Discovery of potent and simplified piperidinone-based inhibitors of the MDM2-p53 interaction

Article abstract of DOI:10.1021/ml500142b

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

Full text of DOI:10.1021/ml500142b

Letter
pubs.acs.org/acsmedchemlett
Discovery of Potent and Simplified Piperidinone-Based Inhibitors of
the MDM2−p53 Interaction
Ming Yu,*,† Yingcai Wang,† Jiang Zhu,† Michael D. Bartberger,§ Jude Canon,∥ Ada Chen,† David Chow,†
John Eksterowicz,† Brian Fox,† Jiasheng Fu,† Michael Gribble,† Xin Huang,⊥ Zhihong Li,†
Jiwen (Jim) Liu,† Mei-chu Lo,† Dustin McMinn,† Jonathan D. Oliner,∥ Tao Osgood,∥ Yosup Rew,†
Anne Y. Saiki,∥ Paul Shaffer,⊥ Xuelei Yan,† Qiuping Ye,‡ Dongyin Yu,§ Xiaoning Zhao,† Jing Zhou,†
Steven H. Olson,† Julio C. Medina,† and Daqing Sun*,†
†Departments of Therapeutic Discovery and ‡Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South
San Francisco, California 94080, United States
§Departments of Therapeutic Discovery and ∥Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California
91320, United States
⊥Department of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Continued optimization of the N-substituent in the
piperidinone series provided potent piperidinone−pyridine inhibitors 6,
7, 14, and 15 with improved pharmacokinetic properties in rats.
Reducing structure complexity of the N-alkyl substituent led to the
discovery of 23, a potent and simplified inhibitor of MDM2. Compound
23 exhibits excellent pharmacokinetic properties and substantial in vivo
antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.
KEYWORDS: MDM2, p53, protein−protein interaction, piperidinone, pyridine
rotein p53 has been recognized as the “guardian of the
1
Pgenome” and is a main cell tumor suppressor. It induces
the cell growth arrest and apoptosis in response to DNA
damage or stress.2,3 In about 50% of human cancers, p53 is
mutated or deleted resulting in loss of it functions. The wild-
type p53 in the remaining 50% of malignancies is regulated by
human murine double minute 2 (MDM2) oncoprotein4,5
a
Figure 1. Chemical structure and potency of 1. IC50 in the HTRF
through three main mechanisms. First, MDM2 directly binds to
and blocks the N-terminal transcriptional activation domain of
p53. Second, MDM2 promotes export of p53 from the nucleus
to the cytoplasm. Finally, MDM2 induces degradation of p53
via ubiquitination through its E3 ligase activity.6 Since these
mechanisms can be blocked by neutralizing the MDM2−p53
interaction, disrupting this interaction has emerged as a
promising strategy to reactivate the p53 pathway.7 To date,
studies with small molecule MDM2 inhibitors have demon-
strated complete tumor regression in vivo.8−10 Several of these
molecules have been tested in the clinic for the treatment of
cancer.11−14
binding assay.16
cell lines in the EdU cell proliferation assay (IC50 = 0.073
μM);16 however, it suffered a high clearance (CL = 3.5 L/h/kg)
and poor bioavailability (%F = 12) in mouse. The high potency
of 1 can be rationalized by the cocrystal structure of 1 bound to
MDM2.15 Compound 1 occupies the three critical binding
pockets of Leu26(p53), Trp23(p53), and Phe19(p53). The C5 aryl
group reaches deep into the Leu26(p53) pocket and engages in a
face-to-face π-stacking interaction with H96, while the C6 aryl
group fills the Trp23(p53) binding cavity. The ethyl group is
directed into the Phe19(p53) pocket by the conformational
We previously reported the discovery of AM-8553 (1)15
(Figure 1) as a potent and selective inhibitor of the MDM2−
p53 interaction. Compound 1 substantially inhibited the
MDM2−p53 interaction in the biochemical HTRF binding
assay (IC50 = 1.1 nM) and the growth of human SJSA-1 tumor
Received: April 8, 2014
Accepted: June 30, 2014
© XXXX American Chemical Society
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constraint induced by the hydroxyethyl moiety. In addition, the
carboxylate anion forms an electrostatic interaction with the
imidazole on the H96 side chain of MDM2. Interestingly, the
hydroxyl group in 1 is found to have no direct interaction with
the protein but project toward the solvent. Analysis of the
cocrystal structure suggested that the hydrophobic region
adjacent to the Phe19 binding pocket and to the right of the N-
alkyl group, might accommodate structure modifications. On
the basis of this structure information, we conducted extensive
modification of the N-alkyl substituent in 1. These efforts led to
the discovery of AMG 232, a new member of piperidinone
MDM2 inhibitors. This compound is currently being evaluated
in human clinical trials for the treatment of cancer.10 In parallel
to these efforts, an alternative strategy was pursued for the
optimization of the N-alkyl substituent to improve metabolic
stability and reduce structure complexity. In this article, we
describe the continued optimization of the N-alkyl substituent,
which led to the discovery of 23, a potent and simplified
MDM2 inhibitor with improved pharmacokinetic properties in
rodents and excellent in vivo efficacy in the SJSA-1
osteosarcoma xenograft mouse model as well.
Table 1. Heterocycle Piperidinone Derivatives
The optimization of the N-alkyl substituent began with
methylation of the secondary alcohol in 1. The resultant
compound 2 (Table 1) exhibited similar potency (HTRF IC50
= 0.002 μM) as the parent 1 in the biochemical assay.
Expanding the methoxy to a tetrahydrofuran (THF) produced
compound 3, which was slightly more potent (HTRF IC50
=
0.001 μM) than 2 in both biochemical and cell-based EdU
assays, suggesting that the region occupied by the hydroxyethyl
element of 1 might accommodate structural diversity (Figure
2). Inspired by tolerance of the THF ring in the region, we
started to search for a heterocycle as a THF replacement in aim
to eliminate the associated chirality and to improve potency.
The optimization of the N-alkyl substituent began with
methylation of the secondary alcohol in 1. The resultant
compound 2 (Table 1) exhibited a similar potency (HTRF IC50
= 0.002 μM) as the parent 1 in the biochemical assay.
Expanding the methoxy to a tetrahydrofuran (THF) produced
compound 3, which was slightly more potent (IC50 = 0.001
μM) than 2 in both biochemical and cell-based EdU assays,
suggesting that the region occupied by the hydroxyethyl
element of 1 might accommodate structural diversity. Inspired
by tolerance of the THF ring in the region, we started to search
for a heterocycle as a THF replacement in aim to eliminate the
associated chirality and improve potency.
a
Values were the means of more than two determinations; standard
b
c
deviation was 30%. See ref 16 for assay protocols. HS = human
d
e
serum. In the presence of 15% human serum. SJSA-1 cell line.
f
g
Dosed iv: 0.5 mg/kg. Not determined.
As summarized in Table 1, a small group of 5- or 6-
membered heterocycles were explored. The oxazole 4 was
considerably less potent than 3 in both biochemical and cell-
based EdU assays, along with a high clearance of 5.7 L/h/kg in
rats. However, compound 5 with a less polar thiazole had a
maintained potency and a significantly low in vivo clearance in
rats (CL = 0.39 L/h/kg). Encouraged by the improved in vivo
metabolic stability of 5 in rats, we set out to optimize the
polarity around the heterocycles. This SAR campaign led to the
discovery of 2-pyridyl compound 6, which had an IC50 of 0.003
μM in the biochemical assay and a reduced potency shift (9-
fold) in the presence of human serum, as well as an IC50 of 0.43
μM in the cell-based EdU assay. Also of note is the fact that the
ostensible zwitterionic character of 6 did not seem to impair its
cellular permeability. This molecule was determined to have
low in vitro intrinsic clearance of 13.0 and 7.0 μL/min/106 cells
in rat and human hepatocyctes, respectively. Moreover,
compound 6 demonstrated excellent in vivo PK properties
Figure 2. Cocrystal structure of 1 bound to human MDM2 (17−111)
at 2.0 Å resolution. White labels indicate positions normally occupied
by key p53 residues. H96 is labeled in yellow. PDB code: 4ERF.
(CL = 0.23 L/h/kg, t1/2 = 3.5 h, and %F = 79) in rats. The 2-
pyrimidyl analogue 7 exhibited a potency (IC50 = 0.42 μM)
B
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comparable to 6 in the cellular EdU assay and reasonable in
vivo PK properties in rats (CL = 0.77 L/h/kg). Analogues with
the nitrogen atom located at distal positions (8 and 9)
maintained potency but yielded no improvement in the in vivo
clearance in rats.
Table 3. Fine Modification of the 2-Pyridyl Ring
With the 2-pyridine identified as the key feature for high
inhibition and good PK properties, numerous pyridine
derivatives were synthesized to define the alkyl group in the
Phe19(p53) pocket. As listed in Table 2, the methyl compound
Table 2. Modifications for the Phe19(p53) Pocket
ab
,
potency (IC50, μM)
HTRF
cellular
e
c
f
(−) HS
EdU
rat CL
d
c
compd
X
(+) HS
10%HS
(iv, L/h/kg)
6
H
0.003
0.028
0.002
0.032
0.43
0.23
14
15
16
17
18
19
6-Me
6-Cl
0.28
0.21
1.82
0.90
0.89
0.31
0.93
1.2
0.0007
0.023
0.005
0.228
0.007
0.123
0.006
0.060
0.005
0.183
ab
,
g
6-cPr
5-Me
4-Me
3-Me
ND
potency (IC50, μM)
HTRF
cellular
EdU
10%HS
0.43
c
e
g
(−) HS
ND
d
c
compd
R
(+) HS
g
6
Et
0.003
0.028
0.010
0.062
0.002
0.025
0.002
0.019
0.006
0.092
ND
g
10
11
12
13
Me
iPr
0.42
0.29
0.68
0.99
ND
a
Values were the means of more than two determinations; standard
b
c
deviation was 30%. See ref 16 for assay protocols. HS = human
cPr
nPr
d
e
serum. In the presence of 15% human serum. SJSA-1 cell line.
f
g
Dosed iv: 0.5 mg/kg. Not determined.
16 (X = 6-cPr), led to a potency decrease (IC50 = 0.005 μM).
Finally, compounds 17−19 illustrated our attempts at the other
positions of the ring as well. Overall, small groups were
tolerated at these positions but offered no advantage over 6.
The cocrystal structure of 16 bound to MDM2 was obtained
by X-ray crystallography to a resolution of 1.7 Å (Figure 3).17
Compound 16 binds to MDM2 in the similar fashion as
a
Values were the means of more than two determinations; standard
b
c
deviation was 30%. See ref 16 for assay protocols. HS = human
serum. In the presence of 15% human serum. SJSA-1 cell line.
d
e
10 was 2-fold less potent (IC50 = 0.010 μM) than 6 in the
biochemical assays but equally active in the cellular EdU assay
(IC50 = 0.42 μM). Expanding the ethyl group to an isopropyl
group (11) marginally improved the biochemical (IC50 = 0.002
μM) and cellular potencies (IC50 = 0.29 μM). Utilizing
cyclopropyl group at this position (12) led to no obvious
improvement in the intrinsic potency but slight decrease in
cellular activity (IC50 = 0.68 μM in the EdU assay). As
exemplified by 13, larger substituents were generally well
tolerated but yielded no noticeable benefit. Overall, this set of
compounds maintained high metabolic stability and generally
exhibited desirable in vivo PK profiles in rats (e.g., CL = 0.20
L/h/kg and t1/2 = 6.9 h for 13).
To further optimize the binding affinity of 6 to MDM2, we
explored a wide range of substitutions for the 2-pyridyl
component in 6. As shown in Table 3, compound 14, featuring
6-methyl substitution, was equipotent to 6 in both biochemical
(IC50 = 0.002 μM) and cellular EdU assays (IC50 = 0.28 μM)
and had a moderate in vivo clearance of 0.93 L/h/kg in rats.
Changing the methyl group (14) to a chlorine (15) at the 6-
position resulted in a noticeable improvement of the intrinsic
potency (IC50 = 0.0007 μM). However, this advantage was not
well preserved in the human serum presented biochemical assay
(IC50 = 0.023 μM) or the cell-based EdU assay (IC50 = 0.21
μM). Increasing the size of the 6-substituent, as exemplified by
Figure 3. Cocrystal structure of 16 bound to human MDM2 (17−
111) at 1.7 Å resolution. White labels indicate positions normally
occupied by key p53 residues. H96 is labeled in yellow. PDB code:
4QO4.
C
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described for 1, occupying the three critical hydrophobic
binding pockets and engaging in interaction with H96 as well.
The 3-chlorophenyl group fills the Leu26(p53) pocket and
engages in a face-to-face π-stacking interaction with H96, the 4-
chlorophenyl group occupies the Trp23(p53) binding cavity. In
addition, the carboxylate of 16 interacts with the imidazole of
the H96 side chain of MDM2. The cyclopropylpyridine of 16
binds to the shelf region between Phe55 and Gly58, with the
cyclopropyl moiety settled at a distance of 3.9 Å from the meta-
chloro phenyl group. Importantly, the pyridyl moiety directs
the ethyl group into the Phe19(p53) pocket to maximize the
hydrophobic contact.
While the explorations discussed so far had led to the
identification of several potent piperidinone−pyridine inhib-
itors (e.g., 6, 7, 14, and 15) with decent in vivo PK properties
in rats, unfortunately, these molecules were found metabolically
unstable in mouse (e.g., iv, CL = 3.1 L/h/kg and t1/2 = 1.9 h for
6). Therefore, they were not suitable for further pharmacody-
namic studies in the SJSA-1 tumor xenografted mouse model.
To eliminate the potential metabolic liability, we decided to
reduce the structural complexity of this class of MDM2
inhibitors. With the N-alkyl substituent being truncated into a
2-pyridyl ring (Table 4), the resulting compound 20 was 3-fold
of the N-alkyl substituent. Replacing the N-phenyl moiety in 21
with a N-3-pentyl or N-isopropyl group resulted in compounds
22 and 23, which were as nearly potent as 6 in the serum-free
biochemical assay and the cell-based EdU assay.18 Furthermore,
both 22 and 23 had a lower intrinsic clearance in rat
hepatocytes (18.8 μL/min/106 cells for 22 and 8.5 μL/min/
106 cells and 23) as compared to 1 (21 μL/min/106 cells).
Consistent with their improved metabolic stability in rat
hepatocytes, compounds 22 and 23 showed low in vivo
clearance (0.45 and 1.04 L/h/kg for 22 and 23, respectively)
and good oral bioavailability (38% and 48% for 22 and 23,
respectively) in rats. Similarly, compounds 22 and 23 displayed
a substantially lower clearance (0.99 and 1.03 L/h/kg for 22
and 23, respectively) and higher oral exposure (42% for 22 and
32% for 23) compared to 1 in the mouse.19
As a result of the cellular potency (EdU IC50 = 0.38 μM) and
excellent oral exposure in mouse, a pharmacodynamic (PD)
assay20 with the SJSA-1 osteosarcoma tumor cells was used to
assess the effects of 23 on the activation of p53 pathway. SJSA-1
tumor cells were implanted in mice 2 weeks prior to treatment.
Compound 23 was orally administered at a single dose of 100
or 200 mg/kg, and p21 mRNA levels, a transcriptional target
and pharmacodynamic readout for p53 activity, were measured
over time, relative to the vehicle control. In this experiment, 14-
fold induction of p21 over the vehicle was observed
approximately 4 h after the 100 mg/kg of 23 was dosed orally,
while the 200 mg/kg dose group achieved 15-fold p21
induction from 4 h postdose out to 8 h (Figure 4a). In a
Table 4. Structure Simplicity-Driven Modifications
a
Values were the means of more than two determinations; standard
b
c
deviation was 30%. See ref 16 for assay protocols. HS = human
d
e
serum. In the presence of 15% human serum. SJSA-1 cell line.
f
g
Dosed iv: 0.5 mg/kg. Not determined.
less potent (IC50 = 0.013 μM) than 6 in the serum-free
biochemical assay. Switching the aromatic ring from 2-pyridyl
to phenyl (21) resulted in a considerable improvement in
binding affinity (HTRF IC50 = 0.004 μM). Also of note is that
compound 21 was fairly comparable (IC50 = 0.055 μM) to 6 in
the human serum mediated biochemical assay as well. These
findings prompted us to further reduce the structure complexity
Figure 4. Pharmacodynamic study: treatment with 23 caused time-
and dose-dependent induction of p21 mRNA in SJSA-1 tumor
xenografts. *p < 0.05. (a) Vehicle and 100 or 200 mg/kg of 23 was
administered orally once. (b) Vehicle and 50, 100, or 200 mg/kg of 23
was administered orally once.
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parallel experiment, a dose-dependent increase in p21 mRNA
induction was also observed (Figure 4b). These data confirm
that compound 23 achieved an on-target activation of the p53
pathway and provided dose-selection guidance for the xenograft
study. Similarly, a time- and dose-dependent increase in p21
mRNA induction was observed with 22 in this PD model (data
not shown).
AUTHOR INFORMATION
■
Corresponding Authors
*(M.Y.) Tel: 650-244-2050. Fax: 650-837-9369. E-mail: yum@
amgen.com.
*(D.S.) Tel: 650-244-2195. E-mail: daqings@amgen.com.
Notes
In the SJSA-1 osteosarcoma xenograft mouse model,19
compound 23 substantially inhibited tumor growth at each
designed dose compared to the vehicle (Figure 5). The 100
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Iain Campuzano, Dr. Manuel Ventura, and
Dhanashri Bagal for the acquisition of high resolution mass
spectra.
REFERENCES
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mg/kg QD and 50 mg/kg BID doses of 23 caused 96% and
82% tumor growth inhibition (TGI), respectively. Tumor
regression was observed in the groups of 200 mg/kg QD and
100 mg/kg BID before animals were taken down due to severe
body weight loss.
The synthetic procedures and characterization data for
compounds 2−23 are provided in the Supporting Information.
In summary, continued optimization of the N-alkyl
substituent in 1 led to the potent piperidinone−pyridine
inhibitor 6 with excellent PK properties in rats. The cocrystal
structure of pyridine analogue 16 bound to MDM2 confirmed
the key interactions of the inhibitor with MDM2. Reducing
structure complexity of the N-alkyl substituent led to the
discovery of 23, a potent and simplified inhibitor of MDM2.
Compound 23 exhibited improved pharmacokinetic properties
in mouse as compared to 1 and 6 and demonstrated substantial
in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft
mouse model, albeit showing signs of toxicity at high doses in
the efficacy study.
ASSOCIATED CONTENT
* Supporting Information
■
S
(i) In vitro biological assays; (ii) in vivo study protocols; (iii)
determination of cocrystal structures of 16 with MDM2; (iv)
pharmacokinetic profiles of 1, 22, and 23 in rats and mouse; (v)
synthetic experimental procedure and characterization data for
compounds 2−23. This material is available free of charge via
the Internet at http://pubs.acs.org.
(13) Ding, Q.; Zhang, Z.; Liu, J. J.; Jiang, N.; Zhang, J.; Ross, T. M.;
Chu, X. J.; Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Zoran M.
Filipovic, Z. M.; Higgins, B.; Glenn, K.; Packman, K.; Vassilev, L. T.;
Graves, B. Discovery of RG7388, a potent and selective p53−MDM2
inhibitor in clinical development. J. Med. Chem. 2013, 56, 5979−5983.
E
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(14) Carry, J.; Garcia-Echeverria, C. Inhibitors of the p53/hdm2
protein−protein interaction: path to the clinic. Bioorg. Med. Chem. Lett.
2013, 23, 2480−2485.
(15) Rew, Y.; Sun, D.; Gonzalez Lopez De Turiso, F.; Bartberger, M.
D.; Beck, H. P.; Canon, J.; Chen, A.; Chow, D.; Deignan, J.; Fox, B.
M.; Gustin, D.; Huang, X.; Jiang, M.; Jiao, X.; Jin, L.; Kayser, F.;
Kopecky, D. J.; Li, Y.; Lo, M.; Long, A. M.; Michelsen, K.; Oliner, J.
D.; Osgood, T.; Ragains, M.; Saiki, A. Y.; Schneider, S.; Toteva, M.;
Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Zhao, X.; Zhou, J.; Medina, J. C.;
Olson, S. H. Structure-based design of novel inhibitors of the MDM2−
p53 interaction. J. Med. Chem. 2012, 55, 4936−4954.
(16) Experimental details of in vitro assays can be found in the
Supporting Information.
(17) The atomic coordinates are deposited in the Protein Data Bank
under an accession code 4QO4.
(18) To evaluate p53 selectivity, we examine the effect of the
piperidinone inhibitors on inhibiting the proliferation of HCT116
p53wt and p53−/− tumor cells in the BrdU assay. All of the
piperidinone MDM2 inhibitors evaluated in the BrdU assay with
HCT116 p53wt and p53−/− tumor cells display significant inhibition of
cell proliferation in the p53wt tumor cells, but no inhibition in p53−/−
tumor cells in concentrations up to 25 μM. For example, potency of 1
in wild-type p53 cells (IC50 = 0.20 μM) was substantially higher than
that in p53 deficient cells (IC50 > 25 μM). No p53 selectivity data was
collected for 23.
(19) Detailed rat and mouse PK profiles can be found in the
Supporting Information.
(20) Experimental details of in vivo studies can be found in the
Supporting Information.
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