Preparative synthesis and pharmacological activity of Albicar racemate and enantiomers

Article abstract of DOI:10.1016/j.mencom.2018.05.030

Racemic Albicar (2,6-diethyl-4,8-dimethylglycoluril) has been synthesized and resolved into enantiomers on a preparative scale by chiral HPLC. A comparison of the pharmacological effects of these compounds has been performed for the first time. It has been demonstrated that the (?)-(1S,5S)-enantiomer exerts a stimulating effect on the central nervous system due to activation of the serotonergic system, since it potentiates the effects of 5-hydroxytryptophan (the serotonin precursor), while the antagonist of serotonin receptors blocks its activity; the (+)-(1R,5R)-enantiomer evinces an inhibitory effect.

Full text of DOI:10.1016/j.mencom.2018.05.030

Mendeleev
Communications
Mendeleev Commun., 2018, 28, 317–319
Preparative synthesis and pharmacological activity
of Albicar racemate and enantiomers
Lada V. Anikina,a Yuri B. Vikharev,a Vladimir V. Baranov,b
Oleg R. Malyshevb and Angelina N. Kravchenko*b
a Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka,
Moscow Region, Russian Federation. Fax: +7 496 524 9508; e-mail: anikina1970@gmail.com
b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow,
Russian Federation. Fax: +7 499 135 5328; e-mail: kani@server.ioc.ac.ru
DOI: 10.1016/j.mencom.2018.05.030
Racemic Albicar (2,6-diethyl-4,8-dimethylglycoluril) has been
synthesized and resolved into enantiomers on a preparative
scale by chiral HPLC. A comparison of the pharmacological
effects of these compounds has been performed for the first
time. It has been demonstrated that the (–)-(1S,5S)-enantiomer
exerts a stimulating effect on the central nervous system due
to activation of the serotonergic system, since it potentiates
the effects of 5-hydroxytryptophan (the serotonin precursor),
while the antagonist of serotonin receptors blocks its activity;
the (+)-(1R,5R)-enantiomer evinces an inhibitory effect.
Me
Et
N
N
Stimulating effect
on the CNS
O
O
O
O
N
N
Me
N
Et
Et
Me
NH2
NH
N
(–)-(1S,5S)
O
O
O
N
N
Me
N
Et
Et
Et
Me
N
Inhibitory effect
on the CNS
Racemate
of Albicar
N
N
Et
Me
(+)-(1R,5R)
Glycolurils represent a class of pharmacologically active com-
pounds.1 One of them, 2,4,6,8-tetramethylglycoluril (Mebicar,
Mebix or Adaptol), is used in Russia for the treatment of
neuroses.2 Yet another compound, 2,6-diethyl-4,8-dimethyl-
glycoluril (Albicar), has passed preclinical trials and has been
recommended for clinical trials as a tranquillizer and an agent for
the treatment of vegetative neuroses.1(c) Unlike achiral Mebicar,
Albicar ‘gull wings’-shaped molecule2(b) is chiral since its C1
and C5 atoms are asymmetric. The pharmacologic tests of Albicar
were performed using a racemate. However, in general only
one enantiomer is useful in racemic pharmaceutical agents (see,
e.g., ref. 3). Moreover, of two enantiomeric diastereomers of
glycolurils obtained from (R)- and (S)-N-carbamoylmethionine
and 4,5-dihydroxyimidazolidin-2-one only one manifests neuro-
tropic activity.1(g),(j)
In search for an agent with optimum properties based on
Albicar, in this study we synthesized an Albicar racemate and
for the first time accomplished its separation into enantiomers.
A comparative study of the pharmacological properties of the
racemate and enantiomers was performed.
Racemic Albicar was synthesized in two stages (Scheme 1)†
by the reaction of ethylurea with glyoxal followed by N-methyla-
tion of intermediate 2,6-diethylglycoluril.2(a),4(a)
Though we have previously synthesized and characterized
Albicar enantiomers,2(a) that method cannot be used to produce
these compounds in amounts sufficient for biological trials (50 mg
of each). Therefore, we were the first to use HPLC for the
preparative separation of the racemate into the enantiomers.Various
columns, eluents and separation conditions were tested. Attempts
to separate the racemate on Chiralpak AD and Chiralcel OJ
columns (Daicel) using propan-2-ol–hexane system in various
ratios were unsuccessful. The best results were achieved on using
two sequentially connected commercial columns Chiralcel OD.‡
Taking into account the published data,2(a) the configurations of
asymmetric bridging carbon atoms were assigned as 1S,5S for
enantiomer 1 (retention time 11 min) and 1R,5R for enantiomer 2
(retention time 28 min).
Et
H
NH2
NH
N
N
Me
N
Et
i
O
O
O
N
N
N
H
O
O
Et
Me
Et
N
N
Et
Et
Me
(–)-(1S,5S)
N
N
ii
iii
O
O
Enantiomer 1
N
N
Me
N
Et
Et
Me
N
†
Racemate
of Albicar
2,6-Diethyl-2,4,6,8-tetraazabicyclo[3.3.0]octane-3,7-dione (2,6-diethyl-
O
O
glycoluril). Yield 60%, mp 286–288°C (lit.,4(a) mp 286–288°C). 1H NMR
(DMSO-d6) d: 1.02 (t, 6H, Me, 3J 7.2 Hz), 2.94–3.06 (AMX3, 2H,
CH2N, 2J 14.2 Hz, 3J 7.1 Hz), 3.15–3.27 (AMX3, 2H, CH2N, 2J 14.2 Hz,
3J 7.1 Hz), 5.22 (s, 2H, CH), 7.48 (s, 2H, 2NH).
N
N
Et
Me
(+)-(1R,5R)
Enantiomer 2
2,6-Diethyl-4,8-dimethyl-2,4,6,8-tetraazabicyclo[3.3.0]octane-3,7-dione
(racemic Albicar). Yield 72%, mp 108–110°C (lit.,4(a) mp 108–110°C).
1H NMR (DMSO-d6) d: 1.08 (t, 6H, Me, 3J 7.1 Hz), 2.81 (s, 6H,
Me), 3.16 (dq, 2H, CH2N, 2J 14.2 Hz, 3J 7.1 Hz), 3.36 (dq, 2H, CH2N,
2J 14.2 Hz, 3J 7.1 Hz), 5.18 (s, 2H, CH) [cf. ref. 4(b)].
Scheme 1 Reagents and conditions: i, glyoxal, H2O, pH 1 (HCl), reflux,
1 h;4(a) ii, Me2SO4, KOH (aq.), 70–75°C, 3 h, then HCl, 20°C, extraction
(CH2Cl2), column chromatography (silica, CH2Cl2) and crystallization from
Et2O;2(a) iii, chiral HPLC on Chiralcel OD.
© 2018 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
– 317 –

Mendeleev Commun., 2018, 28, 317–319
(a)
60
40
20
0
To study the pharmacological effect of the compounds on the
central nervous system (CNS), we used internationally recognized
methods, namely, the ‘open field’ and the ‘elevated plus maze’
tests.5,§
Control
Racemate
EN1
*
*
*
*
EN2
At the first stage, we studied the behavioural reactions of
mice after administration of racemic Albicar in doses from 50 to
300 mg kg–1 in the ‘open field’ integral test and in an effective
dose of 150 mg kg–1 in the ‘elevated plus maze’ test (Figures S1
and S2, Online Supplementary Materials). This ‘dose–effect’
relationship for Albicar was found to be similar to those of
many anxiolytics and antidepressants: small doses show some
stimulating effect, while higher doses cause CNS depression. The
transition to CNS depression by Albicar occurs at doses around
150 mg kg–1. It is expressed as a ca. threefold decrease in the
motional and exploratory activity. Furthermore, upon administering
75 mg kg–1 Albicar, the motional activity of mice did not obey
a normal distribution. Namely, the mice were distinctly divided
into two populations: the behaviour of one half did not change,
whereas the locomotion of the other half decreased abruptly.
This effect may be due to the existing modes of behaviour
(defeatists/tyrants), i.e., the agent has a sedative effect on some
mice and normalizes the behaviour of others.6 In the ‘elevated
plus maze’ test, administering the racemate did not result in
changes in the behavioural reactions of mice in comparison with
the reference group.
30 min
60 min
(b)
20
15
*
Control
Racemate
EN1
*
*
*
10
5
EN2
0
30 min
60 min
Figure 1 Variation of parameters in the ‘open field’test after administering
Albicar racemate and enantiomers (EN1 and EN2).
the motional and exploratory activity by the 30th minute of the
experiment and inhibited both parameters by the 60th minute.
Enantiomer 2 showed an inhibitive effect on the motional and
exploratory activity throughout the entire experiment. Apparently,
the lack of effect of Albicar racemate on the motional and
exploratory activity by the 30th minute of the experiment is due
to the mutually exclusive effects of the two enantiomers, while
both enantiomers act in the same way by the 60th minute.
At the second stage, a comparative in vivo study of the
racemate and enantiomers 1 and 2 in the ‘open field’ test was
performed (Figure 1). The Albicar racemate was administered in
150 mg kg–1 dose, while enantiomers 1 and 2 (EN1 and EN2)
in 75 mg kg–1 dose. The first group of mice was placed into ‘open
field’ 30 min after administering the compounds, the second one
in 60 min, and the third one in 90 min. The data were compared
with those for three reference groups to which physiological
saline was administered abdominally 30, 60 and 90 min before
the test. It was found that Albicar racemate did not affect the
motional and exploratory activity of laboratory animals by the
30th minute of the experiment, but it acted as an inhibitor on
both parameters by the 60th minute. Enantiomer 1 stimulated
Comparison of the behavioural reactions of mice after admi-
nistering racemic Albicar and its enantiomers against the effects
of Yohimbine and Ketanserin was carried out in order to study
the assumed mechanism of action of enantiomers 1 and 2. It is
known that the action of Mebicar (Mebix, Adaptol) is related
to its ability to affect the serotonergic system by enhancing the
action of tryptophane, which is a serotonin precursor.7 To identify
the presence of a serotonergic component and an adrenergic
component intertwined with the former in the mechanism of
Albicar action, we performed experiments to estimate the effects
of Albicar racemate and enantiomers on the action of Yohimbine
and Ketanserin in the ‘open field’ test and on the action of
5-hydroxytryptophan the direct serotonin precursor, which can
pass the hematoencephalic barrier, unlike the mediator itself.8
In our experiments, enantiomer 1 removes the inhibitive effect
ofYohimbine on the motional and exploratory activity of laboratory
animals on the 30th minute of the experiment, i.e., at the peak of
its stimulating action (Figure 2). Racemic Albicar and enantio-
mer 2 throughout the experiment and enantiomer 1 on the 60th
minute of the experiment demonstrate an inhibitive effect on the
behavior of animals. Ketanserin which does not affect the psycho-
‡
Preparative resolution of racemic Albicar. The racemate (100 mg) was
dissolved in propan-2-ol (1 ml), and this solution (20 ml) was injected into
a Rheodyne 7725 loop sampling valve (100 ml) of chromatographic
system comprising Waters 501 pump and RIDK 102 chromatographic
detector (Laboratorni pristroje Praha). The micropreparative separation
was carried out on two sequentially connected commercial Chiralcel OD
4.5×250 mm columns, particle size 10 mm (Daicel, US Department,
Chiral Technologies, Inc., West Chester, PA, USA). Propan-2-ol–hexane
(1:1, v/v) was used as the mobile phase (flow rate 1.5 ml min–1). The
retention times of Albicar enantiomers were 11 min (enantiomer 1) and
28 min (enantiomer 2) with full separation of the peaks to the baseline.
(–)-(1S,5S)-2,6-Diethyl-4,8-dimethyl-2,4,6,8-tetraazabicyclo[3.3.0]-
octane-3,7-dione (enantiomer 1). Mp 138–139°C (lit.,2(a) mp 138–139°C).
[a]2D0 = –41.5° (c 1.6, H2O).
(a)
60
50
40
30
20
10
0
Control
Yohimbine
Yohimbine + racemate
Yohimbine + EN1
Yohimbine + EN2
*
*
*
*
*
*
(+)-(1R,5R)-2,6-Diethyl-4,8-dimethyl-2,4,6,8-tetraazabicyclo[3.3.0]-
octane-3,7-dione (enantiomer 2). Mp 138–139°C (lit.,4(a) mp 138–139°C).
[a]2D0 = +41.7° (c 1.6, H2O).
*
30 min
60 min
§
All experiments involving animals were performed in accordance with
(b)
25
20
15
10
5
Control
Yohimbine
Yohimbine + racemate
Yohimbine + EN1
Yohimbine + EN2
the guidelines set forth by the European Communities Council Directive
of November 24, 1986 (86/609/EEC) and the protocol of experiments
approved by theAnimal Care and Use Committee of IPAC RAS. Mongrel
albino male mice (1.5–2 months old, 20–25 g) were purchased from
the Laboratory Animal Breeding Facility (Branch of M. M. Shemyakin–
Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Puschino, Moscow
Region) and were allowed to acclimate to their environment in the
vivarium for at least 5 days before experiments. Animals were kept in
sawdust-lined plastic cages in a well-ventilated room at 20–22°C in a 12
h light/dark cycle, 40−70% relative humidity, and given ad libitum access
to food and water.
*
*
*
*
*
0
30 min
60 min
Figure 2 Changes in the ‘open field’ test results after administering Albicar
racemate and its enantiomers in the presence of Yohimbine.
– 318 –

Mendeleev Commun., 2018, 28, 317–319
References
Control
Ketanserin
Ketanserin + racemate
Ketanserin + EN1
Ketanserin + EN2
(a)
60
50
40
30
20
10
0
1 (a) A. N. Kravchenko, V. V. Baranov and G. A. Gazieva, Russ. Chem.
Rev., 2018, 87, 89; (b) R. Kamburg, Patent US 227838, 2008 (Chem. Abstr.,
2008, 149, 347537); (c) M. D. Mashkovskii, Lekarstvennye sredstva (Drugs),
NovayaVolna, Moscow, 2016, vol. 1, p. 89 (in Russian); (d) A. S. Berlyand,
O. V. Lebedev and A. A. Prokopov, Pharm. Chem. J., 2013, 47, 176
[Khim.-Farm. Zh., 2013, 47 (3), 52]; (e) A. A. Prokopov, A. S. Berlyand and
O. N. Kazantseva, Pharm. Chem. J., 2003, 37, 132 [Khim.-Farm. Zh.,
2003, 37 (1), 25]; (f) A. S. Berlyand and A. A. Prokopov, Pharm. Chem. J.,
2014, 48, 347 [Khim.-Farm. Zh., 2014, 48 (5), 47]; (g)A. N. Kravchenko,
V. V. Baranov, L. V. Anikina, Yu. B. Vikharev, I. S. Bushmarinov and
Yu. V. Nelyubina, Russ. J. Bioorg. Chem., 2012, 38, 550 (Bioorg. Khim.,
2012, 38, 621); (h) I. S. Ryzhkina, Yu. V. Kiseleva, O. A. Mishina, A. P.
Timosheva, S. Yu. Sergeeva, A. N. Kravchenko and A. I. Konovalov,
Mendeleev Commun., 2013, 23, 262; (i) G. A. Gazieva, D. G. Golovanov,
P. V. Lozhkin, K. A. Lysenko and A. N. Kravchenko, Russ. J. Inorg. Chem.,
2007, 52, 1441 (Zh. Neorg. Khim., 2007, 52, 1539); (j) I. S. Ryzhkina,
S. Yu. Sergeeva, E. M. Masagutova, L. I. Murtazina, O. A. Mishina, A. P.
Timosheva, V. V. Baranov, A. N. Kravchenko and A. I. Konovalov, Russ.
Chem. Bull., Int. Ed., 2015, 64, 2125 (Izv. Akad. Nauk, Ser. Khim., 2015,
2125); (k) G. A. Gazieva, P. V. Lozhkin, V. V. Baranov, A. N. Kravchenko
and N. N. Makhova, Russ. Chem. Bull., Int. Ed., 2010, 59, 642 (Izv. Akad.
Nauk, Ser. Khim., 2010, 628).
*
*
*
*
*
*
*
30 min
60 min
Control
Ketanserin
Ketanserin + racemate
Ketanserin + EN1
Ketanserin + EN2
(b)
20
15
10
5
*
*
*
*
*
*
0
30 min
60 min
Figure 3 Changes in the results of the ‘open field’ test after administering
Albicar racemate and its enantiomers in the presence of Ketanserin.
2 (a) R. G. Kostyanovsky, G. K. Kadorkina, K. A. Lyssenko, V.Yu. Torbeev,
A. N. Kravchenko, O. V. Lebedev, G. V. Grintselev-Knyazev and V. R.
Kostyanovsky, Mendeleev Commun., 2002, 12, 6; (b) V. S. Pletnev,
I. Yu. Mikhailova, A. N. Sobolev, N. M. Galitskii, A. I. Verenich, L. I.
Khmel’nitskii, O. V. Lebedev, A. N. Kravchenko and L. I. Suvorova,
Russ. J. Bioorg. Chem., 1993, 19, 371 (Bioorg. Khim., 1993, 19, 671).
3 (a) J. Chen, K. Natte, A. Spannenberg, H. Neumann, M. Beller and
X. F. Wu, Org. Biomol. Chem., 2014, 12, 5578; (b) B. Satyanarayana,
P. M. Krishna and D. Ramachndran, Int. J. Chem. Tech. Res., 2011, 3,
234; (c) Y. Li, J. K. Coller, M. R. Hutchinson, K. Klein, U. M. Zanger,
N. J. Stanley and A. A. Somogyi, Drug Metab. Dispos., 2013, 41, 1264;
(d) G. W. Muller, W. E. Konnecke, A. M. Smith and V. D. Khetani, Org.
Process Res. Dev., 1999, 3, 139; (e) R.Yokoyama, S. Matsumoto, S. Nomura,
T. Higaki, T. Yokoyama and S. I. Kiyooka, Tetrahedron, 2009, 65, 5181;
(f) S. Robin, J. Zhu, H. Galons, Ch. Pham-Huy, J. R. Claude, A. Tomas and
B. Viossat, Tetrahedron Asymmetry, 1995, 6, 1249; (g) S.Y. Park, B. S. Lee
and S. K. Nangong, J. Int. Eng. Chem., 2002, 8, 515.
4 (a) E. B. Shamuratov, A. S. Batsanov, Yu. I. Struchkov, A. Yu. Tsivadze,
M. G. Tsintadze, L. I. Khmel’nitskii, Yu. A. Simonov, A. A. Dvorkin,
O. V. Lebedev and T. B. Markova, Chem. Heterocycl. Compd., 1991, 27,
745 (Khim. Geterotsikl. Soedin., 1991, 937); (b) E. V. Ivanov, D. V. Batov
and A. N. Kravchenko, J. Chem. Thermodyn., 2016, 97, 341.
5 (a) D. Perals, A. S. Griffin, I. Bartomeus and D. Sol, Animal Behaviour,
2017, 123, 69; (b) E. Zimcikova, J. Simko, I. Karesova, J. Kremlacek and
J. Malakova, Seizure, 2017, 52, 35; (c) E. M. C. Chaves, J. E. R. Honório-
Júnior, C. N. S. Sousa, V. S. Monteiro, D. T. T. Nonato, L. P. Dantas,
A. S. S. C. Lúcio, J. M. Barbosa-Filho, M. C. A. Patrocínio, G. S. B. Viana
and S. M. M. Vasconcelos, Metab. Brain Dis., 2017, 1, 139; (d) I. R. Bossier,
P. Simon and I. M. Zwolf, Thérapie, 1964, 19, 571; (e) S. P. Fernandez,
G. A. R. Johnston, M. Nguyen, T. T. Yow, C. Chu, J. R. Hanrahan and
M. Chebib, Neurochem. Res., 2009, 34, 1867.
pharmacological characteristics of animals blocks the stimulating
properties of enantiomer 1 by the 30th minute and enhances the
inhibitory properties of both enantiomers and Albicar racemate
throughout the entire experiment (Figure 3).
In the tests with 5-hydroxytryptophan, Albicar racemate and
enantiomer 2 did not reliably change the intensity of hyperkinesis
caused by 5-hydroxytryptophan. However, enantiomer 1 reliably
potentiated the action of the dose of 300 mg kg–1 5-hydroxy-
tryptophan: the number of head shakings increased to 50.00±5.33
in comparison with 27.80±2.40 in the reference group of animals
(Figure S3).
In conclusion, we have performed a two-stage synthesis of Albicar
racemate. To obtain enantiomers 1 and 2 in 50 mg amounts, a
chiral HPLC method for racemate separation has been used
for the first time. An in vivo study of Albicar racemate showed
the effective dose to be 150 mg kg–1, which was then used in
the comparative analysis of the effects of Albicar racemate and
enantiomers (the dose of the latter was half that, i.e., 75 mg kg–1).
The lack of an effect of Albicar racemate on the motional and
exploratory activity by the 30th minute of the experiment was
found to be due to the competing mutually exclusive effects
of the two enantiomers (enantiomer 1 has a stimulating effect
on the CNS while enantiomer 2 has an inhibitive effect), whereas
both enantiomers act in an inhibitive way at the 60th minute.
Comparison of the behavioural reactions of mice after administering
racemic Albicar and enantiomers with background action of
Yohimbine and Ketanserin has shown that the action of enantio-
mer 1 is most likely due to the activation of the serotoninergic
system, since it potentiates the effects of 5-hydroxytryptophan,
a serotonin precursor, while the antagonist of serotonin receptors
blocks its activity. The mechanism of action of enantiomer 2
requires further studies.
6 A. V. Val’dman and V. P. Poschivalov, Farmakologicheskaya regulyatsiya
vnutrividovogo povedeniya (Pharmacological Regulation of Intraspecific
Behaviour), Meditsina, Leningrad, 1984 (in Russian).
7 https://amt.allergist.ru/mebicar_l.html.
8 (a) P. Martin, H. Frances and P. Simon, J. Pharmacol. Methods, 1985,
13, 193; (b) G. Sperk, R. M. Stewart, A. Campbell and R. J. Baldessarini,
Brain Res., 1978, 159, 183.
This study was supported by the Russian Science Foundation
(grant no. 17-73-10415).
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2018.05.030.
Received: 11th December 2017; Com. 17/5430
– 319 –