Design, synthesis, and structure-activity relationship studies of a series of [4-(4-carboxamidobutyl)]-1-arylpiperazines: Insights into structural features contributing to dopamine D3 versus D2 receptor subtype selectivity

Article

pubs.acs.org/jmc

Open Access on 08/15/2015

Design, Synthesis, and Structure−Activity Relationship Studies of a

Series of [4-(4-Carboxamidobutyl)]-1-arylpiperazines: Insights into

Structural Features Contributing to Dopamine D3 versus D2

Receptor Subtype Selectivity

Subramaniam Ananthan,*^,†Surendra K. Saini,^†Guangyan Zhou,^†,#Judith V. Hobrath,^†

Indira Padmalayam,^‡Ling Zhai,^‡J. Robert Bostwick,^‡Tamara Antonio,^§Maarten E. A. Reith,^§,∥

Shea McDowell,^⊥Eunie Cho,^⊥Leah McAleer,^⊥Michelle Taylor,^⊥and Robert R. Luedtke^⊥

^†Organic Chemistry Department, Southern Research Institute, Birmingham, Alabama 35205, United States

^‡Lead Generation Biology Laboratory, Southern Research Institute, Birmingham, Alabama 35205, United States

^§Department of Psychiatry, New York University School of Medicine, New York, New York 10016, United States

^∥Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016,

United States

^⊥Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard,

Fort Worth, Texas 76107, United States

S

* Supporting Information

ABSTRACT: Antagonist and partial agonist modulators of the dopamine D3 receptor

(D3R) have emerged as promising therapeutics for the treatment of substance abuse

and neuropsychiatric disorders. However, development of druglike lead compounds

with selectivity for the D3 receptor has been challenging because of the high sequence

homology between the D3R and the dopamine D2 receptor (D2R). In this eﬀort, we

synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and

evaluated their binding and functional activities at the D3 and D2 receptors. Docking

studies and results from evaluations against a set of chimeric and mutant receptors

suggest that interactions at the extracellular end of TM7 contribute to the D3R versus

D2R selectivity of these ligands. Molecular insights from this study could potentially

enable rational design of potent and selective D3R ligands.

amine polydrug abusers.¹²Preclinical studies with a number of

D3R antagonist or partial agonist ligands, such as those shown

in Figure 1 (1−5), have demonstrated that D3R ligands can

INTRODUCTION

■

Dysregulation of the dopaminergic system is implicated in

several pathological conditions including schizophrenia, Par-

eﬀectively suppress motivation to self-administer drugs and

kinson’s disease, depression, and addiction. Dopaminergic

prevent drug-associated cue-induced craving and relapse to

signaling is mediated through two types of receptors termed

D1-like (D1, D5) and D2-like (D2, D3, D4) receptors. Among

drug taking.¹³⁻²⁰

In addition, several lines of evidence indicate that D3

the various approaches, targeting the dopamine D3 receptor

receptors play an important role in the pathophysiology of

(D3R) with antagonist or partial agonist ligands has emerged as

schizophrenia.²¹Elevated levels of D3R expression in the

a promising area for the development of medications for the

treatment of substance abuse and neuropsychiatric disorders.^1,2

mesolimbic regions of the brain of schizophrenic patients have

been demonstrated.²²Overexpression of D3R has been

The D3 dopamine receptor subtype is expressed primarily in

proposed to be responsible for the sensitization to dopamine

mesolimbic regions of the brain including the nucleus

agonists. Inhibition of D3R function may, therefore, attenuate

accumbens and has been implicated in the pathophysiology

of drug addiction.³Studies in animal models have demonstrated

positive symptoms associated with schizophrenia without

causing the extrapyramidal side eﬀects associated with classical

that D3R activation is involved in the reinforcing and

motivational eﬀects of cocaine.⁴⁻⁹Long-term exposure to

D2R antagonists. Moreover, D3R antagonists have been shown

to enhance D3 receptor mediated release of acetylcholine in the

cocaine results in up-regulation of D3 receptors as demon-

strated in post-mortem studies of cocaine-overdose fatal-

ities.^10,11Positron emission tomography (PET) studies have

Received: May 23, 2014

also shown an up-regulation of D3R over D2R in methamphet-

A

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Structures of dopamine D3 receptor selective ligands.

frontal cortex and, therefore, may have beneﬁcial eﬀects on

attention and memory loss associated with schizophrenia.²¹

Indeed, studies with D3R selective or D3R preferring

antagonists have conﬁrmed their eﬃcacy as antipsychotic and

procognitive agents.²³⁻²⁷

In the design and development of novel D3R ligands, a

primary challenge is achieving a high degree of selectivity for

D3R over the highly homologous D2R for ligands with druglike

characteristics. These issues, as well as the progress made in the

development of D3R selective ligands, have been the subject of

several reviews.²⁸⁻³⁴In the search for novel D3R selective

ligands, compounds possessing a 4-phenylpiperazine tethered

to an amide via a four-carbon linker such as that found in

structures 1 and 3 have emerged as a particularly promising

group of ligands. Previous structure−activity relationship

(SAR) studies on this class of compounds, generically

represented in Figure 2, have elucidated the importance of

contribute to D3R selectivity of the ligands that occupy this

site.^40,41The head group of arylpiperazine class of ligands is

accommodated in this region. Hydrophobic substituents on the

head groups could potentially explore diﬀerences in the

residues Val350 (D3R)/Ile365 (D2R) and Thr353 (D3R)/

Ile368 (D2R) at this orthosteric binding site. Therefore, we

pursued a series of ligands possessing various aryl or heteroaryl

head groups with nonpolar aliphatic substituents that may

explore this region (Table 1). In an eﬀort to keep the overall

lipophilicity low, in this series of ligands (6−25) we

incorporated imidazo[1,2-a]pyridine as the tail group.

The use of a heteroaryl functionality such as 2-

indolyl-^{35,36,42−44}and 2-benzofuranyl^{16,35,42−47}as tail groups

has been shown to yield ligands with selectivity for D3 versus

D2 receptors. Recent studies have implicated interaction of

these substituents at a secondary binding pocket (SBP) near

the transmembrane helices 1, 2, and 7 and ELI and ELII as

contributing to the D3R selectivity of such ligands.^38,39To gain

insight into such selectivity conferring tail group interactions,

we pursued another series of compounds possessing isosteric

heterocyclic systems in place of imidazo[1,2-a]pyridine (26−

31). Additional analogues were designed to investigate

potential salt bridge/polar interactions (32−36) and hydro-

phobic interactions (37−39) of the tail group (Table 2). The

results and insights gained from the synthesis, receptor binding,

and molecular modeling studies are presented herein.

RESULTS AND DISCUSSION

■

Synthesis. The target compounds 6−25 listed in Table 1

and 26−34 and 37−39 listed in Table 2 were synthesized using

a general procedure involving the coupling of a carboxylic acid

with the appropriate aminobutylpiperazine, as depicted in

Scheme 1. The coupling of the amine with the acids was

performed using either BOP-Cl or HATU as the coupling

agent. The desired aminobutylpiperazine intermediates 43 were

prepared from the corresponding piperazines 41 by alkylation

with bromobutylphthalimide 40 followed by deprotection of

the resulting intermediate 42 with hydrazine hydrate.

Figure 2. Schematic representation of the generic pharmacophore for

the acylaminobutylpiperazine class of ligands.

the length and composition of the linker, the carboxamide

function, the substituent group on the piperazine (referred to as

the “head group”), and the substituent group on the amide

moiety (referred to as the “tail group”) in modulating the

aﬃnity and intrinsic activity of this class of compounds.³⁵⁻³⁸

Structural comparisons of the D3R crystal structure³⁹and

D2R homology model as well as docking studies suggest that a

putative orthosteric binding site near transmembrane helices

(TM) 5 and 6 and part of extracellular loop II (ELII) may

The 5-hydroxymethyl and 5-(dimethylaminomethyl) com-

pounds 35 and 36 were synthesized as shown in Scheme 2

starting with 2-amino-6-hydroxymethylpyridine (44).

B

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Binding Aﬃnity of Imidazo[1,2-a]pyridinecarboxamides with Head Group Variations

a

b

Partition coeﬃcients (CLogP) was calculated using ChemBioOﬃce Ultra 2010. Displacement of [¹²⁵I]IABN from HEK cell membranes stably

c

expressing human D3R. Displacement of [¹²⁵I]IABN from HEK cell membranes stably expressing human D2_LR. K_ivalues are the mean SEM

from three or more independent experiments. D2R K_i/D3R K_i.

d

Binding Aﬃnities of Head Group Variants. The aﬃnity

of the compounds at D3 and D2 receptors were determined

using previously established displacement binding assays using

substituted phenyl groups, displayed K_ivalues of <10 nM at

D3R with D2R/D3R selectivity ratio in the range of 5.4- to 56-

fold. Compared to the unsubstituted phenylpiperazine

compound 6, the 2-methoxyphenylpiperazine compound 7

displayed ∼3-fold enhancement in aﬃnity at the D3R.

However, it also displayed nearly 10-fold higher aﬃnity at

D2R, thus resulting in a reduction of D2R/D3R selectivity. The

[

¹²⁵I]IABN as the radioligand and membrane preparations from

HEK cells stably expressing human D3 or D2_Lreceptors.^43,48

The data for compounds containing head group variations are

shown in Table 1. Compounds 6−10, possessing phenyl and

C

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

highest D3R aﬃnity (in the subnanomolar range) was displayed

by compound 8, the 2,3-dichlorophenylpiperazine analogue.

Earlier studies have shown that the eﬀect of 2-methoxyphenyl

and 2,3-dichlorophenyl substituents on binding aﬃnity and

selectivity varies depending upon the linker and the nature of

the arylamide moiety.^47,49The improved aﬃnity and selectivity

of compound 8 compared to compound 7, however, are

associated with an increase in lipophilicity (CLogP of 3.6 for 7

vs 5.1 for 8). Interestingly, the introduction of a triﬂuoromethyl

group at the 3-position of the phenyl ring of compound 6 led to

an increase in aﬃnity at D3R without signiﬁcantly aﬀecting

aﬃnity at D2R, thus improving the binding selectivity

(compound 9 D2R K_i/D3R K_i= 56). In addition, the

introduction of an electron withdrawing cyano substituent at

the 5-position (compound 10) led to a modest decrease in

aﬃnity at D3R and D2R as well as a decrease in D3R selectivity.

The introduction of a tert-butyl group at the 3- and 5-position

gave compound 11, which displayed signiﬁcant reductions in

aﬃnity at both the D3R and the D2R. Incorporation of a

bicyclic heterocyclic functionality, such as 4-quinolinyl, 2-

quinolinyl, and 4-quinazolinyl rings, led to compounds (12−

18) with lower aﬃnities at both D3R and D2R. Whereas the

unsubstituted 4-quinolinyl compound 12 and its 7-chloro

analogue 15 displayed moderate aﬃnity at D3R, the 2-

triﬂuoromethyl and 7-triﬂuoromethyl analogues 13 and 14

had much lower aﬃnity at D3R.

Table 2. Binding Aﬃnity of 2-tert-Butyl-6-

triﬂuoromethylpyrimidines with Tail Group Variations

Further, a series of compounds were prepared in which

various substituents were introduced at the 2- and 6-position of

the pyrimidine ring. The isomeric methyl-triﬂuoromethyl

pyrimidines 19 and 20 displayed low aﬃnities at the D3R.

However, the isomeric methyl-tert-butyl compounds 21 and 22

displayed interesting diﬀerences in their binding proﬁles at D3R

and D2R. For example, the 2-methyl-6-tert-butyl isomer 21 had

greatly reduced aﬃnity at both receptors, while the 2-tert-butyl-

6-methyl isomer 22 had K_i< 10 nM at D3R with nearly a 40-

fold selectivity over D2R. Incorporation of a bulky tert-butyl

group at the 2- and 6-position resulted in compound 23 with

lower aﬃnity (D3R K_i= 19 nM) compared to the compound

24 (D3R K_i= 8.4 nM) possessing a tert-butyl and cyclopropyl

group at the 2- and 6-position, respectively. Among the

pyrimidinyl variants containing a tert-butyl group at the 2-

position (compounds 22−25), the most dramatic eﬀect was

observed with the 6-triﬂuoromethyl compound 25, which was

the most potent (D3R K_i= 4.2 nM) and D3R-selective (D2R

K_i/D3R K_iratio =122) compound among the 4-pyrimidinylpi-

perazines. The improved aﬃnity and selectivity of the 2-tert-

butyl compound 25 as compared to the 2-methyl compound 19

a

Partition coeﬃcients (CLogP) was calculated using ChemBioOﬃce

b

Ultra 2010. Displacement of [¹²⁵I]IABN from HEK cell membranes

c

stably expressing human D3R. Displacement of [¹²⁵I]IABN from

HEK cell membranes stably expressing human D2_LR. K_ivalues are the

mean SEM from three or more independent experiments. D2R K_i/

D3R K_i.

d

a

Scheme 1. General Synthesis of Target Compounds

a

Reagents and conditions: (a) K₂CO₃, CH₃CN, reﬂux; (b) N₂H₄·H₂O, MeOH, reﬂux; (c) R′CO₂H, BOP-Cl, Et₃N, CH₂Cl₂, rt or R′CO₂H, HATU,

Et₃N, CH₃CN, rt.

D

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 2. Synthesis of Target Compounds 35 and 36

a

Reagents and conditions: (a) BrCH₂COCO₂Et, EtOH, reﬂux, 3 h (70%); (b) TIPSCl, imidazole, DMF, 70 °C, 2 h (72%); (c) (i) NaOH, MeOH−

THF−H₂O, 2 h, (ii) AcOH, 81%; (d) amine [43, R = 2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl], HATU, Et₃N, CH₃CN, rt, 16 h (22%); (e)

Et₄NF, THF, rt, 16h (62%); (f) (i) MeSO₂Cl, Et₃N, CH₂Cl₂, 1 h, (ii) Me₂NH, THF, rt, 6 h (23%).

highlight the importance of the tert-butyl substituent at the 2-

position of the pyrimidine ring for D3R aﬃnity and selectivity.

To gain insight into the role of tert-butyl substituents in

inﬂuencing binding aﬃnity and D2R/D3R selectivity, we

performed a retrospective analysis of the binding modes of

compounds 19−25.

The docked poses of these compounds display a preference

for two opposite orientations of the pyrimidine ring. The

docked poses of compounds that exemplify these opposite

orientations are shown in Figure 3 for compounds 21 and 22

the more favorable orientation. Binding aﬃnity diﬀerences

between the isomers 21 and 22 or between isomers 19 and 20

support this notion that the “e” orientation is the more

favorable of the two. The docked pose of compound 25 (Figure

4 and Figure S2 in Supporting Information) is very similar to

that obtained for compound 22 with the pyrimidine ring having

the “e” orientation. In this orientation, the triﬂuoromethyl

group forms polar interactions with Thr115 and Ser196 while

the tert-butyl group participates in favorable hydrophobic

interactions with Ile183 (ELII), Val189 (TM5), Val350 (TM6)

in D3R corresponding to Ile184, Val190, and Ile365 in D2R.

This interaction may contribute to the binding potency and

D3R selectivity of compound 25 for the following reasons. (1)

The D2R side chain of residue Ile365 exposed in this pocket is

bulkier than the corresponding V350 residue in the D3R,

contributing to a smaller available space in D2R than in the

D3R. (2) Val190 of D2R participates in an interhelical

interaction with Ile368. Such an interaction is absent in D3R

where the residue corresponding to Ile368 is Thr353 which

hydrogen-bonds to the helical backbone of TM6. This

diﬀerence likely aﬀects the interhelical packing between the

extracellular ends of TM5 and TM6 and may contribute to

D3R/D2R subtype selectivity of ligands.

Binding Aﬃnities of Tail Group Variants. The binding

aﬃnities of this series of compounds are listed in Table 2.

Replacement of the imidazo[1,2-a]pyridine group in compound

25 with a 2-indolyl moiety gave compound 26 that had lower

binding potency at both D3R and D2R (2.6-fold and 1.8-fold,

respectively) and was less selective for D3R (122-fold vs 83-

fold). The 2-benzimidazole compound 27 had D3R binding

potency comparable to that of compound 25 but with reduced

D3R selectivity. The incorporation of a 2-pyridothienyl group

gave compound 28 with reduced binding aﬃnity at the D3R.

Introduction of an additional nitrogen atom in the imidazopyr-

idine system gave the imidazopyrimidine (29), imidazopyrazine

(30), and imidazopyridazine (31) analogues, all of which

showed slightly reduced D3R binding aﬃnity and D3R

selectivity. The amino, dimethylaminomethyl, and hydroxy-

methyl compounds 32−36, designed to explore potential polar

Figure 3. Docked poses of compounds 21 and 22 at the D3 receptor

are illustrated. For clarity, only the side chain orientations of the

reﬁned D3 crystal structure used for docking are shown. Atoms are

colored by atom type (C, light yellow; O, red; N, blue) except for

ligand carbon atoms as shown. The region of the pyrimidine ring

relevant to the “e” and “b” orientations is circled.

and in Figure S1 (Supporting Information) for compounds 19

and 20. In the binding mode of compound 21, the nitrogen at

the 3-position of the pyrimidine ring is buried facing the

intracellular side which we deﬁned as the “b” (buried)

orientation as opposed to the “e” (exposed) orientation

shown for compound 22. Analogously, compound 19 has “b”

as opposed to “e” orientation in compound 20. The “e”

orientation exposes a more polar surface of the pyrimidine ring

to the aqueous environment than “b”, and it is expected to be

E

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. (A) Docked pose of compound 25 in the D3R binding site. Residues forming favorable interactions with the ligand are shown. Atoms are

colored by atom type (C, light yellow; O, red; N, blue) except ligand carbon atoms are colored orange. TM1 and TM5−7 helices are displayed as

ribbons. (B) Schematic representation of the interactions between compound 25 and D3R residues. Polar interactions are indicated with dashed

lines and nonpolar/steric interactions with gray contour lines.

Table 3. Aﬃnity of Selected Compounds at Human Dopamine D2R, D3R, Chimeric, and Mutant Receptors

a

K_iSEM (nM)

b

c

d

e

f

compd

D3R wild type

D2R wild type

D3R-D2R ELII

D2R-D3R ELII

D3R (S182I)

D3R (E90A)

D3R (E90Q)

25

34

36

8.4 0.8 (3)

9.1 3.0 (4)

16 5 (4)

702 167 (3)

191 5 (3)

245 30 (3)

40 9 (3)

27 6 (3)

44 7 (3)

148 10 (3)

72 8 (3)

12 2 (3)

16 2 (3)

24 5 (3)

13 3 (3)

16 5 (4)

12 4 (4)

20 4 (3)

19 3 (3)

12 2 (3)

176 25 (3)

a

Binding aﬃnities derived from competition binding experiments using [¹²⁵I]IABN as the radioligand. The number of independent experiments is

b

c

shown in parentheses. Chimeric D3 receptor possessing extracellular loop II of D2 receptor. Chimeric D2 receptor possessing extracellular loop II

of D3 receptor. D3 receptor with serine 182 mutated to isoleucine. D3 receptor with glutamate 90 mutated to alanine. D3 receptor with glutamate

90 mutated to glutamine.

d

e

f

and salt bridge interactions by these substituents, displayed

moderate binding aﬃnity at D3R with K_ivalues in the range of

25−45 nM and diminished (<21-fold) D3R selectivity.

with Glu90 (D3R)/Glu95 (D2R) or Glu181 (D2R). Binding

aﬃnities of these analogues and lead compound 25 were

evaluated at the wild type receptors and a set of chimeric and

single-point mutant receptors to test for interactions predicted

by these alternative binding modes.

Previous studies have suggested that the occupation of a

secondary binding pocket formed by transmembrane helices

TM1, -2, -7 and extracellular loops ELI and ELII by tail groups

of aminobutylpiperazines contributes to the D3R selectivity.³⁸

Prior to the determination of the D3R crystal structure,

Geneste and co-workers docked a ligand series related to

compound 25 into a D3R model and predicted the positioning

of the amide and tail groups near TM7 to form interactions

with Thr368.⁵⁰⁻⁵²While our docking results predicted a single

preferred orientation for the head group region of compound

25 and its analogues, we found that the arylamide tail group

may adopt three distinct possible orientations within the

secondary pocket formed by transmembrane helices TM1, -2,

-7 and extracellular loops ELI and ELII at D2R and D3R. In

one of these possible orientations the tail group is in proximity

to Glu90 (D3R)/Glu95 (D2R), while a second orientation

places the tail group near the ELII loop residues Val180

(D3R)/Glu181 (D2R) and Ser182 (D3R)/Ile183 (D2R). In

the third possible orientation the tail group forms aromatic

interactions with Tyr365 (D3R)/Tyr379 (D2R) while its

amide group hydrogen-bonds with Thr369 (D3R)/Thr383

(D2R). In order to gain insight as to which might be the most

likely placement of the imidazo[1,2-a]pyridine tail group of

compound 25 and its analogues in the secondary binding

pocket, we designed the two analogues 34 and 36 containing a

charged amine substituent introduced at distinct positions of

the imidazo[1,2-a]pyridine, which could potentially interact

Binding aﬃnities of compound 25 and the designed

analogues 34 and 36 at chimeric receptors D3/D2 ELII

(human D3 receptor with the D2 ELII loop), D2/D3 ELII

(human D2 receptor with the D3 ELII loop) and the single

point D3 receptor mutants Glu90Ala, Glu90Gln, Ser182Ile are

listed in Table 3. All three compounds displayed only a

moderate change in aﬃnities between the wild type D3R and

D3R-D2R ELII, suggesting the lack of signiﬁcant stabilizing

interactions between the positively charged tail groups of

compounds 34 and 36 with Glu181 in D3/D2 ELII and, by

extension, in the D2R. This is further corroborated by the

observation that, compared to aﬃnity at wild type D2R, these

compounds show slight improvement in aﬃnity at the D2/D3

ELII chimeric receptor that has a valine substitution for

Glu181. Furthermore, compounds 25, 34, and 36 did not

display a marked change in their binding aﬃnities at Glu90Ala,

Glu90Gln, Ser182Ile compared to the wild type D3R,

indicating the lack of signiﬁcantly stabilizing salt bridge or

polar interactions between the positively charged amine

substituent of compounds 34 and 36 with Glu90 or Ser182

of D3R.

These results suggest that the side chains of Glu90 (D3)/

Glu95 (D2) or Glu181 (D2)/Val180 (D3) do not signiﬁcantly

contribute to the binding of the arylamide tail groups of

compounds 25, 34, and 36. In docked poses, the tail group

F

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 5. (A) Docked poses of compounds 34 and 36 at the D3R crystal structure. D3R residues are shown with light yellow colored carbons and

underlined residue numbers. D2R residues with sequence diﬀerences from D3R are shown with carbons colored gray. Amino acids included in

mutation experiments and discussed in text are also shown. Ligand carbon atoms are in cyan for 34 and orange for 36. Fluorine atoms are colored

dark green. All other atoms are colored by atom type. (B) Schematic representation of the tail region of the poses shown in (A). Polar interactions

are illustrated with dashed lines, nonpolar/steric interactions with gray contour lines.

orientation that is consistent with these results is shown in

Figure 5. The protonated dimethylamino nitrogen in the

docked poses of compounds 34 and 36 are too distant (6.4 and

8.7 Å, respectively) to engage in salt bridge interactions with

the carboxylate group of Glu90 of the D3R. Binding

interactions of compounds 34 and 36 include hydrogen

bonding between the amide and Thr369 in TM7. The distance

between the tail group’s amide NH of compounds 34 and 36

and the Thr369 side chain oxygen are 2.4 and 2.9 Å,

respectively. Thr369 also hydrogen-bonds with a ring nitrogen

of the imidazo[1,2-a]pyridine in compounds 34 and 36 (heavy

atom distances of 3.2 and 3.1 Å, respectively). These hydrogen

bonds may play a role in the positioning of the imidazopyridine

ring for favorable aromatic interactions with Tyr365 in D3R

(Tyr379 in D2R) in TM7.

In order to test the hypothesis that tail groups engage in

aromatic interactions as predicted by our docked poses, we

designed and evaluated binding aﬃnities of compounds 37−39

Figure 6. Docked pose of compound 39 in the D3R binding site.

(Table 2). Whereas the piperidine-4-carboxamide analogues 37

Residues forming favorable interactions with the ligand are shown.

Atoms are colored by atom type. Fluorine atoms are colored dark

green. Ligand carbon atoms are shown in orange.

and 38 displayed only moderate aﬃnity, the cyclohexane-

carboxamide analogue 39 displayed single digit nanomolar

aﬃnity at D3R. Interestingly, its aﬃnity at D2R was markedly

diminished, thus providing a ligand with >150-fold selectivity

for D3R over D2R. Docking results of compound 39 at D3R

and D2R predict aromatic−aromatic interactions between the

phenyl ring of the 4-phenylcyclohexanecarboxamide group of

compound 39 and the Tyr365 D3R side chain (Figure 6)

corresponding to Tyr379 in D2R. Sequence and structural

comparison between the D3R crystal structure and D2R model

suggests that interactions with this tyrosine in TM7 may

contribute to D3R selectivity of ligands as follows. The

nonconservative amino acid diﬀerence Thr368 (D3R)/Phe382

(D2R) within the interhelical region of TM6TM7 likely

aﬀects interhelical packing near the extracellular end of these

helices leading to structural diﬀerences between D2R and D3R.

Another sequence diﬀerence at the extracellular end of TM7, at

its TM1 interface, is Tyr36 in D3R corresponding to Leu41 in

D2R. Furthermore, the region of the TM7TM1 interface is

rich in sequence diﬀerence amino acids. TM1 is the least

conserved helix, only 65% identical between D2R, D3R

sequences as compared to overall 78% identity within the

seven transmembrane helical region. These diﬀerences may

position the extracellular end of TM7 closer to TM1 and more

distant from TM6 in the case of the D2R compared to D3R.

The D3R selectivity of ligands, therefore, may arise from direct

interactions with residues near the extracellular end of TM7

such as Tyr365 (D3R)/Tyr379 (D2R). Indeed, it has been

suggested that subtle diﬀerences in relative positioning of TM1

and TM7 helices in D2R and D3R may contribute to selectivity

even if the ligands interact with the same amino acid side chains

in D2R and D3R.³⁹Consistent with this notion, docked poses

of ligands with high D3R selectivity (e.g., 25−27 and 39)

display interactions between their pendent aryl group and the

tyrosine residue at the extracellular end of TM7 in both D3R

and D2R. In a recent study, mutagenesis results coupled with

docking and molecular dynamics simulations led to the

identiﬁcation of Gly94 (D3R) in the ELI loop as an important

contributor to D3R/D2R selectivity of ligands through its

ability to modulate the size and shape of the secondary binding

G

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 4. Eﬃcacy of Selected Ligands in Mitogenesis Assay

agonist activity

antagonist activity

a

b

c

compd

D3R % stimulation

D2R % stimulation

D3R IC₅₀(nM)

D2R IC₅₀(nM)

26

selectivity ratio

7

6.4

<20

3.0 1.3

7.2 3.3

120 22

940 150

157 34

208 74

51 21

8

8.7

7.6

8

<20

0

55 16

d

e

12

17

25

27

29

30

31

nd

na

d

e

nd

0

2300 1100

490 150

205 48

15

<20

<1

<20

<1

<20

<1

2.4

4.0

1.7

245 33

637 99

420 120

1160 110

1.8

a

b

Percent stimulation at 10 μM normalized to the maximal stimulation by quinpirole. Results are from two independent experiments. Inhibition

potency against stimulation of mitogenesis by standard agonist quinpirole (30 nM). IC₅₀values are mean SEM from at least three independent

c

d

experiments, each conducted with duplicate determinations. (D2R IC₅₀)/(D3R IC₅₀). Not determined because of weak binding aﬃnity at D2R (K_i

> 500 nM). Not applicable.

e

pocket.⁵³The ELI loop in D3R contains two glycine residues

Table 5. Eﬃcacy of Selected Ligands in Adenylyl Cyclase

(Gly93, Gly94), while this loop in D2R is one residue shorter

containing one glycine residue (Gly98). Interestingly, in the

docked poses of our ligands, Gly94 is in proximity to tail

groups, for example, in the case of compound 25 (Figure S2 in

Supporting Information), the α-carbon of Gly94 is at 3.4 Å

distance from the closest heavy atom in the imidazopyridine

ring of this ligand docked at D3R.

Assay

a

compd

D3R % inhibition

44

66 13

D2R % inhibition

7

6

25

45

32

5

1

4

8

9

70

50

4

6

12

17

25

26

27

29

30

31

34

39

1.4 6.7

19

70 18

6

Functional Activity. In an eﬀort to assess the intrinsic

activity proﬁle of the compounds, functional activity evaluations

were performed using assays that measure various end points

such as the mitogenesis assay, the cyclase assay, β-arrestin

recruitment assay, and the GTPγS binding assay. In the

mitogenesis assay using CHOp cells expressing human D3 and

D2 dopamine receptors, the evaluated compounds did not

display appreciable intrinsic eﬃcacy at D3 or D2 receptors

(Table 4).⁵⁴The maximum stimulation of [³H]thymidine

incorporation at a ligand concentration of 10 μM was <20% of

the full agonist quinpirole. These compounds displayed varying

antagonist potencies in inhibiting mitogenesis induced by the

agonist quinpirole at the D3R and D2R. With the exception of

compound 7 which displayed D3R antagonist IC₅₀value (3.0

nM) similar to its D3R binding K_ivalue (3.5 nM), the D3R

antagonist potencies of all of the evaluated compounds were

weaker than their binding aﬃnities at the D3R. The D2R/D3R

antagonist selectivity of the compounds in the mitogenesis

assay was also much lower than the selectivity in the binding

assay. The pyrimidinyl compound 25 displayed moderate

antagonist potency with IC₅₀of 157 nM at D3R. However, it

displayed 15-fold D3R antagonist selectivity due to its much

weaker potency at D2R.

Selected compounds were evaluated in the adenylyl cyclase

assay which measures the ability of the compounds to inhibit

forskolin-dependent stimulation of adenylyl cyclase activity.^43,48

The evaluations were carried out using HEK 293 cells

expressing human D3 and D2 dopamine receptors (Table 5).

In the adenylyl cyclase inhibition assay, all of the evaluated

compounds displayed a partial agonist proﬁle at D3R with

intrinsic eﬃcacies ranging from 12% (compound 27) to 70%

(compounds 9 and 17). Interestingly, among the compounds

possessing identical arylamide groups (7−9, 12, 17, and 25),

compound 25, which has a 2-tert-butyl-6-triﬂuoromethyl-

pyrimidinyl head group, displayed lower eﬃcacy at the D3R

(28%) than compounds possessing substituted phenyl, 4-

quinolinyl, and 4-quinazolinyl head groups. Among the

28

38

7

4

−1.9 13

b

nd

12 11

51 10

5.6 4.1

9.2 7.3

b

38

32

47

5

7

4

nd

b

nd

8.9 5.0

b

52 13

nd

a

Percent inhibition values were normalized to the percent inhibition of

the full agonist quinpirole at D3R (100 nM) and D2R (1 μM). For D3

receptors the maximum inhibition by quinpirole ranged from 38% to

53%, and for D2 receptors the maximum inhibition was >90%. The

test compounds were used at a concentration equal to approximately

10 times the K_ivalue in the radioligand binding assays. The values are

the mean SEM from three or more independent experiments. Not

determined because of insolubility of the compounds at 10 times their

binding K_ivalues at D2R.

b

compounds possessing a 2-tert-butyl-6-triﬂuoromethyl-

pyrimidinyl head group (25−27, 29−31, 34, and 39), the

benzimidazole-2-carboxamide 27 displayed the lowest eﬃcacy

at the D3R (12%).

Binding of agonists at D3R and D2R is known to lead to the

recruitment of β-arrestin to the receptor. Selected compounds

were evaluated for their activity on β-arrestin-2 recruitment

using a cell-based receptor/β-arrestin interaction assay

(DiscoveRx PathHunter).⁵⁵In this assay, the interaction of β-

arrestin with a GPCR is monitored using β-galactosidase (β-

gal) enzyme fragment complementation. Activation of the

receptor by an agonist results in the translocation of β-arrestin

to active receptor, which leads to the formation of an active β-

galactosidase enzyme. The activity of β-galactosidase is then

measured by addition of chemiluminescent detection reagents.

These assays were performed according to manufacturer’s

protocol using CHO-K1 cells expressing human D2_Lreceptors

and U2OS cells expressing human D3 receptors. Intrinsic

agonist or inverse agonist activity of the compounds was

H

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

determined by evaluating the ability of the compounds to

stimulate or inhibit basal activity. In these evaluations, all of the

tested compounds (Table 6) were found to be devoid of

25, 30, and 39 were selected for evaluation in the [³⁵S]GTPγS

binding functional assays using CHO cell lines expressing

human D3R and D2R.⁵⁶⁻⁵⁸These compounds did not

stimulate [³⁵S]GTPγS binding, and up to 1 μM concentration

these compounds caused less than 30% inhibition of basal

binding at D2R and D3R. Compounds 25, 30, and 39 at 10 μM

caused inhibition of 40−60% of D3R basal [³⁵S]GTPγS

binding, indicating some inverse agonist activity which did

not appear pharmacologically relevant compared with their

binding aﬃnity assessed with [¹²⁵I]IABN. The potencies of

these compounds as antagonists were then determined using

the “shift” experiments⁵⁹to obtain antagonist K_evalues based

on their ability to reduce the apparent potency of dopamine in

stimulating [³⁵S]GTPγS binding in CHO-D2R and CHO-D3R

cells (Table 7). For compound 39, the antagonist potency at

Table 6. Antagonist Potencies of Selected Ligands in the β-

Arrestin-2 Recruitment Assay

D3R

IC₅₀SEM, nM

D2R

IC₅₀SEM, nM

selectivity

a

b

c

compd

ratio

7

8

9

0.7 0.1

0.2 0.1

1.8 0.8

6.6 5.1

9.4

455

33

91

7

60 12

161 78

248 63

261 54

418 80

301 50

37 17

49 12

22 15

311 53

217 10

281 43

>500

12

52

7

3.1

0.6

435

75

17

409 25

0.6 0.1

5.6 1.8

8.5 2.1

0.4 0.2

0.3 0.1

25

26

27

35

Table 7. Antagonist Potencies of Selected Compounds in

29

93

[³⁵S]GTPγS Binding Assay

30

163

73

a

b

31

compd CHO-hD3 K_e(nM)

CHO-hD2 K_e(nM)

selectivity ratio

33

14

7.2 3.7

29

3

22

8

0.0038 0.0013

1.3 0.4

1.1 0.2

111 25

289

85

34

30

25

30

39

36

3

9.7

>73

2.7

0.33 0.05

25

c

6

76

39

6.8 1.8

c

19

4

na

haloperidol

0.06 0.02

0.16 0.02

a

The antagonist K_evalue was calculated from “shift” experiments as

a

IC₅₀values for inhibition of (+)-PD128907-induced arrestin

described in Experimental Section. Dopamine dose−response curves

were generated in the absence or presence of compound 8 (10 nM for

D2R; 0.1 nM for D3R), compound 25 or 39 (1 μM for D2R; 100 nM

for D3R), or compound 30 (50 nM for D2R; 1 nM for D3R). The

ﬁxed concentrations of drug were chosen as being high enough to shift

the dopamine stimulation curves to the right but low enough to not

appreciably inhibit [³⁵S]GTPγS binding below baseline based on the

inverse agonist activity. The K_ewas calculated from the increase in

ED₅₀observed with test compound (see Experimental Section).

b

translocation in U2OS cells expressing D3R. IC₅₀values for inhibition

of pergolide-induced arrestin translocation in CHO-K1 cells expressing

D2_LR. (D2R IC₅₀)/(D3R IC₅₀).

c

agonist activity and displayed very weak inverse agonist activity

at both D3R and D2R. The maximum inhibition of the basal

activity observed was <21% (compound 33) at D3R and <30%

(compound 34) at D2R at 500 nM concentration of the test

compounds. The antagonist potency of the compounds was

assessed by determining the ability of these compounds to

inhibit the agonist activity of ligands PD128907 and pergolide

at D3R and D2R, respectively. Haloperidol was included as a

standard antagonist ligand. The results are presented in Table

6.

Results are the mean

SEM for three to four independent

b

experiments assayed in triplicate. (CHO-hD2 K_e)/(CHO-hD3 K_e).

c

Measurement could not be made with shift protocol: Concentrations

of drug that by themselves did not appreciably inhibit binding below

baseline did not substantially shift the dopamine curve to the right.

Most of the evaluated compounds displayed potent (<1.0

nM) to moderately potent (>1.0 nM) antagonist activity in

inhibiting agonist stimulated recruitment of β-arrestin-2 by D3

receptors. Compounds that displayed subnanomolar antagonist

potency at D3R include 7, 8, 25, 29−31. The antagonist

potency of the compounds at the D3R, in general, correlates

with their binding aﬃnities (Pearson’s correlation coeﬃcient

between binding pK_iand antagonist pIC₅₀, r = 0.77; P = 0.008).

Similar to their binding aﬃnity at D2R, the antagonist potency

of these ligands at D2R was weaker than at D3R. The binding

aﬃnity and antagonist potency at D2R, however, was not as

well correlated, as they were at D3R. In this assay, compounds

8 and 25, both possessing the imidazo[1,2-a]pyridine tail

group, with the 2,3-dichlorophenyl (8) or 2-tert-butyl-6-

triﬂuoromethyl-4-pyrimidinyl (25) head group emerged as

high potency ligands with very high (>400-fold) D3R

selectivity. A comparison of the proﬁle of compound 25 with

compound 26 indicates that incorporation of the imidazo[1,2-

a]pyridine-2-carboxamide group (25) provides a more

favorable D3R binding and D3R antagonist functional

selectivity than the indole-2-carboxamide group (26).

D2R could not be determined in shift experiments, as drug

concentrations substantially higher than 1 μM were required to

shift the dopamine curve; such high concentrations had inverse

agonist activity invalidating the shift method.

Among the compounds possessing the 2-tert-butyl-6-

triﬂuoromethylpyrimidinylpiperazine head group (compounds

25, 30, and 39), compounds 25 and 39 displayed D3R

antagonist potency somewhat similar to their binding potency.

The antagonist potency of imidazopyrazine compound 30 was

moderately higher at D3R and D2R compared to its binding

aﬃnities at these receptors. Interestingly, compound 8 which

contains the 2,3-dichlorphenylpiperazinyl head group displayed

extraordinary potency at D3R in the picomolar range.

Increasing evidence indicates that GPCR ligands including

D3R and D2R ligands can display diﬀering intrinsic eﬃcacy

and/or potency for diﬀerent signaling pathways linked to the

same receptor.^48,60−65These types of eﬀects have been referred

to by various terms which include stimulus traﬃcking,

functional selectivity, collateral eﬃcacy, and biased agonism.

The manifestation of functional selectivity in our series of

compounds is exempliﬁed by the functional activity proﬁle of

compounds 8, 25, and 30. At D3R, these compounds behave as

antagonists in the [³⁵S]GTPγS binding, β-arrestin-2 recruit-

On the basis of the relatively high D3 over D2 antagonist

selectivity in the β-arrestin-2 recruitment assay, compounds 8,

I

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Bruker BIOTOF II in electrospray ionization (ESI) mode. Elemental

analyses were performed by Atlantic Microlab, Inc. (Atlanta, GA) or

by the Spectroscopic and Analytical Laboratory of Southern Research

Institute. Analytical results indicated by elemental symbols were within

0.4% of the theoretical values. Thin layer chromatography (TLC)

was performed on Analtech silica gel GF 0.25 mm plates. Flash column

chromatography was performed with E. Merck silica gel 60 (230−400

mesh). Yields are of puriﬁed compounds and were not optimized. On

the basis of NMR and combustion analysis data, all ﬁnal compounds

reported in the manuscript are >95% pure.

General Amidation Procedures. Procedure A. To a solution of

the carboxylic acid (1 equiv, 1 mmol) in anhydrous CH₂Cl₂(10 mL)

was added BOP-Cl (1 equiv), and the mixture was stirred at room

temperature for 2.5 h under nitrogen atmosphere. Triethylamine (3

equiv) and the appropriate amine (1 equiv) were added, and the

mixture was stirred at room temperature overnight. The reaction

mixture was concentrated under reduced pressure, and the residue was

partitioned between CHCl₃and water. The organic layer was

separated, dried over anhydrous sodium sulfate, ﬁltered, and the

ﬁltrate was evaporated under reduced pressure. The residue obtained

was puriﬁed by column chromatography over silica gel to obtain the

desired product.

Procedure B. To a solution of the carboxylic acid (1 equiv, 1 mmol)

in acetonitrile (12 mL) was added HATU (1 equiv), and the mixture

was stirred at room temperature for 15 min. Triethylamine (3 equiv)

and the appropriate amine (1 equiv) were added, and the mixture was

stirred at room temperature overnight. The mixture was concentrated

under reduced pressure, and the residue obtained was partitioned

between CHCl₃and saturated aqueous sodium bicarbonate. The

organic layer was separated, dried over anhydrous sodium sulfate,

ﬁltered, and the solvent was removed under reduced pressure. The

crude product thus obtained was puriﬁed by chromatography over a

column of silica gel to obtain the desired product.

ment, and mitogenesis assays but function as partial agonists in

the cyclase assay.

Among the compounds studied, compound 25 displayed

favorable D3R binding aﬃnity (K_i< 5 nM) and D2R/D3R

binding selectivity (>100-fold) with antagonist−partial agonist

functional activity proﬁle and was chosen for pharmacological

evaluations in vivo. This compound, coded as SR 21502, was

evaluated in rats and was found to produce signiﬁcant decreases

in cocaine reward, cocaine seeking, preference for cocaine-

associated environments, and cocaine induced locomotor

activity at doses that had no eﬀect on food reward or

spontaneous locomotor activity, indicating that it selectively

inhibits cocaine’s rewarding and stimulant eﬀects.^66,67

SUMMARY AND CONCLUSIONS

■

In an eﬀort to identify dopamine D3 receptor ligands with

selectivity for D3R versus D2R, we designed a series of ligands

based on the acylaminobutylarylpiperazine pharmacophore

incorporating, primarily, aza-aromatic systems on the acyl and

piperazine moieties. Docking of these ligands at the binding

sites of the human D3R crystal structure and our D2R

homology model provide insights into molecular features

contributing to binding aﬃnity and binding selectivity of this

panel of ligands. Among the ligands possessing a 4-

pyrimidinylpiperazine group, the aﬃnity diﬀerences between

isomeric compounds at the D3 receptor could be traced to a

favorable orientation of the pyrimidine ring nitrogen positioned

toward the solvent-exposed extracellular side of the receptor

with simultaneous occupation of a small pocket near helices

TM5 and TM6 formed by Val189, Ile183, and Val350 by a

hydrophobic substituent from the 2-position of the pyrimidine

ring. The results from binding experiments with chimeric and

mutant receptors support the hypothesis that D3 receptor

selectivity over D2 receptor conferred by tail groups such as the

imidazo[1,2-a]pyridine arises through interaction with the

tyrosine residue (D3R Tyr365, D2R Tyr379) at the

extracellular end of TM7, which is a region with signiﬁcant

structural diﬀerences between D2 and D3 receptors. These

structural insights provide a basis for the rational design of

future ligands with improved binding aﬃnity and selectivity for

the dopamine D3 receptors.

N-(4-(4-Phenylpiperazin-1-yl)butyl)imidazo[1,2-a]pyridine-

2-carboxamide (6). To a solution of imidazo[1,2-a]pyridine-2-

carboxylic acid (0.162 g, 1.0 mmol) in anhydrous CH₂Cl₂(10 mL)

was added 0.254 g (1.0 mmol) of BOP-Cl, and the mixture was stirred

at room temperature for 2.5 h under nitrogen atmosphere.

Triethylamine (0.42 mL, 3.0 mmol) and 4-(4-phenylpiperazin-1-

yl)butan-1-amine (0.233 g, 1.0 mmol) were added, and the mixture

was stirred at room temperature overnight. The reaction mixture was

concentrated under reduced pressure, and the residue was partitioned

between CHCl₃and water. The organic layer was separated, dried over

anhydrous sodium sulfate, ﬁltered, and the ﬁltrate was evaporated

under reduced pressure. The residue obtained was puriﬁed by column

chromatography over silica gel (CHCl₃−MeOH, 95:5) to obtain 0.164

g (44%) of the desired product 6 as a colorless solid. Mp 169−171 °C.

Results from functional assays characterized the investigated

class of compounds as antagonists or partial agonists with

varying potencies depending upon the functional end point of

the assay. From this series, compound 25 was selected as a lead

compound and evaluated in rat models of cocaine self-

administration and conditioned place preference. Results from

these studies showed that compound 25 was eﬀective at

signiﬁcantly attenuating cocaine self-administration, cocaine

reward, cue-induced reinstatement of cocaine-seeking and

blocking cocaine-induced place preference.^66,67Further studies

on the development of dopamine D3R selective ligands are in

progress and will be the subject of future publications.

1

TLC R_f= 0.39 (CHCl₃−MeOH, 92.5:7.5). H NMR (DMSO-d₆) δ

1.47−1.63 (m, 4H), 2.34 (t, 2H) 2.44−2.52 (m, 4H), 3.11 (t, 4H),

3.22−3.35 (m, 2H), 6.76 (td, 1H), 6.89 (dd, J = 8.6, 9.0 Hz, 2H), 6.97

(td, 1H), 7.19 (td, 2H), 7.32 (td, 1H), 7.55 (dd, J = 9.0, 9.0 Hz, 1H),

8.31−8.39 (m, 2H), 8.56 (dt, 1H). ESI MS m/z 378 (M + H)⁺. Anal.

(C₂₂H₂₇N₅O) C, H, N.

N-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)imidazo[1,2-

a]pyridine-2-carboxamide (7). Imidazo[1,2-a]pyridine-2-carboxylic

acid (0.243 g, 1.5 mmol) was reacted with 4-(4-(2-methoxyphenyl)-

piperazin-1-yl)butan-1-amine (0.40 g, 1.5 mmol) in the presence of

BOP-Cl (0.42 g, 1.65 mmol) and triethylamine (3.0 mL, 21.6 mmol)

in CH₂Cl₂(15 mL) according to general procedure A. The crude

product was puriﬁed by column chromatography over silica gel

(EtOAc−MeOH, 10:1) to obtain compound 7 (0.141 g, 23%) as a

colorless solid. Mp 169−171 °C. TLC R_f= 0.49 (CHCl₃−MeOH,

EXPERIMENTAL SECTION

General Methods. Melting points were determined in open

■

1

95:5). H NMR (DMSO-d₆) δ 1.42−1.58 (m, 4H), 2.31 (t, J = 6.58

capillary tubes with a Mel-Temp melting point apparatus and are

Hz, 2H), 2.49 (t, 4H), 2.94 (s, 4H), 3.27−3.32 (m, 2H), 3.75 (s, 3H),

6.80−6.99 (m, 5H), 7.28−7.38 (m, 1H), 7.56 (d, 1H), 8.34−8.42 (m,

2H), 8.56−8.60 (m, 1H). ESI MS m/z 408 (M + H)⁺. Anal.

(C₂₃H₂₉N₅O₂·H₂O) C, H, N.

1

uncorrected. H NMR spectra were recorded on a Nicolet 300NB

spectrometer operating at 300.635 MHz. Chemical shifts are expressed

in parts per million downﬁeld from tetramethylsilane. Spectral

assignments were supported by proton decoupling. Mass spectra

were recorded on a Varian MAT 311A double-focusing mass

spectrometer in the fast atom bombardment (FAB) mode or on a

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

[1,2-a]pyridine-2-carboxamide (8). Imidazo[1,2-a]pyridine-2-car-

boxylic acid (0.649 g, 4.0 mmol) was reacted with 4-(4-(2,3-

J

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

dichlorophenyl)piperazin-1-yl)butan-1-amine (1.21 g, 4.0 mmol) in

the presence of BOP-Cl (1.02 g, 4.0 mmol) and triethylamine (2.0 mL,

14.4 mmol) in CH₂Cl₂(30 mL) according to general procedure A.

The crude product was puriﬁed by column chromatography over silica

gel (EtOAc−MeOH, 7:1) to obtain compound 8 (0.268 g, 15%) as a

colorless solid. Mp 122−124 °C. TLC R_f= 0.12 (CHCl₃−MeOH,

95:5). ¹H NMR (DMSO-d₆) δ 1.39−1.57 (m, 4H), 2.24 (t, J = 6.6 Hz,

2H), 2.44 (bs, 4H), 2.98 (s, 4H), 3.21 (m, 2H), 6.95 (t, 1H), 7.09−

7.16 (m, 1H), 7.27−7.37 (m, 3H), 7.56 (dd, J = 9.12, 9.23 Hz, 1H),

8.34 (d, J = 0.66 Hz, 1H), 8.37 (t, 1H), 8.56 (m, 1H). ESI MS m/z

446 (M + H)⁺. Anal. (C₂₂H₂₅Cl₂N₅O·0.25H₂O) C, H, N.

7.99 (d, J = 7.42 Hz, 1H), 8.35 (s, 1H), 8.35 (t, J = 5.93 Hz, 1H), 8.57

(d, J = 6.92 Hz, 1H), 8.66 (d, J = 4.94 Hz, 1H). ESI MS m/z 429 (M +

H)⁺. Anal. (C₂₅H₂₈N₆O·0.25H₂O) C, H, N.

N-(4-(4-(2-(Triﬂuoromethyl)quinolin-4-yl)piperazin-1-yl)-

butyl)imidazo[1,2-a]pyridine-2-carboxamide (13). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 4-(4-(2-(triﬂuoromethyl)quinolin-4-yl)piperazin-1-yl)butan-1-

amine according to general procedure A. The crude product was

puriﬁed by column chromatography over silica gel (CHCl₃−MeOH,

97:3) to aﬀord compound 13 as a colorless solid in 16% yield. Mp

122−124 °C. TLC R_f= 0.56 (CHCl₃−MeOH, 90:10). ¹H NMR

(DMSO-d₆) δ 1.51−1.68 (m, 4H), 2.44 (t, 2H), 2.67 (bs, 4H), 3.26−

3.44 (m, 6H), 6.97 (ddd, J = 1.2, 1.2, 1.0 Hz, 1H), 7.24 (s, 1H), 7.31−

7.65 (m, 1H), 7.56 (dd, J = 9.2, 9.2 Hz, 1H), 7.68−7.74 (m, 1H),

7.78−7.88 (m, 1H), 8.03−8.12 (m, 2H), 8.35 (s, 1H), 8.40 (t, 1H),

8.56−8.60 (m, 1H). ESI MS m/z 497 (M + H)⁺. Anal. (C₂₆H₂₇F₃N₆O·

H₂O) C, H, N.

N-(4-(4-(7-(Triﬂuoromethyl)quinolin-4-yl)piperazin-1-yl)-

butyl)imidazo[1,2-a]pyridine-2-carboxamide (14). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 4-(4-(7-(triﬂuoromethyl)quinolin-4-yl)piperazin-1-yl)butan-1-

amine according to general procedure A. The crude product was

puriﬁed by column chromatography over silica gel (CHCl₃−MeOH,

97:3) to obtain compound 14 as an oﬀ-white solid in 34% yield. Mp

55−58 °C. TLC R_f= 0.35 (CHCl₃−MeOH, 92.5:7.5). ¹H NMR

(DMSO-d₆) δ 1.61−1.73 (bs, 2H), 1.87−1.94 (bs, 2H), 3.18−3.30

(bs, 2H), 3.33−3.48 (m, 2H), 3.55−4.38 (m, 8H), 7.29 (s, 1H), 7.44

(d, J = 6.4 Hz, 1H), 7.74 (bs, 2H), 7.95 (d, J = 8.6 Hz, 1H), 8.41 (d, J

= 8.8 Hz, 1H), 8.58 (s, 1H), 8.74 (bs, 1H), 8.88 (bs, 1H), 8.96 (d, J =

6.1 Hz, 1H), 9.16 (bs, 1H). ESI MS m/z 497 (M + H)⁺. Anal.

(C₂₆H₂₇F₃N₆O·H₂O) C, H, N.

N-(4-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)butyl)imidazo-

[1,2-a]pyridine-2-carboxamide (15). This compound was prepared

from imidazo[1,2-a]pyridine-2-carboxylic acid and 4-(4-(7-chloroqui-

nolin-4-yl)piperazin-1-yl)butan-1-amine according to general proce-

dure A. The crude product was puriﬁed by column chromatography

over silica gel (EtOAc−MeOH, 80:20) to aﬀord compound 15 as an

oﬀ-white solid in 28% yield. Mp 155−157 °C. TLC R_f= 0.67

(CHCl₃−MeOH, 85:15). ¹H NMR (DMSO-d₆) δ 1.47−1.65 (m, 4H),

2.42 (t, 2H), 2.62−2.72 (bs, 4H), 3.14−3.23 (bs, 2H), 3.27−3.46 (m,

4H), 6.96 (d, J = 3.1 Hz, 1H), 6.98−7.18 (m, 1H), 7.28−7.34 (m,

1H), 7.50−7.56 (m, 2H), 7.96 (d, J = 2.2 Hz, 1H), 8.00 (d, J = 9.0 Hz,

1H), 8.34 (s, 1H), 8.38 (t, 1H), 8.58−8.62 (m, 1H), 8.69 (d, J = 4.9

Hz, 1H). ESI MS m/z 463 (M + H)⁺. Anal. (C₂₅H₂₇ClN₆O·0.25H₂O)

C, H, N.

N-(4-(4-(3-(Triﬂuoromethyl)phenyl)piperazin-1-yl)butyl)-

imidazo[1,2-a]pyridine-2-carboxamide (9). To a solution of

imidazo[1,2-a]pyridine-2-carboxylic acid (0.08 g, 0.5 mmol) in

acetonitrile (6 mL) was added HATU (0. 190 g, 0.5 mmol), and

the mixture was stirred at room temperature for 15 min. Triethylamine

(0.21 mL, 1.5 mmol) and 4-(4-(3-(triﬂuoromethyl)phenyl)piperazin-

1-yl)butan-1-amine (0.15 g, 0.5 mmol) were then added, and the

mixture was stirred at room temperature overnight. The mixture was

concentrated under reduced pressure, and the residue obtained was

partitioned between CHCl₃and saturated aqueous sodium bicar-

bonate. The organic layer was separated, dried over anhydrous sodium

sulfate, ﬁltered, and the solvent was removed under reduced pressure.

The crude product thus obtained was puriﬁed by chromatography over

a column of silica gel (CHCl₃−MeOH, 96:4) to obtain 0.065 g (29%)

of the desired product 9 as a colorless solid. Mp 144−146 °C. TLC R_f

= 0.33 (CHCl₃−MeOH, 92.5:7.5). ¹H NMR (DMSO-d₆) δ 1.45−1.62

(m, 4H), 2.35 (t, 2H), 2.47−2.51 (m, 4H), 3.22 (t, 4H), 3.27−3.34

(m, 2H), 6.96−6.99 (m, 1H), 7.04 (d, J = 7.9 Hz, 1H), 7.14 (bs, 1H),

7.19 (dd, J = 8.2, 8.6 Hz, 1H), 7.31−7.34 (m, 1H), 7.37−7.42 (m,

1H), 7.56 (dd, J = 9.0, 9.0 Hz, 1H), 8.30−8.40 (m, 2H), 8.57 (dt, 1H).

ESI MS m/z 446 (M + H)⁺. Anal. (C₂₃H₂₆F₃N₅O·0.25H₂O) C, H, N.

N-(4-(4-(3-Cyano-5-(triﬂuoromethyl)phenyl)piperazin-1-yl)-

butyl)imidazo[1,2-a]pyridine-2-carboxamide (10). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 3-(4-(4-aminobutyl)piperazin-1-yl)-5-(triﬂuoromethyl)-

benzonitrile according to general procedure A. The crude product

was puriﬁed by column chromatography over silica gel (CHCl₃−

MeOH, 97:3) to obtain compound 10 as a colorless solid in 29% yield.

1

Mp 124−126 °C. TLC R_f= 0.40 (CHCl₃−MeOH, 92.5:5). H NMR

(DMSO-d₆) δ 1.45−1.66 (m, 4H), 2.35 (t, 2H), 2.44−2.56 (m, 4H),

3.18−3.35 (m, 6H), 6.93−7.02 (m, 1H), 7.31−7.32 (m, 1H), 7.46 (s,

1H), 7.50 (s, 1H), 7.55−7.62 (m, 1H), 7.64 (s, 1H), 8.34 (s, 1H), 8.43

(t, 1H), 8.54−8.62 (m, 1H). ESI MS m/z 471 (M + H)⁺. Anal.

(C₂₄H₂₅F₃N₆O·0.25H₂O) C, H, N.

N-(4-(4-(Quinolin-2-yl)piperazin-1-yl)butyl)imidazo[1,2-a]-

pyridine-2-carboxamide (16). This compound was prepared from

imidazo[1,2-a]pyridine-2-carboxylic acid and 4-(4-(quinolin-2-yl)-

piperazin-1-yl)butan-1-amine according to general procedure A. The

crude product was puriﬁed by column chromatography over silica gel

(EtOAc−MeOH, 92.5:7.5) to obtain compound 16 as a colorless solid

in 33% yield. Mp 137−139 °C. TLC R_f= 0.63 (CHCl₃−MeOH,

N-(4-(4-(3,5-Di-tert-butylphenyl)piperazin-1-yl)butyl)-

imidazo[1,2-a]pyridine-2-carboxamide (11). This compound was

prepared from imidazo[1,2-a]pyridine-2-carboxylic acid and 4-(4-(3,5-

di-tert-butylphenyl)piperazin-1-yl)butan-1-amine according to general

procedure B. The crude product was puriﬁed by column

chromatography over silica gel (CHCl₃−MeOH, 96:4) to obtain

compound 11 as a colorless solid in 28% yield. TLC R_f= 0.37

(CHCl₃−MeOH, 95:5). ¹H NMR (DMSO-d₆) δ 1.25 (s, 18H), 1.45−

1.64 (bs, 4H), 2.32−2.72 (bs, 6H), 3.10−3.22 (bs, 4H), 3.28−3.38 (m,

2H), 6.72 (d, J = 1.5 Hz, 2H), 6.86 (t, 1H), 6.97 (td, 1H), 7.32 (td,

1H), 7.55 (dt, 1H), 8.34 (d, J = 0.8 Hz, 1H), 8.39 (t, 1H), 8.52 (dt,

1H). ESI MS m/z 490 (M + H)⁺. Anal. (C₃₀H₄₃N₅O·H₂O) C, H, N.

N-(4-(4-(Quinolin-4-yl)piperazin-1-yl)butyl)imidazo[1,2-a]-

pyridine-2-carboxamide (12). This compound was prepared from

imidazo[1,2-a]pyridine-2-carboxylic acid (0.648 g, 4.0 mmol) and 4-

(4-(quinolin-4-yl)piperazin-1-yl)butan-1-amine (1.14 g, 4.0 mmol) in

the presence of BOP-Cl (1.38 g, 5.4 mmol) and triethylamine (1.67

mL 12.0 mmol) in CH₂Cl₂(20 mL) according to general procedure A.

The crude product was puriﬁed by column chromatography over silica

gel (CHCl₃−MeOH, 95:5) to obtain 0.19 g (11%) of compound 12 as

a colorless solid. Mp 126−128 °C. TLC R_f= 0.09 (CHCl₃−MeOH,

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.47−1.63 (m, 4H), 2.35 (bs, 2H),

2.44−2.58 (bs, 4H), 3.31−3.49 (m, 2H), 3.68 (bs 4H), 6.97 (dt, 1H),

7.17−7.25 (m, 2H), 7.31−7.36 (m, 1H), 7.52 (dt, 1H), 7.55−7.78 (m,

2H), 7.68 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 8.34 (s, 1H),

8.38 (t, 1H), 8.56 (dd, J = 6.8, 6.9 Hz, 1H). ESI MS m/z 429 (M +

H)⁺. Anal. (C₂₅H₂₈N₆O·0.25H₂O) C, H, N.

N-(4-(4-(Quinazolin-4-yl)piperazin-1-yl)butyl)imidazo[1,2-a]-

pyridine-2-carboxamide (17). This compound was prepared from

imidazo[1,2-a]pyridine-2-carboxylic acid (0.65 g, 4.0 mmol) and 4-(4-

(quinazolin-4-yl)piperazin-1-yl)butan-1-amine (1.136 g, 4.0 mmol) in

the presence of BOP-Cl (1.38 g, 5.42 mmol) and triethylamine (1.68

mL, 12 mmol) in CH₂Cl₂(20 mL) according to general procedure A.

The crude product was puriﬁed by column chromatography over silica

gel (EtOAc−MeOH, 8:1) to obtain 0.12 g (7%) of compound 17 as a

pale yellow solid. Mp 130−134 °C. TLC R_f= 0.10 (CHCl₃−MeOH,

1

95:5). H NMR (DMSO-d₆) δ 1.46−1.65 (m, 4H), 2.38−2.47 (m,

1

2H), 2.65 (bs, 4H), 3.18 (bs, 4H), 3.26−3.39 (m, 2H), 6.95 (d, J =

5.27 Hz, 1H), 6.99 (d, J = 1.99 Hz, 1H), 7.28−7.39 (m, 1H), 7.50−

7.60 (m, 2H), 7.62−7.72 (m, 1H), 7.92 (dd, J = 8.35, 8.34 Hz, 1H),

95:5). H NMR (DMSO-d₆) δ 1.66−1.78 (m, 4H), 2.45−2.54 (m,

2H), 2.64−2.74 (m, 4H), 3.42−3.55 (m, 2H), 3.82−3.94 (m, 4H),

6.91−7.00 (m, 1H), 7.32−7.40 (m, 1H), 7.50−7.61 (m, 2H), 7.76−

K

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.86 (m, 2H), 7.97−8.05 (m, 2H), 8.27 (d, J = 0.76 Hz, 1H), 8.43−

8.50 (m, 1H), 8.56 (s, 1H). ESI MS m/z 430 (M + H)⁺. Anal.

(C₂₄H₂₇N₇O) C, H, N.

(s, 9H), 1.44−1.60 (m, 4H), 2.23 (s, 3H), 2.23−2.46 (m, 6H), 3.24−

3.44 (m, 2H), 3.58 (bs, 4H), 6.47 (s, 1H), 6.97 (td, 1H), 7.37 (td,

1H), 7.56 (dd, J = 9.4, 9.0 Hz, 1H), 8.34 (s, 1H), 8.37 (t, 1H), 8.56

(dt, 1H). ESI MS m/z 450 (M + H)⁺. Anal. (C₂₅H₃₅N₇O·H₂O) C, H,

N.

N-(4-(4-(2-(tert-Butyl)quinazolin-4-yl)piperazin-1-yl)butyl)-

imidazo[1,2-a]pyridine-2-carboxamide (18). This compound was

prepared from imidazo[1,2-a]pyridine-2-carboxylic acid and 4-(4-(2-

(tert-butyl)quinazolin-4-yl)piperazin-1-yl)butan-1-amine according to

general procedure A. The crude product was puriﬁed by

chromatography over a column of silica gel using CHCl₃−MeOH,

92.5:7.5, as the eluent to obtain compound 18 as a pale yellow solid in

18% yield. Mp 59−61 °C. TLC R_f= 0.44 (CHCl₃−MeOH, 92.5:7.5).

¹H NMR (DMSO-d₆) δ 1.50 (s, 9H), 1.52−1.68 (m, 2H), 1.77−1.88

(m, 2H), 3.17 (bs, 2H), 3.24−3.41 (m, 4H), 3.64 (d, J = 11.1 Hz, 2H),

4.11 (bs, 2H), 4.85 (bs, 2H), 7.32 (t, 1H), 7.64−7.82 (m, 3H), 8.04 (t,

1H), 8.20 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.75 (s, 1H),

8.86 (d, J = 6.8 Hz, 1H), 9.10 (s, 1H). ESI MS m/z 486 (M + H)⁺.

Anal. (C₂₈H₃₅N₇O·0.25H₂O) C, H, N.

N-(4-(4-(2,6-Di-tert-butylpyrimidin-4-yl)piperazin-1-yl)-

butyl)imidazo[1,2-a]pyridine-2-carboxamide (23). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 4-(4-(2,6-di-tert-butylpyrimidin-4-yl)piperazin-1-yl)butan-1-amine

according to general procedure B. The crude product was puriﬁed by

column chromatography over silica gel (CHCl₃−MeOH, 92:8) to

obtain compound 23 as an oﬀ-white solid in 30% yield. Mp 90−94 °C.

1

TLC R_f= 0.54 (CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.24

(s, 9H), 1.27 (s, 9H), 1.45−1.60 (m, 4H), 2.33 (t, 2H), 2.41 (t, 4H),

3.28−3.34 (m, 2H), 3.60 (t, 4H), 6.45 (s, 1H), 6.96 (td, 1H), 7.33 (td,

1H), 7.58 (dd, J = 9.4, 9.0 Hz, 1H), 8.34 (d, J = 0.8 Hz, 1H), 8.38 (t,

1H) 8.57 (dt, 1H). ESI MS m/z 492 (M + H)⁺. Anal. (C₂₈H₄₁N₇O·

0.75H₂O) C, H, N.

N-(4-(4-(2-Methyl-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide

(19). This compound was prepared using general procedure B by

reacting imidazo[1,2-a]pyridine-2-carboxylic acid (0.081 g, 0.5 mmol)

with 4-(4-(2-methyl-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)-

butan-1-amine (0.159 g, 0.5 mmol) in the presence of HATU (0.19 g,

0.5 mmol) and triethylamine (0.13 mL, 0.75 mmol) in acetonitrile (7.0

mL). The crude product was puriﬁed by column chromatography over

silica gel (CHCl₃−MeOH, 96:4) to obtain 0.096 g (42%) of

compound 19 as a crystalline white solid. Mp 156−158 °C. TLC R_f

N-(4-(4-(2-(tert-Butyl)-6-cyclopropylpyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide

(24). This compound was prepared from imidazo[1,2-a]pyridine-2-

carboxylic acid and 4-(4-(2-(tert-butyl)-6-cyclopropylpyrimidin-4-yl)-

piperazin-1-yl)butan-1-amine according to general procedure B. The

crude product was puriﬁed by column chromatography over silica gel

(CHCl₃−MeOH, 92:8) to obtain compound 24 as a colorless solid in

36% yield. Mp 34−37 °C. TLC R_f= 0.19 (CHCl₃−MeOH, 95:5). ¹H

NMR (DMSO-d₆) δ 0.81−0.96 (2m, 4H), 1.28 (s, 9H), 1.45−1.60 (m,

4H), 1.84−1.90 (m, 1H), 2.32 (t, 2H), 2.40 (t, 4H), 3.27−3.33 (m,

2H), 3.58 (t, 4H), 6.49 (s, 1H), 6.97 (td, 1H), 7.32 (td, 1H), 7.56 (dd,

J = 9.4, 9.0 Hz, 1H), 8.37 (t, 2H), 8.56 (dt, 1H). ESI MS m/z 476 (M

+ H)⁺. Anal. (C₂₇H₃₇N₇O·H₂O) C, H, N.

1

= 0.67 (CHCl₃−MeOH, 90:10). H NMR (DMSO-d₆) δ 1.44−1.61

(m, 4H), 2.34 (t, 2H), 2.42 (s, 3H), 2.44−2.50 (m, 4H), 3.25−3.34

(m, 2H), 3.71 (bs, 4H), 6.95−6.99 (m, 1H), 7.05 (s, 1H), 7.31−7.36

(m, 1H), 7.56 (d, J = 9.0 Hz, 1H), 8.31 (s, 1H), 8.37 (t, 1H), 8.57 (d, J

= 6.6 Hz, 1H). ESI MS m/z 462 (M + H)⁺. Anal. (C₂₂H₂₆F₃N₇O·

0.25H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide

(25). Imidazo[1,2-a]pyridine-2-carboxylic acid (5.89 g, 36.6 mmol)

was reacted with 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-

yl)piperazin-1-yl)butan-1-amine (13.14 g, 36.6 mmol) in the presence

of BOP-Cl (9.3 g, 36.6 mmol) and triethylamine (15.0 mL) in CH₂Cl₂

(350 mL) as described in general procedure A. The crude product was

puriﬁed by column chromatography over silica gel (EtOAc−hexane,

2:1) to yield 6.0 g (32%) of the desired product. A solution of the free

base in ether was treated with 1.0 M solution of HCl in Et₂O to obtain

the dihydrochloride salt as a light brown solid. Mp 253−255 °C. TLC

N-(4-(4-(6-Methyl-2-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide

(20). This compound was prepared from imidazo[1,2-a]pyridine-2-

carboxylic acid (0.102 g, 0.63 mmol) and 4-(4-(6-methyl-2-

(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine (0.20 g,

0.63 mmol) in the presence of HATU (0.24 g, 0.63 mmol) and

triethylamine (0.26 mL, 0.189 mmol) in acetonitrile (7.0 mL)

according to general procedure B. The crude product was puriﬁed

by column chromatography over silica gel (CHCl₃−MeOH, 95:5) to

obtain 0.076 g (26%) of compound 20 as a colorless solid. Mp 118−

1

R_f= 0.29 (CHCl₃−MeOH, 90:10). H NMR (DMSO-d₆) δ 1.31 (s,

9H), 1.62−1.69 (m, 2H), 1.80−1.88 (m, 2H), 2.88−3.19 (broad

hump, 1H), 3.10−3.17 (m, 2H), 3.37−3.33 (q, 2H), 3.52−3.60 (m,

4H), 4.32−4.86 (b, 2H), 7.11 (s, 1H), 7.13−7.18 (m, 1H), 7.50−7.57

(t, 1H), 7.65 (dd, J = 9.2, 9.0 Hz, 1H), 8.53 (s, 1H), 8.57 (bs, 1H),

8.68 (d, J = 6.9 Hz, 1H), 11.0−11.8 (b, 1H). ESI MS m/z 504 (M +

H)⁺. Anal. (C₂₅H₃₂F₃N₇O·2HCl·0.25H₂O) Calcd: C, 51.68; H, 5.98;

N, 16.87.Cl, 12.20. Found: C, 51.64; H, 6.00; N, 16.89, Cl, 11.97.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-1H-indole-2-carboxamide (26). This com-

pound was prepared from indole-2-carboxylic acid and 4-(4-(2-(tert-

butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine

according to general procedure B. The crude product was puriﬁed by

column chromatography over silica gel (CHCl₃−MeOH, 92:8) to

obtain compound 26 as a colorless solid in 35% yield. Mp 70−72 °C.

1

120 °C. TLC R_f= 0.60 (CHCl₃−MeOH, 90:10). H NMR (DMSO-

d₆) δ 1.43−1.61 (m, 4H), 2.33 (t, 2H), 2.37 (s, 3H), 2.42 (t, 4H),

3.26−3.34 (m, 2H), 3.65 (bs, 4H), 6.96 (s, 1H), 6.97−6.99 (m, 1H),

7.31−7.36 (m, 1H), 7.56 (m, 1H), 8.31 (d, J = 0.8 Hz, 1H), 8.37 (t,

1H), 8.55−8.59 (m, 1H). ESI MS m/z 462 (M + H)⁺. Anal. (C₂₂H₂₆

F₃N₇O·0.25H₂O) C, H, N.

N-(4-(4-(6-(tert-Butyl)-2-methylpyrimidin-4-yl)piperazin-1-

yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide (21). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 4-(4-(6-(tert-butyl)-2-methylpyrimidin-4-yl)piperazin-1-yl)butan-

1-amine (0.305 g, 1.0 mmol) according to general procedure B. The

crude product was puriﬁed by column chromatography over silica gel

(CHCl₃−MeOH, 96:4) to obtain compound 21 as a colorless viscous

1

oil in 35% yield. TLC R_f= 0.32 (CHCl₃−MeOH, 95:5). H NMR

TLC R_f= 0.54 (CHCl₃−MeOH, 92.5:7.5). H NMR (DMSO-d₆) δ

(DMSO-d₆) δ 1.21 (s, 9H), 1.45−1.64 (m, 4H), 2.22−2.35 (m, 2H),

2.34 (s, 3H), 2.46 (t, 4H), 3.28−3.34 (m, 2H), 3.58 (t, 4H), 6.47 (s,

1H), 6.96 (td, 1H), 7.33 (td, 1H), 7.58 (dd, J = 8.6, 8.2 Hz, 1H), 8.37

(t, 2H), 8.56 (dt, 1H). ESI MS m/z 450 (M + H)⁺. Anal. (C₂₅H₃₅N₇O·

75H₂O) C, H, N.

1.28 (s, 9H), 1.43−1.63 (m, 4H), 2.35 (t, 2H), 2.42 (t, 4H), 3.26−3.34

(m, 2H), 3.71 (bs, 4H), 7.00−7.02 (m, 2H), 7.16−7.19 (m, 2H), 7.41

(d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 8.44 (t, 1H), 7.78 (t,

1H). ESI MS m/z 503 (M + H)⁺. Anal. (C₂₆H₃₃F₃N₆O·0.25H₂O) C,

H, N.

N-(4-(4-(2-(tert-Butyl)-6-methylpyrimidin-4-yl)piperazin-1-

yl)butyl)imidazo[1,2-a]pyridine-2-carboxamide (22). This com-

pound was prepared from imidazo[1,2-a]pyridine-2-carboxylic acid

and 4-(4-(2-(tert-butyl)-6-methylpyrimidin-4-yl)piperazin-1-yl)butan-

1-amine according to general procedure B. The crude product was

puriﬁed by column chromatography over silica gel (CHCl₃−MeOH,

96:4) to obtain compound 22 as a light brown viscous oil in 20% yield.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-1H-benzo[d]imidazole-2-carboxamide

(27). This compound was prepared from benzimidazole-2-carboxylic

acid and 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butan-1-amine according to general procedure A. The

crude product was puriﬁed by column chromatography over silica gel

(CHCl₃−MeOH, 92:8) to obtain compound 27 as a colorless solid in

18% yield. Mp 80−72 °C. TLC R_f= 0.56 (CHCl₃−MeOH, 90:10). ¹H

1

TLC R_f= 0.17 (CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.25

L

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.44−1.66 (m, 4H), 2.36 (t, 2H),

2.43 (t, 4H), 3.28−3.48 (m, 2H), 3.64−3.68 (m, 4H), 7.03 (s, 1H),

7.22−7.36 (m, 2H), 7.52 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 7.5 Hz, 1H),

8.97 (t, 1H), 13.12 (s, 1H). ESI MS m/z 504 (M + H)⁺. Anal.

(C₂₅H₃₂F₃N₇O) C, H, N.

aminothieno[2,3-b]pyridine-2-carboxylic acid (0.109 g, 0.56 mmol)

and 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-

yl)butan-1-amine (0.20 g, 0.56 mmol) in the presence of HATU

(0.213 g, 0.56 mmol) and triethylamine (0.23 mL, 1.68 mmol) in

acetonitrile (10 mL) according to general procedure B. The crude

product was puriﬁed by column chromatography over silica gel

(CHCl₃−MeOH, 96:4) to obtain 0.09 g (30%) of compound 32 as an

oﬀ-white solid. Mp 138−140 °C. TLC R_f= 0.46 (CHCl₃−MeOH,

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)thieno[2,3-b]pyridine-2-carboxamide

(28). This compound was prepared from thieno[2,3-b]pyridine-2-

carboxylic acid (0.10 g, 0.56 mmol) and 4-(4-(2-(tert-butyl)-6-

(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine (0.20 g,

0.56 mmol) in the presence of HATU (0.211 g, 0.56 mmol) and

triethylamine (0.23 mL, 1.68 mmol) in acetonitrile (7.0 mL) according

to general procedure B. The crude product was puriﬁed by column

chromatography over silica gel (CHCl₃−MeOH, 92:8) to yield 0.125 g

(43%) of compound 28 as a colorless solid. Mp 122−124 °C. TLC R_f

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.42−1.62 (m, 4H),

2.33 (t, 2H), 2.43 (t, 4H), 3.24 (q, 2H), 3.71 (s, 4H), 7.03 (s, 1H),

7.14 (s, 2H), 7.44 (q, 1H), 7.71 (t, 1H), 8.4 (dd, J = 1.6, 1.6 Hz, 1H),

8.61 (dd, J = 1.5, 1.5 Hz, 1H). ESI MS m/z 536 (M + H)^+.Anal.

(C₂₅H₃₂F₃N₇OS·0.25H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-3-((dimethylamino)methyl)-1H-indole-2-

carboxamide (33). Step 1. To an ice cold solution of dimethylamine

(15 mL, 2 M solution in THF, 30.0 mmol) was added acetic acid (3.65

mL) followed by 2.20 mL of 37% aqueous formaldehyde solution

(30.0 mmol). Ethyl indole-2-carboxylate (3.0 g, 15.8 mmol) in

methanol (150 mL) was then added, and the resulting solution was

heated under reﬂux for 4 h. The mixture was concentrated to 20% of

its volume in vacuo and diluted with water (50 mL). The aqueous

solution was washed with CHCl₃(2 × 50 mL). The aqueous layer was

separated, chilled, and basiﬁed to pH 12 by addition of 20% aqueous

NaOH. The mixture was extracted with CH₂Cl₂(3 × 50 mL). The

organic extract was dried with sodium sulfate and the solvent was

removed under reduced pressure to obtain ethyl 3-((dimethylamino)-

methyl)-1H-indole-2-carboxylate. Yield 2.8 g (72%). ¹H NMR

(DMSO-d₆) δ 1.34−1.38 (m, 3H), 2.16 (s, 6H), 3.89 (s, 2H),

4.31−4.37 (m, 2H), 7.05−7.42 (m, 4H), 11.59 (s, 1H). ESI MS m/z

247 (M + H)⁺.

Step 2. The above ester (1.0 g, 4.06 mmol) was dissolved in 20 mL

of dioxane−water (90:10), treated with NaOH (0.8 g, 20.0 mmol),

and the mixture was stirred at room temperature for 4 h. The reaction

mixture was diluted with EtOAc (75 mL), and the organic layer was

separated, dried over anhydrous sodium sulfate, and concentrated

under reduced pressure to obtain 3-((dimethylamino)methyl)-1H-

indole-2-carboxylic acid. Yield (0.58 g, 65%). ESI MS m/z 219 (M +

H)⁺The acid thus obtained was used in the next step without further

puriﬁcation.

1

= 0.49 (CHCl₃−MeOH, 92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s,

9H), 1.44−1.62 (m, 4H), 2.34 (t, 2H), 2.45 (t, 4H), 3.33 (q, 2H), 3.71

(bs, 4H), 7.03 (s, 1H), 7.49 (q, 1H), 8.08 (s, 1H), 8.40 (dd, J = 1.6, 1.6

Hz, 1H), 8.65 (dd, J = 1.5, 1.5 Hz, 1H), 8.84 (t, 1H). ESI MS m/z 521

(M + H)⁺. Anal. (C₂₅H₃₁F₃N₆OS·H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyrimidine-2-carboxamide

(29). This compound was prepared from imidazo[1,2-a]pyrimidine-2-

carboxylic acid (0.50 g, 3.07 mmol) and 4-(4-(2-(tert-butyl)-6-

(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine (1.105

g, 3.07 mmol) in the presence of BOP-Cl (0.783 g, 3.08 mmol) and

triethylamine (1.25 mL, 9.0 mmol) in CH₂Cl₂(15 mL) according to

general procedure A. The crude product was puriﬁed by column

chromatography over silica gel (EtOAc−MeOH, 6:1) to obtain 0.776

g (50%) of compound 29 as a colorless solid. Mp 136−138 °C. TLC

1

R_f= 0.47 (CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.80 (s,

9H), 1.37−1.57 (m, 4H), 2.24 (t, J = 5.96 Hz, 2H), 2.44−2.48 (m,

4H), 2.98−3.06 (m, 2H), 3.21−3.26 (m, 2H), 3.62 (bs, 2H), 7.03 (s,

1H), 7.15 (dd, J = 6.92, 6.91 Hz, 1H), 8.29 (s, 1H), 8.57 (t, 1H), 8.63

(dd, J = 4.05 and 4.18 Hz, 1H), 8.98 (dd, J = 6.91, 6.81 Hz, 1H). ESI

MS m/z 505 (M + H)⁺. Anal. (C₂₄H₃₁F₃N₈O·H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)imidazo[1,2-a]pyrazine-2-carboxamide

(30). This compound was prepared from imidazo[1,2-a]pyrazine-2-

carboxylic acid (0.50 g, 3.07 mmol) and 4-(4-(2-(tert-butyl)-6-

(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine (0.75 g,

2.09 mmol) in the presence of BOP-Cl (1.1 g, 4.32 mmol) and

triethylamine (3.0 mL, 21.52 mmol) in CH₂Cl₂(20 mL) according to

general procedure A. The crude product was puriﬁed by column

chromatography over silica gel (CHCl₃−MeOH, 92:8) to obtain 0.412

g (39%) of compound 30 as an oﬀ-white solid. Mp 200−202 °C. TLC

Step 3. The acid (0.091 g, 0.42 mmol) obtained above was coupled

with 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-

1-yl)butan-1-amine (0.151 g, 0.42 mmol) in the presence of HATU

(0.16 g, 0.42 mmol) and triethylamine (0.18 mL, 1.26 mmol) in

acetonitrile (10 mL) according to general procedure B. The crude

product was puriﬁed by column chromatography over silica gel

(CHCl₃−MeOH, 97:3) to obtain 0.10 g (43%) of compound 33 as an

oﬀ-white solid. Mp 174−176 °C. TLC R_f= 0.43 (CHCl₃−MeOH,

1

R_f= 0.25 (CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.28 (s,

9H), 1.47−1.67 (m, 4H), 2.32 (t, J = 5.96 Hz, 2H), 2.44−2.48 (m,

4H), 3.21−3.36 (m, 2H), 3.71 (bs, 4H), 7.04 (s, 1H), 7.95 (d, J = 4.66

Hz, 1H), 8.50 (s, 1H), 8.61−8.66 (m, 2H), 9.12 (d, J = 0.77 Hz, 1H).

ESI MS m/z 505 (M + H)⁺. Anal. (C₂₄H₃₁F₃N₈O·2HCl·0.75H₂O) C,

H, N.

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.56−1.66 (m, 4H),

2.23 (s, 6H), 2.37 (t, 2H), 2.44 (t, 4H), 3.28−3.38 (m, 2H), 3.70 (bs,

4H), 7.03 (s, 1H), 7.04−7.08 (m, 2H), 7.17 (td, 1H), 7.40 (d, 1H, J =

8.2 Hz), 7.65 (d, J = 8.2 Hz, 2H), 10.40 (s, 1H), 11.58 (s, 1H). ESI

MS m/z 560 (M + H)⁺. Anal. (C₂₉H₄₀F₃N₇O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-6-chloroimidazo[1,2-b]pyridazine-2-car-

boxamide (31). This compound was prepared from 6-chloroimidazo-

[1,2-b]pyridazine-2-carboxylic acid (0.591 g, 3.0 mmol) and 4-(4-(2-

(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-

amine (1.078 g, 3.0 mmol) in the presence of BOP-Cl (0.764 g, 3.0

mmol) and triethylamine (1.25 mL, 9.0 mmol) in CH₂Cl₂(20 mL)

using general procedure A. The crude product was puriﬁed by column

chromatography over silica gel (EtOAc−MeOH, 10:1) to obtain 0.242

g (15%) of compound 31 as a colorless solid. Mp 119−121 °C. TLC

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-3-((dimethylamino)methyl)imidazo[1,2-

a]pyridine-2-carboxamide (34). This compound was prepared

from 3-((dimethylamino)methyl)imidazo[1,2-a]pyridine-2-carboxylic

acid dihydrochloride (0.164 g, 0.56 mmol) and 4-(4-(2-(tert-butyl)-

6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-amine (0.20

g, 0.56 mmol) in the presence of HATU (0.213 g, 0.56 mmol) and

triethylamine (0.235 mL, 1.68 mmol) in acetonitrile (7.0 mL)

according to general procedure B. The crude product was puriﬁed

by column chromatography over silica gel (CHCl₃−MeOH, 98:2) to

obtain 0.119 g (38%) of compound 34 as a colorless oil. TLC R_f= 0.38

1

R_f= 0.59 (CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.27 (s,

9H), 1.43−1.62 (m, 4H), 2.29 (t, J = 4.63 Hz, 2H), 2.45 (t, J = 1.81

Hz, 4H), 3.34 (bs, 2H), 3.62 (bs, 4H), 7.03 (s, 1H), 7.46 (d, J = 9.56

Hz, 1H), 8.23 (dd, J = 9.61, 9.56 Hz, 1H), 8.54 (t, J = 5.96 Hz, 1H),

8.67 (d, J = 0.65 Hz, 1H). ESI MS m/z 539 (M + H)⁺. Anal.

(C₂₄H₃₀ClF₃N₈O·0.5H₂O) C, H, N.

1

(CHCl₃−MeOH, 95:5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.41−

1.64 (m, 4H), 2.16 (s, 6H), 2.34 (t, 2H), 2.43 (t, 4H), 3.22−3.36 (m,

2H), 3.72 (bs, 4H), 4.17 (s, 2H), 6.92−7.05 (m, 2H), 7.38 (t, 1H),

7.57 (d, J = 9.0 Hz, 1H), 8.38 (t, 1H), 8.44 (d, J = 6.7 Hz, 1H). ESI

MS m/z 561 (M + H)⁺. Anal. (C₂₈H₃₉F₃N₈O·0.5H₂O) C, H, N.

3-Amino-N-(4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)-

pyrimidin-4-yl)piperazin-1-yl)butyl)thieno[2,3-b]pyridine-2-

carboxamide (32). This compound was prepared from 3-

M

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-5-(hydroxymethyl)imidazo[1,2-a]-

pyridine-2-carboxamide (35). Step 1. A solution of 2-amino-6-

hydroxymethylpyridine (5.0 g, 40.28 mmol) and ethyl 3-bromopyr-

uvate (1.57 g, 80.56 mmol) in ethanol (150 mL) was stirred with

heating under reﬂux for 3 h. The reaction mixture was cooled to room

temperature and concentrated under reduced pressure. The residue

obtained was puriﬁed by chromatography over a column of silica gel

using hexane−EtOAc (75:25) to yield 6.2 g (70%) of ethyl 5-

(hydroxymethyl)imidazo[1,2-a]pyridine-2-carboxylate (45). ESI MS

m/z 221 (M + H)⁺.

Step 2. To a stirred solution of the above compound (3.89 g, 17.66

mmol) and imidazole (3.5 g, 51.4 mmol) in DMF (12 mL) was added

chlorotriisopropylsilane (6.82 g, 35.4 mmol) dropwise, and the

reaction mixture was heated at 70 °C for 2 h. After cooling to room

temperature, the reaction mixture was diluted with EtOAc (200 mL)

and washed with saturated aqueous sodium carbonate (150 mL). The

organic phase was washed with brine, dried over anhydrous sodium

sulfate, and concentrated under reduced pressure. The residue thus

obtained was puriﬁed by silica gel column using CH₂Cl₂−EtOAc (1:1)

as the eluent to obtain 4.8 g (72%) of ethyl 5-(((triisopropylsilyl)-

oxy)methyl)imidazo[1,2-a]pyridine-2-carboxylate (46). ESI MS m/z

377 (M + H)⁺.

Step 3. To a solution of the above silyl ether 4.8 g (12.7 mmol) in

50 mL of methanol/THF/water (2:2:1) was added NaOH (2.1 g).

The mixture was stirred at room temperature for 2 h. The mixture was

neturalized by the addition of acetic acid, and the solvents were

removed under reduced pressure. The residue obtained was puriﬁed

over a column of silica using CH₂Cl₂−MeOH (7:1) to give 3.6 g

(81%) of 5-(((triisopropylsilyl)oxy)methyl)imidazo[1,2-a]pyridine-2-

carboxylic acid (47). ESI MS m/z 349 (M + H)⁺.

Step 4. To a solution of the above acid (1.02 g, 2.92 mmol) in

acetonitrile (15 mL) was added HATU (1.11 g, 2.92 mmol) and Et₃N

(0.6 mL, 4.31 mmol). The mixture was stirred for 10 min, and 4-(4-(2-

(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-

amine (1.05 g, 2.92 mmol) was added. The stirring was continued for

16 h at room temperature. The reaction mixture was concentrated

under reduced pressure, and the residue was dissolved in CHCl₃and

washed with aqueous sodium bicarbonate. The CHCl₃extract was

dried over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The crude product thus obtained was puriﬁed by

chromatography over a column of silica gel (CHCl₃−MeOH 97:3) to

obtain 0.45 g (22%) of N-(4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)-

pyrimidin-4-yl)piperazin-1-yl)butyl)-5-(((triisopropylsilyl)oxy)-

methyl)imidazo[1,2-a]pyridine-2-carboxamide (48).

reduced pressure, and the residue was partitioned between CHCl₃and

saturated aqueous sodium bicarbonate. The organic layers were

separated and dried over anhydrous sodium sulfate. Filtration, removal

of the solvent under reduced pressure, and puriﬁcation by silica gel

column chromatography (CHCl₃−MeOH 97:3) aﬀorded 0.03 g

(23%) of the desired product 36 as a colorless solid. Mp 62−64 °C.

1

TLC R_f= 0.64 (CHCl₃−MeOH, 92.5:7.5). H NMR (DMSO-d₆) δ

1.28 (s, 9H), 1.44−1.61 (m, 4H), 2.21 (s, 6H), 2.34 (t, 2H), 2.43 (t,

4H), 3.09−3.35 (m, 2H), 3.72 (bs, 4H), 3.76 (s, 2H), 6.93 (d, J = 6.7

Hz, 1H), 7.03 (s, 1H), 7.26−7.34 (m, 1H), 7.55 (d, J = 9.0 Hz, 1H),

8.31 (s, 1H), 8.39 (t, 1H). ESI MS m/z 561 (M + H)⁺. Anal.

(C₂₈H₃₉F₃N₈O·H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-1-phenylpiperidine-4-carboxamide (37).

This compound was prepared from 1-phenylpiperidine-4-carboxylic

acid and 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butan-1-amine according to general procedure A. The

crude product was puriﬁed by column chromatography over silica gel

(EtOAc−MeOH, 10:1) to obtain compound 37 as a colorless solid in

26% yield. Mp 178−180 °C. TLC R_f= 0.30 (CHCl₃−MeOH,

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.42−1.46 (m, 4H),

1.58−1.77 (m, 4H), 2.22−2.35 (m, 3H), 2.42 (t, 4H), 2.61−2.69 (m,

2H), 3.06 (dd, J = 11.7, 11.7 Hz, 2H), 3.66−3.72 (m, 6H), 6.71−6.77

(m, 1H), 6.91−6.94 (m, 2H), 7.04 (s, 1H), 7.16−7.22 (m, 2H), 7.79

(t, 1H). ESI MS m/z 547 (M + H)⁺. Anal. (C₂₉H₄₁F₃N₆O·25H₂O) C,

H, N.

1-Benzyl-N-(4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)-

pyrimidin-4-yl)piperazin-1-yl)butyl)piperidine-4-carboxamide

(38). This compound was prepared from 1-benzylpiperidine-4-

carboxylic acid and 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-

4-yl)piperazin-1-yl)butan-1-amine according to general procedure A.

The crude product was puriﬁed by column chromatography over silica

gel (EtOAc−MeOH, 95:5) to obtain compound 38 as a pale yellow

solid in 41% yield. Mp 36−37 °C. TLC R_f= 0.49 (CHCl₃−MeOH,

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.36−1.47 (m, 4H),

1.54−1.68 (m, 2H), 1.84−1.93 (m, 2H), 2.07−2.11 (m, 2H), 2.29 (t,

2H), 2.40 (t, 2H), 2.78 (d, J = 11.6 Hz, 4H), 3.02 (dd, J = 11.8, 11.6

Hz, 2H), 3.42 (s, 2H), 3.60−3.88 (bs, 4H), 7.03 (s, 1H), 7.22−7.33

(m, 5H), 7.69 (t, 1H). ESI MS m/z 561 (M + H)⁺. Anal.

(C₃₀H₄₃F₃N₆O·H₂O) C, H, N.

trans-N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-

yl)piperazin-1-yl)butyl)-4-phenylcyclohexanecarboxamide

(39). This compound was prepared from trans-4-phenylcyclohexane-

carboxylic acid and 4-(4-(2-(tert-butyl)-6-(triﬂuoromethyl)pyrimidin-

4-yl)piperazin-1-yl)butan-1-amine (0.359 g, 1.0 mmol) according to

general procedure A. The crude product was puriﬁed by column

chromatography over silica gel (EtOAc−MeOH, 80:20) to obtain

compound 39 as a colorless solid in 30% yield. Mp 137−139 °C. TLC

R_f= 0.69 (CHCl₃−MeOH, 92.5:7.5). ¹H NMR (DMSO-d₆) δ 1.28 (s,

9H), 1.37−1.55 (m, 8H), 1.78−1.86 (m, 4H), 2.11−2.19 (m, 1H),

2.31 (t, 2H), 2.42 (t, 4H), 2.49−2.51 (m, 1H), 3.06 (dd, J = 11.9, 11.7

Hz, 2H), 3.71 (bs, 4H), 7.04 (s, 1H), 7.13−7.31 (m, 5H), 7.71 (t, 1H).

ESI MS m/z 546 (M + H)⁺. Anal. (C₃₀H₄₂F₃N₅O·25H₂O) C, H, N.

4-(4-(6-(tert-Butyl)-2-methylpyrimidin-4-yl)piperazin-1-yl)-

butan-1-amine (43a). Step 1. A solution of 4-(tert-butyl)-6-chloro-2-

methylpyrimidine (7.98 g, 43.2 mmol) in absolute ethanol (100 mL)

was added dropwise to a boiling solution of piperazine (18.46 g, 215

mmol) in ethanol (175 mL) over a period of 2 h. The reaction mixture

was reﬂuxed for 12 h, cooled to room temperature, and the solvent was

removed under reduced pressure. The residue was treated with ice-

cold water (1 L) and stirred for 15 min. After the mixture was allowed

to stand for an hour, the solid crystalline product obtained was

collected by ﬁltration and dried under reduced pressure over P₂O₅to

obtain 9.09 g (90%) of 4-(tert-butyl)-2-methyl-6-(piperazin-1-yl)-

pyrimidine. ESI MS m/z 235 (M + H)⁺.

Step 5. To a solution of the above compound 0.45 g (0.65 mmol) in

THF (20 mL) was added tetraethylammonium ﬂuoride (0.146 g, 0.98

mmol), and the mixture was stirred at room temperature for 16 h. The

mixture was then concentrated and partitioned between CHCl₃and

water. The organic layer was separated, dried over anhydrous sodium

sulfate, ﬁltered, and the solvent was removed under reduced pressure.

The residue thus obtained was puriﬁed by chromatography over a

column of silica gel (CHCl₃−MeOH, 92:8) to obtain 0.216 g (62%) of

the desired product as a colorless oil. TLC R_f= 0.46 (CHCl₃−MeOH,

1

92.5:7.5). H NMR (DMSO-d₆) δ 1.28 (s, 9H), 1.41−1.60 (m, 4H),

2.34 (t, 2H), 2.44 (t, 4H), 3.01−3.28 (m, 2H), 3.70 (bs, 4H), 4.78 (d,

J = 7.8 Hz, 2H), 5.71−5.78 (m, 1H), 6.94 (dd, J = 6.6, 6.6 Hz, 1H),

7.03 (s, 1H), 7.30−7.40 (m, 1H), 7.53 (d, J = 9.0 Hz, 1H), 8.28 (s,

1H), 8.40 (t, 1H). ESI MS m/z 534 (M + H)⁺. Anal. (C₂₆H₃₄F₃N₇O₂·

75H₂O) C, H, N.

N-(4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butyl)-5-((dimethylamino)methyl)imidazo[1,2-

a]pyridine-2-carboxamide (36). A solution of hydroxymethyl

compound 35 (0.125 g, 0.235 mmol) and triethylamine (0.1 mL,

0.702 mmol) in dichloromethane (5 mL) was cooled in ice bath and

treated dropwise with methanesulfonyl chloride (0.15 g, 1.3 mmol).

The mixture was allowed to warm to room temperature and stirred at

room temperature for 1 h. To the mixture was then added

dimethylamine (0.25 mL, 1 M solution in THF), and the mixture

was stirred at room temperature for 6 h. Volatiles were removed under

Step 2. A mixture of the above piperazine (7.5 g, 32.0 mmol), N-(4-

bromobutyl)phthalimide (9.03 g, 32.0 mmol), and potassium

carbonate (5.30 g, 38.35 mmol) in acetonitrile (200 mL) was stirred

at room temperature overnight. The reaction mixture was ﬁltered, and

the ﬁltrate was concentrated under reduced pressure. The residue thus

N

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

obtained was puriﬁed by ﬂash chromatography over a column of silica

gel using CHCl₃−MeOH 98:2 as the eluent to obtain 11.5 g (82%) of

2-(4-(4-(6-(tert-butyl)-2-methylpyrimidin-4-yl)piperazin-1-yl)butyl)-

isoindoline-1,3-dione. ESI MS m/z 436 (M + H)⁺.

the concentration of the competitive inhibitor ranged over 5 orders of

magnitude. Binding was terminated by addition of cold wash buﬀer

(10 mM Tris-HCl/150 mM NaCl, pH 7.5) and ﬁltration over a glass-

ﬁber ﬁlter (Whatman no. 32, Piscataway, NJ). Filters were washed, and

the radioactivity was measured using a Packard γ counter with an

eﬃciency of 75%. The protein concentration of the membranes was

determined using a BCA reagent (Pierce, Rockford, IL) and BSA as

the protein standard.

Step 3. A mixture of the above (11.5 g, 32.05 mmol) and hydrazine

hydrate (64%, 5.01 g, 64.10 mmol) in methanol (150 mL) was heated

under reﬂux for 2 h. The reaction mixture was cooled to room

temperature, and the volatiles were removed under reduced pressure.

The residue was dissolved in ether (150 mL), ﬁltered, and the ﬁltrate

was concentrated under reduced pressure. The residue obtained was

puriﬁed by chromatography over a column of silica using CHCl₃−

MeOH−NH₄OH (85:13:2) as the eluent to yield 7.6 g (94%) of the

desired product. ¹H NMR (DMSO-d₆) δ 1.22 (s, 9H), 1.37−1.38 (m,

2H), 1.41−1.50 (m, 2H), 2.28 (t, J = 7.0 Hz, 2H), 2.39 (t, J = 5.1 Hz,

2H), 2.54 (t, J = 6.8 Hz, 2H), 3.57 (s, 4H), 6.47 (s, 1H). ESI MS m/z

306 (M + H)⁺.

4-(4-(2-(tert-Butyl)-6-methylpyrimidin-4-yl)piperazin-1-yl)-

butan-1-amine (43b). This compound was prepared from 2-(tert-

butyl)-4-chloro-6-methylpyrimidine using procedures analogous to

those described for compound 43a. Overall yield: 48%. ¹H NMR

(DMSO-d₆) δ 1.25 (s, 9H), 1.26−1.38 (m, 2H), 1.40−1.50 (m, 2H),

2.23 (s, 3H), 2.28 (t, J = 3.7 Hz, 2H), 2.39 (t, J = 5.1 Hz, 2H), 2.51 (t,

J = 6.9 Hz, 2H), 3.57 (t, J = 4.9 Hz, 4H), 6.46 (s, 1H). ESI MS m/z

306 (M + H)⁺.

Estimates of the K_dand maximum binding sites (B_max

)

were

50

obtained using unweighted linear regression analysis of data. Data

from competitive inhibition experiments were modeled using

nonlinear regression analysis to determine the concentration of

inhibitor that inhibits 50% of the speciﬁc binding of the radioligand

(IC₅₀). Since transfected cells expressing receptor were used for this

study, competition curves were modeled for a single site using

B₀

B +

+ B_ns

1 + (L/IC₅₀)

where B is the amount of ligand bound to tissue, B₀is the amount of

ligand bound in the absence of competitive inhibitor, L is the

concentration of the competitive inhibitor, B_nsis the nonspeciﬁc

binding of the radioligand (deﬁned using a high concentration of a

structurally dissimilar competitive inhibitor), and IC₅₀is the

concentration of competitive inhibitor that inhibits 50% of the total

speciﬁc binding. Data from competition dose−response curves were

analyzed using Tablecurve program (Jandel/Systat Software Inc., San

Jose, CA). IC₅₀values were converted to equilibrium dissociation

constants (K_ivalues).

4-(4-(2-(tert-Butyl)-6-cyclopropylpyrimidin-4-yl)piperazin-1-

yl)butan-1-amine (43c). This compound was prepared from 2-(tert-

butyl)-4-chloro-6-cyclopropylpyrimidine using procedures analogous

1

to those described for compound 43a. Overall yield: 28%. H NMR

(DMSO-d₆) δ 0.83−0.88 (m, 2H), 0.92−0.97 (m, 2H), 1.22 (s, 9H),

1.34−1.38 (m, 2H), 1.44−1.49 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.39

(t, J = 4.9 Hz, 2H), 2.56 (t, J = 6.8 Hz, 2H), 3.57 (t, J = 4.9 Hz, 4H),

6.49 (s, 1H). ESI MS m/z 332 (M + H)⁺.

Generation of Chimeric and Mutant Receptors. Four methods

were utilized to construct the D2/D3 receptor chimeras used in these

studies. The methods for the preparation of these chimeric receptor

genes have been previously described.^37,68,69The ﬁrst method exploits

a unique Pst1 restriction site in the ﬁrst intracellular loop (I1) that is

common to both the human D2 and human D3 receptor cDNAs. The

second method involved making chimeric D2/D3 receptors. A human

D3 receptor cDNA and a human D2 receptor cDNA were cloned in

tandem into the pIRESneo2 vector with a unique restriction site

(Hpa1) located between the two receptor cDNAs speciﬁcally within

the junctions at the helical TMS regions TMS IV and TMS V, using

site directed mutagenesis techniques (Quick-Change site-directed

mutagenesis kit, Stratagene/Agilent Technologies, Santa Clara, CA).

Finally, the D2/D3E2 and D3/D2E2 receptor loop chimeras were

prepared using the Quick Change kit strategy with synthetic

oligonucleotides encoding the E2 loop with the appropriate 5′ and

3′ ﬂanking region. The authenticity of the chimeric receptor was

veriﬁed by DNA sequencing, and the expression of the receptor

construct in HEK 293 cells was veriﬁed by radioligand binding using

4-(4-(2-(tert-Butyl)-6-(triﬂuoromethyl)pyrimidin-4-yl)-

piperazin-1-yl)butan-1-amine (43d). This compound was pre-

pared from 2-(tert-butyl)-4-chloro-6-(triﬂuoromethyl)pyrimidine using

procedures analogous to those described for compound 43a. Overall

yield: 33%. ¹H NMR (DMSO-d₆) δ 1.36 (s, 9H), 1.48−1.61 (m, 4H),

2.38 (t, J = 7.25 Hz, 2H), 2.51 (t, J = 5.15 Hz, 2H), 2.72 (t, J = 6.71

Hz, 2H), 3.72 (s, 4H), 6.59 (s, 1H). ESI MS m/z 360 (M + H)⁺.

4-(4-(2,6-Di-tert-butylpyrimidin-4-yl)piperazin-1-yl)butan-1-

amine (43e). This compound was prepared from 2,4-di-tert-butyl-6-

chloropyrimidine using procedures analogous to those described for

1

compound 43a. Overall yield: 46%. H NMR (DMSO-d₆) δ 1.24 (s,

9H), 1.27 (s, 9H), 1.34−1.49 (m, 4H), 2.29 (t, J = 7.5 Hz, 2H), 2.40

(t, J = 5.0 Hz, 2H), 2.53 (t, J = 6.71 Hz, 2H), 3.59 (t, J = 4.8 Hz, 4H),

6.47 (s, 1H). ESI MS m/z 348 (M + H)⁺.

4-(4-(2-(tert-Butyl)quinazolin-4-yl)piperazin-1-yl)butan-1-

amine (43f). This compound was prepared from 2-(tert-butyl)-4-

chloroquinazoline using procedures analogous to those described for

compound 43a. Overall yield: 66%. ESI MS m/z 342 (M + H)⁺.

4-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)butan-1-amine

(43g). This compound was prepared from 2-(tert-butyl)-4-chloroqui-

nazoline using procedures analogous to those described for compound

43a. Overall yield: 66%. ESI MS m/z 319 (M + H)⁺.

Binding Aﬃnity. A ﬁltration binding assay was used to

characterize the binding properties of the D2, D3, chimeric D2/D3,

and mutant dopamine receptors. Direct binding and competition

curves were performed using [¹²⁵I]IABN with dopamine receptors

stably expressed in HEK 293 cells. Stably transfected cells were

harvested by centrifugation. The cell pellet was resuspended in cold (4

°C) homogenization buﬀer (50 mM Tris-HCl, pH 7.4, with 10 mM

EDTA, 150 mM NaCl) by vortexing and then homogenizing with a

Polytron (Brinkmann Instruments, Westbury, NY). The homogenate

was centrifuged at 12 000g at 4 °C and the membrane pellet

resuspended in buﬀer and kept at −80 °C. Tissue homogenates (50

μL) were suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM

EDTA buﬀer, pH 7.5, and incubated with 50 μL of [¹²⁵I]IABN and 50

μL of test ligand at 37 °C for 60 min. Nonspeciﬁc binding was deﬁned

using 2 μM (+)-butaclamol. For competition experiments, the

radioligand concentration was generally equal to the K_dvalue and

[

¹²⁵I]IABN.

Mitogenesis Assays. Agonist stimulation of D2 or D3 dopamine

receptors leads to an increase in mitogenic activity.⁵⁴These assays are

based on [³H]thymidine uptake by cells that are proliferating. CHO

cells expressing human D2 or D3 receptors (CHOp-D2 or CHOp-

D3) were maintained in α-MEM with 10% FBS, 0.05% pen−strep, and

200 μg/mL of G418. To measure stimulation of mitogenesis (agonist

assay) or inhibition of quinpirole stimulation of mitogenesis

(antagonist assay), CHOp-D2 or CHOp-D3 cells were seeded in a

96-well plate at a concentration of 5000 cells/well. The cells were

incubated at 37 °C in α-MEM with 10% FBS. After 48−72 h, the cells

were rinsed twice with serum-free α-MEM and incubated for 24 h at

37 °C. Serial dilutions of test compounds were made by the Biomek

robotics system in serum-free α-MEM. In the functional assay for

agonists, the medium was removed and replaced with 100 μL of test

compound in serum-free α-MEM. In the antagonist assay, the serial

dilution of the putative antagonist test compound was added in 90 μL

(1.1× of ﬁnal concentration) and 300 nM quinpirole (30 nM ﬁnal)

was added in 10 μL. After another 24 h incubation at 37 °C of CHOp-

D2 cells or 16 h incubation of CHOp-D3 cells, 0.25 μCi

[³H]thymidine in α-MEM supplemented with 10% FBS was added

to each well and the plates were further incubated for 2 h at 37 °C.

O

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

The cells were trypsinized by addition of 10× trypsin solution (1%

trypsin in calcium−magnesium-free phosphate-buﬀered saline), and

the plates were ﬁltered and counted as usual. Quinpirole was run as an

internal control, and dopamine was included for comparative purposes.

Butaclamol was run as standard antagonist.

Data Analysis. Data were analyzed using GraphPad Prism, and

agonist potency was expressed as EC₅₀values or % stimulation.

Antagonist potency was expressed as IC₅₀values. In these assays, based

on the % maximum stimulation as compared to the standard agonist

quinpirole (100%), the compounds were classiﬁed into following

categories: ≥90% full agonist; 70−90% partial to full agonist; <70%

partial agonist; 0−20% potential antagonist.

Whole Cell Adenylyl Cyclase Assay. Whole cell cyclic AMP

accumulation was measured by an adaptation of the method of

Shimizu and co-workers.⁷⁰Transfected HEK 293 cells were treated

with serum-free medium containing [2,8-³H]adenine, and cells were

incubated at 37 °C for 75 min. The medium was replaced with serum-

free medium containing 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-

Aldrich, St. Louis, MO). Forskolin (100 μM ﬁnal) was added in the

presence or absence of test drug to a total volume of 500 μL and

incubated at 37 °C for 20 min. The reaction was stopped by addition

of 500 μL of 10% trichloroacetic acid and 1 mM cyclic AMP. After

centrifugation, the supernatants were fractionated using Dowex AG1-

X8 and neutral alumina to separate the [³H]ATP and the [³H]cAMP.

Individual samples were corrected for column recovery by monitoring

the recovery of the cyclic AMP using spectrophotometric analysis at

OD 259 nm. The percent conversion of [³H]ATP into [³H]cAMP was

then calculated as the percent of inhibition of the [³H]cAMP

accumulation relative to the assay performed in the absence of a test

compound, minus basal activity. The classic D2-like dopamine

receptor antagonist and agonist (haloperidol and quinpirole,

respectively) were included in the assay design as reference

compounds. The percent maximum response is the value for the

inhibition normalized to the value obtained for the full agonist

luminescence in the absence of the inhibitors, as the EC₅₀or IC₅₀

values of the compounds were greater than the highest tested

concentration of 5 μM. All of the compounds were tested in the

counterscreen for inhibition of β-galactosidase enzyme using the EFC

BlockDetect kit (DiscoveRx no. 92-0004) in dose response mode and

were found to be devoid of any signiﬁcant inhibitory activity at the

highest tested concentration of 5 μM.

[³⁵S]GTPγS Binding Assays. CHO cells expressing human D2_L

and D3 receptors served as the source for membrane fractions which

were washed and resuspended with assay buﬀer containing Mg²⁺, Na⁺,

EGTA, and bovine serum albumin as in our previous work.⁵⁵⁻⁵⁷The

GTPγS binding assays were performed in a ﬁnal volume of 0.5 mL for

CHO D2 and 1 mL for CHO D3 containing test compound or

dopamine (1 mM for D2 cells, and 100 μM for D3 cells) as indicator

of binding plateau, [³⁵S]GTPγS (0.11 nM for CHO D2 and 0.07 nM

for CHO D3, 1250 Ci/mmol, PerkinElmer, Boston, MA 02118), and

cell membrane suspension (in assay buﬀer and GDP for ﬁnal

concentration of 3 μM for CHO D2 and 6 μM for CHO D3). After

preincubation with test compounds, cell membranes and GDP were

placed for 15 min at 30 °C in a shaking water bath. [³⁵S]GTPγS was

added, and incubation proceeded for an additional 45 min. Cell

membranes were harvested on Brandel GF/B ﬁltermats with a 24-pin

Brandel harvester (Biomedical Research & Development Laboratories,

Inc., Gaithersburg, MD). Nonspeciﬁc binding of [³⁵S]GTPγS

measured in the presence of 10 μM GTPγS was a very small fraction

(5% or less) of basal binding in the absence of test compounds and

does not impact the EC₅₀(concentration producing half-maximal

stimulation) of the test compound estimated by nonlinear logarithmic

ﬁtting (logistics model) with OriginPro 7.0. The plateau binding

(maximal binding stimulation) with test compound was expressed as

percent of maximal binding observed with the full agonist dopamine

(% E_max). The test compounds 8, 25, 30, and 39 did not stimulate

GTPγS binding in three independent experiments. The last three

compounds at high micromolar concentrations showed weak inverse

agonist activity at D3R resulting in [³⁵S]GTPγS binding below

baseline, but this was not further studied in the present work.

Antagonist activity was assessed by testing a ﬁxed concentration that

by itself had little or no inverse agonist eﬀect for its ability to shift the

concentration curve of an agonist stimulating [³⁵S]GTPγS binding as

described for opioid receptors by Sally and co-workers.⁵⁹The ﬁxed

concentrations of test compound were as listed in the Results and

Discussion, and the agonist used was dopamine (0.001 μM to 100 uM

for CHO D2 and 0.1 nM to 10 μM for CHO D3). K_eis the functional

K_i(equilibrium dissociation constant) of an antagonist and is

calculated according to the following equation: [test compound]/

(EC_50‑2/EC_50‑1− 1), where EC_50‑2is the EC₅₀value in the presence of

the test compound and EC_50‑1is the value in the absence of the test

compound.

quinpirole. Values are reported as the mean values

SEM. The

concentrations for all test compounds was ≥10× the K_ivalue of the

compound at human D2 or D3 dopamine receptors, obtained from

competitive radioligand binding experiments.⁶⁹

β-Arrestin Recruitment Assays. Eﬀect of compounds on agonist

induced recruitment of β-arrestin by D3R and D2_LR was measured

using DiscoverRx PathHunter eXpress kits. Brieﬂy, U2OS DRD3

(DiscoveRx no. 93-0591E3) and CHO-K1 DRD2L (DiscoveRx no.

93-0579E2) cells were seeded at a density of 2500 cells/well in 384-

well white, clear-bottom plates (Corning no. 3707) with PathHunter

Cell Plating Reagents, CP0 (DiscoveRx no. 93-0563R0) and CP2

(DiscoveRx no. 93-0563R2), respectively, and incubated at 37 °C for

48 h. For the determination of EC₈₀values of standard agonists, the

cells were treated with multiple concentrations (starting from 190 nM,

1:3 dilution, 12 dose points) of (+)-PD128907 (DiscoveRx no. 92-

1163) for D3R and Pergolide (DiscoveRx no. 92-1162) for D2_LR.

After incubation at 37 °C for 90 min, the DiscoveRx detection reagent

was added, the plates were incubated at room temperature for 60 min,

and the luminescence was measured on a Synergy 4 plate reader

(BioTek). The data were plotted, and the agonist EC₈₀values were

calculated using GraphPad Prism software. For antagonist activity, the

cells were plated as described above and treated with multiple

concentrations (starting from 500 nM, half log dilution, 10 dose

points) of the test compounds in plating reagent with 0.1% DMSO.

Following a 30 min preincubation at 37 °C, the cells were treated with

an EC₈₀dose (8 nM PD128907 for D3R and 6 nM pergolide for D2R)

of the agonist and incubated at 37 °C for an additional 90 min. The

DiscoveRx detection reagent was then added, and the plates were

incubated at room temperature for 60 min. Luminescence was

measured, and the IC₅₀values were calculated. For determination of

agonist or inverse agonist activity, the cells were plated and incubated

for 48 h as described above and treated with multiple concentrations

(starting from 5 μM, half log dilution, 11 dose points) of the test

compounds, incubated at 37 °C for 120 min, treated with detection

reagents, and the luminescence was measured. The activity was

calculated as percent stimulation or inhibition of the basal

Molecular Modeling and Ligand Docking. Reﬁnement of the

Dopamine D3 Receptor Crystal Structure. The human D3R crystal

structure in complex with the antagonist eticlopride at 2.89 Å

resolution³⁹(PDB code 3PBL) was reﬁned using Prime preparation

and reﬁnement tools of the Protein Preparation Wizard implemented

in the Schrodinger software package. After the addition of hydrogens

̈

and detection of disulﬁde bonds the structure was optimized by

applying default parameters of the Impref utility using the OPLS2001

force ﬁeld. Ligand structures were prepared using the LigPrep utility at

neutral pH.

D2R Homology Model Development. A model of the D2 receptor

was developed based on the human D2 receptor sequence

(UniProtKB accession code P14416) and the D3 receptor crystal

structure (PDB code 3PBL) as the homology modeling template. The

generated model includes transmembrane and loop regions except for

intracellular loop 3, which is missing in the D3 crystal structure where

it is replaced by T4-lysozyme. InsightII/Homology modeling tools

were used for initial homology model building. D2 loop conformations

in loop regions containing gap(s) within the D3/D2 sequence

alignment were generated in sets of 10. From these, D2 loop structures

that closely overlap with the corresponding D3 loops in the crystal

structure were selected. Loop side chains were adjusted using a built-in

P

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

rotamer library to avoid steric clashes with the rest of the structure.

Disulﬁde bridges involving any cysteine residues in loops were

reproduced in the D2 model structure except for a disulﬁde bond that

is within extracellular loop III, D3:Cys355-Cys358, which could not be

reproduced in D2 because the loop is shorter in the D2R than in the

D3R. The sequence identity between our D2 model and the D3 crystal

structure is 71.5% (including loops). In order to relax the obtained D2

homology model structure, reﬁnement tools of the Protein Preparation

Wizard were applied. The structure was optimized in the OPLS2001

force ﬁeld using default parameters of the Impref utility, analogous to

the reﬁnement of the D3 receptor crystal structure. The root-mean-

square deviation of α-carbon atoms between the reﬁned D2R model

and D3R crystal structure was 0.7.

receptor; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-

methyluronium hexaﬂuorophosphate; HEK, human embryonic

kidney; IABN, 2,3-dimethoxy-5-iodo-N-(9-benzyl-9-azabicyclo-

[3.3.1]nonan-3-yl)benzamide; TM, transmembrane domain

REFERENCES

■

(1) Joyce, J. N.; Millan, M. J. Dopamine D3 receptor antagonists as

therapeutic agents. Drug Discovery Today 2005, 10, 917−925.

(2) Sokoloff, P.; Diaz, J.; Le Foll, B.; Guillin, O.; Leriche, L.; Bezard,

E.; Gross, C. The dopamine D3 receptor: a therapeutic target for the

treatment of neuropsychiatric disorders. CNS Neurol. Disord.: Drug

Targets 2006, 5, 25−43.

InducedFit Ligand Docking. InducedFit docking combines Glide

docking with Prime structural reﬁnement tools to account for side

chain ﬂexibility in the ligand binding site.⁷¹The center of the docking

grid was deﬁned as the centroid of the cocrystallized eticlopride; the

enclosing box size was determined automatically. InducedFit settings

were at default values except for removing the side chain of a Tyr

(D3:Tyr373, D2:387) in the initial step of the protein preparation

constrained reﬁnement in order to facilitate the initial docking of

ligands longer than eticlopride. In the InducedFit workﬂow this step

was followed by an initial Glide docking run using softened potentials

(van der Waals scaling of 0.70 for the receptor and 0.50 for ligands).

The obtained structures were then reﬁned using Prime side chain

optimization of residues within 5 Å from docked ligand poses. The

derived “induced-ﬁt” receptor structures were then utilized for the ﬁnal

step of Glide re-docking with default parameters, applied to structures

within 30 kcal/mol of the lowest energy structures. The same

InducedFit protocols were applied for ligand docking at both D3 and

D2 receptor structures.

(3) Heidbreder, C. A.; Gardner, E. L.; Xi, Z. X.; Thanos, P. K.;

Mugnaini, M.; Hagan, J. J.; Ashby, C. R., Jr. The role of central

dopamine D3 receptors in drug addiction: a review of pharmacological

evidence. Brain Res. Rev. 2005, 49, 77−105.

(4) Acri, J. B.; Carter, S. R.; Alling, K.; Geter-Douglass, B.; Dijkstra,

D.; Wikstrom, H.; Katz, J. L.; Witkin, J. M. Assessment of cocaine-like

discriminative stimulus effects of dopamine D3 receptor ligands. Eur. J.

Pharmacol. 1995, 281, R7−R9.

(5) Spealman, R. D. Dopamine D3 receptor agonists partially

reproduce the discriminative stimulus effects of cocaine in squirrel

monkeys. J. Pharmacol. Exp. Ther. 1996, 278, 1128−1137.

(6) Nader, M. A.; Mach, R. H. Self-administration of the dopamine

D3 agonist 7-OH-DPAT in rhesus monkeys is modified by prior

cocaine exposure. Psychopharmacology (Berlin, Ger.) 1996, 125, 13−22.

(7) Caine, S. B.; Koob, G. F. Modulation of cocaine self-

administration in the rat through D-3 dopamine receptors. Science

(New York, N.Y.) 1993, 260, 1814−1816.

(8) Self, D. W.; Barnhart, W. J.; Lehman, D. A.; Nestler, E. J.

Opposite modulation of cocaine-seeking behavior by D1- and D2-like

dopamine receptor agonists. Science (New York, N.Y.) 1996, 271,

1586−1589.

(9) Cheung, T. H.; Loriaux, A. L.; Weber, S. M.; Chandler, K. N.;

Lenz, J. D.; Schaan, R. F.; Mach, R. H.; Luedtke, R. R.; Neisewander, J.

L. Reduction of cocaine self-administration and D3 receptor-mediated

behavior by two novel dopamine D3 receptor-selective partial agonists,

OS-3-106 and WW-III-55. J. Pharmacol. Exp. Ther. 2013, 347, 410−

423.

(10) Staley, J. K.; Mash, D. C. Adaptive increase in D3 dopamine

receptors in the brain reward circuits of human cocaine fatalities. J.

Neurosci. 1996, 16, 6100−6106.

(11) Segal, D. M.; Moraes, C. T.; Mash, D. C. Up-regulation of D3

dopamine receptor mRNA in the nucleus accumbens of human

cocaine fatalities. Mol. Brain Res. 1997, 45, 335−339.

ASSOCIATED CONTENT

* Supporting Information

■

S

Figures showing docked poses; Cartesian coordinates of D3R,

D2R residues, and docked compounds. This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: 205-581-2822. Fax: 205-581-2726. E-mail: ananthan@

southernresearch.org.

■

Present Address

^#G.Z.: Department of Basic Science, Touro University

California, Vallejo, California, 94592.

(12) Boileau, I.; Payer, D.; Houle, S.; Behzadi, A.; Rusjan, P. M.;

Tong, J.; Wilkins, D.; Selby, P.; George, T. P.; Zack, M.; Furukawa, Y.;

McCluskey, T.; Wilson, A. A.; Kish, S. J. Higher binding of the

dopamine D3 receptor-preferring ligand [11C]-(+)-propyl-hexahydro-

naphtho-oxazin in methamphetamine polydrug users: a positron

emission tomography study. J. Neurosci. 2012, 32, 1353−1359.

(13) Pilla, M.; Perachon, S.; Sautel, F.; Garrido, F.; Mann, A.;

Wermuth, C. G.; Schwartz, J. C.; Everitt, B. J.; Sokoloff, P. Selective

inhibition of cocaine-seeking behaviour by a partial dopamine D3

receptor agonist. Nature 1999, 400, 371−375.

(14) Stemp, G.; Ashmeade, T.; Branch, C. L.; Hadley, M. S.; Hunter,

A. J.; Johnson, C. N.; Nash, D. J.; Thewlis, K. M.; Vong, A. K.; Austin,

N. E.; Jeffrey, P.; Avenell, K. Y.; Boyfield, I.; Hagan, J. J.; Middlemiss,

D. N.; Reavill, C.; Riley, G. J.; Routledge, C.; Wood, M. Design and

synthesis of trans-N-[4-[2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-

yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB-277011): a potent

and selective dopamine D(3) receptor antagonist with high oral

bioavailability and CNS penetration in the rat. J. Med. Chem. 2000, 43,

1878−1885.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This investigation was supported by NIH Grant R01DA024675

and Contract N01-DA-1-8827 from the National Institute on

Drug Abuse (NIDA). We thank the NIDA Addiction

Treatment Discovery Program for providing mitogenesis

functional assay data through Inter-Agency Agreement Y1-

DA-5007-05. We thank Dr. James M. Riordan, Mark D.

Richardson, Joan C. Bearden, and Jackie W. Truss for analytical

and spectral data. We are grateful to Dr. John A. Secrist III for

his encouragement, valuable comments, and suggestions

throughout the course of this work.

ABBREVIATIONS USED

■

BOP-Cl, bis(2-oxo-3-oxazolidinyl)phosphinic chloride; CHO,

Chinese hamster ovary; CLogP, calculated log of partition

coeﬃcient; D2R, dopamine D2 receptor; D3R, dopamine D3

receptor; EL, extracellular loop; GPCR, G-protein-coupled

(15) Vorel, S. R.; Ashby, C. R., Jr.; Paul, M.; Liu, X.; Hayes, R.;

Hagan, J. J.; Middlemiss, D. N.; Stemp, G.; Gardner, E. L. Dopamine

D3 receptor antagonism inhibits cocaine-seeking and cocaine-

enhanced brain reward in rats. J. Neurosci. 2002, 22, 9595−9603.

Q

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(16) Grundt, P.; Carlson, E. E.; Cao, J.; Bennett, C. J.; McElveen, E.;

Taylor, M.; Luedtke, R. R.; Newman, A. H. Novel heterocyclic trans

olefin analogues of N-{4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl}-

arylcarboxamides as selective probes with high affinity for the

dopamine D3 receptor. J. Med. Chem. 2005, 48, 839−848.

(17) Higley, A. E.; Spiller, K.; Grundt, P.; Newman, A. H.; Kiefer, S.

W.; Xi, Z. X.; Gardner, E. L. PG01037, a novel dopamine D3 receptor

antagonist, inhibits the effects of methamphetamine in rats. J.

Psychopharmacol. 2011, 25, 263−273.

(18) Micheli, F.; Arista, L.; Bonanomi, G.; Blaney, F. E.; Braggio, S.;

Capelli, A. M.; Checchia, A.; Damiani, F.; Di-Fabio, R.; Fontana, S.;

Gentile, G.; Griffante, C.; Hamprecht, D.; Marchioro, C.; Mugnaini,

M.; Piner, J.; Ratti, E.; Tedesco, G.; Tarsi, L.; Terreni, S.; Worby, A.;

Ashby, C. R., Jr.; Heidbreder, C. 1,2,4-Triazolyl azabicyclo[3.1.0]-

hexanes: a new series of potent and selective dopamine D(3) receptor

antagonists. J. Med. Chem. 2010, 53, 374−391.

(19) Roman, V.; Gyertyan, I.; Saghy, K.; Kiss, B.; Szombathelyi, Z.

Cariprazine (RGH-188), a D(3)-preferring dopamine D (3)/D (2)

receptor partial agonist antipsychotic candidate demonstrates anti-

abuse potential in rats. Psychopharmacology (Berlin, Ger.) 2013, 226,

285−293.

(20) Heidbreder, C. Selective antagonism at dopamine D3 receptors

as a target for drug addiction pharmacotherapy: a review of preclinical

evidence. CNS Neurol. Disord.: Drug Targets 2008, 7, 410−421.

(21) Lacroix, L. P.; Hows, M. E.; Shah, A. J.; Hagan, J. J.; Heidbreder,

C. A. Selective antagonism at dopamine D3 receptors enhances

monoaminergic and cholinergic neurotransmission in the rat anterior

cingulate cortex. Neuropsychopharmacology 2003, 28, 839−849.

(22) Gurevich, E. V.; Bordelon, Y.; Shapiro, R. M.; Arnold, S. E.; Gur,

R. E.; Joyce, J. N. Mesolimbic dopamine D3 receptors and use of

antipsychotics in patients with schizophrenia. A postmortem study.

Arch. Gen. Psychiatry 1997, 54, 225−232.

(23) Millan, M. J.; Loiseau, F.; Dekeyne, A.; Gobert, A.; Flik, G.;

Cremers, T. I.; Rivet, J. M.; Sicard, D.; Billiras, R.; Brocco, M. S33138

(N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano-

[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenyl-acetamide), a preferential dop-

amine D3 versus D2 receptor antagonist and potential antipsychotic

agent: III. Actions in models of therapeutic activity and induction of

side effects. J. Pharmacol. Exp. Ther. 2008, 324, 1212−1226.

(24) Millan, M. J.; Buccafusco, J. J.; Loiseau, F.; Watson, D. J.;

Decamp, E.; Fone, K. C.; Thomasson-Perret, N.; Hill, M.; Mocaer, E.;

Schneider, J. S. The dopamine D3 receptor antagonist, S33138,

counters cognitive impairment in a range of rodent and primate

procedures. Int. J. Neuropsychopharmacol. 2010, 13, 1035−1051.

(25) Agai-Csongor, E.; Nogradi, K.; Galambos, J.; Vago, I.; Bielik, A.;

Magdo, I.; Ignacz-Szendrei, G.; Keseru, G. M.; Greiner, I.; Laszlovszky,

I.; Schmidt, E.; Kiss, B.; Saghy, K.; Laszy, J.; Gyertyan, I.; Zajer-Balazs,

M.; Gemesi, L.; Domany, G. Novel sulfonamides having dual

dopamine D2 and D3 receptor affinity show in vivo antipsychotic

efficacy with beneficial cognitive and EPS profile. Bioorg. Med. Chem.

Lett. 2007, 17, 5340−5344.

tics for addictions and related disorders. Ann. N.Y. Acad. Sci. 2010,

1187, 4−34.

(30) Lober, S.; Hubner, H.; Tschammer, N.; Gmeiner, P. Recent

advances in the search for D3- and D4-selective drugs: probes, models

and candidates. Trends Pharmacol. Sci. 2011, 32, 148−157.

(31) Micheli, F. Recent advances in the development of dopamine

D3 receptor antagonists: a medicinal chemistry perspective. Chem-

MedChem 2011, 6, 1152−1162.

(32) Newman, A. H.; Blaylock, B. L.; Nader, M. A.; Bergman, J.;

Sibley, D. R.; Skolnick, P. Medication discovery for addiction:

translating the dopamine D3 receptor hypothesis. Biochem. Pharmacol.

2012, 84, 882−890.

(33) Ye, N.; Neumeyer, J. L.; Baldessarini, R. J.; Zhen, X.; Zhang, A.

Update 1 of: Recent progress in development of dopamine receptor

subtype-selective agents: potential therapeutics for neurological and

psychiatric disorders. Chem. Rev. 2013, 113, PR123−PR178.

(34) Micheli, F.; Heidbreder, C. Dopamine D3 receptor antagonists:

a patent review (2007−2012). Expert Opin. Ther. Pat. 2013, 23, 363−

381.

(35) Leopoldo, M.; Berardi, F.; Colabufo, N. A.; De Giorgio, P.;

Lacivita, E.; Perrone, R.; Tortorella, V. Structure−affinity relationship

study on N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides as potent

and selective dopamine D(3) receptor ligands. J. Med. Chem. 2002, 45,

5727−5735.

(36) Newman, A. H.; Cao, J.; Bennett, C. J.; Robarge, M. J.; Freeman,

R. A.; Luedtke, R. R. N-(4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]-

butyl, butenyl and butynyl)arylcarboxamides as novel dopamine D(3)

receptor antagonists. Bioorg. Med. Chem. Lett. 2003, 13, 2179−2183.

(37) Banala, A. K.; Levy, B. A.; Khatri, S. S.; Furman, C. A.; Roof, R.

A.; Mishra, Y.; Griffin, S. A.; Sibley, D. R.; Luedtke, R. R.; Newman, A.

H. N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-

yl)butyl)arylcarboxamides as selective dopamine D3 receptor ligands:

critical role of the carboxamide linker for D3 receptor selectivity. J.

Med. Chem. 2011, 54, 3581−3594.

(38) Newman, A. H.; Beuming, T.; Banala, A. K.; Donthamsetti, P.;

Pongetti, K.; Labounty, A.; Levy, B.; Cao, J.; Michino, M.; Luedtke, R.

R.; Javitch, J. A.; Shi, L. Molecular determinants of selectivity and

efficacy at the dopamine D3 receptor. J. Med. Chem. 2012, 55, 6689−

6699.

(39) Chien, E. Y.; Liu, W.; Zhao, Q.; Katritch, V.; Han, G. W.;

Hanson, M. A.; Shi, L.; Newman, A. H.; Javitch, J. A.; Cherezov, V.;

Stevens, R. C. Structure of the human dopamine D3 receptor in

complex with a D2/D3 selective antagonist. Science (New York, N.Y.)

2010, 330, 1091−1095.

(40) Hobrath, J. V.; Wang, S. Computational elucidation of the

structural basis of ligand binding to the dopamine 3 receptor through

docking and homology modeling. J. Med. Chem. 2006, 49, 4470−4476.

(41) Wang, Q.; Mach, R. H.; Luedtke, R. R.; Reichert, D. E. Subtype

selectivity of dopamine receptor ligands: insights from structure and

ligand-based methods. J. Chem. Inf. Model. 2010, 50, 1970−1985.

(42) Campiani, G.; Butini, S.; Trotta, F.; Fattorusso, C.; Catalanotti,

B.; Aiello, F.; Gemma, S.; Nacci, V.; Novellino, E.; Stark, J. A.;

Cagnotto, A.; Fumagalli, E.; Carnovali, F.; Cervo, L.; Mennini, T.

Synthesis and pharmacological evaluation of potent and highly

selective D3 receptor ligands: inhibition of cocaine-seeking behavior

and the role of dopamine D3/D2 receptors. J. Med. Chem. 2003, 46,

3822−3839.

(26) Agai-Csongor, E.; Domany, G.; Nogradi, K.; Galambos, J.; Vago,

I.; Keseru, G. M.; Greiner, I.; Laszlovszky, I.; Gere, A.; Schmidt, E.;

Kiss, B.; Vastag, M.; Tihanyi, K.; Saghy, K.; Laszy, J.; Gyertyan, I.;

Zajer-Balazs, M.; Gemesi, L.; Kapas, M.; Szombathelyi, Z. Discovery of

cariprazine (RGH-188): a novel antipsychotic acting on dopamine

D3/D2 receptors. Bioorg. Med. Chem. Lett. 2012, 22, 3437−3440.

(27) Gyertyan, I.; Kiss, B.; Saghy, K.; Laszy, J.; Szabo, G.; Szabados,

T.; Gemesi, L. I.; Pasztor, G.; Zajer-Balazs, M.; Kapas, M.; Csongor, E.

A.; Domany, G.; Tihanyi, K.; Szombathelyi, Z. Cariprazine (RGH-

188), a potent D3/D2 dopamine receptor partial agonist, binds to

dopamine D3 receptors in vivo and shows antipsychotic-like and

procognitive effects in rodents. Neurochem. Int. 2011, 59, 925−935.

(28) Newman, A. H.; Grundt, P.; Nader, M. A. Dopamine D3

receptor partial agonists and antagonists as potential drug abuse

therapeutic agents. J. Med. Chem. 2005, 48, 3663−3679.

(43) Chu, W.; Tu, Z.; McElveen, E.; Xu, J.; Taylor, M.; Luedtke, R.

R.; Mach, R. H. Synthesis and in vitro binding of N-phenyl piperazine

analogs as potential dopamine D3 receptor ligands. Bioorg. Med. Chem.

2005, 13, 77−87.

(44) Newman, A. H.; Grundt, P.; Cyriac, G.; Deschamps, J. R.;

Taylor, M.; Kumar, R.; Ho, D.; Luedtke, R. R. N-(4-(4-(2,3-Dichloro-

or 2-methoxyphenyl)piperazin-1-yl)butyl)heterobiarylcarboxamides

with functionalized linking chains as high affinity and enantioselective

D3 receptor antagonists. J. Med. Chem. 2009, 52, 2559−2570.

(45) Bettinetti, L.; Schlotter, K.; Hubner, H.; Gmeiner, P. Interactive

SAR studies: rational discovery of super-potent and highly selective

(29) Heidbreder, C. A.; Newman, A. H. Current perspectives on

selective dopamine D(3) receptor antagonists as pharmacotherapeu-

R

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

dopamine D3 receptor antagonists and partial agonists. J. Med. Chem.

2002, 45, 4594−4597.

(46) Hocke, C.; Prante, O.; Lober, S.; Hubner, H.; Gmeiner, P.;

Kuwert, T. Synthesis and radioiodination of selective ligands for the

dopamine D3 receptor subtype. Bioorg. Med. Chem. Lett. 2004, 14,

3963−3966.

(47) Leopoldo, M.; Lacivita, E.; De Giorgio, P.; Colabufo, N. A.;

Niso, M.; Berardi, F.; Perrone, R. Design, synthesis, and binding

affinities of potential positron emission tomography (PET) ligands for

visualization of brain dopamine D3 receptors. J. Med. Chem. 2006, 49,

358−365.

(48) Taylor, M.; Grundt, P.; Griffin, S. A.; Newman, A. H.; Luedtke,

R. R. Dopamine D3 receptor selective ligands with varying intrinsic

efficacies at adenylyl cyclase inhibition and mitogenic signaling

pathways. Synapse (New York, N.Y.) 2010, 64, 251−266.

(49) Grundt, P.; Prevatt, K. M.; Cao, J.; Taylor, M.; Floresca, C. Z.;

Choi, J. K.; Jenkins, B. G.; Luedtke, R. R.; Newman, A. H. Heterocyclic

analogues of N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-

arylcarboxamides with functionalized linking chains as novel dopamine

D3 receptor ligands: potential substance abuse therapeutic agents. J.

Med. Chem. 2007, 50, 4135−4146.

(50) Geneste, H.; Backfisch, G.; Braje, W.; Delzer, J.; Haupt, A.;

Hutchins, C. W.; King, L. L.; Kling, A.; Teschendorf, H. J.; Unger, L.;

Wernet, W. Synthesis and SAR of highly potent and selective

dopamine D(3)-receptor antagonists: 1H-pyrimidin-2-one derivatives.

Bioorg. Med. Chem. Lett. 2006, 16, 490−494.

(51) Geneste, H.; Amberg, W.; Backfisch, G.; Beyerbach, A.; Braje,

W. M.; Delzer, J.; Haupt, A.; Hutchins, C. W.; King, L. L.; Sauer, D. R.;

Unger, L.; Wernet, W. Synthesis and SAR of highly potent and

selective dopamine D3-receptor antagonists: variations on the 1H-

pyrimidin-2-one theme. Bioorg. Med. Chem. Lett. 2006, 16, 1934−1937.

(52) Geneste, H.; Backfisch, G.; Braje, W.; Delzer, J.; Haupt, A.;

Hutchins, C. W.; King, L. L.; Lubisch, W.; Steiner, G.; Teschendorf, H.

J.; Unger, L.; Wernet, W. Synthesis and SAR of highly potent and

selective dopamine D(3)-receptor antagonists: quinolin(di)one and

benzazepin(di)one derivatives. Bioorg. Med. Chem. Lett. 2006, 16,

658−662.

(53) Michino, M.; Donthamsetti, P.; Beuming, T.; Banala, A.; Duan,

L.; Roux, T.; Han, Y.; Trinquet, E.; Newman, A. H.; Javitch, J. A.; Shi,

L. A single glycine in extracellular loop 1 is the critical determinant for

pharmacological specificity of dopamine D2 and D3 receptors. Mol.

Pharmacol. 2013, 84, 854−864.

(54) Pilon, C.; Levesque, D.; Dimitriadou, V.; Griffon, N.; Martres,

M. P.; Schwartz, J. C.; Sokoloff, P. Functional coupling of the human

dopamine D3 receptor in a transfected NG 108-15 neuroblastoma-

glioma hybrid cell line. Eur. J. Pharmacol. 1994, 268, 129−139.

(55) Bassoni, D. L.; Raab, W. J.; Achacoso, P. L.; Loh, C. Y.;

Wehrman, T. S. Measurements of beta-arrestin recruitment to

activated seven transmembrane receptors using enzyme complemen-

tation. Methods Mol. Biol. 2012, 897, 181−203.

(56) Johnson, M.; Antonio, T.; Reith, M. E.; Dutta, A. K. Structure−

activity relationship study of N(6)-(2-(4-(1H-indol-5-yl)piperazin-1-

yl)ethyl)-N(6)-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine

analogues: development of highly selective D3 dopamine receptor

agonists along with a highly potent D2/D3 agonist and their

pharmacological characterization. J. Med. Chem. 2012, 55, 5826−5840.

(57) Biswas, S.; Zhang, S.; Fernandez, F.; Ghosh, B.; Zhen, J.;

Kuzhikandathil, E.; Reith, M. E.; Dutta, A. K. Further structure−

activity relationships study of hybrid 7-{[2-(4-phenylpiperazin-1-

yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol analogues:

identification of a high-affinity D3-preferring agonist with potent in

vivo activity with long duration of action. J. Med. Chem. 2008, 51,

101−117.

(59) Sally, E. J.; Xu, H.; Dersch, C. M.; Hsin, L. W.; Chang, L. T.;

Prisinzano, T. E.; Simpson, D. S.; Giuvelis, D.; Rice, K. C.; Jacobson,

A. E.; Cheng, K.; Bilsky, E. J.; Rothman, R. B. Identification of a novel

“almost neutral” mu-opioid receptor antagonist in CHO cells

expressing the cloned human mu-opioid receptor. Synapse (New

York, N.Y.) 2010, 64, 280−288.

(60) Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.;

Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.;

Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B. Functional

selectivity and classical concepts of quantitative pharmacology. J.

Pharmacol. Exp. Ther. 2007, 320, 1−13.

(61) Kobilka, B. K.; Deupi, X. Conformational complexity of G-

protein-coupled receptors. Trends Pharmacol. Sci. 2007, 28, 397−406.

(62) Shukla, A. K.; Xiao, K.; Lefkowitz, R. J. Emerging paradigms of

beta-arrestin-dependent seven transmembrane receptor signaling.

Trends Biochem. Sci. 2011, 36, 457−469.

(63) Tschammer, N.; Elsner, J.; Goetz, A.; Ehrlich, K.; Schuster, S.;

Ruberg, M.; Kuhhorn, J.; Thompson, D.; Whistler, J.; Hubner, H.;

Gmeiner, P. Highly potent 5-aminotetrahydropyrazolopyridines:

enantioselective dopamine D3 receptor binding, functional selectivity,

and analysis of receptor−ligand interactions. J. Med. Chem. 2011, 54,

2477−2491.

(64) Allen, J. A.; Yost, J. M.; Setola, V.; Chen, X.; Sassano, M. F.;

Chen, M.; Peterson, S.; Yadav, P. N.; Huang, X. P.; Feng, B.; Jensen,

N. H.; Che, X.; Bai, X.; Frye, S. V.; Wetsel, W. C.; Caron, M. G.;

Javitch, J. A.; Roth, B. L.; Jin, J. Discovery of beta-arrestin-biased

dopamine D2 ligands for probing signal transduction pathways

essential for antipsychotic efficacy. Proc. Natl. Acad. Sci. U.S.A. 2011,

108, 18488−18493.

(65) Chen, X.; Sassano, M. F.; Zheng, L.; Setola, V.; Chen, M.; Bai,

X.; Frye, S. V.; Wetsel, W. C.; Roth, B. L.; Jin, J. Structure−functional

selectivity relationship studies of beta-arrestin-biased dopamine D(2)

receptor agonists. J. Med. Chem. 2012, 55, 7141−7153.

(66) Galaj, E.; Ananthan, S.; Saliba, M.; Ranaldi, R. The effects of the

novel DA D3 receptor antagonist SR 21502 on cocaine reward,

cocaine seeking and cocaine-induced locomotor activity in rats.

Psychopharmacology (Berlin, Ger.) 2014, 231, 501−510.

(67) Hachimine, P.; Seepersad, N.; Ananthan, S.; Ranaldi, R. The

novel dopamine D3 receptor antagonist, SR 21502, reduces cocaine

conditioned place preference in rats. Neurosci. Lett. 2014, 569, 137−

141.

(68) Luedtke, R. R.; Mishra, Y.; Wang, Q.; Griffin, S. A.; Bell-Horner,

C.; Taylor, M.; Vangveravong, S.; Dillon, G. H.; Huang, R. Q.;

Reichert, D. E.; Mach, R. H. Comparison of the binding and functional

properties of two structurally different D2 dopamine receptor subtype

selective compounds. ACS Chem. Neurosci. 2012, 3, 1050−1062.

(69) Silvano, E.; Millan, M. J.; Mannoury la Cour, C.; Han, Y.; Duan,

L.; Griffin, S. A.; Luedtke, R. R.; Aloisi, G.; Rossi, M.; Zazzeroni, F.;

Javitch, J. A.; Maggio, R. The tetrahydroisoquinoline derivative

SB269,652 is an allosteric antagonist at dopamine D3 and D2

receptors. Mol. Pharmacol. 2010, 78, 925−934.

(70) Shimizu, H.; Daly, J. W.; Creveling, C. R. A radioisotopic

method for measuring the formation of adenosine 3′,5′-cyclic

monophosphate in incubated slices of brain. J. Neurochem. 1969, 16,

1609−1619.

(71) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R.

Novel procedure for modeling ligand/receptor induced fit effects. J.

Med. Chem. 2006, 49, 534−553.

(58) Gogoi, S.; Antonio, T.; Rajagopalan, S.; Reith, M.; Andersen, J.;

Dutta, A. K. Dopamine D(2)/D(3) agonists with potent iron

chelation, antioxidant and neuroprotective properties: potential

implication in symptomatic and neuroprotective treatment of

Parkinson’s disease. ChemMedChem 2011, 6, 991−995.

S

dx.doi.org/10.1021/jm500801r | J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Design, synthesis, and structure-activity relationship studies of a series of [4-(4-carboxamidobutyl)]-1-arylpiperazines: Insights into structural features contributing to dopamine D3 versus D2 receptor subtype selectivity

DOI: 10.1021/jm500801r

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm500801r

Full text of DOI:10.1021/jm500801r

Products guided by the article

R&D Labs maybe for 1596371-68-4

Relevant to this article

Hot Product