Imidazolone-amide bridges and their effects on tubulin polymerization in cis-locked vinylogous combretastatin-A4 analogues: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2011.03.068

A series of novel combretastatin-A4 analogues in which the cis-olefinic bridge is replaced by an imidazolone-amide were synthesized, and their cytotoxicity and tubulin-polymerization inhibitory activities were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors. Compounds 10, 9b and 9c, bearing 3′-NH₂-4′-OCH ₃, 4′-CH₃ and 3′-CH₃-substituted 1-phenyl B-ring, confer optimal bioactivity. The binding modes of these compounds to tubulin were obtained by molecular docking, which can explain the compounds' structure-activity relationship. The studies presented here provide a new structural type for the development of novel antitumor agents.

Full text of DOI:10.1016/j.bmc.2011.03.068

Bioorganic & Medicinal Chemistry 19 (2011) 3579–3584
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Imidazolone–amide bridges and their effects on tubulin polymerization
in cis-locked vinylogous combretastatin-A4 analogues: Synthesis
and biological evaluation
Yao-Wu Li b, , Jia Liu a, , Na Liu a, Duo Shi a, Xiao-Tian Zhou a, Jia-Guo Lv a, Ju Zhu a, Can-Hui Zheng a,
,
⇑
You-Jun Zhou a,
⇑
a School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Air Force General Hospital, Beijing 100036, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel combretastatin-A4 analogues in which the cis-olefinic bridge is replaced by an
imidazolone–amide were synthesized, and their cytotoxicity and tubulin-polymerization inhibitory
activities were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors.
Compounds 10, 9b and 9c, bearing 30-NH2-40-OCH3, 40-CH3 and 30-CH3-substituted 1-phenyl B-ring,
confer optimal bioactivity. The binding modes of these compounds to tubulin were obtained by molec-
ular docking, which can explain the compounds’ structure–activity relationship. The studies presented
here provide a new structural type for the development of novel antitumor agents.
Received 11 February 2011
Revised 27 March 2011
Accepted 30 March 2011
Available online 3 April 2011
Keywords:
Imidazolone–amide
Tubulin polymerization inhibitor
Antitumor agent
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
a wide variety of human cancer cell lines including MDR cancer cell
lines. Unfortunately, CA-4 does not show efficacy in vivo because of
Microtubule polymerization dynamics can greatly affect a cell’s
ability to change and maintain its shape and undergo critical pro-
cesses such as cell signaling and mitosis. At the same time, these
polymerization dynamics can be profoundly affected by even small
chemical changes.1 For this reason, microtubules are attractive
molecular targets for anticancer therapeutics. Three distinct micro-
tubule small-molecule binding sites are well characterized. These
include the vinca, taxane and colchicine sites. The colchicine site
its low aqueous solubility and the isomerization of its cis-double
bond into the more thermally stable, but inactive, trans-isomer.4
However, a more soluble prodrug, CA-4 phosphate disodium
(CA-4P), has displayed promising results in human cancer clinical
trials,5 thus fueling continued research in novel CA-4 analogues.
Over the years, a large number of natural and synthetic CA-4
analogues have been identified as CSIs.2,4–6 However, only a few
of these have been found to have linkers of three or more atoms
between the two aryl rings of the acyclic bridgehead.7–10 Ty et al.
reported that the vinylogous analogues of CA-4, 1 (Scheme 1)
and its (E, Z)-dienic isomer show comparable tubulin polymeriza-
tion inhibitory activity with CA-4. However, these dienic deriva-
tives are prone to isomerization and become more stable but
inactive (E, E)-isomeric derivatives.11 The cyclopropyl group is gen-
erally considered an alkene bioisostere, and a secondary amide
may be considered to have a similar spatial disposition to that of
a trans-alkene. Under such context, a type of configurationally
cis-locked vinylogous CA-4 analogues, characterized by a cyclopro-
pyl–amide unit (structure B in Scheme 2) was synthesized. How-
ever, these compounds showed low tubulin polymerization
inhibition and cytotoxic activity,12 which may be related to the
spatial difference between the cyclopropyl and the cis-alkene
group. To solve the problem, we first wanted to evaluate a number
of amide derivatives whose cyclopropyl group was replaced by a
group more spatially similar to cis-alkene. Because five-membered
is located on monomeric unpolymerized
a/b-tubulin, and inhibi-
tors that bind to tubulins at this site disrupt the polymerization
of tubulin into microtubules.2
Natural products are an important source of colchicine site
inhibitors (CSIs). The first colchicine site inhibitor (CSI), colchicine
(Scheme 1), was extracted from the poisonous meadow saffron
Colchicum autumnale L, but its high toxicity has limited its thera-
peutic applications.3 Combretastatin A-4 (CA-4) (Scheme 1),
isolated from a South African willow tree Combretum caffrum,
binds to the colchicine site and exerts potent cytotoxicity against
Abbreviations: CSI, colchicine site inhibitor; CA-4, combretastatin A-4; CA-4P,
CA-4 phosphate disodium; TMP, trimethoxyphenyl.
⇑
Corresponding authors. Tel.: +86 021 81871240 (C.-H.Z.); tel.: +86 021
81871231 (Y.-J.Z.).
E-mail addresses: canhui_zheng@yahoo.com.cn (C.-H. Zheng), zhouyoujun2006
@yahoo.com.cn (Y.-J. Zhou).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.068

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Y.-W. Li et al. / Bioorg. Med. Chem. 19 (2011) 3579–3584
76.7% yield. Decarboxylation of 3 by NH4OH provided intermediate
4 in 78.2% yield. Compound 4 was treated with NBS in diethyl ether
to provide 5 in 88.2% yield. Then 5 was allowed to react with
CO(NH2)2 in N(C2H5)3/DMF to form the products of cyclization 6
in 62.8% yield. Hydrolysis of 6 by NaOH/H2O yielded 7 in 81.5%
yield. The resulting compound 7 was allowed to react with differ-
ent aromatic amine compounds 8(a–m) and 1-naphthylamine by
EDC/DMAP in DMF to yield9(a–m) and 11 in medium yield. Reduc-
tion of 9m with SnCl2/HCl in water provided 10 in 49% yield. The
structures of the target compounds are reported in Table 1.
Scheme 1. Two-dimensional structures of three colchicine site inhibitors.
heterocycle has been shown to be a good surrogate for the double
bond of CA-4 with regards to retaining the cis-alkene configura-
tion,4–6 it seems likely that CA-4 analogues bearing a five-mem-
bered heterocycle-amide moiety (structure C in Scheme 2) will
confer good bioactivity and improve physicochemical properties.
In this study, a series of cis-locked vinylogous CA-4 analogues char-
acterized by a imidazolone–amide unit were synthesized, and the
cytotoxicity and tubulin-polymerization inhibitory activity of each
analogue was evaluated. Their modes of binding to tubulin were
studied by molecular docking.
2.2. Biological evaluation
The cytotoxicity of compounds 9(a–m), 10, and 11 were evalu-
ated against three human tumor cell lines by MTT assay: the CEM
human leukemia, MDA-MB-435 human breast carcinoma and
LOVO human colon carcinoma cell lines. The cytotoxicity levels
of the target compounds are reported in Table 1.
CA-4 analogues with imidazolone–amide bridges showed more
cytotoxic activity than CA-4 analogues with cyclopropyl–amide
bridges.12 Compound 10, which has a substitution pattern similar
to that of CA-4 and neoflavonoid derivative 1, demonstrated strong
cytotoxicity. Compounds 9b and 9c also showed strong cytotoxic-
ity. Middle to low cytotoxic effects were observed with 9a, 9(d–g),
9(i–k) and 11.
2. Results and discussion
2.1. Synthesis
The chemical synthesis of these compounds is outlined in
Scheme 3. Compounds 2–7 was obtained by our previous proce-
dure.13 Intermediate 2 was prepared in 82.6% yield by treatment
of 3,4,5-trimethoxybenzoic acid with SOCl2. Compound 2 was trea-
ted with CH3COCH2COOC2H5 and C2H5ONa in C2H5OH to afford 3 in
The characteristics of the 1-phenyl B-ring (Table 1) are impor-
tant for the cytotoxic efficacy of these compounds. A 40-hydropho-
bic group on the 1-phenyl B-ring increases cytotoxic activity. This
is evidenced by compounds 9b, 9e and 9f, which each has a 40-CH3,
Scheme 2. Design concept of new configurationally cis-locked vinylogous CA-4 analogues.
Scheme 3. Synthesis of CA-4 analogues with an imidazolone–amide bridge. Reagents and conditions: (a) SOCl2, 75 °C; (b) CH3COCH2COOC2H5/C2H5ONa, rt; (c) NH4OH, rt; (d)
NBS/C2H5OC2H5, rt; (e) CO(NH2)2/N(C2H5)/DMF, 80 °C; (f) NaOH/H2O, 55 °C; (g) 8(a–m)/EDC/DMAP/DMF, rt; (h) SnCl2/HCl/H2O/80 °C; (i) 1-naphthylamine/EDC/DMAP/DMF,
rt.

Y.-W. Li et al. / Bioorg. Med. Chem. 19 (2011) 3579–3584
3581
Table 1
Cytotoxicity of subject compounds
No.
Compound structure
R3
Cytotoxicity (IC50, lM)
R1
R2
CEM
MDA-MB-435
LOVO
O
9a
9b
9c
9d
9e
9f
9g
9h
9i
H
H
Me
Me
H
H
OMe
H
Cl
Cl
Cl
NO2
NO2
H
Me
H
H
tBu
OMe
OMe
Cl
H
Cl
H
H
45.87
0.24
0.18
19.43
38.83
2.30
4.19
>200
4.80
12.09
2.11
>200.0
>200.0
40.37
0.63
0.76
33.78
32.21
17.43
4.87
151.91
4.77
19.23
8.34
44.23
>240
77.64
4.67
HN
H
Me
H
H
H
H
H
H
H
H
H
0.62
23.45
33.99
35.60
11.90
110.44
3.62
4.93
5.46
35.02
43.92
NH
MeO
MeO
NH
O
OMe
R1
R3
R2
9j
9k
9l
Me
H
OMe
9m
O
10
11
NH2
/
OMe
/
H
/
0.33
11.87
0.62
9.32
3.4
3.46
HN
NH
NH
MeO
MeO
O
OMe
Colchicine
/
/
/
0.0172
0.0073
0.101
(a) Control
40-OCH3 and 40-tBu substituted 1-phenyl B-ring. These three com-
pounds are more active than 9a and 9h, which have 40-unsubstitut-
ed and 40-Cl substituted 1-phenyl B-rings. Based on the comparison
of their cytotoxic activities, methyl may be the optimal group for
this position. Another important factor that affects cytotoxicity is
the 30 position of the 1-phenyl B-ring. In this position, several
small-size polar and nonpolar groups increase cytotoxic activity.
This is evidenced by compounds 9c and 9i, which each has a 30-
CH3 and 30-Cl substituted 1-phenyl B-ring and becomes more ac-
tive than 30-unsubstituted 9a, and by compounds 10 and 9g, which
have 30-NH2-4-OCH3- and 30-OCH3-4-OCH3-substituted 1-phenyl
B-rings and are more active than 30-unsubstituted-4-OCH3 com-
pound 9f. However, compounds with larger-sized 30-NO2, are
significantly unfavorable to the cytotoxic activity. The 30-NO2-
substituted compounds 9l and 9m show significantly less activity.
To test whether these compounds bind to tubulin and inhibit its
polymerization, representative compounds 10, 9b, 9c and 9m were
subjected to evaluation by tubulin assembly assay. CA-4 was also
evaluated by this assay as a positive control. Figure 1 shows the ef-
(b) 10 (10μM)
(c) 9b (10μM)
(d) 9c (10μM)
(e) 9m(10μM)
(f) CA-4(10μM)
0.20
0.15
0.10
0.05
0.00
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (min)
Figure 1. Effect of subject compounds on the turbidity time course of in vitro
microtubule assembly. The reaction was started by warming the solution from 0 to
37 °C. The figure shows (a) tubulin at 3 mg/mL and (b–f) aliquots of the same
fects of these compounds in 10 lM solution on the turbidimetry
time course of microtubule assembly from pure tubulin. The final
amount of microtubule was significantly lower in the presence of
10, 9b and 9c than in the blank control experiment, and the extent
of inhibition by these compounds was about half of that of CA-4.
However, the inhibition activity level of compound 9m was signif-
icantly lower than that of 10, 9b or 9c. It showed almost no effect
solution with 10 lM of 10, 9b, 9c, 9m and CA-4, respectively.
To validate the observed structure–activity relationships,
molecular docking studies of the vinylogous analogues of CA-4, 1
and CA-4 analogues with an imidazolone–amide bridge to tubulin
protein were performed. The X-ray crystal structure of the DAMA-
colchicine-tubulin complex (PDB code 1SA0) was used as the tubu-
lin protein template. Based on the predictive binding modes of 1
(Fig. 3B) and 10 (Fig. 3C) to tubulin, the imidazolone–amide bridge
of 10 retains the (Z, E)-dienic configuration of 1 well, which is
consistent with our design concept. The bridge moieties cause
the A-ring and B-ring of these compounds to lie in similar positions
to that of the colchicine site with colchicines, whose binding mode
is derived from X-ray crystal structure 1SA0 (Fig. 3A). The A-ring
trimethoxyphenyl (TMP) moiety of 10 can foster Van der Walls
interactions with the hydrophobic P1 pocket in the interface
on tubulin polymerization in 10 lM solution. Considering that
compounds 10, 9b and 9c, which each showed strong cytotoxicity,
clearly inhibited tubulin assembly, while 9m, which showed little
cytotoxicity, had very little effect in this assay, it seems that tubu-
lin represents a potential target for CA-4 analogues with an
imidazolone–amide bridge.
Because the inhibition of tubulin polymerization has implicated
in G2/M phase cell cycle arrest in various cancer cell lines,14–16 the
effects of compound 10 on the cell cycle progression on LOVO cells
was determined (Fig. 2). The results of flow cytometry analysis
indicate 10 induce a modest accumulation of cells in the G2/M
phase (28% compared to 13% for untreated cells) in
(Fig. 2B), and cause a more evident accumulation (74%) in a higher
concentration 25 M (Fig. 2C).
5 lM
between
a/b-tubulin. The –OCH3 substituted on it can form a
hydrogen bond with Cys241 in b subunit. The 1-phenyl B-ring of
l

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Y.-W. Li et al. / Bioorg. Med. Chem. 19 (2011) 3579–3584
Figure 2. Cell cycle distribution determined by flow cytometry on LOVO cells treated for 24 h without any compounds (A) or with compound 10 in 5 lM (B) and 25 lM (C).
Sub-G0-G1, G0-G1, S, and G2-M cells are indicated in the panel (A).
Figure 3. Binding modes of colchicine (A) and 1 (B) to tubulin protein and the comparison between the binding modes of 1 (purple in C), 10 (light purple in C), 9b (purple in
D) and 9f (light purple in D). The binding mode of colchicine is derived from the X-ray crystal structure of the DAMA-colchicine-tubulin complex (PDB code 1SA0). P1 and P2
are two hydrophobic pockets in the interface between a/b-tubulin. The black dot lines denote the distance between two atoms (Å).
10 and 40-OCH3 on it can foster Van der Walls interactions with the
hydrophobic P2 pocket, which can explain why the 40-hydrophobic
group on the 1-phenyl B-ring is favorable to cytotoxic activity. On
the basis of the comparison between the binding modes of 9b and
9f (Fig. 3D), the compound bearing 40-CH3 1-phenyl B-ring binds
into the colchicine site deeper than when it bears 40-OCH3, which
may explain why methyl may be the optimal group for this posi-
tion. 30-NH2 substituted on the 1-phenyl B-ring of 10 can form
hydrogen bonds with the backbone at Val315 and Lys352 in b sub-
unit. However, this group still lies in a hydrophobic position with
limited space, which can explain why several small-size polar
and nonpolar groups are favorable to cytotoxic activity while the
30-NO2 group of larger size is not.
3. Conclusion
This paper describes the synthesis of a series of novel combre-
tastatin-A4 analogues, in which the cis-olefinic bridge is replaced
by an imidazolone–amide, and the evaluation of their cytotoxicity
and tubulin polymerization inhibitory activity. These compounds
seem to show promise as potential tubulin polymerization inhibi-
tors. Compounds 10, 9b and 9c, bearing 30-NH2-40-OCH3-, 40-CH3-
and 30-CH3-substituted 1-phenyl B-rings, confer optimal bioactiv-
ity. The binding modes of these compounds to tubulin protein were
obtained by molecular docking, which explains the structure–
activity relationships. The studies presented here provide a new
structural type for the development of novel antitumor agents.

Y.-W. Li et al. / Bioorg. Med. Chem. 19 (2011) 3579–3584
3583
4.1.1.6. N-(4-Methoxyphenyl)-2-oxo-5-(3,4,5-trimethoxyphen-
yl)-2,3-dihydro-1H-imidazole- 4-carboxamide (9f). Brown crys-
tal: mp 228–229 °C; 1H NMR (300 MHz, DMSO, d ppm): 3.68 (s, 3H,
Ar–OCH3), 3.72(s, 3H, Ar–OCH3), 3.77 (s, 6H, Ar–OCH3), 6.90 (d, 2H,
J = 9 Hz, Ar–H), 7.36 (br, 2H, imidazolone-NH), 7.58 (d, 2H, J = 9 Hz,
Ar–H), 7.7 (s, 2H, Ar–H), 9.64 (s, 1H, Ar–NHCO); ESI-MS (m/z):
400.40 [M+H]+.
4. Experiments
4.1. Chemistry
Melting points were determined on an electrically heated RK-Z
melting point apparatus and are uncorrected. Mass spectra (MS)
were measured on a Micromass Qtof-Micro LC-MS instrument.
1H NMR spectra were recorded at 300 MHz on a Bruker AC-300P
spectrometer with Me4Si as the internal standard. Chemical shifts
are given in ppm (d), and the spectral data are consistent with the
assigned structures. Silica gel column chromatography was per-
formed with Silica gel 60 G (Qindao Haiyang Chemical, China).
Commercial solvents and reagents were of reagent grade and,
when necessary, were purified and dried by standard protocols. Or-
ganic solutions were dried over anhydrous sodium sulfate. Com-
pounds 2–7 was obtained by our previous procedure.13
4.1.1.7.
N-(3,4-Dimethoxyphenyl)-2-oxo-5-(3,4,5-trimethoxy-
phenyl)-2,3-dihydro-1H- imidazole-4-carboxamide (9g). Light
yellow crystal: mp 216–218 °C; 1H NMR (300 MHz, DMSO, d
ppm): 3.69 (s, 3H, Ar–OCH3), 3.72 (s, 6H, Ar–OCH3), 3.78 (s, 6H,
Ar–OCH3), 6.90 (d, 1H, J = 8.7 Hz, Ar–H), 7.26–7.32 (m, 2H, Ar–H),
7.37 (br, 2H, imidazolone-NH), 7.69 (s, 2H, Ar–H), 9.60 (s, 1H,
Ar–NHCO); ESI-MS (m/z): 430.46 [M+H]+.
4.1.1.8. N-(4-Chlorophenyl)-2-oxo-5-(3,4,5-trimethoxyphenyl)-
2,3-dihydro-1H-imidazole-4- carboxamide (9h). White crystal:
mp 240 °C, dec.; 1H NMR (300 MHz, DMSO, d ppm): 3.69 (s, 3H,
Ar–OCH3), 3.78 (s, 6H, Ar–OCH3), 7.37 (d, 2H, J = 9 Hz, Ar–H), 7.45
(br, 2H, imidazolone-NH), 7.69 (s, 2H, Ar–H), 7.73 (d, 2H, J = 9 Hz,
Ar–H), 9.91 (s, 1H, Ar–NHCO); ESI-MS (m/z): 402.36 [M+H]+.
4.1.1. General procedure for the preparation of 9(a–m), 11
A
mixture of 7 (0.02 mol), 8(a–m) or 1-naphthylamine
(0.02 mol), EDC (0.08 mol), DMAP (0.002 mol) and DMF (60 ml)
was stirred at room temperature for 12 h. Then water (200 ml)
was added, and the resulting solid was filtered and washed by
water twice. After filtration, the filter cake was recrystallized to
yield compounds 9(a–m) and 11 (30–60%).
4.1.1.9. N-(3-Chlorophenyl)-2-oxo-5-(3,4,5-trimethoxyphenyl)-
2,3-dihydro-1H-imidazole-4- carboxamide (9i). Light yellow
crystal: mp 207–208 °C; 1H NMR (300 MHz, CDCl3, d ppm): 3.91
(s, 3H, Ar–OCH3), 3.93 (s, 6H, Ar–OCH3), 5.47 (br, 2H, imidazo-
lone-NH), 7.10–7.13 (m, 1H, Ar–H), 7.25–7.30 (m, 1H, Ar–H),
7.39–7.44 (m, 1H, Ar–H), 7.59 (s, 2H, Ar–H), 7.73 (s, 1H, Ar–H),
7.84 (s, 1H, Ar–NHCO); ESI-MS (m/z): 404.38 [M+H]+.
4.1.1.1. 2-Oxo-N-Phenyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihy-
dro-1H-imidazole-4-carbox-amide (9a). Light yellow crystal:
mp 216–219 °C; 1H NMR (300 MHz, DMSO, d ppm): 3.69 (s, 3H,
Ar–OCH3), 3.72 (s, 6H, Ar–OCH3), 7.08 (d, 1H, J = 9 Hz, Ar–H),
7.29–7.35 (m, 2H, Ar–H), 7.42 (br, 2H, imidazolone-NH), 7.67 (d,
2H, J = 9 Hz, Ar–H), 7.69 (s, 2H, Ar–H), 9.74 (s, 1H, Ar–NHCO);
ESI-MS (m/z): 370.41 [M+H]+.
4.1.1.10.
N-(3,4-Dichlorophenyl)-2-oxo-5-(3,4,5-trimethoxy-
phenyl)-2,3-dihydro-1H-imidazole-4-carboxamide (9j). Brown
crystal: mp 209–211 °C; 1H NMR (300 MHz, DMSO, d ppm): 3.70
(s, 3H, Ar–OCH3), 3.78 (s, 6H, Ar–OCH3), 7.47 (br, 2H, imidazo-
lone-NH), 7.56–7.71 (m, 5H, Ar–H), 8.04 (s, 1H, Ar–NHCO); ESI-
MS (m/z): 438.41 [M+H]+.
4.1.1.2. 2-Oxo-N-p-Tolyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihy-
dro-1H-imidazole-4-carbox-amide (9b). Light yellow crystal:
mp 224–226 °C; 1H NMR (300 MHz, DMSO, d ppm): 2.25 (s, 3H,
Ar–CH3), 3.69 (s, 3H, Ar–OCH3), 3.77 (s, 6H, Ar–OCH3), 7.12 (d,
2H, J = 8.4 Hz, Ar–H), 7.41 (br, 2H, imidazolone-NH), 7.56 (d, 2H,
J = 8.4 Hz, Ar–H), 7.69 (s, 2H, Ar–H), 9.66 (s, 1H, Ar–NHCO); ESI-
MS (m/z): 384.45 [M+H]+.
4.1.1.11. N-(3-Chloro-4-methylphenyl)-2-oxo-5-(3,4,5-trimeth-
oxyphenyl)-2,3-dihydro-1H-imidazole-4-carboxamide
(9k).
Light yellow crystal: mp 233–235 °C; 1H NMR (300 MHz, DMSO,
d ppm): 2.27 (s, 3H, Ar–CH3), 3.69 (s, 3H, Ar–OCH3), 3.78 (s, 6H,
Ar–OCH3), 7.28 (d, 1H, J = 6.3 Hz, Ar–H), 7.43 (br, 2H, imidazo-
lone-NH), 7.52 (dd, 1H, J1=6.3 Hz, J2=2.1 Hz, Ar–H), 7.66 (s, 2H,
Ar–H), 7.83 (d, J = 2.1 Hz, 1H, Ar–H), 9.86 (s, 1H, Ar–NHCO); ESI-
MS (m/z): 418.65 [M+H]+.
4.1.1.3. 2-Oxo-N-m-Tolyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihy-
dro-1H-imidazole-4-carbox- amide (9c). Light yellow crystal:
mp 205–207 °C; 1H NMR (300 MHz, CDCl3, d ppm): 2.36 (s, 3H,
Ar–CH3), 3.91 (s, 3H, Ar–OCH3), 3.94 (s, 6H, Ar–OCH3), 5.38 (br,
2H, imidazolone-NH), 6.95–6.97 (m, 1H, Ar–H), 7.21–7.24 (m, 1H,
Ar–H), 7.35–7.42 (m, 2H, Ar–H), 7.58 (s, 2H, Ar–H), 7.76 (s, 1H,
Ar–NHCO); ESI-MS (m/z): 384.49 [M+H]+.
4.1.1.12. N-(3-Nitrophenyl)-2-oxo-5-(3,4,5-trimethoxyphenyl)-
2,3-dihydro-1H-imidazole-4-carboxamide (9l). Yellow crystal:
mp 240 °C, dec.; 1H NMR (300 MHz, DMSO, d ppm): 3,70 (s, 3H,
Ar–OCH3), 3.79 (s, 6H, Ar–OCH3), 7.49 (br, 2H, imidazolone-NH),
7.62 (t, 2H, J = 8.4 Hz, Ar–H), 7.67 (s, 2H, Ar–H), 7.90–7.94 (m,
1H, J = 8.4 Hz, Ar–H), 8.13 (d, 1H, J = 7.8 Hz, Ar–H), 8.69 (t, 1H,
J = 2.1 Hz, Ar–H); ESI-MS (m/z): 413.42 [M+H]+.
4.1.1.4.N-(3,5-Dimethylphenyl)-2-oxo-5-(3,4,5-trimethoxyphen-
yl)-2,3-dihydro-1H-imidazole-4-carboxamide (9d). Light yel-
low crystal: mp 215–218 °C; 1H NMR (300 MHz, CDCl3, d ppm):
6.06 (s, 6H, Ar–CH3), 3.90 (s, 3H, Ar–OCH3), 3.93 (s, 6H,
Ar–OCH3), 5.21 (br, 2H, imidazolone-NH), 6.78 (s, 1H, Ar–H), 7.20
(s, 2H, Ar–H), 7.51 (s, 2H, Ar–H), 7.68 (s, 1H, Ar–NHCO); ESI-MS
(m/z): 398.38 [M+H]+.
4.1.1.13. N-(4-Methoxy-3-nitrophenyl)-2-oxo-5-(3,4,5-trimeth-
oxyphenyl)-2,3-dihydro-1H-imidazole-4-carboxamide
(9m).
4.1.1.5. N-(4-Tert-butylphenyl)-2-oxo-5-(3,4,5-trimethoxyphen-
yl)-2,3-dihydro-1H-imidazole-4-carboxamide (9e). Light yellow
crystal: mp 202.5–204 °C; 1H NMR (300 MHz, DMSO, d ppm): 1.26
(s, 9H, tert-butyl), 3.69 (s, 3H, Ar–OCH3), 3.78 (s, 6H, Ar–OCH3),
7.33 (d, 2H, J = 8.7 Hz, Ar–H), 7.38 (br, 2H, imidazolone-NH), 7.58
(d, 2H, J = 8.7 Hz, Ar–H), 7.70 (s, 2H, Ar–H), 9.65 (s, 1H, Ar–NHCO);
ESI-MS (m/z): 472.40 [M-H]ꢀ.
Yellow crystal: mp 223–225 °C; 1H NMR (300 MHz, DMSO, d
ppm): 3.69 (s, 3H, Ar–OCH3), 3.78 (s, 6H, Ar–OCH3), 3.90 (s, 3H,
Ar–OCH3), 7.37 (d, 1H, J = 9.3 Hz, Ar–H), 7.45 (br, 2H, imidazo-
lone-NH), 7.67 (s, 2H, Ar–H), 7.93 (dd, 1H, J1=9.3 Hz, J2=2.7 Hz,
Ar–H), 8.29 (d, 1H, J = 2.7 Hz, Ar–H), 10.03 (s, 1H, Ar–NHCO); ESI-
MS (m/z): 445.42 [M+H]+.

3584
Y.-W. Li et al. / Bioorg. Med. Chem. 19 (2011) 3579–3584
4.1.1.14.
N-(Naphthalen-1-yl)-2-oxo-5-(3,4,5-trimethoxyphe-
(11). Brown
(PDB code 1SA0) was used as the tubulin protein template. The
nyl)-2,3-dihydro-1H-imidazole-4-carboxamide
AutoDock program was used.19 A grid of 45 ꢁ 40 ꢁ 40 was used
to ensure that the area probed was adequate for the ligands to ex-
plore all possible binding modes. Docking runs were performed
using the Larmarckian genetic algorithm (LGA) with some modifi-
cations of the docking parameters. Each docking experiment was
derived from 50 different runs that was set to terminate after a
maximum of 10,000,000 energy evaluations or 370,000 genera-
tions, yielding 50 docked conformations. At the end of a docking
job with multiple runs, AutoDock performed cluster analysis.
Docking solutions with ligand all-atom RMSDs within 0.5 Å
of each other were clustered together and ranked by the lowest
energy representative. Low energy binding modes and the lowest
energy conformation from clusters of five or more occurrences
were selected for further refinement. These were then refined by
Sybyl,20 with the binding mode that had the lowest complex
energy after minimisation being selected as the final binding
mode.
crystal: mp 202–203 °C; 1H NMR (300 MHz, DMSO, d ppm): 3.70
(s, 3H, Ar–OCH3), 3.79(s, 6H, Ar- OCH3), 7.40–7.47 (m, 2H, Ar–H),
7.44 (br, 2H, imidazolone-NH), 7.69 (s, 2H, Ar–H), 7.73–7.79 (m,
1H, Ar–H), 7.81–7.88 (m, 3H, Ar–H), 8.30 (d, 1H, J = 2.1 Hz, Ar–H),
9.94 (s, 1H, Ar–NHCO); ESI-MS (m/z): 421.00 [M+H]+.
4.1.2. N-(3-Amino-4-methoxyphenyl)-2-oxo-5-(3,4,5-trim-
ethoxyphenyl)-2,3-dihydro-1H-imidazole-4-carboxamide (10)
Compound 8m (2 g) was added into a solution of SnCl2 (4.1 g) in
hydrochloric acid (40 ml), and the reaction mixture was stirred at
80 °C for 4 h. After cooling, the pH value of the reaction mixture
was adjusted to 8 by sodium hydroxide. Then the solution was ex-
tracted with acetic ether and dried over Na2SO4. Distillation of the
solvent under reduced pressure and recrystallization of the residue
yielded compounds 10 (0.92 g, 49%) as brown crystal (49% yield):
mp 200–202 °C; 1H NMR (300 MHz, DMSO, d ppm): 2.27 (s, 3H,
Ar–CH3), 3.69 (s, 3H, Ar–OCH3), 3.78 (s, 6H, Ar–OCH3), 4.77 (br,
2H, NH2), 6.69–7.75 (m, 2H, Ar–H), 7.00 (d, 1H, J = 2.1 Hz, Ar–H),
7.33 (br, 2H, imidazolone-NH), 7.68 (s, 2H, Ar–H), 9.33 (s, 1H,
Ar–NHCO); ESI-MS (m/z): 418.65 [M+H]+.
Acknowledgments
The work was supported by the Key Program for Basic Research
of Shanghai ‘Technology Innovation Action Plan’ (Grant No.
09JC1417500), Shanghai Natural Science Foundation (Grant No.
09ZR1438800) and National Natural Science Foundation of China
(Grant No. 30901859).
4.2. Biology
4.2.1. In vitro cytotoxicity assay
Cytotoxic effects were examined in the CEM human leukemia,
MDA-MB-435 human breast carcinoma, and LOVO human colon car-
cinoma cell lines. Cells in logarithmic phase were diluted to a density
of 40000–50000 cells/mL in culture medium based on growth char-
Supplementary data
acteristics. For each well of a 96-well microplate, 100 lL of cell dilu-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.068.
tion was seeded, allowed to attach overnight, and then exposed to
varying concentrations (10ꢀ4–10ꢀ9 M) of compounds for 72 h
(37 °C, 5% CO2 atmosphere). The number of living cells was esti-
mated by the MTT assay. Absorbance at 570 nm was recorded on a
Wellscan MK-2 multifunction microplate reader (Labsystems Inc.).
Compounds were tested in triplicate in at least three independent
assays. The IC50 values were determined by a nonlinear regression
analysis. Average values were reported.
References and notes
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4.2.2. In vitro tubulin polymerization assay16–18
Bovine brain tubulin was purchased from cytoskeleton. Tubulin
(>99% pure, 3 mg/mL) in 100 lL of general tubulin buffer (80 mM
Na-pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, and 5%
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For flow cytometric analysis of DNA content, 106 LOVO cells
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cells in the different phases of the cell cycle.
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4.3. Molecular docking
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20. SYBYL, release version 6.9; Tripos Associates, St. Louis, Missouri.
All calculations were performed on an Origin 300 Server. The
X-ray crystal structure of the DAMA-colchicine-tubulin complex