Structure-based design of novel inhibitors of the MDM2-p53 interaction

Article

pubs.acs.org/jmc

Structure-Based Design of Novel Inhibitors of the MDM2−p53

Interaction

Yosup Rew,^†Daqing Sun,*^,†Felix Gonzalez-Lopez De Turiso,^†Michael D. Bartberger,^∥Hilary P. Beck,^†

Jude Canon,^⊥Ada Chen,^†David Chow,^†Jeﬀrey Deignan,^†Brian M. Fox,^†Darin Gustin,^†Xin Huang,^#

Min Jiang,^§Xianyun Jiao,^†Lixia Jin,^§Frank Kayser,^†David J. Kopecky,^†Yihong Li,^†Mei-Chu Lo,^†

Alexander M. Long,^#Klaus Michelsen,^∥Jonathan D. Oliner,^⊥Tao Osgood,^⊥Mark Ragains,^‡

Anne Y. Saiki,^⊥Steve Schneider,^#Maria Toteva,^‡Peter Yakowec,^#Xuelei Yan,^†Qiuping Ye,^§

Dongyin Yu,^⊥Xiaoning Zhao,^†Jing Zhou,^†Julio C. Medina,^†and Steven H. Olson^†

†

‡

§

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: Structure-based rational design led to the discovery

of novel inhibitors of the MDM2−p53 protein−protein

interaction. The aﬃnity of these compounds for MDM2 was

improved through conformational control of both the piperidinone

ring and the appended N-alkyl substituent. Optimization aﬀorded

29 (AM-8553), a potent and selective MDM2 inhibitor with

excellent pharmacokinetic properties and in vivo eﬃcacy.

degradation of both p53 and itself in the cellular 26S

proteasome.^1,2

INTRODUCTION

■

The tumor suppressor protein p53 plays a central role in

preventing tumor development. Cellular stress in the form of

DNA damage, hypoxia, or proapoptotic oncogene activation

stimulates p53, resulting in its accumulation in the nucleus.

Activated p53 up-regulates the transcription of numerous genes

involved in cell cycle arrest, DNA repair, senescence, and

apoptosis.^1,2As a result, inactivation of the p53 pathway in

tumor cells provides a strong selective growth advantage, and it

has been proposed that elimination of the p53 function may be

a requisite step in tumor formation.³Recently, three

independent studies demonstrated that restoring endogenous

p53 function results in tumor regression in vivo and could be an

eﬀective anticancer therapeutic approach.⁴⁻⁷

The MDM2 (murine double minute 2) oncogene is an

important negative regulator of p53. In unstressed cells, a

negative feedback loop maintains both p53 and MDM2 at very

low levels. MDM2 is transcriptionally activated by p53, and the

activity of p53 is regulated by MDM2 through three main

mechanisms. First, MDM2 represses p53 transcriptional activity

by binding to the p53 transactivation domain. Second, MDM2

transports p53 from the nucleus to the cytosol. Finally, MDM2

functions as an E3 ubiquitin ligase and facilitates the

Wild-type p53 is found in approximately 50% of human

cancers,⁸and inhibition of the MDM2−p53 interaction with

nonpeptidic small-molecule MDM2 inhibitors has been shown

to be a tractable mechanism for activation of the p53 pathway.⁹

However, targeting a protein−protein interaction with small

molecule inhibitors still represents a considerable challenge for

drug discovery. The potential binding regions typically involve

vast surface areas that contain poorly deﬁned binding regions.¹⁰

As a result, small molecule inhibitors of protein−protein

interactions tend to have high molecular weight, and when

bound to their target, a considerable fraction of their total

surface area is solvent-exposed. These factors can make lead

optimization quite formidable. Although researchers have

recognized the potential beneﬁts of neutralizing the MDM2−

p53 protein−protein interaction for nearly 20 years, to date,

only a few molecules have advanced into clinical trials.¹¹

Previously, we identiﬁed a class of chromenotriazolopyr-

imidine inhibitors of the MDM2−p53 interaction from high-

throughput screening of our chemical library.^12,13To comple-

Received: March 18, 2012

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ment these eﬀorts, we embarked on a parallel strategy to

design, de novo, a new scaﬀold of MDM2 inhibitors based on

the binding mode of known inhibitors with MDM2 (Figure

1).^12,14,15

RESULTS AND DISCUSSION

■

The crystal structure of the 109-residue amino-terminal domain

of MDM2 bound to a 15-residue transactivation domain

peptide of p53 revealed that MDM2 has three hydrophobic

clefts that bind three critical residues of p53 (Phe19, Trp23,

and Leu26).¹⁷Because of the central role p53 plays in human

cancer and the presence of a relatively well-deﬁned MDM2

binding region, inhibition of the MDM2/p53 protein−protein

interaction with low molecular-weight small molecules has been

considered a compelling therapeutic target in oncology for the

past decade.^9,14,15,18

On the basis of a structural analysis of known MDM2

inhibitors (Figure 1), several novel scaﬀolds were conceived.²⁰

Initial SAR around those scaﬀolds led to the identiﬁcation of

1,3,5,6-tetrasubstituted piperidinone 1 (Figure 2), whose IC₅₀

in the HTRF assay was 2.42 μM in serum free buﬀer. Changing

the cis-diaryl conﬁguration into trans-diaryl, inverting the

stereochemical conﬁguration of the C3 acetic acid substituent,

and resolving/identifying the active enantiomer resulted in a

50- to 70-fold increase in potency (2 vs 1).²⁰

Figure 1. Binding mode of three known MDM2 inhibitors. Blue labels

a

indicate the positions normally occupied by key p53 residues. IC₅₀in

biochemical assay (HTRF, serum free). ^bIC₅₀in biochemical assay

(HTRF, 15% human serum).

The predicted binding mode of compound 2²¹from

docking²²is shown in Figure 3. It was proposed that the

Herein, we describe the discovery of novel scaﬀolds,

exempliﬁed by compounds 1 and 2 in Figure 2, as promising

Figure 2. Discovery of novel piperidinone lead 2 from de novo design

and synthesis. ^aRacemic mixture. ^bSingle enantiomer. ^cIC₅₀in

biochemical assay (HTRF, serum free). ^dIC₅₀in biochemical assay

(HTRF, 15% human serum).

Figure 3. Model of proposed binding mode of compound 2. MDM2

binding pockets are labeled (by p53 side chain) in white, H96 labeled

in yellow.

leads for the inhibition of the MDM2−p53 interaction.

Compound 2 exhibits meaningful potency levels in both

biochemical (HTRF-based neutralization assay measuring

inhibition of the interaction between MDM2 and p53) and

cell-based (p21 induction and cell proliferation assays in SJSA-1

tumor cells) assays (Table 2).¹⁶

Leu26_(p53)pocket contains the C5 m-Cl phenyl substituent,

with the deepest Trp23_(p53)pocket occupied by the C6 p-Cl

phenyl group. The cyclopropylmethyl group occupies the

Phe19_(p53)pocket, while the carboxylate anion forms an

electrostatic interaction with the H96 imidazole side chain of

MDM2 (Figure 3).

Initial optimization of 2 began with the modiﬁcation of the

N-alkyl group. By use of the minimally substituted piperidinone

3 as a template, several primary alkyl derivatives were rapidly

synthesized to optimize the N-alkyl moiety (Table 1,

compounds 4−8).

The N-cyclopropylmethyl derivative 4 was the most potent

compound among the primary N-alkylpiperidinone derivatives,

with a 6-fold improvement over the unsubstituted piperidinone

3. The n-propyl derivative 5 possessed an activity similar to that

of 4. When the size of the alkyl group was larger than the

cyclopropylmethyl or n-propyl, potency decreased (6, 7, and 8).

Resolution of the two enantiomers of 4 demonstrated that only

Systematic optimization of this novel piperidinone series led

to compound 25, a potent and selective MDM2 inhibitor.

Consistent with its mechanism of action, the ability of 25 to

inhibit cell proliferation and increase p21 induction of HCT116

p53^wtvs HCT116 p53^−/−tumor cells conﬁrmed that the

activity of this compound is p53 dependent. In addition,

compound 25 exhibited both dose-dependent p21 induction

and tumor growth inhibition in SJSA-1 tumor xenograft studies.

Further optimization of 25 resulted in the discovery of 29 (AM-

8553), a molecule with improved potency and pharmacokinetic

properties. 29 showed substantially lower intrinsic clearance in

human hepatocytes and excellent projected human pharmaco-

kinetic parameters. In alignment with its improved in vitro

potency, 29 demonstrated superior eﬃcacy in 14-day SJSA-1

tumor xenograft studies.

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Table 1. Exploration of the N-Alkyl Group

Figure 4. Interconversion of two cyclopropylmethyl conformations.

improve the binding aﬃnity to MDM2 by “directing” the highly

ﬂexible N-alkyl group of 2 into the Phe19 pocket. Therefore, a

series of α-substituted N-alkyl derivatives were synthesized, and

the biochemical activities of these compounds in the HTRF

competition assay¹⁶are summarized in Table 1.

B3LYP/6-31G* analysis of the dihedral proﬁle about the N−

C bond of compound 2 demonstrates that the cyclo-

propylmethyl possesses two local minima (Figure 5a). The

Figure 5. B3LYP/6-31G* dihedral proﬁles of (a) unsubstituted and

(b) model α-methylated analogue of 2.

desired, Phe19_(p53)-binding conformer (orange), destabilized by

the adjacent p-chlorophenyl, is found to lie ∼1 kcal/mol higher

in energy than the anti global minimum (cyan). Disubstitution

at the α-carbon eﬀectively raises the energy of the undesired

conformation, aiding equalization of the population (Figure 5b)

and ensuring that one of the substituents will always be directed

toward the p-chlorophenyl and into the Phe19_(p53)pocket.

Compound 9, possessing an ethyl ester “directing group” at

the α-position of the n-propyl substituent, was twice as potent

as 5, while the other diastereomer, compound 10, was 2-fold

less potent than 5 presumably because of the incorrect

positioning of the ethyl group by the ester. Lengthening,

shortening, or deleting the alkyl chain in 9 diminishes potency

(11, 12, and 13). Notably, 9 was the only compound that

showed measurable activity (IC₅₀= 18.7 μM) in the

biochemical assay upon addition of 15% human serum

(Table 1).

a

b

Compounds are racemic. Potency data are reported as the average of

c

at least two determinations. HS = human serum.

the (5R,6S)-4 enantiomer exhibited measurable activity [IC₅₀

0.82 μM; IC₅₀> 30 μM for (5S,6R)-4].

=

The N-cyclopropylmethyl group of 2 (or 4) likely requires a

“downward” orientation into the Phe19_(p53)pocket to maximize

binding aﬃnity (see Figures 3 and 4). This orientation of the

N-alkyl substituent (syn with respect to the nearby p-

chlorophenyl) results in destabilization of the desired binding

conformer.

Attention was then focused on optimizing the substituent at

the C3 position (Table 2). Introduction of the (3R) acetic acid

fragment to the piperidinone core of (5R,6S)-4 improved

It was hypothesized that incorporation of an additional

substituent at the methylene adjacent to the ring nitrogen could

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line SJSA-1 (which displays MDM2 gene ampliﬁcation), were

employed. The cyclin-dependent kinase inhibitor, p21, is a

direct transcription target of activated p53²³and is involved in

both cell cycle arrest²⁴and apoptosis.^25,26Induction of p21 is a

proximal marker of p53 activity.^14,15

Table 2. Modiﬁcation of the Carboxylic Acid

Compound 22, with an ethyl ester directing group on the N-

alkyl fragment (Table 3), was 3−4 times more potent than 2 in

the biochemical assay, the functional p21 induction assay, and

the cell proliferation assay. The tert-butyl ester derivative 23

was even more potent than the ethyl ester 22, with an

additional 2-fold improvement in both biochemical and cell-

based assays. Analogous to previous observations, replacement

of the carboxylic acid in 23 with a tetrazole provided slightly

enhanced potency (24 vs 23).

The X-ray crystal structure of compound 23 bound to human

MDM2 was determined at a resolution of 1.8 Å (Figure 6)²⁷

and was found to be consistent with the proposed binding

mode of 2 based on docking models. As predicted, the ethyl

group occupies the Phe19_(p53)binding pocket, with the tert-

butyl ester directing group extending away from the protein

with the carbonyl oxygen facing out toward solvent. Moreover,

the tert-butyl group makes van der Waals contact with the

surface of the MDM2 protein.

The X-ray cocrystal structure of 23 conﬁrmed that the trans-

C5 and C6 aryl groups reside in a gauche-like orientation when

bound to MDM2. However, quantum mechanical calculations

on the parent core structure (N-methyl, (R)-CH₃at C3; Figure

7a) predict that in the free state, the more stable conformer

instead possesses an anti-like arrangement of the C5 and C6

aryls (ΔE = 0.5 kcal/mol; B3LYP/6-31G*)²⁸unsuitable for

binding. Quaternization of the C3 position via disubstitution

was predicted to stabilize the binding conformation, via a 1,3-

steric interaction with the C5 aryl group when in the undesired,

anti conformation (Figure 7b). The crystal structure of 23

bound to MDM2 shows that such substitution would project

outward toward the solvent and not interfere with MDM2

binding. Quantum mechanical calculations predict a shift in the

equilibrium from a calculated 1:3 ratio of binding to

nonbinding conformer to one in which the binding, gauche

form now dominates (>98%; ΔE = 2.4 kcal/mol).

a

Potency data are reported as the average of at least two

b

determinations. HS = human serum.

Consistent with our hypothesis, it was found that

substitution at C3 with a methyl group aﬀorded compounds

with increased potency. Compounds 25 and 26 were 2−3 times

more potent in the biochemical and cellular assays than the

corresponding C3 des-methyl compounds 23 and 24 (Table 4).

potency in the biochemical assay by more than 20-fold,

presumably because of the electrostatic interaction between the

carboxylate anion and the H96 in MDM2 (IC₅₀for 2 of 0.034

μM vs IC₅₀for (5R,6S)-4 of 0.82 μM). Compound 14, the C3-

epimer of 2, was about 2-fold less potent than 2. The length of

the tether between the carboxylic acid and the piperidinone

core in 2 appeared to be optimized, since the corresponding

homologated acids lost potency (16 and 17 vs 2 and 14).

Additional functional groups were evaluated for their ability to

interact with H96. The terminal alkene (15) and nitrile (20)

analogues were signiﬁcantly less potent (over 70-fold).

Hydrogen bond acceptors including ester (18) and amide

(19) also had substantially reduced potency (over 10-fold).

Only compound 21, the tetrazole derivative with an acidic

proton comparable to that of the carboxylic acid, exhibited

slightly enhanced potency in the biochemical assay compared

to 2.

1

The H NMR coupling constants of 25 and 23 at two

diﬀerent temperatures were also consistent with the theoretical

predictions (Figure 8).²⁹The vicinal coupling constant between

the C5 and C6 methine protons (J_ab) of compound 23 at 298

and 203 K was 6.8 and 0 Hz, respectively. However J_abfor

compound 25 was consistently 10.9 Hz at both temperatures.

These results indicated that both anti and gauche conformations

are present in 23 at 298 K, whereas only the gauche

conformation exists for 25 at 298 K. At low temperature

(203 K), the diﬀerence is even more signiﬁcant. Compound 23

exists exclusively in the anti conformation, while the gauche

conformation of 25 is still predominant.

Further in vitro assessment of these compounds revealed that

the carboxylic acid 25 had less hPXR activation and CYP3A4

inhibition compared to tetrazole analogue 26 (Table 5).

Compound 26, the tetrazole version of 25, had an unfavorable

hPXR proﬁle (65% activation at 2 μM) and inhibited CYP3A4

(80% inhibition at 3 μM). Compound 26 is also a strong time-

Having identiﬁed the key features for the N-alkyl group and

the C3 position, an eﬀort was made to synthesize a series of

fully optimized derivatives and evaluate their activity in both

biochemical and cell-based assays (Table 3). Two diﬀerent cell-

based assays, a functional p21 induction assay¹⁶and a

proliferation assay¹⁶in the human osteosarcoma tumor cell

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Table 3. Fully Functionalized Piperidinone Derivatives

a

b

Potency data are reported as the average of at least two determinations. HS = human serum.

eﬀect on p21 mRNA was observed when 25 was treated with

HCT116 p53^−/−tumor cell lines for 7 h (Figure 9b).

A pharmacodynamic (PD) study³⁰with 25 in the SJSA-1

tumor xenograft model was also completed. SJSA-1 tumor cells

Figure 6. Co-crystal structure of 23 bound to human MDM2 (17−

111) at 1.8 Å resolution. MDM2 binding pockets are labeled (by p53

side chain) in white, H96 labeled in yellow. Cocrystallized water

molecules are shown in red.

dependent inhibitor of CYP3A4. In light of these data,

compound 25 was selected for further evaluation.

To evaluate its selectivity, we examined the eﬀect of 25 on

inhibiting the proliferation of HCT116 p53^wtand p53^−/−tumor

cells in vitro.¹⁶HCT116 p53^wtand p53^−/−tumor cells were

incubated with 25 for 16 h, and the percentage of cell-growth

inhibition was measured in a BrdU proliferation assay. Potency

in wild-type p53 cells (IC₅₀= 0.85

0.23 μM) was

substantially higher than that in p53 deﬁcient cells (IC₅₀> 25

μM) (Figure 9a). Similarly, compound 25 also exhibited a dose-

dependent increase of p21 mRNA in HCT116 p53^wtcells (IC₅₀

= 1.0 0.5 μM) in the p21 induction assay. In contrast, no

Figure 7. B3LYP/6-31G* structures and relative energies (kcal/mol)

for (a) C3 (R)-monosubstituted and (b) C3-disubstituted systems.

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Table 4. C3 Me Piperidinone Derivatives

a

b

Potency data are reported as the average of at least two determinations. HS = human serum.

Table 5. Comparison of 25 and 26 in Selected PKDM Assays

25

26

a

hPXR activation, % of control at 2 μM

27

40

77

65

80

30

CYP3A4, % inhibition at 3 μM

b

TDI, % remaining of CYP3A4 over control after 15 min at

10 μM

a

b

Rifampin used as a positive control. DMSO used as a control.

were implanted in the mice 2 weeks prior to treatment.

Compound 25 was administered orally, and p21 mRNA

induction levels were measured. In this experiment, peak

induction of p21 was observed approximately 6 h after dosing

(15-fold induction at 300 mg/kg) (Figure 10a). In a parallel

experiment, a dose-dependent increase in p21 mRNA induction

(6 h) was also observed (Figure 10b).

Next, the ability of 25 to inhibit tumor growth in the SJSA-1

xenograft model was evaluated.³⁰After a 12-day incubation of

SJSA-1 human cancer cells in the mice, compound 25 was

orally administered once daily for 14 days, and the tumor

volume was monitored. Treatment with 25 showed a dose-

dependent inhibition of tumor growth with a calculated ED₅₀of

118 mg/kg. At 200 mg/kg, 91% tumor growth inhibition was

observed (Figure 11).

Figure 8. Vicinal coupling constant between C5 and C6 methine

protons (J_ab) in compounds 23 and 25 at two diﬀerent temperatures,

298 and 203 K. H proton NMR spectra were obtained in MeOH-d₄.

1

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Figure 9. The cell activity of 25 is p53 dependent. (a) In HCT116

p53^wtand p53^−/−cells, the percentage of BrdU positive cells was

measured 16 h after compound treatment by ﬂow cytometry. DMSO

control was designated as 0% inhibition. (b) In HCT116 p53^wtand

p53^−/−cells, total RNA was extracted 7 h after compound treatment

and p21 mRNA was measured by quantitative RT-PCR.

The demonstration of robust antitumor eﬃcacy in xenograft

studies with compound 25 led us to focus on further

optimization to improve potency and pharmacokinetic (PK)

properties. On the basis of the X-ray cocrystal structure of 23

with the MDM2 protein (Figure 6), in which the carbonyl of

the tert-butyl ester points away from the protein, it was

hypothesized that replacing the tert-butyl ester with other

functional groups would allow us to modulate the phys-

icochemical properties of our molecules without disrupting

critical interactions with the protein. Although the tert-butyl

group does engage in some degree of van der Waals contact

with the protein, we felt that such modiﬁcation could also

potentially oﬀer an opportunity to enhance potency by picking

up better interactions with the protein surface than those

provided by the tert-butyl ester.

A variety of functional groups such as alcohols, amides,

amines, and heterocycles are tolerated in this area of the

molecule (Figure 12). These changes provided diverse

physicochemical properties while maintaining acceptable

potency.³¹For example, replacing the tert-butyl ester in 25

with an ethyl group (27) or a primary alcohol (28) provided

molecules with good biochemical potency (IC₅₀for 27 of 2.8

nM and IC₅₀for 28 of 1.7 nM in Table 4), although the ethyl

derivative (27) showed a greater loss in potency upon addition

of human serum compared to the primary alcohol (IC₅₀for 27

of 36.3 nM vs IC₅₀for 28 of 6.8 nM). Relative to 28,

compound 27 also exhibited a higher IC₅₀in cell-based assays,

apparently because of the protein binding diﬀerences.

Interestingly, addition of a methyl group to the α-position of

the hydroxyl group in 28 boosted potency 2- to 3-fold in the

biochemical and cell-base assays (29 vs 28). On the basis of its

potency, pharmacokinetic properties, and selectivity, the

secondary alcohol derivative 29 was selected for further

development.

Figure 10. PD study results of compound 25 in SJSA1 tumor

xenograft: *p < 0.05. Female athymic nude mice were implanted

subcutaneously with 5 × 10⁶SJSA-1 cells. When tumors reached ∼175

mm³, (a) 25 or vehicle was administered orally once. Mice were

sacriﬁced at 1, 3, 6, 9, 16, and 24 h postdose (n = 5/group). (b)

Vehicle or 30, 100, or 300 mg/kg 25 was administered orally once.

Mice were sacriﬁced at 6 h postdose (n = 5/group). Tumors were

immediately removed and snap-frozen. p21 mRNA levels were

measured by quantitative RT-PCR. Tumors treated with vehicle

served as a negative control and indicated the baseline p21 mRNA

level. Data are represented as mean p21 fold induction over vehicle,

and error bars represent SEM of data from ﬁve mice. Concentrations

in blood plasma were analyzed by LC/MS/MS.

observed with 25. It was also found that 29 had reduced liability

in the hPXR, CYP3A4, and TDI assays compared to 25. In

addition to its suitable pharmacokinetic proﬁle in rat, 29

exhibited low plasma clearance relative to hepatic blood ﬂow in

cynomolgus monkey (CL = 0.72 L h⁻¹kg⁻¹) with a high

volume of distribution (6.2 L/kg) and long half-life (t_1/2= 14

h).

The most signiﬁcant advantage of 29 over 25 is its greatly

improved human hepatocyte intrinsic clearance (3.0 μL/min

per 10⁶cells for 29 vs 26 μL/min per 10⁶cells for 25), which

resulted in extended predicted human half-life (>12 h for 29 vs

0.28 h for 25). Given the improved potency and superior

predicted human PK properties of 29, we decided to evaluate

its pharmacodynamic response and ability to inhibit tumor

growth in the SJSA-1 mouse xenograft model.

The dissociation constant (K_D) for 29 and 25 was

determined in a surface plasmon resonance (SPR) spectroscopy

binding assay (Table 6).¹⁶The K_Dof 29 was 0.4 nM, while that

of 25 was 1.0 nM. Compound 29 is consistently more potent

than 25 in the biochemical and cell-based assays. Evaluation of

29 in the HCT116 p53^wtand HCT116 p53^−/−tumor cell lines

showed >100-fold selectivity and is consistent with the data

The eﬀect of 29 on p21 mRNA induction after dosing q.d.

for 4 days at 150 mg/kg was evaluated in the mouse SJSA-1

tumor model. As shown in Figure 13, 29 caused signiﬁcant and

time-dependent p21 induction over vehicle. A 14-fold induction

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Table 6. Comparison of 25 and 29

25

29

Biochemical Potency

K_D(SPR, nM)

1.0

0.4

IC₅₀(HTRF, nM)

2.2 0.7

1.1 0.5

4.2 1.8

a

IC₅₀(HTRF, nM)

19.9 7.5

Cellular Potency (SJSA-1)

b

p21 IC₅₀(μM)

1.9 0.5

0.76 0.29

b

EdU IC₅₀(μM)

0.19 0.06

0.068 0.016

Speciﬁcity (HCT116)

b

BrdU IC₅₀(p53^wt, μM)

0.85 0.23

>25

0.20 0.12

>25

b

BrdU IC₅₀(p53^−/−, μM)

hPXR Activation

c

Figure 11. Inhibition of SJSA-1 tumor xenograft growth by compound

25: *p < 0.05. SJSA-1 cells (5 × 10⁶) were implanted subcutaneously

into female athymic nude mice. Treatment with vehicle or 25 at 25, 50,

100, or 200 mg/kg q.d. by oral gavage began on day 12 when tumors

had reached ∼200 mm³(n = 10/group). Tumor sizes and body

weights were measured twice per week. Data are represented as mean

tumor volumes, and the error bars represent SEM of data from 10

mice.

% of control at 2 μM

27

40

77

15

13

96

CYP3A4

% inhibition at 3 μM

TDI

d

% remaining of CYP3A4 over control

after 15 min at 10 μM

Rat PK

CL (L h⁻¹kg⁻¹

V_dss(L/kg)

F (%)

)

2.7

1.5

77

1.2

12

100

Mouse PK

CL (L h⁻¹kg⁻¹

V_dss(L/kg)

F (%)

)

1.5

1.3

36

3.5

5.2

12

Hepatocyte Stability

human (μL/min per 10⁶cells)

26

3.0

Projected Human PK

0.55

CL (L h⁻¹kg⁻¹

half-life (h)

)

0.03

>12

0.28

a

b

c

15% human serum. 10% human serum. Rifampin used as a positive

Figure 12. Transformation of 25 into 29.

d

control. DMSO used as a control.

of p21 mRNA was observed 4 h postdose. The AUC of p21

fold induction was about 2-fold greater than that observed with

25 at 300 mg/kg q.d. dosing (230-fold p21 induction·h/day for

29 vs 135-fold p21 induction·h/day for 25). Furthermore, 29

also demonstrated a dose-dependent eﬀect on p21 mRNA

induction in SJSA-1 tumors (data not shown).

C5 aryl group ﬁlls the Leu26_(p53)pocket and engages in a face-

to-face, π-stacking interaction with H96, while the C6 aryl

group occupies the Trp23_(p53)binding cavity. The hydroxyl

Compound 29 was also evaluated for its ability to inhibit

tumor growth in the SJSA-1 mouse xenograft model. As

expected, a dose-dependent response of tumor growth

inhibition was observed with 29. It signiﬁcantly inhibited

tumor growth at 150 and 200 mg/kg q.d. compared to vehicle,

with an ED₅₀of 78 mg/kg (Figure 14). Importantly, 200 mg/kg

q.d. of 29 caused tumor regression (R = 27%). There was no

body weight loss in any of the treatment groups.

Since 29 has a relatively high clearance (CL = 3.5 L h⁻¹kg⁻¹)

and short half-life time (t_1/2= 2.6 h) in mouse, we decided to

explore whether more frequent dosing of 29 would lead to

enhanced tumor growth inhibition in the SJSA-1 xenograft

model. A b.i.d. treatment caused a dose-dependent reduction in

tumor growth with statistically signiﬁcant inhibition at 75 and

100 mg/kg compared to vehicle (Figure 15). Overall, inhibition

with b.i.d. dosing was comparable to that of q.d. dosing at all

dose levels. Again, no signiﬁcant body weight loss was observed

for any of the treated groups compared to the vehicle group.

Finally, the cocrystal structure of 29 with human MDM2

protein was obtained by X-ray crystallography to a resolution of

2.0 Å (Figure 16).³²As was seen with the structure of 23,

compound 29 occupies the three critical binding pockets. The

Figure 13. PD study results of compound 29 in SJSA-1 tumor

xenograft: *p < 0.05. Female athymic nude mice were implanted

subcutaneously with 5 × 10⁶SJSA-1 cells. When tumors reached ∼175

mm³, 29 or vehicle was administered orally once daily (q.d.) for 4

days. Mice were sacriﬁced on day 4 at 1, 2, 4, 8, and 24 h postdose (n =

5/group). Tumors were immediately removed and snap-frozen. p21

mRNA levels were measured by quantitative RT-PCR. Tumors treated

with vehicle served as a negative control and indicated the baseline p21

mRNA level. Data are represented as mean p21 fold induction over

vehicle, and error bars represent SEM of data from ﬁve mice.

Concentrations in blood plasma were analyzed by LC/MS/MS.

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Figure 14. Inhibition of SJSA1 tumor xenograft growth by 29: *p <

0.05. SJSA-1 cells (5 × 10⁶) were implanted subcutaneously into

female athymic nude mice. Treatment with vehicle or 29 at 25, 75,

150, or 200 mg/kg q.d. by oral gavage began on day 11 when tumors

had reached ∼200 mm³(n = 10/group). Tumor sizes and body

weights were measured twice per week. Data are represented as mean

tumor volumes, and the error bars represent SEM of data from 10

mice.

Figure 16. Cocrystal structure of 29 bound to human MDM2 (17−

111) at 2.0 Å resolution. White labels indicate the positions normally

occupied by key p53 residues. H96 is labeled in yellow. Cocrystallized

water molecules are shown in red.

reaction of azide with Me₃P, followed by cyclization under

mildly basic conditions. Deprotonation of 34 with NaH and

treatment with cyclopropylmethyl bromide provided inter-

mediate 35, which was alkylated at C3 with NaHMDS and allyl

bromide (dr = 99:1). Oxidative cleavage of the alkene in 36

with catalytic ruthenium tetroxide provided the desired racemic

piperidinone acid 1 (Scheme 1).

Compounds 3−13 were synthesized according to the

procedures outlined in Scheme 2. The preparation of the

racemic piperidinone core 3 started with mesylation of 32,

followed by displacement with NaN₃, to provide azide 37. The

piperidinone core 3 was obtained via procedures similar to

those described for the synthesis of 34. Alkylation of 3 with the

a

Scheme 1

Figure 15. SJSA-1 cells (5 × 10⁶) were implanted subcutaneously into

female athymic nude mice: *p < 0.05. Treatment with vehicle or 29 at

25, 50, 75, or 100 mg/kg b.i.d. by oral gavage began on day 10 when

tumors had reached ∼200 mm³(n = 10/group). Tumor sizes and

body weights were measured twice per week. Data are represented as

mean tumor volumes, and the error bars represent SEM of data from

10 mice.

group is exposed to solvent and the ethyl group directed into

the Phe19_(p53)pocket by the conformational constraint induced

by the α-substitution. Additionally, the carboxylate of 29

interacts with adjacent and presumably charged imidazole on

the H96 side chain of MDM2. Finally, this cocrystal structure

shows that the C3 methyl group points directly out toward

solvent, supporting our proposal that the potency increase from

C3 methylation (23 vs 25) is conformationally induced and

independent of protein contacts.

CHEMISTRY

■

The synthesis of cis-piperidinone 1 started with the preparation

of ketone 31, which was obtained from the reaction of the

dianion of 2-(3-chlorophenyl)acetic acid (30) with methyl 4-

chlorobenzoate. Subsequent conjugate addition with methyl

acrylate, followed by reduction with NaBH₄provided alcohol

32 as a single diastereomer. Bromination of 32 with CBr₄and

PPh₃, followed by displacement with NaN₃, gave azide 33. The

piperidinone core 34 was obtained from the Staudinger

a

Reagents and conditions: (a) NaHMDS, 4-ClPhCO₂Me, THF, 50%;

(b) methyl acrylate, t-BuOK, THF, 84%; (c) NaBH₄, MeOH, 82%;

(d) CBr₄, PPh₃, DCM, 80%; (e) NaN₃, DMF, 77%; (f) Me₃P, THF/

H₂O; NaHCO₃, MeOH/H₂O, 87%; (g) cyclopropylmethyl bromide,

NaH, THF, 84%; (h) LiHMDS, allyl bromide, 84%; (i) RuCl₃, NaIO₄,

CH₃CN/CCl₄/H₂O, 79%.

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a

Scheme 2

Scheme 3

a

Reagents and conditions: (a) for n = 1, allyl bromide, LiHMDS,

THF, 85%; for n = 2, 4-bromo-1-butene, LiHMDS, THF, 42%; (b)

NaIO₄, RuCl₃, CH₃CN/CCl₄/H₂O, 70−82%; (c) SOCl₂, MeOH,

89%; (d) 7 M NH₃, in MeOH, 78%; (e) (CF₃CO)₂O, TEA, THF,

93%; (f) NaN₃, NH₄Cl, DMF, 90 °C, 41%.

a

Scheme 4

a

Reagents and conditions: (a) MsCl, TEA, DCM; NaN₃, DMF, 56%;

(b) Me₃P, THF/H₂O; NaHCO₃, MeOH/H₂O, 88%; (c) separation

by chiral HPLC; (d) primary alkyl halide, NaH, DMF, 70−90%; (e)

EtOC(O)CHBrR′, NaH, DMF, 75−83%.

corresponding primary alkyl halides provided 4−8 and 13.

Compounds 9−12 were prepared by N-alkylation of 3 with

EtOC(O)-CHBrR′, followed by separation of diastereomers

using silica gel column chromatography (R′ = Me, dr = 2.3:1; R′

= Et, dr = 1.8:1; R′ = n-Pr, dr = 1:1 in favor of the desired S-

isomer). Preparation of (5R,6S)-4³³and (5S,6R)-4³³was

accomplished by separation of the enantiomers in 3 using

normal phase chiral HPLC, followed by N-alkylation of each

enantiomer.

a

Scheme 3 describes the synthesis of compounds 2 and 14−

21. C3 allylation of (5R,6S)-4, followed by separation of

diastereomers gave 15³⁴and its C3 epimer, 38.³⁴Oxidative

cleavage of the alkene in 15 and 38 with catalytic ruthenium

tetroxide provided acid 2 and its C3 epimer 14, respectively. In

a similar manner, the homologated acids 16 and 17 were

synthesized from homoallyl intermediates 39³⁴and 40.³⁴

Compound 2 was converted to its methyl ester 18 by

SOCl₂/MeOH. Direct transformation of ester 18 to primary

amide 19 was achieved using 7 M NH₃in MeOH. Dehydration

of 19 with triﬂuoroacetic acid anhydride and TEA provided

nitrile 20, which was converted to the tetrazole 21 using

sodium azide.

Reagents and conditions: (a) ROC(O)CHBrEt, NaH, DMF, 42−

46%; (b) allyl bromide, LiHMDS, THF, 75−80%; (c) NaIO₄, RuCl₃,

CH₃CN/CCl₄/H₂O, 60−70%; (d) isobutyl chloroformate, N-methyl-

morpholinone, NH₄OH, 90%; (e) (CF₃CO)₂O, TEA, THF, 85%; (f)

NaN₃, NH₄Cl, DMF, 90 °C, 45%.

was subjected to C3 allylation to provide a mixture of two C3

isomers (R = Et, dr = 1.6:1; R = t-Bu, dr = 4.9:1 in favor of the

S-isomer). Again, the desired S-isomer was isolated by silica gel

column chromatography (41, R = Et; 42, R = t-Bu) and treated

with NaIO₄and RuCl₃to aﬀord acids 22 and 23. Further

modiﬁcation of the carboxylic acid in 23 into the tetrazole via

amide coupling with NH₃(43), dehydration, and reaction with

sodium azide yielded 24.

Compounds 22−24³³were synthesized in a straightforward

manner, through the routes illustrated in Scheme 4. After N-

alkylation of (5R,6S)-3 with ROC(O)CHBrEt, the desired S-

isomer was isolated by silica gel column chromatography (R =

Et, dr = 1.8:1; R = t-Bu, dr = 4.6:1 in favor of the S-isomer) and

Compounds 25−29³³were synthesized via the routes

outlined in Scheme 5. Introduction of a DMB (2,4-

dimethoxybenzyl) protecting group to the amide nitrogen of

(5R,6S)-3 with freshly prepared DMBCl³⁵followed by

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a

similar to those described for the synthesis of 23 and 24.

Reduction of 49 with LiBH₄in Et₂O provided the primary

alcohol 50 which was oxidized with Dess−Martin periodinane.

The resulting aldehyde was treated with MeMgBr to give the

secondary alcohol 51 as a mixture of two diastereomers (dr =

40:60 in favor of the R-isomer). Simultaneous oxidation of both

the secondary alcohol and the alkene in 51 with ruthenium

tetroxide provided keto acid 52 in good yield. Finally, reduction

of methyl ketone 52 with L-Selectride at −78 °C gave 29 as the

predominant product (dr = 99:1 in favor of the S-isomer).³⁶

Scheme 5

CONCLUSION

■

In summary, the synthesis and initial SAR of rigid cores capable

of holding two aromatic rings in proximity led to the

identiﬁcation of 1 as a novel inhibitor of the MDM2−p53

protein−protein interaction. Optimization of 1 led to the

discovery of 2, a piperidinone derivative featuring an acetic acid

substituent at C3 capable of forming an electrostatic interaction

with the H96 imidazole side chain of MDM2. Systematic

optimization of the N-alkyl substituent of 2 led to molecules

with highly populated, well-deﬁned free-state conformations

suited for optimal binding. These molecules were designed to

occupy the Phe19 pocket, provide polar functional groups to

interact with solvent, and gain additional van der Waals contact

with the MDM2 protein. In addition, optimization of the

piperidinone ring led to stabilization of the desired trans-diaryl

conformation of these molecules by incorporation of a methyl

group at the C3 position, leading to the discovery of 25.

Further optimization resulted in the discovery of 29 which

demonstrated superior eﬃcacy in 14-day SJSA-1 xenograft

studies. Compound 29 is a highly potent, selective, and orally

bioavailable inhibitor of the MDM2−p53 interaction and is

projected to have low clearance and a long half-life in humans.

EXPERIMENTAL SECTION

■

General Chemistry. All reactions were conducted under an inert

gas atmosphere (nitrogen or argon) using a Teﬂon-coated magnetic

stir bar at the temperature indicated. Commercial reagents and

anhydrous solvents were used without further puriﬁcation. Analytical

thin layer chromatography (TLC) and ﬂash chromatography were

performed on Merck silica gel 60 (230−400 mesh). Removal of

solvents was conducted by using a rotary evaporator, and residual

solvent was removed from nonvolatile compounds using a vacuum

manifold maintained at approximately 1 Torr. All yields reported are

isolated yields. Preparative reversed-phase high pressure liquid

chromatography (RP-HPLC) was performed using an Agilent 1100

series HPLC 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, B = CH₃CN with 0.1% TFA;

gradient, 5−95% B (0.0−15.0 min); ﬂow rate, 1.5 mL/min. Low-

resolution mass spectral data were determined on an Agilent 1100

series LCMS with UV detection at 254 nm and a low resolution

electrospray mode (ESI). High-resolution mass spectra were obtained

on an Agilent 6510 Q-TOF MS instrument with an Agilent 1200 LC

on the front end. ¹H NMR spectra were obtained on a Bruker Avance

III 500 (500 MHz) or Bruker Avance II 400 (400 MHz) spectrometer.

Chemical shifts (δ) are reported in parts per million (ppm) relative to

residual undeuterated solvent as an internal reference. The following

abbreviations were used to explain the multiplicities: s = single; d =

doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet

of triplets, m = multiplet, br = broad. Optical rotations ([α]_D) were

a

Reagents and conditions: (a) DMBCl, NaH, 25 °C, 85%; (b) MeI,

LiHMDS, THF, −78 °C, 91%; (c) allyl bromide, LiHMDS, THF, 40

°C 82%; (d) TFA, 50 °C, 70%; (e) 3-bromopentane, NaH, 40%; (f)

NaIO₄, RuCl₃, CH₃CN/CCl₄/H₂O, 47−80%; (g) R′OC(O)CHBrEt,

NaH, DMF, 40−50%; (h) isobutyl chloroformate, N-methylmorpho-

linone, NH₄OH, 90%; (i) (CF₃CO)₂O, TEA, THF, 80%; (j) NaN₃,

NH₄Cl, DMF, 90 °C, 41%; (k) LiBH₄, Et₂O, 75%; (l) TBDPSCl,

imidazole, 90%; (m) TBAF, 78%; (n) Dess−Martin periodinane, 79%;

(o) MeMgBr, THF, 87%; (p) L-Selectride, −78 °C, 80%.

sequential methylation and allylation of the DMB protected

core (44) gave 45 with the desired C3-epimer as a major

product (dr = 3.9:1 in favor of the S-isomer). Removal of the

DMB group using TFA and separation of the diastereomers by

silica gel column chromatography provided 46. The 3-pentyl

derivative 27 was synthesized by N-alkylation of 46 with neat 3-

bromopentane and NaH, followed by the reaction of 47 with

NaIO₄and RuCl₃, as before. N-Alkylation of 46 with

R′OC(O)-CHBrEt followed by isolation of the desired isomer

by silica gel column chromatography aﬀorded intermediates 48

(R′ = t-Bu) and 49 (R′ = Et) (R′ = t-Bu, dr = 2.2:1; R′ = Et, dr =

1:1 in favor of the desired S-isomer). The carboxylic acid (25)

and the tetrazole (26) were prepared from 48 via procedures

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measured on a JASCO P-1020 polarimeter. Speciﬁc rotations are given

as deg/dm, and the concentrations are reported as g/100 mL of the

speciﬁc solvent and were recorded at the temperature indicated.

2-(3-Chlorophenyl)-1-(4-chlorophenyl)ethanone (31). To a

solution of 30 (13.0 g, 76.0 mmol) in THF (250 mL) was added

NaHMDS (1.0 M in THF, 160 mL, 160 mmol) slowly over 15 min at

−78 °C. After the mixture was stirred at −78 °C for 20 min, a solution

of methyl 4-chlorobenzoate (13.0 g, 76.0 mmol) in THF (50 mL) was

added over 10 min. The resulting solution was allowed to warm to 25

°C and stirred for 2 h. The reaction was quenched (ice-cold water)

and extracted (2 × EtOAc). The combined organic layers were dried

(Na₂SO₄) and concentrated under reduced pressure. Flash column

chromatography (SiO₂, 5−7% EtOAc/hexanes, gradient elution)

( )-(5S,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)piperidin-

2-one (34). To a solution of 33 (195 mg, 0.516 mmol) in THF/H₂O

(4:1, 5 mL) was added trimethylphosphine (1.0 M solution in THF,

0.65 mL, 0.65 mmol). After the mixture was stirred for 1 h at 25 °C,

the residue was basiﬁed (ice-cold 2 M LiOH) and extracted (2 ×

EtOAc). The combined organic layers were washed (2 × brine), dried

(Na₂SO₄), and concentrated under reduced pressure to provide the

crude ( )-(4S,5S)-methyl 5-amino-4-(3-chlorophenyl)-5-(4-

chlorophenyl)pentanoate as a colorless ﬁlm.

The crude amine was dissolved in MeOH/saturated aqueous

NaHCO₃(4:1, 35 mL), and the mixture was reﬂuxed for 3 h. After

MeOH was removed under reduced pressure, the residue was diluted

(water) and extracted (3 × DCM). The combined organic layers were

washed (1 × brine), dried (Na₂SO₄), and concentrated under reduced

pressure. Flash chromatography (SiO₂, 80−100% EtOAc/hexanes,

gradient elution) provided 34 (115 mg, 70% yield) as a whilte soild:

¹H NMR (400 MHz, CDCl₃) δ ppm 7.18−7.23 (1 H, m), 7.10−7.17

1

provided 31 (10.2 g, 50% yield) as a white solid: H NMR (400

MHz, CDCl₃) δ ppm 7.93−7.96 (2 H, m), 7.42−7.48 (2 H, m), 7.23−

7.30 (3 H, m), 7.12−7.16 (1 H, m), 4.24 (2 H, s); MS (ESI) 265.0 [M

+ H]⁺.

(3 H, m), 6.84 (1 H, t, J = 1.8 Hz), 6.62−6.69 (3 H, m), 6.18 (1 H, br

s), 4.77 (1 H, t, J = 4.5 Hz), 3.47−3.55 (1 H, m), 2.73−2.83 (1 H, m),

2.58−2.71 (1 H, m), 2.08−2.21 (1 H, m), 1.81−1.90 (1 H, m); MS

(ESI) 319.9 [M + H]⁺.

( )-(4S,5S)-Methyl 4-(3-Chlorophenyl)-5-(4-chlorophenyl)-5-

hydroxypentanoate (32). To a solution of 31 (5.62 g, 21.2 mmol)

and methyl acrylate (1.91 mL, 21.2 mmol) in THF (60 mL) was

added t-BuOK (1.0 M in THF, 2.12 mL, 2.12 mmol) at 0 °C, and the

mixture was allowed to warm to 25 °C. After being stirred at 25 °C for

1 h, the mixture was concentrated under reduced pressure, diluted

(H₂O), extracted (2 × EtOAc), and washed (brine). The combined

organic layers were dried (Na₂SO₄) and concentrated under reduced

pressure. Flash column chromatography (SiO₂, 10% EtOAc/hexanes)

provided methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-oxopenta-

(

)-(5S,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-

(cyclopropylmethyl)piperidin-2-one (35). To a solution of 34

(300 mg, 0.937 mmol) in DMF (3.8 mL) was added NaH (60% in

mineral oil, 49 mg, 1.22 mmol) at 0 °C. The mixture was stirred at 0

°C for 20 min and then treated with cyclopropylmethyl bromide (152

μL, 1.12 mmol). After the mixture was stirred at 25 °C for 5 h, the

reaction was quenched (saturated aqueous NH₄Cl), extracted (2 ×

EtOAc), and washed (brine). The combined organic layers were dried

(Na₂SO₄) and concentrated under reduced pressure. Flash chroma-

tography (SiO₂, 30−50% EtOAc/hexanes, gradient elution) provided

1

noate (6.23 g, 84% yield) as a colorless liquid: H NMR (500 MHz,

CDCl₃) δ ppm 7.87−7.90 (2 H, m), 7.37−7.41 (2 H, m), 7.26−7.28

(1 H, m), 7.20−7.26 (2 H, m), 7.14−7.17 (1 H, m), 4.64 (1 H, t, J =

7.3 Hz), 3.67 (3 H, s), 2.40−2.48 (1 H, m), 2.31 (2 H, t, J = 7.2 Hz),

2.09−2.19 (1 H, m); MS (ESI) 351.1 [M + H]⁺.

1

35 (294 mg, 84% yield) as a colorless foam: H NMR (400 MHz,

CDCl₃) δ ppm 7.19−7.24 (1 H, m), 7.11−7.17 (3 H, m), 6.86 (1 H, t,

J = 1.8 Hz), 6.65 (1 H, d, J = 7.4 Hz), 6.55 (2 H, d, J = 8.6 Hz), 4.85 (1

H, d, J = 4.7 Hz), 3.91 (1 H, dd, J = 14.1, 6.3 Hz), 3.53 (1 H, ddd, J =

13.4, 5.0, 2.7 Hz), 2.82−2.90 (1 H, m), 2.67−2.78 (1 H, m), 2.44 (1

H, dd, J = 14.1, 7.8 Hz), 2.04−2.20 (1 H, m), 1.78−1.85 (1 H, m),

0.88−0.98 (1 H, m), 0.52 (1 H, tt, J = 8.7, 4.6 Hz), 0.36−0.44 (1 H,

m), 0.05−0.20 (2 H, m); MS (ESI) 373.9 [M + H]⁺.

To a solution of the product above (6.23 g, 17.8 mmol) in MeOH

(60 mL) was added NaBH₄(671 mg, 17.7 mmol) at 0 °C. After the

mixture was stirred at 25 °C for 1 h, the reaction was quenched (ice-

cold H₂O) and extracted (2 × EtOAc). The combined organic layers

were washed (brine), dried (Na₂SO₄), and concentrated under

reduced pressure. Flash column chromatography (SiO₂, 20−30%

EtOAc/hexanes, gradient elution) provided 32 (5.14 g, 82% yield) as a

colorless liquid: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.29−7.34 (2 H,

m), 7.25−7.27 (2 H, m), 7.19−7.24 (3 H, m), 7.03−7.07 (1 H, m),

4.76 (1 H, d, J = 7.4 Hz), 3.58 (3 H, s), 2.81−2.88 (1 H, m), 2.00−

2.13 (2 H, m), 1.74−1.96 (2 H, m); MS (ESI) 375.1 [M + Na]⁺.

(

)-(3R,5S,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophen-

yl)-1-(cyclopropylmethyl)piperidin-2-one (36). To a solution of

35 (275 mg, 0.735 mmol) and allyl bromide (67 μL, 0.77 mmol) in

THF (3.6 mL) was added LiHMDS (1 M in THF, 0.81 mL, 0.81

mmol) at −78 °C dropwise. After the mixture was stirred at −78 °C

for 3 h, the reaction was quenched (saturated aqueous NH₄Cl),

extracted (2 × EtOAc), and washed (brine). The combined organic

layers were dried (Na₂SO₄) and concentrated under reduced pressure.

Flash chromatography (SiO₂, 20−30% EtOAc/hexanes, gradient

elution) provided 36 (256 mg, 84% yield) as a colorless oil: ¹H

NMR (400 MHz, CDCl₃) δ ppm 7.17−7.24 (1 H, m), 7.14 (3 H, t, J =

8.0 Hz), 6.88 (1 H, s), 6.65 (1 H, d, J = 7.4 Hz), 6.54 (2 H, d, J = 8.6

Hz), 5.73−6.02 (1 H, m), 5.05−5.12 (2 H, m), 4.81 (1 H, d, J = 4.7

Hz), 3.92 (1 H, dd, J = 14.1, 6.3 Hz), 3.56−3.64 (1 H, m), 2.77−2.87

(2 H, m), 2.40−2.53 (2 H, m), 2.11−2.22 (1 H, m), 1.71−1.78 (1 H,

m), 0.85−1.00 (1 H, m), 0.47−0.58 (1 H, m), 0.36−0.43 (1 H, m),

0.13−0.21 (1 H, m), 0.02−0.11 (1 H, m); MS (ESI) 414.0 [M + H]⁺.

(

)-(4S,5S)-Methyl 5-Azido-4-(3-chlorophenyl)-5-(4-

chlorophenyl)pentanoate (33). To a solution of 32 (300 mg,

0.849 mmol) in DCM (5 mL) were added carbon tetrabromide (422

mg, 1.27 mmol) and triphenylphosphine (334 mg, 1.27 mmol)

sequentially at 0 °C. After the mixture was stirred at 0 °C for 3 h, the

reaction was quenched (ice-cold H₂O) and extracted (2 × DCM). The

combined organic layers were washed (brine), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash chromatography (SiO₂,

10−15% EtOAc/hexanes, gradient elution) provided ( )-(4S,5R)-

methyl 5-bromo-4-(3-chlorophenyl)-5-(4-chlorophenyl)pentanoate

(283 mg, 80% yield) as a colorless liquid: ¹H NMR (500 MHz,

CDCl₃) δ ppm 7.13−7.18 (2 H, m), 7.08−7.13 (4 H, m), 6.94−6.99

(1 H, m), 6.79−6.86 (1 H, m), 5.02 (1 H, d, J = 10.0 Hz), 3.64 (2 H,

s), 3.13−3.37 (1 H, m), 2.70−2.89 (1 H, m), 2.11−2.22 (2 H, m),

1.91−2.05 (1 H, m).

(

)-2-((3S,5S,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-

(cyclopropylmethyl)-2-oxopiperidin-3-yl)acetic Acid (1). To a

rapidly stirring solution of 36 (27 mg, 0.064 mmol) and RuCl₃hydrate

(1.4 mg, 0.0064 mmol) in H₂O/CCl₄/AcCN (1.5:1:1, 1.3 mL) was

added NaIO₄(55 mg, 0.26 mmol) in 4 portions over 20 min while

maintaining the temperature below 25 °C. After being stirred

vigorously for 3 h, the mixture was ﬁltered through Celite and the

ﬁlter cake was washed (EtOAc). The ﬁltrate was extracted (2 ×

EtOAc) and washed (2 × 10% aqueous NaHSO₃and 1 × brine). The

combined organic layers were dried (Na₂SO₄) and concentrated under

reduced pressure. Puriﬁcation by RP-HPLC (10−90% A/B, gradient

To a solution of the bromide above (280 mg, 0.673 mmol) in DMF

(1.8 mL) was added NaN₃(219 mg, 3.36 mmol). After being stirred at

100 °C for 30 min, the mixture was cooled to ambient temperature,

diluted (water), extracted (2 × EtOAc), and washed (3 × brine). The

combined organic layers were dried (Na₂SO₄) and concentrated under

reduced pressure. Flash chromatography (SiO₂, 5−10% EtOAc/

hexanes, gradient elution) provided 33 (195 mg, 77% yield) as a

colorless liquid: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.32−7.43 (2 H,

m), 7.26 (2 H, d, J = 2.3 Hz), 7.12−7.22 (3 H, m), 6.93−7.09 (1 H,

m), 4.61 (1 H, d, J = 8.2 Hz), 3.59 (3 H, s), 2.91 (1 H, q, J = 8.2 Hz),

1.96−2.23 (2 H, m), 1.82 (2 H, q, J = 7.6 Hz).

1

elution) provided 1 (22 mg, 79% yield) as a white solid: H NMR

(400 MHz, CDCl₃) δ ppm 7.23−7.26 (1 H, m), 7.13−7.19 (3 H, m),

6.87 (1 H, t, J = 1.6 Hz), 6.63 (1 H, d, J = 7.8 Hz), 6.54 (2 H, d, J = 8.6

L

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Hz), 4.87 (1 H, d, J = 5.1 Hz), 3.85 (1 H, dd, J = 14.1, 6.7 Hz), 3.51−

3.58 (1 H, m), 3.33−3.41 (1 H, m), 3.14 (1 H, dd, J = 16.4, 7.0 Hz),

2.72 (1 H, dd, J = 16.6, 6.5 Hz), 2.59 (1 H, dd, J = 14.1, 7.4 Hz), 2.36−

2.50 (1 H, m), 1.70 (1 H, d, J = 13.7 Hz), 0.87−1.01 (1 H, m), 0.51−

0.61 (1 H, m), 0.40−0.50 (1 H, dd, J = 8.2, 4.7 Hz), 0.14−0.24 (1 H,

m), 0.03−0.13 (1 H, m); MS (ESI) 431.9 [M + H]⁺, 429.9 [M − H]⁻.

( )-(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-propyl-

piperidin-2-one (5). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.26−7.30

(2 H, m), 7.14−7.24 (2 H, m), 7.06−7.08 (1 H, m), 6.93−6.97 (2 H,

m), 6.84−6.88 (1 H, m), 4.53 (1 H, d, J = 7.8 Hz), 3.84−3.93 (1 H,

m), 2.93−3.00 (1 H, m), 2.56−2.69 (2 H, m), 2.40−2.50 (1 H, m),

2.01−2.09 (2 H, m), 1.48−1.60 (2 H, m), 0.83 (3 H, t, J = 7.2 Hz);

HRMS (ESI) m/z 362.1060 [M + H]⁺(C₂₀H₂₁Cl₂NO requires

362.1069).

(

)-(4S,5R)-Methyl 5-Azido-4-(3-chlorophenyl)-5-(4-

chlorophenyl)pentanoate (37). To a solution of 32 (5.14 g, 14.5

mmol) and TEA (4.06 mL, 29.1 mmol) in DCM (72 mL) was added

MsCl (1.47 mL, 18.9 mmol) dropwise at 0 °C, and the resulting

solution was stirred at 0 °C for 1 h. The reaction was quenched (ice-

cold water), extracted (3 × DCM), and washed (brine). The

combined organic layers were dried (Na₂SO₄) and concentrated

under reduced pressure to provide a crude mesylated intermediate.

The residue above was dissolved in DMF (40 mL), and NaN₃(4.73

g, 72.8 mmol) was added. After being stirred at 90 °C for 30 min, the

mixture was cooled to 25 °C, diluted (water), extracted (2 × EtOAc),

and washed (3 × brine). The combined organic layers were dried

(Na₂SO₄) and concentrated under reduced pressure. Flash column

chromatography (SiO₂, 5−7% EtOAc/hexanes, gradient elution)

( )-(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-isobu-

1

tylpiperidin-2-one (6). H NMR (400 MHz, CDCl₃) δ ppm 7.25−

7.27 (2 H, m), 7.15−7.24 (2 H, m), 7.03−7.06 (1 H, m), 6.86−6.93 (2

H, m), 6.80−6.83 (1 H, m), 4.49 (1 H, d, J = 8.6 Hz), 3.87−3.96 (1 H,

m), 2.88−2.96 (1 H, m), 2.74−2.82 (1 H, m), 2.60−2.72 (1 H, m),

1.88−2.22 (4 H, m), 0.87 (3 H, d, J = 6.6 Hz), 0.83 (3 H, d, J = 6.3

Hz); HRMS (ESI) m/z 376.1243 [M + H]⁺(C₂₁H₂₃Cl₂NO requires

376.1225).

(

)-(5R,6S)-1-Butyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-

piperidin-2-one (7). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.27−7.30

(2 H, m), 7.15−7.24 (2 H, m), 7.06−7.08 (1 H, m), 6.93−6.97 (2 H,

m), 6.83−6.87 (1 H, m), 4.53 (1 H, d, J = 7.8 Hz), 3.88−3.96 (1 H,

m), 2.93−3.00 (1 H, m), 2.55−2.79 (2 H, m), 2.42−2.51 (1 H, m),

2.02−2.10 (2 H, m), 1.44−1.52 (2 H, m), 1.15−1.30 (2 H, m), 0.86 (3

H, t, J = 7.4 Hz); HRMS (ESI) m/z 376.1216 [M + H]⁺

(C₂₁H₂₃Cl₂NO requires 376.1225).

1

provided 37 (3.10 g, 56% yield) as a colorless liquid: H NMR (400

MHz, CDCl₃) δ ppm 7.19−7.26 (2 H, m), 7.10−7.18 (2 H, m), 7.02−

7.05 (2 H, m), 6.97−7.01 (1 H, m), 6.81−6.86 (1 H, m), 4.58 (1 H, d,

J = 8.2 Hz), 3.62 (3 H, s), 2.84−2.93 (1 H, m), 2.35−2.45 (1 H, m),

2.06−2.18 (2 H, m), 1.87−2.02 (1 H, m).

( )-(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)piperidin-

2-one (3). To a solution of 37 (3.10 g, 8.20 mmol) in THF/H₂O

(4:1, 40 mL) was added Me₃P (1.0 M in THF, 10.2 mL, 10.2 mmol).

After the mixture was stirred for 1 h at 25 °C, the residues were

basiﬁed (ice-cold 2 M aqueous LiOH) and the product was extracted

(3 × EtOAc) and washed (2 × brine). The combined organic layers

were dried (Na₂SO₄) and concentrated under reduced pressure to

provide the crude amine as a colorless ﬁlm.

( )-(5R,6S)-1-Benzyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-

piperidin-2-one (8). ¹H NMR (400 MHz, CDCl3) δ ppm 7.28−7.32

(3 H, m), 7.22−7.25 (2 H, m), 7.05−7.17 (3 H, m), 6.98−7.05 (1 H,

m), 6.86−6.92 (2 H, m), 6.78−6.82 (1 H, m), 6.57−6.61 (1 H, m),

5.55 (1 H, d, J = 14.5 Hz), 4.38 (1 H, d, J = 7.0 Hz), 3.35 (1 H, d, J =

14.5 Hz), 2.91−2.97 (1 H, m), 2.63−2.70 (2 H, m), 1.92−2.10 (2 H,

m); HRMS (ESI) m/z 410.1068 [M + H]⁺(C₂₄H₂₁Cl₂NO requires

410.1069).

( )-Methyl 2-((2S,3R)-3-(3-Chlorophenyl)-2-(4-chlorophen-

yl)-6-oxopiperidin-1-yl)acetate (13). ¹H NMR (400 MHz,

CDCl₃) δ ppm 7.22−7.26 (2 H, m), 7.13−7.21 (2 H, m), 7.08−

7.11 (1 H, m), 6.86−6.94 (3 H, m), 4.63 (1 H, d, J = 9.0 Hz), 4.58 (1

H, d, J = 17.2 Hz), 3.71 (3 H, s), 3.18 (1 H, d, J = 17.2 Hz), 2.93−3.01

(1 H, m), 2.60−2.76 (2 H, m), 2.20−2.32 (1 H, m), 2.06−2.13 (1 H,

m); HRMS (ESI) m/z 392.0847 [M + H]⁺(C₂₀H₁₉Cl₂NO₃requires

392.0811).

The crude amine above was dissolved in MeOH/saturated aqueous

NaHCO₃(4:1, 380 mL), and the mixture was reﬂuxed overnight. After

most of the MeOH was removed under reduced pressure, the residue

was diluted (water) and extracted (3 × EtOAc). The combined

organic layers were washed (1 × brine), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash column chromatography

(SiO₂, 80−100% EtOAc/hexanes, gradient elution) provided 3 (2.30

1

( )-(S)-Ethyl 2-((2S,3R)-3-(3-Chlorophenyl)-2-(4-chlorophen-

yl)-6-oxopiperidin-1-yl)butanoate (9) and ( )-(R)-Ethyl 2-

((2S,3R)-3-(3-Chlorophenyl)-2-(4-chlorophenyl)-6-oxopiperi-

din-1-yl)butanoate (10). To a solution of 3 (442 mg, 1.38 mmol) in

DMF (4.0 mL) was added NaH (60% in mineral oil, 221 mg, 5.52

mmol) at 0 °C. After the mixture was stirred for 20 min at 0 °C, ethyl

2-bromobutyrate (1.02 mL, 6.90 mmol) was added at 0 °C. Then the

mixture was allowed to warm to 25 °C and stirred for 24 h. The

reaction was quenched (saturated aqueous NH₄Cl), extracted (2 ×

EtOAc), and washed (3 × H₂O and 1 × brine). The combined organic

layers were dried (Na₂SO₄) and concentrated under reduced pressure.

Flash column chromatography (SiO₂, 20%, 30%, and 40% EtOAc/

hexanes) provided 9 (320 mg, 53%) as a colorless foam and 10 (181

g, 88% yield) as a white solid: H NMR (400 MHz, CDCl₃) δ ppm

7.20−7.24 (2 H, m), 7.11−7.20 (2 H, m), 7.01−7.04 (1 H, m), 6.95−

6.98 (2 H, m), 6.76−6.80 (1 H, m), 5.83 (1 H, s, br), 4.51 (1 H, d, J =

9.8 Hz), 2.78−2.88 (1 H, m), 2.62−2.68 (2 H, m), 2.20−2.32 (1 H,

m), 2.07−2.19 (1 H, m); HRMS (ESI) m/z 320.0586 [M + H]⁺

(C₁₇H₁₅Cl₂NO requires 320.0601).

(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)piperidin-2-

one [(5R,6S)-3] and (5S,6R)-5-(3-Chlorophenyl)-6-(4-

chlorophenyl)piperidin-2-one [(5S,6R)-3]. Individual stereo-

isomers in 3 were separated by chiral HPLC [ﬂow rate, 18 mL/min

on a Chiralcel OD-H 20 mm i.d. × 250 mm, 5 μm column (Daicel

Inc., Fort Lee, NJ), using 40% isopropyl alcohol/hexanes as the

eluent] to give (5R,6S)-3 (t_R= 8.2 min) and (5S,6R)-3 (t_R= 12.4 min)

as a white solid. (5S,6R)-3: [α]²³_D+158° (c 1.12, MeOH). (5R,6S)-3:

1

mg, 30%) as a white solid successively. Compound 9: H NMR (400

[α]²³−156° (c 1.13, MeOH).

MHz, CDCl₃) δ ppm 7.25 (2 H, d, J = 8.2 Hz), 7.10−7.22 (2 H, m),

7.04−7.06 (1 H, m), 7.01 (2 H, d, J = 8.2 Hz), 6.83 (1 H, d, J = 7.0

Hz), 4.56 (1 H, d, J = 9.0 Hz), 4.05−4.16 (2 H, m), 3.36−3.42 (1 H,

m), 3.00−3.08 (1 H, m), 2.63−2.68 (2 H, m), 2.15−2.31 (2 H, m),

2.05−2.12 (1 H, m), 1.54−1.65 (1 H, m), 1.28 (3 H, t, J = 7.2 Hz),

0.62 (3 H, t, J = 7.4 Hz); HRMS (ESI) m/z 434.1265 [M + H]⁺

D

(

)-(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-

(cyclopropylmethyl)piperidin-2-one (4). Compound 4 was

prepared as a colorless foam from 3 according to a similar procedure

described for the synthesis of 35: ¹H NMR (500 MHz, CDCl₃) δ ppm

7.28−7.32 (2 H, m), 7.19−7.25 (3 H, m), 6.97−7.02 (3 H, m), 4.84 (1

H, d, J = 6.1 Hz), 4.06−4.12 (1 H, m), 2.97−3.04 (1 H, m), 2.45−2.60

(2 H, m), 2.23−2.29 (1 H, m), 2.06−2.14 (1 H, m), 1.95−2.04 (1 H,

m), 0.96−1.05 (1 H, m), 0.52−0.60 (1 H, m), 0.42−0.50 (1 H, m),

0.08−0.22 (2 H, m); HRMS (ESI) m/z 374.1060 [M + H]⁺

(C₂₁H₂₁Cl₂NO requires 374.1069).

1

(C₂₃H₂₅Cl₂NO₃requires 434.1279). Compound 10: H NMR (400

MHz, CDCl₃) δ ppm 7.23 (2 H, d, J = 8.6 Hz), 7.11−7.20 (2 H, m),

7.09 (2 H, d, J = 8.6 Hz), 6.99−7.03 (1 H, m), 6.76−6.80 (1 H, m),

4.50 (1 H, d, J = 9.4 Hz), 4.06−4.20 (2 H, m), 3.33 (1 H, t, J = 7.0

Hz), 2.96−3.04 (1 H, m), 2.63−2.73 (2 H, m), 2.05−2.19 (3 H, m),

1.88−1.98 (1 H, m), 1.26 (3 H, t, J = 7.0 Hz), 1.00 (3 H, t, J = 7.4

Hz); HRMS (ESI) m/z 434.1285 [M + H]⁺(C₂₃H₂₅Cl₂NO₃requires

434.1279).

Compounds (5R,6S)-4 and (5S,6R)-4 were prepared from (5R,6S)-

3 and (5S,6R)-3, respectively, according to a similar procedure

described for the synthesis of 35.

Compounds 5−8 and 13 were prepared from 3 according to a

similar procedure described for the synthesis of 35.

Compounds 11 and 12 were prepared according to a similar

procedure described for the synthesis of 9.

M

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( )-(S)-Ethyl 2-((2S,3R)-3-(3-Chlorophenyl)-2-(4-chlorophen-

yl)-6-oxopiperidin-1-yl)pentanoate (11). ¹H NMR (400 MHz,

CDCl₃) δ ppm 7.25 (2 H, d, J = 8.6 Hz), 7.11−7.20 (2 H, m), 7.04−

7.06 (1 H, m), 7.02 (2 H, d, J = 8.6 Hz), 6.82−6.85 (1 H, m), 4.57 (1

H, d, J = 9.4 Hz), 4.08−4.15 (2 H, m), 3.45−3.49 (1 H, m), 2.99−3.06

(1 H, m), 2.63−2.68 (2 H, m), 2.13−2.28 (2 H, m), 2.04−2.12 (1 H,

m), 1.43−1.52 (1 H, m), 1.28 (3 H, t, J = 7.0 Hz), 1.05−1.17 (1 H,

m), 0.80−0.92 (1 H, m), 0.62 (3 H, t, J = 7.4 Hz); HRMS (ESI) m/z

448.1428 [M + H]⁺(C₂₄H₂₇Cl₂NO₃requires 448.1435).

7.16 (1 H, m), 6.98−7.02 (1 H, m), 6.86 (2 H, d, J = 8.2 Hz), 6.72−

6.75 (1 H, m), 4.63 (1 H, d, J = 10.2 Hz), 3.89−3.96 (1 H, m), 2.93−

3.10 (3 H, m), 2.56−2.63 (1 H, m), 2.31−2.38 (1 H, m), 2.08−2.25 (2

H, m), 0.85−0.95 (1 H, m), 0.48−0.55 (1 H, m), 0.40−0.46 (1H, m),

0.08−0.16 (1H, m), −0.02 to 0.06 (1 H, m); HRMS (ESI) m/z

432.1140 [M + H]⁺(C₂₃H₂₃Cl₂NO₃requires 432.1128).

(3S,5R,6S)-3-(But-3-en-1-yl)-5-(3-chlorophenyl)-6-(4-chloro-

phenyl)-1-(cyclopropylmethyl)piperidin-2-one (39) and

(3R,5R,6S)-3-(But-3-en-1-yl)-5-(3-chlorophenyl)-6-(4-chloro-

phenyl)-1-(cyclopropylmethyl)piperidin-2-one (40). To a sol-

ution of (5R,6S)-4 (630 mg, 1.68 mmol) and 4-bromo-1-butene (1.11

mL, 10.9 mmol) in THF (12 mL) was added LiHMDS (1.0 M in

THF, 10.1 mL, 10.1 mmol) at 0 °C, and the resulting solution was

heated to 40 °C and stirred for 3 h. The reaction was quenched

(saturated aqueous NH₄Cl) and extracted (3 × EtOAc). The

combined organic layers were washed (brine), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash column chromatography

(SiO₂, 4−30% EtOAc/hexanes, gradient elution) provided a mixture

of two diastereomers (304 mg, 42% yield, dr = 1.5/1 in favor of 39).

Individual stereoisomers in a mixture above were separated by chiral

HPLC [ﬂow rate, 18 mL/min on a Chiralcel OD-H 20 mm i.d. × 250

mm, 5 mic column (Daicel Inc., Fort Lee, NJ), using 2% isopropyl

alcohol/hexanes as the eluent] to give 40 and 39 successively.

Compound 39: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.37−7.40 (1 H,

m), 7.33 (2 H, d, J = 8.4 Hz), 7.23−7.26 (2 H, m), 7.12−7.19 (1 H,

m), 7.03 (2 H, d, J = 8.4 Hz), 5.72−5.84 (1 H, m), 4.93−5.04 (3 H,

m), 4.13 (1 H, dd, J = 14.1, 6.5 Hz), 3.11−3.16 (1 H, m), 1.88−2.36

(7 H, m), 1.62−1.70 (1 H, m), 1.03−1.14 (1 H, m), 0.46−0.65 (2 H,

m), 0.13−0.25 (2 H, m); MS (ESI) 428.2 [M + H]⁺. Compound 40:

¹H NMR (400 MHz, CDCl₃) δ ppm 7.11−7.25 (4 H, m), 6.98−7.03

( )-(S)-Ethyl 2-((2S,3R)-3-(3-Chlorophenyl)-2-(4-chlorophen-

yl)-6-oxopiperidin-1-yl)propaneate (12). ¹H NMR (400 MHz,

CDCl₃) δ ppm 7.26 (2 H, d, J = 8.6 Hz), 7.11−7.21 (2 H, m), 7.04−

7.07 (1 H, m), 7.01 (2 H, d, J = 8.6 Hz), 6.81−6.86 (1 H, m), 4.57 (1

H, d, J = 9.0 Hz), 4.15−4.26 (2 H, m), 3.40 (1 H, q, J = 6.8 Hz), 2.93−

3.02 (1 H, m), 2.60−2.66 (2 H, m), 2.14−2.26 (1 H, m), 2.05−2.14 (1

H, m), 1.34 (3 H, d, J = 7.0 Hz), 1.30 (3 H, t, J = 7.4 Hz); HRMS

(ESI) m/z 420.1113 [M + H]⁺(C₂₂H₂₃Cl₂NO₃requires 420.1123).

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-

(cyclopropylmethyl)piperidin-2-one (15) and (3R,5R,6S)-3-

A l l y l - 5 - ( 3 - c h l o r o p h e n y l ) - 6 - ( 4 - c h l o r o p h e n y l ) - 1 -

(cyclopropylmethyl)piperidin-2-one (38). To a solution of

(5R,6S)-4 (433 mg, 1.16 mmol) and allyl bromide (105 μL, 1.22

mmol) in THF (4.6 mL) was added LiHMDS (1 M in THF, 1.27 mL,

1.27 mmol) dropwise at −78 °C. After the mixture was stirred at −78

°C for 3 h, the reaction was quenched (saturated aqueous NH₄Cl),

extracted (2 × EtOAc), and washed (brine). The combined organic

layers were dried (Na₂SO₄) and concentrated under reduced pressure.

Flash column chromatography (SiO₂, 15−20% MTBE/hexanes,

gradient elution) provided less polar minor diastereomer 15 (105

mg, 25% yield) and more polar major diastereomer 38 (246 mg, 60%

yield) as colorless oils. Compound 15: ¹H NMR (400 MHz, CDCl₃) δ

ppm 7.41−7.43 (1 H, m), 7.34 (2 H, d, J = 8.2 Hz), 7.24−7.27 (2 H,

m), 7.16−7.21 (1 H, m), 7.05 (2 H, d, J = 8.2 Hz), 5.70−5.83 (1 H,

m), 5.00−5.09 (3 H, m), 4.11−4.18 (1 H, m), 3.11−3.15 (1 H, m),

2.62−2.70 (1 H, m), 2.42−2.50 (1 H, m), 2.32−2.38 (1 H, m), 2.24−

2.30 (1 H, m), 1.95−2.02 (1 H, m), 1.83−1.90 (1 H, m), 1.08−1.16 (1

H, m), 0.50−0.65 (2 H, m), 0.18−0.27 (2 H, m); HRMS (ESI) m/z

414.1386 [M + H]⁺(C₂₄H₂₅Cl₂NO requires 414.1381). Compound

(1 H, m), 6.80−6.83 (2 H, m), 6.72−6.77 (1 H, m), 5.77−5.89 (1 H,

m), 4.95−5.10 (2 H, m), 4.59 (1 H, d, J = 10.2 Hz), 3.95 (1 H, dd, J =

14.2, 6.6 Hz), 2.85−2.95 (1 H, m), 2.50−2.60 (1 H, m), 1.97−2.30 (6

H, m), 1.62−1.71 (1 H, m), 0.82−0.92 (1 H, m), 0.34−0.53 (2 H, m),

−0.05 to 0.17 (2 H, m); MS (ESI) 428.2 [M + H]⁺.

3-((3S,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)propanoic Acid (16).

Compound 16 was prepared as a white solid from 39 according to a

1

38: H NMR (400 MHz, CDCl₃) δ ppm 7.21 (2 H, t, J = 8.2 Hz),

similar procedure described for the synthesis of 2: H NMR (400

7.17−7.19 (1 H, m), 7.11−7.15 (1 H, m), 7.02−7.04 (1 H, m), 6.85 (2

H, d, J = 8.2 Hz), 6.72−6.77 (1 H, m), 5.77−5.88 (1 H, m), 5.05−5.15

(2 H, m), 4.60 (1 H, d, J = 10.2 Hz), 3.93−3.98 (1 H, m), 2.88−2.96

(1 H, m), 2.78−2.85 (1 H, m), 2.59−2.68 (1 H, m), 2.37−2.45 (1 H,

m), 2.22−2.30 (1 H, m), 1.96−2.09 (2 H, m), 0.83−0.92 (1 H, m),

0.45−0.53 (1 H, m), 0.36−0.44 (1 H, m), 0.09−0.15 (1 H, m), −0.02

to 0.04 (1 H, m); MS (ESI) 414.2 [M + H]⁺.

MHz, CDCl₃) δ ppm 7.33 (2 H, d, J = 8.4 Hz), 7.20−7.27 (3 H, m),

7.01−7.06 (1 H, m), 6.98 (2 H, d, J = 8.4 Hz), 4.90 (1 H, d, J = 5.1

Hz), 4.07 (1 H, dd, J = 14.1, 6.5 Hz), 3.09−3.18 (1 H, m), 2.53−2.63

(3 H, m), 2.27−2.35 (1 H, m), 2.17−2.27 (1 H, m), 2.06−2.15 (1 H,

m), 1.87 − 2.03 (2 H, m), 1.00−1.10 (1 H, m), 0.56−0.65 (1 H, m),

0.46−0.54 (1 H, m), 0.10−0.25 (2 H, m); HRMS (ESI) m/z 446.1283

[M + H]⁺(C₂₄H₂₅Cl₂NO₃requires 446.1291).

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)acetic Acid (2). To a

rapidly stirring solution of 15 (1.20 g, 2.90 mmol) and RuCl₃hydrate

(65.3 mg, 0.290 mmol) in H₂O/CCl₄/AcCN (1.5:1:1, 30 mL) was

added NaIO₄(2.48 g, 11.6 mmol) in four portions over 40 min while

maintaining the temperature below 25 °C. After being stirred

vigorously for 16 h, the mixture was ﬁltered through Celite and the

ﬁlter cake was washed (EtOAc). The ﬁltrate was extracted (2 ×

EtOAc) and washed (2 × 10% aqueous NaHSO₃and 1 × brine). The

combined organic layers were dried (Na₂SO₄) and concentrated under

reduced pressure. Puriﬁcation by RP-HPLC (10−90% A/B, gradient

elution) provided 2 (1.03 g, 78% yield) as a white solid: ¹H NMR (400

MHz, CDCl₃) δ ppm 7.46−7.49 (1 H, s), 7.40 (2 H, d, J = 8.6 Hz),

7.32−7.35 (2 H, m), 7.22−7.26 (1 H, m), 7.13 (2 H, d, J = 8.6 Hz),

5.17 (1 H, s, br), 4.21−4.27 (1 H, m), 3.18−3.23 (1 H, m), 2.80−2.87

(1 H, m), 2.53−2.62 (1 H, m), 2.46−2.53 (1 H, m), 2.32−2.40 (1 H,

m), 2.05−2.15 (1 H, m), 1.83−1.92 (1 H, m), 1.15−1.26 (1 H, m),

0.68−0.76 (1 H, m), 0.58−0.66 (1 H, m), 0.25−0.36 (2 H, m); HRMS

(ESI) m/z 432.1133 [M + H]⁺(C₂₃H₂₃Cl₂NO₃requires 432.1128).

2-((3S,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)acetic Acid (14). Com-

pound 14 was prepared as a white solid from 38 according to a similar

procedure described for the synthesis of 2: ¹H NMR (400 MHz,

CDCl₃) δ ppm 7.24 (2 H, d, J = 8.2 Hz), 7.18−7.22 (1 H, m), 7.12−

3-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)propanoic Acid (17).

Compound 17 was prepared as a white solid from 40 according to a

1

similar procedure described for the synthesis of 2: H NMR (400

MHz, CDCl₃) δ ppm 7.22 (2 H, d, J = 8.2 Hz), 7.11−7.20 (2 H, m),

7.01 (1 H, s), 6.85 (2 H, d, J = 8.4 Hz), 6.75 (1 H, d, J = 7.4 Hz), 4.61

(1 H, d, J = 10.0 Hz), 3.92 (1 H, dd, J = 14.3, 6.5 Hz), 2.90−3.01 (1 H,

m), 2.47−2.78 (3 H, m), 2.21−2.36 (2 H, m), 1.90−2.15 (3 H, m),

0.81−0.93 (1 H, m), 0.45−0.54 (1 H, m), 0.37−0.45 (1 H, m), 0.07−

0.15 (1 H, m), −0.04 to 0.04 (1 H, m); HRMS (ESI) m/z 446.1283

[M + H]⁺(C₂₄H₂₅Cl₂NO₃requires 446.1291).

Methyl 2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-

1-(cyclopropylmethyl)-2-oxopiperidin-3-yl)acetate (18). To a

suspension of 2 (250 mg, 578 μmol) in MeOH (3 mL) was added

thionyl chloride (78 μL, 1.1 mmol) dropwise at 0 °C. After being

stirred at 25 °C overnight, the mixture was diluted (EtOAc), basiﬁed

(saturated aqueous NaHCO₃), extracted (2 × EtOAc), and washed

(brine). The combined organic layers were dried (Na₂SO₄) and

concentrated under reduced pressure to provide 18 (230 mg, 89%

yield) as a colorless liquid: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.48−

7.52 (1 H, m), 7.39 (2 H, d, J = 8.6 Hz), 7.28−7.31 (2 H, m), 7.25−

7.27 (1 H, m), 7.24 (2 H, d, J = 8.6 Hz), 5.12 (1 H, s, br), 4.18−4.26

(1 H, m), 3.69 (3 H, s), 3.16−3.20 (1 H, m), 2.78−2.85 (1 H, m),

2.62−2.68 (1 H, m), 2.53−2.61 (1 H, m), 2.20−2.32 (2 H, m), 1.78−

N

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1.85 (1 H, m), 1.12−1.22 (1 H, m), 0.62−0.70 (1 H, m), 0.52−0.59 (1

H, m), 0.20−0.30 (2 H, m); HRMS (ESI) m/z 446.1285 [M + H]⁺

(C₂₄H₂₅Cl₂NO₃requires 446.1279).

5.76−5.86 (1 H, m), 5.03−5.12 (2 H, m), 4.50 (1 H, d, J = 10.6 Hz),

4.12−4.21 (2 H, m), 3.16−3.22 (1 H, m), 3.05−3.14 (1 H, m), 2.74−

2.82 (1 H, m), 2.62−2.70 (1 H, m), 2.24−2.38 (2 H, m), 1.96−2.14 (2

H, m), 1.60−1.66 (1 H, m), 1.31 (3 H, t, J = 7.2 Hz), 0.60 (3 H, t, J =

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)acetamide (19). In a tube,

18 (60 mg, 130 μmol) was treated with ammonia (7 N in MeOH, 4.8

mL, 34 mmol). The tube was sealed and stirred at 25 °C for 5 days.

Then NaCN (3 mg) was added, and the resulting solution was stirred

at 50 °C for additional 3 days. The mixture was cooled, and excess

NH₃and MeOH were removed under reduced pressure. Puriﬁcation

by RP-HPLC (10−90% A/B, gradient elution) provided 19 (45 mg,

1

7.6 Hz); MS (ESI) 474.0 [M + H]⁺. Compound 41: H NMR (400

MHz, CDCl₃) δ ppm 7.29 (2 H, d, J = 8.6 Hz), 7.17−7.21 (2 H, m),

7.13−7.16 (1 H, m), 7.08 (2 H, d, J = 8.6 Hz), 6.98−7.02 (1 H, m),

5.77−5.87 (1 H, m), 5.10−5.17 (2 H, m), 4.70 (1 H, d, J = 7.4 Hz),

4.07−4.20 (2 H, m), 3.38−3.43 (1 H, m), 3.13−3.20 (1 H, m), 2.68−

2.76 (1 H, m), 2.58−2.65 (1 H, m), 2.47−2.57 (1 H, m), 2.21−2.32 (1

H, m), 2.03−2.18 (2 H, m), 1.55−1.62 (1 H, m), 1.28 (3 H, t, J = 7.0

Hz), 0.66 (3 H, t, J = 7.4 Hz); MS (ESI) 474.0 [M + H]⁺.

(S)-Ethyl 2-((2S,3R,5R)-3-(3-Chlorophenyl)-2-(4-chlorophen-

yl)-5-(hydroxymethyl)-6-oxopiperidin-1-yl)butanoate (22). 22

was prepared from 41 according to a similar procedure described for

the synthesis of 2: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.34−7.40 (3

H, m), 7.27−7.32 (3 H, m), 7.24−7.26 (2 H, m), 4.91 (1 H, d, J = 3.5

Hz), 4.10−4.21 (2 H, m), 3.52−3.56 (1 H, m), 3.16−3.20 (1 H, m),

2.70−2.84 (3 H, m), 2.29−2.38 (2 H, m), 1.95−2.03 (1 H, m), 1.50−

1.60 (1 H, m), 1.27 (3 H, t, J = 7.4 Hz), 0.70 (3 H, t, J = 7.4 Hz);

HRMS (ESI) m/z 492.1328 [M + H]⁺(C₂₅H₂₇Cl₂NO₅requires

492.1333).

1

78% yield) as a white solid: H NMR (400 MHz, CDCl₃) δ ppm

7.47−7.52 (1 H, m), 7.37 (2 H, d, J = 8.2 Hz), 7.28−7.32 (2 H, m),

7.22−7.26 (1 H, m), 7.17 (2 H, d, J = 8.2 Hz), 6.40 (1 H, s, br), 5.42

(1 H, s, br), 5.11 (1 H, s, br), 4.18−4.24 (1 H, m), 3.15−3.20 (1 H,

m), 2.69−2.77 (1 H, m), 2.47−2.59 (2 H, m), 2.24−2.34 (2 H, m),

1.85−1.92 (1 H, m), 1.12−1.22 (1 H, m), 0.63−0.72 (1 H, m), 0.54−

0.60 (1 H, m), 0.18−0.32 (2 H, m); HRMS (ESI) m/z 431.1269 [M +

H]⁺(C₂₃H₂₄Cl₂N₂O₂requires 431.1283).

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(cy-

clopropylmethyl)-2-oxopiperidin-3-yl)acetonitrile (20). A sol-

ution of 19 (36 mg, 83 μmol) and TEA (58 μL, 420 μmol) in THF

(1.3 mL) was treated with (CF₃CO)₂O (29 μL, 210 μmol) at 0 °C.

After the mixture was stirred at 0 °C for 2 h, the reaction was

quenched (10% aqueous citric acid), extracted (2 × EtOAc), and

washed (brine). The combined organic layers were dried (Na₂SO₄)

and concentrated under reduced pressure. Flash column chromatog-

raphy (SiO₂, 20−30% EtOAc/hexanes, gradient elution) provided 20

(S)-tert-Butyl 2-((3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-

chlorophenyl)-2-oxopiperidin-1-yl)butanoate (42). 42 was pre-

pared from (5R,6S)-3 using tert-butyl 2-bromobutyrate according to

1

similar procedures described for the synthesis of 41: H NMR (400

MHz, CDCl₃) δ ppm 7.25−7.29 (2 H, m), 7.12−7.18 (2 H, m), 7.02−

7.09 (3 H, m), 6.88−6.93 (1 H, m), 5.76−5.89 (1 H, m), 5.08−5.17 (2

H, m), 4.64 (1 H, d, J = 8.6 Hz), 3.14−3.21 (1 H, m), 3.09−3.14 (1H,

m), 2.71−2.78 (1 H, m), 2.62−2.69 (1 H, m), 2.45−2.56 (1 H, m),

2.15−2.30 (2 H, m), 1.98−2.04 (1 H, m), 1.50−1.60 (1 H, m), 1.47 (9

H, s), 0.61 (3 H, t, J = 7.6 Hz); MS (ESI) 446.0 [M + H]⁺.

1

(32 mg, 93% yield) as a white foam: H NMR (400 MHz, CDCl₃) δ

ppm 7.52−7.55 (1 H, m), 7.41 (2 H, d, J = 8.6 Hz), 7.28−7.33 (3 H,

m), 7.16 (2 H, d, J = 8.6 Hz), 5.18 (1 H, s, br), 4.20−4.25 (1 H, m),

3.25−3.29 (1 H, m), 2.96−3.03 (1 H, m), 2.64−2.71 (1 H, m), 2.44−

2.52 (1 H, m), 2.24−2.38 (2 H, m), 1.94−2.01 (1 H, m), 1.15−1.22 (1

H, m), 0.65−0.73 (1 H, m), 0.56−0.63 (1 H, m), 0.23−0.33 (2 H, m);

HRMS (ESI) m/z 413.1155 [M + H]⁺(C₂₃H₂₂Cl₂N₂O requires

413.1178).

2-((3R,5R,6S)-1-((S)-1-(tert-Butoxy)-1-oxo-butan-2-yl)-5-(3-

chlorophenyl)-6-(4-chlorophenyl)-2-oxopiperidin-3-yl)acetic

Acid (23). 23 was prepared from 42 according to a similar procedure

1

described for the synthesis of 2: H NMR (400 MHz, CDCl₃) δ ppm

7.33−7.37 (2 H, m), 7.23−7.29 (3 H, m), 7.16−7.21 (3 H, m), 4.86 (1

H, d, J = 4.9 Hz), 3.32−3.37 (1 H, m), 3.13−3.19 (1 H, m), 2.72−2.92

(3 H, m), 2.18−2.34 (2 H, m), 2.05−2.14 (1 H, m), 1.51−1.60 (1 H,

m), 1.44 (9 H, s), 0.67 (3 H, t, J = 7.4 Hz); HRMS (ESI) m/z

520.1635 [M + H]⁺(C₂₇H₃₁Cl₂NO₅requires 520.1645).

(3R,5R,6S)-3-((1H-Tetrazol-5-yl)methyl)-5-(3-chlorophenyl)-

6-(4-chlorophenyl)-1-(cyclopropylmethyl)piperidin-2-one (21).

To a solution of 20 (29 mg, 70 μmol) in DMF (0.18 mL) were added

NaN₃(23 mg, 350 μmol) and NH₄Cl (19 mg, 350 μmol). The

mixture was stirred at 90 °C for 2 days. The mixture was cooled to

ambient temperature, acidiﬁed (10% aqueous citric acid), extracted (2

× EtOAc), and washed (brine). The combined organic layers were

dried (Na₂SO₄) and concentrated under reduced pressure. Puriﬁcation

by RP-HPLC (10−90% A/B, gradient elution) provided 21 (13 mg,

(S)-tert-Butyl 2-((3R,5R,6S)-3-(2-Amino-2-oxo-ethyl)-5-(3-

chlorophenyl)-6-(4-chlorophenyl)-2-oxopiperidin-1-yl)-

butanoate (43). To a solution of 23 (195 mg, 0.374 mmol) and N-

methylmorpholine (58 μL, 0.53 mmol) in THF (1.8 mL) was added

isobutyl chloroformate (59 μL, 0.45 mmol) at 0 °C. The cloudy

colorless solution was stirred at 0 °C for 30 min. Then ammonium

hydroxide (28% ammonia in water, 51 μL, 0.75 mmol) was added to

the mixture at 0 °C, and the resulting mixture was stirred at 0 °C for 1

h. The reaction was quenched (water), extracted (2 × EtOAc), and

washed (brine). The combined organic layers were dried (Na₂SO₄)

and concentrated under reduced pressure. The residue was puriﬁed by

RP-HPLC (10−90% A/B, gradient elution) to give 43 (174 mg, 90%

yield) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.33 (2 H,

d, J = 8.4 Hz), 7.19−7.24 (3 H, m), 7.15 (2 H, d, J = 8.4 Hz), 7.06−

7.12 (1 H, m), 6.72 (1 H, s, br), 4.76 (1 H, d, J = 6.3 Hz), 3.17−3.27

(2 H, m), 2.85−2.93 (1 H, m), 2.74−2.83 (2 H, m), 2.24−2.33 (2 H,

m), 2.13−2.23 (1 H, m), 1.50−1.57 (1 H, m), 1.47 (9 H, s), 0.64 (3 H,

t, J = 7.5 Hz); MS (ESI) 519.1 [M + H]⁺

1

41% yield) as a white solid: H NMR (400 MHz, CDCl₃) δ ppm

7.47−7.50 (1 H, m), 7.35 (2 H, d, J = 8.6 Hz), 7.29−7.33 (2 H, m),

7.22−7.25 (1 H, m), 6.86 (2 H, d, J = 8.6 Hz), 5.14 (1 H, s), 4.19−

4.25 (1 H, m), 3.36−3.43 (1 H, m), 3.20−3.24 (1 H, m), 3.11−3.17 (1

H, m), 2.48−2.56 (1 H, m), 2.30−2.37 (1 H, m), 2.15−2.25 (1 H, m),

1.94−2.02 (1 H, m), 1.15−1.23 (1 H, m), 0.69−0.76 (1 H, m), 0.59−

0.67 (1 H, m), 0.23−0.36 (2 H, m); HRMS (ESI) m/z 456.1361 [M +

H]⁺(C₂₃H₂₃Cl₂N₅O requires 456.1349).

(S)-Ethyl 2-((3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlor-

ophenyl)-2-oxopiperidin-1-yl)butanoate (41). (S)-Ethyl 2-

((2S,3R)-3-(3-chlorophenyl)-2-(4-chlorophenyl)-6-oxopiperidin-1-yl)-

butanoate was prepared from (5R,6S)-3 according to a similar

procedure described for the synthesis of 9.

To a solution of the product above (362 mg, 833 μmol) and allyl

bromide (87 μL, 1.00 mmol) in THF (3.3 mL) was added LiHMDS

(1 M in THF, 875 μL, 875 μmol) dropwise at −78 °C. After the

mixture was stirred at −78 °C for 3 h, the reaction was quenched

(saturated aqueous NH₄Cl), extracted (2 × EtOAc), and washed

(brine). The combined organic layers were dried (Na₂SO₄) and

concentrated under reduced pressure. Flash column chromatography

(SiO₂, 15−20% EtOAc/hexanes, gradient elution) provided the C3

epimer of 41 (129 mg, 33% yield) and 41 (183 mg, 46% yield)

(S)-tert-Butyl 2-((3R,5R,6S)-3-((1H-Tetrazol-5-yl)methyl)-5-(3-

chlorophenyl)-6-(4-chlorophenyl)-2-oxopiperidin-1-yl)-

butanoate (24). 24 was prepared from 43 according to similar

1

procedures described for the synthesis of 21: H NMR (400 MHz,

CDCl₃) δ ppm 7.32 (2 H, d, J = 8.4 Hz), 7.17−7.25 (3 H, m), 7.02−

7.06 (1 H, m), 7.00 (2 H, d, J = 8.4 Hz), 4.77 (1 H, d, J = 6.3 Hz),

3.54−3.61 (1 H, m), 3.36−3.44 (2 H, m), 3.22−3.28 (1 H, m), 2.92−

2.98 (1 H, m), 2.16−2.32 (3 H, m), 1.49−1.51 (1 H, m), 1.46 (9 H, s),

0.66 (3 H, t, J = 7.5 Hz); HRMS (TOF-MS) m/z 544.1879 [M + H]⁺

(C₂₇H₃₁Cl₂N₅O₃requires 544.1871).

1

successively. C3 epimer of 41: H NMR (400 MHz, CDCl₃) δ ppm

7.23 (2 H, d, J = 8.6 Hz), 7.13−7.18 (1 H, m), 7.07−7.13 (1 H, m),

6.97−7.00 (1 H, m), 6.98 (2 H, d, J = 8.6 Hz), 6.73−6.77 (1 H, m),

(5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-(2,4-

dimethoxybenzyl)piperidin-2-one (44). To a suspension of NaH

O

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(60% in mineral oil, 0.25 g, 6.25 mmol) in DMF (8.0 mL) was added

(5R,6S)-3 (1.00 g, 3.12 mmol) in DMF (2.0 mL) at 0 °C. The

resulting reaction mixture was stirred at 0 °C for 45 min. Then freshly

prepared 2,4-dimethoxybenzyl chloride³⁵(1.34 g, 7.18 mmol) in DMF

(3.0 mL) was added, and the resulting solution was slowly allowed to

warm to 25 °C and stirred for 2.5 h. The reaction was quenched (ice-

cold water) at 0 °C, extracted (2 × EtOAc), washed (3 × brine), and

dried (MgSO₄). The combined organic layers were concentrated

under reduced pressure. Flash column chromatography (SiO₂, 45%

and concentrated under reduced pressure. Flash column chromatog-

raphy (SiO₂, 10% EtOAc/hexanes) provided 47 (47 mg, 0.11 mmol,

1

40% yield) as a colorless foam: H NMR (400 MHz, CDCl₃) δ ppm

7.22 (2 H, d, J = 8.2 Hz), 7.07−7.18 (2 H, m), 6.85−7.04 (3 H, m),

6.70 (1 H, d, J = 7.6 Hz), 5.80−5.93 (1 H, m), 5.12−5.21 (2 H, m),

4.30 (1 H, d, J = 10.6 Hz), 3.10−3.20 (1 H, m), 2.69−2.79 (1 H, m),

2.56−2.67 (2 H, m), 1.80−2.06 (4 H, m), 1.48−1.58 (1 H, m), 1.35−

1.47 (1 H, m), 1.27 (3 H, s), 0.93 (3 H, t, J = 7.4 Hz), 0.54 (3 H, t, J =

7.5 Hz); MS (ESI) 444.1 [M + H]⁺.

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-3-

methyl-2-oxo-1-(pentan-3-yl)piperidin-3-yl)acetic Acid (27). 27

was prepared from 47 according to a similar procedure described for

the synthesis of 3: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.24−7.27 (2

H, m), 7.07−7.19 (2 H, m), 6.85−7.05 (3 H, m), 6.68 (1 H, d, J = 7.6

Hz), 4.34 (1 H, d, J = 10.4 Hz), 3.01−3.13 (2 H, m), 2.67−2.78 (2 H,

m), 2.16−2.27 (1 H, m), 1.98−2.04 (1 H, m), 1.85−1.97 (2 H, m),

1.51 (3 H, s), 1.38−1.49 (2 H, m), 0.95 (3 H, t, J = 7.3 Hz), 0.49 (3 H,

t, J = 7.5 Hz); HRMS (ESI) m/z 462.1598 [M + H]⁺(C₂₅H₂₉Cl₂NO₃

requires 462.1604).

1

EtOAc/hexanes) gave 44 (1.25 g, 85% yield) as a white foam: H

NMR (400 MHz, CDCl₃) δ ppm 7.20 (2 H, d, J = 8.4 Hz), 7.03−7.09

(2 H, m), 6.96−7.02 (1 H, m), 6.86 (2 H, d, J = 8.4 Hz), 6.82−6.84 (1

H, m), 6.67 (1 H, d, J = 7.6 Hz), 6.30−6.39 (2 H, m), 5.20 (1 H, d, J =

14.5 Hz), 4.53 (1 H, d, J = 5.7 Hz), 3.72 (3 H, s), 3.59 (3 H, s), 3.53

(1 H, d, J = 14.3 Hz), 2.82−2.88 (1 H, m), 2.33−2.53 (2 H, m), 1.93−

2.04 (1 H, m), 1.75−1.88 (1 H, m); MS (ESI) 470.2 [M + H]⁺.

(5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-(4-

methoxybenzyl)-3-methylpiperidin-2-one (45). To a solution of

44 (525 mg, 1.12 mmol) and MeI (0.087 mL, 1.40 mmol) in THF

(3.7 mL) was added LiHMDS (1 M in THF, 1.23 mL, 1.23 mmol) at

−78 °C. After being stirred at −78 °C for 15 min, the mixture was

allowed to warm to 25 °C and stirred overnight. The reaction was

quenched (saturated aqueous NH₄Cl), extracted (2 × EtOAc), and

washed (brine). The combined organic layers were dried (Na₂SO₄)

and concentrated under reduced pressure. Flash column chromatog-

raphy (SiO₂, 15−35% EtOAc/hexanes, gradient elution) provided

(5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-(2,4-dimethoxyben-

zyl)-3-methylpiperidin-2-one (490 mg, 91% yield) as a colorless foam:

MS (ESI) 484.2 [M + H]⁺.

To a solution of the product above (474 mg, 0.979 mmol) and allyl

bromide (745 μL, 8.81 mmol) in THF (4.8 mL) was added LiHMDS

(1.0 M in THF, 7.83 mL, 7.83 mmol) at 25 °C. After being stirred for

5 min, the mixture was heated at 40 °C for 3 h. The reaction was

quenched (saturated aqueous NH₄Cl), extracted (2 × EtOAc), and

washed (brine). The combined organic layers were dried (Na₂SO₄)

and concentrated under reduced pressure. Flash column chromatog-

raphy (SiO₂, 10−30% EtOAc/hexanes, gradient elution) provided 45

(420 mg, 0.801 mmol, 82% yield, dr = 3.9:1 in favor of the desired

isomer) as a colorless foam: MS (ESI) 524.2 [M + H]⁺.

(S)-tert-Butyl 2-((3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-

chlorophenyl)-3-methyl-2-oxopiperidin-1-yl)butanoate (48).

To a solution of 46 (310 mg, 0.828 mmol) in DMF (1.7 mL) was

added NaH (60% in mineral oil, 133 mg, 3.31 mmol) at 0 °C. After

the mixture was stirred at 0 °C for 20 min, tert-butyl 2-

bromobutanoate (924 mg, 4.14 mmol) was added, and then the

mixture was allowed to warm to 25 °C. After the mixture was stirred

for 4 h, the reaction was quenched (saturated aqueous NH₄Cl),

extracted (2 × EtOAc), and washed (3 × brine). The combined

organic layers were dried (Na₂SO₄) and concentrated under reduced

pressure. Flash column chromatography (SiO₂, 0−20% EtOAc/

hexanes, gradient elution) provided 48 (257 mg, 60%) and Cα

epimer of 48, (R)-tert-butyl 2-((3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-

6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-1-yl)butanoate (120 mg,

1

28%), as colorless foams successively. Compound 48: H NMR (400

MHz, CDCl₃) δ ppm 7.23 (2 H, d, J = 8.4 Hz), 7.07−7.17 (2 H, m),

6.97−7.05 (3 H, m), 6.74−6.77 (1 H, m), 5.81−5.92 (1 H, m), 5.15−

5.22 (2 H, m), 4.57 (1 H, d, J = 10.8 Hz), 3.24−3.33 (1 H, m), 2.88−

2.92 (1 H, m), 2.54−2.66 (2 H, m), 2.22−2.35 (1 H, m), 2.08−2.17 (1

H, m), 1.92−1.98 (1 H, m), 1.49−1.55 (1 H, m), 1.51 (9 H, s), 1.25 (3

H, s), 0.56 (3 H, t, J = 7.5 Hz); MS (ESI) 538.2 [M + Na]⁺. Cα

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-

methylpiperidin-2-one (46). 45 (82 mg, 0.16 mmol) was dissolved

in triﬂuoroacetic acid (1.2 mL, 16 mmol), and the resulting solution

was stirred at 50 °C for 2 h. TFA was removed under reduced

pressure, and the crude product was dissolved (10% MeOH/DCM),

neutralized (saturated aqueous NaHCO₃), and ﬁltered through Celite

to remove insoluble material. Then the ﬁltrate was extracted (2 × 10%

MeOH/DCM) and washed (brine). The combined organic layers

were dried (Na₂SO₄) and concentrated under reduced pressure. Flash

column chromatography (SiO₂, 27−37% EtOAc/hexanes, gradient

elution) provided the C3 epimer of 46 (13 mg, 22% yield) and 46 (41

mg, 70% yield) as white solids successively. C3 epimer of 46: ¹H NMR

(500 MHz, CDCl₃) δ ppm 7.19−7.23 (2 H, m), 7.16−7.18 (1 H, m),

7.10−7.14 (1 H, m), 7.00−7.03 (1 H, m), 6.93−6.97 (2 H, m), 6.76−

6.80 (1 H, m), 5.78−5.86 (1 H, m), 5.69 (1 H, s, br), 5.15−5.22 (2 H,

m), 4.45 (1 H, d, J = 10.8 Hz), 2.96−3.03 (1 H, m), 2.68−2.73 (1 H,

m), 2.39 (1 H, t, J = 13.4 Hz), 2.23−2.29 (1 H, m), 1.71−1.75 (1 H,

1

epimer of 48: H NMR (400 MHz, CDCl₃) δ ppm 7.20 (2 H, d, J =

8.2 Hz), 7.00−7.17 (4 H, m), 6.93−6.95 (1 H, m), 6.68−6.73 (1 H,

m), 5.84−5.96 (1 H, m), 5.14−5.22 (2 H, m), 4.47 (1 H, d, J = 10.6

Hz), 3.18−3.26 (1 H, m), 3.06−3.12 (1 H, m), 2.53−2.68 (2 H, m),

1.88−2.13 (4 H, m), 1.46 (9 H, s), 1.28 (3 H, s), 0.98 (3 H, t, J = 7.6

Hz); MS (ESI) 538.2 [M + Na]⁺.

2-((3R,5R,6S)-1-((S)-1-(tert-Butoxy)-1-oxo-butan-2-yl)-5-(3-

chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-

yl)acetic Acid (25). 25 was prepared from 48 according to a similar

procedure described for the synthesis of 2: ¹H NMR (400 MHz,

CDCl₃) δ ppm 7.24−7.26 (2 H, m), 7.07−7.19 (2 H, m), 6.98−7.06

(3 H, m), 6.73−6.77 (1 H, m), 4.58 (1 H, d, J = 10.6 Hz), 3.20−3.28

(1 H, m), 2.92−3.02 (2 H, m), 2.76−2.82 (1 H, m), 2.22−2.36 (2 H,

m), 2.08−2.15 (1 H, m), 1.47−1.52 (1 H, m), 1.50 (9 H, s), 1.46 (3 H,

s), 0.54 (3 H, t, J = 7.5 Hz); HRMS (ESI) m/z 534.1808 [M + H]⁺

(C₂₈H₃₃Cl₂NO₅requires 534.1801).

1

m), 1.45 (3 H, s); MS (ESI) 374.0 [M + H]⁺. Compound 46: H

(S)-tert-Butyl 2-((3R,5R,6S)-3-((1H-Tetrazol-5-yl)methyl)-5-(3-

chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-1-

yl)butanoate (26). 26 was prepared from 25 according to similar

NMR (500 MHz, CDCl₃) δ ppm 7.19−7.23 (2 H, m), 7.16−7.18 (1

H, m), 7.10−7.14 (1 H, m), 6.99−7.02 (1 H, m), 6.93−6.97 (2 H, m),

6.75−6.78 (1 H, m), 5.84−5.94 (1 H, m), 5.67 (1 H, s, br), 5.15−5.20

(2 H, m), 4.50 (1 H, d, J = 10.8 Hz), 3.03−3.10 (1 H, m), 2.59−2.65

(1 H, m), 2.52−2.56 (1 H, m), 2.02−2.12 (2 H, m), 1.31 (3 H, s); MS

(ESI) 374.0 [M + H]⁺.

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-

methyl-1-(pentan-3-yl) piperidin-2-one (47). To a solution of 46

(100 mg, 0.267 mmol) in 3-bromopentane (1.0 mL, 8.0 mmol) was

added sodium hydride (60% in mineral oil, 75 mg, 1.87 mmol) at 25

°C. After the mixture was heated at 120 °C for 2 days, the reaction was

quenched (saturated aqueous NH₄Cl), extracted (2 × EtOAc), and

washed (brine). The combined organic layers were dried (Na₂SO₄)

1

procedures described for the synthesis of 24: H NMR (400 MHz,

CDCl₃) δ ppm 7.23−7.26 (2 H, m), 7.10−7.19 (2 H, m), 7.00−7.03

(1 H, m), 6.88−6.94 (2 H, m), 6.76−6.78 (1 H, m), 4.60 (1 H, d, J =

10.8 Hz), 3.43−3.60 (2 H, m), 3.22−3.31 (1 H, m), 3.13−3.17 (1 H,

m), 2.29−2.42 (2 H, m), 2.18−2.25 (1 H, m), 1.49−1.52 (9 H, m),

1.34−1.40 (1 H, m), 1.32 (3 H, s), 0.55 (3 H, t, J = 7.4 Hz); HRMS

(ESI) m/z 558.2031 [M + H]⁺(C₂₈H₃₃Cl₂N₅O₃requires 558.2027).

(S)-Ethyl 2-((3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlor-

ophenyl)-3-methyl-2-oxopiperidin-1-yl)butanoate (49). 49 was

prepared from 46 using ethyl 2-bromobutanoate according to a similar

1

procedure described for the synthesis of 48: H NMR (400 MHz,

P

dx.doi.org/10.1021/jm300354j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

CDCl₃) δ ppm 7.23 (2 H, d, J = 8.2 Hz), 7.07−7.17 (2 H, m), 6.92−

7.05 (3 H, m), 6.74−6.77 (1 H, m), 5.80−5.92 (1 H, m), 5.14−5.23 (2

H, m), 4.57 (1 H, d, J = 10.8 Hz), 4.10−4.25 (2 H, m), 3.26−3.33 (1

H, m), 3.07−3.12 (1 H, m), 2.60 (2 H, d, J = 7.4 Hz), 2.22−2.33 (1 H,

m), 2.03−2.18 (1 H, m), 1.94−1.99 (1 H, m), 1.58−1.68 (1 H, m),

1.30 (3 H, t, J = 7.1 Hz), 1.24 (3 H, s), 0.60 (3 H, t, J = 7.5 Hz); MS

(ESI) 488.2 [M + H]⁺.

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-

((S)-1-hydroxybutan-2-yl)-3-methylpiperidin-2-one (50). To a

solution of 49 (3.35 g, 6.86 mmol) in Et₂O (55 mL) was added LiBH₄

(299 mg, 13.7 mmol) at 0 °C. After the mixture was stirred at 0 °C for

30 min, the reaction was quenched (saturated aqueous NH₄Cl),

extracted (2 × EtOAc), and washed (brine). The combined organic

layers were dried (MgSO₄) and concentrated under reduced pressure.

Flash column chromatography (SiO₂, 40−60% EtOAc/hexanes,

gradient elution) provided 50 (2.10 g, 69% yield) as a colorless

foam: ¹H NMR (400 MHz, CDCl₃) δ ppm 7.24 (2 H, d, J = 8.4 Hz),

7.07−7.19 (2 H, m), 6.95−7.04 (3 H, m), 6.68−6.74 (1 H, m), 5.80−

5.92 (1 H, m), 5.13−5.22 (2 H, m), 4.46 (1 H, d, J = 10.2 Hz), 3.58−

3.66 (2 H, m), 3.12−3.24 (2 H, m), 2.61 (2 H, d, J = 7.4 Hz), 1.91−

2.07 (3 H, m), 1.39−1.51 (1 H, m), 1.27 (3 H, s), 0.68 (3 H, t, J = 7.5

Hz); MS (ESI) 446.1 [M + H]⁺.

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-

1-hydroxybutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic

Acid (28). To a solution of 50 (630 mg, 1.41 mmol) and imidazole

(240 mg, 3.52 mmol) in DMF (9 mL) was added tert-butyl

diphenylchlorosilane (485 μL, 1.85 mmol) slowly at 25 °C. After

the mixture was stirred for 16 h, the reaction was quenched (water),

extracted (2 × EtOAc), and washed (3 × brine). The combined

organic layers were dried (MgSO₄) and concentrated under reduced

pressure. Flash column chromatography (SiO₂, 0−10% EtOAc/

hexanes, gradient elution) provided (3S,5R,6S)-3-allyl-5-(3-chloro-

phenyl)-6-(4-chlorophenyl)-1-((S)-1-(2,2-dimethyl-1,1-

diphenylpropoxy)butan-2-yl)-3-methylpiperidin-2-one (850 mg, 90%

yield) as a colorless foam: MS (ESI) 684.2 [M + H]⁺.

To a rapidly stirring solution of the TBDPS protected alcohol above

(388 mg, 2.90 mmol) and RuCl₃hydrate (19 mg, 0.085 mmol) in

H₂O/CCl₄/AcCN (1.5:1:1, 7 mL) was added NaIO₄(606 mg, 2.83

mmol) in four portions over 20 min while maintaining the

temperature below 25 °C. After being stirred vigorously for 4 h, the

mixture was ﬁltered through Celite and the ﬁlter cake was washed

(EtOAc). The ﬁltrate was extracted (2 × EtOAc) and washed (2 ×

10% aqueous NaHSO₃and 1 × brine). The combined organic layers

were dried (Na₂SO₄) and concentrated under reduced pressure.

Puriﬁcation by ﬂash column chromatography (SiO₂, 0−20% MeOH/

DCM, gradient elution) provided 2-((3R,5R,6S)-5-(3-chlorophenyl)-

6-(4-chlorophenyl)-1-((S)-1-(2,2-dimethyl-1,1-diphenylpropoxy)-

butan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid (317 mg, 80%

yield) as a pale brown foam: MS (ESI) 702.2 [M + H]⁺.

washed (2 × saturated aqueous NaHCO₃and 2 × brine). The

combined organic layers were dried (Na₂SO₄) and concentrated under

reduced pressure. Flash column chromatography (SiO₂, 20%, 25%, and

30% EtOAc/hexanes) provided (S)-2-((3S,5R,6S)-3-allyl-5-(3-chlor-

ophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-1-yl)butanal

(4.61 g, 79% yield) as a colorless ﬁlm: ¹H NMR (400 MHz, CDCl₃) δ

ppm 9.50 (1 H, s), 7.24 (2 H, d, J = 8.2 Hz), 7.06−7.17 (2 H, m),

6.93−7.06 (3 H, m), 6.77 (1 H, d, J = 7.4 Hz), 5.80−5.95 (1 H, m),

5.17−5.25 (2 H, m), 4.54 (1 H, d, J = 10.8 Hz), 3.26−3.35 (1 H, m),

3.14−3.19 (1 H, m), 2.62 (2 H, d, J = 7.4 Hz), 2.10−2.28 (2 H, m),

1.96−2.04 (1 H, m), 1.46−1.58 (1 H, m), 1.26 (3 H, s), 0.69 (3 H, t, J

= 7.6 Hz); MS (ESI) 444.1 [M + H]⁺.

To a solution of the aldehyde above (5.20 g, 11.7 mmol) in THF

(117 mL) was added MeMgBr (1.4 M in toluene/THF (75:25), 25

mL, 35 mmol) at 0 °C. The mixture was allowed to warm to 25 °C

and stirred for 1 h. The reaction was quenched (aqueous saturated

NH₄Cl), extracted (2 × EtOAc), and washed (2 × brine). The

combined organic layers were dried (Na₂SO₄) and concentrated under

reduced pressure. Flash column chromatography (SiO₂, 30% and 50%

EtOAc/hexanes) provided 51 (4.70 g, 87% yield) as a pale yellow

foam (dr = 4:6 in favor of the R-isomer): MS (ESI) 460.2 [M + H]⁺.

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-3-

methyl-2-oxo-1-((S)-2-oxopentan-3-yl)-piperidin-3-yl)acetic

Acid (52). To a rapidly stirring solution of 51 (3.14 g, 6.82 mmol) and

RuCl₃·hydrate (31 mg, 0.14 mmol) in water/AcCN/CCl₄(1.5:1:1, 98

mL) was added NaIO₄(8.75 g, 40.9 mmol) in six portions over 1 h

while maintaining the temperature below 25 °C. The mixture was

stirred for an additional 1 h after the ﬁnal addition of NaIO₄. The

mixture was ﬁltered through Celite, and the ﬁlter cake was washed

(EtOAc). The ﬁltrate was extracted (2 × EtOAc) and washed (2 ×

10% aqueous NaHSO₃and 1 × brine). The combined organic layers

were dried (Na₂SO₄) and concentrated under reduced pressure. Flash

column chromatography [SiO₂, 10−50% (15% MeOH/acetone)/

1

hexanes] provided 52 (2.59 g, 80% yield) as a light pink foam: H

NMR (400 MHz, CDCl₃) δ ppm 7.24 (2 H, d, J = 8.2 Hz), 7.06−7.16

(2 H, m), 6.95−7.04 (3 H, m), 6.74−6.76 (1 H, m), 4.47 (1 H, d, J =

10.8 Hz), 3.25−3.33 (1 H, m), 3.12−3.17 (1 H, m), 2.82−2.93 (2 H,

m), 2.28−2.35 (1 H, m), 2.12−2.22 (2 H, m), 2.16 (3 H, s), 1.78−

1.86 (1 H, m), 1.47 (3 H, s), 0.64 (3 H, t, J = 7.5 Hz); HRMS (ESI)

m/z 476.1407 [M + H]⁺(C₂₅H₂₇Cl₂NO₄requires 476.1397).

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-

((2S,3S)-2-hydroxypentan-3-yl)-3-methyl-2-oxopiperidin-3-yl)-

acetic Acid (29). To a solution of 52 (3.86 g, 8.13 mmol) in THF

(102 mL) was added L-Selectride (1 M in THF, 16.3 mL, 16.3 mmol)

dropwise over 5 min at −78 °C. After being stirred at −78 °C for 30

min, the mixture was allowed to warm to 25 °C and stirred for 2 h.

The reaction was quenched (saturated aqueous NH₄Cl), extracted (3

× EtOAc), and washed (3 × ice-cold 1 N aqueous HCl and 3 × brine).

The combined organic layers were dried (Na₂SO₄) and concentrated

under reduced pressure. Flash column chromatography [SiO₂, 10−

30% (15% MeOH/acetone) in hexanes, a gradient elution] and

recrystallization (hexanes/acetone = 3:1) provided 29 (2.90 g, 75%

To a solution of the product above (317 mg, 0.451 mmol) in THF

(13 mL) was added tetrabutylammonium ﬂuoride (1.0 M in THF, 2.2

mL, 2.2 mmol) slowly at 0 °C. After the mixture was stirred at 25 °C

for 16 h, the reaction was quenched (1 N aqueous HCl) and extracted

(2 × EtOAc). The combined organic layers were dried (Na₂SO₄) and

concentrated under reduced pressure, and puriﬁcation by RP-HPLC

(10−90% A/B, gradient elution) provided 28 (1.03 g, 78% yield) as a

white solid: ¹H NMR (500 MHz, CDCl₃) δ ppm 7.24−7.26 (2 H, m),

7.15−7.19 (1 H, m), 7.08−7.13 (1 H, m), 6.94−7.08 (3 H, m), 6.72−

6.77 (1 H, m), 4.56 (1 H, d, J = 9.8 Hz), 3.72−3.81 (1 H, m), 3.60−

3.65 (1 H, m), 3.19−3.27 (1 H, m), 3.08−3.15 (1 H, m), 2.97−3.04 (1

H, m), 2.73−2.78 (1 H, m), 2.16−2.26 (1 H, m), 2.04−2.11 (1 H, m),

1.88−1.99 (1 H, m), 1.43−1.50 (1 H, m), 1.47 (3 H, s), 0.63 (3 H, t, J

= 7.2 Hz); HRMS (ESI) m/z 464.1386 [M + H]⁺(C₂₄H₂₇Cl₂NO₄

requires 464.1397).

1

yield) as a white solid: H NMR (500 MHz, DMSO-d₆) δ ppm 12.42

(1 H, br s), 7.33 (2 H, d, J = 8.4 Hz), 7.17−7.27 (4 H, m), 7.08−7.10

(1 H, m), 6.93−6.95 (1 H, m), 4.80 (1 H, br s), 4.77 (1 H, d, J = 10.9

Hz), 4.01−4.06 (1 H, m), 3.35−3.40 (1 H, m), 2.87 (1 H, d, J = 13.7

Hz), 2.48 (1 H, d, J = 13.7 Hz), 2.29−2.33 (1 H, m), 2.04−2.15 (2 H,

m), 1.55−1.64 (1 H, m), 1.41−1.49 (1 H, m), 1.26 (3 H, s), 1.00 (3 H,

d, J = 6.3 Hz), 0.30 (3 H, t, J = 7.6 Hz); HRMS (ESI) m/z 478.1560

[M + H]⁺(C₂₅H₂₉Cl₂NO₄requires 478.1553); [α]²³+110° (c 0.51,

D

MeOH).

ASSOCIATED CONTENT

■

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-

((3S)-2-hydroxypentan-3-yl)-3-methylpiperidin-2-one (51). To

a solution of 50 (5.87 g, 13.2 mmol) and water (360 μL, 19.7 mmol)

in DCM (167 mL) was added Dess−Martin periodinane (8.37 g, 19.7

mmol) at 25 °C. After the mixture was stirred for 1.5 h, the reaction

was quenched (aqueous 1 M Na₂S₂O₃), extracted (2 × DCM), and

S

* Supporting Information

(i) ¹H NMR spectra of 23 and 25 at 298 and 203 K, (ii) in vitro

biological assays, (iii) in vivo study protocols, (iv) determi-

nation of cocrystal structures of 23 and 29 with MDM2, and

(v) structures and energies of the gauche and anti conformers

Q

dx.doi.org/10.1021/jm300354j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(10) 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.

(11) Information from www.clinicaltrials.gov. (a) RG7112 (Hoﬀ-

mann-La Roche). (b) RO5503781 (Hoﬀmann-La Roche). (c) MK-

8242 (Merck).

in Figure 7. This material is available free of charge via the

Internet at http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

(12) 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 chromeno-

triazolopyrimidines as potent inhibitors of the mouse double minute 2-

tumor protein 53 protein−protein interaction. J. Med. Chem. 2009, 52,

7044−7053.

(13) 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.

(14) 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 (Washington, D.C.) 2004, 303, 844−

848.

*Telephone: 650-244-2195. Fax: 650-837-9369. E-mail:

daqings@amgen.com.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Manuel Ventura, Brent Murphy, J. Preston, Fang Xia,

Jason Simiens, and Kevin Crossley for the puriﬁcation of

(5R,6S)-3; David Bauer, Michele Kubryk, and Larry Miller for

the preparation of 46; Simon Wong for the time-dependent

inhibition (TDI) determinations; and Jonathan Houze and

Larry McGee for helpful discussions.

ABBREVIATIONS USED

■

AcCN, acetonitrile; b.i.d., twice a day dosing; Boc, tert-

butoxycarbonyl; BrdU, 5-bromo-2-deoxyuridine; CL, clearance;

CYP3A4, cytochrome P450 3A4; DCM, dichloromethane;

DMBCl, 2,4-dimethoxybenzyl chloride; DMF, N,N-diemethyl-

formamide; 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; q.d., once a day dosing; qRT-

PCR, quantitative reverse transcription polymerase chain

reaction; SEM, standard error of the mean; SPR, surface

plasmon resonance; TBAF, tetrabutylammonium ﬂuoride; TDI,

time dependent inhibition; TEA, triethylamine; TFA, triﬂuoro-

acetic acid; THF, tetrahydrofuran

(15) Shangary, S; Qin, D.; McEachern, D.; Liu, M.; Miller, R. S.; Qiu,

S.; Nikolovska-Coleska, Z.; Ding, K.; Wang, G.; Chen, J.; Bernard, D.;

Zhang, J.; Lu, Y.; Gu, Q.; Shah, R. B.; Pienta, K. J.; Ling, X.; Kang, S.;

Guo, M.; Sun, Y.; Yang, D.; Wang, S. Temporal activation of p53 by a

speciﬁc MDM2 inhibitor is selectively toxic to tumors and leads to

complete tumor growth inhibition. Proc. Natl. Acad. Sci. U.S.A. 2008,

105, 3933−3938.

(16) Experimental details of the in vitro biological assays can be

found in the Supporting Information.

(17) Kussie, P. H.; Gorina, S; Marechal, 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

(Washington, D.C.) 1996, 274, 948−953.

(18) Popowicz, G. M.; Domling, A.; Holak, T. A. The structure-based

̈

design of Mdm2/Mdmx−p53 inhibitors gets serious. Angew. Chem.,

Int. Ed. 2011, 50, 2680−2688.

(19) When the TFA salt of MI-63 was in organic solvents (MeOH

and DMSO), the quaternary carbon center of the spirolactam system

and the tert-butyl group were slowly isomerized (in-house result).

(20) Detailed analysis of these scaﬀolds and SAR studies leading to

both 1 and 2 will be reported in a separate communication.

(21) The absolute stereochemistry of compound 2 was assigned via

comparison of theoretical and experimental VCD and optical rotations

of the C3-allyl precursors of both compound 2 and its enantiomer.

Detailed studies will be reported in a separate communication.

(22) Docking was performed via alignment of 2 to various MDM2

cocrystallized ligands with the in-house ﬂexible alignment tool

FLAME.³⁷The resultant structures were subject to minimization in

the MDM2 protein using AMBER, version 9.0.³⁸Top-scoring poses

gave rise to the model depicted in Figure 3.

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Supporting Information.

1

(29) H NMR spectra can be found in the Supporting Information.

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S

dx.doi.org/10.1021/jm300354j | J. Med. Chem. XXXX, XXX, XXX−XXX

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Structure-based design of novel inhibitors of the MDM2-p53 interaction

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