Benzofuran-Based Carboxylic Acids as Carbonic Anhydrase Inhibitors and Antiproliferative Agents against Breast Cancer

Article abstract of DOI:10.1021/acsmedchemlett.0c00094

Pursuing our effort for developing effective inhibitors of the cancer-related hCA IX isoform, here we describe the synthesis of novel benzofuran-based carboxylic acid derivatives, featuring the benzoic (9a-f) or hippuric (11a,b) acid moieties linked to 2-

Full text of DOI:10.1021/acsmedchemlett.0c00094

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Letter
Benzofuran-based carboxylic acids as carbonic anhydrase
inhibitors and antiproliferative agents against breast cancer
Wagdy M. Eldehna, Alessio Nocentini, Zainab M. Elsayed, Tarfah Al-Warhi, Nada Aljaeed,
Ohoud J. Alotaibi, Mohammad M. Al-Sanea, Hatem A. Abdel-Aziz, and Claudiu T. Supuran
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00094 • Publication Date (Web): 18 Mar 2020
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Benzofuran-based carboxylic acids as carbonic anhydrase
inhibitors and antiproliferative agents against breast cancer
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Wagdy M. Eldehnaa,b,*, Alessio Nocentinic,*, Zainab M. Elsayedb, Tarfah Al-Warhid,d Nada Aljaeedd,
Ohoud J. Alotaibid, Mohammad M. Al-Saneae, Hatem A. Abdel-Azizf, Claudiu T. Supuranc,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, P.O. Box 33516, Egypt
bScientific Research and Innovation Support Unit, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt
cDepartment of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico, Via U.
Schiff 6, 50019, Sesto Fiorentino, Firenze, Italy
dDepartment of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
eDepartment of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Aljouf 2014, Saudi Arabia
fDepartment of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
KEYWORDS: anticancer; benzofurans; carbonic anhydrases; carboxylic acids; synthesis.
ABSTRACT: Pursuing on our effort for developing effective inhibitors of the cancer-related hCA IX isoform, here we describe
the synthesis of novel benzofuran-based carboxylic acid derivatives, featuring the benzoic (9a-f) or hippuric (11a,b) acid
moieties linked to 2-methylbenzofuran or 5-bromobenzofuran tails via an ureido linker. The target carboxylic acids were
evaluated for the potential inhibitory action against hCAs I, II, IX and XII. Superiorly, benzofuran-containing carboxylic acid
derivatives 9b, 9e and 9f acted as submicromolar hCA IX inhibitors with KIs = 0.91, 0.79 and 0.56 µM, respectively, with
selective inhibitory profile against the target hCA IX over the off-target isoforms hCA I and II (SIs: 2 – > 63 and 4 – 47,
respectively). Compounds 9b, 9e and 9f were examined for their anti-proliferative action against human breast cancer (MCF-
7 and MDA-MB-231) cell lines. In particular, 9e displayed promising anti-proliferative (IC50 = 2.52±0.39 µM), cell cycle
disturbance and pro-apoptotic actions in MDA-MB-231 cells.
Carbonic anhydrases (CAs, EC 4.2.1.1) are considered
as the most widespread metalloenzymes present in the
living organisms that play a vital role in catalyzing the
efficacious inter-conversion between carbon dioxide and
bicarbonate.1 Such a simple CA-catalyzed reaction is crucial
for diverse physiological and pathological events associated
with pH and CO2 homeostasis, electrolyte secretion,
tumorigenicity and others.2 Up to now, fifteen diverse
human (h) CA isoforms (α-CAs) have been described and
identified. Among these, twelve isoforms only are
catalytically active with distinct kinetic properties, tissue
distributions and subcellular localizations; cytosolic (I, II,
III, VII, and XIII), mitochondrial (VA and VB), secreted (VI),
and membrane-bound (IV, IX, XII, and XIV).3
During last decades it was well-established that modulators
of these metalloenzymes represent an important class of
therapeutics such as diuretics,4 anti-epileptics,5 anti-
glaucoma agents,6 and anticancer agents.7,8 Moreover hCA
IX isoform, not considerably expressed in most human
normal tissues, is up-regulated in the hypoxic tumors upon
induction via HIF-1α, and thus considered as a crucial
element in tumor cells proliferation, invasiveness, survival,
and metastasis.8 Accordingly, selective inhibition of hCA IX
emerged out as a valuable therapeutic approach for
targeting and treatment of different human malignancies.8
SLC-0111 (Figure 1) is a front-runner carbonic anhydrase
inhibitor that is currently in Phase II clinical trials for
management of advanced hypoxic tumors, with
a
preferential hCA IX inhibitory action.9
Carboxylic acids are among the most versatile CA inhibitor
(CAI) chemotypes.10 They are capable of interacting with
the CAs through a variety of inhibition mechanisms, such as
coordination to the metal ion likely as carboxylate anions,11
anchoring to the zinc-bound water/hydroxide ion,12
occluding the entrance of the carbonic anhydrase active site
cavity,13 and inhibiting CAs binding out of the active site.14
Recently, Abdelrahman et al. developed a novel series of
benzofuran-based CAIs exhibiting the zinc anchoring
sulfonamide group connected to a benzofuran tail through
hydrazido and hydrazino spacers, compounds I and II
(Figure 1).15 The arylsulfonehydrazones derivatives
exerted effective and selective inhibitory activities toward
target hCA IX and XII isoforms over off-target hCA I and II
isoforms.
Pursuing on our endeavour toward developing efficient
inhibitors for the cancer-related isoform hCA IX,16-20 here
we present the design and synthesis of novel benzofuran-
based carboxylic acid derivatives, featuring the benzoic acid
(9a-f) or hippuric acid (11a, b) moieties linked to 2-
methylbenzofuran or 5-bromobenzofuran tails via an
ureido linker (Figure 1).
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SO2NH2
The slow cytosolic isoform hCA I (mainly considered as an
F
SO2NH2
R
1
2
3
4
5
6
7
8
O
off-target isoform when CAIs are developed as a potential
anticancer agents) was moderately or weakly inhibited by
five of the investigated benzofuran-based carboxylic acids;
9b, 9c, 9e, 9f and 11a which showed KIs spanning in the
range of 4.5 and 64.7 µM, whereas benzofuran derivatives
9a, 9d and 11b could not inhibit the cytosolic isoform hCA
I up to 100 μM.
It is worth stressing that grafting carboxylic acid
functionality at the ortho-position in both 2-
methylbenzofuran (9a) and 5-bromobenzofuran (9d)
scaffolds resulted in a diminished hCA I inhibitory activity.
Moreover, replacement of the benzoic acid moiety with the
hippuric acid one led to a significant worsening (for the 2-
methylbenzofuran scaffold; 11a: KI = 64.7 µM) or led to an
abolished (for the 5-bromobenzofuran scaffold; 11b: KI
>100 µM) inhibitory activity against hCA I.
Inhibition of the most physiologically relevant cytosolic
isoform hCA II was ranged from moderate to weak, with KI
values in the range of 3.1 – 67.1 µM. In particular
benzofuran derivatives 9a, 9c, 9d and 9f were the best
herein reported hCA II inhibitors with single-digit
micromolar inhibitory activity (KIs = 7.9, 3.1, 4.1 and 7.2
µM, respectively), Table 1. It is worth stressing that meta-
substituted 2-methylbenzofuran derivative (9b) exhibited
a slightly reduced inhibitory efficacy (KI = 10.1 µM) than it’s
ortho-substituted (9a: KI = 7.9 µM) and para-substituted
(9c: KI = 3.1 µM) analogues, likewise, the meta-substituted
5-bromobenzofuran derivative (9e) showed a weaker hCA
II inhibitory activity (KI = 37.0 µM) than both ortho-
substituted (9d: KI = 4.1 µM) and para-substituted (9f: KI =
7.2 µM) counterparts. Furthermore, incorporation of the
hippuric acid moiety resulted in a decreased activity for
both 2-methylbenzofuran and 5-bromobenzofuran
scaffolds (11a and 11b: KIs = 25.8 and 67.1 µM,
respectively) in comparison to their benzoic acid-
containing analogues (9c and 9f: KIs = 3.1 and 7.2 µM,
respectively), Table 1. These structure-activity
relationships (SARs) highlighted that appending ortho- and
para-benzoic acids is more advantageous for hCA II
inhibitory action than incorporation of meta-benzoic acid
or hippuric acid.
Br
N
N
H
H
O
N
N
H
O
SLC-0111
I
R = H or SO2-Ph
R
O
R
m
Br
R1
o
OH
p
O
O
SO2NH2
N
N
O
H
N
N
H
H
II
9a-f
9
R = H or SO2-Ph
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OH
O
R
O
R1
N
H
O
O
N
N
H
H
11a, b
Figure 1. Structures for SLC-0111 and CAIs I and II, and target
benzofuran-based carboxylic acid derivatives 9a-f and 11a, b.
Herein reported benzofuran-based carboxylic acid
derivatives (9a-f and 11a, b) were synthesized following
the procedures outlined in Schemes 1 and 2. The ethyl
benzofuran-2-carboxylate derivatives 4a, b were generated
through a cyclocondensation of intermediates 3a, b yielded
by an o-alkylation reaction for sodium phenolate 1a and 2-
hydroxybenzaldehyde 1b with ethyl 2-chloroacetoacetate
2a and ethyl bromoacetate 2b, respectively.21,22 Thereafter,
hydrazinolysis of ethyl benzofuran-2-carboxylates 4a, b
was performed via their refluxing with an excess of 99%
hydrazine hydrate in ethyl alcohol to yield benzofuran-2-
carbohydrazides 5a, b, respectively.
The benzofuran-2-carbonyl azides 6a,b were provided by
stirring of benzofuran-2-carbohydrazides 5a,b with sodium
nitrite (NaNO2) in an ice-cold acetic acid (Scheme 1), which
was subsequently subjected to Curtius rearrangement via
stirring under reflux temperature in dry xylene to furnish
the
corresponding
key
intermediates
isocyanatobenzofuran derivatives 7a,b (Scheme 2).23
Finally, the target benzofuran-based carboxylic acids 9a-f
and 11a,b were obtained through addition of aminobenzoic
acids 8a-c or para-aminohippuric acid 10 to the hot stirred
solution of isocyanatobenzofurans 7a,b in dry xylene, with
moderate yields (68–85%) (Scheme 2).
Elucidation of the structures for the newly synthesized
benzofuran-based carboxylic acids (9a-f and 11a,b) were
supported by the elemental and spectral data which was in
consistence with the postulated structures
Table 1. Inhibition data (KIs) of hCAs I, II, IX and XII with
carboxylic acids 9a-f and 11a,b and AAZ as reference inhibitor
by a stopped-flow CO2 hydrase assay.24
KI (µM)
All the prepared benzofuran-based carboxylic acid
derivatives 9a-f and 11a, b were assessed for their potential
inhibitory actions against the cytosolic hCA I and II, in
addition to the trans membrane cancer-related hCA IX and
XII isoforms, by the use of an instrument of applied
photophysics stopped-flow.24 Thereafter, the anti-
proliferative activities for the best efficient and selective
hCA IX inhibitors in this study were screened toward two
breast cancer (MCF-7 and MDA-MB-231) cell lines.
Furthermore, the impact of benzofuran-based benzoic acid
derivative 9e on distribution of the cell cycle phases in
breast cancer MDA-MB-231 cell line was assessed, in
addition to assessment of its ability to induce the early and
late apoptosis via AnnexinV-FITC/PI binding assay.
Cmp
R
R1
o/m/p
CA I
CA II CA IX
CA XII
>100
32.8
4.5
1.6
0.91
5.1
3.4
2.2
9a
9b
9c
H
H
CH3
CH3
CH3
H
o
m
p
o
m
p
-
7.9
10.1
3.1
0.88
8.0
H
>100
33.2
20.5
64.7
>100
0.25
5.1
9d
9e
9f
Br
Br
Br
H
4.1
0.79
0.56
35.7
19.0
0.02
2.3
H
37.0
7.2
1.6
H
2.7
11a
CH3
H
25.8
67.1
0.01
10.1
11b Br
-
AAZ
-
-
-
0.006
a. Mean data from 3 different assays. SD: standard deviations ranged
from ±5% to ±10% of the indicated KI values.
Inhibition data for the tested hCA isoforms (I, II, IX and XII)
are displayed in Table 1.
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O
R
O
O
R
i
ii
O
O
+
Cl
ONa
O
O
2a
1a
3a
, R = H
R1
R
O
O
5
O
4a, b
6
7
8
9
O
O
O
R
O
R
iii
iv
Br
+
O
O
OH
v
O
2b
1b
3b,
R = Br
10
11
12
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R1
R1
R
O
R
vi
4-6
a
R
R1
O
Me
H
H
O
HN NH2
O
N
N
N
b
Br
5a, b
6a, b
Scheme 1. Reagent and conditions: (i) dry toluene, reflux 4 hrs, 91%; (ii) H2SO4, stirring 2 hrs (0-5 °C), 84%; (iii) NaH, DMF, stirring
2.5 hrs at 0 oC, 80%; (iv) a) EtONa, EtOH, reflux 3 hrs, b) EtOH, H2SO4, reflux 4 hrs, 72%; (v) 99% NH2NH2·H2O, EtOH, reflux 4 hrs,
79-84%; (vi) NaNO2, AcOH, stirring 1 hr (0 °C) then 1.5 hrs (r.t.), 76-81%.
6a, b
i
O
o
m
O
O
OH
H2N
OH
H2N
p
R1
HN
R
8a-c
10
ii
N
C O
ii
O
7a, b
R
O
OH
O
R
O
m
R1
9
a
b
c
d
e
f
R
H
H
H
R1
Me
Me
Me
H
o, m, p
OH
o
R1
p
O
N
H
o
m
p
O
O
N
H
N
H
O
N
N
H
H
11
R
R1
9a-f
Br
Br
Br
o
11a, b
a
b
H
Me
H
m
p
Br H
H
Scheme 2. Conditions and reagent: (i) Dry xylene, reflux 1 hr, 95%; (ii) Dry xylene, reflux 5 hrs, 68-85%.
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The obtained KI values pointed out that the main antitumor
target isoform hCA IX was effectively inhibited by the here
reported benzofuran-based derivatives decorated with
benzoic acid moiety 9a-f with KIs ranging between 0.56 and
5.1 µM, whereas hCA IX was moderately affected by the
benzofuran-based carboxylic acids decorated with hippuric
acid moiety (11a and 11b) with KIs values equal 35.7 and
19.0 µM, respectively. Superiorly, benzofuran-based
carboxylic acids 9b, 9e and 9f emerged as submicromolar
hCA IX inhibitors with KIs = 0.91, 0.79 and 0.56 µM,
respectively.
bromobenzofuran-based regioisomers 9d-f toward was
decreased in the order para isomer (9f) > meta isomer (9e)
> ortho isomer (9d).
The second cancer-related target CA isoform examined here
is hCA XII. As can be seen from the results presented in
Table 1, hCA XII was efficiently inhibited by the herein
reported benzofuran-based acids with KIs in the range of
0.88–3.4 µM, aside from benzofuran derivatives 9d and 11b
whose potency raised at slightly higher concentration (KIs =
8.0 and 10.1 µM, respectively). The 2-methylbenzofuran-
based derivative 9c with KI equals 0.88 µM, is the only
submicromolar CA inhibitor identified toward hCA XII
isoform in this study (Table 1). Remarkably, the deduced
SAR suggested that utilizing of the 2-methylbenzofuran
scaffold elicited an enhancement of effectiveness toward
hCA XII for both benzoic acid-containing derivatives 9a-c
(KIs = 3.4, 2.2 and 0.88 µM, respectively) and hippuric acid-
containing derivative 11a (KI = 2.7 µM) in comparison to
their corresponding 5-bromobenzofuran counterparts 9d-f
(KIs = 8.0, 2.3 and 1.6 µM, respectively) and 11b (KI = 10.1
µM). With regard to the impact of regioisomerism, the order
of potencies within the 2-methylbenzofuran-based
Table 2. Selectivity index for inhibition of isoforms hCA IX and XII over
hCA I and II, for carboxylic acids 9a-f and 11a,b.
Cmp I / IX II / IX I / XII II / XII
9a
9b
9c
> 63
36
-
5
11
-
> 29
15
5
2
5
4
9d
9e
9f
11a
11b
20
42
37
2
-
13
14
13
24
10
-
16
5
10
7
47
13
-
regioisomers
9a-c
and
5-bromobenzofuran-based
5
4
regioisomers 9d-f against hCA XII was lowered as the
following order; para isomers > meta isomers > ortho
isomers.
Concerning the target/off-target CAs selectivity indexes
(SIs) of action for target benzofuran-based carboxylic acid
It is noteworthy to mention that the order of activities
within the 2-methylbenzofuran-based regioisomers 9a-c
toward hCA IX was decreased in the order of meta isomer
(9b) > ortho isomer (9a) > para isomer (9c), whereas, the
order of hCA IX inhibitory activities for the 5-
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upper right: late apoptotic; lower left: viable; upper left:
necrotic).
derivatives (9a-f and 11a,b), all compounds, except 9c,
1
2
3
4
5
6
7
8
exhibited adequate selectivity profiles for hCA IX over hCA I
(SI: 2 – >63). In addition, compounds 9a, 9b, 9e, 9f and 11b
displayed remarkable II/IX inhibitory specificity with SIs
span the range of 4 – 47, Table 2.
Table 3. Anti-proliferative activities of benzofuran-based
carboxylic acids 9b, 9e and 9f against two breast cancer cell
lines; MCF-7 and MDA-MB-231
The promising anticancer effect of benzofuran-based
carboxylic acid derivative 9e toward MDA-MB-231 breast
cancer cell line (Table 3) motivated
a further
examination for its growth inhibitory activity. The effect
of carboxylic acid derivative 9e on cell cycle phases
distribution, upon incubation with breast cancer MDA-
MB-231 cells for 24 hrs at its IC50 concentration (2.52
µM), was assessed by the use of a DNA flow cytometry
assay (Figure 2).
The assay outcomes revealed that MDA-MB-231 cancer
cells treated with benzofuran-based acid derivative 9e
were significantly arrested at the G2-M phase showing a
cell population increase from 10.80 % (for the control
cells) to 32.30 % (in 9e-treated MDA-MB-231 cells). In
addition, the number of cells in the sub-G1 phase was
strikingly increased from 1.43 % (for the control cells) to
25.53 % (in 9e-treated MDA-MB-231 cells), Figure 2.
IC50 (µM)a
Cmp
MCF-7
NAb
MDA-MB-231
37.60 ± 1.86
2.52 ± 0.39
9
9b
9e
9f
10
11
12
13
14
15
16
17
18
19
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60
14.91 ± 1.04
19.70 ± 2.06
1.43 ± 0.12
11.50 ± 1.05
2.36 ± 0.18
Dox
a. IC50 values are the mean ± S.D. of three separate experiments.
b. NA: Derivatives possessing having IC50 value > 100 µM.
Table 4. Distribution of apoptotic MDA-MB-231 cells after
incubation with benzofuran derivative 9e in the AV-FITC/PI
staining assay
Total
%
Early
Apoptosis
Late
Apoptosis %
Cmp
Necrosis
Figure 2. Effect of benzofuran-based carboxylic acid 9e on the
phases of cell cycle in breast cancer MDA-MB-231cells.
9e
34.29
1.46
8.11
0.47
23.77
0.31
2.41
0.68
Benzofuran-based carboxylic acids 9b, 9e and 9f displayed
effective and selective inhibitory actions toward the cancer-
associated hCA IX / XII isoforms over the off-target isoforms
CA I and II (Tables 1 and 2). Therefore, the three
benzofuran derivatives have been chosen to be tested for
their possible in vitro anti-proliferative actions toward the
breast cancer (MCF-7 and MDA-MB-231) cell lines, using
the SRB colorimetric reduction method as reported by
Skehan et al.25 The results from this assay have been
expressed as IC50 values and presented in Table 3.
The obtained IC50 values from the SRB analysis (Table 3)
indicated that the tested benzofuran derivatives 9b, 9e and
9f were more effective toward MDA-MB-231 (IC50 values =
37.60 ± 1.86, 2.52 ± 0.39 and 11.50 ± 1.05 µM, respectively)
than MCF-7 cells (IC50 values = > 100 µM, 14.91 ± 1.04 and
19.70 ± 2.06 µM, respectively). Superiorly, the 5-
bromobenzofuran-based derivative 9e was the most
effective anti-proliferative agent toward MDA-MB-231 cells
with IC50 value = 2.52 ± 0.39 µM, with a comparable potency
to Doxorubicin (Dox), the reference drug, (IC50 = 2.36 ± 0.18
μM).
Ctrl
The AV/PI staining assay has been adopted in order
to investigate the effect of benzofuran-based carboxylic
acid derivative 9e on the early and late-apoptosis
percentages in human breast MDA-MB-231 cancer cells
(Figure 3, Table 4). The results of this assay pointed out
that incubation of MDA-MB-231 cells with acid derivative
9e provoked apoptosis in such cells, evidenced by the
significant increase in the percentages of apoptotic MDA-
MB-231 cells for both the early apoptosis phase (from
0.47 %, for the control, to 8.11 %), and the late apoptosis
phase (from 0.31 %, for the control, to 23.77 %) (Table
4).
In summary, we explored in this letter the design and
synthesis for novel benzofuran-based carboxylic acid
derivatives, featuring the benzoic (9a-f) or hippuric (11a,
b) acid moieties linked to 2-methylbenzofuran or 5-
bromobenzofuran tails via an ureido linker. All the target
carboxylic acid derivatives were tested for their potential
inhibitory action against four hCA isoforms (I, II, IX and XII).
The cancer-related hCA IX isoform was effectively affected
by the all prepared benzofuran derivatives decorated with
benzoic acid moiety 9a-f with KIs ranging between 0.56 and
5.1 µM, Superiorly, compounds 9b, 9e and 9f emerged as
submicromolar hCA IX inhibitors with KIs = 0.91, 0.79 and
0.56 µM, respectively. Moreover, all compounds, except 9d
and 11b, inhibited hCA XII isoform with KIs in the range:
0.88–3.4 µM. Regarding the target/off-target CAs SIs of
action for the target benzofuran-based carboxylic acid
derivatives, compounds 9b, 9e and 9f showed good
selective inhibitory action toward hCA IX over the off-target
hCA I and II (SIs: 2–>63 and 4–47, respectively). The
Figure 3. Influence of benzofuran-based carboxylic acid
derivative 9e over the AV-FITC-positive staining percentages in
breast cancer MDA-MB-231 cells. (Lower right: early apoptotic;
4
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3. Supuran, C. T.; Carbonic anhydrases: from biomedical
concluded SAR revealed that replacement of the benzoic
acid moiety with the hippuric acid one led to a worsening or
abolishment of inhibitory activity against all the tested hCA
isoforms, whereas, utilizing of the 2-methylbenzofuran
scaffold elicited an enhancement of effectiveness toward
hCA XII for both benzoic and hippuric acid-containing
derivatives in comparison to the corresponding 5-
bromobenzofuran-based counterparts 9d-f. Moreover,
compounds 9b, 9e and 9f were tested for their potential
growth inhibitory actions toward two human breast cancer
(MCF-7 and MDA-MB-231) cell lines. In particular, 9e
displayed the best anti-proliferative action toward MDA-
MB-231 cancer cells (IC50 = 2.52 ± 0.39 µM) that is
comparable to Doxorubicin (IC50 = 2.36 ± 0.18 μM).
Furthermore, 9e disrupted the cell cycle (through arrest of
G2-M stage and alteration of Sub-G1 phase), and significantly
boosted the percentage of AV-FITC positive MDA-MB-231
apoptotic cells (from 0.78 to 31.88%).
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ASSOCIATED CONTENT
Supporting Information
0111
enaminone
analogs,
3/4-(3-aryl-3-oxopropenyl)
The Supporting Information is available free of charge on
the ACS Publications website at DOI: synthetic procedures,
compounds characterization, in vitro kinetic method (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*W.M.E. Phone: +201068837640. E-mail:
wagdy2000@gmail.com
*A.N. Phone: +390554573685. Email:
alessio.nocentini@unifi.it.
*C.T.S. Phone: +390554573729. Email:
claudiu.supuran@unifi.it.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding Sources
The authors extend their appreciation to the Deanship of
Scientific Research at Princess Nourah bint Abdulrahman
University for funding this work through the Research Groups
Program Grant No. RGP-1440-0025.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
CA, carbonic anhydrase; AAZ, acetazolamide; SRB, sulfo-
rhodamine-B; AV, annexinV; PI, propidium iodide.
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