Carbonic anhydrases are metalloenzymes that reversibly catalyze the hydration of carbon dioxide to a proton and bicarbonate and are implicated in the pathophysiology of many diseases. In the quest for novel carbonic anhydrase inhibitors with prospective therapeutic effects, a series of benzenesulfonamides-thiourea derivatives was synthesized. The compounds were further assessed for their inhibitory activity against human carbonic anhydrases hCA I, hCA II, hCA IX, and hCA XII, and subsequently evaluated for their in vitro anticancer activity against cancer cell lines overexpressing hCA IX and hCA XII, such as endometrial (ECC-1), lung (A549), colon (Caco-2, HT-29), breast (MCF-7), and glioblastoma (U118-MG) cancer cells. The compounds generally show nanomolar inhibition of all four isoforms, with compound 8a showing the best activity against hCA I (Ki: 2.05 nM) and hCA II (Ki: 0.39 nM), compound 8h against hCA IX (Ki: 17 nM), and compound 8c against hCA XII (Ki: 7.3 nM). In silico studies were conducted to rationalize the hCA inhibition data, revealing that tail modifications contribute to both potency and isoform selectivity. Among the series, compound 8h, which has the most potent hCA IX inhibition, outperformed 5-fluorouracil against MCF-7 and U118-MG cells.

Herein we report the chemical derivatization of the naturally occurring Tropolone (TRP) and its related compound β-Thujaplicin (β-TJP) as well as their in vitro assessment for inhibition of the physio/pathologically relevant hCAs isoforms I, II, VA; VII, IX and XII to obtain a first set of inhibition data useful for driving selected derivatives towards appropriate biomedical exploitation. The selected compound 17β was characterized for its chemical stability and assessed for its antiproliferative activity on a multiple myeloma model and showed potent pro-apoptotic features jointly with a safe toxicity profile on healthy cells. The binding mode of β-TJP within the hCA II was assessed by means of X-ray crystallography of the hCA II/β-TJP complex and showed almost complete superposition with the hCA II/TRP adduct reported in the literature. The data produced were used to elaborate a binding prediction model of such compounds on the hCAs VA, IX, and XII which are directly connected to important diseases. Overall, the achievements reported in this work are in the sustainment of the exploitation of naturally occurring troponoloid-based structures for biomedical purposes and thus contribute to the field in extending the variety of available chemical features.

The development of isoform-selective carbonic anhydrase (CA) inhibitors is an essential strategy for enhancing therapeutic efficacy, reducing off-target interactions, and minimizing side effects. Non-selective CA inhibitors like acetazolamide target multiple isoforms found in various tissues, often causing side effects such as fatigue, GI disturbances, and renal dysfunction. In contrast, selective inhibition of hCA II-the predominant isoform in the ocular ciliary body-effectively lowers intraocular pressure with improved safety, making it a targeted strategy for glaucoma treatment. Herein, we report highly selective human CA II (hCA II) inhibitors with urea-linked benzenesulfonamide scaffolds that exhibit potent and selective inhibitory activities over other isoforms. Compound 8c with 3-chlorophenyl tail group demonstrated the most potent inhibitory activity with a Ki value of 4.3 nM, whereas compound 8f with 3-trifluoromethylphenyl tail group displayed high potency (Ki = 8.2 nM) and exceptional selectivity toward hCA II. This selectivity was achieved through strategic elongation of the tail moiety, as determined through detailed analysis of isoform-specific residues at the outer rim of the active site. Docking studies and molecular dynamics simulations further validated the stable binding and selective recognition of these compounds by hCA II. This approach provides valuable insight and a promising foundation for the development of selective hCA II inhibitors with potential clinical applications, particularly in anti-glaucoma therapies.

Carbonic anhydrases (CAs) have emerged as promising drug targets for cancer therapy. In particular, the human (h) CA IX (hCA IX) isoform is expressed in a wide variety of malignancies and appears tightly regulated by micro-environmental hypoxia. Ongoing efforts aim to identify novel classes of selective CA inhibitors (CAIs) by exploring molecular diversity and discovering original chemotypes and pharmacophores. Previously, we identified a new hit compound (TDP1) carrying a trifluorodihydroxypropanone (TDP) motif as an original zinc-binding function (ZBF), which has undergone structural optimization to generate derivatives with selective inhibition profile toward hCA IX. Herein, we report on the synthesis, biological evaluation, X-ray crystallographic analysis, and computational studies of a series of aromatic-substituted TDP derivatives as novel CAI-directed chemotypes. The most potent compounds selectively inhibited hCA IX, with KI values in the submicromolar to high nanomolar range and exhibited significant antiproliferative activity against representative normoxic and hypoxic pancreatic tumor cell lines. Ultrastructural studies indicated for TDPs a possible interference with the mitochondrial function or iron metabolism. Moreover, X-ray crystallography data provided insights into the CA inhibition mechanism, suggesting that these compounds behave similarly to classical CAIs. In summary, this original TDP pharmacophore effectively inhibits human CAs, with relative selectivity towards hCA IX over cytosolic isoforms, thus providing structural insights for the development of a new class of selective anticancer agents.

The present investigation reports the design, synthesis and structure elucidation via elemental analyses and spectral techniques of six benzoylthioureido derivatives 6a, 6b,7a,7b, 8 and 13. Compounds 7a,7b, 8 and 13 were screened for their biological activity including carbonic anhydrase inhibition, anticancer, antimicrobial and antioxidant properties. The biological evaluation revealed a superior carbonic anhydrase inhibitory activity of compound 8 against hCAI (ki = 58.60 nM) along with a moderate activity toward hCAII (ki = 44.00 nM) compared to acetazolamide AAZ (ki = 250.00 and 12.10 nM), respectively. The anticancer activity did not show any remarkable cytotoxicity. Antimicrobial and antioxidant activities of the newly synthesized compounds in addition to three previously reported sulfonamide-based derivatives 14a-c demonstrated that the tested compounds show a potent antibacterial activity against P. aeruginosa (MIC = 125-250 mu g/mL) except 14a, moreover, 7a, 8 and 13 displayed a potent antifungal activity (MIC = 125 mu g/mL) against C. albicans. Furthermore, compound 14b revealed a potent antioxidant activity in ABTS and DPPH assays. A molecular docking study was performed to correlate hCAI and hCAII inhibition of compound 8 with its binding pattern in the active site of the enzyme. Additionally, ADMET prediction of the synthesized compounds showed favorable physicochemical characteristics.

To investigate the intrinsic relation between carbonic anhydrase inhibition and anticancer activity, we have prepared four sets of diaryl urea molecules and tested for the inhibition of hCA-IX and XII on two breast cancer cell lines. Among 21 compounds, compound J2 (with -SO2NH2 group) and J16 (without -SO2NH2 group) showed the best activity under normoxic and hypoxic conditions. The IC50 values of J16 for MDA-MB-231 and MCF-7 cells, under normoxic condition were 6.3 and 3.7 µM respectively, which are 1.9/3.3 and 15.8 times better than U-4-Nitro and SLC-0111 respectively. Whereas, under the hypoxic condition the corresponding values were 12.4 and 1.1 µM (MDA-MB-231 and MCF-7 cells respectively), which are equal/8 times better than U-4-Nitro. Whereas, J2 showed better IC50 value than U-4-Nitro (6.3 µM) under normoxic condition for both MDA-MB-231 and MCF-7 cells (1.9/2.7 times). Compound J2 inhibits the activity of hCA-IX and XII in nanomolar concentration [Ki values 4.09 and 9.10 nM respectively with selectivity ratio of 1.8 and 0.8 with hCA-II]. The crystal structure and modelling studies demonstrates that the inhibition of CAs arises due to the blocking of the CO2 coordination site of zinc in its catalytic domain. However, J16 was found to be unable to inhibit the activity of hCAs (Ki > 89000 nM). qPCR and western blot analysis showed a significant reduction (1.5 to 20 fold) of the transcription and expression of HIF1A, CA9 and CA12 genes in presence of J2 and J16. Both J2 and J16 found to reduce accumulation of HIF-1α protein by inhibiting the chaperone activity of hHSP70 with IC50 values of 19.4 and 15.3 µM respectively. Perturbation of the hCA-IX and XII activity by binding at active site or by reduced expression or by both leads to the decrease of intracellular pH, which resulted in concomitant increase of reactive oxygen species by 2.6/2.0 (MCF-7) and 2.9/1.8 (MDA-MB-231) fold for J2/J16. Increased cyclin D1 expression in presence of J2 and J16 was presumed to be indirectly responsible for the apoptosis of the cancer cells. Expression of the other apoptosis markers Bcl-2, Bim, caspase 9 and caspase 3 substantiated the apoptosis mechanism. However, decreased transcription/expression of HIF1A/HIF-1α and hCA-IX/XII also implies the inhibition of the extracellular signal-regulated kinase pathway by J2 and J16.

A series of sulfonamide derived quinoxaline 1,4-dioxides were synthesized and evaluated as inhibitors of carbonic anhydrases with antiproliferative potency.

Sulfamide was desymmetrized by a reaction with aldehydes to give N-sulfamoylimines. The latter reagents were successfully introduced into diastereo- and chemoselective transformation with cyclic anhydride using the Castagnoli-Cushman reaction to give unprotected N-sulfamoyl tetrahydroisoquinolonic (THIQ) acids under simple metal-free protocol. Thereby, the first general approach to the direct assembly of six-membered N-sulfamoyl lactams was developed. The synthesized compounds belong to representative, drug-like chemotypes with their well-defined three-dimensional structures and tunable physicochemical properties. In an attempt to probe their pharmacological potential, the newly synthesized lactams were screened against therapeutically relevant human and bacterial carbonic anhydrases, with some derivatives showing low micromolar enzyme inhibitory profiles.

The synthesis of 3H-1,2-benzoxaphosphepine 2-oxides and evaluation of their inhibitory activity against human carbonic anhydrase (hCA) isoforms I, II, IX, and XII are described. The target compounds were obtained via a concise synthesis from commercial salicylaldehydes and displayed low to sub-micromolar inhibition levels against the tumour-associated isoforms hCA IX and XII. All obtained benzoxaphosphepine 2-oxides possess remarkable selectivity for inhibition of hCA IX/XII over the off-target cytosolic hCA isoforms I and II, whose inhibition may lead to side effects.

The three-components one-pot Kabachnik-Fields reaction of sulfapyridine, diethyl phosphite, and aldehyde under thermal catalysis reaction condition in the presence of bismuth (III) triflate as a catalyst afford the corresponding sulfonamide-phosphonates (3a-3p) in good to excellent yields (78%-91%). The structures of the new synthesized compounds were elucidated and confirmed by variable spectroscopic studies. Single crystal X-ray studies for 3a, 3d, and 3i verified the proposed structure. The newly developed sulfonamide-phosphonates were evaluated for their inhibitory properties against four isoforms of human carbonic anhydrase (hCA I, II, IX, and XII). The results demonstrated that they exhibited greater potency in inhibiting hCA XII compared to hCA I, II, and IX, with Ki ranging from 5.1 to 51.1 nM. Compounds 3l and 3p displayed the highest potency, exhibiting selectivity ratios of I/XII >298.7 and 8.5, and II/XII ratios of 678.1 and 142.1, respectively. Molecular docking studies were conducted to explore their binding patterns within the binding pocket of CA XII. The results revealed that the sulfonamide NH group coordinated with the Zn2+ ion, and hydrogen bond interactions were observed with residue Thr200. Additionally, hydrophobic interactions were identified between the benzenesulfonamide phenyl ring and Leu198. Compounds 3p and 3l exhibited an additional hydrogen bonding interaction with other amino acid residues. These supplementary interactions may contribute to the enhanced potency and selectivity of these compounds toward the CA XII isoform.

It has been demonstrated previously that a variety of carbonic anhydrase inhibitors (CAIs) can induce vasodilation in pre-contracted retinal arteriolar segments although with different efficacy and potency. Since the CAIs tested so far are able to permeate cell membranes and inhibit both intracellular and extracellular isoforms of the enzyme, it is not clear whether extra- or intracellular isoforms or mechanisms are mediating their vasodilatory effects. By means of small wire myography, we have tested the effects of four new CAIs on wall tension in pre-contracted retinal arteriolar segments that demonstrably do not enter cell membranes but have high affinity to both cytosolic and membrane-bound isoforms of CA. At concentrations between 10−6 M to 10−3 M, none of the four membrane impermeant CAIs had any significant effect on arteriolar wall tension, while the membrane permeant CAI benzolamide (10−3 M) fully dilated all arteriolar segments tested. This suggests that CAI act as vasodilators through cellular mechanisms located in the cytoplasm of vascular cells.

To discover novel carbonic anhydrase (CA, EC 4.2.1.1) inhibitors for cancer treatment, a series of 4-{4-[(hydroxyimino)methyl]piperazin-1-yl}benzenesulfonamides were designed and synthesized using SLC-0111 as the lead molecule. The developed novel compounds 27-34 were investigated for the inhibition of human (h) isoforms hCA I, hCA II, hCA IX, and hCA XII. The hCA I was inhibited by compound 29 with a Ki value of 3.0 nM, whereas hCA II was inhibited by compound 32 with a Ki value of 4.4 nM. The tumor-associated hCA IX isoform was inhibited by compound 30 effectively with an Ki value of 43 nM, whereas the activity of another cancer-related isoform, hCA XII, was significantly inhibited by 29 and 31 with a Ki value of 5 nM. Molecular modeling showed that drug molecule 30 participates in significant hydrophobic and hydrogen bond interactions with the active site of the investigated hCAs and binds to zinc through the deprotonated sulfonamide group.

An N,N-bis(p-methoxybenzyl)-protected α-acetyl-α-diazo-methane sulfonamide proved to be a useful building block for accessing new 5-methyl-1,2,3-thiadiazole-4-sulfonamide as well as methyl 3-sulfamoyl-1H-pyrazole-5-carboxylate. The latter was further subjected to N-alkylation and N-arylation reactions. All resulting compounds showed potent inhibition of I, II and particularly of cancer-related IX and XII isoforms of human carbonic anhydrase.

A newly introduced diazo reagent, 1-diazo-N,N-bis(4-methoxybenzyl)methanesulfonamide, enables access to a range of azole-based primary sulfonamides via [3+2] cycloaddition followed by protecting group removal. Such compounds are representative of the sulfonamide chemical space highly relevant but hitherto not investigated in the context of inhibition of therapeutically relevant isoforms of carbonic anhydrase enzyme. Using this reagent, three sets of primary sulfonamides based on pyrazole, 1,2,3-triazole and tetrazole cores were synthesized and profiled for inhibition of tumor-associated hCA IX and XII isoforms as well as abundant cytosolic hCA I and II isoforms. Using virtual library design and docking prioritization tool of the Schrödinger suite, one of the promising leads was evolved into a dual hCA IX/XII inhibitor with excellent selectivity over off-target hCA I and II. The new synthetic strategy to access azole-based primary sulfonamides will support the discovery of novel, isoform-selective inhibitors of carbonic anhydrase within the poorly explored azole chemical space.

The present investigation reports the design and synthesis of three series of benzoylthioureido derivatives bearing either benzenesulfonamide 7a-f, benzoic acid 8a-f or ethylbenzoate 9a-f moieties. The synthesised compounds were screened for their carbonic anhydrase inhibitory activity (CAI) against four isoforms hCA I, II, IX, and XII. Compounds 7a, 7b, 7c, and 7f exhibited a potent inhibitory activity towards hCAI (Kis = 58.20, 56.30, 33.00, and 43.00 nM), respectively compared to acetazolamide (AAZ) and SLC-0111 (Kis = 250.00 and 5080.00 nM). Compounds 7a, 7b, 7c, 7e, and 7f elicited selectivity over h CA II (Kis = 2.50, 2.10, 56.60,39.60 and 39.00 nM) respectively, relative to AAZ and SLC-0111(Kis = 12.10 and 960.00 nM). Also, compounds 7c, 7f, and 9e displayed selectivity against the tumour-associated isoform hCA IX (Kis = 31.20, 30.00 and 29.00 nM) respectively, compared to AAZ and SLC-0111 (Kis = 25.70 and 45.00 nM). Additionally, compounds 8a and 8f revealed a moderate to superior selectivity towards hCAXII (Kis = 17.00 and 11.00 nM) relative to AAZ and SLC-0111(Kis = 5.70 and 45.00 nM). Molecular docking and ADME prediction studies were performed on the most active compounds to shed light on their interaction with the hot spots of the active site of CA isoforms, in addition to prediction of their pharmacokinetic and physicochemical properties.

We report the synthesis of twelve novel O-glycosylmethyl isoxazoles incorporating the phenoxyaryl moiety, that were designed to selectively target the cancer-related human carbonic anhydrase (CA, EC 4.2.1.1) isoforms. The O-glycosides have been prepared by cycloaddition of nitrile oxides to O-propargyl glycosides. Compounds were assessed as inhibitors of the physiologically dominant isozymes hCA I and II and the tumor-associated isozyme hCA IX and hCA XII. In this study, several glycosides have been identified as highly selective inhibitors of hCA IX and XII. Glycoconjugation enhances the anticancer activity against human osteosarcoma cell line. Thus, these glycosides may prove interesting lead candidates for a targeted cancer therapy.

Cognitive deficits are enduring and disabling symptoms for many patients with severe mental illness, and these impairments are inadequately addressed by current medications. In this study, we reported the synthesis of β-arylchalcogeno amines bearing sulfurated, selenated, and tellurated moieties (2-4) which are structurally related to amphetamine with good activation properties for Carbonic Anhydrases (CAs) isoforms present in the cortical and hippocampal brain structures (hCA IV and hCA XIV). In addition, these compounds showed selective inhibition against the Monoamine oxidase (MAO) A isoform. In vivo evaluation of two derivatives (2a and 3a) revealed procognitive effects in the object recognition and social discrimination tests. Interestingly, these compounds, despite having a similar structure to amphetamine, did not caused hypophagia or hyperlocomotion, two effects often observed following the administration of amphetamine-like drugs. In this context, β-arylchalcogeno amines may have utility for improving the symptoms of cognitive decline associated with neurodegenerative and psychiatric diseases such as attention deficit disorder, Parkinson's disease-related cognitive dysfunction and cognitive disorders associated with depression.

A series of 1,2,3-triazol-1-ylbenzenesulfonamide derivatives was designed, synthesized and their ability to inhibit several carbonic anhydrase isoforms was evaluated. The basis of our design is to hybridize the benzenesulfonamide moiety widely used as a zinc-binding group, a triazole ring as spacer with a tail of different substituted aryl moieties. The synthesis of these compounds was achieved using Cu(I)-mediated click chemistry between the azide containing the benzenesulfonamide pharmacophore and various aryl acetylenes or 1,6-heptadiyne through copper-catalyzed [3+2] cycloaddition reaction. The ability the new derivatives to inhibit four human carbonic anhydrase isoforms hCA I, II, IX, and XII was evaluated. All the compounds exhibited good potency and high selectivity towards isoforms hCA I and II more than isoforms hCA IX and XII, especially for the derivatives 3c and 3j that displayed K-i of 2.8 and 3.8 nM against hCA II and a high hCA II selectivity ratio ranging from 77.6 to 3571.4 over other isoforms. All the compounds were docked in the active site of the downloaded hCA II active site and their binding pattern confirmed their significant activity by interacting of the sulfonamide moiety with zinc ion in the active site, in addition to its hydrogen bond interaction with Thr199 and Thr200. All the above-mentioned findings pointed out towards the promising activity of the synthesized series that can be presented as a new scaffold to be further optimized as selective antiglaucoma drugs.

Human carbonic anhydrase VII (hCA VII) constitutes a promising molecular target for the treatment of epileptic seizures and other central nervous system disorders due to its almost exclusive expression in neurons. Achieving isoform selectivity is one of the main challenges for the discovery of new hCA inhibitors, since nonspecific inhibition may lead to tolerance and side effects. In the present work, we report the development of a molecular docking protocol based on AutoDock4Zn for the search of new hCA VII inhibitors by virtual screening. The docking protocol was applied to the screening of two sets of compounds: a ZINC15 subset of sulfur-containing structures and an in-house library consisting of synthetic and commercial candidates (including approved drugs). Five compounds were selected from the first screening campaign and three from the second one, and they were tested in vitro against the enzyme. Among the eight selected structures, four showed Ki values in the low nanomolar range. These confirmed hits include three approved drugs: meloxicam, piroxicam, and nitrofurantoin, which also showed good selectivity for hCA VII versus hCA II.

We report for the first time Antibody-Drug-Conjugates (ADCs) containing human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) directed Monoclonal Antibodies (MAbs) linked to low molecular weight inhibitors of the same enzymes by means of hydrophilic peptide spacers. In agreement with the incorporated CA directed MAb fragments, in vitro inhibition data of the obtained ADCs showed sub-nanomolar K-I values for the tumour associated CAs IX and XII which were up to 10-fold more potent when compared to the corresponding unconjugated MAbs. In addition, the introduction of the CA inhibitor (CAI) benzenesulfonamide allowed the ADCs to potently inhibit the housekeeping tumoral off-target human CA II isoform. Such results are supporting the definition of an unprecedented reported class of ADCs able to hit simultaneously multiple hCAs physiologically cooperative in maintaining altered cellular metabolic pathways, and therefore ideal for the treatment of chronic diseases such as cancers and inflammation diseases.