| Focused on the two large series of diseases that we are suffering heavily—one is the muscular dystrophy, a kind of genetically-inherited diseases, which is incurable for the time being, and the other is superbug-associated infectious diseases, we, on the basis of rational drug design, performed innovative medicinal chemistry researches, taking full advantage of modern technologies and strategies from previous drug discovery experiences.The first section of the thesis is about the discovery and structural optimization of small molecules, which can regulate the a-dystroglycan O-mannosylation. Aiming to identify such small molecules, we rooted on the physiological and pathological molecular basis that give rise to dystroglycanopathy, hypothesized that cells can up-regulate glycosylation of a-DG in response to certain chemical stimuli, and employed an cell-based, wash-free AlphaScreen protocol, which was developed by Doctor Xiaohua Wu from Mannose company and amenable to High-throughput Screening (HTS), using Pro5 cell, a kind of Chinese hamster ovary cell line. In our HTS assay, we used an antibody (IIH6) recognizing the functional O-mannosyl glycan on a-DG as the probe to quantitatively monitor O-mannosylation of a secreted form of recombinant a-DG protein in cell culture media. We then screened more than 300,000 structurally diversified small compounds in MLSMR. After filter of PAINS and a secondary screen by immunofluorescent technology using IIH6 as antibody or Laminin-DyLight 594 to detect endogenous functional O-mannosyl glycans (FOG), two compounds were identified with reasonable potency with EC so 30μM and 15μM respectively. We then applied several kinds of drug design strategies to conduct chemical synthesis, including (1) thiophene replacement by aliphatic alkanes or heterocycles, (2) tetrazole isosterism by carboxylic acid, amide or even triazole, (3) side chain size change, such as methyl, ethyl, propyl or even polar group like carbonyl, (4) linker ring rigidity change with ethylene diamine chain and phenyl ring, and (5) functional group modification to benzothiazole ring by installation of fluorine, amine, methoxy and so on. Eventually, the functional group modification led to the discovery of the best compound referring as compound 52, with satisfactory potency (ECso=3μM), a great improvement to original compound 8 of EC50=5μM. Based on the optimized compound 52, two kinds of small molecular probes were developed, which are promising for fishing of target protein and study of mechanism of action.The second section of the thesis is about the design, synthesis and pharmacological assessment of new peptide deformylase inhibitors containing spiro cyclopropane moiety. Firstly, we made a docking study to virtually screen our designed small molecules, leading to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor’s extra hydrophobic binding, and providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds, to find majority of the designed compounds showed very good activity (MIC up to 0.008μM, much lower than that of reference compounds). To mitigate metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vitro rat pharmacokinetic assessment, plasma protein binding assay, methemoglobin formation assay and in vivo efficacy determination. Those findings showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors. |
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