CN106279337A - A kind of dammarane's compounds preparation method and anticancer usage thereof - Google Patents

A kind of dammarane's compounds preparation method and anticancer usage thereof Download PDF

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CN106279337A
CN106279337A CN201510245565.0A CN201510245565A CN106279337A CN 106279337 A CN106279337 A CN 106279337A CN 201510245565 A CN201510245565 A CN 201510245565A CN 106279337 A CN106279337 A CN 106279337A
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dammarane
compound
preparation
formula
cancer
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杨明
杨霁初
王鲁明
董晓林
张秀国
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TUOLIN MEDICINE SCI-TECH Co Ltd BEIJING
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TUOLIN MEDICINE SCI-TECH Co Ltd BEIJING
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Abstract

Invention relates to medicinal chemistry art, it is specifically related to 20 (R)-dammarane-[the 12-hydroxyl-20 of a kind of chemosynthesis, 25-epoxy]-3-0-β-D-pyranglucoside (Formulas I, 20 (R)-DEG), its preparation method and anticancer purposes thereof.The present invention is split by chemosynthesis chiral post HPLC and implements.Pharmacodynamic experiment shows, 20 (R)-DEG anticancer effect is significantly better than racemic modification DEG.

Description

A kind of dammarane's compounds preparation method and anticancer usage thereof
Technical field
The invention belongs to field of pharmaceutical chemistry research.It is specifically related to tetracyclic triterpenoid 20 (R)-dammarane-[12-beta-hydroxy-20,25-epoxy]-3-O-β-D-pyranglucoside of a kind of chemosynthesis.English name:
20 (R)-Dammarane-[12-beta-ol-20,25-epoxy]-3-O-beta-D-glucopyranside (is called for short: 20 (R)-DEG), its preparation method and anti-tumor application thereof.
Technical background
Containing the compound medicine of chirality, the activity of its enantiomer and toxicity often have huge difference, and often its R type has efficient, low toxicity, and the high poison of its S type then poor efficiency (or invalid), vice versa.The example of chipal compounds and medicine thereof is too numerous to enumerate.The most at home and abroad in new drug research, the fractionation of chipal compounds, is common innovative point, significant.
The present inventor is during follow up study's Radix Notoginseng antineoplastic activated saponin, one tetracyclic triterpene monomeric compound of isolated from Folium Notoginseng: dammarane-[12-hydroxyl-20,25-epoxy radicals]-3-O-β-D-pyranglucoside (abbreviation: DEG), and applied for Chinese invention patent (application number: CN 201510067046.X).This compound in 1993 by Zhao's equality isolated from Radix Notoginseng[1. Yunnan plant is studied 1993 : 15(4) : 409-412].In view of natural DEG content is the lowest, in order to expand medicament sources, inventor has carried out chemosynthesis to DEG.
Inventor, during research synthesis DEG, finds that it is a racemic modification, is by C20Optical isomer mixture thing produced by methyl.
In conventional record, the most do not find the statement about DEG optical isomer, not about the C of DEG yet20Methyl causes the record of DEG optical isomer.
Summary of the invention
The invention discloses the optical isomer of a kind of DEG, it may be assumed that Formulas I, 20 (R)-dammarane-[12-beta-hydroxy-20,25-epoxy]-3-O-β-D-pyranglucoside.English name:
20 (R)-Dammarane-[12-beta-ol-20,25-epoxy]-3-O-beta-D-glucopyranside, is called for short 20 (R)-DEG.
DEG is through conventional C18HPLC detects, and presents single mesokurtosis, but chiral post HLPC detects, then present 2 peaks, be shown to be and there is chipal compounds.Split with by chiral for DEG post HPLC, obtained 20 (R)-DEG and another molecular weight is same, it may be possible to the compound of its enantiomer.In vitro cell experiment and zoopery all prove, 20 (R)-DEG has preferable active anticancer.
Specifically, inventor realizes inventing by following method:
A.DEG synthesizes.
With reference to Liu Wei etc.[2. Shenyang Pharmacy College's journal 1988 : 5(1) : 14-15]Synthetic method, by 4 acetyl bromides for beta-D glucose and dammarane-3 beta, 12 beta-diols; it is condensed under Disilver carbonate is catalyzed, obtains dammarane-[12-beta-hydroxyl-20,25-epoxy]-3-O-beta-D-four acetyl glucosaminidase; slough protection group again, obtain DEG.Synthetic route is as follows:
B.DEG splits.
The chiral separation of DEG is to use DAICELAD-H chiral column is achieved.Amylose-three (3 will be applied, 5-xylyl carbamate) silica gel is the semi-preparative chiral column of filler, using normal hexane/isopropanol/diethylamine=80/30/0.1 → 30/15/0.1 as flowing phase, detect with differential refraction detector, carry out HPLC fractionation.In this chiral separation system, DEG has obtained good separation, and obtains high-purity 20 (R)-DEG, such as following formula:
C.20 (R)-DEG structural identification.
By the nuclear magnetic resonance, NMR to 20 (R)-DEG1H-NMR and13The parsing of C-NMR, and HMBC, the integration analysis of HMQC, NOESY.Confirm structure and the spatial configuration thereof of 20 (R)-DEG.The result of study of integration analysis shows, 20 (R)-DEG and DEG is at C17, C18, C21, C23, C27Etc. there is bigger difference, other C then height overlaps.
D.20 (R)-DEG active anticancer.
Pharmacology's cell in vitro horizontal inhibiting tumor assay mtt assay completes；And whole animal tumor inhibition, then complete with NU/NU Female nude mice.Inside and outside the pharmacological results shows, 20 (R)-DEG anticancer effect is considerably better than DEG, thus proves that the active anticancer of DEG mostlys come from 20 (R)-DEG.
In sum, an object of the present invention, is elaboration 20 (R)-DEG preparation method and structure thereof.The two of the object of the invention, are antitumaous effect and the medical usage thereof of elaboration 20 (R)-DEG.
Accompanying drawing explanation
Fig. 1 DEG chiral column HPLC chromatogram.
Fig. 2 20 (R)-DEG chromatogram
Detailed description of the invention
Embodiment 1
The synthesis of DEG:
Take dammarane-3 beta respectively, 12 beta-diols, English: dammarane-3beta, 12beta-diol (has another name called: panoxadiol, Man Site bio tech ltd, Chengdu) 820mg and four acetyl bromides are placed in reaction bulb for glucose (AlfaAesar<Tianjin>Chemical Co., Ltd.) 1420mg, take 30ml absolute ether again and 10ml dichloromethane dissolves and is placed in same reaction bulb, make it dissolve 35 DEG C of constant temperature stirrings, add 1580mg AgCO3, 35 DEG C of isothermal reactions 8 hours under the conditions of lucifuge, filter, filtrate ＜ 45 DEG C is concentrated under reduced pressure to give product.Product is through silica gel column chromatography, and eluant chloroform/methanol/water=60/10/1 → 15/10/1 obtains dammarane-[12-beta-hydroxyl-20,25-epoxy]-3-O-beta-D-four acetyl bromide for glucoside 450mg.
By dammarane-[12-beta-hydroxyl-20, 25-epoxy]-3-O-beta-D-four acetyl bromide is placed in reaction vessel for glucoside 450mg, it is dissolved in 5ml methanol, stir at normal temperatures, slowly dropping distilled water is to slightly cloudy, add 10ml triethylamine, continue stirring reaction 5 hours, silica gel column chromatography is again passed by after product room temperature concentrating under reduced pressure, with chloroform/ethyl acetate/methanol=40/5/4 eluting, access stream part respectively, obtain product DEG (dammarane-[12-beta-hydroxy-20, 25-epoxy]-3-O-β-D-pyranglucoside) 167mg.
The data of DEG are as follows:
13C NMR (400MHz, C5D5N):
δ 39.1,26,8,88.7,40.1,56.2,18.5,35,7,37.0,50.2,39.7,31.3,70.3,49.7,51.4,31.2,25.3,55.0,16.7,16.4,77.1,27.4,35.8,30.0,36.6,73.1,33.3,19.7,28.2,15.8,17.3,107.0,75.7,78.871.9,78.4,63.1.
1H MNR (400MHz, C5D5N):
1.48,3.75,1.26,1.94,0.98,0.89,1.23,1.25,1.19,0.951.03,0.88, δ 4.89.
Embodiment 2
The fractionation of DEG:
By DEG, detect through chiral column, 2 peaks (see accompanying drawing 1) occur.With DEG being split with partly preparing chiral column.Specifically, complete through following method.
Chromatographic condition: U.S. Agilent 1260 type high performance liquid chromatography work station；
Chromatographic column: DAICELAD-H, 20 × 250mm, 5 μm；(being purchased from Beijing green BAICAO development in science and technology company limited, Daicel company of Japan produces)
Detection wavelength: 206nm
Flowing phase: normal hexane/isopropanol/diethylamine=80/30/0.1 → 30/15/0.1, has run gradient in 40min.
Flow velocity: 2.0ml/min
Column temperature: 35 DEG C
Sampling volume: 200 μ l
Method: DEG is dissolved methanol, is made into 1mg/ml methanol solution, and said system is prepared separation, collects by peak, obtains 20 (R)-DEG (see accompanying drawing 2).
Embodiment 3
The Structural Identification of 20 (R)-DEG:
20 (R)-DEG is off-white powder.Mp168～169 DEG C；IR(cm-1) 3410,3243,2955,2932,1452,1389；FAB-MS (m/z), 623 [M+H]+, main fragment peak 495 [623-128 (C8H16O, side chain)]+, 461 [623-162 (Glc)]+, prompting has 6 carbon sugar, speculates that its molecular weight is 622 accordingly, and molecular formula is C36H62O8；20 (R)-DEG's13C NMR、1H MNR, HMBC, HMQC, NOE spectrum warp and DEG comparing, at C17, C21, C23, C27There is bigger difference.It addition, C21-H is respectively and C13-H and C18There is NOESY in-H.Through the hydrocarbon ownership of integration analysis of nuclear magnetic spectrum is correlated with, it is determined that the stereochemical structure of 20 (R)-DEG.
Hydrocarbon ownership and the dependency relation thereof of 20 (R)-DEG nuclear magnetic spectrum are as follows:
13C NMR (400MHz, C5D5N, CPosition): δ 39.1 (C1), 26,8 (C2), 88.7 (C3), 40.1 (C4), 56.4 (C3), 18.5 (C6), 35,9 (C7), 39.7.0 (C8), 50.2 (C9), 37.1 (C10), 31.8 (C11), 70.3 (C12), 49.7 (C13), 51.4 (C14), 31.4 (C15), 25.4 (C16), 55.0 (C17), 15.8 (C18), 16.4 (C19), 76.9 (C20), 19.6 (C21), 35.8 (C22), 16.4 (C23), 36.5 (C24), 73.1 (C25), 33.2 (C26), 27.3 (C27), 28.2 (C28), 16.8 (C29), 17.3 (C30), 106.9 (Cg1), 75.8 (Cg2), 78.7 (Cg3)71.8(Cg4), 78.4 (Cg5), 63.1 (Cg6)
1H MNR (400MHz, C5D5N, CPosition): 4.04 (2H, m, C1), 4.25 (2H, m, C2), 3.37 (1H, dd, C3), 0.83 (1H, C5), 1.47 (2H, m, C6), 1.34 (2H, m, C7), 1.48 (1H, m, C9), 2.10 (2H, m, C11), 3.76 (1H, m, C12), 1.78H, m, C13), 1.46 (2H, m, C15), 1.76 (2H, m, C16), 2.00 (1H, m, C17), 0.99 (3H, s, C18), 0.89 (3H, s C19), 1.21 (3H, s C21), 1.16 (2H, m, C22), 1.53 (2H, m, C23), 1.34 (2H, m, C24), 1.25 (3H, s, C26), 1.21 (3H, s, C27), 1.22 (3H, s, C28), 1.03 (3H, s, C29), 0.88 (3H, s, C30), 6.03 (1H, s, 12-OH), δ 4.90 (1H, d, J=7.8Hz, Cg1)。
HMBC (400MHz, C5D5N, CPosition): C1-H(C2, C3, C5), C2-H(C1, C3, C4), C3-H(C1, C28, C29), C5-H(C1, C28, C29), C6-H(C5, C8), C7-H(C5, C8, C19), C9-H(C11, C14, C18), C11-H(C8, C12, C13), C12-H(C11), C13-H(C12, C14, C17), C15-H(C16, C30), C16-H(C14, C15, C17), C17-H(C13, C16, C20, C21, C22), C18-H(C7, C9), C19-H(C1, C4, C9), C20-H(C17, C20, C22), C22-H(C20, C25), C23-H(C20), C24-H(C23, C26, C27), C26-H(C24, C27), C27-H(C22, C24), C28-H(C3, C5, C29), C29-H(C3, C4, C28), C30-H(C14, C15)。
HMQC (400MHz, C5D5N, CPosition): C3-H(C3), C5-H(C5), C13-H(C13), C19-H(C19), C21-H(C21), C26-H(C26), C27-H(C27), C28-H(C28), C29-H(C29), C30-H(C30), Cg1-H(Cg1), Cg2-H(Cg2), Cg3-H(Cg3), Cg4-H(Cg4), Cg5-H(Cg5), Cg6-H(Cg6)。
NOESY (400MHz, C5D5N, CPosition): when irradiating C21During-H, C13-H and C18-H has substantially reinforcement, shows have NOE effect.As follows:
Embodiment 4
The external inhibitory action to tumor cell of 20 (R)-DEG:
Exist by MTT method, metastatic human colon cancer cell (SW620), human gastric adenocarcinoma (BGC-823), Non-small cell lung carcinoma cell A549, human breast cancer cell (MCF-7), on 5 kinds of tumor cells such as Human Prostate Cancer Cells (PC-3), is evaluated the anticancer effect of 20 (R)-DEG, using Docetaxel and DEG as control drug, and carry out the comparison of anticancer effect.
Experimental technique: take well-grown cell 200 μ l and add in 96 orifice plates, puts in CO2 incubator and cultivates 24 hours.Add test medicine 20 μ l/ hole, cultivate 48 hours.Again 20 μ l MTT are added in 96 orifice plates, react 4 hours, suck supernatant, add 20 μ l DMSO/ holes, at wavelength 570nm, measure the light absorption value in every hole with enzyme-linked immunosorbent assay instrument, measure the medicine lethal effect to cell.Calculate the concentration (IC needed for Drug inhibition 50% growth of tumour cell50).Result such as table 1 below.
Result shows, all shows in five tumor strains, 20 (R)-DEG external anticancer effect IC50Concentration is slightly above Docetaxel, but significantly lower than DEG, judges that the active anticancer of DEG is mostly derived from 20 (R)-DEG with this.
Table 1:20 (the R)-DEG suppression IC to five kinds of cancerous cell50Value
Embodiment 5
20 (the R)-DEG inhibitory action to Mus interior tumor cell
In order to evaluate 20 (the R)-DEG active anticancer to animal, with whole animal nude mice, it is evaluated.
Experimental technique:
Animal: NU/NU Female nude mice, packet, often group 10.
Transplantation tumor: taking well-grown cell, to be seeded in forelimb subcutaneous, after transplantation tumor the 6th～10 day, tumor weight was 100～about 300mg.
It is administered: the single administration next day of lumbar injection, altogether to 7 days.Every other day claim nude mice body weight, calculate tumor weight.Tumour inhibiting rate %=matched group body weight-medicine group body weight/matched group body weight the results are shown in Table 2.
Result: from table 2 it can be seen that the tumor suppression dosage of 20 (R)-DEG is slightly larger than how western taxol.Under same dose, 20 (R)-DEG tumour inhibiting rate is apparently higher than DEG.This result is consistent with cell in vitro tumor suppression, further illustrates the active anticancer of DEG mainly by 20 (R)-DEG contribution.
Table 2 20 (the R)-DEG therapeutical effect to metastatic human colon cancer SW620 transplanted tumor

Claims (7)

1. Compounds of structural formula I or its pharmaceutically acceptable salt.Compound of formula I Chinese name: 20 (R)-dammarane-[12-hydroxyl -20,25-epoxy]-3-O-β-D-pyranglucoside.English name: Dammarane-[12-ol-20,25-epoxy]-3-O-beta-D-glucopyranside.
2. claim 1 compound of formula I or its most acceptable salt and pharmaceutically acceptable excipient carrier thereof.
3. compound of formula I and the most acceptable salt thereof, and pharmaceutically acceptable carrier is used for preparation and cancer phase The medicinal usage of related disorders.
4. the preparation of claim 1 compound, splits through chiral column HPLC and realizes.Including: Daicel AD-H chiral column is achieved.Normal hexane surface is applied amylose-three (3,5-xylyl carbamic acids Ester) silica gel is the semi-preparative chiral column of filler, using normal hexane/isopropanol/diethylamine=80/30/0.1 → 30/15/0.1 as flowing Phase, detects with differential refraction detector, collects by peak and obtains.
5. according to claim 3, the medicinal usage of compound of formula I, including: liquid preparation (injection, oral liquid, nasal drop), Solid preparation (tablet, capsule, granule and slow releasing agent etc.), external preparation (such as: patch, spray etc.), aerosol agent and Smears etc..
6. claim 5 pharmaceutical carrier, includes disintegrating agent, diluent, adhesive, lubricant etc. for constituting the adjuvant of solid drugs With constitute liquid medicine adjuvant include solvent, pH adjusting agent, osmotic pressure regulator, antioxidant, metal chelating agent, preservative, Correctives etc..
7. according to claim 3-6, there is prevention and the pharmaceutical composition for the treatment of cancer, it is characterized in that prevention and treatment cancer, such as: Transitivity colon cancer (SW620), adenocarcinoma of stomach (BGC-823), nonsmall-cell lung cancer (A549), breast carcinoma (MCF-7) and prostatitis Adenocarcinoma PC-3 etc..
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Cited By (1)

* Cited by examiner, † Cited by third party
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Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN104434939A (en) * 2014-11-14 2015-03-25 深圳大学 Antitumor medicament composition of panax notoginseng saponins R7 and oridonin with capabilities of reducing toxicity and enhancing efficacy and application of antitumor medicament composition

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Publication number Priority date Publication date Assignee Title
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