Controlling the selectivity of aminergic GPCR ligands from the extracellular vestibule

Article abstract of DOI:10.1016/j.bioorg.2021.104832

In addition to the orthosteric binding pocket (OBP) of GPCRs, recent structural studies have revealed that there are several allosteric sites available for pharmacological intervention. The secondary binding pocket (SBP) of aminergic GPCRs is located in t

Full text of DOI:10.1016/j.bioorg.2021.104832

Bioorganic Chemistry 111 (2021) 104832
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Controlling the selectivity of aminergic GPCR ligands from the
extracellular vestibule
a
a
a,
b
c
b
´
´
´
´
´
Attila Egyed , Adam A. Kelemen , Marton Vass , Andras Visegrady , Stephanie A. Thee ,
Zhiyong Wangb, Chris de Graafd, Jose Breae, Maria Isabel Lozae, Rob Leursb,
a,*
˝
¨
Gyorgy M. Keseru
a
´
Medicinal Chemistry Research Group, Research Center for Natural Sciences Magyar tudosok krt. 2, Budapest, H-1117, Hungary
b Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan
1108, Amsterdam, 1081 HZ, Netherlands
c
¨
˝
Gedeon Richter plc. Gyomroi út 19-21, Budapest, H-1103, Hungary
d Sosei Heptares, Steinmetz Granta Park, Great Abington, Cambridge CB21 6DG, UK
e Innopharma Screening Platform, BioFarma Research Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de
Compostela, 15782, Santiago de Compostela, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
In addition to the orthosteric binding pocket (OBP) of GPCRs, recent structural studies have revealed that there
are several allosteric sites available for pharmacological intervention. The secondary binding pocket (SBP) of
aminergic GPCRs is located in the extracellular vestibule of these receptors, and it has been suggested to be a
potential selectivity pocket for bitopic ligands. Here, we applied a virtual screening protocol based on fragment
docking to the SBP of the orthosteric ligand-receptor complex. This strategy was employed for a number of
aminergic receptors. First, we designed dopamine D3 preferring bitopic compounds from a D2 selective orthos-
teric ligand. Next, we designed 5-HT2B selective bitopic compounds starting from the 5-HT1B preferring ergoline
core of LSD. Comparing the serotonergic profiles of the new derivatives to that of LSD, we found that these
derivatives became significantly biased towards the desired 5-HT2B receptor target. Finally, addressing the
known limitations of H1 antihistamines, our protocol was successfully used to eliminate the well-known side
effects related to the muscarinic M1 activity of amitriptyline while preserving H1 potency in some of the designed
bitopic compounds. These applications highlight the usefulness of our new virtual screening protocol and offer a
powerful strategy towards bitopic GPCR ligands with designed receptor profiles.
GPCR
Selectivity
Bitopic ligand
Secondary binding pocket
Fragment linking
1. Introduction
drugs target a specific combination of these receptors [4]. However,
sequence and structural conservation of the 42 human aminergic GPCR
G protein-coupled receptors (GPCRs) represent the largest protein
family of drug targets, as 34% of FDA-approved drugs act on 108 GPCRs
[1]. Although many of these medicines were designed to target a single
receptor, it has been increasingly recognized that many drugs act on
more than one target protein [2,3]. The first approach follows the “one
drug, one target” paradigm and requires “magic bullet” compounds that
are highly specific for the particular target. The multitarget approach
needs ligands with optimally designed (or unintentionally discovered)
receptor profiles, which has been recognized as a powerful strategy for
central nervous system pharmacotherapy. Considering only aminergic
GPCRs, 60% of the marketed drugs are “magic bullets”, while 40% of the
subtypes makes the design principles less than obvious for both types of
ligands.
Aminergic GPCRs recognize small biogenic amines such as acetyl-
choline, adrenaline, dopamine, histamine, and serotonin bound to an
orthosteric site of the receptors. In addition to this evolutionarily
conserved orthosteric binding pocket (OBP) formed by 7 trans-
membrane helices, recent structural studies have revealed the avail-
ability of another pocket located at the extracellular vestibule of
aminergic GPCRs. This secondary binding pocket (SBP) may function as
a stable or transient allosteric binding site (see Table S1 in the Supple-
mentary Material for the definition of these binding pockets) [5]. For
* Corresponding author.
˝
E-mail address: keseru.gyorgy@ttk.mta.hu (G.M. Keseru).
https://doi.org/10.1016/j.bioorg.2021.104832
Received 26 October 2020; Received in revised form 18 January 2021; Accepted 15 March 2021
Available online 19 March 2021
0045-2068/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
example, the muscarinic M2 receptor harbours the allosteric modulator
LY2119620 in the crystal structure of the ternary complex [6]. In the
case of the β2 adrenergic receptor, the SBP was transiently occupied
during the simulation of the association/dissociation of the alprenolol
ligand-receptor complex [7], and for the muscarinic M3 receptor, tio-
tropium has been shown in simulations to bind to both binding sites [8].
Analysing the sequence and conformational similarity of both
pockets, SBPs were found to be much less conserved than OBPs, sup-
porting an SBP-focused strategy for designing ligands with specific re-
ceptor profiles for aminergic GPCRs [9] (see Fig. S2 in the
Supplementary Material for the sequence comparison of the binding
pockets of the receptors studied in this paper). Having developed bitopic
compounds bound to both the OBP and the SBP, we and others have
previously confirmed the role of the SBP in both the affinity and selec-
tivity of these ligands [9–13]. Most of the promising bitopic compounds
were, however, identified by trial and error, and the role of the different
pockets was only confirmed retrospectively.
Fig. 1. Bitopic ligands identified to bind to the dopaminergic D2 and D3 re-
ceptors. OBP and SBP binder moieties are coloured red and blue, respectively.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
In this study, we present a prospective strategy for designing com-
pounds for specific receptors using our multiple fragment docking pro-
tocol [10,14]. First, we docked suitable fragments into the OBP of the
receptors and merged these fragments with the protein structure.
Starting from the GPCR structure with a core fragment bound in the
OBP, we performed a second round of virtual fragment screening against
the SBP of the merged protein-ligand complex. The identified hit frag-
ments in the SBP were then linked to the core OBP fragment, resulting in
rationally designed bitopic ligands. These bitopic ligands were redocked
to confirm the conserved binding modes of the OBP and SBP fragments.
Finally, the designed bitopic compounds with the best fragment com-
binations were synthesized and tested experimentally. Using this new
strategy, we were able to reverse the selectivity of the original OBP
fragment for the dopaminergic D2/D3 and serotonergic 5-HT1B/5-HT2B
receptor pairs and designed novel and selective H1 antagonists free of
off-target binding to the muscarinic M1 receptor.
work, starting from D2 preferring 2-methoxyphenyl-piperazine core (1),
we found that attaching the hit fragment N,N-dimethylcyclohexylurea
from the virtual screening against the SBP of the D3 receptor to the OBP
core resulted in balanced D2/D3 bitopic compound 2. Linking the
cyclohexylurea fragment, which was the best scoring fragment for D3,
turned the OBP binder into D3 preferring bitopic derivative 3 (Table 1).
This observation has also been seen with other OBP cores [10] (see Fig. 5
for the proposed binding modes of 3).
2.2. Selectivity between the serotonin 5-HT1B/2B receptors
Next, we challenged the design protocol against the serotonergic 5-
HT1B and 5-HT2B receptors. We chose these targets since they represent
another main group of aminergic GPCRs with multiple X-ray structures
available. The first structures of both receptors were co-crystallized with
the highly potent but non-selective antimigraine drug ergotamine
[21,22]. Comparative analysis of the binding modes revealed that
ergotamine binds to the orthosteric and secondary binding pockets.
While the binding modes and the interaction patterns were very similar
in the OBP, the binding modes in the corresponding SBPs were found to
be significantly different (see Fig. S2 in the Supplementary Material and
Fig. 5 for SBP comparison). First, the SBP available in the 5-HT1B
structure was expanded much more than the 5-HT2B receptor. Further-
more, the bulky M2185x40 residue located in the SBP of the 5-HT2B re-
ceptor is mutated to the much smaller T2095x40 in 5-HT1B, which makes
the 5-HT1B SBP more accessible for larger ligands. More recently, the
psychotropic ergoline derivative LSD has been co-crystallized with the
5-HT2B receptor [23]. Although this crystal structure revealed slight
binding mode differences, the ergoline ring system of ergotamine and
LSD fit into the OBP in both cases. The ethyl groups of LSD do not reach
the SBP and therefore LSD can be considered an OBP binder (Fig. 2).
Comparing the pharmacological profiles of these two compounds,
LSD shows tenfold selectivity towards the 5-HT1B receptor, activity that
is absent for bitopic ergotamine, which has a balanced 5-HT1B/5-HT2B
2. Results and discussion
2.1. Selectivity between the dopamine D2/3 receptors
The first test of our design strategy was directed at the dopamine D2
and D3 receptor pair. The D2 receptor has been established as an
important target for atypical antipsychotics, including risperidone, and
this drug has also been recently co-crystallized with the receptor [15].
This high-resolution X-ray structure confirmed that the tetrahydropyr-
ido[1,2-a]pyrimidin-4-one ring of risperidone binds in the SBP, which
differs considerably from that of the D3 and D4 receptors. Interestingly,
however, risperidone does not show high D2 selectivity over these re-
ceptor subtypes (Ki values are 2.4, 8.0 and 5.8 nM for D2, D3 and D4,
respectively [16]). In our work developing next-generation treatment
options for mental diseases, we discovered cariprazine [17], a new drug
approved by the US FDA and EMEA for psychosis, mania, and depres-
sion. Cariprazine shows a remarkable structural difference relative to all
other marketed antipsychotics with a non-aromatic moiety in place of
the previously indispensable aromatic pharmacophore in the SBP
(Fig. 1). This feature also differentiates cariprazine from aripiprazole,
another D2 partial agonist that shares the identical 2,3-dichlorophenyl-
piperazine core in the OBP. Comparing the dopamine receptor profile of
cariprazine [18] to that of risperidone [19] and aripiprazole [18], car-
iprazine shows a clear preference towards the D3 receptor. This has been
proposed to account for the unprecedented efficacy of cariprazine on the
negative symptoms of schizophrenia [20].
Table 1
D2 and D3 receptor affinities of the OBP binding 2-methoxyphenyl-piperazine
(1) and its bitopic derivatives (2, 3). s.d. values are in parentheses.
SBP fragment
pKi D2
pKi D3
Selectivity
The correlation between the D3 receptor preference and the unique
pharmacophore of cariprazine prompted us to utilize our strategy to
design novel bitopic ligands by fragment linking. In our previous study,
we docked 266 aliphatic fragment candidates into the SBP in the pres-
ence of different core fragments bound to the OBP [10] (see all
Computational Methods in the Supplementary Material). In the current
N/A (1)
6.17
(0.05)
8.67
(0.1)
8.48
(0.1)
5.65
D2 preferred
(0.08)
8.79
(ΔpKi: ꢀ 0.52)
Balanced D2/D3
(ΔpKi: 0.12)
N,N-dimethylcyclohexylurea (2)
cyclohexylurea (3)
(0.13)
9.30
D3 preferred
(0.15)
(ΔpKi: 0.82)
2

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
Fig. 2. Bitopic ligands identified to bind to the serotonergic 5-HT1B and 5-HT2B
receptors. OBP and SBP binder moieties are coloured red and blue, respectively.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 3. Comparative receptor profile of LSD [24], ergotamine [25], and the
designed bitopic compounds (4 and 5) measured against a panel of aminergic
GPCRs. Binding affinities are expressed as pKi values.
profile [24,25] (Table 2).
Our motivation again was to turn the selectivity of the OBP binding
core in the opposite direction. Therefore, we applied our design protocol
using a selected set of SBP fragments extracted from known bitopic 5-
HT2B binders available in the ChEMBL database [26] to improve selec-
tivity towards the 5-HT2B receptor. A set of 119 fragments was docked
into the SBP of the 5-HT2B receptor while keeping the ergoline core in
the OBP. Linking the best-scored fragments to the OBP core through an
amide bond, the resulting bitopic compounds were redocked, and con-
servation of the binding modes was confirmed in both the OBP and the
SBP (see all Computational Methods in the Supplementary Material).
The two best combinations were synthesized and tested for their 5-HT1B
and 5-HT2B activity (Table 2). These results confirmed that linking
carefully selected 5-HT2B preferring SBP fragments to the unchanged
OBP core enabled us to rationally design bitopic compounds 4 and 5
with the desired selectivity profiles, even achieving reversed selectivity
relative to LSD, the starting OBP core. It is worth noting that the SBP
lower affinity towards adrenergic and dopaminergic receptors than
ergotamine. The general serotonergic preference of LSD turned into a
more specific 5-HT2B preferring profile for the designed bitopic com-
pounds. Compound 4 showed almost two orders of magnitude selec-
tivity for 5-HT2B over the other serotonin receptors, while compound 5
was active only on the 5-HT2B and 5-HT7 receptors, having a clear
preference for the former (see Fig. 5 for the proposed binding modes of
5).
2.3. Selectivity between histamine H1 and muscarinic acetylcholine M1
receptors
Finally, we used the developed design protocol to address a thera-
peutic issue associated with first-generation antihistamines. These
compounds were found to be fairly potent muscarinic acetylcholine M1
receptor antagonists that cause reduced secretions in the airways and
saliva [27] (Fig. 4).
fragment of
5 originates from the 5-HT1B-selective compound
CHEMBL1631542; however, in the docked binding poses of 5 and
CHEMBL1631542, the SBP fragments are flipped and have different
interactions with T/M5x40, which are favourable for 5 and Y/T2x63 and
F/L7x34, which are favourable for CHEMBL1631542. This result high-
lights the potential of the proposed methodology to design bitopic li-
gands based on SBP virtual screening. Next, we investigated the impact
of the designed SBP moieties on the broader aminergic receptor profile
of the compounds and compared their profiles to those of LSD and
ergotamine (Fig. 3).
Analysing the sequences and high-resolution crystal structures of the
H1 [28] and M1 [29] receptors revealed that the corresponding OBPs are
highly similar, but their SBPs are considerably different (see Fig. S2).
Most of the key ligand-contacting residues are the same in the OBPs,
with the most prominent change being the F4326x52 to N3826x52 mu-
tation from H1 to M1, which introduces increased polarity and more
specific interactions with the carbonyl group of acetylcholine in the
muscarinic acetylcholine receptors. In contrast, the different sizes and
This comparison revealed that ergotamine is more potent on the
tested receptors than LSD and the less complex bitopic compounds. LSD,
however, is more selective for serotonergic receptors, having much
Table 2
5-HT1B and 5-HT2B receptor affinities of OBP binding LSD and its bitopic de-
rivatives (ergotamine, 4, and 5). s.d. values are in parentheses.
SBP fragment
pKi 5-
pKi 5-
Selectivity
HT1B
HT2B
N/A (LSD) [21]
8.50
7.50
5-HT1B preferred
(ΔpKi: ꢀ 1.00)
cyclic tripeptide (ergotamine)
[22]
9.77
9.50
Balanced 5-HT1B/
2B
(ΔpKi: ꢀ 0.27)
1-(o-tolyl)pyrrolidine-2,5-dione
(4)
6.12
7.24
5-HT2B preferred
(ΔpKi: 1.12)
Fig. 4. Bitopic ligands identified to bind to the histamine H1 and muscarinic
acetylcholine M1 receptors. OBP and SBP binding moieties are coloured red and
blue, respectively. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
(0.05)
<5.00
(0.06)
6.79
3-(o-tolyl)oxazolidin-2-one (5)
5-HT2B preferred
(ΔpKi: >1.79)
(0.05)
3

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
characteristics of the SBPs suggest that connecting specific SBP frag-
ments to the OBP binding core can improve H1 selectivity. Considering
the negatively charged character of the phosphate ion from the crys-
tallization buffer found in the SBP in the X-ray structure of the H1 re-
effects of bitopic binding on selectivity. However, final proof would
need X-ray studies with the present set of compounds. Since an
increasing number of GPCR crystal structures have been elucidated, we
envision that in the future, this method will be applicable not only to
certain receptor pairs but also to design compounds with specific multi-
receptor profiles, ultimately resulting in the desired on-target activity
and reduced side effect profiles.
ceptor, we docked
a
library of commercial, in-house, and
orthophosphate isostere fragments into this pocket in the presence of
amitriptyline bound in the orthosteric pocket. We chose amitriptyline
because this ligand binds only in the OBP, its histaminergic and mus-
carinergic profiles are largely known from the DrugMatrix database
(literature pKi values H1: 9.3, H2: 6.1, M1: 8.6, M2: 7.5, M3: 8.3, M4: 9.1,
M5: 8.2 [30]), and it is easy to derivatize starting from nortriptyline.
Linking the top scoring fragments through the basic amine group of
the OBP core resulted in bitopic compounds that were redocked to
investigate the conservation of the binding modes in the OBP and the
SBP (see all Computational Methods in the Supplementary Material).
The best SBP fragment, a pyrimidin-2-one unit, was linked to nortrip-
tyline with either an ethyl (6) or propyl (7) linker and tested experi-
mentally (Table 3). These results revealed that this design protocol is
able to provide bitopic compounds with remarkable selectivity towards
the preferred histamine H1 target. Amitriptyline shows only 7-fold
selectivity towards the H1 receptor, whereas the designed bitopic com-
pounds show 50- to 80-fold selectivity, with the propyl linker being the
best option for high H1 receptor affinity (Table 3) towards the desired
target, proving to be useful candidates to study the relationship between
selectivity and muscarinic receptor-related side effects (see Fig. 5 for the
proposed binding modes of 7).
4. Experimental section
4.1. Computational Methods
The work on dopamine D2-D3 receptors has been addressed in Vass
et al. [10] Briefly, the crystal structure of the D3 receptor (PDB ID: 3PBL
[37]) and a homology model of the D2 receptor obtained using
¨
Schrodinger Prime 3.2 [38] were used for docking. A focused library of
196 fragments (comprising of a substituted aryl ring and a piperidine/
piperazine moiety connected with a single bond or short linkers) were
¨
prepared with Schrodinger LigPrep 2.6 [38] and docked with
¨
Schrodinger Glide 5.9 [38] into the OBP of both receptors. Poses were
filtered for H-bond interaction with D3x32. The OBP fragments were then
merged with the receptor structures in Maestro [38] and new docking
grids were generated. A second focused fragment library of 266
substituted cyclohexyl amines and piperidines was docked into the SBP
of the merged structures, and best fragment combinations based on
docking scores were selected. Finally, the selected OBP and SBP frag-
ments were linked with ethylene linkers and the resulting bitopic ligands
were docked again in the apo structures of the receptors. In the present
paper ligands were redocked into the crystal structure of the D2 receptor
It should be noted that high selectivities in this case may be attrib-
utable to the larger sequence and structural differences found between
the H1 and M1 receptors, as they belong to different receptor families,
compared to the available differences within a certain receptor family.
¨
(PDB ID: 6CM4 [15]) with Schrodinger Suite 2019-4 [39] using a similar
protocol.
3. Conclusions
In case of the serotonin 5-HT1B-5-HT2B receptors a set of 119 virtual
fragments was compiled by the fragmentation of known active and se-
lective 5-HT1B and 5-HT2B ligands obtained from the ChEMBL 24 data-
base [26]. The tripeptide moiety of ergotamine was cut off, and the
remaining ergoline core was merged with the receptor structures
resulting in four new receptor structures with the SBP as singular
binding pocket. The fragment set was docked into the secondary binding
pocket of these four merged receptor structures. The docking poses were
filtered for occupying the SBP: based on the interactions with the non-
conserved amino-acid pairs at 5-HT1B and 5-HT2B receptor SBP sites
(Y/T2x63, V/L45x52, T/M5x40, M/L6x58, F/L7x34) [9] and based on the
principal interactions of the tripeptide moiety in the SBP. The two
selected fragments (in compounds 4 and 5) originated from the
following compounds: ChEMBL1631542 (pKi (5-HT1B) = 8.7, pKi (5-
HT2B) = 5.9) and ChEMBL1631544 (pKi (5-HT1B) = 7.4, 5-HT2B data not
available) respectively [40]. These best docked fragments were then
linked to the ergoline scaffold by enumerating different regioisomers
(ortho, meta, and para anilines) and (CH2)0-2 linkers between the aniline
and the linker amide NH of ergotamine. All of the derivatives were
docked into the original entire binding pocket of the four GPCR struc-
tures. Docked ligands were evaluated by Glide docking scores and poses
were filtered by the criteria of H-bonding to D3x32, occupation of the SBP
cavity, and RMSD to the ergoline core.
Targeting aminergic GPCRs requires precise selectivity or poly-
pharmacological profiles for drug candidates to achieve the desired
therapeutic effect. While an abundance of bitopic ligands are known for
aminergic GPCRs, the complex receptor profiles of multitarget com-
pounds could hardly be designed. Here, we present a general structure-
based fragment linking methodology to exploit both the conservation of
the orthosteric binding pocket (OBP) and the divergence of the sec-
ondary binding pocket (SBP) to design compounds with the desired af-
finity and selectivity profile against specific receptor pairs. We have
demonstrated the utility of this virtual fragment screening and linking
methodology to design compounds with different selectivity profiles
against D2/D3, 5-HT1B/5-HT2B, and H1/M1 receptor pairs. We have
shown that this general methodology is capable of delivering such
compounds irrespective of the source of the fragments and the compu-
tational tools employed. The fragment and bitopic compound binding
modes, binding pose robustness, and docking scores can also be used to
rationalize the achieved selectivity. Although the computational and
experimental data of the three case studies discussed in this work fully
support the proposed design strategy of bitopic compounds, we should
note that factors other than bitopic binding might also influence the
selectivity observed. Available X-ray structures of the dopamine D2
[15,33–35] and serotonin 5-HT2B [22,23,36] receptors confirmed the
The ligands were prepared for docking by generating possible ioni-
zation states (at a pH range of 7.0 ± 2.0), tautomers, and stereoisomers
¨
using Schrodinger’s Ligand Preparation Wizard with default settings
Table 3
[41]. 2-2 X-ray structures of 5-HT1B (PDB ID: 4IAQ, 4IAR [21]) and 5-
HT2B (PDB ID: 4IB4 [22], 4NC3 [36]) receptors were collected from the
PDB database. The structures were first prepared for docking by keeping
the monomeric receptor structure, removing crystallization agents (fatty
H1 and M1 receptor affinities of OBP binding amitriptyline and its bitopic de-
rivatives (6 and 7). s.d. values are in parentheses.
SBP fragment
pKi H1
pKi M1
Selectivity
N/A (amitriptyline)
9.20
8.32
H1 preferred
¨
acids, etc.) and using Schrodinger’s Protein Preparation Wizard [41]
(0.00)
7.94
(0.06)
6.25
(ΔpKi: 0.88)
1 preferred
with default settings, including the assignment of bond orders, adding
missing side chains, and missing hydrogen atoms, creating disulphide
bonds, removing crystalline waters, applying protomer-optimization,
and restrained minimization with the OPLS_2005 force field.
1-ethylpyrimidin-2-one (6)
1-propylpyrimidin-2-one (7)
H
(0.03)
9.03
(0.15)
7.13
(ΔpKi: 1.69)
H
1 preferred
(0.06)
(0.07)
(ΔpKi: 1.90)
4

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
Fig. 5. Proposed binding modes of select bitopic
compounds and their OBP and SBP fragments in the
respective receptor structures. Compound 3 and its
fragments 1-(2-methoxyphenyl)piperazine (OBP)
and cyclohexylurea (SBP) bound in the dopamine D3
receptor (PDB ID: 3PBL); 3 bound in the dopamine
D2 receptor (PDB ID: 6CM4); 4 and its fragments
ergoline (OBP) and 1-phenylpyrrolidine-2,5-dione
(SBP) bound in the serotonin 5-HT2B receptor (PDB
ID: 4IB4); 4 bound in the serotonin 5-HT1B receptor
(PDB ID: 4IAQ); 5 and its fragments ergoline (OBP)
and 3-phenyl-1,3-oxazolidin-2-one (SBP) bound in
the serotonin 5-HT2B receptor (PDB ID: 4IB4); 5
bound in the serotonin 5-HT1B receptor (PDB ID:
4IAQ); 7 and its fragments amitriptyline (OBP) and
2-pyrimidone (SBP) bound in the histamine H1 re-
ceptor (PDB ID: 3RZE); these same fragments bound
in the muscarinic acetylcholine M1 receptor (PDB
ID: 5CXV). Compound 7 could not be docked in the
M1 structure due to the closed tyrosine lid [31], and
induced fit docking was not attempted. Docked
binding modes were generated as outlined for each
receptor in the Computational Methods section in
the Supplementary Material. The OBP and SBP
fragments are shown in thick orange, linked bitopic
compounds are shown in thick green, protein resi-
dues within 3 Å of the ligands are shown in thin
grey, the protein backbone is shown in rainbow
ribbons from TM1-red to TM7-violet, H-bonds are
shown as yellow dashes, aromatic stacking in-
teractions are shown as blue dashes, ionic in-
teractions are shown as pink dashes, and cation-pi
interactions are shown as green dashes. Specific
interacting residues in the SBP are shown with the
GPCRdb structure-based numbering of each recep-
tor [32]. (For interpretation of the references to
colour in this figure legend, the reader is referred to
the web version of this article.)
¨
Structures were transformed into a receptor grid using Schrodinger
3.59 [43] using the extra ring sampling option. The crystal structures of
H1 (PDB ID: 3RZE [28]) and M1 (PDB ID: 5CXV [29]) were prepared with
MOE 2015 [44]. Docking was performed with PLANTS [45] with IFP
[46] post processing using the co-crystallized doxepin and tiotropium
reference ligands. Standard settings were used for docking. The binding
site was defined as the residues around the reference ligands with a
software package, with the binding pocket centred on the D3x32 residue.
¨
All docking procedures have been conducted using Schrodinger’s Glide
software [41], with precision set to XP (Extra Precision).
In case of the histamine H1 and muscarinic acetylcholine M1 re-
ceptors OBP compounds were selected with available data from the
ChEMBL 22 database [26]. Compounds were required to have
PCHEMBL > 6 value available for the H1 and M1-5 receptors, and all
within a 1.5 log unit interval. This was to ensure starting from active
OBP ligands with a known selectivity profile, of which only four were
found in the database (amitriptyline, benztropine, cyproheptadine, and
orphenadrine). The ligands were protonated at pH 7.4 using ChemAxon
cxcalc 5.1.4 [42] and 3D conformations were generated with Corina
radius of 6.5 Å. Poses were filtered for H-bond interaction with D3x32
.
Docking poses were selected with the lowest PLANTS and highest IFP
similarity scores as in de Graaf et al. 2011 [47]. The OBP fragments were
then merged with the receptor structures in MOE.
The SBP fragment library was collected from several sources: i) the
proprietary IOTA library with 1482 fragments, ii) the commercial
MayBridge fragment screening library [48] with 2500 fragments, and
5

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
iii) orthophosphate bioisosteres from the PDB extracted with the pro-
tocol of Zhang et al. 2017 [49] adapted for the PDB ligand identifier
“PO4” resulting in 393 additional fragments. The SBP fragments were
prepared and docked using the same protocol as the OBP fragments,
with the exception of the reference ligands used for IFP calculation. For
H1 the co-crystallized orthophosphate anion was used as the reference
ligand. For M1 the ligand CHEMBL2313377 was docked to the M1 SBP
using PLANTS, and the binding pose with the lowest RMSD of the
maximum common substructure (MCS) with LY2119620 in the overlaid
M2 crystal structure (PDB ID: 4MQT [6]) was selected and used as a
reference in further docking experiments. This was required as there are
no co-crystallized M1 allosteric modulators available in the PDB.
CHEMBL2313377 is a known allosteric modulator of M1,2,4 [50] of the
same chemotype as LY2119620.
mL of DCM and added dropwise to the previous mixture. The reaction
mixture was stirred at room temperature for 1 h. In the next step,
dimethylamine hydrochloride (410 mg, 5 mmol) and TEA (695 µl, 5
mmol) were added. The reaction was continued for 20 h at room tem-
perature. The reaction mixture was filtered and the filtrate was washed
with water. The phases were separated. The organic phase was dried
over Na2SO4 and evaporated. The crude material was purified by flash
chromatography, eluting with DCM: MeOH (0–5%). 160 mg (33%)
white solid was obtained. 1H NMR (500 MHz, DMSO‑d6) δ 7.05–6.96 (m,
2H), 6.96–6.87 (m, 2H), 3.79 (s, 3H), 3.53 (dd, J = 25.7, 12.3 Hz, 4H),
3.36 (tt, J = 11.5, 3.9 Hz, 1H), 3.24–3.06 (m, 4H), 3.00–2.86 (m, 2H),
2.75 (s, 6H), 1.75 (ddd, J = 24.4, 13.2, 3.6 Hz, 4H), 1.57 (dt, J = 11.6,
6.9 Hz, 2H), 1.21 (qd, J = 12.1, 3.1 Hz, 3H), 1.04–0.91 (m, 2H). 13C
NMR (125 MHz, DMSO‑d6) δ 157.49, 151.80, 139.25, 123.43, 120.77,
118.22, 116.78, 114.46, 111.90, 55.27, 53.84, 51.18, 51.11, 49.13,
46.98, 35.76, 34.38, 32.54, 31.41, 29.99. HRMS (ESI) (M + H)+ calcd
for C22H37N4O+2 , 389.2932; found 389.2939
SBP fragment poses were filtered using a number of criteria. Since
the binding sites and reference ligands for IFP were markedly different,
the scores could not be directly compared, therefore poses in the top
16% (1σ in normal distribution) of the ligands ranked by both PLANTS
score and IFP similarity were selected instead of defining a cut-off. The
SBP fragment poses in the four OBP ligand merged structures had to
show a robust docked binding pose within 1 Å RMSD of each other
(calculated using fconv [51]). Fragments with the largest difference in
average PLANTS score between H1 and M1 were then selected and linked
to the four OBP ligands using (CH2)1-4 linkers at suitable synthetic
handles (heteroatoms with a free H). The bitopic ligands were docked
using the same protocol to the apo protein structures using the OBP
compounds for IFP reference. Finally, the MCS RMSD was calculated
between the SBP fragments and bitopic compounds using fconv [51],
and only the fragments with <1 Å RMSD were kept.
4.2.2.2. 1-((1R,4R)-4-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)
cyclohexyl)urea. 400 mg (1.26 mmol) (1r,4r)-4-{2-[4-(2-methox-
yphenyl)piperazin-1-yl]ethyl}cyclohexan-1-amine was suspended in 50
mL of DCM. 695 µl (5 mmol) of TEA was added to the reaction mixture.
Triphosgene (145 mg, 0.48 mmol) was dissolved in 5 mL of DCM and
added dropwise to the previous mixture. The reaction mixture was
stirred at room temperature for 1 h. Subsequently, ammonia gas was
introduced into the reaction mixture for 2 h. Thereafter, the reaction
mixture was stirred at room temperature for 20 h. The reaction mixture
was filtered and the filtrate was washed with water. The phases were
separated. The organic phase was dried over Na2SO4 and evaporated.
The crude material was purified by flash chromatography, eluting with
DCM: MeOH (0–10%). 180 mg (40%) white solid was obtained. 1H NMR
(300 MHz, DMSO‑d6 + TFA-d1) δ 10.04 (s, 1H), 7.11–6.83 (m, 4H), 3.80
(s, 3H), 3.54 (t, J = 15.1 Hz, 4H), 3.17 (s, 5H), 2.93 (t, J = 12.0 Hz, 2H),
1.77 (dd, J = 30.2, 10.8 Hz, 4H), 1.58 (dt, J = 11.7, 6.6 Hz, 2H), 1.24 (s,
1H), 1.03 (q, J = 12.9 Hz, 4H). 13C NMR (75 MHz, DMSO‑d6 + TFA-d1) δ
158.58, 152.32, 139.81, 123.96, 121.31, 118.74, 112.40, 55.82, 54.38,
51.66, 48.61, 47.52, 34.70, 33.35, 31.70, 30.45. HRMS (ESI) (M + H)+
calcd for C20H33N4O+2 , 361.2603; found 361.2589.
4.2. Chemistry
4.2.1. Synthesis of dopamine D2/D3 ligands
The synthesis procedures of the dopamine D2-D3 receptor ligand
have been reported in Vass et al. [10] The NMR experiments were
performed at 500 MHz (1H) on a Varian VNMR SYSTEM spectrometer.
Chemical shifts are referenced to the residual solvent signals, 2.50 ppm
for 1H in DMSO‑d6. The LC-MS measurements were performed on Shi-
madzu LCMS2020 LC/MS system. Purifications by preparative-HPLC
were performed with Hanbon NS4205 Binary high pressure semi-
preparative HPLC. Thin-layer chromatography was performed on TLC
Silica Gel 60 F254. High resolution mass spectrometry measurements
were performed using a Q-TOF Premier mass spectrometer (Waters
Corporation, Milford, MA, USA) in positive electrospray ionization
mode.
4.2.2.3. (4R,7R)-N-[2-(2,5-dioxopyrrolidin-1-yl)phenyl]-6-methyl-6,11-
diazatetracyclo[7.6.1.02,7.012 16
, ]hexadeca-1(15),2,9,12(16),13-pen-
taene-4-carboxamide (S6)
To a mixture of o-nitroaniline (S1, 1380 mg, 10 mmol) and succinic
acid (S2, 1180 mg, 10 mmol) polyphosphoric acid (PPA, 10 g) was
added. The reaction mixture was stirred at 90 ◦C for 3 h. Afterwards the
reaction was quenched by an ice:water mixture (10 g of ice in 20 mL of
water) and the resulting crude 1-(2-nitrophenyl)pyrrolidine-2,5-dione
(S3) was filtered of as a yellow precipitate. The crude product was dis-
solved in methanol:dimethyl-acetamide (3:1 mixture, 40 mL) in an
autoclave and palladium on charcoal (10% Pd/C, 75 mg) was added to
the solution. The reactor was filled with 5 bar of hydrogen gas, and the
reaction mixture was stirred at room temperature overnight. The
resulting suspension was filtered over Celite (30 g). To the filtrate was
added water (50 mL) and the mixture was extracted with ethyl-acetate
three times (15 mL × 3), and washed with sat. sodium-bicarbonate so-
lution three times (10 mL × 3). To the collected organic phases was
added hydrochloric acid (10% in water, 20 mL) and the aqueous phase
was extracted with ethyl-acetate three times (15 mL × 3). The aqueous
phase was made basic by the use of sodium-hydroxide solution (10% in
water, 30 mL). The precipitate was filtered off and washed with water to
afford the crude 1-(2-aminophenyl)pyrrolidine-2,5-dione (S4) as white
crystals. To a solution of the crude 1-(2-aminophenyl)pyrrolidine-2,5-
dione (S4) in dimethyl-formamide (10 mL) was added lysergic acid (S5,
134 mg, 0.5 mmol), HATU (209 mg, 0.55 mmol) and diisopropyl-
ethylamine (DIPEA, 77 mg, 0.6 mmol, 0.104 mL). The reaction
mixture was stirred overnight at room temperature, quenched by brine
4.2.2. Synthesis of serotonin 5-HT1B/5-HT2B ligands
In case of the serotonin 5-HT1B-5-HT2B receptor ligands, procedures
were based on Kobeissi et al. [52], Zhou et al. [53], and Cheung et al.
[54] The NMR experiments were performed at 500 MHz (1H) on a
Varian VNMR SYSTEM spectrometer. Chemical shifts are referenced to
1
the residual solvent signals, 2.50 ppm for H in DMSO‑d6. The LC-MS
measurements were performed on Shimadzu LCMS2020 LC/MS sys-
tem. Purifications by preparative-HPLC were performed with Hanbon
NS4205 Binary high pressure semi-preparative HPLC. Thin-layer chro-
matography was performed on TLC Silica Gel 60 F254. High resolution
mass spectrometry measurements were performed using a Q-TOF Pre-
mier mass spectrometer (Waters Corporation, Milford, MA, USA) in
positive electrospray ionization mode.
4.2.2.1. 3,3-dimethyl-1-[(1R,4R)-4-{2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl}cyclohexyl]urea. 400 mg (1.26 mmol) (1r,4r)-4-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}cyclohexan-1-amine was sus-
pended in 50 mL of DCM. 695 µl (5 mmol) of TEA was added to the
reaction mixture. Triphosgene (145 mg, 0.48 mmol) was dissolved in 5
6

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
(20 mL) and extracted with ethyl acetate three times (10 mL × 3). The
collected organic phases were evaporated under reduced pressure and
the crude product was chromatographed in reverse phase preparative-
HPLC (Eluent A: 0.1% formic acid in water; eluent B: 0.1% formic
acid in acetonitrile; gradient: 0% to 100% B) to afford the title product
(S6). Yield: 1.18 mg. 1H NMR (500 MHz, DMSO‑d6) δ ppm 10.71 (s, 1H),
9.40 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.39–7.36 (m, 1H), 7.21–7.16 (m,
3H), 7.11–7.05 (m, 3H), 6.51–6.50 (m, 1H), 4.28 (m, 1H), 3.55–3.51 (m,
1H), 3.44–3.40 (m, 1H), 3.20 (m, 2H), 3.14–3.07 (m, 2H), 2.71–2.61 (m,
3H), 2.51 (s, 3H). HR-MS (ESI + ): m/z [M + H]+ calcd. For C26H25N4O3:
441.1927; found: 441.1929.
(m, 3H), 4.02–3.93 (m, 2H), 3.88–3.83 (m, 1H), 3.58–3.54 (m, 1H), 3.19
(m, 2H), 3.08 (m, 1H), 2.58 (s, 3H). HR-MS (ESI + ): m/z [M + H]+
calcd. For C25H25N4O3: 429.1927; found: 429.1922
4.2.3. Synthesis of histamine H1 /muscarinic acetylcholine M1 receptor
ligands
Anhydrous THF and DCM were obtained by elution through an
activated alumina column prior to use. All other solvents and chemicals
were acquired from commercial suppliers and were used as received.
ChemBioDraw Ultra 16.0.1.4 was used to generate systematic names for
all molecules. All reactions were performed under an inert atmosphere
(N2), unless mentioned otherwise. TLC analyses were carried out with
alumina silica plates (Merck F254) using staining and/or UV visualiza-
tion. Column purifications were performed manually using Silicycle
Ultra Pure silica gel or automatically using Biotage equipment. NMR
spectra (1H, 13C, and 2D) were recorded on a Bruker 600 (600 MHz)
spectrometer. Chemical shifts are reported in ppm (δ) and the residual
solvent was used as internal standard. Data are reported as follows:
chemical shift (integration, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, br = broad signal, m = multiplet, app = apparent),
and coupling constants (Hz)). A Bruker microTOF mass spectrometer
using ESI in positive ion mode was used to record HRMS spectra. A
Shimadzu LC-20AD liquid chromatograph pump system linked to a
Shimadzu SPD-M20A diode array detector with MS detection using a
Shimadzu LC-MS-2010EV mass spectrometer was used to perform LC-
MS analyses. An Xbridge (C18) 5 µm column (50 mm, 4.6 mm) was
used. The solvents that were used were the following: solvent B (MeCN
4.2.2.4. (4R,7R)-6-methyl-N-[2-(2-oxo-1,3-oxazolidin-3-yl)phenyl]-6,11-
diazatetracyclo[7.6.1.02,7.012 16
, ]hexadeca-1(15),2,9,12(16),13-pen-
taene-4-carboxamide (S8)
To a solution of 3-(2-aminophenyl)-1,3-oxazolidin-2-one (S7, 107
mg, 0.6 mmol) in dimethyl-formamide (10 mL) was added lysergic acid
(S5, 134 mg, 0.5 mmol), HATU (209 mg, 0.55 mmol) and DIPEA (77 mg,
0.6 mmol, 0.104 mL). The reaction mixture was stirred overnight at
room temperature and was quenched by brine (20 mL). The mixture was
extracted by ethyl-acetate three times (10 mL × 3). The collected
organic phases were evaporated under reduced pressure and the crude
product was chromatographed in reverse phase preparative-HPLC
(Eluent A: 0.1% formic acid in water; eluent B: 0.1% formic acid in
acetonitrile; gradient: 0% to 100% B) to afford the title product (S8).
Yield: 1.03 mg. 1H NMR (500 MHz, DMSO‑d6) δ ppm 10.70 (s, 1H),
10.08 (s, 1H), 8.16 (m, 1H), 7.40 (d, J = 7.66 Hz, 1H), 7.30 (t, J = 7.31
Hz, 1H), 7.23–7.17 (m, 1H), 7.13–7.05 (m, 4H), 6.57 (m, 1H), 4.49–4.36
7

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
with 0.1% formic acid) and solvent A (water with 0.1% formic acid),
flow rate of 1.0 mL/min, start 5% B, linear gradient to 90% B in 4.5 min,
then 1.5 min at 90% B, then linear gradient to 5% B in 0.5 min, then 1.5
min at 5% B; total run time of 8 min. All final compounds have a purity
of ≥95% (unless specified otherwise), calculated as the percentage peak
area of the analyzed compound by UV detection at 254 nm (values are
rounded).
was stirred for 6 d at room temperature, diluted with water (30 mL) and
extracted with EtOAc (3 × 50 mL). The collected organic phases were
dried over Na2SO4, filtered and evaporated under reduced pressure. The
crude product was purified using column chromatography twice (Eluent
A: cyclohexane; eluent B: EtOAc; gradient: 10% to 100% B, followed by
eluent A: DCM; eluent B: MeOH; gradient 3% to 5% B). Basic extraction
with aq. 2.5 M NaOH was performed to afford the title product as a white
solid. Yield: 160 mg. 1H NMR (600 MHz, CDCl3) δ 8.51 (dd, J = 4.1, 2.9
Hz, 1H), 7.64 (dd, J = 6.5, 2.9 Hz, 1H), 7.27 (dd, J = 4.9, 1.9 Hz, 1H),
7.23–7.10 (m, 6H), 7.07–7.01 (m, 1H), 6.03 (dd, J = 6.4, 4.1 Hz, 1H),
5.82 (app t, J = 7.3 Hz, 1H), 3.91 (br app s, 2H), 3.46–3.21 (m, 2H),
3.02–2.91 (br, 1H), 2.84–2.72 (br, 1H), 2.46–2.37 (m, 2H), 2.29 (app t,
J = 7.3 Hz, 4H), 2.10 (s, 3H), 1.94 (app p, J = 6.6 Hz, 2H). 13C NMR
(151 MHz, CDCl3) δ 165.90, 156.45, 149.10, 144.09, 141.20, 140.03,
139.44, 137.13, 130.21, 129.33, 128.57, 128.33, 128.23, 127.70,
127.33, 126.25, 125.92, 103.52, 57.31, 53.55, 49.93, 41.15, 33.87,
32.20, 27.28, 25.06. LC-MS (ESI): tR = 3.49 min, purity: >99% (area %
@ 254 nm), m/z [M + H]+: 400. HR-MS (ESI + ): m/z [M + H]+ calcd.
for C26H29N3O: 400.2383, found: 400.2369.
4.2.3.1. 1-(2-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)
propyl)(methyl) amino)ethyl)pyrimidin-2(1H)-one (6, VUF16435)
The free base of nortriptyline hydrochloride (1.0 g, 3.3 mmol) was
obtained using 2.5 M aq. NaOH (1.3 mL, 3.3 mmol). The resulting so-
lution was extracted with DCM (3 × 30 mL). The collected organic
phases were dried over Na2SO4, filtered and evaporated under reduced
pressure. The crude product was dissolved in THF (20 mL). Et3N (0.93
mL, 6.7 mmol) and 1,2-dibromoethane (0.57 mL, 6.7 mmol) were
added. The reaction mixture was stirred for 12 d at RT. The resulting
mixture was diluted with THF (20 mL). Et3N (0.93 mL, 6.7 mmol) and
pyrimidin-2(1H)-one hydrochloride (442 mg, 3.34 mmol) were added.
The reaction mixture was stirred for 3 d at RT and diluted with water
(30 mL). The mixture was extracted with EtOAc (3 × 30 mL). The
collected organic phases were dried over Na2SO4, filtered and evapo-
rated under reduced pressure. The crude product was purified using
column chromatography (Eluent A: 2% Et3N in cyclohexane; eluent B:
2% Et3N in EtOAc; gradient: 20% to 80% B). Basic extraction with 2.5 M
aq. NaOH and azeotropic distillation with EtOAc and hexane were
performed to afford the title product as a colorless oil that solidifies over
time. The compound retained EtOAc and hexane solvate even after
extensive drying. Yield: 30 mg. 1H NMR (600 MHz, CDCl3) δ 8.42–8.34
(m, 1H), 7.59–7.47 (m, 1H), 7.23–7.11 (m, 6H), 7.07 (d, J = 7.4 Hz, 1H),
7.04 (d, J = 7.3 Hz, 1H), 5.84–5.76 (m, 1H), 5.71 (t, J = 7.3 Hz, 1H),
3.90 (app s, 2H), 3.35–3.23 (m, 2H), 3.00–2.90 (m, 1H), 2.79–2.67 (m,
3H), 2.49–2.40 (m, 2H), 2.24–2.19 (m, 2H), 2.15 (s, 3H). 13C NMR (151
MHz, CDCl3) δ 171.27, 165.85, 156.37, 149.28, 141.24, 139.94, 139.42,
137.14, 130.10, 129.24, 128.66, 128.38, 128.19, 127.64, 127.28,
126.18, 125.91, 103.25, 57.40, 55.48, 49.08, 41.62, 33.89, 32.17,
27.35. LC-MS (ESI): tR = 3.53 min, purity: 98% (area % @ 254 nm), m/z
[M + H]+: 386. HR-MS (ESI + ): m/z [M + H]+ calcd. for C25H27N3O:
386.2227; found: 386.2231.
4.3. Biological assays
4.3.1. Competition binding in human D3 and D2 receptors
For full experimental details see Vass et al. 2014 [10]. Briefly,
membrane aliquots of CHO-K1 cells expressing human recombinant D3
and D2 receptors were thawed and washed in binding buffer and the
◦
assay was performed using tritiated raclopride at 25 C for 120 min.
Nonspecific binding was determined in the presence of excess haloper-
idol. At the end of incubation, samples were filtered on GF/B 96-well
plates (Millipore) and washed four times with ice cold binding buffer.
The plate was dried and 40 µl Microscint-20 scintillation cocktail was
added to each well. Radioactivity was determined in a microplate beta
scintillation counter (TopCount NXT, PerkinElmer, Waltham, MA, USA).
For dopamine D2 receptor competition binding data shown on Fig. 3
experiments were carried out in a polypropylene 96-well plate. In each
well was incubated 10 μg of membranes from CHO-D2 #S20 cell line
prepared in our laboratory (Lot: A002/22-07-2015, protein concentra-
tion = 4185
μ
g/ml), 0.15 nM [3H]-Spiperone (80.2 Ci/mmol, 1 mCi/ml,
Perkin Elmer NET1187001MC) and compounds studied and standard.
Non-specific binding was determined in the presence of Sulpiride 10
(Sigma S112). The reaction mixture (Vt: 250 l/well) was incubated at
25 ◦C for 120 min, 200
l was transferred to GF/C 96-well plate (Mil-
μM
4.2.3.2. 1-(3-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-yli-
μ
dene)propyl)(methyl)
VUF16412)
amino)propyl)pyrimidin-2(1H)-one
(7,
μ
lipore, Madrid, Spain) pre-treated with 0.5% of PEI and treated with
binding buffer (50 mM Tris-HCl,1 mM EDTA, 5 mM MgCl2 , 5 mM KCl,
120 mM NaCl, pH = 7.4), after was filtered and washed four times with
The free base of nortriptyline hydrochloride (1.0 g, 3.3 mmol) was
obtained using 2.5 M aq. NaOH (1.3 mL, 3.3 mmol). The resulting so-
lution was extracted with DCM (3 × 30 mL). The collected organic
phases were dried over Na2SO4, filtered and evaporated under reduced
pressure. The crude product was dissolved in THF (20 mL). Et3N (1.4
mL, 10 mmol) and 1,3-dibromopropane (0.52 mL, 5.0 mmol) were
added. The reaction mixture was stirred for 3 d at RT. To the resulting
reaction mixture Et3N (0.93 µl, 6.7 mmol) and pyrimidin-2(1H)-one
hydrochloride (442 mg, 3.34 mmol) were added. The reaction mixture
was stirred overnight at RT and another portion of pyrimidin-2(1H)-one
hydrochloride (442 mg, 3.34 mmol) was added. The reaction mixture
250 μl wash buffer (50 mM Tris-HCl, 0.9% NaCl, pH = 7.4), before
measuring in a microplate beta scintillation counter (Microbeta Trilux,
PerkinElmer, Madrid, Spain).
4.3.2. Competition binding for human 5-HT1B receptor
Serotonin 5-HT1B receptor competition binding experiments were
carried out in a polypropylene 96-well plate. In each well was incubated
5 μg of membranes from Hela-5-HT1B cell line prepared in our labora-
tory (Lot: A001/14-11-2011, protein concentration = 3179 μg/ml), 1.5
8

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
nM [3H]-GR125743 (83.9 Ci/mmol, 0.1 mCi/ml, Perkin Elmer
NET1172100UC) and compounds studied and standard. Non-specific
buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA, pH = 7.4), after
was filtered and washed four times with 250 μl wash buffer (50 mM Tris-
binding was determined in the presence of GR55562 10
μ
M (TOCRIS
HCl, pH = 7.4), before measuring in a microplate beta scintillation
1054). The reaction mixture (Vt: 250
μ
l/well) was incubated at 25 ◦C for
counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).
90 min, 200 μl was transferred to GF/C 96-well plate (Millipore, Madrid,
Spain) pre-treated with 0.5% of PEI and treated with binding buffer
4.3.6. Competition binding for human 5-HT7 receptor
(Tris-HCl 50 mM, EDTA 1 mM, MgCl2 10 mM, pH = 7.4), after was
Serotonin 5-HT7 receptor competition binding experiments were
carried out in a polypropylene 96-well plate. In each well was incubated
filtered and washed four times with 250 μl wash buffer (Tris-HCl 50 mM,
pH = 7.4), before measuring in a microplate beta scintillation counter
2 μg of membranes from HEK-5-HT7#14 cell line prepared in our lab-
(Microbeta Trilux, PerkinElmer, Madrid, Spain).
oratory (Lot: A006/21-07-2016, protein concentration = 3316 μg/ml),
2 nM [3H]-SB269970 (34.5 Ci/mmol, 0.25 mCi/ml, Perkin Elmer
NET1198U250UC) and compounds studied and standard. Non-specific
4.3.3. Competition binding for human 5-HT2B receptor
Serotonin 5-HT2B receptor competition binding experiments were
carried out in a polypropylene 96-well plate. In each well was incubated
binding was determined in the presence of clozapine 25
μ
M (Sigma
C6305). The reaction mixture (Vt: 250
for 60 min, 200 μl was transferred to GF/C 96-well plate (Millipore,
μ
l/well) was incubated at 37 ◦C
20
μ
g of membranes from CHO-5-HT2B cell line prepared in our labo-
ratory (Lot: A004/19-10-2017, protein concentration = 2775
μ
g/ml),
Madrid, Spain) pre-treated with 0.5% of PEI and treated with binding
buffer (50 mM Tris-HCl, 4 mM CaCl2, 1 mM ascorbic acid, 0.1 mM
pargyline, pH = 7.4), after was filtered and washed four times with 250
0.8 nM [3H]-LSD (82.9 Ci/mmol,
1
mCi/ml, Perkin Elmer
NET638250UC) and compounds studied and standard. Non-specific
binding was determined in the presence of 5-HT 50
μ
M (Sigma
μl wash buffer (50 mM Tris-HCl, 4 mM CaCl2, 1 mM ascorbic acid, 0.1
H9523). The reaction mixture (Vt: 250
μ
l/well) was incubated at 37 ◦C
mM pargyline, pH = 7.4) before measuring in a microplate beta scin-
for 30 min, 200
μ
l was transferred to GF/C 96-well plate (Millipore,
tillation counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).
Madrid, Spain) pre-treated with 0.5% of PEI and treated with binding
buffer (Tris-HCl 50 mM, Ascorbic acid 0.1%, CaCl2 4 mM, pH = 7.4),
4.3.7. Competition binding for human α1A adrenergic receptor
after was filtered and washed four times with 250
μl wash buffer (Tris-
α1A adrenergic receptor competition binding experiments were car-
ried out in a polypropylene 96-well plate. In each well was incubated 20
HCl 50 mM, pH = 7.4), before measuring in a microplate beta scintil-
lation counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).
μg of membranes from HEK-α1A#5 cell line prepared in our laboratory
(Lot: A004/07-06-2017, protein concentration = 4171 μg/ml), 0.4 nM
4.3.4. Competition binding for human 5-HT2C receptor
[3H]-Prazosin (80.6 Ci/mmol, 1 mCi/ml, Perkin Elmer NET823250UC)
and compounds studied and standard. Non-specific binding was deter-
Serotonin 5-HT2C receptor competition binding experiments were
carried out in a polypropylene 96-well plate. In each well was incubated
mined in the presence of prazosin 1 M (Sigma P115). The reaction
μ
3
μ
g of membranes from Hela-5-HT2C cell line prepared in our labora-
mixture (Vt: 250 μ μl was
l/well) was incubated at 25 ◦C for 90 min, 200
tory (Lot: A002/20-06-2011, protein concentration = 2041
μ
g/ml),
transferred to GF/C 96-well plate (Millipore, Madrid, Spain) pre-treated
with 0.5% of PEI and treated with binding buffer (50 mM Hepes, 5 mM
MgCl2, 1 mM CaCl2, 0.2% BSA, pH = 7.4), after was filtered and washed
1.25 nM [3H]-Mesulergine (84.7 Ci/mmol, 1 mCi/ml, Perkin Elmer
NET1148250UC) and compounds studied and standard. Non-specific
binding was determined in the presence of Mianserin 10
μ
M (Sigma
four times with 250 μl wash buffer (50 mM Hepes, 500 mM NaCl, 0.1%
M2525). The reaction mixture (Vt: 250
μ
l/well) was incubated at 27 ◦C
BSA, pH = 7.4) before measuring in a microplate beta scintillation
for 60 min, 200
μ
l was transferred to GF/C 96-well plate (Millipore,
counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).
Madrid, Spain) pre-treated with 0.5% of PEI and treated with binding
buffer (50 mM Tris-HCl, pH = 7.5), after was filtered and washed four
4.3.8. Competition binding for human α2A adrenergic receptor
times with 250
μl wash buffer (50 mM Tris-HCl , pH = 6.6), before
α2A adrenergic receptor competition binding experiments were car-
ried out in a multiscreen GF/C 96-well plate (Millipore, Madrid, Spain)
pre-treated with binding buffer (25 mM NaH2PO4 pH = 7.4). In each
measuring in a microplate beta scintillation counter (Microbeta Trilux,
PerkinElmer, Madrid, Spain).
well was incubated 30 μg of membranes from CHO-α2A cell line pre-
4.3.5. Competition binding for human 5-HT6 receptor
pared in our laboratory (Lot: A001/21-07-2009, protein concentration
Serotonin 5-HT6 receptor competition binding experiments were
carried out in a polypropylene 96-well plate. In each well was incubated
= 3330
μ
g/ml), 0.37 nM [3H]-MK912 (87.5 Ci/mmol, 1 mCi/ml, Perkin
Elmer NET1227250UC) and compounds studied and standard. Non-
specific binding was determined in the presence of norepinephrine
5
μg of membranes from HEK-5-HT6#10p cell line prepared in our
laboratory (Lot: A001/02-03-2010, protein concentration = 2624
μg/
100 μM (Sigma A7257). The reaction mixture (Vt: 200 μl/well) was
ml), 2 nM [3H]-LSD (82.9 Ci/mmol, 1 mCi/ml, Perkin Elmer
NET638250UC) and compounds studied and standard. Non-specific
incubated at 25 ◦C for 30 min after was filtered and washed four times
with 250 μl wash buffer (25 mM NaH2PO4 pH = 7.4), before measuring
binding was determined in the presence of 5-HT 100
μ
M (Sigma
l/well) was incubated at 37 ◦C
l was transferred to GF/C 96-well plate (Millipore,
Madrid, Spain) pre-treated with 0.5% of PEI and treated with binding
in a microplate beta scintillation counter (Microbeta Trilux, Perki-
nElmer, Madrid, Spain).
H9523). The reaction mixture (Vt: 250
for 60 min, 200
μ
μ
9

A. Egyed et al.
Bioorganic Chemistry 111 (2021) 104832
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4.3.9. Competition binding for human H1 receptors
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each well was incubated; 25 µl compound (dissolved to the desired
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(50 mM Tris, pH 7.4) and allowed to dry at 50 ◦C for 1 h. Once dry, 20 µl
of scintillation fluid (PerkinElmer, art. 6013611) was added to each
well, the plate sealed (PerkinElmer, art. 6050185) and measured in a
microplate beta scintillation counter (Wallac Trilux, PerkinElmer).
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μl was transferred to GF/C 96-well plate
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
´
´
´
´
´
´ ´
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´
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Acknowledgements
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tional Brain Research Program (2017-1.2.1‑NKP‑2017‑00002) to GMK
and The Netherlands Organization for Scientific Research (NWO) TOP-
PUNT (“7 ways to 7TMR modulation (7-to-7)”Grant 718.014.002) to RL.
The experimental assistance of Dr Tiffany van der Meer in some of
´
´
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´
´
the binding experiments is highly appreciated. Pal Szabo (RCNS) and
Hans Custers (VUA) are acknowledged for HRMS measurements. Ruben
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Appendix A. Supplementary data
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