Novel Carbohydrate-based Carbonic Anhydrase ? Inhibitors:Design,Synthesis And Biological Activity Research

Carbonic Anhydrases ??CA ??is a tumor-related isoform enzyme which overexpresses in a large variety of hypoxia cancer cells and restrictedly expresses in normal cells.This enzyme plays crucial roles in pH regulation,tumor cell proliferation,adhesion and malignant metastasis,so has been considered as a promising antitumor therapeutic target.The classical fragment for CA inhibitors is a sulfonamide moiety?-SO₂NH₂?which is served as a very reliable anchor with Zn²⁺ in the active site of CA ?.Appending different substitutions on this group has offered an effective method for finding new inhibitors with improved potency and selectivity.It is demonstrated that carbohydrate-based modifications led to highly success on targeting CA?.Furthermore,as the sugar moiety impairs the ability of the glycoinhibitors to diffuse the lipid membranes,the glycosylation may facilitate the preferential inhibition of transmembrane CA ? over the cytosolic isoforms.In addition,certain of special sugar blocks may help to target the tumor cells in the drug delivery systems.This dissertation introduces five series of novel carbohydrate-based derivatives as CA ? inhibitors with high activity,double selectivity between CA ? and its isoforms,and between tumor cells and normal cells.Seventy-three target compounds,together with fifteen intermediates,were characterized by-H NMR,13C NMR and HR-MS?ESI?.All target compounds were assessed for the inhibition on CA isozymes and antiproliferation activities against tumor cell lines.Moreover,the values of cLog P and topological polar surface area?TPSA?of all the compounds and possible binding modes between CA ? and synthesized derivatives were predicted by the method of computer aided drug design.Combining aryl-thiourea tail,glycosyl-thiourea moiety and 6-sulfamoyl-glucopyranose and replacing sulfonamide group to its bioisostere carboxyl group,LA series of compounds were designed,synthesized and investigated for their abilities to inhibit CA?.The inhibition rate showed that only benzenesulfonamide substituted analogs 5h and 6h possessed comparable or better activity than the positive control drug acetazolamide?AZA?and the remaining derivatives had no activity on CA ?.It is obvious that the zinc-binding group is sulfonamide rather than carboxyl group.Besides,the unmarked glycoconjugate 6h functioned selectively on CA? over CA?.According to the activity results of LA series,a collection of thiourea-bridged monosaccharide containing benzenesulfonamide which is used as zinc-binding group were designed and synthesized.The unmarked sugar derivatives showed better selectivity than AZA.Compound 11a-11c and 11f showed better inhibition against CA ? than the positive control.The acetylation on monosaccharide led to reduced inhibition and preference on CA ?.Compared to deprotected pentose substituted group,hexose substituted compounds were more effective.The glucose substituted inhibitor 11a was docked with CA ? structure,and six hydrogen bonds formed were found.The selectivity on CA ? of deprotected glycoinhibitors was elucidated more affinities among the cancer-revlevant isoforms while less interactios with CA?.The cLog P results implied that acetyl group protected carbohydrate-based sulfonamides had potential as prodrugs to facilitate the lipophilicity.TPSA value-based predictions highlighted the selectivity of these carbohydrate-based inhibitors for membrane-associated CA ?.Choosing compound 11a as the lead,we developed the LC seises of compounds?19a-19r?by replacing glucose with glucosamine and introducing the substituted sulfonyl onto the 2-amino of glucosamine to simultaneously form the secondary sulfonamide group to enhance the interactions between inhibitors and CA ?.Meanwhile,by appending the acetyl and trichloroethoxycarbonyl?Troc?in the 2-amino of glucosamine and changing the linker length,derivatives 16a-16c,23a-23c and 24a-24c were also designed.The IC50 values revealed that some conjugates were more potent than AZA.Most of them showed better activities but non-selevtivity against CA ? in comparison with LB series.For the deprotected sugar analogues,the introduction of secondary sulfonamide can significantly increase the inhibition against CA ?,while for the acetyl on the hydroxyl of glycoconjugates,substituted by Troc group 16a-16c,showed preferable inhibition on CA ?.The change of linkage and the presence of acetyl on the hydroxyl of glycoconjugates were influential on CA ? inhibitory properties.It was demonstrated that the most potent compound,19f,showed more affinities with CA ?.Owing to introduce the lipophicity group to the glycoconjugates,the cLog P values displayed apparent improvement.Several compounds may exhibit desirable lipophilicity as their cLog P values range from 0.0785 to 1.0453.The TPSA results indicated that all derivatives with poor membrane permeabilities would enhance the selectivity on CA ?.To avoid the side effect of thiourea linker,LD series presented a new library of CA?inhibitors comprising aromatic sulfonamide and triazole-tethered carbohydrate tails by employing 1,3-dipolar cycloaddition.By using molecular docking method,we confirmed the linkage length and designed four triazolyl derivatives.The enzyme inhibition profiles suggested that the activities of these compounds were weaker than the positive control drug AZA and none of them are CA ? selective.The inhibition on CA ? was decreased as replacing the thiourea linker to triazole linker.The stereochemical diversity within the carbohydrate tails can affect inhibitory activity on CA ?.The binding modes between these compounds and CA ? showed that trizole group and carbohydrate moiety formed interactions with the amino acid residues of the active site.However,the hydrophobic interaction between trizole group and enzyme was weaker than the hydrogen bond between thiourea fragment of LB series and CA?,which led to the decrease of CA ? inhibition of LD series.Compounds 34a-34p were designed by replacing the linker of LC series into triazole.All of these sulfonamides were less effective CA ? inhibitors compared to AZA.And it was illustrated that the secondary sulfonamide group possessed increasing inhibition on CA ?.The substitution of secondary sulfonamide group had the impact on the activity.Molecular docking exhibited that in addition of the triazole group and carbohydrate moiety,the secondary sulfonamide also formed hydrophobic interactions with the amino acid residues of CA ?.With the replacement of linker,the cLog P values of LD and LE series were reduced,but the values of some analogs were still between 0 and 3.Values of TPS A were greater than 140 A2.It is possible for the designed compounds to exhibit limited membrane permeabilities and specifically target CA?.The antiproliferation activities were measured by MTT assay against three cancer cell lines?HT-29,MDA-MB-231 and HCT116?.All compounds with potent CA ? inhibition did not exhibit antitumor activities.This suggests the relationship between CA? and cancer is so complicated that further intensive pharmacological tests should be given sufficient consideration.