Virtual screening-driven discovery of dual 5-HT6/5-HT2A receptor ligands with pro-cognitive properties

Article abstract of DOI:10.1016/j.ejmech.2019.111857

A virtual screening campaign aimed at finding structurally new compounds active at 5-HT₆R provided a set of candidates. Among those, one structure, 4-(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl}ethyl)amino]methyl}furan-2-yl)phenol (1, 5-HT₆R K_i = 91 nM), was selected as a hit for further optimization. As expected, the chemical scaffold of selected compound was significantly different from all the serotonin receptor ligands published to date. Synthetic efforts, supported by molecular modelling, provided 43 compounds representing different substitution patterns. The derivative 42, 4-(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl}ethyl)amino]methyl}furan-2-yl)phenol (5-HT₆R K_i = 25, 5-HT_2AR K_i = 32 nM), was selected as a lead and showed a good brain/plasma concentration profile, and it reversed phencyclidine-induced memory impairment. Considering the unique activity profile, the obtained series might be a good starting point for the development of a novel antipsychotic or antidepressant with pro-cognitive properties.

Full text of DOI:10.1016/j.ejmech.2019.111857

Journal Pre-proof
Virtual screening-driven discovery of dual 5-HT /5-HT receptor ligands with pro-
2A
6
cognitive properties
Jakub Staroń, Rafał Kurczab, Dawid Warszycki, Grzegorz Satała, Martyna Krawczyk,
Ryszard Bugno, Tomasz Lenda, Piotr Popik, Adam S. Hogendorf, Agata Hogendorf,
Krzysztof Dubiel, Mikołaj Matłoka, Rafał Moszczyński-Pętkowski, Jerzy Pieczykolan,
Maciej Wieczorek, Paweł Zajdel, Andrzej J. Bojarski
PII:
S0223-5234(19)31009-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111857
EJMECH 111857
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 August 2019
Revised Date: 17 October 2019
Accepted Date: 4 November 2019
Please cite this article as: J. Staroń, Rafał. Kurczab, D. Warszycki, G. Satała, M. Krawczyk, R. Bugno,
T. Lenda, P. Popik, A.S. Hogendorf, A. Hogendorf, K. Dubiel, Mikoł. Matłoka, Rafał. Moszczyński-
Pętkowski, J. Pieczykolan, M. Wieczorek, Paweł. Zajdel, A.J. Bojarski, Virtual screening-driven
discovery of dual 5-HT /5-HT receptor ligands with pro-cognitive properties, European Journal of
6
2A
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111857.
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© 2019 Published by Elsevier Masson SAS.

Virtual screening-driven discovery of dual 5-HT6/5-HT2A
receptor ligands with pro-cognitive properties
Jakub Starońa, Rafał Kurczaba, Dawid Warszyckia, Grzegorz Satałaa, Martyna
Krawczyka, Ryszard Bugnoa, Tomasz Lendaa, Piotr Popika, Adam S. Hogendorfa, Agata
Hogendorfa, Krzysztof Dubielb, Mikołaj Matłokab, Rafał Moszczyński-Pętkowskib, Jerzy
Pieczykolanb, Maciej Wieczorekb, Paweł Zajdelc, Andrzej J. Bojarskia
a Maj Institute of Pharmacology, Polish Academy of Sciences, 31-343 Kraków, 12 Smętna Street, Poland.
b Celon Pharma S.A., 2A Ogrodowa Street, 05-092 Łomianki, Poland
c
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688
Kraków, Poland.
Keywords : 5-HT6, 5-HT2A, serotonin, procognitive, CNS
Abstract
A virtual screening campaign aimed at finding structurally new compounds active at 5-HT6R provided
a set of candidates. Among those, one structure, 4-(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-
yl}ethyl)amino]methyl}furan-2-yl)phenol (1, 5-HT6R Ki = 91 nM), was selected as a hit for further
optimization. As expected, the chemical scaffold of selected compound was significantly different
from all the serotonin receptor ligands published to date. Synthetic efforts, supported by molecular
modelling, provided 43 compounds representing different substitution patterns. The derivative 42, 4-
(5-{[(2-{5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl}ethyl)amino]methyl}furan-2-yl)phenol (5-HT6R Ki = 25,
5-HT2AR Ki = 32 nM), was selected as a lead and showed a good brain/plasma concentration profile,
and it reversed phencyclidine-induced memory impairment. Considering the unique activity profile,
the obtained series might be a good starting point for the development of a novel antipsychotic or
antidepressant with pro-cognitive properties.
1. Introduction
The subtype six serotonin receptor (5-HT6R) identified in the mid-90s[1,2] is a seven-
transmembrane receptor coupled to Gs and positively coupled to adenylyl cyclase. Its localization,
together with results of behavioural experiments, suggested that it plays an important role in
cognition and consolidation of short and long-term memory.[3–6] Additionally, its presence in the
pharmacological spectra of psychotropic drugs indicated its application in the therapy of different
psychiatric disorders. It was found that ligands of 5-HT6R, especially antagonists, which behave as
inverse agonists and neutral antagonists, enhance cognitive functions.[7–11] Moreover, this receptor
is engaged in the control of neurite growth and plays an important role in the development of neural
tissue, affecting neural plasticity.[12–14] These findings led to the synthesis of several thousands of
5-HT6R ligands, among which some were investigated as possible therapeutics for Alzheimer’s
disease (AD) and cognitive impairments associated with schizophrenia.[15,16] In 2015, a phase 2
clinical trial by Avineuro LLC [17,18] was completed to investigate the synergistic effect of 5-HT6R
antagonist (AVN-211 (Figure 1)) in terms of antipsychotic efficacy in patients with schizophrenia. The
clinical trial showed a tendency towards superiority over the placebo in mean reduction of the
Positive and Negative Syndrome Scale score. Additionally, AVN-211 significantly delayed the
progressive decline in memory function associated with AD, as tested in an in vivo preclinical

evaluation.[17] In turn, Pfizers’ selective 5-HT6R antagonist PF-05212377 was discontinued during
phase 2 clinical trials in 2018 because of a lack of efficacy.[19] Another compound – idalopirdine (Lu
AE58054), investigated by Lundbeck A/S,[20] failed to meet its primary efficacy endpoint vs placebo
in three phase 3 clinical trials in 2016, probably due to the 3-fold decrease in dosage, compared to a
successful phase 2 trial. Similarly, intepirdine (a nonselective 5-HT6R antagonist developed by
Axovant (initially by GSK)) was also discontinued in 2017 after failure in a phase 3 clinical trial.[21]
The failure of 5-HT6R antagonists in improving the condition of patients with AD may lie in the
insufficient understanding of the role of 5-HT6R antagonists in neurodegenerative disorders.[22]
Nevertheless, the 5-HT6 receptor is still a promising target candidate for future medications for
cognitive impairments [23] or other symptoms associated with AD-like anxiety and depression.[24]
11
Additionally, a recent PET study (positron emission tomography) with C-GSK215083 that showed a
significant age-related decline in 5-HT6R availability in the caudate and putamen,[25] regions of the
brain associated with learning, further supports the involvement of 5-HT6R in cognitive dysfunctions.
H C
3
H
CH
3
N
CH
N
3
H
N
N
N
S
CH
N
H C
N
O
3
3
O
O
S
O
N
H
O
F
S
O
F
F
F
F
AVN-211
PF-05212365
Idalopirdine
N
N
O
S
O
N
N
N
N
H
H C
S
CH
3
3
O
O
Intepirdine
GSK-215083
Figure 1. Chemical structures of clinically tested 5-HT6R ligands.
The subtype 2a serotonin receptor (5-HT2aR) is a target of many atypical antipsychotics such
as aripiprazole, olanzapine or ziprasidone, in contrast to typical antipsychotics, which represent
activity mainly at subtype 2 dopamine receptor (D2R). Antagonistic activity of atypical antipsychotics
at the 5-HT2A receptor was shown to induce a marked antidepressant effect in placebo-controlled
trials.[26] Also augmenting the SSRI treatment of depressive patient with these agents (e.g.,
risperidone) gave very promising results by significantly increasing the output. Moreover, in vivo
experiments with selective 5-HT2AR antagonists also confirmed the antidepressant effect of this
mode of action.[27–29] The possibility of reinforcing the antidepressant action of 5-HT2A antagonists
with pro-cognitive properties of 5-HT6 antagonists provide an interesting alternative for currently
applied treatments. Especially, considering that treatment with the majority of current antipsychotics
is often associated with cognitive dysfunction.[30,31]
Considering a general shift in philosophy of drug design that has transformed from “one drug
– one target” approach to “one drug – multiple targets”, new polypharmacology strategies for
psychiatric/neurodegenerative disorders has been initiated.[32–34] Such polypharmacological
substances, directed selectively on two or more networked targets with proven connectivity, might
provide beneficial activity and safety.[35–37] Augmenting the action of 5-HT6R antagonists with
another activity was recently shown in the case of dual 5-HT6R/D3R antagonist. In this case, the

selected compound displayed neuroprotective properties against astrocyte damage induced by
doxorubicin, a feature possibly beneficial in the therapy of cognitive decline.[38] , we present the
discovery and synthesis of dual 5-HT6R/5-HT2AR ligands based on a 2-(1H-indol-3-yl)ethyl[(5-
phenylfuran-2-yl)methyl]amine core. In vitro biological profiles together with preliminary
pharmacokinetic and behavioural studies are also reported.
2. Virtual Screening
The initial campaign was aimed at the development of a selective 5-HT6R ligand as a
candidate procognitive drug for the treatment of cognitive impairments, associated with AD or
schizophrenia. In the study, the Princeton BM (http://www.princetonbio.com/) and ChemBridge
(https://www.chembridge.com/screening_libraries/) databases were used as a source of compounds
for the VS protocol, which consisted of several steps (Figure 2), including 2D fingerprint screening,
basic property and ADMET filtering, 3D pharmacophore-based screening and flexible docking. Ninety-
two compounds were finally selected for in vitro binding affinity evaluation (Supplementary
Information, Page 2). Fourteen compounds were found to exhibit 5-HT6R Ki < 1 000 nM: five had Ki <
100 nM, and two were in the range of 100 < Ki < 200 nM (Figure 2). The most active hit OSSK_759428
(5-HT6R Ki = 7 nM) was optimized in another study and was discontinued because of unfavourable
behavioural effects (unpublished data). Finally, compound OSSK_777444 was selected for further
optimization because of its structural novelty, good synthetic accessibility and moderate affinity.
O
H
C
Cl
3
N
S
N
N
O
O
O
N
N
NH
N
O
N
H
C
3
H
C
3
7247121
OSSK_189819
5-HT6 Ks = 58 nM
5-HT6 Ks = 61 nM
CH
3
H
N
N
O
S
CH
3
N
O
N
CH
3
NH
Cl
OSSK_378922
OSSK_777444
5-HT6 Ks = 20 nM
5-HT6 Ks = 145 nM
O
NH
NH
NH
N
NH
N
N
NH
N
N
N
CH
N
3
H
C
3
OSSK_759428
5-HT6 Ks = 7 nM
OSSL_256422
5-HT6 Ks = 21 nM
Figure 2. On the left is a schematic representation of a Virtual Screening protocol that led to the
identification of hit OSSK_777444 (compound 1). A number of structures that fulfilled the
requirements of each step are indicated. On the right are the structures of the identified hits with Ki <
100 nM plus OSSK_777444.
3. Synthesis

The synthesis of OSSK_777444 derivatives (1-29, 33-43) consisted of a reductive amination of
aldehydes with appropriate amines (tryptamine, 2-(benzofuran-3-yl)ethanamine or 7-azatryptamine),
which was conducted in methanol using NaBH4 as a reducing agent (Scheme 1). The aldehydes were
prepared using Suzuki-Miyaura coupling of a 5-bromofuraldehyde, 5-bromothiophenaldehyde or 5-
bromopyrroladehyde with an appropriate arylboronic acid (Scheme 1).
H
H
HO
R4
R4
X
X
a
Br
B
+
O
O
OH
R4
R3
R3
X
H
R1
NH2
R2
R1
NH
R2
R4
X
b
+
O
Z
Z
Y
Y
Scheme 1. A schematic of the synthesis of the designed compounds with marked modifications of a
core structure. Synthesis of aldehydes a) Pd(PPh3)4, K2CO3, toluene, EtOH, H2O, 70°C, overnight;
synthesis of final compounds b) MeOH, NaBH4, RT. X = O, NH, S; Y = C, N; Z = NH, O. R1 = -OMe, -F, -
CH3, -Cl; R2 = H, -CH3; R3 = H, -CH3; R4 = Aryl.
The coupling conditions utilized
a
classical approach with Pd(PPh3)4 in
a
toluene/ethanol/water medium.[ref] The required amines were either supplied by a vendor or
synthesized by one of four methods. The first method utilized a condensation of
indolecarboxyaldehyde with nitroethane and subsequent reduction with LiAlH4 to yield compounds
t1 and t2 (Scheme 2; Supplementary information, synthesis of t1 and t2). The second method
consisted of several steps: reaction of indole with oxalyl chloride, esterification of the obtained acid
chloride, substitution with ammonia and, finally, reduction of glyoxylamide with LiAlH4 (Scheme 2,
Supplementary information, synthesis of t4 and t5). The synthesis of 2-(benzofuran-3-yl)ethanamines
required the substitution of a methyl 2-hydroxy-benzoate with ethyl bromoacetate, hydrolysis with
NaOH followed by cyclization, condensation with (diethylphosphono)acetonitrile and, finally,
reduction of a nitrile (Scheme 3, Supplementary information, synthesis of t3 (reduction of a nitrile),
t6, t7, t8, t9, t10 and t11). The synthesis of 7-azatryptamines (t12, t13) followed a Larock method for
indoles.[39] First, the hydroxyl group of 4-(trimetylosilylo)-but-3-ynol was substituted with iodine,
which was, in turn, substituted with sodium azide. The azide was reduced with LiAlH4, and the
resulting amine was protected with an acetic anhydride. The obtained N-[4-(trimethylsilyl)but-3-yn-1-
yl]acetamide was coupled with an amino-bromopyridine using Pd(dppf)Cl2·CH2Cl2 as a catalyst, and
the resulting intermediate was deprotected with sodium hydroxide (Scheme 3, Supplementary
information, synthesis of t12 and t13).

NO2
NH2
O
R1
R1
R1
CH3
CH3
a
b
N
H
N
H
N
H
t1
,
t2
R1 = -OMe, -F, -CH3, -Cl;
R2 = -H, -CH3
Cl
CH3
O
O
O
O
O
R1
c
R1
R2
R1
R2
d
R2
N
H
N
H
N
H
NH2
NH2
O
O
R1
R1
f
e
R2
R2
N
H
N
H
t3 - t5
Scheme 2. Synthesis of α-Me-tryptamine and tryptamine intermediates: a) EtNO2, AcONH4, 90°C; b)
LiAlH4, THF, reflux; c) (COCl)2, Et2O, 4°C; d) MeOH, RT; e) aqueous NH3, RT; f) LiAlH4, THF, reflux.
CH3
CH3
O
O
O
O
O
OH
O
R1
OH
O
O
O
g
h
i
R1
R1
O
R1
HO
O
O
,
t6 t7
H3C
CN
R1
R1
NH2
j
k
(Me)3Si
O
O
t10, t11
t8, t9
HO
l
R1
Br
m
n
(Me)3Si
o
(Me)3Si
(Me)3Si
(Me)3Si
+
H3C
O
HO
N3
N
NH2
H2N
NH
CH3
R1 = -OMe, -F, -H
NH
NH2
CH2
R1
R1
p
q
Si(Me)3
N
H
N
H
N
N
t12, t13
Scheme 3. Synthesis of 2-(1-benzofuran-3-yl)ethanamine and 7-azatryptamine intermediates: g)
BrCH2COOEt, K2CO3, acetone, reflux; h) NaOH, H2O, MeOH; i) Ac2O, AcONa; j)
diethylcyanomethylphosphonate, NaH, THF, RT; k) LiAlH4, AlCl3, Et2O; l) PPh3, imidazole, I2, THF, RT;
m) NaN3, DMSO, RT; n) LiAlH4, Et2O, RT; o) Ac2O, TEA, DCM, RT; p) Pd(dppf)Cl2·CH2Cl2, LiCl, Na2CO3,
DMF, MW, 130°C, 30 min q) NaOH, EtOH, reflux.

4. Results
4.1 In vitro pharmacology
Radioligand binding assays were utilized to determine the affinity and selectivity profiles of
the synthesized compounds in competition binding experiments for human serotonin 5-HT6R, 5-
HT2aR, 5-HT1AR, 5-HT7R and dopaminergic D2R, which were stably expressed in HEK293 cells,
according to previously published procedures (Table 1).[40,41] Only for compounds with 5-HT6R Ki <
100 nM was a selectivity profile at the four other (5-HT2aR, 5-HT1AR, 5-HT7R and D2R) targets
assessed. 5-HT6R functional activity was evaluated based on an ability of a ligand to inhibit cAMP
production induced by the agonist 5-CT (100 nM) in HEK293 cells overexpressing 5-HT6 receptors
(Table 3).

Table 1. Structure and in vitro affinity data for the synthesized compounds (a variable substitution at C4, C5 and C6 of indole is marked in blue). Only
compounds with 5-HT6R Ki < 100 nM were evaluated for activity at other targets.
R4
R5
N
R1
X
R3
Y
R2
Z
Ki [nM]a
5-HT6 5-HT2A 5-HT1A
Cmpd
X
Y
Z
R1
R2
R3
R4
R5
5-HT7
1 281
–
–
–
–
–
–
–
–
–
–
871
–
–
–
–
–
–
D2
676
–
–
–
–
–
–
–
–
–
–
n.d.
–
–
–
–
–
–
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
O
S
NH
S
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N-Me
N
N-Me
N
N-Me
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
91
152
264
418
201
693
419
220
185
266
1 463
76
210
486
1 032
247
556
109
303
132
55
461
–
–
–
–
–
–
–
–
–
–
216
–
–
–
–
–
–
125
–
–
–
–
–
–
–
–
–
–
91
–
–
–
–
–
–
1-naphthyl
1-naphthyl
3,4-diMeO-Ph
1-furyl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
3,4-methylenodioxy-Ph
3-Cl-Ph
H
H
3,5-diCl-Ph
4-AcO-Ph
3-MeO-Ph
3,5-diCl-Ph
3-IsoPropO-Ph
3-MeO-Ph
3-MeO-Ph
3-MeO-Ph
Ph
Ph
4-F-Ph
3-OH-Ph
3-imidazole
5-MeO
5-MeO
H
H
4-MeO
H
4-MeO
H
4-MeO
5-MeO
5-MeO
–
–
29
28
–
–
64
146
–
–
–
–
N
N
3 147
1 501
391
1 730
121

23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
-
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
-
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
N
N
-
5-MeO
5-MeO
5-MeO
H
4-MeO
5-MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-quinoline
4-Cl-Ph
4-MeO-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-NO2-Ph
Ph
H
H
H
H
H
H
H
Me
Pr
Me
H
H
H
H
H
H
H
H
H
101
67
90
7
14
3
774
61
334
199
2
2
60
5
475
345
21
1 696
522
25
843
607
35
175
131
41
–
199
–
–
11
96
33
22
–
–
42
–
1 052
218
334
292
732
120
–
775
–
–
1 010
202
431
399
–
9 855
2 170
3 298
1 622
2 095
206
–
525
–
–
895
2 730
n.d.
51
–
–
1 025
571
1 365
757
178
220
–
368
–
–
376
446
n.d.
n.d.
–
–
167
–
H
H
Ph
5-MeO
5-F
6-F
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
4-OH-Ph
-
5-Cl
5-Me
H
5-MeO
5-MeO Me
5-MeO
5-F
5-F
H
H
Me
Me
H
H
H
H
H
-
–
–
–
–
5 316
–
–
13 750
–
8 635
H
H
H
H
-
–
32
–
–
505
–
H
H
-
6 204
–
3 936
531
3.54
Idalopirdine
-
791
1 215
a
Binding affinity, Ki, expressed as the average of at least two independent experiments; the maximum S.D. did not exceed 32% (see
Supplementary Information, page 8); n.d. – not determined. Affinity of the reference drugs: 5-HT6R and 5-HT2AR, olanzapine – Ki = 10.7 ± 2.1 nM
and Ki = 6.2 ± 0.9 nM, respectively; 5-HT1AR, buspirone – Ki = 34.3 ± 4.2 nM; 5-HT7R, clozapine – Ki = 45.5 ± 5.1 nM; D2R, ziprasidone – Ki = 2.1 ±
0.3 nM.

4.2 Structure–Activity Relationship
The first structural element to be optimized was the type of heteroatom X in the five-
member heterocyclic ring; from the three heterocycles containing furyl, thiophene and pyrrole
(compounds 1, 2 and 3), the furyl derivative exhibited the highest affinity for 5-HT6R (Ki = 91 nM).
Second, the effect of the R1 substituent of indole and R4 of furan moiety on the affinity for 5-HT6R
was investigated. To optimize those elements, we used a combinatorial approach supported by
molecular modelling.
A combinatorial library (CL) of chemical structures, which was constructed using all 59
available in-house boronic acids combined with two tryptamine cores (4-methoxy and 5-
methoxytryptamine), enumerated a library consisting of 118 structures. All were pre-filtered with the
ADMETox filter (98 structures passed through), docked to a homology model of 5-HT6R and sorted
according to a GlideScore parameter (gscore, see Supplementary Information page 4), which
indicates the quality of interactions formed with the receptor model. Next, based on the scores, we
selected individual structures from the ranking list, which were directed for synthesis (compounds 4
– 29). The top ranked compound (28, gscore = -9.603), with R4 = 4-OH-phenyl and the methoxy
group at C5 of the indole ring, was found to have the highest affinity for 5-HT6R (Ki = 3 nM);
additionally, compound 28 exhibited high affinity for 5-HT2AR (Ki = 41 nM). In the next step, in an
attempt to optimize the substitution of the amine nitrogen, two different groups were tested: methyl
and propyl (compounds 30 and 31). Substitution with methyl was found to increase affinity (1.5-fold
increase, 5-HT6R Ki = 61 nM) compared with the unsubstituted derivative 1; however, the
introduction of methyl at the amine nitrogen to the most active compound 28 resulted in 67-fold less
active derivative (32, 5-HT6R Ki = 199 nM). Substitution of methoxy at C5 of indole (compound 28)
with fluorine (compound 33) retained the affinity for 5-HT6R (Ki = 2 nM) and increased the affinity for
5-HT2AR 4-fold (Ki = 11 nM). This modification additionally increased affinity for 5-HT2CR (99% inh. at
10-6M, considered as an off-target because of its association with obesity). Shifting the position of
fluorine to C6 of the indole ring (compound 34) did not affect the affinity for 5-HT6R, but 9-fold
lowered the affinity for 5-HT2A (Ki = 96 nM). The derivative with chlorine at C5 (35) was found to be
20-fold less active (5-HT6R Ki = 60 nM) than 28, whereas the derivative with methyl at C5 (36) was
only slightly less active (5-HT6R Ki = 5 nM).
At this point, metabolic stability of four compounds, 26, 28, 32 and 33, was investigated.
Among those compounds, 28 was the most stable with CLint = 63.4 µl/min/mg, whereas 33 was the
least stable with CLint = 250.9 µl/min/mg (Table 2). Unfortunately, compound 28 exhibited low
selectivity over other the investigated receptors. To improve the stability and selectivity, several
structural modifications were applied. First, methylation of the alpha carbon of tryptamine
(compounds 37 and 38) was found to be unfavourable (5-HT6R Ki = 475 nM and 345 nM,
respectively). The introduction of a methyl group at position 2 of the indole ring (compound 39, 5-
HT6R Ki = 21 nM) significantly reduced the stability (CLint = 183.6 µl/min/mg, Table 2). Changing the
indole nitrogen to oxygen resulted, in turn, in a substantial decrease in affinity (40 and 41, 5-HT6R Ki =
1 696 nM and 522 nM, respectively). The introduction of a nitrogen atom instead of a carbon atom at
the C7 position of the indole ring, in the case of the 5C-fluoro substituted derivative, resulted in an
active, moderately stable (CLint = 102.6 µl/min/mg) compound (42, 5-HT6R Ki = 25 nM), which
additionally displayed comparable affinity at 5-HT2AR (Ki = 32 nM); it was also 66-fold less active
towards 5-HT7R and 2-fold less active towards D2R than 28. These excludes potential side-effects
associated with D2R blockade, such as extrapyramidal symptoms. Comparison of the metabolic

stability of 42 with idalopirdine showed that both exhibited comparable clearance (idalopirdine, CLint
= 99.3 µl/min/mg, Table 2).
The lead 42 behaved as antagonist at 5-HT6 receptors (Table 3). Moreover, within the extended panel
of relevant targets, the compound showed only a moderate affinity for 5-HT2C (%inhibition at 1 µM =
59.9, Table 2). Additionally, 42 was found to be brain penetrant (Figure 3), a non-substrate for PgP
and cytotoxic at concentrations greater than Kd (> 50 µM) (Table 4).
Table 2. Metabolic stability measured using rat microsomes.
Cmpd.
CLinta [µl/min/mg]
221.7
t1/2 [min]
26
6
28
32
33
63.4
137.9
250.9
22
10
6
39
183.6
8
42
102.6
99.3
109.4
14.4
14
14
13
96
Idalopirdine
Verapamil
Donepezil
aMetabolic clearance
4.3 The novel object recognition test
To estimate the influence of dual 5-HT6/5-HT2AR ligand 42 on cognitive functions, the novel
object recognition (NOR) test was performed. The NOR test was based on earlier studies of Nikiforuk
[42] and adapted from the original work of Ennaceur and Delacour.[43] In our experiment, PCP was
used to attenuate learning. NMDA receptor-based models are commonly used to mimic some
symptoms of schizophrenia in laboratory animals. Acute administration of phencyclidine (PCP)
evokes a broad range of schizophrenia-like symptoms, including cognitive impairment. Compound 42
prevented PCP-induced memory impairment, and this effect was similar to idalopirdine (Lu
AE58054), which was used as a “positive” control in our experiment (Figure 4). Considering the
cognition deficits as comorbid symptoms of depression/schizophrenia, compounds displaying
complementary modes of action represented by 42, may likely present broad-based efficacy in
relation to compounds acting on core symptoms of CNS diseases.
Table 3. Functional and anti-target profile of lead compound 42
Function at
5-HT6 Kb
[nM]a
Function
5-HT2B
Antagonism
at 5-HT2A
Alpha
1Ab,d
b,d
H1b,d
3.2
M1b,d
5-HT2C
hERGb,d
b,d
c,d
antagonist antagonist
27 -1.3
8.9/19.1
35.5
4
59.9
42.2
a
b
Expresses the functional activity as the average of at least three independent experiments;
%
inhibition of control binding at 10e-6 M; % inhibition of control binding at 10e-6/10e-7 M; tested at
c
d
Eurofins, Le Bois Levêque, 86600 Celle, France, www.eurofins.com
Table 4. Results of the cytotoxicity and CYP inhibition assays for compound 42.

Toxi Light Cell
Cytotoxicity
IC50 [µM]
Cell viability
CYP3A4
IC50 [µM]b
CYP2D6
IC50 [µM]c
PgPa
AMES
IC50 [µM]
Medium
permeability,
not a
5.14
6.2
7.8
8.4
not mutagenic
substrate for
PgP
Papp A2B is equal 8,87 x10-6 cms-1, Papp B2A 3,43 x10-6 cms-1; For reference inhibitor
ketoconazole IC50 for CYP3A4 equals 0.1 [µM]; c For reference inhibitor quinine IC50 for CYP2D6
equals 0.01 [µM].
a
b
Figure 3. Concentration time profiles for 42 in plasma and brain after per os administration to male
Wistar rats at a dose of 10 mg/kg. The concentrations in plasma and brain are expressed as ng/ml
and ng/g, respectively. The brain to plasma ratio at Cmax (brain) is equal to 0.7.
1.00
0.75
###
###
0.50
0.25
0.00
###
###
*
***
PCP
0
0
5
0
5
3
5
5
1
5
3
mg/kg
Compd
10
42
Lu AE58054
Figure 4. Effects of compounds 42 and Lu AE58054 on PCP-induced cognitive impairment in the novel-
object recognition test in rats. The data are presented as the mean ± standard error of the mean of

the discrimination index (DI). N = 8-10 animals per group. Symbols: *p < 0.05, ***p < 0.001,
significant reduction in DI compared with the vehicle-treated group; ### p < 0.001, significant
increase in DI compared with the PCP-treated group.
4.4 Molecular modelling
To investigate the observed structure-activity relationship, an induced fit docking (IFD) was
performed for selected compounds (detailed experimental data are presented in the Supplementary
Information, page 7). Analysis of the obtained ligand-receptor (L-R) complexes showed that the
protonated nitrogen atom formed a charge-assisted hydrogen bond (salt bridge) with aspartic acid
D3.32, while the indole was positioned in proximity to the phenylalanine cluster of F6.51 and F 6.52
(Figure 5A). In turn, the aryl-furan moiety was directed towards the extracellular part of the receptor
protein, with the phenyl ring positioned next to asparagine N2.63, aspartic acid D7.36 and cysteine
C45.50x50 (Figure 5A). This unique location allowed substituents at C4 of the phenyl ring to interact
with these residues. In particular, the hydroxyl group formed hydrogen bonds with the amide side
chain of asparagine and backbone carbonyl of cysteine (Figure 5A). Shifting the hydroxyl to the C3
position (compound 21) moved it out from an optimal orientation, causing an 18-fold drop in affinity
(in comparison to para-substitution).
Figure 5. Comparison of a binding mode (A) of 28 (5-HT6R Ki = 3 nM, green) and 21 (5-HT6R Ki = 55
nM, blue). Both compounds superimpose almost perfectly, with the only visible difference being the
alternative position of hydroxyl groups. In the case of para-substituted 28, the hydroxyl is involved in
hydrogen bonding with N2.64 and C45.50X50, whereas the meta-substituted 21 forms only one
interaction with C45.50X50. Comparison of a binding mode (B) of 33 (5-HT6R Ki = 2 nM, yellow) with
idalopirdine (5-HT6R Ki = 3.54 nM, magenta). The fluoroalkyl chain of idalopirdine lies in proximity to
asparagine N2.63, enabling it to form hydrogen bonds or dipol-type interactions.
The best binder, compound 33, was docked to a 5-HT2AR crystal structure along with idalopirdine
(Figure 6). Both compounds positioned themselves nearly identically in the binding pocket of 5-
HT2AR. The tryptamine part of both molecules was located deep in the binding pocket, with the ‘tail’
reaching the extracellular side of the receptor protein. Similarly, as in the complex with 5-HT6R, the
hydroxyl of compound 33 formed a hydrogen bond with asparagine, but at position 7.36.

Figure 6. Comparison of a binding mode of 33 (5-HT2AR Ki = 11 nM, green) and idalopirdine (5-HT2AR Ki
= 791 nM, magenta), docked to a crystal structure of 5-HT2AR. Both compounds were similarly located
inside the binding pocket with almost identical positions of the tryptamine part of the molecules. The
para hydroxyl of 33 formed a hydrogen bond with asparagine N7.35, similarly to that in the complex
with 5-HT6R. The fluoroalkyl chain of idalopirdine did not form any interactions.
5. Discussion
The structure-activity relationship analysis clearly showed that the para-hydroxyl was crucial
for a high affinity for the 5-HT6 receptor; however, the docking results obtained during the VS and CL
campaigns were insufficient to provide an explanation for this effect. The performed induced-fit
docking experiments revealed that the tryptamine moiety of the synthesized compounds adopted a
similar orientation as the tryptamine part of LSD in the crystal structure of a complex of LSD with 5-
HT2BR.[44] In complex with the 5-HT6R homology model, the obtained compounds assumed an
atypical binding mode compared with the binding modes of previously published 5-HT6R ligands [45–
47]. Our compounds were located at shallower positions than ‘classic’ arylsulfone ligands, with the
aromatic ‘tail’ interacting with asparagine N2.64 and cysteine C45.50X50, from extracellular loop 2. In
most scientific papers published to date, an interaction with tryptophan W6.48 was highlighted as
important for the ligand-5-HT6 receptor interaction. In our case, such an interaction was precluded
because of the shallow location of the compound. Additionally, the hydroxyl group at the C4 position
was positioned in such a way that it could form a net of attractive interactions with neighbouring
residues. This phenomenon was probably responsible for the markedly higher activity of p-hydroxy-
substituted compounds. Our assumption was supported by the observation that m-hydroxy
derivatives exhibited 18-fold lower binding affinity. Of particular interest was the replacement of an
indole nitrogen atom with oxygen (compounds 28 and 40), which caused a significant >500-fold drop
in affinity. The superposition of L-R complexes (see Supplementary Information) of both compounds
showed that both derivatives assumed a nearly identical position in the binding pocket, but no clear
difference was apparent that would explain such a major affinity change. Supposedly, the indole
nitrogen atom of 28 lied in proximity to threonine T5.46 and could potentially form a water-bridged
hydrogen bond. In turn, benzofurane derivative 40 would be unable to form such a stabilizing
interaction.

The reported compounds were structurally similar to another published 5-HT6R ligand–idalopirdine.
The main difference between these two lied in a 3-(2,2,3,3-tetrafluoropropoxy)phenyl moiety
connected to a tryptamine nitrogen, instead of 5-(4-hydroxyphenyl)furanyl. The docking of
idalopirdine to a homology model of 5-HT6R revealed that the fluoroalkyl chain (Figure 5B) formed a
N-H…F interaction with asparagine N2.63, analogously to p-hydroxyl of the presented compounds.
This interaction stabilized the L-R complex and resulted in a similar binding affinity (idalopirdine 5-
HT6R Ki = 3.54 nM). The main difference between idalopirdine and the newly obtained compounds
was in the activity towards 5-HT2AR. Our compounds exhibited relatively high affinity for 5-HT2AR (33,
5-HT2AR Ki = 11 nM), in contrast to idalopirdine, which is selective towards 5-HT6R (5-HT2AR Ki = 791
nM). The low activity of idalopirdine at 5-HT2AR could be explained by the lack of interactions of the
fluoroalkyl chain of idalopirdine with surrounding amino acid residues, whereas the structure of the
presented compounds allowed them to form a stabilizing interaction with asparagine N7.35.
Interestingly, among the studied receptors (5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R and D2R), the
asparagine N2.63 moiety is unique to 5-HT6R (Table 5), while asparagine N7.36 is present only in 5-
HT2AR.
The selected lead structure 42 showed low metabolic stability, similar to a reference
compound with low stability – verapamil. We neglected this unfavourable feature because
idalopirdine, a drug that reached phase 3 clinical trials, also possessed a similar clearance value
(measured in rats). In turn, the lead was revealed to possess favourable properties in terms of target
selectivity, toxicity and pharmacokinetics. Importantly, 42 did not exhibit activity at hERG and 5-HT2B,
supporting its safety for the cardiac system. The compound showed satisfactory bioavailability in
both plasma and brain tissue. The maximum brain concentration was achieved at 0.5 h and gradually
subsided until at 8 h, exceeding the plasma concentration.
Table 5. Comparison of amino acids present in 5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R and D2R at selected
positions.
Receptor
2.63
5-HT1AR
Tyr
5-HT2AR
Thr
Asn
5-HT6R
Asn
Asp
5-HT7R
Thr
Arg
D2R
Leu
Ser
7.36
Ala
Cognitive impairment constitutes an integral part of central nervous system disorders. In
particular, it is the main feature of Alzheimer’s disease and an important aspect of major depressive
disorder and schizophrenia.[48,49] Blockade of NMDA receptor by non-competitive antagonists (such
as ketamine or PCP) evokes, in healthy subjects, symptoms that occur in patients suffering from
schizophrenia (delusions, hallucinations) and produces a recrudescence of psychotic symptomatology
in patients.[50–52] Furthermore, acute administration of ketamine or PCP causes impairment of
cognition in rodents.[53–55] 5-HT6 receptor antagonism was previously recognized as a strategy for
treating cognitive impairment and supported by preclinical and clinical studies.[9,56–58] In our
experiment, the lead compound 42 was active in vivo and successfully reversed PCP-induced
cognitive impairment in the NOR test. The atypical antipsychotic drugs (clozapine, olanzapine) exert
their effects in schizophrenic patients mainly by blockade of the 5-HT2A receptor. This mechanism,
combined with the large body of evidence indicating the role of 5-HT6 receptor in cognition, implies
that 5-HT6/5-HT2A receptor antagonism might be a promising strategy in the treatment of

schizophrenia. Compounds with such a dual pharmacological profile could have the potential to
improve cognitive deficits as well as attenuate psychotic symptoms.
6. Conclusions
The present data provide a good example of a successful virtual screening campaign with
successive hit-to-lead optimization. From among the 14 hit compounds, seven exhibited high to
moderate affinity for 5-HT6R, and one was selected for further development. Successive structure
optimization was enforced with molecular modelling techniques, and the highest scored structure
was the one an optimal substitution pattern. As a result, a concise group of novel tryptamine
derivatives was obtained; compounds exhibited varying binding affinity for a set of receptors (5-
HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R and D2R), with the highest activity at 5-HT6R and 5-HT2AR,
respectively. The most active compounds exhibited nanomolar affinity for 5-HT6R (28, 33, 34; 5-HT6R
Ki = 3, 2 and 2 nM, respectively). Simultaneously, several compounds showed high affinity for 5-HT2AR
(33, 36; 5-HT2AR Ki = 11 and 22 nM, respectively); in the case of the other receptors studied, the
obtained derivatives exhibited low to negligible affinity. Compound 42 was selected as a lead
because of its superior selectivity against 5-HT1AR, 5-HT7R and D2R, acceptable metabolic stability and
good blood-brain concentration profile. Further, 42 was found to be active in vivo in a novel-object
recognition test in rats aimed at investigating procognitive properties. Interestingly, the obtained
compounds were markedly different from any ligands of 5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R and D2R
(stored within the ChEMBL database, release 24, Table 6). However, further in vivo studies are
needed to fully evaluate the hypothesis of beneficial dual activity at 5-HT2AR and 5-HT6R.
Table 6. Similarity between the obtained compounds and structures stored within ChEMBL (release
24). Generally, when the Tanimoto coefficient (Tc) between two structures is greater than 0.85, then
they are considered similar entities. The values presented below indicate a maximum and average Tc
achieved between structures stored within the ChEMBL database and the obtained compounds.
Receptor
Tc
5-HT6
0.66
0.39
5-HT2A
0.66
5-HT1A
0.70
5-HT7
0.66
0.36
D2
maximum
average
0.63
0.37
0.37
0.36
6. Experimental section
In silico drug design
2D Fingerprint-Based Screening
To maximize the screening performance and to obtain structurally diverse compounds, 2D
pharmacophore-based fingerprints and structural (chemical hashed) fingerprints were generated
based on the 58 reference ligands. Two-dimensional (2D) pharmacophore-based and structural
(chemical hashed) fingerprints were generated using the GenerateMD command line tool of JChem
[59] with default settings. The pharmacophore-based fingerprints were constructed from the 2D
molecular structures of the reference ligands by defining the collection of all-atom pharmacophore
feature pairs along with their topological distances. The chemical hashed fingerprints encoded the
topological structure of the ligands into bit strings using atom type and bond type information (linear
and cyclic patterns were detected). The metrics-threshold pair was determined by three optimization
iterations for each query, which were performed using test sets containing compounds from a given

group and randomly selected, assumed inactive compounds from the ZINC database.[60] The
optimization of parametrized Tanimoto or Euclidean metrics and subsequent 2D fingerprint-based
screening were performed using the ScreenMD command line tool of JChem.[61]
Basic Property and ADMET Filtering
First, for all 58 reference ligands, the strongest basic pKa descriptor was calculated using the Instant
JChem calculator plugin.[62] Next, the obtained range of this parameter (pKa =3 – 11.5) was used in
the filtering of compounds that passed the 2D fingerprint-based screening step. Compounds with
unfavourable ADMET profiles were then removed from the dataset using the Schrödinger software
module QikProp.[63] The following ADMET descriptor values were accepted: number of reactive
functional groups (rtvFG = 0−2), predicted aqueous solubility (QPlogS = −6.5 to 0.5 mol·l-1), model for
gut-blood barrier (QPPCaco > 500 nm·s-1), and predicted blood-brain coefficient (QPlogBB = −3.0 to
1.2).
3D Pharmacophore-Based Screening
Eight ligand-based pharmacophore models based on Kim [cite] were reconstructed using the Catalyst
module of Discovery Studio (Accelrys) [64], with the same parameters. The compounds were allowed
to deviate from the pharmacophore models with a maximum of one feature except the positively
ionizable (PI) group. Following the 3D pharmacophore-based screening, overlap analysis was
performed using the Instant JChem tool from ChemAxon[65] to remove repeated structures.
Flexible Docking
The construction of the 5-HT6R homology models has been described in detail elsewhere.[66] The 3-
dimensional structures of the ligands were prepared using LigPrep v3.6 [67], and the appropriate
ionization states at pH = 7.4±1 were assigned using Epik v3.4. Protein Preparation Wizard was used to
assign bond orders, appropriate amino acid ionization states and to check for steric clashes. The
receptor grid was generated (OPLS_2005 force field) by centring the grid box with a size of 12 Å on
D3.32. Automated docking was performed using Glide v7.0 at the SP level with the flexible docking
option turned on [68].
Virtual screening of combinatorial library enumeration
The combinatorial library of 118 compounds was enumerated from an in-house stock of 59 boronic
acids and 2 cores (4-methoxytryptamine and 5-methoxytryptamine) by using CombiGlide software
[69]. The appropriate ionization states generated at pH=7.4 for all the structures used in the
screening procedures were assigned using Epik [67]. The 3D structures were generated in LigPrep by
using default settings (force field of OPLS2005, retention of specified chiralities and generation of
only one low-energy ring conformation per ligand) [70]. The following criteria were used to filter
compounds with unfavourable profiles: number of reactive functional groups (desirable range of
values: 0–2), logarithm of calculated aqueous solubility (-6.5–0.5 mol·l-1), gut-blood barrier (>500
nm·s-1) and blood–brain barrier penetration coefficient (-3.0–1.2). Homology models were generated
as described previously [71]. The model was centred at D3.32 with the grid box size set to 25x25x25
Å. Docking runs were performed in Glide software [68] at the XP level by using default settings
(sampling nitrogen inversion, sampling ring conformations with an energy window equal to 2.5
kcal·mol-1, penalizing nonplanar conformation of amides up to 100 steps during energy minimization
and post-docking optimization).

The synthesized compounds were compared to all compounds fetched from the ChEMBL database
(version 24) [72] with determined activity against 5-HT1AR, 5-HT2AR, 5-HT6R, 5-HT7R or D2R by applying
the IJChem Chemistry Tool [65].
Induced fit docking protocol
The 5-HT6R homology model was optimized using the induced-fit docking (IFD) protocol from
Schrödinger.[73] This method combines flexible ligand docking using the Glide algorithm with
receptor structure prediction and side chain refinement in Prime. The structures of compounds 28
and 33 were used as inputs for IFD. The Extended Sampling Protocol was used, which generates up to
80 poses per ligand by automated docking. In each case, the centroid of the grid box was fixed on
D3.32 and allowed residue refinement within 12 Å of the ligand poses. All obtained L–R complexes
were inspected visually to select those that displayed the closest compliance with the common
binding mode for monoaminergic receptor ligands.[74] Final validation of the selected receptor
conformations was performed by docking (Glide SP mode) the synthetized library, retaining only
those with a coherent binding mode.
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,[75] and the results
are expressed as the means of at least two independent experiments.
5-HT6R functional activity assay
HEK293 cells overexpressing 5-HT6 receptors (prepared using Lipofectamine 2000) were maintained
at 37°C in a humidified atmosphere with 5% CO2 and grown in Dulbecco’s Modified Eagle’s Medium
containing 10% dialyzed foetal bovine serum and 500 mg/ml G418 sulfate. For the functional
experiments, the cells were subcultured in 25-cm2 diameter dishes, grown to 90% confluence,
washed twice with phosphate-buffered saline (PBS) prewarmed to 37°C and centrifuged for 5 min

(160 × g). The supernatant was aspirated, and the cell pellet was resuspended in stimulation buffer (1
× HBSS, 5 mM HEPES, 0.5 mM IBMX, 0.1% BSA). The functional properties of compounds were
evaluated using their ability to inhibit cAMP production induced by agonist 5-CT – 100 nM. Total
cAMP was measured using the LANCE cAMP detection kit (PerkinElmer) according to the
manufacturer’s directions. To quantify the levels of cAMP, the cells (5 µl) were incubated with the
test compounds and 5-CT (5 µl) for 30 min at room temperature in a 384-well white opaque
microtiter plate. After incubation, the reaction was stopped, and the cells were lysed by addition of
10 µl of a working solution (5 µl of Eu-cAMP and 5 µl of ULight-anti-cAMP). The assay plate was
incubated for 1 h at room temperature. Time-resolved fluorescence resonance energy transfer (TR-
FRET) was detected using an Infinite M1000 Pro (Tecan) with instrument settings according to the
LANCE cAMP detection kit manual. Kb values were calculated using the Cheng-Prusoff
equation[75] specific for the analysis of functional inhibition curves: Kb = IC50/(1 + A/EC50), where A is
the agonist concentration, IC50 is the concentration of antagonist that produces a 50% reduction in
response to agonist, and EC50 is the agonist concentration that causes a half maximal response.
Metabolic stability
The tested compounds were incubated in triplicate with a rat liver microsomal fraction at 37°C in the
presence of metabolic phase 1 cofactors (NADP, G6P, G6P dehydrogenase, MgCl2) necessary for the
metabolic transformations. The concentration of a non-metabolized test compound in the reaction
mixture was measured using HPLC-LC/MS at 4 incubation time points: 0, 20, 40 and 60 min. The AUC
of the compounds at those time points was compared with the AUC at time point 0 to determine the
% loss of parental compound. The obtained data were used to calculate the internal clearance
(Cl(int)) and half-life (T½). The metabolic activity of the microsomes was assessed by measuring the
respective stabilities of two standards of low and high metabolic stability, verapamil and donepezil.
Cytotoxicity
The human hepatocellular carcinoma cells (HepG2) were cultured using standard procedures
(protocol from the ATCC). Briefly, the cells were cultured in Dulbecco’s Modified Eagle’s Medium –
high glucose, (DMEM, Life Technologies, 5791 Van Allen Way, 92008 Carlsbad, USA) supplemented
with 10% foetal bovine serum (Life Technologies), 100 IU/ml penicillin (Sigma Aldrich) and 100 μg/ml
streptomycin (Sigma Aldrich), and incubated at 37◦C, 5% CO2. To test the compounds with the
HepG2 cell line, hepatocytes were seeded in a 96-well culture plate (Falcon) at a density of 2x104
cells per well in fresh medium. The cells were grown for 24 h in an incubator (37°C, 5% CO2) before
performing the experiments. After addition of the test compounds, the cells were incubated for an
additional 24 h. Four concentrations of test compounds were tested: 1 µM, 10 µM, 50 µM and 100
µM. PrestoBlue reagent (Invitrogen) to assess cell viability and ToxiLight bioassay (Lonza) to
determine cell membrane damage were used according to the manufacturer’s protocol. The
fluorescence and luminescence signals were measured by using a POLARstar Omega plate reader
(BMG Labtech).
CYP inhibition
The luminescent CYP3A4 P450-Glo™ and CYP2D6 P450-Glo™ assays and protocols were provided by
Promega (Madison, WI, USA). The stock solutions (10 mM) of ketoconazole (KE), quinidine (QD) and
the examined compounds were generated in DMSO. The 4X concentrated dilutions were prepared
before the assays (0.04-100 μM). The enzymatic reactions were conducted in white polystyrene, flat-
bottomed Nunc™ MicroWell™ 96-Well Microplates (Thermo Scientific, Waltham, MA USA). The CYPs,

proluciferin and examined compounds (25 μl/well) were first preincubated for 5 min, followed by
addition of the NADPH Regeneration System (25 μl) to start the reaction. The final concentrations of
KE and the examined compounds ranged from 0.01 μM to 25 μM. The final concentrations of QD
ranged from 0.001 to 10 μM. The control reactions for measuring 100% CYP activity and minus-CYP
negative control reactions for measuring background luminescence were also prepared. The
microplates were incubated at room temperature for 30 min (CYP3A4) or 45 min (CYP2D6). Finally,
LDR was added (50 μl/well), and after 20 min of incubation at room temperature, the luminescence
signal was measured in luminescence mode by using a microplate reader (EnSpire). The signal
produced by CYPs without the presence of compound was considered 100% CYP activity. The IC50
values were calculated using GraphPad Prism 5 Software.
Mini AMES test
The Ames microplate fluctuation protocol (MPF) assay was performed with Salmonella typhimurium
strain TA100, enabling the detection of base-pair substitutions. The bacterial strain as well as
exposure and indicator medium were obtained from Xenometrix AG (Allschwil, Switzerland). The
mutagenic potential of the tested structures was evaluated by incubating the bacteria with the test
compound at a concentration of 1 µM and 10 µM for 90 min (37°C) in exposure medium containing a
limited amount of histidine. After addition of indicator medium, each well of the 24-well plate was
aliquoted into 48 wells of a 384-well plate.
The occurrence of reversion events to histidine prototrophy was observed as growth of bacteria in
the indicator medium without histidine after 72 h of incubation in 37°C. Bacterial growth in 384-well
plates was visualized by a colour change of the medium from violet to yellow due to addition of the
pH indicator dye. The absorbance was measured with a microplate reader (EnSpire) at 420 nm. The
reference mutagen NQNO (0.5 μM) served as a positive control in the experiments. The medium
control baseline (MCB) was calculated, as derived from the mean number of revertants in the
medium control plus one standard deviation. Data points with a fold increase ≥ 2.0 and binomial B-
value ≥ 0.99 served as mutagenic alerts.

Novel-Object-Recognition test
Procedures were based on the earlier studies of Popik et al. [54]. The experiments were conducted in
accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by
the Ethics Committee for Animal Experiments, Institute of Pharmacology.
Male Sprague–Dawley rats (Charles River, Germany) weighing ~250 g at arrival were housed in
standard laboratory cages under standard colony A/C controlled conditions: room temperature
21 ± 2°C, humidity (40–50%), 12-hr light/dark cycle (lights on: 06:00), with ad libitum access to food
and water. Rats were allowed to acclimatize for at least 7 days before the start of the experimental
procedure. During this week, the animals were handled at least 3 times. Behavioural testing was
conducted during the light phase of the light/dark cycle.
At least 1 h before the start of the experiment, the rats were transferred to the experimental room
for acclimation. They were tested in a dimly lit (25 lx) “open field” apparatus made of a dull grey
plastic (66 × 56 × 30 cm). After each measurement, the floor was cleaned and dried.
The procedure consisted of habituation to the arena (without any objects) for 5 min, 24 h before the
test, and the test session comprised two trials separated by an intertrial interval (ITI). For the
phencyclidine (PCP)-induced memory impairment paradigm, a 1-hour ITI was chosen. During the first
trial (familiarization, T1), two identical objects (A1 and A2) were presented in opposite corners,
approximately 10 cm from the walls of the open field. In the second trial (recognition, T2), one of the
objects was replaced with a novel one (A=familiar and B=novel). Both trials lasted 3 min, and the
animals were returned to their home cage after T1. The objects used were glass beakers filled with
gravel and plastic bottles filled with sand. The heights of the objects were comparable (~12 cm), and
the objects were sufficiently heavy that they could not be displaced by the animals. The sequence of
presentations and locations of the objects were randomly assigned to each rat. The animals explored
the objects by looking, licking, sniffing or touching the object while sniffing, but not when leaning
against, standing or sitting on the object. Any rat that spent less than 5 s exploring the two objects
within 3 min of T1 or T2 was eliminated from the study. The exploration time of the objects and
distance travelled were measured using the Any-maze® video tracking system. Based on the
exploration time (E) of the two objects during T2, the discrimination index (DI) was calculated
according to the following formula: DI = (EB–EA)/(EA+AB).
Phencyclidine, used to attenuate learning, was administered at a dose of 5 mg/kg (i.p.) 45 before the
familiarization phase (T1). Compounds 44 and idalopirdine were administered p.o. 120 min before
the familiarization phase (T1).
Phencyclidine (Sigma-Aldrich, Germany) or idalopirdine (provided by Celon Pharma, Poland) were
diluted in distilled water.
There were no significant differences in the time spent exploring two identical objects in the
familiarization phase in any group (data not shown).
In the recognition trial, vehicle-treated, but not phencyclidine (PCP)-treated, rats spent significantly
more time exploring the novel object compared with the familiar one. Thus, the administration of
PCP (5 mg/kg) abolished the ability to discriminate novel and familiar objects. This deficit was
reduced by administration of 44 (3 and 10 mg/kg) and idalopirdine (1 and 3 mg/kg) compounds.

Because the analyses of exploration time during the recognition trial and DI yielded the same results,
only DI data are presented. DI (discrimination index) data were analysed by one-way ANOVA. Post-
hoc analyses (Newman–Keul’s test) revealed that 44 (3 and 10 mg/kg) and 44 (1 and 3 mg/kg)
significantly attenuated the PCP-induced DI reduction.
Synthesis
Unless otherwise stated, the starting materials, reagents, and solvents were purchased as
high-grade commercial products from Avantor, Sigma-Aldrich, Apollo Scientific, Combi-Blocks or
Fluorochem. Analytical thin-layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel
60 F-254) with detection by UV light (λ = 254 nm). Column chromatography was performed on a silica
gel (Kiesegel 60, 0.060 – 0.200 mm). All compounds were obtained as oils or fumarates. NMR spectra
13
were recorded on a Bruker Avance III HD 400 NMR (1H, 400 MHz; C, 100 MHz) instrument at room
temperature. For all final compounds, purity was determined by high-performance liquid
chromatography (HPLC) coupled to mass spectrometry (MS) using a Waters TQD instrument, and
satisfactory chromatograms confirmed a purity of at least 95% for all tested compounds. LC
separation was achieved with a Waters H-Class UPLC using an ACQUITY UPLC BEH C18 1,7 μm 2.1x50
mm column with a solvent gradient starting with 80% water:20% ACN up to 100% ACN for 3 min,
followed by 100% ACN for 1 min and back to 80% water:20% ACN through 2 min, with the solvent
flow set at 0.3 ml/min. MS analysis was performed with an ESI source. Spectra were acquired in
positive ionization mode from 100 to 600 m/z and in UV mode in the wavelength range from 190 −
300 nm.
General procedure for coupling of aldehyde intermediates:
A bromo-aldehyde (1 mmol), boronic acid (1.1-1.3 mmol), tetrakistriphenylphosphine (0.05 mmol),
anhydrous potassium carbonate (3 mmol), water (3 ml), ethanol (4 ml) and toluene (4 ml) were
added to a round-bottomed flask. The reaction mixture was flushed with argon, sealed under septa
and heated at 70°C overnight. After cooling to room temperature, water (50 ml) was added, and
product was extracted with ethyl acetate (3x50 ml). Combined extracts were washed with brine,
dried with anhydrous magnesium sulfate and evaporated under reduced pressure. The product was
purified by column chromatography on silica with chloroform or a mixture of methanol and
chloroform (9:1).
General procedure for reductive amination of tryptamine with an aldehyde
Tryptamine (0.0015 mol) and aldehyde (0.001 mol) were dissolved in MeOH (3 ml) together with a
few pellets of molecular-sieve 3A. The reaction mixture was stirred overnight at room temperature,
followed by the addition of NaBH4 (0.002 mol) and stirring of the reaction for 1 hour. Water (100 ml)
was added, and product was extracted with ethyl acetate (3x50 ml). The combined extracts were
washed with brine, dried over anhydrous magnesium sulfate and evaporated under reduced
pressure. The product was purified by column chromatography SiO2/CHCl3:MeOH (9:1).
Appendix. Supplementary data
Supplementary data associated with this article consist of a table of compounds selected in
the Virtual Screening, a table of structures generated through combinatorial library enumeration,
comparison of the binding mode of 28 (5-HT6R Ki = 3 nM, green) and 40 (5-HT6R Ki = 1 696 nM, blue),
a table of standard deviation parameters for the binding affinities of the synthesized compounds and
detailed spectral and synthetic data for all intermediates and final compounds.

Acknowledgements
The study was partially supported by the grant PBS3/B7/20/2015 from the Polish National Centre for
Research and Development, the grant ProKog UDA-POIG.01.03.01-12-063/09-00 from the European
Union—the European Regional Development Fund within the Operational Programme “Innovative
economy” for 2007–2013 and by the statutory fund of the Department of Medicinal Chemistry.
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Highlights:
1. Serotonin receptor ligands with a unique activity profile have been synthesized.
2. The selected hit showed a dual 5-HT6/5-HT2A receptor activity.
3. Synthesized compound showed procognitive properties in vivo.