Synthesis, Characterization And Biological Screening Of Thiazolidinone Derivatives And Nano-Combinatorial Libraries

The developments of combinatorial chemistry,parallel synthesis,analysis, automatic purification,and high throughput screening techniques have been greatly improved in the past decades.The application of combinatorial chemistry has been widely applied in the discovery and optimization of lead compounds.This study is committed to the development of new anti-cancer drugs,which can kill both the drug-sensitive lung cancer cells H460 and paclitaxel-resistant lung cancer cells H460/TaxR,but do not show toxicity towards normal human fibroblast(NHFB). Using thizaolidinones as lead compounds,solution-phase combinatorial chemistry and fluorous chemistry were adapted to synthesize a series of 4-thiazolidinone libraries.Based on the preliminary structure-activity relationship(SAR) exploration, a lead optimization library was designed and synthesized to further optimize the anti-tumor activity and cytoselective toxicity.A series of potent compounds with less toxicity and a much wider expected therapeutic window for their cytoselective toxicity for both multidrug-sensitive and -resistant cancer cells were identified.A separate pharmacophore for the most active compounds showed a common arrangement of two hydrogen bond acceptors and three hydrophobic regions.These most active compounds were less likely to be P-glycoprotein(P-gp) substrates.A number of conditions are required for an active small molecule to be a drug. Many active compounds in cell screening are abandoned due to low aqueous solubility,low permeability or high toxicity in vivo.The physicochemical properties of small molecules can be changed by chemical modification or encapsulation of small molecules with polymeric micelles as well as dendrimers.However,they are always subjected to operational restrictions,or particle sizes.An alternative new approach to improve the nature of small molecules is small-molecule surface modification on nanoparticles.With the application of nanoparticles in biological systems,the concern about the biocompatibility and toxicity is increasing.Surface chemical modification is one of the methods to address such problems.In this study, combinatorial chemistry technology was firstly applied to the surface modification of multi-walled carbon nanotubes(MWNTs).Through multiple biological screening, such as protein binding assay,cyototoxicity and immune response,the MWNTs with better biocompatibility were identified as well as the structure-biocompatibility relationship.The surface characterization restricts the development of chemical modification of nanoparticles.In the synthesis of MWNT library,we developed several methods for qualitatively and quantitatively determination of surface functional groups on MWNTs,such as FTIR and magic angle spinning NMR(MAS NMR).In order to verify and expand the usage of MAS NMR in the nano-surface chemical analysis,we designed and synthesized another widely studied nanoparticles,gold nanoparticles (GNPs).Through the optimization of NMR conditions,the high quality of ¹H and 2D NMR spectra were obtained.In the ¹H MAS NMR spectrum,the signal density of hydrogen is highly dependent on the distance between the surface of GNPs and protons in the ligand molecule.The closer the distance,the weaker the signal density is.Furthermore,NMR spectra of aromatic protons seem to always have a broad base compared with aliphatic ligands,indicating some degree ofÏ€-Ï€stacking effects.The final parts of this study focus on the design and synthesis of a series of bifunctional GNPs for biological experiments.Partâ… :Design,Synthesis,Cytoselective Toxicity,and Structure Activity Relationships(SAR) of Thiazolidinone DerivativesLung cancer is the number one cause of cancer-related deaths worldwide.More than 80%of bronchogenic malignancy is from non-small cell lung cancer(NSCLC). However the bottleneck to the development of effective anticancer drugs does not lie on an inability to identify chemicals that will kill cancer cells.Instead,the bottleneck lies on our inability to identify chemicals that will kill cancer cells at concentrations that do not harm patients.On the other hand,currently available therapies have been only temporarily successful for most cancer patients because they frequently lead to resistance.Thus,resistance to treatment and dose-limiting toxicity are two primary reasons for the failure of anticancer therapies.Resistance to treatment may arise through multidrug resistance(MDR),which is a phenotype of cross-resistance to multiple drugs.One mechanism underpinning MDR is the overexpression of the MDR-1 gene that encodes the transmembrane,ATP-dependent,drug efflux transporter P-gp in response to chemotherapy.Therefore,it's an important step for the success of cancer therapy to discover new compounds that are not substrates of P-gp and are affective against both drug-sensitive and drug-resistant cancer cells but spare normal human cells.Two thiazolidinone derivatives have been identified to inhibit the growth of paclitaxel-sensitive and -resistant NSCLC cell lines H460 and H460/TaxR.Both of them showed relatively low toxicity toward NHFB.To search more active compounds with an acceptable therapeutic window,thus helping to reveal structure-activity relationship for cytoselective anticancer activity,a series of thiazolidinone derivatives were designed.185 2-arylimino-4-thiazolidinones and 17 2-arylimino-3-substituted-4-thiazolidinones were successfully prepared using solution phase parallel synthesis method and 45 2-aryl-4-thiazolidinones were synthesized using fluorous chemistry method.In the primary screening,all compounds were tested against H460,H460/TaxR and NHFB at a concentration of 10μM.Compounds which exhibited toxicity to both cancer cells but not to normal cells were selected. Primary screening indicated that the nitrogen substitution blocked the cytoselective toxicity of the compounds and the imino group at 2-positon of thiazolidinones was necessary for keeping activities.The confirmation assays identified 11 compounds as potent agents for inducing cytoselective toxicity.To optimized the cytoselectivity and explore the structure-activity relationship of active compounds,the early lead optimization library was designed based on those potent compounds.We synthesized, purified and obtained 170 designed compounds with an average purity of 95%.The single-concentration(10μM) primary screening identified 40 hits.Using a lower concentration(5μM),11 compounds were confirmed and selected for dose-response studies,the 50%inhibitory concentration(IC₅₀) of the 11 compounds for H460 and H460_taxR cells was as low as 300 nM.For most of active compounds,NHFBs did not reach 50%cell killing at the highest compound concentration used(100μM).One of the most potent compounds possessed IC₅₀ against H460 at 500 nM and H460/taxR at 210 nM.Structures of 13 compounds that killed H460,but not drug-resistant H460/taxR, and 11 active compounds that killed both cancer cells,but not normal cells were selected for P-gp pharmacophore modeling studies.9 of 13 molecules(70%) were found to map to the P-gp substrate pharmacophore in group 1.In contrast,only 4 of 11 molecules(36%) in group 2 mapped to the P-gp substrate pharmacophore.This suggested that active compounds were less likely to be P-gp substrates.SAR analysis showed that substitution groups were dominantly electron-donating groups at the 2- or 4-position for both rings.Substitution at phenylimino position allowed groups such as -Me,-Cl at both the 2- and 4-positions.However,Substitution at benzylidene position was restricted to only the -NMe₂ group at the 4-position.Compounds with nitrogen substitutions on the 4-thiazolidinone ring B did not show any cytoselective toxicity. We,therefore,tentatively concluded that nitrogen substitution blocked such activity. Anti-cancer molecule pharmacophore was generated with 11 most active compounds. The results indicated that two hydrogen bond acceptors and three hydrophobic regions were common features for all active compounds.This anticancer compound pharmacophore may be useful in further database searching to scaffold-hop to find novel anticancer molecules.Further studies to determine the mechanism of these anticancer compounds,as well as to optimize anticancer activity within this series of compounds,will be desirable.Partâ…¡:Design and synthesis of nanoparticles with better characterization and biocompatibilityHundreds of thousands of active compounds are screened for best drug candidates each year,however,only a few less of them can be drugs in the end.That's because a lot of factors are needed for an active compound to become a drug,i.e.some active compounds in vitro experiment are restricted by bad aqueous solubility or poor permeability in vivo.Chemical modification and encapsulation of drug molecules with the core of polymeric micelles as well as dendrimers are two common methods to address the problems;however,the methods are time-consuming or restricted by the large sizes.Interestingly,the use of nanoparticulate pharmaceutical carries,which have small size from 1 to 100 nm,to enhance the efficiency of many drugs well were established over the past decades.MWNTs have attracted wide biological and medical interests besides the vast interests in their technological and engineering applications. For applications of MWNTs in biological systems,it is urgent to explore the MWNTs with better biocompatibility.Surface modification is an solution to enhance the biocompatibility of nanoparticles.Combinatorial modifications of MWNTs' surface would enable us to map unknown chemical space more effectively and to rapidly discover biocpmpatible functional MWNTs(f-MWNTs) with reduced toxicity,and reveal structure-activity relationships at the same time.Nano-combinatorial chemistry will accomplish two missions:1) to search for an initial lead candidate through combinatorial synthesis and screening and 2) once a lead is available,to optimize the lead in order to meet a set of criteria for nanomedicine candidate.To discover biocompatible nanotubes without a prior knowledge of targets,we decided to expose multiple biological targets to the maximum surface structural diversity.Starting with carboxylated f-MWNT,a tyrosine molecule is first attached as a linker because of its biocompatibility as an amino acid and its two reacting points(amine and carboxyl groups) for further diversification in two dimensions.8 amines and 9 acylators were selected for library synthesis(80 members including 8 intermediates in library).To confirm the purity of final f-MWNTs,LC/MS method was developed for monitoring the effectiveness of each purification process.FTIR was adapted for characterization of special functional groups on the surface of MWNTs,For example,when pristine-MWNT was oxidized, an FTIR band at 1713 cm^-1 appeared indicating the formation of carboxylic acid groups.We were the first to use MAS NMR with Nano probe to acquire ¹H NMR spectra of nanotube-bond molecules in this investigation.F-MWNTs 26,50,28,and 76 have a benzenesulfonyl group except that there was a nitro group at 3-position in 28 and 76.The existence of nitro group reduced the electron density of the phenyl ring and caused a downfield shift in 28 and 76(7.64-8.71 ppm) compared to 26 and 50(7.29-7.94 ppm).The loading of selected f-MWNTs were calculated by elemental analysis and confirmed by quantification of released Fmoc group from intermediates with UV-Vis spectroscopy.In order to select suitable candidates for further optimization and in vivo study,we studied the protein binding properties,cytotoxicity and immune response of the f-MWNT library.The f-MWNTs with less protein binding,immune response,and cytotoxicity were ranked from 1 to 80.The sum of a f-MWNT's ranking was named multi-assay score.Smaller multi-assay score is superior in term of overall biocompatibility.Structure-biocompatibility relationship analysis showed that three out of the top five lead candidates,contained the building block AC005 and all eight AC005 containing f-MWNTs were in the top 25 in term of multi-assay score.AC008, AC006,AC004,AM004 and AM003 also showed moderate correlation to the multi-assay score.By rapidly approaching to surface molecular diversity on MWNTs and accelerating discovery of biocompatible nanomedicine carriers,we demonstrated the general utility of nano combinatorial chemistry approach in nanomedicine discovery.Encouraged by the primary results of the usage of MAS NMR for surface characterization of MWNTs,we successfully expanded the method to GNPs for full structural characterization.Compared with MWNTs,GNPs had advantages of better biocompatibility,easier synthesis process and unique size distribution.We effectively optimized high resolution MAS NMR(HRMAS NMR) experimental conditions by selecting solvent,temperature,spinning speed and pulse sequence for GNPs.Using the optimized condition,we also successfully obtained the ¹H and 2D HRMAS NMR for GNPs,such as COSY,TOCSY and HSQC.We found that there are significant differences in detection sensitivity depending on the distance between the surface of GNP and protons in the ligand molecule,and the loss of sensitivity for protons closer to the nanoparticle surface is consistent with an early finding.Furthermore,NMR spectra of aromatic protons of ligands attached to GNP seem to have a broad base compared with aliphatic ligands,indicating some degree ofÏ€-Ï€stacking effects. Chemical shift assignment was useful for characterization of more complex structure by 2D spectra.Our results demonstrate that ¹H HRMAS NMR holds great potential for structural elucidation of molecules bound to nanoparticle surface.Recognizing that nanoparticles had the advantages of multifunctionalization,we designed and synthesized double functional GNPs with different ratios of two free ligands.We tried different methods to determine the loading of each ligands, including FTIR and cleavage-LC/MS.The loading of certain ligand increased with the increased usage of the free ligand.The conclusion was confirmed by design and synthesis of another group of double functional GNPs.When FITC and folic acid(FA) were used to construct multifunctional GNPs,the existence of FITC was confirmed by fluorescence spectroscopy of the GNPs.Two cell lines,KB cells over expressing FA receptors and A495 with low content of FA receptors,were used for cell uptake experiments.The difference of Confocal images of two cell lines demonstrated the existence of FA on the surface of GNPs.Using the optimized conditions,a double-functional GNP nano-combinatorial library containing 42 members was successfully synthesized and characterized.The development of new quantitative method for GNPs and the bioassays for the GNP library are on going.