{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:00:58Z","timestamp":1787029258854,"version":"build-2736575974"},"reference-count":79,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T00:00:00Z","timestamp":1768867200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"National Science Centre","doi-asserted-by":"publisher","award":["2018\/31\/B\/NZ7\/02122"],"award-info":[{"award-number":["2018\/31\/B\/NZ7\/02122"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005357","name":"Slovak Research and Development Agency","doi-asserted-by":"publisher","award":["APVV-22-0133"],"award-info":[{"award-number":["APVV-22-0133"]}],"id":[{"id":"10.13039\/501100005357","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background:\u00a0Trypanosoma cruzi, the causative agent of Chagas disease, remains a major public health concern, and there is a continued need for new antitrypanosomal agents. Thiosemicarbazone (TSC) derivatives have emerged as a promising class of compounds with potential antiparasitic activity. Objectives: This study aimed to report the synthesis, characterization, and biological profiling of a novel series of thiosemicarbazone derivatives as antitrypanosomal agents against Trypanosoma cruzi. Methods: Fourteen new compounds and six previously described analogues were prepared and characterized by 1H\/13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As a preliminary in vitro screen, activity was assessed by direct parasite counting in epimastigote and bloodstream trypomastigote forms, as tractable models of replicative and infective stages sharing core metabolic targets with intracellular amastigotes. Epimastigote potency was quantified as half-maximal effective concentrations (EC50) derived from dose\u2013response curves, whereas trypomastigote response was evaluated as percent viability after treatment at a fixed concentration of 20 \u00b5M. Mechanistic profiling included inhibition assays against the cysteine protease cruzipain (CZP) and selected redox defense enzymes, complemented by in silico similarity clustering and binding-pose affinity scoring. Results: A nitro-methoxy-substituted TSC showed potent CZP inhibition but limited trypomastigote efficacy, whereas brominated analogues displayed dual-stage activity independent of CZP inhibition. Tanimoto similarity analysis identified distinct structure\u2013activity clusters, linking nitro-methoxy substitution to epimastigote selectivity and brominated scaffolds to broader antiparasitic profiles, with hydrophobicity and steric complementarity as key determinants. Enzymatic assays revealed no significant inhibition of cytosolic tryparedoxin peroxidase (cTXNPx) or glutathione peroxidase type I (TcGPx-I), suggesting redox disruption is not a primary mode of action. In vitro and in silico analyses showed low or no non-specific cytotoxicity under the tested conditions, supporting further optimization of these derivatives as antitrypanosomal preliminary hits. Key hits included derivative 3e (epimastigote EC50 = 0.36 \u00b1 0.02 \u00b5M) and brominated analogues 2c and 2e (epimastigote EC50 = 3.92 \u00b1 0.13 and 4.36 \u00b1 0.10 \u00b5M, respectively), while docking supported favorable binding-pose affinity (e.g., \u0394GS-pose = \u221220.78 \u00b1 2.47 kcal\/mol for 3e). Conclusions: These results support further optimization of the identified thiosemicarbazone derivatives as preliminary antitrypanosomal hits and provide insight into structure\u2013activity relationships and potential mechanisms of action.<\/jats:p>","DOI":"10.3390\/ph19010182","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T14:57:58Z","timestamp":1768921078000},"page":"182","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Integrated In Vitro and In Silico Profiling of Piperazinyl Thiosemicarbazone Derivatives Against Trypanosoma cruzi: Stage-Specific Activity and Enzyme Inhibition"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2688-1697","authenticated-orcid":false,"given":"H\u00e9ctor A.","family":"Baldoni","sequence":"first","affiliation":[{"name":"Facultad de Qu\u00edmica, Bioqu\u00edmica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, San Luis 5700, Argentina"},{"name":"Instituto Multidisciplinario de Investigaciones Biol\u00f3gicas de San Luis (Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas), Universidad Nacional de San Luis, Av. Ej\u00e9rcito de Los Andes 950, San Luis 5700, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4671-2658","authenticated-orcid":false,"given":"Mar\u00eda L.","family":"Sbaraglini","sequence":"additional","affiliation":[{"name":"Laboratory of Bioactive Research and Development, Medicinal Chemistry, Department of Biological Sciences, Faculty of Exact Sciences, National University of La Plata, La Plata CP1900, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dar\u00edo E.","family":"Balcazar","sequence":"additional","affiliation":[{"name":"Centro de Estudios Parasitol\u00f3gicos y Vectores, Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas, Universidad Nacional de La Plata, La Plata CP1900, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diego G.","family":"Arias","sequence":"additional","affiliation":[{"name":"Laboratorio de Enzimolog\u00eda Molecular, Instituto de Agrobiotecnolog\u00eda del Litoral, Santa Fe 3000, Argentina"},{"name":"Facultad de Bioqu\u00edmica y Ciencias Biol\u00f3gicas, Universidad Nacional del Litoral, Santa Fe 3000, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergio A.","family":"Guerrero","sequence":"additional","affiliation":[{"name":"Laboratorio de Enzimolog\u00eda Molecular, Instituto de Agrobiotecnolog\u00eda del Litoral, Santa Fe 3000, Argentina"},{"name":"Facultad de Bioqu\u00edmica y Ciencias Biol\u00f3gicas, Universidad Nacional del Litoral, Santa Fe 3000, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2724-0421","authenticated-orcid":false,"given":"Catalina D.","family":"Alba Soto","sequence":"additional","affiliation":[{"name":"Instituto de Microbiolog\u00eda y Parasitolog\u00eda M\u00e9dica, Departamento de Microbiolog\u00eda, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1053, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6371-9061","authenticated-orcid":false,"given":"Wioleta","family":"Cieslik","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, University of Silesia, 75 Pu\u0142ku Piechoty 1a, 41-500 Chorzow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3411-5143","authenticated-orcid":false,"given":"Marta","family":"Rogalska","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, University of Silesia, 75 Pu\u0142ku Piechoty 1a, 41-500 Chorzow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7361-2671","authenticated-orcid":false,"given":"Jaroslaw","family":"Pola\u0144ski","sequence":"additional","affiliation":[{"name":"Institute of Chemistry, University of Silesia, 75 Pu\u0142ku Piechoty 1a, 41-500 Chorzow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ricardo D.","family":"Enriz","sequence":"additional","affiliation":[{"name":"Facultad de Qu\u00edmica, Bioqu\u00edmica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, San Luis 5700, Argentina"},{"name":"Instituto Multidisciplinario de Investigaciones Biol\u00f3gicas de San Luis (Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas), Universidad Nacional de San Luis, Av. 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