Design And Synthesis Of Novel Benzimidazoles And Their Antimicrobial Studies

Benzimidazole has larger conjugated system and stronger electron transport abilities,which enables its derivatives to exhibit extensive medicinal applications.Particularly,benzimidazoles could readily interact with active targets in biological system via diverse non-covalent interactions such as hydrogen bond,coordination,ion-dipole,cation-π,π-πstacking,and hydrophobic effect as well as van der Waals force to display a wide range of biological activities including antibacterial and antifungal ones etc.Recently,the increasing incidence of multidrug-resistant strains,intractable pathogenic microorganisms and newly emerging pathogens is a global human threat.Therefore,better understanding the mechanisms of currently available antibiotics and exploring alternative antibacterial drugs have emerged as an important strategy in the development of new antimicrobial therapies.Benzimidazole is structurally similar to purine,and its derivatives could compete with purines,distinctly inhibiting the synthesis of nucleic acids and proteins,thereby killing bacterial strains or inhibiting their growth.In the light of this,the development of benzimidazoles as antibacterial agents has become one of the highly active highlights.In view of this,this paper referred to the related literature in recent years,combined with the work of our research group and reviewed the research and application of benzimidazoles in the field of antimicrobial medicinal chemistry,mainly including anti-bacterial and anti-fungal aspect.The research of benzimidazoles as antimicrobial agents is relatively active.Its recent work in antibacterial and antifungal field involves the following aspects:（1）Five-membered heterocycle containing benzimidazoles as antibacterial andantifungal drugs（2）Six-membered heterocycle containing benzimidazoles as antibacterial and antifungaldrugs（3）Fused heterocycle containing benzimidazoles as antibacterial and antifungal drugs（4）Benzimidazole-based metal complexes as antibacterial and antifungal drugs（5）Other benzimidazoles as antibacterial and antifungal drugsThe successful discovery of quinolone antimicrobials greatly decreased the morbidity and mortality caused by fatal bacterial infections.Their good therapy effectiveness,broad antibacterial spectrum and good safety profile rapidly promoted the development of this class of antibacterial agents as first-line alternative to treat respiratory,urinary tract and bone joint infections.Especially,the synthesis of fluoroquinolone family made great contribution to anti-infective agents such as norfloxacin,ciprofloxacin,levofloxacin and moxifloxacin.Antimicrobial mechanism revealed that this type of drugs could target DNA gyrase or topoisomerase IV by binding with enzyme–DNA binary complexes to form ternary supramolecular complexes,thereby obstructing DNA replication and finally resulting in bacterial death.However,the overuse of quinolones to manage bacterial infections has led to the evolution and widespread distribution of resistant strains such as Streptococcus pneumoniae,Escherichia coli and Staphylococcus aureus.It was reported that resistance to quinolones was mainly attributed to the mutations of some key enzymes in quinolone-resistant pathogens.Particularly,the alterations in the conserved ParC helix a4 residues of topoisomerase IV–DNA complex,positioned in close proximity to the groups at N-1 position of quinolones,directly weakened the binding affinity between quinolone antimicrobials and target enzymes.Accordingly,it is a promising strategy that azole rings were introduced into N-1 position of quinolones to strengthen the binding affinity and overcome resistance,because azole rings could bind with DNA,enzymes and receptors in organism through various weak interactions like coordination bonds,hydrogen bonds,π-πstacking and hydrophobic effect.A great deal of literature has reported that some aromatic azoles in combination with quinolones displayed good antibacterial efficacies,especially towards some drug-resistant bacteria like methicillin-resistant Staphylococcus aureus（MRSA）with strong activity and low toxicity.However,to our best knowledge,the combination of benzimidazole and the quinolone backbone,especially at N-1position has been seldom observed.In view of the above mentioned and as an extension of our previous work,herein a series of novel benzimidazole quinolone hybrids in the chapter 2 were designed through the introduction of benzimidazole moiety to N-1position of quinolones using methylene as a bridge,which allowed the flexible rotation of benzimidazole ring.It is expected that these hybrids would have large potentiality in the treatment of bacteria-infected diseases.The structural design of novel quinolone benzimidazoles is based on the following ideas:（1）A series of novel benzimidazole quinolone hybrids were designed by introducing benzimidazole moiety to the N-1 position of the quinolone,using methylene as a bridge,allowing flexible rotation of the benzimidazole ring;（2）Halogen atoms in the quinolone skeleton can enhance the stability of the protein-ligand and subsequently contribute to the binding affinity;（3）Many studies have shown that various substituents（including aliphatic and arylalkyl groups）were introduced to the N-1 position of the benzimidazole core to study their effect on antimicrobial activity because the N-substituent of azoles can significantly affect the pharmacological properties by adjusting the lipid-water partition coefficient and binding affinity.Purines can be regarded as one of the most ubiquitous and functional N-heterocyclic compounds found in nature.Purine scaffold is the core structural fragment of adenine and guanine in RNA and DNA.Purine nucleotides such as ATP,GTP,cAMP,cGMP,NAD and FAD act as co-factors,substrates or mediators in the function of many proteins.These proteins are estimated to include half of the most pharmaceutically available targets,primarily enzymes and receptors.Some naturally existing and synthetic purines have been applied as antitumor,antiviral and antiparasitic agents and adenosine receptor ligands while the application in antimicrobial aspect has rarely been reported.Therefore,the development of purine-based novel antimicrobial agents is of significant value in medicinal chemistry.In view of the pivotal role of purines in the regulation of many biologicalprocesses,it arouses our immense interest to modify the purine skeleton using isosteric ring systems to develop novel structural molecules and investigate their possibility as new antimicrobial agents.Azoles as nitrogen-containing five-membered aromatic heterocycles with desirable electron rich characteristic can readily bind with a variety of enzymes and receptors in biological systems through diverse weak noncovalent interactions,thereby exhibiting broad bioactivities.Thus,the combination of azoles and the purine nucleus provides a great prospect for medicinal and biological application.They have been developed as antimycobacterial and antiviral drugs and also as adenosine receptor agonists and antagonists.Especially,benzimidazole with special benzene-fused imidazole ring as a key component for various biological activities like antiparasitic,anticancer,anti-inflammatory and antiulcer agents etc.has attracted considerable concern.Recently,extensive biochemical and pharmacological studies revealed that benzimidazoles possessed large potentiality to inhibit the growth of bacterial and fungal strains.Further research disclosed that benzimidazole nucleus structurally resembling with purine scaffold in nucleotides was sometimes addressed as 1,3-dideaza-purine and had the capability of competing with purines,distinctly inhibiting the synthesis of nucleic acids and proteins,thereby killing bacterial strains or inhibiting their growth.Clearly,benzimidazole-basedderivatives possess great potential as new antibacterial and antifungal agents.Reasonably,this structural similarity prompts us for the first time in the chapter 3 to combine benzimidazole moiety and purine backbone to generate a novel type of hybrids as potentially antimicrobial agents and evaluate their antimicrobial potency.Design idea for novel purine benzimidazoles was mainly from three aspects:（I）Structural modification on the benzimidazole nucleus;（II）Structural change on the purine scaffold;（III）The different linkers between benzimidazole and purine rings.The related considerations were given as follows:（1）The C-6 and N-9 positions in the purine scaffold are the most widely exploited anchoring points and the substituted purines are commercially available.Therefore the purine nucleus through the N-9 position with benzimidazoles was hybridized;（2）The incorporated flexible–CH₂–aliphatic linker between benzimidazole and purine nucleus is helpful for regulating the molecular conformation to interact with targets.The N-alkyl benzimidazole moiety was reported to have good performance in exerting bioactivities,the–CH₂CH₂–aliphatic linker was also introduced into target compounds in order to evaluate its contribution towards antimicrobial efficacy;（3）A lot of work revealed that substituents at N-containing heterocycle could significantly influence the pharmacological properties by adjusting lipid-water partition coefficient and binding affinity.Rationally,various aliphatic chains with different lengths and substituted phenyl moieties including chloro and fluoro groups were introduced at N-1 position on benzimidazole moiety to investigate their effect on bioactivities;（4）The substituted groups at C-2,5,6 positions on benzimidazole nucleus had the capacity to modulate the pharmacokinetic properties and enhance the antimicrobial efficacy.The substituents on benzimidazole nucleus were changed to explore their impacts on biological activities;（5）It is well known that saturated nitrogen heterocycles such as morpholine,piperidine and pyrrolidine are beneficial building blocks in drug design and prevalently present in many approved drugs.The above mentioned alicyclic amines were introduced to C-6 position on purine nucleus for investigating the influence of the substituents in the purine nucleus on structure-activity relationship and bioactive profiles.The main work in this thesis is summarized as follows:（1）Preparation of novel benzimidazole quinolone hybrids:Compound II–1 was synthesized by reacting diethyl malonate with triethylorthoformate and acetic anhydride acid under the condition of zinc chloride as catalyst,and then further treatment with3-chloro-4-fluoroaniline in ethanol to give compound II–2.Intermediate II–2 was cyclized in diphenyl ether at 250°C to form quinolone II–3.Benzimidazole quinolone hybrids II-4–6 were prepared by coupling intermediate II–3 with a series of N-substituted chloromethyl benzimidazoles II–8,II–10 and II–12 in acetonitrile.Intermediates II–8,II–10 and II–12 were prepared by cyclization of N-monosubstituted o-phenylenediamines II–7,II–9 and II–11 with chloroacetic acid in hydrochloric acid at reflux.Monosubstituted o-phenylenediamines II–7,II–9 and II–11were obtained by nucleophilic substitution of o-phenylenediamine with a series of alkyl halides,benzyl halides and bromopropene/alkyne（1.2:1）.（2）Preparation of novel purine benzimidazoles:The reaction of 6-chloropurine with morpholine at 80°C provided intermediate III–1,which was then further reacted with substituted benzimidazoles III–8,III–10 and III–12 in N,N-dimethylformamide to give purine benzimidazoles III–2,III–3 and III–4.The starting material 6-chloropurine was also refluxed with piperidine and pyrrolidine respectively in ethanol using triethylamine as a base to give intermediates III–5a and III–5b which were further reacted with N-hexylbenzimidazole III–10c in dimethylformamide at 50°C to afford final products III–6a and III–6b.（3）All the newly synthesized compounds were characterized by ¹H NMR,¹³C NMR,IR,MS and HRMS spectra.（4）The prepared intermediates and target compounds were evaluated for their in vitro antimicrobial activities.The biological assays manifested that some prepared compounds could effectively inhibit the growth of the tested microorganisms and some of the target compounds showed comparable or even better inhibitory ability than the reference drugs clinifloxacin,norfloxacin and fluconazole.The 2-fluorobenzyl derivative II–5b displayed relatively efficient antibacterial activities in comparison with other benzimidazole quinolone hybrids.Especially towards P.aeruginosa,compound II–5b gave a quite low MIC of 1μg/mL,which was 4-and 32-fold more potent than reference drugs norfloxacin and clinafloxacin.In comparison to the aliphatic derivatives II–4,most of halobenzyl ones II–5 exerted relatively better activities in inhibiting the growth of the tested strains which might reveal that the presence of benzyl group is helpful for antimicrobial activity.All benzimidazole quinolone hybrids possessed comparative even better antifungal efficiency（MIC=1–256μg/mL）against the clinical drug-resistant C.tropicalis in contrast to fluconazole（MIC=256μg/mL）,especially compound II–5b（MIC=1μg/mL）,which was 256-fold more active than fluconazole.Furthermore,target molecule II–5b also exhibited good inhibition potency towards C.parapsilosis（ATCC 22019）（MIC=1μg/mL）while fluconazole possessed the higher MIC value（2μg/mL）.Furthermore,preliminary active screening revealed that morpholine derivative III–3c showed better antibacterial efficacy than other alicyclic amine ones III–6a and III–6b,and more importantly hexyl-appended hybrid III–3c exhibited stronger inhibition ability against most of the tested bacterial strains among the all target compounds.（5）The preliminary structure-activity relationships showed that not only the length of the alkyl chain had an important influence on antimicrobial ability of the benzimidazoles,but also the saturation of the carbon chains also exerted a significant effect on the activity.In addition,the type and position of substituents on the benzyl halide and the substituents on the benzimidazole ring could also improve the biological activity.The introduction of benzimidazole fragments was helpful for the enhancement of biological activity.（6）Bacterial resistance studies showed that the active quinolone-benzimidazole molecule was more difficult to induce resistance to P.aeruginosa than clinical drug norfloxacin,and the bactericidal kinetics study results indicated that this compound has a quick killing effect against P.aeruginosa.（7）Molecular docking indicated that the carbonyl group at 4-position of II–5b was in close vicinity to the residue Asp397 of the topoisomerase IV–DNA complex and formed hydrogen bonds.The prepared molecule II–5b could also interact with the residue Gly419 of topoisomerase IV–DNA complex and base DT15 of DNA through hydrogen bonds with distance of 2.4 and 2.1?,respectively through the oxygen atom of ester group.These noncovalent interactions with DNA base might block DNA replication.So,the above results showed that this cooperative binding might be propitious to stabilize the quinolone–enzyme–DNA ternary complexes which enabled compound II–5b to possess the strong inhibitory efficacy against P.aeruginosa.The nitrogen atoms of benzimidazole ring at 3-position and purine ring at 3-position of purine benzimidazoles III–3c were in close vicinity to the residue GLN 197 of Topo IA through hydrogen bonds with distance of 2.8 and 2.0?,respectively,which revealed that the nitrogen atom played important role in biological activity.Besides,electrostatic interactions existed between the aromatic fragment of compound III–3c and residues THR 496,VAL 196 and ILE 500 in Topo IA.Specifically,morpholine ring could also form electrostatic interactions with residue TYR 177.These hydrogen bonds and electrostatic interactions might be responsible for the good inhibitory efficacy of compound III–3c against the tested strains.（8）The active molecules exhibited low toxicity to the cells and had excellent safety by regulating the generation of reactive oxygen species,demonstrating that the compounds possessed good biocompatibility,which is considered to be one of the most important properties of active candidate drugs.（9）Quantum chemistry studies theoretically elucidated that the active molecules showed good antimicrobial activity.A total of eighty nine compounds were synthesized in this thesis,including forty two new compounds,including three benzimidazole intermediates,eighteen alkyl compounds,thirteen halobenzyl compounds and eight purine benzimidazoles.Among them,the inhibition of five target compounds in chapter 2 against clinically resistant P.aeruginosa was stronger than that of the reference drug Clinfloxacin,and the inhibitory activity of eight target compounds in chapter 3 against clinically resistant S.aureus was stronger than that of the reference drug Norfloxacin.Experimental studies demonstrated that Compound II–5b could not intercalate into DNA isolated from drug-resistant P.aeruginosa,but it was able to cleave DNA effectively,which might further block DNA replication to exert the powerful bioactivities.In addition,compound II–5b was found to be a promising antibacterial agent with membrane disruption ability.UV-vis and fluorescence absorption spectroscopy showed that the active molecule III–3c could effectively interact with S.aureus DNA to form 3c–DNA complex through groove binding mode,which might block DNA replication to display their powerful antimicrobial activity.Therefore,these new quinolone benzimidazoles and purine benzimidazole have great potential as antibacterial drugs for further study.