4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2018.09.001

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designa

Full text of DOI:10.1016/j.bmc.2018.09.001

Accepted Manuscript
4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new an-
timitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells
Atziri Corin Chavez Alvarez, Mitra Zarifi Khosroshahi, Marie-France Côté,
Mathieu Gagné-Boulet, Sébastien Fortin
PII:
S0968-0896(18)31159-3
https://doi.org/10.1016/j.bmc.2018.09.001
BMC 14526
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2018
24 August 2018
2 September 2018
Please cite this article as: Alvarez, A.C.C., Khosroshahi, M.Z., Côté, M-F., Gagné-Boulet, M., Fortin, S., 4-(3-
Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by
cytochrome P450 1A1 in breast cancer cells, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.09.001
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4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs
activated by cytochrome P450 1A1 in breast cancer cells
Atziri Corin Chavez Alvarez, Mitra Zarifi Khosroshahi, Marie-France Côté, Mathieu Gagné-Boulet
and Sébastien Fortin*
CHU de Québec-Université Laval Research Center, Oncology Division, Hôpital Saint-François d'Assise,
10 rue de l'Espinay, Quebec City, QC, G1L 3L5, Canada.
Faculty of Pharmacy, Laval University, Quebec City, QC, G1V 0A6, Canada.
*Corresponding author: Sébastien Fortin, Phone: 418-525-4444 ext. 52364, Fax: 418-525-4372, e-
mail: sebastien.fortin@pha.ulaval.ca.
Declarations of interest: none
Abbreviations: phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates, PAIB-SOs; cytochromes
P450, CYPs; 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides, PAIB-SAs; phenyl 4-
(2-oxoimidazolidin-1-yl)benzenesulfonates,
yl)benzenesulfonamides, PIB-SAs.
PIB-SOs;
phenyl
4-(2-oxoimidazolidin-1-

ABSTRACT
The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-
yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to
new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-
1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low
micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells
devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent PAIB-SA 13
bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts
the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble
counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-
SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-
expressing cells into potent antimitotics.
Keywords: Antimicrotubule agents; Antimitotics; CYP1A1-activated prodrugs; 4-(3-Alkyl-2-
oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides; PAIB-SAs

1. Introduction
Breast cancer accounts for 25% of all women cancer cases and 15% of all women cancer deaths, making
it by far the most common cancer and the leading cause of cancer death among women worldwide.1
According to the American Cancer Society, it is estimated in the USA in 2018 that 266,120 new cases
of invasive breast cancer will be diagnosed in women and 41,400 breast cancer deaths will occur.2
Despite advances in the treatment of early-stage disease, approximately 40% of patients will experience
recurrence and develop resistance to antihormone therapy and anticancer agents.3, 4 This results in more
aggressive breast cancer, more difficult to treat and it becomes life threatening.5 Therefore, the 5-year
relative survival rate for women diagnosed with localized breast cancer is 98.6% and it declines to 23.3%
for cancers at distant stages.6 Consequently, breast cancer is among the most pressing health problems
worldwide and for that reason new therapeutic options and strategies are intensively sought.
Cytochromes P450 (CYPs) are a large superfamily of heme-containing monooxygenase enzymes. In
humans, there are 18 families and 43 subfamilies of CYPs encoded by 57 genes.7 CYPs metabolize a
large number of structurally different endogenous and exogenous compounds such as steroids, fatty
acids, prostaglandins and plant metabolites. They also metabolize chemical carcinogens, mutagens and
other environmental contaminants.8 They are the main enzymes involved in drug metabolism accounting
for the metabolism of approximately 75% of marketed drugs.9 CYPs are ubiquitous but are
predominantly expressed in the liver and to a lesser extent in the small intestine, lungs, placenta and
kidneys.10 Some CYPs such as CYP3A4, 2D6, 2C19 and 2C9 are not specific metabolizing a wide variety
of compounds while others such as CYP17A1 and 19A1 are more specific and are expressed in specific
tissues such as ovarian follicles as well as in fetal tissues.11 Therefore, the expression level of CYP varies
according to their roles and tissues in which they are expressed.12, 13 There is also evidence that some
CYPs are differentially expressed in tumors cells versus their healthy counterparts.14-16 CYP isoforms
such as CYP1A1 are expressed in tumors and represent valuable therapeutic targets that may influence

tumor response to current antineoplastics.15-17 To that end, that concept has been applied to benzothiazole
and aminoflavone exemplified by Phortress (1) and NSC710464 (2) that are prodrugs of alkylating agents
activated by CYP1A1 (Fig. 1).18, 19
Figure 1. Molecular structure of CYP1A1-activated benzothiazole Phortress (1) and aminoflavone
NSC710464 (2).
CYP1A1 is one of the three members of the CYP1 family (CYP1A1, 1A2 and 1B1). In normal conditions,
CYP1A1 is predominantly detected in extrahepatic tissues at a very low level but it can be induced
significantly by several xenobiotics under the control of the ligand-activated aryl hydrocarbon receptor.20-
22 Studies have shown that most breast tumors express CYP1A1 but CYP1A1 seems to be detected only
at a low level in the normal surrounding tissues.23-26 Moreover, CYP1A1 is involved in the regulation of
breast cancer cell proliferation and survival and correlates positively with the tumor grade.26 In this
context, the use of CYP1A1 as a druggable target to design anticancer prodrugs represents a promising
and attractive strategy to develop new targeted therapy for breast cancer treatment.
To this end, we recently developed a new family of anticancer agents designated as phenyl 4-(2-oxo-3-
alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs, Fig. 2) represented by the prototypical 3,4,5-
trimethoxyphenyl 4-(3-butyl-2-oxoimidazolidin-1-yl)-benzenesulfonate (CEU-818) that are highly
selective toward several breast cancer cell lines notably MCF7 cells.27 They block the cell cycle
progression in the G2/M phase and disrupt the microtubule network of sensitive breast cancer cells.
PAIB-SOs were rationally designed as CYP1A1-activated prodrugs of antimitotics designated as phenyl

4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs, Fig. 2).28 To the best of our knowledge, PAIB-
SOs are the first antimitotic prodrugs to be reported that are activated by CYP1A1. On the one hand, our
structure-activity relationship studies showed that PAIB-SOs must be substituted by a propyl or butyl
group on the phenylimidazolidinone moiety of the aromatic ring A as well as by methoxyl or halogen
group at positions 3, 4, and/or 5 of the aromatic ring B to maintain both antiproliferative activity and
selectivity toward breast cancer cells.27 On the other hand, we found that the sulfonate group of PIB-SOs,
the cytotoxic antimitotic metabolites of PAIB-SOs, retains its cytocidal and antimicrotubule activities
when replaced by a sulfonamide group. This led to antimitotic PIB-SO analogs designed as phenyl 4-(2-
oxoimidazolidin-1-yl)benzenesulfonamides (PIB-SAs, Fig. 2) represented by the prototypical 4-(2-
oxoimidazolidin-1-yl)-N-(3,4,5-trimethoxyphenyl)benzenesulfonamide (CEU-638).29
Figure 2. Molecular structure of CYP1A1-activated prodrugs phenyl 4-(2-oxo-3-alkylimidazolidin-1-
yl)benzenesulfonates
(PAIB-SOs)
and
4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-
phenylbenzenesulfonamides (PAIB-SAs) as well as antimitotics phenyl 4-(2-oxoimidazolidin-1-

yl)benzenesulfonates (PIB-SOs) and phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB-
SAs).
Herein, we studied the conversion of the sulfonate moiety of PAIB-SOs into its bioisosteric sulfonamide
equivalent on the cytocidal activity and the selectivity toward CYP1A1-expressing MCF7 breast cancer
cells. To that end, a series of 11 PAIB-SAs was designed and prepared where the sulfonate moiety of
PAIB-SOs was replaced by a sulfonamide group. Thereafter, the antiproliferative activity of PAIB-SAs
was assessed on sensitive CYP1A1-expressing MCF7 breast cancer cells and two insensitive cell lines
devoid of CYP1A1; one human breast cancer cell line (MDA-MB-231 carcinoma) and one healthy
primary cell line (HaCaT, epidermal keratinocytes). Then, the most potent and selective compound was
assessed for its ability to block the cell cycle progression in the G2/M phase, to disrupt the microtubule
network and to be activated into its antimitotic counterpart by CYP1A1.
2. Chemistry
The method used for the preparation of PAIB-SAs (3-13) is described in Scheme 1. Compound 16 was
prepared as published previously.27 Briefly, the synthesis of 1-phenylimidazolidin-2-one (14) was
achieved by the nucleophilic addition of aniline to 2-chloroethyl isocyanate in methylene chloride at
25 °C followed by the intramolecular cyclization of the 1-(2-chloroethyl)-3-phenylurea using sodium
hydride in THF at 25 °C. Thereafter, 1-butyl-3-phenylimidazolidin-2-one (15) was obtained by the
addition of 1-iodobutane to a solution of 14 and sodium hydride in tetrahydrofuran. Then, 4-(3-butyl-2-
oxoimidazolidin-1-yl)benzenesulfonyl chloride (16) was prepared by the chlorosulfonation of compound
15 using chlorosulfonic acid in carbon tetrachloride at 0 °C. Finally, PAIB-SAs 3-13 were obtained by
nucleophilic addition of either 2-methoxyaniline, 3-methoxyaniline, 3-fluoroaniline, 3-bromoaniline, 3-
iodoaniline, 3,4-dimethoxyaniline, 3,4-difluoroaniline, 3,5-dimethoxyaniline, 3,5-difluoroaniline, 3,5-

dichloroaniline or 3,4,5-trimethoxyaniline to sulfonyl chloride 16 in the presence of 4-
dimethylaminopyridine in dry acetonitrile at 25 °C.
Scheme 1. Reagent conditions: i) NaH, 1-iodobutane, THF, 0 °C to R.T.; ii) ClSO3H, CCl4, 0 °C; iii)
relevant aniline, DMAP/CH3CN, R.T.
3. Results and discussion
Our previous study showed that MCF7 breast cancer cells are expressing CYP1A1 and are sensitive to
PAIB-SOs while MDA-MB-231 breast cancer cells are devoid of CYP1A1 activity and are insensitive
to PAIB-SOs.27 In this context, the antiproliferative activity of PAIB-SAs was assessed on sensitive
MCF7 cells, on insensitive MDA-MB-231 cells and on healthy primary HaCaT epidermal keratinocyte
cells in the aim to confirm that PAIB-SAs act similarly to PAIB-SOs and that they are not cytotoxic on
healthy cells. Antiproliferative activity was assessed according to the NCI/NIH Developmental
Therapeutics Program.30 The PIB-SA bearing a 3,4,5-trimethoxyl group on ring B (CEU-638) and
combretastatin A-4 (CA-4) were used as positive controls. The results are summarized in Table 1 and
they are expressed as the concentration of drug inhibiting cell growth by 50% (IC50). The structure-

activity relationships related to PAIB-SAs show a similar trend to those obtained with PAIB-SOs. First,
except for compound 12, all PAIB-SAs substituted by methoxyl and halogen groups at positions 2, 3, 4,
and/or 5 of the aromatic ring B exhibit an antiproliferative activity in the submicromolar to low
micromolar range on MCF7 cells (0.25 to 3.1 M) and show a selectivity ratio between > 5.3 and 372
(HaCaT/MCF7 cells). Compounds 6, 8 and 13 bearing either a 3-bromo, 3,4-dimethoxyl or a 3,4,5-
trimethoxyl group on the aromatic ring B are the most potent and selective compounds showing
antiproliferative activity between 0.25 and 0.68 M and displaying selectivity ratio between > 147 and
372 (HaCaT/MCF7 cells). Interestingly, the antiproliferative activity difference between PAIB-SOs and
PAIB-SAs is in the same order of magnitude as the difference between PIB-SOs and PIB-SAs.27-29
Compound 13 bearing a 3,4,5-trimethoxyl group is the most potent and selective on MCF7 cells and it
was selected for further studies to confirm its mechanism of action.

Table 1. Antiproliferative activity (IC50) of PIB-SAs (3-13), CEU-818, CEU-638 and CA-4 on MCF7,
MDA-MB-231 and HaCaT cells as well as their selectivity ratio toward sensitive MCF7 breast cancer
cells compared to primary HaCaT epidermal keratinocytes.
Selectivity Ratio2
HaCaT/
MCF7
> 63
IC50 (M)1
#
MCF7
MDA-MB-231
HaCaT
1.6
1.1
> 100
> 10
> 50
83
> 100
> 100
> 10
> 100
> 50
> 10
27
> 100
> 10
> 50
51
3
4
5
6
7
8
9
10
11
> 9.1
> 64
182
0.78
0.28
0.54
0.68
1.9
21
39
> 100
> 10
58
> 50
> 10
93
0.019
> 10
0.0014
> 147
> 5.3
22
> 16
N.A.
372
0.86
> 1800
0.67
2.6
3.1
> 10
0.25
0.022
0.0055
0.0021
12
13
0.038
6.0
0.0024
CEU-638
CEU-81827
CA-43
1IC50 is expressed as the concentration of drug inhibiting cell proliferation by 50% after 72 h of treatment.
When 100 M is not attained, the maximum concentration assessed represents the maximum solubility
2
concentration reached in complete medium containing DMSO 0.5%. Selectivity ratio is expressed as
the ratio of the IC50 value found in primary HaCaT cells to that measured in sensitive MCF7 cells. N.A.,
not applicable. 3CA-4, combretastatin A-4.
The lag time to the onset of antiproliferative action on cancer cells is an important parameter to be
assessed prior to in vivo assays. The time required by PAIB-SA 13 to induce its antiproliferative activity
was thus evaluated on MCF7 cells. The PIB-SA antimitotic counterpart CEU-638 was used as positive
control. As shown in Fig. 3, the antiproliferative activity of compound 13 at high concentrations is similar
either at 24, 36 or 48 h to CEU-638. Our results confirm that the antiproliferative activity is time-
dependent. The incubation of the drug with the cells for at least 24h is required to affect cell proliferation.

Figure 3. Dose-dependent antiproliferative activity of a) compound 13 and b) its parental antimitotic
drug CEU-638 at 24, 36 and 48 h on MCF7 cells.
On one hand, it is known that PAIB-SOs that are the bioisosteric equivalents of PAIB-SAs and that they
are activated in CYP1A1-expressing cells into the highly potent antimitotics PIB-SOs.27, 28 On the other
hand, PIB-SAs are also known to exhibit potent antimicrotubule effects on a large number of cancer cell
lines.29 Therefore, we hypothesized that PAIB-SAs are activated into PIB-SAs by CYP1A1 relevant
cancer cells leading to G2/M phase arrest and disruption of the microtubule dynamics that triggers
anoikis. To confirm our hypothesis, PAIB-SA 13 was assessed for its effect on cell cycle progression
and cytoskeleton integrity in sensitive MCF7 cells. As shown in Fig. 4, MCF7 cells treated with 0.5%
DMSO were found in subG1, G0/G1, S, and G2/M phases at 2.9, 69, 15.1 and 13%, respectively.
Compound 13 strongly blocks the cell cycle progression in G2/M phase increasing the number of cells
in G2/M phase by 17.2 and 29.4% at 0.20 and 0.50 M, respectively which is similar to that obtained for
CEU-638 and CA-4 used as positive controls where the number of cells in G2/M phase was increased by
33.7 and 32.9%, respectively. As shown in Fig. 5, the treatment of sensitive MCF7 cells by PAIB-SA 13
at 0.20 M for 24 h leads to the disruption of the cytoskeleton equivalently to CEU-638 and CA-4 used
as positive controls.

Figure 4. Effect on the cell cycle progression on MCF7 cells after a 24 h treatment with a) PAIB-SA 13
(0.20 M), b) PAIB-SA 13 (0.50 M), c) CEU-638 (0.010 M), d) CA-4 (0.0010 M) and e) DMSO
(0.5%).
Figure 5. Effect of a) PAIB-SA 13 (0.20 M), b) CEU-638 (0.010 M), c) CA-4 (0.0010 M) and d)
DMSO 0.5% on cytoskeleton integrity in MCF7 cells after 24 h of treatment.
To confirm that the PAIB-SAs exhibit the same mechanism of activation observed for PAIB-SOs and
that they are CYP1A1-activated prodrugs, 1.2 M of PAIB-SA 13 was incubated in presence of CYP1A1

SupersomesTM for 0, 15, 30, 60, 90, 120 and 150 min, respectively. Afterward, the metabolites were
identified by HPLC-UV. CEU-638, the parental antimitotic drug of 13 was used as internal standard. As
shown in Fig. 6, CYP1A1 SupersomesTM activated PAIB-SA 13 into its parental antimitotic CEU-638.
Altogether, these results confirm the bioisosterism of sulfonate and sulfonamide groups and that PAIB-
SAs are a new class of CYP1A1-activated prodrugs exhibiting a similar mechanism of activation that
their PAIB-SOs structural analogs.

Figure 6. The time-course activation of PAIB-SA 13 into PIB-SA CEU-638 by CYP1A1.
4. Conclusion
In summary, we have shown that the newly designed PAIB-SAs are active in the submicromolar to low
micromolar range and selective toward MCF7 breast cancer cells comparatively to MDA-MB-231 and
HaCaT cells. Using the most potent PAIB-SA 13, we found that it blocks the cell cycle progression in

the G2/M phase and disrupts the microtubule dynamics efficiently. Finally, PAIB-SAs are activated by
CYP1A1 confirming that 1) PAIB-SAs are CYP1A1-activated antimitotic prodrugs and 2) that sulfonate
and sulfonamide groups are bioisosteric equivalents. Altogether, these results show that the structural
bridge between the two aromatic rings might be investigated to improve the pharmacological and the
physicochemical properties of this new class of promising antimitotic CYP1A1-activated prodrugs.
5. Experimental section
5.1. Chemistry
5.1.1. General
Proton NMR spectra were recorded on a Bruker AM-300 spectrometer (Bruker, Germany). Chemical
shifts (δ) are reported in parts per million (ppm). Uncorrected melting points were determined on an
electrothermal melting point apparatus. HPLC analyses were performed using a Prominence LCMS-2020
system with binary solvent equipped with an UV/vis photodiode array and an APCI probe (Shimadzu,
Columbia, MD). Compounds were eluted within 25 min on an Alltech Alltima C18 reversed-phase
column (5 mm, 250 mm x 4.6 mm) equipped with an Alltech Alltima C18 precolumn (5 mm, 7.5 mm x
4.6 mm) with a MeOH/H2O linear gradient at 1.0 mL/min. Some HPLC analyses were also performed
using an ACQUITY Arc system (Waters, Mississauga, Ontario). The purity of all final compounds was
>95%. HRMS were recorded by direct injection in a TOF system 6210 series mass spectrometer (Agilent
technologies, Santa Clara, CA). All chemicals were supplied by Aldrich Chemicals (Milwaukee, WI),
Fisher Scientific (Toronto, ON, Canada) or VWR International (Mont-Royal, QC, Canada) and used as
received unless specified otherwise. Liquid flash chromatography was performed on silica gel F60, 60
Å, 40-63 μm supplied by Silicycle (Québec, QC, Canada) using an FPX flash purification system
(Biotage, Charlottesville, VA), and using solvent mixtures expressed as v/v ratios. Solvents and reagents
were used without purification unless specified otherwise. The progress of all reactions was monitored

by TLC on precoated silica gel 60 F254 TLC plates (VWR). The chromatograms were viewed under UV
light at 254 and/or 265 nm.
5.1.2. General procedure for preparation of compounds 14-16
1-Phenylimidazolidin-2-one (14) was obtained by nucleophilic addition of aniline on 2-chloroethyl
isocyanate followed by an intramolecular cyclization using sodium hydride as described previously by
Fortin et al.28, 29 Briefly, 2-chloroethyl isocyanate (1.2 eq.) was added dropwise to a cold solution (ice
bath) of aniline (1.0 eq.) in dry methylene chloride (15 mL per g of aniline). The reaction mixture was
stirred at room temperature for 24 h and the solvent was evaporated under reduced pressure. The white
solid obtained was triturated twice in cold hexanes/methylene chloride 10:1. Thereafter, 1-(2-
chloroethyl)-3-phenylurea (1 eq.) was dissolved in dry tetrahydrofuran under dry nitrogen atmosphere
and the solution is cooled to 0 °C. Then, sodium hydride (3 eq.) was slowly added, the ice bath was
removed after 30 min and the reaction mixture was stirred at room temperature for 5 h. The reaction
mixture was quenched at 0 °C with water and diluted with ethyl acetate. The organic layer was washed
with water and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to
provide 1-phenylimidazolidin-2-one (14), which was used without further purification. 4-(3-Butyl-2-
oxoimidazolidin-1-yl)benzenesulfonyl chloride (16) was synthesized using the method described by
Fortin et al.27. Briefly, sodium hydride (1 eq.) was added slowly to a cold solution (ice bath) of 14 (30
mmol) in dry tetrahydrofuran. The ice bath was removed after 30 min. Thirty-six mmol of 1-iodobutane
were then added slowly and the reaction mixture stirred at room temperature for 20 h. The reaction was
quenched at 0 °C with water and the mixture diluted with EtOAc. The organic layer was washed with
water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue
was purified by flash chromatography CH2Cl2 to CH2Cl2/EtOAc (9:1) to give an off-white solid.
Afterwards, 1-butyl-3-phenylimidazolidin-2-one (15) was added slowly to chlorosulfonic acid (23.1
mmol) in CCl4 (5 mL) at 0 °C for 2 h. Then, the reaction mixture was poured slowly into ice-water and

then filtered to collect the solid thus formed. The latter solid was dried overnight under reduced pressure
to provide 4-(3-butyl-2-oxoimidazolidin-1-yl)benzenesulfonyl chloride (16) as a white solid.
5.1.3. Preparation and characterization of compounds 3-13
Compound 16 (1.00 mmol) was suspended in dry acetonitrile (10 mL) under nitrogen atmosphere. Either
2-methoxyaniline, 3-methoxyaniline, 3-fluoroaniline, 3-bromoaniline, 3-iodoaniline, 3,4-
dimethoxyaniline, 3,4-difluoroaniline, 3,5-dimethoxyaniline, 3,5-difluoroaniline, 3,5-dichloroaniline or
3,4,5-trimethoxyaniline (1.00 mmol) and 4-dimethylaminopyridine (4.00 mmol) were successively
added dropwise to the suspension of 16. The mixture was stirred at room temperature for 48 h and then
diluted with ethyl acetate. The resulting solution was washed with hydrochloric acid 1N, brine, dried
over sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The white solid was
purified by flash chromatography on silica gel.
5.1.2.2. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(2-methoxyphenyl)benzenesulfonamide (3). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 67%; white solid; mp: 141-143 °C. H NMR
(CDCl3): δ 7.67-7.64 (m, 2H, Ar), 7.56-7.53 (m, 2H, Ar), 7.48 (d, 1H, J = 7.7 Hz, Ar), 7.01 (s, 1H, NH),
6.98-6.96 (m, 1H, Ar), 6.85 (t, 1H, J = 7.7 Hz, Ar), 6.71-6.68 (m, 1H, Ar), 3.76-3.71 (m, 2H, CH2), 3.63
(s, 3H, CH3), 3.49-3.43 (m, 2H, CH2), 3.26 (t, 2H, J = 7.3 Hz, CH2), 1.55-1.46 (m, 2H, CH2), 1.39-1.29
13
(m, 2H, CH2), 0.92 (t, 3H, J = 7.3 Hz, CH3). C NMR (CDCl3): δ 157.0, 149.4, 144.6, 131.2, 128.3,
126.0, 125.2, 121.0, 120.8, 116.0, 110.6, 55.7, 43.6, 42.1, 41.3, 29.4, 19.9, 13.7. MS (APSI+) m/z found,
404.25; HRMS (ESI) m/z found, 404.1640; C20H26N3O4S (M+ + H) expected, 404.1645.
5.1.2.3. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3-methoxyphenyl)benzenesulfonamide (4). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 73%; white solid; mp: 165-167 °C. H NMR
(CDCl3): δ 7.71-7.68 (m, 3H, Ar and NH), 7.58-7.55 (m, 2H, Ar), 7.08-7.03 (m, 1H, Ar), 6.73 (brs, 1H,
Ar), 6.66-6.58 (m, 2H, Ar), 3.79-3.74 (m, 2H, CH2), 3.68 (s, 3H, CH3), 3.51-3.46 (m, 2H, CH2), 3.28 (t,

2H, J = 7.2 Hz, CH2), 1.57-1.47 (m, 2H, CH2), 1.38-1.24 (m, 2H, CH2), 0.90 (t, 3H, J = 7.2 Hz, CH3).
13C NMR (CDCl3): δ 160.2, 157.2, 144.5, 138.3, 131.4, 129.9, 128.4, 116.1, 113.5, 110.7, 107.0, 55.2,
43.7, 42.1, 41.4, 29.4, 19.9, 13.7. MS (APSI+) m/z found, 404.20; HRMS (ESI) m/z found, 404.1640;
C20H26N3O4S (M+ + H) expected, 404.1645.
5.1.2.4.
4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3-fluorophenyl)benzenesulfonamide
(5).
Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 61%; white solid; mp: 178-180 °C. H NMR
(CDCl3 and MeOD): δ 8.35 (s, 1H, NH), 7.70-7.67 (m, 2H, Ar), 7.57-7.54 (m, 2H, Ar), 7.14-7.06 (m,
1H, Ar), 6.90-6.80 (m, 2H, Ar), 6.73-6.67 (m, 1H, Ar), 3.80-3.75 (m, 2H, CH2), 3.51-3.46 (m, 2H, CH2),
3.26 (t, 2H, J = 7.2 Hz, CH2), 1.55-1.46 (m, 2H, CH2), 1.37-1.25 (m, 2H, CH2), 0.89 (t, 3H, J = 7.2 Hz,
13
CH3). C NMR (CDCl3 and MeOD): δ 164.6, 161.3, 157.2, 144.6, 131.2, 130.4, 130.3, 128.3, 116.3,
116.3, 116.2, 116.1, 111.5, 111.2, 108.1, 107.8, 43.6, 42.1, 41.3, 29.3, 19.9, 13.7. MS (APSI+) m/z found,
392.20; HRMS (ESI) m/z found, 392.1442; C19H23FN3O3S (M+ + H) expected, 392.1445.
5.1.2.6. N-(3-Bromophenyl)-4-(3-butyl-2-oxoimidazolidin-1-yl)benzenesulfonamide (6). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 49%; white solid; mp: 184-186 °C. H NMR
(CDCl3 and MeOD): δ 7.65-7.63 (m, 2H, Ar), 7.54-7.51 (m, 2H, Ar), 7.19 (brs, 1H, Ar), 7.10-7.07 (m,
1H, Ar), 6.98-6.97 (m, 2H, Ar), 3.78-3.72 (m, 2H, CH2), 3.47-3.42 (m, 2H, CH2), 3.21 (t, 2H, J = 7.2 Hz,
CH2), 1.52-1.42 (m, 2H, CH2), 1.34-1.22 (m, 2H, CH2), 0.87 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (CDCl3
and MeOD): δ 157.2, 144.5, 138.8, 131.3, 130.4, 128.1, 127.3, 123.4, 122.5, 119.0, 116.4, 43.5, 42.1,
41.3, 29.3, 19.8, 13.6. MS (APSI+) m/z found 452.15; HRMS (ESI) m/z found, 452.0634;
C19H23BrN3O3S (M+ + H) expected, 452.0644.
5.1.2.7.
4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3-iodophenyl)benzenesulfonamide
(7).
Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 44%; white solid; mp: 202-204 °C. H NMR
(CDCl3 and MeOD): δ 7.66-7.63 (m, 2H, Ar), 7.56-7.53 (m, 2H, Ar), 7.38 (brs, 1H, Ar), 7.31-7.29 (m,

1H, Ar), 7.04-7.01 (m, 1H, Ar), 6.88-6.83 (m, 1H, Ar), 3.79-3.74 (m, 2H, CH2), 3.49-3.44 (m, 2H, CH2),
3.23 (t, 2H, J = 7.2 Hz, CH2), 1.53-1.43 (m, 2H, CH2), 1.35-1.23 (m, 2H, CH2), 0.88 (t, 3H, J = 7.2 Hz,
13
CH3). C NMR (CDCl3 and MeOD): δ 157.2, 144.5, 133.4, 131.3, 130.5, 129.6, 129.3, 128.2, 119.7,
116.4, 94.1, 43.6, 42.1, 41.3, 29.3, 19.9, 13.6. MS (APSI+) m/z found 500.10; HRMS (ESI) m/z found,
500.0499; C19H23IN3O3S (M+ + H) expected, 500.0506.
5.1.2.8. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,4-dimethoxyphenyl)benzenesulfonamide (8). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 84%; white solid; mp: 130-132 °C. H NMR
(CDCl3): δ 7.67 (s, 1H, NH), 7.64-7.61 (m, 2H, Ar), 7.57-7.54 (m, 2H, Ar), 6.76-6.75 (m, 1H, Ar), 6.64-
6.61 (m, 1H, Ar), 6.56-6.52 (m, 1H, Ar), 3.79-3.72 (m, 8H, CH2 and 2x CH3), 3.51-3.46 (m, 2H, CH2),
3.30-3.25 (m, 2H, CH2), 1.55-1.46 (m, 2H, CH2), 1.37-1.25 (m, 2H, CH2), 0.89 (t, 3H, J = 7.3 Hz, CH3).
13C NMR (CDCl3): δ 157.3, 149.0, 147.0, 144.3, 131.3, 130.1, 128.5, 116.0, 115.4, 111.2, 107.8, 55.9,
55.9, 43.7, 42.1, 41.4, 29.3, 19.9, 13.7. MS (APSI+) m/z found 434.20; HRMS (ESI) m/z found,
434.1744; C21H28N3O5S (M+ + H) expected, 434.1750.
5.1.2.9. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,4-difluorophenyl)benzenesulfonamide (9). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 51%; white solid; mp: 204-206 °C. H NMR
(CDCl3 and MeOD): δ 7.71-7.45 (m, 5H, Ar and NH), 6.92-6.81 (m, 2H, Ar), 6.72-6.65 (m, 1H, Ar),
3.73-3.68 (m, 2H, CH2), 3.47-3.41 (m, 2H, CH2), 3.19-3.17 (m, 2H, CH2), 1.48-1.38 (m, 2H, CH2), 1.29-
1.16 (m, 2H, CH2), 0.89-0.80 (m, 3H, CH3). 13C NMR (CDCl3 and MeOD): δ 157.2, 145.6, 144.4, 144.4,
131.1, 131.1, 130.6, 129.6, 129.6, 128.1, 128.1, 117.4, 117.3, 117.3, 117.2, 117.2, 116.3, 116.3, 116.3,
116.2, 116.1, 111.0, 110.7, 43.5, 42.1, 41.2, 29.2, 19.8, 19.8, 13.5, 13.5. MS (APSI+) m/z found 410.20;
HRMS (ESI) m/z found, 410.1343; C19H22F2N3O3S (M+ + H) expected, 410.1351.
5.1.2.10. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,5-dimethoxyphenyl)benzenesulfonamide (10). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 73%; white solid; mp: 177-179 °C. H NMR

(CDCl3): δ 8.00 (s, 1H, NH), 7.71-7.68 (m, 2H, Ar), 7.55-7.52 (m, 2H, Ar), 6.28 (s, 2H, Ar), 6.11 (s, 1H,
Ar), 3.76-3.71 (m, 2H, CH2), 3.64 (s, 6H, 2x CH3), 3.49-3.43 (m, 2H, CH2), 3.26 (t, 2H, J = 7.2 Hz, CH2),
13
1.45-1.45 (m, 2H, CH2), 1.37-1.25 (m, 2H, CH2), 0.89 (t, 3H, J = 7.2 Hz, CH3). C NMR (CDCl3): δ
161.1, 157.2, 144.5, 139.0, 131.3, 128.3, 116.2, 99.0, 96.8, 55.3, 43.6, 42.1, 41.3, 29.3, 19.9, 13.7. MS
(APSI+) m/z found 434.25; HRMS (ESI) m/z found, 434.1739; C21H28N3O5S (M+ + H) expected,
434.1750.
5.1.2.11. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,5-difluorophenyl)benzenesulfonamide (11). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 7%; white solid; mp: 183-185 °C. H NMR
(CDCl3): δ 7.88 (s, 1H, NH), 7.77-7.74 (m, 2H, Ar), 7.64-7.61 (m, 2H, Ar), 6.72-6.70 (m, 2H, Ar), 6.51-
6.45 (m, 1H, Ar), 3.87-3.82 (m, 2H, CH2), 3.57-3.51 (m, 2H, CH2), 3.31 (t, 2H, J = 7.2 Hz, CH2), 1.58-
1.50 (m, 2H, CH2), 1.41-1.29 (m, 2H, CH2), 0.92 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (CDCl3): δ 164.9,
164.8, 164.2, 164.0, 161.7, 161.5, 160.8, 160.7, 157.2, 156.8, 145.8, 144.8, 139.8, 136.5, 130.9, 130.7,
129.8, 129.8, 128.4, 116.4, 116.3, 116.2, 116.1, 115.5, 115.4, 115.3, 115.2, 115.2, 106.5, 106.2, 105.8,
103.5, 103.4, 103.1, 100.2, 99.8, 99.5, 43.7, 43.7, 42.1, 41.4, 41.3, 29.4, 29.4, 20.0, 13.8, 13.7. MS
(APSI+) m/z found 410.20; HRMS (ESI) m/z found, 410.1325; C19H22F2N3O3S (M+ + H) expected,
410.1351.
5.1.2.12. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,5-dichlorophenyl)benzenesulfonamide (12). Flash
1
chromatography (Hex to Hex/EtOAc (50:50)). Yield, 6%; white solid; mp: 198-200 °C. H NMR
(CDCl3/MeOD): δ 7.71-7.68 (m, 2H, Ar), 7.60-7.57 (m, 2H, Ar), 6.98 (s, 2H, Ar), 6.96 (s, 1H, Ar), 3.81-
3.76 (m, 2H, CH2), 3.51-3.45 (m, 2H, CH2), 3.25 (t, 2H, J = 7.2 Hz, CH2), 1.55-1.45 (m, 2H, CH2), 1.37-
1.27 (m, 2H, CH2), 0.90 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (CDCl3/MeOD): δ 156.8, 145.9, 136.3, 135.0,
130.7, 130.5, 130.2, 129.8, 116.1, 43.7, 42.2, 41.3, 29.4, 20.0, 13.8. MS (APSI+) m/z found 442.15;
HRMS (ESI) m/z found, 442.0755; C19H22Cl2N3O3S (M+ + H) expected, 442.0760.

5.1.2.13. 4-(3-Butyl-2-oxoimidazolidin-1-yl)-N-(3,4,5-trimethoxyphenyl)benzenesulfonamide (13).
Flash chromatography (methylene chloride to methylene chloride/EtOAc (90:10)). Yield, 75%; white
solid; mp: 177-179 °C. 1H NMR (CDCl3): δ 7.85 (s, 1H, NH), 7.69-7.66 (m, 2H, Ar), 7.59-7.56 (m, 2H,
Ar), 6.37 (s, 2H, Ar), 3.80-3.73 (m, 5H, CH2 and CH3), 3.66 (s, 6H, 2x CH3), 3.52-3.47 (m, 2H, CH2),
3.28 (t, 2H, J = 7.2 Hz, CH2), 1.56-1.46 (m, 2H, CH2), 1.37-1.25 (m, 2H, CH2), 0.89 (t, 3H, J = 7.2 Hz,
CH3). 13C NMR (CDCl3): δ 157.2, 153.3, 144.5, 135.3, 133.1, 131.3, 128.5, 116.1, 99.5, 60.9, 56.0, 43.7,
42.1, 41.4, 29.3, 19.9, 13.7. MS (APSI+) m/z found 464.20; HRMS (ESI+) m/z found 464.1849;
C22H30N3O6S (M+ + H) expected, 464.1856.
5.2. Biology
5.2.1. Cell lines culture
MCF7 human breast carcinoma and MDA-MB-231 human breast carcinoma were purchased from the
American Type Culture Collection (Manassas, VA) while HaCaT primary epidermal keratinocytes were
purchased from Thermo Fisher Scientific. All cell lines were cultured in high-glucose Dulbecco’s
minimal essential medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 5% (MCF7
and MDA-MB-231) and 10 % (HaCaT) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific). All
cells were grown at 37 °C in a 5% CO2 moisture-saturated atmosphere.
5.2.2. Antiproliferative activity assay
The antiproliferative activity assay was assessed using the procedure described by the National Cancer
30
Institute for its drug screening program, with slight modifications . Ninety six-well plates (Costar®)
were seeded with 75 µL of the tumor cell suspension containing 3000, 3500 and 4500 cells for MCF7,
MDA-MB-231 and HaCaT cells, respectively. Plates were incubated for 24 h and then the drugs freshly
solubilized in DMSO at 40 mM were added in fresh medium. Thereafter, 75 µL aliquots containing a

range of concentrations of the drug according to the maximum solubility was then added to the plates
and cells were incubated for 24, 36, 48 and/or 72 h. DMSO concentration was kept constant at 0.5% (v/v)
to prevent any related cytoxicity. Cell growth was stopped by the addition of trichloroacetic acid to the
wells (10% (w/v) final concentration) and incubated for at least 30 min at 4 °C. The supernatant was
removed and the plates were washed three times with distilled water and dried overnight. Seventy five
microliters of a sulforhodamine B solution (0.1% (w/v) in 1% acetic acid) were then added to each well,
and the plates were incubated for 15 to 30 min at room temperature. The unbounded dye was removed
by washing the plates thrice with 1% acetic acid. The dye bounded to the cells was solubilized using a
20 mM Tris base solution and incubated for a minimum of 1 h. The absorbance was read with a
SpectraMax® i3x (Molecular Devices) using an optimal wavelength (530-580 nm). 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.
5.2.3. Cell cycle progression analysis
MCF7 cells were seeded in 6-well plates (Costar®) (1.5 x 105 cells per well) and incubated in DMEM
medium for 24h. The cells were treated with compound 13 (0.2 and 0.5 M), CEU-638 (0.01 M) and
CA-4 (0.001 M) for 24 h. DMSO (0.5%) was used as negative control while CEU-638 and CA-4 were
used as positive controls. After 24 h, the cells were trypsinized, washed with PBS, resuspended in 250
L of PBS, fixed by the addition of 750 L of ice-cold EtOH under agitation, and stored at -20 °C. Prior
to FACS analysis, the cells were washed with PBS and resuspended in 500 μL of PBS containing 2
g/mL 4’,6’-diamidino-2-phenylindole (DAPI). Cell cycle distribution of fixed cell suspensions was
evaluated using an LSR II flow cytometer (BD, Biosciences, Franklin Lakes, NJ). The increasing number

of cells in G2/M phase was calculated by subtracting the cellular population of DMSO 0.5% from the
cellular population treated with compound 13, CEU-638 and CA-4.
5.2.4. Immunofluorescence of microtubule
MCF7 cells were seeded at 1.5 × 105 cells per well in 6-well plates (Costar®) containing glass coverslips
(22 mm x 22 mm) coated with fibronectin (10 μg/mL) and incubated for 24 h. Tumor cells were incubated
either with compound 13 (0.2 M), CEU-638 (0.01 M) or CA-4 (0.001 M) for 24 h. DMSO (0.5%)
was used as negative control while CEU-638 and CA-4 were used as positive controls. The cells were
then washed thrice with PBS and fixed with 3.7% formaldehyde in PBS for 10 min. After two washes
with PBS, the cells were permeabilized 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 2 h at room temperature with an anti-β-tubulin
monoclonal antibody (clone TUB 2.1, 1:200) in a solution containing 0.1% saponin and 3% BSA in PBS.
The cells were washed twice with PBS containing 0.05% Tween 20 and the nuclei were then stained
using antimouse IgG Alexa Fluor 488 (Molecular Probes, Eugene, OR; 1:1000) and DAPI (1.25 ng/mL
final concentration) in blocking buffer for 1 h at 37 °C. The cover slides were mounted on a microscope
slide with slow fade reagent (DakoCytomation, Carpinteria, CA) before analysis under an Olympus
BX51 fluorescence microscope. Images were acquired as 8 bit-tagged image format files with a Q
imaging RETIGA EXI digital camera using the Image Pro Express software.
5.2.5. Activation of PAIB-SA 13 into its PIB-SA parental antimitotic CEU-638
Corning® Supersomes™ Human CYP1A1 + oxidoreductase, potassium phosphate (0.5 M), and
NADPH-regenerating system solutions A and B were supplied by Fisher Scientific (Toronto, ON,
Canada). Briefly, compound 13 was freshly solubilized at 60 M in CH3CN. Then, 451.8 L of purified
water, 130 L of potassium phosphate buffer, 32.5 L of NADPH-regenerating system solution A, 6.5
L of NADPH-regenerating system solution B, and 13 L of the substrate (in CH3CN) were mixed and

warmed to 37 °C for 5 min. The enzymatic reaction was next initiated by adding 13.75 μL of freshly
thawed human SupersomesTM (1 nmol/mL) to the mixture. At 0, 15, 30, 60, 90, 120 and 150 min, the
enzymatic reaction was stopped by adding 100 μL of MeOH. Then, the mixture was centrifuged at 12
000 g for 5 min and 20 µL of the supernatant was analyzed by HPLC-UV (Waters ACQUITY Arc System
equipped with 2998 PDA detector). The mixture was eluted using a MeOH/H2O (with 0.1% formic acid)
linear gradient (1.0 mL/min) on a CORTECS C18+ reversed-phase column 30 x 50 mm x 2.7 m
equipped with a CORTECS C18+ VanGuard Cartridges 2.1 x 5 mm x 2.7 m. Wavelength was selected
at 280 nm and the peaks of compound 13 and CEU-638 were confirmed by internal standards and
absorbance spectra (200-600 nm).
Acknowledgments
We acknowledge financial support from Merck Sharpe & Dohm program from the Faculté de médecine
de l’Université Laval, start-up funds from Foundation of CHU de Québec and research infrastructure
program of John R. Evans Leaders Fund from Canada Foundation for Innovation (CFI). We also
acknowledge financial support from studentships from the Fonds de recherche du Québec-Santé (FRQS,
MAGB) and from Fonds d'enseignement et de recherche of the Faculté de pharmacie de l’Université
Laval (ACCA, MZK and MAGB).
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Highlights
PAIB-SAs are new antimitotic prodrugs





PAIB-SAs exhibit antiproliferative activity in the submicromolar range in MCF7
PAIB-SA 13 shows a selectivity ratio of 372 between MCF7 and HaCaT cells
PAIB-SA 13 blocks the cell cycle in G2/M phase and disrupts microtubules
PAIB-SA 13 is metabolized by CYP1A1 into its parental antimitotic CEU-638
Graphical abstract
4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs
activated by cytochrome P450 1A1 in breast cancer cells
Atziri Corin Chavez Alvarez, Mitra Zarifi Khosroshahi, Marie-France Côté, Mathieu Gagné-Boulet
and Sébastien Fortin*