CN111253414A - Synthesis of perfluorobenzyl norcantharidinate carboxylate and anti-tumor application thereof - Google Patents
Synthesis of perfluorobenzyl norcantharidinate carboxylate and anti-tumor application thereof Download PDFInfo
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Abstract
The invention provides a synthesis method of norcantharidin carboxylic acid perfluorobenzyl ester with a structural formula shown as I and an anti-tumor application thereof.
Description
Technical Field
The invention belongs to the field of new drug design and synthesis, and particularly relates to synthesis of norcantharidin carboxylic acid perfluorobenzyl ester and anti-tumor application thereof.
Background
Cantharidin (CA) is an anticancer substance extracted from Mylabris (Mylabris phalerata Linnaeus) of family Meloidae, and has good therapeutic effects on hepatocarcinoma, ovarian cancer, esophageal cancer, etc., especially on primary hepatocarcinoma. However, cantharidin has severe toxicity and serious irritation to urinary system and digestive system of human body, and has certain limitation in clinical application. The artificially synthesized cantharidin derivative Norcantharidin (NCTD) has the same configuration as that of cantharidin, but 2 and 3 methyl groups are replaced by hydrogen, so that the toxic and side effects are obviously reduced, the Norcantharidin derivative Norcantharidin can stimulate bone marrow to have the effect of increasing white blood cells, and has the effects of protecting liver cells and enhancing immunity, but because the Norcantharidin has short detention time in a human body, adverse reactions occur when the Norcantharidin is slightly excessive, and the limitation of the dosage greatly prevents the medicine from exerting curative effects. The sodium norcantharidinate is prepared by hydrolyzing norcantharidin under alkaline conditions, the toxicity of the compound is obviously reduced, the pharmacological action is clear, and the compound can be applied to clinic.
Therefore, the synthesis of the high-efficiency low-toxicity norcantharidin derivative and the development of the variety of cantharidin anticancer drugs have great significance, and the synthesis of the norcantharidin derivative becomes an antitumor research hotspot of modern Chinese medicaments.
"fluorine" is a highly surprising atom with a small atomic radius (between the hydrogen and oxygen atoms) and a maximum electronegativity, so that the selective introduction of a fluorine atom or fluorine-containing group into a drug lead molecule can produce significant changes in the physical, chemical, and biological properties of the parent molecule. According to the statistics of pharmacologists, the introduction of fluorine into the drug lead molecules can improve the drug success rate by ten times, 30-40% of pesticides contain fluorine at present, and 20% of marketed drugs contain fluorine, so that the efficient synthesis of fluorine-containing analogues of some bioactive molecules is an effective means for obtaining high-activity drug lead molecules, and is one of important ways for developing new drugs
At present, research on introducing fluorine-containing groups into cantharidin is less, and in order to search candidates of antitumor drugs with better drug effect and lower toxicity and expand the types of cantharidin antitumor drugs, the fluorine-containing groups are introduced into norcantharidin, so that the fluorine-containing derivatives of norcantharidin are synthesized, and a foundation is laid for subsequent screening of anticancer activity.
Disclosure of Invention
In one of its objects, the present invention provides perfluorobenzyl norcantharidin carboxylate; the structural formula is shown as a formula I,
in another aspect, the present invention provides a method for synthesizing perfluorobenzyl norcantharidin carboxylate I as described above, comprising the steps of: 1) the side chain pentafluorobenzyl alcohol 5 (compound 5) and norcantharidin (compound 4) react in an organic solvent under the action of organic base at a certain temperature to obtain the perfluorobenzyl norcantharidin carboxylate shown in the formula I, and the synthetic route is as follows:
in a preferred embodiment, the organic base described in step 1) above is selected from triethylamine, 4-dimethylaminopyridine (4-DMAP for short) and the like, preferably 4-DMAP.
In a preferred embodiment, the solvent of step 1) above is selected from ethyl acetate, tetrahydrofuran, dichloromethane and the like, preferably dichloromethane (abbreviated as DCM), preferably at a temperature of 60 ℃.
In another aspect, the invention also provides a substrate 4 for synthesizing the perfluorobenzyl norcantharidinate carboxylate shown in formula I and a preparation method thereof, wherein the method comprises the following steps: a) furan is used as a raw material and reacts with maleic anhydride (compound 1) in an organic solvent to obtain 5-alkene norcantharidin 2 (compound 2); b) the 5-alkene-norcantharidin 2 and hydrogen are subjected to addition reaction in an organic solvent to obtain a substrate norcantharidin 4 (compound 4), and the synthetic route is shown as follows:
in a preferred embodiment, the organic solvent used in step a) is an ethereal solvent or a halogenated hydrocarbon, such as: diethyl ether, tetrahydrofuran, dichloromethane or chloroform.
In a preferred embodiment, step a) can be carried out at room temperature, or with suitable heating; preferably room temperature.
In a preferred embodiment, the organic solvent used for the reaction of step b) is ethyl acetate, tetrahydrofuran or dichloromethane;
in a preferred embodiment, the catalyst used in the catalytic hydrogenation of step b) is selected from the group consisting of Pd/C, and Pd (OH)2Palladium carbon and platinum carbon including/C; Pd/C is preferred.
In the above synthesis and preparation methods, the reaction temperature may be appropriately selected according to the type of reaction. The reaction time can be obtained by tracking the reaction condition through monitoring means such as thin layer chromatography TLC, high performance liquid chromatography HPLC or LC-MS liquid mass spectrum combination and the like.
Activity tests prove that the demethylcantharidin carboxylic acid perfluorobenzyl ester shown in the formula I, which is designed and synthesized by the invention, has a good anti-tumor effect, especially aiming at liver cancer; the half inhibitory concentration (IC50) on human liver cancer SMMC-7721 cells can reach 18.55 mu M in 24 hours and 24.53 mu M in 48 hours; the IC50 of the Bel-7402 cell of human liver cancer can reach 14.26 mu M in 24 hours and 19.76 mu M in 48 hours; the activity of the norcantharidin is stronger than that of norcantharidin and sodium norcantharidin which are positive control medicaments or the inhibitory activity is equivalent; can be used as cantharidin antineoplastic agent in clinic.
Therefore, the fourth aspect of the invention provides the application of the perfluorobenzyl norcantharidin carboxylate shown in the formula I in preparing the antitumor drugs; preferably, the application of the compound in preparing anti-liver cancer drugs.
The invention has the advantages that: the invention provides a norcantharidin carboxylic acid perfluorobenzyl ester shown in a formula I, which is a suitable candidate anti-tumor drug, and particularly serves as a candidate anti-liver cancer drug. Compared with positive control drugs namely norcantharidin and sodium norcantharidinate, the compound I introduces carboxyl in the molecular structure, so that the water solubility and the stability are improved; in addition, due to the introduction of the fluorine-containing group, the physical, chemical, and biological activities of the parent molecule can be significantly changed, thereby enhancing the pharmacokinetic efficacy. In addition, the synthesis method of the norcantharidin carboxylic acid perfluorobenzyl ester has the advantages of easily obtained raw materials and very easy operation and implementation.
Detailed Description
The invention will be further illustrated by the following specific examples, which are not intended to limit the scope of the invention. Without departing from the inventive concept, a person skilled in the art may make modifications or combinations of the parameters or conditions of the claims, which modifications or combinations shall also be considered as the protective scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
The furan, maleic anhydride, pentafluorobenzyl alcohol used in the present invention were from sahn chemical technology (shanghai) ltd; the solvent used is from Shanghai Tantake technology, Inc. The reagents used are all chemically pure, unless otherwise specified.
The synthesis method of the lead compound norcantharidin 4 of the invention,
the method specifically comprises the following steps:
1) placing maleic anhydride in a dry round-bottom flask, adding diethyl ether for dissolving, dropwise adding furan 2 after complete dissolution, reacting at room temperature for 24h, performing suction filtration, and drying to obtain a white solid intermediate 5-alkene norcantharidin 3 for later use;
2) sequentially adding the 5-alkene norcantharidin 3 obtained in the step 1) and 10% palladium carbon into a 100mL three-necked bottle, vacuumizing, introducing hydrogen, adding 30mL of ethyl acetate, stirring at room temperature for 24h, filtering after the reaction is finished, and concentrating under reduced pressure to obtain a white solid product norcantharidin 4.
In the above reaction, the progress of the reaction can be monitored by chromatography or HPLC-MS. In the chromatography, thin layer chromatography can be used, and gas chromatography or liquid chromatography such as HPLC can be used instead.
Example 1, preparation of 5-Endecamethylcantharidin 3:
putting a certain amount of maleic anhydride 1(26mmol,2.6g) into a round-bottom flask, adding 20mL of diethyl ether into the flask to completely dissolve the maleic anhydride, slowly dropwise adding 22.9 mL (39mmol) of furan after complete dissolution, reacting at room temperature for 24h, performing suction filtration, and drying to obtain a white solid compound 3, namely 5-alkene norcantharidin, wherein the dry weight is 2.1g, and the yield is 48%.1H NMR(400Hz,DMSO-d6):δ6.58(s,2H),5.35(s,2H),3.31(d,J＝4.0Hz,2H)。
The organic solvent used for dissolving maleic anhydride in step 1 of example 1 may be replaced with any one of dichloromethane, chloroform, and tetrahydrofuran, in addition to diethyl ether.
Example 2 preparation of norcantharidin 4:
at room temperature, the compound 3(10mmol,1.7g) in the step (1) and 10% palladium carbon (0.16g,15 mmol%) are sequentially added into a 100mL three-necked bottle, the bottle is plugged and vacuumized, hydrogen is introduced, then 30mL of ethyl acetate is added, and the mixture is stirred at room temperature for 24 hours. After the reaction is finished, suction filtration is carried out, a filter cake is washed by ethyl acetate for 2-3 times, and the obtained filtrate is decompressed and concentrated to obtain a white solid intermediate 4(1.6g), namely the norcantharidin, with the yield of 97%.1H NMR(400Hz,DMSO-d6):4.86(s,2H),3.39(s,2H),1.65(s,4H)。
Example 3 preparation of perfluorobenzyl 6 norcantharidin carboxylate:
to a 25mL sealed tube, norcantharidin 4(1.0mmol,168mg), DMAP (1.0mmol,244mg) were added, and after three times of replacement with argon, 2.5mL of DCM and pentafluorobenzyl alcohol 5(2.0mmol, 274. mu.L) were added in that order, and the mixture was reacted at 60 ℃ for 14 hours. After the reaction is finished, the reaction product is cooled to room temperature, the reaction product is washed for three times by HCl (1mol/L) and saturated saline solution respectively, organic phases are combined, the organic phases are dried by anhydrous sodium sulfate, and the white solid product 6 is obtained by flash column chromatography, wherein the yield is 28.5%.1H NMR(400MHz,DMSO-d6)δ12.15(s,1H),5.11(s,2H),4.67(t,J＝4.4Hz,2H),3.01(dd,J＝16.8Hz,9.6Hz,2H),1.57-1.49(m,4H).19F NMR(376MHz,DMSO-d6)δ-144.00(d,J＝15.0Hz,2F),-155.55(t,J＝22.6Hz,1F),-162.47—-162.58(m,2F).
Example 4 Activity test of Perfluorobenzyl norcantharidinate carboxylate 6
Cell line and solvent
Human liver cancer SMMC-7721 cells;
human liver cancer Bel-7402 cells;
culturing the cells in RPMI1640 containing 10% fetal bovine serum;
solvent: dimethylsulfoxide (abbreviated as DMSO).
Embodiment for detecting anti-tumor activity of cells by CCK-8 staining method
Selecting the cells with the ratio of the tumor living cells to be detected being more than 90 percent for experiment. Cell proliferation inhibition assay Using EnoGeneCellTMCounting Kit-8 (CCK-8 for short) cell viability detection Kit. Taking human liver cancer SMMC-7721 and Bel-7402 cells in logarithmic growth phase, discarding culture solution, washing with PBS for 2 times, digesting with pancreatin, centrifuging, re-suspending and mixing the cells uniformly in culture solution, 8000 cells/hole, and inoculating in 96-well plate in parallel. The 96-well plate was placed at 37 ℃ in 5% CO2Culturing in incubator for 24 hr, taking out, adding 100 μ L culture solution containing compound to be tested into each well, and simultaneously establishing negative control group, solvent control group, and positive control group (the positive control group respectively adopts norcantharidin and sodium norcantharidinate). 3 replicates per group at 37 ℃ with 5% CO2After the culture is continued for 24 or 48 hours in the incubator, 10 mu L of CCK-8 solution is added into each hole, the culture plate is incubated in the incubator, an enzyme-labeling instrument is used for measuring the light absorption value (OD value) at 450nm, the experiment group and the control group are repeated for 3 times, and the IC50 of each compound acting on the human liver cancer cell SMMC-7721 and the human liver cell Bel-7402 is calculated. The experimental results are detailed in tables 1-2.
TABLE 1 IC50 of human liver cancer SMMC-7721 cells
TABLE 2 IC50 of human hepatoma Bel-7402 cells
The experimental results in tables 1-2 show that the compound I of the present invention has good in vitro anti-tumor activity. When the compound is acted for 24 hours, the IC50 of the compound reaches 18.55 mu M to human liver cancer cell SMMC-7721, and the compound reaches 24.53 mu M in 48 hours, the activity of the compound is obviously stronger than that of norcantharidin and is equivalent to that of sodium norcantharidinate; the IC50 of human liver cancer cell Bel-7402 can reach 14.26 mu M in 24 hours and 19.76 mu M in 48 hours, and the activity of the compound is obviously stronger than that of norcantharidin and sodium norcantharidinate.
In addition, compared with a positive control norcantharidin, the compound I introduces carboxyl into the molecular structure, so that the water solubility and the stability are improved; compared with positive reference products of norcantharidin and sodium norcantharidinate, the biological activity of the norcantharidin and sodium norcantharidinate is obviously changed due to the introduction of fluorine-containing groups. Therefore, the compound I is a suitable candidate drug for resisting tumors, particularly as a candidate drug for resisting liver cancer.
Claims (4)
1. The synthesis and anti-tumor application of the norcantharidin carboxylic acid perfluorobenzyl ester are characterized in that the structural formula is shown as a formula I:
the synthesis method of the norcantharidin carboxylic acid perfluorobenzyl ester I comprises the following steps: the side chain compound (5) and norcantharidin (4) react in an organic solvent under the action of an organic base catalyst to obtain a compound (6), and the synthetic route is as follows:
2. the method for synthesizing perfluorobenzyl norcantharidin carboxylate according to claim 1, wherein the organic base is selected from triethylamine, 4-dimethylaminopyridine abbreviated as 4-DMAP, preferably 4-DMAP; the solvent is selected from ethyl acetate, tetrahydrofuran, dichloromethane abbreviated as DCM, and preferably DCM; the temperature is preferably 60 ℃; a substrate (4) for synthesizing the norcantharidin carboxylic acid perfluorobenzyl ester shown in the formula I and a preparation method thereof comprise the following steps: a) furan is used as a raw material and reacts with maleic anhydride, namely a compound (1), in an organic solvent to obtain 5-alkene norcantharidin (2), namely a compound (2); b) the 5-alkene-norcantharidin (2) and hydrogen are subjected to addition reaction in an organic solvent to obtain a substrate norcantharidin (4), namely a compound (4), and the synthetic route is as follows:
the organic solvent adopted in the step a) is an ether solvent or halogenated hydrocarbon, wherein: diethyl ether, tetrahydrofuran, dichloromethane or chloroform;
the temperature of step a) may be heated; preferably room temperature;
the catalyst used in the catalytic hydrogenation reaction of step b) is selected from the group consisting of Pd/C, and Pd (OH)2Palladium carbon and platinum carbon including/C; Pd/C is preferred.
3. The perfluorobenzyl norcantharidin carboxylate I according to claim 1, wherein the perfluorobenzyl cantharidin carboxylate I is used for preparing an antitumor drug.
4. The use of perfluorobenzyl norcantharidin carboxylate I according to claim 3, wherein said antineoplastic drug is especially predominantly liver cancer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447782A (en) * | 2014-12-30 | 2015-03-25 | 贵州柏强制药有限公司 | Bromo-norcantharidin acid-benzyl ester, and synthetic method and application thereof |
| CN106083879A (en) * | 2016-06-21 | 2016-11-09 | 遵义医学院 | Norcantharidin mono-acid monoester derivates and antitumor application thereof |
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- 2020-03-17 CN CN202010184579.7A patent/CN111253414A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447782A (en) * | 2014-12-30 | 2015-03-25 | 贵州柏强制药有限公司 | Bromo-norcantharidin acid-benzyl ester, and synthetic method and application thereof |
| CN106083879A (en) * | 2016-06-21 | 2016-11-09 | 遵义医学院 | Norcantharidin mono-acid monoester derivates and antitumor application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SAMUEL J. DOMINIANNI等: "Some Derivatives of 7-Oxabicyclo[2.2.1]heptane-exo-cis-2,3-dicarboxylic Acid", 《JOURNAL OF MEDICINAL CHEMISTRY》 * |
| 孔晶晶: "尿嘧啶及去甲斑蝥素的氟烷基化反应研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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