Structure-based Design,Synthesis And Biological Evaluation Of Novel Carbonic Anhydrase Inhibitors

Carbonic anhydrases(CAs)are ubiquitous zinc metalloenzymes that catalyze a very simple physiological reaction,the interconversion between carbon dioxide and the bicarbonate ion.They play relevant roles in a large series of physiological processes such as intracellular and extracellular pH homeostasis,cell proliferation and differentiation,bone resorption,calcification and a variety of biosynthetic reactions(such as gluconeogenesis,lipogenesis,and ureagenesis).The research in this dissertation focus on the design,synthesis and bioactivity evaluation of novel CA inhibitors based on the enzyme structure,to find potent lead compounds for antitumor or antiglaucoma use.Carbohydrate-based sulfonamides are kind of artificial glycoconjugates which can offer new CA inhibitors as therapeutic agents.According to the summary of the progress on CA inhibitors during the past decades,we carried out the structure-based design of novel inhibitors,prepared series of compounds,tested their bioactivity in vitro and in vivo,and evaluated their potential clinic value of antitumor or reducing intraocular pressure.The work in this dissertation consists of three parts.(?).Dual-tail approach was employed to design novel CA IX inhibitors by simultaneously matching the hydrophobic and hydrophilic halves of the active site,which also contains a zinc ion as part of the catalytic center.? The classic sulfanilamide moiety was used as the zinc binding group.An amino glucosamine fragment was chosen as the hydrophilic part and a cinnamamide fragment as the hydrophobic part in order to draw favorable interactions with the corresponding halves of the active site.Therefore,compounds 27a-27g were synthesized and characterized by HRMS,1H NMR and 13C NMR spectra.In comparison with sulfanilamide which is largely devoid of the hydrophilic and hydrophobic interactions with the two halves of the active site,these compounds showed 1000-fold improvement in binding affinity.Most of the compounds inhibited the CA effectively with IC50 values in the range of 7 nM-152 nM.The results proved that the dual-tail approach to simultaneously matching the hydrophobic and hydrophilic halves of the active site by linking hydrophobic and hydrophilic fragments was useful for designing novel CA inhibitors.The effectiveness of these compounds was elucidated by both the experimental data and molecular docking simulations.? In order to improve the inhibitory activity of CA IX,a new round of dual-tail small molecule inhibitors was designed.The classic sulfanilamide moiety was kept as the zinc binding group.A glucuronic acid fragment was chosen as the hydrophilic part and a coumarin fragment as the hydrophobic part in order to draw favorable interactions with the corresponding halves of the active site.Accordingly,compounds 39a-39x were prepared,whose structures were identified by HRMS,1H NMR and 13C NMR spectra.The in vitro CA IX inhibition tests showed these compounds presented 10,000-fold improvement in binding affinity.Eight of the compounds were low nanomolar inhibitors.Furthermore,the antitumor activity of these compounds was also evaluated under two different conditions.Compared to reference drug acetazolamide,compounds 39a-39h showed much increased antiproliferation activity.The work within this area laid a solid foundation for future development of novel and efficacious CA IX inhibitors for cancer treatment.(?).Based on the analysis of the advantages and disadvantages of antiglaucoma agents clinically used nowadays or under reseach,we carried out the expedient synthesis of sulfonamide glycoconjugates.? The novelity of idea is that the carbohydrate tail is linked directly to the benzene sulfonamide fragment via an amide bond.15 novel compounds were prepared and all the target compounds were identified correctly by 1H-NMR,13C-NMR and HRMS.Adjusting the length of hydrophobic chain on the anomeric center of the sugar is helpful to offer lead compounds which own both good water solubility and good corneal permeability.Isozyme CA II was strongly inhibited by some of these compounds,which proved their good selectivity.In hypertensive rabbit model of glaucoma tests,compound 72a showed strong activity of reducing intraocular pressure(IOP),being equivalent to brinzolamide,the clinically agent ? Compounds 78a-78e were prepared by replacement of benzene ring with thiadiazoles.The presence of thiadiazoles can enhance the water solubility compared to compound 72a.In hypertensive rabbit models,compound 78a showed strong and long-lasting property of reducing intraocular pressure(IOP),more effective than brinzolamide.(?).To develop a general strategy for the synthesis of certain glucuronic acid derivatives,a systematic investigation on selective deprotection was carried out.Accordingly,an exceptionally selective condition was found for removing the C6 methyl protecting group by potassium hydroxide in acetone without affecting the benzoyl protecting groups on the C2,C3 and C4 hydroxyl groups in high yields(95-99%).To verify the applicability of this protocol,we tested seven types of different substrates.The high yields up to 99%verified the feasibility and efficiency of this protocol.Therefore,an efficient and reliable procedure was set up for the synthesis of C6-carboxyl glucuronyl acid and its application in the preparation of corresponding glycosyl compounds.Given other advantages of this protocol,including its mild conditions,short reaction time,lower cost and easy operation,it provides rapid access to biologically relevant carbohydrates in a labor-saving and economical manner.In summary,CA has been proved to be one of the effective targets for developing novel antitumor or antiglaucoma drugs.The work in this dissertation aims to achieve novel CA inhibirtors with high affinity and specificity.Series of novel carbohydrate-based CA inhibitors were designed on the basis of the latest released principle,and synthesized via appropriate strategy.Results from this research will afford new and significant approach to CA inhibitors,and also will be beneficial to provide new scientific idea for finding other CA inhibitor leads.