Design, synthesis, and evaluation of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamides as selective dopamine D3 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2019.07.020

As part of our on-going effort to explore the role of dopamine receptors in drug addiction and identify potential novel therapies for this condition, we have a identified a series of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide D₃ ligands. Members of this class are highly selective for D₃ versus D₂, and we have identified two compounds (13g and 13r) whose rat in vivo IV pharmacokinetic properties that indicate that they are suitable for assessment in in vivo efficacy models of substance use disorders.

Full text of DOI:10.1016/j.bmcl.2019.07.020

Accepted Manuscript
Design, synthesis, and evaluation of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-
(thiophen-3-yl)benzamides as selective dopamine D3 receptor ligands
Peng-Jen Chen, Michelle Taylor, Suzy A. Griffin, Armaghan Amani, Hamed
Hayatshahi, Kenneth Korzekwa, Min Ye, Robert H. Mach, Jin Liu, Robert R.
Luedtke, John C. Gordon, Benjamin E. Blass
PII:
S0960-894X(19)30466-4
https://doi.org/10.1016/j.bmcl.2019.07.020
BMCL 26561
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2019
8 July 2019
10 July 2019
Please cite this article as: Chen, P-J., Taylor, M., Griffin, S.A., Amani, A., Hayatshahi, H., Korzekwa, K., Ye, M.,
Mach, R.H., Liu, J., Luedtke, R.R., Gordon, J.C., Blass, B.E., Design, synthesis, and evaluation of N-(4-(4-phenyl
piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamides as selective dopamine D3 receptor ligands, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.07.020
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Design, synthesis, and evaluation of N-(4-(4-phenyl
piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamides as
selective dopamine D3 receptor ligands
Leave this area blank for abstract info.
Peng-Jen Chena, Michelle Taylorb, Suzy A. Griffinb, Armaghan Amanic, Hamed Hayatshahic, Kenneth
Korzekwaa, Min Yea, Robert H. Machd, Jin Liuc, Robert R. Luedtkeb, John C. Gordona, and Benjamin E. Blassa
R
N
O
N
N
H
13a-v
(
)
S

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis, and evaluation of N-(4-(4-phenyl piperazin-1-yl)butyl)-4-
(thiophen-3-yl)benzamides as selective dopamine D3 receptor ligands
Peng-Jen Chena , Michelle Taylorb, Suzy A. Griffinb, Armaghan Amanic, Hamed HayatshahiC, Kenneth
Korzekwaa, Min Yea, Robert H. Machd, Jin Liuc, Robert R. Luedtkeb, John C. Gordon a , and Benjamin E.
Blassa 
a
Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 North Broad Street, Philadelphia, PA 194140 USA
b Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Blvd. Fort Worth, Texas 76107, USA
c Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, 3500 Camp
Bowie Blvd. Fort Worth, Texas 76107, USA
d Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Chemistry Building, 231 S. 34th Street, Philadelphia, PA 19104
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
As part of our on-going effort to explore the role of dopamine receptors in drug addiction and
identify potential novel therapies for this condition, we have a identified a series of N-(4-(4-
phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide D3 ligands. Members of this class are
highly selective for D3 versus D2, and we have identified two compounds (13g and 13r) whose
rat in vivo IV pharmacokinetic properties that indicate that they are suitable for assessment in in
vivo efficacy models of substance use disorders.
Keywords:
Dopamine Receptor
2009 Elsevier Ltd. All rights reserved.
D3
D2
G-protein coupled receptor (GPCR)
Drug addiction
———
 Corresponding author. Tel.: +1-215-707-2218; fax: +1-215-707-5620; e-mail: Benjamin.Blass@Temple.edu

Drug addiction is a major societal and medical issue. The scope
of this issue in the U.S. was quantified in the National Survey on
Drug Use and Health which reported that in 2014 over 27 million
people over the age of 12 had consumed an illicit drug in the past
30 days. The same survey indicated that there were >7.1 million
people in the U.S. suffering from addiction to illicit drugs (e.g.
cocaine, heroin, oxycodone, etc.) and that the annual societal cost
of dealing with this issue exceeds $600 billion.1 The impact on
individual health and wellness is also significant, as illicit drug use
is linked to increased rates of numerous diseases and conditions
including cardiovascular disease, stroke, cancer, lung disease, as
well as HIV and hepatitis infection.2 The impact of drug addiction
extends beyond the patient, as family members, friends, and co-
workers often deal with the impact of the patient’s drug use.
Unfortunately, there are few treatment options available to address
drug addiction, and the substantial risk of relapse further
which revealed that over 500,000 patients required hospital
emergency department services as a result of cocaine use.6
While the magnitude of this problem is clear, therapeutic options
are limited.
Behavioral interventions, such as contingency
management programs provide rewards for abstinence have
produced positive benefits in some patients. Similarly, cognitive-
behavioral therapies that teach recovering addicts to recognize and
avoid situations that will trigger the desire to use cocaine can be
beneficial. These methods, however, are limited by high costs and
the lack of sufficient resources to address the full scope of patients
requiring treatment.7 Ideally, pharmacological treatments could be
paired with behavioral intervention programs, but there are no FDA
approved medicines for the treatment of cocaine addiction. Several
research teams have focused on developing novel therapies or
reapplying existing therapies to the problem of substance abuse.
Disulfiram (2), a medication approved for the treatment of
alcoholism, has shown promise in a limited patient population, but
the mechanism of action of this medication remains a mystery.8
The antiepileptic irreversible inhibitor of GABA transaminase
Vigabatrin (3),9 and the AMPA/Kainate receptor antagonist
Topiramate (4)10 have also been examined in clinical trials, but
neither have been approved for use as treatments for cocaine
addiction. There have also been reports of positive results in
animal studies with compounds that selectively target the
O
O
N
S
O
O
OH
N
S
S
N
NH2
S
1
2
3
Cocaine ( )
Disulfiram ( )
Vigabatrin ( )
O
O
O
NH2
O
HO
HO
S
O
O
O
O
O
NH2
O
O
N
N
4
5
Dopamine ( )
Topiramate ( )
N
H
Figure 1:Structures of Cocaine (1), Disulfiram (2), Vigabatrin (3),
Topiramate (4), and Dopamine (5).
6
WW-III-55, ( )
D3 = 19.8 nM
D2 = >17,000 nM
S
complicates patient care.
O
N
F
Cocaine (1) is one of the most commonly abused illicit drugs.
This addictive psychostimulant was isolated from the coca plant
(Erythroxylon coca) over 100 years ago,3 and while there are some
validated medical uses (e.g. local anesthetic for surgeries of the
eyes, ears, nose, and throat), its primary use is as a recreational,
illicit drug. As of 2014, over 1.5 million people in the U.S.
reported being current user of cocaine, which can be either injected
or snorted as the HCl salt or smoked as the free base (crack
cocaine). Irrespective of the delivery mechanism, once cocaine
enters the brain, it interferes with the mesocorticolimbic dopamine
(MCL-DA) system. Under normal conditions, dopamine is
released by pre-synaptic neurons and interacts with dopamine
receptors on post-synaptic neurons. The dopamine transporters
(DAT) clear dopamine from the synapse.4 Cocaine interferes with
this process by preventing dopamine uptake by DAT, and the
resulting increase in synaptic dopamine amplifies dopamine
signaling and creates the euphoric feeling associated with cocaine
exposure. Chronic cocaine exposure leads to changes in expression
of DAT and dopamine receptors. These changes in dopamine
receptor expression may contribute to the increasing level of
cocaine required to deliver the same euphoric effects as chronic use
continues. In addition to euphoria, cocaine also has negative
impact on the decision making process, which often leads to feeling
of anxiety, panic, paranoia, and violent behavior. It also increases
heart rate, blood pressure, and body temperature.5 As the cycle of
cocaine exposure continues, the dose required by addicts rises, and
the risk of overdose increases. The impact of cocaine overdoses is
evident in the 2011 Drug Abuse Warning Network (DAWN) report
N
O
N
H
LS-3-134 (7)
D3 = 27.7 nM
D2 = 0.17 nM
S
N
O
N
N
F
H
O
9
8
( )
( )
S
D3 = >30,000 nM
D2 = >5,000 nM
D3 = 22.3 nM
D2 = 14.7 nM
Figure 2: Structures of WW-III-55 (6), LS-3-134 (7),
piperzine component (8), and amide component (9).
neurokinin-1 receptor,11 the cysteine glutamate antiporter,12 and the
5-HT2C serotonin receptor.13
As part of our on-going efforts to identify potential novel
therapies for the treatment of cocaine addiction, we have been
exploring the impact of modulating dopamine signaling using
dopamine D3 receptor ligands. Dopamine (5), a neurotransmitter
that is synthesized in the brain and the periphery, has been linked to
a wide range of physiology. In the periphery, for example, this
compound can act as a vasodilator, modulate renal sodium
excretion, and impact urine output. In the central nervous system,
dopamine (5) is known to have a significant impact on learning,
movement, and behavioral motivations.14 At the cellular level,
dopamine initiates signaling via the action of dopamine receptors.

Scheme 1
R
O
a) DMT-MM, EtOH, 23 oC
O
N
NH
Br
N
NH2
12a-v
(
)
HO
H
HO
11
(
)
b) CBr4, PPh3, CH2Cl2, 23 oC
K2CO3, CH3CN, reflux,
S
10
(
)
S
R
R
Scheme 2
Br
Br
N
O
O
DMT-MM, EtOH, 23 oC
N
N
(a) K2CO3, acetone, reflux
N
N
NH
H
NH2
(b) K2CO3, NaI, 1,4-dioxane, reflux
O
13a-v
(
)
15a-v
(
)
R
O
14
(
)
N
NH
S
HO
S
10
(
)
12a-v
(
)
(c) Hydrazine, EtOH ,23 oC
The dopamine family of G-protein coupled receptors (GPCRs) has
5 members designated D1, D2, D3, D4, and D5 that can be further
sub-divided into two classes, D1-like (D1 and D5) and D2-like (D2,
D3 and D4) receptors. These classifications are based on genetic
organization, amino acid homology and pharmacological
properties.15 The D3 receptor has been the subject of intense
interest as a potential therapeutic target for the treatment of cocaine
addiction based on multiple lines of evidence. It is known, for
example, that both chronic and acute exposure to cocaine leads to
increased D3 expression in the nucleus accumbens. This region is
responsible for cognitive processing including behaviors associated
with cocaine addiction.16 In addition, D3 is highly expressed in key
regions of the brain linked to cocaine addiction. Multiple reviews
describing the role of D3 in cocaine addiction are available.17 The
D2 receptor has also been implicated in cocaine addiction. This
target has been explored as a potential therapeutic target,18 but
modulation of D2 signaling is also associated with serious side
effects (e.g. extrapyamidal symptoms, catalepsy).19 These risks
lead us to focus on the development of compounds with a high
degree of selectivity for D3 over the D2 dopamine receptor.
1-ol using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-
4-methyl-morpholinium chloride (DMT-MM), and the resulting
compound is converted to a primary bromide (11) using carbon
tetrabromide and triphenylphosphine. Nucleophilic displacement
of the bromide with piperazines (12a-v) under basic conditions
(K2CO3, CH3CN) provides the desired products (13a-v).
Alternatively, alkylation of phthalimide (14) with 1,4-
dibromobutane under basic conditions (K2CO3, acetone) produces
the corresponding primary alkyl bromide. This is followed by
nucleophilic displacement of the remaining bromide with
piperazines (12a-v) under basic conditions (K2CO3, 1,4-dioxane) in
the presence of sodium iodide. Removal of the phthalimide with
hydrazine provide the primary amine intermediate (15a-v), which
can be coupled with 4-(thiophen-3-yl)benzoic acid (10) using
DMT-MM to provide the desired final compounds (13a-v).
Table 1: 1st tier In vitro pharmacology of (13a-v)
D3
D2
Ki (nM)
D2/D3
RLM T1/2
(min.)
24.6
3.5
28.1
50.2
14.4
18
>60
2.4
9.3
>60
9.4
5.1
11.4
3.7
22.3
7.5
#
R
13a
13b
13c
13d
13e 2,3-Cl
13f 2,4-Cl
13g 3,5-Cl
H
8
0.55
8.3
32.7
0.89
3.8
8434
169
12046
27359
63.1
757
2049
38.6
1832
17098
3449
140
4753
190
16318
24994
92.9
1054
309
1451
836
70.8
198
4615
193
102
862
206
301
349
281
744
692
369
1486
8.4
2-Cl
3-Cl
4-Cl
We have previously identified WW-III-55 (6) as a potent D3
ligand that is highly selective for D3 over D2 (D3 Ki = 19.8 nM, D2
Ki >17,000 nM, D2/D3 > 858)20 and a fluorinated analog (7) that
when radiolabeled with an 18F can be used in PET tracer studies.21
As part of these studies, we hypothesized that the phenylpiperazine
region is critical to binding, while the high degree of selectivity
observed in these compounds was driven by the incorporation of
the phenyl thiophene region. We based these hypotheses on the
observations that the n-butylpiperazine component (8) binds to both
D2 (Ki = 14.7 nM) and D3 (Ki = 22.3 nM) with similar affinity,
while the amide component (9) binds with low affinity at these
dopamine receptors. In addition, our previously reported molecular
dynamics and docking studies of the (7) and (8) show that the
0.44
13h* 2-OCH3 0.20
13i 3-OCH3 18.0
13j* 4-OCH3 19.8
13k* 2-OH
13l 3-OH
16.7
0.47
13.6
0.68
21.9
36.1
0.25
0.50
2,378
1.5
13m 4-OH
13n 2-CH3
13o 3-CH3
13p 4 CH3
13q 2-CN
piperazine nitrogens of these compounds undergo
a key
electrostatic interaction with Asp3.32 that is not available to the
2
amide component (9).22
13r
3-CN
743
>20000
261
99.7
>30000
>60
13.2
4
16.9
>60
13s 4-CN
13t 2-CF3
13u 3-CF3
13v 4-CF3
We recently turned our attention to developing a better
understanding of the impact of the piperazine region of WW-III-55
(6) with the aim of identifying compounds with in vivo
pharmacokinetic properties supportive of oral dosing in rat models
of cocaine addiction. A series of analogs was prepared beginning
with either 4-(thiophen-3-yl)benzoic acid (10, scheme 1) or
phthalimide (14, scheme 2). Specifically, 4-(thiophen-3-yl)benzoic
acid (10) is converted to an amide by reaction with 4-aminobutan-
172
243
384
0.41
78.3
*13h, 13j and 13k were previously by Mach et. al. 20, 23
In vitro competitive D3 and D2 radioligand binding assays and
rat liver microsome (RLM) stability studies were used to identify
potential lead compounds for further examination. As indicated in

Table 1, electron donating and electron withdrawing substituents on
the phenyl ring of the aryl piperazine were well tolerated by the D3
dopamine receptor as indicated by the potent binding observed (Ki
< 100 nM). There are, however, notable differences in D3 binding
affinity. In the 2-position, for example, analogs with electron
donating and withdrawing substituents (13b, 13h, 13n, 13q, 13t)
demonstrated D3 receptor Ki values between 0.2 nM and 2.0 nM
with the notable exception of the 2-OH analog (13k, D3 Ki = 16.7
nM). Sub-nanomolar potency was observed in several analogs with
substituents in the 3-postion of the phenyl ring of the aryl
piperazine (13l, 3-OH, D3 Ki = 0.47 nM, 13r, 3-CN, D3 Ki = 0.50
nM, 13u, 3-CF3 D3 Ki = 0.41 nM), but other analogs bound with
substantially lower affinity (13c, 3-Cl, D3 Ki = 8.3 nM, 13i, 3-
OCH3 D3 Ki = 18.0 nM, 13o, 3-CH3, D3 Ki = 21.9 nM). Analogs
with substituents in the 4-position displayed the lowest level of D3
binding affinity, but the majority of analogs examined had D3
receptor Ki values <100 nM. Electron withdrawing (13d, 4-Cl, D3
Ki = 32.7 nM, 13v, 4-CF3, D3 Ki = 78.3 nM) and electron donating
substituents (13j, 4-OCH3, D3 Ki = 19.8 nM, 13m, 4-OH, D3 Ki =
13.6 nM, 13p, 4-CH3, D3 Ki = 36.1 nM) were well tolerated, but the
4-CN analog (13s) bound with substantially lower affinity (D3 Ki =
2378). This loss of binding affinity may be the result of
unfavorable steric interactions or possibly the result of negative
electrostatic interaction between the highly polarized cyano group
and amino acid residues in the D3 receptor binding pocket.
The impact of the same structural changes on D2 binding
demonstrated some key differences in the structure activity
relationship (SAR) of this receptor in comparison to D3.
Substitution in the 4-position of the phenyl ring of the aryl
piperazine lead to substantial losses in D2 receptor binding
irrespective of the electron character of the substituent. Electron
withdrawing substituents in the 2-position produced (13b, 13q and
13t) moderate affinity (Ki = 169 nM, 92.9 nM, and 261 nM
respectively), while electron donating groups produced mixed
results. The non-polar 2-methyl (13n) and 2-methoxy (13h)
substituents demonstrated moderate D2 receptor binding (Ki = 190
nM and 38.6 nM), but the more polar 2-hydroxy substituent (13k)
Figure 3: Docking pose of (13r) in D2 (top) and D3 (bottom).
Salt bridges are shown as green dashed lines. Only key
hydrogen atoms are shown for clarity.
substituents in the 2-position produces compounds with D2/D3
selectivity profiles ranging from 172 to 369-fold, while some 3-
substituted analogs are >1400-fold selective for D3 over D2 receptor
binding (3-CN: 13r and 3-Cl: 13c). The highest degree of D3/D2
selectivity was observed when two chlorine atoms were appended
to the phenyl ring of the phenyl piperazine, but positioning of these
substituents was critical to selectivity. Specifically, the 2,3-
dichloro analog (13e) is 70-fold selective for D3 vs. D2, the 2,4-
dichloro analog (13f) is 2.8 times more selective (198 fold), and the
3,5-dichloro (13g) analog is over 65 times more selective (4615
fold) than the 2,3-dichloro analog (13e). The differences in D3 and
D2 SAR allowed us to identify multiple compounds with D3
binding potency below 10 nM and >1000 fold selectivity vs. D2
(13a, 13c, 13g, and 13r).
has substantially lower affinity (Ki = 3449 nM).
Finally,
substitution in the 3-position only produced moderate affinity when
an OH (13l, Ki = 140 nM) or CF3 (13u, Ki = 99.7 nM) moiety was
installed. Other substituents, both electron withdrawing and
electron donating lead to decreased in D2 binding affinity (Ki = 743
to 16318 nM). One possible explanation for these observations
may be that the 3-OH and the CF3 group are both acting as
hydrogen bond acceptors in the D2 receptor binding site, thereby
stabilizing the interaction.
The impact of the differences in the SAR of D3 and D2 binding
sites are evident when the binding affinities of individual
compounds at both receptors are considered. While we have
previously demonstrated that the 4-(thiophen-3-yl)benzene moiety
is a significant driver of D2/D3 receptor selectivity, it is clear that
the phenyl piperazine region of the molecule can also have a
significant impact on selectivity. Altering the type and position of
functional groups in this region of the molecule produces
compounds with significantly different D2/D3 binding selectivity
profiles. The 4-methoxy analog (13j), for example, is 862 fold
more potent at D3 vs D2, but the 2-methoxy (13h, previously
reported as OS-3-10620) and 3-methoxy (13i) analogs are
significantly less selective (193 fold and 102 fold D3 vs. D2
selectivity respectively). In addition, varying the selection of
To understand how the pockets of D2 and D3 provide selective
binding of these ligands, we used Autodock vina to dock (13r) on
the crystal structures of D2 (PDB code 6C3824) and D3 (PDB code
3PBL25). The highest energy poses with an interaction between the
protonated nitrogen and Asp 3.32 and orthosteric position of
phenylpiperazine pharmacophore are shown in Figure 3. A source
of better affinity for the ligand for D3 may be the potential
interaction with Tyr 7.35. In D3, the amide oxygen of (13r) could
form a hydrogen bond with Tyr 7.35 in some configurations of the
linker rotatable bonds. In three copies of 300 ns molecular
dynamics simulation of this molecule in complex with D3

haloperidol as a prototypic full agonist and antagonist, respectively.
Compounds were tested for efficacy in these assay at a dose equal
to 10x their D3 Ki values in order to ensure >90% receptor
occupancy and the results were compared with the impact of the
full agonist. While neither compound demonstrated activity in the
β-arrestin binding assay, both compounds demonstrated partial
agonism in the forskolin-dependent adenylyl cyclase inhibition
assay. (13g) and (13r) produced 36.5% (mean ±15.5 S.E.M., n =3)
and 19.4% (mean ±5.4 S.E.M., n=3) of the activity of the maximum
response observed with quinpirole.
In addition, in vitro screening for D1, D4, and D5 binding
indicated that both compounds have a high degree of selectivity for
D3 over these three dopamine receptors (13r has the lowest
selectivity versus D4 but it is >1600-fold selective for D3 over D4).
Further evaluation via the Psychoactive Drug Screening Program
(PDSP) provided in vitro selectivity data for a wide range of targets
(Receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-
HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7, 1A, 1B, 1D, 2A, 2B, 2C,
benzodiazepine, 1, 2, 3, GABA-A, H1, H2, H3, H4, M1, M2, M3,
M4, M5, -Opioid, -Opioid, -Opioid, Sigma-1, Sigma-2.
Figure 4: Histogram of the bending pseudo-angle of the ligand
(13r) in the trajectory of 1 microsecond simulation. Representative
molecular conformations corresponding to each region of the
histogram are shown with asterisks indicating the atoms that form
the pseudo-angle.
Transporters: dopamine, norepinephrine, serotonin.).
Both
compounds were highly selective for D3 over the majority of targets
(Ki’s > 10,000 nM), but (13g) displayed moderate affinity at 5-HT1a
(245 nM, 556 fold vs D3) and (13r) had affinity for 5-HT2C (20 nM,
40 fold vs D3), 5-HT7 (187 nM, 374 fold vs D3), H1, (3112 nM,
6224 fold vs D3), and SERT (118 nM, 236-fold vs D3).
(unpublished results), we observed the formation of this new
interaction. We have also previously reported that the overall ligand
conformation might be important to D3 binding affinity.22 In this
instance, we studied the conformational ensemble of (13r) in a 1
microsecond molecular dynamics (MD) simulation. A bending
pseudo-angle between three atoms in the benzamide region and the
phenyl piperazine region (denoted by asterisk in Figure 4) was
measured as representative of the overall structure. The histogram
of this pseudo-angle shows small population of the folded
conformation in the MD ensemble in comparison to the large
population of the extended and bent conformations (Figure 4),
similar to the conformation population distribution of other high
affinity ligands in our previous work,22 suggesting that extended
conformation of (13r) could explain its high affinity for D3.
Table 3: Rat pharmacokinetic properties of (13g) and (13r)
#
IV Dose
AUC
CL
Vss
T
1/2β*
(mg/kg) (ug/ml*h)
(h)
15.3
10.9
(mL/min/kg)
66.11
(L/kg)
55.67
28.57
13g
13r
1
1
0.21
0.22
74.44
*Terminal T1/2
In vivo pharmacokinetic (PK) assessment of both (13g) and
(13r) was conducted in Sprague Dawley rats. As noted in table 3,
long terminal T1/2’s were observed with both compounds (15.3 and
10.9 hours respectively) when an IV dose of 1 mg/kg was
administered. The volume of distribution of (13g) was nearly
double that of (13r), while clearance and AUC were similar.
Finally, protein binding studies indicated that both compounds are
highly protein bound (>99%).
Table 2: 2nd tier In vitro pharmacology of (13g) and (13r)
HLM
T1/2
CYP
2C9
CYP
2D6
CYP
3A4
D1
D4
D5
#
(min)
Ki (nM)
IC50 (nM)
13g
13r
60 >10000 >10,000 >10000
60 >10000 806 >10000 >10000 >10000 >10000
>10000 >10000
In summary, we have prepared a series of N-(4-(4-phenyl
piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide and evaluated
their properties in a range of in vitro assays. Our studies
demonstrate that the aryl piperazine region of the molecule can
have a substantial impact on D3 vs D2 selectivity, as various
substituent patterns produced selectivity ranging from 70.8- to
4615-fold. In addition, we identified two compounds (13g and 13r)
that are highly selective for D3 across a range of pharmacological
targets. In addition, rat in vivo IV PK studies that indicate that these
compounds are suitable for further study to determine brain
exposure in advanced PK studies followed by assessment in in vivo
efficacy models of substance use disorders.
We further characterized all of the analogs by determining their
stability (T1/2) in rat liver microsomes (RLMs). These in vitro
experiments can be used as predictive tools to identify compounds
that may have an in vivo pharmacokinetic profile capable of
supporting in vivo efficacy studies.26 RLM stability (T1/2) of this
series ranged from as low as 2.4 minutes to as long as 60 minutes.
Only 2 compounds, (13g) and (13r), had RLM T1/2 of 60 minutes
and D3 selectivity over D2 >1000-fold. These compounds were
selected for further evaluation. As seen in Table 2, in vitro human
liver microsome (HLM) studies indicated that both of these
compounds are highly stable (T1/2 = 60 min.) and had minimal
interactions the major metabolic Cyp450 enzymes (Cyp 3A4, 2D6,
and 2C9).
Graphical Abstract
We also examined the activity of (13g) and (13r) in D3
functional assays using a forskolin-dependent adenylyl cyclase
inhibition assay and a β-arrestin binding assay with quinpirole and

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Design, synthesis, and evaluation of N-(4-(4-phenyl
piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamides as
selective dopamine D3 receptor ligands
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Peng-Jen Chena, Michelle Taylorb, Suzy A. Griffinb, Armaghan Amanic, Hamed Hayatshahic, Kenneth
Korzekwaa, Min Yea, Robert H. Machd, Jin Liuc, Robert R. Luedtkeb, John C. Gordona, and Benjamin E. Blassa
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