A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.     

Glycosidic carbonic anhydrase IX inhibitors: A sweet approach against cancer

Targeting tumour associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII is now considered as a pertinent approach for the development of new cancer therapeutics against hypoxic tumours. In the last period, with the help of X-ray crystallography, much progress has been achieved for the drug-design of selective CA IX inhibitors, by considering the three main structural elements that govern both potency and selectivity, that is, a zinc binding group (ZBG), an organic scaffold, and one or more side chains substituting the scaffold. The use of sugar moiety in the structure of sulfonamide-based CA inhibitors (CAIs), has allowed the discovery of very potent CA IX inhibitors able to impair the growth of both primary tumors and metastases. The search for specific CA IX inhibitors by using the sugar approach has become an important research field, leading to sulfonamides, sulfamates, sulfamides and coumarins with excellent in vitro activity and relevant potency in vivo, in animal models of cancer. This paper will review the latest development in this hot topic.     

Synthesis and evaluation of new carbonic anhydrase inhibitors

A series of new sulfamide derivatives have been synthesized, their structures were confirmed by (1)H NMR and ESI-MS. Some target compounds were assessed by the tool of Dock6, and inhibition effects of all the new compounds on carbonic anhydrase II have been investigated. In addition, some compounds have been investigated for their antihypoxic effects in mice. Results indicated that nine target compounds exhibit as effectively as acetazolamide and 10 compounds have more potent inhibition effects on carbonic anhydrase II than acetazolamide. Three of them (I-8, I-18 and I'-3) can prolong markedly the survival time of mice in hypoxia, which are worth carrying out further studies.     

Synthesis and crystallographic analysis of new sulfonamides incorporating NO-donating moieties with potent antiglaucoma action

Several aromatic/heterocyclic sulfonamide scaffolds have been used to synthesize compounds incorporating NO-donating moieties of the nitrate ester type, which have been investigated for the inhibition of five physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms: hCA I (offtarget), II, IV and XII (antiglaucoma targets) and IX (antitumor target). Some of the new compounds showed effective in vitro inhibition of the target isoforms involved in glaucoma, and the X-ray crystal structure of one of them revealed factors associated with the marked inhibitory activity. In an animal model of ocular hypertension, one of the new compounds was twice more effective than dorzolamide in reducing elevated intraocular pressure characteristic of this disease, anticipating their potential for the treatment of glaucoma.     

A nuclear-magnetic-resonance study of the binding of novel N-hydroxybenzenesulphonamide carbonic anhydrase inhibitors to native and cadmium-111-substituted carbonic anhydrase

Various ring- and nitrogen-substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N-Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N-substituted inhibitors. By contrast, N-hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase. Using 111Cd-NMR it has been possible to analyse the molecular interaction of 4-fluoro-N-hydroxybenzenesulphon[15N]amide, with 111Cd-substituted bovine carbonic anhydrase. A large cadmium-111:nitrogen-15 spin-coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4-fluorobenzenesulphon[15N]amide. The 111Cd-chemical shift of the signal for the inhibited enzyme shows that the N-hydroxysulphonamide is bound as its anion. From the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N-hydroxysulphonamide has a longer life-time than that formed with the unsubstituted sulphonamide. By contrast, native zinc-containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N-NMR were unsuccessful, possibly due to a very long relaxation time for the nitrogen nucleus in the N-hydroxysulphonamide when bound to the enzyme leading to loss of the 15N signal.     

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.     

A comparative approach to carbonic anhydrase: the work of Thomas H. Maren

Thomas H. Maren studied carbonic anhydrase (CA) for half a century, venturing into all aspects of this powerful enzyme from active site chemistry to clinical medicine. He was a keen proponent of comparative physiology to illuminate basic principles of the chemistry and biology of CA and spent 47 summers at the Mt. Desert Island Biological Laboratory (MDIBL) studying many non-mammalian species. Following the venerable strategy of selecting the right creature to explore a particular question, Maren derived important insights into the role of CA in ion transport, acid-base regulation and gas exchange. Using the fact that tissue CAs are expressed variably in different species, and that these animals differ in temperature, acid-base status and metabolic rate, he defined the contributions of un-catalyzed and catalyzed CO(2) reactions in many physiological processes. Often this strategy simplified a problem and offered answers not easily obtainable in mammals. As examples, he verified the primary role of HCO(3)(-) as lead ion in CSF formation in fish and extended this to mammals. Using marine fish whose kidneys have very little CA, he uncovered mechanisms of acid-base transfer independent of CA that help to explain why CA inhibition does not lead to total bicarbonate depletion.     

Carbonic anhydrase inhibitors: binding of an antiglaucoma glycosyl-sulfanilamide derivative to human isoform II and its consequences for the drug design of enzyme inhibitors incorporating sugar moieties

N-(4-Sulfamoylphenyl)-alpha-d-glucopyranosylamine, a promising topical antiglaucoma agent, is a potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The high resolution X-ray crystal structure of its adduct with the target isoform involved in glaucoma, CA II, is reported here. The sugar sulfanilamide derivative binds to the enzyme in a totally new manner as compared to other CA-inhibitor adducts investigated earlier. The sulfonamide anchor was coordinated to the active site metal ion, and the phenylene ring of the inhibitor filled the channel leading to the active site cavity. The glycosyl moiety responsible for the high water solubility of the compound was oriented towards a hydrophilic region of the active site, where no other inhibitors were observed to be bound up to now. A network of seven hydrogen bonds with four water molecules and the amino acid residues Pro201, Pro202 and Gln92 further stabilize the enzyme-inhibitor adduct. Topiramate, another sugar-based CA inhibitor, binds in a completely different manner to CA II as compared to the sulfonamide investigated here. These findings are useful for the design of potent, sugar-derived enzyme inhibitors.     

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.     

Attachment of carbohydrates to methoxyaryl moieties leads to highly selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII

The transmembrane isoforms of carbonic anhydrase (hCA IX and XII) have been shown to be linked to carcinogenesis and their inhibition to arrest primary tumor and metastases growth. In this paper, we present a new class of C-glycosides incorporating the methoxyaryl moiety, that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. The glycosides have been prepared by aldol reaction of glycosyl ketones with the appropriate aromatic aldehydes. We also present the inhibition profile of our new glycomimetics, against four isozymes of carbonic anhydrase comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (tumor associated isozymes). In this study, per-O-acetylated glycoside 4, 6 and deprotected compounds 7, 9, 10 and 12 were identified as potent and highly selective inhibitors of hCA IX and XII. These results confirm that attaching carbohydrate moieties to CA methoxyaryl pharmacophore improves and enhances its inhibitory activity. These CA inhibitors have developmental potential to selectively target cancer cells, leading to cell death.     

Potent isothiocyanate inhibitors of carbonic anhydrase: synthesis and evaluation

The reversible hydration of carbon dioxide by carbonic anhydrase (CA) regulates pH and carbon dioxide concentrations in diverse biological systems. Potent irreversible inhibition of CA would facilitate study of the dynamics of CA turnover as well as therapeutic effects due to long-term inhibition of the enzyme. We have synthesized isothiocyanate-containing sulfonamide inhibitors of CA from the corresponding aminosulfonamides. Significant increases in apparent binding of some of the isothiocyanate inhibitors over the amine analogues were consistent with covalent inhibition of the enzyme.     

Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhibitors

In a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfonamides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar concentration. In order to deepen the structure-activity relationships (SARs) and identify novel compounds with improved activity, we synthesized a series of monomethoxy analogues of the previously investigated dimethoxy derivatives. The evaluation of biological profile has been focused on in vitro effects against several CA isoforms. The new monomethoxy derivatives showed higher hCA inhibitory effects against several isoforms compared to the dimethoxy analogues. Particularly, some of these compounds (e.g., 1b and 1h) showed low nanomolar K(I) values and excellent selectivity for hCA IX and hCA XIV versus hCA I and II inhibition.     

Synthesis and characterization of novel dioxoacridine sulfonamide derivatives as new carbonic anhydrase inhibitors

Novel dioxoacridine sulfonamide compounds were synthesized from reaction of cyclic 1,3-diketones, sulfanilamide (4-amino benzene sulfonamide) and aromatic aldehydes. The structures of these compounds were confirmed by using spectral analysis (IR, H-NMR, (13)C-NMR, and mass). Human carbonic anhydrase isoenzymes (hCA I and hCA II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of sulfanilamide, acetazolamide (AAZ), and newly synthesized sulfonamides on hydratase and esterase activities of these isoenzymes have been studied in vitro. The IC(50) values of compounds for esterase activity are 0.71-0.11 μM for hCA I and 0.45-0.12 μM for hCA II, respectively. The K(i) values of these inhibitors were determined as 0,38-0,008 μM for hCA I and 0,19-0,001 μM for hCA II, respectively.     

Sulfonamides incorporating boroxazolidone moieties are potent inhibitors of the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII

A new series of sulfonamides was synthesized by the reaction of the boroxazolidone complex of l-lysine with isothiocyanates incorporating sulfamoyl moieties and diverse organic scaffolds. The obtained thioureas have been investigated as inhibitors of four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. Inhibition between the low nanomolar to the micromolar range has been observed against them, with several low nanomolar and tumor-CA selective inhibitors detected. These boron-containing compounds might be useful for the management of hypoxic tumors overexpressing hCA IX/XII by means of boron neutron capture therapy, a technique not investigated so far with inhibitors of this enzyme.     

Conformational variability of different sulfonamide inhibitors with thienyl-acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydrase isoforms

The X-ray crystal structures of the adducts of human carbonic anhydrase (hCA, EC 4.2.1.1) II complexed with two aromatic sulfonamides incorporating 2-thienylacetamido moieties are reported here. Although, the two inhibitors only differ by the presence of an additional 3-fluoro substituent on the 4-amino-benzenesulfonamide scaffold, their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were rationalized based on the obtained X-ray crystal structures, and their comparison with other sulfonamide CA inhibitors with clinical applications, such as acetazolamide, methazolamide and dichlorophenamide. The conformations of the 2-thienylacetamido tails in the hCA II adducts of the two sulfonamides were highly different, although the benzenesulfonamide parts were superimposable. Specific interactions between structurally different inhibitors and amino acid residues present only in some considered isoforms have thus been evidenced. These findings can explain the high affinity of the 2-thienylacetamido benzenesulfonamides for some pharmacologically relevant CAs (i.e., isoforms II and VII) being also useful to design high affinity, more selective sulfonamide inhibitors of various CAs.     

3H-1,2-benzoxathiepine 2,2-dioxides: a new class of isoform-selective carbonic anhydrase inhibitors

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.     

Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis,molecular docking and anticonvulsant studies

Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.