Synthesis, in vitro and in vivo studies, and molecular modeling of N-alkylated dextromethorphan derivatives as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmc.2014.10.036

9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction.

Full text of DOI:10.1016/j.bmc.2014.10.036

Bioorganic & Medicinal Chemistry 22 (2014) 6846–6856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro and in vivo studies, and molecular modeling of
N-alkylated dextromethorphan derivatives as non-competitive
inhibitors of a3b4 nicotinic acetylcholine receptor
a,
a,
b,c,
a,d
e
Krzysztof Jozwiak , Katarzyna M. Targowska-Duda , Agnieszka A. Kaczor
, Joanna Kozak
,
b
b
b
e
Agnieszka Ligeza , Elzbieta Szacon , Tomasz M. Wrobel , Barbara Budzynska , Grazyna Biala ,
f
c
g
b,
⇑
Emilia Fornal , Antti Poso , Irving W. Wainer , Dariusz Matosiuk
Department of Chemistry, Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, 4a Chodzki St., PL-20093 Lublin, Poland
Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Lab, Faculty of Pharmacy with Division of Medical Analytics,
a Chodzki St., PL-20093 Lublin, Poland
School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, PO Box 1627, FI-70211 Kuopio, Finland
Department of Anatomy, Medical University of Lublin, 4 Jaczewskiego St., PL-20090 Lublin, Poland
Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, 4a Chodzki St., PL-20093 Lublin, Poland
Department of Chemistry, Catholic University of Lublin, al. Krasnicka 102, PL-20718 Lublin, Poland
Laboratory of Clinical Investigation, Division of Intramural Research Programs, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
a
b
4
c
d
e
f
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2014
Revised 3 October 2014
Accepted 24 October 2014
Available online 31 October 2014
9
N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive
inhibitors of 3b4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards 3b4 nicotinic
a
a
acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range.
Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC
membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with
a
3b4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of a3b4
Keywords:
Dextromethorphan
Nicotinic acetylcholine receptors
Non-competitive inhibitors
nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds
for the treatment of addiction.
Ó 2014 Elsevier Ltd. All rights reserved.
a3b4 nicotinic acetylcholine receptor
1
. Introduction
domain, four membrane-spanning helices (M1, M2, M3, and M4),
and a small C-terminal domain.
1
,3
Nicotinic acetylcholine receptors (nAChRs) are members of
In addition to the binding site of endogenous neurotransmitter
and other orthosteric ligands, the receptor contains several alloste-
ric binding sites at which noncompetitive inhibitors (NCIs) may
the ligand gated ion channel family1 and they are distributed at
the synaptic junctions of brain neurons where they regulate the
release of several neurotransmitters. Furthermore they are abun-
dant in the periphery where they mediate synaptic transmission
4
–6
bind.
The most important binding site for NCIs is the luminal
high-affinity domain, which is located on the internal surface of
the ion pore lined predominantly with five M2 helices, each incor-
porated by separate subunit.
2
at the neuromuscular junction and ganglia. In this regard, nAChRs
are involved in several physiologically relevant functions such as
cognition, memory, pain perception, auditory response, muscle
contraction, angiogenesis, and immune response.1
Several subtypes of nAChR pose attractive possibility as drug
targets, one of them,
a3b4 subtype known to be expressed in
nAChRs are members of the Cys-loop ligand-gated ion channel
superfamily and consists of five separate transmembrane proteins
ventral tegmental area, medial habenula, interpeduncular nucleus,
pineal gland, locus coeruleus, dorsolateral tegmentum, basolateral
7
,8
(
subunits), each containing
a
large extracellular N-terminal
amygdale, and hippocampus has been particularly studied as a
target for drug addiction. For example, Toll et al., has recently
reported a novel high affinity and highly selective
a3b4 nAChR
antagonist with potent blocking activity of nicotine self-
⇑
Corresponding author. Tel./fax: +48 81448 7272.
E-mail address: darek.matosiuk@umlub.pl (D. Matosiuk).
Equal contributors.
9
administration in rats. It has been postulated that
a3b4 nAChRs
10
inhibitory activity is related to the anti-addictive properties.
http://dx.doi.org/10.1016/j.bmc.2014.10.036
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
6847
2
.1.1. Demethylation of DM
A solution of 2.68 g (67 mmol) of sodium hydroxide in 50 ml of
water was added to a solution of 25 g (67 mmol) of dextromethor-
phan hydrobromide hydrate in 100 ml of chloroform. The reaction
mixture was stirred for 30 min on a magnetic stirrer. The organic
layer was separated and dried with anhydrous sodium sulfate. The
solvent was evaporated on a rotavap. The obtained freebase of
dextromethorphan was a dense and viscous off-white oil which
solidified upon standing. Yield: 17.95 g (97.98%). Dextromethor-
phan freebase (17.95 g, 63 mmol), 250 ml of toluene and 15.28 g
Figure 1. Morphinan’s derivatives as NCIs of a3b4 nAChR.
Additional studies support the view that b4-containing nAChRs
i.e., 3b4, 3b3b4) play a major role in the appearance of nicotine
withdrawal symptoms as well as in mediating the negative-
(
72.1 mmol) of 2,2,2-trichloroethyl chloroformate was heated
under gentle reflux for 2 h. The reaction mixture was washed with
0 ml of 5% hydrochloric acid and 50 ml of water. The organic layer
(
a
a
5
reinforcing properties.1
1,12
Moreover, there is some evidence that
3b4 nAChRs are involved in the expression of at least two signs
was separated and dried with anhydrous sodium sulfate. Toluene
was removed on a rotatory evaporator under reduced pressure.
The crude product was purified by flash chromatography on silica
gel using hexane/ethyl acetate as a mobile phase. Thus there
was obtained 25.54 g (91.2%) of white solid. The obtained 2,2,2-
a
1
3
of opioid withdrawal.
Dextromethorphan (DM) (Fig. 1) and its metabolite dextror-
phan have been identified to reduce morphine, methamphetamine
14
and nicotine self-administration in rats. These compounds are
primarily known to inhibit NMDA glutamate receptors,1 our pre-
vious studies, however, have determined them, along with other
trichloroethyl (9a,13a,14a)-3-methoxymorphinan-17-carboxylate
5
(
25.54 g, 57.2 mmol) and 250 ml of 90% acetic acid was stirred on
a magnetic stirrer and 11.22 g (171.6 mmol) of zinc powder was
added in portions during 30 min. After 1 h of additional mixing
the remaining zinc was filtered and acetic acid was evaporated on
a rotatory evaporator under reduced pressure. 50 ml of toluene
was added to the obtained dense oil, this was brought to boiling
and the mixture was allowed to cool down in a refrigerator. The
morphinan analogs (Fig. 1), as potent NCIs of the
a
3b4
In affinity chromatography assay DM molecule has
shown high affinity to the luminal binding site of the 3b4 nAChR
6
,16,17
nAChR.
a
receptor immobilized on the stationary phase. Intriguingly, the
opposite enantiomer, levomethorphan has its affinity significantly
reduced in comparison to DM. Non-linear chromatography model
has identified dissociation kinetics of the drug–receptor complex
as responsible for observed affinity difference of the two enantio-
resulting white precipitate of (9
tetraacetozincate was filtered and washed with diethyl ether, yield:
4.84 g (72%). The obtained (9 ,13 ,14 )-3-methoxymorphinan
a,13a,14a)-3-methoxymorphinan
3
a
a
a
1
8
mers to the receptor. Subsequent molecular modeling study has
postulated that the difference lies in the differential orientation of
the two isomeric molecules within the lipophilic cleft formed in
tetraacetozincate (34.84 g, 41.2 mmol–82.4 mmol equiv) was dis-
solved in 50 ml of chloroform and 75 ml of 1 M sodium hydroxide
solution was added. The white precipitate was filtered, washed with
chloroform and organic layer separated. The aqueous layer was
extracted 4 times with 25 ml of chloroform and chloroform layers
were combined and dried with anhydrous sodium sulfate. The solu-
tion was stripped of solvent to afford clear oil of (9a,13a,14a)-3-
methoxymorphinan (nordextromethorphan) which solidified upon
standing. Yield: 12.47 g (55.81%).
6
the channel lumen. As result, the amino moiety of DM molecule
is located in optimized position to form a hydrogen bond with
neighboring serine residues while corresponding moiety of levo-
methorphan lies in further distance preventing such an interac-
tion. Interestingly, due to single point polymorphism present in
the M2 helix of b subunits, the specific cleft binding morphinan
analogs occurs only in nAChRs containing b4 component but not
in b2 containing subtypes.6 The finding has lead us to initiate
rational design of new DM derivatives which while modified at
the amino alkyl moiety seems to be additionally optimized for
selective interactions with the nAChR subtypes containing b4 sub-
2
9
.1.2. Synthesis of N-alkylated derivatives of DM (compounds 1–
), method A
1
.08 g (4 mmol) of (9a,13a,14a)-3-methoxymorphinan, 5 mmol
of bromo- or chloroalkylating agent, 2 g of triethylamine and 20 ml
of anhydrous ethanol or acetonitrile was heated under nitrogen
atmosphere in mild boiling of the solvent for 2–24 h. The solvent
was evaporated under reduced pressure and 20 ml of 20% solution
of sodium carbonate was added to the resulted oil. The obtained
mixture was extracted 4 times with 25 ml of chloroform. The
organic layers were dried with anhydrous sodium sulphate. The
obtained precipitate of hydrobromide or hydrochloride was
washed with diethyl ether. Reactions were routinely monitored
with thin-layer chromatography (TLC) on silica gel (60 F254 Merck
plates) in two eluent systems: 80% acetic acid–chloroform–metha-
units and in particular with natural target for addiction,
nAChR.
a3b4
Current work presents synthesis of novel N-alkylated DM deriv-
atives, and their investigations as NCIs of the 3b4 nAChR. The
a
pharmacological activity of the studied compounds determined
using a patch-clamp technique was in the micromolar range. Fur-
thermore, we applied homology modeling, molecular docking
and molecular dynamics to demonstrate ligand–receptor interac-
tions involving nicotinic receptor ion channel domain. Finally, in
the light of our behavioral studies the novel derivatives have
potential application for the treatment of drug addiction.
4
nol (1:8:1) (A) and 5% NH OH in MeOH–dioxane (5:95) (B) and the
products were visualized with ultraviolet light of 254 nm
wavelength.
2
2
. Materials and methods
.1. Chemistry
2
.1.2.1.
hydrobromide, 1, C19
heated for 11 h and the white crystalline precipitate of 1 was
obtained. Yield: 1.31 g (86.60%). Mp 206–207 °C, R : 0.727 (A)
-DMSO): 8.81 (s, 1H); 7.10–7.12 (m,
H); 6.81–6.84 (m, 2H); 4.73 (s, 2H); 3.73 (s, 3H) 3.54 (s, 1H);
(9
a
,13
a
,14
a
H
)-17-Cyanomethyl-3-methoxymorphinan
25BrN O. The reaction mixture was
2
NMR spectra were acquired using Bruker Avance 400 spectrom-
eter. MS spectra with nanospectrospray ionization (nano ESI) were
recorded with Agilent Technologies 6538 UHD Accurate Mas Q-TOF
LC/MS mass spectrometer on the nano-LC-chip-QTOF system. The
elementary analysis was performed with the application of Per-
kin–Elmer analyzer. Melting points were determined with Boetius
apparatus.
f
1
and 0.663 (B). H NMR (d
6
1
3
2
1
.19–3.21 (m, 1H); 3.14 (s, 1H); 3.02–3.08 (m, 1H); 2.60 (s, 1H);
.43–2.47 (m, 1H); 2.08–2.11 (m, 1H); 1.90 (s, 1H); 1.59–1.62 (m,
H); 1.45–1.52 (m, 3H); 1.29–1.33 (m, 2H); 1.07–1.17 (m, 1H);

6
848
K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
0
1
3
2
6
6
.9–1.00 (m, 1H); 13C NMR (d
28.31, 117.09, 115.59, 110.77, 59.36, 55.29, 46.78, 46.54, 38.46,
24 2
8.35, 37.79, 37.17, 24.75, 23.91, 20.19. MS: calcd for C19H N O
96.1889; found 296.1890, [M+H] 297.1962; Anal. calcd C,
0.48; H, 6.68; N, 7.42; O, 4.24; Br, 21.18; found C, 60.51; H,
.69; N, 7.43; O, 4.25; Br, 21.12.
6
-DMSO): 158.12, 141.80, 130.25,
1.14–1.17 (m, 1H), 0.89–0.96 (m, 1H). 13C NMR (d
-DMSO):
6
158.07, 141.79, 130.64, 128.98, 115.76, 110.75, 103.08, 64.70,
59.45, 55.21, 54.59, 46.75, 44.39, 38.48, 37.85, 37.54, 24.74, 23.93,
+
3
22.21, 20.54. MS: calcd for C21H29NO 343.4631; found 343.4634,
+
[M+H] 344.46.34. Anal calcd C, 59.43; H, 7.13; N, 3.30; O, 11.31;
Br, 18.83; found C, 59.18; H, 7.07; N, 3.28; O, 11.22; Br, 19.25.
2
.1.2.2. (9
a
,13
a
,14
a
)-17-(2-Cyanoethyl])-3-methoxymorphinan
27ClN O. The reaction mixture was
heated for 2 h and the white crystalline precipitate of 2 was
obtained. Yield: 1.06 g (85.40%). Mp 223–226 °C, R : 0.597 (A)
-DMSO): 11.38–11.52 (m, 1H); 7.11–
.12 (m, 1H); 6.81–6.83 (m, 2H); 3.73 (s, 3H); 3.68 (s, 1H); 3.50–
2.1.2.6. (9
oxmorphinan, 6, C22H31NO3.
heated for 13 h and the white crystalline precipitate of 6 was
obtained. Yield: 0.80 g (56.10%). Mp 107–109 °C, R : 0.395 (A)
a
,13
a
,14
a
)-17-[2-(1,3-Dioxolan-2-yl)ethyl]-3-meth-
hydrochloride, 2, C20
H
2
The reaction mixture was
f
f
1
1
and 0.625 (B). H NMR (d
6
and 0.473 (B). H NMR (d -DMSO): 7.01–7.03 (m, 1H); 6.74 (s,
6
7
3
2
1
1H); 6.67–6.69 (m, 1H), 4.81–4.84 (m, 1H), 3.85–3.88 (m, 2H);
3.74–3.76(m, 2H), 3.73 (m, 1H), 3.69 (s, 3H); 2.85 (s, 1H); 2.80(s,
1H); 2.76–2.77 (m, 1H); 2.53–2.56 (m, 1H); 2.42–2.47 (m, 1H);
2.32–2.46 (m, 1H); 1.85–1.92 (m, 1H); 1.60–1.70 (m, 3H); 1.53–
1.58 (m, 1H); 1.45–1.48 (m, 1H); 1.33–1.37(m, 2H), 1.22–1.30(m,
3H), 1.15–1.19(m, 1H), 0.93–1.02 (m, 1H). C NMR (d -DMSO):
157.82, 141.23, 129.51, 128.43, 110.97, 110.53, 102.56, 64.16,
55.53, 54.87, 49.54, 44.94, 44.59, 41.67, 39.53, 37.40, 36.01,
32.07, 26.39, 26.06, 23.76, 21.96. MS calcd for C22H31NO3
357.2304; found 357.2307, [M+H] 358.2380. Anal calcd C, 73.91;
.66 (m, 2H); 3.19–3.27 (m, 2H); 3.14 (s, 2H); 2.92–2.98 (m, 1H);
.43–2.47(m, 2H); 2.25–2.28 (m, 1H); 1.92–2.00 (m, 1H); 1.59–
.62 (m, 1H); 1.42–1.53 (m, 3H); 1.26–1.33 (m, 2H); 1.13–1.23
(
m, 1H); 0.90–1.00 (m, 1H); 13C NMR (d
6
-DMSO): 158.52, 138.60,
1
3
1
3
29.30, 125.84, 117.79, 112.15, 110.58, 57.07, 55.04, 47.83, 40.87,
9.52, 38.27, 35.83, 34.76, 25.35, 25.31, 22.79, 21.41, 12.80, MS:
6
+
calcd for C20
H
26
N
2
O 310.4332, found 310.2048 [M+H] 311.2121.
Anal calcd C, 69.25; H, 7.85; N, 8.08; O, 4.61; Cl, 10.22; found C,
+
6
9.23; H, 7.85; N, 8.08; O, 4.61; Cl, 10.24.
H, 8.74; N, 3.92; O, 13.43; found C, 73.90; H, 8.74; N, 3.92; O, 13.44.
2
.1.2.3. (9
a
,13
a
,14
a
)-17-(3-Cyanopropyl)-3-methoxymorphinan
29BrN O. The reaction mixture was
heated for 13 h and the white crystalline precipitate of 3 was
obtained. Yield: 1.20 g (92.31%). Mp 230–232 °C, R : 0.551 (A)
-DMSO): 9.57–9.74 (m, 1H); 7.12–7.14
m, 1H); 6.81–6.84 (m, 2H); 3.73 (s, 4H); 3.14–3.25 (m, 3H); 3.09
s, 1H); 2.95–3.02 (m, 1H); 2.62–2.66 (m, 2H); 2.42–2.49 (m, 2H),
hydrobromide, 3, C21
H
2
2
.1.2.7.
hydrobromide, 7, C21
heated for 16 h and the white crystalline precipitate of 7 was
obtained. Yield: 0.63 g (48.30%). Mp 184–187 °C, R : 0.539 (A)
-DMSO): 9.40–9.64 (d, 1H); 7.11–7.14
d, 1H); 6.82–6.84 (m, 2H); 4.62–4.75 (m, 1H); 4.51–4.52 (m,
(9
a
,13
a
,14
a
H
)-17-(2-Oxobutyl)-3-methoxymorphinan
30BrNO The reaction mixture was
2
.
f
1
and 0.510 (B). H NMR (d
(
(
6
f
1
and 0.506 (B). H NMR (d
(
6
2
1
.00–2.13 (m, 3H); 1.86–1.94 (m, 1H); 1.60–1.63 (m, 1H); 1.49–
.52 (m, 3H); 1.25–1.32 (m, 2H); 1.10–1.17 (m, 1H); 0.90–0,99
1
1
2
1
H); 3.73 (s, 3H); 3.62 (s, 1H); 3.14–3.27 (m, 2H); 2.95–3.11 (m,
H); 2.52–2.58(m, 2H), 2.38–2.50 (m, 1H), 2.38–2.58 (m, 1H);
.13–2.21 (m, 1H); 1.87–2.03 (m, 1H); 1.60–1.63 (m, 1H); 1.49–
1
3
(
1
3
m, 1H). C NMR (d
6
-DMSO): 158.54, 138.65, 129.22, 125.78,
19.72, 112.17, 110.64, 56.91, 55.05, 51.72, 46.18, 39.52, 38.50,
5.87, 34.79, 25.37, 25.25, 22.63, 21.41, 19.92, 14.03. MS: calcd
.52 (m, 2H); 1.27–1.43 (m, 3H); 1.04–1.18 (m, 1H); 0.98–1.02
13
(
6
m, 3H); 0.86–0.96 (m, 1H). C NMR (d -DMSO): 204.19, 158.54,
+
for C21
28 2
H N O 324.6324; found 3245.6318, [M+H] 325.2640. Anal
1
4
41.72, 130.87, 128.55, 115.56, 110.75, 62.98, 59.37, 55.45, 52.98,
6.74, 38.42, 37.89, 37.19, 32.83, 24.78, 23.94, 22.80, 20.74, 7.89.
calcd C, 62.22; H, 7.21; N, 6.91; O, 3.95; Br, 19.71; found C, 62.12; H
7
.12; N, 6.88; O 3.91; Br 19.97.
327.2198; found 327.2202; [M+H]+
MS: calcd for C21
H29NO
2
3
1
28.2275. Anal calcd C, 61.76; H, 7.40; N, 3.43; O, 7.84; Br,
9.57; found C, 61.80; H, 7.40; N, 3.43; O, 7.86; Br, 19.51.
2
.1.2.4.
(9
a
,13
a
,14
a
)-17-Amidomethyl-3-methoxmorphinan
27BrN The reaction mixture was
heated for 15 h and the white crystalline precipitate of 4 was
obtained. Yield: 0.73 g (58.10%). Mp 247–250 °C, R : 0.727 (A)
-DMSO): 9.88–10.09 (m, 1H); 8.16–
.20 (m, 1H); 7.70–7.76 (m, 1H), 7.10–7.14 (m, 1H); 6.81–6.83
m, 2H); 4.00–4.34 (m, 2H); 3.72 (s, 4H); 3.25–3.30 (m, 1H),
hydrobromide, 4, C19
H
2 2
O .
2
.1.2.8.
(9a,13a,14a)-17-[(4,5-Dihydro-1H-imidazol-2-
f
1
yl)methyl]-3-methoxymorphinan hydrobromide, 8, C21H30BrN3-
O. The reaction mixture was heated for 16 h and the white
crystalline precipitate of 8 was obtained. Yield: 0.52 g (38.10%).
Mp 170–172 °C, R
1
and 0.650 (B). H NMR (d
8
(
6
1
f
: 0.429 (A) and 0.663 (B). H NMR (d
0.02 (s, 1H); 7.09–7.11 (m, 1H); 6.78–6.81 (m, 2H); 3.89 (s, 3H);
.72–3.82 (m, 8H); 2.86–2.98 (m, 3H); 2.41–2.45 (m, 2H);
6
-DMSO):
3
2
.11–3.16 (m, 1H); 2.98–3.04 (m, 1H); 2.77–2.84 (m, 1H); 2.43–
.46 (m, 1H); 2.14–2.38 (m, 1H); 1.90–1.98 (m, 1H); 1.58–1.61
3
(
m, 1H); 1.40–1.52 (m, 3H); 1.25–1.36 (m, 2H); 1.10–1.17 (m,
H); 0.91–0.95 (m, 1H). 1 C NMR (d
3
2.03–2.04 (m, 1H); 1.87 (s, 1H); 1.60–1.63 (m, 1H); 1.50–1.53 (m,
H); 1.27–1.44 (m, 4H); 1.13–1.20 (m, 1H); 0.95–0.98 (m, 1H).
1
1
3
6
-DMSO): 168.72, 158.57,
1
38.72, 129.27, 125.73, 112.16, 110.64, 58.47, 55.07, 52.46, 46.79,
9.53, 38.37, 35.68, 34.77, 27.94, 25.03, 23.39, 21.44. MS: calcd
1
3
C NMR (d
6
-DMSO): 161.68, 158.44, 141.79, 130.18, 128.31,
+
115.56, 110.79, 59.34, 55.26, 52.98, 50.09, 49.36, 48.56, 46.72,
for C19
H
26
N
2
O
2
314.4219, found 314.1999; [M+H] 315.2072. Anal
3
8.41, 37.86, 37.50, 24.79, 23.97, 22.21, 20.98. MS: calcd for
calcd C, 57.72; H, 6.88; N, 7.09; O, 8.09; Br, 20.21; found C, 57.60;
H, 6.80; N, 7.04; O, 8.05; Br, 20.51.
+
C
21
29 3
H N O 339.2709; found 339.2705; [M+H] 340.2715. Anal
calcd C, 60.00; H, 7.19; N, 10.00; O, 3.81; Br, 19.01; found C,
9.60; H, 7.17; N, 9.95; O, 3.80; Br, 19.48.
5
2
.1.2.5. (9
oxymorphinan hydrobromide, 5, C21
mixture was heated for 16 h and the white crystalline precipitate of
was obtained. Yield: 0.66 g (48.10%). Mp 282–284 °C, R : 0.662 (A)
-DMSO): 9.03 (s, 1H); 7.02–7.04 (m, 1H);
.75 (s, 1H); 6.68–6.70 (m, 1H), 4.80–4.82 (m, 1H), 3.84–3.89 (m,
a
,13
a
,14
a
)-17-[(1,3-Dioxolan-2-yl)methyl]-3-meth-
H
30BrNO The reaction
3
.
2.1.3. Synthesis of N-alkylated dextromethorphan derivative
compound 4 (method B)
5
f
1
and 0.620 (B). H NMR (d
6
2 mmol of 1 dissolved in 3 ml DMSO was cooled down in ice
bath and 0.2 g 1.45 mmol of anhydrous potassium carbonate was
added. 0.5 ml of 30% solution of hydrogen peroxide was added
dropwise and after 30 min of mixing on a magnetic stirrer 9 ml
of water was added. The obtained precipitation was filtered and
washed with water.
6
2
2
1
1
H); 3.78–3.81(m, 2H), 3.75 (m, 1H), 3.70 (s, 3H); 2.85 (s, 1H);
.80 (s, 1H); 2.75–2.77 (m, 1H); 2.53–2.56 (m, 1H); 2.42–2.50 (m,
H); 1.88–1.93 (m, 1H); 1.65–1.73 (m, 2H); 1.53–1.61 (m, 1H);
.46–1.49 (m, 1H); 1.33–1.36 (m, 2H), 1.20–1.29 (m, 3H),

K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
6849
2
4
.1.3.1. 2-[(9
a
.
,13
a
,14
a
)-3-Methoxymorphinan-17-ylacetamide,
: 0.729
-DMSO): 7.70–7.76 (m, 1H), 7.10–
using GraphPad Prism Version 5.0 software (GraphPad Software,
Inc., La Jolla, CA, USA).
, C19H
26
N
2
O
2
Yield: 0.22 g (35.00%). Mp 78–80 °C, R
f
1
(
A) and 0.650 (B). H NMR (d
6
7
3
2
.14 (m, 1H); 6.81–6.83 (m, 2H); 4.00–4.34 (m, 2H); 3.72 (s, 4H);
.25–3.30 (m, 1H), 3.11–3.16 (m, 1H); 2.98–3.04 (m, 1H); 2.77–
.84 (m, 1H); 2.43–2.46 (m, 1H); 2.14–2.38 (m, 1H); 1.90–1.98
2.3. Molecular modeling
2.3.1. Homology models
(
m, 1H); 1.58–1.61 (m, 1H); 1.40–1.52 (m, 3H); 1.25–1.36 (m,
The sequences data for the
a3 and b4 nAChRs subunits were
2
1
5
2
3
H); 1.10–1.17 (m, 1H); 0.91–0.95 (m, 1H). 13C NMR (d
6
-DMSO):
obtained from the UniProt database implemented in ExPASy Bioin-
2
0
68.72, 158.57, 138.72, 129.27, 125.73, 112.16, 110.64, 58.47,
5.07, 52.46, 46.79, 39.53, 38.37, 35.68, 34.77, 27.94, 25.03,
formatics Resource Portal. The cryo-electron microscopy struc-
ture of the Torpedo nAChR determined at ꢀ4 Å resolution (PDB ID:
2BG9) was used as a template. Sequence alignment of the whole
3.39, 21.44. MS: calcd for
14.1999, [M+H] 315.2072. Anal calcd C, 72.58; H, 8.33; N, 8.91;
C
19
H
26
N
2
O
2
314.4219; found
+
Torpedo nAChR (i.e.,
and b4 nAChRs subunits was performed using ClustalW2 Server.
Two 1 subunits in the Torpedo template were used to build two
3 subunits in the target, whereas b1, , and d in the template were
a1, a1, b1, c, and d) as well as human (h) a3
21
O, 10.18; found C, 72.56; H, 8.33; N, 8.92; O, 10.19.
a
2
.1.4. Synthesis of N-alkylated dextromethorphan derivatives
compound 9 (method C)
.51 g (2 mmol) of 3-methoxymorphinan and 10 ml of vinyl
a
c
22
used to construct three b4 subunits in the target. Modeller 9.9 was
used to obtain the population of 100 homology models of human
0
ketone was cooled down in ice bath and 0.4 ml of Triton B was
added at temperature of 0–5 °C. The reaction mixture was stirred
on a magnetic stirrer for 3 h. 50 ml of diethyl ether was added
and ethereal solution was decanted and the obtained precipitate
was filtered and washed with diethyl ether.
a3b4 nAChR. The best model was subjected to model quality
assessments, using the MOE Molecular Environment module for
Ramachandran plots (http://www.chemcomp.com/software.htm)
2
3
24
25
and the web-based tools of Annolea, Verify3D and ProCheck.
2
.3.2. Molecular docking and molecular dynamics
DM derivatives were optimized using HF approximation and
2
.1.4.1.
(9
a
,13
a
,14
a
H
)-17-(3-Oxobutyl)-3-methoxymorphinan
30BrNO Yield: 0.15 g (23.10%). Mp
6
: 0.539 (A) and 0.550 (B). H NMR (d -DMSO):
hydrobromide, 9, C21
2
.
6-31G basis set of Spartan 10 V.1.1.0 (Wavefunction, Inc. Irvine,
CA 92612 USA). Spartan was also used to calculate electrostatic
potential. Tautomerization and protonation state at physiological
1
1
9
3
1
2
1
1
40–142 °C, R
f
.19–9.35 (m, 1H); 7.12–7.14 (m, 1H); 6.82–6.84 (m, 2H); 3.73 (s,
H); 3.69 (s, 1H); 3.54–3.55 (m, 1H); 3.14–3.23 (m, 1H); 3.12 (s,
H); 3.04–3.09 (m, 1H); 2.98–3.02 (m, 2H); 2.93–2.95 (m, 1H);
.63–2.70 (m, 1H); 2.32–2.44 (m, 1H); 2.17 (s, 3H); 2.00–2.03 (m,
H); 1.81–1.88 (m, 1H); 1.61–1.63 (m, 1H); 1.46–1.52 (m, 3H);
.24–1.35 (m, 2H); 1.12–1.18 (m, 1H); 0.87–0.99 (m, 1H).
-DMSO): 208.97, 158.12, 141.79, 130.18, 128.31, 115.50,
10.76, 59.37, 55.23, 48.21, 47.96, 46.76, 41.59, 38.44, 37.76,
7.18, 30.67, 24.79, 23.91, 22.22, 20.98. MS: calcd for C21
2
6
27
pH were assessed with LigPrep and Epik modules of Schröding-
er suite of software. Molecular docking was performed with Glide.
The docking space (i.e., Grid definition) covered the ion channel of
studied nAChR model and was selected based on the experimental
1
3
19
C
evidence. Molecular docking was performed with Glide using
NMR (d
6
standard precision (SP) protocol. The complexes subjected to
molecular dynamic (MD) simulations with Desmond v. 3.0.3.128
were selected based on the visual inspection rationalised by earlier
1
3
3
6
7
H29NO
2
+
studied on dextromethorphan binding mode in the a
3b4 nAChR.6
27.2198; found 327.2202; [M+H] 328.2275. Anal calcd C,
1.76; H, 7.40; N, 3.43; O, 7.84; Br, 19.57; found C, 61.60; H,
.37; N, 3.40; O, 7.82; Br, 19.81.
Ligand-receptor complexes were inserted into POPC membrane
and solvated with water. Ions were added to neutralize protein
charges and to the concentration of 0.15 M NaCl. The complexes
embedded in membrane were first minimized and then subjected
to 1 ns MD in NVT ensemble, followed by 10 ns MD in NPT ensem-
2
2
.2. In vitro assay
2
9
30
.2.1. Cell lines and culture
The KXa3b4R2 (expressing rat a3b4 nAChRs) cell lines have
ble. Yasara Structure , PyMol v. 1.5.0.3 and MOE Molecular Envi-
ronment were also used for visualization of results.
19
been previously described. The cell lines were maintained in
MEM supplemented with 10% FBS, 100 units/ml penicillin G,
2.4. Behavioral studies
1
5
00 mg/ml streptomycin and 0.7 mg/ml geneticin at 37 °C with
% CO in a humidified incubator.
2
2.4.1. Animals
The experiments were carried out on naïve male Wistar rats
weighing 250–300 g (Farm of Laboratory Animals, Warszawa,
Poland) or naïve male Swiss mice weighing 20–25 g (Farm of Lab-
oratory Animals, Warszawa, Poland) at the beginning of the exper-
iments. The animals were group-housed, kept under standard
laboratory conditions (12/12-h light/dark cycle) with free access
to tap water, and adapted to the laboratory conditions for at least
one week. The rats had limited access to lab chow (150 g per 8 rats
on 24 h, given as a single meal in the evening) (Agropol, Motycz,
Poland). The rats were handled once a day for 5 days preceding
the experiments. Additionally, all efforts were made to minimize
animal suffering and to use only the number of animals necessary
to produce reliable scientific data. Each experimental group con-
sisted of 7–14 animals. The experiments were performed between
9.00 a.m. and 5.00 p.m. All experiments were carried out according
to the National Institute of Health Guidelines for the Care and Use
of Laboratory Animals and the European Community Council
Directive of 24 November 1986 for Care and Use of Laboratory Ani-
mals (86/609/EEC), and approved by the local ethics committee.
2
.2.2. Whole-cell current recording
Whole cell current was measured using EPC 10 HEKA Instru-
ments amplifier. All drugs were applied by fast perfusion system
DynaFlow, CelletriconAB, Mölndal, Sweden) with 16 channels
chip. Cells were clamped at 60 mV holding potential. KX 3b4R2
cells were collected at 70% confluence and suspended in HEPES
10 mM, pH 7.4] containing 120 mM NaCl, 3.1 mM KCl, 2 mM
CaCl , 5 mM glucose at room temperature. The glass pipettes with
–7 M resistance were filled with HEPES [10 mM, pH 7.2], con-
taining 145 mM CsCl, 1 mM MgCl , 2 mM ATP, 1 mM EGTA. All
a3b4 nAChRs
(
a
[
2
5
X
2
experiments were performed under Visual control using an Olym-
pus CKX41 inverted microscope (Olympus Corporation). Test com-
pounds were applied at concentrations of 0.1, 1, 10, and 100 nM, 1,
1
0 and 100
the presence of 100
For inhibition studies, the decrease in current relative to 100
S)-nicotine (100%) was determined. Raw data was collected using
PatchMaster Version 2.32 software (Celletricon AB) and analysed
l
M in the absence of (S)-nicotine for stimulation or in
l
M (S)-nicotine for inhibition measurements.
lM
(

6
850
K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
2
.4.2. Drugs
The compounds tested were: (ꢁ)-nicotine hydrogen tartrate
Sigma, St. Louis, MO, USA), dextromethorphan (DM), and com-
(10 cm ꢂ 10 cm). The compartments were differentiated by both
visual and tactile cues: one was white with a grooving on the black
floor, another was black with wire mesh on the white floor. The
central, small grey area served as connection and a start compart-
ment. The testing boxes were kept in a soundproof room with neu-
tral masking noise and a dim 40-lꢂ illumination.
(
pounds 1, 5 and 6 (see Fig. 3 and Table 1). The selection of the com-
pounds for behavioural studies was connected with the easiness
and yield of the synthesis as well as with the idea to explore the
spectrum of in vivo effects of compounds with different in vitro
activity. All agents were dissolved in saline (0.9% NaCl). The pH
of the nicotine solution was adjusted to 7.0. Compounds 1, 5 and
2.4.3.2. Locomotor sensitization.
Locomotion was recorded
individually in round actometer cages (32 cm in diameter; Multi-
serv, Lublin, Poland) kept in a sound-attenuated experimental
room. Two photocell beams located across the axis measured the
animal’s movement automatically.
6
(1/10 and 1/20 LD50 previously established) were suspended in
one drop of 1% solution of Tween 80 (Sigma, St. Louis, MO, USA)
and diluted in saline. Fresh drug solutions were prepared on each
day of behavioral testing. All agents were administered intraperito-
neally (ip) in a volume of 5 ml/kg (rats) or 10 ml/kg (mice) and,
except for nicotine, drug doses refer to the salt form. Control
groups received vehicle injections at the same volume and via
the same route of administration.
2.4.4. Procedures
2.4.4.1. Influence of dextromethorphan and compounds 1, 5
and
6
on the expression of nicotine-induced CPP in
rats.
The place conditioning experiment (unbiased design)
The doses of nicotine and DM were chosen based on literature
consisted of pre-conditioning, conditioning and post-conditioning
phases. For pre-conditioning (Day 1), each animal was placed sep-
arately in the neutral area with the guillotine doors removed to
allow access to the entire apparatus. The amount of time the rats
spent in each of the two large compartments was measured (a
baseline preference), and observed on a monitor through a video
camera system for 15 min. Rats that showed a preference for one
of the compartments were considered not to be neutral in prefer-
ence for either side and were excluded from further study (less
than 5% of rats).
The conditioning phase consisted of 30 min sessions, two per
day. In the morning sessions, the rats were injected with vehicle
and confined in one compartment, whereas on afternoon sessions,
they received injections of nicotine (0.175 mg/kg, base, ip) and
were then confined in the opposite compartment. Sessions were
conducted twice each day with an interval of 6–8 h for 3 consecu-
tive days (Days 2–4). Injections were administered immediately
before confinement in one of the two large compartments, as men-
tioned above. Control group received vehicle every day.
3
1–33
data,
whereas the doses of new compounds were chosen
based on preliminary studies.
The general behaviors of the mice were observed continuously
for 1 h after the treatment and intermittently for 4 h during a
period of 24 h. All mice were further observed for any signs of
toxicity, deaths, and the latency of death during a period of one
week. The toxicity of the studied compounds was comparable to
dextromethorphan which is: LD50 in rats 350 mg/kg34 and LD50
3
5
in mice 39 mg/kg. The studied derivatives most probably hit
the same off-targets as dextromethorphan, that is, NMDA receptor
(
it was confirmed experimentally and will be published elsewhere)
and sigma receptor.
2
2
.4.3. Apparatus
.4.3.1. Conditioned place preference (CPP).
Each of six
rectangular boxes (60 cm ꢂ 35 cm ꢂ 30 cm) was divided into three
compartments: two large compartments (20 cm ꢂ 35 cm) were
separated by removable guillotine doors from a small central area
On Day 5, conducted one day after the last conditioning trial,
animals were placed in the neutral area with the guillotine doors
removed and allowed free access to all compartments of appara-
tus for 15 min. The time spent in the vehicle- and drug-paired
compartments was recorded for each animal. To evaluate the
effects of DM and tested compounds on the expression of nico-
tine-induced CPP, on this post-conditioning phase, rats pretreated
with vehicle were injected with vehicle (n = 7) or nicotine
Table 1
The investigated dextromethorphan derivatives and their effect on the (S)-nicotine-
induced current in KXa3b4R2
(
0.175 mg/kg, ip, n = 8) and rats pretreated nicotine (during condi-
tioning, as mentioned above) were injected with nicotine
0.175 mg/kg, ip, n = 10), and DM (5 mg/kg, n = 7 and 10 mg/kg,
(
n = 7) or compounds 1 (1/10, n = 7 or 1/20 LD50, n = 7), 5 (1/10,
n = 8 or 1/20 LD50, n = 7) and 6 (1/10, n = 8 or 1/20 LD50, n = 7),
6
0 min before nicotine injection and were immediately tested
Dextromethorphan derivatives
Substituents (R)
IC50 (lM)
for the expression of CPP.
1
2
3
4
–CH
–CH
–CH
–CH
2
2
2
2
–CN
–CH
–CH
10.47 ± 1.67
176.00 ± 1.1
87.00 ± 1.12
41.40 ± 1.14
2
2
–CN
–CH
2
.4.4.2. Influence of DM, compounds 1, 5 and 6 on the expression
2
–CN
of nicotine-induced locomotor sensitization in mice. This
–CO–NH
2
method was similar to that used in our previous experiments
accordingly to the data indicating that at the dose of 0.175 mg/kg
nicotine produces robust locomotor sensitization in mice under
O
5
1.95 ± 0.13
O
O
33
our laboratory conditions. During the pairing phase (days 1–9),
6
7
7.14 ± 1.0
O
mice received the following injections: vehicle (ip) + vehicle (sc)
or vehicle (ip) + nicotine (0.175 mg/kg, sc) every other day for five
sessions.
–CH
2
–CO–CH
2
–CH
3
2.50 ± 0.19
N
8
6.61 ± 1.06
The mice remained drug free for one week and, on the challenge
day (Day 16), the mice pretreated with vehicle were challenged
with nicotine (0.175 mg/kg, sc n = 8) and the mice pretreated with
nicotine (as mentioned above) were further challenged with
nicotine (0.175 mg/kg, sc, n = 8) or DM (5 mg/kg, n = 8 and
N
H
–CH
9
–CH
–CH
–H
2
2
–CO–CH
3
2.57 ± 0.18
11.05 ± 1.08
3.08 ± 1.03
Dextromethorphan (DM)
Nordextromethorphan (NORDM)
3

K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
6851
1
0 mg/kg, n = 10) and compounds 1 (1/10, n = 8 or 1/20 LD50
n = 10), 5 (1/10, n = 8 or 1/20 LD50, n = 10) and 6 (1/10, n = 8 or 1/
n = 9), 60 min before nicotine challenge injection.
,
2
0 LD50,
Locomotor activity was recorded for 60 min during the pairing
phase (days 1–9) and on the 16th challenge test, immediately after
injections.
2
.4.5. Statistical analysis
For the CPP paradigm, the results are expressed as scores, that
is, the differences (in s) between post-conditioning and pre-condi-
tioning time spent in the drug-associated compartment (means
±
SEM). The data were analyzed by the analysis of variance
(
ANOVA). Locomotor activity was expressed as a number of photo-
cell beam breaks (means ± SEM). For locomotor sensitization, data
were analyzed using repeated measure analysis of variance
(
ANOVA) with treatment as independent factor and days as
repeated measures. The response to drugs on the challenge day
was compared using one-way ANOVA. Post-hoc comparison of
means was carried out with the Tukey’s test for multiple compar-
isons, when appropriate. The confidence limit of p <0.05 was con-
sidered statistically significant.
3
3
. Results and discussion
.1. Chemistry
Figure 2. Demethylation of dextromethorphan.
Compound 1 was previously obtained in the reaction of DM and
3
6,37
trimethylcyanosilane.
These studies were aimed to oxidize ter-
tiary amines and alkaloids through monoelectronic transfer. The
pharmacological activity of compound 1 has been not determined.
Compound 2, obtained from commercial sources was found to
inhibit DM binding sites in guinea pig brain homogenates with
3
8
IC50 of 775 nM.
Compounds 9 has been designed by our research group as NCI
of nAChRs using computer based model3 but it has been never
synthesized. Compounds 3–8 have not been described yet in the
available literature.
9
Demethylation of DM was performed using Peete’s method
4
0
(
Fig. 2). All the compounds 1–9 were obtained from demethylat-
ed DM using various alkylating agents (Fig. 3).
3
.2. Pharmacological activity
It has been previously reported that DM presents a non-
competitive inhibitory activity toward
a3b4 nAChRs, with 20 times
higher binding affinity than levomethorphan to the hydrophobic
cleft the two enantiomers bind.1
6,17,38
In this regard, the effect of
3b4 nAChRs was
studied compounds on the function of the
a
evaluated using patch-clamp technique in the whole-cell configu-
ration. The compounds were first examined for their ability to
induce current activation at the range of 0.1–100 lM concentra-
tions. No measurable response was observed at any tested
concentration, data not shown. The ability of the test compounds
Figure 3. Synthesis of compounds 1–9.
to affect (S)-nicotine-induced current was assessed using 100
lM
(S)-nicotine and appropriate concentration range of studied
dextromethorphan derivatives. Compound 5, 7 and 9 as well as
nordextromethorphan (NORDM) induced ꢀ3–6-fold higher inhibi-
tory activity on (S)-nicotine-induced currents (IC50 values of 1.95,
3.3. Molecular modeling
2
.50, 2.57, and 4.26
l
M, respectively; see Table 1 and Fig. 4) com-
M). Furthermore,
3.3.1. Homology modeling and model quality assessment
The sequence identity of the
pared to dextromethorphan (DM) (IC50 = 12.87
l
a
3 and b4 subunit was ꢀ45–55%
compounds 1, 6, and 8 produce suppression of (S)-nicotine-induced
current comparable to DM (Table 1). The application of compound 2
and 3 had no significant effect on the (S)-nicotine-induced currents
comparing to each subunit of Torpedo nAChR. Homology models
were assessed considering their Discrete Optimized Protein Energy
(DOPE) profiles. The best model was subjected to model quality
assessments, using the following tools: MOE Molecular Environ-
ment module for Ramachandran plots and the web-based tools
(
IC50 values of 176 and 87 lM, respectively; see Table 1) indicating
that both compounds do not produce any effect on the
nAChRs.
a3b4
2
3
24
25
of Annolea, Verify3D and ProCheck.

6
852
K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
along the axis of the channel.41 In the considered ion channel, the
amino acid rings in each
a
3/b4 subunit are named: outer or extra-
0
0
cellular (position 20 ), nonpolar (position 17 ), PHE/VAL (position
0
0
0
0
1
3 ), LEU (position 9 ), SER (position 6 ), THR (position 2 ), interme-
0
0
diate (position ꢁ2 ), and cytoplasmic or inner (position ꢁ5 ).
Accordingly, each b4 subunit carries important change in compari-
4
1
son to most
a
and non-
a
nAChR subunits: that is, phenylalanine
0
that are located at position 13 instead of valine residues.
All the studied compounds have a positively charged nitrogen
atom. This feature enables them to be attracted by the negatively
0
charged residues at the extracellular mouth (i.e., at 20 position)
which results in pulling in the molecule into the ion channel. In
addition, within the binding pocket located deeper in the ion chan-
0
nel (close to position 13 ) there is the b4-Phe255 residue that may
participate in the cation-p interactions with the protonated nitro-
gen. However, the docking results suggested that the protonated
nitrogen atom of studied compounds as well as N-substituted moi-
Figure 4. The effect of the concentration of dextromethorphan (
methorphan (h) as well as compound 5 (d), and 9 (N) on the (S)-nicotine-induced
current in KX 3b4R2 cells determined using whole-cell configuration of the patch-
clamp technique. The calculated IC50 values are included in the Table 1.
r
), nordextro-
a
eties are directed toward the polar ring formed by serine (at posi-
0
tion 6 ) residues. Cation–
p interaction energies are of the same
3
.3.2. Ligand–receptor interactions
order of magnitude as hydrogen bonds or salt bridges and in the
The studied non-competitive antagonists interact with the
considered case hydrogen bonds were favored.
transmembrane region of the
a
3b4 nAChR, which consists of five
In particular, each ligand interacts with the middle portion of
the ion channel between residues that form PHE/VAL (at position
13 ) and SER (at position 6 ) rings (see Fig. 5 and 7) by van der
subunits (i.e., two 3 and three b4), each composed of M1–M4
a
0
0
transmembrane segments. Among the transmembrane segments,
M2 is relatively more hydrophilic and thus, it tends to face the cen-
Waals interactions as well as hydrogen bonding. More specifically,
4
1
0
ter of the ion channel. The amino acid sequences in M2 are highly
conserved among different subunits and species, forming a series of
amino acid rings exposed to the center of the pore and distributed
DM interacted with b4-Phe255 and
a
3-Val254 at position 13 , b4-
0
Ala252 and
a3-Ser251 at position 10 , b4-Leu251 and
a
3-Leu250 at
0
0
position 9 , as well as with b4-Ser248 at position 6 (Fig. 5).
Figure 5. DM molecule in the binding site of a3b4 nAChR model. (A) Scheme of interactions; (B) Side view of the whole model showing the ligand (magenta) docking site; (C)
0
0
Detailed view of the luminal site (i.e., within the ion channel) that is located between PHE/VAL (at position 13 ) and SER (at position 6 ) rings. Residues involved in ligand
binding are shown explicitly in stick mode (element coded mode). DM is rendered in ball (B) or stick (C) mode. For clarity, one b4 subunit is hidden, whereas the M2 segments
are show in yellow. All non-polar hydrogen atoms are hidden.

K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
6853
Figure 6. The map of electrostatic potential distribution for inactive compounds 2 (A), 3 (B) and active compounds 5 (C) and 9 (D).
Figure 7. DM derivatives, including compounds 5, 8 and 9 (shown in magenta) in the binding site within the ion channel of
interactions and detailed view of compound 5 binding site, respectively. This ligand is stabilized in the binding pocket by hydrogen bond interaction, formed between
dioxolane oxygen of the ligand and the hydroxyl group of 3-Ser247; (C and D) present the scheme interactions and detailed view of compound 8 binding site, respectively. In
a3b4 nAChR model. (A and B) show the scheme
a
addition, this ligand is also stabilized in the binding pocket by hydrogen bond interaction, formed between the nitrogen from imidazole ring of compound 8 and hydroxyl
group of the b4-Ser248; (E and F) depict the scheme interactions and detailed view of compound 9 binding site, respectively. Moreover, this ligand is stabilized in the binding
pocket by hydrogen bond interaction, formed between carbonyl oxygen of 3-oxo-butyl moiety of the ligand and hydroxyl group of a3-Ser247. For (B, D, and F) the residues
involved in ligand binding are shown explicitly in stick mode (element coded mode). Each compound is rendered in stick mode and colored in magenta. For clarity, one b4
subunit is hidden, whereas the M2 segments are show in yellow. All non-polar hydrogen atoms are hidden.

6
854
K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
Figure 8. DM derivatives complexes with the
a
3b4 nAChR models obtained after the MD simulation for DM (A) and compounds 5 (B), 8 (C), and 9 (D) (shown in green) as well
as the RMSD values calculated during the MD run for each mentioned before complex. Each ligand is stabilized in the binding pocket by hydrogen bond interaction, formed
between the protonated nitrogen (A), dioxolan oxygen (B), the nitrogen from imidazol-2-yl ring (C) or the carbonyl oxygen of 3-oxo-butyl moiety and the hydroxyl group of
b3-Ser248, respectively. See Figure 7 for other details.

K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
6855
However, the docking results for DM derivatives (i.e., com-
pounds 5, 8 and 9) suggest that they interact with the same resi-
dues as indicated for DM, and these ligands are stabilized by
additional hydrogen bonding. In particular, for compound 5 hydro-
gen bond is formed between oxygen of dioxolane moiety of the
MD run it turned out that the interactions with b4-Ser248 are
favored over interactions with 3-Ser247 (Fig. 8).
a
3.4. Behavioral effects of selected derivatives
ligand and hydroxyl group of
a
3-Ser247 (see Fig. 6). However,
3.4.1. Influence of DM and compounds 1, 5 and 6 on the
expression of nicotine-induced CPP in rats
compounds 8 and 9 are stabilized by hydrogen bonds, formed
between the nitrogen from imidazole ring of compound 8 and
hydroxyl group of the b4-Ser248 and between the carbonyl oxygen
of 3-oxo–butyl moiety of compound 9 and hydroxyl group of the
All rats showed no significant place preference for the drug-
associated compartment before drug conditioning, which indicated
that the CPP apparatus that we used was of a non-biased design.42
Figure 9 indicated the effects of DM and compounds 1, 5 and 6 on
nicotine induced CPP [one-way ANOVA: F(12,77) = 2.687,
p = 0.048]. Administration of nicotine (0.175 mg/kg, four condi-
tioning sessions, days 2–4) induced a clear place preference, shown
as significant increase in time spent in the drug-associated com-
partment during the post-conditioning test phase (Day 5, p
<0.001, post hoc Tukey’s test). Interestingly, post-hoc individual
comparisons indicated a significant inhibitory effect of DM at a
dose of 10 mg/kg (p <0.01) and compound 1 (1/20 LD50, p <0.05)
(Tukey’s test) on nicotine-induced CPP. Both DM and new com-
pounds do not induce significant changes in the place preference
test at the applied doses when administered without nicotine (data
not shown).
a3-Ser247 (see Fig. 7). These a3-Ser247 and b4-Ser248 are located
on the same level in the channel model and equivalent in molecu-
lar docking experiment and it is impossible to point out that only
one of these serine residues is responsible for interactions with
the channel blocker. Indeed, clusters of docking poses where
ligands interacted with either
a3-Ser247 or b4-Ser248 were
obtained and the final pose for a particular ligand was selected
based on Glide scoring and visual inspection. The poses of com-
pounds 6 and 7 are similar to those of compounds 5 and 9.
Molecular docking results do not make it possible to explain the
weak activity of compounds 2 and 3. However, the maps of electro-
static potential distribution (Fig. 6A and B, respectively) show that
in these derivatives the cyano group bears a strong partial negative
charge which is unfavorable regarding negative charge of amino
0
acid ring (position 20 ) at the entrance of the channel. In the case
3.4.2. Influence of DM and compounds 1, 5, and 6 on the
expression of nicotine-induced locomotor sensitization in mice
Concerning the locomotor response after administration of nic-
otine (0.175 mg/kg, sc) or saline during the pairing phase (Day 1
and Day 16—challenge), two-way ANOVA revealed a treatment
effect [F(11,166) = 20.05, p <0.0001], a day effect [F(1,166) = 19.92,
p <0.0001] with interaction between treatment and day [F(11,166) =
13.28, p <0.0001] (Fig. 10). On the Day 1, one-way ANOVA did not
reveal any significant treatment effect [F(11,94) = 1.254, p =
0.2660]. However, on the Day 16, after an additional injection of
nicotine, one-way ANOVA revealed a significant treatment effect
of other derivatives (e.g., compound 5 and 9, Fig. 6C and D, respec-
tively), the molecules do not exhibit highly negative charged
regions and may be easier attracted into the ion channel. In gen-
eral, nAChR ion channels are cation-selective and accommodate
better compounds with a balanced positive charge.
The molecular dynamics simulations showed that the obtained
ligand–receptor complexes were stable regarding potential energy
(Fig. 8). Moreover, ligand RMSD between starting and final com-
plexes after 10 ns MD was in the range of 0.21–1.47 Å which addi-
tionally confirms that the modeled complexes were stable. After
[F(11,94) = 2.797, p <0.0038]. Indeed, after this last nicotine injec-
tion (challenge), a significant difference in response was observed
when compared to either the first nicotine injection (p <0.01) or
in response to nicotine in animals treated repeatedly with vehicle
(p <0.01, Tukey’s test). DM at the dose of 10 mg/kg and adminis-
tered at both used doses before nicotine challenge dose, were
effective in blocking the expression of nicotine sensitization
Figure 9. Effects of dextromethorphan (DM, 5 and 10 mg/kg, ip) and compounds 1,
5, and 6 (1/10 and 1/20 LD50, ip) on the expression of nicotine-induced CPP. Place
preference procedure consisted of pre-conditioning, three conditioning sessions
with nicotine (0.175 mg/kg, ip) and post-conditioning test. During the post-
conditioning phase, rats received injection of vehicle, nicotine (0.175 mg/kg), or
DM (5 and 10 mg/kg) as well as compounds 1, 5, and 6 (1/10 or 1/20 LD50) 60 min
before nicotine injection (0.175 mg/kg), and were immediately tested for the
expression of CPP. Data represent means ± SEM and are expressed as the difference
Figure 10. Effects of dextromethorphan (DM, 5 and 10 mg/kg, ip) and compounds
1, 5, and 6 (1/10 and 1/20 LD50, ip) on the expression of locomotor sensitization to
nicotine in mice. Nicotine (0.175 mg/kg, sc) or vehicle were injected daily for 9 days,
every other day; on day 16 (a test for expression of sensitization) mice were given
nicotine (0.175 mg/kg, sc) or DM as well as compounds 1, 5, and 6, 60 min before
nicotine challenge injection. Data represent means ± SEM; n = 8–10 mice per group.
(
in s) between post-conditioning and pre-conditioning time spent in the drug-
⁄
⁄⁄
##
^^
associated compartment. n = 7–14 rats per group; p <0.05;
p
<0.01 versus
nicotine-conditioned group; # p <0.001 versus vehicle-conditioned control group
Tukey’s test).
p <0.01 versus the first pairing day; p <0.01 versus vehicle-pretreated and
##
⁄
⁄⁄
nicotine-challenged mice;
p
<0.05, p
<0.01, versus nicotine-pretreated and
(
nicotine-challenged mice (Tukey’s test).

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K. Jozwiak et al. / Bioorg. Med. Chem. 22 (2014) 6846–6856
(p <0.05 for compounds 5 and 6, 1/20 LD50; p <0.01 for other
References and notes
compounds, vs nicotine-pretreated and nicotine-challenged mice).
Neither DM nor any compounds tested given acutely or repeatedly,
significantly affected basal locomotor activity of mice (data not
shown).
Compounds 5 and 6 were highly potent in vitro and were also
effective in blocking the expression of the nicotine sensitization
in mice. However, both compounds did not show any significant
inhibitory effect on nicotine-induced CPP in rats. It might be due
to the strain differences in drug metabolism. Moreover, different
pathways as well as various brain areas are involved in CPP and
locomotor sensitization.
In the case of compound 1, which is effective at a dose of 1/20
LD50 only, it follows the trend that a lower dose of all new com-
pounds studied in vivo has greater inhibitory effect on nicotine-
induced CPP even though only the effects elicited by compound 1
is statistically significant. This observation will be explored in more
details and the results reported elsewhere.
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