Design And Synthesis Of Novel Pyrimidine Azole Derivatives And Their Antifungal Evaluation

The high morbidity and mortality of fungal infections have attracted people’s attention.Many clinical drugs are used for the treatment of fungal infections,but the misuse and abuse of antibiotics,the increase of the number of immunosuppressive individuals as well as the emergence of new species of fungi led to serious drug resistance,which made the existing drugs become more limited.Therefore,novel antifungal agents are urgently needed.Pyrimidine derivatives account for a large proportion in both natural and synthetic substances.In recent years,due to the importance of pyrimidines in pharmaceutical chemistry,people have done a lot of pharmacological studies on pyrimidines and their derivatives,especially in antifungal field.Pyrimidines act as antifungal agents by blocking the synthesis of nucleic acids,DNA and RNA.Azoles are widely used in clinical antifungal drugs.They can bind to biological macromolecules through non-covalent bonding,thus interfering with the integrity of fungal cell membranes and hindering DNA synthesis.However,the continuous emergence of drug resistance has brought severe challenges to the clinical use of azole drugs,which has promoted the structural modification of azoles.On the basis of the antimicrobial research progress of pyrimidine and azole recent years in our group,this research designed and synthesized a series of novel pyrimidine azoles.This work evaluated antifungal activity,druggability and preliminary antifungal mechanisms.The main research work is given as follows:1.The development of pyrimidines in antifungal fields,including pyrimidones,aminopyrimidines,mercaptopyrimidines and fused pyrimidines was reviewed,and the research trend of pyrimidines was prospected.2.Synthesis of novel pyrimidine azole compounds:（a）Preparation of novel pyrimidone imidazole alkenes:Starting with barbituric acid and 2-butyl-4-chloro-5-imidazolaldehyde,pyrimidone imidazole alkenes intermediate Ⅱ-2 was obtained by condensation of aldehydes in ethanol catalyzed by triethylenediamine.Target pyrimidone imidazole alkenes Ⅱ-3-5 were obtained by nucleophilic substitution of the intermediate Ⅱ-2 with a series of halogenated compounds.（b）Preparation of novel cyclamine purine thiazoles:Acetyl thiazole Ⅲ-1 was brominated in glacial acetic acid to produce bromide Ⅲ-2.Intermediate Ⅲ-2 was treated with potassium carbonate and reacted with 6-chloropurine Ⅲ-3 in acetonitrile to produce the intermediate purine thiazole Ⅲ-4.Intermediate Ⅲ-4 was reacted with alicyclic amine by catalyst triethylamine in ethanol to produce the corresponding target cyclamine purine thiazoles Ⅲ-6a-d.Intermediate Ⅲ-4 was reacted with piperazine to form piperazine purine thiazole Ⅲ-5,which was then treated with potassium carbonate in acetonitrile through nucleophilic substitution with the halogenated compounds to produce the corresponding target piperazine purine thiazoles Ⅲ-7-11.（c）Preparation of novel purine thiazole oximes:Starting with 6-chlorine purine and piperazine in ethanol reaction,triethylamine as catalyst,and piperazine purine IV-2was obtained.Intermediate IV-2 was reacted with thiobenzothiazole ester catalyzed by triethylamine in ethanol to produce purine thiazole oxime intermediates IV-3.The intermediate IV-3 was reacted with different halogenated compounds by nucleophilic substitution reaction to form a series of corresponding purine thiazole oximes IV-4-6.3.The structures of all new compounds were confirmed by ¹H NMR,¹³C NMR,and HRMS.The purity of some compounds was determined by HPLC.4.The in vitro antifungal activity of the target compounds was tested according to clinical standard test（CLSI）.Further research has shown that some target pyrimidine azoles could significantly inhibit the growth of fungi.In series Ⅱ pyrimidinone imidazole alkenes,some of the compounds such as butyl Ⅱ-3b,allyl Ⅱ-4a,and propargyl Ⅱ-4b showed excellent activity,especially 4-fluoro-benzyl pyrimidinone imidazole alkene Ⅱ-5f displayed the best activity against Candida albicans with MIC value of 1μg/m L,being better than the reference drug fluconazole（MIC=4μg/m L）.In series Ⅲ cyclamine purine thiazole compounds,most of the compounds exhibited good activity against Candida albicans,among which the ethyl acetate piperazine purine thiazole derivative Ⅲ-8c showed good activity against Candida albicans（MIC=1μg/m L）,being better than fluconazole.In series IV purine aminothiazoloxime compounds,m-chlorine and p-chlorobenzylpurine-thiazoloxime IV-6a and IV-6b showed good activity,and the MIC value of p-nitrobenzyl derivative IV-6h against Candida tropicalis reached 1μg/m L,which was better than reference drug fluconazole（MIC=8μg/m L）.Compounds Ⅱ-5f,Ⅲ-8c and IV-6h showed potential antifungal applications,so they were further investigated.5.Druggability study:（a）Development of fungal drug resistance:After 16 days of subculture,the study found that compounds Ⅱ-5f,Ⅲ-8c and IV-6h were more difficult to induce the development of drug resistance towards Candida strains than the reference drug fluconazole.（b）Fungicidal kinetics:The highly active molecules Ⅱ-5f and Ⅲ-8c showed a rapid and more significant effect on Candida albicans than the reference drug fluconazole.（c）Cytotoxicity tests:Compounds Ⅱ-5f and Ⅲ-8c showed low blood toxicity and low hemolysis rate.Compound Ⅱ-5f showed low cytotoxicity to human cervical cancer cells（He La）and human embryonic kidney cells（HEK-293T）.Compound Ⅲ-8c showed low cytotoxicity to human normal embryonic hepatocytes（LO2）.This suggested that the highly active compounds could selectively inhibit the growth of the fungi.（d）Drug combination study:Compound Ⅲ-8c combined with fluconazole showed synergistic effect on Candida albicans and additive effect on Candida tropicalis.6.Antifungal mechanism study:（a）Fungal cell wall destruction:Compound Ⅲ-8c could destroy the fungal cell wall,thus showing good inhibitory action.（b）Damage to fungal cell membrane:Compound Ⅱ-5f,Ⅲ-8c and IV-6h could increased permeability of cell membrane.（c）Interactions with DNA:Compound Ⅱ-5f could be tested by UV-Vis spectroscopy for its interaction with calf thymus DNA.Further studies showed that compound Ⅱ-5f could intercalate into DNA and cause the unhelix of DNA double strands,thus impeding the normal replication of DNA.（d）Molecular docking with CYP51:Compound Ⅱ-5f and IV-6h could form supramolecular interaction with 14α-demethylation enzyme CYP51,thereby inhibiting the synthesis of ergosterol,the main component of fungal cell membrane,thus inhibiting the growth of fungi.（e）Oxidative stress:Compound Ⅲ-8c could cause oxidative imbalance in Candida albicans,increase of ROS and RNS,decrease of GSH activity,eventually leading to oxidative damage and fungal cell death.（f）Fungal cell metabolism:Compound Ⅲ-8c and IV-6h impeded normal metabolism of cells.In this thesis,a total of 76 compounds were synthesized.Among them,69compounds are new,including 66 target compounds:19 pyrimidone imidazoles,31cyclamine purine thiazoles and 16 purine-thiazoximes.The antifungal activities of some compounds were stronger or equipotent compared to the reference drug fluconazole.The highly active compounds with low toxicity hardly developed drug resistance,showed efficient fungicidal ability,interfered the membrane integrity,impeded DNA replication,caused oxidative damage and hindered the normal metabolism.Therefore,these highly active compounds are worthy of further study as candidates for clinical antifungal drugs.