N-Phenyl-N′-(2-chloroethyl)ureas (CEU) as potential antineoplastic agents. Part 2: Role of ω-hydroxyl group in the covalent binding to β-tubulin

Article abstract of DOI:10.1016/j.bmc.2006.11.005

Tubulin is the target of many anticancer drugs, including N-phenyl-N′-(2-chloroethyl)urea (CEU). Unlike most anti-β-tubulin agents, CEUs are protein monoalkylating agents binding through their N′-(2-chloroethyl)urea moiety to an amino acid nearby the colchicine-binding site on β-tubulin isoform-2. Following the previously synthesized and attractive N-(3-ω-hydroxyalkylphenyl)-N′-(2-chloroethyl)urea that exhibited growth inhibitory activity at the nanomolar level, we investigated the importance of lower alkyl and alkoxy groups to evaluate the effect of hydroxylated group and chain length on both cell growth inhibition and the mechanism of action of CEU. Here, we describe the preparation of two new series of CEU and show that the most potent CEU derivatives beside the ω-hydroxylated 1f were 2f and 3e, respectively. We have confirmed that the pentyl substituted CEUs 1f, 2f, and 3e are still covalently binding to β-tubulin and still arrest cell division in G₂/M phase. Crown Copyright

Full text of DOI:10.1016/j.bmc.2006.11.005

Bioorganic & Medicinal Chemistry 15 (2007) 1430–1438
N-Phenyl-N0-(2-chloroethyl)ureas (CEU) as potential
antineoplastic agents. Part 2: Role of x-hydroxyl
group in the covalent binding to b-tubulin
,
Sebastien Fortin, Emmanuel Moreau, Alexandre Patenaude, Michel Desjardins,à
*
´
*
´
Jacques Lacroix, Jean L. C. Rousseau and Rene C-Gaudreault
ˆ
Unite´ des Biotechnologies et de Bioinge´nierie, Centre de recherche, C.H.U.Q., Hopital Saint-Franc¸ois d’Assise,
Universite´ Laval, Que´bec, QC, Canada G01L 3L5
Received 2 August 2006; revised 28 October 2006; accepted 2 November 2006
Available online 10 November 2006
Abstract—Tubulin is the target of many anticancer drugs, including N-phenyl-N0-(2-chloroethyl)urea (CEU). Unlike most anti-b-
tubulin agents, CEUs are protein monoalkylating agents binding through their N0-(2-chloroethyl)urea moiety to an amino acid
nearby the colchicine-binding site on b-tubulin isoform-2. Following the previously synthesized and attractive N-(3-x-hydroxyalkyl-
phenyl)-N0-(2-chloroethyl)urea that exhibited growth inhibitory activity at the nanomolar level, we investigated the importance of
lower alkyl and alkoxy groups to evaluate the effect of hydroxylated group and chain length on both cell growth inhibition and the
mechanism of action of CEU. Here, we describe the preparation of two new series of CEU and show that the most potent CEU
derivatives beside the x-hydroxylated 1f were 2f and 3e, respectively. We have confirmed that the pentyl substituted CEUs 1f,
2f, and 3e are still covalently binding to b-tubulin and still arrest cell division in G2/M phase.
Crown Copyright ꢀ 2006 Published by Elsevier Ltd. All rights reserved.
1. Introduction
and a potent antineoplastic activity on mice bearing co-
lon adenocarcinoma tumors.16 We previously investigat-
ed the effect of several structural parameters such as the
presence of short electron-donating and -withdrawing
groups at different position of the aromatic ring, the
substitution of the aromatic ring by lower alkyl and
branched-alkyl groups,6–8 and the substitution of the
N0-(2-chloroethyl)urea moiety on carbon 2 on the GI50
of CEU13 to improve the biopharmaceutical properties
of these antimitotic agents.
Tubulin is a major target for many anticancer drug can-
didates, including N-phenyl-N0-(2-chloroethyl)urea
(CEU).1–3 Unlike most antimitotic agents, CEUs are
protein monoalkylating agents that covalently bind
through its N0-(2-chloroethyl)urea moiety to an amino
acid neighboring the colchicine-binding site on b-tubulin
isoform-2.4,5 CEUs exhibit cell growth inhibition (GI50)
on numerous cancer cell lines,4,7–16 on tumor cells hav-
ing developed various chemoresistance mechanisms16,
Recently, we published a study demonstrating that GI50
of CEU can be increased by a factor of 5 by substituting
the position 3 of the aromatic ring of CEU by x-hy-
droxylated alkyl chains, while alkynyl chain substitutes
are detrimental to cell growth inhibition.17 Here, we
evaluated the effective role of the hydroxyl functional
group and the side-chain length on both GI50 and the
alkylation of b-tubulin. With the objective to answer
these questions, we have developed CEUs 2a–f and
3e–g with the goal of assessing the role of x-hydroxyl
terminal group of the side chain substituting the aromat-
ic ring in 3-position.
Keywords: Phenyl chloroethylurea; Antimicrotubule agents; Antitub-
ulin agents; Antimitotic agents; Soft alkylating agents; Anticancer
drugs; Colchicine-binding site ligands.
*
Corresponding authors. Tel.: +33 4 73 17 80 00 (E.M), tel.: +1 418
525 4444x52363; fax: +1 418 525 4372 (R.C.); e-mail addresses:
Emmanuel.Moreau@u-clermont1.fr;
ulaval.ca
rene.c-gaudreault@crsfa.
Present address: Inserm, U384, Clermont-Ferrand, F-63001 France;
Univ Clermont 1, UFR Pharmacie, Laboratoire de Chimie Orga-
nique, Clermont-Ferrand, F-63001 France.
´
OmegaChem inc., 8800 Boulevard de la Rive-Sud, Levis, QC,
Canada G6V 9H1.
à
0968-0896/$ - see front matter Crown Copyright ꢀ 2006 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.005

S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
1431
2. Results and discussion
colon carcinoma HT-29, human skin melanoma
M21, and human breast carcinoma MCF-7. Cell
growth inhibition was assessed as described by the
NCI/NIH Developmental Therapeutics Program for
its drug screening program.18 Structure–activity
relationship studies were focused on the aforemen-
tioned structural parameters. The results are summa-
rized in Table 1. GI50 results for 1a–h and 3a–b
were previously described17,9 and are shown for
comparison.
2.1. Chemistry
The homologation of the alkyl side chain at position 3 of
the aromatic ring of CEU (see Scheme 1, pathway 3) was
performed using a Sonogashira reaction involving the
palladium-catalyzed reaction of 3-iodo nitrobenzene 9
with terminal alkynes or x-hydroxyalkynes.17 The reac-
tion was co-catalyzed by Cu (I), a base, and Pd(0)/PPh3
to produce compounds 10d–f17 and 12e–g. The methyl-
ation reaction of x-hydroxylated compounds was per-
formed using a mixture of sodium hydride and methyl
iodide to yield the methoxylated compounds 5a, 8b, c,
and 11d–f in 45–87% yields (Scheme 1, pathways 1, 2,
and 3). Catalytic hydrogenation of nitro derivatives
using Pd/C produced compounds 5b-f and 13e-g, respec-
tively, that were directly reacted with 2-chloroethyliso-
cyanate to obtain the corresponding CEUs 2a–f and
3f–h. Compounds 1a–h17 and 3a, b9 have been previous-
ly described.
We first evaluated GI50 effect of CEUs substituted in 3-
position by lower methoxylated chains. Newly synthe-
sized derivatives 2a–f increased GI50 by 2- to 4-fold as
compared to 1a–f. Similar tendencies were also observed
in the x-alkyl series 3a, b, e–g. However, compound 3e,
which showed an higher GI50 than the x-hydroxy deriv-
atives, may present significant in vivo advantages since it
might be able to resist quick metabolic oxidative inacti-
vation. It is of interest to mention that replacing a
hydroxyl group (1a–h) by a methoxylated group (2a–f)
or an alkyl group (3a, b, e–g) confirmed the importance
of the chain length in the GI50 of CEU. The latter seems
to be an important variable; the GI50 effect being
optimal with a pentyl chain length substituent (see
Fig. 1).
2.2. Tumor cell growth inhibition activity
CEUs’ growth inhibition activity was evaluated
against three human cancer cell lines, namely human
1
O
a
b
Cl
N
N
OMe
H2N
OMe
H2N
OH
H
H
5a
4
2 a
2
n1 = 1, 2
a
c
b
O
OH
n1
OMe
n1
OMe
n1
Cl
OMe
n1
( )
( )
( )
O2N
O2N
H2N
( )
N
N
H
H
6, 7
8 b, c
5 b, c
2 b, c
3
n2 = 1, 2, 3
n3 = 2, 3, 4
a
c
b
O
Cl
O2N
d
O2N
H2N
N
H
N
( )
H
( )
n2
OH
n2
( )
( )
n2
OMe
n2
OMe
OMe
10 d- f
2 d - f
11 d - f
5 d - f
O2N
I
d
9
b
c
O
Cl
H2N
O2N
N
N
H
n
( )
H
( )
n3
CH3
3
n
( )
3
12e - g
13 e - g
3 e - g
CH3
CH3
Scheme 1. Reagents: (a) NaH, Mel/THF; (b) 2-chloroethylisocyanate/DCM; (c) H2, Pd/C/EtOH; (d) K2CO3, Cul, PPh3, alcyne/1,2-DME/water.

1432
S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
Table 1. GI50 values of CEU derivatives and electrophoretic mobility shift assay of alkylated b-tubulin
H
N
H
N
R
Cl
O
Compound
Mp
Yield
(%)
GI50 (µM)
Alkylated tubulin
17 25 48 h
(oC)
2
7
HT-29
10
M21
10
MCF-7
1a
1b
1c
1d
1e
R = OH
117 - 119
106 - 109
109 - 111
96 - 98
96
56
20
19
18
10
R = CH2-OH
10
10
10
R = (CH2)2-OH
R = (CH2)3-OH
R = (CH2)4-OH
10
10
10
5,2
6,0
7,9
2,5
72 - 74
1,1
1,7
1f
R = (CH2)5-OH
R = (CH2)6-OH
R = (CH2)7-OH
92 - 95
83 - 84
86 - 88
19
42
2
0,25
0,47
0,53
0,39
0,49
0,54
0,49
0,58
0,58
1g
1h
2a
2b
2c
2d
R = OMe
81
15
1
> 10
> 10
6,4
> 10
> 10
8,6
> 10
> 10
14
-
R = CH2-OMe
R = (CH2)2-OMe
R = (CH2)3-OMe
84 - 85
-
38
4,6
7,5
> 10
57 - 59
2e
2f
R = (CH2)4-OMe
R = (CH2)5-OMe
43 - 45
1
1,7
2,3
3,9
1,9
-
13
0,84
0,94
3a
3b
R = CH3
38
60
> 10
8,9
> 10
> 10
> 10
> 10
130 - 132
56 - 55
R = CH2-CH3
3e
3f
R = (CH2)4-CH3
R = (CH2)5-CH3
R = (CH2)6-CH3
-
-
-
49
48
41
0,96
1,1
0,87
1,4
1,6
1,0
2,0
3g
1,9
1,7
Figure 1. General formula of 1-(2-chloroethyl)-3-phenyl-urea (CEU) scaffold with key atoms and groups to insure alkylation of b-tubulin and cell
growth inhibition.

S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
1433
Table 2. Quantification of the FACS analyses for compounds 1f, 2f,
and 3e
2.3. Electrophoresis gel assay
The alkylating ability of compounds 1f–h, 2e–f, and
3e–g on b-tubulin was examined using an electropho-
retic mobility shift assay.4,7,14,15,19 The appearance of
a second b-tubulin band (lower band in the gel
shown in Table 1) with an apparent faster mobility
is indicative of a measurable amount of alkylated
b-tubulin4 (see Table 1). The appraisals of the cova-
lent binding of CEU on b-tubulin were performed
using MDA-MB-231 cells. Compounds 1f, 2f, and
3e covalently bound b-tubulin at a concentration of
5 lM. Measurements of the relative intensity of the
alkylated tubulin with respect to the non-alkylated
tubulin (see Table 1) are shown in Figure 2. After
48 h, the extent of the b-tubulin-CEU by-product
was maximal with 1f, followed by its methoxylated
derivative 2f and the alkyl derivative 3e (94%, 81%,
and 90%, respectively). Measurements performed
using 1f at 30 lM indicated an alkylation rate of
50% at 6 h and 100% after 12 h.19 The various alkyl-
ation percentage reported here may reflect CEUs’
affinity for tubulin. However, these results do not
provide any significant information on the kinetics
of b-tubulin’s alkylation by CEUs. Compounds 1f
and 2f are the molecules displaying the highest affin-
ity for b-tubulin. Such results have been reported
previously for other CEU subsets.4,7,14,19 The most
potent CEUs are substituted by five-carbon atom
substituents in 3-position of their aromatic ring. They
are so far our best hit compounds with respect to
b-tubulin alkylation and GI50.
Compound Concn (lM) Apoptotic cells and cell cycle phase
(% of population)
Apoptosis G0/G1
S
G2/M
1f
1.2
4
7.58
9.83
54.35
7.57
5.9
16.65 21.82
12.12 70.24
12.11 10.71
10
10.71
2f
3
10
25
8.5
31.28
7.52
6.74
19.98 40.22
14.37 68.57
13.79 70.84
9.02
8.63
3e
3
10
25
6.16
8.59
7.84
15.06
7.85
8.75
17.75 60.78
13.28 68.06
15.23 68.48
DMSO
3.04
61.36
20.05 15.88
ly growing M21 cells were treated with compounds 1f,
2f, and 3e or DMSO for escalating period of time as de-
scribed in Section 4. Flow cytometric analysis showed
that compounds 1f, 2f, and 3e caused a significant accu-
mulation of cells in G2/M phase. It suggests that 1f, 2f,
and 3e may induce microtubule disruption and conse-
quently prevent mitosis in a similar fashion to colchicine
and vinblastine (see Table 2).
3. Conclusion
We report here the results of our investigation on the
role of terminal x-hydroxyl functional group on biolog-
ical activity of a series of CEUs (compounds 1a–h17).
We have also prepared two new series of CEUs by mod-
ifying the functional group on side chain. Structure–ac-
tivity relationships show that active CEUs, substituted
in 3-position, require for low concentration cell growth
inhibition a lower alkyl substituted or not by either an
hydroxyl or a lower alkoxyl group; the cell growth inhi-
bition being optimal with a five-carbon atom chain. The
best compounds, namely 1f, 2f, and 3e, were still potent
microtubule disruptors by covalently binding to b-tubu-
lin isoform-2. We therefore conclude that other appro-
priate terminal groups might optimize the biological
activity of CEUs. Indeed, it is known that the binding
of colchicinoid derivatives such as MTC, TCB, and
TKB to tubulin relies on the contribution of presence
of carbonyl group on ring C (or C0) to generate a specific
role in the inhibition process.20,21 The results of further
optimization of this series with contribution of carbonyl
terminal group on the side chain will be reported in due
course.
2.4. DNA cell cycle analysis
Antimicrotubule agents such as colchicine and vinblas-
tine are known to block the cell cycle in G2/M phase
through microtubule disruption.4 The analogy of action
between CEUs and microtubule-disrupting agents on
the cytoskeleton prompted us to examine the effects of
compounds 1f, 2f, and 3e on the cell cycle. Exponential-
4. Experimental Section
4.1. Biological assays and reagents
Biochemicals, drugs, and the monoclonal antibody anti-
b-tubulin (clone TUB 2.1) were purchased from Sigma
Chemical (St. Louis, MO). The bovine calf serum was
obtained from Hyclone (Road Logan, UT). The ECL
Figure 2. Extent of b-tubulin alkylation at escalating time of exposure
with the drug as determined from optical density measurement of
electrophoretic mobility shift assay measurements presented in Table 1.

1434
S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
Western blotting detection reagent kit was provided by
Amersham Canada (Oakville, Canada). Mixtures are ex-
pressed as volume/volume ratios unless otherwise indi-
cated. All drugs were dissolved in DMSO, and the
final concentration of DMSO in the culture medium
was maintained at 0.5%.
results. Optical densities of the normal and alkylated
b-tubulin band were integrated using a NIH imager (Sci-
on Corporation, Frederick, MD) and the percentage of
alkylated b-tubulin was calculated.
4.1.3. Cell cycle analysis. After incubation of HT 1080
cells with drug, the cells were harvested, resuspended
in 1 mL PBS, and fixed by the addition of 2.4 mL of
ice-cold anhydrous ethanol. Then, 5 · 105 cells from
each sample were centrifuged for 3 min at 1000g. Cell
pellets were resuspended in PBS containing 50 lg/mL
of propidium iodide and 40 U/mL of RNase A (Boeh-
ringer Mannheim, Laval, Canada). Mixtures were incu-
bated at room temperature for 30 min, and cell cycle
distribution was analyzed using an Epics Elite ESp flow
cytometer (Coulter Corporation, Miami, FL). Quantifi-
cation of the FACS analyses was carried out with the
software developed by Scripps Research Institute
(http://www.scripps.edu/e_index.html).
4.1.1. Cell culture and growth inhibition activity. The
growth inhibition potency of CEUs was assessed using
the procedure described by the National Cancer Insti-
tute for its drug screening program.18 96-Well tissue cul-
ture plates were seeded with 100 lL of tumor cell lines
suspended in high glucose DMEM supplemented with
5% (v/v) defined bovine calf serum iron supplemented
(Hyclone). Plates were incubated at 37 ꢁC, 5% CO2 for
24 h. Freshly solubilized drugs in DMSO were diluted
in fresh medium and aliquots of 100 lL containing
sequential dilution of drugs were added. Final drug con-
centrations ranged from 10 to 0.3 lM. DMSO concen-
tration was maintained lower than 0.5% to avoid
solvent’s cytotoxicity. Plates were incubated for 48 h.
Assays were stopped by addition of cold trichloroacetic
acid to the wells (final concentration was 10%), followed
by incubation for 60 min at 4 ꢁC. Plates were washed
five times with tap 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, unbound dye was removed
by washing five times with 1% acetic acid. Bound stain
was solubilized with 10 mM Tris base, and the absor-
bance was read using a lQuant Universal Microplate
Spectrophotometer (Biotek, Winooski, VT) at 585 nm.
The results were compared with those of a control refer-
ence plate fixed on the treatment day and the growth
inhibition percentage was calculated for each drug con-
tact period. The experiments were performed at least
twice in triplicate. The GI50 assay was considered valid
when the variability among data for a given set of con-
ditions, within the same experiment, was less than 10%
with respect to the mean value.
4.2. Experimental procedures
4.2.1. Chemistry and chemical methods. Proton NMR
spectra were recorded on a Brucker AM-300 spectrom-
eter (Bruker, Germany). Chemical shifts (d) are reported
in parts per million relative to the internal tetramethylsi-
lane standard. IR spectra were recorded on a Unicam
spectrometer. Uncorrected melting points were deter-
mined on an Electrothermal melting point apparatus.
ESIMS spectral analysis was carried out at the Mass
Spectroscopy Laboratory of Molecular Medicine
Research Centre, Medical Sciences Bldg, University of
Toronto (http://www.medresearch.utoronto.ca/pmsc_
home.html). All reactions were conducted under a
rigorously dried nitrogen atmosphere. Chemicals were
supplied by Aldrich Chemical Co. (Milwaukee, WI).
The aromatic nitro alkynols 10d–f and x-hydroxylated
alkyl CEUs 1a–h were prepared as previously de-
scribed.17 Liquid flash chromatography was performed
on silica gel 60 A (American Chemicals Ltd., Montreal,
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 reactions was monitored using
TLC on precoated silica gel plates (Merck Silica Gel
60 F254). The chromatograms were visualized under
UV light at 254 nm. For column chromatography
Merck Silica Gel (70–230 mesh) was used.
4.1.2. Electrophoretic mobility shift assay to evaluate b-
tubulin alkylation. Exponentially growing MDA-MB-
231 cells (2.2 · 105) were plated in 12-well plates and
incubated overnight at 37 ꢁC. The cells were then treated
with a 5 lM test compound solution, except for the con-
trol, for 2, 7, 17, 25, and 48 h, and were harvested direct-
ly in culture medium using a rubber policeman. Cells
were centrifuged, and the pellets were washed with
500 lL of cold PBS. After centrifugation, the pellets
were lysed using Laemmli sample buffer. Samples
(5 · 104 cells) were analyzed by 10% SDS–PAGE using
the Laemmli system. Membranes were then incubated
with PBSMT (PBS, pH 7.4, 5% fat-free dried milk and
0.1% Tween 20TM) for 1 h at room temperature and then
with 1/500 monoclonal anti-b-tubulin (clone TUB 2.1)
for 1 h. Membranes were washed with PBSMT and
incubated with 1/2500 peroxidase-conjugated antimouse
immunoglobulin in PBSMT for 30 min. Detection of the
immunoblot was carried out with the ECL Western
blotting detection reagent kit. All data were obtained
from the same experiment. Duplicate experiments were
conducted on some compounds and showed similar
4.3. General preparation of compounds 1a–h17 and 3a, b9
Syntheses and characterizations of compounds 1a–h17
were previously reported. Briefly, x-hydroxylated CEUs
1a–h were synthetized by nucleophilic addition of the
x-hydroxyl alkylphenylamines on 2-chloroethylisocya-
nate. The x-hydroxylated alkenylphenylamines 10d–h17
were reduced under hydrogen atmosphere to afford the
hydroxylated alkylphenylamines. Compounds 10d–h
were synthesized by a Sonogashira coupling between
1-iodo-3-nitrobenzene and x-hydroxylated alkynyls.
CEUs 3a, b were directly prepared by the nucleophilic
addition of 1-methyl or 1-ethyl-3-aniline to 2-
chloroethylisocyanate.

S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
1435
4.3.1. N-[3-(4-Hydroxybutyl)phenyl]-N0-(2-chloroethyl)
urea (1e). The preparation of compound 1e was previ-
ously reported.17 ESIMS (m/z) 295.2 [M+2+Na]+,
293.2 [M+Na]+, 273.2 [M+2]+, 271.2 [M]+.
7.67 (br s, 1H, NH), 7.15 (m, 3H, Ar), 6.85 (d, 1H,
J = 7.0, Ar), 3.51 (m, 4H, CH2), 3.34 (m, 2H, CH2),
2.58 (m, 2H, CH2), 1.82 (m, 2H, CH2); 13C NMR
(CDCl3) d 156.4, 143.2, 138.6, 129.1, 123.7, 120.5,
118.0, 71.9, 58.5, 44.5, 42.10, 32.3, 31.0.
4.3.2. N-[3-(5-Hydroxypentyl)phenyl]-N0-(2-chloroethyl)
urea (1f). The preparation of compound 1f was previ-
ously reported.17 ESIMS (m/z) 309.2 [M+2+Na]+,
307.2 [M+Na]+, 287.2 [M+2]+, 285.2 [M]+.
4.4.5.
1-(2-Chloroethyl)-3-(3-(4-methoxybutyl)phenyl)
urea (2e). Compound 2e was synthesized from 5e. The
crude product was purified by flash chromatography
(silica gel, ethyl acetate). Yield: 77%; mp 43–45 ꢁC; IR
1
4.3.3. N-[3-(6-Hydroxyhexyl)phenyl]-N0-(2-chloroethyl)
urea (1g). The preparation of compound 1g was previ-
ously reported.17 ESIMS (m/z) 323.2 [M+2+Na]+,
321.2 [M+Na]+, 301.2 [M+2]+, 299.2 [M]+.
(KBr) m 3328, 1639, 1250 cmꢀ1; H NMR (CDCl3) d
7.57 (br s, 1H, NH), 7.14 (m, 3H, Ar), 6.84 (d, 1H,
J = 7.0, Ar), 3.64 (m, 4H, CH2), 3.47 (m, 2H, CH2),
3.35 (s, 3H, CH3), 2.54 (m, 2H, CH2), 1.60 (m, 4H,
CH2), 1.24 (m, 2H, CH2); 13C NMR (CDCl3) d 156.3,
143.7, 138.6, 129.0, 123.7, 120.5, 117.9, 72.7, 58.5,
44.6, 42.1, 35.7, 29.2, 27.8. ESIMS (m/z) 309.2
[M+2+Na]+, 307.2 [M+Na]+, 287.3 [M+2]+, 285.2 [M]+.
4.3.4. N-[3-(7-Hydroxyheptyl)phenyl]-N0-(2-chloroethyl)
urea (1h). The preparation of compound 1h was previ-
ously reported.17 ESIMS (m/z) 337.2 [M+2+Na]+,
335.2 [M+Na]+, 315.2 [M+2]+, 313.2 [M]+.
4.4.6. 1-(2-Chloroethyl)-3-(3-(5-methoxypentyl)phenyl)
urea (2f). Compound 2f was synthesized from 5f. The
crude product was purified by flash chromatography
(silica gel, ethyl acetate). Yield: 27%; IR (NaCl): m
4.4. General preparation of compounds 2a–f and 3e–g
4.4.1. 1-(2-Chloroethyl)-3-(3-methoxyphenyl)urea (2a). 2-
Chloroethylisocyanate (1.640 mmol) was added drop-
wise to a cold solution (ice bath) of 5a (1.370 mmol) in
dry dichloromethane (15 mL/g of aniline). The ice bath
was then removed and the reaction mixture was stirred
at room temperature for 20 h. After completion of the
reaction, the solvent was evaporated under reduced
pressure to give an off-white solid, which was purified
by flash chromatography (silica gel, ethyl acetate:hex-
anes (6/4)). Yield: 91%; IR (KBr) m 3307, 1632,
1
3333, 1640, 1246 cmꢀ1; H NMR (CDCl3) d 8.18 (br s,
1H, NH), 7.14 (m, 3H, Ar), 6.86 (d,, 1H, J = 7.0, Ar),
3.57 (m, 4H, CH2), 3.33 (m, 4H, CH2), 2.55 (t, 2H,
J = 7.0, CH2), 1.59 (m, 4H, CH2); 13C NMR (CDCl3)
d 156.0, 144.0, 138.4, 129.1, 123.9, 120.8, 118.2, 58.5,
44.6, 42.0, 35.9, 31.2, 29.5, 25.9. ESIMS (m/z) 323.2
[M+2+Na]+, 321.2 [M+Na]+, 301.3 [M+2]+, 299.3 [M]+.
4.4.7. 1-(2-Chloroethyl)-3-(3-pentylphenyl)urea (3e).
Compound 3e was synthesized from 13e. The crude
product was purified by flash chromatography (silica
gel, hexanes:ethyl acetate (6/4)). Yield: 89%; IR (KBr)
1
1248 cmꢀ1; H NMR (CDCl3) d 8.12 (br s, 1H, NH),
7.21 (m, 3H, Ar), 6.87 (d, 1H, J = 7.0, Ar), 3.97 (s,
3H, CH3), 3.57 (m, 4H, CH2); 13C NMR (CDCl3) d
156.1, 139.4, 138.0, 129.2, 125.1, 122.1, 118.4, 57.8,
44.5, 42.1.
m 3340, 2932, 1685, 1247 cmꢀ1 1H NMR (CDCl3) d
;
7.94 (br s, 1H, NH), 7.09 (m, 4H, Ar), 6.77 (br s, 1H,
NH), 2.48 (t, 2H, J = 7.5, CH2), 1.53 (m, 2H, CH2),
1.22 (m, 4H, CH2), 0.81 (m, 3H, CH3); 13C NMR
(CDCl3) d 156.4, 143.9, 138.9, 128.7, 123.1, 119.9,
117.2, 36.9, 31.5, 31.0, 22.5, 14.5, 14.0. ESIMS (m/z)
294.2 [M+2+Na]+, 292.2 [M+Na]+, 271.2 [M+2]+,
269.3 [M]+.
4.4.2. 1-(2-Chloroethyl)-3-(3-(1-methoxymethyl)phenyl)
urea (2b). Compound 2b was synthesized from 5b. The
crude product was purified by flash chromatography
(silica gel, ethyl acetate:hexanes (6/4)). Yield: 87%; mp
84–85 ꢁC; IR (KBr) m 3330, 1636, 1245 cmꢀ1
;
1H
NMR (CDCl3) d 7.29 (m, 3H, Ar), 7.04 (m, 1H, Ar),
4.40 (s, 2H, CH2), 3.57 (m, 4H, CH2), 3.38 (s, 3H,
CH3); 13C NMR (CDCl3) d 155.8, 139.4, 138.6, 129.3,
123.1, 120.1, 120.0, 74.4, 58.2, 44.8, 42.2.
4.4.8. 1-(2-Chloroethyl)-3-(4-hexylphenyl)urea (3f). Com-
pound 3f was synthesized from 13f. The crude product
was purified by flash chromatography (silica gel, hex-
anes:ethyl acetate (6/4)). Yield: 86%; IR (KBr) m 3326,
1
4.4.3.
1-(2-Chloroethyl)-3-(3-(2-methoxyethyl)phenyl)
2938, 1698, 1251 cmꢀ1; H NMR (CDCl3) d 7.11 (br s,
urea (2c). Compound 2c was synthesized from 5c. The
crude product was purified by flash chromatography
(silica gel, ethyl acetate). Yield: 81%; IR (NaCl) m
1H, NH), 7.07 (m, 4H, Ar), 6.76 (br s, 1H, NH), 2.49
(m, 2H, CH2), 1.52 (m, 2H, CH2), 1.25 (m, 6H, CH2),
0.78 (m, 3H, CH3); 13C NMR (CDCl3) d 156.0, 144.0,
138.9, 128.7, 123.1, 119.8, 117.1, 35.9, 31.6, 31.3, 29.0,
22.5, 14.0. ESIMS (m/z) 307.3 [M+2+Na]+, 305.3
[M+Na]+, 285.2 [M+2]+, 283.2 [M]+.
1
3329, 1640, 1246 cmꢀ1; H NMR (CDCl3) d 7.87 (br s,
1H, NH), 7.21 (m, 3H, Ar), 6.97 (d, 1H, J = 7.0, Ar),
3.58 (m, 2H, CH2), 3.51 (m, 5H, CH2, CH3), 2.61 (m,
2H, CH2); 13C NMR (CDCl3) d 156.4, 143.2, 138.7,
129.2, 123.4, 120.2, 118.0, 71.9, 44.5, 44.4, 32.2, 29.9.
4.4.9. 1-(2-Chloroethyl)-3-(4-heptylphenyl)urea (3g).
Compound 3g was synthesized from 13g. The crude
product was purified by flash chromatography (silica
gel, hexanes:ethyl acetate (6/4)). Yield: 91%; IR (KBr)
4.4.4. 1-(2-Chloroethyl)-3-(3-(3-methoxypropyl)phenyl)
urea (2d). Compound 2d was synthesized from 5d. The
crude product was purified by flash chromatography
(silica gel, ethyl acetate). Yield: 81%; mp 57–59 ꢁC; IR
m 3326, 2927, 1698, 1250 cmꢀ1 1H NMR (CDCl3) d
;
7.13 (br s, 1H, NH), 7.11 (m, 4H, Ar), 6.79 (br s, 1H,
NH), 2.48 (t, 2H, J = 7.5, CH2), 1.53 (m, 2H, CH2),
1
(KBr) m 3320, 1627, 1237 cmꢀ1; H NMR (CDCl3) d

1436
S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
1.20 (m, 8H, CH2), 0.82 (m, 3H, CH3); 13C NMR
(CDCl3) d 156.0, 144.1, 138.8, 128.8, 123.3, 120.0,
117.2, 36.0, 31.8, 31.4, 29.3, 29.1, 22.6, 14.1. ESIMS
(m/z) 322.3 [M+2+Na]+, 319.2 [M+Na]+, 299.3
[M+2]+, 297.4 [M]+.
2H, CH2); 13C NMR (CDCl3) d 146.3, 143.9, 129.1,
118.9, 114.3, 11.5, 72.8, 58.5, 36.0, 31.3, 29.5, 22.8.
4.5.6. 3-Pentylphenylamine (13e). Compound 13e was
synthesized from 12e. The crude product was purified
by flash chromatography (silica gel, CH2Cl2:EtOH (95/
4.5. General preparation of compounds 5b–f and 13e–g
5)). Yield: 93%; IR (NaCl) m 3335, 297, 1670,
1
1489 cmꢀ1; H NMR (CDCl3) d 7.09 (t, 1H, J = 7.9,
Ar), 6.63 (d, 1H, J = 7.5, Ar), 6.53 (m, 2H, Ar), 3.56
(br s, 2H, NH2), 2.54 (m, 2H, CH2), 1.63 (pent, 2H,
J = 7.0, CH2), 1.36 (m, 4H, CH2), 0.93 (t, 3H, J = 7.0,
CH3); 13C NMR (CDCl3) d 146.3, 144.3, 129.1, 118.9,
115.4, 112.6, 36.0, 31.6, 31.1, 22.6, 14.1.
A mixture of the appropriate alkenyl (0.43 mmol), Pd/C
10% dissolved in ethanol (30 mL) was reduced under
hydrogen atmosphere (38 psi) overnight. The catalyst
was removed by filtration on Celite and the filtrate
was evaporated to dryness. The residue was purified
by flash chromatography on silica gel to afford 5b–f or
13e–g.
4.5.7. 3-Hexylphenylamine (13f). Compound 13f was
synthesized from 12f. The crude product was purified
by flash chromatography (silica gel, CH2Cl2:EtOH (95/
5)). Yield: 97%; IR (NaCl) m 3366, 2927, 1618,
4.5.1. 3-(1-Methoxymethyl)phenylamine (5b). Compound
5b was synthesized from 8b. The crude product was
purified by flash chromatography (silica gel, ethyl ace-
tate:EtOH (9/1)). Yield: 23%; IR (NaCl) m 3323, 2922,
1
1460 cmꢀ1; H NMR (CDCl3) d 7.11 (t, 1H, J = 7.5,
Ar), 6.64 (d, 1H, J = 7.4, Ar), 6.54 (m, 2H, Ar), 3.57
(br s, 2H, NH2), 2.57 (m, 2H, CH2), 1.64 (m, 2H,
CH2), 1.36 (m, 6H, CH2), 0.95 (m, 3H, CH3); 13C
NMR (CDCl3) d 146.3, 144.3, 129.2, 118.9, 115.4,
112.6, 36.1, 31.4, 29.1, 22.7, 14.2.
1
1097 cmꢀ1; H NMR (CDCl3) d 7.25 (s, 1H, Ar), 7.12
(t, 1H, J = 7, Ar), 6.65 (m, 2H, Ar), 4.36 (s, 2H, CH2),
3.65 (br s, 2H, NH), 3.36 (s, 3H, CH3); 13C NMR
(CDCl3) d 146.2, 139.6, 129.4, 118.1, 114.7, 114.4,
72.0, 58.1.
4.5.8. 3-Heptylphenylamine (13g). Compound 13g was
synthesized from 12g. The crude product was purified
by flash chromatography (silica gel, CH2Cl2:EtOH (95/
5)). Yield: 82%; IR (NaCl) m 3374, 2926, 1617,
4.5.2. 3-(2-Methoxyethyl)phenylamine (5c). Compound
5c was synthesized from 8c. The crude product was puri-
fied by flash chromatography (silica gel, ethyl acetate).
1
Yield: 22%; IR (NaCl) m 3359, 2925, 1112 cmꢀ1
;
1H
1459 cmꢀ1; H NMR (CDCl3) d 7.08 (t, 1H, J = 8.0,
NMR (CDCl3) d 7.26 (s, 1H, Ar), 7.15 (m, 2H, Ar),
6.91 (d, 1H, J = 7, Ar), 3.47 (m, 5H, CH2, CH3), 2.41
(m, 2H, CH2); 13C NMR (CDCl3) d 146.1, 143.8,
129.2, 119.1, 115.3, 112.2, 72.0, 58.5, 32.8.
Ar), 6.61 (d, 1H, J = 7.5, Ar), 6.53 (m, 2H, Ar), 3.50
(br s, 2H, NH2), 2.53 (m, 2H, CH2), 1.62 (m, 2H,
CH2), 1.29 (m, 8H, CH2), 0.90 (m, 3H, CH3); 13C
NMR (CDCl3) d 146.0, 144.3, 129.1, 119.1, 115.5,
112.7, 36.0, 31.9, 31.4, 29.4, 29.2, 22.7, 14.1.
4.5.3. 3-(3-Methoxypropyl)phenylamine (5d). Compound
5d was synthesized from 11d. The crude product was
purified by flash chromatography (silica gel, ethyl ace-
4.6. General preparation of compounds 5a, 8b–f, and 11d–f
tate). Yield: 78%; IR (NaCl) m 3310, 2928, 1111 cmꢀ1
;
4.6.1. 3-Methoxybenzenamine (5a). NaH (60%)
(97.40 mg, 4.06 mmol) was suspended in dry THF
(8 mL). A solution of compound 4 (0.98 mmol) in dry
THF (3 mL) was added dropwise at 0 ꢁC. The mixture
was stirred for 15 min at 0 ꢁC. MeI (2.34 mmol) was
added dropwise and the mixture was stirred at room
temperature for 3 h. Saturated solution of NaHCO3
(10 mL) and MeOH (10 mL) were then added. The mix-
ture was extracted with AcOEt (3· 15 mL), dried over
Na2SO4, filtered, evaporated to dryness, and purified
by flash chromatography (silica gel, ethyl acetate).
1H NMR (CDCl3) d 7.07 (t, 1H, J = 7.5, Ar), 6.60 (d,
1H, J = 8, Ar), 6.53 (m, 2H, Ar), 3.68 (br s, 2H, NH),
3.36 (m, 5H, NH, CH2), 2.59 (t, 2H, J = 7, CH2), 1.86
(m, 2H, CH2); 13C NMR (CDCl3) d 146.1, 143.3,
129.3, 119.0, 115.5, 112.9, 72.0, 58.5, 32.3, 31.1.
4.5.4. 3-(4-Methoxybutyl)phenylamine (5e). Compound
5e was synthesized from 11e. The crude product was
purified by flash chromatography (silica gel, ethyl ace-
tate). Yield: 35%; IR (NaCl) m 3390, 2916, 1116 cmꢀ1
;
1H NMR (CDCl3) d 7.04 (t, 1H, J = 7.5, Ar), 6.59 (d,
1H, J = 8, Ar), 6.51 (m, 2H, Ar), 3.62 (br s, 2H, NH),
3.37 (m, 2H, CH2), 3.31 (s, 3H, CH3), 2.53 (t, 2H,
J = 7, CH2), 1.42 (m, 4H, CH2); 13C NMR (CDCl3) d
146.0, 143.7, 129.2, 119.1, 115.5, 112.8, 72.7, 58.5,
35.8, 29.7, 27.8.
Yield: 87%; IR (NaCl) m 1532, 1114 cmꢀ1 1H NMR
;
(DMSO-d6) d 8.18 (s, 1H, Ar), 8.12 (d, 1H, J = 8.0,
Ar), 7.63 (d, 1H, J = 7.5, Ar), 7.51 (t, 1H, J = 8.0, Ar),
3.38 (s, 3H, CH3); 13C NMR (DMSO-d6) d 148.4,
140.6, 133.3, 129.3, 122.5, 122.2, 58.5.
4.6.2. 1-(1-Methoxymethyl)-3-nitrobenzene (8b). Com-
pound 8b was synthesized from 6. The crude product
was purified by flash chromatography (silica gel, ethyl ace-
tate). Yield: 75%; IR (NaCl) m 1535, 1115 cmꢀ1; 1H NMR
(DMSO-d6) d 8.16 (s, 1H, Ar), 8.09 (d, 1H, J = 8.0, Ar),
7.63 (d, 1H, J = 7.5, Ar), 7.48 (t, 1H, J = 8.0, Ar), 4.51
(s, 2H, CH2), 3.40 (s, 3H, CH3); 13C NMR (DMSO-d6)
d 148.4, 140.6, 133.3, 129.3, 122.5, 122.2, 73.3, 58.5.
4.5.5. 3-(1-Methoxypentyl)phenylamine (5f). Compound
5f was synthesized from 11f. The crude product was
purified by flash chromatography (silica gel, ethyl ace-
tate). Yield: 85%; IR (NaCl) m 3366, 2928, 1110 cmꢀ1
;
1H NMR (CDCl3) d 7.07 (t, 1H, J = 7.5, Ar), 6.49 (d,
1H, J = 8, Ar), 6.43 (m, 2H, Ar), 3.31 (m, 5H, CH2,
CH3), 2.51 (m, 2H, CH2), 1.52 (m, 4H, CH2), 1.32 (m,

S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 1430–1438
1437
1
4.6.3. 1-(2-Methoxyethyl)-3-nitrobenzene (8c). Com-
pound 8c was synthesized from 7. The crude product
was purified by flash chromatography (silica gel, ethyl
acetate). Yield: 81%; mp >310 ꢁC; IR (KBr) m 1592,
(NaCl) m 3084, 2934, 2225, 1530, 1350 cmꢀ1; H NMR
(CDCl3) d 8.16 (s, 1H, Ar), 8.06 (d, 1H, J = 8.3, Ar),
7.63 (d, 1H, J = 7.5, Ar), 7.41 (t, 1H, J = 8.0, Ar), 2.37
(t, 2H, J = 7.0, CH2), 1.62 (m, 2H, CH2), 1.01 (m, 3H,
CH3); 13C NMR (CDCl3) d 148.1, 137.3, 129.1, 126.3,
125.9, 122.2, 93.4, 78.6, 22.1, 21.8, 21.3.
1
1162 cmꢀ1; H NMR (DMSO-d6) d 8.13 (s, 1H, Ar),
8.05 (d, 1H, J = 8.0, Ar), 7.72 (d, 1H, J = 8.0, Ar),
7.60 (m, 1H, Ar), 3.62 (t, 2H, J = 7.0, CH2), 3.28 (s,
3H, CH3), 2.92 (t, 2H, J = 7.0, CH2); 13C NMR
(DMSO-d6) d 148.7, 138.1, 129.2, 126.9, 124.3, 123.9,
73.3, 58.5, 14.8.
4.7.2. 1-Nitro-3-hexynylbenzene (12f). Compound 12f
was synthesized from 9 and 1-hexyne. The crude prod-
uct was purified by flash chromatography (silica gel,
hexanes:ethyl acetate (60/40)). Yield: 58%; IR (NaCl) m
1
4.6.4. 1-(3-Methoxyprop-1-ynyl)-3-nitrobenzene (11d).
Compound 11d was synthesized from 10d. The crude
product was purified by flash chromatography (silica
gel, ethyl acetate). Yield: 61%; IR (NaCl) m 2235, 1346
3083, 2933, 2229, 1531, 1350 cmꢀ1; H NMR (CDCl3)
d 8.21 (s, 1H, Ar), 8.09 (d, 1H, J = 8.0, Ar), 7.67 (d,
1H, J = 7.8, Ar), 7.44 (t, 1H, J = 7.9, Ar), 2.42 (t, 2H,
J = 7.0, CH2), 1.61 (m, 2H, CH2), 1.47 (m, 2H, CH2),
0.93 (t,, 3H, J = 7.0, CH3); 13C NMR (CDCl3) d
148.1, 137.4, 129.2, 126.3, 125.8, 122.3, 93.4, 78.6,
22.1, 21.8, 21.5, 21.3.
(NO2), 1091 cmꢀ1 1H NMR (CDCl3) d 8.27 (s, 1H,
;
Ar), 8.15 (d, 1H, J = 8.0, Ar), 7.73 (d, J = 8.0, Ar),
7.49 (t, 1H, J = 8.0, Ar), 7.73 (d, 1H, J = 8.0, Ar), 7.49
(t, 1H, J = 8.0, Ar), 3.59 (s, 2H, CH2), 3.37 (s, 3H,
CH3); 13C NMR (CDCl3) d 148.3, 137.5, 129.5, 127.4,
124.2, 123.3, 87.9, 83.9, 60.2, 57.9, 29.7.
4.7.3. 1-Nitro-3-heptynylbenzene (12g). Compound 12g
was synthesized from 9 and 1-heptyne. The crude prod-
uct was purified by flash chromatography (silica gel,
hexanes:ethyl acetate (60/40)). Yield: 55%; IR (NaCl) m
4.6.5. 1-(4-Methoxybut-1-ynyl)-3-nitrobenzene (11e).
Compound 11e was synthesized from 10e.17 The crude
product was purified by flash chromatography (silica
gel, ethyl acetate). Yield: 67%; IR (NaCl) m 2223, 1353,
3447, 2932, 2227, 1532, 1351 cmꢀ1; H NMR (CDCl3)
1
d 8.17 (s, 1H, Ar), 8.06 (d, 1H, J = 7.9, Ar), 7.63 (d,
1H, J = 7.6, Ar), 7.41 (t, 1H, J = 8.0, Ar), 2.39 (t, 2H,
J = 7.0, CH2), 1.55 (m, 2H, CH2), 1.41 (m, 4H, CH2),
0.91 (t, 3H, J = 7.0, CH3); 13C NMR (CDCl3) d 148.1,
137.3, 129.1, 126.3, 126.0, 122.1, 93.6, 78.5, 31.1, 28.2,
22.2, 19.3, 13.9.
1
1109 cmꢀ1; H NMR (CDCl3) d 8.23 (s, 1H, Ar), 8.10
(d, 1H, J = 8.0, Ar), 7.68 (d, 1H, J = 7.0, Ar), 7.44 (t,
1H, J = 8.0, Ar), 3.59 (t, 2H, J = 7.0, CH2), 3.40 (s,
3H, CH3), 2.70 (t, 2H, CH2); 13C NMR (CDCl3) d
148.1, 137.4, 129.1, 126.5, 125.6, 122.5, 90.0, 79.3,
70.5, 58.8, 19.7.
Acknowledgments
4.6.6. 1-(5-Methoxypent-1-ynyl)-3-nitrobenzene (11f).
Compound 11f was synthesized from 10f.17 The crude
product was purified by flash chromatography (silica
gel, ethyl acetate). Yield: 58%; IR (NaCl) m 2228, 1353,
This research program was realized under research
grants from the National Institute of Health Research
(Grant # MOP-67782), the Cancer Research Society
inc, a sponsorship from IMOTEP inc and the Centre
1
1116 cmꢀ1; H NMR (CDCl3) d 8.18 (s, 1H, Ar), 8.07
ˆ
(d, 1H, J = 8.0, Ar), 7.64 (d, 1H, J = 8.0, Ar), 7.42 (t,
1H, J = 8.0, Ar), 3.49 (t, 2H, J = 7.0, CH2), 3.34 (s,
3H, CH3), 2.49 (t, 2H, J = 7.0, CH2), 1.86 (m, 2H,
CH2); 13C NMR (CDCl3) d 148.1, 137.3, 129.1, 126.3,
125.8, 122.3, 97.7, 78.7, 71.1, 58.6, 29.7, 16.1.
de Recherche de l’Hopital Saint-Franc¸ois d’Assise, and
the Faculty of pharmacy of Laval University (student-
ship to SF). We also acknowledge Mr. Claude Marquis
for his technical help in performing the determination of
b-tubulin alkylation rate with CEU.
4.7. General preparation of compounds 12e–g
References and notes
4.7.1. 1-Nitro-3-pentynylbenzene (12e). To a mixture of
the compound
9
(4.56 mmol), K2CO3 (1.57 g,
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30 mL) were successively added CuI (34 mg,
0.18 mmol), PPh3 (95.80 mg, 0.36 mmol), and Pd/C
10% (97.5 mg, 0.09 mmol). The mixture was stirred at
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1438
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