Synthesis Of Two Novel ?-pinene Derivatives And Their Anticancer Activity

According to the China Cancer Registry Annual Report,the incidence of cancer in China continues to increase,and most anti-cancer treatments have had little success.Expensive anti-cancer treatment has become a major economic burden for many cancer patients.In response to the Nineteenth Medical Benefit-to-People Policy,the development of a wide range of low-cost natural products as anti-hepatoma drugs has become a research hotspot.Alpha-pinene,which originates from various forest plants such as pine,cypress and cassia twig,widely distributed in China,which is convenient for collection and extraction,and is widely used in chemical industry,food and medicine,etc.Alpha-pinene has multiple biological activities such as anti-tumor,anti-fungal and anti-allergic,etc.However,?-pinene also has the disadvantages of structural instability,low affinity,and insufficient activity.Alpha-pinene is originated from a wide variety of forest plants such as pine,cypress and cassia twigs that are widely cultivated in China.The extraction and extraction process is simple.It is widely used in chemical,food and pharmaceutical industries,and has anti-tumor,anti-fungal,anti-allergic properties and other biological activities.However,?-pinene also has the disadvantages of structural instability,low affinity,and insufficient activity.In order to improve the partial inferiority of?-pinene,we designed and introduced active groups to modify the local chemical structure in order to obtain new derivatives with stronger antitumor activity.Firstiy the experiment designed and synthesized?-pinene derivatives according to the design principle of bio-alkylation agents.The synthesis method includes two main steps:?1?On the basis of retaining the double-ring double bond structure of?-pinene,the redox reaction is performed on the atom of carbon number 10 to obtain the intermediate myrtenol;?2?In order to get the target derivative,the alkylating group,such as benzenesulfonyl,p-toluenesulfonyl,trifluoromethyl and etc.having a certain antitumor activity is selected for esterification and hydration.Finally,two target derivatives?6,6-dimethylbicyclo[3,1,1]hept-2-en-2-yl?benzenes ulfonate and?6,6-dimethylbicyclo[3,1,1]Hept-2-en-2-yl?p-toluene sulfonate were synthesized successfully and verified by MS analysis,¹H-NMR analysis and ¹³C-NMR analysis,etc.Meanwhile,the two derivatives of the synthetic route and synthesis conditions were optimized and screened to obtain the better synthesis method.Then,the anti-tumor activity of two derivatives of?-pinene were observed in vitro by MTT assay for hepatoma cells?BEL-7402?,lung cancer cells?A549?,breast cancer cells?MCF-7?,nasopharyngeal carcinoma cells?CNE-2Z?and neuroblastoma cells?PC-12?.The results showedthat?6,6-dimethylbicyclo[3,1,1]hept-2-en-2-yl?p-toluene.Sulfonate inhibited the growth of hepatocellular carcinoma cell line BEL-7402 with the best IC50 of 83.7?mol/L at 24h.Subsequently,the mechanism of action was investigated using flow cytometry and Tunel staining.The results showed that the derivatives could arrest the growth of hepatoma cells in the S phase?DNA synthesis phase?and induce apoptosis in hepatoma cells.According to the results of this study,our research group selected a variety of genes and proteins associated with S-phase arrest and induction of apoptosis as target targets.We used fluorescence quantitative PCR and Western Blot techniques to verify the gene and protein levels of cells.It was shown that the derivativescan down-regulate the expression of C-myc,CDK2 and CyclinE gene and protein,and up-regulate p53.In addition,this subject has also established a tumor bearing model in nude mice to verify the anticancer effect of the derivative in vivo.Derivatives?6,6-Dimethylbicyclo[3,1,1]hept-2-en-2-yl?were found by pathological studies such as observation of tumor size and weight in nude mice and immunofluorescence of tumor tissues.It was shown that the derivatives inhibited the growth of tumors in nude mice and was closely related to the expression of C-myc,CDK2,and CyclinE.The anti-tumor effect of this derivative was preliminarily confirmed.Finally,from the perspective of small molecule level mechanisms and structure-activity relationships,our group used computer simulations of molecular docking and molecular dynamics to establish a docking model using two derivatives as ligands and CDK2 as targets.The results showed that the sulfonate groups derived from both compounds could stably bind to the amino acid Leu83 of the target protein,and(6,6-dimethylbicyclo[3,1,1]hept-2-ene-2-p-toluene sulphonate is more stable and more conducive to the enhancement of the antitumor activity of the derivatives.This result is consistent with the previous screening results.