Ⅰ,Synthesis And Structutre-Activity Relationship Research Of The Derivatives Of Esculentoside A; Ⅱ,Synthesis Of A New Type Activated Acyclic Nucleosides Analogues

The Chinese herb Phytolacca esculenta has been proved to have striking therapeutic effects on a number of diseases such as rheumatoid arthritis, edema, and tumor. Esculentoside A (EsA), a kind of triterpene saponin isolated from roots of Phytolacca esculenta, has been identified as 3-O-[?-D-glucopyranosyl-(1,4)-?-D- xylopyranosyl] Phytolaccagenin. Our previous researches showed EsA had strong inhibition on acute and chronic inflammation in animal models, and the mechanism of its anti-inflammation effect might be associated with the reduction of several key inflammatory mediators. Recently, researches also disclosed that EsA inhibited cyclooxygenase-2 (COX-2) in a dose-dependent manner, but had no effect on COX-1. Although EsA exhibits significant anti-inflammatory activity, it also has great haemolytic activity in our previous researches. Therefore, it is of great interest to us to optimize the structure of EsA and seek for its derivatives with higher potent biological activity and lower toxicity.In the study of structure-activity relationship of triterpene saponin, both the aglycone and the sugar moiety play an important role in the evaluation of biological activity. However, no research has been reported about the SAR of EsA. We herein report the optimization of EsA.The first step of the optimization is to convert the carboxylic acid in the aglycone of EsA (1) into amides. The derivatives 3~23 were prepared by a coupling reaction of EsA with amines. EsA was reacted with HOBt in the presence of DCC in DMF to give the active ester 2, which was also isolated from the reaction mixture by HPLC and identified as the HOBt ester of EsA. Without isolation of 2, amines was added to the reaction mixture. The compounds 4~23 were purified by HPLC. As a by-product isolated from all the reactions, Compound 3 was characterized as the product of EsA coupling with DCU. It seems to be related with the large solubility of DCU in DMF. This suggests DMF is not a favorable solution for this reaction. When DMF-THF (1:2, v:v) was used as the solvent, good yields of the desired amides was finally achieved and compound 3 was not found in the reaction mixture. Three kinds of amines, including aliphatic amines, amino acid esters, and amines bearing phenyl rings, were introduced to EsA by amidization of the C-28 carboxylic acid group. In additional, the derivatives of Phytolaccagenin were also synthesized. All compounds were confirmed by 1H NMR, 13C NMR and HRMS or ESIMS.Being effective, less expensive, and without isotope contamination in contrast to RIA or ELISA, the measurement of the inhibitory effects on reactive oxygen species fluorescence in hCOX-2 expressing sf-9 cells is a rapid method to screen COX-2 inhibitors. The efficacy of all the compounds was determined by this method as described by Zhang. Briefly, recombinant human COX-2 (hCOX-2) was expressed in insect sf-9 cells and harvested cloned sf-9 cells were stored in liquid nitrogen until use. Reactive oxygen species production stimulated by arachidonic acid in sf-9 cells was measured by 2', 7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) fluorescence. DCDHF-DA can rapidly permeate into cells, and been converted into 2', 7'-dichlorodihydrofluorescein (DCDHF). DCDHF does not give out fluorescence itself, but rapidly reacts with reactive oxygen species to produce fluorescence. In order to study the relationship of inhibitory and haemolytic activity, haemolysis activity was also tested.As we can see, compounds 2, 11~16, 23 showed higher potency towards COX-2 than EsA and Celecoxib, but higher haemolytic activity than EsA. With aliphatic amines introduced, compounds 4~9 lost their activity towards COX-2, but the haemolytic activity were decreased too. From the comparison of these two kinds of derivatives, we can make a clear conclusion that the introduction of phenyl ring markedly enhanced the activity towards COX-2, but caused a high haemolytic activity. Most noticeable is compounds 12 and 14, they exhibited the highest inhibition rates, 158.2% and 126.1%, respectively, while EsA and Celecoxib exhibited 21.7% and 40.0%, respectively. However, the haemolytic activities of the two compounds are also the highest. This suggests the C-28 carboxylic acid group plays an important role in their activity.Amino acid esters were introduced in compounds 18~23. Though compounds 18~22 lost their activity towards COX-2, compound 23 showed higher inhibitory activity towards COX-2 than EsA and Celecoxib, and lower haemolytic activity than EsA. This suggests the modification of the C-28 carboxylic acid might be a good objective to seek for high potent COX-2 inhibitors with low haemolytic activity. As a whole, the conversion of the carboxylic acid into amide could enhance not only their inhibitory activity towards COX-2, but also the haemolytic activity. This structure-activity relationship can guide further structure optimization of EsA and search for anti-inflammatory compounds with higher activity and lower toxicity.