Heterocyclic compounds as carbonic anhydrase inhibitor

Abstract

The carbonic anhydrases (CAs, EC 4.2.1.1) constitute interesting targets for the design of pharmacological agents useful in the treatment or prevention of a variety of disorders such as, glaucoma, acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema, and obesity. A quite new and unexpected application of the CA inhibitors (CAIs) is with regard to their potential use in the management (imaging and treatment) of hypoxic tumors. A series of sulfonamides, including some clinically used derivatives like acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug, as well as the sulfamate antiepileptic drug topiramate have been reported to inhibit various human carbonic anhydrase isozyme. Various heterocyclic sulfonamides have been reported in this review with their potency to inhibit different carbonic anhydrases isozymes.

Keywords::

• Carbonic anhydrase
• sulfonamide
• synthesis
• evaluation

Introduction

The carbonic anhydrases (CAs) are a family of metalloenzymes involved in the catalysis of reversible hydration CO₂ (CO2 + H2O ↔ HCO3- + H+) and, as a consequence, play an important part in a number of physiological processes such as pH regulation and electrolyte secretion, and in a number of biosynthetic pathways. From the viewpoint of cancer-drug discovery, perhaps the most interesting CA isoform is CA IX; because CA IX has been reported to be prevalently expressed in several human cancer cells but not in their ‘normal’ counterparts and high expression levels are related to poor patient prognosis¹.

They are involved in crucial physiological processes connected with respiration and transport of CO₂/bicarbonate between metabolizing tissues and the lungs, pH and CO₂ homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as the gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification, tumorigenicity, and many other physiologic or pathologic processes. This enzyme has 16 different isoenzymes presently known in human. Basically, there are several cytosolic forms (CA I, CA II, CA III, and CA VII), four membrane bound forms (CA IV, CA IX, CA XII, and CA XIV), one mitochondrial form (CA V), as well as a secreted CA form (CA VI²). There are three additional “catalytic” CA isoforms (CA VIII, CA X, and CA XI) whose functions remain unclear³. The isozymes CA VA and VB which are present in mitochondria are involved in several biosynthetic processes, such as ureagenesis, gluconeogenesis, and lipogenesis^4,5. The isoenzymes CA IX and XII, are predominantly found in tumor cells and absent (or are present in very limited amount) in normal tissues. Several of these isoenzymes (CA II and CA IV) are expressed in human eyes. The most recently characterized members of this family are cytosolic CA XIII and membrane-bound CA XV. Although the activity of CA XIII is the second lowest reported thus far for any of the human CAs, it may have a role in maintaining the acid-base balance in the kidney and the gastrointestinal and reproductive tracts. CA XV is an exceptional enzyme, as it seems to be active in numerous species, such as rodents, birds and fish, but is absent from humans and chimpanzees. Mouse CA XV is a moderately active enzyme, suggesting that it may play a physiological role at least in the kidney. It is likely that other isozymes have substituted for this protein in humans⁶.

The carbonic anhydrases constitute interesting targets for the design of pharmacological agents useful in the treatment or prevention of a variety of disorders such as, glaucoma, acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema, and obesity^7,8. A quite new and unexpected application of the CA inhibitors (CAIs) is with regard to their potential use in the management (imaging and treatment) of hypoxic tumors^9–16.

Inhibitors of zinc metalloenzymes need precise structural requirements in order to tightly bind to the metal ion(s) present in their active sites. For example, primary sulfonamides (RSO₂NH₂), sulfamates (ROSO₂NH₂), and sulfamides (RNHSO₂NH₂) show high affinity for binding the Zn(II) ion present in α-carbonic anhydrases acting as potent inhibitors with clinical applications as antiglaucoma, diuretic, antiobesity or antitumor drugs. In all of them, the sulfonamide/sulfamate/sulfamide drug binds in deprotonated form to the catalytically critical Zn(II) ion, also participating in extensive hydrogen bond and van der Waals interactions with amino acid residues both in the hydrophobic and hydrophilic halves of the enzyme active site, as shown by X-ray crystallographic studies of enzyme-inhibitor complexes¹⁷. The classical recognition motif for small molecules to bind the active site of CA is an aromatic sulfonamide moiety (ArSO₂NH₂). The deprotonated sulfonamide (ArSO₂NH⁻) coordinates to the CA active site Zn²⁺, and so inhibits the binding of the endogenous substrates (CO₂ and H₂O), thereby reducing the catalytic ability of the enzyme^18,19.

Heterocyclic moieties with carbonic anhydrase inhibitiory activity

Thiadiazole

A small series of 2-substituted-1,3,4-thiadiazole-5-sulfamides were synthesized and assayed for the inhibition of five physiologically relevant isozymes, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I (K_Is of 102 nM–7.42 μM), hCA II (K_Is of 0.54–7.42 μM) and hCA IV (K_Is of 4.32–10.05 μM) but were low-nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2–32 nM and 1.3–74 nM, respectively¹⁹. As extension of their former work C. Temperini et al.²⁰ reported interaction of 2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide [1] with the 12 catalytically active mammalian carbonic anhydrase isozymes, CA I-XIV. The compound was found to be a potent inhibitor of CA IV, VII, IX, XII, and XIII (KIs of 0.61–39 nM), a medium potency inhibitor of CA II and VA (K_Is of 121–438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV) making it a very interesting candidate for situations in which a strong/ selective inhibition of certain isozymes is needed. They included that compound 1 is completely different from those of clinically used sulphonamides like acetazolamide. Indeed compound 1 is one of the most potent CA IV and CA VII inhibitors ever reported, while acting as a much weaker inhibitor of the ubiquitous isoform CA II which is often considered as housekeeping enzyme, whose inhibition is not desired.

5-Amino-1,3,4-thiadiazole-2-sulfonamide has several biological activities and antibacterial properties. Several derivatives of this drug have been synthesized and used in treatment of glaucoma; one of them is acetazolamide which is used systematically (i.e. an oral medication) to reduce intraocular pressure by lowering the humor formation in the eye. A number of side effects of this drug are however experienced such as numbness and tingling in the fingers and toes, taste alterations, blurred vision, kidney stones, and an increase in urination²¹. To overcome these side effects Rahmi et al.²² synthesized various novel pyrazole carboxylic acid derivatives of 5-amino-1,3,4-thiadiazole-2-sulfonamide, [2a-b, 3, 4a-g]. They also reported the inhibitory effects of all newly synthesized amides on hydratase and esterase activities of carbonic anhydrase isoenzymes (hCA-I and hCA-II) in vitro. The comparison of newly synthesized amides to inhibitor 5-amino-1,3,4-thiadiazole-2-sulfonamide and to acetazolamide, indicated that the new derivatives inhibit CA isoenzymes and they are more potent inhibitors than the parent inhibitor. The most effective compounds were 4g for hCA-I, 2a, 4f and 4g for hCA-II.

A series of benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides [5a-g] were synthesized and there binding to two CAse isozymes measured by isothermal titration calorimetry (ITC). Human carbonic anhydrase I (hCAI) and bovine carbonic anhydrase II (bCAII) bound the inhibitors with observed association constants in the range from 1.1 × 10⁶ to 2.6 × 10⁷ M⁻¹. The strongest binder to both isozymes of CAse was compound 5e with the observed Kd of about 0.04μM. The most specific binder was 5d. Compound 5d bound threefold tighter to bCAII that to hCAI²³.

Quinoline

Quinoline derivatives having a substituted or unsubstituted sulfonamide moiety where their design complies with the general pharmacophore of the sulfonamide CA inhibitor, R. Gitto et al.²⁴ synthesized a series of 1-aryl-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-sulfonamides [6a-j]. These newly synthesized compounds incorporate the main features of the above mentioned anticonvulsants and a sulfonamide function capable to inhibit the enzyme carbonic anhydrase, which represents an attractive target in epilepsy. Compound 6h was found to be most interesting, demonstrating K₁ value lower than that of topiramate against hCA XIV, more ever 6h and topiramate inhibited hCA I with similar potency.

Mostafa M. Ghorab et al.²⁵ synthesized some novel 4-(quinoline-1-yl) benzenesulphonamide derivatives [7a-u]. All the newly synthesized compounds were evaluated for their in vitro anticancer activity. Some compounds showed interesting in vitro anticancer activities when compared with doxorubicin as a reference drug. In addition, they also performed docking of the synthesized compounds into carbonic anhydrase isozyme II (CA II) active site in order to give a suggestion about the proposed mechanism of action. They found that quinoline derivative 7h carrying benzo[d][1,3]dioxol at 4-position with IC₅₀ value <22.2 μM, quinoline derivative 7g having 4-methoxyphenyl at 4-position with IC₅₀ = 22.9 μM and quinoline derivative 7e having styryl group at 4-position with IC₅₀ = 23.1 μM showed higher significant cytotoxic activity which was even higher activity than that of the reference drug doxorubicin with IC₅₀ = 69.9 μM.

Triazine

Sulfonamides incorporating triazinyl moieties previously reported were among the most potent and selective hCA IX inhibitors. In continuation of the investigation of this class of derivatives, reporting the synthesis and hCA I, II and IX inhibitory properties of a large series of such novel derivatives, V. Garaj et al.²⁶ reported synthesis of new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide. All the synthesized sulfonamides incorporating triazinyl moieties were screened for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds inhibited hCA I with inhibition constants in the range of 31–8500 nM, hCA II with inhibition constants in the range of 14–765 nM and hCA IX with inhibition constants in the range of 1.0–640 nM. The best hCA I inhibitors in this series were 8a-g, 9a-c and 10a-d, which showed K_Is in the range of 31–95 nM, in the same range as the potent, clinically used inhibitors ethoxzolamide, methazolamide and indisulam. The most effective hCA II inhibitors (with potencies comparable to those of the clinically used sulfonamides were found to be 8a-g, with K_Is in the range of 14–27 nM; The best hCA IX inhibitors were the amino, hydrazine, ethylamino, dimethylamino and amino acid derivatives, which showed inhibition constants in the range of 1–3 nM, being among the most potent hCA IX inhibitors reported up to now.

Vladimir Garaj et al.²⁷ synthesized a series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. All the synthesized sulfonamides incorporating triazinyl moieties were evaluated for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic hCA I and II, and the transmembrane, tumor associated hCA IX. The new compounds reported inhibited hCA I with K_Is in the range of 75–136 nM. The best hCAI inhibitors were the amines 11b-d, the chloroderivative 11a and the ethoxyderivative 11e. Against isozyme hCA II the new derivatives showed a better inhibitory power, with K_Is in the range of 13–278 nM. Against the tumor associated isozyme hCA IX, compound 11e was found to be a potent inhibitor (K_I of 12 nM being more potent than the clinically used sulphonamide including indisulam).

A series of bicyclic 1,3,4-thiadiazolo[3,2-a]triazine-7-sulfonamides [12] and 1,3,4-thiadiazolo[3,2-a]pyrimidine [13a-c] were synthesized by Alan R. Katritzky et al.²⁸ from 5-amino-l,3,4-thiadiazole-2-sulfonamide and evaluated for topical efficacy as ocular hypotensive agents. All compounds tested showed approximately the same activity against the enzyme with IC₅₀ value ranging from 1.4 to 5 × 10⁻⁸ M.

Pyridine sulphonamide

Z. Brzozowski et al.²⁹ synthesized a series of novel 4-substituted-3-pyridinesulfonamides [14–22] and [23a-c] and investigated as inhibitors of five isoforms of zinc enzyme carbonic anhydrase that is, the cytosolic, ubiquitous isozymes CA I and II, and transmembrane isozymes CA IX, XII (cancer-associated) and XIV. The newly synthesized compounds showed inhibition constants in the range of 0.078–11.7 μM. (i) Against the slow cytosolic isoform hCA I, compounds 22e, 23a and 23b showed hCA I inhibitory activity (K_Is = 0.078e1.52 μM) in the same range as the clinically used compounds. (ii) Against the ubiquitous and dominant rapid cytosolic isozyme hCA II, compounds 21d acting as a stronger CA II inhibitors, with K_Is = 9.9nM, of the some order magnitude as most of the clinically used compounds.(iii) A quite larger variation of inhibitory activity was observed for the inhibition of the tumor-associated isoform, hCA IX, compound 15a, 16a, 16b, 16d, 17a-d, 21b, 21c, 21e, 22a-e, 23a and 23b were very effective hCA IX inhibitors (K_Is in range of 4.6–12.0 nM). (iv) A quite good inhibition profile of the second tumor-associated isoform, hCA XII has also been observed, most of the compounds were very effective hCA XII inhibitors (K_Is = 3.4–8.9 nM). (v) All of the investigated sulfonamides showed moderate to weak inhibition properties towards hCA XIV isozyme with K_Is in the range of 50.9–160.0 nM.

A series of 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamide [24–26] had been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase, that is the cytosolic ubiquitous CA I and II, and cancer-associated isozymes CA IX and XII. Against the human isozymes hCA I the new compounds showed inhibition constants in the range of 1.09–12.1 μM, against hCA II in the range of 50.5–172 nM, against hCA IX in the range of 5.2–118 nM, and against hCA XII in the range of 8.7-381 nM, respectively. Compounds 24a, 24b, 24d-j, 25a and 25c-d showed excellent hCA IX inhibitory efficacy, with K_Is = 5.2–11.0 nM, being much more effective as compared to the clinically used sulfonamides AAZ, MZA, EZA, DCP and IND (K_Is = 24–50 nM). Compounds 24a, 24b, 24d-j, 25a and 25c were also very effective hCA XII inhibitors (K_Is = 8.7–45.2 nM) which were comparable or more effective than those clinically used EZA and DCP (K_Is = 22 and 50 nM), respectively³⁰.

Indole

Graham et al.³¹ synthesized indole-2-sulfonamide [27a-d, 28b-c, 29b-c and 30] and all the newly synthesized compounds were evaluated as topically active ocular hypotensive agents. These compounds were found to be excellent inhibitors of carbonic anhydrase and to lower intraocular pressure in a rabbit model of ocular hypertension. Compound 27b and 29b were found to be better intraocular pressure lowering agent with IC₅₀ value 16 nM and 11 nM, respectively.

Özlen Güzel et al.³² observed the effect of indole-sulphonamide derivatives on mammalian carbonic anhydrase isozymes. In their research work they synthesized a series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides [31a-o] possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, or a perfluorophenyl moiety, by reaction with 2,4,6-trimethylpyrylium perchlorate. All the newly sulfonamides were evaluated as inhibitors of four mammalian carbonic anhydrase isoforms, that is, CA I, II (cytosolic), CA IX and XII (transmembrane, tumor-associated forms). Most of these sulfonamides were found to be potent inhibitors of hCA IX, and against hCA XII with some of the new compounds. These compounds were generally less effective inhibitors of hCA II. Being membrane impermeant, these positively-charged sulfonamides were interesting candidates for targeting the tumor-associated CA IX and XII, as possible diagnostic tools or therapeutic agents. Against the cytosolic isoform hCA I compound 31g and 31m were found to be very active with K_Is of 3.2 and 5.1 nM, respectively. Against the ubiquitous and dominant rapid cytosolic isozyme hCA II, compound 31o was found to be most potent with K_Is in subnanomolar range (0.93nM). The tumor associated isoform hCA IX, one of the important targets for obtaining novel antitumor therapies which ultimately emerged, was generally excellently inhibited by the new compounds reported here, many of these derivatives [31c-e, 31g-k and 31m] had KIs < 10 nM, being thus more than 10 times better hCA IX inhibitors compared to lead. Against the second tumor-associated isoform, hCA XII (less widespread as compared to hCA IX) the new compounds reported here showed a mixed behavior, with three of them acting as very effective inhibitors (5, 6 and 9, K_Is in the range of 9.5–12.6 nM), whereas 31a, 31b, 31e and 31f being moderate inhibitors (K_Is in the range of 38–97 nM).

Thiophene

A. Innocenti et al.³³ reported synthesis and screening of some selected benzo[b]thiophene-5- and 6-sulfonamide derivatives for cytotoxic activity and the interactions with several CA isozymes, some of which are known to be involved in tumorigenesis (hCA IX), whereas others are ubiquitously found in many normal tissues (the cytosolic isoforms hCA I and II). They concluded that hCA I was inhibited by unsubstituted sulfonamides with inhibition constants in the range of 63–138 nM, hCA II with inhibition constants in the range of 6.3–8.8 nM, and hCA IX with inhibition constants in the range of 2.8–15 nM, being thus more active than clinically used inhibitors such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide or indisulam. Some of these derivatives also showed some selectivity for the inhibition of the tumor-associated (hCA IX) over the cytosolic isozyme hCA II. Against isozyme hCA I the primary sulfonamides 32a, 32b, 33a-c, and 34a showed moderate inhibitory activity, with inhibition constants in the range of 63–138 nM, Against the ubiquitous, physiologically relevant isozyme hCA II, again the unsubstituted sulfonamides 32a, 32b, 33a-c, and 34a showed very good inhibition, with K_Is in the range of 6.3-8.8 nM, The tumor associated isozyme hCA IX was also inhibited well by the unsubstituted sulfonamides 32a, 32b, 33a-c, and 34a, which showed K_Is in the range of 2.8–15 nM. They concluded that compound 32a, 32c, 32e, 34a, and 34b were cytotoxic.

Hartman et al.³⁴ synthesized a series of 4-substituted thiophene and was found to possess nanomolar-level potency for inhibition of hCA II in vitro. Selected compounds from this group were further evaluated for their potential to act as topically effective ocular hypotensive agents in the ocular normotensive albino rabbit and the ocular α-chymotrypsinized rabbit. A sequence of in vitro, ex vivo, and in vivo studies were used to evaluate the ocular hypotensive profile of this series of thiophene sulfonamides. Several functionalized 4-substituted thiophene- and furan-2-sulfonamides showed sub-10 nM potencies. Specifically, 4-acyl analogs, such as 35b and 35f, bearing monosubstitution in the 3- or 4-positions of the phenyl ring, displayed equivalent or somewhat enhanced activity as compared to their corresponding sulfonyl counterparts, 37b and 37c. Similarly, the thiophene-based Mannich adducts 36a and 36c were of comparable potency to furan adducts. In ex vivo assay for their ability to penetrate the albino rabbit eye and to inhibit the target enzyme in a homogenate of the iris-ciliary body at 1h postdosing. All compounds were screened at 0.5 % (1 drop, 50 pL), while the more potent compounds were screened at lower concentrations. The most potent compounds proved to be 35b, 35d, 37c, and 37e, all of which showed appreciable ex vivo activity at a concentration of 0.1 %. Compounds 36a, 37h, and 37l were found to be most potent for their ability to lower IOP in the α-chymotrypsinized (α -CT) rabbit model of ocular hypertension.

Oxadiazole and triazole

Novel series of mercapto-1,3,4-oxadiazole and -1,2,4-triazole derivatives were synthesized by G. L. Almajan et al.³⁵ by various pathways starting from 4-(4-halogeno-phenylsulfonyl)benzoic acid hydrazides which were reacted with carbon disulfide or isothiocyanates. All the newly synthesized heterocyclic mercaptans were assayed as inhibitors of three physiologically relevant isoforms of the zinc enzyme carbonic anhydrase i.e. the cytosolic CA I and II, and the tumor-associated, transmembrane isozyme CA IX. Interesting biological activity was detected for some of the new mercaptans, with inhibition constants in the low micromolar range. Against the slow cytosolic isozyme hCA I, the heterocycles 38–41 showed good inhibitory activity, with inhibition constants in the range 5.1–9.3 μM, being on the other hand weaker inhibitors as compared to the classical sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), which had a K_I of 0.25 μM. Against the physiologically most important cytosolic isoform, hCA II, the new compounds investigated here showed good inhibitory activity, with K_Is in the range 0.63–31 μM, being again less efficient inhibitors as compared to the sulfonamide acetazolamide (KI of 12 nM). Against the tumor associated isoform hCA IX, compounds 38–41 showed a moderate - weak inhibitory activity, with K_Is in the range of 1.25–59 μM, whereas the bulky triazoles 42 and 43 were quite weak inhibitors, with K_Is in the range 129–476 μM.

Wilkinson et al.¹⁸ reported synthesis of a series of benzene sulfonamides containing triazole-o-glycoside tails by the 1,3-dipolar cycloaddition reaction of 4-azidobenzenesulfonamide with o-propynyl glycosides. These newly synthesized glycoconjugates were assessed for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA I and II and the tumor-associated isozyme hCA IX. Against hCA I these compounds were either micromolar or low-nanomolar inhibitors, while against hCA II and IX inhibition in the range of 6.8–53 and 9.7–107 nM, respectively, was observed. The most potent inhibitor against hCA IX was the galactose derivative 44 (K_I = 9.7 nM); this is so far the most potent glycoconjugate inhibitor reported for the tumor-associated hCA IX. These carbohydrate-tethered sulfonamides may prove interesting lead candidates to target tumor associated CA isozymes, wherein the CA domain is located extracellularly.

Pyrrole-3-carboxamide

S. Gluszok et al.³⁶ reported the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. The inhibitory potency (K_I, nM) of all the derivatives was evaluated against the tumor-associated hCA IX and against the ubiquitous and physiologically relevant hCA I and hCA II. All the compounds exhibited a weak inhibitory activity against hCA I. The K_I values of the R1-phenyl derivatives 45a-e were found between 781 and 2151 nM as observed for the R1-naphthyl molecules 46a-i (427–10,000 nM). The 4-biphenylyl derivatives 47a-i were also weak hCA I inhibitors with K_I values higher than 1736 nM. Against hCA II All the synthesized molecules were found much more potent (K_I: 9-620 nM) than against hCA I. The phenyl 45a-e and the 4-biphenylyl 47a-i derivatives were moderate hCA II inhibitors with Ki values ranging from 127 to 620 nM. The best hCA II inhibitors (K_I≤ 70 nM) belonged to the R1-naphthyl series (46a-e and 46h). The most potent hCA IX inhibitors were the 2-naphthyl compounds 46a, 46b, 46d, and 46h (K_I: 10–22 nM) whereas 46c, 46e-g, and 45i were less active on hCA IX (K_I: 125–147 nM).

Assay of CA activity

C. T. Supuran et al.³⁷ had shown that the pH of the tumor environment can be changed by modulating CA activity, which may favorably influence the anticancer effect of drugs, or other agents, used concomitantly with a CA inhibitor/activator.

CA activity was determined by following the hydration of CO₂ according to the method of Wilbur and Anderson³⁸. In this method, the rate of pH reduction is measured using an electrode as soluble CO₂, in a standardised solution, is converted to HCO₃⁻ by the action of CA, when compared to the uncatalysed reaction. Enzyme units were calculated according to the formula:

where t_o and t_c are the times (seconds) needed for the pH change, without and with enzyme reactions, respectively. Assays were performed at least twice on each lysate and the mean value determined³⁹.

Conclusion

Carbonic anhydrase isozymes are associated with several biological disorders i.e. glaucoma, acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema, obesity and hypoxic tumor. The present review demonstrated that heterocyclic sulphonamide derivatives have potent carbonic anhydrase inhibition activity, so these heterocyclic compounds can be utilized for managing above mentioned disorders. Furthermore, the substitutions to these heterocyclic moieties can be utilized for the development of more potentially active inhibitors with better therapeutic value.

Declaration of interest

The authors declared no conflict of interest.