CN102372630B - Resorcinol derivative with antitumor activity, its preparation method and application - Google Patents
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Abstract
The invention discloses a resorcinol derivative with antitumor activity, its preparation method and application. The resorcinol derivative with antitumor activity is represented formula I, wherein, R is a C10-C15 saturated or non-saturated long-chain substituent. The resorcinol derivative with antitumor activity is obtained by separating the rhizome of short-stem ardisia japonica. In vitro antitumor experiments show that the resorcinol derivative has obvious inhibiting effect on human prostate cancer PC-3, human lung cancer A549, human stomach cancer SGC 7901, human breast cancer MCF-7, human pancreas cancer PANC-1, human liver cancer SMMC-7721, human neuroglioma U 251 and other tumor cell lines, and can be prepared into antitumor drugs.
Description
Technical field
The present invention relates to resorcinol derivatives, relate in particular to from the short stem Japanese Ardisia Herb extract be separated to there is resorcinol derivatives of anti-tumor activity and preparation method thereof, the invention still further relates to the purposes of this resorcinol derivatives in preparing antitumor drug, belong to resorcinol derivatives field.
Background technology
Short stem Japanese Ardisia Herb You Chengxue party is the Myrsinacea plant gland point Japanese Ardisia Herb Ardisa Punctalindl rhizome of (also claim mountain blood red, precious jade language is called short, the golden-rimmed sieve umbrella of Dulong, nine pipe blood).Blood Party spirit is flat, and mildly bitter flavor, pungent is nontoxic.There is blood circulation promoting and enriching, menstruation regulating, remove obstruction in channels to relieve pain, the function of relieving sore throat and diminishing swelling.For menoxenia, postpartum anemia; Treating rheumatic ostealgia, wound; Stomachache, Biliary Calculi; Rhinopharyngitis and carbuncle pyogenic infections from tumour or sore etc.In antitumor herbal medicine screening, find that it has very strong cytotoxic activity.
Summary of the invention
One of the object of the invention is to provide the resorcinol derivatives that a class has anti-tumor activity;
Two of the object of the invention is to provide a kind of method of preparing the resorcinol derivatives of above-mentioned anti-tumor activity;
Three of the object of the invention is that the above-mentioned resorcinol derivatives with anti-tumor activity is applied to prepare antitumor drug;
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One class has the resorcinol derivatives of anti-tumor activity, and its structural formula is shown in general formula I:
General formula I
Wherein R is the saturated or unsaturated long-chain substituting group carbon atom number from ten to 15.
Preferably, described resorcinol derivatives is preferably from compound 1, compound 2 or compound 3, and wherein, the chemical structural formula of compound 1, compound 2 and compound 3 is as follows:
Another object of the present invention is to provide a kind of method of preparing the resorcinol derivatives of above-mentioned anti-tumor activity;
A method of preparing the resorcinol derivatives of above-mentioned anti-tumor activity, comprising:
(1) get the dry rhizome in the short stem Japanese Ardisia Herb, with at room temperature diacolation extraction of methyl alcohol, percolate concentrating under reduced pressure reclaims solvent and obtains methanol extract; (2) get dry methanol extract, be dispersed in water, be extracted with ethyl acetate and obtain ethyl acetate extract; (3) get ethyl acetate extract and carry out silica gel column chromatography, use successively following solvent elution: by volume ratio, be first 1: 0-0: hexane/EtOAc gradient elution of 1; Then the CHCl that is 8: 2 by volume ratio
3/ MeOH wash-out; The CHCl that is 7: 3 by volume ratio again
3/ MeOH wash-out; Thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains altogether 20 cuts, second fraction A B2 wherein, that is: and 50: 1 wash-out parts of hexane/EtOAc are through Sephadex LH-20 column chromatography, the CHCl that volume ratio is 1: 1
3/ MeOH wash-out, thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains 3 sub-fraction A B2-1, AB2-2 and AB2-3; (4) adopt preparative rp-hplc, moving phase is that v/v is the MeOH/H of 95: 5
2o prepares compound 1, compound 2 and compound 3 from sub-fraction A B2-2; Wherein the retention time of compound 1 is 22.5min, and the retention time of compound 2 is 20.2min, and the retention time of compound 3 is 18.6min.
Mutual-through type I compound of the present invention has carried out anti tumor activity in vitro mensuration, anticancer experiment in vitro shows, compound of Formula I has obvious restraining effect to various tumor cell strains such as human prostata cancer PC-3, people's lung cancer A549, people's cancer of the stomach SGC7901, human breast carcinoma MCF-7, human pancreas cancer PANC-1, people's liver cancer SMMC-7721, human glioma U251; In addition, by antibumor molecules Mechanism Study, show, compound 3 can be induced human pancreatic carcinoma PANC-1 cell line generation apoptosis.In a word, compound of Formula I has the effect of obvious inhibition tumour, can be prepared into antitumor drug.
A further object of the present invention is to provide a kind of pharmaceutical composition for the treatment of tumour, above-mentioned compound of Formula I and pharmaceutically acceptable carrier or vehicle that this pharmaceutical composition contains the upper significant quantity for the treatment of, the content of its formula of 1 compound in pharmaceutical composition can be 1-100%.Pharmaceutical composition of the present invention can be prepared into suitable clinically preparation according to conventional medicine formulation method, such as thinking tablet, capsule, oral liquid etc.
Accompanying drawing explanation
Fig. 2 compound 3 causes the apoptotic morphological observation experimental result of PANC-1.
The affect experimental result of Fig. 3 compound 3 on PANC-1 cell caspase-3 and caspase-9 activity.
3 couples of PANC-1 cell death related protein bax of Fig. 4 compound, bcl-2, caspase-3, caspase-9 expresses affects experimental result.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The Isolation and Identification of embodiment 1 the compounds of this invention 1-3
Get the dry rhizome 1.5kg in the short stem Japanese Ardisia Herb, with at room temperature diacolation extraction (5L * 3, seven days) of methyl alcohol, percolate concentrating under reduced pressure reclaims solvent and obtains methanol extract.Get dry methanol extract 120g, be dispersed in 1000mL water, be extracted with ethyl acetate three times (1000mL * 3) and obtain ethyl acetate extract 48g.Get ethyl acetate extract 20g and carry out silica gel column chromatography, use successively following solvent elution: hexane (hexane)/EtOAc, 1: 0-0: 1, v/v; CHCl
3/ MeOH, 8: 2, v/v; CHCl
3/ MeOH, 7: 3, v/v; Thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains altogether 20 cuts (AB1-AB20).Wherein (50: 1 wash-out parts of hexane/EtOAc, 0.15g) through Sephadex LH-20 column chromatography, CHCl for second fraction A B2
3/ MeOH, 1: 1 wash-out, thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains 3 sub-fraction A B2-1, AB2-2 and AB2-3.With preparative rp-hplc, (moving phase is MeOH/H
2o 95: 5, v/v) prepares compound 1 (2.0mg from sub-fraction A B2-2; Rt.22.5min), compound 2 (2.5mg; Rt.20.2min) and compound 3 (15mg; Rt.18.6min).(20: 1 wash-out parts of hexane/EtOAc, 3.2g) can obtain compound 3 (2.5g) by recrystallizing methanol to fraction A B2-3.
Compound 1 (2-methoxy-4-hydroxy-6-(8Z-pentadecenyl)-benzene-1-O-acetate): red oil; UV (MeOH) λ
maxnm (log ε): 223 (3.84), 280 (3.45);
1h-NMR (500MHz, CDCl
3) and
13c-NMR (125MHz, CDCl
3) data are in Table 1; Positive ESI-MS:m/z 391[M+H]
+; Negative ESI-MS:m/z389[M-H]
-.Negative HRTOFMS:389.2714 ([M-H]
-, C
24h
37o
4; Calc.389.2692) .EI-MS:390 (15.5%), 374 (80.3%), 346 (100%), 260 (6.4%), 206 (4.5%).
Compound 2 (2-methoxy-4-hydroxy-6-pentadecyl-benzene-1-O-acetate): red oil; UV (MeOH) λ
maxnm (log ε): 224 (3.754), 280 (3.35);
1h-NMR (500MHz, CDCl
3) and
13c-NMR (125MHz, CDCl
3) data are in Table 1; Positive ESI-MS:m/z 393[M+H]
+; Negative ESI-MS:m/z391[M-H]
-negative HRTOFMS:391.2859 ([M-H]
-, C
24h
39o
4; Calc.391.2848).
Compound 3 (2-methoxy-4-hydroxy-6-tridecyl-benzene-1-O-acetate, Ardisiphenol D): recrystallizing methanol obtains needle crystal; Molecular formula C
22h
36o
4.Positive?ESI-MS：382[M+NH
4]
+；365[M+H]
+。
1h NMR δ 6.24 (1H, d, J=2.7Hz, the H on phenyl ring), 6.18 (1H, d, J=2.7Hz, the H on phenyl ring), 2.29 (3H, s ,-OCOCH
3), 3.71 (3H, s ,-OCH
3), 0.86 (3H, t, J=6.9Hz, long-chain fat alkyl), 1.23-1.49 (18H, m, long-chain fat alkyl), 2.38 (2H, t, J=7.2Hz, long-chain fat alkyl).
13c NMR δ 131.6,153.8,98.1,151.7,107.5,136.5 (C on phenyl ring), 20.5 (OCO
ch
3), 169.8 (O
coCH
3).
Table 1 compound 1 and 2
1h NMR (500MHz) and
13c NMR (125MHz) spectroscopic data (CDCl
3)
The anti tumor activity in vitro experiment of experimental example 1 the compounds of this invention
One, experiment material
1, the compound 1,2 and 3 that test compound: embodiment 1 is separated;
2, cell strain: A549 (people's lung cancer), PANC-1 (human pancreas cancer), PC-3 (human prostata cancer), U251 (human glioma), SMMC-7721 (people's liver cancer), SGC-7901 (people's cancer of the stomach), MCF-7 (human breast carcinoma) (above-mentioned cell strain is all purchased from Chinese Academy of Sciences's Shanghai cell bank).
Two, experimental technique
1, the restraining effect of 1,2 and 3 pairs of tumour cells of compound experiment
The A549 taking the logarithm vegetative period, PANC-1, PC-3, U251, SMMC-7721, SGC-7901, MCF-7 cell is with 0.25% tryptic digestion and make 1 * 10
5the single cell suspension of/mL, is inoculated in 96 porocyte culture plates, every hole 200 μ L.In 37 ℃, cultivate after 24 hours, the compound 1, compound 2 and the compound 3 that add respectively 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, 1.56 μ M, 0.78 μ M, 0.39 μ M, 0.19 μ M, 0.1 μ M, 0.05 μ M, establish blank group, each drug level is established 3 parallel holes simultaneously.In 37 ℃, 5% volume fraction C O
2in incubator, cultivate after 48 hours, every hole adds 5mg/mL MTT solution 8 μ L, continue to cultivate 4 hours, supernatant discarded, every hole adds DMSO 150 μ L, and vibrator fully vibrates after whole dissolving of 10min first a ceremonial jade-ladle, used in libation crystallization to be generated, in microplate reader, measures 570nm absorbance, 630nm is reference wavelength, calculates as follows inhibiting rate: inhibiting rate (%)=(1-A sample sets/A control group) * 100%.And utilize Logit method calculation of half inhibitory concentration (IC
50).
Experimental result is in Table 2.
Restraining effect (the IC of 1,2,3 pairs of tumour cells of table 2. compound
50: μ M)
From experimental result, 1,2,3 pairs of different tumour cells of compound all have significant restraining effect, illustrate that compound 1,2 and 3 all has good anti-tumor activity.
2, the impact of compound 3 on PANC-1 cellular form
It is 2 * 10 that the human pancreatic cancer cell PANC-1 taking the logarithm vegetative period makes cell density
5the single cell suspension of/mL, is inoculated in 6 porocyte culture plates every hole 2mL.After 24 hours, add the compound 3 of different concns, establish blank group simultaneously, establish 3 parallel holes for every group.Put in 37 ℃ of incubators and cultivate 48 hours, use PBS washed cell 2-3 time.Add the PBS solution 20 μ L containing Hoechst 33258 (final concentration is 20 μ g/mL), be placed in the 15min that dyes on ice, observation of cell form taking pictures under inverted fluorescence microscope.
As shown in Figure 2, in blank group, PANC-1 cell all presents circle, even dyeing to experimental result.Compound 3 acted on PANC-1 cell after 48 hours, compared with blank, and cell presents obvious apoptosis feature, comprised that cell volume dwindles, chromatin is condensing, apoptotic body formation etc.
3, the impact of compound 3 on PANC-1 cell caspase-3 and caspase-9 activity
It is 4 * 10 that the human pancreatic cancer cell PANC-1 taking the logarithm vegetative period makes cell count
5the single cell suspension of/mL, be inoculated in (every ware 6mL) in 60mm culture dish, every group establish 3 parallel, after 24 hours, add respectively the compound 3 of 0.39 μ M and 0.19 μ M, establish blank group simultaneously, put in 37 ℃ of incubators and cultivate 1h, 2h, 4h, 8h, after 24h, according to caspase-3 determination of activity test kit specification sheets method, measure, and according to explanation, calculate the enzyme activity level of caspase-3 in cell.Caspase-9 activity is in like manner measured.
Experimental result shows, compound 3 PANC-1 of place cell different time sections, and caspase-3 and caspase-9 are active obviously to raise, and has time-dependent relation (Fig. 3).
4,3 couples of PANC-1 cell bax of compound, bcl-2, caspase-3, the impact that caspase-9 apoptosis-related protein is expressed
The human pancreatic carcinoma PANC-1 cell line of taking the logarithm vegetative period makes 4 * 10
5the single cell suspension of/mL, be inoculated in (every ware 6mL) in 60mm culture dish, the compound 3 that adds different concns after 24 hours, establish blank group simultaneously, put and in 37 ℃ of incubators, cultivate after 48h with PBS washed cell 2 times, cell scraper collecting cell, adds the PBS re-suspended cell containing 1%PMSF, in ultrasonic cell disruption instrument, carry out cytoclasis, by fragmentation completely cell suspension in 10000rmin
-1centrifugal 5min, gets supernatant liquor, with Bradford determination of protein concentration kit measurement total protein concentration, adds 5 * albumen sample-loading buffer of 1/4 volume, boils 5min in boiling water.Get 50 μ g total protein loadings, after row 12%SDS-polyacrylamide gel electrophoresis, albumen electricity consumption swimming method on gel is transferred on nitrocellulose filter, 5% skim-milk sealing is spent the night, TBS-T buffer solution for cleaning film 3 times, each 5min, then adds 4 ℃ of overnight incubation of corresponding primary antibodie solution.By same procedure, wash film, add two anti-solution of horseradish peroxidase-labeled to hatch 1h, same procedure is developed in darkroom with ECL test kit after washing film, photographic fixing.
Claims (1)
1. a method of preparing the resorcinol derivatives of anti-tumor activity, described resorcinol derivatives is compound 1, compound 2 or compound 3; Wherein, the structural formula of compound 1, compound 2 and compound 3 is respectively:
Comprise:
(1) get the dry rhizome in the short stem Japanese Ardisia Herb, with at room temperature diacolation extraction of methyl alcohol, percolate concentrating under reduced pressure reclaims solvent and obtains methanol extract; (2) get dry methanol extract, be dispersed in water, be extracted with ethyl acetate and obtain ethyl acetate extract; (3) get ethyl acetate extract and carry out silica gel column chromatography, use successively following solvent elution: by volume ratio, be first 1: 0-0: hexane/EtOAc gradient elution of 1; Then the CHCl that is 8: 2 by volume ratio
3/ MeOH wash-out; The CHCl that is 7: 3 by volume ratio again
3/ MeOH wash-out; Thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains altogether 20 cuts, wherein second fraction A B2, that is: hexane/EtOAc50: 1 wash-out part is through Sephadex LH-20 column chromatography, the CHCl that volume ratio is 1: 1
3/ MeOH wash-out, thin-layer chromatography launches, 10%H
2sO
4colour developing, shows that the cut of same blob merges, and obtains 3 sub-fraction A B2-1, AB2-2 and AB2-3; (4) adopt preparative rp-hplc, moving phase is that v/v is the MeOH/H of 95:5
2o prepares compound 1, compound 2 and compound 3 from sub-fraction A B2-2; Wherein the retention time of compound 1 is 22.5min, and the retention time of compound 2 is 20.2min, and the retention time of compound 3 is 18.6min.
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| Hsun-Shuo Chang et al..Cytotoxic alkyl benzoquinones and alkyl phenols from Ardisia virens.《Phytochemistry》.2009,第70卷2064–2071. |
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