Diastereomeric spirooxindoles as highly potent and efficacious MDM2 inhibitors

Article abstract of DOI:10.1021/ja3125417

Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new

Full text of DOI:10.1021/ja3125417

Article
pubs.acs.org/JACS
Diastereomeric Spirooxindoles as Highly Potent and Efficacious
MDM2 Inhibitors
Yujun Zhao,† Liu Liu,† Wei Sun,† Jianfeng Lu,† Donna McEachern,† Xiaoqin, Li,‡ Shanghai Yu,†
Denzil Bernard,† Philippe Ochsenbein,§ Vincent Ferey,§ Jean-Christophe Carry,∥ Jeffrey R. Deschamps,⊥
Duxin Sun,‡ and Shaomeng Wang*,†
†Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology and Medicinal Chemistry, and ‡Life Sciences
Institute, University of Michigan, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109, United States
§Sanofi Research & Development, Montpellier, France
∥Sanofi Research & Development, Vitry-sur-Seine, France
⊥Naval Research Laboratory, Code 6930, 4555 Overlook Avenue, Washington, D.C. 20375, United States
S
* Supporting Information
ABSTRACT: Small-molecule inhibitors that block the
MDM2-p53 protein−protein interaction (MDM2 inhibitors)
are being intensely pursued as a new therapeutic strategy for
cancer treatment. We previously published a series of
spirooxindole-containing compounds as a new class of
MDM2 small-molecule inhibitors. We report herein a
reversible ring-opening-cyclization reaction for some of these
spirooxindoles, which affords four diastereomers from a single
compound. Our biochemical binding data showed that the
stereochemistry in this class of compounds has a major effect on their binding affinities to MDM2, with >100-fold difference
between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent
MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent
compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an
animal model of human cancer.
Additionally, spirooxindole-containing compounds have been
reported as actin polymerization inhibitors,4 and as inhibitors of
tubulin polymerization.5
INTRODUCTION
■
Compounds containing a spirocyclic oxindole-pyrrolidine ring
(spirooxindole) system have recently attracted significant
attention because of their unique structure and broad biological
activities (Figure 1).1 For example, the natural product
spirotryprostatin A is a cell cycle regulator,2 while pteropodine
is a modulator of muscarinic M1 and 5-HT2 receptors.3
We previously reported sprirooxindole-containing com-
pounds that are potent and specific small-molecule inhibitors
that block the MDM2-p53 protein−protein interaction.6 For
example, compound MI-219 (Figure 1) binds to MDM2 with
low nanomolar affinity, effectively blocks the MDM2-p53
protein−protein interaction in cells, reactivates p53 in tumor
cells with wild-type p53 in vitro and in vivo, and strongly inhibits
tumor growth in animal models of human cancer.6b Our data
thus suggested that modifications of spirooxindole-containing
compounds may lead to a highly promising MDM2 inhibitor
for the treatment of human cancer by reactivation of the
powerful tumor suppressor function of p53.
Our spirooxindole-containing MDM2 inhibitors were
synthesized using an efficient 1,3-dipolar cycloaddition reaction
(Scheme 1).6d Compound 1a was obtained in high purity and
was found to be relatively stable in aprotic solvents such as
CH2Cl2, THF, EtOAc, and DMSO. Stereochemistry was
assigned to compound 1a and its analogues, including MI-
Figure 1. Examples of spirooxindole-containing compounds with
interesting biological activities.
Received: December 22, 2012
© XXXX American Chemical Society
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219, using the X-ray crystal structures of the key intermediate I
and of compound II (Figure 1 and Scheme 1).7 However, when
a pure sample of 1a was treated with MeOH or CH3CN/H2O,
up to four peaks, all with the same molecular weight, were
observed by analytical HPLC (Supporting Information, SI).
Isolation and purification of the fractions from compound 1a
Scheme 1. Synthesis and Chemical Structures of Our
Previously Reported MDM2 Inhibitors
1
and subsequent analysis by H, DEPT 135, and 13C NMR
showed that individual components possess the same number
of protons, CH, CH2, CH3, and quaternary carbons. The data
suggested that these individual peaks represent different
isomers or stereoisomers.
Based upon these initial observations and on our subsequent
investigations, we hypothesized that reversible ring-opening
and cyclization of the pyrrolidine ring takes place in some of
these compounds. A cascade reaction of this type can generate
four possible diastereomers from one single stereoisomer, as
shown in Table 1. Although reversible ring-opening-cyclization
has been previously observed for certain spirooxindole-
a
Table 1. Isomerization of Selected Spirooxindole-Containing Compounds
a
In cases of 9, 11, and 13, the cis−cis isomers were obtained as the predominant products of ceric ammonium nitrate oxidation without aging
b
c
(equilibrium in CH3CN and H2O at pH 8 for 3 days). Isolated yield after flash column chromatography. Ratio determined by HPLC analysis;
assignments were not made for c and d. The combined yield of a to d in the aging step was determined from the yield in the oxidation step. Yield
d
e
f
g
h
after HPLC separation. Compound 8a−8d was obtained from the hydrolysis by LiOH-THF-H2O of 7a. Hydrolysis yield. Starting from pure 8a.
i
Ratio after crude product was allowed to stand in MeOH for 2 h.
B
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containing alkaloids,8 no detailed investigation has been
reported.
We herein report our detailed investigation of the isomer-
ization of selected spirooxindole-containing compounds. Our
biochemical binding data showed that the stereochemistry in
this class of compounds has a major effect on their binding
affinities to MDM2; with >100-fold difference between the
most potent and the least potent stereoisomers. Importantly,
our investigation has led to the identification of a set of highly
potent MDM2 inhibitors with a stereochemistry that is
different from that of our previously reported compounds.6
One such compound is capable of achieving complete and long-
lasting tumor regression in an animal model of human cancer.
RESULTS AND DISCUSSION
■
We first investigated the isomerization reaction for selected
spirooxindole-containing compounds (entries 1−13, Table 1).
The trans−cis isomers, 1a-7a, were obtained as major products
(67−95%) after ceric ammonium nitrate (CAN) oxidation
(Table 1, entries 1−7).
Figure 2. Interconversion of 5a−5d in MeCN−H2O. Panels A to D
show isomerization of compounds 5a−5d. The proportion of
compounds is on the y axis and reaction time is on the x axis.
Conditions: Trifluoroacetate salts (0.1 mg) of the amine were placed
in 0.25 mL of 50% aqueous MeCN. Purity was analyzed at the
indicated time points by analytical reverse phase HPLC.
However, treatment of the oxidation reaction products
(entries 1−7) with CH3CN and H2O at pH 8 for 3 days
(aging) produced a mixture of four isomers with the cis−cis
isomers (1b to 7b) as the major components. Hydrolysis of 7a
yielded a mixture of four isomers 8a−8d. Interestingly, for
entry 9, after CAN oxidation, a mixture of 9b−9d was obtained
without detectable amount of 9a. Although 10a constituted
56% after oxidation, this isomer rapidly decreased to 10%
within 2 h in MeOH. When R1 = F (Table 1, entries 11 and
13), the cis−cis isomers (11b and 13b) were found to be the
predominant products after oxidation with a small amount of
the other three isomers. For entry 12, a mixture of four isomers
was obtained after oxidation with a similar amount of isomers
12a and 12b. Thus, substitution of an F at the 5-position of the
oxindole ring or removal of a methyl group from the amide
nitrogen seemed to favor isomer b. These data show that for
these compounds, isomerization occurs in CH3CN and H2O or
in MeOH.
To investigate the isomerization in more detail, we obtained
four individual isomers 5a−5d (entry 5) to high purity. Isomer
5a, 5c or 5d each was gradually converted into a mixture of four
isomers over a period of up to four days in CH3CN-H2O
(Figure 2). Isomer 5b, on the other hand, was quite stable in
CH3CN-H2O, only 4% being converted into a mixture of three
other isomers after 12 days.
To determine the absolute stereochemistry of isomer b, we
obtained the crystal structure of 13b, which revealed the
stereochemistry of three hydrophobic groups in a cis−cis
substitution pattern (R-R-S configuration) on the pyrrolidine
ring (Figure 3). The crystal structure of an intermediate after
the ring-opening (14), however, showed that the substitution
pattern of the three hydrophobic groups on the pyrrolidine ring
was trans−cis (R-S-R configuration, Figure 3). Hence, these
data indicate that initial product after oxidative removal of the
chiral auxiliary has a trans−cis configuration on the pyrrolidine
ring, and upon cleavage of the chiral auxiliary, the isomerization
occurs when the final products are exposed to H2O and MeCN
prior to the workup.
Figure 3. Absolute stereochemistry from X-ray crystallography.
reversible retro-Mannich reaction affording the respective ring-
opened transition state (a-TS) in which the pyrrolidine ring has
been opened. Recyclization of a-TS affords four stereoisomers.9
We did not attempt to determine the crystal structures of
stereoisomers c and d because very limited amounts of the
compounds could be obtained, due to their facile conversion to
other isomers. Stereochemistry of isomers c and d were
assigned based on the stability profiles of 5c and 5d in Figure 2
and the stereochemistry for isomers a and b.
On the basis of the X-ray crystallographic data from 13b and
14, we propose the reaction mechanism shown in Scheme 2 for
the observed isomerization. The trans−cis isomers 1a−13a
(Table 1) are the initial products from CAN oxidation. In
aqueous solution, the trans−cis isomers 1a−13a undergo a
We next investigated binding kinetics of isomers 5a−5d to
MDM2 in the binding assay medium using our fluorescence-
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temperature for the whole time range of experiments. Aliquots
of the incubation solutions of 5a−5d were tested at different
time points to determine MDM2 binding affinities. Since
isomerization of 5a−5d is sensitive to the incubation time, total
time used for binding assay plate preparation and incubation
was precisely controlled so that assay plates were read exactly
10 min after aliquots were pipetted from the incubation
solution.
Scheme 2. Proposed Isomerization Mechanism of
Spirooxindole-containing Compounds 1a−13a
In the first time-point, isomer 5b has the highest binding
affinity, followed by 5c, 5a, and 5d (Figure 4). The
stereochemistry has a significant effect on their binding affinity
to MDM2; isomer 5b is >300-times more potent than isomer
5d and is 18-times more potent than 5a. However, after 100 h,
the binding affinities for these four isomers converged (Figure
4).
To understand the binding kinetics of 5a−5d, we
investigated the isomerization kinetics of 5a−5d in binding
assay medium by direct HPLC analysis of a solution of the
individual compound in binding medium at indicated time
points (Figure 5). At the 100 h time-point, there was a similar
polarization (FP) assay (Figure 4). DMSO stock solutions of
5a−5d were freshly prepared from lyophilized powder of 5a−
5d right before the experiments. Then, aqueous incubation
solutions of 5a−5d were prepared by diluting the fresh DMSO
stock solutions in FP binding assay buffer and final DMSO
concentration was 5%. These incubation solutions, in which
isomerization of 5a−5d was taking place, were stored at room
Figure 5. Isomerization of 5a−5d in binding assay medium. Panels A
to D correspond to isomerization studies of isomers 5a−5d. The
proportion of compounds is on the y axis and reaction time is on the x
axis. Conditions: the individual sample 5a, 5b, 5c or 5b (0.1 mg) was
dissolved in binding medium (0.25 mL) and the samples were
incubated at room temperature. The solution of corresponding isomer
in binding medium was directly analyzed in analytical reverse phase
HPLC at the indicated time points.
distribution for each individual isomer, regardless which isomer
was used at the beginning, and isomer 5b and 5a accounted for
approximately 50% and 40%, respectively. Isomer 5b has a slow
isomerization kinetic and was the most stable, followed by
isomer 5d, 5a, and 5c. Isomer 5c had very fast isomerization
kinetics and approximately 20% of it was already isomerized
into 5b within 30 min. Hence, the high binding affinity for
isomer 5c in the beginning (Figure 4) should be interpreted
with caution since there was a significant amount of 5d in the
binding assay medium. Therefore, the binding kinetics of 5a−
5d (Figure 4) is consistent with the observed isomerization
kinetics (Figure 5).
We also investigated the isomerization kinetics for 1a, 1b, 6a,
6b, 12a, and 12b in the binding assay medium. Our data
showed that in general, isomers a isomerized relatively fast,
whereas isomers b were fairly stable (SI). For instance, after 12
days in the medium, 93.9% of 1b remained when started with
Figure 4. Binding kinetics of 5a, 5b, 5c and 5d. The IC50 of MDM2
binding affinity is on the y axis and incubation time is on the x axis. See
Experimental section for binding assay conditions.
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Table 2. Binding Affinity of Selected Spirooxindole Inhibitors to MDM2 Protein
a
,
b
c
d
a
,b
c
d
entry
compound
IC50 (nM)
Ki (nM)
compound
IC50 (nM)
Ki (nM)
1
1a
2a
10700 1300
565 57
1500 180
76.2 7.7
311 24
1b
126 13
30.9 2.9
137 11
25.9 1.5
11.3 1.2
24.6 7.0
721 79
55.1 5.2
22.5 2.2
835 121
112 12
104 16
66.8 7.4
16.1 1.7
3.1 0.3
17.6 1.0
2.4 0.2
1.0
2
2b
b
3
3a
2285 183
387 39
3b
4
4a
5a
51.8 4.2
18.1 1.0
54.9 4.2
2700 500
67.0 5.9
4b
5
141 11
5b
b
6
6a
410 39
6b
2.2 0.5
97.5 10.7
6.4 0.6
1.9 0.2
113 16
14.2 1.1
13.1 1.0
8.0 1.0
7
7a
19500 4000
498 44
7b
8
8a
8b
e
9
9a
n.d.
9b
e
10
11
12
13
14
15
10a
11a
n.d.
10b
11b
12b
13b
e
n.d.
b
12a
180 24
23.5 2.1
e
13a
1d
n.d.
181000 31000
25000 18000
2.2 0.2
5c
24.1 2.3
5d
3100 460
430 62
a
b
c
Purity was >95% unless otherwise stated. Purity of 4a was 94%; of 6a, 93.5% and of 12a, 82%. See Experimental section for binding assay
procedures. Calculation of Ki based on IC50.10 n.d. not determined.
d
e
1b but 53% of 1a converted into 1b and only 17.6% of 1a
remained when started with 1a.
Table 3. Cell Growth Inhibitory Activity of 5a and 5b with
Different Treatment Time
We purified a sufficient amount of cis−cis isomers 1b−13b
and trans−cis isomer 1a−8a and 12a and determined their
binding affinities to MDM2 (Table 2). Upon the basis of the
isomerization kinetics observed for 5a/5b, 1a/1b, 6a/6b and
12a/12b, there was minimal isomerization for these isomers in
the binding assay medium in the initial 20 min. Therefore, the
incubation time in all binding experiments was controlled
precisely at 15 min, resulting in less than 20 min experimental
time in total, including plate preparation and incubation, to
minimize the influence of the isomerization on MDM2 binding
affinity. Our binding data showed that in general, the cis−cis
isomers b have better binding affinities to MDM2 than the
corresponding trans−cis isomers a. For example, 4b and 5b are
>15-times more potent than their respective isomer 4a and 5a.
Interestingly, 12a and 12b have very similar binding affinities.
Our binding data also provided initial structure−activity
relationship for isomers b. Substitution of F at the 2-position of
the phenyl ring of 1b and 2b has no effect on the MDM2
binding affinity (compare 1b versus 3b and 2b versus 4b).
However, substitution of F at the 5-position of the oxindole
ring of 1b and 2b was detrimental to binding, as shown by the
binding affinity difference between 1b and 10b, and between 2b
and 11b. Removal of one methyl from the amide improves the
binding affinity of 1b, 3b, 4b and 10b (compare 1b versus 2b,
3b versus 4b, 4b versus 9b and 10b versus 11b). Changing the
amide of 9b to either an ester (7b) or an acid (8b) decreased
the binding affinity to MDM2.
time
IC50 of 5a (μM)
IC50 of 5b (μM)
Day 1
Day 2
Day 3
Day 4
30.32 6.23
0.95 0.16
0.18 0.02
0.15 0.03
18.06 2.40
0.63 0.04
0.18 0.01
0.14 0.05
Figure 6. Isomerization studies of 5a and 5b in cell culture medium.
Panels A and B depict the isomerization studies of compounds 5a and
5b, respectively. The proportion of compounds is on the y axis and
reaction time is on the x axis. See SI for conditions of the
isomerization study in cell growth inhibition assay medium.
medium (Figure 6, panels 1 and 2). Starting from either 5a or
5b, the distributions of four isomers were very similar after
incubation for one day in the cell culture medium. After
incubation for 2 days, the most potent stereoisomer 5b in the
binding assay accounts for approximately 50% of all 4 isomers,
either starting from 5a or from 5b. Hence, the relatively rapid
isomerization of both 5a and 5b in the cell culture medium
explains why these two isomers show similar activity in the cell
growth inhibitory assay.
We investigated the cell growth inhibitory activity of isomers
5a and 5b in the SJSA-1 osteosarcoma cell line, which has an
amplified MDM2 gene and has been extensively used for
evaluation of MDM2 inhibitors (Table 3).6
Surprisingly, our data showed that despite the difference in
their binding affinities to MDM2, there is no significant
difference in their activity against cellular growth of the SJSA-1
cell line. To investigate this discrepancy between binding
affinities and cellular activities of these two compounds, we
monitored the isomerization of 5a and 5b in cell culture
medium by HPLC analysis of MeCN extracts of cell growth
medium containing 5a or 5b at indicated time points (Figure
6). Both 5a and 5b were found to isomerize in cell growth
We next investigated the in vivo activity of 5a and 5b in a
pharmacodynamic (PD) experiment using SJSA-1 osteosarco-
ma tumor xeonografts in severe combined immunodeficient
(SCID) mice. SCID mice bearing SJSA-1 tumors were treated
with a single dose of 5a or 5b at 200 mg/kg via oral gavage and
the tumor tissues were harvested at indicated time points for
Western blotting analysis of p53 activation. Western blotting
showed that both 5a and 5b effectively activated p53 in tumor
tissue in a time-dependent manner, as shown by the
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accumulation of p53 protein itself, and MDM2 and p21
proteins, two p53 targeted gene products (Figure 7).
induction of p53 activation and of PARP cleavage by 5b than
5a in SJSA-1 tumor tissue (Figure 7) is consistent with the data
that much higher concentrations of 5b were achieved when
dosed with 5b than dosed with 5a in tumor tissue, and 5b is 18-
times more potent than 5a in terms of binding to MDM2.
To further analyze isomerization of compound 5 in vivo, we
determined the ratio of four isomers (5a−5d) when dosed with
either 5a or 5b based upon the HPLC spectra from analysis of
the extracts of plasma and tumor tissue homogenate (Figure 8).
Figure 7. Western blot analysis of p53 activation induced by 5a and 5b
in SJSA-1 osteosarcoma tumor tissue with a single, oral dose of either
compound at 200 mg/kg. FL-PARP, full-length PAPR; Cl-PARP,
cleaved PARP; Pro-Cas3, pro-caspase-3; Cl-Cas3, cleaved caspase-3.
Isomer 5b was clearly more potent than 5a in activating p53
in tumor tissues. At 24 h, 5b resulted in stronger apoptosis
induction than 5a based upon cleavage of poly(ADP-ribose)
polymerase (PARP) and activation of caspase-3, two
biochemical markers of apoptosis. These PD data in tumor-
bearing mice demonstrate that isomer 5b is more potent than
isomer 5a in vivo in activation of p53 and induction of
apoptosis, in contrast to the cell growth data observed in these
two isomers (Table 3).
To further understand the clear difference between isomers
5a and 5b in the in vivo PD experiment in SJSA-1 tumor tissue,
we investigated the isomerization of 5a and 5b in vivo in both
plasma and tumor tissue in SCID mice bearing SJSA-1 tumors.
SCID mice were orally administered with a single, oral dose of
5a or 5b at 100 mg/kg, and samples of plasma and tumor tissue
were collected at 1, 6, and 24 h and analyzed using the LC-MS/
MS method. The data are summarized in Table 4.
Figure 8. Isomerization of 5a and 5b in tumor tissue or plasma. Panels
A and B depict the isomerization of 5a and panels C and D depict the
isomerization of 5b in plasma and tumor tissue accordingly. The
proportion of compounds is on the y axis and the time when samples
were harvested is on the x axis. Bars indicate the standard error.
Our data showed that when dosed with 5b, 5b remained the
dominant isomer at 1 and 6 h time-points and there was only a
small percentage of the other three isomers (<10% when
combined). In contrast, when dosed with 5a, there was rapid
isomerization and isomer 5b became the dominant form at the
6 h time-point. These data show that 5b isomerizes slowly in
vivo whereas 5a isomerizes very fast.
We determined the antitumor activity of 5a and 5b in the
SJSA-1 xenograft model. SCID mice bearing established SJSA-1
tumors were treated with 5a at 200 mg/kg or 5b at 100 mg/kg
and 200 mg/kg daily via oral gavage for 14 days (Figure 9).
Isomer 5a at 200 mg/kg daily dosing inhibited tumor growth
by 41% as compared to vehicle control (p < 0.0001) at the end
of the treatment (day 26). In comparison, 5b at 100 mg/kg
inhibited tumor growth by 67% (p < 0.0001) but shrank tumors
at 200 mg/kg by an average of 91% at the end of treatment
(day 26, see SI). There was no significant weight loss (SI) or
In plasma, the concentration of 5b in mice dosed with 5b was
7-fold higher than that in mice dosed with 5a at 1 h and was 12-
fold at 6 h. In tumor tissue, the concentration of 5b in mice
dosed with 5b was 11-fold higher than that in mice dosed with
5a at 1 h, and was 16-fold at 6 h. Hence, the much stronger
Table 4. Concentration of 5a and 5b in Plasma and Tumor
Tissue in SCID Mice Bearing SJSA-1 Tumors when Dosed
with Either 5a or 5b
dosing
isomer
1 h
6 h
24 h
a
Plasma concentration (ng/mL)
5b
5a
5b
5a
5b
5a
16800 1931
2577 815
47 20
5.9 0.8
b
3343 1385
2380 448
617 97
BLQ
210 141
75 24
BLQ
BLQ
a
Tumor concentration(ng/g)
5b
5a
5b
5a
5b
5a
19133 3775
1963 683
65 24
BLQ
BLQ
BLQ
BLQ
1122 297
1797 527
2973 1036
120 84
27 18
a
b
Three mice per time point. BLQ: below the lower limit of
Figure 9. Antitumor activity of 5a and 5b in the SJSA-1 osteosarcoma
quantification.
tumor xenograft model.
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other signs of toxicity for either compound. The efficacy data
show that isomer 5b has much better in vivo antitumor activity
than isomer 5a, consistent with the much stronger p53
activation and apoptosis induction by 5b than by 5a in tumor
tissues (Figure 7).
Although 5b had a strong antitumor activity and shrank
tumors by 91% in the SJSA-1 xenograft model, it failed to
achieve complete tumor regression. We hypothesized that the
failure of complete tumor regression by 5b may be due to its
less than ideal pharmacokinetic (PK) properties. To this end,
we have performed extensive modifications to spirooxindol-
containing compounds to further improve their in vivo PK
profile, which ultimately led to identification of compound 15b
(MI-888) (Figure 10).12 Compound 15b binds to MDM2 with
compound (SI). The efficacy data thus clearly demonstrate that
isomer 15b is much more efficacious than isomer 15a.
CONCLUSIONS
■
In the present study, we have performed detailed investigations
on the reversible ring-opening-recyclization reaction of selected
spirooxindole-containing MDM2 inhibitors. This reaction
afforded four diastereoisomers from one single stereoisomer.
The stable isomers (isomers b) for these compounds have a
cis−cis configuration and bind to MDM2 with better affinities
than the other three isomers. Importantly, our investigation has
led to the identification of a set of highly potent MDM2
inhibitors with a low nanomolar binding affinity. In particular,
compound 15b (MI-888) has a Ki value of 0.44 nM to MDM2
and is capable of achieving complete and long-lasting tumor
regression in an animal model of human cancer. Our data
clearly suggest that MI-888 is a highly promising MDM2
inhibitor and warrants extensive preclinical investigation as a
potential clinical development candidate.
EXPERIMENTAL SECTION
■
1. Chemistry. All reactions were conducted in round-bottomed
flasks with a Teflon-coated magnet stirring bar. Experiments involving
moisture and/or air sensitive components were performed under an
N2 atmosphere. Commercial reagents and anhydrous solvents were
used without further purification. Crude reaction products were
purified by flash column chromatography on silica gel. Further
purification was performed on a semipreparative HPLC (Waters Delta
600) with 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) and carbon nuclear magnetic resonance (13C NMR)
spectroscopy were performed on a Bruker Advance 300 NMR
spectrometer. Low resolution ESI mass spectra analyses were
determined on a Thermo-Scientific LCQ Fleet mass spectrometer.
The analytical reverse phase HPLC (rp-HPLC) model was Waters
2795 Separation (UV detection at 230 and 254 nm wavelengths). All
final compounds were purified to ≥95% purity as determined by
analytical HPLC analysis using reverse phase column (SunFire, C18−5
μm, 4.6 × 150 mm) unless otherwise stated.
Figure 10. Structures and MDM2 binding affinity of 15a and 15b.
a Ki value of 0.44 nM. In comparison, the corresponding cis−
trans isomer 15a has a Ki value of 4.5 nM in binding to MDM2,
being 10-times less potent than 15b.
To test the therapeutic potential and the potential difference
in their antitumor activity between 15a and 15b, both
compounds were tested in the SJSA-1 tumor xenograft
model. In this experiment, SCID mice bearing established
SJSA-1 tumors were treated with 15a at 100 mg/kg or 15b at
100 mg/kg via oral gavage for 14 days (Figure 11).
The procedures for synthesis of compounds 1a−6a, 12a, and 15a
were adapted from our previously reported methods6d,7 shown in
Scheme 1 with some modifications.6a The trans−cis isomers 1a−6a
and 15a were obtained as major products after ceric ammonium
nitrate (CAN) oxidative cleavage of chiral auxiliary. The trans−cis
isomers 9a, 11a, and 13a were minor isomers after CAN oxidation and
efforts made to obtain 9a−13a in high purity proved futile.
Detailed procedures for the synthesis of 7a and 8a via different
synthetic routes are in the Supporting Information (SI).
Figure 11. Antitumor activity of 15a and 15b in the SJSA-1
osteosarcoma tumor xenograft model.
(2′R,3S,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N,N-dimethyl-2′-
neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
1
(1a, TFA salt). H NMR (300 MHz, CD3OD): 7.40−7.32 (m, 4H),
6.87 (s, 1H), 6.76 (d, J = 8.22 Hz, 1H), 6.55 (d, J = 8.17 Hz, 1H), 5.47
(d, J = 7.88 Hz, 1H), 4.28 (d, J = 6.38 Hz, 1H), 4.17 (d, J = 7.88 Hz,
1H), 2.97 (s, 3H), 2.78 (s, 3H), 2.05 (dd, J = 15.31, 7.63, Hz, 1H),
1.16 (d, J = 15.31 Hz, 1H), 0.84 (s, 9H). 13C (75 MHz, CD3OD):
180.46, 167.42, 145.49, 139.42, 136.92, 136.38, 131.97, 130.50, 130.23,
128.37, 128.30, 124.57, 123.26, 112.09, 63.35, 63.27, 62.84, 55.37,
In contrast to 5b, and other previously reported potent
MDM2 inhibitors, such as MI-219, Nutlin-3a and AM-
8553,6b,13 15b was capable of achieving rapid and complete
tumor regression. After treatments with 15b for 10 days at 100
mg/kg, all tumors of the treated mice completely regressed.
Importantly, the complete regression was long-lasting; all mice
treated with 15b remained completely tumor free for an
additional 64 days. In comparison, while 15a at 100 mg/kg
daily dosing inhibited tumor growth by 78% as compared to
vehicle control (p < 0.0001) at the end of the treatment (day
24), it failed to achieve tumor regression. There was no
significant weight loss or other signs of toxicity for either
42.47, 38.03, 36.96, 30.99, 29.59. ESI-MS calculated for
25
C25H3035Cl2N3O2 [M + H]+ = 474.17, Found: 474.50. [α]D
=
−40.4° (c = 0.0113, MeCN)
(2′R,3S,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N-methyl-2′-neo-
pentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide (2a,
TFA salt). 1H NMR (300 MHz, CD3OD): 7.28−7.16 (m, 3H),
7.16−7.06 (m, 1H), 6.92−6.82 (m, 2H), 6.80−6.76 (m, 1H), 4.92 (d, J
= 10.23, 4.20−4.10 (m, 2H), 2.73 (s, 3H), 1.99 (s, J = 15.32, 6.75 Hz,
1H), 1.47 (dd, J = 15.54, 3.49 Hz, 1H), 0.80 (s, 9H). 13C (75 MHz,
G
dx.doi.org/10.1021/ja3125417 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
CD3OD): 180.04, 167.26, 144.79, 137.00, 136.58, 135.84, 131.40,
129.99, 129.84, 128.18, 128.11, 126.71, 123.42, 111.88, 64.23, 64.23,
62.35, 57.08, 42.71, 30.95, 29.58, 27.00. ESI-MS calculated for
C24H2835Cl2N3O2 [M + H]+ = 460.16, Found: 460.52
29.97, 14.32. ESI-MS calculated for C25H2835Cl2FN2O3 [M + H]+ =
493.15, Found: 493.44
(2′R,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-2′-neo-
pentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxylic acid (8a,
1
TFA salt). H NMR (300 MHz, CD3OD): 7.56−7.46 (m, 1H), 7.46−
(2′R,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N,N-di-
7.36 (m, 1H), 7.26−7.18 (m, 1H), 6.91 (d, J = 1.61 Hz, 1H), 6.76 (dd,
J = 8.10, 1.50 Hz, 1H), 6.47 (d, J = 8.14 Hz, 1H), 5.29 (d, J = 8.67 Hz,
1H), 4.43 (d, J = 8.67 Hz, 1H), 4.11 (d, J = 7.47, 2.13 Hz, 1H), 2.02
(dd, J = 15.36, 7.50 Hz, 1H), 1.16 (dd, J = 15.36, 2.24 Hz, 1H), 0.81
(s, 9H). ESI-MS calculated for C23H2435Cl2FN2O3 [M + H]+ = 465.11,
Found: 465.42;
methyl-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-car-
1
boxamide (3a, TFA salt). H NMR (300 MHz, CD3OD): 7.80−7.70
(m, 1H), 7.50−7.40 (m, 1H), 7.32−7.22 (m, 1H), 6.92−6.88 (m, 1H),
6.74 (dd, J = 8.15, 1.75 Hz, 1H), 6.48 (d, J = 8.11 Hz, 1H), 5.58 (d, J =
7.94 Hz, 1H), 4.38 (d, J = 7.94 Hz, 1H), 4.26 (d, J = 7.55 Hz, 1H),
2.99 (s, 3H), 2.84 (s, 3H), 2.06 (dd, J = 15.42, 7.72 Hz, 1H), 1.13 (d, J
= 15.37 Hz, 1H), 0.89 (s, 9H). 13C (75 MHz, CD3OD): 180.07,
166.93, 157.51 (d, JC−F = 243.98 Hz), 145.59, 137.11, 132.54, 128.77,
127.66, 126.74 (d, JC−F = 4.55 Hz), 125.89 (d, JC−F =13.40 Hz),
123.68, 122.85 (d, JC−F = 10.19 Hz), 123.10 (d, JC−F = 18.37 Hz),
112.07, 63.03, 62.05, 61.76, 42.09, 37.70, 36.89, 30.77, 29.34. ESI-MS
calculated for C25H2935Cl2FN3O2 [M + H]+ = 492.16, Found: 492.44
(2′R,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-methyl-
2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
(4a, TFA salt, 94% purity). 1H NMR (300 MHz, CD3OD): 7.66−7.54
(m, 1H), 7.38−7.28 (m, 1H), 7.22−7.10 (m, 1H), 6.87 (s, 1H), 6.82−
6.72 (m, 2H), 5.21 (d, J = 10.00 Hz, 1H), 4.50 (d, J = 9.93 Hz, 1H),
4.30−4.24 (m, 1H), 2.75 (s, 3H), 2.07 (dd, J = 15.35, 7.35 Hz, 1H),
1.8 (d, J = 14.57 Hz, 1H), 0.80 (s, 9H). 13C (75 MHz, CD3OD):
180.31, 167.24, 157.81 (d, JC−F = 247.78 Hz), 145.44, 136.94, 132.11,
(2′R,3S,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N-((S)-3,4-dihy-
droxybutyl)-5-fluoro-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrroli-
1
dine]-5′-carboxamide (12a, TFA salt, 82% purity). H NMR (300
MHz, CD3OD): 7.32−7.22 (m, 3H), 7.18−7.10 (m, 1H), 7.06 (d, J =
8.74 Hz, 1H), 6.88 (d, J = 6.08 Hz, 1H), 4.97 (d, J = 10.79 Hz, 1H),
4.22 (d, J = 10.79 Hz, 1H), 4.26−4.18 (m, 1H), 3.50−3.20 (m, 5H),
2.03 (dd, J = 15.35, 6.56 Hz, 1H), 1.68−1.52 (m, 2H), 1.50−1.40 (m,
1H), 0.84 (s, 9H). 13C (75 MHz, CD3OD): 179.64, 166.47, 140.40 (d,
JC−F = 2.94 Hz), 136.54, 136.06, 131.64, 130.16, 130.02, 128.14,
127.27 (d, JC−F = 40.14 Hz), 123.05 (d, JC−F = 19.44 Hz), 115.94 (d,
JC−F = 25.54 Hz), 113.09, 70.91, 67.24, 64.19, 64.10, 62.73, 57.52,
42.66, 38.14, 33.85, 30.99, 29.57. ESI-MS calculated for
C27H3335Cl2FN3O4 [M + H]+ = 552.18, Found: 552.75
(2′R,3S,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N-((1s,3S)-3-hy-
droxy-3-methylcyclobutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-
128.66, 127.94, 126.47 (d, JC−F = 4.45 Hz), 125.29, 125.01 (d, JC−F
=
1
pyrrolidine]-5′-carboxamide (15a, TFA salt). H NMR (300 MHz,
13.96 Hz), 123.37, 122.64 (d, JC−F = 18.03 Hz), 122.04, 64.35, 63.69,
61.74, 49.51, 42.34, 30.92, 29.55, 27.10. ESI-MS calculated for
C24H2735Cl2FN3O2 [M + H]+ = 478.15, Found: 478.92
MeOD-d4): 7.40−7.50 (m, 1H), 7.40−7.30 (m, 1H), 7.20−7.10 (m,
1H), 6.85 (s, 1H), 6.83−6.75 (m, 2H), 5.11 (d, J = 10.11 Hz, 1H),
4.47 (d, J = 10.09 Hz, 1H), 4.22 (dd, J = 7.32, 2.72 Hz, 1H), 4.00−3.80
(m, 1H), 2.50−2.30 (m, 2H), 2.15−1.95 (m, 2H), 1.95−1.80 (m, 1H),
1.40−1.20 (m, 1H), 1.29 (s, 3H), 0.80 (s, 9H). 13C NMR (75 MHz,
MeOD-d4): 180.0, 165.9, 157.6 (d, JC−F = 248 Hz), 145.3, 136.7,
(2′R,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-((S)-3,4-
dihydroxybutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-
5′-carboxamide (5a, TFA salt). 1H NMR (300 MHz, CD3OD): 7.63−
7.53 (m, 1H), 7.40−7.30 (m, 1H), 7.23−7.13 (m, 1H), 6.87 (d, J =
1.36 Hz, 1H), 6.86−6.74 (m, 2H), 5.19 (d, J = 10.22 Hz, 1H), 4.49 (d,
J = 10.22 Hz,1H), 4.25 (dd, J = 7.35, 2.71 Hz, 1H), 3.56−3.43 (m,
1H), 3.42−3.30 (m, 4H), 2.07 (dd, J = 15.43, 7.44 Hz,1 H), 1.72−1.58
(m, 1H), 1.56−1.40 (m, 1H), 1.30 (dd, J = 15.43, 2.62 Hz, 1H), 0.80
131.9, 128.6, 127.7, 126.2 (d, JC−F = 4.76 Hz), 125.2, 124.8 (d, JC−F
=
13.8 Hz), 123.1, 122.4 (d, JC−F = 18.1 Hz), 111.8, 64.2, 63.5, 61.4,
49.7, 45.5, 45.4, 42.2, 38.4, 30.7, 29.4, 27.4. ESI-MS calculated for
C28H3335Cl2FN3O3 [M + H]+: 548.17, Found: 548.25. [α]D25 = −30.6°
(c = 0.0154 g/mL in MeOH);
(s, 9H). 13C (75 MHz, CD3OD): 180.26, 166.78, 157.81 (d, JC−F
247.93 Hz), 145.43, 136.90, 132.16, 128.82, 127.96, 126.48 (d, JC−F
=
=
The cis−cis isomers 1b−7b, 10b, 12b, and 15b were obtained as
major products after aging the materials purified by flash column
chromatography in appropriate solvents for 3−4 days. In cases of 9b,
11b, and 13b, the cis−cis isomers were obtained as major products
after the CAN oxidation without aging. Generally, the side isomers c
and d were observed in small amounts after oxidation or after aging.
Standard procedure for aging: 30 mg material obtained from CAN
oxidation (prepurified by flash column chromatography) was placed in
a round bottomed flask with a magnetic stirring bar. MeCN (2.4 mL)
was added to dissolve the product. H2O (2.0 mL) was added to this
MeCN solution followed by 0.4 mL NaHCO3 saturated solution to
adjust the pH to 8. This solution was allowed to stir at room
temperature for 3−4 days. The percentage of isomer b and other
isomers was determined using analytical HPLC. Further purification
was performed by semipreparative or preparative HPLC. Pure MeOH
can also be used as aging solvent.
4.49 Hz), 125.43, 124.94 (d, JC−F = 14.00 Hz), 123.37, 122.63 (d, JC−F
= 18.23 Hz), 112.00, 71.01, 67.25, 64.38, 63.80, 61.69, 50.06, 42.36,
38.30, 33.80, 30.94, 29.55. ESI-MS calculated for C27H3335Cl2FN3O4
25
[M + H]+ = 552.18, Found: 552.92; [α]D = −40.1° (c = 0.0106,
MeOH)
(2′R,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-(2-mor-
pholinoethyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-
carboxamide (6a, TFA salt, 93.5% purity). 1H NMR (300 MHz,
CD3OD): 7.59 (t, J = 6.93 Hz, 1H), 7.41 (td, J = 7.55, 0.97 Hz, 1H),
7.22 (t, J = 7.90 Hz, 1H), 6.90 (d, J = 1.79 Hz, 1H), 6.77 (dd, J = 8.14,
1.79 Hz, 1H), 6.48 (d, J = 8.15 Hz, 1H), 5.21 (d, J = 8.22 Hz, 1H),
4.50 (d, J = 8.19 Hz, 1H), 4.17 (dd, J = 7.63, 2.23 Hz, 1H), 4.10−3.80
(m, 4H), 3.75−3.45 (m, 4H), 3.40−3.00 (m, 4H), 2.08−1.96 (m, 1H),
1.19 (dd, J = 15.32, 2.23 Hz, 1H), 0.81 (s, 9H). 13C (75 MHz,
CD3OD): 180.28, 168.91, 157.75 (d, JC−F = 248.38 Hz), 145.50,
137.13, 132.29, 128.63, 127.67, 126.63 (d, JC−F = 4.50 Hz), 125.74 (d,
JC−F = 13.69 Hz), 124.72, 123.44, 122.83 (d, JC−F = 18.25 Hz), 112.20,
65.16, 64.25, 63.84, 62.22, 58.01, 53.84, 50.05, 42.39, 35.72, 30.95,
29.52. ESI-MS calculated for C29H3635Cl2FN4O3 [M + H]+ = 577.21,
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N,N-dimethyl-2′-
neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
1
(1b, TFA salt). H NMR (300 MHz, CD3OD): 7.58 (d, J = 8.08 Hz,
1H), 7.32−7.10 (m, 5H), 6.78 (d, J = 1.54 Hz, 1H), 5.69 (d, J = 10.03
Hz, 1H), 4.41 (d, J = 6.93 Hz, 1H), 4.14 (d, J = 10.03 Hz, 1H), 2.96 (s,
3H), 2.76 (s, 3H), 1.90 (dd, J = 15.41, 8.26 Hz, 1H), 1.15 (d, J = 15.41
Hz, 1H), 0.89 (s, 9H). 13C (75 MHz, CD3OD): 178.04, 168.67,
145.47, 137.24, 135.99, 135.36, 131.69, 130.50, 129.79, 128.44, 126.49,
124.27, 123.84, 112.31, 65.29, 64.52, 60.50, 57.35, 42.59, 38.06, 37.00,
25
Found: 577.48. [α]D = −43.3° (c = 0.0139, MeCN).
(2′R,3S,4′S,5′R)-Ethyl 6-Chloro-4′-(3-chloro-2-fluorophenyl)-2′-
neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxylate (7a,
1
TFA salt). H NMR (300 MHz, CD3OD): 7.50−7.40 (m, 2H), 7.28−
7.18 (m, 1H), 6.91 (d, J = 1.81 Hz, 1H), 6.78(dd, J = 8.13, 1.87 Hz,
1H), 6.46 (d, J = 8.14 Hz, 1H), 5.30 (d, J = 8.52 Hz, 1H), 4.43 (d, J =
8.52 Hz, 1H), 4.40−4.20 (m, 2H), 4.08 (dd, J = 7.47, 2.43 Hz, 1H),
2.00 (dd, J = 15.32, 7.53 Hz, 1H), 1.22 (t, J = 7.12 Hz, 3H), 1.19 (dd, J
= 15.32, 2.55 Hz, 1H), 0.81 (s, 9H). 13C (75 MHz, CD3Cl, note free
amine): 181.33, 171.91, 156.54 (d, JC−F = 248.02 Hz), 142.85, 134.15,
129.91, 128.37 (d, JC−F = 14.61 Hz), 127.19 (d, JC−F = 3.19 Hz),
31.03, 29.66. ESI-MS calculated for C25H3035Cl2N3O2 [M + H]+
474.17, Found: 474.68. [α]D = 32.1° (c = 0.0126, MeCN).
=
25
(2′R,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N,N-dimethyl-2′-
neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
1
(1d, TFA salt). H NMR (300 MHz, CD3OD): 7.72 (d, J = 8.08 Hz,
1H), 7.28−7.22 (m, 1H), 7.20−7.12 (m, 2H), 7.12−7.08 (m, 1H),
7.04 (d, J = 7.65 Hz, 1H), 6.81 (d, J = 1.79 Hz, 1H), 5.54 (d, J = 10.87
Hz, 1H), 4.37 (dd, J = 6.54, 4.52 Hz, 1H), 4.15 (d, J = 10.85 Hz, 1H),
125.94, 124.94, 124.63 (d, JC−F = 4.50 Hz), 122.10, 121.69 (d, JC−F
=
18.50 Hz), 110.72, 67.16, 65.70, 63.19, 61.74, 51.17, 43.45, 30.33,
H
dx.doi.org/10.1021/ja3125417 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
2.98 (s, 3H), 2.81 (s, 3H), 1.70 (dd, J = 15.24, 6.67 Hz, 1H), 1.20 (dd,
J = 15.26, 4.43 Hz, 1H), 0.91 (s, 9H). 13C (75 MHz, CD3OD): 177.03,
169.16, 144.95, 136.94, 135.68, 135.34, 131.25, 130.20, 130.10, 128.88,
128.33, 124.24, 123.43, 112.43, 65.98, 65.14, 60.60, 60.24, 44.23,
38.07, 37.04, 30.97, 29.77. ESI-MS calculated for C25H3035Cl2N3O2 [M
(m, 1H), 3.56−3.40 (m, 1H), 3.40−3.20 (m, 4H), 1.70−1.40 (m, 3H),
1.10 (dd, J = 14.59, 3.06 Hz, 1H), 0.91 (s, 9H). ESI-MS calculated for
C27H3335Cl2FN3O4 [M + H]+= 552.18, Found: 552.40.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-(2-mor-
pholinoethyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-
1
25
+ H]+ = 474.17, Found: 474.50. [α]D = −132.9° (c = 0.0031,
carboxamide (6b, TFA salt). H NMR (300 MHz, CD3OD): 7.66−
7.48 (m, 2H), 7.42−7.32 (m, 1H), 7.20−7.10 (m, 2H), 6.78 (d, J =
1.68 Hz, 1H), 5.38 (d, J = 11.48 Hz, 1H), 4.65 (d, J = 11.48 Hz, 1H),
4.52 (d, J = 7.59 Hz, 1H), 4.00−3.70 (m, 4H), 3.70−3.60 (m, 2H),
3.50−3.40 (m, 2H), 3.40−3.10 (m, 4H), 1.97 (dd, J = 15.40, 8.62 Hz,
1H), 1.12 (d, J = 15.40 Hz, 1H), 0.88 (s, 9H). 13C (75 MHz,
CD3OD): 177.73, 169.20, 145.19, 137.15, 132.54, 128.69, 126.84,
126.64 (d, JC−F = 4.80 Hz), 124.11, 123.72, 122.57 (d, JC−F = 13.19
Hz), 121.77 (d, JC−F = 13.19 Hz), 112.06, 65.08, 64.59, 64.32, 62.69,
MeCN).
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N-methyl-2′-neo-
pentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide (2b,
TFA salt). 1H NMR (300 MHz, CD3OD): 7.63 (d, J = 8.06 Hz,
1H), 7.30−7.14 (m, 4H), 7.12−7.00 (m, 1H), 6.82−6.76 (m, 1H),
5.29 (d, J = 11.24 Hz, 1H), 4.47 (d, J = 6.68 Hz, 1H), 4.16 (d, J =
11.22 Hz, 1H), 2.73 (s, 3H), 1.92 (dd, J = 15.40, 8.39 Hz, 1H), 1.17
(d, J = 16.85 Hz, 1H), 0.90 (s, 9H). 13C (75 MHz, CD3OD): 177.78,
168.54, 145.38, 137.07, 135.77, 134.60, 131.40, 130.29, 129.48, 128.29,
126.32, 124.24, 124.16, 112.18, 65.11, 64.18, 62.85, 56.95, 43.42,
30.97, 29.66, 26.98. ESI-MS calculated for C24H2835Cl2N3O2 [M + H]+
= 460.16, Found: 460.48.
57.41, 53.70, 48.47, 43.55, 35.61, 31.06, 29.56. ESI-MS calculated for
25
C29H3635Cl2FN4O3 [M + H]+ = 577.21, Found: 577.48. [α]D
=
−24.7° (c = 0.0171, MeOH).
(2′S,3R,4′S,5′R)-Ethyl 6-chloro-4′-(3-chloro-2-fluorophenyl)-2′-
neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxylate (7b,
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N,N-di-
1
TFA salt). H NMR (300 MHz, CD3OD): 7.59 (d, J = 7.41 Hz, 1H),
methyl-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-car-
1
boxamide (3b, TFA salt). H NMR (300 MHz, CD3OD): 7.68−7.57
7.50−7.42 (m, 1H), 7.40−7.32 (m, 1H), 7.17−7.07 (m, 2H), 6.78 (d, J
= 1.65 Hz, 1H), 5.61 (d, J = 12.25 Hz, 1H), 4.56 (d, J = 12.25 Hz,
1H), 4.47 (dd, J = 8.50, 1.50 Hz, 1H), 4.25 (dq, J = 10.77, 7.12 Hz,
1H), 4.13 (dq, J = 10.77, 7.12 Hz, 1H), 1.93 (dd, J = 15.39, 8.67 Hz,
1H), 1.34 (dd, J = 15.39, 1.57 Hz, 1H), 1.10 (t, J = 7.12 Hz, 3H), 0.87
(s, 9H). ESI-MS calculated for C25H2835Cl2FN2O3 [M + H]+ = 493.15,
Found: 493.44.
(m, 2H), 7.45−7.35 (m, 1H), 7.22−7.10 (m, 2H), 6.84−6.77 (m 1H),
5.68 (d, J = 10.24 Hz, 1H), 4.64 (d, J = 10.24 Hz, 1H), 4.48 (dd, J =
8.20, 1.70 Hz, 1H), 2.98 (s, 3H), 2.80 (s, 3H), 1.89 (dd, J = 15.48, 8.26
Hz, 1H), 1.14 (dd, J = 15.48, 1.67 Hz, 1H), 0.89 (s, 9H). 13C (75
MHz, CD3OD): 177.96, 168.37, 152.99 (d, JC−F = 253.32 Hz), 145.26,
137.39, 132.82, 128.82, 126.95, 126.70 (d, JC−F = 4.79 Hz), 124.28,
122.09 (d, JC−F = 13.13 Hz), 118.90, 115.11, 112.24, 64.69, 59.97,
42.64, 37.96, 37.03, 31.03, 29.62. ESI-MS calculated for
C25H2935Cl2FN3O2 [M + H]+ = 492.16, Found: 492.46.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-2′-neo-
pentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxylic acid (8b,
1
TFA salt). H NMR (300 MHz, CD3OD): 7.58−7.42 (m, 2H), 7.36−
7.26 (m, 1H), 7.14−7.02 (m, 1H), 6.75 (s, 1H), 5.43 (d, J = 12.00 Hz,
1H), 4.58 (d, J = 11.97 Hz, 1H), 4.35 (d, J = 8.53 Hz, 1H), 1.87 (dd, J
= 15.08, 9.31 Hz, 1H), 1.09 (d, J = 15.31 Hz, 1H), 0.85 (s, 9H). ESI-
MS calculated for C23H2435Cl2FN2O3 [M + H]+ = 465.11, Found:
465.38.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-2′-neo-
pentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide (9b,
TFA salt). 1H NMR (300 MHz, CD3OD): 7.54−7.44 (m, 2H),
7.36−7.26 (m, 1H), 7.14−7.00 (m, 2H), 6.70 (d, J = 1.76 Hz, 1H),
5.22 (d, J = 11.36 Hz, 1H), 4.50 (d, J = 11.36 Hz, 1H), 4.41 (d, J =
8.23, 1.85 Hz, 1H), 1.81 (dd, J = 15.46, 8.31 Hz, 1H), 1.06 (dd, J =
15.46, 1.90 Hz, 1H), 0.78 (s, 9H). ESI-MS calculated for
C23H2535Cl2FN3O2 [M + H]+ = 464.13, Found: 464.60.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-methyl-
2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
1
(4b, TFA salt). H NMR (300 MHz, CD3OD): 7.65−7.55 (m, 2H),
7.40−7.34 (m, 1H), 7.20−7.10 (m, 2H), 6.84−6.80 (m, 1H), 5.26 (d, J
= 11.15 Hz, 1H), 4.64 (d, J = 11.19 Hz, 1H), 4.45 (d, J = 7.86 Hz,
1H), 2.74 (s, 3H), 1.87 (dd, J = 15.19, 8.58 Hz, 1H), 1.11 (d, J = 15.21
Hz, 1H), 0.90 (s, 9H). 13C (75 MHz, CD3OD): 177.75, 168.20,
159.53 (d, JC−F = 248.25 Hz), 145.22, 137.24, 132.59, 128.58, 126.63
(d, JC−F = 5.25 Hz), 124.16, 123.43, 126.66, 122.59 (d, JC−F = 4.05
Hz), 121.54 (d, JC−F = 12.75 Hz), 112.13, 64.49, 64.40, 62.73, 55.34,
43.33, 31.01, 29.58, 27.06. ESI-MS calculated for C24H2735Cl2FN3O2
[M + H]+ = 478.15, Found: 478.25.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-((S)-3,4-
dihydroxybutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-5-fluoro-N,N-di-
1
5′-carboxamide (5b, TFA salt). H NMR (300 MHz, CD3OD): 7.60
methyl-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-car-
1
boxamide (10b, TFA salt). H NMR (300 MHz, CD3OD): 7.66 (d, J
(d, J = 8.15 Hz, 1H), 7.55 (t, J = 7.12 Hz, 1H), 7.38 (t, J = 7.57 Hz,
1H), 7.20−7.10 (m, 2H), 6.78 (d, J = 1.70 Hz, 1H), 5.27 (d, J = 11.40
Hz, 1H), 4.62 (d, J = 11.40 Hz, 1H), 4.52 (dd, J = 8.33, 1.29 Hz, 1H),
3.45−3.25 (m, 5H), 1.90 (dd, J = 15.46, 8.38 Hz, 1H), 1.64−1.48 (m,
1H), 1.46−1.32 (m, 1H), 1.14 (dd, J = 15.46, 1.34 Hz, 1H), 0.87 (s,
9H). 13C (75 MHz, CD3OD): 177.77, 167.67, 157.90 (d, JC−F = 251.2
Hz), 145.21, 137.26, 132.67, 128.64, 126.89 (d, JC−F = 1.88 Hz),
126.65 (d, JC−F = 4.89 Hz), 124.17, 123.43, 122.60 (d, JC−F = 19.0 Hz),
121.52 (d, JC−F = 13.0 Hz), 112.14, 70.99, 67.25, 64.45, 62.873, 43.39,
38.26, 33.78, 31.02, 29.57. ESI-MS calculated for C27H3335Cl2FN3O4
= 8.49 HZ, 1H), 7.35−7.12 (m, 4H), 6.86 (d, J = 6.02 Hz, 1H), 5.67
(d, J = 10.03 Hz, 1H), 4.41 (dd, J = 8.20, 1.79 Hz, 1H), 4.13 (d, J =
10.03 Hz, 1H), 2.97 (s, 3H), 2.74 (s, 3H), 1.91 (dd, J = 15.46, 8,20 Hz,
1H) 1.17 (dd, J = 15.46, 1.82 Hz, 1H), 0.91 (s, 9H). 13C (75 MHz,
CD3OD):177.80, 168.54, 156.04 (d, JC−F = 243.74 Hz), 141.04,
136.12, 135.16, 131.81, 130.66, 129.73, 128.52, 125.50 (d, JC−F =7.37
Hz), 123.70 (d, JC−F = 19.58 Hz), 114.43 (d, JC−F =25.50 Hz), 113.62,
65.81, 64.45, 60.58, 57.31, 42.59, 38.04, 37.00, 31.04, 29.63. ESI-MS
calculated for C25H2935Cl2FN3O2 [M + H]+ = 492.16, Found: 492.50.
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-5-fluoro-N-methyl-
2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide
(11b, TFA salt). 1H NMR (300 MHz, CD3OD): 7.68 (d, J = 8.47 Hz,
1H), 7.32−7.16 (m, 3H), 7.04 (d, J = 7.63 Hz, 1H), 6.85 (d, J = 6.01
Hz, 1H), 5.23 (d, J = 11.21 Hz, 1H), 4.46 (dd, J = 8.23, 1.76 Hz, 1H),
4.13 (d, J = 11.21 Hz, 1H), 2.71 (S, 3H), 1.90 (dd, J = 15.46, 8.23 Hz,
1H), 1.17 (dd, J = 15.48, 1.75 Hz, 1H), 0.89 (s, 9H). 13C (75 MHz,
CD3OD): 177.57, 168.31, 159.98 (d, JC−F = 244.73 Hz), 140.90,
135.98, 134.31, 131.59, 130.56, 129.45, 128.47, 125.84 (d, JC−F = 7.48
Hz), 123.61 (d, JC−F = 19.45 Hz), 114.33 (d, JC−F = 25.29 Hz), 113.57,
65.62, 64.15, 62.93, 57.04, 63.42, 31.01, 29.60, 27.02. ESI-MS
calculated for C24H2735Cl2FN3O2 [M + H]+ = 478.15, Found: 478.46.
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-N-((S)-3,4-dihy-
droxybutyl)-5-fluoro-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrroli-
dine]-5′-carboxamide (12b, TFA salt). 1H NMR (300 MHz,
CD3OD): 7.68 (d, J = 8.47 Hz, 1H), 7.32−7.17 (m, 3H), 7.12−7.02
25
[M + H]+ = 552.18, Found: 552.42. [α]D = −36.2° (c = 0.0147,
MeOH).
(2′S,3S,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-((S)-3,4-
dihydroxybutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-
1
5′-carboxamide (5c, TFA salt). H NMR (300 MHz, CD3OD): 8.37
(s, broad, NH), 7.62 (d, J = 8.11 Hz, 1H), 7.50−7.34 (m, 2H), 7.36−
7.10 (m, 2H), 6.83 (d, J = 1.83 Hz, 1H), 4.32 (d, J = 12.09 Hz, 1H),
4.24−4.14 (m, 1H), 3.50−3.10 (m, 5H), 1.86−1.72 (m, 2H), 1.58−
1.44 (m, 1H), 1.44−1.26 (m, 1H), 0.80 (s, 9H). ESI-MS calculated for
C27H3335Cl2FN3O4 [M + H]+ = 552.18, Found: 552.42.
(2′R,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-((S)-3,4-
dihydroxybutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-
1
5′-carboxamide (5d, TFA salt). H NMR (300 MHz, CD3OD): 7.49
(d, J = 8.02 Hz, 1H), 7.36−7.24 (m, 1H), 7.11 (dd, J = 8.09, 1.89 Hz,
1H), 7.06−6.96 (m, 1H), 6.96−6.88 (m, 1H), 6.80 (d, J = 1.88 Hz,
1H), 4.89 (d, J = 11.57 Hz, 1H), 4.46 (d, J = 11.57 Hz, 1H), 4.35−4.25
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dx.doi.org/10.1021/ja3125417 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(m, 1H), 6.85 (d, J = 6.00 Hz, 1H), 5.23 (d, J = 11.28 Hz, 1H), 4.46
(dd, J = 8.41, 1.91 Hz, 1H), 4.10 (d, J = 11.28 Hz, 1H), 3.50−3.20 (m,
5H), 1.90 (dd, J = 15.18, 8.62 Hz, 1H), 1.65−1.47 (m, 1H), 1.47−1.33
(m,1H), 1.18 (d, J = 15.18 Hz,1 H), 0.89 (s, 9H). 13C (75 MHz,
CD3OD): 177.56, 167.81, 155.99 (d, JC−F = 243.27 Hz), 140.87,
135.98, 134.28, 131.63, 130.60, 129.51, 128.60, 125.82 (d, JC−F = 7.39
Hz), 123.58 (d, JC−F = 19.68 Hz), 114.30 (d, JC−F = 25.32 Hz), 113.55,
70.86, 67.23, 65.53, 64.16, 63.07, 57.35, 43.43, 38.15, 33.78, 31.01,
29.57. ESI-MS calculated for C27H3335Cl2FN3O4 [M + H]+= 552.18,
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 to assay plates and
incubated at room temperature with gentle shaking. The incubation
time was controlled precisely 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 was 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.10
25
Found: 552.38; [α]D = 1.1° (c = 0.0334, free amine in MeOH).
(2′S,3R,4′R,5′R)-6-Chloro-4′-(3-chlorophenyl)-5-fluoro-N-((1r,4R)-
4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrroli-
dine]-5′-carboxamide (13b, TFA salt). 1H NMR (300 MHz,
CD3OD): 7.66 (d, J = 8.45 Hz, 1H), 7.30−7.16 (m, 3H), 7.10−7.03
(m, 1H), 6.85 (d, J = 5.98 Hz, 1H), 5.22 (d, J = 11.37 Hz, 1H), 4.46
(dd, J = 8.31, 1.68 Hz, 1H), 4.09 (d, J = 11.37 Hz, 1H), 3.70−3.50 (m,
1H), 3.50−3.39 (m, 1H), 2.00−1.84 (m, 3H), 1.82−1.70 (m, 1H),
1.58−1.46 (m, 1H), 1.40−1.12 (m, 4H), 1.08−0.90 (m, 1H), 0.88 (s,
9H). 13C (75 MHz, CD3OD): 177.42, 166.80, 155.82 (d, JC−F
=
243.61 Hz), 140.67 (d, JC−F = 2.77 Hz), 135.73, 134.18, 131.36,
130.33, 129.44, 128.37, 125.77 (d, JC−F = 7.38 Hz), 123.38 (d, JC−F
=
3. Binding Kinetics Studies of 5a, 5b, 5c and 5d and
Isomerization Studies in Binding Medium. Aliquots of freshly
prepared DMSO stock solutions of compounds 5a-5d were diluted in
FP binding assay medium to prepare the aqueous compound
incubation solutions in which compound isomerization was taking
place. Final compound concentration in the incubation solution was
25 μM, and 5% of DMSO was added to enhance the solubility. These
solutions were stored at room temperature for the whole time range of
the experiment. 80 μL aliquots of compound solutions were mixed
with 20 μL of freshly prepared MDM2/PMDM6-F mixture in the
assay plates at different time points. Final concentrations of the
protein, fluorescent probe, and DMSO are the same as those in the
competitive assays described above. Negative and positive controls
were also included in each assay plate. Experimental time was precisely
controlled so that each plate was read exactly 10 min after compound
solution was pipetted from the incubation solution stock, which
typically includes 5 min for plate preparation and 5 min of incubation
at room temperature with gentle shaking. mP values were measured
and IC50 values were determined as described above. Thus, it should
be noted that all IC50 values presented are values actually obtained 10
min after the incubation times presented.
4. Cell Growth Inhibition Assay. The SJSA-1 osteosarcoma
tumor cell line was purchased from the American Type Culture
Collection. Cells were seeded in 96-well flat bottom cell culture plates
at a density of (3−4) × 103 cells/well and grown overnight, then
incubated with compounds at different concentrations. The rate of cell
growth inhibition after treatment with different concentrations of a
compound was determined by assaying with a lactate dehydrogenase-
based WST-8 assay (WST-8; Dojindo Molecular Technologies Inc.,
Gaithersburg, MD). WST-8 solution was added to each well to a final
concentration of 10%, and 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 concentrations of the compounds that
inhibited cell growth by 50% (IC50) were calculated by comparing
absorbance of the untreated cells with the treated cells.
19.54 Hz), 114.06 (d, JC−F = 25.18 Hz), 113.39, 69.97, 65.12, 64.06,
62.94, 57.41, 49.88, 43.37, 34.38, 34.29, 30.98, 30.82, 29.39. ESI-MS
calculated for C29H3535Cl2FN3O3 [M + H]+ = 562.20, Found: 562.36.
(2′S,3R,4′S,5′R)-6-Chloro-4′-(3-chloro-2-fluorophenyl)-N-(cis-3-
hydroxy-3-ethylcyclobutyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-
pyrrolidine]-5′-carboxamide (15b, MI-888, TFA salt). 1H NMR (300
MHz, MeOD-d4): 7.62−7.53 (m, 2H), 7.45−7.35 (m, 1H), 7.20−7.10
(m, 2H), 6.80−6.85 (m, 1H), 5.11 (d, J = 11.07 Hz, 1H), 4.57 (d, J =
11.07 Hz, 1H), 4.40 (d, J = 7.39 Hz, 1H), 4.00−3.80 (m, 1H), 2.50−
2.35 (m, 1H), 2.35−2.20 (m, 1H), 2.10−1.90 (m, 1H), 1.90−1.60 (m,
2H), 1.30 (s, 3H), 1.20−1.05 (m, 1H), 0.88 (s, 9H). 13C NMR (75
MHz, MeOD-d4): 177.85, 167.96, 157.67 (d, JC−F = 249.51 Hz),
144.96, 136.85, 132.22, 128.53, 126.58, 126.34 (d, JC−F = 4.76),
123.91, 123.69, 122.30 (d, JC−F = 18.97 Hz), 122.02 (d, JC−F = 13.10
Hz), 111.84, 67.14, 64.67, 64.53, 62.91, 45.53, 45.47, 43.38, 38.08,
30.84, 29.50, 27.39. ESI-MS calculated for C28H3335Cl2FN3O3 [M +
25
H]+: 548.19, Found: 548.25. [α]D = −16.3° (MeOH, c = 0.0284 g/
mL).
2. 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),11 termed PMDM6-F. The Kd value of PMDM6-F with 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
polarization values were measured using the Infinite M-1000 plate
reader (Tecan U.S., Research Triangle Park, NC) in Microfluor 2 96-
well, black, round-bottomed plates (Thermo Scientific).
In the saturation experiments, 1 nM of 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 incubated at room
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).
5. Pharmacodynamic (PD) Study. The SJSA-1 xenograft tumor
model was used for PD studies. To develop xenograft tumors, 5 × 106
SJSA-1 cancer cells with 50% Matrigel were injected subcutaneously
on the dorsal side of SCID mice (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), 16 mice were treated with a single oral
dose of compound 5a at 200 mg/kg and another 16 mice were treated
with a single oral dose of compound 5b at 200 mg/kg. Mice treated
with vehicle control were sacrificed at 6 h and the tumor tissue was
harvested for Western blot analysis. Two mice treated with 5b were
sacrificed each time at 1, 3, 6, and 24 h and tumor tissue was harvested.
The same schedule was performed on mice treated with 5a.
Ki values of tested compounds were determined in a dose-
dependent competitive binding experiment. Mixtures of 5 μL of a
solution of the tested compound in different concentrations in DMSO
6. Western Blotting. For Western blot analysis, tumor cells or
tumor tissues were lyzed in ice-cold RIPA buffer: 20 mM Tris-HCl
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Journal of the American Chemical Society
Article
(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% sodium
deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophos-
phate, 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).
7. In vivo Experiment. For in vivo studies, the SJSA-1 xenograft
tumor model was used. To develop xenograft tumors, 5 × 106 SJSA-1
cancer cells with 50% Matrigel were injected subcutaneously on the
dorsal side of SCID mice (Charles River), one tumor per mouse.
When xenograft tumors reached a mean of ∼100 mm3 (70−130 mm3)
the mice were randomized into groups of 8 mice. Vehicle Control
(10% PEG400: 3% Cremophor: 87% PBS) was administered p.o. once
per day for 14 days. Compound 5a was administered orally once per
day for 14 days at 200 mg/kg and compound 5b was administered
orally once per day for 14 days at 100 and 200 mg/kg. Tumor sizes
and animal weights were measured three times per week. Data are
represented as mean tumor volumes. Tumor volume (mm3) = (A ×
B2)/2 where A and B are the tumor length and width (in mm),
respectively. Statistical analyses were done by two-way ANOVA and
unpaired two-tailed t test, using Prism (version 4.0, GraphPad). P <
0.05 was considered statistically significant. The same protocol was
applied to compounds 15a and 15b.
8. Pharmacokinetics of 5a and 5b in Tumor Bearing SCID
Mice. The SJSA-1 xenograft tumor model was used for PK studies. To
develop xenograft tumors, 5 × 106 SJSA-1 cancer cells with 50%
Matrigel were injected subcutaneously on the dorsal side of SCID mice
(Charles River), one tumor per mouse. When xenograft tumors
reached a mean of ∼100 mm3 (70−130 mm3), three mice were treated
with vehicle control (10% PEG400: 3% Cremophor: 87% PBS), nine
mice were treated with a single oral dose of compound 5a at 100 mg/
kg and another nine mice were treated with a single oral dose of
compound 5b at 100 mg/kg. Three mice treated with 5a were
sacrificed each time at 1, 6, and 24 h, and the plasma and the tumor
tissue were harvested and frozen at −78 °C immediately. The same
schedule was performed on mice treated with 5b.
The quantitative LC-MS/MS analysis were conducted using an
Agilent 1200 HPLC system coupled to an API 3200 mass
spectrometer (Applied Biosystems, MDS Sciex Toronto, Canada)
equipped with an API electrospray ionization (ESI) source. Aliquots
(5 μL) were injected into a reversed-phase column (5 cm × 4.6 mm
I.D., packed with 3.5 μm Xbridge C18, Waters). The mobile phase
consisted of 0.1% formic acid in water (A) and 0.1% formic acid in
acetonitrile (B). The gradient of mobile phase was as follows: 5% B for
0.5 min, 5% to 60% B over 7.5 min, 60% B for an additional 7.5 min,
and then immediately set back to 5% B for re-equilibration. The flow
rate of the mobile phase was 1.0 mL/min.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and characterization data for all new
compounds, experimental procedures for isomerization studies,
protein binding test, cytotoxicity studies, tumor size measure-
ment, and statistic data of efficacy studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
shaomeng@umich.edu
■
Notes
The authors declare the following competing financial
interest(s): The compounds described in this paper have
been licensed by Ascenta Therapeutics from the University of
Michigan, which was sub-licensed to Sanofi. Dr. Shaomeng
Wang owns stocks in Ascenta and is 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), and Ascenta
Therapeutics, Inc. and Sanofi S.A. We also thank financial
support for J.R.D. provided by NIDA under interagency
agreement number ADA12003-001.
REFERENCES
■
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