Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic Plasma

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX-XXX

Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester

Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic

Plasma

Ken Yamada,*^,†,§Margaret Brousseau,^†Wataru Honma,^§,∥Akiko Iimura,^§,∥Hidetomo Imase,^†,§

Yuki Iwaki,^†,§,⊥Toshio Kawanami,^†,§Daniel LaSala,^‡,#Guiqing Liang,^†Hironobu Mitani,^§,∥

Kazuhiko Nonomura,^§,∇Osamu Ohmori,^§,○Meihui Pan,^†Dean F. Rigel,^‡,◆Ichiro Umemura,^§,∥

Kayo Yasoshima,^†,§Guoming Zhu,^†and Muneto Mogi*^,†,§

^†Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139-4133, United States

^‡Novartis Institutes for BioMedical Research, Novartis Pharmaceuticals Corporation, 1 Health Plaza, East Hanover, New Jersey

07936-1080, United States

^§Novartis Institutes for BioMedical Research, Novartis Pharma K.K., Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan

S

* Supporting Information

ABSTRACT: Herein we describe the discovery and character-

ization of a novel, piperidine-based inhibitor of cholesteryl

ester transfer protein (CETP) with a core structure distinct

from other reported CETP inhibitors. A versatile synthesis

starting from 4-methoxypyridine enabled an eﬃcient explora-

tion of the SAR, giving a lead molecule with potent CETP

inhibition in human plasma. The subsequent optimization

focused on improvement of pharmacokinetics and mitigation

of oﬀ-target liabilities, such as CYP inhibition, whose

improvement correlated with increased lipophilic eﬃciency.

The eﬀort led to the identiﬁcation of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics

in rats and robust eﬃcacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had

only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo.

Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma

cells nor in chronically cannulated rats. On the basis of its preclinical eﬃcacy and safety proﬁle, the compound was advanced into

clinical trials.

with reduced CETP activity are associated with reduced CHD

risk.⁴⁻⁶

INTRODUCTION

■

Coronary heart disease (CHD) is the leading cause of mortality

in the industrialized world. It has long been recognized that

plasma low density lipoprotein (LDL) cholesterol levels

directly correlate with CHD risk. Pharmacological reduction

of LDL cholesterol via 3-hydroxy-3-methylglutaryl-coenzyme A

(HMGCoA) reductase inhibitors (statins) reduces CHD risk

by approximately 30%. However, considerable residual CHD

risk remains.¹Several epidemiological studies have also shown

that plasma high density lipoprotein (HDL) cholesterol levels

inversely correlate with CHD risk although the clinical beneﬁt

of increased HDL cholesterol has yet to be demonstrated.^2,3

Cholesteryl ester transfer protein (CETP) is a 74 kD

circulating glycoprotein secreted mainly by the liver. CETP

mediates the exchange of cholesteryl ester and triglyceride

(TG) between plasma lipoproteins, including LDL, very low

density lipoprotein (VLDL), and HDL.⁴CETP activity has

been correlated with CHD risk in several epidemiological

studies.⁴Moreover, CETP polymorphisms that are associated

Owing to the promise of its therapeutic potential, multiple

CETP inhibitors have been evaluated in clinical trials. Three

CETP inhibitors torcetrapib, dalcetrapib, and evacetrapib, have

failed in phase 3 cardiovascular outcome trials. In 2006,

development of torcetrapib was abruptly terminated based on

increased cardiac events and mortality observed in the

treatment group of the ILLUMINATE trial.⁷It is now

generally accepted that the increased mortality associated

with torcetrapib was due to adverse oﬀ-target eﬀects, including

increased blood pressure and corticosteroid concentrations.⁸⁻¹¹

In contrast, dalcetrapib, which has modest eﬀects on HDL and

LDL cholesterol levels, was terminated due to futility.¹²The

ACCELERATE trial for evacetrapib¹³was also terminated

upon futility analysis despite robust increases in HDL

cholesterol and reductions in LDL cholesterol. The last of

Received: June 18, 2017

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Figure 1. Structures and cLogD_pH7.4of CETP inhibitors that have reached phase 3 clinical trials.

Figure 2. Initial optimization.

Figure 3. SAR of the pyrimidine analogues.

these trials REVEAL (anacetrapib) was expected to provide the

best evidence (i.e., >30000 patients, 5 year follow-up) as to the

relevance of pharmacological CETP inhibition for CHD risk

reduction.¹⁴It was very recently reported that anacetrapib

reduces CHD risk by 9%, while overall mortality was not

aﬀected.¹⁵The reduction in CHD risk is consistent with the

beneﬁt predicted from a 17% reduction in LDL cholesterol,

which suggests a limited role of increased HDL, if any, in CHD

risk reduction.

In our eﬀorts to identify a best-in-class CETP inhibitor, we

noted that most clinical CETP inhibitors suﬀer from high

lipophilicity (Figure 1), arguably owing to the nature of the

binding sites that accommodate highly lipophilic substrates

cholesteryl ester (CE) and TG.¹⁶The most striking example is

anacetrapib, with cLogD_pH7.4(abbreviated hereafter as cLogD

for brevity) of 9.2, which has high distribution to adipose tissue

and an exceptionally long terminal elimination half-life in

humans.¹⁷As for the pressor eﬀects and corticosteroid eﬀects

associated with torcetrapib, we hypothesized that structural

deviation from the tetrahydroquinoline core that has been

associated with the aldosterone risk¹⁰should mitigate these oﬀ-

target liabilities. Thus, we set out to identify a structurally

distinct, novel CETP inhibitor scaﬀold with reduced lip-

ophilicity and without the oﬀ-target liabilities of torcetrapib.

RESULTS AND DISCUSSION

■

In our search for a novel CETP inhibitor that is structurally

distinct from torcetrapib, we explored a number of simpler

scaﬀolds to understand the most essential features of various

CETP inhibitors known at the time. Among many prototypes

prepared, we identiﬁed 1 as a hit (Figure 2). Although weak in

potency (70% inhibition at 100 μM compound concentration),

we were intrigued by the unique piperidine scaﬀold of 1,

presenting a relatively simple and modular structure with high

sp³content, which also seemed amenable to an eﬃcient

structure−activity relationship (SAR) exploration.

We initially explored the synthetically accessible 6-position of

the piperidine. Fortuitously, the mere introduction of an ethyl

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Figure 4. Final optimization.

group at this position to give compound 2 (Figure 2) resulted

in dramatically improved CETP inhibition, with half-maximum

inhibitory concentration (IC₅₀) of 1300 nM in human plasma.

Further improvement was made through introduction of a

benzyl group in place of one of the ethyl groups to give

compound 3 (IC₅₀771 nM). Another boost in potency was

achieved via replacement of the methyl carbamate with a

pyrimidine to give compound 4 (IC₅₀160 nM). The active

enantiomer (eutomer) of racemic 4 was determined to be 5,

whose absolute stereochemistry was conﬁrmed by single

compound X-ray crystallography to be 2S,4S,6R (Supporting

Information, Figure 3). As expected, compound 5 was

approximately 2-fold more potent than the racemate 4, while

the inactive enantiomer (distomer) showed no CETP

inhibition.

Although potent, compound 4 is highly lipophilic, with a

cLogD value exceeding that of anacetrapib (9.7 vs 9.2). Thus,

the next phase of our optimization was focused on reducing

lipophilicity while maintaining or improving potency, using

racemic 4 as the lead (Figure 3). While many attempts were

made to replace the bis-triﬂuoromethyl groups on the N-benzyl

substituent, they all resulted in substantial loss in potency, as

exempliﬁed by the methoxy group in compound 6. The only

exception was chlorine (compound 7, IC₅₀240 nM), which

oﬀered minimal improvement in lipophilicity. Exploration of

the piperidine nitrogen capping group was also accompanied by

reduced potency, as seen with ethyl urea 8 and n-butyl amide 9.

Isopropyl carbamate 10 (IC₅₀200 nM) was equipotent but,

again, with minimal improvement in lipophilicity. The only

point of SAR that showed concomitant improvement in

potency and reduced lipophilicity was at the 5-position of the

pyrimidine. Both 5-methoxypyrimidine compound 11 and 5-

morpholinopyrimidine compound 12 were potent and slightly

less lipophilic (cLogD 9.4 and 9.0, respectively) than

compound 4 (cLogD 9.7).

partitioning of ionized molecules to the octanol phase,²⁰we

calculated a modiﬁed LipE based on cLogD at pH 7.4 instead of

cLogP and refer to the values as LipE_cLogD. By this analysis,

compound 4 has a relatively poor LipE_cLogDvalue of −2.9 and

none of the transformations tested in Figure 3 resulted in

substantial improvement in lipophilic eﬃciency, perhaps with

the marginal exception of the n-butyl amide 9. It was only in the

ﬁnal optimization that the issue of lipophilic eﬃciency was

addressed, which indeed resulted in improved pharmacokinetic

proﬁles, as described below.

Compound 13, the active enantiomer of racemic compound

12, showed robust inhibition of CETP activity (IC₅₀60 nM)

(Figure 4). However, pharmacokinetic (PK) studies in

Sprague−Dawley (SD) rats showed that compound 13 had

very poor oral availability (3%). Reasoning that we needed a

less lipophilic start point, we turned our attention back to the

symmetric 2,6-diethylpiperidine compound 2, which showed

only marginally weaker CETP inhibition than the substantially

more lipophilic 2-benzyl-6-ethylpiperidine analogue 3 (Figure

2). We therefore made the 2,6-diethyl analogue of compound

13 (Figure 4). Gratifyingly, compound 14 retained potent

inhibition of CETP (IC₅₀60 nM), while for the ﬁrst time

substantially improving lipophilic eﬃciency relative to 2-benzyl-

6-ethylpiperidines such as compound 13 (LipE_cLogD−0.3 vs

−1.8). In addition, compound 14 presented a symmetric,

achiral molecule, eliminating the need for chiral resolution or

asymmetric synthesis. Compound 14 showed a much improved

PK proﬁle in SD rats when dosed at 1 mg/kg iv and 3 mg/kg

po, with an area under the curve (AUC) of 2750 nM·h and oral

availability of 35%. However, compound 14 was an inhibitor of

CYP3A4 (2.2 μM) and CYP2C9 (6.6 μM), thereby suggesting

the risk of drug−drug interaction. This is a particular concern

as a CETP inhibitor would likely be coadministered with a

statin (i.e., current standard-of-care), many of which are

metabolized primarily by either CYP3A4 or CYP2C9.²¹

As CYP enzymes often have aﬃnity for lipophilic xeno-

biotics,²²we hypothesized that further reduction in lipophilicity

by introduction of a carboxylic acid motif might mitigate this

liability. Among several carboxylic acid bearing analogues

prepared, we were gratiﬁed to ﬁnd that compound 15 retained

While we did not explicitly use the parameter of lipophilic

eﬃciency (LipE = pIC₅₀− cLogP)^18,19at the time of our

research, it provides a parameter that, in retrospect, illustrates a

main aspect of our compound optimization. Because cLogP

does not take ionization into account and overestimates the

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good CETP inhibition (IC₅₀150 nM) and showed a much

improved CYP inhibition proﬁle compared to compound 14

(CYP3A4 midazolam 25 vs 2.2 μM, CYP2C9 > 50 vs 6.6 μM).

In addition, the introduction of a carboxylic acid resulted in a

dramatically improved rat pharmacokinetic proﬁle with reduced

clearance (0.09 L/h/kg), volume of distribution (0.67 L/kg),

and increased exposure (AUC 28,500 nM·h) from a 3 mg/kg

po dose.

Lastly, we screened a variety of capping groups for the

pyrimidine, as we were concerned with the potential generation

of reactive metabolites from the morpholine motif and wanted

a further boost in potency. We found that the methylpyrazole

derivative, (1r,4r)-4-(((2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-

2,6-diethylpiperidine-1-carbonyl)oxy)cyclohexane-1-carboxylic

acid 16 (TAP311),²³oﬀered the best balance of CETP

inhibition (IC₅₀62 nM), selectivity (>170-fold vs CYPs),

lipophilicity (cLogD 4.9), and rat pharmacokinetic proﬁle (CL

0.09 L/h/kg, V_d0.57 L/kg, AUC 27200 nM·h, F 43%) from 1

mg/kg iv and 3 mg/kg po doses. The compound also had the

best lipophilic eﬃciency (LipE_cLogD2.4) among all compounds

described herein. The protein binding of compound 16 was

determined to be 99.4% in human plasma.

The improvement in the pharmacokinetic proﬁle with

carboxylic acid bearing carbamates was generally consistent

across many analogues. It should be noted that all compounds

were tested as an amorphous form and dosed as a simple

supension in 0.5% methylcellulose, including compound 16.

The high bioavailability observed with these compounds makes

a sharp contrast to other CETP inhibitors such as torcetrapib

and anacetrapib, which in our hands showed bioavailability of

approximately 8−9% in the same suspension formulation.

Experimental assessment of octanol−water distribution at

pH 7.6 revealed a signiﬁcantly lower distribution coeﬃcient

(Log D_pH7.65.2) for 16 compared to the neutral CETP

inhibitors dalcetrapib, torcetrapib, and anacetrapib (Table 1).

increases in HDL cholesterol were observed for 16 and

anacetrapib (Figure 5C), along with dose-dependent reductions

in LDL cholesterol with maximum reductions of 41% and 33%

for 16 and anacetrapib, respectively (Figure 5D).

Consistent with a previous report,²⁴we observed markedly

high adipose and adrenal tissue versus plasma drug

concentration ratios in chow-fed hamsters treated once daily

with anacetrapib (10 mg/kg po) for 14 days (Figure 6). In

sharp contrast, hamsters treated with 16 (10 mg/kg po) for the

same time period had substantially lower adipose and adrenal

tissue versus plasma drug concentration ratios. Liver to plasma

drug concentration ratios were similar for 16 and anacetrapib.

These results suggest that, unlike anacetrapib, 16 does not

preferentially distribute to adipose tissue. This could be an

important diﬀerentiating factor from anacetrapib, which has

been detected in the plasma of patients 2−4 years after

cessation of dosing.²⁵Others have also reported similar

improvements in tissue distribution proﬁle by reducing overall

lipophilicity of the CETP inhibitors.^24,26

We next evaluated the eﬃcacy of 16 and anacetrapib in vitro

using serum samples from patients having a wide range of TG

concentrations. With increasing TG content in this assay,

anacetrapib lost eﬃcacy, while 16 retained eﬃcacy (Figure 7A).

This trend was recapitulated ex vivo when the compounds were

dosed orally in hamsters treated with 4-(1,1,3,3-

tetramethylbutyl)phenol polymer with formaldehyde and

oxirane (Triton WR-1339),²⁷an inhibitor of lipoprotein lipase

activity that results in elevated plasma TG levels.²⁸The

inhibitory eﬀect of 16 on CETP activity in the plasma of the

treated hamsters was largely maintained, while this was not the

case for anacetrapib (Figure 7B). The retained eﬃcacy of 16 in

the setting of high TG is expected to be clinically relevant, as

hypertriglyceridemia is observed in a signiﬁcant portion of

dyslipidemic patients. Overall, 31% of the adult US population

has an elevated plasma TG level (≥150 mg/dL) and high

(≥200 mg/dL) or very high (≥500 mg/dL) fasting TG levels

are found in 16.2% and 1.1% of adults, respectively.²⁹

Combined with the fact that TG concentrations can increase

Table 1. Measured Log D_pH7.6, cLogD_pH7.4, CETP Plasma

IC₅₀, and LipE_cLogDof Clinical CETP Inhibitors and

Compound 16

by more than 100 mg/dL following a high-fat meal,^30,31

a

substantial portion of patients taking a CETP inhibitor may

have a TG level where the observed diﬀerence between 16 and

anacetrapib may be relevant. Interestingly, anacetrapib showed

a trend for greater CHD risk reduction in patients with higher

TG concentrations despite a potential decrease in eﬃcacy,¹⁵

suggesting that compound 16 has an opportunity to further

impact this patient subpopulation.

compd

16

dalcetrapib torcetrapib anacetrapib evacetrapib

a

Log D_pH7.6

>6.0

7.2

>4

>5.9

9.2

5.7

5.2

4.9

cLogD_pH7.4

7.6

CETP

plasma

IC₅₀

4100 nM

18 nM

110 nM

62 nM

Given this observation, we hypothesized that the diﬀerential

eﬃcacy of 16 versus anacetrapib in the hypertriglyceridemic

setting may be related to the compound distribution proﬁle

across diﬀerent lipoprotein fractions, owing to the marked

diﬀerences in their lipophilicity. Indeed, when the compounds

were spiked into serum from human donors with normal TG

levels (TG < 150 mg/dL), 16 was found predominantly in the

HDL and lipoprotein deﬁcient serum (LPDS) fractions (Figure

8A), whereas anacetrapib was more evenly distributed among

HDL, LDL, and triglyceride rich lipoproteins (TRL) (Figure

9A). In serum with higher TG content (300−500 and >750

mg/dL), 16 showed similar distribution among lipoprotein

fractions (Figure 8B,C), while anacetrapib was predominantly

found in TRL fractions (Figure 9B,C). Because CETP is mainly

associated with HDL in the circulation, these observations

corroborate well with the retained potency of 16 in

hypertriglyceridemic plasma.

LipE_cLogD

−1.7

0.2

−1.5

1.8

2.4

a

Concentration in water phase below detection limit.

In fact, for neutral CETP inhibitors, an accurate experimental

log D value could not be determined because compound

concentrations in the aqueous phase were below the detection

limit in our protocol. On the other hand, the calculated

cLogD_pH7.4for carboxylic acid bearing evacetrapib and 16

closely matched their experimental Log D_pH7.6values.

When administered once daily for 2 weeks to chow-fed

hamsters, compound 16 (0.3−10 mg/kg po) showed

comparable exposure to anacetrapib at all doses and statistically

signiﬁcant decreases in CETP activity at all doses compared to

vehicle, while anacetrapib showed statistically signiﬁcant

decreases in CETP activity at 3 and 10 mg/kg doses compared

to vehicle (Figure 5A,B). Furthermore, robust, dose-dependent

D

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Figure 5. Pharmacokinetics and pharmacodynamics of anacetrapib and 16 in hamsters. Compound (0.3, 1, 3, and 10 mg/kg po) or vehicle (Veh)

was dosed once a day for 14 days to hamsters fed a standard chow diet. Blood was withdrawn 24 h post last dose and analyzed for total compound

concentrations (A), CETP inhibition (B), HDL-cholesterol levels (C), and LDL-cholesterol levels (D). Values are mean SEM of six animals. ** P

< 0.01, signiﬁcant versus the vehicle group (Dunnett’s multiple comparisons test following one-way ANOVA).

synthetic routes, we opted for the sequential functionalization

of acylated 4-methoxypyridine that provided a modular

synthetic route for a rapid SAR exploration and compound

optimization. Thus, a variety of 2,4,6-trisubstituted piperidines

were synthesized by 1,4-addition of a Grignard reagent to the

acylated or carbamoylated species of 4-methoxypyridine,

followed by 1,4-addition of alkyl cuprates to the resultant

dihydropiperidones 17b,c to give 2,6-disubstituted piperidine-

4-ones 18b,c (Scheme 1).³²We found that, generally, the cis-

addition of the cuprate was favored when R₂was benzyloxy

compared to tert-butoxy. Subsequent reductive amination gave

the all-cis isomer as the major product. Capping the secondary

Figure 6. Distribution proﬁles of anacetrapib and compound 16 in

amines 19a−c with methyl chloroformate gave 1 and 2, while

various tissues of hamsters administered anacetrapib or compound 16.

further deprotection of 20 and capping with tert-butoxycar-

Anacetrapib and compound 16 were dosed orally once each day for 14

bonyl (BoC) group gave 3.

days to hamsters fed a standard chow diet. Blood and tissues were

collected 24 h after the ﬁnal dose and analyzed for compound

concentrations. Data represent mean total concentration SEM for

seven animals per group.

Pyrimidine capped compounds in Figure 3 were accessed in a

similar manner via ketone 18d (Scheme 2). We initially

investigated the SAR with racemic compounds as shown in

Figure 3, resolving selected compounds of interest at the end

such as with racemic 4 and 12 into eutomers 5 and 13,

respectively. Reductive amination of the ketone 18d gave all-cis

2,4,6-trisubstituted piperidine 21, which was deprotected to

give 22. Capping the primary amine of 22 with 5-bromo-2-

chloropyrimidine gave 23, followed by alkylation with 3,5-

disubstituted benzyl bromide to give 4, 6−7. Deprotection of

the Boc group to 24 was followed by acylation to give 8−10.

The bromopyrimidine 4 was converted to the methoxy

analogue 11 and morpholinyl analogues 12 and 13 through

copper and palladium catalyzed couplings, respectively.

The compounds for ﬁnal optimization (Figure 4) were

prepared from the aforementioned ketone intermediate 18b or

the isopropyl analogue 18e (Scheme 3). The same sequence of

transformation as in Scheme 2 gave the all-cis intermediates

27a,b, which were then elaborated into compounds 14 and 29a

Lastly, given the pressor and corticosteroid eﬀects of

torcetrapib, we evaluated the eﬀects of 16 and torcetrapib on

aldosterone production in H295R cells (Supporting Informa-

tion, Figure 1) and blood pressure and plasma aldosterone

levels in chronically cannulated SD rats (Supporting

Information, Figure 2). We observed a marked increase in

aldosterone secretion upon treatment of H295R cells with

torcetrapib, whereas increases in aldosterone were not observed

with 16 or anacetrapib. In contrast to torcetrapib, 16 did not

increase mean arterial pressure or plasma aldosterone levels in

conscious, cannulated SD rats.

CHEMISTRY

■

While our literature survey for the preparation of various 2,4-

and 2,4,6-substituted piperidines revealed several possible

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Figure 7. Eﬀect of triglyceride level on eﬃcacy of anacetrapib and 16. (A) In vitro CETP activity IC₅₀values of anacetrapib and 16 in serum pooled

from donors with varying TG levels. Values are the mean of two replicates for each serum pool. (B) Ex vivo inhibition of plasma CETP activity by

anacetrapib (Ana) and 16 dosed orally in hamsters treated with saline or Triton WR-1339 (Veh). Doses of Ana and 16 were 3 and 1.5 mg/kg,

respectively. Plasma samples were collected 24 h post dose. Values are mean SEM of ﬁve animals. *P < 0.05, **P < 0.01, signiﬁcant diﬀerence

from each Veh group (Dunnett’s multiple comparisons test following one-way ANOVA).

Figure 8. In vitro distribution proﬁle of compound 16 among serum lipoproteins when spiked into serum from healthy donors with normal (A),

high (B), or very high (C) triglyceride levels. The extraction of compound 16 was done in duplicate for each sample. The absolute mass of 16 was

calculated in each fraction and, as shown in the Y axis, was expressed relative to the total sum of 16 in all lipoprotein pools (mean SEM). One-way

ANOVA was performed to compare the percent distribution of 16 among serum lipoproteins.

through palladium catalyzed coupling with morpholine. In a

similar manner, 29b was prepared by palladium catalyzed

coupling with the pinacol boronate of the methylpyrazole.

Deprotection of the Boc group was followed by acylation with

chloroformate 26, which was prepared by esteriﬁcation of the

acid 25 followed by treatment with triphosgene and base to

form the chloroformate. Finally, hydrolysis of the methyl ester

gave 15 and 16. The all-cis conﬁguration of the piperidine

substituents of 16 was conﬁrmed by single compound X-ray

crystallography (Supporting Information, Figure 4).

to CETP inhibitors that have entered phase 3 clinical trials to

date. On the basis of our preclinical data package, we propose

that the lower lipophilicity of compound 16 contributes to its

lack of accumulation in adipose tissue and retained eﬃcacy

versus anacetrapib in the setting of high plasma triglycerides.

Unlike torcetrapib, compound 16 does not increase blood

pressure or plasma aldosterone levels in vivo. Taken together,

our results indicate that compound 16 is a promising candidate

for targeting residual cardiovascular disease risk. On the basis of

its preclinical eﬃcacy and safety proﬁle, compound 16 was

advanced into clinical trials.³³

CONCLUSION

■

EXPERIMENTAL SECTION

We have identiﬁed 16 as a novel, piperidine-based CETP

inhibitor with excellent pharmacokinetic and eﬃcacy proﬁles.

The compound has a substantially lower log D_pH6.8compared

■

Methods. Software. Figures 5−9 were generated with and

statistical analyses were performed using GraphPad Prism version 7.

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Figure 9. In vitro distribution of anacetrapib among serum lipoproteins when spiked into serum from healthy donors with normal (A), high (B), or

very high (C) triglyceride levels. Anacetrapib extraction was done in duplicate for each sample. The absolute mass of anacetrapib was calculated in

each fraction and, as shown in the Y axis, was expressed relative to the total sum of anacetrapib in all lipoprotein pools (mean SEM). One-way

ANOVA was performed to compare the percent distribution of anacetrapib among serum lipoproteins.

a

Scheme 1

a

Reagents and conditions: (a) PhOCOCl, EtMgBr, tetrahydrofuran, −40 °C; then t-BuOK (for 17b) or BnOCOCl, BnMgBr (for 17c); (b) BF₃·

Et₂O, EtMgBr, CuI, tetrahydrofuran, −78 °C to rt; (c) 3,5-bis(triﬂuoromethyl)benzylamine, Ti(OiPr)₄, MeOH, then NaBH₄, 0 °C to rt (19a), 3,5-

bis(triﬂuoromethyl)benzylamine, NaBH(OAc)₃, acetic acid, dichloroethane, 0 °C to rt (19b,c); (d) MeOCOCl, pyridine, 0 °C to rt (for 1), or

MeOCOCl, 4-dimethylaminopyridine, CH₂Cl₂, rt (for 2, 20); (e) PdCl₂, Et₃SiH, Et₃N, CH₂Cl₂, rt; (f) Boc₂O, 60 °C, no solvent.

Calculated Octanol−Water Distribution Coeﬃcient. Calculated

started by the addition of 14 μL of donor solution diluted with the

assay buﬀer. Fluorescence intensities (relative ﬂuorescence unit, RFU)

were measured every 30 min at 485 nm (excitation) and 535 nm

(emission) for 120 min at 37 °C using ARVO SX+L (PerkinElmer,

Wellesley, MA). CETP activity (RFU/min) was determined from the

changes in ﬂuorescence intensity from 30 to 90 min. The inhibitory

activity of the compound was calculated as % inhibition of enzyme

activity using 1 or 10 μM torcetrapib (as positive control) and DMSO

as 100% and 0% inhibition, respectively. The IC₅₀value of duplicate

wells was obtained by logistic equation (Y = bottom + (top −

cLogD at pH 7.4 was determined using the following approximation:

cLogD_pH= cLogP − Log(1 + 10^pH−cpK

)

a

where cpK_awas determined using FOCUS software³⁴with built-in

MoKa algorithm.³⁵

Lipophilic Eﬃciency.^18,19LipE_cLogD= p[CETP human plasma IC₅₀]

− cLogD_pH7.4

.

CETP IC₅₀Determination in Human Plasma. A pool of

ethylenediaminetetraacetic acid (EDTA)−plasma from healthy male

donors was obtained from New Drug Development Research Center,

Inc. (Hokkaido, Japan). Human dyslipidemic serum (1 lot, lot no.

N83376) was obtained from Uniglobe Research Co. (Reseda, CA). To

evaluate the eﬀect of compounds, 50 μL of human plasma (ﬁnal 50%

plasma), 35 μL of the assay buﬀer, and 1 μL of compound dissolved in

dimethyl sulfoxide (DMSO, ﬁnal 1%, Sigma, St. Louis, MO, catalogue

no. D2650) were added to each well of a 96-well half-area black, ﬂat

bottom polystyrene NBS microplate (Corning, Corning, NY,

catalogue no. 3686). The reaction (ﬁnal volume of 100 μL) was

̂

bottom)/(1 + (x/IC₅₀)Hill slope) using Origin software, version 7.5

SR3 (OriginLab Co., Northampton, MA).

Log D (pH 7.6) Determination. Phosphate buﬀered saline (pH 7.4)

saturated with octanol and 1-octanol saturated with buﬀer were

prepared prior to the start of the log D assay. Aliquots of 10 mM

DMSO compound solution along with an internal reference

compound were dispensed in triplicate in 96-well polypropylene 2

mL plates. The DMSO was removed using a GeneVac HT4X

evaporator for approximately 1 h.

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a

Scheme 2

a

Reagents and conditions: (a) BnNH₂, BF₃·Et₂O, toluene, reﬂux, NaBH₄, MeOH, rt; (b) Pd/C, H₂, EtOH, 55 °C; (c) i-Pr₂NEt, DMF, 120 °C; (d)

NaH, DMF, 0 °C to rt; (e) 4 N HCl in EtOAc, rt; (f) EtNCO, iPr₂NEt, DMF, rt (for 8), or n-PrCOCl, iPr₂NEt, DMF, rt (for 9), or iPrOCOCl,

iPr₂NEt, DMF, rt (for 10); (g) NaOMe, CuI, DMF, 85 °C (for 11), or Pd₂(dba)₃, (2-biphenyl)ditert-butylphosphine, NaOtBu, morpholine, toluene,

100 °C (for 12); (h) chiral HPLC.

a

Scheme 3

a

Reagents and conditions: (a) MeI, K₂CO₃, DMF, 0 °C to rt; (b) triphosgene, pyridine, CH₂Cl₂, 0 °C to rt; (c) benzylamine, Ti(O-i-Pr)_4,MeOH, rt,

then NaBH₄, 0 °C to rt; (d) Pd/C, H₂, EtOH, 60 °C; (e) 2-chloro-5-bromopyrimidine, i-Pr₂NEt, DMF, 120 °C; (f) 3,5-bis(triﬂuoromethyl)-

benzylamine, NaH, DMF, 0 °C to rt; (g) morpholine, Pd₂(dba)₃, (2-biphenyl)di-tert-butylphosphine, t-BuONa, toluene, 100 °C (14 and 29a), 1-

methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh₃)₄, Na₂CO₃, DME-H₂O, 90 °C (29b); (h) 4 N HCl in EtOAc, rt, (i)

26, i-Pr₂NEt, DMF, rt; (j) NaOH, tetrahydrofuran−H₂O, rt.

Dried samples were incubated with 300 μL of octanol and 300 μL of

buﬀer and shaken for at least 4 h. Plates were centrifuged for 15 min at

4000 rpm to separate the octanol and buﬀer layers. Sample preparation

and phase separation were automated using liquid handling work-

stations. Both octanol and buﬀer phases were quantiﬁed using a

tandem mass spectrometer. Log D was derived from the ratio of

compound peak area responses in each phase, adjusted to the internal

standard peak areas. Log P was equivalent to log D when the

compound was un-ionized at pH 7.4.

Statement on Animal Welfare. All procedures were conducted in

accordance with approved Novartis Animal Care and Use Committee

protocols and the Guide for the Care and Use of Laboratory Animals.

Pharmacokinetic Studies. The test article was administered

intravenously (1 mg/mL/kg bolus, in N-methyl pyrrolidin-2-one/

PEG 200) or orally (3 mg/5 mL/kg, suspended in 0.5% aqueous

methylcellulose solution) to conscious 7-week old male Sprague−

Dawley rats (Charles River, Yokohama, Japan). Blood samples were

taken at various time points after administration. The samples were

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treated with methanol for protein precipitation, and the supernatants

were analyzed by liquid chromatography followed by electrospray/

tandem mass spectrometric detection. Pharmacokinetic variables were

estimated using noncompartmental methods. Animals were fed a

standard diet and had free access to tap water throughout the

experiment. This work was conducted with the permission of the

Novartis Tsukuba Research Institute Animal Welfare Committee

(permission no.: AWCJ06054).

Measurement of Human Plasma Protein Binding. Binding of

compound 16 to human plasma proteins was determined by

ultracentrifugation method. Approximately 0.5 mL of human plasma

was prepared in Beckman ultracentrifuge tube for each sample after

spiking with [¹⁴C]compound 16 at multiple concentrations (50−

10000 ng/mL). The remaining samples were centrifuged for 3 h at 37

°C at 306000g using a Sorvall RCM120GX microultracentrifuge and

80K rotor. The centrifuge was allowed to stop without using the brake.

Aliquots (0.05 mL) of the supernatant were carefully removed without

disturbing the separate layers and then transferred to scintillation vials

for radioactivity analysis. The fraction of drug bound to plasma

proteins equals (T_t− T_s)/T_t, where T_tis the total radioactivity in the

uncentrifuged sample and T_sis the radioactivity in the supernatant

after ultracentrifugation.

Hamster Eﬃcacy Study. Ten-week-old, male golden Syrian

hamsters were purchased from Japan SLC Inc. (Shizuoka, Japan).

Animals underwent a one-week acclimation period. Compounds were

prepared in a 0.5% methyl cellulose (MC, methyl cellulose 400 cP,

catalogue no. 138-05072, Wako Pure Chemical Industries, Osaka,

Japan) suspension. Compound 16 (0.3, 1, 3, and 10 mg/kg),

anacetrapib (0.3, 1, 3, and 10 mg/kg), or vehicle (10 mL/kg) was

administrated once a day in the morning for 2 weeks by oral gavage.

Then 24 h after administration of the last dose (16 h fast), whole

blood samples were collected from the abdominal vein by

venipuncture under isoﬂurane (Forane, Abbott Japan Co. Ltd.,

Tokyo, Japan) inhalant anesthesia. After centrifugation at 15000 rpm

(rotor no. AF-2536A, Kubota Co., Tokyo, Japan) for 10 min at 4 °C,

heparinized plasma (ﬁnal concentration about 28.6 U/mL, heparin

sodium injection “Ajinomoto”; Ajinomoto, Tokyo, Japan) or EDTA−

plasma was prepared and stored at −80 °C until use. It should be

noted that some animals in the 0.3 mg/kg anacetrapib group were

mistakenly administered a dose of 1 mg/kg (one animal on day 5 and

two animals on day 7). Because each of the three animals only received

one incorrect dose during the two-week study period, we concluded

that the results would not be signiﬁcantly altered by this error.

Therefore, all animals were included in the data analysis. This study

was performed according to the approval of the Novartis Tsukuba

Research Institute Animal Welfare Committee (permission no.

AWCJ06077).

Lipoprotein cholesterol content in the hamster eﬃcacy study was

assessed using an HPLC-size exclusion chromatography system with

an online enzymatic dual detection system for lipids. Plasma samples

were diluted (5-fold) with saline after ﬁltration (0.45 μm, Millipore

Co, catalogue no. UFC30HV00) and injected into the HPLC system

at a volume of 100 μL (as 20 μL of plasma), every 95 min, using an

autosampler. Plasma lipoproteins were separated using a single

Superose 6 column and ﬁltered phosphate buﬀered saline (0.45 μm,

Millipore Co., Bedford, MA, catalogue no. SJHVM4710; PBS,

Dainippon Pharmaceutical, Osaka, Japan catalogue no. 28-103-05

FN) at a ﬂow rate of 0.5 mL/min. Each enzymatic reagent was

pumped at a ﬂow rate of 0.25 mL/min. Both enzymatic reactions

proceeded at 37 °C in a reactor coil (Teﬂon tube, 15 m × 0.4 mm id)

in the column oven. The color developed after the enzyme reaction

was measured at 580 nm, and the electric signal was monitored (every

0.5 s). A control pooled EDTA-plasma sample was used to standardize

lipoprotein cholesterol levels. Cholesterol levels in control hamster

plasma were measured manually (cholesterol E-test, catalogue no. 439-

17501), and the level was 122 mg/dL. Lipoprotein cholesterol

concentrations were calculated using chromatogram areas observed

with control hamster plasma samples.

(Reseda, CA), Bioreclamation (Hicksville, NY) or from the in-house

donor program at the Novartis Institute for BioMedical Research.

Donors were not fasted before blood collection. Serum was isolated by

centrifugation of samples at 2500 rpm for 20 min and stored at −80

°C until use. Three pools of human serum samples were prepared for

this study. The mean TG concentration (mg/dL) of each pool were as

follows: 122, 347, and 958, respectively, to represent normal, high, and

very high levels. Once thawed, serum samples were gently mixed prior

to incubation with compound. Two μL of each CETP inhibitor (16,

anacetrapib) in dimethyl sulfoxide (2 mg/mL) were added to 4 mL of

serum, mixed well, and incubated in a 37 °C water bath for 1 h.

Lipoprotein fractions were isolated from serum by density gradient

ultracentrifugation, according to Wasan et al.,³⁶with minor

modiﬁcations. NaBr (0.36 g/mL) was added to serum containing

500 ng/mL of CETP inhibitor. Samples were mixed by gentle

inversion to facilitate the dissolution of salt. Density gradient layers

were carefully generated from the bottom of tube as follows: 3 mL

serum/CETPi/NaBr (d = 1.28), 2.8 mL NaBr/H₂O (d = 1.21), 2.8

mL NaBr/H₂O (d = 1.063), and 2.8 mL NaBr/H₂O (d = 1.006).

Tubes were kept in an ice-bath before centrifugation for 18.5 h at 15

°C, using an SW41 Rotor in an L80 ultracentrifuge (Beckman Coulter,

Brea, CA). A fraction recovery system (FRS, Beckman Coulter) was

used to collect fractions (∼0.3 mL) from the bottom of each centrifuge

tube. The density of fractions, relative to water, was measured to

determine fractions for pooling. 2-Propanol was used to recover TRL

adhering to the inner wall of the centrifuge tube. Fractions were

pooled as follows: Lipoprotein-deﬁcient serum (1.21 < d < 1.30), HDL

(1.065<d ≤ 1.21), LDL (1.019<d ≤ 1.065), and TRL (d < 1.019).

For the measurement of lipoprotein drug concentrations, serum and

LPDS were diluted 1:3 using saline. Duplicate 50 μL aliquots of

undiluted TRL, LDL, or HDL and diluted serum or LPDS were

transferred into a 96-well plate (catalogue no. 17P687, Thermo Fisher

Scientiﬁc) for the determination of CETP inhibitor concentration by

LC-MS/MS. All samples were frozen and stored at −20 °C freezer

prior to use. Extraction of CETP inhibitor from lipoprotein pools was

carried out using an acetonitrile protein precipitation method. A

Freedom EVO 150 and a Freedom EVO (Tecan, San Jose, CA) were

used in this procedure. Calibration standards, composed of a CETP

inhibitor (0−10,000 ng/mL) in 50% acetonitrile, and quality control

(QC) samples of a single concentration (50 ng/mL) were prepared.

Then 25 μL of each calibration standard or QC or test sample were

added to a 1 mL 96-well plate. Then 150 μL of acetonitrile with 50

ng/mL glyburide (ISTD) was added to each well. The mixture was

vortexed vigorously, followed by centrifugation at ∼4000 rpm for 5

min. Next, 125 μL of supernatant/well was transferred to a new 1 mL

96-well plate, followed by the addition of 50 μL of water. For each

mixed sample, a 10 μL aliquot was injected into the LC-MS/MS

system.

Tissue Drug Exposure Analysis. A ∼100 mg sample of each tissue

type (adipose, adrenal, liver) was harvested and placed in a 1.5 mL

microcentrifuge tube. All samples were ﬂash frozen and placed at −80

°C for storage. Drug concentrations were determined by LC-MS/MS.

Triton WR-1339 Treated Hamster Study. The eﬀect of acute

hypertriglyceridemia in vivo on the extent of CETP inhibition caused

by anacetrapib and 16 were investigated. Compound or vehicle was

administered once by gavage to hamsters fed a standard chow diet.

Triton WR-1339 (100 mg/kg) or saline was injected intravenously 5 h

after administration of compound. Plasma parameters were measured

3 h after injection of Triton WR-1339. Two doses for each inhibitor

were selected: around 55−75% (lower dose) or 75−80% (higher

dose) inhibition in CETP activity in the saline group. Plasma CETP

activity ex vivo was determined as follows: 50 μL of hamster plasma

(ﬁnal 80% plasma) was mixed with 10 μL of donor solution diluted

with assay buﬀer composed of 50 mM Tris-HCl (pH 7.4) containing

150 mM NaCl and 2 mM EDTA in a 96-well half-area black ﬂat-

bottom plate (catalogue no. 3686, Corning, Corning, NY).

Fluorescence intensity was measured every 15 min at an excitation

wavelength of 485 nm and an emission wavelength of 535 nm for 120

min at 37 °C using ARVO SX+L (PerkinElmer, Wellesley, MA).

Drug Distribution among Serum Lipoprotein Fractions. Human

serum samples were obtained from Uniglobe Research Corporation

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CETP activity (RFU/min) was deﬁned as the change in ﬂuorescence

intensity from 30 to 90 min.

and Boc₂O (909 mg, 4.17 mmol) was allowed to stir at 60 °C for 19 h.

The mixture was cooled and puriﬁed by silica gel column

chromatography (EtOAc/heptane = 6:94 to 60:40) to aﬀord rac-tert-

butyl (2S,4S,6R)-2-benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)-

(methoxycarbonyl)amino)-6-ethylpiperidine-1-carboxylate (3) (410

mg, 61%). MS (ESI⁺) m/z: 603 (M + H)⁺.

Chemical Syntheses. Unless otherwise speciﬁed, all solvents and

reagents were obtained from commercial sources and used without

further puriﬁcation, including intermediates 17c, 18a, and 25. All

reactions were performed under nitrogen atmosphere unless otherwise

noted. Normal-phase ﬂash chromatography was performed using

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpiperidine-1-carbox-

ylate (4). To a suspension of rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((5-

bromopyrimidin-2-yl)amino)-6-ethylpiperidine-1-carboxylate (23)

(0.952 g, 2.00 mmol) in DMF (20 mL) was added NaH (60%

dispersion in oil, 160 mg, 4.00 mmol) at 0 °C under nitrogen. The

mixture was warmed up to rt and stirred for 20 min. To the mixture

was added 3,5-bis(triﬂuoromethyl)benzyl bromide (921 mg, 3.00

mmol) at 0 °C. The reaction mixture was allowed to stir from 0 °C to

rt for 4 h and then quenched with H₂O. The products were extracted

with EtOAc, washed with brine, dried over MgSO₄, ﬁltered, and

concentrated. The obtained residue was puriﬁed by silica gel column

chromatography to aﬀord rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-

bis(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpi-

peridine-1-carboxylate (4) (962 mg, 68%). ESI-MS m/z: 701 [M +

H]⁺.

Chiral tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpiperidine-1-carbox-

ylate (5). rac-Benzyl (2S,6R)-2-benzyl-6-ethyl-4-oxopiperidine-1-car-

boxylate (cis-racemate) (rac-18c) (45 g) was puriﬁed by two

consecutive chiral chromatography runs. The ﬁrst elution with

supercritical carbon dioxide with 5% 2-propanol (isocratic) in a

Daicel Chiralcel OD-H column (30 mm × 250 mm) resulted in good

separation of the enantiomers but with undesired trans-isomer which

coeluted. The second elution with supercritical carbon dioxide with 5%

2-propanol (isocratic) with a Daicel Chiralpak AD-H (30 mm × 250

mm) gave diastereomerically pure enantiomers. The enantiomeric

excess of the separated enantiomers were determined by chiral HPLC

(Daicel Chiralpak AD-H 0.46 cm × 25 cm, n-heptane:EtOH = 80:20,

1 mL/min, 210 nM). T_r= 6.90 min (pro-distomer-18c, 98.3% ee);

8.53 min (pro-eutomer-18c, 95.9% ee).

Pro-eutomer-18c (absolute conﬁguration unknown at this stage)

was converted to compound 5 and pro-distomer-18c to its enantiomer

using the same sequence of reactions as with racemic 4 (Scheme 2,

conditions a, b, c, and d). Compound 5 was shown to be the active

enantiomer by inhibition of CETP activity in human plasma.

Compound 5 was then crystallized in tetrahydrofuran−methanol co-

solvent and the absolute conﬁguration was determined by X-ray

crystallography (Supporting Information, Figure 3) to be 2S,4S,6R.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((5-bromopyrimidin-2-yl)(3-

methoxy-5-(triﬂuoromethyl)benzyl)amino)-6-ethylpiperidine-1-car-

boxylate (6). Compound 6 was synthesized according to a procedure

similar to that described for compound 4, using 3-methoxy-5-

(triﬂuoromethyl)benzyl bromide instead of 3,5-bis(triﬂuoromethyl)-

benzyl bromide. Yield: 16.4 mg (25%). ESI-MS m/z: 663 [M + H]⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((5-bromopyrimidin-2-yl)(3-

chloro-5-(triﬂuoromethyl)benzyl)amino)-6-ethylpiperidine-1-car-

boxylate (7). Compound 7 was synthesized according to a procedure

similar to that described for compound 4, using 3-chloro-5-

(triﬂuoromethyl)benzyl bromide instead of 3,5-bis(triﬂuoromethyl)-

benzyl bromide. Yield: 49 mg (73%). ESI-MS m/z: 667 [M + H]⁺.

rac-(2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-bro-

mopyrimidin-2-yl)amino)-N,6-diethylpiperidine-1-carboxamide (8).

To a mixture of rac-N-((2S,4S,6R)-2-benzyl-6-ethylpiperidin-4-yl)-N-

(3,5-bis(triﬂuoromethyl)benzyl)-5-bromopyrimidin-2-amine hydro-

chloride (24) (50 mg, 0.078 mmol) in DMF (1 mL) were added

ethyl isocyanate (13.7 μL, 0.157 mmol) and DIPEA (41 μL, 0.235

mmol) at rt. The mixture was stirred at rt for 18 h, added to saturated

aqueous NaHCO₃, and extracted with dichloromethane. The

combined organic layers were passed through a phase separator and

concentrated in vacuo. The residue was puriﬁed by preparative HPLC

to give rac-(2S,4S,6R)-2-benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-

bromopyrimidin-2-yl)amino)-N,6-diethylpiperidine-1-carboxamide (8)

(20.5 mg, 39%). ESI-MS m/z: 672 [M + H]⁺.

1

Merck silica gel 60 (230−400 mesh). H and ¹³C NMR spectra were

1

recorded on a Bruker DRX or a Bruker AV400 (400 MHz for H and

100 MHz for ¹³C). Chemical shifts are reported in parts per million

(ppm) downﬁeld from tetramethylsilane (0.00 ppm) or residual peaks

from the corresponding solvent as an internal standard and coupling

constants (J) in Hz. Multiplicity abbreviation are as follows: s = singlet,

d = doublet, t = triplet, q = quartet, m = multiplet, app = apparent, br

= broad. LC-MS analyses were performed on an HPLC system with a

C18 column coupled to a single quad mass spectrometer with

electrospray ionization (ESI). High resolution mass spectra were

obtained with Acquity Xevo G2 QToF system with UPLC (Acquity

UPLC BEH C18 column, 2−98% acetonitrile in water both with 0.1%

formic acid) coupled to time-of-ﬂight detection after electrospray

ionization. Chemical purity of the compounds were assessed by LCMS

and conﬁrmed to have at least 95% purity by UV/ELSD, except

compound 7, which showed 90% purity by UV.

rac-tert-Butyl (2R,4R)-4-((3,5-Bis(triﬂuoromethyl)benzyl)-

(methoxycarbonyl)amino)-2-ethylpiperidine-1-carboxylate (1). To

a mixture of rac-tert-butyl 2-ethyl-4-oxopiperidine-1-carboxylate (18a)

(1 g, 4.4 mmol) and 3,5-bis(triﬂuoromethyl)benzylamine (technical

grade ∼85%, 1.38 g, 4.84 mmol) in MeOH (7.5 mL) and 1,2-

dichloroethane (7.5 mL) was added titanium tetraisopropoxide (∼0.15

mL) at rt under nitrogen. The reaction mixture was allowed to stir at

the same temperature for 6 h and quenched with saturated aqueous

NH₄Cl. The organic products were extracted twice with EtOAc,

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated to

give a crude oil (19a). The crude product was dissolved in pyridine

(10 mL), and methyl chloroformate (2.0 g, 22 mmol) was added at 0

°C. The mixture was gradually warmed up to rt and stirred for 13.5 h.

The reaction was quenched with 1 M aqueous HCl, and the products

were extracted with EtOAc, washed with brine, dried, ﬁltered, and

concentrated to a crude product which was puriﬁed by silica gel

column chromatography to aﬀord rac-tert-butyl (2R,4R)-4-((3,5-

bis(triﬂuoromethyl)benzyl)(methoxycarbonyl)amino)-2-ethylpiperi-

dine-1-carboxylate (1) (1.3 g, 58% in 2 steps). ESI-MS m/z: 513 (M +

H)⁺.

tert-Butyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)-

(methoxycarbonyl)amino)-2,6-diethylpiperidine-1-carboxylate (2).

To a mixture of tert-butyl (2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)-

benzyl)amino)-2,6-diethylpiperidine-1-carboxylate (19b) (20.8 mg,

0.043 mmol) and DMAP (10.6 mg, 0.086 mmol) in DCM (0.3

mL) was added methyl chloroformate (6.1 μL, 0.086 mmol) at rt

under nitrogen. The mixture was stirred overnight and puriﬁed by

preparative TLC (hexane/EtOAc = 2:1) to aﬀord tert-butyl

(2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)(methoxycarbonyl)-

amino)-2,6-diethylpiperidine-1-carboxylate (2) (21.2 mg, 91%). ESI-

MS m/z: 541 (M + H)⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(methoxycarbonyl)amino)-6-ethylpiperidine-1-carboxylate

(3). To a mixture of rac-benzyl (2S,4S,6R)-2-benzyl-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(methoxycarbonyl)amino)-6-ethylpiperidine-

1-carboxylate (20) (710 mg, 1.12 mmol) and PdCl₂(39 mg, 0.22

mmol) in DCM (11 mL) were added Et₃N (61.3 μL, 0.44 mmol) and

Et₃SiH (358 μL, 2.24 mmol) at rt under nitrogen. The mixture was

allowed to stir for 11.5 h. Additional Et₃SiH (1.4 mL, 8.76 mmol) was

added portionwise over 4.5 h. After stirring for a further 3 h, the

reaction mixture was concentrated and puriﬁed by silica gel column

chromatography (DCM/MeOH/NH₃(28% in H₂O) = 300:10:1) to

aﬀord rac-methyl ((2S,4S,6R)-2-benzyl-6-ethylpiperidin-4-yl)(3,5-bis-

(triﬂuoromethyl)benzyl)carbamate (560 mg, 99%). ESI-MS m/z: 503

(M + H)⁺.

A mixture of rac-methyl ((2S,4S,6R)-2-benzyl-6-ethylpiperidin-4-

yl)(3,5-bis(triﬂuoromethyl)benzyl)carbamate (560 mg, 1.11 mmol)

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rac-1-((2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-

bromopyrimidin-2-yl)amino)-6-ethylpiperidin-1-yl)butan-1-one (9).

Compound 9 was synthesized according to a procedure similar to that

described for compound 8, using butyryl chloride instead of ethyl

isocyanate. Yield: 21 mg (40%). ESI-MS m/z: 671 [M + H]⁺.

rac-Isopropyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpiperidine-1-carbox-

ylate (10). Compound 10 was synthesized according to a procedure

similar to that described for compound 8, using isopropyl

chloroformate instead of ethyl isocyanate. Yield: 20 mg (37%). ESI-

MS m/z: 687 [M + H]⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-methoxypyrimidin-2-yl)amino)-6-ethylpiperidine-1-car-

boxylate (11). A mixture of rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-

bis(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpi-

peridine-1-carboxylate (4) (114 mg, 0.16 mmol), sodium methoxide

(25 wt % in methanol, 111 μL, 0.48 mmol), and copper iodide (I) (62

mg, 0.32 mmol) in DMF (1.5 mL) was stirred at 85 °C for 2.5 h and

then allowed to cool to rt. The mixture was diluted with water and

ethyl acetate and then passed through a Celite pad. The ﬁltrate was

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The obtained residue was puriﬁed by preparative HPLC to

aﬀord rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-methoxypyrimidin-2-yl)amino)-6-ethylpiperidine-1-carboxy-

late (11) (26 mg, 25%). ESI-MS m/z: 653 [M + H]⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-morpholinopyrimidin-2-yl)amino)-6-ethylpiperidine-1-

carboxylate (12). To rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpiper-

idine-1-carboxylate (4) (0.038 mmol, 27 mg) in a ﬂask purged with N₂

were added Pd₂(dba)₃(0.0077 mmol, 7 mg), 2-(di-tert-

butylphosphino)biphenyl (0.0154 mmol, 4.6 mg), sodium tert-

butoxide (0.154 mmol, 14.8 mg), and morpholine (0.077 mmol, 7

μL). The ﬂask was purged again with N₂, toluene (0.4 mL) was added,

and the mixture was heated to 100 °C for 4 h. The mixture was cooled,

quenched with silica gel, and the slurry subjected to silica gel column

chromatography to aﬀord rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-

bis(triﬂuoromethyl)benzyl)(5-morpholinopyrimidin-2-yl)amino)-6-

ethylpiperidine-1-carboxylate (12) (13.37 mg, 50%). ESI-MS m/z: 708

[M + H]⁺.

Chiral tert-Butyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(5-morpholinopyrimidin-2-yl)amino)-6-ethylpiperidine-1-

carboxylate (13). The chiral separation of rac-12 was carried out by

chiral HPLC (Daicel Chiralpak AD-H 5 cm × 50 cm, n-hexane:i-PrOH

= 95:5, 1 mL/min, 220 nm) to aﬀord compound 13 and the distomer.

The enantiomeric excess was determined by chiral HPLC (Daicel

Chiralpak AD-H 0.46 cm × 25 cm, n-hexane:i-PrOH = 95:5, 1 mL/

min, 220 nm). T_r= 8.22 min (compound 13, >99% ee); 11.00 min

(the distomer, >99% ee)

0.38 mmol) in 4 M HCl in ethyl acetate (3 mL) was allowed to stir

from 0 °C to rt for 3 h. The reaction mixture was poured into

saturated aqueous NaHCO₃and extracted with ethyl acetate. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo to aﬀord N-(3,5-bis(triﬂuoromethyl)benzyl)-N-((2R,4r,6S)-2,6-

diethylpiperidin-4-yl)-5-morpholinopyrimidin-2-amine (195 mg). The

obtained product was used in the next step without further

puriﬁcation.

To a solution of N-(3,5-bis(triﬂuoromethyl)benzyl)-N-((2R,4r,6S)-

2,6-diethylpiperidin-4-yl)-5-morpholinopyrimidin-2-amine (95 mg,

0.17 mmol) in DMF (0.1 mL) were added methyl (1r,4r)-4-

((chlorocarbonyl)oxy)cyclohexane-1-carboxylate (26) (76 mg, 0.35

mmol) and diisopropylethylamine (68 μL, 0.52 mmol) at rt. The

reaction mixture was allowed to stir at rt for 3 h and then diluted with

DCM and water. The product was extracted with DCM, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue was puriﬁed

by silica gel column chromatography eluting with EtOAc/heptane to

aﬀord (1r,4r)-4-(methoxycarbonyl)cyclohexyl (2R,4r,6S)-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-morpholinopyrimidin-2-yl)amino)-2,6-di-

ethylpiperidine-1-carboxylate (53 mg).

To a solution of (1r,4r)-4-(methoxycarbonyl)cyclohexyl (2R,4r,6S)-

4-((3,5-bis(triﬂuoromethyl)benzyl)(5-morpholinopyrimidin-2-yl)-

amino)-2,6-diethylpiperidine-1-carboxylate (53 mg, 0.073 mmol) in

THF (0.7 mL) and MeOH (0.3 mL) was added 1 M aqueous LiOH

(0.37 mL, 0.37 mL) at rt. The reaction mixture was allowed to stir at rt

for 17 h and then quenched with 1 M aqueous HCl. The mixture was

further diluted with water. The product was extracted with DCM. The

combined organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The obtained crude product was puriﬁed by

silica gel column chromatography eluting with MeOH/DCM to aﬀord

(1r,4r)-4-(((2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-morpho-

linopyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carbonyl)oxy)-

1

cyclohexane-1-carboxylic acid (15) (33 mg, 0.046 mmol, 63%). H

NMR (400 MHz, CDCl₃) δ ppm 0. 84 (t, J = 7.45 Hz, 6H), 1.35−1.53

(m, 6H), 1.55−1.67 (m, 2H) 1.74−1.83 (m, 2H) 2.03−2.18 (m, 6H),

2.30−2.40 (m, 1H), 2.99−3.05 (m, 4H), 3.83−3.87 (m, 4H), 4.10−

4.19 (m, 2H), 4.62−4.76 (m, 2H), 4.79 (s, 2H), 7.69 (s, 2H), 7.73 (s,

1H), 8.09 (s, 2H). ESI-MS m/z: 716.4 [M + H]⁺.

(1r,4r)-4-(((2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-(1-

methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-2,6-diethylpiperi-

dine-1-carbonyl)oxy)cyclohexane-1-carboxylic Acid (16). To a

mixture of tert-butyl (2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)-

(5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-2,6-diethylpiper-

idine-1-carboxylate (29b) (24.6 g) in ethyl acetate (60 mL) was added

dropwise 4 M HCl in ethyl acetate (200 mL) over 15 min, and the

mixture was stirred at rt for 2 h. Precipitates were collected by ﬁltration

and then dissolved in a mixture of saturated NaHCO₃and EtOAc. The

organic layer was washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to aﬀord N-(3,5-bis-

(triﬂuoromethyl)benzyl)-N-((2R,4r,6S)-2,6-diethylpiperidin-4-yl)-5-

(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (18.5 g) as a colorless

solid. ¹H NMR (400 MHz, CDCl₃), δ (ppm): 0.87 (t, 6H), 1.18−1.28

(m, 2H), 1.37−1.45 (m, 4H), 1.63 (bs, 2H), 1.79 (d, 2H), 2.61−2.67

(m, 2H), 3.95 (s, 3H), 4.88 (s, 3H), 7.52 (s, 1H), 7.65 (s, 1H), 7.68 (s,

2H), 7.72 (s, 1H), 8.43 (s, 2H).

To a mixture of N-(3,5-bis(triﬂuoromethyl)benzyl)-N-((2R,4r,6S)-

2,6-diethylpiperidin-4-yl)-5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-

amine (15.0 g, 27.7 mmol) and N,N-diisopropylethylamine (38.0 mL,

218 mmol) in DMF (19 mL) was added a solution of methyl (1r,4r)-

4-((chlorocarbonyl)oxy)cyclohexane-1-carboxylate (26) (12.2 g, 55.1

mmol) in DMF (1 mL). The mixture was stirred for 1 h at room

temperature, then another portion of 26 (2.94 g, 13.3 mmol) was

added. The mixture was stirred for 1 h at rt, and then another portion

of 26 (3.13 g, 14.2 mmol) was added. After the mixture was stirred for

30 min, the reaction mixture was diluted with water and extracted with

EtOAc. The organic layer was washed with water followed by brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography (gradient from 12%

to 80% ethyl acetate in hexane) to aﬀord (1r,4r)-4-(methoxycarbonyl)-

cyclohexyl (2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-(1-meth-

Isopropyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-mor-

pholinopyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate

(14). To

a solution of isopropyl (2R,4r,6S)-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-2,6-diethyl-

piperidine-1-carboxylate (28b) (250 mg, 0.40 mmol) and morpholine

(39 μL, 0.44 mmol) in toluene (5 mL) were added Pd₂(dba)₃(36.6

mg, 0.04 mmol), 2-(di-tert-butylphosphino)biphenyl (12 mg, 0.04

mmol), and sodium tert-butoxide (42 mg, 0.44 mmol). The reaction

mixture was allowed to stir at 70 °C for 1.5 h under nitrogen and then

allowed to cool to rt. The mixture was diluted with brine and extracted

with EtOAc. The organic layer was washed with brine, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The obtained residue was

puriﬁed by silica gel column chromatography eluting with ethyl

acetate/heptane to aﬀord isopropyl (2R,4r,6S)-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-morpholinopyrimidin-2-yl)amino)-2,6-di-

ethylpiperidine-1-carboxylate (14) (126 mg, 0.20 mmol. 50%) as a

white powder. ESI-MS m/z: 632.3 [M + H]⁺.

(1r,4r)-4-(((2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-mor-

pholinopyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carbonyl)-

oxy)cyclohexane-1-carboxylic Acid (15). A solution of tert-butyl

(2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-morpholinopyrimi-

din-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (29a) (250 mg,

K

DOI: 10.1021/acs.jmedchem.7b00900

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

yl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-car-

boxylate (21.3 g). H NMR (400 MHz, CDCl₃), δ (ppm): 0.85 (t,

rac-Benzyl (2S,6R)-2-Benzyl-6-ethyl-4-oxopiperidine-1-carboxy-

late (rac-18c). To copper(I) iodide (2.29 g, 12 mmol) in a ﬂask

was added 1 M EtMgBr in THF (14 mL, 14 mmol) at −78 °C under

nitrogen followed by addition of BF₃·Et₂O (0.75 mL, 6 mmol). After

stirring for 20 min at the same temperature, a solution of rac-benzyl 2-

benzyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (17c) (1.93 g, 6

mmol) in THF (40 mL) was added portionwise at the same

temperature. The mixture was gradually warmed up to rt, stirred for 12

h, and then quenched with saturated aqueous NH₄Cl. The products

were extracted with EtOAc, washed with brine, dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. The crude product was puriﬁed by

silica gel column chromatography to aﬀord a mixture of rac-benzyl

(2S,6R)-2-benzyl-6-ethyl-4-oxopiperidine-1-carboxylate (cis-racemate)

(rac-18c) and the corresponding trans-racemate in a 10:1 ratio (1.60 g,

76%).

rac-tert-Butyl (2S,6R)-2-Benzyl-6-ethyl-4-oxopiperidine-1-carbox-

ylate (rac-18d). A suspension of a 10:1 mixture of rac-benzyl (2S,6R)-

2-benzyl-6-ethyl-4-oxopiperidine-1-carboxylate (rac-18c) (cis-race-

mate) and the trans-racemate, and 10% Pd/C (850 mg) in MeOH

(170 mL) was allowed to stir at rt under hydrogen until complete

consumption of the substrate (monitored by TLC). The reaction

mixture was ﬁltered and concentrated to give a crude product. The

crude product was puriﬁed by silica gel column chromatography

(hexane/EtOAc) to aﬀord rac-(2S,6R)-2-benzyl-6-ethylpiperidin-4-one

(3.88 g, 74%) as a yellow oil. ESI-MS m/z: 218 (M + H)⁺.

A mixture of rac-(2S,6R)-2-benzyl-6-ethylpiperidin-4-one (3.86 g,

17.8 mmol) and Boc₂O (7.84 g, 35.9 mmol) was allowed to stir at 60

°C for 2 h. The mixture was cooled to rt and puriﬁed by silica gel

column chromatography (hexane/EtOAc) to aﬀord rac-tert-butyl

(2S,6R)-2-benzyl-6-ethyl-4-oxopiperidine-1-carboxylate (rac-18d)

(5.37 g, 95%) as a pale-yellow oil. ESI-MS m/z: 262 (M − tBu + 2H)⁺.

Isopropyl (2R,6S)-2,6-Diethyl-4-oxopiperidine-1-carboxylate

(18e). Into a solution of 4-methoxypyridine (53.7 g, 0.492 mol) in

THF (1150 mL) were added isopropyl chloroformate (62 mL, 0.542

mol) and then EtMgBr (1 M in THF, 517 mL, 517 mmol) at −50 °C.

This reaction mixture was stirred for 1 h, warming toward rt. The

reaction mixture was diluted with water (100 mL) at 5 °C and then

ﬁltered. The ﬁlter cake was washed with EtOAc, and the ﬁltrate was

extracted with EtOAc. The combined organic layers were washed with

1 M aqueous HCl and brine, dried over MgSO₄, ﬁltered, and

concentrated in vacuo to aﬀord isopropyl 2-ethyl-4-oxo-3,4-dihydro-

pyridine-1(2H)-carboxylate (102.1 g), which was used in the next step

without further puriﬁcation.

To a suspension of copper(I) iodide (37 g, 195.3 mmol) in THF

(100 mL) cooled to −65 °C were added EtMgBr (195.3 mL, 195.3

mmol), BF₃·Et₂O (12.2 mL, 97.65 mmol), and isopropyl 2-ethyl-4-

oxo-3,4-dihydropyridine-1(2H)-carboxylate (22 g, 97.65 mmol) in

THF (100 mL) at −65 °C in this order. This reaction mixture was

stirred for 16 h, warming to rt. The reaction mixture was diluted with

half-saturated aqueous NH₄Cl and EtOAc and extracted with EtOAc.

The combined organic layers were dried over MgSO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography eluting with ethyl acetate/hexane to aﬀord isopropyl

(2R,6S)-2,6-diethyl-4-oxopiperidine-1-carboxylate (18e) (10 g, 30.65

mmol, 40%).

tert-Butyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)amino)-

2,6-diethylpiperidine-1-carboxylate (19b). To a mixture of tert-

butyl (2R,6S)-2,6-diethyl-4-oxopiperidine-1-carboxylate (18b) (53 mg,

0.21 mmol), 3,5-bis(triﬂuoromethyl)benzylamine (technical grade

∼85%, 61.3 mg, 0.25 mmol), and acetic acid (14 μL, 0.25 mmol) in

1,2-dichloroethane (0.5 mL) was added NaBH(OAc)₃(89 mg, 0.42

mmol) at rt under nitrogen. The mixture was stirred for 20 h and then

quenched with 1 M aqueous NaOH. The organic products were

extracted with DCM. The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated. The crude product was puriﬁed by

preparative TLC (hexane/EtOAc = 2:1) to aﬀord tert-butyl

(2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)amino)-2,6-diethylpi-

peridine-1-carboxylate (19b) (21.2 mg, 20%). ESI-MS m/z: 483 (M +

H)⁺.

1

6H), 1.24−1.27 (m, 1H), 1.34−1.46 (m, 4H), 1.47−1.65 (m, 9H),

1.74−1.84 (m, 2H), 2.01−2.19 (m, 7H), 2.26−2.33 (m, 1H), 3.67 (s,

3H), 3.95 (s, 3H), 4.09−4.20 (m, 2H), 4.61−4.67 (m, 1H), 4.76−4.82

(m, 1H), 4.86 (s, 2H), 7.53 (s, 1H), 7.66 (s, 1H), 7.70 (s, 2H), 7.75 (s,

1H), 8.43 (s, 2H).

To a mixture (1r,4r)-4-(methoxycarbonyl)cyclohexyl (2R,4r,6S)-4-

((3,5-bis(triﬂuoromethyl)benzyl)(5-(1-methyl-1H-pyrazol-4-yl)-

pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (15.4 g,

21.2 mmol) in THF (100 mL) and MeOH (100 mL) was added

aqueous 2 M NaOH (45 mL) at 0 °C. The mixture was stirred at rt for

24 h, cooled to 0 °C, and aqueous 1 M HCl (90 mL) was added

dropwise. The mixture was concentrated and extracted with EtOAc.

The organic layer was washed with water followed by brine, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The obtained solid was

dissolved in a mixture of ethyl acetate (15 mL) and diisopropyl ether

(135 mL) at 80 °C, then gradually cooled to rt. The crystalline solid

was collected by ﬁltration, washed with diisopropyl ether, and then

dried under vacuum at 50 °C to aﬀord (1r,4r)-4-(((2R,4r,6S)-4-((3,5-

bis(triﬂuoromethyl)benzyl)(5-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-

yl)amino)-2,6-diethylpiperidine-1-carbonyl)oxy)cyclohexane-1-carbox-

ylic acid (16) (10.9 g). HRMS calculated exact mass 711.30880,

1

observed 711.30908 [M + H]⁺(Δ0.4 ppm). H NMR (400 MHz,

CDCl₃), δ (ppm): 0.85 (t, 6 H) 1.36−1.70 (m, 7 H) 1.75−1.85 (m,

3H) 2.03−2.20 (m, 6 H) 2.31−2.41 (m, 1 H) 3.95 (s, 3 H) 4.11−4.22

(m, 2 H) 4.61−4.71 (m, 1 H) 4.76−4.88 (m, 1 H) 4.86 (s, 2 H) 7.53

(s, 1 H) 7.66 (s, 1 H) 7.70 (s, 2 H) 7.75 (s, 1 H) 8.43 (s, 2 H). ¹³C

NMR (100 MHz, DMSO-d₆), δ (ppm): 10.7, 12.14, 26.3, 30.4, 30.6,

31.1, 38.7, 40.9, 45.6, 49.3, 52.7, 72.2, 116, 116.3, 120.3, 123.3, 126.8,

127.5, 130.1, 135.4, 144.5, 154.5, 155.1, 159.7, 176.2. ¹⁹F NMR (376

MHz, DMSO-d₆), δ (ppm): −61.3.

rac-tert-Butyl 2-Ethyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxy-

late (17b). To a solution of 4-methoxypyridine (15.6 g, 143 mmol)

in dry THF (1 L) cooled to −35 °C was added phenyl chloroformate

(22.7 g, 144 mmol). After stirring for 1 h, 1 M ethyl magnesium

bromide in THF (150 mL, 150 mmol) was added slowly over 30 min.

The mixture was warmed to 10 °C over 2 h and then quenched with

water. The reaction mixture was extracted twice with Et₂O (1 L). The

combined organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. To a solution of the resulting colorless oil in

dry THF (500 mL) at −78 °C was added potassium tert-butoxide (64

g, 572 mmol). The reaction mixture was stirred from −78 °C to rt

overnight. The reaction mixture was diluted with Et₂O and then

quenched with ice. The organic layer was separated, washed three

times with 1.5 M aqueous NaOH, and then with brine, dried over

MgSO₄, and concentrated in vacuo to aﬀord rac-tert-butyl 2-ethyl-4-

oxo-3,4-dihydropyridine-1(2H)-carboxylate (17b) as a pale-yellow oil

1

(27.8 g, 86% yield). H NMR (400 MHz, CDCl₃), δ (ppm): 0.92 (t,

3H), 1.55 (s, 9H), 1.57−1.75 (m, 3H), 2.44 (d, 1H), 2.80 (dd, 1H),

4.55 (d, 1H), 5.26 (d, 1H), 7.74 (d, 1H). ESI-MS m/z: 226 [M + H]⁺.

tert-Butyl (2R,6S)-2,6-Diethyl-4-oxopiperidine-1-carboxylate

(18b). To copper(I) iodide (0.82 mmol, 156 mg) in a ﬂask purged

with nitrogen was added 1 M EtMgBr in THF (0.82 mmol, 0.82 mL)

at −78 °C. After stirring for 30 min, BF₃·Et₂O (0.41 mmol, 57.9 mg)

was added, and the mixture was stirred for 10 min at the same

temperature. To the suspension was added a solution of rac-tert-butyl

2-ethyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (17b) (0.41

mmol, 92.7 mg) in tetrahydrofuran (3.3 mL) at −78 °C. The mixture

was allowed to stir at the same temperature for 1.5 h and then at −40

°C for 2 h. This mixture was warmed to rt, quenched with saturated

aqueous NH₄Cl, and extracted with EtOAc. The combined organic

layers were washed with brine, dried over MgSO₄, ﬁltered,

concentrated in vacuo, and puriﬁed by silica gel column chromatog-

raphy (hexane/EtOAc = 10:1) to aﬀord tert-butyl (2R,6S)-2,6-diethyl-

1

4-oxopiperidine-1-carboxylate (18b) (50 mg, 50%). H NMR (400

MHz, CDCl₃), δ (ppm): 0.91 (t, 6H), 1.44−1.53 (m, 11H), 1.65−1.70

(m, 2H), 2.35 (dd, 2H), 2.64−2.68 (m, 2H), 4.48 (bs, 2H). ESI-MS

m/z: 200 (M⁺− tBu + 2).

L

DOI: 10.1021/acs.jmedchem.7b00900

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Journal of Medicinal Chemistry

Article

rac-Benzyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)amino)-6-ethylpiperidine-1-carboxylate (19c). To a 10:1

mixture of rac-benzyl (2S,6R)-2-benzyl-6-ethyl-4-oxopiperidine-1-

carboxylate (cis-racemate) (rac-18c) and rac-benzyl (2R,6R)-2-

benzyl-6-ethyl-4-oxopiperidine-1-carboxylate (trans-racemate) (1.60

g, 4.55 mmol), 3,5-bis(triﬂuoromethyl)benzylamine (technical grade

∼85%, 1.55 g, 6.37 mmol), and acetic acid (348 μL, 6.37 mmol) in 1,2-

dichloroethane (10 mL) was added NaBH(OAc)₃(1.93 g, 9.1 mmol)

at rt. The mixture was stirred for 13 h and then quenched with 1 M

aqueous NaOH. The organic products were extracted with DCM. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The crude product was puriﬁed by silica gel column

chromatography (DCM/EtOAc) to aﬀord rac-benzyl (2S,4S,6R)-2-

benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)amino)-6-ethylpiperidine-1-

carboxylate (19c) (990 mg, 38%). ESI-MS m/z: 579 (M + H)⁺.

rac-Benzyl (2S,4S,6R)-2-Benzyl-4-((3,5-bis(triﬂuoromethyl)-

benzyl)(methoxycarbonyl)amino)-6-ethylpiperidine-1-carboxylate

(20). To a mixture of rac-benzyl (2S,4S,6R)-2-benzyl-4-((3,5-bis-

(triﬂuoromethyl)benzyl)amino)-6-ethylpiperidine-1-carboxylate (19c)

(990 mg, 1.71 mmol) and DMAP (420 mg, 3.42 mmol) in DCM (20

mL) was added methyl chloroformate (304 μL, 4.28 mmol) at rt under

nitrogen, The mixture was stirred for 3 h and quenched with brine.

The organic layer was separated and puriﬁed by silica gel column

chromatography (DCM/EtOAc) to aﬀord rac-benzyl (2S,4S,6R)-2-

benzyl-4-((3,5-bis(triﬂuoromethyl)benzyl)(methoxycarbonyl)amino)-

6-ethylpiperidine-1-carboxylate (20) (710 mg, 65%). ESI-MS m/z:

637.0 (M + H)⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-(benzylamino)-6-ethylpiperi-

dine-1-carboxylate (21). A mixture of rac-tert-butyl (2S,6R)-2-benzyl-

6-ethyl-4-oxopiperidine-1-carboxylate (rac-18d) (5.36 g, 16.8 mmol),

benzylamine (2.00 mL, 18.3 mmol) and BF₃·Et₂O (10 μL) in toluene

(200 mL) was reﬂuxed in a Dean−Stark apparatus for 20 min. The

reaction mixture was cooled to rt and concentrated to give a crude oil.

The crude product was dissolved in MeOH (200 mL), and then

NaBH₄(393 mg, 10.4 mmol) was added portionwise at rt. The

reaction mixture was quenched with saturated aqueous NH₄Cl and

then concentrated. The residue was diluted with water and extracted

with dichloromethane. The organic layer was washed with saturated

aqueous NaHCO₃, dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed by silica gel column chromatography

(DCM/MeOH) to aﬀord rac-tert-butyl (2S,4S,6R)-2-benzyl-4-(benzy-

lamino)-6-ethylpiperidine-1-carboxylate (21) (4.18 g, 61%) as a pale-

yellow oil. ESI-MS m/z 409: (M + H)⁺.

rac-tert-Butyl (2S,4S,6R)-4-Amino-2-benzyl-6-ethylpiperidine-1-

carboxylate (22). A mixture of rac-tert-butyl (2S,4S,6R)-2-benzyl-4-

(benzylamino)-6-ethylpiperidine-1-carboxylate (21) (2.89 g, 7 mmol)

and 10% Pd/C (100 mg, 0.09 mmol) in EtOH (80 mL) was allowed

to stir at 40 °C under hydrogen until complete consumption of the

substrate. The reaction mixture was ﬁltered, and the ﬁltrate was

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (DCM/MeOH) to aﬀord rac-tert-butyl (2S,4S,6R)-4-

amino-2-benzyl-6-ethylpiperidine-1-carboxylate (22) (1.85 g, 83%).

ESI-MS m/z: 319 (M + H)⁺.

rac-tert-Butyl (2S,4S,6R)-2-Benzyl-4-((5-bromopyrimidin-2-yl)-

amino)-6-ethylpiperidine-1-carboxylate (23). A mixture of rac-tert-

butyl (2S,4S,6R)-4-amino-2-benzyl-6-ethylpiperidine-1-carboxylate

(22) (1.75 g, 5.5 mmol), 5-bromo-2-chloropyridine (1.6 g, 8.25

mmol), and DIPEA (1.92 mL, 11 mmol) in DMF (16.5 mL) was

heated at 120 °C for 4 h under nitrogen. The mixture was cooled to rt

and diluted with brine. The products were extracted with EtOAc. The

organic layer was washed with saturated aqueous NH₄Cl and water

and then concentrated to give a brown solid. The solid was triturated

with diisopropyl ether to aﬀord rac-tert-butyl (2S,4S,6R)-2-benzyl-4-

((5-bromopyrimidin-2-yl)amino)-6-ethylpiperidine-1-carboxylate as a

colorless solid (23) (1.52 g, 58%).

the obtained residue was added Et₂O, and the solid was collected by

ﬁltration to give rac-N-((2S,4S,6R)-2-benzyl-6-ethylpiperidin-4-yl)-N-

(3,5-bis(triﬂuoromethyl)benzyl)-5-bromopyrimidin-2-amine hydro-

chloride (24) (1.2 mmol, 771 mg, 94% as HCl salt). ESI-MS m/z:

601 [M + H]⁺.

Methyl (1r,4r)-4-Hydroxycyclohexane-1-carboxylate (25). To a

solution of (1r,4r)-4-hydroxycyclohexane-1-carboxylic acid (2.12 g,

14.7 mmol) in DMF (15 mL) were added potassium carbonate (2.44

g, 17.6 mmol) and methyl iodide (1.1 mL, 17.6 mmol) at 0 °C. The

reaction mixture was allowed to stir from 0 °C to rt for 2 h. The

reaction mixture was diluted with water and EtOAc. The organic layer

was separated, dried over Na₂SO₄, ﬁltered, and concentrated in vacuo

to aﬀord methyl (1r,4r)-4-hydroxycyclohexane-1-carboxylate (25)

(1.62 g, 10.29 mmol, 70%). ¹H NMR (400 MHz, CDCl3), δ

(ppm): 1.23−1.33 (m, 2H), 1.48−1.56 (m, 2H), 1.99−2.05 (m, 4H),

2.22−2.30 (m, 1H), 3.58−3.64 (m, 1H), 3.67 (s, 3H).

Methyl (1r,4r)-4-((Chlorocarbonyl)oxy)cyclohexane-1-carboxy-

late (26). To a solution of triphosgene (1.52 g, 5.12 mmol) in

DCM (15 mL) were added a solution of pyridine (870 μL, 10.8

mmol) in DCM (5 mL) and methyl (1r,4r)-4-hydroxycyclohexane-1-

carboxylate (25) (1.62 g, 10.29 mmol) at 0 °C. The reaction mixture

was allowed to stir from 0 °C to rt for 3 h. The reaction mixture was

diluted with aqueous NH₄Cl. The organic layer was separated, dried

over Na₂SO₄, ﬁltered, and concentrated in vacuo to aﬀord methyl

(1r,4r)-4-((chlorocarbonyl)oxy)cyclohexane-1-carboxylate (26) (1.77

1

g, 3.63 mmol, 78%). H NMR (400 MHz, CDCl3), δ (ppm): 1.53−

1.64 (m, 4H), 2.05−2.18 (m, 4H), 2.30−2.37 (m, 1H), 3.68 (m, 3H),

4.77−4.83 (m, 1H).

tert-Butyl (2R,4r,6S)-4-((5-Bromopyrimidin-2-yl)amino)-2,6-dieth-

ylpiperidine-1-carboxylate (27a). To a mixture of tert-butyl (2R,6S)-

2,6-diethyl-4-oxopiperidine-1-carboxylate (18b) (11.1 g, 44 mmol) in

MeOH (150 mL) were added benzylamine (7.1 mL, 65 mmol) and

titanium tetraisopropoxide (26 mL, 87 mmol) at 0 °C, and the mixture

was stirred overnight at rt. After addition of sodium borohydride (2.5

g, 65 mmol), the mixture was stirred for an additional 1 h at rt. H₂O

and EtOAc were added to the mixture, and the resulting precipitate

was removed by ﬁltration. The ﬁltrate was washed sequentially with

saturated aqueous NaHCO₃and brine. The product was extracted

from the aqueous layer with EtOAc, and the combined organic layer

was dried over MgSO₄and concentrated under reduced pressure to

obtain tert-butyl (2R,4r,6S)-4-(benzylamino)-2,6-diethylpiperidine-1-

carboxylate as a clear oil (13.9 g, 92%). The diastereomeric ratio was

typically 10:1 (cis:trans). ¹H NMR (400 MHz, CDCl₃), δ (ppm): 0.87

(t, 6H), 1.17−1.23 (m, 2H), 1.45 (s, 9H), 1.46−1.50 (m, 3H), 1.73−

1.80 (m, 2H), 2.22−2.28 (m, 2H), 2.67−2.71 (m, 1H), 3.78 (s, 2H),

3.98−4.03 (m, 2H).

A mixture of tert-butyl (2R,4r,6S)-4-(benzylamino)-2,6-diethylpi-

peridine-1-carboxylate (4.0 g, 11.4 mmol) and 10% Pd/C (400 mg) in

EtOH (80 mL) was stirred for 5 h at 55 °C under hydrogen. After

removal of the catalyst by ﬁltration through a pad of Celite, the ﬁltrate

was concentrated in vacuo to aﬀord tert-butyl (2R,4r,6S)-4-amino-2,6-

diethylpiperidine-1-carboxylate as clear oil (2.9 g, 99%), which was

used in the next step without further puriﬁcation. ¹H NMR (400 MHz,

CDCl₃), δ (ppm): 0.90 (t, 6H), 1.06−1.12 (m, 3H), 1.38−1.50 (m,

12H), 1.71−1.77 (m, 2H), 2.15−2.21 (m, 2H), 2.84 (m, 1H), 4.00−

4.04 (m, 2H).

A mixture of tert-butyl (2R,4r,6S)-4-amino-2,6-diethylpiperidine-1-

carboxylate (21.1 g, 82.3 mmol), 5-bromo-2-chloropyrimidine (17.1 g,

88.5 mmol), and diisopropylethylamine (28.0 mL, 161 mmol) in DMF

(220 mL) was stirred at 120 °C for 3.5 h. After cooling to rt, the

mixture was diluted with water (300 mL) and extracted with EtOAc

(200 mL × 2). The combined organic layers were washed with water

and brine, dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. To

the obtained solid was added diisopropyl ether (65 mL), and the solid

was dissolved under reﬂux. The resulting solution was cooled to rt,

then n-hexane (65 mL) was added. Precipitates were collected by

ﬁltration and washed with diisopropyl ether to aﬀord tert-butyl

(2R,4r,6S)-4-((5-bromopyrimidin-2-yl)amino)-2,6-diethylpiperidine-

rac-N-((2S,4S,6R)-2-Benzyl-6-ethylpiperidin-4-yl)-N-(3,5-bis-

(triﬂuoromethyl)benzyl)-5-bromopyrimidin-2-amine Hydrochloride

(24). A mixture of rac-tert-butyl (2S,4S,6R)-2-benzyl-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-6-ethylpiper-

idine-1-carboxylate (4) (1.29 mmol, 900 mg) and a solution of 4 M

HCl in EtOAc was stirred for 4 h at rt, then concentrated in vacuo. To

1

1-carboxylate (27a) (12.4 g, 36%). H NMR (400 MHz, CDCl₃), δ

(ppm): 0.89 (t, 6H), 1.19−1.27 (m, 2H), 1.39−1.44 (m, 3H), 1.45 (s,

M

DOI: 10.1021/acs.jmedchem.7b00900

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

9H), 1.73−1.84 (m, 2H), 2.39−2.45 (m, 2H), 3.83−3.92 (m, 1H),

4.09−4.17 (m, 2H), 4.94 (d, 1H), 8.27 (s, 2H).

Isopropyl (2R,4r,6S)-4-((5-Bromopyrimidin-2-yl)amino)-2,6-dieth-

ylpiperidine-1-carboxylate (27b). Compound 27b was synthesized

according to a procedure similar to that described for compound 23,

using compound 18e in 30% yield in three steps.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b00900.

■

S

tert-Butyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-bromo-

pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (28a).

To a solution of tert-butyl (2R,4r,6S)-4-((5-bromopyrimidin-2-yl)-

amino)-2,6-diethylpiperidine-1-carboxylate (27a) (6.05 g, 14.6 mmol)

in DMF (60 mL) was added NaH (60% in oil, 0.70 g, 17.6 mmol) at 0

°C. After the solution was stirred for 1 h, 3,5-bis(triﬂuoromethyl)-

benzyl bromide (3.23 mL, 17.6 mmol) was added, and the reaction

mixture was warmed to rt and stirred for 20 min. After the reaction

was quenched with H₂O at 0 °C, the mixture was extracted with ethyl

acetate, washed with brine, dried over Na₂SO₄, and concentrated in

vacuo. The obtained residue was puriﬁed by silica gel column

chromatography to aﬀord tert-butyl (2R,4r,6S)-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-2,6-diethyl-

Aldosterone and pressor eﬀects of torcetrapib, anace-

trapib, and compound 16; single compound X-ray

crystallography for the conﬁrmation of relative stereo-

chemistry of compound 16 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*For K.Y.: phone, 617-871-8826; E-mail, ken.yamada@

novartis.com.

*For M.M.: E-mail, muneto.mogi@novartis.com.

ORCID

Ken Yamada: 0000-0001-7899-6192

■

1

piperidine-1-carboxylate (28a) (8.02 g, 86%) as yellow oil. H NMR

(400 MHz, CDCl₃), δ (ppm): 0.84 (t, 6H), 1.37−1.56 (m, 5H), 1.47

(s, 9H), 1.73−1.84 (m, 2H), 2.09−2.15 (m, 2H), 4.10 (p, 2H), 4.63−

4.74 (m, 1H), 4.81 (s, 2H), 7.66 (s, 2H), 7.75 (s, 1H), 8.32 (s, 2H).

ESI-MS m/z: 639.2 [M + H]⁺.

Present Addresses

^∥For W.H., A.I., H.M., and I.U.: Novartis Pharma K.K., Minato-

ku, Tokyo 1050-6333, Japan.

^⊥For Y.I.: Janssen Pharmaceutical K.K., Shinjuku, Tokyo 162-

0805, Japan.

Isopropyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-bromo-

pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (28b).

Compound 28b was synthesized according to a procedure similar to

that described for compound 28a, using compound 27b in 74% yield.

tert-Butyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-mor-

pholinopyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate

(29a). To a solution of tert-butyl (2R,4r,6S)-4-((3,5-bis-

(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-2,6-diethyl-

piperidine-1-carboxylate 28a (300 mg, 0.47 mmol) and morpholine

(82 μL, 0.94 mmol) in toluene (5 mL) were added Pd₂(dba)₃(86 mg,

0.09 mmol), 2-(di-t-butylphosphiphino)biphenyl (56 mg, 0.19 mmol),

and t-BuONa (180 mg, 1.88 mmol). The reaction mixture was allowed

to stir at 100 °C for 3 h under nitrogen and then allowed to cool to rt.

The mixture was diluted with brine and extracted with EtOAc. The

organic layer was washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The obtained residue was puriﬁed by silica gel

column chromatography eluting with EtOAc/heptane to aﬀord tert-

butyl (2R,4r,6S)-4-((3,5-bis(triﬂuoromethyl)benzyl)(5-morpholino-

pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (29a) (250

mg, 0.38 mmol. 81%) as a white powder. ESI-MS m/z: 646 [M + H]⁺.

tert-Butyl (2R,4r,6S)-4-((3,5-Bis(triﬂuoromethyl)benzyl)(5-(1-

methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-2,6-diethylpiperi-

dine-1-carboxylate (29b). A mixture of tert-butyl (2R,4r,6S)-4-((3,5-

bis(triﬂuoromethyl)benzyl)(5-bromopyrimidin-2-yl)amino)-2,6-dieth-

ylpiperidine-1-carboxylate (28a) (28.2 g, 44.0 mmol), 1-methylpyr-

azole-4-boronic acid pinacol ester (11.0 g, 53.0 mmol), and Na₂CO₃

(6.99 g, 66.0 mmol) in dimethoxyethane (340 mL) and H₂O (33 mL)

was purged with N₂, then Pd(PPh₃)₄(2.54 g, 2.2 mmol) was added.

The mixture was stirred at 90 °C for 4 h, and then additional amounts

of Pd(PPh₃)₄(2.00 g, 1.7 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-

[1,3,2]dioxaborolan-2-yl)-1H-pyrazole (3.68 g, 18.0 mmol), Na₂CO₃

(2.39 g, 23.0 mmol), and H₂O (11 mL) were added. The mixture was

stirred at 90 °C for a further 16 h, then cooled to rt, and H₂O (400

mL) was added. The product was extracted with EtOAc (200 mL × 2),

and the combined organic layers were washed with water followed by

brine, dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (gradient

from 12% to 60% EtOAc in hexane) to aﬀord tert-butyl (2R,4r,6S)-4-

((3,5-bis(triﬂuoromethyl)benzyl)(5-(1-methyl-1H-pyrazol-4-yl)-

pyrimidin-2-yl)amino)-2,6-diethylpiperidine-1-carboxylate (29b) (ca.

25 g), which was used for the next step without further puriﬁcation. ¹H

NMR (400 MHz, CDCl₃), δ (ppm): 0.84 (t, 6H), 1.38−1.55 (m, 5H),

1.46 (s, 9H), 1.74−1.85 (m, 2H), 2.11−2.19 (m, 2H), 3.90 (s, 3H),

4.08−4.16 (m, 2H), 4.75−4.87 (m, 3H), 7.52 (s, 1H), 7.65 (s, 1H),

7.71 (s, 2H), 7.76 (s, 1H), 8.42 (s, 2H). ESI-MS m/z: 641.6 [M + H]⁺.

^#For D.L.: Insmed, Inc., Bridgewater, New Jersey 07034,

United States.

^∇For K.N.: Mochida Pharmaceutical Co., Ltd., Shinjuku-ku,

Tokyo 160-8515, Japan.

^○For O.O.: Asahi Kasei Pharma Corporation, Izunokuni-shi,

Shizuoka 410-2321, Japan.

^◆For D.F.R.: Rigel BioPharma Consulting LLC, Berkeley

Heights, New Jersey 07922, United States.

Author Contributions

K. Yamada and M.B. prepared the manuscript. M.M., K.

Yamada, O.O., K. Yasoshima, T.K., I.U., Y.I., and H.I. designed

and synthesized the compounds, K.N. and G.Z. developed the

human plasma CETP assay, A.I. and H.M. developed and

performed hamster PD studies, W.H. developed and performed

CYP assays, M.P. and D.L. developed and performed

aldosterone secretion assay in H295R cells, M.P. performed

plasma lipoprotein distribution studies, G.L. performed

pharmacokinetic and tissue distribution studies, D.F.R.

developed and supervised cannulated SD rat studies, and

M.M., H.M., and M.B. directed the discovery program.

Notes

The authors declare the following competing ﬁnancial

interest(s): All research was fully funded by Novartis.

ACKNOWLEDGMENTS

■

We acknowledge Dr. J. M. Elliott for careful review and advice

on this manuscript, Dr. M. Kato for modeling support, Dr. H.

Qin for chemical synthesis support, Drs. T. Miyake, E.

Kawahara, M. Kishida, J. Levell, T. Fujita, K. Konishi, L.

McQuire, and R. Sun for the insights from exploration of

alternative chemotypes, T. Hayashi and S. Raghavan for

performing human plasma CETP assays, Y. Arai and Y.

Murakami for performing hamster PD studies, Dr. H. Fukaya

for supporting PK studies, Dr. L. Shah for formulation support,

Drs. A. Jeng and K. DiPetrillo for the supporting H295R

aldosterone secretion assay, W. Chen, F. Fu, M. Beil, and J. Liu

for conducting the cannulated SD rat studies, J. Leung-Chu and

C.-W. Hu for ex vivo analyses of PAC and PCC from the rat

studies, P. Piechon, Drs. I. Dix and B. Wagner for X-ray

N

DOI: 10.1021/acs.jmedchem.7b00900

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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crystallography of compound 16, and Dr. S. Skolnik and L.

Yang for determining experimental Log D values. We also

acknowledge Drs. G. Iwasaki and A. B. Jones for their guidance

on medicinal chemistry strategy.

ABBREVIATIONS USED

■

CETP, cholesteryl ester transfer protein; LDL, low density

lipoprotein; HDL, high density lipoprotein; CYP, cytochrome

P450; CHD, coronary heart disease; HMGCoA, 3-hydroxy-3-

methylglutaryl-coenzyme A; TG, triglyceride; VLDL, very low

density lipoprotein; TG, triglycerides; LipE, lipophilic eﬃ-

ciency; LPDS, lipoprotein deﬁcient serum; TRL, triglyceride

rich lipoproteins; SD rats, Sprague−Dawley rats; H295R cells,

human adrenal corticocarcinoma cells

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Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic Plasma

DOI: 10.1021/acs.jmedchem.7b00900

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