Rationally designed N-phenylsulfonylindoles as a tool for the analysis of the non-basic 5-HT6R ligands binding mode

Article abstract of DOI:10.1016/j.ejmech.2020.112916

Among all of the monoaminergic receptors, the 5-HT₆R has the highest number of non-basic ligands (approximately 5% of compounds stored in 25th version of ChEMBL database have the strongest basic pKa below 5, calculated using the Instant JChem c

Full text of DOI:10.1016/j.ejmech.2020.112916

Journal Pre-proof
Rationally designed N-phenylsulfonylindoles as a tool for the analysis of the non-basic
5-HT R ligands binding mode
6
Jakub Staroń, Ryszard Bugno, Wojciech Pietruś, Grzegorz Satała, Stefan Mordalski,
Dawid Warszycki, Agata Hogendorf, Adam S. Hogendorf, Justyna Kalinowska-
Tłuścik, Tomasz Lenda, Bogusław Pilarski, Andrzej J. Bojarski
PII:
S0223-5234(20)30888-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112916
EJMECH 112916
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 April 2020
Revised Date: 17 September 2020
Accepted Date: 5 October 2020
Please cite this article as: J. Staroń, R. Bugno, W. Pietruś, G. Satała, S. Mordalski, D. Warszycki,
A. Hogendorf, A.S. Hogendorf, J. Kalinowska-Tłuścik, T. Lenda, B. Pilarski, A.J. Bojarski, Rationally
designed N-phenylsulfonylindoles as a tool for the analysis of the non-basic 5-HT R ligands binding
6
mode, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112916.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Elsevier Masson SAS. All rights reserved.

Simple
Pharmacophore
N
basic
nonbasic
N
N
N
N
CF3
O
?
H
N
N
N
N
N
N
S
N
N
O
O
S
S
O
O
nonbasic
analogs
5-HT6
5-HT6
K = 1.2 nM
K = 4 nM
i
i

Rationally designed N-phenylsulfonylindoles as a tool for the analysis of the non-basic 5-HT6R
ligands binding mode.
Jakub Staroń1, Ryszard Bugno1, Wojciech Pietruś1, Grzegorz Satała1, Stefan Mordalski1, Dawid
Warszycki1, Agata Hogendorf1, Adam S. Hogendorf1, Justyna Kalinowska-Tłuścik2, Tomasz Lenda1,
Bogusław Pilarski3, Andrzej J. Bojarski1
1 Maj Institute of Pharmacology, Polish Academy of Sciences, 31-343 Kraków, 12 Smętna Street, Poland.
2 Department of Crystal Chemistry and Crystal Physic Faculty of Chemistry, Jagiellonian University, Ingardena 3,
30-060 Kraków, Poland;
3 CERKO, 96/98 Zwycięstwa Avenue, 81-525 Gdynia, Poland;
Abstract
Among all of the monoaminergic receptors, the 5-HT6R has the highest number of non-basic ligands
(approximately 5% of compounds stored in 25th version of ChEMBL database have the strongest
basic pKa below 5, calculated using the Instant JChem calculator plugin). These compounds, when
devoid of a basic nitrogen, exhibit high affinity and remarkable selectivity. Despite a decade of
research, no clues have been given for explanation of such an intriguing phenomenon. Here, a series
of analogs of four known 5-HT6R ligands, has been rationally designed to approach this issue. For
each of the synthesized 42 compounds, a binding affinity for 5-HT6R has been measured, together
with a selectivity profile against 5-HT1AR, 5-HT2AR, 5-HT7R and D2R. Performed induced fit docking and
molecular dynamics experiments revealed that no particular interaction was responsible for the
activity of non-basic compounds. In fact, a plain N-phenylsulfonylindole (1e) was found to possess a
moderate (5-HT6R, Ki = 159 nM) affinity. No other monoaminergic receptor has as simple and
selective ligand as this one. Thus, it is stated that it binds to the receptor solely based on its
conformation and as such, possesses a minimum amount of features, required for binding. Also, any
functional group able to form an additional interaction with the receptor increase the binding
affinity, like in the case of two highly active non-basic compounds 3e and 5g (5-HT6R, Ki = 65 nM and
38 nM, respectively).
1. Introduction
The aminergic receptors, i.e., G-Protein Coupled Receptors activated by endogenous amines, are
targets of ~25% of drugs.1 The common mechanism of ligand binding in the aminergic receptors
involves the formation of an ion pair (charge assisted hydrogen bond) between a highly conserved
aminoacid residue D3.32 and a positively ionized atom (fragment) of the ligand.2 This anchoring
interaction is reinforced by several other, in particular, the π-π interactions with residues from the
5th, 6th and 7th helices, formation of hydrogen bonds, van der Waals interactions, halogen bonds as
well as by the entropic driving forces.
4–6
7
There have been reports of non-basic antagonists of 5-HT1B3, 5-HT2A and 5-HT6 receptors in
the recent years. Despite the lack of basic functionality, some of these compounds show very high
binding affinity, even in sub-nanomolar range (Fig. 1). The unique feature of 5-HT6 receptor is the
unusually high abundance of its weakly- or non-basic ligands.7–11

O
O
F
Figure. 1 Non-basic serotonin receptor
antagonists. The pharmacophore features
were marked: aromatic ring - orange,
hydrogen bond acceptor - green.
O
S
F
H
N
HO
O
O
S
O
F
H
O
O
O
N
N
N
O
5-HT1BR Ki = 0.43 nM
Nugiel et. al. 2010
5-HT2AR Ki = 0.1 nM
Ladduwahety et. al. 2006 and 2010
5-HT6R Ki = 0.32 nM
Harris et. al. 2010
The 5-HT6 receptor is a well documented drug target for the treatment of cognitive deficits. The
pharmacological blockade of 5-HT6R can potentially restore the deteriorated cholinergic transmission
in cognitive impairment.12,13 There have been several lead compounds targeting 5-HT6R developed
for the treatment of dementia, Alzheimer’s disease (AD), and to relieve the symptoms of
schizophrenia (Tab. 1).14 The clinical trials conducted so far did not confirm the efficacy of 5-HT6R
antagonists as an augmentative therapy for AD. However, AVN-211 has been shown to be effective in
the treatment schizophrenia, relieving positive symptoms and adding some procognitive (increased
attention) effects to antipsychotic medication.15
Table 1 Selected advanced clinical candidates for the treatment of cognitive impairment that reached
phase III trials or are chemically similar to compounds presented in this manuscript. There have been
no successful phase III trials till date in AD patients, while AVN-211, a non-basic 5-HT6R antagonist
was found effective for the treatment of negative symptoms of schizophrenia. A recent review
summarizes the ongoing trails.16
H
N
H
N
N
H
N
H
N
N
H
N
N
N
N
S
N
N
O
N
N
O
O
S
S
O
O
O
O
N
H
O
S
S
O
F
F
F
O
F
F
Idalopirdine (Lu AE58054)
Ki = 0.8 nM
Cerlapirdine (WAY-262,531)
Ki = 1.3 nM
Intepirdine (SB-742457)
Ki = 0.2 nM
AVN-211
Ki = 1.2 nM
PF-05212377
Ki = 0.53 nM
Idalopirdine
Lundbeck
Cerlapirdine
Intepirdine
AVN-211
Avineuro
PF-05212377
Current
developer
Indication
Pfizer (suspended)
Axovant
Pfizer
AD/Schizophrenia
AD/Schizophrenia
Well tolerated
NCT00726115
AD/dementia with Levy bodies
Well tolerated
Schizophrenia
Well tolerated15
AD
Well tolerated
NCT00948662
Safe and well
tolerated but no
benefit on
Well tolerated
NCT02415907
Phase I
NCT00551772
Some efficacy reported in AD
patients, dose dependent
NCT00710684
Effective for
Effective vs
placebo17
Program suspended
NCT00481520
Phase II
schizophrenia but
not for AD patients
NCT00708552
cognition18
No improvement vs
placebo in AD
No improvement versus
placebo in AD patients in test
patients (lower
dosage used than in
phase II)
scores; the subjective condition
of the patient signifficantly
improved vs placebo
Recrutation
ongoing
Phase III
-
-
NCT02079246
NCT02585934
Most of the 5-HT6R antagonists fall into the category of basic bis-arylsulfones which, in some
cases, may lead to shortcoming in terms of blood-brain barrier permeability, metabolic stability, and
hERG binding.2
The development of novel, rule-breaking 5-HT6 receptor antagonists, which lack the archetypical
basic functionality, emerges as a game changer in the field.2 A new, unexplored chemical space that

can enable the escape from the dead end of classic aryl-amine classes has been opened. An
enhanced blood-brain barrier permeation and better absorption could be expected for weakly basic
compounds due to the fact, that they remain in the unionized state under physiological pH,
moreover in some cases the lack of basic moiety implicates a lower molecular mass, i.e., better
diffusion.
Although the existence of such ligands has questioned the amine-D3.32 residue anchoring
paradigm, there has been little research into this uncommon binding mode. Herein, an effort has
been made to study the mechanism of action of weakly basic antagonists of the 5-HT6 receptor. The
structures of four active phenylsulfonyl indoles, representing analogs with different position of N-
methylpiperazinyl substituent (C3, C4, C5 and C6 of indole carbons), were selected as a starting point
for further modifications.19–22 At first, the N-methyl substituent of piperazine ring was replaced by N-
acetyl and N-2,2,2-trifluoroethyl group, in order to lower the basicity of the distal nitrogen atom.
Next, the N-methylpiperazine ring was simplified to piperidine and dimethylamine. These moieties
were selected to investigate the effect of a distal nitrogen atom cancellation and its replacement to
carbon atom. Finally, aromatic substituents of phenyl and 4-pyridinyl were introduced. Phenyl ring
was selected as a planar hydrophobic group, whereas 4-pyridinyl was selected as a surrogate of
piperazine, because of its basic distal nitrogen atom. Each of the synthesized compounds was
evaluated in terms of activity at 5-HT6R and its virtual interactions with a homology model of the
receptor
were
comprehensively
analyzed.
1. Synthesis
Phenylsulfonyl indole derivatives 1a-1h were synthesized via phase transfer catalyzed reaction
between the appropriate indoles and phenylsulfonyl chloride. Final compounds 2(a-g)-5(a-g) were
obtained by cross-coupling of intermediates 1a-1d (Scheme 1). The introduction of N-Me-piperazine,
N-Ac-piperazine, N-2,2,2-trifluoroethylpiperazine, piperidine and N,N-dimethylamine moieties
(compounds 2(a-e)-5(a-e)) was carried out via Buchwald-Hartwig reaction, whereas coupling to
phenyl and 4-pirydinyl groups (compounds 2(f,g)-5(f,g)) was performed via Suzuki-Miyaura reaction.
Synthesis of compounds 3h-3k was accomplished in two steps (Scheme 2), first by substitution of the
indole with appropriate sulfonyl chlorides or benzyl bromide and then coupling with a
dimethylamine. Compounds 5h-5k were synthesized in a similar, but reversed way (Scheme 3). First
by coupling with 4-pirydinyl moiety at C6 of indole, then by substitution at indole -NH with
appropriate sulfonyl chlorides. Derivative 2l was prepared using sodium hydride and benzyl chloride,
then coupling with 4-pirydinylboronic acid (Scheme 3).

R2
R1
R2 = 3-Br (1a), 75% yield
4-Br (1b), 95% yield
5-Br (1c), 95% yield
6-Br (1d), 96% yield
H (1e), 48% yield
4-OH (1f), 92% yield
4-OMe (1g), 95% yield
5-OMe (1h), 67% yield
a
N
S
N
H
O
O
R1 = H, OAc, OMe, Br
R3
R3 = 3-(N-Me-piperazine) (2a), 83% yield
5-(N-Me-piperazine) (4a), 43% yield
5-(N-Ac-piperazine) (4b), 36% yield
5-(N-2,2,2-trifluoroethylpiperazine) (4c), 31% yield
5-piperidine (4d), 71% yield
5-(N,N-dimethylamine) (4e), 82% yield
6-(N-Me-piperazine) (5a), 83% yield
6-(N-Ac-piperazine) (5b), 84% yield
6-(N-2,2,2-trifluoroethylpiperazine) (5c), 85% yield
6-piperidine (5d), 69% yield
3-(N-Ac-piperazine) (2b), 58% yield
3-(N-2,2,2-trifluoroethylpiperazine) (2c), 52% yield
3-piperidine (2d), 75% yield
3-(N,N-dimethylamine) (2e), 38% yield
4-(N-Me-piperazine) (3a), 76% yield
4-(N-Ac-piperazine) (3b), 81% yield
4-(N-2,2,2-trifluoroethylpiperazine) (3c), 90% yield
4-piperidine (3d), 50% yield
N
S
O
O
Br
6-(N,N-dimethylamine) (5e), 73% yield
4-(N,N-dimethylamine) (3e), 78% yield
b
N
S
O
O
R4
c
R4 = 3-phenyl (2f), 71% yield
R2 = 3-Br (1a
4-Br (1b
)
)
3-(4-pirydynyl) (2g), 41% yield
4-phenyl (3f), 99% yield
4-(4-pirydynyl) (3g), 71% yield
5-phenyl (4f), 45% yield
5-(4-pirydynyl) (4g), 48% yield
6-phenyl (5f), 95% yield
N
S
5-Br (1c
)
O
6-Br (1d
)
O
6-(4-pirydynyl) (5g), 67% yield
Scheme 1. Synthesis of N-phenylsulfonylindole derivatives substituted at C3, C4, C5 or C6 of indole. a)
PhSO2Cl, DCM, 50% NaOH, TBAB; b) R3H, Pd(OAc)2, Cs2CO3, XPhos, toluene; c) R4B(OH)2, Pd(PPh3)4,
K2CO3, toluene, EtOH, H2O.
Br
Br
N
R4 = -SO2Me (1i
-SO2n-Bu (1j
,
3h
)
,
3i
)
a, b
c
-SO2cyclohexyl (1k
-SO2N-piperydynyl (1l
benzyl (
3j
, )
,
3k
)
N
H
N
N
R4
1m 3l
,
)
R4
1i, 81% yield
1j, 81% yield
1k, 46% yield
1l, 87% yield
1m, 88% yield
3h
, % yield
3i
, 91% yield
3j, 65% yield
3k
, 85% yield
3l, 67% yield
Scheme 2. Synthesis of compounds 3h-3l. a) R4SO2Cl, DCM, 50% NaOH, TBAB; b) in case of a benzyl
derivative BnBr, NaH, DMF; c) Me2NH·HCl, Pd(OAc)2, Cs2CO3, XPhos, toluene.

R4 = -SO2Me (5h), 78% yield
-SO2n-Bu (5i), 62% yield
a
b
N
H
N
H
N
Br
-SO2cyclohexyl (5j), 57% yield
-SO2N-piperydynyl (5k), 68% yield
R4
N
N
1n, 70% yield
c
a
N
H
N
N
Br
Br
N
5l, 50% yield
1n, 95% yield
Scheme 3. Synthesis of compounds 5h-5l. a) R2B(OH)2, Pd(PPh3)4, K2CO3, toluene, EtOH, H2O; b)
R4SO2Cl, DCM, 50% NaOH, TBAB; c) BnBr, NaH, DMF.
2. In vitro studies
Radioligand assays were utilized to determine the binding affinity and selectivity profiles of the
synthesized compounds in competition binding experiments for human serotonin 5-HT6R. Selectivity
profiles against 5-HT2AR, 5-HT1AR, 5-HT7R and dopaminergic D2R were presented in Supplementary
Information (SI Table 3). The plain N-phenylsulfonylindole (1e), together with its three analogs (Table
2), exhibited a moderate binding affinity (1e, 5-HT6R Ki = 159 nM). In turn, among the rest of the
obtained compounds, five (out of 24) reached comparable affinity as 1e, whereas another five
exhibited 1.6- to 4-fold higher affinity, with 3e (Table 3, 5-HT6R Ki = 65 nM) and 5g (Table 3, 5-HT6R Ki
= 38 nM) being the best binders. These two substitution patterns were further modified in order to
investigate the importance of the phenylsulfonyl moiety. All modifications of this structural fragment
were
found
to
be
not
tolerated
by
the
receptor
(Table
4).

Table 2. Structure and in vitro affinity data for 5-HT6R for the synthesized N-phenylesulfonylindole and its derivatives.
HO
O
O
N
O
N
O
O
O
N
O
N
O
O
S
S
S
S
O
1e23
1f24
1g25
1h26
Cmpd.
5-HT6
159
108
262
127
Ki [nM]a
a Binding affinity, Ki, expressed as the average of at least two independent experiments; the maximum S.D. did not exceed 10% (see Supplementary
Information, page 9, Table 4); affinity of the reference drugs: 5-HT6R, Olanzapine – Ki = 10.7 ± 2.1 nM.

Table 3. Structure and in vitro affinity data for 5-HT6R for the synthesized compounds, substituted at 3rd, 4th, 5th, and 6th position of indole with either N-Me-
piperazine, N-Ac-piperazine, N-2,2,2-trifluoroethylpiperazine, piperidine, N,N-dimethylamine, phenyl or 4-pyridynyl. Unsubstituted N-phenylsulfonylindole
(1e, Ki = 159 nM), was used as a reference compound. Green color marks the placement of substituent which resulted in 1.6 – 100-fold increase of affinity for
5-HT6R, compared to 1e. Yellow color marks compounds with comparable affinity; orange, compounds 4.4 – 1.6-fold less active and red, compounds 32 – 7.5-
fold less active. A calculated value of pKa is given in parentheses next to the symbol of a compound.
R substituent
CF3
O
N
N
N
N
N
N
N
N
N
5-HT6
Cmpd
(pKa)
Cmpd
(pKa)
5-HT6
Cmpd
(pKa)
5-HT6
Cmpd
(pKa)
5-HT6
Cmpd
(pKa)
5-HT6
Cmpd
(pKa)
5-HT6
Cmpd
(pKa)
5-HT6
R
position
Ki
Ki [nM]a
Ki [nM]a
Ki [nM]a
Ki [nM]a
Ki [nM]a
Ki [nM]a
[nM]a
3
2a (7.4)
3a (7.3)
4a (7.4) 27
5a (8.2)
4
2b (0.6) 97
3b (0.8) 714
4b (0.9) 4 853
5b (-0.4) 97
2c (3.6)
3c (3.9)
4c (3.4)
5c (3.6)
2 029
1 743
5 252
1 201
2d (4.5) 219
3d (5.4) 128
4d (1.9) 360
5d (-0.4) 403
2e (4.2) 474
3e (5.0) 65
4e (3.3) 82
5e (-0.3) 211
2f (-7.5) 146
3f (-6.8) 162
4f (-7.6) 558
5f (-7.3) 178
2g (5.0) 124
3g (4.9) 1 508
4g (4.5) 409
5g (4.9) 38
4
5
6
1
9
a Binding affinity, Ki, expressed as the average of at least two independent experiments; the
maximum S.D. did not exceed 10% (see Supplementary Information, page 9, Table 4). Affinity of
the reference drugs: 5-HT6R, Olanzapine – Ki = 10.7 ± 2.1 nM.
Placement of substituents
which resulted in:
R
4
3
5
6
7.5 - 32 decrease
1.6 - 4.4 decrease
comparable
N
5-HT6R affinity vs unsubstituted reference
compound 1e (R = H, Ki = 159 nM)
O
S
O
1.6 - 100 increase

Table 4. Structure and in vitro affinity data for 5-HT6R for the synthesized derivatives of 3e and 5g, substituted with benzyl and different sulfonyl chlorides.
R2 substituent
4
6
O
O
O
O S
O
S
S
S
N
N
R2
O
O
O
5-HT6
5-HT6
5-HT6
5-HT6
5-HT6
Cmpd
Cmpd
Cmpd
Cmpd
Cmpd
Ki [nM]a
Ki [nM]a
Ki [nM]a
Ki [nM]a
Ki [nM]a
4-(N,N-dimethylamine)
6-(4-pyridine)
3h
5h
4 721
4 050
3i
5i
49 900
1 151
3j
5j
39 070
1 664
3k
5k
4 886
2 183
3l
5l
2 563
3 262
a Binding affinity, Ki, expressed as the average of at least two independent experiments; the maximum S.D. did not exceed 10% (see Supplementary Information,
page 9, Table 4); affinity of the reference drugs: 5-HT6R, Olanzapine – Ki = 10.7 ± 2.1 nM.

3. Molecular modelling
A mass induced fit docking (ifd) of compounds 2(a-g)-5(a-g) to 5-HT6R homology models was
performed. The resulting 28 316 ligand-receptor complexes were filtered regarding the mutual
orientation of phenylsulfonyl and indole moieties so that it corresponded to the existence of a weak,
intramolecular hydrogen bond, which may be important for 5-HT6R ligand binding, according to our
recent finding.27 No strong, specific interaction was observed between any of the non-basic ligands
and the receptor models within the analyzed complexes. However, superposition of the active (Ki <
500 nM) non-basic compounds revealed that they adopt a markedly different position in the binding
pocket than the basic compounds (Figure 2; green vs orange, respectively). A strong interaction
between protonated nitrogen of the piperazine ring and the side chain D3.32 imposed the overall
binding pose. The phenylsulfonyl moiety was positioned close to the helices 6 and 7, forming
interactions with the phenylalanine cluster (F6.51, F6.52), while maintaining contact between
sulfonyl group and N6.55 and S5.43. In turn, the non-basic compounds, free from the constraint of
hydrogen bond with D3.32, head towards the helix 4, either with phenylsulfonyl moiety or with the
non-polar group, the piperazine was substituted for. Despite the different placement, the sulfonyl
moiety could still form interactions with N6.55 and S5.43. Interestingly, only for compounds
representing the 1,6- substitution – 5f and 5g the phenyl ring of the phenylsulfonyl moiety turned in
such a way that it was placed similarly to the piperazine ring of basic compounds. While for 5f such
position gave no gain in binding affinity over other phenyl substituted compounds 2f – 4f, the
pyridine substituted 5g exhibited the highest affinity for 5-HT6R, among all non-basic derivatives.
Compounds substituted with dimethylamine were found to be positioned in another binding pocket,
much closer to the entrance to the receptor binding pocket, surrounded by residues W3.28, V2.57,
P2.60 and A2.61 (for representative ligand-receptor complexes see Supplementary Information).
Next, to further study the investigated binding modes, molecular dynamics experiments were
performed. The best scoring complexes of each synthesized compound, were selected as starting
poses. Numerical analysis of the molecular dynamics was based on the measurement of distances
between sulfonyl group and neighboring amino acid residues: S5.43 and N6.55. As a result, it was
revealed that the average distances calculated for active and non-active compounds did not
correspond with the binding affinity (see Supplementary Information). The position of ligands during
the whole simulation (100 ns) was stable, as evidenced by the low variation of the measured
distances (see Supplementary Information).
Figure 2. Superposition of active (Ki < 500 nM) non-basic compounds (green) with basic compounds
(orange). A – side view; B – view from above. The poses were selected from the top scored ligand-

receptor ifd complexes, so that they represent a corresponding binding motif. The green structures
comprise of compounds: 2b, 2d, 2f, 2g, 3f, 4d, 5b, 5d, 5f and 5g. The orange structures comprise of:
2a, 3a, 4a and 5a. The basic compounds positioned themselves so that an interaction is formed
between the protonated piperazine nitrogen atom and aspartic acid D3.32. Among the non-basic
compounds, only 5f and 5g had a phenyl ring placed in such a way, that it mimicked the position of a
piperazine. An additional comparison between 3a and 5g, together with comparison between 3a and
3e is presented in the supplementary information (Figures S4 and S5, respectively).
4. Discussion
The observed structure-activity relationship is very complex. The simplest compound, N-
phenylsulfonylindole (1e) has no functional groups serving as attachment points, besides sulfonyl
moiety, and yet exhibited moderate binding affinity (5-HT6R Ki = 159 nM). For comparison, this value
will be used as a reference ‘base’ level of affinity. Introduction of a hydroxy and methoxy
substituents at C4 and C5 carbons caused only small changes in the binding affinity value (Table 2).
Also, addition of large substituents like piperidine, N-acetylpiperazine, phenyl and pyridine
surprisingly exhibited a limited influence on binding affinity, in most cases. In turn, introduction of a
2,2,2-trifluoroethylpiperazine was unfavorable in all of the studied substitution patterns, despite the
fact that these four compounds exhibited very similar binding mode in the modelling study, as their
active non-basic analogs.
No specific interaction responsible for the variable activity of non-basic compounds was
identified during a detailed analysis of binding poses obtained from ifd experiments. In a general
view, non-basic compounds interacted with phenylalanine cluster of F6.51 and F6.52 either through
the phenyl group of the phenylsulfonyl moiety or the opposite hydrophobic group. The only aspect
that is common to all of the obtained poses is the relatively stable position of the sulfonyl moiety,
which remained close to the residues N6.55 and S5.43, presumably due to hydrogen bonding.
However, statistical analysis of the PDB (Protein Data Bank) showed that the sulfonyl group is
generally not involved in hydrogen bond formation and usually occupies the hydrophobic pocket of
the protein binding site.28 In turn, mutagenesis studies performed by Harris et al. showed that both
mutations N6.55A and S5.43A had a negative (ΔpKi = -0.4), yet small influence on the binding affinity
of a non-basic ligands. The only mutations that markedly affected the binding affinity of the non-
basic compounds were of hydrophobic residues of W6.48 and F6.51.29 Thus the most probable role of
the sulfonyl group is keeping the mutual orientation of the aromatic systems, and not being a
hydrogen bond acceptor. Thus, the substitution of a sulfonyl with a methylene group should have a
limited effect on the binding affinity. In our previous paper we showed that highly basic, methylene
linked 5-HT6R ligands indeed maintain high affinity.27 However, that was not the case with non-basic
compounds. Substitution of sulfonyl with methylene in two most active non-basic derivatives
dramatically lowered the binding affinity (5l 5-HT6R Ki = 3262 nM; 3l 5-HT6R Ki = 2563 nM,
respectively). The sulfonyl group may thus play a crucial role, by stabilizing the conformation of the
molecules.27 In fact, comparative molecular dynamics of weakly- and highly-basic compounds
showed that they exerted no marked conformational changes. Next, we assumed that in the case of
strong binders, distances to the N6.55 and S5.43 residues should be shorter. In this case, the position
of the ligands would be stabilized, which may have been responsible for the increased activity.
However, the comparison of the average distances between the sulfonyl groups' oxygen atoms and
hydrogen bond donors, did not distinguish actives from non-actives.

Taking into account the importance of a sulfonyl group, we anticipated that crystal structures
containing N-phenylsulfonylindole moiety might provide a probable most optimal, geometrical
parameters of the pharmacophore (Scheme 4). Thus, the average distance between centroids of the
phenyl rings is 5.27 Å, the torsion angle (defined as shown on Scheme 4) is equal 91.06° and the
angle between the planes of the phenyl rings equals 80.48° (diagrams representing the distribution
of the selected values are presented in Supplementary Information page 15). The provided values
define a novel pharmacophore for the ligands of 5-HT6R that contains the indispensable elements
needed for the activity at this target.
ANG (A)
[°]
DIST (D)
[Å]
TOR
[°]
Mean
Median
Min
80.48
82.74
52.50
89.99
5.27
5.26
4.39
6.30
91.06
90.87
45.25
158.74
Max
Scheme 4. On the left the fragment searched within CSD is presented with the defined
geometrical parameters: angle between planes PLN1 (marked in red, defined by the benzene ring of
the indole moiety) and PLN2 (marked in blue, defined by the benzene ring of the phenylsulfonyl
moiety) (ANG), torsion angle N-S-C-C (TOR, marked in red) and distance between centroids of fenyl
ring and the six-membered ring of indole moiety (DIST). On the right a table with calculated mean,
median, minimum and maximum values is shown.
Among all of the obtained non-basic derivatives two compounds stand out, regarding their
binding affinity: 3e, substituted at C4 with dimethylamine, and 5g substituted at C6 with 4-pyridyl.
Detailed analysis of the ifd generated binding modes of 3e showed, that it adopted a markedly
different position, located at the entrance to the binding pocket with both of its methyl groups
staying in the proximity of the W3.28 tryptophan (Figures S2 and S3, Supplementary Information
pages 3-4). That interaction was identified only for the two most active dimethylamine compounds
3e and 4e. We thus assume that this weak C-H…pi interaction might be partially responsible for the
increased affinity of these compounds. However, the most active of the presented non-basic ligands
is the 6-pyridinyl derivative 5g. Comparing its binding mode with the phenyl analog 5f, shows that in
both cases the phenyl/pyridinyl moieties rest near the phenylalanine Phe284, in an almost identical
position (Figure S1, Supplementary Information page 2). Still, the pyridinyl nitrogen atom does not
point towards any amino acid residue so, it is unable to form any strong interaction. However, it was
shown that phenyl-pyridine stacking interaction is much more favorable than the phenyl-phenyl
stacking interactions.30 Thus, in this case, the slightly stronger interaction between the phenyl of
Phe284 and piperidine might be responsible for the 4.7-fold increase of binding affinity.
The removal of basic nitrogen from the structure of 5-HT6R ligand significantly increased the
selectivity over other monoaminergic receptors (see Supplementary Information, page 10). Taking

into consideration two most active nonbasics 3e and 5g, they exert no activity at 5-HT1A, 5-HT2A, 5-
HT7 and D2 receptors. In comparison, the piperazinyl parent of 3e (3a) have high affinity for 5-HT2A
and D2 receptors (5-HT2AR Ki = 41 nM, D2R Ki = 31 nM) and moderate affinity for 5-HT1AR (Ki = 164
nM). In turn, the parent for 5g (5a) is characterized by moderate affinities for 5-HT1AR and D2R (Ki =
247 nM and 241 nM, respectively). Despite higher selectivity, the attenuation of basicity brings some
consequences regarding the drugability of a compound, i.e.: lower solubility in water. These feature
can considerably undermine the possibility of such compounds becoming successful drugs.
5. Conclusions
The N-phenylsulfonylindole seems to possess the minimum number of features that are required
for activity at 5-HT6R. These are: two phenyl rings and a linker that stabilizes them in a defined,
optimal position. Substantially, the sulfonyl linker could be changed to a methylene group, as long as
the defined mutual orientation of both aromatic rings is kept (an example of a non-sulfonyl rigid
linker, stabilizing the mutual orientation of two aromatic ring in 5-HT6R ligands can be found in our
previous paper31). Interestingly, changing the sulfonyl linker to a methylene in basic compounds does
not have a detrimental effect on the affinity for 5-HT6R,27 which is not the case with the non-basic
compounds. The switch from a sulfonyl group to a methylene in the case of the studied non-basic
compounds resulted in a >60-fold drop in binding affinity. We assume, that the main feature defining
the activity at 5-HT6R is the very specific mutual orientation of two aromatic rings (described by a
pharmacophore presented on Scheme 4). The function of the basic nitrogen atom would lie mainly in
the stabilization of the position of the rest of the molecule. Our statement is markedly supported by
the unusual activity of a plain N-phenylsulfonylindole 1e (Table 2) and its close analogs 1f-1h. No
remark of activity at monoaminergic receptors, of such a simple non-basic compound have been
reported so far.
Although there have been several groups of rigorously scrutinized 5-HT6 antagonists, involving
several compounds in the clinical trials32 (Table 1), the success of the future applications may be
dependent upon the finding of new compounds, which would not be limited by the classical
pharmacophore of the aminergic ligands.
6. Acknowledgements
The study was partially supported by the grant OPUS 2014/13/B/NZ7/02210 from the Polish
National Science Centre.
7. Methodology
Synthesis
A general procedure for the functionalization of the indole nitrogen
An indole derivative (0.015 mol) and TBAB (0.5 g) were dissolved in a mixture of dichloromethane
(100 ml) and 50% NaOH (50 ml) then, sulfonyl chloride (1.2 eq.) was added dropwise. Reaction
mixture was vigorously stirred at room temperature for 1 h then, distilled water (200 ml) was slowly
added and reaction mixture was extracted with dichloromethane (3x100 ml). Combined organic
extracts were washed with distilled water (50 ml), brine (30 ml) dried over anhydrous magnesium
sulfate and evaporated under reduced pressure. Product was purified by column chromatography on
silica.

A general procedure for the substitution of indole nitrogen with benzyl
To a solution of indole starting material (2.5 mmol) in dry DMF (2 mL) at 0°C was added NaH (60%
dispersal in mineral oil, 1.5 eq). The mixture was stirred at 0 °C for 10 min, and then it was allowed to
warm to ambient temperature and stirred for 30 min. The solution was cooled to 0 °C, and then
benzyl bromide (1.5 eq) was added. The ice bath was removed, and the reaction mixture was
warmed to 45°C and stirred for 1 h. The mixture was cooled to room temperature, concentrated, and
then diluted with ethyl acetate and saturated NH4Cl. The organic layer was washed with brine, dried
over anhydrous MgSO4, filtered, and concentrated. The residue was purified by column
chromatography on silica
A general procedure for the Buchwald coupling
Starting bromo derivative (0.74 mmol), amine (3 eq.), palladium acetate (8 mg, 0.05 eq.), cesium
carbonate (0.72g, 3 eq.), XPhos (30 mg, 0.083 eq.) and anhydrous toluene (5 ml) were placed in a
round bottom flask and sealed under argon. Reaction mixture was heated at 130°C for 20 h. After
cooling, distilled water was added (100 ml) and reaction mixture was extracted with ethyl acetate
(3x100 ml). Combined organic extracts were washed with distilled water (100 ml), brine (30 ml) dried
over anhydrous magnesium sulfate and evaporated under reduced pressure. Product was purified by
column chromatography on silica.
A general procedure for the Suzuki coupling
A
starting
bromo
derivative
(1.2
mmol),
boronic
acid
(1.6
eq.),
tetrakis(triphenylphosphine)palladium(0) (69 mg, 0.016 eq.), 2M aqueous solution of potassium
carbonate (1.2 mL, 2.0 eq.), toluene (5.0 ml) were sealed under argon. Reaction mixture was heated
at 100°C for 1.5 h in microwave oven (150 W), after cooling to room temperature, distilled water was
added (100 ml) and reaction mixture was extracted with ethyl acetate (3x100 ml). Combined organic
extracts were washed with distilled water (100 ml), brine (30 ml) dried over anhydrous magnesium
sulfate and evaporated under reduced pressure. Product was purified by column chromatography
on silica.
Synthetic procedures, together with MS and NMR spectra, were presented in Supplementary
Information (pages 18-166).
pKa calculation
33–36
pKa values calculations were run in Jaguar
under the default settings (energy change at level of
5x10-05, no SCF level shift, none termal smearing, accuracy level was set as ultrafine) for the lowest
energy conformation for each compound (minimization performed in macroModel37) in water as a
solvent.
Induced Fit Docking
The protocol of homology modelling consisted of three steps and aimed to mimic the conformational
flexibility of the receptor observed in living cells.38
In the first step, the raw models of 5-HT6R were generated based on the available crystal templates
of 5-HT1B and 2B subtypes (PDB: 4IAR and 4IB4, respectively). GPCRdb39 sequence alignments
between target and the templates were used, and water molecules as well as the ligand structure

from the crystal structures were preserved. Schrödinger Prime40 was used to build the model
structures.
The raw models underwent the Simulated Annealing protocol with reference compound
CHEMBL1642416 (ChEMBL ID) docked, (Schrödinger Desmond, default settings) resulting in a batch
of 250 ligand-receptor conformations for each of the templates. Ligand structure was then removed
and the set of compounds were docked into the models. The model used in the third step was
selected based on the number of compounds docked, average Glide score, and consistency of the
obtained docking poses. The latter was evaluated by measuring the distance between fenyl rings of
the docked series of compounds and the residues W6x48, F6x51 and N6x55
.
The last step of the protocol was to generate the conformations of the ligand binding pockets with
Induced Fit Docking protocol. Each series of synthesized compounds was used in the IFD. As a result
of the ifd, a total number of 28 316 ligand-receptor complexes were obtained. These were filtered
regarding the mutual orientation of the phenylsulfonyl and indole moieties, so that it corresponds
with the data published in our previous work.27 4 317 ligand-receptor complexes fulfilled the applied
constraints. From among those, 50 best scored (regarding the Glide Score value) complexes were
selected and visually clustered and analyzed. Instant JChem was used as an additional software for
the compound database management.41
Molecular Dynamics
A Molecular Dynamics (MD) simulations were performed using Schrödinger Desmond software.42
Each ligand–protein complex was immersed into a POPC (300ꢀK) membrane bilayer, which position
was calculated using the System Builder interface. The system was solvated by water molecules
described by the TIP4P potential and the OPLS343 force field parameters were used for all atoms.
0.15ꢀM NaCl was added to mimic the ionic strength inside the cell. Molecular simulations for 100 ns
with a step of 10 ps, using NPAT ensemble class (constant normal pressure, temperature and lateral
surface area of membranes) and OPLS3e44 force field were calculated for each system. Based on
obtained trajectories, the mean geometrical distances between amino acids and ligand were
calculated using Simulation Event Analysis tools in Maestro Schrödinger Suit.
Radioligand binding assays
Cell pellets were thawed and homogenized in 20 volumes of assay buffer using an Ultra Turrax tissue
homogenizer and centrifuged twice at 35 000 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 5-
HT1AR: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 µM pargyline and 0.1% ascorbate; for 5-HT6R:
50 mM Tris–HCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM Tris–HCl, 4 mM 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.
All assays were incubated in a total volume of 200 µl in 96-well microtiter plates for 1 h at 37°C,
excluding 5-HT1AR, which was incubated at room temperature for 1 h. The equilibration process was
terminated by rapid filtration through Unifilter plates with a 96-well cell harvester, and the
radioactivity retained on the filters was quantified using a Microbeta plate reader.

For the displacement studies, the assay samples contained the following as radioligands: 1.5 nM [3H]-
8-OH-DPAT (187 Ci/mmol) for 5-HT1AR, 2 nM [3H]-LSD (85.2 Ci/mmol for 5-HT6R, 0.6 nM [3H]-5-CT
(39.2 Ci/mmol) for 5-HT7bR or [3H]-Raclopride (74.4 Ci/mmol) for D2R.
Non-specific binding was defined using 10 µM of 5-HT in the 5-HT1AR and 5-HT7bR binding
experiments, whereas 10 µM methiothepine or 1 µM (+)butaclamol was used in the 5-HT6R and D2L
assays, respectively. Each compound was tested in triplicate at 7 – 8 concentrations (10−11 – 10−4 M).
The inhibition constants (Ki) were calculated using the Cheng-Prusoff equation,45 and the results are
expressed as the means of at least two independent experiments.
8. References
1.
2.
Rask-Andersen, M., Almén, M. S. & Schiöth, H. B. Trends in the exploitation of novel drug
targets. Nat. Rev. Drug. Discov. 10, 579–90 (2011).
Benhamú, B., Martín-Fontecha, M., Vázquez-Villa, H., Pardo, L. & López-Rodríguez, M. L.
Serotonin 5-HT 6 Receptor Antagonists for the Treatment of Cognitive Deficiency in
Alzheimer’s Disease. J. Med. Chem. 57, 7160–7181 (2014).
3.
4.
5.
6.
Nugiel, D. A. et al. De novo design of a picomolar nonbasic 5-HT(1B) receptor antagonist. J.
Med. Chem. 53, 1876–80 (2010).
Fish, L. R. et al. 4-Fluorosulfonylpiperidines: Selective 5-HT2Aligands for the treatment of
insomnia. Bioorg. Med. Chem. Lett. 15, 3665–3669 (2005).
Ladduwahetty, T. et al. A new class of selective, non-basic 5-HT2A receptor antagonists.
Bioorg. Med. Chem. Lett. 16, 3201–4 (2006).
Ladduwahetty, T. et al. Non-basic ligands for aminergic GPCRs: the discovery and
development diaryl sulfones as selective, orally bioavailable 5-HT2A receptor antagonists for
the treatment of sleep disorders. Bioorg. Med. Chem. Lett. 20, 3708–12 (2010).
7.
Harris, R. N. et al. Highly potent, non-basic 5-HT6 ligands. Site mutagenesis evidence for a
second binding mode at 5-HT6 for antagonism. Bioorg. Med. Chem. Lett. 20, 3436–3440
(2010).
8.
9.
Zhou, P. et al. 4-(2-Aminoethoxy)-N-(phenylsulfonyl)indoles as novel 5-HT6 receptor ligands.
Bioorg. Med. Chem. Lett. 15, 1393–1396 (2005).
van Loevezijn, A. et al. N’-( Arylsulfonyl)pyrazoline-1-carboxamidines as Novel , Neutral 5-
Hydroxytryptamine 6 Receptor (5-HT6R) Antagonists with Unique Structural Features. J. Med.
Chem. 54, 7030–7054 (2011).
10.
Ivachtchenko, A. V, Lavrovsky, Y. & Ivanenkov, Y. A. AVN-211, Novel and Highly Selective 5-
HT6 Receptor Small Molecule Antagonist, for the Treatment of Alzheimer’s Disease. Mol.
Pharmacol. 13, 945–63 (2016).
11.
12.
Staroń, J. et al. Pyrano[2,3,4-cd]indole as a Scaffold for Selective Nonbasic 5-HT6R Ligands.
ACS Med. Chem. Lett. 8, 390–394 (2017).
Marcos, B., Gil-Bea, F. J., Hirst, W. D., García-Alloza, M. & Ramírez, M. J. Lack of localization of
5-HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in
5-HT6 receptor-mediated acetylcholine release. Eur. J. Neurosci. 24, 1299–306 (2006).

13.
Riemer, C. et al. Influence of the 5-HT6 receptor on acetylcholine release in the cortex:
pharmacological characterization of 4-(2-bromo-6-pyrrolidin-1-ylpyridine-4-
sulfonyl)phenylamine, a potent and selective 5-HT6 receptor antagonist. J. Med. Chem. 46,
1273–6 (2003).
14.
15.
de Bruin, N. M. W. J. & Kruse, C. G. 5-HT6 Receptor Antagonists: Potential Efficacy for the
Treatment of Cognitive Impairment in Schizophrenia. Curr. Pharm. Des. 21, 3739–3759 (2015).
Morozova, M. A. et al. Add-on clinical effects of selective antagonist of 5HT6 receptors AVN-
211 (CD-008-0173) in patients with schizophrenia stabilized on antipsychotic treatment: pilot
study. CNS Spectr. 19, 316–23 (2014).
16.
17.
18.
Lalut, J., Karila, D., Dallemagne, P. & Rochais, C. Modulating 5-HT 4 and 5-HT 6 receptors in
Alzheimer’s disease treatment. Futur. Med. Chem. 9, 781–795 (2017).
http://www.avineuro.com/avineuro-completed-phase-ii-clinical-study-of-avn-211-a-selective-
5-ht6-receptor-antagonist/.
Fullerton, T. et al. A Phase 2 clinical trial of PF-05212377 (SAM-760) in subjects with mild to
moderate Alzheimer’s disease with existing neuropsychiatric symptoms on a stable daily dose
of donepezil. Alzheimers Res. Ther. 10, 38 (2018).
19.
Nirogi, R. V. S. et al. Bioorganic & Medicinal Chemistry Letters Indole-3-piperazinyl
derivativesꢀ: Novel chemical class of 5-HT 6 receptor antagonists. Bioorg. Med. Chem. Lett. 21,
346–349 (2011).
20.
21.
22.
23.
24.
Caldirola, P., Johannsson, G. & Nilsson, B. 2-, 3-, 4-, or 5-substituted-N1-
(benzenesulfonyl)indoles and their use in therapy, Patent WO 02/32863. (2002).
Bromidge, S. M. Compounds useful in the treatment of CNS disorders, Patent WO 02/41889.
(2002).
Dunn, R., Nguyen, T. M., Xie, W. & Tehim, A. 6’ substituted compounds having 5-HT6 receptor
affinity, Patent WO 2008/101247. (2008).
Ketcha, D. M. The manganese(III) acetate oxidation of N-protected indolines. Tetrahedron
Lett. 29, 2151–2154 (1988).
Matsumoto, M., Ishoda, Y. & Watanabe, N. Selective Halogenation of 4-Oxo-4,5,6,7-
tetrahydroindoles to 5-Halo-4,5,6,7-tetrahydeoindoles with Copper(II) Halides. Heterocycles
23, 165 (1985).
25.
26.
27.
Ishibashi, H. et al. A new, general entry to 4-substituted indoles. Synthesis of (S)-(−)-pindolol
and (±)-chuangxinmycin. Tetrahedron Lett. 34, 489–492 (1993).
Mahboobi, S. et al. Bis(1 H -2-indolyl)methanones as a Novel Class of Inhibitors of the Platelet-
Derived Growth Factor Receptor Kinase. J. Med. Chem. 45, 1002–1018 (2002).
Kalinowska-Tłuścik, J. et al. The effect of the intramolecular C-H⋯O interactions on the
conformational preferences of bis-arylsulfones-5-HT<inf>6</inf>receptor antagonists and
beyond. RSC Adv. 8, (2018).
28.
29.
Bissantz, C., Kuhn, B. & Stahl, M. A medicinal chemist’s guide to molecular interactions. J.
Med. Chem. 53, 5061–84 (2010).
Harris, R. N. et al. Highly potent , non-basic 5-HT 6 ligands. Site mutagenesis evidence for a
second binding mode at 5-HT 6 for antagonism. Bioorg. Med. Chem. Lett. 20, 3436–3440
(2010).
30.
Hohenstein, E. G. & Sherrill, C. D. Effects of heteroatoms on aromatic π-π interactions:
Benzene-pyridine and pyridine dimer. J. Phys. Chem. A 113, 878–886 (2009).

31.
32.
33.
34.
35.
Staroń, J., Warszycki, D., Kalinowska-Tłuścik, J., Satała, G. & Bojarski, A. J. Rational design of 5-
HT 6 R ligands using a bioisosteric strategy: synthesis, biological evaluation and molecular
modelling. RSC Adv. 5, 25806–25815 (2015).
González-Vera, J. A. et al. A new serotonin 5-HT 6 receptor antagonist with procognitive
activity - Importance of a halogen bond interaction to stabilize the binding. Sci. Rep. 7, 1–10
(2017).
Bochevarov, A. D., Watson, M. A., Greenwood, J. R. & Philipp, D. M. Multiconformation,
Density Functional Theory-Based p K a Prediction in Application to Large, Flexible Organic
Molecules with Diverse Functional Groups. J. Chem. Theory Comput. 12, 6001–6019 (2016).
Yu, H. S., Watson, M. A. & Bochevarov, A. D. Weighted Averaging Scheme and Local Atomic
Descriptor for p K a Prediction Based on Density Functional Theory. J. Chem. Inf. Mod. 58,
271–286 (2018).
Klicić, J. J., Friesner, R. A., Liu, S.-Y. & Guida, W. C. Accurate Prediction of Acidity Constants in
Aqueous Solution via Density Functional Theory and Self-Consistent Reaction Field Methods. J.
Phys. Chem. A 106, 1327–1335 (2002).
36.
37.
38.
Schrödinger Release 2019-4: Jaguar pKa, Schrödinger, LLC, New York, NY, 2019.
Schrödinger Release 2019-4: MacroModel, Schrödinger, LLC, New York, NY, 2019.
Sherman, W., Day, T., Jacobson, M. P., Friesner, R. A. & Farid, R. Novel Procedure for Modeling
Ligand/Receptor Induced Fit Effects. J. Med. Chem. 49, 534–553 (2006).
39.
40.
41.
42.
Pándy-Szekeres, G. et al. GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic
Acids Res. 46, D440–D446 (2018).
Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins 55,
351–367 (2004).
Instant JChem was used for structure database management, search and prediction, Instant
JChem 17.28.0, 2017, ChemAxon (http://www.chemaxon.com).
Bowers, K. J. et al. Molecular dynamics---Scalable algorithms for molecular dynamics
simulations on commodity clusters. in Proceedings of the 2006 ACM/IEEE conference on
Supercomputing - SC ’06 84 (ACM Press, 2006). doi:10.1145/1188455.1188544
43.
44.
45.
Harder, E. et al. OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules
and Proteins. J. Chem. Theory Comput. 12, 281–296 (2016).
Roos, K. et al. OPLS3e: Extending Force Field Coverage for Drug-Like Small Molecules. J. Chem.
Theory Comput. 15, 1863–1874 (2019).
Cheng, Y.-C. & Prusoff, W. H. Relationship between the inhibition constant (KI) and the
concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.
Biochem. Pharmacol. 22, 3099–3108 (1973).

HIGHLIGHTS
Rationally designed N-phenylsulfonylindoles as a tool for the analysis of the non-basic 5-HT6R
ligands binding mode.
Jakub Staroń1, Ryszard Bugno1, Wojciech Pietruś1, Grzegorz Satała1, Stefan Mordalski1, Dawid
Warszycki1, Agata Hogendorf1, Adam S. Hogendorf1, Justyna Kalinowska-Tłuścik2, Tomasz Lenda1,
Bogusław Pilarski3, Andrzej J. Bojarski1
1 Maj Institute of Pharmacology, Polish Academy of Sciences, 31-343 Kraków, 12 Smętna Street, Poland.
2 Department of Crystal Chemistry and Crystal Physic Faculty of Chemistry, Jagiellonian University, Ingardena 3,
30-060 Kraków, Poland;
3 CERKO, 96/98 Zwycięstwa Avenue, 81-525 Gdynia, Poland;
1) The analysis of activity of non-basic serotonin receptor ligands is performed.
2) Substantially simplified ligands still exhibit affinity for the receptor.
3) N-Phenylsulfonyl indole possess all features necessary for the activity.
4) A slight, weak interaction increases the baseline activity.
5) A new pharmacophore for 5-HT6R is proposed.

Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: