Antiviral activity of the products of cyclization of dimethyl 2-[(1-arylamino-1-arylmethylidene)hydrazono]succinate

Article abstract of DOI:10.1016/S0223-5234(00)01201-0

In this research, conditions of cyclization of dimethyl 2-[(1-arylamino-1-arylme-thylidene)hydrazono]succinate 1-5 leading to the formation of 3,4-diaryl-5-carboxymethyl-1,2,4-triazole 6-10 and methyl 2-(5-oxo-3,4-diaryl-1,4,5,6-tetrahydro-1,2,4-triazine-

Full text of DOI:10.1016/S0223-5234(00)01201-0

Eur. J. Med. Chem. 36 (2001) 93–99
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01201-0/SCO
Short communication
Antiviral activity of the products of cyclization of dimethyl
2-[(1-arylamino-1-arylmethylidene)hydrazono]succinate
Boz
ena Modzelewska-Banachiewicza*, Teresa Kamin´skab
;
aDepartment of Organic Chemistry, Medical University, Staszica 6, 20-081 Lublin, Poland
bDepartment of Virology and Immunology, Maria Curie-Sk*lodowska University, Akademicka 19, 20-033 Lublin, Poland
Received 20 April 2000; revised 17 October 2000; accepted 14 November 2000
Abstract – In this research, conditions of cyclization of dimethyl 2-[(1-arylamino-1-arylme-thylidene)hydrazono]succinate 1–5
leading to the formation of 3,4-diaryl-5-carboxymethyl-1,2,4-triazole 6–10 and methyl 2-(5-oxo-3,4-diaryl-1,4,5,6-tetrahydro-1,2,4-
triazine-6-ylidene)acetates 11–15 and the biological activity of some of them have been examined. Their chemical structures were
1
confirmed by IR, H-NMR, EI-MS and elemental analysis. Substances 8 and 13 exhibited moderate virucidal activity and partially
inhibited absorption of the viruses to the susceptible cells. The acute toxicity of compounds 6–10 was established. For compounds
´
8 and 13, the influence on the central nervous system of mice and rats in behavioral tests was examined. © 2001 Editions
scientifiques et me´dicales Elsevier SAS
3,4-diaryl-1,2,4-triazolo-5-carboxylates / 2-(3,4-diaryl-5-oxo-1,2,4-triazine-6-ylidene)acetates / cytotoxicity / antiviral activity / vesicu-
lar stomatitis virus / encephalomiocarditis virus / adenovirus / in vitro study
1. Introduction
structurally based on the 1,2,4-triazole (Ribavirine,
Vibunazole) or 1,2,4-triazine (Azaribine) heterocyclic
Viral infections have for a long time been consid-
systems [3, 4]. This paper presents the results of
ered as non-affectable by chemoterapeutics due to
investigation of antiviral activity of representatives of
innate association of viruses with normal cell struc-
both 1,2,4-triazole and 1,2,4-triazine groups: 3-(2-
tures and biochemistry. Starting from early 1950 with
the discovery of antiviral activity of some thiosemi-
carbazones [1], further findings were launched very
fast. Antiviral chemotherapy began with the advent of
pyridyl)-4-phenyl-5-carboxymethyl-1,2,4-triazole (8)
and methyl 2-(5-oxo-3-(2-py-ridyl)-4-phenyl-1,4,5,6-
tetrahydro-1,2,4-triazine-6-ylidene)-acetates (13) were
synthesized in our laboratory. Their virucidal action
acyclovir in 1977 as the first specific antiviral agent
[2]. With the very fast development of molecular
biology of viruses and their biochemistry, it was pos-
sible to fight them on the different stages of their
activity (cell absorption and penetration, replication)
and every year new biological targets are recognized.
It results in findings of much better treatments for
some viral diseases, especially HIV. The new results in
the search for antiviral agents are still presented in
scientific literature. Some new active compounds are
and effects on the replication of the selected labora-
tory strain viruses RNA (VSV, EMCV) and DNA
(AV-5) were investigated. Some behavioral pharma-
cological tests (acute toxicity and ‘‘writhing test’’)
were also performed to check the influence of the
compounds on the central nervous system of the
laboratory animals.
2. Chemistry
In has been found that the course of reaction of
cyclic amidrazones with dimethyl acetylene dicaboxy-
late (DMAD) depends on the reaction conditions. It
* Correspondence and reprints.
E-mail address: pitucha@panaceum.am.lublin.pl
(B. Modzelewska-Banachiewicz).

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B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
3. Biological investigations
has been reported previously that, when this reaction
is carried out in the presence of triethylamine, deriva-
tives of 5-hydroxy-1,2-pyrrole and 5-oxo-1,2,4-
triazine are formed [5]. We found that carrying
out this reaction in high-boiling solvents (e.g. n-
butanol) in the absence of base can lead to different
products.
The cyclization reaction of dimethyl 2-[(1-ary-
lamino-1-arylmethylidene)hydrazono]succinate when
performed in boiling n-butanol can lead to the forma-
tion of derivatives of 1,2,4-triazole-5-carboxylic acid
6–10 (with concomitant liberation of a molecule of
methyl acetate) and 5-oxo-1,2,4-triazine-6-carboxylic
acid 11–15 (with the liberation of a methanol
molecule) with ratio of ca. 1:1. Separation of both
reaction products was possible based on their differ-
ent solubility in ethyl ether. More detailed data for
compounds 11–15 are given in ref. 6.
Human adenovirus type 5 (AV-5) received from the
National Institute of Hygiene (Warszawa) was propa-
gated in HeLa cells and titrated in human skin fibrob-
lasts (HSF). Vesicular stomatitis virus (VSV) and
encephalomyocarditis virus (EMCV) strain col MM
were obtained from the Institute of Immunology and
Experimental Therapy, Polish Academy of Sciences,
Wroclaw. VSV and EMCV were propagated and ti-
trated in L929 cells. The influence of these com-
pounds on acute toxicity (LD50) and in behavioral
studies was carried out on male and female albino
Swiss mice and male Wistar rats.
4. Results and discussion
The toxicity of the 8 and 13 derivatives were depen-
dent on dose and time of incubation (figures 2–5).
Moreover, the toxicity of both substances depended
In IR spectra, compounds 6–10 exhibit characteris-
tic absorption bands corresponding to the carbonyl of
the amid function in the range of 1702–1729 cm–1. In
1
their H-NMR spectra, only the presence of a singlet
of the CH3 ester group (in 3.3–3.6 ppm range) and a
multiplet of the aromatic hydrogens (in 7.3–8.3 ppm
range) were observed. In contrast, in IR spectra of
compounds 11–15, absorption bands of the carbonyl
of the amide group are present in the 1698–1735 cm–1
1
range. In their H-NMR spectra, besides a singlet of
the CH3 ester group (in the same range as for com-
pounds 6–10) and a multiplet of aromatic hydrogens
(in 7.1–8.2 ppm range), the presence of single signal
of N1 hydrogen in the range of 11.2–11.5 ppm was
observed (table I). Further confirmation of the struc-
tures of compounds 8 and 13 was based on their 13C,
15N spectra and X-ray analyses [7].
To investigate other ways of cyclization, which can
possibly lead to the formation of 1,2,4-triazole deriva-
tives with higher yield, three different media were
used in the cyclization reaction. According to the
method proposed by Le Count and Greer [5], the
respective succinate derivatives were refluxed in (a)
water, (b) acetic anhydride and (c) alloy bath without
any solvent (ca. 90 °C). It was found that all these
methods lead directly to the formation of only the
compounds 11–15, but with lower yields than when
the reaction is performed in methanol in the presence
of triethylamine [6]. The course of the reaction is
presented in figure 1.
Figure 1. Condition-dependent reactions of dimethyl 2-[(1-ary-
lamino-1-arylmethylidene)-hydrazono]succinate.

B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
95
Figure 2. Toxicity of compound 8 for HSF cells.
significantly on the presence of serum. When medium
supplemented with 1% fetal calf serum (FCS) was
used, the toxicity of derivatives was several times
lower than that in medium without FCS (figures 2
and 3). The compounds studied are likely to show
similar interactions with serum proteins such as Vrati-
zoline®, used as a medicine against herpes viruses [8].
Both derivatives used were more toxic for L929 cells
than for HSF; however, both of them at concentra-
tions of 50 mg ml–1 were non-toxic for HSF and
partially toxic for L929 cell cultures.
The experiments revealed that both substances pos-
sessed virucidal activity and during 1 h of contact
Figure 3. Toxicity of compound 13 for HSF cells.

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B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
Figure 4. Toxicity of compound 8 for L929.
Figure 5. Toxicity of compound 13 for L929.
4.1. Pharmacological tests
with virus caused the decrease in titers of viruses by
0.5–1.67 log (table II). Moreover, they were active
against viruses that belong to different families: Ade-
noviridae (AV-5), Rhabdoviridae (VSV) and Picor-
naviridae (EMCV).
Acute toxicity by accessing LD50 dose according to
the Wilcoxone and Litchfield method was investigated in
the Department of Pharmacology and Toxicology, Med-
ical University, Lublin, Poland. Acute toxicity of com-
pounds 6–10 in mice was low and was over 1750 mg
kg–1 i.p. when that of compounds 11–15 (published in
the paper [6]) was over 2000 mg kg–1 i.p. In behavioral
tests, compound 8 exhibited only weak analgesic activity
in the ‘‘writhing test’’ when compound 13 showed no
effect on the central nervous system of mice in all
behavioral tests applied.
Compound 13 from this group also exhibited moder-
ate antibacterial and antifungal activity with MIC val-
ues of 200–300 mg ml–1 against Escherichia coli, Brucella
abortus, Mycobacterium smegmatis, Candida albicans
and Epidermophyton floccosum [6]. Compounds 8 and 13
at a 500 mg ml–1 concentration did not affect the mi-
croflora of the human digestive tract [9] nor, at a
Both derivatives examined present in culture
medium during all steps of virus life-cycle (from ad-
sorption to release) inhibited AV-5 replication and
decreased final virus yield by 0.83 log (13) and 1 log
(8) (table III). It seems likely that both substances can
inhibit early steps of viral replication, as their pres-
ence during adsorption and penetration of viral life-
cycle decreased the final yield of EMCV by 0.59 log
(13) and the final yield of VSV by 1.77 log (table IV).
The level of inhibition of virus titer for compound 8
was about 10% and, for compound 13, about 37%.
When the influence of both derivatives on the eclipse
phase of viral replication (after adsorption and pene-
tration) was examined, no influence of both deriva-
tives on the final yield of VSV and EMCV was
observed (table V).
Table II. Virucidal activity of 8 and 13 derivatives.
Virus titer (CCID50 ml–1)*
Virus
VSV
EMCV
AV-5
Derivative (mg ml–1)
8
13
8
13
8
13
0 (control)
25
50
100
8.0090.45
7.0090.45
7.0090.45
7.0090.45
8.0090.45
7.3390.32
7.3390.36
7.5090.41
8.3390.36
7.6790.37
7.6790.37
7.5090.32
8.3390.36
7.6790.37
7.6790.37
7.6790.37
4.0090.45
3.7790.48
3.0090.45
2.5790.32
3.6790.32
3.0090.32
2.5090.32
2.0090.45
* Incubation of virus with derivatives – 1 h at 37 °C. The virus titers are shown in log9SD.

B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
97
Table III. The influence of derivatives on AV-5 replication in
HSF.
was washed several times with ethyl ether and finally
purified by crystallization from ethanol.
Derivative
Concentration (mg Virus titer (CCID50
ml–1)
ml–1)
5.1.2. General procedure for preparation of compounds
11–15
8
0
5
25
50
5.5090.32
5.0090.45
4.6790.37
4.5090.32
A quantity of 0.01 mol of compound 1–5 was
refluxed in 30 cm3 of water for 4 h. Solution was
concentrated to a volume of 10 cm3. The precipitate was
formed. After collection and purification by crystalliza-
tion, the compounds 11–15 were obtained in a yield of
50–60%; 0.01 mol of compound 1–5 was refluxed in 30
cm3 of acetic anhydride for 6 h. Solvent was removed
under reduced pressure and residue was purified by
crystallization (yield 70%); 0.01 mol of compound 1–5
was heated in the alloy bath in a temperature of ca.
100 °C for 10 h. Formed precipitate was collected and
purified by crystallization (yield 50%); ethyl ether from
combined extracts mentioned in 5.1.1 was distilled off
and the residue was purified by crystallization from
ethanol (yield 30–40%).
13
0
5
25
50
5.5090.41
5.0090.45
4.7790.48
4.6790.37
10–400 mg ml–1 concentration, the morphotic elements
of the green monkey kidney cells [10].
5. Experimental protocols
5.1. Chemistry
5.2. Virucidal investigations
Melting points measured on Boetius apparatus are
given uncorrected. H-NMR spectra were recorded on a
1
Toxicity for cell cultures and antiviral activity of
compounds 8 and 13.
Tesla BS 567A (100 MHz) apparatus in D6-DMSO with
TMS as an external standard. IR spectra were recorded
on a Specord IR-75 spectrometer. Results of elemental
analysis for C, H, and N by the method of microanaly-
sis (performed in the Department of Organic Chemistry,
Medical University in Lublin) were in accordance with
calculated values ( 0.7% for C, 0.75% for N, and 0.9%
for H).
Table IV. The influence of derivatives on viral adsorption and
penetration.
Derivative
Concentration Virus titer (CCID50 ml–1)
(mg ml–1)
VSV
EMCV
The purity of the obtained compounds was examined
by the TLC method. TLC was performed on 10×20-cm
pre-coated plates Si 60 F254 (E. Merck). The silica plates
were activated for 30 min at 110 °C. Compounds were
dissolved in ethanol or acetone. Samples (1 ml at 1 mg
ml–1) were spotted on the plates. The evolution was
carried out in Chromdes (Lublin, Poland) horizontal
sandwich DS-chamber [11, 12] with binary eluent con-
sisting of a mixture of chloroform and ethyl acetate
(70:30 or 75:25). The plates were dried and spots were
visualized under UV light at l=254nm.
8
0
50
4.7790.48
4.3390.36
3.6790.37
3.6790.37
13
0
50
4.7790.48
3.0090.45
3.6790.37
3.0090.45
Table V. The influence of derivatives on eclipse phase of viral
replication.
Derivative
Concentration Virus titer (CCID50 ml–1)
(mg ml–1)
VSV
EMCV
5.1.1. General procedure for preparation of compounds
6–10
8
0
50
8.0090.45
8.3390.36
8.2390.49
8.5090.32
Compounds 1–5 (0.01 mol) were dissolved in 30 cm3
of n-butanol and refluxed for 20 h. After this time,
solvent was removed under reduced pressure; the residue
13
0
50
8.0090.45
8.0090.45
8.2390.49
8.5090.32

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B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
5.2.1. Cell cultures
infected with the diluted virus; immediately after mixing
with derivatives, the cytopathic effect of virus (CPE)
occurring after 72 h of incubation at 37 °C was esti-
mated under a microscope and the titer of the virus was
calculated. The titer of the virus replicating in the
presence of the derivatives was compared to the control
without derivatives.
A strain of HSF was obtained by standard trypsyniza-
tion of a 23-year-old woman skin fragment (1 mm2).
HSF were routinely grown in DMEM (Dulbecco‘s
modified Eagle’s medium, Gibco) supplemented with
10% FCS (Gibco), 100 mg ml–1 of streptomycin, 100 U
ml–1 of penicillin in plastic tissue culture flasks (Nunc,
Denmark).
HeLa cells (human cervix carcinoma) and mouse cell
line L929 were obtained from the Institute of Immunol-
ogy and Experimental Therapy, Polish Academy of
Sciences, Wroclaw. They were grown in Eagle’s Minimal
Essential Medium (MEM, Sigma), supplemented with
10% FCS and 100 mg ml–1 of streptomycin and 100 U
ml–1 of penicillin. Cultures were incubated at 37 °C in
humidified atmosphere with 5% CO2.
5.2.5. The influence on viral adsorption and penetration
Suspension of viruses (VSV and EMCV) was mixed
(1:1 v/v) with derivatives at a concentration of 100 mg
ml–1 (final concentration 50 mg ml–1) prepared in MEM
without serum. L929 cells were immediately infected
with such mixtures and incubated at 37 °C for 1 h.
Controls were infected with the virus suspensions mixed
with medium alone. Cells after viral adsorption were
washed three times with medium to remove the rest of
non-absorbed virus and frozen. After thawing, the
viruses were titrated in L929 cell cultures and the titers
of viruses present in cultures treated with derivatives
were compared to controls.
5.2.2. Toxicity in cell cultures
Both substances examined were dissolved in dimethyl-
sulfoxide (DMSO, Sigma) 10 mg ml–1, and then diluted
in cell culture media supplemented or not with 1% FCS.
HSF or L929 cells were plated into 96-well plastic plates
(Nunc, Denmark) at a cell density of 1×104 (HSF) or
1×105 (L929) cells per well in appropriate media (with
10% FCS) to obtain the confluent growth of cells. After
overnight incubation at 37 °C, the media were removed
and cells were treated with 8 or 13 derivatives, diluted in
media at final concentrations of 500, 200, 100, 50, 25,
10, 5 and 2.5 mg ml–1. Cell cultures were incubated at
37 °C for 24–120 h. The toxicity was estimated by the
MTT method according to the assay described by Take-
nouchi and Munekata [13]. All experiments were done
in triplicates.
5.2.6. The influence on eclipse phase of viral replication
L929 cell cultures were infected with viruses (100
CCID50 ml–1), incubated for 1 h at 37 °C (absorption of
virus) and washed three times with medium. Infected
cell cultures were treated with derivatives (50 mg ml–1)
diluted in medium supplemented with 2% of FCS, incu-
bated at 37 °C for 24 h and frozen. Control cells were
not treated with derivatives. After thawing, viruses were
titrated in L929 cell cultures.
5.3. Pharmacology
5.2.3. Virucidal activity
5.3.1. Behavioral experiments
A suspension of viruses was mixed (1:1 v/v) with
derivatives 8 or 13, which were diluted in media without
FCS (final concentrations of derivatives: 25, 50 and 100
mg ml–1). The viruses’ suspension (VSV, EMCV or
AV-5) with media but without derivatives was a control.
Mixtures were incubated at 37 °C for 1 h and viruses
were titrated in appropriate cell cultures.
Behavioral experiments were carried out on male and
female albino Swiss mice (body weights of 20–25 g) and
male Wistar rats (200–250 g). Investigated compounds
were administered i.p. as suspensions in 3% Tween 80 in
a constant volume of 10 ml kg–1 in mice and 5 ml kg–1
in rats. The compounds were administered in doses
equivalent to 1/10 or 1/20 of LD50. Control animals
received the equivalent volume of solvent. Each experi-
mental group consisted of eight animals.
5.2.4. The influence on AV-5 replication in HSF
AV-5 virus suspension was serially diluted in MEM
supplemented with 1% of FCS and derivatives at final
concentrations of 5, 25, 50 mg ml–1. A suspension of
AV-5 diluted in medium alone was a virus control. The
confluent HSF cells grown in 96-well plastic plates were
5.3.2. Acute toxicity in mice for compounds 6–10
Acute toxicity was assessed by the methods of
Litchfield and Wilcoxon [14] and presented as LD50
calculated from the mortality of mice after 24 h.

B. Modzelewska-Banachiewicz, T. Kamin´ska / European Journal of Medicinal Chemistry 36 (2001) 93–99
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