Design,Synthesis And Activity Evaluation Of Novel Rosin Diterpenoid Derivatives

Glioma is a primary intracranial tumor originating from glial cells,accounting for about 80%of primary malignant brain tumors in adults,and it is the most common heterogeneous brain tumor in the adult central nervous system（CNS）.Among gliomas,the highest-grade malignant glioma is glioblastoma multiforme（GBM）,which is characterized by extremely high invasiveness and metastasis.At present,the clinical standard treatment for glioma is surgery combined with radiotherapy and chemotherapy,but the therapeutic effect is poor and the patients are prone to recurrence.The overall survival rate of glioma patients has not improved significantly for many years.Therefore,the development of high-efficiency and low-toxicity anti-glioma drugs is extremely urgent.Epidermal growth factor receptor（EGFR）and its most common mutant epidermal growth factor receptor variant III（EGFRv III）are abnormally expressed in gliomas.They can participate in the regulation of key signaling pathways such as tumor cells proliferation,migration and survival,and affect chemotherapeutic drugs resistance and poor prognosis.EGFR is one of the targets for the treatment of glioma.Natural products abietic acid（AA）and dehydroabietic acid（DHAA）,which are tricyclic diterpenoid oxygenated compounds obtained from rosin or disproportionated rosin,have a broad spectrum of biological and pharmacological properties,such as antitumor,antibacterial,anti-inflammatory,antiviral and other activities.Studies have shown that rosin diterpenoids and their derivatives can play an excellent antitumor effect on a variety of cancers such as lung cancer,glioma,breast cancer and liver cancer.These compounds can affect a variety of signaling pathways that promote tumorigenesis and development.AA and DHAA are promising antitumor lead compounds.In this study,rosin diterpenoids AA and DHAA,which have good anti-tumor potential and multiple signaling pathways,were used as lead compounds for structural modification.According to the scaffold hopping,bioisosterism and combination principles in the drug design strategy,the pharmacophores commonly used in the classic EGFR inhibitors were introduced,and their structures were properly expanded.Two series of rosin diterpenoid derivatives were designed and synthesized.Subsequently,in vitro bioactivity evaluation and molecular docking studies were carried out on these derivatives,in order to provide lead compounds for the discovery of new and efficient anti-glioma drugs.The contents and results are as follows:（1）Synthesis of target compounds:4-aminophenol and 4-amino-2-chlorophenol as raw materials respectively reacted with 2-（chloromethyl）pyridine hydrochloride through O-alkylation to obtain the intermediate 4-（pyridine-2-methoxy）aniline（1a）and 3-chloro-4-（pyridine-2-methoxy）aniline（2a）.Using 2-chloro-4-nitrophenol and 1-（bromomethyl）-3-fluorobenzene as raw materials,the intermediate 3-chloro-4-（（3-fluorobenzyl）oxy）aniline（3a）was obtained through O-alkylation and reduction reactions.Using aniline containing different substituents（-H,-Cl,-CH3,-Br）as starting materials,the corresponding intermediate N⁴-phenylpyrimidine-4,6-diamine（4a-7a）was obtained by nucleophilic substitution reaction with 6-chloropyrimidin-4-amine.Intermediate 8a is1-phenylpiperazine.Rosin acid or dehydroabietic acid reacted with oxalyl chloride to undergo acylation reaction under the catalysis of N,N-dimethylformamide for obtaining the corresponding acyl chlorides.Then,the acyl chlorides reacted with intermediates 1a-8a by amide condensation reaction to obtain A-series abietic acid derivatives A1-A12 and B-series dehydroabietic acids B1-B12.A13 was obtained by alkylation of rosin acid as raw material.A14 was obtained by further Upjohn dihydroxylation on the basis of A13.In this study,26 target compounds and 7 key intermediates were finally designed and synthesized,and all target compounds were separated and purified by silica gel column chromatography.The compounds were characterized by ¹H-NMR and/or MS and/or ¹⁹F-NMR.（2）Evaluation of biological activity of target compounds:using Temozolomide（TMZ）as a positive control,the antiproliferative activities of rosin diterpene derivatives on human glioma cells T98G,U87MG and U251 were investigated by CCK-8.Then,the representative compounds with better activities were selected for cytotoxicity test on human normal hepatocyte L-02.Based on the above experiments,the compound with the best activity was screened for wound healing experiment to explore the effect of the migration ability of T98G cells.The experimental results showed that for T98G cells,B6,B8,B10 and B12,(IC₅₀=22.17～71.09μM)showed good antiproliferative effects which were far superior to the positive control drug TMZ(IC₅₀=1477.00μM).Among these compounds,B12(IC₅₀=22.17μM)had the strongest inhibitory activity on the proliferation of T98G cells.For U87MG cells,B10(IC₅₀=99.54μM)showed the strongest antitumor activity,which was far better than TMZ(IC₅₀>1600μM).For U251 cells,the antiproliferative activities of the compounds numbered 6,8,10,12 in the series A and B and A13-A14(IC₅₀=11.47～99.88μM)were stronger than TMZ(IC₅₀=322.60μM).Among these compounds,B8(IC₅₀=11.47μM)exhibited the best antiproliferative activities.Based on the above experimental results,B6,B8,B10 and B12 with more prominent antiproliferative activities of glioma cells were screened for the toxicity tests on human normal hepatocyte L-02.The results showed that the cytotoxicity of B12(IC₅₀=136.80μM)was lower than those of B6,B8,B10(IC₅₀=89.04～94.37μM<IC₅₀（U87MG）),and B12 had a certain degree of selectivity to the tested glioma cells.Through comprehensive comparison,B12 was selected as a representative for wound healing experiments.The results showed that B12 attenuated the migration ability of T98G cells with the increase of dose and time in a certain concentration range.（3）p Ka of representative compounds:according to the results of in vitro antiproliferative activities experiments,B6,B8,B10 and B12 with better activity were screened out,and their p Ka（7.17～7.35）were determined by potentiometric titration.The p Ka values were within the ideal parameters for CNS drug design（6<p Ka<10.5）and were all less than8.It showed that these compounds might more easily penetrate the blood-brain barrier to exert the efficacy and were less likely to be P-glycoprotein substrates.（4）Molecular docking study of target compounds:using reverse molecular docking technology,the interaction between representative compounds B6,B8,B10,B12 and EGFR-related target proteins（PDB:4UIP,1M17,1XKK,4HJO,1BNA,4EKZ）were scored and docked.The results indicated that the scores of binding energies of B12 with 1XKK,B10 with 4HJO were the best and both were-10.02 Kcal/mol.B10 and B12were stably bound to the corresponding EGFR target proteins mainly through hydrogen bonding,π-σconjugation and hydrophobic interaction.In conclusion,26 rosin diterpenoid derivatives and 7 key intermediates were designed and synthesized by rational use of drug design strategies,and the preliminary in vitro bioactivity evaluation and molecular docking study of the target compounds were carried out.The experimental results showed that the introduction of 4,6-disubstituted aminopyrimidine fragments into the rosin diterpene scaffold can enhance the anti-proliferative activities of the compounds against glioma cells,and B12 was the most promising anti-glioma candidate compound.This study not only provided an important foundation for subsequent related molecular modification and lead compound research,but also provided important candidate compounds for the exploration and development of new anti-glioma drugs.