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.     

Carbonic anhydrase inhibitors: N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines as topically acting antiglaucoma agents in hypertensive rabbits

A series of N-(p-sulfamoylphenyl)-alpha-D-glycopyranosylamines was prepared by reaction of sulfanilamide with different monosaccharides in the presence of ammonium chloride. The new compounds were investigated for inhibition of the metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1), involved in aqueous humor secretion within the mammalian eye. Isozymes CA I and CA II were strongly inhibited by some of these compounds, which showed inhibition constants in the range of 510-1200 nM against CA I and 10-25 nM against CA II, similarly to clinically used sulfonamides, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide and brinzolamide. The presence of sugar moieties in these molecules induced an enhanced water solubility as compared to other sulfonamides. In hypertensive rabbits (a widely used animal model of glaucoma), two of the new compounds showed strong and long-lasting intraocular pressure (IOP) lowering, being more effective than dorzolamide and brinzolamide, the two clinically used, topically acting antiglaucoma sulfonamides with CA inhibitory properties.     

Carbonic anhydrase inhibitors: topical sulfonamide antiglaucoma agents incorporating secondary amine moieties

Reaction of aromatic/heterocyclic sulfonamides possessing free amino, imino or hydrazino moieties with 7-chloro-4-chloromethylcoumarin afforded a series of N-[(7-chloro-4-coumarinyl)-methyl]- derivatives which showed effective inhibition of three carbonic anhydrase (CA) isozymes. Topical application within the rabbit eye of some of these compounds led to effective intraocular pressure lowering due to CA inhibition within the ocular tissues, and reduced aqueous humor production.     

Carbonic anhydrase inhibitors: synthesis of sulfonamides incorporating dtpa tails and of their zinc complexes with powerful topical antiglaucoma properties

Reaction of diethylenetriamino pentaacetic acid (dtpa) dianhydride with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded bis-sulfonamides containing metal-complexing, polyamino-polycarboxylic acid moieties in their molecule. The corresponding mono-sulfonamide derivatives of dtpa were also obtained by an alternative method, from the free acid. Zn(II) complexes of these new sulfonamides were then prepared. Many of these derivatives showed nanomolar affinity towards isozymes I, II and IV of carbonic anhydrase (CA). Some of the best inhibitors were applied as 2% water solutions/suspensions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with a topically acting antiglaucoma sulfonamide

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with a topically acting antiglaucoma sulfonamide (the 2-N,N-diethylaminoethylamide of 5-(4-carboxybenzenesulfonamido-1,3,4-thiadiazole-2-sulfonamide), has been resolved at a resolution of 1.6A. This compound is a very potent inhibitor of the physiologically most relevant isozyme hCA II for the secretion of aqueous humor within the eye K(I) of 1.4 nM), and in animal models of glaucoma showed very effective intraocular pressure (IOP) lowering after topical administration. Surprisingly, the inhibitor bound within the enzyme active site is in the sulfonylimido-4H- delta(2)-1,3,4-thiadiazoline tautomeric form. The inhibitor is directly bound to the Zn(II) ion of the enzyme through the deprotonated primary sulfonamide moiety, participating to the classical hydrogen bond network involving residues of the zinc-binding function and Thr 199 and Glu 106. The 1,3,4-thiadiazoline fragment of the inhibitor makes two hydrogen bonds with the active site residue Thr 200, the secondary sulfonamide moiety makes two hydrogen bonds involving a water molecule and the residue Gln 92, whereas the phenyl ring of the inhibitor participates to an edge-to-face interaction with the phenyl ring of Phe 131, the two cycles being almost perfectly perpendicular to each other. The tertiary amine fragment of the carboxamido tail and the carboxamido moiety itself make hydrogen bonds with water molecules present at the rim of the active site entrance and van der Waals contacts with His 4, Trp 5, and Phe 20. All these multiple interactions never evidenced previously in CA-sulfonamide complexes, explain the very high affinity of this inhibitor for the hCA II active site and may allow further optimization of this class of inhibitors.     

Carbonic anhydrase inhibitors. Design of anticonvulsant sulfonamides incorporating indane moieties

A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1- and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied. The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very potent inhibitors of the slow red blood cell isozyme hCA I (K(i)s in the range of 1.6-8.5 nM), which usually has a lower affinity for such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (K(i)s in the range of 2.3-12 nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfamate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43-89 nM against the two isozymes, being among the most potent sulfonic acid CA inhibitors reported so far.     

Carbonic anhydrase inhibitors. Preparation of potent sulfonamides inhibitors incorporating bile acid tails

Reaction of TBDMS-protected bile acids (cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic acids) or dehydrocholic acid with aromatic/heterocyclic sulfonamides possessing free amino/hydroxy moieties, in the presence of carbodiimides, afforded after deprotection of the OTBDMS ethers, a series of sulfonamides incorporating bile acid moieties in their molecules. Many such derivatives showed strong inhibitory properties against three isozymes of carbonic anhydrase (CA, EC 4.2.1.1), that is CA I, II and IV, zinc enzymes playing critical roles in many pathologies, and which represent interesting targets for developing diverse pharmacological agents. Some of the most active derivatives, incorporating 1,3,4-thiadiazole-2-sulfonamide or benzothiazole-2-sulfonamide functionalities in their molecules, showed low nanomolar affinity for CA II and CAIV. Furthermore, the bioavailability of these derivatives in rabbits is comparable to that of acetazolamide, being in the range of 85-90%, showing them as promising candidates for systemically acting CA inhibitors.     

Carbonic anhydrase inhibitors with strong topical antiglaucoma properties incorporating a 4-(2-aminopyrimidin-4-yl-amino)-benzenesulfonamide scaffold

Reaction of 4-(2-amino-pyrimidin-4-yl-amino)-benzenesulfonamide with alkyl/aryl-sulfonyl halides, acyl halides or arysulfonyl isocyanates afforded a series of derivatives which were tested for inhibition of three carbonic anhydrase (CA) isozymes. These compounds were designed in such a way as to (i) strongly inhibit several CA isozymes involved in aqueous humor secretion within the eye (such as CA II and CA IV), and (ii) to possess a pharmacological profile that allows easy penetration through the cornea, when administered as eye drops in solution or suspension, constituting thus a valuable therapeutic approach for glaucoma. Several of the obtained inhibitors showed low nanomolar affinities for the two isozymes involved in aqueous humor secretion, CA II and CA IV. Furthermore, in normotensive and hypertensive rabbits, some of them showed an effective and prolonged intraocular pressure (IOP) lowering when administered topically, as 2% suspensions/solutions.     

Carbonic anhydrase inhibitors: 4-sulfamoyl-benzenecarboxamides and 4-chloro-3-sulfamoyl-benzenecarboxamides with strong topical antiglaucoma properties.

Reaction of 4-carboxy-benzenesulfonamide or 4-chloro-3-sulfamoyl benzoic acid with carboxy-protected amino acids/dipeptides, or aromatic/heterocyclic sulfonamides/mercaptans afforded the corresponding benzene-carboxamide derivatives. These were tested as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV. Some of the new derivatives showed affinity in the low nanomolar range for isozymes CA II and IV, involved in aqueous humor secretion within the eye, and were tested as topically acting anti-glaucoma agents, in normotensive and glaucomatous rabbits. Good in vivo activity and prolonged duration of action has been observed for some of these derivatives, as compared to the clinically used drugs dorzolamide and brinzolamide. Some of the 4-chloro-3-sulfamoyl benzenecarboxamides reported here showed higher affinity for CA I than for the sulfonamide avid isozyme CA II.     

Carbonic anhydrase inhibitors: aromatic and heterocyclic sulfonamides incorporating adamantyl moieties with strong anticonvulsant activity

A series of aromatic/heterocyclic sulfonamides incorporating adamantyl moieties were prepared by reaction of aromatic/heterocyclic aminosulfonamides with the acyl chlorides derived from adamantyl-1-carboxylic acid and 1-adamantyl-acetic acid. Related derivatives were obtained from the above-mentioned aminosulfonamides with adamantyl isocyanate and adamantyl isothiocyanate, respectively. Some of these derivatives showed good inhibitory potency against two human CA isozymes involved in important physiological processes, CA I, and CA II, of the same order of magnitude as the clinically used drugs acetazolamide and methazolamide. The lipophilicity of the best CA inhibitors was determined and expressed as their experimental log k' IAM and theoretical ClogP value. Their lipophilicity was propitious with the crossing of the blood-brain barrier (log k' > IAM > 1.35). The anticonvulsant activity of some of the best CA inhibitors reported here has been evaluated in a MES test in mice. After intraperitoneal injection (30 mg kg(-1)), compounds A8 and A9 exhibited a high protection against electrically induced convulsions (> 90%). Their ED50 was 3.5 and 2.6 mg kg(-1), respectively.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Carbonic anhydrase inhibitors: synthesis of water soluble sulfonamides incorporating a 4-sulfamoylphenylmethylthiourea scaffold,with potent intraocular pressure lowering properties

Reaction of thiophosgene with 4-aminomethyl-benzenesulfonamide afforded 4-isothiocyanatomethyl-benzenesulfonamide, which by reaction with amines, amino acids and oligopeptides, lead to a series of new sulfonamides incorporating a 4-sulfamoylphenylmethylthiourea scaffold. These new thioureas showed strong affinities towards isozymes I, II and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory potency was good (in the low nanomolar range) for the derivatives of: amino-benzoic acids, beta-phenyl-serine, alpha-phenyl-glycine, for those incorporating hydroxy- and mercapto-amino acids (Ser, Thr, Cys and Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr; DOPA); dicarboxylic amino acids as well as di-/tri-/tetrapeptides among others. Such CA inhibitors displayed very good water solubility (in the range of 2-3%) as sodium (carboxylate) salts, with pH values for the solutions obtained of 6.5-7.0. Furthermore, in normotensive rabbits, some of them showed an effective and prolonged intraocular pressure (IOP) lowering when administered topically, as 2% solutions.     

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: design of spin-labeled sulfonamides incorporating TEMPO moieties as probes for cytosolic or transmembrane isozymes

A series of spin-labeled sulfonamides incorporating TEMPO moieties were synthesized by a procedure involving the formation of a thiourea functionality between the benzenesulfonamide and free radical fragment of the molecules. The new compounds were tested as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and showed efficient inhibition of the physiologically relevant isozymes hCA II and hCA IX (hCA IX being predominantly found in tumors) and moderate to weak inhibitory activity against hCA I. Some derivatives were also selective for inhibiting the tumor-associated isoform over the cytosolic one CA II, and presented significant changes in their ESR signals when complexed to the enzyme active site, being interesting candidates for the investigation of hypoxic tumors overexpressing CA IX by ESR techniques, as well as for imaging/treatment purposes.     

Carbonic anhydrase inhibitors: aromatic sulfonamides and disulfonamides act as efficient tumor growth inhibitors

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10(-8)-10(-9) M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI50 (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 microM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1,3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

Carbonic anhydrase inhibitory activity of sulfonamides and carboxylic acids incorporating cyclic imide scaffolds

A series of sulfonamides incorporating cyclic imide moieties were investigated as inhibitors of several human α-carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Several carboxylic acids possessing the same scaffolds as the sulfonamides were also included in the study, since the sulfonamidate and the carboxylate are among the frequently used zinc-binding groups (ZBGs) for obtaining zinc enzymes inhibitors. The cytosolic isoform hCA I was moderately inhibited by most of the 30 investigated derivatives; many low nanomolar hCA II inhibitors were detected, whereas some of these compounds were low nanomolar/subnanomolar inhibitors of the transmembrane, tumor-associated isoforms hCA IX and XII. In this series of compounds the SO₂NH⁻ and the COO⁻ ZBGs showed similar efficacy for obtaining potent inhibitors, although some carboxylates had isoform-selective inhibition profiles for the transmembrane CAs.     

Carbonic anhydrase inhibitors: inhibition of the transmembrane isozyme XIV with sulfonamides

The inhibition of the last human carbonic anhydrase (CA, EC 4.2.1.1) isozyme (hCA XIV) discovered has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and zonisamide), as well as the sulfamate antiepileptic drug topiramate. The full-length hCA XIV is an enzyme showing a medium-low catalytic activity, quite similar to that of hCA XII, with the following kinetic parameters at 20 degrees C and pH 7.5, for the CO2 hydration reaction: k(cat) = 3.12 x 10(5) s(-1) and k(cat)/K(M) = 3.9 x 10(7) M(-1) s(-1). All types of activities have been detected for the investigated compounds, with several micromolar inhibitors, including zonisamide, topiramate, and simple sulfanilamide derivatives (K(I)-s in the range of 1.46-6.50 microM). In addition, topiramate and zonisamide were observed to behave as weak hCA XII inhibitors, while zonisamide was an effective hCA IX inhibitor (K(I) of 5.1 nM). Some benzene-1,3-disulfonamide derivatives or simple five-membered heteroaromatic sulfonamides showed K(I)-s in the range of 180-680 nM against hCA XIV, whereas the most effective of such inhibitors, including 3-chloro-/bromo-sulfanilamide, benzolamide-like, ethoxzolamide-like, and acetazolamide/methazolamide-like derivatives, showed inhibition constant in the range of 13-48 nM. The best hCA XIV inhibitor was aminobenzolamide (K(I) of 13 nM), but no CA XIV-selective derivatives were evidenced. There are important differences of affinity of these sulfonamides/sulfamates for the three transmembrane CA isozymes, with CA XII showing the highest affinity, followed by CA IX, whereas CA XIV usually showed the lowest affinity for these inhibitors.     

QSAR study on topically acting sulfonamides incorporating GABA moieties: a molecular connectivity approach

A quantitative Structure-activity relationship study (QSAR) on a set of carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported using first-order valence connectivity index ((1)chi(v)). The inhibitory activity against three isozymes CAI, CAII (cystolic forms), and CAIV (membrane bound form), some of which are involved in important physiological processes, were considered for this purpose. All the three activities were excellently modeled by (1)chi(v) in multi-parametric regression containing indicator parameters. The results are critically discussed on the basis of various regression parameters.     

Carbonic anhydrase activity modulators: synthesis of inhibitors and activators incorporating 2-substituted-thiazol-4-yl-methyl scaffolds

A small series of 2-[4-(4-substituted-phenylsulfonyl)phenyl]-4-chloromethylthiazoles has been used as a scaffold for the preparation of carbonic anhydrase (CA) inhibitors and activators. For obtaining CA inhibitors, zinc-binding functions of the sulfamide and sulfamate type have been introduced into the molecules of these compounds, by reaction of the chloromethyl derivatives with sodium sulfamide/sodium sulfamate. For obtaining CA activators, the primary amino function has been introduced in these molecules by means of the Gabriel syntheses. The new sulfamide/sulfamates were effective CA II and CA IV inhibitors, but showed no inhibitory activity against isozyme I. The new amines on the other hand were much more effective CA I, II and IV activators compared to histamine, the lead compound used for their synthesis.     

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.