Synthesis and In Vitro Neuroprotective Activity of Glycine Analogs of Gk-2 Dimeric Dipeptide Mimetic of Nerve Growth Factor 4th Loop

Article abstract of DOI:10.1007/s11094-020-02168-0

A dimeric dipeptide mimetic of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), was previously developed at V. V. Zakusov State Institute of Pharmacology, activated specific TrkA receptors, and exhibited neuroprotective activity in vitro (10^–5 – 10^–9 M) and in vivo (0.1 – 10 mg/kg i.p. and p.o.). GK-2 was designed based on the beta-turn (-Asp⁹⁴-Glu⁹⁵-Lys⁹⁶-Gln⁹⁷-) of the NGF 4^th loop and preserved the central dipeptide fragment (-Glu⁹⁵-Lys⁹⁶-). The Asp⁹⁴ residue was replaced by its monosuccinyl bioisostere. The dimeric structure of NGF was reproduced using a bivalent hexamethylenediamine spacer. The structure—activity (neuroprotective) relationship for GK-2 was studied in the present work using a glycine scan, i.e., successive replacement of the peptide side groups by H. The bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac), bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenediamides (GK-2-Gly2) were less active with neuroprotective activity in vitro under oxidative stress for HT22 cells at concentrations 10 – 100 times greater than GK-2. The conclusion was drawn that each side radical of GK-2 was important for manifestation of the full neuroprotective activity of dimeric dipeptide GK-2, a mimetic of the NGF 4^th loop. However, removal of any of the side radicals would probably not change the active structure of the beta-turn so that the two remaining side radicals should retain the ability to bind to their TrkA subsites. This could explain the retention of neuroprotective activity in the GK-2 glycine analogs.

Full text of DOI:10.1007/s11094-020-02168-0

DOI 10.1007/s11094-020-02168-0
Pharmaceutical Chemistry Journal, Vol. 54, No. 2, May, 2020 (Russian Original Vol. 54, No. 2, February, 2020)
SYNTHESIS AND IN VITRO NEUROPROTECTIVE ACTIVITY
OF GLYCINE ANALOGS OF GK-2 DIMERIC DIPEPTIDE MIMETIC
OF NERVE GROWTH FACTOR 4TH LOOP
1
N. M. Sazonova, A. V. Tarasyuk, Yu. N. Firsova,
,*
1
1
1
A. A. Zvyagintsev, A. G. Rebeko, P. I. Antipov, S. V. Nikolaev,
1
1
1
1
T. A. Antipova, and T. A. Gudasheva
1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 54, No. 2, pp. 23 – 31, February, 2020.
Original article submitted August 28, 2019.
A dimeric dipeptide mimetic of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-L-lysine)
hexamethylenediamide (GK-2), was previously developed at V. V. Zakusov State Institute of Pharmacology,
–
activated specific TrkA receptors, and exhibited neuroprotective activity in vitro (10 – 10 M) and in vivo
5
–9
9
0.1 – 10 mg/kg i.p. and p.o.). GK-2 was designed based on the beta-turn (-Asp -Glu -Lys -Gln -) of the
4
95
96
97
(
th
95
96
94
NGF 4 loop and preserved the central dipeptide fragment (-Glu -Lys -). The Asp residue was replaced by
its monosuccinyl bioisostere. The dimeric structure of NGF was reproduced using a bivalent
hexamethylenediamine spacer. The structure—activity (neuroprotective) relationship for GK-2 was studied in
the present work using a glycine scan, i.e., successive replacement of the peptide side groups by H. The
bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac), bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and
bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenediamides (GK-2-Gly2) were less active with
neuroprotective activity in vitro under oxidative stress for HT22 cells at concentrations 10 – 100 times greater
than GK-2. The conclusion was drawn that each side radical of GK-2 was important for manifestation of the
th
full neuroprotective activity of dimeric dipeptide GK-2, a mimetic of the NGF 4 loop. However, removal of
any of the side radicals would probably not change the active structure of the beta-turn so that the two remain-
ing side radicals should retain the ability to bind to their TrkA subsites. This could explain the retention of
neuroprotective activity in the GK-2 glycine analogs.
Keywords: mimetic, dipeptide, GK-2, NGF, neuroprotective activity, glycine scan, structure—activity rela-
tionship.
The dimeric dipeptide bis-(N-monosuccinyl-L-gluta-
GK-2 in in vitro experiments at micro- and nanomolar con-
centrations exhibited neuroprotective activity typical of
full-sized NGF [2]. It prevented death caused by H O or
myl-L-lysine) (GK-2) (RU Pat. No. 2,410,392, 2011; US Pat.
No. 9,683,014 B2, 2017; CN Pat. No. 102365294 B, 2016;
EP 2397488, 2019) was previously designed at
V. V. Zakusov State Institute of Pharmacology as a mimetic
2
2
glutamic acid of HT-22 immortalized murine hippocampal
neurons and protected PC-12 rat pheochromocytoma cell
line from the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine). The neuroprotective effect of GK-2 was
confirmed in in vivo experiments using models of Alzhei-
mer’s and Parkinson’s diseases and ischemic cerebral stroke
in rodents [3]. In contrast with full-sized NGF, GK-2 did not
cause hyperalgesia and mass loss [4]. GK-2 has now passed a
full cycle of preclinical studies as a potential neuroprotective
drug for treating cerebral circulatory disorders.
th
of the NGF 4 loop b-turn [1]. The design preserved the cen-
95
96
tral dipeptide fragment of the b-turn (Glu -Lys ) and re-
94
placed the preceding Asp with a bioisosteric monosuccinyl
radical. GK-2 reproduced the homodimeric NGF structure
and was a TrkA receptor agonist, like neurotrophin itself [1].
1
V. V. Zakusov State Institute of Pharmacology, Russian Academy of Med-
ical Sciences, 8 Baltiiskaya, Moscow, 125315 Russia.
*
e-mail: tata-sosnovka@mail.ru
126
0
091-150X/20/5402-0126 © 2020 Springer Science+Business Media, LLC

Synthesis and In Vitro Neuroprotective Activity
127
GK-2
GK-2-Gly1
GK-2-Gly2
GK-2Ac
Fig. 1. Design of GK-2 glycine analogs (GK-2 glycine scan).
Two diastereomers of GK-2 were previously synthesized
to study the stereospecificity of their interactions with the
TrkA receptor [5]. These were bis-(N-monosuccinyl-L-
glutamyl-D-lysine) (GK-2LD) and bis-(N-monosuccinyl-D-
glutamyl-L-lysine) hexamethylenediamides (GK-2DL).
Studies of their neuroprotective effects on HT-22 neuronal
culture with oxidative stress showed that replacing L- by
D-lysine decreased significantly the activity whereas replac-
ing L- by D-glutamic acid destroyed it completely. It was
concluded that the L-configuration of both dipeptide residues
was important for interaction with the TrkA receptor. In con-
tinuation of this research, we studied the effects of the
amino-acid side radicals on GK-2 activity. For this, a glycine
scan was conducted by designing three GK-2 analogs (Fig. 1).
These were bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac),
bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and
bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenedi-
amides (GK-2-Gly2). The compounds were synthesized so
their neuroprotective effects on HT-22 neuronal culture
could be studied in vitro with oxidative stress induced by
H O .
diimide (DCC) at 5 – 10°C. Activated esters Z-L-Lys(Boc)-
OSu and Z-Gly-OSu were condensed with hexamethylenedi-
amine in DMF at room temperature (RT) to give in 93 and
90% yields [Z-L-Lys(Boc)-NH] (CH ) (I) and (Z-Gly-
2
2 6
NH) (CH ) (IX) that were then Z-deblocked via catalytic
2
2 6
hydrogenolysis in the presence of Pd/C (10%). The bis-prod-
ucts [H-L-Lys(Boc)-NH] (CH ) (II) and (H-Gly-
(X) were condensed in DMF with
2
2 6
NH) (CH )
2
2 6
Z-L-Glu(tBu)-OSu; bis-product II also with Z-Gly-OSu, to
produce in 81 – 95% yields the corresponding bis-dipeptides
[Z-L-Glu(tBu)-L-Lys(Boc)-NH] (CH ) (III), [Z-Gly-L-
2
2 6
Lys(Boc)-NH] (CH ) (VI), and [Z-L-Glu(tBu)-Gly-
NH] (CH ) (XI). Catalytic hydrogenolysis in the presence
2
2 6
2
2 6
of Pd/C (10%) produced free a-amines on bis-dipeptides
[H-L-Glu(tBu)-L-Lys(Boc)-NH] (CH ) (IV), [H-Gly-L-
2
2 6
Lys(Boc)-NH] (CH ) (VII), and [H-L-Glu(tBu)-Gly-
NH] (CH ) (XII). Reaction of IV with Ac-OSu in DMF
2
2 6
2
2 6
gave the N-acetyl product [Ac-L-Glu(tBu)-L-Lys(Boc)-
NH] (CH ) (V), which was deprotected by trifluoroacetic
2
2 6
acid (TFA) in CH Cl to give GK-2Ac as the trifluoroacetate
2
2
in 51% total yield calculated for the starting protected lysine.
bis-Dipeptides VII and XII were acylated by succinic anhy-
dride in DMF in 84 and 93% yields to give N-monosuccinyl
2
2
Glycine-containing GK-2 analogs. The glycine GK-2
analogs were synthesized using classical wet methods for
synthesizing peptides from the C-terminus using a Z/Boc-
strategy of protecting groups and activated N-hydroxysucci-
nimide esters (Schemes 1 and 2).
derivatives
[HOOC-CH CH -CO-Gly-L-Lys(Boc)-
2 2
NH] (CH ) (VIII) and [HOOC-CH CH -CO-L-Glu(tBu)-
2
2 6
2
2
Gly-NH] (CH ) (XIII). Finally, removal of tBu- and
2
2 6
Peptides GK-2Ac, GK-2-Gly1, and GK-2-Gly2 were
synthesized starting from commercially available
Z-L-Lys(Boc)-OH, Z-L-Glu(tBu)-OH, and H-Gly-OH.
Z-substituted glycine was prepared in 92% yield by reacting
the sodium salt of glycine with benzyloxycarbonyloxysucci-
Boc-protecting groups by TFA in CH Cl produced the target
2 2
products GK-2-Gly1 (as the trifluoroacetate) and GK-2-Gly2
in total yields of 55 and 48% calculated for starting protected
lysine and glycine, respectively. The target peptides were ho-
mogeneous by TLC and reversed-phase HPLC. The struc-
tures and diastereomeric purity (>98%) of the obtained com-
nimide (Z-OSu) in aqueous Me CO. Activated N-hydroxy-
2
1
pounds were confirmed by PMR and C NMR spectroscopy.
3
succinimide esters of acetic acid, Z/Boc-protected lysine,
Z/tBu-protected glutamic acid, and Z-glycine were synthe-
sized using N-hydroxysuccinimide and dicyclohexylcarbo-
Neuroprotective activity of the synthesized peptides in
–
the concentration range 10 10 M was studied using an
5 –
–8

1
28
N. M. Sazonova et al.
DCHA
a) 5% H SO /EtOAc, 30 min
2
4
DCC/EtOAc, RT, 20 h
DMF, RT, 4 h
DCC
dioxane, 5°C, 1 h
RT, 20 h
H O:Me CO 1:1
2
2
MeOH, RT
DCC
DCC, EtOAc, 10°C, 1 h
RT, 20 h
EtOAc, 20°C, 1 h
RT, 20 h
DMF, RT, 24 h
DMF, RT, 24 h
MeOH, RT
MeOH, RT
DMF, 0°C, 1 h; RT, 24 h
DMF, RT, 24 h
2
TFA/CH Cl2
TFA/CH Cl2
2
RT, 2.5 h
RT, 2.5 h
GK-2-Gly1
Total yield 55%
GK-2Ac
Total yield 51%
Scheme 1. Syntheses of GK-2Ac and GK-2-Gly1
H O -induced oxidative stress model and HT-22 immortal-
istered 24 h before the damaging agent. Table 1 presents the
results.
2
2
ized murine hippocampal cell line [6]. Peptides were admin-

Synthesis and In Vitro Neuroprotective Activity
129
–
8
10
M, i.e., 10 times more active than the two previous
GK-2 analogs but 10 times less than GK-2 itself according to
active concentration and ~5 times by manifestation of ef-
fects.
DMF, RT, 4 h
Thus, both amino-acid residues and the N-terminus
9
4
substituent acting as Asp were important for manifestation
of neuroprotective effects by dimeric dipeptide GK-2, a mi-
th
metic of the NGF 4 loop. The activity, although decreased
by 10 – 100 times, was retained if any two side groups of the
mimetic were preserved. This suggested that the b-turn ac-
tive structure could be retained after removing one of the
side radicals. Apparently, the two remaining side radicals
were still capable of binding to their TrkA receptor subsites.
MeOH, RT
EXPERIMENTAL CHEMICAL PART
DMF, RT, 24 h
Commercially available enantiomerically pure L-amino
acids and their derivatives (Sigma and Fluka) were used in
the work. Melting points were determined in open capillaries
on an Optimelt MPA100 apparatus (Stanford Research Sys-
1
3
tems, USA) and are uncorrected. PMR and C NMR spectra
MeOH, RT
were recorded in DMSO-d solutions with TMS internal
6
standard (0 ppm) on the d-scale on a Bruker Fourier 300 HD
spectrometer (300 and 75 MHz, respectively). Resonances
1
were assigned by analyzing 1D and 2D homonuclear H– H
1
1
13
COSY and heteronuclear H– C COSY spectra (HSQC and
HMBC).
Specific optical rotation was recorded on an ADP 440
automated polarimeter (Bellingham+Stanley Ltd., Great
Britain). TLC used DC Kieselgel 60 G/F254 glass plates
DMF, 10°C, 1 h; RT, 24 h
(
Merck, Germany) and solvent systems CHCl –MeOH (6:1,
3
A); C H –MeOH (1:4, B); CHCl –MeOH– H O–HOAc
6
6
3
2
(15:10:2:3, C); hexane–EtOAc (1:5, D); n-BuOH–HOAc–
H O (4:1:1, E); CHCl –MeOH– HOAc (80:10:1, F); hex-
TFA/CH Cl
2
2
3
2
RT, 2.5 h
ane–EtOAc (1:1, G); C H –MeOH (2:1, I); dioxane–H O
6 6 2
(
9:1, J); CHCl –MeOH (9:1, K).
3
Amine-containing compounds were detected by
ninhydrin; amides, a chlorine-tolidine test; compounds with
a free carboxylic acid, bromocresol green; aromatics, UV
light.
Total yield 48%
HPLC of the dipeptides used a Wellchrom 2001 chroma-
tography system (Knauer, Germany) with a Diasorb-
GK-2-Gly2
Scheme 2. Synthesis of GK-2-Gly2.
110-C16 column (4.0 ´ 250 mm, 5 mm). The loop volume
was 20 mL. Mobile phase A (0.05% aqueous TFA) and mo-
bile phase B (0.05% TFA in MeCN) were used in gradient
mode (0 – 100% B over 30 min). The flow rate was
Table 1 shows that replacing an N-monosuccinyl radical
by N-acetyl (GK-2Ac) and also Glu by Gly (GK-2-Gly1) de-
creased by 100 times the neuroprotective activity according
0.6 mL/min. Detection was made at 220 nm. Analyses were
made at room temperature.
–
9
–7
to the minimum active concentration (from 10 to 10 M)
Solvent purification. DMA was purified by distillation
and by ~3 times according to the effects.
from ninhydrin. Et O was stored over solid NaOH. EtOAc,
2
The GK-2 analog with Lys replaced by Gly (GK-2-Gly2)
CH Cl , CHCl , C H , Me CO, hexane, MeOH, and EtOH
2
2
3
6
6
2
exhibited neuroprotective effects at concentrations down to
(all chemically pure) were used without further purification.

1
30
N. M. Sazonova et al.
Synthesis of starting compounds
N-Benzyloxycarbonylglycine N-hydroxysuccinimide
Z-Gly-OSu) was prepared as before [9] in 96% yield from
(
N-Benzyloxycarbonylglycine (Z-Gly-OH) was pre-
Z-Gly-OH (50.0 g, 0.24 mol) in EtOAc. R 0.83 (EtOAc), Rf
f
pared as before [7] in 92% yield from H-Gly-OH (10.0 g,
0
.43 (G); mp 108 – 110°C. PMR spectrum (DMSO-d ), d,
ppm: 2.81 (s, 4H, -CH CH - -OSu), 4.21 (d, J 7.9 Hz, 2H,
6
0
.13 mol). R 0.15 (EtOAc), R 0.71 (C); mp 116 – 120°C.
f f
2
2
Lit. [8] mp 120°C.
CH Gly), 5.08 (s, 2H, -OCH - Z), 7.36 (m, 5H, C H Z),
2
2
6
5
7
.96 (t, J 7.9 Hz, 1H, NH Gly). Lit. [9]: mp 111 – 115°C.
N-Hydroxysuccinimide ester of acetic acid (Ac-OSu)
was prepared by the literature method [10] in 86% yield from
HOAc (12.0 g, 0.2 mol). R 0.85 (A), R 0.56 (EtOAc), R
f
TABLE 1. Effect of NGF Mimetic GK-2 and Its Analogs GK-2Ac,
GK-2-Gly1, and GK-2-Gly2 on Neuron Viability Under Oxidative
Stress. Peptides Were Administered 24 h Before H O
f
f
0.70 (J); mp 130 – 134°C. PMR spectrum (DMSO-d ), d,
ppm: 2.34 (s, 3H, CH CO-), 2.80 (m, 4H, -CH CH - OSu).
2
2
6
Concentration,
M
MTT assay, D600
,
Activity A,
%
3
2
2
Group
Lit. [10]: mp 130°C.
Z-L-Glu(tBu)-OSu and Z-L-Lys(Boc)-OSu activated
esters and [Z-L-Lys(Boc)-NH] (CH ) (I); [H-L-Lys(Boc)-
mean ± RSD
#
Control
0
0.427 ± 0.071
0.350 ± 0.032
100
0
#
– 3
2
2 6
H
2
O
2
1.5 ´ 10
NH] (CH ) (II); [Z-L-Glu(tBu)-L-Lys(Boc)-NH] (CH )
2 6
2
2 6
2
#
– 9
*
*
NGF
~ 10
0.425 ± 0.047
97
(III), and [H-L-Glu(tBu)-L-Lys(Boc)-NH] (CH ) (IV)
2 2 6
#
– 5
*
*
were previously prepared by us according to the literature
11].
GK-2
10
0.422 ± 0.063
94
[
–
–
8
9
*
*
1
1
0
0
0.416 ± 0.059
96
*
*
Synthesis of [CH
CO-L-Glu-L-Lys-NH]
2 6
(CH ) , GK-2Ac
0.378 ± 0.042
36
3
2
–
10
e
bis-(N-Acetyl-(-tert-butyl-L-glutamyl-N -tert-butylox
1
0
0.358 ± 0.038
0.184 ± 0.005
0.096 ± 0.011^
10
100
0
ycarbonyl-L-lysine) hexamethylenediamide, [CH CO-L-
Control
0
3
Glu(tBu)-L-Lys(Boc)-NH] (CH ) (V). A solution of IV
–
–
–
3
3
3
2
2 6
H
2
O
2
1.5 ´ 10
(
6.0 g, 6.36 mmol) in DMF (40 mL) at RT was treated with
–
–
–
–
5
6
7
8
***
***
*
***
GK-2Ac
10
0.125 ± 0.009
0.124 ± 0.007
33 ± 10
Ac-OSu (2.2 g, 14 mmol) and stirred at RT. When the reac-
tion was finished (TLC monitoring), the DMF was evapo-
***
1
1
1
0
0
0
31 ± 8
25 ± 4
23 ± 8
100
*
rated. The still fluid residue was treated with Me CO
0.118 ± 0.004
0.115 ± 0.008
0.200 ± 0.014
0.118 ± 0.010^
2
(
50 mL) and stored at RT for 2 h. The resulting precipitate
was filtered off and rinsed with Me CO (2 ´ 25 mL). The
white crystals were dried under vacuum (15 mm Hg) in a
2
Control
0
H
2
O
2
1.5 ´ 10
0
desiccator over CaCl to afford V (5.2 g, 79%). R (main
2
f
–
–
–
–
5
6
7
8
*
*
*
spot) 0.60 (A), R 0.83 (E), R 0.81 (J); mp. 194 – 198°C;
GK-2-Gly1
10
0.139 ± 0.015
24 ± 19
22 ± 15
f
f
2
[a]D – 21.8° (s, 1.04; MeOH). PMR spectrum (DMSO-d6),
7
*
1
1
1
0
0
0
0.135 ± 0.012
g
C H C H Lys,
2 2
d
*
*
d, ppm: 1.15 – 1.48 (m, 16H,
2
0.136 ± 0.005
22 ± 5
-
NHCH (CH ) CH NH-), 1.36 (s, 18H, 2-OC(CH ) Boc),
2
2 4
2
3 3
0.134 ± 0.014
0.217 ± 0.009
0.087 ± 0.004^
20 ± 17
100
1
.38 (s, 18H, 2-OC(CH ) tBu), 1.48 and 1.59 (two m, 4H, 2
3 3
b
b
C H Lys), 1.67 and 1.86 (two m, 4H, 2 C H Glu), 1.84 (s,
Control
0
2
2
g
H
2
O
2
1.5 ´ 10
0
6H, CH -CO), 2.20 (t, J 8.0 Hz, 4H, 2 C H Glu), 2.86 (m,
3
2
e
4H, 2 C H Lys), 3.01 (m, 4H, -NHCH (CH ) CH NH-),
–
–
–
–
5
6
7
8
*
*
GK-2-Gly2
10
0.104 ± 0.006
13 ± 5
2 2 2 4 2
a
a
4
.09 – 4.16 (m, 2H, 2 C H Lys), 4.18 – 4.25 (m, 2H, 2 C H
Glu), 6.75 (t, J 5.5 Hz, 2H, 2 NH Lys), 7.78 (t, J 5.5 Hz, 2H,
NH(CH ) NH-), 7.85 (d, J 7.9 Hz, 2H, 2 NH Lys), 8.03 (d, J
***
***
**
*
1
1
1
0
0
0
0.107 ± 0.005
0.106 ± 0.007
15 ± 3
15 ± 5
**
*
-
2
6
7.7 Hz, 2H, 2 NH Glu).
bis-(N-Acetyl-L-glutamyl-L-lysine) hexamethylene-
diamide ditrifluoroacetate, {[CH CO-L-Glu-L-Lys-
NH] (CH ) ·2CF COOH}, GK-2Ac. A suspension of I
0.104 ± 0.007
13 ± 5
Compounds administered 24 h before damage. Statistical signifi-
*
cance of differences: ^, from control (p £ 0.001); from H O
3
2
2
2
2 6
3
*
**
p £ 0.05; p £ 0.01;
***
(
p £ 0.001) Kruskal–Wallis criterion fol-
lowed by Dunn test. n = 12 (GK-2); n = 11 (GK-2Ac); n = 12
(1.00 g, 0.97 mmol) in CH Cl (15 mL) was treated with
2 2
TFA (5 mL) and stirred at RT. When the starting compound
disappeared (TLC monitoring, ~2 h), the mixture was evapo-
(
GK-2-Gly1); n = 10 (GK-2-Gly2). Activity was calculated using
(
Dexp - D
)
H O
2
2
rated. The still fluid residue was triturated with Et O and de-
the formula: A(%) =
×100%, where Dexp is the ex-
2
(
Dcontr - D
)
H O
2
2
canted (3 ´ 20 mL). The precipitate was filtered off using an
apparatus for filtering hygroscopic compounds to afford
perimental solution optical absorption; D
, optical absorption of
2
H O
2
active control (with H O ); Dcontr, optical absorption of passive con-
2
2
trol (without H O ). Literature data [1].
GK-2Ac (0.79 g, 85%) as a white crystalline compound. Rf
#
2
2
0.41 (K); t = 9.0 min; mp 124 – 143°C (dec., did not have a

Synthesis and In Vitro Neuroprotective Activity
131
2
5
sharp melting point, hydroscopic); [a]D – 28.8° (s, 1;
-NHCH (CH ) CH NH-), 3.09 (br.s, 4H, 2 CH Gly), 4.21
2
2 4
2
2
a
m, 2H, 2 C H Lys), 6.74 (t, J 5.0 Hz, 2H, 2 N H Lys), 7.92
a
(
MeOH). PMR spectrum (DMSO-d ), d, ppm: 1.23, 1.37,
b
.51 and 1.68 (four m, 20H, 2 C H C H C H Lys,
6
g
d
(d, 2H, 2 NH Lys), 7.95 (t, 2H, -NH(CH ) NH-).
1
2 6
2
2
2
å
bis-(N-Monosuccinylglycyl-N -tert-butyloxycarbonyl-
b
-
NHCH (CH ) CH NH-), 1.71 – 1.93 (m, 4H, 2 C H Glu),
2 2 4 2 2
g
.85 (s, 6H, 2 CH CO-), 2.24 (t, J 2.26 Hz, 4H, 2 C H Glu),
L-lysine) hexamethylenediamide, [HOOC(CH ) CO-
1
2 2
3
2
e
C H2 Lys), 3.02 (m, 4H,
Gly-L-Lys(Boc)-NH] (CH ) (VIII). A solution of VII
2
.75 (m, 4H,
2
2
2 6
a
NHCH (CH ) CH NH-), 4.12 – 4.23 (m, 4H, 2 C H Lys, 2
(4.26 g, 6.2 mmol) in DMF (60 mL) at 0°C was stirred,
treated at once with succinic anhydride (1.40 g, 14 mmol),
and stirred with cooling for 30 min and at RT for 12 h. When
the reaction was finished (TLC monitoring), the DMF was
evaporated. The residue was treated with MeCN (100 mL)
and left for 40 min. The well-formed precipitate was filtered
-
2 2 4 2
a
C H Glu), 7.76 (6H, br.s, 2 N H3 Lys), 7.78 (t, 2H,
+
-
NH(CH ) NH-), 7.92 (d, J 8.0 Hz, 2H, 2 NH Lys), 8.07 (d, J
2
6
8
.0 Hz, 2H, 2 NH Glu).
Synthesis of [HOOC(CH
GK-2-Gly1
2
)
2
2 2 6
CO-Gly-L-Lys-NH] (CH ) ,
off and rinsed with MeCN (2 ´ 25 mL) and Et O (25 mL).
2
a
bis-(N -Benzyloxycarbonylglycyl-N -tert-butyloxycar
e
The resulting white powder was dried in a vacuum (15 mm
Hg) desiccator over CaCl to afford VIII (4.40 g, 84%). Rf
bonyl-L-lysine)
hexamethylenediamide,
[Z-Gly-L-
2
(
main sport) 0.71 (B), R 0.64 (E); mp 131 – 149°C (dec);
2
Lys(Boc)-NH] (CH ) (VI). A solution of II (10.00 g,
f
2
2 6
4
[
a
]
–
1
0
.
7
°
(
c
,
1
.
0
5
;
M
e
O
H
)
.
P
M
R
s
p
e
c
t
r
u
m
(
D
M
S
O
-
d
)
,
1
7.46 mmol) in DMF (40 mL) was treated with a solution of
Z-Gly-OSu (12.83 g, 41.9 mmol) in DMF (45 mL), stirred at
RT for 12 h, treated with N,N-dimethyl-1-aminopropane
DMAPA, 0.87 mL), stirred for 30 min, and poured slowly
D
6
g
C H C H Lys,
2 2
d
d, ppm: 1.15 – 1.42 (m, 16H,
2
-
NHCH (CH ) CH NH-), 1.36 (s, 18H, 2 -OC(CH ) Boc),
2 2 4 2 3 3
b
.48 and 1.61 (two m, 4H, 2 C H Lys), 2.37 – 2.43 (m, 8H,
(
1
2
2
e
-CH CH - Suc), 2.86 (m, 4H, 2 C H Lys), 3.01 (m, 4H,
into distilled H O (800 mL) to produce a precipitate. The
2
2
2
2
mixture was left overnight. The well-formed precipitate was
-
NHCH (CH ) CH NH-), 3.70 (d, 4H, 2 CH Gly),
2 2 4 2 2
a
.10 – 4.17 (m, 2H, 2 C H Lys), 6.75 (t, J 5.4 Hz, 2H, 2 N H
e
filtered off; rinsed successively with distilled H O to pH 7,
4
2
hexane (100 mL), and Et O (50 mL); and dried in a vacuum
Lys), 7.79 (t, J 5.4 Hz, 2H, -NH(CH ) NH-), 7.85 (d, J
2
2
6
(
15 mm Hg) desiccator over CaCl2 to afford
chromatographically homogeneous VI (15.11 g, 87%) as
white crystals with a light-gray tint. R 0.63 (A), R 0.74 (F);
8
.2 Hz, 2H, 2 NH Lys), 8.17 (t, J 5.7 Hz, 2H, 2 NH Gly),
12.11 (br.s, 2H, 2 -COOH).
bis-(N-Monosuccinylglycyl-L-lysine) hexamethylene-
f
f
27
mp 111 – 117°C (dec); [a]D – 14.8° (s, 1; MeOH). PMR
diamide ditrifluoroacetate, [HOOC(CH ) CO-Gly-L-
2 2
Lys-NH] (CH ) ·2CF COOH, GK-2-Gly1. A suspension
spectrum (DMSO-d ), d, ppm: 1.22 – 1.36 (m, 16H, 2
2
2 6
3
6
g
d
C H C H Lys, -NHCH (CH ) CH NH-), 1.36 (s, 18H, 2
of VIII (1.00 g, 1.1 mmol) in CH Cl (15 mL) was treated
2 2
2
2
2
2 4
2
b
with TFA (5 mL) and stirred at RT. When the starting mate-
rial disappeared (TLC monitoring, ~1 h), the mixture was
-
OC(CH ) Boc), 1.48 and 1.59 (two m, 4H, 2 C H Lys),
3
3
2
e
C H2 Lys), 3.01 (m, 4H,
2
.86 (m, 4H,
2
evaporated. The still fluid residue was triturated with Et O
-
NHCH (CH ) CH NH-), 3.65 (d, J 6.0 Hz, 2H, 2 CH Gly),
2
2
2 4
2
2
a
.14 – 4.21 (m, 2H, 2 C H Lys), 5.03 (s, 4H, 2 -OCH - Z),
4
6
7
and decanted (3 ´ 20 mL). The precipitate was filtered using
a head for filtering hygroscopic compounds to afford
GK-2-Gly1 (0.79 g, 90%) as a white crystalline compound.
Rf (main spot) 0.21 (C); t = 9.1 min, mp 141 – 151°C (dec,
did not have a sharp melting point, hygroscopic);
2
e
.74 (t, J 5.2 Hz, 2H, 2 N H Lys), 7.35 (m, 10H, 2 -C H Z),
6
5
.46 (t, J 6.0 Hz, 2H, 2 NH Gly), 7.87 (t, J 5.3 Hz, 2H,
-
NH(CH ) NH-), 7.91 (d, J 8.2 Hz, 2H, 2 NH Lys).
2 6
e
bis-(Glycyl-N -tert-butyloxycarbonyl-L-lysine) hexa-
methylenediamide, [H-Gly-L-Lys(Boc)-NH] (CH )
VII). A solution of VI (7.00 g, 0.0078 mol) in MeOH
190 mL) was treated with Pd/C (10%, 0.80 g, 50% mois-
2
[a]D – 12.0° (c, 1.1; MeOH). PMR spectrum (DMSO-d ), d,
6
2
2 6
6
(
ppm: 1.23, 1.38, 1.51 and 1.66 (four m, 20H, 2
(
b
C H C H C H Lys, -NHCH (CH ) CH NH-), 2.44 (m, 8H,
g
d
2
2
2
2
2 4
2
ture) and stirred under a H atmosphere at RT. When the
e
2 -CH CH - Suc), 2.75 (m, 4H, 2 C H Lys), 3.02 (m, 4H,
2 2 2
2
starting compound disappeared (TLC monitoring), the cata-
lyst was filtered off and rinsed with MeOH (100 mL). The
MeOH solution was evaporated. The residue was treated
-
NHCH (CH ) CH NH-), 3.70 (d, J 5.7 Hz, 4H, 2 CH Gly),
2 2 4 2 2
a
.13 – 4.19 (m, 2H, 2 C H Lys), 7.71 (br.s, 6H, 2 N H Lys),
+
4
3
7
.80 (t, J 5.5 Hz, 2H, -NH(CH ) NH-), 7.87 (d, J 8.0 Hz, 2H,
2
6
with Et O (60 mL) and left overnight in a refrigerator. The
2
2 NH Lys), 8.20 (t, J 5.7 Hz, 2H, 2 NH Gly).
Et O was decanted. The residue was dried under vacuum
2
(
15 mm Hg, 40°C, ~1 h) to afford VII (4.79 g, 95%) as a
Synthesis of [HOOC(CH
GK-2-Gly2
2 2 2 2 6
) CO-L-Glu-Gly-NH] (CH ) ,
white amorphous compound. R (main spot) 0.69 (C), R 0.21
f
f
2
4
(
B); mp 71 – 79°C; [a]D – 10.4° (s, 1; MeOH). PMR spec-
bis-(N-Benzyloxycarbonylglycine) hexamethylenedi-
amide, (Z-Gly-NH) (CH ) (IX). A solution of Z-Gly-OSu
g
d
trum (DMSO-d ), d, ppm: 1.15 – 1.36 (m, 16H, 2 C H C H
6
2
2
2
2 6
Lys, -NHCH (CH ) CH NH-), 1.36 (s, 18H, 2 -OC(CH )
(10.00 g, 32.6 mmol) in DMF (45 mL) was treated with a so-
lution of hexamethylenediamine (1.60 g, 13.6 mmol) in
DMF (10 mL), stirred at RT for 4 h, left overnight without
2
2 4
2
3 3
b
Boc), 1.49 and 1.58 (two m, 4H, 2 C H Lys), 2.31 (br.s. 2H,
2
e
NH2 Gly), 2.86 (m, 4H, 2 C H2 Lys), 3.02 (m, 4H,

1
32
N. M. Sazonova et al.
stirring, stirred, treated slowly with distilled H O (850 mL)
(DMSO-d6),
d,
ppm:
1.23
1.35
-NHCH CH (CH ) CH CH NH-), 1.38 (s, 18H,
(m,
4H,
4H,
2
2
heated beforehand to 43 – 45°C, stirred until the temperature
reached 20 – 22°C by cooling naturally, and left for 12 h at
-NH(CH ) (CH ) (CH ) NH-),
(s,
2
2
2 2
2 2
2
2
2 2
2
2
b
1
2 – 17°C. The well-formed precipitate was filtered off;
-OC(CH ) tBu), 1.74 and 1.87 (two m, 4H, 2 C H Glu),
3
3
2
g
C H
rinsed with H O (200 mL) to pH ~7, hexane (40 mL), and
2.25 (t, 4H,
2
Glu), 3.03 (m, 4H,
2
2
Et O (40 mL); and dried in air to afford chromatographically
-NHCH (CH ) CH NH-), 3.65 (d, J 8.1 Hz, 4H, 2 CH Gly),
2
2
2 4
2
2
a
3.99 (m, 2H, C H Glu), 5.03 (s, 4H, 2 -OCH -Z ), 7.35 (s,
homogeneous IX (7.36 g, 90%) as white crystals. R 0.56 (A),
f
2
R 0.32 (K); mp 175 – 177°C. PMR spectrum (DMSO-d ), d,
ppm: 1.23 (m, 4H, -NH(CH ) (CH ) (CH ) NH-), 1.37 (m,
10H, 2 C H Z ), 7.54 (d, J 7.6 Hz, 2H, 2 NH Glu), 7.65 (t, J
f
6
6
5
7.8 Hz, 2H, -NH(CH ) NH-), 8.16 (t, J 8.1 Hz, 2H, 2 NH
2
2
2 2
2 2
2 6
4
H, NHCH CH (CH ) CH CH NH), 3.04 (m, 4H,
Gly).
2
2
2 2
2
2
-
NHCH (CH ) CH NH-), 3.57 (d, J 7.4 Hz, 4H, 2 CH Gly),
bis-((-tert-Butyl-L-glutamylglycine) hexamethylene-
diamide, [H-L-Glu(tBu)-Gly-NH] (CH ) (XII). A suspen-
2
2 4
2
2
5
7
.02 (c, 4H, 2 -OCH - Z), 7.35 (s, 10H, 2 C H Z), 7.41 (t, J
2 6 5
2
2 6
.4 Hz, 2H,
2
NH Gly), 7.80 (t,
J
7.8 Hz, 2H,
sion of XI (8.63 g, 9.1 mmol) in MeOH (210 mL) was
-
NH(CH ) NH-).
treated with Pd/C (10%, 0.90 g, 50% moisture) and stirred at
2
6
bis-Glycine
hexamethylenediamide
diacetate,
RT under an H atmosphere. When the starting compound
2
[
(
(
H-Gly-NH] (CH ) ·2CH COOH (X). A suspension of IX
disappeared (TLC monitoring), the catalyst was filtered off
and rinsed with MeOH (70 mL). The solvent was removed
2
2 6
3
7.00 g, 14 mmol) in MeOH (250 mL) was treated with Pd/C
10%, 0.79 g, 50% moisture) and HOAc (50%, 20 mL) and
under vacuum. The residue was re-evaporated with C H6
6
stirred under an H atmosphere at RT. When the starting
(30 mL). The resulting oil was dried under vacuum (9 mm
2
compound disappeared (TLC monitoring), the catalyst was
filtered off and rinsed with MeOH (100 mL). The MeOH so-
lution was evaporated and re-evaporated with toluene
Hg, 40°C, ~1 h) to afford XII (5.78 g, 97%) as an amorphous
white powder with a beige tint. R (main spot) 0.40 (B), Rf
f
0.71 (C), R 0.21 (E); mp (not determined, hygroscopic);
f
2
8
[a]D + 7.2° (s, 1.02; MeOH). PMR spectrum (DMSO-d ), d,
(
3 ´ 50 mL). The oily residue was triturated with anhydrous
Et O (50 mL) and decanted twice. Then Et O (50 mL) was
6
2
2
ppm: 1.24 (m, 4H, -NH(CH ) (CH ) (CH ) NH-), 1.36 (s,
2
2
2 2
2 2
added and the mixture was left for 2 h to form a precipitate,
decanted, and dried in a rotary evaporator at 45°C for 30 min
under vacuum (15 mm Hg, water aspirator) and then under
high vacuum (diaphragm pump, 9 mm Hg, 1 h) to afford X
4
H, -NHCH CH (CH ) CH CH NH-), 1.39 (s, 18H, 2
b
2 2 2 2 2 2
-OC(CH ) tBu), 1.57 and 1.81 (two m, 4H, 2 C H Glu),
3
3
2
g
1
4
.91 (br.s, 4H, 2 NH Glu), 2.26 (t, 4H, 2 C H Glu), 3.04 (m,
a
H, -NHCH (CH ) CH NH-), 3.15 (m, 2H, 2 C H Glu), 3.66
2
2
2
2 4
2
(
4.65 g, 95%) as a white crystalline compound. R 0.25 (C);
f
(br.s, 4H, 2 CH Gly), 7.77 (t, J 7.8 Hz, 2H, -NH(CH ) NH-),
2 2 6
mp 151 – 153°C. PMR spectrum (DMSO-d ), d, ppm: 1.25
(
8.08 (br.t, 2H, 2 NH Gly).
6
m, 4H, -NH(CH ) (CH ) (CH ) NH-), 1.39 (m, 4H,
bis-(N-Monosuccinyl-(-tert-butyl-L-glutamylglycine)-
2
2
2 2
2 2
NHCH CH (CH ) CH CH NH), 1.83 (s, 6H, 2 CH CO),
hexamethylenediamide, [HOOC(CH ) CO-L-Glu(tBu)-
2
2
2 2
2
2
3
2 2
3
.07 (m, 4H, -NHCH (CH ) CH NH-), 3.22 (br.s, 4H, 2 CH
Gly-NH] (CH ) (XIII). A solution of XII (5.64 g,
2
2 4
2
2
2
2 6
+
Gly), 7.09 (br.s, 6H, 2 N H3 Gly), 8.06 (br.t, 2H,
9.4 mmol) in DMF (20 mL) at 10°C was stirred, treated with
succinic anhydride (2.06 g, 21 mmol, 20% excess), stirred
for 30 min, warmed to RT, and stirred for another 12 h. When
the reaction was finished (TLC monitoring), the solvent was
evaporated (50°C). The resulting light-yellow oil was
-
NH(CH ) NH-).
2
6
bis-(N-Benzyloxycarbonyl-(-tert-butyl-L-glutamylgly-
cine) hexamethylenediamide, [Z-L-Glu(tBu)-Gly-
NH] (CH ) (XI). A solution of X (4.30 g, 104 mmol) in
2
2 6
DMF (30 mL) at RT was stirred, treated with N-ethylmor-
pholine (3.9 mL, 24.5 mmol), and stirred for 20 min. The
suspension was treated with a solution of Z-L-Glu(tBu)-OSu
triturated with Et O (80 mL) and decanted. The precipitate
2
on a filter was rinsed with Et O (2 ´ 25 mL) and MeCN
(50 mL) and dried in a vacuum (15 mm Hg) desiccator over
2
(
12.45 g, 28.6 mmol) in DMF (50 mL), stirred for 20 h (dur-
CaCl to afford XIII (7.03 g, 93%) as a finely crystalline
2
ing which the suspension turned to a solution), treated with
DMAPA (0.5 mL, 4 mmol), stirred for 40 min, and poured
light-cream-colored powder. R (main spot) 0.88 (C), R 0.73
26
f
f
(
B), R 0.69 (J); mp 170 – 176°C (dec); [a]
– 2.8° (s 1;
f
D
into H O (400 mL). The oily product that separated was ex-
2
MeOH). PMR spectrum (DMSO-d ), d, ppm: 1.23 (m, 4H,
6
tracted with EtOAc (350 mL). The EtOAc extract was
-
NH(CH ) (CH ) (CH )
NH-),
1.36
(s,
4H,
2
2
2 2
2 2
washed with H O (2 ´ 100 mL) and saturated NaCl solution
(
2
-NHCH CH (CH ) CH CH NH-), 1.39 (s, 18H,
b
2
2
2
2 2
2
2
100 mL) and dried over anhydrous Na SO . The desiccant
2
4
-OC(CH ) tBu), 1.73 and 1.87 (two m, 4H, 2 C H Glu),
3
3
2
was filtered off and rinsed with EtOAc (30 mL). The EtOAc
solution was evaporated. The residue was treated with a mix-
g
.24(t, 4H, 2 C H Glu), 2.40 – 2.45 (m, 8H, 2 -CH CH -
2
2
2
2
Suc), 3.03 (m, 4H, -NHCH (CH ) CH NH-), 3.62 (d, 4H, 2
2
2 4
2
a
ture (50 mL) of petroleum ether and Et O (3:1) and left for 3
2
CH2 Gly), 4.09 – 4.17 (m, 2H, 2 C H Glu), 7.60 (t, J
.75 Hz, 2H, -NH(CH ) NH-), 8.10 (t, 2H, 2 NH Gly), 8.17
h. The precipitate was filtered off, rinsed with petroleum
ether (30 mL), and dried in air to afford XI (8.63 g, 81%) as
a white crystalline compound. R 0.69 (I), R 0.75 (F); mp
7
2
6
(
d, 2H, 2 NH Glu), 12.11 (br.s, 2H, 2 -COOH).
bis-(N-Monosuccinyl-L-glutamylglycine) hexamethyl-
enediamide, [HOOC(CH ) CO-L-Glu-Gly-NH] (CH ) ,
f
f
2
03 – 106°C; [a]D – 2.4° (s, 1.03; MeOH). PMR spectrum
7
1
2
2
2
2 6

Synthesis and In Vitro Neuroprotective Activity
133
GK-2-Gly2. A suspension of XIII (2.0 g, 2.49 mmol) in
Activities in tests for counteraction to oxidative stress
were calculated using the formula:
CH Cl (45 mL) at RT was treated with TFA (15 mL), stirred
2
2
for 2 h, evaporated, and re-evaporated with CH Cl
2
2
(
2 ´ 15 mL). The residue was triturated with anhydrous Et O
(Dexp -DH O )
2
2 2
A(%) =
×100%,
and decanted (2 ´ 20 mL) and left under Et O (20 mL) for 2
h to form a precipitate that was filtered off and dried under
2
(Dcontr -DH O )
2 2
vacuum (15 mm Hg) over CaCl2 to afford GK-2-Gly2
where Dexp is the experimental solution optical absorption;
, optical absorption of active control (with H O );
(
1.5 g, 87%) as white crystals with a light-cream tint. Traces
D
of TFA were removed by lyophilization (3´). R 0.49 (C), Rf
H2O2
2
2
f
Dcontr, optical absorption of passive control (without H O ).
0
.42 (E); t = 9.8 min; mp 85 – 90°C (lyophilizate); –17.1°
2
2
Statistical analysis used standard Statistica 6.0 programs
StatSoft Inc., USA). MTT assay results were analyzed using
nonparametric statistics and qualitative analysis of the data
by the Kruskal–Wallis criterion followed by a Dunn test
ANOVA). Results were considered statistically significant
(
c, 1; H O).
2
(
PMR spectrum (DMSO-d ), d, ppm: 1.23 (m, 4H,
(m,
6
-
NH(CH ) (CH ) (CH ) NH-),
1.36 – 1.38
4H,
2
2
2 2
2 2
-
NHCH CH (CH ) CH CH NH-), 1.75 and 1.89 (two m,
2
2
2 2
2
2
(
b
H, 2 C H Glu), 2.26 (t, 4H, 2 C H Glu), 2.40 – 2.45 (m,
2 2
g
4
8
for p £ 0.05.
H,
2
-CH CH -
Suc),
3.03
(m,
4H,
2
2
-
NHCH (CH ) CH NH-), 3.63 (d, 4H, 2 CH Gly),
2 2 4 2 2
a
.09 – 4.17 (m, 2H, 2 C H Glu), 7.60 (t, J 7.75 Hz, 2H,
ACKNOWLEDGMENTS
4
-
NH(CH ) NH-), 8.10 (t, 2H, 2 NH Gly), 8.16 (d, 2H, 2 NH
2
6
The work was performed under a State Task (topic No.
Glu).
1
3
0
521-2019-0003, Search for pharmacological methods for
C NMR spectrum (DMSO-d ), d, ppm: 174.40 and
74.38 (two s, 4C, 4 COOH, Suc, Glu), 172.2 and 172.0 (two
6
selective activation of signal transduction pathways of tyro-
sine-kinase neurotrophin receptors as design bases for drugs
free of the side effects of native neurotrophins).
1
s, 4C, 4 CO, Glu, Gly), 168.8 (s, 2C, 2 CO Gly), 52.97 (s,
a
C, 2 C Glu), 42.54 (s, 2C, 2 C Gly), 39.99 (s, 2C, 2 C1 of
a
2
g
spacer), 30.59 (c, 2C, C Glu), 30.36 and 29.54 (two s, 4 C
2
b
Suc), 29.44 (c, 2C, C of spacer), 27.35 (c, 2C, 2 C Glu),
REFERENCES
3
6.53 (c, 2C, 2 C of spacer). Characteristic resonances
2
(
quartets) of CF and CO groups of TFA were not observed
1. T. A. Gudasheva, T. A. Antipova, and S. B. Seredenin, Dokl.
Akad. Nauk, 434(4), 549 – 552 (2010).
3
1
in the C NMR spectrum.
3
2
3
4
. T. A. Antipova, T. A. Gudasheva, and S. B. Seredenin, Byull.
Eksp. Biol. Med., 150(11), 537 – 540 (2010).
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S. S. Korsakova, 6, 63 – 70 (2015).
Neuroprotective activity was studied according to the lit-
erature [6] using HT-22 immortalized murine hippocampal
cell line. Cells in DMEM medium (HyClon) were inoculated
into 96-well plates at 3500 cells/well containing fetal bovine
serum (5%, Invitrogen) and L-glutamine (2 mM; ICN) and
. T. A. Gudasheva, P. Y. Povarnina, T. A. Antipova, et al., J.
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7. A. Paquet, Can. J. Chem., 60(8), 976 – 980 (1982).
6
incubated at 37°C in 5% CO until a monolayer formed. Pep-
2
–5
–8
tides at final concentrations from 10 to 10 M were added
4 h before the damaging effect.
Oxidative stress was modeled using H O at a final con-
8
. M. Bergmann and L. Zervas, Berichte, 65, 1192 – 1201 (1932).
. G. W. Anderson, J. E. Zimmerman, and F. M. Callahan,
J. Am. Chem. Soc., 86(9), 1839 – 1842 (1964).
2
9
2
2
centration of 1.5 mM. Cells were incubated with H O in 5%
2
2
1
0. S. Rappoport and Y. Lapidot, Methods Enzymol., 29, 685 – 688
(1974).
1. N. M. Sazonova, A. V. Tarasyuk, D. V. Kurilov, et al., Khim.-
CO at 37°C for 30 min [12]. Then, the medium was changed
2
to the normal one. Cell viability was determined after 4 h us-
1
ing
bromide (MTT, Sigma). Optical density was measured on a
Multiscan EX spectrophotometer (Thermo) at 600 nm.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
farm. Zh., 49(7), 10 – 19 (2015).
12. K. Riveles, L. Z. Huang, and M. Quik, NeuroToxicology, 29(3),
421 – 427 (2008).