5-Arylideneaminouracyls: IV. Phosphorylated derivatives and their biological activity

Article abstract of DOI:10.1134/S1070363209080106

It extension of the studies on the search for the new biologically active 5-aminouracyl derivatives, we synthesized by the reaction of dialkylphosphites with arylideneuracils respective aminomethylphosphonates. The high level of the antiviral and anti-mycobacterial activity of the target compounds correlates well with the physicochemical parameters characterizing their structure.

Full text of DOI:10.1134/S1070363209080106

ISSN 1070-3632, Russian Journal of General Chemistry, 2009, Vol. 79, No. 8, pp. 1645–1649. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © V.I. Krutikov, A.V. Erkin, 2009, published in Zhurnal Obshchei Khimii, 2009, Vol. 79, No. 8, pp. 1287–1291.
5-Arylideneaminouracyls:
IV.1 Phosphorylated Derivatives and Their Biological Activity
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 April 30, 2009
Abstract—It extension of the studies on the search for the new biologically active 5-aminouracyl derivatives,
we synthesized by the reaction of dialkylphosphites with arylideneuracils respective aminomethylphosphonates.
The high level of the antiviral and anti-mycobacterial activity of the target compounds correlates well with the
physicochemical parameters characterizing their structure.
DOI: 10.1134/S1070363209080106
Biological screening of the compounds of a series
of substituted 5-arylidenaminouracyls we synthesized
earlier showed their high activity in vitro with respect
to the Herpes Simplex viruses and different
mycobacteria [2]. Furthermore, these substances
appeared to be the inductors of interferon, demonstrat-
ing thus the ability to strengthen the immunity of
warm-blooded animals. An essential drawback in 5-
arylidenaminouracyls is their low solubility in water.
The synthesis of ammonium and sodium salts of 5-
arylidenaminouracyls allowed to increase several times
the level of the antimicrobial and antiviral activity of
the studied compounds in vitro in comparison with the
parent substances [3]. The basic purpose of the present
investigation was the synthesis of substances, capable
under the conditions of organism to undergo suf-
ficiently easy spontaneous hydrolysis with the forma-
tion of water-soluble substances. Therewith, the rate of
hydrolysis must be rather low, to ensure prolonged
action of the biologically active compounds. We
assumed that (5-amino-2,4-dioxo-1,2,3,4-tetrahydro-
pyrimidin-5-yl)arylmethylphosphonates (I) under the
conditions of spontaneous (nonenzymatic) hydrolysis
would be capable of being converted into the
appropriate substituted phosphonic acids, which will
provide subsequent increase in solubility of the
substance in water and its steady fixation on the
surface of biological object due to the formation of
hydrogen bonds.
The earlier obtained [2] dependences “structure–
biological activity” for substituted 5-arylidenamino-
uracyls led us to a conclusion on a necessity of the
synthesis of new potential biologically active sub-
stances with the higher value of lipophilicity param-
eter, and containing electron-acceptor substituents in
the aromatic fragment of the molecule. This situation
defined the choice of initial aldehydes used in this
work for obtaining the final products (see the scheme
of synthesis and Table 1).
The most convenient of the known methods of
synthesis of the compounds of structure I should be
considered the method based on simultaneous reaction
of dialkylphosphite, aldehyde and amine (the
Kabachnik–Fields reaction) with the removal of the
formed water from the zone of reaction. In the present
work it was necessary to refuse the three-component
system because of the difficulty of selecting the
solvents for creating the homogeneous medium with
the subsequent azeotropic distillation of water. The
reaction in the heterophase system led to the
unreasonable increase in the reaction duration and to
difficulty in purifying the target compounds.
Aminomethylphosphonates I were obtained by a two-
stage scheme.
The features of carrying out the first stage of
process were described in detail in one of our previous
works [2]. At the second stage for creating a
homogenous well stirred reaction mixture we used 9–
10-fold molar excess of phosphite. The compounds of
general formula I are colorless or slightly colored
1
For communication III, see [1].
1645

1646
KRUTIKOV, ERKIN
O
O
O
R'
N
NH2
HN
HN
R'
+
_H2O
O
N
H
O
N
H
O
R'
H
N
(RO)2PHO
HN
P(O)(OR)2
Ia_Is
O
N
H
crystalline substances soluble in dimethylsulfoxide,
which is especially important for the biological testing
in vitro. The yields, melting points, data of TLC and of
elemental analysis are presented in Table 1.
the characteristic signals are as follows: A doublet of
methine proton in the region of 4.9–5.8 ppm, a doublet
of the proton of exocyclic amino group at 4.4–4.7 ppm,
well resolved signals of the protons of endocyclic
amino groups at 10.2 and 11.4 ppm (in contrast to the
degenerated analogous signals in the spectra of
arylideneuracils [2]). Chemical shifts in the 31P NMR
The structure of the synthesized substances is
1
proven by spectral methods. In the H NMR spectra
Table 1. Yields, melting points and the data of TLC and elemental analysis of (5-amino-2,4-dioxo-1,2,3,4-tetrahydro-
pyrimidin-5-yl)arylmethylphosphonates I
Found, %
H
Calculated, %
Comp.
no.
Yield,
%
R
R'
mp, °С
Rf
Formula
C
N
C
H
N
CH3
CH3
4-F
98
84
176
218
0.11
0.10
45.6
4.18
12.0
C13H15FN3O5P
C13H15N4O7P
45.5 4.40
42.2 4.08
12.2
15.1
Iа
4-NO2
42.5
4.17
14.9
Ib
CH3
CH3
2,4-Cl2
31
48
88
64
78
85
83
62
65
67
80
80
83
85
61
60
65
201
224
233
216
148
150
163
179
164
170
175
210
225
160
190
200
165
0.25
0.27
0.37
0.38
0.10
0.54
0.53
0.15
0.43
0.34
0.47
0.53
0.49
0.46
0.54
0.51
0.53
40.0
38.3
51.2
51.3
41.5
43.9
46.0
34.0
33.4
37.3
43.5
49.5
45.4
48.8
46.4
33.6
31.5
3.24
3.29
5.97
6.21
4.33
4.81
5.15
2.61
2.19
2.90
4.28
5.67
5.13
5.49
5.11
3.80
2.75
10.8
10.1
9.64
9.54
9.50
C13H14Cl2N3O5P 39.6 3.58
C13H14Cl2N3O6P 38.1 3.44
C19H27ClN3O5P 51.4 6.13
C19H27ClN3O5P 51.4 6.13
C15H19BrN3O5P 41.7 4.43
10.7
10.2
9.47
9.47
9.72
9.01
8.50
6.95
6.89
7.73
10.2
9.14
8.33
11.9
8.50
6.20
8.42
Ic
Id
Ie
If
2-OH-3,5-Cl2
4-Cl
i-C4H9
i-C4H9
C2H5
3-Cl
4-Br
Ig
Ih
Ii
i-C3H7
C4H9
2-OH-3,5-Cl2
2-OH-3,5-Cl2
8.89 C17H22Cl2N3O6P 43.8 4.72
8.66 C19H26Cl2N3O6P 46.2 5.06
7.10 C17H15BrF8N3O5P 33.8 2.50
7.00 C17H14Cl2F8N3O6P 33.5 2.31
CH2CF2CF2H 4-Br
Ij
CH2CF2CF2H 2-OH-3,5-Cl2
CH2CF2CF2H 4-F
Ik
Il
7.51
10.5
9.17
8.31
11.7
C17H15F9N3O5P 37.6 2.78
C15H19ClN3O6P 43.7 4.37
C19H27ClN3O6P 49.6 5.66
C19H27BrN3O6P 45.2 5.16
C2H5
2-OH-5-Cl
Im
In
Io
Ip
Iq
Ir
Is
i-C4H9
i-C4H9
i-C4H9
i-C4H9
i-C4H9
CH3
2-OH-5-Cl
2-OH-5-Br
2-OH-5-NO2
2-OH-3,5-Cl2
2-OH-3,5-I2
2-OH-3,5-Br2
C19H27N4O8P
48.5 5.53
8.55 C19H26Cl2N3O6P 46.2 5.06
6.30 C19H26I2N3O6P 33.7 3.69
8.50 C13H14Br2N3O6P 31.3 2.81
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 8 2009

5-ARYLIDENEAMINOURACYLS: IV.
1647
spectra of the compounds I fall to the range of 21–
23 ppm, which is inherent in the aminomethyl-
phosphonates containing a heterocyclic fragment [4].
toward the virus of vesicular stomatitis should be
stressed. Even a preliminary analysis of the level of
biological activity of the synthesized compounds
shows the general regularity: a decrease of lipotropy
and an increase in the hydration energy of final
products leads to the loss of biological activity or, at
least, to a significant decrease in its level.
The regularities in the fragmentation of compounds
I under the action of electron impact make it possible
to unambiguously assign these compounds to the
phosphorylated azomethines. This is proved, first, by
the presence of the peaks of molecular ions in the
mass-spectra. Second, to the main peaks in the spectra
of substances I correspond the ions ArCH=N+H–Ura
formed at the cleavage of the bond P–C, which is due
to the stabilizing effect of aromatic ring. Finally, the
structure of the obtained compounds is confirmed by
the special features of the fragmentation of
“phosphoric” part of the molecules. In particular, the
loss of alkoxy groups and olefin molecules from these
groups is the process characteristic of the
fragmentation of the ethers of phosphorus acids.
Therefore it should be regarded as reasonable the
appearance in the spectra of all compounds I of the
peak with m/z = 82, which corresponds to phosphorous
acid.
In the previous publications [2, 3] the importance
of the lipophilicity of arylideneuracyls for their
antimycobacterial and antiviral activity was noted. For
evaluation of the level of lipophilicity of organic
substances, as is known, the parameter of lipophilicity
log P is often used, a calculated or an experimental
value characterizing the relative solubility of the
studied compound in the system of octanol–water. The
values of log P for the same substance, calculated by
us with the use of different software packages (TSAR–
Tool for Structure Activity Relation-ship, Oxford
Molecular [5], and HyperChem™ Release 6.03 for
Windows Molecular Modeling System), are not
indentical. This situation impelled us in the present
work to use experimental Bate–Smith and Westall
constant RM parameter in the system of octanol–water
for evaluating the lipophilicity of substances [6]. A
feature of this value is additivity: it can be calculated
by summation of the increments (ΔRM), which
characterize the individual parts of the molecule.
The target aminomethylphosphonates I were tested
for the biological activity in vitro toward the strains of
yeast-like fungi, pathogenic mycobacteria, and some
viruses. The results of bioassays are represented in
Table 2. The attention is called to the sufficiently high
level of the biological activity of compounds I: the
value log 1/C50 in some cases exceeds 4 (C50 is the
minimum concentration of a substance, which causes
50% oppression of growth of the microorganisms).
The noticeable activity of aminomethyphosphonates I
The Bate–Smith and Westall constants for the
synthesized compounds were determined by TLC. The
calculation of the parameter RM is carried out along the
formula:
Table 2. Correlation parameters for the prognosis of biological activity of aminomethylphosphonates I by Hantzsch method
log 1/C50
Ehydr
,
Rhabdoviride
Vesiculovirus
Mycobacterium
tuberculosis
H37Rv
Comp. no.
RM
kJ mol–1
Candida albicans Aspergillus niger
HSV-1
5.1
6.7
5.1
3.8
5.8
6.1
3.9
4.9
5.4
4.9
7.0
1.08
1.48
1.30
1.27
1.31
1.37
1.28
1.56
1.59
1.60
1.35
3.14
4.12
–
–
–
–
3.91
–
–
±
±
–
–
–
+
–
±
–
+
±
Iа
Id
Ie
If
3.69
3.95
4.09
–
–
–
–
–
3.24
–
3.24
–
–
Ig
Ih
Ii
–
3.75
4.14
3.98
3.99
4.04
3.72
3.30
4.08
4.37
4.04
4.19
–
–
4.08
4.09
–
–
Ij
4.39
–
Ik
Il
–
–
Im
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 8 2009

1648
KRUTIKOV, ERKIN
log 1/C50 = 36.6 – 45.8RM + 15.9(RM)2
RM = log (1/R'f – 1), [6],
4.2
4.1
4.0
3.9
3.8
3.7
r 0.951, s 0.059
where R'f is chromatographic index for the considered
substance in the system of octanol–water.
Inclusion of the values RM into the correlation
equations “structure–bioactivity” confirmed the
importance of lipotropy of aminomethylphosphonates I
in the manifestation by them of antimycobacterial and
antiviral activity. Figures 1 and 2 shoe the parabolic
dependences (by Hantzsch) of the bioactivity of these
compounds toward the herpes simplex virus and yeast-
like fungi on the lipophilicity. The sufficiently high
correlation coefficients for the obtained dependences
attract attention.
1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65
RM
Fig. 1. Effect of lipophilicity of aminomethylphosphonates
I on their activity toward the strain of herpes simplex
virus.
Earlier [2] for describing the mathematical models
of interaction of the derivatives of 5-aminouracyl with
the strains of different pathogenic bacteria and viruses
we noted the importance of such a parameter as
hydration energy Ehydr of substances. In the present
work we obtained one- and two-parameter depen-
dences, which characterize the influence of this param-
eter on the level of bioactivity of aminomethylphos-
phonates toward the herpes simplex virus:
log 1/C50 = 6.12 – 6.23RM + 3.17(RM)2
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
r 0.940, s 0.021
log 1/C50 = 4.68 – 0.142Еhydr
r 0.958, s 0.051, n 9;
,
(1)
log 1/C50 = 4.43 – 0.147Еhydr + 0.193RM,
r 0.970, s 0.047, n 9.
(2)
1.0 1.1 1.2
1.3 1.4 1.5 1.6
RM
A good agreement of experimental and calculated
values of log 1/C50 (Fig. 3) leads to unambiguous
conclusion that the presence in molecule of amino-
methylphosphonate of a labile bond destroyed at the
spontaneous hydrolysis provides the higher level of
bioactivity of the studied substances.
Fig. 2. Effect of lipophilicity of aminomethylphosphonates
I on their activity toward the strain of Candida albicans.
4.2
4.1
4.0
3.9
3.8
3.7
EXPERIMENTAL
1
The H NMR spectra (solutions in DMSO-d6) are
registered on a Bruker spectrometer (operating
frequency 200 MHz, internal reference: the signal of
the residual protons of DMSO, software of Bruker–
Franzen–Analytik GmbH, Version 950801.1).
The 31P NMR spectra were recorded on the
instrument, developed in Saint-Petersburg State
Institure of Technology (operating frequency 16.2 MHz,
external reference 85% H3PO4).
3.6
3.6 3.7 3.8 3.9 4.0 4.1 4.2
log 1/C50, calc.
The mass-spectra were taken on the spectrometer
Finnigan MAT-8430 with the direct introduction of the
substance into the ion source (ionizing electrons
Fig. 3. Comparison of experimental and calculated data on
biological activity of aminomethylphosphonates toward the
strain of Herpes Simplex virus.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 8 2009

5-ARYLIDENEAMINOURACYLS: IV.
1649
energy 70 eV, cathode emission current 0.1 mA,
accelerating voltage 3 kV, source temperature 250°C)
in the Scientific Center of Biological Studies of
Russian Academy of Science (Pushchino-na-Oke).
phonate (Ii). To a flask was placed 1 g of 5-(2-hyd-
roxy-3,5-dichlorobenzylidene)aminopyrimidine-2,4-
(1H,3H)-dione and 7 g of dibutylphosphite. The
mixture was heated at the temperature 80–90°C with
stirring. A colorless precipitate formed. After cooling
the reaction mixture to room temperature it was added
50 ml of diethyl ether. The precipitate formed was
filtered off, washed with ether, and dried over
phosphorus pentaoxide. The yield of product was 1.37 g
(83%). mp 163°C.
The individuality of the substances is proven by
the method of thin-layer chromatography on the plates
Silufol UV-254, eluent carbon tetrachloride– isopropyl
alcohol 9:1.
Hydration energy of compounds was calculated
with the HyperChem™ program package, Release 6.03
for Windows, Molecular Modeling System.
The other aminomethylphosphonates I were ob-
tained analogously.
A procedure of the determination of the
chromatographic parameter RM. On a plate Silufol
UV-254, moistened by water, was dropped of the
investigated substance. Then spot was eluted with
octanol-1. After the desiccation of the plate the value
ACKNOWLEDGMENTS
The authors express deep gratitude to specialists
and to chairman of the Department of microbiology,
virology and immunology of the Pavlov St. Petersburg
State Medical University Professor V.V. Tets for the
estimation of the antibacterial and antiviral activity of
the target products.
of R' was determined and the parameter RM was
f
calculated.
5-(2-Hydroxy-3,5-dichlorobenzylidene)aminopiri-
midine-2,4(1H,3H)-dione. In a flask was placed 1.27 g
of 5-aminouracyl and 150 ml of water. The mixture
was heated with stirring until the complete dissolution
of the parent substance. In parallel, in 50 ml of ethanol
was dissolved 1.91 g of 3,5-dichlorosalicylic aldehyde
and the solution was added dropwise to the solution of
5-aminouracyl. A precipuitate of bright orange color
practically immediately began to fall down. The
reaction mixture was refluxed at stirring for 1 h and
then the stirring was continued for 1 h at room
temperature. Then reaction mixture was left overnight.
The precipitate was then filtered off, washed with
warm water and ethanol, and dried. The yield of the
product was 2.76 g (92%).
REFERENCES
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2009, vol. 79, no. 4, p. 813.
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2009, vol. 79, no. 4, p. 819.
4. Krutikov, V.I., Doctorate Sci. (Chem.) Dissertation, St.
Petersburg, 1998.
5. Viswanadman, V.N., Ghose, A.K., Renankar, G.R., and
Robins, R.K., J. Chem. Inf. Comput., 1989, no. 29, p. 163.
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Shebesta, K., Tomashek, V., Motl, O., Novotnyi, L., and
Shtamberg, Ya., Laboratornoe rukovodstvo po khro-
matograficheskim i smezhnym metodam (Laboratory
Handbook for Chromatography), part 1, Moscow: Mir,
1982.
Other 5-arylideneaminouracyls were synthesized
by analogous procedure.
Dibutyl-(5-amino-2,4-dioxo-1,2,3,4-tetrahydropyri-
midin-5-yl)(2-hydroxy-3,5-dichlorophenyl)methylphos-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 79 No. 8 2009