Multifaceted Sulfonamide-Derived Thiosemicarbazones: Combining Metal Chelation and Carbonic Anhydrases Inhibition in Anticancer Therapy

Academic Editor: David StC Black Received: 7 December 2024 Revised: 24 January 2025 Accepted: 24 January 2025 Published: 30 January 2025 Citation: Martínez-Montiel, M.; Arrighi, G.; Begines, P.; González-Bakker, A.; Puerta, A.; Fernandes, M.X.; Merino-Montiel, P.; Montiel-Smith, S.; Nocentini, A.; Supuran, C.T.; et al. Multifaceted Sulfonamide-Derived Thiosemicarbazones: Combining Metal Chelation and Carbonic Anhydrases Inhibition in Anticancer Therapy. Int. J. Mol. Sci. 2025,26, 1225. https://doi.org/10.3390/ ijms26031225 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Multifaceted Sulfonamide-Derived Thiosemicarbazones: Combining Metal Chelation and Carbonic Anhydrases Inhibition in Anticancer Therapy Mónica Martínez-Montiel 1,2,†, Giulia Arrighi 1,3,†, Paloma Begines 1,3 , Aday González-Bakker 4, Adrián Puerta 4 , Miguel X. Fernandes 4 , Penélope Merino-Montiel 2 , Sara Montiel-Smith 2 , Alessio Nocentini 3 , Claudiu T. Supuran 3, José M. Padrón 4, José G. Fernández-Bolaños 1and Óscar López 1,* 1Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain; [email protected] (M.M.-M.); [email protected] (G.A.); [email protected] (P.B.); [email protected] (J.G.F.-B.) 2Facultad de Ciencias Químicas, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla 72570, PUE, Mexico; [email protected] (P.M.-M.); [email protected] (S.M.-S.) 3NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, University of Florence, 50019 Florence, Italy; [email protected] (A.N.); [email protected] (C.T.S.) 4 BioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Universidad de La Laguna, c/Astrofísico Francisco Sánchez 2, E-38206 La Laguna, Spain; [email protected] (A.G.-B.); [email protected] (A.P.); [email protected] (M.X.F.); jmpadr[email protected] (J.M.P.) *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: The selective inhibition of key enzymes, such as carbonic anhydrases (CAs IX and XII), which are overexpressed in cancer tissues, has emerged as a promising strategy in cancer research. However, a multitarget approach is often preferred to achieve enhanced therapeutic outcomes. In this study, aryl sulfonamides were conjugated with a thiosemicarbazone moiety to enable dual functionality: the inhibition of CAs and the chelation of metal cations. Several structural factors were systematically modified, including the position of the sulfonamido group, the length of the linker, the nature of the aromatic residue, and the type of substituents. Tumor-associated CAs IX and XII inhibition was evaluated using the stopped-flow CO 2 hydrase assay, and the inhibition constants (K i ) were determined. The most promising compounds were further analyzed through molecular docking simulations. Metal chelation capabilities were evaluated using UV–Vis spectroscopy, while antiproliferative activities were measured using the sulforhodamine B (SBR) assay. Additionally, holotomographic 3D microscopy was employed to investigate the mechanisms of cell death. Sulfonamido-derived Schiff bases were synthesized through a three-step procedure that did not require column chromatography purification: (1) isothiocyanation of aminosulfonamides, (2) nucleophilic addition of hydrazine, and (3) acid-promoted condensation with different aldehydes (benzaldehydes or pyridine-2-carboxaldehyde). The synthesized compounds exhibited inhibition of CAs in the low nanomolar to submicromolar range, with selectivity largely influenced by structural features. Notably, the m-sulfonamide derivative 5b, bearing a pyridin-2-yl residue, demonstrated potent and selective inhibition of CA IX (K i = 4.9 nM) and XII (K i = 5.6 nM). Additionally, it efficiently chelated Fe 2+ , Fe 3+ , and Cu 2+ and showed promising antiproliferative activity (GI 50 4.5–10 µ M). Mechanistic studies revealed that apoptosis was involved in its mode of action. Therefore, the synergistic integration of sulfonamides and thiosemicarbazones represents an effective strategy for the development of multimodal anticancer agents. Keywords: carbonic anhydrases; sulfonamides; thiosemicarbazones; docking simulations; metal chelation; antiproliferative activity Int. J. Mol. Sci. 2025,26, 1225 https://doi.org/10.3390/ijms26031225 Int. J. Mol. Sci. 2025,26, 1225 2 of 23 1. Introduction Cancer is the second leading cause of mortality worldwide [ 1 ], representing one of the most significant challenges in biomedical research [ 2 ]. It is among the most complex and devastating diseases of our time [ 3 ]. Despite tremendous advances in early detection [ 4 ], surgery [ 5 ], radiotherapy [ 6 ], immunotherapy [ 7 ], nanotechnology [ 8 ] and other therapeutic approaches, chemotherapy continues to be a cornerstone treatment for many types of cancer [ 9 ]. However, the intricate complexity of cancer, combined with its multifactorial etiology, makes the traditional one-drug, one-target paradigm [ 10 ] insufficient. Consequently, a multitarget strategy [ 11 ] has become essential for combating tumor progression and therapeutic resistance [12]. In this context, our study aimed to develop multimodal therapeutic agents for cancer treatment. Specifically, we sought to design compounds that integrate a pharmacophore targeting enzymes overexpressed in tumors with a metal chelator to address the high levels of certain metals in tumor tissues. Our primary focus was on carbonic anhydrases (CAs), a family of ubiquitous Zn(II)-dependent metalloenzymes. These enzymes catalyze the reversible hydration of CO 2 to produce hydrogen carbonate and a proton [ 13 ]. The Zn(II) cation acts as a Lewis acid, lowering the pK a of the coordinated H 2 O molecule, thereby facilitating its deprotonation under physiological conditions [ 14 ]. CAs are found across almost all domains of life and are classified into eight different genetic families. The ones found in humans ( α -CAs) are in turn divided into 15 different isoforms, which vary in tissue distribution and catalytic activity [ 15 ]. The selective inhibition of the α -CAs IX and XII isoforms has emerged as a promising target in anticancer research [ 16 ], due to their role in acidifying the hypoxic tumor microenvironment, which promotes tumor progression and metastasis [ 17 ]. Alkyl and aryl sulfonamides constitute the largest family of CA inhibitors [ 18 ], which act by binding Zn(II) and thus blocking the enzyme activity. Interestingly, this pharmacophore has also been found to inhibit tyrosine kinases and aromatases [ 19 ], both of which are overexpressed in certain tumors. A notable example is compound SLC-0111, a ureido-containing aryl sulfonamide and a potent CA IX inhibitor currently undergoing clinical trials for advanced solid tumors. SLC-0111 has demonstrated potential as a sensitizer for head and neck squamous cell carcinoma (HNSCC) in combination with cisplatin [ 20 ] and has demonstrated the capacity of reducing hepatoblastoma cell viability and migration [21]. In addition to targeting CAs, we explored the incorporation of a metal-chelating framework linked to the sulfonamido motif. To the best of our knowledge, no prior studies have described anticancer agents designed to simultaneously target CAs and the disruption of metal homeostasis. Metals play crucial roles in healthy cells [ 22 ], contributing to signalling pathways [ 23 ], enzymatic activity, and the structural integrity of cell membranes and the genome [ 24 ]. However, disruptions in metal homeostasis—characterized by either an excess or deficiency—can profoundly affect cellular physiology. Elevated levels of metal cations such as Fe, Cu, and Zn have been linked to oncogenesis and metastasis [ 24 , 25 ]. For instance, high levels of Fe can exacerbate oxidative stress by interacting with H 2 O 2 , causing severe damage to cellular membranes and organelles [26]. Although further research is needed, metal chelators have shown promising results in preclinical anticancer studies [ 27 , 28 ]. Schiff bases and their derivatives, particularly thiosemicarbazones (the scaffold used in this study), have attracted significant attention as metal-binding ligands in anticancer research [ 29 – 31 ], with several compounds entering clinical trials [ 32 ]. Metal complexes derived from thiosemicarbazones have been extensively characterized using diverse analytical techniques, including IR and NMR spectroscopy, Int. J. Mol. Sci. 2025,26, 1225 3 of 23 conductivity measurements, thermogravimetric analysis, and density functional theory (DFT) calculations [33,34]. 2. Results and Discussion 2.1. Drug Design and Chemistry The primary objective of this study was the design, synthesis, and evaluation of multitarget sulfonamide–thiosemicarbazone hybrids (Figure 1) with potential anticancer activity, specifically targeting tumor-related CAs and hazardous high levels of metals. This approach represents a promising strategy for developing multitarget drug candidates with potential application in cancer chemotherapy. It has been suggested that the antineoplastic properties of thiosemicarbazones are enhanced when they exert effective metal-chelating capabilities [ 35 ]. Schiff bases and related compounds, particularly thiosemicarbazones, have been reported to exhibit anticancer properties through various mechanisms [ 36 ], including interactions with DNA [ 37 ], inhibition of topoisomerases [ 38 ], and mitigation of multidrug resistance (MDR) [ 32 ]. Among these mechanisms, their capacity to complex iron is widely recognized as the primary anticancer mechanism [ 39 ], with Fe-dependent ribonuclease reductase (RNR) identified as a key intracellular target. Recent studies on nanomolar anticancer thiosemicarbazones have also highlighted their ability to complex Cu(II) ions, with human serum albumin identified as a potential metal source [ 40 ]. Consequently, thiosemicarbazones are now regarded as more than just simple chelators; they are classified as metal-interacting drugs with multimodal anticancer activity [ 41 ]. A notable example of this pharmacophore is Triapine ® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), which has undergone numerous clinical trials [ 42 ]. In this study, the two key structural moieties investigated were the aryl sulfonamide and the thiosemicarbazone groups. To further enhance biological activity, structural modifications were systematically introduced, including varying the position of the sulfonamido motif, employing different spacers to link the sulfonamide and thiosemicarbazone moieties, incorporating aryl and heteroaryl residues (e.g., pyridine-2-yl) into the imino scaffold, and modifying substituents on the aryl ring. These structural variations provided a robust framework for establishing structure–activity relationships (SARs), facilitating the rational design of more potent and selective anticancer agents. Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 3 of 24 Although further research is needed, metal chelators have shown promising results in preclinical anticancer studies [27,28]. Schiff bases and their derivatives, particularly thiosemicarbazones (the scaffold used in this study), have attracted significant attention as metal-binding ligands in anticancer research [29–31], with several compounds entering clinical trials [32]. Metal complexes derived from thiosemicarbazones have been extensively characterized using diverse analytical techniques, including IR and NMR spectroscopy, conductivity measurements, thermogravimetric analysis, and density functional theory (DFT) calculations [33,34]. 2. Results and Discussion 2.1. Drug Design and Chemistry The primary objective of this study was the design, synthesis, and evaluation of multitarget sulfonamide–thiosemicarbazone hybrids (Figure 1) with potential anticancer activity, specifically targeting tumor-related CAs and hazardous high levels of metals. This approach represents a promising strategy for developing multitarget drug candidates with potential application in cancer chemotherapy. It has been suggested that the antineoplastic properties of thiosemicarbazones are enhanced when they exert effective metalchelating capabilities [35]. Schiff bases and related compounds, particularly thiosemicarbazones, have been reported to exhibit anticancer properties through various mechanisms [36], including interactions with DNA [37], inhibition of topoisomerases [38], and mitigation of multidrug resistance (MDR) [32]. Among these mechanisms, their capacity to complex iron is widely recognized as the primary anticancer mechanism [39], with Fedependent ribonuclease reductase (RNR) identified as a key intracellular target. Recent studies on nanomolar anticancer thiosemicarbazones have also highlighted their ability to complex Cu(II) ions, with human serum albumin identified as a potential metal source [40]. Consequently, thiosemicarbazones are now regarded as more than just simple chelators; they are classified as metal-interacting drugs with multimodal anticancer activity [41]. A notable example of this pharmacophore is Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), which has undergone numerous clinical trials [42]. In this study, the two key structural moieties investigated were the aryl sulfonamide and the thiosemicarbazone groups. To further enhance biological activity, structural modifications were systematically introduced, including varying the position of the sulfonamido motif, employing different spacers to link the sulfonamide and thiosemicarbazone moieties, incorporating aryl and heteroaryl residues (e.g., pyridine-2-yl) into the imino scaffold, and modifying substituents on the aryl ring. These structural variations provided a robust framework for establishing structure–activity relationships (SARs), facilitating the rational design of more potent and selective anticancer agents. Figure 1. Design of multifaceted sulfonamide-derived thiosemicarbazones. Figure 1. Design of multifaceted sulfonamide-derived thiosemicarbazones. The synthetic pathway is outlined in Scheme 1. Commercially available aryl aminosulfonamides 1a–c were transformed into their corresponding isothiocyanates using two different procedures: treatment with thiophosgene under acidic conditions [ 43 ] for derivatives 1a and 1b, or reaction with dicyclohexyl carbodiimide (DCC) and CS 2 in Py [ 44 ] for compound 1c. The resulting heterocumulenes (compounds 2a–c, obtained in good to excellent yields) were subsequently treated with hydrazine hydrate to furnish the corresponding thiosemicarbazides 3a–c, with yields ranging from 42 to 75%. Final condensa- Int. J. Mol. Sci. 2025,26, 1225 4 of 23 tion under acidic conditions with benzaldehydes or pyridine-2-carboxaldehyde yielded sulfonamide-derived thiosemicarbazones 4a–p (47–76%) or 5a–c (31–42%), respectively. The incorporation of a pyridine-2-yl residue in derivatives 5a–c is expected to provide a tridentate ligand, likely capable of complexing relevant cations more efficiently. This property may facilitate the sequestration and removal of metal ions from tumor tissues. The synthetic procedure is efficient and straightforward, with all intermediates and final compounds being crystalline and easily purified by filtration. This eliminates the need for time-consuming column chromatography purifications, making the process highly practical for further development. Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 4 of 24 The synthetic pathway is outlined in Scheme 1. Commercially available aryl aminosulfonamides 1a–c were transformed into their corresponding isothiocyanates using two different procedures: treatment with thiophosgene under acidic conditions [43] for derivatives 1a and 1b, or reaction with dicyclohexyl carbodiimide (DCC) and CS2 in Py [44] for compound 1c. The resulting heterocumulenes (compounds 2a–c, obtained in good to excellent yields) were subsequently treated with hydrazine hydrate to furnish the corresponding thiosemicarbazides 3a–c, with yields ranging from 42 to 75%. Final condensation under acidic conditions with benzaldehydes or pyridine-2-carboxaldehyde yielded sulfonamide-derived thiosemicarbazones 4a–p (47–76%) or 5a–c (31–42%), respectively. The incorporation of a pyridine-2-yl residue in derivatives 5a–c is expected to provide a tridentate ligand, likely capable of complexing relevant cations more efficiently. This property may facilitate the sequestration and removal of metal ions from tumor tissues. The synthetic procedure is efficient and straightforward, with all intermediates and final compounds being crystalline and easily purified by filtration. This eliminates the need for time-consuming column chromatography purifications, making the process highly practical for further development. Scheme 1. Preparation of sulfonamide-derived thiosemicarbazones 4 and 5. (a) CSCl2, aq. HCl; (b) DCC, CS2, Py. As a representative example, Figures 2 and 3 depict the 1Hand 13C-NMR spectra of the pyridine-containing thiosemicarbazone 5b. In the 1H-RNM spectrum, the most notable signals are observed at 12.34 ppm (Ar-NH), 10.51 ppm (NH), and 8.21 ppm (azomethyne proton). Similarly, in the 13C-NMR spectrum, the resonances at 176.7 ppm (thione moiety) and 144.2 ppm (imine moiety) provide further confirmation of the proposed structure. Scheme 1. Preparation of sulfonamide-derived thiosemicarbazones 4and 5. (a) CSCl 2 , aq. HCl; (b) DCC, CS2, Py. As a representative example, Figures 2and 3depict the 1 Hand 13 C-NMR spectra of the pyridine-containing thiosemicarbazone 5b. In the 1 H-RNM spectrum, the most notable signals are observed at 12.34 ppm (Ar-NH), 10.51 ppm (NH), and 8.21 ppm (azomethyne proton). Similarly, in the 13 C-NMR spectrum, the resonances at 176.7 ppm (thione moiety) and 144.2 ppm (imine moiety) provide further confirmation of the proposed structure. Int. J. Mol. Sci. 2025,26, 1225 5 of 23 Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 24 Figure 2. 1H-NMR spectrum of 5b (300 MHz, DMSO-d6). Figure 3. 13C-NMR spectrum of 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessments 2.2.1. CA Inhibition Thiosemicarbazones 4a–p and 5a–c were evaluated as potential inhibitors of tumorassociated CAs IX and XII. For comparison, their synthetic precursors, thiosemicarbazides 3a–c, as well as acetazolamide (AAZ), as a standard positive control, were included in the study. To assess selectivity, the inhibition of cytosolic CAs I and II was also measured. The inhibition constants (Ki, nM), determined using the stopped-flow CO2 hydrase assay, along with the selectivity indexes, are depicted in Table 1. The following SARs were identified: Figure 2. 1H-NMR spectrum of 5b (300 MHz, DMSO-d6). Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 24 Figure 2. 1H-NMR spectrum of 5b (300 MHz, DMSO-d6). Figure 3. 13C-NMR spectrum of 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessments 2.2.1. CA Inhibition Thiosemicarbazones 4a–p and 5a–c were evaluated as potential inhibitors of tumorassociated CAs IX and XII. For comparison, their synthetic precursors, thiosemicarbazides 3a–c, as well as acetazolamide (AAZ), as a standard positive control, were included in the study. To assess selectivity, the inhibition of cytosolic CAs I and II was also measured. The inhibition constants (Ki, nM), determined using the stopped-flow CO2 hydrase assay, along with the selectivity indexes, are depicted in Table 1. The following SARs were identified: Figure 3. 13C-NMR spectrum of 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessments 2.2.1. CA Inhibition Thiosemicarbazones 4a–p and 5a–c were evaluated as potential inhibitors of tumorassociated CAs IX and XII. For comparison, their synthetic precursors, thiosemicarbazides 3a–c, as well as acetazolamide (AAZ), as a standard positive control, were included in the study. To assess selectivity, the inhibition of cytosolic CAs I and II was also measured. The inhibition constants (K i , nM), determined using the stopped-flow CO 2 hydrase assay, along with the selectivity indexes, are depicted in Table 1. The following SARs were identified: Inhibition of CAs IX and XII: all Schiff bases exhibited varying degrees of inhibition of transmembrane CAs IX and XII, with K i values ranging from low nanomolar to sub- Int. J. Mol. Sci. 2025,26, 1225 6 of 23 micromolar (2.3–499 nM), depending on the aryl ring substituents, the position of the sulfonamido moiety, and the presence or absence of an ethylene linker. Preference for CA XII: across all derivatives, including thiosemicarbazide precursors 3, stronger inhibition was consistently observed for CA XII compared with CA IX. This trend is particularly noteworthy, as numerous CA XII inhibitors are also known to inhibit glycoprotein-P (Pg-p), potentially reducing chemoresistance caused by xenobiotic efflux via the Pg-p pump [ 45 ]. An exception to this trend was observed for the pyridinederived thiosemicarbazone 5b, which displayed comparable potency against both isoforms (Ki= 4.9 and 5.6 nM, respectively). mand p-Substituted sulfonamido derivatives: among compounds bearing the sulfonamido motif at the para position, the highest activity against CA XII was observed for unsubstituted aromatic rings (4a,K i = 8.38 nM; 4l,K i = 9.12 nM). The introduction of an ethylene linker (4l–p vs. 4a–f) enhanced selectivity for CA XII, primarily by reducing activity against CA I, with K i values in the micromolar range for derivatives 4m–p. Among the meta regioisomers, derivative 4h, bearing a p-methoxy substituent, exhibited the highest CA XII activity (Ki= 2.3 nM). Pyridine-containing derivatives: thiosemicarbazones containing a pyridine fragment and lacking the ethylene linker (5a,b) displayed strong inhibition of CA XII (K i = 4.9 and 5.6 nM, respectively), with selectivity comparable to or exceeding that of the reference drug AAZ. Additionally, compound 5b exhibited potent inhibition of CA II (K i = 2.5 nM). Notably, CAs IV, XII, and, particularly, II, are validated targets for glaucoma treatment due to their role in mitigating ocular hypertension [ 46 ]. Incorporation of the ethylene linker (5c) preserved strong inhibition of CA XII (K i = 9.3 nM) but significantly decreased CA I/XII selectivity. Selectivity: the meta placement of the sulfonamido moiety on the aromatic ring generally reduced activity against CA I, thereby improving selectivity. This is a critical factor for minimizing off-target effects. Remarkably, the strongest CA XII inhibitor, derivative 4h (K i = 2.3 nM), exhibited the highest CA I/XII selectivity index (S.I. = 325), significantly outperforming AAZ (K i = 5.2 nM for CA XII; S.I. = 43.9). Another noteworthy compound is the m-sulfonamide 5b, which bears a pyridine-2-yl scaffold (S.I. = 109.8 and 96.1 for CA I/IX and CA I/XII, respectively). These findings align with previously reported data, in which many aryl sulfonamides demonstrated significant potency as inhibitors of CA IX and XII isoforms, with K i values in the low nanomolar range. However, their selectivity against off-target isoforms, such as CA I, is usually lower compared with coumarins [ 47 ], another important class of CA inhibitors. Table 1. Inhibition data (K i , nM) of compounds 4and 5against human CAs I, II, IX, and XII [a] using AAZ as a standard drug. Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 23 Inhibition of CAs IX and XII: all Schiff bases exhibited varying degrees of inhibition of transmembrane CAs IX and XII, with Ki values ranging from low nanomolar to submicromolar (2.3–499 nM), depending on the aryl ring substituents, the position of the sulfonamido moiety, and the presence or absence of an ethylene linker. Preference for CA XII: across all derivatives, including thiosemicarbazide precursors 3, stronger inhibition was consistently observed for CA XII compared with CA IX. This trend is particularly noteworthy, as numerous CA XII inhibitors are also known to inhibit glycoprotein-P (Pg-p), potentially reducing chemoresistance caused by xenobiotic efflux via the Pg-p pump [45]. An exception to this trend was observed for the pyridine-derived thiosemicarbazone 5b, which displayed comparable potency against both isoforms (Ki = 4.9 and 5.6 nM, respectively). mand p-Substituted sulfonamido derivatives: among compounds bearing the sulfonamido motif at the para position, the highest activity against CA XII was observed for unsubstituted aromatic rings (4a, Ki = 8.38 nM; 4l, Ki = 9.12 nM). The introduction of an ethylene linker (4l–p vs. 4a–f) enhanced selectivity for CA XII, primarily by reducing activity against CA I, with Ki values in the micromolar range for derivatives 4m–p. Among the meta regioisomers, derivative 4h, bearing a p-methoxy substituent, exhibited the highest CA XII activity (Ki = 2.3 nM). Pyridine-containing derivatives: thiosemicarbazones containing a pyridine fragment and lacking the ethylene linker (5a,b) displayed strong inhibition of CA XII (Ki = 4.9 and 5.6 nM, respectively), with selectivity comparable to or exceeding that of the reference drug AAZ. Additionally, compound 5b exhibited potent inhibition of CA II (Ki = 2.5 nM). Notably, CAs IV, XII, and, particularly, II, are validated targets for glaucoma treatment due to their role in mitigating ocular hypertension [46]. Incorporation of the ethylene linker (5c) preserved strong inhibition of CA XII (Ki = 9.3 nM) but significantly decreased CA I/XII selectivity. Selectivity: the meta placement of the sulfonamido moiety on the aromatic ring generally reduced activity against CA I, thereby improving selectivity. This is a critical factor for minimizing off-target effects. Remarkably, the strongest CA XII inhibitor, derivative 4h (Ki = 2.3 nM), exhibited the highest CA I/XII selectivity index (S.I. = 325), significantly outperforming AAZ (Ki = 5.2 nM for CA XII; S.I. = 43.9). Another noteworthy compound is the m-sulfonamide 5b, which bears a pyridine-2-yl scaffold (S.I. = 109.8 and 96.1 for CA I/IX and CA I/XII, respectively). These findings align with previously reported data, in which many aryl sulfonamides demonstrated significant potency as inhibitors of CA IX and XII isoforms, with Ki values in the low nanomolar range. However, their selectivity against off-target isoforms, such as CA I, is usually lower compared with coumarins [47], another important class of CA inhibitors. Table 1. Inhibition data (Ki, nM) of compounds 4 and 5 against human CAs I, II, IX, and XII [a] using AAZ as a standard drug. Compound CA I CA II CA IX CA XII Selectivity Ratio I/IX//II/IX Selectivity Ratio I/XII//II/XII 3a (n = 0, p-) 327 7600 133 68 2.5//57.1 4.8//111.8 3b (n = 0, m-) 1457 439 185 116 7.9//2.4 12.6//3.8 3c (n = 2, p-) 79.0 8.51 13.0 5.02 6.1//0.65 15.7//1.7 Compound CA ICA II CA IX CA XII Selectivity Ratio I/IX//II/IX Selectivity Ratio I/XII//II/XII 3a (n = 0, p-) 327 7600 133 68 2.5//57.1 4.8//111.8 3b (n = 0, m-) 1457 439 185 116 7.9//2.4 12.6//3.8 3c (n = 2, p-) 79.0 8.51 13.0 5.02 6.1//0.65 15.7//1.7 4a (n = 0, p-, R = H) 504 23.7 16.1 8.38 31.3//1.5 60.1//2.8 Int. J. Mol. Sci. 2025,26, 1225 7 of 23 Table 1. Cont. Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 23 Inhibition of CAs IX and XII: all Schiff bases exhibited varying degrees of inhibition of transmembrane CAs IX and XII, with Ki values ranging from low nanomolar to submicromolar (2.3–499 nM), depending on the aryl ring substituents, the position of the sulfonamido moiety, and the presence or absence of an ethylene linker. Preference for CA XII: across all derivatives, including thiosemicarbazide precursors 3, stronger inhibition was consistently observed for CA XII compared with CA IX. This trend is particularly noteworthy, as numerous CA XII inhibitors are also known to inhibit glycoprotein-P (Pg-p), potentially reducing chemoresistance caused by xenobiotic efflux via the Pg-p pump [45]. An exception to this trend was observed for the pyridine-derived thiosemicarbazone 5b, which displayed comparable potency against both isoforms (Ki = 4.9 and 5.6 nM, respectively). mand p-Substituted sulfonamido derivatives: among compounds bearing the sulfonamido motif at the para position, the highest activity against CA XII was observed for unsubstituted aromatic rings (4a, Ki = 8.38 nM; 4l, Ki = 9.12 nM). The introduction of an ethylene linker (4l–p vs. 4a–f) enhanced selectivity for CA XII, primarily by reducing activity against CA I, with Ki values in the micromolar range for derivatives 4m–p. Among the meta regioisomers, derivative 4h, bearing a p-methoxy substituent, exhibited the highest CA XII activity (Ki = 2.3 nM). Pyridine-containing derivatives: thiosemicarbazones containing a pyridine fragment and lacking the ethylene linker (5a,b) displayed strong inhibition of CA XII (Ki = 4.9 and 5.6 nM, respectively), with selectivity comparable to or exceeding that of the reference drug AAZ. Additionally, compound 5b exhibited potent inhibition of CA II (Ki = 2.5 nM). Notably, CAs IV, XII, and, particularly, II, are validated targets for glaucoma treatment due to their role in mitigating ocular hypertension [46]. Incorporation of the ethylene linker (5c) preserved strong inhibition of CA XII (Ki = 9.3 nM) but significantly decreased CA I/XII selectivity. Selectivity: the meta placement of the sulfonamido moiety on the aromatic ring generally reduced activity against CA I, thereby improving selectivity. This is a critical factor for minimizing off-target effects. Remarkably, the strongest CA XII inhibitor, derivative 4h (Ki = 2.3 nM), exhibited the highest CA I/XII selectivity index (S.I. = 325), significantly outperforming AAZ (Ki = 5.2 nM for CA XII; S.I. = 43.9). Another noteworthy compound is the m-sulfonamide 5b, which bears a pyridine-2-yl scaffold (S.I. = 109.8 and 96.1 for CA I/IX and CA I/XII, respectively). These findings align with previously reported data, in which many aryl sulfonamides demonstrated significant potency as inhibitors of CA IX and XII isoforms, with Ki values in the low nanomolar range. However, their selectivity against off-target isoforms, such as CA I, is usually lower compared with coumarins [47], another important class of CA inhibitors. Table 1. Inhibition data (Ki, nM) of compounds 4 and 5 against human CAs I, II, IX, and XII [a] using AAZ as a standard drug. Compound CA I CA II CA IX CA XII Selectivity Ratio I/IX//II/IX Selectivity Ratio I/XII//II/XII 3a (n = 0, p-) 327 7600 133 68 2.5//57.1 4.8//111.8 3b (n = 0, m-) 1457 439 185 116 7.9//2.4 12.6//3.8 3c (n = 2, p-) 79.0 8.51 13.0 5.02 6.1//0.65 15.7//1.7 Compound CA ICA II CA IX CA XII Selectivity Ratio I/IX//II/IX Selectivity Ratio I/XII//II/XII 4b (n = 0, p-, R = OMe) 215 78 91 49 2.4//0.86 4.4//1.6 4c (n = 0, p-, R = F) 196 44.6 147 51.8 1.3//0.30 3.8//0.86 4d (n = 0, p-, R = Cl) 485 48.8 27.4 12.1 17.7//1.8 40.1//4.0 4e (n = 0, p-, R = Br) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4f (n = 0, p-, R = NO2) 845 820 477 288 1.8//1.7 2.9//2.8 4g (n = 0, m-, R = H) 876 60.5 74.3 50.9 11.8//0.81 17.2//1.2 4h (n = 0, m-, R = OMe) 748 39.4 387 2.3 1.9//0.10 325//17.1 4i (n = 0, m-, R = F) 496 97.1 89.3 49.3 5.6//1.1 10.1//2.0 4j (n = 0, m-, R = Cl) 5186 312 217 85.4 23.9//1.4 60.7//3.7 4k (n = 0, m-, R = Br) 5337 531 206 91.3 25.9//2.6 58.5//5.8 4l (n = 2, p-, R = H) 568 70.2 30.8 9.12 18.4//2.3 62.3//7.7 4m (n = 2, p-, R = OMe) 3479 321 164 35.0 21.2//2.0 99.4//9.2 4n (n = 2, p-, R = F) 2485 590 300 48.5 8.3//2.0 51.2//12.2 4o (n = 2, p-, R = Cl) 6217 773 408 143 15.2//1.9 43.4//2.9 4p (n = 2, p-, R = Br) 6092 813 499 123 12.2//1.6 49.5//6.6 5a (n = 0, p-) 207 156.6 351.9 4.9 0.59//0.45 42.2//31.9 5b (n = 0, m-) 538 2.5 4.9 5.6 109.8//0.49 96.1//0.43 5c (n = 2, p-) 270 83.9 28.5 9.3 9.5//2.9 29.0//9.0 AAZ 250.0 12.0 25.0 5.7 10.0//0.48 43.9//2.1 [a] Mean from three different assays by a stopped flow technique (errors were in the range of ± 5–10% of the reported values). 2.2.2. Docking Simulations The interaction profiles of the selected thiosemicarbazone–sulfonamide hybrids with CA IX and CA XII enzymes were analyzed using ligand–protein docking simulations. The simulations took into account the water molecules surrounding the Zn(II) ion, in line with the widely reported interaction mechanisms for sulfonamide-based CA inhibitors. Additionally, based on extensive literature evidence, the sulfonamido moiety of the hybrids was deprotonated for the simulations. For this study, derivatives 4a and 5b were considered as representative compounds. The binding energies from the docking simulations against CA IX are shown in Table 2. Docking simulations predict that both 4a and 5b interact with the Zn(II) ion through the deprotonated form of the sulfonamido motif and with the gatekeeper residue Thr200. Additionally, the partially negative oxygen atom of 4a also interacts with the Zn(II) ion, while the sulfur atom of the thiosemicarbazone moiety is involved in an H-bond between water and His68. In contrast, for 5b, the same sulfur atom interacts with Gln71 as an electron acceptor, whilst the Gln92 is the acceptor from an interaction with the NH group of the thiosemicarbazone. Moreover, an Hπ stacking interaction occurs between the aromatic moiety of the benzenesulfonamide and Leu199. The heteroatom of the pyridine ring also participates in cooperative H-bonding with water molecules and Pro202 (Figure 4). Int. J. Mol. Sci. 2025,26, 1225 8 of 23 Table 2. Binding energies estimated for compounds 4a and 5b in their interaction with CA IX. Compound Binding Energy (kcal/mol) Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 23 4a (n = 0, p-, R = H) 504 23.7 16.1 8.38 31.3//1.5 60.1//2.8 4b (n = 0, p-, R = OMe) 215 78 91 49 2.4//0.86 4.4//1.6 4c (n = 0, p-, R = F) 196 44.6 147 51.8 1.3//0.30 3.8//0.86 4d (n = 0, p-, R = Cl) 485 48.8 27.4 12.1 17.7//1.8 40.1//4.0 4e (n = 0, p-, R = Br) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4f (n = 0, p-, R = NO2) 845 820 477 288 1.8//1.7 2.9//2.8 4g (n = 0, m-, R = H) 876 60.5 74.3 50.9 11.8//0.81 17.2//1.2 4h (n = 0, m-, R = OMe) 748 39.4 387 2.3 1.9//0.10 325//17.1 4i (n = 0, m-, R = F) 496 97.1 89.3 49.3 5.6//1.1 10.1//2.0 4j (n = 0, m-, R = Cl) 5186 312 217 85.4 23.9//1.4 60.7//3.7 4k (n = 0, m-, R = Br) 5337 531 206 91.3 25.9//2.6 58.5//5.8 4l (n = 2, p-, R = H) 568 70.2 30.8 9.12 18.4//2.3 62.3//7.7 4m (n = 2, p-, R = OMe) 3479 321 164 35.0 21.2//2.0 99.4//9.2 4n (n = 2, p-, R = F) 2485 590 300 48.5 8.3//2.0 51.2//12.2 4o (n = 2, p-, R = Cl) 6217 773 408 143 15.2//1.9 43.4//2.9 4p (n = 2, p-, R = Br) 6092 813 499 123 12.2//1.6 49.5//6.6 5a (n = 0, p-) 207 156.6 351.9 4.9 0.59//0.45 42.2//31.9 5b (n = 0, m-) 538 2.5 4.9 5.6 109.8//0.49 96.1//0.43 5c (n = 2, p-) 270 83.9 28.5 9.3 9.5//2.9 29.0//9.0 AAZ 250.0 12.0 25.0 5.7 10.0//0.48 43.9//2.1 [a] Mean from three different assays by a stopped flow technique (errors were in the range of ±5– 10% of the reported values). 2.2.2. Docking Simulations The interaction profiles of the selected thiosemicarbazone–sulfonamide hybrids with CA IX and CA XII enzymes were analyzed using ligand–protein docking simulations. The simulations took into account the water molecules surrounding the Zn(II) ion, in line with the widely reported interaction mechanisms for sulfonamide-based CA inhibitors. Additionally, based on extensive literature evidence, the sulfonamido moiety of the hybrids was deprotonated for the simulations. For this study, derivatives 4a and 5b were considered as representative compounds. The binding energies from the docking simulations against CA IX are shown in Table 2. Table 2. Binding energies estimated for compounds 4a and 5b in their interaction with CA IX. Compound Binding Energy (kcal/mol) 4a −7.93 5b −7.45 Docking simulations predict that both 4a and 5b interact with the Zn(II) ion through the deprotonated form of the sulfonamido motif and with the gatekeeper residue Thr200. Additionally, the partially negative oxygen atom of 4a also interacts with the Zn(II) ion, while the sulfur atom of the thiosemicarbazone moiety is involved in an H-bond between 4a −7.93 Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 23 4a (n = 0, p-, R = H) 504 23.7 16.1 8.38 31.3//1.5 60.1//2.8 4b (n = 0, p-, R = OMe) 215 78 91 49 2.4//0.86 4.4//1.6 4c (n = 0, p-, R = F) 196 44.6 147 51.8 1.3//0.30 3.8//0.86 4d (n = 0, p-, R = Cl) 485 48.8 27.4 12.1 17.7//1.8 40.1//4.0 4e (n = 0, p-, R = Br) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4f (n = 0, p-, R = NO2) 845 820 477 288 1.8//1.7 2.9//2.8 4g (n = 0, m-, R = H) 876 60.5 74.3 50.9 11.8//0.81 17.2//1.2 4h (n = 0, m-, R = OMe) 748 39.4 387 2.3 1.9//0.10 325//17.1 4i (n = 0, m-, R = F) 496 97.1 89.3 49.3 5.6//1.1 10.1//2.0 4j (n = 0, m-, R = Cl) 5186 312 217 85.4 23.9//1.4 60.7//3.7 4k (n = 0, m-, R = Br) 5337 531 206 91.3 25.9//2.6 58.5//5.8 4l (n = 2, p-, R = H) 568 70.2 30.8 9.12 18.4//2.3 62.3//7.7 4m (n = 2, p-, R = OMe) 3479 321 164 35.0 21.2//2.0 99.4//9.2 4n (n = 2, p-, R = F) 2485 590 300 48.5 8.3//2.0 51.2//12.2 4o (n = 2, p-, R = Cl) 6217 773 408 143 15.2//1.9 43.4//2.9 4p (n = 2, p-, R = Br) 6092 813 499 123 12.2//1.6 49.5//6.6 5a (n = 0, p-) 207 156.6 351.9 4.9 0.59//0.45 42.2//31.9 5b (n = 0, m-) 538 2.5 4.9 5.6 109.8//0.49 96.1//0.43 5c (n = 2, p-) 270 83.9 28.5 9.3 9.5//2.9 29.0//9.0 AAZ 250.0 12.0 25.0 5.7 10.0//0.48 43.9//2.1 [a] Mean from three different assays by a stopped flow technique (errors were in the range of ±5– 10% of the reported values). 2.2.2. Docking Simulations The interaction profiles of the selected thiosemicarbazone–sulfonamide hybrids with CA IX and CA XII enzymes were analyzed using ligand–protein docking simulations. The simulations took into account the water molecules surrounding the Zn(II) ion, in line with the widely reported interaction mechanisms for sulfonamide-based CA inhibitors. Additionally, based on extensive literature evidence, the sulfonamido moiety of the hybrids was deprotonated for the simulations. For this study, derivatives 4a and 5b were considered as representative compounds. The binding energies from the docking simulations against CA IX are shown in Table 2. Table 2. Binding energies estimated for compounds 4a and 5b in their interaction with CA IX. Compound Binding Energy (kcal/mol) 4a −7.93 5b −7.45 Docking simulations predict that both 4a and 5b interact with the Zn(II) ion through the deprotonated form of the sulfonamido motif and with the gatekeeper residue Thr200. Additionally, the partially negative oxygen atom of 4a also interacts with the Zn(II) ion, while the sulfur atom of the thiosemicarbazone moiety is involved in an H-bond between 5b −7.45 Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 of 24 Additionally, the partially negative oxygen atom of 4a also interacts with the Zn(II) ion, while the sulfur atom of the thiosemicarbazone moiety is involved in an H-bond between water and His68. In contrast, for 5b, the same sulfur atom interacts with Gln71 as an electron acceptor, whilst the Gln92 is the acceptor from an interaction with the NH group of the thiosemicarbazone. Moreover, an H-π stacking interaction occurs between the aromatic moiety of the benzenesulfonamide and Leu199. The heteroatom of the pyridine ring also participates in cooperative H-bonding with water molecules and Pro202 (Figure 4). Figure 4. (A,C): docking representation of CA IX (gold)–4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. Similar to the findings observed for CA IX, the deprotonated NH of the sulfonamido moiety in 4a and 5a interacts with the Zn(II) ion and simultaneously with Leu197 and the gatekeeper residue Thr198 in CA XII. Additionally, compound 5b exhibits a direct binding with Ala 129 (Figure 5). The binding energies for the interaction with CA XII are depicted in Table 3. Figure 4. (A,C): docking representation of CA IX (gold)–4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. Similar to the findings observed for CA IX, the deprotonated NH of the sulfonamido moiety in 4a and 5a interacts with the Zn(II) ion and simultaneously with Leu197 and the gatekeeper residue Thr198 in CA XII. Additionally, compound 5b exhibits a direct binding with Ala 129 (Figure 5). The binding energies for the interaction with CA XII are depicted in Table 3. Int. J. Mol. Sci. 2025,26, 1225 9 of 23 Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 9 of 24 Table 3. Binding energies resulting from the docking simulations of the selected molecules against CA XII. Re-docking score of 6R6Y co-crystallized ligand is also included. Compound Binding Energy (kcal/mol) 4a –8.20 5b –7.68 Figure 5. (A,C): docking representation of CAXII (pink)—4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. 2.2.3. Metal Complexation Assays The ability of 5b, as a model compound, to complex metal cations associated with tumorigenesis (Na + , K + , Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ) was analyzed using UV–Vis spectroscopy. To investigate this, UV–Vis spectra of solutions with varying ratios of 5b to metal chlorides (1:0, 4:1, 2:1, 1:1, 1:2, 1:5) were recorded. Additionally, a 1:2 ligand-to-metal spectrum was obtained after 72 h of incubation to account for potential slow complexation. Titration experiments with NaCl and KCl showed no significant spectral changes with increasing amounts of the metals (Figure 6E,F, respectively), indicating that 5b does not Figure 5. (A,C): docking representation of CAXII (pink)—4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. Table 3. Binding energies resulting from the docking simulations of the selected molecules against CA XII. Re-docking score of 6R6Y co-crystallized ligand is also included. Compound Binding Energy (kcal/mol) Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 of 23 water and His68. In contrast, for 5b, the same sulfur atom interacts with Gln71 as an electron acceptor, whilst the Gln92 is the acceptor from an interaction with the NH group of the thiosemicarbazone. Moreover, an H-π stacking interaction occurs between the aromatic moiety of the benzenesulfonamide and Leu199. The heteroatom of the pyridine ring also participates in cooperative H-bonding with water molecules and Pro202 (Figure 4). Figure 4. (A,C): docking representation of CA IX (gold)–4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. Similar to the findings observed for CA IX, the deprotonated NH of the sulfonamido moiety in 4a and 5a interacts with the Zn(II) ion and simultaneously with Leu197 and the gatekeeper residue Thr198 in CA XII. Additionally, compound 5b exhibits a direct binding with Ala 129 (Figure 5). The binding energies for the interaction with CA XII are depicted in Table 3. Table 3. Binding energies resulting from the docking simulations of the selected molecules against CA XII. Re-docking score of 6R6Y co-crystallized ligand is also included. Compound Binding Energy (kcal/mol) 4a –8.20 –7.68 A B C D 4a −8.20 Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 of 23 water and His68. In contrast, for 5b, the same sulfur atom interacts with Gln71 as an electron acceptor, whilst the Gln92 is the acceptor from an interaction with the NH group of the thiosemicarbazone. Moreover, an H-π stacking interaction occurs between the aromatic moiety of the benzenesulfonamide and Leu199. The heteroatom of the pyridine ring also participates in cooperative H-bonding with water molecules and Pro202 (Figure 4). Figure 4. (A,C): docking representation of CA IX (gold)–4a (green) and 5b (light blue) binding complex. (B,D): two-dimensional scheme of the protein–ligand interactions. Similar to the findings observed for CA IX, the deprotonated NH of the sulfonamido moiety in 4a and 5a interacts with the Zn(II) ion and simultaneously with Leu197 and the gatekeeper residue Thr198 in CA XII. Additionally, compound 5b exhibits a direct binding with Ala 129 (Figure 5). The binding energies for the interaction with CA XII are depicted in Table 3. Table 3. Binding energies resulting from the docking simulations of the selected molecules against CA XII. Re-docking score of 6R6Y co-crystallized ligand is also included. Compound Binding Energy (kcal/mol) 4a –8.20 –7.68 A B C D 5b −7.68 2.2.3. Metal Complexation Assays The ability of 5b, as a model compound, to complex metal cations associated with tumorigenesis (Na + , K + , Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ) was analyzed using UV–Vis spectroscopy. To investigate this, UV–Vis spectra of solutions with varying ratios of 5b to metal chlorides (1:0, 4:1, 2:1, 1:1, 1:2, 1:5) were recorded. Additionally, a 1:2 ligand-to-metal spectrum was obtained after 72 h of incubation to account for potential slow complexation. Titration experiments with NaCl and KCl showed no significant spectral changes with increasing amounts of the metals (Figure 6E,F, respectively), indicating that 5b does not chelate these monovalent cations. In contrast, the spectra for divalent and trivalent cations revealed a decrease in absorbance at λmax , accompanied by a significant bathochromic shift. These changes suggest that 5b chelates Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ions. Among these, the most pronounced effect was observed with CuCl 2 (Figure 6B). The addition of stoichiometric or Int. J. Mol. Sci. 2025,26, 1225 16 of 23 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 176.0 (C=S), 163.2 (d, 1 J C,H = 247.6 Hz, C-4 ′′ ), 144.4, 142.1, 140.3 (CH=N, SO 2 NH 2 -Ar-Cp, NH-Ar-Cipso), 130.5 (d, 4 J C,H = 2.9, Hz, C-1 ′′ ), 130.0 (d, 3 J C,H = 8.6 Hz, C-2 ′′ /C-6 ′′ ), 125.6, 125.3 (Ar-C), 115.7 (d, 2 J C,H = 21.9 Hz, C-3 ′′ /C-5 ′′ ) ppm; HRESI-MS m/zcalcd. for C14H13FN4O3S2([M+H]+): 353.0537, found: 353.0536. 1-(4 ′ -Chlorophenylmethylene)-4-(4 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4d). Thiosemicarbazide 3a (200 mg, 0.81 mmol) and 4-chlorobenzaldehyde (114 mg, 0,81 mmol, 1.0 equiv.) were used. Compound 4d was obtained as a white solid. Yield: 223 mg (75%); spectroscopic data are in agreement with those reported [59]. 1-(4 ′ -Bromophenylmethylene)-4-(4 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4e). Thiosemicarbazide 3a (200 mg, 0.81 mmol) and 4-bromobenzaldehyde (87 µ L, 0.81 mmol, 1.0 equiv.) were used. Compound 4e was obtained as a white solid. Yield: 236 mg (71%); spectroscopic data are in agreement with those reported [59]. 1-(4 ′ -Nitrophenylmethylene)-4-(4 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4f). Thiosemicarbazide 3a (200 mg, 0.81 mmol) and 4-nitrobenzaldehyde (128 mg, 0.81 mmol, 1.0 equiv.) were used. Compound 4f was obtained as a yellow solid. Yield: 218 mg (72%). Mp: 238 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 12.27 (s, 1H, NH), 10.46 (s, 1H, Ar-NH), 8.26 (m, 2H, Ar-H), 8.26 (s, 1H, N=CH), 8.20 (m, 2H, Ar-H), 7.80 (m, 4H, Ar-H), 7.35 (brs, 2H, NH 2 ) ppm; 13 C-NMR (125.7 MHz, DMSO-d 6 ) δ 176.3 (CS), 147.7 (C-4 ′′ ), 141.8 (C=N), 140.8, 140.5, 140.2, 128.5, 125.6, 123.7 (Ar-C) ppm; HRESI-MS m/zcalcd. For C 14 H 13 N 5 NaO 4 S 2 ([M+Na]+): 402.0301, found: 402.0296. 1-Phenylmethylene-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4g). Thiosemicarbazide 3b (200 mg, 0.81 mmol) and benzaldehyde (84 µ L, 0.81 mmol, 1.0 equiv.) were used. Compound 4g was obtained as a white solid. Yield: 197 mg (68%); spectroscopic data are in agreement with those reported [59]. 1-(4 ′ -Methoxyphenylmethylene)-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4h). Thiosemicarbazide 3b (200 mg, 0.81 mmol) and 4-methoxybenzaldehyde (98 µ L, 0,81 mmol, 1.0 equiv.) were used. Compound 4h was obtained as a white solid. Yield: 140 mg (48%). Mp: 184 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.84 (s, 1H, NH), 10.2 (s, 1H, Ar-NH), 8.12 (s, 1H, N=CH), 8.05 (brt, 1H, J H,H = 1.9 Hz, H-2 ′′ ), 7.86 (m, 2H, H-2 ′ , H-6 ′ ), 7.84 (m, 1H, Ar-H), 7.64 (dt, 1H, J H,H = 1.4 Hz, J H,H = 8.4 Hz, Ar-H), 7.54 (t, 1H, J H,H = 7.9 Hz, Ar-H), 7.40 (s, 2H, NH 2 ), 6.99 (m, 2H, H-3 ′ , H-5 ′ ), 3.81 (s, 1H, OMe) ppm; 13 C-NMR (75.5 MHz, DMSO-d6) δ 176.1 (CS), 161.1 (C-4 ′ ), 144.4 (C=N), 143.8 (C-3 ′′ ), 140.0 (C-1 ′′ ), 129.8 (C-2 ′ /C-6 ′ ), 129.6, 128.9, 126.9, 123.2, 122.6 (Ar-C), 114.64 (C-3 ′ /C-5 ′ ), 55.8 (OMe) ppm; HRESI-MS m/zcalcd. for C15H16N4NaO3S2([M+Na]+): 387.0556, found: 387.0547. 1-(4 ′ -Fluorophenylmethylene)-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4i). Thiosemicarbazide 3b (200 mg, 0.81 mmol) and 4-fluorobenzaldehyde (87 µ L, 0,81 mmol, 1.0 eq.) were used. Compound 4i was obtained as a white solid. Yield: 198 mg (70%); spectroscopic data are in agreement with those reported [59]. 1-(4 ′ -Chlorophenylmethylene)-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4j). Thiosemicarbazide 3b (200 mg, 0.81 mmol) and 4-chlorobenzaldehyde (114 mg, 0,81 mmol, 1.0 equiv.) were used. Compound 4j was obtained as a white solid. Yield: 225 mg (76%). Mp: 229 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.99 (s, 1H, NH), 10.35 (s, 1H, Ar-NH), 8.15 (s, 1H, N=CH), 8.04 (brt, 1H, J H,H = 1.8 Hz, H-2 ′′ ), 7.97 (m, 2H, H-2 ′ , H-6 ′ ), 7.81 (brd, 1H, J H,H = 8.0 Hz, Ar-H), 7.65 (brd, 1H, J H,H = 7.9 Hz, Ar-H), 7.60–7.49 (m, 3H, Ar-H), 7.38 (brs, 2H, NH 2 ) ppm; 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 176.2 (CS), 144.0, 142.0, 139.5 (CH=N, C-1 ′′ , C-3 ′′ ), 134.6 (C-1 ′ ), 132.9, 129.3, 128.7, 128.5, 122.9, 122.4 (Ar-C) ppm; HRESI-MS calcd. for C14H1335ClN4NaO2S2([M+Na]+): 391.0061, found: 391.0057. 1-(4 ′ -Bromophenylmethylene)-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (4k). Thiosemicarbazide 3b (200 mg, 0.81 mmol) and 4-bromobenzaldehyde (87 µ L, 0,81 mmol, Int. J. Mol. Sci. 2025,26, 1225 17 of 23 1.0 equiv.) were used. Compound 4k was obtained as a white solid. Yield: 250 mg (76%); spectroscopic data are in agreement with those reported [59]. 1-Phenylmethylene-4-[2 ′ -(4 ′′ -sulfonamido)phenyl]ethyl-3-thiosemicarbazone (4l). Thiosemicarbazide 3c (200 mg, 0.73 mmol) and benzaldehyde (74 µ L, 0.73 mmol, 1.0 equiv.) were used. Compound 4l was obtained as a white solid. Yield: 133 mg (50%). Mp: 228 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.56 (s, 1H, NH), 8.59 (t, 1H, J H,H = 6.3 Hz, NH-CH 2 ), 8.10 (s, 1H, N=CH), 7.80 (m, 4H, Ar-H), 7.45 (m, 5H, Ar-H), 7.28 (s, 2H, NH 2 ), 3.81 (brq, 2H, J H,H = 6.5 Hz, NH-CH 2 ), 3.02 (t, 2H, J H,H = 6.5 Hz, CH 2 -Ph) ppm; 13 C-NMR (125.7 MHz, DMSO-d 6 ) δ 177.1 (CS), 143.5, 142.2, 142.1, (CH=N, C-4 ′′ , C-1 ′′ ), 134.2 (C-1, Ph), 129.9, 129.1, 128.7, 127.2, 125,9 (Ar-C), 44.6 (CH2-NH), 34.6 (CH 2 -Ar) ppm; HRESI-MS m/zcalcd. for C16H18N4NaO2S2([M+Na]+): 385.0763, found: 385.0761. 1-(4 ′ -Methoxyphenylmethylene)-4-[2 ′′ -(4 ′′′ -sulfonamido)phenyl]ethyl-3-thiosemicarbazone (4m). Thiosemicarbazide 3c (200 mg, 0.73 mmol) and 4-methoxybenzaldehyde (89 µ L, 0.73 mmol, 1.0 equiv.) were used. Compound 4m was obtained as a white solid. Yield: 186 mg (65%). Mp: 174 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.38 (s, 1H, NH), 8.47 (t, 1H, J H,H = 6.0 Hz, NH-CH 2 ), 8.00 (s, 1H, N=CH), 7.77 (m, 2H, Ar-H), 7.70 (m, 2H, Ar-H), 7.45 (m, 2H, Ar-H), 7.27 (s, NH 2 ), 6.98 (m, 2H, Ar-H), 3.80 (s, 3H, OMe), 3.80 (m, 2H, CH 2 -NH), 3.00 (t, 2H, J H,H = 8.0 Hz, CH 2 -Ar) ppm; 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 176.8 (CS), 160.7 (C-4 ′ ), 143.5, 142.1, 142.0 (N=CH, C-4 ′′′ , C-1 ′′′ ), 129.1, 128.8, 126.7, 125.8, 114.2 (Ar-C), 55.3 (OMe), 44.4 (CH 2 -NH), 34.8 (Ar-CH 2 ) ppm; HRESI-MS m/zcalcd. for C 17 H 20 N 4 NaO 3 S 2 ([M+Na]+): 415.0869, found: 415.0862. 1-(4 ′ -Fluorophenylmethylene)-4-[2 ′′ -(4 ′′′ -sulfonamido)phenyl]ethyl-3-thiosemicarbazone (4n). Thiosemicarbazide 3c (200 mg, 0.73 mmol) and 4-fluorobenzaldehyde (78 µ L, 0.73 mmol, 1.0 equiv.) were used. Compound 4n was obtained as a white solid. Yield: 205 mg (74%). Mp: 242 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.52 (s, 1H, NH), 8.61 (t, 1H, J H,H = 5.6 Hz, NH-CH 2 ), 8.05 (s, 1H, N=CH), 7.84 (m, 2H, Ar-H), 7.78 (m, 2H, Ar-H), 7.46 (m, 2H, Ar-H), 7.28 (m, 4H, Ar-H, NH 2 ), 3.80 (q, 2H, J H,H = 7.0 Hz, CH 2 -NH), 3.01 (t, 1H, J H,H = 7.0 Hz, CH 2 -Ar) ppm; 13 C-NMR (125.7 MHz, DMSO-d 6 ) δ 177.1 (C=S), 163.0 (d, 1 J H,H = 247.1 Hz, C-4 ′ ), 143.5, 142.2, 140.9, (CH=N, C-4 ′′′ , C-1 ′′′ ), 130.8 (d, 4 J H,H = 3.7 Hz, C-1 ′ ), 129.4 (d, 3 J H,H = 7.6 Hz, C-2 ′ /C-6 ′ ), 129.1, 125.9 (Ar-C), 115.8 (d, 2 J H,H = 21.7 Hz, C3 ′ /C-5 ′ ), 44.6 (CH 2 -NH), 34.6 (CH 2 -Ar), ppm; HRESI-MS m/zcalcd. for C 16 H 17 FN 4 NaO 2 S 2 ([M+Na]+): 403.0669, found: 403.0670. 1-(4 ′ -Chlorophenylmethylene)-4-[2 ′′ -(4 ′′′ -sulfonamido)pheny]ethyl-3-thiosemicarbazone (4o). Thiosemicarbazide 3c (200 mg, 0.73 mmol) and 4-chlorobenzaldehyde (103 mg, 0.73 mmol, 1.0 equiv.) were used. Compound 4o was obtained as a white solid. Yield: 150 mg (52%). Mp: 235 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.57 (s, 1H, NH), 8.65 (t, 1H, J H,H = 5.9 Hz, NH-CH 2 ), 8.05 (s, 1H, N=CH) 7.80 (m, 4H, Ar-H), 7.48 (m, 4H, Ar-H), 7.29 (s, 2H, NH 2 ), 3.81 (brq, 2H, J H,H = 6.6 Hz, CH 2 -NH), 3.02 (t, 2H, J H,H = 7.1 Hz, CH 2 -Ar) ppm; 13 C-NMR (125.7 MHz, DMSO-d 6 ) δ 177.1 (CS), 143.5, 142.2, 140.7, (CH=N, C-4 ′′′ , C-1 ′′′ ), 134.3, 133.2, 129.1, 128.9, 128.8, 125.9 (Ar-C), 44.6 (CH 2 -NH), 34.6 (CH 2 -Ar) ppm; HRESI-MS m/zcalcd. for C16H1735ClN4NaO2S2([M+Na]+): 419.0374, found: 419.0367. 1-(4 ′ -Bromophenylmethylene)-4-[2 ′′ -(4 ′′′ -sulfonamido)phenyl]ethyl-3-thiosemicarbazone (4p). Thiosemicarbazide 3c (200 mg, 0.73 mmol) and 4-bromobenzaldehyde (78 µ L, 0.82 mmol, 1.0 equiv.) were used. Compound 4p was obtained as a white solid. Yield: 187 mg (58%). Mp: 244 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.57 (s, 1H, NH), 8.64 (t, 1H, J H,H = 5.8 Hz, NH-CH 2 ), 8.03 (s, 1H, N=CH), 7.76 (m, 4H, Ar-H), 7.63 (m, 2H, Ar-H’), 7.46 (m, 2H, Ar-H), 7.28 (s, 2H, NH 2 ), 3.80 (q, 2H, J H,H = 6.5 Hz, CH 2 -NH), 3.02 (t, 2H, J H,H = 7.0 Hz, CH 2 -Ar) ppm; 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 177.6 (CS), 143.5, 142.2, 140.8 (N=CH, C-4 ′′′ , C-1 ′′′ ), 133.9, 132.1, 129.5, 126.3, 123.5 (Ar-C), 44.6 (CH 2 -NH), 34.6 (CH 2 - Int. J. Mol. Sci. 2025,26, 1225 18 of 23 Ar); HRESI-MS m/zcalcd. for C 16 H 1879 BrN 4 O 2 S 2 ([M+H] + ): 441.0049, found: 441.0049; m/zcalcd. for C16H1881BrN4O2S2([M+H]+): 443.0029, found: 443.0026. 1-(Pyridin-2 ′ -ylmethylene)-4-(4 ′′ -sulfonamidophenyl)-3-thiosemicarbazone(5a). Thiosemicarbazide 3a (200 mg, 0.81 mmol) and pyridine-2-carbaldehyde (74 µ L, 0.81mmol, 1.0 equiv.) were used. Compound 5a was obtained as a yellow solid. Yield: 84 mg (31%). Mp: 178 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 12.48 (s, 1H, NH), 10.52 (s, 1H, Ar-NH), 8.76 (brd, 1H, J 5′,6′ = 5.1 Hz, H-6 ′ ), 8.47 (d, 1H, J 3′,4′ = 8.4 Hz, H-3 ′ ), 8.23 (s, 1H, N=CH), 8.18 (m, 1H, H-4 ′ ), 7.82 (s, 4H, Ar-Ho, Ar-Hm), 7.67 (m, 1H, H-5′), 7.36 (s, 2H, NH2) ppm; 13C-NMR (125.7 MHz, DMSO-d 6 ) δ 176.7 (CS), 150.2 (C-2 ′ ), 146.7 (C-6 ′ ), 141.9 (C=N), 140.9, 139.2, 126.0, 125.8, 125.6, 123.3, 123.3 (Ar-C) ppm; HRESI-MS m/zcalcd. for C 13 H 13 N 5 NaO 2 S 2 ([M+Na] + ): 358.0403, found: 358.0402. 1-(Pyridin-2 ′ -ylmethylene)-4-(3 ′′ -sulfonamidophenyl)-3-thiosemicarbazone (5b). Thiosemicarbazide 3b (200 mg, 0.73 mmol) and pyridine-2-carbaldehyde (74 µ L, 0.81mmol, 1.0 equiv.) were used. Compound 5b was obtained as a yellow solid. Yield: 111 mg (41%). Mp: 186 ◦ C (dec.); 1 H-NMR (300 MHz, DMSO-d 6 ) δ 12.34 (s, 1H, NH), 10.51 (s, 1H, Ar-NH), 8.68 (brd, 1H, J 5′,6′ = 5.1 Hz, H-6 ′′ ), 8.47 (d, 1H, J 3′,4′ = 8.3 Hz, H-3 ′ ), 8.21 (s, 1H, N=CH), 8.07 (t, 1H, JH,H = 7.7 Hz, Ar-H), 8.04 (brt, 1H, J 2′′,4′′ = J 2′′,6′′ = 1.8 Hz, H-2 ′′ ), 7.82 (brd, 1H, J H,H = 7.8 Hz, Ar-H), 7.68 (dt, 1H, J H,H = 1.3 Hz, J H,H = 7.8 Hz, Ar-H), 7.58 (m, 2H, Ar-H), 7.41 (s, 2H, NH 2 ) ppm; 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 176.7 (CS), 150.9 (C-2 ′ ), 147.2 (C-3 ′′ ), 144.2 (C=N), 140.1, 139.6, 139.2, 129.4, 128.7, 125.1, 122.9, 122.7, 122.3 (Ar-C) ppm; HRESI-MS m/zcalcd. For C13H13N5NaO2S2([M+Na]+): 358.0403, found: 358.0399. 1-(Pyridin-2 ′ -ylmethylene)-4-[2 ′′ -(4 ′′′ -sulfonamidophenyl)]ethyl-3-thiosemicarbazone (5c). Thiosemicarbazide 3c (200 mg, 0.73 mmol, 1.0 equiv.) and pyridine-2-carbaldehyde (67 µ L, 0.73 mmol) were used. Compound 5b was obtained as a yellow solid. Yield: 111 mg (42%). Mp: 225 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 12.11 (s, 1H, NH), 8.99 (t, 1H, J H,H = 6.0 Hz, NH-CH 2 ), 8.77 (brd, 1H, J 5′,6′ = 5.1 Hz, H-6 ′ ), 8.35 (brd, 1H, J 3′,4′ = 8.4 Hz, H-3 ′ ), 8.26 (brtd, 1H, J 4′,6′ = 1.4 Hz, J 4′,5′ = 7.8 Hz, H-4 ′ ), 8.13 (s, 1H, N=CH), 7.77 (m, 2H, Ar-H), 7.72 (m, 1H, H-5 ′ ), 7.46 (m, 2H, Ar-H), 7.28 (brs, 2H, NH 2 ), 3.84 (q, 2H, J H,H = 6.5 Hz, NH-CH 2 ), 3.03 (t, 1H, J H,H = 7.1 Hz, CH 2 -Ar) ppm; 13 C-NMR (75.5 MHz, DMSO-d 6 ) δ 178.0 (CS), 155.1 (C-2 ′ ), 148.3 (C-6 ′ ), 144.4 (C=N), 143.7, 143.3, 143.3, 142.2, 134.4, 129.2, 125.8, 124.3 (Ar-C), 44.8 (CH 2 -NH), 34.3 (CH 2 -Ar) ppm; HRESI-MS m/zcalcd. for C 15 H 17 N 5 NaO 2 S 2 ([M+Na]+): 386.0716, found: 386.0720. 3.2. CA Inhibition Assays An applied photophysics stopped-flow instrument was used for assaying the CAcatalyzed CO 2 hydration activity [ 18 ]. Phenol red (at 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) and 20 mM Na 2 SO 4 (for maintaining constant ionic strength), following the initial rates of the CAcatalyzed CO 2 hydration reaction for 10–100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining the initial rate. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilled–deionized water, and appropriate dilutions were done thereafter with distilled– deionized water. AAZ was used as a positive control. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature before assay in order to allow for the formation of the E–I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng–Prusoff equation and represent the mean from at least three different determinations. All CA isoforms were recombinant ones (5–12 nM), obtained in-house. Int. J. Mol. Sci. 2025,26, 1225 19 of 23 3.3. Docking Simulations The crystallographic structure of CAIX and CAXII with 1.82 Å resolution (PDB ID: 5FL4) and 1.38 Å resolution (PDB ID: 4HT2), respectively, were obtained from the Protein Data Bank [ 60 ]. Docking simulations were performed in MOE Software v2019.01 (Chemical Computing Group, Montreal, QC, Canada). Protein optimization was performed following the QuickPrep protocol. Briefly, crystallographic artifacts, non-bonded ligands, and excess copies of the protein are removed. Water molecules were removed except for the ones at a maximum distance of 4.5 Å from the active site. Ligands were built, hydrogens added, and geometry optimized through energy minimization. During docking simulations, ligands were placed in the grid of the co-crystallized ligand. In the placement stage, energy binding calculations used the Triangle Matcher algorithm with the London dG scoring scheme. In the refinement stage, the receptor was kept rigid, and the GBVI/WSA dg scoring scheme was used. 3.4. Metal Complexation Assays The complexation of 5b with different metal chlorides (NaCl, KCl, FeCl 2 , FeCl 3 , ZnCl 2 , and CuCl 2 ) was investigated using UV–Vis spectroscopy (Jasco V-360). The stock solutions of the salts (10 −2 M) and the ligand (2.5 mM) were prepared in pure DMSO and sonicated when necessary. To 20 µ L of a 2.5 mM solution of 5b were added different aliquots of the metal chlorides solutions up to a total volume of 2 mL in quartz cuvettes. The following ligand–metal ratios were used: 1:0.25, 1:0.5, 1:1, 1:2, 1:5, and 1:10. The absorbance was monitored at 25 ◦ C in the range 280–440 nm, either with or without incubation. A blank solution without 5b was also prepared for each concentration of the salt, and its spectrum was subtracted from the ligand–salt spectrum. 3.5. Antiproliferative Assays 3.5.1. Cell Lines and Culture The human cancer cell lines A549, HBL-100, and T-47D, as well as HeLa, were provided by Dr. Raimundo Freire (Hospital Universitario de Canarias, Tenerife, Canary Islands). The lung cancer cell lines SW1573 and WiDr were provided by Prof. G. J. Peters (VU University Medical Center, Amsterdam, The Netherlands). Cells were grown in RPMI-1640 medium containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL of penicillin G, and 0.1 mg/mL of streptomycin at 37 ◦ C in a 95% humidified atmosphere of 5% CO 2 . Cells were maintained in culture in 60 mm cell culture dishes in growth medium (10 mL) and passaged twice weekly. 3.5.2. Antiproliferative Tests The antiproliferative activity of compounds was tested using our implementation of the protocol of the National Cancer Institute (NCI) of the USA. The following seeding densities (cells per well) were used: 2500 (A549, HBL-100, HeLa, and SW1573) and 5000 (T-47D and WiDr). Stock solutions of inhibitors (40 mM) were prepared in pure DMSO (400 times the maximum test concentration). For each test compound, the cells were exposed for a period of 48 h to serial decimal dilutions in cell culture medium of the test compounds (0.001–100 µ M). For each product, GI 50 values were calculated according to the NCI formulas (n = 3; data are expressed as mean ±SD). 3.5.3. Cell Morphology The CX-A imaging platform microscope (Nanolive SA, Lausanne, Switzerland) was used to measure refractive indices, creating a holotomographic 3D image of the cells. SW1573 cells were seeded onto 35 mm cell culture imaging dishes (IBIDI GmbH, Gräfelfing, Int. J. Mol. Sci. 2025,26, 1225 20 of 23 Germany) at a density of 50.000 cells/well. On the next day, treated cells were exposed to the test compounds right before the acquisition of the images. Image data were transferred to FIJI software v2.9.0 (NIH, USA) for image analysis. EVE software v2.2.1.2162 (Nanolive S.A., Tolochenaz, Switzerland)) was used for the analysis of the refractive indices and calculation of the phenotypic parameters. 4. Conclusions In conclusion, we have successfully designed multifunctional antiproliferative agents by combining aryl sulfonamides, which act as pharmacophores for CA inhibition, with a thiosemicarbazone moiety that acts as a metal chelator. Pyridine-2-carbaldehyde thiosemicarbazone 5b, with the sulfonamido motif at the meta position, can be considered as the lead compound. Although the presence of the sulfonamido moiety was found to be deleterious to the antiproliferative activity compared with other α -N-heterocyclic thiosemicarbazones reported in the literature, 5b still preserved good activity (GI 50 values in the low micromolar range), potent inhibition of CA isoforms associated with tumor progression (IX and XII, K i values in the low nanomolar range), and effective chelation of divalent cations Fe 2+ and Cu 2+ , ensuring a multifactorial mechanism of action. The absence of a direct correlation between CA inhibition and antiproliferative effects suggests that additional biological targets may also be involved. The mode of action of 5b was further explored using 3D holotomographic microscopy, which revealed delayed tumor cell division and features indicative of apoptosis. Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms26031225/s1. Author Contributions: Conceptualization, Ó.L.; molecular docking and modelling, A.P., M.X.F. and J.M.P.; data analysis, A.N., M.X.F., J.M.P. and C.T.S.; synthesis and characterization: M.M.-M., G.A. and P.B.; biological assays: A.G.-B., A.P., P.B. and J.M.P.; writing—original draft preparation, Ó.L.; writing—review and editing, P.M.-M., A.N., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; supervision: P.M.-M., S.M.-S., A.N., M.X.F., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; funding acquisition, J.M.P., J.G.F.-B. and Ó.L. All authors have read and agreed to the published version of the manuscript. Funding: J.G.F.-B./Ó.L. and A.G.-B./A.P./J.M.P. thank the Spanish Government (projects PID2020116460RB-I00 funded by MCIN/AEI/10.13039/501100011033 and PID2021-123059OB-I00 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE, respectively) for financial support. J.G.F.-B. and Ó.L. also thank Junta de Andalucía (FQM134) and VII Plan Propio de Investigación (University of Seville). P.M.-M. and S.M.-S. thank VIEP-BUAP (project 100521265-VIEP2024) for financial support. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Dataset available on request from the authors. Acknowledgments: M.M.-M. thanks the Mexican CONAHCYT for the award of a predoctoral fellowship (I1200/311/2023, MOD.ORD. /08/2023). P.B. thanks the European Union (NextGenerationEU) and the Ministerio de Universidades of Spain (Grant Margarita Salas). A.P. thanks the EU Social Fund (FSE) and the Canary Islands ACIISI for a predoctoral grant TESIS2020010055. A.G.-B. thanks the Asociación Española Contra el Cáncer (AECC) de Santa Cruz de Tenerife for the award of a predoctoral grant (PRDTF233958GONZ). We would also like to thank the Servicio de Resonancia Magnética Nuclear, CITIUS (University of Seville), for the performance of NMR experiments. Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. 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