Synthesis and biological evaluation of ethacrynic acid derivatives bearing sulfonamides as potent anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2020.127426

A series of ethacrynic acid (2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxy]acetic acid) (EA, Edecrin) containing sulfonamides linked via three types of linkers namely 1,2-ethylenediamine, piperazine and 4-aminopiperidine was synthesized and subsequently evaluated in vitro against HL60 and HCT116 cancer cell lines. All the EA analogs, excluding 6a and 6c, showed anti-proliferative activity with IC_50s in the micromolar range (less than 4 uM). Three derivatives 6b, 7b and 7e were selected for their interesting dual activity on HL60 cell line in order to be further evaluated against a panel of cancer cell lines (HCT116, A549, MCF7, PC3, U87-MG and SKOV3) as well as on MRC5 as a normal cell line. These compounds displayed IC₅₀ values in nanomolar range against A549, MCF7, PC3 and HCT116 cell lines, deducing the discovery that piperazine or 4-aminopiperidine is the linker's best choice to develop EA analogs with highly potent anti-proliferative activities own up to 24 nM. Besides, in terms of selectivity, those linkers are more suitable offering safety ratios of up to 63.8.

Full text of DOI:10.1016/j.bmcl.2020.127426

Journal Pre-proofs
Synthesis and Biological Evaluation of Ethacrynic Acid Derivatives Bearing
Sulfonamides as Potent Anti-Cancer Agents
Abdelmoula El Abbouchi, Nabil El Brahmi, Marie-Aude Hiebel, Jérôme
Bignon, Gérald Guillaumet, Franck Suzenet, Saïd El Kazzouli
PII:
S0960-894X(20)30537-0
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127426
BMCL 127426
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 June 2020
16 July 2020
18 July 2020
Please cite this article as: El Abbouchi, A., El Brahmi, N., Hiebel, M-A., Bignon, J., Guillaumet, G., Suzenet, F.,
El Kazzouli, S., Synthesis and Biological Evaluation of Ethacrynic Acid Derivatives Bearing Sulfonamides as
Potent Anti-Cancer Agents, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/
j.bmcl.2020.127426
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Synthesis and Biological Evaluation of Ethacrynic Acid Derivatives
Bearing Sulfonamides as Potent Anti-Cancer Agents
Abdelmoula El Abbouchi,a,b Nabil El Brahmi,a Marie-Aude Hiebel,b Jérôme Bignon,c Gérald
Guillaumet,a,b* Franck Suzenet,b* Saïd El Kazzoulia*
a Euromed Research Center, Euromed Institute of Technology, Euromed University of Fes (UEMF)–
Route de Meknès, 30000 Fes, Morocco.
b Institut de Chimie Organique et Analytique, Université d’Orléans, UMR CNRS 7311, BP 6759,
Orléans cedex 2 54067, France
c Institut de Chimie des Substances Naturelles, CNRS, Université Paris-Saclay, Gif-sur-Yvette,
France
Abstract
A series of ethacrynic acid (2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxy]acetic acid)
(EA, Edecrin) containing sulfonamides linked via three types of linkers namely 1,2-
ethylenediamine, piperazine and 4-aminopiperidine was synthesized and subsequently
evaluated in vitro against HL60 and HCT116 cancer cell lines. All the EA analogs, excluding
6a and 6c, showed anti-proliferative activity with IC50s in the micromolar range (less than 4
uM). Three derivatives 6b, 7b and 7e were selected for their interesting dual activity on HL60
cell line in order to be further evaluated against a panel of cancer cell lines (HCT116, A549,
MCF7, PC3, U87-MG and SKOV3) as well as on MRC5 as a normal cell line. These
compounds displayed IC50 values in nanomolar range against A549, MCF7, PC3 and HCT116
cell lines, deducing the discovery that piperazine or 4-aminopiperidine is the linker’s best
choice to develop EA analogs with highly potent anti-proliferative activities own up to 24 nM.
Besides, in terms of selectivity, those linkers are more suitable offering safety ratios of up to
63.8.
O
IC50 = 24.8 nM on PC3
Cl
O
Cl
O
S
O
N
Safety ratios: 4,03 (MRC5/PC3)
IC50 = 64.9 nM on A549
N
OCH3
O
b
6
Cl
Cl
Safety ratios: 1.54 (MRC5/A549)
O
O
O
S
O
O
IC50 = 67.7 nM on A549
HN
N
OCH3
Safety ratios: 63.81 (MRC5/A549)
7b
Cl
Cl
O
O
Cl
O
S
O
O
HN
7e
IC50 = 55.2 nM on A549
N
Safety ratios: 16.3 (MRC5/A549)
H3CO

Chemotherapeutic agents are the primary way to brawl against cancer and represent the first
level of research in cancer therapeutics. Therefore, the development of new anticancer agents
represents an important and challenging field in medicinal chemistry.1–3 Generally, the main
sources of compounds for drug development are natural compounds,4–9 analogues of existing
drugs10–15 and new synthetic products.16–24
A myriad of natural and synthetic α,β-unsaturated carbonyl based compounds including
chalcones, curcumin and their synthetic analogs are identified to exhibit antitumor
activities.25,26 α, β-unsaturated carbonyl unit has an extraordinary advantage in the
development of biologically active and therapeutically relevant compounds in medicinal
chemistry. Recently, a number of derivatives containing α, β-unsaturated carbonyl group has
been reported to display versatile biological activities such as antitumor activity, antimalarial
effects, antiviral activity, and reduce intraocular pressure. Besides, the EA is a well-known drug
27,28
used not only as a diuretic agent
but also as a treatment of high blood pressure and
edema.29,30 The ethacrynic acid’s α,β-unsaturated carbonyl unit acts as a Michael acceptor
involved in the inhibition of the major classes of glutathione S-transferases (GSTs) (α, μ, and
π) that plays a key role in regulating multidrug resistance.31,32 In addition to its modest anti-
proliferative effect against tumor cells,33 the EA was therefore used as an adjuvant to improve
the therapeutic efficacy of several anticancer agents.34–37 Several analogues were synthetized
in order to enhance the biological activities.38–43 All the modification of the α,β-unsaturated unit
by reduction of the double bound and/or the carbonyl have resulted in a total loss of the anti-
proliferative activities.40,41 Similar results are found when one or the two chlorines of the EA
are removed or replaced by other groups. The major improvement relied on the modification
of the carboxylic acid part into ester, amide or heterocycle derivatives.41–43 Recently, the
carboxylic acid in the EA was replaced by either aromatic esters or aromatic amides and the
isolated new compounds exhibited promising anti-proliferative activities especially when the
aromatic amides were used.15,43
Sulfonamide has been confirmed to be an important functional group in drug discovery.44 Drugs
containing sulfonamides were the first chemical substances used to cure and prevent bacterial
infections in humans.45 Similarly, they are known as key parts of many chemotherapeutic
agents. Regarding this, an extensive work has been performed over the last few years the
synthesis, structure-activity relationships (SAR), and the anticancer activities evaluation of
compounds bearing sulfonamides.46–50
In continuation of our recent report on EA derivatives exhibiting anti-proliferative activities by
the activation of caspase cascade which induce the apoptosis,15 we wish to report here the
synthesis and anti-proliferative activities of sulfonamide containing analogues of EA. This
original series of EA-modified compounds was first evaluated in vitro against HL60

(promyelocytic leukemia) and HCT116 (human colon carcinoma) cancer cells. Then, the most
promising analogues were tested on a panel of cancer and non-cancer cells.
The synthesis of compounds 5(a-i), 6(a-i) and 7(a-i) is illustrated in scheme 1. The sulfonamide
moieties were introduced on EA via three types of linkers namely 1,2-ethylenediamine,
piperazine and 4-aminopiperidine. The series of EA sulfonamide derivatives 5(a-i), 6(a-i) and
7(a-i) were synthesized through a two-step sequence starting from commercially available EA
according to scheme 1. First, the key intermediates 2(a-i), 3(a-i) and 4(a-i) were prepared by
reacting a variety of sulfonyl chlorides 1(a-i) with either 1,2-ethylenediamine, piperazine or 4-
(N-Boc-amino) piperidine via an N-sulfonylation reaction in the presence of triethylamine at
room temperature in DCM.51 These intermediates were dried and used directly in the next step
without further purification. Afterwards, a conventional condition of peptide synthesis was
employed using the intermediates 2(a-i), 3(a-i) or 4(a-i) and EA in the presence of EDC/HOBt
as activators in DMF to afford EA derivatives 5(a-i), 6(a-i) and 7(a-i) in moderate to acceptable
yields.
Cl
O
H
O
Cl
O
N
NH2
N
S
H2N
HN
R
NHBoc
NH
HN
Or
R
H
O
O
S
O
1) Et3N, DCM
rt, 4h
2) HCl, dioxane, rt
Et3N, DCM
rt, 4h
O
5(a-i), 44 to 62%
Or
Cl
N
Cl
R
S
O
O
O
O
O
Cl
O
NH2
NH
3 (a-i)
N
R
S
N
,
R
S
Cl
O
R
S
NH3Cl
Or
Cl
O
H
N
O
O
O
Cl
O
O
OH
2 (a-i)
4 (a-i)
N
S
R
O
O
EDC, HOBt, DMF, 0°C to rt, overnight.
6(a-i), 45 to 66%
Or
EA
O
O
R
S
Cl
O
N
O
Cl
O
N
H
7(a-i), 42 to 66%
O
a
b
c
d
e
f
g
h
i
OCH3
OCH3
Cl
N
R
CH3
OCH3
OCH3
NO2
CH3
H3CO
H3CO
Scheme 1: Synthesis of compounds 5(a-i), 6(a-i) and 7(a-i).
All the synthesized compounds, which were purified by column chromatography, were
1
characterized by H and 13C NMR and high-resolution mass spectrometry and were in full
accordance with their depicted structures.
Twenty seven EA derivatives described in this work were tested in vitro to evaluate their anti-
proliferative effects on two different cancer cell lines namely, HL60 (promyelocytic leukemia)
and HCT116 (human colon carcinoma). The most promising compounds were then tested
against a panel of cancer and normal cells.

The anti-proliferative activities are based on the evaluation of the percentage of viability using
the sensitive CellTiter-Glo® luminescent assay, which is a homogeneous method to determine
the number of viable cells in culture and is based on the quantitation of the ATP (an indicator
of metabolically active cells).52 The amount of ATP is directly proportional to the number of
viable cells present in the culture (supporting info). The viability of both HL60 and HCT116
were measured for all compounds at the 10M concentration (Table 1). The results show that
most of the EA analogues, except 6c, inhibited the cell proliferative growth of HL60 (11 to 64%
of cell viability). In the case of HCT116, only compound 6b is very potent with 21% cell viability.
Using the Graph Prism software from polynomial curves (four or five-parameter logistic
equations), the IC50 values were determined for all the compounds induced a reduction of the
viability over 50%. All the EA analogs, excluding 6a and 6c, showed anti-proliferative activity
against HL60 with IC50 ranging between 0.58 and 3.39 M. SAR discussion based on our
previously reported EA analogues15 and the current compounds bearing sulfonamide moiety
SO2NRR' (see lead compound III, figure 1) showed that the introduction of the sulfonamide
group on the previously used linkers maintained the anti-proliferative activity against HL60. For
example, compound 5b with N-(2-aminoethyl)-4-methoxybenzenesulfonamide linker and
compound 6b with 1-((4-methoxyphenyl)sulfonyl)piperazine linker, and containing both of them
a methoxy group on the aromatic ring, display almost the same inhibitory activity in comparison
with their previously reported analogues I and II bearing 2-(4-methoxyphenyl)ethan-1-amine
and 2-(4-methoxyphenyl)ethan-1-amine linker, respectively.15 IC50 values of 5b and 6b are
0.86, 0.59 µM, respectively, while, their corresponding compounds I and II displayed the IC50
values of 1.3 and 0.8 µM, respectively.15 In contrast, compound 5c bearing N-(2-aminoethyl)-
3,4-dimethoxybenzenesulfonamide revealed a sudden drop of potency with IC50 value of 3.38
µM compared to its previously reported analogue IV containing 2-(3,4-dimethoxyphenyl)ethan-
1-amine moiety which displayed an IC50 of 0.8 µM.15
Cl
Cl
O
Cl
O
Cl
N
O
O
N
O
OCH3
HN
II
O
OCH3
I
Cl
Cl
O
Cl
O
Cl
O
O
N
N
O
OCH3
OCH3
HN
O
O
O P
O
IV
O
III (lead compound)
IC50 (HL60) = 0.35
M

Figure 1. Chemical structures of previously reported EA analogues.15
Subsequently, compounds 6b, 7b and 7e (Figure 2) were selected for their interesting dual
activity on both cancer cell lines. The IC50 against HCT116 for these three compounds display
good to very good activity (IC50 values of 0.05, 1.15 and 0.79 M, respectively). Interestingly,

compound 6b exhibited good selectivity towards HCT116 (almost 12 folds more active on
HCT116 cell line than on HL60 cell line). SAR discussion based on the IC50 values of
compounds 6b, 7b and 7e and the previously reported compounds15 upon cancer cell HCT116,
revealed that the piperazine linker along with the presence of the sulfonamide moiety
(compound 6b) are crucial for the achievement of selective and very potent anti-proliferative
EA analogues.
Table 1. Percentage of viability at 10 µM of EA derivatives on HL60 and HCT116 and IC50 of
selected EA analogues
R-
Compounds
Structure
HL60
(%)
HCT116 (%)
IC50 (HL60)
IC50
(HCT116)
(M)
(M)
5a
5b
17±0.8
24±3.0
100±6.0
97±5.0
2.37±1.3
0.86±0.1
H3C
H3CO
5c
19±0.5
97±3.0
3.36±1.0
H3CO
H3CO
OCH
5d
5e
35±0.2
18±0.7
77±2.0
78±6.0
2.79±0.3
1.60±0.4
Cl
O
H
N
O
Cl
O
N
H
S
H3CO
R
O
OCH3
O
Cl
5f
27±3.0
20±0.9
59±0.4
0.58±0.1
1.33±0.4
O2N
5g
100±2.0
N
5h
5i
28±2.0
21±1.0
64±2.0
17±0.3
91±3.0
62±0.4
100±5.0
21±2.0
89±1.0
1.34±0.4
0.94±0.4
CH3
6a
6b
6c
H3C
0.59±0.1
3.19±0.7
0.05±0.001
H3CO
H3CO
100±2.
0
H3CO
OCH
6d
15±0.2
40±2.0
H3CO

Cl
O
OCH3
6e
24±1.0
75±2.0
1.41±0.3
Cl
O
N
O
N
S
R
O
O
Cl
6f
24±0.4
28±0.2
100±3.0
100±1.0
3.26±0.9
2.98±0.9
O2N
6g
N
6h
6i
32±2.0
24±0.3
25±2.0
100±6.0
68±0.3
2.32±1.3
3.39±1.1
0.98±0.02
CH3
7a
100±0.2
H3C
7b
7c
25±2.0
25±0.8
45±1.0
99±1.0
0.63±0.004
1.31±0.3
1.15±0.11
H3CO
H3CO
H3CO
OCH
7d
7e
11±0.8
16±0.8
90±3.0
nc
0.86±0.03
0.70±0.01
O
R
H3CO
S
Cl
O
N
O
Cl
O
N
OCH3
0.79±0.14
H
O
Cl
7f
23±2.0
34±0.5
100±2.0
84±1.0
2.47±0.8
O2N
7g
0.97±0.02
N
7h
7i
41±2.0
26±0.3
61 ± 3.0
73±1.0
2.94±0.8
3.33±0.7
CH3
nc : not calculated
To go further in our study, these compounds were engaged in additional investigations
regarding their anti-proliferative activities on a panel of cancer cell lines. These cancer cell
lines are representative of diverse tissues/organ tumors: human breast cancer (MCF7), human
glioblastoma cancer (U87-MG), human prostate cancer (PC3), ovarian carcinoma (SK-OV3),
human lung epithelial carcinoma (A549), human colon cancer (HCT116) and proliferative
human lung fibroblasts as non-cancer cell line (MCR5) (Table 2).
The data in table 2 show that all compounds display a potent anti-proliferative activity with IC50s
in the nanomolar range (less than 100 nM) on at least one type of cancer cell line. Interestingly,
6b, 7b and 7e inhibited the proliferation of A549 cells with IC50 values of 0.064, 0.067 and
0.055 µM, respectively. These potencies are similar to doxorubicin, which displays an IC50 of

0.056 µM against A549. On the MCF7 cell line, the most potent compounds are 6b with IC50
of 0.056 µM and 7e with IC50 of 0.097 µM. In the case of PC3 and HCT116 cell lines, 6b
showed a very high potency with IC50 of 0.024 µM and 0.050 µM, respectively. Compound 6b
is even more active than doxorubicin on both cell lines. Concerning U87-MG and SKOV3 cell
lines, 6b, 7b and 7e exhibited the same range of activities with more moderate IC50 values
between 0.150 and 0.309 µM.
Encouraged by these very interesting results, we calculated the selectivity index of 6b against
HCT116 compared to compounds 7b and 7e. For compound 6b and 7b having the same
sulfonamide moiety and differentiated only by the type of linker, the selectivity index which is
IC50(7b)/IC50 (6b) show 23 fold. While the selectivity index of 6b versus 7e by calculating
IC50(7e)/ IC50(6b) is 15.8 fold.
It is well known that lipophilicity, which associates both biological and physicochemical
properties, is a crucial property for drug absorption, distribution, potency and elimination. Thus,
using Marvin application ChemAxon, we calculated the membrane-water partition coefficient
(LogP) for the three compounds 6b, 7b and 7e. All the three compounds displayed similar
LogP (between 3.78 and 4.38) suitable for the future development of drug-like compounds.53
O
Cl
O
Cl
O
S
N
N
O
OCH3
6b
O
Cl
Cl
Cl
Cl
O
O
O
O
Cl
O
S
O
O
S
O
HN
HN
N
N
OCH3
O
H3CO
O
7e
7b
Figure 2. The structures of sulfonamide-EA derivatives 6b, 7b and 7e
Table 2. IC50 Values (µM) determined for 6b, 7b, 7e and doxorubicin upon various cancer
human cells.
Compounds
IC50 (M)
6b
7b
7e
Doxorubicin
0.010 ± 0.001
0.056 ± 0.0008
0.120 ± 0.009
HL60
A549
MCF7
0.590 ± 0.100
0.064 ± 0.003
0.561 ± 0.0009
0.630 ± 0.040
0.067 ± 0.004
0.128 ± 0.056
0.700 ± 0.100
0.055 ± 0.001
0.097 ± 0.009
PC3
0.024 ± 0.007
0.112 ± 0.011
0.076 ± 0.003
0.002 ± 0.0001
U87-MG
SKOV3
0.207 ± 0.012
0.219 ± 0.033
0.238 ± 0.047
0.309 ± 0.004
0.307 ± 0.089
0.150 ± 0.060
0.09 ± 0.0023
nd±

HCT116
MRC5
0.050 ± 0.001
0.100 ± 0.050
1.150 ± 0.110
4.320 ± 0.380
0.790 ± 0.140
0.900 ± 0.010
0.090 ± 0.003
0.039 ± 0.001
LogPa
3.82
3.78
4.38
1.27
aPartition coefficient, a measure of lipophilicity.
To select the compounds with the highest selectivity for cancer cells, the safety ratios for
MRC5/cancer cells were calculated for compounds 6b, 7b and 7e. The results summarized in
table 3 show that compound 7b has the highest selectivity for cancer cells A549, PC3 and
MCF7 with safety ratios of 63.8, 38.57 and 33.75, respectively. This compound is also selective
for U87-MG and SKOV3 cell lines with safety ratios of 18.15 and 13.98, respectively.
Interestingly, compound 7b is more selective for all cancer cells than doxorubicin, which
exhibited very low safety ratios (between 19.08 to 0.33 fold). The compound 7e is also more
selective than doxorubicin, except for PC3, but its safety ratios (16.3 to 1.13) are lower than
those obtained with 7b. The compound 6b is the less selective EA analogues for all cancer
cells with low safety ratios. Finally, considering that the structure of compounds 7b and 6b
which only differs by their linkers, those results suggest that 4-aminopiperidine is the best
choice of linker to develop EA analogs with very potent anti-proliferative activities. However,
in terms of selectivity, the 4-aminopiperidine linker is more suitable regarding the fact that EA
analogue 7b offers safety ratios between 3.75 and 63.8.
Table 3. Safety ratios of compounds 6b, 7b, 7e and doxorubicin
Compounds
6b
7b
7e
Doxorubicin
SI
MRC5/HL60
MRC5/A549
MRC5/MCF7
0.16
1.54
1.78
6.85
63.81
33.75
1.28
16.3
9.27
3.98
0.70
0.33
MRC5/PC3
4.03
38.57
11.78
19.08
MRC5/ U87-MG
MRC5/SKOV3
0.48
0.45
18.15
13.98
2.93
6.00
0.40
nd±
MRC5/HCT116
2.00
3.75
1.13
0.44
SI: selectivity index, the ratio of IC50 MRC5/IC50 cancer cell.
In this study, twenty-seven EA-sulfonamide analogues were synthetized, fully characterized
and biologically evaluated. The percentages of viability of HL60 and HCT116 were measured
and IC50 on HL60 were calculated except for two inactive compounds (6a and 6c).

Satisfactorily, the original EA-sulfonamides analogues inhibited HL60 cell line with IC50 values
in the low to sub-micromolar range. From this first screening, three compounds were selected
(6b, 7b and 7e) for further tests on a panel of cancer cell lines as well as on MRC5 as a normal
cell line. In the cases of cancer cell lines A549 and MCF7, compounds 6b, 7b and 7e display
IC50, in the nanomolar range, similar to those obtained with doxorubicin. The IC50 of 6b, 7b and
7e are higher than doxorubicin in the case and HCT116 cell. In contrast, for PC3 cell line, even
if the antiproliferative activities of compounds 6b, 7b and 7e are very interesting 0.0248, 0.112
and 0.0764 µM, respectively, these potencies are lower than doxorubicin (IC50=0.0021 µM).
The second important discovery in this work is distinct abilities of the selected compounds
6b, 7b and 7e to significantly discriminate between cancer cells (e.g. A549, MCF7, PC3 and
HCT116) and normal MRC5 cells. The safety ratios (IC50 MRC5/IC50 cancer cell) calculated for
the selected compounds, are much better than doxorubicin for A549, MCF7, U87-MG and
HCT116 cell lines. The most selective compound for all cancer cells is 7b, with the best safety
ratio of 63.8 on A549 cell line over MRC5 cell line.
Finally, our finding bode well for the future development of drug-like based on EA analogues
capable of differentiating between normal and cancer cells (good safety ratios, especially for
compound 7b and good LogP values).The development of new EA analogues that will show
high antiproliferative activities and selectivities by expanding the chemical domain is
undergoing in our laboratory.
This work was supported by Euromed University of Fes and Campus France and partially
supported by Orléans University, CNRST, CNRS, Labex SynOrg (ANR-11-LABX-0029) and
Labex IRON (ANR-11-LABX-0018-01).

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