Fabrizio Briganti

Carbonic anhydrase inhibitors: Sulfonamides as antitumor agents? ☆

Novel sulfonamide inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) were prepared by reaction of aromatic or heterocyclic sulfonamides containing amino, imino, or hydrazino moieties with N,N-dialkyldithiocarbamates in the presence of oxidizing agents (sodium hypochlorite or iodine). The N,N-dialkylthiocarbamylsulfenamido-sulfonamides synthesized in this way behaved as strong inhibitors of human CA I and CA II (hCA I and hCA II) and bovine CA IV (bCA IV). For the most active compounds, inhibition constants ranged from 10(-8) to 10(-9) M (for isozymes II and IV). Three of the derivatives belonging to this new class of CA inhibitors were also tested as inhibitors of tumor cell growth in vitro. These sulfonamides showed potent inhibition of growth against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. With several cell lines. GI50 values of 10-75 nM were observed. The mechanism of antitumor action with the new sulfonamides reported here remains obscure, but may involve inhibition of CA isozymes which predominate in tumor cell membranes (CA IX and CA XII), perhaps causing acidification of the intercellular milieu, or inhibition of intracellular isozymes which provide bicarbonate for the synthesis of nucleotides and other essential cell components (CA II and CA V). Optimization of these derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents.

Carbonic anhydrase inhibitors. Part 37. Novel classes of isozyme I and II inhibitors and their mechanism of action. Kinetic and spectroscopic investigations on native and cobalt-substituted enzymes

Summary The interaction of Zn(II)- and Co(II)-carbonic anhydrase (CA) with a series of compounds possessing moieties resembling the aromatic sulfonamides, such as sulfamide, sulfamic acid, N -substituted aromatic sulfonamides, sulfenamides, sulfinic and seleninic acids, was investigated using kinetic and spectroscopic techniques. All these compounds inhibit the hydrasic and esterasic activity of the enzyme. Their binding within the active site of isozymes I and II is discussed on the basis of modifications of electronic and 1 H-NMR spectra of their adducts with the Co(II) enzyme. Some of these compounds represent novel classes of CA inhibitors, possessing equal or stronger potencies than the prototypical inhibitors, the unsubstituted sulfonamides. Qualitative structure-activity correlations are discussed.

Carbonic anhydrase inhibitors — Part 52. Metal complexes of heterocyclic sulfonamides: A new class of strong topical intraocular pressure-lowering agents in rabbits†

Abstract A new sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties has been prepared by reaction of 5-amino-1,3,4-thiadiazole-2-sulfonamide with adamantyl-1-carboxylic acid chloride. Metal complexes of the adamantyl derivative, containing di- and trivalent cations, were also obtained and characterized by standard procedures. Although the parent sulfonamide does not possess topical pressure-lowering effects in rabbits when applied as a 2% solution directly into the eye, some of its metal complexes, such as the Zn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals better than dorzolamide, the topical sulfonamide with strong antiglaucoma action, recently introduced in clinical medicine. We propose here that the whole class of metal complexes of heterocyclic sulfonamides (which generally possess much stronger CA inhibitory properties as compared to the original sulfonamides from which they were prepared) might be used for lowering IOP with enhanced efficiencies as compared to those of the simple sulfonamides. Thus, metal complexes of sulfonamides possessing themselves topical antiglaucoma properties might lead to more effective pharmacological agents of this type.

Carbonic anhydrase inhibitors ☆: synthesis of water-soluble, topically effective intraocular pressure lowering aromatic/heterocyclic sulfonamides containing 8-quinoline-sulfonyl moieties: is the tail more important than the ring?

Reaction of 20 aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with 8-quinoline-sulfonyl chloride afforded a series of water-soluble (as hydrochloride or triflate 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 (in nanomolar range), which is the isozyme known to play a critical role in aqueous humor secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were topically applied as 2% water solutions onto the eye of normotensive and glaucomatous albino rabbits, when 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 such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the 8-quinoline-sulfonyl moiety) conferring water solubility to a sulfonamide CA inhibitor is more important than the ring to which the sulfonamido group is grafted, we also prepared a dorzolamide derivative to which the 8-quinoline-sulfonyl moiety was attached. This new compound is quite water soluble as hydrochloride salt, behaves as a strong CA II inhibitor, and fared better than the parent molecule in lowering IOP in experimental animals. Thus, the tail conferring water solubility to such an enzyme inhibitor is more important for its topical activity as antiglaucoma drug than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.

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 53?. Synthesis of substituted-pyridinium derivatives of aromatic sulfonamides: The first non-polymeric membrane-impermeable inhibitors with selectivity for isozyme IV

Abstract Reaction of three aromatic sulfonamides containing a free amino group, i.e., sulfanilamide, homosulfanilamide and 4-(2-aminoethyl)-benzenesulfonamide with di-, tri- or tetra-substituted pyrylium salts afforded three series of cationic sulfonamides, containing a large variety of moieties substituting the pyridinium ring. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Efficient inhibition was observed against all three isozymes, but due to the cationic nature of these inhibitors, in vivo and ex vivo experiments showed that only CA IV is selectively inhibited to a high degree, without affecting the cytosolic isozymes, present in appreciable concentrations in the experimental model used. This is the first example of selective in vivo inhibition of only one physiologically relevant CA isozyme with non-polymeric inhibitors and might lead to more selective drugs from this class of pharmacological agents.

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: Combined actions for the decolorization of different classes of textile dyes

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.

Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens

A catechol 1,2‐dioxygenase (C1,2O) has been purified to homogeneity from Acinetobacter radioresistens grown on phenol as the sole carbon and energy source. The C1,2O appears to be a homodimer, with a molecular mass of 78 000 Da. At relatively high ionic strengths (0.5 M Na2SO4) subunit dissociation occurs and the monomeric unit (38 700 Da) is shown to be active. This phenomenon has never been observed before in dioxygenases. The purified C1,2O contains 0.96 iron(III) ions per unit and spectroscopic measurements suggest the presence of one high‐spin iron(III) ion in an environment characteristic of intradiol cleaving enzymes. The NH2‐terminal amino acid sequence has been determined and compared to the primary structures of intradiol rings cleaving dioxygenases from other Acinetobacter strains revealing 45% homology with the benzoate‐grown A. calcoaceticus ADP‐1 and an identity of only one of the 20 amino acids sequenced for the phenol‐grown A. calcoaceticus NCIB 8250.

The electron-nucleus dipolar coupling in slow rotating systems. 2. The effect of g anisotropy and hyperfine coupling when S = 12 and I = 32

Abstract An equation has been derived for the field dependence of nuclear T1−1 enhancement due to dipolar coupling with an electron of spin 1 2 which takes into consideration g anisotropy under slow rotation conditions. In analogy with the fast rotating systems, g anisotropy effects are found to be relatively small. An equation has also been derived for the S = 1 2 system coupled with a nuclear spin vector I = 3 2 under isotropic coupling conditions. The effect of coupling on the nuclear T1−1 values is dramatic for coupling constant A values larger than ℏτs−1, where τs is the electronic relaxation time. We have derived an analytical solution also for the case A∥ ≠ 0, A⊥ = 0 and I = 3 2 , while for actual cases with A∥ ≠ 0, A⊥ ≠ 0 numerical solutions are given. They are shown to account for previously unexplained experimental data. As an example, the fitting of the field dependence of water proton T1−1 values in solutions of the copper(II)-containing enzyme superoxide dismutase is presented. The experimental curves, including their temperature dependence, are nicely reproduced by using the actual ESR parameters. It is also stressed that for magnetic field values > 5 MHz, i.e., gβeH ⪢ A∥, A⊥, the simple Solomon equation is adequate for fitting the data.

Carbonic anhydrase inhibitors. Part 71. Synthesis and ocular pharmacology of a new class of water-soluble, topically effective intraocular pressure lowering sulfonamides incorporating picolinoyl moieties.

Reaction of 20 aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with picolinic acid in the presence of carbodiimide derivatives afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Efficient inhibition was observed against all three isozymes, but especially against CA II and CA 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 or glaucomatous albino rabbits. Very strong intraocular pressure (IOP) lowering was observed for many of them, and the active drug was detected in eye tissues and fluids. 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 such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the picolinoyl moiety) conferring water solubility to a sulfonamide CA inhibitor is critically important for its topical effectiveness, similarly to the ring to which the sulfonamido group is grafted, we also prepared a dorzolamide derivative to which the picolinoyl moiety was attached. This new compound is more water soluble than dorzolamide (as hydrochloride salt), behaves as a strong CA II inhibitor, and acts similarly to the parent derivative in lowering IOP in experimental animals. Thus, it seems that the tail conferring water solubility is more important for topical activity as antiglaucoma drug, than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.