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

Academic Edi o : Da id S C Black Recei ed: 7 Decembe 2024 Re ised: 24 Janua y 2025 Accep ed: 24 Janua y 2025 Published: 30 Janua y 2025 Ci a ion: Ma ínez-Mon iel, M.; A ighi, G.; Begines, P.; González-Bakke , A.; Pue a, A.; Fe nandes, M.X.; Me ino-Mon iel, P.; Mon iel-Smi h, S.; Nocen ini, A.; Supu an, C.T.; e al. Mul i ace ed Sul onamide-De i ed Thiosemica bazones: Combining Me al Chela ion and Ca bonic Anhyd ases Inhibi ion in An icance The apy. In . J. Mol. Sci. 2025,26, 1225. h ps://doi.o g/10.3390/ ijms26031225 Copy igh : © 2025 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps://c ea i ecommons.o g/ licenses/by/4.0/). A icle Mul i ace ed Sul onamide-De i ed Thiosemica bazones: Combining Me al Chela ion and Ca bonic Anhyd ases Inhibi ion in An icance The apy Mónica Ma ínez-Mon iel 1,2,†, Giulia A ighi 1,3,†, Paloma Begines 1,3 , Aday González-Bakke 4, Ad ián Pue a 4 , Miguel X. Fe nandes 4 , Penélope Me ino-Mon iel 2 , Sa a Mon iel-Smi h 2 , Alessio Nocen ini 3 , Claudiu T. Supu an 3, José M. Pad ón 4, José G. Fe nández-Bolaños 1and Ósca López 1,* 1Depa amen o de Química O gánica, Facul ad de Química, Uni e sidad de Se illa, Apa ado 1203, E-41071 Se ille, Spain; [email p o ec ed] (M.M.-M.); [email p o ec ed] (G.A.); [email p o ec ed] (P.B.); [email p o ec ed] (J.G.F.-B.) 2Facul ad de Ciencias Químicas, Ciudad Uni e si a ia, Benemé i a Uni e sidad Au ónoma de Puebla, Puebla 72570, PUE, Mexico; [email p o ec ed] (P.M.-M.); [email p o ec ed] (S.M.-S.) 3NEUROFARBA Depa men , Sezione di Scienze Fa maceu iche e Nu aceu iche, Uni e si y o Flo ence, 50019 Flo ence, I aly; [email p o ec ed] (A.N.); [email p o ec ed] (C.T.S.) 4 BioLab, Ins i u o Uni e si a io de Bio-O gánica “An onio González” (IUBO-AG), Uni e sidad de La Laguna, c/As o ísico F ancisco Sánchez 2, E-38206 La Laguna, Spain; [email p o ec ed] (A.G.-B.); [email p o ec ed] (A.P.); [email p o ec ed] (M.X.F.); jmpad [email p o ec ed] (J.M.P.) *Co espondence: [email p o ec ed] †These au ho s con ibu ed equally o his wo k. Abs ac : The selec i e inhibi ion o key enzymes, such as ca bonic anhyd ases (CAs IX and XII), which a e o e exp essed in cance issues, has eme ged as a p omising s a egy in cance esea ch. Howe e , a mul i a ge app oach is o en p e e ed o achie e enhanced he apeu ic ou comes. In his s udy, a yl sul onamides we e conjuga ed wi h a hiosemica - bazone moie y o enable dual unc ionali y: he inhibi ion o CAs and he chela ion o me al ca ions. Se e al s uc u al ac o s we e sys ema ically modi ied, including he posi ion o he sul onamido g oup, he leng h o he linke , he na u e o he a oma ic esidue, and he ype o subs i uen s. Tumo -associa ed CAs IX and XII inhibi ion was e alua ed using he s opped- low CO 2 hyd ase assay, and he inhibi ion cons an s (K i ) we e de e mined. The mos p omising compounds we e u he analyzed h ough molecula docking simula ions. Me al chela ion capabili ies we e e alua ed using UV–Vis spec oscopy, while an ip oli - e a i e ac i i ies we e measu ed using he sul o hodamine B (SBR) assay. Addi ionally, holo omog aphic 3D mic oscopy was employed o in es iga e he mechanisms o cell dea h. Sul onamido-de i ed Schi bases we e syn hesized h ough a h ee-s ep p ocedu e ha did no equi e column ch oma og aphy pu i ica ion: (1) iso hiocyana ion o amino- sul onamides, (2) nucleophilic addi ion o hyd azine, and (3) acid-p omo ed condensa ion wi h di e en aldehydes (benzaldehydes o py idine-2-ca boxaldehyde). The syn hesized compounds exhibi ed inhibi ion o CAs in he low nanomola o submic omola ange, wi h selec i i y la gely in luenced by s uc u al ea u es. No ably, he m-sul onamide de i a i e 5b, bea ing a py idin-2-yl esidue, demons a ed po en and selec i e inhibi ion o CA IX (K i = 4.9 nM) and XII (K i = 5.6 nM). Addi ionally, i e icien ly chela ed Fe 2+ , Fe 3+ , and Cu 2+ and showed p omising an ip oli e a i e ac i i y (GI 50 4.5–10 µ M). Mechanis ic s udies e ealed ha apop osis was in ol ed in i s mode o ac ion. The e o e, he syne gis ic in eg a ion o sul onamides and hiosemica bazones ep esen s an e ec i e s a egy o he de elopmen o mul imodal an icance agen s. Keywo ds: ca bonic anhyd ases; sul onamides; hiosemica bazones; docking simula ions; me al chela ion; an ip oli e a i e ac i i y In . J. Mol. Sci. 2025,26, 1225 h ps://doi.o g/10.3390/ijms26031225 In . J. Mol. Sci. 2025,26, 1225 2 o 23 1. In oduc ion Cance is he second leading cause o mo ali y wo ldwide [ 1 ], ep esen ing one o he mos signi ican challenges in biomedical esea ch [ 2 ]. I is among he mos complex and de as a ing diseases o ou ime [ 3 ]. Despi e emendous ad ances in ea ly de ec ion [ 4 ], su ge y [ 5 ], adio he apy [ 6 ], immuno he apy [ 7 ], nano echnology [ 8 ] and o he he apeu- ic app oaches, chemo he apy con inues o be a co ne s one ea men o many ypes o cance [ 9 ]. Howe e , he in ica e complexi y o cance , combined wi h i s mul i ac o ial e iology, makes he adi ional one-d ug, one- a ge pa adigm [ 10 ] insu icien . Conse- quen ly, a mul i a ge s a egy [ 11 ] has become essen ial o comba ing umo p og ession and he apeu ic esis ance [12]. In his con ex , ou s udy aimed o de elop mul imodal he apeu ic agen s o cance ea men . Speci ically, we sough o design compounds ha in eg a e a pha macopho e a ge ing enzymes o e exp essed in umo s wi h a me al chela o o add ess he high le els o ce ain me als in umo issues. Ou p ima y ocus was on ca bonic anhyd ases (CAs), a amily o ubiqui ous Zn(II)-dependen me alloenzymes. These enzymes ca alyze he e e sible hyd a ion o CO 2 o p oduce hyd ogen ca bona e and a p o on [ 13 ]. The Zn(II) ca ion ac s as a Lewis acid, lowe ing he pK a o he coo dina ed H 2 O molecule, he eby acili a ing i s dep o ona ion unde physiological condi ions [ 14 ]. CAs a e ound ac oss almos all domains o li e and a e classi ied in o eigh di e en gene ic amilies. The ones ound in humans ( α -CAs) a e in u n di ided in o 15 di e en iso o ms, which a y in issue dis ibu ion and ca aly ic ac i i y [ 15 ]. The selec i e inhibi ion o he α -CAs IX and XII iso o ms has eme ged as a p omising a ge in an icance esea ch [ 16 ], due o hei ole in acidi ying he hypoxic umo mic oen i onmen , which p omo es umo p og ession and me as asis [ 17 ]. Alkyl and a yl sul onamides cons i u e he la ges amily o CA inhibi o s [ 18 ], which ac by binding Zn(II) and hus blocking he enzyme ac i i y. In e es ingly, his pha macopho e has also been ound o inhibi y osine kinases and a oma ases [ 19 ], bo h o which a e o e exp essed in ce ain umo s. A no able example is compound SLC-0111, a u eido-con aining a yl sul onamide and a po en CA IX inhibi o cu en ly unde going clinical ials o ad anced solid umo s. SLC-0111 has demons a ed po en ial as a sensi ize o head and neck squamous cell ca cinoma (HNSCC) in combina- ion wi h cispla in [ 20 ] and has demons a ed he capaci y o educing hepa oblas oma cell iabili y and mig a ion [21]. In addi ion o a ge ing CAs, we explo ed he inco po a ion o a me al-chela ing ame- wo k linked o he sul onamido mo i . To he bes o ou knowledge, no p io s udies ha e desc ibed an icance agen s designed o simul aneously a ge CAs and he dis up ion o me al homeos asis. Me als play c ucial oles in heal hy cells [ 22 ], con ibu ing o signalling pa hways [ 23 ], enzyma ic ac i i y, and he s uc u al in eg i y o cell memb anes and he genome [ 24 ]. Howe e , dis up ions in me al homeos asis—cha ac e ized by ei he an excess o de iciency—can p o oundly a ec cellula physiology. Ele a ed le els o me al ca ions such as Fe, Cu, and Zn ha e been linked o oncogenesis and me as asis [ 24 , 25 ]. Fo ins ance, high le els o Fe can exace ba e oxida i e s ess by in e ac ing wi h H 2 O 2 , causing se e e damage o cellula memb anes and o ganelles [26]. Al hough u he esea ch is needed, me al chela o s ha e shown p omising esul s in p eclinical an icance s udies [ 27 , 28 ]. Schi bases and hei de i a i es, pa icula ly hiosemica bazones ( he sca old used in his s udy), ha e a ac ed signi ican a en ion as me al-binding ligands in an icance esea ch [ 29 – 31 ], wi h se e al compounds en e ing clinical ials [ 32 ]. Me al complexes de i ed om hiosemica bazones ha e been ex ensi ely cha ac e ized using di e se analy ical echniques, including IR and NMR spec oscopy, In . J. Mol. Sci. 2025,26, 1225 3 o 23 conduc i i y measu emen s, he mog a ime ic analysis, and densi y unc ional heo y (DFT) calcula ions [33,34]. 2. Resul s and Discussion 2.1. D ug Design and Chemis y The p ima y objec i e o his s udy was he design, syn hesis, and e alua ion o mul i a ge sul onamide– hiosemica bazone hyb ids (Figu e 1) wi h po en ial an icance ac i i y, speci ically a ge ing umo - ela ed CAs and haza dous high le els o me als. This app oach ep esen s a p omising s a egy o de eloping mul i a ge d ug candida es wi h po en ial applica ion in cance chemo he apy. I has been sugges ed ha he an ineoplas ic p ope ies o hiosemica bazones a e enhanced when hey exe e ec i e me al-chela ing capabili ies [ 35 ]. Schi bases and ela ed compounds, pa icula ly hiosemica bazones, ha e been epo ed o exhibi an icance p ope ies h ough a ious mechanisms [ 36 ], including in e ac ions wi h DNA [ 37 ], inhibi ion o opoisome ases [ 38 ], and mi iga ion o mul id ug esis ance (MDR) [ 32 ]. Among hese mechanisms, hei capaci y o complex i on is widely ecognized as he p ima y an icance mechanism [ 39 ], wi h Fe-dependen ibonuclease educ ase (RNR) iden i ied as a key in acellula a ge . Recen s udies on nanomola an icance hiosemica bazones ha e also highligh ed hei abili y o complex Cu(II) ions, wi h human se um albumin iden i ied as a po en ial me al sou ce [ 40 ]. Conse- quen ly, hiosemica bazones a e now ega ded as mo e han jus simple chela o s; hey a e classi ied as me al-in e ac ing d ugs wi h mul imodal an icance ac i i y [ 41 ]. A no able example o his pha macopho e is T iapine ® (3-aminopy idine-2-ca boxaldehyde hiosemi- ca bazone), which has unde gone nume ous clinical ials [ 42 ]. In his s udy, he wo key s uc u al moie ies in es iga ed we e he a yl sul onamide and he hiosemica bazone g oups. To u he enhance biological ac i i y, s uc u al modi ica ions we e sys ema - ically in oduced, including a ying he posi ion o he sul onamido mo i , employing di e en space s o link he sul onamide and hiosemica bazone moie ies, inco po a ing a yl and he e oa yl esidues (e.g., py idine-2-yl) in o he imino sca old, and modi ying subs i uen s on he a yl ing. These s uc u al a ia ions p o ided a obus amewo k o es ablishing s uc u e–ac i i y ela ionships (SARs), acili a ing he a ional design o mo e po en and selec i e an icance agen s. In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 3 o 24 Al hough u he esea ch is needed, me al chela o s ha e shown p omising esul s in p eclinical an icance s udies [27,28]. Schiff bases and hei de i a i es, pa icula ly hi- osemica bazones ( he scaffold used in his s udy), ha e a ac ed signi ican a en ion as me al-binding ligands in an icance esea ch [29–31], wi h se e al compounds en e ing clinical ials [32]. Me al complexes de i ed om hiosemica bazones ha e been ex en- si ely cha ac e ized using di e se analy ical echniques, including IR and NMR spec os- copy, conduc i i y measu emen s, he mog a ime ic analysis, and densi y unc ional heo y (DFT) calcula ions [33,34]. 2. Resul s and Discussion 2.1. D ug Design and Chemis y The p ima y objec i e o his s udy was he design, syn hesis, and e alua ion o mul- i a ge sul onamide– hiosemica bazone hyb ids (Figu e 1) wi h po en ial an icance ac- i i y, speci ically a ge ing umo - ela ed CAs and haza dous high le els o me als. This app oach ep esen s a p omising s a egy o de eloping mul i a ge d ug candida es wi h po en ial applica ion in cance chemo he apy. I has been sugges ed ha he an ineo- plas ic p ope ies o hiosemica bazones a e enhanced when hey exe effec i e me al- chela ing capabili ies [35]. Schiff bases and ela ed compounds, pa icula ly hiosemi- ca bazones, ha e been epo ed o exhibi an icance p ope ies h ough a ious mecha- nisms [36], including in e ac ions wi h DNA [37], inhibi ion o opoisome ases [38], and mi iga ion o mul id ug esis ance (MDR) [32]. Among hese mechanisms, hei capaci y o complex i on is widely ecognized as he p ima y an icance mechanism [39], wi h Fe- dependen ibonuclease educ ase (RNR) iden i ied as a key in acellula a ge . Recen s udies on nanomola an icance hiosemica bazones ha e also highligh ed hei abili y o complex Cu(II) ions, wi h human se um albumin iden i ied as a po en ial me al sou ce [40]. Consequen ly, hiosemica bazones a e now ega ded as mo e han jus simple che- la o s; hey a e classi ied as me al-in e ac ing d ugs wi h mul imodal an icance ac i i y [41]. A no able example o his pha macopho e is T iapine® (3-aminopy idine-2-ca boxal- dehyde hiosemica bazone), which has unde gone nume ous clinical ials [42]. In his s udy, he wo key s uc u al moie ies in es iga ed we e he a yl sul onamide and he hiosemica bazone g oups. To u he enhance biological ac i i y, s uc u al modi ica- ions we e sys ema ically in oduced, including a ying he posi ion o he sul onamido mo i , employing diffe en space s o link he sul onamide and hiosemica bazone moie- ies, inco po a ing a yl and he e oa yl esidues (e.g., py idine-2-yl) in o he imino scaffold, and modi ying subs i uen s on he a yl ing. These s uc u al a ia ions p o ided a obus amewo k o es ablishing s uc u e–ac i i y ela ionships (SARs), acili a ing he a- ional design o mo e po en and selec i e an icance agen s. Figu e 1. Design o mul i ace ed sul onamide-de i ed hiosemica bazones. Figu e 1. Design o mul i ace ed sul onamide-de i ed hiosemica bazones. The syn he ic pa hway is ou lined in Scheme 1. Comme cially a ailable a yl amino- sul onamides 1a–c we e ans o med in o hei co esponding iso hiocyana es using wo di e en p ocedu es: ea men wi h hiophosgene unde acidic condi ions [ 43 ] o de i a- i es 1a and 1b, o eac ion wi h dicyclohexyl ca bodiimide (DCC) and CS 2 in Py [ 44 ] o compound 1c. The esul ing he e ocumulenes (compounds 2a–c, ob ained in good o excellen yields) we e subsequen ly ea ed wi h hyd azine hyd a e o u nish he co e- sponding hiosemica bazides 3a–c, wi h yields anging om 42 o 75%. Final condensa- In . J. Mol. Sci. 2025,26, 1225 4 o 23 ion unde acidic condi ions wi h benzaldehydes o py idine-2-ca boxaldehyde yielded sul onamide-de i ed hiosemica bazones 4a–p (47–76%) o 5a–c (31–42%), espec i ely. The inco po a ion o a py idine-2-yl esidue in de i a i es 5a–c is expec ed o p o ide a iden a e ligand, likely capable o complexing ele an ca ions mo e e icien ly. This p ope y may acili a e he seques a ion and emo al o me al ions om umo issues. The syn he ic p ocedu e is e icien and s aigh o wa d, wi h all in e media es and inal compounds being c ys alline and easily pu i ied by il a ion. This elimina es he need o ime-consuming column ch oma og aphy pu i ica ions, making he p ocess highly p ac ical o u he de elopmen . In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 4 o 24 The syn he ic pa hway is ou lined in Scheme 1. Comme cially a ailable a yl amino- sul onamides 1a–c we e ans o med in o hei co esponding iso hiocyana es using wo diffe en p ocedu es: ea men wi h hiophosgene unde acidic condi ions [43] o de i - a i es 1a and 1b, o eac ion wi h dicyclohexyl ca bodiimide (DCC) and CS2 in Py [44] o compound 1c. The esul ing he e ocumulenes (compounds 2a–c, ob ained in good o ex- cellen yields) we e subsequen ly ea ed wi h hyd azine hyd a e o u nish he co e- sponding hiosemica bazides 3a–c, wi h yields anging om 42 o 75%. Final condensa- ion unde acidic condi ions wi h benzaldehydes o py idine-2-ca boxaldehyde yielded sul onamide-de i ed hiosemica bazones 4a–p (47–76%) o 5a–c (31–42%), espec i ely. The inco po a ion o a py idine-2-yl esidue in de i a i es 5a–c is expec ed o p o ide a iden a e ligand, likely capable o complexing ele an ca ions mo e efficien ly. This p ope y may acili a e he seques a ion and emo al o me al ions om umo issues. The syn he ic p ocedu e is efficien and s aigh o wa d, wi h all in e media es and inal compounds being c ys alline and easily pu i ied by il a ion. This elimina es he need o ime-consuming column ch oma og aphy pu i ica ions, making he p ocess highly p ac- ical o u he de elopmen . Scheme 1. P epa a ion o sul onamide-de i ed hiosemica bazones 4 and 5. (a) CSCl2, aq. HCl; (b) DCC, CS2, Py. As a ep esen a i e example, Figu es 2 and 3 depic he 1H- and 13C-NMR spec a o he py idine-con aining hiosemica bazone 5b. In he 1H-RNM spec um, he mos no able signals a e obse ed a 12.34 ppm (A -NH), 10.51 ppm (NH), and 8.21 ppm (azome hyne p o on). Simila ly, in he 13C-NMR spec um, he esonances a 176.7 ppm ( hione moie y) and 144.2 ppm (imine moie y) p o ide u he con i ma ion o he p oposed s uc u e. Scheme 1. P epa a ion o sul onamide-de i ed hiosemica bazones 4and 5. (a) CSCl 2 , aq. HCl; (b) DCC, CS2, Py. As a ep esen a i e example, Figu es 2and 3depic he 1 H- and 13 C-NMR spec a o he py idine-con aining hiosemica bazone 5b. In he 1 H-RNM spec um, he mos no able signals a e obse ed a 12.34 ppm (A -NH), 10.51 ppm (NH), and 8.21 ppm (azome hyne p o on). Simila ly, in he 13 C-NMR spec um, he esonances a 176.7 ppm ( hione moie y) and 144.2 ppm (imine moie y) p o ide u he con i ma ion o he p oposed s uc u e. In . J. Mol. Sci. 2025,26, 1225 5 o 23 In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 o 24 Figu e 2. 1H-NMR spec um o 5b (300 MHz, DMSO-d6). Figu e 3. 13C-NMR spec um o 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessmen s 2.2.1. CA Inhibi ion Thiosemica bazones 4a–p and 5a–c we e e alua ed as po en ial inhibi o s o umo - associa ed CAs IX and XII. Fo compa ison, hei syn he ic p ecu so s, hiosemica bazides 3a–c, as well as ace azolamide (AAZ), as a s anda d posi i e con ol, we e included in he s udy. To assess selec i i y, he inhibi ion o cy osolic CAs I and II was also measu ed. The inhibi ion cons an s (Ki, nM), de e mined using he s opped- low CO2 hyd ase assay, along wi h he selec i i y indexes, a e depic ed in Table 1. The ollowing SARs we e iden- i ied: Figu e 2. 1H-NMR spec um o 5b (300 MHz, DMSO-d6). In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 o 24 Figu e 2. 1H-NMR spec um o 5b (300 MHz, DMSO-d6). Figu e 3. 13C-NMR spec um o 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessmen s 2.2.1. CA Inhibi ion Thiosemica bazones 4a–p and 5a–c we e e alua ed as po en ial inhibi o s o umo - associa ed CAs IX and XII. Fo compa ison, hei syn he ic p ecu so s, hiosemica bazides 3a–c, as well as ace azolamide (AAZ), as a s anda d posi i e con ol, we e included in he s udy. To assess selec i i y, he inhibi ion o cy osolic CAs I and II was also measu ed. The inhibi ion cons an s (Ki, nM), de e mined using he s opped- low CO2 hyd ase assay, along wi h he selec i i y indexes, a e depic ed in Table 1. The ollowing SARs we e iden- i ied: Figu e 3. 13C-NMR spec um o 5b (75.5 MHz, DMSO-d6). 2.2. Biological Assessmen s 2.2.1. CA Inhibi ion Thiosemica bazones 4a–p and 5a–c we e e alua ed as po en ial inhibi o s o umo - associa ed CAs IX and XII. Fo compa ison, hei syn he ic p ecu so s, hiosemica bazides 3a–c, as well as ace azolamide (AAZ), as a s anda d posi i e con ol, we e included in he s udy. To assess selec i i y, he inhibi ion o cy osolic CAs I and II was also measu ed. The inhibi ion cons an s (K i , nM), de e mined using he s opped- low CO 2 hyd ase assay, along wi h he selec i i y indexes, a e depic ed in Table 1. The ollowing SARs we e iden i ied: Inhibi ion o CAs IX and XII: all Schi bases exhibi ed a ying deg ees o inhibi ion o ansmemb ane CAs IX and XII, wi h K i alues anging om low nanomola o sub- In . J. Mol. Sci. 2025,26, 1225 6 o 23 mic omola (2.3–499 nM), depending on he a yl ing subs i uen s, he posi ion o he sul onamido moie y, and he p esence o absence o an e hylene linke . P e e ence o CA XII: ac oss all de i a i es, including hiosemica bazide p ecu so s 3, s onge inhibi ion was consis en ly obse ed o CA XII compa ed wi h CA IX. This end is pa icula ly no ewo hy, as nume ous CA XII inhibi o s a e also known o in- hibi glycop o ein-P (Pg-p), po en ially educing chemo esis ance caused by xenobio ic e lux ia he Pg-p pump [ 45 ]. An excep ion o his end was obse ed o he py idine- de i ed hiosemica bazone 5b, which displayed compa able po ency agains bo h iso o ms (Ki= 4.9 and 5.6 nM, espec i ely). m- and p-Subs i u ed sul onamido de i a i es: among compounds bea ing he sul- onamido mo i a he pa a posi ion, he highes ac i i y agains CA XII was obse ed o unsubs i u ed a oma ic ings (4a,K i = 8.38 nM; 4l,K i = 9.12 nM). The in oduc ion o an e hylene linke (4l–p s. 4a– ) enhanced selec i i y o CA XII, p ima ily by educing ac i i y agains CA I, wi h K i alues in he mic omola ange o de i a i es 4m–p. Among he me a egioisome s, de i a i e 4h, bea ing a p-me hoxy subs i uen , exhibi ed he highes CA XII ac i i y (Ki= 2.3 nM). Py idine-con aining de i a i es: hiosemica bazones con aining a py idine agmen and lacking he e hylene linke (5a,b) displayed s ong inhibi ion o CA XII (K i = 4.9 and 5.6 nM, espec i ely), wi h selec i i y compa able o o exceeding ha o he e e ence d ug AAZ. Addi ionally, compound 5b exhibi ed po en inhibi ion o CA II (K i = 2.5 nM). No ably, CAs IV, XII, and, pa icula ly, II, a e alida ed a ge s o glaucoma ea men due o hei ole in mi iga ing ocula hype ension [ 46 ]. Inco po a ion o he e hylene linke (5c) p ese ed s ong inhibi ion o CA XII (K i = 9.3 nM) bu signi ican ly dec eased CA I/XII selec i i y. Selec i i y: he me a placemen o he sul onamido moie y on he a oma ic ing gene - ally educed ac i i y agains CA I, he eby imp o ing selec i i y. This is a c i ical ac o o minimizing o - a ge e ec s. Rema kably, he s onges CA XII inhibi o , de i a i e 4h (K i = 2.3 nM), exhibi ed he highes CA I/XII selec i i y index (S.I. = 325), signi ican ly ou pe o ming AAZ (K i = 5.2 nM o CA XII; S.I. = 43.9). Ano he no ewo hy compound is he m-sul onamide 5b, which bea s a py idine-2-yl sca old (S.I. = 109.8 and 96.1 o CA I/IX and CA I/XII, espec i ely). These indings align wi h p e iously epo ed da a, in which many a yl sul onamides demons a ed signi ican po ency as inhibi o s o CA IX and XII iso o ms, wi h K i alues in he low nanomola ange. Howe e , hei selec i i y agains o - a ge iso o ms, such as CA I, is usually lowe compa ed wi h couma ins [ 47 ], ano he impo an class o CA inhibi o s. Table 1. Inhibi ion da a (K i , nM) o compounds 4and 5agains human CAs I, II, IX, and XII [a] using AAZ as a s anda d d ug. In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 o 23 Inhibi ion o CAs IX and XII: all Schi bases exhibi ed a ying deg ees o inhibi ion o ansmemb ane CAs IX and XII, wi h Ki alues anging om low nanomola o submi- c omola (2.3–499 nM), depending on he a yl ing subs i uen s, he posi ion o he sul- onamido moie y, and he p esence o absence o an e hylene linke . P e e ence o CA XII: ac oss all de i a i es, including hiosemica bazide p ecu so s 3, s onge inhibi ion was consis en ly obse ed o CA XII compa ed wi h CA IX. This end is pa icula ly no ewo hy, as nume ous CA XII inhibi o s a e also known o inhibi glycop o ein-P (Pg-p), po en ially educing chemo esis ance caused by xenobio ic e lux ia he Pg-p pump [45]. An excep ion o his end was obse ed o he py idine-de i ed hiosemica bazone 5b, which displayed compa able po ency agains bo h iso o ms (Ki = 4.9 and 5.6 nM, espec i ely). m- and p-Subs i u ed sul onamido de i a i es: among compounds bea ing he sul- onamido mo i a he pa a posi ion, he highes ac i i y agains CA XII was obse ed o unsubs i u ed a oma ic ings (4a, Ki = 8.38 nM; 4l, Ki = 9.12 nM). The in oduc ion o an e hylene linke (4l–p s. 4a– ) enhanced selec i i y o CA XII, p ima ily by educing ac- i i y agains CA I, wi h Ki alues in he mic omola ange o de i a i es 4m–p. Among he me a egioisome s, de i a i e 4h, bea ing a p-me hoxy subs i uen , exhibi ed he high- es CA XII ac i i y (Ki = 2.3 nM). Py idine-con aining de i a i es: hiosemica bazones con aining a py idine agmen and lacking he e hylene linke (5a,b) displayed s ong inhibi ion o CA XII (Ki = 4.9 and 5.6 nM, espec i ely), wi h selec i i y compa able o o exceeding ha o he e e ence d ug AAZ. Addi ionally, compound 5b exhibi ed po en inhibi ion o CA II (Ki = 2.5 nM). No ably, CAs IV, XII, and, pa icula ly, II, a e alida ed a ge s o glaucoma ea men due o hei ole in mi iga ing ocula hype ension [46]. Inco po a ion o he e hylene linke (5c) p ese ed s ong inhibi ion o CA XII (Ki = 9.3 nM) bu signi ican ly dec eased CA I/XII selec i i y. Selec i i y: he me a placemen o he sul onamido moie y on he a oma ic ing gen- e ally educed ac i i y agains CA I, he eby imp o ing selec i i y. This is a c i ical ac o o minimizing o - a ge e ec s. Rema kably, he s onges CA XII inhibi o , de i a i e 4h (Ki = 2.3 nM), exhibi ed he highes CA I/XII selec i i y index (S.I. = 325), signi ican ly ou pe o ming AAZ (Ki = 5.2 nM o CA XII; S.I. = 43.9). Ano he no ewo hy compound is he m-sul onamide 5b, which bea s a py idine-2-yl sca old (S.I. = 109.8 and 96.1 o CA I/IX and CA I/XII, espec i ely). These indings align wi h p e iously epo ed da a, in which many a yl sul onamides demons a ed signi ican po ency as inhibi o s o CA IX and XII iso o ms, wi h Ki alues in he low nanomola ange. Howe e , hei selec i i y agains o - a ge iso o ms, such as CA I, is usually lowe compa ed wi h couma ins [47], ano he impo an class o CA inhibi o s. Table 1. Inhibi ion da a (Ki, nM) o compounds 4 and 5 agains human CAs I, II, IX, and XII [a] using AAZ as a s anda d d ug. Compound CA I CA II CA IX CA XII Selec i i y Ra io I/IX//II/IX Selec i i y Ra io 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 Selec i i y Ra io I/IX//II/IX Selec i i y Ra io 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 In . J. Mol. Sci. 2025,26, 1225 7 o 23 Table 1. Con . In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 o 23 Inhibi ion o CAs IX and XII: all Schi bases exhibi ed a ying deg ees o inhibi ion o ansmemb ane CAs IX and XII, wi h Ki alues anging om low nanomola o submi- c omola (2.3–499 nM), depending on he a yl ing subs i uen s, he posi ion o he sul- onamido moie y, and he p esence o absence o an e hylene linke . P e e ence o CA XII: ac oss all de i a i es, including hiosemica bazide p ecu so s 3, s onge inhibi ion was consis en ly obse ed o CA XII compa ed wi h CA IX. This end is pa icula ly no ewo hy, as nume ous CA XII inhibi o s a e also known o inhibi glycop o ein-P (Pg-p), po en ially educing chemo esis ance caused by xenobio ic e lux ia he Pg-p pump [45]. An excep ion o his end was obse ed o he py idine-de i ed hiosemica bazone 5b, which displayed compa able po ency agains bo h iso o ms (Ki = 4.9 and 5.6 nM, espec i ely). m- and p-Subs i u ed sul onamido de i a i es: among compounds bea ing he sul- onamido mo i a he pa a posi ion, he highes ac i i y agains CA XII was obse ed o unsubs i u ed a oma ic ings (4a, Ki = 8.38 nM; 4l, Ki = 9.12 nM). The in oduc ion o an e hylene linke (4l–p s. 4a– ) enhanced selec i i y o CA XII, p ima ily by educing ac- i i y agains CA I, wi h Ki alues in he mic omola ange o de i a i es 4m–p. Among he me a egioisome s, de i a i e 4h, bea ing a p-me hoxy subs i uen , exhibi ed he high- es CA XII ac i i y (Ki = 2.3 nM). Py idine-con aining de i a i es: hiosemica bazones con aining a py idine agmen and lacking he e hylene linke (5a,b) displayed s ong inhibi ion o CA XII (Ki = 4.9 and 5.6 nM, espec i ely), wi h selec i i y compa able o o exceeding ha o he e e ence d ug AAZ. Addi ionally, compound 5b exhibi ed po en inhibi ion o CA II (Ki = 2.5 nM). No ably, CAs IV, XII, and, pa icula ly, II, a e alida ed a ge s o glaucoma ea men due o hei ole in mi iga ing ocula hype ension [46]. Inco po a ion o he e hylene linke (5c) p ese ed s ong inhibi ion o CA XII (Ki = 9.3 nM) bu signi ican ly dec eased CA I/XII selec i i y. Selec i i y: he me a placemen o he sul onamido moie y on he a oma ic ing gen- e ally educed ac i i y agains CA I, he eby imp o ing selec i i y. This is a c i ical ac o o minimizing o - a ge e ec s. Rema kably, he s onges CA XII inhibi o , de i a i e 4h (Ki = 2.3 nM), exhibi ed he highes CA I/XII selec i i y index (S.I. = 325), signi ican ly ou pe o ming AAZ (Ki = 5.2 nM o CA XII; S.I. = 43.9). Ano he no ewo hy compound is he m-sul onamide 5b, which bea s a py idine-2-yl sca old (S.I. = 109.8 and 96.1 o CA I/IX and CA I/XII, espec i ely). These indings align wi h p e iously epo ed da a, in which many a yl sul onamides demons a ed signi ican po ency as inhibi o s o CA IX and XII iso o ms, wi h Ki alues in he low nanomola ange. Howe e , hei selec i i y agains o - a ge iso o ms, such as CA I, is usually lowe compa ed wi h couma ins [47], ano he impo an class o CA inhibi o s. Table 1. Inhibi ion da a (Ki, nM) o compounds 4 and 5 agains human CAs I, II, IX, and XII [a] using AAZ as a s anda d d ug. Compound CA I CA II CA IX CA XII Selec i i y Ra io I/IX//II/IX Selec i i y Ra io 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 Selec i i y Ra io I/IX//II/IX Selec i i y Ra io 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 = B ) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4 (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 = B ) 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 = B ) 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 om h ee di e en assays by a s opped low echnique (e o s we e in he ange o ± 5–10% o he epo ed alues). 2.2.2. Docking Simula ions The in e ac ion p o iles o he selec ed hiosemica bazone–sul onamide hyb ids wi h CA IX and CA XII enzymes we e analyzed using ligand–p o ein docking simula ions. The simula ions ook in o accoun he wa e molecules su ounding he Zn(II) ion, in line wi h he widely epo ed in e ac ion mechanisms o sul onamide-based CA inhibi o s. Addi ionally, based on ex ensi e li e a u e e idence, he sul onamido moie y o he hyb ids was dep o ona ed o he simula ions. Fo his s udy, de i a i es 4a and 5b we e conside ed as ep esen a i e compounds. The binding ene gies om he docking simula ions agains CA IX a e shown in Table 2. Docking simula ions p edic ha bo h 4a and 5b in e ac wi h he Zn(II) ion h ough he dep o ona ed o m o he sul onamido mo i and wi h he ga ekeepe esidue Th 200. Addi ionally, he pa ially nega i e oxygen a om o 4a also in e ac s wi h he Zn(II) ion, while he sul u a om o he hiosemica bazone moie y is in ol ed in an H-bond be ween wa e and His68. In con as , o 5b, he same sul u a om in e ac s wi h Gln71 as an elec on accep o , whils he Gln92 is he accep o om an in e ac ion wi h he NH g oup o he hiosemica bazone. Mo eo e , an H- π s acking in e ac ion occu s be ween he a oma ic moie y o he benzenesul onamide and Leu199. The he e oa om o he py idine ing also pa icipa es in coope a i e H-bonding wi h wa e molecules and P o202 (Figu e 4). In . J. Mol. Sci. 2025,26, 1225 8 o 23 Table 2. Binding ene gies es ima ed o compounds 4a and 5b in hei in e ac ion wi h CA IX. Compound Binding Ene gy (kcal/mol) In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 o 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 = B ) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4 (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 = B ) 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 = B ) 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 om h ee di e en assays by a s opped low echnique (e o s we e in he ange o ±5– 10% o he epo ed alues). 2.2.2. Docking Simula ions The in e ac ion p o iles o he selec ed hiosemica bazone–sul onamide hyb ids wi h CA IX and CA XII enzymes we e analyzed using ligand–p o ein docking simula ions. The simula ions ook in o accoun he wa e molecules su ounding he Zn(II) ion, in line wi h he widely epo ed in e ac ion mechanisms o sul onamide-based CA inhibi o s. Addi- ionally, based on ex ensi e li e a u e e idence, he sul onamido moie y o he hyb ids was dep o ona ed o he simula ions. Fo his s udy, de i a i es 4a and 5b we e consid- e ed as ep esen a i e compounds. The binding ene gies om he docking simula ions agains CA IX a e shown in Table 2. Table 2. Binding ene gies es ima ed o compounds 4a and 5b in hei in e ac ion wi h CA IX. Compound Binding Ene gy (kcal/mol) 4a −7.93 5b −7.45 Docking simula ions p edic ha bo h 4a and 5b in e ac wi h he Zn(II) ion h ough he dep o ona ed o m o he sul onamido mo i and wi h he ga ekeepe esidue Th 200. Addi ionally, he pa ially nega i e oxygen a om o 4a also in e ac s wi h he Zn(II) ion, while he sul u a om o he hiosemica bazone moie y is in ol ed in an H-bond be ween 4a −7.93 In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 o 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 = B ) 574 46.8 23.3 13.6 24.6//2.0 42.2//3.4 4 (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 = B ) 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 = B ) 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 om h ee di e en assays by a s opped low echnique (e o s we e in he ange o ±5– 10% o he epo ed alues). 2.2.2. Docking Simula ions The in e ac ion p o iles o he selec ed hiosemica bazone–sul onamide hyb ids wi h CA IX and CA XII enzymes we e analyzed using ligand–p o ein docking simula ions. The simula ions ook in o accoun he wa e molecules su ounding he Zn(II) ion, in line wi h he widely epo ed in e ac ion mechanisms o sul onamide-based CA inhibi o s. Addi- ionally, based on ex ensi e li e a u e e idence, he sul onamido moie y o he hyb ids was dep o ona ed o he simula ions. Fo his s udy, de i a i es 4a and 5b we e consid- e ed as ep esen a i e compounds. The binding ene gies om he docking simula ions agains CA IX a e shown in Table 2. Table 2. Binding ene gies es ima ed o compounds 4a and 5b in hei in e ac ion wi h CA IX. Compound Binding Ene gy (kcal/mol) 4a −7.93 5b −7.45 Docking simula ions p edic ha bo h 4a and 5b in e ac wi h he Zn(II) ion h ough he dep o ona ed o m o he sul onamido mo i and wi h he ga ekeepe esidue Th 200. Addi ionally, he pa ially nega i e oxygen a om o 4a also in e ac s wi h he Zn(II) ion, while he sul u a om o he hiosemica bazone moie y is in ol ed in an H-bond be ween 5b −7.45 In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 o 24 Addi ionally, he pa ially nega i e oxygen a om o 4a also in e ac s wi h he Zn(II) ion, while he sul u a om o he hiosemica bazone moie y is in ol ed in an H-bond be ween wa e and His68. In con as , o 5b, he same sul u a om in e ac s wi h Gln71 as an elec- on accep o , whils he Gln92 is he accep o om an in e ac ion wi h he NH g oup o he hiosemica bazone. Mo eo e , an H-π s acking in e ac ion occu s be ween he a o- ma ic moie y o he benzenesul onamide and Leu199. The he e oa om o he py idine ing also pa icipa es in coope a i e H-bonding wi h wa e molecules and P o202 (Figu e 4). Figu e 4. (A,C): docking ep esen a ion o CA IX (gold)–4a (g een) and 5b (ligh blue) binding com- plex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. Simila o he indings obse ed o CA IX, he dep o ona ed NH o he sul onamido moie y in 4a and 5a in e ac s wi h he Zn(II) ion and simul aneously wi h Leu197 and he ga ekeepe esidue Th 198 in CA XII. Addi ionally, compound 5b exhibi s a di ec binding wi h Ala 129 (Figu e 5). The binding ene gies o he in e ac ion wi h CA XII a e depic ed in Table 3. Figu e 4. (A,C): docking ep esen a ion o CA IX (gold)–4a (g een) and 5b (ligh blue) binding complex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. Simila o he indings obse ed o CA IX, he dep o ona ed NH o he sul onamido moie y in 4a and 5a in e ac s wi h he Zn(II) ion and simul aneously wi h Leu197 and he ga ekeepe esidue Th 198 in CA XII. Addi ionally, compound 5b exhibi s a di ec binding wi h Ala 129 (Figu e 5). The binding ene gies o he in e ac ion wi h CA XII a e depic ed in Table 3. In . J. Mol. Sci. 2025,26, 1225 9 o 23 In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 9 o 24 Table 3. Binding ene gies esul ing om he docking simula ions o he selec ed molecules agains CA XII. Re-docking sco e o 6R6Y co-c ys allized ligand is also included. Compound Binding Ene gy (kcal/mol) 4a –8.20 5b –7.68 Figu e 5. (A,C): docking ep esen a ion o CAXII (pink)—4a (g een) and 5b (ligh blue) binding complex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. 2.2.3. Me al Complexa ion Assays The abili y o 5b, as a model compound, o complex me al ca ions associa ed wi h umo igenesis (Na + , K + , Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ) was analyzed using UV–Vis spec os- copy. To in es iga e his, UV–Vis spec a o solu ions wi h a ying a ios o 5b o me al chlo ides (1:0, 4:1, 2:1, 1:1, 1:2, 1:5) we e eco ded. Addi ionally, a 1:2 ligand- o-me al spec- um was ob ained a e 72 h o incuba ion o accoun o po en ial slow complexa ion. Ti a ion expe imen s wi h NaCl and KCl showed no signi ican spec al changes wi h inc easing amoun s o he me als (Figu e 6E,F, espec i ely), indica ing ha 5b does no Figu e 5. (A,C): docking ep esen a ion o CAXII (pink)—4a (g een) and 5b (ligh blue) binding complex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. Table 3. Binding ene gies esul ing om he docking simula ions o he selec ed molecules agains CA XII. Re-docking sco e o 6R6Y co-c ys allized ligand is also included. Compound Binding Ene gy (kcal/mol) In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 o 23 wa e and His68. In con as , o 5b, he same sul u a om in e ac s wi h Gln71 as an elec- on accep o , whils he Gln92 is he accep o om an in e ac ion wi h he NH g oup o he hiosemica bazone. Mo eo e , an H-π s acking in e ac ion occu s be ween he a o- ma ic moie y o he benzenesul onamide and Leu199. The he e oa om o he py idine ing also pa icipa es in coope a i e H-bonding wi h wa e molecules and P o202 (Figu e 4). Figu e 4. (A,C): docking ep esen a ion o CA IX (gold)–4a (g een) and 5b (ligh blue) binding com- plex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. Simila o he indings obse ed o CA IX, he dep o ona ed NH o he sul onamido moie y in 4a and 5a in e ac s wi h he Zn(II) ion and simul aneously wi h Leu197 and he ga ekeepe esidue Th 198 in CA XII. Addi ionally, compound 5b exhibi s a di ec binding wi h Ala 129 (Figu e 5). The binding ene gies o he in e ac ion wi h CA XII a e depic ed in Table 3. Table 3. Binding ene gies esul ing om he docking simula ions o he selec ed molecules agains CA XII. Re-docking sco e o 6R6Y co-c ys allized ligand is also included. Compound Binding Ene gy (kcal/mol) 4a –8.20 –7.68 A B C D 4a −8.20 In . J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 o 23 wa e and His68. In con as , o 5b, he same sul u a om in e ac s wi h Gln71 as an elec- on accep o , whils he Gln92 is he accep o om an in e ac ion wi h he NH g oup o he hiosemica bazone. Mo eo e , an H-π s acking in e ac ion occu s be ween he a o- ma ic moie y o he benzenesul onamide and Leu199. The he e oa om o he py idine ing also pa icipa es in coope a i e H-bonding wi h wa e molecules and P o202 (Figu e 4). Figu e 4. (A,C): docking ep esen a ion o CA IX (gold)–4a (g een) and 5b (ligh blue) binding com- plex. (B,D): wo-dimensional scheme o he p o ein–ligand in e ac ions. Simila o he indings obse ed o CA IX, he dep o ona ed NH o he sul onamido moie y in 4a and 5a in e ac s wi h he Zn(II) ion and simul aneously wi h Leu197 and he ga ekeepe esidue Th 198 in CA XII. Addi ionally, compound 5b exhibi s a di ec binding wi h Ala 129 (Figu e 5). The binding ene gies o he in e ac ion wi h CA XII a e depic ed in Table 3. Table 3. Binding ene gies esul ing om he docking simula ions o he selec ed molecules agains CA XII. Re-docking sco e o 6R6Y co-c ys allized ligand is also included. Compound Binding Ene gy (kcal/mol) 4a –8.20 –7.68 A B C D 5b −7.68 2.2.3. Me al Complexa ion Assays The abili y o 5b, as a model compound, o complex me al ca ions associa ed wi h u- mo igenesis (Na + , K + , Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ) was analyzed using UV–Vis spec oscopy. To in es iga e his, UV–Vis spec a o solu ions wi h a ying a ios o 5b o me al chlo ides (1:0, 4:1, 2:1, 1:1, 1:2, 1:5) we e eco ded. Addi ionally, a 1:2 ligand- o-me al spec um was ob ained a e 72 h o incuba ion o accoun o po en ial slow complexa ion. Ti a ion expe imen s wi h NaCl and KCl showed no signi ican spec al changes wi h inc easing amoun s o he me als (Figu e 6E,F, espec i ely), indica ing ha 5b does no chela e hese mono alen ca ions. In con as , he spec a o di alen and i alen ca ions e ealed a dec ease in abso bance a λmax , accompanied by a signi ican ba hoch omic shi . These changes sugges ha 5b chela es Fe 2+ , Fe 3+ , Zn 2+ , and Cu 2+ ions. Among hese, he mos p onounced e ec was obse ed wi h CuCl 2 (Figu e 6B). The addi ion o s oichiome ic o In . J. Mol. Sci. 2025,26, 1225 16 o 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 -A -Cp, NH-A -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 (A -C), 115.7 (d, 2 J C,H = 21.9 Hz, C-3 ′′ /C-5 ′′ ) ppm; HRESI-MS m/zcalcd. o C14H13FN4O3S2([M+H]+): 353.0537, ound: 353.0536. 1-(4 ′ -Chlo ophenylme hylene)-4-(4 ′′ -sul onamidophenyl)-3- hiosemica bazone (4d). Thiosemica bazide 3a (200 mg, 0.81 mmol) and 4-chlo obenzaldehyde (114 mg, 0,81 mmol, 1.0 equi .) we e used. Compound 4d was ob ained as a whi e solid. Yield: 223 mg (75%); spec oscopic da a a e in ag eemen wi h hose epo ed [59]. 1-(4 ′ -B omophenylme hylene)-4-(4 ′′ -sul onamidophenyl)-3- hiosemica bazone (4e). Thiosemica bazide 3a (200 mg, 0.81 mmol) and 4-b omobenzaldehyde (87 µ L, 0.81 mmol, 1.0 equi .) we e used. Compound 4e was ob ained as a whi e solid. Yield: 236 mg (71%); spec oscopic da a a e in ag eemen wi h hose epo ed [59]. 1-(4 ′ -Ni ophenylme hylene)-4-(4 ′′ -sul onamidophenyl)-3- hiosemica bazone (4 ). Thiosemica bazide 3a (200 mg, 0.81 mmol) and 4-ni obenzaldehyde (128 mg, 0.81 mmol, 1.0 equi .) we e used. Compound 4 was ob ained 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, A -NH), 8.26 (m, 2H, A -H), 8.26 (s, 1H, N=CH), 8.20 (m, 2H, A -H), 7.80 (m, 4H, A -H), 7.35 (b s, 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 (A -C) ppm; HRESI-MS m/zcalcd. Fo C 14 H 13 N 5 NaO 4 S 2 ([M+Na]+): 402.0301, ound: 402.0296. 1-Phenylme hylene-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (4g). Thiosemi- ca bazide 3b (200 mg, 0.81 mmol) and benzaldehyde (84 µ L, 0.81 mmol, 1.0 equi .) we e used. Compound 4g was ob ained as a whi e solid. Yield: 197 mg (68%); spec oscopic da a a e in ag eemen wi h hose epo ed [59]. 1-(4 ′ -Me hoxyphenylme hylene)-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (4h). Thiosemica bazide 3b (200 mg, 0.81 mmol) and 4-me hoxybenzaldehyde (98 µ L, 0,81 mmol, 1.0 equi .) we e used. Compound 4h was ob ained as a whi e 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, A -NH), 8.12 (s, 1H, N=CH), 8.05 (b , 1H, J H,H = 1.9 Hz, H-2 ′′ ), 7.86 (m, 2H, H-2 ′ , H-6 ′ ), 7.84 (m, 1H, A -H), 7.64 (d , 1H, J H,H = 1.4 Hz, J H,H = 8.4 Hz, A -H), 7.54 ( , 1H, J H,H = 7.9 Hz, A -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 (A -C), 114.64 (C-3 ′ /C-5 ′ ), 55.8 (OMe) ppm; HRESI-MS m/zcalcd. o C15H16N4NaO3S2([M+Na]+): 387.0556, ound: 387.0547. 1-(4 ′ -Fluo ophenylme hylene)-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (4i). Thiosemica bazide 3b (200 mg, 0.81 mmol) and 4- luo obenzaldehyde (87 µ L, 0,81 mmol, 1.0 eq.) we e used. Compound 4i was ob ained as a whi e solid. Yield: 198 mg (70%); spec oscopic da a a e in ag eemen wi h hose epo ed [59]. 1-(4 ′ -Chlo ophenylme hylene)-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (4j). Thiosemica bazide 3b (200 mg, 0.81 mmol) and 4-chlo obenzaldehyde (114 mg, 0,81 mmol, 1.0 equi .) we e used. Compound 4j was ob ained as a whi e 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, A -NH), 8.15 (s, 1H, N=CH), 8.04 (b , 1H, J H,H = 1.8 Hz, H-2 ′′ ), 7.97 (m, 2H, H-2 ′ , H-6 ′ ), 7.81 (b d, 1H, J H,H = 8.0 Hz, A -H), 7.65 (b d, 1H, J H,H = 7.9 Hz, A -H), 7.60–7.49 (m, 3H, A -H), 7.38 (b s, 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 (A -C) ppm; HRESI-MS calcd. o C14H1335ClN4NaO2S2([M+Na]+): 391.0061, ound: 391.0057. 1-(4 ′ -B omophenylme hylene)-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (4k). Thiosemica bazide 3b (200 mg, 0.81 mmol) and 4-b omobenzaldehyde (87 µ L, 0,81 mmol, In . J. Mol. Sci. 2025,26, 1225 17 o 23 1.0 equi .) we e used. Compound 4k was ob ained as a whi e solid. Yield: 250 mg (76%); spec oscopic da a a e in ag eemen wi h hose epo ed [59]. 1-Phenylme hylene-4-[2 ′ -(4 ′′ -sul onamido)phenyl]e hyl-3- hiosemica bazone (4l). Thiosemica bazide 3c (200 mg, 0.73 mmol) and benzaldehyde (74 µ L, 0.73 mmol, 1.0 equi .) we e used. Compound 4l was ob ained as a whi e solid. Yield: 133 mg (50%). Mp: 228 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.56 (s, 1H, NH), 8.59 ( , 1H, J H,H = 6.3 Hz, NH-CH 2 ), 8.10 (s, 1H, N=CH), 7.80 (m, 4H, A -H), 7.45 (m, 5H, A -H), 7.28 (s, 2H, NH 2 ), 3.81 (b q, 2H, J H,H = 6.5 Hz, NH-CH 2 ), 3.02 ( , 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 (A -C), 44.6 (CH2-NH), 34.6 (CH 2 -A ) ppm; HRESI-MS m/zcalcd. o C16H18N4NaO2S2([M+Na]+): 385.0763, ound: 385.0761. 1-(4 ′ -Me hoxyphenylme hylene)-4-[2 ′′ -(4 ′′′ -sul onamido)phenyl]e hyl-3- hiosemica bazone (4m). Thiosemica bazide 3c (200 mg, 0.73 mmol) and 4-me hoxybenzaldehyde (89 µ L, 0.73 mmol, 1.0 equi .) we e used. Compound 4m was ob ained as a whi e solid. Yield: 186 mg (65%). Mp: 174 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.38 (s, 1H, NH), 8.47 ( , 1H, J H,H = 6.0 Hz, NH-CH 2 ), 8.00 (s, 1H, N=CH), 7.77 (m, 2H, A -H), 7.70 (m, 2H, A -H), 7.45 (m, 2H, A -H), 7.27 (s, NH 2 ), 6.98 (m, 2H, A -H), 3.80 (s, 3H, OMe), 3.80 (m, 2H, CH 2 -NH), 3.00 ( , 2H, J H,H = 8.0 Hz, CH 2 -A ) 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 (A -C), 55.3 (OMe), 44.4 (CH 2 -NH), 34.8 (A -CH 2 ) ppm; HRESI-MS m/zcalcd. o C 17 H 20 N 4 NaO 3 S 2 ([M+Na]+): 415.0869, ound: 415.0862. 1-(4 ′ -Fluo ophenylme hylene)-4-[2 ′′ -(4 ′′′ -sul onamido)phenyl]e hyl-3- hiosemica bazone (4n). Thiosemica bazide 3c (200 mg, 0.73 mmol) and 4- luo obenzaldehyde (78 µ L, 0.73 mmol, 1.0 equi .) we e used. Compound 4n was ob ained as a whi e solid. Yield: 205 mg (74%). Mp: 242 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.52 (s, 1H, NH), 8.61 ( , 1H, J H,H = 5.6 Hz, NH-CH 2 ), 8.05 (s, 1H, N=CH), 7.84 (m, 2H, A -H), 7.78 (m, 2H, A -H), 7.46 (m, 2H, A -H), 7.28 (m, 4H, A -H, NH 2 ), 3.80 (q, 2H, J H,H = 7.0 Hz, CH 2 -NH), 3.01 ( , 1H, J H,H = 7.0 Hz, CH 2 -A ) 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 (A -C), 115.8 (d, 2 J H,H = 21.7 Hz, C- 3 ′ /C-5 ′ ), 44.6 (CH 2 -NH), 34.6 (CH 2 -A ), ppm; HRESI-MS m/zcalcd. o C 16 H 17 FN 4 NaO 2 S 2 ([M+Na]+): 403.0669, ound: 403.0670. 1-(4 ′ -Chlo ophenylme hylene)-4-[2 ′′ -(4 ′′′ -sul onamido)pheny]e hyl-3- hiosemica bazone (4o). Thiosemica bazide 3c (200 mg, 0.73 mmol) and 4-chlo obenzaldehyde (103 mg, 0.73 mmol, 1.0 equi .) we e used. Compound 4o was ob ained as a whi e solid. Yield: 150 mg (52%). Mp: 235 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.57 (s, 1H, NH), 8.65 ( , 1H, J H,H = 5.9 Hz, NH-CH 2 ), 8.05 (s, 1H, N=CH) 7.80 (m, 4H, A -H), 7.48 (m, 4H, A -H), 7.29 (s, 2H, NH 2 ), 3.81 (b q, 2H, J H,H = 6.6 Hz, CH 2 -NH), 3.02 ( , 2H, J H,H = 7.1 Hz, CH 2 -A ) 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 (A -C), 44.6 (CH 2 -NH), 34.6 (CH 2 -A ) ppm; HRESI-MS m/zcalcd. o C16H1735ClN4NaO2S2([M+Na]+): 419.0374, ound: 419.0367. 1-(4 ′ -B omophenylme hylene)-4-[2 ′′ -(4 ′′′ -sul onamido)phenyl]e hyl-3- hiosemica bazone (4p). Thiosemica bazide 3c (200 mg, 0.73 mmol) and 4-b omobenzaldehyde (78 µ L, 0.82 mmol, 1.0 equi .) we e used. Compound 4p was ob ained as a whi e solid. Yield: 187 mg (58%). Mp: 244 ◦ C; 1 H-NMR (300 MHz, DMSO-d 6 ) δ 11.57 (s, 1H, NH), 8.64 ( , 1H, J H,H = 5.8 Hz, NH-CH 2 ), 8.03 (s, 1H, N=CH), 7.76 (m, 4H, A -H), 7.63 (m, 2H, A -H’), 7.46 (m, 2H, A -H), 7.28 (s, 2H, NH 2 ), 3.80 (q, 2H, J H,H = 6.5 Hz, CH 2 -NH), 3.02 ( , 2H, J H,H = 7.0 Hz, CH 2 -A ) 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 (A -C), 44.6 (CH 2 -NH), 34.6 (CH 2 - In . J. Mol. Sci. 2025,26, 1225 18 o 23 A ); HRESI-MS m/zcalcd. o C 16 H 1879 B N 4 O 2 S 2 ([M+H] + ): 441.0049, ound: 441.0049; m/zcalcd. o C16H1881B N4O2S2([M+H]+): 443.0029, ound: 443.0026. 1-(Py idin-2 ′ -ylme hylene)-4-(4 ′′ -sul onamidophenyl)-3- hiosemica bazone(5a). Thiosemi- ca bazide 3a (200 mg, 0.81 mmol) and py idine-2-ca baldehyde (74 µ L, 0.81mmol, 1.0 equi .) we e used. Compound 5a was ob ained 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, A -NH), 8.76 (b d, 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, A -Ho, A -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 (A -C) ppm; HRESI-MS m/zcalcd. o C 13 H 13 N 5 NaO 2 S 2 ([M+Na] + ): 358.0403, ound: 358.0402. 1-(Py idin-2 ′ -ylme hylene)-4-(3 ′′ -sul onamidophenyl)-3- hiosemica bazone (5b). Thiosemi- ca bazide 3b (200 mg, 0.73 mmol) and py idine-2-ca baldehyde (74 µ L, 0.81mmol, 1.0 equi .) we e used. Compound 5b was ob ained 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, A -NH), 8.68 (b d, 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 ( , 1H, JH,H = 7.7 Hz, A -H), 8.04 (b , 1H, J 2′′,4′′ = J 2′′,6′′ = 1.8 Hz, H-2 ′′ ), 7.82 (b d, 1H, J H,H = 7.8 Hz, A -H), 7.68 (d , 1H, J H,H = 1.3 Hz, J H,H = 7.8 Hz, A -H), 7.58 (m, 2H, A -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 (A -C) ppm; HRESI-MS m/zcalcd. Fo C13H13N5NaO2S2([M+Na]+): 358.0403, ound: 358.0399. 1-(Py idin-2 ′ -ylme hylene)-4-[2 ′′ -(4 ′′′ -sul onamidophenyl)]e hyl-3- hiosemica bazone (5c). Thiosemica bazide 3c (200 mg, 0.73 mmol, 1.0 equi .) and py idine-2-ca baldehyde (67 µ L, 0.73 mmol) we e used. Compound 5b was ob ained 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 ( , 1H, J H,H = 6.0 Hz, NH-CH 2 ), 8.77 (b d, 1H, J 5′,6′ = 5.1 Hz, H-6 ′ ), 8.35 (b d, 1H, J 3′,4′ = 8.4 Hz, H-3 ′ ), 8.26 (b d, 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, A -H), 7.72 (m, 1H, H-5 ′ ), 7.46 (m, 2H, A -H), 7.28 (b s, 2H, NH 2 ), 3.84 (q, 2H, J H,H = 6.5 Hz, NH-CH 2 ), 3.03 ( , 1H, J H,H = 7.1 Hz, CH 2 -A ) 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 (A -C), 44.8 (CH 2 -NH), 34.3 (CH 2 -A ) ppm; HRESI-MS m/zcalcd. o C 15 H 17 N 5 NaO 2 S 2 ([M+Na]+): 386.0716, ound: 386.0720. 3.2. CA Inhibi ion Assays An applied pho ophysics s opped- low ins umen was used o assaying he CA- ca alyzed CO 2 hyd a ion ac i i y [ 18 ]. Phenol ed (a 0.2 mM) was used as an indica o , wo king a he abso bance maximum o 557 nm, wi h 20 mM Hepes (pH 7.4) and 20 mM Na 2 SO 4 ( o main aining cons an ionic s eng h), ollowing he ini ial a es o he CA- ca alyzed CO 2 hyd a ion eac ion o 10–100 s. The CO 2 concen a ions anged om 1.7 o 17 mM o he de e mina ion o he kine ic pa ame e s and inhibi ion cons an s. Fo each inhibi o , a leas six aces o he ini ial 5–10% o he eac ion we e used o de e mining he ini ial a e. The unca alyzed a es we e de e mined in he same manne and sub ac ed om he o al obse ed a es. S ock solu ions o inhibi o (10 mM) we e p epa ed in dis illed–deionized wa e , and app op ia e dilu ions we e done he ea e wi h dis illed– deionized wa e . AAZ was used as a posi i e con ol. Inhibi o and enzyme solu ions we e p eincuba ed oge he o 15 min a oom empe a u e be o e assay in o de o allow o he o ma ion o he E–I complex. The inhibi ion cons an s we e ob ained by nonlinea leas -squa es me hods using PRISM 3 and he Cheng–P uso equa ion and ep esen he mean om a leas h ee di e en de e mina ions. All CA iso o ms we e ecombinan ones (5–12 nM), ob ained in-house. In . J. Mol. Sci. 2025,26, 1225 19 o 23 3.3. Docking Simula ions The c ys allog aphic s uc u e o CAIX and CAXII wi h 1.82 Å esolu ion (PDB ID: 5FL4) and 1.38 Å esolu ion (PDB ID: 4HT2), espec i ely, we e ob ained om he P o ein Da a Bank [ 60 ]. Docking simula ions we e pe o med in MOE So wa e 2019.01 (Chemical Compu ing G oup, Mon eal, QC, Canada). P o ein op imiza ion was pe o med ollowing he QuickP ep p o ocol. B ie ly, c ys allog aphic a i ac s, non-bonded ligands, and excess copies o he p o ein a e emo ed. Wa e molecules we e emo ed excep o he ones a a maximum dis ance o 4.5 Å om he ac i e si e. Ligands we e buil , hyd ogens added, and geome y op imized h ough ene gy minimiza ion. Du ing docking simula ions, ligands we e placed in he g id o he co-c ys allized ligand. In he placemen s age, ene gy binding calcula ions used he T iangle Ma che algo i hm wi h he London dG sco ing scheme. In he e inemen s age, he ecep o was kep igid, and he GBVI/WSA dg sco ing scheme was used. 3.4. Me al Complexa ion Assays The complexa ion o 5b wi h di e en me al chlo ides (NaCl, KCl, FeCl 2 , FeCl 3 , ZnCl 2 , and CuCl 2 ) was in es iga ed using UV–Vis spec oscopy (Jasco V-360). The s ock solu ions o he sal s (10 −2 M) and he ligand (2.5 mM) we e p epa ed in pu e DMSO and sonica ed when necessa y. To 20 µ L o a 2.5 mM solu ion o 5b we e added di e en aliquo s o he me al chlo ides solu ions up o a o al olume o 2 mL in qua z cu e es. The ollowing ligand–me al a ios we e used: 1:0.25, 1:0.5, 1:1, 1:2, 1:5, and 1:10. The abso bance was moni o ed a 25 ◦ C in he ange 280–440 nm, ei he wi h o wi hou incuba ion. A blank solu ion wi hou 5b was also p epa ed o each concen a ion o he sal , and i s spec um was sub ac ed om he ligand–sal spec um. 3.5. An ip oli e a i e Assays 3.5.1. Cell Lines and Cul u e The human cance cell lines A549, HBL-100, and T-47D, as well as HeLa, we e p o ided by D . Raimundo F ei e (Hospi al Uni e si a io de Cana ias, Tene i e, Cana y Islands). The lung cance cell lines SW1573 and WiD we e p o ided by P o . G. J. Pe e s (VU Uni e si y Medical Cen e , Ams e dam, The Ne he lands). Cells we e g own in RPMI-1640 medium con aining 5% e al bo ine se um (FBS), 2 mM L-glu amine, 100 U/mL o penicillin G, and 0.1 mg/mL o s ep omycin a 37 ◦ C in a 95% humidi ied a mosphe e o 5% CO 2 . Cells we e main ained in cul u e in 60 mm cell cul u e dishes in g ow h medium (10 mL) and passaged wice weekly. 3.5.2. An ip oli e a i e Tes s The an ip oli e a i e ac i i y o compounds was es ed using ou implemen a ion o he p o ocol o he Na ional Cance Ins i u e (NCI) o he USA. The ollowing seeding densi ies (cells pe well) we e used: 2500 (A549, HBL-100, HeLa, and SW1573) and 5000 (T-47D and WiD ). S ock solu ions o inhibi o s (40 mM) we e p epa ed in pu e DMSO (400 imes he maximum es concen a ion). Fo each es compound, he cells we e exposed o a pe iod o 48 h o se ial decimal dilu ions in cell cul u e medium o he es compounds (0.001–100 µ M). Fo each p oduc , GI 50 alues we e calcula ed acco ding o he NCI o mulas (n = 3; da a a e exp essed as mean ±SD). 3.5.3. Cell Mo phology The CX-A imaging pla o m mic oscope (Nanoli e SA, Lausanne, Swi ze land) was used o measu e e ac i e indices, c ea ing a holo omog aphic 3D image o he cells. SW1573 cells we e seeded on o 35 mm cell cul u e imaging dishes (IBIDI GmbH, G ä el ing, In . J. Mol. Sci. 2025,26, 1225 20 o 23 Ge many) a a densi y o 50.000 cells/well. On he nex day, ea ed cells we e exposed o he es compounds igh be o e he acquisi ion o he images. Image da a we e ans e ed o FIJI so wa e 2.9.0 (NIH, USA) o image analysis. EVE so wa e 2.2.1.2162 (Nanoli e S.A., Tolochenaz, Swi ze land)) was used o he analysis o he e ac i e indices and calcula ion o he pheno ypic pa ame e s. 4. Conclusions In conclusion, we ha e success ully designed mul i unc ional an ip oli e a i e agen s by combining a yl sul onamides, which ac as pha macopho es o CA inhibi ion, wi h a hiosemica bazone moie y ha ac s as a me al chela o . Py idine-2-ca baldehyde hiosemi- ca bazone 5b, wi h he sul onamido mo i a he me a posi ion, can be conside ed as he lead compound. Al hough he p esence o he sul onamido moie y was ound o be dele e ious o he an ip oli e a i e ac i i y compa ed wi h o he α -N-he e ocyclic hiosemica bazones epo ed in he li e a u e, 5b s ill p ese ed good ac i i y (GI 50 alues in he low mic omola ange), po en inhibi ion o CA iso o ms associa ed wi h umo p og ession (IX and XII, K i alues in he low nanomola ange), and e ec i e chela ion o di alen ca ions Fe 2+ and Cu 2+ , ensu ing a mul i ac o ial mechanism o ac ion. The absence o a di ec co ela ion be ween CA inhibi ion and an ip oli e a i e e ec s sugges s ha addi ional biological a ge s may also be in ol ed. The mode o ac ion o 5b was u he explo ed using 3D holo omog aphic mic oscopy, which e ealed delayed umo cell di ision and ea u es indica i e o apop osis. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps://www.mdpi.com/a icle/10.3390/ijms26031225/s1. Au ho Con ibu ions: Concep ualiza ion, Ó.L.; molecula docking and modelling, A.P., M.X.F. and J.M.P.; da a analysis, A.N., M.X.F., J.M.P. and C.T.S.; syn hesis and cha ac e iza ion: M.M.-M., G.A. and P.B.; biological assays: A.G.-B., A.P., P.B. and J.M.P.; w i ing—o iginal d a p epa a ion, Ó.L.; w i ing— e iew and edi ing, P.M.-M., A.N., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; supe ision: P.M.-M., S.M.-S., A.N., M.X.F., J.M.P., C.T.S., J.G.F.-B. and Ó.L.; unding acquisi ion, J.M.P., J.G.F.-B. and Ó.L. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: J.G.F.-B./Ó.L. and A.G.-B./A.P./J.M.P. hank he Spanish Go e nmen (p ojec s PID2020- 116460RB-I00 unded by MCIN/AEI/10.13039/501100011033 and PID2021-123059OB-I00 unded by MCIN/AEI/10.13039/501100011033/FEDER, UE, espec i ely) o inancial suppo . J.G.F.-B. and Ó.L. also hank Jun a de Andalucía (FQM134) and VII Plan P opio de In es igación (Uni e si y o Se ille). P.M.-M. and S.M.-S. hank VIEP-BUAP (p ojec 100521265-VIEP2024) o inancial suppo . Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : Da ase a ailable on eques om he au ho s. Acknowledgmen s: M.M.-M. hanks he Mexican CONAHCYT o he awa d o a p edoc o al ellow- ship (I1200/311/2023, MOD.ORD. /08/2023). P.B. hanks he Eu opean Union (Nex Gene a ionEU) and he Minis e io de Uni e sidades o Spain (G an Ma ga i a Salas). A.P. hanks he EU Social Fund (FSE) and he Cana y Islands ACIISI o a p edoc o al g an TESIS2020010055. A.G.-B. hanks he Asociación Española Con a el Cánce (AECC) de San a C uz de Tene i e o he awa d o a p edoc o al g an (PRDTF233958GONZ). We would also like o hank he Se icio de Resonancia Magné ica Nuclea , CITIUS (Uni e si y o Se ille), o he pe o mance o NMR expe imen s. Con lic s o In e es : The au ho s decla e no con lic o in e es . In . J. Mol. Sci. 2025,26, 1225 21 o 23 Re e ences 1. WHO (Wo ld Heal h O ganiza ion). A ailable online: h ps://www.who.in /heal h- opics/cance # ab= ab_1 (accessed on 3 No embe 2024). 2. Nguyen, O.T. The global challenge o cance . Na . Cance 2020,1, 1–2. 3. G un , T.W.; Valen , P. Cance —A de as a ing disease, bu also an eye-opene and window in o he deep mys e ies o li e and i s o igins. P og ess Biophys. Mol. Biol. 2022,175, 131–139. [C ossRe ] 4. C osby, D.; Bha ia, S.; B indle, K.M.; Coussens, L.M.; Di e, C.; Embe on, M.; Esene , S.; Fi zge ald, R.C.; Gambhi , S.S.; Kuhn, P.; e al. Ea ly de ec ion o cance . Science 2022,375, eaay9040. [C ossRe ] [PubMed] 5. Ache , A.W.; Bleiche , J.; Cannon, A.; Scai e, C. Ad ances in su ge y o panc ea ic cance . J. Gas oin es . Oncol. 2018,9, 1037–1043. [C ossRe ] 6. Nuwe , R. Ad ances in highly a ge ed adia ion ea men o cance ha e igni ed in e es in a once obscu e ield. Na u e 2024, 629, S7–S9. [C ossRe ] 7. Liu, Q.; Wu, P.; Lei, J.; Bai, P.; Zhong, P.; Yang, M.; Wei, P. Old concep s, new icks: How pep ide accines a e eshaping cance immuno he apy? In . J. Biol. Mac omol. 2025,279, 135541. [C ossRe ] 8. Dash, P.; Panda, P.K.; Su, C.; Lin, Y.-C.; Sak hi el, R.; Chena, S.-L.; Chung, R.-J. Nea -in a ed-d i en upcon e sion nanopa icles wi h pho oca alys s h ough wa e -spli ing owa ds cance ea men . J. Ma e . Chem. B 2024,12, 3881–3907. [C ossRe ] 9. Anand, U.; Dey, A.; Chandel, A.K.S.; Sanyal, R.; Mish a, A.; Pandey, D.K.; De Falco, V.; Upadhyay, A.; Kandimalla, R.; Chaudha y, A.; e al. Cance chemo he apy and beyond: Cu en s a us, d ug candida es, associa ed isks and p og ess in a ge ed he apeu ics. Genes Dis. 2023,10, 1367–1401. [C ossRe ] 10. S ebha d , K.; Ull ich, A. Paul Eh lich’s magic bulle concep : 100 yea s o p og ess. Na . Re . Cance 2008,8, 473–480. [C ossRe ] 11. Kabi , A.; Mu h, A. Polypha macology: The science o mul i- a ge ing molecules. Pha macol. Res. 2022,176, 106055. [C ossRe ] 12. Liu, X.-J.; Zhao, H.-C.; Hou, S.-J.; Zhang, H.-J.; Cheng, L.; Yuan, S.; Zhang, L.-R.; Song, J.; Zhang, S.-Y.; Chen, S.-W. Recen de elopmen o mul i- a ge VEGFR-2 inhibi o s o he cance he apy. Bioo g. Chem. 2023,133, 106425. [C ossRe ] [PubMed] 13. Occhipin i, R.; Bo on, W.F. Role o ca bonic anhyd ases and inhibi o s in acid–base physiology: Insigh s om ma hema ical modelling. In . J. Mol. Sci. 2019,20, 3841. [C ossRe ] [PubMed] 14. Kim, J.K.; Lee, C.; Lim, S.W.; Adhika i, A.; And ing, J.T.; McKenna, R.; Ghim, C.-M.; Kim, C.U. Elucida ing he ole o me al ions in ca bonic anhyd ase ca alysis. Na . Commun. 2020,11, 4557. [C ossRe ] 15. Supu an, C.T. Mul i- and poly-pha macology o ca bonic anhyd ase inhibi o s. Pha macol. Re 2025,77, 100004. [C ossRe ] 16. Kuma , S.; Rulhania, S.; Jaswal, S.; Monga, V. Recen ad ances in he medicinal chemis y o ca bonic anhyd ase inhibi o s. Eu . J. Med. Chem. 2021,209, 112923. [C ossRe ] 17. Mboge, M.Y.; McKenna, R.; F os , S.C. Ad ances in an i-cance d ug de elopmen a ge ing ca bonic anhyd ase IX and XII. Top. An icance Res. 2015,5, 3–42. 18. Higazy, S.; Sami , N.; El-Khouly, A.; Gio annuzzi, S.; Begines, P.; Gabe , H.M.; Supu an, C.T.; Abouzid, K.A.M. Iden i ica ion o hienopy imidine de i a i es e he ed wi h sul onamide and o he moie ies as ca bonic anhyd ase inhibi o s: Design, syn hesis and an i-p oli e a i e ac i i y. Bioo g. Chem. 2024,144, 107089. [C ossRe ] 19. Elsayad, K.A.; Elmas y, G.F.; Mahmoud, S.T.; Awadallah, F.M. Sul onamides as an icance agen s: A b ie e iew on sul onamide de i a i es as inhibi o s o a ious p o eins o e exp essed in cance . Bioo g. Chem. 2024,147, 107409. [C ossRe ] 20. Sa nella, A.; Fe a a, Y.; Aule a, L.; Albanese, S.; Ce chia, L.; Al e io, V.; De Simone, G.; Supu an, C.T.; Zanne i, A. Inhibi ion o ca bonic anhyd ases IX/XII by SLC-0111 boos s cispla in e ec s in hampe ing head and neck squamous ca cinoma cell g ow h and in asion. J. Exp. Clin. Cance Res. 2022,41, 122. [C ossRe ] 21. Elo an a, K.; Pihlajoki, M.; Liljes öm, E.; Nousiainen, R.; Soini, T.; Lohi, J.; Cai o, S.; Wilson, D.B.; Pa kkila, S.; Heikinheimo, M. SLC-0111, an inhibi o o ca bonic anhyd ase IX, a enua es hepa oblas oma cell iabili y and mig a ion. F on . Oncol. 2023, 13, 1118268. [C ossRe ] 22. Jomo a, K.; Mako a, M.; Aloma , S.Y.; Alwasel, S.H.; Nepo imo a, E.; Kuca, K.; Rhodes, C.J.; Valko, M. Essen ial me als in heal h and disease. Chem.-Biol. In e ac . 2022,367, 110173. [C ossRe ] [PubMed] 23. K zywoszy´nska, K.; Wi kowska, D.; ´ Swi ˛a ek-Kozłowska, J.; Szebesczyk, A.; Kozłowski, H. Gene al aspec s o me al ions as signaling agen s in heal h and disease. Biomolecules 2020,10, 1417. [C ossRe ] [PubMed] 24. Se a, M.; Columbano, A.; Amma ah, U.; Mazzone, M.; Menga, A. Unde s anding me al dynamics be ween cance cells and mac ophages: Compe i ion o syne gism? F on . Oncol. 2020,10, 646. [C ossRe ] 25. Yang, Y.; Fan, H.; Guo, Z. Modula ion o me al homeos asis o cance he apy. ChemPlusChem 2024,89, e202300624. [C ossRe ] 26. Sousa, L.; Oli ei a, M.M.; Pessôa, M.T.C.; Ba bosa, L.A. I on o e load: E ec s on cellula biochemis y. Clin. Chim. Ac a 2000, 504, 180–189. [C ossRe ] 27. Guan, D.; Zhao, L.; Shi, X.; Ma, X.; Chen, Z. Coppe in cance : F om pa hogenesis o he apy. Biomed. Pha maco he . 2023, 163, 114791. [C ossRe ] In . J. Mol. Sci. 2025,26, 1225 22 o 23 28. Kon oghio ghes, G.J. New i on me abolic pa hways and chela ion a ge ing s a egies a ec ing he ea men o all ypes and s ages o cance . In . J. Mol. Sci. 2022,23, 13990. [C ossRe ] 29. Hassan, A.E.; Albohy, S.A.H.; Elza e , A.S.; El eky, A.S.; El-Fakha any, E.M.; Saleh, A.K.; Mahmoud, A.M.; Elgammal, W.E. Me al complexes wi h hiosemica bazone de i a i e and isa ine: A p omising new class o ma e ials o biomedical and en i onmen al applica ions. J. Pho ochem. Pho obiol. A Chem. 2024,455, 115764. [C ossRe ] 30. Aly, A.A.; Abdallah, E.M.; Ahmed, S.A.; Rabee, M.M.; B äse, S. T ansi ion me al complexes o hiosemica bazides, hioca bo- hyd azides, and hei co esponding ca bazones wi h Cu(I), Cu(II), Co(II), Ni(II), Pd(II), and Ag(I)—A e iew. Molecules 2023, 28, 1808. [C ossRe ] 31. Dömö ö , O.; May, N.V.; Peli an, K.; Kiss, T.; Kepple , B.K.; Kowol, C.R.; Enyedy, E.A. A compa a i e s udy o α -N-py idyl hiosemica bazones: Spec oscopic p ope ies, solu ion s abili y and coppe (II) complexa ion. Ino g. Chim. Ac a 2018,472, 264–275. [C ossRe ] 32. Podolski-Reni´c, A.; Gašpa o i´c, A. ˇ C.; Valen e, A.; López, Ó.; Nunes, J.H.B.; Kowol, C.R.; He e e , P.; Filipo i´c, N.R. Schi bases and hei me al complexes o a ge and o e come (mul id ug) esis ance in cance . Eu . J. Med. Chem. 2024,270, 116363. [C ossRe ] [PubMed] 33. Youse , T.A.; Khai y, M. Syn hesis, cha ac e iza ion, op ical, DFT, TD DFT s udies and in silico ADME p edic ions o hiosemica - bazone ligand and i s Au(III) complex. O ien . J. Chem. 2022,38, 537–546. [C ossRe ] 34. Youse , T.A.; El-Reash, G.M.A. Syn hesis, and biological e alua ion o complexes based on hiosemica bazone ligand. J. Mol. S uc . 2020,1201, 127180. [C ossRe ] 35. de Siquei a, L.R.P.; de Mo aes Gomes, P.A.T.; de Lima Fe ei a, L.P.; de Melo Rêgo, M.J.B.; Lei e, A.C.L. Mul i- a ge compounds ac ing in cance p og ession: Focus on hiosemica bazone, hiazole and hiazolidinone analogues. Eu . J. Med. Chem. 2019, 170, 237–260. [C ossRe ] 36. Shakya, B.; Yada , P.N. Thiosemica bazones as po en an icance agen s and hei modes o ac ion. Mini-Re . Med. Chem. 2020, 20, 638–661. [C ossRe ] 37. de Almeida, S.M.V.; Almeida La aye e, E.; Gomes da Sil a, L.P.B.; da C uz Amo im, C.A.; de Oli ei a, T.B.; Gois Ruiz, A.L.T.; de Ca alho, J.E.; de Mou a, R.O.; Ca nei o Bel ão, E.I.; Al es de Lima, M.d.C.; e al. Syn hesis, DNA binding, and an ip oli e a i e ac i i y o no el ac idine- hiosemica bazone de i a i es. In . J. Mol. Sci. 2015,16, 13023–13042. [C ossRe ] 38. de Almeida, S.M.V.; Ribei o, A.G.; de Lima Sil a, G.C.; Fe ei a Al es, J.E.; Ca nei o Bel ão, E.I.; Fe ei a de Oli ei a, J.; de Ca alho, L.B., J .; Al es de Lima, M.d.C. DNA binding and opoisome ase inhibi ion: How can hese mechanisms be explo ed o design mo e speci ic an icance agen s? Biomed. Pha maco he . 2017,96, 1538–1556. [C ossRe ] 39. Kalinowski, D.S.; Quach, P.; Richa dson, D.R. Thiosemica bazones: The new wa e in cance ea men . Fu . Med. Chem. 2009,1, 1143–1151. [C ossRe ] 40. Schaie , M.; Falcone, E.; P s ek, T.; Vileno, B.; Hage , S.; Kepple , B.K.; He e e , P.; Koellenspe ge , G.; Falle , P.; Kowol, C.R. Human se um albumin as a coppe sou ce o an icance hiosemica bazones. Me allomics 2023,15, m ad046. [C ossRe ] 41. He e e , P.; Pape, V.F.S.; Enyedy, É.A.; Kepple , B.K.; Szakacs, G.; Kowol, C.R. An icance hiosemica bazones: Chemical p ope ies, in e ac ion wi h i on me abolism, and esis ance de elopmen . An ioxid. Redox Signal. 2019,30, 1062–1082. [C ossRe ] 42. Ohui, K.; S epanenko, I.; Besleaga, I.; Babak, M.V.; S a i, R.; Da asio a, D.; Gies e , G.; Pósa, V.; Enyedy, E.A.; Vegh, D.; e al. T iapine de i a i es ac as coppe deli e y ehicles o induce deadly me al o e load in cance cells. Biomolecules 2010,10, 1336. [C ossRe ] [PubMed] 43. Gho ab, M.M.; El Ella, D.A.A.; Heiba, H.I.; Soliman, A.M. Syn hesis o ce ain new hiazole de i a i es bea ing a sul onamide moie y wi h expec ed an icance and adiosensi izing ac i i ies. J. Ma . Sci. Eng. A 2011,1, 684–691. 44. Khobzaoui, M.; Tilleke a ne, L.M.V.; Hudson, R.A. Po en iso hiocyana e inhibi o s o ca bonic anhyd ase: Syn hesis and e alua ion. Biochem. Biophys. Res. Commun. 2004,318, 1–3. [C ossRe ] [PubMed] 45. Sala oglio, I.C.; Mujunda , P.; Anno azzi, L.; Kopecha, J.; Mellai, M.; Schi e , D.; Poulsen, S.A.; Rigan i, C. Ca bonic anhyd ase XII inhibi o s o e come P-glycop o ein-media ed esis ance o emozolomide in glioblas oma. Mol. Cance The . 2018,17, 2598–2609. [C ossRe ] 46. Supu an, C.T.; Al amimi, A.S.A.; Ca a, F. Ca bonic anhyd ase inhibi ion and he managemen o glaucoma: A li e a u e and pa en e iew 2013–2019. Expe Opin. The . Pa . 2019,29, 781–792. [C ossRe ] 47. Ma ínez-Mon iel, M.; Rome o-He nández, L.L.; Gio annuzzi, S.; Begines, P.; Pue a, A.; Ahuja-Casa ín, A.I.; Fe nandes, M.X.; Me ino-Mon iel, P.; Mon iel-Smi h, S.; Nocen ini, A.; e al. Con o ma ionally es ic ed glycoconjuga es de i ed om a ylsul onamides and couma ins: New amilies o umou -associa ed ca bonic anhyd ase inhibi o s. In . J. Mol. Sci. 2023,24, 9401. [C ossRe ] 48. A gibay-O e o, S.; Ca ballo, R.; Vázquez-López, E.M. Coo dina ion chemis y o po en ially S,N,N py - iden a e hiosemica - bazones wi h he {Re(CO)3}+ agmen and o ma ion o hemiaminal de i a i es. Ino g. Chem. 2023,62, 224–237. [C ossRe ] 49. Deng, J.; Yu, P.; Zhang, Z.; Wang, J.; Cai, J.; Wu, N.; Sun, H.; Liang, H.; Yang, F. Designing an icance coppe (II) complexes by op imizing 2-py idine- hiosemica bazone ligands. Eu . J. Med. Chem. 2018,158, 442–452. [C ossRe ] In . J. Mol. Sci. 2025,26, 1225 23 o 23 50. Bai, X.-G.; Zheng, Y.; Qi, J. Ad ances in hiosemica bazone me al complexes as an i-lung cance agen s. F on . Pha macol. 2022, 13, 1018951. 51. Zhang, X.-H.; Wang, B.; Tao, Y.-Y.; Ma, Q.; Wang, H.-J.; He, Z.-X.; Wu, H.-P.; Li, Y.-H.; Zhao, B.; Ma, L.-Y.; e al. Thiosemica bazone- based lead op imiza ion o disco e high-e iciency and low- oxici y an i-gas ic cance agen s. Eu . J. Med. Chem. 2020, 199, 112349. [C ossRe ] 52. Pape, V.F.S.; Tó h, S.; Fü edi, A.; Szebényi, K.; Lo ics, A.; Szabó, P.; Wiese, M.; Szakacs, G. Design, syn hesis and biological e alua ion o hiosemica bazones, hyd azinobenzo hiazoles and a ylhyd azones as an icance agen s wi h a po en ial o o e come mul id ug esis ance. Eu . J. Med. Chem. 2016,117, 335–354. [C ossRe ] [PubMed] 53. Roldán-Peña, J.M.; Pue a, A.; Dini´c, J.; Jo ano i´c S ojano , S.; González-Bakke , A.; Hicke, F.J.; Mish a, A.; Piyasaeng hong, A.; Maya, I.; Wal on, J.W.; e al. Bio inyla ed selenocyana es: Po en and selec i e cy os a ic agen s. Bioo g. Chem. 2023,133, 106410. [C ossRe ] [PubMed] 54. Padilla-Pé ez, M.C.; Sánchez-Fe nández, E.M.; González-Bakke , A.; Pue a, A.; Pad ón, J.M.; Ma ín-Lo o, F.; A oba, A.I.; Ga cía Fe nández, J.M.; Melle , C.O. Fluo o-labelled sp2-iminoglycolipids wi h immunomodula o y p ope ies. Eu . J. Med. Chem. 2023, 255, 115390. [C ossRe ] [PubMed] 55. Pue a, A.; González-Bakke , A.; San os, G.; Pad ón, J.M. Ea ly pha macological p o iling o an ip oli e a i e compounds by li e cell Imaging. Molecules 2022,27, 5261. [C ossRe ] [PubMed] 56. Fulme , G.R.; Mille , A.J.M.; She den, N.H.; Go lieb, H.E.; Nudelman, A.; S ol z, B.M.; Be caw, J.E.; Goldbe g, K.I. NMR Chemical shi s o aceimpu i ies: Common labo a o y sol en s, o ganics, and gases in deu e a ed sol en s ele an o he o ganome allic chemis . O ganome allics 2010,29, 2176–2179. [C ossRe ] 57. Ka alı, N.; Akdemi , A.; Gök a¸s, F.; Elma, P.E.; Angeli, A.; Kızılı mak, M.; Supu an, C.T. No el sul onamide-con aining 2- indolinones ha selec i ely inhibi umo -associa ed alpha ca bonic anhyd ases. Bioo g. Med. Chem. 2017,25, 3714–3718. [C ossRe ] 58. Demi -Yazici, K.; Apaydin, C.B.; Soylu-E e , O.; Oezsoy, N.; Ka ali, N. Syn hesis, molecula modeling and cholines e ase inhibi o y e ec s o 2-indolinone-based hyd azineca bo hioamides. Fu . Med. Chem. 2021,13, 2133–2151. [C ossRe ] 59. T awally, M.; Demi -Yazıcı, K.; Angeli, A.; Kaya, K.; Akdemi , A.; Supu an, C.T.; Güzel-Akdemi , Ö. Thiosemica bazone- benzenesul onamide de i a i es as human ca bonic anhyd ases inhibi o s: Syn hesis, cha ac e iza ion, and in silico s udies. An i-Cance Agen s Med. Chem. 2024,24, 649–667. [C ossRe ] 60. Be man, H.M.; Wes b ook, J.; Feng, Z.; Gilliland, G.; Bha , T.N.; Weissig, H.; Shindyalo , I.N.; Bou ne, P.E. The P o ein Da a Bank. Nucleic Acids Res. 2000,28, 235–242. [C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .