Design, synthesis, and evaluation of bitopic arylpiperazine-phthalimides as selective dopamine D3 receptor agonists

Article abstract of DOI:10.1039/c8md00237a

The dopamine D₃ receptor (D₃R) is a proven therapeutic target for the treatment of neurological and neuropsychiatric disorders. In particular, D₃R-selective ligands that can eliminate side effects associated with dopamine D₂ receptor (D₂R) therapeutics have been validated. However, the high homology in signaling pathways and the sequence similarity between D₂R and D₃R have rendered the development of D₃R-selective ligands challenging. Herein, we designed and synthesized a series of piperazine-phthalimide bitopic ligands based on a fragment-based and molecular docking inspired design. Compound 9i was identified as the most selective D₃R ligand among these bitopic ligands. Its selectivity was improved compared to reference compounds 1 and 2 by 9- and 2-fold, respectively, and it was 21-fold more potent than compound 2. Molecular docking demonstrated that the orientation of Leu^2.64 and Phe^7.39 and the packing at the junction of helices may affect the specificity for D₃R over D₂R. Functional evaluation revealed that D₃R-selective ligand 9i displayed a subpicomolar agonist activity at D₃R with a 199-fold increase in potency compared to quinpirole. These results may be useful for the fragment-based design of bitopic compounds as selective D₃R ligands.

Full text of DOI:10.1039/c8md00237a

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10.1039/C8MD00237A.
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DOI: 10.1039/C8MD00237A
Design, Synthesis, and Evaluation of Bitopic Arylpiperazineꢀphthalimides as Selective
Dopamine D3 Receptor Agonists
Yongkai Caoa,b,c,1, Ningning Sunb,1, Jiumei Zhangc,1, Zhiguo Liud, Yiꢀzhe Tangc, Zhengzhi
Wuc,*, KyeongꢀMan Kimb,* and Seung Hoon Cheonb,∗
a Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou
510632, China
b College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju
500ꢀ757, Republic of Korea
c The Fist Affiliated Hospital of Shenzhen University, Shenzhen 518035,China
d Chemical Biology Research at School of Pharmaceutical sciences, Wenzhou Medical University, Wenzhou
325035, China
Abstract
The dopamine D3 receptor (D3R) is a proven therapeutic target for the treatment of
neurological and neuropsychiatric disorders. In particular, D3Rꢀselecitve ligands that can
eliminate side effects associated with dopamine D2R receptor (D2R) therapeutics have been
validated. However, the high homology in signaling pathways and sequence similarity
between D2R and D3R have rendered the development of D3Rꢀselective ligands challenging.
Herein, we designed and synthesized a series of piperazineꢀphthalimide bitopic ligands based
on a fragmentꢀbased and molecular docking inspired design. Compound 9i was identified as
the most selective D3R ligand among these bitopic ligands. Its selectivity improved reference
compounds 1 and 2 by 9ꢀ and 2ꢀtimes, respectively, and it was 21ꢀfold more potent than
compound 2. Molecular docking demonstrated that the orientation of Leu2.64 and Phe7.39 and
the packing at the junction of helices may affect the specificity at D3R over D2R. Functional
evaluation revealed that D3Rꢀselective ligand 9i displayed subpicomole agonist property at
D3R within a 199ꢀfold increase in potency than quinpirole. These results may be useful for
the fragmentꢀbased design of bitopic compounds as selective D3R ligands.
Keywords: Dopamine D3 receptor; bitopic arylpiperazines; selective ligand; structureꢀactivity
relationship; molecular modeling
1 These authors contribute equally to this work.
∗ Corresponding authors: Tel.: +82625302929, fax: +82625302911, eꢀmail: shcheon@jnu.ac.kr (S.H. Cheon); Tel.:
+82625302936, fax: +82625302949, eꢀmail: kmkim@jnu.ac.kr (K.M. Kim); Tel. and fax: +8675525622938, Eꢀmail:
szwzz001@163.com (Z. Wu).

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1. Introduction
Dopamine, a catecholamine neurotransmitter, exerts its biological effects by binding to five
dopamine receptors, which can be divided into two subfamilies. D1ꢀlike receptors (D1R and
D5R) primarily couple to stimulatory Gsꢀproteins, activating adenylyl cyclase, while D2ꢀlike
receptors (D2R, D3R, and D4R) principally couple to inhibitory Gi/oꢀproteins, inhibiting
adenylyl cyclase.1 D2ꢀlike receptor ligands that mainly target for D2R and D3R are approved
for the treatment of schizophrenia, Parkinson’s disease (PD), drug addiction, and substance
abuse.2ꢀ5 However, these therapies have adverse effects such as hyperprolactinemia,
metabolic syndrome, and extrapyramidal symptoms (EPS), which are believed to arise from
D2R antagonism.6,7 D3Rs are heavily expressed in the brain mesolimbic areas, and are
responsible for emotional, motivational, and cognitive functions.8 Thus, D3Rꢀselective
ligands can avoid these side effects and are expected to treat neuropsychiatric disorders, and
D3Rꢀselective agents may also ameliorate negative symptoms of psychiatric disorders.
Interestingly, the D3Rꢀselective agonists, but not D2Rꢀselective agonists, can reverse PDꢀ
related motivational deficits. Additionally, D3Rꢀselective agonists can attenuate anxietyꢀ and
depressiveꢀlike behaviors. Therefore, the development of a selective and biased D3R ligand is
critically important.
D2R and D3R share ~46% overall sequence homology, 78% sequence identity in
transmembrane domains,9 and the nearꢀidentical binding site residues.1 Indeed, this has
impeded the development of D3Rꢀselective compounds. Although extensive efforts from
medicinal chemists have devoted, and a number of promising D3Rꢀselective ligands have
been developed, few truly selective or biased ligands have approved by Food and Drug
Administration (FDA) or progressed to the clinic trials.10ꢀ15 The compound BP897 has been
shown to display subnanomolar affinity at D3R as well as moderate selectivity (Figure 1).
However, it acts in vivo as either an agonist or an antagonist, and did not indicate clues for
achieving selectivity over the D2R.6,10 The pramipexole bearing aminothiazolyl group also
binds to presynaptic D2R.11 It has been reported that the specificity of SBꢀ277011A is still not
apparent at D3R (<100ꢀfold over D2R). Although GSK598809, a D3R antagonist, exhibited
high D3R selectivity compared to D2R, it induced significant hypertension in dogs in the

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presence of cocaine.12 Compounds 1 and 2 displayed subꢀnanomolar affinity at D3R and
striking selectivity (4682ꢀfold and 55556ꢀfold, respectively, Figure 1).13,14 However, to our
knowledge, no continued investigation on their preclinical evaluation has been reported.
Therefore, onꢀgoing efforts to design more novel D3Rꢀselective ligands is necessary because
none of the FDAꢀapproved drugs have selectively targeted D3R.6,15
Additional selective compounds would provide a better understanding of the physiological
role and the distribution of these two receptor subtypes, and would offer the potential for
improved therapeutics without the aboveꢀmentioned side effects of hyperprolactinemia,
metabolic syndrome, and EPS. Recently, the elucidated D2R crystal structure facilitates the
more rational design of D3Rꢀselective ligands.16 Bitopic ligands that linked orthosteric and
allosteric pharmacophores have been proven to be of particular strategy of enhancing the
selectivity of ligands for dopamine receptors. In the current study, a fragmentꢀbased and
molecular docking inspired design was used to conceive a novel set of bitopic ligands based
on molecular modelling. The radioligand binding assay demonstrated that, among the
arylpiperazineꢀphthalimides, the compound 9i presented 9ꢀ and 2ꢀtimes improvement in
selectivity compared to reference compounds 1 and 2, respectively, in the testing system
which had been validated previously.17,18 The molecular determinants of selectivity at the
D3R were also analyzed based on the molecular docking. Importantly, functional evaluation
demonstrated that D3Rꢀselective ligand 9i exhibited subpicomole agonist activity at D3R
within subpicomolar and 199ꢀfold increase in efficacy compared with quinpirole.
Figure 1. Representative D3Rꢀselective ligands

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2. Results and Discussion
2.1 Molecular docking inspired design
Buspirone is a bitopic preferential D3R antagonist approved by the FDA for the treatment and
shortꢀterm relief of anxiety.19 However, it is subjected to firstꢀpass metabolism and can be
metabolized to 5ꢀhydroxybuspirone and 6’ꢀhydroxybuspirone by cytochrome P450 3A4
(CYP3A4, Figure 2).20 The former metabolite, 5ꢀhydroxybuspirone, is essentially
inactive,21,22 while the affinity of the latter metabolite, 6’ꢀhydroxybuspirone, to D2ꢀlike
receptors decreased significantly (KiD3R=795 nM, KiD2R=5390 nM).20 To obtain high affinity
D3R ligands, we first investigated the pharmacophoric features of D3R ligands. Both
buspirone and compound 1 were docked into the binding cavity of D3R (PDB code: 3PBL)
by the program LeDock2 (http://lephar.com).23 The tertiary amine in the piperazine ring of
both compounds forms a salt bridge to the carboxylate of the strongly conserved Asp110
(Figure 3). This salt bridge is pharmacologically critical for highꢀaffinity ligand binding to
the dopaminergic receptors.1 The pyrimidine motif of buspirone, and particularly the 2,3ꢀ
dichlorophenyl of compound 1, fit tightly within a hydrophobic cavity, the orthosteric
binding site (OBS) delineated by Phe345, Phe346, Ser192, Val111, and Ile183. Considering
that halogen substitution on the phenyl group can achieve good metabolic stability, the
halogen substituted phenyl piperazine was adopted as a primary pharmacophore. Additionally,
phthalimides represent a promising scaffold for antipyschotics without inducing catalepsy.24
SLV310, an antipsychotic candidate bearing phthalimide fragment, displayed high D3R
affinity25 with moderate D2R binding,26 and was predicted to be devoid of EPS, weight gain,
and hyperprolactinaemia.25 Molecular docking demonstrated that arylꢀ3,6ꢀdihydroꢀ2Hꢀ
pyridine from SLV310 was bound in essentially the same OBS of compound 1, while
phthalimide of SLV310 was superimposed with indolꢀ2ꢀylꢀcarboxamide of compound 1
(Figure 3). Namely, both pyrrolidineꢀ2,5ꢀdione and carboxamide form a hydrogen bond with
Thr369; the phenyl fragment from phthalimide was positioned in a hydrophobic cavity of the
allosteric binding pocket occupied by the indole in compound 1. In this regard, the
phthalimide moiety was used as a secondary pharmacophore. As reported previously, a
flexible alkyl linker, such as a butyl spacer, is more beneficial for pronounced dopaminergic

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activities,27 in particular for D3R affinity, molecular conformations, and crystal packing.28 As
such, arylpiperazineꢀphthalimides derivatives were designed as potentially novel D3R ligands.
O
N
N
N
N
HO
N
O
N
N
O
CYP3A4
5-OH BUSP
N
N
N
OH
O
N
O
N
N
Buspirone
N
N
6'-OH BUSP
O
Figure 2. The metabolic pathway of buspirone by CYP3A4
B
A
Figure 3. Docking poses of compound 1 (carbon in green, A and B), buspirone (yellow, A),
and SLV310 (yellow, B) in D3R.
2.2 Chemistry
The target arylpiperazineꢀphthalimides were prepared as shown in Scheme 1, while
arylpiperidineꢀphthalimiedes were synthesized as Scheme 2. All phenylpiperazines were
obtained from substituted anilines and bis(2ꢀchloroethyl)amine hydrochloride according to
the procedures described in the literature.29 Subsequently, the phenylpiperazines were
alkylated with (4ꢀbromobutyl)phthalimide to afford the desired compounds. 5ꢀChloroꢀ1ꢀ(4ꢀ
piperidinyl)ꢀ2ꢀbenzimidazolidinone and 4ꢀ(2ꢀmethoxyphenyl)piperidine were purchased from
Alfa Aesar and SigmaꢀAldrich, respectively. Nucleophilic substitution of the piperidine was
then performed with the (4ꢀbromobutyl)phthalimide furnished bitopic arylpiperidineꢀ
phthalimides 11.

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Scheme 1. Synthetic process of target phenylpiperazineꢀphthalimide compounds
Scheme 2. Synthesis of desired arylpiperidineꢀphthalimides 11
2.3 In vitro binding and structureꢀactivity relationship (SAR) studies
The target compounds were initially screened at a concentration of 100 nM in cellꢀbased
assays with both D2R and D3R.17,18 Human embryonic kidneyꢀ293 (HEKꢀ293) cells were
purchased from the American Type Culture Collection (Manassas, VA, USA). HEKꢀ293 cells
stably expressing human D2R or D3R were used in competition experiments to evaluate the
affinity and selectivity of the target compound for D3R over D2R. The displacement of [3H]ꢀ
sulpiride binding was assessed for each compound using sulpiride as a positive control.
Generally, the orthosteric binding site is primarily responsible for the affinity and efficacy of
a ligand, whereas the allosteric binding site is associated with selectivity.13 Bitopic or
dualsteric ligands that engage both binding sites are expected to increase selectivity and
retain affinity. Indeed, this strategy has been a proven and validated model to develop D3Rꢀ
selective ligands and discriminate their signal transduction. The linker is also a major
contributing factor of D3R selectivity, because the spacer effect and oddꢀeven effect influence
the divergent conformation and packing of a ligand second binding pocket (SBP, generally
aryl amide).28 Furthermore, the protonation of piperazine or piperidine, or even subtle
variations of the head group, can affect SBP and D3R selectivity. Therefore, we investigated
the serial head group variations presented in Table 1 and Figure 4.
A previous quantitative structureꢀselectivity relationship (QSSR) demonstrated that the
electronꢀwithdrawing group attached to the orthosteric phenyl group favored D3R selectivity

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over D2R, but the electronꢀdonating group did not.14 Consequently, in this study we focused
on the electronꢀwithdrawing substituent of phenyl piperazines. Among the monochloro
substituents, the metaꢀ and orthoꢀoccupied derivatives exhibited higher D3R affinity than
those of D2R, while the paraꢀchloro analogue 9a showed a lower affinity for D3R than D2R.
However, the protonation effect of 9a reduced D2R and D3R affinity, which may be due to a
destructive conformational variation, even though the trend of D3R selectivity versus D2R is
consistent with the base formation of the 9a. The metaꢀchloro derivative 9b displayed the
most potent activity for both D2R and D3R. Decorating the ligand with both orthoꢀchloro and
metaꢀchloro yielded a 2,3ꢀdichloro hybrid 9d, which exhibited high activity at both targets,
but no preference for D3R. Similarly, while the incorporation of orthoꢀchloro and paraꢀchloro
afforded the 2,4ꢀdichloro hybrid 9e, this compound could not be dissolved in the test solvent
system, even with dimethyl sulfoxide (DMSO); we therefore converted 9e to the
corresponding salt, the most convenient being a hydrochloride salt. 9e then showed
diminished affinity compared with 9d but slightly greater activity compared with 9a·HCl.
This indicated that orthoꢀchloro and metaꢀchloro substitutions contribute to D3R affinity,
whereas paraꢀchloro substitution is not tolerated for D3R affinity and selectivity over D2R.
Compound 9f, with a trifluoromethyl group attached to the meta position of the head phenyl
group, exhibited moderate affinity but no discrimination between D3R and D2R. We then
investigated the sterically less bulky fluoro group attached to the phenyl group. The para
ꢀ
fluorinated derivative 9g exhibited relatively lower affinity and moderate D3R selectivity
compared to D2R. In contrast, the orthoꢀfluoro analogue 9h preferentially bound to D3R
rather than to D2R with the most potent D3R activity. Grafting a fluoro group onto both the
ortho and meta position of the head group yielded 9i. Compound 9i induced D3R activity and
selectivity comparable to 9h, indicating that the metaꢀfluoro substituent may contribute to
D3R affinity but not to D3R selectivity. However, changing the fluoro substituent from the
meta position to the para position led to a reduction in both the potency and selectivity,
which suggests that the paraꢀfluoro substituent is not tolerated for D3R affinity and
selectivity, nor is it compatible with the orthoꢀfluorinated substituent. In contrast, the 2,6ꢀ
difluoro derivative 9k exhibited relatively low activity and moderate selectivity.

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Incorporating the 2ꢀfluoro and 4ꢀchloro substitutions resulted in a dramatic deactivation at
both D3R and D2R compared with the 2ꢀfluoro derivative 9h and the 2,3ꢀdifluoro substitution
9j. Based on these findings, we postulated that, whether it is a sterically bulky or slim group,
the 4ꢀsubstituent is not tolerated or beneficial for D3R affinity and selectivity. Moreover, the
combination of a 2ꢀfluoro substituent and a 5ꢀtrifluoromethylphenyl head group yielded 9m,
which had almost no activity at either target, illustrating that the 2ꢀ and 5ꢀpositions are not
compatible with D3R affinity and selectivity over D2R.
In addition, the bioisosteric replacement of aryl piperazine with aryl piperidine yielded 11a
and 11b. The orthoꢀmethoxyl phenyl piperidine derivate 11a, bearing an electronꢀdonating
group, exhibited pronounced affinity for both D2R and D3R, resulting in diminished
selectivity. In contrast, the extension of the phenyl group with a benzimidazolidinonyl moiety
along with an electronꢀwithdrawing group produced 11b, which displayed moderate affinity
at D3R but diminished selectivity over D2R.
Table 1. Binding affinities of butyl phthalimides
D3R
Displacement (%)
30.5 ± 12.8
2.5 ± 1.5
D2R
Displacement (%)
59.2 ± 18.3
16.3 ± 4.2
64.8 ± 3.5
28.7 ± 3.3
70.3 ± 2.7
N.D.
Compound
9a
R1
X
4ꢀClphenyl
4ꢀClphenyl
N
N
N
N
N
N
N
N
N
N
N
N
9a·HCl
9b
3ꢀClphenyl
86.7 ± 1.4
43.8 ± 4.5
76.6 ± 3.1
N.D.
2ꢀClphenyl
9c
2,3ꢀDiClphenyl
2,4ꢀDiClphenyl
2,4ꢀDiClphenyl
3ꢀCF3phenyl
3ꢀCF3phenyl
4ꢀFphenyl
9d
9e
9e·HCl
9f
23.0 ± 8.2
51.5 ± 3.3
46.9 ± 1.3
30.8 ± 14.3
82.6 ± 2.7
65.4 ± 5.8
30.2 ± 3.4
44.0 ± 1.6
37.9 ± 2.8
ꢀ6.3 ± 4.0
31.8 ± 3.3
17.7 ± 3.7
9f·HCl
9g
2ꢀFphenyl
9h
2,3ꢀDiFphenyl
9i

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2,4ꢀDiFphenyl
2,6ꢀDiFphenyl
4ꢀClꢀ2ꢀFphenyl
5ꢀCF3ꢀ2ꢀFphenyl
2ꢀOMephenyl
N
N
N
N
C
26.2 ± 2.7
33.9 ± 6.0
7.0 ± 4.6
4.8 ± 6.5
10.3 ± 6.1
2.8 ± 12.0
ꢀ4.7 ± 7.8
92.2 ± 0.5
56.1 ± 4.5
29.6 ± 4.2
22.7 ± 8.5
92.2 ± 0.1
9j
9k
9l
1.6 ± 8.6
9m
85.6 ± 1.2
62.5 ± 3.8
85.4 ± 3.2
57.4 ± 10.7
87.9 ± 1.8
11a
5ꢀClꢀ2ꢀbenzimidazolidinonyl
C
ꢀ
11b
1·HCl
2·HCl
Sulpiride
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
N.D. = Not determined because the compound cannot be dissolved in the test system, even with DMSO
100
D3R
D2R
80
60
40
20
0
Compounds
Figure 4. Graphic binding affinities of butyl phthalimides at D2R and D3R
After screening and exploring the head group, we found that 3ꢀfluoro/2,3ꢀdifluoro phenyl
derivatives as orthosteric modulators were compatible with the phthalimide group as an
allosteric scaffold for D3R selectivity and specificity. We subsequently specified the head
group as an orthoꢀfluoro group, and then investigated the tail group (Table 2 and Figure 5).
The introduction of a reverse amide, as well as the bioisosteric replacement of phthalimide
with benzothiazole, yielded 12a, which decreased both the D2R and D3R affinity but
maintained the differentiation between the two targets. Splitting the benzo moiety and
incorporating a carbonyl group afforded 1,2,4ꢀoxadiazoles 12b and 12c. Compound 12b
displayed slightly reduced affinity for D3R and no selectivity. Unexpectedly, the linker with

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three carbons connecting the piperazine and 1,2,4ꢀoxadiazole destroyed the affinity for D2R
and D3R.
Table 2. Binding profiles of orthoꢀfluorophenyl piperazine derivatives
D3R
D2R
Compound
R2
Displacement (%)
Displacement (%)
82.6 ± 2.7
63.1 ± 3.2
31.8 ± 3.3
17.0 ± 2.5
9h
12a
12b
50.7 ± 2.5
3.2 ± 14.3
69.8 ± 1.5
10.6 ± 7.0
12c
100
80
60
40
20
0
D3R
D2R
e
c
2
1
h
a
b
2
d
9
2
i
1
1
r
i
p
l
u
S
Compounds
Figure 5. Graphic binding affinities of orthoꢀfluorophenyl piperazines at D2R and D3R
After establishing binding profiles and structureꢀactivity relationships, we further
characterized several promising compounds in detail by determining their inhibition constant
(Ki) values; we then compared these compounds with two reference compounds known to be
potent D3Rꢀselective ligands (Table 3 and Figure 6). Because phthalimide 9i and reverse

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amide 12a displayed equivalent potentiation and affinity differentiation between D3R and
D2R, their binding affinities were profiled with Ki values and compared with reference
compounds 1 and 2 which are hydrochloride salts. Interestingly, 9i showed slightly lower
binding affinity at D3R than 12a, but it had markedly higher selectivity than D2R. In fact,
compound 9i displayed more preferential affinity for D3R than the most selective D3R ligand,
12d, among the arypiperazineꢀreverse amides identified. However, 1·HCl exhibited moderate
D3R affinity and selectivity over D2R, whereas 2·HCl showed lower D3R affinity and more
than 59ꢀfold selectivity for D3R compared to D2R. The selectivity of 9i was elevated by 9ꢀ, 2ꢀ
and 2.5ꢀtimes more than reference compound 1, reference compound 2, and compound 12a,
respectively; compound 9i is 21ꢀfold more potent than reference compound 2, but showed
equivalent potency to that of compound 1. As such, 9i was the most potent D3Rꢀselective
ligand among the phthalimides, carboxamides, and reverse amides that were synthesized and
screened in this study.
Table 3. Ki values of selected compounds and reference compounds
Compound
R1
R2
D3R Ki (nM)
19.3
D2R Ki (nM)
2163.1
D2R/D3R
112
9i
2,3ꢀdiF
2ꢀF
3.9
87.6
15.6
407.2
175
5586
45
63.8
13
12a
12d·HCl
1·HCl
2,4ꢀdiCl
2,3ꢀdiCl
2,4ꢀdiCl
202.3
2·HCl
>23889*
>58.7
*Ki value could not be estimated exactly because the doseꢀresponse curve did not pass through of the remaining
50% radioligand even at 30 ꢁM concentration.

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B
A
120
120
100
80
60
40
20
0
D3R
D2R
D3R
100
D2R
80
60
40
20
0
-10
-9
-8
-7
-6
-5
-4
-10
-9
-8
-7
-6
-5
log[12a] (M)
log[9i] (M)
C
D
120
100
80
60
40
20
0
120
100
80
60
40
20
0
D3R
D2R
D3R
D2R
-11 -10 -9
-8
-7
-6
-5
-4
-11 -10 -9
-8
-7
-6
-5
-4
log[1·HCl] (M)
log[2·HCl] (M)
Figure 6. Doseꢀresponse curves of compounds 9i and 12a, and reference compounds 1·HCl
and 2·HCl
2.4 Molecular basis of selectivity over D2R
To shed light on the structural basis of selective ligands at D3R over D2R, compound 9i, the
most selective D3R ligand among the identified compounds, was docked into D3R (PDB code:
3PBL) and D2R (PDB code: 6C38), respectively, with the program LeDock
(www.lephar.com).23 Docking poses were further minimized with the CHARMM force
field.30 Briefly, binding of compound 9i in D3R is characterized by a salt bridge to the
conserved Asp110, hydrophobic burial of the 2,3ꢀdifluorophenyl in the orthosteric site
(Val111, Cys114, Ile183, Ser192, Ser196, Phe345, Phe346, His342) deep in the seven transꢀ
membrane bundle, and extension to the extracellular pocket by the phthalimide terminus
(Figure 7A). Upon binding in D2R, its piperazine linker is well overlaid on the piperidine
linker of the coꢀcrystalized antipsychotic drug risperidone, establishing a salt bridge to
Asp114 (Figure 7B). Similar to risperidone, its head was inserted in the orthosteric site and
its tail extends to the extracellular pocket.
Although residues delineating the binding site in D3R and D2R are nearly identical, their
orientations are significantly different, as revealed by superposition of both structures (Figure
8). Notably, the different orientation of Leu89/94 and Phe365/429 put the phthalimide
terminus in distinct regions in the extracellular pocket. The phthalimide terminus in D3R has

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a tighter interaction with the three residues Val86, Leu89 and Glu90 from the first
extracellular loop (ECL1), and forms a Hꢀbond with Thr369. When it is bound to D2R, this
hydrogen bond was not formed due to a different orientation of the corresponding Thr433.
The different packing at the junction of helices leads to a subtle yet critical difference in the
relative disposition between the orthosteric and extracellular pocket in the D2R and D3R. As a
result, the hydrophobic head of 9i inserts a bit deeper in the orthosteric pocket of D2R, with
the fluorine at the orthoꢀposition facing the aromatic ring of Trp407 at a distance of about 3
Å, slightly shorter than the sum of van der Waalsꢀradii (Figure 7B). Fluorine, which does not
typically feature a σꢀhole,31 thus experiences electrostatic repulsion with the πꢀelectrons of
the aromatic ring. This observation is consistent with the previous SAR analysis that orthoꢀ
fluoro substitution confers selectivity over D2R,32 which is further confirmed in the current
study. Taken together, the selectivity of 9i originates from the subtle but critical difference in
the relative disposition between the orthosteric and extracellular pocket in the D2R and D3R,
leading to distinct interaction features in both sites.
Figure 7. Predicted binding mode of compound 9i in D3R (A) and D2R (B), respectively. For
clarity, the coꢀcrystalized ligand Eticlopride in D3R was not shown. Hydrogen bonds were
illustrated by dashed lines.

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Figure 8. Superposition of compound 9i in the binding site of D3R (carbon shown in gray) and
D2R (carbon shown in green).
2.5 Functional evaluation
To characterize the functional properties of D3Rꢀselective ligand 9i and reference compounds
1 and 2, reporter gene assay based cAMP production assay was conducted as previously
described [33ꢀ34]. Briefly, Cells stably expressing D3R were transfected with firefly luciferase
reporter genes; after seeding, the cells were treated with 2
ꢀM forskolin and varying
concentrations of D3Rꢀselective ligands (quinpirole as a positive control); finally, the cells
were harvested and the relative luciferase expression was measured (Supporting information).
Compared with quinpirole, a full agonist of D3R, the relative efficacies (the maximal
inhibition of the forskolinꢀinduced cAMP production) of reference compounds 1 and 2 were
32.2% and 51.1%, respectively (Figure 9). Thus, reference compounds 1 and 2 were
identified as partial agonists. EC50 (the concentration of half maximal effect) of qupinpirole
was 97 pM, whereas those of reference compounds 1 and 2 were 26 pM and 1.1 nM,
respectively. The efficacy of compound 9i was similar to that of quinpirole but the doseꢀ
response curve of compound 9i was drastically shifted to the left, resulting in about 200ꢀfold
increase in potency (0.48 pM).
Quinpirole
120
9i
1· HCl
100
2· HCl
80
60
40
20
0
-13
-12
-11
-10
-9
-8
Log(M)
Figure 9. Normalized doseꢀresponse curves of the inhibition of forskolinꢀinduced cAMP
production by quinpirole, compounds 9i, 1·HCl, and 2·HCl

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3. Conclusion
In this study, a series of bitopic ligands with preferential affinity for dopamine subtype
receptor D3R over D2R were identified based on molecular docking aided design. The
radioligand binding revealed that 9i was the most potent D3Rꢀselective ligand among our
reverse amides, phthalimides, and carboxamides. The selectivity of 9i is 9ꢀ and 2ꢀtimes
higher than that of reference compounds 1 and 2; the binding affinity of 9i improved 21ꢀfold
compared to reference compound 2. SAR studies demonstrated that an electronꢀwithdrawing
group and a sterically less bulky substituent at the ortho and para position of the head phenyl
group were favorable for D3R specificity. The phthalimide moiety in the tail group tolerated
D3R selectivity over D2R with carboxamide fragments and its reverse amide. Docking of the
most promising D3Rꢀselective ligand, 9i, into the human D3R and D2R crystal structure,
provided insights into the molecular determinants of D3R selectivity. The different orientation
of Leu2.64 and Phe7.39 resulted in a divergent secondary binding site of compound 9i which
may contribute to D3R selectivity over D2R. The different packing of D3R and D2R at the
junction of helices gave rise to a distinctly relative disposition between the orthosteric and
allosteric pockets, which may also be responsible for the D3R selectivity over D2R.
Functional evaluation demonstrated that D3Rꢀselective ligand 9i displayed subpicomole
agonist property at D3R within equivalent efficacy while 199ꢀfold increase in potency as
quinpirole.
Acknowledgments
This work was supported by the National Research Foundation (NRF) of Korea (Basic
Science Research Program, grant No. 2012R1A1A2006613), the Bio & Medical Technology
Development Program of the National Research Foundation (NRF) & funded by the Korean
government (MSIT) (NRFꢀ2017M3A9G2077568), the National Natural Science Foundation
of China (NSFC, Grant No. 81574038 and 81701256), the China Postdoctoral Science
Foundation (Grant No. 2016M600708), the Science and Technology Planning Project of
Guangdong Province (Grant No. 2015B020211001), the Science and Technology Project of
Shenzhen (Grant No. JCYJ20170306171122368), and the Shenzhen Municipal Project of

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Health and Family Planning (SZBC2017015). The authors would like to thank the Chonnam
1
Center For Research Facilities for recording H NMR and 13C NMR, and thank Lephar
research group and Dr. Hongtao Zhao for assistance in molecular modeling.
Conflict Of Interest
The authors declare no competing interests.
Supplementary data
The synthesis procedure, NMR data, and biological evaluations are available from the online
version.
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Graphical Abstract
Design, Synthesis and Evaluation of Bitopic
Arylpiperazine-phthalimide Derivatives as
Selective Dopamine D3 Receptor Agonists
Leave this area blank for abstract info.
Yongkai Caoa,b,c, Ningning Sunb,1, Jiumei Zhangc, Zhiguo Liud, Yi-zhe Tangc, Zhengzhi Wuc,*, Kyeong-Man
Kimb,* and Seung Hoon Cheonb,*
a Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, China
b College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 500-757,
Republic of Korea
c The First Affiliated Hospital of Shenzhen University, Shenzhen Second hospital, Shenzhen University school of
medicine, Shenzhen 518035,China
d Chemical Biology Research at School of Pharmaceutical sciences, Wenzhou Medical University, Wenzhou 325035,
China