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

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 ED₅₀of 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.⁹⁻¹³Consequently, 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.¹Upon

cellular stress, p53 activation leads to the transcription of

multiple downstream genes that regulate cell cycle control,

apoptosis, DNA repair, and senescence.²⁻⁴About 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.⁶⁻⁸Many 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

2963

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

interaction. Our optimization eﬀorts within this series led to the

discovery of AMG 232 (1, Figure 1), which is currently being

binding aﬃnity.^18eThe amide of the oxindole-derived MDM2

inhibitors was reported to engage in similar interactions with

His96.^16,18eThus, 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 CL_int= 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.^17,18Inhibitor 1 is highly potent (K_d= 0.045

nM; SJSA-1 EdU IC₅₀= 9.1 nM)¹⁹with remarkable

pharmacokinetic properties (hHep CL_int= 6.3 μL/min/10⁶

cells) and in vivo eﬃcacy in the SJSA-1 osteosarcoma xenograft

model (ED₅₀= 9.1 mg/kg).^17,20Because 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 MDM2.²⁰This unique

interaction resulted in a nearly 100-fold improvement in

2964

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

2965

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

a

Scheme 4

Scheme 6

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, NaO^tBu, 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, NaO^tBu, DMF, 0 °C, 30 min, 62%; (e) 1,2-dibromoethane,

NaO^tBu, 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.

2966

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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 IC₅₀in

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 IC₅₀for 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).²²Co-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.²³Introduction 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 CL_int= <0.1 μL/min/10⁶cells) compared to the α-

unsubstituted acid 23 (hHep CL_int= 3.4 μL/min/10⁶cells) 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.²¹At 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

2967

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

Table 1. Carboxylic Acid Replacements

a

b

c

IC₅₀in the biochemical assay using serum free buﬀer. IC₅₀in 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 pK_aof the nitrogen in these

2968

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

Table 2. Optimization of the Pyridine Series and Other Six-Membered Heterocyclic Analogues

a

b

c

IC₅₀in the biochemical assay using serum free buﬀer. IC₅₀in 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 pK_a, ∼5.2; pyrimidine pK_a, ∼1.3; and

pyrazine pK_a, ∼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).

2969

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

Table 3. Optimization of the Thioazole Series and Other Five-Membered Heterocyclic Derivatives

a

b

c

IC₅₀in the biochemical assay using serum free buﬀer. IC₅₀in 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).

2970

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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 p53^wtand 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 p53^wt

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).²⁵In this study,

4 caused robust dose-dependent tumor growth inhibition with

the highest dose of 50 mg/kg causing 6% tumor regression.

The calculated ED₅₀was 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.

2971

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

Figure 3. Cell activity of 4 is p53-dependent. (a) In HCT116 p53^wt

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 p53^wtand 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.²⁷Therefore,

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 × 10⁶SJSA-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 (10⁶cells/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.²⁶The acyl glucuronide

metabolite of 4 was not observed in this experiment.

2972

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

Figure 6. Chemical structures and potencies of some known MDM2 inhibitors currently being tested in the clinic: RG7112, RG7388, SAR299155,

a

and 4. IC₅₀in 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.^30,31Compound 4 also shows

robust antitumor activity in the SJSA-1 osteosarcoma xenograft

study with a calculated ED₅₀of 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-

2973

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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-

2974

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

IC₅₀in the biochemical assay using buﬀer containing 15% human serum. ^bCellular 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 MgSO₄and 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 C₂₉H₃₅Cl₂F₃N₂O₆S₂699.1344.

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

2975

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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).³²Part 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]⁺.

2976

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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-

2977

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

C₃₂H₃₆Cl₂N₂O₅S₂663.1521.

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 C₃₂H₃₆Cl₂N₂O₅S₂663.1521.

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

C₃₃H₃₈Cl₂N₂O₅S₂667.1677.

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 C₃₂H₃₅Cl₂FN₂O₅S₂681.1427.

(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

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

= 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

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

C₂₈H₃₆Cl₂N₂O₅S₂615.1521.

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

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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 C₃₅H₄₂Cl₂N₂O₅S₂705.1990.

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. NaHCO₃and

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

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

C₃₃H₃₇Cl₂FN₂O₅S₂695.1583.

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-one²⁸in 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 Na₂SO₄and 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 C₃₃H₃₆Cl₂F₂N₂O₅S₂713.1486.

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)-

2982

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

2983

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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

2984

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

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

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]⁺.

2985

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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.

2986

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

Article

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.

2987

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Journal of Medicinal Chemistry

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.

2988

dx.doi.org/10.1021/jm401911v | J. Med. Chem. 2014, 57, 2963−2988

Article Doi

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

DOI: 10.1021/jm401911v

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm401911v

Full text of DOI:10.1021/jm401911v

Products guided by the article

R&D Labs maybe for 1584732-17-1

Relevant to this article

Hot Product