Design, synthesis and biological activity of selective hCAs inhibitors based on 2-(benzylsulfinyl)benzoic acid scaffold

Article abstract of DOI:10.1080/14756366.2019.1651315

A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives.

Full text of DOI:10.1080/14756366.2019.1651315

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological activity
of selective hCAs inhibitors based on 2-
(benzylsulfinyl)benzoic acid scaffold
Giulia Rotondi, Paolo Guglielmi, Simone Carradori, Daniela Secci, Celeste
De Monte, Barbara De Filippis, Cristina Maccallini, Rosa Amoroso, Roberto
Cirilli, Atilla Akdemir, Andrea Angeli & Claudiu T. Supuran
To cite this article: Giulia Rotondi, Paolo Guglielmi, Simone Carradori, Daniela Secci, Celeste
De Monte, Barbara De Filippis, Cristina Maccallini, Rosa Amoroso, Roberto Cirilli, Atilla Akdemir,
Andrea Angeli & Claudiu T. Supuran (2019) Design, synthesis and biological activity of selective
hCAs inhibitors based on 2-(benzylsulfinyl)benzoic acid scaffold, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1400-1413, DOI: 10.1080/14756366.2019.1651315
To link to this article: https://doi.org/10.1080/14756366.2019.1651315
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1400–1413
https://doi.org/10.1080/14756366.2019.1651315
RESEARCH PAPER
Design, synthesis and biological activity of selective hCAs inhibitors based on
2-(benzylsulfinyl)benzoic acid scaffold
Giulia Rotondia, Paolo Guglielmia, Simone Carradorib , Daniela Seccia, Celeste De Montea, Barbara De Filippisb,
Cristina Maccallinib, Rosa Amorosob, Roberto Cirillic, Atilla Akdemird, Andrea Angelie
and Claudiu T. Supurane
b
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy; Department of Pharmacy, “G. D’Annunzio”,
c
ꢀ
University of Chieti-Pescara, Chieti, Italy; Centro Nazionale per il Controllo e la Valutazione dei Farmaci, Istituto Superiore di Sanita, Rome,
d
Italy; Computer-aided Drug Discovery Laboratory, Faculty of Pharmacy, Department of Pharmacology, Bezmialem Vakif University, Fatih,
Istanbul, Turkey; Neurofarba Department, Section of Pharmaceutical and Nutraceutical Sciences, Universita degli Studi di Firenze, Sesto
e
ꢀ
Fiorentino (Florence), Italy
ABSTRACT
ARTICLE HISTORY
Received 5 July 2019
Revised 26 July 2019
Accepted 26 July 2019
A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and
tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and
XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the dis-
covery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust struc-
ture-activity relationships within this versatile scaffold. HPLC separation of some selected chiral
compounds and biological evaluation of the corresponding enantiomers was performed along with
molecular modelling studies on the most active derivatives.
KEYWORDS
Carbonic anhydrase
inhibitor; sulfoxide
enantioseparation; carbox-
ylic acid; molecular
modelling
1. Introduction
The design of more molecules and scaffolds, and the obtaining
of co-crystals of some of them with hCA enzymes (mostly with
hCA II), prompted the discovery of four different mechanisms
of inhibition for hCAs18,19. The most recently reported one was
observed for the first time with 2(benzylsulfinyl)benzoic acid
(Figure 1) co-crystallized with hCA II20. This compound showed an
atypical mechanism involving the occupancy of a pocket next to
the entrance of the active site, where it established some interac-
tions holding the His64 residue in the “out” conformation. This
amino acid, which exists in two conformations called “in” and
“out”, acts as a proton-shuttle system transferring a proton from
the zinc-coordinated water molecule to the environment, to
reconstitute the catalytic active form. Interfering with this process
(the rate-determining step of the entire catalytic cycle) is equal to
block or strongly reduce the enzymatic activity, obtaining the
enzyme inhibition. Our lead compound was inactive against hCA I
and XII (Ki hCA I/XII >10 mM), while showed inhibitory activity in
the low micromolar range towards both hCA II and IX, with a
slight preference for the former (Ki hCA II ¼ 0.15 mM, Ki hCA IX ¼
1.29 mM). With this in mind, we developed a novel series of deriva-
tives based on the lead compound scaffold, pursuing some
changes on its structure in order to improve activity and selectiv-
ity (Figure 1).
Carbonic anhydrases (CAs, EC 4.2.1.1) are important metalloen-
zymes involved in the hydration of carbon dioxide, a quite simple
reaction whose balance is important in many cellular and physio-
logical processes, spanning from pH homoeostasis and respiration
to biosynthetic pathways including lipogenesis, glucogenesis, and
ureagenesis1–3
.
Human CAs (hCAs) exist in fifteen isoforms, which possess dif-
ferent characteristics as catalytic activity, tissues distribution, cellu-
lar localisation (cytosol, mitochondria and cell membrane) and
sensibility to inhibitors. The researches of the last years showed all
the consequences of their altered activity, either in the case of
excessive or deficient ones4,5. In this regard, investigations on syn-
thetic and natural compounds have been done with the aim to
discover new selective and potent inhibitors and/or activators,
which could restore the normal functions of these enzymes6–13
.
One of the most studied alterations has been observed in
tumours, where some hCA isoforms appear overexpressed; the so-
called tumour-related isoforms, hCA IX and XII which are trans-
membrane enzymes, participate along with cytosolic hCA II in the
complex pH machinery which controls the milieu of hypoxic
tumours, promoting drug resistance as well as proliferation, migra-
tion, invasion and metastasis of tumour cells14–17. To reverse this
condition, efforts have been made to develop inhibitors that act
effectively and selectively towards these targets.
The carboxylic acid moiety (red in Figure 1) was replaced with
methyl ester, amide, N-methyl amide, hydroxamic acid and ketone.
The selected functionalities possess different hydrogen-bond
CONTACT Paolo Guglielmi
Rome, 00185, Italy; Claudiu T. Supuran
paolo.guglielmi@uniroma1.it
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5,
ꢀ
claudiu.supuran@unifi.it
Neurofarba Department, Section of Pharmaceutical and Nutraceutical Sciences, Universita
degli Studi di Firenze, Via U. Schiff 6, Sesto Fiorentino (Florence), 50019, Italy
Supplemental data for this article can be accessed here.
ß 2019 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
1401
Figure 1. Changes performed on the 2-(benzylsulfinyl)benzoic acid scaffold.
acceptor/donator profiles if compared with the carboxylic acid oxidative reaction with meta-chloroperbenzoic acid (mCPBA).
one; furthermore, except for amide and hydroxamic acid moieties Although many routes able to afford sulphur oxidation exist23, we
that still conserve a “deprotonatable” group, the others lack this
feature whose importance has been in this way deepened.
have deliberately chosen this approach to obtain the two oxida-
tion products, the sulfoxide and the sulphone, in the same
reaction.
The benzyl moiety (blue in Figure 1) was challenged through
the insertion of substituents or by its entire substitution with ben-
zoylmethyl and phenylacetic moieties as well as unsaturated
C3–C5 alkyl chains. The aim of these changes was to understand
the importance of the hydrophobic interactions with Phe231 and
Asn232 that the phenyl ring established in the hCA II-inhibitor
adduct. The addition of substituents on this phenyl ring changed
the p-system charge distribution along with its hydro/lipophilicity.
On the contrary, moving the phenyl group away from the sulphur
atom with the addition of a carbonyl group (in the benzoylmethyl
or phenylacetic derivatives) shed light on the importance of this
system in that position and its conformational freedom. Attempts
with unsaturated alkyl chains with increasing lengths have been
done in order to evaluate if the presence of groups, able to give
hydrophobic interactions, could work similarly to the phenyl ring.
The role of sulphur atom was also challenged (yellow, green and
violet in Figure 1), evaluating how both its oxidation state and its
bioisosteric replacement affected the inhibitory activity. In the first
case, we performed sulphide oxidation in order to obtain sul-
phinyl (sulfoxides) and sulphonyl (sulfones) derivatives. This syn-
thesis was performed without chiral auxiliaries, obtaining
sulfoxides in a mixture of enantiomers (racemate) that were
resolved using a chiral HPLC system21,22 and individually tested;
this permitted the evaluation of how chirality could affect inhibi-
tory activity of these derivatives. On the contrary, the bioisosteric
replacement of sulphur atom with oxygen and nitrogen ones or
with methylene group has been done, unravelling the importance
of that atom for the inhibitory activity (violet in Figure 1).
In fact, controlling the amount of oxidant added during the
reaction we obtained both the species that were easily separated
owing to the different chromatographic profiles. Compounds
7–12 and 19–21 were obtained with a nucleophilic substitution
reaction between methyl 2-mercaptobenzoate and (un)substituted
benzyl bromides, followed by the oxidative step. The carboxylic
acid derivatives were obtained through the hydrolysis of com-
pound 7 with aqueous 2 N sodium hydroxide (NaOH) in a mixture
of equal amounts of water and 1,4-dioxane (50/50, v/v) to give 4,
that was subsequently oxidised to
5 and 6 (Scheme 1).
Compounds 13–15 were synthesised performing the first step of
nucleophilic substitution between methyl 2-mercaptobenzoate
and benzoyl bromide at room temperature, with successive oxida-
tion in the same conditions previously seen (Scheme 2). The syn-
thetic pathway of derivatives 22–38 is reported in Scheme 3.
Methyl 2mercaptobenzoate was reacted in the presence of bro-
moalkanes of increasing length, in DMF at reflux in the presence
of K2CO3 to obtain intermediates E2–E5 (E1, methyl 2-(methyl-
thio)benzoate, was commercially available). Only two of the E
intermediates bearing butyl and pentyl chain respectively (E4 and
E5), underwent the oxidative step obtaining the sulphinyl deriva-
tives 22–23. All the E1–E5 intermediates were hydrolysed with
aqueous 2 N sodium hydroxide in a mixture of equal amounts of
water and 1,4-dioxane (50/50, v/v). After synthesis completion, the
reactions were quenched with hydrochloric acid (HCl), giving the
intermediates A1–A5 (Scheme 3). The activation of the carboxylic
acid group for the amide synthesis has been obtained using the
mixed anhydride approach. In this regard, we used ethyl chloro-
formate in tetrahydrofuran (THF) under nitrogen atmosphere (N2)
in the presence of triethylamine (Et3N) excess. Monitoring the
reaction with thin layer chromatography (TLC) we detected the
disappearance of the acidic intermediate, until the completion
of anhydride formation. Then, ammonium chloride (NH4Cl) was
added. The presence of the triethylamine excess involved the “de-
2. Chemistry and HPLC enantioseparation
In order to obtain the novel compounds, we followed the syn-
thetic strategies reported in Schemes 1–4. For the synthesis of
derivatives 1–3 and 16–18 we started from 1–(2-bromophenyl)e-
than-1-one and 2-nitrobenzamide, respectively (Scheme 1); both
of them (individually) underwent an aromatic nucleophilic substi- blocking” of NH3 from NH4Cl, giving the final products (24, 27,
tution with benzyl mercaptan. The reactions were performed in 30, 33, 36).
N,N’dimethylformamide (DMF) at reflux, in the presence of K2CO3.
Although chiral properties are often linked with the presence
The so obtained compounds (1 and 16) were treated in the next of a quaternary carbon atom binding different substituents, even

1402
G. ROTONDI ET AL.
Scheme 1. Synthesis and structures of compounds 1–12 and 16–21.
Scheme 2. Synthesis and structures of compounds 13–15.
the sulphur atom can have chiral behaviour under appropriate
conditions.
In compounds 39–53, the sulphur atom characterising mole-
cules 1–38 was replaced by an isosteric oxygen (39, 44, 47, 52),
The sulfoxides have a pyramidal structure where one vertex nitrogen atom (40, 42, 43, 45, 48, 50, 51, 53), or alkyl chain (46,
contains the sulphur electron pair, which can be considered as 41, 49). Compounds 39–41 and 43 (anthranilic acid) were com-
the fourth substituent bound to the sulphur atom. Therefore, if mercially available and used as purchased.
the two groups bound on the sulphinyl moiety are diverse, there
Compound 42 was obtained starting from anthranilic acid,
is a chiral centre and the presence of two enantiomers. Differing which was treated with sodium hydride (NaH) in dry THF. After
from tertiary amines, which had low pyramidal inversion energy 10 min, the phenylacetyl bromide was added at room temperature
barrier, the sulfoxides possess energy barrier that allows the exist- under nitrogen, giving the desired amide (Scheme 4). Compounds
ence of two stable enantiomers, which can be resolved. Some 44–46 were synthesised starting from the corresponding carbox-
sulfoxides (5, 8, 11, 17, 20) obtained in of the synthetic pathway ylic acids 39–41, which were treated with sulphonyl chloride
shown in Schemes 1–4, were selected and mg-quantities of their (SOCl2) in boiling dry methanol to give the corresponding methyl
pure enantiomeric forms easily isolated by semipreparative enan- esters (Scheme 4)25.
tioselective HPLC on the 250 mm ꢀ 10 mm i.d. Chiralpak IC column
The reaction of the ester 45 with hydroxylamine hydrochloride
in methanol at room temperature for 48 h gave the N-hydroxy
using pure ethanol as a mobile phase24.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1403
Scheme 3. Synthesis and structures of compounds 22–38.
Scheme 4. Synthesis of the amide 42, esters 44–46, hydroxyamide 51 and amides 47–49.
benzamide 5126. Compounds 47–49 were obtained converting ammonium hydroxide (NH4OH), to afford the desired amides
the carboxylic acids 39–41 to the corresponding acyl chlorides by (Scheme 4)27. The amide 50 was obtained by a different route
means of oxalyl chloride in the presence of a catalytic amount of (Scheme 5). At first, it was synthesised the amide from the anthra-
dry DMF. Acyl chlorides were subsequently treated with nilic acid activated by SOCl2 in DMF at room temperature as

1404
G. ROTONDI ET AL.
Scheme 5. Synthesis of amide 50 and ketones 52–53.
previously reported. The addiction of NaH and phenylacetyl chlor- nitrogen (N2) as both the sheath gas and the auxiliary gas. All the
ide in dry THF at room temperature let to the final amide 50.
Finally, ketones 52 and 53 were easily obtained through the
reaction of 2-hydroxy or 2-aminoacetophenone, respectively, with
benzylbromide in the presence of K2CO3 in dry acetone or aceto-
nitrile, respectively (Scheme 5).
characterisation data for each compound were reported as
Supplemental material.
3.2. Enzyme inhibition assays
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalysed CO2 hydration activity28. Phenol red
(0.2 mM) has been used as indicator, working at the absorbance
maximum of 557 nm, with 20 mM Hepes (pH 7.5, for a-CAs) as buf-
fer and 20 mM NaClO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalysed CO2 hydra-
tion 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 constants. In particular, CO2 was
bubbled in distilled deionised water for 30 min till saturation. A
CO2 kit (Sigma, Milan, Italy) was used to measure the concentra-
tion in serially diluted solutions from the saturated one at the
same temperature. For each inhibitor at least six traces of the ini-
tial 5–10% of the reaction have been used for determining the ini-
tial velocity. The uncatalyzed rates were determined in the same
manner and subtracted from the total observed rates. Stock solu-
tions of inhibitor (1 mM) were prepared in distilled-deionised water
and dilutions up to 0.1 nM were done thereafter with the assay
buffer. Inhibitor and enzyme solutions were preincubated together
for 15 min at room temperature prior to assay to allow for the for-
mation of the E-I complex or for the eventual active site mediated
hydrolysis of the inhibitor. The inhibition constants were obtained
by non-linear least-squares methods using PRISM 3 and the
Cheng-Prusoff equation29, and represent the average from at least
three different determinations. All recombinant CA isoforms were
3. Experimental protocols
3.1. General
Solvents were used as supplied without further purification.
Starting materials and other chemicals were purchased by Sigma-
Aldrich (Milan, Italy) and used in the syntheses and in the bio-
logical assays without further purification. All synthesised com-
pounds have been fully characterised by analytical and spectral
data. Column chromatography was carried out using Sigma-
R
AldrichV silica gel (high purity grade, pore size 60 Å, 200–425
mesh particle size). Analytical thin-layer chromatography was car-
R
ried out on Sigma-AldrichV silica gel on TLC aluminium foils with
fluorescent indicator 254 nm. Visualisation was carried out under
UV irradiation (254 and 365 nm). 1H NMR spectra were recorded
on a Bruker AV400 (1H: 400 MHz, 13 C: 101 MHz). Chemical shifts
are quoted in ppm, based on appearance rather than interpret-
ation, and are referenced to the residual non deuterated solvent
peak. Missing signals must be attributed to overlapping peaks.
Infrared spectra of the most representative compounds were
recorded on a Bruker Tensor 27 FTIR spectrometer equipped with
an attenuated total reflectance attachment with internal
calibration.
Absorption maxima (ꢀmax
(4000–400 cmꢁ1). Elemental analyses for C, H, and
recorded on a Perkin-Elmer 240 B microanalyzer obtaining analyt-
)
are reported in wavenumbers
N
were
obtained in-house as previously reported30,31
.
ical results within
0.4% of the theoretical values for all com-
R
pounds. All melting points were measured on a StuartV melting
point apparatus SMP1 and are uncorrected (temperatures are
reported in ꢂC). Where given, systematic compound names are
3.3. Molecular modelling studies
The three-dimensional structures of all ligands were prepared in
their lowest energy conformation using the MOE software pack-
age (v2019.01, Chemical Computing Group, Inc, Montreal,
Canada). The sulphonamide nitrogen atoms of the ligands were
assigned a negative charge (R-SO2NH-) and the ligands were
R
those generated by ChemBioDraw UltraV 12.0 following IUPAC
conventions. Mass spectra on the most representative compounds
were performed on a LCQ (Thermo Finnigan) ion trap mass spec-
trometer (San Jose, CA, USA) equipped with an electrospray ion-
isation (ESI) source. The capillary temperature was set at 300 ꢂC energy minimised (MMFF94x force field). All protein structures
and the spray voltage at 4.25 kV. The fluid was nebulised using were obtained from the RCSB protein databank: hCA I (pdb: 3lxe,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1405
1.90 Å), hCA II (pdb: 4e3d, 1.60 Å), hCA IX (pdb: 3iai; 2.20 Å) and the methyl group, probably enforcing other interactions inside the
hCA XII (pdb: 1jd0; 1.50 Å). The protein atoms and the active site enzyme. Compound 10 was a good inhibitor of hCA IX exhibiting
zinc ions were retained and all other atoms were omitted. The Ki against hCA IX of 1.0 mM, with a high micromolar residual activ-
remaining structure was protonated using the protonate 3 D func- ity against hCA II (Ki hCA IX ¼ 45.7 mM). The oxidation to sulphinyl
tionality of MOE and subsequently, the obtained structure was
energy-minimised (AMBER14:EHT)32. Finally, the obtained protein
models were superposed on the hCA I structure using the back-
bone Ca-atoms and all Zn2þ-ions, zinc-binding histidines and the
overall backbone atoms superposed well (RMSD value: 1.281 Å).
Docking calculations were performed using the FlexX docking tool
(v2.3.2; BioSolveIT GmbH, St. Augustin, Germany) within MOE. The
binding pocket was defined as all residues within 6.5 Å of the ref-
erence ligand acetazolamide. The sulphonamide tail of the ligands
was forced to adopt a similar orientation and interactions to the
Zn2þ ion as observed for acetazolamide using a pharmacophore
model. All ligands were docked fifty times and the best scoring
three poses were subjected to refinement calculations33. To this
end, the ligand and binding pocket residues were energy mini-
mised and rescored using GBVI/WSA force field34.
group ameliorated selectivity, because the two enantiomers (R/S)-
11 were ineffective against both of hCA I and II (Ki hCA I/II
>100 mM). These two optical isomers showed different affinity
towards hCA IX, with the (R)-enantiomer that inhibited this iso-
form better than the (S)-one ((R)-11, Ki hCA IX ¼ 1.4 mM; (S)-11, Ki
hCA IX ¼ 18.1 mM). The further oxidation to sulphone (12) gave a
detrimental effect, reducing both activity against hCA IX
(Ki¼21.1 mM) and selectivity (Ki hCA II ¼ 40.1 mM). The formal inser-
tion of a carbonyl moiety between the methylene group and the
phenyl ring in order to move the latter away from the sulphur
atom, led to the 13–15 derivatives. The presence of this benzoyl
moiety was not tolerated by this scaffold, giving compounds
which did not exhibit effects towards all the tested isoforms
(Ki>100 mM).
The consequence of the amidic moiety insertion was also eval-
uated (16–18). Compound 16, containing a non-oxidised sulphur
atom, was the best inhibitor of the series against hCA IX display-
ing Ki of 0.046 mM. This result was slightly impaired by the oxida-
tion to sulphinyl derivatives ((R)-17, Ki hCA IX ¼ 2.2 mM; (S)-17, Ki
hCA IX ¼ 1.9 mM), while the corresponding sulphone (18) com-
pletely lost the affinity for hCA IX. All the amidic derivatives
showed affinity for hCA II, albeit the sulphide (16) and sulphone
(18) inhibited this isoform better than the sulphinyl enantiomers,
which exhibited Ki values around 50 mM (16, Ki hCA II ¼ 8.22 mM;
18, Ki hCA II ¼ 2.67 mM). The presence of a methyl group on the
amidic moiety was detrimental for the inhibitory activity towards
hCA IX. Indeed, the derivatives 19–21, endowed with monomethyl
amide, were less effective against the cancer-related isoform
(19.8 < Ki hCA IX (mM) <25.8) than the analogues containing pri-
mary amide. Furthermore, the activity against hCA II was also low-
ered in the high micromolar range (36.9 < Ki hCA II (mM)<86.3).
This behaviour was very similar to that observed for molecules
containing a methyl ester moiety, which were less effective com-
pared with their carboxylic acid analogues.
4. Carbonic anhydrase inhibition studies
All the tested compounds had no affinity for the common off-tar-
get hCA I isoform (Ki >100 mM) and some of them were more
active against the tumour-related isoform hCA IX if compared
with the parent drug 2-(benzylsulfinyl)benzoic acid (Table 1). The
derivatives 1–3 bearing the ketone group and with benzyl moiety
bound to the sulphur atom, exhibited affinity and selectivity
against the tested isoforms depending on the sulphur atom oxida-
tion state. Compound 1 exhibited better activity than the lead
compound against hCA IX (Ki hCA IX ¼ 1.1 mM) and was ineffect-
ive against the two off-targets (Ki hCA I/II >100 mM). The oxidation
to sulfoxide (2) impaired the selectivity “restoring” the inhibitory
activity owned by the lead compound for hCA II, although in the
high micromolar range, while the affinity towards hCA IX (2, Ki
hCA IX ¼ 2.0 mM) was slightly inferior than 1. The sulphone 3 dis-
played affinity exclusively for hCA IX, although weaker than 1 and
2 (3, Ki hCA IX ¼ 16.4 mM).
The effect and the importance of the phenyl moiety were also
challenged by substituting it with linear alkyl chains of increasing
length (22–38) and testing the influence against the two off-tar-
get hCA I and II, and the two tumour-related isoforms hCA IX and
XII. The first attempts were done with the two compounds 22 and
23. These derivatives, bearing methyl ester with the sulfoxide
group linked to butyl and pentyl chain respectively, were inactive
against all the tested isoforms, underlying the detrimental effects
of this structural combination. However, better results were
obtained when the amide moiety was placed instead of methyl
ester one (24–38). The derivatives 24–38 displayed their effect
exclusively against hCA IX, without any inhibitory activity against
hCA I, II and XII. Only for derivative 30, bearing propyl chain
bound to the sulphur atom, we observed activity against hCA II,
but with a very low affinity (Ki hCA II ¼ 90.9 mM). Compound 32,
the sulphone analogue of sulphide 30, was the best inhibitor
against hCA IX among the compounds substituted with alkyl
chains, inhibiting the cancer-related isoform with a Ki value of
2.3 mM. Other good results were found with 33 and 36, containing
a sulphur atom and bearing butyl or pentyl chain, respectively
(33, Ki hCA IX ¼ 2.5 mM; 36, Ki hCA IX ¼ 2.7 mM). Except for deriva-
tives with propyl chain (30–32), the compounds containing the
The replacement of 1 methyl group with the hydroxyl one to
give the 4, the sulphide analogue of the lead compound, had det-
rimental effects reducing affinity for hCA IX (Ki hCA IX ¼ 21.8 mM)
but still conserving the selectivity (Ki hCAI/II >100 mM). The oxida-
tion to sulfoxide ((R/S)-5) and sulphone (6) improved inhibitory
activity in the low micromolar range against the tumour-related
isoform. However, while 6 bearing the sulphonyl moiety was
inactive towards the two off-target actings selectively against hCA
IX (6, Ki hCA I/II > 100 mM; Ki hCA IX ¼ 2.3 mM), the two sulphinyl
enantiomers of 5 exhibited their preference for hCA II ((R)-5, Ki
hCA II ¼ 0.21 mM; (S)-5, Ki hCA II ¼ 0.093 mM) rather than hCA IX
((R)-5, Ki hCA IX ¼ 1.4 mM; (S)-5, Ki hCA IX ¼ 1.9 mM). Comparing
the inhibition data against hCA II of 5 enantiomers, it was quite
clear the preference of the enzyme for (S)-5, which was the euto-
mer. Indeed, after incubation with the racemic mixture of 5, only
the adduct between hCA II and (S)-5 was observed20. Compounds
7–9 were endowed with a methyl ester functional group, keeping
constant the benzyl group bound to the sulphur atom (in the dif-
ferent oxidation states). These derivatives showed reduced inhibi-
tory activity against hCA IX, regardless the sulphur oxidation
number. Furthermore, compounds 7 and (R/S)-8 were also weakly
active against hCA II (65.7 < Ki hCA II (mM)<84.9). The unfavour-
able effect of the additional methyl group was mitigated by the non-oxidised sulphur atom showed better inhibitory activity than
insertion of a chloro atom at the para-position of the phenyl ring the related sulfoxide and sulphone analogues (Table 1). In order
(10–12); this substitution counteracted the detrimental effects of to evaluate the role and the importance of sulphur atom in this

1406
G. ROTONDI ET AL.
Table 1 Inhibitory activity of derivatives 1–53 and the reference drug (acetazolamide, AAZ) against the four selected hCA isoforms by a stopped-flow CO2 hydrase
assay28
.
Ki (lM)a
Compound
Structure
hCA I
h‘a II
>100
h‘a IX
h‘a XII
1
>100
1.1
nt
2
3
4
>100
>100
>100
63.2
>100
>100
2.0
nt
nt
nt
16.4
21.8
(R)-5
(S)-5
>100
>100
0.21
0.093
1.4
1.2
nt
nt
6
7
>100
>100
>100
2.3
nt
nt
75.2
15.0
(R)-8
(S)-8
>100
>100
84.9
65.7
15.3
20.0
nt
nt
9
>100
>100
>100
11.4
1.0
nt
nt
10
45.7
(R)-11
(S)-11
>100
>100
>100
>100
1.4
18.1
nt
nt
12
>100
40.1
21.1
nt
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1407
Table 1 Continued.
Ki (lM)a
Compound
Structure
hCA I
h‘a II
>100
h‘a IX
>100
h‘a XII
13
>100
nt
14
15
16
>100
>100
>10
>100
>100
8.22
>100
>100
0.046
nt
nt
2.66
(R)-17
(S)-17
>100
>100
52.2
51.9
2.2
1.9
nt
nt
18
19
>10
2.67
86.3
>10
0.066
nt
>100
19.8
(R)-20
(S)-20
>100
>100
52.2
38.9
22.8
24.6
nt
nt
21
>100
68.2
25.8
nt
22
23
>100
>100
>100
>100
>100
>100
nt
nt
24
25
>100
>100
>100
>100
6.5
>100
>100
34.5
(continued)

1408
G. ROTONDI ET AL.
Table 1 Continued.
Ki (lM)a
Compound
Structure
hCA I
h‘a II
>100
h‘a IX
h‘a XII
>100
26
>100
14.0
27
28
>100
>100
>100
>100
16.7
40.9
>100
>100
29
>100
>100
22.7
>100
30
31
>100
>100
90.9
>100
>100
>100
>100
38.2
32
>100
>100
2.3
>100
33
34
>100
>100
>100
>100
2.5
>100
>100
36.4
35
>100
>100
13.5
>100
36
37
>100
>100
>100
>100
2.7
>100
>100
27.3
38
39
>100
>100
>100
>100
8.7
>100
>100
>100
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1409
Table 1 Continued.
Ki (lM)a
Compound
Structure
hCA I
h‘a II
>100
h‘a IX
>100
h‘a XII
>100
40
>100
41
42
>100
>100
>100
>100
>100
>100
>100
>100
43
44
>100
>100
>100
>100
>100
>100
>100
>100
45
46
47
48
49
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
O
O
51
>100
>100
>100
>100
(continued)

1410
G. ROTONDI ET AL.
Table 1 Continued.
Ki (lM)a
Compound
Structure
hCA I
h‘a II
>100
h‘a IX
>100
h‘a XII
>100
52
>100
53
>100
>100
>100
>100
AAZ
0.25
0.012
0.25
0.006
aMean from three different determinations (errors in the range of 5–10% of the reported values). nt: not tested.
scaffold, we performed its bioisosteric replacement with methy- inactive against all the tested isoforms. Furthermore, some
lene moiety and oxygen or nitrogen atoms (39–53), keeping con- selected compounds were also tested against hCA XII to prelimin-
stant the functional groups constituting the scaffold seen above ary provide information on this target. All the derivatives, except
(e.g. carboxylic acid, amide, etc.). Some of the obtained com- compounds 16 and 18, were inactive. Sulphone 18 was a potent
pounds showed structures completely comparable to the lead and selective hCA XII inhibitor with Ki 0.066 mM, whereas sulphur-
compound (see for example 39, 41). As one can see from Table 1, based 16 was a medium-potency inhibitor.
all the attempts done changing the sulphur atom were ineffective,
giving compounds unable to inhibit all the tested isoforms.
5. Molecular modelling studies
Furthermore, also the influence of the tested functional groups
like the acidic or amidic one, were useless, enforcing and under-
ling the importance that sulphur atom has for the activity of this
scaffold. In light of the above, some considerations can be done.
Among the functional groups tested (carboxylic acid, methyl ester,
etc.) the best hCA IX inhibitor was provided with amidic moiety,
although ketone and carboxylic acid derivatives also showed good
activity. The insertion of a methyl group on the carboxyl acid or
the amide impaired activity. This could be due to the altered
H-bond donator/acceptor profile, or most likely to the increased
steric hindrance. From all the data evaluated, it was quite clear
that the oxidation of sulphur atom did not influence the activity
or selectivity in a specific manner. Furthermore, in most of the
efforts done, the sulphide derivative was the best inhibitor of hCA
IX among the sulphide/sulfoxide/sulphone analogues endowed
with the same substituent. The only exception was for carboxylic
acid derivatives, whose better activity was exhibited by the sul-
phinyl ones.
The chiral resolution of the single sulfoxide enantiomers did
not give a specific trend, with few enantiomers exhibiting better
activity compared with the related optical isomer. The benzyl moi-
ety was essential for the inhibition and the presence of halogen
on the ring improved activity when compared with the unsubsti-
tuted analogues. The replacement of phenyl ring with linear alkyl
chains impaired the activity, while raising the selectivity. The ben-
zoyl moiety was not tolerated by this scaffold, affording com-
pounds ineffective against all the tested isoforms. These outcomes
could be related to the loss of hydrophobic interactions due to
the phenyl ring shift, clashes with amino acids owing to the ring
new position, or both of them. Nevertheless, the reasons of these
5.1. Docking studies into the active site of hCA IX
The lowest KI value for hCA IX was measured for compound 16
(Ki ¼ 46 nM), which is lower compared to the reference compound
acetazolamide (Ki ¼ 250 nM). Two different binding interactions
with the hCA IX active site were suggested. In the first docked
pose, the ligand directly formed an interaction with the Zn2þ-ion
via its carbonyl group (Figure 2(A)). The amine group of the ligand
formed hydrogen bonds with the side chain hydroxyl groups of
Thr199 and Thr200. One of the ligand’s phenyl group interacted
with the side chain of His64 (arene-H interaction). Changing the
amino group or the phenyl group or replacing the S atom by SO
or SO2 groups made it more difficult for the ligands to adopt this
pose. Several compounds (including compounds 1, 5, 10, 11, 24
and 32) can also adopt poses in which a direct interaction with
the Zn2þ is possible. In the second docked pose, the ligand car-
bonyl group formed a hydrogen bond to the zinc-bound water
molecule (Figure 2(B)). In addition, the carbonyl and amine group
of the ligand formed hydrogen bonds with the side chain
hydroxyl group of Thr200. Hydrophobic interactions were formed
with the side chains of His94 and Val121. The replacement of the
amino group with a hydroxyl group or the replacement of the S
atom by SO or SO2 groups may be tolerated according to this
pose. The later may form hydrogen bonds with the side chain of
Gln92. Furthermore, many compounds can adopt docked poses in
which an interaction with the water molecule is possible.
5.2. Docking studies into the active site of hCA XII
data might be other. In fact, the inserted “spacer” (the carbonyl The lowest Ki value for hCA XII was measured for compound 18
group), has an own steric hindrance along with specific bond (Ki ¼ 66 nM) and the second-lowest Ki value was measured for
angles and a stable dipole; all these features could affect nega- compound 16 (Ki ¼ 2.66 mM). Two docked poses have been
tively the interaction with the isoform IX. Finally, the substitution obtained for these compounds: both formed an interaction with
of the sulphur atom with oxygen, nitrogen one, or methylene the Zn2þ-ion via the ligand carbonyl groups (Figure 3). The amino
underlined the importance of the sulphur atom for the activity of group of compound 18 formed hydrogen bonds with the side
these compounds, because all the obtained derivatives were chains of Thr199 and Thr200, while the amino group of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1411
Figure 2. The docked poses of compound 16 (purple) in the active site of hCA IX forming an interaction with either the active site Zn2þ-ion (panel A) or the zinc-
bound water molecule (panel B). Zn2þ is indicated with a turquoise sphere, the water molecule is indicated with a red sphere, hydrogen bonds and interactions to
the Zn2þ-ion are indicated in red dashed lines and arene-H interactions are indicated in yellow dashed lines.
Figure 3. The docked poses of compound 18 (turquoise, left) and compound 16 (purple, right) in the active site of hCA XII forming interactions with the active site
Zn2þ-ion. Zn2þ is indicated with a turquoise sphere, hydrogen bonds and interactions to the Zn2þ-ion are indicated in red dashed lines.
compound 16 only established a hydrogen bond to the side chain
5.3. Docking studies into the active site of hCA I and hCA II
of Thr199. Both ligands formed hydrophobic interactions with
His94. In addition, compound 18 established a hydrophobic inter-
Although the actives sites of the investigated hCA isozymes are
action with the side chain of Val121. No poses have been very similar to each other, important differences still exist that
obtained for these compounds with an interaction with the zinc- most likely influence the binding interactions of the ligands.
bound water molecule.
In hCA I, His200 is present instead of Thr200 and therefore this

1412
G. ROTONDI ET AL.
hydrogen bonding opportunity (see Figures 2 and 3) was no lon-
ger present and the ligands cannot approach the Zn2þ-ion to
form direct interactions or interactions with a zinc-bound water
molecule. This may be responsible for the low activity of these
compounds against hCA I. Similarly, a relatively large Phe131 is
present in hCA II instead of Ala131 in hCA XII. As such, the docked
pose as observed of compound 18 in the active site of hCA XII
(Figure 3(A)) would not be possible due to a clash with Phe131.
et al. pH regulators to target the tumor immune micro-
environment in human hepatocellular carcinoma.
Oncoimmunology 2018;7:e1445452.
6. (a) Ammazzalorso A, Carradori S, Angeli A, et al. Fibrate-
based N-acylsulphonamides targeting carbonic anhydrases:
synthesis, biochemical evaluation, and docking studies. J
Enzyme Inhib Med Chem 2019;34:1051–61; (b) Bouchouit M,
Bouacida S, Zouchoune B, et al. Synthesis, X-ray structure, in
silico calculation, and carbonic anhydrase inhibitory proper-
ties of benzylimidazole metal complexes. J Enzyme Inhib
Med Chem 2018;33:1150–9.
6. Conclusion
€ €
7. (a) Koyuncu I, Tuluce Y, Slahaddin Qadir H, et al. Evaluation
In conclusion, we have explored the chemical space of the main
functional groups of the 2(benzylsulfinyl)benzoic acid, an innova-
tive and atypical hCA inhibitor. We have designed, synthesised,
and tested 53 derivatives against the most important hCA iso-
forms establishing robust SARs within this scaffold. Most of them
were selective hCA IX inhibitors in the low micromolar/high nano-
molar range. The stereochemistry of the sulfoxide group had no
impact on the biological activity, whereas the isosteric replace-
ment of the sulphur atom with oxygen, nitrogen or methylene led
to a total loss of activity. Molecular modelling studies demon-
strated different binding poses of the inhibitors within the active
site, differently from that observed for the parent compound.
of the anticancer potential of a sulphonamide carbonic
anhydrase IX inhibitor on cervical cancer cells. J Enzyme
Inhib Med Chem 2019;34:703–11; (b) Ramya PVS, Angapelly
S, Angeli A, et al. Discovery of curcumin inspired sulfona-
mide derivatives as a new class of carbonic anhydrase iso-
forms I, II, IX, and XII inhibitors. J Enzyme Inhib Med Chem
2017;32:1274–81.
8. (a) Mocan A, Carradori S, Locatelli M, et al. Bioactive isofla-
vones from Pueraria lobata root and starch: Different extrac-
tion techniques and carbonic anhydrase inhibition. Food
Chem Toxicol 2018;112:441–7; (b) Nescatelli R, Carradori S,
Marini F, et al. Geographical characterization by MAE-HPLC
and NIR methodologies and carbonic anhydrase inhibition
of Saffron components. Food Chem 2017;221:855–63.
Disclosure statement
9. Mollica A, Macedonio G, Stefanucci A, et al. Five- and six-
membered nitrogen-containing compounds as selective
Carbonic Anhydrase activators. Molecules 2017;22:2178–90.
10. (a) D’Ascenzio M, Guglielmi P, Carradori S, et al. Open sac-
charin-based secondary sulfonamides as potent and select-
ive inhibitors of cancer-related carbonic anhydrase IX and
XII isoforms. J Enzyme Inhib Med Chem 2017;32:51–9; (b)
The authors declare no conflict of interest.
ORCID
Simone Carradori
Andrea Angeli
Claudiu T. Supuran
http://orcid.org/0000-0002-8698-9440
http://orcid.org/0000-0002-1470-7192
http://orcid.org/0000-0003-4262-0323
ꢁ
Zuvela P, Liu JJ, Yi M, et al. Target-based drug discovery
through inversion of quantitative structure-drug-property
relationships and molecular simulation: CA IX-sulphonamide
complexes. J Enzyme Inhib Med Chem 2018;33:1430–43.
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