Design, synthesis and CoMFA studies of N1-amino acid substituted 2,4,5-triphenyl imidazoline derivatives as p53-MDM2 binding inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.01.003

A series of novel N1-amino-acid substituted 2,4,5-triphenyl imidazoline derivatives was designed and synthesized based on our previous studies. All synthesized target compounds were screened for their p53-MDM2 binding inhibitory activities and anti-proliferative activities against five cancer cell lines. Among them, twelve compounds displayed improved binding inhibitory activities and most compounds showed higher cell growth inhibition activities with IC ₅₀ values in the low micromolar range. Compound 6c exhibited marked p53-MDM2 binding inhibitory activity (IC₅₀ = 0.59 μM) which was eightfold more potent than that of Nutlin-1 (IC₅₀ = 4.78 μM). CoMFA analysis was performed based on obtained biological data and resulted in a statistically significant CoMFA model with high predict abilities (q ² = 0.645, r² = 0.979).

Full text of DOI:10.1016/j.bmc.2012.01.003

Bioorganic & Medicinal Chemistry 20 (2012) 1417–1424
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and CoMFA studies of N1-amino acid substituted 2,4,5-triph-
enyl imidazoline derivatives as p53–MDM2 binding inhibitors
Chunqi Hu a, Xiaoxue Dou a, Yizhe Wu a, Lei Zhang b, Yongzhou Hu a,
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, 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 N1-amino-acid substituted 2,4,5-triphenyl imidazoline derivatives was designed and
synthesized based on our previous studies. All synthesized target compounds were screened for their
p53–MDM2 binding inhibitory activities and anti-proliferative activities against five cancer cell lines.
Among them, twelve compounds displayed improved binding inhibitory activities and most compounds
showed higher cell growth inhibition activities with IC50 values in the low micromolar range. Compound
Received 7 December 2011
Revised 31 December 2011
Accepted 2 January 2012
Available online 9 January 2012
6c exhibited marked p53–MDM2 binding inhibitory activity (IC50 = 0.59
potent than that of Nutlin-1 (IC50 = 4.78 M). CoMFA analysis was performed based on obtained biolog-
ical data and resulted in a statistically significant CoMFA model with high predict abilities (q2 = 0.645,
2 = 0.979).
lM) which was eightfold more
Keywords:
l
p53–MDM2 binding inhibitors
2,4,5-Triphenyl imidazoline
Anti-proliferative activities
CoMFA
r
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
were essential for the potency, and the N-1 substituents displayed a
very important role in remaining the potencies as well.
The tumor suppressor p53 is a potent transcription factor that
controls a major pathway protecting cells from malignant transfor-
mation, and plays a pivotal role in blocking tumor genesis and
development.1,2 The p53 tumor suppressor is controlled by the
murine double minute 23 (MDM2, also frequently referred to as
HDM2 in human), which binds to p53 and negatively regulates
its transcriptional activity and stability.4 In unstressed cells, pro-
tein levels of p53 are tightly controlled by MDM2 protein through
a negative feedback loop.5 Since more types of cancers are tolerant
to elevated levels of wild-type p53,6 the reactivation of p53 by
antagonizing p53–MDM2 interaction offers a new therapeutic
strategy for cancer therapy.
Hence, we designed and synthesized a series of novel Nutlin
analogs, utilizing amino acids, amino-acid esters or amino-acid
amides as the N-1 substituents to discover new p53–MDM2 bind-
ing inhibitors with enhanced p53–MDM2 binding inhibitory activ-
ities and anti-proliferative potencies. Then CoMFA study13 which
was carried out based on the obtained data helped in understand-
ing the interaction between the ligand and the receptor.
2. Results and discussion
2.1. Chemistry
Series of compounds have been developed as p53–MDM2-bind-
ing inhibitors in recent decades.6–12 Nultins were the first potent
and selective p53–MDM2 interaction inhibitors that were discov-
ered through high throughput screening. And Nutlin-39 was one
of the most potent molecules of Nutlins that demonstrated prom-
ising bioactivities in vitro and in vivo.
In our previous study, three new series of imidazoline derivatives
were designed and synthesized using Nultins as lead compounds.12
The imidazoline scaffold of Nutlins was retained as the core struc-
ture with a focus on modifying the phenyl groups on the imidazo-
line ring and the N-1 side chain of Nutlins. Based on the obtained
data, a preliminary SAR was disclosed that the three phenyl groups
The synthetic routes for the imidazoline derivatives are summa-
rized in Scheme 1. Triphenyl imidazoline 5 was obtained according
to previous studies.12 Then, N-formylation reaction of 5 was per-
formed using bis(trichloromethyl)carbonate in dichloromethane
at the presence of triethylamine. The obtained formylchloride
was reacted with corresponding amino-acid ethyl esters to give
6a–e, whose ester groups were further hydrolyzed by 6 N HCl to
form acids 7a–e.14 Amidation of 7a–e with secondary amines in
the presence of DCC-HOBt gave amides 8a–m.
2.2. Biological activity
All synthesized compounds were evaluated for their p53–
MDM2 binding inhibitory activities by fluorescence-polarization
(FP)-based binding assay14 and anti-proliferation activities by
⇑
Corresponding author. Tel./fax: +86 57188208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.003

1418
C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
O
H
O
H
O
H
O
O
OH
Cl
OH
O
6a X=CH2
b
a
O
O
O
6b X=(CH2)2
6c X=(r)CH(CH3)
f
e
O
OH
O
2
NH
N
N
N
X
N
H
O
X
1
g
H2N
COOEt
Cl
Cl
Cl
O
O
H
6d X=(s)CH(CH3)
d
6e X=(CH2)3
6
c
Cl
Cl
5
Cl
Cl
N
NH
O
H2N
NH2
Cl
Cl
h
4
3
Cl
O
O
N
8g X=(CH2)2
R=
R=
N
N
N
N
O
8a X=CH2 R=
8b X=CH2 R=
8c X=CH2 R=
8d X=CH2 R=
8e X=CH2 R=
8f X=(CH2)2 R=
7a X=CH2
N
N
N
N
N
8h X= (CH2)2
7b X=(CH2)2
7c X=(r)CH(CH3)
O
i
O
O
8i X=(s)CH(CH3) R=
8j X=(s)CH(CH3) R=
8k X=(r)CH(CH3) R=
O
N
N
X
N
N
N
X
COOH
N
H
7d X=(s)CH(CH )
N
3
COR
O
H
N
N
N
N
O
7e X=(CH2)3
8l X=(CH2)3
R=
N
Cl
Cl
Cl
Cl
7
8
Scheme 1. Reagents and conditions: (a) (CH3)2SO4, K2CO3, acetone, reflux, 3 h; (b) i-PrBr, K2CO3, acetone, 40 °C, 5 h; (c) CH3COONH4, reflux, 3 h; (d) H2SO4, 120 °C, 3 h; (e)
NBS, CH2Cl2, rt, 24 h; (f) BTC, CH2Cl2, TEA; (g) H2NXCOOEt, TEA, CH2Cl2; h.6 N HCl, refluxing, 1 h; (i) RH, HoBt, DCC, CH2Cl2.
MTT assay in vitro against five human cancer cell lines including
human colon cancer cells HCT116, human lung adenocarcinoma
epithelial cells A549, human colon adenocarcinoma SW620 cells,
human pancreatic carcinoma cells PC3 and human promyelocytic
leukemia cells HL60. Nutlin-1 was employed as the positive
control. The results are presented in Table 1.
in Table 2) as the testing set for model validation. The alignment of
all compounds used in the training set was given in Figure 1. The
pIC50 values (pIC50 = ꢀlogIC50) of these compounds were used as
dependent variables. The CoMFA results are summarized in
Table 3 and the contour maps of CoMFA model were presented in
Figure 2.
As shown in Table 1, twelve compounds (6a–e, 7b–e, 8f–g and
8l–m) displayed improved binding inhibitory activities. Compound
The statistical parameters for the CoMFA models were given in
Table 3. For the CoMFA model, partial least squares (PLS) regres-
sion produced an excellent cross-validated correlation coefficient
(q2) of 0.645 (>0.5) with an optimized component of 5, which
suggested that the model was reliable and it should be a useful tool
for predicting the IC50 values. The noncross-validated PLS analysis
gave a high correlation coefficient (r2) of 0.979, and a low standard
error estimate (SEE) of 0.115. The contributions of steric and elec-
trostatic fields to this model were 0.626 and 0.374, respectively.
The relationship between actual and predicted pIC50 of the training
set and test set compounds of the CoMFA model are illustrated in
Figure 3. The predictive correlation coefficient ðR2predÞ value based
on molecules of the training set was 0.982 for the CoMFA model,
and 0.802 for the test set. The actual and predicted pIC50 values
of the training set and test set by the model are given in Table 3.
The steric contour plot shown in Figure 2A showed a huge green
colored contour near the N-substituent of the imidazoline deriva-
tives, indicating that the bulky substitute near the nitrogen atom
of the linker was favorable to potency. In addition, CoMFA electro-
static contour map as shown in Figure 2B showed a big blue
contour at the end of N-1 substituent, indicating that more posi-
tively charged groups in this region enhanced activity. This model
could well explain why the potent activity of compound 6c was
better than 7c. The ethyl ester was in the green and blue region,
removing the ethyl group to acid made it inaccessible to the green
and blue region.
6c exhibited potent inhibitory activity (IC50 = 0.59
lM), which was
almost eightfold better than that of Nutlin-1(IC50 = 4.78
lM). Com-
paring the various substituents on N-1 revealed that amino-acid
esters possessed significant improved binding inhibitory activities,
while hydrolyzing esters to corresponding acids reduced inhibitory
potencies. When the acids were amidated with amines, the type of
amides influenced the binding inhibitory activities obviously.
Compounds with a 3-aminopropanamide side chain (i.e., 8f and
8g) showed enhanced potencies than molecules with a 2-aminoac-
etamide (i.e., 8a and 8b) or a 2-aminobutanamide (i.e., 8m) the
length of the alkyl linker between the N-1 and amines influenced
the binding inhibitory activities obviously. Interestingly, the intro-
duction of
than that of
D
-alanine (8k–l) as the linker presented better potency
-alanine (8i–j).
L
For the results of MTT assay, nearly all target molecules exhib-
ited enhanced anti-proliferative activities against five tumor cell
lines than that of Nutlin-1, indicating that the introduction of ami-
no acid derivatives as the linkers was beneficial to anti-prolifera-
tive activities.
2.3. 3D-QSAR analysis
3D-QSAR methods, such as CoMFA, are widely used in drug
design because they allow rapid generation of QSAR models, with
which biological activity of newly designed molecules can be pre-
dicted.15 In this study, 30 compounds (including Nutlin-1, 14 ob-
tained molecules in this study and 15 molecules in a previous
study12) with available IC50 values were employed for the 3D-QSAR
analysis. For 3D-QSAR analyses, 20 compounds (unasterisked
molecules in Table 2) were selected as the training set for model con-
struction, and the remaining 10 compounds (asterisked molecules
3. Conclusion
Twenty-three N-1 amino acid substituted 2,4,5-triphenyl imi-
dazoline derivatives were designed and synthesized as p53–
MDM2 binding inhibitors based on previously discovered

C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
1419
Table 1
p53–MDM2 binding inhibitory and anti-proliferative activities of synthesized compounds (6a–e, 7a–e, and 8a–m)
R1
IC50-FPa
(l
M)
IC50 (lM)
HCT116
20.46
A549
24.00
SW620
PC3
HL60
O
Nutlin-1
4.87
4.29
33.72
40.57
19.50
2.93
N
N
O
HN
6a
4.74
9.22
3.63
3.88
9.41
6.89
8.59
O
O
6b
6c
3.36
0.59
14.32
7.82
3.78
HN
HN
O
Ο
Ο
O
O
3.73
0.31
—
5.52
—
2.2
—
b
6d
0.84
—
—
HN
HN
Ο
6e
7a
7b
1.66
6.18
1.07
6.65
6.07
-
3.25
0.48
-
8.02
5.7
-
6.2
3.9
-
5.81
5.73
-
O
OH
HN
O
O
HN
HN
OH
Ο
Ο
OH
7c
3.27
5.95
1.67
9.2
5.59
6.7
OH
7d
7e
3.39
2.05
—
—
—
—
—
HN
HN
OH
O
6.07
0.48
5.7
3.9
5.73
O
N
8a
5.27
5.63
10.92
6.25
7.02
2.99
HN
O
N
N
8b
8c
9.41
5.10
3.99
4.33
3.13
12.35
2.90
5.84
5.87
6.08
6.11
HN
HN
O
O
10.35
O
N
N
8d
5.43
6.41
3.42
8.13
7.10
5.48
N
HN
O
O
N
O
8e
8f
20.65
2.23
0.65
4.49
3.78
6.39
3.62
3.96
4.03
6.17
5.89
3.13
3.55
7.58
7.26
3.29
3.60
4.62
HN
HN
HN
HN
N
N
N
O
N
O
O
8g
8h
8i
5.83
2.36
2.01
5.95
3.31
5.99
4.60
6.83
5.32
2.93
4.22
Ο
N
12.62
HN
(continued on next page)

1420
C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
Table 1 (continued)
R1
IC50-FPa
(lM)
IC50 (lM)
HCT116
—
A549
—
SW620
PC3
—
HL60
—
Ο
Ο
Ο
N
N
N
N
N
8j
12.45
5.01
—
HN
HN
HN
HN
8k
8l
7.53
3.71
>50
8.56
2.74
16.82
6.74
8.48
3.17
5.96
2.97
Ν
8m
4.18
4.26
1.38
6.45
7.54
6.93
O
a
Values are means of three experiments.
NA, no activity.
b
SARs. In vitro biological evaluation for their p53–MDM2 binding
inhibitory activities and anti-proliferative activities revealed that
twelve compounds displayed improved binding inhibitory activi-
ties and most compounds showed enhanced cell growth inhibition
activities. Compound 6c exhibited marked p53–MDM2 binding
(500 MHz, CDCl3) d = 7.60 (d, J = 8.4, 1H), 7.13 (d, J = 7.9, 2H), 7.05
(dd, J = 19.3, 7.9, 4H), 6.95 (d, J = 7.9, 2H), 6.57 (d, J = 8.4, 1H),
6.52 (s, 1H), 5.63 (m, 2H), 5.24 (s, 1H), 4.70 (m, 1H), 4.10 (q,
J = 6.9, 2H), 3.86 (s, 3H), 3.81 (d, J = 4.8, 2H), 1.44 (d, J = 5.9, 3H),
1.26 (d, J = 5.8, 3H), 1.19 (t, J = 7.0, 3H). MS (ESI) m/z = 584 [M+H]+.
inhibitory activity (IC50 = 0.59
tent than that of Nutlin-1 (IC50 = 4.78
l
M) which was eightfold more po-
M). CoMFA model was per-
l
4.1.1.2. Ethyl
methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)
propanoate (6b).
White solid (85%); mp: 94–98 °C. 1H NMR
(500 MHz, CDCl3) d = 7.50 (d, J = 8.4, 1H), 7.10 (d, J = 8.3, 2H), 7.02
(m, 4H), 6.93 (d, J = 8.4, 2H), 6.53 (dd, J = 8.4, 2.1, 1H), 6.49 (d,
J = 2.1, 1H), 5.52 (m, 3H), 4.67 (m, 1H), 4.10 (q, J = 6.9, 2H), 3.84
(s, 3H), 3.46 (m, 2H), 2.29 (m, 2H), 1.44 (d, J = 5.9, 3H), 1.42 (d,
J = 6.0, 3H), 1.29 (d, J = 6.1, 3H). MS (ESI) m/z = 592 [M+H]+.
3-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-
formed based on obtained biological data. The CoMFA model was
statistically significant with high predictabilities (q2 = 0.645,
r2 = 0.979) and explained the potent activity of compound 6c.
4. Experimental sections
4.1. Chemistry
Melting points were determined on a Büchi B-540 apparatus
and are uncorrected. 1H NMR spectra were recorded on a Bruker
500 M spectrometer with CDCl3 as solvent. Chemical shifts were
reported in values (ppm), relative to internal TMS, and J values
were reported in Hertz). Mass spectra were recorded on an Es-
quire-LC-00075 spectrometer. Reagents and solvents were pur-
chased from common commercial suppliers and were used
without further purification. Intermediate 1–5 were synthesized
using previous reported procedures.12
4.1.1.3. (2R)-ethyl 2-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-
4-methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)
propanoate (6c).
(500 MHz, CDCl3) d 7.60 (d, J = 8.4 Hz, 1H), 7.14–7.02 (m, 7H),
6.96 (d, J = 8.4 Hz, 2H), 6.57 (dd, J = 8.5, 2.2 Hz, 1H), 6.51 (d,
J = 2.1 Hz, 1H), 5.08 (s, 4H), 4.74–4.66 (m, 1H), 3.87 (s, 3H), 3.72
(d, J = 7.0 Hz, 1H), 2.34 (td, J = 5.7, 1.5 Hz, 2H), 1.44 (t, J = 5.1 Hz,
3H), 1.32 (d, J = 6.1 Hz, 3H), 1.19 (t, J = 7.1 Hz, 3H). MS (ESI) m/
z = 598[M+H]+.
White solid (87%); mp: 70–74 °C. 1H NMR
4.1.1. Synthesis of N1-amino acid ester substituted imidazoline
derivatives (6a–e): General procedures
4.1.1.4. (2R)-ethyl 2-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-
4-methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)
propanoate (6d).
(400 MHz, CDCl3) d 7.57 (dd, J = 18.0, 8.4 Hz, 1H), 7.08 (m, 6H),
6.96 (dd, J = 8.4, 1.7 Hz, 2H), 6.55 (m, 2H), 5.61 (m, 2H), 5.03 (d,
J = 7.3 Hz, 1H), 4.68 (dt, J = 17.6, 6.0 Hz, 1H), 4.44 (m, 1H), 4.20
(m, 3H), 4.06 (dt, J = 10.8, 5.9 Hz, 2H), 3.85 (m, 3H), 3.27 (m, 1H),
1.37 (m, 3H), 1.29 (m, 3H), 1.15 (dd, J = 5.4, 1.5 Hz, 3H). m/
z = 598[M+H]+.
Bis(trichloromethyl) carbonate (65.0 mg, 0.34 mmol) in dry
methylene chloride (10 mL) was added dropwise to a cooled
(0 °C) mixture of compound 5 (60 mg, 0.132 mmol) and triethyl-
amine (0.13 mL) in methylene chloride (10 mL). The mixture was
stirred for 30 min and then was evaporated under vacuum. The ob-
tained residue was dissolved in dry methylene chloride (10 mL)
and was added dropwise to a solution of the amino-acid ethyl es-
ters (2.56 mmol) in methylene chloride (10 mL). After stirred for
15 min at room temperature, the reaction mixture was worked
up with aqueous sodium bicarbonate and extracted with methy-
lene chloride. The organic extracts were washed with water and
brine, and dried over Na2SO4. The solvent was evaporated and
the residue obtained was purified by chromatography over silica
gel by gradient eluent (petroleum ether/ethyl acetate/triethyl-
amine = 3:1:0.01ꢀ1:1:0.01) to get compound 6a–e.
White solid (78%); mp: 64–48 °C. 1H NMR
4.1.1.5. Ethyl
methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)
butanoate (6e).
Olily stuff (70%); 1H NMR (500 MHz, CDCl3)
4-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-
d = 7.55 (d, J = 8.4, 1H), 7.05 (m, 6H), 6.94 (d, J = 8.4, 2H), 6.57
(dd, J = 8.4, 2.2, 1H), 6.53 (d, J = 2.1, 1H), 5.58 (dd, J = 23.4, 10.3,
2H), 4.81 (t, J = 5.5, 1H), 4.70 (dt, J = 12.2, 6.1, 1H), 4.06 (q, J = 7.1,
2H), 3.85 (s, 3H), 3.05 (dd, J = 9.7, 5.8, 2H), 2.09 (dt, J = 7.3, 3.6,
2H), 1.59 (m, 2H), 1.44 (d, J = 6.0, 3H), 1.31 (d, J = 6.1, 3H), 1.21
(t, J = 7.2, 3H). MS (ESI) m/z = 612[M+H]+.
4.1.1.1. Ethyl
methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxa-
mido)acetate (6a).
White solid (88%); mp: 80–83 °C. 1H NMR
2-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-

C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
1421
Table 2
Experimental and CoMFA-predicted pIC50 values of molecules in both training set and test set
O
O
O
N
N
R1
Cl
Cl
Compds
R1
IC50
(
l
M)
pIC50
6.23
Predict value
5.33
d
Compds
R1
IC50
(
l
M)
pIC50
5.05
Predict Value
5.08
d
O
0.02
9a12
8.99
2.35
0.03
Ο
Ο
H
N
O
O
6c
0.59
6.14
ꢀ0.09
9b⁄
5.63
5.19
ꢀ0.44
HN
HN
HN
Ο
Ο
N
O
6e
1.66
6.18
5.78
5.21
5.69
4.74
ꢀ0.09
ꢀ0.47
9c
2.24
6.23
5.65
5.21
5.55
5.28
ꢀ0.10
Ο
O
O
7a⁄
9d
0.07
ΟΗ
N
N
O
Ο
9e⁄
9f
13.02
1.87
4.89
5.73
4.80
5.01
5.84
4.72
0.12
0.11
OH
7c
3.27
2.05
5.72
5.49
5.69
5.24
5.69
5.72
5.18
0.20
0.03
HN
HN
OH
O
O
7e⁄
8a
O
N
O
HN
N
Ο
O
ꢀ0.06
9g
15.93
ꢀ0.08
N
N
HN
HN
Ο
NH2
O
N
8b
8c
9.41
10.35
5.43
5.03
4.99
5.27
5.02
4.98
4.69
ꢀ0.02
ꢀ0.01
ꢀ0.58
9h
9i⁄
9j
34.34
10.36
4.46
4.98
4.00
4.49
3.66
3.85
0.03
ꢀ1.32
ꢀ0.15
Ο
N
O
N
HN
HN
O
N
N
N
8d⁄
100.89
Ν
N
O
HN
8h⁄
5.83
5.23
4.51
ꢀ0.73
9k
321.29
3.49
3.63
0.14
Ν
N
O
Ο
N
N
N
N
8i
12.62
5.96
5.01
4.90
5.22
5.30
4.78
5.25
5.40
ꢀ0.12
0.03
9l
399.86
3.53
3.40
5.45
5.32
3.48
4.85
5.27
0.08
ꢀ0.60
ꢀ0.05
HN
HN
HN
HN
Ο
Ο
9m⁄
9n
N
N
N
O
8j
8k⁄
0.10
4.77
N
N
8l⁄
4.18
5.38
5.32
ꢀ0.06
9o⁄
5.10
5.29
5.20
ꢀ0.09
N
O
4.1.2. Synthesis of N1-amino acid substituted imidazoline
derivatives (7a–e): General procedures
over silica gel by gradient elution (Dichloromethane/metha-
nol = 9.5:0.5–8:2) to get compounds 7a–e.
A mixture of esters (6a–e) and 5 ml 6 N HCl was heated at
110 °C for 1 h. After cooling to room temperature, the reaction mix-
ture was worked up with aqueous sodium bicarbonate and ex-
tracted with ethyl ester. The organic extracts were combined,
washed with water and brine, and dried over Na2SO4. The solvents
were evaporated and the residue was purified by chromatography
4.1.2.1. 2-(4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
phenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)acetic acid
(7a).
White solid (52%); mp: 89–92 °C. 1H NMR (500 MHz,
CDCl3) d = 7.61 (d, J = 8.4, 1H), 7.17 (d, J = 7.9, 2H), 7.04 (m, 3H),
6.95 (d, J = 8.1, 2H), 6.53 (d, J = 8.4, 1H), 6.48 (s, 1H), 5.57 (t,

1422
C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
Figure 3. Plot of calculated (predicted) activities versus experimental ones for
CoMFA analysis, in which squares indicate values correspond to the 20 compounds
in the training set, triangles that of the 10 compounds in the test set.
Figure 1. Alignment of all compounds used in the training set.
4.1.2.3.
(2R)-2-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-
methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxa-
Table 3
mido)propanoicacid (7c).
White solid (45%); mp: 131–
Cross-validated analyses of the CoMFA models using Nutlin-1 as the structural
template
134 °C. 1H NMR (500 MHz, CDCl3) d 7.60 (d, J = 8.4 Hz, 1H), 7.14–
7.02 (m, 8H), 6.96 (d, J = 8.4 Hz, 2H), 6.57 (dd, J = 8.5, 2.2 Hz, 1H),
6.51 (d, J = 2.1 Hz, 1H), 5.08 (s, 4H), 4.74–4.66 (m, 1H), 3.87 (s,
3H), 3.72 (d, J = 7.0 Hz, 1H), 1.44 (t, J = 5.1 Hz, 3H), 1.32 (d,
J = 6.1 Hz, 3H). MS (ESI) m/z = 570[M+H]+.
Method SE
Components q2
r2
F
Field contribution
Steric Electrostatic
CoMFA 0.115
5
0.645 0.979 156.259 0.626 0.374
q2: Leave one out (LOO) cross-validated correlation coefficient.
ONC: optimum number of components.
r2: non cross-validated correlation coefficient.
SEE: standard error of estimate.
4.1.2.4.
(2R)-2-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-
methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carboxa-
mido)propanoic acid (7d).
White solid (48%); mp: 135–
F: F-test value.
138 °C. 1H NMR (500 MHz, CDCl3) d 7.60 (d, J = 8.2 Hz, 1H), 7.14–
7.04 (m, 8H), 6.96 (d, J = 8.4 Hz, 2H), 6.58 (dd, J = 8.5, 2.2 Hz, 1H),
6.52(d, J = 2.1 Hz, 1H), 5.09 (s, 4H), 4.74–4.66 (m, 1H), 3.88 (s,
3H), 3.72 (d, J = 7.0 Hz, 1H), 1.44 (t, J = 5.1 Hz, 3H), 1.32 (d,
J = 6.1 Hz, 3H). MS (ESI) m/z = 570[M+H]+.
4.1.2.5. 4-(4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
phenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)butanoic
acid (7e).
White solid (40%); mp: 101–104 °C. 1H NMR
(500 MHz, CDCl3) d = 7.55 (d, J = 8.4, 1H), 7.05 (m, 6H), 6.94 (d,
J = 8.4, 2H), 6.57 (dd, J = 8.4, 2.2, 1H), 6.53 (d, J = 2.1, 1H), 5.58 (dd,
J = 23.4, 10.3, 2H), 4.81 (t, J = 5.5, 1H), 4.70 (dt, J = 12.2, 6.1, 1H),
4.06 (q, J = 7.1, 2H), 3.85 (s, 3H), 3.05 (dd, J = 9.7, 5.8, 2H), 1.59 (m,
2H), 1.44 (d, J = 6.0, 3H), 1.31 (d, J = 6.1, 3H). MS (ESI) m/
z = 584[M+H]+.
Figure 2. CoMFA contour maps for the (A) steric field: Green/yellow contours
indicate regions where steric bulky groups increase/decrease activity. Favored and
disfavored levels of these displayed fields are fixed at 80% and 20%, respectively. (B)
Electrostatic field: Red/blue contours indicate regions where negative charge
increase/decrease activity. Favored and disfavored levels of these displayed fields
are fixed at 85% and 15%, respectively.
4.1.3. Synthesis of N1-amide substituted imidazoline
derivatives (8a–l): General procedures
Imidazoline acid derivatives (7a–e, 0.40 mM), DCC (151 mg,
0.80 mM) and HOBt (54 mg, 0.40 mM) was dissolved in anhydrous
DCM (20 mL) under nitrogen and stirred for 30 min. Then the cor-
responding amine (0.80 mM) was slowly added with vigorous stir-
ring at rt for about 3 h. The precipitate was filtered; the filtrate was
washed with brine, and dried over Na2SO4. The solvents were evap-
orated and the residue was purified by chromatography over silica
gel by gradient eluent to get target compounds 8a–l.
J = 7.9, 2H), 5.47 (s, 1H), 4.67 (m, 1H), 3.83 (s, 3H), 3.64 (dd, J = 9.5,
3.6, 2H), 1.42 (d, J = 5.9, 3H), 1.26 (t, J = 6.3, 3H). MS (ESI) m/z = 556
[M+H]+.
4.1.2.2. 3-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
4.1.3.1.
phenyl)-N-(2-morpholino-2-oxoethyl)-4,5-dihydroimidazole-1-
carboxamide (8a).
White solid (82%); mp: 80–82 °C. 1H NMR
(500 MHz, CDCl3) d 7.62 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H),
7.09 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz,
2H), 6.58 (dd, J = 8.5, 2.2 Hz, 1H), 6.52 (d, J = 2.0 Hz, 1H), 5.80 (s,
1H), 5.64 (dd, J = 28.9, 10.3 Hz, 2H), 4.70 (dt, J = 12.2, 6.1 Hz, 1H),
3.92 (dd, J = 17.1, 4.6 Hz, 1H), 3.86 (s, 3H), 3.81 (dd, J = 17.0,
4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
phenyl)-4,5-dihydro-1H-imidazole-1-carboxamido)propanoic
acid (7b).
White solid (60%); mp: 116–121 °C. 1H NMR
(500 MHz, CDCl3) d = 7.48 (d, J = 8.4, 1H), 7.10 (d, J = 8.3, 2H), 7.02
(m, 4H), 6.92 (d, J = 8.4, 2H), 6.54 (dd, J = 8.4, 2.1, 1H), 6.50 (d,
J = 2.1, 1H), 5.52 (m, 3H), 4.67 (m, 1H), 3.84 (s, 3H), 3.46 (m, 2H),
2.29 (m, 2H), 1.44 (d, J = 5.9, 3H), 1.42 (d, J = 6.0, 3H). MS (ESI) m/
z = 570 [M+H]+.

C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
1423
3.7 Hz, 1H), 3.61 (dt, J = 9.7, 4.8 Hz, 4H), 3.52 (t, J = 7.4 Hz, 2H),
3.31–3.23 (m, 2H), 1.44 (d, J = 6.0 Hz, 3H), 1.25 (d, J = 6.1 Hz, 3H).
MS (ESI) m/z = 625 [M+H]+.
85–87 °C. 1H NMR (500 MHz, CDCl3) d = 7.59 (d, J = 8.4, 1H), 7.17
(d, J = 8.3, 2H), 7.08 (d, J = 8.5, 2H), 7.03 (d, J = 8.4, 2H), 6.96 (d,
J = 8.4, 2H), 6.57 (dd, J = 8.4, 2.1, 1H), 6.51 (d, J = 2.0, 1H), 5.70 (t,
J = 3.8, 1H), 5.60 (dd, J = 29.4, 10.2, 2H), 4.69 (dt, J = 12.1, 6.1, 1H),
3.93 (dd, J = 17.1, 4.6, 1H), 3.85 (s, 3H), 3.80 (dd, J = 17.1, 3.4, 1H),
3.53 (s, 2H), 3.28 (m, 2H), 2.31 (t, J = 4.8, 4H), 2.26 (s, 3H), 1.43
(d, J = 6.0, 3H), 1.24 (d, J = 6.0, 3H). MS (ESI) m/z = 638[M+H]+.
4.1.3.2. 4,5-Bis(4-chlorophenyl)-N-(2-(diethylamino)-2-oxoeth-
yl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydroimidazole-
1-carboxamide (8b).
White solid (86%); mp: 101–105 °C. 1H
NMR (500 MHz, CDCl3) d = 7.59 (d, J = 8.4, 1H), 7.18 (d, J = 8.2,
2H), 7.08 (d, J = 8.3, 2H), 7.03 (d, J = 8.3, 2H), 6.96 (d, J = 8.4, 2H),
6.56 (m, 1H), 6.51 (d, J = 1.7, 1H), 5.70 (s, 1H), 5.60 (dd, J = 32.3,
10.2, 2H), 4.69 (dt, J = 12.1, 6.0, 1H), 3.97 (dd, J = 17.0, 4.8, 1H),
3.85 (s, 3H), 3.78 (dd, J = 17.0, 3.1, 1H), 3.30 (dt, J = 9.5, 7.0, 4H),
1.43 (d, J = 6.0, 3H), 1.24 (d, J = 6.0, 3H), 1.07 (m, 7H). MS (ESI) m/
z = 611[M+H]+.
4.1.3.9. 4,5-Bis(4-chlorophenyl)-N-((R)-1-(diethylamino)-1-oxo-
propan-2-yl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydro-
imidazole-1-carboxamide (8i).
White solid (76%); mp: 76–
79 °C. 1H NMR (500 MHz, CDCl3) d 7.60 (dd, J = 22.4, 8.4 Hz, 1H),
7.19–7.00 (m, 6H), 6.95 (d, J = 8.4 Hz, 2H), 6.57 (m, 1H), 6.50 (s,
1H), 5.62 (m, 3H), 4.73–4.63 (m, 1H), 4.63–4.55 (m, 1H), 3.85 (s,
3H), 3.47–3.07 (m, 4H), 1.43 (t, J = 5.8 Hz, 3H), 1.27 (t, J = 6.0 Hz,
3H), 1.16–0.97 (m, 6H). MS (ESI) m/z = 625 [M+H]+.
4.1.3.3.
phenyl)-N-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-4,5-dihydroimidaz-
ole-1-carboxamide (8c).
4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
4.1.3.10.
(2R)-1-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-
White solid (70%); mp: 110–115 °C.
1H NMR (500 MHz, CDCl3) d = 7.59 (d, J = 8.4, 1H), 7.17 (d, J = 8.0,
2H), 7.08 (d, J = 8.5, 2H), 7.03 (d, J = 8.4, 2H), 6.96 (m, 2H), 6.56
(d, J = 8.4, 1H), 6.51 (s, 1H), 5.68 (s, 1H), 5.61 (m, 2H), 4.69 (dt,
J = 12.1, 6.1, 1H), 3.85 (m, 4H), 3.73 (m, 1H), 3.39 (t, J = 6.8, 2H),
3.25 (t, J = 6.8, 2H), 1.91 (m, 2H), 1.81 (m, 2H), 1.43 (d, J = 6.0,
3H), 1.25 (d, J = 6.0, 3H). MS (ESI) m/z = 623 [M+H]+.
methoxyphenyl)-4,5-dihydroimidazol-1-yl)-2-methyl-3-(4-
methylpiperazin-1-yl)propane-1,3-dione (8j).
White solid
(74%); mp: 94–98 °C. 1H NMR (500 MHz, CDCl3) d 7.59 (dd,
J = 37.7, 8.4 Hz, 1H), 7.22–7.13 (m, 3H), 7.12–7.02 (m, 4H), 6.97
(dd, J = 8.5, 2.2 Hz, 2H), 6.59 (ddd, J = 14.5, 8.4, 2.2 Hz, 1H), 6.53
(d, J = 2.1 Hz, 1H), 5.79 (dd, J = 50.3, 7.3 Hz, 1H), 5.66 – 5.53 (m,
2H), 4.68 (ddd, J = 19.2, 12.7, 6.4 Hz, 2H), 3.87 (d, J = 2.2 Hz, 3H),
3.68–3.30 (m, 4H), 2.41–2.30 (m, 5H), 2.28 (s, 3H), 1.45 (dd,
J = 6.0, 2.6 Hz, 3H), 1.29 (dd, J = 10.0, 6.2 Hz, 3H), 1.09 (dd,
J = 15.9, 6.9 Hz, 3H). MS (ESI) m/z = 637[M+H]+.
4.1.3.4.
chlorophenyl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydro-
imidazole-1-carboxamide (8d). White solid (73%); mp: 88–
N-(2-(4-acetylpiperazin-1-yl)-2-oxoethyl)-4,5-bis(4-
90 °C. 1H NMR (500 MHz, CDCl3) d = 7.62 (s, 1H), 7.14 (d, J = 7.7,
2H), 7.08 (d, J = 8.6, 2H), 7.03 (d, J = 8.4, 2H), 6.95 (d, J = 8.0, 2H),
6.57 (d, J = 8.4, 1H), 6.50 (s, 1H), 5.85 (s, 1H), 5.66 (d, J = 25.2,
2H), 4.69 (dt, J = 12.1, 6.0, 1H), 3.88 (m, 5H), 3.56 (s, 3H), 3.28
(dd, J = 14.7, 9.6, 2H), 2.13 (s, 4H), 2.09 (s, 3H), 1.43 (d, J = 6.0,
3H), 1.25 (d, J = 6.0, 3H). MS (ESI) m/z = 666[M+H]+.
4.1.3.11.
oxopropan-2-yl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihy-
droimidazole-1-carboxamide (8k).
Oilly stuff (68%); 1H
NMR (500 MHz, CDCl3) d 7.59 (dd, J = 22.4, 8.4 Hz, 1H), 7.19–6.99
(m, 6H), 6.95 (d, J = 8.4 Hz, 2H), 6.57 (ddd, J = 10.8, 8.5, 2.1 Hz,
1H), 6.50 (s, 1H), 5.62 (m, 3H), 4.73–4.63 (m, 1H), 4.63–4.55 (m,
1H), 3.85 (s, 3H), 3.47–3.07 (m, 4H), 1.43 (t, J = 5.8 Hz, 3H), 1.27
(t, J = 6.0 Hz, 3H), 1.16–0.97 (m, 6H). MS (ESI) m/z = 625 [M+H]+.
4,5-Bis(4-chlorophenyl)-N-((R)-1-(diethylamino)-1-
4.1.3.5.
phenyl)-N-(3-(4-methylpiperazin-1-yl)-3-oxopropyl)-4,5-dihy-
droimidazole-1-carboxamide (8e). White solid (62%); mp:
4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
90–92 °C. 1H NMR (500 MHz, CDCl3) d = 7.53 (d, J = 8.4, 1H), 7.11
(d, J = 8.4, 2H), 7.04 (dd, J = 10.2, 8.7, 4H), 6.95 (d, J = 8.4, 2H),
6.55 (dd, J = 8.4, 2.0, 1H), 6.50 (d, J = 1.8, 1H), 5.55 (m, 3H), 4.68
(dt, J = 12.1, 6.0, 1H), 3.85 (s, 3H), 3.49 (d, J = 4.4, 2H), 3.32 (t,
J = 5.2, 4H), 2.31 (m, 8H), 1.44 (d, J = 6.0, 3H), 1.31 (d, J = 6.0, 3H).
MS (ESI) m/z = 652[M+H]+.
4.1.3.12. 4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
phenyl)-N-((S)-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl)-
4,5-dihydroimidazole-1-carboxamide
(8l).
White solid
(48%); mp: 140–145 °C. 1H NMR (500 MHz, CDCl3) d 7.59 (dd,
J = 22.4, 8.4 Hz, 1H), 7.20–7.00 (m, 6H), 6.95 (d, J = 8.4 Hz, 2H),
6.57 (m, 1H), 6.50 (s, 1H), 5.62 (m, 3H), 4.73–4.63 (m, 1H), 4.63–
4.55 (m, 1H), 3.85 (s, 3H), 3.47–3.07 (m, 4H), 1.43 (t, J = 5.8 Hz,
3H), 1.27 (t, J = 6.0 Hz, 3H), 1.16–0.97 (m, 6H). MS (ESI) m/z = 625
[M+H]+.
4.1.3.6.
phenyl)-N-(3-morpholino-3-oxopropyl)-4,5-dihydroimidazole-
1-carboxamide (8f).
White solid (84%); mp: 95–97 °C. 1H
4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-
NMR (500 MHz, CDCl3) d = 7.52 (d, J = 8.4, 1H), 7.09 (d, J = 8.3, 2H),
7.02 (m, 4H), 6.93 (d, J = 8.4, 2H), 6.53 (dd, J = 8.4, 2.1, 1H), 6.49 (d,
J = 2.1, 1H), 5.52 (m, 3H), 4.67 (m, 1H), 3.84 (s, 3H), 3.59 (dd,
J = 10.9, 5.9, 4H), 3.46 (m, 2H), 3.29 (dd, J = 10.5, 5.3, 4H), 2.29 (m,
2H), 1.42 (d, J = 6.0, 3H), 1.29 (d, J = 6.1, 3H). MS (ESI) m/
z = 639[M+H]+.
4.1.3.13. 4,5-Bis(4-chlorophenyl)-N-(4-(diethylamino)-4-oxobu-
tyl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydroimidazole-
1-carboxamide (8m).
Olily stuff (88%); 1H NMR (500 MHz,
CDCl3) d 7.72 (dd, J = 5.1, 3.7 Hz, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.45
(dd, J = 5.1, 3.8 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.4 Hz,
2H), 6.42 (d, J = 1.8 Hz, 1H), 6.38 (dd, J = 8.5, 2.0 Hz, 1H), 5.74 (d,
J = 10.7 Hz, 1H), 5.69 (s, 1H), 5.59 (d, J = 10.7 Hz, 1H), 4.70–4.61
(m, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.79 (d, J = 6.8 Hz, 3H), 3.24–3.19
(m, 2H), 3.07 (dd, J = 12.9, 6.3 Hz, 2H), 2.35 (t, J = 7.0 Hz, 1H),
2.04 (t, J = 6.0 Hz, 2H), 1.85–1.78 (m, 1H), 1.61–1.53 (m, 2H), 1.43
(t, J = 5.6 Hz, 3H), 1.29 (d, J = 6.0 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H),
1.18 (dd, J = 12.0, 4.8 Hz, 3H). MS (ESI) m/z = 639[M+H]+.
4.1.3.7. 4,5-Bis(4-chlorophenyl)-N-(3-(diethylamino)-3-oxopro-
pyl)-2-(2-isopropoxy-4-methoxyphenyl)-4,5-dihydroimidazole-
1-carboxamide (8g).
White solid (80%); mp: 71–74 °C. 1H
NMR (500 MHz, CDCl3) d = 7.55 (d, J = 8.4, 1H), 7.12 (d, J = 8.3,
2H), 7.05 (dd, J = 12.1, 8.5, 5H), 6.96 (d, J = 8.4, 2H), 6.54 (m, 2H),
5.55 (dd, J = 48.4, 10.3, 3H), 4.69 (dt, J = 12.1, 6.1, 1H), 3.26 (m,
6H), 1.44 (d, J = 6.0, 3H), 1.31 (d, J = 6.0, 3H), 1.06 (m, 6H). MS
(ESI) m/z = 625 [M+H]+.
4.2. Biological evaluation
4.2.1. MDM2. protein expression and purification
MDM2 (1–118) plasmid was provided by Dr. Shaomeng Wang’s
group, and transformed into Escherichia coli BL-21 (DE3). Cultures
4.1.3.8.
phenyl)-N-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4,5-dihy-
droimidazole-1-carboxamide (8h). White solid (88%); mp:
4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-

1424
C. Hu et al. / Bioorg. Med. Chem. 20 (2012) 1417–1424
were grown at 37 °C in TB medium, and induced by 0.4 mM IPTG at
an OD600 of 0.6 at 18 °C for 20 h. Cells were lysed in 50 mM Tries,
pH 7.5 buffer containing 500 mM NaCl and 10% glycerol. MDM2
(1–118) was purified from the soluble fraction using Ni–NTA resin,
and desalted in PBS buffer pH 7.5, 150 mM NaCl and 10% glycerol.
The protein was purified to >95% as judged SDS–PAGE.
a template for superimposition, and the common fragment with
2,4,5-triphenyl imidazoline scaffold was selected for alignment
and all the molecules were aligned on it.
4.3.2. Partial least squares (PLS) analysis and models validation
of 3D-QSAR model
Partial least squares (PLS) analysis was used to construct a lin-
ear correlation between the 3D-fields and the p53–MDM2 binding
inhibitory activities. To select the best model, the cross-validation
analysis was performed using the leave-one-out (LOO) method in
which one compound was removed from the data set and its activ-
ity was predicted using the model built from rest of the data set. It
resulted in the cross-validation correlation coefficient (q2) and the
optimum number of components N. The non-cross-validation was
performed with a column filter value of 2.0 to speed up the analy-
sis and reduce the noise.
4.2.2. Fluorescence polarization competitive binding assay
Measurements were made with an EnVision Multilabel Plate
Reader using a 480 nM excitation filter and a 535 nM emission fil-
ter. Assays were performed in Corning 384-well black plates. Nut-
lin-1 was used as the positive control, while DMSO was used as the
negative control. Assays were performed in duplicate and repeated
at least twice on separate days. Competition experiments were car-
ried out in a total volume of 20 lL 40 mM Tris–HCl, pH 7.5,
150 mM NaCl, and 1 mM DTT, 4% DMSO. Probe peptide was pres-
ent at a final concentration of 1 nM, and MDM2 was present at a
final concentration of 10
lM. Plates were allowed to incubate at
Acknowledgements
room temperature for 1 h prior to measurement.
This study was financially supported by the National Natural
Science Foundation of China (30873163), and partially supported
by the Scholarship Award for Excellent Doctoral Student granted
by Ministry of Education and Zhejiang Innovation Program for
Graduates (YK2010012).
4.2.3. Cell viability assay
HCT-116, A549, SW620, PC3 and HL60 cells were planted in 96-
well plates (4 ꢁ 103/well) for 24 h, and subsequently treated with
different concentrations of compounds 6a–c, 6e, 7a, c, e, and 8a–
l for 72 h. Viable cells were determined using MTT assay. MTT solu-
tion (5.0 mg/mL in RPIM-1640, Sigma, St. Louis, MO) was added
References and notes
(10.0
ll/well), and plates were incubated for a further 4 h at
1. Levine, A. J. Cell 1997, 88, 323.
37 °C. The purple formazan crystals were dissolved in 100.00
l
l
2. Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307.
3. Eldeiry, W. S.; Tokino, T.; Velculescu, V. E.; Levy, D. B.; Parsons, R.; Trent, J. M.;
Lin, D.; Mercer, W. E.; Kinzler, K. W.; Vogelstein, B. Cell 1993, 75, 817.
4. Vu, B. T.; Vassilev, L. Small-Mol. Inhib. Protein–Protein Interact. 2011, 348, 151.
5. Schon, O.; Friedler, A.; Bycroft, M.; Freund, S. M. V.; Fersht, A. R. J. Mol. Biol.
2002, 323, 491.
DMSO. After the crystal dissolved, the plates were read on an auto-
mated microplate spectrophotometer (Bio-Tek Instruments,
Winooski, VT) at 570 nm. The concentration for 50% inhibition of
cells (IC50) was calculated using the software of Dose-Effect Analy-
sis with Microcomputers.
6. Bowman, A. L.; Nikolovska-Coleska, Z.; Zhong, H. Z.; Wang, S. M.; Carlson, H. A.
J. Am. Chem. Soc. 2007, 129, 12809.
7. Fischer, P. M.; Lane, D. P. Trends Pharmacol. Sci. 2004, 25, 343.
8. Issaeva, N.; Bozko, P.; Enge, M.; Protopopova, M.; Verhoef, L. G. G. C.; Masucci,
M.; Pramanik, A.; Selivanova, G. Nat. Med. 2004, 10, 1321.
9. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
844.
10. Hardcastle, I. R.; Ahmed, S. U.; Atkins, H.; Calvert, A. H.; Curtin, N. J.; Farnie, G.;
Golding, B. T.; Griffin, R. J.; Guyenne, S.; Hutton, C.; Kallbad, P.; Kemp, S. J.;
Kitching, M. S.; Newell, D. R.; Norbedo, S.; Northen, J. S.; Reid, R. J.; Saravanan,
K.; Willems, H. M. G.; Lunec, J. Bioorg. Med. Chem. Lett. 2005, 15, 1515.
11. Parks, D. J.; LaFrance, L. V.; Calvo, R. R.; Milkiewicz, K. L.; Gupta, V.; Lattanze, J.;
Ramachandren, K.; Carver, T. E.; Petrella, E. C.; Cummings, M. D.; Maguire, D.;
Grasberger, B. L.; Lu, T. B. Bioorg. Med. Chem. Lett. 2005, 15, 765.
12. Hu, C. Q.; Li, X.; Wang, W. S.; Zhang, L.; Tao, L. L.; Dong, X. W.; Sheng, R.; Yang,
B.; Hu, Y. Z. Bioorg. Med. Chem. 2011, 19, 5454.
13. Liao, S. Y.; Qian, L.; Miao, T. F.; Lu, H. L.; Zheng, K. C. Eur. J. Med. Chem. 2009, 44,
2822.
14. Seltzer, S. J. Am. Chem. Soc. 1990, 112, 4477.
15. Cramer, R. D.; Patterson, D. E.; Bunce, J. D. J. Am. Chem. Soc. 1988, 110, 5959–
5967.
16. Yang, S. H.; Van, H. T. M.; Le, T. N.; Khadka, D. B.; Cho, S. H.; Lee, K. T.; Lee, E. S.;
Lee, Y. B.; Ahn, C. H.; Cho, W. J. Eur. J. Med. Chem. 2010, 45, 5493.
4.3. 3D-QSAR analysis
4.3.1. Data set and alignment
3D-QSAR studies were performed using SYBYL 6.9 molecular
modeling software.16 All parameters used in CoMFA were default
except for explained. Active conformation selection is a key step
for 3D-QSAR analyses. Since the crystal structure of complex of
MDM2 with one of these compounds is not available, conformation
search was performed in Chem3D software to discover the
global energy-minimum conformation for the Nutlin-1 by rotating
rotatable bonds. The rest of the molecules were built by changing
the substitutions of Nutlin-1 and were minimized with the same
way. Finally, Gasteiger-Hückel charges were assigned to all
molecules.
Structural alignment is considered as one of the most sensitive
parameters in CoMFA analysis. The accuracy of the prediction of
CoMFA model and reliability of the contour maps are directly
dependent on the structural alignment rule. Nutlin-1 was used as