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: water-soluble 4-sulfamoylphenylthioureas as topical intraocular pressure-lowering agents with long-lasting effects.

A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory power was good (in the low-nanomolar range) for the derivatives of beta-phenylserine and alpha-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys, Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA), and 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%) mainly as sodium (carboxylate) salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such as the derivatives of Ser, beta-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives, devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed reduced efficacy when administered topically. This may be due to the very hydrophilic nature of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also showed optimal in vivo effects. The interesting pharmacological properties of this new type of CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic applications, make them attractive candidates for developing novel antiglaucoma drugs devoid of major ocular side effects.

Protease inhibitors: Part 4. Synthesis of weakly basic thrombin inhibitors incorporating pyridinium-sulfanilylaminoguanidine moieties.

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of beta-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing KI's around 100-300 nM against thrombin, and 450-1420 nM against trypsin, respectively, the new derivatives showed inhibition constants in the range of 15-50 nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of beta-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO2NHNHC(=NH)NH2 group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.

Carbonic anhydrase inhibitors: synthesis of sulfonamides incorporating 2,4,6-trisubstituted-pyridinium-ethylcarboxamido moieties possessing membrane-impermeability and in vivo selectivity for the membrane-bound (CA IV) versus the cytosolic (CA I and CA II) isozymes.

A new approach is proposed for the selective in vivo inhibition of membrane-bound versus cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes with a class of positively-charged, membrane-impermeant sulfonamides. Aromatic/heterocyclic sulfonamides acting as strong (but unselective) inhibitors of this zinc enzyme were derivatized by the attachment of trisubstituted-pyridinium-ethylcarboxy moieties (obtained from 2,4,6-trisubstituted-pyrylium salts and beta-alanine) to the amino, imino, hydrazino or hydroxyl groups present in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes, i.e., hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound isozyme; h = human; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, ethoxzolamide), are unable to penetrate biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors suggest that: (i) when using the new type of positively-charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected, (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II and IV) occurred.

Protease inhibitors. Part 12. Synthesis of potent matrix metalloproteinase and bacterial collagenase inhibitors incorporating sulfonylated N-4-nitrobenzyl-beta-alanine hydroxamate moieties.

N-4-Nitrobenzyl-beta-alanine was reacted with alkyl/arylsulfonyl halides, followed by conversion of the COOH to the CONHOH group. Structurally related compounds were obtained by reaction of N-4-nitrobenzyl-beta-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzyl-beta-alanine by reaction with arylsulfonyl isocyanates, followed by the introduction of the hydroxamate moiety. The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8 and MMP-9, and of the Clostridium histolyticum collagenase (ChC). Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety. Substitutions leading to the best inhibitors of MMP-1, a short-pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethyl-phenylsulfonyl at P(1') (K(I) of 3-5 nM). For MMP-2, MMP-8 and MMP-9 (deep-pocket enzymes), the best inhibitors were those containing perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, arylsulfonylureido- or arylsulfonylureido-sulfanilyl-/metanilyl moieties at P(1'). Bulkier groups in this position, such as 1- and 2-naphthyl-, substituted-naphthyl or quinoline-8-yl- moieties, among others, led to less effective MMP/ChC inhibitors. The best ChC inhibitors were again those containing pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl P(1') groups. This study demonstrates that the 4-nitrobenzyl moiety, investigated here for the first time, is an efficient P(2') anchoring moiety, whereas the beta-alanyl scaffold can successfully replace the alpha-amino acyl one, for obtaining potent MMP/ChC inhibitors.

Carbonic anhydrase inhibitors; phosphoryl-sulfonamides--a new class of high affinity inhibitors of isozymes I and II.

A series of phosphorylated aromatic/heterocyclic sulfonamides with the general formula ArSO2NHPO3H2 have been prepared by condensing ArSO2NH2 with phosphorus pentachloride, followed by controlled hydrolysis in the presence of formic acid. The new derivatives generally act as stronger inhibitors of two carbonic anhydrase (CA) isozymes, CA I and CA II, as compared to the parent unsubstituted sulfonamides from which they were obtained. The inhibition mechanism by this new class of CA inhibitors, as well as structure activity correlations for the series of investigated derivatives, are also discussed.

Carbonic anhydrase inhibitors: synthesis of membrane-impermeant low molecular weight sulfonamides possessing in vivo selectivity for the membrane-bound versus cytosolic isozymes.

Aromatic/heterocyclic sulfonamides act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1), but the presently available compounds do not generally discriminate between the 14 isozymes isolated in higher vertebrates. Thus, clinically used drugs from this class of pharmacological agents show many undesired side effects due to unselective inhibition of all CA isozymes present in a tissue/organ. Here we propose a new approach for the selective in vivo inhibition of membrane-bound versus cytosolic CA isozymes with a new class of positively charged, membrane-impermeant sulfonamides. This approach is based on the attachment of trisubstituted-pyridinium-methylcarboxy moieties (obtained from 2,4, 6-trisubstituted-pyrylium salts and glycine) to the molecules of classical aromatic/heterocyclic sulfonamides possessing free amino, imino, hydrazino, or hydroxyl groups in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes: i.e., hCA I, hCA II (cytosolic forms), and bCA IV (membrane-bound isozyme) (h = human isozyme; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, and ethoxzolamide), are unable to penetrate through biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors undoubtedly proved that: (i) when using the new type of positively charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected; (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II, and IV) has been evidenced.

Carbonic anhydrase inhibitors. Part 91. Metal complexes of heterocyclic sulfonamides as potential pharmacological agents in the treatment of gastric Acid secretion imbalances.

Zinc, magnesium, aluminum and copper complexes of several potent, clinically used carbonic anhydrase (CA) sulfonamide inhibitors, such as acetazolamide, methazolamide, ethoxzolamide and benzolamide were tested for their possible applications as antacids, in experimental animals. Gastric acid secretion parameters 3 days after treatment with these CA inhibitors (2 x 500 mg, twice a day), in dogs with chronic gastric fistulas, led to the observation that the gastric acid parameters BAO (the basal acid output), and MAO (the maximal acid output after stimulation with histamine) were drastically reduced, as compared to the same parameters in animals that did not receive these enzyme inhibitors. These are promising results for the possible use of metal complexes of heterocyclic sulfonamides as treatment alternatives (alone or in combination with other drugs) for gastric acid secretion imbalances.

Carbonic Anhydrase Inhibitors. Part 55 Metal Complexes of 1,3,4-Thiadiazole-2-Sulfonamide Derivatives: In Vitro Inhibition Studies With Carbonic Anhydrase Isozymes I, II and IV.

Coordination compounds of 5-chloroacetamido-1,3,4-thiadiazole-2-sulfonamide (Hcaz) with V(IV), Cr(lll), Fe(ll), Co(ll), Ni(ll) and Cu(ll) have been prepared and characterized by standard procedures (spectroscopic, magnetic, EPR, thermogravimetric and conductimetric measurements). Some of these compounds showed very good in vitro inhibitory properties against three physiologically relevant carbonic anhydrase (CA)isozymes, i.e., CA I, II, and IV. The differences between these isozymes in susceptibility to inhibition by these metal complexes is discussed in relationship to the characteristic features of their active sites, and is rationalized in terms useful for developing isozyme-specific CA inhibitors.