Synthesis, in silico, and in vitro studies of novel dopamine D2 and D3 receptor ligands

Article abstract of DOI:10.1002/ardp.202000486

Dopamine is an important neurotransmitter in the human brain and its altered concentrations can lead to various neurological diseases. We studied the binding of novel compounds at the dopamine D₂ (D₂R) and D₃ (D₃

Full text of DOI:10.1002/ardp.202000486

Received: 23 December 2020
DOI: 10.1002/ardp.202000486
Revised: 26 January 2021
Accepted: 27 January 2021
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F U L L P A P E R
Synthesis, in silico, and in vitro studies of novel dopamine
D2 and D3 receptor ligands
Milica Elek1
Aleksandra Zivkovic1
| Nemanja Djokovic2
| Annika Frank1
| Slavica Oljacic2
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| Katarina Nikolic2 | Holger Stark1
1Institute of Pharmaceutical and Medicinal
Chemistry, Heinrich Heine University
Düsseldorf, Universitaetsstr. 1, Duesseldorf,
NRW, Germany
Abstract
Dopamine is an important neurotransmitter in the human brain and its altered
concentrations can lead to various neurological diseases. We studied the binding of
novel compounds at the dopamine D2 (D2R) and D3 (D3R) receptor subtypes, which
belong to the D2‐like receptor family. The synthesis, in silico, and in vitro char-
acterization of 10 dopamine receptor ligands were performed. Novel ligands were
docked into the D2R and D3R crystal structures to examine the precise binding
mode. A quantum mechanics/molecular mechanics study was performed to gain
insights into the nature of the intermolecular interactions between the newly in-
troduced pentafluorosulfanyl (SF5) moiety and D2R and D3R. A radioligand dis-
placement assay determined that all of the ligands showed moderate‐to‐low
nanomolar affinities at D2R and D3R, with a slight preference for D3R, which was
confirmed in the in silico studies. N‐{4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butyl}‐4‐
(pentafluoro‐λ6‐sulfanyl)benzamide (7i) showed the highest D3R affinity and
selectivity (pKi values of 7.14 [D2R] and 8.42 [D3R]).
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, University of Belgrade,
Belgrade, Serbia
Correspondence
Holger Stark, Heinrich Heine University
Düsseldorf, Institute of Pharmaceutical and
Medicinal Chemistry, Universitaetsstr.
1, 40225 Duesseldorf, Germany.
Email: stark@hhu.de
Funding information
Deutscher Akademischer Austausch Dienst,
Grant/Award Number: Milica Elek;
Ministarstvo Prosvete, Nauke i Tehnološkog
Razvoja, Grant/Award Number: UB Contract
No. 451‐03‐68/2020‐14/200161; European
Cooperation in Science and Technology,
Grant/Award Numbers: CA15135, CA18133,
CA18240; Deutsche Forschungsgemeinschaft,
Grant/Award Number: INST 208/664‐1 FUGG
K E Y W O R D S
D2 receptor, D3 receptor, ligands, pentafluorosulfanyl, QM/MM
transferase, as well as partly by monoamine oxidase A.[2–5] On the basis
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1
INTRODUCTION
of amino‐acid sequences and similarity in signal transduction, dopamine
Disorders as a result of neurodegenerative diseases, such as schizo-
phrenia, Parkinson's disease, Alzheimer's disease, affective disorder, and
addictive behavior, have been connected to altered concentrations of
neurotransmitters in the human brain, especially dopamine. During the
1960s, dopamine was recognized as an independent neurotransmitter in
addition to other well‐known catecholamines.[1] Biosynthesis of dopa-
mine, epinephrine, and norepinephrine starts from tyrosine. Dopami-
nergic neurons do not contain dopamine β‐hydroxylase enzymes that
convert dopamine to norepinephrine. Catabolic reactions of dopamine
include degradation by monoamine oxidase B,[2] catecholamine‐O‐methyl
receptors are divided into two different classes: dopamine D1‐like re-
ceptors that include D1 and D5 receptors and dopamine D2‐like re-
ceptors that include D2, D3, and D4 receptors.[6–8] All dopamine receptor
subtypes belong to the rhodopsin‐like A class of the largest group of
G‐protein‐coupled receptors (GPCR), characterized by the presence of
seven transmembrane domains. D1‐like receptors express
a long
carboxyl and D2‐like receptors express a short carboxy tail, which is
located intracellularly in both receptor subtypes.[9] D1‐like receptors
signal through Gαs protein and enhance the production of cAMP,
whereas D2‐like receptors activate Gαi that inhibits the production cAMP
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© 2021 The Authors. Archiv der Pharmazie published by Wiley‐VCH Verlag GmbH on behalf of Deutsche Pharmazeutische Gesellschaft
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Arch Pharm. 2021;e2000486.
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in the cell.[10,11] Dopamine D2 receptor (D2R) has also been implicated in
the G protein‐independent GPCR signaling, involving mediation with
β‐arrestin 1 and β‐arrestin 2, which scaffold the different pathways.[12]
D1‐like receptors are mainly located in the corpus striatum, nucleus ac-
cumbens, substantia nigra, olfactory bulb, and frontal cortex.[13,14] D2‐like
receptors are mostly found in substantia nigra, hypothalamus, amygdala,
and hippocampus,[15] and their density, regional distribution, and synaptic
response are affected by various neurological diseases,[16] stress, or
drug abuse.[17] Majority of commercially available D2‐like receptor
ligands have severe side effects due to their low selectivity to the
receptors of interest and high affinity toward other off‐target receptors.
To counter this, efforts have been centered around the design and
synthesis of selective D2‐like receptor ligands.[18] The main challenges in
developing novel ligands as potent pharmacological tools in the treat-
ment of diseases with altered concentration of dopamine are high
homology between receptors subtypes (up to 88% of D2R and D3 re-
ceptor [D3R] in structurally conserved regions)[19] and almost identical
orthosteric binding site (OBS) interaction within two receptors sub-
types.[20–22] Since its revelation, the D3R has been a target of interest in
potential pharmacotherapy of addiction and schizophrenia,[23] due to its
relatively focal localization and its expression in drug‐exposed
brains.[24] In addition, it has also emerged as a new potential target in
the treatment of Parkinson's disease.[25] Although all of the dopamine
receptor subtypes show a high level of similarity, it has been shown that
dopamine itself has a 100‐fold higher affinity at D3R when compared
with those at D1R or D2R.[26] In addition, D3R messenger RNA,
which is localized predominantly in the islands of Calleja and nucleus
accumbens in healthy humans,[27] could be a potential biomarker in
early‐stage Parkinson's patients.[28] Therefore, serious efforts have
been made to find potent, novel, and selective D3R ligands.
to the development of new potent selective ligands.[42–44] These moieties
have, therefore, been used in the synthesis of all 10 reported compounds.
A novel thermally and chemically stable pentafluorosulfanyl (SF5) moiety
that displays high values of electronegativity and lipophilicity[45,46] was
introduced to compare its effects on affinity and selectivity to-
ward receptors of interest. Due to these beneficial chemical properties,
SF5 can be used as a valuable bioisosteric replacement of the tri-
fluoromethyl and under special circumstances of tert‐butyl or nitro
group.[47] Therefore, our main aim is to develop, synthesize, and in vitro
and in silico characterize potent dopamine D2R and D3R ligands, which
could be further optimized and in vivo evaluated.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
To obtain amines 6a–e, two different synthetic methods have been
used. In the first synthetic approach (Route I), compound 1a has been
alkylated with N‐(ω‐bromoalkyl)phthalimide derivatives 2a–c to ob-
tain protected amines 4a–c. Consequently, hydrazine as a cleaving
reagent has been used for the deprotection of amines. To obtain
higher yields as well as to decrease costs of the synthesis, the second
synthetic approach (Route II) was introduced, where compounds
1a,b have been alkylated with 4‐bromobutanenitrile (3a) and
5‐bromovaleronitrile (3b), respectively. Reduction of obtained
nitriles led to crude amines 6c–e. Then, the primary amines 6a–e
were coupled with a corresponding activated carboxylic acid to
amides 7a–j. Both approaches are shown in Scheme 1.
A general pharmacophore of D3R antagonists has been described in
the early 2000s.[29] It contains four regions: The aromatic, the H‐bond
acceptor, the linker, and the amine regions. Piperazine has been de-
scribed as a promising moiety for binding and positioning into OBS of
D3R,[30–32] and therefore, is a structural part not only of many com-
mercially available drugs (e.g., lurasidone and cariprazine)[33–36] but also
of preclinical and clinical candidates.[37–39] The prototype of D3R partial
agonizts is BP897 (Figure 1).[40,41] BP897, which was developed for the
treatment of cocaine abuse, is a potent D3R ligand (Ki = 0.92 nM)
that served as a lead compound for many synthesized ligands in this
study. In addition, it has been established that ligands containing 4‐(2‐
methoxyphenyl)piperazino and 4‐(2,3‐dichlorophenyl)piperazino moieties
could have beneficial properties for D2‐like receptor binding[21] and lead
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2.2
Pharmacology
The affinity at human isoform dopamine D2shortR and D3R was de-
termined by radioligand displacement assays, as described
before.[48–50] In brief, radioligand displacement studies on membranes
prepared from CHO‐K1 cells expressing human dopamine D2shortR or
D3R have been performed using [3H]spiperone as a radioligand and
haloperidol as a standard for unspecific binding. The binding affinities of
the synthesized compounds with the corresponding confidence inter-
vals (CIs) as well as the selectivity index (SI) are shown in Table 1.
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2.3
Molecular docking
Molecular docking was used to access the binding modes of the
synthesized ligands and to obtain atomistic insight into the observed
inhibitory activities. All synthesized compounds were docked into
binding pockets of the cocrystal structures of D2R and D3R. In ad-
dition, docking scores were evaluated in terms of correlation with
observed binding affinities.
N
O
O
N
CH3
N
H
FIGURE 1 Chemical structure of the lead compound BP897

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SCHEME 1 Synthesis of compounds 7a–j. Reagents and conditions: (a) K2CO3, KI, reflux 16 h; (b) H2NH2N, MeOH, reflux, 2 h; 2 M HCl,
reflux, 1 h; (c) Raney‐Ni, NH3/MeOH, H2 5 bar,12 h; (d) HOOC‐R3, HOBt, EDC, R.T., 16 h
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2.3.1
Docking in D3R active site
Asp 1103.32 (OBS) formed a salt bridge with positively charged ni-
trogen from piperazine. This salt bridge interaction is believed to be
crucial in binding of ligands at the OBS of D3R, which is in agreement
with our docking results.[21]
All ligands were docked into the D3R cocrystal structure (PDB ID:
3PBL) in complex with eticlopride[21,51] (a potent D2R/D3R antago-
nist) according to the protocol described in Section 4. The docking
protocol was validated through redocking of eticlopride and calcu-
lation of heavy atoms RMSD (root mean square deviation), which
typically should not exceed 2 Å[52] (RMSD eticlopride = 0.67 Å,
Figure S1). Pearson's correlation coefficient (R2) and Spearman's
rank correlation coefficient (rs) were used to access the correlation
between docking and experimental results. Calculated docking
scores for our set of ligands significantly correlated with experi-
mental affinity (pKi) measurements for D3R (R2 = 0.92, rs = 0.97,
Table S1). High correlation coefficient values justified the reliability
of obtained binding modes for ligands.
Comparison of interacting modes between eticlopride and li-
gand 7i (Figure 1a), as the representative with the highest affinity
to the D3R, indicates a similar interaction profile in OBS. Interest-
ingly, pi–alkyl interaction between arylamine heads of the studied
ligands and Cys 1143.36, absent in the cocrystal structure with
eticlopride (Figure 1a), signifies the importance of this residue in
the binding of our series of phenylpiperazine ligands. Residue Cys
1143.36 (OBS) has been characterized as important for binding of
haloperidol in recent mutagenesis experiments. According to the
predicted poses of docked ligands, arylamide/coumarine tails are
bound to the extracellular vestibule shaped by Tyr 361.39, Val
862.60, Leu 892.63, Glu 902.64, Gly 93, and Ser 3667.35 (Figures 1a
and S2). Tyr 361.39 and Glu 902.64 have been characterized in recent
combined large‐scale high‐throughput molecular dynamics study
and mutagenesis study as important for binding of GSK598809
(dual D2R/D3R antagonist), which further validates predicted
binding modes.[54]
Predicted binding modes indicate that phenylpiperazine moiety
(arylamine head) binds to OBS, with the rest of the structures (ar-
ylamide/coumarine tails) extending to the extracellular vestibule/
second binding pocket (SBP)[21] (Figures 2a and S2). This is in
agreement with previous docking studies of structurally related
compounds.[52,53] For all studied ligands, highly conserved residue

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TABLE 1 Synthesized ligands and their in vitro affinity data
Name
Structure
MW
Ki (D2R) (nM) (95% CI) Ki (D3R) (nM) (95% CI) SI (D2/D3)
Haloperidol
375.9
2.61 (2.02; 3.39)
13.5 (10.4; 17.4)
0.2
(reference compound)
7a
448.4 105.1 (76.5; 144)
184 (99.7; 339)
0.6
7b
7c
7d
462.4
476.4
490.4
152 (68.2; 338);
9.45 (4.71; 19.0)
13.4 (9.16; 19.7)
127 (28.1; 570)
5.67 (1.88; 17.0)
30.7 (12.6; 75.3)
1.2
1.7
0.4
7e
7f
7g
7h
7i
435.5
465.6
493.5
532.2
493.5
532.2
65.5 (42.6; 101)
63.4 (36.5; 110)
54.3 (28.2; 105)
62.3 (25.5; 152)
72.3 (31.3; 167)
163 (90.2; 293)
9.04 (6.87; 11.9)
3.90 (1.57; 9.70)
4.96 (2.51; 9.79)
9.34 (5.45; 16.0)
3.52 (1.46; 8.74)
12.2 (6.69; 22.4)
7.2
16.3
10.9
13.3
20.5
6.7
7j
Abbreviations: MW, molecular weight; SI, selectivity index.

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FIGURE 2 Docking results of 7i into the binding site of (a) D3R and (b) D2R. The left side of the figure corresponds to the three‐dimensional
(3D) representation of binding sites, whereas the right side of the figure corresponds to the comparative 2D interaction plots obtained for 7i
and cocrystal ligands (a, eticlopride; b, risperidone). Encircled residues on 2D interaction plots represent residues that are engaged in
interactions with both ligands
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2.3.2
Docking in the D2R active site
cocrystalized risperidon: arylamino head was docked into OBS,
whereas arylamido/coumarine tail was docked into EBP (Figures 2b
and S4).[22] Comparison of two‐dimensional (2D) interaction profiles
for risperidone and 7i (representative with the highest selectivity
index) indicates high similarity in binding profiles (Figure 2b). A salt
bridge was observed between Asp1143.32 and positively charged
nitrogen from piperazine. This interaction was previously char-
acterized as fundamental for binding to the OBS of D2R. In addition,
two recently reported docking studies on D2R cocrystal struc-
ture further support our results regarding predicted binding modes
of ligands.[57,58]
Atypical antipsychotic risperidone was correctly redocked in PDB ID:
6CM4 using the docking protocol described in Section 4 (heavy
atoms RMSD 0.62 Å, Figure S3). A correlation between docking
scores and experimental affinity (pKi) (R2 = 0.32, rs = 0.73, Table S1)
was not as good as for D3R docking, but still under the range of
medium correlations expected for docking studies.[55] A possible
explanation for better correlation found in D3R docking study could
be found in recently reported higher flexibility of D2R's extended
binding pocket (EBP) as compared with the related part of D3R. This
flexibility was previously seen as the main reason why structure‐
based drug discovery campaigns were less successful in the case of
D2R.[56] All the studied ligands showed a similar binding mode to
In series of p‐monosubstituted SF5 derivatives (7i and 7j),
m‐monosubstituted SF5 derivatives (7g and 7h), and p,m‐disubstituted
derivative with the same linker length (7c), docking results indicate that

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m‐substitution is responsible for achieving an optimal interaction with
Tyr 4087.34 (Figure S5). Our results indicate that p‐substitution with a
voluminous substituent (e.g., SF5 moiety) in series of similar compounds
(four‐methylene groups linker) tends to decrease affinity toward D2R
due to steric hindrance, whereas it does not affect, to a larger extent,
affinity at D3R. This is in accordance with experimental findings re-
garding the distances between OBS and EBS in D2R and D3R, where
this distance is longer in D3R.[21,56]
approaches, where ligand and interacting residues are treated
quantum mechanically, whereas the rest of the system (e.g., mem-
brane, solvent, noninteracting residues, etc.) is treated classically, re-
present viable alternative to overcome limitations of current force
fields in studying noncovalent interactions between SF5 and the bio-
molecule of interest.
To validate predicted docking poses of 7i and to provide more
details on nature of SF5 intermolecular interactions with D3R and D2R,
we designed a multilevel QM/MM approach. The approach presented
here combines a semiempirical level of theory (PM3) with more ad-
vanced and computationally demanding DFT calculations (M06‐2X
functional with def2‐TZVP basis set).[59–61] PM3 calculations are in-
herently faster and can access tens to hundreds of picoseconds (ps) of
dynamics. However, M06‐2X level of theory has an advantage in im-
plementing higher accuracy in dealing with intermolecular interactions.
M06‐2X functional has been shown through benchmarking studies to
produce a good representation of noncovalent interactions.[59,62]
According to our results, poses of 7i obtained through molecular
docking remained stabilized during 100 ps of QM/MM simulations
(Figure 3a,b). Compound 7i was stabilized in D2R and D3R through an
equilibrium between repulsive and attractive noncovalent interactions
(NCIs) (Figure S6a,b). Comparing the initial docking poses and the ones
obtained after QM/MM protocol, 7i in D3R slightly moved SF5 moiety
toward Pro3627.31 residue and established interactions with it
(Figures 3d and S7). This interaction was unseen through molecular
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2.4
Quantum mechanics/molecular mechanics
(QM/MM) calculations
Pentafluorosulfanyl moiety is a relatively novel moiety in medicinal
chemistry and only a limited number of compounds containing this
moiety have been studied in interaction with biological systems.[47]
Furthermore, atomistic details on the interaction between the SF5
group and biological target molecules remain enigmatic, as no co-
crystal/nuclear magnetic resonance (NMR) structures have been de-
scribed so far. Also, there is a lack of detailed molecular modeling
studies on SF5 ligands. To the best of the authors' knowledge, the
most common force fields used for biomolecular simulations (e.g.,
CHARMM and AMBER sets of force fields) do not recognize this
moiety or this specific hypervalent sulfur atom type, which hinders the
application of classical molecular dynamics simulations. Hybrid QM/MM
FIGURE 3 Root mean square deviation (RMSD) of atomic positions during 100 ps of QM/MM (quantum mechanics/molecular mechanics)
(PM3) simulations, (a) D2R:7i system and (b) D3R:7i system and electrostatic potential (ESP) maps calculated on the M06‐2X level of theory for
the QM region after QM/MM minimizations, (c) D2R:7i system and (d) D3R:7i system, and after single‐point calculations. The blue line in (a) and
(b) indicates the RMSD calculated for the ligand atoms, whereas the red line represents the RMSD calculated for the protein backbone

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docking. Contrary to the D3R:7i complex, the D2R:7i complex remained
in a similar pose during the course of QM/MM simulation (Figure S7).
However, the hydrogen bond between Ser4097.35 of D2R and fluorine
of SF5 moiety predicted by molecular docking (Figure 2b) disappeared
after QM/MM, and it was replaced with C–H···F–S interaction (see
below). After QM/MM simulations, we also may note that SF5 moiety of
7i was encircled with interacting D3R residues, whereas upper fluorine
of SF5 was free and solvent‐exposed. Contrary to this, SF5 moiety of 7i
Materials). Results revealed that the protein environment affected
the charge distribution of SF5 moiety, indicating possible in-
tramolecular interactions. Fluorine atoms closer to protein residues
experienced more negative electrostatic potential, which could be
explained with intramolecular interactions between SF5 moiety and
protein residues (Figure 3c,d).
To gain more details of specific spatial regions and the nature of
interactions between proteins and SF5 moiety of 7i, NCI analysis was
performed. Self‐consistent field (SCF) densities for NCI analysis were
obtained from QM/MM calculations on M06‐2X level of theory. The
NCI analysis indicated that all the intramolecular interactions between
SF5 moiety and D2R/D3R appear to be in the spectrum of delocalized
weak interactions, sign(λ2)ρ(r) between 0.01 a.u (Figure 4). No strong
stabilizing interactions (e.g., hydrogen bonds) were detected. In the case
of D2R, C–H···F–S (Figure 4b,c) and S–F···C═O (Figure 4a) were the
most prominent interactions (reduced density gradient, s ≤ 0.3),
whereas for the D3R, C–H···F–S (Figure 4d) and S–F···O (Figure 4f)
intermolecular interactions were observed as most important (reduced
density gradient, s ≤ 0.3). A recent empirical study on fluorine–protein
interactions and 19F NMR isotropic chemical shifts indicated that highly
deshielded fluorines (also seen in SF5 moiety) mainly participate in
was buried in the sub‐pocket of D2R consisting of Tyr4087.34
,
Pro4057.31, and Ser4097.35 (Figure 3c,d), which was in accordance with
molecular docking results (Figure 2). Taken together, results indicate
that molecular docking was reasonably accurate in predicting the pose
of SF5 moiety. Nevertheless, QM/MM protocol is indispensable in
precise characterization of intermolecular interactions of SF5.
Electrostatic potentials (ESP) were derived from electron den-
sities and plotted on molecular van der Waals surfaces, to elucidate
how protein environment affected electronic density in the ligand.
ESP maps obtained after QM/MM calculations of QM regions of
protein/ligand complexes (M06‐2X level of theory) were compared
with ESP maps obtained after single‐point calculations of solo ligands
in the same conformations (Figure 3c,d, cf. Supporting Information
FIGURE 4 Results of the noncovalent interaction (NCI) analysis for D2R:7i (a–c) and D3R:7i (d–f) systems. On three‐dimensional (3D) NCI
plots, green isosurfaces represent delocalized weak attractive interactions (inter‐ and intramolecular), whereas red isosurfaces
represent repulsive interactions calculated from self‐consistent field (SCF) densities (quantum mechanics/molecular mechanics on M06‐2× level
of theory). On 2D NCI plots, sign(λ2)ρ(r) versus reduced density gradient (s), green points represent results from SCF densities for intra‐ and
intermolecular interactions, whereas yellow points represent results from promolecular densities only for intermolecular interactions. Due to
inherent limitations of the NCI approach, it was not possible to omit intermolecular interactions from analysis for SCF densities

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F···C═O orthogonal interactions with carbons from carbonyl groups and
in interactions with aliphatic carbons, whereas F···O interaction was
detected but classified as less common for deshielded fluorines.[63] Our
results are in agreement with these empirical findings.
moiety (7e, 7f) resulted in remaining affinity to both D2R and D3R,
whereas substitution with the 6‐methoxy group at coumarin moiety
(7f) resulted in increased affinities to both D2R and D3R. Compound
7i that contains the novel SF5 moiety showed not only the highest
affinity at D3R, but also the highest selectivity (SI = 20.5). Introduc-
tion of the SF5 moiety (7i) into the para‐position of the western part
of the molecule led to increased selectivity, more than 10‐fold,
toward D3R. When the eastern part was changed to 4‐(2,
3‐dichlorophenyl)piperazino substituents (7j), the selectivity was
reduced in comparison to 7i, but was still more than threefold to-
ward D3R (when compared to the parent compound [7c]). If the
position of SF5 group was changed, from para‐ to meta‐position, with
both groups in the eastern part (2‐methoxy and 2,3‐dichloro) (7g, 7h)
high affinities were obtained at D3R, pKi (D3R) = 8.30 and pKi
(D3R) = 8.03, respectively, with about sixfold increase in selectivity
when compared with that of the parent molecule 7c.
Furthermore, we evaluated a promolecular approach in char-
acterizing NCIs of SF5. The promolecular method (compared with
SCF approach) has an advantage in much faster calculations allowing
us to analyze interactions between the whole ligand and all inter-
acting residues (not limited only to the SF5 moiety). Comparative 2D
NCI plots (Figure 4) indicated that specific intramolecular interac-
tions obtained through SCF calculations are mainly positioned on
similar values of electron density and have similar values of reduced
density gradients as for promolecular NCI calculations. 3D NCI plots
indicated that promolecular approach successfully reproduced the
spatial position of the interactions (Figures 4 and S6). However, in
the case of the 7i:D2R complex, the promolecular method predicted
the existence of stronger interactions (higher values of electron
density) in some cases (Figure 4a,b). After visual inspection of 3D and
2D NCI plots, we concluded that the promolecular approach could be
a viable and faster alternative in the analysis of noncovalent inter-
actions between SF5 moiety and D2R and D3R, but some caveats
regarding bond strengths should be considered.
Our in silico results have confirmed that the protonated phe-
nylpiperazine moiety binds to OBS at both D2R and D3R, forming a
crucially important salt bridge between positively charged nitrogen
on piperazine and Asp110 3.32. The arylamide moiety binds to SBP at
D3R and EBP at D2R, which correlates with previously reported re-
sults. The compound with the highest affinity and selectivity to-
ward D3R (7i) was particularly challenging due to the new SF5 moiety
that is neither synthetically nor computationally fully characterized.
To the best of the authors' knowledge, for the first time, the
QM/MM approach was used to access intermolecular interactions of
SF5 with biomacromolecule. The QM/MM approach revealed that
the protein environment changed electron density distribution in SF5
moiety, whereas the NCI analysis confirmed that all of in-
tramolecular interactions between SF5 moiety and receptors of in-
terest are in the class of week delocalized interactions. In addition, it
has been shown that m‐substituted SF5 derivative was optimal for
interaction with the binding site of D2R, whereas p‐substitution with
this moiety led to decreased affinity at D2R due to steric hindrance,
which is in accordance with in vitro results obtained.
Finally, the promolecular method was used to access whole in-
termolecular interactions between 7i and D2R/D3R (Figure S6).
Considering results from both approaches (promolecular and SCF),
we may conclude that (pentafluorosulfanyl)phenyl moiety of 7i
achieved a larger number of interactions with D2R through SF5
moiety (Figure 4), whereas its interaction with D3R was driven
mainly through phenyl moiety (Figure S6). This is in accordance with
the results of molecular docking where steric effects of –p SF5
moiety of 7i prevented interaction with Tyr 4087.34 in D2R (see
above). In addition, convergence of QM/MM (PM3) simulations was
accessed by integrating promolecular densities for each frame.
Results indicated well‐converged simulations (Figure S6).
On the basis of reported results, we conclude that all of the 10
synthesized compounds represent potent, novel pharmacological
tools in the treatment of various neurological diseases. Compound 7i
that contains a pentafluorosulfanyl moiety has shown the highest in
vitro affinity and interesting binding mode toward receptors of in-
terest in this small series. The SF5 group can be taken as a promising
substituent in dopamine GPCR ligands, and therefore will be further
investigated in other compound classes of aminergic GPCRs.
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3
CONCLUSION
All of the 10 synthesized compounds exhibited nanomolar affinities
at dopamine D2R and D3R. Most of them expressed a slight pre-
ference for D3R. Compound 7c showed the highest affinity at D2R,
pKi (D2R) = 8.02, and 7i showed the highest affinity at D3R, pKi
(D3R) = 8.42. Our studies on the structure–activity relationship have
determined that the prerequisite for the best affinity to-
ward receptors of interest is the four methylene group linker be-
tween the amide and the aryl moiety (7c) pKi (D2R) = 8.02; pKi
(D3R) = 8.25. The compound containing five‐methylene group linker
(7d), pKi (D2R) = 7.87 pKi (D3R) = 7.51, displayed a higher affinity
when compared to the compounds that contain three‐
methylene (7b), pKi (D2R) = 6.82 pKi (D3R) = 6.90, or two‐methylene
linker (7a), pKi (D2R) = 6.98; pKi (D3R) = 6.74. Further optimization
was, therefore, performed with four‐methylene linkers. In vitro data
confirmed that the substitution of benzene ring with the coumarin
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All starting materials were obtained from Sigma Aldrich and Apollo
Scientific and used without further purification. Analytical thin‐layer

ELEK ET AL.
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chromatography was carried out on precoated TLC sheets ALUGRAM®
Xtra SIL G/UV254 (Macherey‐Nagel) with visualization under UV light.
Mass spectra have been determined using Advion Mass Express.
Atmospheric‐pressure chemical ionization (APCI) was used as a method
of ionization, operating in positive mode. Data are shown as [M+H+]+.
Melting points (mps) were determined by Büchi Schmelzpunkt M‐565
(Büchi) with an open capillary tube and were uncorrected. 1H and 13C
NMR spectra of compounds of interest were measured at Bruker
Avance‐III 300 (2010) and Bruker Avance‐III 600 (2011). Deuterated
dimethyl sulfoxide (DMSO‐d6) was used as a solvent for NMR and
tetramethylsilane was used as a standard. Chemical shifts are given as
parts per million (ppm) and reported as follows: s (singlet), d (doublet),
dd (double of doublets), t (triplet), q (quartet), p (pentet), or m (multi-
plet). The coupling constant (J) is given in Hertz (Hz). Purification of
compounds has been accomplished using flash chromatography: Bio-
tage Isolera™ Spektra Systems with ACI™ and Assist (Biotage). SNAP
KP‐Sil and SNAP KP‐Sil ULTRA (Biotage) were used as stationary
phase and dichloromethane (DCM) and MeOH were used as mobile
phase. Solvents have been evaporated using a Rotavapor R II (Büchi)
with a PC 3001 VARIO Chemie‐Vacuum pump (Vacuubrand) and CVC
3000 Vacuum controlling system. The compounds have been dried with
the high‐vacuum pump (Hybrid‐Pumpe RC 6; Vacuubrand). Compound
purities were determined by an elementary analysis Vario MICRO cube
elemental analyzer (Elementar Analysensysteme) and liquid
chromatography‐mass spectrometry (LC‐MS): Elute SP (HPG 700)
Bruker Daltronicsand amaZonspeed ion trap LC/MSn system (ESI‐MS).
Method: Alternating ion polarity: on; scan range: m/z: 80–1200; nebu-
lizer: nitrogen, 15 Psi; dry gas: nitrogen, 8 l/min, 200°C; mass range
mode: UltraScan; column: Intensity Solo 2 C18 (100 × 2.1 mm); tem-
perature: 50°C; mobile phase: A: water hypergrade for LC‐MS with
0.1% formic acid (v/v) (Merck); B: acetonitrile hypergrade for LC‐MS
(for LC‐MS); method of analysis: 0–4‐min 98% A, 4–5 min gradient 95%
A, 5–9 min 95% A, 9–16 min gradient 5% A, 16–17 min gradient to 0%
A, reconditioning: 17–18 min gradient to 98% A, 18–21 min 98% A (see
Supporting Information Materials).
reduced pressure. The crude mixture was purified by flash column
chromatography (sorbent: SiO2, eluent: DCM/MeOH gradient:
100–95%/0–5%) to obtain 4a–c.
2‐{2‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]ethyl}isoindoline‐1,3‐dione
(4a)[64,65]
Yellow solid. Yield: 42%. 1H NMR (300 MHz, DMSO‐d6) δ 7.93–7.81
(m, 4H), 6.97–6.79 (m, 4H), 3.75 (s, 3H), 3.72 (t, J = 6.5 Hz, 2H),
2.88–3.0 (m, 4H), and 2.62–2.52 (m, 6H). MS (APCI[+]): m/z [M+H+]+:
calculated for [C12H24N3O3]+: 366.2, found: 366.1.
2‐{3‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]propyll}isoindoline‐1,3‐
dione (4b)[66]
Yellow solid. Yield: 51%. 1H NMR (300 MHz, DMSO‐d6) δ 7.93–7.77
(m, 4H), 6.96–6.75 (m, 3H), 6.68 (dd, J = 7.6, 1.5 Hz, 1H), 3.72 (s, 3H),
3.67 (t, J = 6.7 Hz, 2H), 2.75–2.62 (m, 4H), 2.44–2.33 (m, 6H), and
1.77 (p, J = 6.6 Hz, 2H). MS (APCI[+]) m/z [M+H+]+: calculated for
[C22H26N3O3]+: 380.1, found: 380.3.
2‐{4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butyl}isoindoline‐1,3‐dione
(4c)[64,66]
Yellow solid. Yield: 96%. 1H NMR (300 MHz, DMSO‐d6) δ 7.93–7.78
(m, 4H), 6.99–6.79 (m, 4H), 3.75 (s, 3H), 3.59 (t, J = 6.9 Hz, 2H),
2.97–2.87 (m, 4H), 2.48–2.41 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 1.61
(p, 2H), and 1.45 (p, J = 7.3 Hz, 2H). MS (APCI[+]) m/z [M+H+]:
calculated for [C23H28N3O3]+: 394.2, found: 394.2.
|
4.1.3
General procedure for the synthesis of
ω‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]alkylamines
(6a–c): Route I
To a stirred solution of N‐{ω‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]
alkyl}phthalimide 4a (0.76 mmol), 4b (2.24 mmol), and 4c (2.82 mmol)
in 30 ml of MeOH, 0.5 ml of hydrazine monohydrate (64–65% aq.
solution) was added and stirred upon reflux for 2 h. After 2 h, 5 ml of
2 M HCl was added to the hot solution and the reaction mixture was
stirred at reflux temperature for another hour. After cooling down to
room temperature, the reaction mixture was filtered, and the filtrate
was concentrated to dryness. Then, 20 ml of 2 M NaOH was added
to the concentrated filtrate and residues were washed with water.
Extraction was performed with EtOAc and water. The organic layer
was separated and the remaining aqueous layer was extracted with
EtOAc (3×) and washed with brine. The combined organic layers
were dried with anhydrous MgSO4, filtered and concentrated under
reduced pressure. Crude products were purified by flash column
chromatography (sorbent: SiO2, eluent: DCM/MeOH/NH3).
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
General procedure for the synthesis
of N‐{ω‐[4‐(2‐methoxyphenyl)piperazin‐1‐y]alkyl}‐
phthalimides (4a–c)
To a stirred solution of suitable N‐(ω‐bromo‐alkyl)phthalimides 2a–c
(1.2 eq.) in acetone, 1‐(2‐methoxyphenyl)piperazine (1a) (1 eq) and
anhydrous K2CO3 (6–12 eq.) were added. The reaction mixture was
stirred at reflux temperature overnight. After cooling down the re-
action mixture to room temperature, inorganic salts were filtered
off and the filtrate was concentrated to dryness. The crude reaction
mixture was partitioned between EtOAc and water. The organic
layer was separated and the remaining aqueous layer was extracted
with EtOAc (3×) and washed with brine. The combined organic layers
were dried over anhydrous MgSO4, filtered and concentrated under
2‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]ethanamine (6a)[64,65,67]
Yellow oil. Yield: 46%. 1H NMR (300 MHz, DMSO‐d6) δ 7.03–6.76 (m,
4H), 3.76 (s, 3H), 3.02–2.89 (br s, 4H), 2.74 (d, J = 6.7 Hz, 2H),
2.46–2.35 (m, 2H), and 1.81–1.73 (m, 6H). MS (APCI[+]) m/z [M+H+]+:
calculated for [C13H22N3O]+: 236.2, found: 236.4.

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3‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]propanamine (6b)[65]
Yellow oil. Yield: 35%. 1H NMR (300 MHz, DMSO‐d6) 7.12–6.78
(m, 4H), 3.76 (s, 3H), 2.95–3.00 (br s, 4H), 2.84–2.75 (m, 2H),
2.42–2.32 (m, 2H), and 2.01–1.60 (m, 8H). MS (APCI[+]) m/z [M+H+]+:
calculated for [C14H24N3O]+: 250.2, found: 250.4.
4.1.5
General procedure for the synthesis of
|
ω‐[4‐(2‐methoxyphenyl)‐piperazinyl]alkylamines and
ω‐[4‐(2,3‐dichlorophenyl)‐piperazinyl]alkylamines
(6c–e): Route II
Obtained nitriles 5a (3.35 mmol), 5b (4.05 mmol), and 5c (5.10 mmol)
were dissolved in 50 ml ammonia solution in methanol and conse-
quently subjected to catalytic hydrogenation using freshly prepared
Raney nickel (from 500 mg of aluminum nickel alloy, as previously de-
scribed).[71] The reaction mixture was reduced with H2 at 5 bar pres-
sure overnight. The reaction mixture was filtered off through celite and
the filtrate was evaporated to dryness. The obtained amines were used
without further purification into the next reaction step.
4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butanamine (6c)[65–68]
Yellow oil. Yield: 69%. 1H NMR (300 MHz, DMSO‐d6) δ 7.10–6.74
(m, 4H), 3.75 (d, J = 3.0 Hz, 3H), 2.94 (t, J = 4.6 Hz, 4H), 2.61–2.53
(m, 2H), 2.40–2.46 (m, 6H), 2.29 (t, J = 6.9 Hz, 2H), and 1.55–1.29
(m, 4H). MS (APCI[+]) m/z [M+H+]+: calculated for [C15H26N3O]+:
264.2, found: 264.1.
|
4.1.4
General procedure for the synthesis of ω‐[4‐
4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butan‐1‐amine (6c)[68,72]
Yellow oil. Yield: 85%. 1H NMR (300 MHz, DMSO‐d6) δ 6.97–6.83 (m,
4H), 3.77 (s, 3H), 3.01–2.87 (br s, 4H), 2.61–2.53 (m, 2H), 2.44–2.52
(m, 6H), 2.30 (t, J = 7.2 Hz, 2H), and 1.53–1.30 (m, 4H). MS (APCI[+])
m/z [M+H+]+: calculated for [C15H26N3O]+: 264.2, found: 265.2.
(2‐methoxyphenyl)piperazinyl]alkylnitriles and ω‐[4‐
(2,3‐dichlorophenyl)piperazin‐1‐yl]alkylnitriles (5a–c)
To a stirred solution of 1‐(2‐methoxyphenyl)piperazine (1 eq.) (1a)
or 1‐(2,3‐dichlorophenyl)piperazine (1b) (1.1 eq.) in acetone, cor-
responding bromo‐alkyl‐nitriles 3a (6.76 mmol, 1 eq.) and 3b
(6.17 mmol, 1 eq.), and anhydrous K2CO3 (6–12 eq.) were added.
The reaction mixture was heated up to reflux for 16 h. After
cooling down, the inorganic salts were filtered off and the filtrate
was concentrated to dryness. The crude product was partitioned
between EtOAc (3×) and water. The organic layer was washed
with a saturated solution of NaHCO3 and brine and dried over
anhydrous MgSO4. Crude products were purified using flash col-
umn chromatography (sorbent: SiO2, eluent: DCM/MeOH
gradient:100–95%/0–5%).
4‐[4‐(2,3‐Dichlorophenyl)piperazin‐1‐yl]butan‐1‐amine (6d)[68]
Light yellow solid. Yield: 59%. 1H NMR (300 MHz, DMSO‐d6) δ
7.34–7.24 (m, 2H), 7.13 (dd, J = 6.3, 3.3 Hz, 1H), 3.02–2.92 (br s, 4H),
2.62–2.51 (m, 4H), 2.45–2.47 (m, 4H), 2.31 (t, J = 7.1 Hz, 2H), and
1.55–1.28 (m, 4H). MS (APCI[+]) m/z [M+H+]+: calculated for
[C14H22Cl2N3]+: 302.1 and 304.1, found: 302.4 and 304.4.
5‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]pentan‐1‐amine (6e)[69]
Yellow oil. Yield: 79%. 1H NMR (300 MHz, DMSO‐d6) δ 7.03–6.79 (m,
4H), 3.76 (s, 3H), 3.07–2.85 (br s, 4H), 2.58–2.52 (m, 2H), 2.40–2.46
(m, 4H), 2.29 (t, J = 8.0, 6.5 Hz, 2H), and 1.52–1.20 (m, 6H). MS (APCI
[+]) m/z [M+H+]+: calculated for [C16H28N3O]+: 278.2, found: 278.5.
4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butannitrile (5a)[67,68]
Yellow solid. Yield: 69%. 1H NMR (300 MHz, DMSO‐d6) δ 7.00–6.85
(m, 4H), 3.78 (s, 3H), 2.97–3.02 (br s, 4H), 2.47–2.50 (m, 6H), 2.41
(t, J = 6.9 Hz, 2H), and 1.76 (p, J = 7.0 Hz, 2H). MS (APCI[+]) m/z [M
+H+]+: calculated for [C15H22N3O]+: 260.2 and 261.2, found: 260.4
and 261.4.
|
4.1.6
General procedure for amide synthesis (7a–j)
To a stirred solution of 6a–e (1 eq.) and corresponding acid (1.1 eq.)
in DCM, HOBt (1.1 eq.) and EDC (1.1 eq.) were added. The reaction
mixture was stirred at room temperature overnight. Into the reaction
mixture, saturated solution of NaHCO3 was added for quenching and
it was stirred for 15 min. The crude product was partitioned between
DCM (3×) and water. The combined organic layers were washed with
a saturated solution of NaHCO3 and brine, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. The
crude mixtures were purified by flash column chromatography
(sorbent: SiO2, eluent: DCM/MeOH gradient: 100–90%/0–10%).
4‐[4‐(2,3‐Dichlorophenyl)piperazin‐1‐yl]butannitrile (5b)[68]
Yellow solid. Yield: 95%. 1H NMR (300 MHz, DMSO‐d6) δ 7.34–7.27
(m, 2H), 7.14 (dd, J = 6.2, 3.5 Hz, 1H), 3.04–2.91 (br s, 4H), 2.57–2.52
(m, 4H), 2.50–2.51 (m, 2H) 2.42 (t, J = 6.8 Hz, 2H), and 1.75
(p, J = 6.9 Hz, 2H). MS (APCI[+]) m/z [M+H+]+: calculated for
[C14H18Cl2N3]+: 298.1 and 300.1, found: 299.0 and 300.0.
5‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]pentannitrile (5c)[69,70]
Transparent oil. Yield: 88%. 1H NMR (300 MHz, DMSO‐d6)
δ
7.05–6.80 (m, 4H), 3.77 (s, 3H), 3.00–2.91 (br s, 4H), 2.56–2.52 (m,
2H), 2.46–2.50 (m, 4H), 2.34 (t, J = 6.5 Hz, 2H), and 1.70–1.47 (m,
4H). MS (APCI[+]) m/z [M+H+]+: calculated for [C16H24N3O]+: 274.2
and 275.2, found: 274.1 and 275.1.
3‐Bromo‐4‐methoxy‐N‐{2‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]-
ethyl})benzamide (7a)
Light yellow solid. Yield: 22%. 1H NMR (300 MHz, DMSO‐d6) δ 8.42
(t, J = 5.6 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H),

ELEK ET AL.
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7.19 (d, J = 8.7 Hz, 1H), 6.97–6.82 (m, 4H), 3.90 (s, 3H), 3.77 (s, 3H),
3.40 (t, J = 6.5 Hz, 2H), 3.02–2.88 (br s, 4H), and 2.63–2.54 (m, 4H).
13C NMR (75 MHz, DMSO‐d6) δ 164.28, 157.47, 151.93, 141.21,
131.75, 128.41, 127.95, 122.31, 120.79, 117.85, 112.07, 111.86,
110.23, 57.01, 56.48, 55.27, 53.01, 50.01, and 36.86. Elemental
analysis (calculated/found): %C 56.26/55.75, %H 5.85/5.87, and %N
9.37/9.50; mp = 163.4°C; Rf = 0.30 (eluent: DCM/MeOH 95:5). MS
(APCI[+]) m/z [M+H+]+: calculated for. [C21H27BrN3O3]+: 448.1;
450.1; and 449.1, found: 449.0; 450.0; and 451.0.
N‐{4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butyl}‐2‐oxo‐2H‐
chromene‐3‐carboxamide (7e)[66,73]
Yellow powder. Yield: 39%. 1H NMR (300 MHz, DMSO‐d6) δ 8.85 (s, 1H),
8.69 (t, J = 5.8 Hz, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (td, J = 8.7, 7.3,
1.7 Hz, 1H), 7.54–7.39 (m, 2H), 6.97–6.79 (m, 4H), 3.76 (s, 3H), 3.35–3.40
(q, 2H), 2.99–2.89 (br s, 4H), 2.45–2.50 (m, 4H), 2.35 (t, J = 6.7 Hz, 2H),
and 1.62–1.38 (m, 4H). 13C NMR (75 MHz, DMSO‐d6) δ 161.01, 160.38,
153.81, 151.93, 147.19, 141.25, 133.97, 130.18, 125.09, 122.28, 120.79,
119.20, 118.47, 117.83, 116.10, 111.87, 57.50, 55.27, 52.99, 50.03,
26.95, and 23.65. LC‐MS (ESI[+]) = 97.14%; mp = 122.5°C; Rf = 0.28
(eluent DCM/MeOH 95:5). MS (APCI[+]) m/z [M+H+]+: calculated for
[C25H30N3O4]+: 436.2 and 437.2, found: 436.1 and 437.1.
3‐Bromo‐4‐methoxy‐N‐{(3‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]-
propyl}benzamide (7b)
White solid. Yield: 30%. 1H NMR (300 MHz, DMSO‐d6) δ 8.49
(t, J = 5.5 Hz, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H),
7.18 (d, J = 8.7 Hz, 1H), 6.99–6.81 (m, 4H), 3.90 (s, 3H), 3.76 (s, 3H),
3.32–3.24 (br s, 2H), 3.00–2.90 (m, 4H), 2.50–2.60 (m, 4H), 2.38
(t, J = 7.0 Hz, 2H), and 1.70 (p, J = 7.0 Hz, 2H). 13C NMR (75 MHz,
DMSO‐d6) δ 164.25, 157.42, 151.93, 141.21, 131.67, 128.41, 128.05,
122.30, 120.79, 117.86, 112.04, 111.86, 110.21, 56.47, 55.79, 55.27,
53.02, 50.04, 37.96, and 26.14. Elemental analysis (calculated/
found): %C 57.15/56.70, %H 6.10/6.06, and %N 9.09/8.82; mp =
127.6°C; Rf = 0.33 (eluent: DCM/MeOH 95:5). MS‐(APCI[+]) m/z
[M+H+]+: calculated for [C22H29BrN3O3]+: 462.1 and 464.1, found:
462.1 and 464.1.
6‐Methoxy‐N‐{4‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]butyl}‐2‐oxo‐
2H‐chromene‐3‐carboxamide (7f)
Light orange solid. Yield: 67%. 1H NMR (300 MHz, DMSO‐d6) δ 8.82
(s, 1H), 8.73 (t, J = 5.7 Hz, 1H), 7.56 (d, J = 3.0 Hz, 1H), 7.46 (d, J = 9.1 Hz,
1H), 7.34 (dd, J = 9.1, 3.0 Hz, 1H), 6.99–6.81 (m, 4H), 3.82 (s, 3H), 3.76
(s, 3H), 3.36 (s, 2H), 2.95 (br s, 4H), 2.51 (m, 4H), 2.36 (s, 2H), and 1.54
(s, 4H). 13C NMR (75 MHz, DMSO‐d6) δ 161.03, 160.53, 155.92, 151.93,
148.31, 147.11, 128.11,122.30, 121.86, 120.79, 119.24, 117.84, 117.23,
117.05, 111.86, 111.79, 56.65, 55.82, 55.25, 52.95, 49.98, 40.54, 26.90,
and 23.60. Elemental analysis (calculated/found): %C 67.08/66.81, %H
6.71/6.67, and %N 9.03/8.90; mp = 140.4°C; Rf = 0.31 (eluent DCM/
MeOH 95:5). MS‐((APCI[+]): m/z [M+H+]+: calculated for
[C26H32N3O5]+: 466.2 and 467.2, found: 466.3 and 467.1.
3‐Bromo‐4‐methoxy‐N‐{4‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]-
butyl}benzamide (7c)
White solid. Yield: 44%. 1H NMR (300 MHz, DMSO‐d6) δ 8.43
(t, J = 5.6 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H),
7.18 (d, J = 8.7 Hz, 1H), 6.96–6.83 (m, 4H), 3.90 (s, 3H), 3.76 (s, 3H),
3.26 (q, J = 6.3 Hz, 2H), 3.00–2.86 (br s, 4H), 2.45–2.50 (m, 4H), 2.33
(t, J = 6.7 Hz, 2H), and 1.60–1.43 (m, 4H). 13C NMR (75 MHz, DMSO‐
d6) δ 164.21, 157.40, 151.93, 141.26, 131.72, 128.40, 128.08,
122.27, 120.79, 117.82, 112.03, 111.87, 110.20, 57.60, 56.46, 55.27,
53.01, 50.04, 27.04, and 23.80. Elemental analysis (calculated/
found): %C 57.99/57.83, %H 6.35/6.38, and %N 8.82/8.68;
mp = 162.0°C; Rf = 0.37 (eluent: DCM/MeOH 95:5). MS (APCI[+]) m/z
[M+H+]+: calculated [C23H31BrN3O3]+: 476.1; 477.1; and 478.1,
found: 476.0; 477.0; and 478.0.
N‐{4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butyl}‐3‐(pentafluoro‐λ6‐
sulfanyl)benzamide (7g)
White solid. Yield: 42%. 1H NMR (300 MHz, DMSO‐d6) δ 8.82
(t, J = 5.6 Hz, 1H), 8.32 (t, J = 1.9 Hz, 1H), 8.19–8.02 (m, 2H), 7.73
(t, J = 8.0 Hz, 1H), 6.99–6.83 (m, 4H), 3.76 (s, 3H), 3.33–3.28 (m, 2H),
3.02–2.88 (br s, 4H), 2.52–2.60 (m, 4H), 2.38 (t, J = 6.8 Hz, 2H), and
1.64–1.45 (m, 4H). 13C NMR (75 MHz, DMSO‐d6) δ 164.01, 152.78,
151.92, 141.16, 135.66, 131.06, 129.77, 128.21, 124.28, 122.33,
120.79, 117.82, 111.86, 57.44, 55.25, 52.92, 49.89, 26.86, and 23.64.
Elemental analysis (calculated/found): %C 53.54/53.24, %H 5.72/
5.87, %N 8.51/8.25, and %S 6.50/6.24; mp = 123.2°C; Rf = 0.49
(eluent DCM/MeOH 9:1). MS (APCI[+]) m/z [M+H+]+: calculated for
[C22H29F5N3O2S]+: 494.2, found: 494.9.
3‐Bromo‐4‐methoxy‐N‐{5‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]‐
pentyl}benzamide (7d)
N‐{4‐[4‐(2,3‐Dichlorophenyl)piperazin‐1‐yl]butyl}‐3‐(pentafluoro‐λ6‐
sulfanyl)benzamide (7h)
White solid. Yield: 30%. 1H NMR (600 MHz, DMSO‐d6) δ 8.42
(t, J = 5.6 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 1H),
7.18 (d, J = 8.7 Hz, 1H), 6.96–6.80 (m, 4H), 3.90 (s, 3H), 3.76 (s, 3H),
3.24 (q, J = 6.6 Hz, 2H), 3.00–2.87 (br s, 4H), 2.45–2.50 (m, 4H), 2.32
(s, 2H), 1.58–1.44 (m, 4H), and 1.36–1.27 (m, 2H). 13C NMR
(150 MHz, DMSO‐d6) δ 164.68, 157.91, 152.43, 141.73, 132.22,
128.91, 128.59, 122.79, 121.29, 118.32, 112.52, 112.37, 110.71,
58.27, 56.97, 55.77, 53.49, 50.47, 40.54, 29.42, 26.39, and 24.81.
LC‐MS (ESI[+]) = 95.08; mp = 110.7°C; Rf = 0.4 (eluent DCM/MeOH
9:1). MS (APCI[+]) m/z [M+H+]+: calculated for [C24H33BrN3O3]+:
490.2 and 492.2, found: 490.2 and 492.1.
Beige solid. Yield: 39%. 1H NMR (300 MHz, DMSO‐d6)
δ 8.80
(t, J = 5.3 Hz, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.22–8.02 (m, 2H), 7.72 (t,
J = 8.0 Hz, 1H), 7.34–7.24 (m, 2H), 7.17– 7.06 (m, 1H), 3.30 (t, J = 5.9 Hz,
2H), 3.06–2.89 (br s, 4H), 2.51–2.55 (m, 4H), 2.36 (t, J = 6.7 Hz, 2H), and
1.68–1.41 (m, 4H). 13C NMR (75 MHz, DMSO‐d6) δ 164.01, 153.1,
151.18, 135.67, 132.58, 131.06, 129.77, 128.38, 128.21, 125.96, 124.50,
124.29, 119.45, 57.39, 52.77, 50.91, 40.03, 26.88, and 23.75. LC‐MS (ESI
[+]) = 95.63%; mp = 104.8°C; Rf = 0.33 (eluent: DCM/MeOH 95:5). MS
(APCI[+]) m/z [M+H+]+: calculated for [C21H25Cl2F5N3OS]+: 532.1; 533.1;
and 534.1, found: 532,1; 533.1; and 534.1.

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ELEK ET AL.
|
N‐{4‐[4‐(2‐Methoxyphenyl)piperazin‐1‐yl]butyl}‐4‐(pentafluoro‐λ6‐
sulfanyl)benzamide (7i)
procedure: For all compounds, selection of dominant microspecies at
physiological pH 7.4 was performed using the Marvin Sketch 5.5.1.0
program. In the next step, the structures of all dominant forms were
preoptimized with the semiempirical/PM3 (parameterized model revi-
sion 3) method.[59] The minimized structures were then refined by using
a more precise quantum chemical Hartree–Fock/3‐21G method[82] for
geometry optimization employing Gaussian 09 software included in
Chem3D Ultra 7 program. Docking procedure: The binding site was
defined as residues within 6 Å from cocrystal ligands, and the number of
genetic algorithm runs was set to 30, with maximum flexibility ac-
counted for ligands. GoldScore was chosen as the scoring function,
according to the lowest RMSD in redocking experiments and 2D
interaction plots were generated using LigPlot+ software.[83]
White solid. Yield: 46%. 1H NMR (300 MHz, DMSO‐d6)
δ 8.74
(t, J = 5.7 Hz, 1H), 8.02 (s, 4H), 6.95–6.83 (m, 4H), 3.76 (s, 3H), 3.29
(t, J = 6.4, 5.1 Hz, 2H), 3.01–2.88 (br s, 4H), 2.45–2.50 (m, 4H), 2.35
(d, J = 8.5 Hz, 2H), and 1.64–1.45 (m, 4H). 13C NMR (75 MHz, DMSO‐d6)
δ 164.43, 151.93, 141.24, 138.17, 128.27, 125.94, 122.28, 120.79,
117.82, 111.87, 57.55, 55.27, 52.99, 50.02, 40.03, 26.90, and 23.74.
Elemental analysis (calculated/found): %C 53.54/53.50, %H 5.72/5.68, %
N 8.51/8.38, and %S 6.50/6.34; mp = 137.7°C; Rf = 0.5 (eluent: DCM/
MeOH 9:1). MS (APCI[+]) m/z [M+H+]+: calculated for [C22H29F5N3O2S]+:
494.2, found: 494.9.
N‐{4‐[4‐(2,3‐Dichlorophenyl)piperazin‐1‐yl]butyl}‐4‐(pentafluoro‐λ6‐
sulfanyl)benzamide (7j)
White solid. Yield: 39%. 1H NMR (300 MHz, DMSO‐d6) δ 8.73
(t, J = 5.4 Hz, 1H), 8.02 (s, 4H), 7.36–7.25 (m, 2H), 7.13 (dd, J = 6.0,
3.6 Hz, 1H), 3.29 (t, 2H), 3.04–2.92 (br s, 4H), 2.50–2.60 (m, 4H), 2.37
(t, J = 6.7 Hz, 2H), and 1.63–1.44 (m, 4H). 13C NMR (150 MHz,
DMSO‐d6) δ 164.01, 151.18, 135.67, 132.58, 131.06, 129.77, 128.38,
125.96, 124.29, 119.45, 57.39, 52.77, 50.91, 26.88, and 23.75.
LC‐MS (ESI[+]) = 96.80%; mp = 129.0°C; Rf = 0.33 (eluent: DCM/
MeOH 95:5). MS (APCI[+]) m/z [M+H+]+: calculated for
[C21H25Cl2F5N3OS]+: 532.1 and 534.1, found: 532.1 and 534.0.
4.3.2
QM/MM calculations
|
Initial protein–ligand complexes were generated through molecular
docking and prepared for simulation as explained above. The system
was minimized and equilibrated through six steps using standard Amber
inputs generated by CHARMM‐GUI (see above). In each equilibration
step, position restraints on protein were gradually reduced (starting
from 10 kcal/mol/Å2), whereas ligand's position restraints were kept
constant (10 kcal/mol/Å2) until the last two stages of equilibration. In
the last two steps (ligand restraints = 10 kcal/mol/Å2 and protein re-
straints = 0.5 kcal/mol/Å2; ligand restraints = 0 kcal/mol/Å2 and protein
restraints = 0.1 kcal/mol/Å2, respectively) of the equilibration protocol,
QM/MM approach was used. QM/MM equilibration phase was per-
formed using the sander suite of Amber 2018 utilizing semiempirical
PM3 Hamiltonian for treatment of the QM region (ligand + residues
located on a distance of 5 Å around the SF5 moiety, Figure S8), whereas
the rest of the system was treated classically using Amber ff14sb. After
equilibration, position restraints were completely removed and 100 ps
of the production run was performed using the same protocol QM/MM
(PM3). Each frame from the last 20 ps of trajectory was additionally
minimized on the same semiempirical PM3 level and the complex with
the minimal total energy was further optimized using QM/MM ap-
proach at the higher level of theory (DFT M06‐2X functional with def2‐
TZUP basis set).[59,61] Each cycle of minimization consisted of 200 cy-
cles of steepest descent and conjugated gradient. For the DFT QM/MM
minimization, Orca 4.2.1 interfaced with the sander utility of Amber
2018 was used.[84,85] These simulations were treated nonperiodically
with an electronic embedding scheme. The SHAKE algorithm was ap-
plied on H atoms in the QM region and the PME approach was used to
calculate long‐range electrostatics.
|
4.2
Pharmacological/biological assays
Radioligand displacement assays hD2R and hD3R have been per-
formed as described previously, including modifications.[47] In
short, the membrane was incubated with test ligands and [3H]spi-
perone. Haloperidol was used to determine nonspecific binding. As-
says were conducted in triplicate and in at Ieast three separate
experiments. Binding data were analyzed with GraphPad Prism using
nonlinear regression. Ki values were obtained from IC50 values.[74]
|
4.3
Molecular modeling
|
4.3.1
Molecular docking
For molecular docking, GOLD 5.6.3 software was used.[75] All ligands
were docked into cocrystal structures of D2R (PDB ID: 6CM4) and D3R
(PDB ID: 3PBL). Protein preparation included the following steps: Ly-
sozyme residues were removed manually and seven alanine residues
were inserted using Modeler software[76]; hydrogen atoms were added
to proteins using the PlayMolecule Protein Prepare procedure[77]
;
|
NCI calculations
proteins were inserted in the POPC membrane using a membrane
builder from CHARMM‐GUI[78]; protein–ligand complexes were sub-
jected to steepest decent energy minimization protocol in sander suite
of Amber 2018 software[79] using Amber ff14sb and GAFF2 force
fields.[80,81] Ligands for docking were prepared by the following
4.3.3
NCIplot 4.0 software[86] was used to interpret interactions between
ligand 7i and D2R and D3R. The NCI approach is based on the analysis
of reduced density gradients (s). Reduced density gradient, given by

ELEK ET AL.
13 of 15
|
Equation (1), represents a simple function of electron density (ρ) and its
gradient. It reflects local inhomogeneity of the electron density through
points of space. In regions far from the molecule, in which the density
decays to zero exponentially, the reduced gradient will have very large
positive values. However, values of RDS approach zero in the cases of
covalent bonds and NCIs. Lower densities and smaller gradients are
usually associated with the NCIs, and higher densities and smaller
gradients correspond to the covalent bonds:
by the Ministry of Education, Science, and Technological Develop-
ment of the Republic of Serbia. Nemanja Djokovic, Slavica Oljacic,
and Katarina Nikolic acknowledge the Ministry of Science and
Technological Development of the Republic of Serbia, Faculty of
Pharmacy UB Contract No. 451‐03‐68/2020‐14/200161.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
|∇ρ(r)|
1
s (r) =
.
(1)
Cs ρ(r)4/3
ORCID
Milica Elek
https://orcid.org/0000-0002-9094-8991
From a practical point of view, NCIplot analyzes only domains of
weak electron density and low reduced density gradients (NCIs).
Types of NCIs (hydrogen bond, van der Waals interactions, or steric
clashes) are determined using Laplacian of the density. Namely, the
sign of the second eigenvalues of the Hessian matrix (λ2) can dis-
criminate between different NCIs. The negative sign λ2 with higher
ρ values (>0.01 a.u.) usually corresponds to the strong stabilizing
interactions (e.g., hydrogen bonds), whereas positive λ2 and ρ va-
lues > 0.01 a.u. usually correspond to strong repulsive interactions.
ρ values around 0 correspond to delocalized weak interactions (e.g.,
van der Waals interactions). Around zero, the sign of λ2 is unstable
and does not reflect the stabilizing or destabilizing nature of such
interactions. Higher density corresponds to the stronger interactions
(stabilizing or destabilizing, depending on the sign of λ2) and vice
versa. NCIs were analyzed in the terms of the 2D NCI plots of
s versus ρ × signλ2, 3D NCI plots (isosurfaces), and integrals of
electron density (∫ρ2). The cut‐off value of s ≤ 0.3 was used to plot
gradients in 3D space and to generate isosurfaces of well‐defined
density values. Considering higher computational time required for
calculation of NCI from SCF calculations (QM/MM), SCF‐derived
gradients were used to specifically access interactions of SF5 moiety
with protein residues and to compare results with a computationally
cheaper promolecular approach. The promolecular approach was
further used to access interactions of whole ligands with proteins.
Integrals of promolecular densities (∫ρ2) of three specific regions, sign
(λ2)ρ(r) between −0.05 and −0.01; sign(λ2)ρ(r) between −0.01 and
0.01, and sign(λ2)ρ(r) between 0.01 and 0.05, across QM/MM tra-
jectory were used to quantitatively access convergence of QM/MM
simulations.[87,88]
Nemanja Djokovic
https://orcid.org/0000-0001-9972-3492
https://orcid.org/0000-0002-1637-2699
https://orcid.org/0000-0001-9128-6072
Annika Frank
Slavica Oljacic
Aleksandra Zivkovic
Katarina Nikolic
https://orcid.org/0000-0001-5034-7916
https://orcid.org/0000-0002-3656-9245
Holger Stark
https://orcid.org/0000-0003-3336-1710
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ACKNOWLEDGMENTS
The authors would like to express their gratitude to the
German Academic Exchange Service (Deutscher Akademischer
Austauschdienst) for Milica Elek. They would like to thank the
European Cooperation in Science and Technology (COST) COST
Actions CA15135, CA18133, CA18240, and German Research
Foundation (Deutsche Forschungsgemeinschaft‐DFG: INST 208/
664‐1 FUGG) for financial support. Numerical simulations were run
on the PARADOX‐IV supercomputing facility at the Scientific
Computing Laboratory, National Center of Excellence for the Study
of Complex Systems, Institute of Physics Belgrade, supported in part
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