Tetrasubstituted thiuronium salts as multitarget compounds affecting brain NMDA and AMPA receptors

Article abstract of DOI:10.1007/s11172-015-1137-6

Tetrasubstituted N′,N′-dibenzylisothioureas were shown to activate AMPA receptors. A number of structural specificities determining activity of the synthesized compounds against NMDA and AMPA receptors were identified.

Full text of DOI:10.1007/s11172-015-1137-6

Russian Chemical Bulletin, International Edition, Vol. 64, No. 9, pp. 2189—2194, September, 2015
2189
Tetrasubstituted thiuronium salts as multitarget compounds
affecting brain NMDA and AMPA receptors*
A. N. Proshin, V. V. Grigor´ev, I. G. Tikhonova, V. A. Palyulin, and S. O. Bachurin
Institute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi prꢀd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Fax: +7 (496) 524 9508. Eꢀmail: proshin@ipac.ac.ru
Tetrasubstituted N´,N´ꢀdibenzylisothioureas were shown to activate AMPA receptors.
A number of structural specificities determining activity of the synthesized compounds against
NMDA and AMPA receptors were identified.
Key words: tetrasubstituted N,Nꢀdibenzylisothioureas, radioligand binding, neuronal NMDA
and AMPA receptors, positive modulators of AMPA receptors, antagonists of NMDA
receptors.aa
The glutamatergic system plays a key role in both
memory consolidation processes1—3 and neurodegeneraꢀ
tive processes leading to the decline in cognitive functions
and memory loss related to the Alzheimer´s disease (AD)
and senile dementia.4,5 An important place in these proꢀ
cesses is taken by ionotropic glutamate receptors directly
bound with ionic channels, namely, the NMDA and
AMPA subtypes of glutamate receptors.6 The synthesis of
new neuroactive ligands of ionotropic glutamate receptors
is one of the important strategies for the development of
efficient agents for treatment and prevention of AD and
other neurodegenerative diseases accompanied by the deꢀ
cline in cognitive functions and memory loss.7—9 A low
affinity noncompetitive antagonist of NMDA receptors
Memantine, which is used for treatment of AD in Europe
from 2002 and in USA from 2004, is the most striking
example of a possibility for the efficient pharmacological
correction of AD through the influence on the glutamaterꢀ
gic system. The neuroprotective mechanism of the Meꢀ
mantine effect is explained by its ability to weaken the
neurodegeneration caused by ꢀamyloid (A) and to
improve the signal/noise ratio in the synaptic transmisꢀ
sion under conditions of the reduced uptake of neuromeꢀ
diator glutamate in the ADꢀtype pathologies.10
retardation of the processes of inactivation and/or desenꢀ
sitization of AMPA receptors.
Earlier, we have found that a number of dibenzyl deꢀ
rivatives of isothiourea improved memory and cognitive
functions in animals with toxicological model of the Alzꢀ
heimerꢀtype dementia.12 From the structural point of view,
dibenzylamines 1 and 2 can be regarded as modified at the
nitrogen atom openꢀchain analogues, isosteric to the agent
MKꢀ801, which is a high affinity blocker of the intrachanꢀ
nel site of the NMDA receptor. At the same time, these
compounds were found to also activate AMPA recepꢀ
tors.13,14
An alternative strategy for the improvement of cogniꢀ
tive functions and memory in AD is an approach dealing
with the stimulation of the AMPA subtype ionotropic
glutamate receptors. The positive modulators of AMPA
receptors enhancing response of the receptors on the effects
of endogenous glutamate and aspartate are of greatest inꢀ
terest.11 It was shown that this effect can be related to the
The purpose of the present work is to synthesize origiꢀ
nal isothiourea derivatives and study a relationship beꢀ
tween the structure of the compounds and their ability to
block NMDA receptors and activate AMPA receptors.
The reaction of the corresponding isothiocyanate
with amine 3 and subsequent Sꢀalkylation of thiourea 4
* Dedicated to Academician of the Russian Academy of Sciences
N. S. Zefirov on the occasion of his 80th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2189—2194, September, 2015.
1066ꢀ5285/15/6409ꢀ2189 © 2015 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015
Proshin et al.
Scheme 1
R1 = Alk, Ar; R2 = H, Me; R3 = H, F; R4 = Alk
(S)ꢀ1b
5: R = Me (b, g, i), Et (c, h, j)

Tetrasubstituted isothioureas
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015 2191
Table 1. Activity toward AMPA receptors and MKꢀ801 binding
site of NMDAꢀreceptors of comparison and synthesized comꢀ
pounds
with alkyl halide (Scheme 1) furnished tetrasubstituted
N´,N´ꢀdibenzylisothioureas racꢀ, (R)ꢀ, (S)ꢀ1b, racꢀ, (R)ꢀ,
(S)ꢀ1c, 2a,b, 5b—j (17 compounds).
Radioligand15,16 and electrophysiological patchꢀ
clamp17,18 methods were used to study the binding of
isothioureas 1b,c, 5b—j with the NMDA and AMPA types
of glutamate receptors, respectively. The studies showed
that most compounds retained their ability to block
NMDA receptors and, at the same time, acquired the
ability to simultaneously activate AMPA receptors.
The introduction of the isothiuronium group in the
dibenzylamine framework in most cases leads to the apꢀ
pearance of the AMPA activating properties, similar
to those in the soꢀcalled "ampakines". If an approach
suggested by R. Sheridan for evaluation of bioequivaꢀ
lence of two different chemical groups19 is used, a short
enough chain of bioisosteric transformations can be built
from a sulfamide group, which is a mandatory structurꢀ
al component of such traditional "ampakines" as cycloꢀ
thiazide and its analogues,20 to the isothiuronium group
(Scheme 2).
Comꢀ
pound
A* (%) for AMPA receptors
at C/mol L–1
IC50,/mol L–1
for NMDAꢀreceptors
1
10
30
1a
—
—
—/
125
115
125
170
130
200
210
290
—/
135.4 10.8
95.6 6.6
49.0 3.2
80.4 8.0
10.9 1.5
4.8 0.9
70.0 2.1
>100
148.0 6.0
111.0 9.5
56.0 4.0
21.6 0.1
>100
7.0 0.1
50.3 3.4
3.3 0.1
1.7 0.1
94.0 5.1
7.1 0.1
racꢀ1b
(R)ꢀ1b
(S)ꢀ1b
racꢀ1c
(R)ꢀ1c
(S)ꢀ1c
2a
2b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
100
100
145
110
100
110
120
115
—
120
110
160
135
120
150
155
170
—
110
165
100
150
120
160
200
135
260
170
500
100
225
195
465
810
470
330
275
1050
90
265
340
700
910
770
360
Scheme 2
* Current amplitudes.
tivity of the racemate racꢀ1c was close to that of more active
Rꢀisomer 1c.
In conclusion, the studies carried out in this work
showed that the introduction of an isothiourea fragment
leads to the appearance of an ability to activate AMPA
receptors. We found a number of structural patterns which
determined activity of the synthesized compounds toward
NMDA and AMPA receptors, that can be used in the
directed design. For the most active compound 5c, we
have performed molecular docking (using the AutoDock
Vina21 and SYBYLꢀX222 programs) in both the model of
binding site of positive allosteric modulators of the dimer
of the glutamateꢀbinding domain of AMPA receptor
(Xꢀray diffraction data, the code in the PDB database is
1lbc) and a model of the openꢀchain form of the ionic
channel of NMDA receptor (which was built earlier23 acꢀ
cording to the homology with the openꢀchain form of
potassium channel Methanothermobacter). The possible
ways of binding of compound 5c are shown in Fig. 1. The
results of docking are in good enough agreement with the
experimental data: though the binding of compound 5c
is possible in both cases, the estimation functions considꢀ
erably favors its interaction with the allosteric site of
AMPA receptor as compared to the ionic channel of
NMDA receptor.
It can be suggested that the ability of compounds unꢀ
der study to activate AMPA receptors is associated with
the "ampakine" activity of the isothiourea fragments.
The ability to compete with MKꢀ801 for the binding
site of the NMDA receptor very strong depends on the
nature of substituents in the isothiourea fragment. A comꢀ
parison of pairs of structural homologues showed that the
presence of an ethyl substituent at the sulfur atom is more
preferable than the methyl one (Table 1, pairs of comꢀ
pounds: 5b and 5c, 5g and 5h, 5i and 5j).
In the series of N,Sꢀsubstituted N´ꢀbenzylꢀN´ꢀ1ꢀ(R,S)ꢀ
phenylethylisothioureas, the ability to block the binding
of the labeled agent [3H]MKꢀ801 depends on the stereoꢀ
isomerism of the compounds. We have synthesized and
studied two groups of stereoisomeric agents (1b and 1c).
The studies showed that in the case of compounds racꢀ1b,
(R)ꢀ1b, (S)ꢀ1b, the activity of racemate racꢀ1b was
close to that of less active Sꢀisomer 1b, while in the
case of three compounds racꢀ1c—(R)ꢀ1c—(S)ꢀ1c, the acꢀ

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Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015
a
Proshin et al.
b
Fig. 1. Possible favorable ways of binding of compound 6c with the "ampakine" site of AMPA receptor (a) and with the ionic channel of
NMDA receptor (b) according to the docking of compounds simultaneously affecting both types of ionotropic glutamate receptors.
C21H24BrFN2S. Calculated (%): C, 57.93; H, 5.56; N, 6.43.
1H NMR, : 3.15 (m, 2 H, SCH2); 4.20 (m, 2 H, NCH2); 4.50
(s, 2 H, CH2Ph); 4.70 (s, 2 H, CH2C6H4—Fꢀ4); 5.05—5.25
(m, 4 H, =CH2); 5.70—5.80 (m, 2 H, —CH=); 7.25—7.40 (m, 14 H,
Harom); 10.25 (br.s, 1 H, NH).
Experimental
1
H spectra NMR were recorded on a BrukerꢀDPXꢀ200 specꢀ
trometer (200 MHz (1H)) for solutions in DMSOꢀd6, chemical
shifts are given in ꢀscale relative to tetramethylsilane as an inꢀ
ternal standard. Melting points were measured on a Boetius heatꢀ
ing stage without correction. Optical rotation was measured on a
Karl Zeiss Jena Polamat A polarimeter. Elemental analysis was
performed on an Elementar Vario MicroCube analyzer (Eleꢀ
mentar GMBH, Germany).
NꢀAllylꢀN´ꢀbenzylꢀN´ꢀ(4ꢀfluorophenylmethyl)ꢀSꢀmethylisoꢀ
thiourea hydrobromide (2b). The yield was 3.4 g (74.6%). M.p.
148—150 C. Found (%): C, 50.22; H, 4.96; N, 6.01. C19H22FIN2S.
1
Calculated (%): C, 50.01; H, 4.86; N, 6.14. H NMR, : 2.85
(s, 3 H, SCH3); 4.20 (m, 2 H, NCH2CH=); 4.45 (s, 2 H, CH2Ph);
4.60 (s, 2 H, CH2C6H4—Fꢀ4); 5.40 (m, 2 H, =CH2); 5.70 (m, 1 H,
—CH=); 7.25—7.40 (m, 9 H, Harom); 10.25 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀNꢀ(3ꢀmethylphenyl)ꢀSꢀmethylisothiourea
hydroiodide (5d). The yield was 3.0 g (61.5%). M.p. 114—116 C.
Found (%): C, 56.09; H, 5.29; N, 6.02. C23H25IN2S. Calculatꢀ
Synthesis of S,N,N´,N´ꢀtetrasubstituted isothiourea derivaꢀ
tives (general procedure). A corresponding isothiocyanate (0.1 mol)
was added dropwise to a solution of secondary amine 3 (0.1 mol)
in diethyl ether (150 mL) with stirring. The crystals of thiourea 4
were filtered off and recrystallized from isopropyl alcohol
(50 mL). A corresponding alkyl halide (0.012 mol) was added to
the thiourea 4 (0.01 mol) in acetone (50 mL). The reaction
mixture was allowed to stand until a precipitate was formed. The
crystals of isothiourea 1b,c, 5b—j were filtered off and recrystalꢀ
lized from PriOH (20 mL).24
NꢀAllylꢀN´,N´ꢀdibenzylꢀSꢀmethylisothiourea hydroiodide
(5b). The yield was 3.5 g (79.8%). M.p. 164—166 C. Found (%):
C, 51.89; H, 5.11; N, 6.41. C19H23IN2S. Calculated (%): C, 52.06;
H, 5.29; N, 6.39. 1H NMR, : 2.30 (s, 3 H, SCH3); 4.30 (m, 2 H,
NCH2); 4.60 (s, 4 H, CH2Ph); 5.25 (m, 2 H, =CH2); 5.80 (m, 1 H,
—CH=); 7.25—7.35 (m, 10 H, Harom); 10.10 (br.s, 1 H, NH).
NꢀAllylꢀN´,N´ꢀdibenzylꢀSꢀethylisothiourea hydroiodide (5c).
The yield was 3.5 g (77.3%). M.p. 150—152 C. Found (%):
C, 52.78; H, 5.65; N, 6.31. C20H25IN2S. Calculated (%):
C, 53.01; H, 5.57; N, 6.19. 1H NMR, : 1.25 (t, 3 H, Me, J = 7.0 Hz);
3.15 (q, 2 H, SCH2, J = 7.0 Hz); 4.25 (d, 2 H, NCH2, J = 4.7 Hz);
4.75 (s, 4 H, CH2Ph); 5.25 (m, 2 H, =CH2); 5.80 (m, 1 H,
—CH=); 7.20—7.35 (m, 10 H, Harom); 10.20 (br.s, 1 H, NH).
N,SꢀDiallylꢀN´ꢀbenzylꢀN´ꢀ(4ꢀfluorophenylmethyl}isothioꢀ
urea hydrobromide (2a). The yield was 3.2 g (70.2%).
M.p. 123—125 C. Found (%): C, 58.12; H, 5.70; N, 6.65.
1
ed (%): C, 56.56; H, 5.16; N, 5.76. H NMR(CDCl3), : 2.15
(s, 3 H, Me); 2.20 (s, 3 H, SMe); 5.20 (s, 4 H, CH2Ph); 7.00—7.50
(m, 14 H, Harom); 11.80 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀNꢀisobutylꢀSꢀmethylisothiourea hydroiodide
(5e). The yield was 3.5 g (71.1%). M.p. 155—157 C. Found (%):
C, 52.46; H, 5.89; N, 6.39. C20H27IN2S. Calculated (%): C, 52.86;
1
H, 6.00; N, 6.16. H NMR, : 1.15 (s, 6 H, Me2); 2.15 (s, 3 H,
SMe); 3.20 (m, 1 H, CH); 3.90 (m, 2 H, NCH2); 5.20 (s, 4 H,
CH2Ph); 7.40—7.50 (m, 10 H, Harom); 10.35 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀSꢀethylꢀNꢀmethallylisothiourea hydroiodide
(5f). The yield was 2,6 g (55.8%). M.p. 156—158 C. Found (%):
C, 54.33; H, 5.89; N, 6.27. C21H27IN2S. Calculated (%): C, 54.08;
H, 5.83; N, 6.01. 1H NMR, : 1.35 (t, 3 H, Me, J = 7.5 Hz); 1.70
(s, 3 H, Me); 3.05 (q, 2 H, SCH2, J = 7.5 Hz); 4.25 (br.s, 2 H,
NCH2C=); 4.70—4.90 (two s, 2 H, =CH2); 5.05 (s, 4 H, CH2Ph);
7.20—7.55 (m, 10 H, Harom); 10.10 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀNꢀcyclopropylꢀSꢀmethylisothiourea hydroꢀ
iodide (5g). The yield was 3.7 g (84.5%). M.p. 142—144 C.
Found (%): C, 51.99; H, 5.21; N, 6.44. C19H23IN2S. Calculated (%):
C, 52.06; H, 5.29; N, 6.39. 1H NMR, : 0.80 (m, 4 H, CH2CH2);
2.70 (s, 3 H, SMe); 3.00 (m, 1 H, CH); 5.10 (s, 4 H, CH2Ph);
7.25—7.40 (m, 10 H, Harom); 9.90 (br.s, 1 H, NH).

Tetrasubstituted isothioureas
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 9, September, 2015 2193
N´,N´ꢀDibenzylꢀNꢀcyclopropylꢀSꢀethylisothiourea hydroꢀ
iodide (5h). The yield was 3.5 g (77.4%). M.p. 152—154 C.
Found (%): C, 52.89; H, 5.61; N, 6.21. C20H25IN2S. Calculatꢀ
(S)ꢀ(–)ꢀNꢀAllylꢀN´ꢀbenzylꢀSꢀethylꢀN´ꢀ(1ꢀphenylethyl)isoꢀ
thiourea hydroiodide ((S)ꢀ1c). The yield was 2.3 g (49.3%).
A crystallizing oil. Found (%): C, 54.22; H, 5.66; N, 5.94.
1
ed (%): C, 53.10; H, 5.57; N, 6.19. H NMR, : 0.90 (m, 4 H,
C21H27IN2S. Calculated (%): C, 54.08; H, 5.83; N, 6.01.
CH2CH2); 1.40 (t, 3 H, Me, J = 1.4 Hz); 3.00 (m, 1 H, CH);
3.30 (q, 2 H, NCH2, J = 7.4 Hz); 5.15 (s, 4 H, CH2Ph);
7.20—7.40 (m, 10 H, Harom); 9.90 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀNꢀcyclopentylꢀSꢀmethylisothiourea hydroꢀ
iodide (5i). The yield was 3.8 g (81.5%). M.p. 143—145 C.
Found (%): C, 54.1; H, 5.71; N, 6.11. C21H27IN2S. Calculatꢀ
ed (%): C, 54.08; H, 5.83; N, 6.01. 1H NMR, : 1.40—1.70 (m, 4 H,
CH2CH2); 1.45—2.00 (m, 4 H, CH2NCH2); 2.75 (s, 3 H, SMe);
4.30 (m, 1 H, CH); 4.85 (s, 4 H, CH2Ph); 7.25—7.40 (m, 10 H,
Harom); 9.50 (br.s, 1 H, NH).
N´,N´ꢀDibenzylꢀNꢀcyclopentylꢀSꢀethylisothiourea hydroꢀ
iodide (5j). The yield was 3.4 g (70.8%). M.p. 132—134 C.
Found (%): C, 54.89; H, 6.11; N, 6.01. C22H29IN2S. Calculatꢀ
ed (%): C, 55.00; H, 6.08; N, 5.83. 1H NMR, : 1.40 (t, 3 H, Me,
J = 7.4 Hz); 1.40—1.60 (m, 2 H, CHCH); 1.75—2.10 (m, 6 H,
CHCH2NCH2CH); 3.10 (q, 2 H, SCH2, J = 7.4 Hz); 4.30 (m, 1 H,
CH); 5.20 (s, 4 H, CH2Ph); 7.30—7.50 (m, 10 H, Harom); 9.75
(br.s, 1 H, NH).
1H NMR, : 1.34 (t, 3 H, Me, J = 7.4 Hz); 1.95 (d, 3 H, Me,
J = 7.4 Hz); 3.08 (q, 2 H, SCH2, J = 7.4 Hz); 4.22 (m, 2 H, NCH2);
4.92 (m, 2 H, NCH2); 5.00—5.20 (m, 2 H, =CH2); 5.26 (m, 1 H,
—CH=); 6.23 (q, 1 H, CH, J = 7.4 Hz); 7.15—7.60 (m, 10 H,
Harom); 9.78 (br.s, 1 H, NH). []D= –86 (c 1, EtOH).
Electrophysiological method. Electrophysiological method
patchꢀclamp was used to study effects of compounds on AMPA
(AMPA is ꢀaminoꢀ3ꢀhydroxyꢀ5ꢀmethylꢀ4ꢀisoxazolepropionic
acid), a subtype of the glutamate receptors of mammalian cenꢀ
tral nervous system.15 Single isolated Purkinje neurons were isoꢀ
lated from cerebellum of 12—16ꢀdayꢀold rats of Wistar breed
according to the modified Kaneda method.16 The 400—600 m
thick cerebrellar slices were placed into a 10ꢀmL thermostated
chamber in the solution of the following composition: 150 mM of
NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgSO4•7H2O, 10 mM
HEPES, 15 mM glucose, pH 7.42 and incubated at 34 C; after
60 min, the buffer was replaced with a similar solution, which
contained pronase (2 mg mL–1) and collagenase (1 mg mL–1),
and incubated for 70 min at 34 C. After washing with the startꢀ
ing buffer for 20 min, the slices were placed in a Petri dish and
mechanically separated using a Pasteur pipette. The solutions
during the isolation of neurons were continuously purged with
100% O2 at 34 C. The neurons were placed in a 0.6ꢀmL working
chamber in buffer A of the following composition: 150 mM NaCl,
5 mM KCl, 2.6 mM CaCl2, 2 mM MgSO4•7H2O, 10 mM HEPES,
15 mM glucose, pH 7.36.
The transmembrane currents were triggered by the activaꢀ
tion of AMPA receptors by application of solutions of these
receptor agonists, kainic acids, by fastꢀperfusion method: a buffer
with the agonist (30 L) (the concentration of the agonist varied
within 10–4—10–3 mol L–1) was added every 2 min to the buffer
washing the neurons at a constant rate. The currents were
registered using borosilicate microelectrodes (resistance of
2.5—5.5 M) filled with a buffer of the following composition:
140 mM KCl, 11 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, 10 mM
HEPES, 5 mM ATP, pH 7.2.
S,N´ꢀDibenzylꢀNꢀphenylꢀN´ꢀ(1ꢀphenylethyl)isothiourea hydroꢀ
bromide (racꢀ1b). The yield was 1.9 g (36.7%). M.p. 126—128 C.
Found (%): C, 67.37; H, 5.32; N, 5.78. C29H29BrN2S. Calculatꢀ
1
ed (%): C, 67.30; H, 5.66; N, 5.66. H NMR, : 1.90 (d, 3 H,
Me, J = 7.4 Hz); 3.80 (m, 2 H, SCH2); 4.90 (m, 2 H, NCH2);
6.05 (q, 1 H, CH, J = 7.4 Hz); 7.15—7.60 (m, 20 H, Harom);
12.60 (br.s, 1 H, NH).
(R)ꢀ(+)ꢀS,N´ꢀDibenzylꢀNꢀphenylꢀN´ꢀ(1ꢀphenylethyl)isoꢀ
thiourea hydrobromide ((R)ꢀ1b). The yield was 1.8 g (34.8%).
M.p. 123—125 C. Found (%): C, 67.32; H, 5.33; N, 5.78.
C29H29BrN2S. Calculated (%): C, 67.30; H, 5.65; N, 5.41.
1H NMR, : 1.80 (d, 3 H, Me, J = 7.4 Hz); 3.80 (m, 2 H, SCH2);
4.90 (m, 2 H, NCH2); 6.00 (q, 1 H, CH, J = 7.4 Hz); 7.15—7.60
(m, 20 H, Harom); 12.50 (br.s, 1 H, NH). []D = 81 (c 1, EtOH).
(S)ꢀ(–)ꢀS,N´ꢀDibenzylꢀNꢀphenylꢀN´ꢀ(1ꢀphenylethyl)isoꢀ
thiourea hydrobromide ((S)ꢀ1b). The yield was 1.7 g (32.9%).
M.p. 121—123 C. Found (%): C, 67.66; H, 6.03; N, 5.70.
C29H29BrN2S. Calculated (%): C, 67.30; H, 5.65; N, 5.41.
1H NMR, : 1.80 (d, 3 H, Me, J = 7.5 Hz); 3.80 (m, 2 H, SCH2);
4.90 (m, 2 H, NCH2); 6.00 (q, 1 H, CH, J = 7.5 Hz); 7.15—7.60
(m, 20 H, Harom); 12.60 (br.s, 1 H, NH). []D= –79 (c 1, EtOH).
NꢀAllylꢀN´ꢀbenzylꢀSꢀethylꢀN´ꢀ(1ꢀphenylethyl)isothiourea
hydrobromide ((R,S)ꢀ1c). The yield was 2.3 g (49.4%). M.p.
92—94 C. Found (%): C, 54.32; H, 6.33; N, 5.78. C21H27IN2S.
To study the influence of compounds on AMPA receptors,
the buffer A washing the neurons was replaced with a similar
solution, which contained a tested compound in the concentraꢀ
tions 10–8, 10–7, 10–6, 10–5, and 3•10–5 mol L–1
.
An EPCꢀ9 instrument (HEKA, Germany) was used for regꢀ
istration. The currents were recorded on a computer using the
Pulse licensed program (HEKA). The results were processed
using the Pulsefit program (HEKA).
1
Calculated (%): C, 54.08; H, 5.83; N, 6.01. H NMR, : 1.35
(t, 3 H, Me, J = 7.4 Hz); 1.95 (d, 3 H, Me, J = 7.5 Hz); 3.05
(q, 2 H, SCH2, J = 7.4 Hz); 4.20 (m, 2 H, NCH2CH=); 4.95
(m, 2 H, NCH2); 5.00—5.20 (m, 2 H, =CH2); 5.25 (m, 1 H,
—CH=); 6.20 (q, 1 H, CH, J = 7.5 Hz); 7.15—7.60 (m, 10 H,
Harom); 9.65 (br.s, 1 H, NH).
(R)ꢀ(+)ꢀNꢀAllylꢀN´ꢀbenzylꢀSꢀethylꢀN´ꢀ(1ꢀphenylethyl)isoꢀ
thiourea hydroiodide ((R)ꢀ1c). The yield was 2.1 g (45.0%). An oil.
Found (%): C, 54.28; H, 6.06; N, 5.89. C21H27IN2S. Calculatꢀ
ed (%): C, 54.08; H, 5.83; N, 6.01. 1H NMR, : 1.35 (t, 3 H, Me,
J = 7.4 Hz); 1.94 (d, 3 H, Me, J = 7.3 Hz); 3.07 (q, 2 H, SCH2,
J = 7.5 Hz); 4.21 (m, 2 H, NCH2CH=); 4.92 (m, 2 H, NCH2);
5.00—5.20 (m, 2 H, =CH2); 5.27 (m, 1 H, —CH=); 6.21 (q, 1 H,
CH, J = 7.3 Hz); 7.15—7.60 (m, 10 H, Harom); 9.82 (br.s, 1 H,
NH). []D = 83 (c 1, EtOH).
Method of radioligand binding. The influence of tested comꢀ
pounds on radioligand binding with NMDA receptors was studꢀ
ied using a modified method.17 A [3H]MKꢀ801 radioactive ligand
(dizocilpine) with specific activity of 210 Ci mol–1 was used.
The membrane agent for the radioligand analysis was prepared
according to the method described earlier.18 The hippocampal
tissue was homogenized in a Potter homogenizer ("Teflon—
glass") in the buffer 1 (5 mM HEPES/4.5 mM Tris buffer, pH 7.6)
containing 0.32 M saccharose in the ratio 1 g of tissue/10 mL of
buffer. The homogenate was diluted with the buffer for studies 2
(5 mM HEPES/4.5 mM Tris buffer, pH 7.6) in the ratio 1 : 50
and centrifuged for 10 min at 1000g. After removal of the superꢀ
natant, the centrifugation was continued for 20 min at 25000g.

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Proshin et al.
The precipitate was homogenized in the buffer 2 in the ratio
1 : 50 and centrifuged for 20 min at 8000g. After removal of the
supernatant and its soft, unsteady layer over the precipitate, the
centrifugation was continued for 20 min at 25000g. The resulting
precipitate was suspended in the buffer 3 (5 mM HEPES/4.5 mM
Tris buffer (pH 7.61)) containing 1 mM Na4EDTA, and the
suspension was centrifuged. This washing procedure was repeatꢀ
ed four times, excluding Na4EDTA from the buffer in the last
washing. The final precipitate was resuspended in the buffer 2 in
the ratio 1 : 5 and storred in liquid nitrogen. The reaction mixꢀ
ture (the final volume 0.5 mL) contained 200 L buffer 2, 50 L
of 50 nM solution of the labeled ligand, and 250 L of protein
suspension. The nonspecific binding was determined in the presꢀ
ence of 50 L of 1 mM nonlabeled ligand.
The reaction mixture was incubated at room temperature for
2 h. After incubation, the samples were filtered through the GF/B
glass fiber filters (Whatman) preꢀsoaked in 0.3% polyethyleneꢀ
imine for 2 h at 4 C. Every testꢀtube was washed once with cold
buffer 2 (4 mL), then the filters were washed thrice with the
same amount of the buffer. The filters were dried in air until
complete dryness and transferred into scintillation vials with scinꢀ
tillation liquid containing diphenyloxazole (PPO) (4 g), dipheꢀ
nyloxazolylbenzene (POPOP) (0.2 g), and toluene (1 L). Radioꢀ
activity was determined on a TriCarb 2800 TR scintillation
counter (Perkin—Elmer, Packard, USA) with about 65% countꢀ
ing efficiency.
3. G. Riedel, B. Platt, J. Micheau, Behav Brain Res., 2003,
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12. S. Bachurin, S. Tkachenko, I. Baskin, N. Lermontova,
T. Mukhina, L. Petrova, A. Ustinov, A. Proshin, V. Grigꢀ
oriev, N. Lukoyanov, V. Palyulin, N. Zefirov, Ann. N. Y.
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V. V. Grigoriev, L. N. Petrova, S. O. Bachurin, J. Med.
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J. Pharmacol. Exp. Ther., 1993, 265, 1380.
The studies of the influence of tested compounds on binding
of [3H]MKꢀ801 with the rat hippocampus membranes was carꢀ
ried out upon addition of studied compounds (50 L) in the
range of concentrations 10–7—10–3 mol L–1 to the incubaꢀ
tion medium. The results of inhibition were used to calculate
IC50 for a particular compound using the GraphPadPrism 4 Demo
program.
In the cases when the inhibition of tested compounds in the
100 mol L–1 concentration was below 50%, the IC50 values
were not determined (n/d).
19. R. J. Sheridan, Chem. Inf. Comput. Sci., 2002, 42, 103.
20. V. V. Grigor´ev, A. N. Proshin, A. S. Kinzinskii, S. O. Baꢀ
churin, Russ. Chem. Rev., 2009, 524.
This work was financially supported by the Russian
Science Foundation (Grant 14ꢀ23ꢀ00160). The work
was performed using facilities of the Community
User Center of the Institute of Physiologically Active
Compounds of Russian Academy of Sciences (Agreeꢀ
ment No. 14.621.21.0008, work identificator
RFMEFI62114X0008).
21. SYBYLꢀX2 http://www.certara.com.
22. AutoDock Vina http://autodock.scripps.edu
23. I. G. Tikhonova, I. I. Baskin, V. A. Palyulin, N. S. Zefirov,
S. O. Bachurin, J. Med. Chem., 2002, 45, 3836.
24. G. L. Perlovich, A. N. Proshin, T. V. Volkova, S. V. Kurkov,
V. V. Grigoriev, L. N. Petrova, S. O. Bachurin, J. Med.
Chem., 2009, 7, 1845.
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Received May 5, 2015;
in revised form June 18, 2015