Studies On Synthesis And Structure-activity Relationship Of Phosphorylated Derivatives Of Flavones

[Backgrounds] Flavones are widely distributed in plants, as an active ingredient in many herbs and fulfilling many pharmacological functions. The structural modification of flavones is usefully way to enhance bio-activity and obtain new activity compounds by. The phosphorylated modification is essentially changes for physical and chemical properties of the parent molecule, and resulting in changes to the intermolecular interaction. Phosphorylated modification is often used as pro-drugs to improve the water solubility and enhance the bio-availability of the agent. Phosphorylated modification of flavones has attracted much attention of medicinal chemists, and has been tested for anti-cancer activity. From now on, there is not reported for pCE as inhibitors, could influence the activity of seroenzymes and the interaction between drugs and serum albumin. The mechanism of action and structure-activity relationship are both not exactly clear.[Objects] The objects of this thesis contain three aspects. Firstly to obtain new derivatives of phosphorylated flavones, modificated way of high yield phosphorylated flavones and screen high activity inhibitors of pCE; Secondly, to understand the effect of phosphorylated flavones on activity of 8 kinds seroenzymes; thirdly, to analysis the combining ability, combining sites, combining ways and combining distances with serum albumin, the mechanism of action and the structure-activity relationship.[Methods] The phosphorylated modification of flavones was studied under various conditions. When the flavones were treated with C1P(O)(OEt)2 as phosphorus acylating agents, Et3N, and DMAP as catalyst in THF, the starting materials such as luteolin, apigenin, chrysin,7-hydroxyflavonoid,6-hydroxyflavonoid with mutihyd-roxy were consumed completely and the products which were totally phosphorylated were isolated by silica column separation in good to excellent yields and structure identified by NMR, MS, IR and element analysis. The inhibition of phosphorylated flavones on the pCE was investigated, and the inhibition mechanism and kinetic characterization were studied. The effect of activity of 8 kinds seroenzymes on phosphorylated flavones were studied by automatic biochemistry analyzer. IC50 of phosphorylated flavones to CHE and their structure-activity relationship were analyzed by same way. Interaction ways and structure-activity relationship between phosphorylated flavones with HSA or BSA like binding ways, binding distances, binding sites, and effect of bind-power on metal ions were studied by using fluorescence spectroscopy and UV-visible absorption differential spectroscopy.[Results](1) In the first part,6 kinds of phosphorylated flavones, such as 2a,2b,2c,2d,2e, 3c and a sodium salt of phosphoric esters of flavones as 3c, were obtained by yield of 90.3%,83.0%,86.3%,80.2%,73.5%,65.8%,57.4% through conditions optimization, and 2a,2b,2e,3a are new compounds among them. It is first time that obtaining high yield fully phosphorylated favones such as luteolin, apigenin.(2) The pCE inhibition activity of flavonoid after phosphorylated were very significant increase sorting as 3c>2d>3c>2a>2b>2e>3a with IC50 as 0.75 nM,2.44 nM,3.76 nM,3.89 nM,26.1 nM,0.39μM and 50.6μM. The results showed that 3c, 2d,3c,2a,2b etc. had a good development prospects as pre-drug of pCE inhibition as mixed inhibitors and 2e as anti-competition inhibitor. All inhibitions to pCE of phosphorylated flavones were irreversible inhibit.2d,2c,3c all can produce stronger polarity intermediate compounds and 2a,2b,2e couldn't produce intermediate compound in combining process with HSA and BSA. Analysis of structure-activity relationship indicated that phosphorylation in 7-site can ehance the pCE inhibition activity, and reduce inhibited activity in 5-site phosphorylation and significantly reduce in 4'-site phosphorylation.(3) The results show that the phosphorylated flavones had little effects on a-HBDH, ALT, y-GT, CK, ALP, LDH, AST with no beyond the normal range value, but they could significantly reduce the activity of CHE with IC50 from 0.00006 nM to 89.45650 nM by inhibition activity sorting as 2c>3c>2a>2b>2d>2e. Study of structure-activity relationship revealed that phosphorylated in 7-site could ehance the CHE inhibition activity and acted the synergistic effect in 5-site phosphorylated, and phosphorylated in ring-B could also improve the inhibition activity by phosphorylated more group and significantly reduce in 6-site phosphorylated.(4) The results showed that 6 kinds of phosphorylated flavones had a strong ability of quench the fluorescence to HSA or BSA with non-radiation energy transfer in the combining process. The adhesion strength between phosphorylated flavones and HSA or BSA was produced by the means of electrostatic attraction. The binding constants (Ka) reduced with temperature rise, and Ka of 3c down the fastest as the most sensitive to the change of temperature. The binding ability of 2c,2d,2e with BSA was stronger than with HSA, which indicated that more modificated sites could cause stronger binding strength. The bind ability with serum albumin enhanced significantly with meanwhile phosphorylation of hydroxide radical in 5-site and 7-site. The bind strength sorted as 5-site phosphorylation> 6-site phosphorylation> 7-site phosphorylation, and 5-site is the key binding site with serum albumin. Results showed the binding site is styleⅠ(the same as the standard medicine binding-site of butazone)The binding distances of phosphorylated flavones as HSA:r2c=5.0877 nm, r2d=3.7137 nm, r2e=5.1431 nm, r3c=4.8682 nm; BSA:r2c=5.5592 rim, r2d=4.0172 nm, r2e=5.2109 nm, r3c=4.1636 nm indicated that 2c or 2e with serum albumin were larger and others were smaller.Phosphorylated modification in 5-site and 6-site of ring-A were in favor of binding with HSA, and modificated in 7-site had small contribution to binding activity, while phosphorylated modification in 7-site was in favour of binding with BSA. Phosphorylation of 3'-site of ring-B was both conducive to bind with HSA and BSA.Results show there exist certain influence on phosphorylated falvones binding with serum albumin by mental ions and most effect by Cu2+, Al3+ and least effect by K+, Zn2+, Mg2+, Ca2+, which compete the binding-site in serum albumin with drugs, extend keeping time of drugs in serum and enhance efficacy.[Conclusions](1) Obtained a general efficient way of phosphorylated falvones by conditons optimization and suitable catalyst selection.(2) The new pCE inhibition activity of flavonoid has got after phosphorylated in strict structure-activity relationship, which become a mew idea of study and development pCE inhibitor.(3) Phosphorylated flavones have little effects on seroenzyme asα-HBDH, ALT,γ-GT, CK, ALP, LDH, AST, but significantly inhibit the activity of CHE with structure-activity relationship.(4) Phosphorylated flavones exist strong adhesion strength with HSA or BSA binding by siteⅠand in great structure-activity relationship. They have the basic characteristics of transportation in blood as drugs.