Novel triple reuptake inhibitors with low risk of CAD associated liabilities: Design, synthesis and biological activities of 4-[(1S)-1-(3,4- dichlorophenyl)-2-methoxyethyl]piperidine and related compounds

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx

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Bioorganic & Medicinal Chemistry

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Novel triple reuptake inhibitors with low risk of CAD associated

liabilities: Design, synthesis and biological activities of 4-[(1S)-1-(3,4-

dichlorophenyl)-2-methoxyethyl]piperidine and related compounds

Yuji Ishichi, Eiji Kimura, Eiji Honda, Masato Yoshikawa, Takashi Nakahata, Yasuko Terao, Atsuko Suzuki,

⇑

Takayuki Kawai, Yuuichi Arakawa, Hiroyuki Ohta, Naoyuki Kanzaki, Hideyuki Nakagawa, Jun Terauchi

Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 2-26-1 Muraoka-higashi, Fujisawa, Kanagawa 251-8555, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:

A novel triple reuptake inhibitor with low potential of liabilities associated with cationic amphiphilic

drug (CAD) was identiﬁed following an analysis of existing drugs. Low molecular weight (MW < ca.

300), low aromatic ring count (number = 1) and reduced lipophilicity (ClogP < 3.5) were hypothesized

to be key factors to avoid the CAD associated liabilities (CYP2D6 inhibition, hERG inhibition and phospho-

lipidosis). Based on the hypothesis, a series of piperidine compounds was designed with consideration of

the common characteristic features of CNS drugs. Optimization of the side chain by adjusting overall lipo-

philicity suggested that incorporation of a methoxymethyl group could provide compounds with a bal-

ance of both potent reuptake inhibition and low liability potential. Compound (S)-3a showed a potent

antidepressant-like effect in the mice tail suspension test (MED = 10 mg/kg, p.o.), proportional mono-

amine transporter occupancies and enhancement of monoamine concentrations in mouse prefrontal

cortex.

Received 25 April 2013

Revised 15 May 2013

Accepted 16 May 2013

Available online xxxx

Keywords:

Monoamine reuptake inhibition

Piperidine compounds

Cationic amphiphilic drug (CAD)

CYP2D6

hERG

PLsis

Tail suspension test

Antidepressant

1. Introduction

take of 5-HT, NE and DA has emerged as a broad-spectrum

antidepressant. TRI has been expected to show high efﬁcacy

Depression is a common mental disease which is characterized

by sadness, loss of interest in activities and decreased energy, and

it affects more than 350 million people worldwide.¹Among a num-

ber of hypotheses for the mechanism of depression, the mono-

amine hypothesis is one of the most widely recognized. Many of

drugs based on the monoamine hypothesis, for example, tricyclic

antidepressants (TCAs), selective serotonin reuptake inhibitor

(SSRI) and serotonin noradrenaline reuptake inhibitor (SNRI), have

been widely used. Among them, SSRI is the most widely prescribed

medication (e.g., Fluoxetine, Citalopram, Paroxetine and Sertra-

line). SSRIs enhance the activities of serotonin (5-HT) at the synap-

tic cleft and are effective with fewer and less severe side effects

than TCAs, however, there are some suggestion about reduced

effectiveness in refractory population and lower remission rates.²

Consequently, adding other monoamines to 5-HT was considered

as a rational approach to improved therapeutic potential. Dual 5-

HT and NE enhancement by SNRI (e.g., Duloxetine, Venlafaxine,

and Milnacipran) have also gained acceptance in the market. Fur-

thermore, ‘triple reuptake inhibitor (TRI)’ which inhibits the reup-

including for refractory population and high remission rate, with

less side effects especially for sexual dysfunction, and early onset

of action.³However, these classes of monoamine reuptake inhibi-

tors have characteristic liabilities, that is, CYP2D6, hERG inhibition

and phospholipidosis (PLsis). These undesirable properties could

be due to their CAD structures responsible for monoamine reup-

take inhibitory activities.

Based on background, our initial direction was to discover a no-

vel TRI compound with low risk of CAD associated liabilities.

2. Design and synthesis

2.1. Liability proﬁles of existing monoamine reuptake inhibitors

Prior to the design of novel compounds, typical existing

monoamine reuptake inhibitors and their liability proﬁles, such

as CYP2D6 inhibition, hERG inhibition and PLsis were evaluated⁴

(Fig. 1). The correlation between liability proﬁles and physico-

chemical properties such as molecular weight (MW) and ClogP⁵

value were investigated. Among the compounds listed in Figure

1, a few drugs with low risk of liability (i.e., Milnacipran and

⇑

Corresponding author. Tel.: +81 466 32 2911; fax: +81 466 29 4461.

E-mail address: jun.terauchi@takeda.com (J. Terauchi).

http://dx.doi.org/10.1016/j.bmc.2013.05.025

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

Figure 1. MW and ClogP values of some existing monoamine reuptake inhibitors (sorted by MW).

Bupropion) were recognized and these results were thought to be

related to their lower molecular weights and lipophilicites. Inter-

estingly, their MWs and three liability scores were correlated well,

and drugs with smaller MW eliminated both hERG and PLsis

potentials. Our MW limit was set at less than 300. Associated with

MW, restriction of the number of aromatic ring to one seemed to

be effective. In addition, it is well known that lipophilicity is a con-

siderable factor for these liabilities.⁶Indeed, drugs with lower

ClogP value seemed to have lower risk of liabilities than those with

higher ClogP even the MWs were comparable (e.g., Escitalopram

vs Paroxetine). Based on our internal analysis, desired ClogP was

set at below <3.5.

In addition to the framework demonstrated above, simple sym-

metrical structures were considered to be a favorable core tem-

plate to penetrate the blood–brain barrier (BBB) as well as

minimization of chiral centers. Effective employment of side chain

to core template is the most important way to adjust its lipophilic-

ity to avoid liabilities.

on the aromatic ring have an important role in potency and bal-

ance of reuptake inhibition. From our preliminary study, 3,4-di-

chloro moiety as R²gave better inhibition potencies to obtain

a TRI proﬁle. Therefore, in this document, mainly these ana-

logues are described.

2.3. Synthesis of piperidine compounds

Syntheses of a series of piperidine compounds are outlined in

Scheme 1. Phenyl acetonitriles 14a–e were condensed with 1-

Boc-4-piperidine 15 under basic conditions. The resulting double

bond of adducts 16a–e were reduced by sodium borohydride to

give intermediate 17a–e which were hydrolyzed by heating with

concentrated hydrobromic acid followed by re-introduction of

Boc group to yield carboxylic acids 18a–e. Partial hydrolysis

from nitrile 17a to the corresponding amide was feasible by

heating with 1 N-NaOH. Subsequent N-methylation afforded the

dimethyl amide 19a. The esters 20a and 21a were prepared by

alkylation of the carboxylic acid 18a with trimethylsilyl diazo-

methane or ethyl iodide respectively. Carboxylic acids 18a–e

were reduced with borane THF complex and the resulting alco-

hols 22a–e were alkylated with alkyl iodide to give ethers

23a–e and 24a. The hydroxy group of compound 22a was trans-

formed to the mesylate 25a, and substitution by hydride was

accomplished by lithium triethylborohydride⁸to give methyl

derivative 26a. Conversion of 25a to thiols 27a and 28a was car-

ried out using potassium thioacetate followed by hydrolysis with

1 N-NaOH and subsequent alkylation to afford sulﬁdes 27a and

28a. Sulfone 29a was synthesized by oxidizing sulﬁde 27a with

meta-chloroperoxybenzoic acid. Carboxylic acid 18a was con-

verted to the activated carbonyl compound 30a by treating with

1,1⁰-carbonyldiimidazole which was converted to ketones⁹31a–

33a by addition of corresponding alkyl magnesium bromides.

Alcohol derivative 22a was subjected to optical resolution by

chiral HPLC column chromatography, and following methylation

of hydroxy group gave optically active methyl ether analogues

((S)- and (R)-23a). Finally, each Boc protected precursors (26a,

22a–24a, 27a–29a, 31a–33a, 23b–e, (S)-23a and (R)-23a) were

deprotected and transformed to corresponding amine hydrochlo-

rides (1a–13a, 3b–e, (S)-3a and (R)-3a) by treating with hydro-

gen chloride in ethanol. Absolute conﬁgurations of (S)-3a was

determined by X-ray analysis and its ORTEP drawing was dem-

onstrated in Figure 3.

Recent antidepressant medication suggests that 5-HT and NE

reuptake inhibitions should be a primary therapy based on the

monoamine hypothesis. DA reuptake inhibition is expected to re-

duce refractory symptom and side-effects to which existing antide-

pressants could not respond well.³Therefore, as a target balance of

reuptake inhibition, DA reuptake inhibition was positioned to ren-

der 5-HT and NE reuptake inhibition.

2.2. Design of piperidine compounds

Considering above mentioned proﬁles, a series of 4-benzyl-

substituted piperidine based novel monoamine reuptake inhibi-

tors was designed (Fig. 2). Piperidine itself is symmetrical and

would be a convenient moiety to pass BBB.⁷The lipophilicity

of the molecule can be modiﬁed by selection of an appropriate

side chain (R¹) located on benzyl side chain. Substituents (R²)

Figure 2. Design of piperidine based core structure.

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

3

Scheme 1. Reagents and conditions: (a) 15, NaOMe, MeOH, reﬂux; (b) NaBH₄, MeOH; (c) concd HBr; (d) Boc₂O, 1 N-NaOH aq, DME; (e) 1 N-NaOH aq, EtOH; (f) NaH, DMF,

then MeI; (g) TMSCHN₂, MeOH; (h) EtI, K₂CO₃, DMF; (i) BH₃THF, THF; (j) NaH, DMF, then R⁴-I; (k) MsCl, Et₃N, THF; (l) LiBH(Et)₃, THF; (m) KSAc, acetone; (n) 1 N-NaOH aq,

EtOH; (o) NaH, DMF, then R⁵-I; (p) mCPBA, CH₂Cl₂; (q) CDI, THF; (r) R⁶MgBr, THF, 0 °C; (s) optical resolution by HPLC; (t) HCl, EtOH.

inhibition, hERG inhibition, PLsis score and minimum effective

dose (mg/kg, p.o.) of mouse tail suspension test (TST). The simple

methyl compound (1a) as a basis of these piperidine series showed

potent and well-balanced S/N/D reuptake inhibition. Introduction

of hydroxy group to the side chain (2a) resulted in signiﬁcant

reduction of each inhibition, though low risk of liabilities and well

balanced molecular proﬁles were observed. Methoxy methyl (3a)

and ethoxy methyl (4a) compounds showed similar potencies to

methyl derivative 1a. Substitution by sulfur atom instead of ether

oxygen increased all the potencies (compounds 5a, 6a) while sig-

niﬁcant loss of potency was observed by oxidizing the sulfur atom

Figure 3. ORTEP drawing and structure of (S)-3a, thermal ellipsoids are drawn at

20% probability.

to sulfone (compound 7a). The order of inhibition potentials ap-

3. Results and discussion

pears to be parallel to the ClogP values. Low potencies of alcohol

(2a), sulfone (7a), and high potencies of sulﬁdes (5a, 6a) could be

explained by their lipophilicites. Retained activities of esters (8a,

9a) and decreased activity of amide (10a) appeared to be correlate

with the ClogP. The ketone series (11a–13a) also exhibited high

activities while relative high potencies on DA reuptake inhibition

were remarkable compared with other compounds. Among these

3.1. SAR of side chain R, and substituents on Ar group

3,4-Dichlorophenyl compounds with various side chain R and

substituents on Ar group are listed in Table 1 together with data

obtain for 5-HT/NE/DA (S/N/D) reuptake inhibition,¹²CYP2D6

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

Table 1

Monoamine reuptake inh., CYP2D6 inh., hERG inh., PLsis score and TST of piperidine compounds with varied Ar and side chain R

Compd^aAr

R

Reuptake inh. IC₅₀(nM), 95% Cl^c

CYP2D6 inhibition^d

(%)

hERG inhibition^e

(%)

PLsis^f

(score)

TST^gMED (p.o.) (mg/ ClogP

kg)

b

SERT

NET

DAT

1a

2a

A

–Me

–CH₂OH

12 (11–14)

79 (65–97)

25 (19–32)

110 (99–

130)

160 (120–210)

2400 (1300–

4200)

27

ꢀ1.5

72

12

4.6

1.8

3

4.6

2.7

3a

4a

5a

6a

7a

A

–CH₂OMe

–CH₂OEt

–CH₂SMe

–CH₂SEt

–

11 (8.8–13)

16 (13–19)

9.1 (7.8–11)

9.2 (8.1–10)

340 (280–

14 (10–18)

28 (20–38)

9.3 (6.6–13)

8.7 (7.0–11)

200 (140–

300)

190 (120–290)

170 (120–250)

79 (56–110)

72 (51–100)

2200 (1400–

3300)

5.7

44

32

55

3.9

42

89

73

94

9.0

4.0

3.7

3.3

4.9

1.5

3

30

3.5

3.8

4.3

4.9

2.0

CH₂SO₂Me 410)

8a

9a

10a

A

–CO₂Me

–CO₂Et

–

CO₂NMe₂

–COMe

–COEt

5.2 (4.5–6.0) 18 (12–25)

9.3 (8.0–1.1) 12 (9.6–16)

220 (170–300)

97 (81–120)

500 (370–680)

13

38

ꢀ8.1

56

110

14

4.0

3.4

1.8

3.1

3.7

2.3

690 (550–

860)

120 (80–

190)

11a

12a

13a

3b

A

B

21 (17–26)

18 (14–22)

37 (29–47)

44 (32–61)

28 (22–37)

22 (16–31)

180 (150–230)

100 (76–150)

26 (19–36)

1100 (790–

1500)

11

6.1

20

22

40

51

72

38

3.4

3.2

4.3

2.2

30

2.7

3.3

3.8

3.3

–COnPr

–CH₂OMe

2.1 (1.8–2.6) 36 (19–67)

3c

3d

3e

C

D

E

–CH₂OMe

180 (150–

220)

120 (95–

160)

98 (54–180) 2600 (1800–

3700)

2.0

23

22

24

1.6

1.7

1.6

2.9

3.0

130 (68–

260)

1700 (1900–

3800)

ꢀ2.8

ꢀ18

32 (26–39)

70 (42–100)

580 (410–830)

a

b

c

d

e

f

All compounds were tested as their racemates.

These values were calculated from the results of two experiments.

95% conﬁdence interval for each IC₅₀value.

CYP2D6 inhibition expressed as its % inhibition value at the drug concentration of 10

hERG inhibition using IonWorks QuattroTM¹⁰, expressed as its% inhibition value at the drug concentration of 10

Phospholipidosis potential score measured by reported method¹¹with NBD-PE and HepG2 cells. In this method, higher score shows increased PLsis potential. Compounds

lM.

in this table exhibited lower potential.

g

Tail suspension test in mice expressed as minimum effective dose.

analogs, the propyl ketone (13a) showed equivalent potencies to-

ward all three reuptake inhibition.

nominated and examined for minimum effective dose (mg/kg).

The methyl (1a) and ether (3a, 4a) substituted compounds exhib-

ited potent antidepressant-like effects. Efﬁcacies of sulﬁde (5a)

and ketone (11a) were modest despite of their potent activities,

which could be due to their poor pharmacokinetic properties and

inadequate drug concentration at the CNS target organ. Among

the compounds, methyl ether derivative (3a) demonstrated the

most well-balanced properties in reuptake inhibition, in vivo

efﬁcacy and liabilities.

In most of cases, CYP2D6 inhibition and PLsis potential were

low. These results were consistent with our initial hypothesis

(small molecule and low lipophilicity). Though similar tendency

was observed for hERG inhibitions, this liability still remained de-

spite of reduction of lipophilicity.

To select compounds as candidates for further evaluation, TST

in mouse (p.o.) was performed to conﬁrm brain penetration as well

as potency for in vivo antidepressant-like efﬁcacies. Compounds

with potent S/N/D reuptake inhibition with lower liabilities were

Based on the results of side chain R, methyl ether compounds

with various aromatic moieties (Ar) were evaluated. 2-Naphthyl

Table 2

Monoamine reuptake inh., CYP2D6 inh., hERG inh., PLsis potential and TST of compound 3a and its optically active derivatives

Compd^aStereo

Reuptake inh. IC₅₀(nM), 95% Cl^c

CYP2D6 inhibition^d(%) hERG inhibition^e(%) PLsis^f(score) TST^gMED (p.o.) (mg/kg) ClogP

b

ꢁ

SERT

NET

DAT

(rac)-3a RS

11 (8.8–13)

14 (10–18) 190 (120–290) 5.7

42

48

46

4.0

2.7

3.1

3

10

3.5

(S)-3a

(R)-3a

S

R

130 (98–160) 15 (11–19) 180 (110–280) 14

5.7 (4.8–6.7) 21 (16–28) 470 (280–780) 13

a

Compounds (rac)-3a is a racemate. Others are optically active compounds possessing indicated absolute conﬁguration at 1-position.

b,c,d,e,f,g

See corresponding footnotes of Table 1.

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compound (3b) showed different balance of S/N/D reuptake inhibi-

tion which is similar to that of SNRI with relatively low DA reup-

take inhibition. Signiﬁcant decrease of each monoamine reuptake

inhibition in 3-Cl (3c) and 4-Cl (3d) was considered to be due to

their lower ClogP values compared with that of compound 3a.

Among the analogs, 3-Cl, 4-F derivative (3e) exhibited low poten-

tial of CYP2D6/hERG inhibition as well as PLsis with suitable S/N/

D balance. However, compound 3e, with ClogP value slightly lower

than that of compound 3a, showed inadequate potencies of

reuptake inhibitions. From these results, compound 3a was sub-

jected to optical resolution and further evaluations.

3.2. Results of optical resolution of compound (3a)

Compound 3a and corresponding optically active enantiomers

are listed in Table 2. 5-HT reuptake inhibition of (R)-3a was ca.

20 times stronger than that of (S)-3a, while NE reuptake property

was similar. DA reuptake inhibition was slightly enhanced in (S)-

Figure 4. Correlation between CYP2D6 (a), hERG (b) and PLsis (c) versus ClogP.

Figure 5. In vivo proﬁle of compound (S)-3a; (a) mouse tail suspension test, (b) monoamine transporter occupancies in mouse frontal cortex and striatum, (c–e) monoamine

concentration (5-HT (c), NE (d), DA (e)) in mouse prefrontal cortex measured via microdialysis.

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

3a compared with racemic 3a or (R)-3a. From these results, (S)-3a

was identiﬁed to be an eutomer toward NE and DA while a distom-

er toward 5-HT reuptake inhibition. Though balance of three

monoamine reuptake inhibition was different from that of (rac)-

3a, compound (S)-3a demonstrated acceptable potencies/balance

of S/N/D reuptake inhibition to exert TRI proﬁle (around 10 times

differences each other as its IC₅₀value) as well as low potential

of CYP2D6/hERG/PLsis.¹³After conﬁrmation of potent antidepres-

sant-like effect by TST in mice, compound (S)-3a was selected as

a candidate of further in vivo evaluation.

(300 MHz) spectrometers. Chemical shifts were given in ppm with

tetramethylsilane as the internal standard. The following abbrevi-

ations were used: s = singlet, d = doublet, t = triplet, q = quartet,

m = multiplet, dd = double doublet, ddd = double double doublet,

dt = double triplet, dq = double quartet, br = broad. Elemental anal-

yses (C, H, N) were measured on Vario EL (EL-04) instrument. Col-

umn chromatography was carried out using Merck silica gel 60

(63–200 lm). TLC was performed on silica gel 60 F₂₅₄(Merck),

and the spots were detected by UV light (wavelength 254 nm).

Abbreviations for solvents are the following: Et₂O, diethyl ether;

EtOH, ethanol; EtOAc, ethyl acetate; Hex, hexane; IPE, diisopropyl

ether; MeOH, methanol; THF, tetrahydrofuran; DMF, N,N-dimeth-

ylformamide; DMSO, dimethyl sulfoxide.

3.3. Relationship between ClogP and liability properties

Correlations between CYP2D6, hERG, PLsis and their ClogP va-

lue are shown in Figure 4a–c. Each value exhibited positive corre-

lation with ClogP value with correlation coefﬁcients of (0.63, 0.69,

0.63, respectively). Meanwhile, potential of monoamine reuptake

inhibition also showed parallel correlation with its ClogP value.

Consequently, activity/liability balance by careful selection of lipo-

philicity is a key point of this study. Safety margins of CYP2D6 and

PLsis appeared to be wide. Among the three liability properties,

hERG inhibition was the most intolerant, so the ClogP value had

to be limited below 3.5 to maintain low risk. Lipophilicity of

compound 3a (ClogP = 3.5) was a ﬁne balance of the activity/liabil-

ity in this series of compounds.

5.1.1. tert-Butyl 4-[cyano(3,4-dichlorophenyl)methylidene]pi

peridine-1-carboxylate (16a)

A

mixture of (3,4-dichlorophenyl)acetonitrile 14a (20.0 g,

108 mmol), tert-butyl 4-oxopiperidine-1-carboxylate 15 (21.5 g,

108 mmol) and 28% sodium methoxide methanol solution

(23.0 g, 119 mmol) in MeOH (250 mL) was stirred at reﬂux for

2 h. After cooling to room temperature, MeOH was removed in va-

cuo, and the residue was extracted with EtOAc, washed with water

and brine. The organic phase was dried over MgSO₄, concentrated

in vacuo. The residue was puriﬁed by column chromatography (sil-

ica gel, eluted with 5–50% EtOAc in hexane) to give 16a as a pale

yellow oil (26.6 g, 67%). ¹H NMR (300 MHz, CDCl₃) d 1.48 (9H, s),

2.41 (2H, t, J = 5.7 Hz), 2.76 (2H, t, J = 5.7 Hz), 3.43 (2H, t,

J = 5.7 Hz), 3.61 (2H, t, J = 5.7 Hz), 7.11 (1H, dd, J = 8.2, 2.1 Hz),

7.38 (1H, d, J = 2.1 Hz), 7.48 (1H, d, J = 8.2 Hz). MS m/z: 311

[M+HꢀtBu]⁺.

3.4. In vivo proﬁle of compound (S)-3a

As a candidate for a potential antidepressant, compound (S)-3a

was subjected to further in vivo evaluation. Dose dependent reduc-

tion of immobility rate in mouse TST¹⁴was conﬁrmed by p.o.

administration of (S)-3a (Fig. 5a). A statistically signiﬁcant de-

crease in immobility rate at 10 mg/kg, p.o. was indicated by para-

metric Williams’ test at P <.025 signiﬁcance level. Monoamine

transporter occupancies¹⁵in mouse frontal cortex and striatum

were evaluated after p.o. administration of (S)-3a (Fig. 5b). Each

occupancy for transporter was increased in dose dependent man-

ner with keeping comparable S/N/D ratio. Monoamine concentra-

tion in mouse prefrontal cortex was monitored by

microdialysis¹⁶(Fig. 5a–c). Each monoamine level was increased

during 120 min after oral administration of (S)-3a. The maximum

monoamine level was each 180% (5-HT), 400% (NE) and 270%

(DA) of basal concentrations. From these results, compound (S)-

3a was estimated to possess potential as an antidepressant.

The following compounds 16b–e were prepared in a similar

manner to that of 16a.

5.1.2. tert-Butyl 4-[cyano(naphthalen-2-yl)methylidene]

piperidine-1-carboxylate (16b)

55%, Colorless powder. ¹H NMR (300 MHz, CDCl₃) d 1.48 (9H, s),

2.51 (2H, t, J = 5.7 Hz), 2.82 (2H, t, J = 5.7 Hz), 3.44 (2H, t, J = 5.7 Hz),

3.64 (2H, t, J = 5.7 Hz), 7.37 (1H, dd, J = 8.2, 1.8 Hz), 7.49–7.56 (2H,

m), 7.75 (1H, d, J = 1.5 Hz), 7.80–7.90 (3H, m). MS m/z: 293

[M+HꢀtBu]⁺.

5.1.3. tert-Butyl 4-[(3-chlorophenyl)(cyano)methylidene]

piperidine-1-carboxylate (16c)

85%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 1.48 (9H, s),

2.38–2.46 (2H, m), 2.71–2.81 (2H, m), 3.39–3.48 (2H, m), 3.62

(2H, t, J = 5.8 Hz), 7.17 (1H, ddd, J = 4.9, 3.8, 1.7 Hz), 7.28 (1H, t,

J = 2.1 Hz), 7.32–7.40 (2H, m). MS m/z: 233 [M+HꢀBoc]⁺.

4. Conclusion

To discover triple reuptake inhibitor with low risk of liability

associated with CAD structure, a series of small molecule piperi-

dine derivatives was designed, synthesized and evaluated. Reup-

take inhibition and the liabilities were enhanced as the

lipophilicity of compounds increased. Lower MW with one aro-

matic ring and adjustment of ClogP value by selecting appropriate

side chain were achieved to satisfy both potent activities and low

liabilities. As the result of optimization and optical resolution,

(S)-3a was discovered as a candidate of triple reuptake inhibitor

which was effective against in vivo test for antidepressant.

5.1.4. tert-Butyl 4-[(4-chlorophenyl)(cyano)methylidene]

piperidine-1-carboxylate (16d)

84%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 1.48 (9H, s), 2.42

(2H, d, J = 6.0 Hz), 2.77 (2H, d, J = 5.8 Hz), 3.42 (2H, d, J = 6.0 Hz),

3.60 (2H, d, J = 6.0 Hz), 7.16–7.31 (2H, m), 7.32–7.46 (2H, m).

5.1.5. tert-Butyl 4-[(3-chloro-4-ﬂuorophenyl)(cyano)methy

lidene]piperidine-1-carboxylate (16e)

84%, Yellow oil. ¹H NMR (300 MHz, CDCl₃) d 1.48 (9H, s), 2.41

(2H, d, J = 6.0 Hz), 2.77 (2H, d, J = 6.0 Hz), 3.43 (2H, d, J = 6.0 Hz),

3.61 (2H, t, J = 5.8 Hz), 7.06–7.22 (2H, m), 7.34 (1H, dd, J = 6.9,

1.8 Hz).

5. Experimental section

5.1. Chemistry

5.1.6. tert-Butyl 4-[(RS)-cyano(3,4-dichlorophenyl)methyl]

piperidine-1-carboxylate (17a)

Melting points were determined on a Yanagimoto micro melt-

ing point apparatus and were uncorrected. ¹H NMR spectra were

taken on Varian Gemini 200 (200 MHz) or Mercury 300

Sodium borohydride (0.73 g, 19.3 mmol) was added portion-

wise to a solution of 16a (7.00 g, 19.1 mmol) in MeOH (100 mL)

at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

7

quenched with water. MeOH was removed in vacuo, and the resi-

due was extracted with EtOAc, washed with water and brine. The

organic phase was dried over MgSO₄, concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel, eluted

with 5–50% EtOAc in hexane) to give 16b as a colorless oil (6.08 g,

86%). ¹H NMR (300 MHz, CDCl₃) d 1.20–1.75 (3H, m), 1.45 (9H, s),

1.80–2.00 (2H, m), 2.50–2.75 (2H, m), 3.62 (1H, d, J = 6.6 Hz),

4.10–4.30 (2H, m), 7.14 (1H, dd, J = 8.2, 2.1 Hz), 7.39 (1H, d,

J = 2.1 Hz), 7.47 (1H, d, J = 8.2 Hz). MS m/z: 313 [M+HꢀtBu]⁺.

The following compounds 17b–e were prepared in a similar

manner to that of 17a.

(1H, m), 2.00–2.20 (1H, m), 2.50–2.80 (2H, m), 3.20 (1H, d,

J = 10.8 Hz), 3.90–4.20 (2H, m), 7.00–8.40 (1H, br), 7.16 (1H, dd,

J = 8.4, 2.4 Hz), 7.40 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 2.4 Hz). MS

m/z: 373 [M+HꢀH₂O]⁺. Anal. Calcd for C₁₈H₂₃Cl₂NO₄: C, 55.68; H,

5.97; N, 3.61. Found: C, 55.67; H, 5.87; N, 3.66.

The following compounds 18b–e were prepared in a similar

manner to that of 18a.

5.1.12. (2RS)-[1-(tert-Butoxycarbonyl)piperidin-4-yl]

(naphthalen-2-yl)ethanoic acid (18b)

68%, Colorless powder. ¹H NMR (300 MHz, CDCl₃) d 0.90–1.50

(3H, m), 1.42 (9H, s), 1.90 (1H, m), 2.24 (1H, m), 2.57 (1H, t,

J = 12.3 Hz), 2.75 (1H, t, J = 12.3 Hz), 3.41 (1H, d, J = 7.5 Hz), 3.80–

4.20 (2H, m), 7.40–7.50 (3H, m), 7.73 (1H, d, J = 1.2 Hz), 7.70–

7.90 (3H, m), 9.00–11.00 (1H, br). MS m/z: 314 [M+HꢀtBu]⁺.

5.1.7. tert-Butyl 4-[(RS)-cyano(naphthalen-2-yl)methyl]

piperidine-1-carboxylate (17b)

92%, Colorless crystals. Mp 130–132 °C (AcOEt–Hexane). ¹H

NMR (300 MHz, CDCl₃) d 1.20–1.70 (3H, m), 1.44 (9H, s), 1.86

(1H, m), 2.00 (1H, m), 2.63 (2H, m), 3.81 (1H, d, J = 7.2 Hz), 4.15

(2H, m), 7.36 (1H, dd, J = 8.4, 1.8 Hz), 7.49–7.58 (2H, m), 7.75

(1H, d, J = 1.5 Hz), 7.80–7.90 (3H, m). MS m/z: 295 [M+HꢀtBu]⁺.

5.1.13. (2RS)-[1-(tert-Butoxycarbonyl)piperidin-4-yl](3-

chlorophenyl)ethanoic acid (18c)

64%, Colorless crystals. Mp 155–156 °C (AcOEt–Hexane). ¹H

NMR (300 MHz, DMSO-d₆) d 0.90 (1H, dq, J = 12.3, 4.1 Hz), 1.01–

1.20 (2H, m), 1.37 (9H, s), 1.73 (1H, d, J = 12.2 Hz), 2.05 (1H, q,

J = 11.1 Hz), 2.54–2.82 (2H, m), 3.30 (1H, s), 3.75–4.03 (2H, m),

7.14–7.47 (4H, m), 12.52 (1H, br).

5.1.8. tert-Butyl 4-[(RS)-(3-chlorophenyl)(cyano)methyl]

piperidine-1-carboxylate (17c)

100%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 1.19–1.40 (2H,

m), 1.45 (9H, s), 1.51–1.59 (1H, m), 1.75–1.98 (2H, m), 2.64 (2H,

br), 3.62 (1H, d, J = 6.8 Hz), 4.06–4.26 (2H, m), 7.14–7.24 (1H, m),

7.28–7.31 (1H, m), 7.34 (2H, d, J = 5.3 Hz). MS m/z: 235

[M+HꢀBoc]⁺.

5.1.14. (2RS)-[1-(tert-Butoxycarbonyl)piperidin-4-yl](4-chloro

phenyl)ethanoic acid (18d)

82%, Colorless crystals. Mp 193–194 °C (AcOEt–Hexane). ¹H

NMR (300 MHz, DMSO-d₆) d 0.77–0.97 (1H, m), 0.99–1.20 (2H,

m), 1.37 (9H, s), 1.73 (1H, d, J = 12.6 Hz), 2.02 (1H, dd, J = 10.1,

7.3 Hz), 2.53–2.82 (2H, m), 3.30 (1H, d, J = 10.5 Hz), 3.82 (1H, d,

J = 12.6 Hz), 3.95 (1H, d, J = 13.6 Hz), 7.24–7.49 (4H, m), 12.49

(1H, br).

5.1.9. tert-Butyl 4-[(RS)-(4-chlorophenyl)(cyano)methyl]

piperidine-1-carboxylate (17d)

84%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 1.27–1.42 (2H,

m), 1.45 (9H, s), 1.51–1.56 (1H, m), 1.74–1.95 (2H, m), 2.63 (2H,

br), 3.62 (1H, d, J = 7.2 Hz), 4.03–4.28 (2H, m), 7.16–7.29 (2H, m),

7.32–7.42 (2H, m). MS m/z: 261 [MꢀtBuO]⁺.

5.1.15. (2RS)-[1-(tert-Butoxycarbonyl)piperidin-4-yl](3-chloro-

4-ﬂuorophenyl)ethanoic acid (18e)

5.1.10. tert-Butyl 4-[(RS)-(3-chloro-4-ﬂuorophenyl)(cyano)

methyl]piperidine-1-carboxylate (17e)

80%, Colorless crystals. Mp 181–182 °C (AcOEt–Hexane). ¹H

NMR (300 MHz, DMSO-d₆) d 0.77–0.99 (1H, m), 0.99–1.23 (2H,

m), 1.37 (9H, s), 1.72 (1H, d, J = 12.2 Hz), 1.90–2.13 (1H, m),

2.53–2.84 (2H, m), 3.36 (1H, s), 3.69–4.06 (2H, m), 7.18–7.45

(2H, m), 7.54 (1H, dd, J = 7.3, 2.0 Hz), 12.60 (1H, br). MS m/z: 271

[MꢀBoc]⁺.

77%, Yellow oil. ¹H NMR (300 MHz, CDCl₃) d 1.25–1.41 (2H, m),

1.45 (9H, s), 1.55 (1H, dt, J = 5.5, 2.7 Hz), 1.75–1.95 (2H, m), 2.64

(2H, br), 3.61 (1H, d, J = 7.2 Hz), 4.04–4.29 (2H, m), 7.18 (2H, dd,

J = 6.7, 1.0 Hz), 7.35 (1H, d, J = 7.0 Hz). MS m/z: 253 [M+HꢀBoc]⁺.

5.1.11. (2RS)-[1-(tert-Butoxycarbonyl)piperidin-4-yl](3,4-dich

lorophenyl)ethanoic acid (18a)

5.1.16. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-

48% Hydrobromic acid (15 mL) was added to 17a (1.64 g,

4.44 mmol) at room temperature. After stirring at reﬂux for 5 h,

the reaction mixture was concentrated in vacuo. The residue was

crystallized from water and 2-propanol to give (2RS)-(3,4-dichloro-

phenyl)(piperidin-4-yl)ethanoic acid hydrobromide as colorless

crystals (1.51 g, 92%). Mp 268–270 °C (H₂O–2-propanol). ¹H NMR

(300 MHz, DMSO-d₆) d 1.15–1.55 (3H, m), 1.80–1.95 (1H, m),

2.15–2.30 (1H, m), 2.70–3.00 (2H, m), 3.10–3.25 (1H, m), 3.25–

3.40 (1H, m), 3.44 (1H, d, J = 10.2 Hz), 7.30–7.40 (2H, m), 7.60–

7.70 (2H, m), 8.20–8.40 (1H, br), 8.55–8.75 (1H, br). MS m/z: 288

[M+H]⁺.

A solution of di-tert-butyl dicarbonate (650 mg, 2.96 mmol) in

1,2-dimethoxyethane (12 mL) was added dropwise to a mixture

of (2RS)-(3,4-dichlorophenyl)(piperidin-4-yl)ethanoic acid hydro-

bromide (1.00 g, 2.71 mmol) and 1 N-NaOH aq (5.42 mL). After

stirring at room temperature for 20 h, the reaction mixture was

concentrated in vacuo, and the pH was adjusted at ca. 3 using

10% citric acid aq. The residue was extracted with EtOAc, washed

with water and brine. The organic phase was dried over MgSO₄,

concentrated in vacuo to give 18a as colorless crystals (0.83 g,

79%). Mp 171–172 °C (EtOAc–Hexane). ¹H NMR (300 MHz, CDCl₃)

d 0.85–1.05 (1H, m), 1.20–1.35 (2H, m), 1.43 (9H, s), 1.80–1.90

(dimethylamino)-2-oxoethyl]piperidine-1-carboxylate (19a)

1 N-NaOH aq (50 mL) was added to a mixture of 17a (3.93 g,

10.6 mmol) and EtOH (50 mL). After being stirred at reﬂux for

4 h, EtOH was removed in vacuo. The residue was extracted with

EtOAc, washed with water and brine. The organic phase was dried

over MgSO₄, concentrated in vacuo to give tert-butyl 4-[(1RS)-2-

amino-1-(3,4-dichlorophenyl)-2-oxoethyl]piperidine-1-carboxyl-

ate as colorless crystals (2.81 g, 68%). Mp 231–232 °C (EtOAc–Hex-

ane). ¹H NMR (300 MHz, CDCl₃) d 0.85–1.05 (1H, m), 1.10–1.30

(2H, m), 1.44 (9H, s), 1.85–1.95 (1H, m), 2.05–2.25 (1H, m), 2.50–

2.80 (2H, m), 2.92 (1H, d, J = 10.2 Hz), 3.90–4.20 (2H, m), 5.35–

5.60 (2H, m), 7.19 (1H, dd, J = 8.4, 2.1 Hz), 7.39 (1H, d, J = 8.4 Hz),

7.44 (1H, d, J = 2.1 Hz). MS m/z: 287 [M+HꢀBoc]⁺. Anal. Calcd for

C₁₈H₂₄Cl₂N₂O₃: C, 55.82; H, 6.25; N, 7.23. Found: C, 55.80; H,

6.27; N, 7.03. NaH (ca. 60% in mineral oil, 250 mg, 6.25 mmol)

was added portionwise to a solution of tert-butyl 4-[(1RS)-2-ami-

no-1-(3,4-dichlorophenyl)-2-oxoethyl]piperidine-1-carboxylate

(600 mg, 1.55 mmol) in DMF (7.0 mL) at 0 °C. After being stirred at

0 °C for 5 min, methyl iodide (0.39 mL, 6.26 mmol) was added. The

mixture was stirred at room temperature overnight, and then

quenched with water. DMF was removed in vacuo, and the residue

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8

Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

was extracted with EtOAc, washed with water and brine. The or-

ganic phase was dried over MgSO₄, concentrated in vacuo. The res-

idue was puriﬁed by column chromatography (silica gel, eluted

with 5–100% EtOAc in hexane) to give 19a as a colorless oil

(422 mg, 66%). ¹H NMR (300 MHz, CDCl₃) d 0.85–1.25 (2H, m),

1.44 (9H, s), 1.70–1.90 (2H, m), 2.15–2.30 (1H, m), 2.50–2.80 (2H,

m), 2.93 (3H, s), 3.00 (3H, s), 3.37 (1H, d, J = 10.2 Hz), 3.90–4.20

(2H, m), 7.17 (1H, dd, J = 8.4, 2.1 Hz), 7.37 (1H, d, J = 8.4 Hz), 7.43

(1H, d, J = 2.1 Hz). MS m/z: 359 [M+HꢀtBu]⁺.

5.1.21. tert-Butyl 4-[(1RS)-1-(3-chlorophenyl)-2-hydroxyethyl]

piperidine-1-carboxylate (22c)

97%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 1.00 (1H, dq, J

=12.4, 4.2 Hz), 1.16–1.40 (3H, m), 1.43 (9H, s), 1.70–1.91 (2H, m),

2.45–2.76 (3H, m), 3.87 (3H, d, J = 8.0 Hz), 4.08–4.23 (1H, m),

6.99–7.11 (1H, m), 7.21 (1H, t, J = 1.9 Hz), 7.22–7.34 (2H, m). MS

m/z: 266 [MꢀtBuO]⁺.

5.1.22. tert-Butyl 4-[(1RS)-1-(4-chlorophenyl)-2-hydroxyethyl]

piperidine-1-carboxylate (22d)

92%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.98 (1H, dq,

J = 12.4, 4.3 Hz), 1.13–1.36 (3H, m), 1.43 (9H, s), 1.65–1.93 (2H,

m), 2.42–2.81 (3H, m), 3.76–4.05 (3H, m), 4.06–4.21 (1H, m),

7.13 (2H, d, J = 8.5 Hz), 7.31 (2H, d, J = 8.3 Hz). MS m/z: 266

[MꢀtBuO]⁺.

5.1.17. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-methoxy-2-

oxoethyl]piperidine-1-carboxylate (20a)

To a solution of 18a (100 mg, 0.257 mmol) in MeOH (3.0 mL)

was added 2.0 M trimethylsilyl diazomethane at 0 °C until the yel-

low color of the reagent was not disappeared. After being stirred at

room temperature for 15 min, the mixture was quenched with

small amount of acetic acid and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with

10–50% EtOAc in hexane) to give 20a as a colorless oil (99.2 mg,

96%). ¹H NMR (300 MHz, CDCl₃) d 0.82–1.06 (2H, m), 1.14–1.32

(2H, m), 1.44 (9H, s), 1.71 (1H, br), 2.05–2.18 (1H, m), 2.50–2.81

(2H, m), 3.20 (1H, d, J = 10.5 Hz), 3.67 (3H, s), 3.92–4.19 (1H, m),

7.16 (1H, dd, J = 8.3, 2.1 Hz), 7.34–7.45 (2H, m). MS m/z: 302

[M+HꢀBoc]⁺.

5.1.23. tert-Butyl 4-[(1RS)-1-(3-chloro-4-ﬂuorophenyl)-2-hydro

xyethyl]piperidine-1-carboxylate (22e)

92%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.99 (1H, dq,

J = 12.4, 4.3 Hz), 1.12–1.37 (3H, m), 1.43 (9H, s), 1.66–1.91 (2H,

m), 2.45–2.60 (2H, m), 2.60–2.78 (1H, m), 3.77–3.94 (2H, m),

4.01 (1H, d, J = 11.5 Hz), 4.07–4.18 (1H, m), 7.01–7.14 (2H, m),

7.24 (1H, dd, J = 7.2, 2.1 Hz). MS m/z: 284 [MꢀtBuO]⁺.

5.1.24. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-methox

yethyl]piperidine-1-carboxylate (23a)

5.1.18. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-ethoxy-2-

oxoethyl]piperidine-1-carboxylate (21a)

NaH (ca. 60% in mineral oil, 87 mg, 2.18 mmol) was added por-

tionwise to a solution of 22a (630 mg, 1.68 mmol) in DMF (5.0 mL)

at 0 °C. After being stirred at room temperature for 10 min, methyl

iodide (0.136 mL, 2.18 mmol) was added. The mixture was stirred

at room temperature for 20 min, and then quenched with water,

extracted with EtOAc, washed with water and brine. The organic

phase was dried over MgSO₄, concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with

5–50% EtOAc in hexane) to give 23a as a colorless oil (480 mg,

73%). ¹H NMR (300 MHz, CDCl₃) d 0.80–1.40 (3H, m), 1.43 (9H, s),

1.70–1.85 (2H, m), 2.45–2.75 (3H, m), 3.28 (3H, s), 3.55–3.65

(2H, m), 3.95–4.25 (2H, m), 7.02 (1H, dd, J = 8.2, 2.1 Hz), 7.28

(1H, d, J = 2.1 Hz), 7.35 (1H, d, J = 8.2 Hz). MS m/z: 374 [M+HꢀMe]⁺.

The following compounds 23b–e, 24a were prepared in a simi-

lar manner to that of 23a.

Anhydrous potassium carbonate (420 mg, 3.04 mmol) and

methyl iodide (0.24 mL, 3.00 mmol) was added to a solution of

18a (900 mg, 2.32 mmol) in DMF (10 mL) at room temperature.

After being stirred at room temperature overnight, the mixture

was quenched with water and concentrated in vacuo. The residue

was extracted with EtOAc, washed with water and brine. The or-

ganic phase was dried over MgSO₄, concentrated in vacuo. The res-

idue was puriﬁed by column chromatography (silica gel, eluted

with 5–100% EtOAc in hexane) to give 21a as a colorless oil

(920 mg, 95%). ¹H NMR (300 MHz, CDCl₃) d 0.85–1.05 (1H, m),

1.10–1.30 (2H, m), 1.22 (3H, t, J = 7.2 Hz), 1.44 (9H, s), 1.70–1.80

(1H, m), 2.00–2.20 (1H, m), 2.50–2.80 (2H, m), 3.18(1H, d,

J = 10.5 Hz), 3.90–4.25 (4H, m), 7.16 (1H, dd, J = 8.4, 2.1 Hz), 7.39

(1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 2.1 Hz). MS m/z: 316 [M+HꢀBoc]⁺.

5.1.19. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-hydrox

yethyl]piperidine-1-carboxylate (22a)

5.1.25. tert-Butyl 4-[(1RS)-2-methoxy-1-(naphthalen-2-yl)

ethyl]piperidine-1-carboxylate (23b)

70%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.95–1.40 (3H,

m), 1.41 (9H, s), 1.80–1.95 (2H, m), 2.45–2.85 (3H, m), 3.29 (3H,

s), 3.65–3.80 (2H, m), 3.85–4.25 (2H, m), 7.32 (1H, dd, J = 8.6,

1.8 Hz), 7.35–7.45 (2H, m), 7.59 (1H, s), 7.75–7.85 (3H, m). MS

m/z: 238 [M+HꢀBoc-MeO]⁺.

To a solution of 18a (3.00 g, 7.73 mmol) in THF (24 mL) was

added 1 M-BH₃/THF complex (24 mL, 24 mmol) at room tempera-

ture. After being stirred at 70 °C for 2 h, the mixture was quenched

with water and NH₄Cl aq, extracted with EtOAc, washed with

water and brine. The organic phase was dried over MgSO₄, concen-

trated in vacuo. The residue was puriﬁed by column chromatogra-

phy (silica gel, eluted with 5–50% EtOAc in hexane) to give 22a as a

colorless oil (2.00 g, 69%). ¹H NMR (300 MHz, CDCl₃) d 0.85–1.35

(3H, m), 1.43 (9H, s), 1.60–1.90 (3H, m), 2.40–2.80 (3H, m), 3.80–

4.20 (4H, m), 7.00–7.10 (1H, m), 7.25–7.30 (1H, m), 7.35–7.40

(1H, m). MS m/z: 318 [M+HꢀtBu]⁺.

5.1.26. tert-Butyl 4-[(1RS)-1-(3-chlorophenyl)-2-methoxyethyl]

piperidine-1-carboxylate (23c)

90%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.99 (1H, dq, J

=12.4, 4.5 Hz), 1.10–1.38 (2H, m), 1.43 (9H, s), 1.70–1.88 (2H, m),

2.47–2.77 (3H, m), 3.29 (3H, s), 3.62 (2H, dd, J = 5.9, 2.8 Hz), 3.98

(1H, br), 4.06–4.18 (1H, m), 7.01–7.08 (1H, m), 7.13–7.23 (3H,

m). MS m/z: 254 [M+HꢀBoc]⁺.

The following compounds 22b–e were prepared in a similar

manner to that of 22a.

5.1.20. tert-Butyl 4-[(1RS)-2-hydroxy-1-(naphthalen-2-yl)

ethyl]piperidine-1-carboxylate (22b)

5.1.27. tert-Butyl 4-[(1RS)-1-(4-chlorophenyl)-2-methoxyethyl]

piperidine-1-carboxylate (23d)

89%, Colorless powder. ¹H NMR (300 MHz, CDCl₃) d 0.95–1.40

(3H, m), 1.42 (9H, s), 1.60–1.95 (3H, m), 2.45–2.80 (3H, m), 3.85–

4.05 (3H, m), 4.05–4.25 (1H, m), 7.31(1H, d, J = 8.4 Hz), 7.40–7.50

(2H, m), 7.62 (1H, s), 7.75–7.85 (3H, m). MS m/z: 238 [M+HꢀBoc-

OH]⁺.

88%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.89–1.05 (1H,

m), 1.12–1.37 (3H, m), 1.38–1.48 (9H, m), 1.67–1.87 (2H, m),

2.46–2.61 (2H, m), 2.68 (1H, t, J = 12.6 Hz), 3.23–3.33 (3H, m),

3.50–3.69 (2H, m), 3.99 (1H, br), 7.06–7.16 (2H, m), 7.19–7.29

(2H, m). MS m/z: 254 [M+HꢀBoc]⁺.

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

9

5.1.28. tert-Butyl 4-[(1RS)-1-(3-chloro-4-ﬂuorophenyl)-2-meth

oxyethyl]piperidine-1-carboxylate (23e)

[(1RS)-2-(acetylsulfanyl)-1-(3,4-dichlorophenyl)ethyl]piperidine-

1-carboxylate (3.70 g, 8.56 mmol) and 1 N-NaOH aq (20 mL,

20 mmol) was stirred at room temperature for 40 min. After re-

moval of EtOH in vacuo, water was added and the mixture was ex-

tracted with EtOAc, washed with water and brine. The organic

phase was dried over MgSO₄, concentrated in vacuo to give tert-bu-

tyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-sulfanylethyl]piperidine-1-

carboxylate as a colorless oil (3.13 g, 94%). ¹H NMR (300 MHz,

CDCl₃) d 0.90–1.60 (4H, m), 1.43 (9H, s), 1.60–1.90 (2H, m), 2.45–

3.00 (5H, m), 3.95–4.25 (2H, m), 6.97 (1H, dd, J = 8.2, 2.1 Hz),

7.22 (1H, d, J = 2.1 Hz), 7.39 (1H, d, J = 8.2 Hz). MS m/z: 334

[M+HꢀtBu]⁺. NaH (ca. 60% in mineral oil, 180 mg, 4.50 mmol)

was added portionwise to a solution of tert-butyl 4-[(1RS)-1-(3,4-

dichlorophenyl)-2-sulfanylethyl]piperidine-1-carboxylate (1.49 g,

3.82 mmol) in DMF (20 mL) at 0 °C. After being stirred at room

temperature for 2 min, methyl iodide (0.285 mL, 4.57 mmol) was

added. The mixture was stirred at room temperature for 1 h, and

then quenched with water, extracted with EtOAc, washed with

water and brine. The organic phase was dried over MgSO₄, concen-

trated in vacuo. The residue was puriﬁed by column chromatogra-

phy (silica gel, eluted with 5–50% EtOAc in hexane) to give 27a as a

colorless oil (923 mg, 60%). ¹H NMR (300 MHz, CDCl₃) d 0.90–1.60

(4H, m), 1.43 (9H, s), 1.60–1.90 (2H, m), 2.00 (3H, s), 2.50–2.80 (3H,

m), 2.93 (1H, dd, J = 12.4, 5.1 Hz), 3.95–4.25 (2H, m), 6.98 (1H, dd,

J = 8.2, 2.1 Hz), 7.23 (1H, d, J = 2.1 Hz), 7.37 (1H, d, J = 8.2 Hz). MS

m/z: 304 [M+HꢀBoc]⁺.

78%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.89–1.08 (1H,

m), 1.10–1.36 (2H, m), 1.40–1.46 (9H, m), 1.68–1.86 (2H, m),

2.47–2.59 (2H, m), 2.59–2.74 (1H, m), 3.29 (3H, s), 3.53–3.67

(2H, m), 3.94–4.07 (1H, m), 4.08–4.18 (1H, m), 6.99–7.08 (2H,

m), 7.22 (1H, dd, J = 7.2, 1.5 Hz). MS m/z: 272 [M+HꢀBoc]⁺.

5.1.29. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-ethoxy

ethyl]piperidine-1-carboxylate (24a)

69%, Pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 0.80–1.60 (3H,

m), 1.14 (3H, t, J = 7.2 Hz), 1.43 (9H, s), 1.70–1.90 (2H, m), 2.45–

2.80 (3H, m), 3.41 (2H, q, J = 7.2 Hz), 3.55–3.70 (2H, m), 3.90–

4.30 (2H, m), 7.02 (1H, dd, J = 8.2, 2.1 Hz), 7.29 (1H, d, J = 2.1 Hz),

7.34 (1H, d, J = 8.2 Hz). MS m/z: 346 [M+HꢀtBu]⁺.

5.1.30. tert-Butyl 4-{(1RS)-1-(3,4-dichlorophenyl)-2-

[(methylsulfonyl)oxy]ethyl}piperidine-1-carboxylate (25a)

Methanesulfonyl chloride (0.92 mL, 11.9 mmol) was added

dropwise to a solution of 22a (4.05 g, 10.8 mmol) in THF (80 mL)

at 0 °C. After being stirred at room temperature for 1 h, the mixture

was quenched with water, extracted with EtOAc, washed with

water and brine. The organic phase was dried over MgSO₄, concen-

trated in vacuo. The residue was puriﬁed by column chromatogra-

phy (silica gel, eluted with 5–50% EtOAc in hexane) to give 25a as a

colorless oil (4.57 g, 94%). ¹H NMR (300 MHz, CDCl₃) d 0.90–1.40

(3H, m), 1.43 (9H, s), 1.75–1.90 (1H, m), 2.50–2.80 (3H, m), 2.87

(3H, s), 3.95–4.25 (2H, m), 4.40–4.45 (3H, m), 7.02 (1H, dd,

J = 8.2, 2.1 Hz), 7.26 (1H, d, J = 2.1 Hz), 7.41 (1H, d, J = 8.2 Hz). MS

m/z: 396 [M+HꢀtBu]⁺.

The following compound 28a was prepared in a similar manner

to that of 27a.

5.1.33. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-(ethylsul

fanyl)ethyl]piperidine-1-carboxylate (28a)

78%, Colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.90–1.60 (3H,

m), 1.19 (3H, t, J = 7.5 Hz), 1.43 (9H, s), 1.60–1.90 (2H, m), 2.42

(2H, q, J = 7.5 Hz), 2.50–2.80 (4H, m), 2.94 (1H, dd, J = 12.6,

5.1 Hz), 3.95–4.25 (2H, m), 6.98 (1H, dd, J = 8.2, 2.1 Hz), 7.23 (1H,

d, J = 2.1 Hz), 7.37 (1H, d, J = 8.2 Hz). MS m/z: 318 [M+HꢀBoc]⁺.

5.1.31. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)

ethyl]piperidine-1-carboxylate (26a)

To a solution of 25a (380 mg, 0.84 mmol) in THF (5.0 mL) was

added lithium triethylborohydride (2.47 mL, 4.20 mmol) dropwise

at room temperature. After stirring at room temperature under ar-

gon gas for 20 min, the mixture was quenched with NH₄Cl aq and

extracted with EtOAc. The organic layer was separated, washed

with water and brine, dried over MgSO₄and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel,

eluted with 0–50% EtOAc in hexane) to give 26a as a colorless oil

(250 mg, 83%). ¹H NMR (300 MHz, CDCl₃) d 0.91–1.19 (3H, m),

1.23 (3H, d, J = 7.2 Hz), 1.28–1.37 (1H, m), 1.38–1.54

(10H, m), 1.80 (1H, d, J = 12.8 Hz), 2.46–2.74 (2H, m), 3.92–4.26

(2H, m), 6.97 (1H, dd, J = 8.3, 1.9 Hz), 7.22 (1H, d, J = 1.9 Hz), 7.35

(1H, d, J = 8.3 Hz). MS m/z: 302 [M+HꢀtBu]⁺.

5.1.34. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-(methyl

sulfonyl)ethyl]piperidine-1-carboxylate (29a)

To a solution of 27a (515 mg, 1.27 mmol) in dichloromethane

(10 mL) was added m-chloroperoxybenzoic acid (ca. 70% with

water, 940 mg, 3.81 mmol) at room temperature. After being stir-

red at room temperature for 1 h, the mixture was quenched with

water, extracted with EtOAc, washed with water and brine. The or-

ganic phase was dried over MgSO₄, concentrated in vacuo. The res-

idue was puriﬁed by column chromatography (silica gel, eluted

with 5–100% EtOAc in hexane) to give 29a as a colorless oil

(420 mg, 75%). ¹H NMR (300 MHz, CDCl₃) d 0.95–1.40 (3H, m),

1.43 (9H, s), 1.60–1.85 (2H, m), 2.45–2.80 (2H, m), 2.51 (3H, s),

3.10 (1H, dt, J = 8.6, 4.5 Hz), 3.30–3.45 (2H, m), 3.95–4.25 (2H,

m), 7.05 (1H, dd, J = 8.2, 2.1 Hz), 7.30 (1H, d, J = 2.1 Hz), 7.44 (1H,

d, J = 8.2 Hz). MS m/z: 380 [M+HꢀtBu]⁺.

5.1.32. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-(methy

lsulfanyl)ethyl]piperidine-1-carboxylate (27a)

A mixture of 25a (4.55 g, 10.1 mmol) and potassium thioacetate

(1.73 g, 15.1 mmol) in acetone (100 mL) was stirred at room tem-

perature for 20 h and at reﬂux for 30 min. After quench with water,

acetone was removed in vacuo and the residue was extracted with

EtOAc, washed with water and brine. The organic phase was dried

over MgSO₄, concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 5–50% EtOAc in

hexane) to give tert-butyl 4-[(1RS)-2-(acetylsulfanyl)-1-(3,4-

dichlorophenyl)ethyl]piperidine-1-carboxylate as a colorless oil

(3.73 g, 85%). ¹H NMR (300 MHz, CDCl₃) d 0.90–1.10 (1H, m),

1.15–1.40 (2H, m), 1.43 (9H, s), 1.60–1.75 (1H, m), 1.85–1.95

(1H, m), 2.26 (3H, s), 2.50–2.80 (3H, m), 2.96 (1H, dd, J = 13.2,

10.8 Hz), 3.48 (1H, dd, J = 13.2, 4.8 Hz), 3.95–4.25 (2H, m), 6.93

(1H, dd, J = 8.2, 2.1 Hz), 7.17 (1H, d, J = 2.1 Hz), 7.36 (1H, d,

J = 8.2 Hz). MS m/z: 332 [M+HꢀBoc]⁺. A mixture of tert-butyl 4-

5.1.35. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-(1H-imida

zol-1-yl)-2-oxoethyl]piperidine-1-carboxylate (30a)

A mixture of 18a (7.00 g, 18.0 mmol) and 1,1⁰-carbonyldiimid-

azole (3.52 g, 21.7 mmol) in THF (100 mL) was stirred at room tem-

perature for 2 h. The solvent was removed in vacuo, and the

residue was puriﬁed by column chromatography (silica gel, eluted

with 5–100% EtOAc in hexane) to give 30a as a colorless oil (6.05 g,

77%). ¹H NMR (300 MHz, CDCl₃) d 0.90–1.30 (3H, m), 1.44 (9H, s),

1.75–1.90 (1H, m), 2.25–2.45 (1H, m), 2.50–2.80 (2H, m),

3.76(1H, d, J = 9.9 Hz), 3.90–4.25 (2H, m), 7.06 (1H, s), 7.15 (1H,

dd, J = 7.8, 2.1 Hz), 7.25 (1H, s), 7.40–7.50 (2H, m), 8.18 (1H, s).

Please cite this article in press as: Ishichi, Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.05.025

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

5.1.36. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-oxopropyl]

piperidine-1-carboxylate (31a)

Anal. Calcd for C₁₉H₂₇Cl₂NO₃: C, 58.77; H, 7.01; N, 3.61. Found: C,

58.81; H, 6.88; N, 3.59.

To a solution of 30a (2.00 g, 4.56 mmol) was added 3 M-methyl

magnesium bromide diethyl ether solution (2.28 mL, 6.84 mmol)

dropwise at 0 °C. After stirring at 0 °C for 5 min, the mixture was

quenched with water, extracted with EtOAc, washed with water,

10% citric acid aq and brine successively. The organic phase was

dried over MgSO₄, concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel, eluted with 5–100% EtOAc

in hexane) to give 31a as a colorless oil (718 mg, 41%). ¹H NMR

(300 MHz, CDCl₃) d 0.85–1.30 (3H, m), 1.43 (9H, s), 1.70–1.80

(1H, m), 2.10–2.25 (1H, m), 2.11 (3H, s), 2.50–2.80 (2H, m), 3.37

(1H, d, J = 9.9 Hz), 3.90–4.25 (2H, m), 7.05 (1H, dd, J = 8.4, 2.1 Hz),

7.31 (1H, d, J = 2.1 Hz), 7.39 (1H, d, J = 8.4 Hz). MS m/z: 286

[M+HꢀBoc]⁺.

5.1.40. 4-[(1RS)-1-(3,4-Dichlorophenyl)-2-methoxyethyl]

piperidine monohydrochloride (3a)

To a solution of 23a (480 mg, 1.24 mmol) in EtOH (10 mL) was

added 10 N-hydrogen chloride in EtOH (10 mL) at room tempera-

ture. After stirring at room temperature for 15 min, the solvent

was removed in vacuo. The residue was crystallized from EtOH–

EtOAc to give 3a as colorless crystals (351 mg, 87%). Mp 187–

188 °C. ¹H NMR (300 MHz, DMSO-d₆) d 1.10–1.50 (3H, m), 1.80–

2.00 (2H, m), 2.60–2.85 (3H, m), 3.05–3.30 (2H, m), 3.19 (3H, s),

3.50–3.65 (2H, m), 7.23 (1H, dd, J = 8.4, 2.1 Hz), 7.51 (1H, d,

J = 2.1 Hz), 7.56 (1H, d, J = 8.4 Hz), 8.50–9.20 (2H, br). MS m/z:

288 [M+H]⁺. Anal. Calcd for C₁₄H₁₉Cl₂NOꢃHCl: C, 51.79; H, 6.21;

N, 4.31. Found: C, 51.81; H, 6.32; N, 4.23.

The following compounds 32–33a were prepared in a similar

manner to that of 31a.

The following compounds 1–2a, 4–13a, 3b–e, (S)-3a and (R)-3a

were prepared in a similar manner to that of 3a from correspond-

ing Boc precursors 26a, 22–22a, 24a, 27–29a, 31–33a, 23b–e, (S)-

23a and (R)-23a.

5.1.37. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-oxobutyl]

piperidine-1-carboxylate (32a)

38%, Pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 0.85–1.30 (3H,

m), 0.98 (3H, t, J = 7.2 Hz), 1.43 (9H, s), 1.65–1.80 (1H, m), 2.10–

2.25 (1H, m), 2.30–2.80 (4H, m), 3.36(1H, d, J = 10.5 Hz), 3.90–

4.25 (2H, m), 7.07 (1H, dd, J = 8.4, 2.1 Hz), 7.33 (1H, d, J = 2.1 Hz),

7.38 (1H, d, J = 8.4 Hz). MS m/z: 344 [M+HꢀtBu]⁺.

5.1.41. 4-[(1RS)-1-(3,4-Dichlorophenyl)ethyl]piperidine

monohydrochloride (1a)

88% from 26a, Colorless crystals. Mp 170–171 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.19 (3H, d, J = 7.2 Hz), 1.21–1.46 (3H, m),

1.58–1.79 (1H, m), 1.90 (1H, d, J = 13.6 Hz), 2.53–2.87 (3H, m),

3.14 (1H, d, J = 12.5 Hz), 3.25 (1H, d, J = 12.5 Hz), 7.22 (1H, dd,

J = 8.3, 2.3 Hz), 7.49 (1H, d, J = 1.9 Hz), 7.57 (1H, d, J = 6.0 Hz),

8.67 (1H, br), 9.01 (1H, br). Anal. Calcd for C₁₃H₁₇Cl₂NꢃHCl: C,

52.99; H, 6.16; N, 4.75. Found: C, 52.93; H, 5.88; N, 4.73.

5.1.38. tert-Butyl 4-[(1RS)-1-(3,4-dichlorophenyl)-2-oxopentyl]

piperidine-1-carboxylate (33a)

43%, Pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 0.80–1.25 (6H,

m), 1.40–1.65 (2H, m), 1.43 (9H, s), 1.65–1.80 (1H, m), 2.10–2.25

(1H, m), 2.30–2.40 (2H, m), 2.50–2.80 (2H, m), 3.35(1H, d,

J = 10.5 Hz), 3.90–4.25 (2H, m), 7.06 (1H, dd, J = 8.4, 2.1 Hz), 7.32

(1H, d, J = 2.1 Hz), 7.38 (1H, d, J = 8.4 Hz). MS m/z: 314 [M+HꢀBoc]⁺.

5.1.42. (2RS)-2-(3,4-Dichlorophenyl)-2-(piperidin-4-yl)ethanol

monohydrochloride (2a)

93% from 22a, Colorless crystals. Mp 223–224 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.10–1.50 (3H, m), 1.80–2.05 (2H, m),

2.40–2.60 (1H, m), 2.60–2.90 (2H, m), 3.05–3.30 (2H, m), 3.60–

3.75 (2H, m), 4.50–4.90 (1H, br), 7.21 (1H, dd, J = 8.4, 2.1 Hz),

7.49 (1H, d, J = 2.1 Hz), 7.55 (1H, d, J = 8.4 Hz), 8.40-9.20 (2H, br).

MS m/z: 274 [M+H]⁺. Anal. Calcd for C₁₃H₁₇Cl₂NOꢃHCl: C, 50.26;

H, 5.84; N, 4.51. Found: C, 50.17; H, 5.88; N, 4.50.

5.1.39. tert-Butyl 4-[(1S)-1-(3,4-dichlorophenyl)-2-methoxy

ethyl]piperidine-1-carboxylate ((S)-23a) tert-butyl 4-[(1R)-1-

(3,4-dichlorophenyl)-2-methoxyethyl]piperidine-1-carboxylate

((R)-23a)

Compound 22a (12.7 g, 33.9 mmol) was separated by HPLC

(CHIRALPAK AD, 50 mmID ꢂ 500 mmL, manufactured by DAICEL

CHEMICAL INDUSTRIES, Ltd, mobile phase: hexane/2-propa-

nol = 900/100, ﬂow rate: 80 mL/min, column temperature 30 °C)

to give tert-butyl 4-[(1S)-1-(3,4-dichlorophenyl)-2-hydroxy-

ethyl]piperidine-1-carboxylate (5.72 g, >99% ee, recovery rate

90%) having a longer retention time and tert-butyl 4-[(1R)-1-(3,4-

dichlorophenyl)-2-hydroxyethyl]piperidine-1-carboxylate (6.05 g,

>99% ee, recovery rate 96%) having a shorter retention time.

NaH (ca. 60% in mineral oil, 795 mg, 19.9 mmol) was added por-

tionwise to a solution of tert-butyl 4-[(1S)-1-(3,4-dichlorophenyl)-

2-hydroxyethyl]piperidine-1-carboxylate (5.72 g, 15.3 mmol) in

DMF (50 mL) at 0 °C. After being stirred at room temperature for

10 min, methyl iodide (1.24 mL, 19.9 mmol) was added. The mix-

ture was stirred at room temperature for 20 min, and then

quenched with water, extracted with EtOAc, washed with water

and brine. The organic phase was dried over MgSO₄, concentrated

in vacuo. The residue was puriﬁed by column chromatography (sil-

ica gel, eluted with 5–50% EtOAc in hexane) to give (S)-23a as col-

orless crystals (3.00 g, 50%). Mp 100–101 °C (AcOEt–Hexane). ¹H

NMR (300 MHz, CDCl₃): identical to that of 23a. Anal. Calcd for

5.1.43. 4-[(1RS)-1-(3,4-Dichlorophenyl)-2-

ethoxyethyl]piperidine monohydrochloride (4a)

82% from 24a, Colorless crystals. Mp 157–158 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.04 (3H, t, J = 6.9 Hz), 1.10–1.50 (3H, m),

1.80–2.00 (2H, m), 2.60–2.85 (3H, m), 3.10–3.30 (2H, m), 3.38

(2H, q, J = 6.9 Hz), 3.55–3.70 (2H, m), 7.23 (1H, dd, J = 8.4, 2.1 Hz),

7.52 (1H, d, J = 2.1 Hz), 7.56 (1H, d, J = 8.4 Hz), 8.20–9.20 (2H, br).

MS m/z: 302 [M+H]⁺. Anal. Calcd for C₁₅H₂₁Cl₂NOꢃHCl: C, 53.19;

H, 6.55; N, 4.14. Found: C, 53.17; H, 6.56; N, 4.09.

5.1.44. 4-[(1RS)-1-(3,4-Dichlorophenyl)-2-(methylsulfanyl)

ethyl]piperidine monohydrochloride (5a)

81% from 27a, Colorless crystals. Mp 176–178 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.10–1.50 (3H, m), 1.80–2.00 (2H, m),

1.96 (3H, s), 2.60–3.00 (5H, m), 3.05–3.30 (2H, m), 7.23 (1H, dd,

J = 8.2, 1.8 Hz), 7.53 (1H, d, J = 1.8 Hz), 7.56 (1H, d, J = 8.2 Hz),

8.40–8.70 (1H, br), 8.90–9.20 (1H, br). MS m/z: 304 [M+H]⁺. Anal.

Calcd for C₁₄H₁₉Cl₂NSꢃHCl: C, 49.35; H, 5.92; N, 4.11. Found: C,

49.27; H, 5.93; N, 3.87.

C₁₉H₂₇Cl₂NO₃: C, 58.77; H, 7.01; N, 3.61. Found: C, 58.75; H,

6.90; N, 3.56.

5.1.45. 4-[(1RS)-1-(3,4-Dichlorophenyl)-2-(ethylsulfanyl)

ethyl]piperidine monohydrochloride (6a)

Compound (R)-23a was prepared from tert-butyl 4-[(1R)-1-

(3,4-dichlorophenyl)-2-hydroxyethyl]piperidine-1-carboxylate in

a similar manner to that of (S)-23a. 69%, Colorless crystals. Mp

100–101 °C. ¹H NMR (300 MHz, CDCl₃): identical to that of 23a.

70% from 28a, Colorless crystals. Mp 167–169 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.11 (3H, t, J = 7.5 Hz), 1.15–1.50 (3H, m),

1.80–2.00 (2H, m), 2.40 (2H, q, J = 7.5 Hz), 2.60–3.00 (5H, m),

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Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

11

3.15–3.30 (2H, m), 7.23 (1H, dd, J = 8.4, 2.1 Hz), 7.52 (1H, d,

5.1.52. (1RS)-1-(3,4-Dichlorophenyl)-1-(piperidin-4-yl)pentan-

J = 2.1 Hz), 7.56 (1H, d, J = 8.4 Hz), 8.60–8.85 (1H, br), 9.05–9.25

(1H, br). MS m/z: 318 [M+H]⁺. Anal. Calcd for C₁₅H₂₁Cl₂NSꢃHCl: C,

50.78; H, 6.25; N, 3.95. Found: C, 50.78; H, 6.27; N, 3.80.

2-one monohydrochloride (13a)

79% from 32a, Colorless crystals. Mp 196–197 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 0.72 (3H, t, J = 7.5 Hz), 1.10–1.30 (2H, m),

1.35–1.55 (3H, m), 1.65–1.80 (1H, m), 2.20–2.40 (1H, m), 2.42

(2H, q, J = 7.5 Hz), 2.65–2.90 (2H, m), 3.05–3.25 (2H, m), 3.82

(1H, d, J = 10.2 Hz), 7.26 (1H, dd, J = 8.4, 2.1 Hz), 7.56 (1H, d,

J = 2.1 Hz), 7.62 (1H, d, J = 8.4 Hz), 8.60–9.10 (2H, br). MS m/z:

314 [M+H]⁺. Anal. Calcd for C₁₆H₂₁Cl₂NOꢃHCl: C, 54.79; H, 6.32;

N, 3.99. Found: C, 54.55; H, 6.12; N, 3.88.

5.1.46. 4-[(1RS)-1-(3,4-Dichlorophenyl)-2-(methylsulfonyl)

ethyl]piperidine monohydrochloride (7a)

91% from 29a, Colorless crystals. Mp 290–292 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.10–1.35 (2H, m), 1.40–1.55 (1H, m),

1.80–2.00 (2H, m), 2.60–2.85 (2H, m), 2.82 (3H, s), 3.10–3.30 (2H,

m), 3.55 (1H, dd, J = 14.7, 3.6 Hz), 3.60–3.85 (1H, m), 3.76 (1H,

dd, J = 14.7, 9.9 Hz), 7.30 (1H, dd, J = 8.4, 2.1 Hz), 7.59 (1H, d,

J = 8.4 Hz), 7.62 (1H, d, J = 2.1 Hz), 8.30–8.50 (1H, br), 8.80–9.00

(1H, br). MS m/z: 336 [M+H]⁺. Anal. Calcd for C₁₄H₁₉Cl₂NO₂SꢃHCl:

C, 45.11; H, 5.41; N, 3.76. Found: C, 45.07; H, 5.61; N, 3.51.

5.1.53. 4-[(1RS)-2-Methoxy-1-(naphthalen-2-yl)ethyl]piperidine

monohydrochloride (3b)

85% from 23b, Colorless crystals. Mp 185–187 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.15–1.35 (1H, m), 1.40–1.55 (2H, m),

1.90–2.10 (2H, m), 2.60–2.90 (3H, m), 3.00–3.30 (2H, m), 3.21

(3H, s), 3.70 (2H, d, J = 3.7 Hz), 7.39 (1H, dd, J = 8.5, 1.2 Hz), 7.40–

7.50 (2H, m), 7.69 (1H, s), 7.80–7.90 (3H, m), 8.60–8.75 (1H, br),

8.80–9.15 (1H, br). MS m/z: 270 [M+H]⁺. Anal. Calcd for

5.1.47. Methyl (2RS)-(3,4-dichlorophenyl)(piperidin-4-yl)

ethanoate monohydrochloride (8a)

44% from 20a, Colorless crystals. Mp 238–240 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.20–1.40 (2H, m), 1.40–1.60 (1H, m),

1.75–1.85 (1H, m), 2.15–2.35 (1H, m), 2.70–2.95 (2H, m), 3.10–

3.30 (2H, m), 3.59 (1H, d, J = 10.2 Hz), 3.62 (3H, s), 7.36 (1H, dd,

J = 8.2, 1.8 Hz), 7.60–7.70 (2H, m), 8.80–9.25 (2H, br). MS m/z:

302 [M+H]⁺. Anal. Calcd for C₁₄H₁₇Cl₂NO₂ꢃHCl: C, 49.65; H, 5.36;

N, 4.14. Found: C, 49.18; H, 5.30; N, 4.14.

C

₁₈H₂₃NOꢃHCl: C, 70.69; H, 7.91; N, 4.58. Found: C, 70.30; H,

7.88; N, 4.37.

5.1.54. 4-[(1RS)-1-(3-Chlorophenyl)-2-methoxyethyl]piperidine

monohydrochloride (3c)

73% from 23c, Colorless crystals. Mp 147–148 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.08–1.29 (1H, m), 1.31–1.49 (2H, m),

1.81–2.04 (2H, m), 2.63–2.90 (3H, m), 3.02–3.28 (3H, m), 3.33

(2H, br), 3.59 (2H, d, J = 7.0 Hz), 7.14–7.23 (1H, m), 7.23–7.40

(3H, m), 8.72 (2H, br). MS m/z: 254 [M+H]⁺. Anal. Calcd for

5.1.48. Ethyl (2RS)-(3,4-dichlorophenyl)(piperidin-4-yl)

ethanoate monohydrochloride (9a)

79% from 21a, Colorless crystals. Mp 198–200 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.15 (3H, t, J = 7.2 Hz), 1.20–1.40 (2H, m),

1.40–1.60 (1H, m), 1.75–1.85 (1H, m), 2.15–2.35 (1H, m), 2.65–

2.90 (2H, m), 3.10–3.30 (2H, m), 3.55 (1H, d, J = 9.9 Hz), 4.00–

4.20 (2H, m), 7.36 (1H, dd, J = 8.2, 1.8 Hz), 7.60–7.70 (2H, m),

8.70–9.30 (2H, br). MS m/z: 316 [M+H]⁺. Anal. Calcd for

C

₁₄H₂₀ClNOꢃHCl: C, 57.94; H, 7.29; N, 4.83. Found: C, 57.96; H,

7.27; N, 4.59.

5.1.55. 4-[(1RS)-1-(4-Chlorophenyl)-2-methoxyethyl]piperidine

monohydrochloride (3d)

C

₁₅H₁₉Cl₂NO₂ꢃHCl: C, 51.08; H, 5.72; N, 3.97. Found: C, 51.08; H,

5.67; N, 3.95.

70% from 23d, Colorless crystals. Mp 158–159 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.11–1.27 (1H, m), 1.29–1.51 (2H, m),

1.79–1.98 (2H, m), 2.59–2.88 (3H, m), 3.04–3.27 (5H, m), 3.57

(2H, d, J = 6.0 Hz), 7.19–7.28 (2H, m), 7.29–7.41 (2H, m), 8.87

(2H, br). MS m/z: 254 [M+H]⁺. Anal. Calcd for C₁₄H₂₀ClNOꢃHCl: C,

57.94; H, 7.29; N, 4.83. Found: C, 57.83; H, 7.27; N, 4.79.

5.1.49. (2RS)-2-(3,4-Dichlorophenyl)-N,N-dimethyl-2-

(piperidin-4-yl)ethanamide monohydrochloride (10a)

79% from 19a, Colorless powder. ¹H NMR (300 MHz, DMSO-d₆)

d 1.10–1.45 (2H, m), 1.75–1.85 (1H, m), 2.15–2.30 (1H, m), 2.65–

2.95 (1H, m), 2.80 (3H, s), 2.99 (3H, s), 3.10–3.30 (2H, m), 3.35–

3.80 (2H, m), 3.85 (1H, d, J = 10.2 Hz), 7.39 (1H, dd, J = 8.2,

2.1 Hz), 7.61 (1H, d, J = 8.2 Hz), 7.67 (1H, d, J = 2.1 Hz), 8.60–9.15

(2H, br). MS m/z: 315 [M+H]⁺.

5.1.56. 4-[(1RS)-1-(3-Chloro-4-ﬂuorophenyl)-2-meth

oxyethyl]piperidine monohydrochloride (3e)

77% from 23e, Colorless crystals. Mp 147–148 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.10–1.31 (1H, m), 1.31–1.54 (2H, m),

1.78–2.02 (2H, m), 2.61–2.87 (3H, m), 3.05–3.29 (5H, m), 3.46–

3.68 (2H, m), 7.17–7.29 (1H, m), 7.29–7.41 (1H, m), 7.46 (1H, dd,

J = 7.3, 2.1 Hz), 8.99 (2H, br). MS m/z: 272 [M+H]⁺. Anal. Calcd for

5.1.50. (1RS)-1-(3,4-dichlorophenyl)-1-(piperidin-4-yl)propan-

2-one monohydrochloride (11a)

79% from 31a, Colorless crystals. Mp 264–266 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 1.10–1.50 (3H, m), 1.70–1.85 (1H, m),

2.09 (3H, s), 2.20–2.40 (1H, m), 2.65–2.95 (2H, m), 3.05–3.30 (2H,

m), 3.83 (1H, d, J = 10.2 Hz), 7.25 (1H, dd, J = 8.4, 2.1 Hz), 7.55

(1H, d, J = 2.1 Hz), 7.63 (1H, d, J = 8.4 Hz), 8.60–9.10 (2H, br). MS

m/z: 286 [M+H]⁺. Anal. Calcd for C₁₄H₁₇Cl₂NOꢃHCl: C, 52.11; H,

5.62; N, 4.34. Found: C, 51.82; H, 5.63; N, 4.24.

C

₁₄H₁₉ClFNOꢃHClꢃ0.1H₂O: C, 54.24; H, 6.57; N, 4.52. Found: C,

54.21; H, 6.59; N, 4.25.

5.1.57. 4-[(1S)-1-(3,4-Dichlorophenyl)-2-methoxyethyl]

piperidine monohydrochloride ((S)-3a)

97% from (S)-23a, Colorless crystals. Mp 191 °C. ½a _D²⁰

ꢄ

+29.8° (c

0.5035, MeOH). ¹H NMR and MS spectra were identical to that of

3a. Anal. Calcd for C₁₄H₁₉Cl₂NOꢃHCl: C, 51.79; H, 6.21; N, 4.31.

Found: C, 51.78; H, 6.26; N, 4.27.

5.1.51. (1RS)-1-(3,4-Dichlorophenyl)-1-(piperidin-4-yl)butan-2-

one monohydrochloride (12a)

85% from 32a, Colorless crystals. Mp 210–212 °C. ¹H NMR

(300 MHz, DMSO-d₆) d 0.85 (3H, t, J = 7.5 Hz), 1.10–1.30 (2H, m),

1.35–1.55 (1H, m), 1.65–1.80 (1H, m), 2.20–2.40 (1H, m), 2.40–

2.60 (2H, m), 2.65–2.90 (2H, m), 3.05–3.25 (2H, m), 3.83 (1H, d,

J = 10.5 Hz), 7.26 (1H, dd, J = 8.4, 2.1 Hz), 7.57 (1H, d, J = 2.1 Hz),

7.62 (1H, d, J = 8.4 Hz), 8.70–9.10 (2H, br). MS m/z: 300 [M+H]⁺.

Anal. Calcd for C₁₅H₁₉Cl₂NOꢃHCl: C, 53.51; H, 5.99; N, 4.16. Found:

C, 53.42; H, 6.00; N, 4.00.

5.1.58. 4-[(1R)-1-(3,4-Dichlorophenyl)-2-methoxyethyl]

piperidine monohydrochloride ((R)-3a)

95% from (R)-23a, Colorless crystals. Mp 191 °C. ½a _D²⁰

ꢀ29.9° (c

ꢄ

0.5140, MeOH). ¹H NMR and MS spectra were identical to that of

3a. Anal. Calcd for C₁₄H₁₉Cl₂NOꢃHCl: C, 51.79; H, 6.21; N, 4.31.

Found: C, 51.76; H, 6.09; N, 4.11.

Please cite this article in press as: Ishichi, Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.05.025

12

Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

5.1.59. X-ray structure analyses of compound (S)-3a

operation. The obtained cells were counted, and diluted to

Crystal data for (S)-3a: [C₁₄H₂₀Cl₂NO]⁺Cl^ꢀ, MW = 324.68; crystal

size, 0.45 ꢂ 0.25 ꢂ 0.10 mm; colorless, prism; monoclinic, space

group P2₁, a = 7.881(3) Å, b = 6.980(2) Å, c = 14.670(5) Å,

3 ꢂ 10⁵cells per 1 mL medium, the mixture was dispensed to a

96 well white plate (Corning) at 100 lL per well, and cultured

overnight in a CO₂incubator. Then, an assay buffer (126 mM NaCl,

4.95 mM KCl, 1.26 mM KH₂PO₄, 1.26 mM MgSO₄, 10 mM HEPES,

2.32 mM CaCl₂, 5.52 mM Glucose, 0.5% BSA) was prepared, the

medium in the cell plate was removed and the assay buffer was

added by 80 lL. A test compound was diluted with the assay buffer

to a 10-fold concentration of the ﬁnal concentration, and the

a = c

= 90°, b = 91.81(2)°, V = 806(1) Å³, Z = 2, Dx = 1.34 g/cm³,

T = 293 K,

l

= 5.076 mm^ꢀ1, k = 1.54186 Å, R₁= 0.040, wR₂= 0.118.

The absolute conﬁguration of (S)-3a was determined as S based

on the Flack parameter,¹⁷0.02(2). Crystals of (S)-3a was obtained

by crystallization from methanol solution.

All measurements were made on a Rigaku R-AXIS RAPID diffrac-

mixture was dispensed to a 96 well polypropylene plate. The di-

tometer using graphite monochromated Cu K

a

radiation. The

luted test compound was dispensed to the cell plate by 10 lL.

structure was solved by direct methods with SIR92¹⁸and was re-

ﬁned using full-matrix least-squares on F²with SHELXL-97.¹⁹All

non-H atoms were reﬁned with anisotropic displacement parame-

ters. CCDC for compound (S)-3a contains the supplementary crys-

tallographic data for this paper (CCDC No.: 895158). These data can

be obtained free of charge from The Cambridge Crystallographic

Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

[³H]-5-Hydroxytryptamine (GE Healthcare) was diluted with the

assay buffer to 200 nM, and the mixture was dispensed to the cell

plate by 10

the assay buffer was removed by suction, and the plate was

washed twice with 150 L of PBS (Invitrogen) per well. Microscinti

20 (PerkinElmer) was dispensed to each well by 100 L, and the

l

L. At 20 min from [³H]-5-hydroxytryptamine addition,

l

mixture was stirred for about 30 min. The radioactivity was mea-

sured by TopCount (PerkinElmer). The inhibitory activity of each

5.2. Monoamine reuptake inhibition

compound (10

lM) was calculated as a relative activity value

based on the inhibitory activity of 10

lM Paroxetine (serotonin

5.2.1. Preparation of human monoamine expressing cell

Human serotonin transporter cDNA was ampliﬁed from human

brain cDNA library by PCR, and inserted into pCRII-TOPO vector

(manufactured by Invitrogen). The base sequence was conﬁrmed

and modiﬁed, and subcloned to pcDNA3.1 vector (manufactured

by Invitrogen), whereby a human serotonin transporter expression

plasmid was constructed.

Human norepinephrine transporter cDNA was purchased from

Invitrogen, and the base sequence was conﬁrm and modiﬁed, and

subcloned to pcDNA3.1 vector, whereby a human norepinephrine

transporter expression plasmid was constructed.

transporter inhibitor) as 100%. IC₅₀and 95% conﬁdence interval

were calculated by XLﬁt (n = 2).

5.2.3. Human norepinephrine transporter inhibitory activity

CHO cells stably expressing human norepinephrine transporter

were used for the measurement of human norepinephrine trans-

porter inhibitory activity. Unless otherwise indicated, these CHO

cells were cultured in Ham/F12 medium (Invitrogen) containing

10% fetal bovine serum (MOREGATE). The cultured cells that

reached almost conﬂuent were rinsed with PBS (Invitrogen), de-

tached with Trypsin/EDTA (Invitrogen), and collected by a centrifu-

gal operation. The obtained cells were counted, and diluted to

3 ꢂ 10⁵cells per 1 mL medium, and the mixture was dispensed to

Human dopamine transporter expression plasmid was prepared

by following procedure. SR

a promoter contained in pTB1411 de-

scribed in JP-A-H5-076385 was cleaved with restriction enzyme

HindIII (manufactured by TAKARA BIO INC.), blunt-ended, further

cleaved with restriction enzyme EcoRI (manufactured by TAKARA

BIO INC.), and fragmented. On the other hand, pCI vector was

cleaved with restriction enzyme BglII (manufactured by TAKARA

BIO INC.), blunt-ended with T4DNA polymerase, and further

cleaved with restriction enzyme EcoRI (manufactured by TAKARA

a 96 well white plate (Corning) at 100 lL per well, and cultured

overnight in a CO₂incubator. Then, an assay buffer (126 mM NaCl,

4.95 mM KCl, 1.26 mM KH₂PO₄, 1.26 mM MgSO₄, 10 mM HEPES,

2.32 mM CaCl₂, 5.52 mM Glucose, 0.5% BSA) was prepared, the med-

ium in the cell plate was removed and the assay buffer was added by

80 lL. A test compound was diluted with the assay buffer to a 10-

fold concentration of the ﬁnal concentration, and the mixture was

BIO INC.). Into this site was inserted a SR

a promoter fragment to

dispensed to a 96 well polypropylene plate. The diluted test com-

give pCI-SRa. Then, pCI-SRa was cleaved with restriction enzyme

ClaI (manufactured by TAKARA BIO INC.) and blunt-ended. Into this

site was inserted a 1.63 Kb fragment obtained by cleaving pGFP-C1

(manufactured by TOYOBO) with restriction enzyme Bsu36I (man-

ufactured by Daiichi Pure Chemicals Co., Ltd) followed by blunt-

pound was dispensed to the cell plate by 10

rine (GE Healthcare) was diluted with the assay buffer to 200 nM,

and the mixture was dispensed to the cell plate by 10 L. At

45 min from [³H]-norepinephrine addition, the assay buffer was re-

moved by suction, and the plate was washed twice with 150 L of

PBS (Invitrogen) per well. Microscinti 20 (PerkinElmer) was dis-

pensed to each well by 100 L, and the mixture was stirred for about

30 min. The radioactivity was measured by TopCount (PerkinEl-

mer). The inhibitory activity of each compound (10 M) was calcu-

lated as a relative activity value based on the inhibitory activity of

10 M DMI (norepinephrine transporter inhibitor) as 100%. IC₅₀

l

L. [³H]-Norepineph-

l

ending, whereby pMSR

a neo was prepared. Human dopamine

transporter cDNA was ampliﬁed from human substantia nigra

cDNA library by PCR, and inserted into the pCRII vector (manufac-

tured by Invitrogen). The base sequence was conﬁrmed, modiﬁed

and subcloned to pMSRa neo, whereby a human dopamine trans-

porter expression plasmid was constructed.

l

The monoamine transporter expression plasmids thus prepared

were introduced into CHO-K1 cells using FuGENE6 (manufactured

by Roche Diagnostics) and according to the attached protocol,

whereby each expressing cell was established.

and 95% conﬁdence interval were calculated by XLﬁt (n = 2).

5.2.4. Human dopamine transporter inhibitory activity

CHO cells stably expressing human dopamine transporter were

used for the measurement of human dopamine transporter inhib-

itory activity. Unless otherwise indicated, these CHO cells were

cultured in Ham/F12 medium (Invitrogen) containing 10% fetal bo-

vine serum (MOREGATE). One day before the assay, the cultured

cells that reached almost conﬂuent were rinsed with PBS (Invitro-

gen), detached with Trypsin/EDTA (Invitrogen), and collected by a

centrifugal operation. The obtained cells were counted, and diluted

to 3 ꢂ 10⁵cells per 1 mL medium, and the mixture was dispensed

5.2.2. Human serotonin transporter inhibitory activity

CHO cells stably expressing a human serotonin transporter

were used for the measurement of human serotonin transporter

inhibitory activity. Unless particularly indicated, these CHO cells

were cultured in a Ham/F12 medium (Invitrogen) containing 10%

fetal bovine serum (MOREGATE). The cultured cells that reached

almost conﬂuent were rinsed with PBS (Invitrogen), detached

with Trypsin/EDTA (Invitrogen), and collected by a centrifugal

to a 96 well white plate (Corning) at 100 lL per well, and cultured

Please cite this article in press as: Ishichi, Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.05.025

Y. Ishichi et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

13

overnight in a CO₂incubator. On the day of the test, an assay buffer

(126 mM NaCl, 4.95 mM KCl, 1.26 mM KH₂PO₄, 1.26 mM MgSO₄,

10 mM HEPES, 2.32 mM CaCl₂, 5.52 mM Glucose, 0.5% BSA) was

prepared, the medium in the cell plate was removed and the assay

ad libitum. Mice were anesthetized with sodium pentobarbital

prior to the stereotaxic implantation of a probe into the medial

prefrontal cortex (+1.8 mm anteroposterior, ꢀ0.6 mm mediolateral

from the bregma, and ꢀ1.0 mm dorsoventral from the skull),

according to the Paxinos and Franklin Atlas (2001). Anchor screws

and acrylic cement are used for ﬁxation of the guide cannulas (AG-

4, Eicom). The mice were allowed to recover from surgery for 1 day,

housed singly in cages. On the day of the experiment, a microdialy-

sis probe (0.5 mm diameter, 4 mm length; A-I-4-02, Eicom) is in-

serted through the guide cannula. The probe is connected via a

channel swivel to a microinjection pump. Perfusion of the microdi-

alysis probe with ﬁltered Ringer solution (147 mM Na⁺, 4 mM K⁺,

2.3 mM, Ca²⁺, 155.6 mM Cl^ꢀ) is begun shortly before insertion of

the probe into the brain and continued for the duration of the

buffer was added by 80 lL. A test compound was diluted with the

assay buffer to a 10-fold concentration of the ﬁnal concentration,

and the mixture was dispensed to a 96 well polypropylene plate.

The diluted test compound was dispensed to the cell plate by

10

buffer to 200 nM, cold dopamine was diluted to 10

mixture was dispensed to the cell plate by 10 L. At 60 min from

[³H]-dopamine addition, the assay buffer was removed by suction,

and the plate was washed twice with 150 L of PBS (Invitrogen)

per well. Microscinti 20 (PerkinElmer) was dispensed to each well

by 100 L, and the mixture was stirred for about 30 min. The radio-

activity was measured by TopCount (PerkinElmer). The inhibitory

activity of each compound (10 M) was calculated as a relative

activity value based on the inhibitory activity of 100 M Nomifen-

l

L. [³H]-Dopamine (GE Healthcare) was diluted with the assay

lM, and the

l

experiment at a constant ﬂow rate of 2 lL/min. After 120 min or

more for stabilization, the experiments are initiated and dialysates

are collected every 20 min. The amounts of 5-HT, DA and NE in the

dialysate samples were quantiﬁed using high-pressure liquid chro-

matography and electrochemical detection (HTEC-500, Eicom).

Each dialysate sample was separated in a liquid chromatography

column. For the assay of 5-HT and DA, Eicompak PP-ODS (Eicom)

was used. The mobile phase for the 5-HT and DA assay contained

0.1 M phosphate buffer (pH 6.0) containing 500 mg/L sodium 1-

decanesulfonate and 50 mg/L EDTA-2Na and 1% methanol. For

the assay of 5-HT, NE and DA, Eicompak CAX (Eicom) was used.

The mobile phase for the 5-HT, NE and DA assay contained 0.1 M

ammonium acetate buffer (pH 6.0) containing 0.05 M sodium sul-

fate, 50 mg/l EDTA-Na2, and 30% methanol. For the assay of NE,

Eicompak CA-50DS (Eicom) was used. The mobile phase for the

NE assay contained 0.1 M phosphate buffer (pH 6.0) containing

500 mg/L sodium 1-octanesulfonate, and 50 mg/L EDTA-Na2 and

5% methanol. Basal levels of 5-HT, NE and DA were obtained from

average concentrations of three consecutive samples when they

were stable. Drugs were dissolved in water and administered orally

in a volume of 10 mL/kg after establishment of stable baseline, and

the dialysate was continuously collected for 120 min.

l

sine (dopamine transporter inhibitor) as 100%. IC₅₀and 95% conﬁ-

dence interval were calculated by XLﬁt (n = 2).

5.3. CYP2D6 Inhibition

Inhibition activity of test compounds of CYP2D6 was evaluated

by incubating 10

from CYP2D6-expressing insect cells (BD Biosciences) in the

presence of 10 mol/L test compound. The incubation mixture

lmol/L bufuralol with 2 nmol/L CYP2D6 derived

l

was allowed to stand for 20 min at 37 °C. The concentration of

1⁰-hydroxybufuralol was measured by LC/MS/MS.

5.4. Evaluation of the antidepressants effects in tail-suspension

test (TST) in mice

Male 7 week-old ICR mice (Clea Japan) were housed in temper-

ature and humidity controlled environment under a 12-h light/

dark cycle (lights on at 07.00 a.m.). Food and water were available

ad libitum. The TST was performed with a computerized device

(BrainScieceIdea). Mice (n = 11/group) were suspended by the tip

of the tail with adhesive tape to a hook connected to a strain gauge

that transmitted movements to computer. Software (TS-2) judged

the immobility using the index of load variation and calculated

the immobility time during a 10 min test. Average immobility ratio

(%) in each group was calculated. Drugs were dissolved in diluted

water (DW) (Otsuka) and administered orally in a volume of

10 mL/kg. Animals received either vehicle (DW) or drugs were

tested in TST 60 min after administration.

Acknowledgement

We thank Ms. Keiko Higashikawa and Mr. Mitsuyoshi Nishitani

for X-ray structure analysis, Ms. Mariko Ishiguro for technical sup-

port in pharmacological experiment, Dr. Yasuyuki, Awasaki for

evaluation of drug safety.

References and notes

1. World Health Organization (WHO). Fact sheet No. 369: Depression, October,

2012. http://www.who.int/mediacentre/factsheets/fs369/en/index.html.

2. Kulkarni, S. K.; Dhir, A. Expert Opin. Investig. Drugs 2009 , 18 (6), 767.

3. (a) Prins, J.; Olivier, B.; Korte, S. M. Expert Opin. Investig. Drugs 2011 , 20 (8),

1107; (b) Golembiowska, K.; Kowalska, M.; Bymaster, F. P. Synapse 2012 , 66 ,

435; (c) Millan, M. J. Neurotherapeutics 2009 , 6 , 53; (d) Skolnick, P.; Krieter, P.;

Tizzano, J.; Basile, A.; Popik, P.; Czobor, P.; Lippa, A. CNS Drug Rev. 2006 , 12 (2),

123; (e) Skolnick, P.; Popik, P.; Janowsky, A.; Beer, B.; Lippa, S. A. Life Sci. 2003 ,

73 , 3175.

5.5. SERT/NET/DAT ex vivo binding assay in mice frontal cortex

and striatum

Compounds were administered orally to male ICR mice. 1 h

after administration, mice were sacriﬁced and the frontal cortex

and striatum were dissected. Ex vivo binding of compounds to

the SERT, NET and DAT was evaluated by incubating aliquots of

the unwashed homogenate with [³H]-citalopram (25 nM), [³H]-

nisoxetine (25 nM) or [³H]-WIN35428 (50 nM) respectively. Sam-

4. Evaluation using in house data.

5. ClogP value was calculated by Daylight Software. (ClogP, version 4.82,

Daylight Software, Daylight Chemical Information Systems, Inc., Aliso Viejo,

CA; http://www.daylight.com).

6. Recent examples of mitigating these liabilities by reducing lipophilicity: (a)

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Novel triple reuptake inhibitors with low risk of CAD associated liabilities: Design, synthesis and biological activities of 4-[(1S)-1-(3,4- dichlorophenyl)-2-methoxyethyl]piperidine and related compounds

DOI: 10.1016/j.bmc.2013.05.025

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