Halogen bonding enhances activity in a series of dual 5-HT6/D2 ligands designed in a hybrid bioisostere generation/virtual screening protocol

Article abstract of DOI:10.1039/c6ra08714k

A novel hybrid bioisostere generation/virtual screening method combined with narrowing of chemical space through similarity to compounds that are active at the second target was successfully applied for the development of structurally new dual 5-HT₆/D₂ receptor ligands. Consequently, a series of derivatives of the found hit 1d (N-[2-(dimethylamino)ethyl]-N-(2-phenylethyl)aniline) was synthesized. The most active 5-HT₆/D₂ ligands also showed affinity for 5-HT₇R and 5-HT_2AR. The para-chloroaniline derivative was identified as a potent dual 5-HT₆/5-HT₇ receptor antagonist (K_i = 24 nM and K_b = 30 nM, K_i = 4 nM and K_b = 1.4 nM, respectively). In the case of halogen-containing compounds, interesting structure-activity relationships were observed at 5-HT₆, D₂ and 5-HT₇ receptors, and the ligand-receptor complexes were subsequently examined using a molecular modelling approach that combined quantum-polarized ligand docking (QPLD) and Molecular-Mechanics-Generalized-Born/Surface Area (MM/GBSA) free-energy calculation, which permitted the identification of putative halogen binding pockets.

Full text of DOI:10.1039/c6ra08714k

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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Halogen bonding enhances activity in a series of dual 5-HT6/D2 ligands designed in a
hybrid bioisostere generation/virtual screening protocol
Jakub Starońa, Dawid Warszyckia, Rafał Kurczaba, Grzegorz Satałaa, Ryszard Bugnoa, Adam Hogendorfa, Andrzej J. Bojarskia,†
A novel hybrid bioisostere generation/virtual screening method combined with narrowing of chemical space through similarity to compounds that are active
at the second target was successfully applied for the development of structurally new dual 5-HT6/D2 receptor ligands. Consequently, a series of derivatives of
the found hit 1d (N-[2-(dimethylamino)ethyl]-N-(2-phenylethyl)aniline) was synthesized. The most active 5-HT6/D2 ligands also showed affinity for 5-HT7R
and 5-HT2R. The para-chloroaniline derivative was identified as a potent dual 5-HT6/5-HT7 receptor antagonist (Ki = 24 nM and Kb = 30 nM, Ki = 4 nM and Kb =
1.4 nM, respectively). In the case of halogen-containing compounds, interesting structure-activity relationships were observed at 5-HT6, D2 and 5-HT7
receptors, and the ligand-receptor complexes were subsequently examined using a molecular modelling approach that combined quantum-polarized ligand
docking (QPLD) and Molecular-Mechanics-Generalized-Born/Surface Area (MM/GBSA) free-energy calculation, which permitted the identification of putative
halogen binding pockets.
development of a series of analogues in hit-to-lead and/or lead
optimization processes.
Introduction
Virtual screening (VS) campaigns are usually aimed at
New drugs can be designed or searched for in silico using a
structure-based or ligand-based approach or both methods
together.1 Structure-based approaches require the structure
(obtained either through crystallographic or NMR methods) of
a biological target (e.g., protein) that is, in certain cases,
difficult to obtain. Indeed, to date, the crystal structures of
most serotonin and dopamine (except 5-HT1BR, 5-HT2BR and
D3) receptors have not been determined. In turn, ligand-based
methods utilize structures of known compounds, both
endogenous and exogenous, that function at a given biological
target. Among the ligand-based methods is bioisoseric
replacement, which includes the modification of the chemical
structure of a drug in a way that a new compound with similar
biological and modified pharmacokinetic properties is
acquired.2,3 Bioisosteric replacement is commonly used for the
identifying compounds that are active at one selected
biological target. It is well known, however, that many drugs
interact with more than one target, such as therapeutics that
are used for mental disorders with a complex aetiology.4–6 The
design of ligands that display a given pattern of activities for
CNS receptors is however a challenging task. Because the
bioisostere generation/virtual screening method was
successfully applied for the development of new 5-HT6R
ligands,7 we assumed that a similar approach combined with
narrowing of the chemical space through similarity to ligands
of the second target may result in the identification of
structurally novel compounds that are active at two given
receptors. This assumption could provide a starting point for
rational design of ligands with a more complex profile of
pharmacological activity. Thus, following the therapeutic
properties revealed by agents of serotonergic and
dopaminergic systems, the 5-HT6 receptor was selected as a
primary target, whereas activity at dopamine D2 receptor was
additionally sought. It should be mentioned, however, that the
verification of the methodological approach, rather than
focusing on development of drug candidates, was the main
objective of the project. Nevertheless such dual 5-HT6/D2
a Department of Medicinal Chemistry, Institute of Pharmacology Polish Academy of
Sciences 12 Smętna Street, 31-343 Kraków, Poland
† Corresponding author, Institute of Pharmacology PAS, Kraków. Tel. +48 12
6623365, email: bojarski@if-pan.krakow.pl
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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activity profile has recently been reported for hybrid produce dozens of thousands of new, unique chemical
compounds that display antidepressant-like effects in animal scaffolds, the majority of which are, however, nonsense. Eight
models together with pro-cognitive properties. Moreover, pro- scaffolds obtained in the first round of the VS protocol were
cognitive efficacy of 5-HT6 antagonists is also suggested as a therefore processed in vBrood and provided 26 791
adjunctive for the primary function of dopamine D2 receptors, bioisosteres, which were subsequently evaluated using the
in antipsychotic drug action.8,9
The 5-HT6 receptor was selected as a primary target because and two bioisosteres, out of these 26 791, passed all VS filters,
its ligands show procognitive properties,10,11 whereas among which four compounds (1d
1g) were selected in terms
additional activity at the D2 receptor was sought because of its of their synthetic accessibility and chemical scaffold novelty
high therapeutic potential.12,13 A similar activity profile has (Table 1). The chemical scaffolds (1a
1c) of the parents were
same virtual screening protocol against 5-HT6R. One hundred
–
–
recently been reported for hybrid compounds that display synthesized rather than the parents themselves because this
antidepressant-like effects in animal models together with study was focused mainly on the effect on activity of the
procognitive properties.14
applied bioisosteric replacement. However, among all of the
Here, we report the design of new ligands with dual 5- chosen bioisosteres, only 1d was active both at 5-HT6R and
HT6R/D2R biological activity through a multi-step in silico D2R. Thus, to investigate the structure-activity relationship, a
screening protocol. The developed group of N-[2- group of its derivatives 4a–4t was obtained.
(dimethylamino)ethyl]-N-(2-phenylethyl)aniline
derivatives
Table 1. Synthesized daughter structures 1d–1g selected from 102 bioisosteres that
was additionally tested for affinity for 5-HT1A, 5-HT2A and 5-HT7
receptors. Among the group of developed derivatives,
halogen-containing compounds showed interesting structure-
activity relationships at 5-HT6, D2 and 5-HT7 receptors, and
thus their complexes were further investigated using hybrid
quantum mechanic/molecular mechanic (QM/MM) methods.
passed through VS together with their parent compounds and main chemical scaffold.
Parent structure
Chemical scaffold
Daughter structure
CMPD_13596
1d
1a
1b
Bioisostere query
(CHEMBL592752)
The applied bioisosteric query was designed to identify 5-
HT6R ligands that are similar to any D2R ligand and thus
potentially exhibit the desired dual 5-HT6R/D2R activity (Figure
1). Initially, a set of 4806 5-HT6R ligands (Ki < 100 nM)
extracted from the ChEMBL database was used for bioisostere
generation with the PipelinePilot program.15 Next, the
obtained group of 165 657 daughter structures was compared
to a set of 3396 D2R ligands that were also obtained from the
ChEMBL database. Only 853 bioisosteres had a Tanimoto
similarity (Tc) higher than 0.9, whereas 53 were identical to
the corresponding D2R ligands. These 906 structures that were
daughters of the 95 parent structures were subsequently
virtually screened for 5-HT6R affinity. The screening protocol
1e
1f
CMPD_14701
(CHEMBL418854)
CMPD_16506
1c
1g
(CHEMBL19589)
Synthesis
consisted of several steps that included Lipinski and Veber The parent compound 1c was synthesized according to a
rules, physicochemical properties, ADME/Tox, 5-HT6R previously described method in patent US 2010/0120792
pharmacophores and docking to 5HT6R homology models.16 As (Scheme 1).19,20 The first step of the synthesis utilized the
a result, a group of 31 bioisosteres that were daughters of 20 reaction of phenylhydrazine with N-methyl-4-piperidone in
5-HT6R ligands was obtained. Visual inspection of these 20 sulfuric acid, which produced N-methyl-2,3,4,5,tetrahydro-1H-
parent structures revealed that they included only 8 chemical pyrido[4,3-b]indole (1a). Next, the product was conjugated to
scaffolds (see Supplementary Information, page 2). The phenylacetylene using KOH, (Bu4N)2SO4 in DMSO and, finally,
specificity of the PipelinePilot program caused that none of the hydrogenated over 10% Pd/C in methanol to afford target
obtained scaffolds was unique because they were mostly product 1c
simple derivatives of their parent compounds. The
PipelinePilot utilizes only bioisosteric replacements that can be
found in bioactive molecules (taken from the BIOSTER
database17), and thus it has a limited ability to create a new
unique chemical scaffold. However, the vBrood program18
provides analyses of the electro- and stereo-topological
properties of molecular fragments and substitutes the
.
Scheme 1. Synthesis of 2-methyl-5-(2-phenylethyl)-1H,2H,3H,4H,5H-pirydo[4,3-
b]indole 1c
fragment with another similar fragment according to
established program settings. This procedure allows vBrood to
.
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Figure 1. Workflow of the applied protocol for developing a dual ligand that acts at 5-HT6R and D2R. From the structures of known 5-HT6R ligands, a database of
bioisosteres was generated (using a PipelinePilot program15), which was subsequently compared with D2R ligands. The obtained structures with a Tanimoto
coefficient >0.9 were evaluated using the 5-HT6R VS protocol. Structures that passed all filters were used in a second bioisostere generation step (using the vBrood
program18), and the obtained structures were evaluated again using the same VS filters. The final hits were selected considering the synthetic accessibility and novelty
of the chemical scaffolds.
acid, followed by the reaction with hydroxylamine and subsequent
reduction with NaBH4. Unfortunately, the attempts to synthesize
bioisostere 1g were unsuccessful, and the synthesis of parent 1c
was also abandoned.
The synthesis of bioisosteres 1d, 4a–4t consisted of two steps
(Scheme 2). First, reaction of the appropriate anilines with an
alkyl chloride (2-chloro-N,N-dimethylethylamine, 2-chloro-N,N-
diethylethylamine or 2-chloro-N,N-dimethylpropylamine) in
isopropanol with K2CO3 provided intermediates 3a–3u.
Second, reaction of the intermediate with phenylacetaldehyde
in DCE with triacetoxyborohydride yielded the final products
1d, 4a–4t.
Scheme 2. Synthesis of N-[2-(dimethylamine)ethyl]-N-(2-phenylehtyl)aniline
derivatives 1d 4a 4t
,
– .
Synthesis of 1b, 1e and 1f was achieved by the conjugation of 5d
with 5i, 5j and 6b in acetonitrile with K2CO3 (Schemes 3 and 4).
Intermediate 5d was synthesized by, first, bromination of ethyl
acrylate, followed by conjugation to catechol, reduction of the ester
group with LiAlH4 and subsequent esterification with tosyl chloride.
Intermediates 5i and 5j were obtained by the reaction of 2,4-
Scheme 3. Synthesis of (2,3-dihydro-1,4-benzodioxin-2-ylmethyl)[(1-phenyl-
piperidin-4-yl)methyl] amine 1b and [(1-cyclohexylpiperidin-4-yl)methyl](2,3-
dihydro-1,4-benzodioxin-2-ylmethyl) amine 1e
.
dinitrochlorobenzene with isonicotinamide, followed
by
Scheme 4. Synthesis of (2,3-dihydro-1,4-benzodioxin-2-ylmethyl)[(4-phenyl-
phenyl)methyl]amine 1f
substitution with aniline. The obtained compound 5f was
hydrogenated under pressure over platinum or palladium to
generate 5g and 5h. The products were reduced using LiAlH4 to
produce 5i and 5j. Intermediate 6b was synthesized by Suzuki-
Miyaura coupling of 4-bromobenzaldehyde with phenylboronic
.
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Pharmacology
Results and discussion
Radioligand binding assays were used to determine the affinity
The Tc among 102 structures, obtained through tandem
and selectivity profiles of the synthesized compounds for bioisosteric queries and the investigation of 5-HT6R and D2R
human serotonin 5-HT1AR, 5-HT2A, 5-HT6R, 5-HT7bR and D2LR, ligand databases, ranged from 0.43 to 0.88. The obtained
which were stably expressed in HEK293 cells. This procedure bioisostere 1d was active at both 5-HT6 and D2 receptors,
was accomplished via the displacement of [3H]-8-OH-DPAT validating the effectiveness of the designed bioisosteric query
(187 Ci/mmol) for 5-HT1AR, [3H]-Ketanserin (47.3 Ci/mmol) for for creating a multi-receptor ligand (Table 2). Consequently,
5-HT2A
carboxyamidotryptamine (5-CT, 39.2 Ci/mmol) for 5-HT7bR and
[3H]-Raclopride (74.4 Ci/mmol) for D2LR. Each compound was but they possessed moderate-to-low affinity for D2R (1e Ki =
tested in triplicate at 7 to 8 different concentrations (10−11
1609 nM, 1f Ki = 110 nM) and high-to-moderate affinity for 5-
,
[3H]-LSD (85.2 Ci/mmol) for 5-HT6R, [3H]-5- compound 1d was chosen as the lead for a SAR series (4a
Bioisosteres 1e and 1f were found to be inactive at 5-HT6R,
–4t).
–
10−4 M). The inhibition constants (Ki) were calculated using the HT1AR (1e Ki = 22 nM, 1f Ki = 101 nM). Interestingly,
Cheng-Prusoff equation,21 and the results are expressed as the substitution of the phenyl group with cyclohexane
mean of at least two independent experiments (Table 2). The (compounds 1b and 1e) resulted in a loss of activity at D2R and
antagonistic properties of 4m on 5-HT6R and 5-HT7R were only a 2-fold decrease in affinity for 5-HT1AR. In contrast,
evaluated based on its ability to inhibit cAMP production substitution of the phenylpiperidine group with a biphenyl
induced by the agonist 5-CT (100 nM in the case of 5-HT6R, 10 group (compounds 1b and 1f) resulted in a 2-fold increase in
nM in the case of 5-HT7R) in HEK293 cells overexpressing 5-HT6 affinity for D2R and a 10-fold decrease in affinity for 5-HT1AR.
or 5-HT7 receptors (Table 3). The experiments were performed This result suggests that aromatic interactions may be more
in triplicate at 8 concentrations (10−11–10−4 M).
important for ligand-receptor (L-R) interactions in D2R
compared with 5-HT1AR.
Table 2. Binding affinitiesa of the synthesized compounds 1a, 1b, 1d, 1e, 1f and 4a–4t.
Substitution
Ki [nM]
Cmpd.
n
R1
-
R2
-
R3
-
R4
-
5-HT6
22
D2
77
5-HT7
8
5-HT1A
217
5-HT2A
29
1a
1b
1d
1e
1f
-
-
-
-
-
-
2280
63
210
476
1609
110
266
55
430
121
3428
857
205
623
31
10
n.d.
233
n.d.
n.d.
128
228
58
1
-
Me
-
H
-
H
H
1682
22
-
-
2675
3760
51
-
-
-
-
-
101
4a
4b
4c
4d
4e
4f
1
2
1
1
1
1
1
2
1
1
1
2
1
1
1
1
1
1
1
1
Et
H
H
H
H
H
H
H
H
H
Cl
H
H
H
Me
H
H
7614
4486
5707
482
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
67
H
F
23
37
H
Me
CF3
OMe
H
25
33
10
70
H
61
274
1147
n.d.
80
30
5341
>10000
605
809
863
777
27
H
234
264
38
141
2253
38
4g
4h
4i
Me
H
F
8969
>10000
>10000
>10000
2140
5960
>10000
4749
>10000
n.d.
F
H
64
594
1176
1738
108
153
n.d.
423
n.d.
505
691
1751
675
748
387
4512
24
312
472
721
204
212
348
621
79
4j
H
Cl
H
597
355
83
4k
4l
OMe
H
Me
Cl
H
4m
4n
4o
4p
4q
4r
H
24
4
H
380
137
21
1040
2037
114
19
naphthyl
H
H
H
F
H
H
H
Cl
F
Br
I
90
295
372
518
311
H
79
10
n.d.
4s
4t
Me
H
Me
H
347
150
597
353
n.d.
n.d.
a Binding affinity, Ki, expressed as the average of at least two independent experiments; the maximum S.D. did not exceed 34% (see Supplementary Information, page
5); n.d. – not determined.
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heavier halogens, bromine and iodine, exhibited an
approximately 4-fold lower affinity (Ki = 90 nM and Ki = 79 nM,
respectively) than compound 4m, suggesting that the putative
halogen binding pocket in 5-HT6R is smaller than that in
5-HT7R.
Table 3. Functional activity of 4m and reference compounds at 5-HT6R and 5-HT7R.
Kb [nM]
Compound
5-HT6R
30 (antagonist)
0.5 (antagonist)
–
5-HT7R
1.4 (antagonist)
–
4m
SB-74245722, a
SB-26997023, b
The binding affinity data suggested that a similar halogen
binding pocket might also be present in D2R. This hypothesis is
supported by the observation that the introduction of a
chlorine increases the affinity 3-fold compared with an
unsubstituted hit 1d. However, introduction of a bromine and
iodine resulted in a decrease in affinity, which might be
explained by the potentially small volume of the putative
halogen binding cavity. This hypothesis is supported by the
observation that compounds containing small groups, such as
0.9 (antagonist)
a
b
a selective, potent 5-HT6R antagonist currently in phase II clinical trials,
selective, potent 5-HT7R antagonist under development by GSK.
a
Compared with the parent compound 1a, the hit 1d
exhibited 3- to 15-fold lower affinity for all targets. However,
the introduction of a fluorine and methyl at position 4 of the
aniline aromatic ring (4c and 4d, respectively) resulted in
compounds that possessed affinity comparable to the parent
1d for 5-HT6R (4c Ki = 23 nM, 4d Ki = 25 nM) and were 2-fold
more active at D2R (4c Ki = 37 nM, 4d Ki =33 nM).
methyl and fluorine (4d, 4c, respectively), showed the highest
affinity for D2R (Ki = 33 nM and 37 nM, respectively) and that
compound containing a 4-trifluoromethyl group (3f) displayed
a lower affinity (D2R Ki = 274 nM) than those with a 4-methyl
group (4d) – with van der Waals volumes equal to 39.8 and
21.6 Å3 for CF3 and CH3, respectively.25
An affinity similar to 5-HT6R was also obtained for
compounds 4m and 4p (Ki = 24 and Ki = 21, respectively), but
they were 2-fold less active at D2R compared with the parent
compound. Compound 1a, in addition to its affinity for 5-HT6R
and D2R, was also highly active at 5-HT7R and 5-HT2A (Ki = 8 and
29 nM, respectively) and moderately active at 5-HT1A (Ki = 217
nM). Only one compound bearing the 3-(N,N-
To gain insight into the observed changes in affinity of the
obtained series of compounds, they were all re-docked to
homology models of 5-HT6, D2 and 5-HT7 receptors. The
combination of QPLD from the Schrödinger Suite with MM-
Generalized-Born/Surface Area (MM/GBSA) calculations was
used to obtain L-R complexes because this approach is able to
describe the anisotropy of the electron density around halogen
dimethylamino)propyl and 4-fluorophenyl group
(4h),
achieved comparable affinity for 5-HT2A (Ki = 27 nM) but was
less active at other targets. Compound 4m, in turn, was twice
as active towards 5-HT7 receptor (Ki = 4 nM) as the parent 1a
,
atoms, which is
a key feature during halogen bond
together with a comparable affinity for 5-HT6R (Ki = 24 nM),
but it exhibited 2- to 27-fold lower affinity for other targets. In
addition, functional activity assays revealed that 4m possessed
antagonistic properties at 5-HT6R and 5-HT7R, with the latter
being comparable to the reference compound SB-269970
(Table 3).
examination. Only the top scored complexes, according to ΔG,
were considered. The binding modes (Figure 2) were proposed
based on the mutual spatial arrangement of particular ligand
fragments and 5-HT6, D2 and 5-HT7 receptor binding pockets.
All of the obtained L-R complexes exhibited very consistent
binding modes (within each receptor type), in line with the
findings of Kooistra et al.26 and potentially associated with the
similar binding pocket construction of these receptors (Table
4). In addition to the crucial charge-assisted hydrogen bond
with Asp3.32, all of the docked ligands formed at least one
specific aromatic interaction (CH-π or π-π stacking) with
Phe6.51 or Phe6.52 residues. Additionally, the second
aromatic ring of a ligand was usually targeted to Phe3.28
(D2R/5-HT7R) and Trp3.28 (5-HT6R).
Analysis of structure-activity relationship in a series of 1d
derivatives revealed a preference for halogens (chlorine,
bromine and iodine), especially in the case of binding to
5-HT7R. It appears that there may be a specific site in the 5-HT7
receptor binding pocket that can form halogen bonds with
a ligand. Among the three mentioned halogens, iodine forms
the strongest halogen bonds.24 However, the iodine-
substituted compound 4r exhibited lower affinity for 5-HT7R (Ki
=
10 nM) than the chlorine-substituted compound 4m
The exchange of a methyl with a methoxy group in the para
position resulted in a 9- (for 5-HT6R) to 35-fold (D2R) decrease
in affinity. Analysis of the binding modes showed that the
methoxy group occupied the same binding cavity as the
methyl group (Figure 2B), which formed many hydrophobic
interactions that stabilized the L-R complex (e.g., Val5.39,
Ser5.42, Phe6.52, and His6.55 in D2R). It should also be noted
that for the methoxy substituent, no hydrogen bonding with
side chain amino acids was detected (e.g., Ser5.42 in D2R), and
thus it destabilized the L-R complex through unfavourable
polar interactions.
(Ki = 4 nM), although its affinity was higher than the bromine
derivative 4q (Ki = 19 nM). This finding indicates that the
putative halogen binding pocket may be large enough to
accommodate chlorine but too small to fully accommodate
larger halogens. Nevertheless, the halogen-bonding properties
of iodine allowed it, in part, to counter this unfavourable steric
hindrance and resulted in a higher affinity for 5-HT7R than for
bromine. A similar observation was obtained for the binding to
5-HT6R; the chlorine-substituted compound (4m) belongs to
derivatives with the highest 5-HT6R affinity, together with
fluorine and methyl-substituted compounds (4c
, 4d). However,
in this case, compounds (4q, 4r) that were substituted with
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Table 4. Sequence alignment of the 5-HT6, D2 and 5-HT7 receptors involving amino acids considered to be responsible for ligand interactions.
Amino
3.28
3.32 3.33 3.36 5.39 5.42 5.43 6.48 6.51
6.52 6.55 7.35 7.39 7.40 7.43
acid no.
5-HT6
Trp
Asp
Asp
Asp
Val
Val
Val
Cys
Cys
Cys
Val
Val
Thr
Ala
Ser
Ser
Ser
Ser
Thr
Trp
Trp
Trp
Phe
Phe
Phe
Phe
Phe
Phe
Asn
His
Ser
Phe
Tyr
Glu
Thr
Thr
Leu
Trp
Trp
Trp
Tyr
Tyr
Tyr
receptor
D2
Phe
Phe
receptor
5-HT7
receptor
Identical amino acids in receptor binding pocket sequences are indicated by a grey background. Amino acids indicated in Figure 1 are
shown in bold.
Figure 2. Representative L-R complexes (top scores based on ΔG) of selected ligands with 5-HT6, D2, and 5-HT7 receptors. Figures A–C show a comparison of docking
poses for compounds 1d (orange) and 4m (yellow), whereas Figures D–F show a comparison for compounds 4d (cyan) and 4f (magenta). Amino acids that were
selected as crucial for the binding of the presented compounds are shown as sticks. A basic nitrogen atom forms a charge-assisted hydrogen bond with aspartic acid
Asp3.32, an aniline aromatic ring (in the case of D2R and 5-HT7R complexes) interacts with Phe6.52, whereas in the case of 5-HT6R, the same interaction is formed by a
phenylethyl moiety. The ∆∆G [kcal/mol] value shows the difference between ∆G of complexes of a particular compound (4m, 4d, 4f) and an unsubstituted analogue
1d. In most cases, the ∆∆G values correspond to the binding affinity; the greatest energy gain was obtained by the introduction of a chlorine atom (4m, yellow),
regardless of the receptor.
The recognized halogen interaction appeared to possess a
highly directional nature. To explain the 4- (D2R) to 88-fold (5-
HT7R) decrease in activity by shifting the chlorine atom from
position 4 to 3 in the phenyl ring, interaction spheres were
plotted onto relevant backbone carbonyl oxygens (Figure 3). In
each case, the 4-chloro substituent was positioned within the
energetically favourable areas of the sphere, whereas in the
meta substitution, the 3-Cl atom pointed outside of the
sphere, indicating that halogen bonding did not occur.
6 | J. Name., 2016, 00, 1-3
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Figure 3. A superposition of the top scored poses of 4-Cl (4m, yellow) and 3-Cl (4t, blue) derivatives against putative halogen binding pocket interaction spheres. The
∆∆G [kcal/mol] value shows the difference between the G of complexes of a particular compound (4m 4t) and an unsubstituted analogue 1d. The higher binding
energy value for 3-Cl than for 4-Cl derivatives illustrates the highly directional nature of the identified halogen bond interaction. The methodology applied has been
∆
,
described by Wilcken et al.27 Geometrical parameters for the weak halogen bonds are: 5-HT6R d(Cl…O) = 3.7 Å,
∡(C-Cl…O) = 141°, D2R d(Cl…O) = 3.2 Å, ∡(C-Cl…O) =
126°, 5-HT7R d(Cl…O) = 2.9 Å, ∡(C-Cl…O) = 131°.
(distance, sigma hole angle) and contribution of hydrophobic
interaction would have required further studies including e.g.
full quantum mechanics optimization of L-R complexes.
Conclusions
An average hit rate for virtual screening to identify new
compounds with affinity for a given biological target seldom
exceeds 10%. The multi-step protocol applied herein differed
from standard virtual screening by the introduction of
additional bioisosteric derivatization steps and a similarity
filter for the ligand of the second target. These modifications
allowed us to obtain new chemical scaffolds that would not be
identified in commonly utilized virtual screening techniques for
commercially available compound databases. Among 102
structures that fulfilled all of the VS filters, only four were
chosen for further evaluation. Among them, one compound
Experimental
1
The H NMR spectra were recorded using a 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
Sigma-Aldrich and Apollo Scientific company and used without
further purification.
Detailed synthetic, biomolecular and molecular modelling
procedures are presented in Supplementary Information.
(
1d) possessed the desired dual 5-HT6R/D2R activity, two (1e,
1f; bioisosteres of the same parent) were inactive at 5-HT6R
and the synthesis of one (1g) was unsuccessful. It is probable
that the remaining screening hits might also exhibit activity
similar to 1d, but their synthesis was determined to be more
challenging.
General procedure 1 for the synthesis of
N-[2-(dialkylamino)alkyl]anilines (3a-3u).
A 100-ml round bottom flask was charged with aniline (0.054
mol), appropriate (2-chloroalkyl)dialkylamine hydrochloride
(0.018 mol), anhydrous potassium carbonate (0.11 mol) and
isopropanol (50 ml). The reaction was maintained at reflux for
12 hours. After completion, the reaction mixture was filtered,
the residuum was washed with isopropanol and the solvent
was evaporated under reduced pressure. The product was
purified by column chromatography on silica using ethyl
acetate:methanol (9:1).
The hybrid QM/MM analysis performed for the series of 1d
derivatives allowed to identify a putative halogen binding
pocket in 5-HT6, D2 and 5-HT7 receptors. It must be stressed
that halogen bonds in complexes with serotonin receptors
have only recently been proposed in the case of 5-HT6R28 and
5-HT7R29 but with different anchoring points: the carbonyl
oxygen of Pro4.60/Thr5.46 and Cys3.36(sulfur)/Thr3.37,
respectively. Here, the characteristics of putative halogen
binding pockets also differed between the investigated
receptors (5-HT6, D2 and 5-HT7) because the affinity gain
resulting from the introduction of the halogen atom was not
equivalent and depended on the volume of the halogen
binding cleft; the most profound gain was observed for the
chloro derivative 4m, which resulted in a 30-fold increase in
affinity for 5-HT7R (compared with the hit 1d).
General procedure 2 for the synthesis of N-[2-(dialkylamino)alkyl]-
N-(2-phenylethyl)anilines (1d, 4a-4t).
A 100-ml round bottom flask was charged with appropriate
N-[2-(dialkylamino)alkyl]aniline (6 mmol), phenylacetaldehyde
(12 mmol), triacetoxyborohydride (9 mmol) and
dichloroethane (20 ml). The reaction was maintained at room
temperature for 24 hours. After completion, distilled water
(100 ml) was added, and the reaction mixture was extracted
with dichloromethane (2x100 ml). The collected organic
extracts were washed with brine, dried over anhydrous
magnesium sulfate and evaporated under reduced pressure.
Because the geometry of the recognized halogen bonds
(see Figure 3) is not optimal, they should be regarded rather as
weak interactions. It has to be stressed, however, that
receptor conformation is not optimized during Schrödinger
QPLD protocol, thus detailed halogen bonding parameters
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23 J. J. Hagan, G. W. Price, P. Je, N. J. Deeks, T. Stean, D. Piper,
M. I. Smith, N. Upton, A. D. Medhurst, D. N. Middlemiss, G. J.
Riley, P. J. Lovell, S. M. Bromidge and D. R. Thomas, Brit. J.
Pharmacol., 2000, 130, 539–548.
The product was purified by column chromatography on silica
using ethyl acetate:methanol (9:1).
24 T. Clark, M. Hennemann, J. S. Murray and P. Politzer, J. Mol.
Model., 2007, 13, 291–296.
Acknowledgements
25 F. Leroux, ChemBioChem, 2004, 5, 644–649.
The study was partially supported by the Polish-Norwegian
Research Programme operated by the National Centre for
Research and Development under the Norwegian Financial
Mechanism 2009–2014 in the frame of the Project
PLATFORMex (Pol-Nor/198887/73/2013) and by the National
Science Center Grant No DEC-2014/15/D/NZ7/01782.
26 A. J. Kooistra, S. Kuhne, I. J. P. de Esch, R. Leurs and C. de Graaf,
Brit. J. Pharmacol., 2013, 170, 101–126.
27 R. Wilcken, M. O. Zimmermann, A. Lange, S. Zahn and F. M.
Boeckler, J. Comp. Aided Mol. Des., 2012, 26, 935–945.
28 B. Benhamú, M. Martín-Fontecha, H. Vázquez-Villa, L. Pardo and
M. L. López-Rodríguez, J. Med. Chem., 2014, 57, 7160–7181.
29 V. Canale, P. Guzik, R. Kurczab, P. Verdie, G. Satała, B. Kubica,
M. Pawłowski, J. Martinez, G. Subra, A. J. Bojarski and P.
Zajdel, Eur. J. Med. Chem., 2014, 78, 10–22.
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ChEMBL
bioisosteres
Virtual
Screening
Hit
Ki [nM]
Ki [nM]
5-HT6 = 24, D2 = 153, 5-HT7 = 4
5-HT6 = 63, D2 = 476 , 5-HT7 = 121