine L): AB is two keys, R
7=-CH
3,
R
9=-OH,
Compound 17 (scutebarbatine M): AB is two keys, R
7=-CH
3, R
8=-OH, R
9=-OH,
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, get silicagel column on the chloroform extract, carry out gradient elution with sherwood oil-acetone, use 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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(c 0.14, MeOH).UV(CDCl3)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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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 ℃,
(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
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
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
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
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
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
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 HT29 cell that contain 5% foetal calf serum.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
A, IC
50Representative makes tumor cell number reduce by 50% drug level.The result with 3 times independently the mean of revision test+-standard deviation represents.
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
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
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.
The result with
Expression is adopted t check carrying out statistics relatively between group, 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 butanedioic acid derivative monopotassium salt of table 11 compound 7 suppresses the test-results of three kinds of knurl strain growths
The butanedioic acid derivative monopotassium salt of table 12 compound 8 suppresses the test-results of three kinds of knurl strain growths
The butanedioic acid derivative monopotassium salt of table 13 compound 16 suppresses the test-results of three kinds of knurl strain growths
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 (2)
1. new NEO-Crow alkane type diterpenoid, its structural formula is as follows:
Formula II
Wherein:
R4 is selected from: OH-,
R5 and R6 independently are selected from: OH-or
Y is selected from:
Wherein, described compound is selected from:
Compound 7:R4=-OH, R5=-OH, R6=-OH,
Compound 8:
R5=-OH,
Compound 9:
R5=-OH,
Compound 10:R4=-OH,
R6=-OH,
Compound 11:
R5=-OH,
Compound 12:R4=-OH, R5=-OH,
Compound 13:R4=-OH, R5=-OH,
2. the application of the described compound of claim 1 in the preparation antitumor drug.
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Cited By (4)
| 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 |
| CN105288603A (en) * | 2015-10-28 | 2016-02-03 | 杨廷稳 | Tumor vaccine for treating liver cancer and preparing method thereof |
| CN110687242A (en) * | 2019-07-03 | 2020-01-14 | 兰州大学 | Method for testing phytotoxicity activity of NEO-clerodane diterpenoid compounds on ryegrass |
| CN113499339A (en) * | 2021-07-06 | 2021-10-15 | 海南精准医疗科技有限公司 | Preparation method and application of traditional Chinese medicine clorine diterpenoid extracted from eleutherococcus senticosus |
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| DE19853476A1 (en) * | 1998-11-19 | 2000-05-31 | Bionorica Arzneimittel Gmbh | Prolactin-lowering agent |
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2007
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Cited By (6)
| 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 |
| CN105288603A (en) * | 2015-10-28 | 2016-02-03 | 杨廷稳 | Tumor vaccine for treating liver cancer and preparing method thereof |
| WO2017071380A1 (en) * | 2015-10-28 | 2017-05-04 | 李淑兰 | Tumor vaccine for use in treating liver cancer and preparation method for vaccine |
| CN107708727A (en) * | 2015-10-28 | 2018-02-16 | 李淑兰 | It is a kind of to be used to treat tumor vaccine of liver cancer and preparation method thereof |
| CN110687242A (en) * | 2019-07-03 | 2020-01-14 | 兰州大学 | Method for testing phytotoxicity activity of NEO-clerodane diterpenoid compounds on ryegrass |
| CN113499339A (en) * | 2021-07-06 | 2021-10-15 | 海南精准医疗科技有限公司 | Preparation method and application of traditional Chinese medicine clorine diterpenoid extracted from eleutherococcus senticosus |
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| CN101948453B (en) | 2012-05-23 |
| CN101948473A (en) | 2011-01-19 |
| CN101948473B (en) | 2012-10-03 |
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