Design, Modification And Bioactivity Evaluation Of Functional Small Molecules Containing 2,3-Dihydrobenzo[B][1,4]Dioxin And/or Pyrazoline

With the constantly progress in medicinal and therapeutic fields, the understanding of human beings on cancer pathogenesis has stepped into cellular and molecular levels. Key signaling pathways, membrane receptors, kinases and other biological macromolecules who play important roles in tumorigenesis and cancer development have been revealed. The possibility of developing high active, high selective and low toxic anti-cancer agents targeting at these specific macromolecules has come consequently. Mitogen activated protein kinase (MAPK) signal transduction pathway is vital in cell growth and survival, while BRAF kinase is a key member of this pathway. The most common valine-to-glutamate transition (V600E) mutation is observed in approximately 8% of human tumors. This kind of mutation constitutively activates downstream MEK-ERK pathway, leading to tumor development, proliferation, invasion and metastasis. Thus, BRAFV600E has been gradually validated as a hotspot of developing anti-cancer agents.β-Ketoacyl-acyl carrier protein synthase III (FabH) controls the initial step of bacterial fatty acid biosynthesis, being the key enzyme of the feedback regulation of the same process. Meanwhile, it is highly conserved among Gram-positive and Gram-negative bacteria. It has been considered as an attractive target in antibacterial researches recently.During the study on drug-like small molecules in our group, 2,3-dihydrobenzo[b][1,4]dioxin moiety has been screened out and introduced into the designing of functional small molecules and the modification of pilot compounds because of its unique properties. Meanwhile, pyrazoline derivatives and similar pyrazole derivatives have drawn much attention all through the world as pilot compounds with multi biological activities.Herein specific BRAFV600E and FabH inhibitors have been designed independently. The correlated structure-activity relation and future designing directions have been then explained. As for BRAFV600E, the possible contradiction between two previous series of diaryl pyrazoline derivatives has firstly been paid attention to. We attempted to reveal that the potency of the series bearing ortho-hydroxyl group was exactly owing to this certain moiety or because the previous SAR model exaggerated the necessity of the defaulting aryl. By building a series of hybrid compounds, we succeeded in comparing the structures of the two previous series. Further biological evaluation was conducted. The result convinced that the introduction of ortho-hydroxyl group on 4,5-dihydro-1H-pyrazole skeleton could cover the disadvantage of defaulting an aryl whereas the defaulting aryl was still important. Meanwhile, as the hybrid series contained the basic advantages of both two previous series, the most potent compound (IC50 BRAFV600E=0.15/μM, GI50 WM266.4=1.75μM) was comparable with positive controls and more potent than the previous best compounds on kinase inhibitory activity. According to the comparison of molecular docking binding patterns and the refined 3D QSAR model, the basic pattern of this series BRAF inhibitors as well as their binding pattern had been settled. Due to a key interaction with PHE583, ring A and ring C which were nearly in one plane squeezed each other while ring B which was almost perpendicular to this plane and squeezing both ring A and ring C. On this basis, one consideration was that the three factors of backbone terminals had been near to or reached the best situation of modification. Although the bioactivity had been quite near to the positive control, the limit of backbone should be faced. That is, the bioactivity might still not be superb to the positive control however we had modified the structure. Thus the limit of backbone should be broken to seek for novel and rational binding patterns. According to the fact that ring A and ring C were nearly on one plane, these two aryl rings were interchanged and the best moiety on ring C was transferred to ring A. Because ring B could cause larger influence than ring A/C could, ring B took the priority to be designed. Moreover, the design range of ring B was extened. Among the new acquired series, other than the ones with similar bioactivity and binding pattern as before, more potent ones via stretching backbone or reversing conformation were gained. The most potent compound among them (IC50 BRAFV600E= 0.04μM, GI50 WM266.4=0.87μM) became one step nearer towards the positive controls with great possibility for improvement. However, this round of modification broke the limit of backbone. Thus, besides the SAR model needed rebuilding and comparing, whether the high selectivity and low toxicity still remained should also be checked for a second time. The representatives of this series showed selectivity against BRAFV600E from wild type BRAF, CRAF who was in the same kinase family and EGFR who was upstream. The selectivity could also been indicated by cell level. Further molecular docking and 3D QSAR results suggested stretching the backbone to outer space or totally reversing the backbone were both potential orientations to break previous limit. Moreover, modification the unsubstituted ring might further improve the bioactivity.As for FabH, remaining acylhydrazone and closing the vanillic acid resulted in a mediocre bioactivity. Abandoning acylhydrazone could acquire potent FabH inhibitory activity after closing the cycle but was harmful for dilevery. Since remaining acylhydrazone and closing the cycle were both important, the design had been set as lengthening two carbon units due to the character of Fatty Acid metabolism. To enrich the SAR discussion, the derive positions of carboxylic acid were expanded to three groups. Thus, three series of 21 arylhydrazones with 2,3-dihydrobenzo[b][1,4]dioxin moiety were synthesized for the first time. Their bioactivities were also reported for the first time. The leading compound indicated superior MIC values against tested strains, being comparable with positive control kanamycin B and showing broad spectrum antimicrobial activity. The E. coli FabH inhibitory potency data (IC50=3.5μM) was comparable with positive control DDCP. Structure-activity relationship and molecular modeling study provided further insight into interactions between the enzyme and ligand, offering valuable information for the design of FabH inhibitors.