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A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzyl-L-alanine was prepared. Related compounds were obtained by reaction of N-2-nitrobenzyl-L-Ala with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in the degradation of extracellular matrix. Many of the obtained hydroxamates proved to be effective bacterial collagenase inhibitors, the main contributor to activity being the substitution pattern at the sulfonamido moiety. The best ChC inhibitors were those containing pentafluorophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P(1)(') groups among others, with affinities in the low nanomolar range. This study also proves that the 2-nitrobenzyl- moiety, similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2000, 43, 1858-1865) is an efficient P(2)(') anchoring moiety for obtaining potent bacterial collagenase inhibitors.

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.

Reaction of perfluoroalkyl/arylsulfonyl chlorides or perfluoroalkyl/arylcarbonyl chlorides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded compounds with the general formula C(x)()F(y)()Z-A-SO(2)NH(2), where Z = SO(2)NH, SO(3), CONH, or CO(2) and A = aromatic/heterocyclic moiety. The sulfonyl chlorides used in synthesis included: CF(3)SO(2)Cl, n-C(4)F(9)SO(2)Cl, n-C(8)F(17)SO(2)Cl, and C(6)F(5)SO(2)Cl, whereas the acyl chlorides were C(8)F(17)COCl and C(6)F(5)COCl. A total of 25 different sulfonamides have been derivatized by means of the above-mentioned perfluorosulfonyl/acyl halides. These new series of sulfonamides showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA). For a given sulfonamide derivatized by the above procedures, inhibitory power was greater for the alkyl/arylsulfonylated compounds, as compared to the corresponding perfluoroalkyl/arylcarbonylated ones. In vitro inhibitory activity generally increased with the number of carbon atoms in the molecule of the acylating/sulfonylating agent, with a maximum for the perfluorophenylsulfonylated and perfluorobenzoylated derivatives. Some of the prepared CA inhibitors displayed very good water solubility (in the range of 2%) and strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. The good water solubility of these new classes of CA inhibitors, correlated with the neutral pH of their solutions used in the ophthalmologic applications, makes them attractive candidates for developing novel types of antiglaucoma drugs devoid of unpleasant ocular side effects.

The three-dimensional structure of a possible intermediate in the hydration reaction of cyanamide to urea catalyzed by human carbonic anhydrase II (hCAII) has been determined by cryocrystallographic techniques. The crystal structure shows that two different adducts are formed under the experimental conditions and that they have different occupancy in the crystal. The high occupancy form consists of a binary hCAII-cyanamide complex where the substrate has replaced the zinc-bound hydroxide anion present in the native enzyme, maintaining the tetrahedral geometry around the metal ion. The second, low-occupancy form consists of a hCAII-cyanamide-water ternary complex where the catalytic zinc ion, still being bound to cyanamide, is approached by a water molecule in a five-coordinate adduct. While the first form can be considered a nonproductive complex, the second form may represent an intermediate state of the catalyzed reaction where the water molecule is about to perform a nucleophilic attack on the zinc-activated cyanamide substrate. The structural evidence is consistent with the kinetic data previously reported about this recently described hydrolytic reaction catalyzed by hCAII, and indicates that a different mechanism with respect to that generally accepted for the physiologic carbon dioxide hydration reaction may be adopted by the enzyme, depending on the substrate chemical properties.

The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physiological functions in these organisms. Unsubstituted sulfonamides act as high-affinity inhibitors for the first type of these enzymes, whereas hydroxamates strongly inhibit the latter ones. Since the active site geometry around the zinc ion in these two types of metalloenzymes is rather similar, we tested whether sulfonylated amino acid hydroxamates of the type RSO(2)NX-AA-CONHOH (X = H, benzyl, substituted benzyl; AA = amino acid moiety, such as Gly, Ala, Val, Leu) with well-known inhibitory properties against MMPs and Clostridium histolyticum collagenase (ChC, another zinc enzyme related to the MMPs) might also act as CA inhibitors. We also investigated whether N-hydroxysulfonamides of the type RSO(2)NHOH (which are effective CA inhibitors) inhibit MMPs and ChC. Here we report several potent sulfonylated amino acid hydroxamate CA inhibitors (with inhibition constants in the range of 5-40 nM, against the human isozymes hCA I and hCA II, and 10-50 nM, against the bovine isozyme bCA IV), as well as preliminary SAR for this new class of non-sulfonamide CA inhibitors. Some N-hydroxysulfonamides also showed inhibitory properties (in the micromolar range) against MMP-1, MMP-2, MMP-8, MMP-9, and ChC. Thus, the SO(2)NHOH group is a new zinc-binding function for the design of MMP inhibitors. Both CA as well as MMPs are involved, among others, in carcinogenesis and tumor invasion processes. On the basis of these findings, we suggest that the mechanism of antitumor action with some hydroxamate inhibitors might also involve inhibition of some CA isozymes (such as CA IX, CA XII, and CA XIV) present only in tumor cell membranes, in addition to collagenases/gelatinases of the MMP type. Our data also suggest that it should be possible to develop dual enzyme inhibitors that would strongly inhibit both these metalloenzymes, CAs and MMPs, based on the nature of the R, AA, and X moieties in the above formula. Compact X (such as H) and AA (such as Gly) moieties favor CA over MMP inhibition, whereas bulkier X (benzyl, substituted benzyl, etc.) and AA (such as Val, Leu) moieties and substituted-aryl R groups are advantageous for obtaining potent MMP and ChC inhibitors, which show lower affinity for CA.

A series of compounds was prepared by reaction of alkyl/arylsulfonyl halides with N-4-nitrobenzylglycine, followed by conversion of the COOH to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-4-nitrobenzylglycine with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzylglycine by reaction with arylsulfonyl isocyanates, followed by conversion of the COOH to 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 best inhibitors of MMP-1, a short pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethylphenylsulfonyl moieties at P(1') (K(I)'s of 3-5 nM). For MMP-2, MMP-8, and MMP-9 (deep-pocket enzymes), best inhibitors were especially those containing long perfluoroalkylsulfonyl and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl, arylsulfonylureido, or arylsulfonylureidosulfanilyl/metanilyl moieties, at P(1'). Bulkier groups in this position, such as 1- and 2-naphthyl, substituted-naphthyl, or quinolin-8-yl moieties among others, led to less effective MMP/ChC inhibitors. Best ChC inhibitors were again those containing pentafluorophenylsulfonyl or 3- and 4-protected-aminophenylsulfonyl P(1') anchoring groups, suggesting that this protease is also a short-pocket wider-neck one (more similar to MMP-1). This study also proves that the 4-nitrobenzyl moiety is an efficient P(2') anchoring moiety and that sulfonylureido, ureido, or carboxythioureido substitutions at P(1') are also tolerated for obtaining potent sulfonylated amino acid hydroxamate-like MMP/ChC inhibitors.

Using benzamidine as a lead molecule, two series of alkyl/aralkyl/arylsulfonylguanidines/sulfonyl-O-methylisoureas+ ++ have been prepared and assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that sulfaguanidine and its corresponding O-methylisourea derivative possess moderate but intrinsically selective thrombin inhibitory properties, with K(I)'s around 100 nM against thrombin and 1350-1500 nM against trypsin. Further elaboration of these two molecules afforded compounds that inhibited thrombin with K(I)'s in the range of 12-50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as L- and D-Phe or L-Pro) or dipeptides (such as Phe-Pro, Gly-His, beta-Ala-His, or Pro-Gly) to the two leads mentioned above, sulfaguanidine and 4-aminobenzenesulfonyl-O-methylisourea. Thus, the present study proposes two novel approaches for the preparation of high-affinity, specific thrombin inhibitors: two novel S1 anchoring moieties in the already large family of arginine/amidine-based inhibitors and novel peptidomimetic scaffolds obtained by incorporating tosylureido amino acids 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 due to facilitated metal (sodium) salts formation (at the relatively acidic SO(2)NHCO protons) as well as increased stability at hydrolysis (in vivo). A QSAR study also explained the activity in terms of global properties of the molecules, electronic properties of the sulfonylguanidine/sulfonylisourea moiety, and novel descriptors, the frontier orbital phase angles (FOPA), that account for the directions of the nodes in the pi orbitals in the aromatic portion of those of the drugs in which the sulfonyl group was bound to a benzene ring. For thrombin inhibition, the size of the molecule was the dominant influence, while for trypsin inhibition the FOPA was the principal determinant of activity. The dependence of activity on the FOPA variables is perhaps the clearest example of a quantum effect in pharmacology and suggests a promising new tool for drug design.

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.

Reaction of 26 aromatic/heterocyclic sulfonamides containing amino, imino, hydrazino, or hydroxyl groups with Boc-Gly, Boc-Sar, TrS-Crt, or Boc-Gly-Gly (Sar = sarcosine, N-Me-Gly; Crt = creatine, N-amidinosarcosine; TrS = tritylsulfenyl; Boc = tert-butoxycarbonyl) in the presence of carbodiimide derivatives afforded after removal of the protecting groups a series of water-soluble compounds (as salts of strong acids, such as hydrochloric, trifluoroacetic, or trifluoromethanesulfonic). The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA) and more precisely of three of its isozymes, CA I, II (cytosolic forms), and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes and especially against CA II and IV (in the nanomolar range), the two isozymes known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as 2% water solutions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. Thus, the aminoacyl/dipeptidyl tail conferring water solubility to these sulfonamide CA inhibitors coupled with strong enzyme inhibitory properties and balanced lipid solubility seem to be the key factors for obtaining compounds with effective topical antiglaucoma activity.

Reaction of several aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino, or hydroxyl group, with 2, 3-pyridinedicarboxylic anhydride or 2,6-pyridinedicarboxylic acid in the presence of carbodiimide derivatives, afforded two series of water-soluble (as hydrochloride, triflate, or carboxylate salts) compounds. The new derivatives were assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA) and more precisely of three of its isozymes, CA I, II (cytosolic forms), and IV (membrane-bound form), involved in important physiological processes. Efficient inhibition was observed against all three isozymes, but especially against CA II and IV (in nanomolar range), the two isozymes known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as 2% water solutions directly into the eye of normotensive and glaucomatous albino rabbits. Very strong and long-lasting intraocular pressure (IOP) lowering was observed with many of them. This result prompted us to reanalyze the synthetic work done by other groups for the design of water-soluble, topically effective antiglaucoma sulfonamides. According to these researchers, the IOP-lowering effect is due to the intrinsic nature of the specific heterocyclic sulfonamide considered, among which the thienothiopyran-2-sulfonamide derivatives represent the best-studied case. Indeed, the first agents developed for topical application, such as dorzolamide, are derivatives of this ring system. To prove that the tail (in this case the pyridinecarboxylic moieties) conferring water solubility to a sulfonamide CA inhibitor is more important than the ring to which the sulfonamido group is grafted, we also prepared dorzolamide derivatives incorporating such moieties. These new compounds possess good water solubility as hydrochloride or carboxylate salts, balanced by a relatively modest lipid solubility. They are strong CA II inhibitors and are able to lower IOP in experimental animals more than the parent derivatives. Our conclusion is that the tail conferring water solubility to such an enzyme inhibitor is more important for topical activity as an antiglaucoma drug, than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.

The interaction of native and Co(II)-substituted isozymes I and II of carbonic anhydrase (CA) with histamine, a well-known activator, was investigated kinetically, spectroscopically, and X-ray crystallographically. This activator is of the noncompetitive type with 4-nitrophenyl acetate and CO2 as substrates for both HCA I and HCA II. The electronic spectrum of the adduct of Co(II)-HCA II with histamine is similar to the spectrum of the Co(II)-HCA II-phenol adduct, being only slightly different from that of the uncomplexed enzyme. This is the first spectroscopic evidence that the activator molecule binds within the active site, but not directly to the metal ion. X-ray crystallographic data for the adduct of HCA II with histamine showed that the activator molecule is bound at the entrance of the active site cavity in a position where it may actively participate in shuttling protons between the active site and the bulk solvent. The role of the activators and the reported X-ray crystal structure of the HCA II-histamine adduct has prompted us to reexamine the X-ray structures of the different CA isozymes in order to find a structural basis accounting for their large differences in catalytic rate. A tentative explanation is proposed on the basis of possible pathways of proton transfer, which constitute the rate-limiting step in the catalytic reaction.