Synthesis, antidepressant evaluation and docking studies of long-chain alkylnitroquipazines as serotonin transporter inhibitors

Article abstract of DOI:10.1111/cbdd.12116

Twelve alkyl analogues (1-12) of the high-affinity serotonin transporter (SERT) inhibitor 6-nitroquipazine (6-NQ) were synthesized and studied using in vitro radioligand competition binding assays to determine their binding affinity (K_i). The p

Full text of DOI:10.1111/cbdd.12116

Chem Biol Drug Des 2013; 81: 695–706
Research Article
Synthesis, Antidepressant Evaluation and Docking
Studies of Long-Chain Alkylnitroquipazines as
Serotonin Transporter Inhibitors
1
2
Mari Gabrielsen , Karol Wołosewicz , Anna
Key words: 6-nitroquipazine alkyl analogues, flexible docking,
NMR spectroscopy, Porsolt forced swim test, radioligand
competition assay, serotonin transporter
3
2
Zawadzka , Jerzy Kossakowski , Gabriel
Nowak , Małgorzata Wolak , Katarzyna
Stachowicz , Agata Siwek , Aina W. Ravna , Irina
4
,5
4
5
4
1
Received 12 July 2012, revised 19 December 2012 and
accepted for publication 15 January 2013
6
7,8
7
Kufareva , Lech Kozerski , El z_ bieta Bednarek ,
7
7
Jerzy Sitkowski , Wojciech Bocian , Ruben
6
5
1
Abagyan , Andrzej J. Bojarski , Ingebrigt Sylte *
and Zdzisław Chilmonczyk
7
The serotonin (5-hydroxytryptamine, 5-HT) transporter
(
SERT) is a putative twelve-transmembrane a-helical pro-
tein that belongs to the neurotransmitter/sodium symporter
NSS) transporter family (1). Located in the membrane of
presynaptic neurons, the transporter plays an important
role in the termination of serotonergic neurotransmission
by removing 5-HT from the synaptic cleft. In addition to
being targeted by psychostimulants such as cocaine and
amphetamines, SERT is targeted by the two main classes
of antidepressant drugs, the tricyclic antidepressants
1
Medical Pharmacology and Toxicology, Department of
Medical Biology, Faculty of Health Sciences, University of
Tromsø, 9037, Tromsø, Norway
Institute of Chemistry, University of Białystok, 1 Hurtowa
(
2
Street, 15-399, Białystok, Poland
Department of Medicinal Chemistry, Warsaw Medical
3
University, Oczki 3, 02-007, Warsaw, Poland
Department of Pharmacobiology, Jagiellonian University
4
Medical College, Medyczna 9, 30-688, Krak oꢀ w, Poland
Institute of Pharmacology, Polish Academy of Sciences,
5
(TCAs) and selective serotonin reuptake inhibitors (SSRIs),
1
2 Smetna Street, 31-343, Krak oꢀ w, Poland
Skaggs School of Pharmacy and Pharmaceutical
Sciences, 9500 Gilman Drive, MC 0747, La Jolla, CA,
z
6
which inhibit the transport of 5-HT into the presynaptic
neuron.
9
2093-0747, USA
7
National Medicines Institute, 30/34 Chełmska Street,
6-Nitroquipazine (6-NQ) is a highly potent and selective
SERT inhibitor with higher affinity for SERT than most
TCAs and SSRIs (2–4). Multiple studies have described
the synthesis and in vitro SERT binding affinities of 6-NQ
analogues (5–10), and several radiolabelled 6-NQ ana-
logues, as well as radiolabelled 6-NQ itself, have also been
developed for use as imaging agents (11–21). Further-
more, dual-acting 6-NQ analogues that inhibit SERT as
well as antagonise the presynaptic autoinhibitory 5-HT1A
receptors have been developed (22). These receptors are
activated by the increases in synaptic 5-HT and upon acti-
vation decrease the serotonergic neurotransmission, hence
causing a delay in the onset of antidepressant action (23).
This delay typically lasts 2–4 weeks until the presynaptic
autoinhibitory receptors have become desensitized and
the firing of the 5-HT neurons is normalized (23). Dual-act-
ing compounds that inhibit SERT and antagonise the 5-
HT1A autoinhibitory receptors may thus reduce the time of
onset of action of antidepressant drugs.
0
0-725, Warsaw, Poland
8
Institute of Organic Chemistry, Polish Academy of
Sciences, Kasprzaka 44, 01-224, Warsaw, Poland
Corresponding author: Ingebrigt Sylte, ingebrigt.sylte@uit.
no
*
Twelve alkyl analogues (1–12) of the high-affinity
serotonin transporter (SERT) inhibitor 6-nitroquip-
azine (6-NQ) were synthesized and studied using in
vitro radioligand competition binding assays to
i
determine their binding affinity (K ). The putative
antidepressant activity of five of the binders with
the highest SERT binding affinities was studied by
the forced swim and locomotor activity mouse tests.
The three-dimensional (3D) structures of 8 and 9
were determined using NOE NMR technique. Flexi-
ble docking of the compounds was undertaken to
illustrate the binding of the compounds in the SERT
model. Our results showed that several of the 6-NQ
analogues are high-affinity SERT inhibitors and indi-
cated that the octyl (8), decyl (10) and dodecyl (12)
In this study, twelve 6-NQ alkyl analogues (2-(4-alkyl-pip-
erazin-1-yl)-6-nitroquinolines, 1–12) were synthesized and
their affinities (K) to SERT and the 5-HT1A receptor were
i
6
-NQ analogues exhibit moderate antidepressant
activity.
determined. The three-dimensional (3D) structure of 8
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12116
695

Gabrielsen et al.
and 9 were determined by NMR spectroscopy (NOE
technique). To illustrate the interaction of the compounds
with SERT, a SERT homology model (24) based on
General preparation procedure of 6-nitro-4′-
alkylquipazine analogues
A 1.1 mol equivalent of sodium hydride (NaH) was added
to an ice-cooled (0 °C) solution of 6-nitroquipazine (0.2 g,
0.77 mmol) in anhydrous tetrahydrofurane (THF). After
30 min, 0.77 mmol of alkyl iodide (RI) was added, and the
mixture was heated under reflux for 70 h. The reaction
mixture was cooled to room temperature, and the excess
of sodium hydride was decomposed with a small amount
of water. Solvents were evaporated under reduced pres-
sure, and the crude products were purified with the aid of
column chromatography on silica gel with 0–4% AcOEt/
hexane (1–8) or 10–30% AcOEt/cyclohexane (9–12).
Figure 1 shows the general synthesis procedure of 1–12.
a
homologous bacterial leucine transporter (LeuT)
X-ray crystal structure (25) was used in flexible
a
docking study of the compounds. Furthermore, the alkyl
analogues with the highest SERT affinities were
screened in vivo using forced swim and locomotor activ-
ity tests.
Materials and Methods
Materials
All reagents were purchased from commercial sources:
iodides were acquired from Aldrich [St. Louis, MO, USA
2-(4-Methyl-piperazin-1-yl)-6-nitroquinoline (1)
(
methyl-octyl)] and from Alfa Aesar [Johnson Matthey
14 16 4 2
C H N O . M = 272.30 g/mol. Yield 72%. Rf: 0.44 (10%
1
Company, Karlsruhe, Germany (nonyl-dodecyl)]. 6-Nitro-
quipazine was obtained from 2-chloroquinoline (ABCR,
Karlsruhe, Germany), piperazine (Aldrich), nitric and sul-
phuric acids (POCh, Gliwice, Poland). NaH was purchased
from Lancaster Synthesis (Alfa Aesar; Johnson Matthey
Company). Solvents (except THF) were purchased from
POCh (Gliwice, Poland). All air and water-sensitive reac-
tions were carried out under argon. THF, purchased
MeOH/DCM); H NMR (200 MHz, CDCl
3
): d 8.53 (d, 1H,
2
= 9.3 Hz), 7.96
J = 2.5 Hz), 8.29 (dd, 1H, J = 2.7 Hz, J
1
(d, 1H, J = 8.6 Hz), 7.66 (d, 1H, J = 9.3), 7.06 (d, 1H,
J = 9.3), 3.87 (t, 4H, J = 5.3), 2.55 (t, 4H, J = 5.3), 2.37
1
3
(s, 3H); C NMR (50.3 MHz, CDCl
141.8, 138.5, 127.0, 124.2, 123.6, 121.0, 110.8, 60.5,
53.0, 44.3; IR (CHCl ): m = 2944.6, 2852.8, 1616.7,
1496.4, 1324.9, 1229.2/cm; anal. calcd. for C14
HCl ∙ 0.25 H O: C, 53.68; H, 5.63; N, 17.88; found: C,
53.80; H, 5.56; N, 17.65%.
3
): d 158.4, 151.5,
3
H N O ∙
16 4 2
ꢀ
from ChemPur (Piekary Slaskie, Poland), was dried
2
z
with sodium and distilled under argon from sodium
benzophenone ketyl. Thin-layer chromatography (TLC)
was performed on 0.2-mm Merck silica gel 60 F254
silica plates (Merck, Darmstadt, Germany), and com-
pounds were visualized under 245-nm ultraviolet irradia-
2
-(4-Ethyl-piperazin-1-yl)-6-nitroquinoline (2)
C H N O . M = 286.33 g/mol. Yield 87%. Rf: 0.52 (10%
1
5 18 4 2
1
MeOH/DCM); H NMR (200 MHz, CDCl
3
): d 8.53 (d, 1H,
tion
chromatography was performed using Baker gel 60
230e400 mesh) (Baker, Deventer, the Netherlands) with
the indicated solvents.
and/or
phosphomolybdic
acid.
Column
J = 2.54 Hz), 8.31 (dd, 1H, J = 2.7 Hz, J = 9.3 Hz),
1
2
7
1
.96 (d, 1H, J = 9.3 Hz), 7.65 (d, 1H, J = 9.3 Hz), 7.06 (d,
H, J = 9.3 Hz), 3.88 (t, 4H, J = 5.3 Hz), 2.59 (t, 4H,
(
J = 5.3 Hz), 2.52 (q, 2H, J = 7.3 Hz), 1.16 (t, 3H,
13
J = 7.3 Hz); C NMR (50.3 MHz, CDCl
3
): d 158.4, 151.5,
1
41.8, 138.5, 127.0, 124.2, 123.5, 120.9, 110.8, 58.7,
1
13
H NMR, C NMR, IR and elemental analysis data
53.0, 44.6, 29.7; IR (CHCl ): m = 2937.0, 2820.1, 1616.0,
1496.4, 1324.9, 1233.5/cm; anal. calcd. for C H N O ∙
HCl: C, 55.81; H, 5.93; N, 17.36; found: C, 55.87; H,
5.98; N, 17.10%.
3
1
13
The H and C NMR spectra were obtained on a Bruker
AVANCE DMX 400WB or at Bruker AVANCE DPX
15 18 4 2
2
00 MHz instrument (Bruker BioSpin, F a€ llanden, Switzer-
land) in CDCl with TMS as an internal reference. Chemi-
cal shifts were expressed in units, and coupling
3
O2N
d
NaH, THF
constants (J) were expressed in hertz (Hz). The following
abbreviations were used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), q (quartet), qt
+
RI
0 o
C
N
N
N
(
quintet), m (multiplet), p (pseudo-) and b (broad-). For the
6
-NQ
H
two-dimensional experiments, the pulse sequences,
acquisition and the processing parameters were taken
from the standard Bruker software library. Infrared spectra
O2N
(KBr) were recorded on a Shimadzu FTIR-8300 spectrom-
eter (Duisburg, Germany). Elemental analyses were per-
formed on a Perkin-Elmer 2400 analyser (Perkin-Elmer,
Norwalk, CT, USA) and were within ꢀ0.4% of the theoreti-
cal values. The infrared spectra were recorded on a Nico-
let Magna 550 IR spectrophotometer (Nicolet, Madison,
WI, USA).
1
R = CH3
-12 R = (CH2)n
n = 1–11
N
N
2
CH3
N
R
Figure 1: General synthesis scheme of 6-NQ aryl analogues 1–
12. RI, alkyl iodide; THF, tetrahydrofurane.
6
96
Chem Biol Drug Des 2013; 81: 695–706

Long Chain Alkylnitroquipazines as SERT Inhibitors
2
-(4-Propyl-piperazin-1-yl)-6-nitroquinoline (3)
HCl: C, 60.27; H, 7.19; N, 14.79; found: C, 60.27; H,
7.15; N, 14.81%.
16 20 4 2
C H N O
. M = 300.36 g/mol. Yield 72%. Rf: 0.53 (10%
1
MeOH/DCM); H NMR (200 MHz, CDCl
3
): d 8.53 (d, 1H,
J = 2.5 Hz), 8.29 (dd, 1H, J = 2.7 Hz, J = 9.3 Hz), 7.96
1
2
(d, 1H, J = 8.6 Hz), 7.66 (d, 1H, J = 9.3 Hz), 7.06 (d, 1H,
2-(4-Heptyl-piperazin-1-yl)-6-nitroquinoline (7)
J = 9.3 Hz), 3.87 (t, 4H, J = 5.3 Hz), 2.58 (t, 4H,
J = 5.3 Hz), 2.38 (t, 2H, J = 7.45 Hz), 1.63–1.52 (m, 2H),
C H N O . M = 356.46 g/mol. Yield 84%. Rf: 0.48 (10%
20 28 4 2
1
MeOH/DCM); H NMR (200 MHz, CDCl ): d 8.51 (d, 1H,
2
J = 2.6 Hz), 8.28 (dd, 1H, J = 2.6 Hz, J = 9.3 Hz), 7.94
3
13
0
1
1
2
.95 (t, 3H, J = 7,31 Hz); C NMR (50.3 MHz, CDCl
58.4, 151.5, 141.8, 138.5, 127.0, 124.2, 123.6, 121.0,
10.8, 60.5, 53.0, 44.6,19.9, 11.8; IR (CHCl ): m = 2930.1,
856.3, 1616.0, 1496.9, 1324.6, 1232.7/cm; anal. calcd.
∙ 2HCl ∙ 0.5 H O: C, 50.27; H, 6.06; N,
4.66; found: C, 50.32; H, 6.34; N, 14.63%.
3
): d
1
(d, 1H, J = 9.3 Hz), 7.64 (d, 1H, J = 9.3 Hz), 7.05 (d, 1H,
J = 9.3 Hz), 3.86 (t, 4H, J = 5.2 Hz), 2.58 (t, 4H,
J = 5.2 Hz), 2.40 (t, 2H, J = 7.3 Hz), 1.58–1.50 (m, 2H),
3
13
for C16
H
20
N
4
O
2
2
1.40–1.20 (m, 8H), 0.89 (t, 3H, J = 6.7 Hz); C NMR
1
(50.3 MHz, CDCl ): d 158.3, 151.4, 141.6, 138.4, 126.9,
3
1
2
24.1, 123.4, 120.9, 110.7, 58.6, 53.0, 44.6, 31.7, 29.1,
7.4, 26.7, 22.5, 14.0; IR (CHCl ): m = 2931.1, 2857.4,
3
2
C
-(4-Butyl-piperazin-1-yl)-6-nitroquinoline (4)
1616.2, 1496.9, 1324.4, 1233.0/cm; anal. calcd. for
20 28 4 2
C H N O ∙ HCl: C, 61.14; H, 7.44; N, 14.26; found: C,
61.22; H, 7.50; N, 14.32%.
17
H
22
N
4
O
2
. M = 314.17 g/mol. Yield 69%. Rf: 0.41 (10%
1
MeOH/DCM); H NMR (200 MHz, CDCl ): d 8.52 (d, 1H,
3
J = 2.6 Hz), 8.29 (dd, 1H, J
1 2
= 3,0 Hz, J = 9.3 Hz), 7.95
(d, 1H, J = 9.3 Hz), 7.64 (d, 1H, J = 9.3 Hz), 7.06 (d, 1H,
J = 9.3 Hz), 3.87 (t, 4H, J = 5.0 Hz), 2.58 (t, 4H,
J = 5.0 Hz), 2.41 (t, 2H, J = 5.5 Hz), 1.54–1.3 (m, 4H),
2-(4-Octyl-piperazin-1-yl)-6-nitroquinoline (8)
21 30 4 2
C H N O . M = 370.49 g/mol. Yield 82%. Rf: 0.42 (10%
13
1
0
1
1
.95 (t, 3H, J = 7.05 Hz); C NMR (50.3 MHz, CDCl ): d
58.4, 151.5, 141.8, 138.5, 127.0, 124.2, 123.4, 121.0,
10.8, 58.4, 53.0, 44.7, 28.9, 20.7, 14.0; IR (CHCl3):
MeOH/DCM); H NMR (200 MHz, CDCl ): d 8.52 (d, 1H,
3
3
1 2
J = 2.6 Hz), 8.29 (dd, 1H, J = 2.6 Hz, J = 9.3 Hz), 7.95
(d, 1H, J = 9.3 Hz), 7.65 (d, 1H, J = 9.3 Hz), 7.06 (d, 1H,
J = 9.3 Hz), 3.87 (t, 4H, J = 5.0 Hz), 2.58 (t, 4H,
J = 5.0 Hz), 2.40(t, 2H, J = 7.3 Hz), 1.58–1.50 (m, 2H),
m = 2930.1, 2856.3, 1616.0, 1496.9, 1324.6, 1232.7/cm;
anal. calcd. for C H N O ∙ HCl: C, 58.20; H, 6.61; N,
17 22 4 2
13
15.97; found: C, 57.99; H, 6.78; N, 15.68%.
1.40–1.20 (m, 10H), 0.89 (t, 3H, J = 6.3 Hz); C NMR
(50.3 MHz, CDCl ):158.3, 151.4, 141.6, 138.4, 126.9,
24.1, 123.4, 120.8, 110.7, 58.6, 53.0, 44.6, 31.7, 29.4,
29.1, 27.4, 26.7, 22.5, 14.0; IR (CHCl ): m = 2930.1,
2856.3, 1616.0, 1496.9, 1324.6, 1232.7/cm; anal. calcd.
for C21 ∙ 2HCl: C, 56.88; H, 7.27; N, 12.64;
d
c
3
1
2-(4-Pentyl-piperazin-1-yl)-6-nitroquinoline (5)
3
C H N O . M = 328.19 g/mol. Yield 79%. Rf: 0.57 (10%
1
8 24 4 2
1
MeOH/DCM); H NMR (200 MHz, CDCl
J = 2.6 Hz), 8.27 (dd, 1H, J = 2.6 Hz, J
d, 1H, J = 9.3 Hz), 7.63 (d, 1H, J = 9.3 Hz), 7.04 (d, 1H,
3
): d 8.49 (d, 1H,
30 4 2
H N O
1
2
= 9.3 Hz), 7.93
found: C, 57.03; H, 7.19; N, 12.08%.
(
J = 9.3 Hz), 3.86 (t, 4H, J = 5.2 Hz), 2.57 (t, 4H,
J = 5.2 Hz), 2.39 (t, 2H, J = 7.3 Hz), 1.65–1.50 (m, 2H),
2-(4-Nonyl-piperazin-1-yl)-6-nitroquinoline (9)
1
3
1.40–1.25 (m, 4H), 0.91 (t, 3H, J = 6.7 Hz); C NMR
22 32 4 2
C H N O . M = 384.51 g/mol. Yield 63%. M.p.: 79–
1
(
50.3 MHz, CDCl ): d 158.4, 151.5, 141.8, 138.5, 127.0,
81 °C. H NMR (400 MHz, CDCl ): d 8.51 (s, 1H, Harom),
3
3
1
2
1
24.2, 123.5, 120.9, 110.8, 58.7, 53.0, 44.6, 29.7, 26.48,
2.6, 14.0; IR (CHCl ): m = 2933.6, 2861.1, 1616.2,
496.9, 1324.6, 1233.0/cm; anal. calcd. for C18
8.28 (d, 1H, J = 8.5 Hz, Harom), 7.95 (d, 1H, J = 9.0 Hz,
3
H
arom), 7.64 (d, 1H, J = 9.0 Hz, Harom), 7.06 (d, 1H,
J = 9.0 Hz, Harom), 3.90 (s, 4H, N(CH (CH NC 19),
2
2.63 (s, 4H, N(CH (CH NC 19), 2.44 (s, 2H, CH ),
H
25
N
4
O
2
∙
2
)
2
2
)
2
9
H
HCl: C, 59.25; H, 6.91; N, 15.36; found: C, 59.42; H,
.05; N, 15.34%.
2
)
2
2
)
2
9
H
7
1.57 (s, 2H, CH ), 1.28 (m, 12H, 6 9 CH ), 0.88 (s, 3H,
2
2
13
CH
3
). C NMR (100 MHz, CDCl
3
): d 158.58, 151.70,
1
42.09, 138.89, 127.33, 124.47, 123.85, 121.26, 111.04,
2
-(4-Hexyl-piperazin-1-yl)-6-nitroquinoline (6)
58.90, 53.17, 44.69, 32.07, 29.73, 29.62, 29.47, 27.70,
26.80, 22.87, 14.32; anal. calcd. for C22H32N4O2: C,
68.72; H, 8.39; N, 14.57%; found C, 68.36; H, 8.15; N,
19 26 4 2
C H N O
. M = 342.44 g/mol. Yield 85%. Rf: 0.55 (10%
1
MeOH/DCM); H NMR (200 MHz, CDCl
J = 2.6 Hz), 8.29 (dd, 1H, J = 2.6 Hz, J
d, 1H, J = 9.3 Hz), 7.65 (d, 1H, J = 9.3 Hz), 7.06 (d, 1H,
3
): d 8.53 (d, 1H,
+
1
2
= 9.3 Hz), 7.96
13.92%. ESI-MS: m/z [%]: 385.2 [M + H] 100.
(
J = 9.3 Hz), 3.87 (t, 4H, J = 5.0 Hz), 2.58 (t, 4H,
J = 5.0 Hz), 2.40 (t, 2H, J = 7.2 Hz), 1.60–1.50 (m, 2H),
2-(4-Decyl-piperazin-1-yl)-6-nitroquinoline (10)
1
3
1
.40–1.20 (m, 6H), 0.90 (t, 3H, J = 3.9 Hz); C NMR
50.3 MHz, CDCl ): d 158.4, 151.5, 141.8, 138.5, 127.0,
24.2, 123.6, 120.9, 110.8, 58.7, 53.0, 44.7, 31.7, 27.2,
6.8, 22.6, 14.0; IR (CHCl ): m = 2932.4, 2859.0, 1616.1,
497.0, 1324.3, 1232.4/cm; anal. calcd. for C H N O ∙
C
23
H
34
N
4
O
2
. M = 398.54 g/mol. Yield 58%. M.p.: 98–
99 °C. H NMR (400 MHz, CDCl ): d 8.50 (s, 1H, Harom),
8.27 (d, 1H, J = 9.2 Hz, Harom), 7.93 (d, 1H, J = 9.2 Hz,
arom), 7.63 (d, 1H, J = 9.2 Hz, Harom), 7.04 (d, 1H,
J = 9.2 Hz, Harom), 3.88 (s, 4H, N(CH ) (CH ) NC H ),
1
(
3
3
1
2
1
3
H
19
26
4
2
2 2
2 2
10 21
Chem Biol Drug Des 2013; 81: 695–706
697

Gabrielsen et al.
1
13
2
.60 (s, 4H, N(CH
J = 7.2 Hz, CH ), 1.55 (br.s, 2H, CH
CH ), 0.87 (t, 3H, J = 6.0 Hz, CH
100 MHz, CDCl ): d 158.83, 151.95, 142.28, 139.08,
27.54, 124.70, 124.07, 121.47, 111.27, 59.16, 53.45,
4.99, 32.33, 30.01, 30.01, 29.85, 29.75, 27.95, 27.13,
2
)
2
(CH
2
)
2
NC10
H
21), 2.41 (br.t, 2H,
was equipped with an inverse H{ C} 5-mm Nalorac
probe with an actively shielded z-gradient coil (ID-PFG)
(Varian).
2
2
), 1.28 (m, 14H,
13
7
(
9
2
3
).
C NMR
3
1
1
4
2
8
H NMR was run using the standard program imple-
mented in the VARIAN software (Varian). Double pulsed field
gradient spin echo–nuclear overhauser effect (DPFGSE-
NOE) experiments were run using a pulse sequence pub-
lished by Stott et al. (26) using shaped selective p soft
pulses generated by a standard Varian program (Varian).
The rsnob p pulse was calibrated for each individual multi-
plet. The Echo–antiecho phase-sensitive gradient-selected
H- C HSQCAD (heteronuclear single quantum coher-
ence adiabatic version) NMR spectra (27) were obtained
with a spectral width of 5000 Hz, 2048 points in the
3.12, 14.56; anal. calcd. for C23
34 4 2
H N O : C, 69.31; H,
.60; N, 14.06%; found C, 70.24; H, 8.56; N, 14.39%.
+
ESI-MS: m/z [%]: 399.2 [M + H] 100.
2
-(4-Undecyl-piperazin-1-yl)-6-nitroquinoline (11)
1
13
C H N O . M = 412.51 g/mol. Yield 55%. M.p.: 89–
2
4 36 4 2
1
9
0 °C. H NMR (400 MHz, CDCl
J = 2.8 Hz, arom), 8.28 (dd, 1H,
J2 = 9.2 Hz, Harom), 7.95 (d, 1H, J = 9.2 Hz, Harom), 7.65
d, 1H, J = 9.2 Hz, Harom), 7.06 (d, 1H, J = 9.6 Hz, Harom),
.91 (s, 4H, N(CH ) (CH ) NC H ), 2.63 (s, 4H, N
3
): d 8.51 (d, 1H,
1
H
J
1
= 2.4 Hz,
H
C
1
3
dimension and 8000 Hz, 800 9 2 increments in the
(
3
1
dimension, 128 transients per t increment, with a relaxa-
1
tion delay of 1 seconds and J(C,H) = 135 Hz. The data
2
2
2 2
11 23
(CH
2
)
2
(CH
2
)
2
NC11
H
23), 2.44 (br.t, 2H, J = 7.2 Hz, CH
2
),
were linearly predicted to 1600 points and zero-filled to
1
.57 (s, 2H, CH ), 1.31 (m, 16H, 8 9 CH ), 0.88 (t, 3H,
4096 points in F before Fourier transformation. The gradi-
2
2
1
13
1
3
1
J = 6.4 Hz, CH
3
). C NMR (100 MHz, CDCl
3
): d 158.59,
ent-selected H- C heteronuclear multiple bond coher-
ence (HMBC) spectra were performed with an acquisition
1
1
2
51.70, 142.15, 138.89, 127.37, 124.45, 123.85, 121.29,
11.04, 58.89, 53.16, 44.68, 32.11, 29.81, 29.77, 29.73,
9.64, 29.54, 27.70, 26.76, 22.88, 14.31; anal. calcd. for
1
time of 0.2 seconds, H – 90° pulse width of 7.8 lsec-
13
onds, C – 90° pulse width of 11.5 lseconds, a spectral
1
C H N O : C, 69.87; H, 8.79; N, 13.58%; found C,
6
width of 5000 Hz, 2048 data points in the H dimension
2
4 36 4 2
13
8.73; H, 8.73; N, 13.11%. ESI-MS: m/z [%]: 413.2
and 25000 Hz, 1024 increments in the C dimension,
relaxation delay of 1.2 seconds. The data were acquired
+
[M + H] 100.
1
as absolute value mode, with 64 transients per t incre-
n
ment. The experiment was optimized for J(C,H) = 8 Hz,
1
2
C
9
-(4-Dodecyl-piperazin-1-yl)-6-nitroquinoline (12)
. M = 426.59 g/mol. Yield 53%. M.p.: 95–
6 °C. H NMR (400 MHz, CDCl ): d 8.51 (d, 1H,
and low-pass filter for J(C,H) = 140 Hz was used. The
25
H
38
N
4
1
O
2
data were linear predicted to 2048 points and zero-filled to
4096 points in F prior to Fourier transformation. The
3
1
1
15
J = 2.0 Hz, Harom), 8.28 (dd, 1H, J
1
= 2.0 Hz, J
.2 Hz, Harom), 7.95 (d, 1H, J = 9.2 Hz, Harom), 7.65 (d,
H, J = 9.2 Hz, Harom), 7.05 (d, 1H, J = 9.2 Hz, Harom),
.91 (s, 4H, N(CH (CH NC12 25), 2.64 (s, 4H, N
2
=
H/ N-HSQC (heteronuclear single quantum coherence)
NMR spectra were obtained using external CH NO
2
9
1
3
3
15
(POCh, Warszawa, Poland) as the N reference (using the
internally locked substitution method) (28). The spectral
parameters were as follows: p/2 pulse lengths 7.2 lsec-
onds ( H) and 21.5 lseconds ( N); acquisition time
0.2 seconds; spectral windows 8000 Hz (F2) and
10 000 Hz (F1); 2048 data points in the H dimension and
256 increments in that of N; 1J(N,H) = 80 Hz; relaxation
delay 1.0 seconds; four transients per increment. The
2
)
2
2
)
2
H
(CH ) (CH ) NC H ), 2.45 (br.t, 2H, J = 7.2 Hz, CH ),
2
2
2 2
12 25
2
1
15
1
3
1
1
2
.57 (br.s, 2H, CH
H, J = 6.0 Hz, CH3).
58.58, 151.70, 142.18, 138.92, 127.39, 124.47, 123.88,
21.31, 111.05, 58.87, 53.14, 44.65, 32.13, 29.87, 29.85,
9.82, 29.78, 29.73, 29.56, 27.69, 26.71, 22.90, 14.33;
2
), 1.28 (m, 18H, 9 9 CH
2
), 0.88 (t,
13
C NMR (100 MHz, CDCl ): d
3
1
15
anal. calcd. for C25
H
38
N
4
O
2
: C, 70.39; H, 8.98; N,
6
spectra were measured in DMSO-d (Dr. Glaser AB,
1
3.13%; found C, 69.82; H, 8.55; N, 12.75%. ESI-MS: m/z
Basel, Switzerland) at 298 K or in DMF-d (Dr. Glaser AB,
Basel, Switzerland) at 253 K using a Varian INOVA 500-
MHz NMR spectrometer (Varian) equipped with an inverse
7
+
[%]: 427.2 [M + H] 100.
1
H{31P/15N} 5-mm Z-SPEC Nalorac probe with an
Nuclear magnetic resonance (NMR) spectroscopy
of 8 and 9
actively shielded z-gradient coil (Varian). The coherence
selection with two pulsed field gradient (PFG) pulses of rel-
ative amplitudes of +(c I+c S): ꢁ(c Iꢁc S) and ꢁ(c Iꢁc S):
+(c I+c S), for heteronuclear echo and antiecho, respec-
tively, was applied. Such implementation enabled us to
obtain nearly pure absorption lineshapes along F1, com-
bined with absolute value mode in F2 dimension. The
spectral parameters were as follows: p/2 pulse lengths
The NMR spectra were recorded at 303 K on Varian INO-
VA 500 spectrometer (Varian, Palo Alto, CA, USA) oper-
1
13
15
ated at 499.8, 125.7 and 50.51 MHz for H, C and
nuclei, respectively. Eight milligrams of the compounds (8
and 9) was dissolved in 0.7 mL DMSO-d (Dr. Glaser AB,
N
6
Basel, Switzerland) and transferred to a 5-mm NMR tube.
Chemical shifts (d, ppm) were referenced against the
internal reference 3-trimethylsilyl-propionic acid (TSPA;
Dr. Glaser AB, Basel, Switzerland). The spectrometer
1
15
7.2 lseconds ( H) and 21.5 lseconds ( N); acquisition
time 0.2 seconds; spectral windows 8000 Hz (F2) and
1
23 000 Hz (F1); 2048 data points in the H dimension and
6
98
Chem Biol Drug Des 2013; 81: 695–706

Long Chain Alkylnitroquipazines as SERT Inhibitors
15
3
2
56 increments in that of N; nJ(N,H) = 3 Hz; relaxation
binding, 100 lL of [ H]8-OH-DPAT and 100 lL of analysed
delay 1.0 seconds; eight transients per increment.
compound were incubated at 37 °C for 15 min. The incuba-
tion was followed by rapid vacuum filtration through Whatman
GF/B glass filters, and the remainder was washed three times
with 5 mL ice-cold buffer (50 mM Tris–HCl, pH 7.7) using a
In vitro binding affinity assay
3
Brandel cell harvester. The final [ H]8-OH-DPAT concentration
SERT
was 1 nM, and the concentrations of the analysed com-
The assay was performed in accordance with the method
described by Owens et al. (2) with slight modifications. Rat
cerebral cortex was homogenized in 30 volumes of ice-
cold 50 mM Tris–HCl containing 150 mM NaCl and 5 mM
KCl, pH 7.7, at 25 °C and centrifuged at 20 000 9 g for
ꢁ10
ꢁ4
pounds ranged from 10
to 10 M.
Homology modelling and docking
SERT
20 min. The supernatant was decanted and pellet was
The construction of the homology model of SERT based
on the LeuT in an outward-facing conformation (PDB id
resuspended in 30 volumes of buffer and centrifuged
again. The resulting pellet was resuspended in the same
quantity of the buffer and centrifuged third time in the
3
F3A) (25) has been described elsewhere (24) and will only
shortly be described here. The LeuT and SERT (UniProt
accession number P31645) sequences were aligned and
adjusted to fit a comprehensive alignment of NSS mem-
bers (31). The homology models were generated using the
BuildModel macro available in ICM version 3.5 and were
energy-refined using the refineModel macro of ICM (32).
3
same conditions. [ H]-citalopram (spec. act. 50 Ci/mmol,
NEN Chemicals, USA) was used for labelling 5-HT trans-
porter. Two hundred and forty microlitres of the tissue sus-
pension, 30 lL of 1 lM imipramine (Sigma, St. Louis, MO,
3
USA) (displacer), 30 lL of 1 nM [ H]-citalopram and
100 lL of the analysed compound were incubated at
2
2 °C for 1 h. The concentrations of analysed compounds
Starting geometries of the ligands were built using ICM (32)
based on the information obtained from the NMR spec-
troscopy data of 8, which showed that the nitroquinoline
and alkyl substituents occupied diequatorial positions in
the piperazine ring and that the alkyl substituent was in
extended conformation and that the piperazine N4′ nitro-
gen atom formed the primary protonation site of the ligand
molecule. The N4′ atom of the ligands was hence charged
using default ECEPP/3 partial charges (33).
ꢁ
10
ꢁ4
to 10 M. Incubations were terminated
ranged from 10
by vacuum filtration through Whatman GF/B filters (What-
man, Maidstone, Kent, UK) and washed two times with
1
00 lL of ice-cold buffer. The radioactivity was measured
using a WALLAC 1409 DSA liquid scintillation counter
Wallac, USA). The assay was performed in duplicate.
(
Radioligand binding data were analysed using iterative
curve-fitting routines (GRAPHPAD/PRISM, version 3.0; San
Diego, CA, USA). Ki values were calculated from the
Cheng-Prusoff equation (29):
A flexible docking protocol (24) was used to dock 6-NQ
and the twelve alkyl analogues. It consisted of (i) detection
of a ligand-binding pocket using ICM POCKETFINDER (34), (ii)
torsional sampling of the side chains of the amino acids
constituting the binding pocket using biased probability
Monte Carlo (BPMC) (35) and (iii) 4D flexible ligand docking
(36). Using this protocol, the side chains of amino acids
Y95, D98, L99, W103, R104, Y107, Y175, Y176, I179,
F335, S336, L337, F341, V343, K399, D400, P403, L405,
L406, F407, S438, T439, E493 and T497 were sampled.
The side-chain sampling resulted in the generation of 47
SERT binding pocket conformations (24) into which 6-NQ
and the twelve analogues were docked using the 4D
docking approach (36). Three parallel docking runs of 6-
NQ and the analogues were performed. Following the 4D
docking, refinement of the best scored complex of each
ligand was performed, in which the side-chain torsional
angles of the amino acids in the vicinity of the ligands were
sampled using the BPMC procedure (35).
IC50
Ki ¼
LO
KD
1
þ
where L
O D
is the labelled ligand concentration and K is the
dissociation constant of labelled ligand.
5
[
-HT1A receptor
3
H]8-Hydroxy-2-(di-n-propylamino)tetralin
(8-OH-DPAT,
spec. act. 106 Ci/mmol, NEN Chemicals) was used for
labelling of the 5-HT1A receptors. The membrane prepara-
tion and assay procedure were carried out according to a
previously described procedure (30) with slight modifica-
tions. In brief, the hippocampus tissue was homogenized
in 20 volumes of 50 mM Tris–HCl buffer (pH 7.7 at 25 °C)
using Ultra-Turraxꢀ
2 000 9 g for 10 min. The supernatant fraction was dis-
carded, the pellet resuspended in the same volume of Tris
HCl buffer and the solution centrifuged again. Before the
third centrifugation, the samples were incubated at 37 °C for
0 min. The final pellet was resuspended in Tris–HCl buffer
containing 10 lM pargyline, 4 mM CaCl and 0.1% ascorbic
acid. Samples containing 1 mL of the tissue suspension
5 mg wet weight), 100 lL of 10 lM serotonin for non-specific
T 25 and then centrifuged at
3
–
The refined SERT–ligand complexes were scored using the
ICM SCANSCOREEXTERNAL MACRO (32). The scoring function
includes terms for steric, entropic, hydrogen bonding,
hydrophobic and electrostatic interactions, together with a
correction term that takes into account the number of atoms
in the ligand to avoid the bias towards larger ligands (37).
1
2
(
Chem Biol Drug Des 2013; 81: 695–706
699

Gabrielsen et al.
-HT1A receptor
5
Forced swim test
A homology model of the 5-HT1A receptor (UniProt acces-
sion number P08908) was constructed using the crystal
The experiment was carried out according to the method of
Porsolt et al. (40). The mice were individually placed in a
glass cylinder (25 cm high, 10 cm in diameter) containing
6 cm of water maintained at 23–25 °C and were left therein
for 6 min. A mouse was regarded as immobile when it
remained floating on water, making only small movements
to keep its head above water. The total duration of immobil-
ity was measured by an experimenter during the final 4 min
of a 6-min test session after a 2-min habituation period.
structure of the human b -adrenergic receptor in complex
2
with alprenolol, a beta-blocker and 5-HT1A receptor antag-
onist (38), as template (PDB id 3NYA (39). The final model
consisted of amino acids S34-K418 except amino acids
2
22–339 of IL3. A disulphide bridge between C109 and
C187 was included. Following the model construction, the
ICM REFINEMODEL MACRO (32) was used to remove possible
close contacts between amino acids in the model and to
relax the structure. A stereochemical quality analysis of the
model and 3NYA template structure was then performed
using the PROCHECK, ERRAT and VERIFY 3D programs
available via the SAVES server (http://nihserver.mbi.ucla.edu/
SAVES/). The results show that the model structure was
of similar stereochemical quality as the template structure.
Locomotor activity test
The spontaneous locomotor activity of mice was recorded in
photoresistor actometers (24 cm in diameter and illuminated
by two light beams), which were connected to a counter for
the recording of light-beam interruptions. The mice were
placed individually in the actometers, and the number of light-
beam crossings was counted twice: during the first 6 min, that
is, at the time equal to the observation period in the forced
swimming test, and during a 30-min experimental session.
ICM POCKETFINDER (tolerance level 3) (34) detected that the
following amino acids constituted the orthosteric binding
site of the 5-HT1A receptor: A93, Y96, Q97, F112, I113,
D116, V117, C120, C187, I189, K191, Y195, T196, S199,
T200, A203, W358, F361, F362, V364, A365, L368, T379,
L381, I385, W387 and Y390. Three parallel docking runs
of 6-NQ, 1–12 and (R)- and (S)-alprenolol into the orthos-
teric binding site using ICM were performed.
Results and Discussion
Synthesis of 6-NQ alkyl analogues and in vitro
binding studies
The general synthesis scheme of the twelve 6-NQ ana-
logues is shown in Figure 1. The results of the in vitro
studies showed that the long-chain analogues (8–12) have
In vivo tests
Based on the results from the in vitro binding studies, 8,
9, 10, 11 and 12 were selected for in vivo tests.
i
the highest binding affinity (K) in SERT, ranging from
1
.8 ꢀ 0.2 to 20.8 ꢀ 3.8 nM, respectively (Table 1). The
Animals and housing
i
Table 1: 6-Nitroquipazine (6-NQ) analogue affinities (K, nM) in
The in vivo experiments were performed on male Albino
Swiss mice (24–28 g). The animals were kept at a room
temperature (20 ꢀ 1 °C) on a natural day–night cycle
serotonin transporter (SERT).
O–
(
April–May) and housed under standard laboratory condi-
N+
O
tions. They had free access to food and tap water before
the experiments. Each experimental group consisted of 6–
N
N
8
animals/dose, and all the animals were used only once.
N
CnH2n+1
Drug treatments and analysis of results
-OH-DPAT (Research Biochemical Inc.) and N-{2-[4-(2-
methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohex-
ane-carboxamidetrihydrochloride (WAY-100635, synthe-
sized by Dr. J. Boksa, Institute of Pharmacology, Polish
Academy of Sciences, Krak oꢀ w, Poland) were used as
8
Compound
-NQ
n
SERT
a
6
0
1
2
3
4
5
6
7
8
9
0.17 ꢀ 0.03
1300 ꢀ 300
86.1 ꢀ 8.5
495.7 ꢀ 34.3
24.0 ꢀ 2.6
80.5 ꢀ 7.4
72.9 ꢀ 8.1
54.1 ꢀ 4.7
20.8 ꢀ 3.8
1.8 ꢀ 0.2
1
2
3
4
5
6
7
8
9
aqueous solutions. 9, 10, 11 and 12 were suspended in a
1
% aqueous solution of Tween-80, whereas 8 was sus-
pended in dimethyl sulphoxide (DMSO). 8-OH-DPAT and
WAY-100635 were injected subcutaneously (sc), whereas
8, 9, 10, 11 and 12 were given intraperitoneally (ip) in vol-
umes of 10 mL/kg. The comparisons of the compounds
were carried out by a one-way analysis of variance (ANOVA)
followed by intergroup comparisons using the Dunnett’s
test (when one drug was administered) or by the Newman
1
0
1
2
10
11
12
4.5 ꢀ 0.7
1
1
12.6 ꢀ 1.3
16.1 ꢀ 1.2
a
–Keuls test (when two drugs were administered).
From reference 6.
7
00
Chem Biol Drug Des 2013; 81: 695–706

Long Chain Alkylnitroquipazines as SERT Inhibitors
binding affinities of the shorter analogues varied more, and
the methyl analogue (1, quipazine) had the lowest affinity
ble to observe resonance position of these atoms neither
in HSQC nor in HMBC experiments.
of the analogues with a K
i
of 1300 ꢀ 300 nM (Table 1).
As 6-NQ analogues may be dual-acting compounds that
inhibit SERT and antagonise the presynaptic 5-HT1A
receptor and hence reduce the onset time of antidepres-
sant action (22), the affinities of the analogues to this
receptor were also determined. The results, however,
showed that they had low 5-HT1A receptor affinity (in the
lM range). The butyl analogue (3) had the highest 5-HT1A
Docking
SERT
The compounds were docked into the ligand-binding
pocket detected by ICM POCKETFINDER (34) in an outward-fac-
ing SERT homology model that had previously been gen-
erated using the X-ray crystal structure of LeuT [PDB id
3F3A (25)] as template (24).
receptor affinity with a K
i
= 842.3 ꢀ 12.6 nM (Supporting
Information).
LeuT, a prokaryotic homologue of SERT, was the first, and
is so far the only, NSS family member to be crystallized.
The LeuT crystal structures published represent three
major conformations of the transport cycle: the outward-
facing conformation [e.g. PDB id 3F3A (25)], in which the
central substrate-binding site is accessible from the extra-
cellular environment but inaccessible to the intracellular
environment, the occluded conformation (e.g. PDB id
2A65 (41)), where a thin extracellular gate and a thick
intracellular gate separate the central substrate-binding site
from either environments, and the inward-facing conforma-
tion (PDB id 3TT3 (42)), where the central substrate-bind-
ing site is inaccessible from the extracellular environment
but accessible from the cytoplasm. In the present study, a
SERT homology model (24) based on the outward-facing
LeuT conformation (25) was chosen for docking as studies
have indicated that inhibitors of SERT stabilize outward-
facing conformations of the transporter (38, 39).
NMR studies
The DPFG NOE results for compound 8 are shown in Fig-
ure 2, while the corresponding results for compound 9 are
shown in the Supplementary Information (Figure S1). The
NMR studies suggested that the piperazine ring of 8 and 9
was in a fixed chair conformation with substituents on
nitrogen atoms occupying the equatorial positions. The
1
high-frequency position of NH signal (11.02 ppm in
H
1
NMR) and J (N, H) = 75 Hz showed that NH did not
exchange and was involved in hydrogen bonding, most
probably with DMSO. The protonation site was confirmed
by DPGSE-NOE experiment, which showed that irradiation
of NH resonance produced NOE enhancements at
a
e
3.06 ppm, that is, the H3′ H3′ and H1″ signals over-
lapped (Figure 2). Both signals were in close vicinity of
piperazine N4 ‘protonation site. Tautomerization of a pro-
ton between the piperazine N1′ and N4′ nitrogen atoms
was excluded. The NMR experiment also suggested that
quinoline N1 and piperazine N1′ atoms could form a sec-
ondary protonation site. The attempts to locate another
hydrogen atom were, however, unsuccessful even at
The binding pocket detected by ICM POCKETFINDER (34) in the
model extended from the putative central substrate-bind-
ing site up towards the extracellular environment (Fig-
ure 3). The lower parts of this pocket corresponded to the
putative substrate-binding site of SERT detected through
experimental studies (43–48). This region also corresponds to
the central high-affinity leucine-binding site of LeuT (41). In
addition to the central substrate-binding site, the vestibular
region extending from the extracellular environment towards
the putative substrate-binding site was also included in the
binding pocket. The vestibular region contained several
charged amino acids (R104, K399, D400 and E493), and EL4
was oriented like a lid over the region (Figure 3).
ꢁ
20 °C in DMF-d . Apparently the exchange of a proton,
7
possibly between quinoline N1 and piperazine N1′, was
very fast. This also may be a reason that it was not possi-
The docking scores of the SERT–ligand complexes are
listed in the Supporting Information and show that docking
scores did not correlate with the binding affinities mea-
sured experimentally (Table 1). The main reason for the
lack of correlation is the structural flexibility of the com-
pounds and putative structural limitations in our SERT
models. Structural flexibility is challenging for sampling the
conformational space of the ligand during docking. The
entropic term of the scoring function is proportional to the
number of rotatable bonds in the ligand. Scoring of struc-
turally flexible compounds during docking is therefore chal-
lenging.
6
Figure 2: DPFG NOE results of 8 9 HCl in DMSO-d solution.
Upper spectrum shows NOE effects, and bottom spectrum
shows a trace of 1H NMR in studied solvent. Irradiation of NH
signal results in NOE enhancements at marked positions.
Chem Biol Drug Des 2013; 81: 695–706
701

Gabrielsen et al.
A
B
C
Figure 3: Docking results. (A)
The binding pocket (ribbon
representation) of the outward-
facing serotonin transporter
homology model, (B) orientation of
8
in the binding pocket, (C)
orientation of 12 in the binding
pocket. Amino acids surrounding
8
and 12 in (B) and (C) are shown
in xstick representation.
Two main orientations of the ligands were observed during
docking. Illustrations of these orientations, 8 and 12 in the
binding pocket, are shown in Figure 3. The nitroquinoline
moiety of 8 was pointing towards EL4 amino acids D400
TM10 (Figure 3). Of the thirteen compounds docked, five
preferred a binding mode similar to that of 12, whereas
the remaining eight were oriented in a similar manner as 8
(Supporting Information). When oriented similar to 8, the
longer alkyl analogues may form favourable hydrophobic
interactions with amino acids located in TM3 and TM8
(A169, A172, A173, F341) of SERT. These hydrophobic
interactions may contribute to more favourable affinity val-
ues of the longer alkyl analogues than the shorter.
(backbone) and P403. The octyl side chain of 8 was
located in the central region of the pocket, surrounded by
the side chains of A169 (TM3), I172 (TM3), A173 (TM3)
and F341 (TM6) (Figure 3). The protonated nitrogen of the
piperazine ring was in the vicinity of the backbone oxygen
of F335 (TM6) (Figure 3). A similar orientation has been
proposed by our group by docking of another series of
long-chain arylpiperazine nitroquipazine analogues (49). In
comparison, 12 had an opposite orientation to that of 8 in
the binding pocket. Its –NO2 moiety was located in the
lower parts of the central binding pocket, pointing towards
S438 in TM8, whereas the protonated piperazine moiety
was located near EL4, possibly forming ionic interactions
with D400 (EL4) and/or E493 (TM10) (Figure 3). Its dodecyl
side chain was located in the extracellular regions of the
binding pocket, juxtaposed between EL4 and TM6 and
5-HT1A receptor
To study the possible interactions of the analogues in the
5-HT1A receptor, a model of the receptor was constructed
2
using the crystal structure of the human b -adrenergic
receptor in complex with alprenolol (39) as template.
Alprenolol is a beta-blocker but also a 5-HT1A receptor
antagonist (38). Docking of the compounds into the or-
thosteric binding site of the 5-HT1A receptor model indi-
cated that the compounds are weak 5-HT1A receptor
7
02
Chem Biol Drug Des 2013; 81: 695–706

Long Chain Alkylnitroquipazines as SERT Inhibitors
binders, which is in agreement with the results of the 5-
HT1A receptor in vitro binding studies’ results (Table S1,
Supporting Information). The experimental 5-HT1A recep-
tor-binding affinities indicated that compound 3 is the
strongest 5-HT1A receptor binder. The best docking score
was likewise obtained with compound 3 (ꢁ4.56 kcal/mol,
Table S1). In comparison, the alprenolol docking scores
were ꢁ14.42 kcal/mol for the R-enantiomer and
known antidepressant drugs (2, 4), and 8–12 were hence
selected for in vivo testing in mice using the forced swim
test developed by Porsolt et al. (40). The forced swim test,
also known as the behavioural despair test, is a common
way to evaluate the antidepressant effect of compounds in
animals (rats or mice). During the test, the animals are
forced to swim in a glass cylinder from which they cannot
escape. After a short time of vigorous activity, the animals
adopt an immobile posture. Administration of antidepres-
sants and psychostimulants reduces the immobility time;
however, psychostimulants additionally cause marked
motor stimulation (40). The forced swim test is thus often
followed by a locomotor activity test.
ꢁ
19.08 kcal/mol for the S-enantiomer. This is also in
agreement with experimental results showing that the S-
enantiomer binds stronger than the R-enantiomer (38). The
orientations of both alprenolol enantiomers were further-
more very similar to that of alprenolol in the b -adrenergic
2
ꢁ
receptor (rmsd approximately 2 A). This suggests that the
model was suitable for docking of 5-HT1A receptor binders.
The results of the forced swim test showed that 8 (20 mg/
kg) and 10 (5 mg/kg) caused a reduction in mice immobil-
ity time by 15% and 12%, respectively, whereas 12
reduced the immobility time by 10% and 8% at doses of 5
and 10 mg/kg, respectively (Figure 4). 9 and 11 were inef-
fective (Figure 4). The results of the locomotor activity test
In vivo tests
The in vitro binding affinities of the 6-NQ analogues with
the longest alkyl side chains were within the range of
A
250
B
250
200
150
100
50
Veh
8 (10)
Veh
9 (5)
9 (10)
*
2
1
1
00
50
00
8
(20)
5
0
0
0
mg/kg
mg/kg
C
250
D
2
2
50
00
Veh
10 (2.5)
Veh
11 (5)
11 (10)
*
2
1
1
00
50
00
1
0 (5)
150
100
50
5
0
0
0
mg/kg
mg/kg
E
250
*
*
2
1
1
00
50
00
Veh
1
1
2 (5)
2 (10)
5
0
0
mg/kg
Figure 4: Effects of 8 (A), 9 (B), 10 (C), 11 (D) and 12 (E) in the forced swim test in Albino Swiss mice. Each bar represents the mean ꢀ
SEM of 9–10 mice. All analogues were injected 30 min before the test. * p < 0.05 versus respective vehicle group (Dunnett’s test).
Chem Biol Drug Des 2013; 81: 695–706
703

Gabrielsen et al.
Table 2: Effect of 8 (20 mg/kg), 10 (5 mg/kg) and 12 (10 mg/kg)
on mice locomotor activity. Number of light-beam cross-
ings ꢀ SEM 8, 10 and 12 were injected ip 30 min before the test.
3. Vaatstra W.J., Deiman-Van Aalst W.M., Eigeman L.
1981) Du 24565, a quipazine derivative, a potent
selective serotonin uptake inhibitor. Eur J Pharma-
col;70:195–202.
(
Locomotor activity
4
5
6
. Owens M.J., Knight D.L., Nemeroff C.B. (2001) Sec-
ond-generation SSRIs: human monoamine transporter
binding profile of escitalopram and R-fluoxetine. Biol
Psychiatry;50:345–350.
Treatment
6 min
30 min
Vehicle
8
554 ꢀ 46
453 ꢀ 41
239 ꢀ 41***
628 ꢀ 55
1514 ꢀ 173
1409 ꢀ 199
897 ꢀ 120**
1774 ꢀ 187
. Gerdes J.M., DeFina S.C., Wilson P.A., Taylor S.E.
1
0
2
(2000) Serotonin transporter inhibitors: synthesis and
1
binding potency of 2′-methyl- and 3′-methyl-6-nitro-
quipazine. Bioorg Med Chem Lett;10:2643–2646.
. Lee B.S., Chu S., Lee B.C., Chi D.Y., Choe Y.S.,
Jeong K.J., Jin C. (2000) Syntheses and binding affini-
ties of 6-nitroquipazine analogues for serotonin trans-
porter. Part 1. Bioorg Med Chem Lett;10:1559–1562.
***p < 0.001, **p < 0.01 versus vehicle group, (n = 10). 6 min: F
(2,27) = 18.47, p < 0.001; 30 min: F(2,27) = 6.505, p < 0.01.
indicated that the reduced immobility time observed in the
forced swim test after administration of 8, 10 and 12 was
not the result of increased spontaneous locomotor activity
7. Lee B.S., Chu S., Lee K.C., Chi D.Y., Choe Y.S., Kim
S.E., Song Y.S., Jin C. (2003) Syntheses and binding
affinities of 6-nitroquipazine analogues for serotonin
transporter: Part 3. A potential 5-HT transporter imag-
ing agent, 3-(3-[18F]fluoropropyl)-6-nitroquipazine. Bio-
org Med Chem;11:4949–4958.
(Table 2).
Conclusion
In this study, a combination of in vitro, in silico and in vivo
approaches has been used to evaluate twelve 6-nitroquip-
azine analogues (1–12). The in vitro binding studies showed
that the analogues with the longest side chains were high-
affinity SERT inhibitors, although their affinity in the 5-HT1A
receptor was low. Three analogues were found to have
moderate antidepressant activity in the in vivo Porsolt forced
swim test (8, 10 and 12). A reason for the moderate antide-
pressant activity may be the polar head group of the com-
pounds that may contribute to low bioavailability and low
concentration of the compounds at the site of action.
8. Lee J.H., Choi Y.H., Lim Y.J., Lee B.S., Chi D.Y., Jin
C. (2007) Syntheses and binding affinities of 6-nitro-
quipazine analogues for serotonin transporter. Part 5:
2′-Substituted
6-nitroquipazines.
Bioorg
Med
Chem;15:3499–3504.
9. Moon B.S., Lee B.S., Chi D.Y. (2005) Syntheses and
binding affinities of 6-nitroquipazine analogues for sero-
tonin transporter. Part 4: 3-Alkyl-4-halo-6-nitroquipa-
zines. Bioorg Med Chem;13:4952–4959.
10. Se Lee B., Chu S., Lee B.S., Yoon Chi D., Song Y.S.,
Jin C. (2002) Syntheses and binding affinities of 6-ni-
troquipazine analogues for serotonin transporter. Part
2
: 4-substituted 6-nitroquipazines. Bioorg Med Chem
Acknowledgments
Lett;12:811–815.
1
1. Biegon A., Mathis C.A., Hanrahan S.M., Jagust W.J.
(1993) [125I]5-iodo-6-nitroquipazine: a potent and selec-
tive ligand for the 5-hydroxytryptamine uptake complex. II.
In vivo studies in rats. Brain Res;619:236–246.
This work was supported by a grant from the Nevronor
program of the Research Council of Norway (project
1
76956/V40), the Polish-Norwegian Research Fund (grant
PNRF-103-AI-1/07) and the University of Tromsø, Norway.
The work was also supported by grants from National
Institutes of Health USA (grant numbers R01 GM071872,
U01 GM094612, U54 GM094618 and RC2 LM 010994).
Mari Gabrielsen gratefully acknowledges support and train-
ing from BioStruct, the Norwegian national graduate
school in Structural biology.
12. Hashimoto K., Goromaru T. (1990) Preparation of [3H]
6-nitroquipazine, a potent and selective 5-hydroxytryp-
tamine uptake inhibitor. Radioisotopes;39:168–169.
13. Hashimoto K., Goromaru T. (1992) 4-Bromo-6-nitro-
quipazine: a new ligand for studying 5-hydroxytrypta-
mine uptake sites in vivo. Neuropharmacology;31:869–
874.
1
4. Jagust W.J., Eberling J.L., Biegon A., Taylor S.E., Van-
Brocklin H.F., Jordan S., Hanrahan S.M., Roberts J.A.,
Brennan K.M., Mathis C.A. (1996) Iodine-123-5-iodo-
References
6
-nitroquipazine: SPECT radiotracer to image the sero-
1
. Saier M.H. Jr (2000) A functional-phylogenetic classifi-
cation system for transmembrane solute transporters.
Microbiol Mol Biol Rev;64:354–411.
tonin transporter. J Nucl Med: official publication, Soci-
ety of Nuclear Medicine;37:1207–1214.
15. Jagust W.J., Eberling J.L., Roberts J.A., Brennan
K.M., Hanrahan S.M., VanBrocklin H., Enas J.D., Bie-
gon A., Mathis C.A. (1993) In vivo imaging of the 5-
hydroxytryptamine reuptake site in primate brain using
single photon emission computed tomography and
2
. Owens M.J., Morgan W.N., Plott S.J., Nemeroff C.B.
(1997) Neurotransmitter receptor and transporter bind-
ing profile of antidepressants and their metabolites. J
Pharmacol Exp Ther;283:1305–1322.
7
04
Chem Biol Drug Des 2013; 81: 695–706

Long Chain Alkylnitroquipazines as SERT Inhibitors
[
123I]5-iodo-6-nitroquipazine.
Eur
J
Pharma-
28. Harris R.K., Becker E.D., De Menezes S.M., Granger P.,
Hoffman R.E., Zilm K.W. (2008) Further conventions for
NMR shielding and chemical shifts (IUPAC Recommen-
dations 2008). Magn Reson Chem;46:582–598.
29. Cheng Y., Prusoff W.H. (1973) Relationship between the
inhibition constant (K1) and the concentration of inhibitor
which causes 50 per cent inhibition (I50) of an enzymatic
reaction. Biochem Pharmacol;22:3099–3108.
30. Middlemiss D.N., Fozard J.R. (1983) 8-Hydroxy-2-(di-
n-propylamino)-tetralin discriminates between subtypes
of the 5-HT1 recognition site. Eur J Pharmacol;90:151
–153.
col;242:189–193.
1
6. Karramkam M., Dolle F., Valette H., Besret L., Bram-
oulle Y., Hinnen F., Vaufrey F., Franklin C., Bourg S.,
Coulon C., Ottaviani M., Delaforge M., Loc’h C., Bot-
tlaender M., Crouzel C. (2002) Synthesis of a fluorine-
1
8-labelled derivative of 6-nitroquipazine, as a radioli-
gand for the in vivo serotonin transporter imaging with
PET. Bioorg Med Chem;10:2611–2623.
7. Lundkvist C., Loc’h C., Halldin C., Bottlaender M., Ot-
taviani M., Coulon C., Fuseau C., Farde L., Maziere B.
1
(
1999) Characterization of bromine-76-labelled 5-
bromo-6-nitroquipazine for PET studies of the seroto-
nin transporter. Nucl Med Biol;26:501–507.
8. Mathis C.A., Taylor S.E., Biegon A., Enas J.D. (1993)
31. Beuming T., Shi L., Javitch J.A., Weinstein H. (2006) A
comprehensive structure-based alignment of prokaryotic
and eukaryotic neurotransmitter/Na+ symporters (NSS)
aids in the use of the LeuT structure to probe NSS struc-
ture and function. Mol Pharmacol;70:1630–1642.
32. Abagyan R., Totrov M., Kuznetsov D. (1994) ICM—A
new method for protein modeling and design: applica-
tions to docking and structure prediction from the dis-
torted native conformation. J Comput Chem;15:488–
506.
1
1
2
[125I]5-iodo-6-nitroquipazine: a potent and selective
ligand for the 5-hydroxytryptamine uptake complex. I.
In vitro studies. Brain Res;619:229–235.
9. Mathis C.A., Taylor S.E., Enas J.D., Akgun E. (1994)
Binding potency of 6-nitroquipazine analogues for the
5-hydroxytryptamine reuptake complex. J Pharm Phar-
macol;46:751–754.
0. Sandell J., Yu M., Emond P., Garreau L., Chalon S.,
N ꢁa gren K., Guilloteau D., Halldin C. (2002) Synthesis, ra-
diolabeling and preliminary biological evaluation of radio-
33. Nemethy G., Gibson K.D., Palmer K.A., Yoon C.N.,
Paterlini G., Zagari A., Rumsey S., Scheraga H. (1992)
Energy parameters in polypeptides. 10. Improved geo-
metrical parameters and nonbonded interactions for
use in the ECEPP/3 algorithm, with application to pro-
line-containing peptides. J Phys Chem;96:6472–6484.
34. An J., Totrov M., Abagyan R. (2005) Pocketome via
comprehensive identification and classification of ligand
binding envelopes. Mol Cell Proteomics;4:752–761.
35. Abagyan R., Totrov M. (1994) Biased probability Monte
Carlo conformational searches and electrostatic calcu-
lations for peptides and proteins. J Mol Biol;235:983–
1002.
36. Bottegoni G., Kufareva I., Totrov M., Abagyan R.
(2009) Four-dimensional docking: a fast and accurate
account of discrete receptor flexibility in ligand dock-
ing. J Med Chem;52:397–406.
37. Schapira M., Abagyan R., Totrov M. (2003) Nuclear
hormone receptor targeted virtual screening. J Med
Chem;46:3045–3059.
38. Kuipers W., Link R., Standaar P.J., Stoit A.R., Van Wi-
jngaarden I., Leurs R., Ijzerman A.P. (1997) Study of
the interaction between aryloxypropanolamines and
Asn386 in helix VII of the human 5-hydroxytrypta-
mine1A receptor. Mol Pharmacol;51:889–896.
39. Wacker D., Fenalti G., Brown M.A., Katritch V., Abag-
yan R., Cherezov V., Stevens R.C. (2010) Conserved
binding mode of human beta2 adrenergic receptor
inverse agonists and antagonist revealed by X-ray
crystallography. J Am Chem Soc;132:11443–11445.
40. Porsolt R.D., Bertin A., Jalfre M. (1977) Behavioral des-
pair in mice: a primary screening test for antidepres-
sants. Arch Int Pharmacodyn Ther;229:327–336.
41. Yamashita A., Singh S.K., Kawate T., Jin Y., Gouaux
E. (2005) Crystal structure of a bacterial homologue of
labeled
5-methyl-6-nitroquipazine,
a
potential
radioligand for the serotonin transporter. Bioorg Med
Chem Lett;12:3611–3613.
1. Guo Y., Chen X., Jia H., Ji X., Liu B. (2008) Synthesis
and biological evaluation of one novel technetium-
2
2
99 m-labeled nitroquipazine derivative as an imaging
agent for serotonin transporter. Appl Radiat
Isot;66:1804–1809.
2. Perrone R., Berardi F., Colabufo N.A., Lacivita E., Lar-
izza C., Leopoldo M., Tortorella V. (2005) Design and
synthesis of long-chain arylpiperazines with mixed
affinity for serotonin transporter (SERT) and 5-HT(1A)
receptor. J Pharm Pharmacol;57:1319–1327.
3. Blier P. (2001) Pharmacology of rapid-onset antide-
pressant treatment strategies. J Clin Psychiatr;62(Sup-
pl 15):12–17.
2
2
4. Gabrielsen M., Kurczab R., Ravna A.W., Kufareva I.,
Abagyan R., Chilmonczyk Z., Bojarski A.J., Sylte I.
(2012) Molecular mechanism of serotonin transporter
inhibition elucidated by a new flexible docking protocol.
Eur J Med Chem;47:24–37.
5. Singh S.K., Piscitelli C.L., Yamashita A., Gouaux E.
2
2
(2008) A competitive inhibitor traps LeuT in an open-
to-out conformation. Science;322:1655–1661.
6. Stott K., Stonehouse J., Keeler J., Hwang T.-L., Shaka
A.J. (1995) Excitation sculpting in high-resolution nuclear
magnetic resonance spectroscopy: application to selec-
tive NOE experiments. J Am Chem Soc;117:4199–4200.
7. Summers M.F., Marzilli L.G., Bax A. (1986) Complete
proton and carbon-13 assignments of coenzyme B12
through the use of new two-dimensional NMR experi-
ments. J Am Chem Soc;108:4285–4294.
2
Chem Biol Drug Des 2013; 81: 695–706
705

Gabrielsen et al.
Na+/Cl–dependent neurotransmitter transporters. Nat-
ure;437:215–223.
2. Krishnamurthy H., Gouaux E. (2012) X-ray structures
of LeuT in substrate-free outward-open and apo
inward-open states. Nature;481:469–474.
3. Adkins E.M., Barker E.L., Blakely R.D. (2001) Interac-
tions of tryptamine derivatives with serotonin transporter
species variants implicate transmembrane domain I in
substrate recognition. Mol Pharmacol;59:514–523.
4. Barker E.L., Moore K.R., Rakhshan F., Blakely R.D.
48. Walline C.C., Nichols D.E., Carroll F.I., Barker E.L.
(2008) Comparative molecular field analysis using
selectivity fields reveals residues in the third transmem-
brane helix of the serotonin transporter associated with
substrate and antagonist recognition. J Pharmacol Exp
Ther;325:791–800.
49. Jaronczyk M., Wolosewicz K., Gabrielsen M., Nowak
G., Kufareva I., Mazurek A.P., Ravna A.W., Abagyan
R., Bojarski A.J., Sylte I., Chilmonczyk Z. (2012) Syn-
thesis, in vitro binding studies and docking of long-
chain arylpiperazine nitroquipazine analogues, as
potential serotonin transporter inhibitors. Eur J Med
Chem;49:200–210.
4
4
4
(1999) Transmembrane domain I contributes to the
permeation pathway for serotonin and ions in the sero-
tonin transporter. J Neurosci;19:4705–4717.
4
5. Celik L., Sinning S., Severinsen K., Hansen C.G., Moller
M.S., Bols M., Wiborg O., Schjøtt B. (2008) Binding of
serotonin to the human serotonin transporter. Molecular
modeling and experimental validation. J Am Chem
Soc;130:3853–3865.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
4
6. Field J.R., Henry L.K., Blakely R.D. (2010) Transmem-
brane domain 6 of the human serotonin transporter
contributes to an aqueously accessible binding pocket
for serotonin and the psychostimulant 3,4-methylene
Figure S1. DPFG NOE results of 9 x HCl in DMSO-d6
solution.
dioxymethamphetamine.
1280.
J
Biol Chem;285:11270–
Table S1. 5-HT1A receptor binding affinities (Ki, nM) and
docking scores (kcal/mol) of 1–12.
1
4
7. Kaufmann K.W., Dawson E.S., Henry L.K., Field J.R.,
Blakely R.D., Meiler J. (2009) Structural determinants
of species-selective substrate recognition in human
and Drosophila serotonin transporters revealed through
computational docking studies. Proteins;74:630–642.
Table S2. SERT docking scores (kcal/mol) and localisation
of the nitroquinolone moiety.
7
06
Chem Biol Drug Des 2013; 81: 695–706