Synthesis and biological evaluation of imidazol-2-one derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2007.11.048

A new series of aryl substituted imidazol-2-one derivatives structurally related to combretastatin A-4 (CA-4) were synthesized and evaluated for their cytotoxic activities in vitro against various human cancer cell lines including MDR cell line. The cytotoxic effects of compounds 7b and 7i proved to be similar to or greater than that of docetaxel. The highly active compound 7b also exhibited excellent inhibitory activity on tumor growth in vivo.

Full text of DOI:10.1016/j.bmc.2007.11.048

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2550–2557
Synthesis and biological evaluation of imidazol-2-one derivatives
as potential antitumor agents
Na Xue,a,c Xiaochun Yang,b Rui Wu,b Jing Chen,a Qiaojun He,b Bo Yang,b
Xiuyang Luc and Yongzhou Hua,*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
bInstitute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
cDepartment of Chemical and Biochemical Engineering, College of Material Sciences and Chemical Engineering, Zhejiang University,
Hangzhou 310027, China
Received 14 October 2007; revised 16 November 2007; accepted 17 November 2007
Available online 22 November 2007
Abstract—A new series of aryl substituted imidazol-2-one derivatives structurally related to combretastatin A-4 (CA-4) were syn-
thesized and evaluated for their cytotoxic activities in vitro against various human cancer cell lines including MDR cell line. The
cytotoxic effects of compounds 7b and 7i proved to be similar to or greater than that of docetaxel. The highly active compound
7b also exhibited excellent inhibitory activity on tumor growth in vivo.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
The structure–activity relationship demonstrated the cis
configuration of double bond and 3,4,5-trimethyloxy-
phenyl group are fundamental.11 The restricted rotation
of rings A and B of CA-4 can be maintained by intro-
ducing suitable conformationally restricted heterocycles
such as Imidazole,12 Isoxazole,13 and Indole14 (Fig. 1).
Many of them showed potent cytotoxicity against vari-
ous cancer cell lines as compared to CA-4. According
to the SAR, we designed and synthesized a series of
cis restricted analogues with imidazol-2-one instead of
the cis double bond in CA-4. In addition, we maintained
3,4,5-trimethoxyphenyl as ring A throughout the pres-
ent investigation and examined several variation of sub-
stituents on ring B.
Combretastatin A-4 (CA-4), a cis-stilbene natural prod-
uct isolated by Pettit et al. in 1989 from the South Afri-
can willow tree Combretum caffrum,1 strongly inhibits
tubulin polymerization by binding to the colchicine site.2
Recently, CA-4, which displayed its potent activity
against a broad spectrum of human cancer cells includ-
ing multidrug resistant cells (MDR),3 has drawn the
significant attention due to its potent and selective effect
on the established tumor vasculature.4 The vascular
disrupting properties of CA-4 and related compounds
represent a new approach to cancer therapy.5,6
A
water-soluble sodium phosphate prodrug (CA-4P) is
currently in phase II and III clinical trials on advanced
cancers based on the vascular shutdown mechanism
of action.7 Another CA-4 derivative AVE80628,9 is
currently under clinical evaluation as tumor vascular
targeting agent.5,10
OCH3
N
N
CH3
H3CO
H3CO
NH
H3CO
H3CO
H3CO
R
To date, many CA-4 analogues were synthesized and
their anticancer activities have been extensively studied.
H3CO OCH3 OCH3
OCH3
NH2
OCH3
OCH3
OH
OCH3
Indole
CA-4: R = OH
CA-4P: R = OPO3Na2
Imidazole
Keywords: Antitumor agents; Combretastatin A-4; Imidazol-2-one
derivatives; Synthesis; Cytotoxicity.
AVE8062: R = NHSer, HCl
*
Figure 1. Structures of combretastatin A-4 and selected analogues of
CA-4.
Corresponding author. Tel./fax: +86 571 88208460; e-mail: huyz@
zjuem.zju.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.11.048

N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
2551
2. Results and discussion
were separated and assigned on the basis of its IR spec-
tra, H NMR, and MS data. Similarly, compounds 4b
1
2.1. Chemistry
and 5b, 4c and 5c, 4d and 5d were achieved by reaction
of the corresponding 2-amino-1-phenylethanone hydro-
chlorides 3b–d with 3,4,5-trimethoxyphenyl isocyanate,
respectively. However, when 2-amino-1-(3-benzyloxy-4-
methoxyphenyl)ethanone hydrochloride 3e was reacted
with 3,4,5-trimethoxyphenyl isocyanate under the same
condition, only compound 5f was isolated in moderate
yield. Reduction of the nitro groups of 4d and 5d using
acetic acid and zinc powder yielded 4e and 5e, respec-
tively. Deprotection of the phenol function of 5f was
carried out by hydrogenation to afford 5g (Scheme 1).
To prepare the imidazol-2-one derivatives, we utilized our
recently developed synthetic protocol15 which was sum-
marized as follows. Bromination of substituted acetophe-
nones 1a–e gave substituted 2-bromo-1-phenylethanones
2a–e, which were treated with hexamethylenetetramine
and then hydrolyzed with concd HCl in EtOH to give
the corresponding 2-amino-1-phenylethanone hydro-
chlorides 3a–e (Scheme 1). Substituted 2-amino-1-phe-
nylethanone hydrochlorides 3f–j were prepared in the
same manner.
On the other hand, acetylation of substituted 2-amino-1-
phenylethanone hydrochlorides 3b–j with acetic
anhydride resulted in the corresponding N-(2-oxo-2-
phenylethyl)acetamides 6a–h16 which were then reacted
with 3,4,5-trimethoxyphenyl isocyanate to yield desired
1-acetyl-1,3-dihydro-3,4-diaryl-2H-imidazol-2-ones 7a–
h. Compound 7i was prepared by using the same
approach as that for 4e (Scheme 2).
Cyclization of 2-amino-1-(4-hydroxyphenyl)ethanone
hydrochloride 3a with 3,4,5-trimethoxyphenyl isocya-
nate in refluxing toluene provided an approximately
1:3 mixture of 1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-
4-(4-hydroxyphenyl)-2H-imidazol-2-one 4a and 2,3-
dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(4-hydroxy-
phenyl)-2-oxo-1H-imidazole-1-carboxamide 5a, which
O
O
O
c
H3CO
H3CO
H3CO
a
b
Br
HCl
NH2
3a-e
CH3
R
R
R
1a-e
2a-e
NCO
For 1, 2, 3
a
b
c
d
e
R = 4-OH
R = 4-OCH3
R = 3,4-(OCH3)2
R = 3-NO2-4-OCH3
R = 3-OBn-4-OCH3
OCH3
O
H3CO
O
O
H3CO
OCH3
NH
N
N
H
H3CO
H3CO
N
A
H3CO
H3CO
N
OCH3
R
B
R
5a R = 4-OH
5b R = 4-OCH3
5c R = 3,4-(OCH3)2
4a R = 4-OH
4b R = 4-OCH3
4c R = 3,4-(OCH3)2
5d R = 3-NO2-4-OCH3
5e R = 3-NH2-4-OCH3
5f R = 3-OBn-4-OCH3
5g R = 3-OH-4-OCH3
4d R = 3-NO2-4-OCH3
4e R = 3-NH2-4-OCH3
d
e
d
Scheme 1. Reagents and conditions: (a) pyridinium hydrobromide perbromide, THF, rt, 3 h; (b) i—hexamethylenetetramine, CHCl3, rt, 1 h; ii—
C2H5OH, HCl aq, rt, 1 h; (c) toluene, reflux, 4 h; (d) AcOH, Zn, rt, 2 h; (e) 10% Pd/C, H2, rt, 2 h.
O
O
H3CO
H3CO
H3CO
O
O
H
a
b
HCl
N
CH3
N
CH3
NH2
N
R
R
O
H3CO
R
H3CO
H3CO
NCO
7a R = H
3b R = 4-OCH3
6a R = H
3c R = 3,4-(OCH3)2
3d R = 3-NO2-4-OCH3
3f R = H
3g R = 4-Br
3h R = 4-Cl
7b R = 4-Br
7c R = 4-Cl
7d R = 4-NO2
7e R = 4-OCH3
7f R = 2,4-2F
6b R = 4-Br
6c R = 4-Cl
6d R = 4-NO2
6e R = 4-OCH3
6f R = 2,4-2F
7g R = 3,4-(OCH3)2
7h R = 3-NO2-4-OCH3
7i R = 3-NH2-4-OCH3
3i R = 4-NO2
3j R = 2,4-2F
6g R = 3,4-(OCH3)2
6h R = 3-NO2-4-OCH3
c
Scheme 2. Reagents and conditions: (a) (Ac)2O, H2O, NaOAc/H2O, 0 ꢁC to rt; (b) toluene, reflux, 4 h; (c) AcOH, Zn, rt, 2 h.

2552
N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
2.2. Biological activity
reports on CA-4 derivatives which suggest that 3-hydroxy
group on the B ring is not essential for cytotoxicity.7,17
The synthesized aryl substituted imidazol-2-ones 4a–e,
5a–e, 5g, 7a–i were tested for their cytotoxic activities
in vitro against several human cancer cell lines including
human myeoloid leukemia cells HL-60, human myeo-
loid leukemia cells K562 and K562R (multidrug resis-
tant cells), human prostate carcinoma cells PC-3,
human breast carcinoma cells MCF-7, human esopha-
geal carcinoma cells ECA-109, human hepatocarcinoma
cells BEL-7402, and human non-small lung cancer cells
A549. Docetaxel was employed as a positive control.
The results are summarized in Table 1.
Consistent with previous reports,18 increased cytotoxic
activity was observed when the nitro groups of 4d, 5d,
and 7h were reduced to the amine compounds 4e, 5e,
and 7i. Besides, a comparison between cytotoxicities
of 7e, 7g, 7h, and 7i revealed that the presence of an
amino group at the C-3 position on the B ring was
beneficial for potency especially against HL-60 and
K562 cells.
Compounds 7a–i with acetyl group at N-1 position of imi-
dazol-2-one ring showed more effective cytotoxic activi-
ties than those with hydrogen atom (4a–e) or
trimethoxyphenyl carbomide group (5a–e, 5g) in overall
activity, suggesting that acetyl group at N-1 position of
imidazol-2-one ring was favorable for cytotoxic activity.
It is worthy to pinpoint that the cytotoxicities of imida-
zol-2-ones showed more sensitivity against leukemia cells
HL-60 as compared to other cell lines over all activity.
As shown in Table 1, compounds 7b and 7i displayed
similar or more potent cytotoxic activities in comparison
with docetaxel. Compound 7b, with a 4-positioned bro-
mine atom on the B ring, exhibited the most potent cyto-
toxic activity, with IC50 values ranging from 0.2 to
10.6 lM against the tested cancer cell lines. However,
replacing 4-positioned bromine atom (7b) with chlorine
or fluorine atom on the B ring (e.g., 7c, 7f), they showed
a drastic loss of activities. Replacement of 4-bromine
atom with 4-methoxy group on the B ring, 7e, displayed
a decrease in the cytotoxic activity by one order of
magnitude.
To gain further insight into the mechanisms of action of
these compounds, the most cytotoxic compound 7b was
further assayed for its effect on cell cycle (by flow cytom-
etry). K562 cells were treated with 7b at different con-
centrations for 48 h. In 0.01 lM 7b treatment group,
31% cells were arrested in G2/M phase and 54% cells
were arrested in S phase, while in control group, 19%
and 1% cells were observed in G2/M phase and S phase,
respectively, (Fig. 2). At higher concentrations of
0.1 lM and 1 lM, compound 7b induced apoptosis in
K562 cells, the percentages of apoptotic cells were
45.6% and 78.6%, respectively.
Compound 5g with 3-hydroxy-4-methoxy groups on the
B ring, identical with B ring of CA-4, showed lower
activity than 5b with only 4-methoxy group on the B
ring. The result implied that the introduction of hydro-
xyl group at the C-3 position of B ring failed to improve
the cytotoxic activity profile in the tested compounds.
This is in agreement with observation in other literature
Table 1. In vitro cytotoxic activities of compounds 4a–e, 5a–e, 5g, 7a–i and docetaxel against eight human cancer cell lines
Compound
Cytotoxicity (IC50, lM)a,b
HL-60
K562
K562R
PC-3
MCF-7
ECA-109
BEL-7402
A549
4a
4b
20.0
19.8
38.8
14.3
10.3
6.3
>50
NDc
ND
ND
49.2
10.4
3.8
>50
>50
>50
>50
>50
>50
3.5
30.2
>50
>50
>50
49.8
3.0
>50
ND
ND
ND
>50
>50
>50
>50
ND
>50
ND
>50
10.6
ND
>50
>50
49.3
>50
>50
43.4
4.0
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3.8
>50
ND
ND
ND
>50
>50
>50
>50
ND
10.1
ND
>50
1.2
>50
>50
>50
>50
>50
2.5
4c
4d
4e
5a
5b
5c
5.4
6.5
49.7
>50
>50
10.0
>50
>50
3.1
>50
>50
>50
11.1
>50
>50
1.3
>50
7.2
>50
>50
9.4
5d
5e
19.7
3.1
5.8
5g
7a
15.4
5.6
>50
>50
1.0
>50
9.6
7b
7c
0.2
4.9
0.9
>50
26.6
0.2
ND
49.6
9.5
ND
32.9
19.7
34.3
>50
30.3
21.4
54.8
>50
>50
>50
>50
>50
30.8
>50
ND
ND
>50
9.5
>50
49.6
17.8
24.1
>50
40.9
1.1
7d
7e
7.2
5.5
7f
7g
7.6
>50
43.4
>50
0.5
>50
>50
>50
49.3
ND
36.2
>50
35.7
0.6
12.2
5.6
7h
7i
Doxetaxel
0.4
ND
8.5
ND
1.7
a IC50, compound concentration required to inhibit tumor cell proliferation by 50%.
b Values are means of three experiments.
c ND, not determined.

N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
2553
Figure 2. Effect of 7b on the cell cycle as determined by flow cytometry. K562 Cells were treated with compound 7b at the concentrations of 0 lM
(control, a), 0.01 lM (b), 0.1 lM (c), and 1 lM (d) for 48 h.
Based on the potent cytotoxic activities of the test com-
pounds in vitro, we chose compound 7b and cyclophos-
phamide (CTX) to investigate their growth inhibitory
activities on murine S-180 and murine H-22 tumor bear-
ing models. After daily administration with different
doses for 10 days, animals were sacrificed and the tu-
mors were excised and weighted. The results of experi-
mental therapeutic efficacy of 7b are shown in Tables
2, 3 and Figures 3, 4.
Compared to the control group, tumor weights of S-
180 in 15 mg/kg 7b group and 30 mg/kg CTX group re-
duced significantly (P < 0.01), and the inhibition rates
were 55.3% and 63.5%, respectively. In H-22 bearing
mice model, the inhibition rates of 20 mg/kg 7b and
30 mg/kg MTX groups were 55.5% (P < 0.01) and
36.2%, respectively. Therefore, comparing with CTX,
compound 7b presented a similar inhibiting effect on
murine S-180 tumor growth and more potent inhibiting
Table 2. Effect of compound 7b and CTX on S-180 tumor bearing mice
Group
Dosage (mg/kg/day)
Ad.
Initial/end
Body weight(g)
Tumor weight (g) X SD
Inhibition rate (%)
Mice (n)
NS
CTX
7b
—
30
15
—
ip
18/18
9/9
9/9
21.2/35.3
21.3/34.0
21.0/35.1
1.07 0.59
0.39 0.14a
0.48 0.19a
—
63.5
55.3
im
a p < 0.01 versus NS group.
Table 3. Effect of compound 7b and CTX on H-22 tumor bearing mice
Group
Dosage (mg/kg/day)
Ad.
Initial/end
Body weight (g)
Tumor weight (g) X SD
Inhibition rate (%)
Mice (n)
NS
CTX
7b
—
30
5
—
ip
18/18
9/9
9/9
18.5/26.7
18.3/26.3
18.2/26.8
18.7/27.6
0.86 0.55
0.55 0.23
0.61 0.41
0.38 0.45a
—
36.2
29.4
55.5
im
im
7b
20
9/9
a p < 0.01 versus NS group.

2554
N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
ESI (positive) were recorded on an Esquire-LC-00075
spectrometer.
4.1. Synthesis
4.1.1. 1,3-Dihydro-3-(3,4,5-trimethoxyphenyl)-4-(4-hydro-
xyphenyl)-2H-imidazol-2-one (4a) and 2,3-Dihydro-N,3-
bis(3,4,5-trimethoxyphenyl)-4-(4-hydroxyphenyl)-2-oxo-
1H-imidazole-1-carboxamide (5a). A mixture of 2-amino-
1-(4-hydroxyphenyl)ethanone hydrochloride 3a (1.5 mmol)
and 3,4,5-trimethoxyphenyl isocyanate (1.65 mmol) in
dry toluene (10 mL) was refluxed for 5 h. After cooling
to room temperature, the reaction mixture was diluted
with water (10 mL) and extracted thoroughly with
CH2Cl2 (3 · 20 mL). Then the CH2Cl2 layer was washed
successively with brine (2 · 20 mL), dried over anhy-
drous Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by silica gel column
chromatography (EtOAc/petroleum ether, 1:1), yielding
pure compounds 4a and 5a. Lower Rf compound 4a,
white solid (0.132 g, 23% yield), mp 207–209 ꢁC; IR
(KBr): 3369, 3272, 3163, 2939, 2839, 1782, 1616, 1572,
Figure 3. Antitumor effect of compound 7b and CTX on S-180 tumor
bearing mice.
1
1513, 1458, 1422 cmÀ1; H NMR (400 MHz, CDCl3) d
3.84 (s, 3H, OCH3), 3.88 (s, 6H, OCH3 · 2), 5.22 (s,
1H, OH), 6.85 (s, 2H, Ar–H), 6.90 (d, J = 8.4 Hz, 2H,
Ar–H), 7.39 (s, 1H, imidazolone H), 7.45 (d,
J = 8.4 Hz, 2H, Ar–H), 9.89 (s, 1H, N–H); MS (ESI):
m/s = 385 [M+1]. Higher Rf compound 5a, white solid
(0.504 g, 61% yield), mp 186–188 ꢁC; IR (KBr): 3306,
3146, 3089, 2935, 2841, 1730, 1680, 1608, 1564, 1507,
Figure 4. Antitumor effect of compound 7b and CTX on H-22 tumor
bearing mice.
1
1458, 1416 cmÀ1; H NMR (400 MHz, CDCl3) d 3.72
(s, 6H, OCH3 · 2), 3.82 (s, 3H, OCH3), 3.84 (s, 9H,
OCH3 · 3), 5.52 (s, 1H, OH), 6.42 (s, 2H, Ar–H), 6.73
(d, J = 8.8 Hz, 2H, Ar–H), 6.88 (s, 2H, Ar–H), 7.00 (d,
J = 8.8 Hz, 2H, Ar–H), 7.17 (s, 1H, imidazolone H),
10.87 (s, 1H, –O@C–N–H–); MS (ESI): m/s = 552
[M+1].
effect on murine H-22 tumor growth (Tables 2, 3 and
Figs. 3, 4).
3. Conclusions
We have synthesized a series of aryl substituted imida-
zol-2-one derivatives and evaluated their biological
activities. Some of these compounds exhibited potent
cytotoxic activities against the tested cancer cell lines
including MDR cancer cell line in vitro. Compounds
7b and 7i displayed comparable cytotoxic activities
with that of docetaxel against the tested human cancer
cell lines. Cell cycle distribution analysis showed that
7b acted on the S phase of the cell cycle arrest at
0.01 lM and induced cell apoptosis at 0.1–1.0 lM.
Compound 7b also showed significant anticancer activ-
ity in two murine tumor bearing models in vivo. Fur-
ther studies of the mechanism of action for 7b are
underway.
In the same manner, compounds 4b and 5b, 4c and 5c, 4d
and 5d, 5f were synthesized by reaction of the correspond-
ing 2-amino-1-phenylethanone hydrochlorides 3b–e with
3,4,5-trimethoxyphenyl isocyanate, respectively.
4.1.2.
1,3-Dihydro-3-(3,4,5-trimethoxyphenyl)-4-(4-
methoxyphenyl)-2H-imidazol-2-one (4b) and 2,3-dihy-
dro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(4-methoxyphenyl)-
2-oxo-1H-imidazole-1-carboxamide (5b). Lower Rf com-
pound 4b, white solid (24% yield), mp 170–172 ꢁC; IR
(KBr): 3272, 3146, 2925, 2855, 1751, 1615, 1567, 1507,
1
1456, 1417 cmÀ1; H NMR (400 MHz, CDCl3) d 3.84
(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.89 (s, 6H,
OCH3 · 2), 6.85 (s, 2H, Ar–H), 6.96 (d, J = 8.8 Hz,
2H, Ar–H), 7.40 (s, 1H, imidazolone H), 7.49 (d, 2H,
J = 8.8 Hz, Ar–H), 9.89 (s, 1H, N–H); MS (ESI): m/
s = 399 [M+1]. Higher Rf compound 5b, white solid
(59% yield), mp 147–149 ꢁC; IR (KBr): 3145, 3087,
2939, 2837, 1730, 1680, 1607, 1563, 1507, 1458,
4. Experimental
Melting points were obtained on a B-540 Buchi melting
point apparatus and are uncorrected. IR spectra, KBr
pellets, 400–4000 cmÀ1, were recorded on a Bruker
1
1416 cmÀ1; H NMR (400 MHz, CDCl3) d 3.79 (s, 6H,
1
VECTOR 22 FTIR spectrophotometer. H NMR spec-
OCH3 · 2), 3.84 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
3.90 (s, 3H, OCH3), 3.91 (s, 6H, OCH3 · 2), 6.49 (s,
2H, Ar–H), 6.86 (d, J = 8.8 Hz, 2H, Ar–H), 6.94 (s,
2H, Ar–H), 7.12 (d, J = 8.8 Hz, 2H, Ar–H), 7.24 (s,
tra was recorded on a Bruker AM 400 instrument at
400 MHz (chemical shifts are expressed as d values rela-
tive to TMS as internal standard). Mass spectra (MS),

N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
2555
1H, imidazolone H), 10.93 (s, 1H, –O@C–N–H–); MS
(ESI): m/s = 569 [M+1].
OCH3), 3.88 (s, 6H, OCH3 · 2), 3.89 (s, 3H, OCH3),
6.81 (d, J = 8.8 Hz, 1H, Ar–H), 6.85 (s, 2H, Ar–H),
6.86 (d, J = 1.6 Hz, 1H, Ar–H), 6.94 (dd, J = 8.8,
1.6 Hz, 1H, Ar–H), 7.34 (s, 1H, imidazolone H), 9.90
(s, 1H, N–H); MS (ESI): m/s = 372 [M+1].
4.1.3.
1,3-Dihydro-3-(3,4,5-trimethoxyphenyl)-4-(3,4-
dimethoxyphenyl)-2H-imidazol-2-one (4c) and 2,3-dihy-
dro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(3,4-dimethoxy-
phenyl)-2-oxo-1H-imidazole-1-carboxamide (5c). Lower
Rf compound 4c, pale yellow solid (21% yield), mp
174–176 ꢁC; IR (KBr): 3280, 3142, 2942, 2838, 1773,
4.1.6. 2,3-Dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(3-
amino-4-methoxyphenyl)-2-oxo-1H-imidazole-1-carbox-
amide (5e). The same procedure as described above was
performed with 5d and gave pure compound 5e as a
white solid (75% yield), mp 179–181 ꢁC; IR (KBr):
3370, 3160, 3093, 2937, 2838, 1727, 1686, 1608, 1569,
1604, 1557, 1511, 1456, 1417 cmÀ1
;
1H NMR
(400 MHz, CDCl3) d 3.84 (s, 3H, OCH3), 3.89 (s, 6H,
OCH3 · 2), 3.92 (s, 3H, OCH3), 3.94 (s, 3H, OCH3),
6.85 (s, 2H, Ar–H), 6.92 (d, J = 8.8 Hz, 1H, Ar–H),
7.02 (d, J = 2.4 Hz, 1H, Ar–H), 7.13 (dd, 1H,J = 8.8,
2.4 Hz, Ar–H), 7.42 (s, 1H, imidazolone H), 9.87 (s,
1H, N–H); MS (ESI): m/s = 387 [M+1]. Higher Rf com-
pound 5c, pale yellow solid (58% yield), mp 178–
180 ꢁC; IR (KBr): 3144, 3091, 2943, 2836, 1729, 1680,
1
1509, 1457, 1417 cmÀ1; H NMR (400 MHz, CDCl3) d
3.80 (s, 6H, OCH3 · 2), 3.87 (s, 6H, OCH3 · 2), 3.90 (s,
9H, OCH3 · 3), 6.49 (m, 3H, Ar–H), 6.58 (d,
J = 1.6 Hz, 1H, Ar–H), 6.69 (d, 1H, J = 8.4 Hz, Ar–H),
6.94 (s, 2H, Ar–H), 7.19 (s, 1H, imidazolone H), 10.94
(s, 1H, –O@C–N–H–); MS (ESI): m/s = 581 [M+1].
1608, 1509, 1459, 1417 cmÀ1
CDCl3)
.
1H NMR (400 MHz,
3.64 (s, 3H, OCH3), 3.72 (s, 6H,
d
4.1.7. 2,3-Dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(3-
benzyloxy-4-methoxyphenyl)-2-oxo-1H-imidazole-1-car-
boxamide (5f). Light yellow solid (48% yield), mp 159–
161 ꢁC; 1H NMR (400 MHz, CDCl3) d 3.81 (s, 6H,
OCH3 · 2), 3.94 (m, 15H, OCH3 · 5), 5.00 (s, 2H,
CH2), 6.50 (s, 2H, Ar–H), 6.70 (s, 1H, Ar–H), 6.82 (d,
J = 8.4 Hz, 1H, Ar–H), 6.88 (d, J = 8.4 Hz, 1H, Ar–
H), 6.96 (s, 2H, Ar–H), 7.23 (s, 1H, imidazolone H),
7.40 (m, 5H, Ar–H), 10.94 (s, 1H, –O@C–N–H–); MS
(ESI): m/s = 672 [M+1].
OCH3 · 2), 3.80 (s, 3H, OCH3), 3.83 (s, 3H, OCH3),
3.84 (s, 9H, OCH3 · 3), 6.46 (s, 2H, Ar–H), 6.59 (s,
1H, imidazolone H), 6.73 (dd, J = 8.0, 2.0 Hz, 2H,
Ar–H), 6.87 (s, 2H, Ar–H), 7.22 (d, J = 8.0 Hz, 1H,
Ar–H), 10.85 (s, 1H, –O@C–N–H–); MS (ESI): m/s =
596 [M+1].
4.1.4. 1,3-Dihydro-3-(3,4,5-trimethoxyphenyl)-4-(3-nitro-
4-methoxyphenyl)-2H-imidazol-2-one (4d) and 2,3-dihy-
dro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(3-nitro-4-methoxy-
phenyl)-2-oxo-1H-imidazole-1-carboxamide (5d). Lower
Rf compound 4d, yellow solid (20% yield), mp 199–
201 ꢁC; IR (KBr): 3286, 3153, 2930, 2848, 1768, 1612,
4.1.8. 2,3-Dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-4-(3-
hydroxy-4-methoxyphenyl)-2-oxo-1H-imidazole-1-car-
boxamide (5g). A mixture of 5f (0.2 mmol) and 10% Pd/
C (20 mg) in THF (5 mL) was stirred at room tempera-
ture under H2 for 12 h. Then the mixture was filtered,
and the filtrate was evaporated to dryness. The residue
was purified by silica gel column chromatography
(EtOAc/petroleum ether, 2:1) to give pure compound
5g as a white solid (45 mg, 39%), mp 175–177 ꢁC; IR
(KBr): 3396, 3160, 3092, 2935, 2841, 1722, 1680, 1605,
1
1547, 1456, 1417 cmÀ1; H NMR (400 MHz, CDCl3) d
3.88 (s, 6H, OCH3 · 2), 3.83 (s, 3H, OCH3), 4.01 (s,
3H, OCH3), 6.84 (s, 2H, Ar–H), 7.18 (d, J = 8.8 Hz,
1H, Ar–H), 7.54 (s, 1H, imidazolone H), 7.71 (dd,
J = 8.8, 2.4 Hz, 1H, Ar–H), 8.04 (d, J = 2.4 Hz, 1H,
Ar–H), 9.80 (s, 1H, N–H); MS (ESI): m/s = 402
[M+1]. Higher Rf compound 5d, yellow solid (61%
yield), mp 162–164 ꢁC; IR (KBr): 3153, 3089, 2930,
1563, 1511, 1457, 1417 cmÀ1 1H NMR (400 MHz,
;
2847, 1735, 1693, 1609, 1567, 1507, 1458, 1416 cmÀ1
;
1H NMR (400 MHz, CDCl3)
d
3.75 (s, 6H,
CDCl3) d 3.79 (s, 6H, OCH3 · 2), 3.81 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 3.89 (s, 6H, OCH3 · 2), 3.90 (s,
3H, OCH3), 5.72 (s, 1H, OH), 6.57 (s, 2H, Ar–H),
6.79 (m, 2H, Ar–H), 6.87 (m, 2H, Ar–H), 6.93 (s, 1H,
Ar–H), 7.29 (s, 1H, imidazolone H), 10.40 (s, 1H,
–O@C–N–H–); MS (ESI): m/s = 582 [M+1].
OCH3 · 2), 3.80 (s, 3H, OCH3), 3.83 (s, 6H,
OCH3 · 2), 3.84 (s, 3H, OCH3), 3.92 (s, 3H, OCH3),
6.44 (s, 2H, Ar–H), 6.85 (s, 2H, Ar–H), 6.96 (d,
J = 8.8 Hz, 1H, Ar–H), 7.22 (dd, J = 8.8, 1.6 Hz, 1H,
Ar–H), 7.29 (s, 1H, imidazolone H), 7.69 (d,
J = 1.6 Hz, 1H, Ar–H), 10.75 (s, 1H, –O@C–N–H–);
MS (ESI): m/s = 611 [M+1].
4.2. General procedure for the preparation of imidazol-2-
ones 7a–h
4.1.5. 1,3-Dihydro-3-(3,4,5-trimethoxyphenyl)-4-(3-ami-
no-4-methoxyphenyl)-2H-imidazol-2-one (4e). To a solu-
tion of nitro compound 4d (0.2 mmol) in AcOH
(4 mL) was added zinc powder (0.5 g). The reaction mix-
ture was stirred at room temperature for 2 h. The mix-
ture was filtered over Celite, and the filtrate was
evaporated to dryness. The residue was purified by silica
gel column chromatography (EtOAc/petroleum ether,
1:1), yielding pure compound 4e as a white solid
(60 mg, 80%), mp 175–177 ꢁC; IR (KBr): 3378, 3286,
3160, 2923, 2853, 1762, 1612, 1564, 1508, 1458,
A mixture of substituted N-(2-oxo-2-phenylethyl)acet-
amide (1.5 mmol) and 3,4,5-trimethoxyphenyl isocya-
nate (1.65 mmol) in dry toluene (10 mL) was refluxed
for 5 h. After cooling to room temperature, the reaction
mixture was diluted with water (10 mL) and extracted
thoroughly with CH2Cl2 (20 mL · 3). Then the CH2Cl2
layer was washed successively with brine (20 mL · 2),
dried over anhydrous Na2SO4, and concentrated under
reduced pressure. Usual workup afforded the crude
product which was further purified by a silica gel col-
umn chromatography.
1
1417 cmÀ1; H NMR (400 MHz, CDCl3) d 3.82 (s, 3H,

2556
N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
4.2.1. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
phenyl-2H-imidazol-2-one (7a). Yellow solid (78% yield),
mp 177–179 ꢁC; IR (KBr): 3144, 2936, 2836, 1720, 1594,
3.67 (s, 3H, OCH3), 3.76 (s, 6H, OCH3 · 2), 3.86 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 6.47 (s, 2H, Ar–H),
6.62 (s, 1H, Ar–H), 6.77 (m, 2H, Ar–H), 7.17 (s, 1H, imi-
dazolone H); MS (ESI): m/s @ 429 [M+1].
1
1506, 1458, 1419 cmÀ1; H NMR (400 MHz, CDCl3) d
2.74 (s, 3H, O@C–CH3), 3.70 (s, 6H, OCH3 · 2), 3.84
(s, 3H, OCH3), 6.40 (s, 2H, Ar–H), 7.14 (m, 2H, Ar–
H), 7.20 (s, 1H, imidazolone H), 7.28 (m, 3H, Ar–H);
MS (ESI): m/s = 369 [M+1].
4.2.8. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(3-nitro-4-methoxyphenyl)-2H-imidazol-2-one (7h). Yel-
low solid (75% yield), mp 231–233 ꢁC; IR (KBr): 3137,
2935, 2841, 1718, 1594, 1530, 1511, 1458, 1416 cmÀ1
;
4.2.2. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(4-bromophenyl)-2H-imidazol-2-one (7b). White solid
(86% yield), mp 175–177 ꢁC; IR (KBr): 3144, 2935,
1H NMR (400 MHz, CDCl3) d 2.80 (s, 3H, O@C–
CH3), 3.83 (s, 6H, OCH3 · 2), 3.93 (s, 3H, OCH3),
4.02 (s, 3H, OCH3), 6.50 (s, 2H, Ar–H), 7.04 (d,
J = 8.0 Hz, 1H, Ar–H), 7.27 (d, J = 8.0 Hz, 1H, Ar–
H), 7.31 (s, 1H, imidazolone H, Ar–H), 7.80 (s, 1H,
Ar–H); MS (ESI): m/s = 444 [M+1].
2841, 1718, 1594, 1557, 1507, 1458, 1416 cmÀ1 1H
;
NMR (400 MHz, CDCl3) d 2.72 (s, 3H, O@C–CH3),
3.72 (s, 6H, OCH3 · 2), 3.79 (s, 3H, OCH3), 6.39 (s,
2H, Ar–H), 7.00 (d, J = 8.8 Hz, 2H, Ar–H), 7.20 (s,
1H, imidazolone H), 7.40 (d, J = 8.8 Hz, 2H, Ar–H);
MS (ESI): m/s = 448 [M+1].
4.2.9. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(3-amino-4-methoxyphenyl)-2H-imidazol-2-one (7i). The
same procedure as described above was performed with
7h and gave pure compound 7i as a white solid (78%
yield), mp 230–232 ꢁC; IR (KBr): 3377, 3131, 2946,
4.2.3. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(4-chlorophenyl)-2H-imidazol-2-one (7c). White solid
(76% yield), mp 147–149 ꢁC; IR (KBr): 3144, 2937,
2838, 1719, 1602, 1557, 1510, 1457, 1416 cmÀ1 1H
;
2841, 1725, 1596, 1503, 1459, 1419 cmÀ1
;
1H NMR
NMR (400 MHz, CDCl3) d 2.68 (s, 3H, O@C–CH3),
3.69 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 6.39 (s, 2H, Ar–H), 6.49
(d, J = 8.4 Hz, 1H, Ar–H), 6.60 (d, J = 8.4 Hz, 1H,
Ar–H), 6.77 (s, 1H, Ar–H), 7.03 (s, 1H, imidazolone
H); MS (ESI): m/s = 414 [M+1].
(400 MHz, CDCl3) d 2.72 (s, 3H, O@C–CH3), 3.72 (s,
6H, OCH3 · 2), 3.85 (s, 3H, OCH3), 6.39 (s, 2H, Ar–
H), 7.06 (d, J = 8.8 Hz, 2H, Ar–H), 7.20 (s, 1H, imidazo-
lone H), 7.26 (m, 2H, Ar–H); MS (ESI): m/s = 403
[M+1].
4.2.4. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(4-nitrophenyl)-2H-imidazol-2-one (7d). Yellow solid
(73% yield), mp 107–109 ꢁC; IR (KBr): 3141, 2925,
4.3. Biology
The human tumor cell lines (HL-60, K562, K562R, PC-
3, MCF-7, ECA-109, BEL-7402, and A549) were ob-
tained from Shanghai Institute of Pharmaceutical
Industry.
2852, 1730, 1598, 1514, 1456, 1416 cmÀ1 1H NMR
;
(400 MHz, CDCl3) d 2.75 (s, 3H, O@C–CH3), 3.75 (s,
6H, OCH3 · 2), 3.88 (s, 3H, OCH3), 6.43 (s, 2H, Ar–
H), 7.32 (d, J = 8.8 Hz, 2H, Ar–H), 7.40 (s, 1H, imidazo-
lone H), 8.15 (d, J = 8.8 Hz, 2H, Ar–H); MS (ESI):
m/s = 414 [M+1].
4.3.1. Cytotoxicity assay. The cytotoxic activity in vitro
was measured using the MTT assay.19 MTT solution
(10.0 ll/well) in RPMI-1640 (Sigma, St. Louis, MO)
was added after cells were treated with drug for 48 h,
and cells were incubated for a further 4 h at 37 ꢁC.
The purple formazan crystals were dissolved in
100.0 ll DMSO. After 5 min, the plates were read on
an automated microplate spectrophotometer (Bio-Tek
Instruments, Winooski, VT) at 570 nm. Assays were
performed in triplicate in three independent experi-
ments. The concentration required for 50% inhibition
of cell viability (IC50) was calculated using the software
‘Dose–Effect Analysis with Microcomputers’. The
tumor cell line panel consisted of HL-60, K562,
K562R, PC-3, MCF-7, ECA-109, BEL-7402, and
A549. In all of these experiments, three replicate wells
were used to determine each point.
4.2.5. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(4-methoxyphenyl)-2H-imidazol-2-one (7e). White solid
(80% yield), mp 177–179 ꢁC; IR (KBr): 3186, 2935,
2841, 1729, 1601, 1509, 1458, 1417 cmÀ1 1H NMR
;
(400 MHz, CDCl3) d 2.71 (s, 3H, O@C–CH3), 3.71 (s,
6H, OCH3 · 2), 3.78 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 6.40 (s, 2H, Ar–H), 6.79 (d, J = 8.8 Hz, 2H,
Ar–H), 7.05 (d, J = 8.8 Hz, 2H, Ar–H), 7.10 (s, 1H, imi-
dazolone H); MS (ESI): m/s = 390 [M+1].
4.2.6. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(2,4-difluorophenyl)-2H-imidazol-2-one (7f). White solid
(77% yield), mp 153–154 ꢁC; IR (KBr): 3177, 2942,
2841, 1723, 1598, 1505, 1456, 1428 cmÀ1 1H NMR
;
(400 MHz, CDCl3) d 2.73 (s, 3H, O@C–CH3), 3.71 (s,
6H, OCH3 · 2), 3.82 (s, 3H, OCH3), 6.37 (s, 2H, Ar–
H), 6.80 (m, 2H, Ar–H), 7.23 (s, 1H, imidazolone H),
7.54 (s, 1H, Ar–H); MS (ESI): m/s = 405 [M+1].
4.3.2. Antitumor activity in S-180 and H-22 tumor
bearing mice. Tumor cells of S-180 and H-22 were inoc-
ulated to mice. After 10 days, tumors were taken out
and cells were harvested. Viable tumor cells (2.5 · 106
cells per mouse) were inoculated to the armpit of mice
by subcutaneous injection and were divided into several
groups: negative control group containing 18 female
mice, treatment groups containing 9 female mice.
Twenty-four hours later, treatment groups were admin-
4.2.7. 1-Acetyl-1,3-dihydro-3-(3,4,5-trimethoxyphenyl)-4-
(3,4-dimethoxyphenyl)-2H-imidazol-2-one (7g). Pale yel-
low solid (78% yield), mp 174–176 ꢁC; IR (KBr): 3144,
2934, 2841, 1719, 1597, 1513, 1460, 1419 cmÀ1 1H
;
NMR (400 MHz, CDCl3) d 2.75 (s, 3H, O@C–CH3),

N. Xue et al. / Bioorg. Med. Chem. 16 (2008) 2550–2557
2557
5. Tozer, G. M.; Kantholl, C.; Baguley, B. C. Nat. Rev.
Cancer 2005, 5, 423.
6. Griggs, J.; Metcalfe, J. C.; Hesketh, R. Lancet Oncol.
2001, 2, 82.
7. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.;
Genazzani, A. A. J. Med. Chem. 2006, 49, 3033.
8. Hori, K.; Saito, S. Br. J. Cancer 2003, 89, 1334.
9. Guffroy, M.; Dally, C.; Vrignaud, P.; Beys, E.; Bissery, M.
C. Proc. Am. Assoc. Cancer Res. 2004, 45, 5438 (abstract).
10. Kelland, L. R. Curr. Cancer Ther. Rev. 2005, 1, 1.
11. Tron, G. C.; Pagliai, F.; Del Grosso, E.; Genazzani, A. A.;
Sorba, G. J. Med. Chem. 2005, 48, 3260.
istered with various doses of 7b (im) or CTX (ip), respec-
tively, once a day for consecutive 10 days. CTX at
30.0 mg/kg was used as a positive control and physiolog-
ical saline as negative control. Tumors were dissected
and weighed, and inhibition rates were calculated on
day 10. The inhibition rate was calculated as follows:
C À T/C · 100, T, average tumor weight of treated
group; C, average tumor weight of negative control
group.
4.3.3. Flow cytometry analysis of apoptosis. For flow
cytometry analysis of DNA content, K562 cells in expo-
nential growth were treated with graded concentrations
of 7b (0.01–1 lM) for 48 h. Cells were washed twice with
PBS and fixed in 70% ethanol at À20 ꢁC. The cell pellet
was resuspended in 100.0 ll of PBS containing 50.0 mg/
ml RNase (Amersco, Solon, OH), then incubated at
37 ꢁC for 1 h. After incubation, the cells were stained
with 200.0 mg/ml propidium iodide (PI, Sigma, St.
Louis, MO) at 4 ꢁC for 30 min. The fluorescence of
2 · 104 cell was measured with FACSCalibur (Becton–
Dickinson, Lincoln Park, NJ).20
12. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey,
R. W.; Hannick, S. M.; Gherke, L.; Gredo Bruce, R.; Hui,
Y. H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielonski-
Mozng, N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J.
Med. Chem. 2002, 45, 1697.
13. Sun, C. M.; Lin, L. G.; Yu, H. J.; Cheng, C. Y.; Tsai,
Y. C.; Chu, C. W.; Din, Y. H.; Chau, Y. P.; Don, M.
J. Bioorg. Med. Chem. Lett. 2007, 17, 1078.
14. Flynn, B. L.; Hamel, E.; Jung, M. K. J. Med. Chem. 2002,
45, 2670.
15. Cheng, Y. F.; Hu, Y. Z. Chin. Chem. Lett. 2004, 15, 1281.
16. Abdalla, G. M.; Soweell, J. W., Sr. J. Heterocycl. Chem.
1987, 24, 297.
17. (a) Cushman, M.; Nagarathnam, D.; Gopal, D.; Chak-
raborti, A. K.; Lin, C. M.; Hamel, E. J. Med. Chem. 1991,
34, 2579; (b) Cushman, M.; Nagarathnam, D.; Gopal, D.;
He, H. M.; Lin, C. M.; Hamel, E. J. Med. Chem. 1992, 35,
2293.
18. LeBlanc, R.; Dickson, J.; Brown, T.; Stewart, M.; Pati, H.
N.; VanDerveer, D.; Arman, H.; Harris, J.; Pennington,
W., ; Holt, H. L., Jr.; Lee, M. Bioorg. Med. Chem. Lett.
2005, 13, 6025.
References and notes
1. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts,
D. S.; Garcia-Kendall, D. Experientia 1989, 45, 209.
2. Lin, C. M.; Singh, S. R.; Chu, P. S.; Dempcy, R. O.;
Schmidt, J. M.; Pettit, G. R.; Hamel, E. Mol. Phamacol.
1988, 34, 200.
3. McGown, A. T.; Fox, B. W. Cancer Chemother. Pharma-
col. 1990, 26, 79.
4. Chaplin, D. J.; Pettit, G. R.; Hill, S. A. Anticancer Res.
1999, 19, 189.
19. Carmichael, J.; DeGraff, W. G.; Gadzar, A. F.; Minna, J.
D.; Mitchell, J. B. Cancer Res. 1987, 47, 936.
20. Yang, B.; Reynolds, C. P. Clin. Cancer Res. 2005, 11,
2774.