Novel inhibitors of the MDM2-p53 interaction featuring hydrogen bond acceptors as carboxylic acid isosteres

Article abstract of DOI:10.1021/jm401911v

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction these inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC₅₀ = 0.1 nM) and cellular potency (SJSA-1 EdU IC₅₀ = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED₅₀ of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

Full text of DOI:10.1021/jm401911v

Article
pubs.acs.org/jmc
Novel Inhibitors of the MDM2-p53 Interaction Featuring Hydrogen
Bond Acceptors as Carboxylic Acid Isosteres
Ana Z. Gonzalez,*,† Zhihong Li,† Hilary P. Beck,† Jude Canon,⊥ Ada Chen,† David Chow,†
Jason Duquette,† John Eksterowicz,† Brian M. Fox,† Jiasheng Fu,† Xin Huang,# Jonathan Houze,†
Lixia Jin,§ Yihong Li,† Yun Ling,§ Mei-Chu Lo,† Alexander M. Long,# Lawrence R. McGee,†
Joel McIntosh,† 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,† Daqing Sun,† and Julio C. Medina†
‡
§
†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 potent and selective morpholinone and piperidinone inhibitors of the
MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction
with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these
acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic
acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable
biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic
properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11
mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2
protein are described herein.
that bind to MDM2 at its p53 active site, impeding MDM2’s
interaction with p53. This results in increased levels of
unbound p53 and reactivation of its pathways. From these
efforts, several inhibitors have recently emerged and are now
being tested in the clinic, most of which were registered within
the past two years.9−13 Consequently, this is arguably one of
the most exciting times in over 30 years of research on the p53
pathway.13−17
INTRODUCTION
■
Activation of the pro-apoptotic protein p53 is a promising and
highly sought out approach toward cancer treatment.1 Upon
cellular stress, p53 activation leads to the transcription of
multiple downstream genes that regulate cell cycle control,
apoptosis, DNA repair, and senescence.2−4 About half of all
human cancers progress either by mutation or deletion of p53.5
However, for the remaining half that retains wild-type p53
activity, survival is achieved by other mechanisms such as
upregulation of its natural antagonists. Amplified in many
tumor tissues, MDM2 (murine double minute 2) has been
identified as p53’s main negative regulator.6−8 Many research
programs, including our own, aim to design small molecules
Recently, we reported on a series of piperidinone-derivatives
as potent and selective inhibitors of the MDM2-p53
Received: December 12, 2013
Published: March 6, 2014
© 2014 American Chemical Society
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interaction. Our optimization efforts within this series led to the
discovery of AMG 232 (1, Figure 1), which is currently being
binding affinity.18e The amide of the oxindole-derived MDM2
inhibitors was reported to engage in similar interactions with
His96.16,18e Thus, we set out to explore this and other acid
replacements within our series. In this article, we described our
efforts to identify acid replacements, which led to the discovery
of 4, an exquisitely potent thiazolyl-containing inhibitor of the
MDM2-p53 interaction. Inhibitor 4 also exhibits excellent
pharmacokinetic properties (hHep CLint = 5.5 μL/min/106
cells) and in vivo efficacy in the SJSA-1 osteosarcoma xenograft
model (ED50 = 11 mg/kg).
CHEMISTRY
■
The route to the isosteres of acid 5, compounds 6−8, and 12 is
depicted in Scheme 1. Many acid-containing heterocycles were
a
Scheme 1
a
Reagents and conditions: (a) trifluoromethanesulfonamide, DIEA,
HATU, DMF, rt, 5 h, 41%; (b) N-methylmorpholine, 28% NH3/H2O,
isobutyl chloroformate, THF, 0 °C, 3 h, 75%; (c) trifluoroacetic
anhydride, TEA, 0 °C, 1 h, 92%; (d) NaN3, DMF, 90 °C, 5 days, 65%;
(e) NaHCO3, NH4OH, MeOH, 70 °C, 12 h, quant.; (f) CDI, DBU,
dioxane, 100 °C, 2 days, 15% (last two steps).
synthesized from common nitrile intermediate 53 (Scheme 2).
From 53, addition of ethanol under acidic conditions resulted
in imidate 54 that was taken directly to iminium 55 by the
addition of ammonia. Intermediate 55 can be transformed to
either imidazole inhibitor 45 or pyrimidine 27 through the
sequential reaction of 55 with ethyl-3-oxo-propionate or 4-
chloroacetic acid methyl ester, respectively, followed by
saponification.
Alternatively, nitrile intermediate 53 could be converted to
thioamide 56 with phosphorus pentasulfide. From 56, a variety
of thiazole isomers could be synthesized by the addition of the
corresponding ester reagent followed by saponification of the
resulting ester to give inhibitors 32, 33, and 37.
Synthesis of pyrazole inhibitor 31 (Scheme 3) commenced
by C3-alkylation of piperidinone intermediate 57 with (2-
chloromethyl)ethyltrimethylsilane giving ether 58. Intermediate
58 was then transformed to primary alcohol 59 with boron
trifluoride and then to mesylate 60 by reaction with
methanesulfonic anhydride. The mesylate moiety of 60 could
then be displaced with hydrazine to give 61 that was converted
to pyrazole 62 by the addition of ethyl-2-formyl-3-oxo-
Figure 1. Co-crystal structures of potent piperidinone inhibitors
bound to human MDM2 (6−110) highlighting their interaction to
MDM2-His96. Coordinates for compounds 2 (PDB code: 4OAB), 3
(PDB code: 4OGN), and 4 (PDB code: 4ODE) bound to MDM2
have been deposited in the PDB.
tested in the clinic.17,18 Inhibitor 1 is highly potent (Kd = 0.045
nM; SJSA-1 EdU IC50 = 9.1 nM)19 with remarkable
pharmacokinetic properties (hHep CLint = 6.3 μL/min/106
cells) and in vivo efficacy in the SJSA-1 osteosarcoma xenograft
model (ED50 = 9.1 mg/kg).17,20 Because 1 is among the most
potent inhibitors of the MDM2/p53 interaction reported to
date and has good pharmacokinetic properties in preclinical
models, with this work, we aim to identify compounds with
distinct metabolic profiles to 1 should a compound with a
different metabolite profile or clearance route is needed.
Crystallographic analysis of analogous inhibitors such as 2
bound to MDM2 shows that the carboxylate interacts with the
imidazole of the His96 side chain of MDM2.20 This unique
interaction resulted in a nearly 100-fold improvement in
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a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) 1-(cyanomethyl)tetrahydro-1-H-thio-
a
Reagents and conditions: (a) (2-chloromethoxy)ethyltrimethylsilane,
LDA, THF, −78 °C, 1 h, 52%; (b) BF3−OEt2, DCM, 0 °C, 3 h, 33%;
(c) methanesulfonic anhydride, NEt3, DCM, 30 min, rt, quant.; (d)
hydrazine, EtOH, 90 °C, 12 h, 40% conversion; (e) ethyl-2-formyl-3-
oxo-propionate, 100 °C, DMA, 2 h, 38%; (f) mCPBA, DCM, 0 °C, 30
min, 30%; (g) LiOH, MeOH/THF/H2O, 50 °C, 1 h, 30% (last two
steps).
phene-1-ium bromide, DIEA, HATU, DCM, rt, 4 h, quant.; then,
oxone, DMF/H2O, rt, 3 h, 64%; (b) NH3, DIEA, HATU, 55 °C, 1 h,
88%; (c) trifluoroacetic anhydride, TEA, 0 °C, 1 h, 90%; (d) HCl,
ethanol, 1 h, rt, quant.; (e) ethyl 3-hydrazinyl-3-oxopropanoate, EtOH,
12 h, 63%; (f) LiOH, MeOH/THF/H2O, 50 °C, 1 h; (g) NH3,
ethanol, 12 h, rt, 100%; (h) ethyl trans-4-oxo-2-butenoate, 130 °C, 1 h,
8%; (i) ethyl-2-formyl-3-oxo-propionate, DMA, 100 °C, 2 h, 66%; (j)
P2S5, ethanol, 70 °C, 12 h, 66%; (k) 4-chloroacetic acid methyl ester,
EtOH, 90 °C, 3 h, 57% (both steps); (l) ethyl-2-chloro-3-
oxopropanoate, toluene, 100 °C, 5 h, 66%; (m) ethyl bromo pyruvate,
dioxane, 3 h, rt; then, pyridine and TFA, 12 h, rt, 22%; (n) oxalyl
chloride, DMF, 1 h; then, tris(trimethylsilyloxy)ethylene, 90 °C, 12 h,
10%.
either methyl iodide or 1,2-dibromoethane resulted in
inhibitors 39 and 40 after saponification of the ethyl ester.
Alternatively, coupling of acid 69 with glycine methyl ester
followed by saponification provided intermediate 70 (Scheme
6). Addition of methyl potassium malonate to 70 followed by
decarboxylation resulted in the formation of β-keto ester 71
that, similar to 67, can undergo cyclization to thiazole 72 in the
presence of Lawesson’s reagent. Reaction of 72 with N-
fluorobenzenesulfonimide resulted in fluorination at the α
carbon to the ester, but the reaction was sluggish and not
chemoselective providing a mixture of 41 and 42 that could be
separated via preparative HPLC. Oxazole 43 was formed from
cyclization of 71 in the presence of Burgess’ reagent.
Morpholinone inhibitors 46 and 47 were synthesized starting
from C2-Me morpholinone 73 (Scheme 7). Alkylation with
methyl (6-bromomethyl)nicotinate yields C2-disubstituted 74
as a nearly 1:1 mixture of epimers at C2. Deprotection of
silylether, Mitsunobu reaction, and oxidation of the resulting
thioether to the corresponding sulfone produced 76 that can be
easily transformed to a mixture of 46 and 47 by saponification.
The isomers were separated by preparative HPLC.
propionate. Finally, inhibitor 31 was obtained through
oxidation of the tert-butyl thioether to its corresponding
sulfone followed by saponification of the ethyl ester to the acid.
Addition of triphenylphosphine and carbon tetrabromide to
alcohol 59 yields alkylbromide 63 (Scheme 4). This
intermediate undergoes displacement by 2-mercaptopyrimidine
to form thioether 64. Oxidation of 64 to bis-sulfone 65
proceeded smoothly with mCPBA. Finally, inhibitor 14 was
formed by reaction of 65 with hydroxylamine-O-sulfonic acid.
Highlighted in Schemes 5 and 6 are the synthetic routes used
to access some of our most potent thiazole inhibitors. Coupling
between acid 9 and ethyl 4-(bis(trimethylsilyl)amino)but-2-
ynoate in the presence of TBAF produced propargylamide 67
that could then undergo one pot cyclization to thiazole 68 by
refluxing in toluene in the presence of Lawesson’s reagent
(Scheme 5). From 68, alkylation α to the ester moiety with
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a
a
Scheme 4
Scheme 6
a
a
Reagents and conditions: (a) glycine methyl ester hydrochloride,
EDC, HOAt, NaHCO3, DMF, 40 °C, 3 h, quant.; (b) LiOH, MeOH/
THF/H2O, 50 °C, 1 h; (c) MgCl2, CDI, methyl potassium malonate,
50 °C, 12 h, 63%; (d) Lawesson’s reagent, toluene, 100 °C, 3 h, 71%;
(e) Burgess’ reagent, DCE, 120 °C, 7 h, 62%; (f) N-fluorobenzene-
sulfonimide, NaOtBu, DMF, 0 °C, 1 h; then step b, 41, 11%; 42, 4%.
Reagents and conditions: (a) triphenylphosphine, CBr4, MeCN, 55
°C, 5 h, 86%; (b) 2-mercaptopyrimidine, K2CO3, DMF, rt, 12 h; (c)
oxone, THF/H2O, rt, 12 h, 48% (last two steps); (d) hydroxylamine-
O-sulfonic acid, K2CO3, MeOH, 12 h, rt, 46%.
a
Scheme 5
a
Scheme 7
a
Reagents and conditions: (a) oxalyl chloride, THF, DMF, rt, 1 h,
a
quant.; (b) ethyl 4-(bis(trimethylsilyl)amino)but-2-ynoate, TBAF,
THF, rt, 12 h; then, HCl/water, 72%; (c) Lawesson’s reagent,
toluene, 60 °C, 3 h, 63%; (d) LiOH, MeOH/THF/H2O, 50 °C, 1 h;
(f) MeI, NaOtBu, DMF, 0 °C, 30 min, 62%; (e) 1,2-dibromoethane,
NaOtBu, DMF, 0 °C, 30 min, 80%.
Reagents and conditions: (a) methyl (6-bromomethyl)nicotinate,
LDA, THF, −78 °C, 2 h, 78%, 1:1 d.r.; (b) TBAF, THF, rt, 12 h, 60%;
(c) BuSH, cyanomethyltri-N-butylphosphorane, 110 °C, 12 h, 61%;
(d) mCPBA, DCM, 30 min, 0 °C, quant.; (e) LiOH, MeOH/THF/
H2O, 50 °C, 1 h.
t
Finally, many of the nicotinic acid analogues were
synthesized through simple alkylation of 57 with the
corresponding alkylbromide (Scheme 8). Alkylation with 5-
bromo-2-(bromomethyl)pyridine results in 78 that can be
functionalized to inhibitors such as 23 through standard Pd-
mediated coupling conditions.
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a
pyrrolidineamide moiety of 15 and 16 bind to MDM2 through
a hydrogen bond interaction with MDM2-His96. Since 15 and
16 are epimers at the stereocenter bearing the carboxylic acid
moiety and have similar potency in the biochemical assay, it was
postulated that this group was not making a specific interaction
with the protein; however, the appending acid facilitated the
decrease in CYP3A4 inhibition. Therefore, it was hypothesized
that heterocyclic derivatives could similarly interact with
MDM2-His96 serving as viable replacements of the carboxylic
acid moiety. Indeed, pyridine 17 has potency similar to that of
the carboxylic acid 9 in the biochemical assay (HTRF IC50 in
the serum free assay for 17 and 9 is 0.30 0.01 and 0.10 0.01
nM, respectively; Table 1). Notably, the phenyl-analogue 18
was 30-fold less potent than 17, presumably due to the loss of
the hydrogen bond interaction between the pyridine nitrogen
and the imidazole moiety on MDM2-His96. Although
encouraged by the intrinsic affinity of 17 toward MDM2 in
the HTRF assay, it was necessary to address its potent
inhibition of CYP3A4. Thus, we introduced a carboxylic acid
with the intention to achieve a similar decrease in CYP3A4
inhibition as previously observed with pyrrolidine amides 15
and 16. This led to the discovery of isonicotinic acid 19, which
shows significant improvement in the biochemical and cell
based assays while displaying no activity in the CYP3A4
inhibition assay. Exploration of other heterocycles led to the
discovery of thiazole 20, a highly potent inhibitor of the
MDM2-p53 interaction in the HTRF and cell proliferation
assays with negligible CYP3A4 inhibition. As a result, we
decided to explore other heterocycles including nicotinic acid
isomers of 19 as well as thiazole inhibitors such as 20.
First, we probed the position of the carboxylic acid on the
pyridine ring. To this end, inhibitors 19, 21, and 3 were
synthesized (Table 2). Notably, the meta (19)- and para-
substituted (3) analogues offer similar potency in the MDM2-
p53 biochemical assay. However, 3 is significantly more potent
than 19 in the cell based assays, including the mechanism based
p21 assay with SJSA-1 cells (p21 IC50 for 3 and 19 of 5 nM and
47 nM, respectively). Unfortunately, 3 also features an
increased potential for drug−drug interactions by time-
dependent inhibition of CYP3A4 (TDI, 61% inhibition at a
concentration of 10 μM; Table 2).22 Co-crystal structure of 3
bound to human MDM2 (17−111) shows a hydrogen bond
interaction between the pyridine moiety and MDM2-His96
confirming our hypothesis for the binding of these inhibitors
(Figure 1). Our measurements show the hydrogen bond
between the pyridine and the imidazole moieties to be classical
in nature (C−C−N-H ∠ 170°) with little to no π-hydrogen
bond contribution.23 Introduction of the meta-fluoro sub-
stituent at the C6-arene (22) resulted in a 10-fold increase in
potency in the serum-free HTRF assay when compared to that
of parent 3. However, this increase in potency did not
materialize when 22 was tested in the same assay run in the
presence of 15% human serum or the cell based assay.
Homologation of the carboxylic acid to acetic acid gave
inhibitor 23, which was 2-fold less potent in the HTRF-serum
free assay and 3-fold less potent in the cell proliferation assay
relative to 3 but had improved stability in the hepatocytes assay
as well as decreased CYP3A4 TDI (Table 2). We sought to
further improve metabolic stability by hindering the carboxylic
acid on 23. Thus, 24 was synthesized and evaluated in vitro for
metabolic stability. Rewardingly, 24 was noticeably more stable
(hHep CLint = <0.1 μL/min/106 cells) compared to the α-
unsubstituted acid 23 (hHep CLint = 3.4 μL/min/106 cells) in
Scheme 8
a
Reagents and conditions: (a) tert-butyl-2-(bromomethyl)-
isonicotinate, LDA, THF, −78 °C, 4 h, 71%, 3:1 d.r.; (b) mCPBA,
DCM, 30 min, 0 °C, quant.; (c) LiOH, MeOH/THF/H2O, 50 °C, 1
h; (d) 5-bromo-2-(bromomethyl)pyridine, LDA, THF, −78 °C, 2 h,
18% (single isomer); (e) Pd(dba)2, Q-phos, (2-(tert-butoxy)-2-
oxoethyl)zinc(II) chloride, THF, 60 °C, 4 h, 37%; (f) formic acid, 6
h, 75%.
RESULTS AND DISCUSSION
■
Piperidinone Inhibitors. Table 1 briefly summarizes our
initial efforts to replace the carboxylic acid moiety with a variety
of functional groups.21 At first, we examined known acid
isosteres or functionalities that could form an electrostatic
interaction with MDM2-His96. Analogues featuring tetrazole
(6) and 1,2,4-oxadiazolone (7) as acid isosteres demonstrated
potency similar to that of the parent carboxylic acid (5) in the
MDM2-p53 biochemical assay (HTRF-based neutralization
assay measuring inhibition of the interaction between MDM2
and p53) and cell proliferation assays (EdU using SJSA-1 tumor
cells). However, unlike 5, compounds 6 and 7 are moderate
inhibitors of CYP3A4. Acylsulfonamide analogues such as 8
showed a substantial reduction in potency in the biochemical
and cell based assays and also displayed moderate CYP3A4
inhibition. Substitution of the carboxylic acid with an α-keto
acid (10) provided an inhibitor with a profile similar to that of
the corresponding acid (9) in the in vitro assays (Table 1) but
also displayed poor pharmacokinetic properties in vivo in
preclinical models (data not shown). The carboxylic acid 5 and
the α-hydroxyl ketone 11 afford similar activity in the HTRF
assay suggesting that an ionized group is not essential for
potent binding to MDM2. Encouraged by this result, we
explored amides as replacements for the carboxylic acid (12 and
13, Table 1). It was reassuring to observe that 13 had potency
similar to that of the analogous carboxylic acid 9 in the HTRF
assay, although 12 and 13 are approximately 10-fold less potent
in the cell proliferation assay compared to their parent acids 5
and 9, respectively, and give rise to the aforementioned
CYP3A4 inhibition liability. A great variety of amides and
amides isosteres, such as sulfonamide 14, provided less potent
compounds and also carried the CYP3A4 inhibition. Our efforts
to improve potency by engaging additional residues on the
MDM2 protein such as Lys94 led us to synthesize pyrrolidines
15 and 16 containing an appended carboxylic acid. These
inhibitors have potency similar to that of the carboxylic acid
analogue (9) in the MDM2-p53 HTRF assay and no activity in
the CYP3A4 inhibition assay. We hypothesized that the
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Table 1. Carboxylic Acid Replacements
a
b
c
IC50 in the biochemical assay using serum free buffer. IC50 in the biochemical assay using buffer containing 15% human serum. Cellular potency in
d
e
SJSA-1 osteosarcoma tumor cells in the presence of 10% human serum. Estimated CYP3A4 inhibition IC50, Midazolam, at 3 μM. Mean and
standard deviation of at least two runs.
our in vitro human hepatocyte assay. Attempts to introduce
methyl substituents on the nicotinic acid core caused a decrease
in potency (25 and 26, Table 2). Replacing the pyridine by
either a pyrimidine (27 and 28, Table 2) or a pyrazine (29)
yielded inhibitors with similar potency in the biochemical and
cellular assays. Notably, the pKa of the nitrogen in these
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Table 2. Optimization of the Pyridine Series and Other Six-Membered Heterocyclic Analogues
a
b
c
IC50 in the biochemical assay using serum free buffer. IC50 in the biochemical assay using buffer containing 15% human serum. Cellular potency in
d
SJSA-1 osteosarcoma tumor cells in the presence of 10% human serum. Time-dependent inhibition, % CYP3A4 activity of Rifampin, 30 min, at 10
μM. Human hepatocyte stability. Mean and standard deviation of at least two runs.
e
f
heterocycles (pyridine pKa, ∼5.2; pyrimidine pKa, ∼1.3; and
pyrazine pKa, ∼0.6) has little to no effect on the CYP3A4 TDI
of the inhibitors. Finally, benzoic acid 30 was 150-fold less
potent than 3 in the serum-free biochemical assay (Table 2),
confirming the importance of the hydrogen bond interaction
observed in the cocrystal structure of 3 between the pyridine
nitrogen and MDM2-His96 (Figure 1). A similar observation
was previously made when we compared the pyridine derivative
17 (HTRF serum free IC50 = 0.30 0.01 nM) to its phenyl
analogue 18 (HTRF serum free IC50 = 137 nM) (Table 1).
We investigated other nitrogen-containing heterocycles,
many of which exhibited good potency (Table 3). For example,
the pyrazole 31 afforded potency similar to that of the
carboxylic acid analogue 9 in the biochemical and cell-based
assays, but again presented a considerable CYP3A4 TDI
liability. Both thiazole-4-carboxylic acid 32 and thiazole-5-
carboxylic acid 33 were synthesized, 33 being 4- and 40-fold
more potent than 32 in the biochemical (HTRF, serum free)
and cell based assays, respectively, showing that the position of
the carboxylic acid substituent plays an important role in
potency (Table 3). As previously observed, hydroxy ketone 34
had similar potency compared to that of the parent acid (33)
suggesting that the ionizable carboxylic acid group was not
essential for affinity to MDM2 or potent cellular activity.
However, 34 has poor metabolic stability in human hepatocytes
and high CYP3A4 inhibition (IC50 = 0.8 μM). The reduced
activity of the unsubstituted thiazole, 36, in the CYP3A4 TDI
assay compared to that of the analogous thiazole-5-carboxylic
acid derivative, 35, suggested that the acid was in part
responsible for the observed time-dependent inhibition of
CYP3A4 and that changes around the carboxylic acid could
help address this potential liability.
Thus, as observed within the nicotinic acid series (3 and 23,
Table 2), homologation of the acid gave inhibitor 37, which is
2-fold more potent than the corresponding thiazole-4-
carboxylic acid (32) in the p53-MDM2 HTRF assay and is
devoid of CYP3A4 TDI activity. Likewise, 38 and 4 show
significantly less CYP3A4 TDI (Table 3).
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Table 3. Optimization of the Thioazole Series and Other Five-Membered Heterocyclic Derivatives
a
b
c
IC50 in the biochemical assay using serum free buffer. IC50 in the biochemical assay using buffer containing 15% human serum. Cellular potency in
d
SJSA-1 osteosarcoma tumor cells in the presence of 10% human serum. Time-dependent inhibition, % CYP3A4 activity of Rifampin, 30 min, at 10
μM. Human hepatocyte stability. Mean and standard deviation of at least two runs.
e
f
A cocrystal structure of 4 bound to human MDM2 (Figure
2) demonstrates that this inhibitor binds to MDM2 filling the
critical pockets naturally occupied by the three key residues of
p53: Leu26, Trp23, and Phe19.9 The 3-chlorophenyl occupies
the Leu26 pocket, engaging in a π−π stacking interaction with
the imidazole of the His96. The 3-fluoro-4-chlorophenyl is
buried in the Trp23 pocket. The small lipophilic cyclopropyl
group projects toward the Phe19 pocket, while the tert-butyl
sulfone is within van der Waals distance from the “glycine
shelf”, named after Gly58 on MDM2, maximizing hydrophobic
contact with the protein. Finally, the thiazole-nitrogen forms a
hydrogen bond (2.9 Å) with the imidazole-NH of His96.
Introducing substituents α to the carboxylic acid of 4 or 38
did not critically alter the overall profile of the inhibitors (39−
42, Table 3). Finally, other heterocycles such as oxazole (43),
triazole (44), and imidazole (45) were also synthesized and
evaluated. While many of these substitutions were tolerated,
they did not lead to increased potency (Table 3).
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CYP3A4, lead to the selection of 4 for further evaluation in our
tumor xenograft model.
First, we confirmed that the cytotoxicity observed with 4 is
attributable to activation of the p53 pathway. Thus, we
evaluated the ability of 4 to inhibit the proliferation of
HCT116 p53wt and p53−/− tumor cells in vitro (Figure 3).24
Compound 4 displayed robust dose-dependent cell growth
inhibitions of HCT116 wild-type p53 cells (IC50 = 11 nM) and
no inhibition of p53 deficient cells at doses <10 μM. Similarly,
4 also exhibited a dose-dependent increase of p21 mRNA, a
direct transcriptional readout of p53 activity, in HCT116 p53wt
cells (IC50 = 58 nM). No induction was observed when
HTC116 p53−/− tumor cells were treated with 4 at
concentrations up to 10 μM.
In a pharmacodynamic in vivo xenograft assay with SJSA-1
osteosarcoma tumor cells, 4 demonstrated significant time-
dependent p21 induction over the vehicle (Figure 4). A
maximum 15-fold induction of p21 mRNA was observed 4 h
after dosing QD for 4 days at 25 mg/kg. These data indicated
that 4 achieved an on-mechanism inhibition of MDM2 and
induction of p53 signaling, and provided dose-selection
guidance for the xenograft study.
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 5).25 In this study,
4 caused robust dose-dependent tumor growth inhibition with
the highest dose of 50 mg/kg causing 6% tumor regression.
The calculated ED50 was 11 mg/kg. Figure 6 compares the
cellular potency of inhibitor 4 to other known inhibitors that
are currently in the clinic for the treatment of cancer.
Figure 2. Co-crystal structure of 4 bound to human MDM2 (6−110).
White labels indicate positions normally occupied by key p53 residues.
MDM2 residues His96 and Gly58 are labeled in yellow. Coordinates
for compound 4 (PDB code: 4ODE) bound to MDM2 have been
deposited in the PDB.
Compounds 3, 4, 33, 35, and 38 were selected for their
potency and evaluated in rodent pharmacokinetic experiments
(Table 4). The majority of these inhibitors exhibit low
clearance in rodent species. The addition of a meta-fluoro
substituent at the C5-arene on 4 and 35 (Table 4) improved
their metabolic stability in rat and mouse and increased oral
bioavailability compared to that of the des-fluoro derivatives.
These results, together with good cellular potency, favorable in
vitro stability in human hepatocytes, and reduced liability to
cause potential drug−drug interactions through inhibition of
Table 4. Rodent PK Profiles of Selected Compounds
a
b
Rat/mouse iv vehicle: 10.0% DMAC, 10.0% EtOH, 30.0% propylene glycol, and 50.0% saline (0.45% NaCl/49.55% water). Mouse iv vehicle: 0.5%
methyl cellulose, 1% Tween 80, and 98.5% water.
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Figure 3. Cell activity of 4 is p53-dependent. (a) In HCT116 p53wt
and p53−/− cells, the percentage of BrdU positive cells was measured
16 h postcompound treatment by flow cytometry. The DMSO control
was designated as 0% inhibition. (b) In HCT116 p53wt and p53−/−
cells, total RNA was extracted 7 h postcompound treatment, and p21
mRNA was measured by quantitative RT-PCR.
Figure 5. SJSA-1 cells (5 × 106) were implanted subcutaneously into
female athymic nude mice. Treatment with vehicle or 4 at 5, 12.5, 25,
or 50 mg/kg QD 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 the mean
tumor volumes, and the error bars represented the SEM of data from
10 mice. * p < 0.005.
Interestingly, this contrasts from 1 for which metabolite
profiles were qualitatively similar across species with only it is
acyl glucuronide observed in these incubates.27 Therefore,
glucuronidation of 1 is predicted to be the primary metabolic
pathway in humans.
Morpholinone Inhibitors. Recently, we disclosed our
efforts toward the optimization of potent morpholinone
inhibitors. From these studies, we concluded that whereas
morpholinone inhibitors are about 3- to 5-fold less potent than
piperidinones, this core substitution had a profound effect on
the in vitro and in vivo metabolic stability of these analogues,
giving them divergent metabolism.28
Thus, we decided to explore some of the most potent acid
replacements encountered thus far on the morpholinone core.
To this end, nicotinic acid inhibitor 46 was synthesized. A
direct comparison between inhibitors 3 and 46 (Table 5) shows
morpholinone 46 to be, as expected, 3- to 5-fold less potent
than piperidinone 3 in the EdU cell assay. Surprisingly, its C2-
epimer 47 turned out to be 2-fold more potent than 46 (EdU
assay, Table 5). The cocrystal structures of 46 and 47 bound to
MDM2 (Figure 8) allowed us to confirm the relative
configuration of these inhibitors. The structure of 46 bound
to MDM2 (Figure 8a) depicts the pyridine nitrogen engaging
in a hydrogen bond interaction with the imidazole-NH of
His96 similar to that observed with inhibitors 3 and 4 (Figure
1). In contrast, the binding of 47 to MDM2 shows interactions
that have yet to been seen in the nicotinic acid series (Figure
8b). In this case, there is no hydrogen bond observed between
the inhibitor and His96. However, there are direct hydrogen
Figure 4. PD study results of 4 in the SJSA-1 tumor xenograft: Female
athymic nude mice (n = 4/group) were implanted subcutaneously with
5 × 106 SJSA-1 cells. When tumors reached ∼175 mm3, 25 mg/kg of 4
or the vehicle was administered orally once daily (QD) 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 the mean p21 fold induction over vehicle, and error
bars represent standard error of the mean (SEM) of data from five
mice. Concentrations in plasma (red dots) were analyzed by LC/MS/
MS. *p < 0.001.
To better understand its metabolism, inhibitor 4 was
incubated in hepatocytes (106 cells/mL) from five different
species and its degradation products were evaluated by LC-
MS/MS (Figure 7). The reported chromatograms show that
oxidative metabolites M1, M2, and M3 are generated across
species, including human hepatocytes.26 The acyl glucuronide
metabolite of 4 was not observed in this experiment.
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Figure 6. Chemical structures and potencies of some known MDM2 inhibitors currently being tested in the clinic: RG7112, RG7388, SAR299155,
a
and 4. IC50 in the EdU cell proliferation assay (SJSA-1 and 10% human serum).
bonds observed between the acid in 47 and the backbone NH
and side chain hydroxyl of Ser17. Furthermore, there is a water
mediated hydrogen bond between this acid and the side chain
of Gln18. The enhanced potency of 47 can be rationalized by
these new interactions offsetting the loss of the hydrogen bond
to His96.
Our previous studies on the morpholinone series showed
that, in general, the C2-R isomer is more potent than its C2-S
epimer. This can be best exemplified by comparing epimeric
morpholinone inhibitors 48 (SJSA-1 EdU IC50 = 38 nM) and
49 (SJSA-1 EdU IC50 = 247 nM) (Figure 8c-d). Notably, in this
case, the carboxylate moiety of both 48 and 49 interacts with
the imidazole moiety of His96.
Although heterocycles are not often thought of as carboxylic
acids isosteres, there is precedence for the successful use of
pyridines as replacements of carboxylic acids.29
Among these new analogues, compound 4 shows excellent
biochemical potency (HTRF IC50 = 0.1 nM, Table 4), cellular
potency (SJSA-1 EdU IC50 = 16 nM), and MDM2 selectivity
over MDMX (MDMX HTRF IC50 > 100 μM), as well as good
pharmacokinetic properties.30,31 Compound 4 also shows
robust antitumor activity in the SJSA-1 osteosarcoma xenograft
study with a calculated ED50 of 11 mg/kg. Compounds 4 and 1
exhibit comparable potency and efficacy. However, while
glucuronidation is the main route of metabolism of 1, inhibitor
4 is cleared primarily by oxidative pathways. Thus, 4 could
provide for a viable alternative to 1 should a compound with a
different metabolic profile or clearance mechanism be desired.
We also synthesized 50 (Table 5), a piperidinone inhibitor
with S-stereochemistry at C3 (Table 5). Crystallographic data
̆
of 50 bound to MDM2 (1.85 A resolution) was obtained, but
we were unable to identify a defined density for the nicotinic
acid moiety. Evidently, in this case, the piperidinone core
precludes the nicotinic acid from picking up the hydrogen
bonds with either His96 or Ser17. This finding is in agreement
EXPERIMENTAL SECTION
■
General Chemistry. Reactions 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 an 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 and B = CH3CN with 0.1% TFA; gradient: 5−95% B (0.0−
15.0 min); and flow rate, 1.5 mL/min. Low-resolution mass spectral
(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 (HRMS) were obtained on an Agilent
with the observed loss in potency of 50 (SJSA-1 EdU IC50
238 nM) compared to 3 (SJSA-1 EdU IC50 = 5 nM).
=
CONCLUSIONS
■
Evaluation of the carboxylic acid moiety of 1 identified known
acid isosteres such as tetrazole (6), oxadiazolone (7), and α-
keto acid (10) as viable replacements, maintaining potency
similar to that of 1 in the biochemical and cell based assays.
However, these analogues are moderate inhibitors of CYP3A4.
Insights into the binding of these inhibitors to MDM2 (2,
Figure 1) led us to hypothesize that the electrostatic interaction
between MDM2 and the carboxylic acid moiety could be
replaced by a dipole−dipole interaction with a hydrogen bond
donor or acceptor. Further studies led to the discovery that
replacement of the acid with hydrogen bond acceptor moieties,
such as amides (13, 15, and 16) and heterocycles like pyridine
(3), pyrimidine (28), pyrazine (29), thiazole (4), pyrazole
(31), oxazole (43), triazole (44), and imidazole (45), provide
potent inhibitors of the MDM2-p53 interaction. Notably,
phenyl analogues 18 and 30, which cannot engage in a
hydrogen bond binding interaction with MDM2-His96, have
significantly less affinity for the protein than their correspond-
ing pyridine analogues (17 and 3 respectively), corroborating
the importance of this interaction for potent binding. The
aforementioned interaction of these novel heterocyclic
inhibitors with MDM2-His96 was confirmed with the MDM2
cocrystal structures of pyridine 3 and thiazole 4 (Figure 1).
1
6510 Q-TOF MS with a 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, and br = broad.
2-((3R,5R,6S)-1-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetamide (12). To a solution of 5 (213 mg, 0.375 mmol) and N-
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Figure 7. Metabolite profiles of 4 from hepatocyte incubation at 10 μM after 2 h.
methylmorpholine (57.7 μL, 0.524 mmol) in THF (1.87 mL) was
added isobutyl chloroformate (58.8 μL, 0.450 mmol) at 0 °C. The
cloudy colorless solution was stirred at 0 °C for 1 h. Then, 28%
ammonia in water (50.6 μL, 0.749 mmol) was added to the mixture at
0 °C, and the resulting mixture was stirred at 0 °C for 3 h. The
reaction mixture was extracted with ethyl acetate and water. The
combined organic layer was dried (Na2SO4) and concentrated under
reduced pressure to provide a crude product as a pale yellow solid.
Purification by RP-HPLC (40% to 55% CH3CN/H2O in 25 min, flow
rate = 45 mL/min) provided 2-((3R,5R,6S)-1-((S)-1-(tert-
butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamide (160 mg, 0.282 mmol, 75%
yield) (12) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.21−7.26
(m, 2H), 7.05−7.21 (m, 3H), 6.96−7.02 (m, 1H), 6.87 (dt, J = 6.4, 2.0
Hz, 1H), 6.79 (br. s., 1H), 6.27 (br. s., 1H), 4.99 (d, J = 10.8 Hz, 1H),
4.08 (dd, J = 13.2, 11.1 Hz, 1H), 3.21−3.37 (m, 2H), 2.74−2.84 (m,
2H), 2.56−2.70 (m, 1H), 2.35 (t, J = 13.7 Hz, 1H), 2.14 (ddd, J =
14.2, 9.8, 7.2 Hz, 1H), 2.02 (dd, J = 13.7, 2.9 Hz, 1H), 1.49 (td, J = 7.0,
2.9 Hz, 1H), 1.44 (s, 9H), 1.43 (s, 3H), 0.41 (t, J = 7.5 Hz, 3H). Mass
spectrum (ESI) m/z = 567.2 [M + H]+. HRMS (ESI) m/z found
567.1841 [M + H]+, calcd for C28H36Cl2N2O4S 567.1851.
2-((3R,5R,6S)-1-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-N-
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Table 5. Comparison between Nicotinic Acid Inhibitors Derived from the Piperidinone and Morpholinone Scaffolds
a
b
Assays conducted in the presence of 10% human serum. Mean and standard deviation of at least two runs.
Figure 8. Co-crystal structures of (a) 46, (b) 47, (c) 48, and (d) 49 bound to human MDM2 (46, 47 MDM2, 6−110; 48, 49 MDM2, 17−111). a
IC50 in the biochemical assay using buffer containing 15% human serum. b Cellular potency in SJSA-1 osteosarcoma tumor cells in the presence of
10% human serum. Coordinates for compounds 46 (PDB code: 4OGT), 47 (PDB code: 4ODF), 48 (PDB code: 4OCC), and 49 (PDB code:
4OGV) bound to MDM2 have been deposited in the PDB.
((trifluoromethyl)sulfonyl)acetamide (8). A solution of 5 (50 mg,
0.088 mmol) and trifluoromethanesulfonamide (52.4 mg, 0.32 mmol)
in DMF (0.281 mL) was treated with HATU (134 mg, 0.352 mmol)
and diisopropylethylamine (0.092 mL, 0.528 mmol) successively.
Then, the reaction was stirred at room temperature for 5 h. After this
period, the crude mixture was diluted in 5 mL of 1 N HCl, then 30 mL
of water, and extracted with diethyl ether (3 × 30 mL). The combined
organic layers were dried over MgSO4 and filtered, and the filtrate was
concentrated under vacuum. Purification by RP-HPLC (25−75%
AcCN/H2O in 30 min) provided 2-((3R,5R,6S)-1-((S)-1-(tert-
butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)-N-((trifluoromethyl)sulfonyl)acetamide
MHz, CDCl3) δ 7.27 (s, 4H), 7.05−7.18 (m, 2H), 6.94 (s, 1H), 6.78−
6.86 (m, 1H), 4.92−5.11 (m, 1H), 3.95−4.13 (m, 1H), 3.30−3.44 (m,
1H), 3.19−3.29 (m, 1H), 2.92−3.08 (m, 1H), 2.77−2.86 (m, 1H),
2.60−2.71 (m, 1H), 2.45−2.58 (m, 1H), 2.11−2.27 (m, 1H), 1.78−
1.89 (m, 1H), 1.51 (s, 3H), 1.45 (s, 9H), 0.42 (t, J = 7.53 Hz, 3H).
Mass spectrum (ESI) m/z = 699.0 [M + H]+. HRMS (ESI) m/z found
699.1346 [M + H]+, calcd for C29H35Cl2F3N2O6S2 699.1344.
2-((3R,5R,6S)-1-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetonitrile (51). A solution of 2-((3R,5R,6S)-1-((S)-1-(tert-
butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamide (12) (180 mg, 0.317 mmol)
and triethylamine (221 μL, 1.586 mmol) in THF (2.64 mL) was
1
(8) (25 mg, 0.036 mmol, 41% yield) as a white foam. H NMR (400
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treated with 2,2,2-trifluoroacetic anhydride (113 μL, 0.793 mmol) at 0
°C. After being stirred at 0 °C for 90 min, the reaction was quenched
(sat. aq. NH4Cl), extracted (2 × EtOAc), and washed (brine). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification of the residue by
combi-flash (24 g SiO2, 30% EtOAc/Hex) provided 2-((3R,5R,6S)-1-
((S)-1-(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetonitrile (51) (160 mg, 0.291
(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophen-
yl)-3-methyl-2-oxopiperidin-3-yl)methyl)-1,2,4-oxadiazol-5(4H)-one
(7) (0.014 g, 0.023 mmol, 15% yield over last two steps). H NMR
1
(500 MHz, CDCl3) δ 10.30 (s, 1H), 7.27 (m, 4H), 7.09−7.20 (m,
2H), 6.96 (s, 1H), 6.85 (d, J = 7.09 Hz, 1H), 5.00 (d, J = 10.76 Hz,
1H), 4.02 (t, J = 12.10 Hz, 1H), 3.34 (m, 1H), 3.22 (d, J = 15.65 Hz,
1H), 3.06 (ddd, J = 13.69, 10.88, 2.57 Hz, 1H), 2.74−2.84 (m, 2H),
2.58 (t, J = 13.82 Hz, 1H), 2.14 (ddd, J = 14.12, 9.84, 7.58 Hz, 1H),
1.87 (dd, J = 13.82, 2.57 Hz, 2H), 1.30−1.54 (m, 12H), 0.42 (t, J =
7.46 Hz, 3H). Mass spectrum (ESI) m/z = 608.2 [M + H]+. HRMS
(ESI) m/z found 608.1758 [M + H]+, calcd for C29H35Cl2N3O5S
608.1753.
3-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
2-oxopropanoic Acid (10).32 Part A: A solution of 2-((3R,5R,6S)-1-
((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-
(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (9) (300
mg, 0.517 mmol) and 1-(cyanomethyl)tetrahydro-1H-thiophen-1-ium
bromide (161 mg, 0.775 mmol) in DCM (5167 μL) was treated with
HATU (236 mg, 0.620 mmol) and DIEA (269 μL, 1.550 mmol) at
room temperature. The reaction was stirred at this temperature for 4 h.
After this period, the reaction was quenched (sat. aq. NH4Cl),
extracted (2 × EtOAc), and washed (brine). The combined organic
layers were dried over Na2SO4, filtered, and concentrated under
reduced pressure. Purification by combi-flash (12 g SiO2, 0% to 70%
acetone/ethyl acetate) provided the intermediate cyanosulfur ylide
(356 mg, 0.516 mmol, 100% yield). Mass spectrum (ESI) m/z = 689.2
[M + H]+.
Part B: To a solution of the sulfur ylide formed in part A (356 mg,
0.516 mmol) in DMF (3.44 mL) and water (1.72 mL) was added
oxone (635 mg, 1.032 mmol) at room temperature. The reaction was
stirred for 2.5 h. After this period, the reaction was quenched (2.5 mL,
1 N aq. HCl), extracted (2 × EtOAc), and washed (brine). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification of the crude
product by RP-HPLC (45 to 65% CH3CN/H2O, both containing
0.1% TFA, in 30 min, flow rate = 45 mL/min) provided 3-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-2-ox-
opropanoic acid (10) (200 mg, 0.329 mmol, 64% yield) as a white
solid. 1H NMR (400 MHz, CDCl3) δ 7.88−8.09 (m, 1H), 7.51 (m, J =
12.5 Hz, 1H), 6.69−7.16 (m, 6H), 4.91 (d, J = 10.6 Hz, 1H), 4.13−
4.21 (m, 1H), 4.03 (d, J = 11.0 Hz, 1H), 3.29−3.41 (m, 1H), 2.85 (d, J
= 12.5 Hz, 1H), 2.66−2.79 (m, 1H), 2.50 (t, J = 13.1 Hz, 1H), 2.23 (d,
J = 11.2 Hz, 1H), 1.67−1.85 (m, 2H), 1.43 (br. s., 3H), 1.41 (s, 9H),
0.25−0.46 (m, 2H), −0.45− −0.24 (m, 1H), −1.19− −1.01 (m, 1H).
Mass spectrum (ESI) m/z = 608.2 [M + H]+. HRMS (ESI) m/z found
608.1630 [M + H]+, calcd for C30H35Cl2NO6S 608.1640.
1
mmol, 92% yield). H NMR (400 MHz, CDCl3) δ 7.21−7.26 (m,
2H), 7.06−7.21 (m, 4H), 6.99 (s, 1H), 6.85−6.90 (m, 1H), 4.99 (d, J
= 10.8 Hz, 1H), 4.07 (dd, J = 13.3, 11.0 Hz, 1H), 3.20−3.36 (m, 2H),
2.95 (d, J = 16.8 Hz, 1H), 2.77 (dd, J = 13.2, 2.2 Hz, 1H), 2.72 (d, J =
16.8 Hz, 1H), 2.42 (t, J = 14.0 Hz, 1H), 2.13 (ddd, J = 14.3, 9.9, 7.3
Hz, 1H), 2.05 (dd, J = 14.1, 2.9 Hz, 1H), 1.47−1.54 (m, 1H), 1.46 (s,
3H), 1.43 (s, 9H), 0.41 (t, J = 7.5 Hz, 3H). Mass spectrum (ESI) m/z
= 549.2 [M + H]+.
(3R,5R,6S)-3-((1H-Tetrazol-5-yl)methyl)-1-((S)-1-(tert-
butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methylpiperidin-2-one (6). To a solution of 2-((3R,5R,6S)-1-((S)-1-
(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophen-
yl)-3-methyl-2-oxopiperidin-3-yl)acetonitrile (51) (57 mg, 0.104
mmol) in DMF (259 μL) was added sodium azide (67.4 mg, 1.037
mmol) and ammonium chloride (55.5 mg, 1.037 mmol). The resulting
mixture was stirred at 90 °C for 5 days. Then, the reaction was
acidified (aq. 10% citric acid), extracted (2 × EtOAc), and washed
(brine). The combined organic layers were dried over Na2SO4, filtered,
and concentrated under reduced pressure. Purification by RP-HPLC
(40% to 55% CH3CN/H2O, 0.1% TFA each, in 25 min, flow rate = 45
mL/min) provided (3R,5R,6S)-3-((1H-tetrazol-5-yl)methyl)-1-((S)-1-
(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophen-
yl)-3-methylpiperidin-2-one (6) (40 mg, 0.068 mmol, 65% yield) as a
white foam. 1H NMR (400 MHz, CDCl3) δ 7.20−7.27 (m, 2H),
7.10−7.17 (m, 2H), 7.00 (d, J = 1.8 Hz, 3H), 6.88 (d, J = 6.7 Hz, 1H),
5.03 (d, J = 10.6 Hz, 1H), 4.10 (t, J = 12.0 Hz, 1H), 3.55−3.62 (m,
1H), 3.42 (d, J = 16.0 Hz, 1H), 3.3−3.39 (m, 1H), 3.14 (t, J = 11.3 Hz,
1H), 2.84 (d, J = 13.3 Hz, 1H), 2.57 (t, J = 13.8 Hz, 1H), 2.10−2.24
(m, 1H), 1.95−2.02 (m, 1H), 1.46 (s, 9H), 1.36−1.43 (m, 1H), 1.32
(s, 3H), 0.42 (t, J = 7.5 Hz, 3H). Mass spectrum (ESI) m/z = 592.2
[M + H]+. HRMS (ESI) m/z found 592.1913 [M + H]+, calcd for
C28H35Cl2N5O3S 592.1916.
2-((3R,5R,6S)-1-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-N′-hy-
droxyacetimidamide (52). A round-bottomed flask equipped with a
reflux condensor was charged with a suspension of 2-((3R,5R,6S)-1-
((S)-1-(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetonitrile (51) (0.100 g, 0.182
mmol) and hydroxyammonium chloride (0.011 mL, 0.273 mmol) in
MeOH (4 mL). Then, sodium hydrogencarbonate (0.023 g, 0.273
mmol) was added. The reaction was refluxed overnight. The reaction
was cooled to room temperature and quenched (sat. aq. NH4Cl),
extracted (2 × EtOAc), and washed (brine). The combined organic
layers were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The compound was used on the next step without
further purification. Mass spectrum (ESI) m/z = 582.2 [M + H]+.
3-(((3R,5R,6S)-1-((S)-1-(tert-Butylsulfonyl)butan-2-yl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)-
1,2,4-oxadiazol-5(4H)-one (7). To a solution of 2-((3R,5R,6S)-1-((S)-
1-(tert-butylsulfonyl)butan-2-yl)-5-(3-chlorophenyl)-6-(4-chlorophen-
yl)-3-methyl-2-oxopiperidin-3-yl)-N′-hydroxyacetimidamide (52)
(0.090 g, 0.154 mmol) in dioxane (2 mL) was added DBU (0.069
mL, 0.463 mmol) via syringe followed by 1,1′-carbonyldiimidazole
(0.075 g, 0.463 mmol). The reaction was refluxed for 2 days. After this
period, the reaction mixture was allowed to cool to room temperature.
The crude was quenched with water (25 mL). Extracted with EtOAc
(100 mL) and the organic layer washed with brine (10 mL). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by reversed phase preparatory HPLC (GeminiTM Prep C18 5 mm
column; Phenomenex, Torrance, CA) (eluent, 40−65% CH3CN/
H2O, 0.1% TFA each, 30 min) to provide 3-(((3R,5R,6S)-1-((S)-1-
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetamide (13). To a solution of 2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (9) (300 mg, 0.517
mmol), HATU (393 mg, 1.033 mmol) and N,N-diisopropylethylamine
(0.270 mL, 1.550 mmol) in DMF (1.00 mL) was added a 7 N
ammonia solution in methanol (0.369 mL, 2.58 mmol). The reaction
was stirred at 40 °C for 1 h. The crude material was concentrated and
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (40 g), eluting with a
gradient of 0% to 40% acetone/MeOH (9:1) in DCM, to provide 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
1
acetamide (13) (264 mg, 0.456 mmol, 88% yield). H NMR (500
MHz, CDCl3) δ 7.27 (s, 2H), 7.04−7.18 (m, 2H), 6.96−7.04 (m, 1H)
6.92−6.96 (m, 1H), 6.71−6.92 (m, 1H), 6.43−6.63 (m, 1H), 5.44−
5.63 (m, 1H), 4.96 (d, J = 10.8 Hz, 1H), 4.13 (q, J = 7.2 Hz, 1H), 3.32
(s, 1H), 2.96 (s, 1H), 2.89 (s, 1H), 2.81 (s, 1H), 2.57−2.80 (m, 3H),
2.38 (s, 1H), 2.05 (s, 2H), 1.97−2.08 (m, 1H), 1.36−1.51 (m, 12H),
1.27 (t, J = 7.2 Hz, 2H). Mass spectrum (ESI) m/z = 579.0 [M + H]+.
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HRMS (ESI) m/z found 579.1851 [M + H]+, calcd for
C29H36Cl2N2O4S 579.1851.
Part B: To a solution of ethyl 2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)pyrimidine-5-car-
boxylate (35 mg, 0.050 mmol) in THF (2 mL) and MeOH (1 mL)
was added lithium hydroxide (1.0 mL, 2 M). The solution was stirred
at 50 °C for 30 min. After this period, the reaction was neutralized (2
mL/1 M HCl), concentrated, and diluted with MeOH (2 mL).
Purification by prep HPLC (25−75% acetonitrile/water both solvents
containing 0.1% TFA, in 30 min, flow rate =45 mL/min) provided 2-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)methyl)pyrimidine-5-carboxylic acid (28) (25 mg, 0.037 mmol,
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetonitrile (53). A solution of 2-((3R,5R,6S)-1-((S)-2-(tert-butylsul-
fonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamide (13) (125 mg, 0.216 mmol)
and triethylamine (0.150 mL, 1.078 mmol) in tetrahydrofuran (3 mL)
was treated with trifluoroacetic anhydride (0.075 mL, 0.539 mmol) at
0 °C. After being stirred at 0 °C overnight, the reaction was quenched
with water (10 mL), extracted (3 × 10 mL EtOAc), and washed with
brine (10 mL). The combined organic layer was dried (MgSO4) and
concentrated under reduced pressure. The crude product was purified
by flash chromatography (acetone/hexanes: 0−50%) to give 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
1
74% yield) as a white amorphous solid. H NMR (400 MHz, CDCl3)
δ 9.24 (s, 2H), 6.95−7.26 (m, 6H), 5.03 (d, J = 10.56 Hz, 1H), 4.37 (t,
J = 11.84 Hz, 1H), 3.71 (d, J = 12.13 Hz, 1H), 3.44−3.56 (m, 1H),
3.38 (s, 1H), 2.91 (d, J = 11.74 Hz, 1H), 2.57−2.72 (m, 1H), 2.42 (t, J
= 13.69 Hz, 1H), 1.81−1.94 (m, 2H), 1.75 (dd, J = 3.23, 13.60 Hz,
1H), 1.44 (s, 3H), 1.42 (s, 9H), 0.25−0.50 (m, 2H), −0.27 (br. s.,
1H), −0.95 (br. s., 1H). Mass spectrum (ESI) m/z = 676.0 [M + H]+.
HRMS (ESI) m/z found 676.1809 [M+H]+, calcd for
C33H36Cl2FN3O5S 676.1815.
1
acetonitrile (53) (92 mg, 0.164 mmol, 90% yield). H NMR (500
MHz, CDCl3) δ 7.27 (s, 2H), 7.11−7.20 (m, 3H), 7.11 (s, 1H), 6.94−
7.05 (m, 1H), 6.86−6.94 (m, 1H), 4.95 (d, J = 10.5 Hz, 1H), 4.13 (d, J
= 7.1 Hz, 1H), 3.20 (s, 1H), 2.98 (s, 1H), 3.01 (s, 1H), 2.80 (s, 1H),
2.84 (m, 2H), 2.48 (s, 1H), 2.05 (s, 1H), 2.03 (dd, J = 13.9, 3.2 Hz,
2H), 1.48 (s, 3H), 1.44 (s, 9H), 1.27 (t, J = 7.2 Hz, 2H). Mass
spectrum (ESI) m/z = 561.2 [M + H]+.
Ethyl 2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetimidate (54). A solution of 2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetonitrile (53) (90 mg, 0.160
mmol) in ethanol (1.603 mL) was bubbled with HCl for 15 min. The
reaction was stirred overnight reaching complete conversion. The
crude was concentrated and used in the next step without further
purification. Mass spectrum (ESI) m/z = 607.2 [M + H]+.
1-Amino-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)ethaniminium (55). A solution of 54 (0.097 g, 0.160 mmol)
in ethanol (1.6 mL) was bubbled with ammonia for 30 min. The
reaction was allowed to stir for 3 h at room temperature when
complete conversion to the desired product was observed. The crude
was concentrated and used in the next step without further
purification. Mass spectrum (ESI) m/z = 578.3 [M + H]+.
2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
ethanethioamide (56). To a solution of 2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetonitrile (53) (100 mg, 0.178
mmol) in ethanol (1.781 mL) was added diphosphorus pentasulfide
(0.076 mL, 0.712 mmol), and the reaction was allowed to stir at 90 °C
overnight. After this period, the crude material was concentrated and
adsorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (40 g), eluting with a
gradient of 0% to 100% acetone in DCM, to provide 2-((3R,5R,6S)-1-
((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-
(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)ethanethioamide (56)
(70 mg, 0.118 mmol, 66% yield). Mass spectrum (ESI) m/z = 595.0
[M + H]+.
2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-
(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)pyrimidine-5-carboxylic Acid (28). Part A: A solution
of 1-amino-2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-ox-
opiperidin-3-yl)ethaniminium (45 mg, 0.075 mmol) and ethyl-2-
formyl-3-oxopropionate (0.109 mL, 0.753 mmol) in dimethylaceta-
mide (1 mL) was heated in the microwave at 100 °C for 2 h. After this
period, the reaction was concentrated, and the crude material was
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (12 g), eluting with a
gradient of 0% to 50% 9:1 acetone/MeOH in DCM to provide ethyl
2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)methyl)pyrimidine-5-carboxylate (35 mg, 0.050 mmol, 66% yield)
as a yellow oil. Mass spectrum (ESI) m/z = 704.2 [M + H]+.
2-(((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)methyl)pyrimidine-5-carboxylic Acid (27). Com-
pound 27 was prepared according to a similar procedure described for
1
the synthesis of 28. H NMR (500 MHz, CDCl3) δ 9.17 (s, 2H),
7.04−7.27 (m, 3H), 6.88−7.04 (m, 2H), 6.69−6.88 (m, 2H), 6.56−
6.69 (m, 1H), 4.56 (d, J = 10.8 Hz, 1H), 4.52 (s, 1H), 3.57 (d, J = 12.7
Hz, 1H), 3.45 (d, J = 12.7 Hz, 1H), 3.17 (br. s., 1H), 3.03−3.14 (m,
1H), 2.88 (d, J = 13.7 Hz, 1H), 2.50 (d, J = 13.4 Hz, 1H), 2.24−2.41
(m, 1H), 1.95−2.18 (m, 3H), 1.26−1.53 (m, 9H), 1.14−1.34 (m, 2H),
0.86−1.05 (m, 1H), 0.63−0.86 (m, 1H). Mass spectrum (ESI) m/z =
658.2 [M + H]+. HRMS (ESI) m/z found 658.1913 [M + H]+, calcd
for C33H37Cl2N3O5S 658.1909.
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)-1H-imidazol-4-yl)acetic Acid (45). To a solution of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetimidamide (55) (80 mg, 0.138 mmol) in acetonitrile (1 mL) was
added ethyl trans-4-oxo-2-butenoate (21.26 μL, 0.166 mmol), and the
mixture was heated to 130 °C for 1 h in the microwave. The reaction
was concentrated and diluted in THF (1 mL) and MeOH (1 mL), and
LiOH (1 mL, 2 M) was added, and the reaction was stirred at 50 °C
for 1 h. After this period, the reaction was cooled and concentrated,
diluted in MeOH, and filtered. Purification by RP-HPLC (25−75%
CH3CN/H2O both solvents containing 0.1% TFA, 30 min, flow rate =
1
45 mL/min) provided 45 (8 mg, 8% yield). H NMR (400 MHz,
CDCl3) δ 7.10−7.56 (m, 1H), 6.87 (d, J = 12.13 Hz, 7H), 4.58−4.83
(m, 1H), 3.92−4.21 (m, 2H), 3.40−3.73 (m, 3H), 3.05−3.32 (m, 2H),
2.49−2.80 (m, 2H), 2.11−2.39 (m, 1H), 1.42−1.68 (m, 2H), 1.07−
1.36 (m, 12H), −0.16−0.28 (m, 2H), −0.74− −0.43 (m, 1H),
−1.55− −1.16 (m, 1H). Mass spectrum (ESI) m/z = 660.2 [M + H]+.
2-(5-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)-4H-1,2,4-triazol-3-yl)acetic Acid (44). Part A: A solution
of ethyl 2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)acetimidate (55) (95 mg, 0.156 mmol) and ethyl 3-hydrazinyl-3-
oxopropanoate (27.4 mg, 0.188 mmol) in EtOH was heated to 75 °C
and stirred overnight. The crude material was concentrated and
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (12 g), eluting with a
gradient of 0% to 40% acetone/MeOH (9:1) in DCM, to provide
ethyl 2-(5-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)-4H-1,2,4-triazol-3-yl)acetate (68 mg, 0.099 mmol,
63% yield) as a light-yellow solid. Mass spectrum (ESI) m/z = 689.0
[M + H]+.
Part B: To a solution of ethyl 2-(5-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
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phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)-4H-1,2,4-triazol-3-yl)-
acetate (68 mg, 0.099 mmol) in THF (0.5 mL)/methanol (0.5 mL)/
water (0.5 mL) was added lithium hydroxide (1 mL, 2 M). The
reaction was stirred for 30 min at 50 °C. After this time, the reaction
was quenched with 10% citric acid solution (5 mL), diluted in water
(10 mL), and washed with ether (3 × 15 mL). The combined organic
layer was dried over MgSO4, filtered, and concentrated. Purification by
RP-HPLC (25−75% CH3CN/H2O both solvents containing 0.1%
TFA, 30 min, flow rate = 45 mL/min) provided 2-(5-(((3R,5R,6S)-1-
((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-
(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)-4H-1,2,4-tria-
Part B: To a solution of methyl 2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazole-5-carboxylate
(77 mg, 0.114 mmol) in THF (2 mL) and MeOH (1 mL) was added
lithium hydroxide (2 mL, 2 M). After this period, the reaction was
neutralized (4 mL, 1 M HCl) and concentrated. Purification of the
crude product by RP-HPLC (45% to 65% AcCN/H2O, both
containing 0.1% TFA, in 30 min, flow rate = 45 mL/min) provided
2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)thiazole-5-carboxylic acid (33) (64 mg, 0.096 mmol, 85%
yield). 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 7.28 (br. s., 4H),
7.11 (d, J = 4.50 Hz, 2H), 7.01 (s, 1H), 6.90 (br. s., 1H), 5.00 (d, J =
10.76 Hz, 1H), 4.29−4.51 (m, J = 13.50 Hz, 1H), 3.72 (d, J = 14.08
Hz, 1H), 3.56 (d, J = 13.89 Hz, 1H), 3.24 (t, J = 12.91 Hz, 1H), 2.95
(d, J = 13.50 Hz, 1H), 2.64−2.79 (m, 1H), 2.35 (t, J = 13.79 Hz, 1H),
1.75−2.06 (m, 2H), 1.40−1.50 (m, 12H), 0.24−0.48 (m, 2H), −0.28
(br. s., 1H), −1.00 (br. s., 1H). Mass spectrum (ESI) m/z = 663.0 [M
+ H]+. HRMS (ESI) m/z found 663.1522 [M + H]+, calcd for
C32H36Cl2N2O5S2 663.1521.
1
zol-3-yl)acetic acid (44) (47 mg, 0.071 mmol, 72% yield). H NMR
(400 MHz, CDCl3) δ 7.31−7.75 (bs, 2H), 6.89−7.25 (m, 6H), 4.96
(d, J = 10.56 Hz, 1H), 4.27 (t, J = 12.03 Hz, 1H), 4.06 (br. s., 3H),
3.34−3.68 (m, 2H), 2.91 (d, J = 12.72 Hz, 1H), 2.64−2.83 (m, 1H),
2.44 (t, J = 14.28 Hz, 1H), 1.75−1.90 (m, 2H), 1.58−1.73 (m, 1H),
1.44 (s, 9H), 1.40 (br. s., 3H), 1.31−1.37 (m, 1H), 0.31−0.49 (m,
1H), 0.05−0.29 (m, 1H), −0.50− −0.18 (m, 1H), −1.48− −0.99 (m,
1H). Mass spectrum (ESI) m/z = 661.0 [M + H]+.
2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)thiazole-4-carboxylic Acid (32). To a mixture of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
ethanethioamide (56) (90 mg, 0.151 mmol) and potassium
bicarbonate (121 mg, 1.209 mmol) was added dry dioxane (1 mL)
and ethyl bromopyruvate (0.0568 mL, 0.453 mmol). The reaction was
allowed to stir under argon for 3 h. After this period, the mixture was
cooled to 0 °C, and a solution of trifluoroacetic acid anhydride (0.084
mL, 0.604 mmol) and pyridine (0.111 mL, 1.360 mmol) in dioxane (1
mL) was added. The reaction was allowed to warm to room
temperature overnight. The crude was washed with brine (30 mL) and
diethyl ether (3 × 30 mL). The combined organic layers were dried
over magnesium sulfate, filtered, and concentrated. The crude material
was absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (12 g), eluting with a
gradient of 0% to 50% acetone in DCM, to provide the intermediate
ester (Mass spectrum (ESI) m/z = 691.2 [M + H]+). This material was
diluted with THF (2 mL), MeOH (1 mL), and LiOH (1 mL, 2 M).
The mixture was stirred at 50 °C for 1 h, then cooled, neutralized with
1 M HCl (2 mL), and concentrated. Purification of the crude product
by RP-HPLC (45 to 65% AcCN/H2O, both containing 0.1% TFA, in
30 min, flow rate = 45 mL/min) provided 2-(((3R,5R,6S)-1-((S)-2-
(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazole-4-carbox-
ylic acid (32) (22 mg, 0.033 mmol, 22% yield over the last two steps).
1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.08−7.17 (m, 1H),
6.99−7.06 (m, 3H), 6.94 (s, 2H), 6.68−6.86 (m, 2H), 4.92 (d, J =
10.56 Hz, 1H), 4.21−4.39 (m, J = 12.52 Hz, 1H), 3.61 (d, J = 14.28
Hz, 1H), 3.41 (d, J = 14.28 Hz, 1H), 3.04−3.19 (m, J = 16.43 Hz, 1H),
2.85 (d, J = 13.89 Hz, 1H), 2.26 (t, J = 13.89 Hz, 2H), 1.65−1.91 (m,
2H), 1.36 (s, 9H), 1.33 (s, 3H), 0.25−0.43 (m, 1H), 0.05−0.23 (m,
1H), −0.54− −0.24 (m, 1H), −1.31− −0.98 (m, 1H). Mass spectrum
(ESI) m/z = 663.0 [M + H]+. HRMS (ESI) m/z found 663.1518 [M +
H]+, calcd for C32H36Cl2N2O5S2 663.1521.
2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)thiazole-5-carboxylic Acid (33). Part A: A solution of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
ethanethioamide (56) (100 mg, 0.168 mmol) and ethyl 2-chloro-3-
oxopropanoate (50.6 mg, 0.336 mmol) in toluene (1 mL) was stirred
at 100 °C in the microwave for 5 h. After this period, the crude was
concentrated and absorbed onto a plug of silica gel and purified by
chromatography through a Redi-Sep prepacked silica gel column (40
g), eluting with a gradient of 0% to 40% acetone in DCM, to provide
ethyl 2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)thiazole-5-carboxylate (77 mg, 0.111 mmol, 66% yield) as a
light yellow solid. Mass spectrum (ESI) m/z = 691.0 [M + H]+.
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)thiazol-4-yl)acetic Acid (37). Part A: To a solution of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
ethanethioamide (56) (70 mg, 0.118 mmol) in ethanol (1.2 mL) was
added 4-chloroacetoacetic acid methyl ester (70.8 μL, 0.470 mmol),
and the mixture was stirred at 90 °C for 3 h. The crude was
concentrated and taken onto the next step without further purification.
Mass spectrum (ESI) m/z = 691.0 [M + H]+.
Part B: To a solution of methyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-4-yl)acetate
(from Part A) in MeOH (1 mL) and THF (1 mL) was added lithium
hydroxide (1 mL, 2 M) and stirred at 50 °C for 1 h. After this period, 2
mL of 1 N HCL was added, and the crude was concentrated. The
crude material was absorbed onto a plug of silica gel and purified by
chromatography through a Redi-Sep prepacked silica gel column (12
g), eluting with a gradient of 0% to 40% MeOH/acetone (1:9 with 1%
acetic acid) and DCM, to provide 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-4-yl)acetic acid
(37) (45 mg, 0.066 mmol, 57% yield). 1H NMR (400 MHz,
CDCl3) δ 6.81−7.25 (m, 8H), 4.97 (d, J = 10.76 Hz, 1H), 4.24−4.55
(m, 1H), 3.86 (d, J = 1.37 Hz, 2H), 3.68 (d, J = 14.09 Hz, 1H), 3.41
(d, J = 14.09 Hz, 1H), 3.13−3.26 (m, 1H), 2.87−2.96 (m, 1H), 2.62−
2.73 (m, 1H), 2.20−2.29 (m, 1H), 2.15−2.19 (m, 1H), 1.76−1.85 (m,
1H), 1.43 (s, 9H), 1.38 (s, 3H), 0.10−0.45 (m, 2H), −0.56− −0.21
(m, 1H), −1.22− −0.95 (m, 1H). Mass spectrum (ESI) m/z = 677.0
[M + H]+. HRMS (ESI) m/z found 677.1682 [M + H]+, calcd for
C33H38Cl2N2O5S2 667.1677.
2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-
(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)thiazole-5-carboxylic Acid (35). Compound 35 was
prepared in a manner similar to a procedure described for the synthesis
1
of 33. H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.09−7.18 (m,
1H), 7.02 (dd, J = 1.37, 3.72 Hz, 2H), 6.90−6.96 (m, 1H), 6.76−6.86
(m, 1H), 6.34−6.73 (m, 3H), 4.91 (d, J = 10.56 Hz, 1H), 4.32 (t, J =
12.03 Hz, 1H), 3.63 (d, J = 13.89 Hz, 1H), 3.43 (d, J = 13.89 Hz, 1H),
3.06 (t, J = 11.64 Hz, 1H), 2.86 (d, J = 12.32 Hz, 1H), 2.60 (t, J = 9.19
Hz, 1H), 2.23 (t, J = 13.89 Hz, 1H), 1.87 (br. s., 1H), 1.36 (s, 9H),
1.35 (br. s., 3H), 0.30 (br. s., 2H), −0.34 (br. s., 1H), −1.05 (br. s.,
1H). Mass spectrum (ESI) m/z = 681.0 [M + H]+. HRMS (ESI) m/z
found 681.1429 [M + H]+, calcd for C32H35Cl2FN2O5S2 681.1427.
(3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-3-(thiazol-2-
ylmethyl)piperidin-2-one (36). Compound 36 was obtained as a side
1
product during the synthesis of 41 and 42. H NMR (400 MHz,
CDCl3) δ 7.81−7.97 (m, 1H), 7.31−7.53 (m, 3H), 7.10 (br. s., 5H),
4.96 (d, J = 10.37 Hz, 1H), 4.26−4.42 (m, J = 9.00 Hz, 1H), 3.77 (d, J
2978
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= 10.76 Hz, 1H), 3.48−3.66 (m, 1H), 3.02−3.25 (m, 1H), 2.92 (d, J =
16.24 Hz, 1H), 2.60−2.76 (m, 1H), 2.18−2.36 (m, 1H), 1.88−2.05
(m, 1H), 1.44 (s, 12H), 0.36 (br. s., 2H), −0.28 (br. s., 1H), −0.97 (br.
s., 1H). Mass spectrum (ESI) m/z = 637.0 [M + H]+. HRMS (ESI)
m/z found 637.1531 [M + H]+, calcd for C31H35Cl2FN2O3S2
637.1528.
2.37 (br. s., 1H), 2.09−2.27 (m, 1H), 1.20−1.41 (m, 12H), 0.36−0.57
(m, 1H), 0.15−0.34 (m, 1H), −0.20− −0.05 (m, 1H), −0.95− −0.67
(m, 1H). Mass spectrum (ESI) m/z = 598.2 [M + H]+.
1-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)-1H-pyrazole-4-carboxylic Acid (31). Part A: To a solution of
((3R,5R,6S)-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl
methanesulfonate (60) (115 mg, 0.192 mmol) in EtOH (2 mL) was
added hydrazine (0.181 mL, 5.76 mmol), and the mixture was stirred
in the microwave at 90 °C for 36 h. The conversion was about 40%
toward the desired product (61). The reaction mixture was
concentrated from toluene (10 mL). The concentrate was taken to
the next step without further purification assuming 40% yield. Mass
spectrum (ESI) m/z = 534.2 [M + H]+.
Part B: A solution of (3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
(hydrazinylmethyl)-3-methylpiperidin-2-one (61) (45 mg, 0.077
mmol) (containing starting material from the last step) and ethyl-2-
formyl-3-oxopropionate (0.111 mL, 0.770 mmol) in dimethylaceta-
mide (1 mL) was heated in the microwave at 100 °C for 2 h. After this
period, the reaction was concentrated, and the crude material was
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (12 g), eluting with a
gradient of 0% to 50% 9:1 acetone/MeOH in DCM to provide ethyl
1-(((3R,5R,6S)-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)-1H-pyrazole-4-carboxylate (62) (19 mg, 0.030 mmol, 38%
yield) as a yellow oil. Mass spectrum (ESI) m/z = 642.1 [M + H]+.
Part C: To a solution of 62 (19 mg, 0.031 mmol) in DCM (1 mL)
was added 3-chloroperoxybenzoic acid, (77%) (13.86 mg, 0.062
mmol) at 0 °C. After stirring for 30 min at this temperature, the
reaction was quenched with sat. aq. sodium bicarbonate (2 mL),
extracted with dichloromethane (3 × 10 mL), and brine (10 mL), and
the combined organic layer was dried over magnesium sulfate. The
crude mixture was concentrated.
(5R,6S)-1-((S)-2-(tert-Butylthio)-1-cyclopropylethyl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-3-((2-(trimethylsilyl)ethoxy)-
methyl)piperidin-2-one (58). In a 25 mL round-bottomed flask
evacuated and filled with argon was added THF (2 mL) and then
diisopropylamine (0.703 mL, 5.02 mmol). This solution was cooled to
−78 °C. At this temperature, butyllithium (2.0 mL, 4.89 mmol, 1 M)
was added, and the mixture was allowed to stir for 30 min. In a
separate flask, also evacuated and filled with argon, (5R,6S)-1-((S)-2-
(tert-butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methylpiperidin-2-one (57) (600 mg, 1.223 mmol) was
placed and diluted with THF (2 mL), and this solution was also cooled
to −78 °C. To this, the solution of freshly produced LDA was added
maintaining the temperature at −78 °C. The solution was allowed to
slowly reach room temperature overnight. After this period, the
mixture was quenched with sat. aq. ammonium chloride (50 mL) and
was washed with diethyl ether (3 × 50 mL). The combined organic
layer was dried over magnesium sulfate, filtered, and concentrated. The
crude material was absorbed onto a plug of silica gel and purified by
chromatography through a Redi-Sep prepacked silica gel column (80
g), eluting with a gradient of 0% to 50% acetone in hexanes, to provide
(5R,6S)-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-3-((2-(trimethylsilyl)ethoxy)-
methyl)piperidin-2-one (58) (393 mg, 0.633 mmol, 52% yield) as
white oil and a 2:1 mixture of isomers. Mass spectrum (ESI) m/z =
620.2 [M + H]+.
(3R,5R,6S)-1-((S)-2-(tert-Butylthio)-1-cyclopropylethyl)-5-(3-chlor-
ophenyl)-6-(4-chlorophenyl)-3-(hydroxymethyl)-3-methylpiperidin-
2-one (59). To a mixture of (5R,6S)-1-((S)-2-(tert-butylthio)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-3-
((2-(trimethylsilyl)ethoxy)methyl)piperidin-2-one (58) (2:1 mixture
of isomers) (0.393 g, 0.633 mmol) in DCM (6.33 mL), under argon,
at 0 °C, was added BF3·OEt2 (0.241 mL, 1.899 mmol). After 3 h, the
crude was diluted with sat. aq. NaHCO3 (20 mL). The aqueous layer
was extracted with DCM (3 × 20 mL). The combined organic layers
were washed with brine (20 mL), dried over magnesium sulfate, and
concentrated. Purification by flash chromatography, (80 g) eluting
with 10% to 20% acetone/hexanes, provided first (3S,5R,6S)-1-((S)-2-
(tert-butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-(hydroxymethyl)-3-methylpiperidin-2-one (56 mg, 0.108
mmol, 17% yield) (fastest eluting isomer) and then (3R,5R,6S)-1-((S)-
2-(tert-butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-(hydroxymethyl)-3-methylpiperidin-2-one (59) (110 mg,
0.211 mmol, 33% yield) (slowest eluting isomer). 1H NMR (400
MHz, CDCl3) δ 7.04−7.41 (m, 7H), 6.90 (d, J = 7.24 Hz, 1H), 4.78
(d, J = 9.59 Hz, 1H), 3.82−4.08 (m, 1H), 3.81−4.15 (m, 1H), 3.38−
3.63 (m, 2H), 2.44−3.01 (m, 2H), 2.21−2.37 (m, 1H), 2.05−2.16 (m,
1H), 1.52−1.99 (m, 2H), 1.37−1.45 (m, 9H), 1.33−1.37 (m, 3H),
0.44−0.61 (m, 1H), 0.29−0.43 (m, 1H), 0.01 (dd, J = 4.30, 8.80 Hz,
1H), −0.69 (br. s., 1H). Mass spectrum (ESI) m/z = 520.2 [M + H]+.
((3R,5R,6S)-1-((S)-2-(tert-Butylthio)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl Methanesulfonate (60). (3R,5R,6S)-1-((S)-2-(tert-Butylthio)-
1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-(hy-
droxymethyl)-3-methylpiperidin-2-one (59) (100 mg, 0.192 mmol)
was dissolved in DCM (0.768 mL). Then, methanesulfonic anhydride
(41.8 mg, 0.240 mmol) and triethylamine (0.0335 mL, 0.240 mmol)
were added at room temperature. After stirring for 30 min, the crude
was diluted with sat. aq. NaHCO3 (10 mL) and extracted with DCM
(2 × 20 mL). The combined organic layer was washed with brine,
dried over magnesium sulfate, and concentrated. The product was
used in the next step without further purification (quantitative yield).
1H NMR (400 MHz, CDCl3) δ 7.03−7.24 (m, 7H), 6.79−6.85 (m,
The crude was then diluted in THF (2 mL) and MeOH (1 mL),
and then lithium hydroxide (2 mL, 2 M) was added. After this period,
the reaction was neutralized (4 mL, 1 M HCl) and concentrated.
Purification of the crude product by RP-HPLC (45% to 65% AcCN/
H2O, both containing 0.1% TFA, in 30 min, flow rate = 45 mL/min)
provided 1-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)-1H-pyrazole-4-carboxylic acid (31) (6 mg, 9.28
1
μmol, 30% yield). H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 9.39
Hz, 2H), 6.83−7.22 (m, 7H), 6.71−6.80 (m, 1H), 4.93 (d, J = 10.56
Hz, 1H), 4.81 (d, J = 13.89 Hz, 1H), 4.29−4.39 (m, 1H), 4.24 (d, J =
13.89 Hz, 1H), 2.92 (d, J = 11.35 Hz, 1H), 2.71 (t, J = 10.27 Hz, 1H),
2.34−2.47 (m, 1H), 2.24 (t, J = 13.79 Hz, 1H), 1.94−2.10 (m, 2H),
1.44 (s, 9H), 1.36−1.40 (m, 3H), 0.37 (d, J = 7.63 Hz, 2H), −0.33 (br.
s., 1H), −0.94 (br. s., 1H). Mass spectrum (ESI) m/z = 646.2 [M +
H]+. HRMS (ESI) m/z found 646.1906 [M + H]+, calcd for
C32H37Cl2N3O5S 646.1909.
(3S,5R,6S)-3-(Bromomethyl)-1-((S)-2-(tert-butylthio)-1-cyclopro-
pylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methylpiperidin-
2-one (63). (3R,5R,6S)-1-((S)-2-(tert-Butylthio)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-(hydroxymethyl)-3-methyl-
piperidin-2-one (59) (326 mg, 0.626 mmol), carbon tetrabromide
(312 mg, 0.939 mmol), and triphenylphosphine (246 mg, 0.939
mmol) were stirred in CH3CN (1.25 mL). After stirring overnight, the
mixture was heated at 55 °C for 5 h. The resulting thick oil was diluted
in DCM and concentrated onto silica gel. This was purified by flash
chromatography (SiO2, 24 g), eluted with 0% to 15% ethyl acetate/
hexanes, to provide (3S,5R,6S)-3-(bromomethyl)-1-((S)-2-(tert-bu-
tylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
1
methylpiperidin-2-one (63) (313 mg, 0.536 mmol, 86% yield). H
NMR (400 MHz, CDCl3) δ 7.05−7.25 (m, 5H), 6.90−7.03 (m, 2H),
6.83 (td, J = 1.49, 7.38 Hz, 1H), 4.68 (d, J = 10.37 Hz, 1H), 4.00 (d, J
= 10.37 Hz, 1H), 3.78 (d, J = 10.37 Hz, 1H), 3.59 (t, J = 11.35 Hz,
1H), 3.05 (dt, J = 5.97, 10.51 Hz, 1H), 2.61 (dd, J = 4.50, 12.13 Hz,
1H), 4.54−4.76 (m, 2H), 4.25 (d, J = 9.78 Hz, 1H), 3.77−3.98 (m,
1H), 3.26−3.44 (m, 1H), 3.06−3.17 (m, 3H), 2.50−2.62 (m, 1H),
2979
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1H), 2.12−2.23 (m, 2H), 1.39−1.43 (m, 3H), 1.33−1.37 (m, 9H),
1.30 (s, 2H), 0.35−0.51 (m, 1H), 0.16−0.30 (m, 1H), −0.15 (qd, J =
4.92, 9.88 Hz, 1H), −0.96− −0.82 (m, 1H). Mass spectrum (ESI) m/z
= 584.0 [M + H]+.
HRMS (ESI) m/z found 615.1523 [M + H]+, calcd for
C28H36Cl2N2O5S2 615.1521.
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)thiazol-5-yl)acetic Acid (38). Part A: To a solution of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetyl
chloride (196 mg, 0.327 mmol) in THF (1.15 mL) was added ethyl 4-
(bis(trimethylsilyl)amino)but-2-ynoate (89 mg, 0.327 mmol) and then
tetra-N-butylammonium fluoride, 1 M solution in THF (0.052 mL,
0.052 mmol). The solution was stirred at room temperature overnight.
After this period, the reaction was acidified with 1 M HCl (5 mL),
then extracted with diethyl ether (3 × 30 mL) and brine (30 mL). The
combined organic layers were dried over magnesium sulfate, filtered,
and concentrated. The crude material was absorbed onto a plug of
silica gel and purified by chromatography through a Redi-Sep
prepacked silica gel column (40 g), eluting with a gradient of 0% to
40% acetone in DCM, to provide ethyl 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)but-2-ynoate
(67) (162 mg, 0.235 mmol, 72% yield) as a white solid. Mass spectrum
(ESI) m/z = 688.3 [M]+.
Part B: A solution of ethyl 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)but-2-ynoate (67)
(162 mg, 0.235 mmol) and Lawesson’s reagent (47.5 mg, 0.117
mmol) in toluene (0.435 mL) was stirred at 60 °C for 3 h. The
reaction was then cooled and concentrated. The crude material was
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (40 g), eluting with
isocratic 15% acetone in DCM to provide ethyl 2-(2-(((3R,5R,6S)-1-
((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-
(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-
acetate (68) (105 mg, 0.149 mmol, 63% yield) as a white solid. Mass
spectrum (ESI) m/z = 704.2 [M]+.
Part C: To a solution of ethyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)acetate
(68) (33 mg, 0.047 mmol) in THF (2 mL) and MeOH (1 mL) was
added lithium hydroxide (1 mL, 2 M). The reaction was stirred at 50
°C for 1 h. Purification of the crude product by RP-HPLC (25 to 75%
CH3CN/H2O, both containing 0.1% TFA, in 30 min, flow rate = 45
mL/min) provided 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)methyl)thiazol-5-yl)acetic acid (38) (18 mg, 0.027
mmol, 57% yield). 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 6.96−
7.26 (m, 6H), 6.79−6.92 (m, 2H), 4.94 (d, J = 10.76 Hz, 1H), 4.24−
4.45 (m, 1H), 3.92 (s, 2H), 3.64−3.77 (m, 1H), 3.55 (br. s., 1H), 3.24
(t, J = 11.93 Hz, 1H), 2.91 (d, J = 12.72 Hz, 1H), 2.71 (br. s., 1H),
2.31 (t, J = 13.69 Hz, 1H), 1.74−1.95 (m, 2H), 1.43 (s, 9H), 1.42 (br.
s., 3H), 0.34 (br. s., 1H), 0.22 (br. s., 1H), −0.35 (br. s., 1H), −1.06
(br. s., 1H). Mass spectrum (ESI) m/z = 677.2 [M + H]+. HRMS
(ESI) m/z found 677.1673 [M + H]+, calcd for C33H38Cl2N2O5S2
677.1677.
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)thiazol-5-yl)-2-methylpropanoic Acid (39). Part A: To a
solution of ethyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)methyl)thiazol-5-yl)acetate (68) (50 mg, 0.071
mmol) in DMF (0.269 mL) was added sodium tert-butoxide (18
mg, 0.203 mmol) at 0 °C. After stirring for 15 min, methyl iodide
(0.00836 mL, 0.135 mmol) was added. The reaction was quenched
with sat. aq. ammonium chloride (5 mL) and extracted with diethyl
ether (3 × 10 mL). The combined organic layers were dried over
magnesium sulfate, filtered, and concentrated. The crude material was
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (40 g), eluting with a
gradient of 0% to 40% acetone in DCM, to provide ethyl 2-(2-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
(3S,5R,6S)-1-((S)-2-(tert-Butylthio)-1-cyclopropylethyl)-5-(3-chlor-
ophenyl)-6-(4-chlorophenyl)-3-methyl-3-((pyrimidin-2-ylthio)-
methyl)piperidin-2-one (64). (3S,5R,6S)-3-(Bromomethyl)-1-((S)-2-
(tert-butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methylpiperidin-2-one (63) (104 mg, 0.178 mmol), 2-
mercapto-pyrimidine (19.99 mg, 0.178 mmol), and potassium
carbonate (29.6 mg, 0.214 mmol) were stirred in DMF (0.5 mL).
After stirring overnight, additional 12 mg of 2-mercaptopyrimidine and
30 mg of potassium carbonate were added. After 2 h, this was diluted
with water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over Na2SO4, and concentrated to provide
(3S,5R,6S)-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-5-(3-chloro-
phenyl)-6-(4-chlorophenyl)-3-methyl-3-((pyrimidin-2-ylthio)methyl)-
piperidin-2-one (64) (quantitative yield). 1H NMR (400 MHz,
CDCl3) δ 8.59 (d, J = 4.89 Hz, 1H), 8.50 (d, J = 4.89 Hz, 2H), 7.01−
7.25 (m, 6H), 6.80 (t, J = 1.86 Hz, 1H), 6.63−6.68 (m, 1H), 4.64 (d, J
= 10.37 Hz, 1H), 4.19 (d, J = 14.09 Hz, 1H), 4.13 (q, J = 7.04 Hz,
1H), 3.52−3.87 (m, 1H), 3.30−3.45 (m, 1H), 2.60 (dd, J = 4.70, 12.13
Hz, 1H), 2.09−2.22 (m, 2H), 1.43 (s, 3H), 1.35 (s, 9H), 1.29 (s, 2H),
0.36−0.47 (m, 1H), 0.17−0.29 (m, 1H), −0.15 (dd, J = 4.70, 9.59 Hz,
1H), −0.91 (d, J = 4.89 Hz, 1H). Mass spectrum (ESI) m/z = 614.2
[M + H]+.
(3S,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-3-((pyrimidin-2-
ylsulfonyl)methyl)piperidin-2-one (65). (3S,5R,6S)-1-((S)-2-(tert-Bu-
tylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-3-((pyrimidin-2-ylthio)methyl)piperidin-2-one (64) (119 mg,
0.194 mmol) was dissolved in THF (2 mL), and water (1 mL) and
oxone (714 mg, 1.162 mmol) were added. After stirring overnight, the
reaction mixture was diluted with water and extracted with ethyl
acetate. The organic layer was washed with brine, dried over Na2SO4,
and concentrated. The crude was purified by preparatory TLC eluting
with 30% acetone in hexanes to provide (3S,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-3-((pyrimidin-2-ylsulfonyl)methyl)piperidin-2-one
(65) (62 mg, 0.091 mmol, 48% yield). 1H NMR (400 MHz, CDCl3) δ
8.98 (d, J = 4.89 Hz, 2H), 7.55 (t, J = 4.89 Hz, 1H), 7.14−7.25 (m,
4H), 7.06−7.12 (m, 2H), 6.89−7.01 (m, 2H), 4.97 (d, J = 10.76 Hz,
1H), 4.25 (t, J = 12.42 Hz, 1H), 4.00−4.18 (m, 1H), 3.59 (dt, J = 4.11,
11.74 Hz, 1H), 2.88 (d, J = 14.48 Hz, 1H), 2.73 (br. s., 1H), 2.31−2.49
(m, 2H), 1.61 (s, 3H), 1.56 (br. s., 2H), 1.43 (s, 9H), 0.14−0.39 (m,
2H), −0.34 (br. s., 1H), −1.07 (br. s., 1H). Mass spectrum (ESI) m/z
= 678.1 [M + H]+.
((3S,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methanesulfonamide (14). (3S,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-
1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
3-((pyrimidin-2-ylsulfonyl)methyl)piperidin-2-one (65) (24 mg, 0.035
mmol) and potassium carbonate (25 mg, 0.181 mmol) were stirred in
MeOH (0.5 mL). After stirring overnight, complete conversion to the
sulfinic acid was observed. Then, hydroxylamine-o-sulfonic acid (31
mg, 0.053 mmol) was added. After stirring overnight, the reaction
mixture was diluted with water and extracted with ethyl acetate. The
organic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated. The crude was purified by preparative TLC eluted with
30% acetone/hexanes to provide ((3S,5R,6S)-1-((S)-2-(tert-butylsul-
fonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)methanesulfonamide (14) (10 mg, 0.016
1
mmol, 46% yield). H NMR (400 MHz, CDCl3) δ 7.05−7.24 (m,
6H), 7.00 (s, 1H), 6.89 (br. s., 1H), 5.38 (br. s., 2H), 4.96 (d, J = 10.56
Hz, 1H), 4.34 (t, J = 11.44 Hz, 1H), 3.45−3.74 (m, 2H), 3.31 (t, J =
11.15 Hz, 1H), 2.91 (d, J = 13.69 Hz, 1H), 2.73 (br. s., 1H), 2.46 (t, J
= 13.60 Hz, 1H), 1.95 (dd, J = 2.93, 13.89 Hz, 1H), 1.90 (br. s., 1H),
1.60 (s, 3H), 1.44 (s, 9H), 0.19−0.47 (m, 2H), −0.32 (br. s., 1H),
−1.08 (br. s., 1H). Mass spectrum (ESI) m/z = 615.1 [M + H]+.
2980
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chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)thiazol-5-yl)-2-methylpropanoate (32 mg, 0.044 mmol, 62%
yield). Mass spectrum (ESI) m/z = 733.2 [M + H]+.
2.21−2.48 (m, 2H), 1.84 (br. s., 1H), 1.44 (s, 9H), 1.37 (s, 3H), 0.19−
0.45 (m, 2H), −0.27 (br. s., 1H), −1.08 (br. s., 1H). Mass spectrum
(ESI) m/z = 655.2 [M + H]+.
Part B: To a solution of ethyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-2-methyl-
propanoate (32 mg, 0.044 mmol) in THF (2 mL) and MeOH (1 mL)
was added lithium hydroxide (1 mL, 2 M). The solution was stirred at
50 °C for 30 min. After this period, the reaction was neutralized (2
mL, 1 M HCl) and concentrated. Purification of the crude product by
RP-HPLC (25 to 75% CH3CN/H2O, both containing 0.1% TFA, in
30 min, flow rate =45 mL/min) provided 2-(2-(((3R,5R,6S)-1-((S)-2-
(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-2-
methylpropanoic acid (39) (23 mg, 0.033 mmol, 75% yield). 1H NMR
(400 MHz, CDCl3) δ 7.71 (s, 1H), 7.02−7.26 (m, 6H), 6.96 (s, 1H),
6.81−6.90 (m, 1H), 4.94 (d, J = 10.76 Hz, 1H), 4.37 (t, J = 11.84 Hz,
1H), 3.73 (d, J = 14.28 Hz, 1H), 3.45 (d, J = 14.28 Hz, 1H), 3.07−3.22
(m, 1H), 2.61−2.77 (m, 2H), 2.27 (t, J = 13.79 Hz, 1H), 1.89−1.99
(m, 1H), 1.84 (d, J = 11.93 Hz, 1H), 1.72 (d, J = 0.98 Hz, 6H), 1.44 (s,
9H), 1.43 (s, 3H), 0.13−0.45 (m, 2H), −0.34 (br. s., 1H), −1.03 (br.
s., 1H). Mass spectrum (ESI) m/z = 705.0 [M + H]+. HRMS (ESI)
m/z found 705.2002 [M + H]+, calcd for C35H42Cl2N2O5S2 705.1990.
1-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)thiazol-5-yl)cyclopropanecarboxylic Acid (40). Com-
pound 40 was synthesized through a procedure similar to that
Methyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopro-
pylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)methyl)thiazol-5-yl)acetate (71). Part A: To a
200-mL round-bottomed flask was added methyl potassium malonate
(4.76 g, 30.5 mmol) and magnesium chloride (2.178 g, 22.88 mmol)
in 30 mL of THF. This mixture was allowed to stir at 50 °C for 4 h.
2-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-
6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)acetamido)acetic acid (70) (5.00 g, 7.63 mmol) was dissolved
in THF (40 mL). Then, 1,1′-carbonyldiimidazole (4.33 g, 26.7 mmol)
was added portionwise to the solution. The mixture was stirred for 2 h
at 50 °C. After this period, the above mixture was added to the methyl
magnesium malonate suspension at 50 °C, and the reaction was
allowed to stir at 50 °C overnight. The reaction mixture was diluted
with EtOAc and washed with 1 M sodium bisulfate. The aqueous layer
was extracted twice with EtOAc and washed with sat. aq. NaHCO3 and
brine. The combined organic layers were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude material was
purified by flash column chromatography (120 g SiO2, 25% to 40%
acetone/hexanes gradient over 15 min) to give methyl 4-(2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetamido)-3-oxobutanoate (71) (3.40 g, 4.78 mmol, 63% yield).
Mass spectrum (ESI) m/z = 711.2 [M + H]+.
Part B: To a 500-mL round-bottomed flask was added methyl 4-(2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetamido)-3-oxobutanoate (71) (4.00 g, 5.62 mmol) and
Lawesson’s reagent (2.273 g, 5.62 mmol) in toluene (50 mL). The
mixture was heated at reflux for 3 h. After this time, the reaction was
cooled and concentrated under reduced pressure. The crude product
was purified by flash column chromatography (330 g SiO2, 35% to
50% EtOAc/hexanes gradient over 20 min) to give methyl 2-(2-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)methyl)thiazol-5-yl)acetate (72) (2.8 g, 71% yield). 1H NMR
(500 MHz, CDCl3) δ 7.62 (s, 1H), 7.03−7.14 (m, 3H), 6.96 (s, 4H),
4.95 (d, J = 10.76 Hz, 1H), 4.38 (t, J = 11.98 Hz, 1H), 3.89 (s, 2H),
3.69 (d, J = 14.18 Hz, 1H), 3.32 (d, J = 14.18 Hz, 1H), 3.01 (br. s.,
1H), 2.93 (d, J = 12.72 Hz, 1H), 2.60−2.76 (m, 1H), 2.23 (t, J = 13.82
Hz, 1H), 2.18 (s, 3H), 2.00 (br. s., 1H), 1.90 (dd, J = 2.32, 13.57 Hz,
1H), 1.44 (s, 9H), 1.40 (s, 3H), 0.26−0.46 (m, 2H), −0.29 (d, J = 3.18
Hz, 1H), −1.00 (br. s., 1H). Mass spectrum (ESI) m/z = 709.2 [M +
H]+.
1
described for the synthesis of 39. H NMR (400 MHz, CDCl3) δ
7.34−7.43 (m, 1H), 6.84 (s, 6H), 6.74 (s, 1H), 6.63 (d, J = 6.26 Hz,
1H), 4.71 (d, J = 10.76 Hz, 1H), 4.01−4.20 (m, J = 8.02 Hz, 1H), 3.49
(d, J = 13.11 Hz, 2H), 3.27 (d, J = 13.89 Hz, 2H), 2.82−3.03 (m, 1H),
2.48 (br. s., 1H), 2.06 (t, J = 13.79 Hz, 1H), 1.68 (br. s., 4H), 1.25−
1.30 (m, 1H), 1.21 (s, 12H), −0.08−0.22 (m, 2H), −0.57 (br. s., 1H),
−1.26 (br. s., 1H). Mass spectrum (ESI) m/z = 703.0 [M + H]+.
2-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)acetamido)acetic Acid (70). Part A: A solution of 2-
((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetic acid (69)17 (22.00 g, 36.8 mmol) and glycine methyl ester
hydrochloride (11.54 g, 92 mmol) in DMF (100.0 mL) was treated
with EDC (17.62 g, 92 mmol), HOAT (12.51 g, 92 mmol), and
sodium hydrogencarbonate (15.44 g, 184 mmol) successively at room
temperature. The reaction was stirred at 40 °C overnight. After this
period, the reaction was diluted (1 N aq. HCl), extracted (2 ×
EtOAc), and washed (1 × sat. aq. NaHCO3, and 2 × brine). The
combined organic layer was dried (Na2SO4) and concentrated under
reduced pressure. The product of this step was used in the next
process without further purification. Mass spectrum (ESI) m/z = 669.2
[M + H]+.
Part B: To a solution of methyl 2-(2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)acetate (see
part A) (24.60 g, 36.7 mmol) in THF (40 mL), MeOH (40 mL),
and H2O (80 mL) was added lithium hydroxide (4.40 g, 184 mmol).
The reaction was stirred at room temperature.
The reaction was quenched with ice-cold 1 N aq. HCl, extracted
with EtOAc, and washed with brine. The combined organic layer were
dried (Na2SO4) and concentrated under reduced pressure. The crude
product crystallized with 60 mL of EtOAc/50 mL hexanes, and the
mother liquor was purified by flash column chromatography: 220g
SiO2, with a gradient of 35% to 50% acetone in hexanes, over 15 min
to give 2-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-ox-
opiperidin-3-yl)acetamido)acetic acid (70) (21.6 g, 32.9 mmol, 90%
yield over the last two steps). 1H NMR (500 MHz, CDCl3) δ 7.45 (br.
s., 1H), 7.14−7.37 (m, 2H), 7.09−7.15 (m, 2H), 7.03 (s, 1H), 6.90−
6.97 (m, 1H), 4.98 (d, J = 10.51 Hz, 1H), 4.29 (t, J = 11.49 Hz, 1H),
4.07−4.18 (m, 2H), 3.25−3.39 (m, 1H), 3.06 (d, J = 13.45 Hz, 1H),
2.87−2.94 (m, 1H), 2.83 (d, J = 13.45 Hz, 1H), 2.68 (br. s., 1H),
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)methyl)thiazol-5-yl)acetic Acid (4). To a solution of
methyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-ox-
opiperidin-3-yl)methyl)thiazol-5-yl)acetate (72) (5.70 g, 8.03 mmol)
in THF (60 mL), MeOH (40 mL), and H2O (80 mL) was added
lithium hydroxide (0.962 g, 40.2 mmol). The reaction was stirred at
room temperature for 1.5 h. The reaction was quenched with ice-cold
1 N aq. HCl, extracted with EtOAc, and washed with brine. The
combined organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by flash column chromatography (220 g SiO2, 40% to 50% acetone/
hexanes gradient over 10 min) to provide 2-(2-(((3R,5R,6S)-1-((S)-2-
(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-
5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-
1
acetic acid (4) (5.10 g, 91% yield). H NMR (500 MHz, CDCl3) δ
7.78 (br. s., 1H), 6.96−7.17 (m, 5H), 6.89 (d, J = 6.60 Hz, 1H), 4.96
(d, J = 10.51 Hz, 1H), 4.34 (t, J = 11.86 Hz, 2H), 3.96 (s, 2H), 3.67−
3.82 (m, 2H), 3.61 (br. s., 1H), 2.91 (d, J = 12.96 Hz, 1H), 2.69 (br. s.,
1H), 2.32 (t, J = 13.94 Hz, 1H), 1.80−1.97 (m, 2H), 1.44 (s, 9H), 1.26
(s, 3H), 0.37 (d, J = 4.16 Hz, 2H), −0.29 (br. s., 1H), −0.98 (br. s.,
1H). 13C NMR (150 MHz, (CD3)2SO) δ 175.4, 171.9, 165.8, 158.9,
2981
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158.6, 144.2, 141.9, 141.6, 133.3, 131.9, 130.9, 127.7, 127.4, 126.6,
119.6, 119.5, 118.7, 116.7, 114.8, 69.9, 59.9, 58.7, 58.6, 44.4, 43.9, 43.6,
41.2, 38.2, 33.4, 26.5, 14.6, 7.3, 4.6. Mass spectrum (ESI) m/z = 695.2
[M + H]+. HRMS (ESI) m/z found 695.1584 [M + H]+, calcd for
C33H37Cl2FN2O5S2 695.1583.
acetate (12 mg, 0.017 mmol) in THF (2 mL) and MeOH (1 mL) was
added lithium hydroxide (1 mL, 2 M). The solution was stirred at 50
°C for 30 min. After this period, the reaction was neutralized (2 mL, 1
M HCl) and concentrated. Purification by preparative HPLC (25−
75% acetonitrile/water both solvents containing 0.1% TFA) provided
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-
(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)oxazol-5-yl)acetic acid (43) (8 mg, 0.012 mmol, 69%
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)methyl)thiazol-5-yl)-2-fluoroacetic Acid (41) and 2-(2-
(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)thiazol-5-yl)-2,2-difluoroacetic Acid (42). Part A: To
a solution of ethyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)acetate (72) (21 mg,
0.029 mmol) in DMF (0.269 mL) was added sodium tert-butoxide
(5.86 mg, 0.061 mmol) at 0 °C. After stirring for 15 min, N-
fluorobenzenesulphonimide (19.21 mg, 0.061 mmol) was added. After
30 min, LCMS showed a mixture of compounds containing unreacted
starting material, mono-, difluorination, and deacetylation products.
The crude mixture was quenched (0.5 mL water), concentrated, and
taken into the next step without further purification.
Part B: The mixture from the previous step was diluted in THF (2
mL), MeOH (1 mL), and 1 mL of 2 M LiOH and heated and 50 °C
for 30 min. After this period, the reaction was cooled, quenched with 1
N HCl (2 mL), and concentrated. Purification by preparative HPLC
(25−75% CH3CN/H2O both solvents containing 0.1% TFA)
provided, in order of elution, first 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-2-
fluoroacetic acid (41) (2.4 mg, 3.36 μmol, 12% yield) as a 1:1 mixture
of isomers; then, 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)methyl)thiazol-5-yl)-2,2-difluoroacetic acid
(42) (0.8 mg, 4% yield, Mass spectrum (ESI) m/z = 731.0 [M + H]+);
and then, (3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropyleth-
yl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-3-(thia-
zol-2-ylmethyl)piperidin-2-one (36) (3 mg, 16% yield), as white
amorphous solids.
1
yield). H NMR (400 MHz, CDCl3) δ 7.29 (s, 2H), 7.14 (d, J = 5.28
Hz, 2H), 7.02 (s, 2H), 6.91−6.98 (m, 1H), 5.00 (d, J = 10.37 Hz, 1H),
4.39 (t, J = 12.03 Hz, 1H), 3.78 (s, 2H), 3.25−3.48 (m, 2H), 3.07−
3.23 (m, 1H), 2.94 (d, J = 13.89 Hz, 1H), 2.68 (t, J = 10.76 Hz, 1H),
2.29 (br. s., 2H), 1.87−2.01 (m, 1H), 1.73 (d, J = 14.09 Hz, 1H), 1.46
(s, 9H), 1.42 (s, 3H), 0.38 (br. s., 2H), −0.26 (br. s., 1H), −0.98 (br.
s., 1H). Mass spectrum (ESI) m/z = 679.0 [M + H]+. HRMS (ESI)
m/z found 679.1812 [M + H]+, calcd for C33H37Cl2FN2O6S 679.1812.
Methyl 6-(((2S,5R,6R)-4-((S)-2-((tert-butyldimethylsilyl)oxy)-1-cy-
clopropylethyl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-
oxomorpholin-2-yl)methyl)nicotinate and methyl 6-(((2R,5R,6R)-4-
((S)-2-((tert-butyldimethylsilyl)oxy)-1-cyclopropylethyl)-6-(3-chloro-
phenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpholin-2-yl)methyl)-
nicotinate (74). Part A: Lithium bis(trimethylsilyl)amide (1.0 M
solution in tetrahydrofuran, 17.3 mL, 17.3 mmol) was added to an
oven-dried, 3-neck round-bottom flask containing (5R,6R)-4-((S)-2-
((tert-butyldimethylsilyl)oxy)-1-cyclopropylethyl)-6-(3-chlorophenyl)-
5-(4-chlorophenyl)morpholin-3-one28 in tetrahydrofuran at −78 °C
under argon. Methyl iodide (1.08 mL, 17.3 mmol) in tetrahydrofuran
was added subsequently. After stirring at −78 °C for 1 h, the reaction
was quenched with sat. aq. ammonium chloride (20 mL), extracted
with ethyl acetate (2 × 30 mL), and washed with brine (30 mL). The
combined organic layer was dried over Na2SO4 and concentrated
under reduced pressure. Purification of the residue by flash column
chromatography (silica gel, gradient elution of 0% to 80% ethyl acetate
in hexanes) afforded (2S,5R,6R)-4-((S)-2-((tert-butyldimethylsilyl)-
oxy)-1-cyclopropylethyl)-6-(3-chloro-phenyl)-5-(4-chlorophenyl)-2-
methylmorpholin-3-one and (2R,5R,6R)-4-((S)-2-((tert-
butyldimethylsilyl)oxy)-1-cyclopropylethyl)-6-(3-chloro-phenyl)-5-(4-
chlorophenyl)-2-methylmorpholin-3-one (73) as a 2:1 mixture of
diastereomers. Mass spectrum (ESI) m/z = 533.2 [M + H]+.
Characterization of 41. 1H NMR (400 MHz, CDCl3) δ 7.89−8.03
(m, 1H), 7.10 (d, J = 1.96 Hz, 7H), 5.95−6.30 (m, 1H), 4.86−5.11
(m, 1H), 4.24−4.44 (m, 1H), 3.64−3.79 (m, 1H), 3.34−3.54 (m, 1H),
3.06−3.21 (m, 1H), 2.88−3.02 (m, 1H), 2.18−2.39 (m, 1H), 1.78−
2.08 (m, 2H), 1.44 (s, 12H), 0.38 (br. s., 2H), −0.26− −0.24 (m, 1H),
−0.38− −0.19 (m, 1H), −0.99 (br. s., 1H). Mass spectrum (ESI) m/z
= 713.0 [M + H]+. HRMS (ESI) m/z found 713.1489 [M+H]+, calcd
for C33H36Cl2F2N2O5S2 713.1486.
Methyl 6-(bromomethyl)nicotinate.
2-(2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopi-
peridin-3-yl)methyl)oxazol-5-yl)acetic Acid (43). Part A: To a
solution of methyl 4-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-
cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-
methyl-2-oxopiperidin-3-yl)acetamido)-3-oxobutanoate (71) (19 mg,
0 . 0 2 7 m m o l ) i n D C E ( 1 . 3 m L ) w a s a d d e d
(methoxycarbonylsulfamoyl)triethyl-ammonium hydroxide (25.4 mg,
0.107 mmol). The solution was stirred for 5 h in the microwave. The
reaction did not reach completion; thus, additional
(methoxycarbonylsulfamoyl)triethyl-ammonium hydroxide (25.4 mg,
0.107 mmol) was added, and the reaction was set again in the
microwave for 2 more hours, after which period only the desired
product was observed. The crude mixture was concentrated and
absorbed onto a plug of silica gel and purified by chromatography
through a Redi-Sep prepacked silica gel column (40 g), eluting with a
gradient of 5% to 60% acetone in hexanes, to provide methyl 2-(2-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)methyl)oxazol-5-yl)acetate (12 mg, 0.017 mmol, 65% yield) as a
white solid. Mass spectrum (ESI) m/z = 693.0 [M + H]+.
In a round-bottomed flask equipped with a reflux condenser, a
mixture of methyl 6-methyl nicotinate (10.0 g, 66.2 mmol), N-
bromosuccinimide (7.1 g, 39.7 mmol), and benzoyl peroxide (1.6 g,
6.62 mmol) in carbon tetrachloride was stirred under nitrogen at 75
°C for 2 days. The cooled reaction mixture was filtered, the filter cake
was washed with dichloromethane, and the filtrate was concentrated
under reduced pressure. Purification of the residue by flash column
chromatography (silica gel column, gradient elution of 5% to 25%
ethyl acetate in hexanes) afforded methyl 6-(bromomethyl)nicotinate
as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.17 (d, J = 1.56 Hz,
1H), 8.31 (dd, J = 2.15, 8.02 Hz, 1H), 7.54 (d, J = 8.22 Hz, 1H), 4.59
(s, 2H), 3.97 (s, 3H). MS (ESI) m/z: 230.0 [M + H]+.
Part B: A three-necked, oven-dried, round-bottomed flask was
cooled under argon and charged with diisopropylamine (2.48 mL, 17.7
mmol) and tetrahydrofuran (20 mL) and cooled to 0 °C. n-
Butyllithium (2.5 M in hexanes, 7.08 mL, 17.70 mmol) was added
dropwise, and the reaction was stirred for 10 min at 0 °C. The reaction
mixture was cooled to −78 °C, and a solution of (2S,5R,6R)-4-((S)-2-
((tert-butyldimethylsilyl)oxy)-1-cyclopropylethyl)-6-(3-chloro-phe-
nyl)-5-(4-chlorophenyl)-2-methylmorpholin-3-one and (2R,5R,6R)-4-
((S)-2-((tert-butyldimethylsilyl)oxy)-1-cyclopropylethyl)-6-(3-chloro-
phenyl)-5-(4-chlorophenyl)-2-methylmorpholin-3-one (73) in tetra-
hydrofuran (30 mL), also at −78 °C, was added. After stirring at −78
°C for 15 min, a solution of methyl 6-(bromomethyl)nicotinate (4.07
Part B: To a solution of ethyl 2-(2-(((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)oxazol-5-yl)-
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g, 17.70 mmol) in tetrahydrofuran (20 mL) at −78 °C was added. The
reaction was stirred at this temperature for 1 h.
was dried over magnesium sulfate, filtered, and concentrated.
Purification of the residue by flash column chromatography (silica
gel column, gradient elution of 0% to 40% acetone in hexanes)
afforded 77a. Mass spectrum (ESI) m/z = 673.0 [M + H]+.
The resulting mixture was quenched with saturated ammonium
chloride (50 mL), extracted with ethyl acetate (3 × 50 mL), and
washed with water (50 mL) and brine (50 mL). The combined
organic layer was dried over magnesium sulfate, filtered, and
concentrated. Purification of the residue by flash column chromatog-
raphy (silica gel column, gradient elution of 0% to 25% acetone in
hexanes) afforded 74.
Methyl 6-(((2R,5R,6R)-4-((S)-2-((tert-butyldimethylsilyl)oxy)-1-cy-
clopropylethyl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-
oxomorpholin-2-yl)methyl)nicotinate (74a, Faster Eluting Isomer).
1H NMR (400 MHz, CDCl3) δ 9.00 (d, J = 2.15 Hz, 1H), 8.10 (d, J =
7.63 Hz, 1H), 7.23 (d, J = 8.22 Hz, 1H), 7.11 (d, J = 8.22 Hz, 2H),
7.00−7.08 (m, 2H), 6.92 (t, J = 7.83 Hz, 1H), 6.71−6.86 (m, 2H),
6.53 (d, J = 7.63 Hz, 1H), 4.85 (d, J = 9.78 Hz, 1H), 4.69 (d, J = 9.98
Hz, 1H), 4.20 (t, J = 9.98 Hz, 1H), 3.84 (s, 3H), 3.77 (d, J = 13.50 Hz,
1H), 3.38−3.43 (m, J = 4.50 Hz, 1H), 3.33 (d, J = 14.28 Hz, 1H),
2.04−2.18 (m, 1H), 1.52 (s, 3H), 0.87 (s, 9H), 0.81 (d, J = 0.78 Hz,
1H), 0.25−0.32 (m, 2H), 0.00 (s, 3H), −0.04 (s, 3H), −0.34− −0.26
(m, J = 8.80 Hz, 1H), −0.63 (dd, J = 4.21, 9.29 Hz, 1H). Mass
spectrum (ESI) m/z = 683.2 [M + H]+.
Part B: Lithium hydroxide (1 mL, 2 M solution) was added to a
solution of 77a (30 mg, 0.045 mmol) in methanol (1 mL) and
tetrahydrofuran (1 mL) at room temperature. The resulting mixture
was heated at 50 °C for 1 h. The reaction mixture was cooled,
quenched with a 10% citric acid solution (5 mL), and extracted with
ethyl acetate (3 × 10 mL). The combined organic layers were dried
over magnesium sulfate, filtered, and concentrated. Purification of the
residue by flash column chromatography (silica gel column, gradient
elution of 0% to 100% ethyl acetate in hexanes) afforded 46. 1H NMR
(400 MHz, CDCl3) δ 9.33 (s, 1H), 8.56 (d, J = 8.61 Hz, 1H), 7.63 (d,
J = 8.22 Hz, 1H), 7.19−7.26 (m, 2H), 7.11−7.18 (m, 3H), 7.04 (t, J =
8.12 Hz, 2H), 6.82 (d, J = 7.63 Hz, 1H), 5.17 (d, J = 9.78 Hz, 1H),
5.06 (d, J = 9.78 Hz, 1H), 4.15−4.27 (m, J = 12.72 Hz, 1H), 3.89 (d, J
= 13.11 Hz, 2H), 3.72 (d, J = 13.30 Hz, 1H), 2.92 (d, J = 13.50 Hz,
1H), 2.71 (t, J = 9.68 Hz, 1H), 1.81−1.97 (m, 1H), 1.68 (s, 3H), 1.40
(s, 9H), 0.31−0.54 (m, 2H), −0.33− −0.20 (m, 1H), −0.79− −0.59
(m, 1H). Mass spectrum (ESI) m/z = 659.0 [M + H]+. HRMS (ESI)
m/z found 659.1748 [M+H]+, calcd for C33H36Cl2N2O6S 679.1749.
6-(((2S,5R,6R)-4-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-
(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpholin-2-
yl)methyl)nicotinic Acid (47). Compound 47 was synthesized in a
manner similar to that for 46 starting from 74b. 1H NMR (400 MHz,
CDCl3) δ 9.33 (s, 1H), 8.56 (d, J = 8.61 Hz, 1H), 7.63 (d, J = 8.22 Hz,
1H), 7.27 (br s, 4H), 7.11−7.17 (m, 2H), 7.04 (t, J = 8.12 Hz, 1H),
6.82 (d, J = 7.63 Hz, 1H), 5.17 (d, J = 9.78 Hz, 1H), 5.06 (d, J = 9.78
Hz, 1H), 4.13−4.28 (m, J = 11.74 Hz, 1H), 3.89 (d, J = 13.11 Hz, 2H),
3.72 (d, J = 13.30 Hz, 1H), 2.91 (d, J = 13.89 Hz, 1H), 2.62−2.79 (m,
1H), 1.80−1.98 (m, 1H), 1.68 (s, 3H), 1.40 (s, 9H), 0.27−0.54 (m,
2H), −0.36− −0.19 (m, 1H), −0.75− −0.59 (m, 1H). Spectrum (ESI)
m/z = 659.0 [M + H]+. HRMS (ESI) m/z found 659.1755 [M + H]+,
calcd for C33H36Cl2N2O6S 679.1749.
Characterization Data for the Slowest Eluting Isomer, Methyl 6-
(((2S,5R,6R)-4-((S)-2-((tert-butyldimethylsilyl)oxy)-1-cyclopropyleth-
yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpho-
1
lin-2-yl)methyl)nicotinate (74b, Slower Eluting Isomer). H NMR
(400 MHz, CDCl3) δ 9.20−9.25 (m, 1H), 8.21−8.28 (m, 1H), 7.43
(d, J = 8.02 Hz, 1H), 7.24 (br s, 2H), 7.11−7.19 (m, 3H), 7.02 (t, J =
7.83 Hz, 1H), 6.92−6.98 (m, 1H), 6.86−6.91 (m, 1H), 6.52 (d, J =
7.83 Hz, 1H), 4.78 (d, J = 9.78 Hz, 1H), 4.62 (d, J = 9.78 Hz, 1H),
3.98−4.06 (m, 1H), 3.96 (s, 3H), 3.89−3.95 (m, 1H), 3.73 (d, J =
13.50 Hz, 1H), 3.46−3.52 (m, J = 5.67, 10.17 Hz, 1H), 1.76 (s, 3H),
1.31−1.43 (m, 1H), 0.91 (s, 9H), 0.28−0.48 (m, 2H), 0.05 (s, 3H),
0.02 (s, 3H), −0.15− −0.02 (m, 1H), −0.56− −0.38 (m, 1H). Mass
spectrum (ESI) m/z = 683.1 [M + H]+.
Methyl 6-(((2R,5R,6R)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-4-
((S)-1-cyclopropyl-2-hydroxyethyl)-2-methyl-3-oxomorpholin-2-yl)-
methyl)nicotinate (75a). Tetrabutylammonium fluoride (1.0 M
solution in tetrahydrofuran, 0.70 mL, 0.70 mmol) was added to a
solution of 74a (300 mg, 0.44 mmol) in tetrahydrofuran (5 mL) and
stirred at ambient temperature overnight. The resulting mixture was
quenched with saturated ammonium chloride (10 mL) and washed
with ethyl acetate (3 × 15 mL). The combined organic layer was dried
over magnesium sulfate, filtered, and concentrated. Purification of the
residue by flash column chromatography (silica gel column, gradient
elution of 0% to 25% ethyl acetate in hexanes) afforded 75a (60%). 1H
NMR (400 MHz, CDCl3) δ 9.10 (dd, J = 0.78, 2.15 Hz, 1H), 8.16−
8.24 (m, J = 5.87 Hz, 1H), 7.29 (d, J = 8.22 Hz, 1H), 7.17−7.25 (m,
3H), 7.11 (t, J = 1.66 Hz, 1H), 7.07 (t, J = 7.92 Hz, 1H), 6.91−7.02
(m, 2H), 6.61 (d, J = 7.63 Hz, 1H), 4.85−4.92 (m, 1H), 4.73−4.82
(m, 1H), 3.95 (s, 3H), 3.82 (s, 1H), 3.56−3.66 (m, 1H), 3.34−3.48
(m, 2H), 3.12−3.25 (m, 2H), 1.60 (s, 3H), 0.68−0.78 (m, 1H), 0.48−
0.68 (m, 2H), 0.21−0.31 (m, 1H), 0.04−0.19 (m, 1H). Mass spectrum
(ESI) m/z = 569.0 [M + H]+.
Methyl 6-(((2R,5R,6R)-4-((S)-2-(tert-butylthio)-1-cyclopropyleth-
yl)-6-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpho-
lin-2-yl)methyl)nicotinate (76a). Cyanomethylenetributylphosphor-
ane (0.356 g, 1.48 mmol) and tert-butanethiol (0.133 g, 1.48 mmol)
were added to a solution of 75a (210 mg, 0.37 mmol) in toluene (2
mL), and the mixture was stirred at 70 °C overnight. The resulting
mixture was cooled and concentrated. Purification of the residue by
flash column chromatography (silica gel column, gradient elution of
0% to 35% ethyl acetate in hexanes) afforded 76a (61%). Mass
spectrum (ESI) m/z = 641.1 [M + H]+.
2-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)isonicotinic Acid (19). Part A: An oven-dried, cooled under
Argon, round-bottomed flask was charged with diisopropylamine
(0.065 mL, 0.459 mmol) and THF (1 mL), and then butyllithium
(0.287 mL, 0.459 mmol) was added at 0 °C. The reaction was stirred
at this temperature for 10 min. The reaction solution was then cooled
to −78 °C. To this, a solution of (5R,6S)-1-((S)-2-(tert-butylthio)-1-
cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methylpi-
peridin-2-one (57) (150 mg, 0.306 mmol) was added in THF (1 mL)
at −78 °C. The reaction was stirred at −78 °C for 15 min, then
warmed up to −20 °C and stirred at this temperature for an additional
15 min. The reaction was then cooled again to −78 °C, and a solution
of tert-butyl 2-(bromomethyl)isonicotinate (125 mg, 0.459 mmol) in
THF (1 mL) was added. The reaction was stirred for 4 h at −78 °C.
After this time, the reaction mixture was diluted with EtOAc and
washed with sat. NH4Cl, water, brine. The crude was purified by
column (silica gel, eluting with ethyl acetate and hexanes) to give a 3:1
mixture of isomers of tert-butyl 2-(((5R,6S)-1-((S)-2-(tert-butylthio)-
1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-
2-oxopiperidin-3-yl)methyl)isonicotinate (147 mg, 0.216 mmol, 71%
yield). Mass spectrum (ESI) m/z = 681.2 [M + H]+.
Part B: To a solution of tert-butyl 2-(((5R,6S)-1-((S)-2-(tert-
butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophen-
yl)-3-methyl-2-oxopiperidin-3-yl)methyl)isonicotinate in N,N-dime-
thylformamide (1.5 mL) was added 3-chloroperoxybenzoic acid,
75% max. (145 mg, 0.647 mmol) at 0 °C. The reaction was allowed to
stir for 40 min after which it was quenched (1.5 mL of 1 M aq.
Na2S2O3), extracted (EtOAc), and washed (1 × sat. aq. NaHCO3, 1 ×
water, and 1 × brine). The combined organic layers were dried
(Na2SO4), filtered, and concentrated under reduced pressure to give a
crude of tert-butyl 2-(((5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclo-
propylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxo-
piperidin-3-yl)methyl)isonicotinate (167 mg, 0.234 mmol, quantitative
6-(((2R,5R,6R)-4-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-
(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-3-oxomorpholin-2-
yl)methyl)nicotinic Acid (46). Part A: 3-Chloroperbenzoic acid (77
wt.%, 0.031 g, 0.14 mmol) was added to a solution of 76a (60 mg, 0.07
mmol) in dichloromethane (3 mL) at 0 °C. The reaction was
quenched after 30 min with 1 N sodium thiosulfate (5 mL) and
washed with ethyl acetate (3 × 10 mL). The combined organic layer
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yield). The crude was used on the next reaction without further
purification. Mass spectrum (ESI) m/z = 713.2 [M + H]+.
Part B: To a vial containing tert-butyl 2-(6-(((3R,5R,6S)-1-((S)-2-
(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)pyridin-3-yl)-
acetate (obtained with a procedure similar to that depicted for the
synthesis of 19, part B) (4.0 mg, 5.50 μmol) was added formic acid
(0.211 μL, 5.50 μmol). The vial was heated on a hot plate at 40 °C for
6 h. The reaction mixture was concentrated. The compound was
loaded onto a Uniplate Silica Gel HLF (catalog number 47521; 250
μm) plate and eluted with 50% acetone/hexanes. The most polar band
was cut and eluted with 6 mL of EtOAc. The eluent was concentrated
to give 2-(6-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)pyridin-3-yl)acetic acid (23) (2.77 mg, 4.12 μmol,
75%) as a white pellet. 1H NMR (500 MHz, CD2Cl2) δ 8.48 (d, J = 2.0
Hz, 1H), 7.62 (dd, J = 2.3, 7.9 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H),
7.23−7.03 (m, 5H), 7.03−6.94 (m, 1H), 6.94−6.81 (m, 1H), 4.92 (d, J
= 10.8 Hz, 1H), 4.36 (br. s., 1H), 3.67 (s, 2H), 3.48 (d, J = 12.7 Hz,
1H), 3.21 (ddd, J = 2.9, 10.8, 13.7 Hz, 1H), 3.10 (d, J = 12.7 Hz, 1H),
2.94 (d, J = 12.5 Hz, 1H), 2.61 (br. s., 1H), 2.20−2.09 (m, 1H), 1.93
(br. s., 1H), 1.81 (dd, J = 2.9, 13.4 Hz, 1H), 1.40 (s, 9H), 1.27 (s, 3H),
0.33 (br. s., 1H), 0.24 (br. s., 1H), −0.32 (br. s., 1H), −1.05 (br. s.,
1H). Mass spectrum (ESI) m/z = 671.2 [M + H]+.
Part C: To a solution of tert-butyl 2-(((5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)isonicotinate (154 mg,
0.216 mmol) in DCM (3 mL) was added TFA (3 mL), and the
reaction was stirred at room temperature for 3 h. Concentration gave a
residue, which was purified by preparative HPLC to give 2-
(((3S,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)isonicotinic acid (22 mg, 0.033 mmol, 16% yield), and then 2-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)isonicotinic acid 19 (96 mg, 0.146 mmol, 68% yield).
1
Characterization for 19: H NMR (400 MHz, CDCl3) δ 8.98 (br. s.,
1H), 8.02−8.19 (m, 2H), 7.05−7.26 (m, 5H), 7.03 (s, 1H), 6.90 (d, J
= 5.28 Hz, 1H), 5.00 (d, J = 10.76 Hz, 2H), 4.33 (t, J = 11.84 Hz, 1H),
3.80 (d, J = 13.50 Hz, 1H), 3.29−3.53 (m, 2H), 2.89 (d, J = 13.50 Hz,
1H), 2.77 (br. s., 1H), 2.45 (t, J = 13.79 Hz, 1H), 1.90 (d, J = 12.72
Hz, 1H), 1.82 (br. s., 1H), 1.42 (s, 9H), 1.38 (s, 3H), 0.33 (d, J = 6.65
Hz, 2H), −0.34 (br. s., 1H), −0.95 (br. s., 1H). Mass spectrum (ESI)
m/z = 657.2 [M + H]+. HRMS (ESI) m/z found 657.1951 [M + H]+,
calcd for C34H38Cl2N2O6S 657.1957.
(3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-3-(pyridin-2-ylmethyl)-
piperidin-2-one (17). Compound 17 was synthesized in a manner
(3R,5R,6S)-3-((5-Bromopyridin-2-yl)methyl)-1-((S)-2-(tert-butylth-
io)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methylpiperidin-2-one (78). An oven-dried 10 mL flask equipped
with a stir bar and rubber inlet was fitted with a needle and argon
balloon. The flask was filled with THF (1.2 mL) and submerged in an
ice−water bath. Diisopropylamine (82 μL, 0.587 mmol) was added,
followed by butyllithium (235 μL, 0.587 mmol). After 2 h, at −78 °C,
a solution of (5R,6S)-1-((S)-2-(tert-butylthio)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methylpiperidin-2-one (58)
(192 mg, 0.391 mmol) in THF (1.2 mL) was added dropwise via a
plastic syringe under an argon atmosphere over 5 min. After 1 h, the
reaction was warmed to −40 °C and a solution of 5-bromo-2-
(bromomethyl)pyridine (147 mg, 0.587 mmol) in THF (1.2 mL) was
added to the reaction mixture. After stirring for 3 h at this temperature,
the reaction mixture was quenched with sat. aq. NH4Cl and
partitioned with EtOAc. The combined organic layers were dried
over sodium sulfate, filtered, and concentrated. The residue was
dissolved in DCM and loaded onto a dry silica gel cartridge and eluted
on a 25 g gold-capped ISCO Redisep silica gel column with 0−40%
1
similar to that described for the preparation of 19. H NMR (500
MHz, CD2Cl2) δ 8.55 (dd, J = 0.9, 4.8 Hz, 1H), 7.65 (dt, J = 1.8, 7.6
Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.25−7.16 (m, 4H), 7.16−7.06 (m,
3H), 7.03−7.00 (m, 1H), 6.91 (td, J = 1.7, 6.8 Hz, 1H), 4.94 (d, J =
10.8 Hz, 1H), 4.45−4.26 (m, 1H), 3.50 (d, J = 12.7 Hz, 1H), 3.32
(ddd, J = 3.2, 10.8, 13.6 Hz, 1H), 3.10 (d, J = 12.7 Hz, 1H), 2.94 (d, J
= 13.2 Hz, 1H), 2.10 (t, J = 13.6 Hz, 1H), 1.93 (br. s., 1H), 1.77 (dd, J
= 2.8, 13.3 Hz, 1H), 1.40 (s, 9H), 1.26 (s, 3H), 0.34 (br. s., 1H), 0.23
(br. s., 1H), −0.31 (br. s., 1H), −1.07 (br. s., 1H). Mass spectrum
(ESI) m/z = 613.2 [M + H]+. HRMS (ESI) m/z found 613.2058 [M +
H]+, calcd for C33H38Cl2N2O3S 613.2058.
4-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-Cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)benzoic Acid (30). Compound 30 was synthesized in a
1
manner similar to that described for the preparation of 19. H NMR
(500 MHz, CD2Cl2) δ 8.06 (d, J = 8.3 Hz, 2 H), 7.42 (d, J = 8.3 Hz, 2
H), 7.26−7.05 (m, 4 H), 6.95−6.78 (m, 3 H), 6.78−6.74 (m, 1 H),
4.89 (d, J = 10.8 Hz, 1 H), 4.37 (br. s., 1 H), 3.42−3.33 (m, 1 H), 3.00
(d, J = 13.2 Hz, 1 H), 2.98−2.90 (m, 2 H), 2.60 (br. s., 1 H), 2.24 (t, J
= 13.8 Hz, 1 H), 1.80 (dd, J = 3.1, 13.8 Hz, 1 H), 1.43−1.36 (m, 10
H), 1.27 (s, 3 H), 0.36 (br. s., 1 H), 0.29 (br. s., 1 H), −0.32 (br. s., 1
H), −1.02 (br. s., 1 H). Mass spectrum (ESI) m/z = 656.2 [M + H]+.
HRMS (ESI) m/z found 656.2014 [M + H]+, calcd for
C35H39Cl2NO5S 656.2004.
1
EtOAc/hexanes to give 79 (45 mg, 18% yield). H NMR (500 MHz,
CD2Cl2) δ 8.58 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 2.4, 8.3 Hz, 1H),
7.20 (d, J = 8.3 Hz, 3H), 7.16−7.10 (m, 2H), 7.08 (s, 1H), 6.95 (br. s.,
2H), 6.88−6.85 (m, 1H), 4.73 (d, J = 10.5 Hz, 1H), 3.47 (t, J = 11.0
Hz, 1H), 3.42 (d, J = 12.7 Hz, 1H), 3.37−3.30 (m, 1H), 3.11 (d, J =
13.0 Hz, 1H), 2.54 (dd, J = 5.1, 12.0 Hz, 1H), 2.41−2.22 (m, 1H),
2.06 (t, J = 13.4 Hz, 1H), 1.85 (dd, J = 3.2, 13.7 Hz, 1H), 1.65−1.59
(m, 1H), 1.31 (s, 9H), 1.25 (s, 3H), 0.47−0.37 (m, 1H), 0.32−0.22
(m, 1H), −0.06− −0.17 (m, 1H), −0.74− −0.89 (m, 1H). Mass
spectrum (ESI) m/z = 661.0 [M + H]+.
(3R,5R,6S)-3-Benzyl-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methylpiperidin-2-one
(18). Compound 18 was synthesized in a manner similar to that
1
described for the preparation of 19. H NMR (500 MHz, CD2Cl2) δ
2-(6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)pyridin-3-yl)acetic Acid (23). Part A: To a vial containing
(3R,5R,6S)-3-((5-bromopyridin-2-yl)methyl)-1-((S)-2-(tert-butylth-
io)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
methylpiperidin-2-one (78) (13.0 mg, 0.020 mmol) was added Q-
Phos (0.839 mg, 1.181 μmol) and tris(dibenzylideneacetone)-
dipalladium(0) (2.163 mg, 2.362 μmol). The solids were diluted in
THF (98 μL), and (2-(tert-butoxy)-2-oxoethyl)zinc(II) chloride (47.2
μL, 0.024 mmol) was added. The vial was placed on a hot plate at 60
°C. After 4 h, the reaction mixture was diluted with DCM and loaded
onto a 4 g gold-capped ISCO silica gel column that had been
preflushed with hexanes. The column was eluted with a gradient of 0−
100% EtOAc/hexanes to give tert-butyl 2-(6-(((3R,5R,6S)-1-((S)-2-
(tert-butylthio)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)pyridin-3-yl)acetate (5.0
mg, 37%). Mass spectrum (ESI) m/z = 695.2 [M + H]+.
7.40−7.29 (m, 6 H), 7.08−7.05 (m, 2 H), 6.86−6.72 (m, 5 H), 4.84
(d, J = 10.8 Hz, 1 H), 4.37 (br. s., 1 H), 3.38 (d, J = 13.2 Hz, 1 H),
2.98−2.84 (m, 2 H), 2.81 (d, J = 13.2 Hz, 1 H), 2.59 (br. s., 1 H),
2.22−2.12 (m, 1 H), 1.83 (dd, J = 2.7, 13.7 Hz, 1 H), 1.40 (s, 10 H),
1.28 (s, 3 H), 0.34 (br. s., 1 H), 0.27 (br. s., 1 H), −0.34 (br. s., 1 H),
−1.04 (br. s., 1 H). Mass spectrum (ESI) m/z = 612.2 [M + H]+.
6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)picolinic Acid (21). Compound 21 was synthesized in a
1
manner similar to that described for the preparation of 19. H NMR
(400 MHz, CDCl3) δ 8.22 (d, J = 7.63 Hz, 1H), 8.02 (t, J = 7.63 Hz,
1H), 7.57−7.67 (m, 1H), 7.61 (d, J = 7.63 Hz, 1H), 7.07−7.25 (m,
6H), 6.97 (s, 1H), 6.76−6.86 (m, 1H), 4.98 (d, J = 10.56 Hz, 1H),
4.38 (t, J = 11.84 Hz, 1H), 3.35−3.55 (m, 2H), 3.12−3.28 (m, 1H),
2.93 (d, J = 13.11 Hz, 1H), 2.70 (br. s., 1H), 2.44 (t, J = 13.79 Hz,
1H), 1.84 (dd, J = 2.35, 13.89 Hz, 2H), 1.44 (s, 9H), 1.32 (s, 3H),
0.23−0.52 (m, 2H), −0.31 (br. s., 1H), −1.05 (br. s., 1H). Mass
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Article
spectrum (ESI) m/z = 657.2 [M + H]+. HRMS (ESI) m/z found
657.1962 [M + H]+, calcd for C34H38Cl2N2O5S 657.1957.
HRMS (ESI) m/z found 671.2119 [M + H]+, calcd for
C34H38Cl2N2O5S 671.2113.
6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)-4-methylnicotinic Acid (26). Compound 26 was synthesized
6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)nicotinic Acid (3). Compound 3 was synthesized in a manner
1
1
in a manner similar to that described for the preparation of 19. H
similar to that described for the preparation of 19. H NMR (400
NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 7.47−7.63 (m, 2H), 6.91−
7.24 (m, 6H), 6.88 (d, J = 5.67 Hz, 1H), 4.97 (d, J = 10.56 Hz, 1H),
4.24 (t, J = 12.03 Hz, 1H), 3.87 (d, J = 13.11 Hz, 1H), 3.43 (t, J =
11.54 Hz, 1H), 3.21 (d, J = 12.72 Hz, 1H), 2.86 (s, 3H), 2.74 (d, J =
10.96 Hz, 3H), 1.86 (d, J = 13.89 Hz, 1H), 1.80 (br. s., 1H), 1.43 (s,
3H), 1.40 (s, 9H), 0.35 (d, J = 6.85 Hz, 2H), −0.35 (br. s., 1H), −0.91
(br. s., 1H). Mass spectrum (ESI) m/z = 671.2 [M + H]+.
5-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)pyrazine-2-carboxylic Acid (29). Compound 29 was synthe-
sized in a manner similar to that described for the preparation of 19.
1H NMR (400 MHz, CDCl3) δ 9.34 (s, 1H), 8.59 (s, 1H), 7.16 (br. s.,
5H), 7.11−7.15 (m, 2H), 7.05 (s, 1H), 6.93−7.01 (m, 1H), 5.02 (d, J
= 10.76 Hz, 1H), 4.36 (t, J = 12.03 Hz, 1H), 3.34−3.65 (m, 3H), 2.91
(d, J = 11.54 Hz, 1H), 2.68 (br. s., 1H), 2.38 (t, J = 13.69 Hz, 1H),
1.81 (br. s., 1H), 1.76 (dd, J = 3.03, 13.60 Hz, 1H), 1.43 (s, 9H), 1.31
(s, 3H), 0.22−0.45 (m, 2H), −0.30 (d, J = 3.52 Hz, 1H), −0.99 (br. s.,
1H). Mass spectrum (ESI) m/z = 658.2 [M + H]+. HRMS (ESI) m/z
found 658.1902 [M + H]+, calcd for C33H37Cl2N3O5S 658.1909.
(3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-((5-(2-hydroxyacetyl)thiazol-2-
yl)methyl)-3-methylpiperidin-2-one (34). Part A: To 2-(((3R,5R,6S)-
1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-
6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)methyl)thiazole-5-
carboxylic acid (33) (0.061 g, 0.091 mmol) in THF (0.913 mL) was
added oxalyl chloride (0.01215 mL, 0.137 mmol) and one drop of
DMF. After 1 h, the crude was concentrated under vacuum and taken
into the next step without further purification.
MHz, CDCl3) δ 9.37 (s, 1H), 8.67 (d, J = 7.63 Hz, 1H), 7.74 (d, J =
8.22 Hz, 1H), 6.98−7.25 (m, 6H), 6.84−6.93 (m, 1H), 5.00 (d, J =
10.76 Hz, 1H), 4.26 (t, J = 12.13 Hz, 1H), 3.92 (d, J = 13.30 Hz, 1H),
3.39−3.55 (m, 1H), 3.28 (d, J = 13.30 Hz, 1H), 2.86 (d, J = 12.91 Hz,
1H), 2.76 (t, J = 9.59 Hz, 1H), 2.52 (t, J = 13.79 Hz, 1H), 1.87 (d, J =
12.52 Hz, 1H), 1.80 (br. s., 1H), 1.41 (s, 13H), 0.35 (d, J = 6.85 Hz,
2H), −0.34 (br. s., 1H), −0.92 (br. s., 1H). Mass spectrum (ESI) m/z
= 657.2 [M + H]+. 13C NMR (150 MHz, (CD3)2SO) δ 175.4, 166.7,
163.5, 162.7, 159.9, 158.6, 158.5, 158.3, 149.9, 144.7, 139.5, 137.7,
132.7, 132.1, 130.9, 128.8, 127.8, 127.2, 126.6, 125.5, 125.3, 118.8,
114.9, 69.1, 59.6, 58.8, 45.5, 44.6, 44.2, 43.7, 38.7, 25.8, 14.6, 2.0.
HRMS (ESI) m/z found 657.1954 [M + H]+, calcd for
C34H38Cl2N2O5S 657.1957.
6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-6-
(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)methyl)nicotinic Acid (22). To a mixture of (3R,5R,6S)-3-
((5-bromopyridin-2-yl)methyl)-1-((S)-2-(tert-butylsulfonyl)-1-cyclo-
propylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-meth-
ylpiperidin-2-one (27 mg, 0.038 mmol), tripotassium phosphate (9.28
mg, 0.044 mmol), palladium(II) acetate (1.450 mg, 6.46 μmol), and
1,3-bis(dicyclohexylphosphino)propane bis(tetrafluoroborate) (4.65
mg, 7.60 μmol) was added DMSO (0.463 mL), followed by DBU
(0.00916 mL, 0.061 mmol) and water (0.021 mL, 1.140 mmol). The
reaction vial was evacuated and backfilled with carbon monoxide 4
times. Then, the reaction was heated at 80 °C overnight. The crude
reaction mixture was filtered using a 25 mm GD/X disposable siringe
filter (Whatman catalog number 6878-2504), washed with DCM, and
concentrated. The concentrated crude was then diluted with MeOH.
Purification by RP-HPLC (25 to 75% CH3CN/H2O in 30 min, both
solvents containing 0.1% TFA, flow rate = 45 mL/min) provided 6-
(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-6-(4-
chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-
Part B: To 2-(((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclo-
propylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxo-
piperidin-3-yl)methyl)thiazole-5-carbonyl chloride (62 mg, 0.091
mmol) was added tris(trimethylsilyloxy)ethylene (90 μL, 0.272
mmol). The reaction was stirred at 90 °C. After stirring overnight,
the reaction was cooled and charged with 1.8 M aqueous HCl (1 mL)
and THF (1 mL). The mixture was diluted with water (10 mL)
extracted with ethyl acetate (2 × 20 mL). The combined organics were
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. Silica gel chromatography (gradient elution 30 to
100% EtOAc in hexanes) afforded (3R,5R,6S)-1-((S)-2-(tert-butylsul-
fonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-
((5-(2-hydroxyacetyl)thiazol-2-yl)methyl)-3-methylpiperidin-2-one
1
3-yl)methyl)nicotinic acid (22) (13 mg, 0.019 mmol, 51% yield). H
NMR (400 MHz, CDCl3) δ 9.33 (s, 1H), 8.58 (d, J = 6.85 Hz, 1H),
7.65 (d, J = 6.65 Hz, 1H), 6.98−7.25 (m, 6H), 6.93 (d, J = 2.93 Hz,
1H), 5.01 (d, J = 10.37 Hz, 1H), 4.16−4.41 (m, 1H), 3.76 (br. s., 1H),
3.28−3.57 (m, 2H), 2.88 (d, J = 13.50 Hz, 2H), 2.71 (d, J = 4.11 Hz,
1H), 2.44 (t, J = 13.60 Hz, 1H), 1.85 (d, J = 12.72 Hz, 2H), 1.41 (s,
9H), 1.40 (br. s., 3H), 0.40 (d, J = 6.26 Hz, 2H), −0.28 (br. s., 1H),
−0.89 (br. s., 1H). Mass spectrum (ESI) m/z = 675.1 [M + H]+.
2-(6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-
5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-
yl)methyl)pyridin-3-yl)-2-methylpropanoic Acid (24). Compound 24
was synthesized in a manner similar to that described for the
preparation of 19. 1H NMR (400 MHz, CDCl3) δ 8.82−8.95 (m, 1H),
8.12 (d, J = 8.80 Hz, 1H), 7.57 (d, J = 8.41 Hz, 1H), 7.07 (s, 6H),
6.80−6.94 (m, 1H), 4.94 (d, J = 10.76 Hz, 1H), 4.30 (t, J = 10.86 Hz,
1H), 3.63 (d, J = 12.91 Hz, 1H), 3.43 (d, J = 13.30 Hz, 1H), 3.33 (t, J
= 11.15 Hz, 1H), 2.82−2.96 (m, 1H), 2.66−2.77 (m, 1H), 2.32−2.43
(m, 2H), 1.74−1.94 (m, J = 19.95 Hz, 2H), 1.69 (s, 6H), 1.42 (s, 9H),
1.37 (s, 3H), 0.29 (br. s., 2H), −0.34 (br. s., 1H), −0.95 (br. s., 1H).
Mass spectrum (ESI) m/z = 699.2 [M + H]+. HRMS (ESI) m/z found
699.2410 [M + H]+, calcd for C37H44Cl2N2O5S 699.2426.
1
(34) (6 mg, 8.85 μmol, 10% yield over the last two steps). H NMR
(400 MHz, CDCl3) δ 8.23 (s, 1H), 7.03 (d, J = 5.28 Hz, 8H), 4.90 (d,
J = 10.76 Hz, 1H), 4.70 (d, J = 1.76 Hz, 2H), 4.30 (t, J = 11.93 Hz,
1H), 3.61 (d, J = 13.89 Hz, 1H), 3.39−3.51 (m, 1H), 3.11−3.25 (m, J
= 2.93 Hz, 1H), 2.84 (d, J = 15.06 Hz, 1H), 2.52−2.68 (m, 1H), 2.24
(t, J = 13.50 Hz, 1H), 1.61−1.70 (m, 2H), 1.36 (s, 9H), 1.32 (s, 3H),
0.25−0.36 (m, 1H), 0.08−0.23 (m, 1H), −0.39 (br. s., 1H), −1.09 (br.
s., 1H). Mass spectrum (ESI) m/z = 677.0 [M + H]+.
3-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
1
2-oxopropanoic Acid (11). H NMR (400 MHz, CDCl3) δ 7.24 (br.
s., 2H), 7.03−7.20 (m, 4H), 6.97 (s, 1H), 6.86 (dt, J = 6.5, 1.9 Hz,
1H), 4.97 (d, J = 10.8 Hz, 1H), 4.22−4.39 (m, 2H), 4.07 (dd, J = 13.2,
10.9 Hz, 1H), 3.14−3.34 (m, 3H), 2.71−2.93 (m, 3H), 2.32 (t, J =
13.8 Hz, 1H), 2.08−2.20 (m, 1H), 2.00 (dd, J = 13.9, 2.9 Hz, 1H),
1.48−1.55 (m, 1H), 1.43 (s, 9H), 1.41 (s, 3H), 0.41 (t, J = 7.5 Hz,
3H). Mass spectrum (ESI) m/z = 582.2 [M + H]+. HRMS (ESI) m/z
found 582.1855 [M + H]+, calcd for C29H37Cl2NO5S 582.1848.
(R)-1-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetyl)pyrrolidine-3-carboxylic Acid (15). Part A: To a solution
of methyl β-dl-prolinate-HCl (133 mg, 1.033 mmol) in DMF (1 mL)
was added 2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperi-
6-(((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropylethyl)-5-
(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
methyl)-2-methylnicotinic Acid (25). Compound 25 was synthesized
1
in a manner similar to that described for the preparation of 19. H
NMR (400 MHz, CDCl3) δ 8.58 (d, J = 7.83 Hz, 1H), 7.48−7.69 (m,
2H), 6.96−7.23 (m, 6H), 6.91 (br. s., 1H), 5.00 (d, J = 10.56 Hz, 1H),
4.17−4.32 (m, 1H), 3.92 (br. s., 1H), 3.47 (t, J = 11.64 Hz, 1H), 3.27
(d, J = 14.09 Hz, 1H), 2.99 (s, 3H), 2.88 (d, J = 12.91 Hz, 1H), 2.78
(br. s., 1H), 2.49 (t, J = 14.28 Hz, 1H), 1.83 (d, J = 13.89 Hz, 2H),
1.41 (s, 9H), 1.38 (br. s., 3H), 0.35 (br. s., 2H), −0.32 (br. s., 1H),
−0.88 (br. s., 1H). Mass spectrum (ESI) m/z = 671.2 [M + H]+.
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din-3-yl)acetic acid (9) (200 mg, 0.344 mmol), HATU (262 mg, 0.689
mmol), and DIEA (0.481 mL, 2.76 mmol). After stirring overnight,
this was diluted with ethyl acetate and washed twice with water. The
mixture of isomers were separated via SFC, 20% isopropanol
(NH4OH)/CO2 (100 bar), IC (2 × 15 cm), and 3 mL/min to
provide in order of elution (stereochemistry arbitrarily assigned); (R)-
methyl 1-(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropy-
lethyl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperi-
din-3-yl)acetyl)pyrrolidine-3-carboxylate (38 mg, 0.055 mmol, 16%,
Mass spectrum (ESI) m/z = 691.3 [M + H]+) and then (S)-methyl 1-
(2-((3R,5R,6S)-1-((S)-2-(tert-butylsulfonyl)-1-cyclopropylethyl)-5-(3-
chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)-
acetyl)pyrrolidine-3-carboxylate (42 mg, 0.061 mmol, 18% yield).
Mass spectrum (ESI) m/z = 691.3 [M + H]+.
ACKNOWLEDGMENTS
■
We thank Simon Wong for the time-dependent inhibition
(TDI) determinations and Manuel Ventura and Dhanashri
Bagal for the acquisition of high resolution mass spectra.
ABBREVIATIONS USED
■
Boc, tert-butoxycarbonyl; BrdU, 5-bromo-2-deoxyuridine; CL,
clearance; CYP3A4, cytochrome P450 3A4; DCM, dichloro-
methane; 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 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; QD, once a day dosing; qRT-PCR, quantitative reverse
transcription polymerase chain reaction; SEM, standard error of
mean; SPR, surface plasmon resonance; TBAF, tetrabutylam-
monium fluoride; TDI, time dependent inhibition; TEA,
triethylamine; TES, triethylsilyl; TESCl, triethylsilyl chloride;
TFA, trifluoroacetic acid; THF, tetrahydrofuran
Part B: To a mixture of (R)-methyl 1-(2-((3R,5R,6S)-1-((S)-2-(tert-
butylsulfonyl)-1-cyclopropylethyl)-5-(3-chlorophenyl)-6-(4-chloro-
phenyl)-3-methyl-2-oxopiperidin-3-yl)acetyl)pyrrolidine-3-carboxylate
(fastest eluting isomer) (38 mg, 0.055 mmol) in MeOH (0.500 mL)
and THF (1 mL) was added 1.0 M aq. LiOH (0.330 mL, 0.330 mmol)
solution. After 90 min, the reaction mixture was acidified with 5% aq.
HCl, diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4, and concentrated to
provide (R)-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)pyrrolidine-3-carboxylic acid (15) (36 mg, 0.053
1
mmol, 97% yield). H NMR (400 MHz, CDCl3) δ 7.23 (br. s., 4H),
7.08 (d, J = 5.09 Hz, 2H), 7.00 (s, 1H), 6.93 (br. s., 1H), 4.94 (dd, J =
3.03, 10.86 Hz, 1H), 4.35 (br. s., 1H), 3.91 (d, J = 7.04 Hz, 1H), 3.83
(t, J = 7.53 Hz, 1H), 3.62−3.75 (m, 1H), 3.31−3.62 (m, 2H), 3.07−
3.29 (m, 1H), 2.77−3.02 (m, 3H), 2.71 (br. s., 1H), 2.16−2.45 (m,
4H), 1.90 (br. s., 1H), 1.44 (s, 9H), 1.41 (d, J = 3.33 Hz, 3H), 0.13−
0.48 (m, 2H), −0.31 (br. s., 1H), −1.04 (br. s., 1H). Mass spectrum
(ESI) m/z = 677.2 [M+H]+). HRMS (ESI) m/z found 677.2223 [M +
H]+, calcd for C34H42Cl2N2O6S 677.2219.
REFERENCES
■
(1) Wells, J. A.; McClendon, C. L. Reaching for high-hanging fruit in
drug discovery at protein−protein interfacese. Nature 2007, 450,
1001−1009.
(2) Levine, A. J. p53, the cellular gatekeeper for growth and division.
Cell 1997, 88, 323−331.
(3) Lane, D. P. p53, guardian of the genome. Nature 1992, 358, 15−
(S)-1-(2-((3R,5R,6S)-1-((S)-2-(tert-Butylsulfonyl)-1-cyclopropyleth-
yl)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-
3-yl)acetyl)pyrrolidine-3-carboxylic Acid (16). Compound 16 was
synthesized in a manner similar to that described for the preparation of
16.
(4) Toledo, F.; Wahl, G. M. Regulating the p53 pathway: in vitro
hypotheses, in vivo veritas. Nat. Rev. 2006, 909−923.
1
15 from the slowest eluting isomer in part A. H NMR (400 MHz,
(5) Hainaut, P.; Hollstein, M. p53 and human cancers: the first ten
thousand mutations. Adv. Cancer Res. 2000, 77, 81−137.
(6) Oliner, J. D.; Kinzler, K. W.; Meltzer, P. S.; George, D.;
Vogelstein, B. Amplification of a gene encoding a p53-associated
protein in human sarcomas. Nature 1992, 358, 80−83.
(7) Chene, P. Inhibiting the p53-MDM2 interaction: An important
target for cancer therapy. Nat. Rev. Cancer 2003, 3, 102−109.
(8) Vogelstein, B.; Lane, D.; Levine, A. J. Surfing the p53 network.
Nature (London) 2000, 408, 307−310.
CDCl3) δ 7.22 (br. s., 4H), 7.08 (d, J = 4.89 Hz, 2H), 6.99 (br. s., 1H),
6.93 (t, J = 4.11 Hz, 1H), 4.94 (d, J = 10.56 Hz, 1H), 4.34 (br. s., 1H),
3.60−4.04 (m, 3H), 3.35−3.60 (m, 2H), 3.07−3.27 (m, 1H), 2.75−
3.00 (m, 3H), 2.71 (br. s., 1H), 2.15−2.40 (m, 4H), 1.90 (br. s., 1H),
1.44 (s, 9H), 1.41 (d, J = 3.13 Hz, 3H), 0.15−0.44 (m, 2H), −0.31 (br.
s., 1H), −1.06 (br. s., 1H). Mass spectrum (ESI) m/z = 677.2 [M +
H]+). HRMS (ESI) m/z found 677.2210 [M + H]+, calcd for
C34H42Cl2N2O6S 677.2219.
(9) Kussie, P. H.; Gorina, S.; Marcchal, V.; Elenbaas, B.; Moreau, J.;
Levine, A. J.; Pavletich, N. P. Structure of the MDM2 oncoprotein
bound to the p53 tumor suppressor transactivation domain. Science
1996, 274, 948−953.
(10) Garcia-Echevarria, C.; Chene, P.; Blommers, M. J. J.; Furet, P.
Discovery of potent antagonists of the interaction between human
double minute 2 and tumor suppressor p53. J. Med. Chem. 2000, 43,
3205−3208.
(11) Arkin, M. R.; Wells, J. A. Small-molecule inhibitors of protein−
protein interactions: progressing towards the dream. Nat. Rev. Drug
Discovery 2004, 3, 301−317.
(12) For other small molecule MDM2-inhibitor scaffolds see
imidazolines; (a) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.;
Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein,
C.; Fotouhi, N.; Liu, E. A. In vivo activation of the p53 pathway by
small molecule antagonists of MDM2. Science 2004, 303, 844−848.
(b) Vu, B. T.; Vassilev, L. Small-molecule inhibitors of the p53-MDM2
interaction. Curr. Top. Microbiol. Immunol. 2011, 348, 151−172.
Benzodiazepinediones: (c) Parks, D. J.; LaFrance, L. V.; Calvo, R. R.;
Milkiewicz, K. L.; Gupta, V.; Lattance, J.; Ramachandren, K.; Carver,
T. E.; Petrella, E. C.; Cummings, M. D.; Maguire, D.; Grasberger, B.
L.; Lu, T. 1,4-Benzodiazepine-2,5-diones as small molecule antagonists
of the HDM2-p53 interaction: discovery and SAR. Bioorg. Med. Chem.
ASSOCIATED CONTENT
■
S
* Supporting Information
In vitro biological assays, in vivo protocols, determination of
cocrystal structures of 3, 4, and 46−49 bound to MDM2. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
Coordinates for compounds 3 (PDB code: 4OGN), 4 (PDB
code: 4ODE), 46 (PDB code: 4OGT), 47 (PDB code: 4ODF),
48 (PDB code: 4OCC), and 49 (PDB code: 4OGV) bound to
MDM2 have been deposited in the Protein Data Bank.
AUTHOR INFORMATION
■
Corresponding Author
*Tel 650-244-2654. Fax: 650-837-9369. E-mail: anagonza@
amgen.com.
Notes
The authors declare no competing financial interest.
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Lett. 2005, 15, 765−770. (d) Parks, D. J.; LaFrance, L. V.; Calvo, R. R.;
Milkiewicz, K. L.; Marugan, J. L.; Raboisson, P.; Schubert, C.; Koblish,
H. K.; Zhao, S.; Franks, C. F.; Lattanze, J.; Carver, T. E.; Cummings,
M. D.; Maguire, D.; Grasberger, B. L.; Maroney, A. C.; Lu, T.
Enhanced pharmacokinetic properties of 1,4-benzodiazepine-2,5-dione
antagonists of the HDM2-p53 protein−protein interaction through
structure-based drug design. Bioorg. Med. Chem. Lett. 2006, 16, 3310−
3314. (e) Grasberger, B. L.; Lu, T.; Schubert, C.; Parks, D. J.; Carver,
T. E.; Koblish, H. K.; Cummings, M. D.; LaFrance, L. V.; Milkiewicz,
K. L.; Calvo, R. R.; Maguire, D.; Lattanze, J.; Franks, C. F.; Zhao, S.;
Ramachandren, K.; Bylebyl, G. R.; 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,
D.; Shangary, S.; Chen, J.; Wang, G.; Ding, K.; McEachern, D.; Qiu, S.;
Nikolovska-Coleska, Z.; Miller, R.; Kang, S.; Yang, D.; Wang, S. Potent
and orally active small molecule inhibitors of the MDM2-p53
interaction. J. Med. Chem. 2009, 52, 7970−7973. (i) Wang, S.; Yu,
S.; Sun, W.; Shangary, S. K.; Sun, D.; Zou, P.; McEachern, D.; Zhao, Y.
Preparation of Spiro-oxindole Derivatives as MDM2 Antagonists.
MK-8242 (Merck). (e) AMG 232 (Amgen) (see ref 17). (f) CGM-
097 (Novartis). (g) DS-3032b (Daiichi Sankyo).
(14) Kamal, A.; Azhar Mohammed, A.; Shaik, T. B. p-53-MDM2
inhibitors: patent review (2009−2010). Expert Opin. Ther. Patents
2012, 22, 95−105.
(15) (a) Tovar, C.; Graves, B.; Packman, K.; Filipovic, Z.; Higgins,
B.; Xia, M.; Tardell, C.; Garrido, R.; Lee, E.; Kolinsky, K.; To, K.-H.;
Linn, M.; Podlaski, F.; Wovkulich, P.; Vu, B.; Vassilev, L. T. MDM2
Small-molecule antagonist RG7112 activates p53 signaling and
regresses human tumors in preclinical cancer models. Cancer Res.
2013, 73, 2587−2597. (b) Vu, B.; Wolkulich, P.; Pizzolato, G.; Lovey,
A.; Ding, Q.; Jiang, N.; Liu, J.-J.; Zhao, C.; Glenn, K.; Wen, Y.; Tovar,
C.; Packman, K.; Vassilev, L. T.; Graves, B. Discovery of RG7112: A
small-molecule MDM2 inhibitor in clinical development. Med. Chem.
Lett. 2013, 4, 466−469.
(16) Ding, Q.; Zhang, Z.; Liu, J.-J.; Jiang, N.; Zhang, J.; Ross, T. M.;
Chu, X.-J.; Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic,
Z. M.; Higgins, B.; Glenn, K.; Packman, K.; Vassilev, L. T.; Graves, B.
Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in
clinical development. J. Med. Chem. 2013, 56, 5979−5983.
(17) Sun, D.; Li, Z.; Rew, Y.; Bartberger, M. D.; Beck, H. P.; Canon,
J.; Chen, A.; Chen, X.; Chow, D.; Deignan, J.; Duquette, J.;
Eksterowicz, J.; Fisher, B.; Fox, B. M.; Fu, J.; Gonzalez, A. Z.;
Gonzalez-Lopez De Turiso, F.; Gribble, M.; Houze, J.; Huang, X.;
Jiang, M.; Jin, L.; Kayser, F.; Liu, J.; Lo, M.-C.; Long, A. M.; Lucas, B.;
McGee, L. R.; McIntosh, J.; Mihalic, J.; Oliner, J. D.; Osgood, T.;
Peterson, M. L.; Roveto, P.; Saiki, A. Y.; Shaffer, P.; Toteva, M.; Wang,
Y.; Wang, Y. C.; Wortman, S.; Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Yu,
M.; Zhao, X.; Zhou, X.; Zhu, J.; Olson, S. H.; Medina, J. C. Discovery
of AMG 232, a potent, selective, and orally bioavailable inhibitor of the
MDM2-p53 interaction. J. Med. Chem. 2014, 57, 1454−1472.
(18) For reports on our team’s efforts see: (a) Allen, J. G.; Bourbeau,
M. P.; Wohlhieter, G. E.; Bartberger, M. D.; Michelsen, K.; Hungate,
R.; Gadwood, R. C.; Gaston, R. D.; Evans, B.; Mann, L. W.; Matison,
M. E.; Schneider, S.; Huang, X.; Yu, D.; Andrews, P. S.; Reichelt, A.;
Long, A. M.; Yakowec, P.; Yang, E. Y.; Lee, T. A.; Oliner, J. D.
Discovery and optimization of chromenotriazolopyrimidines as potent
inhibitors of the mouse double minute 2-tumor protein 53 protein−
protein interaction. J. Med. Chem. 2009, 52, 7044−7053. (b) Beck, H.
P.; DeGraffenreid, M.; Fox, B.; Allen, J. G.; Rew, Y.; Schneider, S.;
Saiki, A. Y.; Yu, D.; Oliner, J. D.; Salyers, K.; Ye, Q.; Olson, S.
Improvement of the synthesis and pharmacokinetic properties of
chromenotriazolopyrimidine MDM2-p53 protein−protein inhibitors.
Bioorg. Med. Chem. Lett. 2011, 21, 2752−2755. (c) Rew, Y.; Sun, D.;
Gonzalez Lopez De Turiso, F.; Bartberger, M. D.; Beck, H. P.; Canon,
J.; Chen, A.; Chow, D.; Deignan, J.; Fox, B. M.; Gustin, D.; Huang, X.;
Jiang, M.; Jiao, X.; Jin, L.; Kayser, F.; Kopecky, D. J.; Li, Y.; Lo, M.;
Long, A. M.; Michelsen, K.; Oliner, J. D.; Osgood, T.; Ragains, M.;
Saiki, A. Y.; Schneider, S.; Toteva, M.; Yakowec, P.; Yan, X.; Ye, Q.;
Yu, D.; Zhao, X.; Zhou, J.; Medina, J. C.; Olson, S. H. Structure-based
design of novel inhibitors of the MDM2-p53 interaction. J. Med. Chem.
2012, 55, 4936−4954. (d) Lucas, B. S.; Fisher, B.; McGee, L. R.;
Olson, S. H.; Medina, J. C.; Cheung, E. An expeditious synthesis of the
MDM2-p53 inhibitor AM-8553. J. Am. Chem. Soc. 2012, 134, 12855−
12860. (e) Gonzalez-Lopez de Turiso, F.; Sun, D.; Rew, Y.; Bartberger,
M. D.; Beck, H. P.; Canon, J.; Chen, A.; Chow, D.; Correll, T. L.;
Huang, X.; Julian, L. D.; Kayser, F.; Lo, M.-C.; Long, A. M.; McMinn,
D.; Oliner, J. D.; Osgood, T.; Powers, J. P.; Saiki, A. Y.; Schneider, S.;
Shaffer, P.; Xiao, S.-H.; Yakowek, P.; Yan, X.; Ye, Q.; Yu, D.; Zhao, X.;
Zhou, J.; Medina, J. C.; Olson, S. H. Rational design and binding mode
of MDM2-p53 inhibitors. J. Med. Chem. 2013, 56, 4053−4070.
(f) Bernard, D.; Zhao, Y.; Wang, S. AM-8553: A novel MDM2
inhibitor with a promising outlook for potential clinical development.
J. Med. Chem. 2012, 55, 4934−4935.
́
US2011/0112052A1, 2011. 3-Imidazolyndole: (j) Furet, P.; Chene,
P.; De Pover, A.; Stachyra Valat, T.; Hergovich Lisztwan, J.; Kallen, J.;
Masuya, K. The central valine concept provides an entry in a class of
non-peptide inhibitors of the p53-MDM2 interaction. Bioorg. Med.
Chem. Lett. 2012, 22, 3498. (k) Zhang, Z.; Chu, X.-J.; Liu, J.-J.; Ding,
Q.; Zhang, J.; Bartkovitz, D.; Jiang, N.; Karnachi, P.; So, S.-S.; Tovar,
C.; Filipovic, Z. M.; Higgins, B.; Glenn, K.; Packman, K.; Vassilev, Z.;
Graves, B. Discovery of potent and orally active p53-MDM2 inhibitors
RO5353 and RO2468 for potential clinical development. Med. Chem.
Lett. 2014, 5, 124−7. (l) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Qju,
S.; Ding, Y.; Gao, W.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Tomita,
Y.; Parrish, D. A.; Deschamps, J. R.; Wang, S. Structure-based desing of
potent non-peptide MDM2 inhibitors. J. Am. Chem. Soc. 2005, 127,
10130−10131. (m) Zhao, Y.; Liu, L.; Sun, W.; Lu, J.; McEachern, D.;
Li, X.; Yu, S.; Bernard, D.; Ochsenbein, P.; Ferey, V.; Carry, J.-C.;
Deschamps, J. R.; Sun, D.; Wang, S. Diastereomeric spirooxindoles as
highly potent and efficacious MDM2 inhibitors. J. Am. Chem. Soc.
2013, 135, 7223−7234. (n) Hardcastle, J. R.; Liu, J.; Valeur, E.;
Watson, A.; Ahmed, S. U.; Blackburn, T. J.; Bennaceur, K.; Clegg, W.;
Drummond, C.; Endicott, J. A.; Golding, B. T.; Griffin, R. J.; Gruber,
J.; Haggerty, K.; Harrington, R. W.; Hutton, C.; Kemp, S.; Lu, X.;
McDonnell, J. M.; Newell, D. R.; Noble, M. E. M.; Payne, S. L.; Revill,
C. H.; Riedinger, C.; Xu, Q.; Lunec, J. Isoindolinone inhibitors of the
Murine Double Minute 2 (MDM2)-p53 protein−protein interaction:
structure−activity studies leading to improved potency. J. Med. Chem.
2011, 54, 1233−1243. (o) Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.;
McEachern, D.; Shargary, S.; Bernard, P.; Li, X.; Zhao, T.; Zou, P.;
Sun, D.; Wang, S. A potent small-molecule inhibitor of the MDM2-
p53 interaction (MI-888) achieve complete and durable tumor
regression in mice. J. Med. Chem. 2013, 56, 5553−5561. (p) Zhuang,
C.; Miao, Z.; Zhu, L.; Dong, G.; Guo, Z.; Wang, S.; Zhang, Y.; Wu, Y.;
Yao, J.; Sheng, C.; Zhang, W. Discovery, synthesis, and biological
evaluation of orally active pyrrolidone derivatives as novel inhibitors of
p53-MDM2 protein-protein interaction. J. Med. Chem. 2012, 55,
9630−9642.
(13) Information from www.clinicaltrials.gov. (a) RG7112
(Hoffmann-La Roche) (see ref 15b.); (b) RG7388 (Hoffmann-La
Roche) (see ref 16.); (c) SAR299155 (Sanofi): Carry, J.-C.; Garcia-
Echeverria, C. Inhibitors of the p53/hdm2 protein-protein interaction-
path to the clinic. Bioorg. Med. Chem. Lett. 2013, 23, 2480−2485. (d)
(19) Experimental details of assays can be found in the Supporting
Information.
(20) For a detailed description of the interactions between MDM2
and 2 please refer to ref 17.
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Article
(21) For reviews on acid bioisosteres see (a) Meanwell, N. A.
Synopsis of some recent tactical application of bioisosteres in drug
design. J. Med. Chem. 2011, 54, 2529−91. (b) Ballatore, C.; Huryn, D.
M.; Smith, A. B. Carboxylic acid (bio)isosteres in drug design. Chem.
Med. Chem. 2013, 8, 385−395.
(22) Time-dependent inhibition, % remaining of CYP3A4 over
positive control, Rifampin, after 30 min at 10 μM.
(23) Shishkin, O V.; Konovalova, I. S. Novel type of mixed O-H···N/
O-H···π hydrogen bonds: monohydrate of pyridine. Struct. Chem.
2009, 20, 37−41.
(24) Experimental details of the BrdU proliferation and HCT116 p21
TaqMan assays can be found in the Supporting Information.
(25) Experimental details of in vivo studies can be found in the
Supporting Information.
(26) Metabolites structures determined by analogy with previous
results; see refs 17 and 28.
(27) Ye, Q.; Jiang, M.; Huang, W. T.; Ling, Y.; Olson, S. H.; Sun, D.
Xu, G.; Yan, X.; Jin, L. Pharmacokinetics and metabolism of AMG 232,
a novel and orally bioavailable inhibitor of the MDM2−p53
Interaction, in Rat, Dog, and Monkey: In vitro-in vivo correlation,
unpublished results.
(28) Gonzalez, A. Z.; Eksterowicz, E.; Bartberger, M. D.; Beck, H. P.;
Canon, J. Chen, A.; Chow, D.; Duquette, J.; Fox, B. M. Fu, J.; Huang,
X. Houze, J.; Jin, L.; Li, Y.; Li, Z.; Ling, Y.; Lo, M.-C.; Long, A. M.;
McGee, L. R.; McIntosh, J.; McMinn, D. L. Oliner, J. D.; Osgood, T.;
Rew, Y.; Saiki, A. Y.; Shaffer, P.; Wortman, S.; Yakowec, P.; Yan, X.;
Ye, Q.; Yu, D.; Zhao, X.; Zhou, J.; Olson, S. H.; Medina, J. C.; Sun, D.
Selective and Potent Morpholinone Inhibitors of the MDM2-p53
Protein-Protein Interaction. J. Med. Chem. [Online early access]. DOI:
10.1021/jm401767k. Published Online: Feb 18, 2014.
(29) Hazuda, D. J.; Anthony, N. J.; Gomez, R. P.; Jolly, S. M.; Wai, J.
S.; Zhuang, L.; Fisher, T. E.; Embrey, M.; Guare, J. P.; Egbertson, M.
S.; Vacca, J. P.; Huff, J. R.; Felock, P. J.; Witmer, M. V.; Stillmock, K.
A.; Danovich, R.; Grobler, J.; Miller, M. D.; Espeseth, A. S.; Jin, L.;
Chen, I.-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.; Wong, B. K.; Xu, W.;
Pearson, P. G.; Schleif, W. A.; Cortese, R.; Emini, E.; Summa, V.;
Holloway, M. K.; Young, S. D. A naphthyridine carboxamide provides
evidence for discordant resistance between mechanistically identical
inhibitors of HIV-1 integrase. Proc. Natl. Acad. Sci. 2004, 101, 11233−
11238.
(30) The MDMX assay is performed exactly as the MDM2 HTRF
assay (in the absence of human serum) described in the Supporting
Information with the exception that MDMX (10 μL) was dispensed to
the reaction plate and incubated with the inhibitor for 20 min before
p53 (20 μL, 1.25 nM) was added.
(31) Riedinger, C.; McDonnell, J. M. Inhibitors of MDM2 and
MDMX: a structural perspective. Future Med. Chem. 2009, 1, 1075−
1094.
(32) This procedure was adapted from: Ju, L.; Lippert, A. R.; Bode, J.
W. Stereoselective synthesis and chemoselective amide-forming
ligation of C-terminal peptide α-ketones. J. Am. Chem. Soc. 2008,
130, 4253−4255.
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