Tetrahydroquinazole-based secondary sulphonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against isoforms I, II, IV, and IX, and computational studies

Article abstract of DOI:10.1080/14756366.2021.1956913

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of not

Full text of DOI:10.1080/14756366.2021.1956913

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Tetrahydroquinazole-based secondary
sulphonamides as carbonic anhydrase inhibitors:
synthesis, biological evaluation against isoforms I,
II, IV, and IX, and computational studies
Emma Baglini, Rahul Ravichandran, Emanuela Berrino, Silvia Salerno,
Elisabetta Barresi, Anna Maria Marini, Monica Viviano, Sabrina Castellano,
Federico Da Settimo, Claudiu T. Supuran, Sandro Cosconati & Sabrina Taliani
To cite this article: Emma Baglini, Rahul Ravichandran, Emanuela Berrino, Silvia Salerno,
Elisabetta Barresi, Anna Maria Marini, Monica Viviano, Sabrina Castellano, Federico Da Settimo,
Claudiu T. Supuran, Sandro Cosconati & Sabrina Taliani (2021) Tetrahydroquinazole-based
secondary sulphonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against
isoforms I, II, IV, and IX, and computational studies, Journal of Enzyme Inhibition and Medicinal
Chemistry, 36:1, 1874-1883, DOI: 10.1080/14756366.2021.1956913
To link to this article: https://doi.org/10.1080/14756366.2021.1956913
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UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1874–1883
https://doi.org/10.1080/14756366.2021.1956913
RESEARCH PAPER
Tetrahydroquinazole-based secondary sulphonamides as carbonic anhydrase
inhibitors: synthesis, biological evaluation against isoforms I, II, IV, and IX, and
computational studies
Emma Baglinia , Rahul Ravichandranb , Emanuela Berrinoc , Silvia Salernoa , Elisabetta Barresia
Anna Maria Marinia , Monica Vivianod , Sabrina Castellanod, Federico Da Settimoa , Claudiu T. Supuranc
Sandro Cosconatib and Sabrina Taliania
,
,
b
c
aDepartment of Pharmacy, University of Pisa, Pisa, Italy; DiSTABiF, University of Campania Luigi Vanvitelli, Caserta, Italy; NEUROFARBA
d
ꢀ
Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto Fiorentino, Italy; Department of
Pharmacy, Epigenetic Med Chem Lab, University of Salerno, Fisciano, Italy
ABSTRACT
ARTICLE HISTORY
Received 9 June 2021
Revised 8 July 2021
A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquina-
zole scaffold was synthesised and biologically evaluated for their inhibitory activity against human car-
bonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar
potency and excellent selectivity for the tumour-related hCA IX isoform. Structure–activity relationship
data attained for various substitutions were rationalised by molecular modelling studies in terms of both
inhibitory activity and selectivity.
Accepted 13 July 2021
KEYWORDS
Carbonic anhydrases
inhibitors; secondary
sulphonamides; tetrahydro-
quinazole derivatives;
structure–activity relation-
ships; tumour-related CA
IX isoform
Introduction
atom11. However, the high sequence homology between hCA iso-
forms, combined with their widespread and ubiquitous tissue
localisation, makes the discovery of CAIs with functional selectivity
for a specific target isoform a major challenge in the development
of potential drugs, free from off-target side effects12.
Carbonic anhydrases (CAs) are ubiquitous zinc enzymes that cata-
lyse the reversible hydration of carbon dioxide to bicarbonate by
a metal hydroxide nucleophilic mechanism1. CAs play key roles in
several physiological processes, such as pH and CO2 homeostasis,
respiration, secretion of electrolytes in a variety of tissues/organs,
biosynthetic reactions, including lipogenesis, gluconeogenesis,
In the last decades, several tricyclic/bicyclic scaffolds, decorated
with a primary sulphonamide moiety, have been exploited by our
research group, leading to the identification of several derivatives
with high CA inhibitory activity and promising selectivity
profiles13–15. In particular, tricyclic primary benzenesulfonamides
featuring the benzothiopyrano[4,3-c]pyrazole and pyridothiopyr-
ano[4,3-c]pyrazole systems (see general formula I, Chart 1) were
initially developed as geometrically constrained analogues of cele-
coxib (CLX, Chart 1) and valdecoxib (VLX, Chart 1), cyclooxyge-
ureagenesis, calcification, bone resorption, and tumorigenicity2–5
.
To date, 15 different isoforms of human CAs (hCAs) have been
identified with different catalytic activities, cellular and subcellular
localisation, and tissue distributions6: CA I, CA II, CA III, CA VII, and
CA XIII isoforms are cytosolic, CA IV, CA IX, CA XII, CA XIV, and CA
XV are transmembrane bound, CA VA and CA VB are mitochon-
drial and CA VI is secreted in saliva and milk7,8. Isoforms VIII, X,
and XI are cytosolic, acatalytic, and preponderantly expressed
within the brain, but their functions remain elusive.
nase 2 (COX-2) specific inhibitors that act also as potent CAIs6,13
.
Based on the encouraging results obtained and the SAR outlined,
our project on CAIs was pursued, always within primary benzene-
sulfonamide-based small molecules, following two different
approaches: (i) investigation of three different, but structurally
related, tricyclic scaffolds, that is benzo/pyrido-thiopyranopyrimi-
dine II and pyrazolodihydroquinazoline III (Chart 1)14,15; (ii) struc-
tural simplification of the core scaffolds of I and II to yield bicyclic
Many hCA isoenzymes are considered promising therapeutic
targets in the development of potentially valuable new small mol-
ecules for the treatment or prevention of many diseases such as
glaucoma, epilepsy, obesity, and cancer9,10. Indeed, CA inhibitors
(CAIs) have been in clinical use for many decades as diuretics and
systemically acting antiglaucoma drugs; many CAIs are primary
sulphonamides (acetazolamide (AAZ, Chart 1), ethoxzolamide,
dichlorphenamide) coordinating the zinc ion present in the cata-
tetrahydroindazole IV and tetrahydroquinazole
V derivatives,
respectively, in which the phenyl- or pyrido-fused moiety was
lytic site through their deprotonated sulphonamide nitrogen removed (Chart 1)15.
CONTACT Silvia Salerno
sandro.cosconati@unicampania.it
silvia.salerno@unipi.it
Department of Pharmacy, University of Pisa, Via Bonanno Pisano, 6, Pisa 56126, Italy; Sandro Cosconati
DiSTABiF, University of Campania Luigi Vanvitelli, Caserta, Italy
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1875
More recently, the tetrahydroindazole scaffold of class IV was General procedure for the synthesis of 4-((5-oxo-5,6,7,8-
further exploited by the development of a library of N-phenyl-sec-
ondary sulphonamides, variously decorated at para- or meta-pos-
ition with groups characterised by different electron-donor/
acceptor capabilities (VI, Chart 1)16. Indeed, although secondary
and tertiary sulphonamides have been recently reported as effi-
cient and selective inhibitors of the cancer-related hCA IX and XII
isoforms,17–25 the chemical space of such CAIs remains so far
rarely explored and their binding mode is still a subject of
investigation26.
tetrahydroquinazolin-2-yl)amino)benzenesulfonamide
derivatives 16a–c
A mixture of the proper 2-(dimethylamino)methylene-1,3-dione
derivative 14a–c (0.45 mmol), N-(4-aminosulfonyl)phenylguanidine
carbonate 15 (0.25 g, 0.90 mmol) and NaOH (54 mg, 1.35 mmol) in
5 ml of n-BuOH was irradiated at a T ¼ 160 ꢁC (pre-stirring time ¼
3 min) for 40 min. After cooling, the obtained crude solid com-
pounds 16a–c were purified by recrystallisation from ethanol.
4-((5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)benzenesulfo-
namide (16a). Yield 66%; m.p. 294–296 ꢁC15.
For what concerns class VI, although, in general, the insertion
of a secondary sulphonamide function on the tetrahydroindazole
scaffold produced inhibitors with a moderate micromolar potency,
lower than the primary sulphonamide parent compounds of series
IV, some interesting structure–activity relationship (SAR) data were
acquired and rationalised through theoretical studies16.
Specifically, the inhibitory profile of the novel indazolyl-N-arylben-
zenesulfonamides VI appears strictly dependent on the substitu-
tion pattern at 6-position, with the 6-phenyl group able to
produce hCA II inhibitors with good potency and selectivity16.
Pursuing our interest in the development of secondary sul-
phonamide CAIs, in the present study, we turned out our atten-
tion to the bicyclic tetrahydroquinazole scaffold of class V. Thus,
derivatives 1–12 (Chart 1) were synthesised and biologically eval-
uated for their enzyme inhibitory activity against four physiologic-
ally relevant CA isoforms, the hCA I, II, IV, and IX. In the novel
compounds, the secondary benzenesulfonamide moiety was deco-
rated at para position with groups of various nature (R3¼H, OCH3,
NO2, CH3) to modulate the acidity of the sulphonamide itself,
keeping in mind the groups conferring the best results, in terms
of potency and selectivity, in series VI; in addition, the same sub-
stituents of the corresponding primary sulphonamides V described
by us15 were maintained at 7-position (R1¼R2¼H; R1¼H,
R2¼C6H5; R1¼R2¼CH3).
4-((5-oxo-7-phenyl-5,6,7,8-tetrahydroquinazolin-2-yl)amino)ben-
zenesulfonamide (16b). Yield 70%; m.p. 214–215 ꢁC15.
4-((5-oxo-7,7-dimethyl-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-
benzenesulfonamide (16c). Yield 72%; m.p. 258–260 ꢁC15.
General procedure for the synthesis of N-aryl-4-((5-oxo-5,6,7,8-
tetrahydroquinazolin-2-yl)amino)benzenesulfonamide
derivatives 1–12
To a solution of the proper 4-(5-oxo-7-substituted-5,6,7,8-tetrahy-
droquinazolin-2-yl)amino)benzenesulfonamide 16a–c (0.94 mmol)
in dry MeCN (10 ml) at room temperature, CuI (8 mg, 0.04 mmol),
anhydrous K2CO3 (0.28 g, 2.07 mmol), the proper aryl iodide
(1.13 mmol), and DMEDA (0.04 ml, 0.38 mmol) were added, and
the resulting mixture was heated at 100 ꢁC for 8–24 h (TLC ana-
lysis). After cooling, the reaction mixture was diluted with water
(20 ml), acidified with 2.0 N HCl to pH ¼ 4, and extracted with
EtOAc (3 ꢀ 20 ml). The organic phase was dried (MgSO4), filtered
and evaporated under reduced pressure. The crude material was
purified by flash chromatography using PE/EtOAc as eluting sys-
tem, to afford compounds 1–12.
Materials and methods
4-((5-Oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-phenylbenze-
nesulfonamide (1)
Chemistry
The uncorrected melting points were determined using a Reichert
Compound 1 was obtained as a solid (0.093 g, 25%) starting from
compound 16a (0.30 g) and iodobenzene (0.13 ml); m.p.
281–283 ꢁC; 1H NMR (400 MHz, DMSO-d6): d 2.04–2.07 (m, 2H),
2.57 (t, J¼ 6.4 Hz, 2H), 2.93 (t, J¼ 6.0 Hz, 2H), 6.99 (t, J¼ 7.2 Hz,
1H), 7.09 (d, J¼ 7.6 Hz, 2H), 7.19–7.23 (m, 2H), 7.70 (d, J¼ 8.8 Hz,
2H), 7.94 (d, J¼ 8.8 Hz, 2H), 8.84 (s, 1H), 10.19 (s, 1H), 10.67 (s, 1H).
13C NMR (100 MHz, DMSO-d6): d 21.0, 31.8, 38.0, 119.3 (2C), 119.5
(2C), 120.2, 124.4, 128.2 (2C), 129.6 (2C), 132.7, 138.1, 143.8, 158.2,
160.4, 174.2, 195.8.
€
Kofler hot-stage apparatus. NMR spectra were obtained on a
Bruker AVANCE 400 (1H, 400 MHz, 13C, 100 MHz) in DMSO-d6.
Chemical shifts are expressed in d (ppm) and coupling constants
(J) in Hertz. Magnesium sulphate was used as the drying agent.
Evaporations were made in vacuo (rotating evaporator). Analytical
TLC have been carried out on Merck 0.2 mm precoated silica gel
aluminium sheets (60 F-254). Silica gel 60 (230–400 mesh) was
used for column chromatography. Microwave (MW) assisted reac-
tions were carried out in BIOTAGE Initiator 2.5 microwave appar-
atus. The purity of the target inhibitors 1–12 was determined,
using a Shimadzu LC-20AD SP liquid chromatograph equipped
with a DDA Detector (Kyoto, Japan) at 220 nm (column C18
(250 mm ꢀ 4.6 mm, 5 mm, Shim-pack)). The mobile phase, deliv-
ered at isocratic flow, consisted of methanol (80%) and water
(20%) and a flow rate of 1.0 ml/min. All the compounds showed
percent purity values of >95%. Reagents, starting materials, and
solvents were purchased from commercial suppliers and used as
received. The following intermediates were obtained according to
previously described procedures: 2-[(dimethylamino)methylene]cy-
clohexan-1,3-dione 14a27, 2-[(dimethylamino)methylene]-5-phenyl-
cyclohexan-1,3-dione 14b27, 2-[(dimethylamino)methylene]-5,5-
dimethylcyclohexan-1,3-dione 14c27, and N-(4-aminosulfonyl)phe-
nylguanidine carbonate 1514.
N-(4-methoxyphenyl)-4-((5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)a-
mino)benzenesulfonamide (2)
Compound 2 was obtained as a solid (0.080 g, 20%) starting from
compound 16a (0.30 g) and 1-iodo-4-methoxybenzene (0.26 g);
m.p. 233–235 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.05–2.08 (m,
2H), 2.58 (t, J¼ 6.2 Hz, 2H), 2.94 (t, J¼ 6.2 Hz, 2H), 3.66 (s, 3H), 6.79
(d, J¼ 9.2 Hz, 2H), 6.98 (d, J¼ 9.2 Hz, 2H), 7.62 (d, J¼ 8.8 Hz, 2H),
7.93 (d, J¼ 9.2 Hz, 2H), 8.85 (s, 1H), 9.81 (s, 1H), 10.67 (s, 1H). 13C
NMR (100 MHz, DMSO-d6): d 20.8, 31.6, 37.8, 55.3, 114.4 (2C),
115.1, 119.0, 119.1 (2C), 123.3 (2C), 126.1, 127.9 (2C), 130.5, 132.6,
143.4, 156.5, 157.8, 160.5, 195.3.

1876
E. BAGLINI ET AL.
H2NO
2S
H2NO
2S
O
N
N
N
NH2
SO2
N
N
S
H
N
O
F
3C
VLX
CLX
AAZ
H2NO
H2NO
2S
2S
R
X
R
N
HN
N
X
N
S
N
S
X = CH, N
X = CH, N
I
II
H2NO
2S
H2NO
2S
H2NO
2S
N
R
R1
R2
R1
R2
N
N
HN
N
N
N
N
N
O
O
III
V
IV
HNO
2S
HNO
2S
R3
R3
R1
R2
R1
R2
HN
N
N
N
N
O
O
1-12
VI
Chart 1. Structures of celecoxib (CLX), valdecoxib (VLX), acetazolamide (AAZ); structures of previously described (I-VI) and newly synthesized (1–12) CAIs.]
N-(4-nitrophenyl)-4-((5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)ami-
no)benzenesulfonamide (3)
4-((5-Oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-(4-tolyl)ben-
zenesulfonamide (4)
Compound 3 was obtained as a solid (0.10 g, 25%) starting from Compound 4 was obtained as a solid (0.077 g, 20%) starting
compound 16a (0.30 g) and 1-iodo-4-nitrobenzene (0.28 g); m.p. from compound 16a (0.30 g) and 1-iodo-4-methylbenzene
264–266 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.05–2.08 (m, 2H), (0.25 g); m.p. 272–274 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
2.58 (t, J¼ 6.4 Hz, 2H), 2.93 (t, J¼ 6.2 Hz, 2H), 7.30 (d, J¼ 9.2 Hz, 2.05–2.08 (m, 2H), 2.17 (s, 3H), 2.58 (t, J¼ 6.8 Hz, 2H), 2.93 (t,
2H), 7.83 (d, J¼ 8.8 Hz, 2H), 7.99–8.02 (m, 2H), 8.13 (d, J¼ 9.2 Hz, J¼ 6.0 Hz, 2H), 6.96–7.03 (m, 4H), 7.66 (d, J¼ 9.2 Hz, 2H), 7.93 (d,
2H), 8.85 (s, 1H), 10.74 (s, 1H), 11.20 (s, 1H). 13C NMR (100 MHz, J¼ 9.2 Hz, 2H), 8.85 (s, 1H), 10.01 (s, 1H), 10.67 (s, 1H). 13C NMR
DMSO-d6): d 21.0, 31.8, 38.0, 118.2 (2C), 119.4, 119.7 (2C), 125.8 (100 MHz, DMSO-d6): d 20.6, 21.0, 31.8, 38.0, 119.2, 119.5 (2C),
(2C), 128.4 (2C), 131.9, 142.8, 144.4, 144.7, 158.1, 160.4, 174.2, 120.8 (2C), 128.1 (2C), 129.9 (2C), 132.8, 133.7, 135.4, 143.6,
180.3, 195.9.
158.1, 160.4, 174.2, 195.9.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1877
4-((5-Oxo-7-phenyl-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-phe- 4-((7,7-Dimethyl-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-
nylbenzenesulfonamide (5)
(4-methoxyphenyl)benzenesulfonamide (10)
Compound 5 was obtained as a solid (0.13 g, 30%) starting from
compound 16b (0.37 g) and iodobenzene (0.23 ml); m.p.
111–113 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.69–2.73 (m, 2H),
2.97–3.10 (m, 2H), 3.52–3.59 (m, 1H), 6.99 (t, J¼ 7.2 Hz, 1H), 7.09
(d, J¼ 8.0 Hz, 2H), 7.19–7.28 (m, 3H), 7.34–7.40 (m, 4H), 7.69 (d,
J¼ 8.8 Hz, 2H), 7.96 (d, J¼ 8.8 Hz, 2H), 8.90 (s, 1H), 10.19 (s, 1H),
10.75 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 38.3, 44.8, 118.6,
119.3, 119.9 (2C), 124.0, 127.0, 127.1 (2C), 127.9, 128.8 (2C), 129.3
(2C), 132.6, 138.0, 143.3, 143.5, 157.8, 160.5, 172.9, 194.7.
Compound 10 was obtained as a solid (0.15 g, 35%) starting from
compound 16c (0.32 g) and 1-iodo-4-methoxybenzene (0.26 g);
1
m.p. 210–212 ꢁC. H NMR (400 MHz, DMSO-d6): d 1.03 (s, 6H), 2.86
(s, 2H), 3.65 (s, 3H), 6.79 (d, J¼ 8.8 Hz, 2H), 6.98 (d, J¼ 8.8 Hz, 2H),
7.62 (d, J¼ 8.8 Hz, 2H), 7.93 (d, J¼ 8.8 Hz, 2H), 8.84 (s, 1H), 9.80 (s,
1H), 10.67 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 27.8, 32.4, 51.2,
55.2, 114.3 (2C), 119.1 (2C), 123.2 (2C), 127.8 (2C), 129.2, 130.5,
138.1, 143.2, 153.0, 153.8, 160.6, 166.4, 195.1.
N-(4-methoxyphenyl)-4-((5-oxo-7-phenyl-5,6,7,8-tetrahydroquinazo- 4-((7,7-Dimethyl-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-
lin-2-yl)amino)benzenesulfonamide (6)
(4-nitrophenyl)benzenesulfonamide (11)
Compound 6 was obtained as a solid (0.094 g, 20%) starting from
compound 16b (0.37 g) and 1-iodo-4-methoxybenzene (0.26 g);
m.p. 103–105 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.67–2.73 (m,
2H), 2.97–3.01 (m, 2H), 3.53–3.57 (m, 1H), 3.65 (s, 3H), 6.79 (d,
J¼ 9.2 Hz, 2H), 6.97 (d, J ¼ 9.2 Hz, 2H), 7.26–7.28 (m, 1H), 7.34–7.40
(m, 4H), 7.61 (d, J¼ 8.8 Hz, 2H), 7.95 (d, J¼ 8.8 Hz, 2H), 8.91 (s, 1H),
9.82 (s, 1H), 10.75 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 38.4,
44.8, 55.2, 114.3 (2C), 116.8, 118.4, 119.1, 123.2 (2C), 127.0, 127.8
(2C), 128.7, 130.6 (2C), 132.6, 143.2, 156.4, 158.0, 160.5, 194.5.
Compound 11 was obtained as a solid (0.13 g, 30%) starting from
compound 16c (0.32 g) and 1-iodo-4-nitrobenzene (0.28 g); m.p.
264–266 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 1.04 (s, 6H), 2.87 (s,
2H), 7.32 (d, J¼ 8.8 Hz, 2H), 7.84 (d, J¼ 8.8 Hz, 2H), 8.02 (d,
J¼ 9.2 Hz, 2H), 8.14 (d, J¼ 9.2 Hz, 2H), 8.85 (s, 1H), 10.74 (s, 1H),
11.19 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 27.7, 32.3, 44.8, 51.0,
117.7 (2C), 117.9, 119.3 (2C), 125.3 (2C), 127.9 (2C), 131.6, 142.3,
144.0, 144.4, 157.2, 160.5, 172.3, 195.0.
N-(4-nitrophenyl)-4-((5-oxo-7-phenyl-5,6,7,8-tetrahydroquinazolin-
2-yl)amino)benzenesulfonamide (7)
4-((7,7-Dimethyl-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-
(p-tolyl)benzenesulfonamide (12)
Compound 7 was obtained as a solid (0.14 g, 30%) starting from
compound 16b (0.37 g) and 1-iodo-4-nitrobenzene (0.28 g); m.p.
118–120 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.67–2.73 (m, 2H),
2.97–3.04 (m, 2H), 3.54–3.56 (m, 1H), 7.26–7.30 (m, 2H), 7.34–7.40
(m, 3H), 7.82 (d, J¼ 8.8 Hz, 2H), 7.96–8.07 (m, 4H), 8.12 (d,
J¼ 9.2 Hz, 2H), 8.91 (s, 1H), 10.82 (s, 1H), 11.21 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 38.3, 44.9, 104.5, 118.0 (2C), 118.7, 119.6
(2C), 125.2 (2C), 125.6 (2C), 127.0, 127.1 (2C), 128.2, 128.9 (2C),
139.0, 143.3, 144.0, 157.9, 160.6, 173.0, 194.8.
Compound 12 was obtained as a solid (0.10 g, 25%) starting from
compound 16c (0.32 g) and 1-iodo-4-methylbenzene (0.25 g); m.p.
246–248 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 1.03 (s, 6H), 2.17 (s,
3H), 2.86 (s, 2H), 6.96–7.03 (m, 4H), 7.66 (d, J¼ 8.8 Hz, 2H), 7.93 (d,
J¼ 8.8 Hz, 2H), 8.84 (s, 1H), 10.01 (s, 1H), 10.67 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 20.4, 27.9, 32.4, 45.9, 51.2, 119.0, 119.2
(2C), 120.4 (2C), 126.1, 127.8 (2C), 129.6 (2C), 130.3, 143.4, 143.8,
144.4, 157.2, 157.4, 195.1.
CA inhibition assays
4-((5-Oxo-7-phenyl-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-(4-
tolyl)benzenesulfonamide (8)
An Applied Photophysics stopped-flow instrument was used for
assaying the CA catalysed CO2 hydration activity28. Phenol red (at
a concentration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as
buffer, 0.1 M Na2SO4 (for maintaining constant ionic strength), fol-
lowing the CA-catalysed CO2 hydration reaction for a period of
10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and inhibition con-
stants. For each inhibitor at least six traces of the initial 5–10% of
the reaction have been used for determining the initial velocity.
The uncatalysed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of inhibi-
tors (10 mM) were prepared in distilled-deionised water with 10%
of DMSO and dilutions up to 0.01 mM were done thereafter with
the assay buffer. The inhibitor and enzyme solutions were preincu-
bated together for 15 min (standard assay at room temperature)
prior to assay, to allow for the formation of the E-I complex. The
inhibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng–Prusoff equation and rep-
resent the mean from at least three different determinations.
Enzyme concentrations in the assay system were in the range
of 5–12 nM.
Compound 8 was obtained as a solid (91 mg, 20%) starting from
compound 16b (0.37 g) and 1-iodo-4-methylbenzene (0.25 g); m.p.
83–85 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 2.10 (s, 3H), 2.67–2.73
(m, 2H), 2.97–3.04 (m, 2H), 3.54–3.56 (m, 1H), 7.26–7.30 (m, 2H),
7.34–7.40 (m, 3H), 7.82 (d, J¼ 9.2 Hz, 2H), 7.96–8.07 (m, 4H), 8.12
(d, J¼ 9.2 Hz, 2H), 8.91 (s, 1H), 10.82 (s, 1H), 11.21 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 20.9, 38.5, 41.7, 45.0, 118.1, 119.4 (2C),
123.1 (2C), 126.4 (2C), 127.2, 127.3 (2C), 129.0 (2C), 130.7 (2C),
132.0, 138.2, 139.8, 142.7, 143.5, 158.1, 161.0, 173.1, 195.1.
4-((7,7-Dimethyl-5-oxo-5,6,7,8-tetrahydroquinazolin-2-yl)amino)-N-
phenylbenzenesulfonamide (9)
Compound 9 was obtained as a solid (0.12 g, 30%) starting from
compound 16c (0.32 g) and iodobenzene (0.23 ml); m.p.
215–217 ꢁC. 1H NMR (400 MHz, DMSO-d6): d 1.03 (s, 6H), 2.46 (s,
2H), 2.86 (s, 2H), 7.00 (t, J¼ 7.2 Hz, 1H), 7.09 (d, J¼ 8.8 Hz, 2H),
7.21–7.24 (m, 2H), 7.70 (d, J¼ 8.8 Hz, 2H), 7.95 (d, J¼ 8.8 Hz, 2H),
8.84 (s, 1H), 10.17 (s, 1H), 10.68 (s, 1H). 13C NMR (100 MHz, DMSO-
d6): d 27.9, 32.4, 44.9, 51.2, 118.0, 119.2 (2C), 120.0, 124.0 (2C),
126.1, 127.9 (2C), 129.2 (2C), 129.8, 132.5, 143.5, 157.4,
160.7, 195.2.

1878
E. BAGLINI ET AL.
Molecular modelling methods
substituted phenyliodide in acetonitrile (MeCN) in the presence of
K2CO3, N,N0-dimethylethylendiamine (DMEDA), and CuI. The reac-
tion mixture was heated at 100 ꢁC and stirred for 8–24 h (TLC ana-
lysis); after cooling, the suspension obtained was filtered under
reduced pressure and the solid was purified by flash chromatog-
raphy, yielding the pure target derivatives 1–12.
Docking calculations were executed using the latest AutoDockGPU
docking software29 along with its GUI AutoDockTools (ADT)30. The
high-resolution X-ray crystal structure of hCA IX having (PDB
5FL4), hCA I (PDB 6EVR), hCA II (PDB 3K34), and hCA IV (PDB
5JN9)31–33 was downloaded and superimposed on the structure of
hCA IX. Before docking, the co-crystal ligands of 5FL4, 6EVR, 3K34,
and 5JN9 were removed. Later, the protein structures were pre-
pared using the Protein Preparation Wizard of the Maestro
suite34,35. The Protein Preparation Wizard deletes water molecules,
adds both bond orders and hydrogen atoms, and also produces
the appropriate protonation states. Compound 3, (S)-5, and (R)-5
were constructed using the 2D sketcher tool of Maestro. The
ligands were considered in all the possible flip conformations and
appropriate protonation states were obtained. The ligand and pro-
tein files were then translated into the AD4 specific format
(PDBQT) utilising the scripts prepare_ligand4.py and prepare_re-
ceptor4.py, respectively, with all the standard settings. Using
AutoDock4Zn forcefield protocol, the script zinc_pseudo.py was
applied to add the tetrahedral pseudo atoms to the receptor
PDBQT file36. The grid boxes were centred on the active site of
the protein. While creating the grid parameter files (GPF), the
zinc-specific non-bonded pairwise potentials were also included.
The dimensions of the grid boxes were defined with a set of grids
of 60 Åꢀ40 Åꢀ50 Å along with the spacing of 0.375 Å. A total of
100 independent docking simulations were achieved for every
possible conformation of compounds 3, (S)-5, and (R)-5. Each
docking calculation comprised 20 million energy evaluations utilis-
ing the Lamarckian genetic algorithm local search (GALS) method.
All the dockings were performed with a population size of 250
and 300 runs of Solis and Wets local search with a probability of
0.6. A rate of mutation of 0.02 and a crossover rate of 0.8 were
used to produce new docking attempts for following generations,
and the best individual from each generation was propagated
over the following generation. The docking modes of every 100
independent docking calculations of the ligand binding to the
metalloenzyme were clustered based on a 2 Å cut-off value based
on Cartesian coordinates of the atom and were also ranked based
on free energy binding (DGAD4). Compounds 3, (S)-5, and (R)-5
best conformations were selected based upon the predicted
DGAD4 as well the cluster population.
CA inhibition assays and structure–activity relationships
All the newly synthesised compounds 1–12 were investigated for
their enzyme inhibitory ability against four physiologically relevant
CA isoforms, namely the human hCA I, II, IV, and IX (Table 1), by a
stopped-flow CO2 hydrase assay using acetazolamide (AAZ,
5-acetamido-1,3,4-thiadiazole-2-sulphonamide, Chart 1) as the
standard drug28. Table 1 also reports the data referring to the par-
ent primary sulphonamides 16a–c15 for comparison purposes.
In particular, the inhibitory profile of the tetrahydroquinazolin-
2-yl-amino-benzenesulfonamides 1–12 appears strictly dependent
on the substitution pattern at 7-position, with few exceptions.
Unsubstituted compounds (1–4) exhibit high potency against hCA
IX (Ki values ranging from 0.42 lM to 1.04 lM), combined with an
excellent selectivity with respect to the other isoforms tested. This
trend appears to be independent of the decoration of the pen-
dant phenyl ring (R3).
The inhibitory potency of 7-phenyl derivatives (5–8) appears to
be closely related to the decoration on the benzenesulfonamide
moiety (R3). hCA IX inhibition potency was maintained high when
position 1 is unsubstituted (hCA IX, 5 Ki 0.45 vs. 1 Ki 0.42), and
slightly decreased when R3¼OCH3 or NO2 (hCA IX, 6 Ki 1.73 and 7
Ki 1.33 vs. 2 Ki 0.46 and 3 Ki 0.42, respectively). The hCA IX-select-
ivity profile varied from excellent to good moving from R3¼H to
R3¼NO2 and R3¼OCH3. Specifically, the best performing com-
pound of the subseries in terms of hCA IX inhibitory potency and
selectivity is the unsubstituted (R3¼H) compound 5; the p-nitro-
phenyl-substituted analogue 7 (R3¼NO2) results in a quite potent
(Ki 1.33 mM) and selective (Ki ratio CAI/CAIX >75.2, CAII/CAIX 50.7,
CAIV/CAIX >75.2) hCA IX inhibitor; the p-methoxy-substituted 6
(R3¼OCH3), retains the hCA IX inhibitory potency of the same
order of magnitude of 7 (Ki 1.73 mM), but the selectivity towards
this isoform is considerably reduced (Ki ratio CAI/CAIX >19.6, CAII/
CAIX 6.6, CAIV/CAIX >57.8). The presence of a methyl group at R3
produced a marked reduction in hCA IX inhibitory potency (hCA
IX: 8 Ki 15.7 vs. 4 Ki 1.04), and a loss of selectivity, being 8 equally
potent on hCA II and hCA IX.
Results and discussion
Insertion of a double methyl substitution at position 7 pro-
duced a dramatic drop in hCA IX inhibition potency and a general
Chemistry
The key intermediates for the synthesis of all the new target com- gain in activity towards the other hCAs tested, particularly for hCA
pounds 1–12 were the bicyclic tetrahydroquinazoline derivatives II. The 7,7-dimethyl-quinazolinyl-N-arylbenzenesulfonamides 9–12
16a–c already described by us15 belonging to class V (Chart 1). inhibited all the CAs tested with micromolar potency (with few
However, the synthetic procedure to obtain compounds 16a–c exceptions), showing a moderate selectivity for the hCA II isoform
has been here improved by exploiting MW irradiation as a valu- (Ki values from 5.1 to 22.4 mM). Only compound 12 (R3¼CH3) did
able alternative to conventional heating, allowing to reach higher not register a gain in selectivity for the hCA II isoform showing
yields and shorter reaction times (40 min vs. 16 h). In detail, the comparable Ki values for all the tested hCA isoforms.
commercially available 5-substituted-1,3-cyclohexandiones 13a–c
All in all, these data highlighted the identification of several
were reacted with an excess of N,N-dimethylformamide-dimethyl hCA IX inhibitors with high potency and selectivity, featuring a
acetal (DMF-DMA) at 100 ꢁC for 1 h, to furnish the intermediates secondary benzenesulfonamide on the tetrahydroquinazoline scaf-
14a–c. Then, MW-assisted condensation reaction between the fold. The best-performing compounds are represented by 3
proper bielectrophyle 2-[(dimethylamino)methylene]cyclohexan- (R1¼R2¼H, R3¼NO2) and 5 (R1¼H, R2¼C6H5, R3¼H), that are the
1,3-dione 14a–c15 and the binucleophile guanidine 1514 was per- most potent (3 Ki 0.42 mM; 5 Ki 0.45 mM) and selective (Ki ratio
formed to obtain crude compounds 16a–c which were purified by CAI/CAIX, CAII/CAIX, CAIV/CAIX >238.1 and >222.2 for 3 and 5,
recrystallisation from ethanol (Scheme 1).
respectively) hCA IX inhibitors of this new series of derivatives.
It should be outlined that, comparing the most interesting new
The quinazolyl-N-arylbenzenesulfonamides 1–12 were then
obtained by reacting compounds 16a–c with the proper p- compounds 1–5 with the parent primary tetrahydroquinazole

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1879
Scheme 1. Reagents and conditions: (i) DMF-DMA, 100 ꢁC, 1 h; (ii) conc HCl/50% cyanamide water solution, 100 ꢁC, 0.5 h; (iii) n-butanol, NaOH, M.W. (40 min, 160
ꢁC); (iv) MeCN, K2CO3, DMEDA, CuI, 100 ꢁC, 8–24 h.
sulphonamides 16a–c, despite the lower hCA IX inhibitory Molecular modelling studies
potency of the secondary 1–5, their hCA IX selectivity is greater
To rationalise the SAR data attained for various substitutions of
than that observed for primary sulphonamides 16a–c. This greater
newly designed secondary sulphonamides (Table 1), a series of
selectivity is especially evident with respect to isoform II with Ki
molecular modelling studies were performed on compounds 3
ratios CAII/CAIX ranging from 7.6 to 9.45 for 16a–c and from 85.8
and 5, as they are the most potent inhibitors of hCA IX as well as
the most selective over the other tested hCAs (I, II, and IV) of the
whole set. In this process, the AutoDock4Zn docking protocol,
to >238 for 1–5.
Furthermore, a comparison of the obtained results for 1–4
with those of the corresponding tetrahydroindazole-based sec-
devised for docking experiments on zinc-chelating ligands, was
ondary sulphonamides of series VI (VIa–d in Table 2) recently
employed as implemented in our previous reports on hCAIs15,37
.
described by us16, highlighted that, by keeping the same R3-
substituents (R3¼H, p-OCH3, p-NO2, p-CH3) and changing the
scaffold from tetrahydroindazole (VIa–d) to tetrahydroquinazole
(1–4), the good potency (Ki 1.4–5.7 mM) and selectivity towards
the hCA I isoform shifted to high potency (Ki 0.42–1.04 mM)
and excellent selectivity towards the hCA IX isoform. In particu-
lar, by comparing the p-nitro-substituted derivatives 3 and VIc
(R1¼R2¼H, R3¼NO2) which are the best performing compounds
in the two subseries, a Ki value of 5.7 mM for VIc against hCA I
with slight selectivity (Ki ratio CAII/CAI 8.8, CAIV/CAI 13.3, CAIX/
CAI 14.7), and a Ki value of 0.42 mM against the hCA IX isoform
with a high selectivity (Ki ratio CAI/CAIX, CAII/CAIX, CAIV/CAIX
>238.1) for 3 are highlighted.
The best docking solution, i.e. the one having the lowest pre-
dicted binding free energy, achieved for compound 3 in the hCA
IX (PDB 5FL4)31 binding site is shown in Figure 1.
Mainly, the sulphonamide chelates the catalytic zinc ion
through its negatively charged nitrogen atom in a geometry that
is consistent with the other CAIs. Additionally, one of the sul-
phonamide oxygen atoms is well-positioned to accept an H-bond
from the backbone NH of Thr201. Apart from these interactions,
which were consistent with our previous reports on hCAs15,37, it is
also interesting to see that the phenyl ring, attached to the sul-
phonamide negative nitrogen, forms a p–p interaction with His98
and places its p-NO2 group in a hydrophilic protein region at H-

1880
E. BAGLINI ET AL.
Table 1. Inhibition of hCA isoforms I, II, IV, and IX with tetrahydroquinazoline derivatives 1–12, 16a–c, and AAZ as the reference standard by a stopped-flow CO2
hydrase assay.
Ki (mM)a
Compound
R1
R2
R3
hCA I [CA I/CA IX]b
hCA II [CA II/CA IX]b
hCA IV [CA IV/CA IX]b
hCA IX
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
H
H
CH3
H
H
H
H
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
H
H
OCH3
NO2
CH3
H
OCH3
NO2
CH3
H
OCH3
NO2
CH3
–
89.1 [185.6]
>100 [>217]
>100 [>238]
>100 [>96]
>100 [>222]
34.0 [19.6]
>100 [>75.2]
46.1
41.2 [85.8]
50.3 [109.3]
>100 [> 238]
>100 [> 96]
>100 [> 222]
11.5 [6.6]
67.5 [50.7]
13.0
>100 [>208]
>100 [>217]
>100 [>238]
>100 [>96]
>100 [>222]
>100 [>57.8]
>100 [>75.2]
>100
0.48
0.46
0.42
1.04
0.45
1.73
1.33
15.7
47.3
48.1
>100
19.3
0.00055
0.00079
0.00089
0.026
9
70.5
24.7
62.0
84.9
7.4
5.1
6.9
22.4
91.1
78.1
>100
62.1
10
11
12
16ac
16bc
16cc
AAZ
0.0639 [116]
>100 [869]
>100 [712]
0.25
0.0052 [9.45]
0.0060 [7.6]
0.0082 [9.2]
0.012
0.073 [132.7]
0.078 [98.7]
0.3194 [359]
0.074
C6H5
CH3
–
–
aMean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
bKi ratio for the indicated enzyme isoforms.
cData from ref.15
Table 2. Inhibition of hCA isoforms I, II, IV, and IX with tetrahydroquinazoline (1–4) tetrahydroindazole (VIa–d)16 and secondary sulphonamides.
Ki (mM)a
Compound
R3
hCA I
hCA II
hCA IV
hCA IX
1
2
3
4
H
89.1 [CA I/CA IX]b¼185.6
41.2 [CA II/CA IX]b¼85.8
50.3 [CA II/CA IX]b¼109.3
>100 [CA II/CA IX]b>238
>100 [CA II/CA IX]b>96
31.0 [CA II/CA I]b¼5.4
5.9 [CA II/CA I]b¼4.2
50.3 [CA II/CA I]b¼8.8
8.4 [CA II/CA I]b¼1.71
0.012
>100 [CA IV/CA IX]b>208
>100 [CA IV/CA IX]b>217
>100 [CA IV/CA IX]b>238
>100 [CA IV/CA IX]b>96
59.6 [CA IV/CA I]b¼10.4
79.3 [CA IV/CA I]b¼56.6
75.8 [CA IV/CA I]b¼13.3
52.5 [CA IV/CA I]b¼10.7
0.074
0.48
0.46
0.42
1.04
OCH3
NO2
CH3
H
OCH3
NO2
CH3
>100 [CA I/CA IX]b>217
>100 [CA I/CA IX]b>238
>100 [CA I/CA IX]b>96
VIac
VIbc
VIcc
VIdc
AAZ
5.7
1.4
5.7
4.9
0.25
97.9 [CA IX/CA I]b¼17.2
56.5 [CA IX/CA I]b¼40.3
83.7 [CA IX/CA I]b¼14.7
39.2 [CA IX/CA I]b¼8.0
0.026
aMean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
bKi ratio for the indicated enzyme isoforms.
cData from ref.16
bonding distance to multiple residues such as Asn66, His68, and and Leu199 while the attached pyrimidine ring of the tetrahydro-
Gln71. These latter interactions might explain why compound 4, quinazoline scaffold forms an H-bond with Gln92 residue. This lat-
featuring the lipophilic p-CH3 group, by establishing less favour- ter interaction might explain why the tetrahydroquinazolines
able interactions with the enzyme counterpart, is less active than described in the present work are generally more potent than the
compounds 1–3. Interestingly, the same trend was recorded for previously reported tetrahydroindazoles16. Moreover, the oxygen
the phenyl-substituted analogues 5–8. The benzene ring of the atom enclosed in tetrahydroquinazoline is pointing towards the
benzenesulfonamide forms van der Waals interactions with Val121 “selectivity hotspot” of the hCA IX binding site.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1881
Dockings of both isomers of 5 allow postulating the absence of a IV, no steric clashes were evidenced, while unfavourable electro-
stereoselective recognition by hCAIX as, in both cases, the predicted static interactions between the hydrophilic “selectivity hotspot” of
hCA IV and the ligands could be detected (Figures 3(C) and 4(D)).
This should explain while compounds 3 and 5 are unable to effi-
ciently inhibit hCA I, hCA II, and hCA IV.
lowest energy conformation places the negatively charged sulphona-
mide nitrogen to chelate the Zn2þ (Figure 2) with an overall orienta-
tion resembling the one achieved for 3.
In particular, the (S)-5 docking suggests that the negative nitro-
gen connected to the secondary sulphonamide is chelating the zinc
ion, while a sulphonamide oxygen is forming an H-bond with the
backbone NH of Thr201. If compared to the binding pose achieved
for 3, the presence of the phenyl ring at position 7 of the tetrahy-
droquinazoline ring seems to induce a partial relocation of the lig-
and inducing the loss of the H-bond interaction with Gln92, thereby
explaining why compounds 5–8 are less active than their unsubsti-
tuted counterparts 1–4. In (S)-5, this loss seems to be counterbal-
anced by the presence of several van der Waals contacts between
the phenyl ring and Leu91, Gln92, and Val130 enzyme residues in
the “selectivity hotspot”. (R)-5 docking calculations indicate the
presence of a similar interaction pattern (Figure 2(B)).
To explain the selectivity profiles of 3, (S)-5, and (R)-5, docking
calculations of these compounds were also attempted employing
the published structures of hCA I (PDB 6EVR), hCA II (PDB 3K34),
and hCA IV (PDB 5JN9)32,33. Interestingly, analysis of the obtained
results revealed a certain difficulty in predicting a binding pose
featuring the chelation of the Zn2þ ion. To explain these theoret-
ical results, the binding pose obtained for 3, (S)-5, and (R)-5 into
the hCA IX structure and conducive of the Zn2þ chelation, was
rigidly translated into the aligned binding sites of hCA I, hCA II,
and hCA IV. From this analysis, it is clear that such a ligand orien-
tation is unfeasible into the hCA I, hCA II as it would give rise to
multiple steric clashes (Figures 3(A,B) and 4(A,B)). Concerning hCA
Figure 3. 3/hCA IX theoretical binding pose translated into hCA I (PDB 6EVR, A),
hCA II (PDB 3K34, B), and hCA IV (PDB 5JN9, C) structures. The proteins are
shown in blue, sea-green, and coral ribbons and sticks, respectively, with their
molecular surface shaded in white. The ligand is depicted in pink.
Figure 1. 3/hCA IX theoretical complex (PDB 5FL4) computed by docking calcula-
tions. The protein is shaded in salmon and all the critical residues are labelled.
The ligand is shown in pink colour while the H-bonds are shown in black
dashed lines.
Figure 2. (S)-5/hCA IX (A) and (R)-5/hCA IX theoretical complexes (PDB 5FL4) computed by docking calculations. The protein is shaded in salmon and all the critical
residues are labelled. The ligand is shown in yellow and cyan, respectively, while the H-bonds are shown in black dashed lines.

1882
E. BAGLINI ET AL.
inhibitory properties, may lead to relevant medicinal chemistry
discoveries. Moreover, very recently Kennedy et al.38 investigated
inflammasome and pyroptotic pathways and identified a novel
photoaffinity alkyne-tagged probe for such processes, compound
belonging to the secondary sulphonamide class, which is also tar-
geting efficiently CA II, confirming thus our earlier data16,25,39 that
secondary sulphonamides effectively inhibit these enzymes, by a
mechanism which starts to be elucidated through this and the
above-mentioned works.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by University of Campania Luigi
Vanvitelli under Grant VALERE: Vanvitelli per la Ricerca, ANIMA
and VALEREPlus projects, Campania and Regional Government
Technology Platform Lotta alle Patologie Oncologiche under Grant
iCURE, University of Pisa under Grant PRA_2020_58 and Italian
MIUR under Grant PRIN 2017, 2017XYBP2R.
ORCID
Emma Baglini
http://orcid.org/0000-0002-8378-4052
Rahul Ravichandran
Emanuela Berrino
http://orcid.org/0000-0002-1908-6161
http://orcid.org/0000-0002-4258-0678
Silvia Salerno
Elisabetta Barresi
http://orcid.org/0000-0002-6072-4698
http://orcid.org/0000-0002-9814-7195
Anna Maria Marini
Monica Viviano
Federico Da Settimo
Claudiu T. Supuran
Sandro Cosconati
http://orcid.org/0000-0001-8664-375X
http://orcid.org/0000-0003-1118-790X
http://orcid.org/0000-0002-7897-7917
http://orcid.org/0000-0003-4262-0323
http://orcid.org/0000-0002-8900-0968
Sabrina Taliani
http://orcid.org/0000-0001-8675-939X
Figure 4. (S)-5 and (R)-5/hCA IX theoretical binding pose translated into hCA I
(PDB 6EVR, A), hCA II (PDB 3K34, B), and hCA IV (PDB 5JN9, C) structures. The
proteins are shown in blue, sea-green, and coral ribbons and sticks, respectively,
with their molecular surface shaded in white. (S)-5 and (R)-5 are depicted in yel-
low and cyan sticks. All the pictures were rendered using UCSF chimera molecu-
lar visualisation software40.
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Within our project aimed at investigating the chemical space of
secondary sulphonamide CAIs, we herein reported the design, syn-
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that has attracted significant interest as a putative cancer thera-
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