N-Phenyl-N′-(2-chloroethyl)urea analogues of combretastatin A-4: Is the N-phenyl-N′-(2-chloroethyl)urea pharmacophore mimicking the trimethoxy phenyl moiety?

Article abstract of DOI:10.1016/j.bmcl.2007.01.023

A series of novel N-phenyl-N′-(2-chloroethyl)urea derivatives potentially mimicking the structure of combretastatin A-4 were synthesized and tested for their cell growth inhibition and their binding to the colchicine-binding site of β-tubulin. Compounds 2a, 3a, and 3b were found to inhibit cell growth at the micromolar level on four human tumor cell lines. Flow cytometric analysis indicates that the new compounds act as antimitotics and arrest the cell cycle in G₂/M phase. Covalent binding of 2a, 3a, and 3b to the colchicine-binding site of β-tubulin was confirmed also using SDS-PAGE and competition assays.

Full text of DOI:10.1016/j.bmcl.2007.01.023

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2000–2004
N-Phenyl-N0-(2-chloroethyl)urea analogues of combretastatin A-4:
Is the N-phenyl-N0-(2-chloroethyl)urea pharmacophore mimicking
the trimethoxy phenyl moiety?
a
b,c,d,
a
b,c,d
*
´
Sebastien Fortin, Emmanuel Moreau,
Jacques Lacroix, Jean-Claude Teulade,
a
a,
*
´
Alexandre Patenaude and Rene C-Gaudreault
a
ˆ
Unite´ des Biotechnologies et de Bioinge´nierie, Centre de recherche, C.H.U.Q., Hopital Saint-Franc¸ois d’Assise,
Universite´ Laval, Que., Canada G1L 3L5
bUniv Clermont 1, UFR Pharmacie, Laboratoire de Chimie Organique, Clermont-Ferrand F-63001, France
cInserm, U484, Clermont-Ferrand F-63001, France
dCentre Jean Perrin, Clermont-Ferrand, F63001, France
Received 13 November 2006; revised 8 January 2007; accepted 8 January 2007
Available online 19 January 2007
Abstract—A series of novel N-phenyl-N0-(2-chloroethyl)urea derivatives potentially mimicking the structure of combretastatin A-4
were synthesized and tested for their cell growth inhibition and their binding to the colchicine-binding site of b-tubulin. Compounds
2a, 3a, and 3b were found to inhibit cell growth at the micromolar level on four human tumor cell lines. Flow cytometric analysis
indicates that the new compounds act as antimitotics and arrest the cell cycle in G2/M phase. Covalent binding of 2a, 3a, and 3b to
the colchicine-binding site of b-tubulin was confirmed also using SDS–PAGE and competition assays.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Microtubules are cytoskeletal components present in all
eukaryotic cells and are recognized as playing key roles
in intracellular transport, secretion, and maintenance of
shape and scaffolding. They are also involved in cell
division by forming the mitotic spindle during mitosis.1
Drugs interfering with the microtubule dynamics such as
vinca and taxus alkaloids are widely used in the manage-
ment of several cancers.2 Unfortunately, their effect is
often hampered by chemoresistance and they exhibit
biopharmaceutical properties suitable for the treatment
of only a limited number of cancers.3 Combretastatins
are a very interesting class of alternative antimitotic
agents of natural origin which have received much atten-
tion due to their simple diaryl structure and their high
potency as colchicine-binding agonists.4 Despite their
very potent antitubulin activities, their therapeutic
applications are limited by a high general toxicity.5
Therefore, several good candidates such as CA4-P,6
CA1-P,7 AVE 8062,8 and phenstatin9 were in phase
Keywords: Phenyl chloroethylureas; Combretastatin analogues; Anti-
microtubule agents; Anti-b-tubulin agents; Antimitotic agents; Soft
alkylating agents; Anticancer drugs; Colchicine-binding site ligands.
*
Figure 1. A molecular model of the docking of 4-tBCEU into the
colchicine-binding site on b-tubulin that were generated with Sybyl 7.0
using an Octane 2 linked to a Onyx3800 supercomputer. The protein
structure was obtained from protein data bank number 1SA0.20
Corresponding authors. Tel.: +33 4 73 17 80 13; fax: +33 4 73 17 80
00 (E.M.), tel.: +1 418 525 4444x52363; fax: +1 418 525 4372
(R.C.-G.); e-mail addresses: Emmanuel.Moreau@u-clermont1.fr;
rene.c-gaudreault@crsfa.ulaval.ca
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.023

S. Fortin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2000–2004
2001
I/II clinical trials on advanced cancers and the results
have been published.10 In this context, the promise to
discover therapeutically useful colchicine site inhibitors
(CSIs) has fueled innovative research programs of con-
tinued research.11,12
Starting from commercially available nitrobenzyl bro-
mides 4a and 4b, the triphenylphosphonium bromide
salts were prepared by addition of triphenyphosphine
in m-xylene to yield compound 6a20 and 6b.21 The phe-
nolic group of 5 was protected with the tert-butyldim-
ethylsilyl group (TBDMS) using tert-butyldimethylsilyl
chloride, imidazoles as a base in DMF to yield com-
pound 7.22 The conjugation of 4a, b, and 7 to 8a, b,
and 9a, b using the Wittig-coupling was performed as
described by Cushman et al.23 Following the careful sep-
aration of the isomers by flash chromatography, the ni-
tro group of 8a, 8b and 9a, 9b was reduced with zinc
dust in acetic acid8 into the corresponding silylated
E- and Z-amino stilbenes. The nitro and the alkenyl
groups of 9a, b were reduced simultaneously by catalytic
hydrogenation using H2 and Pd/C.17 The 2-chloroeth-
ylurea moiety was added by the nucleophilic addition
of 2-chloroethylisocyanate onto the corresponding ani-
lines. Removal of TBDMS group was carried out using
TBAF23 to yield compounds 1a, b,24 2a, b,25 and 3a, b.26
Over the years, we developed new antimicrotubule
N-phenyl-N0-(2-chloroethyl)ureas
agents
so-called
(CEU). CEU covalently bind to the colchicine-binding
site through a nucleophilic substitution involving the
N0-(2-chloroethyl)urea pharmacophore, whereas com-
bretastatins bind through electrostatic interactions.13
Recently, we showed using mass spectrometry that
4-tert-butyl-CEU covalently bind to b-tubulin isoform
5 by esterifying on the glutamic-198 residue (Glu198).14
Glu198 is part of a pocket that is slightly behind the
two cysteine Cys239 and Cys354 residues, that are ma-
jor keys for the binding of typical-CSIs involving the
trimethoxyphenyl (TMP) moiety of these drugs.12
Esterification of Glu198 by CEU may physically im-
pede or alter the neighboring cysteine’s pKa, similarly
to what was described by Carvalho et al. for
thioredoxin.15
The cell growth inhibitory activity of CA-4-CEU hy-
brids 1a, b, 2a, b, 3a, b, CA-4, and 10 was assessed on
human colon carcinoma (HT-29), skin melanoma
(M21), breast carcinoma (MCF-7), and breast hor-
mone-independent adenocarcinoma (MDA-MB-231).
As shown in Table 1, the 1,2-diarylethenyl Z-isomers
3a and 3b are significantly more active than the E-isomer
1a and 1b, and the saturated 1,2-diarylethanyl deriva-
tives 2a and 2b. A similar effect of the special conforma-
tion of the biaryl bridge on the biological activity is
already well documented.12,19,27 Unexpectedly CEU
substituted in position 4 showed a better cell growth
inhibition than CEU substituted in position 3. It could
be that the covalent binding of the drugs with b-tubulin
introduced spatial constraints that are partly compen-
sated by shifting the (3-hydroxy-4-methoxyphenyl)eth-
enyl moiety from position 3 to position 4 of the
phenyl moiety of CEU (see Scheme 1).
Molecular modeling experiments based on the assump-
tion that CEU are esterifying Glu198 suggested that the
phenyl group of CEU might be positioned in the vicinity
of Cys239 and Cys354 in a similar manner as reported
with colchicinoids and combretastatin; where the 2-
and the 3-methoxyl groups are anchored nearby Cys239
and Cys354, respectively11,12,16 (Fig. 1). It has been long
propounded that the presence of the trimethoxyphenyl
ring (TMP) moiety is crucial to obtain relevant cytotoxic
and antitubulin responses12 but different biological
results have been obtained by other groups over the
aromatic cycle.16,19 Moreover, structural similarities
between 3-(5-hydroxypentyl)CEU17,18 (10, Fig. 2), CA-4,
and several colchicinoids11 raised the hypothesis that
the N-phenyl-N0-(2-chloroethyl)urea moiety of CEU
may replace the TMP ring of CA-4. We therefore car-
ried out the synthesis of the designed hybrids of CA-4
and CEU with the aim to induce both cell growth inhi-
bition and irreversible colchicine-binding site inactiva-
tion (Fig. 2).
CA-4 is known to block cell cycle in G2/M phase due to
microtubule depolymerization and cytoskeleton disrup-
tion.17 The cell growth inhibitory potency of CA-4-
CEU hybrid derivatives prompted us to evaluate their
O
R: Iodo, tButyl, lower
O
O
O
A
Cl
akyl chains
N
N
H
A
A
O
H
R
O
O
B
O
B
O
B
O
Cl
OH
OH
N
H
N
H
O
OH
3a
CA-4
O
O
O
A
O
O
O
Cl
N
H
N
H
A
O
A
O
NH
NH
B
B
B
O
Cl
O
OH
N
N
3b
O
H
H
C
OH
C
O
O
10
O
O
CEU analogs to CA-4
3-(5-hydroxypentyl)CEU
Colchicine
Figure 2. Structure homologies between 10, CA-4, and colchicinoids.

2002
S. Fortin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2000–2004
H
H
N
a = 3-substituted on the ring A
b = 4-substituted on the ring A
N
OH
Cl
A
O
O
B
NO2
O2N
NO2
OTBS
O
1a, b (from 8a, b)
a
b
Br-
Ph3+P
8a, b
Br
H
N
H
4a, b
6a, b
N
OH
c
+
g, e, f
Cl
Cl
A
H
O
O
H
O
B
O
O2N
2a, b (from 9a, b)
H
H
HO
N
N
TBSO
O
A
O
O
TBSO
5
7
O
3a, b (from 9a, b)
HO
B
9a, b
O
Scheme 1. Reagents and conditions: (a) PPh 3, m-xylene, reflux, 16 h; (b) TBDMSiCl, imidazole, DMF, rt, 16 h; (c) NaH, DCM, rt, 16 h; (d) Zn,
AcOH, rt, 2 h; (e) 2-chloroethylisocyanate, DCM, rt, 16 h; (f) TBAF 1 M, THF, rt, 16 h; (g) Pd/C 10%, H2 (38 PSI), EtOH, rt, 8 h.
Table 1. Cell growth inhibition of 1a, b, 2a, b, 3a, b, 10, and CA-4
*
effect on the cell cycle.28 As shown in Table 2, the treat-
ments of M21 cells with CEU derivatives showed that
compounds 2a, 3a, 2b, and 3b caused a significant accu-
mulation of cells in G2/M, suggesting that these CEU,
like combretastatin, might inhibit the tubulin polymeri-
zation and consequently initiate apoptosis.
cells treated with compounds 2a, 3a, and 3b exhibited
a second immunoreactive band of b-tubulin as report-
ed previously with other antimicrotubule CEU,13
strongly suggesting the nucleophilic addition of com-
pounds 2a, 3a, and 3b, and 10 to b-tubulin. No cova-
lent binding to tubulin was observed for compounds
1a, b, and 2b. The competition assay between colchi-
cine and the CEU shows the disappearance of the
band corresponding to the CEU-tubulin by-product,
suggesting that compounds 2a, 3a, and 3b are cova-
lently binding to the colchicine-binding site. Combre-
tastatin A4 did not compete efficiently with CEU for
To confirm our hypothesis that the N-(2-chloroeth-
yl)phenylurea moiety may mimic the TMP ring of
CA-4, we have assessed the potential of 1a, b, 2a, b,
3a, b, combretastatin, and 10 to covalently bind to
b-tubulin using SDS–PAGE.29 As shown in Table 1,

S. Fortin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2000–2004
Table 2. Cell cycle evaluation of M21 cells treated with 1a, b, 2a, b, 3a,
b, 10, and CA-4
2003
References and notes
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1991, 52, 35.
Compound Concn (lM)
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Apoptosis G1
S
G2/M
1a
24
80
8.3
5.3
4.5
37.2 16.6 22.5
38.1 18.7 21.3
40.3 19.0 19.6
200
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2a
24
80
12.5
18.7
6.9
23.8 20.7 27.5
15.2 16.4 34.9
13.5 15.7 42.6
200
3a
24
80
44.8
15.0
21.7
22.1 12.3 12.5
18.4 16.4 35.5
14.7 10.5 41.3
7. Pettit, G. R.; Lippert, J. W. Anti-Cancer Drug Des. 2000,
15, 203.
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9. Pettit, G. R.; Toki, B.; Hamel, E.; Pettit, R. K. J. Med.
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1b
24
80
9.9
5.5
38.1 12.9 19.5
38.8 15.6 21.0
37.4 21.9 12.4
200
11.9
10. Hsieh, H. P.; Liou, J. P.; Mahidroo, N. Curr. Pharm. Des.
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14. Bouchon, B.; Chambon, C.; Mounetou, E.; Papon, J.;
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2b
24
80
2.8
2.3
2.6
37.8
9.9 30.2
15.5 13.4 45.1
7.2 16.3 47.9
200
3b
6
21
53
10.7
17.6
9.7
11.4 15.8 43.3
6.8 9.7 47.2
8.1 12.5 48.0
CA-4
DMSO
0.015
0.050
0.125
10.7
15.8
11.6
9.4 15.0 46.4
10.1 14.6 40.6
8.2 14.7 45.4
2.6
45.8 19.4 17.6
15. Carvalho, A. T.; Fernandes, P. A.; Ramos, M. J. J. Phys.
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E.; C-Gaudreault, R. Bioorg. Med. Chem. 2005, 13, 6703.
18. Fortin, S.; Moreau, E.; Patenaude, A.; Desjardins, M.;
Lacroix, J.; Rousseau, J. L.; C-Gaudreault, R. Bioorg.
Med. Chem. 2007, 15, 1430.
the colchicine-binding site. This might be related to
the fact that the rate constant for dissociation of com-
bretastatin (4.8 · 10ꢀ3 sꢀ1
)
is significantly lower that
8
for colchicine, which is essentially irreversible.30 As
expected, vinblastine did not compete either with com-
pounds 2a, 3a, 3b; vinblastine binding to tubulin using
a different binding site.
In summary, we have shown that new CA-4-CEU hy-
brid derivatives are cytotoxic on tumor cells through
their nucleophilic covalent binding to b-tubulin in the
colchicine-binding site. These CA-4-CEU antimitotic
agents that arrest the cell cycle in G2/M phase might
be an alternative to the TMP ring such as an atypic-
CSI. We therefore conclude that other structural bridges
between the two aromatic rings might be investigated
such as sulfonamides, sulfonates, amine or amide deriv-
atives, cyclic or heterocyclic moiety. These results of fur-
ther optimized biological activities of CA-4-CEU hybrid
derivatives will be reported in due course.
19. Maya, A. B. S.; Perez-Melero, C.; Mateo, C.; Alonso, D.;
Fernandez, J. L.; Gajate, C.; Moliinedo, F.; Pelaez, R.;
Cabellero, E.; Medarde, M. J. Med. Chem. 2005, 48, 556.
20. Gorsane, M.; Defay, N.; Martin, R. H. Bull. Soc. Chim.
Belg. 1985, 94, 215.
21. Nonoyama, N.; Oshima, H.; Shoda, C.; Suzuki, H. Bull.
Soc. Chim. Belg. 2001, 74, 2385.
22. Pettit, G. R.; Singh, S. B.; Cragg, G. M. J. Org. Chem.
1985, 50, 3404.
23. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti,
A. K.; Lin, C. M.; Hamel, E. J. Med. Chem. 1991, 34,
2579.
24. (E)-1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxystyryl)-
phenyl)urea (1a): mp 191–193 ꢁC; 1H NMR (200MHz,
DMSO-d6) d 9.02 (s, 1H), 8.67 (s, 1H), 7.59 (s, 1H), 7.06 (m,
7H), 6.42 (s, 1H), 3.80 (s, 3H), 3.68 (t, 2H, J = 6.0 Hz), 3.45
(m, 2H); 13C NMR (50 MHz, DMSO-d6) d: 155.1, 146.7,
140.6, 137.8, 130.1, 129.0, 128.3, 126.3, 119.0, 119.5, 118.5,
116.9, 115.5, 113.0, 112.3, 55.7, 44.4, 41.3. ESIMS (m/z)
371.2 [M+2+Na]+, 369.2 [M+Na]+, 349.2 [M+3]+, 347.2
[M+1]+, 346.1 [M]+.31 (E)-1-(2-Chloroethyl)-3-(4-(3-hy-
droxy-4-methoxystyryl)phenyl)urea (1b): mp 208–210 ꢁC;
1H NMR (200 MHz, DMSO-d6) d 8.14 (s, 1H), 7.46 (m,
4H), 7.10 (s, 1H), 6.96 (m, 4H), 6.13 (s, 1H), 3.85 (s, 3H), 3.69
(t, 2H, J = 6.0 Hz), 3.67 (m, 2H); 13C NMR (50 MHz,
DMSO-d6) d: 155.7, 148.0, 147.5, 140.5, 132.2, 127.4, 127.1,
Acknowledgments
This work was supported by a grant from the Canadian
Health Research Institute (RCG, Grant #MOP-79334)
and a scholarship from the Faculty of Pharmacy, Uni-
´
versite Laval (SF). The authors thank Dr. Lakshmi P.
Kotra for molecular modeling experiments, Dr. Philippe
Labrie for advice for the preparation of the drugs, and
Dr. Jean L. C. Rousseau for critical review of this
manuscript.

2004
S. Fortin et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2000–2004
127.0, 119.2, 119.0, 118.9, 113.0, 112.4, 56.2, 44.8, 42.5.
371.1 [M+2+Na]+, 369.1 [M+Na]+, 349.1 [M+3]+, 347.1
[M+1]+, 346.1 [M]+.31
droxy-4-methoxystyryl)phenyl)urea (3b): mp 119–121 ꢁC;
1H NMR(200 MHz, DMSO-d6) d 8.15(s, 1H), 7.42(m, 2H),
7.17 (m, 2H), 6.74 (m, 3H), 6.16 (m, 2H), 3.82 (s, 3H), 3.69 (t,
2H, J = 6.0 Hz), 3.53 (m, 2H); 13C NMR (50 MHz, DMSO-d6)
d: 155.8, 147.5, 147.0, 140.1, 131.7, 131.6, 130.0, 129.4,
129.1, 121.2, 118.5, 116.1, 112.1, 56.1, 44.8, 42.5. ESIMS (m/
z) 371.2 [M+2+Na]+, 369.2 [M+Na]+, 349.2 [M+3]+, 347.3
[M+1]+, 346.1 [M]+.31
25. 1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxyphenethyl)-
phenyl)urea (2a): mp 134–136 ꢁC; 1H NMR (200 MHz,
CDCl3 and CD3OD) d 7.09 (m, 3H), 6.75 (m, 1H), 6.65 (m,
2H), 6.55 (m, 1H), 3.76 (s, 3H), 3.53 (m, 4H), 2.73 (m, 4H);
13C NMR (50 MHz, CDCl3 and CD3OD) d: 156.3, 145.5,
145.3, 142.8, 138.8, 135.0, 128.8, 123.2, 119.7, 119.6, 117.2,
115.0, 111.1, 55.9, 44.4, 41.7, 37.9, 36.9. ESIMS (m/z) 373.1
[M+2+Na]+, 371.1 [M+Na]+, 351.2 [M+3]+, 349.2 [M+1]+,
348.1 [M]+.311-(2-Chloroethyl)-3-(4-(3-hydroxy-4-meth-
oxyphenethyl)phenyl)urea (2b): mp 139–140ꢁC; 1H NMR
(200 MHz, DMSO-d6) d 7.99 (s, 1H), 7.39 (m, 2H), 7.09 (m,
2H), 6.81 (m, 1H), 6.72 (m, 1H), 6.62 (m, 1H), 6.08 (s, 1H),
3.71 (s, 3H), 3.69 (m, 2H), 3.52 (m, 2H), 2.76 (m, 4H); 13C
NMR (50 MHz, DMSO-d6) d: 155.9, 147.2, 146.5, 139.0,
136.0, 135.7, 129.3, 120.0, 119.0, 116.0, 112.3, 56.2, 44.8,
38.0, 37.9, 30.6. ESIMS (m/z) 373.1 [M+2+Na]+, 371.1
[M+Na]+, 351.2 [M+3]+, 349.2 [M+1]+, 348.1 [M]+.31
26. (Z)-1-(2-Chloroethyl)-3-(3-(3-hydroxy-4-methoxystyryl)-
phenyl)urea (3a): mp 97–98 ꢁC; 1H NMR (200 MHz,
CDCl3 and CD3OD) d 7.12 (m, 1H), 6.98 (m, 2H), 6.77
(m, 1H), 6.60 (m, 3H), 6.25 (m, 2H), 3.67 (s, 3H), 3.35 (m,
4H); 13C NMR (50 MHz, CDCl3 and CD3OD) d: 156.4,
146.6, 145.2, 138.8, 138.2, 130.1, 129.9, 128.6, 123.4, 121.0,
119.8, 118.2, 115.3, 110.9, 55.5, 44.0, 41.5. ESIMS (m/z)
371.1 [M+2+Na]+, 369.1 [M+Na]+, 349.3 [M+3]+, 347.3
[M+1]+, 346.2 [M]+.31(Z)-1-(2-chloroethyl)-3-(4-(3-hy-
27. Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.;
Schmidt, J. M.; Pettit, G. R.; Hamel, E. Mol. Pharmacol.
1988, 34, 200.
28. Exponentially growing M21 cells were treated with 3-, 10-,
and 25-fold the GI50 of CEU and CA-4 for 24 h followed
by evaluation of cell cycle distribution by flow cytometry
using propidium iodide.13
29. M21 cells were incubated for 24 and 48 h, respectively,
with 10-fold the GI50 concentrations of the studied
compounds. The competition assays were performed with
drugs having shown significant binding to b-tubulin. M21
cells were treated with the drug (10-fold the GI50) and
colchicine (5 lM), CA-4 (50 nM), and vinblastine (5 lM).
The cells were harvested and the proteins were quantified
and analyzed using SDS–PAGE.13
30. Ray, K.; Bhattacharyya, B.; Biswas, B. B. Eur. J. Biochem.
1984, 142, 577.
31. ESIMS spectra were carried out in the Mass Spectroscopy
Laboratory of Molecular Medicine Research Centre,
Medical Sciences Bldg, University of Toronto (http://
www.medresearch.utoronto.ca/pmsc_home.html).