Synthesis And Anti-tuberculosis Activity Of New Isatin Hybrids

Tuberculosis（TB）is still a major public health problem that poses a serious threat to human life and health.About 10 million people contract TB each year,and millions die.At present,the first-line anti-tuberculosis drugs used in clinical practice mainly include isoniazid,rifampicin,ethambutol,pyrazinamide,etc.,which once had a very high cure rate for drug-sensitive tuberculosis patients.But the evolution of MTB new virulent forms such as drug-resistant TB（DR-TB）and multidrug-resistant TB（MDR-TB）making these drugs more and more ineffective.Therefore,new anti-TB agents are urgent needed to control this plague.Compared with the corresponding single pharmacophore drug molecules,heterozygous molecules often have the advantages of higher biological activity,lower toxic side effects,broader antimicrobial spectrum and lower drug resistance.Isatin has a variety of biological activities.In this thesis,heteronuclear and homonuclear isatin dimers hybrids、isatin-triazole-coumarin hybrids and ciprofloxacin-isatin hybrids connected by different linkers at the N-1 position were prepared by substitution reaction.The anti-tuberculosis activity and the cell toxicity of the substituent on the C-3 and C-5 of isatin molecular structure and the linkers between two isatin nuclei on anti-tuberculosis activity had been investigated.The chemical structure of the hybrids was tested by ¹H NMR,¹³C NMR,SEI-MS and HR-MS.The tuberculosis activity were studied by testing the Minimal Inhibitory Concentration,and determined their cytotoxicity.The results showed that,The antituberculosis activity of series I isatin dimers hybrids monosubstituted at the C-3 position of isotin unit was generally superior to that of series I isatin dimers hybrids disubstituted at the C-3 position of isotin unit.The sequence of influence of substituents at the C-3 position of isotin unit investigated on anti-tuberculosis activity was-NNHCSNH2>-O>-NOMe>-NOEt>-NOH.The anti-tuberculosis activity of isatin dimers hybrids was improved by-F at the C-5 position of isotin unit.The structure activity relationship showed that the anti-tuberculosis activity was greatly influenced by substituents at C-3 position and C-5 position of isotin unit.The sequence of influence of substituents at the C-3position of isotin unit investigated on the anti-tuberculosis activity was-NNHCSNH2>-NNHCONH2>-NOEt>-O>-NOH.The reason that the anti-tuberculosis activity of thioureido imino group at C-3 position of isotin unit was greater was chelation of thiourea imino group with metal ions,S atoms as the active site of thioureas alkylation and the cumulative effect of imino as the target of the complexes.The sequence of influence of substituents at the C-5 position of isotin unit investigated on the anti-tuberculosis activity was-F>-H>-Me.The electro-withdraw substituents at C-5 position of isotin unit had greater effect on improving the anti-tuberculosis activity than the electro-donating substituents at C-5position of isotin unit.The anti-tuberculosis activity of the heteronuclear isatin dimers hybrids was higher than that of homonuclear isatin dimers hybrids.The sequence of influence of substituents at the C-3 position of isotin unit investigated on the anti-tuberculosis activity was-NNHCONH2>-NOMe>-NOEt>-O>-NOH.The sequence of influence of substituents at the C-5 position of isotin unit investigated on the anti-tuberculosis activity was-F>-H>-Me.The sequence of influence of substituents was the same as the homonuclear isatin dimers hybrids.The anti-tuberculosis MTB H37Rv and MDR-TB activity of the heteronuclear isatin dimers hybrids III-3 and VIII-4 were the best,which was 2～>8 times of the anti-tuberculosis MDR-TB activity of rifampicin and isoniazid.The anti-tuberculosis MTB H37Rv and MDR-TB activity of the homonuclear isatin dimers hybrids V-5was the best,which was>4 times of the anti-tuberculosis MDR-TB activity of rifampicin and isoniazid.The structure-toxicity relationship of each series showed that when-F substituted at the C-5 of isatin unit,the cytotoxicity was significantly increased.The CC50 concentration of V-5 was 15.6μg/m L,and the CC50concentration of VIII-4 was 62.5μg/m L.The anti-tuberculosis activity of the tetraethylene glycol-linked hybrids was higher than that of the rigid ethylidene-linked hybrids,because the ether bond on the tetraethylene glycol could form non-covalent bond interactions such as hydrogen bond.Twenty isatin-triazole-coumarin hybrids were synthesized using 1,2,3-triazole as linkers.The structure was confirmed by ¹H NMR、¹³C NMR and ESI-MS.The anti-tuberculosis activity of 20 isatin-triazole-coumarin hybrids was tested by Minimal Inhibitory Concentration,and cytotoxicity was also tested.The results showed that,the linker between isatin and triazole had obvious effect on cytotoxicity.The cytotoxicity of long-chain propylidyne-linked hybrids was lower than that of corresponding ethylidene-linked hybrids,that was to say prolonging the linker’s length could reduce cytotoxicity.The electron-absorbing substituents-F and-Cl at the C-5 position of isotin unit were beneficial to improve the anti-tuberculosis activity of the hybrids,and the cytotoxicity of the hybrids was also significantly increased.The structure activity relationship showed that the electron-withdrawing substituents at C-5 position of isotin unit was beneficial to improve the anti-tuberculosis activity than the electron-donating substituents.Although the anti-tuberculosis activity of these hybrids against MTB H37Rv was much lower than that of the first-line anti-tuberculosis drug rifampicin and isoniazid,the anti-tuberculosis activity of some hybrids against MDR-TB was better than that of the reference drugs,and the cytotoxicity was lower.Among them,the hybrid XIV-2 was not only less toxic（CC50:128μg/m L）,but also had the same activity（MIC:50μg/m L）to MDR-TB as rifampicin,and was>2 times of isoniazid.It had no cross resistance to first-line anti-tuberculosis drugs,and could be further optimized as a lead compound.The strategy of introducing isatin into ciprofloxacin at C-7 of isotin unit could undoubtedly improve the lipophilicity,which may further improve the anti-tuberculosis activity.Based on this,novel ciprofloxacin-isatin hybrids XVI-1～6and ciprofloxacin-bis-isatin hybrids XVII-1～6 were designed and synthesized,and the structure of the hybrids was confirmed by ¹H NMR、¹³C NMR and ESI-MS.The anti-tuberculosis activity of the hybrids was tested by Minimal Inhibitory Concentration,and cytotoxicity were also tested.The results showed that,the anti-tuberculosis activity of ciprofloxacin-isatin hybrids XVI-1～6 was better than that of ciprofloxacin-bis-isatin hybrids XVII-1～6.The structure activity relationship showed that alkyl hydroxamic substituent at C-3 position and electron-donating substituent-Me at C-5 position of isatin unit was not beneficial to the anti-tuberculosis activity.The Minimal Inhibitory Concentration of the hybrid XVI-2to MTB H37Rv and MDR-TB was 0.1μg/m L and 0.5μg/m L,the activity to MTB H37Rv was 4 and 8 times that of the parent drug ciprofloxacin and rifampicin,and the activity to MDR-TB was 4～>256 times that of ciprofloxacin,rifampicin and isoniazid.Furthermore,the cytotoxicity（CC50:64μg/m L）of the hybrid was low and had the potential for further study.The research work of this thesis not only synthesized the heteronuclear isatin dimers hybrids,selected multiple potential anti-tuberculosis activity especially to multi-drug resistant mycobacterium tuberculosis activity hybrids,but also enriched the structure-activity relationship of isatin dimers hybrids,isatin-coumarin hybrids and ciprofloxacin-isatin hybrids,which provided theoretical reference for further rational design.To obtain anti-tuberculosis activity drugs that can be used clinically,further optimization on the molecular structure of the selected drugs with high anti-tuberculosis activity should be carried out to gain access to clinical research and contribute to the development of new anti-tuberculosis drugs.