Design,Synthesis,Anti-LOH And Anti-inflammatory Activities Evaluation Of Tricyclic Diterpenoid Analogs

In this dissertation,phenotypic screening of tricyclic e diterpenoid analogs for anti-LOH(Late-onset hypogonadism)or anti-inflammatory agents was performed to find the hit compounds.Based on the hits,further structure modifications and activity evaluation were displayed,which mainly consisted of two parts:Part 1.Design,synthesis of 2-methylpyrimidine-fused tricyclic diterpene analogs and investigation of their anti-LOH activity.Late-onset hypogonadism(LOH)is reported as one of the most common age-related diseases occurred in middle-aging men,which is mainly caused by testosterone deficiency.In recent years,it has gradually become a hotspot in the research of drugs.The 2-methylpyrimidine-fused tricyclic diterpene analog 2-7 was afforded as anti-LOH hit,which was screened out from our small synthetic library.At the concentration of 20?M,the activity of the hit was higher than that of icariin,which was used as the positive control.Based on the hit,67 derivates were designed,synthesized,NMR,HRMS and HPLC were used for characterization and analysis.Then their effects in promoting testosterone production and cytotoxicities were evaluated in mouse TM3 Leydig cells and preliminary SAR was summarized as follows:The activity of the compounds decreased obviously when carbonyl,hydroxyl,oxime,and other substituents were introduced into the B ring.Methoxyl was the dominant substituent at C-12 position.When acyl or amides were introduced into the C-13 position,the N-methylamide derivative 2-37 was most potent and safe.2-37increased the testosterone production to 174.6%of that of normal TM3 cells.In addition,it exhibited little toxicity to TM3 and other normal cells(CC₅₀>200?M).Then further modifications of the N-methylamide group in 2-37 were obtained.When the N-methylamide group was replaced by N,N-dimethylamide formohydrazide,amino,N-methylmethanamine,N-hydroxyamide and N-acetamide,the comprehensive evaluation of the activity and safety of the compounds was not as good as 2-37.Then we investigated the anti-LOH mechanism of 2-37 in vitro.Resluts showed that 2-37could significantly promote the expression of the key testosterone synthesis-related enzymes(St AR and 3?-HSD)in a concentration-dependent manner.Further studies discovered that 2-37 could stimulate autophagy in TM3 cells by promoting the expression of LC3 through regulating AMPK/m TOR signaling pathway.In vivo experiments disclosed that 2-37 significantly improved the serum total and free testosterone levels in PADAM rats.Moreover,it effectively overcomed the side effects of TRT on testis and sperm.The result of Hematoxylin and Eosin(H&E)staining assay showed that the arrangement of the Sertoli cells was ameliorated obviously and the seminiferous tubules was only slight swelling in 2-37-treated rats.In vivo anti-LOH mechanism research of 2-37 indicated that the compound could also enhance testosterone synthesis by up-regulation of the steroidogenic-related gene and inducing autophagy in Leydig cells.The results of initial pharmaco-kinetics evaluation showed that 2-37 has good pharmacokinetic properties.Part 2.Design,synthesis of 3-(carbamyl oxide)tricyclic diterpene analogs and investigation of their anti-macrophage inflammatory activity.Macrophage inflammation is a non-specific immune process.When macrophages overreact to inflammation,it can lead to systemic inflammatory response syndrome(SIRS).Existing treatment methods have limited therapeutic effects on SIRS.In this thesis,we screened our synthetic library of tricyclic diterpene analogs for anti-macrophage inflammatory activity and selected the hit compound QW60.Based on the hit,we designed and synthesized 21 3-(carbamyl oxide)tricyclic diterpene analogs and investigated their anti-inflammatory activity in LPS-induced macrophage inflammatory model of RAW264.7 cells.Derivative 4-18 with a N-propargyl carbamyl oxide substituent at C-3 position was seleted for its good anti-inflammatory activity and further structure modifications were carried out based on it.Then we modified the structure of 4-18 at C-7,C-12 and C-13 positions,designed and synthesized a total of21 derivatives for activity screening.When the acetyl group at C-13 was removed(4-22),the activity increased significantly.In modifications of the methoxy group at C-12position,we found that the activity increased significantly when the methyl group was replaced by the n-propyl group and finally obtained compound 4-30 with high anti-inflammatory activity.MTT assay and LDH release assay were displayed to determine the toxicity of 4-30 in RAW264.7 cells.Resluts showed that 4-30 has little toxic effects on cell viability under the concentration of 20?M.Meanwhile,westernbolt and RT-PCR results showed that 4-30 has significant inhibitory effect on the expression of TNF-?and IL-1?.