CN104926912B - Synthesis and purpose of glycyrrhetinic acid derivative - Google Patents
Synthesis and purpose of glycyrrhetinic acid derivative Download PDFInfo
- Publication number
- CN104926912B CN104926912B CN201510084887.1A CN201510084887A CN104926912B CN 104926912 B CN104926912 B CN 104926912B CN 201510084887 A CN201510084887 A CN 201510084887A CN 104926912 B CN104926912 B CN 104926912B
- Authority
- CN
- China
- Prior art keywords
- enoxolone
- derivative
- glycyrrhetinic acid
- norcantharidin
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
Abstract
The invention designs and synthetizes a lead compound by using a glycyrrhetinic acid derivative as a carrier and being connected with norcantharidin and a derivative thereof through ester bonds, the obtained lead compound expects to have the advantages of having liver targeting action, prolonging action time, reducing toxic and side effects and improving the curative effect of resisting liver cancer, and a model compound is provided for the research of a liver targeting medicine for the liver cancer. Firstly, two positions of C11 and C30 in glycyrrhetinic acid molecules are reduced and synthetized respectively, methyl ester is chemically modified into glycyrrhetinic acid (GA) and 18 alpha-glycyrrhetinic acid as well as a derivative thereof, the glycyrrhetinic acid (GA) and 18 alpha-glycyrrhetinic acid as well as the derivative thereof are used as framework molecules, through a series of reactions, the framework molecules and the norcantharidin (NCTD) as well as a derivative thereof are condensed to form ester as a prodrug, a cell activity screening test is performed on 5 object compounds, 13 object compounds in 3 series are synthetized, and structural characterization is performed on a carbon spectrum, a hydrogen spectrum and a mass spectrum; the influence of different concentrations of 5 object compounds on HepG2 cell proliferation of the liver cancer is inspected by a method, results show that inhibitory action presents time-dose dependence, and the suppression ratio of 14 mu g/mL is the highest.
Description
Technical field
The present invention relates to a kind of preparation method and its usage of Enoxolone derivative, in particular to one kind with sweet
Careless hypo acid is the liver target anticancer medicine of the Norcantharidin of carrier.
Background technology
Radix Glycyrrhizae (Glycyrrhiza root) belongs to legume, is distributed in the European countries such as China western part and Russia,
And it is widely medicinal in many countries.Its main pharmacological activity material be glycyrrhizic acid (glycyrrhizic acid, abbreviation GL) and
(Fig. 1) such as its aglycon enoxolones (glycyrrhetinic acid, abbreviation GA).Radix Glycyrrhizae acids medicine main Jing in human body
GRD beta-glucuronidase is decomposed into enoxolone in hydrochloric acid in gastric juice hydrolysis or Jing livers, then enteral interior bacterium service portion is mitogenetic in hepato-enteric circulation
There is pharmaceutically active into 3- tables-enoxolone and a small amount of 3- dehydrogenations enoxolone.Therefore the effect of Radix Glycyrrhizae acids medicine is substantial
It is the effectiveness of enoxolone performance, there is GA antibacterial, anti-inflammatory, antiviral, antitumor, anti-oxidant, anti-arrhythmia and immunity to adjust
Various effects such as section, therefore be widely studied.
Negishi et al. is confirmed in rat hepatocytes membrane component containing a large amount of GA specific binding sites, GA and the site
Combination be in saturability, high degree of specificity, and site tool protein properties.In recent years Enoxolone derivative is used as one kind
Well Liver targeting material becomes the focus of research, Shiro etc. and reports and participates in glycyrrhizic acid and enoxolone liver active transport
Carrier is probably organic anion transhipment polypeptide (OATP), can if the 3- hydroxyls or 30- carboxyls in GA molecules is performed the derivatization
Can be favourable to the affinity of acceptor, improve the hepatic targeting energy of carrier.Jin Hui etc. has synthesized enoxolone and 11- deoxidations Radix Glycyrrhizae time
The 30- positions amide derivatives of sour (DGA), by Wistar Isolated Rat Liver cellular uptakes GA experiments, GA derivatives to liver cell
The Inhibition test of intake GA, have studied uptake pattern of the isolated rat liver cell to GA, as a result show, the GA derivatives of synthesis can
The emulative intake for suppressing isolated rat liver cell to GA, shows the 30- amide derivatives and GA competition cell films of GA and DGA
Identical acceptor, therefore can be used as Liver targeting carrier；Additionally, derivative shows 30- carboxylics to the inhibition constant result that GA is absorbed
Base is connected with amino-acid ester and 30- carboxyls are connected with the compound of amino acid and the suppression that GA is absorbed is not significantly different from, and illustrates in GA
In molecule, anionic group is whether there is near 30- carboxyls affects less to the transhipment of GA, should be in 30- carboxyls when connecting other molecules
Place's bonding.Ma Shuyan etc. is by the active anticancer of 3-hydroxyl of 18 β-and 18 α-methyl glycyrrhetate and cancer therapy drug endoxan
Structure fragment --- Glyciphosphoramide dichloro is connected, and makes the β of Liver targeting prodrug 18 with anticancer potential-and 18 α-sweet first phosphorus nitrogen
Mustard, expects to reach the effect of active Liver targeting.
Norcantharidin (Norcantharidin, NCTD) is the derivative (Fig. 2) of cantharidin, is China's independent research
With the new type antineoplastic medicine compared with the effect such as powerful antitumor activity and unique increasing leukocyte, protection liver cell, regulation immunity,
NCTD has comprehensive antitumor action, and it can suppress DNA replication dna to suppress tumour cell to increase by cell cycle regulation
Grow；Activation mitochondria pathway, it is many that adjustment promotees the ratio induced tumor apoptosis of apoptosis and anti-apoptotic genes expression and albumen etc.
Individual aspect collective effect reaches antitumous effect, and its toxicity is substantially low than cantharidin, and antitumor action is better than cantharidin, clinical
The upper treatment for being used for liver cancer or auxiliary treatment, show preferable curative effect.Norcantharidin (NCTD) is main with tablet and injection
Agent is applied to clinic, Wei Chunming etc. and have studied pharmacokinetics and Tissue distribution result of study in Norcantharidin mouse body to show
Show, Mouse oral 3H- Norcantharidins absorb rapid, liver distribution less and eliminates fast, kidney is then distributed many, but elimination is also fast.
In vivo distribution results explanation Norcantharidin is low for treating liver cancer arrival target organ concentration, therefore not only reduces drug effect and increasing
The toxicity of other organs is added.Preferably to play its curative effect, toxic and side effect is reduced, extend action time.
The hepatic targeting of medicine mainly realizes that a kind of approach is that medicine is passed through into special carrier such as by 2 kinds of approach
Liposome or nano-carrier, are realized using the phagocytosis of hepatic endothelial network；Another kind is by little point by covalent bond
The medicine of son is bonded on targeted molecular, realizes that receptor mediated endocytosis is realized using the acceptor of stem cell surface,
Such as the pro-drug of asialoglycoprotein receptor mediation.
Fan Feng etc. with Norcantharidin as raw material, with various small molecules it is amino acid modified after, through acylation, hydrogenolysis, glucosides
Change and deacetylation, synthesized Norcantharidin-galactolipin (NCTD-Gal) derivative, expect to obtain before liver target anticancer
Medicine, and anti-tumor experiment in preliminary mouse body has been carried out to product a, as a result shows, the middle and high dosage group tumour inhibiting rates of a apparently higher than
NCTD groups, show that galactosylation improves to NCTD antitumaous effects, and hepatic targeting needs further research.Hu Zhanhong
Norcantharidin and lactobionic acid connection the Norcantharidin of pro-drug lactosylation one has been synthesized into Deng by linking arm of ethylenediamine
(1actosyl-norcantharidin, Lac-NCTD), expecting can be by internal liver using the galactose residue on molecular structure
The AS-GP-R specific recognitions of cell, reach active hepatic targeting.Studies on acute toxicity result shows the toxicity of Lac-NCTD
It is a kind of noval chemical compound of safety non-toxic much smaller than NCTD.It is (sweet that the superfine utilization film dispersion method of Wu prepares Enoxolone derivative
Careless hypo acid tristearin alcohol ester -3-O- galactosides, Gal-GAOSt) after modification Norcantharidin liposome, Jing mouse tail veins are given
Medicine, detects concentration of the Norcantharidin in Mouse Liver kidney over time, with the Norcantharidin aqueous solution using HPLC methods
Group is compared, and to target index TI=(AUC)NC-SOL/(AUC)Gal-GAOStNC-LPTo judge that Enoxolone derivative modification is gone
Targeting of the norcantharidin liposome (Gal-GAOStNC-LP) to major organs.As a result show, Gal-GAOStNC-LP has
Hepatic targeting, and adorned study on targeting of liposome is substantially, can improve curative effect of medication, reduces toxic and side effect.
The content of the invention
The present invention with C3 positions hydroxyl and Norcantharidin and goes first using enoxolone and Enoxolone derivative as carrier
Cantharidin derivative obtains being acted on liver active targeting into 3 list of target compound (Fig. 3-7) of Lipase absobed, prolongation effect
Time, toxic and side effect is reduced, increase the lead compound of anti-liver cancer and anti-curative effect, be the pharmacological screening and structure-activity relationship of Liver targeting prodrug
Research lays the foundation.
To improve fat-soluble and cell-penetrating power, the false aldosterone side effect of parent is reduced, in enoxolone molecule
C11, C30Two positions are chemically modified, by C11- carbonyl reduction and by C30- COOH prepares deoxy-glycyrrhetinic acid into methyl esters, sweet
The carrier of careless hypo acid methyl esters and deoxy-glycyrrhetinic acid methyl esters as prodrug；Enoxolone has two kinds of optical isomers, wherein 18
The hepatic tissue distribution capability of α-enoxolone is more higher than 18 β-enoxolone, and the present invention is by enoxolone (containing 97%18 β-sweet
Careless hypo acid) configuration conversion is carried out, 18 α of its optical isomer-enoxolone and its methyl esters are made as the carrier of prodrug.
The structure activity study of cantharidin and Norcantharidin shows that epoxy bridge is its anticancer pharmacophore, and the present invention is protecting
Stay the Norcantharidin and its mono ethyl ester (3- ethoxycarbonyl -7- oxabicyclos of epoxy bridge【2,2,1】Heptane -2- formic acid), it is suitable
7- oxabicyclos【2,2,1】- heptane -5- alkene -2,3- dicarboxylic acid anhydrides, as anti-cancer function group；The anticancer that the present invention will synthesize
Functional group respectively with carrier enoxolone and its methyl esters (M1 is serial), deoxy-glycyrrhetinic acid and its methyl esters (M2 is serial), 18 α-
Enoxolone and its methyl esters (M3 is serial) 3- positions hydroxyl are into Lipase absobed M1～3 three list of target compound.Wherein, M-1 is serial：
Enoxolone and its methyl esters are carrier families；M-2 is serial：Deoxy-glycyrrhetinic acid and its methyl esters are carrier families；M-3 is serial：18
α-enoxolone and its methyl esters are carrier families.
Present invention relates particularly to a class compound and its stereoisomer, wherein compound are selected from table 1：
The compound of table 1 (M-1～3 are serial) structure and title
The present invention compound by proton nmr spectra (1H-NMR), carbon-13 nmr spectra (13C-NMR), mass spectrum (MS)
Confirm, target compound is investigated using mtt assay after synthesis the cytoactive of tumor cell line HepG2 is acted on.
The invention further relates to a kind of composition, it includes above-claimed cpd and pharmaceutically acceptable carrier.
The invention further relates to purposes of the above-mentioned composition in treatment liver cancer, by the group for giving patient therapeuticallv's effective dose
Compound.
Description of the drawings
Fig. 1：The chemical structural formula of glycyrrhizic acid and enoxolone
Fig. 2：The structure of Norcantharidin
Fig. 3：The design of compound
Fig. 4：The synthetic route of Enoxolone derivative
Fig. 5：3- ethoxycarbonyl -7- oxabicyclos【2,2,1】The synthetic route of heptane -2- formic acid
Fig. 6：Along 7- oxabicyclos【2,2,1】The synthetic route of-heptane -5- alkene -2,3- dicarboxylic acid anhydrides
Fig. 7：Compound synthesis route
Fig. 8：Compound proton nmr spectra (1H-NMR), carbon-13 nmr spectra (13C-NMR), mass spectrum (MS) collection of illustrative plates
Fig. 9：The structural formula of compound M-1-b
Figure 10：5 kinds of compounds of 24 hours variable concentrations act on proliferation inhibition rate curve to hepatoma Hep G 2 cells
Figure 11：5 kinds of compounds of 48 hours variable concentrations act on proliferation inhibition rate curve to hepatoma Hep G 2 cells
Specific embodiment
The compound synthesis of embodiment 1. are tested
1.1 experiment material
1.1.1 major experimental instrument
XT4A type binocular micro-meldometers, temperature is not calibrated；RE-52A type rotary evaporators；CCA-20 type recirculated waters
Pump；CL-2 type magnetic stirring apparatus；ZF-1 type uv analyzers；KQ-250DE type numerical control ultrasonic cleaning machines；Agilent 1100LC-
MSD-Trap-SL mass spectrographs；The type nuclear magnetic resonance chemical analysers of Bruker 400 and the type nuclear magnetic resonance chemical analysers of avanceIII 500,
Solvent is DMSO-d6Or CDCl3, TMS is internal standard.
1.1.2 main agents and material
Enoxolone (contain 97%18 β-enoxolone), Norcantharidin, tetrahydrofuran (anhydrous), triethylamine (anhydrous),
Acetic anhydride (anhydrous), N- methylmorpholines, oxalyl chloride, natrium carbonicum calcinatum, DCC (dicyclohexylcarbodiimide), DMAP (4- diformazans
Aminopyridine), DMF (N,N-dimethylformamide), zinc powder, 4A molecular sieves, VLC：Haiyang Chemical Plant, Qingdao produces silica gel H 200-
300 mesh and 300-400 mesh.
1.2 experimental procedure
1.2.1 the preparation of 18 α-enoxolone
2.0g (35.71m mol) KOH and a certain amount of diethylene glycol (DEG) are added in three-necked bottle, after stirring 10min at 100 DEG C,
5g (10.64m mol) enoxolone is added in above-mentioned system, flow back 2h.Room temperature is cooled to, a certain amount of distilled water is added, it is dense
Salt acid for adjusting pH value is extracted, while hot suction filtration, filter after vacuum drying to faintly acid, suction filtration, filter cake distillation water washing with hot acetone
White powder sterling 2.3g (4.89m mol) is recrystallizing repeatedly so as to obtain with absolute ethyl alcohol after cake vacuum drying, yield 46% melts
Point：325-328℃.
1.2.2 the synthesis of methyl glycyrrhetate
In 1000mL three-necked bottles, enoxolone 14.0g (0.03mol), absolute methanol 700mL is added to start stirring, treat
After enoxolone is completely dissolved, the 21mL concentrated sulfuric acids are added dropwise with dropping funel.Completion of dropping, is heated to reflux 24h.After completion of the reaction,
By reactant liquor natural cooling, have and crystallize after precipitation, refrigeration in refrigerator there are a large amount of solids to separate out after standing half an hour, suction filtration is used and fitted
Amount distillation water washing filter cake, drains, and is dried, and obtains white solid powder 11.3g, yield 88%.Fusing point：254-256℃.
Prepare with method：18- α methyl glycyrrhetates, after completion of the reaction, reactant liquor is transparent, and vacuum distillation removes solvent and obtains
White solid, yield 68%.Fusing point：274-276℃.
1.2.3 the synthesis of deoxy-glycyrrhetinic acid
Extracting liquorice hypo acid 0.88g (0.0019mol) is dissolved in 200mL absolute ethyl alcohols, adds zinc powder 18g (0.28mol), plus
Heat backflow, is added dropwise 160mL concentrated hydrochloric acids in 2 hours, after reacting 2h, continuation is added dropwise absolute ethyl alcohol and clarifies to solution, continues backflow 10
Minute, make fully reaction, heat filtering remove unreacted zinc powder, filtrate is collected, place to room temperature, precipitation is separated out, place 24 little
When after suction filtration, obtain white precipitate 0.65g (thick), recrystallizing methanol obtains sterling, the ﹪ of yield 76, fusing point：327-329℃.
Prepare with method：Deoxy-glycyrrhetinic acid methyl esters, white solid, yield 72%, fusing point：288-291℃.
1.2.4 3- ethoxycarbonyls -7- oxabicyclos【2,2,1】The synthesis of heptane -2- formic acid
Norcantharidin 5g (0.03mol), absolute ethyl alcohol 250mL are added in 500mL round-bottomed flasks, flow back 12h, reaction
Liquid is transparent, and reaction is finished and is cooled to room temperature, and vacuum distillation removes solvent, obtains white solid 5.71g, yield 88.9%, fusing point：
109-111℃。
1.2.5 3- ethoxycarbonyls -7- oxabicyclos【2,2,1】The synthesis of heptane -2- formyl chlorides
3g (0.014mol) demethylcantharidin acid mono ethyl ester, 35mL dichloromethane, DMF 2 is added to drip in 100mL there-necked flasks,
2.38mL (0.028mol) oxalyl chloride is slowly added dropwise under the conditions of ice salt bath, drop finishes, is stirred at room temperature 4 hours, and reaction is finished, and decompression is steamed
Solvent is removed in distillation, obtains faint yellow solid 2.02g, yield 62%.
1.2.6 3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- carboxyls]-epoxide-enoxolone (M-1-a)
Synthesis
In 150mL three-necked bottles add enoxolone 0.94g (0.002mol), Norcantharidin 2.00g (0.012mol),
DMAP0.32g (0.002mol), dichloromethane 40mL.It is heated to reflux 20 hours, TLC monitoring reaction [solvents：V (chloroform)：V
(methyl alcohol)].After completion of the reaction, room temperature is cooled to, reactant liquor is concentrated into yellow oily liquid after washing three times with 10% citric acid.
Methanol-water is recrystallized, and suction filtration obtains white solid.By this white solid ethyl alcohol recrystallization, white powder is obtained.Jing silica gel column layers
Analysis is separated, and eluant, eluent is dichloromethane-methyl alcohol, and gradient elution obtains white needles.Fusing point：302℃-305℃.Same legal system
It is standby：3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- carboxyls]-epoxide-methyl glycyrrhetate (M-1-b)：Jing silica gel
Column chromatography for separation, eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.Fusing point：194℃-196
℃；
3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -5- alkene -3- carboxyls]-epoxide-enoxolone (M-1-e)：
Jing silica gel column chromatographies are separated, and eluant, eluent is dichloromethane-methyl alcohol, and gradient elution obtains white powdery solids；
3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -5- alkene -3- carboxyls]-epoxide-methyl glycyrrhetate (M-1-
f)：Jing silica gel column chromatographies are separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.
3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- carboxyls]-epoxide-deoxy-glycyrrhetinic acid (M-2-g)：Jing
Silica gel column chromatography is separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.Fusing point：287℃-
290℃；
3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- carboxyls]-epoxide-deoxy-glycyrrhetinic acid methyl esters (M-2-
h)：Jing silica gel column chromatographies are separated, and eluant, eluent is dichloromethane-methyl alcohol, and gradient elution obtains white powdery solids.Fusing point：
197-199℃；
3 β-[- 2- formyl-7- oxabicyclos [2,2,1] heptane-3- carboxyls]-8 α of Oxy-1-enoxolone (M-3-j)：Jing
Silica gel column chromatography is separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.Fusing point：283℃-
288℃；
3 β-[- 2- formyl-7- oxabicyclos [2,2,1] heptane-3- carboxyls]-8 α of Oxy-1-methyl glycyrrhetate (M-3-
k)：Jing silica gel column chromatographies are separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.Fusing point：
176℃-179℃；
1.2.7 3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- ethoxycarbonyls]-epoxide-enoxolone
(M-1-c) synthesis
Weigh enoxolone 0.282g (0.006mol) to be dissolved in 12mL dichloromethane, be placed in 50mL single port bottles, go first
Cantharidic acid mono ethyl ester list acyl chlorides 0.42g (0.018mol), N- methylmorpholine 0.4mL (0.0036mol) is dissolved in 20mL dichloromethanes
In alkane, in being placed in dropping funel, it is slowly added dropwise under the conditions of ice salt bath, is heated to reflux 10 hours, TLC monitoring reaction [solvents：
V (chloroform)：V (methyl alcohol)], after completion of the reaction, room temperature is cooled to, 10% citric acid solution of reactant liquor is washed 3 times, and removing layer (has
Machine layer), concentration, Methanol-water recrystallization, suction filtration obtains white solid.Jing silica gel column chromatographies separate, eluant, eluent be dichloromethane-
Methyl alcohol, gradient elution obtains white powdery solids, fusing point：284-287℃.
Prepare with method：3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- ethoxycarbonyls]-epoxide-Radix Glycyrrhizae time
Sour methyl esters (M-1-d)：Ethyl alcohol recrystallization, obtains white powdery solids, fusing point：243-245℃.
3 β-[- 2- formyl -7- oxabicyclos [2,2,1] heptane -3- ethoxycarbonyls]-epoxide-deoxy-glycyrrhetinic acid methyl esters
(M-2-i)：Jing silica gel column chromatographies are separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids.
3 β-[- 2- formyl-7- oxabicyclos [2,2,1] heptane-3- ethoxycarbonyls]-8 α of Oxy-1-enoxolone (M-
3-l)：DMAP is catalyst, and Jing silica gel column chromatographies are separated, and eluant, eluent is dichloromethane-methyl alcohol, and gradient elution obtains white powder
Shape solid, fusing point：266-269℃.
3 β-[- 2- formyl-7- oxabicyclos [2,2,1] heptane-3- ethoxycarbonyls]-8 α of Oxy-1-methyl glycyrrhetate
(M-3-m)：Jing silica gel column chromatographies are separated, and eluant, eluent is petroleum ether-ethyl acetate, and gradient elution obtains white powdery solids,
Fusing point：261-263℃.
Embodiment .2 compound pharmacological evaluation
2.1 main agents and material
HepG2 cell lines are purchased from the Chinese Academy of Medical Sciences of Chinese Academy of Medical Sciences Shanghai cell bank center, DMEM
Culture medium, hyclone (Hyclone, Hyclone companies of the U.S.), trypsase (Huamei Bio-Engrg Co.), dimethyl is sub-
Sulfone (sigma companies), phosphate buffer (PBS), 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides (MTT)
Deng.
2.2 experimental implementation
2.2.1 solution is prepared
1. MTT solution (5g/L)
250mgMTT is weighed, in being put into small beaker, plus 50mLPBS (0.01mol/L, pH7.4) is on electromagnetic force agitator
Stirring 30min, the filtering with microporous membrane with 0.22 μm is degerming, dispenses under aseptic condition, 4 DEG C of preservations.In two weeks effectively.
2. DMEM nutrient solutions of 10% hyclone
90mL DMEM nutrient solutions are drawn, 10mL hyclones are added, is mixed standby.
3. 0.25% trypsase
Trypsase 0.25g is weighed, in being added to volumetric flask, PBS 80mL is added so as to dissolve, 4 DEG C are placed in
Refrigerator overnight, secondary daily PBS is settled to 100mL, and the filtering with microporous membrane with 0.22 μm is degerming, lower point of aseptic condition
Dress, -20 DEG C of preservations.
4. PBS
The dissolving of PBS by specification prepares rear autoclave sterilization sterilizing.
5. 75% alcohol
The distilled water of 250mL is measured, in being added to the absolute ethyl alcohol of 750mL, is mixed, it is standby.
2.2.2.MTT method detects the propagation of Drug inhibition hepatoma Hep G 2 cells
1. cell recovery and culture:The frozen HepG2 cells in liquid nitrogen are taken out, is placed in 37 DEG C of water-bath and is solved rapidly
Freeze, 1000 turns/min, be centrifuged 5min, abandoning supernatant, add 10% inactivated fetal bovine serum DMEM nutrient solutions, be placed in 37 DEG C, 5%
CO2Culture in incubator.
2. the Secondary Culture of cell and cell count:Cell is covered with after Tissue Culture Flask, is passed on, and is sucked with liquid-transfering gun
Nutrient solution in blake bottle, adds PBS 3mL washed once, and is rounded to most cells with 0.25% Trypsin Induced
Afterwards, the DMEM culture mediums containing serum are added to terminate digestion.Single cell suspension is gently blown and beaten into liquid-transfering gun, is centrifuged, plus DMEM
Nutrient solution is resuspended to be poured in blake bottle.
Cell count：The μ L of cell suspension 20 are drawn in 1.5mL sterile centrifugation tubes, after adding the μ L of nutrient solution 180 to mix, will
10ul cell suspensions are taken after dilution it is added drop-wise on clean cell counting count board and counted, finally with 6 × 105The density of/mL connects
Plant into 96 porocyte culture plates (parallel to do 3 holes), per the μ L of pore volume 100, be placed in 37 DEG C, 5% CO2Cultivate in incubator
12h。
3. dosing:Next day, observation of cell has 98% cell growth state good, in being paved with 96 porocyte culture plates, respectively
Addition has diluted the liquid of variable concentrations per each 100 μ L in hole, medicine final concentration be divided into 14 μ g/mL, 7 μ g/mL, 3.5ug/mL,
Totally 14 groups of 1.4ug/mL, four dosage groups and blank control group, solvent control group (0.1%DMSO), each concentration group sets 3 again
Hole, then it is placed in 37 DEG C, 5% CO2Cultivate in incubator.
4. plus MTT colour developing:Respectively be incubated 24h, 48h, after, add 20 37 DEG C of μ L/ hole MTT (5g/L), 5% CO2Culture
4h is incubated in case, is taken out from incubator and is discarded stoste, add 150 μ L/ hole DMSO, vibrate 3min, crystal is fully dissolved.
5. OD values are surveyed:At 490nm on ELIASA, OD values are determined.Take the mean value of 3 multiple holes experiments.According to following public affairs
Formula calculates the inhibiting rate of cell growth：Inhibiting rate %=(blank control wells OD490- experimental port OD490)/control wells OD490 ×
100%.
Embodiment 3：Compound synthesis and the pharmacological results
Synthesized 13 target compounds for having no document report, by proton nmr spectra (1H-NMR), nuclear magnetic resonance
Carbon spectrum (13C-NMR), mass spectrum (MS) confirms structural formula (Fig. 8).Gained target compound is easy to molten tetrahydrofuran, acetic acid second
Ester, is dissolved in methyl alcohol, ethanol, is practically insoluble in water.
3.1 compound wave spectrum analysis
With target compound M-1-b (molecular formula C39H56O8, molecular weight 652.40, structural formula is shown in Fig. 9) as a example by, it is related to it
Spectrum data is parsed.
3.1.1 proton nmr spectra1H-NMR：
1H-NMR(400MHz,CDCl3)δ:5.69 (1H, s, H-12), 4.89-5.05 (3H, m, 3CH), 4.52-4.54
(1H,m,CH),4.13-4.15(1H,m,H-3),3.01-3.08(3H,m,COOCH3),2.37(1H,s,H-9),2.08(1H,
m,H-18),1.86(2H,m,CH2),1.22-1.70(17H,m,H-2,H-6,H-7,H-15,H-16,H-19,H-21,H-22,
H-27),1.63(2H,m,CH2),0.90(3H,m,-CH3)。
3.1.2 carbon-13 nmr spectra13C-NMR:
13C-NMR(100MHz,CDCl3)δ:200.3(C-11),176.9(C-30),174.9(8’),170.4(9’),
169.5(C-13),128.4(C-12),81.9(C-3),61.7C-9),51.8(C-31)。
The chemical shift of carbonyl generally has 4 carbonyls in 160～220ppm, target compound M-1-b molecules, chemistry
Displacement be respectively 200.3,176.9,174.9,170.4, δ 200.3 be attributed to alkylene connection carbonyl carbons chemical shifts, δ
170.4 chemical shifts for being attributed to carboxylic acid carbonyl's carbon, δ 176.9,174.9 is attributed to the chemical shift of the carbonyl carbon of carboxylate, δ
169.5,128.4 it is attributed to the chemical shift of two carbon of alkylene.
3.1.3 mass spectrum ESI-MS:
In the ESI-MS of target compound M-1-b, m/z M+Na+(675), it is known that molecular weight is 652, with target chemical combination
The molecular weight of thing M-1-b is consistent.
To sum up, pass through1H-NMR、13C-NMR, ESI-MS, it is target compound M-1- that can confirm synthesized compound
b。
The structure determination of other target compounds is similar, specifically1H-NMR、13C-NMR, MS spectral data is shown in Table 2,
Table 3, table 4.
3.2 partially synthetic intermediates and target product proton nmr spectra (1H-NMR), carbon-13 nmr spectra (13C-
NMR), mass spectrum (MS) data are shown in Table respectively 2, table 3, table 4.
The target product of table 21H-NMR spectral datas
The M-3 series compounds of table 313C-NMR data
Mass spectrum (MS) data of the target compound of table 4
3.3 target compound M-1-b, M-1-d, M-2-g, M-1-e, M-1-f act on the propagation of hepatoma Hep G 2 cells
Inhibitory action
Impact of five compounds of variable concentrations to proliferation of hepatocellular carcinoma HepG 2 cell line the results are shown in Table 5 and table 6；Positive drug and
The same drug concentration of five kinds of medicines acts on respectively 24h, 48h, 2 time points, i.e., increase with the prolongation inhibiting rate of time, with
48h inhibiting rate highests.Variable concentrations group acts on Hepg2 cells, increases with the increase inhibiting rate of concentration, with 14 μ g/mL suppression
Rate highest.Inhibitory action is in time and dose dependent.
All data are processed using SPSS13.0 statistics softwares, all data are represented using X, compared between OD value groups
Relatively using one way-ANOVA methods analysis, with P<0.05 represents that difference is statistically significant.
Table 5：Five compounds of variable concentrations act on hepatoma Hep G 2 cells the inhibited proliferation (OD of different time
Value) (X)
Table 6：The proliferation inhibition rate (%) that five kinds of medicines of variable concentrations act on different time to hepatoma Hep G 2 cells is (X)
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (4)
1. a kind of compound and its stereoisomer, the compound is selected from
2. one kind treats liver cancer composition, and it includes compound and pharmaceutically acceptable carrier of claim 1.
3. the compound of claim 1 or the composition of claim 2 are in the purposes for preparing treatment liver-cancer medicine.
4. in claim 1 compound preparation method, it is characterised in that：Using enoxolone and Enoxolone derivative as load
Body, with C3 positions hydroxyl with Norcantharidin and norcantharidin derivative into Lipase absobed, wherein, enoxolone and enoxolone spread out
Biology goes first selected from enoxolone and its methyl esters, deoxy-glycyrrhetinic acid and its methyl esters and 18 α-enoxolone and its methyl esters
Cantharidin and norcantharidin derivative are the Norcantharidin and its mono ethyl ester for retaining epoxy bridge.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510084887.1A CN104926912B (en) | 2015-02-16 | 2015-02-16 | Synthesis and purpose of glycyrrhetinic acid derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510084887.1A CN104926912B (en) | 2015-02-16 | 2015-02-16 | Synthesis and purpose of glycyrrhetinic acid derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104926912A CN104926912A (en) | 2015-09-23 |
| CN104926912B true CN104926912B (en) | 2017-05-10 |
Family
ID=54114356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510084887.1A Expired - Fee Related CN104926912B (en) | 2015-02-16 | 2015-02-16 | Synthesis and purpose of glycyrrhetinic acid derivative |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104926912B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110028544B (en) * | 2019-05-05 | 2022-01-04 | 湖南中医药大学 | 11-deoxy glycyrrhetinic acid stearyl ester, derivative, cantharidin liposome, preparation method and application |
| CN111789854A (en) * | 2020-07-27 | 2020-10-20 | 大理大学 | Medical application of 2-alkene-1-alcohol glycyrrhetinic acid methyl ester in preparation of antiviral hepatitis B |
| CN115677813A (en) * | 2021-03-25 | 2023-02-03 | 河北中医学院 | Application of compound in TMEM16A agonist |
| CN113651867B (en) * | 2021-09-17 | 2022-09-16 | 山东大学 | Sulfonamide 18 beta-glycyrrhetinic acid derivative and preparation method and application thereof |
-
2015
- 2015-02-16 CN CN201510084887.1A patent/CN104926912B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 具有抗肿瘤活性的几类中药结构修饰研究进展;吕东峰等;《天津药学》;20131231;第25卷(第6期);41-44 * |
| 去甲斑蝥素主动肝靶向性药物设计研究进展;赵岩等;《环球中医药》;20141130;第7卷(第11期);889-892 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104926912A (en) | 2015-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104926912B (en) | Synthesis and purpose of glycyrrhetinic acid derivative | |
| US20030153512A1 (en) | Curcumin derivatives with improved water solubility compared to curcumin and medicaments containing the same | |
| CN104039796B (en) | 1-oxo/acylated Oridonin derivative acylated for-14-, and its preparation method and application | |
| JP2009530405A (en) | Synthetic pentacyclic triterpenoids and derivatives of betulinic acid and betulin | |
| CN102229598B (en) | Mapping-agathis dammara type diterpene compound, and preparation method and application thereof | |
| US10214558B2 (en) | Natural and synthetic compounds for treating cancer and other diseases | |
| CN110028547B (en) | Diosgenin 3-OH derivative and preparation method and medical application thereof | |
| CN103570792A (en) | Bufalin derivative as well as preparation method, pharmaceutical composition and application thereof | |
| CN109575099A (en) | Dammarane saponins member derivative and its preparation method and application | |
| WO2023160354A1 (en) | Novel acid-sensitive aptamer triptolide conjugate and application | |
| CN111333686A (en) | Baicalin derivative and preparation method and application thereof | |
| Wang et al. | Synthesis and cytotoxic activity of the N-acetylglucosamine-bearing triterpenoid saponins | |
| CN113845551B (en) | Pt (II) complex with photodynamic anti-triple-negative breast cancer activity and preparation method and application thereof | |
| EP2460812A1 (en) | Sterol derivatives and their synthesis and use | |
| Yamada et al. | Syntheses of a series of lacto-N-neotetraose clusters using a carbosilane dendrimer scaffold | |
| CN110964078A (en) | Hederagenin compound H-X with anti-lung cancer effect and preparation method and application thereof | |
| CN102391352B (en) | Amino acid derivatives of rotundic acid and application of derivatives in preparation of antitumor medicines | |
| Cai et al. | Synthesis and biological activity evaluation of podophyllotoxin-linked bile acid derivatives as potential anti-liver cancer agents | |
| Coetzee et al. | Structure and synthesis of the first procassinidin dimers based on epicatechin, and gallo-and epigallo-catechin | |
| CN114907438A (en) | Lupeol coupling triphenylphosphine derivative and preparation and application thereof | |
| CN110759961B (en) | Ursolic acid indolyquinone amide derivatives and preparation method and application thereof | |
| CN111518157B (en) | Triptolide derivative and preparation method and application thereof | |
| CN100341888C (en) | Anticancer new compound of Xiacaogan I, preparation method, and application in pharmacy | |
| CN108341850B (en) | Saponin derivative, and pharmaceutical composition and application thereof | |
| CN110698533B (en) | Ursolic acid indoquinone derivative and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170510 |