Potent and orally active small-molecule inhibitors of the MDM2-p53 interaction

Article abstract of DOI:10.1021/jm901400z

We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a K _i of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits

Full text of DOI:10.1021/jm901400z

7970 J. Med. Chem. 2009, 52, 7970–7973
DOI: 10.1021/jm901400z
Potent and Orally Active Small-Molecule
Inhibitors of the MDM2-p53 Interaction
Shanghai Yu,†,§ Dongguang Qin,†,§ Sanjeev Shangary,†
Jianyong Chen,† Guoping Wang,† Ke Ding,†
Donna McEachern,† Su Qiu,† Zaneta Nikolovska-Coleska,†
Rebecca Miller,† Sanmao Kang,‡ Dajun Yang,‡ and
Shaomeng Wang*,†
†Comprehensive Cancer Center and Departments of Internal
Medicine, Pharmacology, and Medicinal Chemistry, University of
Michigan, 1500 E. Medical Center Drive, Ann Arbor, Michigan
48109, and ‡Ascenta Therapeutics Inc.,101 Lindenwood Drive,
Malvern, Pennsylvania 19355. §Equal contributions.
Received September 20, 2009
Abstract: We report herein the design of potent and orally active
small-molecule inhibitors of the MDM2-p53 interaction. Com-
pound 5 binds to MDM2 with a Ki of 0.6 nM, activates p53 at
concentrations as low as 40 nM, and potently and selectively inhibits
cell growth in tumor cells with wild-type p53 over tumor cells with
mutated/deleted p53. Compound 5 has a good oral bioavailability
and effectively inhibits tumor growth in the SJSA-1 xenograft
model.
Figure 1. Chemical structures of potent MDM2 inhibitors.
Table 1. Binding Affinities of MDM2 Inhibitors to MDM2 and Their
Activity in Cancer Cell Lines with Wild-Type p53 Status and Selectivity
in Cancer Cell Lines with p53 Deletion
binding affinity
to MDM2
IC50 (μM) in cell
growth inhibition assay
cellular
Saos-2
(p53
deleted)
selectivity
(SJSA-1/
Saos-2)
The p53 tumor suppressor is an attractive cancer therapeu-
tic target because its tumor suppressor activity can be stimu-
lated to eradicate tumor cells.1-3 In tumor cells with wild-type
p53, the p53 activity is effectively inhibited by its endogenous
inhibitor, the human MDM2 protein, through direct binding
to p53.4,5 The interaction site between MDM2 and p53
proteins is mediated by a well-defined pocket in MDM2 and
a short helix from p53, making this site attractive for the
design of small-molecule inhibitors to block the MDM2-p53
protein-protein interaction.6,7 Reactivation of p53 by block-
ing the MDM2-p53 interaction using a small-molecule in-
hibitor is being pursued as an exciting, new cancer therapeutic
strategy.8-22
We have recently reported the design of spirooxindoles as a
new class of potent, selective, cell permeable, nonpeptidic,
small-moleculeinhibitorsof theMDM2-p53 interaction.9-11
Using a structure-based approach, we have obtained 1
(MI-63, Figure 1) as a potent and cell-permeable MDM2
inhibitor. Compound 1 binds to MDM2 protein with a low
nanomolar affinity in our fluorescence-polarization (FP)
based, competitive, biochemical binding assay.10 Consistent
with its mode of action, 1 potently inhibits cell growth in
cancer cellswith wild-type p53 and is selective over cancer cells
with mutated/deleted p53. In our subsequent pharmaco-
kinetic (PK) evaluations, 1 was found to have a poor PK
profile and a modest oral bioavailability (Table 2). Hence, 1 is
not a suitable candidate for drug development.
IC50 ( SD Ki ( SD
SJSA-1
(p53 WT)
compd
(nM)
(nM)
1
2
3
4
5
6
7
8
9
28.3 ( 5.5 1.7 ( 0.5
25.7 ( 4.3 1.5 ( 0.4
32.4 ( 6.0 2.0 ( 0.5
32.4 ( 6.0 0.8 ( 0.3
16.4 ( 0.3 0.6 ( 0.1
56.0 ( 4.7 4.0 ( 0.4
162 ( 75
157 ( 13
189 ( 56
0.5 ( 0.1 14.1 ( 1.3
0.3 ( 0.0 4.6 ( 0.7
0.3 ( 0.0 3.3 ( 0.3
0.7 ( 0.2 14.2 ( 3.4
0.2 ( 0.0 18.2 ( 0.8
0.6 ( 0.2 28.8 ( 4.7
28
15
11
20
91
48
22
24
18
13.1 ( 6.4 0.7 ( 0.1 15.4 ( 0.8
12.6 ( 1.1 1.7 ( 0.1 40.5 ( 3.8
15.4 ( 4.8 1.0 ( 0.2 17.8 ( 1.4
inhibitors of the MDM2-p53 interaction with excellent oral
bioavailability.
Analysis of the predicted binding model for 1 showed that
the morpholinyl group is partially exposed to solvent.10 This
suggested that the morpholinyl group in 1 may be replaced by
other groups without a detrimental effect on binding to
MDM2 and cellular activity. We have therefore carried out
chemical modifications of this region to investigate the struc-
ture-activity relationship on binding, cellular activity, and
PK parameters.
We first designed and synthesized 2 and 3 (Figure 1), in
which the morpholinyl group in 1 is replaced by a methylpi-
perazinyl group or a methylpiperidinyl group, respectively.
Our binding experiments showed that these two compounds
bind to MDM2 with Ki of 1.5 and 2.0 nM, respectively
(Table 1). Consistent with their high binding affinities to
MDM2, they potently inhibit cell growth in the cancer cell
lines with wild-type p53 and display excellent selectivity over
cancer cell lines with deleted p53 (Table 1 and Supporting
Information). However, PK testing showed that while 2 and 3
have an improved PK profile over 1, the cmax and AUC values
are still relatively low with oral dosing (Table 2).
Herein, we report our design, synthesis, and evaluation of
new analogues of 1 with a goal to discover potent MDM2
inhibitors with improved oral bioavailability and overall PK
parameters. Our efforts yielded new, potent small-molecule
*To whom correspondence should be addressed. Phone: 734-615-
0362. Fax: 734-647-9647. E-mail: shaomeng@umich.edu.
r
pubs.acs.org/jmc
Published on Web 11/24/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7971
Table 2. PK Parameters of MDM2 Inhibitors in Rats with Oral
Dosinga
oral
dose
Cmax
(ng/mL)
AUC (0ft)
(h μg/L)
T1/2
(h)
F
(%)
compd (mg/kg)
3
1
2
3
4
5
6
7
8
9
25
25
25
25
50
50
25
25
25
248 ( 176
274 ( 82
120 ( 112
97 ( 28
1514 ( 905
805 ( 158
3751 ( 1068 7677 ( 328
3548 ( 934
2067 ( 467
321 ( 154
1888 ( 515
1078 ( 891
145 ( 30
8769 ( 5178
4110 ( 1872
1.3 ( 0.2
3.8 ( 0.5
7.1 ( 1.8
1.2 ( 0.2
3.9 ( 1.6
3.0 ( 0.4
1.4 ( 0.1
10
31
14
5
21
19
65
31
18
11746 ( 2337 3.4 ( 0.5
5094 ( 1533 3.2 ( 2.8
Figure 2. Dose-dependent p53 activation induced by 5 in SJSA-1
and HCT-116 cell lines and its specificity in HCT-116 isogenic p53
knockout cell line.
a Cmax: maximum concentration of the compound detected in plasma.
AUC: area under the curve. T1/2: terminal half-life. F: oral bioavail-
ability.
of cell growth in the SJSA-1 and HCT-116 cell lines with wild-
type p53 (Table 1 and Supporting Information). Compound
7, however, has a much improved PK profile with oral dosing
over 6. Compound 7 at 25mg/kg oraldosingachieves acmax of
In 1, 2, and 3, the morpholinyl group, the methylpiperazinyl
group, and the methylpiperidinyl group are protonated and
positively charged at physiological conditions. We hypothe-
sized that these charged groups may contribute to the low cmax
and AUC values with oral dosing for these compounds. We
therefore designed 4 and 5 to test if replacement of a positively
charged group with a neutral group can improve the oral
bioavailability without compromising binding affinity to
MDM2 and cellular activity.
Compounds 4 and 5 have Ki of 0.8 and 0.6 nM to MDM2
protein in our binding assay (Table 1). Both compounds also
potently inhibit cancer cell growth in cancer cell lines with
wild-type p53 and display good selectivity over cancer cell
lines with deleted p53 (Table 1 and Supporting Information).
For example, 5 achieves an IC50 of 0.2 μM in inhibition of cell
growth in the SJSA-1 osteosarcoma cell line with wild-type
p53 and displays a selectivity of 91 times over Saos-2 osteo-
sarcoma cell line with deleted p53 in the same assay (Table 1).
Despite their similar potencies in binding and cellular
assays, 4 and 5 have very different PK profiles with oral
dosing. Compound 4 with a 1-ethoxy-2-methoxyethanyl tail
has a very poor PK profile, as indicated by its low AUC and
cmax values. In contrast, 5 with a butyl-1,2-diol tail has a
significantly improved PK profile over 1, 2, and 3. Its cmax at
50 mg/kg oral dosing reaches 1514 ng/mL, and it has an AUC
3751 ng/mL (6.4 μM), AUC of 7677 h mg/L, and an oral
3
bioavailability of 65%.
Using 7 as the template, we performed additional modifica-
tions on the butyl-1,2-diol tail to further explore the struc-
ture-activity relationship at this site on binding, cellular
activity, and PK parameters. Change of the chiral center in
the tail from the S-configuration to R-configuration leads
to 8. Compound 8 has the same binding affinity to MDM2 as
7 but is slightly less potent than 7 in cell growth inhibition in
the SJSA-1 and HCT-116 cell lines (Table 1 and Supporting
Information). Compounds 7 and 8 also have very similar PK
profiles in terms of their cmax and AUC values at 25 mg/kg
oral dosing. These data indicate that the chirality does not
have a significant impact on the binding affinity to MDM2,
cellular activity, and PK parameters.
We next examined the effect of shortening the tail from
butyl-1,2-diol to propyl-1,2-diol, which leads to 9. Compound
9 is as potent as 7 and 8 in binding to MDM2 and in cell
growth inhibition in SJSA-1 and HCT-116 cancer cell lines
with wild-type p53 (Table 1 and Supporting Information).
Our PK studies, however, showed that9 is slightly inferior to 7
and 8 in terms of cmax and AUC values.
of 8769 h mg/L. We did not evaluate 5 at 25 mg/kg oral
We tested 5 for its ability to activate p53 in cancer cells by
Western blot analysis (Figure 2). Compound 5 effectively and
dose-dependently activates p53 in the SJSA-1 cancer cell line
with wild-type p53 and overexpressed MDM2 at concentra-
tions as low as 40 nM, as evident by the robust increase of p53
protein, as well as the MDM2 and p21 proteins, two p53
targeted gene products. Compound 5 also activates p53 and
induces an increase of MDM2 and p21 proteins in the HCT-
116 cancer cell line with wild-type p53 in a dose-dependent
manner but not in the isogenic HCT-116 cell line with deleted
p53 (Figure 2).
We next compared 5, 7, and racemic nutlin-3 for their
ability to activate p53 in the SJSA-1 cell line (Figure 3A).
Both 5 and 7 effectively and dose-dependently activate p53 in
the SJSA-1 cancer cell lines with wild-type p53, evident by
robust increase of p53 protein, as well as MDM2 and p21
proteins. Compound 5 is more potent than 7, and both
compounds are more potent than racemic nutlin-3, the first
bona fide potent MDM2 inhibitor reported by Vassilev and
colleagues.8 The levels of p53 activation by 5 at 0.5 μM are
similar to those observed by 7 at2.5 μM and by 10 μM racemic
nutlin-3. In contrast, MI-61 at 10 μM, a previously reported
3
dosage, thereby preventing us from direct comparison of the
improvement over 1. However, if we assume a dose-linearity
for the PK parameters of 5, its cmax and AUC have improved
by 3 and 10 times, respectively, over 1.
Using 5 as the new lead compound, we next designed and
synthesized 6 to evaluate the effect of the 2-F substitution in
the phenyl ring on in vitro activity and PK parameters. While
6 still potently binds to MDM2, it is 7 times less potent than 5
(Table 1). Consistent with its weaker binding affinity to
MDM2, 6 is 2-3 times less potent than 5 in cell growth
inhibition inthe SJSA-1 andHCT-116 cell lines withwild-type
p53 (Table 1 and Supporting Information). PK evaluations
showed that AUC and cmax for 6 are 2 times lower than those
for 5. Hence, we conclude that the 2-F substitution in the
phenyl ring makes a positive impact on binding, cellular
activity, and PK parameters in 5.
We next designed 7 based on the chemical structure of 6 to
examine the effect of a 4-F substitution in the oxindole ring on
binding, cellular activity, and PK parameters. In direct com-
parison, 7 is4 times less potent than 6 inits binding toMDM2.
Interestingly, 7 is only slightly less potent than 6 in inhibition

7972 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Yu et al.
Figure 3. Western blot analysis of p53 activation induced by 5 and
7. MI-61 was used as an inactive control, whereas racemic nutlin-3
was used as a positive control.
Figure 5. Antitumor activity of 5 alone and in combination with
irinotecan in the SJSA-1 xenograft model: (A) tumor volume; (B)
animal weight.
death at the same concentration. The cell death induction is
specific and p53-dependent, since both compounds at 10 μM
have minimal effect on the cell viability in the Saos-2 cell line
with deleted p53 (Figure 4). The effective cell-death induction
by 5 is also consistent with its ability to up-regulate Puma and
Noxa, two potent proapoptotic proteins.
To further test the therapeutic potential for 5, we evaluated
its ability to inhibit tumor growth in the SJSA-1 xenograft
model (Figure 5). Compound 5 is highly effective in inhibition
of tumor growth as an oral agent. At the end of the treatment
(day 26), tumors treated with the vehicle control grew to a
mean volume of 1328 mm3 from 95 ( 21 mm3 at the start of
the treatment (day 13). In comparison, tumors treated with
daily, oral dose of 5 at 300 mg/kg for 14 days only grew to 308
( 283 mm3 at the end of the treatment from 100 ( 33 mm3.
The T/C (treatment/control) for 5 is 19% when the mean
tumor volume in the control group reached 750 mm3
(Supporting Information). Irinotecan at a weekly 100 mg/kg
intraperitoneal dose for 2 weeks, a near maximum tolerated
dose, also achieved significant tumor growth inhibition and a
T/C of 20.5% when the mean tumor volume in the control
group reached 750 mm3 (Supporting Information). The com-
bination of 5 and irinotecan is statistically more effective than
either agent alone (Supporting Information). The combina-
tion is able to completely inhibit tumor growth during treat-
ment; the mean tumor volume at the end of treatment is 98 (
47 mm3, which is the same as that at the start of the treatment
(97 ( 29 mm3). There is no significant weight loss in animals
treated with 5 alone and in combination with irinotecan,
compared to those in the vehicle control group (Figure 5B).
There are no other signs of toxicity in animals treated with 5
throughout the experiment.
Figure 4. Induction of cell death by 5 and 7 in the SJSA-1 cancer
cell line with wild-type p53 status and in the aOS-2 cancer cell line
with deleted p53.
inactive control of compound 7,11 has little effect in induction
of an accumulation of p53, MDM2, and p21 compared to
untreated control, indicating the specific effect by 5 and 7.
Compounds 5 and 7 fail to induce MDM2 and p21 in the
Saos-2 cell line with deleted p53 (Figure 3B), consistent with
their mechanism of action as potent and specific inhibitors of
the MDM2-p53 interaction (Figure 3B).8,11 Compound 5
also effectively induces an increase of Bax, Puma, and Noxa
in the SJSA-1 cancer cells, which are three other p53-targeted
gene products and are all proapoptotic Bcl-2 members, in a
dose-dependent manner (Figure 3C). A robust increase of
Noxa and Puma proteins is observed with 0.37 and 3.3 μM of
5, respectively (Figure 3C).
Activation of p53 by potent and cell-permeable MDM2
inhibitors can effectively induce tumor cells to undergo cell
death.8,11 Indeed, 5 and 7 are capable of inducing cell death in
the SJSA-1 cell line with wild-type p53 in a dose-dependent
manner (Figure 4). Compound 5 is more potent than 7,
consistent with their potencies in cell growth inhibition and
in induction of p53 activation. Compound 5 at 1.25 μM is able
to induce 50% of cells to undergo cell death in the SJSA-1 cell
line for 2-day treatment, whereas 7 only induces 20% of cell
The synthesis of these new compounds is similar to that
reported for 1,10 andthe detailsareprovided in the Supporting
Information.
We have previously performed extensive biological and
pharmacological studies for 7.11 Our data demonstrated that
7 activates the p53 function in tumor cells with wild-type p53

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7973
by blocking the MDM2-p53 protein-protein interaction.
While 7 also activates p53 in normal cells, it selectively induces
tumor cells with wild-type p53 to undergo cell death and
apoptosis but not in normal cells. In vivo, 7 induces p53
activation in xenograft tumor tissues with wild-type p53.
While 7 also induces p53 activation in normal mouse tissues,
it is selectively toxic to tumor tissues but not to normal mouse
tissues, even after a total of 28 doses. In contrast, irinotecan
and irradiation are toxic to certain normal mouse tissues.
Collectively, our current and previous studies have provided
strong evidence that reactivation of p53 using small-molecule
inhibitors is a promising new cancer therapeutic strategy.
Although targeting protein-protein interaction using non-
peptidic, small molecules has proven to be a very challenging
task in modern drug discovery and medicinal chemistry, our
present study has provided solid proof that it is feasible to
design potent, cell-permeable, and orally active small-mole-
cule inhibitors of the MDM2-p53 interaction. Compound 5
is arguably the most potent, specific, cell-permeable, and
orally active small-molecule inhibitor discovered to date and
represents a promising lead compound for further evaluation
as a new class of anticancer drug.
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Acknowledgment. We are grateful for financial support from
the National Cancer Institute, National Institutes of Health
(Grants R01CA121279, P50CA06956, and P50CA097248),
the University of Michigan Cancer Center (Core Grant
P30CA046592), the Prostate Cancer Foundation, the Leukemia
and Lymphoma Society, and Ascenta Therapeutics, Inc.
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Ramachandren, K.; Bylebyl, G. R.; Zhang, M.; Manthey, C. L.;
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Supporting Information Available: Experimental section,
including chemical data for 2-9, details of the fluorescence
polarization-based binding assay, cell growth and cell viability
assays, Western blot analysis, and in vivo animal experiment.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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