In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and II, with a series of salicylic acid derivatives was investigated by using the esterase method with 4-nitrophenyl acetate as substrate. IC(50) values for sulfasalazine, diflunisal, 5-chlorosalicylic acid, dinitrosalicylic acid, 4-aminosalicylic acid, 4-sulfosalicylic acid, 5-sulfosalicylic acid, salicylic acid, acetylsalicylic acid (aspirin) and 3-metylsalicylic acid were of 3.04 microM, 3.38 microM, 4.07 microM, 7.64 microM, 0.13 mM, 0.29 mM, 0.42 mM, 0.56 mM, 2.71 mM and 3.07 mM for hCA I and of 4.49 microM, 2.70 microM, 0.72 microM, 2.80 microM, 0.75 mM, 0.72 mM, 0.29 mM, 0.68 mM, 1.16 mM and 4.70 mM for hCA II, respectively. Lineweaver-Burk plots were also used for the determination of the inhibition mechanism of these substituted phenols, most of which were noncompetitive inhibitors with this substrate. Some salicylic acid derivatives investigated here showed effective hCA I and II inhibitory activity, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.     

Investigation of the effects of some sulfonamides on acetylcholinesterase and carbonic anhydrase enzymes

Human carbonic anhydrase I and II isoenzymes (hCA I and II) and acetylcholinesterase (AChE) are important metabolic enzymes that are closely associated with various physiological and pathological processes. In this study, we investigated the inhibition effects of some sulfonamides on hCA I, hCA II, and AChE enzymes. Both hCA isoenzymes were purified by Sepharose‐4B‐L‐Tyrosine‐5‐amino‐2‐methylbenzenesulfonamide affinity column chromatography with 1393.44 and 1223.09‐folds, respectively. Also, some inhibition parameters including IC₅₀ and K_i values were determined. Sulfonamide compounds showed IC ₅₀ values of in the range of 55.14 to 562.62 nM against hCA I, 55.99 to 261.96 nM against hCA II, and 98.65 to 283.31 nM against AChE. K_i values were in the range of 23.40 ± 9.10 to 365.35 ± 24.42 nM against hCA I, 45.87 ± 5.04 to 230.08 ± 92.23 nM against hCA II, and 16.00 ± 45.53 to 157.00 ± 4.02 nM against AChE. As a result, sulfonamides had potent inhibition effects on these enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly in the treatment of some disorders.     

Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II

The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC(50) values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC(50) concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 μM and 4250 μM and for hCA II 950 μM and 6200 μM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes.     

Comparison of the inhibitory potential towards carbonic anhydrase,acetylcholinesterase and butyrylcholinesterase of chalcone and chalcone epoxide

Chalcones and chalcone epoxides are important synthetic intermediates in organic and medicinal chemistry. Chalcones possess a broad spectrum of biological activities; however, 1,3‐diphenyl‐2‐propenone or chalcone has not been given the attention it deserve as its substituted derivatives. In this study, the inhibition effects of chalcone and its epoxidated derivative chalcone epoxide against human carbonic anhydrase isozymes I and II (hCA I and hCA II), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were evaluated. The results obtained showed that both compounds exhibited potent inhibitory activity, with IC₅₀ values less than 10 µM. IC ₅₀ values in the submicromolar (hCA I and hCA II) to low micromolar range (AChE and BuChE) were observed for both compounds. The mechanism of inhibition and the inhibitory constants ( K _i values) for each compound were also determined. Furthermore, chalcone epoxide was docked within the active sites of hCA I, hCA II, AChE, and BuChE to explore its binding mode with the enzymes.     

NO-releasing esters show carbonic anhydrase inhibitory action against human isoforms I and II

Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) are a class of pharmaceuticals used as antiglaucoma agents, diuretics, antiepileptics, in the management of mountain sickness, gastric and duodenal ulcers, neurological disorders, or osteoporosis. We report here the inhibitory capacities of some organic nitrates against two human (hCA) isozymes, hCA I and hCA II. The IC₅₀ values of compounds 1–12 against hCA I ranged between 7.13 mM and 124 mM, and against hCA II between 65.1 μM and 0.79 mM. Nitrate esters are thus interesting hCA I and II inhibitors, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.     

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).     

Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors

Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, (13)C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of sulfanilamide, acetazolamide (AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been studied in vitro. The IC(50) values of compounds for esterase activity are 0.71-0.11 μM for hCA I and 0.45-0.12 μM for hCA II, respectively. The K(i) values of these inhibitors were determined as 0,38-0,008 μM for hCA I and 0,19-0,001 μM for hCA II, respectively.     

Synthesis and bioactivity of several new hetaryl sulfonamides

1-(4-Methylsulfonyl)-2-thione-4-aryl-5-Z-6-methyl and oxyalkyl-imidazoles were synthesized from different tetrahydropyrimidinethiones and aryl sulfonyl chloride. These compunds were tested for metal chelating effects and to determine the phrase in which inhibition occured between two physiologically pertinent compunds and carbonic anhydrase (CA) isozymes I and II (hCA I and II), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). AChE was detected in high concentrations in the brain and red blood cells. BChE is another enzymes that is abundant available in the liver and released into the blood in a soluble form. Newly synthesized hetaryl sulfonamides exhibited impressive inhibition profiles with Ki values in the range of 1.42–6.58?nM against hCA I, 1.72–7.41?nM against hCA II, 0.20–1.14?nM against AChE and 1.55–5.92?nM against BChE. Moreover, acetazolamide showed Ki values of 43.69?±?6.44?nM against hCA I and 31.67?±?8.39?nM against hCA II. Additionally, tacrine showed Ki values of 25.75?±?3.39?nM and 37.82?±?2.08 against AChE and BChE, respectively.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).     

The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives

Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66–4.14?nM) and hCA II inhibitors (Kis of 1.37–3.12?nM) and perfect AChE inhibitors (Kis in the range of 1.87–7.53?nM) compared to acetazolamide as CA inhibitor (Ki: 6.76?nM for hCA I and Ki: 5.85?nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64?nM).     

(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors

In this study, we have synthesised (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and a series of its derivatives (5, 13–16) and tested the ability of these compounds to inhibit two metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and hCA II. The synthesised compounds showed inhibitory effect on hCA I and hCA II isozymes. The results showed that synthesised compounds (5, 13–16) demonstrated the best inhibition activity against hCA I (IC₅₀: 3.22–54.28 μM) and hCA II (IC₅₀: 18.52–142.01 μM). The compound 14 showed the highest inhibiton effect against hCA I (IC₅₀: 3.22 μM; K_i: 1.19?±?1.4 μM). On the other hand, the compound 13 showed the highest inhibiton effect against hCA II (IC₅₀: 18.52 μM; K_i: 3.25?±?1.13 μM).     

Synthesis of some tetrahydropyrimidine-5-carboxylates,determination of their metal chelating effects and inhibition profiles against acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase

2-(Methacryloyloxy)ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate, is a cyclic urea derivative synthesized from urea, 2-(methacryloyloxy) ethyl acetoacetate and substituted benzaldehyde, and tested in terms of the inhibition of two physiologically relevant carbonic anhydrase (CA) isozymes I and II. Acetylcholinesterase (AChE) is found in high concentrations in the red blood cells and brain. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Also, they were tested for the inhibition of AChE and BChE enzymes and demonstrated effective inhibition profiles with Ki values in the range of 429.24–530.80?nM against hCA I, 391.86–530.80?nM against hCA II, 68.48–97.19?nM against AChE and 104.70–214.15?nM against BChE. On the other hand, acetazolamide clinically used as CA inhibitor, showed Ki value of 281.33?nM against hCA I, and 202.70?nM against hCA II. Also, Tacrine inhibited AChE and BChE showed Ki values of 396.03 and 209.21?nM, respectively.     

Inhibition of carbonic anhydrase isozymes I and II with natural products extracted from plants, mushrooms and honey

Different natural products and secondary metabolites from mushrooms, teas, honeys, mosses, plants and seaweeds were investigated for their in vitro inhibitory effects on human carbonic anhydrase (hCA, E.C.4.2.1.1) isoforms I and II. Inhibition data were correlated with the total phenol content in the extract and investigated with the pure compounds believed to be responsible for this activity. Methanolic extracts were prepared for 17 such pure chemicals present in the natural products and for 41 diverse natural products. The IC(50) values were in the range of 0.11-66.50 μg/mL against hCA I and of 0.09-54.54 μg/mL against hCA II, respectively. The total phenol content was in the range of 0.02-1318.96 (as milligrams of gallic acid equivalents) per gram of sample. These data offer new insights on possible novel classes of CA inhibitors based on natural products, possessing a range of chemical structures not present in the classical inhibitors with pharmacological applications, such as the sulfonamides and sulfamates.     

Novel sulphonamides incorporating triazene moieties show powerful carbonic anhydrase I and II inhibitory properties

Abstract

A series of compounds incorporating 3-(3-(2/3/4-substituted phenyl)triaz-1-en-1-yl) benzenesulfonamide moieties were synthesised and their chemical structure was confirmed by physico-chemical methods. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the compounds were evaluated against human isoforms hCA I and II. K_I values of these sulphonamides were in the range of 21?±?4–72?±?2?nM towards hCA I and in the range of 16?±?6–40?±?2?nM against hCA II. The 4-fluoro substituted derivative might be considered as an interesting lead due to its effective inhibitory action against both hCA I and hCA II (K_Is of 21?nM), a profile rarely seen among other sulphonamide CA inhibitors, making it of interest in systems where the activity of the two cytosolic isoforms is dysregulated.     

Intermolecular amination of allylic and benzylic alcohols leads to effective inhibitions of acetylcholinesterase enzyme and carbonic anhydrase I and II isoenzymes

In this study, we aimed to determine the inhibition effects of novel synthesized sulfamates ( 2a–g ), sulfonamides ( 3b–f ), carbonyl sulfonamides ( 3h and i ), and carbonyl sulfamates ( 4h and 4i ), which were tested against two human cytosolic carbonic anhydrase I and II isozymes (hCA I and II) and acetylcholinesterase (AChE) enzyme. For inhibition properties of allylic sulfamates, the half maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) were calculated for each novel compounds. The allylic sulfamates showed that K_i values are in the range of 187.33–510.31 pM for hCA I, 104.22–290.09 pM against hCA II, and 12.73–103.63 pM against AChE. The results demonstrated that all newly synthesized compounds had shown effective inhibition against hCA I and II isoenzymes and AChE enzyme.     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

In vitro effects of some anabolic compounds on erythrocyte carbonic anhydrase I and II

The in vitro effects of the anabolic compounds, zeranol, 17 β-estradiol, diethylstilbestrol (DES), and trenbolone, on the activity of purified human carbonic anhydrase I and II were evaluated. In vitro CA enzyme activity was determined colorimetrically using the CO? hydration method of Maren. IC?? values of the compounds that caused inhibition were determined by means of activity percentage diagrams. The IC?? concentrations of zeranol, 17 β-estradiol, DES and trenbolone on hCA I were 94, 55, 10, 898 μM and for hCA II 89, 159, 439 and 101 μM, respectively.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Synthesis and biological evaluation of benzenesulphonamide-bearing 1,4,5-trisubstituted-1,2,3-triazoles possessing human carbonic anhydrase I,II, IV,and IX inhibitory activity

A library of benzenesulphonamides incorporating 1,2,3-triazole rings functionalised with ester, carboxylic acid, carboxamide, carboxyhydrazide, and hydroxymethyl moieties were synthesised. The carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV, and hCA IX. Among them, hCA II and IV are anti-glaucoma drug targets, being involved in aqueous humour secretion within the eye. hCA I was inhibited with Ki’s ranging between 8.3?nM and 0.8737?µM. hCA II, the physiologically dominant cytosolic isoform, was excellently inhibited by these compounds, with Ki’s in the range of 1.6–9.4?nM, whereas hCA IV was effectively inhibited by most of them, with Ki’s in the range of 1.4–55.3?nM. Thirteen of the twenty sulphonamides were found to be excellent inhibitors of tumour associated hCA IX with Ki’s?≤?9.5?nM. Many of the new compounds reported here showed low nM inhibitory action against hCA II, IV, and IX, isoforms involved in glaucoma and some tumours, making them interesting candidates for further medicinal chemistry/pharmacologic studies.     

Carbonic anhydrase inhibitors: inhibition of human cytosolic isozyme II and mitochondrial isozyme V with a series of benzene sulfonamide derivatives

Among the 14 human isozymes of carbonic anhydrase (CA, EC 4.2.1.1) presently known, the cytosolic hCA II is the most active and plays a host of physiological functions, whereas the mitochondrial hCA V is unique due to its role in several biosynthetic reactions. An inhibition study of these isozymes with a series of sulfonamides is reported here, with the scope to detect lead molecules for the design of isozyme-specific CA inhibitors (CAIs) targeting the mitochondrial isoform. Indeed, recently it has been shown that CA V is a novel target for the drug design of anti-obesity agents among others. Compounds included in this study were mainly ortho-, meta-, and para-substituted-benzenesulfonamides, together with several halogeno-substituted sulfanilamides and disubstituted-benzene-1,3-disulfonamide derivatives. Isozyme V showed an inhibition profile with these sulfonamides different of that of hCA II. Thus, IC(50) values in the range of 80 nM to 74 microM against hCA II, and 0.78-63.7 microM against hCA V with these derivatives have been obtained. Only one compound, 2-carboxymethyl-benzenesulfonamide, was more active against hCA V over hCA II (selectivity ratio of 1.39), whereas all other derivatives investigated here were much better hCA II inhibitors (selectivity ratios CA II/CA V in the range of 0.0008-0.73) than hCA V inhibitors.