High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice

Article

pubs.acs.org/jmc

High Aﬃnity Dopamine D Receptor (D R)-Selective Antagonists

3

Attenuate Heroin Self-Administration in Wild-Type but not D₃R

Knockout Mice

Comfort A. Boateng, Oluyomi M. Bakare, Jia Zhan,^†^,^#Ashwini K. Banala, Caitlin Burzynski,

†

,⊥

†,⊥

†

†,‡

∥

§

Elie Pommier, Thomas M. Keck, Prashant Donthamsetti, Jonathan A. Javitch, Rana Rais,

§

†

,†

Barbara S. Slusher, Zheng-Xiong Xi, 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 Chemistry & Biochemistry, College of Science and Mathematics, Department of Biomedical & Translational

Sciences, School of Biomedical Science & Health Professions, Rowan University, 201 Mullica Hill Road, Glassboro, New Jersey

0

8028, United States

§

Department of Neurology, Brain Science Institute, The Johns Hopkins University School of Medicine, 855 North Wolfe Street,

Baltimore, Maryland 21205, United States

∥

Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, and Division of Molecular

Therapeutics, New York State Psychiatric Institute, New York, New York 10032, United States

*

S Supporting Information

ABSTRACT: The dopamine D receptor (D R) is a promising target for the development

3

of pharmacotherapeutics to treat substance use disorders. Several D R-selective antagonists

3

are eﬀective in animal models of drug abuse, especially in models of relapse. Nevertheless,

poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in

translating these drug candidates to clinical use. Herein, we report a series of D R-selective

3

4

-phenylpiperazines with improved metabolic stability. A subset of these compounds was

evaluated for D R functional eﬃcacy and oﬀ-target binding at selected 5-HT receptor

3

subtypes, where signiﬁcant overlap in SAR with D R has been observed. Several high

3

aﬃnity D R antagonists, including compounds 16 (K = 0.12 nM) and 32 (K = 0.35 nM),

3

i

showed improved metabolic stability compared to the parent compound, PG648 (6).

Notably, 16 and the classic D R antagonist SB277011A (2) were eﬀective in reducing self-

3

administration of heroin in wild-type but not D R knockout mice.

3

1

7−19

INTRODUCTION

upon chronic exposure to cocaine and methamphetamine.

Inspired by early preclinical studies using lead compounds

■

The dopamine D -like family of receptors, comprising the D ,

2

20,21

22−24

BP897 (1)

and SB277011A (2),

intensive eﬀorts

toward discovering D R-selective antagonists and partial

agonists as potential therapeutics for substance use disorders

have been underway for nearly two decades.

Although early D R-selective partial agonists, such as 1, and

antagonists such as 2, NGB2904 (3), and PG01037 (4), (Chart

) have served as highly useful preclinical tools, none of these

compounds have progressed to the clinic due to poor

bioavailability and the advancement of newer generation

D , and D₄receptor subtypes (D R, D R, and D R,

respectively), regulate physiological functions such as move-

ment, emotion, and cognition. Numerous medications for the

treatment of neuropsychiatric disorders, such as schizophrenia,

target this important family of receptors. The D R subtype is

3

2

3

4

3

1

3

localized in key neurocircuits that underlie motivation and

1

cognition, and in contrast to D R, do not appear to play a major

2

,3

role in movement. Hence, the D R has been proposed as a

3

promising target for development of psychostimulant addiction

4

−7,25−27

2

,4−9

analogues.

One example of a D R-selective compound

tested in humans is GSK598,809 (5) (Chart 1), which was ﬁrst

reported in 2007. Recently, [ C]PHNO PET imaging studies

3

and relapse pharmacotherapy.

Additionally, seminal reports

11

10

using [ C]raclopride for PET imaging in both humans and

25

11

nonhuman primates have demonstrated that overall D -like

2

demonstrated a correlation between this compound’s D R

receptor availability in the basal ganglia is signiﬁcantly reduced

after chronic exposure to cocaine. Interestingly, the converse

appears to be true for the D R subtype: recent human PET

3

2

8,29

occupancy and eﬃcacy toward smoking cessation.

Although clinical studies have been conducted with 5 for

Received: May 21, 2015

3

1

12,13

studies with the D R-preferential PET ligand [ C]PHNO

3

conﬁrmed earlier reports in post-mortem brains of cocaine

1

4−16

overdose victims

that D R availability is actually increased

3

©

XXXX American Chemical Society

A

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Chart 1. Selected Lead D R-Selective Antagonists and Partial Agonists

3

smoking cessation, ⁸⁻³¹it is unlikely that this compound will be

developed as a medication to treat other substance use

disorders, as GlaxoSmithKline terminated this research and

development program.

2

in mouse microsomes, two lead candidates were selected for

behavioral evaluation in murine models of heroin self-

administration and compared to the classic D R antagonist, 2.

3

Although D R antagonists have largely been developed

3

One of our lead compounds, R-PG648 (R-6) binds with

500-fold selectivity for D R (K = 0.53 nm) over D R (K =

3 i 2 i

26,27,32,33

toward nicotine, cocaine, and methamphetamine abuse, they

∼

have shown eﬃcacy in other preclinical models of drug and

37−39

2

95 nm) in vitro

and is an antagonist/weak partial

alcohol abuse.

For example, the D R antagonist 2 has been

3

4

agonist. Using molecular simulations based on the D R X-ray

3

shown to block the acquisition and expression of heroin

3

5

40

crystal structure, we demonstrated that the 4-phenylpiper-

azine moiety binds to the structurally similar D R/D R

conditioned place preference in rats. Nevertheless, to our

2

3

knowledge, D R antagonists have not been evaluated for their

3

orthosteric binding site (OBS) in which dopamine and the

D R/D R antagonist eticlopride bind, whereas the arylamide

terminus binds in a secondary binding pocket (SBP) that is

topographically divergent between D R and D R.

early preclinical promise, R-6 and its racemate (6) proved to be

unsuitable for translation to clinical study.

The development of D R antagonists with suﬃcient

metabolic stability and eﬃcacy as medications to treat

substance use disorders remains a major challenge. Hence the

purpose of the present study was to extend D R SAR by

exploring modiﬁcations to all three segments (Chart 1) of the

-phenylpiperazine class of molecules to identify ligands that

demonstrate high D R binding aﬃnity and subtype selectivity,

with improved bioavailability and metabolic stability. Speciﬁ-

cally, we replaced the 2,3-diCl-substitution of the 4-phenyl-

piperazine in lead compound 6 with a 2-OMe,3-Cl or a 2,3-

naphthyl-substituent. Additionally, we explored bioisosteric

replacement of the indolylamide of 6 with various hetero-

arylamides as well as further investigating the 3-hydroxy (3-

OH) substituent in the 4-carbon linker. After assessing in vitro

binding and functional eﬃcacy proﬁles, and metabolic stability

eﬀects on heroin self-administration. As D R antagonists are

3

2

3

typically not eﬀective in attenuating cocaine or methamphet-

amine self-administration under low ﬁxed-ratio (FR) schedules

34−36

Despite

41,42

2

3

(

e.g., FR1, FR2),

we hypothesized that in the presence of

33

these psychostimulants, high extracellular dopamine (DA)

levels may compete at D R and thus render these D R-selective

3

antagonists ineﬀective. Indeed, most reports show D₃R

antagonists as being most eﬀective in models of relapse when

extracellular DA levels would be substantially lower than during

3

2,33

active exposure to psychostimulants.

In comparison to

psychostimulant drugs, heroin induces relatively moderate

increases in extracellular DA within the accumbens during

4

3

43−46

active drug-taking,

thus we posited that D R antagonists

3

may be more eﬀective in reducing heroin self-administration

than psychostimulant use. If the data support our hypothesis,

this would represent an alternative strategy to treating opioid

abuse and addiction. Herein, we evaluate our lead compounds

in both wild-type (WT) and D R knockout (KO) mice to not

3

only assess eﬀectiveness in attenuating heroin self-adminis-

tration but also to determine the role of D R in these behaviors.

3

B

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 1. Synthesis of 4-(4-Arylpiperazine)butylamine Intermediates

a

Reagents and conditions: (a) 3-chloro-2-methoxyaniline or naphthalen-1-amine, K CO , bis(2-chloroethyl)amine HCl, diglyme, reﬂux, 48 h; (b)

2

3

appropriate 4-arylpiperazine, 2-propanol, reﬂux, overnight; (c) hydrazine (anhydrous), EtOH, reﬂux, 2−3 h.

a

Scheme 2. Synthesis of Target Compounds 14−37

a

Reagents and conditions: (a) (i) 1,1′-carbonyldiimidazole (CDI), THF, room temperature, 2 h; (ii) appropriate 4-arylpiperazine amine, THF, 0 °C

to room temperature, overnight.

4

8

CHEMISTRY

previously reported via a nucleophilic substitution reaction

with naphthalen-1-amine. The synthesis of the 4-(4-

arylpiperazine)butylamine intermediates 10a−10d and 13a−

■

The synthesis of 24 ﬁnal compounds and their common

intermediates are outlined in Schemes 1 and 2. In Scheme 1,

1

3d, with or without a 3-OH substituent, respectively, was

achieved starting with 7a, 7b, or commercially available 1-(2-

4

7

7a

was synthesized using 3-chloro-2-methoxyaniline and

bis(2-chloroethyl)amine HCl, in diglyme under reﬂux con-

ditions. 1-(Naphthalene-1-yl)piperazine, 7b, was synthesized as

methoxyphenyl)piperazine and 1-(2,3-dichlorophenyl)-

piperazine. Speciﬁcally, an epoxide ring-opening reaction of

49

C

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Human Dopamine D -Like Receptor Binding Data in HEK Cells for Ligands with Varying Arylpiperazine and

2

a

Arylamide Moieties

a

3

K values determined by competitive inhibition of [ H]N-methylspiperone binding in membranes harvested from HEK 293 cells stably expressing

i

hD R or hD R.

2

3

8

with the corresponding 4-arylpiperazines under reﬂux

situ N,N′-carbonyldiimidazole coupling reaction. All 3-OH-

substituted compounds were racemic mixtures. As considerable

enantioselectivity has so far not been demonstrated with either

the 3-OH or 3-F substitution in this class of compounds,

no attempt to separate enantiomers was made.

conditions yielded the 3-OH-phthalimides 9a−9d, which

were subjected to phthalimide deprotection using hydrazine

to give the 3-OH-substituted butylamine intermediates 10a−

26,27

1

0d. The synthesis of intermediate compounds 9c, 9d, 10c, and

0d was previously reported using microwave conditions;

49

however, the methods reported herein use conventional (oil

bath) heating methods. To generate the 4-(4-arylpiperazine)-

butylamine intermediates 13a−13d, commercially available N-

PHARMACOLOGICAL RESULTS AND DISCUSSION

■

SAR at D R and D R. The objective of this study was to

2

3

design metabolically stable ligands with high D R binding

3

(

4-bromobutyl)phthalimide 11 was reacted with the appro-

aﬃnity and subtype selectivity. We extended our previous SAR

studies by exploring modiﬁcations to all three segments of the

lead compound 6 (Chart 1: arylamide moiety, 4-carbon linker,

and 4-phenylpiperazine moiety) in an attempt to overcome

bioavailabilty and metabolic shortcomings of previously

priate 4-arylpiperazine to give phthalimides 12a−12d, which

were then subjected to deprotection with hydrazine to give the

desired 4-(4-arylpiperazine)butylamines 13a−13d. While inter-

mediates 12b−12d and 13a−13d have been previously

3

4,50

33

reported,

from or improve upon these methods. The ﬁnal ligands 14−37

Scheme 2) featuring the arylpiperazine, four-carbon linker

our synthetic methods either diﬀer completely

reported preclinical candidates.

We recently evaluated the binding aﬃnities and selectivities

(

at D R and D R of a library of synthons along with their full-

3

2

3

4

chain (with or without a 3-OH substituent), and arylamide

moieties were synthesized via a general amidation procedure

length ligands. Computational modeling studies demonstra-

ted that the 3-Cl of the 2,3-diCl-phenylpiperazine moiety

makes polar contacts with transmembrane domain 5 (TM5)

5

0,51

(

method A),

in which various arylcarboxylic acids were

5

.43

coupled to corresponding primary amines 10 or 13 via an in

serine (Ser) amino acid residues (particularly Ser193 ), which

D

DOI: 10.1021/acs.jmedchem.5b00776

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Journal of Medicinal Chemistry

Article

Table 2. Human Dopamine D -Like Receptor Binding Data in HEK Cells for N-(4-(4-(2-Methoxyphenyl)piperazin-1-

2

a

yl)butyl)quinoline Carboxamide Ligands

a

3

K values determined by competitive inhibition of [ H]N-methylspiperone binding in membranes harvested from HEK 293 cells stably expressing

i

hD R or hD R.

2

3

4

tion) in nonhuman primates,⁵³⁻⁵⁵Although a recent clinical

trial testing buspirone for cocaine dependence treatment

correlates with observed partial agonist actions in vitro.

However, when the 2,3-diCl substituent was replaced with a 2-

OMe, there were no polar contacts with TM5, which

corresponds to the much weaker partial agonist eﬀects observed

56

proved unsuccessful, developing novel D R antagonists/5-

3

HT₁_Apartial agonists might also be an interesting combination

for compounds with therapeutic potential.

34

with the 2-OMe-phenylpiperazine-substituted ligands. In view

of these data, we explored the eﬀects of incorporating a hybrid

To evaluate the binding aﬃnities of this series of compounds,

we performed competition binding studies with [ H]N-

3

2

-OMe,3-Cl-phenylpiperazine substituent as the primary

pharmacophore. This 2-OMe,3-Cl- substitution pattern is

methylspiperone using membranes prepared from HEK293

present in the nonselective high aﬃnity D R/D R receptor

antagonist/inverse agonist, eticlopride. Thus, we hypothesized

that this substitution would lead to an increase in aﬃnity at

cells expressing either the human D R or D R (Tables 1−3.) In

2

3

2

3

addition, cLogP values are provided as a relative measure of

lipophilicity. Compounds 14−21 displayed moderate to high

binding aﬃnities for D R (K = 8−330 nM; Table 1).

D R compared to the 2,3-diCl-phenylpiperazine.

3

2

i

We also replaced the substituted phenyl ring on the

piperazine with a naphthalene moiety, a bioisosteric replace-

ment for the 2,3-diCl-phenyl ring, and predicted that this

substituent might also result in compounds with high-aﬃnity

Moreover, all of these analogues, with the exception of 21

(K = 1.8 nM), displayed subnanomolar aﬃnities at D R (K =

i

3

i

0.1−0.9 nM), demonstrating that the 2-OMe,3-Cl- and the 2,3-

naphthyl-piperazines, as predicted, were well tolerated as the

primary pharmacophores in this series. For example, the 2-

for D R. The naphthylpiperazine moiety is a well-described

3

pharmacophore for serotonin receptors and speciﬁcally for 5-

OMe,3-Cl-phenylpiperazine (e.g., 14; K = 0.39 nM) and the

i

HT₁_Aagonists, potentially reducing D R selectivity of these

2,3-naphthyl-piperazine (e.g., 16; K = 0.12 nM) indolylamide

3

i

4

8,52

analogues.

Notably, buspirone, a 5-HT partial agonist

analogues generally showed higher D R binding aﬃnities

1

A

3

53

that also antagonizes D R, was recently reported to reduce

compared to lead compound 6 (K = 1.9 nM), although

3

i

cocaine and nicotine self-administration (alone or in combina-

neither was as D R selective. We also explored the bioisosteric

3

E

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 3. Human Dopamine D -Like Receptor Binding Data in HEK Cells for Quinoline-3-carboxamide Ligands with Varying

2

a

Arylpiperazines and 4-Carbon Linker Chains

a

3

K values determined by competitive inhibition of [ H]N-methylspiperone binding in membranes harvested from HEK 293 cells stably expressing

i

hD R or hD R.

2

3

replacement of the indole moiety (as the secondary

pharmacophore) with benzofuran and benzothiophene moi-

(N), that is 2-quinolinyl (compounds 22−25), 3-quinolinyl

(compounds 26−30), and 7-quinolinyl (compound 31)

positions, had any eﬀect on binding aﬃnity and/or selectivity.

We also compared quinolinylamide analogues with or without a

3-OH substituent in the 4-carbon linker between the arylamide

terminus and the 4-phenylpiperazine moiety.

eties, substitutions that have previously demonstrated high D R

3

7

,34,51,57,58

aﬃnities, in related series.

As shown in Table 1, all these benzothienyl and benzofuranyl

analogues displayed high D R aﬃnities, with K values between

3

i

0

8

.13 and 0.91 nM and good selectivity for D R over D R (61−

Binding aﬃnities for the 2-OMe-phenylpiperazine quinoli-

nylamide analogues 22−31 are reported in Table 2. Compared

to the indolylamide, benzofuran, and benzothiophene ana-

logues (Table 1), the quinoline analogues generally displayed

lower aﬃnities at D R and D R as well as decreased selectivity

3

2

0-fold). The presence of an electron-donating OMe group on

the indole ring, in compound 17, was well-tolerated and

resulted in high aﬃnity at D R and 80-fold selectivity for D R

over D R. The presence of an electron-withdrawing F group on

the indole ring in compound 20 also resulted in high D₃R

aﬃnity (K = 0.43 nM), and D R selectivity was improved with

3

2

3

50

for the D R over D R. Moreover, quinolinylamide analogues

3 2

with a 3-OH substituent in the 4-carbon linker chain displayed

i

3

the addition of a 3-OH in the linking chain (21). A small

reduction in selectivity for D R over D R in comparison with 6

reduced D R binding aﬃnities and in contrast to previous

3

reports, reduced D R-selectivity over D R as compared to the

3

2

3

2

26,49

was observed with compound 21 due to a ∼2-fold improve-

unsubstituted analogues.

Speciﬁcally, compounds 22, 24,

ment in D R aﬃnity.

and 27, without a 3-OH substituent in the linker chain,

displayed low nanomolar aﬃnities at D R (K = 3.5, 2.5, and 2.1

2

In summary, for the ligands reported in Table 1, compounds

without the 3-OH substituent in the 4-carbon linker chain

possessed higher cLogP values (i.e., were more lipophilic/

3

i

nM, respectively) and showed equal or higher selectivity for

D R compared to their corresponding 3-OH substituted

3

hydrophobic) and displayed higher aﬃnities at both D R and

analogues 23, 25, and 28 (K = 97.9, 53.1 and 44.8 nM,

2

i

D R but lower selectivity for D R over D2R (e.g., compound

respectively). The position of the quinolinyl N did not appear

3

20 vs 21 in Table 1). Bioisosteric replacement of the indole

to strongly inﬂuence binding aﬃnity and/or selectivity at D R,

3

moiety with other [5,6]-heteroaromatic moieties, such as

benzofuran and benzothiophene, did not signiﬁcantly inﬂuence

D R binding aﬃnity or selectivity.

3

although there appeared to be a slight preference for 3- and 7-

quinolinyl positions over the 2-quinolinyl position (e.g.,

compounds 26 and 31 compared to 23). While aﬃnities at

To improve chemical and metabolic stability, we opted to

further explore other heteroaromatic groups such as quinoline

that does not have an acidic hydrogen or hydrogen-bond donor

group like the N−H group of the indole. To this end,

compounds 22 −31 were prepared that contained the 2-OMe-

phenylpiperazine moiety along with either electron with-

drawing or donating substituents on the quinoline moiety.

We explored whether the position of the quinoline nitrogen

D R remained similarly low for compounds 23, 26, and 31,

2

there was a 3-fold decrease in aﬃnity at D R and selectivity for

3

D R over D R for compound 23 compared to compounds 26

3

2

and 31. Hence, D R aﬃnity and selectivity for the 2-, 3-, and 7-

3

5

9

quinolinyl analogues follow the order 2- < 3- = 7-quinoline. Of

note, within the 3-quinoline series (compounds 26−30), the

presence of an electron-withdrawing or donating substituent on

the quinoline moiety did not substantially aﬀect aﬃnity or

F

DOI: 10.1021/acs.jmedchem.5b00776

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Journal of Medicinal Chemistry

Article

Table 4. Functional Data for Selected Compounds Using Stimulation or Inhibition of Quinpirole-Stimulated Mitogenesis in

a

CHO Cells with Human Dopamine D R or D R

2

3

D R mitogenesis assay

2

D R mitogenesis assay

3

agonist

EC₅₀± SEM

(nM)

antagonist

IC₅₀± SEM

(nM)

agonist

EC₅₀± SEM

(nM)

antagonist

IC₅₀± SEM

(nM)

%

compd

stimulation

b

c

b

6

170 ± 61

76 ± 14

22

4400 ± 60

87 ± 20

>10000

ND

30 ± 5.8

8.0 ± 1.9

52 ± 1.2

47 ± 11

7.4 ± 2.5

72 ± 12

59 ± 24

16

20

26

32

33

34

4.5

140 ± 51

25

c

d

c

d

>10000

ND

564.40 ± 109

>10000

ND

c

ND

0.95 ± 0.39

20 ± 4.6

13 ± 1.9

16 ± 6.4

11

31

36

29

170 ± 52

200 ± 28

>10000

18

8.2

<7

29 ± 8.7

720 ± 190

63 ± 13

a

b

d

Data were obtained through the NIDA Addiction Treatment Discovery Program contract (ADA151001) with Oregon Health & Science University.

26

c

Previously published data. ND = not determined. Functional assays were not conducted if the K value for the binding assay was >500 nM.

i

50

Previously published data.

a

Table 5. Additional in Vitro Binding and Functional Data for Selected Compounds at 5-HT , 5-HT , and 5-HT Receptors

1

A

2A

2C

5

-HT₁_A

S]GTPγS

binding

[³⁵

5

-HT₁_A

5-HT

2C

125

2A

I]DOI

[³

H]-8-OH-DPAT

[

125

[

I]DOI

agonist

EC₅₀± SEM (nM)

compd

K_i± SEM (nM)

% stimulation

b

c

6

100 ± 24

48 ± 13

55 ± 12

>10000

86 ± 0.61

73 ± 16

990 ± 310

1,800 ± 130

3.8 ± 1.4

44 ± 5.0

ND

108

16

20

26

32

33

34

84 ± 7.1

540 ± 48

68 ± 9.8

2.5 ± 0.35

9.0 ± 3.2

21 ± 5.4

250 ± 67

c

ND

94

350 ± 50

0.33 ± 0.12

2.4 ± 0.77

1.7 ± 0.60

340 ± 91

9.0 ± 3.0

17 ± 5.7

43 ± 11

108

104

106

14 ± 2.6

a

Data were obtained through the NIDA Addiction Treatment Discovery Program contract (ADA151001) with Oregon Health & Science University.

b

26 c

Previously published data. ND = Not determined. Functional assays were not conducted if the K value for the binding assay was >250 nM.

i

selectivity at D R, although it appeared that electron-with-

more selective than the unsubstituted quinolines in this series.

These data suggest that among the quinoline-3-carboxamides,

both the 2,3-diCl-phenylpiperazine and the 2,3-naphthyl-

piperazine are good replacements for the 2-OMe-phenyl-

piperazine moiety. Addition of the 3-OH group in the linker in

the 6-F-quinoline compound, 37, showed somewhat lower

aﬃnity and selectivity as compared to its 5-F-indole analogue,

21. Among the quinoline-3-carboxamide analogues, compound

32 with the 2,3-naphthyl-piperazine and 36 with 2,3-diCl-

3

drawing substituents (e.g., F) induced less pronounced loss of

aﬃnity compared to electron-donating substituents (e.g.,

OMe). In general, with the 2-OMe-phenylpiperazine moiety,

the quinolinylamide analogues were less potent and selective

for D R over D R. On the basis of relative SAR within the

3

2

quinoline−carboxamide analogues, we decided to explore the 3-

quinoline moiety as a bioisosteric replacement for indole in the

arylamide terminus in combination with the 2-OMe,3-Cl-

phenylpiperazine, or 2,3-naphthyl-piperazine. In addition, we

prepared the 2,3-diCl analogues for comparison.

phenylpiperazine showed the highest D R aﬃnity and their 3-

3

OH-analogues, 33 and 37, respectively, displayed highest D R-

3

The D R and D R binding aﬃnities of ligands that

selectivity.

2

3

incorporate the quinoline-3-carboxamide moiety with either

-OMe,3-Cl-phenylpiperazine, 2,3-naphthyl-piperazine, or 2,3-

diCl-phenylpiperazine (compounds 32−37) are shown in

Table 3. Comparison of these compounds and the quinoline-

Functional Data and Serotonergic Actions. Six

analogues were selected to compare with the parent compound

6 for their ability to antagonize quinpirole-induced activation of

hD R or hD R in a cell-based mitogenesis assay (Table 4.) All

2

3

3-carboxamide analogues in Table 2 revealed improvement in

of the analogues were moderately potent antagonists in the

binding aﬃnities at the D R. While the 2-OMe,3-Cl-phenyl-

D R mitogenesis assay (IC = 7.4−72 nM), with compounds

3

50

piperazine analogue 35 (K = 31.6 nM; D R/D R ratio = 28) is

16 and 32 being the most potent. Of note these compounds, as

well as 26, 33, and 34, showed weak partial agonist proﬁles at

higher concentrations. In general, functional potencies of these

analogues were lower than their binding aﬃnities. Most of the

analogues were more potent at D R than at D R in this assay,

i

2

3

essentially equipotent at D R and D R as compound 26 (Table

2

3

2

; K = 33.8 nM; D R/D R ratio = 27), the equivalent 2,3-

i 2 3

naphthyl-piperazine analogue, 33, displayed ∼10-fold increase

in aﬃnity (K = 3.63 nM) and ∼2-fold more D R selective

i

3

2

(

D R/D R ratio = 71) for D R over D R than 26. Compound

although selectivities varied.

2

3

2

3

2 had the highest D R aﬃnity (K = 0.35 nM) of the 2,3-

These analogues were also tested for binding at 5-HT , 5-

3

i

1A

naphpthyl or 2-OMe,3-Cl-analogues. Moreover, the 2,3-diCl-

phenylpiperazine analogues with a 6-F-quinoline (D R/D R

ratio = 69 and 103 for 36 and 37, respectively) were slightly

HT , and 5-HT receptors as well as for functional activity at

5-HT₁_A(Table 5). Although all the analogues had aﬃnity

toward these 5-HT receptor subtypes, compounds 32−34

2A

2C

2

3

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Figure 1. (a) Phase I metabolism data for all compounds with an unsubstituted linker chain. (b) Phase I metabolism data for all compounds with a 3-

OH substituted linker chain. (c) Phase I metabolism data for all quinoline compounds. (d) Phase I metabolism data for all indole compounds.

Figure 2. Eﬀects of compound 2 on heroin self-administration in WT and D KO mice. All the data are expressed as mean ± SEM. (A) Total

3

numbers of heroin infusions after injection of each dose of compound 2. (B) Normalized data showing drug-induced % change in heroin self-

administration over new basal levels immediately before each test day. One-way ANOVA with repeated measures over drug dose revealed a

signiﬁcant drug treatment main eﬀect in WT mice ((A) F₃_,₂₄= 10.93, p < 0.001; (B) F₃_,₂₄= 13.06, p < 0.001) but not in D KO mice ((A) F

=

3

3,12

2.21, p > 0.05; (B) F₃_,₁₂= 1.46, p > 0.05). *p < 0.05, ***p < 0.001, compared to vehicle.

exhibited the highest aﬃnities, especially at 5-HT₁_Aand 5-

parent compound 6 following procedures previously de-

60

HT . All these analogues were full agonists at 5-HT

scribed. Compound stability over a 1 h incubation is

presented in Figure 1. In general, the compounds with the

unsubstituted linker, regardless of the primary or secondary

pharmacophores, were more metabolically stable (see Table S3

in Supporting Information). Of the quinoline analogues, 32 was

the most stable, with calculated in vitro rate constant (k) of

−0.017 and in vitro half-life (t ) of 41.8 min, whereas 16 with

2

A

1A

receptors, which was not surprising based on previously

48,52

reported SAR for 5-HT .

However, with the exception

1

A

of 32, all analogues were still signiﬁcantly D R-selective.

3

Interestingly, 32 showed relatively high aﬃnity for all the 5-

HT receptor subtypes tested, providing a unique proﬁle among

this series.

Microsomal Metabolism Results. Metabolic stability

assays were conducted in mouse liver microsomes to predict

in vivo stability after oral administration. The analogues were

tested for phase I metabolism in mouse liver microsomal

incubations in the presence of NADPH and were compared to

1/2

the k value of −0.010 and in vitro t₁_/₂of 67.5 min was the most

stable of the indoles and signiﬁcantly more stable than the

parent compound, 6, with k of −0.037 and t₁of 18.8 min.

/2

Heroin Self-Administration Studies in WT and D KO

3

Mice. On the basis of their pharmacological proﬁle and

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Figure 3. Eﬀects of compound 16 on heroin self-administration in WT and D R-KO mice. All the data are expressed as mean ± SEM. (A) Total

3

numbers of heroin infusions after injection of each dose of compound 16. (B) Normalized data showing drug-induced % change in heroin self-

administration over new baseline immediately before each test day. One-way ANOVA with repeated measures over drug dose revealed a statistically

signiﬁcant drug treatment main eﬀect in WT mice ((A) F₃_,₁₈= 2.13, p > 0.05; (B) F₃_,₁₈= 9.09, p < 0.001) but not in D KO mice ((A) F = 0.63, p

3

3,18

>

0.05; (B) D KO, F = 1.78, p > 0.05). We note that one-way ANOVA did not reveal a signiﬁcant treatment main eﬀect for the data shown in (A)

3 3,18

WT mice. However, the direct two group comparison between the vehicle and 10 mg/kg 16 groups revealed a statistically signiﬁcant reduction ((A)

WT, paired t test, q = 5.07, p < 0.05). These may be related to the relatively smaller group size and/or the relatively variable basal levels of heroin

self-administration in diﬀerent subjects. Therefore, the renormalized data (% change over baseline) are provided ((B) in this ﬁgure; the same as in

Figures 2B and 4B) in this study. *p < 0.05, ***p < 0.001, compared to vehicle.

Figure 4. Eﬀects of compound 32 on heroin self-administration in WT and D KO mice. All the data are expressed as mean ± SEM. (A) Total

3

numbers of heroin infusions after injection of each dose of compound 32. (B) Normalized data showing drug-induced % change in heroin self-

administration over new baseline immediately before each test day. One-way ANOVA with repeated measures over drug dose revealed a statistically

signiﬁcant drug treatment main eﬀect in WT mice ((A) F₃_,₁₈= 0.83, p > 0.05; (B) F₃_,₁₈= 3.34, p < 0.05) and D KO mice ((A) F = 3.63, p < 0.05;

3

3,15

(

B) F₃_,₁₅= 4.39, p < 0.05) *p < 0.05, compared to vehicle.

22−24

microsomal stability, compounds 16 and 32 were chosen for

2

in both WT and D KO mice. Figure 2A shows that

3

behavioral studies in WT and D KO mice. Of note, compound

pretreatment with 2 (25, 50, 100 mg/kg, i.p., 30 min prior to

test) dose-dependently decreased the total number of infusions

of heroin self-administered by the WT mice, with 100 mg/kg

(ip) as the most eﬀective dose.

Given the observation that the basal levels of heroin self-

administration are variable in individual subjects, we further

normalized drug-induced percent changes in self-administration

over new basal levels immediately before each test day in each

animal (Figure 2B) to more accurately reveal drug eﬀects. We

found that pretreatment with 2 produced a dose-dependent

reduction in heroin self-administration in WT mice (Figure

3

1

6 was D R selective over the 5-HT receptor subtypes in

3

contrast to compound 32. Moreover, these two analogues were

the most metabolically stable in this series. Mice were trained

on an FR1 schedule of reinforcement, where every lever press

results in an injection, initially with 0.1 mg/kg (iv) heroin,

followed by 0.05 mg/kg and ﬁnally 0.025 mg/kg, to obtain

more robust lever pressing behavior via a compensatory

response to a reduced reinforcer (e.g., a lower dose of heroin).

Both WT and D KO mice acquired heroin self-administration

3

after 1−2 weeks of training. Overall, D KO mice display higher

3

total number of heroin infusions taken over 3 h than WT mice

2B). Of note, although the D KO mice self-administered heroin

3

although the diﬀerence was not statistically signiﬁcant (Figures

similarly to the WT mice, this behavior was not attenuated by

2

A, 3A, 4A). After stable heroin self-administration was

2, suggesting that this D R antagonist reduces heroin self-

3

achieved, we evaluated the eﬀects of the classic D R antagonist

administration through a D R-mediated mechanism. Likewise,

3

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in Figure 3, compound 16, at the dose range of 1−10 mg/kg,

D R antagonists have largely been developed as potential

3

also dose dependently attenuated heroin self-administration in

therapeutic agents for the treatment of nicotine or

psychostimulant abuse. However, they are typically unable to

block self-administration under a low FR schedule of

reinforcement and are primarily eﬀective in models of

the WT but not in the D KO mice. Of note, the eﬀective dose

3

of 16 was 5−10-fold lower than that of 2, suggesting that 16 is

more potent than 2 in this model. In our binding assays,

2

,4,33

compound 16 displayed ∼70-fold higher aﬃnity for D R than 2

relapse.

We hypothesized that D R antagonists might be

3

(

K = 0.12 and 13.9 nM, respectively), which is consistent with

able to block self-administration of addictive drugs, like heroin,

that more modestly stimulate dopamine release in the

i

its higher potency in vivo.

Compound 32 also dose dependently attenuated the

accumbens. Indeed, behavioral studies in WT and D KO

3

rewarding eﬀects of heroin, with 3 mg/kg being the most

mice that were trained to self-administer heroin demonstrated

eﬀective dose in both WT and D KO mice (Figure 4),

that D

drug self-administration in WT but not D

an eﬀect that is mediated through D R. In addition, compound

3

R-selective antagonists 2 and 16 signiﬁcantly decreased

3

reﬂecting the lack of D R selectivity of this compound. It is

KO mice, suggesting

3

enticing to speculate that the 5-HT , 5-HT , and/or 5-HT

3

1

A

2A

2C

receptors may be playing a role in the behavioral eﬀects of this

16 was 5−10 times more potent than 2, a classic D R

3

drug as these serotonergic receptor subtypes have all been

antagonist, in its pharmacological eﬀect in vivo. Compound 32

61−64

implicated in both drug reward and impulsivity.

However,

also attenuated heroin self-administration in the WT mice but

additional experiments must be conducted in order to conﬁrm

this. Nevertheless, compound 32 eﬀectively blocked self-

administration of heroin more potently than either 2 or 16,

thus follow-up studies are underway.

appeared to be less selective for D R than either compounds 2

3

or 16, as at the most eﬀective dose of 3 mg/kg, self-

administration behavior was reduced in both the WT and

D KO mice. Its binding proﬁle suggests that actions at the 5-

3

HT receptor subtypes might also play a role in the behavioral

actions of this compound. Additional behavioral assessment is

naturally required, however, these are the ﬁrst studies to show

CONCLUSION

■

In summary, we have synthesized a novel series of analogues of

wherein the 2,3-diCl-phenyl piperazine was replaced with a 2-

OMe, a 2-OMe,3-Cl-, or a 2,3-naphthyl-substituent and a

bioisosteric replacement of the indole moiety. In addition, we

also explored analogues with either the unsubstituted or the 3-

OH substituted 4-carbon linker between the arylpiperazine and

the eﬀectiveness of D R antagonists in heroin self-admin-

3

6

istration and suggest an alternate medication strategy to

methadone maintenance or buprenorphine, the current

pharmacotherapeutic treatments for heroin addiction.

EXPERIMENTAL METHODS

■

aryl amide to investigate its eﬀects on D R aﬃnity, selectivity,

3

Synthesis. Reaction conditions and yields were not optimized.

Anhydrous solvents were purchased from Aldrich and were used

without further puriﬁcation except for tetrahydrofuran, which was

freshly distilled from sodium-benzophenone ketyl. All other chemicals

and reagents were purchased from Sigma-Aldrich Co. LLC, Combi-

Blocks, TCI America, OChem Incorporation, Acros Organics,

Maybridge, and Alfa Aesar. All amine ﬁnal products were converted

into either the oxalate or HCl salt. Spectroscopic data and yields refer

to the free base form of compounds. Teledyne ISCO CombiFlash Rf

or glass ﬂash column chromatography were performed using silica gel

eﬃcacy, and metabolic stability. By varying the arylpiperazine

substitution, we found several ligands that displayed high

aﬃnity and selectivity proﬁles at the D R versus D R. Overall,

we identiﬁed indolylamide analogues, e.g., 16 (K = 0.12 nM),

which displayed higher D R binding aﬃnity compared to lead

3

2

i

3

compound 6 (K = 1.9 nM) and >100-fold selectivity for D R

i

3

over D R. Although in general, the 2-OMe-phenylpiperazine-

2

quinolinylamides had lower aﬃnities at D R, when substituted

3

with the 2-OMe,3-Cl or the 2,3-naphthyl substituents, D₃R

aﬃnity was increased ∼10-fold. Also the 3-OH substituted

analogues showed an increase in selectivity for D R over D R,

as well as a decrease in lipophilicity, as described for other 4-

phenylpiperazine-butylarylamides.

On the basis of the D R/D R binding proﬁles, a subset of

1

13

(

EMD Chemicals, Inc.; 230−400 mesh, 60 Å). H and C 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 according to deuterated

3

2

26,49

1

solvent for H spectra (CDCl , 7.26, CD OD, 3.31, or DMSO-d ,

3

6

13

2

3

2.50) and C spectra (CDCl , 77.2, CD OD, 49.0, or DMSO-d ,

3

6

analogues was evaluated in a cell-based D R or D R functional

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

2

3

assay. These analogues were potent antagonists in the D₃R

mitogenesis assay (e.g., 16 and 32; IC₅₀= 8.0 and 7.4 nM,

respectively) but at higher concentrations demonstrated weak

partial agonist proﬁles. They were also evaluated for activities at

5

-HT , 5-HT , and 5-HT receptors. Among the selected

1A 2A 2C

ligands, in addition to its high binding aﬃnity to D R (K = 0.35

nM), 32 displayed high aﬃnity for the 5-HT receptor subtypes

K = 2.46 nM for 5-HT , 0.33 nM for 5-HT , 3.80 nM for 5-

3

i

2

50 and 280 °C, respectively, and the oven temperature gradient used

was as follows: the initial temperature (100 °C) was held for 3 min and

then increased to 295 °C at 15 °C/min over 13 min and ﬁnally

maintained at 295 °C for 10 min. Combustion analysis was performed

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

(

i

1A

2A

HT ) and was a potent 5-HT agonist.

2C

1A

We then selected compounds based on their in vitro

pharmacology proﬁles and evaluated them for metabolic

stability. Analogues with or without the 3-OH substitution

were compared; 16 and 32 were discovered to be the most

metabolically stable in the series − 37% and 54%, respectively,

remained after 60 min compared to the parent compound 6

11% remaining after 60 min) in mouse microsomes. Of note,

all the compounds with the 3-OH substituent were

metabolically less stable.

±

0.4% of calculated values. cLogP values were calculated using

ChemBioDraw Ultra 4.0. Melting point determination was conducted

using a Thomas−Hoover melting point apparatus and are uncorrected.

On the basis of NMR and combustion data, all ﬁnal compounds are

≥

95% pure.

-(3-Chloro-2-methoxyphenyl)piperazine (7a). K CO (3.31 g,

47

1

2

3

(

2

3.98 mmol) was added to a solution of 3-chloro-2-methoxyaniline

(3.15 g, 19.98 mmol) and bis(2-chloroethyl)amine HCl (4.30 g, 23.98

mmol) in diglyme (20 mL). The resulting mixture was stirred at reﬂux

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for 48 h. The reaction mixture was cooled to room temperature, and

diglyme was removed under vacuum. The crude mixture was diluted

with water (100 mL) and EtOAc (150 mL) and then extracted with

EtOAc (3 × 100 mL). The combined organic layer was dried over

the reaction mixture under an argon atmosphere overnight, the solvent

was reduced in vacuo to give a clear, dark-orange solution. Ice-cold 2-

propanol (30 mL) was added to the orange solution at room

temperature, and the mixture was left to stir overnight at room

temperature, resulting in precipitation of the desired product.

Compound 9c (2.87 g, 73% yield) was recovered after ﬁltration as

MgSO , ﬁltered, and concentrated. The crude compound was puriﬁed

4

by ﬂash column chromatography (90% CMA) to give the pure

1

compound 7a (2.53 g, 56% yield). H NMR (CDCl ) δ 7.01 (dd, J =

an oﬀ-white solid and washed with 2-propanol; mp 108−109 °C. H

3

8

1

.0, 2.0 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.81 (dd, J = 8.0, 2.0 Hz,

H), 3.88 (s, 3H), 3.10−3.03 (m, 8H), 1.89 (br s, 1H). C NMR

NMR (CDCl ) δ 7.86−7.84 (m, 2H), 7.72−7.70 (m, 2H), 7.02−6.98

3

13

(m, 1H), 6.93−6.91 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H), 3.94−3.83 (m,

2H), 3.85 (s, 3H), 3.80−3.76 (m, 1H), 3.57 (br s, 1H), 3.07 (br s,

(

CDCl ) δ 148.9, 147.2, 128.9, 124.8, 123.5, 117.3, 59.2, 51.7, 46.6.

3

+

GC-MS (EI) m/z 226 (M ).

-(Naphthalen-1-yl)piperazine (7b). Compound 7b was synthe-

4H), 2.86−2.82 (m, 2H), 2.61−2.58 (m, 2H), 2.45−2.35 (m, 2H),

4

8

13

1

1.79 (q, J = 7.2, 2H). C NMR (CDCl ) δ 168.5, 152.2, 141.2, 133.9,

3

sized as described for 7a, using naphthalen-1-amine (4.00 g, 27.94

mmol), bis(2-chloroethyl)amine HCl (5.98 g, 33.52 mmol), and

K CO (4.63 g, 33.52 mmol) in diglyme (25 mL). The product 7b

132.2, 123.2, 122.99, 120.99, 118.2, 111.2, 64.5, 63.9, 55.4, 53.4, 50.7,

35.2, 33.6.

2

3

2-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-

49

(

3.40 g, 57% yield) was converted to the HCl salt in 2-propanol/

isoindoline-1,3-dione (9d). Compound 9d was synthesized as

described for 9c using 1-(2,3-dichlorophenyl)piperazine (900 mg, 3.89

mmol) and 8 (850 mg, 3.91 mmol) in 2-propanol (20 mL). The pure

1

acetone to form a gray solid; mp >250 °C (HCl salt). H NMR

(

CDCl ) δ 8.24−8.21 (m, 1H), 7.84−7.81 (m, 1H), 7.55 (d, J = 8.4

3

Hz, 1H), 7.51−7.44 (m, 2H), 7.41 (t, J = 8.4 Hz, 1H), 7.09 (dd, J =

product, 9d (1.37 g, 78% yield), was recovered as a white solid; mp

1

7

1

.6, 1.2 Hz, 1H), 3.16 (t, J = 4.8 Hz, 4H), 3.09 (br s, 4H), 1.99 (br s,

127.5−129 °C. H NMR (CDCl

3

) δ 7.88−7.83 (m, 2H), 7.74−7.69

H). ¹³C NMR (CDCl ) δ 149.75, 134.4, 128.6, 128.0, 125.5, 125.4,

(m, 2H), 7.17−7.12 (m, 2H), 6.94 (dd, J = 6.8, 2.8 Hz, 1H), 3.96−

3.83 (m, 2H), 3.81−3.75 (m, 1H), 3.52 (br s, 1H), 3.04 (br s, 4H),

2.84−2.81 (m, 2H), 2.60−2.59 (m, 2H), 2.47−2.36 (m, 2H), 1.79 (q, J

3

+

24.9, 123.2, 123.1, 114.3, 54.1, 46.3. GC-MS (EI) m/z 212 (M ).

2

-(2-(Oxiran-2-yl)ethyl)isoindoline-1,3-dione (8). Phthalimide po-

= 13.4 Hz, 2H). ¹³C NMR (CDCl ) δ 168.5, 151.1, 134.1, 133.9,

tassium salt (6.47 g, 34.9 mmol) was suspended in DMF (60 mL)

under an argon atmosphere and stirred at room temperature for 10

min. A solution of 2-(2-bromoethyl)oxirane) (5.82 g, 38.50 mmol) in

DMF (15 mL) was added dropwise, and the reaction mixture was left

to stir at room temperature for 20 h. The white precipitate formed was

ﬁltered and the ﬁltrate diluted with EtOAc (175 mL). The white

precipitate collected was then dissolved in water and extracted with

EtOAc (3 × 75 mL), and the extract was combined with the original

diluted ﬁltrate. The combined EtOAc extracts were consecutively

washed with water (100 mL) and brine (100 mL), dried over Na SO ,

3

132.2, 127.5, 127.4, 124.7, 123.2, 118.6, 64.5, 63.8, 51.4, 35.1, 33.6.

4-Amino-1-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)butan-

2-ol (10a). Anhydrous hydrazine (0.16 mL, 5.07 mmol) was added to

a suspension of 9a (0.45 g, 1.01 mmol) in anhydrous ethanol (20 mL),

and the mixture was stirred at reﬂux for 3 h under an argon

atmosphere. Solvents were removed, and the recovered residue was

partitioned between chloroform (50 mL) and a 20% aqueous K CO

2 3

(50 mL) solution. The layers were separated, and the organic layer was

washed with water (50 mL) and brine (50 mL). The combined

2

4

and ﬁltered. The pure product, 8 (7.48 g, 98% yield), was obtained

organic layer was dried over MgSO

4

, ﬁltered, and the solvent removed

1

after solvent removal as an oﬀ-white solid; mp 84−86 °C. H NMR

in vacuo to give 10a (270 mg, 85% yield), which was used without

1

(

2

CDCl ) δ 7.87−7.85 (m, 2H), 7.73−7.71 (m, 2H), 3.96−3.83 (m,

further puriﬁcation. H NMR (CDCl ) δ 7.01−6.92 (m, 2H), 6.80−

3

H), 3.02−2.98 (m, 1H), 2.72 (t, J = 5.0 Hz, 1H), 2.45 (dd, J = 4.8, 2.4

6.77 (dd, J = 8, 2 Hz, 2H), 3.90−3.87 (m, 1H), 3.85 (s, 3H), 3.84−

3.81 (m, 1H), 3.12 (br s, 5H), 3.07−2.93 (m, 2H), 2.78 (br m, 2H),

13

Hz, 1H), 2.04−1.96 (m, 1H), 1.90−1.83 (m, 1H). C NMR (CDCl )

δ 168.3, 133.9, 132.1, 123.3, 50.2, 46.4, 35.2, 31.6.

3

1

3

2.59 (br, 2H), 2.41−2.36 (m, 2H), 1.62−1.57 (m, 2H). C NMR

2

-(3-Hydroxy-4-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)-

(CDCl ) δ 148.6, 146.6, 128.7, 124.6, 123.3, 117.1, 66.3, 64.5, 59.0,

3

+

butyl)isoindoline-1,3-dione (9a). To a solution of 7a (0.40 g, 1.77

mmol) in 2-propanol (7 mL) was added a solution of compound 8

58.9, 53.8, 50.3, 39.5, 37.2. GC-MS (EI) m/z 313 (M ).

4-Amino-1-(4-(4-(naphthalen-1-yl)piperazin-1-yl)butan-2-ol

(10b). Compound 10b was prepared as described for 10a using 9b

(0.35 g, 0.82 mmol) and anhydrous hydrazine (0.13 mL, 4.08 mmol)

in anhydrous ethanol (20 mL). The resulting pure product 10b (190

(

0.39 g, 1.77 mmol) in 2-propanol (15 mL). The reaction mixture was

then stirred at reﬂux at 82 °C overnight under an argon atmosphere.

After cooling to room temperature, the solvent was removed in vacuo,

and the crude product was puriﬁed by ﬂash column chromatography

1

mg, 78% yield) was used without further puriﬁcation. H NMR

1

(

50% EtOAC:hexanes) to give 9a (450 mg, 57% yield). H NMR

(CDCl ) δ 8.18−8.15 (m, 1H), 7.81−7.78 (m, 1H), 7.53 (d, J = 8.4

3

CDCl ) δ 7.85−7.82 (m, 2H), 7.73−7.68 (m, 2H), 7.01−6.91 (m,

Hz, 1H), 7.48−7.42 (m, 2H), 7.40−7.35 (m, 1H), 7.09−7.03 (m, 1H),

3.92 (m, 1H), 3.11 (br s, 8H), 3.02−2.93 (m, 2H), 2.92 (br s, 2H),

3

2

3

2

1

5

H), 6.79−6.76 (dd, J = 8.4, 1.6 Hz, 1H), 3.95−3.84 (m, 2H), 3.83 (s,

1

3

H), 3.81−3.74 (m, 1H), 3.10 (br s, 4H), 2.78 (br, 2H), 2.55 (br, 2H),

2.73−2.64 (m, 2H), 2.45−2.42 (m, 1H), 1.67−1.55 (m, 2H).

C

13

.44−2.34 (m, 2H), 1.79 (q, J = 6.4, 2H). C NMR (CDCl ) δ 168.3,

NMR (CDCl ) δ 149.3, 134.7, 128.8, 128.8, 128.4, 125.8, 125.8, 125.3,

3

48.7, 146.5, 133.9, 132.2, 128.7, 124.6, 123.3, 123.2, 117.1, 64.5, 63.9,

9.0, 53.7, 50.3, 35.1, 33.6.

123.5, 114.6, 77.2, 66.2, 64.5, 53.0, 39.4, 36.9. GC-MS (EI) m/z 299

+

(M ).

N-(3-Hydroxy-4-(4-(naphthalen-1-yl)piperazin-1-yl)butyl)-

4-Amino-1-(4-(2-methoxyphenyl)piperazin-1-yl)butan-2-ol

49

isoindoline-1,3-dione (9b). Compound 9b was synthesized as

described for 9a using 7b (0.29 g, 1.38 mmol) and 8 (0.30 g, 1.38

mmol) in 2-propanol (8 mL). The pure product 9b (400 mg, 68%

(10c). Compound 10c was prepared as described for 10a using 9c

(2.87 g, 7.01 mmol) and anhydrous hydrazine (0.33 mL, 10.51 mmol)

in ethanol (35 mL). The pure product, 10c (quantitative), was

1

yield) was isolated after puriﬁcation by ﬂash column chromatography

recovered as a yellow oil that solidiﬁed over time. H NMR (CDCl

3

) δ

1

(

50% EtOAC:hexanes). H NMR (CDCl ) δ 8.18−8.15 (m, 1H),

7.02−6.98 (m, 1H), 6.95−6.90 (m, 2H), 6.86 (d, J = 8.0 Hz, 1H),

3.92−3.86 (m, 4H), 3.10 (br s, 4H), 3.00−2.90 (m, 2H), 2.87−2.83

(m, 2H), 2.63−2.61 (br m, 2H), 2.44−2.38 (m, 2H), 2.12 (br s, 3H),

3

7

(

.86−7.84 (m, 2H), 7.83−7.80 (m, 1H), 7.73−7.69 (m, 2H), 7.55−

.53 (d, J = 8.0 Hz, 1H), 7.48−7.43 (m, 2H), 7.39 (t, J = 8.0 Hz, 1H),

.07−7.05 (dd, J = 7.2, 1.2 Hz, 1H), 3.96−3.87 (m, 2H), 3.85−3.80

1

3

1.62−1.51 (m, 2H). C NMR (CDCl ) δ 152.3, 141.3, 123.0, 121.0,

3

m, 1H), 3.12 (br s, 4H), 2.92 (br s, 2H), 2.69 (br s, 2H), 2.53−2.41

118.2, 111.2, 66.1, 64.5, 55.4, 53.6, 50.8, 39.6, 37.6.

m, 2H), 1.82 (q, J = 6.4, 2H). ¹³C NMR (CDCl ) δ 168.5, 149.5,

4-Amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol

3

49

1

34.7, 133.9, 132.2, 128.8, 128.4, 125.8, 125.8, 125.3, 123.54, 123.50,

23.2, 114.6, 64.6, 63.9, 53.0, 35.1, 33.7.

(10d). Compound 10d was synthesized as described for 10a using

9d (1.36 g, 3.03 mmol) and anhydrous hydrazine (0.145 mL, 4.62

2

-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

mmol) in ethanol (16 mL). The pure product, 10d (740 mg, 77%

4

9

1

isoindoline-1,3-dione (9c). A solution of 8 (2.09 g, 9.62 mmol) in 2-

propanol (5 mL) was added to a solution of 1-(2-methoxyphenyl)-

piperazine (1.85 g, 9.62 mmol) in 2-propanol (40 mL). After reﬂuxing

yield), was recovered as a viscous light-yellow oil. H NMR (CDCl ) δ

3

7.17−7.12 (m, 2H), 6.95 (dd, J = 6.8, 3.2 Hz, 1H), 3.93−3.87 (m,

2H), 3.07 (br s, 4H), 3.01−2.88 (m, 2H), 2.85−2.82 (m, 2H), 2.63−

K

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2

6

.62 (m, 2H), 2.46−2.39 (m, 2H), 2.17 (br s, 2H), 1.62−1.50 (m,

for 13a, using 12b (3.20 g, 7.75 mmol) and anhydrous hydrazine (0.73

H). ¹³C NMR (CDCl ) δ 151.2, 134.0, 127.5, 127.4, 124.6, 118.6,

mL, 23.25 mmol) in ethanol (50 mL). H NMR (CDCl ) δ 8.21−8.19

1

3

6.3, 64.5, 53.4, 51.4, 39.7, 37.5.

(m, 1H), 7.83−7.81 (m, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.47−7.43 (m,

2H), 7.40 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 7.6 Hz, 2H), 3.15 (br s, 4H),

2.75 (t, J = 6.8 Hz, 4H), 2.49 (t, J = 7.6 Hz, 2H), 1.65−1.48 (m, 4H).

2

-(4-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-

isoindoline-1,3-dione (12a). K CO (2.21 g, 16.00 mmol) was added

2

3

13

to a solution of 7a (904 mg, 4.00 mmol) and 11 2-(4-bromobutyl)-

isoindoline-1,3-dione (2.26 g, 8.00 mmol) in acetone (25 mL). The

resulting solution was stirred at reﬂux overnight. After cooling the

reaction mixture to room temperature, K CO₃was removed via

ﬁltration and the ﬁltrate was concentrated. The crude product was

puriﬁed by ﬂash column chromatography (50−70% EtOAC:Hexanes)

to give pure 12a (1.40 g, 82% yield). H NMR (CDCl ) δ 7.84 (dd, J

=

C NMR (CDCl ) δ 149.8, 134.8, 128.9, 128.5, 125.9, 125.8, 125.4,

3

1

2

23.7, 123.5, 117.7, 58.7, 53.9, 53.1, 42.3, 31.9, 24.5. GC-MS (EI) m/z

+

83 (M ).

6

5

2

4-(4-(2-Methoxyphenyl)piperazin-1-yl)butan-1-amine (13c).

Compound 13c was synthesized as described for 13a using 12c

(2.62 g, 6.66 mmol) and anhydrous hydrazine (0.32 mL, 10.03 mmol)

1

3

in ethanol (25 mL). The pure product, 13c (850 mg, 48% yield), was

1

5.6, 3.2 Hz, 2H), 7.71 (dd, J = 5.6, 3.6 Hz, 2H), 7.0−6.91 (m, 2H),

obtained as a yellow oil. H NMR (CDCl ) δ 7.02−6.91 (m, 3H), 6.86

3

6.79 (dd, J = 7.6, 1.2 Hz, 1H), 3.85 (s, 1H), 3.73 (t, J = 7.2 Hz, 2H),

(dd, J = 8.0, 1.2 Hz, 1H), 3.86 (s, 3H), 3.10 (br s, 4H), 2.73 (t, J = 6.8

3

.11 (br s, 4H), 2.58 (br s, 4H), 2.42 (t, J = 7.6 Hz, 2H), 1.78−1.70

Hz, 2H), 2.65 (br s, 4H), 2.42 (t, J = 7.6 Hz, 2H), 1.60−1.46 (m, 6H).

13

(

1

m, 2H), 1.61−1.55 (m, 2H). C NMR (CDCl ) δ 168.6, 148.8,

C NMR (CDCl ) δ 152.3, 141.4, 122.9, 120.99, 118.2, 111.2, 58.6,

3

46.8, 134.0, 132.3, 128.8, 124.7, 123.4, 123.3, 123.3, 117.2, 59.1, 58.2,

3.8, 50.3, 37.9, 26.7, 24.3.

5

5.3, 53.5, 50.7, 42.2, 31.9, 24.3.

-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butan-1-amine

5

4

50,66

2

-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)isoindoline-1,3-

(13d)..

Compound 13d was synthesized as described for 13a using

65

dione (12b). Compound 11b (3.00 g, 77% yield) was synthesized as

described for 12a using 7b (2.01 g, 9.48 mmol), 2-(4-bromobutyl)-

isoindoline-1,3-dione (5.35 g, 18.96 mmol), and K CO (5.24 g, 37.92

1

2d (1.46 g, 3.37 mmol) and anhydrous hydrazine (0.32 mL, 10.19

mmol) in ethanol (33 mL). The pure product, 13d (1.01 g, 99%

yield), was obtained as a light-yellow oil. H NMR (CDCl ) δ 7.16−

7.11 (m, 2H), 6.96 (dd, J = 6.8, 3.2 Hz, 1H), 3.07 (br s, 4H), 2.73 (t, J

1

2

3

1

mmol) in acetone (50 mL). H NMR (CDCl ) δ 8.20−8.17 (m, 1H),

3

7.86−7.82 (m, 3H), 7.72−7.70 (m, 2H), 7.53 (d, J = 8.0 Hz, 1H),

7.47−7.44 (m, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.07 (dd, J = 7.2, 1.2 Hz,

1H), 3.75 (t, J = 7.6 Hz, 2H), 3.13 (br s, 4H), 2.73 (br s, 4H), 2.51 (t,

=

6.6 Hz, 2H), 2.64 (br s, 4H), 2.43 (t, J = 7.6 Hz, 2H), 1.62−1.46 (m,

13

6H). C NMR (CDCl ) δ 151.3, 134.0, 127.5, 127.4, 124.5, 118.6,

3

58.5, 53.3, 51.3, 42.1, 31.7, 24.3.

13

J = 7.6 Hz, 2H), 1.81−1.73 (m, 2H), 1.66−1.58 (m, 2H). C NMR

General Amidation Procedure, Method A. 1,1′-Carbon-

yldiimidazole (1 equiv) was added to the solution of carboxylic acid

(1 equiv) in THF (10 mL/mmol). The resulting mixture was stirred at

room temperature for 2 h. The solution was cooled to 0 °C, and the

amine substrate (1 equiv) in THF (3 mL/mmol) was added dropwise.

The reaction mixture was allowed to warm to room temperature and

stirred overnight. The reaction mixture was concentrated, and the

(

CDCl ) δ 168.6, 149.8, 134.9, 134.1, 134.0, 132.3, 128.5, 126.0, 125.9,

3

1

25.4, 123.6, 123.5, 123.3, 114.8, 58.3, 53.9, 53.1, 38.0, 26.81, 24.4.

-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)isoindoline-1,3-

2

65

dione (12c). Compound 12c was synthesized as described for 12a

using K CO (19.64 g, 142.10 mmol), 1-(2-methoxyphenyl)piperazine

2

3

HCl (8.11 g, 35.46 mmol), and 2-(4-bromobutyl)isoindoline-1,3-dione

(10.01 g, 35.48 mmol) in acetonitrile (110 mL). The crude product

crude product was diluted with CHCl (20 mL/mmol) and washed

3

was puriﬁed by column chromatography (10−90% EtOAc:hexanes) to

give pure 12c (2.74 g, 20% yield) as a thick-yellow oil. H NMR

with saturated aqueous NaHCO solution (2 × 10 mL). The organic

3

1

layer was dried over MgSO , ﬁltered, and concentrated. The crude

4

(

CDCl ) δ 7.86−7.82 (m, 2H), 7.31−7.69 (m, 2H), 7.01−6.97 (m,

3

product was puriﬁed by ﬂash column chromatography (EtOAc

1

3

7

H), 6.95−6.88 (m, 2H), 6.85 (dd, J = 7.8, 1.4 Hz, 1H), 3.85 (s, 3H),

followed by 8% MeOH:CHCl ), or as indicated.

3

.73 (t, J = 7.0 Hz, 2H), 3.08 (br s, 4H), 2.64 (br s, 4H), 2.44 (t, J =

.6 Hz, 2H), 1.78−1.70 (m, 2H), 1.62−1.54 (m, 2H). C NMR

N-(4-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-1H-in-

dole-2-carboxamide (14). Compound 14 was synthesized according

to general method A, using 13a (490 mg, 1.65 mmol) and

commercially available 1H-indole-2-carboxylic acid (266 mg, 1.65

mmol). The pure product, 14 (450 mg, 62% yield), was converted to

13

(

CDCl ) δ 168.4, 152.3, 141.4, 133.9, 132.2, 123.2, 122.8, 120.9, 118.2,

3

1

11.1, 58.1, 55.3, 53.4, 50.6, 37.9, 26.7, 24.2.

-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)isoindoline-1,3-

2

50,66

1

dione (12d)..

Compound 12d was synthesized as described for

the HCl salt in 2-propanol/acetone; mp 235−236 °C (HCl salt). H

1

2a using K CO₃(2.50 g, 18.09 mmol), 1-(2,3-dichlorophenyl)-

NMR (DMSO-d ) δ 11.50 (br s, 1H), 8.42 (br s, 1H), 7.57 (d, J = 8.0

2

6

piperazine (1.03 g, 4.45 mmol), and 2-(4-bromobutyl)isoindoline-1,3-

dione (2.51 g, 8.89 mmol) in acetone (31 mL). The crude product was

puriﬁed by column chromatography (20−80% EtOAc:hexanes

Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.08 (s,

1H), 7.01−6.98 (m, 3H), 6.87−6.85 (m, 1H), 3.74 (s, 3H), 3.29 (m,

2H), 3.01 (br s, 4H), 2.50 (br s, 4H), 2.34 (t, J = 7.2 Hz, 2H), 1.53 (m,

4H). ¹³C NMR (CDCl ) δ 161.9, 148.8, 146.7, 136.5, 131.0, 128.8,

gradient) to give 12d (1.60 g, 83% yield) as a white solid; mp 121−

3

1

7

2

1

3

23 °C. H NMR (CDCl ) δ 7.86−7.82 (m, 2H), 7.73−7.69 (m, 2H),

127.7, 124.8, 124.5, 123.4, 121.9, 120.7, 117.2, 112.2, 101.9, 59.1, 58.2,

3

.15−7.11 (m, 2H), 6.94 (dd, J = 6.0, 3.2 Hz, 1H), 3.73 (t, J = 7.0 Hz,

H), 3.05 (br s, 4H), 2.62 (br s, 4H), 2.45 (t, J = 7.6 Hz, 2H), 1.80−

53.9, 50.3, 39.7, 27.7, 24.5. Anal. (C H ClN O ·HCl) C, H, N.

24

29

4

2

N-(4-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-

benzofuran-2-carboxamide (15). Compound 15 was synthesized

according to general method A, using 13a (420 mg, 1.41 mmol) and

commercially available benzofuran-2-carboxylic acid (227 mg, 1.41

mmol). The pure product, 15 (385 mg, 62% yield), was converted to

13

.71 (m, 2H), 1.61−1.54 (m, 2H). C NMR (CDCl ) δ 168.6, 151.4,

3

34.1, 134.0, 132.3, 127.6, 127.5, 124.6, 123.3, 118.7, 58.1, 53.4, 51.4,

7.99, 26.7, 24.4.

4

-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)butan-1-amine

47

(

13a). Anhydrous hydrazine (0.28 mL, 9.00 mmol) was added to a

the oxalate salt in 2-propanol/acetone; mp 118−120 °C (oxalate salt).

1

solution of 12a (1.28 g, 3.00 mmol) in ethanol (30 mL). The solution

was stirred at reﬂux for 3 h. The reaction mixture was cooled and

concentrated. The crude reaction mixture was partitioned between

CHCl (50 mL) and 20% K CO (20 mL) solution, and the organic

H NMR (CDCl ) δ 7.68−7.65 (m, 1H), 7.47−7.45 (m, 2H), 7.42−

3

7

(

.37 (m, 1H), 7.31−7.26 (m, 1H), 7.0 (dd, J = 8.0, 1.6 Hz, 2H), 6.94

t, J = 8.0 Hz, 1H), 6.79 (dd, J = 8.0, 1.6 Hz, 1H), 3.86 (s, 3H), 3.53

3

2

3

(q, J = 6.0 Hz, 2H), 3.17 (br s, 4H), 2.64 (br s, 4H), 2.47 (t, J = 7.2

1

3

layer was collected and dried over MgSO . The organic layer was

Hz, 2H), 1.75−1.65 (m, 4H). C NMR (CDCl ) δ 159.0, 154.8,

4

3

ﬁltered and concentrated to give 13a (855 mg, 96% yield), which was

149.1, 148.8, 146.7, 128.8, 127.8, 126.9, 124.7, 123.8, 123.3, 122.8,

117.2, 111.7, 110.4, 59.1, 58.1, 53.9, 50.3, 39.3, 27.7, 24.5. Anal.

(C H ClN O ·C H O ·EtOAc) C, H, N.

1

used without further puriﬁcation. H NMR (CDCl ) δ 7.01−6.92 (m,

3

2

H), 6.81 (dd, J = 8.0, 1.2 Hz, 1H), 3.86 (s, 3H), 3.14 (br s, 4H), 2.73

24

28

3

2

4

(

t, J = 6.8 Hz, 2H), 2.61 (br s, 4H), 2.41 (t, J = 7.2 Hz, 2H), 1.60−1.46

N-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)-1H-indole-2-car-

boxamide (16). Compound 16 was synthesized according to general

method A, using 13b (580 mg, 2.05 mmol) and 1H-indole-2-

carboxylic acid (330 mg, 2.05 mmol). The pure product, 16 (524 mg,

m, 4H). ¹³C NMR (CDCl ) δ 148.7, 146.7, 128.8, 124.7, 123.3,

3

1

17.2, 59.0, 58.6, 53.8, 50.3, 42.0, 31.6, 24.3.

-(4-(Naphthalen-1-yl)piperazin-1-yl)butan-1-amine (13b).

Compound 13b (1.88 g, 87% yield) was synthesized as described

6

7

4

1

60% yield), was converted to the HCl salt; mp >250 °C (HCl salt). H

L

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

NMR (DMSO-d ) δ 11.52 (br s, 1H), 8.45 (t, J = 5.8 Hz, 1H), 8.08 (d,

J = 10.0 Hz, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.57 (t, J = 8.8 Hz, 2H),

N-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)quinoline-2-car-

6

59

boxamide (22). Compound 22 was synthesized according to general

method A using 13c (762 mg, 2.89 mmol) and commercially available

quinoline-2-carboxylic acid (500 mg, 2.89 mmol). The crude product

was puriﬁed by column chromatography as described for general

method A to give 22 (1.02 g, 84% yield) as a gummy dark-yellow

7

.52−7.46 (m, 2H), 7.40 (t, J = 8.0 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H),

7

.12−7.09 (m, 2H), 7.01 (t, J = 7.6 Hz, 1H), 3.39−3.32 (m, 2H), 3.01

(

br s, 4H), 2.64 (br s, 4H), 2.43 (t, J = 6.8 Hz, 2H), 1.61−1.57 (m,

4

1

H). ¹³C NMR (DMSO-d ) δ 161.0, 149.3, 136.3, 134.3, 131.9, 128.3,

6

28.1, 127.1, 126.0, 125.8, 125.3, 123.3, 123.1, 122.97, 121.4, 119.6,

solid; the pure product was converted to the oxalate salt in 2-

1

14.5, 112.2, 102.2, 57.6, 53.2, 52.7, 38.7, 27.2, 23.8. Anal.

propanol/acetone; mp 183−185 °C (oxalate salt). H NMR (CDCl

3

)

3

(

C H N O·HCl· / H O) C, H, N.

δ 8.34 (br s, 1H), 8.31 (s, 2H), 8.11 (d, J = 8.4 Hz, 1H), 7.88 (dd, J =

8.0, 0.8 Hz, 1H), 7.76 (td, J = 8.6, 1.5 Hz, 1H), 7.62 (td, J = 8.2, 1.2

Hz, 1H), 7.01−6.97 (m, 1H), 6.95−6.83 (m, 2H), 6.85 (dd, J = 7.6,

1.2 Hz, 1H), 3.86 (s, 3H), 3.58 (q, J = 13.2 Hz, 2H), 3.11 (br s, 4H),

27

34

4

2

5

-Methoxy-N-(4-(4-(naphthalen-1-yl)piperazin-1-yl)butyl)-1H-in-

dole-2-carboxamide (17). Compound 17 was synthesized according

to general method A, using 13b (490 mg, 1.73 mmol) and

commercially available 5-methoxy-1H-indole-2-carboxylic acid (330

1

3

2.67 (br s, 4H), 2.49 (t, J = 7.2 Hz, 2H), 1.81−1.65 (m, 4H).

C

mg, 1.73 mmol). The pure product, 17 (457 mg, 58% yield), was

NMR (CDCl ) δ 164.6, 152.4, 150.1, 146.6, 141.5, 137.6, 130.2, 129.8,

3

1

converted to the HCl salt; mp >250 °C (HCl salt). H NMR (CDCl )

129.4, 127.9, 127.9, 122.9, 121.1, 119.0, 118.3, 111.3, 58.4, 55.5, 53.6,

3

δ 9.44 (s, 1H), 8.19 (d, J = 9.6 Hz, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.54

50.8, 39.6, 27.9, 24.6; Anal. (C H N O ·C H O ) C, H, N.

25

30

4

2

4

(

d, J = 8.0 Hz, 1H), 7.49−7.43 (m, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.33

N-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

quinoline-2-carboxamide (23). Compound 23 was synthesized

according to general method A using 10c (648 mg, 2.32 mmol) and

commercially available quinoline-2-carboxylic acid (401 mg, 2.31

mmol). The crude product was recrystallized in hot 2-propanol, and

23 (761 mg, 76% yield) was isolated as a pure-yellow solid that was

(

d, J = 8.8 Hz, 1H), 7.06 (d, J = 6.0 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H),

6.96 (dd, J = 9.0, 2.4 Hz, 1H), 6.76 (d, J = 1.6 Hz, 1H), 6.56 (t, J = 5.4

Hz, 1H), 3.83 (s, 3H), 3.57−3.52 (m, 2H), 3.16 (br s, 4H), 2.75 (br s,

1

3

4

H), 2.54 (t, J = 6.8 Hz, 2H), 1.77−1.69 (m, 4H). C NMR (CDCl )

3

δ 161.9, 154.7, 149.6, 134.7, 131.6, 131.4, 128.9, 128.4, 127.99, 125.9,

1

5

25.8, 125.3, 123.55, 123.51, 115.7, 114.6, 112.8, 102.3, 101.4, 58.1,

converted to the oxalate salt in 2-propanol/acetone; mp 151−153 °C

3

1

5.7, 53.8, 52.9, 39.6, 27.6, 24.4. Anal. (C H N O ·HCl· / H O) C,

(oxalate salt). H NMR (CDCl

3

) δ 8.73 (t, J = 5.4 Hz, 1H), 8.33−8.29

28

32

4

2

H, N.

(m, 2H), 8.12 (dd, J = 8.4, 0.8 Hz, 1H), 7.88 (dd, J = 8.0, 1.6 Hz, 1H),

7.76 (td, J = 7.7, 1.7 Hz, 1H), 7.61 (td, J = 7.7, 1.1 Hz, 1H), 7.02−6.98

(m, 1H), 6.96−6.90 (m, 2H), 6.86 (d, J = 8.2 Hz, 1H), 3.96−3.83 (m,

6H), 3.66−3.58 (m, 1H), 3.10 (br s, 4H), 2.90−2.85 (br m, 2H),

2.64−2.62 (br m, 2H), 2.49−2.42 (m, 2H), 1.92−1.84 (m, 1H), 1.76−

N-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)benzofuran-2-car-

boxamide (18). Compound 18 was synthesized according to general

method A, using 13b (500 mg, 1.77 mmol) and commercially available

benzofuran-2-carboxylic acid (287 mg, 1.77 mmol). The pure product,

1.67 (m, 1H). ¹³C NMR (CDCl ) δ 164.8, 152.3, 149.9, 146.6, 141.2,

1

8 (491 mg, 65% yield), was converted to the oxalate salt in 2-

3

1

propanol/acetone; mp 168−170 °C (oxalate salt). H NMR (CDCl )

δ 7.19 (d, J = 7.6 Hz, 1H), 7.84−7.81 (m, 1H), 7.67 (dd, J = 8.0, 1.2

Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.48−7.44 (m, 4H), 7.42−7.36 (m,

137.4, 129.98, 129.8, 129.3, 127.8, 127.7, 123.0, 120.9, 118.9, 118.2,

3

111.2, 65.2, 64.0, 55.4, 53.5, 50.8, 37.1, 34.4. Anal. (C H N O ·

2

5

30

4

3

C H O ) C, H, N.

2

4

2

4

1

H), 7.32−7.26 (m, 1H), 7.09−7.05 (m, 2H), 7.56 (q, J = 6.0 Hz,

6-Fluoro-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

H), 3.20 (br s, 4H), 2.83 (br s, 4H), 2.60 (t, J = 7.2 Hz, 2H), 1.75 (m,

quinoline-2-carboxamide (24). Compound 24 was synthesized

according to general method A using 13c (691 mg, 2.62 mmol) and

commercially available 6-ﬂuoroquinoline-2-carboxylic acid (501 mg,

2.62 mmol). The crude product was puriﬁed by column chromatog-

raphy as described for general method A to give 24 (326 mg, 28%

yield) that was converted to the oxalate salt (beige-colored solid) in 2-

H). ¹³C NMR (CDCl ) δ 159.1, 154.8, 149.5, 149.1, 134.8, 128.9,

28.5, 127.8, 126.9, 125.9, 125.9, 125.5, 123.8, 123.7, 123.6, 122.8,

14.9, 111.8, 110.4, 57.9, 53.6, 52.6, 39.2, 27.6, 24.0. Anal.

3

1

(

C H N O ·C H O · / H O) C, H, N.

27

29

3

2

4

2

N-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)benzo[b]-

1

thiophene-2-carboxamide (19). Compound 19 was synthesized

according to by general method A, employing 13b (540 mg, 1.91

mmol) and commercially available benzo[b]thiophene-2-carboxylic

acid (340 mg, 1.91 mmol). The pure product, 19 (575 mg, 68% yield),

propanol/acetone; mp 166−168 °C (sublimes; oxalate salt). H NMR

(CDCl ) δ 8.33 (d, J = 8.4 Hz, 1H), 8.28−8.24 (m, 1H), 8.25 (d, J =

3

8.4 Hz, 1H), 8.11 (dd, J = 9.2, 5.2 Hz, 1H), 7.56−7.47 (m, 2H), 7.01−

6.97 (m, 1H), 6.95−6.89 (m, 2H), 6.86 (dd, J = 7.8, 1.0 Hz, 1H), 3.86

was converted to the oxalate salt in 2-propanol/acetone; mp 183−185

(s, 3H), 3.58 (q, J = 13.0 Hz, 2H), 3.11 (br s, 4H), 2.68 (br s, 4H),

1

13

°

C (oxalate salt). H NMR (CDCl ) δ 8.18−8.15 (m, 1H), 7.87−7.81

2.49 (t, J = 7.2 Hz, 2H), 1.80−1.65 (m, 4H). C NMR (CDCl ) δ

3

1

4

(

m, 4H), 7.55 (d, J = 8.4 Hz, 1H), 7.48−7.35 (m, 5H), 7.03 (dd, J =

.2, 0.8 Hz, 1H), 6.86 (br s, 1H), 3.54 (q, 5.2 Hz, 2H), 3.18 (br s, 4H),

.84 (br s, 4H), 2.63 (t, J = 7.2 Hz, 2H), 1.77−1.74 (m, 4H).

NMR (CDCl ) δ 162.6, 149.4, 140.9, 139.3, 138.9, 134.9, 128.9, 128.6,

164.2, 161.2 ( J = 251 Hz), 152.3, 149.5, 143.5, 141.3, 136.8 ( J

5.2 Hz), 132.2 ( J = 9.6 Hz), 130.0 ( J = 10.4 Hz), 122.9, 120.9,

120.5 ( J = 26.1 Hz), 119.7, 118.2, 111.2, 110.8 ( J = 22.0 Hz),

CF CF

=

CF

3

7

2

CF CF

13

2

C

3

58.3, 55.3, 53.5, 50.6, 39.5, 27.8, 24.4. Anal. (C H FN O ·C H O )

25 29 4 2 2 2 4

1

26.3, 125.9, 125.6, 125.2, 125.1, 125.0, 123.9, 123.5, 122.9, 114.9,

C, H, N.

1

57.9, 53.6, 52.4, 39.9, 27.4, 23.9. Anal. (C H N OS·C H O · / H O)

6-Fluoro-N-(3-hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)-

butyl)quinoline-2-carboxamide (25). Compound 25 was synthesized

according to general method A using 10c (596 mg, 2.14 mmol) and

commercially available 6-ﬂuoroquinoline-2-carboxylic acid (407 mg,

2.14 mmol). The crude product was puriﬁed by column chromatog-

raphy as described for general method A to give 25 (330 mg, 34%

yield) as a gummy yellow anhydrous solid which was converted to the

27

29

3

2

4

2

C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-5-

ﬂuoro-1H-indole-2-carboxamide (21). Compound 21 was synthe-

sized according to general method A employing 10d (398 mg, 1.25

mmol) and commercially available 5-ﬂuoroindole-2-carboxylic acid

(

222 mg, 1.24 mmol). The crude product was puriﬁed by column

chromatography as described in general method A to give 21 (320 mg,

4% yield) as a light-brown solid; the pure product was converted to

1

oxalate salt in 2-propanol/acetone; mp 115−118 °C (free base). H

5

NMR (CDCl ) δ 8.69 (t, J = 5.6 Hz, 1H), 8.33 (dd, J = 8.4, 0.8 Hz,

3

the oxalate salt in 2-propanol/acetone; mp 225−228 °C (oxalate salt).

1H), 8.25 (d, J = 8.4 Hz, 1H), 8.13 (dd, J = 9.2, 5.6 Hz, 1H), 7.56−

7.47 (m, 2H), 7.03−6.98 (m, 1H), 6.97−6.90 (m, 2H), 6.86 (d, J = 8.2

Hz, 1H), 3.96−3.80 (m, 6H), 3.68−3.57 (m, 1H), 3.10 (br s, 4H),

2.90−2.86 (m, 2H), 2.64−2.62 (m, 2H), 2.49−2.42 (m, 2H), 1.91−

1

H NMR (DMSO-d ) δ 11.6 (s, 1H), 8.51 (t, J = 5.6 Hz, 1H), 7.42

6

(

dd, J = 9.0, 4.6 Hz, 1H), 7.35 (dd, J = 9.8, 2.6 Hz, 1H), 7.29−7.23 (m,

2

4

1

H), 7.10−6.99 (m, 3H), 4.67 (s, 1H), 3.46−3.30 (m, 3H), 2.94 (br s,

H), 2.57 (br s, 4H), 2.39−2.30 (m, 2H), 1.80−1.72 (m, 1H), 1.57−

.48 (m, 1H). ¹³C NMR (DMSO-d ): δ 161.4, 157.6 ( J = 231 Hz),

1

3

1.83 (m, 1H), 1.75−1.66 (m, 1H). C NMR (CDCl ) δ 164.5, 161.2

3

1

4

( J = 251 Hz), 152.3, 149.5 ( J = 3.0 Hz), 143.6, 141.2, 136.8,

6

CF

3

2

51.6, 133.8, 133.5, 133.0, 128.9, 127.6 ( J = 10.6 Hz), 126.3, 124.7,

19.9, 113.9 ( J = 9.9 Hz), 112.4 ( J = 26.5 Hz), 106.0 ( J = 22.8

136.7, 132.5, 132.4, 130.0 ( J = 10.3 Hz), 123.0, 121.0, 120.5 ( J

25.7 Hz), 119.7, 118.2, 111.2, 110.7 ( J = 22.1 Hz), 65.4, 63.9, 55.4,

=

CF

3

2

CF

4

Hz), 102.8 ( J = 5.3 Hz), 65.9, 64.6, 53.7, 51.2, 36.5, 35.5. Anal.

5 3 . 5 , 5 0 . 8 , 3 7 . 2 , 3 4 . 3 . A n a l . ( C ₂₅H ₂₉F N O ·

CF

4

3

1

(

C H Cl FN O ·C H O ) C, H, N.

C H O · / H O· / C H O) C, H, N.

23

25

2

4

2

4

2 2 4 2 2 2 3 8

M

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

quinoline-3-carboxamide (26). Compound 26 was synthesized

according to general method A using 10c (420 mg, 1.50 mmol) and

commercially available quinoline-3-carboxylic acid (250 mg, 1.44

mmol). The crude product was puriﬁed by column chromatography as

described for general method A, and 26 (460 mg, 73% yield) was

commercially available 6-methoxyquinoline-3-carboxylic acid (211 mg,

1.04 mmol). The crude product was puriﬁed by column chromatog-

raphy as described for general method A to give 30 (320 mg, 66%

yield) as a gummy yellow solid; the pure product was converted to the

oxalate salt in 2-propanol/acetone; mp 83 °C (sublimes at >97 °C;

1

oxalate salt). H NMR (CDCl ) δ 9.13 (d, J = 2.0 Hz, 1H), 8.51 (d, J =

3

obtained as a beige-colored solid from 2-propanol; mp 127−129 °C

1.6 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.73 (br s, 1H), 7.43 (dd, J =

9.2, 2.8 Hz, 1H), 7.14 (d, J = 2.8 Hz, 1H), 7.04−6.99 (m, 1H), 6.95−

6.92 (m, 2H), 6.87 (d, J = 7.8 Hz, 1H), 4.00−3.93 (m, 2H), 3.94 (s,

3H), 3.87 (s, 3H), 3.56−3.48 (m, 1H), 3.11 (br s, 4H), 2.93−2.88 (m,

2H), 2.66−2.61 (m, 2H), 2.51−2.42 (m, 2H), 1.91−1.84 (m, 1H),

1

(

free base). H NMR (CDCl ) δ 9.31 (sd, J = 2.0 Hz, 1H), 8.61 (sd, J

3

=

2.0 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.92 (dd, J = 8.0, 1.2 Hz, 1H),

7

1

4

1

3

.79 (td, J = 8.4, 1.4 Hz, 1H), 7.76 (br s, 1H), 7.60 (td, J = 8.2, 1.1 Hz,

H), 7.04−6.99 (m, 1H), 6.95−6.93 (m, 2H), 6.87 (d, J = 8.2 Hz,

H), 4.01−3.95 (m, 2H), 3.87 (s, 3H), 3.56−3.47 (m, 1H), 3.11 (br s,

H), 2.93−2.88 (m, 2H), 2.64−2.62 (m, 2H), 2.50−2.42 (m, 2H),

1

3

1.72−1.62 (m, 1H). C NMR (CDCl ) δ 165.6, 158.4, 152.4, 145.9,

3

145.6, 141.2, 134.4, 130.9, 128.4, 127.7, 124.1, 123.3, 121.2, 118.4,

111.4, 105.97, 67.0, 63.8, 55.8, 55.5, 53.5, 50.9, 39.0, 33.1. Anal.

13

.92−1.84 (m, 1H), 1.72−1.62 (m, 1H). C NMR (CDCl ) δ 165.3,

3

52.3, 149.2, 148.4, 141.1, 135.4, 131.0, 129.4, 128.8, 127.34, 127.27,

27.0, 123.1, 121.0, 118.2, 111.2, 66.9, 63.7, 55.4, 53.4, 50.8, 38.97,

2.9. Anal. (C H N O ) C, H, N.

(C H N O · / C H O ·H O) C, H, N.

26

32

4

2

4

2

N-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

quinoline-7-carboxamide (31). Compound 31 was synthesized

according to general method A using 10c (420 mg, 1.50 mmol) and

commercially available quinoline-7-carboxylic acid (250 mg, 1.44

mmol). The crude product was puriﬁed by column chromatography as

described for general method A to give 31 (180 mg, 29% yield) as a

light-yellow oil; the pure product was converted to the HCl salt in a

25

30

4

3

6

-Fluoro-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-

quinoline-3-carboxamide (27). Compound 27 was synthesized

according to general method A using 13c (427 mg, 1.62 mmol) and

commercially available 6-ﬂuoroquinoline-3-carboxylic acid (301 mg,

1.57 mmol). The crude product was puriﬁed by column chromatog-

raphy as described for general method A to give 27 (410 mg, 60%

CHCl /ether solvent mixture; mp >220 °C (decomposes; HCl salt).

3

1

yield) as a beige-colored solid; mp 125−127 °C (free base). H NMR

H NMR (CDCl ) δ 8.98 (s, 1H), 8.46 (s, 1H), 8.19 (d, J = 8.4 Hz,

3

(

CDCl ) δ 9.21 (d, J = 2.4 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.15 (dd,

1H), 8.07 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.65 (br s,

1H), 7.47−7.45 (m, 1H), 7.01−6.99 (m, 1H), 6.95−6.91 (m, 2H),

6.87 (d, J = 8.4 Hz, 1H), 3.99−3.94 (m, 2H), 3.87 (s, 3H), 3.54−3.49

(m, 1H), 3.11 (br s, 4H), 2.89 (br s, 2H), 2.62 (br s, 2H), 2.49−2.41

3

J = 9.4, 5.4 Hz, 1H), 7.57 (td, J = 9.6, 2.1 Hz, 1H), 7.50 (dd, J = 8.6,

.4 Hz, 1H), 7.32 (br s, 1H), 6.98 (td, J = 7.8, 1.7 Hz, 1H), 6.88−6.82

m, 2H), 6.75 (dd, J = 8.0, 1.6 Hz, 1H), 3.84 (s, 3H), 3.56 (q, J = 11.8

Hz, 2H), 3.01 (br s, 4H), 2.66 (br s, 4H), 2.50 (t, J = 6.8 Hz, 2H),

2

(

1

3

(m, 2H), 1.87−1.84 (m, 1H), 1.71−1.63 (m, 1H). C NMR (CDCl )

3

1

3

1

.84−1.68 (m, 4H). C NMR (CDCl ): δ 165.7, 160.9 ( J = 250

δ 166.6, 152.3, 151.2, 147.7, 141.1, 135.9, 135.7, 129.7, 128.3, 127.7,

3

CF

4

3

Hz), 152.2, 147.7, 146.3, 140.96, 134.8 ( J = 5.2 Hz), 132.0 ( J

.9 Hz), 128.4, 127.7 ( J = 10.3 Hz), 123.0, 121.4 ( J = 25.8 Hz),

=

125.3, 123.1 122.2, 121.0, 118.3, 111.2, 66.8, 63.8, 55.4, 53.4, 50.8,

CF

3

2

5

8

1

2

38.8, 33.1. Anal. (C H N O ·3HCl· / H O) C, H, N.

CF

25 30

4

3

2

20.9, 118.0, 111.5 ( J = 22.1 Hz), 111.1, 58.0, 55.3, 53.4, 50.4, 40.2,

N-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)quinoline-3-car-

boxamide (32). Compound 32 was synthesized according to general

method A, using 13b (250 mg, 0.88 mmol) and commercially available

quinoline-3-carboxylic acid (153 mg, 0.88 mmol). The pure product,

32 (250 mg, 65% yield), was then converted to the oxalate salt in 2-

CF

1

7.4, 24.5. Anal. (C H FN O · / H O) C, H, N.

2

5

29

4

2

6

-Fluoro-N-(3-hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)-

butyl)quinoline-3-carboxamide (28). Compound 28 was synthesized

according to general method A using 10c (450 mg, 1.61 mmol) and

commercially available 6-ﬂuoroquinoline-3-carboxylic acid (300 mg,

propanol/acetone and isolated as a cream-colored solid; mp 178−179

1

1.57 mmol). The crude product was puriﬁed by column chromatog-

°C (oxalate salt). H NMR (CDCl ) δ 9.28 (d, J = 2.4 Hz, 1H), 8.59

3

raphy as described for general method A to give 28 (520 mg, 73%

(d, J = 2.4 Hz, 1H), 8.16−8.13 (m, 2H), 7.90 (d, J = 8.4 Hz, 1H),

7.82−7.78 (m, 2H), 7.61 (t, J = 6.0 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H),

7.45−7.43 (m, 2H), 7.20 (s, 1H), 6.88 (dd, J = 7.2, 0.8 Hz, 1H) 3.59

(q, t, J = 6.4 Hz, 2H), 3.06 (br s, 4H), 2.75 (br s, 4H), 2.57 (t, J = 7.2

1

yield) as a beige-colored solid; mp 161−161.5 °C (free base). H

NMR (CDCl ) δ 9.26 (d, J = 2.4 Hz, 1H), 8.55 (sd, J = 2.0 Hz, 1H),

3

8

7

4

2

1

(

1

=

.15 (dd, J = 9.0, 5.4 Hz, 1H), 7.78 (br s, 1H), 7.59−7.52 (m, 2H),

.04−6.99 (m, 1H), 6.96−6.92 (m, 2H), 6.87 (d, J = 7.6 Hz, 1H),

.03−3.94 (m, 2H), 3.87 (s, 3H), 3.55−3.47 (m, 1H), 3.11 (br s, 4H),

.93−2.88 (m, 2H), 2.66−2.61 (m, 2H), 2.52−2.42 (m, 2H), 1.91−

1

3

Hz, 2H), 1.82−1.73 (m, 4H). C NMR (CDCl ) δ 165.6, 149.3,

3

149.2, 148.3, 135.5, 134.7, 131.9, 129.5, 128.8, 128.7, 128.4, 127.7,

127.5, 126.9, 125.8, 125.8, 125.3, 123.5, 123.43, 114.6, 58.1, 53.8, 52.7,

13

1

.84 (m, 1H), 1.71−1.61 (m, 1H). C NMR (CDCl ) δ 164.9, 160.8

40.2, 27.5, 24.5. Anal. (C H N O·C H O · / H O) C, H, N.

3

28 30

4

2

4

2

1

4

J

= 250 Hz), 152.3, 147.8, 146.3, 141.0, 134.7 ( J = 5.9 Hz),

31.9 ( J = 8.9 Hz), 128.0, 127.8 ( J = 10.4 Hz), 123.2, 121.3 ( J

N-(3-Hydroxy-4-(4-(naphthalen-1-yl)piperazin-1-yl)butyl)-

quinoline-3-carboxamide (33). Compound 33 was synthesized

according to general method A, using 10b (150 mg, 0.50 mmol)

and commercially available quinoline-3-carboxylic acid (87 mg, 0.50

CF

3

2

CF

2

26.6 Hz), 121.0, 118.2, 111.6 ( J = 22.0 Hz), 111.2, 67.0, 63.6,

CF

5

5.4, 53.4, 50.8, 39.1, 32.8. Anal. (C H FN O ) C, H, N.

25 29 4 3

N-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-6-

mmol). The pure product, 33 (100 mg, 44% yield), was isolated as a

1

methylquinoline-3-carboxamide (29). Compound 29 was synthe-

sized according to general method A using 10c (458 mg, 1.64 mmol)

and commercially available 6-methylquinoline-3-carboxylic acid (301

mg, 1.61 mmol). The crude product was puriﬁed by column

chromatography as described for general method A to give 29 (400

brown solid; mp 90−92 °C (free base). H NMR (CDCl ) δ 9.32 (d, J

3

= 2.4 Hz, 1H), 8.62 (d, J = 1.6 Hz, 1H), 8.19−8.13 (m, 2H), 7.92 (dd,

J = 8.4, 1.2 Hz, 1H), 7.83−7.76 (m, 3H), 7.62−7.55 (m, 2H), 7.50−

7.44 (m, 2H), 7.40 (t, J = 8.4 Hz, 1H), 7.08 (dd, J = 7.6, 1.2 Hz, 1H)

4.03−3.95 (m, 2H), 3.57−3.49 (m, 1H), 3.16 (br s, 4H), 2.98 (br s,

2H), 2.72−2.71 (m, 2H), 2.58−2.47 (m, 2H), 1.92−1.86 (m, 1H),

1

mg, 56% yield) as a yellow solid; mp 52−55 °C (free base). H NMR

1

3

(

=

1

4

CDCl ) δ 9.23 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.03 (d, J

1.73−1.67 (m, 1H). C NMR (CDCl ) δ 165.3, 149.3, 149.2, 148.4,

3

8.8 Hz, 1H), 7.71 (br s, 1H), 7.67 (s, 1H), 7.62 (dd, J = 8.6, 1.8 Hz,

135.5, 134.8, 131.0, 129.4, 128.8, 128.7, 128.4, 127.33, 127.3, 127.0,

125.9, 125.8, 125.4, 123.7, 123.4, 114.7, 77.22, 66.9, 63.7, 59.0, 58.9,

38.8, 33.0. HRMS (MALDI-TOF) m/z 455.2443 (M + H) .

H), 7.04−6.99 (m, 1H), 6.96−6.93 (m, 2H), 6.87 (d, J = 8.2 Hz,

H), 4.02−3.92 (m, 2H), 3.87 (s, 3H), 3.55−3.47 (m, 1H), 3.11 (br s,

H), 2.93−2.88 (m, 2H), 2.66−2.61 (m, 2H), 2.56 (s, 3H), 2.50−2.42

+

N-(4-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-

quinoline-3-carboxamide (34). Compound 34 was synthesized

according to general method A, using 13a (140 mg, 0.47 mmol)

and commercially available quinoline-3-carboxylic acid (81 mg, 0.47

mmol). The pure product, 34 (120 mg, 56% yield), was then

converted to the oxalate salt in 2-propanol/acetone and isolated as a

1

3

(

m, 2H), 1.91−1.84 (m, 1H), 1.71−1.61 (m, 1H). C NMR (CDCl )

3

δ 165.5, 152.3, 147.9, 147.5, 141.1, 137.3, 134.8, 133.6, 129.0, 127.5,

1

3

27.2, 127.1, 123.1, 121.0, 118.2, 111.2, 66.9, 63.7, 55.4, 53.4, 50.8,

5

8.9, 32.9, 21.6. Anal. (C H N O · / H O) C, H, N.

26

32

4

3

4

2

N-(3-Hydroxy-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-6-me-

thoxyquinoline-3-carboxamide (30). Compound 30 was synthesized

according to general method A using 10c (290 mg, 1.04 mmol) and

1

cream-colored solid; mp 149−151 °C (oxalate salt). H NMR

(CDCl ) δ 9.25 (d, J = 2.4 Hz, 1H), 8.57 (d, J = 1.6 Hz, 1H), 8.14

3

N

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.82−7.77 (m, 1H),

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 MgCl ·6H O, 5 mM Tris,

pH 7.4 at 4 °C) and centrifuged at 20000 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),

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.

7

6

3

6

1

.62−7.58 (m, 1H), 7.11−7.10 (m, 1H), 6.98 (dd, J = 7.6, 1.6 Hz, 1H)

.88 (t, J = 8.0 Hz, 1H), 6.63 (dd, J = 8.0, 1.2 Hz, 1H) 3.83 (s, 3H),

.56 (q, J = 6.4 Hz, 2H), 3.06 (br s, 4H), 2.60 (br s, 4H), 2.48 (t, J =

2

1

3

.8 Hz, 2H), 1.81−1.66 (m, 4H). C NMR (CDCl ) δ 165.9, 154.7,

3

49.2, 148.6, 148.3, 146.4, 135.5, 131.1, 129.4, 128.7, 127.6, 127.5,

26.9, 124.6, 123.3, 116.9, 58.9, 58.0, 53.7, 50.0, 40.2, 27.4, 24.5. Anal.

3

(

C H ClN O ·C H O · / H O) C, H, N.

25

29

4

2

4

2

N-(4-(4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl)-3-

hydroxybutyl)quinoline-3-carboxamide (35). Compound 35 was

synthesized according to general method A, using 10a (250 mg, 0.79

mmol) and commercially available quinoline-3-carboxylic acid (138

Radioligand competition binding experiments were conducted using

thawed membranes. Test compounds were freshly dissolved in 30%

mg, 0.79 mmol). The pure product, 35 (150 mg, 40% yield), was

DMSO and 70% H O to a stock concentration of 100 μM. To assist

2

1

isolated as a sticky light-yellow solid (free base). H NMR (CDCl ) δ

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 13 half-log serial dilutions using 30% DMSO vehicle; ﬁnal

test concentrations ranged from 10 μM to 10 pM. Previously frozen

3

9.29 (d, J = 2.4 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 8.0 Hz,

1H), 7.85 (d, J = 7.6 Hz, 2H), 7.76−7.72 (m, 1H), 7.57−7.53 (m,

1H), 6.99 (dd, J = 8.0, 1.6 Hz, 1H), 6.92 (t, J = 8.0 Hz, 1H), 6.76 (dd,

J = 7.6, 1.6 Hz, 1H) 3.97−3.87 (m, 2H), 3.83 (s, 3H), 3.54−3.46 (m,

membranes were diluted in fresh EBSS to a 100 μg/mL (for hD R or

2

1

2

H), 3.45 (s, 1H), 3.11 (br s, 4H), 2.84−2.80 (m, 2H), 2.58−2.56 (m,

hD R) or 200 μg/mL (hD R) stock for binding. Radioligand

3

4

1

3

H), 2.45−2.42 (m, 2H), 1.86−1.80 (m, 1H), 1.69−1.60 (m, 1H).

C

competition experiments were conducted in glass tubes containing

300 μL of fresh EBSS buﬀer with 0.2 mM sodium metabisulﬁte, 50 μL

of diluted test compound, 100 μL of membranes (10 μg total protein

NMR (CDCl ) δ 165.4, 149.1, 148.7, 148.4, 146.4, 135.5, 135.4, 131.0,

3

1

5

29.3, 128.8, 127.3, 127.2, 126.9, 124.7, 123.4, 117.1, 66.7, 63.7, 59.0,

3

8.9, 53.6, 50.3, 38.7, 33.2. Anal. (C H ClN O ·H O) C, H, N.

for hD R or hD R, 20 μg total protein for hD R), and 50 μL of [ H]N-

25

29

4

3

2

3

4

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)-6-ﬂuoroquino-

methylspiperone (0.4 nM ﬁnal concentration; PerkinElmer). Non-

speciﬁc binding was determined using 10 μM butaclamol (Sigma-

Aldrich, St. Louis, MO), and total binding was determined with 30%

DMSO vehicle. All compound dilutions were tested in triplicate and

the reaction incubated for 1 h at room temperature. The reaction was

terminated by ﬁltration through Whatman GF/B ﬁlters, presoaked for

1 h in 0.5% polyethylenimine, using a Brandel R48 ﬁltering manifold

(Brandel Instruments, Gaithersburg, MD). The ﬁlters were washed 3

times with 3 mL of ice-cold EBSS buﬀer and transferred to scintillation

vials. Then 3 mL of CytoScint liquid scintillation cocktail (MP

Biomedicals, Solon, OH) was added and vials were counted using a

PerkinElmer Tri-Carb 2910 TR liquid scintillation counter (Waltham,

MA). IC₅₀values for each compound were determined from dose−

line-3-carboxamide (36). Compound 36 was synthesized according to

general method A employing 13d (340 mg, 1.12 mmol) and

commercially available 6-ﬂuoroquinoline-3-carboxylic acid (213 mg,

1.11 mmol). The crude product was puriﬁed by column chromatog-

raphy as described in general method A and was then converted to the

oxalate salt in 2-propanol/acetone to give 36 (280 mg, 53% yield) as a

1

cream-colored solid; mp 234−236 °C (oxalate salt). H NMR

(

CDCl ) δ 9.20 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 8.15

3

(

dd, J = 9.0, 5.4 Hz, 1H), 7.57 (td, J = 9.0, 2.9 Hz, 1H), 7.51 (dd, J =

8

.6, 2.6 Hz, 1H), 7.16−7.13 (m, 2H), 7.08 (t, J = 8.0 Hz, 1H), 6.77

(

dd, J = 7.8, 2.2 Hz, 1H), 3.57 (q, J = 12.2 Hz, 2H), 2.98 (br s, 4H),

2

1

.63 (br s, 4H), 2.51 (t, J = 6.8 Hz, 2H), 1.80−1.75 (m, 2H), 1.74−

13 1

.69 (m, 2H). C NMR (CDCl ) δ 165.6, 160.9 ( J = 249 Hz),

response curves, and K values were calculated using the Cheng−

3

CF

i

4

3

69

51.0, 147.6 ( J = 3.0 Hz), 146.3, 134.9 ( J = 5.8 Hz), 134.0, 132.0

Prusoﬀ equation; these analyses were performed using GraphPad

CF

3

2

(

J_C_F= 9.1 Hz), 128.4, 127.7, 127.5, 127.4, 124.7, 121.4 ( J = 25.8

Prism version 5.00 for Windows (GraphPad Software, San Diego,

CA). Reported K_ivalues were determined from least three

independent experiments.

CF

2

Hz), 118.5, 111.5 ( J = 21.2 Hz), 58.0, 53.3, 51.1, 40.3, 27.4, 24.5.

CF

Anal. (C H Cl FN O·C H O ) C, H, N.

24

25

2

4

2

4

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-6-

ﬂuoroquinoline-3-carboxamide (37). Compound 37 was synthesized

according to general method A employing 10d (381 mg, 1.20 mmol)

and commercially available 6-ﬂuoroquinoline-3-carboxylic acid (226

mg, 1.18 mmol). The crude product was puriﬁed by column

chromatography as described in general method A to give 37 (310

mg, 53% yield) as a cream-colored solid that was converted to the

Mouse Microsomal Stability Assay. Phase I metabolic stability

assays were conducted in mouse liver microsomes as previously

6

0

described with minor modiﬁcations. In brief, the reaction was carried

out with 100 mM potassium phosphate buﬀer, pH 7.4, in the presence

of NADPH regenerating system, (compound ﬁnal concentration was 1

μM; and 0.2 mg/mL microsomes). Negative controls without

cofactors were assessed to determine the non-CYP mediated

metabolism. Positive controls for phase I metabolism (testosterone)

were also evaluated. Compound disappearance was monitored over

time using a liquid chromatography and tandem mass spectrometry

(LC/MS/MS) method. All reactions were performed in triplicate.

Chromatographic analysis was performed using an Accela ultra

high-performance system consisting of an analytical pump and an

autosampler coupled with TSQ Vantage mass spectrometer (Thermo

Fisher Scientiﬁc Inc., Waltham, MA). Separation of the analyte from

potentially interfering material was achieved at ambient temperature

using Agilent Eclipse Plus column (100 mm × 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

oxalate salt in a 2-propanol/acetone solvent mixture; mp 201−203 °C

1

(

oxalate salt). H NMR (CDCl ) δ 9.25 (d, J = 2.0 Hz, 1H), 8.56 (d, J

3

=

(

2

1.6 Hz, 1H), 8.15 (dd, J = 9.2, 5.2 Hz, 1H), 7.76 (br s, 1H), 7.57

dd, J = 8.4, 2.8 Hz, 1H), 7.53 (dd, J = 7.4, 2.6 Hz, 1H), 7.20−7.14 (m,

H), 6.96 (dd, J = 6.6, 2.6 Hz, 1H), 4.03−3.94 (m, 3H), 3.55−3.47

(

2

m, 1H), 3.09 (br s, 4H), 2.92−2.88 (m, 2H), 2.64−2.62 (m, 2H),

1

3

.53−2.43 (m, 2H), 1.91−1.84 (m, 1H), 1.71−1.60 (m, 1H).

C

=

1

4

NMR (CDCl ): δ 164.9, 160.8 ( J = 24.8 Hz), 150.9, 147.7 ( J

3

CF

4

3

1

.0 Hz), 146.3, 134.8 ( J = 6.1 Hz), 134.1, 131.9 ( J = 9.1 Hz),

27.98, 127.8 ( J = 10.6 Hz), 127.6, 127.5, 124.8, 121.3 ( J = 25.8

CF CF

3

2

CF

2

Hz), 118.6, 111.6 ( J = 22.0 Hz), 66.9, 63.6, 53.2, 51.4, 39.0, 32.9.

CF

Anal. (C H Cl FN O ·C H O ·H O) C, H, N.

24

25

2

4

2

4

2

Radioligand Binding Assays. Binding at dopamine D -like

H O with gradient elution, starting with 10% (organic) linearly

2

6

8

receptors was determined using previously described methods.

increasing to 99% up to 2.5 min, maintaining at 99% (2.5−3.5 min),

and reequlibrating to 10% by 4.5 min. The total run time for each

analyte was 4.5 min. The mass transitions used for compounds for

LC/MS/MS analysis are given in Table S1 (Supporting Information).

The calculations of in vitro half-lives were performed as described

Membranes were prepared from HEK293 cells expressing human D R,

2

D R, or D R, grown in a 50:50 mix of DMEM and Ham’s F12 culture

3

4

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

7

0

previously. Brieﬂy, for the determination of in vitro half-life (t₁_/₂),

the analyte/IS peak area ratios were converted to percentage drug

remaining, using the T = 0 area ratio values as 100%. The slope of the

linear regression from log percentage remaining versus incubation time

and 5% CO . Upon reaching 80−90% conﬂuence, cells were harvested

2

using premixed Earle’s Balanced Salt Solution (EBSS) with 5 μM

O

DOI: 10.1021/acs.jmedchem.5b00776

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

relationships (−k) was used in the conversion to in vitro t₁_/₂, using the

AUTHOR INFORMATION

■

*

equation t₁= −0.693/k.

/2

Corresponding Author

Phone: (443)-740-2887. Fax: (443)-740-2111. E-mail:

anewman@intra.nida.nih.gov.

Heroin Self-Administration. Animals. Male WT and D KO mice

3

with a C57BL/6J genetic background were bred at the National

Institute on Drug Abuse from three D₃^±breeding pairs purchased

from Jackson Laboratory. This strain of D KO mice expresses a

3

Present Address

For J.Z.: Department of Anesthesiology, Zhongnan Hospital,

truncated D R, including the extracellular N-terminal, the ﬁrst

#

3

intracellular loop, and part of the second intracellular loop (a total

of 148 residues) but lacks downstream sequences from the second

intracellular loop (from residue 149) (Song et al., 2011). Genotyping

was performed in our laboratory in accordance with a protocol of

Charles River Laboratories. All mice used in the present experiments

were matched for age (8−14 wk) and weight (25−35 g). They were

housed individually in a climate-controlled animal colony room on a

reversed light−dark cycle (lights on at 7:00 PM, lights oﬀ at 7:00 AM)

with free access to food and water. All experimental procedures were

conducted in accordance with the National Research Council’s Guide

for the Care and Use of Laboratory Animals and were approved by the

National Institute on Drug Abuse’s Animal Care and Use Committee.

Heroin Self-Administration. Intravenous catheterization surgery

and the self-administration procedures were performed as described

Wuhan University Wuhan, Hubei 430071, China

Author Contributions

⊥

C.A.B. and O.M.B. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Support for this research was provided by the National Institute

on Drug AbuseIntramural Research Program C.A.B., O.M.B.,

J.Z., A.K.B., C.B., E.P., T.M.K., Z.-X.X., and A.H.N.) and the

Johns Hopkins Brain Science Institute (R.R. and B.S.),

DA022413, and MH54137 (P.D. and J.A.J.). We acknowledge

Alessandro Bonifazi and Catherine Schweppe for their technical

assistance with the binding assays and Rachel Slack for a critical

reading of an earlier version of this manuscript.

previously (Xi et al., 2011; Song et al., 2012). Brieﬂy, WT or D KO

3

mice were initially trained for oral sucrose (5% solution) self-

administration for 2−3 days to learn operant lever pressing for sucrose

reward. After lever pressing behavior was achieved, animals were

prepared for iv catheterization surgery. After 5−7 days of recovery

from surgery, each mouse was placed into a test chamber (Med

Associates, St. Albans, VT) and allowed to lever-press for iv heroin,

beginning with 0.1 mg/kg/infusion for a week, and then the heroin

dose was lowered to 0.05 and 0.025 mg/kg/infusion in the following

weeks. Each heroin dose was maintained for 5−7 days until stable self-

administration was achieved. A progressively decreased heroin dose

regimen was chosen based on the fact that total numbers of heroin

infusions at higher doses (0.1 mg/kg/infusion) are usually very low

ABBREVIATIONS USED

■

DA, dopamine; SAR, structure activity relationship; TM,

transmembrane; D R, dopamine D receptor; D R, dopamine

2

3

D₃receptor; D R, dopamine D₄receptor; PET, positron

4

emission tomography; PHNO, (+)-4-propyl-9-hydronaphthox-

azine; WT, wildtype; KO, knockout; FR, ﬁxed ratio; OBS,

orthosteric binding site; SBP, secondary binding pocket; 5-HT,

(∼20). Lowering the heroin dose results in a signiﬁcant increase in the

5

-hydroxytryptamine (serotonin); CMA, chloroform/metha-

numbers of heroin infusions or active lever presses by a compensatory

mechanism that signiﬁcantly increases “system sensitivity” to detect

the eﬀects of experimental drugs on heroin self-administration and

prevent potential bottom eﬀects. Each lever press led to a delivery of

nol/ammonium hydroxide

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High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice

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