Synthesis, Inhibition and In-Silico Studies of Carbonic Anhydrase Inhibitors

Abstract

Synthesis, Inhibition and In-Silico Studies of Carbonic Anhydrase Inhibitors Carbonic anhydrases are zinc-metalloenzymes that catalyze reversible hydration of carbon dioxide and bicarbonate ion inside living organisms at the rate of 104 – 106 s -1 . They perform numerous physiological functions such as blood buffering and homeostasis, respiration, electrolytes secretion, body fluids production, renal reabsorption, bone resorption and calcification, gustation and olfaction, and involved in several metabolic pathways. Carbonic anhydrases are categorized into eight distinct (genetically different) families which includes α, β, γ, δ, ζ, η, θ and ι carbonic anhydrase. There are sixteen different isozymes of α-CAs, present in human being with varying degree of catalytic activity, subcellular localization, expression level and tissue distribution. Inhibition of CAs have high therapeutic importance in various physiological disorders such as epilepsy, glaucoma, pulmonary edema, obesity, hypoxic tumors and bacterial infections. To increase therapeutic potential and drug likeness of carbonic anhydrase inhibitors (CAIs), research is focused to design isozyme selective inhibitors. As the active sites of most of the CAs are highly conserved therefore, it is difficult to synthesized isozyme selective CAIs. Recently, several CA isozymes selective inhibitors have been reported by applying different techniques including “Tail” approach. The aim of present research work was the discovery of new potent and selective inhibitors of CAs by applying in-vitro and in-silico studies. Keeping in view, we designed and synthesized novel benzylaminoethylureido benzenesulfonamide derivatives by applying “Tail” approach, with variety of functional groups substitutions present on the ring structure and tail. These substitutions located on para- and meta- positions with respect to benzenesulfonamides warheads. The inhibition potential and selective nature of resultant compounds were determined against physiologically dominant, cytosolic human isozymes hCA I and hCA II, and trans-membrane tumor associated human isozymes hCA IX and hCA XII. Selective inhibition of these compounds was observed towards hCA II, with most active compounds having Kis of 2.8 - 9.2 nM, respectively as compared to acetazolamide with Ki of 12 nM. Compound 34; 3-(3-(2-((4-bromo-2- xii hydroxybenzyl)amino)ethyl)ureido) benzenesulfonamide, was found most active (Ki = 2.8 nM) and selective inhibitor of hCA II. X-ray crystallographic analysis of these sulfonamide derivatives in complex with hCA I and molecular docking studies with hCA II and hCA IX provided understanding of their binding mode and selective nature. Unveiling the selective nature of these compounds towards hCA II, molecular docking studies indicated that they interact with unique residues in hCA II active site. Inhibition potential of benzylaminoethylureido benzenesulfonamides were also investigated against bacterial CAs including Vibrio cholerae carbonic anhydrases (VhCA α, VhCA β and VhCA γ) and Burkholderia pseudomallei carbonic anhydrases (BpsCA β and BpsCA γ). Selective inhibitory profile towards VhCA γ and BpsCA β was observed. The most active compounds with inhibition constants (Ki) in the range of 82.4-97.5 nM were observed against VhCA γ and inhibition constants (Ki) ranging between 394.0-742.8 nM, were observed against BpsCA β as compared to acetazolamide with Ki of 473 and 745 nM, respectively. ADMET profile and Drug like properties of these compounds were assessed through SwissADME and PreADMET online tools which revealed excellent drug-like properties of these compounds according to provided international standards and may qualify for becoming LEAD compounds. Inhibition profile of 221 synthetic compounds belonging to different series of functional groups, obtained from various sources (including but not limited to synthetic chemistry labs of COMSATS University Islamabad, Abbottabad Campus) were investigated by in-vitro high-throughput library screening. Several phenols, oxadiazoles, hydrazides and tin-based compounds showed IC50 values in the range of 98 to 400 μM. Best inhibitor of CA II was (4-(3-hydroxyphenyl)-6-phenyl-2-thioxo 1,2,3,4-tetrahydro-pyrimidin-5-yl)phenyl)methan-one with IC50 value of 98.0 μM. Molecular docking studies of phenolic compounds from Sclerochloa dura represented excellent inhibition potential against hCAs indicating anchoring of -OH groups of sulfonic acid and glucose moiety with zinc-bound water molecule. Similarly, molecular docking studies of anti-proliferative agents; triterpenic monomers and dimers, also represented excellent inhibition potential against hCA IX. Here, several CAIs have been reported with varying degrees of CAs inhibition but more interestingly novel benzylaminoethylureido benzenesulfonamides represented xiii more selectivity towards hCA II as compared to hCA I, IX and XII and more potency as compared to standard inhibitor acetazolamide. On the other hand, they were more potent and selective towards VhCA γ and BpsCA β of pathogenic bacteria. Our results indicated that these compounds can be potential lead compounds to discover new drugs for the treatment of human and bacterial diseases related to carbonic anhydrases.