A potent small-molecule inhibitor of the MDM2-p53 interaction (MI-888) achieved complete and durable tumor regression in mice

Article abstract of DOI:10.1021/jm4005708

We previously reported the discovery of a class of spirooxindoles as potent and selective small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors). We report herein our efforts to improve their pharmacokinetic properties and in vivo antitumor activity. Our efforts led to the identification of 9 (MI-888) as a potent MDM2 inhibitor (K_i = 0.44 nM) with a superior pharmacokinetic profile and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration and represents the most potent and efficacious MDM2 inhibitor reported to date.

Full text of DOI:10.1021/jm4005708

Article
pubs.acs.org/jmc
A Potent Small-Molecule Inhibitor of the MDM2−p53 Interaction (MI-
888) Achieved Complete and Durable Tumor Regression in Mice
Yujun Zhao,†,§ Shanghai Yu,†,§ Wei Sun,†,§ Liu Liu,† Jianfeng Lu,† Donna McEachern,† Sanjeev Shargary,†
Denzil Bernard,† Xiaoqin Li,‡ Ting Zhao,‡ Peng Zou,‡ Duxin Sun,‡ and Shaomeng Wang*,†
†Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, University of
Michigan, Ann Arbor, Michigan 48109, United States
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
ABSTRACT: We previously reported the discovery of a class of
spirooxindoles as potent and selective small-molecule inhibitors of the
MDM2−p53 interaction (MDM2 inhibitors). We report herein our
efforts to improve their pharmacokinetic properties and in vivo antitumor
activity. Our efforts led to the identification of 9 (MI-888) as a potent
MDM2 inhibitor (Ki = 0.44 nM) with a superior pharmacokinetic profile
and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid,
complete, and durable tumor regression in two types of xenograft models
of human cancer with oral administration and represents the most potent
and efficacious MDM2 inhibitor reported to date.
219)8 and 221 (Figure 1) bind to MDM2 with low nanomolar
affinities (Table 1) and are orally active in preclinical animal
INTRODUCTION
■
Tumor suppressor protein p53 is a transcription factor and
plays a key role in regulation of cell cycle, apoptosis, DNA
repair, senescence, and angiogenesis, among other cellular
processes.1−3 In approximately half of human cancers, TP53,
the gene encoding p53 protein, is deleted or mutated, which
renders loss of the tumor suppressor function of p53.4 In the
other half of human cancers, p53 retains wild-type status but its
tumor suppressor function is effectively inhibited by its primary
endogenous inhibitor, the murine double minute 2 protein
(MDM2, or HDM2 in humans).5 MDM2 inhibits the function
Figure 1. Two of our previously reported potent and orally active
of p53 by multiple mechanisms, but all are mediated by their
MDM2 inhibitors.
direct protein−protein interaction.5 The cocrystal structure of
MDM2 in complex with p53 has shown that their binding
models of human cancer. While both compounds inhibited
involves primarily three key residues in p53 (Phe19, Trp23, and
Leu26) and a well-defined, surface binding pocket in MDM2,6
suggesting the possibility of designing non-peptide, small-
molecule inhibitors to block the MDM2−p53 protein−protein
interaction (hereafter called MDM2 inhibitors). Such small-
tumor growth in the SJSA-1 xenograft model in mice, high oral
doses (200−300 mg/kg) were needed. Furthermore, these
MDM2 inhibitors failed to achieve complete tumor regres-
sion.8,21 Collectively, these data suggest that the pharmacoki-
netics (PK) properties and in vivo efficacy for 1 and 2 need to
be further improved for clinical development.
molecule inhibitors may have a promising therapeutic potential
for the treatment of human cancer by reactivation of the
powerful tumor suppressor function of p53.7−10 Vassilev and
Since 2 has a higher binding affinity to MDM2 and more
potent cellular activity in cancer cells than 1,21 we have focused
his colleagues from Hoffmann-La Roche were the first to report
potent, selective, and orally bioavailable, small-molecule MDM2
our modifications on 2 with the goal of improving its PK
inhibitors, as exemplified by nutlin-3.7 In the past few years,
properties and in vivo antitumor activity. Our efforts have now
yielded a highly potent MDM2 inhibitor 9 (MI-888)21 with
there has been intense research interest in the design of new
improved oral PK properties and in vivo antitumor activity. In
fact, 9 was capable of achieving complete and long-lasting
small-molecule MDM2 inhibitors, from both academia and
industry.8,11−21
Our group has previously reported structure-based design of
a series of spirooxindoles as a new class of potent and selective
Received: April 18, 2013
Published: June 20, 2013
MDM2 inhibitors.8,11−13,21 Two of such compounds, 1 (MI-
© 2013 American Chemical Society
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We tested 1−7 for their ability to inhibit cell growth in SJSA-
1 osteosarcoma and RS4;11 acute leukemia cell lines, which
have wild-type p53 and are sensitive to MDM2 inhibitors. The
results are summarized in Table 1. Compounds 3 and 6 have
IC50 values of 0.24 μM in the SJSA-1 cell line and 0.12 μM in
the RS4;11 cell line and are as potent as 2. Compounds 4, 5,
and 7 are slightly less potent than 2, 3, and 6 in these two
cancer cell lines.
Table 1. MDM2 Binding Affinity and Cell Growth Inhibition
Data for 1−10
IC50 (μM)
binding IC50
(nM)
SJSA-1 cell
line
RS4;11 cell
line
compd
Ki (nM)
1
2
3
4
5
6
7
8
9
10
44.6 2.7
9.8 2.7
7.1 1.5
8.2 2.3
8.8 1.4
8.4 1.1
16.3 2.1
11.1 2.1
6.8 0.9
7.6 0.9
10.5 2.0
0.86 0.11
0.61 0.10
1.1 0.3
0.89 0.21
0.25 0.04
0.24 0.05
0.41 0.04
0.32 0.06
0.20 0.02
0.35 0.06
0.60 0.16
0.08 0.02
0.25 0.04
0.54 0.14
0.10 0.01
0.12 0.01
0.30 0.06
0.23 0.04
0.10 0.02
0.21 0.05
0.43 0.07
0.06 0.01
0.10 0.01
Compounds 3−7 were evaluated for their metabolic stability
in mouse, rat, and human microsomes, in direct comparison
with 1 and 2 (Table 2). In mouse microsomes, 1, 2, 3, 6, and 7
0.94 0.07
0.62 0.05
2.4 0.5
Table 2. Stability of MDM2 Inhibitors in Mouse, Rat, and
Human Microsomes
0.97 0.3
0.44 0.22
0.62 0.19
T1/2 (min)
compd
mouse microsome
rat microsome
human microsome
1
>60
>60
>60
20.6
1.5
20.2
16.3
19.1
22.1
3.8
>60
>60
54.1
30.8
4.0
tumor regression in animal models of human cancer upon oral
administration.
2
3
4
RESULTS AND DISCUSSION
5
■
6
>60
>60
>60
>60
>60
21.1
>60
>60
>60
>60
55.1
>60
>60
>60
53.3
Both 1 and 2 have a flexible 1,2-diol side chain, which was
shown to enhance their binding affinities to MDM2 by several
times and to play a role in modulating their oral
pharmacokinetics properties in our previous studies.11,21 Our
metabolic studies of 2 revealed that the major metabolic soft
spots are located in the 1,2-diol side chain (data not shown).
We hypothesized that the overall oral PK profile of 2 can be
improved by conformationally constraining the 1,2-diol side
chain, thus reducing the number of rotatable bonds in the
molecule, and by further improving the metabolic stability.
Although the side chain in 1 and in 2 contains two hydroxyl
groups, we retained only one hydroxyl group in 3−7 containing
a conformationally constrained side chain for consideration of
synthetic feasibility (Figure 2). Their binding affinities to
MDM2 were determined using our optimized fluorescence-
polarization (FP) binding assay,21 and the results are
summarized in Table 1.
7
8
9
10
have similar T1/2 values (T1/2 > 60 min) and are stable.
However, 4 has modest stability (T1/2 = 20.6 min) and 5 is very
unstable (T1/2 = 1.5 min). In rat microsomes, compounds 1, 2,
3, and 6 have a shorter T1/2 than in mouse microsomes, but 7 is
quite stable (T1/2 >60 min), whereas 5 is very unstable (T1/2
=
3.8 min). In human microsomes, the trend is very similar to
that in mouse microsomes, and 1, 2, 3, 6, and 7 have good
microsomal stability.
The excellent microsomal stability for 7 in mouse, rat, and
human microsomes suggested that trisubstituted-alcohol-
containing side chain may block or retard the potential
oxidation by cytochrome P450 enzymes. To test this
hypothesis, we installed a methyl group on the hydroxyl-
substituted carbon of 3 and 4, which resulted in 8 and 9 both
tethering a trisubstituted-alcohol-containing side chain. We also
replaced the hydroxyl group with a methylsulfonamide group in
The binding data showed that 3−6 with a cyclic alcohol side
chain bind to MDM2 with high affinities (Ki = 0.61−1.1 nM).
These compounds are as potent as 2 but 10 times more potent
than 1. However, 7 with a tert-butanol side chain is 3 times less
potent than 2 in binding to MDM2.
Figure 2. Chemical structures of compounds with constrained side chains.
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3, which yielded 10. Microsomal stability evaluation confirmed
that 8−10 are very stable in mouse, rat, and human microsomes
(Table 2). Furthermore, our binding data (Table 1) showed
that 8−10 have essentially the same high binding affinity to
MDM2 as 2. In the cell growth inhibition assay, 8 is 2−4 times
less potent than 2, whereas 9 is 2 times more potent than 2,
and 10 has the same potency as 2 in both cell lines (Table 1).
Therefore, 9 is the most potent MDM2 inhibitor among these
compounds based upon their potencies in inhibition of cell
growth in these two cancer cell lines.
Upon the basis of the high binding affinity, potent cellular
activity, and excellent microsomal stability, we selected 9 for
evaluation of its oral PK properties in rats, in comparison to 2.
Our PK data showed that 9 has an improved PK profile in rats
over 2 (Figure 3 and Table 3). The Cmax and AUC values for 9
Figure 4. Western blot analysis of p53 activation and PARP cleavage
in the SJSA-1 cancer cell line by 2, 9, and nutlin-3a. Cells were treated
with different concentrations of the compounds for 24 h, and Western
blot analysis was performed using specific antibodies: FL-PARP, full-
length PARP; Cl-PARP, cleaved PARP; Pro-Cas3, pro-caspase-3; Cl-
Cas3, cleaved caspase-3.
had an IC50 of >10 μM in the HCT-116 p53−/− cell line, thus
displaying >100-fold selectivity for the HCT-116 cell line with
wild-type p53 over its isogenic p53 deleted cell line.
Encouraged by its promising in vitro activity and excellent
oral PK profile, we evaluated 9 for its antitumor activity in the
SJSA-1 osteosarcoma and RS4;11 acute leukemia xenograft
models in SCID mice.
Figure 3. Oral pharmacokinetics of 2 and 9 in Sprague−Dawley rats.
In the efficacy experiment using the SJSA-1 xenograft model,
SJSA-1 tumors were grown to approximately 100 mm3 and 9
was administered via oral gavage to mice daily for 2 weeks at 10,
30, and 100 mg/kg (Figure 5). While 9 had no significant
activity at 10 mg/kg, it effectively inhibited tumor growth at 30
Table 3. Summary of Oral Pharmacokinetic Data for 2 and 9
in Sprague−Dawley Rats
dose
AUC (0 → t)
compd (mg/kg) route Cmax (μg/L)
(μg L−1 h−1
)
T1/2 (h)
2
9
25
25
oral
oral
675 274
2138 775
3.4 0.5
3.2 0.5
1726 100
14874 2491
are 2.6- and 7-fold higher than those for 2, respectively, at the
same dose, and these two compounds have a similar T1/2 value
(3.4 and 3.2 h for 2 and 9, respectively).
Next, we examined p53 activation by 9 in the SJSA-1
osteosarcoma cell line by Western blot analysis and included 2
and nutlin-3a7 as control compounds (Figure 4). All three
compounds effectively and dose-dependently activate p53, as
evidenced by the accumulation of the p53 protein, as well as
MDM2 and p21, two p53 targeted gene products. In addition,
treatment of three compounds dose-dependently diminished
full-length poly (ADP-ribose) polymerase (PARP) at 24 h,
which indicated dose-dependent apoptosis induction. Treat-
ment of 9 activated p53 at concentrations as low as 30 nM, as
evident by clear accumulation of p21 and p53. In comparison, 9
is at least 3 times more potent than 2 in p53 activation and is
10 times more potent than nutlin-3a.
Upon the basis of the molecular mechanism of action, the
cellular activity of a specific MDM2 inhibitor should be
dependent upon the activation of wild-type p53. To firmly
assess the cellular specificity of 9, we employed the HCT-116
p53+/+ colon cell line and its isogenic p53 knockout (HCT-116
p53−/−) cell line. Compound 9 potently inhibited cell growth in
the HCT-116 p53+/+ colon cell line with an IC50 of 92 nM and
Figure 5. Antitumor activity of 9 in the SJSA-1 osteosarcoma tumor
xenograft model in mice: (A) tumor volume; (B) animal weight
change.
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mg/kg. Impressively, 9 at 100 mg/kg achieved rapid and
complete tumor regression. After 5 days of daily dosing, the
average tumor volume was decreased by >70%, and after 10
days of dosing, all the mice (8 out of 8 mice) treated with 9 had
undetectable tumor. The complete tumor regression was
durable; all the mice remained tumor free for 60 days after
the last dose. There was no significant difference in animal
weight between the vehicle control group of mice and the three
groups of mice treated with 9. Furthermore, there was minimal
weight loss and no sign of toxicity observed in mice treated
with 9 at all doses during the entire experiment. Collectively,
these data showed that 9 was well tolerated in mice at all the
doses tested.
To gain an insight into the in vivo mechanism of action of 9,
we tested its ability and kinetics in activation of p53 and
induction of apoptosis in the SJSA-1 xenograft tissue (Figure
6). Mice bearing SJSA-1 xenograft tumors were given a single
Figure 7. Antitumor activity of 9 in the RS4;11 acute lymphoblastic
leukemia xenograft model: (A) tumor volume; (B) animal weight
change.
respectively. Complete tumor regression was observed in all
the animals after 7 days of dosing with 9 at 200 mg/kg. The
complete tumor regression was also durable; 26 days after the
last dose, all mice treated with 9 at 200 mg/kg still remained
tumor free. There was no significant weight loss or other signs
of toxicity for all the mice treated with 9 during the entire
experiment.
CHEMISTRY
■
Figure 6. Activation of p53 and cleavage of caspase-3 and PARP in
SJSA-1 tumor tissue by 9. Mice bearing SJSA-1 tumors were dosed
with a single oral dose of 9 at 100 mg/kg, and tumor tissue was
harvested at different time points for Western blot analysis using
specific antibodies: FL-PARP, full-length PARP; Cl-PARP, cleaved
PARP; Pro-Cas3, pro-caspase-3; Cl-Cas3, cleaved caspase-3.
The synthesis of 3−10 is shown in Scheme 1. The synthesis of
9 has been reported in a previous communication.21
Compound 12 was synthesized using previously reported
methods.11−13,21 Most amines required for the synthesis of
intermediate 13 were commercially available with the exception
of 8, 9, and 10. The intermediate 14 was synthesized according
to previously reported procedures.11−13,21 Compound 14 was
isomerized in MeCN−H2O or MeOH for 3−4 days, affording
final compounds in 50−60% yield as determined by analytical
HPLC or UPLC.
The trans-N-(3-aminocyclobutanyl)methylsulfonamide 17
used for the synthesis of 10 was synthesized from commercially
available material 15 (Scheme 2). Compound 15 was converted
into mesylate 16 in 84% yield and the N-Boc group was cleaved
in TFA−CH2Cl2 solution, affording 17. The amine 17 was
neutralized using 2 N NaOH aqueous solution prior to use.
oral dose of 9 at 100 mg/kg. Mice were then sacrificed at
different time points, and tumors were harvested for Western
blot analysis. Our Western blot data showed that 9 induced
robust up-regulation of p53, as well as p21 and MDM2 proteins
at 3 and 6 h time points, indicative of strong p53 activation in
tumor tissue. The levels of p53, p21, and MDM2 proteins were
significantly diminished at the 24 h time point, suggesting that
p53 activation was transient in tumor tissue. Interestingly,
cleavage of PARP and caspase-3 was minimal at the 1, 3, and 6
h time points but became very clear at the 24 h time point,
indicating that while p53 was activated by 9 in tumor tissue
quickly, apoptosis induction occurred at later time points.
In the efficacy experiment using the RS4;11 acute leukemia
xenograft model, when tumors grew to an average volume of
150 mm3, compound 9 was administered at 100 and 200 mg/kg
via oral gavage daily for 3 weeks (Figure 7). At both 100 and
200 mg/kg, 9 achieved rapid and complete tumor regression.
After 4 days of dosing, tumor volume was decreased by 61%
and 80% with the treatment at 100 and 200 mg/kg,
CONCLUSION
■
In the present study, we designed and synthesized a set of new
and potent MDM2 inhibitors with the goal to improve the oral
pharmacokinetics profile over 2 by constraining the con-
formation of the 1,2-diol moiety and blocking the metabolic
oxidative site. Among these new compounds, 9 is a highly
potent MDM2 inhibitor and has an excellent oral pharmaco-
kinetics profile. Significantly, 9 is capable of achieving complete
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a
Scheme 1. General Synthetic Route for Final Compounds
a
Reagents and conditions: (a) free amine, 1.0−2.0 equiv, THF, rt, 12 h; (b) 2.1 equiv of ammonium cerium(IV) nitrate (CAN), MeCN/H2O = 1:1,
rt, 1 h; (c) NaHCO3, MeOH−H2O = 1:1, 3−4 days.
a
Scheme 2. Synthesis of trans-N-(3-Aminocyclobutanyl)methylsulfonamide 17
a
Reagents and conditions: (a) MsCl, Et3N, THF, 0 °C to rt, 84%; (b). TFA, CH2Cl2, rt, 1 h; (c) NaOH, MeOH−H2O.
isomers as determined by UPLC analysis. Final purification of 3 was
performed using semipreparative or preparative reverse phase HPLC.
The isomerization and purification of 4−8 and 10 followed the same
isomerization procedure.
and durable tumor regression in both SJSA-1 osteosarcoma and
RS4;11 acute lymphoblastic leukemia tumor xenograft models.
Our in vitro and in vivo data show that 9 is a promising MDM2
inhibitor and warrants extensive evaluation as a potential
clinical development candidate for the treatment of human
cancer.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
(trans-3-hydroxycyclobutyl)-2′-neopentyl-2-oxospiro-
[indoline-3,3′-pyrrolidine]-5′-carboxamide (3). 1H NMR (300
MHz, MeOD-d4): 8.77 (d, J = 5.78 Hz, 1H, NH), 7.64−7.52 (m, 2H),
7.41 (t, J = 6.85 HZ, 1H), 7.23−7.13 (m, 1H), 7.14 (dd, J = 8.11, 1.82
Hz, 1H), 6.79 (d, J = 1.77 Hz, 1H), 5.21 (d, J = 11.34 Hz, 1H), 4.58
(d, J = 11.34 Hz, 1H), 4.50 (dd, J = 8.22, 1.94 Hz, 1H), 4.34−4.22 (m,
1H), 4.20−4.06 (m, 1H), 2.30−2.06 (m, 3H), 1.94−1.80 (m, 2H),
1.15 (dd, J = 15.13, 1.70 Hz, 1H), 0.87 (s, 9H). 13C NMR (75 MHz,
MeOD-d4): 177.87, 167.38, 157.87 (d, JC−F = 245.04 Hz), 145.18,
137.29, 132.68, 128.70, 126.79, 126.63 (d, JC−F = 4.91 Hz), 124.22,
123.47, 122.63 (d, JC−F = 18.68 Hz), 121.64 (d, JC−F =13.39 Hz),
112.17, 65.20, 64.67, 64.27, 62.88, 43.46, 42.97, 40.40, 39.83, 31.01,
29.59. ESI-MS calculated for C27H3135Cl2FN3O3 [M + H]+: 534.17.
Found: 534.14.
To a round-bottom flask, 12 (2.9 g, 4.50 mmol), cis-3-amino-
cyclobutanol (0.40 g, 4.56 mmol), and THF (30 mL) were added, and
the solution was stirred at room temperature for 12 h. THF was
removed, and the crude material was purified on flash column
chromatography. The desired ring-opening adduct was isolated in 2.13
g (2.93 mmol). The ring-opening adduct and MeCN (20 mL) were
placed in a round-bottom flask. CAN (3.37 g, 6.15 mmol) and water
(20 mL) were subsequently added, and the mixture was stirred at
room temperature for 1 h before quenching with NaHCO3 saturated
solution. The aqueous layer was extracted with ethyl acetate (50 mL ×
3), and the combined organic layers were washed with water and brine
and dried over anhydrous Na2SO4. The solvent was removed, and the
crude material was purified on flash column chromatography. The
corresponding intermediate 14 was isolated in 624 mg (1.17 mmol,
26% yield over two steps). 4 constituted 56% of all four isomers upon
isomerization of 14 (Scheme 1, R = cis-3-hydroxycyclobutyl) for 3
days.
EXPERIMENTAL SECTION
■
General Chemistry. All reactions were conducted in a round-
bottom flask equipped with a Teflon-coated magnetic stirring bar.
Experiments involving moisture and/or air sensitive components were
performed under a nitrogen atmosphere. Commercial reagents and
anhydrous solvents were used without further purification. The crude
reaction product was purified by flash column chromatography packed
with silica gel. Further purification was performed on a semipreparative
HPLC instrument (Waters Delta 600) with a SunFire C18 reverse
phase column (19 mm × 150 mm). The mobile phase was a gradient
flow of solvent A (water, 0.1% of TFA) and solvent B (CH3CN, 0.1%
of TFA or MeOH, 0.1% of TFA) at a flow rate of 10 mL/min. Proton
nuclear magnetic resonance (1H NMR) spectroscopy and carbon
nuclear magnetic resonance (13C NMR) spectroscopy were performed
in Bruker Advance 300 NMR spectrometers. Low resolution ESI mass
spectrum analysis was performed on a Thermo-Scientific LCQ Fleet
mass spectrometer. The analytical HPLC model was a Waters 2795
Separation (UV detection at 230 and 254 nm wavelengths) using the
reverse phase column (SunFire, C18-5 μm, 4.6 mm × 150 mm). The
analytical UPLC model was Waters Acquity H class (UV detection at
230 and 254 nm wavelengths). The reverse phase column used was the
Acquity UPLC BEH (C18-1.7 μm, 2.1 mm × 50 mm). All final
compounds were purified to ≥95% purity as determined by analytical
UPLC or HPLC analysis. The optical rotation was measured on an
Autopol III automatic polarimeter (Rudolph Research Analytical). The
specific optical rotation was calculated by application of the following
formula: [α]TD = α/(lc), where α is the observed angular rotation, l is
the length of the cell in decimeters (l = 1 dm), and c is the
concentration in grams per milliliter (g/mL).
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
(cis-3-hydroxycyclobutyl)-2′-neopentyl-2-oxospiro[indoline-
3,3′-pyrrolidine]-5′-carboxamide (4). 1H NMR (300 MHz,
MeOD-d4): 8.77 (d, J = 6.95 Hz, 1H), 7.62−7.51 (m, 2H), 7.37 (d,
J = 7.00 Hz, 1H), 7.20−7.12 (m, 1H), 7.12 (dd, J = 8.02, 1.74 Hz, 1H),
6.78 (d, J = 1.68 Hz, 1H), 5.23 (d, J = 11.33 Hz, 1H), 4.60 (d, J =
11.33 Hz, 1H), 4.50 (dd, J = 8.24, 1.49 Hz, 1H), 3.98−3.84 (m, 1H),
3.84−3.70 (m, 1H), 2.64 (ddd, J = 12.94, 12.71, 6.64 Hz, 1H), 2.50
(ddd, J = 12.46, 12.50, 6.55 Hz, 1H), 1.90 (dd, J = 15.46, 8.33 Hz,
Synthesis of 3 from the corresponding intermediate 14 (Scheme 1,
R = trans-3-hydroxycyclobutyl) was through isomerization: 14 (60
mg) obtained from CAN oxidation was placed in a round-bottom flask
equipped with magnetic stirring bar. MeOH (5 mL) was added to
dissolve 14, followed by water (4 mL) and saturated NaHCO3
solution (0.5 mL) to set the pH at 8−9. This solution was stirred at
room temperature for 3−4 days, and 3 constituted 52% of all four
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1H), 1.80 (dd, J = 19.42, 8.81 Hz, 1H), 1.53 (dd, J = 19.42, 8.81 Hz,
1H), 1.13 (dd, J = 15.46, 1.46 Hz, 1H), 0.86 (s, 9H). 13C NMR (75
MHz, MeOD-d4): 177.78, 166.96, 157.86 (d, JC−F = 249.82 Hz),
145.19, 137.24, 132.58, 128.67, 126.79, 126.58 (d, JC−F = 4.98 Hz),
124.15, 123.47, 122.55 (d, JC−F = 18.94 Hz), 121.58 (d, JC−F = 12.97
Hz), 112.15, 64.55, 64.26, 62.72, 61.22, 43.37, 41.44, 41.36, 38.05,
30.97, 29.56. ESI-MS calculated for C27H3135Cl2FN3O3 [M + H]+:
534.17. Found: 534.28. [α]2D5 −16.7° (MeOH, c 0.0160 g/mL).
To a round-bottom flask, 12 (3.2 g, 4.95 mmol), (1R,3S)-3-
aminocyclopentanol (0.5 g, 4.95 mmol), and THF (25 mL) were
added, and the solution was stirred at room temperature for 12 h. THF
was removed, and the crude material was purified on flash column
chromatography. The desired ring-opening adduct was isolated in 2.68
g (3.46 mmol). The ring-opening adduct and MeCN (20 mL) were
placed in a round-bottom flask. CAN (3.98 g, 7.27 mmol) and water
(20 mL) were subsequently added, and the mixture was stirred at
room temperature for 1 h before quenching with NaHCO3 saturated
solution. The aqueous layer was extracted with ethyl acetate (50 mL ×
3), and the combined organic layers were washed with water and brine
and dried over anhydrous Na2SO4. The solvent was removed, and the
crude material was purified on flash column chromatography. The
corresponding intermediate 14 was isolated in 811 mg (1.48 mmol,
30% yield over two steps). 5 constituted 64% of all four isomers upon
isomerization of 14 (Scheme 1, R = (1S,3R)-3-hydroxycyclopentyl) for
3 days.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
((1S,3R)-3-hydroxycyclopentyl)-2′-neopentyl-2-oxospiro-
[indoline-3,3′-pyrrolidine]-5′-carboxamide (5). 1H NMR (300
MHz, MeOD-d4): 8.24 (d, J = 7.50 Hz, 1H, NH), 7.63−7.52 (m, 2H),
7.37 (t, J = 6.96 Hz, 1H), 7.19−7.11 (m, 1H), 7.12 (dd, J = 8.10, 1.78
Hz, 1H), 6.78 (d, J = 1.70 Hz, 1H), 5.28 (d, J = 11.52 Hz, 1H), 4.60
(d, J = 11.52 Hz, 1H), 4.51 (dd, J = 8.35, 1.65 Hz, 1H), 4.26−4.08 (m,
2H), 2.20−2.06 (m, 1H), 1.91 (dd, J = 15.44, 8.35 Hz, 1H), 1.84−1.60
(m, 2H), 1.56−1.40 (m, 2H), 1.38−1.24 (m, 1H), 1.14 (dd, J = 15.37,
1.48 Hz, 1H), 0.87 (s, 9H). 13C NMR (75 MHz, MeOD-d4): 177.78,
166.74, 157.91 (d, JC−F = 249.49 Hz), 145.18, 137.23, 132.26, 128.67,
room temperature for 1 h before quenching with NaHCO3 saturated
solution. The aqueous layer was extracted with ethyl acetate (50 mL ×
3), and the combined organic layers were washed with water and brine
and dried over anhydrous Na2SO4. The solvent was removed, and the
crude material was purified on flash column chromatography. The
corresponding intermediate 14 was isolated in 690 mg (1.29 mmol,
28% over two steps). 7 constituted 62% of all four isomers upon
isomerization of 14 (Scheme 1, R = 2-hydroxy-2-methylpropyl) for 3
days.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-(2-
hydroxy-2-methylpropyl)-2′-neopentyl-2-oxospiro[indoline-
3,3′-pyrrolidine]-5′-carboxamide (7). 1H NMR (300 MHz,
MeOD-d4): 7.65−7.56 (m, 2H), 7.35 (t, J = 7.04 Hz, 1H), 7.20−
7.14 (m, 1H), 7.12 (dd, J = 8.02, 1.66 Hz, 1H), 6.78 (d, J = 1.64 Hz,
1H), 5.42 (d, J = 11.51 Hz, 1H), 4.64 (d, J = 11.51 Hz, 1H), 4.54 (dd,
J = 8.22, 1.27 Hz, 1H), 3.40−3.30 (m, 1H), 2.98 (d, J = 13.44 Hz, 1H),
1.93 (dd, J = 15.42, 8.38 Hz, 1H), 1.14 (dd, J = 15.45, 1.18 Hz, 1H),
0.97 (s, 3H), 0.87 (s, 9H), 0.83 (s, 3H). 13C NMR (75 MHz, MeOD-
d4): 177.70, 168.15, 157.90 (d, JC−F = 249.83 Hz), 145.20, 137.21,
132.57, 128.83, 126.83, 126.68 (d, JC−F = 4.69 Hz), 124.13, 123.51,
122.66 (d, JC−F = 18.96 Hz), 121.63 (d, JC−F = 13.25 Hz), 112.16,
75.58, 71.16, 64.48, 64.43, 62.85, 51.51, 43.45, 31.01, 29.58, 27.41,
26.65. ESI-MS calculated for C27H3335Cl2FN3O3 [M + H]+: 536.19.
Found: 536.44. [α]2D5 −24.0° (MeOH, c 0.0186 g/mL).
To a round-bottom flask, 12 (643 mg, 1.0 mmol), trans-3-(tert-
butyldimethylsilyloxy)-3-methylcyclobutanamine21 (0.10 g, 0.46
mmol), and THF (10 mL) were added, and the solution was stirred
at room temperature for 12 h. THF was removed, and the crude
material was purified on flash column chromatography. The desired
ring-opening adduct was isolated in 0.36 g (0.41 mmol). The ring-
opening adduct and MeCN (5 mL) were placed in a round-bottom
flask. CAN (450 mg, 0.82 mmol) and water (5 mL) were subsequently
added, and the mixture was stirred at room temperature for 1 h before
quenching with NaHCO3 saturated solution. The aqueous layer was
extracted with ethyl acetate (50 mL × 3), and the combined organic
layers were washed with water and brine and dried over anhydrous
Na2SO4. The solvent was removed, and the crude material was purified
on flash column chromatography. The corresponding intermediate 14
was isolated in 62 mg (0.11 mmol, 24% yield over two steps), and
(2′S,3R,4′S,5′R)-N-((1r,3R)-3-((tert-butyldimethylsilyl)oxy)-3-methyl-
cyclobutyl)-6-chloro-4′-(3-chloro-2-fluorophenyl)-1′-((1R,2S)-2-hy-
droxy-1,2-diphenylethyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrro-
lidine]-5′-carboxamide was also isolated (chiral-auxiliary-cleaved
product with the hydroxy group protected by TBS, 53 mg, 0.08
mmol, 17% yield over two steps). 8 constituted 64% of all four isomers
upon isomerization of 14 (Scheme 1, R = trans-3-hydroxy-3-
methylcyclobutyl) for 3 days.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
(trans-3-hydroxy-3-methylcyclobutyl)-2′-neopentyl-2-
oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide (8). 1H
NMR (300 MHz, MeOD-d4): 8.68 (d, J = 5.95 Hz, 1H, NH),
7.64−7.52 (m, 2H), 7.41 (t, J = 7.56 Hz, 1H), 7.24−7.14 (m, 1H),
7.14 (dd, J = 8.06, 1.64 Hz, 1H), 6.79 (d, J = 1.76 Hz, 1H), 5.19 (d, J =
11.28 Hz, 1H), 4.58 (d, J = 11.28 Hz, 1H), 4.50 (dd, J = 8.06, 1.65 Hz,
1H), 4.37−4.20 (m, 1H), 2.44−2.22 (m, 2H), 1.96−1.82 (m, 2H),
1.62 (dd, J = 11.69, 5.79 Hz, 1H), 1.15 (dd, J = 15.43, 1.66 Hz, 1H),
1.09 (s, 3H), 0.87 (s, 9H). 13C NMR (75 MHz, MeOD-d4): 177.84,
167.28, 157.90 (d, JC−F = 249.25 Hz), 145.17, 137.28, 132.66, 128.70,
126.74, 126.67 (d, JC−F = 4.93 Hz), 124.15, 123.50, 122.62 (d, JC−F
=
19.14 Hz), 121.61 (d, JC−F = 12.95 Hz), 112.16, 73.04, 64.43, 64.30,
62.82, 51.66, 43.41, 42.00, 34.65, 31.79, 30.99, 29.56. ESI-MS
calculated for C28H3335Cl2FN3O3 [M + H]+: 548.19. Found: 548.20.
[α]2D5 −18.7° (MeOH, c 0.0174 g/mL).
The same procedure for the synthesis of 5 was followed using
(1S,3S)-3-aminocyclopentanol as the reactant. The CAN oxidation
furnished the corresponding intermediate 14 in 32% over two steps. 6
constituted 70% of all four isomers upon isomerization of 14 (Scheme
1, R = (1S,3S)-3-hydroxycyclopentyl) for 3 days.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
((1S,3S)-3-hydroxycyclopentyl)-2′-neopentyl-2-oxospiro-
[indoline-3,3′-pyrrolidine]-5′-carboxamide (6). 1H NMR (300
MHz, MeOD-d4): 8.47 (d, J = 7.23 Hz, 1H, NH), 7.68−7.54 (m, 2H),
7.41 (t, J = 6.94 Hz, 1H), 7.24−7.14 (m, 1H), 7.16 (dd, J = 8.13, 1.82
Hz, 1H), 6.81 (d, J = 1.74 Hz, 1H), 5.23 (d, J = 11.38 Hz, 1H), 4.61
(d, J = 11.38 Hz, 1H), 4.54 (dd, J = 8.22, 1.64 Hz, 1H), 4.42−4.28 (m,
1H), 4.28−4.19 (m, 1H), 2.04−1.86 (m, 3H), 1.80−1.60 (m, 2H),
1.56−1.42 (m, 1H), 1.17 (dd, J = 15.34, 1.39 Hz, 1H), 1.14−1.00 (m,
1H), 0.89 (s, 9H). 13C NMR (75 MHz, MeOD-d4): 177.81, 167.10,
157.86 (d, JC−F = 248.99 Hz), 145.17, 137.22, 132.57, 128.75, 126.79,
126.61 (d, JC−F = 4.71 Hz), 124.18, 123.54, 122.54 (d, JC−F = 18.86
Hz), 121.63 (d, JC−F = 12.93 Hz), 112.16, 72.88, 64.59, 64.13, 62.89,
51.52, 51.41, 43.44, 42.29, 34.30, 31.61, 30.99, 29.57. ESI-MS
calculated for C28H3335Cl2FN3O3 [M + H]+: 548.19. Found: 548.32.
[α]2D5 −22.9° (MeOH, c 0.0112 g/mL).
To a round-bottom flask, 12 (3.0 g, 4.67 mmol), 1-amino-2-
methylpropan-2-ol (0.41 g, 4.60 mmol), and THF (25 mL) were
added, and the solution was stirred at room temperature for 12 h. THF
was removed, and the crude material was purified on flash column
chromatography. The desired ring-opening adduct was isolated in 2.19
g (2.99 mmol). The ring-opening adduct and MeCN (10 mL) were
placed in a round-bottom flask. CAN (3.44 g, 6.28 mmol) and water
(10 mL) were subsequently added, and the mixture was stirred at
126.79, 126.68 (d, JC−F = 4.96 Hz), 124.20, 123.41, 122.65 (d, JC−F
=
18.90 Hz), 121.63 (d, JC−F = 12.76 Hz), 112.16, 70.79, 64.65, 64.25,
62.84, 44.80, 43.73, 43.39, 40.89, 30.97, 29.56, 29.43. ESI-MS
calculated for C28H3335Cl2FN3O3 [M + H]+: 548.19. Found: 548.56.
To a round-bottom flask, 12 (1.286 g, 2.0 mmol), 17 (1.8 mmol
based on 16), and THF (15 mL) were added, and the solution was
stirred at room temperature for 12 h. THF was removed, and the
crude material was purified on flash column chromatography. The
desired ring-opening adduct was isolated in 0.776 g (0.96 mmol). The
ring-opening adduct and MeCN (10 mL) were placed in a round-
bottom flask. CAN (1.053 g, 1.84 mmol) and water (10 mL) were
subsequently added, and the mixture was stirred at room temperature
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for 1 h before quenching with NaHCO3 saturated solution. The
aqueous layer was extracted with ethyl acetate (50 mL × 3), and the
combined organic layers were washed with water and brine and dried
over anhydrous Na2SO4. The solvent was removed, and the crude
material was purified on flash column chromatography. The
corresponding intermediate 14 was isolated in 626 mg (1.023 mmol,
56% yield over two steps). 10 constituted 62% of all four isomers upon
isomerization of 14 (Scheme 1, R = trans-3-(methylsulfonamido)-
cyclobutyl) for 3 days.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-
(trans-3-(methylsulfonamido)cyclobutyl)-2′-neopentyl-2-
oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide (10). 1H
NMR (300 MHz, MeOD-d4): 8.96 (d, J = 6.37 Hz, 1H, NH),
7.64−7.54 (m, 2H), 7.36 (t, J = 6.96 Hz, 1H), 7.20−7.16 (m, 1H),
7.11 (dd, J = 8.13, 1.79 Hz, 1H), 6.78 (d, J = 1.75 Hz, 1H), 5.32 (d, J =
11.51 Hz, 1H), 4.63 (d, J = 11.51 Hz, 1H), 4.52 (d, J = 8.47, 1.49 Hz,
1H), 4.28−4.14 (m, 1H), 3.83 (quintet, J = 3.83 Hz, 1H), 2.84 (s,
3H), 2.40−2.30 (m, 2H), 2.30−2.20 (m, 1H), 2.06−1.96 (m, 1H),
1.93 (dd, J = 15.47, 8.42 Hz, 1H), 1.13 (dd, J = 15.47, 1.23 Hz, 1H),
0.86 (s, 9H). 13C NMR (75 MHz, MeOD-d4): 177.73, 167.29, 157.80
(d, JC−F = 249.64 Hz), 145.13, 137.16, 132.63, 128.69, 126.75, 126.61
(d, JC−F = 4.84 Hz), 124.13, 123.52, 122.51 (d, JC−F = 18.77 Hz),
121.55 (d, JC−F = 13.01 Hz), 112.15, 64.51, 64.11, 62.70, 46.60, 43.43,
40.93, 38.47, 38.11, 30.97, 29.54. ESI-MS calculated for
C28H3435Cl2FN4O4S [M + H]+: 611.17. Found: 611.42. [α]2D5 −13.8°
(MeOH, c 0.0208 g/mL).
N-(trans-3-Aminocyclobutyl)methanesulfonamide (17). To a
round-bottom flask, 15 (400 mg, 2.15 mmol, 1.0 equiv), Et3N (1 mL,
7.5 mmol, 3.5 equiv), and THF (25 mL) were added. The reaction
mixture was cooled in an ice−water bath, and methanesulfonyl
chloride (MsCl, 492 mg, 4.3 mmol, 2.0 equiv) was added dropwise via
a syringe. The mixture was stirred at room temperature for 12 h before
quenching with water. The aqueous layer was extracted with ethyl
acetate (70 mL × 2), and the combined organic layers were washed
with brine (50 mL) and dried over anhydrous Na2SO4. The solvents
were removed, and the crude product was purified on flash column
chromatography. 16 was obtained as a white solid (0.475 g, 84%). 16:
1H NMR (MeOD-d4, 300 MHz) 4.10−3.92 (m, 2H), 2.87 (s, 3H),
temperature for 15−30 min with gentle shaking to ensure equilibrium.
The polarization values in millipolarization units (mP) were measured
at an excitation wavelength of 485 nm and an emission wavelength of
530 nm. Equilibrium dissociation constants (Kd) were then calculated
by fitting the sigmoidal dose-dependent FP increases as a function of
protein concentrations using Graphpad Prism 5.0 software (Graphpad
Software, San Diego, CA). Ki values of tested compounds were
determined in a dose-dependent competitive binding experiment.
Mixtures of 5 μL of the tested compound in different concentrations in
DMSO and 120 μL of preincubated protein/fluorescent probe
complex with fixed concentrations in the assay buffer (100 mM
potassium phosphate, pH 7.5, 100 μg/mL bovine γ-globulin, 0.02%
sodium azide, with 0.01% Triton X-100) were added into assay plates
and incubated at room temperature with gentle shaking. The
incubation time was precisely controlled at 15 min to minimize the
influence of the initial isomerization to the binding affinities. Final
concentrations of the protein and fluorescent probe in the competitive
assays were 10 and 1 nM, respectively, and final DMSO concentration
is 4%. Negative controls containing protein/fluorescent probe complex
only (equivalent to 0% inhibition), and positive controls containing
free fluorescent probe only (equivalent to 100% inhibition), were
included in each assay plate. FP values were measured as described
above. IC50 values were determined by nonlinear regression fitting of
the sigmoidal dose-dependent FP decreases as a function of total
compound concentrations using Graphpad Prism 5.0 software
(Graphpad Software, San Diego, CA). Ki values of tested compounds
to the MDM2 protein were calculated using the measured IC50 values,
the Kd value of the fluorescent probe to the protein, and the
concentrations of the protein and fluorescent probe in the competitive
assays.23
Cell Growth Inhibition Assay. The SJSA-1 osteosarcoma cell line
and RS4;11 leukemia cell line were purchased from the American
Type Culture Collection (Manassas, VA). HCT-116 p53+/+ colon cell
line (HCT-116 p53+/+) and its isogenic p53 knockout (HCT-116
p53−/−) cell line are kind gifts from Dr. Bert Vogelstein, Johns
Hopkins University at Baltimore, MD. All cell lines were grown in
PRMI 1640 medium (Gibco, Life Technologies, Grand Island, NY)
supplemented with 10% FBS (Invitrogen, Carlsbad, CA) in a
humidified tissue culture incubator at 37 °C, 5% CO2. For cell growth
inhibition assay, cells were seeded in 96-well, flat-bottom cell culture
plates at a density of (3−4) × 103 cells/well and allowed to grow
overnight, then incubated with a compound at different concen-
trations. The effect of cell growth inhibition of a compound was
determined with a lactate dehydrogenase based WST-8 assay (WST-8;
Dojindo Molecular Technologies Inc., Gaithersburg, MD). For the
RS4;11 cell line, WST-8 solution was directly added to culture
medium in each well to a final concentration of 10% upon the end of
2.30 (t, J = 6.71 Hz, 4H), 1.43 (s, 9H) 13C (MeOD-d4, 75 M Hz):
158.07, 80.52, 46.61, 43.58, 40.90, 38.90, 28.91. ESI-MS: calculated for
C10H20N2NaO4S [M + Na]+ = 287.10; obtained, 287.16.
To a round-bottom flask, 16 (0.475 g, 1.8 mmol), Et3SiH (0.1 mL),
and CH2Cl2 (15 mL) were added. TFA (4 mL) was added via a
syringe, and the mixture was stirred at room temperature for 1 h. The
reaction mixture was concentrated to remove solvent and TFA.
MeOH (15 mL) was added, and the pH was adjusted to be 10−11 by
NaOH (2 N aqueous solution). MeOH and water were then removed
affording crude free amine 17, which was directly used for the next
step without further purification.
treatment. For SJSA-1, HCT-116 p53+/+, and HCT-116 p53−/−
,
culture medium in each well was discharged and fresh RPMI 1640
medium with no phenol red (Gibco, life Technologies, Grand Island,
NY) containing 10% WST-8 solution was added to each well in the
presence of 5% FBS upon the end of treatment. Then the plates were
incubated at 37 °C for 2−3 h. The absorbance of the samples was
measured at 450 nm using a TECAN ULTRA reader. The effect of
each treatment was calculated by the equation [(absorbance value of
treatment) − (absorbance value of medium with FBS + WST solution
only)]/[(absorbance value of untreated control) − (absorbance value
of medium with FBS + WST solution only)]. The IC50 value of a
compound is the concentration that inhibits cell growth by 50% over
the control.
Pharmacodynamics (PD) Study. For PD studies, the SJSA-1
xenograft tumor model was used. To develop xenograft tumors, 5 ×
106 SJSA-1 cancer cells with 50% Matrigel were injected sc on the
dorsal side of SCID mice from Charles River, one tumor per mouse.
When xenograft tumors reached a mean of ∼100 mm3 (70−130 mm3),
two mice were treated with vehicle control (10% PEG400/3%
Cremophor/87% PBS) and eight mice were treated with a single dose
of 9 at 100 mg/kg via oral gavage. Mice treated with vehicle control
were sacrificed at 6 h, and tumor tissue was harvested for Western blot
FP-Based Protein Binding Assay. The binding affinity of MDM2
inhibitors was determined by an optimized, sensitive, and quantitative
fluorescence-polarization-based (FP-based) binding assay using a
recombinant human His-tagged MDM2 protein (residues 1−118)
and a FAM tagged p53-based peptide as the fluorescent probe. The
design of the fluorescent probe was based upon a previously reported
high affinity p53-based peptidomimetic compound (5-FAM-βAla-
βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2).22 This
tagged peptide was named PMDM6-F. The Kd value of PMDM6-F to
the MDM2 protein was determined to be 1.4 0.3 nM by monitoring
the total fluorescence polarization of mixtures composed with the
fluorescent probe at a fixed concentration and the MDM2 protein with
increasing concentrations up to full saturation. Fluorescence polar-
ization values were measured using the Infinite M-1000 plate reader
(Tecan U.S., Research Triangle Park, NC) in Microfluor 2 96-well,
black, round-bottom plates (Thermo Scientific). In the saturation
experiments, 1 nM PMDM6-F and increasing concentrations of
proteins were added to each well to a final volume of 125 μL in the
assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL
bovine γ-globulin, 0.02% sodium azide [Invitrogen], with 0.01% Triton
X-100 and 4% DMSO). Plates were mixed and incubated at room
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analysis. Two mice treated with 9 were sacrificed at 1, 3, 6, and 24 h
(two mice at each time point), and tumor tissue was harvested.
Western Blotting. For Western blot analysis, tumor cells or tumor
tissues were lysed in ice-cold RIPA buffer: 20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% sodium deoxycholate,
2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM
sodium orthovanadate, and 1 μg/mL leupeptin. The expressions of
indicated proteins in the whole cell lysates were detected by Western
blot analysis using the following antibodies: anti-p53 (OP43,
Calbiochem), anti-MDM2 (sc-965, Santa Cruz), anti-p21 (556431,
BD Biosciences), anti-PARP (9542, Cell Signaling Technology),
anticaspase-3 (AAP-113, Stressgen Bioreagents), and HRP-conjugated
anti-GAPDH (sc-5778, Santa Cruz).
In Vivo Efficacy Experiments. For in vivo efficacy experiments
using the SJSA-1 tumor xenograft model, 5 × 106 SJSA-1 cancer cells
with 50% Matrigel were injected sc on the dorsal side of SCID mice
from Charles River, one tumor per mouse. When xenograft tumors
reached a mean of ∼100 mm3 (70−130 mm3), mice were randomized
into groups of eight mice. Vehicle control (10% PEG400/3%
Cremophor/87% PBS) was administered orally (po) once per day
for 14 days. Compound 9 was administered orally once per day for 14
days at 10, 30, and 100 mg/kg. Tumor sizes and animal weights were
measured three times per week. Data are represented as mean tumor
volumes. Tumor volume (mm3) = (AB2)/2 where A and B are the
tumor length and width (in mm), respectively. Statistical analyses were
done using two-way ANOVA and unpaired two-tailed t test, using
Prism (version 4.0, GraphPad). P < 0.05 was considered statistically
significant.
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AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 734-615-0362. Fax: 734-647-9647. E-mail:
shaomeng@umich.edu.
Author Contributions
§Y.Z., S.Y., and W.S. provided equal contributions.
(13) Yu, S.; Qin, D.; Shangary, S.; Chen, J.; Wang, G.; Ding, K.;
McEachern, D.; Qiu, S.; Nikolovska-Coleska, Z.; Miller, R.; Kang, S.;
Yang, D.; Wang, S. Potent and orally active small-molecule inhibitors
of the MDM2-p53 interaction. J. Med. Chem. 2009, 52, 7970−7973.
(14) Rew, Y.; Sun, D. Q.; De Turiso, F. G. L.; Bartberger, M. D.;
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Notes
The authors declare the following competing financial
interest(s): Dr. Shaomeng Wang is an inventor on the
compounds described in this study. The technology has been
licensed by Ascenta and Sanofi for clinical development, and
Dr. Shaomeng Wang receives royalty from the licensing. Dr.
Wang is also a consultant for Ascenta.
ACKNOWLEDGMENTS
■
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), Ascenta Ther-
apeutics, Inc., and Sanofi S.A.
ABBREVIATIONS USED
■
C
AUC, area under the curve; CAN, ceric ammonium nitrate;
max, the maximum compound concentration from oral dosing;
Cl, clearance; CYP, cytochrome P450; CH2Cl2, dichloro-
methane; EtOAc, ethyl acetate; H2O, water; LC/MS, liquid
chromatography−mass spectrometry; MDM2, murine double
minute 2; MeCN, acetonitrile; NaOH, sodium hydroxide; PD,
pharmacodynamics; PK, pharmacokinetics; po, oral adminis-
tration; qD, once a day dosing; SCID, severe combined
immunodeficient; T1/2, half-life; TFA, trifluoroacetic acid; Tmax
the time at which Cmax is reached
,
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