CN101879159B

CN101879159B - Application of 3,5-dimethylpsoralen in preparing anti-tumor medicaments

Info

Publication number: CN101879159B
Application number: CN2010101467272A
Authority: CN
Inventors: 刘非燕; 孙建国; 吴平; 黄志真; 郭添德; 冯国培
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2010-04-15
Filing date: 2010-04-15
Publication date: 2011-11-30
Anticipated expiration: 2030-04-15
Also published as: CN101879159A

Abstract

The invention belongs to the field of pharmacy and relates to application of 3,5-dimethylpsoralen in preparing antitumor medicaments. The molecular formula of the 3,5-dimethylpsoralen is C13H10O3, and the molecular weight is 214.22. The 3,5-dimethylpsoralen has broad-spectrum antitumor activity and strong suppression effect on various human tumor cells in vitro. The mechanism of suppressing the proliferation of hepatocarcinoma HepG2 is cancer cell apoptosis induction, thus the 3,5-dimethylpsoralen can be combined with medicinal dressings and applied to preparing medicaments for resisting tumor activities, particularly to preparing the medicaments for resisting the liver cancer, the breast cancer, the cervical cancer, the skin cancer, the nasopharyngeal carcinoma and the colon cancer. The invention provides more choices for treating the cancers, and the 3,5-dimethylpsoralen has small toxicity to normal cells and is beneficial to clinical application.

Description

3, the application of 5-dimethyl psoralen in the preparation antitumor drug

Technical field

The present invention relates to medical technical field, be specifically related to 3, the application of 5-dimethyl psoralen in the preparation antitumor drug.

Background technology

3,5-dimethyl psoralen (being called for short DMFC), molecular formula is C ₁₃H ₁₀O ₃Be the psoralen derivant, its synthetic method document 1 that sees reference has the structure of formula (I),

Up to now, do not have bibliographical information 3,5-dimethyl psoralen is used in the preparation antitumor drug.Malignant tumor is to threaten the most serious disease of human physical and mental health now, and the mortality rate height does not still have the antitumor drug report of high-efficiency low-toxicity at present, so is badly in need of this class medicine of exploitation.

Summary of the invention

The purpose of this invention is to provide a kind of 3, the application of 5-dimethyl psoralen in the preparation antitumor drug.

3,5-dimethyl psoralen (being called for short DMFC), structural formula with formula (I): the present invention prepared 3,5-dimethyl psoralen medicine can be in the application in the medicines such as preparation treatment hepatocarcinoma, breast carcinoma, cervical cancer, skin carcinoma, nasopharyngeal carcinoma, colon cancer.

Of the present invention 3,5-dimethyl psoralen can be made medicine with any one adjuvant or the drug excipient that pharmaceutically allow, its preparation can be any one dosage form that pharmaceutically allows, and includes but not limited to liquid preparation, granule, tablet, electuary, soft capsule, soft capsule, drop pill or injection.

Medicine provided by the invention, its form of medication mainly comprises oral administration or drug administration by injection.

Experimental results show that provided by the invention 3,5-dimethyl psoralen, the effect with extracorporeal suppression tumor cell propagation detects through tetramethyl azo azoles salt (abbreviation mtt assay), handled tumor cells in 48 hours after, the half of hepatocarcinoma HepG2 is suppressed appreciation rate IC ₅₀Be about 8.46 ± 0.28 μ M, breast carcinoma MCF-7 is 33.5 ± 4.95 μ M, is 68.5 ± 9.19 μ M to cervical cancer SIHA, epithelial cancer A431 is 32.33 ± 4.21 μ M, nasopharyngeal carcinoma CNE2 is 105.5 ± 8.91 μ M, is 124.5 ± 7.62 to colon cancer CaCo2, and 3, the mechanism that 5-dimethyl psoralen kills hepatocarcinoma HepG2 cell is to induce it to produce apoptosis, therefore itself and medical dressing combination can be treated in the malignant tumor medicine in preparation and be used.

Advantage of the present invention: the invention discloses 3, the application of 5-dimethyl psoralen in the preparation antitumor drug, especially the application in anti-hepatocarcinoma, anti-breast cancer, anti-cervical cancer, anti-epithelial cancer, anti-nasopharyngeal carcinoma and the resistive connection bowelcancer medicine, for above-mentioned treatment for cancer provides more more options, and 3,5-dimethyl psoralen is little to Normocellular toxicity, helps clinical practice.

Description of drawings

Fig. 1 is the cell toxicity test of DMFC to hepatocarcinoma HepG2 cell.

Fig. 2 produces the dna ladder shape band gel electrophoresis figure of apoptosis for DMFC induces hepatocarcinoma HepG2 cell.

Fig. 3 produces the two streaming figure that dye of AnnexinV-PI of apoptosis for DMFC induces hepatocarcinoma HepG2 cell.

Fig. 4 is that Caspase-8 and Caspase-9 inhibitor reverse the effect of the inductive HepG2 apoptosis of DMFC.

The specific embodiment

Below with reference to specific embodiments and the drawings the present invention is further elaborated, these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.

Embodiment 1, the synthetic and evaluation of 5-dimethyl psoralen (DMFC) ^[1]

(1) the synthesizing coumarin derivant 1

Resorcinol (3.96 grams, 36 mMs), ethyl acetoacetate (5.59 grams, 43 mMs) and the mixture of 12M sulphuric acid (60 milliliters) at room temperature stirred 15 hours, should pour in the frozen water (50 milliliters) by reacted mixed liquor then, precipitate after filtration with the washing after, get pure coumarin derivative 1 (5.70 grams, 90% productive rate) with recrystallizing methanol.Product is the white powder solid, fusing point: 182-184 ℃.

(2) the synthesizing coumarin derivant 2

Above-mentioned coumarin derivative 1 (5.46 grams, 31 mMs) is dissolved in exsiccant acetone (75 milliliters) and dichloromethane (75 milliliters), adds potassium carbonate (8.55 grams, 62 mMs).Stir and drip acetone dichloride (5.7 grams, 62 mMs) down, drip off back mixture stirring and refluxing 24 hours.After reacting end, mixed liquor cooling, filtration use acetone and washed with dichloromethane filtering residue respectively once.Filtrate merges, is spin-dried for, and gets pure coumarin derivative 2 (5.61 grams, 78% productive rate) with recrystallizing methanol or column chromatography (petroleum ether: ethyl acetate=silica gel was immobile phase as eluent in 1: 1).Product is the white powder solid, fusing point: 147-149 ℃.

(3) synthetic psoralen derivant (DMFC) I

The above-mentioned coumarin derivative 2 (5.33 grams, 23 mMs) and the mixed liquid of 1.0M sodium hydroxide (140 milliliters) were refluxed 24 hours under nitrogen protection.Reaction mixture cooling back 2.0M hcl acidifying, precipitate after filtration with the washing after, (petroleum ether: ethyl acetate=1: 2 is as eluent with recrystallizing methanol or column chromatography, silica gel is immobile phase) pure psoralen derivative I, according to hydrogen nuclear magnetic resonance data and fusing point, contrast list of references [2] knows that promptly it is 3,5-dimethyl psoralen (4.04 grams, 82% productive rate).Product is the white powder solid, fusing point: 220-222 ℃; Hydrogen nuclear magnetic resonance (300MHz, CDCl ₃) δ (ppm): 7.67 (s, 1H), 7.46 (s, 1H), 7.39 (s, 1H), 6.25 (s, 1H), 2.52 (s, 3H), 2.29 (s, 3H).

Embodiment 23, and 5-dimethyl psoralen is tested the human tumor cells proliferation inhibition activity

Experimental technique:

Hepatoma carcinoma cell HepG2, breast cancer cell MCF-7, cervical cancer cell SIHA, epithelial cancer A431, nasopharyngeal carcinoma CNE2, colon cancer CaCo2 are all available from U.S. American Type Culture Collection.Cell culture adds 5% calf serum (Invitrogene) at DMEM culture medium (Invitrogene), 2mM glutamine, 37 ℃, 10%CO ₂Incubator.

Tetramethyl azo azoles salt (abbreviation mtt assay) detection of drugs is to the influence of tumor cell proliferation: select the exponential phase cell, density with 7000/ hole is inoculated in 96 orifice plates, put 37 ℃, cultivate the medicine that adds variable concentrations after 24 hours in 10% incubator, each concentration is established 6 in multiple hole, cultivate 24 respectively, 48 and 72 hours, culture fluid in the sucking-off orifice plate, adding concentration in every hole is the MTT 50 μ l of 1mg/ml, put into incubator and continue to cultivate 3-4 hour, take out 96 orifice plates that added MTT, every hole adds the DMSO of 150 μ l, light shaking orifice plate 30min, after the crystal for the treatment of hole bottom dissolves fully, orifice plate is put into microplate reader survey the absorbance value of wavelength, the record result in each hole of 570nm, test triplicate at least, use IC ₅₀Computed in software half propagation inhibition concentration IC ₅₀

After accompanying drawing 1a result showed that 24,48 and 72 hours DMFC handles, the IC50 of hepatocarcinoma HepG2 cell was respectively 16.33 ± 0.13 μ M for 24h, 8.46 ± 0.28 μ M for 48h and, 5.97 μ M ± 0.55.As seen DMFC shows as the dependency of dosage and time to the cytotoxicity of hepatoma carcinoma cell HepG2 cell, and be 40.75 ± 1.06 μ M (accompanying drawing 1b) to the cytotoxicity IC50 of normal liver cell MIHA, little 5 times than hepatoma carcinoma cell, show that DMFC is little to Normocellular toxicity, can use clinically.

In addition, DMFC handled 48 hours, the IC50 of breast carcinoma MCF-7 is 33.5 ± 4.95 μ M, is 68.5 ± 9.19 μ M to the IC50 of cervical cancer SIHA, the IC50 of epithelial cancer A431 is 32.33 ± 4.21 μ M, the IC50 of nasopharyngeal carcinoma CNE2 is 105.5 ± 8.9 μ M, is 124.5 ± 7.62 to show that DMFC can use in the above-mentioned malignant tumor of preparation treatment to the IC50 of the CaCo2 of colon cancer.

Embodiment 33, and 5-dimethyl psoralen induces hepatoma carcinoma cell HepG2 to produce apoptosis

(1) dna ladder shape band experiment: tumor cell HepG2 is collecting cell after the DMFC of variable concentrations handles 48h, add lysate at 37 ℃ of cracking (5mM Tris-HCl that spends the night, 100mM EDTA, 1% (w/v) SDS, the and E.C. 3.4.21.64), the extracting of genomic DNA usefulness phenol/chloroform/isoamyl alcohol (25: 24: 1), 70% ethanol-20 ℃ following precipitation, after removing RNA with the RNA enzyme, add 40 μ g DNA electrophoresis and take pictures on 1.2% agarose gel through the gel imaging instrument.

One of most typical feature of apoptosis, be that cell is after accepting drug effect, DNA can be cut off in the nucleosome junction by nuclease, produce the DNA fragment of 180-200bp or its integral multiple size, present well-regulated scalariform body band during agarose gel electrophoresis, this is to distinguish apoptosis and downright bad important symbol.Accompanying drawing 2 results present well-regulated scalariform body band when showing agarose gel electrophoresis, DMFC handles with

concentration

0,8 μ M, 16 μ M, 32 μ M and 64 μ M, and visible DMFC can induce HepG2 to produce typical apoptosis band, and is dose dependent.

(2) AnnexinV-PI pair is dyed experiment: about 1.5x10 ⁵The hepatocarcinoma HepG2 cell and the DMFC of individual exponential phase are hatched 48h jointly, the trypsinization collecting cell, PBS cleans once, the AnnexinV-FITC binding buffer liquid that adds 500 μ l, contain the AnnexinV-FITC of 2.5 μ l and the PI buffer of 0.5 μ l, lucifuge is hatched 15min then, with FACSCanto flow cytometer (Becton Dickinson) analysis result.

Apoptosis is early stage, Phosphatidylserine on the cell membrane (PS) can be turned to outside the cell membrane in cell membrane, AnnexinV can combine with it, and the cell membrane of apoptotic cell still is kept perfectly, dyestuff PI can not enter cell and dye, therefore dye experiment by AnnexinV-PI is two, can distinguish apoptosis and non-viable non-apoptotic cell.5 of the HepG2 cell

different DMFC concentration

0,8 μ M, 16 μ M, 32 μ M and 64 μ M handle among the accompanying drawing 3a.The lower right corner percent of every figure is all represented the apoptosis percentage rate of this cell under corresponding drug level, and upper right corner percent is then represented the dead percent under the same terms.As we can see from the figure, DMFC can induce the HepG2 cell to produce apoptosis, and this effect has presented dose dependent.Fig. 3 b is the quantitative analysis figure of Fig. 3 a, clearly illustrates that among the figure that HepG2 apoptosis percentage rate rises to 24% along with DMFC concentration increases to 64 μ M from 0.

Embodiment 43, and 5-dimethyl psoralen induces hepatoma carcinoma cell HepG2 to produce the signal path that apoptosis relates to

(1) Caspase-8 and Caspase-9 inhibitor reverse the effect of the inductive HepG2 apoptosis of DMFC

In apoptotic signal conductive process, caspase (cysteine hydrolytic enzyme) plays critical regulating action, therefore their activation is the key character of apoptosis, and it is death receptor path and the logical startup person of mitochondrion that two main caspase family member caspase-8 and caspase-9 have represented two apoptotic signal paths respectively.

About 1.5x10 ⁵The HepG2 cell of individual exponential phase was hatched 48 hours jointly with DMFC/caspase-8 inhibitor (Z-IETD-FMK) or DMFC/caspase-9 inhibitor (Z-LEHD-FMK) respectively, the trypsinization collecting cell, PBS cleans once, the Annexin V-FITC binding buffer liquid that adds 500 μ l, contain the AnnexinV-FITC of 2.5 μ l and the PI buffer of 0.5 μ l, lucifuge is hatched 15min then, with FACSCanto flow cytometer (Becton Dickinson) analysis result.

Among the accompanying drawing 4a, the contrast of HepG2 cell, DMFC 32 μ M individual processing, DMFC adds caspase-8 inhibitor (8I) combined treatment and DMFC adds caspase-9 inhibitor (9I) combined treatment.The lower right corner percent of every figure is all represented the apoptosis percentage rate of this cell under alignment processing.As can be seen, DMFC 32 μ M act on the HepG2 inducing cell and produce apoptosis 16.19%, and add caspase-8 and-9 inhibitor, can reverse this apoptosis induced, apoptotic cell percentage ratio is corresponding to be adjusted downward to 8.67%, 9.8%, and accompanying drawing 4b is the quantitative analysis figure of Fig. 4 a.

As seen, DMFC induces HepG2 cell generation apoptosis to relate to two different paths really, i.e. (death receptor) and extracellular (mitochondrion) path in the cell.

To sum up embodiment is described, provided by the invention 3,5-dimethyl psoralen (DMFC) is to hepatocarcinoma HepG2, breast carcinoma MCF-7, cervical cancer SIHA, epithelial cancer A431, nasopharyngeal carcinoma CNE2, colon cancer CaCo2 all have intensive proliferation inhibiting effect, the mechanism that DMFC kills hepatocarcinoma HepG2 cell is by (death receptor) and extracellular (mitochondrion) two path inducing apoptosis of tumour cell in the cell, and 3,5-dimethyl psoralen is little to Normocellular toxicity, helps clinical practice.The medicine that lacks at present high-efficiency low-toxicity clinically, so 3,5-dimethyl psoralen has the application prospect of good preparation antitumor drug.

The partial reference document that the present invention relates to:

1.O.Gia，S.M.Magno，H.Gonazalez-Diaz，E.Quezada，L.Santana，E.Uniarte，L.D.Via，Bioorg.Med.Chem.2005，13，809.

2.Q.Shen，Q.Peng，J.Shao，X.Liu，Z.Huang，X.Pu，L.Ma，Y.M.Li，A.S.C.Chan，L.Gu，Synthesis?and?biological?evaluation?of?functionalized?coumarins?asacetylcholinesterase?inhibitors，European?Journal?of?Medicinal?Chemistry，2005，40(12)，1307-1315.

Claims

1. one kind 3, the application of 5-dimethyl psoralen in the preparation anti-breast cancer medicines, described chemical compound has the structure of formula (I),

2. one kind 3, the application of 5-dimethyl psoralen in the anti-medicine for nasopharyngeal of preparation, described chemical compound has the structure of formula (I),