CN112358527A - Triptolide acrylate, preparation method and application thereof - Google Patents

Abstract

The invention discloses a novel triptolide derivative, which is shown in a formula I. The invention also discloses a preparation method of the compound and medical application of the compound in preparing anti-cancer drugs. The triptolide acrylate and the pharmaceutically acceptable salt thereof have anticancer activity, and animal in vivo experiments can effectively inhibit the tumor growth of animals. A plurality of in vitro experiments prove that the protein expression quantity of p53 can be obviously increased, the apoptosis of tumor cells is promoted, the growth of the tumor cells is effectively inhibited, and the function of inhibiting the metastasis of cancer cells is achieved. More importantly, the toxicity to normal cells is less than that of triptolide.

Description

Triptolide acrylate, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a triptolide derivative, a preparation method and a medical application thereof.

Background

Triptolide is also called triptolide and triptolide, is an epoxy diterpene lactone compound extracted from root, leaf, flower and fruit of tripterygium wilfordii belonging to Celastraceae, and forms the main active ingredient of the tripterygium wilfordii extract together with alkaloids such as wilforine, wilfordine, etc., and is insoluble in water, and soluble in methanol, dimethyl sulfoxide, anhydrous ethanol, ethyl acetate, chloroform, etc. The present research shows that it has the functions of resisting oxidation, resisting rheumatoid disease, resisting senile dementia, resisting cancer, etc. Modern research shows that: triptolide has effects of resisting rheumatoid diseases, senile dementia, and cancer.

However, triptolide has good activity and strong toxicity, and clinical tests show that triptolide has strong toxic and side effects in the aspects of digestive system, urinary system, cardiovascular system, blood system, anaphylactic reaction, nervous system, reproductive system and the like. On the basis of ensuring certain activity, weakening the toxicity is an important direction for researching triptolide derivatives. The toxicity of triptolide is related to the epoxy rings at the 12 and 13 positions, and the group can be easily combined with various proteins to generate various biological effects.

Disclosure of Invention

An object of the present invention is to provide a triptolide derivative to solve at least one of the above technical problems. Furthermore, the invention also provides a preparation method and medical application of the compound.

It is still another object of the present invention to provide a method for preparing triptolide derivatives, so as to solve at least one of the above technical problems.

The invention provides a triptolide derivative, in particular to acrylic triptolide and pharmaceutically acceptable salts thereof, and the structural formula of the triptolide derivative is shown as a formula I.

The synthetic route of the triptolide acrylate is as follows:

adding triptolide and acylating agent (acryloyl chloride, acryloyl bromide, acrylic acid glycoside, acrylic acid or equivalent 3-chloropropionyl chloride) into organic solvent (anhydrous dichloromethane, chloroform, tetrahydrofuran, diethyl ether), adding organic base such as triethylamine, trimethylamine, pyridine, Diisopropylethylamine (DITEA), 1, 8-diazabicycloundecane-7-ene (DBU), 2, 6-dimethylpyridine, 4-Dimethylaminopyridine (DMAP) as acid binding agent, adding 4-dimethylaminopyridine, Dicyclohexylcarbodiimide (DCC), 1-hydroxy-7-azabenzotriazole (HOAT), 1-Hydroxybenzotriazole (HOBT), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), O-benzotriazol-N, n, N ', N' -tetramethyluronium tetrafluoroborate (TBTU) and the like are used as catalysts, the mixture is stirred for 1 to 4 hours (specifically 2 hours) at room temperature, a saturated sodium bicarbonate aqueous solution is used for quenching reaction, dichloromethane (or other organic polar solvents with poor water compatibility) is used for extraction, a water layer can be extracted twice with dichloromethane (or other organic polar solvents with poor water compatibility), dichloromethane (or other organic polar solvents with poor water compatibility) extract liquor is combined, saturated sodium chloride aqueous solution is used for washing most of water in the extract liquor, then drying agents (one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium chloride and the like) are used for drying, reduced pressure evaporation is carried out, and the compound of the formula I is obtained after silica gel column chromatography purification.

The triptolide acrylate and the pharmaceutically acceptable salt thereof have anticancer activity, and animal in vivo experiments can effectively inhibit the tumor growth of animals. A plurality of in vitro experiments prove that the protein expression quantity of p53 can be obviously increased, the apoptosis of tumor cells is promoted, the growth of the tumor cells is effectively inhibited, and the function of inhibiting the metastasis of cancer cells is achieved. More importantly, the toxicity to normal cells is less than that of triptolide.

The invention introduces a functional group with specific selectivity on the C14-hydroxyl of triptolide, and the functional group is preferentially combined with a target protein, thereby enhancing the selectivity and weakening the toxicity. Meanwhile, as the introduced functional group is a rotatable flexible group with moderate size, a certain steric hindrance effect is generated on the 12 and 13 epoxy rings, the selectivity of the epoxy resin can be improved, and the toxicity is weakened.

Drawings

FIG. 1 shows the preparation of triptolide acrylate¹H NMR spectrum;

FIG. 2 shows the preparation of triptolide acrylate¹³C NMR spectrum;

FIG. 3 is DEPT135 spectrum of triptolide acrylate;

FIG. 4 is a graph of fluorescence signals in nude mice;

FIG. 5 shows subcutaneous transplanted tumors observed on the skin surface of nude mice;

FIG. 6 shows tumor body tissues stripped after sacrifice of nude mice;

FIG. 7 shows the effect of triptolide acrylate on apoptosis-related protein expression;

FIG. 8 is a graph of the toxic effect of different concentrations of drug on LO2 cells;

FIG. 9 shows the effect of different concentrations of drug on the proliferation rate of hepatoma cells;

FIG. 10 shows the effect of triptolide acrylate with different concentrations on the induction of apoptosis of hepatoma cells;

FIG. 11 shows the inhibition of migration of hepatoma cells by triptolide acrylate.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.

Dissolving 103.4mg (0.287mmol) of triptolide and 1.75mg (0.01435mmol) of 4-dimethylaminopyridine in 5mL of anhydrous dichloromethane, adding 319.5mg (3.157mmol) of triethylamine, carrying out ice bath to about 0 ℃, dropwise adding 259.7mg (2.87mmol) of acryloyl chloride, gradually returning to room temperature after dropwise adding, stirring for 2h, detecting complete reaction by TLC, stopping stirring, quenching the reaction by using a saturated sodium bicarbonate aqueous solution, extracting dichloromethane, extracting a water layer twice by using dichloromethane, combining dichloromethane extract, washing most of water in the dichloromethane extract by using a saturated sodium chloride aqueous solution, drying by using anhydrous sodium sulfate, carrying out reduced pressure evaporation to dryness, separating by using a thin-layer silica gel plate, using petroleum ether-ethyl acetate (2:1-1:1) as a developing agent to obtain 32.1mg of colorless transparent oily matter with the yield of about 27.0%. Through detection, the structural formula of the compound is shown as a formula I, namely the triptolide acrylate.

As shown in figures 1-3, compounds of formula I, formula C₂₃H₂₆O₇，ESI-MS m/z:414.1679[M+H]⁺(theoretical value).

¹H NMR(600MHz,CDCl3)δ6.54(d,J＝16.1Hz,1H),6.23(dd,J＝17.3,10.4Hz,1H),5.94(d,J＝10.4Hz,1H),5.15(s,1H),4.77–4.61(m,2H),3.84(d,J＝3.2Hz,1H),3.52(dd,J＝30.8,4.2Hz,2H),2.70(d,J＝13.3Hz,1H),2.32(d,J＝18.3Hz,1H),2.18(d,J＝26.5Hz,2H),1.90(d,J＝39.2Hz,2H),1.59(dd,J＝4.8,16.0Hz,1H),1.24(m,1H),1.06(s,3H),0.97(d,J＝7.0Hz,3H),0.85(d,J＝6.9Hz,3H).

¹³C NMR(151MHz,CDCl3)δ173.24,165.52,159.98,132.46(CH₂),127.78,125.63,71.13,69.98(CH₂),63.64,63.40,61.11,59.78,55.36,55.09,40.40,35.71,29.86(CH₂),28.33,23.47(CH₂),17.58,17.08(CH₂),16.76,13.74.

Inhibition test of triptolide acrylate on nude mouse HepG2 subcutaneous transplantation tumor

1 materials of the experiment

1.1 Experimental animals: nude mice, male, weighing about 18-20g for 4-6 weeks, were purchased from Peking Witonglihua laboratory animal technology Co., Ltd. (certification number: 11400700270675).

1.2 cells: HepG2 cell line; medicine preparation: triptolide acrylate synthesized in the above example.

1.3 other experimental reagents and consumables:

sterile saline, surgical scissors, forceps, display reading vernier caliper (maka technologies, guangzhou), 1mL syringe, cotton swab. RPMI-1640 medium, DMEM medium (Gbico, USA); fetal bovine serum (Gibco, north america); streptomycin (diabody), 0.25% trypsin (EDTA-containing); apoptosis kit and cell cycle kit (Hangzhou Union Biotechnology, Inc.); cell culture flasks, cell culture dishes (corning, new york, usa); 2mL of cryopreserved tubes (Coring, los Angeles, USA); 96-well cell culture plates, 6-well cell culture plates (corning, los angeles, usa). RIPA lysate (strong), PMSF protease inhibitor, phosphatase protein complex inhibitor (guangzhou dingguo biotechnology company); tween 20(ST825, bi yun, guangzhou wei jia science co., ltd.), SDS-PAGE gel kit (bi yun, guangzhou wei jia science co., ltd.), 5 × Loading Buffer (bi yun, guangzhou wei jia science co., ltd.); prism Protein Marker (Thermo, usa); ECL chemiluminescent solution (P0018A, bi yun, guangzhou weijia science and technology ltd); PVDF membranes (bi yun day, guangzhou weijia science and technology ltd); thin filter paper, sponge, 1.5 μm plate (Bio-Rad, USA); methanol (national chemical group chemical Co., Ltd.); glycine (Qingdao Biotechnology, Inc.), SDS (Beijing Byledi Biotechnology, Inc.), Tris Base (Shanghai Baiyan Biotechnology, Inc.), TBS powder (Biyun, Guangzhou Weijia technology, Inc.); skimmed milk powder (BD, UK).

1.4 Experimental instrumentation

One in ten thousand balance (Beijing Redox Liscow instruments Co., Ltd.), carbon dioxide incubator (Shanghai Bochen industries Co., Ltd.); cell superclean bench (yi si high technology ltd, singapore); low speed bench centrifuge (DT5-3, Beijing times Beili centrifuge, Inc.); microplate reader type victrorx 5 (Perkinelmer, usa); liquid nitrogen tank (Locator PLUS, USA). Multifunctional refrigerated centrifuge (5430R, eppendorf, Eppendorf Co., Ltd., China), electrophoresis, membrane-rotating apparatus (Bio-Rad, USA), speed-adjustable oscillator (HS260, IKA Shanghai Saint instruments, Ltd.), constant temperature metal bath (Q872), gel imaging system (XR +) (Bio-Rad, Shanghai laboratories, Ltd.), shaker (SK-L330-Pro).

2 course of experiment

2.1 establishment of animal models

Establishment of HepG2-Luc cell line stably expressing luciferase: taking He in logarithmic growth phasepG2 cells at 1X 10⁵The cells were plated in 24-well plates and cultured overnight to allow for adequate adherence. Replacing the original medium with 2mL of fresh medium containing 6. mu.g/mL polybrene approximately 1X 10 times the amount of recombinant lentiviral particles stably expressing Luciferase (Luciferase) was added⁵Transfection units, incubated at 37 ℃ for 4h, and diluted polybrene by adding 2mL of fresh medium. The culture was continued, and the virus-containing medium was replaced with fresh medium. And (3) continuing culturing, replacing a culture medium containing puromycin (puromycin) for resistance screening, selecting drug-resistant clones, and continuing screening for two weeks to finally obtain the cell strain HepG2-Luc capable of stably expressing luciferase.

HepG2-Luc cell line was used to construct lotus mice: the frozen HepG2-Luc cells are recovered to 100cm²And (3) culturing in a culture bottle in vitro until the logarithmic phase is reached, digesting by using 0.25% trypsin containing EDTA, collecting cells, centrifuging at room temperature at 1000rpm for 3min, removing supernatant, washing the cells by using serum-free DMEM (DMEM) culture medium, and detecting the cell survival rate by using trypan blue dye exclusion experiment (the proportion of living cells exceeds 95% and meets the experiment requirement). A small amount of serum-free DMEM medium was added to resuspend the cells and the cells were counted. Sterilizing the back skin of nude mouse with 75% alcohol, collecting the solution containing 1 × 10⁷About 200 mu L of the cell suspension is inoculated to the right anterior axilla of the nude mouse, the nude mouse is continuously raised for a week under an aseptic condition, and the naked eye can see whether subcutaneous planting tumors exist or not.

Detecting tumor growth by using a small animal living body imaging system: injecting 150 mu L of 30mg/kg fluorescein substrate into the abdominal cavity of each nude mouse, observing for 15min, anesthetizing the nude mouse by ether in an induction box for 5min, quickly transferring the anesthetized nude mouse into the observation box, aligning and fixing the head of the nude mouse in a conical nose plug, setting parameters for fluorescence imaging (if a tumor grows, fluorescence signals with different intensities can be detected at corresponding positions), transferring the nude mouse into the induction box again after imaging is finished, and opening an oxygen valve to resuscitate the nude mouse.

2.2 animal grouping and administration

Grouping: subcutaneous lumps were observed in all nude mice after 1 week, and tumor body pair growth was verified by in vivo imaging technique of small animals, so nude mice were sorted and numbered according to body weight, 18 random numbers were generated by Excel software, the random numbers and nude mice numbers were respectively assigned one-to-one, and the nude mice were equally divided into a model group, a low dose group (100 μ g/kg), a medium dose group (200 μ g/kg) and a high dose group (400 μ g/kg) according to the random number. Weighing the nude mice and measuring the tumor volume after random grouping, and checking the difference between the nude mice weight and the tumor volume among all groups by using a statistical test, wherein no difference exists among all groups, and the grouping is correct due to better balance.

Administration: the administration is carried out by intraperitoneal injection, and the acrylic triptolide solution is injected into the abdominal cavity by using a disposable sterile syringe. The nude mice of the model group are given physiological saline, and the administration group is given the triptolide acrylate solution according to the dosage. The administration is 1 time daily and is continued for 13 days.

2.3 Observation and recording

After the administration, the nude mice are observed regularly every day, including mental state, activity condition, diet condition, skin color and fecal character, the nude mice body weight is measured and recorded 2 times a week, the size of transplanted tumor is measured, living body imaging is carried out 1 time a week, the growth of tumor body and the remote metastasis condition are monitored, and the administration is continued for 3 weeks until the end of the administration.

2.4 taking materials

After the administration, the animals were anesthetized, the cervical vertebrae were sacrificed, the tumor body was completely detached, and the tumor tissue size was measured and recorded.

2.5 Western Blot

After thawing the tumor tissue on ice, 50mg of the tissue homogenate was placed in a tube and 500. mu.L of a tissue lysate containing protease inhibitor and phosphatase inhibitor was added (1 tablet/10 mL of Roche brand tablet was used). After high-speed homogenization, centrifuging at 12000rpm/min for 15min, taking the supernatant, determining the protein concentration of the sample by using a BCA method, adjusting the concentration of each sample to be consistent, adding a protein loading buffer solution 5Xloading buffer, and performing denaturation at 100 ℃ for 10min and preserving at-80 ℃.

Selecting a 10% premixed polyacrylamide gel preparation solution suitable for brands, and then preparing the gel used in the experiment according to the use instructions (including separating gel and laminating gel, about 2 hours is needed); preparing SDS-PAGE gel electrophoresis liquid in advance in the process of gel solidification, putting the prepared gel in an electrophoresis tank, adding the electrophoresis liquid, adding 10-30 mu L of sample in each hole of the sample adding tank, after the sample runs through the laminated gel at low pressure of 80V, adjusting the voltage to be 100V to enable the sample to run through the whole gel, and finishing the SDS-PAGE gel electrophoresis operation; next, a membrane transfer operation (PVDF membrane, pre-wetted in methanol for 5min in advance) was performed under the following conditions: 300mA, 120-150min (corresponding to the boundary time selection of molecular weight 100kDa, wherein the selective transmembrane time of the separated protein with molecular weight less than 100kDa is 120min, and the selective transmembrane time with molecular weight more than 100kDa is 150 min); after the membrane conversion is finished, sealing the membrane with 5% skim milk for 2h, and incubating the membrane in a refrigerator at 4 ℃ for one night according to corresponding strips; the next day, unbound primary antibody was washed off with TBST wash (5 washes for 5min each); the secondary antibody was incubated at 37 ℃ for 2h, and unbound secondary antibody was washed away with TBST wash (5 washes 5 times for 5min each); and (4) taking ECL luminous liquid as a target strip of substrate exposure, analyzing, recording and counting the result.

3 results and analysis

3.1 the experiment establishes a liver cancer cell line HepG2-Luc capable of stably expressing luciferase, and the cell is utilized to construct a nude mouse subcutaneous liver cancer tumor implantation model.

In the experiment, nude mice were randomly divided into four groups: the Model group (Model), the low dose group (TPO-L), the medium dose group (TPO-M) and the high dose group (TPO-H) were sacrificed and the tumor-bearing tissues and tissue specimens were obtained 13 days after the continuous administration. In the experiment, the tumor growth condition is monitored by using a small animal living body imaging technology, as shown in fig. 4, compared with a model group, the fluorescence signals in the tumor of the nude mice of each dose administration group are obviously weakened and are dose-dependent; and the attenuation of the high-dose group is more obvious, which indicates that the triptolide acrylate (the compound of the formula I) can obviously inhibit the proliferation of hepatoma carcinoma cells in tumor bodies. As shown in FIG. 5, it can be seen from the skin surface and subcutaneous transplanted tumor of nude mice that the tumor volume of the administration group (especially the high dose group) is significantly smaller than that of the model group, and is dose-dependent. Similarly, as shown in fig. 6, tumor body volumes of tumor body tissues stripped after the nude mice are sacrificed and administered groups are smaller than those of the model group, so that the in vivo anti-liver cancer effect of triptolide acrylate is further verified.

3.2 the effect of triptolide acrylate on the expression of important apoptosis-related proteins was also observed in this experiment.

p53 is a very important cancer suppressor gene, and has many biological functions of promoting gene repair, regulating cell cycle progression and inducing apoptosis, among which phosphorylation at serine site has the effect of promoting apoptosis. Caspase-8 and Caspase-3 are at the core positions on the initiator and the executor respectively in the Caspase cascade reaction, and are the key steps for cell apoptosis and the common pathway of all apoptosis signals. As shown in fig. 7, the protein expression level of p53 was significantly increased in the high dose group, while no significant change was observed in the low and medium dose groups. The middle dose group can significantly increase the protein expression level of caspase-8, while the low and high doses can reduce the expression level. The protein expression level of caspase-3 can be obviously increased in the medium and high dose groups, and the protein expression level can be reduced in the low dose group.

Second, the effect of triptolide acrylate on the proliferation and apoptosis of liver cancer cells

1 materials of the experiment

1.1 cell lines and drugs

LO2, HepG2, Hep3B, SMMC-7721, and BEL-7402 cells used in this experiment were purchased from ATCC. The cells used contained 10% fetal bovine serum, 1X 10⁵DMEM complete medium of U.L-1 penicillin and 100 mg.L-1 streptomycin in 5% CO₂The culture was carried out at 37 ℃ and saturated humidity.

1.2 reagents

Fetal bovine serum, DMEM medium, penicillin, streptomycin double antibody (Gibco, usa); annexin V-FITC/PI apoptosis detection kit, ECL detection kit (Kaikyi organism); an MMT kit, a BCA protein concentration determination kit, an SDS-PAGE protein loading buffer solution, PMSF and NP40 lysate (Biyun day); beta-actin, Caspase-3, clear-Caspase-3, PARP, clear-PARP antibodies and secondary antibodies (CST, USA).

1.3 instrument health Force biosafety cabinet, NEWBrunwinck carbon dioxide incubator, Roche biochemical analyzer, inverted microscope, BD flow cytometer, Bio-rad protein electrophoresis system, BioTek Epoch microplate reader, etc.

2 method of experiment

2.1 cytotoxicity assay

Log-grown LO2 cells were taken at 1X 10 per well⁴One was inoculated in a 96-well plate. Cells treated with drugs at final concentrations of 0, 10, 50, 100nM, respectively, were used, and culture supernatants were collected after 24 hours and assayed for LDH activity using a Roche biochemical analyzer.

2.2 MMT method for detecting cell proliferation

When the liver cancer cells grow to logarithmic phase, the single cell suspension is added at 1 × 10 per hole⁴One was inoculated in a 96-well plate. The blank group was DMEM medium containing 10% fetal bovine serum, the control group was a solvent-added control (DMSO) cell group, and the experimental group was a drug-treated cell group at various final concentrations (10, 25, 50, 100 nM). After 24 hours of treatment, the medium supernatant was carefully aspirated off, and a final concentration of 0.5 mg. multidot.L diluted with medium was added to each well^-1After culturing the cells for 4 hours, the medium was discarded, 150. mu.L of DMSO solution was added to each well, the mixture was placed on a shaker in the dark and shaken at a low speed for 10 minutes, and after the crystals were sufficiently dissolved, the absorbance (A) of each well was measured at 490nm using a microplate reader. Cell proliferation inhibition rate [ (control group a)₄₅₀Blank group A₄₅₀) - (Experimental group A)₄₅₀Blank group A₄₅₀) /(control group A)₄₅₀Blank group A₄₅₀)]×100％。

2.3 detection of apoptosis by flow cytometry

Collecting hepatocarcinoma cells at logarithmic growth phase at a rate of 5 × 10 per well⁵One was inoculated in 12-well plates. Cells treated with triptolide acrylate (TPO) and triptolide triol (TP-3-OH) at final concentrations of 0, 50, 100 nM. After 24 hours of treatment, the harvested cells were digested with 0.25% EDTA-free pancreatin, washed 2 times with pre-chilled PBS, and the cells were resuspended with 300. mu.L of 1 XBinding Buffer. mu.L of FITC Annexin V and 5. mu.L of PI were added to each tube of cell suspension, incubated for 5 minutes at room temperature in the dark and detected within 1 hour using a flow cytometer.

2.4 Western blot detection of apoptosis-related proteins

Taking BEL-7402 cells in logarithmic growth phase at 5X 10/well⁵One was inoculated in 12-well plates. TPO treated cells were used at final concentrations of 0, 50, 100 nM. After 24 hours of treatment, harvestAfter cell collection, lysate is added, and total protein is extracted after full lysis on ice. After the protein concentration was determined using the BCA method, Loading buffer was added and the protein was thoroughly denatured by boiling at 100 ℃ for 10 minutes. The denatured sample was subjected to SDS-PAGE gel electrophoresis and electrically transferred to a PVDF membrane. Sealing the transferred PVDF membrane for 1 hour at room temperature by using a 5% BSA solution prepared by TBST, incubating at the corresponding primary antibody at 4 ℃ overnight, washing the TBST, incubating at the secondary antibody for 2 hours at room temperature, washing the TBST again, finally adding ECL chemiluminescence liquid to enable the membrane strip to emit light, tabletting, developing and fixing a photosensitive film to obtain an electrophoresis result, and observing the change trend of apoptosis-related proteins among different treatment groups.

2.5 scratch test

Huh7 cells were harvested at logarithmic growth phase at 5X 10 per well⁵One was inoculated in a 24-well plate. After the growth is completed, scratching is carried out by using a sterile gun head, the cells are washed for 3 times by using PBS after scratching, the scratched cells are removed, a serum-free culture medium containing 50nM TPO is added, the culture is continued for 24 and 48 hours, and photographing is carried out. And analyzing the scratch picture by using Image J software, calculating the scratch distance and evaluating the healing condition of the scratch.

3 result processing and analysis

3.1 statistical analysis

Results were analyzed using SPSS 16.0 software. Statistical data are expressed as mean ± standard deviation (x ± s). The data are subjected to a normality test and a homogeneity test of variance, a single-factor analysis of variance or a t test is adopted for comparison among groups, and the statistical significance is achieved when P is less than 0.05.

3.2 comparison of toxicity of TPL (triptolide), TP-3-OH, TPO on Normal hepatocytes

The cytotoxicity experiment results in FIG. 8 show that LDH activity of LO2 cell culture supernatant treated by TPL is significantly increased, while LDH activity of supernatant of TP-3-OH and TPO treated group is significantly lower than that of TPL treated group, which indicates that toxicity of TP-3-OH and TPO to liver cells is significantly lower than that of triptolide.

3.3 TP-3-OH, TPO inhibition of hepatoma cell proliferation

FIG. 9MTT experimental results show that TPO has obvious inhibition effect on the proliferation of four liver cancer cells, namely HepG2, Hep3B, SMMC-7721 and BEL-7402, and shows a dose-dependent effect, which indicates that TPO has inhibition effect on the proliferation of liver cancer cells. TP-3-OH has no obvious inhibition effect on the proliferation of liver cancer cells.

3.4 Induction of apoptosis of liver cancer cells by TPO

FIG. 10 shows the flow results that the ratio of Annexin V and PI positive cells of Hep3B and BEL-7402 hepatoma cells is obviously increased after TPO treatment (A); FIG. 10Western blot result shows that cleavage of Caspase-3 and PARP in BEL-7402 cells is significantly increased after TPO treatment (B), indicating that TPO has the effect of inducing apoptosis of liver cancer cells. TP-3-OH has no obvious effect on inducing the apoptosis of the liver cancer cells (results are not shown).

3.5 inhibitory Effect of TPO on migration of liver cancer cells

FIG. 11 shows that TPO can significantly inhibit the migration of Huh7 cells, and the difference is statistically significant (P <0.05), suggesting that TPO may have the effect of inhibiting the metastasis of hepatoma cells.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims (10)

1. The triptolide acrylate is characterized in that the triptolide acrylate is a compound shown in a formula I or a pharmaceutically acceptable salt thereof,

2. the method for preparing triptolide acrylate according to claim 1, comprising the steps of:

(1) adding triptolide and an acylation reagent into an organic solvent, stirring for 1-4h at room temperature by taking 4-dimethylaminopyridine as a catalyst and triethylamine as an acid-binding agent, and quenching the reaction by using a saturated sodium bicarbonate aqueous solution;

(2) extracting with dichloromethane, washing the extractive solution with saturated sodium chloride water solution, drying with desiccant, evaporating to dryness under reduced pressure, and purifying with silica gel column chromatography to obtain triptolide acrylate.

3. The method for preparing triptolide acrylate according to claim 2, wherein the step (1) is:

dissolving triptolide and 4-dimethylaminopyridine in an organic solvent, adding triethylamine, carrying out ice bath to about 0 ℃, dropwise adding an acylating reagent, returning to room temperature after dropwise adding, stirring for 1-4h, detecting complete reaction by TLC, stopping stirring, and quenching the reaction by using a saturated sodium bicarbonate aqueous solution.

4. The method for preparing triptolide acrylate according to claim 2, wherein the step (2) is:

extracting with dichloromethane, extracting the water layer with dichloromethane twice, mixing the three dichloromethane extracts, washing with saturated sodium chloride solution, drying with desiccant, evaporating to dryness under reduced pressure, and purifying with silica gel column chromatography to obtain triptolide acrylate.

5. The method for preparing triptolide acrylate according to any one of claims 2-4, wherein the acylating agent is one or more of acryloyl chloride, acryloyl bromide, acrylic acid glycoside, acrylic acid, or 3-chloropropionyl chloride.

6. The method of claim 5, wherein the organic solvent is selected from the group consisting of anhydrous dichloromethane, chloroform, tetrahydrofuran and diethyl ether.

7. The method for preparing triptolide acrylate according to claim 6, wherein the acid-binding agent is one or more of triethylamine, trimethylamine, pyridine, DITEA, DBU, 2, 6-lutidine, and 4-dimethylaminopyridine.

8. The method of claim 7, wherein the catalyst is one or more selected from the group consisting of 4-dimethylaminopyridine, DCC, HOAT, HOBT, HBTU and TBTU.

9. The method for preparing triptolide acrylate according to claim 8, wherein the drying agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride.

10. The use of triptolide acrylate according to claim 1 in the preparation of an anticancer agent.

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