5-Arylideneaminouracils: II. Synthesis of sodium and ammonium salts

Article abstract of DOI:10.1134/S1070363209050211

In continuation of studies on the design of new 5-arylideneaminouracil derivatives possessing antimicrobial and antiviral properties, the corresponding ammonium and sodium salts were synthesized. The antimicrobial activity of water-soluble salts of substituted aminouracils in vitro was shown to be several times higher than that of parent neutral compounds.

Full text of DOI:10.1134/S1070363209050211

ISSN 1070-3632, Russian Journal of General Chemistry, 2009, Vol. 79, No. 5, pp. 991–995. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © V.I. Krutikov, A.V. Erkin, 2009, published in Zhurnal Obshchei Khimii, 2009, Vol. 79, No. 5, pp. 819–823.
5-Arylideneaminouracils: II.1 Synthesis of Sodium
and Ammonium Salts
V. I. Krutikov and A. V. Erkin
St. Petersburg State Institute of Technology, Moskovskii pr. 26, St. Petersburg, 190013 Russia
e-mail: kruerk@yandex.ru
Received May 29, 2008
Abstract—In continuation of studies on the design of new 5-arylideneaminouracil derivatives possessing
antimicrobial and antiviral properties, the corresponding ammonium and sodium salts were synthesized. The
antimicrobial activity of water-soluble salts of substituted aminouracils in vitro was shown to be several times
higher than that of parent neutral compounds.
DOI: 10.1134/S1070363209050211
In the previous communication [1] we reported on
the synthesis of 5-arylideneaminouracils and showed
that their water-soluble derivatives characterized by
large lipophilicity parameters log P should exhibit
enhanced biological activity. In addition, introduction
of electron-withdrawing substituents in the aromatic
fragment of their molecules is desirable.
mycobacteria to the action of acids [2]. On the basis of
the results obtained previously [1], we selected most
potent (from the viewpoint of antimicrobial activity) 5-
arylideneaminouracils and synthesized their water-
soluble sodium and ammonium salts.
The target compounds were prepared in two steps.
In the first step, 5-aminouracil was converted into its
sodium or ammonium salt, and in the second step the
salt thus obtained was brought into condensation with
the corresponding aldehyde in alcohol. 5-Arylidene-
aminouracil sodium and ammonium salts I and II are
brightly colored substances which melt with
decomposition at about 300°C (ammonium salts melt
above 300°C).
We believed that the most appropriate way to obtain
such derivatives is the synthesis of 5-arylidene-
aminouracil sodium and ammonium salts. On the one
hand, such salts should be readily soluble in water,
and, on the other, the presence of ammonium cations in
biological medium should increase antimicrobial
activity, taking into account resistance of some
CHO
O
O
O
R
NH2
N
NH2
Na+ (NH4+)
HN
NaOH (NH4OH)
HN
HN
_
R
_
Na+ (NH4+)
O
N
O
N
O
N
H
Ia_If, IIb, IId, IIe, IIg
R = 2-НО-5-O2N (a), 2-HO-3,5-I2 (b), 2-OH-3-Br-5-O2N (c), 2,4-Cl2 (d), 2-HO-5,6-benzo (f), 2-HO (g).
We failed to obtain satisfactory elemental analyses
of the isolated sodium salt because of their high ash
content. Therefore, we proposed a procedure for their
qualitative identification and quantitative determination.
No qualitative test for arylideneaminouracils has been
reported. For this purpose, we used Brady’s reagent
(2,4-dinitrophenylhydrazine) which is known to react
with carbonyl-containing compounds (aldehyde and
ketones) with formation of the corresponding colored
crystalline hydrazones. We anticipated that the reaction
1
For communication I, see [1].
991

992
KRUTIKOV, ERKIN
of arylideneaminouracil sodium salts with Brady’s
reagent may be accompanied by hydrolysis of the
azomethine moiety with formation of parent aldehyde.
In fact, sodium salt Ie in ethanol reacted with a
solution of 2,4-dinitrophenylhydrazine with formation
of 3,5-dichloro-2-hydroxybenzaldehyde 2,4-dinitro-
phenylhydrazone which separated from the solution as
yellow crystals. This reaction can also be used for
quantitative determination of arylideneaminouracil (from
the weight of the colored product).
HO
Cl
O
O
HO
Cl
Cl
N
NH2
H
O
H2SO4
HN
HN
_
+
+ Na2SO4
Na+
Cl
O
N
O
N
H
NO2
NH2
O2N
N
H
NO2
HO
Cl
O2N
N
N
H
Cl
In the IR spectra of ammonium and sodium salts I
The structure of salts I and II was confirmed by the
1H NMR spectra. 5-Arylideneaminouracil sodium salts
and II, stretching vibrations of the azomethine N=CH
bond appeared at 1670 cm–1, a broad band at 3400 cm–1
was assigned to OH stretching vibrations, N–H groups
in the pyrimidine gave rise to absorption bands at
about 3100 cm–1, carbonyl absorption was observed at
1640 cm–1, and C–Cl stretching vibrations had a
frequency of 800 to 700 cm–1. The electronic spectra of
5-arylideneaminouracil sodium and ammonium salts
contained absorption maxima in the region λ 280–300 nm
due to electron transitions in the uracil and aryl frag-
ments, and the entire conjugation system was
characterized by absorption band in the visible region
(λmax 400 nm; Fig. 1).
1
I characteristically showed in the H NMR spectra a
singlet at δ 9.4–9.6 ppm from the N=CH proton, NH
signal at δ 11.3–11.5 ppm, and a singlet from 6-H in
the pyrimidine ring and signals from aromatic protons
1
in the region δ 7.7–8.1 ppm (Table 1). The H NMR
spectra of ammonium salts II were almost identical to
those of sodium salts I (Table 2). The only difference
was the shape of signals from exchangeable protons:
these protons (NH and OH) appeared in the spectra of
ammonium salts II as broadened signals with strongly
reduced intensity.
Table 1. Yields, melting points, Rf values, and 1H NMR spectra of 5-arylideneaminouracil sodium salts Ia–If
1H NMR spectrum, δ, ppm
Compound
no.
Yield, %
mp,a °C
Formula
Rfb
CH=N
9.4
NH
6-H
7.9
92
282
С11H6N3NaO5
0.46
11.4
Iа
92
98
268
300
С11H6I2N3NaO3
С11H6BrN4NaO5
0.52
0.55
9.5
9.6
11.5
11.4
7.8
8.0
Ib
Ic
92
96
98
295
290
298
С11H6O2Cl2N3Na
С11H6Cl2N3NaO3
С15H10N3NaO3
0.45
0.54
0.51
9.5
9.4
9.4
11.3
11.4
11.3
8.1
7.7
8.1
Id
Ie
If
a
S razlozheniem. b Chloroform–ethanol, 7:1.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 5 2009

5-ARYLIDENEAMINOURACILS: II.
993
ε, l mol–1 cm–1
2000
ε, l mol–1 cm–1
4000
1
3000
2000
1000
0
1500
2
pH 5.5
pH 6.86
1000
3
500
pH 9.18
0
200
250
300
350
400
450
250
300
350
400
450
500
λ, nm
λ, nm
Fig. 1. Electron absorption spectra of (1) 5-aminouracil, (2)
3,5-dichloro-2-hydroxybenzaldehyde, and (3) 5-(3,5-di-
chloro-2-hydroxybenzylideneamino)uracil sodium salt (Ie).
Fig. 2. Electronic absorption spectra of 5-(3,5-dichloro-2-
hydroxybenzylideneamino)uracil ammonium salt (IIe) at
different pH values.
It was important to examine the behavior of 5-
arylideneaminouracil salts in aqueous solution at
different pH values with a view to estimate their
stability in biological media. The UV spectra of salts I
and II were measured in buffer solutions at different
pH values (tartrate, pH 3.56; phthalate, pH 5.5;
phosphate, pH 6.86; borate Na2B4O7·H2O, pH 9.18
[4]). We found that salts I and II decomposed at pH
3.5: long-wave absorption maximum at λ 400 nm
disappeared from the UV spectra. Ammonium salts II
were stable at pH ≥5.5. Figure 2 shows the electron
absorption spectra of 5-(3,5-dichloro-2-hydroxyben-
zylideneamino)uracil ammonium salt (IIe) at different
pH values. The presence of an isosbestic point and a
weak absorption band at λ 340 nm must be noted.
diiodobenzylideneamino)uracil ammonium salt (IIb)
should be noted, especially with respect to S. aureus
29213. The Hantzsch curve in Fig. 3 demonstrates
nonlinear relation between biological activity of 5-
arylideneaminouracil sodium salts and their
lipophilicity. The plot clearly has exponential shape, in
keeping with the correlations drawn by us previously
[1].
We presumed in [1] that the presence of electron-
withdrawing substituents in the aromatic fragment is a
necessary condition for high antimicrobial activity of
5-arylideneaminouracil derivatives. This assumption
was generally confirmed; nevertheless, the scope of the
obtained correlation is fairly limited. It seemed
interesting to estimate biological activity of 5-
arylideneaminouracils having an a fortiori pharma-
cophoric fragment. For this purpose we synthesized 8-
hydroxyquinoline-5-carbaldehyde and its condensation
product with 5-aminouracil. It is known that many
antimicrobial agents contain a 8-hydroxyquinoline
fragment. However, the antimicrobial activity of 5-(8-
5-Arylideneaminouracil sodium and ammonium
salts I and II were tested for biological activity. It was
found that their antimicrobial effect exceeds the effect
of the corresponding neutral compounds by a factor of
6 to 10. The results are given in Tables 3 and 4. High
and versatile antibacterial activity of 5-(2-hydroxy-3,5-
Table 2. Yields, Rf values, and 1H NMR spectra of 5-arylideneaminouracil ammonium salts IIb, IId, IIe, and IIg
1H NMR spectrum, δ, ppm
Compound
Yield, %
Formula
Rf
no.
N=CH
NH
6-H, Harom
OH
14
76
61
77
C11H10I2N4O3
C11H10Cl2N4O2
C11H10Cl2N4O3
C11H12N4O3
0.24b
0.66
0.34а
0.33c
9.9
9.9
9.5
9.5
11.6
11.2
10.9
11.2
7.1–8.1
7–8
12.1
–
IIb
IId
IIe
IIg
7–8
13.8
12.8
6.8–7.8
a
Chloroform–methanol, 9:1. b Chloroform–methanol, 15:1. c CCl4–propan-2-ol, 7:6.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 5 2009

994
KRUTIKOV, ERKIN
Table 3. Antibacterial activity of 5-arylideneaminouracil ammonium salts IIb, IId, IIe, and IIg
Minimal concentration ensuring 100% inhibition, μg ml–1
Comp.
no.
bacteria
S. aureus 29213
6.2
>100
3.1
50
fungi
mycobacteria
E. coli 25922
Candida Albicans
Aspergillus Niger
M. Smegmatis
>100
>100
100
>100
>100
6.2
>100
>100
12.5
>100
>100
6.2
IIb
IId
IIe
IIg
>100
>100
>100
>100
Table 4. Antibacterial activity of 5-arylideneaminouracil sodium salts Ib–If and Ih
Minimal concentration ensuring 100% inhibition, μg ml–1
Comp. no.
bacteria
fungi
mycobacteria
S. aureus 29213
E. coli 25922
Candida Albicans
Aspergillus Niger
M. Smegmatis
3.1
3.1
100
>100
>100
>100
>100
50
6.2
>100
>100
>100
>100
>100
12.5
>100
>100
>100
50
6.2
100
Ib
Ic
Id
Ie
If
50
>100
>100
50
6.2
25
>100
>100
>100
Ih
hydroxyquinolin-5-ylmethylideneamino)uracil sodium
salt (Ih) turned out to be only moderate, and this
compound showed no diverse biological effect: it was
active in vitro only against E. coli 25922.
cophoric fragment did not enhance antimicrobial
activity. These findings confirm structure–activity
correlations obtained by us previously for 5-aryl-
ideneaminouracils and suggest unique mechanism of
their biological action.
In fact, taking into account the correlations drawn
for antibacterial effect of 5-arylideneaminouracils [1],
such parameters of 5-(8-hydroxyquinolin-5-ylmethyl-
ideneamino)uracil as logP, V, and S imply only
medium level of biological activity (logP = 1.47, S =
267 Å2, V = 424 Å3). Thus introduction of a pharma-
EXPERIMENTAL
1
The H NMR spectra were recorded from solutions
in DMSO-d6 on a Bruker spectrometer (Bruker-
Franzen Analytik GmbH, version 950801.1) at 200 MHz
using residual proton signal of the solvent as reference.
The electronic absorption spectra were measured on an
SF-26 spectrophotometer (MicroCal Origin, version
3.0) from solutions in water with a concentration of
10–4 M. The IR spectra were obtained on a Specord M-
80 instrument from samples prepared as KBr pellets.
The purity of the isolated compounds was checked by
thin-layer chromatography on Silufol UV-254 plates.
log (1/C50) = 4.52 – 0.326 log P + 0.143(log P)2;
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
r = 0.932, s = 0.32.
5-Arylideneaminouracil sodium and ammonium
salts I and II were tested for antibacterial and antiviral
activity against standard strains of microorganisms at
the Microbiology Department, Pavlov St. Petersburg
State Medical University.
–2
–1
0
1
2
3
4
5
log P
5-(3,5-Dichloro-2-hydroxybenzylideneamino)pyr-
imidine-2,4(1H,3H)-dione sodium salt (Ie). A solu-
tion of 0.113 g of sodium hydroxide in 10 ml of water
Fig. 3. Plot of log (1/C50) versus logP for antibacterial activity
of 5-arylideneaminouracil sodium salts against S. aureus
29213.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 5 2009

5-ARYLIDENEAMINOURACILS: II.
995
was added to 0.356 g of 5-aminouracil, and the
mixture was heated on a boiling water bath until
complete dissolution of 5-aminouracil. A solution of
0.541 g of 3,5-dichloro-2-hydroxybenzaldehyde in 7 ml
of hot ethanol was added in portions over a period of
30 min under stirring on heating on a boiling water
bath. An orange–red solid separated, the mixture was
stirred for 30 min while heating on a boiling water bath
and allowed to cool down to room temperature, and the
precipitate was filtered off, washed with 15 ml of
ethanol, and dried. Yield 98%. The other 5-aryl-
ideneaminouracil sodium salts were synthesized in a
similar way.
solution was formed, and the solution was made
alkaline by adding solid sodium carbonate to pH 8–9.
The undissolved material was filtered off and re-
peatedly washed with a 2 N solution of sodium carbo-
nate. The alkaline solution was carefully acidified with
acetic acid, and the precipitate was filtered off, washed
with a small amount of water, recrystallized from
ethanol, and dried in a vacuum desiccator. Yield 1.5 g
(3%), mp 179–180°C; published data [3]: mp 180°C.
Qualitative analysis of 5-arylideneaminouracil
salts. 2,4-Dinitrophenylhydrazine, 1 g, was dissolved
in 7 ml of concentrated sulfuric acid, and the resulting
solution was added under stirring to a mixture of 7 ml
of water and 23 ml of 95% ethanol. Mixing of 3 ml of
Brady’s reagent thus obtained with a solution of 20 mg
of sodium salt I in 2 ml of 95% ethanol resulted in the
formation of a yellow solid.
5-(3,5-Dichloro-2-hydroxybenzylideneamino)pyr-
imidine-2,4(1H,3H)-dione ammonium salt (IIe). A
mixture of 0.4 g of 5-aminouracil and 30 ml of
aqueous ammonia was stirred at room temperature
until it became homogeneous. A solution of 0.6 g of
3,5-dichloro-2-hydroxybenzaldehyde in 25 ml of
ethanol was added dropwise under stirring. The
originally yellow solution turned dark brown. The
mixture was heated for 1 h on a water bath, and the
brick-red precipitate was filtered off and dried in a
desiccator. Yield 61%. The other 5-arylidene-
aminouracil ammonium salts were synthesized in a
similar way.
ACKNOWLEDGMENTS
The authors thank Prof. V.V. Tets and co-workers
(Pavlov St. Petersburg State Medical University) for
performing microbiological studies and also A.K.
Ivanova and E.V. Polozova for their participation in the
present work.
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dropwise to the resulting transparent solution, and the
mixture was heated for 4 h under reflux. The alcohol
and excess chloroform were distilled off under reduced
pressure, the residue was diluted on cooling with
concentrated hydrochloric acid until a transparent
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