Novel and High Affinity 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues as Atypical Dopamine Transporter Inhibitors

Article

pubs.acs.org/jmc

Novel and High Aﬃnity 2‑[(Diphenylmethyl)sulﬁnyl]acetamide

(Modaﬁnil) Analogues as Atypical Dopamine Transporter Inhibitors

Jianjing Cao,^†Rachel D. Slack,^†Oluyomi M. Bakare,^†Caitlin Burzynski,^†,‡Rana Rais,^‡Barbara S. Slusher,^‡

Theresa Kopajtic,^§Alessandro Bonifazi,^†Michael P. Ellenberger,^†Hideaki Yano,^†Yi He,^†Guo-Hua Bi,^†

Zheng-Xiong Xi,^†Claus J. Loland,^∥and Amy Hauck Newman*^,†

^†Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, Intramural Research Program, National

Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States

^‡Department of Neurology, Johns Hopkins Drug Discovery, The Johns Hopkins University School of Medicine, 855 N. Wolfe Street,

Baltimore, Maryland 21205, United States

^§Psychobiology Section, Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Intramural Research Program,

National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, United States

^∥Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and

Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark

S

* Supporting Information

ABSTRACT: The development of pharmacotherapeutic treatments of psychostimulant abuse has remained a challenge, despite

signiﬁcant eﬀorts made toward relevant mechanistic targets, such as the dopamine transporter (DAT). The atypical DAT

inhibitors have received attention due to their promising pharmacological proﬁles in animal models of cocaine and

methamphetamine abuse. Herein, we report a series of modaﬁnil analogues that have an atypical DAT inhibitor proﬁle. We

extended SAR by chemically manipulating the oxidation states of the sulfoxide and the amide functional groups, halogenating the

phenyl rings, and/or functionalizing the terminal nitrogen with substituted piperazines, resulting in several novel leads such as

11b, which demonstrated high DAT aﬃnity (K_i= 2.5 nM) and selectivity without producing concomitant locomotor stimulation

in mice, as compared to cocaine. These results are consistent with an atypical DAT inhibitor proﬁle and suggest that 11b may be

a potential lead for development as a psychostimulant abuse medication.

liability of cocaine and have not been developed beyond

preclinical studies.⁸One DAT inhibitor, GBR 12909 [1, 1-(2-

(bis(4-ﬂuorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)-

piperazine, Figure 1] was taken on into a Phase 1 clinical trial

but failed before being tested for eﬃcacy in the cocaine-abusing

population.⁹These results dampened interest in the DAT as a

potential therapeutic target for psychostimulant abuse and,

indeed, negatively impacted the development of any drugs

toward this target, despite the great success of methylphenidate,

for example, a DAT inhibitor that has been used for decades to

treat Attention Deﬁcit Hyperactivity Disorder (ADHD).^10,11

INTRODUCTION

■

With millions of people worldwide suﬀering from substance use

disorders, the development of pharmacotherapeutics for the

treatment of addiction is imperative. While medications exist

for treating alcohol and opiate addiction, there remains no

FDA-approved medication to treat psychostimulant use

disorder.^1,2Psychostimulant drugs of abuse, such as cocaine

and methamphetamine, bind to the dopamine transporter

(DAT), inhibiting the reuptake of dopamine (DA) into the

presynaptic neuron, increasing extracellular dopamine levels,

and resulting in the behavioral activation and euphoria that can

lead to addiction.³⁻⁷Although tremendous eﬀorts have been

made to develop dopamine uptake inhibitors to block the

eﬀects of cocaine and methamphetamine, most of these

compounds share the psychostimulant eﬀects and addictive

Received: September 19, 2016

A

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has demonstrated limited eﬀectiveness in treating cocaine

abuse,^{14−16,20,21}although a recent clinical study showed

promising results in nonalcohol-dependent cocaine abusers.²²

( )-Modaﬁnil and its (R)-enantiomer display unique

pharmacological proﬁles, suggesting they may bind to a

conformation of the DAT dissimilar to the cocaine-bound

conformation and more similar to a class of “atypical” DAT

inhibitors, based on benztropine (e.g., JHW 007, 3, (3-(bis(4-

ﬂuorophenyl)methoxy)-8-butyl-8-azabicyclo[3.2.1]octane,²³

Figure 1).¹³An independent study on modaﬁnil²⁴supported

these ﬁndings and led us to further investigate (R)-modaﬁnil in

rodent models of nicotine abuse.²⁵These studies and previous

investigations of several benztropine analogues suggested that

atypical DAT inhibitors might attenuate the psychostimulant

eﬀects of cocaine and methamphetamine but may not have

abuse liability, thereby leading us to further explore (R)-

modaﬁnil for the treatment of psychostimulant abuse.

Figure 1. Chemical structures of known DAT inhibitors.

Modaﬁnil is essentially insoluble in water and has relatively

low aﬃnity for DAT (K_i= 2 μM), which in some ways makes it

an excellent therapeutic, as it cannot be illicitly diverted into an

injectable and/or abused drug; however, clinical studies in

cocaine addicts who also abuse other substances suggest that

modaﬁnil is not an ideal medication for this patient population,

especially for those who also abuse alcohol.²⁰⁻²²One possibility

for its lack of eﬀectiveness in reducing cocaine-taking in this

patient population might be due to modaﬁnil’s low aﬃnity for

the DAT relative to cocaine, precluding it from eﬃciently

blocking cocaine’s rapid inhibition of dopamine uptake.

Moreover, its low DAT aﬃnity and poor water solubility

make modaﬁnil a challenging compound to study preclinically,

and thus, our previous eﬀorts^26,27were aimed at improving its

pharmacological proﬁle via a structure−activity relationship

(SAR) analyses at the DAT, norepinephrine transporter

(NET), and serotonin transporter (SERT). Several compounds

Another DAT inhibitor, ( )-modaﬁnil (2, 2-

[(diphenylmethyl)sulﬁnyl]acetamide, Figure 1) and its (R)-

enantiomer (armodaﬁnil) are FDA-approved to treat narco-

lepsy and other sleep disorders; however, they are also used oﬀ-

label as cognitive enhancers, with little evidence of abuse

liability in humans.¹²Though modaﬁnil primarily targets the

DAT, it does not share the same psychostimulant and abuse

liability proﬁle of cocaine.¹³Parallels to its atypical behavior in

both humans¹⁴⁻¹⁶and psychostimulant abuse animal models,¹⁷

along with its unique DAT binding proﬁle have piqued interest

for further drug development. In addition, as sleep and

cognitive disorders have been linked to chronic drug abuse

and the inability to remain abstinent,^18,19modaﬁnil has

additional therapeutic attributes that may be useful in

preventing relapse. Nevertheless, in clinical trials, modaﬁnil

a

Scheme 1. Synthesis of Compounds 8a−8k and 9a−9i

a

Reagents and conditions: (a) (i) TFA, CH₂Cl₂, r.t. overnight; (ii) K₂CO₃, H₂O/acetone, overnight; (b) PPh₃, CBr₄, CH₃CN, r.t. overnight; (c)

appropriate piperazine, K₂CO₃, acetone, reﬂux, overnight; (d) H₂O₂, AcOH/MeOH, 40 °C, overnight.

B

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a

Scheme 2. Synthesis of Compounds 10a,b and 11a,b

a

Reagents and conditions: (a) 2-benzyloxirane, isopropanol, reﬂux, overnight; (b) H₂O₂, AcOH/MeOH, 40 °C, overnight.

a

Scheme 3. Synthesis of Compounds 13a−g and 14 a−g

a

Reagents and conditions: (a) (i) THF, CDI, r.t. 2 h; (ii) substituted piperazine analogue, THF, r.t. overnight; (b) (i) SOCl₂, reﬂux, 2 h; (ii)

appropriate piperazine; (c) H₂O₂, AcOH/MeOH, 40 °C, overnight.

from those libraries showed improved water solubility and

higher aﬃnity for DAT (K_i< 1 μM) relative to modaﬁnil; one

lead compound arose and is currently being evaluated in

behavioral models of cocaine and methamphetamine abuse (4,

JJC8-016, N-(2-((bis(4-ﬂuorophenyl)methyl)thio)ethyl)-3-

phenylpropan-1-amine, Figure 1).²⁷

CHEMISTRY

■

Syntheses of novel sulfenylethanamine (8a−8k, 10a,b) and

sulﬁnylethanamine analogues (9a−9i, 11a,b) were achieved as

depicted in Schemes 1 and 2. Dehydration of commercially

available benzhydrols 5a−c with 2-mercaptoethan-1-ol in the

presence of triﬂuoroacetic acid followed by treatment with

K₂CO₃in H₂O/acetone provided sulﬁde alcohols 6a−6c in

61−95% yield. Appel reaction on 6a−6c with triphenylphos-

phine and tetrabromomethane gave bromides 7a−7c in 61−

82% yield. Next, alkylation with the appropriate piperazine

provided the sulfenylethanamine analogues 8a−8k in 50−87%

yield. Epoxide ring opening of 2-benzyloxirane using

unsubstituted piperazine analogues 8e and 8f gave alcohols

10a and 10b, respectively (84% and 86% yield, respectively).

Lastly, oxidation of the appropriate sulfenylethanamine was

achieved using hydrogen peroxide in an acetic acid/methanol

solution to give sulﬁnylethanamine (9a−9i, 11a,b) in 43−78%

yield.

Piperazine-substituted sulfenylacetamides 13a−13g were

generated via either synthetic routes a or b, outlined in Scheme

3. Compounds 13a−13c and 13g were synthesized through

route a by amidation of carboxylic acids 12a−12c using 1,1′-

carbonyldiimidazole (CDI) and the appropriate amines in 71−

90% yield. Synthetic route b utilized thionyl chloride to form

the acid chloride intermediate of carboxylic acids 12a,b,

followed by coupling with the respective amine to aﬀord

13d−13f in 54−67% yield. Oxidation of the sulfenyl moiety

gave the desired sulﬁnylacetamide 14a−14g in 11−72% yield.

All ﬁnal compounds were puriﬁed and characterized in their

free base form and then converted to the oxalate salts for

testing. Note that all ﬁnal compounds with sulfoxide groups

and/or hydroxylated linking chains are the racemic and/or

diastereomeric mixtures.

In order to further improve DAT binding aﬃnity and

selectivity, we designed a series of compounds that extended

SAR by chemically manipulating the oxidation states of the

sulfoxide and the amide functional groups, halogenating the

phenyl rings, and/or functionalizing the terminal nitrogen,

borrowing the piperazine ring from 1, as part of the

scaﬀold.²⁸⁻³⁴All ﬁnal compounds were evaluated for DAT,

NET, and SERT binding in rat brains, and a subset was tested

for binding to the σ1 receptor, as it has been previously posited

that a dual DAT/σ1 proﬁle may have therapeutic advantages

over a highly DAT selective compound.³⁵⁻⁴¹This subset was

also tested for binding aﬃnities at the dopamine D₂, D₃, and D₄

receptor subtypes and for eﬃcacy in a D₂Gi₁Bioluminescence

Resonance Energy Transfer (BRET) assay. In addition, we

tested this subset of analogues for inhibition of [³H]DA uptake

and in a binding assay previously shown to determine if a

compound possesses a classical or an atypical DAT binding

proﬁle, as reported for ( )-modaﬁnil and its (R)-enantiomer.¹³

These data suggest that the analogues do indeed appear to bind

the DAT in this desired atypical fashion like modaﬁnil and the

benztropines, but unlike cocaine. This subset of compounds

was also evaluated for metabolic stability in mouse liver

microsomes, and the compound with highest DAT aﬃnity and

selectivity in this series, 11b, was tested for its eﬀects on

locomotor activity in mice.

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a

Table 1. Binding Data for Sulfenyl- and Sulﬁnylacetamide, Sulfenyl-, and Sulﬁnylethanamine Analogues

K_i(nM) SEM

compound

X

Y

Z

R

clogP

DAT

NET

SERT

b

GBR12909, 1

1.77 0.181

497 17.0

104 11.4

c

d

modaﬁnil, 2

3

2520 204

IA

d

10.5 0.748

116 16.3

3.58 0.157

4.50 0.344

49.6 4.31

16.7 1.22

7.40 1.04

4.98 0.716

26.6 2.09

19.3 1.91

143 19.2

3.17 0.112

2.92 0.383

636 14.7

289 43.0

27.2 1.29

9.67 1.37

39.4 1.45

7.62 0.900

403 23.8

2.54 0.233

6.72 0.977

3.43 0.499

2.53 0.250

47.2 5.56

28.6 1.52

214 20.7

277 12.5

53.7 1.89

58.7 5.05

915 84.5

33.0 2.83

37.6 1.86

752 87.4

279 23.4

74.3 9.64

56.3 6.60

1,380 191

1670 232

IA

4

6.43

5.87

6.12

3.13

3.42

6.86

6.92

7.74

7.57

4.56

3.91

4.20

1.21

1.49

4.93

5.00

5.81

5.64

2.63

4.70

5.00

2.78

3.06

6.37

6.66

4.03

5.36

6.16

6.74

5.17

4.67

5.00

2.33

3.66

4.47

5.04

3.47

3,848 21.7

988 22.8

360 48.3

8a

H

S

C

3-phenylpropyl

2-OH-propyl

benzyl

1,050 152

285 35.4

26,700 2,630

1,770 234

949 122

8b

4,4′-diF

H

1,890 116

44,500 2400

17,800 885

8,240 1,240

6,850 633

15,400 1,950

7,410 102

31,300 2,980

5,540 808

4281 343

8c

8d

4,4′-diF

3,3′-diCl

H

8g

8h

4-F-benzyl

463 69.1

2,020 266

905 121

8i

4-CF₃-benzyl

4-Cl-benzyl

8j

8k

2-OH-propyl

3-phenylpropyl

2-OH-propyl

benzyl

10,800 1,030

7,360 881

678 66.1

9a

SO

S

9b

4,4′-diF

H

e

9c

ND

e

9d

4,4′-diF

3,3′-diCl

H

ND

50,300 5,760

11,200 195

8,520 349

6,800 468

4,940 665

58,900 7,310

1,630 169

213 13.2

9e

45,700 8,480

>50 μM

9f

4-F-benzyl

9g

4-CF₃-benzyl

4-Cl-benzyl

>50 μM

9h

>50 μM

9i

2-OH-propyl

2-OH-3-phenylpropyl

3-phenylpropyl

2-OH-propyl

propyl

>50 μM

10a

10b

11a

11b

13a

13b

13c

13d

13e

13f

13g

14a

14b

14c

14d

14e

14f

14g

1,430 118

1,950 227

25,300 2,040

15,000 575

22,600 3,010

20,200 1,410

4,4′-diF

H

S

SO

S

21,700 2,020

4,610 562

9,320 932

3,170 275

11,400 902

5,970 497

6,210 579

2,200 177

4,4′-diF

H

CO

4,4′-diF

3,3′-diCl

H

S

e

S

ND

e

S

ND

e

S

4-F-benzyl

ND

e

S

4-Cl-benzyl

ND

e

S

2-OH-propyl

3-phenylpropyl

2-OH-propyl

propyl

>50 μM

ND

SO

54,300 3,210

12,000 1,430

15,200 1,100

1,320 152

4,4′-diF

3,3′-diCl

e

ND

32,800 4,430

14,500 1,430

23,600 1,030

9,460 386

e

ND

e

4-F-benzyl

ND

4-Cl-benzyl

63,500 5,290

e

2-OH-propyl

>50 μM

ND

a

Each K_ivalue represents data from at least three independent experiments, each performed in triplicate. K_ivalues were analyzed by PRISM. Binding

b

c

d

assays are described in detail in Experimental Procedures. Previously reported in Cao et al.³⁵Previously reported in Cao et al.²⁶IA; less than 50%

e

inhibition at 100 μM. ND; not determined.

halogenating the benzhydrol, and (4) varying the amide/amine

substituent. The binding aﬃnities (K_ivalues) are presented in

Table 1, where, in general, these novel analogues displayed low

nanomolar DAT aﬃnities and showed >100-fold selectivity for

DAT over NET and/or SERT.

In general, the sulfoxides had similar aﬃnities at DAT relative

to their sulﬁde counterparts; however, aﬃnities at SERT and

NET generally decreased, resulting in more DAT-selective

sulfoxides. The largest sulﬁde to sulfoxide improvement in

BIOLOGICAL RESULTS AND DISCUSSION

■

SAR at DAT, SERT, and NET. All ﬁnal compounds (8, 9,

11, 13, and 14) were evaluated for binding at the monoamine

transporters (DAT, SERT, and NET) in rat brain membranes

and compared to known DAT inhibitors 1−4 (Figure 1).

Changes in DAT binding aﬃnity and selectivity as a result of

modiﬁcations to the modaﬁnil structure were evaluated; SAR

analyses explored the eﬀects of (1) the sulfur oxidation state,

(2) reducing the terminal amide to a secondary amine, (3)

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a

Table 2. Oﬀ Target Binding Aﬃnities for a Selected Subset of Analogues

K_iSEM (nM)

a

compd

(R)-2

DAT

D₂R

D₃R

D₄R

σ₁

σ₁/DAT

3050 258

10.5 0.748

116 16.3

16.7 1.22

289 43.0

2.53 0.25

>100,000

39,000 1050

34.4 6.47

65.9 12.1

424 117

480 184

652 105

0.11 0.03

>100,000

2.4

>300

5

3

68.1 7.81

228 27.6

693 159

298 61.1

78.6 16.6

0.26 0.06

583 128

28.1 5.22

6300 1460

3820 876

1750 593

70.4 2.33

4

159 22.7

4.03 0.22

1010 134

336 42.2

1.4

8d

0.24

3.5

9d

11b

133

b

eticlopride

ND

a

Each K_ivalue represents data from at least three independent experiments, each performed in duplicate. K_ivalues were analyzed by PRISM. Binding

b

assays are described in detail in Experimental Procedures. ND; not determined.

DAT selectivities was seen for the N-bearing 2-hydroxypropyl

and 2-hydroxyphenylpropyl derivatives (compared to their

deshydroxy counterparts). In particular, oxidation of hydroxyl-

containing sulﬁde 10b to its sulfoxide 11b gave nominal

improvement in DAT aﬃnity (K_i= 6.72 to 2.53 nM,

respectively), but a 7- and 20-fold decrease in SERT and

NET aﬃnities, respectively, was observed for sulfoxide 11b,

improving DAT selectivity. In contrast, the deshydroxy

counterparts sulﬁde 8b and sulfoxide 9b showed no signiﬁcant

diﬀerence in DAT K_ivalues, while SERT/NET selectivities

decreased only ∼2-fold for the oxidized compound.

To prevent potential metabolism on the benzhydrol moiety,

4,4′-diﬂuorination was probed, resulting in varying eﬀects on

the monoamine transporter binding aﬃnities. When the

unsubstituted 8a, 9a, 10a, and 11a were 4,4′-diﬂuoro-

substituted to give 8b, 9b, 10b, and 11b, respectively, no

appreciable change was observed in DAT and SERT K_ivalues,

although NET aﬃnity improved between 5- and 10-fold. DAT

aﬃnity (Ki = 143−1380 nM) was lost when the benzhydrol was

3,3′-dichloro-substituted in 8k, 9i, 13g, and 14g.

(R)-modaﬁnil [(R)-2], 3, and 4. As can be seen in Table 2, (R)-

modaﬁnil is highly selective for DAT compared to these oﬀ-

target receptors but has relatively low aﬃnity for the DAT. In

contrast, the atypical DAT inhibitor in the benztropine class, 3,

has relatively high aﬃnities for dopamine D₂and D₃receptors

and has higher aﬃnity for σ1 than for DAT (σ1/DAT = 0.2).

Likewise, our previous lead compound 4²⁷had low aﬃnity for

D₂receptors but moderate aﬃnities for D₃and D₄and had

comparable aﬃnities at σ1 and DAT (σ1/DAT = 1.4).

Compound 8d was selective for DAT over the D₂-like

receptors but had the highest aﬃnity for σ1 in the series (K_i

= 4 nM) resulting in a σ1/DAT ratio similar to 3. Compound

9d had the lowest aﬃnity for DAT in the group (although ∼10-

fold higher than (R)-2), and its selectivity across the oﬀ-target

sites was relatively low. Nevertheless, compared to that of the

classic D₂antagonist, eticlopride, the D₂-like receptor aﬃnities

are very low. Indeed, in Table 3, it can be seen that although all

of these compounds were inhibitors of quinpirole-stimulated

D₂receptor activation of Gi₁, all of them had very low potency.

Hence, we conclude that they are very weak antagonists at D₂

receptors and that this oﬀ-target activity would likely not aﬀect

their behavioral proﬁles. Finally, in this series, compound 11b

emerged as the highest aﬃnity and most selective DAT

inhibitor (Tables 2 and 3).

The phenylpropyls (8a, 8b, 9a, 9b, 13a, 13b, 14a, and 14b)

and 2-hydroxyphenylpropyls (10a, 10b, 11a, and 11b)

generally demonstrated high binding aﬃnities for DAT, with

DAT K_i= 2.5−47 nM. The removal of the phenyl group

resulted in a loss of aﬃnity at DAT, implying that an N-arylalkyl

substituent may be optimal. For example, when the phenyl

group is removed from 10a and 11a (K_i= 2.5 and 3.4,

respectively) to give 8c and 9c, respectively, DAT aﬃnity

decreases by 25- and 187-fold. In general, the N-benzyl

analogues (e.g., 8g−8j and 9e−9h) also demonstrated high

DAT aﬃnities, but metabolic instability was problematic in this

subset (see below).

The 2-hydroxy analogues were prepared based on previous

SAR⁴²to give compounds with lower cLogP values (Table 1).

While 2-hydroxylation of the propyl and phenylpropyl

substitutions did not appreciably change K_ivalues at DAT,

the 2-hydroxy sulfoxides 9d, 11a, 11b, and 14c resulted in a

signiﬁcant decrease in NET and SERT aﬃnities. In contrast, 2-

hydroxylation of the linker in sulﬁdes 10a and 10b gave only

nominal loss in NET or SERT K_ivalues. Of note, cLogP values

were typically in the >5-range for 4,4′-diﬂuoro substituted-

analogues without the sulfoxide function or hydroxylated

terminal N-substituents.

Table 3. Inhibition of Dopamine D₂Receptor Gi₁Activation

a

Using 1 μM Quinpirole

compound

IC₅₀(nM)

(R)-2

>10,000

3

2000 730

>10,000

4

8d

>10,000

9d

>10,000

11b

>10,000

eticlopride

2.1 0.8 nM

a

Results were obtained from G protein activation-BRET experiments

in HEK-293T cells transiently transfected with Gαi1-RLuc8 and γ2-

GFP10 in cells expressing D₂R. Data were ﬁt by nonlinear regression

to a sigmoidal dose−response relationship against the ligand

concentration (prestimulated with 1 μM quinpirole). The IC₅₀values

are the mean SEM of 5 experiments performed in triplicate.

Oﬀ-Target Binding Proﬁles: Dopamine D₂, D₃, D₄, and

σ1 Receptors. On the basis of the DAT, SERT, and NET

binding results and cLogP values, a subset of analogues (8d, 9d,

and 11b) was then evaluated for binding aﬃnities at dopamine

D₂, D₃, and D₄receptors in HEK 293 cells, as well as σ1

receptors in rat brains and compared to known compounds

Molecular Pharmacology and Mutagenesis Studies.

To assess the eﬀect on DAT function, this subset of analogues

(4, 8d, 9d, and 11b) was tested for inhibition of [³H]DA

uptake. Accordingly, COS7 cells transiently expressing DAT

were employed. The analogues were added in increasing

concentrations followed by a ﬁxed concentration of [³H]DA to

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allow transport. The reaction was stopped after 5 min, and the

amount of [³H]DA taken up by the COS7 cells was determined

by scintillation counting and plotted as a function of the

concentration of added analogue (Figure 2). All analogues from

this subset possessed higher inhibition potency than ( )-mod-

aﬁnil. In line with the binding data from rat brain membranes,

compound 11b emerged as the DA uptake inhibitor with the

highest potency (K_i= 12 [10;14] nM, mean [SEM interval], n

= 3). Compound 8d had an inhibitory potency similar to that of

cocaine (K_i= 200 [140;290] nM and 200 [110;350] nM,

respectively, mean [SEM interval], n = 3−4), whereas both

compounds 4 and 9d had signiﬁcantly lower K_ivalues than

cocaine (890 [810;980] nM and 1730 [1370;2170] nM,

respectively, mean [SEM interval], n = 6−8). Although all DAT

inhibitors by deﬁnition block DA uptake, cocaine’s binding

preference to an outward facing conformation of DAT has been

described experimentally, computationally,^43,44and more

recently demonstrated in the X-ray crystal structure of the

drosophila DAT.⁴⁵It has been proposed that the stabilization

of the outward facing DAT conformation by cocaine is, at least

in part, mediated by its interference with a H-bond that forms

between the OH-group of Tyr156 in TM3 and Asp79 in TM1.

This has been substantiated experimentally by showing that the

binding mode of cocaine is independent of the OH-group in

Tyr156;⁴⁴however, when the DAT moves to a more outward

occluded and ultimately closed conformation, a critical H-bond

forms between Y156 and D79, closing the outer “gate.” This

conformation seems to be preferred by substrates, such as

dopamine, amphetamine, and MDMA and also the atypical

DAT inhibitors, such as the benztropines^44,46[e.g., 3].

Accordingly, in contrast to cocaine, the binding of modaﬁnil

and other atypical DAT inhibitors does depend on the presence

of the OH-group since the Y156F mutation aﬀects their binding

aﬃnity.^13,43To assess whether or not compounds 4, 8d, 9d,

and/or 11b demonstrated an atypical DAT inhibitor binding

mode, their binding aﬃnities were evaluated in wild type (WT)

DAT and the Y156F mutant (Figure 3 and Table 4). The

results were compared to the eﬀects of ( )-modaﬁnil (2), 3,

and cocaine (Figure 3 and Table 4). Inhibition of [³H]-

WIN35,428 binding on COS7 cells transiently expressing WT

DAT or Y156F was determined. In contrast to cocaine,

( )-modaﬁnil (2) and all the tested analogues showed a

signiﬁcant decrease in binding aﬃnity for Y156F relative to WT

DAT. Remarkably, 11b was particularly aﬀected with a Y156F/

WT ratio of ∼15, similar to that found for [(R)-2] (Figure 3

and Table 4). Also, 9d shows a marked change (7.6-fold, Table

4), whereas 4 and 8d were aﬀected to a lesser degree (2.8 and

2.6-fold, respectively, Figure 4 and Table 4). Interestingly, the

sulfoxide function was present in the three compounds [(R)-2,

9d and 11b)] that were most impacted by the Y156F mutation.

For comparison, the classical atypical DAT inhibitor, 3, had a

Y156F/WT ratio of 5.5.

Figure 2. Inhibition potency of [³H]DA uptake by modaﬁnil

analogues and cocaine. COS7 cells transiently expressing DAT wild

type were incubated with [³H]DA at the indicated concentrations of 4

(green), 8d (brown), 9d (magenta), 11b (black), and cocaine (cyan).

The inhibition potency of 2 as assessed previously¹³is inserted for

comparison (dotted line). Data are the means

experiments performed in triplicate.

SEM of 3−10

phase I metabolism with only ∼4% remaining in microsomes

fortiﬁed with NADPH at 1 h, suggesting CYP dependent

metabolism of the compound and a relatively short in vitro half-

life of 13 min. Of note, compounds 9f, 9g, 9h, 11a, 13c, 14b,

and 14e were all metabolized in <30 min in the mouse liver

microsomes.

Locomotor Activity in Mice Compared to Cocaine.

Figure 5 shows the eﬀects of (R)-modaﬁnil and its analogues 4

and 11b on locomotor activity compared to cocaine. Systemic

administration (i.p.) of cocaine (3, 10 mg/kg) produced a

robust dose-dependent increase in locomotion (Figure 5A,

cocaine treatment main eﬀect, F_2,21= 10,82, p < 0.001, two-way

ANOVA), while (R)-modaﬁnil and compounds 4 and 11b, at

the same dose (10 mg/kg), produced no signiﬁcant or a very

mild locomotor response. When the dose was increased to 30

mg/kg, (R)-modaﬁnil and 11b produced a signiﬁcant dose-

dependent increase, while 4 did not. Two-way ANOVA for

repeated measures over time revealed a statistically signiﬁcant

treatment main eﬀect for (R)-modaﬁnil (Figure 5B, F_2,21

10.01, p < 0.001) and for compound 11b (Figure 5D, F_2,21

4.35, p < 0.005) but not for compound 4 (Figure 5C, F_2,21

=

0.65, p = ns). Although the assay did not show a signiﬁcant

treatment main eﬀect for compound 4, it revealed a signiﬁcant

time main eﬀect (F_11,231= 5.11, p < 0.001) and treatment ×

time interactions (F_21,231= 1.73, p < 0.05). Therefore, posthoc

individual group comparisons revealed statistically signiﬁcant

diﬀerences between the diﬀerent dose groups at certain time

points (Figure 5C).

Most typical DAT inhibitors increase locomotor activity in

mice; indeed, this is one behavioral hallmark of this class of

agents. In contrast, we and others have reported “atypical” DAT

inhibitors that, despite binding with high aﬃnity and selectivity

to the DAT and inhibiting DA uptake, are not eﬃcacious

locomotor stimulants and do not exhibit other cocaine-like

behaviors. ( )-Modaﬁnil and its (R)-enantiomer display unique

pharmacological proﬁles^12,13,24,48that suggest they may be

atypical. Herein, compound 11b displayed the highest DAT

aﬃnity in the series with good selectivity over other oﬀ-targets

(Tables 2 and 3). As seen in Figure 5, compound 11d, does

increase locomotor activity in mice, but it is far less eﬃcacious

than cocaine suggesting that this modaﬁnil analogue is an

atypical DAT inhibitor. In addition, 11b demonstrated the

highest Y156F/WT ratio in the mutagenesis study (Table 4,

Metabolic Stability in Mouse Microsomes. On the basis

of their in vitro proﬁles, compounds 8d, 9d, and 11b were

tested for phase I metabolism following procedures previously

described⁴⁷to predict the susceptibility to metabolism

following in vivo administration. As depicted in Figure 4,

compounds 8d and 9d exhibited good stability with

approximately 50% of both compounds remaining in mouse

microsomes fortiﬁed with NADPH. Corresponding in vitro

half-lives for 8d and 9d were calculated to be ∼60 min

conﬁrming compounds stable to CYP dependent phase I

metabolism. In contrast, compound 11b undergoes substantial

F

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Figure 3. Assessment of DAT binding proﬁles by modaﬁnil analogues. As demonstrated previously,^13,43a characteristic feature for atypical DAT

inhibitors is that, in contrast to cocaine, their binding depends on the presence of the OH-group on Tyr156. Inhibition of [³H]WIN35,428 binding

to DAT WT (black) or the Y156F (green) mutant by (A) cocaine, (B) 2, (C) 4, and (D) 11b. The fold change in IC₅₀values between WT and

Y156F is 1.4, 10, 2.8, and 14.9 for cocaine, 2, 4, and 11b, respectively. Binding assays are performed as triplicates on COS7 cells transiently

expressing DAT WT or Y156F (n = 3−6). Data are shown as the means SEM.

Table 4. Assessment of Atypical Binding Properties for

a

Selected Analogues

WT

Y156F

Y156F/WT

compd

K_i(nM)

aﬃnity ratio

2

927 [834; 1030]

647 [593;704]

1960 [1700;2270]

47 [33;66]

9250 [7350; 11700]

8920 [6930;11500]

5910 [4640; 7520]

260 [229;296]

10.0

13.8

3.0

(R)-2

(S)-2

3

5.5

4

720 [661;784]

101 [82;125]

2050 [1840;2280]

267 [227; 314]

2.8

8d

2.6

9d

1200 [824;1760]

6.5 [5.1;8.3]

9170 [7680; 10900]

97 [67;140]

7.6

11b

14.9

cocaine

223 [160;309]

321 [235; 438]

1.4

Figure 4. Phase I metabolic stability of selected analogues in mouse

liver microsomes. Compounds 8d, 9d, and 11b were tested in mouse

liver microsomes fortiﬁed with NADPH. Compounds 8d and 9d both

showed slow metabolism with a half-life of approximately 60 min

suggesting the compounds to be stable. Compound 11b, on the

contrary, had a much shorter half-life of 13 min suggesting rapid

metabolism. Note: control experiments without cofactors were

conducted in parallel, and in all cases, >95% compound remained at

60 min suggesting that the compounds were undergoing Cyp

dependent metabolism. Testosterone was used as a positive control.

a

Inhibition of [³H] WIN35,428 binding to DAT WT or the Y156F

mutant. COS7 cells transiently expressing DAT WT or Y156F were

assessed for aﬃnity (K_i) for the indicated compounds. Data were

analyzed by nonlinear regression analysis using Prism 6.0 (GraphPad).

The IC₅₀values were calculated from means of pIC₅₀values and the

SEM interval from pIC₅₀SEM. The K_ivalues were calculated from

the IC₅₀-values using the equation K_i= IC₅₀/(1 + (L +K_d), where K_dis

the aﬃnity for WIN35,428, and L is the concentration of added

[³H]WIN35,428). All data are performed in triplicate, n = 3−6.

Figure 3) suggesting that it binds the DAT in a more occluded

conformation, unlike cocaine.

this series and were tested for binding to σ1, as well as

dopamine D₂, D₃, and D₄receptor subtypes, and for eﬃcacy in

a D₂Gi₁BRET assay. In addition, we tested this subset of

analogues in cell-based [³H]DA uptake and binding assays for

aﬃnities at both the WT and the Y156F DAT mutant, in order

to determine if they demonstrated an atypical binding proﬁle,

as previously reported for ( )-modaﬁnil and its (R)-

enantiomer.¹³These data suggest that all three lead compounds

did indeed bind the DAT in a conformation that was more like

the atypical benztropines and unlike cocaine, with 11b having

the highest DAT aﬃnity and selectivity in this series. We

CONCLUSION

■

In summary, we have designed and synthesized a series of

modaﬁnil analogues that have higher DAT aﬃnity than the

parent molecule and have extended SAR at the DAT by

manipulating the oxidation states of the sulfoxide and the

amide, halogenating the phenyl rings, and/or functionalizing

the terminal nitrogen with N-substituted piperazines. Com-

pounds 8d, 9d, and 11b were selected as lead compounds from

G

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Figure 5. Locomotor eﬀects of (R)-modaﬁnil [(R)-2] and its analogues 4 and 11b compared to cocaine in mice. Systemic administration of cocaine

(3, 10 mg/kg, i.p.) produced a robust dose-dependent increase in locomotion (A). However, (R)-2 and its analogues 4 and 11b, at the same dose of

10 mg/kg, did not (C,D) or produced a very mild increase (B) in locomotion. When the dose was increased to 30 mg/kg, (R)-2 and 11b produced a

signiﬁcant dose-dependent increase in locomotion, while its analogue 4 did not. *p < 0.05, **p < 0.01, and ***p < 0.001, compared to the vehicle

control group at each time point marked.

1

according to deuterated solvent for H NMR spectra (CDCl₃, 7.26 or

DMSO-d₆, 2.50) and ¹³C NMR spectra (CDCl₃, 77.2 or DMSO-d₆,

39.5). Gas chromatography−mass spectrometry (GC/MS) data were

acquired (where obtainable) using an Agilent Technologies (Santa

Clara, CA) 6890N GC equipped with an HP-5MS column (cross-

linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. × 0.25 μm ﬁlm

thickness) and a 5973 mass-selective ion detector in electron-impact

mode. Ultrapure grade helium was used as the carrier gas at a ﬂow rate

of 1.2 mL/min. The injection port and transfer line temperatures were

250 and 280 °C, respectively. Combustion analysis was performed by

Atlantic Microlab, Inc. (Norcross, GA), and the results agree within

0.5% of calculated values. Melting point determination was

conducted using a Thomas-Hoover melting point apparatus and is

uncorrected. On the basis of NMR and combustion data, all ﬁnal

compounds are >95% pure.

discovered, that similar to previously reported atypical DAT

inhibitors,⁴⁹⁻⁵³compound 11b produced only moderate

locomotor stimulation in mice and was substantially less

eﬃcacious than cocaine. These results are consistent with an

atypical DAT inhibitor proﬁle and suggest that 11b may be a

potential lead for development as a psychostimulant abuse

therapeutic. Further investigations of this compound in rodent

models of cocaine and methamphetamine abuse, along with

compounds 4, 8d, and 9d, are underway to extend testing of

the atypical DAT inhibitor hypothesis, as well as to further

investigate the role of σ1 receptors in the behavioral proﬁle of

these agents. Finally, although 11b showed signiﬁcant

metabolism in mouse microsomes, preliminary data show a

rapid and high brain to plasma ratio, suggesting that 11b can

penetrate the blood−brain barrier suﬃciently to block DA

uptake via the DAT. Nevertheless, eﬀorts to modify this

structural template in order to improve metabolic stability are

also ongoing and will be reported in due course.

2-(Benzhydrylthio)ethan-1-ol (6a). 2-Mercaptoethan-1-ol (7.8 g,

100 mmol) was added to commercially available diphenylmethanol

(3.7 g, 20 mmol) in TFA (40 mL) and CH₂Cl₂(40 mL) at 0 °C. The

solution was warmed to room temperature and stirred overnight. The

solvent was removed, K₂CO₃(11 g, 80 mmol), H₂O (7 mL) and

acetone (25 mL) were added to the reaction residue, and the mixture

stirred at room temperature overnight. The solvent was removed, H₂O

(100 mL) was added to the residue obtained, and the aqueous mixture

was extracted with ethyl acetate (3 × 100 mL). The organic layer was

dried over MgSO₄, and the solvent was removed in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (hexane/ethyl

EXPERIMENTAL PROCEDURES

■

1

Synthesis. H and ¹³C NMR spectra were acquired using a Varian

Mercury Plus 400 spectrometer at 400 and 100 MHz, respectively.

Chemical shifts are reported in parts-per-million (ppm) and referenced

H

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acetate = 6:4) to give 6a (3.0 g, 61% yield) as a clear oil. GC/MS (EI)

methanol to give a white solid. Mp 209−210 °C; ¹H NMR (400 MHz,

CDCl₃) δ 7.41−7.43 (m, 4H), 7.19−7.32 (m, 6H), 5.21 (s, 1H),

3.77−3.82 (m, 1H), 3.41 (br, 1H), 2.17−2.67 (m, 14H), 1.11−1.14

(m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 141.4, 128.6, 128.5, 128.3,

127.2, 65.6, 62.2, 57.9, 54.5, 54.4, 53.1, 29.3, 20.0; Anal. (C₂₂H₃₀N₂OS·

2C₂H₂O₄·0.25H₂O) C, H, N.

1

m/z 244 (M⁺). H NMR (400 MHz, CDCl₃) δ 7.43−7.45 (m, 4H),

7.30−7.34 (m, 4H), 7.21−7.26 (m, 2H), 5.21 (s, 1H), 3.64−3.69 (m,

2H), 2.62−2.65 (m, 2H).

2-((Bis(4-ﬂuorophenyl)methyl)thio)ethan-1-ol (6b). Compound

6b was prepared as described for 6a using bis(4-ﬂuorophenyl)-

methanol (6.6 g, 30 mmol) to give the product (6.9 g, 82% yield) as a

1-(4-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)piperazin-1-yl)-

propan-2-ol (8d; JJC 8-089). Compound 8d was prepared as

described for 8a using 7b (950 mg, 2.76 mmol) and 1-(piperazin-1-

yl)propan-2-ol (398 mg, 2.76 mmol) to give the product (880 mg,

79% yield) as a yellow oil. The free base was converted to the oxalate

salt and recrystallized from methanol to give a white solid. Mp 205−

1

colorless oil. H NMR (400 MHz, CDCl₃) δ 7.35−7.39 (m, 4H),

6.99−7.03 (m, 4H), 5.20 (s, 1H), 3.68−3.70 (m, 2H), 2.59−2.62 (m,

2H).

2-((Bis(3-chlorophenyl)methyl)thio)ethan-1-ol (6c). Compound

6c was synthesized as described for compound 6a using bis(3-

chlorophenyl)methanol (2.84 g, 11.2 mmol) to give the product (3.34

g, 95% yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (s,

2H), 7.31−7.22 (m, 6H), 5.14 (s, 1H), 3.71 (t, J = 5.8 Hz, 2H), 2.63

(t, J = 6.0 Hz, 2H), 1.93 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ

142.5, 134.6, 130.0, 128.3, 127.9, 126.4, 60.6, 52.8, 35.3.

1

206 °C; H NMR (400 MHz, CDCl₃) δ 7.33−7.37 (m, 4H), 6.96−

7.02 (m, 4H), 5.19 (s, 1H), 3.77−3.82 (m, 1H), 3.41 (br,1H), 2.18−

2.69 (m, 14H), 1.11−1.13 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ

163.1, 160.7, 137.0, 129.8, 129.7, 115.7, 115.6, 115.5, 115.4, 65.5, 62.2,

57.8, 53.1, 52.9, 29.4, 20.0; Anal. (C₂₂H₂₈F₂N₂OS·2C₂H₂O₄) C, H, N.

1-(2-(Benzhydrylthio)ethyl)piperazine (8e). A mixture of 7a (1.4 g,

4.6 mmol), piperazine (2.35 g, 27.3 mmol), and K₂CO₃(1.05 g, 9.12

mmol) in acetonitrile (25 mL) was reﬂuxed overnight. The solvent

was removed, H₂O (100 mL) was added to the residue, and the

aqueous mixture was extracted with ethyl acetate (3 × 100 mL). The

organic layer was dried over MgSO₄, and solvent was removed in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (CHCl₃/MeOH/NH₄OH = 90/10/0.5) to give 8e (710 mg,

50% yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41−7.43

(m, 4H), 7.19−7.32 (m, 6H), 5.22 (s, 1H), 2.83−2.85 (m, 4H), 2.52−

2.54 (m, 4H), 2.34−2.37 (m, 4H).

Benzhydryl(2-bromoethyl)sulfane (7a). Triphenylphosphine (1.4

g, 5.3 mmol) was added to a solution of 6a (890 mg, 3.64 mmol) in

CH₃CN (12 mL), followed by the addition of carbon tetrabromide

(1.77 g, 5.34 mmol). The reaction was stirred at room temperature

overnight. The solvent was removed, and the crude product was

puriﬁed by ﬂash column chromatography (hexane/ethyl acetate = 5:1)

to give 6a (850 mg, 76% yield) as a colorless oil. ¹H NMR (400 MHz,

CDCl₃) δ 7.23−7.45 (m, 10H), 5.23 (s, 1H), 3.33−3.39 (m, 2H),

2.80−2.90 (m, 2H); GC/MS (EI) m/z 307 (M⁺).

(Bis(4-ﬂuorophenyl)methyl)(2-bromoethyl)sulfane (7b). Com-

pound 7b was prepared as described for 7a using 6b (6.9 g, 25

1

mmol) to give the product (7.0 g, 83% yield) as a light yellow oil. H

1-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)piperazine (8f). Com-

pound 8f was prepared as described for 8e using 7b (1.03 g, 3.00

NMR (400 MHz, CDCl₃) δ 7.34−7.37 (m, 4H), 6.00−7.04 (m, 4H),

5.21 (s, 1H), 3.36−3.40 (m, 2H), 2.81−2.85 (m, 2H); GC/MS (EI)

m/z 343 (M⁺).

1

mmol) to give the product (910 mg, 87% yield) as a yellow oil. H

NMR (400 MHz, CDCl₃) δ 7.34−7.37 (m, 4H), 6.97−7.26 (m, 4H),

5.20 (s, 1H), 2.84−2.86 (m, 4H), 2.50−2.52 (m, 4H), 2.35−2.37 (m,

4H).

(Bis(3-chlorophenyl)methyl)(2-bromoethyl)sulfane (7c). 7c was

prepared as described for 7a using 6c (3.23 g, 10.3 mmol) to give

1

product as colorless oil (3.03 g, 78% yield). H NMR (400 MHz,

1-Benzyl-4-(2-((bis(4-ﬂuorophenyl)methyl)thio)ethyl)piperazine

(8g). Compound 8g was prepared as described as 8a using 7b (687

mg, 2.00 mmol) and 1-benzylpiperazine (353 mg, 2.00 mmol) to give

the product as a yellow oil (530 mg, 60%). The free base was

converted to the oxalate salt and recrystallized from methanol to give a

CDCl₃) δ 7.38 (s, 2H), 7.28−7.22 (m, 6H), 5.14 (s, 1H), 3.39 (t, J =

8.0 Hz, 2H), 2.86 (t, J = 8.0 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ

142.3, 134.9, 130.2, 128.5, 128.2, 126.5, 53.6, 34.5, 30.2.

1-(2-(Benzhydrylthio)ethyl)-4-(3-phenylpropyl)piperazine (8a). A

mixture of 7a (850 mg, 2.76 mmol), commercially available 1-(3-

phenylpropyl)piperazine (564 mg, 2.76 mmol), K₂CO₃(1.52 g, 11.0

mmol), and KI (catalytic) in acetone (30 mL) was reﬂuxed overnight.

The solvent was removed, H₂O (50 mL) was added to the residue, and

the aqueous mixture was extracted with ethyl acetate (3 × 50 mL).

The organic layer was dried over MgSO₄, the solvent was removed in

vacuo, and the crude product was puriﬁed by ﬂash column

chromatography [ethyl acetate/triethylamine = 95:5] to give 8a

(810 mg, 61% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from methanol to give a white solid.

1

white solid. Mp 224−226 °C; H NMR (400 MHz, CDCl₃) δ 7.22−

7.38 (m, 9H), 6.96−7.02 (m, 4H), 5.20 (s, 1H), 3.45−3.51 (m, 2H),

2.43−2.68 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 163.1, 160.4,

138.0, 137.0, 129.8, 129.7, 129.2, 128.2, 127.0, 115.7, 115.6, 115.5,

115.4, 63.0, 57.9, 53.1, 53.0, 52.9, 29.4. Anal. Calc.: C, 58.24, H, 5.21,

N, 4.53. Found: C, 57.98, H, 5.16, N, 4.63. Anal. (C₂₆H₂₈F₂N₂S·

2C₂H₂O₄) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)-4-(4-ﬂuorobenzyl)-

piperazine (8h). Compound 8h was prepared as described as 8a using

7b (515 mg, 1.50 mmol) and 1-(4-ﬂuorobenzyl)piperazine (194 mg,

1.50 mmol) to give the product as a yellow oil (450 mg, 65.7%). The

free base was converted to the oxalate salt and recrystallized from

methanol to give a white solid Mp 221−223 °C; ¹H NMR (400 MHz,

CDCl₃) δ 7.33−7.37 (m, 4H), 7.23−7.27 (m, 2H), 6.95−7.00 (m,

6H), 5.18 (s, 1H), 3.44−3.46 (m, 2H), 2.41−2.56 (m, 12H); ¹³C

NMR (100 MHz, CDCl3) δ 163.1, 160.4, 137.0, 133.8,130.6, 130.5,

129.8, 115.6, 115.4, 115.1, 114.9, 62.2, 57.9, 53.0, 52.9, 29.4; GC/MS

(EI) m/z 456 (M⁺). Anal. Calc.: C, 56.6, H, 4.91, N, 4.40. Found: C,

56.35, H, 4.91, N, 4.38. Anal. (C₂₆H₂₇F₃N₂S·2C₂H₂O₄) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)-4-(4-

(triﬂuoromethyl)benzyl)piperazine (8i). Compound 8i was prepared

as described as 8a using 7b (515 mg, 1.50 mmol) and commercially

available 1-(4-(triﬂuoromethyl)benzyl)piperazine (367 mg, 1.50

mmol) to give the product as yellow oil (500 mg, 66%). The free

base was converted to the oxalate salt and recrystallized from methanol

to give a white solid Mp 223−224 °C; ¹H NMR (400 MHz, CDCl₃) δ

7.55−7.57 (m, 2H), 7.41−7.43 (m, 2H), 7.33−7.37 (m, 4H), 6.97−

7.01 (m, 4H), 5.19 (s, 1H), 3.53−3.54 (m, 2H), 2.43−2.55 (m, 12H);

¹³C NMR (100 MHz, CDCl3) δ 163.1, 160.7, 142.4, 137.0, 129.8,

1

Mp 210 °C (dec.); H NMR (400 MHz, CDCl₃) δ 7.16−7.43 (m,

15H), 5.22 (s, 1H), 2.33−2.64 (m, 16H), 1.78−1.82 (m, 2H); ¹³C

NMR (100 MHz, CDCl₃) δ 142.1, 141.4, 128.5, 128.4, 128.3, 127.2,

125.7, 58.0, 54.5, 54.4, 53.1, 53.0, 33.7, 29.3, 28.6; Anal. (C₂₈H₃₄N₂S·

2C₂H₂O₄·0.25H₂O) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)-4-(3-phenylpropyl)-

piperazine (8b). Compound 8b was prepared as described for 8a using

7b (950 mg, 2.76 mmol) to give the product (940 mg, 73% yield) as a

yellow oil. The free base was converted to the oxalate salt and

recrystallized from methanol to give a white solid. Mp 216−217 °C;

¹H NMR (400 MHz, CDCl₃) δ 7.34−7.37(m, 4H), 7.24−7.29(m,

2H), 7.15−7.19(m, 3H), 6.97−7.01(m, 4H), 5.20 (s, 1H), 2.33−2.64

(m, 16H), 1.67−1.82 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.1,

160.7, 142.1, 137.0, 129.8, 129.7, 128.4, 128.3, 125.7, 115.6, 115.3,

58.0, 53.1, 52.9, 52.8, 33.7, 29.4, 28.6; Anal. (C₂₈H₃₂F₂N₂S·2C₂H₂O₄)

C, H, N.

1-(4-(2-(Benzhydrylthio)ethyl)piperazin-1-yl)propan-2-ol (8c).

Compound 8c was prepared as described for (8a) using 7a (848

mg, 2.76 mmol) and 1-(piperazin-1-yl)propan-2-ol (398 mg, 2.76

mmol) to give the product (850 mg, 83% yield) as a yellow oil. The

free base was converted to the oxalate salt and recrystallized from

129.7, 129.2, 125.2, 125.1, 115.6, 115.4, 62.4, 57.8, 53.0, 52.9, 29.4;

GC/MS (EI) m/z 506 (M⁺). Anal. Calc.: C, 53.87, H, 4.59, N, 4.05.

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Found: C, 53.78, H, 4.69, N, 4.05. Anal. (C₂₇H₂₇F₅N₂S·2C₂H₂O₄·

0.25H₂O) C, H, N.

1-Benzyl-4-(2-((bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)-

piperazine (9e). Compound 5e was prepared as described for 9a using

8g (307 mg, 0.700 mmol) to give the product (150 mg, 47.1% yield)

as a yellow oil. The free base was converted to the oxalate salt and

recrystallized from methanol to give a white solid. Mp 219−220 °C

(dec.); ¹H NMR (400 MHz, CDCl₃) δ 7.37−7.44 (m, 4H), 7.24−7.33

(m, 5H), 7.05−7.11 (m, 4H), 5.30 (s, 1H), 3.50 (s, 2H), 2.45−2.80

(m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 165.9, 163.7, 161.5, 159.3,

138.0, 131.8, 129.2, 127.1, 115.6, 69.7, 63.0, 52.9, 50.9, 48.1; Anal.

(C₂₆H₂₈F₂N₂OS·2C₂H₂O₄) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)-4-(4-chlorobenzyl)-

piperazine (8j). Compound 8j was prepared as described as 8a using

7b (515 mg, 1.50 mmol) and commercially available1-(4-

chlorobenzyl)piperazine (316 mg, 1.50 mmol) to give the product

as a yellow oil (500 mg, 67.7%). The free base was converted to the

oxalate salt and recrystallized from methanol to give a white solid Mp

1

224−225 °C; GC/MS (EI) m/z 473 (M⁺); H NMR (400 MHz,

CDCl₃) δ 7.33−7.37 (m, 4H), 7.22−7.28 (m, 4H), 6.97−7.01 (m,

4H), 5.19 (s, 1H), 3.44−3.45 (m, 2H), 2.41−2.54 (m, 12H); ¹³C

NMR (100 MHz, CDCl3) δ 163.1, 160.6, 137.0, 136.6, 132.8, 130.4,

129.8, 129.7, 128.3, 115.7, 115.6, 115.5, 115.4, 62.2, 57.8, 52.9, 29.4.

Anal. Calc.: C, 54.80, H, 4.83, N, 4.26. Found: C, 54.61, H, 4.80, N,

4.28. Anal. (C₂₆H₂₇ClF₂N₂S·2C₂H₂O₄·0.25H₂O) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)-4-(4-

ﬂuorobenzyl)piperazine (9f). Compound 9f was prepared as

described for 9a using 8h (251 mg, 0.549 mmol) to give the product

(100 mg, 38.5% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from methanol to give a white solid.

1

Mp 216−218 °C (dec.); H NMR (400 MHz, CDCl₃) δ 7.37−7.43

1-(4-(2-((Bis(3-chlorophenyl)methyl)thio)ethyl)piperazin-1-yl)-

propan-2-ol (8k). Compound 8k was prepared as described for 8a

using 7c (866 mg, 2.30 mmol) and commercially available 1-

(piperazin-1-yl)propan-2-ol (335 mg, 2.32 mmol) to give the product

as a yellow oil (650 mg, 64% yield). The free base was converted to

the oxalate salt in a 2-propanol/acetone solvent mixture and recovered

as a white solid. Mp 119−121 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.39

(s, 2H), 7.29−7.18 (m, 6H), 5.15 (s, 1H), 3.85−3.76 (m, 1H), 2.72−

2.62 (br m, 2H), 2.59−2.18 (m, 13H), 1.13 (d, J = 6.4 Hz, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ 142.8, 134.5, 129.96, 128.4, 127.7, 126.4,

65.5, 62.2, 60.4, 57.8, 53.5, 53.1, 29.5, 19.9; Anal. (C₂₂H₂₈Cl₂N₂OS·

2C₂H₂O₄) C, H, N.

(m, 4H), 7.24−7.27 (m, 2H), 6.96−7.11 (m, 6H), 4.94 (s, 1H), 3,46

(s, 2H), 2.44−2.78 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 164.0,

163.7, 163.2, 161.5, 161.3, 160.8, 133.5, 131.8, 131.7, 131.0, 130.7,

130.6, 130.5, 130.4, 130.3, 116.4, 116.2, 115.9, 115.6, 115.1, 114.9,

69.7, 62.1, 53.0, 52.7, 50.8, 48.1; Anal. (C₂₆H₂₇F₃N₂OS·2C₂H₂O₄·

0.5H₂O) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)-4-(4-

(triﬂuoromethyl)benzyl)piperazine (9g). Compound 9g was prepared

as described for 9a using 8i (304 mg, 0.601 mmol) to give the product

(100 mg, 47.8% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from methanol to give a white solid.

1

Mp 216−218 °C (dec.); H NMR (400 MHz, CDCl₃) δ 7.55−7.57

1-(2-(Benzhydrylsulﬁnyl)ethyl)-4-(3-phenylpropyl)piperazine (9a).

Compound 9a was prepared as previously described⁸using 8a (431

mg, 1.00 mmol) to give the product (250 mg, 56% yield) as a yellow

oil. The free base was converted to the hydrochloride salt and

recrystallized from methanol to give a white solid. Mp 210 °C (dec.);

¹H NMR (400 MHz, CDCl₃) δ 7.15−7.50 (m, 15H), 4.96 (s, 1H),

(m, 2H), 7.37−7.43 (m, 6H), 7.04−7.11 (m, 4H), 4.94 (s, 1H), 3.54

(s, 2H), 2.45−2.83 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 164.0,

163.8, 161.6, 161.3, 142.3, 131.8, 131.0, 130.5, 130.4, 130.3, 129.5,

129.2, 128.6, 125.6, 125.2, 125.1, 122.9, 116.4, 116.2, 115.9, 115.669.8,

62.3, 53.0, 52.9, 50.8, 48.2; Anal. (C₂₇H₂₇F₅N₂OS·2C₂H₂O₄) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)-4-(4-

chlorobenzyl)piperazine (9h). Compound 9h was prepared as

described for 9a using 8j (360 mg, 0.761 mmol) to give the product

(160 mg, 43.0% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from methanol to give a white solid.

2.33−2.82 (m, 16H), 1.76−1.84 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 142.1, 136.0, 135.2, 129.3, 129.2, 128.7, 128.4, 128.3, 128.2,

125.8, 72.1, 57.9, 53.4, 53.0, 51.0, 48.2, 33.7, 28.6; Anal. (C₂₈H₃₄N₂OS·

2HCl·0.5H₂O) C, H, N.

1-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)-4-(3-

phenylpropyl)piperazine (9b). Compound 9b was prepared as

described for 9a using 8b (466 mg, 1.00 mmol) to give the product

(290 mg, 60% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from methanol to give a white solid.

Mp 204 °C (dec.); ¹H NMR (400 MHz, CDCl₃) δ 7.37−7.40(m, 4H),

7.24−7.28(m, 2H), 7.16−7.19(m, 3H), 7.05−7.11(m, 4H), 4.95 (s,

1H), 2.34−2.79 (m, 16H), 1.76−2.04 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 164.0, 163.8, 161.6, 142.1, 131.0, 130.6, 130.3, 128.4, 128.3,

125.8, 116.4, 116.2, 115.9, 115.6, 68.8, 68.2, 65.8, 57.9, 53.0, 50.9, 48.1,

33.7, 28.5; Anal. (C₂₈H₃₂F₂N₂OS·2C₂H₂O₄·H₂O) C, H, N.

1-(4-(2-(Benzhydrylsulﬁnyl)ethyl)piperazin-1-yl)propan-2-ol (9c).

Compound 9c was prepared as described for 9a using 8c (556 mg,

1.50 mmol) to give the product (450 mg, 78% yield) as a white solid.

The free base was converted to the oxalate salt and recrystallized from

methanol to give a white solid. Mp 195−197 °C (dec.); ¹H NMR (400

MHz, CDCl₃) δ 7.30−7.49 (m, 10H), 4.95 (s, 1H), 3.78−3.82 (m,

1H), 3.39 (br s, 1H), 2.18−2.83 (m, 14H), 1.11−1.12 (m, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ 136.0, 135.1, 129.3, 129.2, 128.7, 128.6,

128.3, 72.2, 65.5, 62.2, 53.1, 50.9, 48.3, 20.0; Anal. (C₂₂H₃₀N₂O₂S·

2C₂H₂O₄·0.25H₂O) C, H, N.

1

Mp 217−219 °C (dec.); H NMR (400 MHz, CDCl₃) δ 7.37−7.43

(m, 4H), 7.22−7.26 (m, 4H), 7.04−7.10 (m, 4H), 4.94 (s, 1H), 3.45

(s, 2H), 2.44−2.80 (m, 12H); ¹³C NMR (100 MHz, CDCl₃δ 164.4,

164.0, 163.8, 161.5, 161.3, 136.4, 132.8, 131.7, 131.7, 131.0, 130.5,

130.4, 130.3, 128.4, 116.4, 116.2, 115.9, 115.6, 69.8, 62.1, 52.9, 52.8,

50.8, 48.1; Anal. (C₂₆H₂₇ClF₂N₂OS·2C₂H₂O₄·0.25H₂O) C, H, N.

1-(4-(2-((Bis(3-chlorophenyl)methyl)sulﬁnyl)ethyl)piperazin-1-yl)-

propan-2-ol (9i). Compound 9i was prepared as described for 9a

using 8k (400 mg, 1.00 mmol) The free base (190 mg, 46% yield) was

obtained as a yellow oil, which was converted to the oxalate salt in a 2-

propanol/acetone solvent mixture and recovered as a cream-colored

solid. Mp 192−194 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.42−7.31 (m,

8H), 4.91 (s, 1H), 3.85−3.80 (m, 1H), 2.84−2.21 (m, 15H), 1.12 (d, J

= 6.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 137.5, 136.4, 135.3,

134.7, 130.6, 130.1, 129.3, 128.80, 128.77, 127.5, 126.7, 70.4, 65.5,

62.3, 53.2, 50.7, 48.3, 19.9; Anal. (C₂₂H₂₈Cl₂N₂O₂S·2C₂H₂O₄) C, H,

N.

1-(4-(2-(Benzhydrylthio)ethyl)piperazin-1-yl)-3-phenylpropan-2-

ol (10a). A solution of compound 8e (710 mg, 2.27 mmol) and 2-

benzyloxirane (304.6 mg, 2.27 mmol) in isopropanol (24 mL) was

reﬂuxed overnight. Solvent was removed, and the reaction residue was

puriﬁed by ﬂash column chromatography (hexane/ethyl acetate/

triethylamine = 49:49:2) to give 10a (850 mg, 84% yield) as a yellow

oil. The free base was converted to the oxalate salt and recrystallized

from hot isopropanol to give a white solid. Mp 210−211 °C; ¹H NMR

(400 MHz, CDCl₃) δ 7.40−7.43 (m, 4H), 7.20−7.32 (m, 11H), 5.22

(s, 1H), 3.88−3.93 (m, 1H), 3.45 (br s, 1H), 2.27−2.83 (m, 16H); ¹³C

NMR (100 MHz, CDCl₃) δ 141.4, 138.3, 129.3, 128.6, 128.5, 128.3,

127.3, 127.2, 126.3, 67.2, 63.4, 57.9, 54.4, 53.1, 41.3, 29.3; Anal.

(C₂₈H₃₄N₂OS·2C₂H₂O₄·0.5H₂O) C, H, N.

1-(4-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)piperazin-1-yl)-

propan-2-ol (9d; JJC8-091). Compound 9d was prepared as described

for 9a using 8d (610 mg, 1.50 mmol) to give the product (340 mg,

54% yield) as a yellow oil. The free base was converted to the oxalate

salt and recrystallized from methanol to give a white solid. Mp 190−

1

191 °C (dec.); H NMR (400 MHz, CDCl₃) δ 7.36−7.43 (m, 4H),

7.04−7.12 (m, 4H), 4.92 (s, 1H), 3.77−3.83 (m, 1H), 2.22−2.84 (m,

14H), 1.11−1.12 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.0,

163.8, 161.6, 161.3, 131.7, 131.0, 130.6, 130.5, 130.4, 130.3, 116.4,

116.2, 115.9, 115.6, 69.9, 69.8, 65.5, 62.3, 53.1, 50.8, 48.3, 20.0; Anal.

(C₂₂H₂₈F₂N₂O₂S·2C₂H₂O₄) C, H, N.

1-(4-(2-((Bis(4-ﬂuorophenyl)methyl)thio)ethyl)piperazin-1-yl)-3-

phenylpropan-2-ol (10b). Compound 10b was prepared as described

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for 10a using 8f (455 mg, 1.31 mmol) to give the product (540 mg,

86% yield) as a yellow oil. The free base was converted to the oxalate

salt and recrystallized from hot acetone to give a white solid. Mp 206−

CDCl₃) δ 167.2, 163.2, 160.7, 136.2, 130.0, 115.6, 115.4, 65.5, 62.4,

53.2, 52.7, 52.4, 46.4, 41.9, 33.2, 21.1; Anal. (C₂₂H₂₆F₂N₂O₂S·

C₂H₂O₄) C, H, N.

1

207 °C; H NMR (400 MHz, CDCl₃) δ 7.19−7.37 (m, 9H), 6.96−

2-((Bis(4-ﬂuorophenyl)methyl)thio)-1-(4-propylpiperazin-1-yl)-

ethan-1-one (13d). Compound 12b (294 mg, 1.00 mmol) was

reﬂuxed in SOCl₂(3 mL) for 2 h. The solvent was removed. The

reaction mixture was added to 1-propylpiperazine (128 mg, 1 mmol),

NaHCO₃(500 mg, 6.00 mmol) in amylene-stabilized CHCl₃(10 mL)

and H₂O (5 mL) at 0 °C and stirred at room temperature for 2 h. H₂O

(5 mL) was added to the reaction mixture, and the aqueous mixture

was extracted with chloroform (3 × 10 mL). The organic layer was

dried over MgSO₄, and the solvent was removed in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (ethyl acetate/

triethylamine = 95:5) to give 13d (220 mg, 54% yield) as a yellow oil.

The free base was converted to the oxalate salt and recrystallized from

7.02 (m, 4H), 5.18 (s, 1H), 3.88−3.93 (m, 1H), 3.45 (br s, 1H), 2.31−

2.81 (m, 16H); ¹³C NMR (100 MHz, CDCl₃) δ 163.1, 160.7, 138.2,

137.0, 129.8, 129.7, 129.3, 128.3, 126.3, 115.6, 115.4, 67.2, 63.4, 57.8,

53.1, 52.9, 41.3, 29.4; Anal. (C₂₈H₃₂F₂N₂OS·2C₂H₂O₄·0.25H₂O) C, H,

N.

1-(4-(2-(Benzhydrylsulﬁnyl)ethyl)piperazin-1-yl)-3-phenylpropan-

2-ol (11a). Compound 11a was prepared as described for 9a using 10a

(534 mg, 1.20 mmol) to give the product (340 mg, 61% yield) as a

yellow oil. The free base was converted to the oxalate salt and

recrystallized from hot methanol to give a white solid. Mp 198−200

1

°C (dec.); H NMR (400 MHz, CDCl₃) δ 7.20−7.49 (m, 15H), 4.95

(s, 1H), 3.88−3.92 (m, 1H), 2.31−2.83 (m, 16H); ¹³C NMR (100

MHz, CDCl₃) δ 138.2, 136.0, 135.2, 129.3, 129.2, 128.9, 128.7, 128.6,

128.4, 128.3, 126.3, 72.1, 67.3, 63.4, 53.0, 50.8, 48.2, 41.3; Anal.

(C₂₈H₃₄N₂O₂S·2C₂H₂O₄·0.25H₂O) C, H, N.

1

methanol and acetone to give a white solid. Mp 167−168 °C; H

NMR (400 MHz, CDCl₃) δ 7.36−7.40 (m, 4H), 6.98−7.03 (m, 4H),

5.34 (s, 1H), 3.58−3.60 (m, 2H), 3.40−3.42 (m, 2H), 3.16 (s, 2H),

2.37−2.41 (m, 4H), 2.28−2.31 (m, 2H), 1.47−1.52 (m, 2H), 1.13−

1.27 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 163.2, 160.7,

136.3, 130.0, 115.6, 115.4, 60.4, 53.2, 52.7, 52.4, 46.3, 41.9, 33.3, 19.9,

11.9; Anal. (C₂₂H₂₆F₂N₂OS·C₂H₂O₄) C, H, N.

1-(4-(2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)ethyl)piperazin-1-yl)-

3-phenylpropan-2-ol (11b; JJC 8-088). Compound 11b was prepared

as described for 11a using 10b (400 mg, 0.83 mmol) to give the

product (280 mg, 68% yield) as a yellow oil. The free base was

converted to the oxalate salt and recrystallized from hot methanol to

2-((Bis(4-ﬂuorophenyl)methyl)thio)-1-(4-(4-ﬂuorobenzyl)-

piperazin-1-yl)ethan-1-one (13e). Compound 13e was prepared as

described for 13d using 12b (589 mg, 2.00 mmol) and 1-(4-

ﬂuorobenzyl)piperazine (389 mg, 2.00 mmol) to give the product

(600 mg, 64% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from acetone to give a white solid.

Mp 168−170 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.35−7.39 (m, 4H),

7.25−7.28 (m, 2H), 6.98−7.03 (m, 6H), 5.34 (s, 1H), 3.57−3.60 (m,

2H), 3.46 (s, 2H), 3.38−3.41 (m, 2H), 3.15 (s, 2H), 2.37−2.40 (m,

4H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 163.3, 163.2, 160.9,

160.7, 136.3, 133.3, 133.2, 130.6, 130.5, 130.0, 115.6, 115.4, 115.3,

115.1, 62.0, 52.8, 52.5, 52.4, 46.3, 41.9, 33.3; Anal. (C₂₆H₂₅F₃N₂OS·

C₂H₂O₄·1.25H₂O) C, H, N.

1

give a white solid. Mp 198−200 °C (dec.); H NMR (400 MHz,

CDCl₃) δ 7.20−7.43 (m, 9H), 7.05−7.11 (m, 4H), 4.93 (s, 1H),

3.88−3.92 (m, 1H), 2.29−2.84 (m, 16H); ¹³C NMR (100 MHz,

CDCl₃) δ 161.6, 161.3, 138.2, 131.8, 131.0, 130.9, 130.5, 130.3, 129.3,

128.4, 126.3, 116.4, 116.2, 115.9, 115.6, 69.4, 67.3, 63.4, 53.1, 50.8,

48.2, 41.3, Anal. (C₂₈H₃₂F₂N₂O₂S·2C₂H₂O₄) C, H, N.

2-(Benzhydrylthio)-1-(4-(3-phenylpropyl)piperazin-1-yl)ethan-1-

one (13a). A mixture of CDI (583 mg, 3.60 mmol) and 12a²⁷(775

mg, 3.00 mmol) in THF (24 mL) was stirred at room temperature

under argon. After 2 h of reaction time, 1-(3-phenylpropyl)piperazine

(613 mg, 3.00 mmol) in THF (15 mL) was added, and the reaction

mixture was stirred overnight. Solvent was removed, and the reaction

residue was puriﬁed by ﬂash column chromatography (ethyl acetate/

triethylamine 95:5) to give 13a (1.2 g, 90% yield) as a yellow oil. The

free base was converted to the oxalate salt and recrystallized from hot

isopropanol to give a white solid. Mp 92−95 °C; ¹H NMR (400 MHz,

CDCl₃) δ 7.42−7.45 (m, 4H), 7.17−7.33(m, 11H), 5.34 (s, 1H),

3.57−3.60 (t, 2H, J = 5.0 Hz), 3.37−3.40 (t, 2H, J = 5.2 Hz), 3.18 (s,

2H), 2.62−2.66 (m, 2H), 2.34−2.39 (m, 6H), 1.78−1.84 (m, 2H); ¹³C

NMR (100 MHz, CDCl₃) δ 167.2, 141.9, 140.7, 128.6, 128.5, 128.4,

128.3, 127.3, 125.8, 57.7, 54.1, 53.2, 52.7, 46.3, 41.9, 33.5, 28.4; Anal.

(C₂₈H₃₂N₂OS·C₂H₂O₄·0.5H₂O) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)thio)-1-(4-(3-phenylpropyl)-

piperazin-1-yl)ethan-1-one (13b). Compound 13b was prepared as

described for 13a using 12b²⁷(588 mg, 2.00 mmol) and 1-(3-

phenylpropyl)piperazine (408 mg, 2.00 mmol) to give the product

(650 mg, 71% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from hot methanol to give a white

solid. Mp 123−125 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36−7.39 (m,

4H), 7.26−7.30 (m, 2H), 7.17−7.20 (m, 3H), 6.98−7.02 (m, 4H),

5.34 (s, 1H), 3.58−3.60 (t, 2H, J = 5.0 Hz), 3.39−3.42 (t, 2H, J = 4.8

Hz), 3.15 (s, 2H), 2.62−2.66 (m, 2H), 2.34−2.41 (m, 6H), 1.79−1.83

(m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 163.2, 160.7, 141.9,

136.3, 130.0, 128.4, 125.9, 115.6, 115.4, 57.7, 53.1, 52.7, 52.4, 46.4,

41.9, 33.5, 33.3, 28.4; Anal. (C₂₈H₃₀F₂N₂OS·C₂H₂O₄·0.25H₂O) C, H,

N.

2-((Bis(4-ﬂuorophenyl)methyl)thio)-1-(4-(4-chlorobenzyl)-

piperazin-1-yl)ethan-1-one (13f). Compound 13f was prepared as

described for 13d using 12b (589 mg, 2.00 mmol) and 1-(4-

chlorobenzyl)piperazine (411 mg, 2.00 mmol) to give the product

(650 mg, 67% yield) as a yellow oil. The free base was converted to

the oxalate salt and recrystallized from acetone/ethyl ether to give a

1

white solid. Mp 165−167 °C; H NMR (400 MHz, CDCl₃) δ 7.36−

7.40 (m, 4H), 7.25−7.28 (m, 4H), 6.98−7.02 (m, 4H), 5.33 (s, 1H),

3.58−3.59 (m, 2H), 3.38−3.46 (m, 4H), 3.15 (s, 2H), 2.37−2.40 (m,

4H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 163.2, 160.7, 136.3,

136.1, 133.0, 130.3, 130.0, 128.5, 115.6, 115.4, 62.0, 52.9, 52.6, 52.4,

46.3, 41.8, 33.3; Anal. (C₂₆H₂₅ClF₂N₂OS·C₂H₂O₄) C, H, N.

2-((Bis(3-chlorophenyl)methyl)thio)-1-(4-(2-hydroxypropyl)-

piperazin-1-yl)ethan-1-one (13g). Compound 13g was prepared as

described for 13a using 12c²⁷(654 mg, 2.00 mmol) and 1-(piperazin-

1-yl)propan-2-ol (288 mg, 2.00 mmol) to give the product (770 mg,

75% yield) as a yellow oil. The free base was converted to the oxalate

salt and recrystallized from hot methanol to give a white solid. Mp

1

160−161 °C; H NMR (400 MHz, CDCl₃) δ 7.28−7.41 (m, 2H),

7.22−7.24 (m, 6H), 5.30 (s, 1H), 3.82−3.87 (m, 1H), 3.56−3.62 (m,

2H), 3.40−3.43 (m, 2H), 3.20−3.22 (m, 3H), 2.59−2.65 (m, 2H),

2.23−2.40 (m, 4H), 1.13−1.15 (m, 3H); ¹³C NMR (100 MHz,

CDCl₃) δ 166.9, 142.1, 134.6, 130.0, 128.4, 127.9, 126.7, 65.5, 62.3,

53.1, 52.9, 52.7, 46.4, 41.9, 33.2, 19.9; Anal. (C₂₂H₂₆Cl₂N₂O₂S·

C₂H₂O₄) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)thio)-1-(4-(2-hydroxypropyl)-

piperazin-1-yl)ethan-1-one (13c). Compound 13c was prepared as

described for 13a using 12b (588 mg, 2.00 mmol) and 1-(piperazin-1-

yl)propan-2-ol (288 mg, 2.00 mmol) to give the product (630 mg,

75% yield) as a yellow oil. The free base was converted to the oxalate

salt and recrystallized from hot methanol to give a white solid. Mp

2-(Benzhydrylsulﬁnyl)-1-(4-(3-phenylpropyl)piperazin-1-yl)ethan-

1-one (14a). Compound 14a was prepared as described for 9a using

13a (667 mg, 1.50 mmol) to give the product (500 mg, 72%) as a

yellow oil. The free base was converted to the oxalate salt and

recrystallized from hot acetone to give a white solid. Mp 180−182 °C;

¹H NMR (400 MHz, CDCl₃) δ 7.49−7.55 (m, 4H), 7.16−7.43 (m,

11H), 5.30 (s, 1H), 3.27−3.70 (m, 6H), 2.61−2.65 (m, 2H), 2.33−

2.47 (m, 6H), 1.75−1.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ

163.1, 141.9, 136.0, 133.6, 130.0, 129.1, 129.0, 128.7, 128.5, 128.4,

1

163−165 °C; H NMR (400 MHz, CDCl₃) δ 7.36−7.37 (m, 4H),

6.99−7.03 (m, 4H), 5.33 (s, 1H), 3.82−3.87 (m, 1H), 3.57−3.61 (m,

2H), 3.40−3.43 (m, 2H), 3.22 (s, 1H), 3.16 (s, 2H), 2.59−2.60 (m,

2H), 2.23−2.38 (m, 4H), 1.13−1.27 (m, 3H); ¹³C NMR (100 MHz,

K

DOI: 10.1021/acs.jmedchem.6b01373

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

128.3, 128.2, 125.8, 70.1, 70.0, 57.5, 53.1, 52.6, 46.4, 42.0, 33.5, 28.4;

Anal. (C₂₈H₃₂N₂O₂S·C₂H₂O₄·0.25H₂O) C, H, N.

162.7, 137.4, 135.1, 134.8, 134.7, 130.4, 130.1, 130.0, 129.1, 129.0,

128.7, 128.1, 127.1, 68.1, 65.4, 62.4, 53.2, 53.1, 52.8, 52.7, 52.6, 46.4,

46.2, 42.0, 19.9; Anal. (C₂₂H₂₆Cl₂N₂O₃S·C₂H₂O₄) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)-1-(4-(3-phenylpropyl)-

piperazin-1-yl)ethan-1-one (14b). Compound 14b was prepared as

described for 9a using 13b (480 mg, 1.00 mmol) to give the product

(200 mg, 40%) as a yellow oil. The free base was converted to the

oxalate salt and recrystallized from hot acetone to give a white solid.

Mp 135−136 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.45−7.52 (m, 4H),

7.06−7.30 (m, 9H), 5.36 (s, 1H), 3.54−3.69 (m, 3H), 3.27−3.45 (m,

3H), 2.61−2.65 (m 2H), 2.35−2.45 (m, 6H), 1.76−1.84 (m, 2H); ¹³C

NMR (100 MHz, CDCl₃) δ 164.2, 163.8, 162.8, 162.7, 161.7, 161.3,

160.2, 141.8, 131.8, 131.7, 130.7, 130.6, 129.0, 128.4, 125.9, 116.2,

116.0, 115.9, 115.7, 67.7, 57.5, 53.1, 52.7, 52.6, 46.4, 42.0, 33.5, 28.3;

Anal. (C₂₈H₃₀F₂N₂O₂S·2C₂H₂O₄) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)-1-(4-(2-hydroxypropyl)-

piperazin-1-yl)ethan-1-one (14c). Compound 14c was prepared as

described for 9a using 13c (600 mg, 1.43 mmol) to give the product

(70 mg, 16%) as a yellow oil. The free base was converted to the

oxalate salt and recrystallized from hot acetone to give a white solid.

Mp 162−164 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.44−7.51 (m, 4H),

7.06−7.13 (m, 4H), 5.36 (s, 1H), 3.83−3.88 (m, 1H), 3.24−3.67 (m,

7H), 2.60−2.67 (m, 2H), 2.26−2.42 (m, 4H), 1.13−1.14 (m, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ 164.2, 163.8, 162.9, 161.7, 161.3, 131.7,

131.6, 131.5, 130.7, 129.1, 116.3, 116.1, 116.0, 65.4, 62.4, 53.3, 53.2,

52.7, 52.6, 52.4, 52.3, 46.4, 42.0, 20.0; Anal. (C₂₂H₂₆F₂N₂O₃S·

C₂H₂O₄) C, H, N.

Radioligand Binding Assays. DAT Binding Assay. Striata were

dissected from male Sprague−Dawley rat brains (supplied on ice from

Bioreclamation (Hicksville, NY) and prepared by homogenizing

tissues in 20 volumes (w/v) of ice cold modiﬁed sucrose phosphate

buﬀer (0.32 M sucrose, 7.74 mM Na₂HPO₄, and 2.26 mM NaH₂PO₄,

pH adjusted to 7.4) using a Brinkman Polytron (Setting 6 for 20 s)

and centrifuged at 20,000 rpm for 10 min at 4 °C. The resulting pellet

was resuspended in buﬀer, recentrifuged, and suspended in buﬀer

again to a concentration of 10 mg/mL, original wet weight (OWW).

Experiments were conducted in assay tubes containing 0.5 mL of

sucrose phosphate buﬀer, 0.5 nM [³H]WIN 35,428⁵⁴(K_dvalue = 5.53,

speciﬁc activity = 84 ci/mmol; PerkinElmer Life Sciences, Waltham,

MA), 1.0 mg of tissue OWW, and various concentrations of the

inhibitor. The reaction was started with the addition of tissue, and

tubes were incubated for 120 min on ice. Nonspeciﬁc binding was

determined using 100 μM cocaine HCl.

SERT Binding Assay. Membranes from frozen brain stems dissected

from male Sprague−Dawley rat brains (supplied on ice from

Bioreclamation, Hicksville, NY) were homogenized in 20 volumes

(w/v) of 50 mM Tris buﬀer (120 mM NaCl and 5 mM KCl, adjusted

to pH 7.4) at 25 °C using a Brinkman Polytron (at setting 6 for 20 s).

The tissue was centrifuged at 20,000 rpm for 10 min at 4 °C. The

resulting pellet was suspended in buﬀer and centrifuged again. The

ﬁnal pellet was resuspended in cold buﬀer to a concentration of 15

mg/mL OWW. Experiments were conducted in assay tubes containing

0.5 mL of buﬀer, 1.4 nM [³H]citalopram (K_dvalue = 1.94 nM, speciﬁc

activity = 83 ci/mmol; PerkinElmer Life Sciences, Waltham, MA), 1.5

mg of brain stem tissue, and various concentrations of the inhibitor.

The reaction was started with the addition of the tissue, and the tubes

were incubated for 60 min at room temperature. Nonspeciﬁc binding

was determined using 10 μM ﬂuoxetine.

NET Binding Assay. Membranes from frozen frontal cortex

dissected from male Sprague−Dawley rat brains (supplied on ice

from Bioreclamation, Hicksville, NY) were homogenized in 20

volumes (w/v) of 50 mM Tris buﬀer (120 mM NaCl and 5 mM

KCl, adjusted to pH 7.4) at 25 °C using a Brinkman Polytron (at

setting 6 for 20 s). The tissue was centrifuged at 20,000 rpm for 10

min at 4 °C. The resulting pellet was suspended in buﬀer and

centrifuged again. The ﬁnal pellet was resuspended in cold buﬀer to a

concentration of 80 mg/mL OWW. Experiments were conducted in

assay tubes containing 0.5 mL of buﬀer, 0.5 nM [³H]nisoxetine (K_d

value = 1.0 nM, speciﬁc activity = 82 ci/mmol; PerkinElmer Life

Sciences, Waltham, MA), 8 mg of frontal cortex tissue, and various

concentrations of the inhibitor. The reaction was started with the

addition of the tissue, and the tubes were incubated for 180 min at 0−

4 °C. Nonspeciﬁc binding was determined using 1 μM desipramine.

The solvent used to dissolve the various analogues of modaﬁnil was

typically methanol and was present at a ﬁnal concentration of 5%.

Extensive studies previously in this and other laboratories determined

that methanol has no eﬀect on binding at the DAT and SERT.

However, there is an eﬀect of methanol on binding at the NET, and

therefore, methanol concentration was controlled in all tubes in that

assay. When compounds were not soluble in methanol, we used either

ethanol or DMSO at ﬁnal concentrations of no greater than 5 or 6%,

respectively. Previous studies found no eﬀect of either of these solvents

at these concentrations on binding at any of the sites. For all three

MAT binding assays, incubations were terminated by rapid ﬁltration

through Whatman GF/B ﬁlters, presoaked in 0.3% (SERT) or 0.05%

(DAT, NET) polyethylenimine, using a Brandel R48 ﬁltering manifold

(Brandel Instruments Gaithersburg, Maryland). The ﬁlters were

washed twice with 5 mL of cold buﬀer and transferred to scintillation

vials. Cytoscint (MP Biomedicals, OH) (3.0 mL) was added, and the

vials were counted the next day using a Beckman 6000 liquid

scintillation counter (Beckman Coulter Instruments, Fullerton,

California) or a Tri-Carb 2910-B liquid scintillation counter

(PerkinElmer Life Sciences, MA). The K_ivalues for the modaﬁnil

derivatives were obtained using nonlinear least-squares regression

2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)-1-(4-propylpiperazin-1-yl)-

ethan-1-one (14d). Compound 14d was prepared as described for 9a

using 13d (120 mg, 0.297 mmol) to give the product (40 mg, 32%) as

a yellow oil. The free base was converted to the oxalate salt and

recrystallized from hot acetone and methanol to give a white solid. Mp

1

170−171 °C; H NMR (400 MHz, CDCl₃) δ 7.45−7.52 (m, 4H),

7.06−7.13 (m, 4H), 5.36 (s, 1H), 3.36−3.67 (m, 6H), 2.30−2.45 (m,

6H), 1.47−1.53 (m, 2H), 0.90−0.92 (m, 3H); ¹³C NMR (100 MHz,

CDCl₃) δ 164.2, 163.8, 162.8, 161.7, 161.3, 131.8, 131.7, 130.7, 130.6,

129.0, 116.2, 116.0, 115.9, 115.7, 67.7, 60.2, 53.1, 52.6, 46.3, 41.9, 19.8,

11.8; Anal. (C₂₂H₂₆F₂N₂O₂S·C₂H₂O₄) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)-1-(4-(4-ﬂuorobenzyl)-

piperazin-1-yl)ethan-1-one (14e). Compound 14e was prepared as

described for 9a using 10e (510 mg, 1.09 mmol) to give the product

(120 mg, 23%) as a yellow oil. The free base was converted to the

oxalate salt and recrystallized from hot acetone and methanol to give a

1

white solid. Mp 158−159 °C; H NMR (400 MHz, CDCl₃) δ 7.44−

7.48 (m, 4H), 7.24−7.26 (m, 2H), 6.98−7.13 (m, 6H), 5.36 (s, 1H),

3.27−3.68 (m, 8H), 2.34−2.45 (m, 4H); ¹³C NMR (100 MHz,

CDCl₃) δ 166.2, 164.2, 163.8, 162.8, 161.7, 161.3, 160.9, 133.1, 131.7,

130.7, 130.6, 130.5, 129.0, 116.2, 116.0, 115.9, 115.7, 115.3, 115.1,

67.7, 61.8, 52.9, 52.7, 52.4, 46.4, 42.0; Anal. (C₂₆H₂₅F₃N₂O₂S·C₂H₂O₄·

1.5H₂O) C, H, N.

2-((Bis(4-ﬂuorophenyl)methyl)sulﬁnyl)-1-(4-(4-chlorobenzyl)-

piperazin-1-yl)ethan-1-one (14f). Compound 14f was prepared as

described for 9a using 13f (487 mg, 1.00 mmol) to give the product

(200 mg, 40%) as a yellow oil. The free base was converted to the

oxalate salt and recrystallized from hot acetone and methanol to give a

1

white solid. Mp 157−158 °C; H NMR (400 MHz, CDCl₃) δ 7.44−

7.52 (m, 4H), 7.22−7.30 (m, 4H), 7.06−7.13 (m, 4H), 5.36 (s, 1H),

3.30−3.65 (m, 8H), 2.37−2.43 (m, 4H); ¹³C NMR (100 MHz,

CDCl₃) δ 164.2, 163.8, 162.8, 161.7, 161.3, 136.0, 133.1, 131.7, 130.7,

130.3, 129.1, 129.0, 128.5, 116.2, 116.0, 115.9, 115.7, 67.7, 61.9, 52.9,

52.7, 52.4, 46.4, 42.0; Anal. (C₂₆H₂₅ClF₂N₂O₂S·C₂H₂O₄·1.5H₂O) C,

H, N.

2-((Bis(3-chlorophenyl)methyl)sulﬁnyl)-1-(4-(2-hydroxypropyl)-

piperazin-1-yl)ethan-1-one (14g). Compound 14g was prepared as

described for 9a using 13g (590 mg, 1.15 mmol) to give the product

(70 mg, 11%) as a yellow oil. The free base was converted to the

oxalate salt and recrystallized from hot acetone and methanol to give a

1

white solid. Mp 169−170 °C; H NMR (400 MHz, CDCl₃) δ 7.31−

7.43 (m, 8H), 5.33 (s, 1H), 3.82−3.84 (m, 1H), 3.59−3.67 (m, 3H),

3.33−3.47 (m, 3H), 3.16−3.19 (m, 1H), 2.61−2.66 (m, 2H), 2.24−

2.42 (m, 4H), 1.13−1.14 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ

L

DOI: 10.1021/acs.jmedchem.6b01373

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(using GraphPad Prism Software, San Diego, CA) of the displacement

data giving IC₅₀values, from which aﬃnities (K_ivalues) were

calculated using the Cheng−Prusoﬀ equation.⁵⁵

temperature and then terminated by ﬁltration through PerkinElmer

UniFilter-96 GF/B ﬁlters, presoaked for 1 h in 0.5% polyethylenimine,

using a Brandel 96-Well Plates Harvester Manifold (Brandel

Instruments, Gaithersburg, MD). The ﬁlters were washed 3 times

with 3 mL (3 × 1 mL/well) of ice cold EBSS buﬀer. 65 μL of

PerkinElmer MicroScint 20 Scintillation Cocktail was added to each

well, and ﬁlters were counted using a PerkinElmer MicroBeta

Microplate Counter. IC₅₀values for each compound were determined

from dose−response curves, and K_ivalues were calculated using the

Cheng−Prusoﬀ equation.⁵⁵These analyses were performed using

GraphPad Prism version 6.00 for Macintosh (GraphPad Software, San

Diego, CA). Reported K_ivalues were determined from at least three

independent experiments, each with duplicate determinations.

Bioluminescence Resonance Energy Transfer (BRET) Assay.

The Gα−γ protein activation assay uses the RLuc8-fused Gα protein

subunit and GFP10-fused Gγ protein for a resonance energy transfer

pair. Flag-tagged receptor and untagged Gβ constructs were

cotransfected. The BRET assays were performed as described

previously.⁵⁸Brieﬂy, human embryonic kidney cells (HEK-293T)

were transfected, using a constant amount of plasmid DNA but various

ratios of plasmids encoding the fusion protein partners. Expression of

GFP10 fusion proteins was estimated by measuring ﬂuorescence at

515 nm following excitation at 405 nm. Expression of RLuc8 fusion

proteins was estimated by measuring the luminescence of the cells

after incubation with 5 μM coelenterazine 400a. In parallel, BRET was

measured as the ﬂuorescence of the cells at 535 nm at the same time

points using a Mithras LB940 reader (Berthold). In order to measure

the antagonist activity, 10 min of preincubation of 1 μM quinpirole

precedes the 10 min of incubation of tested compounds before the

sample reading.

σ₁Receptor Binding Assay. σ₁receptor binding was performed as

previously reported.⁵⁶Brieﬂy, frozen whole-guinea pig brains (minus

cerebellum) were thawed on ice, weighed, and homogenized (with a

glass and Teﬂon homogenizer) in 10 mM Tris-HCl with 0.32 M

sucrose at pH 7.4 (10 mL/g tissue). The homogenate was centrifuged

at 1000g for 10 min at 4 °C. The supernatant was collected into a clean

centrifuge tube, and the remaining pellet was resuspended by vortex in

10 mL of buﬀer (tissue) and centrifuged again at 50,000g for 15 min at

4 °C. The resulting pellet was resuspended in experimental buﬀer to

80 mg/mL original wet weight (OWW).

Ligand binding experiments were conducted in polypropylene assay

tubes containing 0.5 mL of 50 mM Tris-HCl buﬀer at pH 8.0. Each

tube contained 3 nM [³H](+)-pentazocine (PerkinElmer Life and

Analytical Sciences, Waltham, MA) and 8.0 mg of tissue OWW.

Nonspeciﬁc binding was determined using 10 mM haloperidol. The

reaction was started with the addition of tissue, and the tubes were

incubated for 120 min at room temperature.

Incubations for all binding assays were terminated by rapid ﬁltration

through Whatman GF/B ﬁlters, presoaked in polyethylenimine, using

a Brandel R48 ﬁltering manifold (Brandel Instruments, Gaithersburg,

MD). The ﬁlters were washed twice with 5 mL of ice-cold buﬀer and

transferred to scintillation vials. Beckman Ready Safe (3.0 mL) was

added, and the vials were counted the next day using a Beckman 6000

liquid scintillation counter (Beckman Coulter Instruments, Fullerton,

CA) at 50% eﬃciency. Assays were typically conducted in at least three

independent experiments, each performed in triplicate.

For the displacement of radioligand binding, IC₅₀values were

computed using a nonlinear, least-squares regression analysis (Prism;

GraphPad Software Inc., San Diego, CA). Aﬃnities (K_ivalues) were

calculated using the concentration of radioligand used in the assay.

D₂-Like Binding Assay. Binding at dopamine D₂-like receptors was

determined using previously described methods.⁵⁷Membranes were

prepared from HEK293 cells stably expressing human D₂R, D₃R, or

D₄R, grown in a 50:50 mix of DMEM and Ham’s F12 culture media,

supplemented with 20 mM HEPES, 2 mM ^L-glutamine, 0.1 mM

nonessential amino acids, 1× antibiotic/antimycotic, 10% heat-

inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life

Technologies, Grand Island, NY) and kept in an incubator at 37 °C

and 5% CO₂. Upon reaching 80−90% conﬂuence, cells were harvested

using premixed Earle’s Balanced Salt Solution (EBSS) with 5 mM

EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at

21 °C. The supernatant was removed, and the pellet was resuspended

in 10 mL of hypotonic lysis buﬀer (5 mM MgCl₂·6 H₂O, 5 mM Tris,

pH 7.4 at 4 °C) and centrifuged at 20,000 rpm for 30 min at 4 °C. The

pellet was then resuspended in fresh EBSS buﬀer made from 8.7 g/L

Earle’s Balanced Salts without phenol red (US Biological, Salem, MA)

and 2.2 g/L sodium bicarbonate, pH to 7.4. A Bradford protein assay

(Bio-Rad, Hercules, CA) was used to determine the protein

concentration, and membranes were diluted to 500 μg/mL and

stored in a −80 °C freezer for later use. Radioligand competition

binding experiments were conducted using thawed membranes. Test

compounds were freshly dissolved in 30% DMSO and 70% H₂O to a

stock concentration of 1 mM or 100 μM. To assist the solubilization of

free-base compounds, 10 μL of glacial acetic acid was added along with

the DMSO. Each test compound was then diluted into 11 half-log

serial dilutions using 30% DMSO vehicle; ﬁnal test concentrations

ranged from 100 μM to 100 pM or from 10 μM to 10 pM. Previously

frozen membranes were diluted in fresh EBSS to a 200 μg/mL (for

hD₂R or hD₃R) or 300 μg/mL (hD₄R) stock for binding. Radioligand

competition experiments were conducted in 96-well plates containing

300 μL of fresh EBSS buﬀer with 0.2 mM sodium metabisulﬁte, 50 μL

of diluted test compound, 100 μL of membranes (20 μg total protein

for hD₂R or hD₃R, 30 μg of total protein for hD₄R), and 50 μL of [³H]

N-methylspiperone (0.4 nM ﬁnal concentration; PerkinElmer).

Nonspeciﬁc binding was determined using 10 μM butaclamol

(Sigma-Aldrich, St. Louis, MO), and total binding was determined

with 30% DMSO vehicle. The reaction was incubated for 1 h at room

Molecular Pharmacology. Site-Directed Mutagenesis. Synthetic

cDNA encoding the human DAT (synDAT) was subcloned into

pcDNA3 (Invitrogen, Carlsbad, CA). The Y156F mutation was

introduced using QuickChange (adapted from Stratagene, La Jolla,

CA) and conﬁrmed by restriction enzyme mapping and DNA

sequencing. DAT WT and Y156F cDNA containing plasmids were

ampliﬁed by transformation into XL1 blue competent cells

(Stratagene) and grown in LB media overnight at 37 °C in an orbital

incubator (Infors) at 200 rpm. Plasmids were harvested using the maxi

prep kit (Qiagen) according to the manufacturer’s manual.

Cell Culture and Transfection. COS7 cells were grown in

Dulbecco’s modiﬁed Eagle’s medium 041 01885 supplemented with

10% fetal calf serum, 2 mM ^L-glutamine, and 0.01 mg/mL gentamicin

at 37 °C in 10% CO₂. DAT WT and Y156F were transiently

transfected into COS7 cells with Lipo2000 (Invitrogen) according to

the manufacturer’s manual using a cDNA/Lipo2000 ratio of 3:6.

[³H]DA Uptake Experiments. Uptake assays were performed on

intact COS7 cells essentially as described⁵⁹using 3,4-[Ring-2,5,6-³H]-

dihydroxyphenylethylamine ([³H]DA) (30−60 Ci/mmol) (Perki-

nElmer). Brieﬂy, transfected COS7 cells were plated in 24-well dishes

(10⁵cells/well) coated with poly ornithine (Sigma) to achieve an

uptake level of no more than 10% of total added [³H]DA. The uptake

assays were carried out 2 days after transfection in uptake buﬀer (UB)

(25 mM HEPES, 130 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM

MgSO₄, 1 mM L-ascorbic acid, 5 mM D-glucose, and 1 μM of the

catechol-O-methyltransferase inhibitor Ro 41-0960 (Sigma), pH 7.4).

Prior to the experiment, the cells were washed once in 500 μL of UB,

and the nonlabeled compound was added to the cells in the indicated

concentrations in a total volume of 500 μL. The assay was initiated by

the addition of 6−10 nM [³H]DA. Nonspeciﬁc uptake was determined

with 1 μM nomifensine (Sigma-Aldrich). After 5 min of incubation at

room temperature, the cells were washed twice with 500 μL of ice cold

UB, lysed in 250 μL (24 well) of 1% SDS and left for >30 min at 37 °C

on gentle shaking. All samples were transferred to 24-well counting

plates (PerkinElmer, Waltham, MA), 500 μL (24 well) of Opti-phase

Hi Safe 3 scintillation ﬂuid (PerkinElmer) was added followed by

counting of the plates in a Wallac Tri-Lux β-scintillation counter

(PerkinElmer). All experiments were carried out with 12 determi-

nations of DA or inhibitor concentrations ranging from 1 nM to 1 mM

performed in triplicate.

M

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

[³H]WIN35,428 Binding Experiments. Binding assays were carried

out essentially as described for the uptake experiments on whole cells

only using [³H]2β-carbomethoxy-3β-(4-ﬂuorophenyl)tropane ([³H]-

WIN35,428) (76−87 Ci/mmol) (PerkinElmer). Previous to the

binding experiment, cells were washed once in ice cold UB, and after

the addition of unlabeled compound in the indicated concentrations

and [³H]WIN35,428, the reactions were incubated at 5 °C until

equilibrium were obtained (>90 min). All experiments were carried

out with 12 determinations with inhibitor concentration range from 1

nM to 1 mM, performed in triplicate.

Mouse Microsomal Stability Assay. The phase I metabolic

stability assay was conducted in mouse liver microsomes as described

previously⁴⁷with minor modiﬁcations. Brieﬂy, the reaction was carried

out using potassium phosphate buﬀer (100 mM, pH 7.4), in the

presence of an NADPH regenerating system, (compound ﬁnal

concentration was 10 μM; and 0.5 mg/mL microsomes). Testosterone

was used as a positive control. Compound disappearance was

monitored over time using a liquid chromatography and tandem

mass spectrometry (LC/MS/MS) method.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

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

chem.6b01373.

■

S

Elemental analysis results (PDF)

SMILES data (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: 443-740-2887. Fax: 443-740-2111. E-mail: anewman@

intra.nida.nih.gov.

■

ORCID

Amy Hauck Newman: 0000-0001-9065-4072

Notes

The authors declare no competing ﬁnancial interest.

Chromatographic analysis was performed using an Accela ultra

high-performance system consisting of an analytical pump and an

autosampler coupled with a TSQ Vantage mass spectrometer

(Thermo Fisher Scientiﬁc Inc., Waltham, MA). Separation of analyte

was achieved at ambient temperature using Agilent Eclipse Plus

column (100 × 2.1 mm i.d.) packed with a 1.8 μm C18 stationary

phase. The mobile phase used was composed of 0.1% formic acid in

acetonitrile and 0.1% formic acid in H₂O with gradient elution, starting

with 10% (organic) linearly increasing to 99% up to 2 min,

maintaining at 99% (2−2.5 min) and reequlibrating to 10%. The

total run time was 4.5 min. The mass transitions used for compounds

for LC/MS/MS analysis are given in Table S1 (Supporting

Information).

Locomotor Activity Studies in Mice. Thirty-two male mice with

a C57BL/6J genetic background and body weight of 22−25 g were

purchased from the Charles River Laboratories (Raleigh, NC). After

arrival, they were group housed (4 per cage) in the animal facility

under a reversed 12 h light-dark cycle (light on at 7:00 PM) with free

access to food and water, and allowed to acclimate to the new

environment for 7 days prior to initiating the experiment. All

procedures were in accordance with the “Principles of Laboratory

Animal Care” outlined by National Institutes of Health (NIH

publication 86−23, 1996).

Locomotor activity tests were conducted in open-ﬁeld chambers

(43 × 43 × 30 cm³) (Accuscan Instruments, Inc., Columbus, OH,

USA). Before testing, the mice were habituated to the locomotor

chamber (2 h/day for 3 consecutive days) and then were randomly

divided into 4 groups (n = 8 per group) to examine the eﬀects of

cocaine, (R)-modaﬁnil [(R)-2], and compounds 4 and 11b on

locomotor activity, respectively. On habituation days, the animals were

moved to the test room, acclimated there for about 30 min, and then

placed in their assigned locomotor chambers. On test day 1, each

group of mice was ﬁrst placed in the locomotor chambers for 1 h of

habituation, and then they received either vehicle (25% of 2-

hydroxypropyl-β-cyclodextrin) or one dose of the corresponding test

compound. The animals were then immediately placed into the test

chambers. Their locomotor activities were recorded every 10 min for 2

h. Each animal was tested 3 times with 3 diﬀerent doses, with the

intertest interval of 3−5 days. The order of the drug doses was

counterbalanced.

The locomotor behavioral data are expressed as means ( SEM).

The traveled distance (cm) every 10 min (Figure 5) was used to

evaluate the locomotor eﬀects of each test compound in mice. Two-

way ANOVA with repeated measures over time was used to evaluate

the statistical signiﬁcance of the changes in locomotor activity after

each drug administration. Posthoc Fisher’s least signiﬁcant diﬀerence

was used for multiple comparisons. p < 0.05 was considered

statistically signiﬁcant.

ACKNOWLEDGMENTS

■

Support for this research was provided to A.H.N., J.C., R.D.S.,

O.M.B., C.B., T.K., A.B., M.P.E., H.Y., Y.H., G.-H.B., and Z.-

X.X. by the National Institute on Drug Abuse, Intramural

Research Program. C.J.L. is supported by the Lundbeck

Foundation and the Danish Council for Independent Research

Sapere Aude programme.

ABBREVIATIONS USED

■

DA, dopamine; DAT, dopamine transporter; SERT, serotonin

transporter; NET, norepinephrine transporter; CDI, N,N′-

carbonyldiimidazole; IA, inactive; ND, not determined;

NADPH, nicotinamide adenine dinucleotide phosphate

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