Design and Synthesis and Biological Evaluation of New Thiazoles as Potential Antimicrobial Agents

Author:Gao Wei Wei

Supervisor:zhou cheng he


Degree Year:2018





Thiazole ring is a quite important five-membered aromatic heterocycle containing nitrogen and sulfur atoms with both electron donating(-S-)and accepting(C=N)groups.The unique structural feature is beneficial for thiazole derivatives to readily interact with various small molecules and macromolecules such as enzymes,proteins,nucleic acid and receptors in biological system through various weak interactions like hydrogen bonds,coordination,ion-dipole,cation-π,π-πstacking,electrostatic and hydrophobic effects,van der Waals force etc to exhibit a variety of biological activities.Thus,thiazole fragment has been being extensively employed to design and develop a broad range of medicinal molecules.Numerous thiazole-based drugs have been successfully developed,marketed and extensively used in clinic,such as antibacterial cephalosporins,anticancer dasatinib,anti-inflammatory toxicam and the antiparasitic niazonide,which have almost been expanded to the whole range of medicinal field.Particularly,an increasing number of thiazole-based compounds are becoming clinical drug candidates in actively ongoing research and development.More importantly,thiazole compounds play a very important role in the antibacterial and antifungal fields.There have been a lot of investigations on thiazole-based compounds with multi-targeting ability to possess broad spectrum,high activity,low drug resistance develorment,low toxicity and high bioavailability.All the above mentions strongly point out the great developmental value and spacious potentiality of thiazole-based medicinal research and application.On the basis of previous research on antibacterial and antifungal thiazoles in our group and referring to current situation in the researches on thiazoles,a series of novel thiazole-based compounds were designed and synthesized based on the following ideas,which were expected to obtain novel thiazole compounds with better biological activity and multi-targeting ability,thus possibly overcoming the severe resistance.These novel compounds were evaluated for their antimicrobial activity,and structure-activity relationships were also discussed and summarized.To identify the safety profile,the cytotoxic tests for the highly active compounds were done against human cells.The further experimental studies including bactericidal kinetic assay,drug resistance development,bacterial membrane permeabilization and theoretical exploration of molecular modeling were also performed to verify the research values and foreground of the thiazole-based derivatives.Also,the combination use of the highly active molecules with clinically drugs was evaluated to further excavate their potential in enhancing the antimicrobial efficiency and overcoming drug resistance.Moreover,the genomic DNA was isolated from the sensitive resistant strains in order to explore the possible antibacterial mechanism by the use of UⅤ-visible absorption spectra and along with Agarose gel electrophoresis.Finally,the transportation ability of human serum albumin(HSA)to highly active target compounds was assessed.The main work was summarized as follows:1、Design ideas(1)Design of berberine thiazoles:Berberine is widely used in clinic as a natural antimicrobial drug.In particular,it plays an important role in the treatment of intestinal infections and gastroenteritis.A great deal of work has been done to develop berberine derivatives with medical values.Natural berberine is a well-known natural isoquinoline alkaloid with a quaternary nitrogen and desirable largeπ-conjugated backbone.It has been reported that berberine and its derivatives can target DNA and cell membrane.On the basis of this,herein we have overwhelming interest in combining the naturally antibacterial berberine backbone and the important antibacterial structural thiazole to develop a novel series of potentially antibacterial hybrids.The new hybrids are expected to exert multi-targeting properties including the validated berberine targeting DNA and membrane activity.(2)Design of coumarin thiazoles:Compared to berberine,coumarin ring is also an important natural antimicrobial parent with a smaller conjugate system of benzopyranone.In recent years,a lot of synthetic coumarin compounds have been reported to be with good and broad spectrum of antimicrobial efficiency,especially against MRSA.These exciting results attract a number of researchers to be engaged in the development of coumarin-based antimicrobials.They can target not only DNA,but also topoismerase Ⅱ or DNA gyrase to interfere the superhelix,reproduction and transcription of DNA as well as the isolation of chromosome,which further inhibit the growth of bacteria.Therefore,we have overwhelming interest in introducing thiazole ring into coumarin skeleton to develop a series of coumarin thiazole derivatives with structural novelty,and compares the antimicrobial activity of them with that of berberine thiazoles.(3)Design of quinolone thiazoles:Compared to coumarin,quinolone ring(benzopyridyl acid)with a similar structure to coumarin(benzopyranone)can also interact with various active sites in organisms through non-covalent bonds.Quinolones mainly target topoismerase or DNA-gyrase complexes.However,many of the severe side effects of quinolone drugs such as gastroenteritis,vomiting,and cartilage damage have been shown to be mainly induced by the 3-carboxyl group.Therefore,we have overwhelming interest in replacing the carboxyl group of quinolone with a thiazole fragment to produce a series of novel quinolone thiazole derivatives.The antimicrobial activity of them with was investigated and expected to change the classic binding model of quinolone with enzyme-DNA complexes to overcome the resistance and side effects caused by the carboxyl group.(4)Design of organophosphorus imidazoles:Imidazole ring has also been identified as an attractive isostere of thiazole and extensively used to design and develop various bioactive molecules.A large number of imidazole-based compounds as antimicrobials(metronidazole,ornidazole,ketoconazole and oxiconazole)have been used in clinical with high therapeutic potency,which have shown the huge development value in anti-infectious field.More importantly,imidazole ring with multiple binding sites is capable of coordinating with a variety of inorganic metal ions or interacting with organic molecules via non-covalent bonds to produce supramolecular drugs,which may have not only bioactivities of imidazoles themselves but also the advantages of numerous supramolecular drugs,possibly exerting double action mechanisms that are helpful to overcome drug resistance.Additionally,the introduction of organophosphorus moiety would play important roles in regulating the physicochemical properties of target molecules through changing the intermolecular forces and molecular polarity.Herein,a series of novel organophosphorus imidazoles were designed and synthesized to investigate the introduction of less aromatic benzene ring and the effect of imidazole as an isostere on the antimicrobial activity.2、Preparation(1)Preparation of novel berberine thiazoles:Commercially available berberine chloride Ⅱ-1 was easily converted into the important intermediate Ⅱ-4 through demethylation,reduction and formylation.The condensation of compound Ⅱ-4 with hydrazinecarbothioamide in anhydrous alcohol conveniently afforded carbothioamide derivative Ⅱ-5,and then further cyclization with 2-chloroacetaldehyde efficiently produced target hydroxyl aminothiazolyl berberine derivative Ⅱ-6.The substitution of compound Ⅱ-4 with a series of aliphatic bromides or chlorides in DMF at 80°C using potassium carbonate as the base gave the 9-position substituted berberine derivatives Ⅱ-7a-g,Ⅱ-10 and Ⅱ-13.These prepared intermediates were further condensed with hydrazinecarbothioamide to afford aliphatic carbothioamide precursors Ⅱ-8a-g,Ⅱ-11and Ⅱ-14,respectively,which were further reacted with 2-chloroacetaldehyde in anhydrous ethanol at 80°C to produce the desirable aliphatic aminothiazolyl berberine derivatives Ⅱ-9a-g,Ⅱ-12 and Ⅱ-15,respectively.The target phenyl series of aminothiazolyl berberine derivative Ⅱ-18a-g was also obtained under the similar reaction condition starting from intermediate Ⅱ-4,the substituted benzyl chlorides,hydrazinecarbothioamide and 2-chloroacetaldehyde.(2)Preparation of novel coumarin thiazoles:The key intermediates Ⅲ-2a-c were easily obtained via direct formylation of compounds Ⅲ-1a-c.Methyl coumarin Ⅲ-1c was constructed by using commercially available resorcinol Ⅲ-5 and ethyl acetoacetate.The thiocarbamide derivatives Ⅲ-3a-c and target hydroxyl aminothiazolyl coumarins Ⅲ-4a-c were produced by the above mentioned methods.Similarly,the target aliphatic series of aminothiazolyl coumarins Ⅲ-8a-e and phenyl ones Ⅲ-11a-e were also obtained according to the above mentioned methods starting from intermediate Ⅲ-2c,aliphatic bromides or substituted benzyl chlorides,thiosemicarbazide and2-chloroacetaldehyde.(3)Preparation of novel quinolone thiazoles:Compound Ⅳ-2 was easily prepared by the reaction of commercial triethoxymethane,ethyl 3-oxobutanoate and acetic anhydride.The synthesized intermediate Ⅳ-2 was reacted with a series of substituted phenylamines in the absence of solvent to afford Ⅳ-3a-e in almost quantitative yields,and then were further cyclized in phenoxybenzene under reflux to produce the quinolones Ⅳ-4a-e.Compounds Ⅳ-4a-e were brominated by bromine in acetic acid to produce corresponding 3-(2-bromoacetyl)quinolones Ⅳ-5a-e and then further cyclized with thiourea or ethanethioamide in ethanol readily gave the target quinolone thiazoles Ⅳ-6a-j.Compounds Ⅳ-6a-j were then reacted with commercial chloracetone to yield target N-(2-oxopropyl)derivatives Ⅳ-7a-j.Finally,the target thiocarbamide derived quinolone thiazoles Ⅳ-8a-j were produced by the above mentioned methods.In addition,the N1-position of intermediate Ⅳ-4a was substituted by propionyl group,then brominated and cyclized to obtain target quinolone dithiazole compound Ⅳ-11.Furthermore,the target oxime derived quinolone thiazole Ⅳ-12 was obtained from compound Ⅳ-7a and hydroxylamine hydrochloride.In order to investigate the important effect of thiazole ring in 3-position of quinolone on the antimicrobial activity,the 3-carboxylate quinolone derivatives Ⅳ-17a-b were also obtained according to the above mentioned methods.(4)Preparation of novel organophosphorus imidazoles:Alkyl imidazoles Ⅴ-2a-b were prepared from commercially available compound Ⅴ-1,diethyl phosphonate and aliphatic amines in toluene.Phenyl type of imidazole compounds Ⅴ-3a-i were obtained under the same reaction condition starting from a series of anilines,and then further hydrolyzed in concentrated hydrochloric acid to afford imidazole phosphonic acids Ⅴ-4a-f and Ⅴ-5a-c.Pyrimidine-based molecule Ⅴ-6 was obtained under the same reaction condition starting from 2-aminopyrimidine.Also,coumarin derivative Ⅴ-7 was efficiently synthesized in good yield by the Mannish reaction of 3-aminocoumarin which was easily prepared from the one-pot cyclization of 2-hydroxyl benzaldehyde and methyl glycinate hydrochloride.3、CharacterizationThe newly synthesized compounds were characterized by 1H NMR,13C NMR,IR and HRMS spectra.4、Biological activity(1)The antimicrobial assays in vitro indicated that most of intermediates and target compounds in the series Ⅱ,Ⅲ,Ⅳ and V could significantly inhibit the growth of all the tested microorganisms including drug-resistant ones.Especially,target berberine thiazole derivatives Ⅱ-9c and Ⅱ-18a gave strong inhibitory activities against drug-resistant Gram-negative A.baumanii with low MIC values of 2 nmol/mL,superior to the current clinical drugs.Coumarin thiazole derivatives Ⅲ-8b(MIC=1μg/mL)and Ⅲ-11b(MIC=1μg/mL)exhibited bactericidal nature(MBC/MIC=2)toward clinically drug-resistant E.faecalis.The target quinolone thiazole Ⅳ-8a showed stronger antimicrobial activity than the reference drug norfloxacin,especially against drug-resistant S.aureus(MIC=2μg/mL).Notably,the antimicrobial activities revealed that imidazole,the isostere of thiazole,derived organophosphorus compound Ⅴ-3f showed superior activities against MRSA and S.cerevisiae(MIC=2mg/mL)to clinical drugs.(2)The above mentioned highly active compounds exhibited rapidly bactericidal effects and quite slow resistance development toward the sensitive pathogens.(3)The highly active compounds showed low toxicities to human cells,which might be concluded that the antibacterial activities exhibited by thiazole derivatives were not due to the cytotoxic effects.(4)The combination use of the active molecules Ⅱ-18a and Ⅴ-3f with clinically antibacterial or antifungal drugs,respectively,could enhance the efficiency,broaden antimicrobial spectrum and effectively combat drug resistance.(5)Molecular docking showed that hydrogen bonds existed in the supramolecular interaction between DNA gyrase and the active berberine thiazole molecule Ⅱ-9c or Ⅱ-18a.Coumarin thiazole derivatives Ⅲ-8b and Ⅲ-11b could interact with gyrase-DNA complex through hydrogen bonds and hydrophobic supramolecular interactions,and the similar supramolecular interactions also existed between the quinolone thiazole Ⅳ-8a and topoismerase Ⅳ-DNA complex to exhibit good biological activity.(6)Experimental data revealed that HSA could form ground-state complexes with organophosphorus imidazole Ⅴ-3f through hydrophobic interactions and hydrogen bonds with the spontaneous binding process.Competitive interactions suggested that the participation of K+,Na+,Mg2+,Cu2+,Ca2+,Zn2+,Ni2+and Ag+ions in Ⅴ-3f-HSA system could increase the concentration of free compound Ⅴ-3f,shorten its storage time and half-life in the blood to improve the maximum antimicrobial efficacy,or possibly produce Ⅴ-3f-ion supramolecular complexes to exhibit biological activities.5、Preliminary antibacterial mechanism study(1)The interactive investigation with cell membrane revealed that berberine thiazole molecules Ⅱ-9c and Ⅱ-18a were found to be active toward drug-resistant A.baumanii membrane.Coumarin thiazole derivative Ⅲ-11b treated drug-resistant E.faecalis cells were unable to sustain under the collective effects,which resulted in severe disruption of cell surface integrity and membrane permeabilization.Quinolone thiazole Ⅳ-8a could effectively pass through the cell membrane of drug-resistant S.aureus.Organophosphorus imidazole derivative Ⅴ-3f could effectively interact with the membranes of both Gram-positive(MRSA)and Gram-negative(P.aeruginosa)bacteria.(2)The supramolecular interactive investigation with DNA revealed that the highly active compounds Ⅱ-9c,Ⅲ-11b and Ⅳ-8a could effectively intercalate/cleave DNA,isolated from the sensitive pathogens,to inhibit the DNA replication of bacterial strains,thus exhibiting antimicrobial effects with multi-targeting property.(3)Quinolone thiazole Ⅳ-8a could inhibit topoisomerase Ⅳ effectively with lower LLE value than norfloxacin.There are 184 compounds successfully synthesized in this thesis,and 163 compounds are new,including 62 berberine thiazoles,40 coumarin thiazoles,59 quinolone thiazoles and 23 organophosphorus imidazoles.The antimicrobial evaluation indicated that 83compounds exerted better antibacterial effects against some strains than reference drugs ciprofloxacin,norfloxacin,chloromycin and/or fluconazole.The higly active compounds were further studied,and the results demonstrated that these compounds displayed low cytotoxicity to human cells and rapidly bactericidal effects with quite slow resistance development.Also,these active compounds could intercalate and/or cleave DNA,be active toward cell membrane and inhibit the enzyme.All the above indicated that this type of compounds exerted great potentiality in the treatment of microbial infections.