Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction

Article abstract of DOI:10.1021/jm401767k

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC₅₀ = 0.4 nM) and cellular potency (SJSA-1 EdU IC₅₀ = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED₅₀ of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Full text of DOI:10.1021/jm401767k

Article
pubs.acs.org/jmc
Selective and Potent Morpholinone Inhibitors of the MDM2−p53
Protein−Protein Interaction
Ana Z. Gonzalez,*,† John Eksterowicz,† Michael D. Bartberger,∥ Hilary P. Beck,† Jude Canon,⊥
Ada Chen,† David Chow,† Jason Duquette,† Brian M. Fox,† Jiasheng Fu,† Xin Huang,#
Jonathan B. Houze,† Lixia Jin,§ Yihong Li,† Zhihong Li,† Yun Ling,§ Mei-Chu Lo,† Alexander M. Long,#
Lawrence R. McGee,† Joel McIntosh,† Dustin L. McMinn,† Jonathan D. Oliner,⊥ Tao Osgood,⊥
Yosup Rew,† Anne Y. Saiki,⊥ Paul Shaffer,# Sarah Wortman,‡ Peter Yakowec,# Xuelei Yan,† Qiuping Ye,§
Dongyin Yu,⊥ Xiaoning Zhao,† Jing Zhou,∥ Steven H. Olson,† Julio C. Medina,† and Daqing Sun†
†
‡
§
Departments of Therapeutic Discovery, Pharmaceutics, and Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans
Boulevard, South San Francisco, California 94080, United States
Departments of ∥Therapeutic Discovery and ⊥Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California
91320, United States
#Department of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: We previously reported the discovery of AMG 232, a
highly potent and selective piperidinone inhibitor of the MDM2−
p53 interaction. Our continued search for potent and diverse
analogues led to the discovery of novel morpholinone MDM2
inhibitors. This change to a morpholinone core has a significant
impact on both potency and metabolic stability compared to the
piperidinone series. Within this morpholinone series, AM-8735
emerged as an inhibitor with remarkable biochemical potency
(HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25
nM), as well as pharmacokinetic properties. Compound 4 also
shows excellent antitumor activity in the SJSA-1 osteosarcoma
xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences
between the morpholinone and the piperidinone series will be described herein.
bind to MDM2 in the p53 binding region.5,11−14 Several small
INTRODUCTION
■
molecule inhibitors of the MDM2−p53 interaction are now
Recognized as the “guardian of the genome” the transcription
factor p53 is the cell’s main tumor suppressor and one of the
being tested in the clinic.15−18
We recently described the structure-based design of a novel
most frequently inactivated genes in human cancers.1,2 Over 30
class of potent and selective piperidinone inhibitors of the
years of research has shown that upon cellular stress, p53
MDM2−p53 interaction exemplified by AM-8553 (1, Figure 1)
response triggers the expression of multiple genes that control
and AMG 232 (2, Figure 1).19−22 While 1 is a potent inhibitor
cell cycle arrest, apoptosis, senescence, and DNA repair.3 In
(SJSA-1 EdU IC50 = 72 nM),23 continued investigation of the
about 50% of cancer cells p53 inactivation takes place through
N-alkyl substituent in 1 resulted in a substantial improvement
mutations within the p53 gene (TP53) or by translational
in the biochemical and cellular potency of our inhibitors.21
These efforts led to the discovery of AMG 232 (2), an
exquisitely potent and selective piperidinone inhibitor.21
Compound 2 demonstrated significant inhibition in both
biochemical (HTRF) and cellular (SJSA-1, EdU) assays with
an IC50 of 0.6 and 9.2 nM, respectively.23 Furthermore, 2 has an
outstanding pharmacokinetic profile and shows robust in vivo
antitumor activity inhibiting tumor growth in an SJSA-1
osteosarcoma xenograft model with an ED50 of 9.2 mg/kg.21
modifications of its gene product.4 In those tumors that retain
wild-type p53 function, loss of activity occurs by other means,
such as direct inhibition by its natural inhibitors.5 Discovered in
1987, the oncogene MDM2 has been identified as p53’s main
inhibitor and is found to be amplified in many tumor tissues.6−8
Research has shown that overexpression of MDM2 blocks p53-
mediated cell cycle arrest and apoptosis.9 As a result, disruption
of MDM2 binding to p53 has emerged as an attractive strategy
for the reactivation of the p53 pathway.10 Despite the inherent
challenges associated with this task, the relatively small
hydrophobic interface between MDM2 and p53 suggested
the possibility of designing a small molecule inhibitor that could
Received: November 14, 2013
Published: February 18, 2014
© 2014 American Chemical Society
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overlap between the two, suggesting that 2 binds to MDM2 in a
low energy conformation that contributes to its high potency.21
Initial efforts on morpholinone compounds indicated that
this scaffold could be a template for the design of very potent
MDM2−p53 inhibitors.25 In this article, we report how our
continued quest to find molecules with diverse structures that
maintain the favorable efficacy of 2 led to the discovery of novel
morpholinone-derived inhibitors of the MDM2−p53 inter-
action. Among these, AM-8735 (4, Figure 1), a potent and
selective MDM2 inhibitor (SJSA-1 EdU IC50 = 25 nM), shows
remarkable pharmacokinetic properties and in vivo efficacy in a
SJSA-1 osteosarcoma xenograft model (ED50 = 41 mg/kg). In
addition, we will discuss the significant differences in metabolic
stability observed between the morpholinone inhibitors and
their piperidinone counterparts.
RESULTS AND DISCUSSION
■
We commenced our research in this area by synthesizing
morpholinone compounds using knowledge gained from the
piperidinone series. From this effort, two findings became
evident that are best exemplified by comparing morpholinone 5
and the aforementioned piperidinone 3 (Table 1): whereas
Figure 1. Potent morpholinone and piperidinone inhibitors of the
MDM2−p53 interaction.
The exceptional potency of 2 can be understood by the
cocrystal structure of analogue 3 (Figure 2).19 Compound 3
Table 1. Comparison between Piperidinone 3 and
Morpholinone 5
Biochemical Potency
KD (SPR, nM)
0.036
Potency in SJSA-1 Cells
18.4 0.6
0.392
ac
,
p21 IC50 (nM)
EdU IC50 (nM)
240 90
37 19
bc
,
3.2 1.2
Hepatocyte Intrinsic Clearance (mL/min per 106 cells)
human
rat
15.0
6.7
1.9
1.8
Figure 2. Cocrystal structure of 3 bound to human MDM2 (17−111)
at 1.7 Å resolution. White labels indicate positions normally occupied
by key p53 residues. MDM2 residues His96 and Gly58 are labeled in
yellow. The coordinates of 3 with MDM2 have been deposited in the
PDB with accession code 4OAS.
mouse
dog
18.0
68.0
12.0
15.5
a
b
Assay conducted in the absence of human serum. Assay conducted
in the presence of 10% human serum. Mean and standard deviation of
at least two runs.
c
occupies three critical binding sites on MDM2, namely, the
Leu26(p53), Trp23(p53), and Phe19(p53) pockets. The carboxylate
of 3 presumably engages in a salt bridge with the imidazole
moiety on His96. The C3-Me points directly toward solvent,
supporting our hypothesis that the improvement in potency
observed with this substitution in the piperidinone series is due
to the stabilization of the gauche conformation rather than from
an additional contact with the MDM2 protein.24 Additionally,
the sulfone moiety plays an important role in directing the
small lipophilic ethyl group on 3 toward the Phe19(p53) binding
pocket while placing the tert-butyl moiety in close contact with
the protein, in a shelf region located above Gly58. Direct
comparison of the single X-ray crystal structure of 2 with the
structure of analogue 3 bound to MDM2 shows a nearly perfect
morpholinone inhibitors are 5- to 10-fold less potent than their
piperidinone counterparts, most morpholinones are more
stable in hepatocytes, a finding that is consistent across
preclinical species and human.26 Given that the observed
metabolism of most analogues is driven by glucuronidation of
the parent acid (<10% turnover in human microsomes), we
hoped that the morpholinone series could provide a
pharmacokinetic advantage over the piperidinones that would
compensate for the observed loss in potency. Besides this core
change, there is another key difference between inhibitors 3 and
5. Piperidinone inhibitor 3 requires the C3-methyl substituent
to enforce a conformational equilibrium in favor of the gauche
binding conformation (Figure 3; C3-monosubstituted,
ΔEanti‑gauche = −0.5 kcal/mol, 3:1 anti/gauche; C3-disubstituted,
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Figure 3. B3LYP/6-31G* structures and relative energies (kcal/mol) for model mono- and disubstituted piperidinone and morpholinone inhibitors
of the MDM2−p53 interaction.
ΔEanti‑gauche = 2.8 kcal/mol, 1:46 anti/gauche), resulting in a 2-
to 3-fold improvement in potency.24 Conversely, analogous
quantum mechanical calculations on the morpholinone core
predict that the MDM2-binding gauche conformer is preferred,
even in the absence of a stabilizing methyl substituent (Figure
3). These calculations are in agreement with the observed in
vitro data showing that the C2-Me-substituted morpholinone 6
(Figure 4) exhibits similar potency (HTRF HS IC50 = 0.6/
2.3 nM, EdU IC50 = 32 nM) to its des-Me counterpart 5
(HTRF HS IC50 = 0.5/2.0 nM, EdU IC50 = 37 nM).23
show minimal liabilities in these assays, many sulfonamides
demonstrate considerable time dependent inhibition of
CYP3A4 (e.g., 9, 70%; 13, 74%; 14, 0.4%; 15, 3%; 5, 23%).27
With the potent tert-butyl sulfone in place we studied the
interaction of the small alkyl group with the Phe19(p53) pocket
in the MDM2 protein. As shown in Table 3, increasing the
group size from methyl to ethyl resulted in a 3-fold
improvement in cellular potency (entries 1 and 2). However,
attempts to further improve potency by increasing the size of
this substituent to isopropyl or tert-butyl were not successful
(Table 3, entries 3−5). Gratifyingly, the cyclopropyl function-
ality provided a good balance for this position, yielding the
most potent analogue with an IC50 of 25 nM in the SJSA-1 EdU
assay (Table 3, entry 6) and a decrease in PXR activity.
Importantly, all analogues studied within this series maintained
favorable stability in human hepatocytes. The majority of
inhibitors within this group exhibit low clearance in rats. On the
basis of its good potency and stability in human hepatocytes, 4
was selected for further evaluation.
First, we evaluated the selectivity of 4 by examining its ability
to inhibit the proliferation of HCT116 p53wt and p53−/− tumor
cells in vitro (Figure 5).28 Compound 4 displayed substantial
Figure 4. C2-Me-substituted morpholinone 6.
growth inhibition of wild-type p53 cells (BrdU, 10% HS, IC50
63 nM) and no growth inhibition of p53-deficient cells (IC50
=
>
With these data in hand, we began our research efforts by
exploring modifications around the N-alkyl substituent. Thus, a
series of sulfonamides, reverse sulfonamides, and sulfones were
synthesized (Table 2). From these, incorporation of N-
arylsulfonamide substituents provided some of our most potent
morpholinone inhibitors (Table 2, entries 2−7). Introducing a
fluorine at the ortho position of the arene moiety resulted in
increased potency and decreased PXR activity (Table 2, entry
3). The para-fluoro substitution also resulted in decreased PXR
activation (Table 2, entry 4 vs entry 2). With the potent ortho-
phenyl substituent in place, the size of the S-alkyl substituent
proved to be inconsequential to potency but did affect stability
in human hepatocytes (Table 2, entries 6 and 7 vs entry 3). In
the case of the sulfone substituent, increasing the size of the
group from ethyl to tert-butyl resulted in a significant
improvement in both biochemical and cellular potency
(Table 2, entries 8−10). Nonetheless, further increasing the
size of this small lipophilic group did not lead to more potent
analogues and in most cases was also detrimental to the
hepatocyte stability (Table 2, entries 11−13). Finally,
substituents such as sulfonamide 19 and oxetanesulfone 20
(Table 2, entries 14−15) provided inhibitors with reduced
cellular potency when compared to 5.
25 μM). Similarly, 4 exhibited a dose-dependent increase of
p21 mRNA, a direct transcriptional readout of p53 activity, in
HCT116 p53wt cells (p21, 10% HS, IC50 = 160 nM). No
induction of p21 was observed in HTC116 p53−/− tumor cells
treated with 4 at concentrations up to 10 μM. Compound 4
also demonstrated significant time and concentration depend-
ent p21 mRNA induction in vivo in a pharmacodynamic assay
in SJSA-1 osteosarcoma tumors (Figure 6).19,21 A maximum
p21 induction of 8-fold was observed 2 h after dosing on day 4
at 25 mg/kg. These data indicate in vivo p53-mediated activity
displayed by 4 and provided dose-selection guidance for a
xenograft efficacy study.
Thus, we tested the ability of 4 to inhibit tumor growth in a
mouse xenograft model bearing the same SJSA-1 osteosarcoma
tumor used in the pharmacodynamic assay (Figure 7).29 In this
study, SJSA-1 tumor cells were implanted subcutaneously and
grown in mice for 9 days to an average volume of 200 mm3.
After this period, 4 was administered by oral gavage (as a
solution in 15% HPβCD, 1% Pluronic F68, pH 8) at 5, 25, 50,
and 100 mg/kg q.d. for a period of 2 weeks, and tumor volume
was quantified. This compound caused robust dose-dependent
tumor growth inhibition with the highest dose of 100 mg/kg
resulting in tumor stasis. The calculated ED50 was 41 mg/kg
(95% confidence interval of 34.11−50.25).
Representative sulfonamides and sulfones in Table 2 were
evaluated for their potential to cause drug−drug interactions by
inhibition of the CYP enzymes. Whereas a majority of sulfones
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Table 2. Optimization of the N-Alkyl Group: Sulfonamides, Reverse Sulfonamides, and Sulfones
a
b
c
IC50 in biochemical assay using serum free buffer. IC50 in biochemical assay using buffer containing 15% human serum. Cellular potency in SJSA-1
d
e
f
in the presence of 10% human serum. Human PXR activation, % of control, rafampin, at 2 μM. Human hepatocyte stability. Mean and standard
deviation of at least two runs.
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Table 3. Modification to the R Group that Occupies the MDM2 Phe19p53 Pocket
af
,
b
c
g
HTRF Serum Free
HTRF 15% HS
SJSA-1 EdU
hPXR
df
hHep CL
Rat PK
f
f
f
e
,
Entry Compd
R
IC50 (nM)
IC50 (nM)
IC50 (nM)
(%)
(μL/min per 106 cells) CL (L h−1 kg−1)/t1/2 (h)
1
2
3
4
5
6
21
5
Me
Et
1.1 0.1
0.5 0.3
0.4 0.2
2.0
6.9 2.1
2.0 0.1
2.1 0.6
2.5
129 54
37 19
22 11
50 21
0.7
1.9
4.7
1.7
2.1
1.4
0.23/4.1
0.43/4.0
0.11/6.0
22
23
24
4
CH2CF3
i-Pr
33
8
12
2
5
9
67 14
42
t-Bu
0.5 0.1
0.4 0.1
5.1 1.6
1.8 0.9
120 16
49
c-Pr
25
9
19.2
0.35/5.7
a
b
c
IC50 in biochemical assay using serum free buffer. IC50 in biochemical assay using buffer containing 15% human serum. Cellular potency in SJSA-1
d
e
f
in the presence of 10% human serum. Human PXR activation, % of control, rafampin, at 2 μM. Human hepatocyte stability. Mean and standard
deviation of at least two runs. Rat PK, iv, 0.5 mg/kg.
g
Figure 6. PD study results of 4 in SJSA-1 tumor xenograft: (∗) p <
0.05. Female athymic nude mice (n = 5/group) were implanted
subcutaneously with 5 × 106 SJSA-1 cells. When tumors reached ∼175
mm3, 25 mg/kg 4 or vehicle was administered orally once daily (q.d.)
for 4 days. Mice were sacrificed on day 4 at 1, 2, 4, 8, and 24 h
postdose. 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 (blue bars) + SEM of data from five mice. Concentrations in
plasma were analyzed by LC/MS/MS and plotted as red dots against
the right y-axis.
Figure 5. Cell activity of 4 is p53 dependent. (a) In HCT116 p53wt
M1 and acylglucuronide M2 are generated across species,
and p53−/− cells, the percentage of BrdU positive cells was measured
16 h after compound treatment by flow cytometry. DMSO control was
designated as 0% inhibition. (b) In HCT116 p53wt and p53 −/− cells,
total RNA was extracted 7 h after compound treatment and p21
mRNA was measured by quantitative RT-PCR.
including human hepatocytes (Figure 8b).30 Formation of the
acylglucuronide of 25 in dog and human hepatocytes was
qualitatively more significant than that in hepatocytes of other
species.
In contrast, the oxidative metabolite of 4 was the major
metabolite in mouse and rat hepatocytes. Although both the
oxidative metabolite and acylglucuronide of 4 were detected in
other species, they were barely detected in dog and human
hepatocytes (Figure 8a). It appeared that this seemingly simple
core substitution is enough to change the formation rates of
metabolites. Moreover it impacted their intrinsic clearances
across species, especially in human hepatocytes.
Potency and hepatocyte stability comparisons between
piperidinone 25 and morpholinone 4 are shown in Table 4.
The most significant advantage morpholinone 4 has over
piperidinone 25 is its improved stability in hepatocytes across
all tested species. This observation supports what was observed
with compounds 3 and 5 (Table 1).
In order to better understand their metabolic pathways,
analogues 4 and 25 were incubated separately in hepatocytes
(106 cells/mL) from five different species and their degradation
products evaluated by LC−MS/MS (Figure 8). In the case of
25, the chromatograms show that both the oxidative metabolite
In an X-ray cocrystal with human MDM2 (Figure 9),
compound 4 binds to the MDM2 protein in a similar fashion to
several piperidinone inhibitors (Figure 2).31 The C6-m-Cl-
phenyl, C5-p-Cl-phenyl, and cyclopropyl moieties occupy the
Leu26(p53), Trp23(p53), and Phe19(p53) pockets, respectively. The
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trans-amino alcohol 31 which could be subsequently hydro-
lyzed to amino alcohol 32 hydrochloride.
Alternatively, amino alcohol 32 was also synthesized through
a Henry reaction between nitroalkane 34 and 3-chlorobenzal-
dehyde followed by in situ protection of the secondary carbinol
to its corresponding silyl ether 35 (Scheme 2). Reduction of
the nitro group in 35 followed by chiral resolution of 32
provided the desired amino alcohol 36 in 97% ee.
With the desired amino alcohol at hand, the secondary
alcohol was converted to the TBS ether 37 (Scheme 3). This
transformation can be accomplished in good yields starting
from either the free-base amino alcohol 36 or its hydrochloride
salt 32. Ytterbium-catalyzed addition of the free amine on the
least hindered side of a chosen epoxide generated intermediates
38a−e. This transformation proved to be tolerant to a wide
variety of monosubstituted epoxides. Moreover, starting from
the corresponding enantiomerically enriched epoxide allowed
for diastereoselective formation of 38 with retention of
configuration at the carbinol center. Aziridines 39a−d could
be formed under standard Mitsunobu conditions in good yields.
However, for 38e, the tert-butyl moiety proved to be too
hindered for these conditions, and thus, mesylate formation
followed by an intramolecular displacement of the mesylate
yielded aziridine 39e albeit in low yields.32
Indium-catalyzed aziridine opening with the appropriate thiol
occurred smoothly.33 Alkylation of the free alcohol with
bromoacetic acid produced acids 41 that could then undergo
an intramolecular amide coupling reaction to form morpholi-
nones 42. Interestingly, attempts to produce this amide bond in
an intermolecular fashion with either acid chlorides or
carboxylic acids failed with a variety of reagents. Thus,
proceeding in an intramolecular fashion was pivotal for the
success of this synthetic route. Finally, allylation at C2 followed
by oxidation of both the thioether and the terminal alkene to
their corresponding sulfones and carboxylic acids, respectively,
afforded the desired inhibitors as a nearly 1.5:1 mixture of
diastereoisomers in favor of the desired C2-R-isomer.25
Since reaction of 37 with 1-cyclopropyloxirane gave a
complex mixture of products under the ytterbium-catalyzed
reaction conditions depicted in Scheme 3, an alternative
synthetic route was devised to access the cyclopropyl-
substituted analogues (Scheme 4). First, formation of the
morpholinone core was achieved by addition of chloroacetyl
chloride to 36 under basic conditions.25 From 43, N-alkylation
proceeded smoothly to 44, yielding a 45:55 mixture of isomers
favoring 44b.
However, after isolation of the desired isomer 44a, 44b could
be easily re-equilibrated to a 45:55 mixture of 44a/44b that
could subsequently be separated to give more of the desired
isomer. Compound 44a could then be reduced to the primary
alcohol with superhydride. Alcohol 45 could easily be
transformed to a thioether under Mitsunobu conditions and
then to inhibitor 4 following a similar alkylation/oxidation
sequence as that depicted previously in Scheme 2.34
Given the versatility of the Mitsunobu reaction, intermediate
49 was synthesized to quickly access a variety of sulfone- and
sulfonamide-substituted inhibitors (Scheme 5). To this end,
enantiomerically pure brosylate 47 was synthesized from R-2-
ethoxyoxirane and 3,4-dimethoxybenzyl alcohol. N-Alkylation
of 43 with 47 formed isomerically pure 48. Subsequently, DDQ
effected the formation of the desired primary carbinol 49 that
served as a common intermediate toward the synthesis of both
sulfonamide- and sulfone-containing analogues.
Figure 7. SJSA-1 cells (5 × 106) were implanted subcutaneously into
female athymic nude mice. Treatment with vehicle or 4 at 5, 25, 50, or
100 mg/kg q.d. by oral gavage began on day 9 when tumors had
reached ∼200 mm3 (n = 10/group). Tumor sizes and body weights
were measured twice per week. Data are represented as mean tumor
volumes, and the error bars represent SEM of data from 10 mice. No
significant (>5%) body weight loss was observed in any of the groups:
(∗) p < 0.05.
Table 4. Comparison between Morpholinone 4 and
Piperidinone 25
Biochemical Potency
ab
,
IC50 (HTRF, nM)
IC50 (HTRF, 15% HS, nM)
0.4 0.1
1.8 0.9
0.10 0.01
1.1 0.6
b
Rat PK (iv, 0.5 mg/kg)
CL (L h−1 kg−1
)
0.35
5.7
1.1
3.8
t1/2 (L/kg)
Hepatocyte Stability (μL/min per 106 cells)
human
rat
1
16
24
25
3
mouse
dog
6
13
24
a
b
IC50 in biochemical assay using serum free buffer. Mean and
standard deviation of at least two runs.
carboxylate appears to engage in a salt bridge with the
imidazole moiety on His96, a residue that also engages in a
face-to-face, π-stacking interaction with the C6-arene. The tert-
butyl moiety on the sulfone projects onto the glycine shelf
region maximizing the hydrophobic contact with the MDM2
protein.
CHEMISTRY
■
Synthesis of amino alcohol 32 commenced with commercially
available amino acid 27 (Scheme 1). Reduction of 27 with
lithium aluminum hydride followed by Boc protection gave
carbamate 29. Dess−Martin periodinane-mediated oxidation of
the alcohol produced aldehyde 30. Addition of 3-chlorophe-
nylmagnesium bromide at room temperature to 30 yielded
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Figure 8. Metabolite profiles of 4 (a) and 25 (b) from hepatocyte incubation at 10 μM.
a
Scheme 1
a
Reagents and conditions: (a) LiAlH4, THF, reflux, 3 h, quant; (b)
Boc2O, TEA, DCM, 12 h, 73%; (c) DMP, DCM, rt, 2 h, 95%; (d) 3-
chlorophenylmagnesium bromide, THF, rt, 12 h, 47%; (e) HCl/
dioxane, 2 h, quant.
Figure 9. Cocrystal structure of 4 bound to human MDM2 (17−111).
White labels indicate positions normally occupied by key p53 residues.
MDM2 residues His96 and Gly58 are labeled in yellow. The
coordinates of 4 with MDM2 have been deposited in the PDB with
accession code 4OBA.
easily converted to inhibitor 7 utilizing our standard oxidation
conditions.
Finally, tetrasubstituted inhibitor 6 was synthesized from 42b
by two subsequent alkylation reactions followed by our
standard oxidative conditions (Scheme 7).
CONCLUSION
■
Cyclopropylsulfonamide inhibitor 7 could be prepared from
intermediate 48 by alkylation at C2 followed by deprotection of
the 3,4-dimethoxybenzyl ether to yield 52 (Scheme 6). Dess−
Martin periodinane oxidation of 52 produced aldehyde 53
which, after a quick workup, was converted to methylamine 54
through a reductive amination. Addition of cyclopropanesul-
fonyl chloride to 54 rendered sulfonamide 55 that could be
Novel, potent, and selective morpholinone inhibitors of the
MDM2−p53 interaction were described. The morpholinone
inhibitors are less efficacious but have improved hepatocyte
stability relative to piperidinone inhibitors. Among these,
compound 4 shows excellent biochemical potency (HTRF
IC50 = 0.4 nM, Table 4), MDM2 selectivity over MDMX
(MDMX HTRF IC50 > 100 μM), cellular potency (SJSA-1 EdU
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a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) AgNO2, Et2O, 0 °C, 62%; (b) 3-
chlorobenzaldehyde, pyridine, alumina, TESCl, 55 °C, 12 h; (c) Zn/
HCl, 40 °C, 2 h, 20% over two steps; (d) (+)-di-p-toluoyl-D-tartaric
acid, EtOH/water, 38% (97% ee).
IC50 = 25 nM), as well as good pharmacokinetic properties
(Table 4).35 Importantly, compound 4 shows a marked
improvement in hepatocyte stability compared to piperidinone
inhibitor 25. Oxidation is the main metabolic pathway for the
morpholinone series and the morpholinone inhibitors have
significantly less formation of the acylglucuronide metabolite
than the piperidinones across preclinical species. This molecule
also demonstrates robust antitumor activity in the SJSA-1
osteosarcoma xenograft study with a calculated ED50 of 41 mg/
kg.
EXPERIMENTAL SECTION
■
General Chemistry. Reactions, unless otherwise stated, were
conducted under an inert gas atmosphere (nitrogen or argon) at the
temperature indicated. Commercial reagents and anhydrous solvents
were used without further purification. Analytical thin layer
chromatography (TLC) was performed on Analtech silica gel with
organic binder 250 μm TLC plates. 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 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
[A, H2O with 0.1% trifluoroacetic acid (TFA); B, CH3CN with 0.1%
TFA] with UV detection at 220 nm. All final compounds were purified
to ≥95% purity as determined by a Agilent 1100 series HPLC 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, A = H2O with
0.1% TFA, B = CH3CN with 0.1% TFA; gradient 5−95% B (0−15
min); flow rate, 1.5 mL/min. Low-resolution mass spectrometry (MS)
data were determined on an Agilent 1100 series LCMS with UV
detection at 254 nm and a low resolution electrospray mode (ESI).
High-resolution mass spectra were obtained on an Agilent 6510 Q-
a
Reagents and conditions: (a) TBSCl, imidazole, DCM 25 °C, 90%;
(b) Yb(OTf)3, CH3CN, 6 h, 130 °C, 79−93%; (c) DEAD, PPh3, THF,
40 °C, 12 h, 69−82% (d) t-BuSH, InCl3, DCM, 25 °C, 12 h, 88−98%;
(e) MsCl, TEA, DCM, −78 °C to rt, 12 h, 31%; (f) TBAF, THF, 15
min, 25 °C, 73−95%; (g) bromoacetic acid, NaH, THF, 25 °C, 12 h,
100% conversion; (h) HATU, DMF, DIPEA, 25 °C, 1 h, 61−90% (last
two steps); (i) LiHMDS, allyl bromide, −78 °C, 1 h, 85%, 2:1 mixture
of isomers; (j) NaIO4, RuCl3, CH3CN/CCl4/H2O, 32−75% combined
yield for both isomers.
1
TOF MS instrument with an Agilent 1200 LC on the front end. H
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 = single, 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 deg/dm, and the concentrations are reported as g/100 mL of the
specific solvent and were recorded at the temperature indicated.
Absolute stereochemistry of inhibitors 1−5 was unambiguously
determined through crystallographic analysis; for the rest of the
inhibitors stereochemistry at C2 was assigned as R in agreement with
that observed from inhibitors 1−5.
(R)-2-Amino-2-(4-chlorophenyl)ethanol (28). A solution of 1
M LiAlH4 in THF was diluted with 460 mL of anhydrous THF and
was heated to 75 °C under a nitrogen atmosphere. (R)-2-Amino-2-(4-
chlorophenyl)acetic acid (27) (25.0 g, 135 mmol; Asta Tech, Inc.,
Bristol, PA) was added in several portions. The reaction mixture was
heated to reflux for about 3 h, cooled to room temperature, and
quenched by adding water (8.0 mL), aqueous 15% NaOH (8.0 mL),
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a
a
Scheme 4
Scheme 5
a
Reagents and conditions: (a) chloroacetyl chloride, TEA, 0 °C, THF,
2 h; then NaH, rt, 12 h, 86%; (b) ethyl 2-bromo-2-cyclopropylacetate,
NaH, DMF, rt, 12 h, 82%; (c) NaOEt/EtOH, rt, 4 h, 57%; (d)
LiEt3BH, THF, rt, 30 min, 90%; (e) t-BuSH, cyanomethyltributyl-
phosphorane, toluene, 110 °C, 12 h, 43%. (f) LiHMDS, allyl bromide,
−78 °C, 1 h, 85%, 2:1 mixture of isomers; (g) NaIO4, RuCl3, CH3CN/
CCl4/H2O, 32−75% combined yield for both isomers.
and water (25.6 mL), successively. The mixture was stirred vigorously
for 30 min. The solids were filtered off, rinsed with THF and the
combined organics were concentrated in vacuo to provide 28 as a
yellow solid (>98% yield, 23 g). 1H NMR (400 MHz, CDCl3) δ 7.31−
7.35 (m, 2H), 7.29 (m, 2 H), 4.05 (br s, 1H), 3.69−3.78 (m, 1H), 3.53
(dd, J = 10.56, 8.02 Hz, 1H), 2.48 (br s, 1H), 1.59 (br s, 2 H). MS
(ESI) m/z = 172.0 [M + H]+ .
(R)-tert-Butyl 1-(4-Chlorophenyl)-2-hydroxyethylcarbamate
(29). A solution of di-tert-butyl dicarbonate (36.6 g, 168 mmol) in
DCM (50 mL) was added dropwise to a solution of 28 (23 g, 135
mmol) in DCM (200 mL) at 0 °C. After the addition was completed,
the ice bath was removed and the reaction mixture was stirred at room
temperature for 1.5 h. The reaction mixture was concentrated in vacuo
and the crude material was triturated with 10% DCM/hexane (850
mL) to provide 29 as a white powder (73% yield, 27 g). 1H NMR (400
MHz, (CD3)2SO) δ 7.33−7.39 (m, 2 H), 7.27−7.32 (m, 2 H), 7.24 (d,
J = 8.02 Hz, 1 H), 4.75−4.84 (m, 1 H), 4.43−4.56 (m, 1 H), 3.41−
3.52 (m, 2 H), 1.36 (s, 9 H). MS (CI+) m/z = 565.0 [2M + Na]+.
(R)-tert-Butyl 1-(4-Chlorophenyl)-2-oxoethylcarbamate (30).
1,1,1-Tris(acetoxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (“Dess−
Martin periodinane”) (17.4 g, 41.1 mmol) was added in several
portions to a stirred suspension of 29 (5.5 g, 20.5 mmol) in wet DCM
(40 mL of DCM/40 μL of water) at room temperature. The mixture
was stirred for 2 h, diluted with diethyl ether (40 mL), and quenched
by adding a solution of Na2S2O3 (10 equiv) in saturated aqueous
NaHCO3 solution (80 mL) at room temperature. The mixture was
stirred vigorously for 10 min, and the layers were separated. The
aqueous layer was extracted with diethyl ether (40 mL). The organics
were pooled, washed with saturated aqueous NaHCO3 solution and
brine, dried over MgSO4, filtered, and the filtrate was concentrated.
The crude material was used in the next step without further
a
Reagents and conditions: (a) 3,4-dimethoxybenzyl alcohol, NaH,
DMF, 60 °C, 2 h, 44%; (b) 4-bromobenzenesulfonyl chloride, DMAP,
DCM, rt, 78%; (c) 47, NaOtBu, DMF, 85 °C, 12 h, 66%; (d) DDQ,
CH2Cl2/H2O, 0 °C, 2 h, 87%; (e) cyanomethyltributylphosphorane,
toluene, 110 °C, 12 h, 10−85%; (f) LiHMDS, allyl bromide, −78 °C, 1
h, 85%, 2:1 mixture of isomers; (g) NaIO4, RuCl3, CH3CN/CCl4/
H2O, 32−75% combined yield for both isomers.
tert-Butyl (1R,2R)-2-(3-Chlorophenyl)-1-(4-chlorophenyl)-2-
hydroxyethylcarbamate (31). A 500 mL three-neck flask was
charged with a magnetic stir bar, magnesium (2.163 g, 89 mmol), a
crystal of iodine, and anhydrous diethyl ether (100 mL). The flask was
equipped with an addition funnel and a reflux condensor. The addition
funnel was charged with a solution of 1-bromo-3-chlorobenzene (89
mmol) in anhydrous ether. An amount of 15 mL of this solution was
added dropwise into the flask under vigorous stirring. Then the
reaction mixture was heated to reflux for about 5 min until the color of
the iodine was no longer visible. The heating source was removed, and
the remainder of the 1-bromo-3-chlorobenzene solution was added
dropwise at a rate to maintain gentle reflux. After the addition was
completed, the reaction mixture was maintained at reflux temperature
for another 30 min. The reaction mixture turned light brown during
the heating. The oil bath was removed, and without cooling, a solution
of 30 (4.80 g, 17.80 mmol) in anhydrous diethyl ethyl ether (66 mL)
1
purification. H NMR (400 MHz, CDCl3) δ 9.46 (br s, 1H), 7.31−
7.37 (m, 2H), 7.17−7.24 (m, 2H), 5.58−5.92 (m, 1H), 5.25 (d, J =
3.72 Hz, 1H), 5.14−5.34 (m, 1H), 1.27−1.45 (m, 9H).
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g). 1H NMR (500 MHz, CDCl3) δ 7.16−7.32 (8H, m), 5.38 (1H, d, J
= 4.4 Hz), 4.85 (1H, d, J = 5.0 Hz), 1.32 (9H, s). MS (ESI) m/z =
404.0 [M + Na]+.
a
Scheme 6
(1R,2R)-2-Amino-1-(3-chlorophenyl)-2-(4-chlorophenyl)-
ethanol Hydrochloride (32). Compound 31 (3.0 g, 7.49 mmol) was
treated with 20 mL of 4 N HCl in dioxane at room temperature for 2
h. The solvent was evaporated. The residue was triturated with diethyl
ether, filtered, washed with diethyl ether, and dried under vacuum to
1
give 32 (>98% yield, 2.4 g). H NMR (400 MHz, CD3OD) δ 7.36
(2H, d, J = 12 Hz), 7.27 (5H, m), 7.07 (1H, d, J = 8 Hz), 4.90 (1H, d,
J = 8 Hz), 4.36 (1H, d, J = 12 Hz). MS (ESI) m/z = 282.0 [M + H]+.
1-Chloro-4-(nitromethyl)benzene (34). A suspension of AgNO2
(392 g) in diethyl ether (1.6 L) was cooled to 0 °C, and a solution of
4-chlorobenzyl bromide (395 g, 1.92 mol) in diethyl ether (1.6 L) was
added dropwise over 1 h (temperature maintained below 3 °C during
addition). The reaction mixture was stirred for 16 h at 0 °C in the
dark. Then the mixture was filtered and the solids were washed with
diethyl ether (3×). The combined filtrates were concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel (eluent, 0−10% EtOAc in heptane; gradient
elution) to give 34 (62% yield, 205 g). 1H NMR (300 MHz, CDCl3) δ
7.41 (s, 4H), 5.42 (s, 2H). MS (ESI) m/z = 172.0 [M + H]+.
( )-2-Amino-1-(3-chlorophenyl)-2-(4-chlorophenyl)ethanol
(( )-32). To a flask containing 3-chlorobenzaldehyde (135 mL, 168 g,
1.19 mol) were added 34 (205 g, 1.19 mol), alumina (135 g), pyridine
(96 mL, 1.19 mol), and chlorotriethylsilane (200 mL, 180 g, 1.19
mol). The flask was covered in aluminum foil and spun for 16 h in the
dark at room temperature on a rotary evaporator. The resulting thick
paste was then filtered and washed with isopropanol. The filtrate was
divided into two equal portions. The procedure for each portion is as
follows: To each solution was added 1 M HCl (7 L, 7 mol), and then
Zn powder (800 g, 12.3 mol) was added in several portions. The
reaction mixture was stirred until the observed exothermic reaction (to
35 °C) was complete (90 min). Then the mixture was cooled to 0 °C
and basified with 30% NaOH to approximately pH 10. The suspension
was filtered through a pad of Celite and washed with DCM. The
filtrate was transferred to a separatory funnel, and the layers were
separated. The aqueous layer was extracted with DCM. The combined
organic layers were dried over Na2SO4, filtered, and the filtrate was
concentrated under reduced pressure. The residue was dissolved in
MTBE (1.5 L) and cooled to 0 °C. Then 4 N HCl in dioxane (375
mL, 1.5 mol) was added dropwise. The solid was collected by
filtration. The solid was purified by crystallization from dioxane/
ethanol to give a racemic mixture of 32 (20% overall yield, 76 g). MS
(ESI) m/z = 281.9 [M + H]+.
a
Reagents and conditions: (a) LHMDS, allyl bromide, −78 °C, 1 h,
85%, 2:1 mixture of isomers; (b) DDQ, CH2Cl2/H2O, 0 °C, 2 h, 87%;
(c) DMP, DCM, 1 h, rt, quant; (d) MeNH2, Na(OEt)3BH, DCE, 12
h, 83%; (e) cyclopropanesulfonyl chloride, TEA, DCM, rt, 24 h, 38%
(three steps); (f) NaIO4, RuCl3, CH3CN/CCl4/H2O, 65%, combined
yield for both isomers.
a
Scheme 7
(R,R)-2-Amino-1-(3-chlorophenyl)-2-(4-chlorophenyl)-
ethanol ((R,R-)36). trans-32 (900 g, 2.8 mols) (>99.5% purity) was
basified with 1 M NaOH (8 L) and extracted with EtOAc (6 L x 2).
The EtOAc layers were combined, washed with water and brine, dried
and concentrated, affording 800 g of free amine. The free amine was
taken in EtOH (32 L) and (+)-di-p-toluoyl-D-tartaric acid (1095 g)
was added. The mixture was heated to reflux and water was added until
the solution became clear (2.4 L). The solution was allowed to cool
overnight. The solid was filtered, washed with EtOH, and dried,
affording 1.2 kg of wet salt (74% ee). The salt was taken in EtOH (24
L) and water (1.8 L), heated to reflux, and cooled overnight. The solid
was filtered, washed with EtOH, and dried, affording 900 g of wet salt
(92% ee). The salt was taken in EtOH (18 L) and water (1.2 L),
heated to reflux, and cooled overnight. The solid was filtered, washed
with EtOH, and dried, affording 600 g of wet salt (>97% ee). The salt
was basified with 2 M NaOH (8 L) and extracted with EtOAc (4 L x
2). The EtOAc layers were combined, dried over Na2SO4, and
concentrated in vacuo. 36 (>95% purity, > 97%ee) was obtained as a
white solid (38% yield, 302 g). 1H NMR (300 MHz, CD3OD) δ 7.11−
7.21 (m, 7H), 6.96−6.98 (m, 1H), 4.67 (d, J = 7.8 Hz, 1H), 3.92 (d, J
= 7.8 Hz, 1H). MS (ESI) m/z = 282.0 [M + H]+. Enantiomeric excess
determined via chiral HPLC (OD-H, 250 × 4.6 mm, hexane:IPA =
a
Reagents and conditions: (a) LiHMDS, MeI, −78 °C, 1 h, 85%,
∼1.5:1 mixture of isomers; (b) LiHMDS, allyl bromide, −30 °C, 12 h,
45%, ∼1.5:1 mixture of isomers; (c) NaIO4, RuCl3, CH3CN/CCl4/
H2O, 49%, combined yield for both isomers.
was added over a period of 30 min. The mixture was stirred for an
additional 2 h at room temperature. The reaction mixture was poured
into 200 mL of a cold saturated aqueous NH4Cl solution. The two
layers were separated, and the aqueous layer was extracted with ethyl
acetate (2 × 100 mL). The combined organic layers were washed with
water and then brine, dried over Na2SO4, filtered, and the filtrate was
concentrated. The residue was washed with 100 mL of hexanes,
filtered, and dried to provide the title compound as a mixture of two
diastereomers (anti/syn = 9:1 by NMR). The crude material was used
in the next step without further purification (49% combined yield, 3.2
90:10, 215, and 230 nm, 20 min method) 11.3 min (major, 99.8%) and
23.5
15.9 min (minor, 0.2%). Optical rotation for major isomer: [α]D
+92.7° (c = 0.385, in MeOH).
=
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(1R,2R)-2-((tert-Butyldimethylsilyl)oxy)-2-(3-chlorophenyl)-
1-(4-chlorophenyl)ethanamine (37). To a solution of (1R,2R)-2-
amino-1-(3-chlorophenyl)-2-(4-chlorophenyl)ethanol (36) (1.0 g,
3.54 mmol) and imidazole (0.483 g, 7.09 mmol) in DCM (3.54
mL) was added tert-butyldimethylchlorosilane (0.588 g, 3.90 mmol) at
room temperature. The mixture was stirred overnight at room
temperature. After this period the reaction mixture was diluted with
water (10 mL) and extracted with DCM (3 × 20 mL). The organic
extract was dried over MgSO4. The solution was filtered and
concentrated under vacuum to give the crude material as a light-
yellow oil. The crude material was adsorbed onto a plug of silica gel
and purified by chromatography through a Redi-Sep prepacked silica
gel column (120 g), eluting with isocratic 70/25/5 DCM/acetone/
MeOH with 0.1% of triethylamine to provide 37 (90% yield, 1.26 g) as
stirred for 1 h at this temperature. The reaction was quenched with
water (10 mL). The mixture was extracted with diethyl ether (3 × 10
mL), and the organic extracts were dried over MgSO4. The solution
was filtered and concentrated under vacuum to give the crude material
as a light-yellow oil.
The crude material was adsorbed onto a plug of silica gel and
purified by chromatography through a Redi-Sep prepacked silica gel
column (40 g), eluting with three-step isocratic 8−20% acetone in
hexanes to provide (1R,2R)-2-(((S)-1-(tert-butylthio)butan-2-yl)-
amino)-1-(3-chlorophenyl)-2-(4-chlorophenyl)ethanol (73.1% yield,
1.8 g). MS (ESI) m/z = 426.2 [M + H]+.
Step B. To a 100 mL round-bottomed flask were added (1R,2R)-2-
(((S)-1-(tert-butylthio)butan-2-yl)amino)-1-(3-chlorophenyl)-2-(4-
chlorophenyl)ethanol (1.74 g, 4.08 mmol) and bromoacetic acid
(0.680 g, 4.90 mmol) in THF (13.60 mL). To this solution sodium
hydride, 60% in oil (0.408 g, 10.20 mmol), was added in portions over
a period of 10 min. The mixture was allowed to stir at room
temperature overnight. After this period the crude mixture was diluted
in water (10 mL) and extracted with diethyl ether (3 × 20 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated. The residue was used in the next step without further
purification. MS (ESI) m/z = 484.2 [M + H]+.
1
a light-yellow oil. H NMR (400 MHz, CD3OD) δ 7.12−7.30 (m,
7H), 7.07 (dt, J = 1.76, 4.11 Hz, 1H), 4.71 (d, J = 5.87 Hz, 1H), 3.97
(d, J = 5.67 Hz, 1H), 0.86−0.90 (m, 9H), −0.13 to −0.06 (m, 3H),
−0.31 to −0.21 (m, 3H). MS (ESI) m/z = 396.2 [M + H]+.
(R)-1-(((1R,2R)-2-((tert-Butyldimethylsilyl)oxy)-2-(3-chloro-
phenyl)-1-(4-chlorophenyl)ethyl)amino)butan-2-ol (38b). A
solution of (1R,2R)-2-((tert-butyldimethylsilyl)oxy)-2-(3-chlorophen-
yl)-1-(4-chlorophenyl)ethanamine (37) (1.22 g, 3.08 mmol), (R)-
(+)-1,2-epoxybutane (0.288 mL, 4.00 mmol), and ytterbium(III)
trifluoromethanesulfonate (0.345 mL, 0.462 mmol) in acetonitrile (10
mL) was heated at 130 °C for 3 h in a microwave. This was diluted
with water and extracted with ethyl acetate (3×). The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated to provide 38b (1.39 g, 96% yield). 1H NMR (400 MHz,
CDCl3) δ 7.19 (d, J = 8.61 Hz, 4H), 7.14−7.22 (m, 1H), 7.06−7.13
(m, 2H), 7.01 (d, J = 8.41 Hz, 2H), 6.80 (d, J = 7.63 Hz, 1H), 4.55 (d,
J = 7.24 Hz, 1H), 3.73 (d, J = 7.24 Hz, 1H), 3.42−3.54 (m, 1H), 2.56
(d, J = 11.93 Hz, 1H), 1.31−1.49 (m, 3H), 0.91 (s, 9H), −0.04 (s,
3H), −0.23 (s, 3H). MS (ESI) m/z = 468.1 [M + H]+.
Step C. To a solution of 2-((1R,2R)-2-(((S)-1-(tert-butylthio)butan-
2-yl)amino)-1-(3-chlorophenyl)-2-(4-chlorophenyl)ethoxy)acetic acid
(1.98 g, 4.09 mmol) and N,N-diethylpropan-2-amine (1.273 mL, 8.17
mmol) in DMF (13.62 mL) was added HATU (1.865 g, 4.90 mmol).
The mixture was stirred overnight at room temperature. After this
period, the crude mixture was quenched with water and extracted with
diethyl ether (3 × 30 mL). The organic extracts were dried over
MgSO4, filtered, and concentrated. The crude material was adsorbed
onto a plug of silica gel and purified by chromatography through a
Redi-Sep prepacked silica gel column (40 g), eluting with three-step
isocratic 5−15% acetone in hexanes to provide 42b (70% yield over
1
(S)-1-((1R,2R)-2-((tert-Butyldimethylsilyl)oxy)-2-(3-chloro-
phenyl)-1-(4-chlorophenyl)ethyl)-2-ethylaziridine (39b). To a
solution of triphenylphosphine (2.55 g, 9.72 mmol) and 38b (4.14 g,
8.84 mmol) dissolved in THF (15 mL) was added diethyl
azodicarboxylate, 40 wt % solution in toluene (3.83 mL, 9.72
mmol). The mixture was stirred at 40 °C overnight. The crude mixture
was concentrated, adsorbed onto a plug of silica gel, and purified by
chromatography through a Redi-Sep prepacked silica gel column (120
g), eluting with isocratic 5−10% EtOAc in hexanes to provide 39b
(69% yield, 3.0 g) as a light-yellow oil. 1H NMR (500 MHz, CDCl3) δ
7.29−7.38 (m, 3H), 7.12 (d, J = 7.83 Hz, 2H), 7.03 (d, J = 16.38 Hz,
1H), 6.96 (d, J = 8.31 Hz, 1H), 6.73−6.78 (m, 1H), 4.84 (d, J = 6.36
Hz, 1H), 3.72−3.87 (m, 1H), 3.49−3.66 (m, 1H), 2.50 (d, J = 6.36
Hz, 1H), 1.79 (d, J = 3.42 Hz, 1H), 1.64 (d, J = 6.36 Hz, 1H), 1.26−
1.30 (m, 2H), 0.85−0.90 (m, 9H), 0.68 (t, J = 7.34 Hz, 3H), 0.10 (s,
3H), −0.18 (s, 3H). MS (ESI) m/z = 450.2 [M + H]+.
the last two steps, 1.34 g) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.30 (d, J = 8.41 Hz, 2H), 7.23−7.27 (m, 1H), 7.12−7.20
(m, 2H), 7.08 (d, J = 8.41 Hz, 2H), 6.83 (d, J = 6.65 Hz, 1H), 4.82 (d,
J = 8.80 Hz, 1H), 4.56 (d, J = 9.00 Hz, 1H), 4.43 (q, J = 16.43 Hz,
2H), 3.27 (dd, J = 9.98, 12.52 Hz, 1H), 2.87−3.03 (m, 1H), 2.58 (dd, J
= 4.50, 12.52 Hz, 1H), 1.94−2.22 (m, 1H), 1.55−1.61 (m, 1H), 1.31−
1.37 (m, 9H), 0.64 (t, J = 7.53 Hz, 3H). MS (ESI) m/z = 466.2 [M +
H]+.
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-6-(3-
chlorophenyl)-5-(4-chlorophenyl)-3-oxomorpholin-2-yl)acetic
Acid (5). Compound 5 was prepared from 42b as an amorphous solid
through a procedure similar to that described elsewhere.25 1H NMR
(400 MHz, CDCl3) δ 7.18−7.27 (m, 5H), 7.11−7.17 (m, 1H), 7.07 (t,
J = 7.82 Hz, 1H), 6.97 (d, J = 7.63 Hz, 1H), 5.06 (d, J = 6.46 Hz, 1H),
4.86 (d, J = 6.65 Hz, 1H), 4.63 (t, J = 5.97 Hz, 1H), 3.85 (dd, J = 9.00,
13.69 Hz, 1H), 3.23−3.39 (m, 1H), 3.00 (s, 2H), 2.87 (d, J = 13.69
Hz, 1H), 1.97−2.17 (m, 1H), 1.45−1.64 (m, 1H), 1.31−1.40 (m, 9H),
0.45 (t, J = 7.53 Hz, 3H). MS (ESI) m/z = 556.0 [M + H]+. HRMS
(ESI) m/z found 556.1334 [M + H]+, calcd for C26H32Cl2NO6S
556.1322.
(S)-N-((1R,2R)-2-((tert-Butyldimethylsilyl)oxy)-2-(3-chloro-
phenyl)-1-(4-chlorophenyl)ethyl)-1-(tert-butylthio)butan-2-
amine (40b). To a solution of (S)-1-((1R,2R)-2-((tert-butyl-
dimethylsilyl)oxy)-2-(3-chlorophenyl)-1-(4-chlorophenyl)ethyl)-2-
ethylaziridine (39b) (170 mg, 0.377 mmol) in DCM (1.9 mL) at
room temperature were added 2-methyl-2-propanethiol (85 μL, 0.755
mmol) and indium(III) chloride (0.835 mg, 3.77 μmol). The light
yellow slurry mixture was stirred at room temperature overnight. The
mixture was concentrated to give quantitatively 40b (>98% yield, 206
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)propan-2-yl)-6-(3-
chlorophenyl)-5-(4-chlorophenyl)-3-oxomorpholin-2-yl)acetic
Acid (21). Compound 21 was prepared as a single isomer from 37
(11% overall yield for the last eight steps) through similar procedures
1
to those described for the synthesis of 5 in Scheme 2. H NMR (400
1
mg). H NMR (400 MHz, CDCl3) δ 7.04 (br s, 7H), 6.57−6.81 (m,
MHz, CDCl3) δ 7.31 (d, J = 8.41 Hz, 2H), 7.20−7.22 (m, 1H), 7.04−
7.16 (m, 4H), 6.81 (d, J = 7.82 Hz, 1H), 5.02 (d, J = 9.59 Hz, 1H),
4.73 (dd, J = 7.04, 4.69 Hz, 1H), 4.64 (d, J = 9.78 Hz, 1H), 4.15 (dd, J
= 13.40, 9.68 Hz, 1H), 3.57−3.71 (m, 1H), 3.26 (dd, J = 16.24, 7.24
Hz, 1H), 2.97 (dd, J = 16.24, 4.70 Hz, 1H), 2.85 (dd, J = 13.40, 3.42
Hz, 1H), 1.44 (br s, 9 H), 1.37 (d, J = 6.85 Hz, 3H). MS (ESI) m/z =
542 [M + H]+. HRMS (ESI) m/z found 542.1177 [M + H]+, calcd for
C25H30Cl2NO6S 542.1165.
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)-4,4,4-trifluorobu-
tan-2-yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-oxomor-
pholin-2-yl)acetic Acid (22). Compound 22 was prepared as a
single isomer from 37 (2% overall yield for the last eight steps)
1H), 4.34−4.57 (m, 1H), 3.69−3.98 (m, 1H), 2.46−2.68 (m, 2H),
2.21−2.44 (m, 2H), 1.45 (bs, 2H), 1.32 (br s, 9H), 0.94 (s, 9H), 0.89
(t, J = 7.35 Hz, 3H), 0.05 (br s, 3H), −0.21 (s, 3H). MS (ESI) m/z =
540.3 [M + H]+.
(5R,6R)-4-((S)-1-(tert-Butylthio)butan-2-yl)-6-(3-chlorophen-
yl)-5-(4-chlorophenyl)morpholin-3-one (42b). Step A. To a 250
mL round-bottomed flask were added (S)-N-((1R,2R)-2-((tert-butyl-
dimethylsilyl)oxy)-2-(3-chlorophenyl)-1-(4-chlorophenyl)ethyl)-1-
(tert-butylthio)butan-2-amine 40b (3.07 g, 5.68 mmol) and
tetrabutylammonium fluoride, 1.0 M in tetrahydrofuran (14.19 mL,
14.19 mmol) in THF (10 mL), at room temperature. The mixture was
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through procedures similar to those described for the synthesis of 5 in
Scheme 2. 1H NMR (400 MHz, CDCl3) δ 7.31−7.36 (m, 2H), 7.25−
7.30 (m, 2H), 7.20−7.24 (m, 2H), 7.13 (t, J = 8.12 Hz, 1H), 6.88−
7.04 (m, 1H), 5.18 (d, J = 7.83 Hz, 1H), 5.04 (d, J = 7.83 Hz, 1H),
4.82 (dd, J = 6.85, 4.11 Hz, 1H), 4.05−4.19 (m, 1H), 3.81−3.94 (m,
1H), 3.22 (dd, J = 16.92, 6.94 Hz, 2H), 2.92−3.14 (m, 2H), 2.26−2.42
(m, 1H), 1.36−1.51 (m, 9H). MS (ESI) m/z = 610 [M + H]+. HRMS
(ESI) m/z found 610.1050 [M + H]+, calcd for C26H29Cl2NO6S
610.1039.
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)-3-methylbutan-2-
yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-oxomorpholin-2-
yl)acetic Acid (23). Compound 23 was prepared as a single isomer
from 37 (3% overall yield for the last eight steps) through procedures
similar to those described for the synthesis of 5 in Scheme 2. 1H NMR
(400 MHz, CDCl3) δ 7.49 (d, J = 8.22 Hz, 2H), 7.33−7.42 (m, 3H),
7.22 (d, J = 0.98 Hz, 3H), 5.28−5.39 (m, 1H), 5.16 (s, 1H), 4.66 (t, J
= 6.16 Hz, 1H), 3.77 (dd, J = 7.43, 14.09 Hz, 1H), 3.30 (t, J = 7.53 Hz,
1H), 3.09−3.22 (m, J = 3.23, 6.16 Hz, 3H), 2.33−2.49 (m, 1H), 1.49
(s, 9H), 0.78 (d, J = 6.65 Hz, 3H), 0.53 (d, J = 6.85 Hz, 3H). MS
(ESI) m/z = 570.0 [M + H]+. HRMS (ESI) m/z found 570.1490 [M +
H]+, calcd for C27H34Cl2NO6S 570.1478.
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)-3,3-dimethylbu-
tan-2-yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-oxomor-
pholin-2-yl)acetic Acid (24). Compound 24 was prepared as a
single isomer from 37 (2% overall yield for the last eight steps)
through procedures similar to those described for the synthesis of 5 in
Scheme 2. 1H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.41 Hz, 2H),
7.27−7.30 (m, 3H), 7.08−7.23 (m, 3H), 5.34 (d, J = 7.24 Hz, 1H),
5.20 (d, J = 7.43 Hz, 1H), 4.76 (t, J = 6.26 Hz, 1H), 3.95 (dd, J = 9.59,
13.69 Hz, 1H), 3.60 (d, J = 2.35 Hz, 1H), 3.51 (s, 1H), 3.12 (d, J =
6.26 Hz, 1H), 2.99 (dd, J = 2.35, 13.69 Hz, 1H), 1.48 (s, 9H), 0.78 (s,
9H). MS (ESI) m/z = 584.0 [M + H]+. HRMS (ESI) m/z found
584.1642 [M + H]+, calcd for C28H36Cl2NO6S 584.1635.
acid (10 mL), 5% NaHCO3 (10 mL), water (10 mL), brine (10 mL).
The combined organic extracts were dried with MgSO4. The solvent
was evaporated under reduced pressure, and the residue (a 45:55 44a/
44b mixture of epimers) was purified by flash chromatography on
silica gel (120 g), eluting with 5% ethyl acetate in DCM to give first
44a (40% yield, 2.4 g) and then 44b (48% yield, 2.9 g).
Characterization of (S)-Ethyl 2-((2R,3R)-2-(3-Chlorophenyl)-
3-(4-chlorophenyl)-5-oxomorpholino)-2-cyclopropylacetate
(44a). 1H NMR (500 MHz, CDCl3) δ 7.28−7.31 (m, 2H), 7.21−7.27
(m, 2H), 7.12−7.20 (m, 3H), 6.90 (d, J = 7.83 Hz, 1H), 4.88 (d, J =
7.34 Hz, 1H), 4.73 (d, J = 7.34 Hz, 1H), 4.35−4.54 (m, 2H), 4.13 (q, J
= 7.09 Hz, 1H), 3.95−4.09 (m, 1H), 3.53 (d, J = 10.51 Hz, 1H), 1.30−
1.42 (m, 1H), 1.23 (t, J = 7.09 Hz, 3H), 0.65−0.77 (m, 1H), 0.56 (dd,
J = 4.89, 7.83 Hz, 1H), 0.33 (dd, J = 5.26, 9.90 Hz, 1H), −0.05 (dd, J =
5.87, 9.54 Hz, 1H). MS (ESI) m/z = 448.0 [M + H]+.
Characterization of (R)-Ethyl 2-((2R,3R)-2-(3-Chlorophenyl)-
3-(4-chlorophenyl)-5-oxomorpholino)-2-cyclopropylacetate
1
(44b). H NMR (500 MHz, CDCl3) d 7.28 (s, 1H), 7.24−7.27 (m,
2H), 7.21 (t, J = 1.83 Hz, 1H), 7.09−7.18 (m, 3H), 6.83 (d, J = 7.83
Hz, 1H), 4.83 (d, J = 8.07 Hz, 1H), 4.65 (d, J = 8.07 Hz, 1H), 4.46 (d,
J = 3.42 Hz, 2H), 4.09−4.28 (m, 2H), 2.85 (d, J = 10.03 Hz, 1H),
1.36−1.48 (m, 1H), 1.28−1.32 (m, 3H), 0.68 (d, J = 8.56 Hz, 1H),
0.53 (d, J = 7.82 Hz, 1H), 0.10−0.20 (m, 2H). MS (ESI) m/z = 448.0
[M + H]+.
(S)-Ethyl 2-((2R,3R)-2-(3-Chlorophenyl)-3-(4-chlorophenyl)-
5-oxomorpholino)-2-cyclopropylacetate (44a) from (R)-Ethyl
2-((2R,3R)-2-(3-Chlorophenyl)-3-(4-chlorophenyl)-5-oxomor-
pholino)-2-cyclopropylacetate (44b). Sodium ethoxide (0.144
mL, 0.386 mmol) was added to a solution of (R)-ethyl 2-((2R,3R)-2-
(3-chlorophenyl)-3-(4-chlorophenyl)-5-oxomorpholino)-2-cyclopro-
pylacetate (0.346 g, 0.772 mmol) in anhydrous ethanol (1.543 mL) at
room temperature. After 4 h, the reaction mixture was neutralized with
3 M HCl (0.13 mL). The mixture was partitioned between water and
EtOAc, and the layers were separated. The aqueous layer was extracted
with EtOAc twice, and the organics were pooled and washed with
brine. The combined organic phase was dried over Na2SO4, filtered,
and concentrated in vacuo to provide a crude that was subsequently
purified by flash chromatography on silica gel, eluting with 5% ethyl
acetate in DCM to provide 44b (57% yield, 0.2 g) (see experimental
above for characterization of 44b).
(5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)morpholin-3-
one (43). To a solution of (1R,2R)-2-amino-1-(3-chlorophenyl)-2-(4-
chlorophenyl)ethanol (36) (0.47 g, 1.7 mmol) and triethylamine
(0.349 mL, 2.5 mmol) in THF at 0 °C was added chloroacetyl chloride
(0.16 mL, 2.0 mmol). The reaction mixture was stirred at 0 °C for 1 h.
After this time, saturated aqueous NH4Cl solution and ethyl acetate
were added. The layers were separated and the combined organic
layers were washed with water (3 × 10 mL), dried over MgSO4,
filtered and the filtrate was concentrated in vacuum to give 2-chloro-N-
((1R,2R)-2-(3-chlorophenyl)-1-(4-chlorophenyl)-2-hydroxyethyl)-
acetamide as a light yellow oil which was taken to the next step
without further purification. MS (ESI) m/z 380.0 [M + Na]+.
The product above was dissolved in THF (15 mL) and treated with
several portions of sodium hydride (60% dispersion in mineral oil,
0.167 g, 4.16 mmol) over a period of 5 min. The mixture was stirred at
room temperature for 5 h. After this time, saturated aqueous NH4Cl
solution and ethyl acetate were added. The organic extracts were
separated and the combined organic layers were washed with water (3
× 10 mL), dried over MgSO4, filtered and the filtrate was concentrated
in vacuo to give the crude material as a yellow oil. The crude was
absorbed onto a plug of silica gel and purified by silica gel column
chromatography, eluting with a gradient of 0−30% acetone in hexanes
(5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-cy-
clopropyl-2-hydroxyethyl)morpholin-3-one (45). To a solution
of 44a (3.6 g, 8.29 mmol) in THF (8.29 mL) was added superhydride
(lithium triethylborohydride, 1.0 M solution in THF) (17.4 mL, 17.4
mmol) at 0 °C. After 15 min, LCMS showed complete conversion to
the desired product. MeOH (3 mL) was added dropwise over a period
of 1 min. Then potassium peroxymonosulfate (Oxone) (15.29 g, 24.87
mmol) in water (60 mL) was added dropwise over a period of 10 min.
After 1 h, saturated aqueous NaHSO3 (9 mL) was added at room
temperature. Extraction was with diethyl ether (2 × 60 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated under a vacuum. The crude material was adsorbed
onto a plug of silica gel and purified by flash chromatography through
a silica gel column (330 g), eluting with isocratic 20% acetone in
1
hexanes to provide 45 as an off-white solid (90% yield, 3.0 g). H
1
to provide 43 (86% yield, 0.49 g). H NMR (400 MHz, CDCl3) δ
NMR (400 MHz, CDCl3) δ 7.04−7.25 (m, 7H), 6.79 (d, J = 7.63 Hz,
1H), 4.87 (d, J = 7.43 Hz, 1H), 4.52 (d, J = 7.43 Hz, 1H), 4.26−4.42
(m, 2H), 3.50−3.59 (m, 1H), 3.13−3.36 (m, 2H), 2.88 (br s, 1H),
0.79 (ddd, J = 3.03, 4.94, 7.87 Hz, 1H), 0.40−0.57 (m, 2H), 0.11−0.23
(m, 1H), −0.10 to 0.06 (m, 1H). MS (ESI) m/z = 405.4 [M + H]+.
2-((2R,5R,6R)-4-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-oxomorpholin-2-
yl)acetic Acid (4). Compound 4 was prepared as a single isomer from
45 (24% yield, last three steps) through a procedure similar to that
described for the synthesis of 5 (for Mitsunobu reaction conditions see
7.47−7.54 (m, 1H), 7.46−7.54 (m, 1H), 7.34 −7.42 (m, 2H), 7.29−
7.32 (m, 2H), 7.26−7.29 (m, 1H), 7.13−7.25 (m, 1H), 6.98 (d, J =
8.41 Hz, 1H), 4.57−4.65 (m, 1H), 4.45 (d, J = 9.98 Hz, 1H), 4.00 (d, J
= 5.67 Hz, 2H). MS (ESI) m/z = 322.2 [M]+.
Ethyl 2-((2R,3R)-2-(3-Chlorophenyl)-3-(4-chlorophenyl)-5-
oxomorpholino)-2-cyclopropylacetate (44). To a solution of
(5R,6R)-6-(3-chlorophenyl)-5-(4-chlorophenyl)morpholin-3-one (43)
(4.3 g, 13.35 mmol) in DMF (26.7 mL) was added sodium hydride
(1.068 g, 26.7 mmol) at 0 °C, and the mixture was stirred at this
temperature for 30 min. To the mixture was added racemic ethyl 2-
bromo-2-cyclopropylacetate (3.71 mL, 26.7 mmol) in DMF (40 mL)
dropwise and was stirred at room temperature for 2 h. The reaction
was quenched with saturated aqueous NH4Cl (10 mL) and diluted
with diethyl ether (10 mL). The solution was washed with 10% citric
1
synthesis of 50a below and ref 34). H NMR (400 MHz, CDCl3) δ
7.28 (s, 5H), 7.06−7.17 (m, 3H), 5.10 (d, J = 4.70 Hz, 1H), 4.86 (d, J
= 4.70 Hz, 1H), 4.50 (t, J = 6.16 Hz, 1H), 3.74−3.98 (m, 1H), 2.90−
3.16 (m, J = 5.67 Hz, 2H), 2.01−2.08 (m, 1H), 1.35 (s, 9H), 1.09−
1.28 (m, 1H), 0.22−0.43 (m, 2H), −0.13 to 0.07 (m, 1H), −0.69 to
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−0.66 (bs, 1H). MS (ESI) m/z = 568.1 [M + H]+. HRMS (ESI) m/z
found 568.1331 [M + H]+, calcd for C27H32Cl2NO6S 568.1322.
(R)-1-((3,4-Dimethoxybenzyl)oxy)butan-2-ol (46). To a sus-
pension of sodium hydride (60% dispersion in mineral oil, 8.91 g, 223
mmol) in DMF (300 mL) at 60 °C was added dropwise a solution of
3,4-dimethoxybenzyl alcohol (29.4 mL, 202 mmol) in DMF (100 mL)
over a period of 30 min. The mixture was stirred at 60 °C for 0.5 h
until H2 evolution ceased. The mixture was cooled to 45 °C, and then
(R)-2-ethyloxirane (17.61 mL, 202 mmol) was added dropwise. The
mixture was allowed to stir at 45 °C overnight. The reaction mixture
was diluted with saturated aqueous NaHCO3 solution (300 mL) and
extracted with diethyl ether (3 × 300 mL). The organic extract was
washed with water (3 × 300 mL) and dried over MgSO4. The solution
was filtered and concentrated under reduced pressure. The residue was
adsorbed onto a plug of silica gel and purified by chromatography
through a silica gel column (330 g, RediSep Rf, Teledyne Isco,
Lincoln, NE), eluting with a gradient of 10−40% acetone in hexanes to
afforded 49 (87% yield, 89 mg). 1H NMR (400 MHz, CDCl3) δ
7.22−7.26 (m, 2H), 7.17−7.21 (m, 1H), 7.01−7.13 (m, 4H), 6.75 (d, J
= 7.83 Hz, 1H), 4.54 (d, J = 4.70 Hz, 2H), 4.35 (d, J = 2.74 Hz, 2H),
3.53−3.62 (m, 2H), 3.41 (q, J = 7.04 Hz, 1H), 3.26−3.37 (m, 1H),
1.76−1.92 (m, 1H), 1.41 (ddd, J = 6.06, 7.53, 13.79 Hz, 1H), 1.14 (t, J
= 6.94 Hz, 1H), 0.62−0.70 (m, 3H). MS (ESI) m/z = 394.2 [M + H]+.
(5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-
(ethylthio)butan-2-yl)morpholin-3-one (50a). To a solution of 49
(300 mg, 0.761 mmol) and ethanethiol (0.225 mL, 3.04 mmol) in 0.2
mL of toluene was added cyanomethylenetributylphosphorane (0.735
mL, 3.04 mmol). The resulting solution was stirred at 110 °C
overnight. After this period, the mixture was concentrated, and the
crude material absorbed onto a plug of silica gel and purified by
chromatography on silica gel, eluting with 20% acetone in hexanes,
provided 50a as a light yellow oil (85% yield, 283 mg). 1H NMR (400
MHz, CDCl3) δ 7.21−7.34 (m, 3H), 7.11−7.18 (m, 2H), 7.07 (d, J =
8.41 Hz, 2H), 6.79 (d, J = 7.83 Hz, 1H), 4.77 (d, J = 9.00 Hz, 1H),
4.56 (d, J = 9.00 Hz, 1H), 4.44 (q, J = 16.43 Hz, 2H), 3.06−3.15 (m,
2H), 2.44−2.62 (m, 3H), 2.04 (td, J = 7.36, 14.43 Hz, 1H), 1.56−1.68
(m, 1H), 1.23−1.31 (m, 3H), 0.66 (t, J = 7.43 Hz, 3H). MS (ESI) m/z
= 438.2 [M + H]+
1
provide 46 as an off-white oil (44% yield, 21.4 g). H NMR (400
MHz, CDCl3) δ 6.81−6.93 (m, 3H), 4.51 (s, 2H), 3.91 (d, J = 1.00
Hz, 6H), 3.69−3.82 (m, 1H), 3.52 (dd, J = 3.03, 9.49 Hz, 1H), 3.33
(dd, J = 8.02, 9.39 Hz, 1H), 1.44−1.58 (m, 2H), 0.98 (t, J = 7.53 Hz,
3H). MS (ESI) m/z = 263.2 [M + Na]+.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-3-oxo-
4-((S)-1-(N-phenylcyclopropanesulfonamido)butan-2-yl)-
morpholin-2-yl)acetic Acid (8). Compound 8 was prepared as a
single isomer from 49 (6% combined yield for the last three steps)
through procedures similar to those described for the synthesis of 5.
The isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, both solvents containing 0.1%
(R)-1-((3,4-Dimethoxybenzyl)oxy)butan-2-yl 4-
Bromobenzenesulfonate (47). To DMAP (14.54 g, 119 mmol)
and (R)-1-((3,4-dimethoxybenzyl)oxy)butan-2-ol (46) (13.0 g, 54.1
mmol) in CH2Cl2 (180 mL) was added 4-bromobenzene-1-sulfonyl
chloride (20.74 g, 81 mmol). The mixture was stirred at room
temperature overnight. The crude material was partitioned between
ethyl acetate (50 mL) and saturated aqueous sodium bicarbonate (100
mL). The organics were sequestered, and the aqueous portion was
extracted further with ethyl acetate (50 mL). The organics were
combined and washed with 0.1 M HCl (2 × 50 mL). The organics
were then washed with brine (50 mL), dried over MgSO4, filtered, and
the filtrate was concentrated under vacuum. The residue was adsorbed
onto a plug of silica gel and purified by chromatography through a
silica gel column (330 g, RediSep Rf, Teledyne Isco, Lincoln, NE),
eluting with a three-step gradient of 10−30% acetone in hexanes which
afforded 47 (78% yield, 19 g). 1H NMR (400 MHz, CDCl3) δ 7.75 (d,
J = 8.56 Hz, 2H), 7.59 (d, J = 8.31 Hz, 2H), 6.66−6.89 (m, 3H), 4.67
(quin, J = 5.62 Hz, 1H), 4.25−4.45 (m, 2H), 3.90 (s, 3H), 3.89 (s,
3H), 3.42−3.58 (m, 2H), 1.64−1.82 (m, 2H), 0.88 (t, J = 7.46 Hz,
3H). MS (ESI) m/z = 481.0 [M + Na]+.
(5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-((3,4-
dimethoxybenzyl)oxy)butan-2-yl)morpholin-3-one (48). A sol-
ution of 43 (260 mg, 0.807 mmol), (R)-1-((3,4-dimethoxybenzyl)-
oxy)butan-2-yl 4-bromobenzenesulfonate (47) (556 mg, 1.210 mmol),
and sodium tert-butoxide (116 mg, 1.210 mmol) in 1,4-dioxane (2
mL) was stirred at 85 °C overnight. After this period the reaction
mixture was poured into saturated aqueous NaHCO3 solution (10
mL) and brine (10 mL) and extracted with 2.5% MeOH in CH2Cl2 (3
× 30 mL). The combined organics were dried over sodium sulfate,
filtered, and the filtrate was concentrated under vacuum. Silica gel
chromatography (gradient elution with 10−40% acetone in hexanes)
afforded 48 (66% yield, 289 mg). 1H NMR (400 MHz, CDCl3) δ 7.29
(s, 4H), 7.21−7.25 (m, 1H), 7.18 (t, J = 1.66 Hz, 1H), 7.11 (t, J = 7.92
Hz, 1H), 7.06 (d, J = 8.41 Hz, 1H), 6.78−6.92 (m, 3H), 4.77 (d, J =
7.83 Hz, 1H), 4.58 (d, J = 7.83 Hz, 1H), 4.45 (s, 2H), 4.37−4.40 (m,
2H), 3.96 (t, J = 9.49 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 1.80−1.95
(m, J = 7.14, 14.77 Hz, 1H), 1.61 (bs, 2H), 1.64−1.72 (m, 1H), 0.63
(t, J = 7.53 Hz, 3H). MS (ESI) m/z = 566.2 [M + Na]+.
1
TFA, 30 min method) to provide 8 as the first eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.43−7.54 (m, 4H), 7.33−7.43 (m, 2H),
7.31 (d, J = 1.37 Hz, 1H), 7.16−7.27 (m, 2H), 6.95−7.14 (m, 3H),
4.79 (d, J = 12.52 Hz, 2H), 4.44−4.62 (m, 1H), 4.33 (s, 1H), 3.72−
3.92 (m, 1H), 2.91−3.16 (m, 3H), 2.30−2.47 (m, 1H), 1.83−2.07 (m,
1H), 1.45−1.67 (m, 1H), 1.06 (d, J = 4.70 Hz, 2H), 0.93 (d, J = 6.46
Hz, 2H), 0.51 (t, J = 7.53 Hz, 3H). MS (ESI) m/z = 631.1 [M + H]+.
HRMS (ESI) m/z found 631.1434 [M + H]+, calcd for
C31H33Cl2N2O6S 631.1431.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-(2-fluorophenyl)cyclopropanesulfonamido)butan-2-yl)-3-
oxomorpholin-2-yl)acetic Acid (9). Compound 9 was prepared as a
single isomer from 49 (16% combined yield for the last three steps)
through procedures similar to those described for the synthesis of 5.
The isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, both solvents containing 0.1%
1
TFA, 30 min method) to provide 9 as the first eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.52 (dt, J = 1.56, 7.82 Hz, 1H), 7.35−
7.43 (m, 1H), 7.29−7.34 (m, 2H), 7.26−7.28 (m, 1H), 7.25 (s, 1H),
7.14−7.24 (m, 5H), 7.07 (d, J = 7.43 Hz, 1H), 4.90−4.98 (m, 1H),
4.82−4.89 (m, 1H), 4.44 (t, J = 6.36 Hz, 1H), 4.34 (dd, J = 8.90, 14.77
Hz, 1H), 3.81 (dd, J = 4.60, 14.77 Hz, 1H), 3.15 (br s, 1H), 3.04 (d, J
= 6.46 Hz, 2H), 2.41−2.55 (m, 1H), 1.83−2.00 (m, J = 7.04, 8.22 Hz,
1H), 1.47−1.65 (m, J = 2.25, 7.14 Hz, 1H), 0.87−1.12 (m, 4H), 0.49
(t, J = 7.53 Hz, 3H). MS (ESI) m/z = 649.0 [M + H]+. HRMS (ESI)
m/z found 649.1349 [M + H]+, calcd for C31H32Cl2FN2O6S 649.1337.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-(4-fluorophenyl)cyclopropanesulfonamido)butan-2-yl)-3-
oxomorpholin-2-yl)acetic Acid (10). Compound 10 was prepared
as a single isomer from 49 (6% combined yield for the last three steps)
through procedures similar to those described for the synthesis of 5.
The isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, both solvents containing 0.1%
(5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-hy-
droxybutan-2-yl)morpholin-3-one (49). To a 0 °C solution of 48
(140 mg, 0.257 mmol) in CH2Cl2 (2.4 mL) and water (129 μL) was
added DDQ (70.0 mg, 0.309 mmol). The mixture was stirred at 0 °C.
After 2 h the reaction contents were poured into saturated aqueous
NaHCO3 solution (50 mL) and CH2Cl2 (30 mL). The layers were
separated, and the aqueous layer was extracted further with CH2Cl2 (2
× 20 mL). The combined organics were dried over sodium sulfate,
filtered, and the filtrate was concentrated under vacuum. Silica gel
chromatography (gradient elution 10−40% acetone in hexanes)
1
TFA, 30 min method) to provide 10 as the first eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.39−7.46 (m, 2H), 7.32 (d, J = 8.61 Hz,
2H), 7.19−7.28 (m, 3H), 7.10−7.18 (m, 4H), 7.06 (d, J = 7.43 Hz,
1H), 4.84 (td, J = 7.43, 9.19 Hz, 2H), 4.33−4.48 (m, J = 8.80 Hz, 2H),
3.80 (dd, J = 4.79, 14.38 Hz, 2H), 2.95−3.14 (m, 2H), 2.31−2.42 (m,
1H), 1.84−2.00 (m, 1H), 1.51−1.65 (m, 1H), 1.00−1.10 (m, 2H),
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0.89−1.00 (m, 2H), 0.50 (t, J = 7.53 Hz, 3H). MS (ESI) m/z = 649.0
[M + H]+. HRMS (ESI) m/z found 649.1336 [M + H]+, calcd for
C31H32Cl2FN2O6S 649.1337.
similar to those described for the synthesis of 5. The isomers at C2
were separated by reverse phase preparatory HPLC (Gemini Prep C18
5 μm column; Phenomenex, Torrance, CA; gradient elution of 25−
75% MeCN in water, both solvents containing 0.1% TFA, 30 min
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-(2,4-difluorophenyl)cyclopropanesulfonamido)butan-2-
yl)-3-oxomorpholin-2-yl)acetic Acid (11). Compound 11 (4%)
was prepared as a single isomer through procedures similar to those
described for the synthesis of 5. The isomers at C2 were separated by
reverse phase preparatory HPLC (Gemini Prep C18 5 μm column;
Phenomenex, Torrance, CA; gradient elution of 25−75% MeCN in
water, both solvents containing 0.1% TFA, 30 min method) to provide
1
method) to provide 15 as the first eluting isomer. H NMR (400
MHz, CDCl3) δ 7.32−7.39 (m, 3H), 7.30 (s, 2H), 7.21−7.27 (m, 2H),
7.17 (s, 1H), 6.99−7.07 (m, 1H), 5.07−5.17 (m, 1H), 4.95 (s, 1H),
4.74 (s, 1H), 3.11 (d, J = 6.06 Hz, 3H), 2.86−2.98 (m, 1H), 2.05−2.21
(m, 1H), 1.55−1.69 (m, 1H), 1.43 (dd, J = 4.30, 6.85 Hz, 6H), 0.56 (t,
J = 7.53 Hz, 3H). MS (ESI) m/z = 542.0 [M + H]+. HRMS (ESI) m/z
found 542.1177 [M + H]+, calcd for C25H30Cl2NO6S 542.1165.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(neopentylsulfonyl)butan-2-yl)-3-oxomorpholin-2-yl)acetic
Acid (16). Compound 16 was prepared as a single isomer from 49
(4% combined yield for the last three steps) through procedures
similar to those described for the synthesis of 5. The isomers at C2
were separated by reverse phase preparatory HPLC (Gemini Prep C18
5 μm column; Phenomenex, Torrance, CA; gradient elution of 25−
75% MeCN in water, both solvents containing 0.1% TFA, 30 min
1
11 as the first eluting isomer. H NMR (500 MHz, CDCl3) δ 7.38−
7.54 (m, 1H), 7.13−7.37 (m, 7H), 7.03−7.13 (m, 1H), 6.90−7.03 (m,
2H), 4.96 (d, J = 7.09 Hz, 1H), 4.87 (d, J = 7.34 Hz, 1H), 4.48 (t, J =
6.48 Hz, 1H), 4.20−4.34 (m, 1H), 3.69−3.82 (m, 1H), 3.20 (br s,
1H), 2.97−3.15 (m, 2H), 2.42−2.53 (m, 1H), 1.85−1.94 (m, 1H),
1.48−1.64 (m, 1H), 0.87−1.09 (m, 4H), 0.49 (t, J = 7.46 Hz, 3H). MS
(ESI) m/z = 667 [M + H]+.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-(2-fluorophenyl)propan-2-ylsulfonamido)butan-2-yl)-3-
oxomorpholin-2-yl)acetic Acid (12). Compound 12 was prepared
as a single isomer from 49 (28% combined yield for the last three
steps) through procedures similar to those described for the synthesis
of 5. The isomers at C2 were separated by reverse phase preparatory
HPLC (Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA;
gradient elution of 25−75% MeCN in water, both solvents containing
0.1% TFA, 30 min method) to provide 12 as the first eluting isomer.
1H NMR (400 MHz, CDCl3) δ 7.67 (dt, J = 1.47, 7.97 Hz, 1H), 7.34−
7.43 (m, J = 1.47, 4.79 Hz, 1H), 7.31 (d, J = 8.41 Hz, 2H), 7.26−7.28
(m, 1H), 7.13−7.25 (m, 6H), 7.04 (d, J = 7.63 Hz, 1H), 4.89−4.98
(m, 1H), 4.77−4.87 (m, 1H), 4.37 (t, J = 6.16 Hz, 2H), 3.78 (dd, J =
4.50, 14.87 Hz, 1H), 2.92−3.28 (m, 4H), 1.90 (quint, J = 7.53, 15.06
Hz, 1H), 1.47−1.63 (m, 1H), 1.39 (d, J = 6.85 Hz, 6H), 0.45 (t, J =
7.53 Hz, 3H). MS (ESI) m/z = 651.2 [M + H]+.
1
method) to provide 16 as the first eluting isomer. H NMR (400
MHz, CDCl3) δ 7.23−7.28 (m, 2H), 7.21 (s, 3H), 7.14−7.17 (m, 1H),
7.05−7.12 (m, 1H), 6.95 (d, J = 7.63 Hz, 1H), 5.00 (d, J = 6.46 Hz,
1H), 4.85 (d, J = 6.65 Hz, 1H), 4.60 (t, J = 6.06 Hz, 1H), 3.82−3.97
(m, 1H), 3.01 (d, J = 6.46 Hz, 2H), 2.77−2.95 (m, 3H), 1.95−2.10
(m, 1H), 1.41−1.56 (m, 1H), 1.16 (s, 10H), 0.38−0.52 (m, 3H). MS
(ESI) m/z = 570.2 [M + H]+.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(cyclopentylsulfonyl)butan-2-yl)-3-oxomorpholin-2-yl)acetic
Acid (17). Compound 17 was prepared as a single isomer from 49
(12% combined yield for the last three steps) through similar
procedures to those described for the synthesis of 5. The isomers at
C2 were separated by reverse phase preparatory HPLC (Gemini Prep
C18 5 μm column; Phenomenex, Torrance, CA; gradient elution of
25−75% MeCN in water, both solvents containing 0.1% TFA, 30 min
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-(2-fluorophenyl)methylsulfonamido)butan-2-yl)-3-oxo-
morpholin-2-yl)acetic Acid (13). Compound 13 was prepared as a
single isomer from 52 (20% combined yield for the last two steps)
through procedures similar to those described for the synthesis of 5.
The isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, both solvents containing 0.1%
1
method) to provide 17 as the first eluting isomer. H NMR (400
MHz, CDCl3) δ 7.22 (ddd, J = 6.46, 8.61, 12.52 Hz, 5H), 7.12−7.17
(m, J = 1.17, 1.96 Hz, 1H), 7.08 (t, J = 7.82 Hz, 1H), 6.95 (d, J = 7.63
Hz, 1H), 5.02 (d, J = 6.65 Hz, 1H), 4.85 (d, J = 6.65 Hz, 1H), 4.62 (t, J
= 6.06 Hz, 1H), 3.89 (dd, J = 9.00, 13.50 Hz, 1H), 3.19−3.39 (m, 2H),
2.98−3.04 (m, 2H), 2.84 (dd, J = 2.64, 13.99 Hz, 1H), 1.87−2.12 (m,
5H), 1.75 (br s, 2H), 1.60 (d, J = 4.11 Hz, 3H), 0.46 (t, J = 7.53 Hz,
3H). MS (ESI) m/z = 568.1 [M + H]+. HRMS (ESI) m/z found
568.1332 [M + H]+, calcd for C27H33Cl2NO6S 568.1322.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-3-oxo-
4-((S)-1-(phenylsulfonyl)butan-2-yl)morpholin-2-yl)acetic Acid
(18). Compound 18 was prepared as a single isomer from 49 (37%
combined yield for the last three steps) through procedures similar to
those described for the synthesis of 5. The isomers at C2 were
separated by reverse phase preparatory HPLC (Gemini Prep C18 5 μm
column; Phenomenex, Torrance, CA; gradient elution of 25−75%
MeCN in water, both solvents containing 0.1% TFA, 30 min method)
to provide 18 as the first eluting isomer. 1H NMR (400 MHz, CDCl3)
δ 7.96 (d, J = 7.24 Hz, 2H), 7.56−7.77 (m, 3H), 7.14−7.46 (m, 7H),
7.02 (d, J = 7.63 Hz, 1H), 4.88−5.12 (m, 2H), 4.63 (dd, J = 4.89, 6.85
Hz, 1H), 4.21 (dd, J = 9.39, 14.28 Hz, 1H), 3.42 (br s, 1H), 2.98−3.21
(m, 3H), 2.09 (ddd, J = 7.43, 9.44, 14.23 Hz, 1H), 1.57 (ddd, J = 4.11,
7.58, 13.94 Hz, 1H), 0.48 (t, J = 7.43 Hz, 3H). MS (ESI) m/z = 576.0
[M + H]+. HRMS (ESI) m/z found 576.1020 [M + H]+, calcd for
C28H28Cl2NO6S 576.1009.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N,N-dimethylsulfamoyl)butan-2-yl)-3-oxomorpholin-2-yl)-
acetic Acid (19). Compound 8 was prepared as a single isomer from
42 (>1% combined yield from 42) through procedures similar to those
described for the synthesis of 5. 1H NMR (400 MHz, CDCl3) δ 7.31−
7.36 (m, 2H), 7.20−7.26 (m, 4H), 7.12−7.19 (m, 1H), 7.01 (d, J = 7.6
Hz, 1H), 5.03 (d, J = 7.0 Hz, 1H), 4.93 (d, J = 7.0 Hz, 1H), 4.77 (t, J =
6.0 Hz, 1H), 3.87 (dd, J = 13.7, 9.4 Hz, 1H), 3.29 (br s, 1H), 3.11 (d, J
= 6.1 Hz, 2H), 2.89 (s, 3H), 2.89 (s, 3H), 2.82−2.87 (m, 1H), 2.05−
2.20 (m, 1H), 1.63 (ddd, J = 13.9, 7.7, 4.2 Hz, 1H), 0.56 (t, J = 7.5 Hz,
3H). MS (ESI) m/z = 543.2 [M + H]+.
1
TFA, 30 min method) to provide 13 as the first eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.46−7.52 (m, J = 1.57 Hz, 1H), 7.35−
7.44 (m, 1H), 7.32 (d, J = 8.41 Hz, 3H), 7.14−7.26 (m, 6H), 7.05 (d, J
= 7.63 Hz, 1H), 4.89−4.98 (m, 1H), 4.87 (s, 1H), 4.41 (d, J = 12.72
Hz, 1H), 4.29 (dd, J = 8.71, 14.77 Hz, 1H), 3.73 (dd, J = 4.70, 14.87
Hz, 1H), 3.03 (dd, J = 3.23, 6.36 Hz, 3H), 2.93 (br s, 3H), 1.80−1.98
(m, 1H), 1.49−1.64 (m, 1H), 0.48 (t, J = 7.53 Hz, 3H). MS (ESI) m/z
= 623.1 [M + H]+.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(ethylsulfonyl)butan-2-yl)-3-oxomorpholin-2-yl)acetic Acid
(14). Compound 14 was prepared as a single isomer from 49 (14%
combined yield for the last three steps) through procedures similar to
those described for the synthesis of 5. The isomers at C2 were
separated by reverse phase preparatory HPLC (Gemini Prep C18 5 μm
column; Phenomenex, Torrance, CA; gradient elution of 25−75%
MeCN in water, both solvents containing 0.1% TFA, 30 min method)
to provide 14 as the first eluting isomer. 1H NMR (400 MHz, CDCl3)
δ 7.33−7.38 (m, 2H), 7.25 (td, J = 2.15, 3.91 Hz, 4H), 7.14−7.21 (m,
1H), 7.02 (d, J = 7.63 Hz, 1H), 5.09 (d, J = 7.04 Hz, 1H), 4.95 (d, J =
7.04 Hz, 1H), 4.79 (t, J = 5.87 Hz, 1H), 4.05 (br s, 1H), 3.44 (br s,
1H), 3.01−3.16 (m, 4H), 2.97 (d, J = 12.91 Hz, 1H), 2.14 (ddd, J =
7.34, 9.44, 14.13 Hz, 1H), 1.60 (ddd, J = 4.21, 7.53, 13.89 Hz, 1H),
1.38−1.50 (m, 3H), 0.57 (t, J = 7.43 Hz, 3H). MS (ESI) m/z = 528.0
[M + H]+. HRMS (ESI) m/z found 528.1021 [M + H]+, calcd for
C24H28Cl2NO6S 528.1009.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(isopropylsulfonyl)butan-2-yl)-3-oxomorpholin-2-yl)acetic
Acid (15). Compound 15 was prepared as a single isomer from 49
(3% combined yield for the last three steps) through procedures
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2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(oxetan-3-ylsulfonyl)butan-2-yl)-3-oxomorpholin-2-yl)acetic
Acid (20). Compound 20 was prepared as a single isomer from 39b
(10% combined yield for the six three steps) through procedures
similar to those described for the synthesis of 5 in Scheme 3. The
isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, both solvents containing 0.1%
residue was purified by flash chromatography on silica gel (12 g
column, gradient elution with 5−70% ethyl acetate in hexanes) to
1
provide 55 (38% yield, 75 mg). H NMR (400 MHz, CDCl3) (1.5:1
mixture of isomers at C2) δ 7.33−7.19 (m, 5.5H), 7.10−7.02 (m,
5.5H), 6.82 (d, J = 8 Hz, 1H), 5.98−5.93 (m, 2H), 5.24−5.11 (m,
4H), 4.72 (m, J = 12H, 1H), 4.61 (m, J = 12 Hz, 2H), 4.40−4.36 (m,
2H), 2.97 (s, 4.5H), 2.93 (s, 3H), 2.40−2.22 (m, 2H), 1.96−1.94 (m,
2H), 1.69−1.61 (m, 1.5H), 1.27−1.10 (m, 3.5H), 0.99−0.96 (m,
3.5H), 0.67−0.57 (m, 5H). MS (ESI) m/z = 551.0 [M + H]+.
2-((2R,5R,6R)-6-(3-Chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(N-methylcyclopropanesulfonamido)butan-2-yl)-3-oxomor-
pholin-2-yl)acetic Acid (7). Compound 7 was prepared as a single
isomer from 55 (39% yield) through a procedure similar to that
described for the synthesis of 5. The isomers at C2 were separated by
reverse phase preparatory HPLC (Gemini Prep C18 5 μm column;
Phenomenex, Torrance, CA; gradient elution of 25−75% MeCN in
water, where both solvents contain 0.1% TFA, 30 min method) to
provide 7 as the faster eluting isomer. 1H NMR (400 MHz, CDCl3) δ
7.28−7.38 (m, 4H), 7.14−7.27 (m, 4H), 4.82−4.98 (m, 2H), 4.71 (t, J
= 6.16 Hz, 1H), 3.11 (dd, J = 6.26, 9.00 Hz, 3H), 2.88 (s, 3H), 2.33 (s,
1H), 1.77−1.96 (m, 1H), 1.53−1.73 (m, 1H), 1.08−1.29 (m, 2H),
0.85−1.07 (m, 4H), 0.57 (t, J = 7.53 Hz, 3H). MS (ESI) m/z = 569.2
[M + H]+. HRMS (ESI) m/z found 569.1279 [M + H]+, calcd for
C26H31Cl2NO6S 569.1274.
1
TFA, 30 min method) to provide 20 as the first eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.35 (s, 2H), 7.30 (s, 2H), 7.23−7.26 (m,
2H), 7.19 (t, J = 7.82 Hz, 1H), 7.04 (d, J = 7.63 Hz, 1H), 4.86−5.13
(m, 5H), 4.74 (dd, J = 5.67, 6.65 Hz, 1H), 4.33−4.45 (m, 1H), 3.91−
4.05 (m, 1H), 3.39−3.54 (m, 1H), 3.04−3.19 (m, 2H), 2.86 (d, J =
16.43 Hz, 1H), 2.04−2.17 (m, 1H), 1.50−1.70 (m, 1H), 0.58 (t, J =
7.43 Hz, 3H). MS (ESI) m/z = 556.0 [M + H]+. HRMS (ESI) m/z
found 556.0966 [M + H]+, calcd for C25H28Cl2NO7S 556.0958.
(2S)-2-((5R,6R)-2-Allyl-6-(3-chlorophenyl)-5-(4-chlorophen-
yl)-3-oxomorpholino)butanal (53). To a solution of a 2:1.3
mixture of (2R,5R,6R)-2-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-
4-((S)-1-hydroxybutan-2-yl)morpholin-3-one and (2S,5R,6R)-2-allyl-
6-(3-chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-hydroxybutan-2-yl)-
morpholin-3-one (52) (163 mg, 0.375 mmol, synthesized from 48
through procedures similar to those described in Scheme 5) in DCM
(1.8 mL) was added Dess−Martin periodinane (175 mg, 0.413 mmol)
and water (7.44 μL, 0.413 mmol). After 30 min, saturated aqueous
sodium thiosulfate was added. The mixture was extracted with DCM.
The combined organic layers were washed with saturated aqueous
NaHCO3, dried over Na2SO4, and concentrated to provide 53 (>98%
yield, 159 mg). 1H NMR (400 MHz, CDCl3) (1.5:1 mixture of
isomers at C2) δ 9.54 (s, 1H), 9.42 (s, 1.5H), 8.27 (d, J = 12 Hz, 1H),
8.01 (d, J = 12 Hz, 1H), 7.94 (t, J = 6.3 Hz, 1H), 7.36 (t, J = 6 Hz,
1H), 7.34−7.22 (m, 10H), 7.16−7.05 (m, 7.5H), 6.71 (d, J = 8 Hz,
1.5H), 5.99−5.93 (m, 2.5H), 5.31−5.16 (m, 6H), 4.96 (d, J = 8 Hz,
1H), 4.75 (d, J = 12 HZ, 1.5H), 4.71 (d, J = 8 Hz, 1H), 4.70−4.68 (M,
1H), 4.57 (d, J = 12 Hz, 1.5H), 4.37 (m, 1H), 3.80 (t, J = 8 Hz, 1.5H),
3.59 (m, J = 8 Hz, 1H), 2.84−2.80 (m, 5H), 2.23−2.15 (m, 2.5H),
0.84−0.79 (m, 7.5H). MS (ESI) m/z = 432.2 [M + H]+.
(5R,6R)-2-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-4-((S)-
1-(methylamino)butan-2-yl)morpholin-3-one (54). To a solution
of (2S)-2-((5R,6R)-2-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-
oxomorpholino)butanal (53) (188 mg, 0.435 mmol) in DCE (4
mL) were added methanamine (1.305 mL, 2.61 mmol) 2.0 M solution
in THF, sodium triacetoxyborohydride (276 mg, 1.305 mmol), and 3
drops of HOAc. After being stirred overnight, the reaction mixture was
quenched with water and saturated aqueous NaHCO3. The reaction
mixture was extracted with ethyl acetate. The organic layer was washed
with brine, dried over Na2SO4, and concentrated to provide (5R,6R)-
2-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-
(methylamino)butan-2-yl)morpholin-3-one (54) (83% yield, 162 mg),
used without further purification. 1H NMR (400 MHz, CDCl3) (1.5:1
mixture of isomers at C2) δ 7.34−7.22 (m, 8H), 7.13−7.09 (m, 4H),
6.71 (d, J = 12 Hz, 1H), 6.05−5.87 (m, 1.5H), 5.31−5.15 (m, 4H),
4.90−4.88 (m, 1H), 4.61 (d, J = 8 Hz, 1.5H), 4.50 (t, J = 8 Hz, 1H),
3.74 (s, 1H), 2.83−2.79 (m, 3H), 2.42 (m, J = 12 Hz, 6H), 1.89−1.75
(m, 2H), 1.66−1.52 (m, 3H), 0.71−0.64 (m, 7.5H). MS (ESI) m/z =
447.1 [M + H]+.
2-((2R,5R,6R)-4-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-6-(3-
chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpholin-
2-yl)acetic Acid (6). Compound 6 was prepared from 42b (10%
combined yield for the last three steps) as a single isomer through
procedures similar to those described for the synthesis of 5. The
isomers at C2 were separated by reverse phase preparatory HPLC
(Gemini Prep C18 5 μm column; Phenomenex, Torrance, CA; gradient
elution of 25−75% MeCN in water, where both solvents contain 0.1%
1
TFA, 30 min method) to provide 6 as the faster eluting isomer. H
NMR (400 MHz, CDCl3) δ 7.27−7.34 (m, 2H), 7.08 (d, J = 16.43 Hz,
5H), 6.81 (d, J = 7.83 Hz, 1H), 5.07 (q, J = 9.59 Hz, 2H), 4.01 (dd, J =
10.56, 13.30 Hz, 1H), 3.32 (dd, J = 7.24, 9.39 Hz, 2H), 3.18 (d, J =
15.85 Hz, 1H), 3.01 (d, J = 15.65 Hz, 1H), 2.80 (d, J = 12.32 Hz, 1H),
2.12−2.33 (m, 1H), 1.69−1.78 (m, 3H), 1.43 (s, 9H), 0.53 (t, J = 7.43
Hz, 3H). MS (ESI) m/z = 570.2 [M + H]+. HRMS (ESI) m/z found
570.1479 [M + H]+, calcd for C27H34Cl2NO6S 570.1478.
ASSOCIATED CONTENT
* Supporting Information
■
S
(i) In vitro biological assays, (ii) in vivo protocols, and (iii)
determination of cocrystal structures of 4 bound to MDM2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
The coordinates of 4 with MDM2 have been deposited in the
PDB with accession code 4OBA.
AUTHOR INFORMATION
Corresponding Author
■
*Telephone: 650-244-2654. Fax: 650-837-9369. E-mail:
anagonza@amgen.com.
N-((2S)-2-((5R,6R)-2-Allyl-6-(3-chlorophenyl)-5-(4-chloro-
phenyl)-3-oxomorpholino)butyl)-N-methylcyclopropane-
sulfonamide (55). To a solution of (2R,5R,6R)-2-allyl-6-(3-
chlorophenyl)-5-(4-chlorophenyl)-4-((S)-1-(methylamino)butan-2-
yl)morpholin-3-one and (2S,5R,6R)-2-allyl-6-(3-chlorophenyl)-5-(4-
chlorophenyl)-4-((S)-1-(methylamino)butan-2-yl)morpholin-3-one
(54) (162 mg, 0.362 mmol) and pyridine (0.070 mL, 0.869 mmol) in
DCM (4 mL) was added cyclopropanesulfonyl chloride (0.044 mL,
0.435 mmol). After 90 min, DMAP (5.0 mg, 0.041 mmol) was added.
After 1 h, cyclopropanesulfonyl chloride (0.044 mL, 0.435 mmol) and
pyridine (0.070 mL, 0.869 mmol) were added. After being stirred for 3
days, the mixture was quenched with water and extracted with DCM
(2×), and saturated aqueous NH4Cl was added. The combined
organic layers were dried over Na2SO4, and concentrated. The crude
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AcCN, acetonitrile; Boc, tert-butoxycarbonyl; BrdU, 5-bromo-
2-deoxyuridine; CL, clearance; CYP3A4, cytochrome P450
3A4; DCM, dichloromethane; DMBCl, 2,4-dimethoxybenzyl
chloride; DMF, N,N-diemethylformamide; DMP, Dess−Martin
periodinane; DMSO, dimethylsulfoxide; dr, diastereoselectivity
ratio; EdU, 5-ethynyl-2′-deoxyuridine; EtOAc, ethyl acetate;
FACS, fluorescence-activated cell sorting; hPXR, human
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Journal of Medicinal Chemistry
Article
D.; LaFrance, L. V.; Milkiewicz, K. L.; Calvo, R. R.; Maguire, D.;
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Zhang, M.; Manthey, C. L.; Petrella, E. C.; Pantoliano, M. W.;
Deckman, I. C.; Spurlino, J. C.; Maroney, A. C.; Tomczuk, B. E.;
Molloy, C. J.; Bone, R. F. Discovery and co-crystal structure of
benzodiazepinedione HDM2 antagonists that activate p53 in cells. J.
Med. Chem. 2005, 48, 909−912. (f) Huang, Y.; Wolf, S.; Bista, M.;
Meireles, L.; Camacho, C.; Holak, T. A.; Domling, A. 1,4-
Thienodiazepine-2,5-diones via MCR (I): synthesis, virtual space
and p53-Mdm2 activity. Chem. Biol. Drug Des. 2010, 76, 116−129.
Spiro-oxoindoles: (g) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Wang,
G.; Qiu, S.; Shangary, S.; Gao, W.; Qin, D.; Stuckey, J.; Krajewski, K.;
Roller, P. P.; Wang, S. Structure-based design of spiro-oxindoles as
potent, specific small-molecule inhibitors of the MDM2-p53
interaction. J. Med. Chem. 2006, 49, 3432−3435. (h) Yu, S.; Qin,
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Preparation of Spiro-oxindole Derivatives as MDM2 Antagonists.
pregnane X receptor; HTRF, homogeneous time-resolved
fluorescence; LiHMDS, lithium bis(trimethylsilyl)amide;
MDM2, murine double minute 2; MsCl, methanesulfonyl
chloride; NaHMDS, sodium bis(trimethylsilyl)amide; NMO,
N-methylmorpholinine N-oxide; q.d., once a day dosing; qRT-
PCR, quantitative reverse transcription polymerase chain
reaction; SEM, standard error of mean; SPR, surface plasmon
resonance; TBAF, tetrabutylammonium fluoride; TDI, time
dependent inhibition; TEA, triethylamine; TES, triethylsilyl;
TESCl, triethylsilyl chloride; TFA, trifluoroacetic acid; THF,
tetrahydrofuran
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