Insertion of metal carbenes into the anilinic N–H bond of unprotected aminobenzenesulfonamides delivers low nanomolar inhibitors of human carbonic anhydrase IX and XII isoforms

Article abstract of DOI:10.1016/j.ejmech.2021.113352

Herein we report the synthesis of a set of thirty-four primary sulfonamides generated via formal N–H-insertion of metal carbenes into anilinic amino group of sulfanilamide and its meta-substituted analog. Obtained compounds were tested in vitro as inhibitors of four physiologically significant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Many of the synthesized sulfonamides displayed low nanomolar K_i values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Provided the promising activity profiles of the substances towards tumor-associated hCA IX and XII isozymes, single-concentration MTT test was performed for the entire set. Disappointingly, most of the discovered hCA inhibitors did not significantly suppress the growth of cancer cells either in normoxia or CoCl₂ induced hypoxic conditions. The only two compounds exerting profound antiproliferative effect turned out to be modest hCA inhibitors. Their out of the range activity in cells is likely attributive to the presence of Michael acceptor substructure which can potentially act either through the inhibition of Thioredoxin reductases (TrxRs, EC 1.8.1.9) or nonspecific covalent binding to cell proteins.

Full text of DOI:10.1016/j.ejmech.2021.113352

European Journal of Medicinal Chemistry 218 (2021) 113352
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Insertion of metal carbenes into the anilinic NeH bond of unprotected
aminobenzenesulfonamides delivers low nanomolar inhibitors of
human carbonic anhydrase IX and XII isoforms
a
a
a
b
Tatiana Sharonova , Polina Paramonova , Stanislav Kalinin , Alexander Bunev ,
b
c
a
a
Rovshan Е. Gasanov , Alessio Nocentini , Vladimir Sharoyko , Tatiana B. Tennikova ,
Dmitry Dar’in , Claudiu T. Supuran
Saint Petersburg State University, Saint Petersburg, 199034, Russian Federation
Medicinal Chemistry Center, Togliatti State University, Togliatti, 445020, Russian Federation
Neurofarba Department, Universita degli Studi di Firenze, Florence, 50019, Italy
a
c
,
**
, Mikhail Krasavin a
, *
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 February 2021
Received in revised form
Herein we report the synthesis of a set of thirty-four primary sulfonamides generated via formal NeH-
insertion of metal carbenes into anilinic amino group of sulfanilamide and its meta-substituted analog.
Obtained compounds were tested in vitro as inhibitors of four physiologically significant isoforms of the
metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Many of the synthesized sulfonamides
1
March 2021
Accepted 1 March 2021
Available online 16 March 2021
i
displayed low nanomolar K values against therapeutically relevant hCA II, IX, and XII, whereas they did
not potently inhibit hCA I. Provided the promising activity profiles of the substances towards tumor-
associated hCA IX and XII isozymes, single-concentration MTT test was performed for the entire set.
Disappointingly, most of the discovered hCA inhibitors did not significantly suppress the growth of
Keywords:
Carbonic anhydrase
Enzyme inhibitors
Diazo compounds
The ‘SAFE’ protocol
Metal carbenes
2
cancer cells either in normoxia or CoCl induced hypoxic conditions. The only two compounds exerting
profound antiproliferative effect turned out to be modest hCA inhibitors. Their out of the range activity in
cells is likely attributive to the presence of Michael acceptor substructure which can potentially act either
through the inhibition of Thioredoxin reductases (TrxRs, EC 1.8.1.9) or nonspecific covalent binding to cell
proteins.
NeH insertion
Primary sulfonamides
Stopped-flow assay
Nanomolar inhibition
Cancer cells
©
2021 Elsevier Masson SAS. All rights reserved.
MTT-Test
Hypoxic environment
1
. Introduction
activity and subcellular localization [8,9]. 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 [10,11]. Some hCAs play pivotal roles in fundamental
Carbonic anhydrases (CA, EC 4.2.1.1) is a well-explored super-
family of methalloenzymes that catalyze the interconversion be-
tween carbon dioxide and bicarbonate ion (CO
2
þ H
þ H ) in living organisms [1,2]. Eight evolutionary unrelated
gene families have been identified so far, including -, -, -, -, -,
-, , and -CAs [3e7]. In human, CAs are represented by 15 isoforms
which differ significantly in terms of tissue distribution, catalytic
2
O #
2
physiological processes, such as CO -homeostasis, pH-regulation,
ꢀ
þ
HCO
3
electrolyte secretion, gluconeogenesis, lipogenesis, bone resorp-
tion, etc. [12,13]. Furthermore, their abnormal activity or expression
profiles have been linked to a range of diseases, such as glaucoma,
edema, obesity, neuropathic pain and cancer [14e20]. Accordingly,
many hCA isoforms have been validated as valuable therapeutic
targets and novel links to pathological disorders continue to be
discovered [21e23].
a
b g d z
h
q
i
*
Corresponding author.
Primary sulfonamides are the most investigated class of hCA
inhibitors [24]. Indeed, many aromatic sulfonamides exert
** 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.2021.113352
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
profound pan-isoform inhibitory activity against hCAs (Fig. 1А)
parallel and diversity-oriented synthesis of small molecules which
is of utmost importance for medicinal chemistry [45]. Indeed, such
approach allowed us to cover hitherto unexplored chemical space
by involving diazo compounds in diverse reactions yielding novel
derivatives of various privileged scaffolds (Fig. 2) [46e50].
[
25]. In the meantime, the design of therapeutically relevant
isozyme-selective agents traditionally relies on the ‘tail-approach’
26]. This strategy implies decoration of the aromatic sulfonamide
[
warhead with various ‘tails’ capable of interacting with non-
conservative amino acid residues on the middle and outer parts
of the active site cavity, thus conferring improved ligand binding
and isozyme selectivity [27e31]. It is this approach which gave rise
to a range of highly potent and selective inhibitors reported in last
decades which are currently under extensive evaluation in pre-
clinical or clinical settings (Fig. 1B) [32e35].
Despite the fact, that numerous X-ray structures of hCAs with
small molecule ligands are available nowadays, rational approaches
to the structure-based design of isozyme-selective inhibitors are
still in their infancy [38e40]. Therefore, screening of small mole-
cule compound libraries (preferably meeting the ‘tail-approach’
requirements) remains the main source of the insights on the
structure-activity relationship (SAR) of inhibitors targeting various
therapeutically relevant hCA isoforms [41]. Thus, extensive syn-
thetic efforts furnishing versatile drug-like sulfonamides are of
importance in medicinal chemistry as they serve to further deci-
pher the design principles of the isozyme selective hCA inhibitors
as well as investigate the translation of their inhibitory properties
into the desired response in cell culture/animal disease models and
in human [42].
Among other findings, we showed that Rh
2 2
(esp) -catalyzed
coupling of -diazo- -butyrolactams with aromatic amines can be
a
g
performed with moderate to good yields [51]. Remarkably the re-
action displayed tolerance to the presence of such functional
groups as primary sulfonamide (Scheme 2) [51]. This gave us an
idea that diazo compounds can be employed in the generation of
small molecule sulfonamide libraries within our research program
aiming at development of hCA inhibitors.
Herein we report the results of our study including preparation
of novel sulfonamide-bearing small molecules from mono- and
dicarbonyl diazo compounds as well as evaluation of their hCA
inhibitory and antiproliferative profile.
2. Results and discussion
2.1. Synthesis of a set of primary sulfonamides
The coupling of
aminobenzenesulfonamide 12a was performed as reported earlier
51]. Briefly, -diazo- -butyrolactams 14a-c were dissolved in
anhydrous 1,4-dioxane and 12a was added along with Rh (esp)
a-diazo-g-butyrolactams 14a-c with 4-
[
a
g
Diazo compounds are valuable building blocks furnishing metal
carbenoid intermediates for the synthesis of vast variety of
medicinally relevant scaffolds [43]. However, the generation of
diazo compounds has been traditionally associated with sulfonyl
azide-based diazo transfer reagents known to be unsafe in handling
due to the high potential of explosion hazards [44]. This limitation
often motivated researchers to look for alternative synthetic routes
2
2
catalyst (1 mol%). The reaction was complete within 10 min at room
temperature and furnished corresponding sulfonamide-bearing
lactams 15a-c in modest to good yields (Scheme 3).
While
a-diazo-g-butyrolactams reacted with 12a at ambient
conditions (their high reactivity apparently stems from the certain
ring strain and decreased conjugation of the carbene anion with the
carbonyl group), linear a-diazo monocarbonyl compounds 16 were
(
which is especially true in the context of industrial applications)
and hampered the exploration of chemical space areas accessible
via diazo chemistry [44]. To address the said inconveniences, we
recently reported the sulfonylazide-free (‘SAFE’) protocol for the
generation of diazo compounds in basic aqueous medium (Scheme
less reactive and hence somewhat more forcing reaction conditions
were required. After brief experimentation, we modified the reac-
tion protocol using the mixture of 1,4-dioxane and toluene (3:4)
which ensured both reasonable heating and dissolution of the re-
agents. This allowed for the preparation of compounds 17a-l using
1
) [45].
Furthermore, we have demonstrated that using this protocol,
diazo compounds generated in situ can be further employed for
3
3
sulfonamides 12a (R ¼ 4-SO
2
NH
2
2 2
) and 12b (R ¼ 3-SO NH ) in
modest to moderate yields (Scheme 4).
Fig. 1. (A) Examples of aromatic sulfonamide-based pan-isoform hCA inhibitors 1 [36] and 2 [37]. (B) Examples of potent and isozyme selective hCA inhibitors 3 [32], 4 [33], 5 [34]
and SLC-0111 [35] designed using the ‘tail-approach’.
2

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
Scheme 1. ‘SAFE’ approach for the in situ generation of diazo compounds [45].
Fig. 2. Examples of privileged scaffold-based small molecules 6 [46], 7 [47], 8 [48], 9 [49], and 10 [50] synthesized in our group with the use of diazo compounds obtainable via the
‘
SAFE’ protocol.
Noticeably, when linear 1,3-dicarbonyl diazo compounds 18
the NeH insertion, double bond migration was observed under the
reaction conditions, furnishing maleimides 22a-c in good yields
(Scheme 6).
were introduced to the reaction with 12a,b in some cases the Wolf
rearrangement [34] was observed under the same conditions. In-
dividual compounds were then isolated using column chromatog-
raphy. Thus, not only NeH insertion products 19a-j were obtained
in modest to good yields, but also Wolf rearrangement products
2.2. Inhibitory profiles of the synthesized compounds against a
panel of hCAs
2
1
0a-f as shown in Scheme 5. It should be noted that for compounds
1
9f and 20e the H NMR spectra display two partially overlapping
We investigated the hCA inhibitory activities of the synthesized
compounds by means of the stopped-flow carbon dioxide hydra-
tase assay in comparison to acetazolamide (AAZ) as reference CAI
sets of signals due to keto-enol tautomerism (see Experimental
Section).
Finally, (E)-3-arylidene-4-diazopyrrolidine-2,5-diones 21a-c
were employed in the same reaction. Interestingly, in addition to
[
52]. The panel of enzymes included four physiologically significant
isoforms, the cytosolic hCA I and II, as well as the transmembrane,
3

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
2 2
Scheme 2. Rh (esp) -catalyzed coupling of a-diazo-g-butyrolactam 11 with 4-aminobenzenesulfonamide 12a [51].
Scheme 3. Synthesis of sulfonamide-bearing g-butyrolactam derivatives 15a-c.
Scheme 4. Coupling of linear a-diazo monocarbonyl compounds 16 with sulfonamides 12a,b.
tumor-associated hCA IX and XII. The inhibitory profile of small
molecules against those isozymes is of interest for drug discovery in
a number of therapeutic areas [8,53,54]. For instance, hCA II and XII
are upregulated in the eyes of patients suffering from glaucoma and
are validated targets for intraocular pressure-lowering agents [19].
In contrast, hCA IX (and hCA XII) proved valuable extracellular tu-
mor biomarkers and their inhibitors attract growing attention for
the needs of cancers treatment and prognosis [14,55,56]. Finally,
cytosolic hCA I is considered an off-target enzyme for both anti-
glaucoma and anticancer therapeutic applications of CAIs [57]. The
results of the assay are outlined in Table 1.
The following structure-activity relationship trends can be un-
veiled from the inhibition data represented in Table 1.
4

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
Scheme 5. Reaction of linear 2-diazo 1,3-dicarbonyl compounds with sulfonamides 12a,b leading to NeH insertion products 19a-j or Wolf rearrangement products 20a-f.
Scheme 6. Reaction of (E)-3-Arylidene-4-diazopyrrolidine-2,5-diones with sulfonamides 12a,b.
(
i) Many of the generated compounds tend to possess moderate
or weak inhibitory activity against the cytosolic isoform hCA
order of magnitude more potent hCA I inhibitors as
compared to the m-substituted counterparts. This can be
3
I. Indeed, inhibition constants (K
i
’s) for sulfonamides 17i,17k,
exemplified with cases like the following: 19f (R ¼ 4-
3
19i, and 20e ranged between 1357 and 5876 nM, whereas for
H
2
NSO
2
C
6
H
4
, K
i
245.4 nM) vs 19g (R ¼ 3-H
2 2 6 4 i
NSO C H , K
3
2
2a-c they exceeded 10 000 nM. Most of the remaining
1566 nM); 20d (R ¼ 4-H
2
NSO
2 6 4
C H , Ki 182.8 nM) vs 20e
NSO C H , Ki 1357 nM) and so on. Meanwhile, this
tendency was not observed among monocarbonyl de-
3
representatives blocked the enzyme’s activity at sub-
micromolar concentrations, similarly to the standard AAZ (Ki
(R ¼ 3-H
2 2 6 4
2
50 nM). However, 15a, 17a, 17g, 19d, 19e, and 20a displayed
rivatives 17a-l. Among all tested compounds 17a turned out
double-digit nanomolar (i.e. 53.3e85.8 nM) K values thus
i
i
to show the highest activity against hCA I with the K value of
outperforming the reference drug. Noticeably, within the
homologous range of compounds 19a-c the inhibitory ac-
tivity against hCA I increased with the growth of lipophilicity
53.3 nM.
(ii) Inhibitory activity of the tested substances against the
physiologically dominant hCA II isoform was generally
higher than that versus hCA I. In fact, the K ’s of the vast
i
majority of the compounds laid in the double-digit nano-
molar region. Encouragingly, three derivatives 15b, 19j, and
20d turned out to be low nanomolar inhibitors of hCA II
1
3
1
2
with K
i
’s 166.5 nM (R , R ¼ OMe), 134.9 nM (R , R ¼ OEt),
1
2
and 105.3 nM (R , R ¼ O-i-Pr) respectively. Furthermore,
1
2
compound 19d (R , R ¼ O(CH
2
)
2
OMe) showed even better
of 85.8 nM. Interestingly,
inhibition of this isozyme with K
i
among dicarbonyl derivatives 19a-j, 20a-f p-amino-
benzenesulfonamide bearing compounds were nearly one
displaying K
with 19f (K
i
’s 8.1, 5.9, and 6.2 nM correspondingly. Together
11.3 nM) these frontrunners outperformed the
i
5

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
Table 1
Inhibition data of human hCA isoforms hCA I, II, IX and XII for compounds 15a-c, 17a-l, 19a-j, 20a-f and 22a-c using acetazolamide (AAZ) as a reference.
Cmpd
R1
R2
R3
K
i
(nM)a
hCA I
hCA II
hCA IX
hCA XII
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
5a
5b
5c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
9a
9b
9c
9d
9e
9f
9g
9h
9i
Ph
3-CF
2-thiazolyl
4-MeC
4-MeC
4-MeOC
4-MeOC
t-Bu
H
H
H
H
H
H
H
H
4-SO
4-SO
4-SO
4-SO
3-SO
4-SO
3-SO
3-SO
4-SO
3-SO
4-SO
4-SO
4-SO
3-SO
4-SO
4-SO
4-SO
4-SO
4-SO
4-SO
4-SO
3-SO
4-SO
3-SO
4-SO
4-SO
3-SO
4-SO
4-SO
3-SO
4-SO
4-SO
3-SO
4-SO
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
NH
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
62.0
36.9
8.1
9.5
24.9
13.8
18.8
67.4
208.4
81.7
189.5
24.1
33.2
83.7
63.0
35.7
11.3
36.1
49.2
72.4
57.2
61.4
45.0
24.7
21.8
82.9
16.5
167.6
26.5
66.2
113.5
41.6
35.6
59.6
22.6
128.5
1067
87.9
5.7
3
C
6
H
4
462.9
137.6
53.3
293.8
124.5
919.7
343.4
242.7
70.6
134.8
5876
146.4
3951
202.3
166.5
134.9
105.3
85.8
19.7
36.8
15.2
63.9
10.1
54.3
41.7
89.1
45.9
54.3
6.5
25.8
43.4
94.9
38.6
123.9
81.5
144.1
123.3
154.7
73.5
78.1
409.6
91.6
71.8
27.6
20.3
18.5
55.2
11.3
277.6
28.6
161.3
5.9
66.3
87.5
57.2
6.2
98.7
48.0
2751
3163
4635
12.5
6
H
H
4
6
4
6
H
4
4
6
H
Et
Et
2
N
N
H
H
2
OEt
OEt
4-ClC
3,4-(MeO) C H
2 6 3
H
H
6
H
4
4-ClC
4-ClC
4-ClC
OMe
OEt
6
6
6
H
H
H
4
CH
4
CH
4
CH
2
2
2
-piperazine
- piperazine
- piperazine
21.2
116.1
6.4
H
OMe
OEt
O-i-Pr
58.1
52.4
28.1
36.8
64.1
18.0
58.6
12.5
91.9
25.6
32.0
70.1
61.5
30.1
71.1
34.3
81.9
635.2
68.0
25.0
O-i-Pr
O(CH
OMe
OEt
OEt
OEt
OEt
OMe
OEt
OEt
OEt
Ph
Ph
4-ClC
4-MeC
4-MeC
Ph
2
)
2
OMe
O(CH
2
)
2
OMe
CH
Ph
Ph
4-MeOC
4-MeOC
2-MeOC
i-Pr
i-Pr
Ph
i-Pr
i-Pr
2
OMe
69.8
245.4
1566
313.9
2547
828.8
56.9
345.8
310.5
182.8
1357
215.2
>10 000
>10 000
>10 000
250
6
6
6
H
H
H
4
4
4
9j
0a
0b
0c
0d
0e
0f
2a
2b
2c
6
H
4
Me
4-ClC
4-ClC
6
H
H
4
4
6
H
H
4
4
6
6
2-MeOC
6
H
4
AAZ
-
1
2
*
*R , R ¼ e(CH
2 5
) e.
a
Mean from three different assays, by a stopped flow technique (errors were in the range of ± 5e10% of the reported values).
reference drug AAZ whose K
i
value was 12.5 nM. In contrast,
the
differences
between
m-
and
p-amino-
maleimides 22a-c again showed negligible inhibition of the
enzyme, as their Ki’s ranged between 2751 and 4635 nM. As
in the case of hCA I, the inhibition activity against hCA II
benzenesulfonamide bearing derivatives. Indeed, most of the
reported compounds showed very efficient inhibitory activ-
ity also against these isozymes with double-digit nanomolar
increased in the range of compounds 19a-d with K
i
’s
K
i
values prevailing throughout the whole set. These values
1
3
1
2
1
7
R
1.8 nM (R , R ¼ OMe), 27.6 nM (R , R ¼ OEt), 20.3 nM (R ,
were comparable to the reference drug AAZ (K 25.0 nM).
i
2
1
2
¼ O-i-Pr), and 18.5 nM (R , R ¼ O(CH
2
)
2
OMe) respec-
Therefore, it can be concluded that the synthesized mole-
cules are flexible enough to efficiently adopt the catalytic
cavity of hCA IX and no substantial hindrance is conferred
with the introduced substituents. Meanwhile, several most
potent inhibitors can be highlighted, such as 15a, 17c, 17i, 17l,
and 19h exhibiting Ki’s in the range between 6.4 and
12.5 nM. Interestingly, three of them (17c, 17i, 19h) incor-
porated alkoxy-substituted aromatic moieties as the ‘tail’
motif. In contrast, the only compound exhibiting less pro-
nounced, submicromolar activity toward hCA IX isoform
turned out to be 22b, a maleimide derivative endowed with
the m-aminobenzenesulfonamide warhead.
tively. It should be also noted that similarly to hCA I, hCA II
isoform was sensitive to the substitution pattern of the
dicarbonyl derivatives 19a-j, 20a-f with clear one order of
magnitude drop of the Ki values for the m-amino-
benzenesulfonamide bearing analogs. Thus, meta-position of
the sulfonamide function within the benzenesulfonamide
warhead can be considered generally detrimental pattern for
the compounds’ inhibitory activity against cytosolic hCA I
and II isoforms.
(
iii) As opposed to the cytosolic isozymes, tumor-associated
transmembrane protein hCA IX was nearly insensitive to
6

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
(
iv) Turning to another transmembrane isozyme e hCA XII,
known as promising target in the context of glaucoma
management as well as therapy of solid tumors, again,
remarkable activity of many of generated compounds could
2.3. Antiproliferative activity against cancer and normal cell lines
Among the potential clinical applications of hCA inhibitors, tu-
mor diagnostic and treatment has drawn the most significant
attention in the last decade [59]. Indeed, numerous studies showed
that overexpression of hCA IX and XII isoforms can serve as a robust
marker of malignant tissues [60,61]. Therefore, a wide range of
fluorescent and radiolabeled agents for tumor-visualization have
been developed, basing on either hCA IX/XII targeting biomolecules
or sulfonamide-bearing small molecular ligands [55,62]. Further-
more, the catalytic activity of hCA IX and XII largely ensures the
adaptation of tumor cells to unfavorable growth conditions char-
acterized by hypoxia and lack of nutrients [63]. In particular, the
catalytic activity of the extracellular domain of these enzymes plays
a key role in the functioning of a number of transport proteins and
helps avoid intracellular acidosis (maintaining the slightly alkaline
intracellular pH) [64,65]. At the same time, the extracellular acid-
ification induced by hCA IX (on average the microenvironment of
hypoxic tumors is characterized by pH 6.5e6.8) [66] can contribute
to the disruption of intercellular adhesion contacts by suppressing
the adhesion protein E-cadherin, as well as transformation of ma-
lignant cells into a less differentiated state [67] In this case, cancer
cells acquire the so-called “stem-like” phenotype (an increase in
the proportion of cancer stem cells in the tumor), which makes the
tumor phenotype more aggressive and resistant to many chemo-
therapeutic drugs [60]. Thus, recent data indicate the multiple roles
that hCA IX and XII isoforms play in the survival and proliferation of
cancer cells.
It should be stressed, however, that the discovery of anticancer
agents based on the small molecule hCA inhibitors is not a
straightforward task. Despite the fact that several clinical studies
are in progress employing well-known hCA inhibitors AAZ [68,69]
or SLC-0111 [35] as antineoplastic agents in combined therapy of
aggressive tumors, certain aspects remain unraveled in the process
of identification of novel promising drug candidates. Our recent
literature data analysis highlighted that potent inhibitory activities
of sulfonamides against isolated recombinant hCA IX and XII is
often not translated into antiproliferative properties at the stage of
2D cell culture models [42]. Considering that multiple reasons can
be assumed in each particular case (e. g., insufficient metabolic
stability of the molecule, it’s binding to off-target proteins etc.),
further screening efforts and biochemical studies are highly desired
in order to shed more light on this problem.
be observed. Specifically, 15b, 17j, and 19h exhibited K
between 11.3 and 16.5, therefore approaching the efficiency
level of AAZ (K 5.7 nM). Three compounds with K values
i
’s
i
i
above 100 nM, (17d, 19i, 22b) incorporated m-amino-
benzenesulfonamide warhead which is apparently undesir-
able in the context of hCA XII inhibition as well.
(
v) Taking a closer look at the compounds’ isoform-selectivity
profiles the following trends can be highlighted. Most of
compounds were less potent against hCA I compared to the
other hCA isoforms which is considered beneficial for the
medicinal applications of CAIs. It can be seen from Table 1
that the m-aminobenzenesulfonamide bearing compounds
were generally less active against all isoforms in question in
comparison to the p-aminobenzenesulfonamide-substituted
counterparts. However, the activity drop for the meta-
substituted derivatives was somewhat uneven. Indeed, such
substitution pattern tended to confer higher selectivity to-
ward transmembrane hCA IX and XII over cytosolic enzymes.
Compounds 19f and 19g can serve as examples for this trend.
Thus, upon the shift from the p-to the m-substituted war-
heads a 6.4-fold (from 245.4 to 1566 nM) activity decrease
was observed against hCA I, and 24.6-fold (from 11.3 to 277.6)
against hCA II. On the other hand, for the transmembrane
hCAIX and hCA XII isozymes only 3.3 (from 18.0 to 58.6 nM)
and 3.8 (form 21.8e82.9 nM) fold activity drop was observed,
respectively, thus showing that 19g is selective to hCAIX and
hCA XII. Aryl-substituted monocarbonyl derivatives 17c, 17d,
17i and 17l displayed certain level of selectivity towards hCA
IX. Of these substances, compound 17l incorporating cyclo-
heptan-2-one fragment was the most potent and displayed
the most attractive profile with the K ratios of 31.6 (hCA I/
i
hCA IX) and 14.3 (hCA II/hCA IX). Interestingly, m-amino-
benzenesulfonamide-bearing compound 17k acted as potent
and selective hCA XII inhibitor, while para-substituted 17j
also strongly affected hCA IX. Diazocarbonyl compound 19g
decorated with m-aminobenzenesulfonamide proved selec-
tive towards hCA IX/XII over the cytosolic isozymes. In
contrast, 19j demonstrated selectivity toward hCA II. The
latter profile was very close to that of 20d, whereas Wolf
rearrangement products 20a-c, 20e and 20f were generally
double-digit nanomolar inhibitors of hCA II, IX and XII and
displayed certain variations in their affinity to the off-target
hCA I. Finally, it should be noted that despite their modest
Provided our compounds revealed remarkable potency against
tumor-associated hCA IX and hCA XII we became interested in
evaluating their antiproliferative activity. To this end, we per-
formed the well-established MTT test in 96-well plate format.
Since, the expression of hCA IX in tumor cells is controlled by
inhibitory
activities,
p-aminobenzenesulfonamide
substituted maleimide derivatives 22a and 22c showed
hypoxia-inducible factor 1a (HIF-1a) [50] we employed both nor-
remarkable selectivity towards tumor associated isoforms.
moxic conditions and CoCl -induced hypoxia to perform the study
2
For instance, the K
i
ratios of 22c were found to be 147.1 (hCA
[70]. The results of a single-concentration screening against human
I/hCA IX) and 68.2 (hCA II/hCA IX). Summing up, many of
generated compounds displayed attractive selectivity pro-
files, in contrast to the reference inhibitor AAZ. In particular,
the low-nanomolar activity of compounds 15b, 19j, and 20d
against glaucoma-related hCA II and hCA XII, in combination
with their high selectivity over the off-target hCA I render
them promising in the context of continuing search for
intraocular-pressure lowering agents with enhanced safety
level and duration of action [16,58]. On the other hand,
several potent and selective inhibitors of hCA IX/XII such as
breast cancer cell lines MDA-MB-231 and MCF-7 are presented in
Figs. 3 and 4, respectively.
As evident from the screening results, compounds 15a-c, 17a-l,
19a-i, and 20a-e exhibited either no or mild antiproliferative ac-
tivity against both cancer cell lines at 100 mМ concentration. Of
note, no significant difference was observed between the com-
pounds’ profiles under normoxic and hypoxic conditions. Discour-
agingly, many of the discovered highly potent hCA IX/XII inhibitors
did not exert noticeable antiproliferative activity. From the entire
subset only four compounds 17h (K (hCA IX) 54.3 nM; K (hCA XII)
i
i
17i, 17l, and 19h, demonstrated attractive profiles for the
i i i
63.0 nM), 17l (K (hCA IX) 6.4 nM; K (hCA XII) 49.2 nM), 19g (K (hCA
anticancer drug development as these isoforms play crucial
roles in a range of cancer-related pathological processes [50].
i i i
IX) 58.6 nM; K (hCA XII) 82.9 nM), and 20f (K (hCA IX) 34.3 nM; K
(hCA XII) 22.6 nM) induced at least 30% decrease of the MDA-MB-
31 cell line viability. Furthermore, MCF-7 was only sensitive to 17c
2
7

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
Fig. 3. Cell viability MTT assay results for compounds 15a-c, 17a-l, 19a-i, 20a-e and 22a-c at 100 mМ concentration against MDA-MB-231 cell line under normoxic (shown in green)
and hypoxic conditions (100 M CoCl , shown in red). Values are displayed in % as the mean ± SEM of three experiments relative to control (0 M, 100% viability, not shown).
m
2
m
Fig. 4. Cell viability MTT assay results for compounds 15a-c, 17a-l, 19a-i, 20a-e and 22a-c at 100 mМ concentration against MCF-7 cell line under normoxic (shown in green) and
hypoxic conditions (100 M CoCl , shown in red). Values are displayed in % as the mean ± SEM of three experiments relative to control (0 M, 100% viability, not shown).
m
2
m
(
K
i
(hCA IX) 10.1 nM; K
i
(hCA XII) 81.7 nM) and 20f. On the other
Michael acceptors are known to block the activity of Thioredoxin
reductases (TrxRs, EC 1.8.1.9) which are another class of anticancer
drug targets attracting growing attention in recent years [69]. We
have previously hypothesized that combination of CAI and TrxR
inhibitory motifs within one chimeric compound can be a valuable
strategy for the design of antiproliferative agents [69]. Further-
more, there is a range of examples present in literature where such
combinations occasionally emerged, leading to increased cytotox-
icity [71e74]. On the other hand, Michael acceptors has been
known as so-called pan-assay interference compounds (PAINS)
[75]. Despite the fact that the presence of this motif in the CAIs
apparently does not lead to the false positive results within the
hand, maleimide derivatives 22a-c revealed profound anti-
proliferative action. Moreover, it was not the relatively potent hCA
inhibitor 22c showing better anticancer properties. Instead, 22a
and especially 22b with micromolar to submicromolar hCA Ki
values decreased cancer cells viability to the level of 10% and below.
Altogether, the obtained results confirmed the observation that
i
low K values against recombinant hCA IX/XII cannot be considered
as robust predictive factor regarding the anticancer potential of
sulfonamides. Indeed, neither of the low nanomolar hCA IX/XII
inhibitors identified in this study exerted substantial influence at
cancer cells growth under normoxic or hypoxic conditions, with
best results comprising only ca. 30% of cell viability reduction. In
the meantime, treatment of cancer cells with less anti-hCA active
derivatives 22a and 22b caused dramatic drop in the cancer cell
survival. High Ki values against the enzymes in question suggest
that the observed anticancer activity could unlikely be attributed to
the inhibition of hCA IX/XII. It should be taken into account that
these compounds incorporated another cytotoxicity-associated
motif, i.e. mild Michael acceptor electrophile. On the one hand,
2
stopped-flow CO hydratase assay [71e74], cell-based screening
results for such compounds should be taken with great caution
[75]. With these considerations in mind we made additional efforts
to validate our observations. In particular, IC50 values were deter-
mined for two most active compounds 22a and 22b against MDA-
MB-231 and MCF-7 cells as well as against normal cell line WI-26
VA4 (Table 2).
8

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
IC50 values of both 22a and 22b laid within the range between
carbon peaks. HPLC preparative chromatography was performed
1
0.84 and 37.21 mМ against all used cell lines either under normoxic
with Agilent PrepHT XDB-C18 preparative cartridge 21.2 ꢁ 150 mm
or hypoxic conditions. In contrast to what can be seen expected
from the single-concentration screening results, EC50 values of 22a
were slightly lower than those of 22b. In the meantime, no signif-
icant gap between the sensitivity of malignant or healthy cell cul-
tures towards the tested agents was observed. Thus, despite the fact
that antiproliferative action of 22a and 22b turned out to be dose-
dependent (see Supporting information for the dose-response
curves), these compounds did not show any selectivity towards
malignant cells over healthy once and therefore their potential for
medicinal application remains unclear.
2
5 mm. Product 15c was purified by HPLC eluting with H OeMeCN
with addition of 0.1% TFA (elution gradient MeCN: 40 / 60%); flow
ꢂ
rate 12 mL/min, column temperature 40 C.
4.1.1. General procedure (GP 1) for the preparation of sulfonamides
15a-c
Aminobenzenesulfonamide 12a,b (0.29 mmol) and corre-
sponding diazo lactam 14 (0.29 mmol) were dissolved in dry 1,4-
dioxane (5 mL). Rh
emission of nitrogen was observed. The mixture was stirred for
0 min and concentrated in vacuo. Obtained residues were purified
2 2
esp (1 mol-%) was added as catalyst and the
1
3
. Conclusion
by column chromatography on silica gel using chloroform/MeOH as
an eluent to afford compounds 15a-c. Compound 15c was purified
by HPLC as described above.
We demonstrated that diazo compounds can be efficiently used
for the generation of structurally diverse small molecule sulfon-
amides which are highly desired in the context of hCA inhibition-
related research domain. Inhibitory profiles of 34 newly synthe-
sized compounds against a panel of four physiologically significant
hCA isoforms (hCA I, II, IX and XII) have been determined. Inter-
estingly, the obtained molecules tended to possess selectivity to-
ward the therapeutically relevant hCA or hCA IX/XII over hCA I
which is considered an off-target protein. Furthermore, certain
4.1.1.1. 4-((2-Oxo-1-phenylpyrrolidin-3-yl)amino)benzenesulfona-
ꢂ
1
mide (15a). Yield 55 mg (31%). Beige solid, m.p. 228.1e229.5 C. H
NMR (400 MHz, DMSO‑d
7.72 (d, J ¼ 8.0 Hz, 2H, ArH), 7.54 (d,
J ¼ 8.7 Hz, 2H, ArH), 7.41 (t, J ¼ 7.9 Hz, 2H, ArH), 7.17 (t, J ¼ 7.4 Hz,1H,
ArH), 6.94 (s, 2H, NH
), 6.79 (d, J ¼ 8.8 Hz, 2H, ArH), 6.70 (d,
J ¼ 7.5 Hz, 1H, NH), 4.61e4.45 (m, 1H, CH- pyrrolid-2-one),
3.93e3.75 (m, 2H, CH
- pyrrolid-2-one), 2.62 (dtd, J ¼ 12.0, 8.2,
4.7 Hz, 1H, CH
- pyrrolid-2-one), 1.94 (dq, J ¼ 12.3, 9.4 Hz, 1H, CH
6
) d
2
2
substances displayed low nanomolar K
i
values against one or
2
2
-
13
several of these isoforms. Considering the promising inhibitory
activity of the generated molecules with respect to tumor-
associated hCA IX and XII, their antiproliferative properties have
been evaluated. Discouragingly, human breast cancer cell lines
MDA-MB-231 and MCF-7 displayed only a modest decrease in cell
viability when treated with 100 mМ of the obtained low nanomolar
hCA inhibitors. In contrast, a couple of least active hCA blockers
exhibited profound antiproliferative properties, which is likely
attributive of the mild Michael acceptor motif present in their
structures. In the meantime, the potential of the synthesized
compounds as intraocular pressure lowering agents remains to be
evaluated and will be reported in due course.
6
pyrrolid-2-one). C NMR (101 MHz, DMSO‑d ) d 172.69, 151.31,
139.99, 131.27, 129.24, 127.69, 124.72, 119.75, 112.03, 55.00, 44.90,
þ
þ
26.52. HRMS (ESI) m/z [MþNa] calculated for C16
17 3 3
H N O SNa
354.0876, found 354.0883.
4.1.1.2. 4-((2-Oxo-1-(3-(trifluoromethyl)phenyl)pyrrolidin-3-yl)
amino)benzenesulfonamide (15b). Yield 31 mg (61%). White amor-
1
phous solid. H NMR (400 MHz, DMSO‑d
6
)
d
8.24 (s, 1H, ArH), 7.89
(d, J ¼ 8.0 Hz, 1H, ArH), 7.67 (t, J ¼ 8.1 Hz, 1H, ArH), 7.54 (dd, J ¼ 7.9,
3.5 Hz, 3H, ArH), 6.95 (s, 2H, NH
), 6.79 (d, J ¼ 8.8 Hz, 2H, ArH), 6.72
(d, J ¼ 7.7 Hz, 1H, NH), 4.64e4.53 (m, 1H, CH-pyrrolid-2-one),
3.98e3.83 (m, 2H, CH -pyrrolid-2-one), 2.70e2.58 (m, 1H, CH
pyrrolid-2-one), 2.05e1.90 (m, 1H, CH
2
2
2
-
13
2
-pyrrolid-2-one). C NMR
C NMR (101 MHz, DMSO) 172.94, 150.72,
13
4
. Experimental section
(101 MHz, DMSO‑d
6
)
d
d
1
40.10, 130.92, 130.07, 129.44 (d, J ¼ 31.7 Hz), 127.20, 124.04 (q,
4.1. General experimental procedures
J ¼ 272.6 Hz), 122.57, 120.46 (q, J ¼ 3.7 Hz), 115.47 (q, J ¼ 4.2 Hz),
þ
1
11.58, 54.47, 44.35, 25.76. HRMS (ESI) m/z [MþNa] calculated for
þ
17 16 3 3 3
C H F N O SNa 422.0755, found 422.0757.
Diazo compounds used in this study were prepared as previ-
ously reported [45,76,77] other reagents were obtained from
commercial sources and used without additional purification. 1,4-
Dioxane and toluene were distilled from suitable drying agents.
Mass spectra were recorded with a Bruker Maxis HRMS-ESI-qTOF
spectrometer (electrospray ionization mode). NMR spectroscopic
data were recorded with Bruker Avance 400 spectrometer
4.1.1.3. 4-((2-Oxo-1-(thiazol-2-yl)pyrrolidin-3-yl)amino)benzene-
sulfonamide (15c). Yield 13 mg (15%). Beige solid, m.p. >300 C. 1H
NMR (400 MHz, DMSO‑d ) d 7.59e7.50 (m, 3H, ArH, CH-thiazole),
6
7.35 (d, J ¼ 3.5 Hz, 1H, NH), 6.95 (s, 2H, NH2), 6.78 (d, J ¼ 8.8 Hz,
3H, ArH, CH-thiazole), 4.84e4.68 (m, 1H, CH-pyrrolid-2-one), 4.20
(ddd, J ¼ 10.6, 9.1, 1.8 Hz, 1H, CH2- pyrrolid-2-one), 3.98e3.83 (m,
1H, CH2-pyrrolid-2-one), 2.66 (ddd, J ¼ 14.0, 9.8, 7.4 Hz, 1H, CH2-
pyrrolid-2-one), 2.15e1.96 (m, 1H, CH2-pyrrolid-2-one). 13C NMR
ꢂ
1
13
19
(
400.13 MHz for H, 100.61 MHz for C and 376.50 MHz for F) and
were referenced to residual solvent proton peaks and solvent
(
1
101 MHz, DMSO‑d
14.96, 112.14, 54.38, 44.94, 26.51. HRMS (ESI) m/z [MþH]þ calcu-
lated for C13H15N4O3S2þ 339.0579, found 339.0580.
6
) d 172.49, 157.54, 151.03, 138.18, 131.65, 127.74,
Table 2
IC50 values determined for maleimides 22a and 22b against cancer cell lines MDA-
MB-231 and MCF-7 and one normal cell line WI-26 VA4.
Compounds IC50
MCF7
Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia
,
mМa
4
.1.2. General procedure (GP 2) for the preparation of sulfonamides
MDA-MB-231
WI-26 VA4
17a-l, 19a-i, 20a-e and 22a-c
Aminobenzenesulfonamide 12a,b (0.29 mmol) was dissolved in
5
mL of dry mixture of 1,4-dioxane and toluene (3:4) under
refluxing. Then corresponding diazo compound 16, 18, 21a-c
0.35 mmol) and the catalyst Rh (OAc) (1 mol-%) were added and
2
2
2a
2b
10.84
14.90
11.52
15.28
16.55
37.21
15.61
36.77
21.91
22.03
26.15
35.61
(
2
4
a
Compound concentration required to reduce cell proliferation by 50%, as
the emission of nitrogen was observed. The mixture was refluxed
overnight and concentrated in vacuo. Obtained residues were pu-
rified by column chromatography on silica gel using chloroform/
determined with the MTT method, under conditions allowing untreated controls to
undergo at least two consecutive rounds of multiplication. Data represent mean
values from three independent determinations.
9

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
MeOH or DCM/EA as an eluent. Some of the resulting compounds
4.1.2.7. N,N-diethyl-2-((3-sulfamoylphenyl)amino)acetamide (17g).
Yield 197 mg (68%). White solid, m.p. 225.1e226.8 C. 1H NMR
ꢂ
(
19a, 19c, 22a-c) precipitated and did not require additional
purification.
(400 MHz, DMSO‑d
6
)
d
7.24 (t, J ¼ 7.9 Hz, 1H, ArH), 7.17 (s, 2H, NH2),
7
.07 (s, 1H, ArH), 7.02 (d, J ¼ 7.6 Hz, 1H, ArH), 6.84 (d, J ¼ 8.5 Hz, 1H,
ArH), 6.04 (t, J ¼ 4.5 Hz, 1H, NH), 3.92 (d, J ¼ 5.2 Hz, 2H, CH2),
3.43e3.24 (m, 4H, CH2eEt), 1.17 (t, J ¼ 7.0 Hz, 3H, CH3), 1.06 (t,
4.1.2.1. 4-((2-Oxo-2-(p-tolyl)ethyl)amino)benzenesulfonamide (17a).
ꢂ
Yield 89 mg (23%). Beige solid, m.p. 220.0e221.9 C. 1H NMR
J ¼ 7.0 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO‑d
6
) d 168.09, 148.97,
(
400 MHz, DMSO‑d
J ¼ 8.9 Hz, 2H, ArH), 7.38 (d, J ¼ 7.9 Hz, 2H, ArH), 6.93 (s, 2H, NH2),
.75 (d, J ¼ 8.9 Hz, 2H, ArH), 6.58 (t, J ¼ 5.5 Hz, 1H, NH), 4.74 (d,
J ¼ 5.5 Hz, 2H, CH2), 2.41 (s, 3H, Me). 13C NMR (101 MHz, DMSO‑d
195.96, 151.45, 144.58, 132.99, 131.15, 129.81, 128.48, 127.66,
11.89, 49.78, 21.69. HRMS (ESI) m/z [M ꢀ H]þ calculated for
C15H15N2O3Sþ 303.0788, found 303.0798.
6
)
d
7.98 (d, J ¼ 8.1 Hz, 2H, ArH), 7.53 (d,
1
(
45.20, 129.63, 115.89, 113.30, 109.39, 44.69, 14.55, 13.47. HRMS
ESI) m/z [MþNa]þ calculated for C12H19N3O3SNaþ 308.1025,
found 308.1039.
6
6
)
d
1
4.1.2.8. Ethyl 2-(4-chlorophenyl)-2-((4-sulfamoylphenyl)amino)ace-
ꢂ
tate (17h). Yield 46 mg (29%). White solid, m.p. 170.0e172.1 C. 1H
NMR (400 MHz, DMSO‑d
J ¼ 8.0 Hz, 1H, NH), 6.93 (s, 2H, NH2), 6.76 (d, J ¼ 8.8 Hz, 2H, ArH),
.42 (d, J ¼ 8.0 Hz, 1H, CH), 4.24e4.04 (m, 2H, CH2), 1.14 (t,
J ¼ 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO‑d 171.24, 149.94,
36.78, 133.29, 132.17, 129.89, 129.12, 127.59, 112.62, 61.75, 58.95,
4.41. HRMS (ESI) m/z [MþH]þ calculated for C16H18ClN2O4Sþ
6
) d 7.56e7.44 (m, 6H, ArH), 7.09 (d,
4
.1.2.2. 3-((2-Oxo-2-(p-tolyl)ethyl)amino)benzenesulfonamide (17b).
Yield 68 mg (18%). Beige amorphous solid. 1H NMR (400 MHz,
DMSO‑d
7.97 (d, J ¼ 7.8 Hz, 2H, ArH), 7.38 (d, J ¼ 7.8 Hz, 2H, ArH),
.23 (t, J ¼ 7.9 Hz, 1H, ArH), 7.15 (s, 2H, NH2), 7.11 (s, 1H, ArH), 7.01
d, J ¼ 7.5 Hz, 1H, ArH), 6.89e6.79 (m, 1H, ArH), 6.35 (t, J ¼ 5.3 Hz,
H, NH), 4.69 (d, J ¼ 5.4 Hz, 2H, CH2), 2.40 (s, 3H, Me). 13C NMR
101 MHz, DMSO‑d 196.18, 149.03,145.23,144.52,133.06,129.81,
29.66, 128.44, 115.79, 113.36, 109.43, 49.97, 21.69. HRMS (ESI) m/z
5
6
) d
6
) d
1
1
7
(
1
(
369.0677, found 369.0670.
6
) d
4.1.2.9. Ethyl 2-(3,4-dimethoxyphenyl)-2-((4-sulfamoylphenyl)
1
amino)acetate (17i). Yield 61 mg (41%). Beige amorphous solid. 1H
NMR (400 MHz, CDCl3)
7.66e7.57 (m, 2H, ArH), 6.99 (dd, J ¼ 8.2,
[
3
M
ꢀ H]þ calculated for C15H15N2O3Sþ 303.0788, found
d
03.0798.
2
.1 Hz, 1H, ArH), 6.94 (d, J ¼ 2.1 Hz, 1H, ArH), 6.82 (d, J ¼ 8.3 Hz, 1H,
ArH), 6.53 (d, J ¼ 8.9 Hz, 2H, ArH), 5.53 (d, J ¼ 5.8 Hz, 1H, NH),
5.06e4.95 (m, 3H, NH2, CH), 4.18 (ddq, J ¼ 35.4, 10.8, 7.1 Hz, 2H,
CH2), 3.83 (s, 6H, OMe), 1.21 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR
4
.1.2.3. 4-((2-(4-Methoxyphenyl)-2-oxoethyl)amino)benzenesulfo-
ꢂ
namide (17c). Yield 88 mg (24%). Beige solid, m.p. 207.8e208.8 C.
1
H NMR (400 MHz, DMSO‑d
J ¼ 8.6 Hz, 2H, ArH), 7.07 (d, J ¼ 8.7 Hz, 2H, ArH), 6.93 (s, 2H, NH2),
.73 (d, J ¼ 8.5 Hz, 2H, ArH), 4.69 (s, 2H, CH2), 3.85 (s, 3H, OMe).13C
NMR (101 MHz, DMSO‑d 194.76, 163.96, 151.48, 131.04, 130.72,
28.33, 127.66, 114.49, 111.88, 56.07, 49.49. HRMS (ESI) m/z
M ꢀ H]þ calculated for C15H15N2O4Sþ 319.0758, found 319.0747.
6
)
d
8.05 (d, J ¼ 8.7 Hz, 2H, ArH), 7.51 (d,
(101 MHz, CDCl3) d 171.34, 149.41, 149.30, 149.23, 130.11, 128.82,
128.30, 119.65, 112.63, 111.40, 109.94, 62.18, 59.77, 55.97, 55.89,
14.03. HRMS (ESI) m/z [MþNa]þ calculated for C18H22N2O6SNaþ
417.1095, found 417.1091.
6
6
) d
1
[
4.1.2.10. 4-((2-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-oxoethyl)
amino)benzenesulfonamide (17j). Yield 67 mg (44%). Beige amor-
phous solid. 1H NMR (400 MHz, DMSO‑d
ArH), 7.40 (d, J ¼ 8.5 Hz, 2H, ArH), 7.35 (d, J ¼ 8.6 Hz, 2H, ArH), 6.92
s, 2H, NH2), 6.71 (d, J ¼ 8.9 Hz, 2H, ArH), 6.30 (t, J ¼ 5.2 Hz,1H, NH),
.98 (d, J ¼ 5.2 Hz, 2H, CH2), 3.51 (s, 6H, CH2, CH2-piperazine), 2.37
6
)
d
7.51 (d, J ¼ 8.8 Hz, 2H,
4
.1.2.4. 3-((2-(4-Methoxyphenyl)-2-oxoethyl)amino)benzenesulfo-
namide (17d). Yield 24 mg (7%). Beige amorphous solid. 1H NMR
400 MHz, acetone-d6)
8.11 (d, J ¼ 8.3 Hz, 2H, ArH), 7.28 (d,
J ¼ 9.2 Hz, 2H, ArH), 7.16 (d, J ¼ 7.8 Hz, 1H, ArH), 7.08 (d, J ¼ 8.4 Hz,
H, ArH), 6.99 (t, J ¼ 8.2 Hz, 1H, ArH), 6.29 (s, 2H, NH2), 5.63 (s, 1H,
NH), 4.73 (d, J ¼ 4.8 Hz, 2H, CH2), 3.92 (s, 3H, OMe). 13C NMR
101 MHz, acetone-d6) 193.27, 164.15, 148.50, 145.14, 130.14,
30.12, 129.34, 128.26, 116.01, 113.98, 109.66, 55.12, 49.49. HRMS
(
3
(
(
d
d, J ¼ 31.6 Hz, 4H, CH2-piperazine). 13C NMR (101 MHz, DMSO‑d
6
)
2
d
167.59, 151.32, 137.42, 132.03, 131.13, 131.05, 128.67, 127.60, 111.86,
6
1.35, 53.08, 52.59, 44.56, 44.43, 41.93. HRMS (ESI) m/z [MþH]þ
(
1
d
calculated for C19H24ClN4O3Sþ 423.1253, found 423.1252.
(
3
ESI) m/z [MþH]þ calculated for C15H17N2O4Sþ 321.0914, found
4
.1.2.11. 3-((2-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-oxoethyl)
21.0904.
amino)benzenesulfonamide (17k). Yield 48 mg (41%). Beige solid,
m.p. 239.1e240.9 C. 1H NMR (400 MHz, DMSO‑d
ꢂ
6
)
d
7.39 (d,
4
(
.1.2.5. 3-((3,3-Dimethyl-2-oxobutyl)amino)benzenesulfonamide
J ¼ 8.2 Hz, 2H, ArH), 7.34 (d, J ¼ 8.4 Hz, 2H, ArH), 7.22 (t, J ¼ 7.9 Hz,
ꢂ
17e). Yield 30 mg (25%). Beige solid, m.p. 157.1e159.0 C. 1H NMR
1
H, ArH), 7.15 (s, 2H, NH2), 7.06 (s, 1H, ArH), 7.00 (d, J ¼ 7.5 Hz, 1H,
(
400 MHz, CD3OD)
d
7.28 (t, J ¼ 7.9 Hz, 1H, ArH), 7.20e7.15 (m, 1H,
ArH), 6.86e6.78 (m, 1H, ArH), 6.06 (t, J ¼ 5.0 Hz, 1H, NH), 3.94 (d,
J ¼ 5.0 Hz, 2H, CH2), 3.50 (s, 6H, CH2, CH2-piperazine), 2.37 (d,
ArH), 7.12 (t, J ¼ 2.1 Hz, 1H, ArH), 6.85 (ddd, J ¼ 8.3, 2.5, 1.1 Hz, 1H,
ArH), 4.25 (s, 2H, CH2), 1.26 (s, 9H, CH3). 13C NMR (101 MHz,
J ¼ 34.4 Hz, 4H, CH2-piperazine). 13C NMR (101 MHz, DMSO‑d
6
)
CD3OD)
2.76, 25.38. HRMS (ESI) m/z [MþNa]þ calculated for
C12H18N2O3Sþ 293.0921, found 293.0930.
d 211.93,148.44,144.14,129.22,116.21,113.84,109.01, 47.93,
d
1
(
4
167.80, 148.95, 145.20, 137.44, 132.03, 131.12, 129.63, 128.68,
15.84,113.33,109.43, 61.36, 53.08, 52.60, 44.78, 44.48, 41.94. HRMS
ESI) m/z [MþH]þ calculated for C19H24ClN4O3Sþ 423.1253, found
23.1252.
4
4
.1.2.6. N,N-diethyl-2-((4-sulfamoylphenyl)amino)acetamide (17f).
ꢂ
Yield 50 mg (25%). White solid, m.p. 189.2e191.4 C. 1H NMR
4.1.2.12. 4-((2-Oxocycloheptyl)amino)benzenesulfonamide
Yield 80 mg (20%). Beige solid, m.p. 153.9e155.7 C. 1H NMR
(17l).
ꢂ
(
400 MHz, DMSO‑d
NH2), 6.71 (d, J ¼ 8.8 Hz, 2H, ArH), 6.28 (t, J ¼ 5.0 Hz, 1H, NH), 3.96
d, J ¼ 5.1 Hz, 2H, CH2), 3.40e3.29 (m, 4H, CH2eEt), 1.16 (t,
J ¼ 7.1 Hz, 3H, CH3), 1.05 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR (101 MHz,
DMSO‑d 167.91, 151.32, 131.02, 127.61, 111.86, 44.48, 14.53, 13.46.
6
)
d
7.51 (d, J ¼ 8.8 Hz, 2H, ArH), 6.92 (s, 2H,
(400 MHz, (СD3)2СO)
d
7.62 (d, J ¼ 8.8 Hz, 2H, ArH), 6.71 (d,
(
J ¼ 8.8 Hz, 2H, ArH), 6.15 (s, 2H, NH2), 5.86 (s, 1H, NH), 4.47e4.39
(m, 1H, CH-cycloheptanone), 2.69 (dt, J ¼ 16.8, 4.7 Hz, 1H, CH2-
cycloheptanone), 2.48 (ddd, J ¼ 16.8, 10.2, 4.5 Hz, 1H, CH2-
cycloheptanone), 2.14e2.05 (m, 2H, CH2-cycloheptanone),
1.98e1.72 (m, 6H, CH2-cycloheptanone), 1.55 (dtd, J ¼ 14.1, 9.6,
6
) d
HRMS (ESI) m/z [MþH]þ calculated for C12H20N3O3Sþ 286.1218,
found 286.1220.
10

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
4
.0 Hz, 1H, CH2-cycloheptanone), 1.47e1.34 (m, 1H, CH2-
cycloheptanone).13C NMR (101 MHz, (СD3)2СO) 209.41, 149.69,
31.31, 127.87, 111.86, 61.05, 41.05, 31.25, 26.98, 23.19. HRMS (ESI)
13.85. More number of proton peaks are due to the keto-enol
tautomerism (17:1). HRMS (ESI) m/z [MþNa]þ calculated for
C17H18N2O5SNaþ 385.0828, found 385.0829.
d
1
m/z [M ꢀ H]þ calculated for C13H17N2O3Sþ 281.0944, found
2
81.0954.
4.1.2.19. Ethyl 3-oxo-3-phenyl-2-((3-sulfamoylphenyl)amino)prop-
anoate (19g). Yield 74 mg (23%). White amorphous solid. 1H NMR
4
.1.2.13. Dimethyl 2-((4-sulfamoylphenyl)amino)malonate (19a).
(400 MHz, (CD3)2CO) d 8.20e8.14 (m, 2H, ArH), 7.76e7.68 (m, 1H,
ꢂ
Yield 63 mg (33%). Beige solid, m.p. 208.7e210.0 C. 1H NMR
ArH), 7.59 (t, J ¼ 7.7 Hz, 2H, ArH), 7.42e7.36 (m, 1H, ArH), 7.32 (t,
J ¼ 7.9 Hz, 1H, ArH), 7.25e7.18 (m, 1H, ArH), 7.06 (dd, J ¼ 8.1, 1.6 Hz,
1H, ArH), 6.43 (s, 2H, NH2), 6.14 (d, J ¼ 8.5 Hz, 1H, NH), 6.02 (d,
J ¼ 8.4 Hz,1H, CH), 4.14 (q, J ¼ 7.1 Hz, 2H, CH2), 1.09 (t, J ¼ 7.1 Hz, 3H,
(
400 MHz, acetone-d6)
d
7.66 (d, J ¼ 14.3 Hz, 2H, ArH), 6.91e6.80
(
m, 2H, ArH), 6.23 (s, 2H, NH2), 6.17 (d, J ¼ 8.0 Hz, 1H, NH), 5.07 (d,
J ¼ 8.1 Hz, 1H, CH), 3.79 (s, 6H, OMe).13C NMR (101 MHz, acetone-
d6) 168.40, 150.10, 133.93, 128.72, 113.15, 60.29, 53.43. HRMS (ESI)
d
CH3).13C NMR (101 MHz, (CD3)2CO) d 193.09, 168.84, 147.80,
m/z [MþNa]þ calculated for C11H14N2O6SNaþ 325.0470, found
146.09, 135.52, 135.05, 130.50, 129.99, 129.68, 117.20, 116.14, 11.57,
63.40, 62.56, 14.21. HRMS (ESI) m/z [MþNa]þ calculated for
C17H18N2O5SNaþ 385.0833, found 385.0829.
3
25.0465.
4.1.2.14. Diethyl
2-((4-sulfamoylphenyl)amino)malonate
(19b).
ꢂ
Yield 28 mg (35%). White solid, m.p. 146.4e147.4 C. 1H NMR
4.1.2.20. Ethyl 3-(4-methoxyphenyl)-3-oxo-2-((4-sulfamoylphenyl)
(
400 MHz, DMSO‑d
6
)
d
7.53 (d, J ¼ 8.7 Hz, 2H, ArH), 6.97 (d,
amino)propanoate (19h). Yield 30 mg (19%). White amorphous
solid. 1H NMR (400 MHz, (CD3)2СO) d 8.24e8.16 (m, 2H, ArH),
J ¼ 6.9 Hz, 3H, NH2, NH), 6.80 (d, J ¼ 8.8 Hz, 2H, ArH), 5.16 (d,
J ¼ 8.6 Hz, 1H, CH), 4.20 (dtq, J ¼ 10.3, 6.9, 3.4 Hz, 4H, CH2), 1.21 (t,
7.75e7.64 (m, 2H, ArH), 7.15e7.08 (m, 2H, ArH), 6.96 (d, J ¼ 8.8 Hz,
2H, ArH), 6.32 (d, J ¼ 8.2 Hz, 1H, NH), 6.23 (s, 2H, NH2), 6.06e6.02
(m, 1H, CH), 4.16 (q, J ¼ 7.1 Hz, 2H, CH2), 3.95 (s, 3H, OMe), 1.12 (t,
J ¼ 7.1 Hz, 6H, CH3). 13C NMR (101 MHz, DMSO‑d
6
) d 167.67, 149.68,
1
32.60, 127.65, 112.49, 62.30, 59.80, 14.34. HRMS (ESI) m/z
[
3
MþNa]þ calculated for C13H18N2O6SNaþ 353.0784, found
J ¼ 7.1 Hz, 3H, CH3).13C NMR (101 MHz, (CD3)2СO)
d 189.98,167.91,
53.0778.
164.65, 149.47, 132.78, 131.66, 127.82, 127.29, 114.02, 112.48, 61.78,
6
1.65, 55.24, 13.35. HRMS (ESI) m/z [MþNa]þ calculated for
4.1.2.15. Diisopropyl 2-((4-sulfamoylphenyl)amino)malonate (19c).
C18H20N2O6SNaþ 415.0933, found 415.0934.
Yield 30 mg (18%). White amorphous solid. 1H NMR (400 MHz,
DMSO‑d
6
)
d
7.53 (d, J ¼ 8.8 Hz, 2H, ArH), 6.97 (s, 2H, NH2), 6.89 (d,
4.1.2.21. Ethyl 3-(4-methoxyphenyl)-3-oxo-2-((3-sulfamoylphenyl)
amino)propanoate (19i). Yield 84 mg (28%). White amorphous
J ¼ 8.5 Hz, 1H, NH), 6.80 (d, J ¼ 8.9 Hz, 2H, ArH), 5.12e4.92 (m, 3H,
CH, CH-i-Pr), 1.24 (d, J ¼ 6.2 Hz, 6H, CH3), 1.20 (d, J ¼ 6.3 Hz, 6H,
solid. 1H NMR (400 MHz, CDCl3)
d
8.16 (d, J ¼ 8.3 Hz, 2H, ArH),
CH3). 13C NMR (101 MHz, DMSO‑d
6
)
d
167.17, 149.61, 132.11, 127.70,
7.42e7.24 (m, 3H, ArH), 7.01 (d, J ¼ 8.3 Hz, 2H, ArH), 6.93 (d,
J ¼ 6.7 Hz, 1H, ArH), 5.66 (s, 2H, CH, NH), 4.84 (s, 2H, NH2), 4.17 (q,
J ¼ 6.4 Hz, 2H, CH2), 3.93 (s, 3H, OMe), 1.14 (t, J ¼ 6.8 Hz, 3H, CH3).
112.57, 79.24, 78.91, 78.59, 70.54, 54.90, 21.67, 21.56. HRMS (ESI) m/
z [MþNa]þ calculated for C15H22N2O6SNaþ 381.1102, found
3
81.1091.
13C NMR (101 MHz, CDCl3) d 189.19, 168.00, 164.75, 146.33, 143.01,
131.79, 130.25, 126.77, 117.78, 116.05, 114.16, 110.53, 77.22, 62.52,
4
.1.2.16. Bis(2-methoxyethyl)
2-((4-sulfamoylphenyl)amino)malo-
55.64, 13.91. HRMS (ESI) m/z [MþH]þ calculated for
ꢂ
nate (19d). Yield 112 mg (70%); white solid, m.p. 113.1e115.4 C. 1H
NMR (400 MHz, DMSO‑d
C18H21N2O6Sþ 393.1104, found 393.1115.
6
)
d
7.54 (d, J ¼ 8.8 Hz, 2H, ArH), 7.00 (d,
J ¼ 8.0 Hz, 3H, NH, NH2), 6.82 (d, J ¼ 8.9 Hz, 2H, ArH), 5.23 (d,
4.1.2.22. Methyl 3-(2-methoxyphenyl)-3-oxo-2-((4-sulfamoylphenyl)
J ¼ 8.6 Hz,1H, CH), 4.32e4.23 (m, 4H, CH2), 3.59e3.46 (m, 4H, CH2),
amino)propanoate (19j). Yield 100 mg (31%). Beige solid, m.p.
ꢂ
3
.26 (s, 6H, OMe). 13C NMR (101 MHz, DMSO‑d
6
)
d
167.63, 149.62,
203.6e205 C. 1H NMR (400 MHz, CD3OD)
d
7.84 (d, J ¼ 8.8 Hz, 2H,
132.76, 127.59, 112.56, 69.90, 65.28, 59.71, 58.54. HRMS (ESI) m/z
ArH), 7.75 (t, J ¼ 9.6 Hz, 2H, ArH), 7.33 (t, J ¼ 7.3 Hz, 2H, ArH), 7.02 (d,
J ¼ 8.3 Hz, 1H, ArH), 6.99e6.93 (m, 1H, ArH), 3.85 (s, 3H, OMe), 3.75
[M ꢀ H]þ calculated for C15H21N2O8Sþ 389.1008, found 389.1013.
(
s, 3H, OMe). 13C NMR (101 MHz, CD3OD) d 169.89, 167.56, 157.12,
4
.1.2.17. Methyl
4-methoxy-3-oxo-2-((4-sulfamoylphenyl)amino)
141.85, 138.67, 129.14, 128.87, 126.81, 122.32, 120.29, 119.19, 110.48,
54.77, 51.69. HRMS (ESI) m/z [M
H]þ calculated for
C17H17N2O6Sþ 377.0806, found 377.0802.
butanoate (16e). Yield 21 mg (12%). White amorphous solid,. 1H
NMR (400 MHz, DMSO‑d
ꢀ
6
)
d
7.53 (d, J ¼ 8.8 Hz, 2H, ArH), 6.96 (s, 2H,
NH2), 6.70 (d, J ¼ 8.9 Hz, 3H, ArH, NH), 4.54 (td, J ¼ 8.0, 5.5 Hz, 1H,
NH), 3.65 (s, 3H, OMe), 3.62 (s, 3H, OMe), 2.92 (dd, J ¼ 16.4, 5.6 Hz,
4.1.2.23. Ethyl
3-methyl-2-((4-sulfamoylphenyl)carbamoyl)buta-
1
H, CH2), 2.81 (dd, J ¼ 16.4, 7.5 Hz, 1H, CH2). 13C NMR (101 MHz,
noate (20a). Yield 135 mg (38%). White amorphous solid. 1H NMR
(400 MHz, CD3OD)
DMSO‑d
2.33, 52.22, 36.76. HRMS (ESI) m/z [MþH]þ calculated for
C12H16N2O6Sþ 317.0812, found 317.0802.
6
)
d
172.49, 170.93, 150.49, 132.00, 127.78, 112.08, 52.66,
d
7.87 (d, J ¼ 8.8 Hz, 2H, ArH), 7.77 (d, J ¼ 8.9 Hz,
5
2H, ArH), 4.22 (qd, J ¼ 7.1, 1.4 Hz, 2H, CH2), 3.21 (d, J ¼ 9.8 Hz, 1H,
CH), 2.47 (ddt, J ¼ 13.4, 9.9, 6.7 Hz, 1H, CH- i-Pr), 1.28 (t, J ¼ 7.1 Hz,
3
H, CH3), 1.04 (dd, J ¼ 12.2, 6.7 Hz, 6H, CH3-i-Pr). 13C NMR
4
.1.2.18. Ethyl 3-oxo-3-phenyl-2-((4-sulfamoylphenyl)amino)prop-
anoate (19f). Yield 111 mg (66%). Beige amorphous solid. 1H NMR
400 MHz, CDCl3)
(101 MHz, CD3OD) d 169.42, 168.00, 141.60, 138.80, 126.83, 119.28,
61.08, 60.97, 29.09, 19.61, 19.06, 13.05. HRMS (ESI) m/z [MþNa]þ
(
d
13.03 (s, 0.06H, OH), 8.16 (dd, J ¼ 8.4, 1.2 Hz, 2H,
calculated for C14H20N2O5SNaþ 351.0998, found 351.0985.
ArH), 7.77 (d, J ¼ 8.8 Hz, 2H, ArH), 7.76e7.65 (m, 1.28H, ArH), 7.56 (t,
J ¼ 7.8 Hz, 2H, ArH), 7.45e7.37 (m, 0.10H, ArH), 7.39e7.31 (m, 0.23H,
ArH), 6.78 (d, J ¼ 8.9 Hz, 2H, ArH), 6.68 (d, J ¼ 8.8 Hz, 0.17H, ArH),
4.1.2.24. Ethyl
3-methyl-2-((3-sulfamoylphenyl)carbamoyl)buta-
noate (20b). Yield 135 mg (38%). Dirty pink amorphous solid. 1H
NMR (400 MHz, (CD3)2CO), 9.60 (s, 1H, NH), 8.31 (s, 1H, ArH), 7.79
(d, J ¼ 8.0 Hz,1H, ArH), 7.62 (d, J ¼ 7.8 Hz,1H, ArH), 7.49 (t, J ¼ 7.9 Hz,
1H, ArH), 6.63 (s, 2H, NH2), 4.16 (tt, J ¼ 10.6, 5.3 Hz, 2H, CH2), 3.19
(d, J ¼ 9.6 Hz, 1H, CH), 2.46 (ddt, J ¼ 13.3, 9.0, 6.6 Hz, 1H, CH-i-Pr),
1.22 (t, J ¼ 7.1 Hz, 3H, CH3), 1.01 (d, J ¼ 6.7 Hz, 6H, CH3-i-Pr). 13C
5
.84 (d, J ¼ 7.1 Hz, 1H, NH), 5.71 (d, J ¼ 7.2 Hz, 1H, CH), 5.14 (s, 0.07H,
NH2), 4.75 (s, 2H, NH2), 4.27 (q, J ¼ 7.1 Hz, 0.28H, CH2), 4.18 (q,
J ¼ 7.1 Hz, 2H, CH2), 1.21 (t, J ¼ 7.1 Hz, 0.33H, CH3), 1.12 (t, J ¼ 7.1 Hz,
3
H, CH3). 13C NMR (101 MHz, CDCl3) d 190.77, 167.38, 149.08,
134.70, 133.77, 130.73, 129.26, 128.95, 128.70, 112.85, 62.73, 62.64,
11

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
NMR (101 MHz, (CD3)2CO)
d
170.05, 167.47, 145.83, 140.21, 130.28,
4.1.2.30. 3-((4-(4-Chlorobenzyl)-2,5-dioxo-1-(p-tolyl)-2,5-dihydro-
1
23.32, 122.04, 117.83, 62.17, 61.59, 20.89, 20.47, 14.46. HRMS (ESI)
1H-pyrrol-3-yl)amino)benzenesulfonamide (22b). Yield 96 mg
(69%). Yellow solid, m.p. 149.3e151.6 C. 1H NMR (400 MHz, (CD3)
ꢂ
m/z [MþNa]þ calculated for C14H20N2O5SNaþ 351.0985, found
3
51.0985.
2CO)
ArH), 7.10e7.03 (m, 2H, ArH), 6.77 (d, J ¼ 8.4 Hz, 2H, ArH), 6.72 (s,
H, NH2), 3.62 (s, 2H, CH2), 2.37 (s, 3H, Me). 13C NMR (101 MHz,
CD3)2CO) 13C NMR (101 MHz, Acetone) 172.79, 167.86, 145.71,
40.12, 138.42, 137.78, 131.92, 130.96, 130.56, 130.26, 130.08, 128.94,
27.51, 126.86, 122.98, 121.43, 103.65, 28.70, 21.07. HRMS (ESI) m/z
d 8.59 (s, 1H, NH), 7.71e7.62 (m, 2H, ArH), 7.37e7.23 (m, 6H,
2
4.1.2.25. Ethyl 3-oxo-2-phenyl-3-((4-sulfamoylphenyl)amino)prop-
(
d
d
anoate (20c). Yield 163 mg (41%). Beige amorphous solid. 1H NMR
400 MHz, DMSO‑d
1
1
(
6
)
d
10.68 (s, 1H, NH), 7.76 (d, J ¼ 8.8 Hz, 2H,
ArH), 7.71 (d, J ¼ 8.7 Hz, 2H, ArH), 7.46e7.31 (m, 5H), 7.25 (s, 2H,
[
4
MþH]þ calculated for C24H21ClN3O4Sþ 482.0938, found
NH2), 4.93 (s, 1H, CH), 4.15 (q, J ¼ 7.0 Hz, 2H, CH2), 1.18 (t, J ¼ 7.1 Hz,
82.0936.
3
6
H, CH3). 13C NMR (101 MHz, DMSO‑d ) d 168.28, 166.27, 141.48,
1
38.90, 133.88, 127.79, 126.77, 118.87, 61.13, 58.32, 13.97. HRMS (ESI)
4
1
.1.2.31. 4-((4-(2-Methoxybenzyl)-2,5-dioxo-1-phenyl-2,5-dihydro-
H-pyrrol-3-yl)amino)benzenesulfonamide (22c). Yield 148 mg
m/z [MþNa]þ calculated for C17H18N2O5SNaþ 385.0829, found
3
85.0829.
(51%). Yellow amorphous solid. 1H NMR (400 MHz, DMSO‑d
6
) d9.51
(
s, 1H, NH), 7.62 (d, J ¼ 8.6 Hz, 2H, ArH), 7.52e7.47 (m, 2H, ArH), 7.39
4
.1.2.26. 2-Benzoyl-3-methyl-N-(4-sulfamoylphenyl)butanamide
ꢂ
(dd, J ¼ 15.5, 7.5 Hz, 3H, ArH), 7.24 (s, 2H, NH2), 7.10 (dd, J ¼ 12.2,
.9 Hz, 3H, ArH), 6.89 (d, J ¼ 6.6 Hz, 1H, ArH), 6.76 (dd, J ¼ 17.7,
.9 Hz, 2H, ArH), 3.62 (s, 3H, OMe), 3.51 (s, 2H, CH2). 13C NMR
101 MHz, DMSO‑d 171.32, 166.71, 156.60, 142.31, 139.13, 138.33,
32.08, 128.79, 128.59, 127.33, 127.27, 126.44, 126.14, 125.34, 121.05,
19.97, 110.17, 105.66, 64.91, 55.08, 45.76, 23.15, 15.16, 8.68. HRMS
(
20d). Yield 51 mg (32%). White solid, m.p. 211.4e212.9 C. 1H NMR
8
7
(
1
(
400 MHz, DMSO‑d
6
)
d
10.63 (s, 1H, NH), 8.07 (d, J ¼ 7.3 Hz, 2H,
ArH), 7.75 (d, J ¼ 8.6 Hz, 2H, ArH), 7.69 (m, J ¼ 9.0 Hz, 3H, ArH), 7.56
6
) d
(
2
d, J ¼ 7.7 Hz, 2H, ArH), 7.23 (s, 2H, NH2), 4.37 (d, J ¼ 9.4 Hz, 1H, CH),
.57 (dt, J ¼ 9.1, 6.7 Hz, 1H, CH-i-Pr), 1.00 (d, J ¼ 6.8 Hz, 3H, CH3),
.93 (d, J ¼ 6.6 Hz, 3H, CH3).13C NMR (101 MHz, DMSO‑d 195.13,
1
0
6
) d
(
4
ESI) m/z [MþH]þ calculated for C24H22N3O5Sþ 464.1273, found
1
6
67.65, 141.84, 139.38, 137.14, 134.04, 129.38, 128.61, 127.19, 119.50,
2.82, 29.14, 21.61, 20.38. HRMS (ESI) m/z [MþNa]þ calculated for
C18H20N2O4SNaþ 383.1037, found 383.1036.
64.1275.
4
.2. Carbonic anhydrase inhibition assay
4
(
(
9
(
6
.1.2.27. 2-Benzoyl-3-methyl-N-(3-sulfamoylphenyl)butanamide
20e). Yield 65 mg (41%). White amorphous solid. 1H NMR
400 MHz, acetone-d6) 14.93 (s, 0.01H, OH), 9.73 (s, 0.38H, NH),
.63 (s, 1H, NH), 8.25 (s, 1,2H, ArH), 8.13 (d, J ¼ 7.4 Hz, 1H, ArH), 8.09
d, J ¼ 7.3 Hz, 0.19H, ArH), 7.79 (d, J ¼ 8.1 Hz, 1.59H), 7.53e7.67 (m,
.24H, ArH), 7.47 (t, J ¼ 7.9 Hz, 2.08H, ArH), 7.31e7.40 (m, 1.21H,
ArH), 6.59 (s, 2H, NH2), 4.37 (d, J ¼ 9.6 Hz, 1H, CH), 2.92 (dt, J ¼ 13.7,
.9 Hz, 0.28H, CH), 2.72 (dqd, J ¼ 13.4, 6.7, 3.1 Hz, 1H, CH), 1.10 (d,
J ¼ 6.7 Hz, 3.31H, CH3), 1.05 (d, J ¼ 6.9 Hz, 0.65H, CH3), 0.98 (d,
J ¼ 3.0 Hz, 3H, CH3). 13C NMR (101 MHz, acetone-d6) 196.46,
67.75, 145.76, 140.11, 138.22, 134.37, 130.38, 130.26, 129.72, 129.67,
29.32, 129.28, 129.20, 123.41, 123.31, 122.09, 117.90, 117.81, 65.26,
4.47, 30.59, 21.62, 21.47, 20.54, 18.93, 18.80. Higher number of
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO hydration activity [52].
2
Phenol red (at a concentration of 0.2 mM) has been used as indi-
cator, working at the absorbance maximum of 557 nm, with 20 mM
d
Hepes (pH 7.5) as buffer, and 20 mM Na
constant the ionic strength), following the initial rates of the CA-
catalyzed CO hydration reaction for a period of 10e100 s.15 The
CO concentrations ranged from 1.7 to 17 mM for the determination
2 4
SO (for maintaining
2
6
2
of the kinetic parameters and inhibition constants. For each in-
hibitor 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 temper-
ature prior to assay, in order to allow for the formation of the EꢀI
complex. The inhibition constants were subsequently obtained by
d
1
1
6
proton peaks is attributed to the keto-enol tautomerism (7:1).
HRMS (ESI) m/z [MþNa]þ calculated for C18H20N2O4SNaþ
3
83.1028, found 383.1036.
4.1.2.28. 3-(4-Chlorophenyl)-2-methyl-3-oxo-N-(4-sulfamoylphenyl)
propenamide (20f). Yield 44 mg (28%). White solid, m.p.
2
8
nonlinear least-squares methods using PRISM
3 and the
ꢂ
23.6e225.1 C. 1H NMR (400 MHz, DMSO‑d
6
)
d
10.63 (s, 1H, NH),
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
.00 (d, J ¼ 8.6 Hz, 2H, ArH), 7.75 (d, J ¼ 8.8 Hz, 2H, ArH), 7.69 (d,
J ¼ 8.9 Hz, 2H, ArH), 7.63 (d, J ¼ 8.7 Hz, 2H, ArH), 7.24 (s, 2H, NH2),
.64 (q, J ¼ 6.8 Hz, 1H, CH), 1.39 (d, J ¼ 6.9 Hz, 3H, Me). 13C NMR
101 MHz, DMSO‑d 195.91, 169.91, 142.20, 139.16, 138.77, 135.00,
4
(
[
78e81].
6
) d
1
30.51,129.47, 127.19,119.29, 50.07, 14.54. HRMS (ESI) m/z [M ꢀ H]þ
4.3. Сell culture
calculated for C16H14ClN2O4Sþ 365.0373, found 365.0357.
MCF7 breast carcinoma cells, MDA-MB-231 mammary gland/
4
1
.1.2.29. 4-((4-(4-Chlorobenzyl)-2,5-dioxo-1-(p-tolyl)-2,5-dihydro-
breast cancer cell, WI-26 VA4 lung epithelial-like cells were pur-
chased from the ATCC. MCF7 cells were maintained in Advanced
MEM (Gibco, UK) supplemented with 5% fetal bovine serum (FBS,
H-pyrrol-3-yl)amino)benzenesulfonamide (22a). Yield 100 mg
ꢂ
(
71%). Yellow solid, m.p. 260.0e261.9 C. 1H NMR (400 MHz, (CD3)
ꢀ1
ꢀ1
2
7
2
CO)
.28 (d, J ¼ 8.3 Hz, 2H, ArH), 7.26e7.21 (m, 2H, ArH), 7.14e7.09 (m,
H, ArH), 6.82 (d, J ¼ 8.4 Hz, 2H, ArH), 6.60 (s, 2H, NH2), 3.65 (s, 2H,
CH2), 2.37 (s, 3H, Me). 13C NMR (101 MHz, (CD3)2CO) 172.66,
67.86, 142.90, 140.72, 139.93, 138.21, 137.81, 132.02, 130.89, 130.67,
30.08, 129.01, 127.81, 126.85, 123.56, 105.18, 28.91, 21.06. HRMS
ESI) m/z [MþH]þ calculated for C24H20ClN3O4Sþ 480.0790,
found 480.0790.
d
8.62 (s, 1H, NH), 7.88e7.79 (m, 2H, ArH), 7.32 (m, 2H, ArH),
Gibco, UK), penicillin (100 UI mL ), streptomycin (100
human recombinant insulin (10 mg mL , Capricorn Scientific,
m
g mL ),
ꢀ
1
Germany), and GlutaMax (1.87 mM, Gibco, UK). MDA-MB-231 cells
were maintained in Advanced DMEM/F12 (Gibco, UK) supple-
mented with 5% fetal bovine serum (FBS, Gibco, UK), penicillin (100
d
1
1
(
ꢀ1
ꢀ1
UI mL ), streptomycin (100 mg mL ), and GlutaMax (2.5 mM,
Gibco, UK). WI-26 VA4 cells were maintained in Advanced MEM
(Gibco, UK) supplemented with 5% fetal bovine serum (FBS, Gibco,
12

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
UK), penicillin (100 UI mLꢀ1), streptomycin (100
m
g mL ), and
ꢀ1
Appendix A. Supplementary data
GlutaMax (1.87 mM, Gibco, UK). All cells line cultivation under a
ꢂ
humidified atmosphere of 95% air/5% CO
2
at 37 C. Subconfluent
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113352.
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.
References
[1] C.T. Supuran, Structure and function of carbonic anhydrases, Biochem. J. 473
(2016) 2023e2032, https://doi.org/10.1042/BCJ20160115.
[2] C.D. Boone, M. Pinard, R. McKenna, D. Silverman, Catalytic mechanism of a-
4
.3. Antiproliferative assay (end point)
class carbonic anhydrases: CO2 hydration and proton transfer, Subcell. Bio-
chem. 75 (2014) 31e52, https://doi.org/10.1007/978-94-007-7359-2_3.
3] C.T. Supuran, C. Capasso, An overview of the bacterial carbonic anhydrases,
Metabolites 7 (2017) 56, https://doi.org/10.3390/metabo7040056.
[
The effects of the synthesized compounds on cell viability were
determined using the MTTcolorimetric test. All examined cells were
diluted with the growth medium (or medium containing 100 mМ
[4] A. Angeli, M. Pinteala, S.S. Maier, S. Del Prete, C. Capasso, B.C. Simionescu,
C.T. Supuran, Inhibition of -, -, -, -, - and -class carbonic anhydrases
a
b
g
d
z
h
from bacteria, fungi, algae, diatoms and protozoans with famotidine, J. Enzym.
Inhib. Med. Chem. 34 (2019) 644e650, https://doi.org/10.1080/
14756366.2019.1571273.
CoCl
2
for chemically-induced hypoxia) to 3.5 ꢁ 104 cells per mL and
the aliquots (7 ꢁ 103 cells per 200
m
L) were placed in individual wells
[5] E. Berrino, M. Bozdag, S. Del Prete, F.A.S. Alasmary, L.S. Alqahtani, Z. AlOthman,
in 96-multiplates (Eppendorf, Germany) and incubated for 24 h. The
next day the cells were then treated with synthesized compounds
C. Capasso, C.T. Supuran, Inhibition of
from bacteria and diatoms with N -aryl- N -hydroxy-ureas, J. Enzym. Inhib.
a
-,
b
-,
g
-, and
d
-carbonic anhydrases
0
ꢂ
separatelyat 100 mМ concentration and incubated for 72 h at 37 C in
Med.
4756366.2018.1490733.
6] V. De Luca, A. Petreni, A. Nocentini, A. Scaloni, C.T. Supuran, C. Capasso, Effect
of sulfonamides and their structurally related derivatives on the activity of
carbonic anhydrase from burkholderia territorii, Int. J. Mol. Sci. 22 (2021)
e13, https://doi.org/10.3390/ijms22020571.
7] S. Del Prete, V. De Luca, G. De Simone, C.T. Supuran, C. Capasso, Cloning,
expression and purification of the complete domain of the -carbonic anhy-
drase from Plasmodium falciparum, J. Enzym. Inhib. Med. Chem. 31 (2016)
54e59, https://doi.org/10.1080/14756366.2016.1217856.
8] R. McKenna, C.T. Supuran, Carbonic anhydrase inhibitors drug design, Subcell.
Biochem. 75 (2014) 291e323, https://doi.org/10.1007/978-94-007-7359-2_
Chem.
33
(2018)
1194e1198,
https://doi.org/10.1080/
1
5
% CO
incubation, the cells were then treated with 40
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 L) was added
2
atmosphere. Each compound was tested in triplicate. After
[
[
[
mL MTT solution (3-
i-
(
1
m
h
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.
15.
[9] C.T. Supuran, Structure-based drug discovery of carbonic anhydrase inhibitors,
J. Enzym. Inhib. Med. Chem. 27 (2012) 759e772, https://doi.org/10.3109/
1
4756366.2012.672983.
[10] K.M. Gilmour, Perspectives on carbonic anhydrase, Comp. Biochem. Physiol. -
Mol. Integr. Physiol. 157 (2010) 193e197, https://doi.org/10.1016/
4.4. MTT assay
A
j.cbpa.2010.06.161.
All examined cells were diluted with the growth medium (or
[
11] M.E. McDevitt, L.A. Lambert, Molecular evolution and selection pressure in
alpha-class carbonic anhydrase family members, Biochim. Biophys. Acta
Protein Proteonomics 1814 (2011) 1854e1861, https://doi.org/10.1016/
j.bbapap.2011.07.007.
12] S.C. Frost, Physiological functions of the alpha class of carbonic anhydrases,
Subcell. Biochem. 75 (2014) 9e30, https://doi.org/10.1007/978-94-007-7359-
medium containing 100 mМ CoCl
2
for chemically-induced hypoxia)
L)
to 3.5 ꢁ 104 cells per mL and the aliquots (7 ꢁ 103 cells per 200
m
were placed in individual wells in 96-multiplates (Eppendorf,
Germany) and incubated for 24 h. Triplicate wells were treated with
[
[
2_2.
test compounds starting at 200.0
mM concentration and diluted
13] R. McKenna, S.C. Frost, Overview of the carbonic anhydrase family, Subcell.
Biochem. 75 (2014) 3e5, https://doi.org/10.1007/978-94-007-7359-2_1.
[14] S. Zamanova, A.M. Shabana, U.K. Mondal, M.A. Ilies, Carbonic anhydrases as
1
1 at various concentrations or DMSO (Sigma, USA) as control with
ꢂ
final concentration 0.1%. Plates were incubated for 72 h at 37 C in
% CO atmosphere. After incubation, the cells were then treated
with 40 MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, 5 mg mL-1 in PBS) and incubated
h. After an additional 4 h incubation, the medium with MTT was
removed and DMSO (150 L) was added to dissolve the crystals
5
2
disease markers, Expert Opin. Ther. Pat. 29 (2019) 509e533, https://doi.org/
1
0.1080/13543776.2019.1629419.
15] C. Supuran, Carbonic anhydrases as drug targets - an overview, Curr. Top.
Med. Chem. (2007) 825e833, https://doi.org/10.2174/
156802607780636690.
mL
[
[
7
4
16] S. Kalinin, A. Valtari, M. Ruponen, E. Toropainen, A. Kovalenko, A. Nocentini,
m
M. Gureev, D. Dar’in, A. Urtti, C.T. Supuran, M. Krasavin, Highly hydrophilic
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.
1
,3-oxazol-5-yl benzenesulfonamide inhibitors of carbonic anhydrase II for
reduction of glaucoma-related intraocular pressure, Bioorg. Med. Chem. 27
2019), https://doi.org/10.1016/j.bmc.2019.115086.
(
[
17] A. Nocentini, C.T. Supuran, Carbonic anhydrase inhibitors for the treatment of
neuropathic pain and arthritis, in: Carbon. Anhydrases Biochem. Pharmacol.
An Evergr. Pharm. Target, Elsevier, 2019, pp. 367e386, https://doi.org/
1
0.1016/B978-0-12-816476-1.00017-4.
Declaration of competing interest
[
[
[
[
[
[
18] C.T. Supuran, Carbonic anhydrase inhibitors as emerging drugs for the treat-
ment of obesity, Expet Opin. Emerg. Drugs 17 (2012) 11e15, https://doi.org/
10.1517/14728214.2012.664132.
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.
19] A. Scozzafava, C.T. Supuran, Glaucoma and the applications of carbonic
anhydrase inhibitors, Subcell. Biochem. 75 (2014) 349e359, https://doi.org/
10.1007/978-94-007-7359-2_17.
20] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for in-
hibitors and activators, Nat. Rev. Drug Discov. 7 (2008) 168e181, https://
doi.org/10.1038/nrd2467.
Acknowledgements
21] C.T. Supuran, Experimental carbonic anhydrase inhibitors for the treatment of
hypoxic tumors, J. Exp. Pharmacol. 12 (2020) 603e617, https://doi.org/
This research was supported by the Russian Federation Gov-
ernment Megagrant 14.W03.031.0025. We are grateful to the
Centre for Chemical Analysis and Materials Research of Saint
Petersburg State University Research Park for the high-resolution
mass-spectrometry data. T.S. is grateful to the Russian Foundation
for Basic Research (project grant 19e33-90017).
1
0.2147/JEP.S265620.
22] I.S. Ismail, The role of carbonic anhydrase in hepatic glucose production, Curr.
Diabetes Rev. 14 (2018) 108e112, https://doi.org/10.2174/
573399812666161214122351.
1
23] P. Blandina, G. Provensi, M.B. Passani, C. Capasso, C.T. Supuran, Carbonic
anhydrase modulation of emotional memory. Implications for the treatment
of cognitive disorders, J. Enzym. Inhib. Med. Chem. 35 (2020) 1206e1214,
13

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
10.1038/nchem.2034.
https://doi.org/10.1080/14756366.2020.1766455.
[
[
24] C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist?
[44] F.W. Bollinger, L.D. Tuma, Diazotransfer reagents 1, Synlett. 1996 (1996)
407e413, https://doi.org/10.1055/s-1996-5440.
[45] D. Dar’in, G. Kantin, M. Krasavin, A “sulfonyl-azide-free” (SAFE) aqueous-
phase diazo transfer reaction for parallel and diversity-oriented synthesis,
Chem. Commun. 55 (2019) 5239e5242, https://doi.org/10.1039/c9cc02042j.
[46] I. Shershnev, D. Dar’in, S. Chuprun, G. Kantin, O. Bakulina, M. Krasavin, The use
J. Enzym. Inhib. Med. Chem. 31 (2016) 345e360, https://doi.org/10.3109/
14756366.2015.1122001.
25] J.-Y. Winum, J.-L. Montero, A. Scozzafava, C.T. Supuran, Zinc binding functions
in the design of carbonic anhydrase inhibitors, in: Drug Des. Zinc-Enzyme
Inhib., John Wiley & Sons, Inc., Hoboken, NJ, USA, 2009, pp. 39e72, https://
doi.org/10.1002/9780470508169.ch3.
of f-diazo-g-butyrolactone in the Büchner-Curtius-Schlotterbeck reaction of
[
[
26] M. Ferraroni, B. Cornelio, J. Sapi, C.T. Supuran, A. Scozzafava, Sulfonamide
carbonic anhydrase inhibitors: zinc coordination and tail effects influence
inhibitory efficacy and selectivity for different isoforms, Inorg. Chim. Acta. 470
cyclic ketones: a facile entry into spirocyclic scaffolds, Tetrahedron Lett. 60
(2019) 1800e1802, https://doi.org/10.1016/j.tetlet.2019.06.008.
[47] J. Synofzik, D. Dar’In, M.S. Novikov, G. Kantin, O. Bakulina, M. Krasavin,
-diazoacetates in transition-metal-free -lactam synthesis, J. Org. Chem. 84
(2019) 12101e12110, https://doi.org/10.1021/acs.joc.9b02030.
a-Acyl-
(
2018) 128e132, https://doi.org/10.1016/j.ica.2017.03.038.
a
b
27] Z. Hou, B. Lin, Y. Bao, H. ning Yan, M. Zhang, X. wei Chang, X. xin Zhang, Z. jie
Wang, G. fei Wei, M. sheng Cheng, Y. Liu, C. Guo, Dual-tail approach to dis-
covery of novel carbonic anhydrase IX inhibitors by simultaneously matching
the hydrophobic and hydrophilic halves of the active site, Eur. J. Med. Chem.
[48] M. Gecht, G. Kantin, D. Dar’in, M. Krasavin, A novel approach to biologically
relevant oxazolo[5,4-d]pyrimidine-5,7-diones via readily available diazo-
barbituric acid derivatives, Tetrahedron Lett. 60 (2019) 151120, https://
doi.org/10.1016/j.tetlet.2019.151120.
132 (2017) 1e10, https://doi.org/10.1016/j.ejmech.2017.03.023.
[
[
28] A. Bonardi, A. Nocentini, S. Bua, J. Combs, C. Lomelino, J. Andring, L. Lucarini,
S. Sgambellone, E. Masini, R. McKenna, P. Gratteri, C.T. Supuran, Sulfonamide
inhibitors of human carbonic anhydrases designed through a three-tails
approach: improving ligand/isoform matching and selectivity of action,
[49] D. Dar’in, G. Kantin, O. Bakulina, M. Krasavin, Facile one-pot access to
a-Diazo-
b
-ketosulfones from sulfonyl chlorides and -haloketones, Synth. Met. 52
a
(2020) 2259e2266, https://doi.org/10.1055/s-0040-1707525.
[50] A. Bubyrev, D. Dar’in, G. Kantin, M. Krasavin, Synthetic studies towards CH-
diazomethane sulfonamides: a novel type of diazo reagents, Eur. J. Org
Chem. 2020 (2020) 4112e4115, https://doi.org/10.1002/ejoc.202000446.
J.
Med.
Chem.
63
(2020)
7422e7444,
https://doi.org/10.1021/
acs.jmedchem.0c00733.
29] M. Bozdag, M. Ferraroni, E. Nuti, D. Vullo, A. Rossello, F. Carta, A. Scozzafava,
C.T. Supuran, Combining the tail and the ring approaches for obtaining potent
and isoform-selective carbonic anhydrase inhibitors: solution and X-ray
crystallographic studies, Bioorg. Med. Chem. 22 (2014) 334e340, https://
doi.org/10.1016/j.bmc.2013.11.016.
[51] D. Zhukovsky, D. Dar’in, M. Krasavin, Rh2(esp)2-Catalyzed coupling of
Diazo- -butyrolactams with aromatic amines, Eur. J. Org Chem. 2019 (2019)
4377e4383, https://doi.org/10.1002/ejoc.201900565.
a-
g
[52] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase,
J. Biol. Chem. 246 (1971) 2561e2573. http://www.jbc.org/content/246/8/
2561.
[53] S. Pastorekova, S. Parkkila, J. Pastorek, C.T. Supuran, Carbonic anhydrases:
current state of the art, therapeutic applications and future prospects,
J. Enzym. Inhib. Med. Chem. 19 (2004) 199e229, https://doi.org/10.1080/
14756360410001689540.
[54] 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.
[
30] M.A. Pinard, B. Mahon, R. McKenna, Probing the surface of human carbonic
anhydrase for clues towards the design of isoform specific inhibitors, Biomed.
Res. Int. 2015 (2015), https://doi.org/10.1155/2015/453543.
[
31] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Multiple
binding modes of inhibitors to carbonic anhydrases: how to design specific
drugs targeting 15 different isoforms? Chem. Rev. 112 (2012) 4421e4468,
https://doi.org/10.1021/cr200176r.
[
32] M. Krasavin, M. Korsakov, Z. Zvonaryova, E. Semyonychev, T. Tuccinardi,
S. Kalinin, M. Tanç, C.T. Supuran, Human carbonic anhydrase inhibitory profile
of mono- and bis-sulfonamides synthesized via a direct sulfochlorination of 3-
and 4-(hetero)arylisoxazol-5-amine scaffolds, Bioorg. Med. Chem. 25 (2017)
[55] V. Burianova, S. Kalinin, C.T. Supuran, M. Krasavin, Radiotracers for positron
emission tomography (PET) targeting tumour-associated carbonic anhydrase
1914e1925, https://doi.org/10.1016/j.bmc.2017.02.018.
[
33] S. Kalinin, S. Kopylov, T. Tuccinardi, A. Sapegin, D. Dar’in, A. Angeli,
C.T. Supuran, M. Krasavin, Lucky Switcheroo: dramatic potency and selectivity
improvement of imidazoline inhibitors of human carbonic anhydrase VII, ACS
Med. Chem. Lett. (2017), https://doi.org/10.1021/acsmedchemlett.7b00300.
34] G. La Regina, A. Coluccia, V. Famiglini, S. Pelliccia, L. Monti, D. Vullo, E. Nuti,
V. Alterio, G. De Simone, S.M. Monti, P. Pan, S. Parkkila, C.T. Supuran,
isoforms,
j.ejmech.2020.113046.
Eur.
J.
Med.
Chem.
(2020),
https://doi.org/10.1016/
[56] S. Dong Soo, P. Won Young, K. Ki Hyung, K. Ahrong, K. Young Keum,
K. Kyungbin, C. Kyung Un, Differential regulation and prognostic significance
of endogenous hypoxic markers in endometrial carcinomas, Eur. J. Gynaecol.
Oncol. 39 (2018) 773e778.
[57] C.T. Supuran, Therapeutic applications of the carbonic anhydrase inhibitors,
Therapy 4 (2007) 355e378, https://doi.org/10.2217/14750708.4.3.355.
[58] B. Cvenkel, M. Kolko, Current medical therapy and future trends in the
management of glaucoma treatment, J. Ophthalmol. 2020 (2020), https://
doi.org/10.1155/2020/6138132.
[
0
A. Rossello, R. Silvestri, Discovery of 1,1 -biphenyl-4-sulfonamides as a new
class of potent and selective carbonic anhydrase XIV inhibitors, J. Med. Chem.
58 (2015) 8564e8572, https://doi.org/10.1021/acs.jmedchem.5b01144.
[
35] A study of SLC-0111 and Gemcitabine for Metastatic Pancreatic Ductal cancer
in Subjects positive for CAIX - Full Text View - ClinicalTrials.gov, (n.d.). https://
www.clinicaltrials.gov/ct2/show/NCT03450018?term¼SLC-0111
(accessed
[59] C.T. Supuran, V. Alterio, A. Di Fiore, K. D’Ambrosio, F. Carta, S.M. Monti, G. De
Simone, Inhibition of carbonic anhydrase IX targets primary tumors, metas-
tases, and cancer stem cells: three for the price of one, Med. Res. Rev. 38
(2018) 1799e1836, https://doi.org/10.1002/med.21497.
[60] P. Ebbesen, E.O. Pettersen, T.A. Gorr, G. Jobst, K. Williams, J. Kieninger,
R.H. Wenger, S. Pastorekova, L. Dubois, P. Lambin, B.G. Wouters, T. Van Den
Beucken, C.T. Supuran, L. Poellinger, P. Ratcliffe, A. Kanopka, A. G o€ rlach,
10.02.21).
€
[
36] S. Durdagi, M. Entürk, D. Ekinci, H.T. Balaydin, S. G o€ ksu, O.I. Küfreviolu,
A. Innocenti, A. Scozzafava, C.T. Supuran, Kinetic and docking studies of
phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evi-
dence a new binding mode within the enzyme active site, Bioorg. Med. Chem.
19 (2011) 1381e1389, https://doi.org/10.1016/j.bmc.2011.01.016.
[
37] C. Chazalette, B. Masereel, S. Rolin, A. Thiry, A. Scozzafava, A. Innocenti,
C.T. Supuran, Carbonic anhydrase inhibitors. Design of anticonvulsant sul-
fonamides incorporating indane moieties, Bioorg. Med. Chem. Lett 14 (2004)
M. Gasmann, A.L. Harris, P. Maxwell, A. Scozzafava, Taking advantage of tumor
cell adaptations to hypoxia for developing new tumor markers and treatment
strategies, J. Enzym. Inhib. Med. Chem. 24 (2009) 1e39, https://doi.org/
10.1080/14756360902784425.
5781e5786, https://doi.org/10.1016/j.bmcl.2004.09.061.
[
38] S. Kalinin, A. Nocentini, A. Kovalenko, V. Sharoyko, A. Bonardi, A. Angeli,
P. Gratteri, T.B. Tennikova, C.T. Supuran, M. Krasavin, From random to
rational: a discovery approach to selective subnanomolar inhibitors of human
carbonic anhydrase IV based on the Castagnoli-Cushman multicomponent
reaction, Eur. J. Med. Chem. 182 (2019), https://doi.org/10.1016/
j.ejmech.2019.111642.
[61] A. Waheed, W.S. Sly, Carbonic anhydrase XII functions in health and disease,
Gene 623 (2017) 33e40, https://doi.org/10.1016/j.gene.2017.04.027.
[62] C.T. Supuran, Carbonic anhydrase inhibitors as emerging agents for the
treatment and imaging of hypoxic tumors, Expet Opin. Invest. Drugs 27
(2018) 963e970, https://doi.org/10.1080/13543784.2018.1548608.
[63] R. Baghban, L. Roshangar, R. Jahanban-Esfahlan, K. Seidi, A. Ebrahimi-Kalan,
M. Jaymand, S. Kolahian, T. Javaheri, P. Zare, Tumor microenvironment
complexity and therapeutic implications at a glance, Cell Commun. Signal. 18
(2020), https://doi.org/10.1186/s12964-020-0530-4.
[64] S.H. Lee, J.R. Griffiths, How and why are cancers acidic? Carbonic anhydrase ix
and the homeostatic control of tumour extracellular ph, Cancers (Basel) 12
(2020) 1e23, https://doi.org/10.3390/cancers12061616.
[65] H.M. Becker, Carbonic anhydrase IX and acid transport in cancer, Br. J. Canc.
122 (2020) 157e167, https://doi.org/10.1038/s41416-019-0642-z.
[66] S.H. Lee, D. McIntyre, D. Honess, A. Hulikova, J. Pacheco-Torres, S. Cerd aꢀ n,
[
[
39] G. Poli, S. Galati, A. Martinelli, C.T. Supuran, T. Tuccinardi, Development of a
cheminformatics platform for selectivity analyses of carbonic anhydrase in-
hibitors, J. Enzym. Inhib. Med. Chem. 35 (2020) 365e371, https://doi.org/
10.1080/14756366.2019.1705291.
40] S. Galati, D. Yonchev, R. Rodríguez-P eꢀ rez, M. Vogt, T. Tuccinardi, J. Bajorath,
Predicting isoform-selective carbonic anhydrase inhibitors via machine
learning and rationalizing structural features important for selectivity, ACS
Omega (2021), https://doi.org/10.1021/acsomega.0c06153.
[
[
41] C.T. Supuran, Advances in structure-based drug discovery of carbonic anhy-
drase inhibitors, Expet Opin. Drug Discov. 12 (2017) 61e88, https://doi.org/
P. Swietach, A.L. Harris, J.R. Griffiths, Carbonic anhydrase IX is a pH-stat that
sets an acidic tumour extracellular pH in vivo, Br. J. Canc. 119 (2018) 622e630,
https://doi.org/10.1038/s41416-018-0216-5.
10.1080/17460441.2017.1253677.
42] M. Krasavin, S. Kalinin, T. Sharonova, C.T. Supuran, Inhibitory activity against
carbonic anhydrase IX and XII as a candidate selection criterion in the
development of new anticancer agents, J. Enzym. Inhib. Med. Chem. 35 (2020)
[67] G. Ilardi, N. Zambrano, F. Merolla, M. Siano, S. Varricchio, M. Vecchione,
G. Rosa, M. Mascolo, S. Staibano, Histopathological determinants of tumor
resistance: a special look to the immunohistochemical expression of carbonic
anhydrase IX in human cancers, Curr. Med. Chem. 21 (2014) 1569e1582,
https://doi.org/10.2174/09298673113209990227.
1555e1561, https://doi.org/10.1080/14756366.2020.1801674.
[43] G. Karageorgis, S. Warriner, A. Nelson, Efficient discovery of bioactive scaffolds
by activity-directed synthesis, Nat. Chem. 6 (2014) 872e876, https://doi.org/
14

T. Sharonova, P. Paramonova, S. Kalinin et al.
European Journal of Medicinal Chemistry 218 (2021) 113352
[
[
[
[
68] Inhibition of carbonic anhydrase in combination with Platinum and
Etoposide-based Radiochemotherapy in patients with localized small cell lung
cancer - Full Text View - ClinicalTrials.gov, (n.d.). https://clinicaltrials.gov/ct2/
show/NCT03467360 (accessed February 10.02.21.).
anhydrase ix inhibitors endowed with antitumor activity: synthesis, biological
evaluation and in silico insights, Int. J. Mol. Sci. 20 (2019), https://doi.org/
10.3390/ijms20102484.
[75] J.B. Baell, J.W.M. Nissink, Seven year itch: pan-assay interference compounds
(PAINS) in 2017 - utility and limitations, ACS Chem. Biol. 13 (2018) 36e44,
https://doi.org/10.1021/acschembio.7b00903.
69] Trial to determine Optimal phase II dose of the oral dual CAIX inhibitor/
radiosensitizer - Full Text View - ClinicalTrials.gov, (n.d.). https://clinicaltrials.
gov/ct2/show/NCT02216669?term¼carbonicþanhydrase&draw¼4
[76] D. Zhukovsky, D. Dar’in, G. Kantin, M. Krasavin, Synthetic exploration of
diazo -butyrolactams, Eur. J. Org. Chem. 2019 (2019) 2397e2400, https://
doi.org/10.1002/ejoc.201900133.
[77] D. Dar’in, G. Kantin, M. Krasavin, Practical application of the aqueous “sulfo-
nyl-azide-free” (SAFE) diazo transfer protocol to less -C-H acidic ketones and
a-
(
accessed 10.02.21.).
g
70] C.Y. Chu, Y.T. Jin, W. Zhang, J. Yu, H.P. Yang, H.Y. Wang, Z.J. Zhang, X.P. Liu,
Q. Zou, CA IX is upregulated in CoCl2-induced hypoxia and associated with
cell invasive potential and a poor prognosis of breast cancer, Int. J. Oncol. 48
a
(
2016) 271e280, https://doi.org/10.3892/ijo.2015.3253.
esters, Synth. Met. 51 (2019) 4284e4290, https://doi.org/10.1055/s-0039-
1690613.
[78] B.L. Wilkinson, L.F. Bornaghi, T.A. Houston, A. Innocente, C.T. Supuran,
S.A. Poulsen, A novel class of carbonic anhydrase inhibitors: glycoconjugate
benzene sulfonamides prepared by “click-tailing”, J. Med. Chem. 49 (2006)
6539e6548, https://doi.org/10.1021/jm060967z.
[79] M. Lopez, A.J. Salmon, C.T. Supuran, S.-A. Poulsen, Carbonic anhydrase in-
hibitors developed through ‘click tailing’, Curr. Pharmaceut. Des. 16 (2010)
3277e3287, https://doi.org/10.2174/138161210793429869.
[80] B.L. Wilkinson, L.F. Bornaghi, T.A. Houston, A. Innocenti, D. Vullo, C.T. Supuran,
S.A. Poulsen, Carbonic anhydrase inhibitors: inhibition of isozymes I, II, and IX
with triazole-linked O-glycosides of benzene sulfonamides, J. Med. Chem. 50
(2007) 1651e1657, https://doi.org/10.1021/jm061320h.
71] W.M. Eldehna, A. Nocentini, S.T. Al-Rashood, G.S. Hassan, H.M. Alkahtani,
A.A. Almehizia, A.M. Reda, H.A. Abdel-Aziz, C.T. Supuran, Tumor-associated
carbonic anhydrase isoform IX and XII inhibitory properties of certain isatin-
bearing sulfonamides endowed with in vitro antitumor activity towards colon
cancer, Bioorg. Chem. 81 (2018) 425e432, https://doi.org/10.1016/
j.bioorg.2018.09.007.
[
[
[
72] H.I. Gul, C. Yamali, H. Sakagami, A. Angeli, J. Leitans, A. Kazaks, K. Tars,
D.O. Ozgun, C.T. Supuran, New anticancer drug candidates sulfonamides as
selective hCA IX or hCA XII inhibitors, Bioorg. Chem. 77 (2018) 411e419,
https://doi.org/10.1016/j.bioorg.2018.01.021.
73] M. Krasavin, R. Zalubovskis, A. Grandane, I. Domraceva, P. Zhmurov,
C.T. Supuran, Sulfocoumarins as dual inhibitors of human carbonic anhydrase
isoforms IX/XII and of human thioredoxin reductase, J. Enzym. Inhib. Med.
Chem. 35 (2020) 506e510, https://doi.org/10.1080/14756366.2020.1712596.
74] M.A. Abdelrahman, W.M. Eldehna, A. Nocentini, S. Bua, S.T. Al-Rashood,
G.S. Hassan, A. Bonardi, A.A. Almehizia, H.M. Alkahtani, A. Alharbi, P. Gratteri,
C.T. Supuran, Novel diamide-based benzenesulfonamides as selective carbonic
ꢁ
ꢂ
ꢁ
[81] N. Pala, L. Micheletto, M. Sechi, M. Aggarwal, F. Carta, R. McKenna,
C.T. Supuran, Carbonic anhydrase inhibition with benzenesulfonamides and
tetrafluorobenzenesulfonamides obtained via click chemistry, ACS Med.
Chem. Lett. 5 (2014) 927e930, https://doi.org/10.1021/ml500196t.
15