Styryl- N -phenyl- N′ -(2-chloroethyl)ureas and styrylphenylimidazolidin-2-ones as new potent microtubule-disrupting agents using combretastatin A-4 as model

Article abstract of DOI:10.1016/j.ejmech.2015.05.034

Combretastatin A-4 (CA-4) is a well-studied and attractive molecular template to develop new antimitotics. Several thousand of modifications were performed on the ring B and the ethenyl bridge of CA-4 but only a few involved the trimethoxyphenyl moiety (TMP, ring A) often considered essential to the antiproliferative and antimicrotubule activities. In this study, we described the design, the preparation, the characterization and the biological evaluation of three new series of CA-4 analogs namely styryl-Nphenyl- N'-ethylureas (SEUs), styryl-N-phenyl-N'-(2-chloroethyl)ureas (SCEUs) and styrylphenylimidazolidin-2-ones (SIMZs) bearing a 3-Cl (series a), 3,5-Me (series b) and TMP (series c) substituents, respectively. All SCEU and SIMZ Z-isomers were active in the high and the low nanomolar range, respectively. Conversely to SEUs and their E-isomers that were significantly less active or inactive. Interestingly, the TMP moiety is giving rise to derivatives exhibiting the lowest antiproliferative activity in the SCEU series (10c) and the most active compound in the SIMZ series (12c). Moreover, SIMZ Zisomers bearing either a 3-Cl (12a) or a 3,5-Me (12b) exhibited antiproliferative activities that are also in the same order of magnitude as 12c. All SCEU and SIMZ Z-isomers also arrested the cell cycle progression in G2/M phase, bound to the colchicine-binding site and disrupted the cytoskeleton of cancer cells. In addition to the promising and innovative microtubule-disrupting properties of SCEUs and SIMZs, these results show that the TMP moiety is not essential for the cytocidal activity of these new CA-4 analogs.

Full text of DOI:10.1016/j.ejmech.2015.05.034

Accepted Manuscript
Styryl-N-phenyl-N’-(2-chloroethyl)ureas and Styrylphenylimidazolidin-2-ones as New
Potent Microtubule-Disrupting Agents Using Combretastatin A-4 as Model
Mathieu Gagné-Boulet, Sébastien Fortin, Jacques Lacroix, Carole-Anne Lefebvre,
Marie-France Côté, René C. Gaudreault
PII:
S0223-5234(15)30061-1
10.1016/j.ejmech.2015.05.034
EJMECH 7913
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 April 2015
Revised Date: 21 May 2015
Accepted Date: 22 May 2015
Please cite this article as: M. Gagné-Boulet, S. Fortin, J. Lacroix, C.-A. Lefebvre, M.-F. Côté, R.C.
Gaudreault, Styryl-N-phenyl-N’-(2-chloroethyl)ureas and Styrylphenylimidazolidin-2-ones as New Potent
Microtubule-Disrupting Agents Using Combretastatin A-4 as Model, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.05.034.
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ACCEPTED MANUSCRIPT
O
O
O
S
O
O
Cl
N
N
O
X
Cl
N
H
N
H
H
H
N
R1
N
HN
R1
OH
HN
R1
O
SCEUs
0a: R =3-Cl (130-200 nM)
CEU analog to CA-4
(860-2400 nM)
PIB-SOs: X = O (4-220 nM)
PIB-SAs: X = NH (13-810 nM)
SIMZs
12a: R =3-Cl (25-38 nM)
1
0b: R =3,5-Me (510-860 nM)
1
1
1
0c: R =3,4,5-OMe (680-1400 nM)
12b: R =3,5-Me (8.4-23 nM)
12c: R =3,4,5-OMe (1.7-2.6 nM)
1
1
1
O
O
1
1
1
0a
10a
12b
12b
5
4
3
2
1
00 SubG1: 2.2
350 SubG1: 8.2
00
00
00
00
0
G0/G1: 2.1
S: 3.7
G2/M: 92.0
300
250
200
G0/G1: 5.4
S: 8.0
G2/M: 78.4
O
OH
150
1
00
50
O
0
50
100
150
200
250
50
100
150
200
250
DAPI-A (x 1000)
CA-4 (1.6-560 nM)
DAPI-A (x 1000)

ACCEPTED MANUSCRIPT
Styryl-N-phenyl-N’-(2-chloroethyl)ureas and Styrylphenylimidazolidin-2-ones as New
Potent Microtubule-Disrupting Agents Using Combretastatin A-4 as Model
a,†
a,b,†,*
a
a,‡
Mathieu Gagné-Boulet, Sébastien Fortin,
Jacques Lacroix, Carole-Anne Lefebvre,
a
a,c,*
Marie-France Côté, René C.-Gaudreault
a
Centre de recherche du CHU de Québec, axe oncologie, Hôpital Saint-François d’Assise,
Québec, QC, Canada G1L 3L5.
b
Faculté de pharmacie, Université Laval, Pavillon Vandry, Québec, QC, Canada G1V 0A6.
c
Faculté de médecine, Département de médecine moléculaire, Université Laval, Pavillon
Vandry, Québec, QC, Canada G1V 0A6.
†
These authors contributed equally.
‡
Present address: Département de chimie, Université Laval, Pavillon Vachon, Québec, QC,
Canada G1V 0A6.
*Corresponding authors: Sébastien Fortin; Phone: 418-525-4444 ext. 52364, Fax: 418-525-
4372, e-mail: sebastien.fortin@ulaval.ca or René C.-Gaudreault; Phone: 418-525-4444 ext.
52363, Fax: 418-525-4372, e-mail: rene.c-gaudreault@crchudequebec.ulaval.ca. Centre de
recherche du CHU de Québec, axe oncologie, Hôpital Saint-François d’Assise, Québec, QC,
Canada G1L 3L5.
Abbreviations List: combretastatin A-4, CA-4; chorioallantoic membrane assay, CAM
assay; colchicine-binding site, C-BS; N-phenyl-N’-(2-chloroethyl)urea, CEU; N,N'-ethylene-
bis(iodoacetamide), EBI; N-phenyl-N’-ethylurea, EU; phenylimidazolidin-2-one, IMZ; phenyl
4-(2-oxoimidazolidin-1-yl)benzenesulfonamide, PIB-SA; phenyl 4-(2-oxoimidazolidin-1-
yl)benzenesulfonate, PIB-SO; structure-activity relationships, SAR; SCEU, styryl-N-phenyl-
1

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N’-(2-chloroethyl)urea; styryl-N-phenyl-N’-ethylurea, SEU; SIMZ, styrylphenylimidazolidin-
2-one; trimethoxyphenyl, TMP.
2

ACCEPTED MANUSCRIPT
Abstract
Combretastatin A-4 (CA-4) is a well-studied and attractive molecular template to develop
new antimitotics. Several thousand of modifications were performed on the ring B and the
ethenyl bridge of CA-4 but only a few involved the trimethoxyphenyl moiety (TMP, ring A)
often considered essential to the antiproliferative and antimicrotubule activities. In this study,
we described the design, the preparation, the characterization and the biological evaluation of
three new series of CA-4 analogs namely styryl-N-phenyl-N’-ethylureas (SEUs), styryl-N-
phenyl-N’-(2-chloroethyl)ureas (SCEUs) and styrylphenylimidazolidin-2-ones (SIMZs)
bearing a 3-Cl (series a), 3,5-Me (series b) and TMP (series c) subtituents, respectively. All
SCEU and SIMZ Z-isomers were active in the high and the low nanomolar range,
respectively. Conversely to SEUs and their E-isomers that were significantly less active or
inactive. Interestingly, the TMP moiety is giving rise to derivatives exhibiting the lowest
antiproliferative activity in the SCEU series (10c) and the most active compound in the SIMZ
series (12c). Moreover, SIMZ Z-isomers bearing either a 3-Cl (12a) or a 3,5-Me (12b)
exhibited antiproliferative activities that are also in the same order of magnitude as 12c. All
SCEU and SIMZ Z-isomers also arrested the cell cycle progression in G2/M phase, bound to
the colchicine-binding site and disrupted the cytoskeleton of cancer cells. In addition to the
promising and innovative microtubule-disrupting properties of SCEUs and SIMZs, these
results show that the TMP moiety is not essential for the cytocidal activity of these new CA-4
analogs.
Keywords:
Styrylphenylimidazolidin-2-ones;
styryl-N-phenyl-N’-(2-chloroethyl)ureas;
styryl-N-phenyl-N’-ethylureas; combretastatin A-4; colchicine-binding site inhibitors;
antimitotics.
3

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1. Introduction
Microtubules are heterodimers composed of α- and β-tubulin isoforms present in all
eukaryotic cells. Microtubules are not only involved in cell shape but also in numerous
cellular mechanisms such as cell motility and division mitosis, intracellular vesicle transport,
organization, and positioning of membranous organelles [1]. When natural or synthetic
ligands such as combretastatin A-4 (CA-4, 1), podophyllotoxin (2) and phenstatin (3, Fig. 1)
interact with microtubule dynamics, they disrupt the cytoskeleton, block mitosis into the
G2/M phase and initiate an apoptotic program [2]. It is why molecules acting on the cellular
microtubule dynamics form one of the largest groups of effective chemotherapeutics used
against cancers. However, the efficacy and the clinical usefulness of currently available
antimicrotubule agents are impeded by the occurrence of chemoresistance, systemic toxicity,
and poor biopharmaceutical properties [3, 4]. Therefore, the search for new antimicrotubule
agents exhibiting improved biopharmaceutical profiles and pharmacodynamics is still
recognized as of utmost importance. To that end, tens of thousands of derivatives and analogs
based on modifications of the ring B and the ethenyl bridge of CA-4 were synthesized and
screened for anticancer activity [5, 6]. However, only a few modifications were made on the
trimethoxyphenyl (TMP) moiety (ring A, Fig. 1) of CA-4, notably its replacement by either a
pentafluorophenyl (T138067, 4) [7], a trimethylphenyl or a dimethylphenyl moiety [8].
Indeed, the TMP moiety present in CA-4 and in numerous other antimicrotubule agents has
long been considered as essential to their binding to the colchicine-binding site (C-BS), their
inhibition of the microtubule assembly and their antiproliferative activity [9].
Based on the computational models published by Nogales [10] and others [11, 12] we have
designed and developed a N-phenyl-N’-(2-chloroethyl)urea (CEU) fragment that can be used
as a “bioisosteric equivalent” of TMP that acylates the C-BS on the glutamic acid residue in
4

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position 198 [13, 14]. This CEU fragment was used to prepare several antimicrotubule agents
substituted at position 4 of the aromatic ring such as 1-(4-(tert-butyl)phenyl)-3-(2-
chloroethyl)urea (tBCEU, 5a) [15] and 1-(4-(sec-butyl)phenyl)-3-(2-chloroethyl)urea
(sBCEU, 5b) [15], and substituted at position 3 of the aromatic ring represented by 1-(2-
chloroethyl)-3-(4-(5-hydroxypentyl)phenyl)urea (5c) [16]. This was also confirmed by
replacing the TMP moiety of CA-4 by the CEU group resulting in molecules referred to as
(Z)-1-(2-chloroethyl)-3-(4-(3-hydroxy-4-methoxystyryl)phenyl)ureas (CEU analogs to CA-4,
5
d-g, Fig. 1) exhibiting antiproliferative activity (IC ) at the micromolar level and that are
5
0
still acylating the C-BS [14, 17]. Of note, only the methoxylated phenolic moieties of CA-4 at
positions 3 and 4 have been studied so far. Recently our structure-activity relationships (SAR)
studies showed that the modification of the ethenyl bridge by its sulfonate or sulfonamide
bioisosteric equivalents followed by the cyclisation of the CEU moiety into the corresponding
phenylimidazolidin-2-ones (IMZs) lead to potent new classes of antimicrotubule agents
designated as phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs, 6) [18] and
phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB-SAs, 7) [19]. Compounds 6 and
7
exhibited antiproliferative activity in the nanomolar range similarly to CA-4, which
represents an increase by up to 3 logs in magnitude of the antiproliferative activity when
compared to compounds 5a-g. Compounds 6 and 7 exhibited also high potency against human
fibrosarcoma HT-1080 tumors grafted onto the chorioallantoic membrane of developing chick
embryos (CAM assay) and low toxicity to the embryos. The SAR studies of these compounds
showed that the nature of the bridge linking the 2 phenyl groups and the substituent on ring B
of compounds 6 and 7 had a significant impact on the biological activity. Due to their
molecular similarity, we hypothesized that the IMZ moiety of PIB-SOs and PIB-SAs is a
“bioisosteric equivalent” of the TMP ring of CA-4 as is the CEU moiety. However, our recent
molecular modeling experiments show that the IMZ moiety group might be docked in the C-
5

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BS in such way that it would mimic the tropolone and the methoxylated phenolic moieties of
colchicine and CA-4, respectively instead the TPM moiety [20]. This might be due to the
steric hindrance that may prevent the binding of the IMZ moiety to the adjacent pocket behind
CYS239 and 354 residues of β-tubulin [20, 21].
Based on these premises, we hypothesized that the sulfonate and the sulfonamide bridges of
PIB-SO and PIB-SA analogs are bioisoteric equivalents of the ethenyl bridge. Moreover, we
postulated that the TMP moiety is not essential for the antiproliferative activity and the
affinity for the C-BS. Finally, we postulated also that the chloro group and the methoxylated
phenolic moieties are not optimal and essential for the activity of the CEU analogs of CA-4.
To validate our hypothesis, we designed, prepared and biologically evaluated three series of
CA-4 analogs named styryl-N-phenyl-N’-ethylureas (SEUs, 8 and 9), styryl-N-phenyl-N’-(2-
chloroethyl)ureas (SCEUs, 10 and 11) and styrylphenylimidazolidin-2-ones (SIMZs, 12 and
13) each one comprising the E- and Z-isomers. The substituents on the aromatic ring B
namely, 3-Cl (series a) and 3,5-Me (series b) were selected for their potent antiproliferative
activity in the PIB-SOs and PIB-SAs series and their dissimilarity providing a maximum of
information for the SAR studies. The TMP moiety (series c) was selected to evaluate its
importance on the antiproliferative and antimicrotubule activities on these new CA-4 analogs.
These compounds were assessed for their antiproliferative activity on human cancer cell lines
namely, HT-29, M21, MCF-7 and HT-1080. Finally, the most potent compounds were
assessed for their effect on cell cycle progression, cytoskeleton disruption and affinity to the
C-BS.
2. Chemistry
The general synthesis of SEUs, SCEUs and SIMZs started from triphenylphosphonium
bromide salts (14) which were prepared by addition of triphenyphosphine to 4-nitrobenzyl
6

ACCEPTED MANUSCRIPT
bromides in toluene followed by the conjugation of 3-chlorobenzaldehyde, 3,5-
dimethylbenzaldehyde or 3,4,5-trimethoxybenzaldehyde using the Wittig-coupling that was
performed as described by Cushman et al. [22] and Fortin et al. [17]. Briefly, sodium hydride
was added slowly to a stirring mixture of 14 and the relevant benzaldehyde in dry methylene
chloride to yield 15a-c. Afterward, the nitro group of compounds 15a-c was reduced with iron
dust with a catalytic amount of concentrated hydrochloric acid in a mixture of ethanol-water
(10: 1) into the corresponding E- and Z-amino stilbenes 16a-c and 17a-c. Of note, the E- and
Z-isomers of series b were carefully separated at the conjugation step (compounds 15b) while
series a and c were separated at the reduction step (16a, c and 17a, c). For the preparation of
SEUs 8a, c and 9a, c, ethylisocyanate was added to the corresponding E- or Z-amino stilbenes
1
6a, c and 17a, c in methylene chloride at room temperature while SEUs 8b and 9b were
prepared by addition of ethylisocyanate in acetonitrile at 90 °C under pressure. SCEUs 10a-
1c were obtained by addition of 2-chloroethylisocyanate to E- or Z-amino stilbenes 16a-17c
1
in methylene chloride at room temperature. Finally, SIMZs 12a-13c were prepared by
cyclisation of the CEU moiety of SCEUs 10a-11c into IMZ in presence of sodium hydride in
dry tetrahydrofuran.
3
3
. Results/Discussion
.1 SCEUs and SIMZs Exhibit Antiproliferative Activity on Human Tumor Cell lines
First, SEUs (8a-9c), SCEUs (10a-11c) and SIMZs (12a-13c) were assessed for their
antiproliferative activity on human colon carcinoma (HT-29), skin melanoma (M21), breast
carcinoma (MCF-7), and human fibrosarcoma (HT-1080) according to the NCI/NIH
Developmental Therapeutics Program [23]. The results are summarized in Table 1 and
expressed as the concentration of drug inhibiting cell growth by 50% (IC ). SIMZs 12a-c are
5
0
active in the low nanomolar range (1.7-38 nM) and SCEUs 10a-c are active at 130 to 1400
7

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nM while SEUs are either inactive or several logs less active than CA-4 analogs (11 000 to >
20 000 nM). As expected, the antiproliferative activity of the stilbene Z-isomers was up to 3
logs higher than their respective stilbene E-isomers similarly as observed with CA-4
derivatives and analogs. These SAR studies confirm several important elements for the
activity and the design of these new CA-4 analogs. First, our studies show that the Z-ethenyl
bridge is a bioisoteric equivalent of the sulfonate and the sulfonamide moieties. Indeed, this
modification globally increase by approximately two-fold the activity of SIMZs
comparatively to PIB-SO and PIB-SA analogs bearing the same substituents. These results
strongly suggest and corroborate our molecular models showing that the sulfonate and the
sulfonamide groups in PIB-SO and PIB-SA analogs must adopt a cisoid conformation to be
active. Interestingly, the antiproliferative activities of SIMZs 12a-c are in the low nanomolar
range and are very similar to CA-4. In addition, they are at least 10-times more potent on HT-
2
9 cells than CA-4. Moreover, although that SIMZ 12c bearing a TMP moiety is the most
active compound (1.7 to 2.6 nM), SIMZs bearing a 3-Cl (compound 12a, 25 to 38 nM) or a
,5-Me substituents (compound 12b, 8.4 to 23 nM) are also in the same order of magnitude.
3
This suggests that the TMP group is not essential for the antiproliferative activity of SIMZ
derivatives. The antiproliferative activity of SIMZs increases in the following order of
substitution of ring B: 3,4,5-OMe > 3,5-Me > 3-Cl.
The modification of the IMZ group by a CEU group lead to SCEUs that are active in the mid
to high nanomolar range. On one hand, SCEUs are among the most potent CEUs prepared so
far. Their antiproliferative activity is approximately 10-times higher than CEUs substituted at
position 4 (e.g. 5a, b and d) and in the same order of magnitude as CEUs substituted at
position 3 (e.g. compound 5c). They are approximately 2 to 10-times more active than CEU
analogs to CA-4 that is bearing a methoxylated phenolic group. Moreover, SCEU 10c bearing
a 3-Cl substituent is the most potent CEU prepared to date and it exhibits an antiproliferative
8

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activity ranging between 130 and 200 nM. In addition, compounds 10b and 10c are almost as
potent as CA-4 on HT-29 cells. However, SCEUs are approximately 100-times less active
than SIMZs. SAR studies show that the chloro group in the CEU moiety is essential for the
antiproliferative activity; SEU derivatives without the chloro group are significantly less
active or inactive even those bearing a TMP moiety (compound 8c, 11 000 to 35 000 nM).
Moreover, the antiproliferative activity of SCEUs increases according to the following order
of substitution of ring B: 3-Cl > 3,5-Me > 3,4,5-OMe which are in the opposite direction of
SIMZs. These results confirm that the TMP group is not essential for the antiproliferative
activity of these CA-4 analogs comparatively to electrophilic chlorine atom present in CEUs.
3
.2 SCEUs and SIMZs Arrest the Cell Cycle Progression in G /M Phase
2
The cell growth inhibitory potency of SCEUs (10a-c) and SIMZs 12a-c prompted us to
evaluate their effect on cell cycle progression and on the microtubules and cytoskeleton
integrity on M21 cells. For cell cycle progression analysis, M21 cancer cells were treated at
approximately 5-times the respective IC of SCEUs and SIMZs for 24 h. As shown in Figure
5
0
2, SCEUs 10a-c and SIMZs 12a-c arrested efficiently the cell cycle progression in G2/M
phase; increasing the percentage of cells in G2/M phase by 33 to 70%. These results are
comparable to CA-4 and colchicine used as positive controls that increase the percentage of
cells in G2/M phase by 59 and 36%, respectively.
3.3 SCEUs and SIMZs Disrupt the Cytoskeleton of Cancer Cells
In the aim to assess the effect of SCEUs 10a-c and SIMZs 12a-c on microtubules and
cytoskeleton integrity, M21 cells were treated for 16 h with 10a-c and 12a-c at 5-times their
respective IC , and cellular microtubule structures were visualized by indirect
5
0
immunofluorescence using an anti-β-tubulin monoclonal antibody. As shown in Figure 3,
9

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10a-c and 12a-c lead to microtubule depolymerization and cytoskeleton disruption on M21
cancer cells similarly to CA-4 and colchicine.
3.4 SCEUs Acylate the Glutamic Acid Residue in Position 198 of the C-BS
We finally assessed whether the binding of SCEUs and SIMZs on β-tubulin was the same as
CA-4 and colchicine that bind to the C-BS. As mentioned earlier, CEUs such as tBCEU [24]
and sBCEU [25] are acylating the glutamic acid residue in position 198 of the C-BS [13, 14].
This adduct is easily detectable by Western blot as a second immunoreacting band of β-
tubulin [16, 21, 26-29]. As seen in Figure 4, SCEUs 10a and 10b induce a significant second
immunoreacting band at 1 500 and 3 100 nM, respectively while SCEUs 10c bearing a TMP
moiety induces only a very week second immunoreacting band at 8 200 nM. These results
confirm that SCEUs act on the C-BS and acylate the glutamic acid 198 residue of β-tubulin.
These results also show that that the TMP moiety is not essential for the affinity and the
acylation of the C-BS. The weak antiproliferative activity and the poor acylation of β-tubulin
by SCEUs bearing a TMP group (compound 10c) may be explained by the high affinity of the
TMP for the hydrophobic pocket where is located the cysteine 239 residue disfavoring the
conformation for an acylation by the glutamic acid at position 198.
3.5 SIMZs Inhibit EBI Binding to the C-BS
We recently develop a quick and simple detection technique to assess the binding of
antimicrotubule agents that do not acylate/alkylate the C-BS such as SIMZs [30]. This assay
is based on N,N'-ethylene-bis(iodoacetamide) (EBI) that has the ability to crosslink the
cysteine residues at position 239 and 354 in the C-BS. Similarly to CEUs, EBI forms an
adduct easily detectable by Western blot as a second immunoreacting band of β-tubulin.
Antimicrotubule agents acting in the C-BS prevent the formation of the EBI: β-tubulin
10

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adduct. Briefly, M21 cells were treated with SIMZs 12a, 12b, 12c at 100-times their
respective IC as well as SIMZ 13c at 100 000 nM; the maximum concentration used for this
5
0
assay. Colchicine at 10, 100, 1 000 and 10 000 nM was used as positive controls. As shown in
Figure 5, SIMZs 12a, 12b, 12c and 13c inhibit the formation of the EBI: β-tubulin adduct
similarly to colchicine as shown by the disappearance of the second immunoreacting band of
β-tubulin and the appearance of the native β-tubulin band (first immunoreacting band of β-
tubulin). Interestingly, the experiment also shows that a large concentration of E-isomer of
SIMZ bearing a trimethoxyl group (compound 13c, approximately 100-times less potent than
the Z-isomer SIMZ) inhibits also the formation of the EBI: β-tubulin adduct. First, these
results show that IMZ and TMP groups have a significant affinity for the C-BS and explains
the antiproliferative activity of SIMZ 13c (370-550 nM) and the disruption of the
cytoskeleton; E-isomers are usually weak or inactive compounds exhibiting in parallel only a
weak affinity for the C-BS. Moreover, they show that the TMP group is not essential for the
affinity of SIMZs to C-BS.
4. Conclusions
In summary, our studies identified new cytocidal molecules designed as SIMZs and SCEUs
that are active in the low and high nanomolar ranges, respectively, as their parent compounds.
They block the cell cycle progression in the G2/M phase, bind to the C-BS leading to the
depolymerization of microtubules and disruption of the cytoskeleton. In addition, this study
shows that the sulfonate and sulfonate bridges are bioisosteric equivalents of the ethenyl
bridge of CA-4. Moreover, it shows that the TMP group is not essential for the
antiproliferative and the antimicrotbule activities of SIMZs and SCEUs. Furthermore, SCEUs
are among the most potent CEUs prepared so far and are 2 to 10-times more potent than CEU
analogs to CA-4. Finally, SIMZs and SCEUs are new molecular scaffolds to design new
potent antimicrotubule agents and could be used as alternative to circumvent some
11

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biopharmaceutical problems that might be encountered during the clinical development of
CA-4.
5
5
5
. Experimental Protocols
.1 Biological Methods
.1.1 Cell Lines Culture
HT-29 human colon carcinoma, MCF7 human breast carcinoma, HT-1080 human
fibrosarcoma were purchased from the American Type Culture Collection (Manassas, VA).
M21 human skin melanoma cells were kindly provided by Dr. David Cheresh (University of
California, San Diego School of Medicine, USA). Cells were cultured in DMEM medium
containing sodium bicarbonate, high glucose concentration, glutamine and sodium pyruvate
(Hyclone, Logan, UT) supplemented with 5% of fetal bovine serum (FBS, Invitrogen,
Burlington, ON) and were maintained at 37 ºC in a moisture-saturated atmosphere containing
5% CO2.
5.1.2 Antiproliferative Activity Assay
The antiproliferative activity assay of all compounds was assessed using the procedure
recommended by the National Cancer Institute for its drug screening program with slight
modifications [23]. Briefly, 96-well microtiter plates were seeded with 75 µL of a suspension
3
3
3
3
of HT-29 (4.0 x 10 ), M21 (3.0 x 10 ), MCF-7 (2.5 x 10 ) or HT-1080 (2.5 x 10 ) cells per
well in DMEM and incubated for 24 h. Drugs freshly solubilized in DMSO were diluted in
fresh DMEM, and 75 µL aliquots containing serially diluted concentrations of the drug were
added. Final drug concentrations ranged from 50 µM to 1 nM. DMSO was maintained at a
concentration of < 0.5% (v/v) to avoid any related cytoxicity. Plates were incubated for 48 h.
Afterward, cell growth was stopped by addition of cold trichloroacetic acid to the wells (10%
12

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w/v, final concentration), followed by a 1 h incubation at 4 °C. Plates were then washed 5-
times with water. Then, 75 µL of a sulforhodamine B solution (0.1% w/v) in 1% acetic acid
was added to each well, and the plates were incubated for 15 min at room temperature. After
staining, unbound dye was removed by washing 5-times with 1% acetic acid. Bound dye was
solubilized in 20 mM Tris base, and the absorbance was read using an optimal wavelength
(530−568 nm) with a ꢀQuant Universal microplate spectrophotometer (Biotek, Winooski,
VT). Readings obtained from treated cells were compared with measurements from control
cell plates fixed on treatment day, and the percentage of cell growth inhibition was calculated
for each drug. The experiments were performed at least twice in triplicate. The assays were
considered valid when the coefficient of variation for a given set of conditions and within the
same experiment was < 10%.
5.1.3 Cell Cycle Analysis
5
After incubation of 2.5 x 10 M21 cells with the drugs at 2- and 5-times their respective IC ,
5
0
for 24 h, the cells were trypsinized, washed with Phosphate Buffered Saline (PBS) and
resuspended in 250 µL of PBS. Cells were fixed by the addition of 750 µL of ice-cold EtOH
under agitation and stored at -20 °C until analysis. Prior to fluorescence-activated cell sorting
analysis, cells were washed with PBS and resuspended in 500 µL of PBS containing 2 µg/mL
DAPI. Cell cycle distribution of fixed cell suspensions was analyzed using an LSR II flow
cytometer (BD Biosciences, Franklin Lakes, NJ).
5.1.4 Immunofluorescence
Cover slides (22 mm × 22 mm) sterilized with 70% (v/v) EtOH were placed in six-well plates.
To promote cell adhesion, cover slides were treated with 1.5 mL of a fibronectin solution in
5
PBS (5 µg/mL) for 1 h at 37 °C. Slides were then rinsed twice with PBS. M21 cells (1 x 10 )
13

ACCEPTED MANUSCRIPT
were seeded onto the plates and incubated for 24 h. Cells were then incubated with the
compound to be tested at 2- and 5-times their respective IC for 16 h. The control solution
5
0
was DMSO (0.5%) diluted in the culture medium (0.5%, v/v). Cells were fixed using 1 mL of
formaldehyde at 3.7% and permeabilized by addition of a saponin solution (0.1% in PBS)
containing 3% (w/v) BSA (saponin-BSA). Cells were incubated with mouse anti-H2AX
pS139 antibody (1/4000 in saponin-BSA, Millipore, Billerica, MA). Cover slides were next
incubated for 2 h at room temperature and then washed twice with PBS supplemented with
0.05% (v/v) Tween 20 (PBST). Saponin-BSA containing goat anti-mouse IgG conjugated to
AlexaFluor 594 (1/1000, Invitrogen, Burlington, Ontario, Canada), and DAPI (0.3 µg/mL,
Sigma, Oakville, Ontario, Canada) was then added. The cover slides were incubated for 1 h at
room temperature and then washed twice with PBST and twice with PBS. The cover slides
were mounted with polyvinyl alcohol-1,4-diazobicyclo[2.2.2]octane (10-2.5%, v/v) in buffer
(5% (v/v) glycerol and 25 mM Tris buffer, pH 8.7) (Sigma, Oakville, Ontario, Canada). Cells
were visualized using an epifluorescence microscope (Olympus BX51, Center Valley, PA)
with a Qimaging RETIGA EXi camera (Qimaging, Surrey, British Columbia, Canada).
5.1.5 β-tubulin Acylation Assay
5
M21 cells (4.0 x 10 ) were plated in 6-well plates and incubated overnight. The cells were
treated for 24 h at 2, 5 and 10-times the respective IC for SCEUs, at 30 000 nM of CEU-022
5
0
and CEU-071 (used as positive controls) and at 0.5% of DMSO (used as negative control).
5.1.6 Competition and Inhibition of EBI Binding to β-Tubulin
5
M21 cells (7.5 x 10 ) were plated in 6-well plates and incubated overnight. The cells were
treated for 1.5 h at 100-times the IC for SIMZs (cutoff at 100 000 nM for 13a) and at 10,
5
0
100, 1 000 and 10 000 nM for colchicine. Then, 100 µL of EBI (Toronto Research Chemicals,
14

ACCEPTED MANUSCRIPT
North York, Ontario, Canada) in PBS (100 µM, final concentration) were then added for 1.5 h
without changing the culture medium, which contains the drug tested.
5.1.7 Gel Electrophoresis and Immunoblot.
For both assays, after incubation of the cells with the drugs, the supernatant was collected and
the cells were trypsinized (trypsine-EDTA 0.5%), harvested and centrifuged for 5 min at 12
000 rpm. The pellets were washed with 500 µL of cold PBS and stored at -80 °C until use.
The cells pellets were resuspended in buffer (0.32M sucrose, 1 mM EDTA at pH 8, 10 mM
Tris at pH 7.4 and protease inhibitor).
The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad
laboratories, Mississauga, Canada). Samples were prepared to obtain protein at a
concentration of 2 mg/ml in Laemmli [31] sample (60 mM Tris-Cl at pH 6.8, 2% SDS, 10%
glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and boiled for 5 min. Forty
micrograms of proteins from the protein extracts were subjected to electrophoresis using 10%
polyacrylamide gels. The proteins were transferred onto nitrocellulose membranes that were
incubated with TBSTM (Tris-Buffered Saline + 0.1% (v/v) Tween-20 with 5% fat-free dry
milk) for 2 h at room temperature, and then with the anti-β-tubulin (clone TUB 2.1) (Sigma-
Aldrich, St. Louis, MO) primary antibody 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, 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, Canada).
Detection of bands was performed with Super Rx medical X-Ray film (Fujifilm, Tokyo
Japan).
15

ACCEPTED MANUSCRIPT
5
5
.2 Chemical Methods
.2.1 General.
Proton NMR spectra were recorded on a Bruker AM-300 spectrometer (Bruker, Germany).
Chemical shifts (δ) are reported in parts per million. 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 23 min on an Alltech Alltima C18 reversed-phase column (5 µm, 250 mm × 4.6 mm)
equipped with an Alltech Alltima C18 precolumn (5 µm, 7.5 mm × 4.6 mm) with a
MeOH/H O linear gradient at 1.0 mL/min. Purity of the final compounds was > 95%. All
2
reactions were performed under a dried Ar atmosphere. All chemicals were supplied by
Sigma-Aldrich Canada (Oakville, Ontario, Canada) or VWR International (Mont-Royal,
Québec, Canada) and used as received unless specified otherwise. Liquid flash
chromatography was performed on silica gel F60, 60A, 40−63 µm supplied by Silicycle
(Québec, Canada) using a 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 the reactions was monitored by TLC
using precoated silica gel 60 F254 TLC plates (VWR International, Ville Mont-Royal,
Québec). The chromatograms were viewed under UV light at 254 and/or 265 nm.
5.2.2 General Preparation of Compounds 8a-c and 9a-c.
Method A. Ethyl isocyanate (1.2-2 Eq) was added to a solution of the appropriate aniline
16a, c and 17a, c, 1 Eq) in methylene chloride (15 mL). The reaction mixture was stirred at
(
16

ACCEPTED MANUSCRIPT
room temperature for 3-5 days. The solvent was evaporated under reduced pressure, and the
compound was purified by flash chromatography using a mixture of hexane/ethyl acetate.
Method B. Ethyl isocyanate (3 Eq) was added to the appropriate aniline (16b and 17b, 1 Eq)
dissolved in acetonitrile (2 mL). The reaction mixture was heated to 90 °C in a sealed tube
and stirred for 48 h. Afterward, the mixture was cooled to room temperature, SiO was added
2
and the solvent evaporated to dryness under reduced pressure. The residue adsorbed on SiO2
was purified by flash chromatography on silica gel using a mixture of hexane/ethyl acetate.
5
5
.2.3 Characterization of Compounds 8a-c and 9a-c
.2.3.1 (Z)-1-(4-(3-Chlorostyryl)phenyl)-3-ethylurea (8a). Method A, flash chromatography
1
(
hexane/ethyl acetate (75:25)). Yield: 57%; fade yellow solid; mp: 121-123 °C; H RMN
(
DMSO-d ): δ 8.50 (s, 1H, NH), 7.31-7.19 (m, 6H, Ar), 7.09 (d, 2H, J = 8.6 Hz, Ar), 6.61 (d,
6
1H, J = 12.2 Hz, CH), 6.48 (d, 1H, J = 12.2 Hz, CH), 6.13 (t, 1H, J = 5.4 Hz, NH), 3.14-3.10
1
3
(
m, 2H, J = 7.1 Hz, CH ), 1.05 (t, 3H, J = 7,1 Hz, CH ); C NMR (DMSO-d ): δ 155.0,
2 3 6
1
40.1, 139.5, 133.0, 131.3, 130.2, 129.1, 128.7, 128.1, 127.1, 126.9, 126.7, 117.3, 34.0, 15.4;
+
MS (ES+) found 301.10; C H ClN O (M + H) requires 301.11.
17
17
2
5.2.3.2 (Z)-1-(4-(3,5-Dimethylstyryl)phenyl)-3-ethylurea (8b). Method B, flash
chromatography (1. hexane to hexane/ethyl acetate (65:35) and 2. methylene chloride to
1
methylene chloride/ethyl acetate (90:10)). Yield: 81%; white solid; mp: 147-149 °C; H RMN
(
CDCl ): δ 7.20 (d, 2H, J = 8.7 Hz, Ar), 7.12 (d, 2H, J = 8.5 Hz, Ar), 6.89 (s, 2H, Ar), 6.84 (s,
3
1
H, Ar), 6.52-6.42 (m, 2H, CH), 3.27 (q, 2H, J = 7.2 Hz, CH ), 2.22 (s, 6H, CH ), 1.12 (t, 3H,
2 3
1
3
J = 7.2 Hz, CH ); C NMR (CDCl ): 155.8, 137.7, 137.4, 137.3, 132.9, 129.9, 129.1, 128.8,
3
3
+
1
26.5, 120.5, 35.2, 21.2, 15.4; MS (ES+) found 295.15; C H N O (M + H) requires 295.18.
19 22 2
17

ACCEPTED MANUSCRIPT
5.2.3.3 (Z)-1-Ethyl-3-(4-(3,4,5-trimethoxystyryl)phenyl)urea (8c). Method A, flash
chromatography (hexane/ethyl acetate (75:25)). Yield: 21%; brown-orange solid; mp: 104-
1
1
06 °C; H RMN (CDCl ): δ 7.23-7.17 (m, 4H, Ar), 7.09 (s, 1H, NH), 6.50-6.47 (m, 3H, Ar
3
and CH), 6.42 (d, 1H, J = 12.3 Hz, CH), 5.26 (t, 1H, J = 5.4 Hz, NH), 3.84 (s, 3H, CH ), 3.67
3
1
3
(
s, 6H, CH ), 3.28-3.19 (m, 2H, CH ), 1.10 (t, 3H, J = 7.2 Hz, CH ); C NMR (CDCl ): δ
3 2 3 3
1
1
55.8, 152.9, 138.3, 136.7, 133.1, 131.7, 129.8, 129.6, 129.1, 119.3, 105.9, 61.0, 55.9, 35.0,
+
5.4; MS (ES+) found 357.20; C H N O (M + H) requires 357.18.
20
24
2
4
5.2.3.4 (E)-1-(4-(3-Chlorostyryl)phenyl)-3-ethylurea (9a). Method A, flash chromatography
1
(
hexane/ethyl acetate (75:25)). Yield: 49%; fade orange solid; mp: 208-210 °C; H RMN
(
DMSO-d ): δ 8.58 (s, 1H, NH), 7.66 (s, 1H, Ar), 7.54-7.37 (m, 6H, Ar), 7.30-7.25 (m, 2H,
6
Ar and CH), 7.09 (d, 1H, J = 16.2 Hz, CH), 6.16 (t, 1H, J = 5.3 Hz, NH), 3.15-3.08 (m, 2H,
13
CH ), 1.07 (t, 3H, J = 7.2 Hz, CH ); C NMR (DMSO-d ): δ 155.0, 140.7, 139.9, 133.6,
2
3
6
1
3
30.5, 130.1, 129.4, 127.3, 126.7, 125.6, 124.8, 124.2, 117.6, 34.0, 15.5; MS (ES+) found
+
01.10; C H ClN O (M + H) requires 301.11.
17
17
2
5.2.3.5 (E)-1-(4-(3,5-Dimethylstyryl)phenyl)-3-ethylurea (9b). Method B, flash
chromatography (1. hexane to hexane/ethyl acetate (65:35) and 2. methylene chloride to
1
methylene chloride/ethyl acetate (90:10)). Yield: 50%; white solid; mp: 216-217 °C; H RMN
(
CDCl and MeOD): δ 7.12 (d, 2H, J = 8.6 Hz, Ar), 7.05 (d, 2H, J = 8.7 Hz, Ar), 6.82 (s, 2H,
3
Ar), 6.75 (d, 1H, J = 15 Hz, CH), 6.64 (d, 1H, J =16.3 Hz, CH), 6.58 (s, 1H, Ar), 2.96 (q, 2H,
1
3
J = 7.3 Hz, CH ), 2.03 (s, 6H, CH ), 0.87 (t, 3H, J = 7.3 Hz, CH ); C NMR (CDCl and
2
2
3
3
MeOD): δ 156.4, 138.6, 137.6, 137.1, 131.4, 128.6, 127.4, 126.6, 126.5, 123.7, 118.6, 34.2,
+
2
0.5, 14.5; MS (ES+) found 295.18; C H N O (M + H) requires 295.18.
19
22
2
5.2.3.6 (E)-1-Ethyl-3-(4-(3,4,5-trimethoxystyryl)phenyl)urea (9c). Method A, flash
chromatography (methylene chloride/ethyl acetate (85:15)). Yield: 69%; white solid; mp:
18

ACCEPTED MANUSCRIPT
1
1
85-187 °C; H RMN (DMSO-d ): δ 7.36 (d, 2H, J = 8.6 Hz, Ar), 7.30 (d, 2H, J = 8.6 Hz,
6
Ar), 6.92 (m, 2H, CH), 6.66 (s, 2H, Ar), 3.85 (s, 6H, CH ), 3.82 (s, 3H, CH ), 3.21 (q, 2H, J =
3
3
1
3
7
1
1
.2 Hz, CH ), 1.09 (t, 3H, J = 7.3 Hz, CH ); C NMR (CDCl and MeOD): δ 156.2, 153.3,
2 3 3
38.9, 137.4, 133.5, 131.6, 127.9, 127.1, 126.8, 119.5, 119.4, 103.3, 61.0, 56.0, 34.9, 34.7,
+
5.3; MS (ES+) found 357.20; C H N O (M + H) requires 357.18.
20
24
2
4
5.2.4 General Preparation of Compounds 10a-c and 11a-c. 2-Chloroethyl isocyanate (1.2
Eq) was added to a solution of the appropriate aniline (16a-c and 17a-c, 1 Eq) in methylene
chloride (15 mL). The reaction mixture was stirred at room temperature for 1-3 days. The
solvent was evaporated under reduced pressure, and the compound was purified by flash
chromatography.
5
5
.2.5 Characterization of Compounds 10a-c and 11a-c
.2.5.1
(Z)-1-(2-Chloroethyl)-3-(4-(3-chlorostyryl)phenyl)urea
(10a).
Flash
chromatography (methylene chloride/ethyl acetate (75:25)). Yield: 75%; white solid; mp:
1
1
00-102 °C; H RMN (DMSO-d ): δ 8.75 (s, 1H, NH), 7.33-7.20 (m, 6H, Ar), 7.10 (d, 2H, J
6
=
8.5 Hz, Ar), 6.62 (d, 1H, J = 12.2 Hz, CH), 6.50 (d, 1H, J = 12.2 Hz, CH), 6.45 (t, 1H, J =
1
3
5
1
4
.7 Hz, NH), 3.66 (t, 2H, J = 6.1 Hz, CH ), 3.45-3.41 (m, 2H, CH ); C NMR (DMSO-d ): δ
2 2 6
54.9, 139.8, 139.5, 133.0, 131.3, 130.2, 129.1, 129.0, 128.1, 127.1, 126.9, 126.8, 117.4, 44.4,
+
1.2; MS (ES+) found 335.10; C H Cl N O (M + H) requires 335.07.
17
16
2
2
5.2.5.2
(Z)-1-(2-Chloroethyl)-3-(4-(3,5-dimethylstyryl)phenyl)urea
(10b).
Flash
chromatography (methylene chloride/methanol (96:4)). Yield: 87%; white solid; mp: 103-105
1
°
C; H RMN (CDCl ): δ 7.22 (d, 2H, J = 8.5 Hz, Ar), 7.14, (d, 2H, J = 8.6 Hz, Ar), 6.89-6.75
3
(m, 4H, Ar and NH), 6.51 (d, 1H, J = 12.4 Hz, CH), 6.45 (d, 1H, J = 11.4 Hz, CH), 5.43 (br,
1
3
1
H, NH), 3.63-3.58 (m, 4H, CH ), 2.22 (s, 6H, CH ); C NMR (CDCl ): δ 155.5, 137.8,
2 3 3
19

ACCEPTED MANUSCRIPT
137.3, 136.9, 133.2, 130.1, 130.0, 129.0, 128.8, 126.5, 120.7, 44.8, 42.1, 21.3; MS (ES+)
+
found 329.15; C H ClN O (M + H) requires 329.14.
1
9
21
2
5.2.5.3 (Z)-1-(2-Chloroethyl)-3-(4-(3,4,5-trimethoxystyryl)phenyl)urea (10c). Flash
chromatography (methylene chloride/ethyl acetate (90:10)). Yield: 41%; dark red solid; mp:
1
6
8
6
7-69 °C; H RMN (CDCl ): δ 7.82 (s, 1H, NH), 7.22 (d, 2H, J = 8.7 Hz, Ar), 7.16 (d, 2H, J =
3
.6 Hz, Ar), 6.50 (s, 2H, Ar), 6.47 (d, 1H, J = 12.5 Hz, CH), 6.40 (d, 1H, J = 12.2 Hz, CH),
.11 (t, 1H, J = 5.6 Hz, NH), 3.83 (s, 3H, CH ), 3.65 (s, 6H, CH ), 3.56-3.49 (m, 4H, CH );
3
3
2
1
3
C NMR (CDCl ): δ 156.0, 152.8, 138.3, 136.6, 133.2, 131.6, 129.7, 129.7, 129.0, 118.9,
3
+
1
05.9, 61.0, 55.9, 44.4, 41.9; MS (ES+) found 391.15; C H ClN O4 (M + H) requires
2
0
23
2
391.14.
5.2.5.4
(E)-1-(2-Chloroethyl)-3-(4-(3-chlorostyryl)phenyl)urea
(11a).
Flash
chromatography (methylene chloride/ethyl acetate (90:10)). Yield: 97%; fade yellow solid;
1
mp: 192-193 °C; H RMN (DMSO-d ): δ 8.81 (s, 1H, NH), 7.65-7.25 (m, 9H, Ar and CH),
6
7
3
1
.10 (d, 1H, J = 16.4 Hz, CH), 6.47 (t, 1H, J = 5.7 Hz, NH), 3.68 (t, 2H, J = 6.2 Hz, CH2),
1
3
.47-3.41 (m, 2H, CH ); C NMR (DMSO-d ): δ 154.9, 140.4, 139.9, 133.6, 130.5, 130.0,
2
6
29.8, 127.3, 126.8, 125.6, 124.9, 124.5, 117.8, 44.4, 41.3; MS (ES+) found 335.10;
+
C H Cl N O (M + H) requires 335.07.
17
16
2
2
5.2.5.5
(E)-1-(2-Chloroethyl)-3-(4-(3,5-dimethylstyryl)phenyl)urea
(11b).
Flash
1
chromatography (hexane/ethyl acetate (75:25)). Yield: 59%; white solid; mp: 194-195 °C; H
RMN (DMSO-d ): δ 8.78 (s, 1H, NH), 7.47 (d, 2H, J = 8.5 Hz, Ar), 7.42 (d, 2H, J = 8.4 Hz,
6
Ar), 7.18-7.11 (m, 3H, Ar and CH), 7.01 (d, 1H, J = 16.4 Hz, CH), 6.89 (s, 1H, Ar), 6.46 (t,
1
H, J = 5.5 Hz, NH), 3.68 (t, 2H, J = 5.9 Hz, CH ), 3.47-3.41 (m, 2H, CH ), 2.29 (s, 6H,
2 2
13
CH ); C NMR (DMSO-d ): δ 155.0, 139.9, 137.6, 137.3, 130.3, 128.8, 127.9, 127.0, 126.1,
3
6
20

ACCEPTED MANUSCRIPT
+
1
24.1, 117.8, 44.5, 41.2, 21.0; MS (ES+) found 329.15; C H ClN O (M + H) requires
19 21 2
329.14.
5.2.5.6 (E)-1-(2-Chloroethyl)-3-(4-(3,4,5-trimethoxystyryl)phenyl)urea (11c). Flash
chromatography (methylene chloride/ethyl acetate (90:10)). Yield: 68%; white solid; mp:
1
1
61-163 °C; H RMN (CDCl ): δ 7.98 (s, 1H, NH), 7.38 (d, 2H, J = 8.6 Hz, Ar), 7.32 (d, 2H,
3
J = 8.5 Hz, Ar), 6.94 (d, 1H, J = 16.6 Hz, CH), 6.88 (d, 1H, J = 16.4 Hz, CH), 6.68 (s, 2H,
13
Ar), 6.24 (t, 1H, J = 5.7 Hz, NH), 3.87 (br, 9H, CH ), 3.62-3.52 (m, 4H, CH ); C NMR
3
2
(
CDCl ): δ 156.3, 153.3, 138.6, 137.3, 133.6, 132.0, 127.8, 127.2, 119.9, 103.3, 61.1, 56.1,
3
+
4
4.3, 42.0; MS (ES+) found 391.15; C H ClN O (M + H) requires 391.14.
20
23
2
4
5.2.6 General Preparation of Compounds 12a-c and 13a-c.
Sodium hydride (3 Eq) was added slowly to a cold solution of 2-chloroethylurea derivatives
compounds 10a-c and 11a-c, 1 Eq) in dry tetrahydrofuran under Ar atmosphere. The ice bath
(
was then removed after 30 min and the reaction mixture was stirred at room temperature for 4
h. Excess of sodium hydride was quenched by the addition of water (20 mL) at 0 °C.
Tetrahydrofuran from the reaction mixture was evaporated under reduced pressure. The
aqueous phase was extracted thrice by methylene chloride (20 mL). The combined organic
phases were washed with brine (40 mL), dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness under vacuum. The residue was purified by flash chromatography on
silica gel.
5
5
.2.7 Characterization of Compounds 12a-c and 13a-c.
.2.7.1 (Z)-1-(4-(3-Chlorostyryl)phenyl)imidazolidin-2-one (12a). Flash chromatography
1
(
hexane/ethyl acetate (75:25)). Yield: 85%; white sticky solid; H RMN (DMSO-d ): δ 7.48
6
(d, 2H, J = 8.7 Hz, Ar), 7.32-7.15 (m, 6H, Ar), 7.01 (s, 1H, NH), 6.65 (d, 1H, J = 12.2 Hz,
21

ACCEPTED MANUSCRIPT
CH), 6.53 (d, 1H, J = 12.2 Hz, CH), 3.84 (t, 2H, J = 7.4 Hz, CH ), 3.43-3.31 (m, 2H, CH
2
2
1
3
hidden by the water peak); C NMR (DMSO-d ): δ 158.9, 140.2, 139.5, 133.1, 131.1, 130.3,
6
1
29.1, 128.9, 128.1, 127.1, 127.0, 116.5, 44.3, 36.5; MS (ES+) found 299.10; C H ClN O
1
7
15
2
+
(
M + H) requires 299.10.
5.2.7.2
(Z)-1-(4-(3,5-Dimethylstyryl)phenyl)imidazolidin-2-one
(12b).
Flash
chromatography (methylene chloride to methylene chloride/ethyl acetate (75:25)). Yield:
1
9
7
6
1
9%; white solid; mp: 169-171 °C; H RMN (CDCl and MeOH): δ 7.35 (d, 2H, J = 9.2 Hz),
3
,22 (d, 2H, J = 8.7 Hz), 6.89 (s, 2H, Ar), 6.84 (s, 1H, Ar), 6.48 (s, 2H, CH), 3.91 (t, 2H, J =
1
3
.0 Hz, CH ), 3.53 (t, 2H, J = 8.7 Hz, CH ), 2.22 (s, 6H, CH ); C NMR (CDCl ): δ 160.1,
2
2
3
3
38.4, 137.1, 137.0, 131.3, 129.0, 128.8, 128.7, 128.0, 125.9, 117.0, 44.9, 36.7, 20.2; MS
+
(
ES+) found 293.15; C H N O (M + H) requires 293.17.
1
9
20
2
5.2.7.3
(Z)-1-(4-(3,4,5-Trimethoxystyryl)phenyl)imidazolidin-2-one
(12c).
Flash
chromatography (methylene chloride to methylene chloride /ethyl acetate (50/50)). Yield:
1
4
9%; yellow oil; H RMN (CDCl ): δ 7.40 (d, 2H, J = 7.8 Hz, Ar), 7.25 (d, 2H, J = 8,0 Hz,
3
Ar), 6.49-6.40 (m, 4H, Ar and CH), 6.11 (s, 1H, NH), 3.87-3.82 (m, 5H, CH and CH ), 3.66
2
3
1
3
(
s, 6H, CH ), 3.52 (t, 2H, J = 7.6 Hz, CH ); C NMR (CDCl ): δ 160.1, 152.9, 139.2, 137.1,
3 2 3
1
32.9, 131.2, 129.5, 129.2, 117.2, 105.9, 60.9, 56.0, 45.2, 37.5; MS (ES+) found 355.15;
+
C H N O (M + H) requires 355.17.
20
22
2
4
5.2.7.4 (E)-1-(4-(3-Chlorostyryl)phenyl)imidazolidin-2-one (13a). Flash chromatography
1
(
hexane/ethyl acetate (75:25)). Yield: 97%; white solid; mp: 249-251 °C; H RMN (DMSO-
d6): δ 7.68 (br, 1H, NH), 7.62-7.53 (m, 5H, Ar), 7.42-7.29 (m, 3H, Ar), 7.17-7.06 (m, 2H,
13
CH), 3.89 (t, 2H, J = 7.3 Hz, CH ), 3.43 (t, 2H, J = 8.0 Hz, CH ); C NMR (DMSO-d ): δ
2
2
6
1
3
58.9, 140.7, 139.8, 133.6, 130.5, 129.9, 129.9, 127.1, 126.8, 125.7, 124.9, 124.8, 116.9, 44.4,
+
6.6; MS (ES+) found 299.10; C H ClN O (M + H) requires 299.10.
17
15
2
22

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5.2.7.5
(E)-1-(4-(3,5-Dimethylstyryl)phenyl)imidazolidin-2-one
(13b).
Flash
chromatography (methylene chloride to methylene chloride/ethyl acetate (75:25)). Yield:
1
9
5%; white solid; mp: 207-209 °C; H RMN (DMSO-d ): δ 7.60-7.52 (m, 4H, Ar), 7.58 (d,
6
2
H, J = 8.9 Hz, Ar), 7.53 (d, 2H, J = 8.9 Hz, Ar), 7.19-7.01 (m, 5H, Ar and CH), 6.89 (br, 1H,
1
3
NH), 3.91-3.85 (m, 2H, CH ), 3.45-3.40 (m, 2H, CH ), 2.30 (s, 6H, CH ); C NMR (DMSO-
2
2
3
d6): δ 158.9, 140.2, 137.6, 137.2, 130.4, 128.9, 127.8, 126.8, 126.5, 124.1, 117.0, 44.4, 36.6,
+
2
1.0; MS (ES+) found 293.15; C H N O (M + H) requires 293.17.
19
20
2
5.2.7.6
(E)-1-(4-(3,4,5-Trimethoxystyryl)phenyl)imidazolidin-2-one
(13c).
Flash
chromatography (methylene chloride/ethyl acetate (50:50)). Yield: 99%; white solid; mp:
1
1
7
3
1
3
57-159 °C; H RMN (CDCl ): δ 7.54 (d, 2H, J = 8.8 Hz, Ar), 7.48 (d, 2H, J = 8.8 Hz, Ar),
3
.02-6.91 (m, 2H, CH), 6.73 (s, 2H, Ar), 5.40 (br, 1H, NH), 3.97-3.92 (m, 8H, CH and CH ),
2
3
1
3
.87 (s, 3H, CH ), 3.58 (t, 2H, J = 8.3 Hz, CH ); C NMR (CDCl ): δ 159.7, 153.4, 139.6,
3
2
3
37.7, 133.3, 131.6, 127.7, 127.3, 127.0, 117.9, 103.4, 61.0, 56.1, 45.3, 37.5; MS (ES+) found
+
55.15; C H N O (M + H) requires 355.17.
20
22
2
4
5.2.8 General Preparation of Compounds 15a-c.
Compounds 15a-c were synthesized using the methods described by Cushman [22] and Fortin
17]. First, compound 14 was prepared according to the procedure of Fortin et al. [17].
[
Briefly, triphenyphosphine (1.0 Eq, 13.9 mmol) was added to a solution of 4-nitrobenzyl
bromides (1.0 Eq, 13.9 mmol) in toluene (60 mL) and heated to reflux for 12 h. The mixture
was then cooled at room temperature and the precipitate was filtrate and triturate with toluene
to give a white solid in quantitative yield. Afterwards, sodium hydride (3 Eq, 12.5 mmol) was
added portion-wise to a well-stirred suspension of 4-nitrobenzylphosphonium bromide 14 (2
Eq, 8.4 mmol) and a relevant benzaldehyde (2 Eq, 8.4 mmol) in methylene chloride (90 mL)
under an argon atmosphere at 0 °C. Subsequently, the mixture was warmed to room
23

ACCEPTED MANUSCRIPT
temperature. After further stirring for 24 h, excess sodium hydride was quenched by the
addition of water (20 mL) at 0 °C. The aqueous phase was extracted thrice by methylene
chloride (20 mL). The combined organic phases were washed with brine (40 mL), dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness under vacuum. The residue was
purified by flash chromatography on silica gel with a mixture of hexane/methylene chloride
for 15a and hexane/ethyl acetate for 15b, c. The geometric isomers of compound 15b (yield,
Z-isomer 33% and E-isomer 50%) were separated at this step whereas the geometric isomers
of compounds 15a, c were used as a mixture for the next chemical reaction (yield, 83% for
1
5
5a and 98% for the 15c).
.2.9 General Preparation of Compounds 16a-c and 17a-c.
The appropriate nitro compound (1.0 Eq, 2.1 mmol) was dissolved in a mixture of EtOH and
H O (10:1, 60 mL). Powdered iron (7.3 Eq, 15.1 mmol) and a few drops of concentrated
2
hydrochloric acid (8 drops) were added and the mixture was refluxed for 4h. After cooling,
the mixture was filtered on Celite® and rinsed with methylene chloride. Then, the ethanol and
methylene chloride were evaporated and the aqueous phase was extracted thrice by methylene
chloride (50 mL). The combined organic phases were washed with brine (60 mL), dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness under vacuum. The residue was
purified by flash chromatography on silica gel using a mixture of hexane/methylene chloride
for 16a (yield: 20%) and 17a (yield: 44%), hexane/ethyl acetate for 16b (yield: 54%) and 17b
(
yield: 72%), and methylene chloride/ethyl acetate for 16c (yield: 26%) and 17c (yield: 27%).
All the geometric isomers were isolated at this step.
Acknowledgment
This work was supported by a grant from the Canadian Health Research Institute (RCG, Grant
#MOP-79334 and #MOP-89707).
24

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