Effective Carbonic Anhydrase Inhibitors Research Articles

Exploring heterocyclic scaffolds in carbonic anhydrase inhibition: a decade of structural and therapeutic insights.

Heterocyclic compounds represent a prominent class of molecules with diverse pharmacological activities. Among their therapeutic applications, they have gained significant attention as carbonic anhydrase (CA) inhibitors, owing to their potential in the treatment of various diseases such as epilepsy, cancer and glaucoma. CA is a widely distributed zinc metalloenzyme that facilitates the reversible interconversion of carbon dioxide and bicarbonate. This reaction is essential for numerous physiological and pathological processes. In humans, CA exists in sixteen different isoforms, labeled hCA-I to hCA-XV, each distributed across various tissues and organs and involved in crucial physiological functions. Clinically utilized CA inhibitors, such as brinzolamide, dorzolamide and acetazolamide, exhibit poor selectivity, leading to undesirable side effects. A significant challenge in designing effective CA inhibitors is achieving balanced isoform selectivity, prompting the exploration of new chemotypes. This review compiles recent strategies employed by various researchers in developing CAIs across different structural classes, including pyrazoline, quinoline, imidazole, oxadiazole, pyrimidine, coumarin, chalcone, rhodanine, phthalazine, triazole, isatin, and indole. Additionally, the review summarizes structure-activity relationship (SAR) analyses, isoform selectivity evaluations, along with mechanistic and in silico investigations. Insights derived from SAR studies provide crucial directions for the rational design of next-generation heterocyclic CA inhibitors, with improved therapeutic efficacy and reduced side effects. To the best of our knowledge, for the first time, we have comprehensively summarized all known isoforms of CA in relation to various heterocyclic motifs. This review examines the use of different heterocycles as CA inhibitors, drawing on research published over the past 11 years. It offers a valuable resource for early-career researchers, encouraging further exploration of synthetic heterocycles in the development of CA inhibitors.

Exploring Penetration Ability of Carbonic Anhydrase Inhibitor-Loaded Ultradeformable Bilosome for Effective Ocular Application.

Brinzolamide is an effective carbonic anhydrase inhibitor widely used in glaucoma therapy but limits its application due to inadequate aqueous solubility and permeability. The aim of the present research work is the development and characterization of brinzolamide-loaded ultradeformable bilosomes to enhance the corneal permeation of the drug. These ultradeformable bilosomes were prepared by ethanol injection method and evaluated for physicochemical properties, particle size, morphology, drug release, ultra-deformability, corneal permeation, and irritation potential. The optimized formulation exhibited an average particle size of 205.4 ± 2.04nm with mono-dispersity (0.109 ± 0.002) and showed entrapment efficiency of 75.02 ± 0.017%, deformability index of 3.91, and release the drug in a sustained manner. The brinzolamide-loaded ultradeformable bilosomes released 76.29 ± 3.77% of the drug in 10h that is 2.25 times higher than the free drug solution. The bilosomes were found non-irritant to eyes with a potential irritancy score of 0 in Hen's egg-chorioallantoic membrane assay. Brinzolamide-loaded ultradeformable bilosomes showed 83.09 ± 5.1% of permeation in 6h and trans-corneal permeability of 8.78 ± 0.14cm/h during the ex vivo permeation study. The acquired findings clearly revealed that the brinzolamide-loaded ultradeformable bilosomes show promising output and are useful in glaucoma therapy.

Isoxazole Derivatives against Carbonic Anhydrase: Synthesis, Molecular Docking, MD Simulations, and Free Energy Calculations Coupled with In Vitro Studies.

Heterocyclic compounds with a five-membered ring as a core, particularly those containing more than one heteroatom, have a wide spectrum of biological functions, especially in enzyme inhibition. In this study, we present the synthesis of five-membered heterocyclic isoxazole derivatives via sonication of ethyl butyrylacetate with aromatic aldehyde in the presence of a SnII-Mont K10 catalyst. The synthesized compounds were characterized using sophisticated spectroscopic methods. In vitro testing of the compounds reveals three derivatives with significant inhibitory action against carbonic anhydrase (CA) enzyme. The compound AC2 revealed the most promising inhibitory activity against CA among the entire series, with an IC50 = 112.3 ± 1.6 μM (%inh = 79.5) followed by AC3 with an IC50 = 228.4 ± 2.3 μM (%inh = 68.7) compared to the standard with 18.6 ± 0.5 μM (%inh = 87.0). Molecular docking (MD) study coupled with extensive MD simulations (400 ns) and MMPBSA study fully supported the in vitro enzyme inhibition results, evident from the computed ΔGbind (AC2 = −13.53 and AC3 = −12.49 kcal/mol). The in vitro and in silico studies are also augmented by a fluorescence-based enzymatic assay in which compounds AC2 and AC3 showed significant fluorescence enhancement. Therefore, on the basis of the present study, it is inferred that AC2 and AC3 may serve as a new framework for designing effective CA inhibitors.

Synthesis and in vitro carbonic anhydrases and acetylcholinesterase inhibitory activities of novel imidazolinone-based benzenesulfonamides.

New imidazolinone-based benzenesulfonamides 3a-e and 4a-e were synthesized in three stepsand their chemical structures were confirmed by 1 H NMR (nuclear magnetic resonance), 13 C NMR, and high-resolution mass spectrometry. The benzenesulfonamides used were sulfacetamide (3a, 4a), sulfaguanidine (3b, 4b), sulfanilamide (3c, 4c), sulfadiazine (3d, 4d), sulfamerazine (3e), and sulfathiazole (4e). The compounds were evaluated against carbonic anhydrase (CA) and acetylcholinesterase (AChE) enzymes to obtain possible drug candidate/s. The lead compounds of the series were 3a and 4a against human CA (hCA) I, whereas3d and 4a were leads against hCA II in terms of Ki values. Series 4 includes more effective CAs inhibitors than series 3 (except 3d). Series 4 compounds having a nitro group (except 4d) were 3.3-4.8 times more selective inhibitors than their corresponding analogues 3a-d in series 3, in which hydrogen was located in place of the nitro group, by considering Ki values against hCA II. Compounds 3c and 4c, where the sulfanilamide moiety is available, were the leads in terms of AChE inhibition with the lowest Ki values. The use of secondary sulfonamides was a more effective modification on CA inhibition, whereas the primary sulfonamide was the effective substitution in terms of AChE inhibitory potency.

Synthesis, characterization and biological evaluation of tertiary sulfonamide derivatives of pyridyl-indole based heteroaryl chalcone as potential carbonic anhydrase IX inhibitors and anticancer agents

In the quest for novel effective carbonic anhydrase inhibitors, some sulfonamide derivatives of pyridyl-indole based chalcone were synthesized and screened in vitro for inhibitory activity against human carbonic anhydrase IX isoform. Among all the synthesized compounds (SC2 -SC11), only three compounds SC3, SC7 and SC10 were found to have better binding affinity as shown by molecular docking and fluorescence binding studies. Further, the enzyme inhibition assay and in vitro anti-tumor evaluation against MCF-7 and HepG-2 cell lines revealed that the compounds SC3, SC7 and SC10 inhibited the CA IX selectively, possessed predominant anti-proliferative potential and significantly induced apoptosis in cancerous cells.

An Update on Natural Products with Carbonic Anhydrase Inhibitory Activity.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological processes. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. It is more than 70 years that synthetic compounds, mainly sulfonamides, have been used in clinical practice as diuretics and systemic acting antiglaucoma drugs. Recent studies using natural product libraries and isolated constituents from natural sources (such as fungi and plants) have disclosed novel chemotypes possessing carbonic anhydrase inhibition activities. These natural sources offer new opportunities in the search for new and more effective carbonic anhydrase inhibitors, and may serve as new leads for the design and development of future drugs. This review will discuss the most recent advances in the search of naturally occurring products and their synthetic derivatives that inhibit the CAs and their mechanisms of action at molecular level. Plant extracts are not considered in the present review.

Development and Validation of a Simple and Reliable LC-MS/MS Method for the Determination of Acetazolamide, an Effective Carbonic Anhydrase Inhibitor, in Plasma and its Application to a Pharmacokinetic Study

A simple, rapid and accurate liquid chromatography - tandem mass spectrometry method was developed and validated for the quantification of acetazolamide in beagle plasma using sulfadiazine as the internal standard. After extraction by acetonitrile, the analytes were separated by a rapid gradient elution with acetonitrile and water as the mobile phase on a SHIMADZU VP-ODS C18 column and then detected by an API 4000 triple-quadrupole mass spectrometer using electrospray ionization in negative ion mode. Multiple reaction monitoring was performed on the ion transitions of m/z 220.9→83.3 (acetazolamide) and m/z 248.9→185.0 (sulfadiazine). The plasma concentration of acetazolamide in beagle dogs showed good linearity over the range of 0.20~50 μg/mL, and the intra- and inter-day variations were small with high accuracy and absolute recovery. Both analytes can maintain stable during the whole experimental process. The developed method was successfully applied to the pharmacokinetic study of acetazolamide extended-release capsules after oral administration to beagle dogs.

Carbonic Anhydrase Inhibitors: Aliphatic N-phosphorylated Sulfamates--A Novel Zinc-anchoring Group Leading to Nanomolar Inhibitors

A small library of phosphorylated sulfamates (N-(O-alkylsulfamoyl)-phosphoramidic acids) incorporating long aliphatic chains (C8-C16) has been synthesized and investigated for their interaction with two physiologically relevant carbonic anhydrase (CA) isozymes. These compounds behaved as very potent inhibitors of both isozymes, with inhibition constants in the range of 8.2-16.1 nM against isozyme hCA I, and 5.3-11.9 nM against isozyme hCA II. Activity was optimal for the n-octyl derivative (similarly with that of the corresponding unsubstituted sulfamates) and gradually decreased for the longer chain derivatives. Some of these compounds are much more effective CA inhibitors as compared to the clinically used derivatives acetazolamide, sulfanilamide or topiramate, which are used as standards for the enzymatic determinations. The phosphorylated sulfamate moiety represents a novel zinc-binding group for the design of effective CA inhibitors.

Determination of MK-507, a novel topically effective carbonic anhydrase inhibitor, and its de-ethylated metabolite in human whole blood, plasma, and urine by high-performance liquid chromatography

Sensitive methods for the determination of a novel topically effective carbonic anhydrase inhibitor (CAI) I, MK-507, and its de-ethylated metabolite II, in human whole blood, plasma and urine were developed. These methods were based on liquid—liquid extraction of I and II from biological matrices, back extraction into acid, and analysis by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm). The assays were fully validated in the concentration range of 5 to 500 ng/ml, and the limit of quantification (LOQ) for I and II, defined as the lowest concentration on the standard curve for which precision (coefficient of variation, C.V.) is < 10%, was 5 ng/ml in whole blood, plasma, and urine. These methods were applied for the analyses of biological fluid samples from a variety of clinical pharmacokinetic studies. In addition, a method in whole blood based on column-switching HPLC with UV detection and with an LOQ of 50 ng/ml was also developed. The switching valve was used to eliminate interferences from late eluting peaks extracted from whole blood. The details of these assays, together with some representative data from a human study, are presented.

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3- b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [( S) and ( R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4 H-thieno[2,3- b]thiopyran-2-sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25–250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single ( S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5–100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.

ChemInform Abstract: Synthesis and Physicochemical Properties of Thiadiazolo(3,2‐a)pyrimidinesulfonamides and Thiadiazolo(3,2‐a)triazinesulfonamides as Candidates for Topically Effective Carbonic Anhydrase Inhibitors.

AbstractStarting with the thiadiazole (I), the bicyclic sulfonamides (V), (VII), (IX), and (XI) are prepared by cyclocondensation reactions as described in the reaction scheme.

Synthesis and physicochemical properties of thiadiazolo[3,2-a]pyrimidinesulfonamides and thiadiazolo[3,2-a]triazinesulfonamides as candidates for topically effective carbonic anhydrase inhibitors

A series of bicyclic 1,3,4-thiadiazolo[3,2-a]pyrimidine- and 1,3,4-thiadiazolo[3,2-a]triazine-7-sulfonamides were synthesized from 5-amino-1,3,4-thiadiazole-2-sulfonamide and evaluated for topical efficacy as ocular hypotensive agents. The compounds were tested for the physicochemical properties of sulfonamide pKa, free acid water solubility, CHCl3/buffer partition, and transcorneal penetration (kin), as well as for activity against carbonic anhydrase (I50). A number of these compounds exhibited lower sulfonamide pKa and higher water solubility than those of acetazolamide (1) and methazolamide (2), and one, 12, brought about a small reduction in IOP in the normal rabbit eye.

Quaternary ammonium sulfanilamide: a membrane-impermeant carbonic anhydrase inhibitor.

A novel carbonic anhydrase (CA) inhibitor, quaternary ammonium sulfanilamide (QAS), was tested for potency as a CA inhibitor and for its ability to be excluded from permeating biological membranes. Inhibitor titration plots of QAS vs. pure bovine CA II and CA from the gills of the blue crab, Callinectes sapidus, yielded Ki values of approximately 15 microM; thus QAS is a relatively weak but effective CA inhibitor. Permeability of the QAS was directly tested by two independent methods. The inhibitor was excluded from human erythrocytes incubated in 5 mM QAS for 24 h as determined using an 18O-labeled mass spectrometer CA assay for intact cells. Also QAS injected into the hemolymph of C. sapidus (1 or 10 mM) did not cross the basal membrane of the gill. The compound was cleared from the hemolymph by 96 h after injection, and at no time during that period could the QAS be detected in homogenates of gill tissue. Total branchial CA activity was only slightly reduced following the QAS injection. These data indicate that QAS is a CA inhibitor to which biological membranes are impermeable and that can be used in vivo or in vitro in the study of membrane-associated CA.

Metal ion dependent binding of sulphonamide to carbonic anhydrase.

ACETAZOLAMIDE (2-acetylamino-1,3,4-thiadiazole-5-sulphonamide, ‘Diamox’) is the most potent known inhibitor of the zinc enzyme carbonic anhydrase. This communication reports the direct demonstration that binding of acetazolamide to human carbonic anhydrase requires the presence of a metal ion at the active site and that binding depends on the species of divalent metal ion present. Zinc (II) and cobalt (II) ions are the only ions which induce the formation of very stable acetazolamide carbonic anhydrase complexes and are also the ions which most effectively catalyse the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate. Metal-binding monodentate ions, CN−, HS−, OCN−, and N3−, known as effective carbonic anhydrase inhibitors, compete for the acetazolamide binding site of the zinc enzyme.