Novel 3-substituted coumarins as selective human carbonic anhydrase IX and XII inhibitors: Synthesis, biological and molecular dynamics analysis

Article abstract of DOI:10.1016/j.ejmech.2020.112897

In this study, diverse series of coumarin derivatives were developed as potential carbonic anhydrase inhibitors (CAIs). A “tail” approach was adopted by selecting the coumarin motif as a tail that is connected to the ZBG benzenesulfonamide moiety via a hy

Full text of DOI:10.1016/j.ejmech.2020.112897

European Journal of Medicinal Chemistry 209 (2021) 112897
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Novel 3-substituted coumarins as selective human carbonic anhydrase
IX and XII inhibitors: Synthesis, biological and molecular dynamics
analysis
a
a
b, c
Mohamed A. Abdelrahman , Hany S. Ibrahim , Alessio Nocentini
,
Wagdy M. Eldehna d , Alessandro Bonardi , Hatem A. Abdel-Aziz , Paola Gratteri ,
, Claudiu T. Supuran
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, 11829, Egypt
,
*
b
,
c
e
c
f
,
**
b, ***
Sahar M. Abou-Seri
a
b
Department of NE.UROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico, Via U. Schiff 6, 50019, Sesto
Fiorentino, Firenze, Italy
c
Department of NEUROFARBA e Pharmaceutical and Nutraceutical Section, Laboratory of Molecular Modeling Cheminformatics & QSAR, University of
Firenze, Via U. Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
d
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, P.O. Box 33516, Egypt
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo, 12622, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, P.O. Box 11562, Egypt
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 May 2020
Received in revised form
In this study, diverse series of coumarin derivatives were developed as potential carbonic anhydrase
inhibitors (CAIs). A “tail” approach was adopted by selecting the coumarin motif as a tail that is con-
nected to the ZBG benzenesulfonamide moiety via a hydrazine (4a,b) or hydrazide (5a,b) linker.
Thereafter, an aryl sulfone tail was incorporated to afford the dual tailed coumarin-sulfonamide aryl-
sulfonehydrazones (13a-d) and hydrazides (14a,b). Then, the ZBG were removed from compounds 13 and
25 September 2020
Accepted 26 September 2020
Available online 1 October 2020
14 to furnish coumarin arylsulfonehydrazones (11a-d) and hydrazides (12a,b). Coumarin-sulfonamides 4
and 5 emerged as non-selective CAIs as they displayed good inhibitory activities toward all the examined
CA isozymes (I, II, IX and XII) in the nanomolar ranges. Interestingly, the “dual-tail” approach (com-
pounds 13 and 14) succeeded in achieving a good activity and selectivity toward CA IX/XII over the
physiologically dominant CA I/II. In particular, compounds 13d and 14a were the most selective
coumarin-sulfonamide counterparts. Concerning non-sulfonamide coumarin derivatives, coumarins 8
exhibited excellent activity and selectivity profiles against the target hCA IX/XII, whereas, coumarins 11
Keywords:
Coumarins
Sulfonamides
Molecular dynamics
Carbonic anhydrase inhibitors
Tail approach
I
and 12 reported excellent selectivity profile, but they barely inhibited hCA IX/XII with K s spanning in the
micromolar ranges. Furthermore, molecular modelling studies were applied to get a deep focus about the
feasible affinities and binding interactions for target coumarin-sulfonamides 4, 5, 13 and 14 with the
active site for CA II, IX and XII isoforms.
©
2020 Elsevier Masson SAS. All rights reserved.
ꢀ
1
. Introduction
CO
2
and dehydration of HCO
3
[1e3]. Seven families have been
-CAs exist in
vertebrates, the cytoplasm of green plants, bacteria, and algae [4].
Regarding hCAs, fifteen different CA isozymes belonging to the
discovered within carbonic anhydrases from which
a
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, car-
rying a zinc (II) ion which is essential for catalysis of hydration of
a
class have been identified, among them isozymes hCA IX and XII are
important in the cancer cells pH regulatory machine, as their in-
hibition was considered to be an effective antitumor strategy [3].
The expression of hCA IX is strongly related to poor prognosis in
cancers, whereas hCA XII isozyme is expressed in plenty of normal
tissues and organs, like endometrium, eye kidney, and colon and
*
Corresponding author.
*
*
* Corresponding author.
** Corresponding author.
E-mail addresses: wagdy2000@gmail.com (W.M. Eldehna), sahar.shaarawy@
pharma.cu.edu.eg (S.M. Abou-Seri), claudiu.supuran@unifi.it (C.T. Supuran).
https://doi.org/10.1016/j.ejmech.2020.112897
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
overexpressed in a wide range of human cancers. Additionally, hCA
I and II are ubiquitous cytosolic isozymes and their inhibition is to
be avoided to overcome the upsetting side effects when treatment
of cancer is the main target [5].
substituted phenyl and isatin tails, in CAIs IV and V, by a coumarin
motif to furnish target coumarin-sulfonamide hydrazones (4a,b)
and hydrazides (5a,b) (Fig.1). Thereafter, a “dual-tail” approach was
utilized through merging an aryl sulfone tail within compounds 4
Classical CAIs are primarily sulfonamide-based compounds. The
sulfonamide function is the most widely used zinc-binding group
and 5 to afford target coumarin-sulfonamide arylsulfonehy-
drazones (13a-d) and hydrazides (14a,b), respectively, which ex-
pected to modulate the interactions at both hydrophobic and
hydrophilic regions of CA active site. Moreover, the ZBG were
removed from compounds 13 and 14 affording target coumarin
arylsulfonehydrazones (11a-d) and hydrazides (12a,b), respec-
tively, in order to explore the CA inhibitory impact of the coumarin
motif only. In addition, the coumarin moiety was decorated with 6-
bromo substituent in order to furnish a lipophilic environment
which should be suitable for the hydrophobic nature of the hCA IX
active site and to explore a valuable SAR.
(
ZBG) among the CAIs which include the long-lasting clinically used
CAI- drugs as acetazolamide, furosemide and indisulam. Non-
classical CAIs are on contrast not based on ZBG. Non-classical
CAIs as polyamines, phenols, carboxylic acids and coumarins
could inhibit CA catalytic activity by binding directly to the catalytic
zinc moiety, through anchoring the zinc-bound water/hydroxide
ion or binding to the amino acid residues outside the active site,
occluding the entry of substrate and preventing catalysis [6].
In 2009, Supuran CT. et al., have reported coumarin derivatives I
as a promising novel class of non-zinc mediated CAIs and suggested
that the product of coumarin hydrolysis, cis-2-hydroxy-cinnamic
acid, but not the coumarin moiety bound with the CA active site [7].
Interestingly, the high selective inhibitory action toward cancer-
related isozymes (hCA IX and XII) exerted by coumarin-based
CAIs is attributed to the binding of the coumarin hydrolysis prod-
uct at the entrance for the active site cavity; the region which
significantly differ amongst the diverse hCAs [8,9]. The discovery of
this CAI class paved the way to develop a considerable number of
structurally diverse coumarin-based CAIs with efficient and selec-
tive profiles toward the cancer-associated isozymes IX and XII over
the ubiquitous CA I and II isozymes [10e12], such as umbelliferone
II and compound III [13,14] (Fig. 1).
2. Result and discussion
2.1. Chemistry
The proposed synthetic approaches to develop the target cou-
marins in this manuscript have been shown in Schemes 1, 2 and 3.
First, synthesis of coumarin-sulfonamide hydrazones (4a,b) and
hydrazides (5a,b) has been accomplished by condensation of 4-
hydrazinobenzenesulfonamide 2 or 4-sulfamoylbenzoic acid hy-
drazide 3 with 3-acetylcoumarins 1a,b in boiling glacial acetic acid
(Scheme 1).
In Scheme 2, 3-acetylcoumarins 1a,b were subjected to bromi-
Recently our research group adopted the “tail” approach, a
successful strategy to design isoform-selective CAIs, to develop
several sulfonamide-based CAIs with good potency and selectivity
profiles toward the cancer-related isozymes hCA IX and XII [15e18],
such as compounds IV [19] and V [20,21] (Fig. 1).
In this research work, our aim is to explore the CA inhibitory
activities for novel series of coumarin-based small molecules. Our
design is based on bioisosteric replacement for the para-
nation through reaction with Br
2
in glacial acetic acid to yield 3-
(bromoacetyl)coumarins 6a,b, which subsequently treated with
sodium benzenesulfinates 7a,b in refluxing absolute ethanol to
afford 3-(2-(phenylsulfonyl)acetyl)coumarin derivatives 8a-d.
Thereafter, reaction of compounds 8a-d with phenylhydrazine 9 or
benzoic acid hydrazide 10 in absolute ethyl alcohol with addition of
catalytic amount of glacial acetic acid furnished coumarin arylsul-
fonehydrazones (11a-d) and hydrazides (12a,b), respectively, in
Fig. 1. Chemical structures for some coumarin-based (I-III) and sulphonamide-based (IV and V) CAIs, in addition to the structures of the target coumarin derivatives 4, 5, 11, 12, 13
and 14.
2

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
Scheme 1. Reagents and condition: (i) Glacial acetic acid/reflux 2 h.
Scheme 2. Reagents and condition: (i) Br
2
/Glacial acetic acid/Stirring r.t 5 h; (ii) Absolute ethyl alcohol/reflux 3 h; (iii) Ethyl alcohol/Acetic acid/reflux 5 h.
3

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
7
4e82% yield.
Finally, condensation of 3-(2-(phenylsulfonyl)acetyl)coumarins
a-d with 4-hydrazinobenzenesulfonamide and 4-
between 1.35 and 3.96
micromolar potency (K
arylsulfones 8a-d, 11a-d and 12a,b failed to inhibit CA I isoform
up to 100 M (Table 1).
The in vitro kinetic data presented in Table 1 suggested that
inhibition profile for the physiologically dominant CA II isozyme is
analogous to the inhibition profile for CA I. Coumarin-sulfonamide
hydrazones (4a,b) and hydrazides (5a,b) potently inhibited CA II
m
M, except 13a which displayed sub-
I
¼ 0.82 M). On the contrary, coumarin-
m
8
2
sulfamoylbenzoic acid hydrazide 3 produced target coumarin-
sulfonamide arylsulfonehydrazones (13a-d) and hydrazides
m
(
14a,b), respectively, in 74e78% yield (Scheme 3).
Postulated structures for the target coumarins were in agree-
ment with their various spectral and analytical data.
I
with K values equal 29.1, 40.0, 26.3 and 12.1 nM, respectively,
2
2
.2. Biological evaluation
whereas coumarin-arylsulfone-sulfonamide hydrazones (13a-d)
and hydrazides (14a,b) displayed moderate hCA II inhibitory action
.2.1. Carbonic anhydrase inhibition
Target coumarins 4, 5, 8, 11, 12, 13 and 14 were assessed for their
with K
the other hand, coumarin-arylsulfones 8a-d, 11a-d and 12a,b didn’t
inhibit hCA II isoform up to 100 M.
I
values in the high nanomolar range (224.8e559.7 nM). On
CA inhibitory actions toward the ubiquitous CA I and II (cytosolic)
and CA IX and XII (tumor-related) isozymes by the use of a stopped
flow CO hydrase assay, utilizing acetazolamide (AAZ) as reference
2
CAI. Several structure activity relationships (SARs) were high-
lighted from the inhibition data displayed in Table 1.
The ubiquitous cytosolic hCA I isozyme was affected by the
synthesized coumarin derivatives in a variable degree. While
coumarin-sulfonamide hydrazones (4a,b) and hydrazides (5a,b)
showed potent hCA I inhibitory action with inhibition constant (K
values in the nanomolar range (K
respectively), coumarin-arylsulfone sulfonamide hydrazones (13a-
d) and sulfonamide hydrazides (14a,b) weakly inhibited hCA I
m
It is worthy to mention that, presence of sulfonamide func-
tionality is essential for inhibitory activity toward both hCA I and II
isoforms. Moreover, replacement of the hydrazine linker in
coumarin-sulfonamide 4a,b with the hydrazide one furnished
coumarin-sulfonamide 5a,b with slightly enhanced hCA I and hCA II
inhibitory actions, whereas, incorporation of the arylsulfone moiety
illustrated a lowering of effectiveness toward hCA I and hCA II
isoforms both for the hydrazones 13 and the hydrazides 14 in
comparison to their counterparts 4 and 5 (Table 1).
I
)
I
s ¼ 98.8, 159.7, 77.6 and 92.5 nM,
The investigated coumarin-sulfonamide hydrazones/hydrazides
(4a,b and 5a,b), coumarin-arylsulfones (8a-d) and coumarin-
arylsulfone-sulfonamide hydrazones/hydrazides (13a-d and 14a,
I
isoform with K values in the low micromolar range, as mentioned,
Scheme 3. Reagent and conditions: (i) Ethanol/Acetic acid/reflux 5 h.
4

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
Table 1
Inhibition data for hCA isozymes I, II, IX and XII for target coumarins 4, 5, 8, 11, 12, 13, 14 using AAZ as a standard CAI.
Cmp
R
R
1
X
K
I
(nM)a
CA I
CA II
CA IX
CA XII
4
4
5
5
8
8
8
8
1
1
1
1
1
1
1
1
1
1
1
1
a
b
a
b
H
Br
H
Br
H
H
Br
Br
H
e
e
98.8
159.7
77.6
29.1
40.0
26.3
12.1
43.8
24.2
31.6
19.8
41.4
66.3
76.6
87.2
1.50
3.94
4.64
8.11
16.0
8.86
89.1
76.0
63.6
29.2
18.6
42.1
25.0
10.1
55.8
19.7
34.6
60.1
51.5
45.7
83.7
1.81
1.05
7.21
4.77
6.59
9.34
74.4
91.1
36.2
12.0
32.3
76.8
5.7
e
e
e
C¼O
C¼O
e
e
92.5
b
a
H
CH
H
CH
H
CH
H
CH
H
H
H
CH
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
824.2
2596
3455
3962
1537
1352
250.0
m
m
m
m
m
m
m
m
m
m
M
M
M
M
M
M
M
M
M
M
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
442.4
559.7
276.0
538.3
224.8
365.2
12.0
m
m
m
m
m
m
m
m
m
m
M
M
M
M
M
M
M
M
M
M
b
b
3
3
3
3
e
b
c
e
b
d
1a
1b
1c
1d
2a
2b
3a
3b
3c
3d
4a
4b
e
b
b
e
m
M
M
M
M
M
M
m
m
m
m
m
m
M
M
M
M
M
M
H
e
m
m
m
m
m
b
Br
Br
H
Br
H
e
b
e
b
b
C¼O
C¼O
e
H
3
3
3
e
Br
Br
H
H
e
e
CH
H
CH
e
e
C¼O
C¼O
e
AAZ
a
Mean from three different assays (errors were calculated in the range of ± 5e10% of the aforementioned values).
Incubation time of 6 h (for coumarin derivatives only).
b
b) stood out as efficient inhibitors for the cancer-related hCA IX
isozyme (K values: 18.6e89.1 nM), whereas coumarin arylsulfone-
hydrazones/hydrazides (11a-d and 12a,b) exerted weak hCA IX
inhibitory action with values in the micromolar range
1.5e16.0 M). In particular, hydrazides 5b and 14a stood out as the
best CA IX inhibitors in the current study with low nanomolar
potencies (K
s ¼ 19.8 and 18.6 nM, respectively).
Remarkably, the deduced SAR pointed out that utilizing the
hydrazide linker (compounds 5a, 5b and 14a, 14b; K
s ¼ 31.6, 19.8,
8.6 and 42.1 nM, respectively) is more profitable for inhibitory
action toward hCA IX than the hydrazine linker (compounds 4a, 4b
and 13a, 13b; K
s ¼ 43.8, 24.2, 89.1 and 76.0 nM, respectively),
Table 1. Notably, the increased activities of 6-Br substituted cou-
marins 4b, 5b, 13c and 13d (K
s ¼ 24.2, 19.8, 63.6 and 29.2 nM,
respectively) than their corresponding unsubstituted derivativess
a, 5a, 13a and 13b (K
likened to the unsubstituted derivatives.
I
Regarding target/off-target hCAs selectivity ratio of action for
herein reported coumarin derivatives, all the examined coumarins,
except 4 and 5, displayed significant I/IX and I/XII inhibitory
selectivity, with calculated SI spanning the ranges 9.3 e >2415.5
and 11.1 e >2188.2, respectively, Table 2. Moreover, all compounds,
except 4a, b and 5a, b, exhibited a preferred inhibition action for
target CA IX and XII isozymes over the main off-target CA II isoform
with SIs spanning between 4.3 and >2415.5 and 4.8 e >2188.2,
respectively, Table 2.
Investigation of the calculated SIs for coumarin-sulfonamides 4,
5, 13 and 14 (Table 2) revealed that the dual tailed compounds 13d
and 14a were the most selective counterparts among these series
toward hCA IX/XII isoforms. Concerning non-sulfonamide
coumarin derivatives, coumarins 8a-d stood out as the most effi-
cient tumor-associated isoforms hCA IX/XII inhibitors reported here
in terms of both activity and selectivity. Coumarins 8a-d could not
K
I
(
m
I
I
1
I
I
4
I
s ¼ 43.8, 31.6, 89.1 and 76.0 nM, respectively),
highlighted that grafting of 6-Br group within coumarin moiety is
advantageous for hCA IX inhibitory activity. The obtained SAR to-
ward hCA II and IX isoforms was illustrated in Fig. 2.
inhibit hCA I and II lower than 100 mM, whereas, hCA IX and XII
were inhibited in low nanomolar ranges: 41.4e87.2 and
45.7e83.7 nM, respectively and possessed excellent selectivity
profile (SI ¼ >1146.8 e >2415.5). In contrast, coumarin arylsulfone-
hydrazones/hydrazides 11a-d and 12a,b reported good selectivity
profile (SI ¼ >6.3 e >95.2), but they barely inhibited hCA IX/XII
The second cancer-associated isozyme hCA XII was potently
inhibited by all coumarin derivatives prepared here with K
spanned in the range 10.1e91.1 nM, except coumarins 11 and 12
which barely inhibited hCA XII (K s: 1.05e9.34 M). Superiorly,
compound 4a exerted the best hCA XII inhibition here reported
with K
I
s
I
m
with values in the micromolar ranges: 1.5e16.0 and
K
I
I
¼ 10.1 nM.
1.05e9.34 mM, respectively.
On the contrary to the hCA IX inhibition profile, 6-Br substitu-
tion of the coumarin scaffold resulted in a worsening of effective-
ness toward hCA XII within coumarin-sulfonamide hydrazones/
hydrazides 4 and 5, whereas such substitution within coumarin-
arylsulfone- sulfonamide hydrazones 13 led to an improvement
of the activity with about 2- and 7.5-fold potency enhancement
3
4
. In silico analysis
Investigation on the binding mode of coumarin-sulfonamides
a,b, 5a,b, 13a-d and 14a,b within the active sites for hCA II (pdb
code 5LJT), hCA IX (pdb code 5FL4) and hCA XII (pdb code 1JD0) was
5

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
Fig. 2. SAR for the target CAIs toward hCA II and IX isoforms.
Table 2
Selectivity index (SI) calculated for hCA (IX and XII) over off-targets hCA isozymes (I
and II) as ratio between K s.
I
Cmpd
I/IX
I/XII
II/IX
II/XII
4
4
5
5
8
8
8
8
1
1
1
1
1
1
1
1
1
1
1
1
a
b
a
b
a
b
c
d
1a
1b
1c
1d
2a
2b
3a
3b
3c
3d
4a
4b
2.3
6.6
2.5
4.7
>2415.5
>1508.3
>1305.5
>1146.8
>66.7
>25.4
>21.6
>12.3
>6.3
>11.3
9.3
34.2
54.3
135.7
82.6
9.8
2.9
3.9
2.7
0.7
1.7
0.8
0.6
2.9
0.7
1.3
0.3
>1663.9
>1941.7
>2188.2
>1194.7
>55.2
>95.2
>13.9
>21.0
>15.2
>10.7
11.1
28.5
95.4
330.2
47.6
17.6
>2415.5
>1508.3
>1305.5
>1146.8
>66.7
>25.4
>21.6
>12.3
>6.3
>11.3
5.0
7.4
4.3
18.4
12.1
8.7
0.5
>1663.9
>1941.7
>2188.2
>1194.7
>55.2
>95.2
>13.9
>21.0
>15.2
>10.7
5.9
6.1
7.6
44.9
7.0
4.8
2.2
Fig. 3. Predicted binding orientation of 4a (green) and 13d (orange) within CA II active
site. H-bonds and p-p interactions are represented as black and blue dashed lines. (For
interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
32.1
10.0
AAZ
43.9
To study the solvation effect and conformational behavior over
the time, for the most selective coumarin-sulfonamides, against the
tumor-associated isoforms (13d and 14a), the docking was per-
formed with a 100 ns long molecular dynamic (MD) simulations
performed on the predicted binding poses. While the proteins are
even subjected to small conformational diversions, the plots in
Fig. 4 (red line) and Fig. 6 (violet) report variable dynamic evolution
of the docked ligands complexed with CA IX and XII during the 100
ns.
carried out by docking simulations. In general, all docking solutions
revealed that benzenesulfonamide moiety was located in-depth
into the CA active sites, where the sulfamoyl group was coordi-
nated to the zinc ion trough the deprotonated nitrogen atom and it
engaged two hydrogen bonds with the OG1 and backbone NH of
T199. Moreover, the phenyl moiety located in an area defined by
V121, V143, L198 residues where it was stabilized by the formation
of hydrophobic contacts.
The reduced size of the tail of derivatives 4a,b and 5a,b enables a
better accommodation of the ligand in the narrow binding site of
CA II, when compared to the bulkier dual tailed 13a-d and 14a,b,
inducing a greater CA II inhibition (Fig. 3).
In CA IX and XII, the coumarin moiety is placed in a cleft lined by
the amino acids V131, L91, Q92, Q71 and A131, T91, Q92 and K67,
respectively, while the arylsulfonyl moiety is oriented toward the
hydrophilic residues Q92, Q71, N67, H64, W5 (CA IX) and Q92, K67,
In particular, 13d reached a stable conformation (pose 5, Figs. 4A
and 5A) within CA IX active site after 50.0 ns with RMSD fluctuating
around 4.5 Å (Fig. 4A). As observed in Fig. S1A (Supporting Infor-
mation), the two tails of the ligand undergo small fluctuations
induced by the conformational evolution of the active site: from 50
ns onwards, the coumarin framework stably accommodates in the
hydrophobic area of the cavity, whereas the sulfonyltolyl portion
fits into the area between the metal-coordination pattern and
residues Q92, Q71, N67, H64, W5.
2
N62, H64, W5 (CA XII) engaging H-bonds with the eSO - group.
6

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
Fig. 4. RMSD analysis of ligand 13d heavy atoms and A) CA IX and B) CA XII backbone over the 100 ns MD simulation. The ligand color darkens over the dynamic simulation. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5. Final stable binding mode of ligand 13d into A) CA IX and B) CA XII active site after dynamic simulations of 100 ns. Water molecules are represented as red spheres. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Likewise, from about 50 ns onwards the binding conformation
of 13d stably settles within CA XII active site (Figs. 4Be5B, RMSD:
.8 Å). In detail, the coumarin and tolyl rings move to stack one onto
the other additionally forming VdW interactions with the CA XII
area lined by S135, A131 and T91 and a H-bond with Q92 (Fig. S1B).
Two main binding conformations, which quickly interchange
one into the other for the entire dynamic simulation, are found for
As well, in CA XII, the conformation of derivative 14a alternates
between pose 1 and 2 and then stabilizes definitively in pose 3
(Figs. 6B and 7B, RMSD: 2.9 Å). In this latter pose the coumarin
scaffold restores a docking-like conformation engaging a H-bond
with the OH of S135 side chain whereas the methyl-
enesulfonylphenyl pendant folds accommodating in the hydro-
philic pocket nearby H64. Moreover, a water-bridged H-bond
3
1
4a within CA IX active site (Fig. 6A, lower RMSD: 2.7 Å). In all poses
the hydrazide NH engages a water-bridged H-bond with the side
chain C]O of Q92, while the NH donates a H-bond to the exocyclic
2
occurs between the S]O group and N204 side chain NH (Fig. 7B).
2
4. Conclusion
coumarin C]O (Fig. S2A). It is worth noting that moving from the
hydrazone (13d) to hydrazide (14a) there is an inversion in the
positioning of the two ligand tails within CA IX active site (Figs. 5A
and 7A), as the coumarin framework of 14a accommodates into the
hydrophilic area previously occupied by 13d.
In summary, the current research work described the design and
synthesis of novel coumarin-sulfonamides (4a,b, 5a,b, 13a-d and
4a,b) and coumarins (8a-d, 11a-d, and 12a,b) as efficient hCAIs. All
the tested CA isoforms were affected by the synthesized coumarin
1
7

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
Fig. 6. RMSD analysis of ligand 14a heavy atoms and A) CA IX and B) CA XII backbone over the 100 ns MD simulation. The ligand color darkens over the dynamic simulation. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7. Final stable binding mode of ligand 14a into A) CA IX and B) CA XII active site after dynamic simulations of 100 ns. Water molecules are represented as red spheres. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
derivatives in variable degrees with K
CA I,12.1 nMe100 M for CA II, 19.8 nMe8.86
nMe9.34 M for XII. Concerning selectivity of our target coumarins,
all compounds except 4a,b and 5a,b showed good selectivity to-
ward CA IX and XII over CA I (SI ranges: 6.3e2415.5 and
I
ranges: 77.6 nMe100
m
M for
the narrow binding site of hCA I and II. Furthermore, removal of the
ZBG from series 13 and 14 resulted in coumarins 11 and 12 that
m
mM for CA XI, and 10.1
m
barely inhibited hCA IX/XII with K
In addition, utilizing the hydrazide linker (compounds 5a, 5b and
14a, 14b; K
s ¼ 31.6, 19.8, 18.6 and 42.1 nM, respectively) was found
to be more profitable for inhibitory action against hCA IX than the
hydrazine linker (compounds 4a, 4b and 13a, 13b; K
s ¼ 43.8, 24.2,
I
values in the micromolar ranges.
I
10.7e2188.2, respectively, respectively) and over CA II (SI ranges:
4
.3e2415.5 and 4.8e2188.2, respectively). The SAR outcomes
I
highlighted that incorporation of the arylsulfone moiety led to a
worsening of effectiveness against CA I and II isoforms for the dual
tailed hydrazones 13 and hydrazides 14 in comparison to their
counterparts 4 and 5, which resulted in a better selectivity profile
for series 13 and 14. This worsening of effectiveness could be
attributed to the worse accommodation of the bulkier dual tailed 13
and 14 than the reduced size coumarin-tailed derivatives 4 and 5 in
89.1 and 76.0 nM, respectively). The molecular modeling for 13d
revealed that the coumarin tail stably accommodated in the hy-
drophobic area of the cavity, whereas, the aryl sulfone tail oriented
toward the hydrophilic pocket. Replacement of hydrazino linker
(13) with the hydrazide one (14) resulted in an inversion in the
positioning of the ligand’s two tails within CA IX active site where
the coumarin framework accommodated into the hydrophilic area.
8

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
5
5
5
. Experimental
5.1.3. Synthesis of 3-(bromoacetyl)coumarins 6a, b
-Acetylcoumarins 1a, b were brominated according to the re-
ported procedures [22,23].
3
.1. Chemistry
.1.1. General
5.1.4. General procedures for preparation of key intermediate of 8a-
d
NMR spectra have been recorded by Varian Gemini-400BB
1
13
4
4
00 MHz spectrometer. H and C spectra were carried out at
00 and 100 MHz, respectively, in deuterated dimethylsulfoxide
The appropriate sodium benzenesulfinate derivative 7a,b
(15 mmol) was added to a hot stirred solution of 3-(bromoacetyl)
coumarin derivatives 6a,b (15 mmol) in absolute ethanol (15 mL),
then the resulting mixture was stirred under reflux for 3 h. The
formed precipitate was collected by filtration while hot, washed
with methyl alcohol, dried and recrystallized dioxan/ethanol to
yield 3-(2-(phenylsulfonyl)acetyl)coumarins 8a-d.
(
6 H
DMSO‑d ). Chemical shifts (d ) are reported relative to TMS as
internal standard. All coupling constant (J) values are given in hertz.
IR spectra have been recorded with a Bruker FT-IR spectropho-
tometer as KBr disks and expressed in wave number (cm ). Unless
otherwise noted, all solvents and reagents were commercially
available and used without further purification.
ꢀ1
5
.1.4.1. 3-(2-(Phenylsulfonyl)acetyl)-2H-chromen-2-one
(8a).
ꢁ
Gray crystals (yield 86%), m.p. 138e140 C; IR: 1727, 1689 (2C]O)
and 1318, 1144 (SO ); H NMR d ppm: 5.42 (s, 2H, CH ), 7.43 (d, 1H,
2 2
5.1.2. General procedures for synthesis of coumarin-sulfonamide
1
hydrazones/hydrazides 4a,b and 5a,b
To a solution of 3-acetylcoumarin derivatives 1a,b (1 mmol) in
glacial acetic acid (5 mL), 4-hydrazinobenzenesulfonamide 2
J ¼ 7.9 Hz, AreH), 7.55e7.58 (m, 3H, AreH), 7.68 (t, 1H, J ¼ 7.9 Hz,
AreH), 7.89e7.99 (m, 4H, AreH), 8.15 (s, 1H, H-4 coumarin); Anal.
12 5
calcd. for C17H O S (328.34): C, 62.19; H, 3.68. Found C, 62.04; H,
(
1
0.187 g, 1 mmol) or 4-sulfamoylbenzoic acid hydrazide 3 (0.215 g,
mmol) was added. The resulting reaction mixture was allowed to
3.71.
stir under reflux for 2 h. The precipitated solid was collected by
filtration while hot, washed with cold ethanol, dried and recrys-
tallized from dioxan to produce the titled coumarin-based sulfon-
amides 4a,b and 5a,b.
5
.1.4.2. 3-(2-Tosylacetyl)-2H-chromen-2-one (8b). Gray crystals
ꢁ
(
(
(
yield 73%), m.p. 152e154 C; IR: 1733, 1684 (2C]O) and 1320, 1146
SO
m, 4H, AreH), 7.65e7.69 (m, 2H, AreH), 7.79e7.81 (m, 2H, AreH),
1
2 3 2
); H NMR d ppm: 2.39 (s, 3H, CH ), 5.41 (s, 2H, CH ), 7.30e7.37
8
6
.17 (s, 1H, H-4 coumarin); Anal. calcd. for C18
3.15; H, 4.12. Found C, 63.29; H, 4.10.
14 5
H O S (342.37): C,
5
.1.2.1. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)ethylidene)hydrazineyl)
benzenesulfonamide (4a). Yellow crystals (yield 88%), m.p.
ꢁ
2
23e225 C; IR: 3437, 3324, 3242 (NH, NH
2
), 1725 (C]O) and 1334,
ppm: 2.28, (s, 3H, CH ), 7.12 (s, 1H, AreH),
.36e7.45 (m, 5H, AreH and NH O exchangeable), 7.61e7.70 (m,
H, AreH), 7.86 (d, 1H, J ¼ 7.8 Hz, AreH), 8.26 (s, 1H, H-4 coumarin),
.99 (s, 1H, NH D O exchangeable); Anal. calcd. for C17
1
5.1.4.3. 6-Bromo-3-(2-(phenylsulfonyl)acetyl)-2H-chromen-2-one
1168 (SO
2
); H NMR
d
3
ꢁ
(
(
(
8c). Gray crystals (yield 82%), m.p. 188e190 C; IR: 1734, 1683
2C]O) and 1349, 1156 (SO
d,1H, J ¼ 8.9 Hz, AreH), 7.63e7.68 (m, 2H, AreH), 7.73e7.76 (m,1H,
7
3
9
2 2
D
1
2 2
); H NMR d ppm: 5.29 (s, 2H, CH ), 7.46
2
15
H N
3
O
4
S
AreH), 7.91e7.96 (m, 3H, AreH), 8.22 (d, 1H, J ¼ 2.4 Hz, AreH), 8.68
(
357.38): C, 57.13; H, 4.23; N,11.76. Found C, 57.23; H, 4.19; N, 11.67.
13
(s, 1H, H-4 coumarin); C NMR
2
d ppm: 64.95 (CH ), 117.13, 119.04,
1
20.25, 124.60, 128.53, 129.79, 133.35, 134.62, 137.89, 139.87, 147.76,
5
.1.2.2. 4-(2-(1-(6-Bromo-2-oxo-2H-chromen-3-yl)ethylidene)
154.19, 158.26, 186.27; Anal. calcd. for C17
H11BrO
5
S (407.23): C,
hydrazineyl)benzenesulfonamide (4b). Yellow crystals (yield 77%),
50.14; H, 2.72. Found C, 50.31; H, 2.69.
ꢁ
m.p. 232e234 C; IR: 3421, 3329, 3243 (NH, NH
2
), 1720 (C]O) and,
ppm: 2.27, (s, 3H, CH ), 7.12 (s, 2H,
AreH), 7.37e7.42 (m, 3H, AreH and NH O exchangeable), 7.68
d, 2H, J ¼ 8.6 Hz, AreH), 7.75 (d, 1H, J ¼ 8.8 Hz, AreH), 8.13 (s, 1H,
AreH), 8.25 (s, 1H, H-4 coumarin), 10.04 (s, 1H, NH
exchangeable); Anal. calcd. for C17 14BrN S (336.28): C, 46.80; H,
.23; N, 9.63. Found C, 46.73; H, 3.26; N, 9.71.
1
1341, 1163 (SO
2
); H NMR
d
3
5.1.4.4. 6-Bromo-3-(2-tosylacetyl)-2H-chromen-2-one
(8d).
2
D
2
ꢁ
Gray crystals (yield 75%), m.p. 168e170 C; IR: 1740, 1686 (2C]O)
(
1
and 1350, 1158 (SO
CH
), 7.41 (d, 2H, J ¼ 8.0 Hz, AreH), 7.45 (d, 1H, J ¼ 8.9 Hz, AreH),
.76 (d, 2H, J ¼ 8.4 Hz, AreH), 7.91 (dd, 1H, J ¼ 2.5 Hz, J ¼ 8.8 Hz,
AreH), 8.19 (d, 1H, J ¼ 2.4 Hz, AreH), 8.63 (s, 1H, H-4 coumarin);
Anal. calcd. for C18 13BrO S (421.26): C, 51.32; H, 3.11. Found C,
2 3
); H NMR d ppm: 2.36 (s, 3H, CH ), 5.24 (s, 2H,
2
D O
2
H
3 4
O
7
3
H
5
5
.1.2.3. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)ethylidene)hydrazine-1-
51.23; H, 3.13.
carbonyl)benzenesulfonamide (5a). Yellow crystals (yield 78%), m.p.
2
and 1344, 1166 (SO
ꢁ
20e222 C; IR: 3427, 3321, 3246 (NH, NH
2
), 1724, 1655 (2C]O)
ppm: 2.26, 2.33 (2s, 3H, CH ),
2
O exchangeable), 7.64e7.73 (m,
5.1.5. General procedures for preparation of target coumarins 11a-
1
2
); H NMR
d
3
d, 12a, b, 13a-d and 14a, b
7
1
8
.38e7.52 (m, 5H, AreH and NH
2
D
To a hot solution of the appropriate key intermediate 3-(2-
H, AreH), 7.78e7.96 (m, 2H, AreH), 8.01e8.07 (m, 2H, AreH), 8.17,
.25 (2s, 1H, H-4 coumarin), 10.91, 10.98 (2s, 1H, NH D
S (385.38): C, 56.10; H,
.92; N, 10.90. Found C, 56.23; H, 3.89; N, 10.97.
(phenylsulfonyl)acetyl)coumarins 8a-d (4 mmol) in absolute
2
O
ethanol (10 mL) containing few drops of glacial acetic acid, phe-
nylhydrazine 9 (0.43 g, 4 mmol) or benzoic acid hydrazide 10
15 3 5
exchangeable); Anal. calcd. for C18H N O
3
(0.54 g, 4 mmol) or 4-hydrazinobenzenesulfonamide 2 (0.74 g,
4
mmol) or 4-sulfamoylbenzoic acid hydrazide 3 (0.86 g, 4 mmol)
5
.1.2.4. 4-(2-(1-(6-Bromo-2-oxo-2H-chromen-3-yl)ethylidene)hy-
was added. The resulting reaction mixture was heated under reflux
for 5 h with stirring. Thereafter, the reaction mixture was filtered
while hot, and the collected precipitate was washed with ethyl
alcohol and recrystallized from ethanol/DMF mixture (5:1) to
afford the target coumarins 11a-d, 12a,b, 13a-d, and 14a,b,
respectively.
drazine-1-carbonyl)benzenesulfonamide (5b). Yellow crystals (yield
ꢁ
76%), m.p. 230e232 C; IR: 3430, 3353, 3249 (NH, NH
2
), 1722, 1639
ppm: 2.25, 2.32 (2s, 3H,
O exchangeable), 7.77 (m,
H, AreH), 7.89e7.93 (m, 2H, AreH), 7.98e8.03 (m, 2H, AreH), 8.14,
.20 (2s, 1H, H-4 coumarin), 10.91, 10.98 (2s, 1H, NH D
14BrN S (464.29): C, 46.57; H,
.04; N, 9.05. Found C, 46.73; H, 3.00; N, 9.10.
1
(
2C]O) and 1320, 1193 (SO
2
); H NMR d
CH ), 7.40e7.52 (m, 4H, AreH and NH D
1
8
exchangeable); Anal. calcd. for C18
3
3
2 2
2
O
H
3
O
5
5.1.5.1. 3-(1-(2-Phenylhydrazono)-2-(phenylsulfonyl)ethyl)-2H-chro-
ꢁ
men-2-one (11a). Orange crystals (yield 81%), m.p. 210e212 C; IR:
9

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
1
3
(
452 (NH), 1712 (C]O) and 1349, 1149 (SO
s, 2H, CH
), 6.85 (t, 1H, J ¼ 7.2 Hz, AreH), 7.15 (d, 2H, J ¼ 8.0 Hz,
AreH), 7.24e7.28 (m, 2H, AreH), 7.37e7.41 (m, 2H, AreH),
.46e7.49 (m, 3H, AreH), 7.61e7.64 (m, 1H, AreH), 7.83e7.86 (m,
H, AreH), 8.11 (s, 1H, H-4 coumarin), 10.17 (s, 1H, NH D
2
); H NMR
d
ppm: 5.29
5.1.5.7. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)-2-(phenylsulfonyl)ethyl-
idene)hydrazinyl)benzenesulfonamide (13a). Yellow crystals (yield
2
ꢁ
77%), m.p. 255e257 C; IR: 3421, 3327, 3257 (NH, NH
2
), 1747 (C]O),
1
7
3
1310 and 1156 (SO
NH
2
); H NMR
d
ppm: 5.31 (s, 2H, CH ), 7.15 (s, 2H,
2
2
O
2
D
2
O exchangeable), 7.25 (d, 2H, J ¼ 8.9 Hz, AreH), 7.39e7.43
13
exchangeable); C NMR
25.26, 125.29, 126.24, 128.54, 129.38, 129.50, 132.37, 134.45,
39.36, 140.80, 144.55, 153.16, 160.07; Anal. calcd. for C23
418.47): C, 66.02; H, 4.34; N, 6.69. Found C, 66.10; H, 4.39; N, 6.61.
d
ppm: 52.51, 113.79, 116.18, 119.58, 121.10,
(m, 2H, AreH), 7.45e7.51 (m, 3H, AreH), 7.63 (t, 1H, J ¼ 8.0 Hz,
1
1
(
AreH), 7.69e7.72 (m, 2H, AreH), 7.83e7.89 (m, 3H, AreH), 8.18 (s,
13
18
H N
2
O
4
S
1H, H-4 coumarin), 10.48 (s, 1H, NH D
(DMSO‑d ppm: 52.73 (CH ), 113.17, 116.27, 119.49, 124.93, 125.29,
27.71, 128.60, 129.13, 129.57, 129.61, 132.67, 134.53, 135.95, 139.27,
141.67, 147.20, 153.33, 159.89; Anal. calcd. for C23
2
O exchangeable); C NMR
6
)
d
2
1
5
(
1
5
.1.5.2. 3-(1-(2-Phenylhydrazono)-2-tosylethyl)-2H-chromen-2-one
11b). Orange crystals (yield 79%), m.p. 215e217 C; IR: 3423 (NH),
19 3 6 2
H N O S
ꢁ
(497.54): C, 55.52; H, 3.85; N, 8.45. Found C, 55.43; H, 3.90; N, 8.51.
1
725 (C]O) and 1347, 1150 (SO
.23 (s, 2H, CH
), 6.85 (t,1H, J ¼ 7.3 Hz, AreH), 7.16 (d, 2H, J ¼ 8.0 Hz,
AreH), 7.20 (d, 2H, J ¼ 7.9 Hz, AreH), 7.25 (t, 2H, J ¼ 7.7 Hz, AreH),
2 3
); H NMR d ppm: 1.99 (s, 3H, CH ),
2
5.1.5.8. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)-2-tosylethylidene)hydra-
zinyl)benzenesulfonamide (13b). Yellow crystals (yield 74%), m.p.
ꢁ
7
2
.37 (t, 2H, J ¼ 7.8 Hz, AreH), 7.59 (t, 1H, J ¼ 7.8 Hz, AreH), 7.66 (d,
240e242 C; IR: 3430, 3340, 3259 (NH, NH
2
), 1731 (C]O), 1320 and
), 5.25 (s, 2H, CH ), 7.19
2 2
D O exchangeable), 7.25 (d, 2H,
1
H, J ¼ 7.9 Hz, AreH), 7.81 (d, 1H, J ¼ 7.6 Hz, AreH), 8.00 (s, 1H, H-4
1148 (SO
2
); H NMR
d
ppm: 2.00 (s, 3H, CH
3
2
13
coumarin), 10.17 (s, 1H, NH D
2
1
1
2
O exchangeable); C NMR
d
ppm:
(d, 4H, J ¼ 7.6 Hz, AreH and NH
1.11 (CH
25.06,125.24,126.52,128.67,129.31,129.48,129.69,129.83,130.04,
32.26, 136.28, 140.89, 144.58,145.38, 153.11,160.00; Anal. calcd. for
3
), 52.63 (CH
2
),113.77,116.14,119.57,119.98,120.33,121.02,
J ¼ 8.8 Hz, AreH), 7.39 (t, 2H, J ¼ 8.0 Hz, AreH), 7.61 (dd, 1H,
J ¼ 1.7 Hz, J ¼ 8.0 Hz, AreH), 7.66 (d, 2H, J ¼ 8.2 Hz, AreH), 7.71 (d,
2H, J ¼ 8.8 Hz, AreH), 7.83 (d, 1H, J ¼ 9.4 Hz, AreH), 8.08 (s, 1H, H-4
13
C
4
24 20
H N
2
O
4
S (432.49): C, 66.65; H, 4.66; N, 6.48. Found C, 66.58; H,
coumarin), 10.51 (s, 1H, NH D
21.15 (CH ), 52.78 (CH
27.69, 128.72, 129.35, 130.07, 132.57, 135.84, 136.14, 141.77, 145.46,
147.21, 153.25, 159.84; Anal. calcd. for C24 (511.57): C,
56.35; H, 4.14; N, 8.21. Found C, 56.43; H, 4.19; N, 8.16.
2
O exchangeable); C NMR d ppm:
2
), 113.14, 11,621, 119.46, 124.66, 125.29,
.69; N, 6.55.
3
1
5
.1.5.3. 6-Bromo-3-(1-(2-phenylhydrazono)-2-(phenylsulfonyl)
ethyl)-2H-chromen-2-one (11c). Orange crystals (yield 77%), m.p.
30e232 C; IR: 3428 (NH), 1705 (C]O) and 1350, 1141 (SO
21 3 6 2
H N O S
ꢁ
1
2
2
); H
NMR ppm: 5.27 (s, 2H, CH
d
2
), 6.86 (t, 1H, J ¼ 7.2 Hz, AreH), 7.16 (d,
5.1.5.9. 4-(2-(1-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-(phenyl-
2
H, J ¼ 8.0 Hz, AreH), 7.24 (t, 2H, J ¼ 7.7 Hz, AreH), 7.35 (d, 1H,
sulfonyl)ethylidene)hydrazineyl)benzenesulfonamide
(13c).
ꢁ
J ¼ 8.8 Hz, AreH), 7.49e7.51 (m, 3H, AreH), 7.74 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 8.8 Hz, AreH), 7.81e7.84 (m, 2H, AreH), 8.12e8.13 (m, 2H, H-4
Yellow crystals (yield 78%), m.p. 215e217 C; IR: 3420, 3330, 3249
1
(NH, NH
2 2
), 1721 (C]O), 1332 and 1146 (SO ); H NMR d ppm:
coumarin and AreH), 10.23 (s, 1H, NH D
2
O exchangeable); Anal.
5.30e5.36 (m, 2H, CH
2
), 7.04 (d, 2H, J ¼ 8.5 Hz, NH
2 2
D O
calcd. for C23
H17BrN
2
O
4
S (497.36): C, 55.54; H, 3.45; N, 5.63. Found
exchangeable), 7.21 (s, 2H, AreH), 7.46e7.53 (m, 2H, AreH), 7.63 (t,
2H, J ¼ 7.7 Hz, AreH), 7.71e7.75 (m, 3H, AreH), 7.91e7.96 (m, 2H,
AreH), 8.20e8.22 (m, 1H, AreH), 8.69 (s, 1H, H-4 Coumarin), 10.48
C, 55.57; H, 3.49; N, 5.71.
5
.1.5.4. 6-Bromo-3-(1-(2-phenylhydrazono)-2-tosylethyl)-2H-chro-
2 3 6 2
(s, 1H, NH D O exchangeable); Anal. calcd. for C23H18BrN O S
(574.44): C, 47.92; H, 3.15; N, 7.29. Found C, 47.87; H, 3.19; N, 7.37.
ꢁ
men-2-one (11d). Orange crystals (yield 74%), m.p. 233e235 C; IR:
3
1
437 (NH), 1722 (C]O) and 1348, 1143 (SO
2
); H NMR d ppm: 2.04
(
2
s, 3H, CH ), 5.21 (s, 2H, CH
H, J ¼ 8.0 Hz, AreH), 7.21 (d, 2H, J ¼ 8.0 Hz, AreH), 7.25 (t, 2H,
3
2
), 6.86 (t, 1H, J ¼ 7.2 Hz, AreH), 7.16 (d,
5.1.5.10. 4-(2-(1-(6-Bromo-2-oxo-2H-chromen-3-yl)-2-
tosylethylidene)hydrazineyl)benzenesulfonamide
(13d). Yellow
ꢁ
J ¼ 7.7 Hz, AreH), 7.35 (d, 1H, J ¼ 8.8 Hz, AreH), 7.65 (d, 2H,
crystals (yield 75%), m.p. 250e252 C; IR: 3378, 3321, 3272 (NH,
1
J ¼ 8.0 Hz, AreH), 7.73 (dd, 1H, J ¼ 2.4 Hz, J ¼ 8.8 Hz, AreH), 8.00 (s,
NH
(2s, 3H, CH
O exchangeable), 7.36e7.48 (m, 2H, AreH), 7.65e7.84 (m, 5H,
AreH), 8.08e8.18 (m, 2H, H-4 coumarin and AreH), 10.56 (s, 1H, NH
O exchangeable); Anal. calcd. for C24 20BrN (590.46): C,
2
), 1721 (C]O), 1330 and 1149 (SO
2
); H NMR d ppm: 1.99, 2.04
1
D
5
H, H-4 coumarin), 8.09 (d, 1H, J ¼ 2.4 Hz, AreH), 10.22 (s, 1H, NH
3
), 5.24 (s, 2H, CH ), 7.21e7.28 (m, 5H, AreH and NH
2
2
2
O exchangeable); Anal. calcd. for C24
H19BrN
2
O
4
S (511.39): C,
D
2
6.37; H, 3.75; N, 5.48. Found C, 56.43; H, 3.70; N, 5.54.
D
2
H
3 6 2
O S
5
.1.5.5. N’-(1-(2-Oxo-2H-chromen-3-yl)-2-(phenylsulfonyl)ethyl-
48.82; H, 3.41; N, 7.12. Found C, 48.92; H, 3.36; N, 7.24.
idene)benzohydrazide (12a). Yellow crystals (yield 81%), m.p.
ꢁ
2
(
70e272 C; IR: 3428 (NH), 1641, 1630 (2C]O) and, 1337, 1160
5.1.5.11. 4-(2-(1-(2-oxo-2H-chromen-3-yl)-2-(phenylsulfonyl)ethyl-
1
SO
AreH), 7.54e7.56 (m, 4H, AreH), 7.64e7.71 (m, 3H, AreH),
.78e7.80 (m, 2H, AreH), 7.84e7.91 (m, 3H, AreH), 8.12 (s, 1H, H-4
coumarin), 11.31 (s, 1H, NH D O exchangeable); Anal. calcd. for
S (446.48): C, 64.56; H, 4.06; N, 6.27. Found C, 64.49; H,
.10; N, 6.33.
2
); H NMR
d
ppm: 5.43 (s, 2H, CH
2
), 7.42 (d, 2H, J ¼ 7.7 Hz,
idene)hydrazine-1-carbonyl)benzenesulfonamide
(14a). Yellow
ꢁ
crystals (yield 78%), m.p. 225e227 C; IR: 3422, 3287, 3206 (NH,
1
7
NH
(s, 2H, CH
NH O exchangeable), 7.67e7.71 (m, 1H, AreH), 7.90e8.01 (m,
7H, AreH), 8.15 (s, 1H, H-4 coumarin), 11.42 (s, 1H, NH D
2
), 1718, 1691 (2C]O), 1318 and 1156 (SO
2
); H NMR d ppm: 5.43
2
2
), 7.40e7.44 (m, 2H, AreH), 7.54e7.60 (m, 5H, AreH and
C
4
23
18
H N
2
O
4
2 2
D
2
O
13
exchangeable); C NMR
2
d ppm: 53.72 (CH ), 116.46, 119, 124.52,
5
.1.5.6. N’-(1-(6-bromo-2-oxo-2H-chromen-3-yl)-2-(phenylsulfonyl)
125.51, 126.21, 128.66, 129.32, 129.68, 129.96, 131.66, 133.50, 134.41,
134.87,136.43, 136.90,138.96,143.92,143.96, 153.71, 159.70,163.87;
ethylidene)benzohydrazide (12b). Yellow crystals (yield 82%), m.p.
above 300 C; IR: 3431 (NH), 1649, 1628 (2C]O) and 1334, 1154
ꢁ
19 3 7 2
Anal. calcd. for C24H N O S (525.55): C, 54.85; H, 3.64; N, 8.00.
(
SO
AreH), 7.54e7.66 (m, 4H, AreH), 7.74e7.77 (m, 2H, AreH),
.79e7.90 (m, 2H, AreH), 7.98e8.01 (m, 3H, AreH), 8.13 (s, 1H, H-4
coumarin), 11.39 (s, 1H, NH D O exchangeable); Anal. calcd. for
17BrN S (525.37): C, 54.87; H, 3.26; N, 5.33. Found C, 54.77;
H, 3.31; N, 5.41.
2
); 1H NMR
d
ppm: 5.41 (s, 2H, CH
2
), 7.50 (d, 2H, J ¼ 8.0 Hz,
Found C, 54.72; H, 3.59; N, 8.11.
7
5.1.5.12. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)-2-tosylethylidene)hydra-
2
zine-1-carbonyl)benzenesulfonamide (14b). Yellow crystals (yield
ꢁ
C
24
H
2
O
5
75%), m.p. 240e242 C; IR: 3421, 3298, 3220 (NH, NH
2
), 1696, 1663
1
(2C]O), 1319 and 1159 (2SO
2
); H NMR
d
ppm: 2.03 (s, 3H, CH ),
3
10

M.A. Abdelrahman, H.S. Ibrahim, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112897
J. Enzym. Inhib. Med. Chem. 31 (2016) 345e360.
10] A. Nocentini, F. Carta, M. Ceruso, G. Bartolucci, C.T. Supuran, Click-tailed
coumarins with potent and selective inhibitory action against the tumor-
associated carbonic anhydrases IX and XII, Bioorg. Med. Chem. 23 21 (2015)
6955e6966.
5
.39 (s, 2H, CH
NH
O exchangeable), 7.61 (s, 2H, AreH), 7.66 (t, 1H, J ¼ 7.2 Hz,
AreH), 7.73 (d, 2H, J ¼ 7.9 Hz, AreH), 7.84e7.85 (m, 1H, AreH),
.94e8.03 (m, 5H, AreH and H-4 coumarin), 11.41 (s, 1H, NH D
2
), 7.26 (d, 2H, J ¼ 7.9 Hz, AreH), 7.41e7.44 (m, 2H,
[
2
D
2
7
2
O
13
[11] B.Z. Kurt, F. Sonmez, D. Ozturk, A. Akdemir, A. Angeli, C.T. Supuran, Synthesis
of coumarin-sulfonamide derivatives and determination of their cytotoxicity,
carbonic anhydrase inhibitory and molecular docking studies, Eur. J. Med.
Chem. 183 (2019) 111702.
exchangeable); C NMR
16.57, 118.98, 124.14, 125.49, 126.18, 128.72, 129.28, 129.86, 130.16,
30.40, 133.40, 135.91, 136.48, 136.91, 140.27, 144.00, 145.09 145.18,
45.81, 147.55, 153.64, 159.64, 163.80; Anal. calcd. for
21BrN (439.58): C, 55.65; H, 3.92; N, 7.79. Found C, 55.53;
H, 3.89; N, 7.87.
3 2
d ppm: 21.20 (CH ), 53.73 (CH ), 116.42,
1
1
1
C
ꢁ
ꢂ
ꢁ
[
12] 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.
25
H
3 7 2
O S
[
13] A. Maresca, A. Scozzafava, C.T. Supuran, 7,8-Disubstituted-but not 6,7-
disubstituted coumarins selectively inhibit the transmembrane, tumor-
associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I
and II in the low nanomolar/subnanomolar range, Bioorg. Med. Chem. Lett 20
5.2. Biological evaluation
(
2010) 7255e7258.
The experimental procedures adopted for CA inhibitory assay
for the target compounds were performed as descriped eariler
24,25], and presented in the Supplementary Materials.
[
[
[
14] M. Bozdag, M. Ferraroni, F. Carta, D. Vullo, L. Lucarini, E. Orlandini, A. Rossello,
E. Nuti, A. Scozzafava, E. Masini, Structural insights on carbonic anhydrase
inhibitory action, isoform selectivity, and potency of sulfonamides and cou-
marins incorporating arylsulfonylureido groups, J. Med. Chem. 57 (2014)
[
9
152e9167.
5.3. In silico study
15] A. Petreni, A. Bonardi, C. Lomelino, S.M. Osman, Z.A. ALOthman, W.M. Eldehna,
R. El-Haggar, R. McKenna, A. Nocentini, C.T. Supuran, Inclusion of a 5-
fluorouracil moiety in nitrogenous bases derivatives as human carbonic
anhydrase IX and XII inhibitors produced a targeted action against MDA-MB-
The procedures adopted for the in silico were presented in the
Supplementary Materials.
2
31 and T47D breast cancer cells, Eur. J. Med. Chem. 190 (2020) 112112.
16] W.M. Eldehna, M.F. Abo-Ashour, A. Nocentini, R.S. El-Haggar, S. Bua,
A. Bonardi, S.T. Al-Rashood, G.S. Hassan, P. Gratteri, H.A. Abdel-Aziz,
Enhancement of the tail hydrophobic interactions within the carbonic anhy-
drase IX active site via structural extension: synthesis of novel N-substituted
isatins-SLC-0111 hybrids as carbonic anhydrase inhibitors and antitumor
agents, Eur. J. Med. Chem. 162 (2019) 147e160.
17] W.M. Eldehna, M.F. Abo-Ashour, E. Berrino, D. Vullo, H.A. Ghabbour, S.T. Al-
Rashood, G.S. Hassan, H.M. Alkahtani, A.A. Almehizia, A. Alharbi, SLC-0111
enaminone analogs, 3/4-(3-aryl-3-oxopropenyl) aminobenzenesulfonamides,
as novel selective subnanomolar inhibitors of the tumor-associated carbonic
anhydrase isoform IX, Bioorg. Chem. 83 (2019) 549e558.
Declaration of competing interest
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
[
[
18] M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, H.S. Ibrahim, S. Bua, H.A. Abdel-
Aziz, S.M. Abou-Seri, C.T. Supuran, Novel synthesized SLC-0111 thiazole and
thiadiazole analogues: determination of their carbonic anhydrase inhibitory
activity and molecular modeling studies, Bioorg. Chem. 87 (2019) 794e802.
19] H.A. Allam, S.H. Fahim, M.F. Abo-Ashour, A. Nocentini, M.E. Elbakry,
M.A. Abdelrahman, W.M. Eldehna, H.S. Ibrahim, C.T. Supuran, Application of
hydrazino and hydrazido linkers to connect benzenesulfonamides with hy-
drophilic/phobic tails for targeting the middle region of human carbonic
anhydrases active site: selective inhibitors of hCA IX, Eur. J. Med. Chem. 179
Ministero dell’istruzione, dell’universit aꢀ e della ricerca (MIUR) is
gratefully acknowledged for the grant PRIN 2017XYBP2R.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112897.
(2019) 547e556.
[
20] M.F. Abo-Ashour, W.M. Eldehna, A. Nocentini, A. Bonardi, S. Bua, H.S. Ibrahim,
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11