Composition on the basis of phosphonoacetic acid. Synthesis and antiviral activity

Article abstract of DOI:10.1134/S1070363215100394

A manufacturable technology for the production of an antiviral composition containing phosphonoacetic acid, involing PH-alkylation of dimethyl phosphite with methyl chloroacetate under phase-transfer conditions and subsequent in situ hydrolysis of the intermediate product, was developed. The composition showed expressed antiviral effect against herpesviral infections caused by herpes simplex virus 1 and 2 and cytomegalovirus. The composition in the effective concentration range was found to exhibit no cytotoxic effect on VERO green monkey kidney and M-19 human diploid cell cultures. The resulting data suggest potential uses of the composition as an antiviral agent in practical medicine and as an antiviral additive to synthetic detergents for sanitation in health care and food production facilities.

Full text of DOI:10.1134/S1070363215100394

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 10, pp. 2441–2448. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © L.M. Alimbarova, A.V. Kharlamov, N.A. Bondarenko, I.F. Barinskii, 2014, published in Rossiiskii Khimicheskii Zhurnal, 2014,
Vol. 58, No. 1, pp. 23–30.
Composition on the Basis of Phosphonoacetic Acid.
Synthesis and Antiviral Activity
L. M. Alimbarovaa, A. V. Kharlamovb, N. A. Bondarenkob, and I. F. Barinskiia
a Ivanovskii Research Institute of Virology, ul. Gamalei 16, Moscow, 123098 Russia
b State Research Institute of Chemical Reagents and Special Purity Chemical Substances (IREA),
ul. Bogorodskii val 3, Moscow, 107076 Russia
e-mail: katamin@mail.ru; bond039@mail.ru
Received January 1, 2014
Abstract―A manufacturable technology for the production of an antiviral composition containing
phosphonoacetic acid, involing РН-alkylation of dimethyl phosphite with methyl chloroacetate under phase-
transfer conditions and subsequent in situ hydrolysis of the intermediate product, was developed. The
composition showed expressed antiviral effect against herpesviral infections caused by herpes simplex virus 1
and 2 and cytomegalovirus. The composition in the effective concentration range was found to exhibit no
cytotoxic effect on VERO green monkey kidney and М-19 human diploid cell cultures. The resulting data
suggest potential uses of the composition as an antiviral agent in practical medicine and as an antiviral additive
to synthetic detergents for sanitation in health care and food production facilities.
DOI: 10.1134/S1070363215100394
INTRODUCTION
in this process also belongs to contaminated equipment
and materials [8]. Therefore, the use of disinfectants in
hand hygiene of medical staff, as well as in sanitary
treatment of surfaces plays an important role in the
prevention of hospital-acquired infections.
The problem of herpesviral infections (HIs) has not
lost urgency. Even though great progress has been
reached in the understanding their etiolog y,
pathogenesis, and therapy, HIs still cause some the
common infectious diseases [1–5]. The main causative
agents of HIs are representatives of the Herpesviridae
family, specifically herpes simplex viruses 1 and 2
(HSV-1 and HSV-2), as well as cytomegalovirus
(CMV), which, depending on conditions, can cause
patholog ical processes with different localization and
degree of expression, capable of retaining activity in
body cells for a long time, and tending to recur. From
60% to 95% of the world population is infected with
HSV, and the prevalence of CMV in the total
population of different countries makes reaches 20–
75% [1, 3]. The clinical course of diseases caused be
His features asymptomatic forms in 70–80% cases, and
clinical symptoms of herpesviral diseases are observed
in 20–30% of infected patients [1].
The antiviral activity of phosphonoacetic acid
(HO)2P(O)CH2COOH (1, FAA) was discovered in 1973
[9], about 50 years later than it had been first
synthesized [10]. This discovery prompted intense
research into the biolog ical activity of FAA [11–17],
which showed that the acid and a number of its
derivatives strongly inhibit replication of many viruses,
including hepatitis, immunodeficiency, and human
herpes viruses. The inhibitory activity of PAA and its
derivatives against herpes viruses, in particular, HSV,
CMV, and Epstein–Barr virus, is associated with the
fact that these compounds, being analog antimetabolites
of pyrophosphates, are capable of inhibiting DNA
polymerase responsible for replication of herpes viruses
[1, 3]. Furthermore, PAA is a respresentaive of
phosphonic acids which are widely used as a component
of synthetic detergents. Therefore, aiming at developing
technolog ies for provoding safety for humans and
animals (including design of antiviral agents for
medicine and veterinary), we have developed a method
of synthesis of a PAA-containing composition to be
Pathogens are best destroyed by means of
disinfection [6, 7]. The hospital environment is one of
the main sources of infections, because most pathogens
are transmitted via direct contact with patients (for
example, via hand contact), even though a certain role
2441

2442
ALIMBAROVA et al.
Table 1. Chemical shifts of signals in the 31Р{1H} solutions
of phosphonoacetic acid 1 and its derivatives 2–8 prepared
by independent synthesis
O
+ K2CO3 + KHCO3 + H2O
O
(OMe)2P
COOMe
2
Comp.
no.
Formula
Solvent δР, ppm
20−30oC
−MeOH
(OMe)2P
COOK
1
(HO)2P(O)CH2COOH
CD3OD
Н2О
D2O
17.7
15.4
16.2
3
O
MeO
P
100−105oC
−MeOH
COOK
2
(MeO)2P(O)CH2COOMe
(MeO)2P(O)CH2COOK
CDCl3
D2O
H2O
22.3
26.0
25.0
KO
4
The progress of the conversion of trimethyl ester 2
into phosphonoacetic acid 1 was monitored by 31Р{1H}
NMR spectroscopy of solutions of the reaction
mixtures in deuterated solvents.
3
4
5
6
7
8
D2O
29.0
20.6
18.1
13.2
16.2
13.8
(MeO)(KO)P(O)CH2COOK D2O
(MeO)(HO)P(O)CH2COOH D2O
The reaction mixture obtained at the first stage was
diluted with water and left to stand for 15 h at room
temperature. Comparative analysis of the 31Р{1H}
NMR data for the solution of the reaction mixture in
D2O and independently synthesized derivatives of
phosphonoacetic acid (Table 1) showed that under
these conditions trimethyl ester 2 converts into potas-
sium (dimethoxyphosphinoyl)methylacetate 3 (δР =
29.0 ppm, 92.1%), whereas dimethyl phosphite converts
into potassium methyl phosphite (δР = 8.6 ppm, 7.9%).
(HO)2P(O)CH2COOK
(KO)(HO)P(O)CH2COOK D2O
(KO)2P(O)CH2COOK D2O
D2O
used as an antiviral additive to detergents for sanitation
in health care and food production facilities.
Synthesis of the Composition
The composition is synthesized in two stages. The
first stage involves the synthesis of trimethyl
phosphonoacetate 2 by the previously developed facile
and convenient РН-alkylation of dimethyl phosphite
with methyl chloroacetate in the presence of potassium
carbonate in the heterogeneous system “organic liquid
phase–inorganic solid phase” [18] without using
phase-transfer catalysts. The role of the latter is played
to success by the starting and final phosphoryl-con-
taining compounds [19–21].
O
O
H2O
MeO
(MeO)2P−H + K2CO3 + KHCO3
P−H
−MeOH
KO
The subsequent 4-h boiling of the reaction mixtures
gives dipotassium salt of (hydroxymethoxyphosphi-
noyl)acetic acid 4 (δР = 20.6 ppm, 89.2%), implying
hydrolysis of only one methoxy groups on phosphorus
in salt 3. Potassium methyl phosphite remains unchanged.
The last methoxy group in dipotassium salt 4 could
only be hydrolyzed under acid catalysis. The 31Р{1H}
spectrum of a D2O solution of the mixture of
monoester 4 and potassium methyl phosphite acidified
with conc. HCl at room temperature no longer showed
the signals of the components at δР = 20.6 and
8.2 ppm, respectively; instead, a signal of acid 5 (δР =
18.1 ppm) appeared.
O
(MeO) P−H +
Cl
COOMe
2
O
2K2CO3
+ K2CO3 + KHCO3.
COOMe
(OMe)2P
−KCl
2
At the second stage trimethyl ester 2 is hydrolyzed
in situ under the action of potassium carbonate and
bicarbonate, which are present in the reaction mixture.
As shown previously, the reaction proceeds via con-
secutive dealkylation of the methoxycarbonyl group
[22–24] and one of the methoxy groups on phosphorus
[10, 23, 25] to form mono- and dipotassium salts of
phosphonoacetic acid 3 and 4.
O
P
O
P
HCl conc.
MeO
KO
MeO
HO
COOK
COOH
−2KCl
4
5
O
H3O+
(HO)2P
COOH
−MeOH
1
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COMPOSITION ON THE BASIS OF PHOSPHONOACETIC ACID
2443
O
was confirmed by comparative analysis of the chemical
shifts of signals in the 31Р NMR spectra of the reaction
mixtures with those in the spectra of CDCl3, CD3OD,
Н2О, and D2O solutions of phosphonoacetic acid
derivatives 3–8 (Table 1) prepared by independent
synthesis (see scheme below). As the composition was
intended to be used as an antiviral additive to
detergents which are basic in nature, the reaction
mixture was neutralized with 40% aqueous KOH to
рН = 8 and then filtered to remove the precipitate of
KCl. The resulting antiviral composition (PAAC) is an
aqueous solution of a mixture of di- and trisalts of
[hydroxy(methoxy)phosphinoyl]acetic (4) and phos-
phonoacetic (7, 8) acids (21% per phosphonoacetic
acid, ratio 1 : 2), as well as phosphoric acid (1.6% per
phosphoric acid) and KCl.
O
O
H3O+
HCl conc.
MeO
HO
MeO
KO
(HO)2P−H
P−H
P−H
−MeOH
−KCl
The subsequent 12-h boiling of the mixture with
distillation of the water–methanol fraction at 90°С led
to dealkylation of the methoxy groups in acid 5 and
methyl dihydrogen phosphite to form phosphonoacetic
acid 1 and phosphorous acid. According to 31Р{1H}
NMR data, the resulting mixture contained 58.1% of
acid 1 (δР = 16.4 ppm), 29.5% of unhydrolyzed mono-
ester 5 (δР = 20.5 ppm), and 12.4% of phosphorous
acid (δР = 3.0 ppm).
The identification of the intermediate products
formed in the course of hydrolysis of trimethyl ester 2
O
O
O
b
MeO
KO
a
P
COOK
(OMe)2P
COOMe
(OMe)2P
COOK
COOK
3
2
4
d
c
O
O
O
P
e
MeO
HO
(HO)2P
(HO)2P
COOH
COOH
6
1
5
f
g
O
P
O
KO
HO
COOK
(KO)2P
COOH
7
8
а: KOH/MeOH, 50°C, 1 h [22]; b: aq. KOH, 100°C, 10 h [23]; c: HCl conc., 20°C; d: H2O, 150°C, 10 h [18]; e, f, g:
1 equiv., 2 equaiv., 3 equiv. 1 N aq. NaOH, respectively.
Assessment of the Antiviral Activity
of the PAA Composition
HSV-1 and HSV-2 were cultured and titered in a
multilayer VERO green monkey kidney cell culture and
CMV in an М-19 human diploid cell culture. The
infectious virus titers were determined by standard by
standard Reed–Muench assay and expressed in
log TCD50/0.1 mL.
Procedure of Antiviral Activity Assessment
The antiviral activity of PAAC was assessed in
vitro against experimental herpesviral infections by
standard procedure [26–29] using freshly prepared
serial samples obtained by successive half-dilutions of
the starting sample (from 1 : 2 to 1 : 512) in a serum-
free Eagle’s nutrient medium. The test viruses were
human herpes viruses HSV-1 (strains K1 and L2),
HSV-2 (strain BH), and CMV (strain AD-169),
obtained from the State Collection of Viruses, Ivanovo
Research Institute of Virolog y, Ministry of Health of
the Russian Federation.
The cytotoxicity of PAAC was tested against to the
cell cultures used for infectious activity testing: VERO
(Ivanovo Research Institute of Virology) and М-19
(Chumakov Institute of Poliomyelitis and Viral
Encephalitis, Russian Academy of Medical Sciences).
Cells were cultured in Eagle’s growth medium
containing 10% inactivated fetal bovine serum (FBS,
PanEko, Russia), 2 mM of L-glutamine (Sigma, USA),
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 10 2015

2444
ALIMBAROVA et al.
and antibiotics (100 units/mL of penicillin, 100 µg/mL
of streptomycin, and 80 µg/mL of gentamycin). The
maintenance medium contained all the above-
mentioned ingredients plus 2% of FBS. Cells were
grown in plastic flasks or plates (Сostar, USA) and
incubated in a thermostat at +37°С in a humid
atmosphere containing 5% of СO2. A 24-h monolayer
of cell cultures was used.
cultures (VERO or М-19) had been inoculated with test
viruses (HSV-1, HSV-2, CMV). The infection
procedure was as follows: cells were cultured in 96-well
plates, the growth medium was removed, and cells were
washed with 3 portions of Hanks’s solution, inoculated
with test viruses in the dose 100 TCD50, and incubated
for 1 h in a thermostat at 37°С. The inoculate was
removed, cell cultures were washed with 3 portions of
Hanks’s solution, after which serial PAAC solutions
were added. The positive controls were cell cultures
inoculated with test viruses, to which the maintenance
medium containing no PAAC was added. The negative
controls were intact cell cultures with added pure
maintenance medium. The cytotoxicity controls were
intact cell cultures with added maintenance medium and
PAAC . Cells were incubated for 4 (with HSV-1 and
HSV 2) or 14 days (with CMV); the virus-specific CPE
in the test and control cell cultures was recorded daily
by optical microscopy.
According to methodical recommendations on
preclinical specific activity testing of medicines, at the
first stage we assessed the cytotoxicity of PAAC with
respect to intact cell cultures in terms of its effect on
the morpholog y and viability of cells [26]. To this
end, 1 : 2–1 : 512 composition samples in Eagle’s
nutrient medium were introduced into 24-h VERO or
М-19 cell cultures preliminarily freed from the growth
medium and washed with three portions of Hanks’
solution. For each dilution we used four wells. The
nutrient medium without PAAC was used as control.
Culturing was performed in a thermostat for 96 h, as
described above. Cytodestructive and morpholog ical
changes in the cell monolayer were monitored every
day by means of optical microscopy.
The antiviral activity study of PAAC in cell
cultures also included assessment of the pathogenicity
of the virus formed in the presence of the composition.
To this end, at the 4th (with HSV-1 and HSV-2) or
14th day (with CMV) of incubation of infected cell
cultures and PAAC samples the virus-containing fluid
(VCF) from the test and control wells was taken and
tested for virus pathogenicity using subculture cell
lines. Before measurements the samples were stored at
–70°С. Immediately before measurements 10-fold
serial dilutions of the VCF samples (10–1–10–8) were
prepared on Eagle’s nutrient medium and used to
infect the 24-h monolayer of cell cultures after removal
of the growth medium and washing with Hanks’s
solution. Cell cultures inoculated with HSV or CMV
(positive control) and intact cell cultures (negative
control) were used as reference. Incubation was
performed at 37°С for 4 (with HSV-1 and HSV-2) or
14 days (with CMV). During this time the
accumulation of test viruses in control samples reached
a maximum and a 100% CPE was observed.
Viable cell counting was performed daily using the
vital dye exclusion assay with Trypan Blue which
colors nonviable cells blue. To this end, cells were
removed with a trypsin–versene mixture and stained
vitally with a 0.4% solution with Trypan Blue for
5 min at +37°С. Cell viability was assessed by the
percentage of uncolored cells in the total cell popula-
tion. For statistically reliable results each experimental
series was repeated in triplicate.
The concentration of the composition, which
caused microscopically visible changes in morpholog y
in 50% of cells in the monolayer or death of 50% of
cells in the population was taken as 50% cytotoxic
concentration (CTC50) [26, 28]. Along with this
parameter we also determined the maximum tolerable
concentration (MTC), specifically, the highest con-
centration of the composition that did not cause visible
cytodestructive changes in the cell monolayer or cell
death. In further research, the composition was used in
concentrations not higher than the MTC.
The antiviral effect was evaluated by the inhibition
of virus accumulation under the action of the composi-
tion compared to the control samples (∆log TCD50), as
well as by the inhibition coefficient (IC) [26, 28]. At
∆log TCD50 1.0–1.5 the sample was considered to
possess a low antiviral activity with respect to HSV or
CMV and at ∆log TCD50 1.5 – 2.0 and >2.0, moderate
and expressed antiviral activity, respectively.
The antiviral activity of the composition was
determined by commonly accepted criteria, i.e. ability
to prevent virus-induced cytopathic effect (CPE) and
inhibit virus replication in the cell culture [26–29].
The lowest concentration of PAAC, which
prevented death of 50% of cells in the infected
Activity assessment was performed using a treat-
ment scheme. Treatment was initiated 1 h after test
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 10 2015

COMPOSITION ON THE BASIS OF PHOSPHONOACETIC ACID
2445
Table 2. Cytotoxic effect of the composition on intact
subculture Vero and М-19 cell lines
population or decreased the virus-induced CPE by
50% compared to 100% in the control infected culture
4–14 days after infection was taken as the minimum
inhibitory concentration (MIC50) of the composition.
Cytotoxic effect, %
Dilution of the starting sample
Vero
100.0
75.0
50.0
0.0
М-19
100.0
100.0
50.0
We also calculated the chemotherapeutic index
(CTI) as the ratio of CTC50 (which relates to the effect
on the cell morphology) to MTC50 (relates to the
antiviral effect)
1 : 2
1 : 4
1 : 8
Statistical treatment was performed using Microsoft
Excel-50 and Statistika 5.0 programs. Statistical signi-
ficance was evaluated by Student’s t-test at p < 0.05.
from 1 : 16 to 1 : 256
0.0
tolerable concentration or at higher dilutions, scarcely
differed from those of control cells incubated in the
absence of the composition.
Results of Antiviral Activity Assessment
of the Composition
First of all we studied the cytotoxic effect of the
composition on the morpholog y and viability of intact
cells of the subculture VERO cell line and М-19
human diploid cell culture. The resulting data are
presented in Table 2.
The 1 : 16 dilution was considered as a dilution
corresponding to the MTC of the composition, which
is nontoxic for 100% of cells in the monolayer. In
further experiments we used PACC samples in
dilutions not lower than 1 : 16.
In was found that 1 : 2 to 1 : 4 dilutions of the
composition after 24-h incubation caused visible
destructive changes in 100% and 75% of VERO cells
and 100% of М-19 cells, respectively, and a 1 : 8
dilution changed morpholog y in 50% in both VERO
and М-19 cells on the third day of observations. The
1 : 8 dilution of the composition was taken as CTC50.
The PAAC samples diluted from 1 : 16 to 1 : 512
showed no cytotoxic effect on VERO and M-19 cells
over the entire observation period (by optical
microscopy and Trypan Blue assay). The viability
characteristics of VERO and М-19 cells incubated for
96 h both in the presence of PAAC at the maximum
The results of the assessment of the antiviral activity
of PAAC with respect to the His caused by HSV-1,
HSV-2, and CMV are summarized in Tables 3–5.
As follows from Table 3, PAAC efficiently
inhibited HSV-1 replication when added 1 h after the
cell monolayer had been infected.
Thus, PAAC diluted 1 : 8–1 : 32 completely
inhibited replication of HSV-1 (strain K1), and its
1 : 8–1 : 16 inhibited replication of HSV-1 (strain L2);
the IC was 100%.
The effect of 1 : 64 and higher dilutions on the
HSV-1 strains was statistically significant. The
Table 3. Antiviral activity of the composition against the experimental herpesviral infection caused by herpes simplex virus 1
Infectious titers of HSV-1
strain K1
strain L2
inhibition of virus
Scheme of
study
Dilution
virus
inhibition of virus
replication,
virus
accumulation,
log TCD50/0.1 mL
IC,
%
IC,
%
accumulation,
log TCD50/0.1 mL
replication,
∆log TCD50
∆log TCD50
1 h post
inoculation
1 : 8
1 : 16
1 : 32
1 : 64
1 : 128
1 : 256
1 : 512
0
0±0
6.0
6.0
6.0
5.5
4.5
4.0
3.0
–
100.0
100.0
100.0
91.6
75.0
66.7
50.0
–
0±0
6.0
6.0
5.5
5.0
4.5
3.5
3.0
–
100.0
100.0
91.6
83.3
75.0
58.3
50.0
–
0±0
0±0
0±0
0.5± 0.25
1.0±0.1
1.5±0.3
2.5±0.1
3.0±0.1
6.0±0.5
0.5± 0.25
1.5±0.2
2.0±0.2
3.0±0.2
6.0±0.5
Control
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 10 2015

2446
ALIMBAROVA et al.
Table 4. Antiviral activity of the composition against the experimental herpesviral infection caused by herpes simplex virus 1
and cytomegalovirus
Infectious titers of viruses
HSV-2, strain ВН
CMV, strain AD-169
inhibition of virus
Scheme of
study
Dilution
inhibition of virus
virus accumulation,
log TCD50/0.1 mL
IC, virus accumulation,
%
IC,
%
replication,
replication,
log TCD50/0.1 mL
∆log TCD50
∆log TCD50
1 h post
inoculation
1 : 8
0.25 ± 0.15
1.0 ± 0.1
1.0 ± 0.3
1.0 ± 0.2
2.25 ±0 .1
3.25 ± 0.4
4.5 ± 0.25
6.0 ± 0.5
5.75
5.00
5.00
5.00
3.50
3.75
1.75
–
95.8
83.3
83.3
83.3
62.5
45.8
25.0
–
–
–
–
1 : 16
1 : 32
1 : 64
1 : 128
1 : 256
1 : 512
0
1.25 ± 0.15
1.5 ± 0.2
3.0 ± 0.2
4.75 ± 0.3
6.25 ± 0.1
7.0 ± 0.4
7.5 ± 1.0
6.25
6.00
4.50
2.75
1.50
0.50
–
83.3
80.0
60.0
36.7
16.7
6.7
Control
–
composition reduced the pathogenicity of HSV-1 by
3.0–5.5 log TCD50 compared to control. The infectious
titers of HSV-1 (strain K1) in the inoculated but not
PAAC -treated cell culture in 24, 48, 72, and 96 h were
3.5, 4.5, 5.25, and 6.0 log TCD50/0.1 mL, respectively.
The same trend was observed with HSV-1 (strain L2).
With the 1 : 8 to 1 : 64 dilutions, the specific CPE in the
cell culture inoculated сwith HSV-1 was only observed
96 h post inoculation, whereas in the control cultures,
already 24 h post inoculation. The inhibition coefficient
of the HSV-1 strains under the action of PAAC diluted
1 : 8–1 : 512 was 50–100%.
50log TCD50/0.1 mL in 72 h, and 6.0log TCD50/0.1 mL
in 96 h. The infectious activity of HSV-2 in the
presence of PAAC diluted 1 : 16 – 1 : 64 in 96 h after
inoculation was lower by 5.0log TCD50 compared to
the respective characteristic in control (р < 0.05). The
infectious titers of HSV-2 were not higher than 1.0log
TCD50/0.1 mL. It should be noted that the 1 : 256–
1 : 512 dilutions of the composition statistically
significantly inhibited HSV-2 replication (by 1.75–
3.75log TCD50).
The infectious activity of HSV-2 under the action
of PAAC in the dilution range 1 : 8–1 : 512 decreased
by 1.75–5.75log TCD50, the IC was 25.0–95.8%.
Thus, PAAC in the dilution range from 1 : 8 to
1 : 512, applied by the treatment scheme 1 h after
inoculation, shows a statistically significant antiviral
effect with respect to certain HSV-1 strains (K1 and
L2), the strength of which depends on the dose of the
composition.
Similar results were obtained with CMV. The
experimental results in Table 3 show that PAAC
diluted from 1 : 8 to 1 : 256 exhibits a high antiviral
activity with respect to CMV, significantly inhibits
CMV reproduction in the М-19 cell culture. The
infectious titers of CMV in the presence of PAAC
were 1.5–6.25log TCD50 lower than the respective
values in control. The IC was 16.7–83.3%.
It was found that PAAC in the dilution range from
1 : 8 to 1 : 64 strongly inhibited HSV-2 replication in
cell cultures (Table 4).
In these cases the virus-induced CPE in the cell
culture inoculated with HSV-2 was observed only 96 h
post inoculation, whereas in the control cultures, already
in 24 h. The highest IC (95.8%) was observed with the
1 : 8 dilution.
Comparative data on the antiviral activity of the
composition on the basis of phosphonoacetic acid
against the experimental herpesviral infection caused
by HSV-1, HSV-2, and CMV are presented in Table 5.
Analysis of these data shows that PAAC exhibits
expressed chemotherapeutic effect on the test viruses.
The CTI of the composition with respect to HSV-1/
HSV-2 and CMV was 64 and 32, respectively, which
The infectious titers of HSV-2 in the absence of
the composition were 2.0log TCD50/0.1 mL in 24 h
after inoculation, 4.0log TCD50/0.1 mL in 48 h,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 10 2015

COMPOSITION ON THE BASIS OF PHOSPHONOACETIC ACID
2447
Table 5. Comparison of the antiviral activity of the composition against the experimental herpesviral infections 1 h after
tripling
Cytotoxic
concentration (CTC50)
Minimum inhibitory
concentration (MIC)
Herpes virus
Chemotherapeutic index (CTI)
HSV-1 (strains K1 and L2)
HSV-2 (strain BH)
1 : 8
–
1 : 512
1 : 512
1 : 256
64
64
32
CMV (strain AD-169)
1 : 8
allows us to conclude that it holds promise both as a
specific antiviral agent and as antiviral additive to
preparations used in sanitary practice.
and 0.3 mL to the first, second, and third tube,
respectively). The solutions were diluted with D2O to
standard volumes (Table 1).
The 31P NMR spectra in CDCl3 and D2O were
measured on a Bruker Avanсe 300 spectrometer at
121.495 MHz, external reference 85% H3PO4.
EXPERIMENTAL
Synthesis of the composition. Methyl chloro-
acetate, 35.8 g (29.0 mL, 330 mmol), was added
dropwise with vigorous stirring at 20–30°С to a
mixture of 36.6 g (30.4 mL, 330 mmol) of dimethyl
phosphite and 91.0 g (660 mmol) of powdered K2CO3.
The reaction mixture was stirred at 20–30°С for 18 h,
diluted with 50 mL of water, heated under reflux for
4 h, and let to cool down to room temperature.
Concentrated HCl, 85 mL (990 mmol) , was then
added with stirring, and the mixture was boiled for 6 h
with distilletion of the water–metanol fraction up to bp
90°С. The still residue at 20–30°С was neutralized
with 40% aqueous solution of 35.3 g (630 mmol) of
KOH to рН 8, the mixture was stirred for 1 h at room
temperature, and the precipitate of KCl that formed
was filtered off to obtain 215.8 g of aqueous solution
of di- and trisalts of [hydroxy(methoxy)phosphinoyl]-
acetic (4) and phosphonoacetic (7, 8) acids (21% per
phosphonoacetic acid, ratio 1 : 2), as well as
phosphoric acid (1.6% per phosphoric acid) and KCl.
CONCLUSIONS
A manufacturable technolog y for the production of
an antiviral composition containing phosphonoacetic
acid, involing РН-alkylation of dimethyl phosphite
with methyl chloroacetate under phase-transfer
conditions and subsequent in situ hydrolysis of the
intermediate product, was developed.
The composition showed expressed antiviral effect
against HSV-1 and HSV-2, as well as CMV. In vitro
experiments revealed the ability of the composition to
inhibit replication of test viruses in different cell
lines, leading to a statistically significant reduction of
their infectious activity. The effect is the stronger the
higher is the concentration of the composition.
Furthermore, the composition in the effective con-
centration range was found to exhibit no cytotoxic
effect on VERO green monkey kidney and М-19
human diploid cell cultures.
Phosphonoacetic acid (1) [18], its trimethyl ester 2
[18], monopotassium salt of (dimethoxyphosphinoyl)-
acetatic acid 3 [22], and dipotassium salt of [hydroxyl-
(methoxy)phosphinoyl]acetic acid 4 [23] were syn-
thesized by previously described procedures (Table 1).
The resulting data suggest potential use of the
composition in the development of human and animal
safety technolog ies, including its uses as an antiviral
agent in practical medicine and as an antiviral additive
to synthetic detergents for sanitation in health care and
food production facilities.
[Hydroxy(methoxy)phosphinoyl]acetic acid (5).
The aqueous solution of salt 4, prepared from 1.0 g of
triester 2 [23], was neutralized with 1 mL of conc. HCl
(Table 1).
ACKNOWLEDGMENTS
The work was financially supported by the Ministry
of Education and Science of the Russian Federation in
the framework of the “Research and Development in
Priority Fields of Science and Technology Complex of
Mono-, di-, and tripotassium salts of phosphono-
acetic acid (6–8). Three 0.0144 g portions of acid 1
[18] were placed in NMR tubes, and 1 N aquoues
solution of NaOH was added to each tube (0.1, 0.2,
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2448
ALIMBAROVA et al.
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