Pyrroloquinoline scaffold-based 5-HT6R ligands: Synthesis, quantum chemical and molecular dynamic studies, and influence of nitrogen atom position in the scaffold on affinity

Article abstract of DOI:10.1016/j.bmc.2018.05.033

Based on pyrroloquinoline scaffold bearing 5-HT_2C agonists, a series of arylsulfonamide derivatives of 1H-pyrrolo[2,3-f]quinoline and 1H-pyrrolo[3,2-h]quinoline, substituted at position 3 with tetrahydropyridine, were synthesized and evaluated in vitro for their affinity for 5-HT₆ receptors. A structure–activity relationship study showed that the 1H-pyrrolo[3,2-h]quinoline scaffold was more favorable for 5-HT₆R binding than the 1H-pyrrolo[2,3-f]quinoline one, suggesting dependence upon the type of condensation of the pyrrole and quinoline rings. As revealed by quantum-chemical calculations and molecular dynamic studies, position of the quinoline nitrogen atom in the planar pyrroloquinoline skeleton might affect the spatial orientation of the arylsulfonyl fragment, as a result of structure stabilization by internal hydrogen bonds.

Full text of DOI:10.1016/j.bmc.2018.05.033

Accepted Manuscript
Pyrroloquinoline scaffold-based 5-HT R ligands: synthesis, quantum chemical
6
and molecular dynamic studies, and influence of nitrogen atom position in the
scaffold on affinity
Katarzyna Grychowska, Rafał Kurczab, Paweł Śliwa, Grzegorz Satała,
Krzysztof Dubiel, Mikołaj Matłoka, Rafał Moszczyński-Pętkowski, Jerzy
Pieczykolan, Andrzej J. Bojarski, Paweł Zajdel
PII:
S0968-0896(18)30641-2
https://doi.org/10.1016/j.bmc.2018.05.033
BMC 14375
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 March 2018
17 May 2018
22 May 2018
Please cite this article as: Grychowska, K., Kurczab, R., Śliwa, P., Satała, G., Dubiel, K., Matłoka, M., Moszczyński-
Pętkowski, R., Pieczykolan, J., Bojarski, A.J., Zajdel, P., Pyrroloquinoline scaffold-based 5-HT R ligands:
6
synthesis, quantum chemical and molecular dynamic studies, and influence of nitrogen atom position in the scaffold
on affinity, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.05.033
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Pyrroloquinoline scaffold-based 5-HT R ligands: synthesis, quantum
6
chemical and molecular dynamic studies, and influence of nitrogen
atom position in the scaffold on affinity
a
b
c
b
Katarzyna Grychowska, Rafał Kurczab, Paweł Śliwa, Grzegorz Satała,
d
d
d
d
Krzysztof Dubiel, Mikołaj Matłoka, Rafał Moszczyński-Pętkowski, Jerzy Pieczykolan,
b
a,
Andrzej J. Bojarski, Paweł Zajdel *
a
Department of Medicinal Chemistry, Jagiellonian University Medical College,
9
Medyczna Str. 30-688 Kraków, Poland
b
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of
Sciences, 12 Smętna Str. 31-343 Kraków, Poland
c
Faculty of Chemical Engineering and Technology, Cracow University of Technology,
4 Warszawska Str. 31-155 Kraków, Poland
2
d
Innovative Drugs Research & Development Department, Celon Pharma S.A., Mokra 41A,
Kiełpin, 05–092 Łomianki, Poland
*Corresponding author:
Paweł Zajdel
Department of Medicinal Chemistry
Jagiellonian University Medical College
E-mail: pawel.zajdel@uj.edu.pl
Tel.: +48 126205450; fax: +48 126205405
1

Abstract
Based on pyrroloquinoline scaffold bearing 5-HT2C agonists, a series of arylsulfonamide
derivatives of 1H-pyrrolo[2,3-f]quinoline and 1H-pyrrolo[3,2-h]quinoline, substituted at
position 3 with tetrahydropyridine, were synthesized and evaluated in vitro for their affinity
for 5-HT
6
receptors. A structure–activity relationship study showed that the 1H-pyrrolo[3,2-
R binding than the 1H-pyrrolo[2,3-
h]quinoline scaffold was more favorable for 5-HT
6
f]quinoline one, suggesting dependence upon the type of condensation of the pyrrole and
quinoline rings. As revealed by quantum-chemical calculations and molecular dynamic
studies, position of the quinoline nitrogen atom in the planar pyrroloquinoline skeleton might
affect the spatial orientation of the arylsulfonyl fragment, as a result of structure stabilization
by internal hydrogen bonds.
Keywords
1
H-pyrrolo[2,3-f]quinoline, 1H-pyrrolo[3,2-h]quinoline, 5-HT
6
receptor, arylsulfonyl-
pyrroloquinoline derivatives, quantum chemical calculations, molecular dynamic, halogen
bonding, hydrogen bonding
2

1
. Introduction
Pyrroloquinoline scaffolds, which have been found among many biologically active
compounds, constitute privileged structures in medicinal chemistry. Several natural 3H-
1
pyrrolo[2,3-c]quinolines displayed antimalarial activity, whereas 3H-pyrrolo[3,2-f]quinoline,
7
H-pyrrolo[2,3-h]quinoline and 3H-pyrrolo[2,3-f]quinoline derivatives were investigated as
2
–4
potential anticancer drugs (Figure 1). Some pyrroloquinolines were explored as templates
for CNS acting agents, particularly by modulating the function of serotonin (5-HT) through
5
,6
interaction with specific 5-HT receptors (5-HTRs).
Figure 1. Structures of 3H-pyrrolo[2,3-c]quinoline (I), 7H-pyrrolo[2,3-h]quinoline (II) and
H-pyrrolo[3,2-f]quinoline (III, IV) derivatives displaying antimalarial and anticancer
activity.
3
Recently, Adams et al. reported on the design and biological evaluation of 1H-
pyrrolo[2,3-f]quinoline, 1H-pyrrolo[3,2-h]quinoline, 1H-pyrrolo[2,3-f]isoquinoline and 1H-
5
pyrrolo[3,2-h]isoquinoline derivatives. These compounds displayed high-to-moderate
affinity for 5-HT2CR and showed moderate selectivity over 5-HT2A and 5-HT2B receptors. The
most potent compound V (VER-2692) behaved as an agonist of 5-HT2CR (K
EC50 = 2.9 nM), and showed anti-obesity properties, while causing reduced food intake
Figure 2). Pyrroloquinoline V and VI showed higher affinity for 5-HT2CR (K = 1.6 nM and
= 37 nM, respectively) than their pyrroloisoquinoline counterparts VII and VIII
i
= 1.6 nM,
(
i
K
i
(
K
i
= 72 nM and K
i
= 86 nM, respectively). Furthermore, among pyrroloquinoline derivatives,
3

compound V (1H-pyrrolo[2,3-f]quinoline derivative) showed higher affinity for 5-HT2CR than
compound VI (1H-pyrrolo[3,2-h]quinoline derivative). This suggested preference for the 1H-
pyrrolo[2,3-f]quinoline scaffold, with nitrogen atom outside the bay formed by the three
annelated rings, over its 1H-pyrrolo[3,2-h]quinoline congener containing nitrogen localized
inside the bay, for interaction with 5-HT2CR.
Figure 2. Pyrroloquinoline (V, VI) and pyrroloisoquinoline (VII, VIII) derivatives classified
as 5-HT2CR agonists.
Our previous work disclosed 1H-pyrrolo[3,2-c]quinoline as the central core for providing
5
-HT R modulators, among which compound IX (CPPQ ((S)-1-[(3-chlorophenyl)sulfonyl]-4-
6
(
pyrrolidine-3- yl-amino)-1H-pyrrolo[3,2-c]quinoline) behaved as a neutral antagonist of 5-
7
HT R (Figure 3).
6
Figure 3. Structure of CPPQ, a neutral 5-HT R antagonist.
6
The quest for specific modulators of 5-HT
6
R resulted because of association of 5-HT R
6
8
with diverse signaling pathways, involving adenylyl cyclase, extracellular signal-regulated
4

9
10
kinase (ERK)1/2, cyclin-dependent kinase (Cdk)5 and mammalian target of rapamycin
1
1
12
(
mTOR), and its engagement in cognition and neurogenesis processes. The unique
properties of 5-HT R also result from its exclusive localization in the central nervous system
and the fact that it displays high level of constitutive activity.
compounds with antagonist and inverse agonists properties for 5-HT
6
1
0,13
It was reported that,
6
R might improve
13–15
cognitive decline associated with neurodegenerative and genetic disorders.
Because the type of condensation of pyrrole and quinoline rings was a crucial feature in
determining the biological activity of the pyrroloquinolines V–VIII at 5-HT2CR, the primary
aim of the work was to investigate the effect of this phenomenon on 5-HT
small series of 1H-pyrrolo[2,3-f]quinoline and 1H-pyrrolo[3,2-h]quinoline derivatives (Figure
).
6
R binding using a
4
Figure 4. Strategy toward design of 1H-pyrrolo[2,3-f]quinolines and 1H-pyrrolo[3,2-
h]quinolines as 5-HT R ligands.
6
For this purpose the newly synthesized compounds 4–12 were evaluated for their affinity
for 5-HT R in the radioligand binding assays, and the influence of localization of nitrogen
6
atom in the quinoline system on biological activity was studied using quantum chemical
calculations and molecular dynamics.
5

2
. Chemistry
The designed compounds were obtained via a multistep procedure (Scheme 1).
-Nitroquinoline or 8-nitroquinoline reacted with 4-chlorophenoxyacetonitrile, in the presence
5
of potassium tert-butoxide in dimethylsulfoxide (DMSO) at –16°C yielding 2-(5-
nitroquinolin-6-yl)acetonitrile (1a) and 2-(8-nitroquinolin-7-yl)acetonitrile (1b), respectively.
This kind of reaction, namely vicarious nucleophilic substitution, has been extensively used
1
6
by Mąkosza, and it was previously described by Vlachou for the synthesis of 1H-
1
7
pyrrolo[2,3-f]quinoline and 1H-pyrrolo[3,2-h]quinoline scaffolds. Subsequently, compounds
a and 1b were hydrogenated using palladium on activated carbon in the presence of acetic
1
acid in ethanol/water (1/7, v/v) to give 1H-pyrrolo[2,3-f]quinoline 2a and 1H-pyrrolo[3,2-
h]quinoline 2b. These were then reacted with Boc-piperidone in sodium methanolate at 67°C
giving Boc-protected 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-f]quinoline 3a and 3-
(
1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-h]quinoline 3b.
Scheme 1. Reagents and conditions. (i) 4-Chlorophenoxyacetonitrile, t-BuOK, DMSO,
°
–
16 C, 2h, 80% (ii) Pd/C, H
2
, AcOH, EtOH/H O (1/7, v/v), RT, 48 h, 30%, (iii) 1-Boc-4-
2
piperidone, MeONa/MeOH, 67°C, 24 h, 65%, (iv) Aryl sulfonyl chloride, BTPP, DCM, 0°C
6

3
0 min or 3-chlorobenzyl bromide, BTPP, DCM, 30°C, 16 h, 70%-80%, (v) 1M HCl/MeOH,
RT, 16 h, 100%.
Coupling of pyrroloquinolines 3a and 3b with selected arylsulfonyl chlorides in the
presence of the strong phosphazene base P -t-Bu-tris(tetramethylene) (BTPP) at 0°C or with
-chlorobenzyl bromide at 30°C provided the respective arylsulfonyl and 3-chlorobenzyl
derivatives of Boc-protected 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-f]quinolines
1
3
18
(
4–7) and 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-h]quinolines (8–12). These
compounds, upon removal of the Boc-group in acidic conditions, yielded the final products 4–
1
2 as hydrochloride salts.
3
. Results and discussion
The strategy for designing 5-HT R ligands involved modifications of 1H-pyrrolo[2,3-
6
f]quinoline and 1H-pyrrolo[3,2-h]quinoline scaffolds with rationally selected substituents. 3-
Aminopyrrolidine, present in CPPQ, was replaced with 4-(1,2,3,6)-tetrahydropyridine, and the
latter amine moiety was shifted to the C3 position of pyrroloquinoline (Figure 4). Then, an
arylsulfonyl fragment was introduced on the nitrogen atom of pyrrole. Taking into account the
7
results obtained from the 1H-pyrrolo[3,2-c]quinoline series, as well as the impact of halogen
1
9
bonds on receptor affinity, the substitution pattern in the phenyl ring was limited to halogen
atoms in the meta position.
Our initial interest was focused on the way of fusion of the pyrrole and quinoline rings. 1H-
Pyrrolo[3,2-h]quinoline derivatives (8–12), with nitrogen atom in the quinoline system
directed into the bay formed by three annelated rings, displayed 2–8 fold higher affinity for 5-
HT
6
R than their 1H-pyrrolo[2,3-f]quinoline counterparts (4–7), with nitrogen atom directed
outside the bay (Table 1).
7

Table 1. Binding data of compounds 4–12 at the 5-HT
6
R.
K
i 5-HT6
Cmpd
Core structure
Z
R
a
[
nM] ± SEM
4
5
6
7
8
9
1H-pyrrolo[2,3-f]quinoline
1H-pyrrolo[2,3-f]quinoline
1H-pyrrolo[2,3-f]quinoline
1H-pyrrolo[2,3-f]quinoline
1H-pyrrolo[3,2-h]quinoline
1H-pyrrolo[3,2-h]quinoline
1H-pyrrolo[3,2-h]quinoline
1H-pyrrolo[3,2-h]quinoline
1H-pyrrolo[3,2-h]quinoline
Intepirdine (SB-742457)
-SO
-SO
-SO
-SO
-SO
-SO
-SO
-SO
2
2
2
2
2
2
2
2
-
-
-
-
-
-
-
-
-H
-F
711 ± 55
365 ± 23
427 ± 31
137 ± 5
131 ± 9
45 ± 3
-Cl
-Br
-H
-F
1
1
1
0
1
2
-Cl
-Br
-Cl
49 ± 4
61 ± 4
-CH
2
-
269 ± 11
1.4 ± 0.2
a
i
Mean K values, based on three independent binding experiments.
Replacement of the sulfonyl group with a methylene group significantly decreased the
affinity for 5-HT R (10 vs 12). Additionally, compound 12 (K = 269 nM) bearing 3-
6
i
chlorobenzyl fragment in position 1 displayed the lowest affinity in the 1H-pyrrolo[3,2-
h]quinoline series, confirming the importance of the sulfonyl fragment for the interaction with
5
-HT
6
R.
To further investigate the effect of the position of nitrogen in the pyrroloquinoline core
and the role of the sulfonyl group, representative ligands 6 (3-chlorophenylsulfonyl derivative
of 1H-pyrrolo[2,3-f]quinoline), 10 (3-chlorophenylsulfonyl derivative of 1H-pyrrolo[3,2-
h]quinoline) and 12 (3-chlorobenzyl derivative of 1H-pyrrolo[3,2-h]quinoline) were evaluated
using quantum methods. Full optimization at the density functional theory (DFT) level led to
the corresponding energy minimum a (Figure 5A and 5B). For sulfones (6, 10), the obtained
conformation is stabilized by intramolecular hydrogen bonds between oxygen atoms of the
8

SO group and hydrogen of the m-chlorophenyl moiety (H2 on Figure 5A) and hydrogen from
2
the indole ring (H1 on Figure 5A). This arrangement stabilized the conformation of the
chlorobenzene ring. The results of the potential energy scan through rotation of S–N bond
(
for 6 and 10) or C–N (for 12), indicated the two symmetrical conformations a and b being
almost in thermodynamic equilibrium in water. Sulfones have an additional less stable c
minimum (Figure 5B). The highest energy barrier occurred for the rotation from a to b
minima via the second transition state (TS2) for sulfones or TS1 for 12, associated with
unfavorable alignment of the pyrroloquinoline and chlorobenzene rings. Nevertheless, it
appears that in both the less active compounds i.e., 6 and 12 the arylsulfonyl fragment could
relatively freely rotate in the opposite direction through either TS3 and TS1 for 6, or only TS2
for 12. The largest rotational limitation was observed for the most active 1H-pyrrolo[3,2-
h]quinoline derivative 10. This might be the result of the presence of the sulfone group and
the pyrroloquinoline moiety in the h conformation in its structure.
9

Figure 5. (A) Structures of representative ligands 6 (3-chlorophenylsulfonyl derivative of 1H-
pyrrolo[2,3-f]quinoline), 10 (3-chlorophenylsulfonyl derivative of 1H-pyrrolo[3,2-
h]quinoline) and 12 (3-chlorobenzyl derivative of 1H-pyrrolo[3,2-h]quinoline) optimized
using DFT method in water. (B) A relaxed potential energy surface scan of rotation of bond
N1–S (6, 10) or N1–C2 (12). The X-axis denotes the dihedral C1–N1–S–C2 (6, 10) or C1–
N1–C2–C3 (12) and values range ±180 degree from optimal as in conformation a.
10

In the next step, the molecular mechanism of action of the synthesized compounds in
their binding with the 5-HT R was studied using the molecular docking method. The binding
6
mode was coherent (Figure 6), i.e., the protonated tetrahydropyridine moiety created a salt
bridge with D3.32, the pyrroloquinoline ring formed CH–π interaction with F6.52/F6.51, the
sulfonamide group formed hydrogen bond with N6.55 and the terminal substituted phenyl
ring expanded into a hydrophobic cavity between transmembrane domains (TMs) 3, 4, 5, and
the extracellular loop 2 (ECL2).
Figure 6. A comparison of the binding mode of compound 10 (A, green) and 6 (B, cyan) with
its most populated conformation obtained on the clustering of the MD trajectory (yellow and
pink for compound 10 and 6, respectively). For each binding pose of 10 and 6, the changes of
C1–N1–S–C2 dihedral through the 100 ns long MD simulations are shown.
Unfortunately, the analysis of the binding modes did not explain the change of the
affinity upon the position of nitrogen atom in the pyrroloquinoline ring. Therefore, a series of
1
00 ns molecular dynamic (MD) simulations was performed for complexes of compounds 6
and 10 (selected via the molecular docking analysis) with 5-HT R. The MD results showed
6
11

that the conformation of compound 10 through the whole simulation time remained almost
unchanged (Figure 6A); the value of the C1-N1-S-C2 dihedral angle during the simulation
varied slightly around the initial value of 74º, which corresponds to the conformation of
compound 10 in minimum a (Figure 5). However, the complex of compound 6 with 5-HT R
6
during the MD simulations was unstable. After the initial 10–15 ns the C1–N1–S–C2 dihedral
angle drastically changed, and at the end of the simulation it shifted by ±175º (Figure 6B),
which in this case corresponds to the higher energy minimum c (Figure 5). These findings are
in line with the potential energy surface scan, and showed that due to a lower energy barrier
between the most stable conformation a or b and less stable c, compound 6 can pass the
barrier significantly easier than 10, which is stabilized by stronger intermolecular hydrogen
bond (Figure 5).
Next, the role of a halogen substituent on the affinity for 5-HT
Unsubstituted arylsulfonyl pyrroloquinoline derivatives 4 and 8 showed the lowest affinity for
-HT R within 1H-pyrrolo[2,3-f]quinolines and 1H-pyrrolo[3,2-h]quinolines, respectively.
Introduction of a halogen atom increased affinity for 5-HT R by 1.5–5 fold in both series of
6
R was evaluated.
5
6
6
compounds. The analysis of binding modes suggested that the halogen substituent forms two
halogen bonding contacts, i.e., with carbonyl oxygen of A4.56 and of S4.57; however,
measured geometric parameters (for S5.47: d(Cl···O) = 3.3 Å, sigma hole angle (C–Cl···O) =
1
69°, and for A4.56:d(Cl···O) = 3.4 Å, sigma hole angle (C–Cl···O) = 124°) indicated the
dominant role of S5.47 in the formation of halogen bonding.
This observation was also supported by MD simulations showing that compounds 10 and
6
adopted more often appropriate geometrical parameters for halogen bonding with S5.47
than A5.46 (Figure 7). In the 1H-pyrrolo[2,3-f]quinoline set (4–7), a bromine was the most
favorable modification among all halogens. On the other hand, among the 1H-pyrrolo[3,2-
h]quinoline series (8–11) introduction of fluorine was the most beneficial.
12

Figure 7. The halogen bonding distance–sigma hole angle dependencies measured for A4.56
and S4.57 during the 100 ns MD simulations performed for complexes of compound 6 and 10
with 5-HT R.
6
4
. Conclusions
In summary, 1H-pyrrolo[2,3-f]quinoline- and 1H-pyrrolo[3,2-h]quinoline-based
compounds were, for the first time, designed and evaluated for affinity at 5-HT R. SAR
studies demonstrated that arylsulfonyl 1H-pyrrolo[3,2-h]quinolines displayed 2–8 fold higher
affinity for 5-HT R than their 1H-pyrrolo[2,3-f]quinoline congeners, demonstrating that the
type of condensation of pyrrole and quinoline rings and the respective geometry of
pyrroloquinoline scaffold is a crucial feature in determining 5-HT R affinity. Quantum-
6
6
6
chemical calculations and molecular dynamic studies revealed that this observation was
explained by the stabilization of the structure by a network of internal hydrogen bonds, which
impact the spatial orientation of the arylsulfonyl fragment. Furthermore, the sulfonyl group
was required for the interaction with the receptor while substituents in the phenyl ring
additionally increased 5-HT R affinity via halogen bonding.
6
13

5
5
5
. Experimental part
.1. Chemistry
.1.1.General methods
The synthesis was carried out at ambient temperature, unless indicated otherwise. Organic
solvents (from Aldrich and Chempur) were of reagent grade and were used without
purification. The reagents were purchased from Sigma-Aldrich and Fluorochem.
1
13
H NMR and C NMR spectra were obtained in a Varian BB 200 spectrometer using TMS
(
(
(
0.00 ppm) and were recorded at 300 MHz and 75 MHz respectively; J values are in hertz
Hz), and splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), m
multiplet).
UPLC/MS were carried out on a system consisting of a Waters Acquity UPLC, coupled to
a Waters TQD mass spectrometer. All the analyses were carried out using an Acquity UPLC
BEH C18, 100 × 2.1 mm column, at 40°C. A flow rate of 0.3 mL/min and a gradient of (0–
1
00)% B over 10 min was used. Eluent A: water/0.1% HCOOH; eluent B: acetonitrile/0.1%
HCOOH. Retention times t were given in minutes. The UPLC/MS purity of all the test
R
compounds and key intermediates was determined to be >99%.
High-resolution MS measurements were performed on a Bruker Impact II mass
spectrometer (Bruker Corporation, Billerica, USA). Electrospray ionization (ESI) was used in
the positive ion mode. Mass accuracy was within 2 ppm error in full-scan mode. The
optimized MS parameters were the following: ion spray voltage 4 kV; capillary temperature
2
40 ºC, dry gas flow rate 4 l/min. High-purity nitrogen as the nebulizing gas was used.
Samples of 50 µM concentration were prepared from tested compounds using an eluent of
ACN + H O (80:20) + 1% cc. HCOOH.
Melting points were determined with a Büchi apparatus and are uncorrected.
2
14

5
.1.2. Synthetic procedures
Procedure for preparation of compounds (1a) and (1b)
A mixture of 5-nitroquinoline or 8-nitroquinoline (15 g, 86 mmol, 1 eq) and 4-
chlorophenoxyacetonitrile (21 g, 129 mml, 1.5 eq) in 270 ml DMSO was added dropwise to a
solution of potassium tert-butoxide (19 g, 172 mmol, 2 eq) in 200 ml DMSO at -16°C. The
appearing red solution was stirred for 3 hours at room temperature. Subsequently, the mixture
was diluted with AcOEt and washed with 1M KHSO
organic phase was dried over Na SO and evaporated. The crude product was purified on
silica gel using AcOEt/Hex 4/6 (1a) and AcOEt/Hex 6/4 (1b) as developing solvent.
4
, three times with water and brine. The
2
4
2
-(5-nitroquinolin-6-yl)acetonitrile (1a)
1
Yellow solid, yield 80%, t
R
= 4.41, MW 213.20. H NMR (300 MHz, CDCl
3
) δ ppm 4.04 (s,
2
1
H), 7.61 – 7.67 (m, 1H), 7.94 (d, J = 8.72 Hz, 1H), 8.22 – 8.26 (m, 1H), 8.38 (d, J = 8.72 Hz,
+
H), 9.07 (dd, J = 4.23, 1.67 Hz, 1H). Monoisotopic mass 213.05, [M+H] = 214.0.
2
-(8-nitroquinolin-7-yl)acetonitrile (1b)
1
Yellow solid, yield 80%, t
R
= 4.59, MW 213.20. H NMR (300 MHz, CDCl
3
) δ ppm 3.99 (s,
2
H), 7.60 (dd, J = 8.34, 4.23 Hz, 1H), 7.79 (d, J = 8.72 Hz, 1H), 8.03 – 8.10 (m, 1H), 8.28
(
[
dd, J = 8.46, 1.54 Hz, 1H), 9.04 (dd, J = 4.23, 1.67 Hz, 1H). Monoisotopic mass 213.05,
+
M+H] = 214.0.
Procedure for preparation of compounds (2a) and (2b)
The respective nitroquinolinylacetonitrile derivative 1 (5 g, 23 mmol, 1 eq) was suspended
in a mixture of ethanol/water 1/7 (160 ml) and 10% Pd/C (70% weight) and glacial acetic acid
were subsequently added. The mixture as stirred under hydrogen atmosphere at room
temperature for 48 h. Then the mixture was filtered through Celite, which was rinsed with
methanol. The subsequent evaporation yielded a brown oil.
1
H-pyrrolo[2,3-f]quinoline (2a)
1
Brown oil, yield 30%, %, t = 3.02, MW 168.20. H NMR (300 MHz, DMSO-d ) δ ppm 6.64
R
6
(
dd, J = 2.95, 1.92 Hz, 1H), 7.49 – 7.60 (m, 3H), 7.91 (s, 1H), 8.73 – 8.78 (m, 2H), 12.22 (bs,
+
1
H). Monoisotopic mass 168.07, [M+H] = 169.2.
15

1
H-pyrrolo[3,2-h]quinoline (2b)
1
Brown oil, yield 30%, %, t
R
= 2.33, MW 168.20, H NMR (300 MHz, DMSO-d
6
) δ ppm
6
.60 – 6.68 (m, 4H), 6.63 (dd, J = 2.82, 2.05 Hz, 1H), 7.41 – 7.47 (m, 3H), 7.75 (d, J = 8.46
Hz, 1H), 8.33 (dd, J = 8.21, 1.54 Hz, 1H), 8.81 (dd, J = 4.36, 1.54 Hz, 1H), 12.27 (bs, 1 H).
+
Monoisotopic mass 168.07, [M+H] = 169.2.
Procedure for preparation of compounds (3a) and (3b)
Compounds 2a and 2b (1 g, 6 mmol, 1 eq) were suspended in sodium methanolate and 1-
Boc piperidone (2.5 g, 12.5 mmol, 2.5 eq) was added as a solid. The mixture was stirred at
6
7°C for 24 h. The solution was poured onto ice and the appearing yellow precipitate was
filtered and dried under vacuum. The crude product was subsequently purified on silica gel
using AcOEt/Hex as a developing solvent.
Tert-butyl
3a)
4-(1H-pyrrolo[2,3-f]quinolin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate
(
1
Yellow solid, yield 65%, t
R
= 6.65, MW 349.43, H NMR (300 MHz, CDCl
3
) δ ppm 1.48 –
1
.52 (m, 9H) 2.60 (d, J = 1.76 Hz, 2H) 3.70 (t, J = 5.57 Hz, 2H) 4.08 – 4.18 (m, 2H) 4.16 (d,
J = 2.93 Hz, 4H) 7.29 (d, J = 2.93 Hz, 1H) 7.40 (d, J = 4.10 Hz, 1H) 7.80 (d, J = 8.79 Hz, 1H)
8
.17 (d, J = 8.79 Hz, 1H) 8.46 (d, J = 8.21 Hz, 1H) 8.82 (dd, J = 4.40, 1.47 Hz,1H) 9.90 –
+
1
0.00 (m, 1H). Monoisotopic mass 389.1. [M+H] = 349.2
Tert-butyl
3b)
4-(1H-pyrrolo[3,2-h]quinolin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate
(
1
Yellow solid, yield 65%, t
R
= 6.65, MW 349.43, H NMR (300 MHz, CDCl
3
) δ ppm 1.51 (s,
9
H) 2.62 (bs, 2H) 3.70 (t, J = 5.86 Hz, 2H) 4.16 (bs, 2H) 6.15 – 6.22 (m, 1H) 7.49 (d,
J = 8.79Hz, 1H) 8.03 (d, J = 8.79 Hz, 1H) 8.27 (dd, J = 8.21, 1.76 Hz, 1H) 8.80 (dd, J = 4.40,
+
1
.47 Hz, 2H) 10.44 – 10.60 (m, 1H). Monoisotopic mass 389.1. [M+H] = 349.2.
General procedure for preparation of compounds 4–11
Compounds 3a and 3b were dissolved in DCM (100 mg, 0.29 mmol, 1 eq) and BTPP was
added (160 µl, 0.52 mmol, 1.8 eq). The mixture was placed in an ice-bath, arylsulfonyl
chloride (0.52 mmol, 1.8 eq) was added and the reaction mixture was stirred for 30 min.
Subsequently the mixture was evaporated and the remaining yellow crude product was
16

purified on silica gel using DCM/MeOH as a developing solvent. The obtained compounds
were treated with 1M HCl solution in methanol to give the final product as HCl salt.
1
-(Phenylsulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-f]quinoline
hydrochloride (4)
1
Yellow solid, yield 80%, t
R
= 7.94, MW 425.93, H NMR (300 MHz, Methanol-d ) δ ppm
4
2
2
9
.91 – 2.97 (m, 2H), 3.57 (s, 2H), 3.98 (bs, 2H), 6.30 – 6.38 (m, 1H), 7.50 (d, J = 8.21 Hz,
H), 7.59 – 7.72 (m, 2H), 7.81 – 7.89 (m, 2H), 8.14 – 8.26 (m, 3H), 8.37 (s, 1H), 8.63 (s, 1H),
+
.13 – 9.21 (m, 1H), 10.22 – 10.30 (m, 1H). Monoisotopic mass 389.1, [M+H] = 490.2.
HRMS calcd for C22
H
19
N
3
2
O S, 389.1198, found 390.1273.
1
-((3-Fluorophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-
f]quinoline hydrochloride (5)
Yellow solid, yield 70%, t = 3.96, MW 443.92, H NMR (300 MHz, DMSO-d
bs, 2H), 3.34 (bs, 2H), 6.34 (bs, 1H), 7.50 – 7.67 (m, 3H), 7.82 – 7.95 (m, 2H), 8.15 (d,
1
R
6
) δ ppm 2.81
(
J = 9.23 Hz, 1H), 8.25 (s, 1H), 8.38 (d, J = 9.23 Hz, 1H), 9.05 (d, J = 3.59 Hz, 1H), 9.44 (bs,
+
2
H), 9.54 (d, J = 8.72 Hz, 1H). Monoisotopic mass 407.1, [M+H] = 408.2; HRMS calcd for
C
22
H18FN
3
2
O S, 407.1104, found 408.1174.
1
-((3-Chlorophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-
f]quinoline hydrochloride (6)
Yellow solid, yield 70%, t = 4.32, MW 460.37, H NMR (300 MHz, Methanol-d
.88 – 2.97 (m, 2H), 3.54 – 3.61 (m, 2H), 3.95 – 3.99 (m, 2H), 6.31 – 6.40 (m, 1H), 7.41 –
1
R
4
) δ ppm
2
7
.50 (m, 1H), 7.60 – 7.79 (m, 3 H), 7.90 – 7.96 (m, 1H), 8.17 – 8.29 (m, 2H), 8.38 (s, 1H),
8
.62 – 8.70 (m, 1H), 9.17 – 9.22 (m, 1H), 10.22 – 10.30 (m, 1H). Monoisotopic mass 423.1,
+
[
M+H] = 424.1; HRMS calcd for C22
H18ClN
3
2
O S, 423.0808, found 424.0883.
1
-((3-Bromophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-
f]quinoline hydrochloride (7)
Yellow solid, yield 73%, t = 5.63, MW 504.83, H NMR (300 MHz, Methanol-d
.87 – 2.98 (m, 2H), 3.53 – 3.59 (m, 2H), 3.91 – 4.01 (m, 2H), 6.31 – 6.38 (m, 1H), 7.32 –
1
R
4
) δ ppm
2
7
.42 (m, 1H), 7.73 – 7.82 (m, 2H), 8.02 – 8.08 (m, 1H), 8.13 – 8.27 (m, 3H), 8.36 – 8.40 (m,
1
4
H), 8.61 – 8.67 (m, 1H), 9.14 – 9.22 (m, 1H), 10.18 – 10.27 (m, 1H). Monoisotopic mass
+
67.0, [M+H] = 468.1; HRMS calcd for C22
H18BrN
3
2
O S, 467.0303, found 468.0379.
17

1
-(Phenylsulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-h]quinoline
hydrochloride (8)
1
Yellow solid, yield 82%, t
R
= 9.14, MW 425.93, H NMR (300 MHz, Methanol-d ) δ ppm
4
2
.81 – 2.91 (m, 2H), 3.48 – 3.57 (m, 2H), 3.90 – 3.98 (m, 2H), 6.26 – 6.34 (m, 1H), 7.31 –
7
.57 (m, 3H), 7.60 – 7.68 (m, 1H), 7.88 – 7.97 (m, 2H), 8.03 – 8.11 (m, 1H), 8.13 – 8.22 (m,
1
3
H), 8.27 – 8.36 (m, 2H), 9.12 – 9.21 (m, 1H), 9.26 – 9.33 (m, 1H). Monoisotopic mass
+
89.1. [M+H] = 490.2; HRMS calcd for C22
H
19
N
3
O S, 389.1198; found 390.1273.
2
1
-((3-Fluorophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-
h]quinoline hydrochloride (9)
1
Yellow solid, yield 75%, t
R
= 4.99, MW 443.92, H NMR (300 MHz, DMSO-d
6
) δ ppm 2.85
(
bs, 2H), 3.35 (bs., 2H), 6.39 (bs, 1H), 7.48 (dd, J = 8.21, 4.36 Hz, 1H), 7.55 (dd, J = 8.72,
1
.54 Hz, 1H), 7.64 (m, 1H), 7.84 (d, J = 8.98 Hz, 1H), 7.99 (d, J = 8.21 Hz, 1H), 8.05 – 8.12
(
(
m, 2H), 8.22 (s, 1H), 8.40 (dd, J = 8.34, 1.67 Hz, 1H), 8.69 (dd, J = 4.23, 1.67 Hz, 1H), 9.42
+
bs, 2H). Monoisotopic mass 407.1, [M+H] = 408.2; HRMS calcd for C22
H18FN
3
2
O S,
4
07.1104, found 408.1181.
1
-((3-Chlorophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-
h]quinoline hydrochloride (10)
Yellow solid, yield 77%, t = 5.64, MW 460.37, H NMR (300 MHz, Methanol-d
.88 (bs, 2H), 3.53 – 3.58 (m, 2H), 3.96 (bs, 2H), 6.27 – 6.32 (m, 1H), 7.46 (s, 1H), 7.61 (d,
1
R
4
) δ ppm
2
J = 2.34 Hz, 1H), 7.70 – 7.75 (m, 2H), 7.83 – 7.92 (m, 2H), 8.01 – 8.06 (m, 2H), 8.20 (d,
J = 8.79 Hz, 1H), 8.29 (s, 1H), 8.91 – 8.96 (m, 1H), 9.13 (d, J = 3.52 Hz, 1H). Monoisotopic
+
mass 423.1, [M+H] = 424.1; HRMS calcd for C22
H18ClN
3
2
O S, 423.0808, found 424.0885.
1
-((3-Bromophenyl)sulfonyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-
h]quinoline hydrochloride (11)
Yellow solid, yield 73%, t
1
R
= 4.49, MW 504.83, H NMR (300 MHz, Methanol-d
4
) δ ppm
2
.90 – 3.01 (m, 2H), 3.52 – 3.59 (m, 2H), 3.93 – 4.00 (m, 2H), 6.34 – 6.41 (m, 1H), 7.41 –
7
.48 (m, 2H), 7.74 – 7.80 (m, 2H), 7.99 – 8.07 (m, 2H), 8.21 – 8.26 (m, 1H), 8.30 – 8.36 (m,
+
1
H), 8.37 – 8.41 (m, 1H), 8.67 – 8.74 (m, 1H). Monoisotopic mass 467.0, [M+H] = 468.0;
HRMS calcd for C22
H18BrN
3
2
O S, 467.0303, found 468.0379.
18

1
-(3-Chlorobenzyl)-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[3,2-h]quinoline
hydrochloride (12)
Compound 3b (100 mg, 0.29 mmol, 1 eq) was dissolved in DCM and BTPP was added (160
µl, 0.52 mmol, 1.8 eq). The solution was heated at 30 °C, and 3-chlorobenzyl bromide (0.52
mmol, 1.8 eq) was added dropwisely. The reaction mixture was stirred for 16 h. Subsequently
the mixture was evaporated and the remaining yellow crude product was purified on silica gel.
1
Yellow solid, yield 75%, t
R
= 4.78, MW 410.34, H NMR (300 MHz, Methanol-d
4
) δ ppm
2
1
8
.88 (bs, 2H), 3.53 – 3.58 (m, 2H), 3.96 (bs, 2H), 5.53 (s, 2H), 6.27 – 6.32 (m, 1H), 7.46 (s,
H), 7.61 (d, J = 2.34 Hz, 1H), 7.70 – 7.75 (m, 2H), 7.83 – 7.92 (m, 2H), 8.01 – 8.06 (m, 2H),
.20 (d, J = 8.79 Hz, 1H), 8.29 (s, 1H), 8.91 – 8.96 (m, 1H), 9.13 (d, J = 3.52 Hz, 1H).
+
Monoisotopic mass 373.13, [M+H] = 374.2; HRMS calcd for C23
H
20ClN , 373.1346, found
3
3
74.1416.
5
.2. Quantum-chemical calculation
To evaluate the effect of the nitrogen position in the pyrroloquinoline moiety and the role
of the sulfon bridge, we performed the quantum-chemical calculations for representative
2
0
ligands 6, 10 and 12 using methods implemented in Gaussian 09 software. The potential
energy surfaces (PES) scan of rotation appropriate bond (N–S or N–C) was performed using
relaxed approach with geometry optimization at each point and with the possibility of
2
1
symmetry breaking. Geometry optimization was performed at CAM-B3LYP/6-31G* level
2
2
with the polarizable continuum model (PCM) (solvent=water). The obtained extremes were
then reoptimized without any constrains and further confirmed by the absence or presence of
imaginary frequencies. For global minima, the electrostatic potential surfaces (ESP) were
calculated as a SCF density at the same level of theory. The preparation of the input file and
2
3
24
results analysis were done with GaussView 5. The figures were prepared using PYMOL.
.3. Molecular docking
The 3-dimensional structures of the ligands were prepared using LigPrep v3.6, and the
5
2
5
2
6
appropriate ionization states at pH=7.4±1.0 were assigned using Epik v3.4. Compounds
with unknown absolute configuration were docked in R and S configurations. One low energy
ring conformation per ligand was generated. The Protein Preparation Wizard was used to
assign the bond orders, appropriate amino acid ionization states and to check for steric
19

2
7
clashes. The receptor grid was generated (OPLS3 force field) by centering the grid box with
a size of 12 Å on Asp3.32 side chain. Automated flexible docking was performed using Glide
2
8
v6.9 at the SP level.
5
.4. Molecular Dynamic simulations
A 100 ns Molecular Dynamics (MD) simulations were performed using Schrödinger
2
9
Desmond software. Each ligand–protein complex was immersed into a POPC (300K)
membrane bilayer, which position was calculated using the PPM web server (accessed 15-08-
30
2
017). The system was solvated by water molecules described by the TIP4P potential and
the OPLS3 force field parameters were used for all atoms. 0.15 M NaCl was added to mimic
the ionic strength inside the cell. The output trajectories were hierarchically clustered into 10
clusters according to the ligand using trajectory analysis tools available in the Maestro
Schrödinger Suit.
5
.5. In vitro pharmacology
5.5.1. Cell culture and preparation of cell membranes for radioligand binding assays
HEK293 cells with stable expression of human 5-HT
6
receptors (prepared with the use of
and
Lipofectamine 2000) were maintained at 37°C in a humidified atmosphere with 5% CO
2
grown in Dulbecco’s Modifier Eagle Medium containing 10% dialyzed fetal bovine serum
2
and 500 µg/ml G418 sulfate. For membrane preparation, cells were subcultured in 150 cm
flasks, grown to 90% confluence, washed twice with prewarmed to 37°C phosphate buffered
saline (PBS) and pelleted by centrifugation (200 g) in PBS containing 0.1 mM EDTA and 1
mM dithiothreitol. Prior to membrane preparation, pellets were stored at -80°C.
5
.5.2. Radioligand binding assays
Cell pellets were thawed and homogenized in 10 volumes of assay buffer using an Ultra
Turrax tissue homogenizer and centrifuged twice at 35.000 g for 15 min at 4°C, with
incubation for 15 min at 37°C in between. The incubation buffer consisted of 50 mM Tris
HCl, 0.5 mM EDTA and 4 mM MgCl
2
. Each compound was tested in triplicate at 7
-10
-4
3
concentrations (10 –10 M) and 2 nM [ H]-LSD (PerkinElmer, USA) was used as
radioligand. Non-specific binding was defined with 10 µM of methiothepine. Assay was
20

incubated in a total volume of 200 µL in 96-well microtitre plate for 1 h at 37°C. The process
of equilibration was terminated by rapid filtration through Unifilter plate with a 96-well cell
harvester and radioactivity retained on the filter was quantified on a Microbeta plate reader
(
PerkinElmer, USA). The inhibition constants (K ) were calculated from the Cheng-Prusoff
i
3
1
equation. Results were expressed as means of at least two separate experiments.
Acknowledgements
The authors thank Dr Krzysztof Marciniec for performing HR MS analyses. This project was
supported from the grant no. PBS3/B7/20/2015 from the Polish National Centre for Research
and Development and the grant no. 2016/21/B/NZ7/01742 from the National Science Center,
Poland.
21

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24