Phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates and phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamides as new antimicrotubule agents targeting the colchicine-binding site

Article abstract of DOI:10.1016/j.ejmech.2020.113136

We recently designed and prepared new families of potent antimicrotubule agents designated as N-phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB–SOs) and phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB–SAs). Our previous structure-activity relationship studies (SAR) focused on the aromatic ring B of PIB-SOs and PIB-SAs leaving the impact of the phenylimidazolidin-2-one moiety (ring A) on the binding to the colchicine-binding site (C-BS) poorly studied. Therefore, the aim of the present study was to evaluate the effect of replacing the imidazolidin-2-one (IMZ) group by a pyrrolidin-2-one moiety. To that end, 15 new phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (PYB–SO) and 15 phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamide (PYB-SA) derivatives were designed, prepared, chemically characterised and biologically evaluated. PYB-SOs and PYB-SAs exhibit antiproliferative activity in the low nanomolar to low micromolar range (0.0087–8.6 μM and 0.056–21 μM, respectively) on human HT-1080, HT-29, M21 and MCF7 cancer cell lines. Moreover, they block cell cycle progression in G2/M phase. Immunofluorescence, tubulin affinity and tubulin polymerisation assays show that they cause microtubule depolymerisation by docking the C-BS. In addition, docking assays with the most potent derivatives show binding affinity toward the C-BS and they also exhibit weak or no toxicity toward chick embryos. Finally, physicochemical properties calculated using the SwissADME algorithm show that PYB-SOs and PYB-SAs are promising new families of antimicrotubule agents.

Full text of DOI:10.1016/j.ejmech.2020.113136

European Journal of Medicinal Chemistry 213 (2021) 113136
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates and phenyl 4-(2-
oxopyrrolidin-1-yl)benzenesulfonamides as new antimicrotubule
agents targeting the colchicine-binding site
a
,
,
, b
Mathieu Gagne-Boulet b, Chahrazed Bouzriba a b, Atziri Corin Chavez Alvarez a
,
ꢀ
a, b, *
ꢀ
Sebastien Fortin
a
ꢀ
ꢀ
^
ꢀ
Centre de recherche du CHU de Quebec - Universite Laval, Axe oncologie, Hopital Saint-François d’Assise, 10 rue de l’Espinay, Quebec, QC, G1L 3L5, Canada
Faculte de pharmacie, Universite Laval, Quebec, QC, G1V 0A6, Canada
b
ꢀ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently designed and prepared new families of potent antimicrotubule agents designated as N-
phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIBeSOs) and phenyl 4-(2-oxoimidazolidin-1-yl)
benzenesulfonamides (PIBeSAs). Our previous structure-activity relationship studies (SAR) focused on
the aromatic ring B of PIB-SOs and PIB-SAs leaving the impact of the phenylimidazolidin-2-one moiety
(ring A) on the binding to the colchicine-binding site (C-BS) poorly studied. Therefore, the aim of the
present study was to evaluate the effect of replacing the imidazolidin-2-one (IMZ) group by a pyrrolidin-
2-one moiety. To that end, 15 new phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (PYBeSO) and 15
phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamide (PYB-SA) derivatives were designed, prepared,
chemically characterised and biologically evaluated. PYB-SOs and PYB-SAs exhibit antiproliferative ac-
Received 13 October 2020
Received in revised form
23 December 2020
Accepted 24 December 2020
Available online 6 January 2021
Keywords:
Anticancer agents
Antimicrotubule agents
Antimitotics
tivity in the low nanomolar to low micromolar range (0.0087e8.6 mM and 0.056e21 mM, respectively) on
human HT-1080, HT-29, M21 and MCF7 cancer cell lines. Moreover, they block cell cycle progression in
G2/M phase. Immunofluorescence, tubulin affinity and tubulin polymerisation assays show that they
cause microtubule depolymerisation by docking the C-BS. In addition, docking assays with the most
potent derivatives show binding affinity toward the C-BS and they also exhibit weak or no toxicity to-
ward chick embryos. Finally, physicochemical properties calculated using the SwissADME algorithm
show that PYB-SOs and PYB-SAs are promising new families of antimicrotubule agents.
© 2020 Elsevier Masson SAS. All rights reserved.
Colchicine-binding site inhibitors
Phenyl 4-(2-oxopyrrolidin-1-yl)
benzenesulfonates
Phenyl 4-(2-oxopyrrolidin-1-yl)
benzenesulfonamides
1. Introduction
used in clinical trials still have significant deleterious effects,
limited effectiveness and are prone to induce chemoresistance. In
Cancer is a large class of diseases characterised by an uncon-
trolled and abnormal cellular growth causing serious health issues
and ultimately leading to death if untreated. To this day, it is still
one of the most important health problems in the world [1]. Indeed,
cancer was responsible for approximately 9.6 million deaths
worldwide in 2018 [2]. Despite major progress achieved in the past
decades, many cancers are still having poor prognoses and high
mortality rates [3]. In addition, some anticancer agents currently
this context, the development of new antimitotic drugs that aim to
improve both the life expectancy and the quality of life of cancer
patients is of uttermost importance [4].
Microtubules are the main constituents of the cytoskeleton in
eukaryotic cells. They are composed of a, b-tubulin heterodimers
which are in constant dynamic equilibrium between their poly-
merisation and depolymerisation states [5]. They participate in
diverse cellular functions such as cell replication, maintenance of
cell shape, mitosis, motility and cellular transport [6]. Therefore,
microtubules are an important target in cancer chemotherapy.
Antimicrotubule agents are divided into two main classes based on
their effect on microtubules: microtubule-stabilising agents and
microtubule-destabilising agents. Microtubule-stabilising agents
bind to microtubules and stabilise the polymer while microtubule-
destabilising agents bind to the tubulin heterodimers and
Abbreviations: Combretastatin A-4, CA-4; N-phenyl 4-(2-oxoimidazolidin-1-yl)
benzenesulfonate, PIB-SOs; phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates,
PYB-SOs; phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamides, PYB-SAs.
ꢀ
ꢀ
ꢀ
* Corresponding author. Faculte de pharmacie, Universite Laval, Quebec, QC, G1V
0A6, Canada.
E-mail address: sebastien.fortin@pha.ulaval.ca (S. Fortin).
https://doi.org/10.1016/j.ejmech.2020.113136
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
destabilise microtubules. Antimicrotubule agents such as paclitaxel
(1, Fig. 1A) have been part of the armamentarium of physicians for
treating cancer patients since the 1990s [7,8]. New antimicrotubule
agents are still of interest for several reasons, notably to reduce
their toxicity, to improve their effectiveness, to improve their
selectivity for given tumours and to reduce their production costs.
To that end, new antimicrotubule agents are in preclinical devel-
opment and are currently undergoing clinical trials [9e11]. Some
well-known previously studied compounds include combretastatin
A-4 disodium phosphate (CA-4DP, 2, Fig. 1B) [12] and T138067
sodium (3, Fig. 1C) [13].
To circumvent these impediments, our research group previ-
ously designed and developed two new families of antimicrotubule
agents designated as phenyl 4-(2-oxoimidazolidin-1-yl)-benzene-
sulfonates (PIBeSOs, Fig. 1D) [14] and phenyl 4-(2-
oxoimidazolidin-1-yl)-benzenesulfonamides (PIBeSAs, Fig. 1E)
[15]. The molecular structures of PIB-SOs and PIB-SAs comprise two
aromatic rings (A and B) linked either by a sulfonate or a sulfon-
amide bridge. The aromatic ring A is substituted by an imidazoli-
din-2-one (IMZ) moiety at position 4 while the aromatic ring B
bears either a methoxyl group, short alkyl chain or halogen groups
in position 3, 4 and/or 5. PIB-SO and PIB-SA derivatives exhibit
antiproliferative activity in the nanomolar to low micromolar range
on numerous human cancer cell lines, notably HT-1080 fibrosar-
coma, HT-29 colon adenocarcinoma, M21 skin melanoma and
MCF7 oestrogen-dependent breast adenocarcinoma. Moreover,
they induce arrest of the cell cycle progression in G2/M phase and
they cause microtubule depolymerisation and cytoskeleton
disruption by targeting the colchicine-binding site (C-BS) located at
high-binding affinity for the C-BS. SAR studies on the aromatic ring
A evidenced that the IMZ moiety cannot be modified easily without
losing the biological activity [14e17]. In addition, our molecular
modeling experiments using PIB-SOs and PIB-SAs suggested that
the substituent shape is more important than hydrogen bonds and
electrostatic forces since the binding pocket is clearly hydrophobic
and mostly driven by van der Waals forces [16]. Consequently, the
importance of the NH group of the IMZ moiety for the interaction of
PIB-SOs and PIB-SAs with the C-BS remains to be studied.
In this study, we investigate the impact of the NH group of the
IMZ moiety of PIB-SOs and PIB-SAs on the biological activity by
replacing the NH group by a CH2 group. Therefore, a new series of
phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates (PYBeSOs,
Fig. 1E) as well as a new series of 4-(2-oxopyrrolidin-1-yl)-N-phe-
nylbenzenesulfonamides (PYBeSAs, Fig. 1F) were prepared. PYB-
SOs and PYB-SAs were first assessed for their antiproliferative ac-
tivity on HT-1080, HT-29, M21 and MCF7 human cancer cell lines.
The most potent compounds were assessed for their potential to
arrest M21 cell cycle progression. Afterwards, they were evaluated
for their potency to disrupt the cytoskeleton and inhibit microtu-
bule polymerisation. Furthermore, we assessed our most potent
derivatives for their binding affinity for the C-BS and their toxicity
on chick embryos. Finally, their biopharmaceutical properties were
assessed using SwissADME free web tool [18].
2. Chemistry
The preparation of PYB-SOs (4-18) and PYB-SAs (19e33) is
depicted in Scheme 1 and was achieved within 4 steps. Briefly, the
general synthesis started with the nucleophilic addition of aniline
on 4-chlorobutanoyl chloride in presence of triethylamine in
methylene chloride followed by the intramolecular cyclization of 4-
chloro-N-phenylbutanamide (34) into 1-phenylpyrrolidin-2-one
(35) by addition of sodium hydride in tetrahydrofuran.
the interface of the a, b-tubulin heterodimer [14,15].
Previous structure-activity relationship (SAR) studies were
focusing almost exclusively on the aromatic ring B and showed that
the substitution in positions 3, 3,5 or 3,4,5 is required to maintain
both the antiproliferative activity in the nanomolar range and the
Fig. 1. Molecular structures of antimicrotubule agents A) paclitaxel (1), B) combretastatin A-4 disodium phosphate (CA-4DP, 2), C) T138067 sodium (3), D) N-phenyl 4-(2-
oxoimidazolidin-1-yl)benzenesulfonates (PIBeSOs), E) phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIBeSAs) F) phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates
(PYBeSOs, 4e18) and phenyl 4-(2-oxopyrrolidin-1-yl)-N-phenylbenzenesulfonamides (PYBeSAs, 19e33).
2

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Scheme 1. Reagents and conditions: (i) 4-chlorobutanoyl chloride, CH2Cl2, rt, 24 h, 78%; (ii) NaH, THF, 0 ꢀC to rt, 24 h, 86%; (iii) ClSO3H, 0 ꢀC to rt, overnight, 72%; (iv) relevant
phenol, TEA, CH3CN, 120 ꢀC under microwaves, 8 h, 31e97% or relevant aniline, DMAP, CH3CN, 120 ꢀC under microwaves, 8 h, 18e67%.
Chlorosulfonation of 35 with chlorosulfonic acid gave the 4-(2-
oxopyrrolidin-1-yl)benzenesulfonyl chloride (36). Finally, PYB-SOs
(4-18) and PYB-SAs (19e33) were prepared by the nucleophilic
addition of a relevant phenol in presence of triethylamine or a
relevant aniline in presence of 4-dimethylaminopyridine to 36 in
acetonitrile under microwaves.
PYB-SAs 31 are the most potent derivatives with IC50 ranging from
8.7 to 11 nM and 56e130 nM, respectively. Furthermore, the most
active PYB-SOs, bearing either a 3,5-dimethoxyl (14), a 3,5-dibromo
(15) or a 3,4,5-trimethoxyl (16) groups exhibited antiproliferative
activities (8.7e39 nM) similar to CA-4 (1.9e5.1 nM), CEU-602
(4.0e5.9 nM) and CEU-638 (13e21 nM) used as positive controls.
Finally, these results confirm that the NH group of IMZ moiety of
PIB-SOs is not essential to the antiproliferative activity.
3. Results/discussion
3.1. PYB-SOs and PYB-SAs exhibit antiproliferative activity on
human cancer cells
3.2. PYB-SO and PYB-SA derivatives arrest the cell cycle progression
in G2/M phase
The antiproliferative activity of PYB-SOs (4-18) and PYB-SAs
(19e33) was assessed on the HT-1080 fibrosarcoma, HT-29 colon
adenocarcinoma, M21 skin melanoma and MCF7 oestrogen-
dependent breast adenocarcinoma using the sulforhodamine B
colorimetric assay and the results are listed in Table 1 and corre-
spond to the concentration of drug needed to inhibit 50% of cell
growth (IC50). Combretastatin A-4 (CA-4), PIB-SO (CEU-602) and
PIB-SA (CEU-634) bearing a 3,4,5 trimethoxyl group on ring B that
were the most potent PIB-SO and PIB-SA prepared so far were used
as positive controls [14,15]. PYB-SOs and PYB-SAs exhibit anti-
proliferative activity ranging from the low nanomolar to the low
Antimicrotubule agents are known to arrest cell cycle progres-
sion during the G2/M phase. To verify the mechanism of action
underlying the antiproliferative activity of PYB-SOs and PYB-SAs
with PIB-SOs and PIB-SAs that were used as molecular scaffolds,
we first assessed their effect on the cell cycle progression of
M21 cells [14,15]. To that end, PYB-SOs 11, 12 and 14e16, and PYB-
SAs 21, 25, 27 and 29e31 were incubated with M21 cells at 2 and 5-
times their respective IC50 for 24 h. The results depicted in Fig. 2
show the optimal concentration (either 2 or 5-times the IC50) for
a maximal arrest of cell cycle progression in the G2/M phase. As
shown in Fig. 2, cells treated with 0.5% DMSO (negative control)
were at 5.0, 65.5, 16.1 and 13.4% in subG1, G0/G1, S and G2/M
phases, respectively. CA-4 was used as positive control and showed
a population increase in G2/M phase by 66.1%. Except PYB-SA 21
bearing a phenyl substituent in position 2, all PYB-SOs and PYB-SAs
assessed increased the cell population in G2/M phase by
28.8e68.4%. Furthermore, PYB-SOs and PYB-SAs bearing the same
substituent have a similar effect on the cell cycle progression which
indicates that the modification of the sulfonate bridge by a sul-
fonamide moiety does not affect their ability to arrest the cell cycle
micromolar levels on all cancer cell lines tested (0.0087e8.6
mM for
PYB-SOs and 0.056e21 M for PYB-SAs, respectively). Globally,
m
PYB-SOs are more potent than their PYB-SA counterparts. The
sensitivity of cell lines to PYB-SOs and PYB-SAs usually decreases
according to the following order: MCF7 > HT-29 z M21 >HT-1080.
The disubstitution of the aromatic ring B in positions 3 and 5 by
methoxyl and bromo groups and trisubstitution in positions 3, 4
and 5 by methoxyl groups are the most favourable substitutions
leading to maximal antiproliferative activities. PYB-SOs 15 and
3

ꢀ
M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Table 1
are antimicrotubule agents inducing cytoskeleton disruption
similarly as reported for CA-4, CEU-602 and CEU-638 [14,15].
Antiproliferative activity (IC50) of PYB-SOs (4-18) and PYB-SAs (19e33) on HT-1080,
HT-29, M21 and MCF7 human cancer cell lines.
3.4. PYB-SOs and PYB-SAs inhibit microtubule polymerisation
To confirm the effect of PYB-SOs and PYB-SAs on microtubules,
we assessed the effect of PYB-SOs 14 and 15 and PYB-SAs 30 and 31
on dynamic polymerisation of tubulin to microtubules [19]. The
curves shown in Fig. 4 are representative of 2 separated experi-
ments. As shown in Fig. 4, microtubule formation rates are dis-
played over a period of 60 min at 37 ꢀC. 15
destabilising agent (CA-4) and 3 M of a microtubule-stabilising
agent (paclitaxel) were used as positive controls while DMSO
(0.5%) was used as negative control. The concentrations of PYB-SOs
mM of a microtubule-
#
X
R
IC50 (nM)a
HT-1080
m
HT-29
M21
MCF7
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CEU-602
CEU-638
CA-4b
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
H
920
840
370
1300
190
810
180
80
40
590
26
11
11
8500
2600
4900
21000
1200
3200
1300
840
680
1300
860
2250
990
590
130
8500
8800
5.9
480
780
350
1200
120
310
120
60
59
690
39
10
16
4800
2900
3400
12000
730
2400
860
5400
680
590
390
890
1300
390
80
460
910
190
1300
120
300
110
40
88
650
12
8.7
13
7500
2300
5600
9900
430
3000
940
2300
770
950
330
5100
390
280
56
650
500
210
890
100
370
140
60
22
290
21
11
17
4600
1700
2800
16000
960
1800
1100
680
560
830
600
2600
690
520
88
2-iprop
2-Ph
2-OMe
3-OMe
3-F
3-Cl
3-Br
3-I
3,4-OMe
3,5-OMe
3,5-Br
3,4,5-OMe
4-OMe
4-Ph
14 (30
mM) and 15 (3 mM) as well as PYB-SAs 30 (3 mM) and 31
(15 M) were determined by maximal solubility in the buffer so-
m
lution. Our results show that CA-4 inhibits tubulin polymerisation
while paclitaxel stabilises it. PYB-SOs 14 and 15 and PYB-SAs 31
strongly inhibit tubulin polymerisation similarly to CA-4 while the
inhibition of tubulin polymerisation by PYB-SA 30 is slightly lower.
PYB-SA 31 show the most potent inhibition of tubulin polymeri-
sation. These results strongly indicate that the mechanism of action
of PYB-SOs and PYB-SAs is the inhibition of polymerisation of
tubulin to microtubules.
H
3.5. PYB-SOs and PYB-SAs bind to the colchicine-binding site
2-iprop
2-Ph
2-OMe
3-OMe
3-F
3-Cl
3-Br
3-I
3,4-OMe
3,5-OMe
3,5-Br
3,4,5-OMe
4-OMe
4-Ph
3,4,5-OMe
3,4,5-OMe
N/A
It is known that antimicrotubule agents can interact with mi-
crotubules or tubulin heterodimers through different binding sites
[20]. Moreover, PIB-SOs, PIB-SAs and CA-4 are known to dock into
the C-BS [14,15]. In this context, we used a detection technique
developed by our laboratory to study the binding of PYB-SOs and
PYB-SAs with the C-BS [21]. This assay is based on the N,N0-
ethylene-bis(iodoacetamide) (EBI) which crosslinks cysteines in
positions 239 and 354 located in the C-BS. This adduct is conve-
niently detectable by Western blot as a second immunoreacting
4800
5400
4.0
21
5.1
7500
4700
4.0
13
2.2
4600
7000
5.0
18
1.9
band of
b
-tubulin. Antimicrotubule compounds that bind to the C-
-tubulin adduct with EBI. To that
BS inhibits the formation of the
b
end, we also used PYB-SOs 14e16, and PYB-SAs 27, 30, and 31 at 100
and 1000-times their respective IC50. The maximum concentration
NH
N/A
17
2.2
a
used in this experiment was 100
SOs and PYB-SAs tested inhibited the formation of the EBI:
tubulin adduct similarly to CA-4 and illustrated by the loss of the
second immunoreacting band of -tubulin and the presence of the
native -tubulin band [21]. These results show that both PYB-SO
and PYB-SA derivatives bind efficiently to the C-BS leading to the
depolymerisation of microtubules, disruption of the cytoskeleton
and also the arrest of cell cycle progression in G2/M phase. This
evidences the bioisosterism between 2-oxopyrrolidin-1-yl and the
IMZ moieties of PIB-SOs and PIB-SAs and also that the nitrogen
atom of the IMZ moiety is not involved in indispensable in-
teractions in the C-BS.
mM. As displayed in Fig. 5, all PYB-
IC50: concentration of drug inhibiting cell growth by 50%.
CA-4: Combretastatin A-4.
b
b-
b
progression in the G2/M phase. In general, PYB-SOs and PYB-SAs
bearing multiple substitutions have higher potential to arrest the
cell cycle in G2/M phase than those bearing only one substitution.
Finally, PYB-SO 16 exhibits the most potent cell cycle arrest in G2/M
phase showing a population increase in G2/M phase by 68.4%
which is similar to CA-4.
b
3.3. PYB-SOs and PYB-SAs induce cytoskeleton disruption
Since PYB-SOs and PYB-SAs arrest cell cycle progression in the
G2/M phase, we evaluated their ability to induce cytoskeleton
disruption by interfering with microtubules. To that end, we
selected our 3 most potent PYB-SOs (14e16) and PYB-SAs (27, 30
and 31) for their effect on microtubules and cytoskeleton integrity
of M21 cells when they are incubated at 5-times their respective
IC50 for 24 h. Cellular microtubule structures were visualised with
3.6. PYB-SOs 14 and 15 and PYB-SAs 30 and 31 dock into the C-BS
In the aim to predict the active conformation of PYB-SOs and
PYB-SAs and to identify the key amino acids involved in their in-
teractions with the C-BS, molecular docking studies were per-
formed to prepare a binding model derived from tubulin (protein
data bank (PDB): 1SA0) [22] using the docking tool in the Molecular
Operating Environment (MOE) software. The C-BS used to build our
computational model is constituted of GTP and 32 amino acids
distributed in the vicinity of the colchicine and GTP. The energy
docking results of the 5 most favourable poses of PYB-SOs 14 and 15
and PYB-SAs 30 and 31 in the C-BS are shown in Table 2. Firstly, the
indirect immunofluorescence using an anti-b-tubulin monoclonal
antibody. CA-4 and paclitaxel were used as positive controls while
DMSO (0.5%) was used as negative control. As depicted in Fig. 3,
PYB-SOs and PYB-SAs disrupt microtubules and induce mitotic
abnormalities similarly to CA-4. Therefore, PYB-SOs and PYB-SAs
4

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 2. Effect of PYB-SOs 11, 12, 14e16 and PYB-SAs 21, 25, 27, 29e31 on the cell cycle progression of M21 cells after 24 h of treatment. CA-4 and DMSO were used as controls.
most favourable docking poses of PYB-SOs 14 and 15 and PYB-SAs
30 and 31 showing the lowest S-score (ꢁ7.49, ꢁ7.29, ꢁ7.08
and ꢁ7.90 kcal/mol, respectively) are depicted in Fig. 6. In addition,
the superposition of these most favourable docking poses is illus-
trated in Fig. 7. Our results indicate that the molecular structure of
selected PYB-SOs and PYB-SAs has the ability to dock into the C-BS.
PYB-SO 14 and PYB-SA 30 are positioned into the C-BS similarly
while PYB-SO 15 and PYB-SA 31 adopt different poses. Fig. 8 illus-
trates 2D interaction diagrams of the most favourable PYB-SO and
PYB-SA poses and Table 3 summarises their key interactions with
amino acids present into the C-BS. The aromatic ring B of com-
pound 14 performs a pi-H interaction with Leu248, while com-
pound 15 is generating two distinct H-donor interactions with
Ala317 and Val238, respectively through its bromine atoms and it
also forms an H-acceptor interaction with Lys352 via its ketone
moiety. In our model, the most favourable poses for PYB-SAs 30 and
31 are not driven by pi-H, hydrogen bonds or halogen bonds but
driven by van der Waals interaction with the C-BS. These results
indicate that both amino acids (Leu248, Ala317, Val238 and Lys352)
and conformation poses are important for the interactions of PYB-
SOs and PYB-SAs with the C-BS.
3.7. PYB-SOs and PYB-SAs have low to no toxicity toward chick
embryos
The toxicity of the most potent PYB-SOs 14 and 15 and PYB-SAs
5

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 3. Effect of PYB-SOs 14e16 and PYB-SAs 27, 30 and 31 on cytoskeleton integrity of M21 cells after 24 h of treatment (400-times total magnification). CA-4 and paclitaxel were
used as positive controls while DMSO was used as negative control.
Fig. 4. Effect of PYB-SOs 14 and 15 and PYB-SAs 30 and 31 on tubulin assembly. CA-4 and paclitaxel were used as positive controls while DMSO was used as negative control.
6

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 5. Effect of PYB-SOs 14e16 and PYB-SAs 27, 30 and 31 on the binding of EBI to the colchicine-binding site at 100 and 1000-times their respective IC50. CA-4 was used as positive
control while DMSO and EBI were used as negative and positive controls, respectively.
Table 2
Docking energy scores of the 5 most favourable poses of PYB-SOs 14 and 15 and PYB-SAs 30 and 31 in the colchicine-binding site.
14
15
30
31
Pose
S (kcal/mol)
RMSD refine (Å)
S (kcal/mol)
RMSD refine (Å)
S (kcal/mol)
RMSD refine (Å)
S (kcal/mol)
RMSD refine (Å)
1
2
3
4
5
ꢁ7.49
ꢁ7.30
ꢁ7.25
ꢁ7.23
ꢁ7.04
1.13
1.79
2.02
1.03
1.85
ꢁ7.29
ꢁ7.12
ꢁ7.02
ꢁ6.91
ꢁ6.89
1.49
2.67
2.75
1.19
1.99
ꢁ7.08
ꢁ7.05
ꢁ6.86
ꢁ6.79
ꢁ6.78
1.19
0.91
2.08
1.77
1.56
ꢁ7.90
ꢁ7.84
ꢁ7.79
ꢁ7.76
ꢁ7.73
1.90
1.96
1.13
2.82
1.61
Fig. 6. Docking of the most stable poses of PYB-SOs and PYB-SAs superposed with colchicine (purple). A) 14 (red), B) 15 (white), C) 30 (yellow) and D) 31 (black) into the colchicine-
binding site. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
30 and 31 was assessed on chick embryos. A mixture of 1.0% DMSO,
6.2% cremophor EL™, 6.2% ethanol (99%) and 86.6% PBS was used as
significant difference between the untreated and the excipient
groups (100% vs 95%, F value of 0.58 compared to a critical F value of
4.11). Second, the percentage of weight of embryos treated by PYB-
SOs 14 and 15 and PYB-SAs 30 and 31 relative to the weight of
embryos untreated are 95, 93, 83 and 93%, respectively. Third, the
percentage of death embryos of untreated, excipient, PYB-SOs 14
and 15 and PYB-SAs 30 and 31 are 17, 18, 18, 18, 25 and 8%,
respectively. Although PYB-SA 30 has a lower percentage of em-
bryos weight and a higher death rate, these percentage differences
are not statistically significant with the excipient. Our results show
excipient to administrate PYB-SOs 14 (20
mg/egg) and 15 (10
mg/
egg) and PYB-SAs 30 (10 g/egg) and 31 (1
m
mg/egg). A group of
untreated chick embryos as well as another group of chick embryos
that were exclusively treated with the excipients were used as
negative controls. Drugs were injected at their maximum solubility
in the formulation. The percentages of weight of different groups
relative to the weight of untreated embryos are depicted in Fig. 9.
First, the formulation is not toxic since there is no statistically
7

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 7. Superposition of the docking of the most stable poses of PYB-SOs and PYB-SAs into the colchicine-binding site. Color coding: 14 (red), 15 (white), 30 (yellow) and 31 (black).
(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
that PYB-SOs and PYB-SAs have very low to no toxicity toward chick
embryos and present important features for the future develop-
ment of this new family of antimicrotubule agents.
Muegge filters, which means that these compounds have the po-
tential to be absorbed through the gastrointestinal tract. Therefore,
PYB-SOs and PYB-SAs display high theoretical druglikeness prop-
erties and bioavailability scores despite chemical and structural
modulations showing that PYB-SOs and PYB-SAs are promising for
in vivo experiments.
3.8. PYB-SOs and PYB-SAs have drug-like properties
Pharmacokinetics, physicochemical and drug-likeness proper-
ties are important in the development of new medicinal entities. To
that end, we used the SwissADME free online tool to estimate the
effect of structural modifications of our new PYB-SOs and PYB-SAs
on their theoretical pharmacokinetics, physicochemical and drug-
likeness properties [18]. PYB-SOs 11, 12 and 14e16 and PYB-SAs 21,
25, 27 and 29e31 were selected for analysis and the results are
summarised in Table 4. First, PYB-SOs and PYB-SAs have molecular
weights ranging from 377.41 to 475.15 and 350.82e474.17 g/mol,
respectively. The number of rotatable bonds varies from 4 to 7 for
both classes of compounds. PYB-SO derivatives have 4 to 7 H-bond
acceptors and 0 H-bond donor while PYB-SA derivatives have 3 to
6 H-bond acceptors and 1 H-bond donor. The topological polar
surface area (TPSA) ranges from 72.06 to 99.75 and 74.86 to
102.55 Å2 for PYB-SOs and PYB-SAs, respectively. The CLogP varies
from 2.54 to 3.78 and 1.98 to 3.37 for PYB-SOs and PYB-SAs,
respectively while LogS varies from ꢁ5.04 to ꢁ3.93 and ꢁ4.76
to ꢁ3.39. In general, compounds bearing a 3,5-dimethoxyl or a
3,4,5-trimethoxyl group are more soluble than their halogenated
counterparts regardless of the presence of a sulfonate or a sulfon-
amide bridge. Globally, all compounds exhibit roughly similar
ClogPs and LogSs and are either in the moderately soluble or sol-
uble class. Moreover, all PYB-SOs and PYB-SAs evaluated so far have
a high probability of gastrointestinal absorption (GIA). Beside
compounds 11,12, 25 and 27, all PYB-SOs and PYB-SAs are predicted
to be able to diffuse through the blood-brain barrier (BBB). PYB-SOs
and PYB-SAs at the exception of compounds 29 and 31 should not
be substrates of the p-glycoprotein, an efflux protein that is
responsible for multidrug resistance. Of interest, PYB-SOs and PYB-
SAs do not exhibit violation of either the Lipinski, Veber, Egan or
4. Conclusion
In conclusion, we reported here the synthesis and the biological
activity of 30 new PYB-SO and PYB-SA derivatives. They are active
in the low nanomolar to the low micromolar levels on the four
human cancer cell lines assessed. They arrest the cell cycle pro-
gression in G2/M phase, inhibit polymerisation of tubulin to mi-
crotubules, bind to the C-BS, and exhibit low to no toxicity toward
chick embryos. In addition, as determined by the SwissADME al-
gorithm they possess druglikeness properties and bioavailability
scores suitable for in vivo experiments. Therefore, PYB-SOs and
PYB-SAs are a new family of promising antimicrotubule agents.
5. Experimental protocols
5.1. Biological methods
5.1.1. Cell lines culture
HT-1080 human fibrosarcoma, HT-29 human colon carcinoma,
and MCF7 human breast carcinoma were purchased from the
American Type Culture Collection (Manassa, VA, USA). M21 human
skin melanoma cells were provided by Dr. David Cheresh (Univer-
sity of California, San Diego School of Medicine, CA). Cells were
cultured in DMEM medium containing sodium bicarbonate, high
glucose concentration, glutamine and sodium pyruvate (Hyclone,
Logan, UT, USA) supplemented with either 5 or 10% of fetal bovine
serum (FBS, Invitrogen, Burlington, ON, Canada) and were main-
tained at 37 ꢀC in a moisture-saturated atmosphere containing 5%
CO2.
8

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 8. 2D interaction diagrams of the most stable poses of PYB-SOs and PYB-SAs into the colchicine-binding site A) 14, B) 15, C) 30 and D) 31.
Table 3
for its drug screening program with slight modifications [23].
Briefly, ninety-six-well Costar microtiter clear plates were seeded
Interactions summary of PYB-SOs 14,15 and PYB-SAs 30 and 31 with the amino acids
of the colchicine-binding site.
with 75 m
L of a suspension of either HT-1080 (2.5 x 103), HT-29 (4.0
x 103), M21 (3.0 x 103) or MCF7 (2.5 x 103) cells per well in DMEM
supplemented with 5 or 10% FBS. Freshly solubilised drugs in DMSO
Ligand Receptor Interaction
Distance Energy
Atom
Atom
Amino Acid Type
(Å)
(Kcal/mol)
(40 mM) were diluted in fresh supplemented DMEM and 75
aliquots containing serially diluted concentrations of the drug were
added. Final drug concentrations ranged from 100 M to 0.39 nM.
mL
14 6-ring
15 BR
CD1
O
O
NZ
e
Leu248
Ala317
Val238
Lys352
e
pi-H
H-donor
H-donor
H-acceptor 3.20
e
3.53
3.50
3.39
ꢁ0.5
ꢁ0.6
ꢁ1.5
ꢁ6.2
e
m
BR
O
DMSO concentration was kept constant at 0.5% (v/v) to prevent any
related toxicity. Plates were incubated for 48 h, after which growth
was stopped by the addition of cold trichloroacetic acid to the wells
(10% w/v, final concentration). Afterwards, plates were incubated at
4 ꢀC for 1 h. Then, plates were washed 5-times with distilled water
and a sulforhodamine B solution (0.1% w/v) in 1% acetic acid was
added to each well. After 15 min at room temperature, the
exceeding dye was removed and the plates were washed 5-times
with a solution of 1% acetic acid. Bound dye was solubilised in
30
31
e
e
e
e
e
e
e
e
5.1.2. Antiproliferative activity assay
The antiproliferative activity of PYB-SOs 4e18 and PYB-SAs
19e33 was assessed using the procedure described by the Na-
tional Cancer Institute (NCI) Developmental Therapeutics Program
9

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
Fig. 9. Effect of PYB-SOs 14 and 15 and PYB-SAs 30 and 31 on the weight and the mortality of chick embryos. Grey bars represent the percentage of wet weight embryos relative to
untreated wet weight embryos. Black bars represent the percentage of chick embryo mortality.
Table 4
Physicochemical properties, pharmacokinetic properties and druglikeness of PYB-SOs 11, 12, 14e16, and PYB-SAs 21, 25, 27, 29e31 calculated using the web-based SwissADME
application.
#
Rba
HeBab
H-BDc
TPSAd (Å2)
CLogPe
LogSf
Sclassg
GIAh
BBBPi
Pgpj
Drug-like (#viol.)k
11
12
14
15
16
21
25
27
29
30
31
4
4
6
4
7
5
4
4
6
4
7
4
4
6
4
7
3
3
3
5
3
6
0
0
0
0
0
1
1
1
1
1
1
72.06
72.06
90.52
72.06
99.75
74.86
74.86
74.86
93.32
74.86
102.55
3.15
3.20
2.56
3.78
2.54
3.37
2.60
2.75
2.10
3.31
1.98
ꢁ4.32
ꢁ4.27
ꢁ3.93
ꢁ5.04
ꢁ4.09
ꢁ4.76
ꢁ3.72
ꢁ3.74
ꢁ3.39
ꢁ4.50
ꢁ3.55
MS
MS
S
MS
MS
MS
S
S
S
MS
S
H
H
H
H
H
H
H
H
H
H
H
Yes
Yes
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
Yes
No
Yes
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
Yes (0)
a
Rb: number of rotatable bonds.
b
c
d
e
f
H-Ba: number of H-bond acceptors.
H-BD: number of H-bond donors.
TPSA: topological polar surface area.
CLogP: consensus Log P (average of iLOGP, XLOGP3, WLOGP, MLOGP and Silicos-IT Log P).
LogS: Ali topological method Log S.
g
h
i
SClass: Ali solubility class (insoluble (IS)<ꢁ10<poorly soluble (PS)<ꢁ6<moderately soluble (MS)<ꢁ4<soluble (S)<ꢁ2<very soluble (VS) < 0<highly soluble (HS)).
GIA: gastrointestinal absorption (H means high).
BBBP: blood-brain barrier permeability.
Pgp: p-glycoprotein substrates.
Drug-like: drug-likeness indices (bioavailability) from Lipinski, Ghose, Veber, Egan and Muegge filters. # viol: number of violations of the 5 filters.
j
k
20 mM Tris base and the absorbance was read using an optimal
wavelength (520e580 nm) with a SpectraMax® i3x (Molecular
Devices, San Jose, CA, USA). Data obtained from treated cells were
compared to the control cell plates fixed on the treatment day and
the percentage of cell growth was thus calculated for each drug. The
experiments were done at least twice in triplicate. The assays were
considered valid when the coefficient of variation was <10% for a
given set of conditions within the same experiment.
10

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
5.1.3. Cell cycle progression analysis
trypsinised (trypsin-EDTA 0.5%), harvested and centrifuged for
After incubation of 2.5 x 105 M21 cells with selected PYB-SOs
and PYB-SAs at 2- and 5-times their respective IC50 for 24 h, the
5 min at 1200 rpm. The pellets were washed with 500 mL by cold
PBS and stored at ꢁ80 ꢀC until analysis.
cells were trypsinised, washed with 750
mL of phosphate buffered
saline (PBS) and resuspended in 250 L of PBS. Cells were fixed by
m
5.1.7. Gel electrophoresis and immunoblot
the addition of 750
m
L of ice-cold ethanol under agitation and
The cell pellets were resuspended in a buffer containing 0.32 M
sucrose, 1 mM EDTA at pH 8, 10 mM Tris at pH 7.4 and protease
inhibitor. The protein concentration was assessed using the Bio-Rad
protein assay (Bio-Rad laboratories, Mississauga, ON, Canada).
Samples were prepared and diluted to obtain proteins at 4 mg/mL
in Laemmli sample buffer [25] (60 mM Tris-Cl at pH 6.8, 2% SDS,
stored at ꢁ20 ꢀC until analysis. Prior analysis, cells were washed
with 750 mL of PBS and resuspended in 300 mL of PBS containing
2 mg/mL DAPI. Cell cycle distribution of fixed cell suspensions was
analysed using an LSR II flow cytometer (BD Biosciences, Franklin
Lakes, NJ, USA).
10%, glycerol, 5%
boiled for 5 min. Then, 40
b
-mercaptoethanol, 0.01% bromophenol blue) and
g of proteins from the protein extracts
5.1.4. Immunofluorescence of microtubules
m
MCF7 cells were seeded at 2.5 x 105 cells per well in 6-well
plates (Costar®) containing glass cover slides (22 mm ꢂ 22 mm)
were used for the electrophoresis using 10% polyacrylamide gels.
The proteins were transferred onto nitrocellulose membranes that
were incubated with TBSTM (Tris-Buffered Saline þ 0.1% (v/v)
Tween-20 with 5% fat-free dry milk) for 2 h at room temperature.
coated with fibronectin (10 mg/mL) and incubated for 24 h. Tumour
cells were treated either with PYB-SOs 14 (58 nM) and 15 (44 nM),
PYB-SAs 30 (1400 nM) and 31 (560 nM), paclitaxel (25 nM) or CA-4
(10 nM) for 24 h. DMSO (0.5%) was used as negative control while
paclitaxel and CA-4 were used as positive controls. The cells were
then washed thrice with 2 mL of PBS and fixed with 3.7% formal-
dehyde in PBS for 10 min. After two washes with PBS, the cells were
permeabilised with saponin (0.1% in PBS) and blocked with 3% (w/
v) BSA in PBS for 45 min at 37 ꢀC. The cells were then incubated for
Next, the anti-b-tubulin (clone TUB 2.1, Sigma-Aldrich, St. Louis,
MO, USA) primary antibody was incubated in TBSTM (1:500) for
2 h at room temperature. Membranes were washed with TBST
(Tris-Buffered Saline with 0.1% (v/v) Tween 20) and incubated with
peroxidase conjugated anti-mouse immunoglobulin (Amersham
Canada, Oakville, ON, Canada) in TBSMT (1:5000) for 1 h at room
temperature. After washing the membranes with TBST, detection of
the immunoblot was carried out using Clarity Western enhanced
chemiluminescence reagents (Bio-Rad laboratories, Mississauga,
ON, Canada). Detection of bands was performed with Super Rx
medical X-Ray film (Fujifilm, Tokyo, Japan).
2 h at room temperature with an anti-b-tubulin monoclonal anti-
body (clone TUB 2.1, 1:300) in a solution containing 0.1% saponin
and 3% BSA in PBS. The cells were washed thrice with PBS con-
taining 0.05% Tween 20 and stained using antimouse IgG Alexa
Fluor 488 (Molecular Probes, Eugene, OR, USA; 1:1000) and DAPI
(1.25 ng/mL final concentration) in blocking buffer for 1 h at 37 ꢀC.
5.1.8. Toxicity toward chick embryos
The cover slides were mounted using 25
m
L of Fluoromount-G®
Fertilised eggs were purchased from Couvoir La Coop (Victor-
iaville, QC, Canada). They were incubated for a 10-days period in a
Pro-FI egg incubator (Lyon Electric, Chula Vista, CA, USA) with an
automatic turning system. Compounds were solubilised in a
formulation containing 1.0% DMSO, 6.2% cremophor EL™, 6.2%
ethanol (99%) and 86.6% PBS. After the incubation period, the veins
of the chorioallantoic membrane were identified on the sides of the
egg shells. The eggs were opened using a hobby drill (Dremel,
Racine, WI) to obtain a 1.5 ꢂ 1.5 cm opening directly over the
location of the veins. Groups of 10e12 eggs were formed by ran-
domisation and the drugs were intravenously administered at their
(SouthernBiotech, Birmingham, AL, USA) before analysis under an
Olympus BX51 fluorescence microscope (Olympus BX51, Center
Valley, PA, USA). Images were acquired as 8 bit-tagged image
format files with a Q imaging RETIGA EXI digital camera (Qimaging,
Surrey, BC, Canada) using the Image Pro Express software at a total
magnification of 400ꢂ.
5.1.5. Microtubule polymerisation assay
The effect of PYB-SOs 14 (30
mM) and 15 (3 mM) and PYB-SAs 30
(3
m
M) and 31 (15 M) on dynamic polymerisation of tubulin to
m
microtubules was analysed with the fluorescence based Tubulin
polymerisation assay using >99% pure tubulin (Cytoskeleton, Inc.,
Denver, CO, USA). Drugs were solubilised at the required concen-
maximum solubility in our excipient (100 mL/egg). The openings
were closed using adhesive tape and the eggs were then incubated
in a static incubator (Lyon Electric, Chula Vista, CA, USA) for a 7-
days period at 37 ꢀC. The embryos were euthanised at 4 ꢀC for at
least 2h. The wet weight of each embryo and the number of dead
embryos were registered. A group of untreated embryos and a
group of embryos that were treated only with the excipient were
used as negative controls. A one-way ANOVA analysis was used to
determine the statistical significance of the results.
tration in DMSO, then 5 mL of drug solutions were diluted in 325 mL
of nanopure water to reach 10x of their final concentration. The
assay was performed according to the manufacturer guidelines
[24]. Briefly, tubulin >99% (2.0 mg/mL) was dissolved in general
tubulin buffer (80 mM PIPES pH 6.9, 0.5 mM EGTA, 2.0 mM MgCl2)
and incubated with drugs at 37 ꢀC. The polymerisation was
measured over 60 min with fluorescent excitation at 360 nM and
emission at 450 nM in kinetic mode with a SpectraMax® i3x
(Molecular Devices, San Jose, CA, USA). The curves shown in Fig. 8
are representative of 2 separated experiments.
5.2. Chemical methods
5.2.1. General
Proton NMR spectra were recorded on a Bruker AM-300 spec-
5.1.6. In vitro competition binding assay of EBI to the C-BS
trometer (Bruker, Germany). Chemical shifts (d) are reported in
M21 cells were seeded at a concentration of 7.5 x 105 M21 cells/
well and were incubated overnight in 6-well plates. Then, cells
were treated with either PYB-SOs 14 and 15 or PYB-SAs 30 and 31
for 1.5 h at 100- and 1000-times their respective IC50 for 2 h. The
maximum concentration of PYB-SOs and PYB-SAs used for the ex-
parts per million (ppm). Reactions requiring microwave heating
were performed at 200 W with an initiator system (Biotage, Char-
lottesville, VA, USA). Uncorrected melting points were obtained on
a
MPA100 automated melting point system (SRS Standford
Research Systems, Sunnyvale, CA, USA). UHPLC analyses were
performed using an ACQUITY Arc system (Waters, Mississauga, ON,
Canada) equipped with a 2998 PDA detector. The samples were
obtained by solubilisation of the compounds in DMSO and a sub-
sequent solution was produced by diluting the compounds at 1%
periments was set at 100 mM. Next, 100 mL of EBI (100 mM, final
concentration, Toronto Research Chemicals, North York, ON, Can-
ada) in PBS were added to each well for 1.5 h. After the incubation,
the supernatant was withdrawn and the cells in the plates were
11

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
DMSO in a mixture of MeOH/H2O 50:50. These samples were then
eluted using a mixture of MeOH/H2O with a linear mobile phase
gradient (1.0 mL/min) on a CORTECS C18þ reversed-phase column
oxopyrrolidin-1-yl)benzenesulfonyl chloride (36, 0.10 g) in aceto-
nitrile (3.0 mL) in presence of triethylamine (0.16 mL, 3.0 Eq). The
reaction mixture was stirred at 120 ꢀC under microwave radiation
for 8 h. After completion of the reaction, the mixture was cooled at
room temperature and the solvent was evaporated under reduced
pressure. The residue was diluted within AcOEt (10 mL) and was
washed twice with HCl 1M (10 mL), NaOH 1M (10 mL) and brine
(10 mL). The resulting solution was dried over anhydrous sodium
sulfate, filtered and evaporated to dryness under reduced pressure.
The residue was purified by flash chromatography on silica gel.
3.0 ꢂ 50 mm ꢂ 2.7
mm (Waters, Mississauga, ON, Canada). Wave-
length was selected at 280 nm and the percentage of all the present
compounds was determined. Purity was confirmed by UHPLC and
was equal or greater than 95%. All chemicals were supplied by
Aldrich Chemicals (Milwaukee, WI, USA), VWR International
(Mont-Royal, QC, Canada), Fisher Scientific (Montreal, QC, Canada).
Liquid flash chromatography was performed on silica gel F60, 60 Å,
40e63 mm supplied by Silicycle (Quebec, QC, Canada) using an FPX
flash purification system (Biotage, Charlottesville, VA, USA), and
using solvent mixtures expressed as v/v ratios. Solvents and re-
agents were used without purification unless specified otherwise.
The progress of all reactions was monitored by TLC on precoated
silica gel 60 Å F254 TLC plates provided by VWR International
(Mont-Royal, QC, Canada). The chromatograms were viewed under
UV light at 254 and/or 275 nm. HRMS were recorded by direct in-
jection in a TOF system 6210 series mass spectrometer (Agilent
technologies, Santa Clara, CA, USA).
5.2.6. Characterization of compounds 4e18
5.2.6.1. Phenyl
4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(4).
Flash chromatography (methylene chloride to methylene chloride/
ethyl acetate (95:5)). Purity: 100.0%; yield: 83%; white solid; mp:
133e135 ꢀC; 1H NMR (CDCl3):
d 7.82e7.72 (m, 4H, Ar), 7.28e7.21 (m,
3H, Ar), 6.96e6.93 (m, 2H, Ar), 3.86 (t, 2H, J ¼ 7.0 Hz, CH2), 2.63 (t,
2H, J ¼ 8.0 Hz, CH2), 2.23e2.13 (m, 2H, CH2); 13C NMR (CDCl3):
d
175.0, 149.6, 144.5, 129.7, 129.5, 129.4, 127.2, 122.3, 118.7, 48.3,
32.9, 17.7; HRMS (ESI) m/z found, 318.0802; C16H16NO4S (Mþ þ H)
expected, 318.0801.
5.2.2. Preparation of compound 34
4-Chlorobutanoyl chloride (6.1 mL, 1.0 Eq) was added to a so-
lution of aniline (5.0 mL, 1.0 Eq) in methylene chloride (100 mL) in
presence of triethylamine (9.2 mL,1.2 Eq). The reaction mixture was
stirred at room temperature for 24 h. After completion of the re-
action, the mixture was washed twice with HCl 1M (100 mL) and
brine (100 mL). The resulting organic solution was dried over
anhydrous sodium sulfate, filtered and evaporated to dryness un-
der reduced pressure. The product 4-chloro-N-phenylbutanamide
(34) was used without further purification with a yield of 86%. The
1H and 13C NMR are equivalent to the NMR characterisation pub-
lished previously [26].
5.2.6.2. 2-Isopropylphenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(5). Flash chromatography (hexanes/methylene chloride (50:50) to
methylene chloride). Purity: 99.7%; yield: 61%; white solid; mp:
127e129 ꢀC; 1H NMR (CDCl3):
d 7.84 (brs, 4H, Ar), 7.27e7.18 (m, 2H,
Ar), 7.12e7.07 (m, 1H, Ar), 7.01e6.98 (m, 1H, Ar), 3.89 (t, 2H,
J ¼ 7.0 Hz, CH2), 3.16e3.10 (m, 1H, CH), 2.66 (t, 2H, J ¼ 8.0 Hz, CH2),
2.26e2.16 (m, 2H, CH2), 1.07 (d, 6H, J ¼ 6.9 Hz, CH3); 13C NMR
(CDCl3):
d 174.9, 147.0, 144.4, 141.8, 130.5, 129.4, 127.3, 127.2, 126.7,
121.9, 118.8, 48.4, 32.8, 26.7, 23.1, 17.7; HRMS (ESI) m/z found,
360.1269; C19H22NO4S (Mþ þ H) expected, 360.1269.
5.2.6.3. [1,10-Biphenyl]-2-yl 4-(2-oxopyrrolidin-1-yl)benzenesulfo-
nate (6). Flash chromatography (methylene chloride). Purity:
96.5%; yield: 97%; white solid; mp: 157e159 ꢀC; 1H NMR (acetone-
5.2.3. Preparation of compound 35
Compound 35 was prepared as published previously with slight
modifications [27]. Briefly, sodium hydride (in oil [60%], 2.2 g, 1.2
Eq) was dissolved in an ice-cold solution of 4-chloro-N-phenyl-
butanamide (34, 9.0 g, 1.0 Eq) in dry THF (250 mL) under argon
atmosphere. The ice bath was removed after 10 min. The reaction
was stirred at room temperature for 24 h. The reaction was
quenched at 0 ꢀC with water (50 mL) and the organic solvent was
evaporated. The aqueous solution was extracted with AcOEt
(250 mL). The organic solution washed twice with brine (250 mL),
dried over anhydrous sodium sulfate, filtered and evaporated to
dryness under reduced pressure. The residue was purified by flash
chromatography on silica gel (hexanes/AcOEt (75:25) to hexanes/
AcOEt (66:33) to obtain 1-phenylpyrrolidin-2-one (35) with a yield
of 78%. The 1H and 13C NMR are equivalent to the NMR character-
isation published previously [28].
d6):
J ¼ 6.9 Hz, CH2), 2.59 (t, 2H, J ¼ 7.9 Hz, CH2), 2.26e2.16 (m, 2H, CH2);
13C NMR (acetone-d6):
179.9, 151.7, 150.1, 141.8, 140.6, 136.4, 134.3,
d 7.66e7.64 (m, 2H, Ar), 7.47e7.13 (m, 11H, Ar), 3.94 (t, 2H,
d
134.3, 133.9, 133.9, 133.3, 132.8, 132.6, 129.0, 123.5, 53.2, 37.6, 22.7;
HRMS (ESI) m/z found, 394.1110; C22H20NO4S (Mþ þ H) expected,
394.1109.
5.2.6.4. 2-Methoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(7). Flash chromatography (methylene chloride to methylene
chloride/ethyl acetate (97:3)). Purity: 96.5%; yield: 70%; white
solid; mp: 157e158 ꢀC; 1H NMR (CDCl3):
d 7.83e7.76 (m, 4H, Ar),
7.20e7.11 (m, 2H, Ar), 6.89e6.81 (m, 2H, Ar), 3.88 (t, 2H, J ¼ 7.0 Hz,
CH2), 3.55 (s, 3H, CH3), 2.64 (t, 2H, J ¼ 8.1 Hz, CH2), 2.24e2.14 (m,
2H, CH2); 13C NMR (CDCl3):
d 174.9, 151.8, 144.3, 138.3, 130.5, 129.6,
128.1, 123.9, 120.6, 118.5, 112.7, 55.6, 48.4, 32.8, 17.7; HRMS (ESI) m/z
5.2.4. Preparation of compound 36
found, 348.0902; C17H18NO5S (Mþ þ H) expected, 348.0906.
Compound 36 was prepared as published previously with slight
modifications [29]. Briefly, 1-phenylpyrrolidin-2-one (35, 5.0 g, 1.0
Eq) was dissolved in ice cold chlorosulfonic acid (2.1 mL, 7.7 Eq)
under a dry argon atmosphere. The ice bath was removed after
15 min. The reaction mixture was stirred at room temperature for
16 h. After completion of the reaction, the solution was poured on
iced water dropwise. The solid was filtered, rinsed with water
(100 mL) and dried for 1 h under vacuum. The resulting white 4-(2-
oxopyrrolidin-1-yl)benzenesulfonyl chloride (36) was used
without further purification with a yield of 72%.
5.2.6.5. 3-Methoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(8). Flash chromatography (methylene chloride/ethyl acetate
(97:3)). Purity: 99.6%; yield: 71%; pale solid; mp: 122e124 ꢀC; 1H
NMR (CDCl3):
d 7.83e7.76 (m, 4H, Ar), 7.16e7.11 (m, 1H, Ar),
6.77e6.74 (m, 1H, Ar), 6.56e6.50 (m, 2H, Ar), 3.87 (t, 2H, J ¼ 7.0 Hz,
CH2), 3.70 (s, 3H, CH3), 2.64 (t, 2H, J ¼ 8.0 Hz, CH2), 2.24e2.14 (m,
2H, CH2); 13C NMR (CDCl3):
d 175.0, 160.4, 150.4, 144.5, 129.9, 129.5,
129.5, 118.7, 114.2, 113.0, 108.3, 55.5, 48.4, 32.8, 17.7; HRMS (ESI) m/z
found, 348.0899; C17H18NO5S (Mþ þ H) expected, 348.0906.
5.2.5. General preparation of compounds 4e18
5.2.6.6. 3-Fluorophenyl
4-(2-oxopyrrolidin-1-yl)benzenesulfonate
The relevant phenol (1.5 Eq) was added to a solution of the 4-(2-
(9). Flash chromatography (methylene chloride). Purity: 99.8%;
12

ꢀ
M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
yield: 60%; white solid; mp: 120e122 ꢀC; 1H NMR (CDCl3):
473.9003; C16H14Br2NO4S (Mþ þ H) expected, 473.9009.
d
7.86e7.77 (m, 4H, Ar), 7.28e7.20 (m, 1H, Ar), 6.99e6.93 (m, 1H,
Ar), 6.80e6.74 (m, 2H, Ar), 3.89 (t, 2H, J ¼ 7.0 Hz, CH2), 2.66 (t, 2H,
5.2.6.13. 3,4,5-Trimethoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzene-
sulfonate (16). Flash chromatography (hexanes/ethyl acetate
(80:20) to (60:40)). Purity: 96.5%; yield: 67%; pale solid; mp:
J ¼ 8.0 Hz CH2), 2.26e2.16 (m, 2H, CH2); 13C NMR (CDCl3):
d 175.0,
164.3,161.0,150.2,150.1,144.7,130.5,130.4,129.5,129.2,118.8,118.2,
118.1, 114.5, 114.2, 110.6, 110.3, 48.3, 32.8, 17.7; HRMS (ESI) m/z
found, 336.0696; C16H15FNO4S (Mþ þ H) expected, 336.0707.
129e131 ꢀC; 1H NMR (CDCl3):
d 7.77 (brs, 4H, Ar), 6.17 (s, 2H, Ar),
3.84 (t, 2H, J ¼ 7.0 Hz, CH2), 3.73 (s, 3H, CH3), 3.65 (s, 6H, CH3), 2.60
(t, 2H, J ¼ 8.0 Hz, CH2), 2.18e2.13 (m, 2H, CH2); 13C NMR (CDCl3):
5.2.6.7. 3-Chlorophenyl
4-(2-oxopyrrolidin-1-yl)benzenesulfonate
d 175.0, 153.3, 145.4, 144.5, 136.7, 129.6, 129.3, 118.8, 99.8, 60.9, 56.2,
(10). Flash chromatography (methylene chloride/ethyl acetate
48.3, 32.8, 17.7; HRMS (ESI) m/z found, 408.1115; C19H22NO7S
(99:1) to (95:5)). Purity: 100.0%; yield: 79%; white solid; mp:
(Mþ þ H) expected, 408.1119.
102e104 ꢀC; 1H NMR (CDCl3):
d 7.85e7.76 (m, 4H, Ar), 7.26e7.16 (m,
2H, Ar), 7.04 (brs, 1H, Ar), 6.87e6.84 (m, 1H, Ar), 3.88 (t, 2H,
J ¼ 7.0 Hz, CH2), 2.65 (t, 2H, J ¼ 8.0 Hz, CH2), 2.25e2.15 (m, 2H, CH2);
5.2.6.14. 4-Methoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(17). Flash chromatography (methylene chloride/ethyl acetate
(98:2)). Purity: 100.0%; yield: 42%; white solid; mp: 120e121 ꢀC; 1H
13C NMR (CDCl3):
d 175.0, 149.9, 144.8, 134.9, 130.4, 129.5, 129.1,
127.5, 123.0, 120.6, 118.8, 48.3, 32.8, 17.7; HRMS (ESI) m/z found,
NMR (CDCl3): d 7.83e7.74 (m, 4H, Ar), 6.88e6.85 (m, 2H, Ar),
352.0393; C16H15ClNO4S (Mþ þ H) expected, 352.0411.
6.76e6.73 (m, 2H, Ar), 3.89 (t, 2H, J ¼ 7.0 Hz, CH2), 3.75 (s, 3H, CH3),
2.65 (t, 2H, J ¼ 8.0 Hz, CH2), 2.25e2.15 (m, 2H, CH2); 13C NMR
5.2.6.8. 3-Bromophenyl
4-(2-oxopyrrolidin-1-yl)benzenesulfonate
(CDCl3): d 175.0, 158.2, 144.4, 143.0, 129.6, 129.5, 123.3, 118.7, 114.5,
(11). Flash chromatography (methylene chloride/ethyl acetate
55.6, 48.4, 32.8, 17.7; HRMS (ESI) m/z found, 348.0898; C17H18NO5S
(99:1) to (95:5)). Purity: 99.5%; yield: 74%; white solid; mp:
(Mþ þ H) expected, 348.0906.
89e92 ꢀC; 1H NMR (CDCl3):
d 7.85e7.76 (m, 4H, Ar), 7.38e7.35 (m,
1H, Ar), 7.20e7.11 (m, 2H, Ar), 6.91e6.88 (m, 1H, Ar), 3.88 (t, 2H,
J ¼ 7.0 Hz, CH2), 2.65 (t, 2H, J ¼ 8.0 Hz, CH2), 2.25e2.15 (m, 2H, CH2);
5.2.6.15. [1,10-Biphenyl]-4-yl 4-(2-oxopyrrolidin-1-yl)benzenesulfo-
nate (18). Flash chromatography (methylene chloride/ethyl acetate
(90:10)). Purity: 99.9%; yield: 36%; white solid; mp: 145e147 ꢀC; 1H
13C NMR (CDCl3):
d 175.0, 149.9, 144.8, 130.7, 130.4, 129.5, 129.1
125.8, 122.5, 121.1, 118.8, 48.3, 32.8, 17.7; HRMS (ESI) m/z found,
NMR (CDCl3): d 7.85e7.79 (m, 4H, Ar), 7.52e7.31 (m, 7H, Ar),
395.9898; C16H15BrNO4S (Mþ þ H) expected, 395.9906.
7.05e7.02 (m, 2H, Ar), 3.88 (t, 2H, J ¼ 7.0 Hz, CH2), 2.65 (t, 2H,
J ¼ 8.0 Hz, CH2), 2.24e2.17 (m, 2H, CH2); 13C NMR (CDCl3):
d 175.0,
5.2.6.9. 3-Iodophenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (12).
Flash chromatography (methylene chloride to methylene chloride/
ethyl acetate (95:5)). Purity: 97.6%; yield: 31%; white solid; mp:
148.9, 144.6, 140.2, 139.7, 129.6, 129.5, 128.9, 128.3, 127.7, 127.1,
122.6, 118.8, 48.4, 32.9, 17.7; HRMS (ESI) m/z found, 394.1103;
C
22H20NO4S (Mþ þ H) expected, 394.1109.
103e105 ꢀC; 1H NMR (CDCl3):
d 7.86e7.77 (m, 4H, Ar), 7.59e7.56
(m, 1H, Ar), 7.38 (s, 1H, Ar), 7.03e6.92 (m, 2H, Ar), 3.90 (t, 2H,
J ¼ 7.0 Hz, CH2), 2.66 (t, 2H, J ¼ 8.0 Hz, CH2), 2.26e2.16 (m, 2H, CH2);
5.2.7. General preparation of compounds 19e33
The relevant aniline (1.5 Eq) was added to a solution of the 4-(2-
oxopyrrolidin-1-yl)benzenesulfonyl chloride (36, 0.10 g) in aceto-
nitrile (3.0 mL) in presence of 4-dimethylaminopyridine (0.14 g, 3.0
Eq). The reaction mixture was stirred at 120 ꢀC under microwave
radiations for 8 h. The mixture was cooled at room temperature and
the solvent was evaporated under reduced pressure. The residue
was diluted with AcOEt (10 mL) and washed twice with HCl 1M
(10 mL) and brine (10 mL). The resulting solution was dried over
anhydrous sodium sulfate, filtered and evaporated to dryness un-
der reduced pressure. The residue was purified by flash chroma-
tography on silica gel.
13C NMR (CDCl3):
d 175.0, 149.6, 144.7, 136.3, 131.5, 130.9, 129.6,
129.2, 121.7, 118.8, 93.5, 48.4, 32.8, 17.7; HRMS (ESI) m/z found,
443.9757; C16H15INO4S (Mþ þ H) expected, 443.9767.
5.2.6.10. 3,4-Dimethoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzenesul-
fonate (13). Flash chromatography (methylene chloride/ethyl ace-
tate (97:3) to (95:5)). Purity: 97.6%; yield: 55%; white solid; mp:
152e153 ꢀC; 1H NMR (CDCl3):
d 7.85e7.78 (m, 4H, Ar), 6.71e6.44
(m, 3H, Ar), 3,90 (t, 2H, J ¼ 7.0 Hz, Ar), 3.84 (s, 3H, CH3), 3.77 (s, 3H,
CH3), 2.67 (t, 2H, J ¼ 8.0 Hz, CH2), 2.27e2.17 (m, 2H, CH2); 13C NMR
(CDCl3):
d 175.0, 149.3, 147.9, 144.5, 143.1, 129.7, 129.5, 118.7, 113.8,
110.7, 106.5, 56.1, 56,1, 48.4, 32.9, 17.8; HRMS (ESI) m/z found,
5.2.8. Characterization of compounds 19e33
378.0991; C18H20NO6S (Mþ þ H) expected, 378.1012.
5.2.8.1. 4-(2-Oxopyrrolidin-1-yl)-N-phenylbenzenesulfonamide (19).
Flash chromatography (methylene chloride to methylene chloride/
ethyl acetate (90:10)). Purity: 100.0%; yield: 38%; white solid; mp:
5.2.6.11. 3,5-Dimethoxyphenyl 4-(2-oxopyrrolidin-1-yl)benzenesul-
fonate (14). Flash chromatography (methylene chloride to methy-
lene chloride/ethyl acetate (97:3)). Purity: 99.4%; yield: 39%; white
184e186 ꢀC; 1H NMR (DMSO‑d6):
d 10.20 (s, 1H, NH), 7.81e7.70 (m,
4H, Ar), 7.22e7.17 (m, 2H, Ar), 7.08e6.96 (m, 3H, Ar), 3.79 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.48 (t, 2H, J ¼ 7.8 Hz, CH2), 2.06e1.96 (m, 2H, CH2);
solid; mp: 119e121 ꢀC; 1H NMR (CDCl3):
d 7.82 (brs, 4H, Ar), 6.31
(brs, 1H, Ar), 6.16e6.15 (m, 2H, Ar), 3.88 (t, 2H, J ¼ 7.0 Hz, CH2), 3.68
13C NMR (DMSO‑d6):
d 175.2, 143.5, 138.2, 134.0, 129.6, 128.1, 124.4,
(s, 6H, CH3), 2.65 (t, 2H, J ¼ 7.4 Hz, CH2), 2.25e2.15 (m, 2H, CH2); 13
C
120.4, 119.1, 48.3, 32.9, 17.7; HRMS (ESI) m/z found, 317.0957;
NMR (CDCl3):
d
174.9, 161.0, 151.0, 144.5, 129.7, 129.5, 118.7, 100.8,
C
16H17N2O3S (Mþ þ H) expected, 317.0961.
99.3, 55.5, 48.4, 32.8, 17.7; HRMS (ESI) m/z found, 378.0998;
C
18H20NO6S (Mþ þ H) expected, 378.1012.
5.2.8.2. N-(2-Isopropylphenyl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (20). Flash chromatography (methylene chloride/ethyl
acetate (95:5)). Purity: 96.8%; yield: 39%; white solid; mp:
5.2.6.12. 3,5-Dibromophenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfo-
nate (15). Flash chromatography (hexanes/ethyl acetate (80:20) to
(60:40)). Purity: 99.5%; yield: 54%; white solid; mp: 138e139 ꢀC; 1H
179e181 ꢀC; 1H NMR (DMSO‑d6):
d 9.60 (s, 1H, NH), 7.89e7.86 (m,
2H, Ar), 7.71e7.69 (m, 2H, Ar), 7.31e7.18 (m, 2H, Ar), 7.09e7.05 (m,
1H, Ar), 6.90e6.87 (m, 1H, Ar), 3.86 (t, 2H, J ¼ 6.9 Hz, CH2),
3.30e3.25 (m, 1H, CH), 2.56 (t, 2H, J ¼ 8.0 Hz, CH2), 2.11e2.06 (m,
NMR (CDCl3):
3.87 (t, 2H, J ¼ 7.0 Hz, CH2), 2.63 (t, 2H, J ¼ 8.0 Hz, CH2), 2.24e2.16
(m, 2H, CH2); 13C NMR (CDCl3):
175.1, 149.9, 145.0, 133.0, 129.5,
128.6, 124.6, 122.9, 118.9, 48.3, 32.8, 17.7; HRMS (ESI) m/z found,
d 7.86e7.75 (m, 4H, Ar), 7.51 (s,1H, Ar), 7.11 (s, 2H, Ar),
d
2H, CH2), 1.00 (d, 6H, J ¼ 6.7 Hz, CH3); 13C NMR (DMSO‑d6):
d 175.1,
146.3, 143.4, 135.3, 133.6, 128.1, 127.8, 127.7, 126.8, 126.3, 119.1 48.5,
13

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M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
32.9, 27.2, 24.0, 17.7; HRMS (ESI) m/z found, 359.1423; C19H23N2O3S
(Mþ þ H) expected, 359.1429.
118.6, 48.3, 32.9, 17.7; HRMS (ESI) m/z found, 395.0049;
C
16H16BrN2O3S (Mþ þ H) expected, 395.0066.
5.2.8.3. N-([1,10-Biphenyl]-2-yl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (21). Flash chromatography (methylene chloride to
methylene chloride/ethyl acetate (95:5)). Purity: 98.0%; yield: 52%;
5.2.8.9. N-(3-Iodophenyl)-4-(2-oxopyrrolidin-1-yl)benzenesulfona-
mide (27). Flash chromatography (methylene chloride/ethyl ace-
tate (95:5)). Purity: 99.4%; yield: 54%; pale solid; mp: 188e190 ꢀC;
pale yellow solid; mp: 104e106 ꢀC; 1H NMR (DMSO‑d6):
d 9.42 (s,
1H NMR (DMSO‑d6):
d 10.41 (s, 1H, NH), 7.84e7.73 (m, 4H, Ar),
1H, NH), 7.77e7.74 (m, 2H, Ar), 7.57e7.54 (m, 2H, Ar), 7.34e7.21 (m,
8H, Ar), 7.08e7.05 (m, 1H, Ar), 3.83 (t, 2H, J ¼ 6.9 Hz, CH2),
2.55e2.50 (m, 2H, CH2), 2.10e2.02 (m, 2H, CH2); 13C NMR
7.42e7.32 (m, 2H, Ar), 7.13e6.97 (m, 2H, Ar), 3.79 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.48 (t, 2H, J ¼ 7.9 Hz, CH2), 2.05e1.96 (m, 2H, CH2); 13C NMR
(DMSO‑d6):
d 175.2, 143.8, 139.7, 133.6, 132.9, 131.6, 128.1, 128.1,
(DMSO‑d6):
d 175.1, 143.3, 139.0, 139.0, 135.5, 133.8, 131.4, 129.7,
119.2, 119.0, 95.3, 48.3, 32.9, 17.7; HRMS (ESI) m/z found, 442.9908;
C
16H16IN2O3S (Mþ þ H) expected, 442.9927.
128.5, 128.4, 127.8, 127.5, 127.1, 126.9, 119.1, 48.4, 32.9, 17.7; HRMS
(ESI) m/z found, 393.1266; C22H21N2O3S (Mþ þ H) expected,
393.1269.
5.2.8.10. N-(3,4-Dimethoxyphenyl)-4-(2-oxopyrrolidin-1-yl)benze-
nesulfonamide (28). Flash chromatography (methylene chloride/
ethyl acetate (95:5)). Purity: 97.2%; yield: 67%; pale solid; mp:
5.2.8.4. N-(2-Methoxyphenyl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (22). Flash chromatography (methylene chloride to
methylene chloride/ethyl acetate (95:5)). Purity: 100.0%; yield:
184e187 ꢀC; 1H NMR (DMSO‑d6):
d 9.84 (s, 1H, NH), 7.80e7.77 (m,
39%; pale solid; mp: 190e192 ꢀC; 1H NMR (CDCl3):
d 7.74e7.65 (m,
2H, Ar), 7.69e7.66 (m, 2H, Ar), 6.77e6.69 (m, 2H, Ar), 6.54e6.51 (m,
1H, Ar), 3.79 (t, 2H, J ¼ 6.8 Hz, CH2), 3.63e3.62 (m, 6H, CH3), 2.49 (t,
2H, J ¼ 8.0 Hz, CH2), 2.06e1.97 (m, 2H, CH2); 13C NMR (DMSO‑d6):
4H, Ar), 7.51e7.49 (m, 1H, Ar), 7.05e6.98 (m, 2H, Ar and NH),
6.89e6.84 (m, 1H, Ar), 6.73e6.70 (m, 1H, Ar), 3.81 (t, 2H, J ¼ 7.0 Hz,
CH2), 3.64 (s, 3H, CH3), 2.60 (t, 2H, J ¼ 8.0 Hz, CH2), 2.19e2.09 (m,
d
175.1, 149.2, 146.4, 143.4, 134.0, 131.1, 128.1, 119.0, 113.7, 112.4,
2H, CH2); 13C NMR (CDCl3):
d 174.8, 149.4, 143.3, 133.8, 128.2, 125.8,
106.7, 56.0, 55.8, 48.3, 32.9, 17.7; HRMS (ESI) m/z found, 377.1158;
C
18H21N2O5S (Mþ þ H) expected, 377.1172.
125.3, 121.1, 120.9, 118.7, 110.6, 55.7, 48.4, 32.8, 17.7; HRMS (ESI) m/z
found, 347.1058; C17H18N2O4S (Mþ þ H) expected, 347.1066.
5.2.8.11. N-(3,5-Dimethoxyphenyl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (29). Flash chromatography (methylene chloride/ethyl
acetate (95:5)). Purity: 96.6%; yield: 60%; white solid; mp:
5.2.8.5. N-(3-Methoxyphenyl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (23). Flash chromatography (methylene chloride/ethyl
acetate (95:5)). Purity: 99.8%; yield: 62%; brown solid; mp:
177e179 ꢀC; 1H NMR DMSO‑d6):
d 10.23 (s, 1H, NH), 7.82e7.75 (m,
173e175 ꢀC; 1H NMR (DMSO‑d6):
d
10.24 (s, 1H, NH), 7.82e7.73 (m,
4H, Ar), 6.28e6.13 (m, 3H, Ar), 3.76 (t, 2H, J ¼ 6.8 Hz, CH2), 3.62 (s,
4H, Ar), 7.12e7.06 (m, 1H, Ar), 6.67e6.54 (m, 3H, Ar), 3.77 (t, 2H,
J ¼ 6.9 Hz, CH2), 3.63 (s, 3H, CH3), 2.47 (t, 2H, J ¼ 7.8 Hz, CH2),
6H, CH3), 2.47 (t, 2H, J ¼ 7.8 Hz, CH2), 2.02e1.97 (m, 2H, CH2); 13C
NMR (DMSO‑d6): d 175.2, 161.2, 143.6, 140.1, 133.9, 128.2, 119.1, 98.1,
2.02e1.97 (m, 2H, CH2); 13C NMR (DMSO‑d6):
d
175.2, 160.1, 143.6,
95.5, 55.5, 48.3, 32.8, 17.7; HRMS (ESI) m/z found, 377.1154;
139.5, 134.0, 130.4, 128.1, 119.1, 112.2, 109.3, 106.0, 55.4, 48.3, 32.8,
17.7; HRMS (ESI) m/z found, 347.1057; C17H19N2O4S (Mþ þ H) ex-
pected, 347.1066.
C
18H21N2O5S (Mþ þ H) expected, 377.1172.
5.2.8.12. N-(3,5-Dibromophenyl)-4-(2-oxoimidazolidin-1-yl)benze-
nesulfonamide (30). Flash chromatography (methylene chloride/
ethyl acetate (95:5)). Purity: 96.9%; yield: 18%; white solid; mp:
5.2.8.6. N-(3-Fluorophenyl)-4-(2-oxopyrrolidin-1-yl)benzenesulfo-
namide (24). Flash chromatography (hexanes/ethyl acetate
(63:37)). Purity: 99.6%; yield: 39%; white solid; mp: 206e208 ꢀC;
244e246 ꢀC; 1H NMR (DMSO‑d6):
d 10.77 (s, 1H, NH), 7.88e7.85 (m,
2H, Ar), 7.79e7.76 (m, 2H, Ar), 7.44 (s, 1H, Ar), 7.25 (s, 2H, Ar), 3.82
(t, 2H, J ¼ 6.9 Hz, CH2), 2.50e2.48 (m, 2H, CH2), 2.07e1.98 (m, 2H,
1H NMR (DMSO‑d6):
d 10.53 (s, 1H, NH), 7.84e7.75 (m, 4H, Ar),
CH2); 13C NMR (DMSO‑d6):
d 175.3, 144.0, 141.2, 133.1, 128.8, 128.1,
7.27e7.20 (m, 1H, Ar), 6.91e6.78 (m, 3H, Ar), 3.79 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.48 (t, 2H, J ¼ 7.9 Hz CH2), 2.05e1.95 (m, 2H, CH2); 13C NMR
123.2, 120.8, 119.3, 48.3, 32.9, 17.7; HRMS (ESI) m/z found, 472.9156;
C
16H15Br2N2O3S (Mþ þ H) expected, 472.9169.
(DMSO‑d6):
d 175.2, 164.3, 161.1, 143.8, 140.2, 140.1, 133.6, 131.5,
131.3, 128.1, 119.2, 115.7, 115.6, 110.9, 110.7, 106.7, 106.4, 48.3, 32.9,
17.7; HRMS (ESI) m/z found, 335.0859; C16H16FN2O3S (Mþ þ H)
expected, 335.0866.
5.2.8.13. 4-(2-Oxopyrrolidin-1-yl)-N-(3,4,5-trimethoxyphenyl)ben-
zenesulfonamide (31). Flash chromatography (methylene chloride/
ethyl acetate (95:5)). Purity: 97.1%; yield: 47%; pale solid; mp:
5.2.8.7. N-(3-Chlorophenyl)-4-(2-oxopyrrolidin-1-yl)benzenesulfo-
namide (25). Flash chromatography (methylene chloride/ethyl ac-
etate (95:5)). Purity: 99.7%; yield: 43%; white solid; mp:
199e201 ꢀC; 1H NMR (DMSO‑d6):
d 10.07 (s, 1H, NH), 7.83e7.75 (m,
4H, Ar), 6.39 (s, 2H, Ar), 3.78 (t, 2H, J ¼ 6.7 Hz, CH2), 3.64 (s, 6H,
187e189 ꢀC; 1H NMR (DMSO‑d6):
d 10.52 (s, 1H, NH), 7.84e7.74 (m,
CH3), 3.53 (s, 3H, CH3), 2.48 (t, 2H, J ¼ 7.8 Hz, CH2), 2.03e1.98 (m,
2H, CH2); 13C NMR (DMSO‑d6):
d 175.2, 153.4, 143.6, 134.4, 134.2,
4H, Ar), 7.26e7.20 (m, 1H, Ar), 7.10e7.03 (m, 3H, Ar), 3.81 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.48 (t, 2H, J ¼ 7.9 Hz, CH2), 2.06e1.98 (m, 2H, CH2);
133.9, 128.3, 119.1, 98.0, 60.5, 56.2, 48.3, 32.8, 17.7; HRMS (ESI) m/z
found, 407.1269; C19H23N2O6S (Mþ þ H) expected, 407.1278.
13C NMR (DMSO‑d6):
d 175.2, 143.8, 139.9, 133.8, 133.5, 131.4, 128.1,
124.1, 119.3, 119.2, 118.3, 48.3, 32.9, 17.7; HRMS (ESI) m/z found,
351.0561; C16H16ClN2O3S (Mþ þ H) expected, 351.0571.
5.2.8.14. N-(4-Methoxyphenyl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (32). Flash chromatography (methylene chloride/ethyl
acetate (95:5)). Purity: 97.1%; yield: 18%; pale brown solid; mp:
5.2.8.8. N-(3-Bromophenyl)-4-(2-oxopyrrolidin-1-yl)benzenesulfo-
namide (26). Flash chromatography (methylene chloride/ethyl ac-
etate (95:5)). Purity: 100.0%; yield: 56%; pale solid; mp:
206e209 ꢀC; 1H NMR (DMSO‑d6):
d 9.83 (s, 1H, NH), 7.80e7.62 (m,
4H, Ar), 6.97e6.76 (m, 4H, Ar), 3.80 (t, 2H, J ¼ 6.9 Hz, CH2), 3.64 (s,
195e198 ꢀC; 1H NMR (DMSO‑d6):
d 10.50 (s, 1H, NH), 7.84e7.74 (m,
3H, CH3), 2.52e2.47 (m, 2H, CH2), 2.07e2.00 (m, 2H, CH2); 13C NMR
4H, Ar), 7.24e7.09 (m, 4H, Ar), 3.79 (t, 2H, J ¼ 6.9 Hz, CH2), 2.48 (t,
(DMSO‑d6): d 175.2, 156.9, 143.4, 134.0, 130.7, 128.1, 123.8, 119.0,
2H, J ¼ 7.9 Hz, CH2), 2.06e1.98 (m, 2H, CH2); 13C NMR (DMSO‑d6):
114.7, 55.6, 48.4, 32.9, 17.7; HRMS (ESI) m/z found, 347.1061;
d
175.2, 143.8, 140.0, 133.5, 131.7, 128.1, 127.0, 122.2, 122.2, 119.2,
C
17H19N2O4S (Mþ þ H) expected, 347.1066.
14

ꢀ
M. Gagne-Boulet, C. Bouzriba, A.C. Chavez Alvarez et al.
European Journal of Medicinal Chemistry 213 (2021) 113136
5.2.8.15. N-([1,10-biphenyl]-4-yl)-4-(2-oxopyrrolidin-1-yl)benzene-
sulfonamide (33). Flash chromatography (methylene chloride/ethyl
acetate (95:5) to (85:15)). Purity: 98.2%; yield: 54%; white solid;
respectively. A.C. Chavez Alvarez is recipient of studentships from
Fonds d’enseignement et de recherche of the Faculty of pharmacy
ꢀ
of Universite Laval.
mp: 177e179 ꢀC; 1H NMR (DMSO‑d6):
d 10.35, (s, 1H, NH), 7.83e7.76
(m, 4H, Ar), 7.56e7.51 (m, 4H, Ar), 7.41e7.26 (m, 3H, Ar), 7.19e7.16
(m, 2H, Ar), 3.78 (t, 2H, J ¼ 6.9 Hz, CH2), 2.50e2.47 (m, 2H, CH2),
Appendix B. Supplementary data
2.05e1.95 (m, 2H, CH2); 13C NMR (DMSO‑d6):
d 175.2, 143.6, 139.7,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113136.
137.7, 136.0, 134.1, 129.3, 128.1, 127.8, 127.6, 126.7, 120.5, 119.2, 48.3,
32.9, 17.7; HRMS (ESI) m/z found, 393.1256; C22H21N2O3S (Mþ þ H)
expected, 393.1269.
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Declaration of competing interest
[17] M. Zarifi Khosroshahi, A.C. Chavez Alvarez, M. Gagne-Boulet, R.C. Gaudreault,
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versite Laval and Fonds de recherche du Quebec-Sante (FRQS),
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16