Design, Synthesis And Anticancer Activity Of Pyridinylisoxazole And Pyridinylisozazoline Derivatives

This dissertation is divided into three chapters, describing the design ofpyridinylisoxazoles and pyridinyl-4,5-2H-isoxazoles as potential allosteric kinaseinhibitors based on the X-ray co-crystal structures of reported inhibitors bound totheir corresponding protein kinases, the establishment of a library of small-moleculecompounds and their anticancer activities as well as their preliminarystructure-activity relationships.The first chapter summarizes the current status of protein kinases and proteinkinase inhibitors and provides the background information and rationales for thisresearch project.Protein kinases are responsible for the phosphorylation of the substrate proteinsand play a vital role in the transduction cellular signals. The dis-regulation of proteinkinase activities have been linked to many diseases such as cancer, immunesystem disorders, certain metabolic diseases, inflammation and so on. For thesereasons, protein kinases have become one of the most comprehensive classes oftherapeutic targets. Many of them have been validated in clinical studies to beeffective treatments for various diseases. Compared to the traditional therapies, thesetargeted approaches can greatly improve the efficacy and safety for the patients.With the progress of this research area, many new protein kinase inhibitors arebeing identified. They can be divided into five categories according to theirinteraction with the kinase targets: type Ⅰ, type Ⅱ and type Ⅲ inhibitors bind to the ATP binding site or thereabouts; type Ⅳ inhibitors bind to the region which is awayfrom the substrate binding site; type Ⅴ inhibitors are a kind of bivalent inhibitors.There are more than40small molecule protein kinase inhibitors have been marketed,and most of them are used for the treatments of cancers. Protein kinase inhibitorshave been widely recognized by both cancer patients and medical researchers fortheir characteristics of high efficiency, low toxicity, highly selective with fewer sideeffects.Based on the preliminary studies of our research group, a series of novelpyridinylisoxazole and pyridinyl-4,5-2H-isoxazole derivatives have been designedand synthesized. We examined their anticancer activities and exploredthe preliminary structure-activity relationships. This provides a base for furtheroptimization of the lead compounds based on the central structures ofpyridinylisoxazole and pyridinyl-4,5-2H-isoxazole and forms the foundation for thesubsequent development of novel kinase inhibitors.The second chapter describes the design and synthesis of pyridinylisoxazolederivatives and evaluation of their anticancer activity.The pyridinylisoxazole derivatives are designed based a A-B bis-cyclicpharmacophore model. We expected one end of the molecule will extend to theoriginal adenine binding region. The other one binds in a hydrophobic pocketvacated by a crankshaft-like180°rotation of the DFG-fragment in the activationloop.During the development of the synthetic route, we selected2-methyl-5-nitronicotinate as starting material. After exploring a series of reactionconditions, we obtained the crucial intermediate with the central structure ofpyridinylisoxazole through eight steps with the overall yield of33.1percent finally.We firstly screened amide products for anticancer biological activity againsthuman breast cancer cell line MCF-7with MTS method. However, the result was not satisfactory. For this result, we supposed the molecule could not be able to extend tothe original adenine binding region. Then we synthesized sulfonamide and ureaproducts and the result have improved. Finally we introduced pyrimidine fragmentsand the result showed that most of them are worth noting and the IC50value of thebest activity compound could reach6.2μM.In chapter three, we screened anticancer activity of the library ofpyridinyl-4,5-2H-isoxazole derivatives.First of all, we separated the hit compound into three parts: Region1extendsinto a hydrophobic cavity created by the rotation of DFG-fragment; Region2bindsthe original adenine binding region. For about Region3, we replacedpyridinylisoxazole with pyridinyl-4,5-2H-isoxazole. This change could increase theflexibility of the whole molecule, so that it could bind the kinase more tightly.Secondly, based on the synthetic route explored in the second chapter, we gotthe important intermediate by replacing tert-butyl allylcarbamate with tert-butylprop-2-yn-1-ylcarbamate. We tried to introduce substituted groups on pyrimidine andexamined their anticancer activity against MCF-7. The result showed that most ofthe compounds exhibited good anticancer activities and the best IC50value couldreach1.5μM. The anticancer activity increased after changing the central structurefrom pyridinylisoxazole into pyridinyl-4,5-2H-isoxazole. The chain aliphatic aminesand aromatic amines on pyrimidine were suitable and the methyl on pyrimidineplayed an important role in the anticancer activity.After that, we shifted our attentions on Region1. In order to reduce theworkload of derivatization on Region1, we improved the synthetic route explored inthe second chapter. We introduced the phthalimide protecting group and optimizedthe reaction conditions. Then five kinds of pharmaceutical molecular fragments hadbeen introduced and the biological activity evaluation results suggested that onlyhydrophobic groups were suitable, which was consistent with our original idea. Finally, to further study the anticancer activity, seven compounds were selectedagainst another two other types of human cancer cell lines: liver cancer cell lineHepG2and cervical cancer cell line Hela. The result showed that most of themexhibited good anticancer activities against these cell lines. It was a amazingdiscovery that two of them exhibited more stable biology activity among this threehuman cancer cell lines. This broad-spectrum anti-proliferative activity wasencouraging and further investigation on biological targets and mechanism-of-actionwere still underway.