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
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Licensee MDPI, Basel, Swi ze land.
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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
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