Design, Synthesis, Structure And Biological Evaluation Of Ferrocenyl Prazole-fused Heterocyclone Compounds

Chemical genetics has been an emerging cross-discipline with the rapid development of structural biology and molecular biology during the end of the 20th century. Now, as the indispensable research tool in the field of life process and drug discovery on the molecular level, chemical genetics studies a gene's role by altering the activity (inhibition or stimulation) of the cognate protein using small molecule compounds. Chemical genetics is divided into two approaches, forward and reverse. According to screening libraries with variety cell models, forward chemical genetics can discover novel active small molecule compounds and the corresponding target proteins. It studies changes in phenotype(s) such as morphology, growth, or behavior resulting from random genomic DNA mutations and then identifies the gene responsible through mutation mapping.Organic synthetic chemistry plays an important role to set up the libraries which can be defined as the collections of complex and various compounds and are the absolute starting point for any chemical genetics study or discussion. According to testing or screening for their biological activities of the libraries with different biological model systems, some active small molecule compounds as protein or cell function regulators can be obtained. Then using the active compounds as probes, we could reveal the basic law of life and discover drug candidates.Under the guidance of forward chemical genetics, we had established a library of ferrocenyl prazole-fused heterocyclone compounds including 6,7-dihydro pyrazole-fused pyrazinones, pyrazole-fused pyrazinones and pyrazole-fused oxazinones derivatives. The diversity-oriented synthesis and biological activities of the library were studied. Basing the important biological application of ferrocene, conjugates of ferrocene with prazole-fused heterocyclone were designed. In the future, new target protein and biological mechanism may be found because of remarkable structure and nature of ferrocene. Through the screening with A549 lung cancer cell model, several active small molecule compounds were discovered, the 8d, 8e and 8f compounds which were chemical tools in chemical genetics showed prominent antitumor activity. The biological results showed that 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives 8d, 8e and 8f could effectively induce apoptosis in A549 lung cancer cells.In this paper, three aspects were included:Partâ… : Synthesis, structure characterization and preliminary biological evaluation of novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives.A series of novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H )-one derivatives were synthesized by the reaction of ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-carboxylate with non-aromatic primary amines in one-pot procedure and characterized by IR,¹H NMR, ¹³C NMR, HRMS and X-ray diffraction analysis. The one-pot procedure included two stages: nucleophilic substitution of halogenated hydrocarbons and amine acylation reaction. The effects of all the compounds on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells. Especially, the inhibitory effects of the compounds 8d, 8e, 8f were much better than the ones of the 5-Fu which was the staple and clinical drug. When we synthesized the critical intermediate product 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-carboxylate, we isolated two positional isomers of pyrazole N-alkylation and for the first time confirmed the exact target isomer structure by single crystal X-ray diffraction. Then we could accurately compare the two isomers differences in the ¹H NMR spectra. Partâ…¡: Synthesis and structural characterization and preliminary biological evaluation of novel 2-ferrocenyl-pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives.A series of novel ferrocenyl pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of ethyl 1-(2-aryl-2-oxoethyl)-3-ferrocenyl-1H-pyrazole -5-carboxylate with nonaromatic primary amines in one-pot procedure and characterized by ¹H NMR, ¹³C NMR, IR, HRMS and X-ray diffraction analysis. The reactions of ethyl 1-(2-aryl-2-oxoethyl)-3-ferrocenyl-1H-pyrazole-5-carboxylate with nonaromatic primary amines in toluene were carried out in the sealed tube which was heated to high temperature(160-190℃). The effects of all the compounds on A549 cell growth were investigated. The results showed that some compounds had excellent inhibitory effects on the growth of A549 cells.Partâ…¢: Synthesis and structural characterization of novel 2-ferrocenyl-6-arkyl or aryl-4H-pyrazolo[5,1-c][1,4]oxazin-4-one derivatives.A series of novel ferrocenyl pyrazolo[5,1-c][1,4]oxazin-4-one derivatives were synthesized by the reaction of ethyl 1-(2-aryl or alkyl -2-oxoethyl)-3-ferrocenyl-1H -pyrazole-5-carboxylate itself with the soft Lewis acid MgBr ? Et2O and characterized by ¹H NMR, ¹³C NMR, IR, HRMS and X-ray diffraction analysis. The methodology used in the cyclization step to construct oxazinone ring system was applied for the first time for the synthesis of ferrocenyl-substituted pyrazolo-oxazinone systems.In conclusion, we have developed novel and effective synthesis methods to build a diversity-oriented library of ferrocenyl prazole-fused heterocyclone compounds and screen the library with the A549 lung cancer cells. Several active small molecule compounds were discovered and could be used to chemical genetics research as essential chemical tools. In this paper, the results could provide the material basis of related chemical genetics. It had created the starting point and original methods of chemical genetics about pyrazole-fused heterocyclones. In the future, the further related research may be necessary because such compounds have very remarkable biological activities.