Neuroprotective activity of (+)-(S)-2-[(1S,5R)-(3,7-dioxo-2,4,6,8- tetraazabicyclo[3.3.0]oct-2-yl)]-4-methylthiobutanoic acid

Article abstract of DOI:10.1134/S106816201205007X

A study of the neurotropic, neuroprotective, and antioxidant action of the enantiomers and racemate of 2-[(3,7-dioxo-2,4,6,8-tetraazabicyclo[3.3.0]oct-2- yl)]-4-methylthiobutanoic acid synthesized in a stereoselective reaction of (R)-, (S)-, or (R,S)-N-carbamoylmethionine with 4,5-dihydroxyimidazolidine-2-one showed that only (+)-(S)-2-[(1S,5R)-(3,7-dioxo-2,4,6,8-tetraazabicyclo[3.3.0] oct-2-yl)]-4-methylthiobutanoic acid had neuroprotective properties. X-ray structure analysis showed that the predominating racemate of glycolurils is crystallized from aqueous solutions as a conglomerate. Antioxidant activity was not detected. Pleiades Publishing, Ltd., 2012.

Full text of DOI:10.1134/S106816201205007X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 5, pp. 550–557. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © A.N. Kravchenko, V.V. Baranov, L.V. Anikina, Yu.B. Vikharev, I.S. Bushmarinov, Yu.V. Nelyubina, 2012, published in Bioorganicheskaya Khimiya, 2012,
Vol. 38, No. 5, pp. 621–628.
Neuroprotective Activity of (+)ꢀ(S)ꢀ2ꢀ[(1S,5R)ꢀ(3,7ꢀDioxoꢀ2,4,6,8ꢀ
Tetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀMethylthiobutanoic Acid
, 1
A. N. Kravchenkoa , V. V. Baranova, L. V. Anikinab, Yu. B. Vikharevb,
I. S. Bushmarinovc, and Yu. V. Nelyubinac
a Federal StateꢀFunded Research Organization N.D. Zelinsky Institute of Organic Chemistry
of the Russian Academy of Sciences, Leninsky pr. 47, Moscow, 119991 Russia
b Federal StateꢀFunded Research Organization Institute of Technical Chemistry of the Ural Branch
of the Russian Academy of Sciences, Perm, Russia
c Federal StateꢀFunded Research Organization N.A. Nesmeyanov Institute of Organoelement Compounds
Chemistry of the Russian Academy of Sciences, Moscow, Russia
Received September 21, 2011; in final form, February 1, 2012
Abstract—A study of the neurotropic, neuroprotective, and antioxidant action of the enantiomers and raceꢀ
mate of 2ꢀ[(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid synthesized in a
stereoselective reaction of (
one showed that only (+)ꢀ(
R
S
)ꢀ, (
)ꢀ2ꢀ[(1
S
)ꢀ, or (R,S)ꢀ
Nꢀcarbamoylmethionine with 4,5ꢀdihydroxyimidazolidineꢀ2ꢀ
S,5R)ꢀ(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthioꢀ
butanoic acid had neuroprotective properties. Xꢀray structure analysis showed that the predominating raceꢀ
mate of glycolurils is crystallized from aqueous solutions as a conglomerate. Antioxidant activity was not
detected.
Keywords: enantiomers, racemate, (R)ꢀ, (S)ꢀ, and (R,S)ꢀNꢀcarbamoylmethionine, 4,5ꢀdihydroxyimidazoliꢀ
dineꢀ2ꢀone, diastereoselective synthesis, glycolurils, neuroprotective activity
DOI: 10.1134/S106816201205007X
1
INTRODUCTION
We have recently shown that introduction of a carboxꢀ
ypropyl fragment to the nitrogen atom of a glycoluril
(compound (II)) confers neuroprotective properties
to this compound [6].
It is well known that usually only one of the enanꢀ
tiomers composing racemic drugs has the required
effect. The wellꢀknown examples of ketamine, peniꢀ
cillamine, and thalidomide confirm this fact [1, 2]:
(
S
)ꢀketamine is an anesthetic, while (
hallucinogenic, ( )ꢀpenicillamine is tuberculoplastic,
while ( )ꢀpenicillamine causes blindness, and ( )ꢀ
)ꢀthalidomide is
R)ꢀketamine is
Me
Me
Me
S
H
N
R
S
N
N
N
*
thalidomide is a teratogen, while (
R
O
O
O
O
an antidepressant.
N
N
N
N
*
Me
Me
Me
A new class of neurotropic organic compounds—
2,4,6,8ꢀtetraazabicyclo[3.3.0]octaneꢀ3,7ꢀdiones, usually
referred to under the trivial name of glycolurils in
chemical literature—has been identified at the
N.D. Zelinsky Institute of Organic Chemistry. A
member of this class, 2,4,6,8ꢀtetramethylꢀ2,4,6,8ꢀtetꢀ
raazabicyclo[3.3.0]octaneꢀ3,7ꢀdione (the therapeutic
(I
)
(II
)
COOH
Due to the rigidity of the heterocyclic framework
and cisꢀjoining of the imidazolidine rings, glycoluril
molecules have the shape of a halfꢀopen book or a
seagull’s wings [7, 8] and therefore asymmetrically
substituted glycolurils are chiral compounds [4, 7, 9–
15]. Most of the pharmacological activity studies were
performed with glycoluril racemates. Therefore, the
preparation of pure glycoluril enantiomers and a comꢀ
parative analysis of their pharmacological activity
seemed a task of considerable interest.
drug mebicar (
izer for 30 years [3]. The range of medical application
of ꢀalkyl glycolurils is constantly expanding [4–6].
I)) has been used as a daytime tranquilꢀ
N
Abbreviations: DHI, 4,5ꢀdihydroxyimidazolidineꢀ2ꢀone, XRA, Xꢀ
ray analysis.
Corresponding author: phone: +7(499)135ꢀ8817; eꢀmail
kani@server.ioc.ac.ru.
1
550

NEUROPROTECTIVE ACTIVITY
551
Our recent studies showed that pure glycoluril
RESULTS AND DISCUSSION
enantiomers can be synthesized in diastereoselective
and diastereospecific reactions of 4,5ꢀdihydroxyimiꢀ
Pure enantiomers of 2ꢀ[(3,7,dioxoꢀ2,4,6,8ꢀtetꢀ
raazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid,
glycolurils (III)–(VI), were synthesized by cycloconꢀ
dazolidineꢀ2ꢀones with
(
S
)ꢀ
N
ꢀcarbamoylꢀ
α
amino
acids and some )ꢀ ꢀcarbamoylꢀ
(R
N
α
amino acids [16–
18]. The aim of the present work was to synthesize
enantiomers and racemate of 2ꢀ[(3,7ꢀdioxoꢀ2,4,6,8ꢀ
tetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic
acid from (R)ꢀ, (S)ꢀ, or (R,S)ꢀNꢀcarbamoylmethioꢀ
nine and characterize their effects on brain homogeꢀ
nates and behavioral reactions of mice.
densation of 4,5ꢀdihydroxyimidazolidineꢀ2ꢀone (VII
)
(DHI) with )ꢀ or )ꢀ ꢀcarbamoylmethionine
(
R
(S N
((VIII) and (IX), respectively) under conditions favorꢀ
ing the diastereoselective formation of bicyclic comꢀ
pounds (pH 1, 90°С, 2 h) [16] (Scheme 1).
H2N
O
HN
H2N
O
HN
(
S)
(R)
HOOC
HOOC
S
VIII)
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
S
(
IX)
(
OH
Me
O
O
O
Me O
O
O
O
+
+
O
O
N
H
N
N
H
N
N
H
OH
N
H
N
N
H
N
(
S)
(
VII)
(
S
)
(
R
)
(R)
HOOC
HOOC
HOOC
HOOC
H2N
HN
S
S
O
S
S
(
III
)
(IV
)
(V
)
(VI)
Me
Me
Me
Me
*
HOOC
S
(X)
Me
H
H
N
H
H
N
H
N
H
N
H
N
H
N
N
N
+
+
O
+
O
O
O
O
O
O
O
N
H
N
N
H
N
N
H
N
N
H
N
(S)
(R)
(S)
(
R)
HOOC
HOOC
HOOC
HOOC
S
S
S
S
Me
Me
Me
Me
(
XI
)
(XII)
Scheme 1.
1
The major diastereomers (III) and (V) precipitate used). H NMR analysis of the compounds obtained
from the reaction mixture after the end of the reaction.
The minor diastereomers (IV) and (VI) are isolated at
the end of the crystallization process. The physicoꢀ
showed that both diastereoselective and diastereospeꢀ
cific condensation reactions result in the formation of
a mixture of two glycoluril racemates (XI) and (XII) in
a 3 : 1 ratio, i.e., the diastereospecific reaction does not
occur. The racemate ratio was determined from the
ratio of the integrated signal intensities of the amino
acid fragment CHꢀgroup protons in the H NMR
spectra: dd, 4.47 ppm for the major racemate (XI) and
dd, 4.07 ppm for the minor racemate (XII). Signals
chemical properties of the stereoisomers (III)–(V)
correspond to those we reported previously [16, 17].
The stereoisomer (VI) has been isolated and characꢀ
terized for the first time in the present work.
1
The reaction of (R,S)ꢀNꢀcarbamoylmethionine (X)
with DHI has not been reported and therefore it was
performed both under conditions favoring diastereoꢀ
selectivity and under the conditions ensuring diasteꢀ
reospecific formation of the bicyclic compound; these
conditions differ by the amount of the acid used (in the
first case the acid is added dropwise until pH 1 is
from the protons of the nonꢀtransformed
carbamoylmethionine ( ) and the wellꢀknown thioꢀ
hydantoins (XIIIa,b)—side products formed upon the
cyclization of ( R,S)ꢀ ꢀcarbamoylmethionine (
(R,S)ꢀNꢀ
X
(
N
X)
reached, while in the second case 2 mol/eq acid are and DHI dehydration (Scheme 2)—were detected in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

552
KRAVCHENKO et al.
mixture of enantiomer crystals, i.e., as a conglomerate
(spatial group 212121). We have previously deterꢀ
mined the structure of the enantiomer ( ) [16] in a
P
C12
V
coordination compound with nickel, namely,
[Ni(H2O)6]2+ ((
V
)–)2 (H2O)4
.
H2N
O
HN
S1
C11
H
N
H
N
OH
OH
O
R
O
O
N
H
N
H
*
C10
HOOC
O2
S
(
X)
(
XIIIa,b
)
(VII)
Me
R = H– (
C9
O1
N8
O4
a
), (CH2)2SMe– (
b)
C7
N2
C1
N4
Scheme 2.
C13
The uniformity of the conglomerate (XI), i.e., the
absence of contamination by the other racemate
N6
C3
(
XII), was confirmed by powder Xꢀray diffraction
analysis. Analysis of the diffractogram of the sample
XI) (Fig. 2) by the method of Pauly allowed us to
ascribe it to a single phase of rhombic syngony with the
following elementary cell parameters: = 9.1016
= 10.8675 and = 12.5763 Å. The systematic
extinctions observed were consistent with assignment
to the space group 212121. The elemental cell paramꢀ
C5
O3
(
a
Å,
Fig. 1. Structure of the molecule (V), the crystal sample for
Xꢀray analysis was selected from the crystals of the comꢀ
pound (XI). The asymmetric C1, C5, and C9 atoms are in
ꢀ, Rꢀ, and Rꢀconfiguration, respectively.
b
Å
,
c
S
P
eters obtained correspond to those obtained in the
monocrystal experiment, and the deviations observed
are caused by thermal expansion of the crystal.
1H NMR spectra of the residue obtained after filtrate
evaporation.
Since mebicar (I) and glycoluril (II) have neurotroꢀ
Increasing the amount of acid resulted in a
pic and neuroprotective activity [6, 19], we investiꢀ
gated similar effects of the compounds (III)–(VI) and
the racemate (XI).
decrease in racemate yield. Racemates (XI) and (XII
)
were separated by six steps of crystallization from
aqueous solution. Xꢀray structure analysis (XRA) of
the product (XI) (Fig. 1) showed that the structure of
Comparative assessment of the neurotropic activity
the crystal analyzed corresponds to that of the enantiꢀ of the enantiomers was performed in vivo on randomꢀ
omer ( ), a component of the racemate (XI) with ( )ꢀ bred white mice in an openꢀfield test and elevated
2ꢀ[(1 ,5 )] configuration of the asymmetric carbon plusꢀmaze test before and after hypoxia [20, 21]. The
V
R
S
R
atoms. Therefore, this racemate is crystallized as a compound (XI) was shown not to affect the behavior
50000
40000
30000
20000
10000
0
10
20
30
40
50
60
70
2
θ
, deg
Fig. 2. Experimental (circles), calculated (solid black line), and subtractive (gray line) diffractograms for the sample (XI).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 5 2012

NEUROPROTECTIVE ACTIVITY
553
Table 1. Effects of the compounds on the behavioral reactions of animals
Openꢀfield test
Elevated plus maze test
number
of squares
crossed
number
of holes
explored
number
of entries into
the open arms
number
of rearings and of grooming
head dippings
number
Compound
total number
of entries
time spent
in open arms
episodes
(III)
63.40 3.58 12.10 1.56
66.70 6.15 17.20 2.08
68.40 8.65 17.70 3.55
60.40 4.57 25.80 2.34
71.20 6.51 23.10 2.20
96.90 12.03 26.80 2.24
47.00 5.32 21.50 2.74
59.30 5.95 25.50 3.95
60.50 6.05 24.00 2.80
2.90 0.31 6.80 0.79 74.50 11.39 8.20 1.56
2.60 0.48 5.20 0.73 76.80 20.68 7.30 1.40
1.80 0.39 4.10 1.21 113.90 19.69 4.80 1.34
1.60 0.37 3.70 0.78 99.40 18.95 6.10 0.80
3.10 0.23* 6.00 0.70* 93.30 17.53 12.50 2.25
4.60 0.60 9.30 1.18 62.30 8.26 14.80 2.87
1.70 0.49 3.80 1.30 93.80 13.69 9.50 3.25
2.30 0.95 6.00 1.29 91.00 11.11 7.50 2.58
1.60 0.26 4.40 0.78 66.80 13.85 9.50 2.11
1.00 0.30
0.90 0.18
0.50 0.17
0.30 0.21
0.30 0.15
0.40 0.16
0.20 0.20
0.80 0.48
1.30 0.45
Control 1
(IV)
Control 2
(V)
Control 3
(VI)
(XI)
Control 4
Mebicar
Control 5
71.00 9.62 11.90 1.98* 2.40 0.56 4.80 1.07 80.50 19.20* 7.60 1.86* 1.60 0.34*
78.50 4.75 25.20 5.51
2.50 0.31 6.60 0.85 37.90 5.96 20.30 2.57
0.60 0.16
*
p ≤ 0.05 relatively to control.
of mice before hypoxia and to stimulate the motor and
explorative activity of the animals in the openꢀfield
test after hypoxia (Tables 1 and 2). This effect is obviꢀ
ously due to the action of the stereoisomer (III) only,
since this substance does not affect intact mice and
stimulates the motor activity of the animals in the eleꢀ
vated plusꢀmaze and their explorative activity in both
tests after hypoxia. The stereoisomer (IV) does not
affect the behavior of animals before or after hypoxia.
The present work resulted in the synthesis of pure
glycoluril enantiomers with predetermined configuraꢀ
tions of asymmetric carbon atoms {(+)ꢀ(
[(1 ,5 )] (III), (–)ꢀ( )ꢀ2ꢀ[(1 ,5 )] (IV), (–)ꢀ(
[(1 ,5 )] ( ), and (+)ꢀ( )ꢀ2ꢀ[(1 ,5 )] (VI)} and
S
)ꢀ2ꢀ
R
S
S
S
R
R)ꢀ2ꢀ
S
R
V
R
R S
racemates (XI) + (XII); the effects of the stereoisoꢀ
mers on mouse behavior were investigated. It has been
shown that only the diastereomer (III) has neuroproꢀ
tective properties, increasing the exploratory and
motor activity under conditions of hypoxic stress.
The stereoisomer (V) decreases the motor activity of
intact mice in the elevated plusꢀmaze and suppresses
both the motor and explorative activity of the animals in
EXPERIMENTAL
the openꢀfield test after hypoxia. The stereoisomer (VI
)
Commercially available (R)ꢀ, (S)ꢀ, and (R,S)ꢀ
methionine, urea, and a 40% aqueous solution of glyꢀ
oxal supplied by Acros were used in the present work.
does not affect intact mice, but has a certain suppresꢀ
sive effect on the central nervous system, decreasing
the motor activity of mice after hypoxia in both tests.
According to the data obtained, the profile of comꢀ
pound (XI) is similar to that of compound (III), and
(
R
)ꢀ,
VIII)–(
)ꢀ, ( )ꢀ, or (R,S)ꢀmethionine with KOCN accordꢀ
ing to the procedure we developed previously [23].
4,5ꢀDihydroxyimidazolidineꢀ2ꢀone (VII DHI) was
(
X
S
)ꢀ
,
and
(
R,S)ꢀ
N
ꢀcarbamoylmethionines
(
) were synthesized by
Nꢀcarbamoylation of
(R
S
the suppressive activity of component (V) is maniꢀ
fested only as a slight decrease of the stimulatory effect
after hypoxia compared to compound (III). Thereꢀ
fore, only one of the four enantiomers obtained,
namely, compound (III), can compensate for the
deviations in animal behavior caused by hypoxia.
,
synthesized in a reaction of urea and glyoxal according
to the procedure described in [24]. 1Hꢀ and 13C NMR
spectra were registered in DMSOꢀd6 on Bruker AMꢀ250
(
1H, 250 MHz) and Bruker AMꢀ300 (13С, 75.5 MHz)
spectrometers; the chemical shifts are reported in the
scale relative to the internal standard Ме4Si. The
melting temperature was determined on a GALLENꢀ
KAMP device (Sanyo).
Since the compounds exhibiting neurotropic activꢀ
ity often have antioxidant properties, and some wellꢀ
known antioxidants, for example, tocopherol, emoxꢀ
ipin, and probucol, are used for the treatment of
hypoxic conditions, we have studied the antioxidant
effects of the compounds synthesized on lipid peroxiꢀ
dation triggered by ironꢀascorbate treatment in mouse
brain homogenates [22]. The study showed that comꢀ
δ
Xꢀray diffraction data. Crystals of the compound
XI) (C9H14N4O4S, M 274.30) were rhombic,
(
belonged to the P212121 space group at 120 K, and had
the following parameters: = 9.0258(6)
10.7539(7) Å, and = 12.6020(9)
= 1223.18(14) Å3
= 4 ( (Mo
' = 1), dcalc = 1.490 g cm–3
a
Å, b =
pounds (III)–(V) do not possess antioxidant properꢀ
ties.
c
Å,
V
,
Z
Z
,
μ
K ) =
α
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NEUROPROTECTIVE ACTIVITY
555
2.79 cm–1, F(000) = 576. The intensities of 12476 18.50
(c 2; 1M NaOH). Found, %: C 39.45; H 5.09;
°
reflections were measured in a Bruker SMART 1000 N 20.46; S 11.64; С9Н11N4О4S. Calculated, %: C
13
39.41; H 5.14; N 20.43; S 11.69. 1Нꢀ and C NMR
CCD diffractometer [
λ
(Mo
K
) = 0.71072
Å
, ꢀscanꢀ
ω
α
ning, < 56 ], and 2934 independent reflections [Rint =
2
θ
°
spectra corresponded to the reported data [16].
0.0281] were used in the subsequent refinement. The
structure was solved using the direct method and
refined using the fullꢀmatrix LSM by F2 in the anisoꢀ
tropicꢀisotropic approximation. Hydrogen atoms of
the NH and OH groups were localized from the subꢀ
tractive Fourier syntheses of the electron density. The
(–)ꢀ(S)ꢀ2ꢀ[(1S,5R)ꢀ(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazaꢀ
bicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid
(IV). The yield equaled 0.08 g (3%), the melting temꢀ
perature was 233–235
°
С
(with decomposition),
[α
]2D0(–) 77.77
° (c 2; 1 M NaOH). Found, %: C 39.33;
positions of H(C) hydrogen atoms were calculated H 5.01; N 20.61; S 11.75; С9Н11N4О4S. Calculated,
13
%: C 39.41; H 5.14; N 20.43; S 11.69. 1Нꢀ and C
geometrically. The positions of all hydrogen atoms
were adjusted in the isotopic approximation using the
“rider” model. The final value of the uncertainty facꢀ
NMR spectra corresponded to the reported data [16].
(–)ꢀ(R)ꢀ2ꢀ[(1S,5R)ꢀ(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazaꢀ
tor for the compound
1.005 for all the independent reflections (R1 = 0.0456
was calculated according to the structure factor for
2392 observed reflections with > 2 ). All calculaꢀ
(XI): wR2 = 0.1087 and GOF =
bicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid
(V). The yield equaled 1.01 g (37%), the melting temꢀ
F
perature was 256–258
°
С
(with decomposition),
I
σ(I)
[α
]2D0(–) 18.50
°
(c 2; 1M NaOH) [17]. Found, %: C
tions were performed using the SHELXTL PLUS 5.0
software [25].
The diffractogram of the sample (XI) was registered
on a Bruker D8 Advance Vario diffractometer using
39.47; H 4.92; N 20.33; S 11.51; С9Н11N4О4S. Calcuꢀ
lated, %: C 39.41; H 5.14; N 20.43; S 11.69. 1Нꢀ and
13C NMR spectra corresponded to the reported data
[17].
Cu
K
radiation (Ge {111} monochromator) and the
α
1
positionꢀsensitive Bruker LynxEye detector. The meaꢀ
surements were performed in the transmission mode;
the sample was layered on Kapton film. The measureꢀ
(+)ꢀ(R)ꢀ2ꢀ[(1R,5S)ꢀ(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazabiꢀ
cyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid (VI).
The yield equaled 0.08 g (3%), the melting temperaꢀ
ment range was 4.5–70
°
2
θ
;
θ
/2
θ
scanning with a step
ture was 233–235
°С
(with decomposition),
[
]2D0(+)
α
of 0.02 was used. The Pauly method refinement
°
2
θ
77.77 ; 1M NaOH). Found, %: C 39.55; H 5.03;
° (c 2
was performed using the Bruker TOPAS 4.2 software.
The final values of the deviance factor were the followꢀ
N 20.49; S 11.61; С9Н11N4О4S. Calculated, %: C
1
39.41; H 5.14; N 20.43; S 11.69. H NMR spectrum:
ing:
Rp = 5.83%, Rwp = 8.77%, Rexp = 2.24%, GOF =
2.10–2.19 (5H, m, CH3 + CH2), 2.51–2.61 (2H, m,
3.92.
CH2), 4.07 (1H, dd,
J
4.89,
J
10.36 CH), 5.29 (1H,
8.16 0.03
Since we have not reported the diastereoselective
synthesis procedures, element analysis results, and
some physicalꢀchemical characteristics of the glyꢀ
colurils (III)–(VI), (XI), and (XII) in the previous
short reports [16, 17], we report them in the following
sections.
br.d., 8.16 0.03, H1), 5.34 (1H, br.d.,
J
J
,
H5), 7.28 (1H, s, 1NH), 7.38 (1H, s, 1 NH), 7.51 (1H,
s, NH), and 12.75 (1H, br.s, OH).
Synthesis of the racemate (XI)
(R,S)ꢀNꢀcarbamoꢀ
ylmethionine ( ) and DHI were taken in equal
X
Synthesis of the diastereomers (III)–(VI).
(
S
)ꢀ (IX
)
)
amounts (0.01 mol, corresponding to 1.92 g and 1.18 g,
respectively), mixed with 20 mL of water and 0.02 mL
of strong hydrochloric acid (36% aqueous solution),
or ( )ꢀ ꢀcarbamoylmethionine (VIII) and DHI (VII
R
N
were taken in equal quantities (0.01 mol, corresponding
to 1.92 g and 1.18 g, respectively), mixed with 20 mL
of water and strong hydrochloric acid was added dropꢀ
wise until the mixture pH was 2–1; the reaction mixꢀ
incubated at 80
and filtered to remove the precipitate of the 3 : 1 raceꢀ
mate mixture (XI XII). The compound (XI) was
°С for 2 h, cooled to 20°С, left for 36 h,
+
ture was incubated at approximately ~80
cooled to 20 , left for 12 h, and filtered to remove the
precipitate of the predominating diastereomer (III) or
). The filtrate was concentrated to half of the initial
°
С for 1 h,
obtained in six steps of crystallization from aqueous
solutions.
°
С
The yield of the compounds (XI
ratio equaled 1.23 g (45%).
+ XII) in a 3 : 1
(V
volume by vacuum evaporation, left for 12 h, and filꢀ
tered to remove the precipitate of the diastereomer
Racemate (XI) ꢀ [(+)ꢀ(
S
)ꢀ2ꢀ[(1
R,5S)ꢀ(3,7,dioxoꢀ
mixtures (III
+ IV) or (V + VI). The filtrate was left at
2,4,6,8ꢀtetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthioꢀ
20 for 24 h and filtered to remove the precipitated
°С
butanoic acid (III) and (–)ꢀ(
R)ꢀ2ꢀ[(1S,5R)ꢀ
minor diastereomer. Products (III)–(VI) were crystalꢀ
lized from water.
(3,7,dioxoꢀ2,4,6,8ꢀtetraazabicyclo[3.3.0]octꢀ2ꢀyl)]ꢀ
4ꢀmethylthiobutanoic acid (V)]. The yield equaled 0.27 g
(10%), the melting temperature was 256–258
°С
(with
(+)ꢀ(S)ꢀ2ꢀ[(1R,5S)ꢀ(3,7ꢀdioxoꢀ2,4,6,8ꢀtetraazabiꢀ
]2D00
cyclo[3.3.0]octꢀ2ꢀyl)]ꢀ4ꢀmethylthiobutanoic acid (III).
decomposition),
[α °(c 2; 1M NaOH). Found, %:
The yield equaled 1.01 g (37%), the melting temperaꢀ
C 39.51; H 5.00; N 20.44; S 11.70; С9Н11N4О4S. Calꢀ
culated, %: C 39.41; H 5.14; N 20.43; S 11.69.
20
D
ture was 256–258
°С
(with decomposition),
[α]
+
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 5
2012

556
KRAVCHENKO et al.
Neuroprotective properties of the compounds experimental precision. A sample in which malonic
(III)–(VI) and (XI) were assessed in white randomꢀ dialdehyde formation was induced was used as a posiꢀ
bred male mice with a body mass of 22–24 g. The mice tive control. The experiment was performed in sextuꢀ
were divided into isolated groups of ten animals. All plicate.
groups were housed similarly and belonged to progeny
of close siblings obtained at the same time. The behavꢀ
ioral reactions were assessed in “openꢀfield” [20] and
“elevated plus maze” [21] tests. Motor activity assessꢀ
ment was based on the number of squares crossed in
the open field and on the total number of entries to the
open and closed arms of the maze. Exploratory activꢀ
ity assessment was based on the number of holes
explored in the open field and on the number of rearꢀ
ings and head dippings in the plus maze.
Statistical analysis of all data was performed using
the Student’s tꢀtest. The difference was considered
significant at
р
< 0.05.
ACKNOWLEDGMENTS
Financial support for the present work was proꢀ
vided by the “Biomolecular and Medicinal Chemisꢀ
try” program of the Chemistry and Material Science
Division of the Russian Academy of Sciences.
Assessment of the anxiety level of the mice was
based on the number of entries to the open arms of the
maze and time spent there. The control animals were
given an intraperitoneal injection of physiological
saline, the experimental animals received injections of
the drugs tested (100 mg/kg in physiological saline),
and mebicar (100 mg/kg) was used as the reference
drug. In order to maintain the drug treatment duration
exactly the same for all drugs, the time delay between
the injection of physiological saline mebicar solution
and the drugs tested to mice of each group equaled
6 min. Exactly 1 h after the injection each mouse was
placed into the “open field” apparatus for 3 min and then
into the “elevated plus maze” apparatus immediately for
3 min. All the experiments were performed between
14.00 and 16.00; ten animals were tested every day.
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