Selective alkylation of βII-tubulin and thioredoxin-1 by structurally related subsets of aryl chloroethylureas leading to either anti-microtubules or redox modulating agents

Article abstract of DOI:10.1016/j.bmc.2008.06.028

Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as β_II-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G₀/G₁ phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to β-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G₀/G₁. On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating β_II-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward β-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.

Full text of DOI:10.1016/j.bmc.2008.06.028

Bioorganic & Medicinal Chemistry 16 (2008) 7277–7290
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective alkylation of bII-tubulin and thioredoxin-1 by structurally related
subsets of aryl chloroethylureas leading to either anti-microtubules
or redox modulating agents
a
a
a,
Jessica S. Fortin a,c, , Marie-France Côté , Jacques Lacroix , Michel Desjardins , Éric Petitclerc a,b
,
*
René C.-Gaudreault a,b,
*
a Unité des Biotechnologies et de Bioingénierie, CHUQ, Hôpital Saint-François d’Assise, Québec, QC, Canada G1L 3L5
b Département de Médecine, Faculté de Médecine, Université Laval, Québec, QC, Canada G1K 7P4
c Faculté de Pharmacie, Université Laval, Québec, QC, Canada G1K 7P4
a r t i c l e i n f o
a b s t r a c t
Article history:
Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins
such as bII-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alky-
lates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumula-
tion of cells in G0/G1 phase. We have evaluated the effects of various substituents and their position on
the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle pro-
gression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to b-tubulin. The same
experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety
is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results
suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cyclo-
alkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression
in G0/G1. On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused ali-
phatic ring are alkylating bII-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea deriv-
atives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward b-
tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1
translocation.
Received 15 May 2008
Revised 14 June 2008
Accepted 17 June 2008
Available online 20 June 2008
Keywords:
Aryl chloroethylurea
bII-tubulin
Thioredoxin-1
Alkylation
Selectivity
Chemo sensitizers
Radio sensitizers
Cancer
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
points and the actin stress fibers, cell rounding, detachment of
the cells from the extracellular matrix and death by anoikis.9
N-Aryl-3-(2-chloroethyl)ureas (CEUs) are potential anti-cancer
drugs that have been developed in our laboratory over the past
two decades.1–12 At first, structure–activity relationship studies
have identified a subset of CEU binding covalently to the colchi-
cine-binding site on bII-tubulin through the acylation of the
glutamic acid-198 residue.3,4 The covalent binding of CEU to
b-tubulin results in a complex sequence of events initiated by
microtubule depolymerization and leading to cytoskeleton disrup-
tion, arrest of the cell cycle progression in G2/M phase.3–5,9,11 These
events are paralleled by disorganization of the focal adhesion
CEUs of the first generation are active anti-microtubule agents
on tumor cells having developed chemoresistance through mecha-
nisms such as increased P-glycoprotein expression, increased DNA
repair, increased intracellular glutathione-S-transferase activity,
alteration of topoisomerase II activity and even cell adhesion-med-
iated drug resistance.2,5 Two prototypical CEUs, namely tBCEU and
ICEU, were shown to inhibit angiogenesis in vitro in Boyden’ cham-
bers and in vivo in the CAM assay and in mice using the Matrigel
assay.5 Furthermore [14C]tBCEU and [125I]ICEU were also shown
to be preferentially biodistributed in organs of the gastro-intestinal
tract, notably, duodenum and colon. ICEU was demonstrated as ac-
tive as 5-fluorouracil on mice bearing CT-26 mouse colon
carcinoma.11,12
* Corresponding authors. Tel.: +1 418 525 4485; fax: +1 418 525 4372 (J.S.F.); tel.:
+1 418 525 4444x52363; fax: +1 418 525 4372 (R.C.G).
E-mail addresses: jessica.fortin.1@ulaval.ca (J.S. Fortin), rene.c-gaudreault@
crsfa.ulaval.ca (R. C.-Gaudreault).
Present address: OmegaChem Inc., 8800, Boulevard-de-la-Rive-Sud, Lévis, QC,
Canada G6V 9H1.
Recently, the widening of our structure–activity relationship
studies uncovered a compound designated as 1-(2-chloroethyl)-
3-(4-cyclohexylphenyl)urea, (compound 36) that exhibited a dra-
matic effect on the cell cycle progression when compared to tBCEU
or ICEU.13–16 The latter molecules arrested the cell cycle in G2/M
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.028

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J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
phase as the majority of anti-microtubules agents do while 36 was
arresting the cell cycle in G0/G1 phase instead.13–16 Interestingly,
using [14C]36, we confirmed that the drug does not covalently bind
to the colchicine-binding site.13–15 However, the drug was shown
to covalently bind to thioredoxin isoform-1 (Trx-1) and to the
aspartic acid residue in position 40 of prohibitin.14 Compound 36
is binding also, to a lesser extent, to galectin-1 and -3 and to the
mitochondrial voltage-dependent anion channel.
chloroacetylureas (CAUs) and 2-ethylureas (EUs), respectively.
These compounds were prepared by the nucleophilic addition of
either 2-chloroethylisocyanate, ethylisocyanate, or 2-chloroacety-
lisocyanate, respectively, on the corresponding anilines (Scheme
3).3,6–8,10,15,16 Anilines substituted by cycloalkyl moieties were pre-
pared using the Friedel–Craft alkylation followed by the nitration
of the aromatic moiety (Scheme 1). Anilines substituted by cyclo-
alkyloxy moieties were prepared using the Williamson’ reaction
followed by the reduction of the aromatic nitro group using SnCl2
(Scheme 2).15,16 OXA derivatives were prepared from the catalytic
cyclization of CEU in presence of KF adsorbed on SiO2 (ratio 4:6) in
refluxing acetonitrile overnight (Scheme 3).
Thioredoxin-1 (Trx-1) represents an important target for the
development of new anti-cancer drugs. Trx-1 is a 12-kDa protein
involved in the control of the redox homeostasis of the cell.17–30
Trx-1 is located normally in the cytoplasm and the nucleus.18 How-
ever, Trx-1 is known to translocate to the nucleus when the cell is
under a number of physiological and stress conditions such as UV
radiations, H2O2, and cisplatinum leading to the stimulation of
2.2. Anti-proliferative activity
various transcription factors such as Nf-
j
B and AP-1.17–19 In addi-
The anti-proliferative activity of CEU, OXA, CAU, and EU was
evaluated on human cell lines: MCF-7 breast carcinoma, M21 skin
melanoma, and HT-29 colon carcinoma cells. Cell growth inhibi-
tion was assessed according to the sulforhodamine method used
at the NCI/NIH Developmental Therapeutics Program.31 As de-
picted in Tables 1 and 2 compounds 28–37 and 39–56 have exhib-
ited significant cell growth inhibition. CEU 27, 38, and EU 57
tion, Trx binds to PTEN and ASK-1 and contributes significantly to
the cell survival signaling.23–25 Trx-1 is overexpressed in a large
number of cancer cell lines and correlates with poor patient prog-
nostics.19–22,25 Moreover, Trx-1 is involved in the chemoresistance
to anti-neoplastic agents and radioactive radiations.19–22 In that
context, several studies showed that abrogating the cellular activ-
ity of Trx-1 reversed the chemoresistance to anti-neoplastics, such
as cisplatinum, mitomycine C, doxorubicin, etoposide, and doce-
taxel, and raised the sensibility of tumor cells to radiotherapy.18–22
Such information prompted us to expand our structure-rela-
tionship studies to develop a new subset of CEU based on the
molecular structure of the prototypical compound 36 that arrests
the cell cycle in G0/G1 and covalently binds to Trx-1. Therefore,
in this study, we have evaluated the nature and the position of dif-
ferent substituents on the aromatic ring of a series of derivatives of
compound 36 on (i) the anti-proliferative activity, (ii) the cell cycle
progression, (iii) the nuclear translocation of Trx-1, and (iv) their
covalent binding to b-tubulin. We have also assessed the effects
of modulating the electrophilic character of the 2-chloroethyl ami-
no moiety of selected derivatives on the same parameters.
displayed a GI50 higher than 50
inactive.
lM and thus were considered as
Ethyl urea derivatives, exemplified in Table 1 by compound 57,
are the non-electrophilic counterparts of CEU and have no notice-
able impacts on cell growth and on the intracellular localization of
Trx-1.10,15,16 This confirmed that the 2-chloroethyl amino moiety
of CEU is prerequisite to the anti-proliferative activity of these mol-
ecules. The replacement of the 2-chloroethyl amino moiety by the
bioisosteric 2-aminooxazolinyl moiety (e.g., compounds 58–62)
was conducted similarly to anti-proliferative molecules (Table 2).
The bioisosteric OXA derivatives 58–62 exhibited GI50 that were
generally similar to their CEU counterparts 54, 36–38. However,
CAU derivatives 63–67 bearing the potent electrophilic 2-chloro-
acetylamino moiety were 10 times more potent than their corre-
sponding CEU and OXA derivatives. The increase of the
electrophilic character of CEU through the replacement of the 2-
chloroethyl amino group by a 2-chloroacetylamino moiety (e.g.,
compounds 27, 36–38) led to molecules having a higher anti-pro-
liferative activity than CEU and OXA.
2. Results and discussion
2.1. Chemistry
The aromatic ring of CEU must be substituted to initiate any
anti-proliferative activity. To that end, the aryl 3-(2-chloroeth-
Schemes 1–3 illustrate the synthesis of CEU, N-aryl amino-2-
oxazolines (4,5-dihydro-N-phenyloxazol-2-amines, OXAs) and 2-
NO2
4
NO2
R
5
NO2
NO2
NO2
NH2
NH2
b
c
6
7
R
a
OH
OH
1
2
R= aliphatic substituent
10-15
8
NO2
OH
9
3
Scheme 1. Synthesis of selected anilines, Part 1. Reagents: (a) benzene, H2SO4; (b) acetic anhydride, HNO3; (c) SnCl2ꢀ2H2O, EtOH, reflux.

J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
7279
OH
O
O
a
c
NO2
Br
NH2
+
n
NO2
n=1,2,3,4
16-19
n=1,2,3,4
22-25
n=1,2
HO
b
c
B
+
HO
20
21
Br
NO2
NO2
NH2
O
NO2
NH2
d
26
Scheme 2. Synthesis of selected anilines, Part 2. Reagents: (a) NaOH, DMF, reflux; (b) NiI2, trans-2-aminocyclohexanol, NaHMDS, N2, 2-PrOH; (c) SnCl2ꢀ2H2O, EtOH, reflux; (d)
AcOH, H2SO4, Pd/C, ethanol, H2.
CEU 34–36, 40, 41, 42, 43, 46, 47, 50–52 arrested cell in G0/G1. The
treatment of cells with the aforementioned CEU resulted in the in-
crease of cells population in G0/G1 phase by 4–16%. Figure 1 illus-
trates the accumulation of cells in G0/G1 induced by 36 and
compound 64. Conversely, the anti-microtubule CEUs 29, 31, 32,
33, 49, 50, and 55–57 increased the percentage of cells in G2/M
by 11–55%.
O
O
R
Cl
Cl
R
N
N
b
N
N
H
H
H
27-56
58-62
R
O
a
O
NH2
R
R
N
N
Treatment of M21 cells with compounds 54 and 55 for 24 h re-
sulted in their accumulation in G2/M, destabilization of the cyto-
skeleton, interruption of the b-tubulin entry in the mitotic
spindle, and apoptosis.3–5,14 On the other hand, 36 arrested the cell
cycle in G0/G1 phase. Compound 36 has been shown using mass
spectrometry to covalently bind to Trx-1. CEUs 28, 29, 30, 32, 33,
49, 56 target b-tubulin, leading to the arrest of the cell cycle pro-
gression in G2/M. CEUs 36, 39, 40, 41, 42, 46, 47, 50–53, and OXA
59 lead to the abrogation of the nuclear translocation of Trx-1
and in the accumulation of cells in G0/G1 phase. These phenomena
might be related to the fact that nuclear Trx-1 induces cell tran-
scription and proliferation through the reduction of Ref-1 and the
induction in parallel of AP-1 activity, which is a powerful transcrip-
tion factor promoting cell growth and survival.33–35 AP-1 is a key
element in G0 to G1 transition via the AP-1-responsive cyclin D1
gene.33–35 We hypothesized that CEUs, which confine Trx-1 to
the cytoplasmic compartment, inhibit important pathways of nu-
clear transcriptional activity required for cell cycle regulation and
cell proliferation.
R= Substituted ring
H
H
10-15, 21-26
63-67
O
N
N
H
H
57
Scheme 3. Synthesis of aryl-substituted ethylureas. Reagents: (a) 2-chloroethyl-
isocyanate, 2-chloroacetylisocyanate, or ethylisocyanate, CH2Cl2; (b) SiO2ꢀKF,
CH3CN.
yl)urea 27 does not exhibit any anti-proliferative activity (Table 1).
In addition, previous studies have clearly established that substitu-
tion on either position 2 and 6 abrogates also the biological activ-
ity.1,6–8,10 From this study, it is not possible to predict any potential
advantages to substitute the aromatic ring in position 3 versus po-
sition 4. However, the substitution of the aromatic ring on these
positions by cycloalkyl or oxocycloalkyl groups having more than
four carbon atoms leads to a significant anti-proliferative activity
on all tumor cell lines tested. However, monosubstituted CEU ap-
peared less active than ‘bisubstituted’ CEUs on positions 3 and 4
through non-heterocyclic fused rings such as the 2,3-dihydro-1H-
indenyl 28, the 1,2,3,4-tetrahydronaphthalenyls 29 and 30, the
6,7,8,9-tetrahydro-5H-benzo7annulenyl 31, the 9H-fluorenyl 33,
and the phenyl ring 32. These CEUs exhibited similar anti-prolifer-
ative activities to several CAU tested. The presence of atoms such
as oxygen and nitrogen in the alicyclic ring (e.g., 38) decreases dra-
matically the anti-proliferative activity.
2.4. b-Tubulin alkylation on M21-treated human melanoma
cancer cells
Previous studies had shown that b-tubulin is a major target for
tBCEU (54) and ICEU (55).3–5,14 The covalent binding of CEU to b-
tubulin increases the electrophoretic mobility of the bII-tubulin
by-product protein on SDS–PAGE electrophoresis, resulting in the
appearance of a second band of bII-tubulin band exhibiting an
apparent lower molecular weight.3
To assess the intracellular alkylation of the colchicine-binding
site on bII-tubulin by selected CEU (28–38, 32–33, 34–37, 39–
41, 42–57, 59, 60, 62–67), M21 human melanoma cancer cells
were treated with two times the GI50 of CEU for 16, 24 and
2.3. Effects of aromatic chloroethylureas and derivatives on the
cell cycle
48 h using molecules having GI50 ranging from 1 to 50 lM. Pro-
Table 1 shows percentage of MCF-7 cells in G0/G1, S, and G2/M
phases, respectively, after treatment with CEUs 27–57 and EU 58
for 24 h at two times their respective GI50. Control cells (DMSO)
were 59%, 22%, and 19% in G0/G1, S, and G2/M phases, respectively.
teins were extracted and separated by SDS–PAGE. Tables 1 and 2
show Western blot analysis using a monoclonal anti-bII-tubulin
antibody. Compound 30 induced the production of the alkylated

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J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
Table 1
Evaluation of the anti-proliferative activity on HT-29, M21, and MCF-7 cells and the effect of cell cycle progression on MCF-7 cells treated with 2-times the GI50 of selected CEU
derivatives, PX-12, cisplatinum, colchicine, vinblastine and paclitaxel
O
R
Cl
N
H
N
H
Compound
Substituent
GI50
(l
M)
Cell cycle progression (%) on MCF-7a
b-Tubulin alkylationM21a
Trx-1 localization in M21a
HT-29
n.a.
M21
MCF-7
n.a.
G0/G1
S
G2/M
16, 24, and 48 h
277,41
n.a.
59
22
19
n.e.
286,7,10
2942
30
3.6
2.0
1.3
3.2
1.9
8.0
3.7
2.6
7.2
4.1
14.1
7.3
4.2
12
31
10
6
29
49
20
24
38
40
41
74
43
45
31
33
17
326,7
5.6
3310
2.3
19
5.1
27
14.2
22
11
63
57
29
32
8
3410
35
18
27
26
63
24
13
367,15,16
3743
3844
39
12.6
11.7
n.a.
7.8
21
18
n.a.
16
21
20
71
52
n.e
60
16
32
n.e
9
13
16
n.e
31
n.a.
18
n.e.
O
N

J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
7281
Table 1 (continued)
Compound
Substituent
GI50
(
lM)
Cell cycle progression (%) on MCF-7a
b-Tubulin alkylationM21a
Trx-1 localization in M21a
HT-29
9.1
M21
MCF-7
17
G0/G1
S
G2/M
16, 24, and 48 h
40
19
66
17
17
41
18
31
36
38
23
49
73
72
14
19
13
9
4215,16
4315,16
9.9
23
22
36
35
45
64
57
24
29
12
14
4415,16
4515,16
7.9
20
23
25
21
59
63
33
13
8
46
16
25
O
47
19
32
5.2
16
33
9.1
24
65
15
44
20
20
26
15
65
30
O
4815,16
2.4
5.6
4915,16
5015,16
5115,16
52
13
18
20
26
30
19
21
18
23
75
72
70
61
19
22
14
29
6
6
10.7
14
16
10
O
O
53
20
(continued on next page)

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J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
Table 1 (continued)
Compound
Substituent
GI50
(l
M)
Cell cycle progression (%) on MCF-7a
b-Tubulin alkylationM21a
Trx-1 localization in M21a
HT-29
2.3
M21
MCF-7
6.2
G0/G1
S
G2/M
16, 24, and 48 h
546,7,10 tBCEU
4.3
17
24
58
556,7,10 ICEU
1.9
2.9
3.9
3.8
6.0
25
16
29
53
19
9
33
41
13
27
19
11
23
22
42
43
58
20
62
80
22
19
566,7
6.5
PX-1230
cDDP
25
8.3
8.3
10.1
0.004
0.015
n.a.
12.9
6.4
COL
0.015
0.009
0.054
n.a.
n.a.
Pac
0.037
5715,16
n.a.
55
59
DMSO
n.a.
n.a.
In addition, the drug was assessed for b-tubulin alkylation using SDS–PAGE and the thioredoxin-1 intracellular localization was performed using a monoclonal anti-Trx-1
antibody.
n.a., non-active; n.e., not evaluated; cDDP, cisplatinum; Col, colchicine; Pac, paclitaxel.
a
Drugs were tested at two times their respective GI50
.
band of bII-tubulin after 24 h while CEUs 28, 29, 32, 33, 49, 54–
56 needed 48 h of incubation with M21 cells to exhibit a similar
level of alkylation. Interestingly, the other molecules tested did
not show the production of any alkylated bII-tubulin by-products
on SDS–PAGE.
a partial inhibition of the nuclear translocation of Trx-1. Com-
pounds 27, 30–34, 48, 49, 54, 56–58, 61–63, colchicine, vinblastine,
paclitaxel, and DMSO did not affect the intracellular location of
Trx-1. Cisplatinum, as expected, favor significantly the nuclear
translocation of Trx-132 but unexpectedly compounds 28, 38, and
PX-12 also stimulated the nuclear translocation of Trx-1 from the
cytosol. The mechanisms by which PX-12, 28 and 29 stimulate
the nuclear translocation of Trx-1 are still unknown.
2.5. Changes in the intracellular location of thioredoxin-1
We previously showed that compound 36 inhibited the translo-
cation of Trx-1 from the cytosol to the nucleus.15,16 The influence of
the new subset of CEU derivatives on the translocation of Trx-1
was assessed by immunocytofluorescence on M21 melanoma cells
for treated for 16 h with concentrations two times the GI50 of the
selected CEUs. Tables 1 and 2 show that compounds 29, 36, 37,
39–42, 44, 46, 47, 50–53, OXA 59, 60, CAU 64, 65, and 67 inhibited
significantly the translocation of Trx-1 from the cytosol into the
nucleus. Compounds 35, 32, 34, 35, 43, 45, 55, and 66 induced only
2.6. Selectivity of CEU toward Trx-1 versus b-tubulin
Our results obtained from previous and current experiments
demonstrate that slight modifications of the structure of the
substituents on the aromatic ring of CEU may lead to dramatic
modification of the pattern of the proteins alkylated by the
drugs.1,3,6–10,13–16 For example (i) aliphatic and branched aliphatic
CEUs having between
1 and 6 carbon atoms were shown

Table 2
Evaluation of the anti-proliferative activity, the ability to alkylate b-tubulin, and the intracellular localization of thioredoxin-1 of selected CEU, OXA, and CAU derivatives on M21 cells
R
O
O
O
R
O
N
R
R
Cl
N
Cl
H
N
H
N
H
N
H
N
H
Mol #
GI50
n.a.
(l
M)
b-tubulin alkylation
Trx-1 localization
Mol #
GI50
(l
M)
b-tubulin alkylation
Trx-1 localization
Mol #
GI50
6.9
(l
M)
b-tubulin alkylation
Trx-1 localization
16, 24, and 48 h
16, 24, and 48 h
16, 24, and 48 h
277,41
n.a.
58
81
n.a.
63
3615,16
21
5916
18
6416
0.9
2.4
8.3
1.1
3743
18
60
45
65
3844
n.a.
4.3
n.a.
61
n.a.
2.4
n.a.
66
O
N
5410
6210
6710
n.a., non-active.

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J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
36
59
64
A
B
CTRL
10 µM
50 µM
100 µM
CTRL
CTRL
50 µM
100 µM
200 µM
50 µM
100 µM
200 µM
59
36
64
CTRL
10 µM
50 µM
100 µM
CTRL
CTRL
50 µM
100 µM
200 µM
50 µM
100 µM
200 µM
Figure 1. Effect of CEU 36, OXA 59 and CAU 64 on cell cycle progression. Exponentially growing (A) MCF-7 or (B) HT-29 cells were incubated in absence or presence of 36, 59
at 50, 100 and 200 M (approximately two, four, and eight times their respective GI50) or 64 at 10 or 50 M for 24 h at 37 °C. The cell cycle was evaluated by flow cytometry
l
l
using propidium iodide staining.
previously to covalently bind to bII-tubulin,1,3,6–10 (ii) cyclization of
the n-hexyl CEU derivative into its cyclohexyl isomer led predom-
inantly to the covalent binding of the molecule to Trx-1 without
binding to bII-tubulin (Table 3),14–16 and (iii) homologation of the
indanyl CEU 28, that favors the nuclear translocation of Trx-1, into
its tetrahydronaphthalenyl homolog 29 decreased the nuclear
translocation of Trx-1 while both molecules are weakly alkylating
bII-tubulin. Tables 1 and 2 illustrate that CEUs derived from com-
pound 36 are exhibiting a strong inhibition of the translocation
of Trx-1 from the cytosol into the nucleus without any apparent
covalent binding to bII-tubulin.
sition 1 of the alicyclic ring. Modifications of the electrophilic char-
acter of CEU by replacing the 2-chloroethyl amino portion of the
pharmacophore by a 2-aminooxazolinyl or a 2-chloroacetylamino
moiety led also to compounds exhibiting slightly higher anti-pro-
liferative activity without significantly modifying the end-points
assessed in this study. We believe that besides sodium dichro-
mate23 these CEUs are among the first molecules reported to alkyl-
ate Trx-1 and to exhibit dramatic effects on its intracellular
distribution. Thioredoxin-1 being comprised in powerful mecha-
nisms of chemoresistance involving its translocation into the nu-
cleus, this new subset of CEU could be useful as adjuvant chemo-
or radio-sensitizing agents for cancer chemotherapy.
3. Conclusion
4. Experimental
This study aimed to understand the importance of the electro-
philicity and the presence of substituting groups on the aromatic
moiety of CEU on the ‘specificity’ of CEU toward b-tubulin and thi-
oredoxin-1, respectively, and also their effect on cell cycle progres-
sion. At first, our results confirmed our previous observation10 that
non-electrophilic EU derivatives were pharmacologically inactive
showing no effect on cell proliferation, cell cycle progression, and
thioredoxin translocation and b-tubulin alkylation. Previous exper-
iments had clearly established10 that CEUs are substituted in posi-
tion 3 or 4 or disubstituted in positions 3 and 4 by alkyl groups
bearing between 1 and 6 carbon atoms and their bioisosteres are
potent alkylators of the colchicine-binding site on tubulin. Unex-
pectedly, the cyclization of the n-hexyl group of 56 into 36 changed
a molecule that is arresting the cell cycle in G2/M phase, alkylating
b-tubulin without modifying significantly the intracellular locali-
zation of thioredoxin-1 into a compound that is arresting the cell
cycle progression at the G0/G1 phase, abrogating the nuclear trans-
location of thioredoxin-1, and that does not alkylate b-tubulin.
These results suggest that three simple structural modifications
of the aryl 2-chloroethyl pharmacophore may lead to the alkyl-
ation and the modulation of the activity of two different proteins
or groups of proteins and their respective signaling pathways
and then exhibiting different pharmacological properties. Our re-
sults suggest also that the inhibition of thioredoxin translocation
by CEU requires the presence of an alicyclic ring alone or linked
to the aromatic ring by an ether group either on position 3 or 4
of the aromatic ring. These CEU subsets are also active when
substituted by lower alkyl groups having 1–3 carbon atoms on po-
4.1. Chemistry and chemical methods
All drugs were dissolved in DMSO and used at final concentra-
tions lower than 0.12% (v/v) to avoid any potential toxicity. tBCEU,
ICEU, and cHCEU were prepared as published previously.10,15,16
Vinblastine sulfate, cisplatinum, colchicine, and paclitaxel were
obtained from Sigma Chemicals (St. Louis, MO). 1-methylpropyl
2-imidazolyl disulfide (PX-12) was prepared as described by Kirk-
patrick.30 Proton NMR spectra were recorded on a Bruker AM-300
spectrometer (Bruker, Germany). Chemical shifts (d) are reported
in parts per million, relative to the internal tetramethylsilane stan-
dard. IR spectra were recorded on a Unicam spectrometer. Uncor-
rected melting points were determined on an electrothermal
melting point apparatus. Mass spectra were performed on the most
potent CEU derivatives, at the University of Montréal, Montréal,
Canada. Chemicals and reagents were supplied by Aldrich Chemi-
cals (Milwaukee, WI). 7-nitro-1-tetralone, 1,2,3,4-tetrahydronaph-
thalene and 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene,
( )-trans-2-aminocyclohexanol hydrochloride were purchased
from Alfa Aesar (Ward Hill, MA, USA). Liquid flash chromatography
was performed on silica gel 60 A (American Chemicals Ltd, Mon-
treal, Canada), using the indicated solvent mixture expressed as
volume/volume ratios. Solvents and reagents were used without
purification unless specified otherwise. The progress of all reac-
tions was monitored using TLC on precoated silica gel plates
(Merck Silica Gel 60 F254). The chromatograms were viewed under
UV light at 254 nm.

J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
7285
4.2. General preparation of cycloalkylbenzenes (1–3)
CH2), 1.77–1.46 (m, 6H, 3ꢁ CH2); 13C NMR (CDCl3) d: 145.0,
138.7, 126.4, 115.2, 40.6, 29.7, 21.8.
A solution of 1-n-methylcyclohexanol or 1-n-ethylcyclohexanol
or 1-n-propylcyclohexanol (20 mmol) in benzene (15 mL) was
added dropwise at 0 °C to 10 mL of H2SO4 (12 M).36 The mixture
was stirred at room temperature for 3 h. The organic layer was sep-
arated, washed with brine and water, dried over anhydrous MgSO4,
and evaporated. The crude product was purified by liquid flash
chromatography using petroleum ether.
4.3.4. 1-(1-Methylcyclohexyl)-4-nitrobenzene (7)36
Compound 7 was synthesized from the nitration of compound
1. Yield: 88%; 1H NMR (CDCl3) d: 8.14 (d, 2H, J = 8.9 Hz, Ar), 7.51
(d, 2H, J = 8.8 Hz, Ar), 1.98 (m, 2H, CH2), 1.51 (m, 8H, 4ꢁ CH2),
1.38 (s, 3H, CH3); 13C NMR (CDCl3) d: 149.5, 145.8, 128.2, 120.5,
37.9, 30.7, 26.5, 22.9.
4.2.1. 1-(1-Methylcyclohexyl)benzene (1)36
4.3.5. 1-(1-Ethylcyclohexyl)-4-nitrobenzene (8)36
Compound 1 was synthesized from the alkylation of benzene
with 1-n-methylcyclohexanol. Yield: 68%; 1H NMR (CDCl3) d:
7.55 (d, 2H, J = 8.2 Hz, Ar), 7.50 (m, 2H, Ar), 7.36 (t, 1H, J = 7.6 Hz,
Ar), 2.18 (m, 2H, CH2), 1.76 (m, 8H, 4ꢁ CH2), 1.39 (s, 3H, CH3);
13C NMR (CDCl3) d: 150.0, 147.0, 128.4, 125.7, 125.4, 38.2, 37.5,
30.8, 26.9, 22.9.
Compound 8 was synthesized from the nitration of compound
2. Yield: 84%; 1H NMR (CDCl3) d: 8.15 (d, 2H, J = 7.3 Hz, Ar), 7.48
(d, 2H, J = 8.8 Hz, Ar), 2.19 (m, 2H, CH, CH2), 1.82–0.80 (m, 9H,
CH, CH2), 0.54 (t, 3H, J = 7.5 Hz, CH3); 13C NMR (CDCl3) d: 155.4,
145.7, 128.0, 123.2, 42.4, 37.7, 35.8, 26.7, 22.5, 7.8.
4.3.6. 1-Nitro-4-(1-propylcyclohexyl)benzene (9)36
4.2.2. 1-(1-Ethylcyclohexyl)benzene (2)36
Compound 9 was synthesized from the nitration of com-
pound 3. Yield: 75%; 1H NMR (CDCl3) d: 8.12 (d, 2H,
J = 8.7 Hz, Ar), 7.47 (d, 2H, J = 8.9 Hz, Ar), 2.06 (m, 3H, CH,
CH2), 1.85–0.86 (m, 11H, CH, CH2), 0.71 (t, 3H, J = 6.9 Hz,
CH3); 13C NMR (CDCl3) d: 155.8, 145.7, 127.8, 123.2, 46.2,
42.3, 36.1, 25.5, 22.3, 16.6, 14.6.
Compound 2 was synthesized from the alkylation of benzene
with 1-n-ethylcyclohexanol. Yield: 41%; 1H NMR (CDCl3) d: 7.39
(m, 5H, Ar), 2.17 (m, 3H, CH, CH2), 1.79–0.99 (m, 9H, CH, 4ꢁ
CH2), 0.64 (t, 3H, J = 7.3 Hz, CH3); 13C NMR (CDCl33) d: 146.9,
127.1, 126.9, 126.5, 41.5, 38.0, 35.9, 26.9, 22.6, 8.1.
4.2.3. 1-(1-Propylcyclohexyl)benzene (3)36
4.4. General preparation of 1-cycloalkyloxy-4-nitrophenols (16,
18) and 1-cycloalkyloxy-3-nitrophenols (17, 19)
Compound 3 was synthesized from the alkylation of benzene
with 1-n-propylcyclohexanol. Yield: 56%; 1H NMR (CDCl3) d: 7.43
(m, 5H, Ar), 2.20 (m, 3H, CH, CH2), 2.1–0.93 (m, 11H, CH, CH2),
0.88 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3) d: 147.3, 128.4,
127.1, 124.9, 46.6, 40.1, 36.5, 26.9, 22.6, 16.8, 14.6.
The appropriate cycloalkylbromide (2 equiv, 14.4 mmol) was
added to a solution of 3-nitrophenol or 4-nitrophenol (1 equiv,
7.19 mmol) and NaOH (2.5 equiv, 17.9 mmol) in 10 mL of DMF.38
The mixture was stirred under reflux for 48 h. After completion
of the reaction (TLC), water (200 mL) was added. The mixture
was extracted with dichloromethane. The organic phase was
washed with 1 N NaOH, water, and 1 N HCl, water, respectively.
Afterward, the solution was dried over anhydrous Na2SO4, filtered,
and evaporated under reduced pressure.
4.3. General preparation of cycloalkylnitrobenzenes (4–9)
To a solution of either 1, 2, 3, 1,2,3,4-tetrahydronaphthalene,
1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene, or 1-cyclo-
pentylbenzene (10 mmol) in 10 mL of acetic anhydride was
added dropwise a solution of HNO3 (0.05 M) in 1 mL of acetic
anhydride at 0 °C.36 The mixture was stirred at room tempera-
ture for 2 h, poured on ice, and extracted with ether. The com-
bined ether extracts were washed with a saturated NaHCO3
solution, water, and dried over anhydrous MgSO4. The solvent
was evaporated under reduced pressure, and the oily crude prod-
uct was purified by flash liquid chromatography with petroleum
ether: CH2Cl2 (3:1).
4.4.1. 1-(4-Methylcyclohexyloxy)-4-nitrobenzene (16)
Compound 16 was synthesized from the nucleophilic substitu-
tion of 4-nitrophenol to 1-bromo-4-methylcyclohexane (cis + -
trans). Yield: 14%; 1H NMR (CDCl3) d: 8.16 (m, 2H, J = 9.0 Hz, Ar),
6.92 (m, 2H, J = 8.9 Hz, Ar), 4.28 (m, 1H, CH), 2.10–1.24 (m, 7H,
CH, CH2), 1.17–0.94 (m, 5H, CH, CH3); 13C NMR (CDCl3) d: 163.2,
141.1, 125.9, 115.4, 73.1, 34.8, 32.2, 30.8, 22.2.
4.3.1. 1,2,3,4-Tetrahydro-6-nitronaphthalene (4)
4.4.2. 1-(4-Methylcyclohexyloxy)-3-nitrobenzene (17)
Compound 17 was synthesized from the nucleophilic substitu-
tion of 3-nitrophenol to 1-bromo-4methylcyclohexane (cis + trans).
Yield: 12%; 1H NMR (CDCl3) d: 7.73 (d, 1H, J = 7.8 Hz, Ar), 7.71 (s,
1H, Ar), 7.40 (t, 1H, J = 8.1 Hz, Ar), 7.18 (d, 1H, J = 7.2 Hz, Ar), 4.19
(m, 1H, CH), 2.10–1.34 (m, 7H, CH, CH2), 1.12–0.89 (m, 5H, CH,
CH3); 13C NMR (CDCl3) d: 158.5, 149.3, 129.9, 122.8, 115.4, 110.2,
72.9, 34.2, 32.9, 31.6, 22.3.
Compound 4 was synthesized from the nitration of 1,2,3,4-tet-
rahydronaphthalene. Yield: 55%; 1H NMR (CDCl3) d: 7.96 (s, 1H,
Ar), 7.67 (d, 1H, J = 8.1 Hz, Ar), 7.33 (d, 1H, J = 7.5 Hz, Ar), 2.88
(m, 4H, 2ꢁ CH2), 1.85 (m, 4H, 2ꢁ CH2); 13C NMR (CDCl3) d:
145.2, 137.2, 133.8, 129.9, 125.8, 121.8, 29.7, 26.1, 22.6, 22.4.
4.3.2. 1,2,3,4-Tetrahydro-1,1,4,4-tetramethyl-6-
nitronaphthalene (5)37
Compound 5 was synthesized from the nitration of 1,2,3,4-tet-
rahydro-1,1,4,4-tetramethylnaphthalene. Yield: 77%; 1H NMR
(CDCl3) d: 7.50 (s, 1H, Ar), 7.32 (d, 1H, J = 8.7 Hz, Ar), 7.17 (d, 1H,
J = 8.7 Hz, Ar), 1.58 (m, 4H, 2ꢁ CH2), 1.40 (s, 12H, 4ꢁ CH3); 13C
NMR (CDCl3) d: 157.8, 152.8, 149.8, 126.5, 125.1, 123.1, 31.5,
29.0, 22.5.
4.4.3. (4-Nitrophenoxy)cyclooctane (18)
Compound 18 was synthesized from the nucleophilic substitu-
tion of 4-nitrophenol to bromocyclooctane. Yield: 49%; 1H NMR
(CDCl3) d: 8.13 (d, 2H, J = 9.0 Hz, Ar), 6.84 (d, 2H, J = 9.2 Hz, Ar),
4.50 (m, 1H, CH), 1.86 (m, 4H, 2ꢁ CH2), 1.59 (m, 10 H, 5ꢁ CH2);
13C NMR (CDCl3) d: 163.2, 140.9, 125.9, 115.4, 78.9, 31.5, 27.0,
25.6, 23.0.
4.3.3. 1-Cyclopentyl-4-nitrobenzene (6)
Compound 6 was synthesized from the nitration of 1-cyclopen-
tylbenzene. Yield: 34%; 1H NMR (CDCl3) d: 8.20 (d, 2H, J = 8.7 Hz,
Ar), 7.79 (d, 2H, J = 8.6 Hz, Ar), 2.75 (m, 1H, CH), 1.93 (m, 2H,
4.4.4. (3-Nitrophenoxy)cyclooctane (19)
Compound 19 was synthesized from the nucleophilic substitu-
tion of 3-nitrophenol to bromocyclooctane. Yield: 33%; 1H NMR

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J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
(CDCl3) d: 7.75 (d, 1H, J = 9.0 Hz, Ar), 7.67 (s, 1H, Ar), 7.37 (t, 1H,
J = 8.2 Hz, Ar), 7.16 (d, 1H, J = 8.1 Hz, Ar), 4.49 (m, 1H, CH), 1.89
(m, 4H, 2ꢁ CH2), 1.63 (m, 10H, 5ꢁ CH2); 13C NMR (CDCl3) d:
158.4, 149.3, 129.9, 122.8, 115.3, 110.2, 78.9, 31.4, 27.0, 25.6,
23.0.
4.6.5. 4-(1-Ethylcyclohexyl)benzenamine (14)36
Compound 14 was synthesized by the reduction of 8. Yield:
98%; IR (KBr) v: 3323 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.11 (d, 2H,
;
J = 8.6 Hz, Ar), 6.68 (d, 2H, J = 8.6 Hz, Ar), 3.48 (s, 1H, NH), 2.01
(m, 3H, CH, CH2), 1.71–091 (m, 9H, CH, CH2), 0.54 (t, 3H,
J = 7.9 Hz, CH3); 13C NMR (CDCl3) d: 143.4, 136.9, 126.4, 115.0,
40.6, 38.1, 35.9, 26.7, 22.5, 7.9.
4.5. Preparation of 1-cyclohexyl-3-nitrobenzene (20)
Nickel iodide (0.06 mmol), trans-2-aminocyclohexanol hydro-
chloride (0.06 mmol), 3-nitrophenylboronic acid (1.2 mmol), and
NaHMDS (2 mmol) were placed in a 10-mL round-bottomed
flask.39 The flask was purged with dry nitrogen for 5 min. 2-PrOH
(5 mL) was added using a syringe, and the resulting mixture was
stirred for 5 min at room temperature.39 Cyclohexylbromide
(1 mmol) was added using a syringe, and the flask was heated at
60 °C in an oil bath for 6 h. The reaction mixture was cooled to
room temperature, and then filtered through a short pad of silica
gel. Silica gel was washed with 200 mL of a mixture of hexanes/
ether (1:1). The filtrate was concentrated under reduced pressure,
and the residue was purified by flash liquid chromatography using
a mixture of petroleum ether/CH2Cl2 (3:1).
4.6.6. 4-(1-Propylcyclohexyl)benzenamine (15)36
Compound 15 was synthesized by the reduction of 9. Yield:
82%; IR (KBr) v: 3372 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.08 (d, 2H,
;
J = 8.6 Hz, Ar), 6.65 (d, 2H, J = 8.3 Hz, Ar), 3.50 (s, 1H, NH), 2.01
(m, 3H, CH, CH2), 1.78–0.88 (m, 11H, CH, CH2), 0.76 (s, 3H,
J = 7.1 Hz, CH3); 13C NMR (CDCl3) d: 143.4, 137.4, 127.7, 115.0,
40.5, 36.4, 26.7, 22.5, 16.7, 14.8.
4.6.7. 3-Cyclohexylbenzenamine (21)
Compound 21 was synthesized by the reduction of 20. Yield:
75%; IR (KBr) v: 3356 (NH) cmꢂ1 1H NMR (DMSO-d6) d: 7.11 (t,
;
1H, J = 7.5 Hz, Ar), 6.65 (d, 1H, J = 7.5 Hz, Ar), 6.53 (m, 2H, Ar),
3.56 (s, 1H, NH), 2.44 (m, 1H, CH), 1.80 (m, 5H, CH, CH2), 1.39
(m, 5H, CH, CH2); 13C NMR (DMSO-d6) d: 149.5, 146.4, 129.2,
117.4, 113.8, 112.8, 44.7, 34.4, 27.0, 26.3.
Yield: 5%; 1H NMR (CDCl3) d: 7.96 (m, 2H, Ar), 7.70 (t, 1H,
J = 7.6 Hz, Ar), 7.54 (t, 1H, J = 7.8 Hz, Ar), 2.44 (m, 1H, CH), 1.76
(m, 5H, CH, CH2), 1.36 (s, 5H, CH, CH2); 13C NMR (CDCl3) d:
153.6, 140.4, 133.1, 130.3, 123.3, 122.1, 45.3, 33.4, 25.1, 24.6.
4.6.8. 4-(4-Methylcyclohexyloxy)benzenamine (22)
Compound 22 was synthesized by the reduction of 16. Yield:
4.6. General preparation of the anilines 10–15 and 21–25
42%; IR (KBr) v: 3421 (NH) cmꢂ1 1H NMR (CDCl3) d: 6.77 (d, 2H,
;
J = 8.3 Hz, Ar), 6.62 (d, 2H, J = 8.5 Hz, Ar), 3.97 (m, 1H, CH), 3.42
(s, 1H, NH), 2.07–1.23 (m, 7H, CH, CH2), 1.02–0.83 (m, 5H, CH,
CH3); 13C NMR (CDCl3) d: 150.8, 140.3, 118.2, 116.4, 73.4, 34.4,
33.3, 32.2, 22.0.
Nitro derivatives 4–9, 16–19, and 21 (1 equiv, 4.97 mmol)
were added to a solution of SnCl2ꢀ2H2O (6 equiv, 29.8 mmol) in
10 mL of ethanol. The mixture was stirred under reflux for 12 h.
Then, 1 N NaOH (20 mL) was added. The mixture was extracted
with ether. The organic phases were pooled, and washed with
brine and afterward with water. The ether solution was dried
over anhydrous MgSO4, filtered, and evaporated under reduced
pressure.
4.6.9. 3-(4-Methylcyclohexyloxy)benzenamine (23)
Compound 23 was synthesized by the reduction of 17. Yield:
84%; IR (KBr) v: 3416 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.04 (t, 1H,
;
J = 7.5 Hz, Ar), 6.28 (m, 3H, Ar), 4.11 (m, 1H, CH), 3.62 (s, 1H,
NH), 2.13–1.27 (m, 7H, CH, CH2), 1.05–0.87 (m, 5H, CH, CH3); 13C
NMR (CDCl3) d: 147.8, 130.0, 107.7, 106.2, 103.3, 71.6, 34.2, 32.8,
30.3, 22.3.
4.6.1. 5,6,7,8-Tetrahydronaphthalen-2-amine (10)
Compound 10 was synthesized by the reduction of 4. Yield:
75%; IR (KBr) v: 3353 (NH) cmꢂ1 1H NMR (CDCl3) d: 6.96 (d, 1H,
;
J = 6.2 Hz, Ar), 6.67 (s, 1H, Ar), 6.62 (d, 1H, J = 6.2 Hz, Ar), 3.58 (s,
1H, NH), 2.80 (m, 4H, 2ꢁ CH2), 1.97 (m, 4H, 2ꢁ CH2); 13C NMR
(CDCl3) d: 144.0, 137.2, 129.9, 125.5, 119.7, 115.6, 29.5, 23.5.
4.6.10. 4-(Cyclooctyloxy)benzenamine (24)
Compound 24 was synthesized by the reduction of 18. Yield:
98%; IR (KBr) v: 3411 (NH) cmꢂ1 1H NMR (CDCl3) d: 6.72 (d, 2H,
;
J = 8.8 Hz, Ar), 6.62 (d, 2H, J = 8.7 Hz, Ar), 4.24 (m, 1H, CH), 3.41
(s, 1H, NH), 1.90 (m, 4H, 2ꢁ CH2), 1.58 (m, 10H, 5ꢁ CH2); 13C
NMR (CDCl3) d: 150.8, 140.0, 117.9, 116.4, 79.1, 31.7, 27.2, 25.7,
23.1.
4.6.2. 5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalen-2-
amine (11)37
Compound 11 was synthesized by the reduction of 5. Yield:
98%; IR (KBr) v: 3392 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.31 (d, 1H,
;
Ar), 7.15 (s, 1H, Ar), 6.65 (d, 1H, J = 8.2 Hz, Ar), 3.50 (s, 1H, NH),
1.51 (m, 4H, 2ꢁ CH2), 1.39 (s, 12H, 4ꢁ CH3); 13C NMR (CDCl3) d:
149.8, 143.6, 137.7, 127.4, 114.1, 112.9, 31.8, 28.7, 22.8.
4.6.11. 3-(Cyclooctyloxy)benzenamine (25)
Compound 25 was synthesized by the reduction of 19. Yield:
61%; IR (KBr) v: 3431 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.05 (t, 1H,
;
J = 8.0 Hz, Ar), 6.29 (m, 3H, Ar), 4.38 (m, 1H, CH), 3.64 (s, 1H, NH),
1.91 (s, 4H, 2ꢁ CH2), 1.60 (m, 10H, 5ꢁ CH2); 13C NMR (CDCl3) d:
159.0, 147.9, 130.1, 107.7, 106.1, 103.3, 77.6, 31.9, 27.2, 25.8, 23.2.
4.6.3. 4-Cyclopentylbenzenamine (12)
Compound 12 was synthesized by the reduction of 6. Yield:
53%; IR (KBr) v: 3362 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.37 (d, 1H,
;
J = 8.5 Hz, Ar), 7.15 (d, 1H, J = 7.8 Hz, Ar), 3.55 (s, 1H, NH), 2.80
(m, 1H, CH), 1.98 (m, 2H, CH2), 1.72–1.45 (m, 6H, 3ꢁ CH2); 13C
NMR (CDCl3) d: 140.2, 139.8, 129.3, 116.4, 41.1, 32.8, 24.0.
4.7. Preparation of 6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-
amine (26)40
Palladium on 10% charcoal (55 mg) was added to a solution of 7-
nitro-1-tetralone, 1,2,3,4-tetrahydronaphthalene (1 mmol) in
10 mL of acetic acid containing four drops of H2SO4 (12 M).40 The
suspension was shaken under 40 psi of hydrogen in a Parr apparatus
for 12 h. The mixture was filtered on Celite, and the filtrate was
evaporated under reduced pressure. The crude product was mixed
with HCl (1 N) and washed with ethyl acetate. The aqueous phase
was neutralized at pH 8 with a solution of NaOH (2 N). The product
4.6.4. 4-(1-Methylcyclohexyl)benzenamine (13)36
Compound 13 was synthesized by the reduction of 7. Yield:
98%; IR (KBr) v: 3362 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.16 (d,
;
1H, J = 8.5 Hz, Ar), 6.66 (d, 2H, J = 8.4 Hz, Ar), 3.56 (s, 1H, NH),
1.96 (m, 2H, CH2), 1.48 (m, 8H, 4ꢁ CH2), 1.35 (s, 3H, CH3); 13C
NMR (CDCl3) d: 143.6, 140.3, 126.7, 116.1, 38.0, 37.0, 30.6,
26.5, 22.7.

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was extracted with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous Na2SO4, and evaporated under vacuum.
Yield: 76%; IR (KBr) v: 3445 (NH) cmꢂ1; 1H NMR (CDCl3) d: 6.95
(d, 1H, J = 7.8 Hz, Ar), 6.53 (s, 1H, Ar), 6.47 (d, 1H, J = 7.7 Hz, Ar),
3.50 (s, 1H, NH), 2.76 (t, 4H, J = 4.6 Hz, 2ꢁ CH2), 1.88 (m, 2H,
CH2), 1.71 (m, 4H, 2ꢁ CH2); 13C NMR (CDCl3) d: 144.5, 144.4,
133.8, 129.9, 116.5, 112.4, 37.0, 35.9, 32.9, 29.0, 28.6.
(KBr) v: 3320 (NH), 1638 (C@O) cmꢂ1
;
1H NMR (DMSO-d6) d:
8.49 (s, 1H, NH), 7.13 (s, 1H, Ar), 7.09 (s, 1H, Ar), 6.95 (d, 1H,
J = 7.9 Hz, Ar), 6.38 (s, 1H, NH), 3.66 (t, 2H, J = 6.2 Hz, CH2), 3.41
(m, 2H, CH2), 2.70 (m, 4H, 2ꢁ CH2), 1.77 (m, 2H, CH2), 1.56 (m,
4H, 2ꢁ CH2); 13C NMR (DMSO-d6) d: 155.3, 143.3, 137.8, 136.2,
129.1, 118.9, 115.4, 44.5, 41.2, 36.1, 35.2, 32.1, 28.4, 28.1. MS
(ESI) m/z: 267.1 (M+1)+.
4.8. General procedure for the preparation of substituted N-
phenyl-N0-(2-chloroethyl)ureas (CEUs, 27–56), N-aryl amino-2-
oxazolines (4,5-dihydro-N-phenyloxazol-2-amine: OXA
derivatives, 58–62), N-phenyl-N0-(2-chloroacetyl)urea
derivatives (CAU, 63–67)
4.8.5. 1-(2-Chloroethyl)-3-(4-cyclopentylphenyl)urea (35)
Compound 35 was synthesized from the nucleophilic addition
of 12 to 2-chloroethylisocyanate. Yield: 65%; mp: 154–157 °C; IR
(KBr) v: 3324 (NH), 1642 (C@O) cmꢂ1 1H NMR (DMSO-d6) d:
;
8.57 (s, 1H, NH), 7.30 (d, 2H, J = 8.3 Hz, Ar), 7.10 (d, 2H, J = 8.3 Hz,
Ar), 6.38 (s, 1H, NH), 3.60 (t, 2H, J = 6.1 Hz, CH2), 3.43 (m, 2H,
CH2), 2.89 (m, 1H, CH), 1.96 (m, 2H, CH2), 1.73 (m, 2H, CH2), 1.68
(m, 2H, CH2), 1.46 (m, 2H, CH2); 13C NMR (DMSO-d6) d: 155.2,
138.7, 138.0, 127.1, 117.9, 44.7, 44.4, 41.3, 29.3, 25.0. MS (ESI) m/
z: 267.1 (M+1)+.
EU 57,15,16 CEUs 28,6,7,10 32,6,7 33,10 34,10 36 (cHCEU),7,15,16 42–
45,15,16 48–51,15,16 54 (tBCEU),6,7,10 55 (ICEU),6,7,10 OXA 59,16 62,10
and CAU 64,16 6710 were prepared as published previously. The cor-
responding isocyanate (3.6 mmol), either ethylisocyanate, 2-chloro-
ethylisocyanate, or 2-chloroacetylisocyanate, was added dropwise
to a stirred solution of the relevant aniline (3 mmol) in dichloro-
methane (15 mL). The reaction mixture was stirred under a dried
nitrogen atmosphere overnight at ambient temperature. The result-
ing precipitate was filtered, washed with cold ether, and purified by
recrystallization from ethanol and water. For the preparation of the
2-amino oxazoline derivatives, a mixture of SiO2.KF (60:40%)
(3.5 mmol) was added to a stirred solution of the appropriate N-phe-
nyl-N0-(2-chloroethyl)urea derivatives (0.35 mmol) in acetonitrile
(10 mL). The suspension was refluxed overnight. After cooling, the
mixture was filtered and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on silica
gel (dichloromethane/methanol 95:5).
4.8.6. 1-(2-Chloroethyl)-3-(3-cyclohexylphenyl)urea (37)43
Compound 37 was synthesized from the nucleophilic addition
of 21 to 2-chloroethylisocyanate. Yield: 70%; mp: 156–160 °C; IR
(KBr) v: 3323 (NH), 1660 (C@O) cmꢂ1 1H NMR (DMSO-d6) d:
;
8.61 (s, 1H, NH), 7.31 (s, 1H, Ar), 7.15 (m, 2H, Ar), 6.76 (d, 1H,
J = 7.2 Hz, Ar), 6.41 (s, 1H, NH), 3.67 (t, 2H, J = 6.1 Hz, CH2), 3.44
(m, 2H, CH2), 2.43 (m, 1H, CH), 1.77 (m, 5H, CH, CH2), 1.40 (m,
5H, CH, CH2); 13C NMR (DMSO-d6) d: 155.1, 148.1, 140.3, 128.5,
119.7, 116.2, 115.4, 44.4, 44.0, 41.3, 29.1, 26.4, 25.7. MS (ESI) m/
z: 281.1 (M+1)+.
4.8.7. 1-(2-Chloroethyl)-3-(4-morpholinophenyl)urea (38)44
Compound 38 was synthesized from the nucleophilic addition
of 4-morpholinobenzenamine to 2-chloroethylisocyanate. Yield:
4.8.1. 1-(2-Chloroethyl)-3-phenylurea (27)7,41
Compound 27 was synthesized from the nucleophilic addition
of aniline to 2-chloroethylisocyanate. Yield: 36%; mp: 126–
60%; mp: 184–189 °C; IR (KBr) v: 3330 (NH), 1639 (C@O) cmꢂ1
;
1H NMR (DMSO-d6) d: 8.40 (s, 1H, NH), 7.26 (d, 2H, J = 8.5 Hz,
Ar), 6.85 (d, 2H, J = 8.4 Hz, Ar), 6.30 (s, 1H, NH), 3.74 (m, 4H, 2ꢁ
CH2), 3.64 (m, 2H, CH2), 3.43 (m, 2H, CH2), 3.01 (m, 4H, 2ꢁ CH2);
13C NMR (DMSO-d6) d: 155.3, 146.1, 132.8, 119.2, 116.0, 66.2,
49.4, 44.5, 41.4. MS (ESI) m/z: 284.1 (M+1)+.
129 °C; IR (KBr) v: 3320 (NH), 1640 (C@O) cmꢂ1 1H NMR (CDCl3)
;
d: 7.30 (m, 4H, Ar), 7.09 (m, 1H, Ar), 6.96 (s, 1H, NH), 5.57 (s, 1H,
NH), 3.65 (m, 4H, 2ꢁ CH2); 13C NMR (CDCl3) d: 155.9, 138.3,
129.4, 124.1, 121.2, 44.7, 42.0. MS (ESI) m/z: 199.1 (M+1)+.
4.8.2. 1-(2-Chloroethyl)-3-(1,2,3,4-tetrahydronaphthalen-6-
yl)urea (29)42
4.8.8. 1-(2-Chloroethyl)-3-(4-(1-methylcyclohexyl)phenyl)urea
(39)
Compound 29 was synthesized from the nucleophilic addition
Compound 39 was synthesized from the nucleophilic addition
of 13 to 2-chloroethylisocyanate. Yield: 62%; IR (KBr) v: 3315
of 10 to 2-chloroethylisocyanate. Yield: 15%; mp: 175–183 °C; IR
(KBr) v: 3334 (NH), 1644 (C@O) cmꢂ1
;
1H NMR (DMSO-d6) d:
(NH), 1699 (C@O) cmꢂ1 1H NMR (CDCl3) d: 7.95 (s, 1H, NH), 7.20
;
8.44 (s, 1H, NH), 7.10 (d, 1H, J = 7.1 Hz, Ar), 7.06 (s, 1H, Ar), 6.90
(d, 1H, J = 8.2 Hz, Ar), 6.33 (s, 1H, NH), 3.66 (t, 2H, J = 6.1 Hz,
CH2), 3.40 (m, 2H, CH2), 2.64 (m, 4H, CH2), 1.71 (m, 4H, CH2); 13C
NMR (DMSO-d6) d: 155.2, 137.6, 136.6, 129.5, 129.0, 118.1, 115.7,
44.5, 41.2, 29.1, 28.2, 23.0, 22.8. MS (ESI) m/z: 253.1 (M+1)+.
(s, 4H, Ar), 6.30 (s, 1H, NH), 3.55 (m, 4H, 2ꢁ CH2), 1.50 (m, 10H,
5ꢁ CH2), 1.29 (s, 3H, CH3); 13C NMR (CDCl3) d: 156.8, 144.3,
136.3, 126.2, 119.6, 43.9, 41.9, 38.2, 37.8, 30.4, 26.4, 22.7. MS
(ESI) m/z: 295.2 (M+1)+.
4.8.9. 1-(2-Chloroethyl)-3-(4-(1-ethylcyclohexyl)phenyl)urea
(40)
4.8.3. 1-(2-Chloroethyl)-3-(1,2,3,4-tetrahydro-1,1,4,4-
tetramethylnaphthalen-6-yl)urea (30)
Compound 40 was synthesized from the nucleophilic addition
of 14 to 2-chloroethylisocyanate. Yield: 63%; IR (KBr) v: 3333
Compound 30 was synthesized from the nucleophilic addition
of 11 to 2-chloroethylisocyanate. Yield: 23%; IR (KBr) v: 3337
(NH), 1711 (C@O) cmꢂ1 1H NMR (CDCl3) d: 7.72 (s, 1H, NH), 7.15
;
(NH), 1660 (C@O) cmꢂ1
;
1H NMR (CDCl3) d: 7.60 (d, 1H,
(m, 4H, Ar), 6.11 (s, 1H, NH), 3.52 (m, 4H, 2ꢁ CH2), 1.23 (m, 12H,
6ꢁ CH2), 0.50 (t, 3H, J = 7.4 Hz, CH3); 13C NMR (CDCl3) d: 156.4,
135.9, 127.6, 119.9, 44.7, 42.1, 36.2, 26.7, 22.4, 7.9. MS (ESI) m/z:
309.2 (M+1)+.
J = 8.3 Hz, Ar), 7.41 (s, 1H, Ar), 7.12 (s, 1H, NH), 7.00 (d, 1H,
J = 8.0 Hz, Ar), 6.02 (s, 1H, NH), 3.51 (m, 4H, 2ꢁ CH2), 1.52 (m,
4H, 2ꢁ CH2), 1.39 (s, 12H, 4ꢁ CH3); 13C NMR (CDCl3) d: 156.4,
145.1, 140.7, 135.7, 126.6, 120.5, 119.1, 44.5, 42.1, 31.9, 29.0,
22.6. MS (ESI) m/z: 309.2 (M+1)+.
4.8.10. 1-(2-Chloroethyl)-3-(4-(1-propylcyclohexyl)phenyl)urea
(41)
4.8.4. 1-(2-Chloroethyl)-3-(6,7,8,9-tetrahydro-5H-
benzo7annulen-3-yl)urea (31)
Compound 31 was synthesized from the nucleophilic addition
of 26 to 2-chloroethylisocyanate. Yield: 98%; mp: 159–165 °C; IR
Compound 41 was synthesized from the nucleophilic addition
of 15 to 2-chloroethylisocyanate. Yield: 71%; IR (KBr) v: 3372
(NH), 1714 (C@O) cmꢂ1 1H NMR (CDCl3) d: 7.81 (s, 1H, NH),
;
7.24 (m, 4H, Ar), 6.11 (s, 1H, NH), 3.54 (m, 4H, CH2), 1.97–

7288
J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
0.88 (m, 14H, CH2), 0.66 (t, 3H, J = 7.7 Hz, CH3), 3.45, 2.21 (s, 3H,
CH3); 13C NMR (CDCl3) d: 156.4, 142.4, 135.8, 127.4, 120.1, 44.7,
42.1, 40.9, 36.2, 26.6, 22.3, 16.7, 14.7. MS (ESI) m/z: 323.2
(M+1)+.
4.8.17. N-(3-Cyclohexylphenyl)-4,5-dihydrooxazol-2-amine
(60)
Compound 60 was synthesized from 37 and SiO2. Yield: 50%; IR
(KBr) v: 3323 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.35 (s, 1H, Ar), 7.25
;
(m, 2H, Ar), 7.08 (d, 1H, J = 7.1 Hz, Ar), 6.41 (s, 1H, NH), 4.43 (t, 2H,
J = 8.0 Hz, CH2), 3.70 (t, 2H, J = 8.0 Hz, CH2), 2.45 (m, 1H, CH), 1.82
(m, 5H, CH, CH2), 1.38 (m, 5H, CH, CH2); 13C NMR (CDCl3) d: 156.5,
149.0, 139.6, 128.9, 120.8, 119.2, 117.3, 66.5, 47.4, 44.2, 34.4, 26.7,
26.2. MS (ESI) m/z: 245.2 (M++1).
4.8.11. 1-(4-(4-Methylcyclohexyloxy)phenyl)-3-(2-
chloroethyl)urea (46)
Compound 46 was synthesized from the nucleophilic addition
of 22 to 2-chloroethylisocyanate. Yield: 70%; mp: 112–118 °C; IR
(KBr) v: 3304 (NH), 1635 (C@O) cmꢂ1 1H NMR (CDCl3) d: 7.30 (s,
;
1H, NH), 7.13 (d, 2H, J = 8.7 Hz, Ar), 6.81 (d, 2H, J = 8.3 Hz, Ar),
5.80 (s, 1H, NH), 4.05 (s, 1H, CH), 3.51 (m, 4H, 2ꢁ CH2), 2.06–
1.22 (m, 7H,CH, CH2), 1.08–0.86 (m, 5H, CH, CH3); 13C NMR (CDCl3)
d: 156.8, 154.8, 131.0, 123.7, 116.9, 72.9, 44.6, 42.0, 33.2, 32.0, 31.4,
22.2. MS (ESI) m/z: 311.2 (M+1)+.
4.8.18. 4,5-Dihydro-N-(4-morpholinophenyl)oxazol-2-amine
(61)
Compound 61 was synthesized from 38 and SiO2. Yield: 93%;
mp: 149–157; IR (KBr) v: 3367 (NH) cmꢂ1 1H NMR (CDCl3) d:
;
7.19 (d, 2H, J = 8.9 Hz, Ar), 6.84 (d, 2H, J = 8.9 Hz, Ar), 5.62 (s, 1H,
NH), 4.36 (t, 2H, J = 8.3 Hz, CH2), 3.83 (m, 6H, 3ꢁ CH2), 3.07 (t,
4H, J = 4.7 Hz, 2ꢁ CH2); 13C NMR (CDCl3) d: 157.8, 147.1, 134.7
121.1, 116.8, 66.9, 66.8, 50.2, 50.1. MS (ESI) m/z: 248.1 (M++1).
4.8.12. 1-(3-(4-Methylcyclohexyloxy)phenyl)-3-(2-
chloroethyl)urea (47)
Compound 47 was synthesized from the nucleophilic addition
of 23 to 2-chloroethylisocyanate. Yield: 98%; IR (KBr) v: 3353
4.8.19. 1-(2-Chloroacetyl)-3-phenylurea (63)
(NH), 1710 (C@O) cmꢂ1
;
1H NMR (CDCl3) d: 7.62 (s, 1H, NH), 7.13
Compound 63 was synthesized from the nucleophilic addition
of aniline to 2-chloroacetylisocyanate. Yield: 73%; mp: 164–
(t, 1H, J = 8.1 Hz, Ar), 7.00 (s, 1H, Ar), 6.78 (t, 1H, J = 6.6 Hz, Ar),
6.57 (d, 1H, J = 6.5 Hz, Ar), 6.00 (s, 1H, NH), 4.08 (s, 1H, CH), 3.46
(m, 4H, 2ꢁ CH2), 2.06–1.21 (m, 7H, CH, CH2), 0.97–0.82 (m, 5H,
CH, CH3); 13C NMR (CDCl3) d: 158.2, 156.2, 139.9, 129.8, 112.5,
111.3, 108.5, 72.2, 44.4, 42.0, 32.0, 31.8, 31.4, 22.2. MS (ESI) m/z:
311.2 (M+1)+.
166 °C; IR (KBr) v: 3240 (NH), 1715 (C@O) cmꢂ1 1H NMR (CDCl3)
;
d: 10.25 (s, 1H, NH), 9.25 (s, 1H, NH), 7.51 (d, 2H, J = 7.9 Hz, Ar),
7.36 (t, 2H, J = 8.1 Hz, Ar), 7.16 (t, 1H, J = 7.4 Hz, Ar), 4.20 (s, 2H,
CH2); 13C NMR (CDCl3) d: 167.9, 150.0, 136.6, 129.2, 124.9, 120.5,
42.5. MS (ESI) m/z: 163.1 (M+1)+.
4.8.13. 1-(2-Chloroethyl)-3-(4-(cyclooctyloxy)phenyl)urea (52)
Compound 52 was synthesized from the nucleophilic addition
of 24 to 2-chloroethylisocyanate. Yield: 62%; mp: 106–110 °C; IR
4.8.20. 1-(2-Chloroacetyl)-3-(3-cyclohexylphenyl)urea (65)
Compound 65 was synthesized from the nucleophilic addition
of 21 to 2-chloroacetylisocyanate. Yield: 70%; mp: 119–124 °C; IR
(KBr) v: 3293 (NH), 1710 (C@O) cmꢂ1
;
1H NMR (CDCl3) d: 7.36 (s,
(KBr) v: 3226 (NH), 1717 (C@O) cmꢂ1 1H NMR (DMSO-d6) d:
;
1H, NH), 7.12 (d, 2H, J = 8.8 Hz, Ar), 6.75 (d, 2H, J = 8.8 Hz, Ar),
5.85 (s, 1H, NH), 4.31 (m, 1H, CH), 3.48 (m, 4H, 2ꢁ CH2), 1.71 (m,
6H, 3ꢁ CH2), 1.60 (m, 8H, 4ꢁ CH2); 13C NMR (CDCl3) d: 156.9,
154.7, 130.9, 123.6, 116.8, 78.4, 44.4, 42.0, 31.7, 27.1, 25.7, 23.1.
MS (ESI) m/z: 325.2 (M+1)+.
10.89 (s, 1H, NH), 10.15 (s, 1H, NH), 7.34 (d, 2H, J = 9.3 Hz, Ar),
7.25 (t, 1H, J = 7.1 Hz, Ar), 6.97 (d, 1H, J = 7.5 Hz, Ar), 4.42 (s, 2H,
CH2), 2.52 (m, 1H, CH), 1.74 (m, 5H, CH, CH2), 1.36 (m, 5H, CH,
CH2); 13C NMR (DMSO-d6) d: 168.7, 150.2, 148.6, 137.4, 128.9,
122.3, 118.1, 117.4, 43.8, 43.2, 33.9, 26.4, 25.6. MS (ESI) m/z:
295.1 (M+1)+.
4.8.14. 1-(2-Chloroethyl)-3-(3-(cyclooctyloxy)phenyl)urea (53)
Compound 53 was synthesized from the nucleophilic addition
of 25 to 2-chloroethylisocyanate. Yield: 86%; mp: 98–103 °C; IR
4.8.21. 1-(2-Chloroacetyl)-3-(4-morpholinophenyl)urea (66)
Compound 66 was synthesized from the nucleophilic addition
of 4-morpholinobenzenamine to 2-chloroacetylisocyanate. Yield:
(KBr) v: 3392 (NH), 1710 (C@O) cmꢂ1 1H NMR (CDCl3) d: 7.94 (s,
;
1H, NH), 7.10 (t, 1H, J = 8.1 Hz, Ar), 6.97 (s, 1H, Ar), 6.75 (d, 1H,
J = 7.6 Hz, Ar), 6.51 (d, 1H, J = 8.1 Hz, Ar), 6.23 (s, 1H, NH), 4.34
(m, 1H, CH), 3.47 (m, 4H, 2ꢁ CH2), 1.87 (m, 6H, 3ꢁ CH2), 1.72 (m,
8H, 4ꢁ CH2); 13C NMR (CDCl3) d: 158.6, 156.4, 140.1, 129.7,
112.2, 110.9, 108.3, 78.0, 44.2, 41.9, 31.6, 27.1, 25.7, 23.1. MS
(ESI) m/z: 325.2 (M+1)+.
39%; mp: 175–179 °C; IR (KBr) v: 3362 (NH), 1702 (C@O) cmꢂ1
;
1H NMR (DMSO-d6) d: 10.84 (s, 1H, NH), 9.99 (s, 1H, NH), 7.39 (d,
2H, J = 8.8 Hz, Ar), 6.94 (d, 2H, J = 8.8 Hz, Ar), 4.39 (s, 2H, CH2),
3.75 (t, 4H, J = 4.7 Hz, 2ꢁ CH2), 3.08 (t, 4H, J = 4.41 Hz, 2ꢁ CH2);
13C NMR (DMSO-d6) d: 168.5, 150.2, 147.7, 129.5, 121.1, 115.7,
66.1, 48.9, 43.2. MS (ESI) m/z: 298.1 (M+1)+.
4.8.15. 1-(2-Chloroethyl)-3-(4-hexylphenyl)urea (56)6,7
Compound 56 was synthesized from the nucleophilic addition
of 4-hexylbenzenamine to 2-chloroethylisocyanate. Yield: 14%;
4.9. Biological assays
4.9.1. Materials and reagents
mp: 121–123 °C; IR (KBr) v: 3300 (NH), 1640 (C@O) cmꢂ1
;
1H
The monoclonal antibody anti-bII-tubulin (clone TUB 2.1) and
the mouse anti-thioredoxin (clone 2G11) were obtained from Sig-
ma Chemicals (St. Louis, MO) and BD Transduction Laboratories
(Mississauga, ON, Canada), respectively. The anti-mouse Alexa
488 was purchased from Invitrogen (Carlsbad, CA). The horseradish
peroxidase-conjugated anti-mouse IgG was supplied by GE Health-
care (Little Chalfont, Buckinghamshire, UK).
NMR (DMSO-d6) d: 8.53 (s, 1H, NH), 7.30 (d, 2H, J = 8.3 Hz, Ar),
7.03 (d, 2H, J = 8.3 Hz, Ar), 6.40 (s, 1H, NH), 3.66 (t, 2H, J = 6.0 Hz,
CH2), 3.42 (m, 2H, CH2), 2.48 (t, 2H, J = 7.4 Hz, CH2), 1.52 (m, 2H,
CH2), 1.26 (m, 6H, 3ꢁ CH2), 0.86 (m, 3H, CH3); 13C NMR (DMSO-
d6) d: 155.1, 138.0, 135.1, 128.4, 117.9, 44.4, 41.3, 34.5, 31.1,
28.3, 22.1, 13.9. MS (ESI) m/z: 283.2 (M+1)+.
4.8.16. 4,5-Dihydro-N-phenyloxazol-2-amine (58)
4.9.2. Cell culture
Compound 58 was synthesized from 27 and SiO2.KF. Yield: 98%;
Human cell lines HT-29 (colon carcinoma), MCF-7 (breast carci-
noma) and M21 (skin melanoma) cells were purchased from the
American Type Culture Collection (Manassas, VA). The cells were
cultured in DMEM medium supplemented with 2.2 g/L sodium
IR (KBr) v: 3309 (NH) cmꢂ1 1H NMR (CDCl3) d: 7.24 (m, 4H, Ar),
;
6.99 (m, 1H, Ar), 6.58 (s, 1H, NH), 4.37 (t, 2H, J = 8.3 Hz, CH2),
3.80 (t, 2H, J = 8.3 Hz, CH2); 13C NMR (CDCl3) d: 157.9, 129.0,
122.5, 120.1, 67.4, 49.2. MS (ESI) m/z: 163.1 (M+1)+.
bicarbonate, 4.5 g/L glucose, 100 lg/mL streptomycin sulfate A,

J. S. Fortin et al. / Bioorg. Med. Chem. 16 (2008) 7277–7290
7289
100 U of penicillin G, 292
um (Hyclone laboratories, Logan, UT). Cells were maintained in a
l
g/mL glutamine, and 5% bovine calf ser-
tagged image format files with a Q imaging RETIGA EXI digital
camera driven by Image Pro Express software.
moisture saturated atmosphere at 37 °C in 5% CO2.
4.9.6. Western blot analysis
Prior to drug exposure, approximately 5 ꢁ 105 M21 cells were
seeded into 6-well plates and incubated for 12 h. Exponentially
growing M21 cells were incubated in the presence of two times
the GI50 of the selected CEU; vinblastine sulfate, colchicine, and
paclitaxel were tested at 50 nM for 16, 24, and 48 h at 37 °C. The
controls consisted of the final concentration of DMSO in the culture
medium (maintained under 0.12% (v/v)). Afterward, floating and
the adherent M21 cells were pooled, washed with ice-cold PBS,
4.9.3. Cell growth inhibition assay
The growth inhibition potency of CEUs, OXAs and CAUs was as-
sessed using the procedure described by the National Cancer Insti-
tute for its drugs screening program.31 96-well microtiter plates
were seeded with either 5 ꢁ 103 HT-29, 3.5 ꢁ 103 M21, or
3.5 ꢁ 103 MCF-7 cells suspended in 100
lL of calf serum. Freshly
solubilized drugs in DMSO were diluted in culture medium and ali-
quots of 100
l
L containing sequential dilution of drugs were
M.
and then lysed by addition of 100 lL Laemmli sample buffer. The
added. Final drug concentrations ranged from 100 to 0.3
l
cell extracts were boiled for 5 min. The protein concentration
was determined using the Lowry method. Twenty micrograms of
proteins from the protein extracts were subjected to electrophore-
sis using 0.1% SDS and 10% polyacrylamide gels. The proteins were
transferred onto nitrocellulose membranes that were incubated
with TBSMT (TBS, pH 7.4, 5% fat-free dry milk or BSA or TBST
and 0.1% Tween 20TM) for 1 h at 37 °C, and then with the b-tubulin
(1:500, 5% fat-free dry milk) primary antibody for 1 h at room tem-
perature. Membranes were washed with TBST and incubated with
1:2500 peroxidase-conjugated anti-mouse immunoglobulin in
TBSMT for 1 h at room temperature. After washing the membranes
with TBST, detection of the immunoblot was carried out with an
enhanced chemiluminescence (ECL) detection reagent kit.
DMSO concentration was maintained at 0.12% to avoid growth
inhibition. Plates were incubated for 48 h. The incubation was
stopped by addition of cold trichloroacetic acid to the wells (10%
final concentration), followed by incubation for 1 h at 4 °C. Plates
were washed five times with water. Sulforhodamine B solution
(50 lL) at 0.1% (w/v) in 1% acetic acid was added to each well,
and plates were incubated for 15 min at room temperature. After
staining, unbounded dye was removed by washing five times with
1% acetic acid. Bonded dye was solubilized with 10 mM Tris base,
and the absorbance was read using a
l Quant Universal Microplate
Spectrophotometer (Biotek, Winooski, VT) at 585 nm. A back-
ground OD from a control reference plate fixed on the day of treat-
ment was subtracted from the OD obtained with the 48-h growth
period. The growth inhibition percentage was calculated with ref-
erence to DMSO-treated cells for each drug concentrations. The re-
sults were obtained from at least three separated experiments. The
GI50 assay was considered valid when the variability among data
for a given set of conditions, within the same experiment, was less
than 10% with respect to the mean value.
Acknowledgments
This work was supported by grants from le Fonds de la Recher-
che en Santé du Québec, Junior II (E.P.) and Canadian Institute for
Health Research (R.C.-G., E.P.; Grant #MOP-79334). Jessica S. Fortin
is recipient of a studentship from the Fonds de la Recherche en
Santé du Québec. We thank the University of Montreal for their
professional services of mass spectrometry. We are very grateful
to Mr. Maurice Dufour for FACS analysis and technical advice.
4.9.4. Cell cycle analysis
After incubation of 3.5 ꢁ 105 MCF-7 cells with compounds 32–
37, 39–57, 59, and 64 at two, four, and eight times their respective
GI50 or DMSO for 24 h, the cells were trypsinized, washed with PBS,
resuspended in 1 mL of PBS, and fixed by the addition of 2.4 mL of
ice-cold anhydrous ethanol. Then, cells were centrifuged for 5 min
References and notes
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