Investigation of 3-sulfamoyl coumarins against cancer-related IX and XII isoforms of human carbonic anhydrase as well as cancer cells leads to the discovery of 2-oxo-2H-benzo[h]chromene-3-sulfonamide – A new caspase-activating proapoptotic agent

Article abstract of DOI:10.1016/j.ejmech.2021.113589

Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives. Prepared compounds were tested in vitro for inhibition of four physiologically relevant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed low nanomolar K_I values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of A431 cell growth with low nanomolar IC₅₀ values. We demonstrated that 10q possessed a concentration-dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable lead for further development of new-generation anticancer agents.

Full text of DOI:10.1016/j.ejmech.2021.113589

European Journal of Medicinal Chemistry 222 (2021) 113589
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Investigation of 3-sulfamoyl coumarins against cancer-related IX and
XII isoforms of human carbonic anhydrase as well as cancer cells leads
to the discovery of 2-oxo-2H-benzo[h]chromene-3-sulfonamide e A
new caspase-activating proapoptotic agent
Dmitry Dar'in a, Grigory Kantin a, Stanislav Kalinin a, Tatiana Sharonova a,
Alexander Bunev b, Gennady I. Ostapenko b, Alessio Nocentini c, Vladimir Sharoyko a,
,
, *
Claudiu T. Supuran c **, Mikhail Krasavin a
a Saint Petersburg State University, Saint Petersburg, 199034, Russian Federation
b Medicinal Chemistry Center, Togliatti State University, Togliatti, 445020, Russian Federation
c Neurofarba Department, Universita Degli Studi di Firenze, Florence, 50019, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives.
Prepared compounds were tested in vitro for inhibition of four physiologically relevant isoforms of the
metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed
low nanomolar KI values against therapeutically relevant hCA II, IX, and XII, whereas they did not
potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative
action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In
the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line
Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-
benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of
A431 cell growth with low nanomolar IC50 values. We demonstrated that 10q possessed a concentration-
dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic
anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects
likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable
lead for further development of new-generation anticancer agents.
Received 30 April 2021
Received in revised form
23 May 2021
Accepted 27 May 2021
Available online 10 June 2021
Keywords:
Carbonic anhydrase
Enzyme inhibitors
Antiproliferative agents
Primary sulfonamides
Coumarins
Stopped-flow assay
Nanomolar inhibition
Cancer cells
© 2021 Elsevier Masson SAS. All rights reserved.
MTT-Test
Hypoxic environment
Apoptosis induction
Caspase activation
1. Introduction
and therefore govern the major physiological buffering system
[1e3]. These enzymes have been found across all kingdoms of life
Carbon dioxide and bicarbonate ion are crucial species in
biochemical processes and their interconversion has paramount
significance in all living organisms [1]. Carbonic anhydrases (CA, EC
4.2.1.1) comprise a superfamily of metalloenzymes that catalyze the
reversible hydration of carbon dioxide (CO2 þ H2O # HCOꢀ3 þ Hþ)
and eight evolutionary unrelated gene families have been identified
so far, including -, -, -, -, -, -, , and -CAs [4e8]. In human,
CAs are represented by 15 isoforms (incorporating Zn2þ as catalytic
ion and attributed to the -class) which differ significantly in their
catalytic activity, tissue distribution and subcellular localization
[9,10]. Thus, cytosolic (I, II, III, III, VII, XIII), mitochondrial (VA, VB)
secreted (VI), and membrane-associated (IV, IX, XII, XIV) human (h)
CA isozymes have been described [11,12]. Therefore, these enzymes
are essential for a wide spectrum of biological processes requiring
acid-base balance be it in subcellular compartments or across the
plasma membrane [13]. Interestingly, aberrant expression/activity
a
b
g
d
z
h
q
i
a
* Corresponding author.
** Corresponding author.
E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), m.krasavin@spbu.ru
(M. Krasavin).
https://doi.org/10.1016/j.ejmech.2021.113589
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
of many hCAs is associated to disease states, which include glau-
coma, edema, obesity, neuropathic pain, osteoporosis, etc. [14e20].
Hence, many hCA isoforms have been validated as valuable thera-
peutic targets and novel links to pathological disorders continue to
be discovered [21e23]. Particularly, recent decades were marked
with validation of the membrane-associated isozymes hCA IX and
XII as anticancer therapeutic targets [24]. In fact, it has been
demonstrated that these surficial proteins are largely overex-
pressed in hypoxic solid tumors where they play pivotal roles in
microenvironment regulation, thus being instrumental in the sur-
vival, proliferation, metastasis and development of drug resistance
of malignant cells [13,25e27]. In the light of these facts, intensive
efforts are being made to bring such agents to clinical use for the
needs of diagnostic, theranostic, and combination therapy of solid
tumors [28,29]. Significant progress has been achieved in the
development of diagnostic tools based on hCA IX/XII targeting bi-
ologics and small molecules, furnishing several drug candidates on
advanced stages of clinical trials [30,31]. In the meantime, cancer
therapeutic agents blocking the activity of these enzymes are
somewhat lagging behind with only SLC-0111 hCA IX/XII inhibitor
entering phase II of clinical studies [24]. Therefore, ongoing efforts
focused on the discovery of hCA IX/XII-blocking small molecules
are of significant importance [24]. Moreover, supremely desired is
the development of isoform-selective inhibitors i.e. uncomplicated
with high affinity to the ubiquitous cytosolic isozymes hCA I and II
[32].
The vast majority of highly potent and selective hCA IX/XII in-
hibitors known so far (including those studied in preclinical and
clinical settings) belong to two dominant classes of hCA-blocking
compounds. Thus, most of isoform-selective nanomolar inhibitors,
such as hCA IX/XII specific SLC-0111 and indane sulfonamide 1
(Fig. 1), represent the class of ‘Zn-binders’ which exert their hCA
inhibitory effect via coordination to the catalytic Zn2þ ion situated
at the bottom of the enzyme's active site. Such mechanism is
characteristic to primary sulfonamide-based hCA inhibitors, as well
as their bioisosters (sulfamides, sulfamates) [33]. It has been shown
that while the Zinc binding warhead anchors in depth of the cat-
alytic cleft (whose structure is highly conservative across all alpha-
CA isoforms), molecular periphery of efficient inhibitors is capable
of binding at the entrance of the active site [9,34,35]. Noticeably
this outer rim of the catalytic cavity is outlined with certain number
of non-conservative amino acid residues and therefore isoform
selectivity can be achieved on the way to safe and efficient thera-
peutic agents [36]. Another well explored class of hCA inhibitors is
represented by coumarin-based “prodrugs” which were found to
occlude the entrance of the catalytic cleft after being catalytically
hydrolyzed within the hCA active site [37]. Importantly, the
resulting derivatives of 2-hydroxycinnamic acid interact with the
outer rim of the cavity (mainly forming van der Waals contacts with
hydrophobic amino acid residues [38]) rendering this class of in-
hibitors sensitive to the differences in the active site architecture
between different enzyme isoforms [39]. Schematic representation
of the described mechanisms of hCA inhibition is outlined in Fig. 1
(A, B) along with hCA IX/XII-specific compounds SLC-0111, 1, 2, and
3 exemplifying the corresponding chemotypes (C) [37,40e42].
Interestingly, while analyzing the emerging publications on this
issue, we have noticed that a number of efficient hCA inhibitors
have been recently reported which were designed through linking
of sulfonamide and coumarin warheads within one molecule
[43e47]. Furthermore, recent review highlighted the anticancer
potential of such chimeric compounds which is often claimed to be
attributive of the inhibition of tumor-associated hCA IX and XII
isoforms (Fig. 2) [48].
bulky linker fragments, which can be considered unfavorable for
further medicinal chemistry optimization of such hit-compounds
[49,50]. In this context, we found it attractive to perform direct
merging of sulfonamide moiety to the coumarin scaffold which
appeared to be feasible via introduction of 2-sulfamoylacetic acid
esters into the classical Knoevenagel condensation with salicy-
laldehyde derivatives [51]. Thus, herein we report the preparation,
hCA inhibitory profiling and anticancer properties evaluation of
seventeen hitherto undescribed coumarin derivatives incorpo-
rating primary sulfonamide group at their 3 position. In addition,
detailed characterization of anticancer potential of the synthesized
compounds is provided.
2. Results and discussion
2.1. Chemistry
The proposed synthetic approach furnishing the desired 3-
sulfamoylcoumarins relied on the introduction of ethyl 2-
sulfamoylacetate 7 into the canonical reaction of
boxylic acid derivatives with salicylaldehydes 8 [52]. To this end,
reagent was synthesized from commercial ethyl 2-(chlor-
a-CH-acidic car-
7
osulfonyl)acetate 9 according to a modified literature procedure
[53] (Scheme 1).
Thus obtained reagent 7 was then mixed with variously
substituted salicylaldehydes 8a-q in n-butanol to give the target 3-
sulfamoylcoumarines 10a-q after stirring for 2e6 h at 110 ꢁC under
basic conditions (Scheme 2).
Following the developed protocol, seventeen variously
substituted 3-sulfamoylcoumarines 10a-q were generated which
represented a novel type of potential hCA inhibitors. Interestingly,
the products’ yields exceeded 60% for the unsubstituted 10a, as well
as for the derivatives bearing electron donating substituents. In the
meantime, several compounds were only obtained in modest to
medium yields, which included 6-nitro- (10j, 41%), 6,8-dichloro
(10k, 30%), and naphthalene (10q, 33%) derivatives.
2.2. Carbonic anhydrase inhibition
With regards to evaluate hCA inhibitory activities of the ob-
tained compounds 10a-q the stopped-flow carbon dioxide hydrase
assay was employed, and acetazolamide (AAZ) was used as a
reference drug [54]. The profiling was performed on a panel of four
physiologically significant hCA isoforms, which comprised two
ubiquitously expressed cytosolic proteins hCA I and II and two
transmembrane tumor-associated enzymes hCA IX and XII. The
inhibitory profile of small molecules against those isozymes is of
interest with regards to different fields of medicinal chemistry
[9,24,55]. Thus, hCA IX (and XII) proved valuable extracellular tu-
mor biomarkers and their inhibitors attract growing attention for
the needs of cancers treatment and prognosis (vide supra)
[14,31,56]. On the other hand, hCA II and XII are known to govern
the aqueous humor secretion in eye and therefore represent vali-
dated targets for intraocular pressure-lowering agents [19].
Otherwise, cytosolic hCA I is considered an off-target protein for
both antiglaucoma and anticancer therapeutic applications of CAIs
[57]. Inhibition constant (KI) values of the synthesized compounds
against these four proteins are presented in Table 1.
Analysis of the inhibition data outlined in Table 1 highlighted
the following SAR trends:
(i) As evident from Table 1, unsubstituted 3-sulfamoylcoumarin
10a displayed medium inhibitory potency against hCA I, II
and XII with KI values of 481.6, 232.4, and 137.5 nM corre-
spondingly. Meanwhile, higher affinity was observed for 10a
However, the majority of these agents turned out to have rather
high molecular weights, partly due to the presence of somewhat
2

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Fig. 1. (A) Schematic representation of CA inhibition via coordination to the catalytic Zn2þ ion. ZBG e zinc binding group. Reproduced from [33]. (B) Schematic representation of CA
inhibition via occlusion of the catalytic site entrance; SLG e Sticky Lipophilic Group [33]. (C) Examples of highly potent and selective sulfonamide and coumarin based hCA IX/XII
inhibitors described in literature [37,41,42].
Scheme 1. Preparation of ethyl 2-sulfamoylacetate 7 [53].
5756, 10,060, and 2975 nM. In contrast, the activity of 10b
against hCA XII decreased insignificantly, compared to that of
10a, reaching 307.2 nM. Similarly, compound 10c bearing
methyl substituents in the positions 5 and 7 of the coumarin
substructure was characterized with micromolar range of
KI's versus the cytosolic hCA I and II (2727 and 2098 nM
respectively). Furthermore, 10c was more potent inhibitor of
hCA IX (KI of 824.6 nM) than 10b, and the Ki value against
hCA XII was not influenced by the substitution pattern in this
case retaining at the level of 302.6 nM.
(ii) In contrast to the discussed derivatives 10b and 10c, 6-
substituted 3-sulfamoylcoumarins 10d-j displayed pro-
nounced activity versus tumor-associated hCA IX and XII
isozymes and some representatives showed selectivity over
the cytosolic hCA I and II. Thus, both 5-methoxy- and 5-
methyl substituted derivatives 10d and 10e exerted potent
inhibition towards hCA IX and XII with KI's ranging between
47.3 and 77.4 nM. On the other hand, hCA I and II isoforms
were only blocked at submicromolar concentrations
Fig. 2. Recently reported chimeric molecules 4e8 incorporating both primary sul-
fonamide and coumarin warheads, and their inhibition of hCA IX/XII [43e45].
towards hCA IX characterized by KI of 65.1 nM. Interestingly,
introduction of a methoxy group into the position 5 of the
coumarin scaffold caused dramatic drop in the affinities to
hCA I, II and especially IX. Actually, the KI values of the de-
rivative 10b towards the latter isozymes corresponded to
3

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Scheme 2. Synthesis of 3-sulfamoylcoumarins 10a-q via treatment of salicylaldehydes 8a-q with ethyl 2-sulfamoylacetate 7.
Table 1
Inhibitory profiles compounds 10a-q against hCA isoforms hCA I, II, IX and XII. Acetazolamide (AAZ) was employed as standard drug.
N
R1
R2
R3
R4
KI (nM)*
ꢁ
CA I
CA II
CA IX
CA XII
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
AAZ
H
H
H
H
OMe
Me
OH
F
H
H
Me
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
F
OH
OMe
OEt
H
481.6
5756
2727
543.0
835.1
238.5
257.8
920.9
1450
946.9
1406
330.2
167.5
725.1
258.5
398.4
75,100
250
232.4
10,060
2098
114.8
149.9
53.9
65.1
2975
824.6
58.1
47.4
66.4
32.6
176.9
317.1
147.7
518.2
40.5
41.1
98.7
87.9
51.5
6371
25
137.5
307.2
302.6
54.4
77.4
26.5
OMe
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
**
96.7
35.8
Cl
618.1
1588
414.1
1249
63.1
69.4
155.2
85.4
127.9
266.2
100.7
253.6
53.8
26.6
64.8
39.7
94.5
Br
NO2
Cl
H
H
H
H
H
**
93.4
>100
m
M
7990
5.7
e
12.5
*Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5e10% of the reported values). ** R1, R2 ¼ -(-CH]CHeCH]CH-)-.
between 114.8 and 835.1 nM by 10d and 10e and hence
certain level of isoform-selectivity was observed for these
molecules. It is worth mentioning that the introduction of
hydroxyl group (10f) or fluorine atom (10g) at the position 6
of the coumarin scaffold furnished the derivatives possessing
equipotent double-digit nanomolar activity against hCA II, IX
and XII isoforms. It was only hCA I which was slightly less
sensitive to 10f,g with KI values of 238.5 and 257.8 nM
respectively. Noticeably, replacing fluorine atom with larger
halogens, such as chlorine (10h) and bromine (10i) clearly
reduced the affinity of the corresponding counterparts to all
hCA isoforms in question. Indeed for 10h KI values for against
the selected panel of enzymes ranged between 127.9 and
920.9 nM, whereas for 10i the interval was between 266.2
and 1588 nM. However, both compounds displayed selec-
tivity towards hCA IX and XII over the cytosolic isozymes. The
inhibitory profile of the 6-nitro- derivative 10j was very close
to that of chlorine bearing analog 10i. In the meantime, 6,8-
dichloro-3-sulfamoylcoumarin 10k showed micromolar ac-
tivities against hCA I and II, while being nearly one order of
magnitude more potent inhibitor of hCA IX and XII.
4

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
(iii) Compound 10l bearing methoxy group in the position 7 of
the coumarin substructure exhibited significant potency
against hCA II, IX and XII (KI values between 40.5 and
63.1 nM) while being somewhat less active with regards to
hCA I (KI of 330.2). Likewise, 8-substituted derivative 10m-p
generally showed double-digit nanomolar potencies against
hCA II, IX and XII, whereas hCA I was affected at concentra-
tions spanning the range between 167.5 and 725 nM. Of this
latter subset 8-fluorosubstituted analog 10m showed the
lowest KI values against hCA IX and XII, namely 41.1 and
26.6 nM. Of note, contrary to compounds 10f,g, fluoro- and
hydroxysubstituted counterparts 10m and 10n did not
possess substantial similarity in their inhibitory profiles, as
10m was at least two times more potent against all employed
isozymes.
hCA XII just as against hCA IX. Therefore many of synthesized
3-sulfamoylcoumarins exerted considerable activities to-
wards hCA II and more importantly against tumor-associated
hCA IX/XII with some derivatives proved selective inhibitors
of these latter isozymes.
2.3. Antiproliferative activity against cancer and normal cell lines
Development of highly potent and selective hCA inhibitors holds
a great promise in the fields of diagnostic and management of solid
tumors [59]. In fact, hostile tumor microenvironment endowed
with hypoxia (due to the poor and chaotic vascularization) and
acidosis (associated with glycolytic metabolism) gives rise to
massive molecular and phenotypic changes and results into selec-
tion of highly aggressive types of malignant cells [60]. It has been
shown, that cancer cell survival and proliferation is largely ensured
by upregulation of exofacial hCA IX and hCA XII isoforms [61,62].
Not only these proteins impact significantly into pH regulation
across the plasma membrane via facilitating CO2 interconversion,
but also enhance the functioning of a wide range of transport
proteins [13]. Additionally, the contribution of these enzymes into
pHi and pHe maintenance is considered crucial in disrupting
intercellular adhesion contacts ensuring cancer cell invasion and
metastasis [63]. However, despite the fact that several clinical
studies are in progress employing well-known hCA inhibitors
Acetazolamide (AAZ) [64,65] or SLC-0111 [66] as antiproliferative
agents in combined therapy of aggressive tumors, the discovery of
novel anticancer drug candidates with such mechanism is not a
straightforward endeavor. This is mainly related to the frequently
observed insufficient translation of hCA IX and XII inhibitory ac-
tivities of small molecules into antiproliferative effect on cell cul-
tures, which may have multiple reasons including compounds’ off-
target binding, metabolic instability, etc. [67]. Therefore, charac-
terization and analysis of anticancer properties for the newly
discovered hCA IX/XII inhibitors is of interest to further advance the
drug discovery efforts in this field [68]. To this end the well-
established MTT test in 96-well multiplate format has been per-
formed for the synthesized compounds 10a-q. Since, the expres-
sion of hCA IX in tumor cells is controlled by hypoxia-inducible
(iv) Finally, the investigation of the naphthalene derivative 10q
revealed that incorporation of extra aromatic rings may be
detrimental
for
the
anti-hCA
activity
of
3-
sulfamoylcoumarins as it showed only micromolar KI
values against all studies enzyme isoforms.
(v) As can be seen from Table 1, all the synthesized compounds
possess moderate or weak inhibitory activity against the
cytosolic isoform hCA I. Thus, compounds 10b, 10c, 10i, 10k
and 10q displayed only micromolar affinity to this isozyme,
whereas other representatives showed submicromolar KI
values. The most potent hCA I inhibitor turned out to be 8-
fluorosubstituted 10m with KI 167.5 nM. In the context of
therapeutic applications of CAIs, such as anticancer or anti-
glaucoma action, inhibition of hCA I is considered undesired,
as it causes a range of unpleasant side-effects [52,58].
Therefore, the observed modest affinity of the generated
molecules toward this enzyme can be deemed beneficial for
their medicinal potential.
(vi) On the other hand, the physiologically dominant hCA II
turned out to be rather sensitive to the 3-
sulfamoylcoumarins 10a-q. Interestingly, as evident from
the assay results, the presence of bulky substituents in po-
sitions 5 or 6 of the coumarin scaffold was unfavorable for
the hCA II inhibition. In fact, compound 10b, 10i, and 10q
showed neglectable activity toward this enzyme. In contrast
the introduction of small polar substituents in the position 6
(hydroxyl group for 10f or fluorine atom for 10g) significantly
enhanced the hCA II blocking effect of the compounds as
compared to the parent structure 10a. The same was true for
the 7- and 8-substituted counterparts 10l-p, which showed
hCA II inhibition in double-digit nanomolar range of con-
centrations with exception of 10n possessing KI as high as
155.2 nM.
(vii) In the meantime, two tumor-associated hCA isoforms,
namely hCA IX and XII were significantly affected by many of
the prepared substances. Particularly, compounds 10a, 10d,
10e, 10g, and 10l-p exerted double-digit nanomolar KI values
against hCA IX, being nearly equipotent to the reference drug
AAZ:. Similarly to hCA II, hCA IX turned out to be rather
sensitive to the presence of substituents in the position 5 and
6 of the coumarin scaffold, as can be exemplified by the
decreased activities of 10b, 10c, 10h, 10h-k, and especially
10q. Interestingly, the compounds' potencies against another
transmembrane isoform, hCA XII were remarkably close to
those displayed versus hCA IX. Thus, with exception of 10a,b,
KI values against these two isoforms were in the same orders
of magnitude. Finally, two front running inhibitors stood out
with respect to hCA XII, 10f and 10m showing KI values of
26.5 nM. In turn, molecules 10a-c, 10i-k exhibited sub-
micromolar activities whereas 10q was ineffective versus
factor 1a (HIF-1a) [13], we employed CoCl2-iniduced hypoxic
conditions in this study [69]. At the first stage the obtained com-
pounds were screened in concentrations of 30, 100 and 300 mМ
against the following cancer cell lines: A431 human epidermoid
carcinoma, RT4 human bladder cancer, and U-87MG human glio-
blastoma. Additionally, Bj-5ta hTERT immortalized human foreskin
fibroblasts were employed to control nonspecific cytotoxicity. The
results expressed as a percentage of cells viability in comparison
the control survival level (inhibitor concentration 0 mМ) set as 100%
are presented in Figs. 3e6 (tabulated values can be found in Sup-
plemental Materials, S21).
Thus, U87 MG was practically insensitive to the treatment with
10a-q. In fact, at 30 and 100
mM concentrations of the CAIs, no
significant inhibition of cell growth was observed, and furthermore,
increased proliferation rates were detected in many cases. The only
two compounds that approached 50% inhibition levels at 300 mM
concentration turned out to be 10k and 10m (in presence of these
compounds observed cell viability amounted to 54.13 and 57.93). Of
note, the hCA IX/XII inhibitory activities of these hit compounds
differed significantly, as 10m was one of the most potent agent
against these isozymes (KI's of 41.1 and 26.6 nM respectively),
whereas 10k displayed one order of magnitude lower KI values
(518.2 and 253.6 nM correspondingly). In addition, compounds 10n
and 10q exerted decreased U87 MG cell survival rates to ca. 70%
levels (more precisely 67.7 and 67.92) at 300 mM concentrations.
5

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Fig. 3. Cell viability MTT assay results for compounds 10a-q at 30 (green), 100 (blue) and 300 (red) mМ concentrations against U-87MG cell line under hypoxic conditions (100
CoCl2). Values are displayed in % as the mean SEM of three experiments relative to control (0 M, 100% viability, not shown).
mM
m
Fig. 4. Cell viability MTT assay results for compounds 10a-q at 30 (green), 100 (blue) and 300 (red) mМ concentrations against RT4 cell line under hypoxic conditions (100 mM CoCl2).
Values are displayed in % as the mean SEM of three experiments relative to control (0 mM, 100% viability, not shown).
Again, while 10n was rather potent hCA IX/XII inhibitor (KI values of
98.6 and 64.8), 10q was the least active blocker of these isoforms
with respect to all tested compounds (KI values of 6371 and
7990 nM). Therefore, despite 3-sufamoyl coumarins 10a-q showed
U87 MG growth suppressing effect, if any, only at very high con-
centrations, the results highlighted the fact that hCA IX/XII inhibi-
tion may not be the only mechanism of the observed
antiproliferative effect, as hCA inhibitory profiles of the hit com-
pounds were polar opposite to each other.
Unlike U-87 MG, RT-4 cell line exhibited more pronounced
response when treated with 3-sulfamoylcoumarins 10a-q. Indeed,
at least one compound 10k displayed clear dose-dependent anti-
proliferative effect on this cell culture with 43.54 and 15.05% cell
viability levels ranged between 19.70 and 25.42%), yet their anti-
proliferative effect was not observed at lower concentration ranges.
Of all examined cancer cell lines A431 cells turned out to be the
most sensitive to the compounds 10a-q. To our delight, four de-
rivatives 10h, 10i, 10k and 10q exerted profound antiproliferative
effect on these cells. Intriguingly, as in the case of RT-4 potent hCA
IX/XII inhibitor 10k caused distinct response with cell survival rates
of 38.76 and 2.84% at 100 and 300 mM concentrations respectively.
On the other hand, derivatives 10h and 10i possessing sub-
micromolar KI values against the tumor-associated hCA isoforms
exhibited nearly identical levels of A431 cell survival at all three
tested concentrations (e.g. 36.48 and 39.33% at 300 mM concen-
trations correspondingly). Meanwhile, 10q that was found inactive
versus hCA IX and XII isoforms also showed marked effect on the
A431 cell growth with percentage of living cells of 72.95 and
23.41 at 100 and 300 mM concentrations.
With a view to control nonspecific cytotoxicity of the synthe-
sized compounds 10a-q, their effect on the growth of normal
viability levels at 100 and 300 mM concentrations respectively. As
discussed above 10k demonstrated potent inhibition of hCA IX and
XII and therefore its anticancer effect could be at least partially
related to the blocking of hCA enzymatic activity. Compounds 10h,
10i and 10n, in turn, influenced RT-4 cell growth at 300 mM (cell
6

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Fig. 5. Cell viability MTT assay results for compounds 10a-q at 30 (green), 100 (blue) and 300 (red) mМ concentrations against A431 cell line under hypoxic conditions (100
CoCl2). Values are displayed in % as the mean SEM of three experiments relative to control (0 M, 100% viability, not shown).
mM
m
Fig. 6. Cell viability MTT assay results for compounds 10a-q at 30 (green), 100 (blue) and 300 (red) mМ concentrations against Bj-5ta hTERT cell culture under hypoxic conditions
(100 M CoCl2). Values are displayed in % as the mean SEM of three experiments relative to control (0 M, 100% viability, not shown).
m
m
human cells has been evaluated. Specifically, Bj-5ta hTERT
immortalized human foreskin fibroblasts have been employed for
this purpose. Reassuringly, derivatives 10a-q were generally well
tolerated by Bj-5ta cells, which were only sensitive to at 300 mМ
concentrations of 10d (cell viability 70.97%), 10f (65.75%), 10n
(54.76%) and 10p (55.97). It is important to note that the front
running compounds 10h, 10i, 10k and 10q exhibiting remarkable
antiproliferative activity against malignant cell lines (vide supra)
did not show any detectable effect on the survival of healthy human
cells. The latter observation renders these agents attractive in terms
of more detailed investigation of their anticancer properties.
In order to further characterize the anticancer activity of the
four discovered hit compounds 10h, 10i, 10k and 10q we proceeded
to determine their IC50 values. To this end, dose-response curves
have been obtained against the most sensitive A431 cancer cell line
(Supplementary materials, S22-25). Moreover, since A341 cells
originate from human lungs, WI-26 AV4 healthy human lung fi-
broblasts cell culture was employed as a control in this experiment
(Table 2).
As evident from Table 2 compounds 10h, 10i and 10k possessed
A431 IC50 values in double-digit micromolar range, with limited
deviations observed at different incubation times. In contrast,
compound 10q displayed clearly growing IC50 values (from 9.73 to
70.14 mM) with increasing the incubation time. Furthermore, while
pronounced cumulative effect was observed for 10h, 10i and 10k
against the normal WI-26 AV4 cells, 10q showed the opposite
behavior and the IC50 values grew consistently reaching more than
350 mM after incubating for 72 h. It is worth noting that unlike 10h,
10i and 10k, naphthalene derivative 10h exerted nearly no inhibi-
tion of tumor-associated hCA IX and XII, yet its antiproliferative
properties were distinguished with a clear time- and dose-
dependent character. Unexpectedly, both A431 and WI-26 AV4
cell lines demonstrated progressive desensibilization to the incu-
bation with 10q, which was not observed for the other tested 3-
sulfamoylcoumarins. It should be additionally stressed that com-
pound 10q retained its selectivity toward the A431 cell line over the
non-tumorigenic normal human fibroblast cell line WI-26 AV4. The
latter trend is vividly illustrated in Fig. 7 representing the observed
7

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Table 2
IC50 values of the hit compounds 10h, 10i, 10k and 10q as well as anticancer kinase inhibitor Gefitinib at different incubation times against A431 and WI-26 AV4 cell lines.
Compound
A431
IC50
24 h
WI-26 AV4
IC50
24 h
,
mM
, mM
48 h
72 h
48 h
72 h
10h
10i
10k
10q
51.91 8.85
38.43 5.60
26.89 4.36
9.73 3.13
32.17 3.44
29.10 3.57
24.7 3.99
20.30 1.91
25.91 5.86
24.55 2.58
57.80 10.00
49.20 9.63
35.24 3.40
70.14 26.06
16.02 2.87
89.15 7.82
76.57 7.13
73.97 30.04
41.68 7.84
51.18 7.44
45.11 1.92
42.05 2.08
9.54 1.03
84.48 17.92
42.95 6.55
5.87 1.69
21.5 2.48
14.35 1.70
>350
Gefitinib
38.76 4.37
Fig. 7. Shifts in IC50 values at increasing incubation times for compounds 10h and 10q against A431 and WI-26 AV cells.
shifts in the IC50 values for 10h and 10q against the said cell lines.
Such strikingly different modes of action for 10q and the
remaining hit compounds could be hypothetically related to
differing mechanisms of the observed anticancer effects exerted by
these compounds. In fact, as the only compound in the set based on
the planar 2H-benzo [h]chromen-2-one nucleus, 10q could poten-
tially bind a wide range of essential biomolecules, including DNA,
which is unlikely the case for the other obtained compounds
bearing low-bulk non-cyclic substituents at their coumarin cores.
Being structurally similar to polycyclic aromatic hydrocarbon DNA
intercalators [70e72], should 10q exert genotoxicity and induce
apoptosis, which is typical for classical DNA-intercalating chemo-
therapeutics [73].
slightly decreased under the same conditions from 3.06% to 2.31%,
and the percentage of dead cells grew from 0.14% to 0.41%. Thereby,
dose-dependent induction of apoptosis was displayed by com-
pound 10q after incubation with A431 cancer cells (Fig. 8).
Apoptosis is known to be a highly regulated and controlled
biochemical process associated with caspase enzyme activity [80].
To further establish the cell death pathway induced by 10q the
activation of caspases 3/7 was evaluated. Expectedly, in a full
accordance with the obtained morphological data, dose-dependent
caspase 3/7 activation was vividly displayed by the compound 10q
as can be seen from Fig. 9 (Fig. 9).
In this manner the results of the study showed that apoptosis
was promoted by compound 10q through mediation of caspases in
the А431 cells.
2.4. Apoptosis detection by Annexin V-FITC and caspase 3/7 assays
3. Conclusion
Apoptosis, a physiological process of programmed cell death, is
disrupted in malignant cells [74]. Hence, apoptosis induction by
various chemotherapeutic agents has been widely exploited in the
context of cancer treatment [75]. Noticeably, sulfonamide based
hCA inhibitors are known to possess cytotoxic effect to malignant
cells by inducing apoptosis [76,77]. The same is true for a range of
recently reported sulfonamide bearing coumarins for which the
mechanism of anticancer action remains unraveled [78,79]. In this
context, Annexin V-FITC/propidium iodide (AV/PI) dual staining
assay was performed to examine the impact of 3-
In summary, hCA IX and XII are promising therapeutic targets
with regards to management of hypoxic solid tumors. A range of
primary sulfonamide and coumarin based inhibitors display
remarkable potency and isoform-selectivity toward these iso-
zymes. We have designed and synthesized a novel type of chimeric
molecules comprising primary sulfonamide group directly attached
to the coumarin core at its 3 position. The synthesis was performed
via introduction of ethyl 2-sulfamoylacetate into Knoevenagel
condensation with diversely substituted salicylaldehydes,
furnishing target molecules in moderate to high yields. Inhibitory
profiles of seventeen newly synthesized compounds against a panel
of four physiologically relevant hCA isoforms (hCA I, II, IX and XII)
have been determined. Noticeably, the obtained molecules were
generally potent hCA inhibitors, with many compounds possessing
KI values in double-digit nanomolar range. Encouragingly, tumor-
associated isoforms hCA IX and XII were significantly affected by
sulfamoylcoumarin 10q in 5e25
late apoptosis levels in A431 cells (Fig. 8). The obtained morpho-
logical data indicated that treatment of A431 cells with 25 M 10q
mM concentrations on early and
m
resulted into a substantial increase in the percent of Annexin V-
FITC-positive early apoptotic cells from 6.10% to 18.56% after 24 h
incubation which corresponds to 3-fold total increase as compared
to control. In the meantime, the fraction of late apoptotic cells
8

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
Fig. 8. Apoptosis induction caused by different concentrations of hit compound 10q against A431 cell line.
9

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
determined in open capillary tubes on Stuart SMP50 Automatic
Melting Point Apparatus. Analytical thin-layer chromatography
was carried out on UV-254 silica gel plates using appropriate elu-
ents. Compounds were visualized with short-wavelength UV light.
4.1.1. Preparation of ethyl 2-sulfamoylacetate (11) [53]
To a solution of ethyl 2-(chlorosulfonyl)acetate 14 [81,82]
(1.87 g, 10 mmol) in dry DCM (8 mL) under ice-cooling was slowly
added hexamethyldisilazane (2.2 mL, 10.5 mmol). Mixture was
stirred under cooling for 30 min, and then 30 min without bath.
Solvent was removed in vacuo. The oily residue was cooled in ice
bath, dissolved in ethanol (5 mL), and stirred for 45 min. Evapo-
ration of solvent gave white solid, 1.35 g (81%). 1H NMR (400 MHz,
CDCl3)
J ¼ 7.1 Hz, 3H).
d
5.25 (br.s, 2H), 4.31 (q, J ¼ 7.2 Hz, 2H), 4.14 (s, 2H), 1.35 (t,
4.1.2. General procedure for the preparation of sulfamoyl coumarins
10a-q
A
mixture of ethyl 2-sulfamoylacetate (10a-q) (167 mg,
Fig. 9. Caspase 3/7 activation exerted by 10q after incubation with A431 cells for 24 h
1.0 mmol) and corresponding salicylaldehyde (1.0 mmol) in n-
butanol (2 mL) was stirred in a closed vessel at 110 ꢁC for 2e6 h
(controlled by TLC). Upon cooling to ambient temperature, the
precipitate was collected, washed with ether (3 ꢂ 1.5 mL) and dried
in vacuo to afford the target coumarins 10a-q.
at different concentrations.
several hit compounds displaying selectivity over the widespread
cytosolic hCA II. In addition, weak inhibition was exerted towards
another cytosolic isozyme hCA I which is considered an off-target
protein for the therapeutically relevant hCA inhibitors. In this
context, antiproliferative effect of the obtained compounds has
been measured against three human cancer cell lines and one non-
tumorigenic healthy human cell line under hypoxic conditions.
Thus, while, U-87MG human glioblastoma cells were insensitive to
the treatment with the synthesized 3-sulfamoylcoumarins, RT4
human bladder cancer and especially A431 human epidermoid
carcinoma cells were efficiently inhibited by several newly ob-
tained derivatives in a dose-dependent manner. In fact, four hit
compounds 10h, 10i, 10k, and 10q have been discovered displaying
low nanomolar IC50 values against A431 cell line. Unexpectedly, the
behavior of the most potent and selective antiproliferative agent
10q turned out to drastically differ from the remaining hits. Thus,
nearly 10-fold shifts in IC50 values was observed for 10q at 24 and
72 incubation times, against both cancer and non-tumorigenic
human cells. Consequently, 10q was characterized as potent cyto-
toxic agent inducing apoptosis in A431 cells. Furthermore, dose-
dependent caspase 3/7 activation has been shown in presence of
this compound. Thus, as contrasted to hCA inhibitory compounds
10h, 10i, 10k which displayed poorly selective anticancer profile,
10q devoid of hCA IX/XII blocking activity, exhibited selective time-
and dose-dependent cytotoxic properties against A431 cell culture.
The mechanism of the observed cytotoxic effect requires further
investigation which will be reported in due course.
4.1.2.1. 2-Oxo-2H-chromene-3-sulfonamide (10a). Prepared from
salicylaldehyde according to the general procedure; yield: 181 mg
(80%). White solid; m. p. 255.1e256.5 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
7.3,1.6 Hz,1H), 7.51 (d, J ¼ 7.8 Hz,1H), 7.50 (br.s, 2H), 7.46 (td, J ¼ 7.5,
1.0 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO‑d6)
155.8, 154.6, 144.7,
d
8.75 (s,1H), 8.01 (dd, J ¼ 7.8,1.6 Hz,1H), 7.77 (dd, J ¼ 8.7,
d
134.9, 131.1, 129.9, 125.7, 117.9, 116.8. HRMS (ESI), m/z calcd for
C9H7NNaO4S [MþNa]þ 247.9988 found 247.9993.
4.1.2.2. 5-Methoxy-2-oxo-2H-chromene-3-sulfonamide
(10
b).
Prepared from 6-methoxysalicylaldehyde according to the general
procedure; yield: 138 mg (54%). White solid; m. p. 250.6e251.4 ꢁC.
1H NMR (400 MHz, DMSO‑d6)
d
8.61 (d, J ¼ 0.7 Hz, 1H), 7.73 (t,
J ¼ 8.4 Hz, 1H), 7.51 (br.s, 2H), 7.09e7.03 (m, 2H), 3.99 (s, 3H). 13
C
{1H} NMR (101 MHz, DMSO‑d6)
d 157.6, 155.6, 155.5, 138.8, 136.3,
128.2, 108.9, 107.9, 107.3, 57.1. HRMS (ESI), m/z calcd for
C
10H9NNaO5S [MþNa]þ 278.0094 found 278.0094.
4.1.2.3. 5,7-Dimethyl-2-oxo-2H-chromene-3-sulfonamide
(10c).
Prepared from 4,6-dimethylsalicylaldehyde according to the gen-
eral procedure; yield: 149 mg (59%). White solid; m. p.
227.9e229.8 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
8.55 (s, 1H), 7.49
(br.s, 2H), 7.17 (s, 1H), 7.15 (s, 1H), 2.53 (s, 3H), 2.41 (s, 3H). 13C{1H}
NMR (101 MHz, DMSO‑d6) 155.8, 155.4, 146.2, 141.2, 138.8, 128.1,
d
128.0, 114.8, 114.1, 21.9, 18.2. HRMS (ESI), m/z calcd for
4. Experimental section
C
11H11NNaO4S [MþNa]þ 276.0301 found 276.0304.
4.1. General experimental procedures
4.1.2.4. 6-Methoxy-2-oxo-2H-chromene-3-sulfonamide
(10
d).
Prepared from 5-methoxysalicylaldehyde according to the general
All commercial reagents were used without purification. NMR
spectrum were recorded using Bruker Avance III spectrometer in
DMSO‑d6 (1H: 400.13 MHz; 13С: 100.61 MHz; 19F: 376.50 MHz);
procedure; yield: 214 mg (84%). Pale yellow solid; m. p.
242.4e243.6 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 8.70 (s, 1H), 7.60 (d,
J ¼ 3.0 Hz, 1H), 7.50 (br.s, 2H), 7.46 (d, J ¼ 9.1 Hz, 1H), 7.36 (dd,
chemical shifts are reported as parts per million (d, ppm); the re-
J ¼ 9.1, 3.0 Hz, 1H), 3.83 (s, 3H). 13C{1H} NMR (101 MHz, DMSO‑d6)
sidual solvent peak was used as internal standard: 2.50 ppm for 1H,
39.52 ppm for 13C; multiplicities are abbreviated as follows:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet,
br ¼ broad, dd ¼ doublet of doublets, dt ¼ doublet of triplets;
coupling constants, J, are reported in Hz. Mass spectrum were
recorded using Bruker microTOF spectrometer (ionization by
electrospray, positive ions detection). Melting points were
d 156.4, 156.0, 149.0, 144.5, 130.1, 122.5, 118.3, 117.9, 112.9, 56.3.
HRMS (ESI), m/z calcd for C10H9NNaO5S [MþNa]þ 278.0094 found
278.0098.
4.1.2.5. 6-Methyl-2-oxo-2H-chromene-3-sulfonamide
(10e).
Prepared from 5-methylsalicylaldehyde according to the general
procedure; yield: 163 mg (68%). White solid; m. p. 240.5e243.6 ꢁC.
10

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
1H NMR (400 MHz, DMSO‑d6)
d
8.65 (s, 1H), 7.77 (d, J ¼ 2.1 Hz, 1H),
procedure; yield: 156 mg (61%). Beige solid; m. p. 191.6e193.8 ꢁC.
7.57 (dd, J ¼ 8.5, 2.2 Hz, 1H), 7.50 (br.s, 2H), 7.39 (d, J ¼ 8.5 Hz, 1H),
1H NMR (400 MHz, DMSO‑d6)
d
8.67 (s, 1H), 7.92 (d, J ¼ 8.7 Hz, 1H),
7.40 (br.s, 2H), 7.11 (d, J ¼ 2.4 Hz, 1H), 7.06 (dd, J ¼ 8.7, 2.4 Hz, 1H),
3.91 (s, 3H). 13C{1H} NMR (101 MHz, DMSO‑d6)
165.0, 156.9, 156.2,
2.38 (s, 3H). 13C{1H} NMR (101 MHz, DMSO‑d6)
d
156.0, 152.8, 144.5,
135.8, 135.1, 130.4, 129.8, 117.5, 116.5, 20.7. HRMS (ESI), m/z calcd for
d
C
10H9NNaO4S [MþNa]þ 262.0145 found 262.0148.
144.9, 132.3, 126.1, 114.0, 111.2, 101.1, 56.7. HRMS (ESI), m/z calcd for
C
10H9NNaO5S [MþNa]þ 278.0094 found 278.0095.
4.1.2.6. 6-Hydroxy-2-oxo-2H-chromene-3-sulfonamide
(10f).
Prepared from 5-hydroxysalicylaldehyde according to the general
4.1.2.13. 8-Fluoro-2-oxo-2H-chromene-3-sulfonamide
(10
m).
procedure; yield: 181 mg (75%). Beige solid; m. p. >290 ꢁC. 1H NMR
Prepared from 3-fluorosalicylaldehyde according to the general
(400 MHz, DMSO‑d6)
7.36 (d, J ¼ 8.9 Hz, 1H), 7.30 (d, J ¼ 2.9 Hz, 1H), 7.19 (dd, J ¼ 9.0,
2.9 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO‑d6)
156.1, 154.7, 148.0,
d 9.82 (br.s, 1H), 8.67 (s, 1H), 7.52 (br.s, 2H),
procedure; yield: 175 mg (72%). White solid; m. p. 262.4e263.6 ꢁC.
1H NMR (400 MHz, DMSO‑d6)
d
8.78 (d, J ¼ 1.4 Hz, 1H), 7.83 (dt,
d
J ¼ 7.9, 1.2 Hz, 1H), 7.72 (dd, J ¼ 10.9, 8.3, 1.4 Hz, 1H), 7.60 (br.s, 2H),
144.7, 129.8, 123.0, 118.4, 117.7, 114.7. HRMS (ESI), m/z calcd for
C9H7NNaO5S [MþNa]þ 263.9937 found 263.9941.
7.44 (td, J ¼ 8.0, 4.7 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO‑d6)
d
154.7, 148.7 (d, J ¼ 248.7 Hz), 144.2 (d, J ¼ 2.8 Hz), 142.5 (d,
J ¼ 11.5 Hz), 130.7, 126.5 (d, J ¼ 3.7 Hz), 125.7 (d, J ¼ 6.9 Hz), 120.8 (d,
4.1.2.7. 6-Fluoro-2-oxo-2H-chromene-3-sulfonamide
(10
g).
J ¼ 16.9 Hz), 119.9. 19F{1H} NMR (376 MHz, DMSO‑d6)
d
ꢀ134.80.
Prepared from 5-fluorosalicylaldehyde according to the general
HRMS (ESI), m/z calcd for C9H6FNNaO4S [MþNa]þ 265.9894 found
procedure; yield: 182 mg (75%). White solid; m. p. 251.8e253.0 ꢁC.1H
265.9896.
NMR (400 MHz, DMSO‑d6)
7.65 (td, J ¼ 8.9, 3.1 Hz, 1H), 7.59 (br.s, 2H), 7.57 (dd, J ¼ 9.2, 4.5 Hz,
3H). 13C{1H} NMR (101 MHz, DMSO‑d6)
d
8.73 (s, 1H), 7.91 (dd, J ¼ 8.4, 3.0 Hz,1H),
4.1.2.14. 8-Hydroxy-2-oxo-2H-chromene-3-sulfonamide
(10n).
d
158.6 (d, J ¼ 241.8 Hz),
Prepared from 3-hydroxysalicylaldehyde according to the general
155.6, 151.1 (d, J ¼ 1.8 Hz), 143.8 (d, J ¼ 2.9 Hz), 130.8, 122.2 (d,
J ¼ 24.9 Hz), 118.9 (d, J ¼ 8.7 Hz), 118.8 (d, J ¼ 10.0 Hz), 116.0 (d,
procedure; yield: 166 mg (69%). Beige solid; m. p. >290 ꢁC
(decomp.). 1H NMR (400 MHz, DMSO‑d6)
d
8.67 (s, 1H), 7.81 (br.s,
J ¼ 24.6 Hz). 19F{1H} NMR (376 MHz, DMSO‑d6)
d
ꢀ116.92. HRMS
3H), 7.43e7.38 (m, 1H), 7.28e7.23 (m, 2H). 13C{1H} NMR (101 MHz,
(ESI), m/z calcd for C9H6FNNaO4S [MþNa]þ 265.9894 found
DMSO‑d6) d 155.8, 145.1, 145.0, 143.2, 129.7, 125.7, 121.1, 120.8, 118.8.
265.9896.
HRMS (ESI), m/z calcd for C9H7NNaO5S [MþNa]þ 263.9937 found
263.9935.
4.1.2.8. 6-Chloro-2-oxo-2H-chromene-3-sulfonamide
(10
h).
Prepared from 5-chlorosalicylaldehyde according to the general
4.1.2.15. 8-Methoxy-2-oxo-2H-chromene-3-sulfonamide
(10ꢁ).
procedure; yield: 177 mg (68%). White solid; m. p. 271.2e272.3 ꢁC.
Prepared from 3-methoxysalicylaldehyde according to the general
1H NMR (400 MHz, DMSO‑d6)
d
8.73 (s, 1H), 8.15 (d, J ¼ 2.6 Hz, 1H),
7.80 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.57 (br.s, 2H), 7.55 (d, J ¼ 8.9 Hz, 1H).
13C{1H} NMR (101 MHz, DMSO‑d6)
155.5,153.3,143.5,134.3,130.9,
procedure; yield: 189 mg (74%). White solid; m. p. 244.6e245.5 ꢁC.
1H NMR (400 MHz, DMSO‑d6)
d
8.71 (s, 1H), 7.54 (dd, J ¼ 7.8, 1.5 Hz,
1H), 7.51 (br.s, 2H), 7.45 (dd, J ¼ 8.2, 1.5 Hz, 1H), 7.38 (t, J ¼ 7.9 Hz,
1H), 3.94 (s, 3H). 13C{1H} NMR (101 MHz, DMSO‑d6)
155.5, 146.8,
d
129.9, 129.3, 119.3, 118.8. HRMS (ESI), m/z calcd for C9H6ClNNaO4S
[MþNa]þ 281.9598 found 281.9599.
d
144.9, 143.9, 130.0, 125.7, 121.9, 118.4, 117.0, 56.7. HRMS (ESI), m/z
calcd for C10H9NNaO5S [MþNa]þ 278.0094 found 278.0097.
4.1.2.9. 6-Bromo-2-oxo-2H-chromene-3-sulfonamide
(10i).
Prepared from 5-bromosalicylaldehyde according to the general
4.1.2.16. 8-Ethoxy-2-oxo-2H-chromene-3-sulfonamide
(10p).
procedure; yield: 204 mg (67%). Pale yellow solid; m. p.
Prepared from 3-ethoxysalicylaldehyde according to the general
281.8e283.6 ꢁC (decomp.). 1H NMR (400 MHz, DMSO‑d6)
d
8.72 (s,
procedure; yield: 167 mg (62%). Pale tan solid; m. p.178.0e179.8 ꢁC.
1H), 8.28 (d, J ¼ 2.4 Hz, 1H), 7.91 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.57 (br.s,
1H NMR (400 MHz, DMSO‑d6)
d
8.71 (s, 1H), 7.53 (dd, J ¼ 7.7, 1.4 Hz,
2H), 7.48 (d, J ¼ 8.9 Hz, 1H). 13C{1H} NMR (101 MHz, DMSO‑d6)
1H), 7.51 (br.s, 2H), 7.44 (dd, J ¼ 8.3, 1.5 Hz, 1H), 7.38e7.34 (m, 1H),
d
155.4, 153.7, 143.5, 137.1, 132.9, 130.9, 119.8, 119.1, 117.2. HRMS (ESI),
4.21 (q, J ¼ 6.9 Hz, 2H), 1.42 (t, J ¼ 7.0 Hz, 3H). 13C{1H} NMR
m/z calcd for C9H6BrNNaO4S [MþNa]þ 325.9093 found 325.9097.
(101 MHz, DMSO‑d6) d 155.6, 146.1, 145.0, 144.0, 130.0, 125.7, 121.9,
118.5, 117.9, 65.1, 15.0. HRMS (ESI), m/z calcd for C11H11NO5S
4.1.2.10. 6-Nitro-2-oxo-2H-chromene-3-sulfonamide
(10j).
[MþH]þ 270.0431 found 270.0431.
Prepared from 5-nitrosalicylaldehyde according to the general
procedure; yield: 111 mg (41%). Beige solid; m. p. 241.4e243.6 ꢁC.
4.1.2.17. 3-Oxo-3H-benzo[f]chromene-2-sulfonamide
(10q).
1H NMR (400 MHz, DMSO‑d6)
d
9.03 (d, J ¼ 2.7 Hz, 1H), 8.94 (s, 1H),
8.53 (dd, J ¼ 9.2, 2.8 Hz, 1H), 7.72 (d, J ¼ 9.2 Hz, 1H), 7.65 (br.s, 2H).
13C{1H} NMR (101 MHz, DMSO‑d6)
158.2, 155.0, 144.4, 143.7, 131.6,
Prepared from 2-hydroxy-1-naphthaldehyde according to the
general procedure; yield: 91 mg (33%). Yellow solid; m. p. >290 ꢁC.
d
1H NMR (400 MHz, DMSO‑d6)
d
9.25 (s, 1H), 8.53 (d, J ¼ 8.4 Hz, 1H),
129.0, 126.9, 118.5, 118.4. HRMS (ESI), m/z calcd for C9H6N2NaO6S
[MþNa]þ 292.9839 found 292.9845.
8.34 (d, J ¼ 9.0 Hz, 1H), 8.10 (dd, J ¼ 8.2, 1.3 Hz, 1H), 7.80 (dd, J ¼ 8.4,
7.0, 1.4 Hz, 1H), 7.68 (dd, J ¼ 8.0, 6.9, 1.0 Hz, 1H), 7.64 (d, J ¼ 9.1 Hz,
1H), 7.58 (br.s, 2H). 13C{1H} NMR (101 MHz, DMSO‑d6)
d 155.8,
4.1.2.11. 6,8-Dichloro-2-oxo-2H-chromene-3-sulfonamide (10 k).
Prepared from 3,5-dichlorosalicylaldehyde according to the general
procedure; yield: 88 mg (30%). Pale yellow solid; m. p.
155.2, 139.9, 136.5, 130.4, 129.7, 129.6, 129.5, 128.9, 127.1, 122.6,
117.0,111.9. HRMS (ESI), m/z calcd for C13H10NO4S [MþH]þ 276.0325
found 276.0328.
217.7e218.8 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 8.73 (s, 1H), 8.14 (d,
J ¼ 2.5 Hz, 1H), 8.09 (d, J ¼ 2.3 Hz, 1H), 7.65 (br.s, 2H). 13C{1H} NMR
4.2. Carbonic anhydrase inhibition assay
(101 MHz, DMSO‑d6)
d 154.7, 149.2, 143.3, 133.6, 131.6, 129.2, 129.0,
121.5, 120.5. HRMS (ESI), m/z calcd for C9H5Cl2NNaO4S [MþNa]þ
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity [54]. Phenol red
(at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
315.9209 found 315.9211.
4.1.2.12. 7-Methoxy-2-oxo-2H-chromene-3-sulfonamide
(10
l).
Prepared from 4-methoxysalicylaldehyde according to the general
11

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
strength), following the initial rates of the CA-catalyzed CO2 hydra-
tion reaction for a period of 10e100 s. The CO2 concentrations ranged
from 1.7 to 17 mM for the determination of the kinetic parameters
and inhibition constants. For each inhibitor at least six traces of the
initial 5e10% of the reaction have been 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 inhibitor (0.1 mM) were prepared in distilledꢀdeionized 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 assay, in order to allow for
the formation of the EꢀI complex. The inhibition constants were
subsequently obtained by nonlinear least-squares methods using
PRISM 3 and the ChengꢀPrusoff equation, as reported earlier, and
represent the mean from at least three different determinations. All
CA isoforms were recombinant ones obtained in-house as reported
earlier [83e86].
to 3.5 ꢂ 104 cells per mL and the aliquots (7 ꢂ 103 cells per 200
mL)
were placed in individual wells in 96-multiplates (Eppendorf,
Germany) and incubated for 24 h. Triplicate wells were treated with
test compounds starting at 200.0 mM concentration and diluted at
various concentrations or DMSO (Sigma, USA) as control with final
concentration 0.1%. Plates were incubated for 72 h (or 24 h, 48 h) at
37 ꢁC in 5% CO2 atmosphere. After incubation, the cells were then
treated with 40 mL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, 5 mg mL-1 in PBS) and incubated
4 h. After an additional 4 h incubation, the medium with MTT was
removed and DMSO (150 mL) was added to dissolve the crystals
formazan. The plates were shaken for 10 min. The optical density of
each well was determined at 560 nm using a microplate reader
GloMax Multiþ (Promega, USA). Each of the tested compounds was
evaluated for cytotoxicity in three separate experiments.
4.6. Apoptosis assay
4.3. Сell culture
For the detection of apoptosis, the cells were plated at 6-well
culture plates (Eppendorf, Germany) and allowed to grow over-
night. After the cells reached subconfluency, the medium was
A431 epidermoid carcinoma, RT4 urinary bladder cancer cell, WI-
26 VA4 lung epithelial-like cells were purchased from the ATCC.
Primary fibroblasts were purchased from Cell Application Inc. A431
cells and Primary fibroblast cells were maintained in Advanced
DMEM (Gibco, UK) supplemented with 5% fetal bovine serum (FBS,
replaced with tested compounds (5 … 25 mM). The exposed cells
were placed at 37 ꢁC in a 5% CO2 incubator for 24 h. The cultured
cells were washed twice with PBS and resuspended in 1 ꢂ binding
buffer (AnnexinV-FITC kit, Invitrogen, USA) at a concentration
Gibco, UK), penicillin (100 UI mLꢀ1), streptomycin (100
m
g mLꢀ1) and
1 ꢂ 106 mLꢀ1. Annexin FITC (5
mL) and propidium iodide (PI, 2 mL)
were added to 100 mL of the cell suspension and incubated for
GlutaMax (3 mM, Gibco, UK). RT4 cells were maintained in McCoy's
5 A media (Gibco, UK) supplemented with 10% fetal bovine serum
(FBS, Gibco, UK), penicillin (100 UI mLꢀ1), streptomycin
15 min at room temperature (25 ꢁC) in the dark. After incubation
400
L of 1 ꢂ binding buffer was added to each tube and the stained
m
(100
m
g mLꢀ1), and GlutaMax (1.5 mM, Gibco, UK). WI-26 VA4 cells
cells were analyzed within 1 h using CytoFlex (Beckham Culture,
USA) and CytExpert 2.1 program. Since, Annexin V FITC staining
precedes the loss of membrane integrity that accompanies the later
stage identified by PI, Annexin FITC positive, PI negative indicates
early apoptosis, while the viable cells are Annexin V FITC negative,
PI negative. The cells that are in late apoptosis, or dead are both
Annexin V FITC and PI positive.
were maintained in Advanced MEM (Gibco, UK) supplemented with
5% fetal bovine serum (FBS, Gibco, UK), penicillin (100 UI mLꢀ1),
streptomycin (100 m
g mLꢀ1), and GlutaMax (1.87 mM, Gibco, UK). All
cells line cultivation under a humidified atmosphere of 95% air/5%
CO2 at 37 ꢁC. Subconfluent monolayers, in the log growth phase,
were harvested by a brief treatment with TrypLE Express solution
(Gibco, UK) in phosphate buffered saline (PBS, Capricorn Scientific,
Germany) and washed three times in serum-free PBS. The number of
viable cells was determined by trypan blue exclusion.
4.7. Caspase 3/7 assay
For the detection of caspase 3/7 activation, the cells were plated
at 6-well culture plates (Eppendorf, Germany) and allowed to grow
overnight. After the cells reached subconfluency, the medium was
4.4. Antiproliferative assay (end point)
The effects of the synthesized compounds on cell viability were
determined using the MTT colorimetric test. All examined cells
were diluted with the growth medium (or medium containing 100
mМ CoCl2 for chemically-induced hypoxia) to 3.5 ꢂ 104 cells per mL
replaced with tested compounds (5 … 25 mM). The exposed cells
were placed at 37 ꢁC in a 5% CO2 incubator for 24 h. The cultured
cells were washed twice with PBS and resuspended in 1 mL fresh
PBS at a concentration 1 ꢂ 106 mLꢀ1. Diluted the CellEvent™
Caspase-3/7 Green Detection Reagent into cells suspension to a
and the aliquots (7 ꢂ 103 cells per 200
mL) were placed in individual
wells in 96-multiplates (Eppendorf, Germany) and incubated for
24 h. The next day the cells were then treated with synthesized
compounds separately concentration 100 mМ and incubated for
24 h at 37 ꢁC in 5% CO2 atmosphere. Each compound was tested in
final concentration of 4 m
M and incubated for 30 min at 37 ꢁC in a
5% CO2 incubator. After incubation, stained cells were analyzed
within 20 min using CytoFlex (Beckham Culture, USA) and CytEx-
pert 2.1 program.
triplicate. After incubation, the cells were then treated with 40
mL
MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-
Declaration of competing interest
diphenyltetrazolium bromide, 5 mg mL-1 in PBS) and incubated
4 h. After an additional 4 h incubation, the medium with MTT was
removed and DMSO (150 mL) was added to dissolve the crystals
formazan. The plates were shaken for 10 min. The optical density of
each well was determined at 560 nm using a microplate reader
GloMax Multiþ (Promega, USA). Each of the tested compounds was
evaluated for cytotoxicity in three separate experiments.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This research was supported by the Russian Science Foundation
(project grant 21-73-20264). We are grateful to the Centre for
Chemical Analysis and Materials Research of Saint Petersburg State
University Research Park for the high-resolution mass-spectrom-
etry data.
4.5. MTT assay
All examined cells were diluted with the growth medium (or
medium containing 100 mМ CoCl2 for chemically-induced hypoxia)
12

D. Dar'in, G. Kantin, S. Kalinin et al.
European Journal of Medicinal Chemistry 222 (2021) 113589
https://doi.org/10.1080/14756366.2020.1766455.
[24] A. Angeli, F. Carta, A. Nocentini, J.-Y. Winum, R. Zalubovskis, A. Akdemir,
V. Onnis, W.M. Eldehna, C. Capasso, G. De Simone, S.M. Monti, S. Carradori,
W.A. Donald, S. Dedhar, C.T. Supuran, Carbonic anhydrase inhibitors targeting
metabolism and tumor microenvironment, Metabolites 10 (2020) 412,
https://doi.org/10.3390/metabo10100412.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113589.
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bonic anhydrases, Int. J. Mol. Sci. 22 (2021) 1e17, https://doi.org/10.3390/
ijms22063171.
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