Discovery of 1-arylpyrrolidone derivatives as potent p53-MDM2 inhibitors based on molecule fusing strategy

Article abstract of DOI:10.1016/j.bmcl.2014.04.063

Introducing an aryl moiety to our previous pyrrolidone scaffold by molecule fusing strategy afforded two sets of isopropylether-pyrrolidone and α-phenylethylamine-pyrrolidone derivatives. Two novel compounds 8b and 8g of the latter serial showed potent p5

Full text of DOI:10.1016/j.bmcl.2014.04.063

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2648–2650
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-arylpyrrolidone derivatives as potent p53–MDM2
inhibitors based on molecule fusing strategy
Jin Li a, , Yuelin Wu a,b, , Zizhao Guo a, Chunlin Zhuang a, Jianzhong Yao a, Guoqiang Dong a,
Zhiliang Yu a, Xiao Min a, Shengzheng Wang a, Yang Liu a, Shanchao Wu a, Shiping Zhu a,
a,
a,
Chunquan Sheng a, , Zhenyuan Miao , Wannian Zhang
⇑
⇑
⇑
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
b School of Chemical Engineering, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 December 2013
Revised 2 April 2014
Accepted 17 April 2014
Available online 26 April 2014
Introducing an aryl moiety to our previous pyrrolidone scaffold by molecule fusing strategy afforded two
sets of isopropylether–pyrrolidone and
a-phenylethylamine–pyrrolidone derivatives. Two novel com-
pounds 8b and 8g of the latter serial showed potent p53–MDM2 inhibitory activities with Ki values of
90 nM which were three-time higher than that of the parent compound. We also confirmed compound
8b can activate p53 proteins in lung cancer A549 cells. The results offered us valuable information for
further lead optimization.
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Arylpyrrolidone
p53–MDM2 inhibitor
Molecule fusing
Synthesis
The human tumor suppressor p53, as one of the most potent
transcriptional activator proteins, plays a protective role in normal
tissues. The p53 is also short-lived in normal cells under physiolog-
ical conditions and is mainly regulated by the murine double min-
ute 2 protein (MDM2).1–3 However, in approximately 50% of all
human cancers p53 has been found to be inactivated. Overexpres-
sion of MDM2 was discovered as the main reason for p53 function
impairment.4 Recently, restoration of p53 activity by inhibiting the
p53–MDM2 interaction has been demonstrated as a successful
strategy for anticancer drug development.5,6 To date, an increasing
number of small molecule inhibitors have been generated and
six excellent compounds RG7112, RO5503781, MK-8242,
SAR405838, CGN097 and DS-3032b have entered clinical trials.7–14
In 2012, Zhuang et al. discovered a novel lead structure with
pyrrolidone scaffolds by virtual screening method based on co-
crystal structures of p53–MDM2 interaction.15,16 Further hit opti-
mization and SAR led to the discovery of two compounds Z41
(Ki = 260.0 nM) and Z60a (Ki = 150.0 nM) which showed potent
antitumor efficacy both in vitro and vivo (Fig. 1). These optimized
compounds Z41 and Z60a showed the similar interaction manner
of MDM2 protein with benzoyl group and 4-bromophenyl group
located in the Trp23 pocket and Leu26 pockets. However, imidaz-
ole group could not work well in concert with Phe19 pocket.
According to binding mode of RG7112 which was the first genera-
tion small molecule inhibitor in clinical trial, two 4-chlorophenyl
groups respectively entered Trp23 pocket and Leu26 pocket, while
the 2-ethoxy-4-butylphenyl group projected into the Phe19 pocket
(PDB code 4IPF).7 In this paper, we present two novel classes of
small-molecule inhibitors of p53–MDM2 protein–protein interac-
tion based on pyrrolidone scaffold by a molecule fusing strategy
(Fig. 2). Fourteen compounds with substituted phenyl groups on
N1 position had synthesized and insight into the SAR of pyrroli-
dones was also revealed for further research.
The general synthetic route of target compounds is outlined in
Scheme 1. Benzylamines 3a–3d were prepared by the etherifica-
tion of 2-fluoro-4-(trifluoro-methyl)benzonitrile 1 and four ali-
phatic alcohols, and subsequently reduction by LiAlH4 in dried
ether in good yield. With these benzylamines in hand, compounds
6a–6g were readily obtained by an efficient three-component
coupling reaction.15 Followed Mitsunobu reaction and Leuckart–
Wallach reaction afforded isopropylether–pyrrolidone derivatives
7a–7g and
a-phenylethylamine–pyrrolidone derivatives 8a–8g,
respectively.
The binding constants Ki of all target compounds were mea-
sured by fluorescence polarization (FP) binding assay method. Nut-
lin-3 was used as the reference drug who was one of the most
active small-molecule p53–MDM2 inhibitors. The results were
⇑
Corresponding authors. Tel./fax: +86 21 81871241.
E-mail addresses: shengcq@hotmail.com (C. Sheng), miaozhenyuan@hotmail.
com (Z. Miao), zhangwnk@hotmail.com (W. Zhang).
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.04.063
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2648–2650
2649
O
S
O
Cl
Cl
O
O
O
O
N
HN
N
N
N
O
N
O
N
O
Br
Br
N
N
N
Z60a
RG7112
Z41
N
Figure 1. The first small molecule inhibitor RG7112 of p53–MDM2 in clinical trial and active pyrrolidones developed by our group.
O
S
O
Trp 23
Cl
Cl
O
N
N
O
O
O
HN
N
N
Z41
Z60a
O
O
N
N
O
R
R
Phe 19
Br
Br
Leu 26
RG7112
Figure 2. The molecule fusing strategy of 2-arylpyrrolidone derivatives.
R2
F
R1
CN
CN
NH2
ii
i
F3C
F3C
F3C
O
2a,3a:R1=OC(CH3)3
2b,3b:R1=OCH(CH3)2
2a-2d
3a-3d
1
1
2c,3c
2d,3d
:R =OCH2CH3
1
:R =OCH3
R1
O
OH
O
OH
CHO
O
NH2
iii
O
+
+
R2
X
N
Br
3a-3g
4
5
R2
Br
X
R1
3a,6a,7a,8a:R1=OC(CH3)3, R2=CF3, X=C
6a-6g
3b,6b,7b,8b:R1=OCH(CH3)2, R2=CF3, X=C
iv
1
2
3c,6c,7c,8c
3d,6d,7d,8d
:R =OCH2CH3, R =CF3, X=C
:R =OCH3, R =CF3, X=C
v
1
2
3e,6e,7e,8e:R1=F, R2=CF3, X=C
3f,6f,7f,8f:R1=H, R2=CF3, X=C
3g,6g,7g,8g:R1=H, R2=H, X=N
O
O
HN
N
O
O
O
N
R2
R2
Br
Br
X
X
R1
R1
7a-7g
8a-8g
Scheme 1. The synthetic route of 2-arylpyrrolidone derivatives 7a–7g and 8a–8g. Reagents and conditions: (i) NaH, ROH, 0 °C–25 °C, 55–64%; (ii) LiAlH4, Et2O, rt, 60–66%;
(iii) 1,4-dioxane, rt, 35–55%; (iv) PPh3, DIAD, THF, i-PrOH, rt, 41–59%; (v) CH3COOH, R-
a
-phenylethylamine, microwave, 120 °C, 12–15%.
Table 1
Table 2
MDM2 binding affinity of isopropylether–pyrrolidones 7a–7g
MDM2 binding affinity of
Compds Nutlin-3 8a
Ki ( M) 0.09 8.18 0.09 3.02 >100 >100 37.7 0.09 0.26
a
-phenylethylamine–pyrrolidones 8a–8g
Compds Nutlin-3 7a
7b
7c
7d
7e
7f
7g
Z41
8b 8c 8d 8e 8f
8g
Z60a
Ki ( M) 0.09 1.37 >100 0.72 0.71 >100 1.72 0.32 0.26
l
l
summarized in Tables 1 and 2. As expected, most isopropylether-
pyrrolidone derivatives indicated potent inhibitory activity with
derivatives showed potent p53–MDM2 inhibitory activity except
for 8d and 8e. To our delight, compounds 8b and 8g displayed
excellent p53–MDM2 inhibitory activity with Ki values of 90 nM
which were 3-time than that of the compound Z60a. As shown
in Figure 3, three aromatic substituents of compound 8g mimic
Ki values ranging from 0.26
compounds 7b and 7e failed to show p53–MDM2 binding inhibitor
activity. Similarly, all the -phenylethylamine–pyrrolidone
lM to 1.72 lM. However, two
a

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J. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2648–2650
affect of MDM2 proteins in A549 cells (Fig. 4), indicating a good
activation of p53.
In summary, a series of 2-arylpyrrolidone derivatives which
was designed by a molecule fusing strategy were prepared in four
steps with good yields. Among all targeted fourteen compounds,
two phenylethylamine–pyrrolidone derivatives 8b and 8g were
obtained with 3-time enhanced activities compared to parent com-
pound Z60a. It was noted that fluorine on position 3 of N1 phenyl
revealed disappear activity. In particular, compound 8b was
confirmed as a p53 proteins activator in A549 cells. Therefore,
this study offered us valuable information for further lead
optimization.
Acknowledgments
Figure 3. Binding modes of compound 8g in three key subpockets of MDM2. The
figure was generated using PyMol (http://pymol.sourceforge.net/).
This work was supported in part by the National Natural Sci-
ence Foundation of China (Grant 81373331), and the Bio-Pharma-
ceutical Project of Science and Technology of Shanghai (No.
13431900302).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.063.
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Phe19, Trp23 and Leu26 residues of p53 which is similar to the
binding mode of RG7112.7,15 Furthermore, both of the compounds
showed equivalent inhibitory activity compared to the most
famous active Nutlin-3. Structure–activity relationship analysis
demonstrated that the fluorine substitution of pyrrolidone reduced
inhibitory activity. For example, compounds 7e and 8e with fluo-
rine on position 3 of N1 phenyl group indicated absent activity at
100 lM.
The inhibition of the p53–MDM2 interaction is expected to acti-
vate p53, resulting in an increased level in the cells with wild-type
p53. To confirm whether target compounds can activate p53 and
increase the levels of MDM2 proteins in wild-type cells, compound
8b, as one of the most two active pyrrolidones, was selected for
Western blot assay. Consistent with the predictions, compound
8b caused a dose dependent increase in the levels of p53 and slight