Design,Synthesis And Biological Activities Of Novel Derivatives Based On Two Diterpenoid Skeletons

Natural products are considered to be one of the main sources of medicines.There are many abundant natural products in nature and these kinds of natural products have the characteristics of large content,variety,complex structure,cheap and easy to get.Meanwhile,the added value of these natural products is low.Terpenoids are widely distributed in nature and are the most natural products.Among them,diterpenoids are the most abundant type,and have the pharmacological activity such as anti-tumor,anti-microbial and cardiovascular.Many diterpenoids are listed as clinical drugs,such as paclitaxel,andrographolide and salvia miltiorrhiza.Based on the complex skeleton structure and extensive biological activity of natural products,researchers have always been interested.On the one hand,with the natural products as raw materials,the structural modifications are directly carried out to improve the biological activity,reduce the side effects and enhance the drug-like properties.On the other hand,it can focus on the complex structure and expensive small molecule drugs,looking for natural products with similar structure to targeted structural modification.By this means,it may be possible to improve the probability of finding new lead structures.In addition,this concept could be used in the construction and preparation of complex molecules in a more convenient and efficient way.Protease-activated receptor I(PAR1),a G-protein-coupled thrombin receptor,is the most potent cell surface inducer of platelet activation.The PARI antagonist could be used as potent antiplatelet agent for the prevention of thrombosis and restenosis.Additionally,because PAR1 antagonists target the cellular effector of thrombin,thrombin-mediated fibrin generation is not affected,and therefore normal hemostasis should be maintained when platelet activation is induced.Therefore,a PAR1 antagonist is likely to result in a lower risk of bleeding than conventional antithrombotic agents.So far,only vorapaxar,derived from the natural product himbacine,has been approved by the US Food and Drug Administration as the first-in-class anti-platelet drug targeting PAR1 to reduce the risk of recurrent cardiovascular events.Although vorapaxar exhibits excellent anticoagulant activity,there are still many deficiencies including a complicated structure with seven chiral centers,a long synthetic route with sixteen linear synthetic steps,a low overall yield and high preparation cost.In addition,patients receiving vorapaxar were more likely to experience bleeding events compared with those receiving placebo,which has limited its clinical use.However,this adverse effect is attributed to the long elimination time(126-269 h)of vorapaxar rather than to the PAR1 target itself.Andrographolide,an easily accessible molecule isolated from the leaves of Andrographis paniculata,is an important active natural product.The two fused six-membered rings of andrographolide adopt the chair conformation and,importantly,the key chiral centers on its scaffold are fully consistent with those in vorapaxar.Based on these results,the use of the chiral fragment of andrographolide instead of the tricyclic ring of Vorapaxar while maintaining the key chiral conformation is a convenient approach to quickly develop new PAR1 antagonists.In this study,we prepared two series of novel PAR1 inhibitors by "natural-product hybridization",and"borrowed" the two fused six-membered ring moiety from the natural product andrographolid,which led the discovery of the lead compound I-13a.The further structural optimization was guided by the metabolic stability evaluation and finally resulted in the most potent thrombin receptor antagonist ?-39,which also displayed potent anti-platelet activity both in vitro and ex vivo.Compound ?-39 inhibited human platelet aggregation induced by thrombin(0.5 U/mL)or TRAP(15?M)in a concentration-dependent manner,with IC50 values of 0.65?M and 1.6 ?M,respectively.Meanwhile,the platelet aggregation response following stimulation of human PRP with ADP(10 ?M)and collagen(5 ?g/mL),was not affected by ?-39,up to a concentration of 10 ?M.These data demonstrated that compound ?-39 could selectively inhibit PAR1-mediated platelet aggregation.The compound ?-39 exhibited an excellent oral bioavailability of 52.5%and the oral half-life was 3.1 h.Importantly,with the help of the natural product skeleton,the synthetic route of compound ?-39 was relatively short and resulted in a high yield.Therefore,compound I-39 could be obtained at low cost,which is an advantage in developing compound ?-39 as a PAR1 antagonist.These studies suggest that compound 1-39 has a potential to be developed as a new generation of PARI antagonists.In our previous research on the varied natural products with antifungal activities from bryophytes and endolichenic fungi,we found that many tetracyclic diterpenes are active against C.albicans strains,and most of them have ?,?-unsaturated ketone motif.However,the activity of these diterpenoids is not good enough and the limited amounts restrict us to directly modify these agents.Isosteviol,easily obtained by acid hydrolysis of stevioside,is a tetracyclic diterpenoid,which has the similar skeleton structure.The MIC assay revealed that the activity of isosteviol was weak.In previous studies,researchers have shown that exo-methylene group is pivotal for the anti-bacterial activity.For this reason,we tried to build up a crucial structure fragment of exo-methylene cyclopentanone in the ring D of isosteviol.And the activity was four-fold more potent than isosteviol with MIC 64 ?g/mL.Based on the skeleton,the TPP cation was further connected to target the mitochondria.And the activity was further improved eight-fold with MIC 8 ?g/mL.To obtain more active derivatives,several derivatives with different linkers but the same ?,?-unsaturated ketone core structure were synthesized,and finally resulted in the most potent antifungal compound ?-31 with MIC 0.5?g/mL.In addition,we observed that compound ?-31 was also active against other types of azole-resistant C.albicans strains suggesting that compound ?-31 could address the drug-resistance problems resulting from efflux pumps and other azole-resistance mechanisms.Flow cytometry analysis revealed that 11-31 remarkably increase the MMP of C.albicans and stimulate the generation of ROS,suggesting that the mitochondrial membrane was impaired.Confocal laser scanning microscopy(CLSM)observation based on Tom70-GFP imaging revealed that non-drug-treated cells had an extensive network of mitochondria displaying a tubular structure,whereas those in ?-31-treated cells presented an aggregated distribution and a disrupted tubular structure.Most Candida infections are associated with the formation of biofilms,which are highly recalcitrant to antifungal agents,on inert or biological surfaces.Our findings here showed that the compound 11-31 could not only prevent C.albicans biofilm formation but also kill preformed mature biofilms.Using Caenorhabditis elegans as an infectious model,compound ?-31 was observed to prolong the survival of C.albicans-infected C.elegans.In short,based on the skeleton of isosteviol,series of derivatives were synthesized with potent antifungal activity.The further structural optimization was guided by activity and finally resulted in the most potent antifungal compound 11-31.Further investigations revealed that compound 11-31 not only elicited mitochondria dysfunction and dramatically increased intracellular reactive oxygen species,but also it exhibited comparable ability to amphotericin B to eradicate preformed C.albicans biofilms.These studies suggest that compound 11-31 has a potential to be developed as a new generation of antifungal drug.The previous research of our group found that tetracyclic diterpenes with?,?-unsaturated ketone motif have potent antitumor activity.The antitumor activity of compound 11-14 was evaluated and we found that it has a moderate activity with IC5010 ?M.Take it as a substrate,some derivatives were synthesized by forming a dimmer or by connecting a variety of unsaturated ketone groups.Meanwhile,we further access diverse nitrogen-containing electrophiles for targeting lysosome to improve anticancer activity.Although the activity of some derivatives were improved,it still haven't met our expectation,and the structure needs to be further optimized.To date,little chemical effort has been directed toward modifications of the A-ring of isosteviol.Herein,we,for the first time,introduce ?,?-unsaturated ketone or a five-membered unsaturated lactone in isosteviol A-ring and resulted in the lead compound III-32 with potent anticancer activity.In addition,modifications of the D-ring with Baeyer-Villiger rearrangement,Beckmann rearrangement and Mannich reaction monoconfigurational generated a series of diverse heterocyclic compounds.Not surprisingly,these new molecules have demonstrated potent antiproliferative effects against various human cancer cell lines,especially,compound ?-53 with the IC50 value of 0.29 ?M against A549 cell lines.Further mechanisms research on compound III-53 proved that it damaged lysosome and induced LMP to cause cell death and had a potential to be developed as a noval anticancer agent.In conclusion,we synthesized several series of novel derivatives in a more convenient and efficient way by borrowing the skeleton of andrographolide and isosteviol,two easily accessible natural molecules.After structural optimization,several compounds with potent activity and powerful druggability were found,and higher value application of bulk natural products was realized.