{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:48:11Z","timestamp":1790038091646,"version":"4.0.1"},"reference-count":40,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T00:00:00Z","timestamp":1776643200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003947","name":"Universidad Nacional de La Plata","doi-asserted-by":"crossref","award":["11X\/927"],"award-info":[{"award-number":["11X\/927"]}],"id":[{"id":"10.13039\/501100003947","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003947","name":"Universidad Nacional de La Plata","doi-asserted-by":"crossref","award":["11\/X958"],"award-info":[{"award-number":["11\/X958"]}],"id":[{"id":"10.13039\/501100003947","id-type":"DOI","asserted-by":"crossref"}]},{"award":["11X\/927"],"award-info":[{"award-number":["11X\/927"]}],"id":[{"id":"https:\/\/ror.org\/01tjs6929","id-type":"ROR","asserted-by":"publisher"}]},{"award":["11\/X958"],"award-info":[{"award-number":["11\/X958"]}],"id":[{"id":"https:\/\/ror.org\/01tjs6929","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002923","name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas","doi-asserted-by":"publisher","award":["PIP1695"],"award-info":[{"award-number":["PIP1695"]}],"id":[{"id":"10.13039\/501100002923","id-type":"DOI","asserted-by":"publisher"}]},{"award":["PIP1695"],"award-info":[{"award-number":["PIP1695"]}],"id":[{"id":"https:\/\/ror.org\/03cqe8w59","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Coumarins represent a distinctive class of non-classical carbonic anhydrase inhibitors that interact with the entrance region of the catalytic pocket rather than directly coordinating the catalytic Zn2+ ion. In this study, a series of glycosylated coumarins was synthesized through a copper-catalyzed multicomponent reaction involving propargyl glycosides, salicylaldehyde, and tosyl azide, providing efficient access to iminocoumarin-based glycosides derived from natural carbohydrates. The inhibitory activity of the synthesized compounds was evaluated against human carbonic anhydrase isoforms I, II, IX, and XII using a stopped-flow CO2 hydrase assay. The compounds showed negligible inhibition of the cytosolic isoforms hCA I and hCA II, while displaying moderate activity toward the tumor-associated isoforms hCA IX and hCA XII, with Ki values ranging from 12.9 to 41.8 \u03bcM. Among the series, 6-O-(2H-chromene-2-one-3-yl-methyl)-D-galactopyranose (10a) emerged as the most potent inhibitor of hCA IX and XII. Structure\u2013activity relationship analysis indicated that deprotected glycosyl derivatives exhibit improved inhibitory activity compared to protected analogues. To rationalize these observations, molecular docking followed by molecular dynamics simulations and MM-GBSA binding free energy calculations were performed for both anomeric forms of compound 10a. The computational results revealed a clear preference for the \u03b2-anomer, particularly in hCA IX and XII, where favorable interactions with catalytic threonine residues and isoform-specific aromatic residues stabilize the ligand within the active-site entrance. These findings provide a molecular explanation for the experimentally observed selectivity and highlight glycosyl coumarins as potential starting points for further optimization toward selective inhibitors of tumor-associated carbonic anhydrases.<\/jats:p>","DOI":"10.3390\/ijms27083659","type":"journal-article","created":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T12:01:22Z","timestamp":1776686482000},"page":"3659","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Glycosyl Coumarins as Selective Inhibitors of Tumor-Associated Carbonic Anhydrase IX and XII: Synthesis, Structure\u2013Activity Relationships, and Molecular Modeling"],"prefix":"10.3390","volume":"27","author":[{"given":"Macarena S.","family":"Le Pors","sequence":"first","affiliation":[{"name":"CEDECOR (UNLP-CICBA), Departamento de Qu\u00edmica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, La Plata 1900, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ignacio","family":"Aznar","sequence":"additional","affiliation":[{"name":"CEDECOR (UNLP-CICBA), Departamento de Qu\u00edmica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, La Plata 1900, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0669-7481","authenticated-orcid":false,"given":"Simone","family":"Giovannuzzi","sequence":"additional","affiliation":[{"name":"Section of Pharmaceutical Chemistry, NEUROFARBA Department, Universit\u00e0 Degli Studi di Firenze, via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4262-0323","authenticated-orcid":false,"given":"Claudiu T.","family":"Supuran","sequence":"additional","affiliation":[{"name":"Section of Pharmaceutical Chemistry, NEUROFARBA Department, Universit\u00e0 Degli Studi di Firenze, via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8678-4237","authenticated-orcid":false,"given":"Martin J.","family":"Lavecchia","sequence":"additional","affiliation":[{"name":"CEQUINOR (UNLP-CONICET, CCT-La Plata, Associated with CIC), Departamento de Qu\u00edmica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata Blvd. 120, La Plata 1465, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7688-3063","authenticated-orcid":false,"given":"Pedro A.","family":"Colinas","sequence":"additional","affiliation":[{"name":"CEDECOR (UNLP-CICBA), Departamento de Qu\u00edmica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, La Plata 1900, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Annunziata, F., Pinna, C., Dallavalle, S., Tamborini, L., and Pinto, A. (2020). An Overview of Coumarin as a Versatile and Readily Accessible Scaffold with Broad-Ranging Biological Activities. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21134618"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1462","DOI":"10.1080\/14756366.2020.1788009","article-title":"Coumarin Carbonic Anhydrase Inhibitors from Natural Sources","volume":"35","author":"Supuran","year":"2020","journal-title":"J. Enzym. Inhib. Med. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/nrd2467","article-title":"Carbonic Anhydrases: Novel Therapeutic Applications for Inhibitors and Activators","volume":"7","author":"Supuran","year":"2008","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1889","DOI":"10.1021\/acsmedchemlett.5c00443","article-title":"Human Carbonic anhydrase Inhibitors","volume":"16","author":"Supuran","year":"2025","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3057","DOI":"10.1021\/ja809683v","article-title":"Non-Zinc Mediated Inhibition of Carbonic Anhydrases: Coumarins Are a New Class of Suicide Inhibitors","volume":"131","author":"Maresca","year":"2009","journal-title":"J. Am. Chem. Soc."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1021\/jm901287j","article-title":"Deciphering the Mechanism of Carbonic Anhydrase Inhibition with Coumarins and Thiocoumarins","volume":"53","author":"Maresca","year":"2010","journal-title":"J. Med. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1021\/jm901524f","article-title":"The Coumarin-Binding Site in Carbonic Anhydrase Accommodates Structurally Diverse Inhibitors","volume":"53","author":"Temperini","year":"2010","journal-title":"J. Med. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Paoletti, N., Giovannuzzi, S., and Supuran, C.T. (2025). Hybrid and Chimeric Heterocycles for the Inhibition of Carbonic Anhydrases. Pharmaceuticals, 18.","DOI":"10.3390\/ph18091387"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Saleem, B.A.A., Qurtam, A.A., Ahmed, M., Al-Aouadi, R.F.A., Alrikabi, A.A.A., Hetta, H.F., Br\u00e4se, S., Alotaibi, G., Alkhammash, A., and Farhan, S.M. (2026). Discovery of a Novel Coumarin\/Thiazole Chalcone Hybrid as a Potent Dual Inhibitor of Tubulin and Carbonic Anhydrases IX & XII with Promising Anti-Proliferative Activity. Molecules, 31.","DOI":"10.3390\/molecules31060917"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e70094","DOI":"10.1002\/ardp.70094","article-title":"Design and Synthesis of New Multi-Target Coumarin Analogues Acting as Carbonic Anhydrases IX\/XII and Topoisomerase II Inhibitors with Apoptotic Potential","volume":"358","author":"Yousef","year":"2025","journal-title":"Arch. Pharm."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Fusco, M., Pic\u00f3n, C.R., Diaz, M.A., Bayo, J., Benitez, P.C.A., Piccioni, F., G\u00f3mez, N., Stinco, M., Martinez, J.M., and Minatta, J.N. (2025). 4-Methylumbelliferone Modulates CAIX to Mitigate Hypoxia-Driven Dysregulation and Enhance PD-1 Immunotherapy in Lung Cancer. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms262110427"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8271","DOI":"10.1021\/jm200983e","article-title":"Glycosyl Coumarin Carbonic Anhydrase IX and XII Inhibitors Strongly Attenuate the Growth of Primary Breast Tumors","volume":"54","author":"Touisni","year":"2011","journal-title":"J. Med. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Chu, N., Wang, Y., Jia, H., Han, J., Wang, X., and Hou, Z. (2022). Design, Synthesis and Biological Evaluation of New Carbohydrate-Based Coumarin Derivatives as Selective Carbonic Anhydrase IX Inhibitors via \u201cClick\u201d Reaction. Molecules, 27.","DOI":"10.3390\/molecules27175464"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1900","DOI":"10.1002\/anie.200802036","article-title":"New Principles for Glycoside-Bond Formation","volume":"48","author":"Zhu","year":"2009","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2004","DOI":"10.1002\/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5","article-title":"Click Chemistry: Diverse Chemical Function from a Few Good Reactions","volume":"40","author":"Kolb","year":"2001","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"109244","DOI":"10.1016\/j.carres.2024.109244","article-title":"Sugar Functionalized Coumarin Motifs: Synthesis and Applications","volume":"544","author":"Dwivedi","year":"2024","journal-title":"Carbohydr. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3516","DOI":"10.1016\/j.bmcl.2014.05.063","article-title":"Synthesis and evaluation of iminocoumaryl and coumaryl derivatized glycosides as galectin antagonists","volume":"24","author":"Rajput","year":"2014","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1841","DOI":"10.1055\/s-0031-1289763","article-title":"An Efficient Copper-Catalyzed Three-Component Synthesis of 3-C-Linked Glycosyl Iminocoumarins","volume":"44","author":"Palanichamy","year":"2012","journal-title":"Synthesis"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1021\/acs.molpharmaceut.0c00917","article-title":"Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates","volume":"18","author":"Sokka","year":"2021","journal-title":"Mol. Pharm."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3415","DOI":"10.1016\/S0040-4039(00)84810-8","article-title":"Iodine, a novel catalyst in carbohydrate reactions I. -isopropylidination of carbohydrates","volume":"27","author":"Kartha","year":"1986","journal-title":"Tetrahedron Lett."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1569","DOI":"10.1055\/s-1999-3579","article-title":"Synthesis of Enantiopure Pyranoisoxazole and Oxepanoisoxazole Derivatives from O-Alkynyl Carbohydrate Ethers by the Application of Intramolecular Nitrile Oxide Cycloaddition","volume":"10","author":"Pal","year":"1999","journal-title":"Synthesis"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"14527","DOI":"10.1002\/chem.201102583","article-title":"Formal Homo-Nazarov and Other Cyclization Reactions of Activated Cyclopropanes","volume":"17","author":"Saget","year":"2011","journal-title":"Chem. Eur. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2561","DOI":"10.1016\/S0021-9258(18)62326-9","article-title":"The carbon dioxide hydration activity of carbonic anhydrase: I. Stop-flow kinetic studies on the native human isoenzymes B and C","volume":"246","author":"Khalifah","year":"1971","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3099","DOI":"10.1016\/0006-2952(73)90196-2","article-title":"Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction","volume":"22","author":"Cheng","year":"1973","journal-title":"Biochem. Pharmacol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2187","DOI":"10.1021\/jm021123s","article-title":"Carbonic Anhydrase Inhibitors. Inhibition of Tumor-Associated Isozyme IX by Halogenosulfanilamide and Halogenophenylaminobenzolamide Derivatives","volume":"46","author":"Ilies","year":"2003","journal-title":"J. Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"963","DOI":"10.1016\/j.bmcl.2004.12.053","article-title":"Carbonic Anhydrase Inhibitors. Inhibition of the Transmembrane Isozyme XII with Sulfonamides\u2014A New Target for the Design of Antitumor and Antiglaucoma Drugs?","volume":"15","author":"Vullo","year":"2005","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1016\/S0223-5234(97)86179-X","article-title":"Carbonic Anhydrase Inhibitors. Part 37. Novel Classes of Isozyme I and II Inhibitors and Their Mechanism of Action","volume":"31","author":"Briganti","year":"1996","journal-title":"Eur. J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1093\/molbev\/msp259","article-title":"SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building","volume":"27","author":"Gouy","year":"2010","journal-title":"Mol. Biol. Evol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1021\/ci100031x","article-title":"Conformer Generation with OMEGA: Algorithm and Validation Using High Quality Structures from the Protein Databank and Cambridge Structural Database","volume":"50","author":"Hawkins","year":"2010","journal-title":"J. Chem. Inf. Model."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1021\/ci100436p","article-title":"FRED Pose Prediction and Virtual Screening Accuracy","volume":"51","author":"McGann","year":"2011","journal-title":"J. Chem. Inf. Model."},{"key":"ref_32","unstructured":"(2026, March 02). OEDOCKING, Available online: http:\/\/www.eyesopen.com."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1781","DOI":"10.1002\/jcc.20289","article-title":"Scalable molecular dynamics with NAMD","volume":"26","author":"Phillips","year":"2005","journal-title":"J. Comput. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3696","DOI":"10.1021\/acs.jctc.5b00255","article-title":"ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB","volume":"11","author":"Maier","year":"2015","journal-title":"J. Chem. Theory Comput."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1157","DOI":"10.1002\/jcc.20035","article-title":"Development and testing of a general amber force field","volume":"25","author":"Wang","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"10269","DOI":"10.1021\/j100142a004","article-title":"A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: The RESP model","volume":"97","author":"Bayly","year":"1993","journal-title":"J. Phys. Chem."},{"key":"ref_37","unstructured":"Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Montgomery, J.A., Vreven, T., Kudin, K.N., and Burant, J.C. (2004). Gaussian 03, Revision C.02, Gaussian, Inc."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2935","DOI":"10.1021\/ct1002626","article-title":"Structural Survey of Zinc-Containing Proteins and Development of the Zinc AMBER Force Field (ZAFF)","volume":"6","author":"Peters","year":"2010","journal-title":"J. Chem. Theory Comput."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3314","DOI":"10.1021\/ct300418h","article-title":"MMPBSA.py: An Efficient Program for End-State Free Energy Calculations","volume":"8","author":"Miller","year":"2012","journal-title":"J. Chem. Theory Comput."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1186\/s13321-021-00548-6","article-title":"ProLIF: A library to encode molecular interactions as fingerprints","volume":"13","author":"Bouysset","year":"2021","journal-title":"J. Cheminform."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/8\/3659\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T09:02:07Z","timestamp":1776848527000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/8\/3659"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,20]]},"references-count":40,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ijms27083659"],"URL":"https:\/\/doi.org\/10.3390\/ijms27083659","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,20]]}}}