Synthesis, antiproliferative activity evaluation and structure-activity relationships of novel aromatic urea and amide analogues of N-phenyl-N′- (2-chloroethyl)ureas

Article abstract of DOI:10.1016/j.ejmech.2010.03.018

Seven subsets of aromatic urea and amide analogues of N-phenyl-N′-(2- chloroethyl)ureas (CEU) have been synthesized by nucleophilic addition of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl

Full text of DOI:10.1016/j.ejmech.2010.03.018

European Journal of Medicinal Chemistry 45 (2010) 2928e2937
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original Article
Synthesis, antiproliferative activity evaluation and structureeactivity
relationships of novel aromatic urea and amide analogues of
N-phenyl-N0-(2-chloroethyl)ureas
,
b
,
c
a
a
a
Sébastien Fortin a , Emmanuel Moreau , Jacques Lacroix , Marie-France Côté , Éric Petitclerc ,
**
*
René C.-Gaudreault a
,d,
a Unité des Biotechnologies et de Bioingénierie, Centre de recherche, C.H.U.Q., Hôpital Saint-François d’Assise, Quebec G1L 3L5, Canada
b Faculté de Pharmacie, Université Laval, Pavillon Vandry, Quebec G1V 0A6, Canada
c UMR INSERM/UdA 990, Clermont-Ferrand cedex, France
d Faculté de Médecine, Université Laval, Pavillon Vandry, Quebec G1V 0A6, Canada
a r t i c l e i n f o
a b s t r a c t
Seven subsets of aromatic urea and amide analogues of N-phenyl-N0-(2-chloroethyl)ureas (CEU) have
been synthesized by nucleophilic addition of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, eth-
ylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlorobutanoyl chloride, and
acryloyl chloride, respectively, to selected anilines or benzylamines to afford 3-chloropropylureas (1,
CPU), 2-chloroacetylureas (2, CAU), ethylureas (3, EU), 2-chloroacetamides (4, CA), 3-chlor-
opropionamides (5, CPA), 4-chlorobutyramides (6, CBA) and acrylamides (7, Acr). The molecular structure
of these compounds has been confirmed by IR, 1H and 13C NMR, and MS spectra and their purity also
confirmed by HPLC. The CEU analogues were evaluated for their antiproliferative activity against three
human tumor cell lines, namely human colon carcinoma HT-29, human skin melanoma M21, and human
breast carcinoma MCF-7. CAU (2c to 2g), CA (4a to 4d, 4f and 4g), CPA (5a) and Acr (7a and 7b) had IC50
Article history:
Received 10 February 2010
Received in revised form
15 March 2010
Accepted 17 March 2010
Available online 25 March 2010
Keywords:
Aromatic ureas
Aromatic amides
N-phenyl-N0-(2-chloroethyl)ureas
CEU
ranging from 1.4 to 25 mM. CAU, CA, CPA and Acr exhibited interesting antiproliferative activity through
Anticancer drugs
Antimitotic agents
mechanism(s) of action unrelated to the acylation of glutamic acid at position 198 on
characterizing CEU.
b-tubulin that is
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
vitro on a large number of tumor cell lines. They block also cell cycle
progression in different phases and are leading to cell death. Finally,
CEU block angiogenesis and inhibit tumor growth in three distinct
animal models [4,5,16,17].
Cancer is the second cause of death after cardiovascular diseases
in Western countries [1]. Novel molecules selectively targeting
cancer tumor cells are in constant need to improve both life
expectancy and the quality of life of patients [2,3]. It is in that
context that our laboratory develops novel antineoplasic agents
designated as N-phenyl-N0-(2-chloroethyl)ureas (CEU). Several
subclasses of CEU are known to target selectively key proteins of
cancer cells including microtubules [4e9], thioredoxin-1 [10e12],
prohibitin-1 [13] and the voltage-dependent anion channel-1
[13,14]. CEU exhibit a unique and unprecedented mechanism of
action by covalently and selectively binding to specific acidic amino
acid residues [13,15]. CEU exhibit potent antiproliferative activity in
Our research program aims to the understanding of the struc-
tureeactivity relationships ruling the antimitotic properties of CEU
to improve their biological and biopharmaceutical properties. To
that end, CoMFA and CoMSIA studies have confirmed that CEU are
constituted of 2 main moieties: (i) an N-aryl-N0-(2-chloroethyl)urea
pharmacophore responsible for the antiproliferative activity and
(ii) an auxophore group substituting the aromatic ring responsible
for the selectivity of the drug and the biopharmaceutical properties
(Fig. 1). Moreover, computational experiments conducted on anti-
microtubule CEU showed that the distances between the 2-chlor-
oethylurea moiety of CEU and the nucleophilic carboxyl group of
the glutamic-198 residue on b-tubulin (5.0e8.5 Å) were unfavor-
able to an efficient acylation of the protein [18,19]. Of note, the
optimal nucleophile/electrophile distances for an efficient acylation
should be between 3.2 and 3.6 Å [20,21]. Based on our computa-
tional studies on the binding of CEU to the colchicine-binding site
* Corresponding author. Tel.: þ1 418 525 4444x53787; fax: þ1 418 525 4372.
** Corresponding author. Tel.: þ1 418 525 4444x52363; fax: þ1 418 525 4372.
E-mail addresses: sebastien.fortin.1@ulaval.ca (S. Fortin), rene.c-gaudreault@
crsfa.ulaval.ca (R. C.-Gaudreault).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.018

S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
2929
Fig. 1. The pharmacophore, the auxophore and the 2-chloroethylurea groups of CEU together with the auxophore group of the most potent CEU previously synthesized.
(C-BS), the adjacent pocket to the C-BS where the 2-chlor-
oethylurea moiety reacts is narrow and around 8 Å deep. In addi-
tion, our studies have shown interesting characteristics of the
stabilization of the CEU-protein complex, notably: (1) the impor-
tance of the formation of a hydrogen bond between the carbonyl
group of the urea moiety of CEU and the thiol group of the cysteine
29 colon carcinoma, M21 skin melanoma and MCF-7 breast carci-
noma cells. HT-29, M21, and MCF-7 cell lines were selected, as they
are good representatives of tumor cells originating from the three
embryological germ layers. Cell growth inhibition was assessed
according to the NCI/NIH Developmental Therapeutics Program
[23]. The results are summarized in Table 2 and expressed as the
concentration of drug inhibiting cell growth by 50% (IC50).
residue in position 239 of
b-tubulin, (2) initiation of a covalent
interaction between the carbon atom bearing the chlorine atom
and the glutamic acid residue at position 198 and (3) stabilization of
the auxophore group within the C-BS. These observations promp-
ted us to evaluate the importance of a urea and a chloro group in
the 2-chloroethylurea moiety of CEU on the antiproliferative
activity and the selectivity of the derivative for the C-BS. To that
end, we first modified the urea moiety into an amide or an acety-
lurea group, respectively maintaining approximately the same
length of that portion of the pharmacophore group. Afterward, we
have modified the structure and the properties of the 2-chlor-
oethylurea moiety either by introducing an acrylamidyl or an eth-
ylurea group to assess the importance of the chlorine atom. We
have also modulated the length of the carbon chain between the
urea or the amide group and the chlorine atom to optimize in each
family the length of pharmacophoric moiety. Finally, we have
assessed the contribution of the resonance effect provided by the
aromatic ring and the location of the urea group by adding
a methylenyl group between the phenyl ring and the urea moiety.
In this study, we report the synthesis and the antiproliferative
activity of seven novel analogues of CEU namely: 3-chlor-
opropylureas (1, CPU), 2-chloroacetylureas (2, CAU), ethylureas (3,
EU), 2-chloroacetamides (4, CA), 3-chloropropionamides (5, CPA),
4-chlorobutyramides (6, CBA) and acrylamides (7, Acr). The aux-
ophore groups were selected from 4-iodo, 4-tert-butyl, 3-(n-pen-
tyl), 3-(pentan-1-ol), 3-(1-methoxypentyl) and 3-(hexan-2-one);
groups that have previously exhibited the most potent anti-
proliferative activities (Fig. 1 and Table 1) [4,6e8].
3.2. Structureeactivity relationships
Structureeactivity relationships show that compounds bearing
an amide group when compared to compounds bearing either
a urea or an acetylurea group are exhibiting significantly lower
antiproliferative activities. When the amide group is constituted of
a chain of only one carbon atom (CA), which increases significantly
the electrophilicity of the carbon atom bearing the chlorine atom,
then the antiproliferative activity decreases weakly when
compared to CEU. The presence of a CAU haptophoric moiety had
a similar effect on the antiproliferative activity. When the hapto-
phore group is devoid of the chlorine atom, as exemplified by Acr
and EU derivatives, the antiproliferative activity decreases
dramatically. CPA, CBA and CPU analogues bearing a substituting
chain having more than 2 carbon atoms exhibit also lowered
antiproliferative activities when compared to CEU. Finally, most
derivatives of the “c” series bearing a methylenyl group between
the phenyl ring and the proton acceptor groups had their anti-
proliferative activity also significantly lowered except for
compound 2c (CAU serie). These results suggest that either the
resonance between the phenyl group and the proton acceptor
group or the position of the proton acceptor group might be
important for the antagonistic and irreversible binding of the drug
to its target. Consequently, minor modifications of the 2-chlor-
oethylurea moiety of CEU lead to decreasing antiproliferative
activity. However, CA, CAU, CPA and Acr analogues of CEU are still
interesting antiproliferative agents since they had IC50 ranging
2. Chemistry
from 1.4 to 25 mM. These results confirm that the constitution and
the spatial conformation of the pharmacophore and the hapto-
phore groups together with the electrophilic character of the
molecules are critical parameters to maintain the antiproliferative
activity of the drug. Moreover, increasing the length of the chain
substituting the aromatic ring of CEU is not an optimal approach to
improve the antiproliferative activity.
CPU, CAU, EU, CA, CPA, CBA, and Acr analogues of CEU were
prepared by nucleophilic addition of 3-chloropropylisocyanate, 2-
chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-
chloropropanoyl chloride, 4-chlorobutanoyl chloride, or acryloyl
chloride, respectivelytoselectedanilinesorbenzylamines(Scheme 1)
[9]. Anilines substituted in position 3 either by pentyl, pentan-1-ol,1-
methoxypentyl or hexan-2-one groups were prepared as previously
described [6e8] using a Sonogashira coupling [22] followed by the
concomitant reduction of the aromatic nitro and the alkynyl group by
catalytic hydrogenation using Pd/C (Scheme 1).
3.3. Covalent binding of CEU analogues to the colchicine-binding
site on b-tubulin
Antimicrotubule CEU have been shown to selectively acylate the
glutamic acid-198 (Glu-198) residue located in the vicinity of the
3. Results and discussion
colchicine-binding site on b-tubulin [4,5,8,9,24,25]. Therefore, we
compared the binding of CPU, CAU, CA, CPA and Acr derivatives
onto the glutamic acid-198 residue of molecules exhibiting IC50
3.1. Antiproliferative activity
lower than 25 mM on M21 skin melanoma tumor cells. Previous
Cell growth inhibition screening for CPU, CAU, EU, CA, CPA, CBA,
and Acr was assessed on three human cancer cell lines, namely, HT-
studies using antimicrotubule CEU derivatives have shown that the
acylation of CEU by Glu-198 is clearly demonstrated when using

2930
S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
Table 1
Antiproliferative activity of most potent CEU previously synthesized and their ability to covalently bind to
b
-tubulin.
IC50
Comp.
ICEU
Name
Structures
(
m
M)
b
-tubulin acylationa
Ctrl 24h 48h
HT-29
2.9
M21
MCF-7
6.6
Cl
O
N-(4-Iodophenyl)-N0-2-
chloroethylurea
NH
4.4
I
NH
Cl
Cl
O
N-(4-tert-Butylphenyl)-N0-
2-chloroethylurea
NH
tBCEU
NH
3.0
21
3.6
15
5.1
24
HN
HN
1-(4-tert-butylbenzyl)-3-
(2-chloroethyl)urea
O
tBBCEU
O
Cl
Cl
Cl
Cl
1-(2-Chloroethyl)-3-
(3-pentylphenyl)urea
( )4
N
N
PCEU
1.1
1.4
0.39
0.94
2.1
1.0
0.49
1.90
3.9
H
H
O
HO
1-(2-Chloroethyl)-3-[3-
(5-hydroxypentyl)phenyl]urea
( )5
( )5
( )4
N
N
H
H
HPCEU
MPCEU
0.25
0.84
0.90
O
O
O
O
1-(2-Chloroethyl)-3-[3-
(5-methoxypentyl)phenyl]urea
N
H
N
H
1-(2-Chloroethyl)-3-[3-
(5-oxohexyl)phenyl]urea
N
H
N
H
MKBCEU
a
M21 cells were incubated for 24 and 48 h in the presence of most potent CEU previously synthesized at 10-times their respective IC50.
SDS-Page and a monoclonal anti-
CEU by Glu-198 gives rise to the production of a
migrating faster (apparent lower molecular weight) than the native
-tubulin band [4,5,8,9,24,25]. CAU (2ce2g), CA (4ae4d, 4f and 4g),
CPA (5a) and Acr (7a and 7b) did not show any immunoreacting
b
-tubulin antibody. Acylation of
tubulin band migrating faster than the native b-tubulin. Conse-
b-tubulin band
quently, these compounds are unable to acylate Glu-198 suggesting
that the covalent binding of the drug to the C-BS is not involved in
the mechanism of action of these new compounds and that they are
most likely acting through still undetermined mechanisms of
b
b
-
Scheme 1. Synthesis of CPU, CAU, EU, CA, CPA, CBA, and Acr analogues. Reagents: (a) 3-chloropropylisocyanate, CH2Cl2; (b) 2-chloroacethylethylisocyanate, CH2Cl2; (c) ethyl-
isocyanate, CH2Cl2; (d) 2-chloroacetyl chloride, CH2Cl2 or 2-chloroacetyl chloride, K2CO3, CH3CN; (e) 3-chloropropanoyl chloride, K2CO3, CH3CN; (f) 4-chlorobutanoyl chloride,
K2CO3, CH3CN; (g) acryloyl chloride, K2CO3, CH3CN.

S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
2931
Table 2
Antiproliferative activity of compounds 1aeg (CPU), ICAU, tBCAU, 2ceg (CAU), IEU, tBEU, 3ceg (EU), 4aed, 4f and 4g (CA), 5aeg (CPA), 6aeg (CBA) and 7aeg (Acr) on HT-29,
M21 and MCF-7 cell lines and their ability to covalently bind to -tubulin.
b
R
R1
( )n
Comp.
R
R1
n
IC50
(m
M)
b
-tubulin acylationa
HT-29
M21
MCF-7
Ctrl 24h 48h
1a
1b
4-Iodo
4-tert-Butyl
CPU
CPU
0
0
90
28
>100
35
>100
49
n.e.
n.e.
1c
4-tert-Butyl
CPU
CPU
1
0
19
28
11
24
17
26
1d
3-(n-Pentyl)
1e
1f
1g
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
CPU
CPU
CPU
0
0
0
19
56
41
35
51
36
>100
50
71
n.e.
n.e.
n.e.
ICAU
tBCAU
2c
4-Iodo
CAU
CAU
CAU
CAU
CAU
CAU
CAU
0
0
1
0
0
0
0
11
6.5
7.1
8.4
10
8.1
1.4
2.4
3.5
4.6
4.2
4.2
4.9
2.0
2.8
8.3
5.7
6.1
4.1
4-tert-Butyl
4-tert-Butyl
2d
3-(n-Pentyl)
2e
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
2f
10
2g
9.9
IEU
tBEU
3c
3d
3e
4-Iodo
4-tert-Butyl
4-tert-Butyl
3-(n-Pentyl)
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
EU
EU
EU
EU
EU
EU
EU
0
0
1
0
0
0
0
>100
96
>100
16
>100
>100
40
>100
53
>100
36
>100
>100
>100
>100
>100
>100
67
>100
>100
>100
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
3f
3g
4a
4b
4-Iodo
CA
CA
0
0
9.7
15
5.1
5.4
6.2
5.4
4-tert-Butyl
(continued on next page)

2932
Table 2 (continued)
S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
Comp.
R
R1
CA
n
IC50
(
m
M)
b
-tubulin acylationa
HT-29
18
M21
MCF-7
23
Ctrl 24h 48h
4c
4-tert-Butyl
1
16
4d
4f
3-(n-Pentyl)
CA
0
0
0
0
12
13
7.0
18
1.4
6.9
4.9
5.7
12
3-(1-Methoxypentyl)
3-(Hexan-2-one)
4-Iodo
CA
4g
5a
CA
4.9
26
CPA
20
74
> 100
54
>100
>100
>100
>100
>100
69
>100
>100
>100
>100
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
4-tert-Butyl
4-tert-Butyl
3-(n-Pentyl)
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
4-Iodo
4-tert-Butyl
4-tert-Butyl
CPA
CPA
CPA
CPA
CPA
CPA
CBA
CBA
CBA
CBA
CBA
CBA
CBA
0
1
0
0
0
0
0
0
1
0
0
0
0
88
87
46
49
67
54
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
>100
>100
>100
>100
>100
72
>100
>100
>100
>100
>100
>100
> 100
>100
>100
92
>100
>100
>100
>100
3-(n-Pentyl)
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
6g
7a
7b
4-Iodo
Acr
Acr
0
0
15
24
11
22
17
29
4-tert-Butyl
7c
7d
7e
7f
4-tert-Butyl
3-(n-Pentyl)
3-(Pentan-1-ol)
3-(1-Methoxypentyl)
3-(Hexan-2-one)
Acr
Acr
Acr
Acr
Acr
1
0
0
0
0
> 100
31
> 100
40
72
27
98
48
33
>100
49
>100
>100
99
n.e.
n.e.
n.e.
n.e.
n.e.
7g
29
a
M21 cells were incubated for 24 and 48 h in the presence of CPU, CAU, CA, CPA and Acr derivatives at 10-times their respective IC50; n.e., not evaluated.
action. The ability of these compounds to covalently bind to other
proteins is currently under evaluation.
Finally, the presence of a methyl group between the phenyl ring
and the proton acceptor group of the “c” series of compounds
significantly decreases the antiproliferative activity. These results
suggest that either the resonance between the phenyl group and
the proton acceptor group or the position of the proton acceptor
may play an important role in the antiproliferative activity of the
drug. Finally, CAU (2ce2g), CA (6ae6d, 6f and 6g), CPA (7a) and Acr
4. Conclusions
The aim of this study was to evaluate the effect of modifying the
2-chloroethylurea moiety of CEU on the antiproliferative activity of
HT-29 colon carcinoma, M21 skin melanoma and MCF-7 breast
carcinoma cell lines. To that end, we have prepared and charac-
terized seven subclasses of compounds deriving from CEU. First, the
replacement of the urea group by an amide group decreased
dramatically the antiproliferative activity. Moreover, the CA and
CAU groups exhibiting higher electrophilic characters maintained
a good antiproliferative activity. Furthermore, Acr and Eu deriva-
tives, devoid of leaving groups, are dramatically less cytocidal.
(9a and 9b) had IC50 ranging from 1.4 to 25 mM. These compounds
did not acylate Glu-198 suggesting that the covalent binding of the
drug to the C-BS is not involved in the mechanism of action and that
they are most likely acting through still undetermined mechanisms
of action. In conclusion, urea and the chloro groups involved in the
2-chloroethylurea moiety of CEU can be modulated to maintain
interesting antiproliferative activity at the expense of the acylation
of Glu-198 and at the expense of the selectivity towards b-tubulin.

S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
2933
In addition, it appears that there is little room for structural
modifications of the 2-chloroethylurea moiety to design novel
antimicrotubule CEU exhibiting improved pharmacological and
biopharmaceutical properties.
4.21 (d, 2H, J ¼ 5.4 Hz, CH2), 3.46 (t, 2H, J ¼ 6.4 Hz, CH2), 3.19 (q, 2H,
J ¼ 6.2 Hz, CH2), 1.82 (apparent quint, 2H, J ¼ 6.3 Hz, CH2), 1.29 (s,
9H, 3ꢂ CH3); 13C NMR (CDCl3)
d: 158.9, 150.2, 136.3, 127.0, 125.5,
43.9, 42.5, 37.5, 34.5, 32.9, 31.4; MS (ESI) m/z: 283.1 [M þ H]þ.
5. Experimental
5.2.4. 1-(3-Chloropropyl)-3-(3-pentylphenyl)urea (1d)
Flash chromatography (silica gel, methylene chloride to meth-
5.1. Chemistry
ylene chloride/ethyl acetate (90:10)). Yield: 57%; mp: 56e57 ꢀC; IR
n
: 3288 (NH), 1634 (C]O) cmꢁ1 1H NMR (CDCl3)
; d: 7.84 (brs, 1H,
Proton NMR spectra were recorded on a Bruker AM-300 spec-
trometer (Bruker, Germany). Chemical shifts (d) are reported in
NH), 7.16e7.07 (m, 3H, Ar), 6.84 (d, 1H, J ¼ 7.0 Hz, Ar), 6.13 (t, 1H,
J ¼ 5.6 Hz, NH), 3.48 (t, 2H, J ¼ 6.3 Hz, CH2), 3.28 (q, 2H, J ¼ 6.2 Hz,
CH2), 2.49 (t, 2H, J ¼ 7.9 Hz, CH2), 1.85 (apparent quint, 2H,
J ¼ 6.5 Hz, CH2), 1.59e1.50 (m, 2H, CH2), 1.34e1.25 (m, 4H, 2ꢂ CH2),
parts per million. IR spectra were recorded on a Magna FT-IR
spectrometer (Nicolet instrument corporation, Madison, WI, USA).
Uncorrected melting points were determined on an electrothermal
melting point apparatus. HPLC-MS spectra were recorded on
Quattro PremierÔ XE tandem quadrupole mass spectrometer
(Waters, Milford, MA, USA). All reactions were conducted under
a dried nitrogen atmosphere. Chemicals were supplied by Aldrich
Chemicals (Milwaukee, WI, USA) or VWR international (Mont-
Royal, Qc, Canada). Liquid flash chromatography was performed on
0.88 (t, 3H, J ¼ 6.8 Hz, CH3); 13C NMR (CDCl3)
d: 157.1, 144.1, 138.8,
128.9, 123.4, 120.4, 117.7, 42.3, 37.4, 35.9, 32.8, 31.6, 31.1, 22.5, 14.0;
MS (ESI) m/z: 283.1 [M þ H]þ.
5.2.5. 1-(3-Chloropropyl)-3-[3-(5-hydroxypentyl)phenyl]urea (1e)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (60:40)). Yield: 36%; mp: 49e50 ꢀC; IR
silica gel F60, 60 A, 40e63
mm supplied by Silicycle (Québec, QC,
n d: 7.82
: 3322 (NH), 1635 (C]O) cmꢁ1; 1H NMR (CDCl3 and MeOD)
Canada) using a FPX flash purification system (Biotage, Charlot-
tesville, VA, USA) and 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 60 F254 (VWR international, Mont-Royal, Qc, Canada). The
chromatograms were viewed under UV light at 254 and/or 265 nm.
(brs, 1H, NH), 7.04 (s, 1H, Ar), 7.00e6.96 (m, 2H, Ar), 6.66e6.65 (m,
1H, Ar), 5.93 (t, 1H, J ¼ 5.8 Hz, NH), 3.44e3.39 (m, 4H, 2ꢂ CH2), 3.18
(t, 2H, J ¼ 6.5 Hz, CH2), 2.40 (t, 2H, J ¼ 7.7 Hz, CH2), 1.80 (apparent
quint, 2H, J ¼ 6.4 Hz, CH2), 1.51e1.36 (m, 4H, 2ꢂ CH2), 1.25e1.18 (m,
2H, CH2); 13C NMR (CDCl3 and MeOD)
d: 156.8, 143.4, 139.1, 128.6,
122.6, 119.4, 116.7, 62.0, 42.2, 36.9, 35.7, 32.6, 32.2, 30.9, 25.2; MS
(ESI) m/z: 299.0 [M þ H]þ.
5.2. General procedure for synthesis of compounds 1ae1g, ICAU,
tBCAU, 2ce2g, IEU, tBEU, 3ce3g
5.2.6. 1-(3-Chloropropyl)-3-[3-(5-methoxypentyl)phenyl]urea (1f)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (75:25)). Yield: 86%; mp: 55e56 ꢀC; IR
Compounds ICAU, tBCAU, IEU and tBEU were prepared as pub-
lished previously [4,9], 3-chloropropylisocyanate, 2-chlor-
oacetylisocyanate or ethylisocyanate (1.1 mmol) was added
dropwise to a stirred solution of the selected aniline or benzyl-
amine (1.0 mmol) in anhydrous methylene chloride (10 mL). The
reaction mixture was stirred overnight at ambient temperature.
The solution was evaporated and the resulting crude precipitate
was purified by recrystallization or trituration in the appropriate
solvent or by flash chromatography on silica gel.
n ; d: 7.66 (brs, 1H,
: 3334 (NH), 1633 (C]O) cmꢁ1 1H NMR (CDCl3)
NH), 7.14e7.04 (m, 3H, Ar), 6.81 (d, 1H, J ¼ 7.1 Hz, Ar), 5.94 (t, 1H,
J ¼ 5.7 Hz, NH), 3.49 (t, 2H, J ¼ 6.2 Hz, CH2), 3.36e3.26 (m, 7H, 2ꢂ
CH2 and CH3), 2.49 (t, 2H, J ¼ 7.8 Hz, CH2), 1.87 (apparent quint, 2H,
J ¼ 6.5 Hz, CH2), 1.61e1.51 (m, 4H, 2ꢂ CH2), 1.38e1.30 (m, 2H, CH2);
13C NMR (CDCl3)
d: 156.8, 143.8, 138.8, 128.9, 123.3, 120.3, 117.6,
72.8, 58.5, 42.4, 37.4, 35.9, 32.8, 31.2, 29.5, 25.9; MS (ESI) m/z: 313.1
[M þ H]þ.
5.2.7. 1-(3-Chloropropyl)-3-[3-(5-oxohexyl)phenyl]urea (1g)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (80:20)). Yield: 32%; mp: 61e62 ꢀC; IR
5.2.1. 1-(3-Chloropropyl)-3-(4-iodophenyl)urea (1a)
Trituration in methylene chloride. Yield: 74%; mp: 173e175 ꢀC;
IR
n
: 3315 (NH), 1632 (C]O) cmꢁ1; 1H NMR (DMSO-d6)
d: 8.59 (brs,
n d: 7.76 (brs,1H, NH), 7.12e7.03
: 1711 (C]O) cmꢁ1; 1H NMR (CDCl3)
1H, NH), 7.54 (d, 2H, J ¼ 8.7 Hz, Ar), 7.26 (d, 2H, J ¼ 8.7 Hz, Ar), 6.31
(t, 1H, J ¼ 5.5 Hz, NH), 3.68 (t, 2H, J ¼ 6.4 Hz, CH2), 3.21 (q, 2H,
J ¼ 6.2 Hz, CH2), 1.89 (apparent quint, 2H, J ¼ 6.6 Hz, CH2); 13C NMR
(m, 3H, Ar), 6.77 (d, 1H, J ¼ 7.1 Hz, Ar), 6.00 (t, 1H, J ¼ 5.7 Hz, NH),
3.48 (t, 2H, J ¼ 6.3 Hz, CH2), 3.28 (q, 2H, J ¼ 6.2 Hz, CH2), 2.47 (t, 2H,
J ¼ 6.6 Hz, CH2), 2.38 (t, 2H, J ¼ 6.5 Hz, CH2), 2.08 (s, 3H, CH3), 1.86
(DMSO-d6)
d
: 155.1, 140.4, 137.2, 120.0, 83.6, 43.1, 36.6, 32.7; MS
(apparent quint, 2H, J ¼ 6.5 Hz, CH2), 1.53e1.51 (m, 4H, 2ꢂ CH2); 13
C
(ESI) m/z: 338.9 [M þ H]þ.
NMR (CDCl3) d: 209.7, 156.8, 143.2, 139.0, 128.9, 123.1, 119.9, 117.4,
43.5, 42.4, 37.3, 35.7, 32.8, 30.7, 29.9, 23.4; MS (ESI) m/z: 311.1
5.2.2. 1-(4-tert-Butylphenyl)-3-(3-chloropropyl)urea (1b)
[M þ H]þ.
Trituration in a mixture of ether/hexane (5:1). Yield: 75%; mp:
139e140 ꢀC; IR
n
: 2967 (NH), 1644 (C]O) cmꢁ1; 1H NMR (CDCl3)
d:
5.2.8. 1-(4-tert-Butylbenzyl)-3-(2-chloroacetyl)urea (2c)
7.61 (brs, 1H, NH), 7.26 (d, 2H, J ¼ 8.5 Hz, Ar), 7.16 (d, 2H, J ¼ 8.5 Hz,
Ar), 5.98 (t, 1H, J ¼ 5.5 Hz, NH), 3.51 (t, 2H, J ¼ 6.4 Hz, CH2), 3.31 (q,
2H, J ¼ 6.2 Hz, CH2), 1.87 (apparent quint, 2H, J ¼ 6.4 Hz, CH2), 1.27
Trituration in a mixture of hexane/ether (6:1). Yield: 95%; mp:
149e150 ꢀC; IR : 3331 (NH), 1673 (C]O) cmꢁ1; 1H NMR (CDCl3)
n d:
9.99 (brs, 1H, NH), 8.62 (t, 1H, J ¼ 5.4 Hz, NH), 7.38 (d, 2H, J ¼ 8.2 Hz,
(s, 9H, 3ꢂ CH3); 13C NMR (CDCl3)
d: 157.0, 146.4, 136.0, 126.0, 120.5,
Ar), 7.25 (d, 2H, J ¼ 8.2 Hz, Ar), 4.48 (d, 2H, J ¼ 5.4 Hz, CH2), 4.09 (s,
42.4, 37.4, 34.3, 32.8, 31.4; MS (ESI) m/z: 269.1 [M þ H]þ.
2H, CH2),1.32 (s, 9H, 3ꢂ CH3); 13C NMR (CDCl3)
d: 167.9,153.6,150.7,
134.5, 127.4, 125.7, 43.6, 42.5, 34.5, 31.3; MS (ESI) m/z: 283.3
5.2.3. 1-(4-tert-Butylbenzyl)-3-(3-chloropropyl)urea (1c)
[M þ H]þ.
Recrystallization from a mixture of methylene chloride/ether
(1:50). Yield: 86%; mp: 114e115 ꢀC; IR
n
: 3338 (NH), 1627 (C]O)
: 7.30 (d, 2H, J ¼ 8.2 Hz, Ar), 7.15 (d, 2H,
J ¼ 8.2 Hz, Ar), 5.61 (t,1H, J ¼ 5.3 Hz, NH), 5.47 (t,1H, J ¼ 5.4 Hz, NH),
5.2.9. 1-(2-Chloroacetyl)-3-(3-pentylphenyl)urea (2d)
Trituration in a mixture of hexane/ether (10:1) and flash chro-
matography (silica gel, methylene chloride to methylene chloride/
cmꢁ1
;
1H NMR (CDCl3)
d

2934
S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
ethyl acetate (90:10)). Yield: 43%; mp: 115e116 ꢀC; IR
1700 (C]O) cmꢁ1; 1H NMR (CDCl3)
: 10.27 (s, 1H, NH), 9.84 (s, 1H,
n
: 2955 (NH),
J ¼ 7.1 Hz, Ar), 6.06 (t, 1H, J ¼ 4.8 Hz, NH), 4.36 (t, 1H, J ¼ 4.9 Hz, OH),
3.42e3.34 (m, 4H, 2ꢂ CH2), 3.11 (apparent quint, 2H, J ¼ J ¼ 6.4 Hz,
CH2), 1.59e1.50 (m, 2H, CH2), 1.47e1.41 (m, 2H, CH2), 1.35e1.27 (m,
d
NH), 7.37e733 (m, 2H, Ar), 7.26 (t, 1H, J ¼ 7.7 Hz, Ar), 6.99 (d, 1H,
J ¼ 7.7 Hz, Ar), 4.19 (s, 2H, CH2), 2.61 (t, 2H, J ¼ 7.9 Hz, CH2), 1.63
(apparent quint, 2H, J ¼ 7.5 Hz, CH2), 1.39e1.32 (m, 4H, 2ꢂ CH2),
2H, CH2), 1.06 (t, 3H, J ¼ 7.1 Hz, CH3); 13C NMR (DMSO-d6)
d: 155.9,
143.5, 141.3,129.2, 121.8, 118.3,115.9, 61.4, 36.2, 34.7, 33.2, 31.6, 26.0,
0.91 (t, 3H, J ¼ 6.8 Hz, CH3); 13C NMR (CDCl3)
d
: 168.2, 150.7, 144.2,
16.3; MS (ESI) m/z: 251.1 [M þ H]þ.
136.5, 129.0, 125.1, 120.5, 117.8, 42.5, 35.9, 31.5, 31.0, 22.5, 14.0; MS
(ESI) m/z: 283.3 [M þ H]þ.
5.2.16. 1-Ethyl-3-[3-(5-methoxypentyl)phenyl]urea (3f)
Flash chromatography (silica gel, methylene chloride to meth-
5.2.10. 1-(2-Chloroacetyl)-3-[3-(5-hydroxypentyl)phenyl]urea (2e)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (50:50)). Yield: 39%; mp: 112e113 ꢀC;
ylene chloride/ethyl acetate (75:25)). Yield: 85%; mp: 69e70 ꢀC; IR
n
: 3312 (NH), 1646 (C]O) cmꢁ1; 1H NMR (CDCl3)
d: 7.95 (s, 1H, NH),
7.12e7.05 (m, 3H, Ar), 6.77 (d, 1H, J ¼ 6.2 Hz, Ar), 6.04 (t, 1H,
J ¼ 5.4 Hz, NH), 3.35e3.30 (m, 5H, CH2 and CH3), 3.21e3.12 (m, 2H,
CH2), 2.47 (t, 2H, J ¼ 7.5 Hz, CH2), 1.59e1.49 (m, 4H, 2ꢂ CH2),
1.37e1.29 (m, 2H, CH2),1.03 (t, 3H, J ¼ 7.3 Hz, CH3); 13C NMR (CDCl3)
IR n d: 10.28 (s,
: 2979 (NH), 1703 (C]O) cmꢁ1; 1H NMR (acetone-d6)
1H, NH), 9.90 (s, 1H, NH), 7.44e7.42 (m, 2H, Ar), 7.23 (t, 1H,
J ¼ 7.8 Hz, Ar), 6.97 (d, 1H, J ¼ 7.8 Hz, Ar), 4.43 (s, 2H, CH2), 3.54 (t,
2H, J ¼ 6.4 Hz, CH2), 2.94 (s, 1H, OH), 2.62 (t, 2H, J ¼ 7.8 Hz, CH2),
1.70e1.60 (m, 2H, CH2), 1.58e1.51 (m, 2H, CH2), 1.46e1.36 (m, 2H,
d
: 156.9, 143.5, 139.4, 128.7, 122.7, 119.8, 117.1, 72.8, 58.5, 35.9, 34.8,
31.2, 29.5, 25.9, 15.4; MS (ESI) m/z: 265.1 [M þ H]þ.
CH2); 13C NMR (acetone-d6)
d: 169.5, 150.7, 144.5, 138.5, 129.5,
124.8, 120.5, 117.9, 62.2, 43.6, 36.4, 33.4, 32.0, 26.2; MS (ESI) m/z:
5.2.17. 1-Ethyl-3-[3-(5-oxohexyl)phenyl]urea (3g)
299.0 [M þ H]þ.
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (80:20)). Yield: 49%; mp: 52e53 ꢀC; IR
5.2.11. 1-(2-Chloroacetyl)-3-[3-(5-methoxypentyl)phenyl]urea (2f)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (50:50)) and trituration in cold ether.
n d: 7.90 (s, 1H, NH), 7.13 (s, 1H,
: 1706 (C]O) cmꢁ1; 1H NMR (CDCl3)
Ar), 7.06 (d, 2H, J ¼ 4.6 Hz, Ar), 6.75e6.72 (m, 1H, Ar), 5.97 (t, 1H,
J ¼ 5.4 Hz, NH), 3.21e3.12 (m, 2H, CH2), 2.45 (t, 2H, J ¼ 6.8 Hz, CH2),
2.35 (t, 2H, J ¼ 6.4 Hz, CH2), 2.07 (s, 3H, CH3), 1.51e1.49 (m, 4H, 2ꢂ
Yield: 51%; mp: 96e97 ꢀC; IR
NMR (CDCl3)
n ;
: 2939 (NH), 1699 (C]O) cmꢁ1 1H
: 10.28 (s, 1H, NH), 10.06 (s, 1H, NH), 7.34e7.21 (m,
d
CH2), 1.03 (t, 3H, J ¼ 7.2 Hz, CH3); 13C NMR (CDCl3)
d: 209.6, 156.8,
3H, Ar), 6.96 (d, 1H, J ¼ 6.9 Hz, Ar), 4.16 (s, 2H, CH2), 3.38e3.32 (m,
143.1,139.4, 128.8, 122.6, 119.6,117.1, 43.5, 35.7, 34.8, 30.7, 29.9, 23.4,
5H, CH2 and CH3), 2.59 (t, 2H, J ¼ 7.0 Hz, CH2), 1.66e1.57 (m, 4H, 2ꢂ
15.4; MS (ESI) m/z: 263.1 [M þ H]þ.
CH2), 1.43e1.35 (m, 2H, CH2); 13C NMR (CDCl3)
d: 168.4, 151.0, 143.9,
136.5, 129.0, 125.0, 120.5, 117.9, 72.8, 58.5, 42.6, 35.8, 31.2, 29.5,
5.3. General procedure for the synthesis of compounds 4ae4c
25.8; MS (ESI) m/z: 313.0 [M þ H]þ.
2-Chloroacetyl chloride (1.0 mmol) was added slowly to a solu-
tion of the selected aniline (1.0 mmol) or benzylamine in methy-
lene chloride (10 mL). The reaction was stirred overnight at room
temperature. The solvent was evaporated under reduced pressure
and the residue was purified by trituration in hexane.
5.2.12. 1-(2-Chloroacetyl)-3-[3-(5-oxohexyl)phenyl]urea (2g)
Flash chromatography (silica gel, methylene chloride to meth-
ylene chloride/ethyl acetate (80:20)). Yield: 23%; mp: 89e90 ꢀC; IR
n
: 2935 (NH), 1706 (C]O) cmꢁ1 1H NMR (CDCl3)
; d: 10.25 (s, 1H,
NH), 9.78 (s, 1H, NH), 7.31e7.21 (m, 3H, Ar), 6.96 (d, 1H, J ¼ 7.4 Hz,
Ar), 4.18 (s, 2H, CH2), 2.63e2.59 (m, 2H, CH2), 2.47e2.42 (m, 2H,
CH2), 2.12 (s, 3H, CH3), 1.63e1.58 (m, 4H, 2ꢂ CH2); 13C NMR (CDCl3)
5.3.1. 2-Chloro-N-(4-iodophenyl)acetamide (4a)
Yield: 36%; mp: 198e199 ꢀC; IR : 3184 (NH), 1675 (C]O) cmꢁ1
n ;
d
: 209.1, 168.2, 150.6, 143.4, 136.6, 129.1, 125.0, 120.5, 118.0, 43.5,
1H NMR (DMSO-d6)
d
: 10.41 (brs, 1H, NH), 7.69 (d, 2H, J ¼ 8.6 Hz,
42.6, 35.7, 30.7, 29.9, 23.4; MS (ESI) m/z: 311.0 [M þ H]þ.
Ar), 7.44 (d, 2H, J ¼ 8.6 Hz, Ar), 4.26 (s, 2H, CH2); 13C NMR (DMSO-
d6) d: 164.8, 138.5, 137.6, 121.6, 87.49, 43.6; MS (ESI) m/z: 295.8
5.2.13. 1-(4-tert-Butylbenzyl)-3-ethylurea (3c)
[M þ H]þ.
Trituration in cold hexane. Yield: 99%; mp: 104e105 ꢀC; IR
n:
3338 (NH), 1625 (C]O) cmꢁ1
;
1H NMR (CDCl3)
d
: 7.28 (d, 2H,
5.3.2. N-(4-tert-Butylphenyl)-2-chloroacetamide (4b)
J ¼ 8.3 Hz, Ar), 7.14 (d, 2H, J ¼ 8.3 Hz, Ar), 5.83 (t, 1H, J ¼ 5.3 Hz, NH),
5.51 (t, 1H, J ¼ 4.6 Hz, NH), 4.20 (d, 2H, J ¼ 5.3 Hz, CH2), 3.08e2.99
(m, 2H, CH2), 1.28 (s, 9H, 3ꢂ CH3), 0.98 (t, 3H, J ¼ 7.1 Hz, CH3); 13C
Yield: 86%; mp: 113e114 ꢀC; IR : 3246 (NH), 1656 (C]O) cmꢁ1
n ;
1H NMR (CDCl3)
d
: 8.24 (brs,1H, NH), 7.46 (d, 2H, J ¼ 8.7 Hz, Ar), 7.35
(d, 2H, J ¼ 8.7 Hz, Ar), 4.17 (s, 2H, CH2), 1.32 (s, 9H, CH3); 13C NMR
NMR (CDCl3)
d
: 159.1, 149.9, 136.7, 127.0, 125.4, 43.8, 35.0, 34.4, 31.4,
(CDCl3) d: 163.8, 148.3, 134.1, 126.0, 120.1, 42.9, 34.5, 31.3; MS (ESI)
15.5; MS (ESI) m/z: 235.1 [M þ H]þ.
m/z: 226.1 [M þ H]þ.
5.2.14. 1-Ethyl-3-(3-pentylphenyl)urea (3d)
5.3.3. N-(4-tert-Butylbenzyl)-2-chloroacetamide (4c)
Trituration and recrystallization from cold ether. Yield: 70%; mp:
Yield: 90%; mp: 181e183 ꢀC; IR : 2957 (NH), 1644 (C]O) cmꢁ1
n ;
95e96 ꢀC; IR
n
: 3313 (NH), 1648 (C]O) cmꢁ1
;
1H NMR (CDCl3)
d
:
1H NMR (CDCl3)
Ar), 6.90 (brs,1H, NH), 4.46 (d, 2H, J ¼ 5.7 Hz, CH2), 4.08 (s, 2H, CH2),
d: 7.39 (d, 2H, J ¼ 8.1 Hz, Ar), 7.24 (d, 2H, J ¼ 8.1 Hz,
7.60 (s, 1H, NH), 7.16e7.11 (m, 3H, Ar), 6.84e6.81 (m, 1H, Ar), 5.78 (t,
1H, J ¼ 5.4 Hz, NH), 3.20 (apparent quint, 2H, J ¼ 6.3 Hz, CH2), 2.49
(t, 2H, J ¼ 7.8 Hz, CH2), 1.59e1.50 (m, 2H, CH2), 1.29e1.28 (m, 4H, 2ꢂ
1.33 (s, 9H, CH3); 13C NMR (CDCl3)
d: 165.8, 150.9, 134.3, 127.7, 125.8,
43.6, 42.6, 34.6, 31.3; MS (ESI) m/z: 240.1 [M þ H]þ.
CH2), 1.06 (t, 3H, J ¼ 7.1 Hz, CH3), 0.88 (t, 3H, J ¼ J ¼ 6.5 Hz, CH3); 13
C
NMR (CDCl3)
d
: 156.7, 144.0, 139.0, 128.8, 123.2, 120.4, 117.7, 35.9,
5.4. General procedure for the synthesis of compounds 4d, 4fe4g,
5ae5g, 6ae6g and 7ae7g
34.9, 31.6, 31.0, 22.5, 15.4, 14.0; MS (ESI) m/z: 235.2 [M þ H]þ.
5.2.15. 1-Ethyl-3-[3-(5-hydroxypentyl)phenyl]urea (3e)
2-Chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlor-
obutanoyl chloride or acryloyl chloride (2.2 mmol) was added
slowly to a stirred suspension of the relevant aniline (2.0 mmol)
and sodium carbonate (4.0 mmol) in acetonitrile (10 mL). The
Trituration in methylene chloride. Yield: 79%; mp: 103e104 ꢀC;
IR
1H, NH), 7.24 (s, 1H, Ar), 7.20e7.08 (m, 2H, Ar), 6.72 (d, 1H,
n ; d: 8.33 (s,
: 3307 (NH), 1646 (C]O) cmꢁ1 1H NMR (DMSO-d6)

S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
2935
reaction mixture was stirred overnight at room temperature. The
mixture was filtered and the solvent was evaporated under reduced
pressure. The residue was purified by trituration in hexane,
recrystallization in the appropriate solvent or by flash chromatog-
raphy on silica gel.
7.45 (s, 1H, NH), 7.38 (s, 1H, Ar), 7.33e7.19 (m, 2H, Ar), 6.95 (d, 1H,
J ¼ 7.6 Hz, Ar), 3.88 (t, 2H, J ¼ 6.3 Hz, CH2), 2.80 (t, 2H, J ¼ 6.3 Hz,
CH2), 2.58 (t, 2H, J ¼ 7.9 Hz, CH2), 1.65e1.55 (m, 2H, CH2), 1.33e1.30
(m, 4H, 2ꢂ CH2), 0.89 (t, 3H, J ¼ 6.9 Hz, CH3); 13C NMR (CDCl3)
d:
167.7, 144.2, 137.4, 128.9, 124.9, 120.1, 117.4, 40.6, 39.9, 35.9, 31.5,
31.1, 22.5, 14.0; MS (ESI) m/z: 254.1 [M þ H]þ.
5.4.1. 2-Chloro-N-(3-pentylphenyl)acetamide (4d)
Trituration in hexane. Yield: 70%; mp: 83e84 ꢀC; IR
n
: 3267
5.4.8. 3-Chloro-N-[3-(5-hydroxypentyl)phenyl]propionamide (5e)
(NH), 1667 (C]O) cmꢁ1
;
1H NMR (CDCl3)
d: 8.20 (brs, 1H, NH),
Flash chromatography (silica gel, methylene chloride/ethyl
7.39e7.37 (m, 2H, Ar), 7.26 (t, 1H, J ¼ 7.8 Hz, Ar), 7.00 (d, 1H,
J ¼ 7.8 Hz, Ar), 4.19 (s, 2H, CH2), 2.61 (t, 2H, J ¼ 7.9 Hz, CH2),
1.67e1.57 (m, 2H, CH2), 1.37e1.30 (m, 4H, CH2), 0.90 (t, 3H,
acetate (80:20)). Yield: 11%; mp: 89e90 ꢀC; IR
n
: 3305 (NH), 1647
(C]O) cmꢁ1 1H NMR (CDCl3 and CH3OD)
; d: 7.35 (s, 1H, Ar),
7.30e7.27 (m, 1H, Ar), 7.16 (t, 1H, J ¼ 7.7 Hz, Ar), 6.87 (d, 1H,
J ¼ 7.7 Hz, Ar), 3.81 (t, 2H, J ¼ 6.6 Hz, CH2), 3.53 (t, 2H, J ¼ 6.6 Hz,
CH2), 3.00 (brs, 2H, OH and NH), 2.76 (t, 2H, J ¼ 6.5 Hz, CH2), 2.54
J ¼ 7.0 Hz, CH3); 13C NMR (CDCl3)
d: 163.7, 144.3, 136.6, 129.0, 125.4,
120.1, 117.5, 42.9, 35.9, 31.5, 31.1, 22.5, 14.0; MS (ESI) m/z: 240.1
[M þ H]þ.
(m, 2H, CH2), 1.63e1.47 (m, 4H, 2ꢂ CH2), 1.37e1.27 (m, 2H, CH2); 13
C
NMR (CDCl3 and CH3OD) d: 168.5, 143.5, 137.7, 128.7, 124.6, 120.2,
5.4.2. 2-Chloro-N-[3-(5-methoxypentyl)phenyl]acetamide (4f)
117.5, 62.4, 40.0, 39.9, 35.7, 32.3, 30.9, 25.2; MS (ESI) m/z: 270.1
Flash chromatography (silica gel, methylene chloride/ethyl
[M þ H]þ.
acetate (90:10)). Yield: 30%; mp: 71e72 ꢀC; IR
n
: 3265 (NH), 1665
(C]O) cmꢁ1
;
1H NMR (CDCl3)
d: 8.28 (brs, 1H, NH), 7.36e7.35 (m,
5.4.9. 3-Chloro-N-[3-(5-methoxypentyl)phenyl]propionamide (5f)
2H, Ar), 7.24 (m, 1H, Ar), 6.97 (d, 1H, J ¼ 7.6 Hz, Ar), 4.15 (s, 2H, CH2),
3.35 (t, 2H, J ¼ 6.6 Hz, CH2), 3.31 (s, 3H, CH3), 2.59 (t, 2H, J ¼ 7.9 Hz,
CH2), 1.67e1.54 (m, 4H, 2ꢂ CH2), 1.42e1.34 (m, 2H, CH2); 13C NMR
Flash chromatography (silica gel, methylene chloride/ethyl
acetate (90:10)). Yield: 75%; mp: 57e58 ꢀC; IR
n
: 3310 (NH), 1663
(C]O) cmꢁ1 1H NMR (CDCl3)
; d: 8.51 (brs, 1H, NH), 7.36e7.29 (m,
(CDCl3)
d
: 163.9, 143.9, 136.7, 129.0, 125.4, 120.1, 117.6, 72.8, 58.5,
2H, Ar), 7.15 (t, 1H, J ¼ 7.6 Hz, Ar), 6.88 (d, 1H, J ¼ 7.6 Hz, Ar), 3.80 (t,
2H, J ¼ 6.4 Hz, CH2), 3.37e3.30 (m, 5H, CH2 and CH3), 2.75 (t, 2H,
J ¼ 6.4 Hz, CH2), 2.51 (t, 2H, J ¼ 7.7 Hz, CH2), 1.60e1.51 (m, 4H, 2ꢂ
42.9, 35.8, 31.2, 29.5, 25.8; MS (ESI) m/z: 270.0 [M þ H]þ.
5.4.3. 2-Chloro-N-[3-(5-oxohexyl)phenyl]acetamide (4g)
CH2), 1.38e1.30 (m, 2H, CH2); 13C NMR (CDCl3)
d: 168.4, 143.6, 137.8,
Flash chromatography (silica gel, methylene chloride/ethyl
128.8, 124.7, 120.3, 117.8, 72.8, 58.5, 40.1, 40.0, 35.8, 31.2, 29.4, 25.8;
acetate (80:20)). Yield: 46%; mp: 52e53 ꢀC; IR
n
: 3271 (NH), 1708
MS (ESI) m/z: 284.1 [M þ H]þ.
(C]O) cmꢁ1 1H NMR (CDCl3)
; d: 8.46 (brs, 1H, NH), 7.35e7.32 (m,
2H, Ar), 7.20 (t, 1H, J ¼ 7.6 Hz, Ar), 6.93 (d, 1H, J ¼ 7.6 Hz, Ar), 4.13 (s,
2H, CH2), 2.56 (t, 2H, J ¼ 4.7 Hz, CH2), 2.41 (t, 2H, J ¼ 4.8 Hz, CH2),
5.4.10. 3-Chloro-N-[3-(5-oxohexyl)phenyl]propionamide (5g)
Flash chromatography (silica gel, methylene chloride/ethyl
2.09 (s, 3H, CH3), 1.58e1.54 (m, 4H, 2ꢂ CH2); 13C NMR (CDCl3)
d:
acetate (85:15)). Yield: 39%; mp: 64e65 ꢀC; IR
n
: 3333 (NH), 1718
209.5, 164.3, 143.4, 136.9, 129.0, 125.3, 120.2, 117.8, 43.5, 43.0, 35.6,
(C]O) cmꢁ1; 1H NMR (CDCl3)
d: 8.40 (brs, 1H, NH), 7.39 (s, 1H, Ar),
30.7, 29.9, 23.4; MS (ESI) m/z: 268.1 [M þ H]þ.
7.33e7.27 (m, 1H, Ar), 7.16 (t, 1H, J ¼ 7.7 Hz, Ar), 6.88 (d, 1H,
J ¼ 7.7 Hz, Ar), 3.82 (t, 2H, J ¼ 6.4 Hz, CH2), 2.78 (t, 2H, J ¼ 6.4 Hz,
CH2), 2.52 (t, 2H, J ¼ 6.1 Hz, CH2), 2.40 (t, 2H, J ¼ 6.4 Hz, CH2), 2.10 (s,
5.4.4. 3-Chloro-N-(4-iodophenyl)propionamide (5a)
Recrystallization from a mixture of methylene chloride/hexane
3H, CH3), 1.57e1.52 (m, 4H, 2ꢂ CH2); 13C NMR (CDCl3)
d: 209.6,
(1:5). Yield: 92%; mp: 158e159 ꢀC; IR
n
: 3240 (NH), 1659 (C]O)
168.3, 143.2, 137.8, 128.8, 124.6, 120.2, 117.7, 43.5, 40.2, 40.0, 35.6,
cmꢁ1
;
1H NMR (DMSO-d6)
d: 10.20 (brs, 1H, NH), 7.66 (d, 2H,
30.7, 30.0, 23.4; MS (ESI) m/z: 282.1 [M þ H]þ.
J ¼ 8.6 Hz, Ar), 7.47 (d, 2H, J ¼ 8.6 Hz, Ar), 3.89 (t, 2H, J ¼ 6.2 Hz,
CH2), 2.84 (t, 2H, J ¼ 6.2 Hz, CH2); 13C NMR (DMSO-d6)
d
: 168.2,
5.4.11. 4-Chloro-N-(4-iodophenyl)butyramide (6a)
138.8, 137.4, 121.4, 86.7, 40.7; MS (ESI) m/z: 310.1 [M þ H]þ.
Recrystallization from methylene chloride/hexane (1:10). Yield:
67%; mp: 139e140 ꢀC; IR : 3295 (NH), 1664 (C]O) cmꢁ1; 1H NMR
n
5.4.5. N-(4-tert-butylphenyl)-3-chloropropionamide (5b)
(DMSO-d6)
d
: 10.17 (brs, 1H, NH), 7.63 (d, 2H, J ¼ 8.4 Hz, Ar), 7.46 (d,
Flash chromatography (silica gel, methylene chloride/ethyl
acetate (90:10)). Yield: 71%; mp: 123e124 ꢀC; IR
: 3275 (NH), 1656
(C]O) cmꢁ1 1H NMR (CDCl3)
: 8.57 (brs, 1H, NH), 7.47 (d, 2H,
2H, J ¼ 8.4 Hz, Ar), 3.71 (t, 2H, J ¼ 6.4 Hz, CH2), 2.50 (t, 2H, J ¼ 7.0 Hz,
n
CH2), 2.04 (apparent quint, 2H, J ¼ 6.7 Hz, CH2); 13C NMR (DMSO-
;
d
d6) d: 170.4, 139.1, 137.3, 121.3, 86.4, 45.0, 33.5, 27.9; MS (ESI) m/z:
J ¼ 8.6 Hz, Ar), 7.32 (d, 2H, J ¼ 8.6 Hz, Ar), 3.83 (t, 2H, J ¼ 6.4 Hz,
323.9 [M þ H]þ.
CH2), 2.80 (t, 2H, J ¼ 6.4 Hz, CH2), 1.31 (s, 9H, 3ꢂ CH3); 13C NMR
(CDCl3)
d
: 168.6, 147.7, 131.1, 125.8, 120.5, 40.1, 34.4, 31.4; MS (ESI)
5.4.12. N-(4-tert-butylphenyl)-4-chlorobutyramide (6b)
m/z: 240.1 [M þ H]þ.
Flash chromatography (silica gel, methylene chloride/ethyl
acetate (95:5)). Yield: 43%; mp: 95e96 ꢀC; IR
n: 3275 (NH), 1646
5.4.6. N-(4-tert-butylbenzyl)-3-chloropropanamide (5c)
(C]O) cmꢁ1; 1H NMR (CDCl3)
d
:, 7.44 (d, 2H, J ¼ 8.5 Hz, Ar), 7.34 (d,
Recrystallization from methylene chloride/hexane (1:20). Yield:
2H, J ¼ 8.5 Hz, Ar), 3.65 (t, 2H, J ¼ 6.1 Hz, CH2), 2.54 (t, 2H, J ¼ 7.0 Hz,
55%; mp: 88e89 ꢀC; IR
n
: 3291 (NH), 1648 (C]O) cmꢁ1
;
1H NMR
CH2), 2.19 (apparent quint, 2H, J ¼ 6.5 Hz, CH2), 1.30 (s, 9H, CH3); 13
C
(CDCl3)
d
: 7.37 (d, 2H, J ¼ 8.3 Hz, Ar), 7.22 (d, 2H, J ¼ 8.3 Hz, Ar), 6.05
NMR (CDCl3) d: 170.0, 147.5, 135.1, 125.8, 119.8, 44.5, 34.4, 34.1, 31.4,
(brs, 1H, NH), 4.42 (d, 2H, J ¼ 5.6 Hz, CH2), 3.81 (t, 2H, J ¼ 6.5 Hz,
28.0; MS (ESI) m/z: 254.1 [M þ H]þ.
CH2), 2.64 (t, 2H, J ¼ 6.5 Hz, CH2), 1.31 (s, 9H, 3ꢂ CH3); 13C NMR
(CDCl3)
d
: 169.3, 150.7, 134.8, 127.7, 125.7, 43.5, 40.2, 39.6, 34.5, 31.3;
5.4.13. N-(4-tert-butylbenzyl)-4-chlorobutanamide (6c)
MS (ESI) m/z: 254.1 [M þ H]þ.
Flash chromatography (silica gel, methylene chloride/ethyl
acetate (80:20)). Yield: 45%; mp: 71e72 ꢀC; IR
: 3266 (NH), 1640
(C]O) cmꢁ1; 1H NMR (CDCl3)
n
5.4.7. 3-Chloro-N-(3-pentylphenyl)propionamide (5d)
d
: 7.37 (d, 2H, J ¼ 8.0 Hz, Ar), 7.21 (d,
Recrystallization from ether/hexane (1:20). Yield: 50%; mp:
2H, J ¼ 8.0 Hz, Ar), 5.86 (brs, 1H, NH), 4.40 (d, 2H, J ¼ 5.4 Hz, CH2),
92e93 ꢀC; IR : 3313 (NH), 1664 (C]O) cmꢁ1 1H NMR (CDCl3)
n ; d:
3.61 (t, 2H, J ¼ 6.1 Hz, CH2), 2.38 (t, 2H, J ¼ 6.9 Hz, CH2), 2.13

2936
S. Fortin et al. / European Journal of Medicinal Chemistry 45 (2010) 2928e2937
(apparent quint, 2H, J ¼ 6.5 Hz, CH2), 1.31 (s, 9H, CH3); 13C NMR
NMR (CDCl3)
d
: 7.38 (d, 2H, J ¼ 8.2 Hz, Ar), 7.25 (d, 2H, J ¼ 8.2 Hz,
(CDCl3)
d: 171.4, 150.7, 135.1, 127.7, 125.7, 44.5, 43.4, 34.5, 33.2, 31.3,
Ar), 6.34e6.28 (m, 1H, ]CH or ]CH2), 6.15e6.06 (m, 1H, ]CH or ]
CH2), 5.96 (brs, 1H, NH), 5.67e5.63 (m, 1H, ]CH or ]CH2), 4.50 (d,
28.2; MS (ESI) m/z: 268.1 [M þ H]þ.
2H, J ¼ 5.7 HZ, CH2), 1.32 (s, 9H, 3ꢂ CH3); 13C NMR (CDCl3)
d: 165.3,
5.4.14. 4-Chloro-N-(3-pentylphenyl)butyramide (6d)
150.7, 135.0, 130.8, 127.8, 126.6, 125.7, 43.4, 34.5, 31.3; MS (ESI) m/z:
Flash chromatography (silica gel, methylene chloride/ethyl
218.1 [M þ H]þ.
acetate (85:15)). Yield: 88%; IR
NMR (CDCl3) : 8.21 (brs, 1H, NH), 7.37e7.32 (m, 2H, Ar), 7.18 (t, 1H,
n
: 2955 (NH), 1651 (C]O) cmꢁ1; 1H
d
5.4.21. N-(3-pentylphenyl)acrylamide (7d)
J ¼ 7.7 Hz, Ar), 6.92 (d, 1H, J ¼ 7.7 Hz, Ar), 3.61e3.55 (m, 2H, CH2),
Recrystallization from cold hexane. Yield: 29%; mp: 58e60 ꢀC;
2.56e2.49 (m, 4H, 2ꢂ CH2), 2.18e2.06 (m, 2H, CH2), 1.62e1.55 (m,
IR n d: 7.93 (s, 1H, Ar
: 3298 (NH), 1663 (C]O) cmꢁ1; 1H NMR (CDCl3)
2H, CH2),1.34e1.29 (m, 4H, 2ꢂ CH2), 0.88 (t, 3H, J ¼ 6.9 Hz, CH3); 13
C
or NH), 7.47 (s, 1H, Ar or NH), 7.41e7.39 (m, 1H, Ar), 7.21 (t, 1H,
J ¼ 7.7 Hz, Ar), 6.94 (d, 1H, J ¼ 7.7 Hz, Ar), 6.44e6.25 (m, 2H, ]CH or
]CH2), 5.73e5.69 (m, 1H, ]CH or ]CH2), 2.56 (t, 2H, J ¼ 7.9 Hz,
CH2),1.59 (apparent quint, 2H, J ¼ 7.7 Hz, CH2),1.35e1.27 (m, 4H, 2ꢂ
NMR (CDCl3) d: 170.9, 144.0, 137.8, 128.7, 124.7, 120.4, 117.7, 44.4,
35.9, 34.2, 31.5, 31.1, 28.2, 22.5, 14.0; MS (ESI) m/z: 268.1 [M þ H]þ.
5.4.15. 4-Chloro-N-[3-(5-hydroxypentyl)phenyl]butyramide (6e)
CH2), 0.88 (t, 3H, J ¼ 6.7 Hz, CH3); 13C NMR (CDCl3)
d: 163.8, 144.0,
Flash chromatography (silica gel, methylene chloride/ethyl
137.8, 131.4, 128.8, 127.5, 124.7, 120.2, 117.5, 35.9, 31.5, 31.0, 22.5,
acetate (80:20)). Yield: 19%; IR
n
: 3343 (NH), 1606 (C]O) cmꢁ1; 1H
14.0; MS (ESI) m/z: 218.1 [M þ H]þ.
NMR (CDCl3) : 7.97 (brs, 1H, NH), 7.38 (s, 1H, Ar), 7.30e7.15 (m, 2H,
d
Ar), 6.90 (d, 1H, J ¼ 7.4 Hz, Ar), 3.62e3.58 (m, 4H, 2ꢂ CH2),
2.57e2.48 (m, 4H, 2ꢂ CH2), 2.15e2.10 (m, 2H, CH2), 1.62e1.50 (m,
5.4.22. N-[3-(5-hydroxypentyl)phenyl]acrylamide (7e)
Flash chromatography (silica gel, methylene chloride to methy-
4H, 2ꢂ CH2), 1.40e1.30 (m, 2H, CH2); 13C NMR (CDCl3)
d: 170.5,
lene chloride/ethyl acetate (75:25)). Yield: 12%; IR
n:3287 (NH),1665
143.6, 137.8, 128.8, 124.5, 120.1,117.4, 62.7, 44.5, 35.8, 34.1, 32.5, 31.0,
(C]O) cmꢁ1 1H NMR (CDCl3)
;
d: 8.54 (s, 1H, NH), 7.45 (s, 1H, Ar),
28.0, 25.3; MS (ESI) m/z: 284.0 [M þ H]þ.
7.39e7.26 (m,1H, Ar), 7.16 (t,1H, J ¼ 7.5 Hz, Ar), 6.88 (d,1H, J ¼ 7.5 Hz,
Ar), 6.42e6.27 (m, 2H, ]CH or ]CH2), 5.69e5.66 (m,1H, ]CH or ]
CH2), 3.58 (t, 2H, J ¼ 6.5 Hz, CH2), 2.60 (brs, 1H, OH), 2.52 (t, 2H,
J ¼ 7.5 Hz, CH2), 1.62e1.49 (m, 4H, 2ꢂ CH2), 1.37e1.28 (m, 2H, CH2);
5.4.16. 4-Chloro-N-[3-(5-methoxypentyl)phenyl]butyramide (6f)
Flash chromatography (silica gel, methylene chloride/ethyl
acetate (90:10)). Yield: 35%; IR
n
: 2857 (NH), 1770 (C]O) cmꢁ1; 1H
13C NMR (CDCl3)
d: 164.2,143.5,137.9,131.4,128.8,127.5,124.7,120.3,
NMR (CDCl3) : 7.87 (brs, 1H, NH), 7.35e7.26 (m, 2H, Ar), 7.18 (t, 1H,
d
117.7, 62.6, 35.7, 32.5, 30.9, 25.3; MS (ESI) m/z: 234.2 [M þ H]þ.
J ¼ 7.1 Hz, Ar), 6.90 (d, 1H, J ¼ 7.1 Hz, Ar), 3.61 (t, 2H, J ¼ 6.3 Hz, CH2),
3.35 (t, 2H, J ¼ 6.6 Hz, CH2), 3.31 (s, 3H, CH2), 2.57e2.48 (m, 4H,
CH2), 2.15 (apparent quint, 2H, J ¼ 6.6 Hz, CH2), 1.64e1.53 (m, 4H,
5.4.23. N-[3-(5-methoxypentyl)phenyl]acrylamide (7f)
Flash chromatography (silica gel, methylene chloride to meth-
CH2), 1.40e1.32 (m, 2H, CH2); 13C NMR (CDCl3)
d: 170.3, 143.7, 137.9,
ylene chloride/ethyl acetate (90:10)). Yield: 45%; IR
n: 3276 (NH),
128.8, 124.5, 120.0, 117.4, 72.8, 58.5, 44.5, 35.9, 34.1, 31.2, 29.5, 28.0,
1663 (C]O) cmꢁ1; 1H NMR (CDCl3)
d
: 8.39 (s, 1H, NH), 7.45 (s, 1H,
25.8; MS (ESI) m/z: 298.1 [M þ H]þ.
Ar), 7.40 (d, 1H, J ¼ 7.7 Hz, Ar), 7.17 (t, 1H, J ¼ 7.7 Hz, Ar), 6.90 (d, 1H,
J ¼ 7.7 Hz, Ar), 6.43e6.26 (m, 2H, ]CH or ]CH2), 5.69e5.66 (m,
1H, ]CH or ]CH2), 3.35 (t, 2H, J ¼ 6.6 Hz, CH2), 3.31 (s, 3H, CH3),
2.53 (t, 2H, J ¼ 7.8 Hz, CH2),1.61e1.52 (m, 4H, 2ꢂ CH2),1.39e1.31 (m,
5.4.17. 4-Chloro-N-[3-(5-oxohexyl)phenyl]butyramide (6g)
Flashchromatography (silica gel, methylenechloride/ethylacetate
(85:15)). Yield: 27%; mp: 46e48 ꢀC; IR
n
: 3345 (NH), 1685 (C]O)
2H, CH2); 13C NMR (CDCl3)
d: 164.0, 143.6, 138.0, 131.5, 128.8, 127.4,
cmꢁ1; 1H NMR (CDCl3)
d: 8.17 (brs,1H, NH), 7.38e7.28 (m, 2H, Ar), 7.15
124.6, 120.2, 117.6, 72.8, 58.5, 35.9, 31.1, 29.4, 25.8; MS (ESI) m/z:
(t, 1H, J ¼ 7.6 Hz, Ar), 6.86 (d, 1H, J ¼ 7.6 Hz, Ar), 3.58 (t, 2H, J ¼ 6.3 Hz,
CH2), 2.52e2.47 (m, 4H, 2ꢂ CH2), 2.40 (t, 2H, J ¼ 6.1 Hz, CH2),
2.17e2.09 (m, 5H, CH2 and CH3),1.57e1.53 (m, 4H, 2ꢂ CH2); 13C NMR
248.1 [M þ H]þ.
5.4.24. N-[3-(5-oxohexyl)phenyl]acrylamide (7g)
(CDCl3)
d
: 209.4,170.5,143.1,138.1,128.8,124.3,120.0,117.5, 44.5, 43.5,
Flash chromatography (silica gel, methylene chloride to methy-
35.7, 34.1, 30.8, 29.9, 28.1, 23.4; MS (ESI) m/z: 296.1 [M þ H]þ.
lene chloride/ethyl acetate (80:20)). Yield: 25%; mp: 51e52 ꢀC; IR
n:
3298 (NH), 1703 (C]O) cmꢁ1 1H NMR (CDCl3)
; d: 8.51 (s, 1H, NH),
5.4.18. N-(4-iodophenyl)acrylamide (7a)
7.46 (s, 1H, Ar), 7.39 (d, 1H, J ¼ 7.7 Hz, Ar), 7.16 (t, 1H, J ¼ 7.7 Hz, Ar),
6.88 (d, 1H, J ¼ 7.7 Hz, Ar), 6.42e6.28 (m, 2H, ]CH or ]CH2),
5.69e5.65 (m,1H, ]CH or]CH2), 2.52(t, 2H, J ¼ 6.5 Hz, CH2), 2.40(t,
Recrystallization from methylene chloride/hexane (1:10). Yield:
77%; mp: 204e206 ꢀC; IR : 3113 (NH), 1662 (C]O) cmꢁ1 1H NMR
n ;
(DMSO-d6)
d
: 10.27 (brs,1H, NH), 7.68 (d, 2H, J ¼ 8.6 Hz, Ar), 7.53 (d, 2H,
2H, J ¼ 6.5 Hz, CH2), 2.09 (s, 3H, CH3),1.57e1.52 (m, 4H, 2ꢂ CH2); 13
C
J ¼ 8.6 Hz, Ar), 6.49e6.40 (m, 1H, ]CH or ]CH2), 6.31e6.25 (m, 1H, ]
NMR (CDCl3) d: 209.5, 164.1, 143.1, 138.1, 131.5, 128.8, 127.4, 124.5,
CHor ]CH2), 5.80e5.77 (m,1H, ]CH or ]CH2);13C NMR (DMSO-d6)
d:
120.1,117.7, 43.5, 35.7, 30.7, 29.9, 23.4; MS (ESI) m/z: 246.1 [M þ H]þ.
163.3,138.9,137.5,131.7,127.3,121.6, 87.1; MS (ESI) m/z: 293.9 [M þ H]þ.
5.5. Evaluation of the antiproliferative activity
5.4.19. N-(4-tert-butylphenyl)acrylamide (7b)
Flash chromatography (silica gel, methylene chloride to meth-
Human HT-29 colon carcinoma, human M21 skin melanoma,
and human MCF-7 breast carcinoma cells were purchased by the
American Type Culture Collection. The cells were cultured in DMEM
medium (Hyclone) supplemented calf serum iron containing
ylene chloride/ethyl acetate (90:10)). Yield: 21%; mp: 97e98 ꢀC; IR
n
: 3255 (NH), 1663 (C]O) cmꢁ1; 1H NMR (CDCl3)
d: 8.73 (s, 1H, NH),
7.58 (d, 2H, J ¼ 8.5 Hz, Ar), 7.32 (d, 2H, J ¼ 8.5 Hz, Ar), 6.41e6.39 (m,
2H, ]CH or ]CH2), 5.70e5.66 (m, 1H, ]CH or ]CH2), 1.31 (s, 9H,
NaHCO3 (2.2 g/L), glucose (4.5 g/L), and glutamine (292 mg/mL). The
3ꢂ CH3); 13C NMR (CDCl3)
d
: 164.3, 147.5, 135.5, 131.6, 127.2, 125.8,
cells were maintained at 37 ꢀC in a moisture-saturated atmosphere
containing 5% CO2. The growth inhibition potency of aromatic urea
and aromatic amide analogue of CEU was assessed using the
procedure described by the National Cancer Institute for its drug
screening program [23]. 96-well microtiter plates were seeded
120.3, 34.4, 31.4; MS (ESI) m/z: 204.2 [M þ H]þ.
5.4.20. N-(4-tert-butylbenzyl)acrylamide (7c)
Recrystallization from methylene chloride/hexane (1:100).
Yield: 72%; mp: 85e86 ꢀC; IR
n
: 3300 (NH), 1681 (C]O) cmꢁ1
;
1H
with 75 mL of tumor cell (5000 cells of HT-29, 3500 cells of M21 and

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7 500 cells of MCF-7) in DMEM medium (Hyclone) supplemented
calf serum. Plates were incubated at 37 ꢀC, 5% CO2 for 24 h. Freshly
solubilized drugs in DMSO were diluted in fresh medium and
aliquots of 75
were added. Final drug concentrations ranged from 100 to
0.078 M. DMSO concentration was maintained lower than 0.5% to
mL containing escalating concentrations of the drug
m
avoid any related toxicity. Plates were incubated for 48 h. Assays
were 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 mL) 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. Bonded dye was solubilized in 10 mM Tris base,
and the absorbance was read at 585 nm using a mQuant Universal
Microplate Spectrophotometer (Biotek, Winooski, VT). A back-
ground OD from a control reference plate fixed on the day of
treatment was subtracted from the OD obtained with the 48 h.
growth period. The growth inhibition percentage was calculated in
reference to the control DMSO-treated cells for each drug concen-
tration. The experiments were performed at least in triplicate. The
IC50 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.
5.6. Western blot analysis of b-tubulin monomer
Prior to drug exposure, 3ꢂ 105 M21 cells were seeded into six-
well plates and incubated for 24 h. Then, cells were incubated in the
presence of 10ꢂ IC50 of the tested compound, except for controls,
for 24 and 48 h at 37 ꢀC. The control consisted of DMSO in the
culture medium at 0.5% (v/v). After the treatments, floating and
adherent M21 cells were harvested and were pooled, centrifuged
for 5 min at 8000 rpm, washed in ice-cold PBS, and then lysed in
Laemmli sample buffer. The cell extracts were boiled for 5 min,
separated on 10% SDS-PAGE electrophoresis gel, and transferred
onto nitrocellulose membrane. The membranes were incubated
with TBSMT (TBS, pH 7.4, 5% fat-free dried milk, and 0.1% Tween
20Ô) for 1 h at 37 ꢀC, and then with 1:500 monoclonal anti-
b-
tubulin for 1 h at room temperature. 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 immu-
noblot was carried out with an enhanced chemiluminescence (ECL)
detection reagent kit.
Acknowledgements
This work was supported by the Canadian Institute for Health
Research (R.C.G; Grant #MOP-79334 and #MOP-89707). S. Fortin is
recipient of a studentship from the Canadian Institute for Health
Research (CGD-83623). We also acknowledge the technical exper-
tise of Dr. Michel Déry for HPLCeMS experiments.
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