Design,Synthesis And Anti-Inflammatory And Anti-Tumor Activities Of Hespretin 7-O-Triazole Derivatives

Hesperetin is a natural flavonoid,which is widely distributed in nature and is found in a variety of vegetables,fruits,and Chinese herbal medicines,such as rutaceae Citrus,bergamot,banana,lemon,quinoa and citron etc_,herbaceous,cruciferous amaranth,dried tangerine or orange peel.And a large number of studies have shown that it has many pharmacological effects,such as anti-inflammatory,anti-tumor,anti-oxidant,anti-free radical,liver protection and cardiovascular protection.Hesperetin is widely used in food,cosmetics and health products.For exarnple,it is used as a food additive with a vitamin p-like effect in Japan;for cosmetics,such as night cream of Clarins,hesperetin is added;in health products,many Amway’s health products contain hesperetin,Vitality 50 the best-selling health product in the United States and its active ingredient is hesperetin.In the early stage,our group was committed to the research on the structural modification and pharmacological activity of hesperetin.7,3’-dimethoxy hesperetin was synthesized,which had good anti-inflammatory activity and inhibits the production of rat peritoneal macrophage cytokines in the adjuvant arthritis（adjuvant arthritis,AA）model by regulating gene and protein levels.The synthesized 5,7,3’-triacetylhesperetin had a good anti-inflammatory effect in vitro In vivo,the results of pharmacokinetics and pharmacodynamics studies showed that delaying the peak time,extending the half-life,increasing its bioavailability,and significantly improving the swelling of the mouse feet;inhibiting the proliferation of MCF-7 cells by inducing DNA degradation.The synthetic hesperetin manich base derivatives were screened.And hesperetin derivative HDND-7 significantly improved liver fibrosis in mice in carbon tetrachloride model.The study of pharmacological mechanism showed that it reduced the damage degree of liver fibrosis by inhibiting the activation and proliferation of liver stellate cells induced by PDGF.The 7-O-cinnamamide-based hesperetin derivative inhibits NO（nitric Oxide）release and TNF-α（tumor necrosis factor）,IL-6（interleukin-6）secretion at 40 μM to improve CCl4 induced acute liver injury（ALI）.The releases of NO,inflammatory factor of IL-1β（interleukin-1β;）and IL-6 can be inhibited by 7-O-ether group and 7-ethylamide-based hesperetin derivatives at 10 μM concentrations,and the 7-ethylamide-based hesperetin derivatives were more active.In order to further improve the activity of hesperetin derivatives and explore their deeper value,this paper continues the structural modification of hesperetin to find more active hesperetin derivatives.The specific research work of this paper is as follows:1.Synthesis of 7-O-triazole hesperetin derivatives:Based on the structure-activity relationship of the anti-inflammatory activity of 7-O-acetamido hesperetin derivatives synthesized earlier,the group of triazole was used instead of amide group,whether increasing its anti-inflammatory activity by increasing hydrophobic interactions?And the series of a1-a14,b1-b5,c1-c11 30 7-O-triazole hesperetin derivatives and 2 intermediates were synthesized.The 7-OH of hesperetin is facing the hydrophobic pocket inside the receptor,and whether its opposite 4 carbonyl is facing the hydrophilic region outside the receptor.The hydrophilic oxime group was introduced at the 4 carbonyl group to imrove their anti-inflammatory activity by increasing hydrophilic interactions.Based on the above considerations,we have synthesized the series of dl-d9 4-oxime-7-O-triazole hesperetin derivatives.The anti-inflammatory activity was screened for structure-activity relationship analysis.2.The screening for anti-inflammatory activity:（1）In vitro:mouse peritoneal macrophages（RAW264.7 cell line）as the test subject,and the anti-inflammatory activities of the series of a1-a14,b1-b5,c1-c11,d1-d9 7-O-triazole hesperetin derivatives were screening.Seting up blank control group,positive drug control group,LPS model group and different concentrations（20,10,5,2.5,1-25 μM）in the experimental group,the releasing contents of NO,TNF-α,IL-1β and IL-6 were measured.The four series of hesperetin derivatives showed significantly inhibitory activity of the releasing of NO compared with the positive control group.The further ELISA experiments showed that the four series of hesperetin derivatives reduced the secretions of TNF-α,IL-1β and IL-6.Among them,the effect of d5 with 7-O-triazole and 4-oxime groups was superior.In general,the anti-inflammatory activities of the series of triazole were more significant than 7-O-acetamido hesperetin derivatives.（2）The levels of protein:d5 with the most significant anti-inflammatory activity,was used for research of protein expression.The express of iNOS（inducible nitric oxide synthase enzymes）,COX-2（cyclooxygenase-2）,and NF-κB signaling pathway-related proteins were inhibited by d5.（3）In vivo:The model of mice acute liver injury induced by CCl4（Acute liver injury,ALI）was used to evaluate the anti-inflammatory activity of d5 at different dose groups（low,medium and high dose groups）,compared with control group,silymarin group and Model group Histopathological results,serological indicators,and serum-related inflammatory factors indicated that d5 could attenuate liver injury in mice.3.Anti-tumor screening:through the screening of anti-tumor and CDK6 enzyme inhibitory activity,a1-a14 series compounds have certain CDK6 inhibitory activity,among which compound a8 has the best activity（CDK6:IC50=0.19±0₀6 μM;MCF-7:IC50=5.83 ± 1.06μM;MDA-MB-231:IC50=7₂3±0₉3μM）.Through DS（Discovery Studio）and structure-activity relationship analysis,compound a8 forms a double hydrogen bond with the hinge residues Val101 and Asp102 at one end through the 3’hydroxyl group,At the other end,the π-π stacking effect formed by the terminal substituted benzene ring and Phe90,and the anchoring effect at both ends,make the hesperetin of compound a8 firmly occupy the front fissure of the ATP active pocket The terminal substituted The benzene ring forms a hydrophobic interaction with Val27,Ala41,Val77,Phe98,etc.,the meta-substitution of chlorine atoms extends into the rear crack of the binding pocket and interacts with the rear crack.We preliminarily explored its structure-activity relationship,which laid a theoretical foundation for subsequent studies.