Discovery of AM-7209, a potent and selective 4-amidobenzoic acid inhibitor of the MDM2-p53 interaction

Article abstract of DOI:10.1021/jm501550p

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (K_D from ITC competition was 38 pM, SJSA-1 EdU IC₅₀ = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED₅₀ = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED₅₀ = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Full text of DOI:10.1021/jm501550p

Article
pubs.acs.org/jmc
Discovery of AM-7209, a Potent and Selective 4‑Amidobenzoic Acid
Inhibitor of the MDM2−p53 Interaction
Yosup Rew,*,† Daqing Sun,† Xuelei Yan,† Hilary P. Beck,† Jude Canon,∥ Ada Chen,† Jason Duquette,†
John Eksterowicz,† Brian M. Fox,† Jiasheng Fu,† Ana Z. Gonzalez,† Jonathan Houze,† Xin Huang,⊥
Min Jiang,§ Lixia Jin,§ Yihong Li,† Zhihong Li,† Yun Ling,§ Mei-Chu Lo,† Alexander M. Long,⊥
Lawrence R. McGee,† Joel McIntosh,† Jonathan D. Oliner,∥ Tao Osgood,∥ Anne Y. Saiki,∥ Paul Shaffer,⊥
Yu Chung Wang,∥ Sarah Wortman,‡ Peter Yakowec,⊥ Qiuping Ye,§ Dongyin Yu,∥ Xiaoning Zhao,†
Jing Zhou,† Julio C. Medina,† and Steven H. Olson†
†Department of Therapeutic Discovery, ‡Department of Pharmaceutics, and §Department of Pharmacokinetics and Drug Metabolism,
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
∥Department of Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
⊥Department of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Structure-based rational design and extensive structure−activity relationship studies led to the discovery of AMG
232 (1), a potent piperidinone inhibitor of the MDM2−p53 association, which is currently being evaluated in human clinical
trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid,
afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM),
remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50
=
2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses
distinct mechanisms of elimination compared to 1.
ition of the MDM2−p53 interaction with small-molecule
MDM2 inhibitors has been shown to be a tractable strategy for
reactivation of the p53 pathway in tumors with wild-type
p53,14,15 which is present in approximately 50% of human
cancers.16 The potential benefits of neutralizing the MDM2−
p53 protein−protein interaction have been recognized for
nearly 20 years, and recently, several small molecules
interdicting the target have advanced into clinical trials.17−21
We discovered AMG 232 (1) (Figure 1), a novel
piperidinone inhibitor of the MDM2−p53 interaction via
structure-based rational design using conformational control
and extensive SAR studies.21−23 Herein, we report the
continued investigation of 1, which led to the discovery of
AM-7209 (25), a novel 4-amidobenzoic acid-containing
MDM2 inhibitor that possesses structural differentiation and
distinct in vivo mechanisms of elimination compared to 1.
INTRODUCTION
■
p53 is a short-lived transcription factor which plays a critical
role in preventing tumor development. It is stabilized by
cellular stress and accumulates in the nucleus. Activated p53
binds to DNA and increases transcription of numerous genes
involved in cell cycle arrest, DNA repair, senescence, and
apoptosis.1,2 Accordingly, loss of p53 function in tumor cells
provides a strong selective growth advantage, and it has been
proposed that dysfunction in the p53 pathway may be a
required step in tumor development.3 Multiple independent
studies have demonstrated that loss of p53 function contributes
to tumor progression. Furthermore, restoring endogenous p53
function in established tumors results in tumor regression in
vivo and could be an effective anticancer therapeutic
approach.4−7
The MDM2 (murine double minute 2) oncogene is a key
cellular negative regulator of p53. MDM2 is transcriptionally
activated by p53 and, in turn, inhibits p53 by controlling its
activity,8 subcellular location,9−11 and degradation.12,13 Inhib-
Received: October 7, 2014
© XXXX American Chemical Society
A
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Figure 1. Chemical structures and potencies of 1 and 25. MDM2
binding pockets are labeled (by p53 side chain) in blue. Notes: (a)
IC50 in the HTRF biochemical assay (serum free),24 (b) IC50 in the
EdU cell proliferation assay (SJSA-1, 10% human serum).24
Figure 2. Binding mode of compound 1 based on the cocrystal
structures of its analogues bound to human MDM2 (17−111).21
MDM2 binding pockets are labeled (by p53 side chain) in white.
MDM2 residues Phe55, Gly58, Asn59, Met62, and His96 are labeled
in yellow.
RESULTS AND DISSCUSION
■
As previously reported,21b compound 2 (Table 1), the tert-butyl
sulfone analogue having a cyclopropyl group at the α position
of the N-alkyl group, was extremely potent due to the favorable
interactions of the cyclopropyl group and the tert-butyl group
with MDM2. However, 2 exhibited rapid turnover in human
hepatocytes, resulting in a high projected clearance in humans.
Adding fluorine to the C5 or C6 aryl group furnished even
more potent tert-butyl sulfones 3 and 4, but yielded no
improvement of metabolic stability in human hepatocytes.
The crystal structure of the 109-residue amino-terminal
domain of MDM2 bound to a 15-residue transactivation
domain peptide of p53 disclosed that MDM2 has three
hydrophobic clefts that bind three critical residues of p53
(Phe19, Trp23, and Leu26).25 The binding modes of
compounds 1−4 were predicted from the X-ray cocrystal
structures of related analogues (Figure 2).21,22
substitution. The isopropyl (for 1) or tert-butyl (for 2−4)
moiety attached to the sulfonyl group lies in the “Gly58 shelf”
region (Phe55, Gly58, Asn59, and Met62) and engages in a
hydrophobic interaction with this region, while the sulfone
function is close to the α-carbon of Gly58, inducing a CH···O-
type interaction with this residue. Lastly, the carboxylic acid is
located on top of the His96 side chain of MDM2 and interacts
with the adjacent, and presumably protonated, imidazole of
His96.
Preliminary metabolite profiling studies indicated that
compound 2 was mainly cleared through glucuronidation of
the carboxylic acid in human hepatocytes (data not shown).
Thus, we hypothesized that replacing the carboxylic acid with
other functional groups to reduce glucuronidation but maintain
an interaction with the imidazole of His96 in MDM2 could
improve the metabolic stability of the cyclopropyl analogues
(2−4) without diminishing their superb potency.
The C6 p-Cl-phenyl occupies the deepest Trp23(p53) pocket,
and the C5 m-Cl phenyl reaches into the Leu26(p53) pocket.
The latter interaction is augmented by a face-to-face π-stacking
interaction with His96. The C3 methyl drives the trans C5 and
C6 aryl groups to reside in a gauche-like orientation, the
favored conformation for binding to MDM2. The isopropyl
group (for 1) or the cyclopropyl group (for 2−4) at the α
position of the N-alkyl group is forced into the Phe19(p53)
pocket by the conformational constraint induced by α-
Our investigation started with the conversion of the
carboxylic acid into amides (Table 2). Both primary amide 5
and phenyl amide 6 were well tolerated in the biochemical
assay (5 and 6 vs 2), suggesting that an amide can also have a
hydrogen bond interaction with the side chain of His96. This
result was consistent with the previous findings in the X-ray
Table 1. Potent tert-Butyl Sulfone Derivatives
1
2
3
4
biochemical potency
a
IC50 (HTRF) (nM)
0.63 0.44
0.10 0.01
0.10 0.03
<0.10
cellular potency (SJSA-1)
a
b
p21 IC50 (10% HS ) (nM)
47 15
9.2 3.1
6.3
18.1 4.2
1.6 0.8
16
15.5 9.2
1.2 0.3
26
6.4 3.5
0.80 0.25
27
a
b
EdU IC50 (10% HS ) (nM)
human hepatocyte CLint (μL/min/106 cells)
a
b
Potency data are reported as the mean and standard deviation of at least two determinations. HS = human serum.
B
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was significantly improved (7 vs 6) and compound 7 was
almost equipotent with 2. Furthermore, compound 7 exhibited
significantly enhanced stability in human hepatocytes [human
hepatocyte CLint (μL/min/106 cells) = 3.2 for 7 vs 16 for 2,
Table 4]. Recently, an approach has been reported where a p-
benzoic acid side chain was added to pyrrolidine-derived
MDM2 inhibitors to improve their pharmacokinetic (PK)
parameters.19
Table 2. Amidobenzoic Acid Derivatives
Encouraged by the superior potency and improved metabolic
stability of 7 in human hepatocytes, further investigation of the
benzoic acid moiety was conducted (Table 2). Adding a
methoxy group at the ortho-position was tolerated (8), but
shifting it into the meta-position diminished the potency (9).
Moving the amino group from the para-position to the meta-
and ortho-positions reduced the potency, particularly in the cell
proliferation assay (10 and 11 vs 7). Converting the carboxylic
acid into a primary amide or a nitrile resulted in a significant
reduction in potency (12 and 13). Changing the benzoic acid
to a phenylacetic acid or replacing the phenyl ring with a
pyridine ring was well tolerated (14 and 15), while N-
methylation resulted in loss of potency (16).
Replacement of the phenyl ring between the acid and the
amide nitrogen with a nonaromatic ring was also explored
(Table 3). The trans-cyclohexanecarboxylic acid analogue 17
exhibited a slightly decreased activity in both the biochemical
and cell proliferation assays compared to 7. The cis analogue 18
was even less potent in the cell proliferation assay. The potency
of the bicyclo[2.2.2]octane analogue 19, which has a linear
topology, was close to the potency of 7. The piperidinecarbox-
ylic acid derivatives 20 and 21 exhibited potency comparable to
that of the trans-cyclohexanecarboxylic acid analogue 17.
Decreasing the ring size from a six-membered ring to a four-
membered ring resulted in no potency improvement in
biochemical or cell proliferation assays (17, 18, and 20 vs
22−24).
Adding a fluorine to the C5 or C6 aryl group of the
piperidinone core in 8 provided two extremely potent
compounds, 25 and 26, which were derived from 3 and 4,
respectively (Table 4). The amide-containing compounds 7, 8,
19, 25, and 26 retained their potency in both biochemical and
cell-based assays compared to the simple carboxylic acids 2−4
in Table 1. These compounds were further profiled for CYP
inhibition, PXR activation, and CYP time-dependent inhibition
(TDI). All were found to have minimal liabilities in these
assays, and furthermore, the metabolic stability of these
compounds in human hepatocytes was greatly improved
[human hepatocyte CLint (μL/min/106 cells) = 1.5−4.6 for 7,
8, 19, 25, and 26 vs 16−27 for 2−4]. On the basis of PK
profiles in dog, predicted human PK, and rodent toxicology
studies (data not shown), 25 was selected for further
evaluation.
The reported dissociation constant (KD) of 1 was 45 pM in a
surface plasmon resonance (SPR) spectroscopy binding assay,21
while 25 had a KD that was too low to be determined under
identical assay conditions. In an isothermal titration calorimetry
(ITC) competition assay, the KD for 25 and 1 was measured as
38 and 257 pM, respectively.24 Compound 25 was consistently
more potent than 1 in both the HTRF biochemical assay (IC50
= <0.10 nM vs 0.60 nM) and the cell proliferation assay in the
MDM2 amplified SJSA-1 osteosarcoma cell line (IC50 = 1.6 nM
vs 9.2 nM). In addition to its significant in vitro potency,
compound 25 showed pharmacokinetic properties both in
mouse (iv CL = 0.026 L/h/kg, po F = 30%) and in dog (iv CL
a
Potency data are reported as the mean and standard deviation of at
b
least two determinations. HS = human serum.
cocrystal structures of the oxindole inhibitors with MDM2 by
us23 and others.19 However, amides 5 and 6 were significantly
less potent than carboxylic acid 2 in the cell proliferation assay
(20−50-fold), presumably due to the poor permeability.
Extensive investigation on carboxylic acid replacements
including various amide modifications was carried out to
modulate the properties of the amide derivatives.26 Interest-
ingly, when an additional carboxylic acid moiety was introduced
at the para-position of the phenyl group in 6, cellular potency
C
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An in vivo pharmacodynamic (PD) study28 with 25 in an
SJSA-1 tumor xenograft mouse model showed a dose-
dependent induction of p21 mRNA, a direct transcriptional
marker of p53 activity.29 This outcome clearly indicated an on-
mechanism activation of the p53 pathway by 25 (Figure 5). A
concentration-dependent peak induction of p21 mRNA was
observed approximately 4 h after dosing (30-fold induction at
10 mg/kg and 10-fold induction at 3 mg/kg).
Table 3. Replacement of the Phenyl Ring with a
Nonaromatic Ring
Next, compound 25 was evaluated for its ability to inhibit
tumor growth in an SJSA-1 mouse xenograft model.28
Consistent with the findings of the PD study, robust dose-
dependent tumor growth inhibition was observed. Compound
25 completely inhibited tumor growth at 10 mg/kg QD
compared to vehicle with an ED50 of 2.6 mg/kg (Figure 6). The
calculated unbound EC50 associated with the ED50 was 2.0
nM.30 Notably, both the 25 mg/kg QD dose and the 50 mg/kg
QD dose of 25 caused complete tumor regression in 9 of 10
mice and 10 of 10 mice, respectively. Tumors in the 50 mg/kg
group continued to show complete regression for 30 days after
treatment (data not shown). There was no significant body
weight loss or other signs of toxicity observed in any of the
treatment groups (≤ 5%).
Moreover, in the HCT-116 colorectal carcinoma mouse
xenograft model, treatment with 25 caused a dose-dependent
reduction in tumor growth with an ED50 of 10 mg/kg. At a
dose of 100 mg/kg QD, 100% tumor growth inhibition was
observed (Figure 7).28 There was also no body weight loss in
any of the treatment groups. The effective inhibition of tumor
growth in the p53wt HCT-116 tumors with normal MDM2
expression confirmed that the neutralization of the MDM2−
p53 interaction with a small-molecule MDM2 inhibitor could
be an efficacious therapeutic strategy against tumors that are
p53wt.
What clearly differentiated 25 from 1 was its different
metabolic profile. When incubated in rat, dog, cyno, and human
hepatocytes,31 compound 1 is converted exclusively to the acyl
glucuronide (M1) (Figure 8a). In contrast, hepatocyte
incubation of compound 25 provided oxidative metabolite
M2 as the major product, although phenol M3, produced via
oxidative demethylation, and acylglucuronide M1 were also
detected (Figure 8b). The reported structural modifications
alter the metabolic fate of the molecules, which was an
important consideration for this backup program.
a
Potency data are reported as the mean and standard deviation of at
b
least two determinations. HS = human serum.
CHEMISTRY
= 0.69 L/h/kg, po F = 45%) (iv = intravenous, po = per os)
sufficient to support further development.
■
The synthesis of target compounds started with the preparation
of lactone intermediates 30a−30c. The synthesis of 30a was
previously reported.32 Lactones 30b and 30c were prepared
according to the procedures described for the synthesis of 30a,
replacing ketone 27a21,32 with the appropriate ketones 27b and
27c, respectively (Scheme 1). Michael addition of 27b and 27c
to methyl methacrylate provided δ-keto esters 28b and 28c,
which were subjected to a dynamic kinetic resolution (DKR)
using a Noyori catalyst. Following a two-step hydrolysis/
lactonization sequence, lactones 29b and 29c were obtained as
a mixture of epimers at C3 [er > 83:17 in favor of the (5R,6R)-
isomer]. Diasteroselective allylation at C3 of cis-5,6-disubsti-
tuted δ-lactones 29b and 29c gave C3 quaternized lactones 30b
and 30c with excellent selectivity (dr > 95:5). Recrystallization
of the lactones 30b and 30c from DCM in hexanes easily
improved the er to >99:1.
The cocrystal structure of 25 with MDM2 (PDB code
4WT2) revealed that it binds to the protein in a manner
consistent with the predicted binding mode of compound 1
(Figure 3). Notably, the amide oxygen of 25 interacts with the
His96 imidazole in a manner analogous to that of the
carboxylate of compound 1. In addition, the cocrystal structure
shows that the carboxylic acid in 25 and the amine of Lys94 are
in close proximity, but solution NMR studies failed to provide
evidence of an interaction.27
To evaluate its selectivity, the effect of 25 on inhibiting the
proliferation of HCT-116 p53wt and p53−/− tumor cells in vitro
was examined (Figure 4).24 In accordance with the data
observed for 1 and other piperidinone analogues,21,22 25
potently inhibited cell growth on-target in the tumor cell line
with wild-type p53 cells (IC50 = 2.0 nM) and exhibited more
than 12500-fold selectivity over those with p53-deficient cells
(off-target IC50 > 25 μM).
Compounds 2−4 were synthesized from lactones 30a−30c
utilizing the bicyclic iminium ether chemistry shown in Scheme
D
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Table 4. Potent Amide-Containing Derivatives
2
7
8
19
<0.10
25
<0.10
26
biochemical potency
a
IC50 (HTRF) (nM)
0.10 0.01
1.1 0.6
0.18 0.06
0.60 0.03
0.13 0.07
0.50 0.19
<0.10
a
b
IC50 (HTRF, 15% HS ) (nM)
cellular potency (SJSA-1)
0.70 0.14
0.60 0.13
0.50 0.22
a
b
p21 IC50 (10% HS ) (nM)
18.1 4.2
1.6 0.8
<10
10.4 2.8
13.9 7.0
26.5 4.7
13.7 5.2
6.1 2.0
a
b
EdU IC50 (10% HS ) (nM)
2.9 0.6
1.9 0.5
5.9 0.8
1.6 0.5
1.0 0.2
CYP3A4 inhibition (%) at 3 μM
<10
0
14
14
85
<10
0
<10
0
14
0
c
hPXR activation (%) compared to the control at 2 μM
28
remaining activity (TDI) (%) of CYP3A4 compared to the
75
78
75
63
72
d
control after 30 min at 10 μM
human hepatocyte CLint (μL/min/106 cells)
16
3.2
4.6
3.7
1.5
1.7
a
b
c
Potency data are reported as the mean and standard deviation of at least two determinations. HS = human serum. Rifampin used as a positive
d
control. DMSO used as the control.
Figure 4. Cell activity of 25 is p53 dependent. In HCT-116 p53wt and
p53−/− cells, the percentage of BrdU positive cells was measured 16 h
post compound treatment by flow cytometry. The DMSO control was
designated as 0% inhibition.
Figure 3. Cocrystal structure of 25 bound to human MDM2 (6−110)
at 1.4 Å resolution (PDB code 4WT2). MDM2 binding pockets are
labeled (by p53 side chain) in white. MDM2 residues Lys94 and His96
are labeled in yellow.
the tert-butyl sulfide group, 32a−32c were hydrolyzed with
aqueous NaHCO3 to afford primary alcohols 33a−33c, which
were purified using silica gel column chromatography. The
resulting alcohols 33a−33c were converted again into iminium
ether tosylate salts 32a−32c, which were subjected to the
nucleophilic ring opening reaction with 2-methyl-2-propane-
thiol in the presence of LiHMDS. Subsequent oxidative
cleavage of the allyl moiety using catalytic ruthenium tetroxide
afforded carboxylic acids 2−4.
2.21,32 The amide intermediates 31a−31c were obtained
quantitatively by the ring opening of lactones 30a−30c with
(S)-2-amino-2-cyclopropylethanol. The reaction of 31a−31c
with Ts2O and 2,6-lutidine at reflux resulted in the formation of
iminium ether tosylate salts 32a−32c.33 Prior to installation of
E
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Figure 5. PD study results of compound 25 in an SJSA-1 tumor
xenograft: *p < 0.0001; **p = 0.01. Female athymic nude mice were
implanted subcutaneously with 5 × 106 SJSA-1 cells. When the tumors
reached ∼250 mm3, vehicle or 10 or 3 mg/kg 25 was administered
orally once daily (QD) for 4 days. The mice were sacrificed at 1, 2, 4,
8, and 24 h postdose (n = 4 per group). The tumors were immediately
removed and snap-frozen. p21 mRNA levels were measured by
quantitative RT-PCR. Tumors treated with vehicle served as a negative
control and indicated the baseline p21 mRNA level. Data are
represented as mean p21 fold induction over vehicle, and error bars
represent the SEM of data from four mice. Concentrations in blood
plasma were analyzed by LC/MS/MS. The red dots denote the total
plasma concentration of 25.
Figure 7. Inhibition of HCT-116 tumor xenograft growth by 25: *p <
0.005. HCT-116 cells (2 × 106) were implanted subcutaneously into
female athymic nude mice. Treatment with vehicle or 25 at 5, 15, 50,
or 100 mg/kg QD by oral gavage began on day 10 when the tumors
had reached ∼200 mm3 (n = 10 per group). The tumor sizes and body
weights were measured twice per week. Data are represented as mean
tumor volumes, and error bars represent the SEM of data from 10
mice.
vivo antitumor activity and PK profile, 25 is a very promising
candidate for the treatment of human cancer.
EXPERIMENTAL SECTION
■
General Chemistry. All reactions were conducted under an inert
gas atmosphere (nitrogen or argon) using a Teflon-coated magnetic
stir bar at the temperature indicated. Commercial reagents and
anhydrous solvents were used without further purification. Analytical
thin layer chromatography (TLC) and flash chromatography were
performed on Merck silica gel 60 (230−400 mesh). Removal of
solvents was conducted by using a rotary evaporator, and residual
solvent was removed from nonvolatile compounds using a vacuum
manifold maintained at approximately 1 Torr. All yields reported are
isolated yields. Preparative reversed-phase high-pressure liquid
chromatography (RP-HPLC) was performed using an Agilent 1100
series HPLC instrument and Phenomenex Gemini C18 column (5
μm, 100 mm × 30 mm i.d.), eluting with a binary solvent system (A
and B) using a gradient elusion [solvent A, H2O with 0.1%
trifluoroacetic acid (TFA); solvent B, CH3CN with 0.1% TFA] with
UV detection at 220 nm. All final compounds were purified to ≥95%
purity as determined by the Agilent 1100 series HPLC instrument with
UV detection at 220 nm using the following method: Zorbax SB-C8
column (3.5 μm, 150 mm × 4.6 mm i.d.); mobile phase, solvent A =
H2O with 0.1% TFA, solvent B = CH3CN with 0.1% TFA; gradient,
5−95% B (0.0−15.0 min); flow rate, 1.5 mL/min. Low-resolution
mass spectrometry (MS) data were determined on an Agilent 1100
series LC/MS system with UV detection at 254 nm in low-resolution
electrospray ionization (ESI) mode. High-resolution (HR) mass
spectra were obtained on an Agilent 6510 Q-TOF mass spectrometer
with an Agilent 1200 liquid chromatograph on the front end. 1H NMR
spectra were obtained on a Bruker Avance III 500 (500 MHz) or
Bruker Avance II 400 (400 MHz) spectrometer. Chemical shifts (δ)
are reported in parts per million (ppm) relative to residual
undeuterated solvent as an internal reference. The following
abbreviations were used to explain the multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet
of triplets, m = multiplet, br = broad. Optical rotations ([α]D) were
measured on a JASCO P-1020 polarimeter. Specific rotations are given
as degrees per decimeter, and the concentrations are reported as grams
per 100 mL of the specific solvent and were recorded at the
temperature indicated.
Figure 6. Treatment with 25 inhibited the growth of SJSA-1 tumors in
vivo: *p < 0.05. SJSA-1 cells (5 × 106) were implanted subcutaneously
into female athymic nude mice. Treatment with vehicle or 25 at 1, 2.5,
5, 10, 25, or 50 mg/kg QD by oral gavage began on day 11 when the
tumors had reached ∼200 mm3 (n = 10 per group). The tumor sizes
were measured twice per week. Data represent mean tumor volumes,
and error bars represent the SEM of data from 10 mice.
Finally, acids 2−4 were converted to amides 5−26 via an
EDC-mediated coupling reaction, followed by the hydrolysis of
ester to acid if necessary (Scheme 3).
CONCLUSION
■
In summary, structural modifications of 1 by (a) replacing the
isopropylsulfonyl group with a tert-butylsulfonyl group, (b)
switching the isopropyl group for a cyclopropyl group for the
interaction with the Phe19(p53) pocket in the MDM2 protein,
(c) introducing an m-fluoro group at the C6 phenyl group, and
(d) changing the carboxylic acid to a secondary amide with 4-
amido-2-methoxybenzoic acid led to the discovery of 25, the
most potent and selective MDM2 inhibitor reported to date,
with structural differentiation and distinct mechanisms of
elimination compared to 1. Consistent with its remarkable in
1-(4-Chloro-3-fluorophenyl)-2-(3-chlorophenyl)ethanone
(27b). To a solution of 4-chloro-3-fluorobenzoic acid (450 g, 2.59
mol) in MeOH (4.5 L) was added thionyl chloride (450 mL, 6.19
F
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a
Scheme 1
a
Reagents and conditions: (a) methyl methacrylate, t-BuOK, THF,
74−78%; (b) RuCl2[((S)-xylBINAP)((S)-DAIPEN)], t-BuOK, i-
PrOH, H2, 50 psi, 65−75%; (c) LiOH, THF/MeOH/H2O, 100%;
(d) PPTS, toluene, reflux, 72−75%; (e) LiHMDS, allyl bromide, 69−
73%; (f) recrystallization in hexanes/DCM, 88−92%.
a
Scheme 2
a
Reagents and conditions: (a) (S)-2-amino-2-cyclopropylethanol,
Figure 8. Representative HPLC mass chromatogram of 1 (a) and 25
(b) obtained following incubation in rat, dog, cyno, and human
hepatocytes.
100%; (b) Ts2O, 2,6-lutidine; NaHCO3, H2O/DCE, 75−77%; (c)
TsOH, toluene, 100%; (d) 2-methyl-2-propanethiol, LiHMDS, THF,
79−92%; (e) NaIO4, RuCl3, CH3CN/EtOAc/H2O, 80−92%.
mol) dropwise over 30 min at 0 °C. After the mixture was stirred for
12 h at ambient temperature, the reaction was concentrated under
reduced pressure, quenched (1.0 M aqueous NaHCO3, 0.50 L), and
extracted (DCM, 2 × 5.0 L). The combined organic layers were
washed (brine, 2.5 L), dried (Na2SO4), and concentrated under
reduced pressure to afford a crude methyl 4-chloro-3-fluorobenzoate
(450 g, 93%) as a light brown solid. The crude compound was used in
the next step without further purification.
To a solution of 2-(3-chlorophenyl)acetic acid (250 g, 1.47 mol) in
anhydrous THF (1.8 L) was added NaHMDS (1 M in THF, 4.00 L,
4.00 mol) over 1 h at −78 °C, and the resulting mixture was stirred for
another 1 h at −78 °C. Then a solution of the crude ester above (221
G
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a
To a solution of the hydroxy ester above (240 g, 581 mmol) in
THF (1.9 L) and methanol (0.48 L) was added LiOH (2.5 M aqueous
solution, 0.48 L) at 25 °C. After the mixture was stirred at 25 °C for 12
h, most organic solvents were removed under reduced pressure. The
residue was acidified (2 N aqueous HCl, pH ≈ 5−6) and extracted
(EtOAc, 2 × 1.0 L). The combined organic layers were washed (brine,
0.75 L), dried (Na2SO4), and concentrated under reduced pressure to
give crude (4R,5R)-5-(4-chloro-3-fluorophenyl)-4-(3-chlorophenyl)-5-
hydroxy-2-methylpentanoic acid (216 g, quantitatively) as a dark
colored liquid, which was used in the next step without further
purification.
Scheme 3
To a solution of the hydroxy acid above (25.4 g, 68.4 mmol) in
toluene (274 mL) was added pyridinium p-toluenesulfonate (0.516 g,
2.05 mmol). After the mixture was heated to reflux under Dean−Stark
conditions for 1 h, the reaction was cooled to 25 °C and concentrated
under reduced pressure. The reaction was basified (saturated aqueous
NaHCO3, 150 mL) and extracted (EtOAc, 2 × 150 mL). The
combined organic layers were washed (brine, 105 mL), dried
(Na2SO4), and concentrated under reduced pressure. Flash column
chromatography (SiO2, 0−30% acetone/hexanes) provided 29b (a
mixture of two diastereomers at C3, 18.1 g, 75%) as a pale yellow
solid: MS (ESI) m/z 435.0 [M + Na]+.
(3S,5R,6R)-3-Allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chloro-
phenyl)-3-methyltetrahydro-2H-pyran-2-one (30b). To a sol-
ution of 29b (18.0 g, 51.0 mmol) and 3-bromoprop-1-ene (11.0 mL,
127 mmol) in THF (100 mL) was added LiHMDS (1 M in THF, 56.1
mL, 56.1 mmol) dropwise at −40 °C. The reaction was allowed to
warm to −10 °C and stirred at the same temperature for 3 h. Then the
reaction was quenched (saturated aqueous NH4Cl, 10 mL), diluted
(water, 150 mL), and extracted (Et2O, 2 × 150 mL). The combined
organic layers were washed (brine, 100 mL), dried (MgSO4), and
concentrated under reduced pressure. Flash column chromatography
(SiO2, 0% to 30% acetone/hexanes) provided 30b (84% ee, 13.8 g,
69%) as a white solid.
a
Reagents and conditions: (a) amide coupling reactions with amines
for 5−26; (b) MeI, NaH, THF only for 16; (c) LiOH, MeOH/THF/
H2O for 7−12 and 14−26; (d) NH4Cl, EDC, NaHCO3, DMF only
for 12.
g, 1.18 mol) in THF (0.50 L) was added over 1 h at −78 °C. After the
mixture was stirred at the same temperature for 2 h, the reaction was
quenched (2 N aqueous HCl, 2.5 L) and extracted (EtOAc, 2 × 2.5 L).
The combined organic layers were washed (brine, 2.5 L), dried
(Na2SO4), and concentrated under reduced pressure. Flash column
chromatography (SiO2, 2% EtOAc/hexanes) provided 27b (180 g,
54%) as a white solid: 1H NMR (400 MHz, CDCl3) δ (ppm) 7.74 (2
H, ddd, J = 10.1, 8.9, 1.8 Hz), 7.56−7.48 (1 H, m), 7.26 (3 H, t, J = 6.4
Hz), 7.12 (1 H, d, J = 5.7 Hz), 4.22 (2 H, s); MS (ESI) m/z 282.9 [M
+ 1]+.
2-(3-Chloro-2-fluorophenyl)-1-(4-chlorophenyl)ethanone
(27c). Compound 27c was prepared from 1-bromo-3-chloro-2-
fluorobenzene and 4′-chloroacetophenone according to a procedure
similar to that described for the synthesis of 27a utilizing Buchwald’s
ketone α-arylation conditions:32 1H NMR (400 MHz, CDCl3) δ
(ppm) 7.96 (2 H, d, J = 8.5 Hz), 7.47 (2 H, d, J = 8.5 Hz), 7.33 (1 H,
dd, J = 10.6, 4.3 Hz), 7.13 (1 H, t, J = 6.2 Hz), 7.06 (1 H, t, J = 7.8
Hz), 4.31 (2H, s); MS (ESI) m/z 282.9 [M + 1]+.
Methyl 4-(3-Chloro-2-fluorophenyl)-5-(4-chlorophenyl)-2-
methyl-5-oxopentanoate (28b). To a solution of 27b (327 g,
1.16 mol) in THF (2.6 L) was added methyl methacrylate (136 g, 1.16
mol), followed by t-BuOK (1 M in THF, 115 mL, 115 mmol), at 0 °C.
After being stirred at 0 °C for 1 h, the mixture was allowed to warm to
ambient temperature and stirred for 12 h. Then the reaction was
quenched (water, 1.0 L) and extracted (EtOAc, 2 × 2.5 L). The
combined organic layers were washed (brine), dried (Na2SO4), and
concentrated under reduced pressure. Flash column chromatography
(SiO2, 4% EtOAc/hexanes) provided 28b (1:1 mixture of two
Individual enantiomers of 30b were analyzed by analytical chiral
HPLC [flow rate 1 mL/min on a Chiralcel OD-H 4.5 mm i.d. × 250
mm, 5 μm column (Daicel Inc., Fort Lee, NJ) using 20% i-PrOH/
hexanes as the eluent] to separate the minor enantiomer (tR = 5.74
min) and the major enantiomer (tR = 6.73 min). Enantiomeric excess
was upgraded to >99% through recrystallization of 30b from hexanes
in DCM: 1H NMR (400 MHz, CDCl3) δ (ppm) 7.17−7.24 (3 H, m),
6.94 (1 H, s), 6.80 (1 H, d, J = 7.5 Hz), 6.48 (1 H, dd, J = 10.0, 1.9
Hz), 6.40 (1 H, d, J = 8.3 Hz), 5.76−5.90 (1 H, m), 5.69 (1 H, d, J =
5.2 Hz), 5.13−5.20 (2 H, m), 3.81 (1 H, dd, J = 13.9, 6.9 Hz), 2.62 (1
H, dd, J = 13.8, 7.6 Hz), 2.50 (1 H, dd, J = 13.8, 7.3 Hz), 1.96 (2 H, d,
J = 8.4 Hz), 1.40 (3 H, s); MS (ESI) m/z 393.1 [M + H]+; [α]D
−209 (T = 24 °C, c = 1.10, DCM); mp 96−98 °C.
=
1
(3S,5R,6R)-3-Allyl-5-(3-chloro-2-fluorophenyl)-6-(4-chloro-
phenyl)-3-methyltetrahydro-2H-pyran-2-one (30c). Compound
30c with 80% ee was prepared as a white solid from 27c according to a
procedure similar to that described for the synthesis of 30b. Individual
enantiomers of 30c were analyzed by analytical chiral HPLC [flow rate
1 mL/min on a Chiralcel OD-H 4.5 mm i.d. × 250 mm, 5 μm column
(Daicel Inc., Fort Lee, NJ) using 20% i-PrOH/hexanes as the eluent]
to separate the minor enantiomer (tR = 4.63 min) and the major
enantiomer (tR = 5.18 min). Enantiomeric excess was upgraded to
>99% through recrystallization of 30c from hexanes in DCM: 1H
NMR (400 MHz, CDCl3) δ (ppm) 7.30 (1 H, t, J = 7.5 Hz), 7.16 (2
H, d, J = 8.4 Hz), 6.86 (1 H, t, J = 7.8 Hz), 6.66 (2 H, d, J = 8.4 Hz),
6.44 (1 H, t, J = 6.8 Hz), 5.78−5.89 (2 H, m), 5.13−5.22 (2 H, m),
4.16 (1 H, dd, J = 7.3, 4.4 Hz), 2.64 (1 H, dd, J = 13.8, 7.5 Hz), 2.52 (1
H, dd, J = 13.8, 7.2 Hz), 1.97−2.05 (1 H, m), 1.86−1.92 (1 H, m),
1.40 (3 H, s); MS (ESI) m/z 393.1 [M + H]+; [α]D = −194 (T = 24
°C, c = 1.09, DCM); mp 99−102 °C.
diastereomers, 330 g, 74%) as a light yellow liquid: H NMR (400
MHz, CDCl3) δ (ppm) 7.61−7.74 (4 H, m), 7.40−7.47 (2 H, m),
7.18−7.28 (6 H, m), 7.10−7.16 (2 H, m), 4.56 (2 H, m), 3.68 (3 H, s),
3.60 (3 H, s), 2.39−2.50 (2 H, m), 2.25−2.37 (2 H, m), 2.02−2.10 (1
H, m), 1.94 (1 H, ddd, J = 13.6, 9.1, 4.2 Hz), 1.21 (3 H, d, J = 7.0 Hz),
1.15 (3 H, d, J = 7.0 Hz); MS (ESI) m/z 383.0 [M + 1]+.
(5R,6R)-6-(4-Chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
methyltetrahydro-2H-pyran-2-one (29b). In a 2 L hydrogenation
vessel, 28b (138 g, 360 mmol) was dissolved in 2-propanol (500 mL)
and treated with t-BuOK (16.2 g, 144 mmol). After the mixture was
stirred at 25 °C for 30 min, a solution of RuCl2[((S)-xylBINAP)((S)-
DAIPEN)] (1.76 g, 1.44 mmol) in toluene (30 mL) was added, and
the resulting mixture was vigorously stirred at 25 °C for 2 h. Then the
vessel was purged with hydrogen five times, pressurized to 50 psi, and
allowed to stir at 25 °C. The reaction was recharged with additional
hydrogen as needed. After 3 d, the reaction was quenched (water, 1.5
L) and extracted (EtOAc, 2 × 2.5 L). The combined organic layers
were washed (brine, 1.5 L), dried (Na2SO4), and concentrated under
reduced pressure. Flash column chromatography (SiO2, 12% EtOAc/
hexanes) provided (4R,5R)-isopropyl 5-(4-chloro-3-fluorophenyl)-4-
(3-chlorophenyl)-5-hydroxy-2-methylpentanoate (a mixture of two
diastereomers, 102 g, 69%) as a dark colored liquid: MS (ESI) m/z
435.0 [M + Na]+.
(2S)-2-((2R)-3-(4-Chloro-3-fluorophenyl)-2-(3-chlorophenyl)-
3-hydroxypropyl)-N-((S)-1-cyclopropyl-2-hydroxyethyl)-2-
methylpent-4-enamide (31b). A solution of 25% sodium
methoxide in methanol (60.7 mL, 265 mmol) was added to a solution
of (S)-2-amino-2-cyclopropylethanol hydrochloride (36.5 g, 265
mmol; NetChem Inc., Ontario, Canada) in MeOH (177 mL) at 0
H
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°C. A precipitate formed during the addition. After the addition was
complete, the reaction mixture was allowed to warm to 25 °C. The
reaction mixture was filtered in vacuo, and the solid phase was washed
with DCM. The filtrate was concentrated under reduced pressure to
provide a cloudy brown oil. The oil was taken up in DCM (150 mL)
and filtered in vacuo and the solid phase washed with DCM to provide
a clear orange solution. The solution was concentrated in vacuo to
provide (S)-2-amino-2-cyclopropylethanol (26.7 g, 100%) as a light
brown liquid.
(S)-2-Amino-2-cyclopropylethanol (26.7 g, 265 mmol) was
combined with 30b (32.0 g, 81.4 mmol), and the suspension thus
obtained was heated at 100 °C overnight. The reaction mixture was
cooled to 25 °C, diluted (EtOAc and 1 N aqueous HCl), and extracted
(2 × EtOAc). The combined organic layers were washed (2 × 1 N
aqueous HCl, 1 × water, 1 × brine), dried (MgSO4), and concentrated
under reduced pressure to provide 31b (40.2 g, 100%) as a white solid:
1H NMR (500 MHz, CDCl3) δ (ppm) 7.23 (1 H, dd, J = 10.0, 5.0
Hz), 7.17 (1 H, m), 7.10−7.14 (2 H, m), 6.88−6.90 (2 H, m), 6.77 (1
H, dd, J = 8.3, 2.2 Hz), 6.01 (1 H, d, J = 7.1 Hz), 5.83 (1 H, m), 5.00−
5.07 (2 H, m), 4.74 (1 H, d, J = 5.1 Hz), 3.66 (1 H, dd, J = 11.1, 3.1
Hz), 3.51 (1 H, dd, J = 11.0, 5.9 Hz), 3.08 (1 H, m), 2.97 (1 H, m),
2.39 (1 H, dd, J = 13.7, 7.1 Hz), 2.12 (1 H, dd, J = 14.4, 7.8 Hz), 2.05
(1 H, m), 1.96 (1 H, dd, J = 14.4, 7.8 Hz), 1.13 (3 H, s), 0.82 (1 H,
dtd, J = 13.0, 4.9, 4.9, 3.2), 0.54 (1 H, m), 0.47 (1 H, m), 0.21−0.30 (2
H, m); MS (ESI) m/z 494.2 [M + H]+.
0.18 (1 H, m), −0.25 to −0.10 (2 H, m); MS (ESI) m/z 458.2 [M +
H]+.
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)acetic Acid (3). 2-Methyl-2-propanethiol (15.3
mL, 135 mmol) (dried over activated 4 Å molecular sieves) was added
to LiHMDS (1 M in THF, 135 mL, 135 mmol) at 25 °C under Ar,
and the reaction mixture was heated to 60 °C. After 30 min, a solution
of 32b (77.6 g, 123 mmol) in THF (100 mL) was added dropwise.
The reaction mixture was heated at 60 °C for 3 h and then cooled to
25 °C. The reaction mixture was quenched (water) and extracted (3 ×
EtOAc). The combined organic layers were washed (brine), dried
(MgSO4), and concentrated under reduced pressure to provide a
yellow foam. Purification of the residue by flash column chromatog-
raphy (SiO2, 5−30% EtOAc/hexanes, a gradient elution) provided
(3S,5R,6S)-3-allyl-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methylpiperidin-2-one
1
(62.2 g, 92%) as an off-white foam: H NMR (400 MHz, CDCl3) δ
(ppm) 7.19−7.26 (1 H, m), 7.12−7.18 (2H, m), 7.05−7.07 (1 H, m),
6.84−6.98 (1 H, m), 6.80−6.83 (1 H, m), 6.65−6.80 (1 H, m), 5.82−
5.93 (1 H, m), 5.19−5.21 (1 H, m), 5.16 (1 H, s), 4.70 (1 H, d, J =
10.1 Hz), 3.62 (1 H, t, J = 11.1 Hz), 3.09 (1 H, dt, J = 10.4, 3.1 Hz),
2.60−2.63 (3 H, m), 2.17−2.23 (1 H, m), 2.16 (1 H, t, J = 13.7 Hz),
1.86 (1 H, dd, J = 13.5, 3.1 Hz), 1.70−1.77 (1 H, m), 1.35 (9 H, s),
1.28 (3 H, s), 0.41−0.48 (1 H, m), 0.27−0.34 (1 H, m), −0.15 to
−0.09 (1 H, m), −0.89 to −0.80 (1 H, m); MS (ESI) m/z 548.2 [M +
H]+.
(3S,5R,6S)-3-Allyl-6-(4-chloro-3-fluorophenyl)-5-(3-chloro-
phenyl)-1-((S)-1-cyclopropyl-2-hydroxyethyl)-3-methylpiperi-
din-2-one (33b). To a solution of Ts2O (66.3 g, 203 mmol) in DCM
(220 mL) was added a solution of 31b (40.2 g, 81.3 mmol) in DCM
(80 mL) at 0 °C, and the reaction mixture was stirred at the same
temperature for 10 min. To the reaction mixture was added 2,6-
lutidine (43.6 mL, 374 mmol) dropwise at 0 °C for 10 min. The
resulting solution was allowed to warm to 25 °C and then heated at
reflux for 24 h. After the formation of (3S,5S,6R,8S)-8-allyl-6-(3-
chloro-2-fluorophenyl)-5-(4-chlorophenyl)-3-cyclopropyl-8-methyl-
2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium 4-methylbenzene-
sulfonate was confirmed by LC/MS, a solution of NaHCO3 (68.3 g,
814 mmol) in water (600 mL) and 1,2-dichloroethane (300 mL) was
added to the reaction mixture successively. The reaction mixture was
then heated at reflux for 1 h and cooled to 25 °C. The layers were
separated, and the aqueous layer was extracted (DCM). The combined
organic layers were washed (1 × 1 N aqueous HCl, 1 × water, 1 ×
brine) and concentrated under reduced pressure. Purification of the
residue by flash column chromatography (SiO2, 10−50% EtOAc/
hexanes, a gradient elution) provided 33b (29.8 g, 77%) as a white
To a rapidly stirring solution of the product above (62.2 g, 113
mmol) and NaIO4 (24.7 g, 115 mmol) in water/EtOAc/CH3CN
(1.5:1:1, 760 mL) was added RuCl3·H2O (0.562 mg, 2.49 mmol) at 20
°C. The temperature quickly rose to 29 °C. The reaction mixture was
cooled to 20 °C, and NaIO4 (24.7 g, 115 mmol) was added in portions
over 2 h while the internal reaction temperature was maintained below
25 °C. LC/MS 30 min after the final NaIO4 addition indicated that the
reaction was incomplete. Additional NaIO4 (13.0 g, 60.8 mmol) was
added. The temperature increased from 20 to 25 °C. LC/MS after 1.5
h indicated that the reaction was complete. The reaction mixture was
filtered in vacuo, and the filter cake was washed (EtOAc). The
combined filtrates were separated, and the aqueous layer was extracted
(2 × EtOAc). The combined organic layers were washed (brine), dried
(MgSO4), and concentrated under reduced pressure to provide a dark
green foam. Purification of the residue by flash column chromatog-
raphy (SiO2, 0−20% IPA/hexanes, a gradient elution) followed by
recrystallization in 15% EtOAc/heptanes provided 3 (62.2 g, 92%) as a
1
white solid: H NMR (500 MHz, CDCl3) δ (ppm) 7.16−7.25 (1 H,
1
solid: H NMR (500 MHz, CDCl3) δ (ppm) 7.29 (1 H, t, J = 10.0
m), 7.11−7.16 (2 H, m), 7.02−7.09 (1 H, m), 6.98 (1 H, s, br), 6.88−
6.90 (1 H, m), 6.75−6.87 (1 H, m), 4.98 (1 H, d, J = 10.8 Hz), 4.30 (1
H, t, J = 13.5 Hz), 3.11 (1 H, dt, J = 11.0, 2.7 Hz), 3.07 (1 H, d, J =
14.9 Hz), 2.93 (1 H, dd, J = 13.7, 2.0 Hz), 2.78 (1 H, d, J = 14.9 Hz),
2.69−2.73 (1 H, m), 2.46 (1 H, t, J = 13.7 Hz), 1.89−1.95 (1 H, m),
1.87 (1 H, dd, J = 13.7, 2.7 Hz), 1.50 (3 H, s), 1.45 (9 H, s), 0.38−0.43
(1 H, m), 0.27−0.37 (1 H, m), −0.30 to −0.22 (1 H, m), −1.10 to
−1.00 (1 H, m); HRMS (ESI) m/z 598.1597 [M + H]+
(C29H34Cl2FNO5S requires 598.1592).
Hz), 7.21 (1 H, dd, J = 10.0, 1.6 Hz), 7,16 (1 H, dd, J = 10.0, 7.7 Hz),
7.02 (1 H, t, J = 2.0 Hz), 6.80−6.90 (2 H, m), 6.77 (1 H, dd, J = 7.7,
1.6 Hz), 5.87 (1 H, m), 5.16−5.19 (2 H, m), 4.86 (1 H, d, J = 10.0
Hz), 3.63 (1 H, dd, J = 11.0, 4.6 Hz), 3.36 (1 H, td, J = 10.3, 4.6 Hz),
3.19 (1 H, t, J = 10.0 Hz), 3.09 (1 H, ddd, J = 11.8, 9.8, 4.8 Hz), 2.57−
2.65 (2 H, m), 1.85−2.20 (2 H, m), 1.25 (3 H, s), 0.85 (1 H, m),
0.57−0.67 (2H, m), 0.26 (1H, m), 0.06 (1H, m); MS (ESI) m/z 476.2
[M + H]+.
(3S,5S,6R,8S)-8-Allyl-6-(3-chloro-2-fluorophenyl)-5-(4-chlor-
o p h e n y l ) - 3 - c y c l o p r o p y l - 8 - m e t h y l - 2 , 3 , 5 , 6 , 7 , 8 -
hexahydrooxazolo[3,2-a]pyridin-4-ium 4-Methylbenzenesul-
fonate (32b). To a solution of 33b (73.6 g, 154 mmol) in toluene
(386 mL) was added TsOH·H2O (30.3 g, 159 mmol). The reaction
was heated at reflux under Dean−Stark conditions for 4 h. Then the
reaction was cooled and concentrated under reduced pressure to
provide crude 32b (97.1 g, 100%) as a pale yellow syrup. The crude
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chloro-2-fluorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)acetic Acid (4). Compound 4 was prepared as
a white foam from 30c according to a procedure similar to that
described for the synthesis of 3 from 30b: 1H NMR (400 MHz,
CDCl3) δ (ppm) 7.14−7.27 (6 H, m), 6.98−7.04 (1 H, m), 5.05 (1 H,
d, J = 10.8 Hz), 4.30 (1 H, t, J = 11.9 Hz), 3.65−3.75 (1 H, m), 3.16 (1
H, d, J = 15.7 Hz), 2.93 (1 H, d, J = 12.1 Hz), 2.72−2.84 (2 H, m),
2.44 (1 H, t, J = 13.7 Hz), 1.85 (2 H, dd, J = 13.7, 2.7 Hz), 1.50 (3 H,
s), 1.45 (9 H, s), 0.33−0.42 (1 H, m), 0.20−0.29 (1 H, m), −0.35 to
−0.26 (1 H, m), −1.15 to −1.04 (1 H, m); HRMS (ESI) m/z
598.1598 [M + H]+ (C29H34Cl2FNO5S requires 598.1592).
1
product was used in the next step without further purification: H
NMR (500 MHz, CDCl3) δ (ppm) 7.87 (2 H, d, J = 8.3 Hz). 7.25−
7.32 (2 H, m), 7.16−7.20 (5 H, m), 7.08 (1 H, s), 6.80 (1 H, s, br),
5.75−5.93 (2 H, m), 5.47 (1 H, dd, J = 10.0, 9.1 Hz), 5.37 (1 H, d, J =
10.5 Hz), 5.33 (1 H, d, J = 17.1 Hz), 4.57−4.63 (1 H, m), 4.51 (t, J = 1
H, 8.6 Hz), 3.29 (1 H, m), 2.93 (1 H, t, J = 13.7 Hz), 2.72 (1 H, dd, J
= 7.6 and 13.7 Hz), 2.63 (1 H, dd, J = 13.7, 7.3 Hz), 2.37 (3 H, s), 1.92
(1 H, dd, J = 13.9, 3.7 Hz), 1.57 (3 H, s), 0.33−0.50 (2 H, m), 0.08−
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamide (5). Oxalyl chloride (33 μL, 0.38 mmol) was
added to a solution of 224 (200 mg, 0.344 mmol) in DCM (1.5 mL) at
I
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Journal of Medicinal Chemistry
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25 °C. The reaction mixture was stirred at the same temperature for 1
h and then concentrated under reduced pressure to provide the acid
chloride as a white foam. To a solution of the acid chloride in THF
(0.5 mL) was added LiHMDS (1.0 M in THF, 0.52 mL, 0.52 mmol) at
25 °C. The reaction mixture was stirred for 6 h and was then diluted
(aqueous 1 N HCl) and extracted (3 × EtOAc). The combined
organic layers were washed (brine), dried (Na2SO4), and concentrated
under reduced pressure. Purification of the residue by flash column
chromatography (SiO2, 35−100% EtOAc/hexanes, a gradient elution)
Compounds 8−11, 14, 15, and 17−24. The title compounds
were prepared from 224 according to a procedure similar to that
described for the synthesis of 7.
Data for 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
1
peridin-3-yl)acetamido)-2-methoxybenzoic acid (8): H NMR (500
MHz, CDCl3) δ (ppm) 9.66 (1 H, s, br), 8.14 (1 H, d, J = 8.6 Hz),
8.06−8.12 (1 H, m), 7.06−7.25 (5 H, m), 6.85−7.01 (4 H, m), 4.98 (1
H, d, J = 10.8 Hz), 4.27−4.39 (1 H, m), 4.13 (3 H, s), 3.23 (1 H, ddd,
J = 13.6, 10.8, 2.7 Hz), 3.06 (1 H, d, J = 14.7 Hz), 2.96 (1 H, d, J =
12.7 Hz), 2.83 (1 H, d, J = 14.7 Hz), 2.68−2.79 (1 H, m), 2.49 (1 H, t,
J = 13.6 Hz), 2.03 (1 H, dd, J = 13.6, 2.3 Hz), 1.80−1.94 (1 H, m),
1.49 (3 H, s), 1.46 (9 H, s), 0.32−0.45 (1 H, m), 0.06−0.19 (1 H, m),
−0.36 to −0.24 (1 H, m), −1.21 to −1.10 (1 H, m); HRMS (ESI) m/
z 729.2178 [M + H]+ (C37H42Cl2N2O7S requires 729.2163).
1
provided 5 (40 mg, 20%) as an off-white foam: H NMR (500 MHz,
CDCl3) δ (ppm) 7.12−7.29 (2 H, m), 7.06−7.11 (3 H, m), 7.00 (2 H,
s), 6.90−6.91 (1 H, m), 6.64 (1 H, s, br), 5.63 (1 H, s, br), 4.96 (1 H,
d, J = 10.8 Hz), 4.30−4.38 (m, 1 H), 3.31 (1 H, dt, J = 10.8, 2.9 Hz),
2.90−2.96 (1 H, m), 2.73−2.80 (2 H, m), 2.65−2.75 (1 H, m), 2.39 (1
H, t, J = 13.7 Hz), 2.00 (1 H, dd, J = 13.5, 2.7 Hz), 1.85−1.92 (1 H,
m), 1.44 (9 H, s), 1.43 (3 H, s), 0.30−0.38 (1 H, m), 0.17−0.26 (1 H,
m), −0.38 to −0.25 (1 H, m), −1.10 to −1.00 (1 H, m); HRMS (ESI)
m/z 579.1853 [M + H]+ (C29H36Cl2N2O4S requires 579.1846).
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)-N-phenylacetamide (6). To a solution of 224 (119
mg, 0.204 mmol) and aniline (20 μL, 0.23 mmol) in pyridine (0.5 mL)
was added EDC (117 mg, 0.612 mmol) at 0 °C. The reaction was
allowed to warm to 25 °C. After the mixture was stirred for 19 h, the
reaction was quenched (ice-cold 1 N aqueous HCl), extracted (2 ×
Et2O), and washed (brine). The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. Flash chroma-
tography (SiO2, 15−100% EtOAc/hexanes, gradient elution) provided
Data for 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
1
peridin-3-yl)acetamido)-3-methoxybenzoic acid (9): H NMR (400
MHz, CDCl3) δ (ppm) 8.68 (1 H, d, J = 8.4 Hz), 8.53 (1 H, s), 7.86
(1 H, dd, J = 8.5, 1.5 Hz), 7.59 (1 H, d, J = 1.8 Hz), 6.93−7.25 (7 H,
m), 6.89 (1 H, td, J = 4.3, 1.6 Hz), 4.94 (1 H, d, J = 10.8 Hz), 4.35 (1
H, t, J = 11.6 Hz), 3.89 (3 H, s), 3.31−3.41 (1 H, m), 3.14 (1 H, d, J =
13.3 Hz), 2.92 (1 H, d, J = 13.1 Hz), 2.64−2.76 (2 H, m), 2.31−2.41
(1 H, m), 2.16−2.25 (1 H, m), 1.88−2.00 (1 H, m), 1.47 (3 H, s), 1.45
(9 H, s), 0.25−0.37 (1 H, m), −0.10 to +0.02 (1 H, m), −0.39 to
−0.26 (1 H, m), −1.30 to −1.15 (1 H, m); HRMS (ESI) m/z
729.2174 [M + H]+ (C37H42Cl2N2O7S requires 729.2163).
Data for 3-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamido)benzoic acid (10): 1H NMR (500 MHz,
CDCl3) δ (ppm) 9.83 (1 H, s), 8.65 (1 H, dd, J = 8.2, 0.9 Hz), 8.25 (1
H, t, J = 1.7 Hz), 7.84 (1 H, d, J = 8.1 Hz), 7.43−7.49 (1 H, m), 7.15−
7.35 (4 H, m), 6.91−7.15 (4 H, m), 5.01 (1 H, d, J = 10.8 Hz), 4.35 (1
H, t, J = 11.6 Hz), 3.37−3.45 (1 H, m), 3.34 (1 H, d, J = 13.0 Hz),
3.01 (1 H, d, J = 13.0 Hz), 2.98 (1 H, d, J = 13.4 Hz), 2.71−2.80 (1 H,
m), 2.55 (1 H, dd, J = 13.4, 2.4 Hz), 2.31 (1 H, t, J = 13.6 Hz), 1.88−
1.94 (1 H, m), 1.47 (9 H, s), 1.46 (3 H, s.), 0.33−0.50 (2 H, m),
0.18−0.28 (1 H, m), −0.33 to −0.21 (1 H, m), −1.16 to −1.07 (1 H,
m); HRMS (ESI) m/z 699.2062 [M + H]+ (C36H40Cl2N2O6S requires
699.2057).
Data for 2-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamido)benzoic acid (11): 1H NMR (400 MHz,
CDCl3) δ (ppm) 11.31 (1 H, s), 8.78 (1 H, d, J = 8.6 Hz), 8.12 (1 H,
dd, J = 7.9, 1.5 Hz), 7.58−7.66 (1 H, m), 7.02−7.25 (6 H, m), 6.83−
7.02 (3 H, m), 4.96 (1 H, d, J = 10.8 Hz), 4.31−4.45 (1 H, m), 3.34−
3.46 (1 H, m), 3.17 (1 H, d, J = 13.3 Hz), 2.93 (1 H, d, J = 12.9 Hz),
2.84 (1 H, d, J = 13.5 Hz), 2.66−2.75 (1 H, m), 2.26−2.40 (2 H, m),
1.83−1.95 (1 H, m), 1.52 (3 H, s), 1.44 (9 H, s), 0.26−0.39 (1 H, m),
0.09−0.21 (1 H, m), −0.27 to −0.27 (1 H, m), −0.40 to −0.26 (1 H,
m), −1.23 to −1.05 (1 H, m); HRMS (ESI) m/z 699.2067 [M + H]+
(C36H40Cl2N2O6S requires 699.2057).
Data for 6-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamido)nicotinic acid (14): 1H NMR (500 MHz,
CDCl3) δ (ppm) 12.12 (1 H, s, br), 8.89 (1 H, s, br), 8.74 (1 H, d, J =
8.6 Hz), 8.59 (1 H, d, J = 8.3 Hz), 7.15−7.42 (3 H, m), 6.85−7.14 (5
H, m), 4.96 (1 H, d, J = 10.5 Hz), 4.35 (1 H, t, J = 11.6 Hz), 3.38 (1
H, t, J = 11.5 Hz), 3.14 (1 H, d, J = 13.2 Hz), 2.97 (1 H, d, J = 13.4
Hz), 2.91 (1 H, d, J = 13.0 Hz), 2.71−2.79 (1 H, m), 2.46 (1 H, t, J =
13.6 Hz), 1.97 (1 H, d, J = 12.2 Hz), 1.80−1.91 (1 H, m), 1.57 (3 H,
s), 1.44 (9 H, s), 0.28−0.37 (1 H, m), 0.17−0.27 (1 H, m), −0.39 to
−0.28 (1 H, m), −1.11 to −0.96 (1 H, m); HRMS (ESI) m/z
700.2022 [M + H]+ (C35H39Cl2N3O6S requires 700.2009).
1
6 (74 mg, 55%) as a white foam: H NMR (500 MHz, CDCl3) δ
(ppm) 8.46 (1 H, s, br), 7.66 (2 H, d, J = 7.8 Hz), 7.38 (2 H, t, J = 8.3
Hz), 7.17 (1 H, t, J = 7.3 Hz), 7.02−7.09 (6 H, m), 6.99 (1 H, s),
6.86−6.89 (1 H, m), 4.94 (1 H, d, J = 10.8 Hz), 4.31 (1 H, t, J = 11.7
Hz), 3.30 (1 H, dt, J = 11.0, 2.7 Hz), 2.95 (2 H, t, J = 13.5 Hz), 2.67−
2.73 (2 H, m), 2.39 (1 H, t, J = 13.7 Hz), 2.07 (1 H, dd, J = 13.7, 2.7
Hz), 1.94 (1 H, s, br), 1.47 (3 H, s), 1.45 (9 H, s), 0.30−0.40 (1 H,
m), −0.28 to +0.10 (1 H, m), −0.40 to −0.28 (1 H, m), −1.32 to
−1.20 (1 H, m); HRMS (ESI) m/z 655.2161 [M + H]+
(C35H40Cl2N2O4S requires 655.2159).
4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxo-
piperidin-3-yl)acetamido)benzoic Acid (7). A solution of 224 (500
mg, 0.861 mmol) and methyl 4-aminobenzoate (260 mg, 1.72 mmol)
in DMF (2.5 mL) was treated with EDC (495 mg, 2.58 mmol), HOAt
(352 mg, 2.58 mmol), and NaHCO3 (217 mg, 2.58 mmol)
successively at rt. After the mixture was stirred at 40 °C for 18 h,
the reaction was quenched (1 N aqueous HCl), extracted (2 ×
EtOAc), and washed (1 × saturated aqueous NaHCO3, 2 × brine).
The combined organic layers were dried (Na2SO4) and concentrated
under reduced pressure. Purification by RP-HPLC (45−75% A/B,
gradient elution) provided methyl 4-(2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)benzoate (490 mg,
80%) as a white solid: MS (ESI) m/z 713.2 [M + H]+.
To a solution of the ester above (490 mg, 0.687 mmol) in THF/
MeOH/H2O (1:1:2, 20 mL) was added LiOH (2 N in H2O, 1.03 mL,
2.06 mmol). After the mixture was stirred at 50 °C for 30 min, the
reaction was quenched (ice-cold 1 N aqueous HCl), extracted (2 ×
EtOAc), and washed (brine). The combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. Purification by
RP-HPLC (45−65% A/B, gradient elution) provided 7 (400 mg, 83%)
1
as a white solid: H NMR (500 MHz, CDCl3) δ (ppm) 9.47 (1 H, s,
br), 8.14 (2 H, d, J = 8.8 Hz), 7.84 (2 H, d, J = 8.6 Hz), 7.02−7.23 (5
H, m), 6.85−7.02 (3 H, m), 4.98 (1 H, d, J = 10.8 Hz), 4.34 (1 H, t, J
= 11.5 Hz), 3.28−3.38 (1 H, m), 2.89−3.07 (3 H, m), 2.69−2.80 (1 H,
m), 2.42 (1 H, t, J = 13.7 Hz), 2.18 (1 H, dd, J = 13.7, 2.4 Hz), 1.81−
1.95 (1 H, m), 1.49 (3 H, s), 1.46 (9 H, s), 0.31−0.44 (1 H, m), 0.01−
0.15 (1 H, m), −0.37 to −0.25 (1 H, m), −1.26 to −1.11 (1 H, m);
HRMS (ESI) m/z 699.2058 [M + H]+ (C36H40Cl2N2O6S requires
699.2057).
Data for 2-(4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclo-
propylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-ox-
1
opiperidin-3-yl)acetamido)phenyl)acetic acid (15): H NMR (500
MHz, CDCl3) δ (ppm) 8.78 (1 H, s, br), 7.57 (2 H, d, J = 8.3 Hz),
7.27 (2 H, d, J = 8.3 Hz), 6.76−7.20 (8 H, m), 4.95 (1 H, d, J = 10.8
Hz), 4.25−4.35 (1 H, m), 3.70 (2 H, s), 3.34 (1 H, t, J = 11.2 Hz),
2.89−3.01 (2 H, m), 2.83 (1 H, d, J = 13.7 Hz), 2.67−2.76 (1 H, m),
J
dx.doi.org/10.1021/jm501550p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.37 (1 H, t, J = 13.6 Hz), 2.18 (1 H, d, J = 12.5 Hz), 1.86 (1 H, s, br),
1.45 (3 H, s), 1.45 (9 H, s), 0.39−0.49 (1 H, m), 0.28−0.38 (1 H, m),
0.01−0.12 (1 H, m), −0.40 to −0.28 (1 H, m), −1.30 to −1.17 (1 H,
m); HRMS (ESI) m/z 713.2225 [M + H]+ (C37H42Cl2N2O6S requires
713.2213).
Data for (1R,4r)-4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)acetamido)cyclohexanecarboxylic acid (17): 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.14−7.36 (4 H, m), 6.83−7.13 (5
H, m), 4.96 (1 H, d, J = 10.5 Hz), 4.24−4.36 (1 H, m), 3.77−3.88 (1
H, m), 3.30 (1 H, t, J = 11.7 Hz), 2.93 (1 H, d, J = 13.2 Hz), 2.77−
2.88 (2 H, m), 2.68−2.76 (1 H, m), 2.28−2.42 (2 H, m), 1.98−2.20 (5
H, m), 1.78−1.89 (1 H, m), 1.53−1.68 (2 H, m), 1.45 (9 H, s), 1.40 (3
H, s), 1.28−1.36 (2 H, m), 1.25−1.27 (1 H, m), 0.40−0.54 (2 H, m),
0.32−0.40 (1 H, m), 0.15−0.26 (1 H, m), −0.35 to −0.25 (1 H, m),
−1.14 to −1.04 (1 H, m); HRMS (ESI) m/z 705.2530 [M + H]+
(C36H46Cl2N2O6S requires 705.2526).
Data for (1S,4s)-4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)acetamido)cyclohexanecarboxylic acid (18): 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.76 (1 H, d, J = 5.1 Hz), 7.13−
7.39 (3 H, m), 6.85−7.12 (5 H, m), 4.96 (1 H, d, J = 10.8 Hz), 4.29 (1
H, t, J = 11.5 Hz), 4.09−4.17 (1 H, m), 3.41 (1 H, td, J = 11.3, 4.3
Hz), 2.89−3.00 (2 H, m), 2.85 (1 H, d, J = 13.2 Hz), 2.67−2.76 (1 H,
m), 2.47−2.57 (1 H, m), 2.20−2.35 (2 H, m), 1.97−2.06 (1 H, m),
1.85−1.96 (2 H, m), 1.67−1.84 (5 H, m), 1.45 (9 H, s), 1.40 (3 H, s),
0.39−0.52 (1 H, m), 0.30−0.39 (1 H, m), 0.14−0.23 (1 H, m), −0.38
to −0.26 (1 H, m), −1.19 to −1.07 (1 H, m); HRMS (ESI) m/z
705.2538 [M + H]+ (C36H46Cl2N2O6S requires 705.2526).
H, m); HRMS (ESI) m/z 677.2221 [M + H]+ (C34H42Cl2N2O6S
requires 677.2213).
(1S,3s)-3-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiper-
idin-3-yl)acetamido)cyclobutanecarboxylic acid (23): 1H NMR
(500 MHz, CDCl3) δ (ppm) 8.00 (1 H, d, J = 6.1 Hz), 7.14−7.34
(3 H, m), 6.83−7.13 (5 H, m), 4.95 (1 H, d, J = 10.8 Hz), 4.52−4.62
(1 H, m), 4.26 (1 H, t, J = 11.5 Hz), 3.36 (1 H, t, J = 10.8 Hz), 2.86−
3.01 (2 H, m), 2.60−2.85 (5 H, m), 2.44 (1 H, q, J = 9.8 Hz), 2.19−
2.36 (3 H, m), 1.71−1.82 (1 H, m), 1.44 (9 H, s), 1.35 (3 H, s), 0.29−
0.39 (1 H, m), 0.12−0.22 (1 H, m), −0.41 to −0.26 (1 H, m), −1.21
to −1.06 (1 H, m); HRMS (ESI) m/z 677.2229 [M + H]+
(C34H42Cl2N2O6S requires 677.2213).
Data for 1-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
1
peridin-3-yl)acetyl)azetidine-3-carboxylic acid (24): H NMR (500
MHz, CDCl3) δ (ppm) 7.05−7.27 (6 H, m), 6.99 (1 H, s), 6.91 (1 H,
d, J = 3.2 Hz), 4.95 (1 H, d, J = 10.5 Hz), 4.46−4.53 (2 H, m), 4.24−
4.37 (3 H, m), 3.31−3.52 (2 H, m), 2.93 (1 H, d, J = 13.0 Hz), 2.67−
2.79 (2 H, m), 2.50−2.61 (1 H, m), 2.26−2.35 (1 H, m), 2.09−2.20 (1
H, m), 1.83−1.94 (1 H, m), 1.44 (9 H, s), 1.40 (3 H, s), 0.31−0.40 (1
H, m), 0.18−0.28 (1 H, m), −0.37 to −0.26 (1 H, m), −1.12 to −1.00
(1 H, m); HRMS (ESI) m/z 663.2062 [M + H]+ (C33H40Cl2N2O6S
requires 663.2057).
4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxo-
piperidin-3-yl)acetamido)-2-methoxybenzamide (12). A solu-
tion of 8 (30 mg, 0.041 mmol) in DMF (0.3 mL) was treated with
EDC (24 mg, 0.12 mmol), HOAt (17 mg, 0.12 mmol), and NaHCO3
(10 mg, 0.12 mmol) successively at 25 °C. After the mixture was
stirred at 40 °C for 30 min, NH3 (7 N solution in MeOH) was added,
and the resulting mixture was stirred at 40 °C for 30 min. The reaction
was quenched (1 N aqueous HCl), extracted (2 × EtOAc), and
washed (1 × saturated aqueous NaHCO3, 2 × brine). The combined
organic layers were dried (Na2SO4) and concentrated under reduced
pressure. Purification by RP-HPLC (45−65% A/B, gradient elution)
Data for 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamido)bicyclo[2.2.2]octane-1-carboxylic acid (19):
1H NMR (400 MHz, CDCl3) δ (ppm) 7.15−7.31 (3 H, m), 6.83−7.13
(5 H, m), 6.46 (1 H, s), 4.94 (1 H, d, J = 10.8 Hz), 4.24−4.40 (1 H,
m), 3.24−3.36 (1 H, m), 2.92 (1 H, d, J = 13.5 Hz), 2.58−2.80 (3 H,
m), 2.32 (1 H, t, J = 13.6 Hz), 2.08 (1 H, dd, J = 13.3, 2.7 Hz), 1.89−
2.04 (12 H, m), 1.77−1.87 (1 H, m), 1.44 (9 H, s), 1.36 (3 H, s),
0.30−0.43 (1 H, m), 0.14−0.27 (1 H, m), −0.40 to −0.25 (1 H, m),
−1.16 to −0.98 (1 H, m); HRMS (ESI) m/z 731.2670 [M + H]+
(C38H48Cl2N2O6S requires 731.2683).
1
provided 12 (19 mg, 63%) as a white solid: H NMR (500 MHz,
CDCl3) δ (ppm) 9.44 (1 H, s, br), 8.16 (1 H, d, J = 8.6 Hz), 8.02−
8.16 (2 H, m), 7.03−7.29 (6 H, m), 6.78−7.03 (4 H, m), 4.97 (1 H, d,
J = 10.8 Hz), 4.27−4.40 (1 H, m), 4.06 (3 H, s), 3.22 (1 H, ddd, J =
13.7, 10.9, 2.8 Hz), 2.92−3.03 (2 H, m), 2.84 (1 H, d, J = 14.4 Hz),
2.68−2.77 (1 H, m), 2.48 (1 H, t, J = 13.7 Hz), 2.00 (1 H, dd, J = 13.6,
2.1 Hz), 1.84−1.94 (1 H, m), 1.49 (3 H, s), 1.46 (9 H, s), 0.33−0.42
(1 H, m), 0.05−0.14 (1 H, m), −0.35 to −0.24 (1 H, m), −1.22 to
−1.12 (1 H, m); HRMS (ESI) m/z 728.2325 [M + H]+
(C37H43Cl2N3O6S requires 728.2322).
Data for 1-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
1
peridin-3-yl)acetyl)piperidine-4-carboxylic acid (20): H NMR (500
MHz, CDCl3) δ (ppm) 7.13−7.28 (3 H, m), 6.83−7.13 (5 H, m), 4.94
(1 H, d, J = 10.5 Hz), 4.48 (1 H, t, J = 14.1 Hz), 4.29−4.37 (1 H, m),
4.02−4.14 (1 H, m), 3.24−3.43 (2 H, m), 2.87−3.02 (4 H, m), 2.59−
2.75 (2 H, m), 2.25−2.35 (1 H, m), 2.11−2.21 (1 H, m), 2.03 (2 H,
dd, J = 13.1, 3.1 Hz), 1.85−1.95 (1 H, m), 1.69−1.84 (2 H, m), 1.44
(9 H, s), 1.38 (3 H, d, J = 6.6 Hz), 0.30−0.41 (1 H, m), 0.19−0.28 (1
H, m), −0.37 to −0.25 (1 H, m), −1.13 to −1.01 (1 H, m); HRMS
(ESI) m/z 691.2386 [M + H]+ (C35H44Cl2N2O6S requires 691.2370).
Data for 1-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetyl)-4-methylpiperidine-4-carboxylic acid (21): 1H
NMR (400 MHz, CDCl3) δ (ppm) 7.14−7.27 (3 H, m), 6.83−7.12 (5
H, m), 4.94 (1 H, d, J = 10.8 Hz), 4.26−4.41 (2 H, m), 3.93 (1 H, t, J
= 13.5 Hz), 3.30−3.48 (2 H, m), 2.81−3.16 (4 H, m), 2.65−2.78 (1 H,
m), 2.09−2.36 (4 H, m), 1.83−1.97 (1 H, m), 1.40−1.55 (2 H, m),
1.44 (9 H, s), 1.38 (3 H, d, J = 12.5 Hz), 1.30 (3 H, d, J = 3.1 Hz),
0.29−0.40 (1 H, m), 0.17−0.28 (1 H, m), −0.39 to −0.25 (1 H, m),
−1.12 to −0.99 (1 H, m); HRMS (ESI) m/z 705.2544 [M + H]+
(C36H46Cl2N2O6S requires 705.2526).
Data for (1R,3r)-3-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)acetamido)cyclobutanecarboxylic acid (22): 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.13−7.28 (3 H, m), 6.82−7.13 (6
H, m), 4.96 (1 H, d, J = 10.0 Hz), 4.63−4.76 (1 H, m), 4.22−4.35 (1
H, m), 3.24−3.35 (1 H, m), 3.13−3.22 (1 H, m), 2.94 (1 H, d, J = 13.2
Hz), 2.66−2.90 (5 H, m), 2.30−2.47 (3 H, m), 2.02 (1 H, d, J = 13.2
Hz), 1.78−1.89 (1 H, m), 1.45 (9 H, s), 1.43 (3 H, s), 0.32−0.44 (1 H,
m), 0.18−0.27 (1 H, m), −0.34 to −0.23 (1 H, m), −1.16 to −1.04 (1
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)-N-(4-cyanophenyl)acetamide (13). A solution of 224
(50 mg, 0.086 mmol) and 4-aminobenzonitrile (15 mg, 0.13 mmol) in
DMF (0.3 mL) was treated with EDC (50 mg, 0.26 mmol), HOAt (35
mg, 0.26 mmol), and NaHCO3 (22 mg, 0.26 mmol) successively at 25
°C. After the mixture was stirred at 40 °C for 18 h, the reaction was
quenched (1 N aqueous HCl), extracted (2 × EtOAc), and washed (1
× saturated aqueous NaHCO3, 2 × brine). The combined organic
layers were dried (Na2SO4) and concentrated under reduced pressure.
Purification by RP-HPLC (45−75% A/B, gradient elution) provided
1
13 (15 mg, 26%) as a white solid: H NMR (500 MHz, CDCl3) δ
(ppm) 9.60 (1 H, s, br), 7.79 (2 H, d, J = 8.8 Hz), 7.65 (2 H, d, J = 8.6
Hz), 7.05−7.24 (5 H, m), 6.81−7.02 (3 H, m), 4.97 (1 H, d, J = 10.8
Hz), 4.27−4.37 (1 H, m), 3.19−3.28 (1 H, m), 3.04 (1 H, d, J = 14.7
Hz), 2.95 (1 H, d, J = 13.7 Hz), 2.82 (1 H, d, J = 14.7 Hz), 2.68−2.78
(1 H, m), 2.49 (1 H, t, J = 13.8 Hz), 1.99 (1 H, dd, J = 13.7, 2.7 Hz),
1.80−1.93 (1 H, m), 1.49 (3 H, s), 1.45 (9 H, s), 0.34−0.40 (1 H, m),
0.02−0.10 (1 H, m), −0.35 to −0.25 (1 H, m), −1.24 to −1.14 (1 H,
m); HRMS (ESI) m/z 680.2113 [M + H]+ (C36H39Cl2N3O4S requires
680.2111).
4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxo-
piperidin-3-yl)-N-methylacetamido)benzoic Acid (16). To a
solution of the methyl ester precursor for the synthesis of 7, methyl
K
dx.doi.org/10.1021/jm501550p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetamido)benzoate (40 mg, 0.056 mmol), in DMF (1 mL) was added
NaH (60% in mineral oil, 21 mg, 0.53 mmol). After the mixture was
stirred at 25 °C for 10 min, MeI (28 μL, 0.45 mmol) was added, and
the mixture was stirred at 25 °C for 1 h. Then the reaction was
quenched by adding water (1 mL).
ACKNOWLEDGMENTS
■
We thank Simon Wong for the time-dependent inhibition
determinations and Manuel Ventura and Dhanashri Bagal for
the acquisition of high-resolution mass spectra.
ABBREVIATIONS USED
■
To the quenched reaction above were added THF (0.5 mL),
MeOH (0.5 mL), and LiOH (2 N in H2O, 84 μL, 0.168 mmol), and
the resulting mixture was stirred at 50 °C for 30 min. The reaction was
quenched (ice-cold 1 N aqueous HCl), extracted (2 × EtOAc), and
washed (brine). The combined organic layers were dried (Na2SO4)
and concentrated under reduced pressure. Purification by RP-HPLC
(45−65% A/B, gradient elution) provided 16 (22 mg, 56%) as an off-
BrdU, 5-bromo-2-deoxyuridine; CYP3A4, cytochrome P450
3A4; CL, clearance; DCE, 1,2-dichloroethane; DCM, dichloro-
methane; DKR, dynamic kinetic resolution; DMF, N,N-
diemethylformamide; dr, diastereoselectivity ratio; EDC, N-
(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochlor-
ide; EdU, 5-ethynyl-2′-deoxyuridine; er, enantioselectivity
ratio; EtOAc, ethyl acetate; HOAt, 1-hydroxy-7-azabenzotria-
zole; hPXR, human pregnane X receptor; HTRF, homogeneous
time-resolved fluorescence; i-PrOH, isopropyl alcohol; ITC,
isothermal titration calorimetry; iv, intravenous; LiHMDS,
lithium bis(trimethylsilyl)amide; po, per os; LiOH, lithium
hydroxide; MDM2, murine double minute 2; NaH, sodium
hydride; NaHMDS, sodium bis(trimethylsilyl)amide; PPTS,
pyridinium p-toluenesulfonate; QD, once a day dosing; qRT-
PCR, quantitative reverse transcription polymerase chain
reaction; SAR, structure−activity relationship; SEM, standard
error of the mean; SPR, surface plasmon resonance; t-BuOK,
potassium tert-butoxide; TDI, time-dependent inhibition; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; Ts2O, 4-toluene-
sulfonic anhydride; TsOH, 4-toluenesulfonic acid
1
white solid: H NMR (500 MHz, CDCl3) δ (ppm) 8.17 (2 H, d, J =
8.3 Hz), 7.34 (2 H, d, J = 8.3 Hz), 7.05−7.25 (5 H, m), 6.80−7.03 (3
H, m), 4.86 (1 H, d, J = 10.5 Hz), 4.29 (1 H, t, J = 11.7 Hz), 3.36 (3
H, s, br), 3.24 (1 H, t, J = 11.2 Hz), 2.89 (1 H, d, J = 13.2 Hz), 2.83 (1
H, d, J = 14.4 Hz), 2.61−2.76 (2 H, m), 2.21−2.32 (1 H, m), 2.13−
2.19 (1 H, m), 1.82−1.92 (1 H, m), 1.42 (9 H, s), 1.34 (3 H, s), 0.26−
0.37 (1 H, m), 0.16−0.25 (1 H, m), −0.39 to −0.30 (1 H, m), −1.17
to −1.07 (1 H, m); HRMS (ESI) m/z 713.2220 [M + H]+
(C37H42Cl2N2O6S requires 713.2213).
4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropy-
lethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic
Acid (25). Compound 25 was prepared as a white solid from 3
according to a procedure similar to that described for the synthesis of
7: 1H NMR (500 MHz, DMSO-d6) δ (ppm) 12.29 (1 H, s, br), 10.44
(1 H, s),. 7.68 (1 H, d, J = 8.6 Hz), 7.54 (1 H, s, br), 7.40−7.50 (1 H,
m), 7.22−7.26 (3 H, m), 7.19 (1 H, d, J = 8.8 Hz), 6.90−6.93 (3 H,
m), 4.89 (1 H, d, J = 10.5 Hz), 4.05−4.20 (1 H, m), 3.77 (3 H, s),
3.40−3.50 (1 H, m), 3.02−3.16 (2 H, m), 2.66 (1 H, d, J = 13.7 Hz),
2.50−2.60 (1 H, m), 2.08−2.18 (2 H, m), 1.75−1.86 (1 H, m), 1.34 (9
H, s), 1.30 (3 H, s), 0.25−0.38 (1 H, m), 0.14−0.25 (1 H, m), −0.30
to −0.13 (1 H, m), −1.30 to −1.12 (1 H, m); HRMS (ESI) m/z
747.2063 [M + H]+ (C37H41Cl2FN2O7S requires 747.2068).
4-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chloro-2-fluorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic
Acid (26). Compound 26 was prepared as a white solid from 4
according to a procedure similar to that described for the synthesis of
7: 1H NMR (400 MHz, CDCl3) δ (ppm) 9.50 (1 H, s, br), 8.17 (1 H,
s, br), 8.13 (1 H, d, J = 8.4 Hz), 6.95−7.27 (7 H, m), 6.90 (1 H, d, J =
7.2 Hz), 5.06 (1 H, d, J = 11.0 Hz), 4.24−4.39 (1 H, m), 4.13 (3 H, s),
3.75 (1 H, t, J = 11.5 Hz), 2.84−3.09 (3 H, m), 2.68−2.80 (1 H, m),
2.46 (1 H, t, J = 13.7 Hz), 1.98−2.06 (1 H, m), 1.82−1.97 (1 H, m),
1.49 (3 H, s), 1.46 (9 H, s), 0.32−0.46 (1 H, m), 0.08−0.21 (1 H, m),
−0.37 to −0.23 (1 H, m), −1.21 to −1.07 (1 H, m); HRMS (ESI) m/
z 747.2085 [M + H]+ (C37H41Cl2FN2O7S requires 747.2068).
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ASSOCIATED CONTENT
* Supporting Information
■
S
In vitro biological assays, in vivo study protocols, determination
of cocrystal structures of 25 with MDM2, and metabolite
profiling of compounds 1 and 25 from hepatocyte incubation.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Accession Codes
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AUTHOR INFORMATION
Corresponding Author
*Phone: 650-244-2682. E-mail: yrew@amgen.com or
yosuprew@yahoo.com.
■
Notes
The authors declare no competing financial interest.
L
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dx.doi.org/10.1021/jm501550p | J. Med. Chem. XXXX, XXX, XXX−XXX