Heteroaromatic and aniline derivatives of piperidines as potent ligands for vesicular acetylcholine transporter

Article

pubs.acs.org/jmc

Heteroaromatic and Aniline Derivatives of Piperidines As Potent

Ligands for Vesicular Acetylcholine Transporter

Junfeng Li,^†Xiang Zhang,^†Zhanbin Zhang,^†Prashanth K. Padakanti,^†Hongjun Jin,^†Jinquan Cui,^†

Aixiao Li,^†Dexing Zeng,^†Nigam P. Rath,^‡Hubert Flores,^§Joel S. Perlmutter,^†,§Stanley M. Parsons,^∥

and Zhude Tu*^,†

^†Department of Radiology, Washington University School of Medicine, 510 South Kingshighway Boulevard, St. Louis, Missouri

63110, United States

^‡Department of Chemistry and Biochemistry and Center for Nanoscience, University of MissouriSt. Louis, One University

Boulevard, St. Louis, Missouri 63121, United States

^§Department of Neurology, Washington University School of Medicine, 660 South Euclid, St. Louis, Missouri 63110, United States

^∥Department of Chemistry and Biochemistry and the Graduate Program in Biomolecular Science and Engineering, University of

California, Santa Barbara, California 93106, United States

S

* Supporting Information

ABSTRACT: To identify suitable lipophilic compounds having

high potency and selectivity for vesicular acetylcholine transporter

(VAChT), a heteroaromatic ring or a phenyl group was introduced

into the carbonyl-containing scaﬀold for VAChT ligands. Twenty

new compounds with ALogD values between 0.53 and 3.2 were

synthesized, and their in vitro binding aﬃnities were assayed. Six of

them (19a, 19e, 19g, 19k, and 24a−b) displayed high aﬃnity for

VAChT (K_i= 0.93−18 nM for racemates) and moderate to high

selectivity for VAChT over σ₁and σ₂receptors (K_i= 44−4400-fold).

These compounds have a methyl or a ﬂuoro substitution that

provides the position for incorporating PET radioisotopes C-11 or

F-18. Compound (−)-[¹¹C]24b (K_i= 0.78 nM for VAChT, 1200-

fold over σ receptors) was successfully synthesized and evaluated in

vivo in rats and nonhuman primates. The data revealed that (−)-[¹¹C]24b has highest binding in striatum and has favorable

pharmacokinetics in the brain.

of cognitive dysfunction and monitoring the eﬃcacy of

cholinergic therapies for dementia in neurodegenerative

disorders.

INTRODUCTION

■

Dementia is a clinical syndrome characteristic of loss or decline

in memory and other cognitive abilities that result in functional

impairment in the elderly. It is a global public health

problem.^1,2The prevalence in the USA has been recently

estimated at 5.4 million individuals, including 5.2 million over

the age of 65 and 200,000 individuals under the age of 65.¹

Increasing lifespan of people will lead to increased number of

patients with dementia. The severity of dementia in neuro-

degenerative diseases is linked to loss of cholinergic neurons

and synapses in the central nervous system (CNS). The use of

¹¹C-labeled Pittsburgh Compound-B ([¹¹C]PIB) to assess β-

amyloid plaques and diagnose Alzheimer’s disease (AD) was a

signiﬁcant breakthrough.³However, [¹¹C]PIB cannot speciﬁ-

cally assess the loss of cholinergic neurons and synapses in the

brain, which correlates to severity of cognitive dysfunction in

AD.⁴⁻⁹For Parkinson’s disease associated with dementia

(PDD), the cholinergic deﬁcit in cortical regions is more

severe than for nondemented patients with Parkinson’s disease

(PD).¹⁰A PET tracer that can assess the loss of cholinergic

synapses would provide a useful tool for assessing the severity

Vesicular acetylcholine transporter (VAChT) and choline

acetyltransferase (ChAT) are essential for a cholinergic

neuron.¹¹VAChT is localized in cholinergic terminals, and it

transports acetylcholine (ACh) from the cytoplasm into the

synaptic vesicles. Anti-ChAT preferentially stains cell bodies,

whereas anti-VAChT preferentially stains nerve terminals.^4,12

Anti-ChAT is more useful for monitoring the death of

cholinergic cells, whereas anti-VAChT is more useful for

monitoring changes in the density of cholinergic terminals. It is

widely accepted that VAChT is a reliable biomarker to study

cholinergic function in the brain. Currently, (−)-5-[¹²³I]-

iodobenzovesamicol ([¹²³I]IBVM) is the only radiotracer

used for imaging VAChT levels in living human brain using

single-photon emission computed tomography (SPECT). The

relative distribution for speciﬁc binding of [¹²³I]IBVM in the

Received: May 5, 2013

Published: June 26, 2013

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Figure 1. Structures of VAChT compounds.

human brain corresponds well with post-mortem results

reported for ChAT.^13,14When [¹²³I]IBVM was used to assess

cholinergic deﬁciency in patients with dementia, it was found

that PDD and AD patients have globally reduced cortical

binding.¹⁵In addition, a signiﬁcant decrease in [¹²³I]IBVM

binding (47−62%) in cingulate cortex and parahippocampal-

amygdaloid in AD subjects compared to control patients has

been observed.¹⁶However, the slow binding kinetics of

fully radiosynthesized and used to conduct in vivo evaluation in

rats and monkeys; the initial results were very promising.¹⁹The

possibility that (−)-[¹⁸F]7 can serve as a clinical PET tracer for

quantifying the level of VAChT in vivo is under investigation.

The current manuscript focuses on: (1) Optimizing the

structures of this new class of VAChT ligand to identify highly

potent ligands having lipophilicity suitable to eﬃciently cross

the BBB. (2) Separate the enantiomers of the optimal

compound, and radiosynthesize with carbon-11 PET isotope.

(3) In vivo evaluate the new carbon-11 radiotracer in rodent

and nonhuman primate. The strategies to achieve optimization

include: (1) replacing the thiophenyl group in (1-((2S,3S)-3-

hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)-

(thiophen-2-yl)methanone 9 with N-methyl pyrrole⁴(the

methyl group provides a position for conveniently labeling

with carbon-11 isotope), (2) replacing the 4-ﬂuorophenyl

group in 7⁴with pyridine, substituted pyridines, and pyrroles

and optimizing the substitution so that radiolabeling with

carbon-11 or ﬂuorine-18 can be achieved, and (3) converting

the primary amino group of compound 6 to monomethyl

amino or dimethyl amino, which will provide access for

radiolabeling with carbon-11. This investigation was inspired

by: (1) the observation that a series of carbonyl group

containing analogues have high aﬃnity for VAChT and low

aﬃnity for σ receptors,^4,19,34(2) our in vivo validation of

(−)-[¹⁸F]7¹⁹and its analogue (−)-[¹¹C]8,³⁵which showed

high binding in the striatum, the VAChT enriched regions in

the brain, and (3) the high demand for a clinically suitable PET

probe for investigating the correlation between loss of cognitive

function and loss of cholinergic synapses, which will help

improve the early diagnosis of dementia and monitor the

therapeutic eﬃcacy of treating Alzheimer’s and other neuro-

degenerative diseases.

[

¹²³I]IBVM requires scanning for approximately 6 h post-

injection, which can be stressful for patients. Positron emission

tomography (PET) imaging will be able to carry out scans with

higher sensitivity and spatial resolution (3−5 mm) compared to

SPECT (10 mm).^17,18The demand to provide higher accuracy

in clinical imaging of VAChT levels in humans makes the

identiﬁcation of a PET tracer for VAChT very important. To

date, this goal has not been achieved because of the lack of

suitable PET radiotracers.

In eﬀorts to develop a PET tracer for VAChT, investigators

have put tremendous eﬀort into optimizing the structure of

vesamicol analogues with the goal of identifying highly potent

and selective ligands.^4,19−27Among various radioligands

developed, only a small number have been evaluated in vivo

in nonhuman primates and humans.^19,27−29Despite promising

in vitro, ex vivo, and initial in vivo studies, most of the ligands

are unsuitable for clinical use due to poor selectivity over σ

receptors in brain, low extraction from the blood, slow brain

kinetics, or fast metabolism. Among the physicochemical

properties of ligands, lipophilicity is a one of the key properties

that plays a pivotal role in absorption, distribution, metabolism,

and elimination of ligands.³⁰For central nervous system drugs,

it was found that the blood−brain barrier (BBB) penetration is

optimal with the ALogD values in the range of 1.5−3.0, with a

mean value of 2.5.³¹⁻³³Although other properties of

compounds aﬀect the BBB penetration, those ligands with

moderate lipophilicity often exhibit highest brain uptake.³⁰

Highly lipophilic radiotracers are usually cleared slowly from

the brain.

Our group has reported a new class of VAChT inhibitors

containing a carbonyl group attached to the 4-position of the

piperidine ring and discussed the structure−activity relationship

(SAR) of this new class of compounds.^4,19,34,35Among them

(as shown in Figure 1), compounds 5, 7, and 8 displayed high

potencies and good selectivity for VAChT in vitro.^4,19In

particular, a ﬂuorine-18 labeled version of (−)-2-hydroxy-3-(4-

(4-ﬂuorobenzoyl)piperidino)tetralin, (−)-[¹⁸F]7, was success-

RESULTS AND DISCUSSION

■

Chemistry. The synthesis of a new series of vesicular

acetylcholine transporter inhibitors was accomplished accord-

ing to Schemes 1−5. t-Boc-protected piperidine-4-carboxylic

acid (10) was treated with 1,1′-carbonyldiimidazole (CDI) and

N,O-dimethylhydroxylamine to aﬀord Weinreb amide 11,

which served as a versatile intermediate for synthesizing

piperidines bearing a substituted heteroaromatic carbonyl

group at the 4-position. Bromo substituted heteroaromatic

compounds were lithiated with n-butyllithium (n-BuLi) and

reacted with Weinreb amide to aﬀord intermediates 16a−i.

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a

Scheme 1

a

Reagents and conditions: (a) CDI, N,O-dimethylhydrocylamine hydrochloride, NEt₃, CH₂Cl₂, rt, overnight; (b) ArLi, THF; (c) H₂SO₄/NaNO₂,

0−5 °C, 2 h; (d) CH₃l, K₂CO₃, acetone, 80 °C, 4 h; (e) 11, BuLi, THF, −78 °C; (f) TFA, CH₂Cl₂, rt, 4 h; (g) NEt₃, EtOH, 75 °C, 36 h.

When synthesizing N-methyl pyrrole derivative 16a, via

treatment of N-methylpyrrole with n-BuLi³⁶followed by

compound 11, it was observed that the yield was much higher

when the reaction vessel was precooled to −78 °C while adding

n-BuLi. To synthesize N-methyl pyridine derivatives 16h and

16i, commercially available 4- or 6-bromine substituted pyridin-

2-amines 12h and 12i were ﬁrst converted to 2-hydroxypyr-

idines 13h and 13i or 2-pyridones 14h and 14i with sodium

nitrite in sulfuric acid,^37,38followed by methylation using

iodomethane in acetone to form bromo substituted N-methyl-

2-pyridones 15h and 15i as major products.³⁹These pyridones

reacted with 11 to aﬀord the corresponding carbonyl group

containing intermediates 16h and 16i. Triﬂuoroacetic acid

(TFA) was used to remove the t-Boc group in 16a−i to aﬀord

the key intermediates 17a−i. Treatment of 17a−i with epoxide

1a,2,7,7a-tetrahydronaphtho[2,3-b]oxirene (18)⁴aﬀorded the

target compounds 19a−i as shown in Scheme 1. To conﬁrm

whether the methyl group was on the N- or O-atom of 19h and

19i, the X-ray crystal structure of the oxalate salt of 19i was

obtained as shown in Figure 2. The X-ray crystal structure

revealed that the methyl group is on the nitrogen.

acetylcholine transporter inhibitors,^19,40it proved challenging

when synthesizing 19j and 19k due to the low solubility of

substituted heteroaromatic piperidines in commonly used

solvents such as methanol, ethanol, dichloromethane, DMF,

and DMSO. Thus, an alternative approach was followed in

which the t-Boc group in compound 11 was removed ﬁrst, and

the resulting piperidine was treated with 18 to aﬀord

compound 20. The free hydroxyl group of compound 20 was

protected with the tert-butyldimethylsilyl group by reacting

with tert-butyldimethylsilyl chloride to aﬀord compound 21.

Commercially available 5-bromo-2-ﬂuoropyridine (17j) or 3-

bromo-2-ﬂuoropyridine (17k) was treated with n-BuLi in THF

followed by compound 21, and further removal of TBDMS

group aﬀorded target compounds 19j and 19k as shown in

Scheme 2.

Compounds 24a and 24b were synthesized as depicted in

Scheme 3. Compound 6 was synthesized following the reported

procedure.¹⁹Direct dimethylation of the aromatic amine in

compound 6 with iodomethane in the presence of NaH

aﬀorded compound 24a very easily. However, the synthesis of

the N-monomethylaniline analogue 24b was more complicated.

The free hydroxyl group and the primary aniline group of 6

were protected by treating with acetic anhydride to form

Although direct condensation of substituted piperidines and

epoxide 18 was a common procedure for synthesizing vesicular

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To make aminobenzovesamicol (ABV) analogues containing

substituted heteroaromatic carbonyl groups, namely 30a, 30c−

e and 31a, the corresponding piperidine precursors 17a, 17c−e

containing substituted heteroaromatic carbonyl groups were

reacted with 2,2,2-triﬂuoro-N-(1a,2,7,7a-tetrahydronaphtho-

[2,3-b]oxiren-3-yl)acetamide (29), which was synthesized by

following the literature protocol.⁴The triﬂuoroacetyl protection

group was then removed via hydrolysis in the presence of

sodium hydroxide to aﬀord the regioisomers 30a, 30c−e and

31a as shown in Scheme 5.

In vitro binding studies revealed that 24b was highly potent.

Therefore, the (−)-24b and (+)-24b were obtained by

separating the enantiomers on HPLC using chiralcel OD

column. The precursor (−)-33 for the radiolabeling of

(−)-[¹¹C]24b was synthesized as shown in Scheme 6. Brieﬂy,

the enantiomeric separation of 6 was performed on chiral

HPLC using Chiralcel OD column to give (+)-6 and (−)-6.

The (−)-6 isomer was treated with Boc anhydride in the

presence of triethylamine to give the tri-Boc protected

intermediate (−)-32. 4-Dimethylaminopyridine (DMAP) was

used in stoichiometric amount in this reaction. The tri-Boc

protected compound upon treatment with potassium carbonate

in methanol under reﬂux selectively deprotected one Boc group

on the aniline nitrogen to give the precursor (−)-33, which was

used for radiosynthesis of (−)-[¹¹C]24b.

Figure 2. Chemical structure and X-ray crystal structure of 19i.

diacetylated compound 22.⁴¹N-Methylation of aromatic amide

22 gave intermediate 23, which upon hydrolysis aﬀorded the

target compound 24b.

The radiosynthesis of (−)-[¹¹C]24b was successfully

accomplished in two steps from the precursor as shown in

Scheme 7. The precursor was ﬁrst reacted with [¹¹C]CH₃I to

obtained the intermediate [¹¹C]methylated Boc protected

compound. This intermediate was then treated with triﬂuoro-

acetic acid in situ to obtain the Boc deprotected product

(−)-[¹¹C]24b in high radiochemical yield (40−50%). The new

PET radiotracer obtained was subjected to biodistribution

evaluations in Sprague−Dawley rats and microPET imaging

studies in nonhuman primates.

Synthesis of ﬂuoroethoxy analogues 26a and 26b followed

two diﬀerent approaches as shown in Scheme 4. The ﬁrst

approach was to demethylate 19e in the presence of boron

tribromide (BBr₃) or trimethylsilyl iodide (TMSI) to generate

the corresponding alcohol 25, which was reacted with 1-bromo-

2-ﬂuoroethane to aﬀord the target O-alkylated compound 26a.

However, use of a similar approach to synthesize compound

26b was not successful with either BBr₃or TMSI to remove the

methyl group in 19f. To overcome this challenge, an alternative

approach was used. Commercially available 2-bromopyridin-3-

ol (27) was reacted with 1-bromo-2-ﬂuoroethane via O-

alkylation to aﬀord 2-bromo-3-(2-ﬂuoroethoxy)pyridine (28).

This compound was lithiated and treated with compound 11,

followed by 18 to aﬀord target compound 26b following the

procedure described for synthesis of compounds 19j and 19k.

In Vitro Binding Studies. VAChT binding was assayed

using membrane homogenates from PC12 cells which over-

express human VAChT, as previously described.¹⁹Apparent

equilibrium dissociation constants (K_i, nM) are reported in

Table 1. The σ₁and σ₂binding aﬃnities were assayed in rat

brain and in guinea pig membranes, respectively. Apparent

equilibrium dissociation constants (K_i, nM) are reported in

Table 1. The ligand selectivity is deﬁned in terms of an index

a

Scheme 2

a

Reagents and conditions: (a) TFA, CH₂Cl₂, rt, 4 h; (b) 1,4-dihydronaphthalene oxide, NEt₃, EtOH, 75 °C, 36 h; (c) TBDMSCI, imidazole,

CH₂Cl₂, rt, overnight; (d) n-BuLi, THF, −78 °C, 4 h; (e) 12N HCl, THF, rt.

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a

Scheme 3

a

Reagents and conditions: (a) CH₃I, NaH, THF, rt, 0.5 h; (b) (CH₃CO)₂O, Et₃N, CH₂Cl₂, rt; (c) concentrated HCl, ethylene glycol, reﬂuxed; (d)

separation of enantiomers of 24b on HPLC: column, Chiralcel OD; mobile phase, 35% 2-propanol in hexane; ﬂow rate, 4.0 mL/min;

(+)-enantiomer at 15 min and (−)-enantiomer at 30 min.

a

Scheme 4

a

Reagents and conditions: (a) BBr₃or TMSI; (b) Br(CH₂)₂F, K₂CO₃, DMF, rt, 72 h; (c) tert-butyl 4-(methoxy(methyl)carbamoyl) piperidine-1-

carboxylate (11), n-BuLi, THF, −78 °C; (d) TFA, CH₂Cl₂rt, 4 h; (e) 1,4-dihydronaphthalene oxide, NEt₃, EtOH, 75 °C, 36 h.

that is the K_ifor σ₁or σ₂receptors divided by K_ifor VAChT

(K_{i σ1}/K_{i VAChT}or K_{i σ2}/K_{i VAChT}). A larger number represents

good selectivity for binding to VAChT.

(thiophen-2-yl)methanone (9), which replaces the phenyl

group in the structure of compound 5 with a 2-thiophene

moiety, displays comparable aﬃnity for VAChT, with a K_ivalue

In the previous studies, we had reported that a new class of

carbonyl group containing analogues displayed high potency

and high selectivity for VAChT;⁴particularly, compound (1-(3-

hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)-

of 5.00

1.20 nM. In the current study, to explore the

structure−activity relationship, we ﬁrst replaced the thiophen-2-

yl group in compound 9 with 1-methyl-1H-pyrrol-2-yl or 1-

methyl-1H-pyrrol-3-yl group to obtain compounds 19a and

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a

Scheme 5

a

Reagents and conditions: (a) NEt₃, EtOH, 60 °C, 48 h; (b) 1N NaOH, EtOH, 60 °C, overnight.

a

Scheme 6. Synthesis of the Precusor (−)-33

a

Reagents and conditions: (a) separation of enantiomers of 6 on HPLC: column, Chiralcel OD; mobile phase, 34% 2-propanol in hexane; ﬂow rate,

4.0 mL/min; (+)-enantiomer at 20.8 min and (−)-enantiomer at 33 min; (b) (Boc)₂O, DMAP, Et₃N, rt, 1.5 h; (c) K₂CO₃, MeOH, reﬂux, overnight.

Scheme 7. Radiosynthesis of (−)-[¹¹C]24b

19b. Both of these compounds displayed moderately potent

binding toward VAChT with the K_ivalues of 18.4 2.5 and

methoxy group, or ﬂuoride group, the order of the binding

potency for VAChT is −OCH₃> −F ≈ −CH₃, with K_ivalues

of 8.36 0.68, 26.1 2.4, and 27.9 8.0 nM for compounds

19e, 19j, and 19d, respectively. This demonstrates that an

electron donating group, −OCH₃, at the 6-position of pyridin-

3-carbonyl results in increased aﬃnity for VAChT. When

further replacing the methoxy in 19e (K_ivalue of 8.36 0.68

nM) with ﬂuoroethoxy to aﬀord compound 26a (K_ivalue of

38.0 3.8 nM), the aﬃnity for VAChT decreased 4.5-fold.

When the substitution pattern in the pyridine ring of 19e is

changed from the 6-methoxy-pyridin-3-carbonyl to that of 3-

methoxy-pyridin-2-carbonyl (19f), binding aﬃnity (K_ivalue)

for VAChT dropped 15-fold from 8.36 0.68 to 121 29 nM.

Replacing the methoxy group in 19f with a ﬂuoroethoxy group

to obtain compound 26b resulted in slight improvement in

VAChT binding (K_ivalue of 76.4 8.3 nM). The aﬃnities of

compounds 19f and 26b are lower than those of the structural

isomers 19e and 26a.

26.6

3.8 nM for compounds 19a and 19b, respectively

(Table 1), for binding to VAChT. Compared to 9, compounds

19a and 19b displayed about 4−5-fold decrease in aﬃnity for

VAChT. However, the selectivity of 19a and 19b for VAChT

versus σ₁receptor reached at least 71-fold and 114-fold,

respectively. More importantly, these modiﬁed structures (with

a methyl group on the N-atom of the pyrrole) provide a site for

labeling with carbon-11 via [¹¹C]methyl iodide.

When the benzoyl group was replaced with pyridin-3-

carbonyl, 6-methyl-pyridin-3-carbonyl, 6-methoxy-pyridin-3-

carbonyl, or 6-ﬂuoro-pyridin-3-carbonyl, compounds 19c,

19d, 19e, and 19j were obtained. The pyridine-3-carbonyl

analogue 19c (K_i= 24.1

2.8 nM) displayed 5-fold lower

aﬃnity for VAChT compared to its benzoyl counterpart 5 (K_i=

4.30 1.00 nM). When the hydrogen on the 6-position of the

pyridine-3-carbonyl group is replaced with a methyl group,

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Table 1. Binding Aﬃnities of New Analogues for VAChT, σ₁Receptor, and σ₂Receptor

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Table 1. continued

a

b

K_ivalues (mean SEM) were determined in at least three experiments for VAChT, σ₁and σ₂binding sites. The VAChT binding assay used

c

d

expressed human VAChT. The σ₁binding assay used membrane preparations of guinea pig brain. The σ₂binding assay used homogenates of rat

liver. Calculated value at pH 7.4 by ACD/Laboratories, version 7.0 (Advanced Chemistry Development, Inc., Canada).

e

The only diﬀerence among compounds 19g, 19j, and 19k

was the position of ﬂuoro substitution in the pyridin-3-carbonyl

group. The order of binding aﬃnity toward VAChT is 19g ≈

19k > 19j, in which the ﬂuorine atom is at 5-, 2-, and 6-

positions of the pyridine-3-carbonyl ring and the dissociation

constants for VAChT are 10.1 1.5, 12.7 1.1, and 26.1 2.4

nM, respectively. Compounds 19h and 19i are N-methyl-2-

pyridone derivatives. The only diﬀerence between them is the

position of the bridging carbonyl group located at the 4-

position in 19h and at the 6-position in 19i. The binding

aﬃnities for VAChT are 66.2 10.2 nM for 19h and 182 65

nM for 19i.

Compound (4-aminophenyl)(1-(3-hydroxy-1,2,3,4-tetrahy-

dronaphthalen-2-yl)piperidin-4-yl)methanone (6) has higher

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potency for VAChT (K_i= 1.68 0.14 nM) and has suitable

lipophilicity (ALogD = 1.56) for crossing the BBB. Introducing

methyl group(s) on the aniline nitrogen by N-methylation is a

conventional way to convert compound 6 into carbon-11

radiolabeled tracer using [¹¹C]methyl iodide. Monomethylation

and dimethylation aﬀorded 24a (K_i= 0.93 0.09 nM) and 24b

to cross the blood−brain barrier (BBB), rats were pretreated

with cyclosporine A (CycA) 30 min prior to injection of the

radiotracer dose and then euthanized at 30 min post injection

of the radiotracer. The distribution data obtained in this study

were shown in Figures 3 and 4. For the normal rats, the uptake

(K_i= 3.03

0.48 nM). Their VAChT binding aﬃnities are

comparable to that of compound 6 (K_i= 1.68 0.14 nM). The

ALogD values of 24a and 24b are 3.24 and 2.61, respectively,

which suggests they have suitable lipophilicity for the BBB

penetration. We experimentally measured the Log D value of

24b and found to be 2.60.

To test whether an amino group in the 5′-position or 8′-

position of the 3′-hydroxy-1′,2′,3′,4′-tetrahydronaphthalen-2′-

yl group aﬀects aﬃnity for VAChT, compounds 30a, 30c−e

and 31a were synthesized and their binding aﬃnities were

determined. Compound 30a (K_i= 38.7 7.0 nM) displayed

60-fold higher VAChT binding aﬃnity than that of 31a (K_i=

2310 390 nM). This result is consistent with the reported

results in which the 8′-amino analogues displayed much higher

potency than that of 5′-amino analogues.⁴When aniline

compounds 30a, 30c, 30d, and 30e are compared with the

nonaniline counterparts, 19a, 19c, 19d, and 19e, there is no any

signiﬁcant change in binding aﬃnities toward VAChT. This

suggests that introducing the 5′-amino group into hetero-

aromatic carbonyl containing analogues may not be beneﬁcial

to VAChT binding aﬃnity. Nevertheless, a large diﬀerence in

binding aﬃnity between the regioisomeric pairs 30a and 31a

was observed.⁴

Figure 3. The biodistribution of (−)-[¹¹C]24b in Sprague−Dawley

rats (185−205 g). Control rats were euthanized at 5 and 30 min. Rats

pretreated with 25 mg/kg of CycA 30 min prior to injection of the

radiotracer. The stars (*) in the ﬁgure denote that the %ID/g value

from heart, muscle, spleen, kidney, and brain were signiﬁcantly

diﬀerent between non-CycA treated and CycA treated group (n = 4,

student t test, p-value <0.05).

These substituted heteroaromatic carbonyl containing

VAChT analogues have moderate to high VAChT binding

aﬃnities. Consequently, we screened their σ receptor binding

aﬃnity. The in vitro data suggest that these new analogues had

very low aﬃnities for the σ receptors. Except for compound 24a

with K_i= 40.9 8.2 nM, all other new analogues exhibit greater

than 300 nM for the σ₁receptor, as shown in Table 1. Among

these new analogues, six compounds 19a, 19e, 19g, 19k, 24a,

and 24b have K_ivalues less than 20 nM toward VAChT.

Compounds 19a and 19g have >70-fold selectivity ratios for

VAChT vs either σ receptor type. Importantly, compounds

19e, 19k, and 24b not only display high aﬃnity toward VAChT

with K_ivalues of 8.36 0.68, 12.7 1.1, and 3.03 0.48 nM,

respectively, but they also display greater than 100-fold

selectivity ratios for VAChT versus σ receptors. Compound

24a has the highest potency for VAChT (0.93 0.09 nM) and

is highly selective for VAChT versus σ₁(44-fold) and σ₂(4400-

fold) receptors. Thus, compound 24a can be a good blocking

agent for validating the selective binding of other VAChT

radioligands.

Figure 4. The brain regional uptake of (−)-[¹¹C]24b in Sprague−

Dawley rats at 30 min in control rats and rats pretreated with 25 mg/

kg of CycA 30 min prior to injection of the radiotracer. The stars (*)

in the ﬁgure denote that the % ID/g value from all brain regions were

signiﬁcantly diﬀerent between non-CycA treated and CycA treated

group (n = 4, student t test, p-value <0.05).

To further test the feasibility of the lead compounds reported

in this manuscript to serve as a PET tracer for imaging VAChT

in vivo in living animals, racemic compound 24b was resolved

by HPLC using chiralcel OD column to obtain the (+)-24b and

of radioactivity at 5 min was 0.34, 0.49, 1.77, 0.22, 0.18, 0.89,

1.19, 1.47, 3.45, and 0.57% injected dose per gram (%ID/g) in

blood, heart, lung, muscle, fat, pancreas, spleen, kidney, liver,

and brain, respectively, post intravenous (iv) injection, and the

liver has the highest uptake; the radioactivity decreased in all

the tissues that were collected in the studies from 5 to 30 min

as shown, and the liver retained the highest uptake as 2.44 at 30

min. For the rats pretreated with CycA, except the uptake (%

ID/g) in kidney displayed a slight increase (0.77 in control rats,

1.21 in rats pretreated with CycA), the changes in other

peripheral tissues were negligible (Figure 3). For the brain

regions of interest, the uptake (%ID/g) at 30 min in

cerebellum, striatum, brain stem, cortex, thalamus, hippo-

(−)-24b. The in vitro data revealed that (−)-24b (K_i‑VAChT

=

0.78 nM) is the more potent isomer than the (+)-24b (K_i‑VAChT

= 19.0 nM). This is consistent to that ligand binding to VAChT

is stereoselective.

In Vivo Evaluation in Rats. To check the in vivo

distribution of (−)-[¹¹C]24b and its washout kinetics from

brain regions of interest and peripheral tissues, ex-vivo

biodistribution was conducted in male Sprague−Dawley rats

(185−205 g). Rats were euthanized at 5 and 30 min post iv

injection of (−)-[¹¹C]24b. To test the ability of (−)-[¹¹C]24b

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Figure 5. MicroPET imaging studies of (−)-[¹¹C]24b in cynomolgus monkey. MicroPET image (top left), coregistered image (top middle), MRI

image (top right), tissue time−activity curve (left bottom).

campus, and brain were 0.157, 0.593, 0.245, 0.262, 0.293, 0.265,

and 0.272, respectively, for the control rats; for the rats

pretreated with CycA, the uptakes (%ID/g) were 0.404, 1.020,

0.577, 0.637, 0.600, 0.562, and 0.596, respectively, for

corresponding brain regions of interest. The uptake in brain

was observed to increase 2.2-fold when the rats were pretreated

with CycA. P-glycoprotein (P-gp) is a 170 kDa protein that is

able to bind with compounds having diversiﬁed structures, and

it has widespread tissue distribution belonging to the adenosine

triphosphate (ATP)-binding cassette (ABC) transporters^42,43

and it is expressed in the capillary endothelial cells which

comprise the blood−brain barrier.⁴²CycA is a modulator/

inhibitor of the adenosine triphosphate (ATP)-binding cassette

(ABC) transporters including P-gp. It was frequently used to

evaluate the ABC transporter-mediated eﬄux of PET radio-

tracers, and it was suggested that ABC transporter-mediated

eﬄux of PET radiotracers has species diﬀerence.⁴⁴If a

radioligand is a substrate of P-gp, rats pretreated with CycA

will results in a signiﬁcant increase of the uptake in the brain of

animals, for example, radioligands, [¹¹C]verapramil,^45,46[¹¹C]-

GR218231⁴⁷and benzamide D₃radioligands.⁴⁸For radioligand,

(−)-[¹¹C]24b, using CycA pretreatment, the rats displayed

only 2.2-fold increase in the brain uptake of the rats.

Considering the brain uptake (%ID/g) of radioactivity reached

0.57 at 5 min, we concluded that the P-gp modulating the brain

uptake of (−)-[¹¹C]24b was very weak and (−)-[¹¹C]24b was

able to penetrate the BBB and displayed suﬃcient uptake in the

brain of the rats.

nontarget (cerebellum) reaches >2.5-fold after 70 min post

injection (Figure 5). This data suggests that (−)-[¹¹C]24b is a

promising PET tracer to quantify VAChT in vivo.

CONCLUSION

■

In the present study, we successfully synthesized a series of new

VAChT inhibitors by replacing the benzoyl group in 5 with a

heteroaromatic carbonyl group or replacing the 4-amino-

benzoyl group with the 4-methylaminobenzoyl group or the

4-N,N-dimethylaminobenzoyl group. The six compounds 19a,

19e, 19g, 19k, 24a, and 24b displayed high VAChT binding

aﬃnities (K_ivalues ranging from 0.93 to 18.4 nM) and good

selectivity for VAChT over σ receptors. In particular,

compounds 19e, 19k, and 24b are very potent for VAChT

with K_ivalues of 8.36 0.68, 12.7 1.1, and 3.03 0.48 nM,

respectively, and binding selectivity ratios equal to or greater

than 100-fold for VAChT over σ receptors. The racemate 24b

was successfully resolved on HPLC to obtain the minus and

plus isomers. In vivo validation of (−)-[¹¹C]24b in rodents and

nonhuman primates demonstrated that (−)-[¹¹C]24b is able to

penetrate the BBB and has highest accumulation in the

striatum, the VAChT enriched area in the brain. The time

tissue-activity of (−)-[¹¹C]24b in the brain of cynomolgus

monkey revealed that it has favorable washout pharmacoki-

netics in the brain. Further imaging studies of (−)-[¹¹C]24b in

nonhuman primate are warranted to test the feasibility of

(−)-[¹¹C]24b to be a promising candidate for assessing the

level of VAChT in the brain.

MicroPET Studies in Monkeys. To further conﬁrm that

(−)-[¹¹C]24b is able to bind with VAChT in the brain in vivo,

microPET studies of (−)-[¹¹C]24b in the brain of male

cynomolgus monkeys were performed (n = 3). The microPET

studies demonstrated that (−)-[¹¹C]24b is able to enter the

brain and has highest accumulation in striatum, and the VAChT

enriched area in the regions of interest in the brain (Figure 5).

(−)-[¹¹C]24b is able to give suﬃcient contrast for the striatum

versus other nontarget regions. The tissue time−activity curves

post injection of (−)-[¹¹C]24b revealed that the radioactivity

accumulation was the highest at 10 min postinjection. The ratio

of radioactivity accumulation in the target (striatum) and

EXPERIMENTAL SECTION

■

General. All reagents and chemicals were purchased from

commercial suppliers and used without further puriﬁcation unless

otherwise stated. All anhydrous reactions were carried out in oven-

dried glassware under an inert nitrogen atmosphere unless otherwise

stated. When the reactions involved extraction with methylene

chloride (CH₂Cl₂), chloroform (CHCl₃), or ethyl acetate (EtOAc),

the organic solutions were dried with anhydrous Na₂SO₄and

concentrated on a rotary evaporator under reduced pressure. Melting

points were determined on the MEL-TEMP 3.0 apparatus and left

1

uncorrected. H NMR spectra of majority of the compounds were

recorded at 300 MHz on a Varian Mercury-VX spectrometer with

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CDCl₃as solvent, and tetramethylsilane (TMS) was used as the

internal standard. Varian 400 MHz NMR was used for some

compounds and was reported in the Experimental Section where

appropriate. Elemental analyses (C, H, N) were determined by

Atlantic Microlab, Inc. and were found to be within 0.4% of theoretical

values. Chiralcel OD column was used for normal phase HPLC to

resolve enantiomers. Phenomenex Luna C18 column was used for

reverse phase HPLC conditions to purify the radioactive product on

HPLC, and Phenomenex Prodigy was used for QC analysis of

radiotracer.

Resolution of (4-Aminophenyl)(1-(3-hydroxy-1,2,3,4-tetra-

hydronaphthalen-2-yl)-piperidin-4-yl)metha-none (6) to Ob-

tain the Minus Isomer (−)-6 and the Plus Isomer (+)-6.

Approximately 105.0 mg of racemate 6 was resolved on HPLC using a

Chiralcel OD column (250 mm × 10 mm) under isocratic conditions

(34% 2-propanol in hexane) at a ﬂow rate of 4.0 mL/min and UV

wavelength at 254 nm to give 41.0 mg of (+)-6 (R_t= 20.8 min) and

48.0 mg of (−)-6 (R_t= 33 min). The speciﬁc rotation was determined

on an automatic polarimeter (Autopol 111, Rudolph Research,

Flanders, NJ). The optical rotation was [α]_D= −50° for (−)-6 and

[α]_D= +63° for (+)-6 at the concentration of 1.0 mg/mL in

dichloromethane at 20 °C.

tert-Butyl 4-(1-Methyl-1H-pyrrole-4-carbonyl)piperidine-1-

carboxylate (16b). To a solution of 3-bromo-1-methyl-1H-pyrrole

(2.19 g, 13.8 mmol) in anhydrous THF at −78 °C, n-BuLi (1.6 M, 10

mL) was added. The solution was stirred for 45 min at −78 °C. The

resulting mixture was cannulated into a solution of 11 (2.5 g, 9.2

mmol) in THF (80 mL) at the same temperature, and the reaction

mixture was stirred at −78 °C for 4 h, then quenched with satd

aqueous NH₄Cl and allowed to warm to room temperature. The

mixture was washed with brine and dried over Na₂SO₄, ﬁltered, and

evaporated under reduced pressure to give the crude product. The

crude compound was puriﬁed by silica gel chromatography to obtain

1

the desired compound 16b (1.07 g, 40%). H NMR (CDCl₃): δ 1.47

(s, 9H), 1.66−1.77 (m, 4H), 2.83 (s, 2H), 3.15−3.18 (m, 1H), 3.70 (s,

3H), 4.13 (s br, 2H), 6.57−6.61 (m, 2H), 7.26−7.29 (m, 1H).

tert-Butyl 4-Nicotinoylpiperidine-1-carboxylate (16c). To a

solution of 3-bromopyridine (1.13 g, 7.2 mmol) in anhydrous THF

(45 mL) at −40 °C under argon atmosphere, lithium dibutyl-

(isopropyl)magnesate (5.2 mL, 0.7 M, 3.6 mmol) was added. Stirring

was continued at the same temperature for 1 h. The resulting mixture

was cannulated into a solution of 11 (1.5 g, 5.5 mmol) in THF (50

mL) at −78 °C. The solution was maintained at −78 °C for 1 h and

quenched with saturated aqueous NH₄Cl and allowed to warm to

room temperature. The organic layer was separated, and the aqueous

layer was extracted with ethyl acetate. The combined organic layers

were washed with water, dried over Na₂SO₄, ﬁltered, and evaporated

under reduced pressure to give the crude product. The crude

compound was puriﬁed by silica gel chromatography to obtain the

tert-Butyl 4-(Methoxy(methyl)carbamoyl)piperidine-1-car-

boxylate (11). To a solution of 10 (2.5 g, 10.9 mmol) in CH₂Cl₂

(30 mL) at room temperature, CDI (1.77 g, 11.0 mmol) was added.

The mixture was stirred at room temperature for 1 h, and N,O-

dimethylhydroxyamine hydrochloride (1.3 g, 13.3 mmol) followed by

Et₃N were added. The reaction mixture was stirred overnight and was

washed with aqueous Na₂CO₃, water, dried, and concentrated under

reduced pressure. The residue was puriﬁed by silica gel column

chromatography to give 11 as a white solid (2.70 g, 90%); mp 54 °C.

¹H NMR (CDCl₃): δ 1.46 (s, 9H), 1.64−1.72 (m, 4H), 2.78 (s br,

1

desired compound 16c (0.49 g, 31%). H NMR (CDCl₃): δ 1.47 (s,

9H), 1.63−1.89 (m, 4H), 2.92 (m, 2H), 3.38 (m, 1H), 4.20 (m, 2H),

7.44 (ddd, J = 8.1, 5.1, 0.9 Hz, 1H), 8.22 (dt, J = 8.1, 2.1 Hz, 1H), 8.79

(dd, J = 5.1, 1.8 Hz, 1H), 9.16 (d, J = 1.8 Hz, 1H).

tert-Butyl 4-(6-Methylnicotinoyl)piperidine-1-carboxylate

(16d). Compound 16d was prepared from 5-bromo-2-methylpyridine

following the procedure described above for the preparation of 16c.

3H), 3.18 (s, 3H), 3.71 (s, 3H), 4.13 (s br, 2H).

Procedure A: General Method for the Synthesis of

Compounds 15h−i. 4-Bromo-1-methylpyridin-2(1H)-one (15h). A

solution of 4-bromopyridin-2-amine 12h (0.60 g, 3.5 mmol) in a

mixture of 2 N H₂SO₄(20 mL) and 2 N NaNO₂(10 mL) was stirred

at 0−5 °C for 2 h. The reaction mixture was extracted with CH₂Cl₂.

Crude product was used in the next step without further puriﬁcation.

To the above crude product, potassium carbonate (0.50 g, 3.6 mmol)

and methyl iodide (0.53 g, 3.7 mmol) were added and heated at 80 °C

in acetone (100 mL) in a sealed tube for 4 h. The reaction mixture was

cooled, and potassium carbonate was ﬁltered oﬀ. Acetone was

evaporated, and a small amount of water was added to the residue.

This solution was extracted with CH₂Cl₂, and the product was puriﬁed

with silica gel column chromatography to aﬀord 15h as a yellow solid

(355 mg, 57%). ¹H NMR (CDCl₃): δ 3.49 (s, 3H), 6.31 (d, J = 6.0 Hz,

1H), 6.82 (s, 1H), 7.14 (d, J = 6.0 Hz, 1H).

1

Yield, 42%. H NMR (CDCl₃): δ 1.49 (s, 9H), 1.68−1.87 (m, 4H),

2.63 (s, 3H), 2.90 (m, 2H), 3.35 (m, 1H), 4.18 (s br, 2H), 7.28 (d, J =

8.4 Hz, 1H), 8.11 (dd, J = 8.4, 2.1 Hz, 1H), 9.04 (d, J = 2.1 Hz, 1H).

tert-Butyl 4-(6-Methoxynicotinoyl)piperidine-1-carboxylate

(16e). Compound 16e was prepared from 5-bromo-2-methoxypyr-

idine following the procedure described above for the preparation of

1

16b. Yield 61%. H NMR (CDCl₃): δ 1.47 (s, 9H), 1.62−1.86 (m,

4H), 2.88 (m, 2H), 3.31 (m, 1H), 4.01 (s, 3H), 4.19 (s br, 2H), 6.81

(d, J = 8.7 Hz, 1H), 8.13 (dd, J = 8.7, 2.4 Hz, 1H), 8.79 (d, J = 2.4 Hz,

1H).

tert-Butyl 4-(3-Methoxypicolinoyl)piperidine-1-carboxylate

(16f). Compound 16f was prepared from 2-bromo-3-methoxypyridine

following the procedure described above for the preparation of 16b.

1

Yield 36%. H NMR (CDCl₃): δ 1.47 (s, 9H), 1.66−1.87 (m, 4H),

6-Bromo-1-methylpyridin-2(1H)-one (15i). Compound 15i as a

yellow solid was synthesized starting with compound 12i as described

in procedure A; mp 105 °C; yield 54%. ¹H NMR (CDCl₃,): δ 3.73 (s,

3H), 6.46−6.52 (m, 2H), 7.10−7.16 (m, 1H).

2.89 (s, 2H), 3.67−3.81 (m, 1H), 3.90 (s, 3H), 4.07 (s br, 2H), 7.35−

7.45 (m, 2H), 8.23−8.25 (m, 1H).

tert-Butyl 4-(3-Fluoronicotinoyl)piperidine-1-carboxylate

(16g). Compound 16g was prepared from 3-bromo-5-ﬂuoropyridine

following the procedure described above for the preparation of 16b.

tert-Butyl 4-(1-Methyl-1H-pyrrole-2-carbonyl)piperidine-1-

carboxylate (16a). To a Schlenk ﬂask containing N-methylpyrrole

(0.49 g, 6.08 mmol), 20 mL of THF was added while stirring at room

temperature until a clear solution was formed. The solution was cooled

down to −78 °C for 15 min, and 12.0 mL (6.08 mmol) of n-BuLi was

added. The solution was allowed to warm up to 0 °C for 20 min,

resulting in the formation of the yellow lithium intermediate. The

resulting mixture was added via cannula into a solution of 11 (0.83 g,

3.04 mmol) in THF (20 mL) at the same temperature, and the

reaction mixture was stirred at −78 °C for 4 h, then quenched with

saturated aqueous NH₄Cl and allowed to warm to room temperature.

The mixture was washed with brine and dried over Na₂SO₄, ﬁltered,

and evaporated under reduced pressure to give the crude product. The

crude compound was puriﬁed by silica gel chromatography to obtain

1

Yield 41%. H NMR (CDCl₃): δ 1.44 (s, 9H), 1.56−1.61 (m, 2H),

1.86−1.90 (m, 2H), 2.87 (s, 2H), 3.17−3.24 (m, 1H), 4.06 (s br, 2H),

7.54−7.58 (m, 1H), 8.53−8.60 (m, 2H).

tert-Butyl 4-(1-Methyl-2-oxo-1,2-dihydropyridine-4-

carbonyl)piperidine-1-carboxylate (16h). Compound 16h was

prepared from compound 15h following the procedure described

1

above for the preparation of 16b. Yield 38%. H NMR (CDCl₃): δ

1.47 (s, 9H), 1.56−1.87 (m, 4H), 2.82 (s, 2H), 3.05−3.07 (m, 1H),

3.50 (s, 3H), 4.17 (s br, 2H), 6.49 (d, J = 6.0 Hz, 1H), 6.73 (d, J = 6.0

Hz, 1H), 7.36 (s, 1H).

tert-Butyl 4-(1-Methyl-6-oxo-1,6-dihydropyridine-2-

carbonyl)piperidine-1-carboxylate (16i). Compound 16i was

prepared from compound 15i following the procedure described

1

the desired compound 16a (0.61 g, 34%). H NMR (CDCl₃): δ 1.47

above for the preparation of 16b. Yield 35%. H NMR (CDCl₃): δ

(s, 9H), 1.66−1.80 (m, 4H), 2.83 (s, 2H), 3.17 (m, 1H), 3.94 (s, 3H),

4.18 (s br, 2H), 6.15 (dd, J = 4.2, 2.4 Hz, 1H), 6.84 (pseudo t, J = 1.8

Hz, 1H), 6.99 (dd, J = 4.2, 1.5 Hz, 1H).

1.45 (s, 9H), 1.58−1.87 (m, 4H), 2.81 (s, 2H), 3.05−3.07 (m, 1H),

3.47 (s, 3H), 4.14 (s br, 2H), 6.44−6.47 (m, 1H), 6.69−6.72 (m, 1H),

7.30−7.36 (m, 1H).

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Procedure B: General Method for Synthesis of Compounds

17a−i. (1-Methyl-1H-pyrrol-2-yl)(piperidin-4-yl)methanone (17a).

To a solution of 16a (0.67 g, 2.3 mmol) in CH₂Cl₂(30 mL), TFA (2

mL) was added. The reaction mixture was stirred at room temperature

for 4 h. The solvent was removed under reduced pressure, and the

residue was neutralized with 1 N NaOH solution and extracted with

CH₂Cl₂(2 × 15 mL). The organic layer was washed with brine, dried,

and concentrated to give crude product. The crude product was

puriﬁed by silica gel column chromatography to aﬀorded 17a (0.32 g,

72%). ¹H NMR (CDCl₃): δ 1.76−1.87 (m, 4H), 2.81 (m, 2H), 3.17−

3.23 (m, 4H), 3.94 (s, 3H), 6.14 (m, 1H), 6.83 (m, 1H), 6.98 (m, 1H).

(1-Methyl-1H-pyrrol-3-yl)(piperidin-4-yl)methanone (17b). Com-

pound 17b was prepared from compound 16b as described in

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(1-methyl-1H-pyrrol-3-yl)methanone (19b). Compound 19b was

prepared from compound 17b as described in procedure C. Yield 50%.

¹H NMR (CDCl₃, free base): δ 1.83−2.00 (m, 4H), 2.38 (m, 1H),

2.76−3.13 (m, 8H), 3.29−3.35 (m, 1H), 3.71 (s, 3H), 4.32 (s br, 1H),

6.61 (m, 2H), 7.12−7.16 (m, 4H), 7.30−7.31 (m, 1H). The free base

was converted to the oxalate salt; mp 212.6 °C (decomposed). Anal.

(C₂₁H₂₆N₂O₂·H₂C₂O₄·0.25H₂O) C, H, N.

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)-

(pyridin-3-yl)methanone (19c). Compound 19c was prepared from

1

compound 17c as described in procedure C. Yield 40%. H NMR

(CDCl₃, free base): δ 1.77−1.98 (m, 4H), 2.43 (m, 1H), 2.75−3.02

(m, 7H), 3.20−3.38 (m, 2H), 3.88 (m, 1H), 7.08−7.15 (m, 4H),

7.42−7.46 (m, 1H), 8.21−8.25 (m, 1H), 8.77−8.79 (m, 1H), 9.16 (s,

1H). The free base was converted to the oxalate salt; mp 214 °C

(decomposed). Anal. (C₂₁H₂₄N₂O₂·H₂C₂O₄) C, H, N.

1

procedure B. Yield 80%. H NMR (CDCl₃): δ 1.76−1.80 (m, 4H),

2.65−2.73 (m, 2H), 3.01−3.17 (m, 4H), 3.68 (s, 3H), 6.57−6.59 (m,

2H), 7.25 (s, 1H).

Piperidin-4-yl(pyridin-3-yl)methanone (17c). Compound 17c was

prepared from compound 16c as described in procedure B. Yield 85%.

¹H NMR (CDCl₃): δ 1.62−1.77 (m, 2H), 1.86−1.89 (m, 3H), 2.79

(td, J = 12.6, 2.7 Hz, 2H), 3.21 (dt, J = 12.9, 3.0 Hz, 2H), 3.37 (tt, J =

11.4, 3.9 Hz, 1H), 7.43 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H), 8.22 (ddd, J =

4.8, 2.1, 1.5 Hz, 1H), 8.78 (dd, J = 4.5, 1.5 Hz, 1H), 9.16 (dd, J = 2.4,

0.9 Hz,1H).

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(6-methoxypyridin-3-yl)methanone (19d). Compound 19d was

prepared from compound 17d as described in procedure C. Yield 44%.

¹H NMR (CDCl₃, free base): δ 1.80−2.02 (m, 4H), 2.43 (m, 1H),

2.62 (s, 3H), 2.78−3.06 (m, 7H), 3.20−3.38 (m, 2H), 3.89 (m, 1H),

6.83 (d, J = 8.7 Hz, 1H), 7.11−7.18 (m, 4H), 8.17 (dd, J = 8.7, 2.4 Hz,

1H), 8.82 (d, J = 2.4 Hz, 1H). The free base was converted to the

oxalate salt; mp 223 °C (decomposed). Anal. (C₂₂H₂₆N₂O₂·2H₂C₂O₄)

C, H, N.

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(6-methylpyridin-3-yl) methanone (19e). Compound 19e was

prepared from compound 17e as described in procedure C. Yield 42%.

¹H NMR (CDCl₃, free base): δ 1.74−1.96 (m, 4H), 2.41 (m, 1H),

2.75−3.01 (m, 7H), 3.26−3.33 (m, 2H), 3.86 (m, 1H), 4.04 (s, 3H),

4.17 (s br, 1H), 7.06−7.14 (m, 4H), 7.27 (d, J = 8.1 Hz, 1H), 8.11 (dd,

J = 8.1, 2.1 Hz, 1H), 9.03 (d, J = 2.1 Hz, 1H). The free base was

converted to the oxalate salt; mp 240 °C (decomposed). Anal.

(C₂₂H₂₆N₂O₃·H₂C₂O₄) C, H, N.

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(3-methoxypyridin-2-yl)methanone (19f). Compound 19f was

prepared from compound 17f as described in procedure C. Yield 36%.

¹H NMR (CDCl₃, free base): δ 1.72−1.96 (m, 4H), 2.33−2.41 (m,

1H), 2.72−2.96 (m, 7H), 3.26−3.33 (m, 1H), 3.55−3.63 (m, 1H),

3.81−3.90 (m, 3H), 4.30 (s br, 1H), 7.06−7.14 (m, 4H), 7.26−7.39

(m, 2H), 8.23−8.25 (m, 1H). The free base was converted to the

ox ala te salt; mp 146. 6 °C (d ecomposed). A nal.

(C₂₂H₂₆N₂O₃·H₂C₂O₄·0.25H₂O) C, H, N.

(6-Methylpyridin-3-yl)(piperidin-4-yl)methanone (17d). Com-

pound 17d was prepared from compound 16d as described in

1

procedure B. Yield 70%. H NMR (CDCl₃): δ 1.62−1.87 (m, 5H),

2.63 (s, 3H), 2.77 (m, 2H), 3.16−3.22 (m, 2H), 3.33 (m, 1H), 7.27 (d,

J = 8.4 Hz, 1H), 8.12 (dd, J = 8.1, 2.4 Hz, 1H), 9.04 (d, J = 2.4 Hz,

1H).

(6-Methoxypyridin-3-yl)(piperidin-4-yl)methanone (17e). Com-

pound 17e was prepared from compound 16e as described in

1

procedure B. Yield 77%. H NMR (CDCl₃): δ 1.65−1.89 (m, 4H),

2.40 (s, 1H), 2.78 (td, J = 12.0, 2.7 Hz, 2H), 3.21 (dt, J = 9.0, 3.6 Hz,

2H), 3.31 (m, 1H), 4.00 (s, 3H), 6.79 (d, J = 8.4 Hz, 1H), 8.13 (dd, J

= 8.4, 2.4 Hz, 1H), 8.78 (d, J = 2.4 Hz, 1H).

(3-Methoxypyridin-2-yl)(piperidin-4-yl)methanone (17f). Com-

pound 17f was prepared from compound 16f as described in

1

procedure B. Yield 78%. H NMR (CDCl₃): δ 1.51−1.81 (m, 4H),

2.69 (s, 2H), 3.08−3.25 (m, 3H), 3.49−3.58 (m, 1H), 3.83 (s, 3H),

7.27−7.36 (m, 2H), 8.16−8.18 (m, 1H).

(5-Fluoropyridin-3-yl)(piperidin-4-yl)methanone (17g). Com-

pound 17g was prepared from compound 16g as described in

1

procedure B. Yield 79%. H NMR (CDCl₃): δ 1.56−1.61 (m, 2H),

1.86−1.90 (m, 2H), 2.87 (m, 2H), 3.17−3.21 (m, 4H), 7.56 (s, 1H),

8.53−8.60 (m, 2H).

(5-Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphtha-

len-2-yl)piperidin-4-yl)methanone (19g). Compound 19g was

prepared from compound 17g as described in procedure C. Yield

1-Methyl-5-(piperidine-4-carbonyl)pyridin-2(1H)-one (17h).

Compound 17h was prepared from compound 16h as described in

1

44%. H NMR (CDCl₃, free base): δ 1.70−2.04 (m, 4H), 2.34−2.43

1

procedure B. Yield 79%. H NMR (CDCl₃): δ 1.57−1.88 (m, 4H),

(m, 1H), 2.75−3.34 (m, 8H), 3.82−3.90 (m, 1H), 4.13 (s br, 1H),

7.06−7.11 (m, 4H), 7.57−7.61 (m, 1H), 8.56−8.62 (m, 2H). The free

base was converted to the oxalate salt; mp 203.7 °C (decomposed).

Anal. (C₂₁H₂₃FN₂O₂·H₂C₂O₄) C, H, N.

2.83 (s, 2H), 3.03−3.09 (m, 4H), 3.50 (s, 3H), 6.50 (d, J = 6.0 Hz,

1H), 6.72 (d, J = 6.0 Hz, 1H), 7.38 (s, 1H).

1-Methyl-6-(piperidine-4-carbonyl)pyridin-2(1H)-one (17i). Com-

pound 17i was prepared from compound 16i as described in

4-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-

carbonyl)-1-methylpyridin-2(1H)-one (19h). Compound 19h was

prepared from compound 17h as described in procedure C. Yield 37%.

¹H NMR (CDCl₃, free base): δ 1.72−1.99 (m, 4H), 2.33−2.39 (m,

1H), 2.75−3.01 (m, 6H), 3.27−3.34 (m, 2H), 3.50 (s, 3H), 3.82−3.91

(m, 1H), 4.08 (s br, 1H), 6.45 (d, J = 6.0 Hz, 1H), 6.72 (d, J = 6.0 Hz,

1H), 7.12−7.13 (m, 4H), 7.35 (s, 1H). The free base was converted to

the oxalate salt; mp 93.8 °C (decomposed). Anal.

(C₂₂H₂₆N₂O₃·H₂C₂O₄·1.5H₂O) C, H, N.

1

procedure B. Yield 75%. H NMR (CDCl₃): δ 1.58−1.87 (m, 4H),

2.82 (s, 2H), 3.02−3.10 (m, 4H), 3.47 (s, 3H), 6.42−6.48 (m, 1H),

6.67−6.72 (m, 1H), 7.31−7.36 (m, 1H).

Procedure C: General Method of Preparing (19a−i) and

Their Corresponding Oxalates. (1-(3-Hydroxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)piperidin-4-yl)(1-methyl-1H-pyrrol-2-yl)-

methanone (19a). A mixture of 17a (0.27 g, 1.4 mmol), 18 (0.10 g,

0.68 mmol), and Et₃N (0.3 mL, 2.2 mmol) in ethanol (5 mL) was

stirred at 75 °C for 36 h. After cooling to room temperature, the

reaction mixture was poured into water and extracted with EtOAc (3 ×

15 mL). The residue was puriﬁed by silica gel column chromatography

6-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidine-4-

carbonyl)-1-methylpyridin-2(1H)-one (19i). Compound 19i was

prepared from compound 17i as described in procedure C. Yield

1

to give 19a as a white solid (0.11 g, 47%). H NMR (CDCl₃, free

1

39%. H NMR (CDCl₃, free base): δ 1.72−2.04 (m, 4H), 2.32−2.39

base): δ 1.81−2.03 (m, 4H), 2.37 (m, 1H), 2.76−3.13 (m, 8H), 3.30

(m, 1H), 3.88 (m, 1H), 3.95 (s, 3H), 4.45 (s br, 1H), 6.14 (m, 1H),

6.83 (m, 1H), 6.98 (m, 1H), 7.09−7.16 (m, 4H). Free base was

converted to the corresponding oxalate salt by adding oxalic acid in

ethyl acetate to 19a in CH₂Cl₂; mp 212 °C (decomposed). Anal.

(C₂₁H₂₆N₂O₂·H₂C₂O₄) C, H, N.

(m, 1H), 2.81−3.01 (m, 6H), 3.27−3.35 (m, 2H), 3.50 (s, 3H), 3.82−

3.89 (m, 1H), 4.06 (s br, 1H), 6.43−6.46 (m, 1H), 6.70−6.73 (m,

1H), 7.06−7.14 (m, 4H), 7.31−7.37 (m, 1H). The free base was

converted to the oxalate salt; mp 122.4 °C (decomposed). Anal.

(C₂₂H₂₆N₂O₃·H₂C₂O₄·1.5H₂O) C, H, N.

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Procedure D: General Method for the Synthesis of

Compounds (19j−k) and Their Corresponding Oxalates. (6-

Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)piperidin-4-yl)methanone (19j). To a solution of 17j (2.42 g, 13.8

mmol) in anhydrous THF at −78 °C, n-BuLi (1.6 M, 10 mL) was

added. The solution was stirred for 45 min at −78 °C. The resulting

mixture was cannulated into a solution of 21 (3.97 g, 9.2 mmol) in

THF (80 mL) at the same temperature, and the reaction mixture was

stirred at −78 °C for 4 h, then quenched with satd aqueous NH₄Cl

and allowed to warm to room temperature. The mixture was washed

with brine and dried over Na₂SO₄, ﬁltered, and evaporated under

reduced pressure to give the crude product. The crude compound was

puriﬁed by chromatography on silica gel to obtain the TBDMS-

protected intermediate (1-(3-(tert-butyldimethylsilyloxy)-1,2,3,4-tetra-

hydronaphthalen-2-yl)piperidin-4-yl)(6-ﬂuoropyridin-3-yl) methanone

(1.55 g, 36%). ¹H NMR (CDCl₃): δ 0.12 (s, 6H), 0.91 (s, 9H), 1.65−

1.77 (m, 4H), 2.46−3.05 (m, 9H), 4.13 (s br, 2H), 7.04−7.12 (m,

4H), 8.23−8.37 (m, 2H), 8.78 (s, 1H).

A mixture of the intermediate (1-(3-(tert-butyldimethylsilyloxy)-

1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(6-ﬂuoropyridin-3-yl)

methanone (1.55 g, 3.3 mmol) and concentrated HCl (5 mL) in THF

were stirred for 4 h at room temperature until TLC indicated that

deprotection was complete, and then it was carefully neutralized with 1

N NaOH and extracted with CH₂Cl₂. The crude product was puriﬁed

by column chromatography to aﬀord 19j as white solid (0.92 g, 79%).

¹H NMR (CDCl₃, free base): δ 1.84−2.03 (m, 4H), 2.38−2.46 (m,

1H), 2.75−3.02 (m, 6H), 3.21−3.34 (m, 2H), 3.82−3.89 (m, 1H),

4.30 (s br, 1H), 7.03−7.11 (m, 4H), 8.33−8.39 (m, 2H), 8.79 (s, 1H).

Free base was converted to the corresponding oxalate salt by adding

oxalic acid in ethyl acetate to 19j in CH₂Cl₂; mp 204.3 °C

(decomposed). Anal. (C₂₁H₂₃FN₂O₂·H₂C₂O₄·0.5H₂O) C, H, N.

(2-Fluoropyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydronaphtha-

len-2-yl)piperidin-4-yl)methanone (19k). Compound 19k was

prepared from compound 17k as describe in procedure D to give

TBDMS-protected intermediate (1-(3-(tert-butyldimethylsilyloxy)-

1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(2-ﬂuoro-pyridin-3-

yl) methanone (1.68 g, 39%). ¹H NMR (CDCl₃): δ 0.11 (s, 6H), 0.90

(s, 9H), 1.63−1.78 (m, 4H), 2.45−3.08 (m, 9H), 4.13 (s br, 2H),

7.04−7.11 (m, 4H), 7.31−7.39 (m, 1H), 8.23−8.30 (m, 1H), 8.38−

8.40 (m, 1H). Removal of TBDMS with HCl gave 19k (0.87 g, 75%).

¹H NMR (CDCl₃, free base): δ 1.84−2.03 (m, 4H), 2.38−2.46 (m,

washed with brine and 1 N NaH₂PO₄(30 mL × 3). The organic layer

was dried and concentrated to a residue. The crude product was

puriﬁed by silica gel column chromatography to aﬀord 21 as a white

solid (0.38 g, 70%). ¹H NMR (CDCl₃,): δ 0.12 (s, 6H), 0.91 (s, 9H),

1.65−1.77 (m, 4H), 2.46−3.05 (m, 9H), 3.17 (s, 3H), 3.69 (s, 3H),

4.13 (s br, 2H), 7.04−7.12 (m, 4H).

3-(4-(4-Acetamidobenzoyl)piperidin-1-yl)-1,2,3,4-tetrahydro-

naphthalen-2-yl acetate (22). Acetic anhydride (1.89 mL, 18.5

mmol) was added dropwise over 15 min to a magnetically stirred

solution of 6 (0.54 g, 1.54 mmol) and Et₃N (3.12 g, 30.8 mmol) in dry

CH₂Cl₂(10 mL) overnight. The reaction was monitored by TLC.

After the reaction was complete, the solvent was evaporated under

reduced pressure. Water was added to wash the resulting solid and the

residue was puriﬁed by silica gel column chromatography (ethyl

1

acetate:hexane = 3:2) to give 22 (0.55 g, 82%). H NMR (CDCl₃): δ

1.24−1.28 (m, 2H), 1.70−1.84 (m, 2H), 2.12 (s, 3H), 2.22 (s, 3H),

2.44−2.60 (m, 2H), 2.84−3.05 (m, 6H), 3.16−3.23 (m, 2H), 5.27−

5.34 (m, 1H), 7.05−7.16 (m, 4H), 7.49 (s br, 1H), 7.62 (d, J = 8.4 Hz,

2H), 7.92 (d, J = 8.4 Hz, 2H).

N-(4-(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidine-4-carbonyl)phenyl)-N-methylacetamide (23). Sodium

hydride (96 mg, 2.4 mmol) was added to 3-(4-(4-acetamidobenzoyl)-

piperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-yl acetate 22 (0.87 g, 2

mmol) in anhydrous THF, and iodomethane (0.34 g, 2.4 mmol) was

added dropwise to the mixture, which was maintained below 5 °C for

0.5 h and then stirred at room temperature for 1 h. The reaction was

monitored by TLC. After the reaction was complete, the mixture was

partitioned between saturated aqueous NH₄Cl and ethyl acetate. The

organic layer was separated, and the aqueous layer was extracted with

ethyl acetate. The combined organic layers were dried and

concentrated. The crude product was puriﬁed by silica gel column

chromatography (ethyl acetate:hexane = 3:2) to give 23 (0.63 g, 78%).

¹H NMR (CDCl₃): δ 1.77−1.90 (m, 4H), 1.96 (s, 3H), 2.44 (t, J =

10.3 Hz, 1H), 2.76−3.03 (m, 7H), 3.27−3.35 (m, 2H), 3.31 (s, 3H),

3.84−3.89 (m, 1H), 4.21 (s br, 1H), 7.09−7.28 (m, 4H), 7.32 (d, J =

8.1 Hz, 2H), 8.01 (d, J = 7.8 Hz, 2H).

(4-(Dimethylamino)phenyl)(1-(3-hydroxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)piperidin-4-yl)methanone (24a). Sodium hydride

(96 mg, 2.4 mmol) was added to 6 (0.87 g, 2 mmol) in anhydrous

THF, and iodomethane (4.8 mmol) was added dropwise to the

mixture, which was stirred at room temperature for 0.5 h. The reaction

was monitored by TLC. After the reaction was complete, the mixture

was partitioned between saturated aqueous NH₄Cl and ethyl acetate.

The organic layer was separated, and the aqueous layer was extracted

with ethyl acetate. The combined organic layers were dried and

contentrated. The crude product was puriﬁed by silica gel column

1H), 2.75−2.99 (m, 6H), 3.25−3.33 (m, 2H), 3.80−3.90 (m, 1H),

4.31 (s br, 1H), 7.03−7.13 (m, 4H), 7.30−7.41 (m, 1H), 8.22−8.31

(m, 1H), 8.38−8.40 (m, 1H). The free base was converted to the

oxalate salt; mp 205. 8 °C (dec omposed). Anal.

(C₂₁H₂₃FN₂O₂·H₂C₂O₄·H₂O) C, H, N.

1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-N-methoxy-N-

methylpiperidine-4-carboxamide (20). TFA (2 mL) was added into a

solution of 11 (0.38 g, 1.4 mmol) in CH₂Cl₂(30 mL). The reaction

mixture was stirred at room temperature for 4 h. The solvent was

removed under reduced pressure, and the residue was neutralized with

1 N NaOH solution and extracted with CH₂Cl₂(2 × 15 mL). The

organic layer was washed with brine, dried, and concentrated to give

the crude compound, which was used in the next step without further

puriﬁcation. Crude compound, 18 (0.10 g, 0.68 mmol), and Et₃N (0.3

mL, 2.2 mmol) in ethanol (5 mL) were stirred at 75 °C for 36 h. After

cooling to room temperature, the reaction mixture was poured into

water and extracted with EtOAc (3 × 15 mL). The organic layer was

dried and concentrated under reduced pressure. The residue was

puriﬁed by silica gel column chromatography to give 20 as a white

solid (86.4 mg, 40%). ¹H NMR (CDCl₃): δ 1.81−1.93 (m, 4H), 2.28−

2.31 (m, 1H), 2.75−2.97 (m, 8H), 3.27−3.20 (s, 3H), 3.27−3.34 (m,

1H), 3.72 (s, 3H), 3.85−3.90 (m, 1H), 4.27 (s br, 1H), 7.09−7.13 (m,

4H).

1

chromatography to give 24a as a white solid (0.63 g, 83%). H NMR

(CDCl₃, free base): δ 1.81−1.98 (m, 4H), 2.36−2.44 (m, 1H), 2.74−

3.06 (m, 13H), 3.23−3.33 (m, 2H), 3.83−3.91 (m, 1H), 4.46 (s br,

1H), 6.66 (d, J = 9 Hz, 2H), 7.09−7.15 (m, 4H), 7.88 (d, J = 9 Hz,

2H). Free base was converted to the corresponding oxalate salt by

adding oxalic acid in ethyl acetate to 24a in CH₂Cl₂; mp 228.9 °C

(decomposed). Anal. (C₂₄H₃₀N₂O₂·H₂C₂O₄) C, H, N.

(1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(4-(methylamino)phenyl)methanone (24b). Concentrated HCl

(12 M, 0.25 mL) was added to a stirred solution of 23 (0.4 g, 1.0

mmol) in ethylene glycol (0.75 mL). The reaction mixture was heated

to reﬂux for 3 h, and the reaction was monitored by TLC. When the

reaction was complete, the mixture was partitioned between water and

ethyl acetate. The organic layer was separated, and the aqueous layer

was extracted. The combined organic layers dried and contentrated.

The crude product was puriﬁed by silica gel column chromatography

(ethyl acetate/hexane, 1/4) to give 24b (0.3 g, 82%). ¹H NMR

(CDCl₃): δ 1.85−1.92 (m, 4H), 2.40−2.44 (m, 1H), 2.77−2.99 (m,

8H), 3.27−3.34 (m, 2H), 3.81−3.89 (m, 2H), 4.19−4.23 (m, 1H),

4.46 (s br, 1H), 6.59 (d, J = 8.1 Hz, 2H), 7.09−7.15 (m, 4H), 7.86 (d,

J = 8.1 Hz, 2H). Free base was converted to the corresponding oxalate

salt by adding oxalic acid in ethyl acetate to 24b in CH₂Cl₂; mp 215.7

°C (decomposed). Anal. (C₂₃H₂₈N₂O₂·H₂C₂O₄) C, H, N.

1-(3-((tert-Butyldimethylsilyl)oxy)-1,2,3,4-tetrahydronaphthalen-

2-yl)-N-methoxy-N-methylpiperidine-4-carboxamide (21). To a

solution of 20 (0.4 g, 1.25 mmol) and imidazole (0.4 g, 5.88 mmol)

in CH₂Cl₂(50 mL), TBDMSCl (0.38 g, 2.5 mmol) was added. The

reaction mixture was stirred overnight at room temperature until TLC

indicated that the reaction was complete. The reaction mixture was

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Resolution of (1-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(4-(methylamino)phenyl)methanone (24b) to Obtain

the Entantiomerically Minus Isomer (−)-24b and Plus Isomer

(+)-24b. Approximately 200 mg of ( )-24b was separated on chiral

HPLC using a Chiralcel OD column (250 mm × 10 mm). The mobile

phase used was 35% 2-propanol in hexane at a ﬂow rate of 4.0 mL/min

to give 83.3 mg of (+)-24b (R_t= 15 min) and 94.9 mg of (−)-24b (R_t

= 30 min). The speciﬁc rotation was determined on an automatic

polarimeter (Autopol 111, Rudolph Research, Flanders, NJ). The

optical rotation of (−)-24b was [α]_D= −30.5° at the concentration of

room temperature for 4 h. The solvent was removed under reduced

pressure, and the residue was neutralized with 1 N NaOH solution and

extracted with CH₂Cl₂(2 × 15 mL). The organic layer was washed

with brine, dried, and concentrated to give crude 4-(3-(2-

ﬂuoroethoxy)picolinoyl)piperidine. The crude product was used in

the next step without further puriﬁcation.

A mixture of the above compound, 18 (0.10 g, 0.68 mmol), and

Et₃N (0.3 mL, 2.2 mmol) in ethanol (5 mL) was stirred at 75 °C for

36 h. After cooling to room temperature, the mixture was poured into

water and extracted with EtOAc (3 × 15 mL). The organic layer was

dried and concentrated under reduced pressure. The residue was

puriﬁed by silica gel column chromatography to give 26b (0.12 g,

1.8 mg/mL in dichloromethane and that of (+)-24b was [α]_D

=

+21.8° at the concentration of 1.1 mg/mL in dichloromethane at 20

°C. The (−)-24b and (+)-24b were converted to oxalates by treating 1

equiv of (−)-24b or (+)-24b with 1 equiv of oxalic acid. The salt

obtained was used for the in vitro studies; mp of (+)-24b, 199.8 °C

(decomposed); mp of (−)-24b, 200.2 °C (decomposed).

1

43%). H NMR (CDCl₃, free base): δ 1.73−1.98 (m, 4H), 2.32−2.40

(m, 1H), 2.71−2.96 (m, 8H), 3.31−3.33 (m, 1H), 3.49−3.56 (m, 1H),

3.80−3.89 (m, 1H), 4.24−4.36 (m, 2H), 4.69−4.87 (m, 2H), 7.09−

7.25 (m, 4H), 7.34−7.38 (m, 2H), 8.27−8.29 (m, 1H). The

corresponding oxalate salt was obtained by adding oxalic acid in

ethyl acetate the solution of 26b in CH₂Cl₂and then recrystallized.

For the oxalate salt, mp 119.9 °C (decomposed). Anal.

(C₂₃H₂₇FN₂O₃·H₂C₂O₄·H₂O) C, H, N.

(1--3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-

yl)(6-hydroxypyridin-3-yl)methanone (25). Into an oven-dried argon-

purged 50 mL round-bottom ﬂask was placed 19e (73.3 mg, 0.2

mmol), and 3.0 mL of chloroform was added followed by TMSI (57

μL, 0.4 mmol). The reaction mixture was heated to 55 °C for 1 h until

TLC (EtOAc/hexane, 1/1) indicated the disappearance of starting

material. The reaction mixture was cooled to room temperature, and

0.6 mL of methanol was added. The reaction mixture was

concentrated. Ethyl acetate was added followed by aqueous sodium

bicarbonate solution. The ethyl acetate layer was separated, and the

aqueous layer was extracted with ethyl acetate. Combined organic

layers were washed with brine, dried over anhydrous sodium sulfate,

and concentrated to give compound 25 as a white solid (69 mg, 97%).

¹H NMR (CDCl₃): δ 1.40−2.00 (m, 3H), 2.20−2.25 (m, 1H), 2.60−

2-Bromo-3-(2-ﬂuoroethoxy)pyridine (28). A mixture of 2-bromo-

pyridin-3-ol 27 (0.17 g, 1.0 mmol), 1-bromo-2-ﬂuoroethane (0.25 g,

2.0 mmol), and K₂CO₃(0.55 g, 4.0 mmol) in acetonitrile were stirred

at reﬂux for one day until TLC indicated that the reaction was

complete. The reaction mixture was ﬁltered, dried, and concentrated

under reduced pressure. The crude product was puriﬁed by silica gel

column chromatography to aﬀord 28 (0.16 g, 68%). ¹H NMR

(CDCl₃): δ 4.25−4.34 (m, 2H), 4.73−4.89 (m, 2H), 7.20−7.22 (m,

2H), 8.01 (s, 1H).

Procedure E: General Method of Preparing 30a, 30c−d, and

31a. (1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(1-methyl-1H-pyrrol-2-yl)methanone (30a). A mixture

of 29 (0.20 g, 0.78 mmol), 17a (0.44 g, 2.3 mmol), and Et₃N (0.5 mL)

in ethanol (10 mL) was stirred at 60 °C for 48 h. To the mixture, 1 N

NaOH (3 mL) was added and the stirring was continued overnight.

The solvent was removed, the residue was extracted with EtOAc (3 ×

30 mL), and the organic layer was washed with aqueous Na₂CO₃

solution, dried, and concentrated. The crude product was puriﬁed by

column chromatography to give 30a (0.11 g, 37%). ¹H NMR (CDCl₃,

free base): δ 1.85−1.96 (m, 4H), 2.43−2.53 (m, 2H), 2.67−2.89 (m

5H), 2.96−3.28 (m, 3H), 3.60 (s br, 2H), 3.85 (m, 1H), 3.95 (s, 3H),

6.14 (m, 1H), 6.57 (m, 2H), 6.83 (s br, 1H), 6.96−7.02 (m, 2H). Free

base was converted to the corresponding oxalate salt by adding oxalic

acid in ethyl acetate to 30a in CH₂Cl₂; mp 131 °C (decomposed).

Anal. (C₂₁H₂₇N₃O₂·2H₂C₂O₄·2H₂O) C, H, N.

(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(pyridin-3-yl)methanone (30c). Compound 30c was

prepared from compound 17c as described in procedure E. Yield 61%.

¹H NMR (CDCl₃, free base): δ 1.80−2.05 (m, 4H), 2.36−2.53 (m,

2H), 2.68−2.92 (m, 5H), 3.01−3.35 (m, 4H), 3.61 (s br, 2H), 3.87

(m, 1H), 6.54−6.60 (m, 2H), 6.99 (td, J = 7.5, 3.3 Hz, 1H), 7.45 (dd, J

= 8.1, 4.8 Hz, 1H), 8.24 (dt, J = 8.1, 1.8 Hz, 1H), 8.80 (dd, J = 4.8, 1.8

Hz, 1H), 9.17 (d, J = 2.1 Hz, 1H). The free base was converted to the

o x a l a t e s a l t ; m p 1 0 9 ° C ( d e c o m p o s e d ) . A n a l .

(C₂₁H₂₅N₃O₂·2H₂C₂O₄·0.5H₂O) C, H, N.

(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(6-methylpyridin-3-yl)methanone (30d). Compound

30d was prepared from compound 17d as described in procedure E.

Yield 44%. ¹H NMR (CDCl₃, free base): δ 1.79−1.99 (m, 5H), 2.35−

2.53 (m, 2H), 2.64 (s, 3H), 2.67−3.28 (m, 8H), 3.62 (s br, 2H), 3.87

(m, 1H), 6.57 (m, 2H), 6.99 (m, 1H), 7.29 (d, J = 8.1 Hz, 1H), 8.13

(dd, J = 8.1, 2.1 Hz, 1H), 9.04 (d, J = 2.1 Hz, 1H). The free base was

converted to the oxalate salt; mp 98 °C (decomposed). Anal.

(C₂₄H₂₉N₃O₄·H₂C₂O₄) C, H, N.

(1-(8-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(6-methoxypyridin-3-yl)methanone (30e). Compound

30e was prepared from compound 17e as describe in procedure E.

Yield 43%. ¹H NMR (CDCl₃, free base): δ 1.65 (s br, 1H), 1.80−2.02

(m, 4H), 2.42−2.53 (m, 2H), 2.67−3.03 (m, 6H), 3.21−3.28 (m, 2H),

3.61 (s br, 2H), 3.86 (m, 1H), 4.02 (s, 3H), 6.57 (m, 2H), 6.82 (d, J =

3.10 (m, 8H), 3.30 (dd, J = 6.2, 13.9 Hz, 1H), 3.80−4.00 (m, 1H),

4.20−4.25 (m, 1H), 6.60 (d, J = 9.6 Hz, 1H), 7.00−7.20 (m, 4 H),

8.05 (dd, J = 2.7, 9.6 Hz, 1H), 8.16 (s, 1H).

(6-(2-Fluoroethoxy)pyridin-3-yl)(1-(3-hydroxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)-piperidin-4-yl)methanone (26a). To the mixture of

25 (69 mg, 0.195 mmol) and 1-bromo-2-ﬂuoroethane (30 μL, 0.4

mmol) in DMF (2 mL) was added potassium carbonate (60 mg, 0.43

mmol). The resultant reaction mixture was stirred at the room

temperature for 72 h. Ethyl acetate was added to the reaction mixture

and washed with aqueous sodium bicarbonate solution. The organic

layer was dried over anhydrous sodium sulfate and concentrated. The

crude product was puriﬁed by silica gel chromatography to give

1

colorless sticky solid (40 mg, 51%). H NMR (CDCl₃): δ 1.75−1.99

(m, 1H), 2.20−2.50 (m, 1H), 2.70−3.10 (m, 11H), 3.30 (dd, J = 16.0,

6.0 Hz, 1H), 3.80−3.95 (m, 1H), 4.27 (t, J = 4.5 Hz, 1H), 4.36 (t, J =

4.5 Hz, 1H), 4.67 (t, J = 4.5 Hz, 1H), 4.83 (t, J = 4.5 Hz, 1H), 6.61 (d,

J = 9.6 Hz, 1H), 7.00−7.20 (m, 4H), 7.88 (dd, J = 2.7, 9.6 Hz, 1H),

8.15 (s, 1H). Free base was converted to the corresponding oxalate salt

by adding oxalic acid in ethyl acetate to 26a in CH₂Cl₂; mp 84 °C

(decomposed). Anal. (C₂₄H₂₇FN₂O₃·H₂C₂O₄·H₂O) C, H, N.

(3-(2-Fluoroethoxy)pyridin-2-yl)(1-(3-hydroxy-1,2,3,4-tetrahydro-

naphthalen-2-yl)piperidin-4-yl)methanone (26b). To a solution of

28 (1.60 g, 7.3 mmol) in THF was added n-BuLi (1.6 M, 7 mL) at

−78 °C under N₂atmosphere. The solution was stirred at −78 °C for

1 h, and a solution of tert-butyl-4-(methoxy(methyl)carbamoyl)-

piperidine-1-carboxylate 11 (1.0 g, 3.6 mmol) in THF was added. The

solution was maintained at −78 °C for 4 h and then quenched with

saturated aqueous NH₄Cl that was allowed to warm to room

temperature with stirring. The organic layer was separated, and the

aqueous layer was extracted with ethyl acetate. The combined extracts

were washed with water, dried over Na₂SO₄, ﬁltered, concentrated, and

the residue chromatographed on a silica gel column with ethyl

acetate:hexane (1:4, v/v) to give the t-Boc protected compound tert-

butyl-4-(3-(2-ﬂuoroethoxy)picolinoyl)piperidine-1-carboxylate (0.51

1

g, 40%). H NMR (CDCl₃): δ 1.47 (s, 9H), 1.62−1.70 (m, 4H),

2.83 (s, 2H), 3.64−3.73 (m, 1H), 4.17 (s br, 2H), 4.25−4.36 (m, 2H),

4.68−4.87 (m, 2H), 7.23−7.26 (m, 1H), 7.38−7.40 (m, 1H), 8.25−

8.30 (m, 1H).

To a solution of the above compound (0.49 g, 1.4 mmol) in CH₂Cl₂

(30 mL), TFA (2 mL) was added. The reaction mixture was stirred at

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8.7 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 8.15 (dd, J = 8.7, 2.4 Hz, 1H),

8.80 (d, J = 2.4 Hz, 1H). The free base was converted to the oxalate

s a l t ; m p 8 1 . 4 ° C ( d e c o m p o s e d ) . A n a l .

(C₂₂H₂₇N₃O₃·2H₂C₂O₄·1.5H₂O) C, H, N.

mm × 4.6 mm; mobile phase, 37% acetonitrile in 0.1 M ammonium

formate buﬀer pH = 4.5; ﬂow rate, 1.2 mL/min; the retention time for

(−)-[¹¹C]-24b was 4.9 min. The entire process was completed in

approximately 1 h. The radiochemical yield was 40−50% (decay

corrected to EOB) with the radiochemical purity of >99% and the

speciﬁc activity was >2000 Ci/mmol (decay corrected to end of

synthesis).

Log D Measurement. Partition coeﬃcient was measured by

mixing the (−)-[¹¹C]24b sample with 3 mL each of 1-octanol and

buﬀer that is 0.1 M phosphate and pH equals 7.4 in a test tube. The

mixture in the test tube were vortexed for 20 s followed by

centrifugation for 1 min at room temperature. Then 2 mL of organic

layer was transferred to a second test tube, and 1 mL of 1-octanol and

3 mL of PBS buﬀer were added. The resulting mixture was vortexed

for 20 s, followed by centrifugation for 1 min at room temperature.

Then 1 mL of organic and aqueous layer were taken separately for

measurement. The radioactivity content values (count per minute) of

two samples (1 mL each) from the 1-octanol and buﬀer layers were

counted in a gamma counter. The partition coeﬃcient Log D_7.4was

determined by calculating as the decimal logarithm the ratio of cpm/

mL of 1-octanol to that of buﬀer. The measurements were repeated

three times. The value of the partition coeﬃcient is 2.60.

In Vitro Biological Evaluation. Vesicular Acetylcholine Trans-

porter Binding Assays. In vitro binding assays to VAChT were

conducted with human VAChT permanently expressed in PC12 cells

at about 50 pmol/mg of crude extract. No signiﬁcant amounts of σ₁or

σ₂receptors were present. The radioligand used was 5 nM

(−)-[³H]vesamicol, and the assay was conducted at ﬁnal concen-

trations of 10⁻¹¹M to 10⁻⁵M novel compounds.^4,19Unlabeled

(−)-vesamicol was used as an external standard, for which K_i= 15 nM,

and the mixture was allowed to equilibrate at 23 °C for 20 h. Duplicate

data were averaged and ﬁtted by regression of a rectangular hyperbola

to estimate the K_ivalue of the novel compounds.

(1-(5-Amino-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidin-4-yl)(1-methyl-1H-pyrrol-2-yl)methanone (31a). Com-

pound 31a was prepared from compound 17a as describe in

1

procedure E. Yield 20%. H NMR (CDCl₃, free base): δ 1.85−1.96

(m, 4H), 2.36−2.45 (m, 2H), 2.76−2.89 (m 5H), 2.96−3.16 (m, 3H),

3.62 (s br, 2H), 3.92 (m, 1H), 3.96 (s, 3H), 6.15 (m, 1H), 6.56 (m,

2H), 6.83 (s br, 1H), 6.96−7.02 (m, 2H). The free base was converted

to the oxalate salt; mp 114 °C (decomposed). Anal.

(C₂₁H₂₇N₃O₂·2H₂C₂O₄·1.5H₂O) C, H, N.

tert-Butyl (4-(3-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-

piperidine-4-carbonyl)phenyl)-carbamate, (−)-33. To a solution

of (−)-6 (153.6 mg, 0.438 mmol) in THF was added di-tert-butyl

dicarbonate (Boc₂O, 287.0 mg, 1.314 mmol), Et₃N (0.5 mL), and

DMAP (5.4 mg, 0.044 mmol). The reaction mixture was stirred at

room temperature while monitoring by TLC. After 1.5 h, the starting

material completely disappeared, at which point the reaction mixture

was concentrated on a rotary evaporator and the residue partitioned

with brine and ethyl acetate. The aqueous phase was washed with ethyl

acetate. The organic phase was dried over sodium sulfate,

concentrated, and puriﬁed on a silica gel column (8:1 to 6:1

hexanes:ethyl acetate) to give the tri-Boc protected intermediate

(−)-32, which was dissolved in methanol, and excess K₂CO₃was

added and reﬂuxed overnight. The product was partitioned between

brine and dichloromethane. Aqueous phase was washed twice with

dichloromethane. The organic phase was dried over sodium sulfate

1

and concentrated to give (−)-33 as a white solid in 28% yield. H

NMR (400 MHz, CDCl₃): δ 1.55 (s, 9H), 1.78−1.95 (m, 4H), 2.42 (t,

J = 12.0 Hz, 1H), 2.78−3.05 (m, 7H), 3.25−3.35 (m, 2H), 3.83−3.90

(m, 1H), 6.73 (s, 1H), 7.08−7.07 (m, 4H), 7.46 (d, J = 11.6 Hz, 2H),

7.92 (d, J = 12.4 Hz, 2H).

Sigma Receptor Binding Assays. The compounds were dissolved

in DMF, DMSO, or ethanol and diluted in 50 mM Tris-HCl buﬀer

containing 150 mM NaCl and 100 mM EDTA at pH 7.4 prior to

performing the σ₁and σ₂receptor binding assays. The detailed

procedures for performing the binding assays have been described.^19,49

For the σ₁receptor assays, guinea pig brain membrane homogenates

(∼300 μg protein) were the receptor resource and ∼5 nM (+)- [³H]

pentazocine (34.9 Ci/mmol, Perkin-Elmer, Boston, MA) was the

radioligand. The incubation was performed in 96-well plates for 90

min at room temperature. Nonspeciﬁc binding was determined from

samples that contained 10 μM of nonradioactive haloperidol. After 90

min, the reaction was quenched by addition of 150 μL of ice-cold wash

buﬀer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). The harvested

samples were ﬁltered rapidly through a 96-well ﬁberglass ﬁlter plate

(Millipore, Billerica, MA) that had been presoaked with 100 μL of 50

mM Tris-HCl buﬀer at pH 8.0 for 60 min. Each ﬁlter was washed 3 ×

200 μL of ice-cold wash buﬀer and the ﬁlter counted in a Wallac 1450

MicroBeta liquid scintillation counter (Perkin-Elmer, Boston, MA).

The σ₂receptor binding assays were determined using rat liver

membrane homogenates (∼300 μg protein) and ∼5 nM [³H]DTG

(58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1 μM

of (+)-pentazocine to block σ₁sites. The incubation time was 120 min

at room temperature. Nonspeciﬁc binding was determined from

samples that contained 10 μM of nonradioactive haloperidol. All other

procedures were the same as those described for the σ₁receptor

binding assay above.

The IC₅₀value was determined using nonlinear regression analysis.

Competitive curves were best ﬁt with a one-site model and gave

pseudo-Hill coeﬃcients of 0.6−1.0. K_ivalues were calculated using the

method of Cheng and Prusoﬀ⁵⁰and are presented as the mean ( 1

SEM). For these calculations, we used a K_dvalue of 7.89 nM for

[³H](+)-pentazocine binding to σ₁receptor in guinea pig brain and a

K_dvalue of 30.7 nM for [³H]DTG binding to σ₂receptor in rat liver.

Biodistribution in Rats. All animal experiments were conducted

in compliance with the Guidelines for the Care and Use of Research

Animals established by Washington University’s Animal Studies

Committee. For the biodistribution studies, ∼350 μCi of (−)-[¹¹C]

Radiochemistry. [¹¹C]CH₃I was produced at our institution from

[¹¹C]CO₂using a GE PETtrace MeI Microlab. Up to 1.4 Ci of

[¹¹C]CO₂was produced from Washington University’s JSW BC-16/8

cyclotron by irradiating a gas target of 0.5% O₂in N₂for 15−30 min

with a 40 μA beam of 16 MeV protons. The GE PETtrace MeI

microlab coverts the [¹¹C]CO₂to [¹¹C]CH₄using a nickel catalyst

(Shimalite-Ni, Shimadzu, Japan PN221-27719) in the presence of

hydrogen gas at 360 °C; it was further converted to [¹¹C]CH₃I by

reaction with iodine that was held in a column in the gas phase at 690

°C. Several hundred millicuries of [¹¹C]CH₃I were delivered as a gas at

approximately 12 min after end of bombardment (EOB) to the hot

cell where the radiosynthesis was accomplished.

Radiosynthesis of (−)-[¹¹C]24b. Approximately 1.2−1.5 mg of

(−) enantiomer of the precursor was placed in a reaction vessel, and

0.2 mL of DMF was added followed by 3.0 μL of 5N aqueous sodium

hydroxide. The mixture was thoroughly mixed on a vortex. [¹¹C]CH₃I

was bubbled into the reaction vessel. The reaction mixture was heated

at 85 °C for 5 min. The reaction vessel was removed from heating, and

200 μL of triﬂuoroacetic acid was added. The reaction vessel was

mixed well and heated again at 85 °C for 5 min and then quenched by

adding 1.4 mL of HPLC mobile phase (29% acetonitrile in 0.1 M

ammonium formate buﬀer pH 4.5) and 200 μL of 5N aqueous NaOH

to neutralize the reaction mixture. The solution was loaded on to

reverse phase C-18 column (Phenomenex Luna C18, 250 mm × 9.6

mm, 10 μm with UV wavelength at 254 nm); using the above HPLC

mobile phase, at a ﬂow rate of 4.0 mL/min, the radioactive product

was collected between 17.0 and 19.0 min and diluted with 50 mL of

sterile water. The aqueous solution was passed through C18 Sep-Pak

Plus (to trap the product) by applying nitrogen pressure. The

collection bottle was rinsed by adding 10 mL of water and passed

through Sep-Pak plus. The product was collected in a vial by eluting

C18 Sep-Pak plus with 0.6 mL of EtOH and 5.4 mL of saline to

formulate the injection dose. The injection dose sample was

authenticated using an analytical HPLC system by coinjecting with

cold standard compound (−)-24b. The HPLC system for quality

control is: column, Phenomenex Prodigy C18 analytical column, 250

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24b in about 175 μL of 10% ethanol/saline solution (v/v) was injected

via the tail vein into mature male Sprague−Dawley rats (185−205 g)

under anesthesia (2.5% isoﬂurane in oxygen at a ﬂow rate of 1 mL/

min). A group of at least four rats were used for each time point. For

the control group, at 5 and 30 min post injection, the rats were

anesthetized and euthanized. For the CycA pretreated group, CycA

(Sandimmune diluted 1:1 with saline) at a dose of 25 mg/kg were

administrated by intravenously (iv) 30 min prior to radioligand

injection; at 30 min post injection of the radioligand, (−)-[¹¹C]24b,

the rats were euthanized. The whole brain was quickly harvested and

various organs dissected comprising cerebellum, brain stem, cortex,

striatum, thalamus, hippocampus, and the brain. The remainder of the

brain was also collected in order to determine total brain uptake.

Simultaneously, samples of blood, heart, lung, liver, spleen, pancreas,

kidney, muscle, fat, and tail were dissected. All the tissue samples were

collected in the tared tubes and counted in an automated gamma

counter (Beckman Gamma 8000 well counter) along with the standard

solution of (−)-[¹¹C]24b prepared by diluting the injectate. The

counted tissues were then weighed, and the %ID/g was calculated.

MicroPET Studies in Nonhuman Primate Brain. A microPET

Focus 220 scanner (Concorde/CTI/Siemens Microsystems, Knoxville,

TN) was used for the imaging studies of (−)-[¹¹C]24b in male

cynomolgus monkey (4−6 kg). The animals were fasted for 12 h

before the study. The animals were initially anesthetized using an

intramuscular injection with ketamine (10 mg/kg) and glycopyrulate

(0.13 mg/kg) and intubated with an endotracheal tube under

anesthesia (maintained at 0.75−2.0% isoﬂurane in oxygen) throughout

the PET scanning procedure. After intubation, a percutaneous venous

catheter was placed for radiotracer injection. A 10 min transmission

scan was performed to check the positioning; once conﬁrmed, a 45

min transmission scan was obtained for attenuation correction. After

that, the animal was administrated 5−7 mCi of (−)-[¹¹C]-24b via the

venous catheter, Subsequently, a 100 min dynamic PET scan (3 × 1

min, 4 × 2 min, 3 × 3 min, and 20 × 5 min) was acquired. During the

whole procedure, core temperature was kept constant at 37 °C with a

heated water blanket. In each microPET scanning session, the head

was positioned supine in the adjustable head holder with the brain in

the center of the ﬁeld of view. For each subject, at least three

independent PET studies were performed (n = 3).

Box 8225, 510 South Kingshighway Boulevard, St. Louis,

Missouri 63110, United States.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Research reported in this publication was supported by the

National Institute Of Neurological Disorders and Stroke of the

National Institutes of Health under award no. R01NS075527,

NS061025, The National Institute of Mental Health (NIMH)

under award no. MH092797, and McDonnell Center for

Systems Neuroscience of Washington University at St. Louis.

ABBREVIATIONS USED

■

ADME, absorption, distribution, metabolism, and excretion;

ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alz-

heimer’s disease; CNS, central nervous system; ABV, amino-

benzovesamicol; Anal, analysis; BBB, blood−brain barrier;

BBr₃, boron tribromide; n-BuLi, n-butyllithium; CDI, 1,1′-

carbonyldiimidazole; ChAT, choline acetyltransferase; CycA,

cyclosporine A; DMF, N,N-dimethylformamide; DMSO,

dimethyl sulfoxide; ( )FBBV, ( )-trans-2-hydroxy-3-(4-(4-

[¹⁸F]ﬂuorobenzoyl)piperidino)tetralin; IC₅₀, half-maximum

inhibitory constant;

[

^{1 2 3}I]IBVM, (−)-5-[^{1 2 3}I]-

iodobenzovesamicol; NBS, N-bromosuccinimide; PD, Parkin-

son’s Disease; PDD, Parkinson’s disease associated with

dementia; PET, positron emission tomography; P-gp, perme-

ability glycoprotein; σ₁receptor, sigma-1 receptor; σ₂receptor,

sigma-2 receptor; SPECT, single photon emission computed

tomography; TBDMSCl, tert-butyldimethylsilyl chloride; t-Boc,

tert-butyloxycarbonyl; THF, tetrahydrofuran; TLC, thin layer

chromatography; TFA, triﬂuoroacetic acid; TMSI, trimethylsilyl

iodide; VAChT, vesicular acetylcholine transporter

MicroPET Image Data Processing and Analysis. PET image

reconstructed resolution was <2.0 mm full width half-maximum for all

three dimensions at the center of the ﬁeld of view. Emission scans

were corrected using individual attenuation and model-based scatter

correction and reconstructed using ﬁltered back projection as

described previously.⁵¹The ﬁrst baseline PET image for each animal

acted as the target image with the MPRAGE MRI scan and subsequent

PETs coregistered to it using the automated image registration

program AIR.^52,53All MPRAGE-based volume of interest (VOI)

analyses were done by investigators blinded to the clinical status of the

monkeys.⁴For quantitative analyses, three-dimensional regions of

interest (ROI) (cerebellum, frontal, occipital, striatum, temporal, white

matter, midbrain, and hippocampus) were transformed to the baseline

PET space and then overlaid on all reconstructed PET images to

obtain time−activity curves. Activity measures were standardized to

body weight and dose of radioactivity injected to yield standardized

uptake value (SUV).

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ASSOCIATED CONTENT

■

S

* Supporting Information

Elemental analyses of new analogues and X-ray structural data

of 19i. This material is available free of charge via the Internet

at http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: 1-314-362-8487. Fax: 1-314-362-8555. E-mail: tuz@

mir.wustl.edu. Address: Zhude Tu, Ph.D., Department of

Radiology, Washington University School of Medicine, Campus

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NOTE ADDED AFTER ASAP PUBLICATION

■

After this paper was published online July 18, 2013, a correction

was made to Table 1. The corrected version was reposted July

22, 2013

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Heteroaromatic and aniline derivatives of piperidines as potent ligands for vesicular acetylcholine transporter

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