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.     

Carbonic anhydrase inhibitors: novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX

A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide. The prepared dichlorotriazinyl-benzenesulfonamides were subsequently derivatized by reacting them with various nucleophiles, such as ammonia, hydrazine, primary and secondary amines, amino acid derivatives or phenol. The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumour-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. Structure-activity relationship was straightforward and rather simple in this class of CA inhibitors, with the compounds incorporating compact moieties at the triazine ring (such as amino, hydrazino, ethylamino, dimethylamino or amino acyl) being the most active ones, and the derivatives incorporating such bulky moieties (n-propyl, n-butyl, diethylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy) being less effective hCA I, II and IX inhibitors. Some of the new derivatives also showed selectivity for inhibition of hCA IX over hCA II (selectivity ratios of 23.33-32.00), thus constituting excellent leads for the development of novel approaches for the management of hypoxic tumours.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K(I)s in the range of 75-136nM, hCA II with K(I)s in the range of 13-278nM, and hCA IX with K(I)s in the range of 0.12-549nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K(I)s in the range 0.12-0.34nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.     

Investigation of piperazines as human carbonic anhydrase I,II, IV and VII activators

Four human (h) carbonic anhydrase isoforms (CA, EC 4.2.1.1), hCA I, II, IV, and VII, were investigated for their activation profile with piperazines belonging to various classes, such as N-aryl-, N-alkyl-, N-acyl-piperazines as well as 2,4-disubstituted derivatives. As the activation mechanism involves participation of the activator in the proton shuttling between the zinc-coordinated water molecule and the external milieu, these derivatives possessing diverse basicity and different scaffolds were appropriate for being investigated as CA activators (CAAs). Most of these derivatives showed CA activating properties against hCA I, II, and VII (cytosolic isoforms) but were devoid of activity against the membrane-associated hCA IV. For hCA I, the K_As were in the range of 32.6–131?µM; for hCA II of 16.2–116?µM, and for hCA VII of 17.1–131?µM. The structure-activity relationship was intricate and not easy to rationalize, but the most effective activators were 1-(2-piperidinyl)-piperazine (K_A of 16.2?µM for hCA II), 2-benzyl-piperazine (K_A of 17.1?µM for hCA VII), and 1-(3-benzylpiperazin-1-yl)propan-1-one (K_A of 32.6?µM for hCA I). As CAAs may have interesting pharmacologic applications in cognition and for artificial tissue engineering, investigation of new classes of activators may be crucial for this relatively new research field.     

Inhibition of carbonic anhydrase isozymes with benzene sulfonamides incorporating thio,sulfinyl and sulfonyl glycoside moieties

A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthesized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four human carbonic anhydrases (hCA): isozymes I, II and tumour-associated isozymes IX and XII. The oxidation state of the sulfur in the carbohydrate tail moiety did not influence either enzyme inhibition potency or isozyme selectivity even though presenting opportunities for differing interactions with the target isozymes.     

3-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I,II and IV

We describe the synthesis of a series of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a–k) acting as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. Reaction of alkyl/aryl isothiocyanates with 3-aminobenzenesulfonamide afforded a series of the title compounds incorporating a variety of short as well as highly lipophilic long tails. The newly synthesized sulfonamides were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IV, and IX). Several compounds showed interesting inhibitory activity. The tumor-associated hCA IX was the most sensitive isoform to inhibition with these compounds, with K_Is in the range of 21.5–44.0 nM and selectivity ratios over the major cytosolic isoform hCA II in the range of 3.35–37.3. The sulfonamides incorporating the phenylacetylthioureido and pentadecanoylthioureido moieties were the most hCA IX-selective inhibitors detected in this work, making them of interest for further investigations.     

Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs as inhibitors of the physiological carbonic anhydrase isozymes I,II and IV and tumor-associated isozyme IX

A new series of thirty s-triazinyl-substituted aminoalkylbenzenesulfonamides, incorporating a symmetric pair of amino acid moieties, is reported, together with inhibition studies of physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II, transmembrane hCA IV and the tumor-associated, membrane-bound hCA IX.The compounds were prepared by nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) using environmentally friendly water-based synthetic conditions. The products yields ranged in the interval of 43–97%. Purity of the products was verified by the HPLC-DAD-ESI-Q-TOF MS method. Identity of the products was confirmed by the same method plus NMR and IR.The products showed weak inhibition of the cytosolic, off-target isozyme hCA II, but some of them were low nanomolar (i.e. strong) inhibitors of the tumor-associated hCA IX. The series offered representatives selective towards isozymes hCA I, IV and IX. 2,2′-((6-((4-sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid demonstrated highest selectivity to the tumor-associated isoform hCA IX over off-target isozymes, with impressive K_I ratio (hCA II/hCA IX) 213.9 and inhibition constant equal to acetazolamide (K_I = 25.8 nM). Although the selectivities of some other products, e.g. those conjugating Leu and Glu, were a bit lower (188.7 and 84.3, respectively) their inhibition constants were similar to acetazolamide too (24.0 and 27.1, respectively).The selected most impressive results from the inhibition study were interpreted via molecular modeling experiment (docking in Glide) revealing different inter-molecular enzyme-substrate interaction of 2,2′-((6-((4-sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid within specific hCA IX and hCA II microregions. Therefore, several selected compounds from this study can be considered as highly effective and selective inhibitors of hCA IX, worthy to further (preclinical) investigation.     

Sulfonamides incorporating piperazine bioisosteres as potent human carbonic anhydrase I,II, IV and IX inhibitors

Starting from the molecular simplification of (R) 4-(3,4-dibenzylpiperazine-1-carbonyl)benzenesulfonamide 9a, a compound endowed with selectivity for human Carbonic Anhydrase (hCA) IV, a series of piperazines and 4-aminopiperidines carrying a 4-sulfamoylbenzamide moiety as Zn-binding group have been designed and tested on human isoforms hCA I, II, IV and IX, using a stopped flow CO₂ hydrase assay. The aim of the work was to derive structure-activity relationships useful for designing isoform selective compounds. These structural modifications changed the selectivity profile of the analogues from hCA IV to hCA I and II, and improved potency. Several of the new compounds showed subnanomolar activity on hCA II. X-ray crystallography of ligand-hCAII complexes was used to compare the binding modes of the new piperazines and the previously synthesized 2-benzyl-piperazine analogues, explaining the inhibition profiles.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with Schiff's bases incorporating chromone and aromatic sulfonamide moieties, and their zinc complexes

A series of Schiff's bases was prepared by reaction of 3-formyl-chromone or 6-methyl-3-formyl-chromone with aromatic sulfonamides, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide, a pyrimidinyl-substituted sulfanilamide derivative, sulfaguanidine and 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide. The zinc complexes of these sulfonamides have also been obtained. The new derivatives and their Zn(II) complexes were investigated for the inhibition of four physiologically relevant isozymes of carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms I and II, as well as the tumor-associated, transmembrane isozymes CA IX and XII. Except for the sulfaguanidine-derived compounds which were devoid of activity against all isozymes, the other sulfonamides and their metal complexes showed interesting inhibitory activity. Against isozyme CA I, the inhibition constants were in the range of 13-100 nM, against isozyme CA II in the range of 1.9-102 nM, against isozyme CA IX in the range of 6.3-48nM, and against CA XII in the range of 5.9-50nM. Generally, the formyl-chromone derived compounds were better CA inhibitors as compared to the corresponding 6-methyl-chromone derivatives, and for the simple, benzenesulfonamide derivatives activity increased with an increase of the spacer from sulfanilamide to homosulfanilamide and 4-aminoethylbenzenesulfonamide derivatives, respectively. Some of these compounds may show applications for the development of therapies targeting hypoxic tumors in which CA IX and XII are often highly overexpressed.     

Discovery of new ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as carbonic anhydrase I,II, IX and XII inhibitors

A series of twenty novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties substituted on one side with aromatic amines and on the other side with dimethylamine, morpholine and piperidine is reported. The compounds were synthesized from the 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido)benzensulfonamide (1) by using stepwise nucleophilic substitution of the chlorine atoms of cyanuric chloride. The intermediates 2(a-e) and final compounds 3(a-o) were tested for their efficiency as carbonic anhydrase (CA) inhibitors against four selected physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, namely, the cytosolic ones hCA I and II, and the transmembrane, tumor associated ones hCA IX, and XII. The compounds 2a, 2e and 3m showed the highest activity for hCA IX with K_is in the range of 11.8–14.6?nM. Most of the compounds showed high hCA IX selectivity over the abundant off-target isoforms hCA I and II. Since hCA IX is a validated drug target for anticancer/antimetastatic agents, these isoform-selective and potent inhibitors may be considered of interest for further medicinal/pharmacologic studies.     

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.     

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.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hypoxic tumors and appears to be involved in acidification of the tumor microenvironment, a process correlated with cancer progression and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic primary amines or hydrazines, leading to the corresponding thioureas. Some of these compounds showed excellent inhibitory properties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor therapies based on the inhibition of the CA isozymes overexpressed in hypoxic tumors. Due to the highest expression of CA IX in clear renal cell carcinoma and its chemo/radioresistance, our efforts are first of all directed to generate effective therapeutic strategies for the cure of this malignancy.     

Synthesis and biological evaluation of histamine Schiff bases as carbonic anhydrase I,II, IV,VII, and IX activators

A series of 20 histamine Schiff base was synthesised by reaction of histamine, a well known carbonic anhydrase (CA, E.C 4.2.2.1.) activator pharmacophore, with substituted aldehydes. The obtained histamine Schiff bases were assayed as activators of five selected human (h) CA isozymes, the cytosolic hCA I, hCA II, and hCA VII, the membrane-anchored hCA IV and transmembrane hCA IX. Some of these compounds showed efficient activity (in the nanomolar range) against the cytosolic isoform hCA VII, which is a key CA enzyme involved in brain metabolism. Moderate activity was observed against hCA I and hCA IV (in the nanomolar to low micromolar range). The structure–activity relationship for activation of these isoforms with the new histamine Schiff bases is discussed in detail based on the nature of the aliphatic, aromatic, or heterocyclic moiety present in the aldehyde fragment of the molecule, which may participate in diverse interactions with amino acid residues at the entrance of the active site, where activators bind, and which is the most variable part among the different CA isoforms.     

Carbonic anhydrase inhibitors: synthesis and inhibition against isozymes I, II and IV of topically acting antiglaucoma sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties

Sulfonamides incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido moieties in their molecules were prepared by reaction of cis-5-norbornene-endo-2,3-dicarboxylic anhydride with aromatic/heterocyclic sulfonamides possessing free amino, hydrazino, or imino groups. Some of these compounds showed very good CA II and CA IV inhibitory properties, with affinities for the enzymes in the low nanomolar range. Some of the most active CA II inhibitors reported here have been formulated as aqueous solutions for topical administration as antiglaucoma agents in normotensive rabbits. Some of the derivatives incorporating cis-5-norbornene-endo-3-carboxy-2-carboxamido and aromatic sulfonamide moieties (as sodium salts) showed effective and longer lasting intraocular pressure (IOP) lowering as compared to dorzolamide, a widely used topical antiglaucoma drug. Compounds incorporating cis-5-norbornene-endo-2,3-carboximido moieties, although stronger in vitro CA inhibitors as compared to the corresponding cis-5-norbornene-endo-3-carboxy-2-carboxamido-;derivatives, showed no topical IOP lowering properties, probably due to their very poor water solubility.     

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.     

Carbonic anhydrase activators. The selective serotonin reuptake inhibitors fluoxetine,sertraline and citalopram are strong activators of isozymes I and II

The selective serotonin reuptake inhibitors (SSRI) fluoxetine, sertraline and citalopram have been investigated for their ability to activate two carbonic anhydrase (CA) isozymes, hCA I and hCA II, in parallel with two standard activators for which the X-ray structure (in complex with isozyme II) has been resolved: histamine and phenylalanine. All three SSRI activated both isozymes with potencies comparable to that of the standards although the profile was different: for hCA I, best activators were fluoxetine and histamine, with citalopram and sertraline showing weaker activity. For hCA II, the best activators were phenylalanine and citalopram, and the weakest histamine and sertraline, whereas fluoxetine showed an intermediate behavior. These results suggest that SSRI efficacy in major depression complicating Alzheimer's disease may be partly due to their ability to activate CA isozymes and may lead to the development of potent activators for the therapy of diseases associated with significant decreases in brain CA activity.     

Novel sulfonamide incorporating piperazine,aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I,II and IX inhibitory action

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with K_Is in the range of 8.5–2679.1 nM, hCA II with K_Is in the range of 4.8–380.5 nM and hCA IX with K_Is in the range of 0.4–307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5–18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.     

Carbonic anhydrase activators: amino acyl/dipeptidyl histamine derivatives bind with high affinity to isozymes I, II and IV and act as efficient activators

Reaction of histamine (Hst) with tetrabromophthalic anhydride and protection of its imidazole moiety with tritylsulfenyl chloride, followed by hydrazinolysis, afforded N-1-tritylsulfenyl-histamine, a key intermediate which was further derivatized at its aminoethyl moiety. Reaction of the key intermediate with N-Boc-amino acids/dipeptides (Boc-AA) in the presence of carbodiimides afforded, after deprotection of the imidazolic and amino moieties, a series of compounds with the general formula AA-Hst (AA=amino acyl; dipeptidyl). The new derivatives were assayed as activators of three carbonic anhydrase (CA) isozymes, hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound form). Efficient activation was observed against all three isozymes, but especially against hCA I and bCA IV, with affinities in the nanomolar range for the best compounds. hCA II was, on the other hand, activatable with affinities around 10–20 nM. This new class of CA activators might lead to the development of drugs/diagnostic agents for the CA deficiency syndrome, a genetic disease of bone, brain and kidneys.