Novel Berberine-derived Azoles:Design,Synthesis And Evaluation Of Antimicrobial Activities

The traditional medicine herb Rhizoma coptidis?Huanglian in Chinese?was widely used for the treatment of various diseases in ancient China.Around 2000 years ago,it was recorded in Shennong's Classic of Materia Medica,the earliest monograph on Chinese medicine,that Rhizoma coptidis possessed multiple curative effects on dysentery,hypomnesia,and blurred vision.Natural berberine is a kind of quaternary benzylisoquinoline alkaloid with specially fused aromatic structure and central N⁺-containing moiety,which enables it to bind with various receptors,enzymes,proteins and signal pathways through non-covalent interactions including hydrogen bonds,electrostatic interaction,cation-?,?-?stacking,ion-dipole hydrophobic effect and so on,and thus exhibiting potentially therapeutic effects for various diseases through multiple mechanism of actions.The related researches of berberines,including antibacterial,antifungal,antiviral,antitubercular,anticancer,anti-diabetic,anti-inflammatory,anti-Alzheimer and Cardiovascular agents,have been an extremely active topic.Azoles?imidazoles,triazoles,benzimidaozles,tetrazoles?could easily interact with various biological targets due to their special structures,thus exhibiting broaden bioactivities.lots of researches suggested that the introduction of azole moieties to natural products could extremely increase solubility and permeability.Therefore,this thesis firstly designed and synthesized a series of berberine azole compounds and evaluated their antimicrobial activities according to our previous researches on berberine.The highly active compounds were further studied for the pharmacology,toxicology and pharmacokinetics,and the preliminary antibacterial mechanism was also investigated.The main work was summarized as follows:?1?This thesis systematically gave a comprehensive review in current developments of berberines in the whole range of structural modifications and as medicinal agents including antibacterial and antifungal.?2?Preparation of novel berberine azoyl ethanols:Berberine was employed to produce berberrubine II-1 through heating under vacuum for 45 min,and the latter was further reduced by excessive sodium borohydride to generate structurally bended derivative II-2.Compound II-2 was further formylated by hexamethylenetetramine?HMTA?using trifluoroacetic acid?TFA?as solvent to conveniently and efficiently produce the corresponding aldehyde II-3.The nucleophilic substitution was employed by epichlorohydrin to produce the important intermediate II-4,and latter was reacted with various azole compounds to obtain the target molecules II-5-8.The hydroxylamine derivative II-9 was conveniently synthesized by the condensation of hydroxylamine and compound II-5a.?3?Preparation of novel berberine-derived nitroimidazoles:Berberine was employed to produce berberrubine III-1 through heating under vacuum for 45 min,and the latter was further reduced by excessive sodium borohydride to generate structurally bended derivative III-2.Compound III-2 was further formylated by hexamethylenetetramine?HMTA?using trifluoroacetic acid?TFA?as solvent to conveniently and efficiently produce the corresponding aldehyde III-3.The condensation of intermediate III-3 and cyanoethyl nitroimidazole III-5,which was readily prepared from 2-methyl-5-nitroimidazole,could afford compound III-6 in the presence of piperidine,and the further O-alkylation of hydroxyl compound III-6 with various aliphatic and aromatic halides was performed to achieve target compounds III-7-8.?4?All the newly synthesized compounds were characterized by ¹H NMR,¹³C NMR,IR,MS and HRMS spectra.?5?The in vitro antimicrobial evaluation displayed that some target molecules exhibited moderate to good inhibitory activities against the tested bacteria and fungi including clinical drug-resistant strains,which were isolated from infected patients.Particularly,berberine nitroimidazolyl derivative II-5a not only gave strong antibacterial and rapid bactericidal activity against drug-resistant E.faecalis in the exponential phase,with MIC value of 0.002 mM,172-fold more active than clinical norfloxacin,but also exhibited low toxicity toward RAW 264.7 cells and less propensity to trigger resistance.The drug combination study suggested that compound II-5a displayed synergistic effect with clinical drugs norfloxacin and berberine.The further exploration of antibacterial mechanism revealed that the highly active compound II-5a could effectively intercalate into DNA isolated from resistant E.faecalis to form II-5a-DNA complex,which might block DNA replication to show the powerful bioactivities.?6?The antimicrobial evaluation showed that some target molecules exhibited moderate to good inhibitory activities against the tested bacteria and fungi including clinical drug-resistant strains isolated from infected patients.Especially,2-fluorobenzyl derivative III-8f not only gave strong activity against drug-resistant E.coli with MIC value of 0.003 mM,33-fold more active than norfloxacin,but also exhibited low toxicity toward RAW 264.7 cells and less propensity to trigger resistance.The aqueous solubility and ClogP values of target compounds were investigated to elucidate the structure-activity relationships.Molecular docking and quantum chemical studies for compound III-8f rationally explained its antibacterial effect.The further exploration of antibacterial mechanism revealed that the highly active compound III-8f could effectively permeabilize E.coli cell membrane and intercalate into DNA isolated from resistant E.coli to form III-8f-DNA complex that might block DNA replication to exert the powerful bioactivities.Compound III-8f could also selectively address resistant E.coli from a mixture of various strains.In this thesis,the small library of berberine derived azoles was constructed,and thirty four compounds were synthesized.The antimicrobial evaluation indicated that 27compounds exerted better bioactivities than berberine and 24 compounds exhibited stronger antibacterial effects than clinical norfloxacin and 22 target compound showed better antifungal activities than fluconazole.