Discovery, synthesis, and biological evaluation of orally active pyrrolidone derivatives as novel inhibitors of p53-MDM2 protein-protein interaction

Article abstract of DOI:10.1021/jm300969t

The p53-MDM2 interaction has been proved to be a valuable target to develop effective antitumor agents. Novel p53-MDM2 inhibitors bearing pyrrolidone scaffolds were successfully identified by structure-based design. The nanomolar inhibitor 5 possessed good p53-MDM2 inhibitory activity (K_i = 780 nM) due to its hydrophobic and hydrogen bonding interactions with MDM2. Further hit optimization led to the discovery of a number of highly potent pyrrolidone derivatives with improved p53-MDM2 inhibitory activity and in vitro antiproliferative potency. Compounds 41 (K_i = 260.0 nM) and 60a (K_i = 150.0 nM) showed good and selective activity against tumor cells with deleted p53. In addition, these two compounds also effectively inhibited the tumor growth in the A549 xenograft model. Interestingly, compound 41 was proved to be a potent MDM2/MDMX dual inhibitor. The novel pyrrolidone p53-MDM2 inhibitors represent promising lead structures for the development of novel antitumor agents.

Full text of DOI:10.1021/jm300969t

Article
pubs.acs.org/jmc
Discovery, Synthesis, and Biological Evaluation of Orally Active
Pyrrolidone Derivatives as Novel Inhibitors of p53−MDM2 Protein−
Protein Interaction
Chunlin Zhuang,† Zhenyuan Miao,† Lingjian Zhu, Guoqiang Dong, Zizhao Guo, Shengzheng Wang,
Yongqiang Zhang, Yuelin Wu, Jianzhong Yao, Chunquan Sheng,* and Wannian Zhang*
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
S
* Supporting Information
ABSTRACT: The p53−MDM2 interaction has been proved to
be a valuable target to develop effective antitumor agents. Novel
p53−MDM2 inhibitors bearing pyrrolidone scaffolds were
successfully identified by structure-based design. The nanomolar
inhibitor 5 possessed good p53−MDM2 inhibitory activity (Ki =
780 nM) due to its hydrophobic and hydrogen bonding
interactions with MDM2. Further hit optimization led to the
discovery of a number of highly potent pyrrolidone derivatives
with improved p53−MDM2 inhibitory activity and in vitro
antiproliferative potency. Compounds 41 (Ki = 260.0 nM) and
60a (Ki = 150.0 nM) showed good and selective activity against
tumor cells with deleted p53. In addition, these two compounds
also effectively inhibited the tumor growth in the A549 xenograft
model. Interestingly, compound 41 was proved to be a potent MDM2/MDMX dual inhibitor. The novel pyrrolidone p53−
MDM2 inhibitors represent promising lead structures for the development of novel antitumor agents.
chlorofusins,17 norbornanes,10 nutlins,18 sulfonamides,19 ben-
INTRODUCTION
■
zodiazepinediones,20 isoindolinones,19 terphenyls,21 spiro-oxin-
The p53 tumor suppressor is a potent transcription factor that
plays a crucial role in DNA repair, differentiation, senescence,
doles,22 and quinolinols.23 However, the majority of them
showed weak inhibitory activity of the p53−MDM2 interaction.
Among them, the most potent p53−MDM2 inhibitors are the
nutlins (e.g., nutlin-3a, Figure 1), benzodiazepines (e.g.,
TDP222669, Figure 1), and spiro-oxindoles (e.g., MI-219,
Figure 1).
apoptosis, and cell-cycle arrest.1,2 It is also the most frequently
inactivated protein in human cancer. In more than 50% of all
human cancers, the p53 gene is deleted or mutated.3,4 For the
remaining half of the cancers, the p53 that retains its wild-type
form is tightly controlled by the human murine double minute
2 (MDM2) oncoprotein through an autoregulatory feedback
loop.5−9 Inhibitors of the p53−MDM2 binding interaction are
expected to restore normal p53 activity in MDM2-over-
expressing cells and thus exert antitumor effects.3
The nutlins were identified by high throughput screening at
Roche.18 Further optimization led to a promising candidate
with potent in vitro and in vivo activities.18 The X-ray crystal
structures revealed that nutlins bound to the p53 binding site of
MDM2 by mimicking the p53 peptide to a high degree.18
Recently, Roche reported on its web site that one of its
inhibitors (RG7112) has entered phase I clinical trials.13 Spiro-
oxindoles were discovered by structure-based de novo design22
and represented another class of promising inhibitors that are
in clinical development. Initially, Wang’s group discovered MI-
219 as a potent, specific, cell-permeable, and orally active small-
molecule inhibitor. Further optimization of MI-219 led to a
more potent analogue (MI-319).24 This compound also
exhibited potent activities against follicular lymphoma that
retains wild-type p53 both in vitro and in vivo. In addition, MI-
319 in combination with cisplatin induced cell growth
However, the inhibition of protein−protein interactions is a
challenging task for drug discovery because most protein−
protein interactions occur over large surface areas with
relatively flat topologies and nondescript surfaces.6−10 For the
p53−MDM2 interface, it is a narrow and deep cleft which is
different from many other protein−protein interfaces.11 The α-
helix of p53 (residues 15−29) is inserted into the groove of
MDM2 and primarily makes hydrophobic contacts with
MDM2. There are three relatively small binding pockets (i.e.,
Phe19 pocket, Trp23 pocket, Leu26 pocket) on MDM2 for p53
binding, which are suitable for the design of small-molecule
inhibitors.12 Because of the importance of p53−MDM2 as a
therapeutic target, several nonpeptide inhibitors have been
reported in recent years.13 These small-molecule inhibitors
mainly include chalcones,14,15 piperazine-4-phenyl derivatives,16
Received: June 24, 2012
© XXXX American Chemical Society
A
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Figure 1. Representative small molecule p53−MDM2 inhibitors.
a
Scheme 1.
a
Reagents and conditions: (a) ethyl oxalate, CH3ONa/CH3OH, reflux, 3 h; (b) R2CHO, N-(3-aminopropyl)imidazole, 1,4-dioxane, room
temperature, 12 h, yield: 14.2−80.1%.
inhibition and apoptosis in pancreatic cancer cells irrespective
of their p53 mutational status.25 Encouragingly, a clinical
candidate derived from MI-319 has entered phase I clinical
trials. Very recently, a novel class of a piperidinone scaffold has
been designed using a structure-based de novo design strategy.
Among the optimized MDM2 inhibitors, AM-8553 (Figure 1)
has a promising outlook for potential clinical development.26,27
The structures of the nutlins, spiro-oxindoles, and
benzodiazepines seem quite different, but they all share
common structural features: (1) two cis-phenyl groups to
ensure the hydrophobic interactions, and (2) the functional
groups that are capable of forming hydrogen-bonding
interactions. Meanwhile, cocrystal structures and docking
studies indicated that their binding mode mimicked that of
p53 peptide.28 So far, small-molecule inhibitors of MDM2 are
all designed to mimic the residues Phe19, Trp23, and Leu26 in
p53 and their interactions with MDM2.20 Although the
potential benefits for the inhibition of the p53−MDM2
protein−protein interaction have been recognized for nearly
20 years, there are currently still limited classes of highly potent
small-molecule inhibitors that show potent antitumor efficacy.29
Thus, it is highly desirable to discover novel chemotypes that
possess high p53−MDM2 inhibitory activities and suitable
drug-like properties. Continuing our efforts in designing novel
inhibitors of the p53−MDM2 interaction,30 we herein report
the discovery and optimization of novel pyrrolidone inhibitors
that showed both nanomolar inhibitory activities toward p53−
MDM2 interaction and potent in vivo antitumor efficacy.
Interestingly, several highly active pyrrolidone derivatives were
found to act by dual inhibition of p53−MDM2 and p53−
MDMX interactions.
the lead optimization stage, the ether-pyrrolidone derivatives
40−47 were synthesized by Mitsunobu reaction between
compound 32 and various alcohols (Scheme 2). The amino-
substituted pyrrolidone derivatives 48−60 were obtained by
Leuckart−Wallach reaction under microwave conditions
(Schemes 3 and 4).
a
Scheme 2.
a
Reagents and conditions: (a) PPh3, DIAD, THF, R3OH, room
temperature, 12 h, yield: 21.3−67.6%.
RESULTS AND DISCUSSION
■
Virtual Screening and p53−MDM2 Binding Assay.
Virtual screening offers a practical strategy to identify new leads
for drug discovery.34 Previously, Wang’s group successfully
discovered quinolinol inhibitors of p53−MDM2 interaction by
computational screening of the NCI 3D database.23 Encour-
aged by the results, we performed a structure-based virtual
screening study to search a new molecular database, namely the
Specs database,35 to find novel types of p53−MDM2 inhibitors
(see Supporting Information for details of virtual screening).
The binding Ki constants of the 25 selected candidate
compounds were measured by the fluorescence polarization
(FP) binding assay.22,23,36 Nutlin-3a, one of the most active
small-molecule p53−MDM2 inhibitors, was used as the
reference drug.18 To our delight, nine hits were identified to
possess p53−MDM2 inhibitory activity with Ki values ranging
from 0.57 μM to 85.97 μM (Figure 2; the binding curves can be
found in the Supporting Information). Interestingly, the
pyrrolidone framework turned out to be a privileged scaffold
among the identified hits. Six out of nine hits, including two
CHEMISTRY
■
As depicted in Scheme 1, the pyrrolidone derivatives were
synthesized via a simple two-step procedure. In the first step,
substituted methyl pyruvates 11 were obtained by Claisen
condensation of aryl methyl ketones 10 with ethyl oxalate.31,32
Then, pyruvates 11 were reacted with N-(3-aminopropyl)-
imidazole and aldehydes to afford authentic compounds 12−39
by a highly efficient three-component coupling reaction.33 In
B
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a
Scheme 3.
a
Reagents and conditions: (a) NH4COOH, 2-methoxyethanol, microwave, 150 °C, 1 h, yield: 32.0%; (b) R4NH2, CH3COOH, microwave, 120 °C,
2 h, yield: 17.9−55.9%.
a
nanomolar inhibitors (5 and 9), have a common pyrrolidone
scaffold, which was completely different from the reported
p53−MDM2 inhibitors. Moreover, the pyrrolidone analogues
were recently designed as inhibitors of the Annexin A2-
S100A10 protein−protein interaction,37 highlighting the
importance of the pyrrolidone scaffold for the design of
small-molecule inhibitors of protein−protein interactions.
Among the identified hits, compounds 5 and 9 displayed
excellent p53−MDM2 inhibitory activity, with Ki values of 780
nM and 570 nM, respectively, which were comparable to that
of the positive drug nutlin-3a.
Scheme 4.
To obtain the accurate binding mode of these hits, molecular
dynamics (MD) simulations were performed on the docking
model of compounds 1, 5, 6, and 9. As shown in Figure 3, the
binding of these compounds with MDM2 is primarily mediated
a
Reagents and conditions: (a) R-α-phenylethylamine, CH3COOH,
microwave, 120 °C, 2 h, yield: 6.93%, 10.3%.
Figure 2. Chemical structures and p53−MDM2 inhibitory activities of hits from structure-based virtual screening.
C
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Figure 3. Binding modes of four hits in three key subpockets of MDM2: (A) Compound 1, (B) compound 5, (C) compound 6, (D) compound 9.
The figure was generated using PyMol (http://pymol.sourceforge.net/).
by extensive hydrophobic interactions. Three aromatic
substituents of the scaffolds mimic the three key residues
(Phe19, Trp23, and Leu26) of p53. For example, compounds 5
and 9 interact with MDM2 by filling its Trp23 subpocket with
their 5-substituted group. The N-substituted and 4-substituted
groups are located in the Phe19 and Leu26 pockets,
respectively. Compounds 1 and 6 did not form direct hydrogen
bonds with MDM2. In contrast, the pyrrolidone derivatives 5
and 9 can form additional hydrogen bonding interaction with
the backbone of Gly16. As a result, these two compounds
showed relatively higher inhibitory activity.
Hit Optimization and Structure−Activity Relation-
ships. The pyrrolidone p53−MDM2 inhibitors represent a
novel chemotype for the development of highly potent p53−
MDM2 inhibitors. Because the solubility of compound 5 was
better than that of compound 9, compound 5 was chosen for
further structural optimization. The optimization studies were
focused on improving the affinity with MDM2 as well as the
physicochemical properties. First, various substitutions were
introduced on the C5-phenyl of the pyrrolidone derivative 5
(compounds 12−32). The fluorescence polarization assay
revealed that seven compounds in this series (12, 20, 23, 26,
27, 29, 32) possessed potent activity with IC50 values lower
than 1 μM (Table 1). Particularly, the 4-bromo derivative 32
(Ki = 0.29 μM) showed high binding affinity comparable to the
positive drug nutlin-3a (Ki = 0.23 μM). Structure−activity
relationship (SAR) analysis demonstrated that the type and
position of the substitutions were important for the inhibitory
activity. The replacement of the methyl of group of hit 5 by a
bromine (compound 32) or nitro group (compound 29, Ki =
0.70 μM) led to the increased activity. The 4-fluoro derivative
12 (Ki = 0.88 μM) and the 4-trifluoromethyl derivative 26 (Ki =
0.81 μM) also showed activity comparable to that of compound
5. However, decreased inhibitory activity was observed for the
4-chloro derivative 14 (Ki = 23.35 μM) and 4-tert-butyl
derivative 25 (Ki = 10.90 μM), and the 4-methoxyl derivative
16 was totally inactive. When the bromine atom of compound
5 was moved to position 2 (compound 27), excellent inhibitory
activity was also observed (Ki = 0.43 μM). Similarly, movement
of the 4-nitro group of compound 29 to position 3 generated
compound 21 (Ki = 0.83 μM), with comparable activity.
Because the phenyl group of compound 5 was located in the
hydrophobic Phe19 pocket, the introduction of a hydrophilic
hydroxyl group on the phenyl ring (compounds 19 and 20)
resulted in the loss of the activity. Two 2,4-disubstituted
compounds, 13 and 21, only showed moderate activity. In
contrast, good inhibitory activity was retained for 3,5-dimethyl
derivative 23 (Ki = 0.91 μM). When the phenyl group of
compound 5 was replaced by various heterocycles or other
aromatic groups, such as pyridine (15, 22, 24), thiophene (17,
28), furan (31), and naphthalene (18), the loss of p53−MDM2
inhibitory activity was observed for them. The results
highlighted the importance of the phenyl group of compound
5 for the binding with MDM2.
Because the 4-bromo group was found to be the most
favorable substitution for the C5-phenyl group, compound 32
was further optimized by introducing various substitutions on
its C4-benzoyl group. Unfortunately, compounds 33−39
showed decreased activity. The 4-chloro derivative 37 was
more active than the 4-fluoro (38), 4-nitro (34), 4-iodo (39),
and 4-bromo (33) derivatives, but it was less potent than the
unsubstituted compound 32. Next, optimization studies were
focused on introducing substituents on the C3-hydroxyl group
of the pyrrole ring. A series of ether-pyrrolidone derivatives
(compounds 40−47) were designed and synthesized. The in
vitro assay revealed that most of them showed nanomolar
binding activity to MDM2 (Table 2). Particularly, the p53−
MDM2 inhibitory activity of compound 41 was comparable to
that of the positive drug nutlin-3a. SAR analysis indicated that
the aliphatic substituents, except for the propyl group, were
more favorable than the benzyl group.
Encouraged by the results, we further replaced the hydroxyl
group of compound 32 by the amine group to generate
compound 48. Interestingly, the inhibitory activity of
D
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Table 1. MDM2 Binding Affinity of Compounds 12−39
Table 2. MDM2 Binding Affinity of Compounds 40−47
a
compound
R1
R2
Ki (μM)
a
compound
R3
Ki (μM)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
nutlin-3a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-F-Ph
0.88
10.7
23.35
n.a.
32
−
0.29
0.69
0.26
0.34
0.47
0.49
2.07
1.34
6.97
0.23
2,4-di-CH3-Ph
4-Cl-Ph
40
CH2CH3
41
CH(CH3)2
(CH2)4CH3
(CH2)3CH3
cyclohexyl
CH2Ph
6-Cl-pyridin-3-yl
4-OCH3-Ph
thiophen-3-yl
naphthalen-2-yl
3-OH-Ph
3-NO2-Ph
2,4-diCl-Ph
pyridin-2-yl
3,5-di-CH3-Ph
pyridin-3-yl
4-C(CH3)3-Ph
4-CF3-Ph
2-Br-Ph
42
n.a.
43
n.a.
44
n.a.
45
n.a.
46
CH2CH2OCH3
CH2CH2CH3
−
0.83
39.43
n.a.
47
nutlin-3a
a
Values were determined by fluorescence polarization assay.
0.91
n.a.
Table 3. MDM2 Binding Affinity of Compounds 48−60
10.90
0.81
0.43
n.a.
thiophen-2-yl
4-NO2-Ph
2-OH-Ph
furan-2-yl
4-Br-Ph
0.70
n.a.
n.a.
0.29
24.87
2.54
15.29
n.a.
4-Br
4-NO2
2-NO2
2-Cl
4-Cl
4-F
4-Br-Ph
a
compound
R4
Ki (μM)
4-Br-Ph
4-Br-Ph
32
−
H
0.29
0.048
0.14
0.14
0.35
0.43
0.27
0.19
0.23
0.12
0.15
0.15
0.47
0.15
n.a.
4-Br-Ph
48
4-Br-Ph
0.91
1.46
3.59
0.23
49
Ph
4-Br-Ph
50
4-CH3-Ph
4-OCH2CH3-Ph
4-F-Ph
4-I
4-Br-Ph
51
−
−
52
a
53
4-I-Ph
Values were determined by fluorescence polarization assay. n.a. = not
active.
54
4-Br-Ph
55
4-Cl-Ph
56
4-OCF3-Ph
3-Br-Ph
compound 48 was significantly improved (Ki = 48.0 nM, Table
3), whose activity was 5 times higher than that of compound
32. The predicted binding mode indicated that the amino
group formed an additional hydrogen bond with His96 (Figure
4A). When the amino group of compound 48 was substituted
by various aromatic groups (compounds 49−59), the p53−
MDM2 inhibitory activity was slightly decreased. However,
compounds 49−59 were also active in the nanomolar range (Ki
range: 0.12 μM to 0.47 μM), and most of them were more
potent than nutlin-3a. Moreover, they were more soluble than
compound 48. Interestingly, when compound 32 was treated
with (R)-α-phenylethylamine, two diastereoisomers (60a and
60b) could be obtained. In the p53−MDM2 binding assay,
these two diastereoisomers showed different activity. Com-
pound 60a had a Ki value of 0.15 μM, whereas compound 60b
was totally inactive. The above results demonstrated that
conformational restriction may be an important factor for
improving biological activities of this class of compounds. To
determine the absolute configuration of the diastereoisomers,
electronic circular dichroism (ECD) was applied for configura-
tional and conformational analysis. The overall predicted ECD
57
58
2-Br-Ph
59
CH2Ph
60a
60b
nutlin-3a
(R)-1-phenylethyl
(R)-1-phenylethyl
−
0.23
a
Values were determined by fluorescence polarization assay.
spectrum of 60a was subsequently compared with the
experimental data, which revealed a good agreement between
the calculated and measured ECD curves (see Supporting
Information for details). Thus, the configuration of compound
60a was established as (R,R). Further docking studies revealed
that the amine group of compound 60a formed a new
hydrogen bond with Ser17 instead of His96 (Figure 4B).
In Vitro Antiproliferative Activity. To investigate the in
vitro antiproliferative activity of the potent p53−MDM2
inhibitors, four human tumor cell lines, namely U-2 OS
(wild-type p53), A549 (wild-type p53), Saos-2(p53 null), and
NCI-H2119 (p53 null), were chosen for assaying. Nutlin-3a
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Figure 4. Schematic representation of the proposed binding mode for compound 48 (A) and compound 60a (B). The figure was generated using
PyMol (http://pymol.sourceforge.net/).
was used as a reference agent. The obtained IC50 values for the
optimized nanomolar p53−MDM2 inhibitors are summarized
in Table 4. In general, these p53−MDM2 inhibitors displayed
p53), which was much higher than that of nutlin-3a (IC50 =
15.12 μM). Furthermore, its IC50 value against the lung cancer
NCI-H1299 cell line (p53 null) was 14.36 μM, demonstrating
the selectivity of compound 60a over cancer cell lines with
deleted p53. Among the synthesized pyrrolidone derivatives,
compounds 41 and 60a showed both high p53−MDM2
inhibitory activities and potent in vitro antiproliferative
activities with selectivity over cancer cell lines with deleted
p53. Moreover, suitable solubility was also observed for them,
and thus they were chosen for further mechanism and in vivo
studies.
Western Blot Assay. The inhibition of the p53−MDM2
interaction is expected to activate p53, resulting in an increased
level in the cells with wild-type p53.38−40 In addition, activation
of p53 can also lead to an increase in the levels of p21cip1/waf and
MDM2 proteins.18 To test these predictions, cells were lysed at
24 h and protein extracts were analyzed by Western
immunoblotting for the levels of p53, MDM2, and p21cip1/waf
proteins in A549 cancer cells that were treated with compound
60a and nutlin-3a. Consistent with the predictions, compound
60a caused a dose-dependent increase in the levels of p53,
MDM2, and p21cip1/waf proteins in A549 cells (Figure 5),
indicating a good activation of p53. Especially for MDM2,
compound 60a was more active than nutlin-3a.
Table 4. In Vitro Antiproliferative Activities of the Selected
Compounds
a
IC50 (μM)
compound
Saos-2
U-2 OS
A549
NCI-H1299
32
31.62
31.68
22.97
9.99
n.a.
21.88
24.83
9.14
73.06
35.25
8.72
19.99
28.86
18.87
10.26
17.11
19.88
168.5
21.18
25.76
19.95
25.44
19.35
19.25
25.04
11.48
11.54
15.62
25.28
14.36
17.00
20.48
40
41
42
7.29
43
11.63
29.68
106.9
24.38
31.87
30.85
32.69
23.38
23.75
24.76
17.87
9.09
32.35
22.43
148.67
25.67
24.12
26.17
30.01
12.01
12.99
22.8
18.52
31.2
44
48
n.a.
49
24.14
29.38
20.21
27.35
24.52
22.23
29.95
20.82
15.77
20.07
52.77
1.97
50
51
52
53
54
55
56
11.51
11.86
16.91
32.48
28.11
30.83
14.31
57
MDMX Binding Assay. MDMX, a homologue of MDM2,
has a distinct but complementary mode of action with MDM2
58
19.62
31.6
59
60a
60b
nutlin-3a
27.45
13.17
31.62
15.70
15.12
a
Values were measured with the MTT method. n.a. = not active.
moderate to good activity against cancer cell lines with wild-
type p53. As compared with compound 32, the ether-
pyrrolidone derivatives showed improved antiproliferative
activities. Particularly, compounds 41 (IC50 = 8.72 μM) and
42 (IC50 = 7.29 μM) showed better activity against the A549
cell line than nutlin-3a (IC50 = 15.12 μM). Compound 42 was
also more active against the U-2 OS cell line (IC50 = 9.14 μM)
than nutlin-3a (IC50 = 14.31 μM).
However, the antitumor activity of compound 48, the most
active p53−MDM2 inhibitor, was weak mainly because of its
poor solubility. Fortunately, its amine-substituted derivatives
showed enhanced in vitro antiproliferative activities. Com-
pounds 53, 54, 56, and 57 revealed better inhibitory activities
against the U-2 OS cell line with wild-type p53 than nutlin-3a.
In particular, compound 60a showed the best inhibitory effect
(IC50 = 1.97 μM) against lung cancer A549 cell line (wild-type
Figure 5. Cellular activity of nutlin-3a and 60a for the p53 pathway
activation detected by Western blotting (A549 cells, 24 h treatment).
F
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in the regulation of the pro-apoptotic activity of p53.41 Recent
data indicated that the development of dual inhibitors of the
two oncogenic proteins could result in more effective antitumor
agents.42−45 Although many MDM2 inhibitors have been
reported, no potent small molecule inhibitor has been reported
to tightly bind to MDMX despite the slight differences between
MDM2 and MDMX pockets (RMSD for main chain atoms:
1.22 Å).46−48 To investigate the MDMX inhibitory efforts of
the pyrrolidone derivatives, compounds 41 and 60a were
subjected to MDMX binding assay (Table 5). Interestingly,
Effect of Pyrrolidone Derivatives on Apoptosis in
A549 Cells. The effect of compound 60a and nutlin-3a on the
induction of A549 cell apoptosis was evaluated by fluorescence-
activated cell sorting (Figure 7). The results indicated that
compound 60a and nutlin-3a caused a dose-dependent increase
in their apoptotic effect. When treated with 5 μM of 60a and
nutlin-3a, there were no obvious differences compared with the
control group and DMSO group. After 48 h treatment, the
percentage of apoptotic cells for compound 60a and nutlin-3a
was only 0.63% and 0.90%, respectively. At the concentration of
10 μM, the percentage of apoptotic cells for compound 60a and
nutlin-3a was increased to 4.02% and 6.46%, respectively. Visual
assessment of the immunostaining suggested that some A549
cells became small, round, and floating. When the concen-
tration was increased to 20 μM, a remarkable apoptotic effect
was observed for compounds 60a (17.85% apoptotic cells) and
nutlin-3a (15.49% apoptotic cells).
In Vivo Antitumor Potency. To further test the
therapeutic potential for the novel pyrrolidone p53−MDM2
inhibitors, compounds 41 and 60a were chosen to evaluate
their ability to inhibit tumor growth in the A549 xenograft
model because of their potent p53−MDM2 inhibitory activity
and antiproliferative activity. The results revealed that no
toxicity or drug-related death was observed in the nude mice
that were treated with the two compounds (200 mg/kg, orally,
once daily) for 14 days. They were effective as oral agents,
producing a tumor volume inhibition of about 40% (Figure
8A). Unlike irinotecan and doxorubicin, compounds 41 and
60a had little effect on the body weight of the nude mice
(Figure 8B).
Table 5. MDMX Binding Affinity of the Selected
Compounds
a
a
compound
Ki (μM)
compound
Ki (μM)
32
40
41
42
43
44
n.a.
38.91
2.68
n.a.
45
n.a.
46
2.11
9.32
n.a.
47
48
n.a.
60a
nutlin-3a
n.a.
17.93
5.86
a
Values were determined by fluorescence polarization assay. n.a. = not
active.
compound 57 showed good MDMX inhibitory activity (Ki =
2.68 μM), which was more potent than nutlin 3a (Ki = 5.86
μM). In comparison, compound 60a was inactive against
MDMX. Furthermore, the unsubstituted amine derivative 48
was assayed, and it was also inactive. Inspired by the results, the
analogues of compound 41 were tested for MDMX inhibitory
activities. The results revealed that compounds 40, 44, 46, and
47 showed moderate to good binding activity to MDMX,
indicating that they were dual inhibitors of p53−MDM2/
MDMX protein−protein interaction.
To further validate the mechanism of MDMX binding for
compound 41, Western immunoblotting was also used for
analyzing the levels of p53, MDMX, and p21cip1/waf proteins in
MCF-7 cancer cells (MDMX overexpression). Consistent with
the result of the fluorescence polarization binding assay,
compound 41 also caused a dose-dependent increase in the
levels of p53, MDMX, and p21cip1/waf proteins after 24 h
treatment in MCF-7 cells (Figure 6). The results also indicated
a superior activation of p53 but no activity to phosphorylation-
p53 (p-p53).
Next, the ability of compound 60a at 300 mg/kg to inhibit
tumor growth in the A549 xenograft model was evaluated. After
a 13-day incubation of A549 human cancer cells in the mice,
compound 60a was orally administered once daily for 14 days,
and the tumor volume was monitored. The results revealed that
compound 60a is highly effective in inhibition of tumor growth
as an oral agent. At 300 mg/kg, 52.53% tumor growth
inhibition was achieved (Figure 9A). Moreover, there is no
significant weight loss in animals treated with 60a compared
with those in the vehicle control group (Figure 9B).
CONCLUSION
■
A series of novel pyrrolidone inhibitors of p53−MDM2
protein−protein interaction were successfully identified by
structure-based virtual screening. Further structural optimiza-
tion and SAR studies led to the discovery of a number of
nanomolar inhibitors. Results of biological evaluations high-
lighted the promising future of compounds 41 and 60a as novel
antitumor leads: (1) They represent a novel class of p53−
MDM2 inhibitors with nanomolar inhibitory activity and
druglike properties. (2) They showed in vitro antitumor
potency better than that of nutlin-3a, with good selectivity
against tumor cells with deleted p53. (3) They were orally
active and able to effectively inhibit the tumor growth in the
A549 xenograft model. (4) Compound 41 was proved to be a
dual inhibitor of p53−MDM2/MDMX interaction. Further
optimization studies are in progress.
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All starting materials were
commercially available and analytical pure. Melting points were
measured on an uncorrected X-5 digital melting point apparatus
Figure 6. Cellular activity of 41 for the p53 pathway activation
detected by Western blotting (MCF-7 cells, 24 h treatment).
(Gongyi City Yuhua Instrument Co., Ltd.; China). H NMR and 13C
1
G
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Figure 7. Pyrrolidone derivative-induced cell apoptosis. (A) A549 cells were treated with DMSO and 10 μM of compound 60a for 48 h. (B) The
effect of evodiamine derivatives on human cancer cell morphology. A549 cells were treated with DMSO and 20 μM of compound 60a and nutlin-3a
for 48 h, and representative photographs were captured under a light microscope. (C) A549 cells were treated with DMSO and 20 μM of compound
60a for 48 h. Apoptosis was examined by flow cytometry (n = 3). Representative photographs from three independent experiments were displayed.
Figure 8. Inhibition of tumor growth for compounds 41 and 60a (200 mg/kg, administered intragastrically) against A549 xenografts in nude mice:
(A) relative tumor volume (RTV); (B) animal body weight.
NMR spectra were recorded on a Bruker AVANCE 300 spectrometer,
a BRUKER AVANCE 500 spectrometer or a Bruker AVANCE 600
spectrometer (Bruker Company, Germany), using TMS as an internal
standard and CDCl3 or DMSO-d6 as solvents. Chemical shifts (δ
values) and coupling constants (J values) are given in ppm and hertz,
respectively. Microwave reactions were performed on a Biotage
microwave reactor. Elemental analyses were performed with a MOD-
1106 instrument and were consistent with theoretical values within
0.4%. TLC analysis was carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China). Flash column chromatography
was carried out on silica gel 300−400 mesh using a Biotage
instrument. Anhydrous solvent and reagents were all analytical pure
and dried through routine protocols.
mmol) in methanol (10 mL) was added dropwise to a fresh prepared
solution of CH3ONa in CH3OH (2.6 mL of 25% w/v, 12.0 mmol),
and the reaction was allowed to proceed under reflux for another 3 h.
After cooling to room temperature, the reaction mixture was poured
into water (40 mL) for 1 h, acidified with HCl (1 mL of 37% w/v) to
reach pH 3−4, and extracted with diethyl ether (100 mL × 3). The
combined organic extracts were washed with brine (30 mL), dried
over Na2SO4, and filtered, and the solvent was removed in vacuum to
afford the crude product 11 which was directly subjected to general
procedure B.
General Procedure B: Synthesis of Compounds 12−39. Aldehyde
(10 mmol) and N-(3-aminopropyl)imidazole (10 mmol) were mixed
in 1, 4-dioxane (10 mL). After stirred for 15 min, a solution of
compound 11 (10 mmol) in 1,4-dioxane (5 mL) was dropped slowly
into the solvent. Then, the mixture was stirred for another 12 h. The
General Procedure A: Synthesis of Compound 11. A solution of
diethyl oxalate (12.0 mmol) and appropriate acetophenone (10, 6.0
H
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Figure 9. Inhibition of tumor growth for compound 60a (300 mg/kg, administered intragastrically) against A549 xenografts in nude mice: (A)
relative tumor volume (RTV); (B) animal body weight.
resulting precipitate was filtered off and recrystallized from CH3OH to
give compounds 12−39.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
butoxy-1H-pyrrol-2(5H)-one (43). Yield 67.6%, white solid, mp: 117−
1
118 °C. H NMR (600 MHz, DMSO-d6) δ: 7.70 (m, 2H), 7.61 (m,
General Procedure C: Synthesis of Compounds 40−47. To a
solution of compound 32 (1.0 mmol) in dry THF (20 mL) were
added triphenylphosphine (1.5 mmol) and DIAD (1.5 mmol) at 0 °C
under nitrogen atmosphere. The solution was stirred for 15 min at 0
°C, and then alcohol (10 mL) was added. The reaction mixture was
stirred at room temperature overnight and concentrated under
reduced pressure. The residue was purified by flash column
chromatography (CH2Cl2:CH3OH = 100: 1, v/v) to give compounds
40−47 (yields: 21.3−67.6%).
2H), 7.52 (d, 2H, J = 8.4 Hz), 7.46 (t, 2H, J = 7.8 Hz), 7.33 (d, 2H, J =
8.1 Hz), 7.13 (s, 1H), 6.86 (s, 1H), 5.55 (s, 1H), 4.38 (m, 2H), 3.96
(m, 2H), 3.53 (m, 1H), 2.69 (m, 1H), 1.89 (m, 2H), 1.41 (m, 2H),
1.07 (m, 2H), 0.73 (t, 3H, J = 7.4 Hz). 13C NMR (125 MHz, DMSO-
d6) δ: 190.19, 164.97, 151.29, 138.18, 137.66, 135.27, 133.44, 132.12,
130.51, 129.14, 128.71, 126.16, 122.12, 119.57, 71.24, 60.56, 43.97,
38.14, 31.57, 29.40, 18.46, 13.79. ESI-MS (m/z): 522.26 [M + 1]+.
Anal. (C27H28BrN3O3) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
General Procedure D: Synthesis of Compounds 49−60. A
solution of compound 32 (1 mmol) and various amines (5 mmol)
in acetic acid (5 mL) was subjected to microwave irradiation (120 °C,
2 h). After completion of reaction, the solution mixture was washed
with water and then extracted by dichloromethane. The organic layer
was washed with water and 2% aq NaHCO3, separated, dried over
Na2SO4, and evaporated to dryness. The residue was purified by flash
column chromatography (CH2Cl2:CH3OH = 100:1, v/v) to give
compounds 49−60 (yields: 17.9−55.9%).
(cyclohexyloxy)-1H-pyrrol-2(5H)-one (44). Yield 52.5%, light yellow
1
solid, mp: 77−78 °C. H NMR (600 MHz, DMSO-d6) δ: 7.68 (m,
2H), 7.59 (m, 2H), 7.53 (d, 2H, J = 8.5 Hz), 7.46 (t, 2H, J = 7.7 Hz),
7.33 (d, 2H, J = 8.5 Hz), 7.12 (s, 1H), 6.86 (s, 1H), 5.58 (s, 1H), 5.52
(m, 1H), 3.93 (m, 2H), 3.53 (m, 1H), 2.69 (m, 1H), 1.88 (m, 2H),
1.78 (m, 2H), 1.60 (m, 2H), 1.31 (m, 6H). 13C NMR (125 MHz,
DMSO-d6) δ: 190.10, 165.03, 150.50, 138.17, 137.66, 135.38, 133.34,
132.16, 130.45, 129.18, 128.77, 128.60, 127.72, 122.09, 119.57, 77.99,
60.47, 43.98, 38.18, 32.00, 29.41, 25.09, 22.47. ESI-MS (m/z): 548.17
[M + 1]+. Anal. (C29H30BrN3O3) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
ethoxy-1H-pyrrol-2(5H)-one (40). Yield 33.9%, yellow solid, mp: 92−
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-3-(benzyloxy)-5-(4-bro-
mophenyl)-1H-pyrrol-2(5H)-one (45). Yield 40.1%, yellow solid, mp:
72−73 °C. 1H NMR (600 MHz, DMSO-d6) δ: 7.68 (m, 2H), 7.59 (m,
2H), 7.52 (d, 2H, J = 8.5 Hz), 7.42 (t, 2H, J = 7.8 Hz), 7.32 (d, 2H, J =
8.5 Hz), 7.27 (m, 3H), 7.13 (s, 1H), 7.10 (m, 2H), 6.87 (s, 1H), 5.59
(s, 1H), 5.53 (m, 2H), 3.95 (m, 2H), 3.57 (m, 1H), 2.72 (m, 1H), 1.89
(m, 2H). 13C NMR (125 MHz, DMSO-d6) δ: 189.96, 165.06, 150.70,
138.01, 137.68, 136.44, 135.10, 133.49, 132.14, 130.51, 129.21, 128.79,
128.76, 128.68, 128.55, 128.15, 127.45, 122.19, 119.56, 72.44, 60.60,
43.95, 38.13, 29.41. ESI-MS (m/z): 556.19 [M + 1]+. Anal.
(C30H26BrN3O3) C, H, N.
1
95 °C. H NMR (600 MHz, DMSO-d6) δ: 7.72 (m, 2H), 7.60 (m,
2H), 7.51 (m, 2H), 7.47 (m, 2H), 7.32 (m, 2H), 7.13 (s, 1H), 6.86 (s,
1H), 5.55 (s, 1H), 4.49 (m, 1H), 4.41 (m, 1H), 3.92 (m, 2H), 3.52 (m,
1H), 2.68 (m, 1H), 1.86 (m, 2H), 1.07 (t, 3H, J = 7.1 Hz). 13C NMR
(125 MHz, DMSO-d6) δ: 190.17, 164.97, 151.06, 137.97, 137.70,
135.22, 133.62, 132.12, 130.53, 129.26, 128.75, 126.04, 122.14, 119.62,
67.63, 60.61, 43.96, 38.11, 29.41, 15.61. ESI-MS (m/z): 494.19 [M +
1]+. Anal. (C25H24BrN3O3) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
isopropoxy-1H-pyrrol-2(5H)-one (41). Yield 40.3%, white solid, mp:
1
122−123 °C. H NMR (600 MHz, DMSO-d6) δ: 7.67 (s, 1H), 7.66
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
(m, 1H), 7.59 (m, 2H), 7.52 (d, 2H, J = 8.5 Hz), 7.46 (t, 2H, J = 7.7
Hz), 7.31 (d, 2H, J = 8.5 Hz), 7.13 (s, 1H), 6.86 (s, 1H), 5.56 (s, 1H),
5.47 (m, 1H), 3.95 (m, 2H), 3.52 (m, 1H), 2.69 (m, 1H), 1.89 (m,
2H), 1.15 (d, 3H, J = 6.1 Hz), 1.01 (d, 3H, J = 6.1 Hz). 13C NMR (150
MHz, DMSO-d6) δ: 190.14, 165.03, 150.46, 137.97, 137.67, 135.33,
133.38, 132.16, 130.45, 129.21, 128.74, 128.60, 127.32, 122.10, 119.58,
74.14, 60.50, 43.97, 38.19, 29.40, 22.96, 22.86. ESI-MS (m/z): 508.15
[M + 1]+. Anal. (C26H26BrN3O3) C, H, N.
(2-methoxyethoxy)-1H-pyrrol-2(5H)-one (46). Yield 21.3%, white
1
solid, mp: 62−63 °C. H NMR (600 MHz, DMSO-d6) δ: 7.70 (m,
2H), 7.61 (m, 2H), 7.53 (d, 2H, J = 8.5 Hz), 7.47 (t, 2H, J = 7.8 Hz),
7.32 (d, 2H, J = 8.5 Hz), 7.14 (s, 1H), 6.87 (s, 1H), 5.56 (s, 1H), 4.68
(m, 1H), 4.54 (m, 1H), 3.96 (m, 2H), 3.54 (m, 1H), 3.44 (m, 1H),
3.38 (m, 1H), 2.69 (m, 1H), 1.88 (m, 2H). 13C NMR (125 MHz,
DMSO-d6) δ: 189.98, 164.94, 150.94, 137.94, 137.62, 135.25, 133.52,
132.12, 130.43, 129.24, 128.72, 128.54, 126.17, 122.12, 119.62, 71.20,
70.27, 60.52, 58.45, 43.98, 38.09, 29.37. ESI-MS (m/z): 524.20 [M +
1]+. Anal. (C26H26BrN3O4) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
(pentyloxy)-1H-pyrrol-2(5H)-one (42). Yield 34.7%, white solid, mp:
1
131−132 °C. H NMR (600 MHz, DMSO-d6) δ: 7.70 (m, 2H), 7.61
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
(t, 2H, J = 8.2 Hz), 7.52 (d, 2H, J = 7.2 Hz), 7.46 (t, 2H, J = 7.8 Hz),
7.34 (d, 2H, J = 8.5 Hz), 7.13 (s, 1H), 6.86 (s, 1H), 5.56 (s, 1H), 4.42
(m, 2H), 3.95 (m, 2H), 3.55 (m, 1H), 2.69 (m, 1H), 1.88 (m, 2H),
1.42 (m, 2H), 1.16 (m, 2H), 1.00 (m, 2H), 0.74 (t, 3H, J = 7.3 Hz).
13C NMR (125 MHz, DMSO-d6) δ: 190.15, 164.96, 151.29, 138.19,
137.64, 135.27, 133.41, 132.10, 130.49, 129.13, 128.68, 126.21, 122.10,
119.55, 71.51, 60.52, 43.95, 38.11, 29.40, 29.23, 27.43, 22.00, 14.12.
ESI-MS (m/z): 536.29 [M + 1]+. Anal. (C28H30BrN3O3) C, H, N.
propoxy-1H-pyrrol-2(5H)-one (47). Yield 64.1%, light yellow solid,
1
mp: 101−103 °C. H NMR (600 MHz, DMSO-d6) δ: 7.71 (m, 2H),
7.61 (t, 2H, J = 7.7 Hz), 7.52 (d, 2H, J = 8.4 Hz), 7.47 (t, 2H, J = 7.8
Hz), 7.33 (d, 2H, J = 8.4 Hz), 7.13 (s, 1H), 6.86 (s, 1H), 5.56 (s, 1H),
4.39 (m, 2H), 3.96 (m, 2H), 3.54 (m, 1H), 2.70 (m, 1H), 1.90 (m,
2H), 1.45 (m, 2H), 0.64 (t, 3H, J = 7.4 Hz). 13C NMR (125 MHz,
DMSO-d6) δ: 190.15, 164.96, 151.23, 138.14, 137.64, 135.26, 133.47,
132.10, 130.48, 129.15, 128.71, 128.66, 126.16, 122.11, 119.58, 73.04,
I
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60.56, 43.98, 38.13, 29.39, 22.97, 10.16. ESI-MS (m/z): 508.42 [M +
1]+. Anal. (C26H26BrN3O3) C, H, N.
128.41, 128.14, 123.65, 122.10, 121.82, 119.80, 116.03, 62.78, 44.40,
38.53, 29.41. ESI-MS (m/z): 667.40 [M + 1]+. Anal. (C29H24BrIN4O2)
C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-3-amino-4-benzoyl-5-(4-bromo-
phenyl)-1H-pyrrol-2(5H)-one (48). To a solution of compound 32 (1
mmol) in 2-methoxyethanol (5 mL) was added ammonium formate (3
mmol) in a sealed tube. The solution was heated under microwave
irradiation (150 °C, 1 h). The mixture was concentrated under
vacuum, and the residue was triturated in ether. After filtration, the
solid was washed with ether to give the crude compound, which was
purified by flash column chromatography (CH2Cl2:CH3OH = 100: 1,
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((4-bromophenyl)amino)-1H-pyrrol-2(5H)-one (54). Yield 43.5%,
1
bright yellow solid, mp: 104−107 °C. H NMR (300 MHz, DMSO-
d6) δ: 9.11 (s, 1H), 7.59 (s, 1H), 7.34−7.41 (m, 5H), 7.21 (d, 2H, J =
6.9 Hz), 7.13 (m, 5H), 7.10 (m, 3H), 5.61 (s, 1H), 3.93 (m, 2H), 3.56
(m, 1H), 2.71 (m, 1H), 1.85 (m, 2H). 13C NMR (125 MHz, DMSO-
d6) δ: 190.48, 166.26, 140.45, 139.54, 139.43, 137.68, 136.53, 132.34,
131.88, 131.28, 130.44, 128.79, 128.41, 128.31, 123.21, 121.68, 119.60,
117.50, 114.87, 62.65, 44.02, 38.45, 29.42. ESI-MS (m/z): 619.15 [M
+ 1]+. Anal. (C29H24Br2N4O2) C, H, N.
1
v/v) to give compound 48, yield 32.0%, mp: 222−226 °C. H NMR
(500 MHz, DMSO-d6) δ: 10.05 (s, 1H), 8.76 (s, 1H), 7.57 (s, 1H),
7.42 (m, 1H), 7.30−7.33 (m, 4H), 7.27 (d, 2H, J = 7.8 Hz), 7.10 (s,
1H), 6.85 (s, 1H), 6.63 (d, 2H, J = 7.9 Hz), 5.41 (s, 1H), 3.81−3.94
(m, 2H), 3.47 (m, 1H), 2.55 (m, 1H), 1.77−1.82 (m, 2H) . 13C NMR
(125 MHz, DMSO-d6) δ: 179.97, 164.59, 163.61, 138.34, 137.64,
134.14, 131.30, 130.70, 129.86, 128.70, 127.99, 120.88, 119.59, 105.16,
58.47, 44.01, 38.31, 29.02. ESI-MS (m/z): 465.09 [M + 1]+. Anal.
(C23H21BrN4O2) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((4-chlorophenyl)amino)-1H-pyrrol-2(5H)-one (55). Yield 43.6%,
1
bright yellow solid, mp: 92−94 °C. H NMR (600 MHz, DMSO-
d6) δ: 9.08 (s, 1H), 7.56 (s, 1H), 7.39 (d, 2H, J = 8.4 Hz), 7.34 (m,
3H), 7.10−7.30 (m, 5H), 7.00 (d, 2H, J = 9.0 Hz), 6.84 (m, 3H), 5.60
(s, 1H), 3.97 (m, 2H), 3.56 (m, 1H), 3.03 (m, 1H), 1.90 (m, 2H). 13C
NMR (125 MHz, DMSO-d6) δ: 190.49, 166.27, 140.02, 139.65,
139.45, 137.69, 136.57, 132.32, 131.88, 130.44, 128.79, 128.40, 128.31,
126.87, 122.86, 121.68, 119.61, 117.32, 62.64, 44.02, 38.46, 29.42. ESI-
MS (m/z): 575.75 [M + 1]+. Anal. (C29H24BrClN4O2) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((4-(trifluoromethoxy)phenyl)amino)-1H-pyrrol-2(5H)-one (56).
Yield 17.9%, bright yellow solid, mp: 70−71 °C. 1H NMR (600
MHz, DMSO-d6) δ: 9.05 (s, 1H), 7.58 (s, 1H), 7.41 (d, 2H, J = 8.4
Hz), 7.30 (m, 3H), 7.14 (m, 5H), 6.93 (m, 4H), 6.84 (s, 1H), 5.59 (s,
1H), 3.96 (m, 2H), 3.57 (m, 1H), 2.75 (m, 1H), 1.87 (m, 2H). ESI-
MS (m/z): 625.82 [M + 1]+. Anal. (C30H24BrF3N4O3) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((3-bromophenyl)amino)-1H-pyrrol-2(5H)-one (57). Yield 42.0%,
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
(phenylamino)-1H-pyrrol-2(5H)-one (49). Following general proce-
dure D, compound 32 was reacted with phenylamine to afford
1
compound 49 as dark yellow solid (Yield 52.1%), mp: 79−82 °C. H
NMR (500 MHz, DMSO-d6) δ: 9.04 (s, 1H), 7.61 (s, 1H), 7.42 (d,
2H, J = 8.4 Hz), 7.34 (m, 3H), 7.11−7.17 (m, 5H), 6.77−6.98 (m,
6H), 5.63 (s, 1H), 3.96 (m, 2H), 3.58 (m, 1H), 2.74 (m, 1H), 1.90 (m,
2H). 13C NMR (125 MHz, DMSO-d6) δ: 190.63, 166.33, 140.86,
139.85, 139.50, 137.68, 136.73, 132.13, 131.87, 130.41, 128.73, 128.58,
128.37, 128.27, 123.22, 121.64, 121.24, 119.63, 116.68, 62.59, 44.06,
38.45, 29.44. ESI-MS (m/z): 541.57 [M + 1]+. Anal. (C29H25BrN4O2)
C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
(p-tolylamino)-1H-pyrrol-2(5H)-one (50). Yield 55.9%, dark yellow
solid, mp: 90−92 °C. 1H NMR (300 MHz, DMSO-d6) δ: 9.04 (s, 1H),
7.59 (s, 1H), 7.30−7.39 (m, 6H), 7.06−7.20 (m, 5H), 6.78−6.85 (m,
4H), 5.61 (s, 1H), 3.94 (m, 2H), 3.54 (m, 1H), 2.49 (m, 1H), 2.11 (s,
3H), 1.82 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ: 190.56,
166.15, 140.63, 139.73, 138.08, 137.68, 136.83, 132.49, 131.94, 131.81,
130.38, 128.99, 128.78, 128.26, 128.19, 121.72, 121.56, 119.59, 115.81,
62.52, 44.02, 38.41, 29.43, 20.76. ESI-MS (m/z): 555.33 [M + 1]+.
Anal. (C30H27BrN4O2) C, H, N.
1
bright yellow solid, mp: 102−104 °C. H NMR (300 MHz, DMSO-
d6) δ: 9.05 (s, 1H), 7.61 (s, 1H), 7.44 (d, 2H, J = 8.1 Hz), 7.35 (m,
3H), 7.18 (m, 5H), 6.91 (m, 5H), 5.63 (s, 1H), 3.96 (m, 2H), 3.58 (m,
1H), 2.74 (m, 1H), 1.86 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ:
190.61, 166.42, 142.98, 139.21, 138.86, 137.69, 136.48, 132.41, 131.93,
130.45, 128.75, 128.46, 128.22, 125.46, 123.37, 121.75, 121.60, 119.89,
119.63, 118.04, 62.63, 44.02, 38.43, 29.44. ESI-MS (m/z): 619.70 [M
− 1]−. Anal. (C29H24Br2N4O2) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((2-bromophenyl)amino)-1H-pyrrol-2(5H)-one (58). Yield 47.1%,
1
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
bright yellow solid, mp: 89−91 °C. H NMR (600 MHz, DMSO-
((4-ethoxyphenyl)amino)-1H-pyrrol-2(5H)-one (51). Yield 31.8%,
d6) δ: 8.81 (s, 1H), 7.63 (s, 1H), 7.35−7.40 (m, 5H), 7.21 (m, 2H),
7.10−7.18 (m, 5H), 6.88 (m, 2H), 5.69 (s, 1H), 3.95 (m, 2H), 3.55
(m, 1H), 2.71 (m, 1H), 1.89 (m, 2H). 13C NMR (125 MHz, DMSO-
d6) δ: 190.88, 165.33, 141.12, 139.36, 138.47, 137.65, 136.26, 132.77,
132.10, 131.89, 130.48, 128.56, 128.49, 128.05, 127.99, 126.30, 125.11,
121.72, 119.70, 117.82, 117.40, 62.34, 44.09, 38.39, 29.41. ESI-MS (m/
z): 619.15 [M + 1]+. Anal. (C29H24Br2N4O2) C, H, N.
1
dark yellow solid, mp: 179−182 °C. H NMR (300 MHz, DMSO-
d6) δ: 9.04 (s, 1H), 7.59 (s, 1H), 7.39 (d, 2H, J = 8.4 Hz), 7.32 (m,
3H), 7.17 (m, 3H), 7.10 (d, 2H, J = 8.4 Hz), 6.83 (m, 3H), 6.56 (d,
2H, J = 9.0 Hz), 5.59 (s, 1H), 3.93 (m, 2H), 3.86 (q, 2H), 3.56 (m,
1H), 2.70 (m, 1H), 1.86 (m, 2H), 1.22 (m, 3H). 13C NMR (125 MHz,
DMSO-d6) δ: 190.51, 166.06, 155.13, 141.35, 139.95, 137.67, 137.00,
133.53, 131.79, 130.38, 128.79, 128.25, 128.16, 123.64, 121.51, 119.60,
114.67, 114.57, 63.56, 62.43, 44.04, 38.39, 29.43, 15.07. ESI-MS (m/
z): 585.40 [M + 1]+. Anal. (C31H29BrN4O3) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-3-(benzylamino)-5-(4-
bromophenyl)-1H-pyrrol-2(5H)-one (59). Yield 36.0%, light yellow
solid, mp: 66−68 °C. 1H NMR (500 MHz, DMSO-d6) δ: 7.58 (s, 1H),
7.34−7.39 (m, 8H), 7.24−7.29 (m, 5H), 7.11 (s, 1H), 6.87 (s, 1H),
6.76 (d, 2H, J = 8.4 Hz), 5.61 (s, 1H), 5.03 (s, 1H), 3.94 (m, 2H), 3.48
(m, 2H), 3.39 (m, 1H), 2.60 (m, 1H), 1.85 (m, 2H). ESI-MS (m/z):
555.48 [M + 1]+. Anal. (C30H27BrN4O2) C, H, N.
(R)-1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-
3-(((R)-1-phenylethyl)amino)-1H-pyrrol-2(5H)-one (60a). Yield
6.93%, light yellow solid, mp: 92−93 °C. 1H NMR (300 MHz,
DMSO-d6) δ: 7.53 (s, 1H), 7.34−7.39 (m, 7H), 7.24−7.29 (m, 5H),
7.06 (s, 1H), 6.83 (s, 1H), 6.77 (d, 2H, J = 8.1 Hz), 6.01 (bar, 1H),
5.63 (s, 1H), 3.90 (m, 2H), 3.47 (m, 1H), 2.57 (m, 1H), 1.82 (m, 1H),
1.79 (m, 2H), 1.59 (d, 3H, J = 6.9 Hz). 13C NMR (75 MHz, DMSO-
d6) δ: 192.17, 165.69, 146.18, 141.87, 139.05, 138.27, 132.94, 132.27,
131.60, 130.50, 130.18, 130.04, 129.80, 129.03, 128.72, 127.84, 122.68,
120.98, 113.46, 62.63, 52.22, 45.33, 39.42, 30.62, 25.45. ESI-MS (m/
z): 569.15 [M + 1]+. Anal. (C31H29BrN4O2) C, H, N. [α]D = −298.9°
(c = 0.540 in CH3OH).
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((4-fluorophenyl)amino)-1H-pyrrol-2(5H)-one (52). Yield 41.9%,
1
bright yellow solid, mp: 85−87 °C. H NMR (600 MHz, DMSO-
d6) δ: 9.03 (s, 1H), 7.60 (s, 1H), 7.41 (d, 2H, J = 8.5 Hz), 7.34 (t, 3H,
J = 9.1 Hz), 7.21 (d, 2H, J = 7.7 Hz), 7.15 (m, 3H), 6.99 (m, 3H), 6.81
(t, 2H, J = 8.7 Hz), 5.60 (s, 1H), 3.98 (m, 2H), 3.58 (m, 1H), 2.75 (m,
1H), 1.91 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ: 190.73,
166.39, 140.61, 139.74, 137.43, 136.84, 132.31, 131.99, 130.54, 128.86,
128.52, 128.40, 123.78, 123.72, 121.77, 121.38, 121.32, 119.79, 116.03,
62.72, 44.20, 38.59, 29.51. ESI-MS (m/z): 559.50 [M + 1]+. Anal.
(C29H24BrFN4O2) C, H, N.
1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-3-
((4-iodophenyl)amino)-1H-pyrrol-2(5H)-one (53). Yield 23.5%, dark
yellow solid, mp: 85−87 °C. 1H NMR (600 MHz, DMSO-d6) δ: 9.10
(s, 1H), 7.72 (s, 1H), 7.34−7.41 (m, 12H), 6.93 (m, 1H), 6.71 (m,
2H), 5.63 (s, 1H), 3.96 (m, 2H), 3.57 (m, 1H), 2.73 (m, 1H), 1.91 (m,
2H). 13C NMR (125 MHz, DMSO-d6) δ: 190.80, 166.37, 140.95,
139.67, 139.51, 137.62, 137.25, 136.55, 132.47, 132.00, 130.54, 128.53,
(R)-1-(3-(1H-Imidazol-1-yl)propyl)-4-benzoyl-5-(4-bromophenyl)-
3-(((S)-1-phenylethyl)amino)-1H-pyrrol-2(5H)-one (60b). Yield
J
dx.doi.org/10.1021/jm300969t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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10.3%, light yellow solid, mp: 75−76 °C. 1H NMR (300 MHz,
DMSO-d6) δ: 7.99 (s, 1H), 7.21−7.33 (m, 13H), 7.00 (s, 1H), 6.72
(m, 2H), 6.77 (d, 2H, J = 8.1 Hz), 5.87 (bar, 1H), 5.64 (s, 1H), 3.99
(m, 2H), 3.39 (m, 1H), 2.50 (m, 1H), 2.07 (m, 1H), 1.85 (m, 2H),
1.53 (d, 3H, J = 6.9 Hz). 13C NMR (75 MHz, DMSO-d6) δ: 192.22,
166.01, 145.84, 141.82, 139.83, 133.00, 132.52, 131.55, 130.47, 130.15,
129.99, 129.85, 129.01, 128.91, 127.77, 122.79, 122.19, 120.92, 113.86,
62.92, 52.79, 46.12, 39.23, 30.47, 25.78. ESI-MS (m/z): 569.15 [M +
1]+. Anal. (C31H29BrN4O2) C, H, N. [α]D = −45.93° (c = 0.540 in
CH3OH).
20 μM for 48 h. The cells were then harvested by trypsinization and
washed twice with cold PBS. After centrifugation and removal of the
supernatants, cells were resuspended in 400 μL of 1 × binding buffer
which was then added to 5 μL of annexin V-FITC and incubated at
room temperature for 15 min. After addition of 10 μL of PI, the cells
were incubated at room temperature for another 15 min in the dark.
The stained cells were analyzed by a flow cytometer (BD Accuri C6).
In Vivo Antiproliferative Assay. The in vivo antitumor activity of
compounds 41 and 60a was evaluated. Irinotecan (IRT) and
doxorubicin (DOX) were used as reference drugs. BALB/C nude
male mice (Certificate SCXK2003-0003, weighing 18−20 g) were
obtained from Shanghai Experimental Animal Center, Chinese
Academy of Sciences. A549 cancer cell suspensions were implanted
subcutaneously into the right axilla region of the mice. Treatment
began when implanted tumors had reached a volume of 100−300 mm3
(after 13 days). The animals were randomized into appropriate groups
(6 animals per treatment and 10 animals for the control group) and
administered by gavage once daily for 14 consecutive days from day 13
after implantation of the cells. Tumor volumes were monitored by
caliper measurement of the length and width and then calculated using
the formula of TV = 1/2 × a × b2, where a is the tumor length and b is
the width. Tumor volumes and bodyweights were monitored every 4
days over the course of treatment. Mice were sacrificed on day 29 or
33 after implantation of cells, and tumors were removed and recorded
for analysis. Irinotecan (0.5 mg/kg, Jiangsu Aosaikang Pharmacetical,
China) was administered intravenously once daily for 5 consecutive
days. Doxorubicin (10 mg/kg, Pfizer Italia S.r.l) was administered
intravenously once.
Computational Protocols. Molecular docking was used to
predict the binding mode of the p53−MDM2 inhibitors. The crystal
structure of MDM2 (PDB code: 1T4E)20 was prepared by removing
the ligand and adding hydrogen atoms in Discovery Studio 2.549
software package. GOLD23,50,51 with the GoldScore fitness function
was used for molecular docking with default parameters. The reliability
of the docking poses was further validated by MD simulations.
Desmond52−54 was used to carry out the entire simulation, while the
systems were modeled with the OPLS-AA force field. The docked
conformations of compounds (1, 5, 6, 9) and MDM2 complexes were
respectively neutralized by adding salt counterion, and the
concentration was set to 0.15 using a system builder package. As a
result, complexes were immersed in a cubic box (15 Å) containing
about 11 689 TIP3P water molecules, respectively. Other parameters
were default values. The systems were then simulated for 2 ns at 300 K
and 1 atm. System coordinates were saved every 4.8 ps for further
analysis, and heavy atom RMSD from the energy-minimized model
structure was calculated for each MD snapshot. After 2 ns of MD
calculations, stable MDM2−inhibitor complexes were obtained. The
MD results indicated that the binding poses of the hits were very
similar to those obtained from the molecular docking model (RMSD <
2 Å).
Fluorescence Polarization Binding Assay. All fluorescence
experiments were performed as described in the literature.22,23,36
Briefly, the fluorescence polarization experiments were read on Biotek
Synergy H4 with the 485 nm excitation and 535 nm emission filters.
The fluorescence intensities parallel (Intparallel) and perpendicular
(Intperpedicular) to the plane of excitation were measured in black 96-
well NBS assay plates (Corning #3993) at room temperature. The
background fluorescence intensities of blank samples containing the
reference buffer were subtracted, and steady-state fluorescence
polarization was calculated using the following equation: P = 1000
× (Intparallel − GIntperpedicular)/(Intparallel + GIntperpedicular),
and the correction factor G (G = 0.998 determined empirically) was
introduced to eliminate differences in the transmission of vertically and
horizontally polarized light. All fluorescence polarization values were
expressed in millipolarization units (mP). The dose-dependent
binding experiments were carried out with serial dilution in DMSO
of compounds. A 5 μL sample of the test sample, preincubated (for 30
min) MDM2 binding domain (1−118) (10 nM) or MDMX binding
domain (1−137) (120 nM), and PMDM-F peptide (Anaspec) (10
nM) in assay buffer (100 mM potassium phosphate, pH 7.5; 100 mg/
mL bovine gamma globulin; 0.02% sodium azide) were added to
microplates to produce a final volume of 115 μL. For each assay, the
controls contained the MDM2/X binding domain and PMDM-F.
Plates were read at 1 h after mixing all assay components. Binding
constant (Ki) and inhibition curves were fitted using GraphPad Prism
software. Nutlin-3a (Sigma-Aldrich) was used as reference compound
for validating the assay in each plate. Details of protein expression and
purification can be found in Supporting Information.
In Vitro Antiproliferative Assay. The cellular growth inhibitory
activity was determined using two human osteosarcoma cell lines [U-2
OS (wild type p53) and Saos-2 (p53 null)] and two human lung
cancer cell lines [A549 (wild-type p53) and NCI-H1299 (p53 null)].
An amount of 5−6 × 104 cells per well was transferred to 96-well
plates. After culturing for 24 h, the test compounds were added to
triplicate wells at different concentrations and 0.1% DMSO for control.
After 72 h of incubation, 20 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) solution (5 mg/mL) was added to
each well, and after the samples were shaken for 1 min, the plate was
incubated further for 4 h at 37 °C. Pyrrolidones were dissolved in 100
μL of DMSO. The absorbance (OD) was quantitated with the
microplates using Biotek Synergy H4 at 570 nm. Wells containing no
drugs were used as blanks. The concentration of the compounds that
inhibited cell growth by 50% (IC50) was calculated.
ASSOCIATED CONTENT
* Supporting Information
■
S
Western Blot Assay. A549 and MCF-7 cancer cell lines with wild-
type p53 were grown in the recommended media supplemented with
10% FBS (Invitrogen) in a humidified environment with 5% CO2.
After 48 h treatment with various concentrations of compounds, cells
were lysed and the protein extract was denatured and run on 5% Tris-
glycine polyacrylamide gels (Invitrogen). Gels were electroblotted
onto nitrocellulose membranes, and Western detection was carried out
using 5% milk buffer (5% nonfat dry milk in TBS/0.1% tween-20)
throughout. Proteins were detected by ECL chemiluminescence
reagents (Amersham, Piscataway, NJ) using antibodies specific for
human p53 (Calbiochem, cat. no. OP43T), p-p53 (Ser-15) (Cell
Signaling, cat. no. 9286S), p21 (BD Biosciences, cat. no. 556430),
MDM2 (Millipore, cat. no. 07−575), MDMX (abcam, cat. no.
ab16058), and GAPDH(2A8)Mouse mAb (abmart, cat. no.
M20028S).
Computational details for virtual screening, structural character-
ization for compounds 12−39, ECD calculations, binding mode
for the two diastereoisomers of compound 60, experimental
details for protein expression and purification, reversibly
binding assay, aqueous solubility of the selected inhibitors,
and analysis of the selectivity of the pyrrolidone p53−MDM2
inhibitors over A2-S100A10 protein−protein interaction. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(W.N.Z.) Phone/fax: 86-21-81871243; e-mail: zhangwnk@
hotmail.com. (C.Q.S.) Phone/fax: 86-21-81871239; e-mail:
shengcq@hotmail.com.
Cell Apoptosis Assay. A549 cells (5 × 105 cells/mL) were seeded
in six-well plates and treated with compounds at concentrations of 5−
K
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Article
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Author Contributions
†These two authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (grant 81222044, 30930107), the 863 Hi-
Tech Program of China (grant 2012AA020302), Key Project of
Science and Technology of Shanghai (no. 09431901700),
Shanghai Rising-Star Program (grant 12QH1402600), and
Shanghai Municipal Health Bureau (grant XYQ2011038). We
thank Dr. Chuanxu Liu from School of Medicine, Shanghai Jiao
Tong University, for technical assistance on the p53−MDM2/
X binding assay.
ABBREVIATIONS USED
■
MDM2, murine double minute 2; FP, fluorescence polar-
ization; MD, molecular dynamics; SAR, structure−activity
relationship; ECD, electronic circular dichroism; DIAD,
diisopropyl azodicarboxylate; IC50, 50% inhibitory concen-
tration; IRT, irinotecan; DOX, doxorubicin; p-p53, phosphor-
ylation-p53; FBS, fetal bovine serum; RMSD, root-mean-square
deviation; NBS, nonbinding surface; RTV, relative tumor
volume
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