Rational design of 5-HT6R ligands using a bioisosteric strategy: Synthesis, biological evaluation and molecular modelling

Article abstract of DOI:10.1039/c5ra00054h

A bioisosteric strategy was successfully implemented with a screening protocol for new, potent 5-HT₆R ligands. Initially, 2-[5-(4-methylpiperazin-1-yl)-2-nitrophenyl]-1,2,3,4-tetrahydroisoquinoline (9) was found in commercial databases using a

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5-HT6R
ligands
Lead
database
Bioisosteric
substituton
5-HT6R
Ki = 6 nM
Bioisostere
query
Hit
compound
5 commercial
compounds
databases
Crystallographic and
molecular modelling
studies

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ARTICLE TYPE
Rational design of 5ꢀHT6R ligands using a bioisosteric strategy:
Synthesis, biological evaluation and molecular modelling
Jakub Staroń,a Dawid Warszyckia, Justyna KalinowskaꢀTłuścik, b Grzegorz Satałaa and Andrzej J.
Bojarskia*
5
aDepartment of Medicinal Chemistry, Institute of Pharmacology Polish Academy of Sciences
12 Smętna Street, 31ꢀ343 Kraków, Poland
bDepartment of Crystal Chemistry and Crystal Physic, Jagiellonian University Faculty of Chemistry
3 R. Ingardena Street, 30ꢀ060 Kraków, Poland
*Corresponding author: Institute of Pharmacology PAS, Kraków. Tel. +48 12 6623365, email: bojarski@ifꢀpan.krakow.pl
10 Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A bioisosteric strategy was successfully implemented with a screening protocol for new, potent 5ꢀHT6R ligands. Initially, (2ꢀ[5ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ1,2,3,4ꢀtetrahydroisoquinoline (9) was found in commercial databases using a bioisosteric query
(screening 5ꢀHT6R Ki = 128 nM). Then, the hit compound was bioisosterically modified (ring alteration) leading to a novel, high affinity
15 (Ki = 6 nM) 5ꢀHT6R ligand (10). Extensive docking studies followed by structural interaction fingerprint analysis supported by singleꢀ
crystal Xꢀray structures of the investigated ligands suggest different binding modes with 5ꢀHT6R models for compounds with varying
activity. An alternative anchoring point for protonated amine (D7.36) that has not been previously reported was identified.
Keywords: virtual screening, bioisosteric substitution, serotonin receptor, 5ꢀHT6 receptor, 5ꢀHT6R ligands
45 The 5ꢀHT6 receptor, a member of the GPCR superfamily, shows
significant therapeutic potential and is a promising target for
Introduction
novel psychotropic drugs.8 This receptor is responsible primarily
20 Isosteric or, in the case of biologically active compounds,
for motor control, memory and learning, and its antagonists have
bioisosteric substitution is one of the most commonly used
several applications, e.g., improving cognitive function and
methods for developing new compounds with required
50 memory in cognitive impairments.9–11 These antagonists are also
characteristics, e.g., drugs with improved pharmacodynamics
potential antiobesity drugs.12,13 The 5ꢀHT6R and some other
and/or pharmacokinetic properties.1 The first known bioisosteric
members of serotonergic receptor family have been investigated
25 replacement that revolutionized drug development and medicine
in our laboratories for a long time, and this research has included
was the discovery of Prontosil.2 Prontosil was the first antibiotic
the synthesis of novel ligands, in vitro testing, homology
of the sulfonamide family. Its active metabolite, sulfanilamide, is
55 modeling, pharmacophore filtering and multistep virtual
an actual bioisostere of pꢀaminobenzoic acid, an intermediate in
screening.14–18
the bacterial synthesis of folate. Since then, this strategy, usually
In this study, we present an implementation of bioisosteric query
30 in the context of structureꢀactivity relationship studies, has been
to the virtual screening (VS) of potential 5ꢀHT6R ligands in a 5.4
widely used in the search for novel active compounds to different
million database of commercial compounds and subsequent
biological targets, among others for the modification of serotonin
receptor ligands.
60 bioisosteric modification of the VS hit. All alterations of the hit
structure were ring modifications which resulted in the discovery
One of the first studies concerning bioisosterism in serotonin
of a new, potent 5ꢀHT6 receptor ligand. To explain differences in
35 receptor ligands was published in 1987 and utilized indazoles as
5ꢀHT6R ligands affinity, the single crystal Xꢀray structure
indole substitutes in 5ꢀHT3R antagonists.3 Another report from
analysis and docking studies were performed.
1992 described the bioisosteric replacement of indole with
benzofuran in a series of 5ꢀHT2R agonists.4 Twelve years later, a
65 Initial screening – selection of a hit compound
5ꢀHT1fR agonist with an indole–furopyridine ring substitution
The hit compound was found from a multistep ligandꢀbased
40 was reported.5 The most recent paper, published in 2010, reported
virtual screening cascade performed on compound collections
the replacement of a benzene ring in haloperidol with [2,2]ꢀ
from five vendors with a query based on bioisosteres of 5ꢀHT6R
paracyclophane.6 In spite of the large number of reports of
ligands (Figure 1.). The ChEMBL database version 13, extended
bioisosterism, none discuss explicitly the application of
70 by data from ca. 30 patents, was used as a source of compounds
nonclassical bioisosteric strategy to 5ꢀHT6 receptor ligands.7
with known 5ꢀHT6 affinity.19 For each of the 4298 compounds
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from the final set, all possible bioisosteres were generated in
with Tanimoto similarity coefficients (Tc) higher than or equal to
0.9 to any previously generated bioisostere were fetched, and the
resulting set of 1718 analogues was subjected to further filtering
steps.
Pipeline Pilot.20 After removal of duplicates, a collection of
137 951 unique compounds was obtained. Next, from the
database of 5.4 million commercially available structures
(Chembridge, Chemdiv, UORSY, Vitas M and Enamine), those
5
10
Figure 1 Workflow for the virtual screening protocol with bioisostere query for the identification of a hit compound.
The applied protocol followed the hierarchical scheme reported
previously and consisted of four filters: physicochemical, ADME,
15 3D pharmacophore and visual inspection.21 First, the Lipinski
rules of 5, the Veber rules and the criteria of the strongest basic
pKa > 5 were applied (Calculator Plugins, JChem).22 After
generation of 3D structures (Ligprep), the ADME descriptors
(QikProp) were calculated, and only compounds fulfilling all
20 ADME requirements (see experimental) were considered.23,24 In
Scheme 1 Synthesis of N-benzyl-5-(4-methylpiperazin-1-yl)-2-nitroaniline
45 (4). Reagents and conditions: (a) H2O, reflux, 1,5 h; (b) KHMDS, Boc2O,
THF, 0 °C; (c) Pd(OAc)2, JohnPhos, K3PO4•H2O, N-Me-piperazine, DME,
100 °C, 20 h; (d) MeOH, conc. HCl, reflux, 3 h.
the next phase,
a linear combination of four different
pharmacophore models, developed using four of the most
populated chemical scaffolds of 5ꢀHT6R antagonists (indoles,
indoleꢀlike, monocyclic arylpiperazines and polycyclic
25 arylpiperazines), were applied.17 Finally, 129 compounds, each
matching at least one of the pharmacophore models, were
evaluated by team members to select structures with the most
diverse chemotypes for biological investigation. As a result, a set
of 11 compounds (Supplementary data) was acquired from Vitas
30 M, Chembridge and Enamine and tested for 5ꢀHT6 receptor
activity at two concentrations (see experimental). Of the tested
Syntheses of the hit compound 9 and its two isosteres (2ꢀ[5ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ2,3ꢀdihydroꢀ1Hꢀisoindole 8
50 and
2ꢀ[5ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ2,3,4,5ꢀ
tetrahydroꢀ1Hꢀ2ꢀbenzazepine 10 were performed according to the
synthetic route shown in scheme 2. The general procedure
utilized coupling of 4ꢀchloroꢀ2ꢀfluoronitrobenzene with amines at
the 2ꢀposition and subsequent addition of NꢀMeꢀpiperazine
55 through the BuchwaldꢀHartwig reaction.
compounds,
(2ꢀ[5ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ
1,2,3,4ꢀtetrahydroisoquinoline), STK148877 (9), was the most
potent (screening 5ꢀHT6R Ki = 128 nM) and was selected as a hit
35 compound for further modification.
Chemistry
The parent of the hit compound, Nꢀbenzylꢀ5ꢀ(4ꢀmethylpiperazinꢀ
1ꢀyl)ꢀ2ꢀnitroaniline (4), was previously reported by Tasler et al..25
A modified route of its resynthesis, where instead of coupling of
40 2ꢀbromoꢀ4ꢀchloronitrobenzene with Bocꢀprotected benzylamine,
a coupling of 5ꢀchloroꢀ2ꢀnitroaniline with benzylbromide with
subsequent Boc protection was employed. (scheme 1).26
Scheme 2 Synthesis of 4-methylpiperazin-1-yl-2-nitroanilines (8, 9, 10).
Reagents and conditions: (a) DMSO, K2CO3, 120 °C, 2 h; (b) Pd(OAc)2,
JohnPhos, K3PO4•H2O, N-Me-piperazine, DME, 100 °C, 20 h.
60
2
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HT1AR; [3H]ꢀLSD (85.2 Ci/mmol) for 5ꢀHT6R; [3H]ꢀ5ꢀCT (39.2
Ci/mmol) for 5ꢀHT7bR and [3H]ꢀRaclopride (74.4 Ci/mmol) for
10 D2LR. Each compound was tested in triplicate at 7 to 8 different
concentrations (10−11–10−4 M). The inhibition constants (Ki) were
calculated from the ChengꢀPrusoff equation.27 The results are
expressed as the mean of at least two separate experiments (Table
1).
Pharmacology
In vitro evaluation
Radioligand binding assays were employed to determine the
affinity and selectivity profiles of the synthesized compounds for
human serotonin 5ꢀHT1AR, 5ꢀHT6R, 5ꢀHT7bR and D2LR, which
were stably expressed in HEK293 cells. This was accomplished
by displacement of [3H]ꢀ8ꢀOHꢀDPAT (187 Ci/mmol) for 5ꢀ
5
15 Table 1 Affinities of the synthesized compounds.
e
Ki [nM]
Function at
5ꢀHT6
KB
R
Cmpd
e
a
b
c
d
[nM]
5ꢀHT1A
5ꢀHT6
22
5ꢀHT7
2783
D2
4
8
1278
557
1396
227
antagonist
antagonist
antagonist
antagonist
79
1000
340
29
2292
3740
1015
245
62
6
5117
4148
5181
9f
10
358
a buspirone as a reference Ki = 20 nM, b olanzapine as a reference Ki = 7 nM, cclozapine as a reference Ki = 18 nM, dolanzapine as a reference Ki = 7 nM,
emethiotepine as a reference KB = 3.1 nM, fresynthesized STK148877.
The most potent and selective 5ꢀHT6R ligand, 2ꢀ[5ꢀ(4ꢀ 25 as compounds 8–10, exhibit antagonistic properties of different
methylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ2,3,4,5ꢀtetrahydroꢀ1Hꢀ2ꢀ
20 benzazepine 10, was further investigated for other activities by
CEREP (Table 2). It is noteworthy that compound 10 was found
to be 60ꢀfold selective, versus only 25ꢀfold selectivity for
potency, proportional to 5ꢀHT6 affinity values. Tasler et al.
reported the function of analogue of parent compound 4 with
unmethylated piperazine to be full agonist.25 The function of
methylated parent compound 4 was not reported but it was shown
compound 4, against D2R. Antagonist effect at 5ꢀHT6R was 30 that similar compound with 2ꢀfurane moiety instead of phenyl
determined by CEREP (Table 1). The parent compound 4, as well
ring possessed partial agonistic properties.
Table 2 % Inhibition of Specific Binding at a compound concentration of 1 ꢁM.
serotonin
5ꢀHT2C
72
serotonin
5ꢀHT3
25
Receptor
dopaminergic D3
89
adrenergic α1
12
adrenergic α2C
12
histamine H1
85
muscarinic M1
3
% Inhibition
35 Crystal structure analysis of ligands
allows the two aromatic fragments to be closer, which is reflected
by centroids distance (Cg1ꢀCg2) of 5.1–5.5 Å. This conformation
also results in a shorter distance between the basic nitrogen atom
55 N(4) and the centroid (Cg2) of the aromatic ring C(12)ꢀC(17),
which varies between 6.8 to 7.7 Å for the most active ligands,
whereas it is approximately 10 Å for the two less active
compounds (Figure 2). Additionally, the close proximity of the
C(12)ꢀC(17) πꢀelectron system influences the mutual orientation
60 of both rings of the arylpiperazine moiety, which can be defined
by the torsion angle C(3)ꢀC(4)ꢀN(3)ꢀC(7). This angle for both
active compounds is approximately ±30°. The opposite sign of
this value is related to the opposite orientation of the aromatic
C(12)ꢀC(17) ring with respect to the piperazine ring. This distinct
65 orientation is a result of the packing in the crystal structure. It is
potentially based upon the formation of weak interaction C(13)ꢀ
H(13)...O(2) in the crystal structure of 4, which is not observed in
10. For the latter structure, similar interactions occur between the
To obtain the protonated form of the synthesized compounds,
several salts were considered. However, crystallization of the
salts were unsuccessful. Good quality single crystals of free bases
40 were obtained instead.
The crystal structures of two of the most potent 5ꢀHT6R ligands
(4 and 10) revealed a similar hairpinꢀlike conformation of the
molecules, which differs from the bent conformation of the less
active bioisosteres (8 and 9). The distinct orientation of the pꢀ
45 nitroaryl fragment and the second aromatic ring of the molecules
is represented by the torsion angle C(2)ꢀN(2)ꢀC(11)ꢀC(12) (Table
3). The hairpin conformation of 4 is a result of intramolecular
hydrogen bond formation N(2)ꢀH(2)...O(1) (Figure 2, Table 4),
while in the case of 10 it is related to the conformation of the
50 introduced sevenꢀmembered ring.
The hairpin conformation of the two most active bioisosteres
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two methylene groups of the sevenꢀmembered ring and the
oxygen atom O(1) as a bifurcated acceptor of weak hydrogen
bond (see Table 4).
to the aromatic ring C(1)ꢀC(6). For compounds 8, 9 and 10, this
coꢀplanarity is not observed. The pꢀNO2 substituent demonstrated
different inductive effects in these molecules. The nitro group
exhibits an electron withdrawing effect in 8 and 10 and a slightly
Within the crystal structure of the presented compounds, weak
5
hydrogen bonds CꢀH...O and CꢀH...N are primarily observed. The 15 electron donating effect in 9. The electron withdrawing effect is
only exception is the crystal structure of 4, in which the presence
of the strong donor N(2) allowed centrosymmetric dimer
formation through hydrogen bonding system (Table 3). The
intramolecular interaction N(2)ꢀH(2)...O(1) mentioned above
shown by the elongation of the N(1)ꢀC(1) bond and the increased
C(2)ꢀC(1)ꢀC(6) angle (Table 3). The differing properties of the
nitro group are related to the number of intermolecular
interactions in which the oxygen atoms are involved.
10 results in a coꢀplanar arrangement of the nitro group with respect
20
Figure 2 Molecular geometry and common geometrical features (distance in A and torsion angle value in o) that determine arrangement of molecules in
the crystal environment. For 4, an intramolecular hydrogen bond is highlighted. The displacement ellipsoids of the non-hydrogen atoms are drawn at
the 30% probability level. H atoms are presented as small spheres with an arbitrary radius.
25 Table 3 Selected geometrical values for the presented crystal structures [Å and °].
Compound
N(1)ꢀC(1)
N(2)ꢀC(2)
4
8
9
10
1.429(2)
1.352(2)
1.398(2)
118.5(1)
120.1(1)
1.443(1)
1.380(1)
1.385(1)
118.4(1)
121.0(1)
1.439(2)
1.388(2)
1.372(2)
116.6(1)
119.9(1)
1.444(1)
1.380(1)
1.385(1)
117.98(8)
121.00(8)
N(3)ꢀC(4)
C(3)ꢀC(4)ꢀC(5)
C(2)ꢀC(1)ꢀC(6)
C(2)ꢀN(2)ꢀC(11)ꢀ
C(12)
ꢀ69.1(2)
ꢀ173.34(9)
ꢀ165.0(1)
90.7(1)
O(1)ꢀN(1)ꢀC(1)ꢀC(2)
O(2)ꢀN(1)ꢀC(1)ꢀC(6)
C(3)ꢀC(4)ꢀN(3)ꢀC(7)
C(5)ꢀC(4)ꢀN(3)ꢀC(9)
4.0(2)
3.4(2)
30.76(2)
ꢀ8.5(2)
ꢀ36.9(2)
ꢀ39.7(1)
ꢀ1.9(2)
ꢀ22.6(2)
ꢀ27.5(2)
ꢀ6.5(2)
ꢀ33.5(1)
ꢀ37.5(1)
ꢀ30.01(1)
3.7(1)
35.5(2)
21.3(2)
4
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Table 4 Geometry of strong and weak hydrogen bonds in the crystal structures [Å and °].
Cmpd
DꢀH...A
d(DꢀH)
d(H...A)
d(D...A)
<(DHA)
N(2)ꢀH(2)...O(1)
0.88(2)
0.88(2)
0.99
1.95(2)
2.31(2)
2.52
2.639(2)
2.994(2)
3.419(2)
3.505(2)
133(2)
134(1)
151
N(2)ꢀH(2)...O(1)_1
C(10)ꢀH(10A)...O(2)_2
C(13)ꢀH(13)...O(2)_3
4
0.95
2.56
175
C(11)ꢀH(11A)...O(1)
C(11)ꢀH(11A)...N(1)
C(7)ꢀH(7A)...O(1)_4
C(9)ꢀH(9B)...O(2)_5
C(5)ꢀH(5)...O(2)_5
0.99
0.99
0.99
0.99
0.95
2.12
2.52
2.57
2.59
2.61
2.870(1)
3.051(2)
3.301(1)
3.537(2)
3.549(1)
130
113
130
161
171
8
C(11)ꢀH(11B)...N(4)_6
C(18)ꢀH(18B)...O(1)
C(19)ꢀH(19A)...N(1)
0.99
0.99
0.99
2.61)
2.55
2.47
3.293(2)
3.164(2)
3.124(2)
126
120
123
9
C(11)ꢀH(11B)...O(1)_7
C(18)ꢀH(18B)...O(1)_7
C(20)ꢀH(20B)...N(1)
0.99
0.99
0.99
2.49
2.48
2.35
3.437(1)
3.419(1)
3.004(1)
161
159
123
10
Symmetry transformations used to generate equivalent atoms: _1: ꢀx,ꢀy,ꢀz; _2: ꢀx,y+1/2,ꢀz+1/2; _3: ꢀx,1ꢀy,ꢀz; _4: xꢀ1,y,z; _5: x,ꢀy+3/2,z+1/2 _6: ;ꢀ
x,yꢀ1/2,ꢀz+1/2; _7: ꢀx+1,ꢀy+1,ꢀz+1
TM6 participated in edgeꢀtoꢀface contacts with F6.51 and F6.52.
Docking studies
20 In case of compound 4, the benzyl moiety was near EL2 and
participated in a hydrophobic interaction with L164. On the other
hand, benzazepine moiety of 10 was situated deep into the
subpocket near TM5 and interacted with F5.38, A5.42, S5.43
(statistically, the most important TM5 residue) and T5.46.
25 Surprisingly, both of the less potent compounds (8 and 9) formed
hydrogen bonds between the positive, ionized nitrogen atom and
D7.36 (instead of D3.32). Thus, they were located shallowly in
the binding pocket, close to TM6 and TM7; consequently, these
compounds had almost no interaction with TM3 (Figure 3B, C).
30 The central nitrophenyl group did not contact F6.52, but
participated in faceꢀtoꢀedge stacking with F6.51 and strongly
interacted with EL2 (especially L164). Despite the fact that the
side phenyl moiety was positioned toward TM5 deeper into the
receptor, it did not interact with S5.43 and T5.46.
5
Compounds 4, 8, 9 and 10 were ionized and docked without any
restraints to 200 homology models of 5ꢀHT6R (aligned on an β2ꢀ
adrenergic template) developed in our laboratory employing a
previously utilized approach (see experimental).14,28,29 For each
compound, only the best docking pose per receptor was
10 considered, and the 100 best scoring complexes were transformed
into bitstrings by applying SIFt formalism to statistically describe
the interactions between ligand and receptor.30,31 The most potent
compounds 4 and 10 (Figure 3A, D) were classically docked near
TM3 and TM5. Crucial chargeꢀreinforced hydrogen bonds of
15 protonated nitrogen were formed with D3.32, and hydrophobic
interactions were created with W3.28, T3.29 and C3.36. The
methyl group of the Nꢀmethylꢀarylpiperazine also interacted with
TM7 (Y7.43). The central nitrophenyl group directed toward
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Figure 3. Computational models of the best scoring complexes between ligand 4 (A), 8 (B), 9 (C), 10 (D) and 5-HT6R. The important residues that are
statistically crucial for the more potent ligands are marked in cyan. Residues (D7.36 and L164) that interact with the less active compounds are indicated
in orange. Hydrogen bonds with D3.32 or D7.36 are shown in red. Both 4 and 10 are situated deeper into the receptor cavity and strongly interact with
TM3, TM5 and TM6, whereas the less potent 8 and 9 are located shallowly and interact with TM7 and EL2.
5
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structures 4 and 10, the distance values are decreased by 2ꢀ3 Å.
35 Similar orientations of the rings in the arylpiperazine fragment
for both of the more active compounds may be critical for ligandꢀ
receptor recognition and may play an important role in selective
ligandꢀreceptor binding. The semiꢀrigid molecule 10 represents
optimal spatial orientation of the crucial molecular fragments,
40 thus allowing more precise definition of the pharmacophore. The
same conformation is also possible for the most flexible molecule
4, although this was not observed in the crystalline form because
of the additional interactions formed in the crystal lattice. This
favorable conformation cannot be attained by 8 and 9 because of
45 their increased rigidity, which explains the lower binding affinity
of these compounds for the 5ꢀHT6 receptor.
The statistical analysis of multiple ligandꢀreceptor complexes
(Table 5) emphasizes the importance of L163, F6.51 and F7.35 in
ligand binding. In addition, the more potent compounds, 4 and
50 10, interacted classically with D3.32, S5.43, T5.46 and F6.52,
whereas the binding modes of the rigid, less potent compounds 8
and 9 indicated atypical interactions with D7.36 (instead of
D3.32). Interestingly, of the entire serotonin GPCR family of 13
receptors, the D7.36 residue is present only in 5ꢀHT1B and 5ꢀ
55 HT1D receptors. As far as we know, this alternative side
interaction of a protonated nitrogen with D7.36 has not been
previously reported. This information can be used as a potential
starting point for the design of novel, potent, selective serotonin
receptor ligands.
Table 5 Averaged SIFt profiles calculated for the100 best scoring
complexes. Only values higher than 0.50 were selected as significant.
Compound
Residue
4
8
9
10
0.74
0.64
0.98
0.96
0.90
–
–
0.99
–
0.98
0.99
0.99
0.95
0.72
0.85
1.00
0.99
1.00
0.50
–
W3.28
T3.29
D3.32
V3.33
C3.36
L4.61
R162
L163
0.79
0.61
0.99
0.85
0.88
–
–
0.56
–
0.79
–
0.51
–
0.96
0.60
0.83
0.89
0.93
0.76
–
–
0.96
–
–
–
–
0.84
–
–
–
0.57
0.98
0.64
0.86
0.82
0.82
0.68
–
0.98
0.59
0.87
0.63
1.00
0.65
0.95
0.63
1.00
0.98
0.99
0.67
–
L164
A165
F5.38
V5.39
A5.42
S5.43
T5.46
F6.51
F6.52
N6.55
V6.58
P7.32
F7.35
D7.36
T7.39
Y7.43
–
0.95
0.56
0.86
0.58
0.50
1.00
0.77
0.75
–
0.86
–
–
0.99
–
1.00
0.94
0.99
0.58
0.74
0.54
1.00
–
1.00
0.93
60 Conclusion
Discussion
In conclusion, bioisosteric exchange based on ring modification
was successfully applied to the design of potent 5ꢀHT6R ligands
with a modified selectivity profile. Whereas a rigid conformation
between two aromatic moieties in compounds 8 and 9 resulted in
65 decreased affinity, the introduction of a sevenꢀmembered ring in
compound 10 conserved the active spatial orientation of the
important pharmacophore features. These observations were
confirmed and explained by crystallographic and computational
studies. Compared to the previously reported compound 4, the
70 most potent compound 10 exhibited greater selectivity towards 5ꢀ
HT1A (threeꢀfold), 5ꢀHT7 (sevenꢀfold) and D2 (twoꢀfold). These
differences in biological activity suggest that 5ꢀHT6 receptors are
sensitive to subtle changes in the orientation of the aromatic
moieties. Additionally, analysis of the ligandꢀreceptor complexes
75 indicated the possibility of the formation of an unusual
interaction with D7.36, which has not been previously reported.
5
Common VS campaigns use the structures of active compounds
as direct queries to search databases; however, here, we
introduced an additional step of bioisostere generation to cover
more biologically relevant chemical space. Indeed, the similarity
between the 11 VS hits and the 5ꢀHT6R ligand database ranged
10 from Tc = 0.57–0.83. Particularly, Tc equaled 0.79 for the hit
compound (9), which means that this compound would not have
been selected using a standard VS procedure with the criteria of
Tc ≥ 0.8. Further bioisosteric modification of 9 based on ring
alteration produced two additional compounds with significantly
15 different affinities for 5ꢀHT6R (Ki = 245 and 6 nM for 8 and 10,
respectively).
The
parent
compound
Nꢀbenzylꢀ5ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitroaniline 4 was previously reported by
Tasler et al. in a series of 5ꢀHT6R ligands developed by
modifying virtual screening hits.25 The most potent compound of
20 a group of obtained nitrophenylpiperazines displayed affinity
Ki = 6 nM towards 5ꢀHT6R.
Experimental protocols
Crystallographic studies of synthesized bioisosteres revealed that
both compounds with higher binding affinities (4 and 10) adopt a
similar molecular arrangement. The mutual orientation of both
25 aromatic moieties, and consequently, the shorter distance
between the C(12)ꢀC(17) ring and the basic nitrogen atom N(4)
seems to play an important role. Interestingly, the binding poses
of the docked ligands resemble crystalline structures. Distances
between the aromatic rings are 1 Å greater, on average, in less
30 potent compounds than in compounds 4 and 10, similar to the
intervals between the N(4) atoms of the Nꢀmethylpiperazine
moiety and the terminal aromatic system. These substructures are
relatively distant for compounds 8 and 9, whereas in the
Synthesis
1
Melting points were determined on an Electrothermal 9100. H
80 NMR spectra were recorded on Bruker Avance III HD 400 NMR
spectrometer. Mass spectra were recorded using a TQD Waters
LC/MS spectrometer with electrospray ionization. Substrates and
solvents were purchased from SigmaꢀAldrich and Apollo
Scientific and were used without further purification.
85 NꢀBenzylꢀ5ꢀchloroꢀ2ꢀnitroaniline (1).
5ꢀChloroꢀ2ꢀnitroaniline (1.0 g) was added to water (12.0 ml),
benzyl bromide (1.19 g) was added, and the reaction was heated
to reflux for 1.5 h. After cooling to RT, a saturated sodium
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bicarbonate solution (10.0 ml) was added to the reaction mixture,
126.32; 126.87; 128.07; 128.71; 133.17; 134.37; 138.89; 139.78;
and the product was extracted 3 times with ethyl acetate (3×20 60 146.34.
ml). The combined extracts were washed with brine, dried over
anhydrous MgSO4 and evaporated under reduced pressure.
Purification by column chromatography on silica eluting with
chloroform/hexane 1:1 afforded a yellow solid (0.71 g). Yield
45%.
2ꢀ(5ꢀChloroꢀ2ꢀnitrophenyl)ꢀ2,3,4,5ꢀtetrahydroꢀ1Hꢀ2ꢀ
benzazepine (7).
5
A red solid was obtained (0.32 g). Yield 18%. LCMS [M+1] =
303.08 m/z (302.08 calcd). 1H NMR (CDCl3, 400.17 MHz) δ
65 (ppm): 2.09 – 1.98 (m, 2H; 2.99 – 2.92 (m, 2H); 3.45 – 3.37 (m,
2H); 4.50 (s, 2H); 6.76 (dd, J = 8.7, 2.1 Hz, 1H); 7.03 (d, J = 2.1
Hz, 1H); 7.31 – 7.12 (m, 4H); 7.67 (d, J = 8.7 Hz, 1H).
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 27.53; 33.72; 55.49;
56.65; 118.37; 119.45; 126.68; 127.78; 127.82; 128.22; 129.83,
1
LCMS [M+1] = 263.09 m/z (262.05 calcd). H NMR (CDCl3,
400.17 MHz) δ (ppm): 4.54 (d, J = 5.5 Hz, 2H); 6.66 (dd, J = 9.1,
10 2.1 Hz, 1H); 6.86 (d, J = 2.1 Hz, 1H); 7.48 – 7.27 (m, 5H); 8.22 –
8.12 (m, 1H); 8.47 (d, J = 6.1 Hz, 1H).
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 47.27; 113.60; 116.32; 70 137.36; 137.97; 139.18; 140.85; 146.21.
127.17; 127.99; 128.27; 129.07; 130.89; 136.9; 142.86; 145.62.
tertꢀButyl NꢀbenzylꢀNꢀ(5ꢀchloroꢀ2ꢀnitrophenyl)carbamate (2).
15 NꢀBenzylꢀ5ꢀchloroꢀ2ꢀnitroaniline (1) (0.66 g) was dissolved in
anhydrous THF (10 ml), and the solution was cooled in an ice
General procedure for the coupling of NꢀMeꢀpiperazine with
aryl chloride.
An ovenꢀdried roundꢀbottom flask was charged with the aryl
chloride (3.6 mmol), palladium acetate (0.1 eq.), biphenyl tertꢀ
bath. Then, a 0.5 M solution (5.82 ml) of KHMDS in toluene was 75 butyl phosphine (JohnPhos) (0.2 eq.) and potassium phosphate
added dropwise. After standing for 30 min, a solution of Boc2O
(0.64 g) in anhydrous THF (10 ml) was added dropwise. The
20 reaction mixture was allowed to warm to RT and was stirred until
completion (as indicated by TLC). The reaction was quenched by
monohydrate (1.4 eq.). The flask was evacuated under vacuum
and filled with argon three times. Then, DME (2 ml / 1 mmol)
and Nꢀmethylpiperazine (1.3 eq.) were added via syringe. The
reaction mixture was heated at 100 °C for 20 h. After completion,
the addition of distilled water (50 ml), and the product was 80 the reaction mixture was filtered through celite, and the solvent
extracted
with
chloroform.
Purification
by column
was evaporated. The product was purified by column
chromatography on silica eluting with CHCl3:MeOH (19:1)
NꢀBenzylꢀ5ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitroaniline (4).
A red oil was obtained (0.21 g). Yield 51%. LCMS [M+1] =
chromatography on silica eluting with chloroform afforded a red
25 solid (0.35 g). Yield 40%. The substance could not be ionized
under ESI conditions. H NMR (CDCl3, 400.17 MHz) δ (ppm):
1
1.56 (s, 9H); 4.54 (d, J = 5.5 Hz, 2H); 6.66 (dd, J = 9.1, 2.1 Hz, 85 327.24 m/z (426.23 calcd); the compound undergoes deprotection
1H); 6.86 (d, J = 2.1 Hz, 1H); 7.48 – 7.29 (m, 5H); 8.17 (dd, J =
9.1, 0.3 Hz, 1H).
during ionization. tertꢀButyl NꢀbenzylꢀNꢀ[5ꢀ(4ꢀmethylpiperazinꢀ
1ꢀyl)ꢀ2ꢀnitrophenyl]carbamate (3) (0.20 g) was dissolved in
MeOH (10 ml) with a few drops of concentrated hydrochloric
acid. The reaction was heated to reflux for 3 h, and distilled water
90 (50 ml) and 15% NaOH solution (5 ml) were added. The products
were extracted with chloroform (3×20 ml), and the extracts were
dried over anhydrous MgSO4 and evaporated under reduced
pressure. The obtained substance was dissolved in diethyl ether
and converted to the hydrochloride salt by addition of a 20%
30 13C NMR (CDCl3, 400.17 MHz) δ (ppm): 27.43; 47.27; 85.17;
113.60; 116.32; 127.16; 127.98; 128.27; 129.07; 136.58; 142.85;
145.62; 146.75.
General procedure for the coupling of 4ꢀchloroꢀ2ꢀfluoroꢀ1ꢀ
nitrobenzene with heterocyclic secondary amines.
35 4ꢀChloroꢀ2ꢀfluoroꢀ1ꢀnitrobenzene (5.7 mmol) and the appropriate
amine (5.7 mmol) were dissolved in DMSO (10 ml), and
anhydrous potassium carbonate (14.2 mmol) was added. The 95 solution of HCl in diethyl ether. The collected solids were
reaction mixture was heated to 120 °C for 2 h. After cooling to
RT, the reaction mixture was poured into distilled water (100 ml)
40 and extracted with ethyl acetate (3×30 ml). The combined
extracts were washed with distilled water (2×100 ml) and brine
recrystallized from ethanol to afford an orange solid (0.094 g).
Yield 55%.
For the purpose of crystallographic studies, the free base was
crystallized from hexane/acetone.
(20 ml), dried over anhydrous MgSO4 and evaporated under 100 Hydrochloride mp. 206ꢀ208 °C. LCMS [M+1] = 327.17 m/z
1
reduced pressure. Purification was performed by flash
chromatography on silica gel eluting with chloroform.
45 2ꢀ(5ꢀChloroꢀ2ꢀnitrophenyl)ꢀ2,3ꢀdihydroꢀ1Hꢀisoindole (5).
A red solid was obtained (1.2 g). Yield 76%. LCMS [M+1] =
(326.17 calcd). H NMR (CDCl3, 400.17 MHz) δ (ppm): 2.31 (s,
3H), 2.47 – 2.42 (m, 4H), 3.32 – 3.27 (m, 4H), 4.50 (d, J = 5.4
Hz, 2H), 5.84 (d, J = 2.6 Hz, 1H), 6.22 (dd, J = 9.7, 2.6 Hz, 1H),
7.33 – 7.24 (m, 1H), 7.39 – 7.30 (m, 4H), 8.08 (d, J = 9.7 Hz,
275.05 m/z (274.05 calcd). 1H NMR (CDCl3, 400.17 MHz) δ 105 1H), 8.79 (t, J = 5.5 Hz, 1H).
(ppm): 4.68 (s, 4H); 6.77 (dd, J = 8.7, 2.0 Hz); 7.03 (d, J = 2.0
Hz); 7.40 – 7.25 (m, 4H); 7.67 (d, J = 8.7 Hz).
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 46.02; 46.80; 47.08;
47.20; 54.48; 94.42; 104.48; 124.46; 127.12; 127.59; 128.88;
128.94; 137.62; 147.44; 155.73.
50 13C NMR (CDCl3, 400.17 MHz) δ (ppm): 55.67, 116.00, 116.38,
122.25; 127.72; 127.84; 135.95; 136.36; 139.02, 142.07.
2ꢀ[5ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ2,3ꢀdihydroꢀ1Hꢀ
2ꢀ(5ꢀChloroꢀ2ꢀnitrophenyl)ꢀ1,2,3,4ꢀtetrahydroisoquinoline (6). 110 isoindole (8).
A red solid was obtained (1.34 g). Yield 82%. LCMS [M+1] =
289.06 m/z (288.06 calcd). 1H NMR (CDCl3, 400.17 MHz) δ
55 (ppm): 3,05 (t, 2H); 3.43 (t, J = 6.2, 5.4 Hz, 2H); 4,32 (s, 2H);
6.92 (dd, J = 8.7, 2.1 Hz, 1H); 7.15 – 7.08 (m, 1H); 7.30 – 7.15
An orange solid was obtained (0.5 g). Hydrochloride mp. 208ꢀ
210 °C. LCMS [M+1] = 339.17 m/z (338.17 calcd). 1H NMR
(CDCl3, 400.17 MHz) δ (ppm): 2.39 (s, 3H); 2.66 – 2.55 (m, 4H);
3.47 – 3.37 (m, 4H); 4.68 (s, 4H); 6.26 (d, J = 2.5 Hz, 1H); 6.36
(m, 4H); 7.82 (d, J = 8.7 Hz, 1H). 13C NMR (CDCl3, 400.17 115 (dd, J = 9.3, 2.4 Hz, 1H); 7.30 (s, 4H); 7.83 (d, J = 9.3 Hz, 1H).
MHz) δ (ppm): 28.72; 49.78; 52.12; 119.12; 119.58; 126.30;
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 45.92; 47.37; 54.56;
8
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55.90; 99.52; 104.47; 122.14; 127.40; 129.39; 130.17; 136.62;
144.46; 154.51.
2ꢀ[5ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ1,2,3,4ꢀ
tetrahydroisoquinoline (9).
5
An orange solid was obtained (0.7 g). Hydrochloride mp. 157ꢀ
159 °C. LCMS [M+1] = 353.17 m/z (352.19 calcd). 1H NMR
(CDCl3, 400.17 MHz) δ (ppm): 2.39 (s, 3H); 2.62 – 2.55 (m, 4H);
3.10 (t, J = 5.8 Hz, 2H); 3.41 (ddd, J = 7.7, 5.5, 4.5 Hz, 6H); 4.34
(d, J = 1.0 Hz, 2H); 6.50 – 6.40 (m, 2H); 7.26 – 7.07 (m, 4H);
10 8.10 – 8.00 (m, 1H).
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 28.92; 46.04; 47.15;
50.89; 52.56; 54.60; 102.64; 106.33; 125.96; 126.27; 126.56;
128.83; 129.91; 131.54; 133.92; 134.75; 148.97; 154.92.
2ꢀ[5ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenyl]ꢀ2,3,4,5ꢀ
15 tetrahydroꢀ1Hꢀ2ꢀbenzazepine (10).
An orange solid was obtained (0.8 g). Hydrochloride mp. 136ꢀ
138 °C. LCMS [M+1] = 367.22 m/z (366.20 calcd). 1H NMR
(CDCl3, 400.17 MHz) δ (ppm): 2.05 – 1.94 (m, 2H); 2.33 (s, 3H);
2.51 – 2.42 (m, 4H); 2.99 – 2.89 (m, 2H); 3.23 – 3.16 (m, 4H);
20 3.45 – 3.36 (m, 2H); 4.57 (s, 2H); 6.35 – 6.24 (m, 2H); 7.32 –
7.09 (m, 4H); 7.87 (dd, J = 9.0, 0.5 Hz, 1H).
13C NMR (CDCl3, 400.17 MHz) δ (ppm): 28.00; 34.06; 45.94;
47.03; 47.17; 54.44; 54.52; 56.59; 56.68; 103.08; 105.43; 126.26;
126.30; 127.41; 128.01; 129.49; 129.99; 130.39; 139.12; 141.23;
25 148.76; 154.43.
Xꢀray structure determination
Single crystals of all compounds were obtained from a 1:1
acetone:hexane mixture by slow evaporation of the solvent under
ambient conditions. Xꢀray diffraction data for crystals of 4, 8 and
30 10 were collected with MoKα radiation (λ=0.71073 Å) using a
Nonius Brucker KappaCCD diffractometer with the software
COLLECT.32 Data were processed with HKL SCALEPACK and
HKL DENZO.33 For the selected crystal 9, intensities of the
diffracted Xꢀray beam were collected with CuKα radiation
35 (λ=1.54184 Å) using SuperNova diffractometer. Data were
processed with CrysAlisPro.34 Experiments were performed at
100(2) K for all compounds except 9. For 9, the temperature was
set at 110(2) K. The phase problem was solved by direct methods
with SHELXSꢀ97 program. The model parameters were refined
40 by fullꢀmatrix leastꢀsquares on F2 using SHELXLꢀ97.35 All nonꢀ
hydrogen atoms were refined anisotropically. In the structure of
4, the hydrogen atom attached to N2 was localized on the
difference Fourier map and refined without restraints. For all
compounds, the positions of the hydrogen atoms attached to C
45 atoms were constrained with CꢀH = 0.95 Å for aromatic protons,
CꢀH = 0.99 Å for methylene protons, and CꢀH = 0.98 Å for
methyl groups and were refined using the riding model with the
isotropic displacement parameter Uiso = 1.2 Ueq and Uiso = 1.5 Ueq
(methyl groups only) of the parent atom.
50 All crystallographic data for the presented structures are shown in
Table 6.
Unit cell layout is presented in Supplementary Infromation
Figures
WinGX software was used to prepare the materials for
55 publication.36 The figures showing the asymmetric units of the
structures were obtained using Mercury CSD 3.3.37
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Table 6 Crystal data and structure refinement.
Identification code
4
8
9
10
Molecular formula
Formula weight
Wavelength
C18H22N4O2
326.39
0.71073 Å
Monoclinic
C19H22N4O2
338.40
0.71073 Å
Monoclinic
C20H24N4O2
352.43
1.54184 Å
Monoclinic
C21H26N4O2
366.46
0.71073 Å
Triclinic
Crystal system
Space group
P 21/c
P 21/c
P 21/c
P
a = 9.5413(2) Å
b = 10.1771(2) Å
c = 11.0759(2) Å
α = 105.049(2)°
β = 92.969 (2)°
γ = 114.710 (1)°
927.62(3)
a = 14.8320(4) Å
b = 4.8941(2) Å
c = 23.9340(7) Å
β = 108.015(2)°
a = 9.2516(3) Å
b = 18.4177(5) Å
c = 12.6426(3) Å
β = 128.998(2)°
a = 14.6428(3) Å
b = 7.5669(1) Å
c = 16.1976(3) Å
β = 100.828(2)°
Unit cell
dimensions
Volume [Å3]
Z
1652.15(10)
1674.18(9)
1762.75(6)
4
4
4
2
Dcalc [g/cm3]
Absorption
coefficient [mmꢀ1]
F(000)
1.312
1.343
1.328
1.312
0.088
696
0.090
0.707
0.086
720
752
392
Crystal size [mm3] 0.50 x 0.30 x 0.05
0.50 x 0.40 x 0.20
2.35 ꢀ 27.50
ꢀ11<=h<=12,
ꢀ21<=k<=23,
ꢀ15<=l<=16
0.35 x 0.25 x 0.05
3.07 ꢀ 71.24
ꢀ17<=h<=17,
ꢀ9<=k<=9,
0.50 x 0.50 x 0.30
2.79 ꢀ 27.45
ꢀ12<=h<=11,
ꢀ12<=k<=13,
ꢀ14<=l<=14
θ range [°]
2.62 ꢀ 28.68
ꢀ19<=h<=19,
ꢀ6<=k<=4,
Index ranges
ꢀ32<=l<=32
ꢀ19<=l<=18
Reflections
collected
10299
9948
23634
7289
Independent
reflections
Completeness % 99.6 (to θ=25.24°)
4234
[R(int)= 0.0329]
3819
[R(int)= 0.0185]
99.5 (to θ=25.24°)
3397
[R(int)= 0.0483]
100.0 (to θ=67.68°)
4191
[R(int)= 0.0107]
99.4 (to θ=25.24°)
Data/restraints/par
4234 / 0 / 221
ameters
3819 / 0 / 226
1.038
3397 / 2 / 235
1.021
4191 / 0 / 244
1.027
Goodnessꢀofꢀfit on
1.016
F2
Final R indices
[I>2σ(I)]
R1 = 0.0469,
wR2 = 0.1076
R1 = 0.0736,
wR2 = 0.1199
R1 = 0.0378,
wR2 = 0.0949
R1 = 0.0444,
wR2 = 0.0993
0.321/ ꢀ0.268
R1 = 0.0385,
wR2 = 0.0977
R1 = 0.0465,
wR2 = 0.1051
0.200/ ꢀ0.211
R1 = 0.0352,
wR2 = 0.0938
R1 = 0.0376,
wR2 = 0.0961
0.298/ ꢀ0.245
R indices (all data)
ꢂρmax/ꢂρmin [e.Åꢀ3] 0.243/ ꢀ0.292
MgCl2, 10 µM pargyline and 0.1% ascorbate; for dopamine
D2LR: 50 mM Tris–HCl, 1 mM EDTA, 4 mM MgCl2, 120 mM
NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate.
30 All assays were incubated in a total volume of 200 µl in 96ꢀwell
microliter plates for 1 h at 37 °C, except for 5ꢀHT1AR, which was
In vitro pharmacology
5
Cell culture and preparation of cell membranes
HEK293 cells with stable expression of human serotonin 5ꢀ
HT1AR, 5ꢀHT6, 5ꢀHT7bR or dopamine D2LR (prepared with the
use of Lipofectamine 2000) were maintained at 37 °C in a
humidified atmosphere with 5% CO2 and were grown in
10 Dulbecco’s Modifier Eagle Medium containing 10% dialyzed
fetal bovine serum and 500 µg/ml G418 sulfate. For membrane
preparations, cells were subcultured in 10 cm diameter dishes,
grown to 90% confluence, washed twice with phosphate buffered
saline (PBS), preꢀwarmed to 37 °C and pelleted by centrifugation
15 (200 g) in PBS containing 0.1 mM EDTA and 1
mM dithiothreitol. Prior to membrane preparations, the pellets
were stored at –80 °C.
incubated at room temperature for
1 h. The process of
equilibration was terminated by rapid filtration through Unifilter
plates with a 96ꢀwell cell harvester, and the radioactivity retained
35 on the filters was quantified on a Microbeta plate reader.
For displacement studies, the assay samples contained the
following as radioligands: 1.5 nM [3H]ꢀ8ꢀOHꢀDPAT (187
Ci/mmol) for 5ꢀHT1AR; 2 nM [3H]ꢀLSD (85.2 Ci/mmol for 5ꢀ
HT6R; 0.6 nM [3H]ꢀ5ꢀCT (39.2 Ci/mmol) for 5ꢀHT7bR or [3H]ꢀ
40 Raclopride (74.4 Ci/mmol).
Nonꢀspecific binding was defined with 10 µM of 5ꢀHT in 5ꢀ
HT1AR and 5ꢀHT7bR binding experiments, whereas 10 µM
methiothepine and 1 µM of (+)butaclamol were used in the 5ꢀ
HT6R and D2L assays, respectively. Each compound was tested in
45 triplicate at 7 to 8 different concentrations (10−11–10−4 M). The
inhibition constants (Ki) were calculated from the ChengꢀPrusoff
equation [27]. The results are expressed as the means of at least
two separate experiments.
Radioligand binding assays
Cell pellets were thawed and homogenized in 20 volumes of
20 assay buffer using an Ultra Turrax tissue homogenizer and
centrifuged twice at 35000 g for 20 min at 4 °C, with incubation
for 15 min at 37 °C in between rounds of centrifugation. The
composition of the assay buffers was as follows: for 5ꢀHT1AR: 50
mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM pargyline
25 and, 0.1% ascorbate; for 5ꢀHT6R: 50 mM Tris–HCl, 0.5 mM
EDTA and 4 mM MgCl2, for 5ꢀHT7bR: 50 mM Tris–HCl, 4 mM
Initial screening experiments were performed using the same
50 conditions with two compound concentrations: 10–6 and 10−7 M.
Molecular modeling
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Preparation of ChEMBL database
Notes and references
Due to a large diversity of activity measures, only the compounds
with defined Ki (IC50 – assumed as 2×Ki, pKi or pIC50 were
converted to Ki), as assayed on human cloned receptors or on rat
cloned or native receptors, were considered. In case there were
multiple data for one ligand, the Ki and human receptors were
given preference; a median value was used if there were many
biological results.
† Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
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This research was supported by the following projects:
Interdisciplinary PhD Studies “Molecular sciences for medicine”
45 (coꢀfinanced by the European Social Fund within the Human
Capital Operational Programme) and UDAꢀPOIG.01.03.01ꢀ12ꢀ
063/09ꢀ00 “Antagonists of 5ꢀHT6 receptor as advanced
antipsychotics with proꢀcognitive properties” coꢀfinanced by
European Union from the European Fund of Regional
50 Development (EFRD).
The crystallographic research was carried out with equipment
purchased using financial support of the European Regional
Development Fund in the framework of the Polish Innovative
Economy Operational Program (contract no. POIG.02.01.00ꢀ12ꢀ
55 023/08).
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