Discovery Of Anti-HIV-1 And Anti-tumor Lead Compounds From Click Chemistry And Privileged Sturcture-based Combinatorial Library

Fragment-based rapid assembly using the facile Cu(?)-catalyzed alkyne-azide cycloaddition(CuAAC)click chemistry and rapid screening are emerging as a highly robust and efficient tool to identify novel bioactive inhibitors quickly against a number of biological targets.The resulting 1,4-disubstituted 1,2,3-triazoles are similar to amide bonds in terms of distance and planarity,and often used as a scaffold in the discovery of enzyme inhibitors.In this paper,using this methodology,structurally diverse compound libraries were constructed and screened with the aim to develop novel potent inhibitors against HIV-1 infection and malignant tumors.This work could be divided into the following three parts.Discovery of Novel HIV-1 Capsid Protein Inhibitors from Click-Chemistry-based Privileged Fragment Combinatorial LibraryHIV-1 capsid protein(CA)is one kind of essential structure protein that shields the genome materials that are critical to HIV-1 infection.Meanwhile,stability and integrity of CA are critical to the normal life cycle and infectivity of the virus particle.Therefore,HIV-1 CA has become an attractive and novel target for HIV-1 therapy.In the second chapter,taking the most widely studied HIV-1 CA inhibitor PF-74 as lead compound,according to its binding mode with target,the phenylalanine core skeleton in the structure is retained,the substituents are modified by CuAAC reaction.Finally,the 71-member 1,2,3-triazole containing combinatorial library are constructed by parallel synthesis.These newly synthesized compounds were assayed in vitro for their anti-HIV-1 activities and cytotoxicity in MT-4 or TZM-bl cells.The results demonstrated that most compounds exhibited moderate anti-HIV-1 activities.In particular,IA-13m(EC50=4.33 ± 0.83 ?M)turned out to be the most potent inhibitor against HIV-1 NL4-3virus,which was superior to that of PF-74(EC50 = 5.95 ± 1.32 pM).Surface plasmon resonance(SPR)binding assays showed that IA-13m interacted robustly with recombinant HIV-1 CA.Besides,it was demonstrated that IA-13m exhibited antiviral activity in both the early(integration,IC50 = 7.0 ± 0-8 ?M)and late stages(assembly,pre-integration,and infection,IC50=31±11?M)of HIV-1 replication,which was similar to that of PF-74.Furthermore,the molecular dynamics stimulation was conducted to postulate inhibitor-protein interactions to account for the experimental results.Overall,IA-13m could act as a novel lead compound for further optimization.Discovery of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors Basedon Click-Chemistry-Based Micro Synthesis and In Situ Screening TechniqueHIV-1 nonnucleoside reverse transcriptase inhibitors(NNRTIs),when used in combinations in the highly active antiretroviral therapy(cART),have proven to be highly effective in reducing AIDS-related mortality throughout the world.However,the development of drug resistance and toxic side effects associated with cART have clearly underline a pressing need for innovative NNRTIs with distinct resistance profiles and safety.In the third Ghapter,based on an established common pharmacophore of the second-generation FDA-approved NNRTIs Etravirine and Rilpivirine,a 280-member triazole library was assembled,by reacting alkyne core fragments with diverse azide building blocks using CuAAC reaction.Screening of these triazole derivatives against RT led to the identification of 10 potent hit compounds(C2N38,C5N19,C5N36,C5N39,C6N34,C6N36,C2N37,C6N38,C6N39 and C6N49,IC50:0.02?0.30 ?1M),with equipotent to or more potent inhibitory activity than ETV(IC50 0.15±0.15?M).Then,these hit compounds were prepared at the milligram level.At present,their antiviral activity evaluation in MT4 cells are being carried out.Discovery of Novel CDC25 Protein Phosphatases Inhibitors Based onClick-Chemistry-Based Micro Synthesis and In Situ ScreeningThe cell division cycle 25(CDC25)family of proteins is a group of highly conserved dual-specificity phosphatases.The CDC25 protein phosphatases represents an attractive therapeutic target in oncology.In the fourth chapter,the click chemistry and in situ screening technology was used to discovery of novel CDC25 protein phosphatase inhibitors for cancer therapy.Taking the widely reported CDC25 inhibitors(NSC 663284,NSC 668394 and Cpd5)as lead compounds,the common p-benzoquinone was selected and modified to be alkyne-bearing privileged fragments,then diverse azide azide-modified peripheral substituents were installed rapidly on this scaffold using CuAAC click chemistry.Then,a 96-member 1,2,3-trizole-1,4-naphthoquinone/quinolinedione library was constructed,followed by direct biological screening for the identification of potential'hits'.The preliminary screening results demonstrated that M2N1,M2N2 and M2N12 showed effective inhibitory activity on CDC25A,CDC25B and CDC25C.Then the three potent compounds were prepared at the milligram level and the secondary screening results demonstrated that M2N12 exhibited the most significant inhibitory activity on CDC25C(IC50= 0.09 ?M),which was about 9-fold potent than that of NSC 663284(IC50=0.76?M).And the inhibitory effect of M2N12 on CDC25C was about 6-fold and 15-fold potent than that on CDC25A(IC50 = 0.53?M)and CDC25B(IC50 = 1.39 ?M),respectively.In addition,their activity on tumor cells in vitro were evaluated by SRB assay.The results indicated that the activity of M2N1,M2N2 and M2N12 on human alveolar basal epithelial cells(A549),human oral epidermoid carcinoma cells(KB)and human breast cancer cells(MCF-7/MDA-MB-231)were less potent than those of paclitaxel(PXL)and NSC 663284.Particularly,M2N12(IC50 = 6.81 ?M)showed a significant inhibitory effect on drug-resistant human oral epithelial cancer cells(KB-VIN)comparable to that of PXL(IC50 = 3145.83 nM)and NSC663284(IC50 = 4.87?M),which can be selected as a lead compound for further development.It should be noted that,the structure of alkyne-bearing key intermediate M2 was confirmed by X-ray diffraction.Altogether,viral capsid protein,reverse transcriptase and human CDC25 phosphatase,which are critical to HIV-1 life cycle and tumor proliferation respectively,were considered important therapeutic targets.In this thesis,fragment-based rapid assembly using the facile CuAAC click chemistry and rapid screening is used as an efficient tool to identify novel bioactive inhibitors against these biological targets.As a result,several potential HIV-1 CA inhibitors,RT inhibitors and CDC25 phosphatase inhibitors with high potency were identified as promising lead compounds through rapid screening of the constructed combinatorial libraries.We envisioned that this universal mothodology would greatly contribute to the discovery of more bioactive molecules with other therapeutic applications.