Sulfonamide-incorporated bis(α-aminophosphonates) as promising carbonic anhydrase inhibitors: Design, synthesis, biological evaluation, and X-ray crystallographic studies.

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate) showed a KI of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a KI of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ.

The dopamine D2 receptors antagonist Veralipride inhibits carbonic anhydrases: solution and crystallographic insights on human isoforms.

The inhibitory effects of veralipride, a benzamide-class antipsychotic acting as dopamine D2 receptors antagonist incorporates a primary sulfonamide moiety and was investigated for its interactions with carbonic anhydrase (CA) isoforms. In vitro profiling using the stopped-flow technique revealed that veralipride exhibited potent inhibitory activity across all tested hCA isoforms, with exception of hCA III. Comparative analysis with standard inhibitors, acetazolamide (AAZ), and sulpiride, provided insights for understanding the relative efficacy of veralipride as CA inhibitor. The study reports the X-ray crystal structure analysis of the veralipride adduct with three human (h) isoforms, hCA I, II, and CA XII mimic, allowing the understanding of the molecular interactions rationalizing its inhibitory effects against each isoform. These findings contribute to our understanding of veralipride pharmacological properties and for the design of structural analogs endowed with polypharmacological properties.

Synthetic Approaches to Novel Human Carbonic Anhydrase Isoform Inhibitors Based on Pyrrol-2-one Moiety.

New dihydro-pyrrol-2-one compounds, featuring dual sulfonamide groups, were synthesized through a one-pot, three-component approach utilizing trifluoroacetic acid as a catalyst. Computational analysis using density functional theory (DFT) and condensed Fukui function explored the structure-reactivity relationship. Evaluation against human carbonic anhydrase isoforms (hCA I, II, IX, XII) revealed potent inhibition. The widely expressed cytosolic hCA I was inhibited across a range of concentrations (KI 3.9-870.9 nM). hCA II, also cytosolic, exhibited good inhibition as well. Notably, all compounds effectively inhibited tumor-associated hCA IX (KI 1.9-211.2 nM) and hCA XII (low nanomolar). Biological assessments on MCF7 cancer cells highlighted the compounds' ability, in conjunction with doxorubicin, to significantly impact tumor cell viability. These findings underscore the potential therapeutic relevance of the synthesized compounds in cancer treatment.

Novel Carbonic Anhydrase Inhibitors with Dual-Tail Core Sulfonamide Show Potent and Lasting Effects for Glaucoma Therapy.

Glaucoma, a leading cause of irreversible vision loss worldwide, is characterized by elevated intraocular pressure (IOP), a well-established risk factor across all its forms. We present the design and synthesis of 39 novel carbonic anhydrase inhibitors by a dual-tailed approach, strategically crafted to interact with distinct hydrophobic and hydrophilic pockets of CA active sites. The series was investigated against the CA isoforms implicated in glaucoma (hCA II, hCA IV, and hCA XII), and the X-ray crystal structures of compounds 25a, 25f, and 26a with CA II, along with 14b in complex with a hCA XII mimic, were determined. Selected compounds (14a, 25a, and 26a) underwent evaluation for their ability to reduce IOP in rabbits with ocular hypertension. Derivative 26a showed significant potency and sustained IOP-lowering effects, surpassing the efficacy of the drugs dorzolamide and bimatoprost. This positions compound 26a as a promising candidate for the development of a novel anti-glaucoma medication.

Ureidobenzenesulfonamides as Selective Carbonic Anhydrase I, IX, and XII Inhibitors.

Sulfonamides remain an important class of drugs, especially because of their inhibitory effects on carbonic anhydrases. Herein, we have synthesized several sulfonamides and tested them for their inhibitory activity against carbonic anhydrases hCA I, hCA II, hCA IX, and hCA XII, respectively. Thereby, biphenyl- and benzylphenyl-substituted sulfonamides showed high selectivity against hCA IX and hCA XII; these enzymes are common targets in the treatment of hypoxic cancers, and noteworthy inhibitory activity was observed for several compounds toward hCA I that might be of interest for future applications to treat cerebral edema. Compound 3 (4-[3-(2-benzylphenyl)ureido]benzenesulfonamide) held an exceptionally low Ki value of 1.0 nM for hCA XII.

First-in-Class Dual Targeting Compounds for the Management of Seizures in Glucose Transporter Type 1 Deficiency Syndrome.

The genetic disorder glucose transporter type 1 deficiency syndrome (GLUT1-DS) heavily affects the main intake of energy in tissues and determines the most relevant outcomes at the central nervous system (CNS) district, which is highly dependent on glucose. Herein, we report the design and development of a set of compounds bearing the glucosyl and galactosyl moieties. We assessed their ability to enhance the GLUT1 mediated glucose intake in non-small-cell lung cancer (NSCLC) cells and to inhibit the carbonic anhydrase (CA; EC 4.2.1.1) isoforms implicated in the physiopathology of uncontrolled seizures associated to epilepsy (i.e., I, II, IV, VA, VB, and XII). The binding mode of 8 in adduct with hCA II was determined by X-ray crystallography. Among the selected derivatives, compound 4b proved effective in suppressing the occurrence of uncontrolled seizures on the in vivo induced maximal electroshock (MES) model and thus gives sustainment of an unprecedently reported pharmacological approach for the management of GLUT1-DS associated diseases.

Crystal structure of the monocupin ring-cleaving dioxygenase 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp.

5-Nitrosalicylate 1,2-dioxygenase (5NSDO) is an iron(II)-dependent dioxygenase involved in the aerobic degradation of 5-nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5-nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5-nitrosalicylate, the enzyme is also active towards 5-chlorosalicylate. The X-ray crystallographic structure of the enzyme was solved at 2.1 Šresolution by molecular replacement using a model from the AI program AlphaFold. The enzyme crystallized in the monoclinic space group P21, with unit-cell parameters a = 50.42, b = 143.17, c = 60.07 Å, ß = 107.3°. 5NSDO belongs to the third class of ring-cleaving dioxygenases. Members of this family convert para-diols or hydroxylated aromatic carboxylic acids and belong to the cupin superfamily, which is one of the most functionally diverse protein classes and is named on the basis of a conserved ß-barrel fold. 5NSDO is a tetramer composed of four identical subunits, each folded as a monocupin domain. The iron(II) ion in the enzyme active site is coordinated by His96, His98 and His136 and three water molecules with a distorted octahedral geometry. The residues in the active site are poorly conserved compared with other dioxygenases of the third class, such as gentisate 1,2-dioxygenase and salicylate 1,2-dioxygenase. Comparison with these other representatives of the same class and docking of the substrate into the active site of 5NSDO allowed the identification of residues which are crucial for the catalytic mechanism and enzyme selectivity.

Mono- and three-tailed sugar and iminosugar decorated benzenesulfonamide carbonic anhydrase inhibitors.

A collection of novel mono- and three-tailed derivatives based on a sugar (glucose) or an iminosugar (trihydroxy piperidine) featuring a terminal benzenesulfonamide were synthesized to investigate the so-called "sugar" and "azasugar" approach with the aim of exploring the activity and selectivity towards the inhibition of human carbonic anhydrases (hCAs). The synthetic approach relies on a general copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction followed by an amine-isothiocyanate coupling. Biological assays were used to collect subtle information on the role of these single or multiple hydrophilic chains. Among the sugar-based inhibitors, the single-tailed compound 10 was identified as a better inhibitor than the reference compound (AAZ) towards three different hCAs, while, among the three sugar tailed derivatives, potent and selective inhibition was found for compounds 25 and 26. A promising and selective inhibitory activity was discovered for the iminosugar single-tailed compound 31 towards hCA VII (Ki = 9.7 nM).

Diversely N-substituted benzenesulfonamides dissimilarly bind to human carbonic anhydrases: crystallographic investigations of N-nitrosulfonamides.

Carbonic anhydrases (CAs) are a zinc metalloenzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton, pivotal for a wide range of biological processes. CAs are involved in numerous pathologies and thus represent valuable drug targets in the treatments of several diseases such as glaucoma, obesity, tumour, neuropathic pain, cerebral ischaemia, or as antiinfectives. In the last two decades, several efforts have been made to achieve selective CA inhibitors (CAIs) employing different drug design approaches. However, N-substitutions on primary sulphonamide groups still remain poorly investigated. Here, we reported for the first time the co-crystallisation of a N-nitro sulphonamide derivative with human (h) CA II pointing out the binding site and mode of inhibition of this class of CAIs. The thorough comprehension of the ligand/target interaction might be valuable for a further CAI optimisation for achieving new potent and selective derivatives.

First-in-Class Dual Hybrid Carbonic Anhydrase Inhibitors and Transient Receptor Potential Vanilloid 1 Agonists Revert Oxaliplatin-Induced Neuropathy.

Here, we report for the first time a series of compounds potentially useful for the management of oxaliplatin-induced neuropathy (OINP) able to modulate the human Carbonic Anhydrases (hCAs) as well as the Transient Receptor Potential Vanilloid 1 (TRPV1). All compounds showed effective in vitro inhibition activity toward the main hCAs involved in such a pathology, whereas selected items reported moderate agonism of TRPV1. X-ray crystallographic experiments assessed the binding modes of the two enantiomers (R)-37a and (S)-37b within the hCA II cleft. Although the tails assumed diverse orientations, no appreciable effects were observed for their hCA II affinity. Similarly, the activity of (R)-39a and (S)-39b on TRPV1 was not influenced by the stereocenters. In vivo evaluation of the most promising derivatives (R)-12a, (R)-37a, and the two enantiomers (R)-39a, (S)-39b revealed antihypersensitivity effects in a mouse model of OINP with potent and persistent effect up to 75 min after administration.

Design, synthesis, biological evaluation and crystal structure determination of dual modulators of carbonic anhydrases and estrogen receptors.

Multi-target compounds have become increasingly important for the development of safer and more effective drug candidates. In this work, we devised a combined ligand-based and structure-based multi-target repurposing strategy and applied it to a series of hexahydrocyclopenta[c]quinoline compounds synthesized previously. The in silico analyses identified human Carbonic Anhydrases (hCA) and Estrogen Receptors (ER) as top scoring candidates for dual modulation. hCA isoforms IX and XII, and ER subtypes ER⍺ and/or ERß are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⍺ is the primary target of anti-estrogen therapy in breast cancer, and the hCA IX isoform is a therapeutic target in triple-negative breast cancer. ER⍺-mediated transcriptional programs and hCA activity in cancer cells promote favorable microenvironments for cell proliferation. Interestingly, several lines of evidence indicate that the combined modulation of these two targets may provide significant therapeutic benefits. Moving from these first results, two additional hexahydrocyclopenta[c]quinoline derivatives bearing a sulfonamide zinc binding group (hCA) and a phenolic hydroxyl (ER) pharmacophoric group placed at the appropriate locations were designed and synthesized. Interestingly, these compounds were able to directly modulate the activities of both hCA and ER targets. In cell-based assays, they inhibited proliferation of breast and prostate cancer cells with micromolar potency and cell type-selective efficacy. The compounds inhibited hCA activity with nanomolar potency and isoform-selectivity. In transactivation assays, they reduced estrogen-driven ER activity with micro-molar potency. Finally, crystal structures of the synthesized ligands in complex with the two targets revealed that the compounds bind directly to the hCA active site, as well as to the ER ligand-binding domain, providing structural explanation to the observed activity and a rationale for optimization of their dual activity. To the best of our knowledge, this work describes the design, synthesis and biological characterization of the first dual modulators of hCA and ER, laying the ground for the structure-based optimization of their multi-target activity.

Probing the Efficiency of 13-Pyridylalkyl Berberine Derivatives to Human Telomeric G-Quadruplexes Binding: Spectroscopic, Solid State and In Silico Analysis.

The interaction between the series of berberine derivatives 1-5 (NAX071, NAX120, NAX075, NAX077 and NAX079) and human telomeric G-quadruplexes (G4), which are able to inhibit the Telomerase enzyme's activity in malignant cells, was investigated. The derivatives bear a pyridine moiety connected by a hydrocarbon linker of varying length (n = 1-5, with n number of aliphatic carbon atoms) to the C13 position of the parent berberine. As for the G4s, both bimolecular 5'-TAGGGTTAGGGT-3' (Tel12) and monomolecular 5'-TAGGGTTAGGGTTAGGGTTAGGG-3' (Tel23) DNA oligonucleotides were considered. Spectrophotometric titrations, melting tests, X-ray diffraction solid state analysis and in silico molecular dynamics (MD) simulations were used to describe the different systems. The results were compared in search of structure-activity relationships. The analysis pointed out the formation of 1:1 complexes between Tel12 and all ligands, whereas both 1:1 and 2:1 ligand/G4 stoichiometries were found for the adduct formed by NAX071 (n = 1). Tel12, with tetrads free from the hindrance by the loop, showed a higher affinity. The details of the different binding geometries were discussed, highlighting the importance of H-bonds given by the berberine benzodioxole group and a correlation between the strength of binding and the hydrocarbon linker length. Theoretical (MD) and experimental (X-ray) structural studies evidence the possibility for the berberine core to interact with one or both G4 strands, depending on the constraints given by the linker length, thus affecting the G4 stabilization effect.

Development of Praziquantel sulphonamide derivatives as antischistosomal drugs.

The almost empty armamentarium to treat schistosomiasis, a neglected parasitic disorder caused by trematode flatworms of the genus Schistosoma, except Praziquantel (PZQ), urged to find new alternatives to fight this infection. Carbonic Anhydrase from Schistosoma mansoni (SmCA) is a possible new target against this nematode. Here, we propose new PZQ derivatives bearing a primary sulphonamide group in order to obtain hybrid drugs. All compounds were evaluated for their inhibition profiles on both humans and Schistosoma CAs, X-ray crystal data of SmCA and hCA II in adduct with some inhibitors were obtained allowing the understanding of the main structural factors responsible of activity. The compounds showed in vitro inhibition of immature and adult S. mansoni, but further optimisation is required for improved activity.

Benzoselenoates: A novel class of carbonic anhydrase inhibitors.

A series of benzoselenoates has been prepared and their inhibitory properties against the most relevant human Carbonic Anhydrases (CAs) isoforms, among which hCA I, II, IV, VII, IX, and XII were investigated. These inhibitors were designed considering the carboxylates and mono-/dithiocarbamates as lead and led to the observation that the COSe- is a new zinc-binding group (ZBG) for metalloenzymes possessing zinc ions at their active site. The substitution pattern on aromatic ring of the benzoselenoates is the crucial structural element influencing selectivity towards various isoforms. We elucidated the binding mode of benzoselenoates to hCA I and hCA II by using X-ray crystallography. The negatively charged selenium atom from the new ZBG was observed coordinated to the zinc ion from the CA active site at a distance of 2.30-2.40 Å from it. Overall, these data might be useful for the development of new inhibitors with higher selectivity and efficacy for various hCAs.

Diversely substituted sulfamides for fragment-based drug discovery of carbonic anhydrase inhibitors: synthesis and inhibitory profile.

A series of sulfamide fragments has been synthesised and investigated for human carbonic anhydrase inhibition. One of the fragments showing greater selectivity for cancer-related isoforms hCA IX and XII was co-crystalized with hCA II showing significant potential for fragment periphery evolution via fragment growth and linking. These opportunities will be identified in the future via the screening of this fragment structure for co-operative carbonic anhydrase binding with other structurally diverse fragments.[Figure: see text].

Inhibition of Schistosoma mansoni carbonic anhydrase by the antiparasitic drug clorsulon: X-ray crystallographic and in vitro studies.

Clorsulon is an anthelmintic drug that is clinically used against Fasciola hepatica. Due to the presence of two sulfonamide moieties in its core nucleus, which are well recognized as zinc-binding groups, it was proposed that it may be efficacious in the inhibition of parasite carbonic anhydrases (CAs). Proteomic analyses revealed the presence of CA in the tegument of Schistosoma mansoni, and recently the druggability of this target was explored by testing the inhibitory activities of several sulfonamide-based derivatives. According to the principles of drug repurposing, the aim was to demonstrate a putative new mechanism of action of clorsulon and thus widen its antiparasitic spectrum. For this purpose, the inhibitory activity and isoform selectivity of clorsulon was studied using human CA I and S. mansoni CA, revealing different modes of binding of clorsulon that explain its inhibitory potency against the two enzymes. The information obtained in this study could be crucial in the design of more active and selective derivatives.

Selenocarbamates As a Prodrug-Based Approach to Carbonic Anhydrase Inhibition.

A study on the activity of selenocarbamates as a novel chemotype acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. Undergoing CA-mediated hydrolysis, selenocarbamates release selenolates behaving as zinc binding groups and effectively inhibiting CAs. A series of selenocarbamates characterised by high molecular diversity and complexity have been studied against different human CA isoforms such as hCA I, II, IX and XII. Selenocarbamates behave as masked selenols with potential biological applications as prodrugs for CAs inhibition-based strategies. X-ray studies provided insights into the binding mode of this novel class of CA inhibitors.

One-Pot Procedure for the Synthesis of Asymmetric Substituted Ureido Benzene Sulfonamides as Effective Inhibitors of Carbonic Anhydrase Enzymes.

We report a one-pot procedure for the synthesis of asymmetrical ureido-containing benzenesulfonamides based on in situ generation of the corresponding isocyanatobenezenesulfonamide species, which were trapped with the appropriate amines. A library of new compounds was generated and evaluated in vitro for their inhibition properties against a representative panel of the human (h) metalloenzymes carbonic anhydrases (EC 4.2.1.1), and the best performing compounds on the isozyme II (i.e., 7c, 9c, 11g, and 12c) were screened for their ability to reduce the intraocular pressure in glaucomatous rabbits. In addition, the binding modes of 7c, 11f, and 11g were assessed by means of X-ray crystallography.

Calixarenes Incorporating Sulfonamide Moieties: Versatile Ligands for Carbonic Anhydrases Inhibition.

Carbonic anhydrases (CAs) continue to represent a relevant pharmaceutical target. The need of selective inhibitors and the involvement of these metalloenzymes in many multifaceted diseases boost the search for new ligands able to distinguish among the different CA isoforms, and for multifunctional systems simultaneously able to inhibit CAs and to interfere with other pathological events by interacting with additional targets. In this work, we successfully explored the possibility of preparing new CAs ligands by combining calixarenes with benzensulfonamide units. Inhibition tests towards three human CA isoforms evidenced, for some of the ligands, Ki values in the nanomolar range and promising selectivity. X-ray and molecular modeling studies provided information on the mode of binding of these calixarene derivatives. Thanks to the encouraging results and the structural features typical of the calixarene scaffold, it is then possible to plan for the future the design of multifunctional inhibitors for this class of widely spread enzymes.

Chalcogenides-incorporating carbonic anhydrase inhibitors concomitantly reverted oxaliplatin-induced neuropathy and enhanced antiproliferative action.

Platinum-based chemotherapy is widely used for the treatment of different tumors but is associated with serious side effects, among which neuropathic pain. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have recently been validated as therapeutic agents in neuropathic pain and as antitumor agents. We report the synthesis of new organochalcogenides bearing the benzensulfonamide moiety acting as potent inhibitors of several human CA isoforms and, in particular, against hCA II and VII endowed with potent neuropathic pain attenuating effects. Moreover, in combination with cisplatin or doxorubicin, some of the new CA inhibitors enhanced the effects of the anticancer drugs capability in counteracting breast cancer MCF7 cell viability. The concomitant anti-neuropathic pain and antiproliferative effects of the new chalcogenide-based CA inhibitors represent an innovative approach for the counteraction and management of side effects associated with clinically platinum drugs as antitumor agents.