Discovery And Optimization Of Antifungal And Antitumor Lead Compounds

Discovery and optimization of lead compounds play an important role in drugdevelopment. In this thesis, a variety of strategies were used to discover and optimizeantifungal and antitumor lead compounds:(1) Using structure-based drug designtechnique to optimize the azole antifungal lead compound, which was previouslyreported by our laboratory;(2) Using cell-based phenotypic screening technique tofind novel β-carboline antifungal lead compound. After rational structuralmodification, some derivatives showed potent antifungal activity and new antifungalmechanism;(3) Systematic structural modification and biological evaluation wereconducted on natural product evodiamine. Some derivatives showed excellent in vitroand in vivo antitumor activity, showing the potential as antitumor drug candidates;(4)Using organocatalytic asymmetric cascade reactions to develop new methodologies toconstruct three sulfur-containing scaffolds, and constructed a drug-like molecularlibrary. Through biological screening, a novel lead structure with broad-spectrumantitumor activity was discovered.1. Design, synthesis and biological evaluation of novelantifungal lead compounds.1.1Homology modeling of Candida albicans CYP51andoptimization of azole antifungal lead compound.Fungal sterol14α-demethylase (CYP51) is an important target for antifungalagents. Azole CYP51inhibitors are currently the most widely used agents inantifungal chemotherapy. However, fungal CYP51s are membrane-associated proteins,and solving their crystal structures remains a challenge. In order to obtain morestructural information of fungal CYP51, homology modeling of Candida albicansCYP51(CA-CYP51) was conducted using human CYP51as the template. Aftermolecular dynamic optimization, detailed model evaluation (Pro-check andProfiles-3D), molecular docking validation and enrichment testing were performed toassess the accuracy of CA-CYP51model. The results showed that the CA-CYP51model had convincing accuracy, and could be used to guide the rational design of newazole antifungals. In our previous study, azole compounds containing N-methyl side chain showedexcellent in vitro antifungal activity. In order to get more structure-activityrelationships (SARs), structural modification was performed to investigate the effectof diverse substitutions attached to the nitrogen side chain on antifungal activity. Atotal of25new compounds was designed and synthesized. SARs showed thatsubstitutions such as hydrogen and methyl group were more suitable to enhance theantifungal activity. Moreover, they played an important role for the conformation ofthe side chain in the active site of CA-CYP51. Compounds A1and A14showedcomparable or superior antifungal activity to fluconazole (FLC). Molecular dockingwas performed to clarify the binding mode between azole compounds and CA-CYP51,providing reasonable explanation for the SARs and useful information for further leadoptimization.1.2Novel carboline derivatives as potent antifungal leadcompounds: discovery, optimization and biological evaluation.Cell-based phenotypic screening plays an important role for lead discovery.Different with target-based approaches, phenotypic screening has the advantage of thewhole organism being exposed to the compound and thus all the targets and biologicalpathways associated with it. Cell penetration and access to targets in their “natural”environment are taken into account. In recent years, phenotypic screening has beenused successfully to identify novel lead compounds.Herein, fungi cell-based phenotypic screening was conducted on our in-housecompound library. Interestingly, compound1with novel β-carboline scaffold showedbroad-spectrum antifungal activity. After rational structural modification, a total of27new derivatives were designed and synthesized. Most compounds showed improvedin vitro antifungal activity compared with lead compound1. Among them, compoundC27showed comparable in vitro antifungal activity to FLC. It also exhibited goodfungicidal activity against both FLC-sensitive and-resistant C. albicans cells, and hadpotent inhibition activity against C. albicans biofilm formation and hyphal growth.Moreover, C27showed good synergistic antifungal activity in combination with FLCagainst FLC-resistant Candida species. In order to get more information of antifungalmechanism, transmission electron microscopy and GC-MS method were conducted,and the results indicated that C27might act by inhibiting the synthesis of fungal cellwall, which was different with FLC. Due to potent antifungal activity and novel antifungal mechanism of the β-carboline derivatives, they are good starting points forresolving fungal resistance problem.2. Novel evodiamine derivatives as potent antitumor drugcandidates: design, synthesis and biological evaluation.Natural products have long been as an important source of drugs and leads. Inour previous study, evodiamine was identified as a novel topoisomerase I inhibitor bystructure-based virtual screening. Despite the promising properties of evodiamine as agood starting point for the development of novel antitumor agents, its antitumoractivity is relatively low and molecular targets are poorly understood.Systematic structural modification was conducted on evodiamine to investigatethe effects of diverse substitutions and molecular scaffolds on antitumor activity. Atotal of139new derivatives were designed and synthesized. In vitro antitumor assayshowed that most derivatives demonstrated broad-spectrum and improved antitumoractivity compared with evodiamine, and several compounds exhibited potentantitumor activity with GI50values lower than3nM. Moreover, some derivatives alsodemonstrated potent in vivo antitumor activity in nude colon and lung cancerxenograft models. For instance, compound E135reduced50.39%tumor growth at thedose of2mg/kg, and showed low toxicity and good tolerance. Furthermore,compounds E38, E112and E133could effectively induce the apoptosis and G2/Marrest of A549cells. Preliminary investigation of the molecular targets revealed thatevodiamine derivatives were the “first-in-class” Top1/Top2/tubulin triple inhibitors. Inparticular, compounds E112and E135showed better tubulin inhibitory activities (IC50=5.3μM and4.5μM) than colchicines (IC50=10.8μM). The multi-targetingantitumor feature of the evodiamine derivatives highlights the significance forimproving the antitumor effectiveness and overcome tumor resistance problem.3. Organocatalytic asymmetric synthesis of drug-likescaffolds and discovery of antitumor lead compound.In recent years, organocatalytic asymmetric cascade reactions have becomepowerful approaches to facile and stereoselective assembly of complex naturalproducts or synthetic building blocks in ‘one-pot’operation. They commonly possessgood yield, excellent stereoselective control, environmental friendly and atomeconomy features, and have been becoming a research hot spot in organic synthesis. 3,4-Dihydro-2H-thiopyrans are widely distributed in a number of biologicallyactive compounds. An unprecedented formal thio [3+3] cycloaddition process wasdeveloped to create this valuable scaffold in good to high yields (51%-84%) and withgood diastereo-(up to>20:1dr) and enantio-selectivities (up to>99%ee). Thisreaction proceeded smoothly under mild reaction conditions with two contiguousstereogenic centers in a one-pot operation. Moreover, novel scaffold with fourstereogenic centers could be obtained through Nazarov reaction.Furthermore, an organocatalytic highly enantioselective Michael-Michaelcascade reaction for the creation of structurally diverse tetrahydrothiopyrans wasdeveloped. Heavily functionalized tetrahydrothiopyran molecular architectures withfour consecutive stereogenic centers were assembled in good to high yield (58%-78%)and with good diastereo-(up to5.2:1dr) and excellent enantioselectivities (up to>99%ee) in ‘one-pot’ operation under mild reaction conditions. Moreover, simplesynthetic elaboration of the Michael-Michael adducts lead to structurally diverse,complex scaffolds.Spirooxindole is a privileged scaffold in natural product and bioactive molecules.Organocatalytic asymmetric Michael-Michael cascade reaction was developed toconstruct oxindole-spiro-tetrahydrothiopyran scaffold with four consecutivestereogenic centers in good yield (55%-74%), and excellent stereoselective control(dr>30:1; ee≥99%). Moreover, simple chemical transformation of the adducts leadto more novel and complex scaffold.A drug-like compound library was built on the basis of these sulfur-containingscaffolds. Through antitumor activity testing, the oxindole-spiro-tetrahydrothiopyranderivatives showed broad-spectrum in vitro antitumor activity. Among them,compound3b showed comparable antitumor activity compared with nutlin-3, andcould be used as antitumor lead structure for further optimization. This studycombined organocatalytic asymmetric synthesis with medicinal chemistry, andprovided a new strategy for antitumor lead discovery. Moreover, the activecompounds can be used as molecular probes for chemical biology studies.4. ConclusionIn summary, various strategies of lead discovery and optimization were used tofind novel antifungal and antitumor lead compounds. A total of191new derivativeswere designed and synthesized. Through biological evaluation, four kinds of novelantifungal and antitumor lead compounds were successfully discovered. The novelty of this thesis was focused on:(1) Novel carboline derivatives with potent antifungalactivity and new mode of action were identified for the first time. In particular, highlyactive compound C27showed the potential to overcome fungal resistance.(2)Systematic structural modification was conducted on natural product evodiamine, andnovel antitumor candidates with low toxicity and broad-spectrum antitumor activitywere discovered. Moreover, evodiamine derivatives were identified to be“first-in-class” Top1/Top2/tubulin triple inhibitors.(3) Novel organocatalyticasymmetric cascade reactions were developed to assemble three sulfur-containingscaffolds. Among them, oxindole-spiro-tetrahydrothiopyran derivatives showed broadspectrum antitumor activity. This work demonstrated the advantages by combinationof organocatalytic methodology and medicinal chemistry, and provided new strategyfor lead discovery. This thesis provided basis for the development of novel antifungaland antitumor agents.