Chromatographia

https://doi.org/10.1007/s10337-018-3599-9

SHORT COMMUNICATION

Isolation, Identification, and Chromatographic Separation of N-Methyl

Derivatives of Glycoluril
D. A. Kurgachev1 · O. A. Kotelnikov1 · D. V. Novikov1 · V. R. Kusherbaeva1 · S. I. Gorbin1 · E. V. Tomilova1 ·
A. Zhaksynbaeva1 · N. B. Dementeva1 · V. S. Malkov1 · A. A. Bakibaev1

Received: 18 May 2018 / Revised: 22 August 2018 / Accepted: 22 August 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Mono-, di-, and tetramethylglycolurils were synthesized, isolated, and purified. For the first time, the cis- and the trans-
isomers of N,N-dimethylglycoluril were isolated as individual substances by semi-preparative HPLC method. The structures
of the synthesized compounds were confirmed by 1H NMR, 13C NMR, and HR–HPLC–MS. The EI mass spectra of indi-
vidual substances were obtained by the GC–MS. Retention and resolution of N-methyl glycolurils were investigated in the
reversed-phase HPLC mode for different stationary phases: ­C18, SB–Aq, and Luna 5u PFP(2). The retention of N-methyl
glycolurils depended on the amount of ­CH3 groups and distance between the ­CH3 groups in the structure. The stationary
phases provided different selectivity for glycoluril and its N-methyl derivatives due to different shape selectivity. Complete
separation of the N-methyl derivatives of glycoluril was achieved in 4.5 min on the stationary phase with pentafluorophenyl
propyl ligand in a gradient mode.
Graphical abstract

Keywords Column liquid chromatography · Mass spectrometry · Nuclear magnetic resonance spectroscopy · Methyl
glycoluril · Mebicar

* D. V. Novikov
novikov.tsu@gmail.com
1
Tomsk State University, 36 Lenin Avenue, 634050 Tomsk,
Russia

13
Vol.:(0123456789)

Introduction
N-Methylglycolurils (tetrahydroimidazo[4,5-d]imidazole-
2,5(1H,3H)-diones) are the condensation products of gly-
oxal and urea (Fig. 1).
Tetramethylglycoluril 6 is an active pharmaceutical
ingredient of the drug “Mebicar” (INN: tetraazabicyclooc-
tanedione), which is used to treat brain, heart, and blood
vessel diseases [1–4]. The substances 2–5 formed during
the synthesis of the substance 6 are potential impurities
in “Mebicar” [2, 3]. The substances 2–5 are also used to
synthesize the bambusurils, a promising class of supramo-
lecular compounds [5]. The structure and physical–chemi-
cal properties of the bambusurils depend on the spatial
structure and purity of the initial N-methyl-derivatives of
the glycoluril [6–10]. The presence of the isomers can lead
to unpredictable effects on the spatial configuration of the
produced bambusurils [14].
There is an analysis of substances 1–6 using NMR
[4–10, 13], X-ray analysis of the substances 3–5 [7, 12],
and volt-amperometric analysis of the substance 6 [8]. The
preparation of 3 by a condensation of monomethylurea and
glyoxal, and also the investigation of the reaction products
by enantioselective HPLC were carried out in [16–18], but
there is no information about the substance 5 that is inevi-
tably formed in the reaction mixture by a condensation of
monomethyl urea and glyoxal [7, 15].
The chromatographic analysis conditions that can be
used to separate and analyze the substances 1–6 are not
described. The EI mass spectra, NMR spectra, and meth-
ods of separation and isolation as individual isomers 3, 4,
5 are not described in the literature.
The aim of this research is to develop the analytical
HPLC separation of the substances 1–6 and to obtain
the reference EI mass spectra (GC–MS). We have also
Fig. 1  Investigated substances: 1, glycoluril (tetrahydroimidazo[4,5-d]
imidazole-2,5(1H,3H)-dione); 2, methylglycoluril (1-methyltetrahydro
imidazo[4,5-d]imidazole-2,5(1H,3H)-dione); 3–2, 6-dimethylgly-
coluril (1,4-dimethyltetrahydroimidazo[4,5-d]imidazole-2,5(1H,3H)-
dione); 4–2, 4-dimethylglycoluril (1,3-dimethyltetrahydroimidazo
13
D. A. Kurgachev et al.
obtained the substances 2–6 in the pure state and con-
firmed their structure using the NMR and HPLC–MS.
Materials and Methods
Chemicals and Reagents
The HPLC gradient-grade methanol and HPLC gradient-
grade acetonitrile were supplied by PanReac AppliChem
(Darmstadt, Germany). The ultrapure water used in all
experiments was purified with the Milli-Q Simplycity
ultrapure water purification system (Millipore, France). The
deuterium oxide with 99.9% atom D was used. For synthesis,
glycoluril and its N-methyl derivatives used urea (98.0%,
Sigma-Aldrich), methylurea (97.0%, Acros), dimethylurea
(98.0%, Acros), and 40% glyoxal solution (BASF).
Synthesis of N‑Methyl Derivatives of Glycoluril
The substances 2 and 4 were synthesized by acid-catalyzed
condensation of methylurea with 4,5-dihydroxyimidazoli-
din-2-one, which was prepared by the reaction of glyoxal
with methylurea [12]. The mixture of the isomers 3, 5 was
synthesized by the reaction of glyoxal with methylurea in
accordance with the method described in [18]. Glycoluril
1 was synthesized by the condensation of urea with glyoxal
according to procedure described in Ref [19]. Tetramethyl-
glycoluril 6 was prepared by the reaction of dimethylurea
with glyoxal as in [18].
The structures of the synthesized substances were con-
firmed by 1H, 13С NMR, and HR HPLC–MS.
1
1 Н NMR (­ D2O, δ, ppm, J/Hz): 5.24 (s, 2H, СН), 7.16 (s,
2H, NH); 13C-NMR ­(D2O, δ, ppm): 64.60 (СН), 160.30
(CO)
[4,5-d]imidazole-2,5(1H,3H)-dione); 5–2, 8-dimethylglycoluril (1,6-
dimethyltetrahydroimidazo[4,5-d]imidazole-2,5(1H,3H)-dio
ne); 6–2, 4,6,8-tetramethylglycoluril (1,3,4,6-tetramethyltetrahydro
imidazo[4,5-d]imidazole-2,5(1H,3H)-dione)
Isolation, Identification, and Chromatographic Separation of N-Methyl Derivatives of…
1
2  Н NMR (d6-DMSO, δ, ppm, J/Hz): 2.60 (s, 3H, ­CH3),
5.14 (d, 1H, СН, J = 8.0), 5.19 (d, 1H, СН J = 7.6), 7.30
(s, 2H, NH) 7.47 (s, 1H, NH); 13C NMR (d6-DMSO,
δ, ppm): 27.56 (СН3), 62.54 (СН), 69.89 (СН), 159.75
(CO), 161.79 (CO); HR HPLC–MS: theoretical
m/z = 157.0720 (M + H)+, measured m/z = 157.0726
(M + H)+, δm/z = 3.8 ppm
1
3  Н NMR (d6-DMSO, δ, ppm): 2.64 (s, 6H, ­CH3), 5.12
(s, 2H, СН), 7.54 (s, 2H, NH); 13C NMR (d6-DMSO,
δ, ppm): 28.21 (СН3), 76.67 (СН), 158.22 (CO); HR
HPLC–MS: theoretical m/z = 171.0877 (M + H)+, meas-
ured m/z = 171.0880 (M + H)+, δm/z = 7.8 ppm
1
4  Н NMR (d6-DMSO, δ, ppm): 2.60 (s, 6H, C ­ H3), 5.10
(s, 2H, СН), 7.40 (s, 2H, NH); 13C NMR (d6-DMSO,
δ, ppm): 27.42 (СН3), 67.39 (СН), 159.66 (CO); HR
HPLC–MS: theoretical m/z = 171.0877 (M + H)+, meas-
ured m/z = 171.0882 (M + H)+, δm/z = 2.9 ppm
1
5  Н NMR (d6-DMSO, δ, ppm): 2.78 (s, 6H, ­CH3), 5.11
(d, 1H, СН, J = 8.4), 5.18 (d, 1H, СН, J = 8.4), 7.40 (s,
2H, NH); 13C NMR (d6-DMSO, δ, ppm): 29.68 (СН3),
60.63 (СН), 75.63 (СН), 160.19 (CO); HR HPLC–
MS: theoretical m/z = 171.0877 (M + H)+, measured
m/z = 171.0877 (M + H)+, δm/z = 0.0 ppm
1
6  Н NMR (d6-DMSO, δ, ppm): 2.82 (s, 12H, ­CH3),
5.09 (s, 2H, СН); 13C NMR (d6-DMSO, δ, ppm):
30.44 (СН3), 71.92 (СН), 159.05 (CO); HR HPLC–
MS: theoretical m/z = 199.1190 (M + H)+, measured
m/z = 199.1190 (M + H)+, δm/z = 0.0 ppm.
Preparative HPLC
The preparative separation was performed on the Kromasil
­C18 column 250 × 20 mm, 5 µm particle size (AkzoNobel,
Bohus, Sweden) and the column temperature was set at
25 °C (± 1 °C). The mobile phase consisted of the water–ace-
tonitrile mixture (94:6). The flow rate was 5 mL min− 1 in the
isocratic mode, and the detection wavelength of UV detector
was 195 nm. The samples were dissolved in water (1:5).
NMR Spectroscopy
The NMR analysis was carried out using an NMR spec-
trometer Bruker AVANCE 400 III HD (Bruker, Billerica,
MA, USA). The one-dimensional spectra were recorded on
the nuclei of 1H atoms (a frequency was 400.17 MHz) and
13
C (a frequency was 100.63 MHz) to confirm the structure.
Dimethylsulfoxide (DMSO D-6) with 99.9% atom D and
heavy water ­(D2O) were used as solvents.
HR HPLC–MS
The experiments were carried out using an Agi-
lent 1260 Infinity Liquid Chromatography System
(Agilent Technologies, Santa Clara, CA, USA) equipped
with a Poroshell EC-C18 reverse-phase 120 column
2.1 × 100 mm with 2.7 µm particle size (Agilent Tech-
nologies, Santa Clara, CA, USA). The column was main-
tained at 35 °C; the injected sample volume was 1 µL.
Solvent A was water; solvent B was acetonitrile. Gradient
conditions were as follows: 0–3 min 1% B, 3–8 min linear
gradient from 1 to 10% B, and 8–9 min return to 1% B,
from 9 to 13 min. column equilibration with 1% B. The
flow rate was 0.3 mL min− 1. The MS experiments were
performed on an Agilent 6550 iFunnel Q-TOF LC-MS
system (Agilent Technologies, Santa Clara, CA, USA)
equipped with an electrospray ionization source. An
electrospray interface was operated in positive ion mode.
The conditions for the acquisition parameters were fol-
lows: the gas temperature was 200 °C, the drying gas
was 14 mL min− 1, the nebulizer pressure was 35 psi, the
sheath gas temperature was 350 °C, the sheath gas flow
was 11 mL min− 1, and the capillary voltage was 3.5 kV.
The scan range was 100–500 with a 2 Hz sampling rate.
GC–MS
The analysis was performed on a gas chromatography–mass
spectrometer Shimadzu QP 2020 (Shimadzu, Kyoto,
Japan). The ion source was operated at 290 °C. The GC
separations were carried out using an HP-5MS column of
30 × 0.25 mm (Agilent Technologies, Santa Clara, CA, USA)
in the following temperature mode: (1) 1 min—150 °С, (2)
10 min—250 °C, and (3) 17 min—290 °C. The flow rate was
1.44 mL min− 1 and the carrier gas was helium. The refer-
ence EI mass spectra were taken on a mass-spectrometer
detector with an electron ionization of 0.2 kV. The samples
were dissolved in methanol at the ratio of 1:1000.
Analytical HPLC
The substances 1–6 in were separated from the mixture
using the HPLC columns PerfectSil Target ODS-3 HD
250 × 4.6 mm, 5 µm particle size (MZ-Analysentechnik,
Mainz, Germany) and Zorbax SB-Aq 150 × 4.6 mm, 5 µm
particle size (Agilent Technologies, Santa Clara, CA, USA).
The complete selective separation of the substances 1–6
was carried out the using stationary-phase Luna 5u PFP(2)
100 Å 150 × 4.6 mm, 5 µm particle size (Phenomenex, Tor-
rance, CA, USA). The mobile phase consisted of water with
acetonitrile in a gradient mode: 0 min—5% of acetonitrile,
1.5 min—25% of acetonitrile, and 4 min—25% of acetoni-
trile. The summary time was 4.5 min; the temperature of
column was 30 °С; flow rate was 1.5 mL min− 1; detection
wavelength of UV detector was 195 nm. The samples were
dissolved in water at the ratio of 1:1000. A chromatographic
13

purity of the substances 2–6 was controlled by the peak area
normalization under optimal conditions. The 2 mg mL− 1
aqueous solutions of the substances were analyzed.
Results and Discussion
Purification and Identification
The compounds 2, 6 were purified by the fractional crystal-
lization. The chromatographic purity of the substances 2 and
6 was 98.9% and 99.3%, respectively. N-dimethyl glycolurils
form both the cis- 5 and the trans-isomer 3 that cannot be
Fig. 2  Mass spectra of compounds 2–6
13
D. A. Kurgachev et al.
separated from each other using the standard purification
methods (extraction, fractional crystallization, etc.) [7, 15].
Accordingly, we separated the cis- and the trans-isomers by
the semi-preparative HPLC. The compound 3 was also puri-
fied by the semi-preparative HPLC from the impurity of the
substance 4. The chromatographic purities of substances 3,
5, and 4 were 98.2%, 99.6%, and 99.5%, respectively. Com-
pounds 1–6 were identified by their 1H, 13С NMR, and HR
HPLC–MS spectral data.
Isolation, Identification, and Chromatographic Separation of N-Methyl Derivatives of…

GС–MS Spectra

The EI mass spectrum contains a molecular radical ion ­M+•

and the fragment radical ions (Fig. 2).
The mass spectra of substances 2–6 contain molecu-
lar radical cations: m/z = 156 (2), m/z = 170 (3, 4, 5), and
m/z = 198 (6), and the fragment radical cations that are char-
acteristic for the compounds 1–6: m/z = 69, 83–85, 98, and
112–113. The substances 2–6 have different ways of frag-
mentation: the mass spectra of 3, 5, and 6 contain fragment
radical cation m/z = 141 that is absent in the mass spectra
of 2 and 4.
It was observed that the dimethyl glycolurils 3, 4, 5 had
different fragmentation. The base fragment ions of the
compounds 3, 5 were at m/z = 98, and for the substance 4 at
m/z = 84. The spectrum for the compound 4 does not contain
the fragment ion at m/z = 141. Furthermore, the substance 4
contains fewer fragment ions of the compounds 3, 5.
The obtained mass spectra can be used as a reference to
identify the compounds 2–6.

Analytical HPLC

It is necessary to develop a chromatographic system that

allows fast and effective separation of glycolurils 1–6. The
maxima of the UV absorbance of compounds 1–6 are at
a wavelength below 200 nm. Nevertheless, the residual
absorption at 195 nm allows detecting the compounds using
a UV spectrophotometric detector.
Using normal-phase and HILIC modes of HPLC is
impractical, because glycoluril 1 is practically insoluble in
any of the organic solvents. Based on the solubility and the
structure of glycolurils 1–6, the reversed-phase mode was
chosen to separate them. The substances 1, 2, 6 differ from
each other in hydrophobicity. Their retention in the reverse-
phase mode will occur through the distributing mechanism
due to ­CH3 and CH–CH groups. Despite the fact that the
substances 3–5 do not differ in hydrophobicity, their ­CH3
groups have different steric availability. Therefore, the reten-
tion factor of the substances 1–6 in the reverse-phase mode
should be proportional to the amount and steric availability
of methyl groups.
The compound 6 is not eluted in the water–acetoni-
trile eluent (99:1) on С 18 PerfectSil Target ODS-3 HD Fig. 3  Chromatogram of a mixture of substances 1 and 6 was
250 × 4.6 mm, 5 µm particle size. Complete elution and sep- obtained using column Target ODS-3 HD, 250 × 4.6 mm, 5 µm with
the water–acetonitrile eluent 95:5 vol. (a), Chromatogram of a mix-
aration of the substances 1–6 are achieved using the water ture of substances 1–6 was obtained using column Zorbax SB-Aq,
and acetonitrile eluent in the ratio of 95:5: the retention fac- 150 × 4.6 mm, 5 µm in gradient mode (b), chromatogram of a mix-
tor of the compound 1 is less than 0.5, but the compound 6 ture of substances 1–6 was obtained using column Luna 5u PFP(2),
has a very high retention factor value (Fig. 3a). 150 × 4.6 mm, 5 µm in gradient- mode (c)
The analysis was transferred to short reversed-phase
column Zorbax SB-Aq (150 × 4.6 mm, 5 µm particle size) The gradient elution stage was added after the com-
to reduce the analysis time and to avoid phase dewetting. pounds 1–4 were eluted to minimize the retention of com-
pounds 5, 6. The optimal gradient profile in which the

13

mixture of substances 1–6 was eluted from the column in
4.5 min (Fig. 3b) was as follows: 0 min—5% of acetoni-
trile, 1.5 min—25% of acetonitrile, and 4 min—25% of
acetonitrile.
The dead time value (tM) at the specified flow rate was
determined by the first baseline disturbance (Fig. 3b) and
the chromatogram of the urea solution: tM value was 1 min
for column 150 × 4.6 mm, 5 µm. As shown in Fig. 3b, there
was a minimum resolution for peaks of substances 3 and
4. The low resolution for compounds 3 and 4 was due to
the lack of shape selectivity with respect to the regioi-
somers. A stationary phase based on silica gel with pen-
tafluorophenyl propyl ligand was used to solve this prob-
lem (Fig. 3c). This phase provided higher shape selectivity
due to the lesser thickness of the modified layer.
Elution order of N-methyl derivatives of glycolurils
does not change for different stationary phases [Luna 5u
PFP(2), SB-Aq] in the reversed-phase mode. These sta-
tionary phases demonstrate an equal retention for N-methyl
derivatives of glycoluril. The retention is influenced by the
amount of ­CH3 groups in the structure, and is related to the
distance between the C ­ H3 groups. Glycoluril 1 has a mini-
mal retention factor, because its retention only depends on
the CH–CH groups.
As shown in the obtained chromatograms (Fig. 3b, c),
the stationary phases provided different shape selectivity
and ­CH3 selectivity for glycoluril and its N-methyl deriva-
tives. The complete separation of the compounds 1–6 was
achieved on stationary phase with pentafluorophenyl propyl
ligand (Luna 5u PFP(2) column) in 4.5 min. The system pro-
vided a minimum resolution for peaks of compounds 2–3,
but the resolution is not less than 1.5 for all peaks.
The validation parameters were evaluated under the
optimal chromatographic conditions (Fig. 3c). The limit
of quantification was estimated on the signal-to-noise
ratio (S/N) of at least 10:1. The quantification limit was
1.60 µg·mL− 1 for the compound 1 and 1.60 µg·mL− 1 for
the compound 6. The linearity of the method was shown
for the substances 1 and 6. The linearity observed from
the limit of quantification to the upper bound of the range:
C = 1.03 × 10− 7 · S + 0.016, R2 = 0.9990 for the compound
1, and C = 7.81 × 10− 8 · S + 0.051, R2 = 0.9998 for the sub-
stance 6. The repeatability relative standard deviation (RSD)
at the quantification limit level, mid (80.0 µg·mL− 1), and the
upper bounds of the range (2.0 mg mL− 1) does not exceed
2.0% for the compound 1, and does not exceed 1.0% for the
compound 6. The chromatographic efficiency under optimal
conditions was not less than 5000 for the peak of compound
2. The tailing factor for all peaks was not more than 1.2. The
retention factor of the compound 1 was about 0.45.
The suitability test parameters were recommended for the
chromatographic analysis of the mixture of substances 1–6.
13
D. A. Kurgachev et al.
• A minimal resolution between peaks of substances 2 and
3 should be at least 1.5.
• A retention factor of the compound 1 should be at least
0.3.
• A tailing factor for any detectable peak on the chromato-
gram should be not more than 1.2.
• A chromatographic efficiency should be at least 4000 for
a peak of the substance 1.
Conclusions
1. The retention factors on different stationary phases in
the reversed-phase mode are depending on the amount
of ­CH3 groups and the distance between ­CH3 groups in
structure.
2. Stationary phases C18, SB-Aq, and provided different
selectivity for glycoluril and its N-methyl derivatives
due to different shape selectivity.
3. The optimal separation of the N-methyl derivatives of
glycoluril is achieved in 4.5 min on stationary-phase
Luna 5u PFP(2) with a water-based mobile phase in a
gradient mode. This method can be used to determine
the ratio of regioisomers 3–5 in the synthesis products,
analyze the purity of compounds 1–6, and quantify of
impurities 1–5 in the substance “Mebicar”.
4. The obtained EI mass spectra of the substances 1–6 can
be used as library materials for identification.
Acknowledgements This research was supported by “The Tomsk State
University competitiveness improvement programme” under Grant No.
8.2.10.2018.
Compliance with ethical standards
Conflict of interest All authors declare no conflict of interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors. No compli-
ance with ethical standards was involved.
References
1. Ministry of Health of the Russian Federation (2017) FS000189-
280911 Tetramethyltetraazabicyclooctandione. State Register of
Drugs, Moscow
2. Novikov SS, Khmelnitsky LI, Lebedev OI (1977) 2,4,6,8-Tetra-
methyl-2,4,6,8-tetraazabicyclo-[3.3.0]-octanedi-3,7-one in treat-
ing psychic disorders, Patent US 4,004,013 A, 18 Jan 1977
3. Kamburg R (2008) Method for neuroprotection with glycoluril
derivatives, Patent US 0,227,838 A1, 18 Sep 2008
4. Fiala T, Sindelar V (2016) Supramolecular complexes of bambu-
surils with dialkyl phosphates. Supramol Chem 28:810–816
Isolation, Identification, and Chromatographic Separation of N-Methyl Derivatives of…
5. Havel V, Svec J, Wimmerova A at al (2011) Bambus[n]
urils: a new family of macrocyclic anion receptors. Org Lett
13(15):4000–4003
6. Havel V (2017) Bambusuril derivatives: synthesis and supramo-
lecular properties. PhD Dissertation, Masaryk University
7. Stancl A, Svec J, Sindelar V (2011) Novel supramolecular hosts
based on linear and cyclic oligomers of glycoluril. Isr J Chem
51:592–599
8. Fiala T, Ludvikova L, Heger D et al (2017) Bambusuril as a one
electron donor for photoinduced electron transfer to methyl violo-
gen in mixed crystals. J Am Chem Soc 97(7):2597–2603
9. Havel V, Babik M (2017) Modulation of Bambusuril anion affinity
in water. Chem A Eur J 23(37):8963–8968
10. Solel E, Singh M, Reany O, Keinan E (2016) Enhanced anion
binding by heteroatom replacement in bambusurils. Phys Chem
Chem Phys 18:13180–13185
11. Veniamin MP, Vakirtzi-Lemonias C (1970) Chemical basis of the
carbamidodiacetyl micromethod for estimation of urea, citrulline,
and carbamyl derivatives. Clin Chem 16(1):3–6
12. Grillon E, Gallo R, Pierrota M, Boileaub J, Wimmerb E (1987)
Isolation and X-ray structure of the intermediate dihydroxyimi-
dazolidine (DHI) in the synthesis of glycoluril from glyoxal and
urea. Tetrahedron Lett 29(9):1015–1016
13. Correia HD, Cicolani RS, Moral RF, Demets GJF (2017)
Easy synthesis of trans-4,5-dihydroxy-2-imidazolidinone and
2,4-dimethylglycoluril. Synthesis 48:210–212
14. Ajami D, Rebek J (2013) Unexpected consequences of methyl
substitutions in supramolecular chemistry. Supramol Chem
25:574–580
15. Kravchenko AN, Sigachev AS, Maksareva EY et al (2005) Syn-
thesis of new chiral mono-, di-, tri-, and tetraalkylglycolurils. Rus
Chem Bull Int Ed 54(3):691–704
16. Kravchenko AN, Strelenko YA (2013) Synthesis of 2,4,6-trialkyl-
8-(2,3-epoxypropyl)glycolurils. Mendeleev Commun 23:104–105
17. Lyubavskaya SS, Chernov YN, Batishcheva GA, Ushakov IB,
Goncharova NY (2016) Complex therapy of chronic pancreatitis
complicated by anxio-depressive disorders in railroad workers.
Res Result Pharmacol Clin Pharmacol 2(4):73–86
18. Nematollahi J, Ketcham R (1963) The reaction of ureas with gly-
oxal. Tetrahydroimidazo[4,5-d]imidazole-2,5-diones. Imidazoimi-
dazoles 28:2378–2380
19. Slezak FB, Bluestone H, Magee TA et al (1962) Preparation of
substituted glycolurils and their N-chlorinated derivatives. J Org
Chem 27:2181–2183
13