Chromene-containing aromatic sulfonamides with carbonic anhydrase inhibitory properties

Article abstract of DOI:10.3390/ijms22105082

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of chromene-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I, hCA II and the transmembrane hCA IX and XII. Several of the investigated derivatives showed interesting inhibition activity towards the tumor associate isoforms hCA IX and hCA XII. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

Full text of DOI:10.3390/ijms22105082

International Journal of
Molecular Sciences
Article
Chromene-Containing Aromatic Sulfonamides with Carbonic
Anhydrase Inhibitory Properties
Andrea Angeli 1,2,
*
, Victor Kartsev 3, Anthi Petrou 4 , Mariana Pinteala 2 , Volodymyr Brovarets 5,
Sergii Slyvchuk 5, Stepan Pilyo 5, Athina Geronikaki 4,
*
and Claudiu T. Supuran 1
1
Neuro Farba Department, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze,
Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; claudiu.supuran@unifi.it
2
Centre of Advanced Research in Bionanoconjugates and Biopolymers, Petru Poni Institute of Macromolecular
Chemistry, Aleea Grigore Ghica-Voda, No. 41A, 700487 Iasi, Romania; pinteala@icmpp.ro
InterBioScreen, Chernogolovka 142432, Moscow Region, Russia; vkartsev@ibscreen.chg.ru
Department of Pharmacy, School of Health, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
anthi.petrou.thessaloniki1@gmail.com
Department of Chemistry of Bioactive Nitrogen-Containing Heterocyclic Bases, V.P. Kukhar Institute of
Bioorganic Chemistry and Petrochemistry, NAS of Ukraine 1, Murmanska St, 02094 Kyiv, Ukraine;
brovarets@bpci.kiev.ua (V.B.); s.slivchyk@mail.enamine.net (S.S.); stepanpilyo@ukr.net (S.P.)
Correspondence: andrea.angeli@unifi.it (A.A.); geronik@oharm.auth.gr (A.G.)
3
4
5
*
Abstract: Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO2 hydration in
all living organisms, being actively involved in the regulation of a plethora of patho/physiological
conditions. A series of chromene-based sulfonamides were synthesized and tested as possible
CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA
I, hCA II and the transmembrane hCA IX and XII. Several of the investigated derivatives showed
interesting inhibition activity towards the tumor associate isoforms hCA IX and hCA XII. Furthermore,
computational procedures were used to investigate the binding mode of this class of compounds,
within the active site of hCA IX.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Angeli, A.; Kartsev, V.;
Petrou, A.; Pinteala, M.; Brovarets, V.;
Slyvchuk, S.; Pilyo, S.; Geronikaki, A.;
Supuran, C.T. Chromene-Containing
Aromatic Sulfonamides with
Carbonic Anhydrase Inhibitory
Properties. Int. J. Mol. Sci. 2021, 22,
5082. https://doi.org/10.3390/
ijms22105082
Keywords: carbonic anhydrase; inhibitors; metalloenzymes; Chromene
1. Introduction
Academic Editor: Christo Z. Christov
The carbonic anhydrases (CA’s) are metalloenzymes that catalyze the formation of
bicarbonate from carbon dioxide in two steps [1–4]. Sixteen isoforms have been known up
to now in humans, five of which CAs are cytosolic (CA I–III, VII, and XIII), two are mito-
chondrial (CA VA and VB), one is secreted (CA VI), and the others are membrane-bound
Received: 13 April 2021
Accepted: 6 May 2021
Published: 11 May 2021
(CA IV, IX, XII, XIV and XV) [
pharmacological agents useful, for example, as antiglaucoma, anticonvulsant, antiurolithic,
and for the treatment of obesity [ ]. Isoforms CA IX and CA XII were discovered to be
overexpressed and associated with many tumors, where they are involved in processes
related with cancer progression [ ]. Hence, small molecules targeting CA IX/XII in-
5]. Many CA subtypes are interesting targets for the design of
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
6
7–9
hibition may be an attractive strategy against cancer. However, due to high homology
among the human isoforms, selectivity towards one of them is of the highest importance to
prevent off-target related side effects. The classical CA inhibitors (CAIs) are the primary
sulfonamides, which coordinate the zinc ion with their terminal deprotonated nitrogen
atom and have been in clinical use for more than 70 years as diuretics and systemically
acting antiglaucoma drugs [10]. Chromenes have attracted the interest of the scientific
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
community due to their wide range of biological activities, such as antimicrobial [11
anticancer [13 14], anti-inflammatory [15 16], and CA inhibitors [17–21], nematicidal [22],
antiallergic [23] Some sulfonamides also show antimicrobial [24 25], anticancer [26 27],
,12],
,
,
,
,
antidiabetic [28], anti-inflammatory [15
,29], and antitubercular [30] activities. Taking into
Int. J. Mol. Sci. 2021, 22, 5082. https://doi.org/10.3390/ijms22105082
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 5082
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account all of the above-mentioned, here we report the synthesis of (4-sulfamoylphenyl)-
4H-chromene-2-carboxamide derivatives for its incorporation into one scaffold chromene
and sulfonamide moieties. The experimental studies were developed together with in silico
techniques, aimed to propose a reliable binding disposition for this class of CA Is.
2. Results
2.1. Chemistry
All the target compounds have been synthesized using the well-known strategy depicted
in Scheme 1. It implies the synthesis of diverse 4-oxo-4H-chromene-2-carboxylic acids as
well as amides mini array, which contains a streptocid moiety. The synthesis was started
with commercially available 20-hydroxyacetophenones 1a
–j, applying Fernando Cagide’s
optimized synthetic route [31]. The desired carboxylic acids (2a ) were synthesized using
–j
microwave irradiation in the presence of diethyl oxalate and sodium methoxide with very
high yields (93–97%). To reach our final amides, we used two different methodologies that
have a common characteristic—the formation of acyl imidazoliums. These highly reactive acyl
transfer intermediates have been recognized as important species for amide bond formation.
Scheme 1. General synthetic strategy followed to obtain target compounds (5a–j) and (6a–k).

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As a part of the ongoing studies that are focusing on the synthesis of big libraries
for biological screening, we discovered that the combination of N,N,N0,N0-tetramethyl-
chloroformamidinium hexafluoro-phosphate (TCFH) and N-methylimidazole (NMI) is
a mild method for in situ generation of highly reactive acyl imidazoliums, allowing
for the formation of the amides in high yields. This approach is especially efficient
for electron-deficient amines. Because of the low nucleophilicity of 4-aminobenzene-
sulfonamide
or DIPEA, as a base in DMF, are not suitable for amide synthesis. Using the TCFH-NMI
method, we have managed to access anilides 5a in very good yields. In order to expand
3, common coupling agents such as CDI, HATU, EDC, and EDCI with TEA
–j
our screening library and to investigate an impact of aliphatic linker on bioactivity, we
synthesized a set of amides based on 4-(aminomethyl)benzenesulfonamide 4a and 4-(2-
aminoethyl)benzenesulfonamide 4b. In this case, we can use the simple amide coupling
agent CDI, considering the high nucleophilic properties of both amines. In this work, we
synthesized two new 4-oxo-4H-chromene-2-carboxylic acids using modern microwave
methodology, in which a set of amides have been subsequently produced.
2.2. Evaluation of CA Inhibitory Activity
All compounds were evaluated for their inhibitory activity against human CA iso-
forms, hCA I, hCA II, hCA IX, and hCA XII, and results are presented in Table 1. The
result of biological evaluation revealed that all compounds showed activity against all
isoforms, here tested, with different ranges of inhibition constants. The Ki values of com-
pounds against hCA I ranged from 213.7 to 5314 nM. The best activity was achieved
for compounds 6i (Ki = 213.6 nM) and 6e (Ki = 246.7 nM), higher than that of acetazo-
lamide (AAZ) used as reference drug. On the other hand, the lowest activity was observed
for compound 5b with Ki = 5314 nM followed by compound 6j (Ki = 4412 nM). The
structure activity relationships (SAR) study revealed that, in general, derivatives of 4-
oxo-N-(4-sulfamoylphenethyl)chroman-2-carboxamide are more potent hCA I inhibitors
than 4-oxo-N-(4-sulfamoylphenyl)chroman-2-carboxamide and derivatives 4-oxo-N-(4-
sulfamoylbenzyl)chroman-2-carboxamide. The presence of methyl groups in positions 7
and 8 of 4H-chromen-4-one moiety of -sulfamoylphenethyl derivatives is beneficial for
hCA inhibitory activity, while the presence of 7-Me group at 4H-chromen-4-one moiety in
case of sulfamoylphenyl derivatives was detrimental. As far as cytosolic hCA II isoform
is concerned, the compounds showed, in general, better activity than against hCA I. Two
compounds, 5f and 6f, exhibited excellent activity on isoform hCA II, demonstrating Ki
values of 9.3 and 7.5 nM, higher than the reference drug AAZ (Ki = 12.1 nM). Compound
6d with Ki value of 16.6 nM was comparable with the reference drug. The SAR revealed
that the presence of a methyl group in position 7 of 4H-chromene-4-one, as well as CH2CH2
group between 4-oxo-4H-chromene-2–carboxamide and benzensulfonamide (6f), is benefi-
cial for CA II inhibitory activity. Removal of a methyl group from position 7 of compound
6f led to compound 6d being twice less active than 6f. On the other hand, the addition of
one more methyl group in position 8 of the previous compound and removal of CH2CH2
group resulted in compound 5f, with decreased activity compared to 6f, but still more
active than AAZ.
Removal of both methyl groups from compound 5f led to compound 5a (Ki = 35.1 nM),
which is almost four times less active than compound 5f, while removal of Me group from
position 8 of compound 5f, decreased more the activity (Ki = 39.5 nM). The presence
of 6-Cl, 7-Me substitution on 4H-chromen-4-one moiety of sulfamoylphenyl derivatives
was found to be very negative for hCA II inhibitory activity. In the case of hCA IX,
compounds showed activity with Ki ranging from 16.6 to 3285 nM. Three compounds,
4-oxo-N-(4-sulfamoylphenyl)chroman-2-carboxamide 5a (Ki = 16.6 nM); 7,8-dimethyl–4-
oxo-N-(4-sulfamoylphenyl)chroman-2-carboxamide 5f (Ki = 19.5 nM); and 5,8-dimethyl-4-
oxo-N-(4-sulfamoylphenyl)chroman-2-carboxamide 5h (Ki = 22.5 nM) displayed excellent
activity superior compared to AAZ (Ki = 25.7 nM).

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Table 1. Inhibition data of human CA isoforms hCA I, II, IX, and XII with compounds 1–14 and the standard sulfonamide
inhibitor acetazolamide (AAZ).
Ki (nM) *
hCA II
Ki (nM) *
hCA II
N
5a
hCA I
hCA IX
16.6 ± 1.5
77 ± 6.5
hCA XII
20.1 ± 1.7
64.8 ± 6.1
50.8 ± 4.3
619.1 ± 36.7
76.3 ± 6.0
46.4 ± 4.4
26.8 ± 1.4
424.7 ± 41.0
155.3 ± 10.1
34.0 ± 2.2
5.7 ± 0.4
N
6b
6c
hCA I
hCA IX
2039 ± 189.0
3285 ± 302
81.0 ± 7.7
72.7 ± 4.3
64.8 ± 3.4
182.6 ± 16.1
51.4 ± 4.5
84.5 ± 4.3
91.1 ± 8.9
1917 ± 146
25.7 ± 2.1
hCA XII
320.4 ± 23.2
59.4 ± 5.0
211.6 ± 13.7
439.3 ± 32.2
337.3 ± 27.4
147.9 ± 8.1
22.6 ± 1.5
525.2 ± 44.4
5314 ± 272
3563 ± 228
2891 ± 161
341.1 ± 29.6
681.5 ± 40.6
504.8 ± 27.0
2698 ± 265
580.3 ± 50.5
627.7 ± 35.5
250 ± 13.0
35.1 ± 3.3
95.9 ± 8.0
39.5 ± 3.2
311.4 ± 28.3
9.3 ± 0.5
61.4 ± 4.4
46.6 ± 2.9
66.2 ± 5.8
900.7 ± 75.4
219.3 ± 11.8
12.1 ± 0.6
612.3 ± 49.5
2646 ± 227
321.9 ± 27.9
246.7 ± 24.3
333.5 ± 17.5
971.0 ± 82.4
547.3 ± 39.2
213.6 ± 15.9
4412 ± 347
926.9 ± 68.9
250 ± 13.0
81.7 ± 4.2
678.8 ± 45.0
16.6 ± 1.2
36.4 ± 2.1
7.5 ± 0.5
279.9 ± 15.5
51.2 ± 3.6
39.2 ± 3.7
143.0 ± 12.8
91.6 ± 7.0
12.1 ± 0.6
5b
5c
63.4 ± 6.3
298.6 ± 18.4
19.5 ± 1.6
54.7 ± 5.0
22.5 ± 1.9
51.0 ± 4.8
515.5 ± 37.2
855.2 ± 49.2
25.7 ± 2.1
6d
6e
5e
5f
6f
5g
5h
5i
6g
6h
6i
132.2 ± 11.0
31.4 ± 2.6
41.4 ± 3.4
5j
6j
6a
6k
AAZ
AAZ
5.7 ± 0.4
* Mean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
The structure–activity relationships revealed that unsubstituted 4-oxo-N-(4-
sulfamoylphenyl)chroman-2-carboxamide (5a) was beneficial for hCA IX inhibition. The
introduction of two methyl group at position 7 and 8 of the previous compound revealed
a decrease in activity, leading to compound 5f still being more active than AAZ. Shifting
the 7-Me group to position 6 (5h) decreased the activity even more, but the compound
remained still more active than AAZ. Introduction to unsubstituted compound (5a) chlo-
rine at position 6 resulted in three times lesser active compound (5i). The presence of 6-Cl
substituent at 4H-chromen-4-one moiety and methyl group between 4-oxo-4H-chromene-2-
carboxamideand benzensulfonamide (6e) appeared to be detrimental for hCA IX inhibitory
activity. For hCA XII, the inhibition constant ranged from 20.1 to 619.1 nM. The best
activity against this isoform was exhibited by compounds 5a, 6h, and 5h with Ki val-
ues of 20.1, 22.6, and 26.8 nM, respectively, compared to AAZ (Ki = 5.7 nM). None of
compounds did not exert the activity of AAZ against hCA XII. Nevertheless, the unsub-
stituted 4-oxo-N-(4-sulfamoylphenyl)chroman-2-carboxamide was positive for hCA XII
inhibitory activity, while the substitution by a 7-Me group of 4H-chromen-4-one moiety of
-sulfamoylphenethyl derivatives is unfavorable for this kind of activity. Finally, it should
be mentioned that compound 5a was the most active one hCA IX with a selectivity index
(SI) of 31.67 compared to hCA I, while compound 6f was the most active hCA II with SI
44.47 compared to hCA I and 44.97 to hCA XII.
2.3. Molecular Modeling Studies
In order to explain the inhibition mechanism of the tested compounds, molecular
docking studies were performed. For the in-silico experiments, ligands 5a, 5h, 6h, 6f, 6d,
and 6i were selected as representatives of the whole set. Docking studies showed that
all tested compounds bind in a deprotonated form, as anions (negative nitrogen of the
sulfonamide group), chelating the Zn(II) ion of the active site of the enzymes [32]. It is
known that all the active isoforms of human CAs have a similar active site architecture,
consisting of three conserved His residues (His94, His96 and His119) acting as zinc ligands
and another two conserved residues Thr199 and Glu105, acting as “gate keepers” [33–36].
Nevertheless, these isoforms differ in the amino acids mostly in the middle and to the exit
of the active site cavity.
According to docking studies, these differences in the active site of the enzymes are
the reason for the selectivity of the compounds against each isoform. The N-C=O linker
allow compounds to adopt several conformations and interactions within the enzyme
active site. These conformations, depending on the nature of the amino acids of the active

Int. J. Mol. Sci. 2021, 22, 5082
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site cavity, can affect the inhibition profile of the ligands. For instance, compound 5a
which has a Ki for hCA II of 35.1 nM and a lower Ki for hCA IX of 16.6 nM, adopts a
much different conformation upon binding both hCAs. This is probably due to bulky
hydrophobic residue Phe131 in hCA II enzyme, unlike that of the smaller residue Val131 in
hCA IX, which allows ligands to freely enter the active site in a conformation that favors
interactions with residues in the hydrophobic pocket and increases selectivity (Figure 1).
The superposition of the two structures of hCAs bound to compound 5a, revealed that this
residue (Phe131) could indeed cause steric hindrance (Figure 1C). Therefore, 5a adopts a
different conformation within the active site of hCA II enzyme with less interactions and
therefore less stability of the complex and probably explains the experimental lower Ki for
hCA II. It is north worthy to mention that the sulfonamide is involved in H-bond formation
with the backbone of Thr199 of both isoforms and in an additional H-bond formation with
the backbone of His94, which further stabilizes the complex ligand hCA IX and contributes
to the high inhibition potency of the compound (Figure 1A,B and Table 2).
,
On the other hand, compounds 6h, 6f, 6d, and 6i differ from compounds 7 and 11
mainly by the presence of an ethylene linker between the benzimidazole scaffold and the
side phenyl ring. This ethyl linker provides flexibility to the compounds and maximizes the
favorable interactions in hCA IX and hCA XII, increasing the inhibition potency (compound
6h) but, on the other hand, it also allows a better fit into the hCA I and hCA II isoform
structures, affecting the ligand selectivity profile (compounds 6f, 6d, and 6i). In the case of
compound 6f, the flexible ethyl linker leads the compound to adopt a fold conformation
inside the larger active site cavity of hCA IX, unlike to the extended conformation of
the hCA II isoform structure (Figure 2C). In both structures, negative nitrogen of the
sulphonamide group chelates the Zn (II) ion and the interaction forms a H-bond with the
side chain of conserved residue Thr199, but the extended conformation of the compound
into the hCA II isoform structure allows the formation of hydrophobic interactions between
the benzene ring of the compound and the residues Phe131 and Ile91 (Figure 2A,B and
Table 2). These interactions further stabilize the complex ligand-enzyme and positively
impacts the selectivity profile of the compound (Ki for hCA II of 7.5 nM and a lower Ki for
hCA IX of 64.8 nM).
Figure 1.
(
A) 2D interaction diagram of compound 7 docking pose interactions with the key amino acids in hCA II, (
B) in
hCA IX, and (
C
) Superposition of compound 7 bound to hCA IX (grey) in comparison to hCA II (blue), with specific residues
labeled. Active site zinc is shown as a blue sphere, red dotted and green arrows indicate H-bond, and yellow spheres
indicate hydrophobic interactions. Brown double-headed arrow indicates the direction of conformational change of the
compound bound to hCA IX- in comparison to hCA II.

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Table 2. Molecular docking free binding energies (kcal/mol) and interactions of tested compounds on hCA I, II, IX, and
XII isoforms.
hCA
Isoform
Estimated Free Binding
Energy (Kcal/mol)
Chelating the
Zn(II) Ion
Residues Involved in
H-Bond Interactions
Residues Involved in
Hydrophobic Interactions
No.
5a
hCA I
hCA II
hCA IX
−6.49
−7.42
−9.25
Yes
Yes
Yes
Thr199
Thr199
Ala121, Leu198
Ile91, Val121, Leu198, Thr200
Val121, Val195, Leu198
His94, Thr199
His94, His96, Thr199,
Thr200
hCA XII
−9.61
Yes
Val121, Leu198
hCA I
hCA II
hCA IX
hCA XII
hCA I
−5.17
−7.11
−8.74
−9.02
−6.77
−9.14
−6.44
−4.81
−6.62
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
-
Ala121, Ala135
Ile91, Leu198, Thr200
Val121, Leu198
Thr199
5h
6d
His94, Thr199
His94, Thr200
Thr199
Val121, Leu198
Ala121, Ala135, Leu198
Ile91, Val121, Phe131, Thr200
Val121, Leu198, Thr200
Val121
hCA II
hCA IX
hCA XII
hCA I
His94, Thr199
Thr199
-
Thr199
Ala135, Leu198
Ile91, Val121, Phe131, Leu198,
Thr200
hCA II
−9.34
Yes
His94, Thr199
6f
6h
6i
hCA IX
hCA XII
hCA I
−6.81
−4.72
−5.29
−7.87
−6.53
−9.52
Yes
No
Yes
Yes
Yes
Yes
Thr199
Val121, Leu198, Thr200
Val121, Thr200
-
-
His94
Ala135, Leu198
hCA II
Val121, Phe131, Leu198
Val121, Thr200
hCA IX
hCA XII
Thr199
Ala131, Ser132, Thr200
Trp5, Val121, Leu198
Leu131, Ala135, Ala132,
Thr202, Leu198
hCA I
−8.15
Yes
Gln92, Thr199
hCA II
hCA IX
hCA XII
−7.14
−6.27
−4.35
Yes
Yes
No
Thr200
Thr199
-
Val121, Phe131, Thr200
Val121, Thr200
Val121, Leu198
Leu198, Thr199, His200, Pro
201, Trp209
hCA I
hCA II
hCA IX
hCA XII
Yes
Yes
Yes
Yes
Gln92
Val121, Phe131, Leu198,
Trp209
Thr199, Thr200
Thr199, Thr200
Thr199, Thr200
AAZ
Val121, Val143, Val131,
Leu198, Trp209
Val121, Val143, Leu198,
Trp209
- Means that there is no residues involved in H –bond formation.

Int. J. Mol. Sci. 2021, 22, 5082
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Figure 2.
(
A
) 2D interaction diagram of compound 17 docking pose interactions with the key amino acids in hCA II, (
B) in
hCA IX, and (
C) superposition of compound 17 bound to hCA IX (grey) in comparison to hCA II (blue), with specific
residues labeled. Active site zinc is shown as a blue sphere, red dotted and green arrows indicate H-bond, and yellow
spheres indicate hydrophobic interactions. Brown circle.
3. Materials and Methods
3.1. Chemistry
Reagents, chemicals, starting materials (1a–j, 3, 4a,b), and solvents were obtained
from commercial sources and used without further purification. Melting points were
determined on an Microquimica MQAPF-302 apparatus (Palhoça, Brazil). NMR spectra
were recorded on a Avance III HD Bruker spectrometer (Rheinstetten, Germany). with
chemical shifts values (δ) in ppm relative to TMS using the residual DMSO-d6 signal as
an internal standard. High-resolution mass spectra (HRMS) were recorded on an LTQ
Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). This
system combines an LTQ XL linear ion-trap mass spectrometer and an Orbitrap mass
analyzer. The analyses were performed by direct infusion of the sample in DMSO (flow
rate of 10 mL/min) in a positive-ion mode, using electrospray ionization (ESI). For the
elemental composition, the calculations used the specific tool included in the Qual Browser
module of Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. Microwave-assisted
synthesis was performed in a Biotage®Initiator Microwave Synthesizer (Biotage, Uppsala,
Sweden). The progress of reactions was monitored by thin-layer chromatography (TLC)
using precoated TLC sheets with Ultraviolet (UV) fluorescent silica gel (Merck, 60F254,
Germany. HPLC (Fuji Silysia Chemical Ltd., Aichi-ken, Japan) purification method: mobile
phase, system 20–70% 0–5min H2O/MeOH, flow: 30 mL/min (loading pump 4 mL/min
methanol); column: Chromatorex 18 SMB100-5T 100 × 19 mm 5 µm.
3.1.1. General Procedure for Synthesis of Compounds (2a–j)
The appropriate 20-hydroxyacetophenone (1a
dioxane (2 mL) in a MW vial. Then diethyl oxalate (3.49 mmol, 474
of MeONa in MeOH (2.32 mmol, 531
–
j
) (1.16 mmol, 1 eq.) was dissolved in
L, 3 eq.) and a solution
L, 25% w/w, 2 eq.) were added. The resulting
µ
µ
solution was heated to 120 ◦C for 20 min. Then, a solution of HCl (18 mmol, 3 mL, 6 M)
was added and the reaction was heated to 120 ◦C for 40 min. The reaction mixture was
poured into water (50 mL) and the solid formed was filtered and washed with water. The
solid was then dried, washed with dichloromethane, and dried again. Carboxylic acids
2a–e, h–j had been synthesized previously by means of different methods and their QC
data did not differ from ones we obtained [34–37].

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3.1.2. 7,8-Dimethyl-4-oxo-4H-chromene-2-carboxylic Acid (2f)
◦
93% yield. MP = 259–261 C. 1H NMR (400 MHz, DMSO-d6)
δ
: 14.20 (bs, 1H, COOH),
7.81 (d, J = 7.7 Hz, 1H, Ar), 7.42 (d, J = 7.6 Hz, 1H, Ar), 6.85 (s, 1H, Ar), 2.47 (s, 3H, CH3),
2.41 (s, 3H, CH3). MS (ESI): m/z (%) = 219.1 (13.2) [M + H]+. Anal. Calcd. for C12H10O4
(%): C, 66.05; H, 4.62. Found (%): C, 66.18; H, 4.59.
3.1.3. 5,7-Dimethyl-4-oxo-4H-chromene-2-carboxylic Acid (2g)
◦
97% yield. MP = 264–266 C. 1H NMR (400 MHz, DMSO-d6)
δ: 14.25 (bs, 1H, COOH),
7.61 (s, 1H, Ar), 7.38 (s, 1H, Ar), 6.87 (s, 1H, Ar), 2.55 (s, 3H, CH3), 2.39 (s, 3H, CH3). MS
(ESI): m/z (%) = 219.2 (11.4) [M + H]+. Anal. Calcd. for C12H10O4 (%): C, 66.05; H, 4.62.
Found (%): C, 66.09; H, 4.65.
3.1.4. General Procedure for Synthesis of Compounds 5a–j
To the 4-oxo-4H-chromene-2-carboxylic acid (2a–j) (1.52 mmol, 1.0 eq.), 4-aminobenzene-
sulfonamide 3 (0.314 g, 1.82 mmol, 1.2 eq.) and N-methylimidazole (0.42 mL, 5.33 mmol,
3.5 eq.) combined and dissolved in MeCN (4 mL)) TCFH (0.517 g, 1.83 mmol, 1.2 eq.) was
added in a single portion. The reaction was stirred until complete by LCMS (21 h). The
reaction mixture was then diluted with ethyl acetate (6 mL) and water (4 mL). The layers
were separated, the aqueous layer was extracted with ethyl acetate (4 mL), and the combined
organics were washed with water (4 mL), dried with MgSO4, filtered, and concentrated before
purification by HPLC to afford final amides with excellent yields.
3.1.5. 4-Oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5a)
◦
93% yield. MP = 296–298 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.92 (s, 1H, NH),
8.08 (dd, J = 7.8, 1.5 Hz, 1H, Ar), 7.98 (d, J = 8.7 Hz, 2H, Ar), 7.93–7.81 (m, 4H, Ar), 7.54 (t,
J = 7.8 Hz, 1H, Ar), 7.23 (s, 2H, SO2NH2), 6.99 (s, 1H, Ar). MS (ESI): m/z (%) = 345.1 (17.6)
[M + H]+. Anal. Calcd. for C16H12N2O5S (%): C, 55.81; H, 3.51; N, 8.14; S, 9.31. Found (%):
C, 55.87; H, 3.49; N, 8.18; S, 9.28.
3.1.6. 6-Methyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5b)
◦
89% yield. MP = 307–308 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.97 (s, 1H, NH),
8.02–7.86 (m, 5H, Ar), 7.74 (s, 2H, Ar), 7.34 (s, 2H, SO2NH2), 6.97 (s, 1H, Ar), 2.45 (s, 3H,
CH3). MS (ESI): m/z (%) = 359.1 (20.3) [M + H]+. Anal. Calcd. for C17H14N2O5S (%): C,
56.98; H, 3.94; N, 7.82; S, 8.95. Found (%): C, 57.09; H, 3.99; N, 7.76; S, 9.08.
3.1.7. 7-Methyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5c)
◦
91% yield. MP = 309–311 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.87 (s, 1H, NH),
7.99–7.84 (m, 5H, Ar), 7.61 (s, 1H, Ar), 7.34 (d, J = 8.4 Hz, 1H, Ar), 7.25 (s, 2H, SO2NH2),
6.95 (s, 1H, Ar), 2.53 (s, 3H, CH3). MS (ESI): m/z (%) = 359.1 (20.4) [M + H]+. Anal. Calcd.
for C17H14N2O5S (%): C, 56.98; H, 3.94; N, 7.82; S, 8.95. Found (%): C, 57.07; H, 3.98; N,
7.78; S, 8.93.
3.1.8. 6-Ethyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5d)
◦
84% yield. MP = 297–299 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.92 (s, 1H, NH),
7.98 (d, J = 6.9 Hz, 2H, Ar), 7.87–7.83 (m, 4H, Ar), 7.74 (d, J = 1.5 Hz, 1H, Ar), 7.25 (s,
2H, SO2NH2), 6.97 (s, 1H, Ar), 2.78 (q, J = 7.6 Hz, 2H, CH2CH3), 1.29 (t, J = 7.6 Hz, 3H,
CH2CH3). 13C NMR (151 MHz, DMSO-d6)
δ: 177.68, 158.66, 155.55, 154.00, 142.43, 140.94,
140.41, 135.58, 127.09, 123.96, 123.36, 121.19, 119.34, 111.65, 40.51, 27.98, 15.79. MS (ESI):
m/z (%) = 373.1 (21.4) [M + H]+. Anal. Calcd. for C18H16N2O5S (%): C, 58.05; H, 4.33; N,
7.52; S, 8.61. Found (%): C, 58.07; H, 4.37; N, 7.58; S, 8.63.
3.1.9. 6,7-Dimethyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5e)
◦
87% yield. MP = 310–312 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.95 (s, 1H, NH),
7.98 (d, J = 8.7 Hz, 2H, Ar), 7.87 (d, J = 8.7 Hz, 2H, Ar), 7.80 (s, 1H, Ar), 7.60 (s, 1H, Ar),

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7.35 (s, 2H, SO2NH2), 6.93 (s, 1H, Ar), 2.41 (s, 3H, CH3), 2.34 (s, 3H, CH3). MS (ESI):
m/z (%) = 373.1 (21.4) [M + H]+. Anal. Calcd. for C18H16N2O5S (%): C, 58.05; H, 4.33; N,
7.52; S, 8.61. Found (%): C, 58.03; H, 4.32; N, 7.51; S, 8.65.
3.1.10. 7,8-Dimethyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5f)
◦
90% yield. MP = 328–330 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.85 (s, 1H, NH), 7.96
(d, J = 8.7 Hz, 2H, Ar), 7.87 (d, J = 8.7 Hz, 2H, Ar), 7.81 (d, J = 8.1 Hz, 1H, Ar), 7.38–7.36
(m, 3H, (1H, Ar + SO2NH2), 7.00 (s, 1H, Ar), 2.48 (s, 3H, CH3), 2.43 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ: 177.86, 162.72, 159.18, 155.74, 153.92, 144.78, 141.02, 140.36, 128.04,
127.10, 126.54, 122.18, 122.02, 121.02, 111.44, 40.86, 40.48, 36.22, 31.21, 20.51, 12.04. MS (ESI):
m/z (%) = 373.1 (21.2) [M + H]+. Anal. Calcd. for C18H16N2O5S (%): C, 58.05; H, 4.33; N,
7.52; S, 8.61. Found (%): C, 58.09; H, 4.35; N, 7.48; S, 8.67.
3.1.11. 5,7-Dimethyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5g)
◦
76% yield. MP = 304–306 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.86 (s, 1H, NH), 7.98
(d, J = 8.7 Hz, 2H, Ar), 7.87 (d, J = 8.7 Hz, 2H, Ar), 7.46 (s, 1H, Ar), 7.31 (s, 2H, SO2NH2),
7.12 (s, 1H, Ar), 6.85 (s, 1H, Ar), 2.73 (s, 3H, CH3), 2.44 (s, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6) δ: 179.43, 158.61, 157.08, 153.85, 145.14, 140.96, 140.35, 139.94, 130.04, 127.08,
121.12, 120.26, 116.97, 113.00, 22.42, 21.54. MS (ESI): m/z (%) = 373.1 (21.4) [M + H]+. Anal.
Calcd. for C18H16N2O5S (%): C, 58.05; H, 4.33; N, 7.52; S, 8.61. Found (%): C, 58.07; H, 4.37;
N, 7.56; S, 8.64.
3.1.12. 6,8-Dimethyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5h)
◦
88% yield. MP = 309–311 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.87 (s, 1H, NH), 7.93
(d, J = 8.7 Hz, 2H, Ar), 7.88 (d, J = 8.7 Hz, 2H, Ar), 7.48 (s, 1H, Ar), 7.42 (s, 2H, SO2NH2), 7.23
(s, 1H, Ar), 6.87 (s, 1H, Ar), 2.63 (s, 3H, CH3), 2.42 (s, 3H, CH3). MS (ESI): m/z (%) = 373.1
(21.5) [M + H]+. Anal. Calcd. for C18H16N2O5S (%): C, 58.05; H, 4.33; N, 7.52; S, 8.61.
Found (%): C, 58.11; H, 4.30; N, 7.51; S, 8.70.
3.1.13. 6-Chloro-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5i)
◦
92% yield. MP = 296–298 C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.92 (s, 1H, NH),
8.00–7.84 (m, 7H, Ar), 7.23 (s, 2H, SO2NH2), 7.02 (s, 1H, Ar). 13C NMR (151 MHz, DMSO-
d6) δ: 176.66, 158.30, 155.88, 154.12, 140.84, 140.49, 135.47, 131.09, 127.12, 125.26, 124.40,
121.90, 121.21, 111.72, 40.51. MS (ESI): m/z (%) = 380.0 (39.2) [M + H]+. Anal. Calcd. for
C16H11ClN2O5S (%): C, 50.73; H, 2.93; Cl, 9.36; N, 7.40; S, 8.47. Found (%): C, 50.81; H, 2.95;
Cl, 9.38; N, 7.45; S, 8.53.
3.1.14. 6-Chloro-7-methyl-4-oxo-N-(4-sulfamoylphenyl)-4H-chromene-2-carboxamide (5j
)
◦
92% yield. MP = 305–307 C. 1H NMR (400 MHz, DMSO-d6)
δ
: 10.97 (s, 1H, NH),
8.00–7.85 (m, 6H, Ar), 7.35 (s, 2H, SO2NH2), 7.00 (s, 1H, Ar), 2.52 (s, 3H, CH3). 13C NMR
(151 MHz, DMSO-d6) δ: 176.46, 158.38, 155.69, 153.94, 144.05, 140.89, 140.45, 131.91, 127.13,
124.64, 123.35, 121.53, 121.11, 111.66, 40.51, 20.75. MS (ESI): m/z (%) = 394.0 (39.5) [M + H]+.
Anal. Calcd. for C17H13ClN2O5S (%): C, 51.98; H, 3.34; Cl, 9.03; N, 7.13; S, 8.16. Found (%):
C, 52.03; H, 3.27; Cl, 9.08; N, 7.15; S, 8.21.
3.1.15. General Procedure for Synthesis of Compounds 6a–k
The corresponding 4-oxo-4H-chromene-2-carboxylic acid (2a–j) (1.52 mmol, 1 eq.) was
dissolved in DMF (10 mL), and 1,1-carbonyldiimidazole (0.320◦g, 1.98 mmol, 1.3 eq.) was
added in one portion. The reaction mixture was stirred at 80–90 C for 1.2 h; afterward, the
corresponding amine (4a,b) (1.82 mmol, 1.2 eq.) in DMF (7 mL) was added and the mixture
was continually heated for another 1 h. After cooling to room temperature, the reaction
mixture was treated with water (20 mL) and extracted with ethyl acetate (2 × 30 mL),
washed with brine, and dried with Na2SO4. The solvent was evaporated under reduced
pressure and the residue was purified by HPLC to afford the desired amide (6a–k).

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3.1.16. 4-Oxo-N-(4-sulfamoylbenzyl)-4H-chromene-2-carboxamide (6a)
◦
96% yield. MP = 284–286 C. 1H NMR (400 MHz, CF3COOD)
δ
: 8.31 (d, J = 6.7 Hz, 1H,
Ar), 8.08–7.95 (m, 3H, Ar), 7.77 (m, 4H, Ar), 7.56 (s, 1H, Ar), 4.88 (s, 2H, CH2). 13C NMR
(126 MHz, DMSO-d6) δ: 177.74, 159.47, 156.02, 155.51, 143.71, 142.64, 135.46, 129.60, 126.47,
126.23, 125.39, 124.07, 119.19, 110.89, 40.90, 34.86. MS (ESI): m/z (%) = 359.1 (20.3) [M + H]+.
Anal. Calcd. for C17H14N2O5S (%): C, 56.98; H, 3.94; N, 7.82; S, 8.95. Found (%): C, 57.02;
H, 3.87; N, 7.79; S, 8.94.
3.1.17. 6-Methyl-4-oxo-N-(4-sulfamoylbenzyl)-4H-chromene-2-carboxamide (6b)
91% yield. MP = 293–295 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.75 (t, J = 6.0 Hz, 1H,
NH), 7.84 (d, J = 2.1 Hz, 1H, Ar), 7.83–7.78 (m, 2H, Ar), 7.70 (dd, J = 8.6, 2.3 Hz, 1H, Ar),
7.61 (d, J = 8.6 Hz, 1H, Ar), 7.57–7.50 (m, 2H, Ar), 7.34 (s, 2H, SO2NH2), 6.84 (d, J = 1.5 Hz,
1H, Ar), 4.58 (d, J = 6.0 Hz, 2H, CH2), 2.43 (s, 3H, CH3). 13C NMR (151 MHz, DMSO-d6)
δ:
177.67, 159.81, 155.75, 153.81, 143.34, 142.97, 136.50, 136.15, 128.22, 126.23, 124.63, 123.83,
118.99, 110.97, 42.83, 40.51, 20.91. MS (ESI): m/z (%) = 373.1 (21.5) [M + H]+. Anal. Calcd.
for C18H16N2O5S (%): C, 58.05; H, 4.33; N, 7.52; S, 8.61. Found (%): C, 58.10; H, 4.29; N,
7.50; S, 8.71.
3.1.18. 6-Chloro-4-oxo-N-(4-sulfamoylbenzyl)-4H-chromene-2-carboxamide (6c)
◦
1
84% yield. MP = 299–301 C. H NMR (400 MHz, DMSO-d6) δ: 9.74 (s, 1H, NH),
8.01–7.89 (m, 2H, Ar), 7.78 (ddd, J = 18.4, 8.6, 1.8 Hz, 3H, Ar), 7.53 (d, J = 7.9 Hz, 2H, Ar),
7.29 (s, 2H, SO2NH2), 6.89 (d, J = 1.7 Hz, 1H, Ar), 4.59 (d, J = 6.0 Hz, 2H, CH2). 13C NMR
(126 MHz, DMSO-d6) δ: 176.72, 159.49, 156.09, 154.12, 143.36, 142.87, 135.38, 130.91, 128.23,
126.23, 125.23, 124.39, 121.69, 111.05, 42.83. MS (ESI): m/z (%) = 394.0 (39.5) [M + H]+. Anal.
Calcd. for C17H13ClN2O5S (%): C, 51.98; H, 3.34; Cl, 9.03; N, 7.13; S, 8.16. Found (%): C,
52.04; H, 3.28; Cl, 9.09; N, 7.16; S, 8.22.
3.1.19. 4-Oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6d)
◦
95% yield. MP = 248–250 C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.24–9.17 (m, 1H, NH),
8.05 (dd, J = 8.0, 1.5 Hz, 1H, Ar), 7.89 (ddd, J = 8.5, 7.1, 1.7 Hz, 1H, Ar), 7.77 (d, J = 7.9 Hz,
2H, Ar), 7.71 (d, J = 8.5 Hz, 1H, Ar), 7.53 (t, J = 7.6 Hz, 1H, Ar), 7.46 (d, J = 8.0 Hz, 2H), 7.27
(s, 2H, SO2NH2), 6.82 (s, 1H, Ar), 3.58 (q, J = 6.9 Hz, 2H, NCH2), 2.98 (t, J = 7.4 Hz, 2H,
CH2Ar). MS (ESI): m/z (%) = 373.1 (21.5) [M + H]+. Anal. Calcd. for C18H16N2O5S (%): C,
58.05; H, 4.33; N, 7.52; S, 8.61. Found (%): C, 58.10; H, 4.29; N, 7.50; S, 8.71.
3.1.20. 6-Methyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6e)
◦
84% yield. MP = 257–259 C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.18–9.14 (m, 1H, NH),
7.82 (s, 1H, Ar), 7.89 (d, J = 8.5 Hz, 2H, Ar), 7.77–7.61 (m, 2H, Ar), 7.42 (d, J = 8.5 Hz, 2H,
Ar), 7.19 (s, 2H, SO2NH2), 6.77 (s, 1H, Ar), 3.54 (q, J = 6.9 Hz, 2H, NCH2), 2.96 (t, J = 7.4 Hz,
2H, CH2Ar), 2.46 (s, 3H, CH3). MS (ESI): m/z (%) = 387.1 (22.5) [M + H]+. Anal. Calcd. for
C19H18N2O5S (%): C, 59.06; H, 4.70; N, 7.25; S, 8.30. Found (%): C, 59.10; H, 4.79; N, 7.23;
S, 8.33.
3.1.21. 7-Methyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6f)
◦
87% yield. MP = 253–255 C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.23 (t, J = 6.9 Hz,
1H, NH), 7.92 (d, J = 8.4 Hz, 1H, Ar), 7.75 (d, J = 8.0 Hz, 2H, Ar), 7.50 (s, 1H, Ar), 7.44 (d,
J = 8.0 Hz, 2H, Ar), 7.35 (d, J = 8.4 Hz, 2H, Ar), 7.30 (s, 2H, SO2NH2), 6.76 (s, 1H, Ar), 3.55
(q, J = 6.9 Hz, 2H, NCH2), 2.96 (t, J = 7.4 Hz, 2H, CH2Ar), 2.47 (s, 3H, CH3). MS (ESI): m/z
(%) = 387.1 (22.5) [M + H]+. Anal. Calcd. for C19H18N2O5S (%): C, 59.06; H, 4.70; N, 7.25; S,
8.30. Found (%): C, 59.12; H, 4.73; N, 7.24; S, 8.35.
3.1.22. 6-Ethyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6g)
◦
1
90% yield. MP = 257–259 C. H NMR (400 MHz, DMSO-d6)
δ: 9.21 (s, 1H, NH),
7.85 (d, J = 2.2 Hz, 1H, Ar), 7.80–7.71 (m, 3H, Ar), 7.63 (d, J = 8.6 Hz, 1H, Ar), 7.46 (d,

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J = 8.3 Hz, 2H, Ar), 7.28 (s, 2H, SO2NH2), 6.79 (s, 1H, Ar), 3.56 (q, J = 6.8 Hz, 2H, NCH2),
2.97 (t, J = 6.9 Hz, 2H, CH2Ar), 2.75 (q, J = 7.6 Hz, 2H, CH2Ar), 1.23 (t, J = 7.6 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6)
δ: 177.72, 159.53, 155.90, 153.94, 143.71, 142.64, 142.27,
135.48, 129.59, 126.22, 123.87, 123.37, 119.10, 110.72, 40.89, 34.86, 27.97, 15.83. MS (ESI):
m/z (%) = 387.1 (22.5) [M + H]+. Anal. Calcd. for C20H20N2O5S (%): C, 59.99; H, 5.03; N,
7.00; S, 8.01. Found (%): C, 59.96; H, 5.07; N, 7.04; S, 8.05.
3.1.23. 6,7-Dimethyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6h)
78% yield. MP = 282–284 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.20 (t, J = 5.7 Hz, 1H,
NH), 7.76 (dd, J = 6.3, 1.9 Hz, 3H, Ar), 7.46 (d, J = 8.2 Hz, 3H, Ar), 7.31 (s, 2H, SO2NH2),
6.74 (d, J = 1.2 Hz, 1H, Ar), 3.56 (q, J = 6.8 Hz, 2H, NCH2), 2.96 (t, J = 7.3 Hz, 2H, CH2Ar),
2.38 (s, 3H, CH3), 2.32 (s, 3H, CH3). 13C NMR (151 MHz, DMSO-d6)
δ: 177.42, 159.61,
155.65, 153.99, 145.63, 143.71, 142.64, 135.60, 129.58, 126.22, 124.84, 121.97, 118.95, 110.74,
40.85, 40.51, 34.85, 20.47, 19.36. MS (ESI): m/z (%) = 387.1 (22.5) [M + H]+. Anal. Calcd. for
C20H20N2O5S (%): C, 59.99; H, 5.03; N, 7.00; S, 8.01. Found (%): C, 59.94; H, 5.05; N, 7.02;
S, 8.03.
3.1.24. 7,8-Dimethyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6i)
82% yield. MP = 280–282 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 8.75 (t, J = 6.0 Hz, 1H,
NH), 7.77–7.74 (m, 3H, Ar), 7.43 (d, J = 8.1 Hz, 2H, Ar), 7.30 (d, J = 8.4 Hz, 1H, Ar), 7.20 (s,
2H, SO2NH2), 6.75 (d, J = 1.2 Hz, 1H, Ar), 3.60 (q, J = 6.0 Hz, 2H, NCH2), 2.96 (t, J = 7.3 Hz,
2H, CH2Ar), 2.52 (s, 3H, CH3), 2.48 (s, 3H, CH3). MS (ESI): m/z (%) = 387.1 (22.5) [M + H]+.
Anal. Calcd. for C20H20N2O5S (%): C, 59.99; H, 5.03; N, 7.00; S, 8.01. Found (%): C, 59.91;
H, 5.08; N, 7.05; S, 8.00.
3.1.25. 6,8-Dimethyl-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6j)
80% yield. MP = 283–285 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 8.81 (t, J = 6.8 Hz, 1H,
NH), 7.77 (d, J = 8.0 Hz, 2H, Ar), 7.65 (s, 1H, Ar), 7.53 (s, 1H, Ar), 7.47 (d, J = 8.0 Hz, 2H,
Ar), 7.27 (s, 2H, SO2NH2), 6.76 (s, 1H, Ar), 3.58 (q, J = 6.8 Hz, 2H, NCH2), 2.97 (t, J = 7.2 Hz,
2H, CH2Ar), 2.49 (s, 3H, CH3), 2.38 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ: 177.84,
159.87, 156.03, 152.33, 143.72, 142.66, 137.18, 135.53, 129.64, 128.33, 126.21, 123.82, 122.17,
110.72, 40.94, 34.85, 20.85, 15.73. MS (ESI): m/z (%) = 387.1 (22.5) [M + H]+. Anal. Calcd. for
C20H20N2O5S (%): C, 59.99; H, 5.03; N, 7.00; S, 8.01. Found (%): C, 60.03; H, 5.04; N, 7.02;
S, 8.06.
3.1.26. 6-Chloro-4-oxo-N-(4-sulfamoylphenethyl)-4H-chromene-2-carboxamide (6k)
73% yield. MP = 285–287 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 9.26 (t, J = 5.6 Hz, 1H,
NH), 7.99–7.88 (m, 2H, Ar), 7.75 (t, J = 8.9 Hz, 3H, Ar), 7.46 (d, J = 8.1 Hz, 2H, Ar), 7.29 (s,
2H, SO2NH2), 6.83 (s, 1H, Ar), 3.62–3.51 (m, 2H, NCH2), 2.96 (t, J = 7.3 Hz, 2H, CH2Ar). 13C
NMR (126 MHz, DMSO-d6) δ: 176.70, 159.21, 156.20, 154.08, 143.66, 142.65, 135.35, 130.90,
129.59, 126.23, 125.19, 124.38, 121.65, 110.81, 40.91, 34.84. MS (ESI): m/z (%) = 408.0 (39.7)
[M + H]+. Anal. Calcd. for C18H15ClN2O5S (%): C, 53.14; H, 3.72; Cl, 8.71; N, 6.89; S, 7.88.
Found (%): C, 53.04; H, 3.78; Cl, 8.79; N, 7.16; S, 7.82.
3.2. Carbonic Anhydrase Inhibition
An Applied Photophysics stopped-flow instrument was used for assaying the CA
catalyzed CO2 hydration activity [38]. Phenol red (at a concentration of 0.2 mM) was used
as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4)
as a buffer, and 20 mM Na2SO4 (for maintaining constant ionic strength), following the
initial rates of the CA-catalyzed 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 constants [39]. The non-catalyzed CO2 hydration was not subtracted from
these curves and accounts for the remaining observed activity even at a high concentration
of an inhibitor, being in the range of 16–25%. However, the background activity from the

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uncatalyzed reaction is always subtracted when IC50 values are obtained by using the data
analysis software for the stopped flow instrument. Enzyme concentrations ranged between
5–12 nM. For each inhibitor, at least six traces of the initial 5–10% of the reaction were
used for determining the initial velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates. Stock solutions of the inhibitor
(0.1 mM) were prepared in distilled, deionized water, and dilutions up to 0.01 nM were
done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated
together for 15 min at room temperature prior to the assay, allowing 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 as reported earlier, which represent the
mean from at least three different determinations. All CA isoforms were recombinant
proteins obtained in house, as reported earlier [40–42].
3.3. Molecular Modeling Studies (Experimental Part)
Docking calculations were carried out using the AutoDock 4.2 software [43]. The free
energy of binding (DG) of the cytosolic isoforms hCA I and II as well as the transmembrane
tumor-associated ones IX and XII in complex with the tested compounds was generated
using this molecular docking program. The crystal structures of hCA I (PDB code 3W6H),
hCA II (PDB code 3HS4), hCA IX (PDB code 3IAI), and hCA XII (PDB code 1JD0) were
obtained from the Protein Data Bank [44]. For the enzymes’ preparation, all water molecules
were eliminated polar hydrogens, which were added, and the co-crystallized ligands were
removed from each enzyme’s active site, while for the preparation of the inhibitors, the
charges were added and the rotatable bonds determined. Grid maps have been calculated
utilizing Autogrid algorithm and must contain the area to be connected. A set of grids
of 60 Å
×
50 Å
×
50 Å with 0.375 Å spacing was calculated, considering the docking
area for all the ligand atom types employing AutoGrid4. Three-dimensional structures of
all compounds were constructed using Chem3Dultra 12.0 software (Chemical Structure
Drawing Standard; Perkin Elmer Informatics, Waltham, MA, USA). For the present system,
the Lamarckian genetic algorithm was applied for minimization using default parameters.
The pitch was 1.0 Å, while the quaternion and pivot angle were set to 5 and 0 degrees.
For each compound, 200 configurations were produced. The results from the Autodock
calculations were grouped using a root mean standard deviation (RMSD) value of 1.5 Å,
while the lowest-energy configuration of the largest population group was chosen as
the most likely tethering configuration. The LigandScout software program (inte:ligand,
Vienna, Austria) was used to display the results and process of the configurations with
the highest tie rating. Finally, the docking protocol was verified by re-docking of the co-
crystallized ligand acetazolamide (AAZ) in the vicinity of the active sites of each enzyme
with RMSD values 0.885, 0.966, 1.034, and 1.176 Å for hCA I, II, IX, and XII, respectively.
4. Conclusions
In conclusion, we synthetized and investigated two novel series of chromene-containing
aromatic sulfonamides for their effective inhibition against different and most relevant
human carbonic anhydrase isoforms such as the ubiquitous hCA I, hCA II, and the tumor
associate isoforms hCA IX and XII, which are involved in a variety of diseases such as
glaucoma, retinitis pigmentosa, epilepsy, and tumors. Compound 5a showed a good
selectivity index of 31.67 on hCA IX compared to hCA I and over two times compared to
hCA II. On the other hand, compound 6f showed to be the most active hCA II with an
SI of 44.47 compared to hCA I and 44.97 compared to hCA XII. In addition, the methy-
lene linker among the chromene scaffold and sulfonamide moiety played a crucial role in
the modulation of potency and selectivity inhibition against different isoforms. Indeed,
compound 5b showed a Ki of 77 nM against hCA IX and the analog 6b, with a methylene
linker, and dramatically decreased the potency to 2039 nM, losing the selective inhibition
against this isoform. These interesting features make them good candidates for preclinical
evaluation in glaucoma or various tumors in which the two enzymes (hCA II and hCA IX)

Int. J. Mol. Sci. 2021, 22, 5082
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are involved. Furthermore, computational procedures were used to investigate the binding
mode of this class of compounds within the active site of hCA IX.
Author Contributions: Conceptualization, A.G. and V.K.; methodology, C.S.; software, A.P.; valida-
tion, A.P.; formal analysis, S.S., and S.P.; investigation. V.B., A.A. and M.P; data curation, V.B., C.S.;
writing–original draft preparation, V.B., A.A., C.S. and A.G.; writing-review and editing, A.A., C.S.
and A.G.; supervision, C.S. and A.G. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: We have no studies involved humans or animals in this paper.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: we have no supporting information.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CA
Carbonic Anhydrase
CAI Carbonic Anhydrase Inhibitors
SAR Structure Activity Relationships
SI
Selectivity Index
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