Alkylation potency and protein specificity of aromatic urea derivatives and bioisosteres as potential irreversible antagonists of the colchicine-binding site

Article abstract of DOI:10.1016/j.bmc.2007.04.028

A number of N-phenyl-N′-(2-chloroethyl)ureas (CEUs) have been shown to be potent antimitotics through their covalent binding to the colchicine-binding site on intracellular β-tubulin. The present communication aimed to evaluate the role of the electrophilic 2-chloroethyl amino moiety of CEU on cell growth inhibition and the specificity of the drugs as irreversible antagonists of the colchicine-binding site. To that end, several N-phenyl-N′-(2-ethyl)urea (EU), N-phenyl-N′-(2-chloroethyl)urea (CEU), N-aryl amino-2-oxazoline (OXA), and N-phenyl-N′-(2-chloroacetyl)urea (CAU) derivatives were prepared and tested for their antiproliferative activity, their effect on the cell cycle, and their irreversible binding to β-tubulin. EU derivatives were devoid of antiproliferative activity. CEUs (2h-2i, 2k, 2l, OXA 3e, 3h, 3i, 3k, 3l, tBCEU, and ICEU), OXA (3h, 3i, 3k, 3l, tBOXA, and IOXA), and CAU (4a-4m, tBCAU, and ICAU) had GI₅₀ between 1.7 and 10 μM on three tumor cell lines. Cytotoxic CEU and OXA arrested the cell cycle in G₂/M phase, while the corresponding CAU were not phase specific. Finally, Western blot analysis clearly showed that only CEUs 2h, 2k, 2l, tBCEU, ICEU and OXA 3h, 3i, 3k, 3l, tBOXA,and IOXA were able to bind irreversibly to the colchicine-binding site. Our results suggest that increasing the potency of the electrophilic moiety of the aromatic ureas enhances their antiproliferative activity but decreases significantly their capacity to covalently bind to the colchicine-binding site.

Full text of DOI:10.1016/j.bmc.2007.04.028

Bioorganic & Medicinal Chemistry 15 (2007) 4456–4469
Alkylation potency and protein specificity of aromatic
urea derivatives and bioisosteres as potential irreversible
antagonists of the colchicine-binding site
Jessica S. Fortin,a,c, ,ꢀ Jacques Lacroix,a Michel Desjardins,a,ꢀ Alexandre Patenaude,a,b
*
a,b
a,b,c,
*
´
´
Eric Petitclerc and Rene C.-Gaudreault
a
ˆ
´
Unite´ des Biotechnologies et de Bioinge´nierie, CHUQ, Hopital Saint-Franc¸ois d’Assise, Quebec, Que., Canada G1L 3L5
bDe´partement de Me´decine, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Que., Canada G1K 7P4
cFaculte´ de Pharmacie, Universite´ Laval, Que´bec, Que., Canada G1K 7P4
Received 17 January 2007; revised 4 April 2007; accepted 15 April 2007
Available online 22 April 2007
Abstract—A number of N-phenyl-N0-(2-chloroethyl)ureas (CEUs) have been shown to be potent antimitotics through their covalent
binding to the colchicine-binding site on intracellular b-tubulin. The present communication aimed to evaluate the role of the elec-
trophilic 2-chloroethyl amino moiety of CEU on cell growth inhibition and the specificity of the drugs as irreversible antagonists of
the colchicine-binding site. To that end, several N-phenyl-N0-(2-ethyl)urea (EU), N-phenyl-N0-(2-chloroethyl)urea (CEU), N-aryl
amino-2-oxazoline (OXA), and N-phenyl-N0-(2-chloroacetyl)urea (CAU) derivatives were prepared and tested for their antiprolif-
erative activity, their effect on the cell cycle, and their irreversible binding to b-tubulin. EU derivatives were devoid of antiprolifer-
ative activity. CEUs (2h–2i, 2k, 2l, OXA 3e, 3h, 3i, 3k, 3l, tBCEU, and ICEU), OXA (3h, 3i, 3k, 3l, tBOXA, and IOXA), and CAU
(4a–4m, tBCAU, and ICAU) had GI50 between 1.7 and 10 lM on three tumor cell lines. Cytotoxic CEU and OXA arrested the cell
cycle in G2/M phase, while the corresponding CAU were not phase specific. Finally, Western blot analysis clearly showed that only
CEUs 2h, 2k, 2l, tBCEU, ICEU and OXA 3h, 3i, 3k, 3l, tBOXA ,and IOXA were able to bind irreversibly to the colchicine-binding
site. Our results suggest that increasing the potency of the electrophilic moiety of the aromatic ureas enhances their antiproliferative
activity but decreases significantly their capacity to covalently bind to the colchicine-binding site.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ecules between aromatic nitrogen mustards and nitrosou-
reas.10–18 CEUs are cytotoxic on a large number of cancer
cell lines and remain active on most chemoresistant
cells.11 Prototypical CEU such as 4-tert-butyl-[3-(2-chlo-
roethyl)ureido]benzene (tBCEU) and 4-iodo-3-[(2-chlo-
roethyl)]ureido]benzene (ICEU) (Fig. 1) were shown to
covalently bind to the colchicine-binding site on human
b-tubulin isoform 2.12,13 Consequently, CEUs disrupt
the cytoskeleton, block the cell cycle progression at the
G2/M transition phase, and induce anoikis.12–14 Interest-
ingly, ICEU exhibited potent antineoplastic activity on
mice bearing CT-26 murine colon carcinoma tumors
and its [125I]-derivative is biodistributed preferentially
into organs of the gastrointestinal tract, notably the
colon.
A major hindrance of chemotherapy origins from the
intrinsic and acquired chemoresistance of cancer cells to
chemotherapeutic agents.1–4 To circumvent the lack of
tissue selectivity of anticancer agents and to alleviate their
deleterious effects, new molecules have to be designed.5–9
In that context, our ongoing research program focuses on
the design and the development of new small-molecule
anticancer drugs based on 1-aryl-3-(2-chloroethyl)ureas
(CEUs).10–14 CEUs were initially designed as hybrid mol-
Keywords: Phenyl chloroethylurea; N-Aryl 2-amino-oxazoline; Anti-
tubulin agents; Alkylating agents; Anticancer drugs; Colchicine-bind-
ing site ligands.
*
Corresponding authors. Tel.: +1 418 525 4444x52363; fax: +1 418 525
4372 (R.C.-G.); tel.: +1 418 525 4485; fax: +1 418 525 4372
(J.S.F.); e-mail: rene.c-gaudreault@crsfa.ulaval.ca
The present work aimed to understand the structure–
activity relationships involved in the antimitotic proper-
ties of CEU and to improve their biological and
biopharmaceutical properties. In that context, we have
ꢀ
Present address: OmegaChem Inc., 8800, Boulevard de la Rive-Sud,
Le´vis, Que., Canada G6V 9H1.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.028

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4457
described by Jiang and known to bind to the colchicine-
binding site on b-tubulin.19–21
O
Cl
2. Results and discussion
2.1. Chemistry
N
N
H
H
tBCEU
Scheme 1 illustrates the synthesis of CEU derivatives,
EU derivatives, and CAU derivatives performed by
nucleophilic addition of either 2-chloroethylisocyanate,
ethylisocyanate or 2-chloroacetylisocyanate, respec-
tively, on the corresponding anilines (see Scheme
1).11,12 OXA derivatives were prepared from the cata-
lytic cyclization of CEU in presence of KF on SiO2 in
acetonitrile at room temperature (Scheme 1).
I
O
Cl
N
N
H
H
ICEU
Figure 1. Chemical structures of 4-tert-butyl-[3-(2-chloroethyl)ure-
ido]benzene (tBCEU) and 4-iodo-3-[(2-chloroethyl)]ureido]benzene
(ICEU).
2.1.1. Tumor cell growth inhibition activity. Tumor cell
growth inhibition activity of CEU, EU, CAU, and
OXA was evaluated on three human cell lines: breast
carcinoma MCF-7, skin melanoma M21, and colon car-
cinoma HT-29 cells. Cell growth inhibition was assessed
according to the NCI/NIH Developmental Therapeutics
Program.18 The results are summarized in Tables 1–3
and expressed as the concentration of drug inhibiting
cell growth by 50% (GI50).
prepared and assessed the antiproliferative activity and
the specificity for the colchicine-binding site on b-tubu-
lin of a series of CEU derivatives and analogues,
namely, N-phenyl-N0-(2-ethyl)ureas (EU), N-phenyl-
N0-(2-chloroacetyl)urea derivatives (CAU), and N-aryl
amino-2-oxazoline
(4,5-dihydro-N-phenyloxazol-2-
amines: OXA) derivatives. Two main sets of structural
modifications were achieved. On one hand, we have var-
ied the nature and the position of the substituents on the
aromatic ring. On the other hand, the 2-amino chloro-
ethyl moiety was modified to assess the importance of
the electrophilic character on the cell growth inhibition
and the specificity toward the colchicine-binding site.
To that end, the 2-chloroethyl amino moiety of CEU
was converted into a 2-ethylamino group, a 2-amino
oxazolinyl group, and a 2-chloroacetylamino group,
respectively. EU were prepared as biologically inactive
and non-electrophilic derivatives of CEU. OXA deriva-
tives were obtained from the intramolecular cyclization
of CEU in presence of KF and SiO2. They are consid-
ered as weak electrophilic entities. CAU are potent elec-
trophilic compounds based on the aryl 2-haloacetamides
The results show that the antiproliferative activity of the
drugs is related to the presence of an electrophilic group
on the urea moiety of the molecules. Accordingly, EU
derivatives, which are devoid of electrophilicity, did
not inhibit the growth of the cell lines tested in this study
at 100 lM (data not shown). However, all CAU deriva-
tives (4a–4m) bearing the potent electrophilic 2-chloro-
acetylamino moiety displayed potent antiproliferative
activity on all tumor cells tested. Conversely, only CEUs
2h, 2i, 2k, 2l along with tBCEU and ICEU exhibited a
significant proliferation inhibition of tumor cells. The
bioisosteric OXA were less active than CAU but gener-
ally slightly more potent than their corresponding CEU.
It is of interest to point out that the substitution on
the 2-position for the OXA derivatives (3d–3g)
O
O
R
N
Cl
N
H
H
a
4a-m
R
NH2
Aniline
O
O
d
R
b
c
R
Cl
R= Substituted phenyl
4 or 5-indanyl
N
N
H
N
H
N
H
2a-m
2 or 9-fluorenyl
3a-m
O
R
N
H
N
H
1a-m
Scheme 1. Reagents: (a) 2-chloroacetylisocyanate, CH2Cl2; (b) 2-chloroethylisocyanate, CH2Cl2; (c) ethylisocyanate, CH2Cl2; (d) SiO2ÆKF, CH3CN.

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J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
Table 1. Growth inhibition activity of compounds 2a–2m (CEU), tBCEU, ICEU, colchicine, vinblastine, and paclitaxel on breast carcinoma MCF-7,
skin melanoma M21, and colon carcinoma HT-29 cells and their covalent binding to b-tubulin
O
R
Cl
N
H
N
H
Compound
Substituent
GI50 (lM)
b-Tubulin alkylation
48 36 24 16 8 0 h
HT-29
n.e.
M21
MCF-7
n.e.
X
2a
2b
2c
2d
n.e.
81
39
n.e.
44
X
16
33
X
n.e.
n.e.
n.e.
X
X
2e
n.e.
n.e.
n.e.
X
X
2f
2g
2h
2i
78
n.e.
n.e.
8.4
16
n.e.
n.e.
12
67
X
4.2
8.5
31
X
X
2j
95
n.e.
8.9
n.e.
17
X
2k
4.7
2l
2.3
16
5.1
38
14.2
21
X
2m
X
DMSO
tBCEU
ICEU
n.e.
2.3
1.9
n.e.
4.3
3.9
n.e.
6.2
6.0
X
I
X

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4459
Table 1 (continued)
Compound
Substituent
GI50 (lM)
b-Tubulin alkylation
48 36 24 16 8 0 h
HT-29
0.004
M21
MCF-7
0.009
Colchicine
Vinblastine
Paclitaxel
0.015
0.002
0.015
0.010
0.037
0.002
0.054
demonstrated a weak but significant improvement of the
antiproliferative activity when compared with the corre-
sponding CEU derivatives.
and IOXA were induced the production of the alkylated
band of b-tubulin after 16 to 24 h, while other CEUs
such as 2l needed 48 h of incubation. In contrast,
CAU (4a–4m) and some OXA (3e and 3f) did not alkyl-
ate b-tubulin but they were cytotoxic on all tumor cell
lines suggesting that their covalent binding to the colchi-
cine-binding site is not involved in the mechanism of ac-
tion of these compounds.
2.1.2. Cell cycle analysis. Previous studies have shown
that prototypical CEU derivatives such as tBCEU and
ICEU arrested the cell cycle in G2/M phase possibly
through their covalent binding to the colchicine-binding
site.12 We have evaluated whether the new antiprolifera-
tive compounds inhibit the cell cycle in G2/M phase such
as CEUs do. Cell cycle analysis was performed using
compounds 2k, 2l, 3k, 3l, 4k, and 4l that were the most
potent antiproliferative CEU, OXA, and CAU, respec-
tively. The experiments were conducted on M21 cells
at 3, 10, and 30 times the respective GI50.
3. Conclusion
This study aimed to understand the importance of the
electrophilic moiety of CEU derivatives on the antipro-
liferative activity, the specificity of the covalent binding
of the drug to the colchicine-binding site, and the effect
on cell cycle blockage specificity. Our results show that,
as expected, the non-electrophilic EU derivatives were
pharmacologically inactive. Interestingly, most OXA
and CAU derivatives (4a–4m) were antiproliferative,
while only CEUs 2h–2l, tBCEU, and ICEU have shown
significant activity on the three tumor cell lines tested.
Experiments conducted to compare the effect of the ac-
tive prototypical indanyl substituted 2k, 3k, 4k and flu-
orenyl substituted 2l, 3l, and 4l on the cell cycle showed
that CEU and OXA derivatives were accumulating the
cells in G2/M phase, while the equivalent CAU did not
exhibit arrest of the cell division in any specific phases,
suggesting that CAU exhibit antiproliferative activity
through a different mechanism of action. Interestingly,
several derivatives were tested using SDS–PAGE on
b-tubulin. The results displayed that compounds 2h,
2k, 2l, 3h, 3i, 3k, 3l, tBCEU, ICEU, tBOXA, and IOXA
were able to covalently bind to b-tubulin. All other mol-
ecules seemed cytotoxic through mechanism(s) of action
unrelated to colchicine-binding site antagonism. Our re-
sults suggest that the nature of the electrophilic moiety
bonded to similar biofunctional groups is of utmost
importance for the specificity of the molecule toward
the colchicine-binding site. Furthermore, compounds
having the same biofunctional groups, bearing equiva-
lent electrophilic moieties and exhibiting similar antipro-
liferative activity, might kill the cells through different
mechanism of action.
Figure 2 illustrates that the incubation of M21 cells with
compound 4k or 4l for 24 h did not induce any signifi-
cant accumulation of cells in specific phases of the cell
cycle. Conversely, CEUs 2k, 2l and OXA 3k and 3l
caused a significant accumulation of the cells in the
G2/M, as reported previously with several CEU
derivatives.12,14
2.1.3. Covalent binding to b-tubulin. b-Tubulin is one of
the primary pharmacological targets of tBCEU and
ICEU.12,14 Therefore, we compared the binding of
EU, CEU, CAU, and OXA derivatives to b-tubulin
using molecules having GI50 ranging from 1 to
20 lM.12,14 Previous studies showed that the covalent
binding of CEU to b-tubulin increases significantly the
electrophoretic mobility of the protein in SDS–PAGE
conditions.12,14 The alkylation of b-tubulin by the vari-
ous molecules was assessed on human intracellular
b-tubulin present in living cells not on purified b-tubulin.
As previously reported, the covalent binding of active
CEU on the colchicine-binding site of b-tubulin was
identified by the appearance of an additional immunore-
active band exhibiting an apparent lower molecular
weight of b-tubulin. In addition, the appearance of the
b-tubulin by-product was time-dependent.12,14 We
showed also that compounds 2h, 2k, tBCEU, and
ICEU, and their corresponding OXA 3h, 3k, tBOXA,

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J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
Table 2. Growth inhibition activity of compounds 3a–3m (OXA), tBOXA, and IOXA on breast carcinoma MCF-7, skin melanoma M21, and colon
carcinoma HT-29 cells and their covalent binding to b-tubulin
R
O
N
N
H
Compound
Substituent
GI50 (lM)
b-Tubulin alkylation
48 36 24 16 8 0 h
HT-29
13
M21
MCF-7
21
X
3a
3b
3c
3d
18
61
22
27
33
13
15
52
27
23
X
X
X
X
3e
3f
9.0
15
17
26
73
15
23
72
X
X
3g
44
X
3h
3i
2.7
7.0
16
5.1
12
5.7
19
X
X
3j
30
26
X
3k
4.9
8.1
9.1
3l
1.9
15
3.2
17
3.7
11
X
3m
X
tBOXA
IOXA
1.5
1.3
2.4
2.0
3.0
2.9
X
I
X
4. Experimental
4.1. Chemistry and chemical methods
(d) are reported in parts per million, relative to the inter-
nal tetramethylsilane standard. IR spectra were re-
corded on
a Unicam spectrometer. Uncorrected
melting points were determined on an electrothermal
melting point apparatus. Mass spectra were recorded
on the best antiproliferative compounds, by the proteo-
Proton NMR spectra were recorded on a Bruker AM-
300 spectrometer (Bruker, Germany). Chemical shifts

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4461
Table 3. Growth inhibition activity of compounds 4a–4m (CAU), tBCAU, and ICAU on breast carcinoma MCF-7, skin melanoma M21, and colon
carcinoma HT-29 cells and their covalent binding to b-tubulin
O
O
R
Cl
N
H
N
H
Compound
Substituent
GI50 (lM)
b-Tubulin alkylation
48 36 24 16 8 0 h
HT-29
9.8
M21
MCF-7
10.6
X
4a
4b
4c
4d
4e
4f
6.9
4.0
4.9
6.3
4.7
7.4
3.8
4.3
3.7
4.9
1.7
4.8
9.0
10
6.1
7.5
7.4
6.5
8.6
5.8
5.5
4.3
2.2
4.0
5.0
X
X
6.6
6.0
11
X
X
X
X
4g
4h
4i
6.7
6.9
6.1
3.2
7.0
13
X
X
X
4j
X
4k
4l
X
4m
8.6
3.3
6.9
3.4
1.1
4.2
4.8
4.8
6.8
X
tBCAU
ICAU
X
I
X
mic and mass spectrometry centre, University of Toron-
to. All reactions were conducted under a dried nitrogen
atmosphere. Chemicals were supplied by Aldrich Chem-
icals (Milwaukee, WI). 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 spec-
ified otherwise. The progress of all reactions was moni-
tored using TLC on precoated silica gel plates (Merck
Silica Gel 60 F254). The chromatograms were viewed un-
der UV light at 254 nm.

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J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
2k
3k
4k
2l
Ctrl
Ctrl
22.2 µM
88.7 µM
200.0 µM
12.8 µM
51.2 µM
128.0 µM
0
0
0
100
200
200
200
300
300
300
400
400
400
500
500
500
0
100
200
300
400
500
3l
Ctrl
Ctrl
8.0 µM
20.3 µM
81.0 µM
200.0 µM
32.0 µM
80.0 µM
100
0
100
200
300
400
500
4l
Ctrl
Ctrl
12.1 µM
48.3 µM
120.7 µM
4.3 µM
17.0 µM
42.5 µM
100
0
100
200
300
400
500
Figure 2. Effect of 2k, 3k, 4k, 2l, 3l, and 4l (CEU, OXA, CAU derivatives) on the cell cycle. Exponentially growing M21 cells were incubated in the
absence or presence of the drug for 24 h at 37 ꢁC. The cell cycle was evaluated using propidium iodide staining and flow cytometry analysis.
4.2. General preparation of compounds 1a–1m
3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3) d: 151.7, 133.9,
133.6, 123.6, 118.5, 115.7, 112.1, 29.7, 16.2, 10.2.
Ethylisocyanate (3.6 mmol) was added dropwise to a
stirred solution of the relevant aniline (3 mmol) in
dichloromethane (15 mL). The reaction mixture was
stirred overnight at ambient temperature. The resulting
crude precipitate was filtered, washed with cold ether,
and purified by recrystallization from ethanol and water.
4.2.3. 1-Ethyl-3-p-tolylurea (1c). Compound 1c was syn-
thesized from the nucleophilic addition of p-toluidine to
ethylisocyanate. Yield: 10%; mp: 145–148 ꢁC; IR (KBr)
1
m: 3324 (NH), 1716 (C@O) cmꢀ1; H NMR (DMSO-d6)
d: 8.28 (s, 1H, NH), 7.28 (d, 2H, J = 8.3 Hz, Ar), 7.02 (d,
2H, J = 8.2 Hz, Ar), 6.03 (br s, 1H, NH), 3.10 (m, 2H,
CH2), 2.22 (s, 3H, CH3), 1.05 (t, 3H, J = 7.1 Hz, CH3);
13C NMR (DMSO-d6) d: 155.3, 138.1, 129.6, 129.0,
117.8, 34.0, 20.3, 15.5.
4.2.1. 1-Ethyl-3-o-tolylurea (1a). Compound 1a was syn-
thesized from the nucleophilic addition of o-toluidine to
ethylisocyanate. Yield: 82%; mp: 166–168 ꢁC; IR (KBr)
1
m: 3326 (NH), 1631 (C@O) cmꢀ1; H NMR (DMSO-d6)
d: 7.85 (d, 1H, J = 8.0 Hz, Ar), 7.60 (s, 1H, NH), 7.11
(m, 2H, Ar), 6.88 (t, 1H, J = 7.3 Hz, Ar), 6.52 (br s, 1H,
NH), 3.14 (m, 2H, CH2), 2.20 (s, 3H, CH3), 1.09 (t, 3H,
J = 7.3 Hz, CH3); 13C NMR (DMSO-d6) d: 155.4, 138.3,
130.0, 126.7, 126.1, 121.8, 120.5, 34.0, 17.9, 15.4.
4.2.4. 1-Ethyl-3-(2,6-dimethylphenyl)urea (1d). Com-
pound 1d was synthesized from the nucleophilic addi-
tion of 2,6-dimethylaniline to ethylisocyanate. Yield:
93%; mp: 236–238 ꢁC; IR (KBr) m: 3317 (NH), 1628
(C@O) cmꢀ1 1H NMR (DMSO-d6) d: 7.41 (s, 1H,
;
NH), 7.04 (s, 3H, Ar), 5.97 (br s, 1H, NH), 3.09 (m,
2H, CH2), 2.17 (s, 6H, 2 · CH3), 1.05 (t, 3H,
J = 7.2 Hz, CH3); 13C NMR (DMSO-d6) d: 156.0,
136.2, 135.6, 127.7, 125.6, 34.3, 18.3, 15.7.
4.2.2. 1-Ethyl-3-m-tolylurea (1b). Compound 1b was syn-
thesized from the nucleophilic addition of m-toluidine to
ethylisocyanate. Yield: 100%; mp: 96–98 ꢁC; IR (KBr) m:
3328 (NH), 1706 (C@O) cmꢀ1; 1H NMR (CDCl3) d: 7.79
(s, 1H, NH), 7.09 (m, 3H, Ar), 6.79 (m, 1H, Ar), 5.93 (s,
1H, NH), 3.18 (m, 2H, CH2), 2.22 (s, 3H, CH3), 1.06 (t,
4.2.5. 1-Ethyl-3-(2,3-dimethylphenyl)urea (1e). Com-
pound 1e was synthesized from the nucleophilic addition

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4463
of 2,3-dimethylaniline to ethylisocyanate. Yield: 94%;
mp: 181–184 ꢁC; IR (KBr) m: 3317 (NH), 1633 (C@O)
(m, 2H, CH2), 1.08 (t, 3H, J = 6.9 Hz, CH3); 13C
NMR (DMSO-d6) d: 155.2, 144.2, 136.5, 131.4, 126.6,
117.3, 116.1, 33.9, 32.8, 29.9, 24.4, 15.4.
1
cmꢀ1; H NMR (DMSO-d6) d: 7.61 (s, 1H, NH), 7.53
(d, 1H, J = 8.0 Hz, Ar), 6.98 (t, 1H, J = 7.71 Hz, Ar),
6.83 (d, 1H, J = 7.4 Hz, Ar), 6.38 (br s, 1H, NH), 3.12
(m, 2H, CH2), 2.24 (s, 3H, CH3), 2.08 (s, 3H, CH3),
1.08 (t, 3H, J = 7.2 Hz, CH3); 13C NMR (DMSO-d6)
d: 155.6, 137.9, 136.3, 126.8, 125.1, 124.2, 34.1, 20.4,
15.5, 13.5.
4.2.11. 1-Ethyl-3-(2,3-dihydro-1H-inden-5-yl)urea (1k).
Compound 1k was synthesized from the nucleophilic
addition of 5-aminoindan to ethylisocyanate. Yield:
47%; mp: 136–139 ꢁC; IR (KBr) m: 3323 (NH), 1631
(C@O) cmꢀ1 1H NMR (DMSO-d6) d: 8.26 (s, 1H,
;
NH), 7.33 (s, 1H, Ar), 7.08 (m, 2H, Ar), 6.04 (br s,
1H, NH), 3.12 (m, 2H, CH2), 2.78 (m, 4H, 2 · CH2),
1.98 (m, 2H, CH2), 1.06 (t, 3H, J = 7.2 Hz, CH3); 13C
NMR (DMSO-d6) d: 155.4, 144.0, 138.8, 136.0, 124.0,
116.0, 114.1, 34.0, 32.6, 31.7, 25.2, 15.5.
4.2.6. 1-Ethyl-3-(2,4-dimethylphenyl)urea (1f). Com-
pound 1f was synthesized from the nucleophilic addition
of 2,4-dimethylaniline to ethylisocyanate. Yield: 97%;
mp: 185–192 ꢁC; IR (KBr) m: 3328 (NH), 1633 (C=O)
1
cmꢀ1; H NMR (DMSO-d6) d: 7.64 (d, 1H, J = 8.1 Hz,
Ar), 7.51 (s, 1H, NH), 6.91 (m, 2H, Ar), 6.39 (br s,
1H, NH), 3.12 (m, 2H, CH2), 2.22 (s, 3H, CH3), 2.15
(s, 3H, CH3), 1.07 (t, 3H, J = 7.1 Hz, CH3); 13C NMR
(DMSO-d6) d: 155.5, 135.7, 130.7, 130.6, 127.2, 126.5,
121.1, 34.0, 20.3, 17.8, 15.5.
4.2.12. 1-Ethyl-3-(9H-fluoren-2-yl)urea (1l). Compound
1l was synthesized from the nucleophilic addition of 2-
aminofluorene to ethylisocyanate. Yield: 64%; mp:
239–242 ꢁC; IR (KBr) m: 3329 (NH), 1638 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 8,55 (s, 1H, NH), 7.75
(t, 3H, J = 8.1 Hz, Ar), 7.52 (d, 1H, J = 7.4 Hz, Ar),
7.35 (m, 2H, Ar), 7.23 (t, 1H, J = 7.4 Hz, Ar), 6.17 (br
s, 1H, NH), 3.87 (s, 2H, CH2), 3.17 (m, 2H, CH2),
1.10 (t, 3H, J = 7.2 Hz, CH3); 13C NMR (DMSO-d6)
d: 155.3, 143.9, 142.5, 141.4, 139.9, 134.4, 126.7, 125.6,
124.9, 120.1, 119.0, 116.6, 114.5, 36.5, 34.1, 15.5.
4.2.7. 1-Ethyl-3-(2,5-dimethylphenyl)urea (1g). Com-
pound 1g was synthesized from the nucleophilic addi-
tion of 2,5-dimethylaniline to ethylisocyanate. Yield:
100%; mp: 176–180 ꢁC; IR (KBr) m: 3329 (NH), 1634
(C@O) cmꢀ1 1H NMR (DMSO-d6) d: 7.66 (s, 1H,
;
NH), 7.52 (s, 1H, Ar), 7.00 (d, 1H, J = 7.5 Hz, Ar),
6.70 (d, 1H, J = 7.3 Hz, Ar), 6.49 (br s, 1H, NH), 3.12
(m, 2H, CH2), 2.23 (s, 3H, CH3), 2.14 (s, 3H, CH3),
1.08 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (DMSO-d6)
d: 155.4, 138.1, 134.9, 129.9, 123.7, 122.5, 121.2, 34.0,
21.0, 17.5, 15.4.
4.2.13. 1-Ethyl-3-(9H-fluoren-9-yl)urea (1m). Compound
1m was synthesized from the nucleophilic addition of 9-
aminofluorene to ethylisocyanate. Yield: 80%; IR (KBr)
1
m: 3336 (NH), 1655 (C@O) cmꢀ1; H NMR (DMSO-d6)
d: 7.85 (d, 2H, J = 7.4 Hz, Ar), 7.55 (d, 2H, J = 7.3 Hz,
Ar), 7.42 (t, 2H, J = 7.2 Hz, Ar), 7.34 (t, 2H, J = 7,3 Hz,
Ar), 6.42 (br s, 1H, NH), 5.84 (d, 2H, J = 8.2 Hz, CH,
NH), 3.16 (t, 2H, J = 6.3 Hz, CH2), 1.07 (t, 3H,
J = 7.1 Hz, CH3); 13C NMR (DMSO-d6) d: 158.6,
146.0, 139.8, 128.2, 127.6, 124.9, 120.1, 55.2, 34.4, 15.7.
4.2.8. 1-Ethyl-3-(3,4-dimethylphenyl)urea (1h). Com-
pound 1h was synthesized from the nucleophilic addi-
tion of 3,4-dimethylaniline to ethylisocyanate. Yield:
21%; mp: 145–146 ꢁC; IR (KBr) m: 3332 (NH), 1635
(C@O) cmꢀ1 1H NMR (DMSO-d6) d: 8.20 (s, 1H,
;
NH), 7.13 (m, 2H, Ar), 6.95 (d, 1H, J = 8.1 Hz, Ar),
6.01 (br s, 1H, NH), 3.10 (m, 2H, CH2), 2.14 (d, 6H,
J = 8.6 Hz, 2 · CH3), 1.05 (t, 3H, J = 7.2 Hz, CH3);
13C NMR (DMSO-d6) d: 155.3, 138.3, 136.1, 129.5,
128.4, 119.1, 115.3, 33.9, 19.6, 18.6, 15.5.
4.2.14. 1-(4-tert-Butylphenyl)-3-ethylurea (tBEU). tBEU
was synthesized from the nucleophilic addition of 4-tert-
butylaniline to ethylisocyanate. Yield: 65%; mp: 133–
1
137 ꢁC; IR (KBr) m: 3318 (NH), 1640 (C@O) cmꢀ1; H
NMR (DMSO-d6) d: 8.30 (s, 1H, NH), 7.30 (d, 2H,
J = 8.6 Hz, Ar), 7.23 (d, 2H, J = 8.5 Hz, Ar), 6.03 (br
s, 1H, NH), 3.11 (m, 2H, CH2), 1.25 (s, 9H, 3 · CH3),
1.09 (t, 3H, J = 7.0 Hz, CH3); 13C NMR (DMSO-d6)
d: 156.6, 145.8, 132.8, 125.3, 122.2, 40.7, 36.3, 31.4, 15.7.
4.2.9. 1-Ethyl-3-(3,5-dimethylphenyl)urea (1i). Com-
pound 1i was synthesized from the nucleophilic addition
of 3,5-dimethylaniline to ethylisocyanate. Yield: 77%;
mp: 149–151 ꢁC; IR (KBr) m: 3342 (NH), 1656 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 8.24 (s, 1H, NH), 7.03
4.2.15. 1-Ethyl-3-(4-iodophenyl)urea (IEU). IEU was
synthesized from the nucleophilic addition of 4-iodoan-
iline to ethylisocyanate. Yield: 58%; mp: 238–240 ꢁC; IR
(s, 2H, Ar), 6.53 (s, 1H, Ar), 6.07 (br s, 1H, NH), 3.12
(m, 2H, CH2), 2.21 (s, 6H, 2 · CH3), 1.06 (t, 3H,
J = 7.2 Hz, CH3); 13C NMR (DMSO-d6) d: 155.2,
140.5, 137.5, 122.6, 115.5, 34.0, 21.1, 15.5.
(KBr) m: 3319 (NH), 1634 (C@O) cmꢀ1 1H NMR
;
(DMSO-d6) d: 8.55 (s, 1H, NH), 7.53 (d, 2H,
J = 8.6 Hz, Ar), 7.25 (d, 2H, J = 8.6 Hz, Ar), 6.16 (br
s, 1H, NH), 3.10 (m, 2H, CH2), 1.05 (t, 3H,
J = 7.1 Hz, CH3); 13C NMR (DMSO-d6) d: 154.1,
140.5, 137.1, 119.9, 83.4, 34.5, 15.5.
4.2.10. 4,5-Dihydro-N-(2,3-dihydro-1H-inden-4-yl)oxa-
zol-2-amine (1j). Compound 1j was synthesized from
the nucleophilic addition of 4-aminoindan to ethylisocy-
anate. Yield: 78%; mp: 168–170 ꢁC; IR (KBr) m: 3321
(NH), 1636 (C@O) cmꢀ1
;
1H NMR (DMSO-d6) d:
4.3. General preparation of compounds 2a–2m
7.75 (d, 1H, J = 8.1 Hz, Ar), 7.66 (s, 1H, NH), 6.99 (t,
1H, J = 7.6 Hz, Ar), 6.81 (d, 1H, J = 7.3 Hz, Ar), 6.47
(s, 1H, NH), 3.12 (m, 2H, CH2), 2.86 (t, 2H,
J = 7.4 Hz, CH2), 2.75 (t, 2H, J = 7.3 Hz, CH2), 2.02
CEUs 2a–2I, 2k–2l and tBCEU and ICEU were pre-
pared as published previously.15,16 2-chloroethylisocya-
nate (3.6 mmol) was added dropwise to a stirred

4464
J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
solution of the relevant aniline (3 mmol) in dichloro-
methane (15 mL). The reaction mixture was stirred over-
night at ambient temperature. The resulting precipitate
was filtered, washed with cold ether, and purified by
recrystallization from ethanol and water.
addition of 2,4-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 76%; mp: 170–172 ꢁC; IR (KBr) m: 3309
(NH), 1634 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
7.80 (s, 1H, NH), 7.63 (d, 1H, J = 8.1 Hz, Ar), 6.89
(m, 2H, Ar), 6.81 (br s, 1H, NH), 3.67 (t, 2H,
J = 6.1 Hz, CH2), 3.44 (m, 2H, CH2), 2.22 (s, 3H,
CH3), 2.17 (s, 3H, CH3); 13C NMR (DMSO-d6) d:
155.5, 135.4, 131.1, 130.7, 127.6, 126.5, 121.4, 44.6,
41.4, 20.3, 17.9.
4.3.1. 1-(2-Chloroethyl)-3-o-tolylurea (2a). Compound
2a was synthesized from the nucleophilic addition of
o-toluidine to 2-chloroethylisocyanate. Yield: 66%; mp:
148–151 ꢁC; IR (KBr) m: 3336 (NH), 1632 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 7.84 (m, 2H, Ar, NH),
4.3.7. 1-(2-Chloroethyl)-3-(2,5-dimethylphenyl)urea (2g).
Compound 2g was synthesized from the nucleophilic
addition of 2,5-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 74%; mp: 170–175 ꢁC; IR (KBr) m: 3353
7.12 (m, 2H, Ar), 6.91 (m, 2H, Ar, NH), 3.69 (t, 2H,
J = 6.2 Hz, CH2), 3.45 (m, 2H, CH2), 2.21 (s, 3H,
CH3); 13C NMR (DMSO-d6) d: 155.4, 138.0, 130.1,
127.1, 126.1, 122.2, 120.8, 44.6, 41.3, 18.0.
(NH), 1638 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
7.80 (s, 1H, NH), 7.66 (s, 1H, Ar), 7.00 (d, 1H,
J = 7.6 Hz, Ar), 6.89 (br s, 1H, NH), 6.72 (d, 1H,
J = 7.6 Hz, Ar), 3.68 (t, 2H, J = 6.2 Hz, CH2), 3.43 (m,
2H, CH2), 2.23 (s, 3H, CH3), 2.15 (s, 3H, CH3); 13C
NMR (DMSO-d6) d: 155.4, 137.8, 134.9, 129.9, 124.0,
122.8, 121.5, 44.6, 41.3, 20.9, 17.5.
4.3.2. 1-(2-Chloroethyl)-3-m-tolylurea (2b). Compound
2b was synthesized from the nucleophilic addition of
m-toluidine to 2-chloroethylisocyanate. Yield: 26%;
mp: 228–229 ꢁC; IR (KBr) m: 3320 (NH), 1636 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 8.60 (s, 1H, NH), 7.25
(s, 1H, Ar), 7.20 (d, 1H, J = 8.4 Hz, Ar), 7.12 (t, 1H,
J = 7.9 Hz, Ar), 6.74 (d, 1H, J = 7.2 Hz, Ar), 6.42 (br
s, 1H, NH), 3.67 (t, 2H, J = 6.2 Hz, CH2), 3.44 (t, 2H,
J = 5.9 Hz, CH2), 2.26 (s, 3H, CH3); 13C NMR
(DMSO-d6) d: 155.1, 140.3, 137.8, 128.5, 122.0, 118.3,
115.0, 44.5, 41.3, 21.3.
4.3.8. 1-(2-Chloroethyl)-3-(3,4-dimethylphenyl)urea (2h).
Compound 2h was synthesized from the nucleophilic
addition of 3,4-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 32%; mp: 149–151 ꢁC; IR (KBr) m: 3329
(NH), 1642 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
8.49 (s, 1H, NH), 7.17 (t, 2H, J = 7.9 Hz, Ar), 6.98 (d,
1H, J = 8.1 Hz, Ar), 6.40 (br s, 1H, NH), 3.67 (t, 2H,
J = 6.1 Hz, CH2), 3.44 (m, 2H, CH2), 2.16 (d, 6H,
J = 9.5 Hz, 2 · CH3); 13C NMR (DMSO-d6) d: 155.2,
138.0, 136.2, 129.6, 128.8, 119.3, 115.5, 44.5, 41.3,
19.6, 18.6. MS (ESI) m/z: 227.2 (M+ + 1).
4.3.3. 1-(2-Chloroethyl)-3-p-tolylurea (2c). Compound 2c
was synthesized from the nucleophilic addition of p-
toluidine to 2-chloroethylisocyanate. Yield: 48%; mp:
178–183 ꢁC; IR (KBr) m: 3326 (NH), 1641 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 8.58 (s, 1H, NH), 7.32
(d, 2H, J = 8.4 Hz, Ar), 7.04 (d, 2H, J = 8.3 Hz, Ar),
6.41 (br s, 1H, NH), 3.67 (t, 2H, J = 6.2 Hz, CH2),
3.46 (t, 2H, J = 5.9 Hz, CH2), 2.23 (s, 3H, CH3); 13C
NMR (DMSO-d6) d: 155.2, 137.8, 130.0, 129.3, 129.1,
118.0, 44.5, 41.3, 20.3. MS (ESI) m/z: 213.1 (M++1).
4.3.9. 1-(2-Chloroethyl)-3-(3,5-dimethylphenyl)urea (2i).
Compound 2i was synthesized from the nucleophilic
addition of 3,5-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 54%; mp: 152–154 ꢁC; IR (KBr) m: 3325
(NH), 1640 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
4.3.4. 1-(2-Chloroethyl)-3-(2,6-dimethylphenyl)urea (2d).
Compound 2d was synthesized from the nucleophilic
addition of 2,6-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 44%; mp: 165–168 ꢁC; IR (KBr) m: 3303
8.54 (s, 1H, NH), 7.05 (s, 2H, Ar), 6.56 (s, 1H, Ar),
6.42 (br s, 1H, NH), 3.67 (t, 2H, J = 6.1 Hz, CH2),
3.45 (t, 2H, J = 5.8 Hz, CH2), 2.22 (s, 6H, 2 · CH3);
13C NMR (DMSO-d6) d: 155.1, 140.2, 137.6, 122.9,
115.6, 44.4, 41.3, 21.1. MS (ESI) m/z: 227.2 (M++1).
(NH), 1634 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
7.74 (s, 1H, NH), 7.04 (s, 3H, Ar), 6.39 (s, 1H, NH),
3.64 (t, 2H, J = 6.3 Hz, CH2), 3.40 (m, 2H, CH2), 2.18
(s, 3H, CH3); 13C NMR (DMSO-d6) d: 155.9, 135.9,
135.7, 127.7, 125.8, 44.5, 41.5, 18.2.
4.3.10.
1-(2-Chloroethyl)-3-(2,3-dihydro-1H-inden-4-
yl)urea (2j). Compound 2j was synthesized from the
nucleophilic addition of 4-aminoindan to 2-chloroethy-
lisocyanate. Yield: 72%; mp: 147–149 ꢁC; IR (KBr) m:
1
4.3.5. 1-(2-Chloroethyl)-3-(2,3-dimethylphenyl)urea (2e).
Compound 2e was synthesized from the nucleophilic
addition of 2,3-dimethylaniline to 2-chloroethylisocya-
nate. Yield: 62%; mp: 148–151 ꢁC; IR (KBr) m: 3321
3327 (NH), 1632 (C@O) cmꢀ1; H NMR (DMSO-d6)
d: 7.91 (s, 1H, NH), 7.75 (d, 1H, J = 8.1 Hz, Ar), 7.03
(t, 1H, J = 7.6 Hz, Ar), 7.76 (d, 2H, J = 6.7 Hz, Ar,
NH), 3.69 (t, 2H, J = 6.0 Hz, CH2), 3.45 (m, 2H,
CH2), 2.87 (t, 2H, J = 7.4 Hz, CH2), 2.77 (t, 2H,
J = 7.3 Hz, CH2), 2.03 (m, 2H, CH2); 13C NMR
(DMSO-d6) d: 155.1, 144.3, 136.2, 131.7, 126.7, 117.7,
116.3, 44.7, 41.3, 32.8, 29.9, 24.4.
(NH), 1634 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
7.92 (s, 1H, NH), 7.52 (d, 1H, J = 8.0 Hz, Ar), 7.00 (t,
1H, J = 8.2 Hz, Ar), 6.86 (d, 1H, J = 7.4 Hz, Ar), 6.78
(br s, 1H, NH), 3.68 (t, 2H, J = 6.1 Hz, CH2), 3.44 (m,
2H, CH2), 2.31 (s, 3H, CH3), 2.11 (s, 3H, CH3); 13C
NMR (DMSO-d6) d: 155.6, 137.6, 136.5, 127.2, 125.2,
124.5, 120.3, 44.6, 41.4, 20.4, 13.6.
4.3.11.
1-(2-Chloroethyl)-3-(2,3-dihydro-1H-inden-5-
yl)urea (2k). Compound 2k was synthesized from the
nucleophilic addition of 5-aminoindan to 2-chloroethyl-
isocyanate. Yield: 47%; mp: 155–157 ꢁC; IR (KBr) m:
4.3.6. 1-(2-Chloroethyl)-3-(2,4-dimethylphenyl)urea (2f).
Compound 2f was synthesized from the nucleophilic
3330 (NH), 1637 (C@O) cmꢀ1; H NMR (DMSO-d6)
1

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4465
d: 8.56 (s, 1H, NH), 7.35 (s, 1H, Ar), 7.12 (m, 2H, Ar),
6.40 (br s, 1H, NH), 3.66 (t, 2H, J = 5.9 Hz, CH2), 3.41
(t, 2H, J = 5.9 Hz, CH2), 2.81 (m, 4H, 2 · CH2), 2.00 (t,
2H, J = 7.3 Hz, CH2); 13C NMR (DMSO-d6) d: 155.3,
144.1, 138.5, 136.4, 124.1, 116.2, 114.2, 44.5, 41.3,
31.8, 25.4. MS (ESI) m/z: 239.2 (M++1).
4.4.1. 4,5-Dihydro-N-o-tolyloxazol-2-amine (3a). Com-
pound 3a was synthesized from 2a and SiO2ÆKF. Yield:
1
79%; IR (KBr) m: 3356 (NH), 1648 (C@O) cmꢀ1; H
NMR (CDCl3) d: 7.36 (d, 1H, Ar), 7.17 (m, 2H, Ar),
6.95 (t, 1H, J = 7.3 Hz, Ar), 6.02 (s, 1H, NH), 4.34 (t,
2H, J = 8.0 Hz, CH2), 3.70 (t, 2H, J = 8.0 Hz, CH2),
2.22 (s, 3H, CH3); 13C NMR (CDCl3) d: 157.1, 142.2,
130.3, 129.1, 126.5, 122.9, 121.7, 67.1, 47.3, 17.9. MS
(ESI) m/z: 177.2 (M++1).
4.3.12. 1-(2-Chloroethyl)-3-(9H-fluoren-2-yl)urea (2l).
Compound 2l was synthesized from the nucleophilic addi-
tion of 2-aminofluorene to 2-chloroethylisocyanate.
Yield: 75%; mp: 203–206 ꢁC; IR (KBr) m: 3330 (NH),
1635 (C@O) cmꢀ1; 1H NMR (DMSO-d6) d: 8.77 (s, 1H,
NH), 7.76 (t, 3H, J = 7.7 Hz, Ar), 7.53 (d, 1H,
J = 7.4 Hz, Ar), 7.34 (t, 2H, J = 7.1 Hz, Ar), 7.24 (t, 1H,
J = 7.3 Hz, Ar), 6.46 (br s, 1H, NH), 3.88 (s, 2H, CH2),
3.70 (t, 2H, J = 6.2 Hz, CH2), 3.46 (m, 2H, CH2); 13C
NMR (DMSO-d6) d: 155.2, 143.9, 142.5, 141.4, 139.5,
134.7, 136.7, 125.7, 125.0, 120.2, 119.2, 116.7, 114.6,
44.5, 41.3, 36.5. MS (ESI) m/z: 287.2 (M++1).
4.4.2. 4,5-Dihydro-N-m-tolyloxazol-2-amine (3b). Com-
pound 3b was synthesized from 2b and SiO2ÆKF. Yield:
29%; mp: 92–99 ꢁC; IR (KBr) m: 3343 (NH), 1687 (C@O)
1
cmꢀ1; H NMR (CDCl3) d: 7.14 (m, 3H, Ar), 6.83 (d,
1H, J = 7.1 Hz, Ar), 5.24 (s, 1H, NH), 4.38 (t, 2H,
J = 8.2 Hz, CH2), 3.86 (t, 2H, J = 8.3 Hz, CH2), 2.32
(s, 3H, CH3); 13C NMR (CDCl3) d: 157.7, 141.3,
138.8, 128.7, 123.4, 120.3, 116.7, 67.3, 49.8, 21.5.
4.4.3. 4,5-Dihydro-N-p-tolyloxazol-2-amine (3c). Com-
pound 3c was synthesized from 2c and SiO2ÆKF. Yield:
51%; mp: 138–142 ꢁC; IR (KBr) m: 3257 (NH), 1652
4.3.13. 1-(2-Chloroethyl)-3-(9H-fluoren-9-yl)urea (2m).
Compound 2m was synthesized from the nucleophilic
addition of 9-aminofluorene to 2-chloroethylisocyanate.
Yield: 45%; mp: 228–230 ꢁC; IR (KBr) m: 3353 (NH),
(C@O) cmꢀ1
;
1H NMR (CDCl3) d: 7.13 (d, 2H,
J = 7.9 Hz, Ar), 7.06 (d, 2H, J = 8.3 Hz, Ar), 6.20 (br
s, 1H, NH), 4.35 (t, 2H, J = 8.3 Hz, CH2), 3.81 (t, 2H,
J = 8.4 Hz, CH2), 2.30 (s, 3H, CH3); 13C NMR (CDCl3)
d: 157.9, 139.1, 131.8, 129.1, 119.8, 67.3, 49.63, 21.0. MS
(ESI) m/z: 177.1 (M++1).
1622 (C@O) cmꢀ1 1H NMR (DMSO-d6) d: 7.85 (d,
;
2H, J = 7.5 Hz, Ar), 7.56 (d, 2H, J = 7.3 Hz, Ar), 7.43
(t, 2H, J = 7.2 Hz, Ar), 7.34 (t, 2H, J = 7,3 Hz, Ar),
6.70 (br s, 1H, NH), 6.21 (br s, 1H, NH), 5.86 (d, 1H,
J = 8.6 Hz, CH), 3.70 (t, 2H, J = 6.3 Hz, CH2), 3.48
(m, 2H, CH2); 13C NMR (DMSO-d6) d: 158.5, 145.7,
139.8, 128.3, 127.6, 124.9, 120.1, 55.2, 44.6, 41.7. MS
(ESI) m/z: 287.2 (M++1).
4.4.4. 4,5-Dihydro-N-(2,6-dimethylphenyl)oxazol-2-amine
(3d). Compound 3d was synthesized from 2d and
SiO2ÆKF. Yield: 64%; IR (KBr) m: 3285 (NH), 1702
(C@O) cmꢀ1
;
1H NMR (CDCl3) d: 7.02 (d, 2H,
4.3.14.
1-(4-tert-Butylphenyl)-3-(2-chloroethyl)urea
J = 7.4 Hz, Ar), 6.91 (t, 1H, J = 7.1 Hz, Ar), 6.02 (s, 1H,
NH), 4.35 (t, 2H, J = 7.5 Hz, CH2), 3.57 (t, 2H,
J = 7.7 Hz, CH2), 2.17 (s, 6H, 2 · CH3); 13C NMR
(CDCl3) d: 156.1, 143.4, 131.5, 127.8, 123.3, 67.1, 44.1,
18.3.
(tBCEU).16 tBCEU was synthesized from the nucleo-
philic addition of 4-tertbutylaniline to 2-chloroethyliso-
cyanate. Yield: 60%; mp: 130–134 ꢁC; IR (KBr) m: 3376
(NH), 1649 (C@O) cmꢀ1; H NMR (DMSO-d6) d: 8.56
1
(s, 1H, NH), 7.30 (m, 4H, Ar), 6.37 (br s, 1H, NH), 3.67
(t, 2H, J = 6.2 Hz, CH2), 3.43 (m, 3H, CH3), 1.26 (s, 9H,
3 · CH3); 13C NMR (DMSO-d6) d: 156.7, 146.8, 135.6,
126.0, 120.8, 44.2, 42.0, 34.3, 31.4.
4.4.5. 4,5-Dihydro-N-(2,3-dimethylphenyl)oxazol-2-amine
(3e). Compound 3e was synthesized from 2e and SiO2Æ
KF. Yield: 56%; IR (KBr) m: 3357 (NH), 1648 (C@O)
cmꢀ1; 1H NMR (CDCl3) d: 7.12 (d, 1H, J = 7.8 Hz, Ar),
7.04 (t, 1H, J = 7.53 Hz, Ar), 6.87 (d, 1H, J = 7.3 Hz,
Ar), 5.80 (s, 1H, NH), 4.36 (t, 2H, J = 8.0 Hz, CH2),
3.69 (t, 2H, J = 8.0 Hz, CH2), 2.28 (s, 3H, CH3), 2.14 (s,
3H, CH3); 13C NMR (CDCl3) d: 157.2, 142,5, 137.3,
128.5, 125.7, 125.0, 120.2, 67.1, 46.6, 20.6, 13.7. MS
(ESI) m/z: 191.3 (M++1).
16
4.3.15. 1-(2-Chloroethyl)-3-(4-iodophenyl)urea (ICEU).
ICEU was synthesized from the nucleophilic addition of
4-iodoaniline to 2-chloroethylisocyanate. Yield: 81%;
mp: 195–198 ꢁC; IR (KBr) m: 3324 (NH), 1632 (C@O)
1
cmꢀ1; H NMR (DMSO-d6) d: 8.79 (s, 1H, NH), 7.55
(d, 2H, J = 8.5 Hz, Ar), 7.27 (d, 2H, J = 8.5 Hz, Ar),
6.46 (br s, 1H, NH), 3.67 (t, 2H, J = 6.1 Hz, CH2),
3.46 (m, 2H, CH2); 13C NMR (DMSO-d6) d: 155.4,
140.2, 137.4, 120.1, 83.9, 44.2, 42.0.
4.4.6. 4,5-Dihydro-N-(2,4-dimethylphenyl)oxazol-2-amine
(3f). Compound 3f was synthesized from 2f and SiO2Æ
KF. Yield: 54%; IR (KBr) m: 3378 (NH), 1694 (C@O)
1
4.4. General preparation of compounds 3a–3m
cmꢀ1; H NMR (CDCl3) d: 7.23 (m, 1H, Ar), 6.97 (m,
2H, Ar), 6.03 (s, 1H, NH), 4.34 (t, 2H, J = 8.0 Hz,
CH2), 3.70 (t, 2H, J = 8.0 Hz, CH2), 2.28 (s, 3H, CH3),
2.19 (s, 3H, CH3); 13C NMR (CDCl3) d: 157.5, 139.2,
132.5, 131.1, 129.3, 127.0, 122.0, 67.2, 47.5, 20.8, 17.8.
MS (ESI) m/z: 191.3 (M+ + 1).
To a stirred solution of the appropriate N-phenyl-N0-(2-
chloroethyl)urea derivatives (CEUs) (0.35 mmol) in ace-
tonitrile (10 mL) was added a mixture of SiO2ÆKF
(60:40%) (3.5 mmol). The suspension was refluxed over-
night. After cooling, the mixture was filtered and the sol-
vent was evaporated under reduced pressure. The
residue was purified by flash chromatography on silica
gel (dichloromethane/methanol 95:5).
4.4.7. 4,5-Dihydro-N-(2,5-dimethylphenyl)oxazol-2-amine
(3g). Compound 3g was synthesized from 2g and
SiO2ÆKF. Yield: 75%; mp: 105–110 ꢁC; IR (KBr) m:

4466
J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
1
3179 (NH), 1682 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.19 (s, 1H, Ar), 7.03 (d, 1H, J = 7.6 Hz, Ar), 6.78 (d,
1H, J = 7.4 Hz, Ar), 6.36 (s, 1H, NH), 4.34 (t, 2H,
J = 8.0 Hz, CH2), 3.71 (t, 2H, J = 8.0 Hz, CH2), 2.31
(s, 3H, CH3), 2.19 (s, 3H, CH3); 13C NMR (CDCl3) d:
157.2, 141.9, 136.1, 130.1, 126.0, 123.8, 122.4, 67.1,
47.4, 21.2, 17.5.
4.4.13. N-(9H-Fluoren-9-yl)-4,5-dihydrooxazol-2-amine
(3m). Compound 3m was synthesized from 2m and
SiO2ÆKF. Yield: 48%; mp: 155–158 ꢁC; IR (KBr) m:
1
3175 (NH), 1648 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.64 (m, 4H, Ar), 7.31 (m, 4H, Ar), 5.77 (s, 1H, NH),
4.62 (s, 1H, CH), 4.28 (t, 2H, J = 8.4 Hz, CH2), 3.78
(t, 2H, J = 8.5 Hz, CH2); 13C NMR (CDCl3) d: 161.9,
144.9, 140.2, 128.6, 127.6, 125.3, 119.8, 68.3, 58.4,
52.4. MS (ESI) m/z: 251.2 (M++1).
4.4.8. 4,5-Dihydro-N-(3,4-dimethylphenyl)oxazol-2-amine
(3h). Compound 3h was synthesized from 2h and
SiO2ÆKF. Yield: 95%; mp: 135–140 ꢁC; IR (KBr) m:
4.4.14. N-(4-tert-Butylphenyl)-4,5-dihydrooxazol-2-amine
(tBOXA). tBOXA was synthesized from tBCEU and
SiO2ÆKF. Yield: 47%; mp: 150–154 ꢁC; IR (KBr) m:
1
3198 (NH), 1691 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.02 (m, 3H, Ar), 6.59 (s, 1H, NH), 4.36 (t, 2H,
J = 8.3 Hz, CH2), 3.80 (t, 2H, J = 8.4 Hz, CH2), 2.21
(d, 6H, J = 5.6 Hz, 2 · CH3); 13C NMR (CDCl3) d:
158.0, 139.3, 137.0, 130.5, 130.0, 121.1, 117.2, 67.3,
49.9, 19.9, 19.0. MS (ESI) m/z: 191.2 (M++1).
1
3186 (NH), 1698 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.28 (d, 2H, J = 8.7 Hz, Ar), 7.19 (d, 2H, J = 8.7 Hz,
Ar), 5.52 (s, 1H, NH), 4.38 (t, 2H, J = 8.3 Hz, CH2),
3.82 (t, 2H, J = 8.3 Hz, CH2); 13C NMR (CDCl3) d:
156.7, 143.4, 138.7, 125.1, 117.6, 65.9, 51.3, 38.4, 31.3.
4.4.9. 4,5-Dihydro-N-(3,5-dimethylphenyl)oxazol-2-amine
(3i). Compound 3i was synthesized from 2i and
SiO2ÆKF. Yield: 77%; mp: 118–125 ꢁC; IR (KBr) m:
4.4.15. 4,5-Dihydro-N-(4-iodophenyl)oxazol-2-amine
(IOXA). IOXA was synthesized from ICEU and
SiO2ÆKF. Yield: 56%; mp: 126–131 ꢁC; IR (KBr) m:
1
3246 (NH), 1712 (C@O) cmꢀ1; H NMR (CDCl3) d:
1
6.92 (s, 2H, Ar), 6.86 (s, 1H, Ar), 6.65 (s, 1H, NH),
4.36 (t, 2H, J = 8.3 Hz, CH2), 3.85 (t, 2H, J = 8.4 Hz,
CH2), 2.28 (s, 6H, 2 · CH3); 13C NMR (CDCl3) d:
157.9, 141.3, 138.5, 124.2, 117.3, 67.3, 50.1, 21.4. MS
(ESI) m/z: 191.2 (M++1).
3185 (NH), 1679 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.48 (d, 2H, J = 8.6 Hz, Ar), 6.94 (m, 2H, J = 8.7 Hz,
Ar), 4.35 (t, 2H, J = 8.2 Hz, CH2), 3.69 (t, 2H,
J = 8.3 Hz, CH2), 3.65 (s, 1H, NH); 13C NMR (CDCl3)
d: 157.1, 140.5, 137.1, 128.8, 87.5, 65.5, 51.4.
4.4.10. 4,5-Dihydro-N-(2,3-dihydro-1H-inden-4-yl)oxa-
zol-2-amine (3j). Compound 3j was synthesized from 2j
and SiO2ÆKF. Yield: 78%; mp: 72–79 ꢁC; IR (KBr) m:
4.5. General preparation of compounds 4a–4m
2-Chloroacetylisocyanate (3.6 mmol) was added drop-
wise to a stirred solution of the relevant aniline (3 mmol)
in dichloromethane (15 mL). The reaction mixture was
stirred overnight at ambient temperature. The resulting
crude precipitate was filtered, washed with cold ether,
and purified by recrystallization from ethanol and water.
1
3360 (NH), 1701 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.37 (d, 1H, J = 7.8 Hz, Ar), 7.11 (t, 1H, J = 7.6 Hz,
Ar), 6.91 (d, 2H, J = 7.3 Hz, Ar), 5.91 (s, 1H, NH),
4.35 (t, 2H, J = 8.2 Hz, CH2), 3.80 (t, 2H, J = 8.2 Hz,
CH2), 2.93. (t, 2H, J = 7.4 Hz, CH2), 2.79 (t, 2H,
J = 7.3 Hz, CH2), 1.27 (m, 2H, CH2); 13C NMR
(CDCl3) d: 157.3, 145.1, 138.4, 133.9, 127.1, 118.9,
117.7, 67.2, 49.3, 33.3, 30.1, 24.8. MS (ESI) m/z: 203.2
(M++1).
4.5.1. 1-(2-Chloroacetyl)-3-o-tolylurea (4a). Compound
4a was synthesized from the nucleophilic addition of
o-toluidine to 2-chloroacetylisocyanate. Yield: 63%;
mp: 145–146 ꢁC; IR (KBr) m: 3235 (NH), 1709 (C@O)
4.4.11. 4,5-Dihydro-N-(2,3-dihydro-1H-inden-5-yl)oxa-
zol-2-amine (3k). Compound 3k was synthesized from
2k and SiO2ÆKF. Yield: 68%; mp: 130–134 ꢁC; IR (KBr)
cmꢀ1 1H NMR (DMSO-d6) d: 11.06 (s, 1H, NH),
;
10.18 (s, 1H, NH), 7.94 (d, 1H, J = 8.0 Hz, Ar), 7.23
(m, 2H, Ar), 7.06 (t, 1H, J = 7.3 Hz, Ar), 4.42 (s, 2H,
CH2), 2.27 (s, 3H, CH3); 13C NMR (DMSO-d6) d:
169.1, 150.3, 135.8, 130.4, 127.7, 126.5, 124.1, 121.0,
43.2, 17.5. MS (ESI) m/z: 227.0 (M++1).
1
m: 3352 (NH), 1679 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.15 (s, 1H, Ar), 7.10 (d, 1H, J = 7.5 Hz, Ar), 6.99 (d,
1H, J = 7.9 Hz, Ar), 4.36 (t, 2H, J = 8.3 Hz, CH2), 3.82
(t, 2H, J = 8.4 Hz, CH2), 2.85 (m, 4H, 2 · CH2), 2.05
(m, 2H, CH2); 13C NMR (CDCl3) d: 158.1, 145.1, 139.8,
138.2, 124.4, 118.0, 116.1, 67.3, 49.8, 33.1, 32.3, 25.6.
MS (ESI) m/z: 203.2 (M++1).
4.5.2. 1-(2-Chloroacetyl)-3-m-tolylurea (4b). Compound
4b was synthesized from the addition of m-toluidine to
2-chloroacetylisocyanate. Yield: 95%; mp: 227–228 ꢁC;
IR (KBr) m: 3249 (NH), 1706 (C@O) cmꢀ1; H NMR
1
4.4.12. N-(9H-Fluoren-2-yl)-4,5-dihydrooxazol-2-amine
(3l). Compound 3l was synthesized from 2l and
SiO2ÆKF. Yield: 74%; mp: 168–172 ꢁC; IR (KBr) m:
(DMSO-d6) d: 10.91 (s, 1H, NH), 10.13 (s, 1H, NH),
7.35 (m, 2H, Ar), 7.23 (t, 1H, J = 7.8 Hz, Ar), 6.93 (d,
1H, J = 7.4 Hz, Ar), 4.41 (s, 2H, CH2), 2.30 (s, 3H,
CH3); 13C NMR (DMSO-d6) d: 168.7, 150.2, 138.4,
137.4, 128.8, 124.6, 120.2, 116.9, 43.2, 21.1. MS (ESI)
m/z: 227.0 (M++1).
1
3208 (NH), 1687 (C@O) cmꢀ1; H NMR (CDCl3) d:
7.68 (m, 3H, Ar), 7.50 (d, 1H, J = 7.4 Hz, Ar), 7.35 (t,
1H, J = 7.4 Hz, Ar), 7.23 (m, 2H, Ar), 5.24 (br s, 1H,
NH), 4.41 (t, 2H, J = 9.0 Hz, CH2), 3.89 (t, 4H,
J = 9.0 Hz, 2 · CH2); 13C NMR (CDCl3) d: 157.5,
144.5, 143.0, 141.6, 140.3, 136.6, 126.7, 125.9, 124.9,
120.2, 119.2, 118.5, 116.3, 67.3, 49.9, 37.0. MS (ESI)
m/z: 251.2 (M++1).
4.5.3. 1-(2-Chloroacetyl)-3-p-tolylurea (4c). Compound
4c was synthesized from the nucleophilic addition of p-
toluidine to 2-chloroacetylisocyanate. Yield: 59%; mp:
189–192 ꢁC; IR (KBr) m: 3238 (NH), 1720 (C@O)

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4467
cmꢀ1; 1H NMR (DMSO-d6) d: 10.89 (s, 1H, NH), 10.11
(s, 1H, NH), 7.42 (d, 2H, J = 8.2 Hz, Ar), 7.15 (d, 2H,
J = 8.1 Hz, Ar), 4.40 (s, 2H, CH2), 2.27 (s, 3H, CH3);
13C NMR (DMSO-d6) d: 168.7, 150.2, 134.9, 132.9,
129.4, 119.8, 43.2, 20.4. MS (ESI) m/z: 227.0 (M++1).
4.5.9. 1-(2-Chloroacetyl)-3-(3,5-dimethylphenyl)urea (4i).
Compound 4i was synthesized from the nucleophilic
addition of 3,5-dimethylaniline to 2-chloroacetylisocya-
nate. Yield: 75%; mp: 178–183 ꢁC; IR (KBr) m: 3256
(NH), 1703 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
10.89 (s, 1H, NH), 10.09 (s, 1H, NH), 7.16 (s, 2H,
Ar), 6.76 (s, 1H, Ar), 4.40 (s, 2H, CH2), 2.26 (s, 6H,
2 · CH3); 13C NMR (DMSO-d6) d: 168.8, 150.1, 138.2,
137.3, 125.4, 117.4, 43.2, 21.0. MS (ESI) m/z: 241.1
(M++1).
4.5.4. 1-(2-Chloroacetyl)-3-(2,6-dimethylphenyl)urea (4d).
Compound 4d was synthesized from the nucleophilic
addition of 2,6-dimethylaniline to 2-chloroacetylisocya-
nate. Yield: 100%; mp: 203–205 ꢁC; IR (KBr) m: 3285
(NH), 1730 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
10.90 (s, 1H, NH), 9.52 (s, 1H, NH), 7.12 (s, 3H, Ar),
4.41 (s, 2H, CH2), 2.20 (s, 6H, 2 · CH3) ; 13C NMR
(DMSO-d6) d: 168.6, 150.7, 135.1, 133.9, 127.8, 126.8,
43.2, 18.1. MS (ESI) m/z: 241.1 (M++1).
4.5.10. 1-(2-Chloroacetyl)-3-(2,3-dihydro-1H-inden-4-yl)-
urea (4j). Compound 4j was synthesized from the nucle-
ophilic addition of 4-aminoindan to 2-chloroacetylisocy-
anate. Yield: 74%; mp: 203–205 ꢁC; IR (KBr) m: 3238
(NH), 1716 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
4.5.5. 1-(2-Chloroacetyl)-3-(2,3-dimethylphenyl)urea (4e).
Compound 4e was synthesized from the nucleophilic
addition of 2,3-dimethylaniline to 2-chloroacetylisocy-
anate. Yield: 69%; mp: 168–170 ꢁC; IR (KBr) m: 3245
11.04 (s, 1H, NH), 10.16 (s, 1H, NH), 7.79 (d, 1H,
J = 8.0 Hz, Ar), 7.14 (t, 1H, J = 7.6 Hz, Ar), 7.00 (d,
1H, J = 7.9 Hz, Ar), 4.41 (s, 2H, CH2), 2.91 (t, 2H,
J = 7.3 Hz, CH2), 2.81 (t, 2H, J = 7.2 Hz, CH2), 2.07
(m, 2H, CH2); 13C NMR (DMSO-d6) d: 169.1, 150.1,
144.8, 133.7, 133.0, 127.1, 119.9, 117.1, 43.2, 32.8,
29.6, 24.3. MS (ESI) m/z: 253.1 (M++1).
(NH), 1716 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
10.98 (s, 1H, NH), 10.10 (s, 1H, NH), 7.68 (d, 1H,
J = 8.0 Hz, Ar), 7.09 (t, 1H, J = 7.7 Hz, Ar), 6.99
(d, 1H, J = 7.4 Hz, Ar), 4.41 (s, 2H, CH2), 2.28 (s,
3H, CH3), 2.14 (s, 3H, CH3); 13C NMR (DMSO-d6)
d: 169.0, 150.5, 136.9, 135.5, 127.3, 126.1, 125.6,
120.0, 43.2, 20.3, 13.3. MS (ESI) m/z: 241.1 (M++1).
4.5.11. 1-(2-Chloroacetyl)-3-(2,3-dihydro-1H-inden-5-yl)-
urea (4k). Compound 4k was synthesized from the
nucleophilic addition of 5-aminoindan to 2-chloroacety-
lisocyanate. Yield: 24%; mp: 170–174ꢁC; IR (KBr) m:
1
4.5.6. 1-(2-Chloroacetyl)-3-(2,4-dimethylphenyl)urea (4f).
Compound 4f was synthesized from the nucleophilic
addition of 2,4-dimethylaniline to 2-chloroacetylisocya-
nate. Yield: 20%; mp: 171–173 ꢁC; IR (KBr) m: 3235
3257 (NH), 1704 (C@O) cmꢀ1; H NMR (DMSO-d6)
d: 10.89 (s, 1H, NH), 10.11 (s, 1H, NH), 7.44 (s, 1H,
Ar), 7.18 (m, 2H, Ar), 4.39 (s, 2H, CH2), 2.85 (m, 4H,
2 · CH2), 2.01 (m, 2H, CH2); 13C NMR (DMSO-d6) d:
168.7, 150.2, 144.5, 139.2, 135.5, 124.4, 117.9, 116.0,
43.2, 32.5, 31.8, 25.2. MS (ESI) m/z: 253.1 (M++1).
(NH), 1698 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
11.00 (s, 1H, NH), 10.08 (s, 1H, NH), 7.77 (d, 1H,
J = 8.2 Hz, Ar), 7.03 (m, 2H, Ar), 4.40 (s, 2H, CH2),
2.24 (d, 6H, J = 11.7 Hz, 2 · CH3); 13C NMR
(DMSO-d6) d: 169.0, 150.3, 133.20, 133.17, 130.9,
127.8, 126.9, 121.2, 43.1, 20.4, 17.4. MS (ESI) m/z:
241.1 (M++1).
4.5.12. 1-(2-Chloroacetyl)-3-(9H-fluoren-2-yl)urea (4l).
Compound 4l was synthesized from the nucleophilic
addition of 2-aminofluorene to 2-chloroacetylisocyanate.
Yield: 78%; mp: 235–236 ꢁC; IR (KBr) m: 3234 (NH), 1712
(C@O) cmꢀ1; 1H NMR (DMSO-d6) d: 10.97 (s, 1H, NH),
10.29 (s, 1H, NH), 7.85 (d, 3H, J = 8.8 Hz, Ar), 7.57 (d,
1H, J = 7.29 Hz, Ar), 7.49 (d, 1H, J = 8.1 Hz, Ar), 7.37
(t, 1H, J = 7,3 Hz, Ar), 7.29 (t, 1H, J = 7.3 Hz, Ar), 4.43
(s, 2H, CH2), 3.92 (s, 2H, CH2); 13C NMR (DMSO-d6)
d: 155.3, 136.8, 130.6, 129.4, 127.4, 123.6, 122.9, 113.3,
112.9, 111.6, 106.9, 106.2, 105.2, 103.2, 29.8, 23.1. MS
(ESI) m/z: 301.1 (M++1).
4.5.7. 1-(2-Chloroacetyl)-3-(2,5-dimethylphenyl)urea (4g).
Compound 4g was synthesized from the nucleophilic
addition of 2,5-dimethylaniline to 2-chloroacetylisocya-
nate. Yield: 90%; mp: 178–179 ꢁC; IR (KBr) m: 3241
(NH), 1711 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
11.04 (s, 1H, NH), 10.14 (s, 1H, NH), 7.79 (s, 1H,
Ar), 7.11 (d, 1H, J = 7.6 Hz, Ar), 6.86 (d, 1H,
J = 7.4 Hz, Ar), 4.41 (s, 2H, CH2), 2.28 (s, 3H, CH3),
2.21 (s, 3H, CH3); 13C NMR (DMSO-V) d: 169.1,
150.2, 135.6, 135.5, 130.1, 124.6, 124.5, 121.4, 43.1,
20.9, 17.1. MS (ESI) m/z: 241.1 (M++1).
4.5.13. 1-(2-Chloroacetyl)-3-(9H-fluoren-9-yl)urea (4m).
Compound 4m was synthesized from the nucleophilic
addition of 9-aminofluorene to 2-chloroacetylisocya-
nate. Yield: 48%; mp: 205–208 ꢁC; IR (KBr) m: 3324
4.5.8. 1-(2-Chloroacetyl)-3-(3,4-dimethylphenyl)urea (4h).
Compound 4h was synthesized from the nucleophilic
addition of 3,4-dimethylaniline to 2-chloroacetylisocya-
nate. Yield: 39%; mp: 148–150 ꢁC; IR (KBr) m: 3239
(NH), 1729 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
10.91 (s, 1H, NH), 8.44 (s, 1H, NH), 7.88 (d, 2H,
J = 7.4 Hz, Ar), 7.60 (d, 2H, J = 7.3 Hz, Ar), 7.46 (t,
2H, J = 7.3 Hz, Ar), 7.36 (t, 2H, J = 7,3 Hz, Ar), 5.98
(d, 1H, J = 8.0 Hz, CH), 4.36 (s, 2H, CH2); 13C NMR
(DMSO-d6) d: 168.5, 153.5, 144.2, 140.0, 128.8, 127.9,
124.8, 120.4, 54.7, 43.1. MS (ESI) m/z: 301.1 (M++1).
(NH), 1700 (C@O) cmꢀ1 1H NMR (DMSO-d6) d:
;
10.88 (s, 1H, NH), 10.07 (s, 1H, NH), 7.27 (m,
2H, Ar), 7.08 (d, 1H, J = 7.8 Hz, Ar), 4.39 (s, 2H,
CH2), 2.19 (d, 6H, J = 8.1 Hz, 2 · CH3); 13C NMR
(DMSO-d6) d: 168.7, 150.2, 136.7, 135.1, 131.7, 129.8,
120.9, 117.2, 43.2, 19.5, 18.7. MS (ESI) m/z: 241.1
(M++1).
4.5.14. 1-(4-tert-Butylphenyl)-3-(2-chloroacetyl)urea
(tBCAU). tBCAU was synthesized from the nucleophilic
addition of 4-tertbutylaniline to 2-chloroacetylisocyanate.

4468
J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
Yield: 60%; mp: 170–173 ꢁC; IR (KBr) m: 3240 (NH),
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 concentration. The
experiments were performed at least in triplicate. 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.
1
1714 (C@O) cmꢀ1; H NMR (DMSO-d6) d: 10.22 (s,
1H, NH), 9.63 (s, 1H, NH), 7.35 (m, 4H, Ar), 4.18 (s,
2H, CH2), 1.32 (s, 9H, 3 X CH3); 13C NMR (DMSO-
d6) d: 168.6, 150.2, 146.3, 134.8, 125.6, 119.7, 43.2,
34.1, 31.2.
4.5.15. 1-(2-Chloroacetyl)-3-(4-iodophenyl)urea (ICAU).
ICAU was synthesized from the nucleophilic addition
of 4-iodoaniline to 2-chloroacetylisocyanate. Yield:
81%; mp: 235–240 ꢁC; IR (KBr) m: 3237 (NH), 1718
4.6.4. Cell cycle analysis. After incubation of 3.5 · 105
M21 cells with compounds (CAU, CEU or OXA) at
three concentrations (3, 10, and 30 · GI50) or DMSO
for 24 h, the cells were harvested by trypsinization,
washed with PBS, resuspended in 1 mL of PBS, and
fixed by the addition of 2.4 mL of ice-cold ethanol
(anhydrous). Then, cells were centrifuged for 5 min at
1000g. Cell pellets were resuspended in PBS containing
50 lg/mL of propidium iodide and 200 lg/mL of
RNase. Mixtures were incubated at room temperature
for 30 min, and cell cycle distribution was analyzed
using an Epics Elite ESP flow cytometer (Coulter Cor-
poration, Miami, FL).
(C@O) cmꢀ1 1H NMR (DMSO-d6) d: 10.93 (s, 1H,
;
NH), 10.16 (s, 1H, NH), 7.66 (d, 2H, J = 8.7 Hz, Ar),
7.37 (d, 2H, J = 8.7 Hz, Ar), 4.38 (s, 2H, CH2); 13C
NMR (DMSO-d6) d: 168.7, 156.3, 137.0, 128.0, 83.6, 66.0.
4.6. Biological assays
4.6.1. Materials and reagents. Vinblastine sulfate, colchi-
cine, paclitaxel, and the monoclonal antibody anti-b-
tubulin (clone TUB 2.1) were obtained from Sigma
Chemicals (St. Louis, MO). The peroxidase-conjugated
anti-mouse immunoglobulin and ECL Western blotting
detection reagent kit were purchased from Amersham
Canada (Oakville, Canada). Propidium iodide and
RNase were purchased from (Boehringer Mannheim,
Laval, Canada).
4.6.5. Western blot analysis of b-tubulin monomer. Prior
to drug exposure, approximately 5 · 105 M21 human
melanoma cells were seeded into six-well plates and
incubated for 24 h. Exponentially growing M21 human
melanoma cells were incubated in the presence of 25
lM test compound, except for controls, for 0, 6, 12,
and 24 h at 37 ꢁC. The control consisted of the final con-
centration of DMSO in the culture medium (maintained
under 0.5% (v/v)). Vinblastine sulfate, colchicine, and
paclitaxel were tested at a concentration of 50 nM. After
the treatments, floating and adherent M21 cells were
pooled, washed in ice-cold PBS, and then lysed in
100 lL Laemmli sample buffer 1·. The cell extracts were
boiled for 5 min, separated on 10% SDS–PAGE electro-
phoresis gel, and transferred onto nitrocellulose mem-
brane. The membranes were incubated with TBSMT
(TBS, pH 7.4, 5% fat-free dried milk, and 0.1% Tween
20) for 1 h at 37 ꢁC, and then with 1:500 monoclonal
anti-b-tubulin (clone TUB 2.1) for 1 h at room temper-
ature. Membranes were washed with TBST and incu-
bated with 1:2500 peroxidase-conjugated anti-mouse
immunoglobulin in TBSMT for 1 h at room tempera-
ture. After washing the membranes with TBST, detec-
tion of the immunoblot was carried out with an
enhanced chemiluminescence (ECL) detection reagent
kit.
4.6.2. Cell lines and tissue culture. HT-29 human colon
carcinoma, M21 human skin melanoma, and MCF-7
human breast carcinoma cells were purchased from the
American Type Culture Collection. The cells were cul-
tured in calf serum iron supplemented (Hyclone) med-
ium containing NaHCO3 (2.2 g/L), glucose (4.5 g/L),
and glutamine (292 lg/mL). The cells were maintained
at 37 ꢁC in a moisture-saturated atmosphere containing
5% CO2.
4.6.3. Tumor cell growth inhibition activity. The growth
inhibition potency of CEUs was assessed using the pro-
cedure described by the National Cancer Institute for
its drug screening program.22 96-well microtiter plates
were seeded with 100 lL of tumor cell lines in calf ser-
um iron supplemented (Hyclone) medium. Plates were
incubated at 37 ꢁC, 5% CO2 for 24 h. Freshly solubi-
lized drugs in DMSO were diluted in fresh medium
and aliquots of 100 lL containing sequential dilution
of drugs were added. Final drug concentrations ranged
from 10 to 0.3 lM. DMSO concentration was main-
tained lower than 0.5% to avoid growth inhibition.
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 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 stain-
ing, unbound 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 lQuant Universal Microplate Spectrophotometer
(Biotek, Winooski, VT) at 585 nm. A background
OD from a control reference plate fixed on the day
Acknowledgments
This work was supported by grants from le Fonds de la
´
´
Recherche en Sante du Quebec Junior II (E.P.) and
IRSC (RCG, Grant #MOP-79334). Jessica S. Fortin is
a recipient of a studentship from the Fonds de la
´
´
Recherche en Sante du Quebec. We thank the Proteomic
and Mass Spectrometry Centre of the University of Tor-
onto for their professional services. We are very grateful
´
to Mrs. Josee Boulet for the conception of some of the
illustrations.

J. S. Fortin et al. / Bioorg. Med. Chem. 15 (2007) 4456–4469
4469
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J.; Miot-Noirault, E.; C.-Gaudreault, R.; Madelmont,
J.-C.; Degoul, F. Mol. Pharmacol. 2005, 68, 1415–
1422.
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