E. Procházková et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6405–6409
6407
uct of a successive reaction of 2a, the yellow 2b (⁄, 8.9 and
9.0 ppm). The low intensity of these signals is caused by the low
solubility of 2b in DMSO.
(see Scheme 3). Compound 1c was also prepared (for the compar-
ison of spectral properties) by a known oxidation reaction of 5-
aminouracil with potassium ferricyanide.11
We isolated the two products of the reaction (2a and 2b). The
absorption spectra of the isolated products are shown in Figure
3. The absorption maxima of the individual products (2a and 2b)
are in agreement with the maxima observed in the reaction mix-
ture after 11 days at room temperature, that is at 347 and
550 nm for 2a and 397 nm for 2b.
Another studied compound was 5-aminobarbituric acid (4). The
reaction was observed by UV/VIS spectroscopy and 1H NMR spec-
troscopy. In the DMSO solution, the ammonium cation released
(observed by 1H NMR) and the ‘open form’ 4a (i.e. a metalochromic
indicator murexide, see Scheme 3) was detected, but no subse-
quent intramolecular condensation was observed. This is not sur-
prising, because no aminogroup is present in 4a. Product 4a has
its absorption maximum at 532 nm and by comparison with the
commercially available sample of murexide, its structure was
unambiguously confirmed.
The DMSO oxidation of 5,6-diamino-4-oxo-2-thiopyrimidine (5)
was also studied. The changes in the UV/VIS spectra and the pres-
ence of ammonia cation observed in 1H NMR suggest that similar
oxidation and condensation reactions take place. However, a com-
plex mixture of products appeared in the solution and the isolation
of the products failed. Some unidentified subsequent reactions
involving the reactive SH group (e.g. oxidation of the SH group)
probably took place. The DMSO solution of 2,5-diamino-4,6-
dimethoxypyrimidine (6) slowly changed its colour to orange. How-
ever, the only change we were able to detect in the 1H NMR spectra
was the hydrolysis of one of the methoxy groups (signals of metha-
nol appeared in the spectra together with an amidic proton and one
methoxy group). Apparently, the hydrolysed product may undergo
further reactions (hydrolysis of the other methoxy group, oxidation
and condensation), which leads to colour changes, but the subse-
quent reactions were so slow that the products were below the
NMR detection limit even after two months. The DMSO solution of
2,5-diamino-4,6-dichloropyrimidine (7) slowly changed its colour
to dark red, which was further converted into yellow. However, both
the NMR and MS spectra revealed that a complicated mixture of
products was formed. The chlorine atoms might have been hydroly-
sed and/or substituted, because the typical isotopic pattern of chlo-
rinated compounds was not observed in the MS spectra.
To support further the proposed reaction mechanism and the
structures of the condensation products, we performed condensa-
tion reactions of compounds 1, 2, 3, 4 and 6 with alloxan (2,4,5,6-
tetraoxopyrimidine). The reactions were performed in DMSO by
mixing equimolar amounts of alloxan with a substituted 5-amino-
pyrimidine. Compound 1 gave bipyrimidine 1d, which gave pyrim-
idopteridine 1b after heating to 120 °C. Similarly, we obtained
bipyrimidine 2d and pyrimidopteridine 2e from compound 2.
The product 2e was hydrolysed with sodium nitrite to 1b.
We were not able to isolate the bipyrimidine derivative from
the reaction mixture of compound 3 with alloxan; the only product
of the reaction was pyrimidopteridine 3e, which was also hydroly-
sed with sodium nitrite to 1b. 5-Aminobarbituric acid (4) gave
murexide (4a) after reaction with alloxan, and compound 6 con-
densed with alloxan to a bipyrimidine derivative 6d.
The high-resolution mass spectra (HRMS) revealed the composi-
tions of the products 2a and 2b to be C8H10N9O2 and C8H8N9O,
respectively. The hydrolysis of compound 2b with sodium nitrite
and dilute hydrochloric acid yielded compound 1b of molecular for-
mula C8H4N6O4, the UV/VIS spectrum of which was identical with
the previously published spectrum of bis-alloxazine.10 The struc-
tures of compounds 2a and 2b together with the proposed mecha-
nism of the oxidation reaction are depicted in Scheme 2. First,
compound 2 is oxidised by DMSO to the pyrimidine-quinone-imine
while an ammonium cation (which was observed in the 1H NMR
spectra) is released. The quinone-imine can subsequently condense
with the 5-aminogroup of another molecule 2, after which the
intermediate 2a can undergo a new ring closure to give 2b as the fi-
nal product of the reaction. In the 13C NMR spectrum of compound
2a, we observed four signals, which is in agreement with the pro-
posed structure of 2a, where the iminogroup of one ring can be in
fast tautomer exchange equilibrium with the amino group of the
second ring. Unfortunately, the very low solubility of pyrimidopter-
idines did not allow us to acquire the 13C NMR spectrum of com-
pound 2b. Interestingly, we did not observe the formation of a
condensation product analogical to the compound 1c.
We performed the reaction also in dried DMSO under an inert
atmosphere, and the reaction rates were unchanged. When the
reaction was performed in larger quantities, the smell of dimethyl
sulphide (DMS) was apparent. These observations are in agreement
with the proposed mechanism. Interestingly, when the DMSO solu-
tion was alkalised with a drop of NaOH solution, we were not able
to detect the purple intermediate 2a and the reaction to 2b was
completed in 24 h. Conversely, when a drop of HCl was added to
the DMSO solution, the reaction was slowed down.
The solution of tetraaminopyrimidine 3 in DMSO changed from
colourless to orange overnight. The oxidation products of com-
pound 3, two constitutional isomers 3b (yellow) and 3c (red), were
isolated and their absorption spectra were compared with the
reaction mixture in DMSO (see Fig. S3 in SI). The molecular formula
C8H8N10 was determined by HRMS for both compounds 3b and 3c.
The ‘open form’ similar to the intermediate 2a was not detected in
this case because the second condensation reaction is probably too
fast. The reason for the different reactivity might be in different
acido-basic properties of the two compounds. After hydrolysis of
the amino groups of both isomers with sodium nitrite in dilute
hydrochloric acid, we obtained compounds 1b and 1c, respectively
We have also studied a series of disubstituted 5-aminopyrimi-
dines (with hydrogen atom in position 2 or 6). They were oxidised
more slowly than the trisubstituted derivatives 1–6. In the UV/VIS
spectra of these compounds, we observed similar changes like for
compounds 1–6. However, we were not able to isolate the oxida-
tion products for the majority of the disubstituted 5-aminopyrim-
idines in sufficient quantity for proper characterisation. The full
discussion of these compounds is given in the SI.
For comparison, we dissolved 2,6-diamino-4-oxopyrimidine
(i.e. compound 2 without the 5-aminogroup) in DMSO and we
did not observe any changes in the UV/VIS or NMR spectra. Obvi-
ously, the 5-aminogroup is crucial for the oxidation reaction. Nei-
ther did we observe any reaction of this compound with alloxan.
All of the reactions were monitored by UV/VIS spectroscopy;
the spectra are shown in the SI. The isolated products were charac-
O
N
O
O
NH2
NH2
DMSO
HN
HN
H2N
2
-DMS
-NH3
HN
NH2
N
2
O
N
NH2
N
O
N
NH2
N
N
HN
HN
HN
H2N
N
N
2b
N
NH2
NH2
O
N
H
NH2
2a
Scheme 2. The proposed mechanism of compound
2 oxidation and self-
condensation.